| ObjectiveObesity is associated with metabolic abnormalities such as disorders of glucolipid metabolism.It has been found that plant polysaccharides have the physiological activity of regulating disorders of glucolipid metabolism.Based on this study,crude polysaccharides of Euryale ferox Salisb.were extracted from the dried seed kernels of Euryale ferox Salisb.,isolated and purified,and structurally characterized.Finally,an animal model was used to study how Euryale ferox Salisb.polysaccharide could improve the disorders of glucolipid metabolism in obese mice by regulating intestinal flora.An obese mouse model was constructed to investigate the effect of Euryale ferox Salisb.polysaccharide on improving disorders of glucolipid metabolism and the association with the composition and abundance of intestinal flora.MethodThe crude polysaccharides of Euryale ferox Salisb.were extracted from the dried seed kernels of Euryale ferox Salisb.by hot water extraction,and separated on a diethylaminoethylcellulose 52(DEAE-52)chromatographic column,and structurally characterized by infrared spectroscopy,Congo red experiment,liquid chromatography,gas chromatography-mass spectrometry,scanning electron microscopy and nuclear magnetic resonance.A high-fat diet was used to construct an obese mouse model to study the ameliorative effect of Euryale ferox Salisb.polysaccharide on disorders of glucolipid metabolism.Fifty 8-week-old C57BL/6J mice were randomly divided into control group,model group,and Euryale ferox Salisb.polysaccharide intervention group(400 mg/kg d,600 mg/kg d,800 mg/kg d),and 10 mice in each group were gavaged at 3 pm daily for 8 weeks.Before the end of the intervention,OGTT glucose tolerance test was performed.At the end of the intervention,serum,liver,epididymal fat,perirenal fat,subcutaneous fat and cecum contents were collected,glucose and insulin were measured separately,and insulin resistance coefficient(HOMA-IR)was calculated;serum levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDLc)and high-density lipoprotein(HDL-c)were measured;the weight of adipose tissue was weighed and the Adipose/body mass index;HE staining of epididymal fat and HE and PAS staining of liver;detection of liver TG;RT-PCR for liver Foxol,G6pc,Pparg,Fasn,Cebpa,Srebf-1c mRNA expression;detection of intestinal flora of cecum contents using 16s rRNA.Results1.The dried seed kernels of Euryale ferox Salisb.were extracted by hot water,de-starch by high temperature resistant α-amylase,and crude polysaccharide was obtained after protein removal by Sevag method,alcohol precipitation and dialysis,with the content of 70.15%,protein content and total phenol content of 0.02%and 0.66%,respectively;the gradient chromatography of crude polysaccharide of Euryale ferox Salisb.was performed by DEAE-52 chromatographic column to obtain the pure water fraction,which had 81.45%polysaccharide content and it almost did not contain phenols and proteins.The structural characterization results showed that the fraction of Euryale ferox Salisb.polysaccharide had a molecular weight of 3319 Da and was mainly composed of glucose(99.53%).The conformational results showed that the fraction of Euryale ferox Salisb.polysaccharide had no triple helix structure,and the overall arrangement was disorganized,with the surface showing fish scale projections and clear but irregular edges.The structure of the fraction of Euryale ferox Salisb.polysaccharide was further characterized by methylation and NMR structure,and the results showed that the glycosidic bonds of the fraction of Euryale ferox Salisb.polysaccharide were all glucose residues,and their linkages were mainly t-Glcp,4Glc(p),6-Glc(p)and 4,6-Glc(p),and the primary structure was a glucan with→4)-αD-Glcp-(1→as the main chain,α-D-Glcp-(1→ is attached at position 6.The results of in vitro digestive enzyme inhibition experiments showed that the crude polysaccharide of Euryale ferox Salisb.had an inhibitory effect on the activity of digestive enzymes,in which the inhibitory effect on α-amylase activity was higher than that of α-glucosidase.2.The results of animal experiments showed that in terms of glucose metabolism,fasting blood glucose regressed and the area under the OGTT curve decreased in obese mice;the crude Euryale ferox Salisb.polysaccharide intervention group could significantly reduce the insulin resistance coefficient of mice(P<0.01)and improve glucose metabolism homeostasis by significantly reducing the mRNA(P<0.05)expression of Foxol and G6pc,genes associated with glucose metabolism abnormalities in the liver.Regarding lipid metabolism,compared with the obesity model group,the crude Euryale ferox Salisb.polysaccharide intervention group showed significantly slower weight gain(P<0.01),significantly lower serum triglyceride(TG)and low-density lipoprotein content(LDL-c)(P<0.05),and significantly increased high-density lipoprotein(HDL-c)content(P<0.05);fat accumulation such as liver was significantly improved,probably by significantly decreasing liver Pparg mRNA expression(P<0.01)to reduce lipid formation.The results related to glucolipid metabolism suggested that Euryale ferox Salisb.polysaccharide has a certain ameliorative effect on the disorders of glucolipid metabolism in obese mice.3.16s rRNA sequencing results showed that the crude Euryale ferox Salisb.polysaccharide intervention improved the Shannon index and Simpson index and increased the diversity of intestinal flora in obese mice.LFfSe analysis showed that there were 43 dominant flora in the control group,2 dominant flora in the model group,and a total of 30 dominant flora in the crude Euryale ferox Salisb.polysaccharide intervention group,with the dominant flora Roseburia,Ruminococcaceae,Oscillibacter,Veillonella,Intestinimonas,Pseudoflavonifractor and Clostridium-IV can have beneficial effects on the organism by breaking down hard-to-degrade substances such as lignin and producing intestinal flora metabolites.ConclusionEuryale ferox Salisb.polysaccharide is a glucan composed of glucose with αconfiguration and β-configuration.Euryale ferox Salisb.polysaccharide can improve the disorders of glucolipid metabolism in obese mice by reducing impaired glucose tolerance,maintaining homeostasis of glucolipid metabolism and fat accumulation.The possible mechanism is that Euryale ferox Salisb.polysaccharide improves the disorders of glucolipid metabolism in obese mice by increasing the diversity of intestinal flora in obese mice,increasing the dominant flora of short-chain fatty acid producing Roseburia,Intestinimonas and Ruminococcus etc.,and ameliorate the disorders of glucolipid metabolism in obese mice. |
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