| Type 2 diabetes is a type of chronic disorder of glucose metabolism disorder,which seriously affects the health and quality of life of patients.In addition to existing clinical drug treatments such as metformin,the development of new natural products and derivatives with the prevention or adjuvant treatment of diabetes has become current research hotspots.Chitooligosaccharide have a certain hypoglycemic effect,but have different effects and mechanisms according to their degree of polymerization.The development of chitooligosaccharide derivatives with more significant auxiliary treatment for diabetes through molecular modification or modification has great market demand.The purpose of this study was to synthesize chitooligosaccharide guanidine hydrochloride with a corresponding degree of polymerization using chitooligosaccharide monomers with a polymerization degree of 2-4,and evaluate the hypoglycemic activity and its mechanism of action using an insulin resistance cell model.The functional modification of sugar provides technical basis for expanding the application field.Firstly,chitooligosaccharide guanidine hydrochloride with a degree of polymerization of 2-4 was synthesized.Using chitobiose(COS2),chitotriose(COS3),chitotetraose(COS4)and dicyandiamide as reaction raw materials,under high temperature and acidic conditions,nucleophilic addition reactions occur respectively to generate chitobiose guanidine hydrochloride(COS2G),chitotriose guanidine hydrochloride(COS3G),chitotetraose guanidine hydrochloride(COS4G).NMR,FT-IR and Elemental analysis were used to characterize and identify the synthesized products.The results showed that the c chitooligosaccharide monomers with the polymerization degree of 2-4 were all successfully connected with guanidine groups,and the reaction proceeded successfully.Secondly,the synthesis process of chitooligosaccharide guanidine hydrochloride with polymerization degree of 2-4 was optimized.Taking the yield of the product and the degree of substitution of guanidine groups as the measurement indicators,the effects of reaction time,reaction pH,molar ratio of raw materials and reaction temperature on the synthesis were analyzed and discussed,and the best synthesis process was selected.The results show that the best process for the synthesis of COS2G:the reaction time is 4 h,the reaction pH is 1.0,the reaction molar ratio is chitooligosaccharide monomer:dicyandiamide=1:3,the reaction temperature is 100℃;the best process for the synthesis of COS3G:Reaction time is 3 h,reaction pH is 1.0,reaction molar ratio is chitooligosaccharide monomer:dicyandiamide=1:3,reaction temperature is 100℃;the best process for synthesis of COS4G:reaction time is 3 h,reaction The pH is 1.5,the reaction molar ratio is chitooligosaccharide monomer:dicyandiamide=1:3,and the reaction temperature is 100℃.Again,the glucose-lowering activity of chitooligosaccharide guanidine hydrochloride with a polymerization degree of 2-4 was evaluated using a high glucose and high fat induced insulin resistance cell model.In this model,the difference in hypoglycemic effect of chitooligosaccharide guanidine hydrochloride with different degrees of substitution was compared.The results showed that the chitooligosaccharide guanidine hydrochloride with high degree of substitution had better hypoglycemic effect.Then,the products with high degree of substitution were selected for the evaluation of hypoglycemic activity.The results showed that chitooligosaccharide guanidine hydrochloride with a polymerization degree of 2-4 at 600 μg/mL can increase glucose absorption,increase cellular glycogen content,and reduce Nitric oxide synthase(iNOS)activity,of which COS4G has the best effect.Finally,the mechanism of hypoglycemic action of COS4G was explored.Fluorescence quantitative PCR was used to determine the changes in the expression levels of key genes related to gluconeogenesis.The results showed that compared with the model group,the expression of phosphoenolpyruvate carboxykinase(PEPCK)and glucose-6-phosphatase(G6Pase)genes in the COS4G intervention group were significantly reduced(p<0.05).Western Blot was used to detect changes in protein expression levels on related signaling pathways.The results showed that compared with the model group,the p-IRS-1 and p-NF-κB p65(Ser536)protein expression levels of the COS4G intervention group were significantly reduced,and the p-Akt(Ser473)protein expression level was significantly increased(p<0.05).It can be inferred that COS4G reduces insulin by reducing the mRNA expression of PEPCK and G6Pase genes and inhibiting the expression of p-IRS-1(Ser307),p-NF-κB p65(Ser536)and activating p-Akt(Ser473)protein resistance.This study shows that chitooligosaccharide guanidine hydrochloride with a degree of polymerization of 2-4 respectively has a certain improvement effect on diabetes,and provides strong support for the development of new drugs with prevention or adjuvant treatment of diabetes. |
PDF Full Text Request