Experimental Research On Mechanism Of The Effective Fraction Of Flavone In The Portulaca Oleracea L Improving Insulin Resistance

[Objective]:On the basis of previous investigations, in the present study, to extract total flavones from Portulaca Olerace L and determine its content by UV-spectrophotometry and to develop RP-HPLC method for the determination of genistin in flavone extractive from Portulaca Olerace L.[Methods]:Using NaNO2-Al (NO3) 3-NaOH spectrophotometry to determine the total flavones in Portulaca Olerace L with a detection wavelength at 510nm. Developping RP-HPLC method for the determination of genistin in flavone extractive from Portulaca Olerace L.The chromatographic separation was performed on a Sunfire C18column (250 mm×4.0 mm,5.0μm)and eluted with a mobile phase of MeOH-HAC-H2O(45:1:55). The detection wavelength was set at 259 nm and column temperature was set at 25℃. The flow rate was 0.8 mL.min-1.[Results]:Good linear relationship with absorbability occurred when the concentration range of rutin was 8-48mg/L(r=0.9999). The content of total flavones was 7.67%, callback was 97.6%, RSD was 1.43%(n=5).Stable linear relation between the peak area and injected amount existed when the amount was within 6.25 ng.mL-1-200.00 ng.mL-1 for genistin, r=0.9999. The average recovery was 98.3% and the RSD was 0.57%(n=5).[Conclusion]:The UV-spectrophotometry can be used for the determination of the total flavones in portulaca olerace L. It is a convenient and simple method with high accuracy.The HPLC method was accurate and sensitive for the determination of genistin in flavone extractive from Portulaca Olerace L. [Objective]:To investigate the effects of genistin on improving insulin resistance induced by free fatty acids (FFAs) in HepG2 hepatocytes.[Methods]:The model of insulin resistance in HepG2 cells was established by adding palmitic acid (0.5 mmol/L) to the culture medium.Various concentration genistin(1,2,4μmol/L) or rosiglitazone(1μmol/L) treatment was performed at the same time. The MTT assay was used to determine the effect of the drugs used on cell growth. Glucose comsumption (GC) was determined by glucose oxidase method. The level of glucose transporter1(GLUT-1) proteins was detected by Western blotting.[Results]:After the intervention of palmitic acid for 24 h, the insulin-stimulated glucose transport in HepG2 cells was inhibited, the glucose comsuption was reduced greatly, meanwhile, GLUT1 was obviously reduced, as compared with those in normal control. However, the above indexes, which indicated the existence of insulin resistance, were reversed by genistin 1-4μmol/L in a dose-dependent manner. GC of HepG2 cells was increased in every group compared with model group(P<0.05 or P<0.01), the maximal GC was 0.35mM in presence of 4μmol/L genistin.[Conclusion]:Insulin resistance induced by FFAs in HepG2 hepatocytes can be improved by Genistin. [Objective]:To investigate the effects and molecular mechanisms of genistein on improving free-fatty-acid-induced insulin resistance in HepG2 hepatocytes.[Methods]:Insulin resistance in HepG2 cells was induced by exposure to 0.5 mmol/L palmitic acid for 24 hours. Genistein supplementation to the culture medium at various concentrations was in progress at the same time. Then the changes of glucose consumption and expressions of proteins involved in c-jun N-terminal kinase (JNK) signaling pathway in HepG2 cells were observed. Glucose consumption was determined by glucose oxidase method. The expressions of c-jun N-terminal kinase (JNK) phosphorylation, insulin receptor substrate-1(IRS-1) Ser307 phosphorylation, JNK, IRS-1, phosphatidylinositol-3-kinase p85 (PI-3K p85) and glucose transporter 1(GLUT1) proteins were detected by Western blot assay.[Results]:After the intervention of palmitic acid for 24 hours the insulin-stimulated glucose transport in HepG2 cells was inhibited as well as the glucose consumption was reduced significantly. Meanwhile, the expressions of IRS-1, PI-3K p85 and GLUT 1 proteins were obviously reduced but the expressions of JNK phosphorylation, IRS-1 Ser307 phosphorylation and JNK proteins were significantly increased compared with those in normal control. However, genistein, at the concentration between 1 to 4μmol/L, reverses the above alterations of HepG2 cells in a dose-dependent manner. [Conclusion]:Free-fatty-acid-induced insulin resistance in HepG2 hepatocytes could be improved by genistein. The molecular mechanisms might be associated with modulating the expressions of relevant proteins involved in JNK signaling pathway.