| Diabetes mellitus is one kind of chronic comprehensive disease characterized by the absolute or relative defieieney of insulin, which can be induced by genetic factor or environmental factor. There are lots of patients in the world and most of them belong to type2diabetes(T2DM). Typical performance of T2DM is hyperglycemia.In tranditional Chinese medicine, we call T2DM as "xiaokezheng". Insulin resistance (IR) is the main mechanism of T2DM. Therefore, finding drugs which can treat IR effectively very important clinical significance.Stigma maydis is a traditional Chinese herbal medicine, which has been widely used for hundreds of years. Response surface methodology (RSM) is a particular set of mathematical and statistical methods for designing experiments, building, models, evaluating the effects of variables and searching optimum conditions of parameters to predict targeted responses. Recently, it has been used for an effective statistic technique for optimizing complex processes. The main advantage of RSM is the reduced number of experimental trials needed to evaluate multiple factors and their interactions. It has been successfully demonstrated that RSM can be used to optimize the polysaccharide from many medicine plants. In the current thesis, the influence of some extraction parameters on stigma maydis polysaccharide (SMP) conducted. A Box-Behnken design was performed in order to optimize the optimal extraction conditions of SMP by ultrasonic-assisted enzymatic hydrolysis method.SMP are total polysaccharides which are extracted from corn silk, stigma maydis polysaccharide5(SMP-5) is a new monomer polysaccharide which is separated from SMP.It has been evidenced the crude SMP has hypoglycemic effect on mice induced by alloxan. Crude SMP also can repair diseased pancreas and increase the content of liver glycogen. SMP-5is one of a monomer component of SMP. However, the study about the effect of SMP-5on hypoglycemic effect has not been reported. In the thesis, firstly we study the hypoglycemic action of SMP-5by adrenaline model experiment, glucose tolerant test and T2DM model induced by alloxan and high fat diet experiment. Secondly, we established T2DM model induced by alloxan and high fat to study on the effect of SMP-5on IR and PI3K/AKT pathways. The main study are as follows:1. Optimization technological parameter of SMP extraction from stigma maydis by RSMThe SMP extraction from stigma maydis was further optimized by the RSM using a4-factor,3-level Box-Behnken design (BBD). The four factors are enzymatic quantities, hyperacoustic time, enzymatic hydrolysis temperature, and pH..The optimized combination of response function was enzymatic quantities210U, enzymatic hydrolysis time28min, enzymatic hydrolysis temperature51℃, and pH4.5, and under such conditions, the rate of extraction was12.84%, which increased31.93percent compared with orthogonal experiment.2. The effect of SMP-5on hypoglycemic actionThe hypoglycemic action of SMP-5was observed by adrenaline model experiment and glucose tolerant test. T2DM model was induced by alloxan and high fat diet experiment in order to observe the effect of SMP-5on T2DM. Testing index as follows:determine the FBG of mice every week. When the experiment was finished, the rats blood serum were separated. Determine TC, TQ HDL-c, LDL-c in blood serum by automatic biochemical analyzer, determine the blood serum level of insulin, C-peptide, TNF-a by enzyme-linked immuno sorbent assay(ELISA) and calculate the index of ISI, HOMA-IR and MOMA-β, determine the content of FFA and MD A, the activity of SOD in mice and the content of liver glycogen and mucule glycogen using spectrophotometer. HE staining was used to observe pancreas pathological changes.The results shown that SMP-5has hypoglycemic effect on mice induced by adrenaline glucose. SMP-5also has hypoglycemic effect on T2DM rats. SMP-5could reduce the content of TQ TC, LDL-c, regulate lipid metabolism, increase the content of liver glycogen and mucule glycogen, decrease the content of FFA and TNF-a. The level of SOD was increased and MDA was decreased. SMP-5could decrease serum insulin level, change the situation of high blood glucose and high insulin, decrease the index of HOMA-IR and decrease the index of ISI and MOMA-p. Pathological photos show that SMP-5could repaired diseased pancreas. From thoese results we can speculate that SMP-5do have hypoglycemic effect and its effect may be related to the effect of IR.3. The mechanism of SMP-5on hypoglycemic actionThe IR model was established by alloxan and high fat diet in order to continue study SMP-5effect on IR and PI3K/AKT pathways. T2DM IR model was induced by alloxan and high fat diet experiment in order to observe the effect of SMP-5on T2DM. Testing index as follows:determine the FBG of mice every week. When the experiment was finished, the mice blood serum were separated. Determine TC, TQ HDL-c, LDL-c in blood serum by automatic biochemical analyzer, determine the blood serum level of insulin, TNF-a by ELISA and calculate the index of ISI, HOMA-IR and MOMA-β. The protein expression of p-KAT, Bcl-2and Bax were detected by immunohistochemistry. The level of Akt2, PKCX,GSK3α,GSK3β and GLUT4gene were determined by RT-PCR. The protein expression of p-KAT p-GSK3and GLUT4were detected by Western Blot.The immunohistochemical results shown that SMP-5can increase p-AKT and Bcl-2expression, decrease the expression of Bax, increase the ratio of Bcl-2/Bax of pancreas.The RT-PCR results shown that SMP-5can up-regulate Akt2, PKCX and GLUT4gene level, and reduced GSK3α,GSK3β gene level. Western Blot results demonstrated that SMP-5can up-regulate p-AKT and p-GSK3levels and increase the movement of GLUT4to cell membrane. The results above manifested that SMP-5can improve liver IR and muscle IR. The mechanism of its effect may be related to the activition of pathway of PI3K/AKT.In summary, the optimized combination of response function was enzymatic quantities210U, enzymatic hydrolysis time28min, enzymatic hydrolysis temperature51℃, and pH4.5, and under such conditions, the rate of extraction was12.84%, which increased31.93percent compared with orthogonal experiment. SMP-5do have hypoglycemic effect and improve IR effect. The mechanism of its effect may be related to the activition of pathway of PI3K/AKT.
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