BEAN Plant Active Ingredient Whole Grain And Improve Insulin Resistance HepG2 Cells With AMPK Pathway In

Objective:(1) The extractions, content determination, comparison of various kinds of dietary fiber(DF), flavonoids(FN), phenolic acid(PAD) and phytosterol(PS)(derived from the cooked and non-cooked whole grain-soybean package)were studied to investigate the basis of how to these biological effects occur. The antioxidant capacity of different sources of active element extracts in vitro were also measured.(2) The influences, which different sources of active element extracts from the cooked and non-cooked whole grain-soybean package had on the glucose consumption and the content of triglyceride(TG), malondialdehyde(MDA), superoxide dismutase(SOD) and glutathione peroxidase(GSH-PX) were researched in palmitic acid(PA) induced HepG2insulin resistance cytoplasm, to explore the different effects of these active element extracts on the insulin resistance cell.(3) The effects of active element extracts from the cooked and non-cooked whole grain-soybean package on AMP-activated protein kinase a2(AMPKa2), acetyl-CoA carboxylasel(ACCl), fatty acid synthase(FAS), glucose transporter2(GLUT2) and peroxisome proliferators-activated receptor-γ coactivator-1(PGC-1) were detected in the insulin resistance cell at mRNA level to study the mechanism, which indicated how to these different kinds of these active element extracts on the insulin resistance HepG2cells.(4) Comparison the difference between cooking and non-cooking and the effects of active element extracts from the whole grain-soybean package to improve insulin resistance in HepG2cells.Methods:(1) Using enzyme-chemical methods to extract DF from the cooked and non-cooked whole grain-soybean package, The content of dietary fiber was measured according to the GB method. The FN from the cooked and non-cooked whole grain-soybean package were extracted by extraction-reflux method and their content were determined by colorimetry. The PAD and PS from the cooked and non-cooked whole grain-soybean package were extracted by ultrasonic extraction method and their content were determined by colorimetry. The antioxidant capacity of different sources of active element extracts in vitro was measured with DPPH and T-AOC methods.(2) Vitro insulin resistance model was established by PA-induced HepG2cell. The concentration of PA used was determined by MTT, oil red O staining, the glucose consumption with GOD-POD method and the detection of cytoplasm TG content. (3) After the insulin resistance of HepG2cell induced by PA, adding different sources of active element extracts from the cooked and non-cooked whole grain-soybean package, the glucose consumption and the changes of cytoplasm TG, MDA, SOD and GSH-PX content were observed. Also the expressions of intracellular AMPKa2, ACC1, GLUT2, FAS and PGC-1mRNA were assayed.Results:(1) The dietary fiber content was17.5g/100g from non-cooked whole grain-soybean package and21.6g/100g from cooked whole grain-soybean package. The flavonoids content was2.28g/100g from non-cooked whole-soygrain package and3.51g/100g from cooked whole grain-soybean package. The phenolic acid content was0.62g/100g from non-cooked whole grain-soybean package and0.52g/100g from cooked whole grain-soybean package. The phytosterol content was4.60g/100g from non-cooked whole grain-soybean package and5.88g/100g from cooked whole grain-soybean package. The different sources of active element extracts from the cooked and non-cooked whole grain-soybean package were all had strong abilities to eliminate DPPH free radical and their total antioxidant capacity were also strong.(2)0.25mM PA decreased the glucose intake ability and led to intracellular TG significant increase and lipid droplet visible accumulation, furthermore, it had no significant influence on cell viability, it suggested that insulin resistance model of HepG2cell in vitro was successful induced by0.25mM PA.(3) In the MTT experiment, we can observe200μg/ml concentration of different sources of active element extracts had certain protective effects on insulin resistance of HepG2cells induced by palmitic acid. While comparing to FN groups、PS groups and DF groups, PAD groups have not obvious protection. Oil red O staining results also showed that palmitic acid can cause intracellular free fatty acids increase, different sources of active element extracts can reduce the formation of intracellular free fatty acids.200μg/ml active element extracts from the cooked and non-cooked whole grain-soybean package enhanced the glucose intake abilities. There was no significant difference between negative control group and intervention groups in glucose intake ability (P>0.05), and the glucose intake ability of the intervention groups were much lower than that of PA group (P<0.05). Compared to the PA group, active element extracts decreased the contents of TG and MDA, and increased the level of SOD and GSH-PX in cell supernatant (P<0.05). this showed these active element extracts from the cooked and non-cooked whole grain-soybean package had a certain improvement on the insulin resistance of HepG2cells.(4) Compared to the PA group, the expression of AMPKa2, GLUT2and PGC-1mRNA in HepG2cells in FN groups, PAD groups, PS groups and DF groups were significantly higher and the expression of ACC1and FAS mRNA in HepG2cells in FN groups, PAD groups, PS groups and DF groups were significantly lower(P<0.05). Exception PAD groups, there was no signiticant difference between negative control group and other intervention groups in the improvement of genes expression(P>0.05).Conclusion:(1) The whole grain-soybean package is rich in dietary fiber, flavonoids, phenolic acid, phytosterol and other antioxidative substances. These active element extracts have a strong antioxidant capacity in vitro.(2) HepG2cell, incubated with0.25mM PA for24hours, decreased the glucose intake ability and formed the insulin resistance of HepG2cell model.(3) The active element extracts from the cooked and non-cooked whole grain-soybean package could increase the expression of AMPKα2, GLUT2and PGC-1mRNA and decrease the expression of ACC1and FAS to prevent the occurrence of insulin resistance in HepG2cells which were induced by PA. This may be one of the mechanisms that the improvement of body’s insulin resistance by the whole grain-soybean package.(4) The improvement of insulin resistance in HepG2cells, the effects of FN groups best, followed by DF groups and PS groups, while the PAD groups ineffective, especially the PAD group after cooking. Possible reason is that PAD is the substance that can not stand high temperature, the high temperature will accelerate the oxidation and decomposition of the PAD.