| Objective: To extract, Isolate and Determinate the Anti-oxidative damage effective parts in Equisetum and to observe its effects on inflammatory reaction in HUECV. To further lay the foundation for clarifying effective parts and mechanism of Equisetum anti-atherosclerosis .Methods:The First part1 extraction and Isolation of the Anti-oxidative damage effective parts in EquisetumWeigh modest Equisetum decoction Pieces and join 12 times 70% ethanol, Heating to extract three times, After filtration of the extractive,it was Isolated and Purified by distilled water, 30% ethanol and 70% ethanol in the macroporous . Eluate collection of 70% ethanol as an effective test sample part.2 Determination of Equisetum effective partsQuercetin and ferulic acid would be diluted into multiple different concentrations and measured by ultraviolet absorption spectrophotometer. Draw a standard curve and calculate the regression equation. According to the regression equation obtained the effective parts (quercetin and ferulic acid) content. Precision would take the test sample volume and drying until constant weight, measured by the effective use site content compared with the purpose of calculating the effective parts ,then contained purity.3 preparation of drug solutionextractive would be fully dissolved by DMSO before adding DMEM dubbed concentration of 1000μg/ml ,500μg/ml, 250μg/ml of fluid, -20℃preservation reserve.The Second part1 isolation, identification and oxidation of Low-density lipoproteinThe chemical precipitation was used to extract low-density lipoprotein. Determine its concentration using Coomassie brilliant blue law. LDL was placed in 10μmol / L CuSO4 solution in 37℃for 24 h , the PH7.4 PBS dialysis for 24 h, filtrate, obtain ox-LDL, preservate at 4℃.Using UV spectrophotometry to monitor the process and the level of oxidation.2 Culture and divide Human Umbilical Vein Endothelial CellsThe HUVEC was cultured in DMEM containing 15% calf serum. Subculture by Trypsin digestion method. Cells from the logarithmic growth phase were randomly divided into normal control group, injury group, 1000μg/ml Equisetum extract group, 500μg/ml Equisetum extract group, 250μg/ml Equisetum extract group.3 detect the targetsMorphological changes of cells were observed under inverted light and electron microscopes. the levels of NO in the culture media and activity of NOS in human endoderlial cells (HUVEC) were measured sepretallly by nitrate reductase assay and chemical colorimetry assay. the levels of IL-8 was measured by radio immunoassay. The rate of VCAM-1 protein related in endothelium cells were detected by flow cytometry. iNOSmRNA,IL-8,VCAM-1 mRNA, expression of endothelial cells were detected by semi-quantitative RT-PCR.Results:The First partThe Quercetin average levels: 1.69 mg / g, the ferulic acid average levels: 1.23 mg / g, the Equisetum effective parts average levels: 2.92 mg / g. the purity of Equisetum effective parts is 63.28%. The Second part1 Electrophoresis of human blood serum,LDL,ox-LDLThe Electrophoresis of human blood serum,LDL,ox-LDL prompts that the isolation, identification and oxidation of Low-density lipoprotein has succeed.2 Curve plot of in vitro oxidization of LDLThe oxidization of LDL can be obviously divided into three periods:lag period, fast reaction period and breakdown period.At about 10h it was completely oxidized. 3 Inverted microscope observationIn Inverted microscope, the cells in control group grow in good condition, the more firmly adherent cells were closely inter-connected, a single paving stone mosaic-like arrangement. the cells in injury group have obvious morphological changes: shrinkage, smaller variable round cell, loose connections, and more loss cells.4 Electron microscope observationIn Electron microscope the cell surface bubbly empty formation processes and dents, and most apoptosis. the state of cells in Equisetaceae extract group had been markedly improved.5 The effects of Equisetum effective parts on the levels of NO in the culture mediathe expression of NO in injury group was lower than that in control group(P<0.01). the expression of NO in Equisetum groups was increased compared with those in injury group (P<0.01).6 The effects of Equisetum effective parts on the activity of NOS in human endoderlial cellsthe expression of NOS in injury group was lower while iNOS was higher than that in control group(P<0.01). the expression of NOS in Equisetum groups was increased while iNOS was decreased compared with those in injury group (P<0.01).7 The effects of Equisetum effective parts on the levels of IL-8 in the culture mediathe expression of IL-8 in injury group was higher than that in control group(P<0.01). the expression of IL-8 in Equisetum groups was decreased compared with those in injury group (P<0.01).8 The effects of Equisetum effective parts on expression of VCAM-1 protein in human endoderlial cellsThe expression of VCAM-1 protein in injury group was higher than that in control group (p<0.01). And it was decreased evidently in 50μg/ml Equisetum group (p<0.01).9 The effects of Equisetum effective parts on expression of iNOS mRNA in human endoderlial cellsThe expression of iNOSmRNA in injury group was higher than that in control group (p<0.01). And it was decreased evidently in 50μg/ml Equisetum group (p<0.01).10 The effects of Equisetum effective parts on expression of IL-8 mRNA in human endoderlial cellsThe expression of IL-8mRNA in injury group was higher than that in control group (p<0.01). And it was decreased evidently in 50μg/ml Equisetum group (p<0.01).11 The effects of Equisetum effective parts on expression of VCAM-1 mRNA in human endoderlial cellsThe expression of VCAM-1 mRNA in injury group was higher than that in control group(P<0.01). the expression of VCAM-1 mRNA in Equisetum groups was decreased compared with those in injury group (P<0.01).Conclusion: Through this process,we have a higher purity of ferulic acid and quercetin, Comply with the standards as effective parts. Through its intervention cell experiments it confirmed that Equisetum effective parts through various channels to inhibit endothelial cell damage by ox-LDL. many mechanisms play a important role in anti-Early AS. |
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