| Diabetes mellitus has become a harmful disease with the highest incidence and the highest mortality in the world,which pose a serious threat to our health and quality of life.Type 2 diabetes mellitus is a heterogeneous endocrine disorder in which hyperglycemia is the unifying feature.Insulin resistance syndrome,hypertension and dyslipidemia are the common complications.Moreover,cardiovascular disease or stroke account for most of the morbidity and mortality associated with diabetes.Currently the conventional treatment of diabetes mellitus are synthetic drugs and insulin.The side effects of synthetic drugs and the hidden danger of injected insulin can not be ignored.In recent years,numerous studies found that polysaccharides which were extracted from natural products have good antidiabetic activity with nontoxicity,and it is valuable in the advanced development and research.Therefore,this paper aimed to seek the Ophiopogon japonicus polysaccharide(OJP)component of optimal anti-type 2 diabetes activity and a preliminary study was conducted to explore the structure-activity relationship.However,the low bioavailability seriously limited the clinical application of OJP.Hence,the aim of this study is to develop OJP loaded multivesicular liposomes to improve anti-type 2 diabetes activities.Chapter Ⅰ ReviewThis part reviewed the research progress of synthetic drugs,insulin and natural polysaccharide used in the treatment of type 2 diabetes mellitus,and summarized the mechanism,merits and demerits.The polysaccharides from Ophiopogon japonicus with anti-type 2 diabetes activities were mainly introduced,and multivesicular liposome was considered as a promising delivery system to improve anti-type 2 diabetes activity.Chapter Ⅱ Extraction,separation and purification of Ophiopogonjaponicus polysaccharide from different producing areasThis chapter describes the refined polysaccharides were extracted from roots of Ophiopogon japonicus(Sichuan and Hubei producing areas)via water extraction,alcohol precipitation and protein removal.OJP-S-1,OJP-H-1 and OJP-H-2 were purified by D101 macroporous resin and DEAE-52 cellulose column.The purity of these three components were identificated by UV spectra and High-performance gel filtration chromatography(HPGFC).The results showed no interference of protein or nucleic acid and the protein content was less than 0.2 %.Besides,they prossess high purity and high total glucose content(97 %),thus laying a good foundation for screening higher anti-type 2 diabetes activity.Chapter Ⅲ Anti-type 2 diabetes activities and structure analysis of different Ophiopogon japonicus polysaccharidesIn this chapter,the STZ induced type 2 diabetic mice model was successfully established in vivo and the hypoglycemic activities of OJP-S-1,OJP-H-1 and OJP-H-2 at the same dose(200 mg/kg)were studied.Compared with the model group,OJP-S-1,OJP-H-1 and OJP-H-2 could significantly decreased the fasting blood glucose(FBG)level(p<0.01)in type 2 diabetic mice,and effectively improved the oral glucose tolerance(OGTT)(p<0.01),as well as the insulin resistance,which indicated that these three components have certain hypoglycemic effects.The results also proved OJP-S-1 had more obvious hypoglycemic effect than OJP-H-1 and OJP-H-2.The molecular weight,composition of monosaccharide,glycosidic linkage,branch chain,carbonoid,advanced constructs have a high degree of correlation with its pharmacological activity.The primary structure of these three components were determined and the structureactivity relationship was preliminarily analyzed based on its Anti-type 2 diabetes activities.Chapter Ⅳ Preparation and evaluation of Ophiopogon japonicus polysaccharide-loaded multivesicular liposomesThe poor stability,low bioavailability and poor permeability of water-soluble polysaccharide,has limited its clinical application.Therefore,the multivesicular liposome which is suitable for water-soluble macromolecules was promoted in this paper.This part established and validated phenol-sulfuric acid method with an ultraviolet spectrophotometer to analyze OJP-S-1.Results demonstrated that the established analytical method had good system applicability.Linearity range of the calibration curves was 0.1-1 mg/m L.The intra-day/inter-day precisions(RSD < 2 %)and recoveries(RSD < 3 %)met the acceptance criterion,demonstrating the established analytical method was suitable for quantitative analysis of OJP-S-1.Ophiopogon japonicus polysaccharide-loaded multivesicular liposomes(OJP-MVLs)were prepared by multiple emulsion technique,the optimum formulation with high entrapment efficiency(81.24 ± 2.11 %)was screened by single factor method and Box-Behnken response surface design,the optimized prescription was OJP-S-1 24 mg,vitamin E 5 mg,DOPC 20 mg,DPPG 4 mg,cholesterol 16 mg,triolein 10 mg,Tween 80 10 mg.The prepared OJP-MVLs was spherical,homogeneous,and the multi-chamber structure was observed through microscope.The size and distribution profile of OJPMVLs revealed a mean particle diameter of 15.37 μm and a narrow range of particle size distribution.And the zeta potential gave-38.38 ± 3.26 m V.Storage stability showed that prepared OJP-MVLs had an acceptable stability without flocculation,precipitation and obvious EE and size changes.Chapter Ⅴ Anti-type 2 diabetes effect of Ophiopogon japonicuspolysaccharide-loaded multivesicular liposomesThe OJP-MVLs could enhance the in vivo stability and circulation time of OJP,which can improve the bioavailability and pharmacological activity.This chapter systematically evaluated the anti-type 2 diabetes effect of OJP-S-1 and OJP-MVLs.OJP-MVLs(100 mg/kg),OJP-MVLs(200 mg/kg)and OJP-S-1(200 mg/kg)could significantly reduced the level of FBG(p<0.01)in type 2 diabetic mice,improved the oral glucose tolerance(p<0.01)and the symptoms of insulin resistance(p<0.01),and OJP-MVLs(high and low dose)showed better effects than OJP-S-1(200 mg/kg).Both the OJP-S-1 and OJP-MVLs(high and low doses)could significantly reduced TC,TG and LDL(P<0.01),and increased HDL and HDL/LDL(P<0.01)in varying degrees.Remarkably,OJP-MVLs can be more effective than metformin in improving lipid metabolic disorder.It is proved that OJP-MVLs can effectively relieve the symptoms of dyslipidemia,prevent arteriosclerosis and thrombosis,prevent the occurrence of cardiovascular diseases and relieve the symptoms.In addition,OJP-S-1 and OJP-MVLs also could significantly increase the activity of liver glucokinase(GK)(P<0.01),the content of liver glycogen(P<0.01)and reduce the activity of G-6-Pase(P<0.01)in type 2 diabetic mice.The ability of OJP-MVLs to regulate glycometabolism was superior to OJP-S-1,especially its high dose group is better than metformin group.It is confirmed that OJP-MVLs could accelerate glucose metabolism and inhibit gluconeogenesis,as well as enhance the ability of liver to transform blood sugar into glycogen.Liver is the main organ and important place of lipid metabolism and glucose metabolism.Therefore,a well-behaved liver is also essential for diabetics.The OJP-MVLs possess strong ability to repair liver damage caused by STZ induced type 2 diabetes which can be seen from the slices of liver tissues. |
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