| The latest research reported that the number of patients with diabetes in China had reached 114 million, of which 90-95% was type 2 diabetes. Type 2 diabetes has become one of the mainly non-infectious diseases that affected people health. Since most of the current therapeutic drugs are expensive and have certain side effects, the intervention studies about food on type 2 diabetes have great significance. Wheat bran is a by-product of flour processing in our country, and it is rich resource and low price. Recent epidemiological surveys showed that wheat bran had positive role in prevention of type 2 diabetes, but the hypoglycemic functional factors had not been fully elucidated. In this paper, a-glucosidase (one of therapeutic targets of type 2 diabetes) was selected as detected targets for screening a-glucosidase inhibitors from wheat bran, then the intervertion of a-glucosidase inhibitors on type 2 diabetes were studied. The main research contents and results were as follows:(1) Separation and purification of a-glucosidase inhibitors from wheat bran. The results showed that the 95% ethanol extract of wheat bran (WBE) had high inhibitory activities on a-glucosidase in vitro, then the WBE was extracted by petroleum ether, ethyl ether, ethyl acetate and n-butanol saturated with water successively, and the yields and inhibitory activities on a-glucosidase of petroleum ether extract (WBEP) were both better than the other four extracts. Furthermore, WBEP were loaded and purified by silica gel column and Sephadex LH-20 column, and the active components (F3-17 and F13-26) were identified as apigenin(AP) and alkylresorcinols (ARs) respectively by liquid chromatography/mass spectrometry technology (LC/MS).(2) Inhibition kinetics of AP and ARs on a-glucosidase were studied.pNPG, sucrose and maltose were used as substrates respectively, and the inhibition of AP and ARs on a-glucosidase were determined, and the half inhibition concentrations (IC50) of AP were 14.48,137.32,165.35 μg/mL respectively, the IC50 values of ARs were 37.58, 566.78,542.90 μg/mL respectively. Michaelis-Menten equation and double reciprocal plots (Lineweaver-Burk plot) were used to study inhibition kinetics of AP and ARs. a-Glucosidase(p-NPG, sucrose, maltose as substrates) was competitively inhibited by AP, and the inhibition constants Ki were 12.08,36.65,28.55 μg/mL, respectively. α-Glucosidase(p-NPG, maltose as substrates) was noncompetitively inhibited by ARs, and the Ki were 17.24,591.19μg/mL. a-Glucosidase(sucrose as substrates) was mixed inhibited by ARs, the Ki and Ksi values were 1808.63 and 1158.63 μg/mL.(3) The molecular mechanisms of AP and ARs on a-glucosidase were studied by fluorescence spectroscopy along with molecular docking technique. The results displayed that both AP and 5-n-heneicosylresocinol (whose content is the highest in wheat bran) could quench the intrinsic fluorescence of a-glucosidase through a static quenching procedure. The quenching constants between AP and a-glucosidase were 0.8594×104 L/mol(298 K),0.6177×104L/mol(304 K),0.4865×104 L/mol(310 K), and the thermodynamic parameters of the reaction showed that AP combined a-glucosidase mainly by hydrophobic interaction. The quenching constants between 5-n-heneicosylresocinol and a-glucosidase were 0.2012×104 L/mol(298 K),0.0237 X 104L/mol(304 K),0.0103 ×104 L/mol(310 K), and the thermodynamic parameters of the reaction showed that 5-n-heneicosylresocinol combined a-glucosidase mainly by hydrophobic interaction. The results from molecular docking showed the binding sites of AP and 5-n-heneicosylresocinol on a-glucosidase which elucidated the inhibition molecular mechanisms of AP and 5-n-heneicosylresocinol on a-glucosidase. The results also investigated the interactions between AP,5-n-heneicosylresocinol and a-glucosidase were hydrophobic interaction and hydrogen bond.(4) The type 2 diabetic mice model was established through high fat and high sugar diet combined with streptozotocin (STZ) injection, and the effects of AP, ARs on type 2 diabetic mice were studied. The results showed that AP and ARs both had significant anti-diabetic effects as a marked decrease of fasting blood glucose (FBG) and a significantly improvement on glucose tolerance(p<0.05). In addition, AP, ARs treatments can significantly reduce total cholesterol(TC), triglyceride(TG), low-density lipoprotein(LDL) levels, while significantly increased high-density lipoprotein(HDL) levels in type 2 diabetic mice(p<0.05).(5) The intervention mechanism of AP and ARs on type 2 diabetes were studied. The results displayed that:First, AP and ARs both could significantly reduce type 2 diabetic mice fasting serum insulin levels (FINS) and insulin resistance index (HOMA-IR)(p<0.05). Secondly, AP and ARs could protect pancreas, kidney, spleen in type 2 diabetes mice. Electron microscopy of pancreatic tissue ultramicrotome displayed that AP and ARs had played the roles of protection on pancreatic cell structure. Thirdly, AP and ARs regulated glycogen metabolism in liver and muscle of type 2 diabetic mice. ARs significantly increased liver and muscle glycogen content(p <0.05), and also very significantinly creased the liver glucokinase(GK) activities(p< 0.01). AP increased the liver GK activities significantly(p<0.05). Finally, AP and ARs improved the antioxidant ability of type 2 diabetes mice. AP and ARs significantly increased serum, liver and skeletal muscle total antioxidant capacity(T-AOC) (p< 0.05), and high concentration ARs very significantly increased liver T-AOC(p<0.01). AP and ARs could very significantly reduce serum and liver malondialdehyde (MDA) contents (p<0.01), while significantly reduce skeletal muscle MDA contents. High concentration AP significantly increased liver superoxide dismutase(SOD) activities(p <0.05), ARs very significantly increased serum, liver and skeletal muscle SOD activities(p<0.01). AP and ARs significantly increased serum and skeletal muscle catalase(CAT) activities(p<0.05), very significantly increased liver CAT activities(p< 0.01). AP very significantly increased serum and liver glutathione peroxidase (GSH-Px) activities(p<0.01), significantly increased skeletal muscle GSH-Px activities(p<0.05). ARs very significantly increased serum, liver and skeletal muscle GSH-Px activities(p <0.01) in type 2 diabetic mice. In conclusion, AP and ARs intervened glucose and lipid metabolism in type 2 diabetes mice by improving insulin resistance, protecting the damaged tissues, promoting the conversion of glucose in the liver and increasing antioxidant abilities. |
PDF Full Text Request