Isolation,Purification,Structural Identification And Hypoglycemic Activity Of Polysaccharides From Hovenia Dulcis

Diabetes is a chronic metabolic disease,which is also a syndrome with typical characteristics of sustained hyperglycemia caused by relative or absolute insufficient insulin secretion resulting in disorder of glycometabolism,lipid metabolism and protein metabolism.The World Health Organization(WHO)has classified diabetes mellitus into four groups: type 1 diabetes mellitus(T1DM),type 2 diabetes mellitus(T2DM),gestational diabetes mellitus(GDM),and other diabetes mellitus.According to the latest statistics from the International Diabetes Federation(IDF),there were about 463 million diabetic patients in the world in 2019 and approximately 116 million of them were in China,the highest in the world.On current trends,it is easy to speculate that the number of diabetic patients in the world will reach 700 million by 2045.At present,insulin injection and oral hypoglycemic drugs are the most effective therapies for diabetes and diabetic complications,while most oral hypoglycemic drugs have a certain toxic side effects.Therefore,it is quite crucial to find out a convenient and effective drug with little or no side effects for prevention and treatment of diabetes.Hovenia dulcis belongs to genus Hovenia in the family Rhamnaceae,whose edible part is pedicel.The research indicated that pedicel of Hovenia dulcis was rich in polysaccharide,flavonoids,triterpenoid saponins,and alkaloids and other active components.In recent years,efficiency of Hovenia dulcis in nutrition and healthcare is more and more popular.However,development in Hovenia dulcis resources was mainly focused on Hovenia dulcis Thunb,t edible part(pedicel)of Hovenia dulcis was mainly used for the developing fruit vinegar,fruit wine,and fruit juice,Few studies on extraction,separation,purification and functional properties of bioactive small molecular substance(such as flavonoids)in Hovenia dulcis were reported.The problem with low added value of Hovenia dulcis industrial products were still existed caused by no in-depth study,resulting in huge waste of resources.Therefore,it is highly necessary to improve value-added of Hovenia dulcis and reduce the waste of resources in the Hovenia dulcis industy.Based on above,we make pedicel of Hovenia dulcis as the object of this study and explore active component of plant polysaccharides.First,we extracted Hovenia dulcis polysaccharide by three kinds of extraction methords,screened the polysaccharide samples with the highest hypoglycemic activity in vitro.Then separation,purification and structural analysis of Hovenia dulcis were conducted.Finally,we investigated hypoglycemic effects and mechanism of purified components in Hovenia dulcis polysaccharide on T1 DM and T2 DM.The main results of the study were as follows:(1)Effects of three extraction methods on physicochemical properties,structural properties and biological activity of polysaccharides from Hovenia dulcisThe Hovenia dulcis polysaccharide(HDPs)was extracted respectively by hot water extraction,accelerated solvent extraction and ultrasonic assisted extraction,named as HWE-HDPs,ASE-HDPs and UAE-HDPs.Then,effects of the three extraction processes on physicochemical properties,structural properties and biological activities of Hovenia dulcis polysaccharide were discussed.The results showed that the three extraction processes had obvious effects on basic chemical composition.The average molecular weight of HWE-HDPs was significantly higher than that of ASE-HDPs and UAE-HDPs.The monosaccharide compositions of HWE-HDPs mainly contained galacturonic acid,galactose and arabinose.The monosaccharide compositions of ASE-HDPs and UAE-HDPs mainly contained rhamnose,glucose,galactose,and arabinose.In addition,all the three kinds of polysaccharides all had a certain hypoglycemic activity in vitro.The inhibition ability of ?-glucosidase and improvement of insulin resistance on HEP-G2 cells of HWE-HDPs was significantly higher than that of ASE-HDPs and UAE-HDPs.(2)Isolation,purification,structure identification of polysaccharides from Hovenia dulcisThe Cellulose DEAE-52 anion exchange column chromatography method and Sephaex G-100 size exclusion column chromatography method were used for isolation and purification of HDPs.Then the purity and molecular weight distribution of the crude polysaccharides were determined.Finally,chemical analysis and modern instrumental analysis were used to identify the structure of crude polysaccharides.The results showed that three polysaccharide composition(HDPS-1,HDPs-2,and HDPs-3)were obtained by separation and purification of DEAE-52 anion exchange column chromatography,among them,HDPs-2 had highest purification yield and ?-glycosidase inhibition.Then,Sephadex G-100 column chromatography was used for the separation and purification of HDPs-2 to obtain a single polysaccharide component,HDPs-2A,and the yield was 19.63 of the crude polysaccharide HDPs.HDPs-2A was a pure polysaccharide with a uniform molecular weight.The average molecular weight was 372.91 k Da,,total sugar content was 84.22%,uronic acid content was 5.35%,and no protein was contained.HDPs-2A was mainly composed of Man,Rha,Glc A,Gal A,Glc,Gal,Ara,and molar percentages were 3.64%,1.41%,4.67%,5.16%,3.01%,60.02%,and 22.09%,respectively.The results of periodate oxidation,Smith degradation,and NMR analysis showed that HDPs-2A was made up of eight glycoside bonds,including ?-L-Araf-(1???3,5)-?-L-Araf-(1? ? ?3)-?-L-Araf-(1? ? ?3,6)-?-D-Manp-(1? ? ?3)-?-D-Galp A-(1? ??6)-?-D-Galp-(1? ? ?-D-Glcp A-(1?,and ?6)-?-D-Glcp-(1?.The result of atomic force microscopy(AFM)showed that HDPs-2A presented irregular polymer particles in water.The result of X-RD indicated that HDPs-2A exists as a single crystal structure.(3)Hypoglycemic effect and mechanism of HDPs-2A on type 1 diabetes mellitusThe HDPs-2A was taken as research object.T1 DM rats model was induced by streptozotocin(STZ),and the experimental rats were randomly divided into 6 groups(8 in each group): blank control group(NG),model group(DM),positive control group(MET),low dosage of HDPs-2A group(L-PA),medium dosage of HDPs-2A group(M-PA),and high dosage of HDPs-2A group(H-PA).All experimental rats were gavaged for 4 weeks.The results showed that high and medium doses of HDPs-2A could increase body weight,serum insulin and liver glycogen levels in T1 DM rats,decrease blood glucose levels and improve oral glucose tolerance in T1 DM rats.Moreover,medium and high dose of HDPs-2A could also partially restore pancreatic ?-cell damage,reduce oxidative stress response of pancreas,inhibit the synthesis of serum proinflammatory factors.The hypoglycemic effect of H-PA on T1 DM was not significantly different from that of MET.The results of Real-time quantitative PCR and Western Blotting showed that: In M-PA and H-PA groups,1)Expression of PDX-1 in pancreatic was significantly up-regulated,and then expression of IRS2 was activated and up-regulated,so as to regulate apoptosis and regeneration of pancreatic ?-cell,restoring functional damage of pancreatic ?-cell;at the same time,expressions of GK and GLUT2 in pancreas were up-regulated to improve insulin secretion capacity of pancreatic ?-cells,improving disorder of glycometabolism.2)Expression of GK in liver was significantly up-regulated and expression of G6 Pase was significantly down-regulated,so as to improve ability of liver glycogen synthesis and inhibit liver gluconeogenesis,thereby improving disorder of glycometabolism in liver.In summary,hypoglycemic effect of HDPS-2A on T1 DM may be as follows: expressions of PDX-1,IRS2,GK,and GLUT2 in pancreatic were up-regulated to regulate apoptosis and regeneration of pancreatic ?-cells,promoting insulin secretion.Meanwhile,expression of GK was up-regulate and expression of G6 Pase down-regulated in liver to improve glycometabolism disorder.Finally,function of improving T1 DM was achieved.(4)Hypoglycemic effect and mechanism of HDPs-2A on type 2 diabetes mellitusThe HDPs-2A was as research object.T2 DM rats model was induced by high fat and high sugar combined with low dose STZ.The experimental rats were divided into 6 groups as well as the group in hypoglycemic experiment of T1 DM rats,and all rats were intervened by gavage for 4 w.The results indicated that medium and high doses of HDPs-2A increased body weight,reduced blood glucose levels,improved oral glucose tolerance,and increased serum insulin and liver glycogen levels in T2 DM rats.In addition,medium and high dose of HDPs-2A could also partially restored tissue damage of liver,reduced oxidative stress of liver,and finally adjusted disorder of glycometabolism in T2 DM rats,and increase short chain fatty acid(SCFAs)levels in T2 DM rat feces.There was no significant difference between hypoglycemic effect of H-PA on T2 DM rats and that of the MET group.The results of Real-time quantitative PCR and Western Blotting showed that: In M-PA and H-PA groups,1)They could significantly increase expression of Ins R and IRS2 in liver of T2 DM rat,and the PI3 K was activated.Thus,expression of Akt,a key signaling molecule downstream of PI3 K,was activated and up-regulated.Furthermore,expression of the GLUT4 was up-regulated to promote the absorption and utilization of glucose in liver of T2 DM rats,improve the insulin sensitivity of liver,and reduce the insulin resistance of liver.2)Expression of p-AMPK in liver of T2 DM rats was significantly up-regulated,then the AMPK pathway was activated,and expression of G6 Pase and PEPCK,which were mediated by AMPK pathway,were down-regulated,so as to inhibit hepatic gluconeogenesis and improve disorder od glycometabolism.3)Expression of glycogen synthase kinase(GSK-3?)was significantly down-regulated in liver of T2 DM rats,and then expression of GS was activated and up-regulated to promote glycogen synthesis of liver.It also significantly down-regulated expression of Fox O1,a key regulator of hepatic gluconeogenesis,so as to inhibits hepatic gluconeogenesis,reduces hepatic glucose output,and improves hepatic insulin resistance.4)The expression of PPAR? and PGC-1? were significantly up-regulated.Activation of the PPAR /PGC-1 signaling pathway would lead to up-regulation of PI3K-P85 and GLUT4,as well as activation of AMPK and glycometabolism-related kinases,improved glucose transport,enhanced synthesis of liver glycogen,improved disorder of glycometabolism in liver,and reduced insulin resistance of liver.That is,hypoglycemic effect of HDPs-2A on T2 DM was may be as follows: by activating the upstream and downstream related genes of the PI3K/Akt signal transduction pathway,the insulin resistance of liver was reduced.The AMPK pathway and glycometabolism-related enzymes were activated to improve disorder of glycometabolism in liver.Regulation of GS/SGK-3? signal transduction and down-regulation of Fox O1 expression to improve disorder of glycometabolism and insulin resistance in liver.Regulation of the PPAR? /PGC-1? signaling pathway,which in turn regulates the expression of other related signaling factors,to improve disorder of glycometabolism and reduce insulin resistance of liver.In conclusion,HDPs-2A could improve T2 DM by improving disorder of glycometabolism and reducing insulin resistance of liver,and the two mechanisms could adjust each other.Conclusion: In this study,the hypoglycemic activity of HDPs was taken as starting point.Firstly,the polysaccharide was extracted,isolated and purified,and its structure was analyzed,then its hypoglycemic mechanism for type 1/Type 2 diabetes was discussed.The experimental results were as follows: three extraction processes were used to extract polysaccharide,among which polysaccharide extracted by hot water had strong hypoglycemic activity in vitro.Therefore,HDPS-2A with high hypoglycemic activity was isolated and purified,which mainly contained 8 glycoside bonds,?-L-Araf-(1? ? ?3,5)-?-L-Araf-(1? ? ?3)-?-L-Araf-(1? ??3,6)-?-D-Manp-(1? ? ?3)-?-D-Galp A-(1? ? ?6)-?-D-Galp-(1? ? ?-D-Glcp A-(1?,and ?6)-?-D-Glcp-(1?.And then,HDPS-2A was used as the research object,and the hypoglycemic effect mechanism of HDPS-2A on T1 DM was found to be: by regulating expression of pancreatic related genes in T1 DM rats,the apoptosis and regeneration of pancreatic ?-cells and the promotion of insulin secretion were regulated.By regulating expression of enzymes related to liver glycometabolism,the disorder of liver glycometabolism was improved in T1 DM rats.And for the hypoglycemic effect of HDPS-2A on T2 DM was as follows: by activating PI3K/Akt signal and expression of liver glycometabolism-related pathways or genes,it could improve disorder of liver glycometabolism and reduce insulin resistance of liver,and two mechanisms could regulate each other to improve T2 DM..