| ObjectiveIn this study,we investigated the effects of different doses of Rubusoside,Chitosan Oligosaccharides(COS)and the combination of the two groups on diabetic mice,further discussed the potential mechanisms,and provided the theoretical supports on diabetes treatments in clinical practice.In this study,we compared four extracted technologies of Rubuso side from Sweet tea by their results of extraction rate of Rubusoside,and explored the best extraction methods and conditions.Methods1 Study on the effects of Rubusoside,COS,and the combination of the two groups on diabetic mice.310 adult male Kunming mice were purchased.After five days adaptive feeding,the mice were injected fresh Streptozotocin after random selection of 13 mice as a blank control group.Mice with blood sugar level higher than 14mmol/L and less than 25mmol/L was hyperglycemia model animal.The hyperglycemia model animals were randomly divided into 11 groups:one model control group,one standard control group(Metformin),and 9 dose groups,including three groups of Rubusoside(low-dose group,middle-dose group and high-dose group),three groups of COS(low-dose group,middle-dose group and high-dose group)and three groups of combination(low-dose group,middle-dose group and high-dose group).Continuous intragastric administration for 12 weeks,we recorded the general situation of animals.After the end of the experiment,we measured fasting blood glucose(FBG)and blood glucose curve area.We extracted the eyeball,collecting blood to measure glycosylated hemoglobin(HbAlc),insulin(INS),total cholesterol(TC),triglyceride(TG),high-density lipoprotein(HDL),low density lipoprotein(LDL),blood urea nitrogen(BUN),creatinine(Cre),liver glycogen(HG),Alanine aminotransferase(ALT),Aspartate aminotransferase(AST).2 Study on the exploration of the extraction methods and conditions of RubusosideUsing water as the extraction solvent,four extraction technologies were used to extract Rubusoside from Sweet tea.We explored the effect of extraction time,extraction temperature and solid-liquid ratio on extraction rate in ultrasonic extraction,the effect of extraction time,solid-liquid ratio and extraction times on extraction rate in reflux extraction,the effect of enzyme concentration on extraction rate in enzyme digestion,and the effect of the solution pH,the power of microwave digestion instrument,and extraction time in microwave digestion.The content of Rubusoside was determined by HPLC and the standard curve was drawn.Took the extraction rate as the evaluation index and explored the best extraction methods and conditions.Results1 Study on the effects of Rubusoside,COS,and the combination of the two groups on diabetic mice.1.1 Diabetic mice obviously showed the symptoms of disease.The weights of mice in diabetic model groups were significantly lower than those in blank control group(P<0.05).1.2 Compared with the model control group,groups of Rubusoside and combination can reduce the level of FBG,HbA1c and blood glucose curve area.COS and combination can reduce the level of TG、TG and LDL-c,and increase the level of HDL-c(P<0.05).High-dose Rubusoside,and high and middle-dose combination can increase the level of HG(P<0.05).High-dose groups of COS and combination can decrease the level of AST and ALT(P<0.05).High-dose Rubusoside can reduce the level of LDL-c and AST,and high and middle-dose COS can reduce the level of blood glucose curve area(P<0.05).There was no abnormality in the level of serum Cre and BUN in individual intervention groups.1.3 Compared with individual groups,the levels of TG and ALT in high-dose group of combination were significantly lower than those in high-dose group of Rubusoside,and the levels of FBG.HbAlc and blood glucose curve area were lower than those in high-dose group of COS(P<0.05).The levels of HbAlc and blood glucose curve area in medium-dose of group of combination were lower than those in middle-dose group of COS,and the levels of TC and LDL-c were higher than those in medium-dose group of COS(P<0.05).The level of HbAlc in low-dose group of combination was higher than that in low-dose group of Rubusoside,and TC and LDL-c were significantly higher than those of low-dose group Rubusoside(P<0.05).The level of HbA1c and blood glucose curve area in low-dose group of combination were significantly lower than those in low-dose group of COS(P<0.05).There was no statistical significance among other groups(P>0.05).1.4 Compared with the model control group,hepatic cord structures were clear,and the liver cells were still tidy arrangement.Liver cell injuries were mitigated,the liver cells with steatosis were decreased,and the number of cells with vacuolation was significantly reduced,even disappeared in individual intervention groups.Renal histopathology reveled that,glomerular structural integrity,and the volume of glomerular cells was decreased.A small amount of neutrophils infiltration in the renal interstitium,and tubular epithelial oedema was improved.The lumen open,and the number of cells with degeneration was significantly reduced,even disappeared in individual intervention groups.In individual intervention groups,islets of pancreas were less wounded than that in the model control group,and the shapes of the islets were more regular and larger,and with more cells.The amount of neutrophils infiltration was small,and the number of cells with degeneration was significantly reduced,even disappeared.After each intervention,the dose-effect relationships on organs were obvious.2 Study on the exploration of the extraction methods and conditions of RubusosideUltrasonic extraction,reflux extraction,enzymatic extraction and microwave extraction are effective methods for the extraction of Rubusoside.The longer the extraction time,the higher the solid-liquid ratio,and the higher the extraction rate in Ultrasonic extraction.There were no significant differences in the extraction rate between the reflux extraction method,the solid-liquid ratio and the extraction times.Extraction rate was reduced with the increase of the cellulase in enzymatic extraction.The lower the pH value and the higher the power,the higher the extraction rate in microwave extraction.Compared with other extracted technologies,microwave extraction can reduce the extraction time and increase the extraction rate.The orthogonal tests on the the optimal extraction condition have revealed that the following experiment conditions were selected as optimum:in acid solution,extraction time 30min,repeated twice,with an extraction rate of 92.43%under the power of 1200W in microwave digestion instrument.Conclusions1 Rubusoside and COS effectively improved the symptoms of experimental diabetes mice,by promoting the secretion of insulin,liver glycogen synthesis to control blood sugar levels,changed the dysregulation of lipid metabolism,and had protective effects on liver,kidney and pancreas.The intervention effects of Rubusoside and COS in high and middle-dose groups were more obvious.2 The combination of Rubusoside and COS can combine their respective advantages.It had a certain protective effect on the control of blood sugar and islet beta cells,as well as regulated the disorder of lipid metabolism and reduced the pathological damage on the organs.The effect of the combination on the improvement of the diabetes was more comprehensive than any individual intervention,and achieved a better therapeutic effect on diabetes in general.3 Compared with the other three extraction methods,microwave extraction can effectively improve the extraction rate of Rubusoside,reduce the extraction time,have better safety and tightness,avoid the damage on the environment and protect the laboratory personnel from injury.It is an ideal method to extract Rubusoside. |
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