| Objective: Extracted, Isolated and Determinated the Anti-oxidative damage effective parts in Equisetum. And observed its effect of proliferation and apoptosis on HUVEC injured by oxidized low-density lipoprotein. To further lay the foundation for clarifying effective parts and mechanism of Equisetum anti-atherosclerosis.Methods:The first part1. Extraction and Isolation of Equisetum effective partsWeigh modest Equisetum decoction Pieces and joined 12 times volume of 70% ethanol. Heated to extract three times. After filtrating the macro porous resin were used to isolated and purified Equisetum extract by distilled water, 30% ethanol, 70% ethanol. Collection of 70% ethanol eluate as an test sample.2. Determination of Equisetum effective partsQuercetin and ferulic acid were diluted into multiple different concentrations and measured by ultraviolet absorption spectrophotometer. Drawed a standard curve and calculated the regression equation. The content of total flavonoids and total phenolic acid was obtained according to the regression equation. The Precise test sample volume was took and dried until constant weight. The purity was measured through the comparison between the content of effective parts and the net weight of extract.3. Preparation of drug solutionThe extract was fully dissolved in DMSO, then added DMEM dubbed to make its concentration be 500μg/ml, -20℃preservation.The second part1. Isolation, oxidation and identification of LDLLDL was extract by the chemical precipitation method and determinated its concentration by Coomassie brilliant blue law. LDL was placed in 10μmol/L CuSO4 solution in 37℃for 24 hours, dialysis in PBS for 24 hours, After filtration and sterilization, perseverated at 4℃. LDL and ox-LDL were authenticated by nagarose gel electrophoresis. UV- spectrophot- ometry to monitor the process and the level of oxidation.2. Human umbilical vein endothelial cells in culture and divisionAfter recovery, the cell strain was cultured in DMEM containing 15% newborn calf serum. Subcultured by Trypsin digestion method. Cells in the logarithmic growth phase were randomly divided into normal control group, injury group and Equisetum extract group. After six hours of culturing in serum-free DMEM, injury group and control group were added the equal volume of serum-free DMEM, Equisetum extract group Equisetum added extract. After continuing to culture one hour, injury group and Equisetum extract group were added final concentration of 80μg/ml ox-LDL, control group added serum-free DMEM. 12 hours later, collected cells and culture media to detect.3. Observation and detectionMorphological changes of cells were observed under inverted light and electron microscopes. The rate of apoptosis and Caspase-3 protein in endothelium cells were detected by flowcytometry. The expression of Caspase-3 mRNA, Bax mRNA and Bcl-2 mRNA in endothelial cells were detected by semi-quantitative RT-PCR.Results:The first partThe total flavonoids average levels: 1.69mg/g, the total phenolic acid average levels: 1.23mg/g, the Equisetum effective parts average levels: 2.92mg/g. The purity of Equisetum effective parts is 63.28%. The second part1. Morphological observationIn Inverted microscope, the cells in control group grew in good condition, the more firmly adherent cells closely inter-connected, a single paving stone mosaic-like arrangement. The cells in injury group obvious morphological changed, cells shrank, varied round and smaller, loose connections, more cell lost. In Electron microscope the surface of cells in injury group bubbly empty and hollow, most cells were apoptosis. The formation of cells in Equisetum extract group markedly improved and the quantity of cells increased.2. The effects of Equisetum effective parts on cell cycle in endotheliumMost cells retained in G0/G1 phase and a little cells retained in S and G2/M phase (p<0.01) in injury group. The ratio of S and G2/M phase of cells in all the treatment groups were higher than that in injury the group (p<0.01). The PI in injury group were significantly lower than that in the control group(P<0.01) and in Equisetum extract group increased significantly(P<0.01).3. The effects of Equisetum effective parts on the apoptosis rate in endotheliumThe apoptosis rate in injury group was higher than that in control group (p<0.01)and it was decreased evidently in Equisetum extract group compared with the injury group (p<0.01).4. The effects of Equisetum effective parts on expression of Caspase-3 protein and Caspase-3mRNA in endotheliumThe expression of Caspase-3 protein and Caspase-3 mRNA in injury group ware higher than that in control group (p<0.01). And both were decreased evidently in Equisetum extract group (p<0.01). 5. The effects of Equisetum effective parts on expression of Bax mRNA,Bcl-2 mRNA in endotheliumThe expression of Bax mRNA in injury group was higher than that in control group (p<0.01). Compared with the injury group, the expressions in Equisetum extract group was markedly lower(P<0.05). The trend of Bcl-2 mRNA was contrary to the above. The differences of Bax/Bcl-2 were more obvious among groups, the ration was the highest in injury group (P<0.01), the ratio in Equisetum extract group was significantly lower than that in injury group (P <0.01).Conclusion: The higher purity of flavonoids and phenolic acid was made through this process. And its purity complied with the standards as effective part. A conclusion was made in the experiments that Equisetum effective parts inhibit damage of ox-LDL-induced HUVEC through various channels. |
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