Separation And Purification Of Polysaccharide From Dendrobium Officinale And Regulation Of Glucose Metabolism Disorders On Mice Of T2DM

DM(diabetes mellitus) is permanent metabolic disease,characterized by chronic hyperglycemia,which is recognized as one of the three diseases that could cause serious damage to the human health currently.According to the world health organization(WHO),more than 90% diabetes patients are type 2 diabetes mellitus(T2DM),which has obvious rising trend and is great threaten to people's health.As the traditional treatment of diabetes drugs have many severe side effects,such as liver and kidney toxicity,hypoglycemia,gastrointestinal,so the development of prevention and treatment of hyperglycemia natural drugs and health food is of great significance.On the process of looking for the resources of new drug,Chinese herbal polysaccharide have many advantages to the tradition drug,which made more and more attention to the discovery.Recent studies show that Dendrobium rich in polysaccharide, Dendrobium bases, amino acids, stilbene and derivatives thereof and other active ingredients, has a hypoglycemic, lipid-lowering, anti-inflammatory, anti-aging, enhance immunity and other effects, and Dendrobium polysaccharide is the main antidiabetic active component of the Dendrobium officinale Kimura et Migo.At present, Dendrobium officinale polysaccharide and its prescription for the prevention and treatment of diabetes have reported, but the molecular mechanism of hypoglycemic.almost have no reports.Therefore,further study of the hypoglycemic activity of Dendrobium officinale polysaccharide to clarify its hypoglycemic mechanism becomes important.In this study, the Dendrobium officinale polysaccharide was extracted from the stem of the Dendrobium officinale Kimura et Migo and then purified. And on this basis, we try to explore the effect on treatment and mechanism of diabetes to provide a new theoretical and experimental basis for the development of clinical applications of Dendrobium officinale polysaccharide and health products.Part I Extraction and purification of Dendrobium Officinale PolysaccharideThe crude Dendrobium officinale polysaccharide was extracted by the method of Water extraction and alcohol precipitation,and then bleached by low concentrations of hydrogen peroxide,after that Freezing and thawing to remove tannins acid and protein.At last,after dialysis and separated by G50 gel column,clear Dendrobium officinale polysaccharide solution was obtained.The total sugar content was about 105% measured by phenol-sulfuric acid method. The molecular weight was 1.23×105 Da measured by HPGPC. Monosaccharide composition was glucose and mannose measured by PMP-HPLC.UV scan showed that DOP almost has no acids and proteins.These results show that the DOP meet the follow-up experiment requirements.Part II The therapeutic effect of DOP on type? diabetic miceT2DM model was induced by repeated low dose injection of STZ(66mg/kg) after four weeks high fed diet.50 mice were randomly selected and fed with HFD,Another 10 mice were randomly selected and kept with regular chow as normal control group(NC).After 4 weeks,the mice that fed with HFD were given injection of STZ(i.p.,q.w.d.,66mg/kg) and after another 4 weeks,FBG were determined.Using fasting blood glucose FBG>11.1mmol/L as a model for the successful establishment of type 2 diabetic model,the success rate of model was 90%.Type 2 diabetic mice were randomly divided into 4 groups with 10 mice in each group. respectively divided as diabetes control group(DC, i.g.,equal volume of normal saline,b.l.d.×28),low dose of DOP group(DOP-L,i.g.,b.l.d.×28,100mg/kg),with the middle dose of DOP group and high dose of DOP group(DOP-H,),the dose of DOP were 200mg/kg?400mg/kg,and other the same with DOP-L.During the experiment, the active state of mice was observed, and the weight and blood glucose were recorded.Oral glucose tolerance test(OGTT) were taken in twenty days.And in twenty-eighth days, the serum insulin(FINS),blood lipid,muscle and liver glycogen were measured,and superoxide enzyme(SOD),malondialdehyde(MDA),glutathione(GSH),GSH-PX) also been determined.Experimental results show that the mice that given DOP were in good mental condition,the trend of weight loss was significantly decreased and FBG significantly lower the DC group.At the same time, the oral glucose tolerance test(OGTT) and insulin resistance index(HOMA-IR) confirmed that DOP could significantly improve the glucose tolerance of diabetic mice, increase the serum insulin level in diabetic mice and reduce insulin resistance index.In addition,DOP can significantly reduce total cholesterol and triglyceride levels in diabetic mice and performed a obvious dose dependence.TO some extend,that indicated that DOP has effection on regulation of lipid metabolism in diabetic mice.These results confirmed that the Dendrobium officinale polysaccharide can improve diabetic mice in vivo antioxidant enzyme activity and reduce the content of lipid peroxide(LPO), so as to enhance the scavenging free radical capacity and reduce the generation of reactive oxygen species(ROS), played against oxidative stress, prevent the oxidative damage. SO we expect that oxidative stress may be the Dendrobium officinale polysaccharide anti diabetic and one of the ways to alleviate insulin resistance.Part III Research on the regulation of glucose metabolism disorders of DOP in T2 DM miceTo explore the mechanism of DOP of regulation glucose metabolism disorders,we employed HE staining and PAS staining of liver tissue of diabetic mice to observe the morphological changes of liver tissue and liver glycogen content detection.With HE,we noticed that the liver of NC group mice is complete and clear. Liver cells are arranged in a regular pattern, and the cytoplasm is even and the shape present normally;while the DC group,behaviors the disorder of liver cells,there are obvious fatty degeneration and phenomennon of nuclear and cytoplasmic separation;And compared with the DC group,the liver structure of the mice treated with DOP is clear,the liver structure was clear, the cell arrange regularly, the hepatic steatosis is significantly of great reduction.With PAS,we observed that the cytoplasm of hepatic cells of DC group mice has less purple granules and the cell arrange loosely, while the NC group,the cytoplasm of the cells increased obviously with the increase of the red particles.Experimental results show that after the treatment of the Dendrobium officinale polysaccharide, type II diabetes in mice liver tissue damage in a certain extent can repair and the content of glycogen in liver tissue were significantly increased. To reveal the Dendrobium officinale polysaccharide to the protective effect on diabetic hepatic tissue and increase liver glycogen synthesis to regulate glucose metabolism.In addition, to explore the mechanism of regulation of glucose metabolism of Dendrobium officinale polysaccharide from the molecular level,we adopted Western blot to determine the expression of the key protein in the insulin signal pathway,such as IRS-??IRS1,JNK?PI3K and Glut2 protein in the liver tissue of diabetic mice.According to the results,DOP could significantly inhibit the expression of JNK protein in liver tissue of diabetic mice,inhibition rates were35.31%, 68.77%, 84.88% and there was a significant dose-dependent manner.Meanwhile, DOP can significantly improve the expression of protein IRS-?, PI3 K, AKT and Glut2 in the liver tissue of diabetic mice.This part of the experiment confirmed,DOP decreased the expression of JNK in liver tissue of diabetic mice by anti-oxidative stress,thereby promoting the combination of IRS1 and IRS-?,increasing the expression of PI3 K,so that affecting the entire PI3 K pathway,to make the increase of Glut2.ultimately against diabetes and insulin resistance, thereby improving the regulation of glucose metabolism effect.