| Objective: In this paper,polysaccharides were extracted and separated from the root of Burdock.The relationship between the molecular weight of Burdock root polysaccharides(BP)and the inhibition of α-glucosidase and antioxidant activity was studied.The animal model of diabetes mellitus(DM)was established to study the BP.The hypoglycemic activity of BP in vivo was further explored to investigate the effect of BP on visceral and glucose and lipid metabolism in diabetic rats.Method: 1.The single factor analysis and orthogonal optimization experiments were used to determine the best extraction process of BP.The effects of temperature,acid and alkali,light,redox and metal ions on the stability of BP were preliminarily investigated.2.The different polar components in BP were separated by stepwise alcohol precipitation and analyzed by HPLC.The molecular weight of each component was analyzed by size exclusion chromatography combined with RID-GPC software to explore the differences in in vitro antioxidant activity.3.The α-glucosidase inhibition assay was used to study the optimal enzyme inhibitory activity of BP in vitro.4.80 male SD rats were randomly divided into normal group and high-fat group,fed with normal feed and high-sugar and high-fat diet,respectively.After 7 weeks,the rats in the high-fat group were fasted for 12 hours,and the rats whose body weight did not exceed 10%in the normal group were removed.A diabetic model was established by intraperitoneal injection of 40 mg/kg Streptozotocin(STZ).One week after blood sugar stabilization,fasting blood glucose(FBG)≥ 11.1 mmol/L,the model rats were randomly divided into model group(normal saline),acarbose group(200mg/kg),BP low,middle and high dose groups(200mg/kg,400mg/kg,800mg/kg).Dosing once daily for 6 weeks.After the last administration,an oral glucose tolerance test(OGTT)was performed to calculate the area under curve of glucose(AUC).The rats in each group were fasted for 12 hours after fasting and anesthetized anesthetized.Abdominal aorta was used to take blood to prepare plasma.Triglyceride(TG)and Total Cholesterol(TC)were detected by the kit method.Low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),blood urea nitrogen(BUN),creatinine(CRE),Malondialdehyde(MDA),Nitric oxide synthase(NOS),Superoxide dismutase(SOD)content;Enzyme linked immunosorbent assay(ELISA)assay Insulin(INS),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)levels in rat plasma;HE staining for rat pancreas,kidneys Histopathological morphology of the liver;MASSON staining analysis of renal fibrosis in diabetic rats;oil red O staining of rat liver fat content;The expression levels of AMPK alpha1,SREBP1 and AKT protein in rat liver were detected by Western blot.Result: 1.The optimal extraction conditions for BP are solid-liquid ratio 1:25,extraction temperature 80°C,and extraction time 90 min.The maximum yield under this condition is 67.54%.BP has good thermal stability,is relatively stable under light conditions,is easily oxidized,and has poor stability in p H>8 or p H<4 and in solutions containing Cu2+ and Fe3+.2.In this experiment,the weight-average molecular weight of BP was distributed below 5500 Da.In the stepwise alcohol precipitation,the molecular weight of polysaccharide was precipitated at 40%-80%,and the molecular weight of the polysaccharide was 5258,5201,4695,3074,1728 Da.The 70% alcohol precipitation polysaccharide component had the best free hydroxyl radical and DPPH free scavenging activity and reached 94.52% and 72.17% at2 mg/m L,respectively.3.Compared with the normal group,the blood sugar of the model rats was significantly higher(p<0.01);after six weeks of continuous treatment,the FBG of the BP-M group was reduced to 19.84 mmol/L(p< 0.05),BP-H group rats FBG reduced to 19.02 mmol / L(p <0.01),BP-M,BP-H OGTT and AUC were significantly lower than the model group rats(p <0.01).4.Compared with the normal group,the levels of TC,TG,LDL-C,NOS,MDA,CRE,BUN,TNF-α and IL-6 in plasma of the model group were significantly increased(p<0.01).HDL-C,SOD The INS level was significantly lower(p<0.01).Compared with the model group,BP significantly reduced the plasma levels of TC,TG,and LDL-C in diabetic rats(p<0.05 or p<0.01)and increased HDL-C levels(p<0.05 or p<0.01)to improve hyperlipidemia in diabetic rats;BP can significantly reduce the plasma levels of NOS and MDA in diabetic rats(p<0.01)and increase SOD content(p<0.05 or p<0.01).Improve the level of oxidative stress in rats;BP can reduce the level of CRE,BUN in plasma of diabetic rats(p<0.05 or p<0.01),improve the damage of renal function in rats;BP can reduce the plasma level of TNF-α in diabetic rats.The content of IL-6(p<0.05 or p<0.01)decreased the levels of inflammatory factors in the body;BP could significantly improve the insulin resistance in diabetic rats(p<0.05),but had no significant effect on plasma INS levels.5.Compared with the model group,the HE staining results showed that BP had no significant effect on the islets after the destruction of diabetic rats;BP treatment improved tubular atrophy,vacuolar degeneration and other lesions;BP can improve the cause of diabetes Liver cell necrosis and steatosis.MASSON staining results showed that BP can reduce the level of renal fibrosis in diabetic rats.Oil red O staining results show that BP can significantly reduce the fat content in the liver of diabetic rats.Western blot results showed that BP up-regulated the expression of AMPK alpha1 protein(p<0.05 or p<0.01),improved the imbalance of glucose metabolism in diabetic rats,BP up-regulated the expression of AKT protein(p<0.05),and improved diabetic rats.Livercell viability;BP up-regulated SREBP1 protein expression level(p<0.05)and reduced hepatic intracellular cholesterol content in diabetic rats.conclusion: 1.BP has a number of molecular weight components,can inhibit the inhibition of α-glucosidase activity in vitro,and its biological activity is related to the size of the molecular weight;2.BP can reduce fasting blood glucose in diabetic rats and has a protective effect on liver and kidney.Its hypoglycemic effect may be through the inhibition of α-glucosidase activity in vivo,slowing down the absorption of carbohydrates;up-regulation of AKT,AMPK alpha1,SREBP1 protein expression levels improve glucose and lipid metabolism. |
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