| Objective:（1）Alcohol extract of the Chinese herbal compound of Jiuwei Tongqiao decoction was divided into different extracting parts by gradient extraction with four kinds of organic solvents and component analysis was conducted on each extracting parts.Select the effective parts via pharmacodynamic experiments in vitro,explore the anti-glioma-effective extraction site of Jiuwei Tongqiao decoction,provide experimental basis for further study on the mechanism of the whole party.（2）The Chinese herbal compound of Jiuwei Tongqiao decoction was divided into different prescriptions and the optimal prescription was selected via pharmacodynamic experiments in vitro,Alcohol extract of the optimal prescription was divided into different extracting parts by gradient extraction with four kinds of organic solvents and component analysis was conducted on each extracting parts.Explore the anti-glioma-effective extraction site of Jiuwei Tongqiao decoction and provide experimental basis for further study on the mechanism of the whole party.（3）The anti-glioma mechanism of Jiuwei Tongqiao decoction,group partiesⅣ and quercetin was initially studied in U251 cells.Methods:（1）After alcohol extract enrichment,gradient extraction with petroleum ether,chloroform,ethyl acetate and water saturated n-butyl alcohol was to get the petroleum ether extract（partⅠ）,chloroform extract（partⅡ）,ethyl acetate extract（part Ⅲ）,water saturated n-butyl alcohol extract（partⅣ）and the residual liquid water extract（partⅤ）.Using HPLC,and calycosin-7-glucoside（calycosin-7-O-β-Dglucoside）,ononin（formononetin-7-O-β-D-glucoside）,calycosin and formononetin as the indicative components,each component in every extract system was detected.Finally,using U251 cells of anthropogenic brain glioma,MTT was applied to determine the effect of extraction parts for cell survival.（2）Jiuwei Tongqiao decoction was separated to seven prescriptions based on the principles of monarch,After alcohol extract enrichment,using U251 cells of anthropogenic brain glioma,MTT was applied to determine the effect of different prescriptions for cell survival.As alcohol extract of the optimal prescription was concentrated,gradient extraction with petroleum ether,chloroform,ethyl acetate and water saturated n-butyl alcohol was to get the petroleum ether extract（partⅠ）,chloroform extract（partⅡ）,ethyl acetate extract（part Ⅲ）,water saturated n-butyl alcohol extract（partⅣ）and the residual liquid water extract（partⅤ）,and MTT was applied to determine the effect of extraction parts for cell survival.Using HPLC,and calycosin-7-glucoside（calycosin-7-O-β-D-glucoside）,calycosin,formononetin and ononin（formononetin-7-O-β-D-glucoside）as the indicative components,each component in every extract system was detected.（3）Human glioma U251 cells was selected to track and analyze Jiuwei Tongqiao decoction,group parties Ⅳ and quercetin,Flow cytometry analysis of apoptosis and using the Bradford method for quantifying cell protein,and developing a standard curve.Selection of Bcl-2,P53 gene protein as a representative,Western-blot experiments was used to analyze the content changes in cellular protein.The experimental operation steps were accorded with preparation of glue,electrophoresis,transmembrane,blocking,primary antibody incubation,secondary antibody incubation,exposure imaging.using Image J software to get the gray-striped analysis,results were repeated three times,while GAPDH as a measure.Results:（1）The content ratio of calycosin-7-glucoside,calycosin,ononin is 1.0︰10.8︰8.2,and IC50 is 46.89 g·L-1.In comparison with other parts,the index ingredients in part Ⅲ were more comprehensive and concentration of them were higher,inhibitory effect on U251 cells was more obvious and appeared a certain concentration-dependent.（2）In comparison with other parts,inhibition of U251 cells of prescriptionⅣ was more obvious,its IC50 is 80.88 g·L-1 and appeared a certain concentration-dependent.the index ingredients in part Ⅲ were more comprehensive,concentration of them were higher and inhibition of cells showed a certain linear.（3）Previous studies showed that the dose-response curve of different concentrations of quercetin with U251 cells is y = 0.012 x + 0.2331,R2 = 0.941,IC50 was 0.067 g·L-1 at 72 h.0.067 g·L-1 quercetin as a positive control,apoptosis rates of prescription Ⅳ,all parties and quercetin were:（62.53 ± 0.72）%,（69.06 ± 0.81）% and（52.83 ± 0.95）%,all parties and prescription Ⅳ were significantly higher than the control group of quercetin.Bcl-2 / GAPDH-1 : 1.03（blank）,0.41（prescription Ⅳ）,0.34（all parties）,0.67（quercetin）.P53 / GAPDH-2: 0.06（blank）,0.44（prescription Ⅳ）,0.71（all parties）,0.40（quercetin）.It has significant changes of Bcl-2,P53 levels in all parties,group parties Ⅳ and quercetin.Conclusion: PrescriptionⅣ was the optimal prescription,Part Ⅲ is the most effective parts of the anti-proliferative effect of U251 glioma cells.Jiuwei Tongqiao decoction regulated the protein content changes of Bcl-2 and P53,disturbed cell proliferation,and thus induced apoptosis.This paper selected the most efficient parties and the most effective part of Jiuwei Tongqiao decoction.initially revealed the anti-brain-glioma mechanism of Jiuwei Tongqiao decoction in vitro. |
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