Mechanism And Composition Analysis Of The Mechanism Of Improving Insulin Resistance In Ethyl Acetate Of Pseudosciaena Chinensis

Objective:To explore mechanisms of ethyl acetate fraction of ethanol extract from Gentianella acuta on insulin resistance in IR-HepG2 cells and to analysis chemical components of ethyl acetate fraction of ethanol extract from G.acuta.The ultimate aim is to study medicinal material base in G.acuta,to research and development new drugs for diabetes,and then enhance the value of ethnic drug.Methods:Part one,the effective part(ethyl acetate fraction)was obtained from the ethanol extract of G.acuta.IR-HepG2 cell model could be established with high insulin condition(10-6 mol/L)for 36 hours.CCK-8 was used to select suit drug concentration and to test the activity of HepG2 cells.Consumed glucose of IR-HepG2 cells was measured by the glucose oxidase-peroxidase method.The experiment groups were divided by consumed glucose:control group,IR group,IR+ ethyl acetate fraction(50 ?g/mL)groups.IR+ethyl acetate fraction(500 ?g/mL)group.The influence of ethyl acetate fraction on the IR-HepG2 cells apoptosis were detected 24 hours after administration by Annexin V-FITC/PI double-staining method.The influence of ethyl acetate fraction on TNF-a of IR-HepG2 cells was detected 24 hours after administration by enzyme linked immunosorbent assay.The expression of IRS1,PI3Kp85,PDK1,Akt mRNA insulin signaling pathway of IR-HepG2 cells was detected 24 hours after administration by Real-time PCR technique.Phosphorylation level of p-InsR?Tyr1135/1136,p-IRS1 ser307,p-PDK1 ser241,p-Akt Thr308 and level of protein PI3Kp85 in IR-HepG2 cells was detected 30 minute after administration by Western blotting.Part two,high resolution HPLC-MS technique was used to analysis chemical components of ethyl acetate fraction of ethanol extract from G.acuta.HPLC-DAD was conducted to establish a method for quantitative determination of demethylbellidifolin and bellidifolin meanwhile.Results:Part one,filtering to get the best model conditions:IR-HepG2 cell model could be established with high insulin condition(10-6 mol/L)for 36 hours and the model would not exist after 48 hours.Cytotoxicity experiments show:the survival rate of the cells is qualified when concentration of ethyl acetate fraction of ethanol extract from G.acuta is 500 ?g/mL and under this concentration.There are different effects of different concentration(500 ?g/mL,250 ?g/mL,100 ?g/mL,50 ?g/mL)of ethyl acetate fraction to promote the glucose consumption(P<0.01).The hypoglycemic effect is greater with higher concentration.The hypoglycemic effect is equivalent to metformin hydrochloride when the concentration is 500?g/mL.There is no effect of low concentration(10 ?g/mL)to promote the glucose consumption(P>0.05).The ethyl acetate fraction has no effect of apoptosis.Elisa results show:the level of TNF-a is lower with higher concentration of ethyl acetate fraction(P<0.01),which suggests ethyl acetate fraction has an effect to reduce TNF-a level.Molecular biology experiments show:compared with IR group,IR+ethyl acetate fraction(50 ?g/mL)group and IR+ ethyl acetate fraction(500 ?g/mL)group can increase the expression of IRS1,PI3Kp85,PDK1,Akt mRNA(P<0.01 or P<0.05),increase the phosphorylation level of p-InsR?Tyr1135/1136,p-PDK1 ser241,p-Akt Thr308(P<0.01),reduce the phosphorylation level of p-IRS1 ser307(P<0.01),increase the level of PI3Kp85(P<0.05).Part two,we have identified nine compounds,including four xanthones(1,6-dihydroxy-3,4-dim ethoxy xanthone,demethylbellidifolin,swertiabisxanthone-I,bellidifolin),three xanthone glycosides(norswer-tianolin,swertianolin,5,8-dihydroxy-1,4dimetho xy xanthone-3-O-?-D-glucoside),one flavone glycoside(isorientin),one triterpenoid(oleanolic acid).We have established a method for quantitative determination of demethylbellidifolin and bellidifolin meanwhile.The quantity of dimethylbellidifolin and bellidifolinis 103.93?106.33 mg/g and 374.45?379.22 mg/g respectively.Conclusion:The ethyl acetate fraction of ethanol extract from G.acuta can promote the glucose consumption and improve glucose metabolism of IR-HepG2 cells clearly.The mechanism may be correlated with regulating the expression of IRS1,PI3Kp85,PDK1,Akt genes in insulin IRS/PI3K/Akt signaling pathway,regulating the phosphorylation level of p-InsR? Tyr1135/1136,p-PDK1 ser241,p-Akt Thr308,and regulating the level of protein PI3Kp85.Reducing TNF-a level may be one of the mechanism.The main components of the ethyl acetate fraction are xanthones and xanthone glycosides.In order to determine the content of its main components control of the ethyl acetate fraction,a method for quantitative determination of two components meanwhile has been established.