| The method based on cell model evaluates the biological activity and studies mechanism of natural product not only simulating the human body physiological environment but also working on multiple targets at the same time, compared with chemical method; not only saving the time but also greatly reducing the test cost, compared with the experiments on animals. So the evaluation method based on cell model has been widely used in the biological activity of natural products by researchers. In this paper, the hypoglycemic activity of different fractions from Oleaeuropaea L. leaves and the antitumor activity of8-NH2-isocorydine were evaluted, the possible antioxidant mechanism of protect the H2O2-induced PC12cells was studied by metabolomics, A new method was developed for evaluation bioactive natural products in vitro cell model. The contents of this dissertation are the following:(1) The study on hypoglycemic activity of Oleaeuropaea L. leaves. The hypoglycemic activity of six kinds of extract fractions A-F from Oleaeuropaea L leaves were evaluated by the model of ilinsulin resistance to HepG2cells. The cell model was developed by high concentrations of insulin (5.8×10-3mg/mL) induced HepG2cells (glucose consumption of unit cell:normal group,2.224±1.122mmol/L model group2.003±1.567mmol/L, P<0.05), cell glucose consumption was detected by glucose oxidase assay and the survival rate of cell was detected by MTT assay. The results show that all the fractions A-F from Oleaeuropaea L. leaves improved insulin resistance, glucose consumption of unit cell were4.577±1.178,5.338±1.070,6.687±1.384,7.083±1.405,5.633±1.449,6.350±0.880mmol/L, respectively. It was found that fraction D had the significantly higher activity than model group2.003±1.567mmol/L (P<0.01). and had higher activity than positive control group of metformin6.853±0.609mmol/L (P<0.01).(2) The study on anti-tumor activity of8-NH2-isocorydine.8-NH2-isocorydine was obtained by modifying isocorydine which is a main alkaloid from Dicranostigma leptopodum. In this paper, cell growth inhibitions of8-NH2-isocorydine on six kinds of tumor cells (MGC80-3, BGC-823, SGC-7901, HL-60, A549, HepG2) were detected by MTT assay and the effects of8-NH2-isocorydine on the cell cycle were studied by flow cytometer. The results show that8-NH2-isocorydine has inhibit proliferation effect on tumor cells, especially on gastric cancer cells (IC50:5.68±0.78,7.13±0.67,7.62±1.11,9.22±1.57,17.72±2.86,24.33±2.06μg/mL), and its anti-tumor activity function is not implemented by blocking the cell cycle.(3) Research of methodology about antioxidant activity evaluation by metabonomics on cell.Preliminary study on the combination method about metabolomics and antioxidant cell model. In this paper, the antioxidant protective effect of the H2O2-induced PC12cells pretreated with extracts from Herba Cynomorii by metabonomics. The cells were quenched by60%cold methanol (-20℃) and broken by High-speed Shear Dispersing Emulsifier (HSDE) with19000rpm×3min. Cell metabolites were concentrated(40℃,0.08Mpa)and analysised by Chromatography(HPLC), pattern recognition analysis the date of14common peaks from6times parallel experiments by partial least squares discriminant analysis (PLS-DA). The results showed that the extracts from Herba Cynomorii has antioxidant activity, but weaker than VE, and found4potential biomarkers which involved in cell oxidative damage process. The results show that the method is feasible for evaluation the activity and explore mechanism of natural products.(4) The study on scavenging free radicals activity of Oleaeuropaea L. leaves. DPPH and ABTS+·assay were employed to evaluate the active fractions of scavenging free radical of Oleaeuropaea L. leaves, respectively. Furtherly, active component in the most active fractions of scavenging free radical was traced by HPLC. The results show that fraction C is the best active fraction of scavenging free radical (IC50DPPH·:0.0149±0.0051mg/mL, ABTS+·:0.1976±0.0993mg/mL), and oleuropein is one of the main active components. |
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