| Objective: The active of five extraction parts of Polygonum chinensis was determined by the active site for the treatment of breast hyperplasia in the M C F-1 0 A(h u m a n n o r m a l b r e a s t h y p e r p l a s i a c e l l l i n e).T h e n,t h e active parts were separated and purified by phytochemical separation in order to obtain monomeric compounds,and the cell model was used to study the in vitro activity.Method:1.The preparation of alcohol extract and five extraction sites of Polygonum chinensis The dried grass was dried and extracted with 95% ethanol.The obtained extract was concentrated and added with silica gel.The samples were eluted with different polar organic solvents such as petroleum ether,chloroform,ethyl acetate,acetone,and methanol.Concentrated and dry solution.2.The screening of the active site of breast hyperplasia The effect of five extraction sites on the cell proliferation of MCF-10A(human normal breast hyperplasia cell line)was detected by MTT assay.3.The separation and purification of the active site of breast hyperplasia and its structural identification.The active components were separated and purified by silica gel column chromatography,ODS reversed-phas chromatographic column,Sephadex LH-20 gel column and MCI pore adsorption resin,lc-3000 medium pressure preparation and other chromatographic techniques The corresponding monomer components were obtained.The physical and chemical properties of the compounds were analyzed by HPLC,MS and NMR spectroscopy.The isolated monomeric compounds were identified.4.The effect of 5,4 '-2 hydroxy-6,7,3'-trimethoxyflavone and apigenin isolated from the active site of breast hyperplasia on the cell proliferation of MCF-10 A cells.The effects of monomers on cell proliferation were investigated by MTT assay in vitro activity on MCF-10 A.The real-time PCR was used to detect the expression of cmyc?cyclin D1?ER? m RNA expression levels.Results:1.Through the active of five extraction parts of Polygonum chinensis was determined by the active site for the treatment of breast hyperplasia in the MCF-10A(human normal breast hyperplasia cell line)screening found that ethyl acetate and acetone had obvious inhibitory effects on MCF-10 A cells,and showed a tendency of concentration dependence and time dependence.2.A total of 19 compounds were isolated from ethyl acetate and acetone.14 compounds were identified by hydrogen spectrum,carbon spectrum and mass spectrometry.Including 4 flavonoids: quercetin(pce-1)?5,4 '-dihydroxy-6,7,3'-trimethoxy flavone(pce-8)?luteolin(pce-15)?apigenin(pce-16);1 coumarin compound: isofraxidin(pce-12);4 organic acids: ortho two benzoic acid(pce-2)?palmitic acid(pce-3)?3,4-dihydroxybenzoic acid(pce-7)?oleic acid(pce-11);5 phenolic acids: gallate gallate(pce-5)?gallic acid(pce-13)?caffeic acid(pce-14)?corilla(pce-17)?3-O-methyl ellagic acid-4 '-mouse liposide(pce-19).Among them,Pce-2,pce-3,pce-8,pce-12,pce-17 and pce-19 were all isolated from the plant for the first time.3.The expression of cmyc ? cyclin D1 ? ER? in MCF-10 A cells was down-regulated by 5,4 '-2 hydroxy-6,7,3'-trimethoxyflavone and apigenin(P<0.01,P <0.05).Conclusion:1.Both ethyl acetate and acetone can inhibit MCF-10 A cells,and present a trend of concentration dependence and time dependence2.12 compounds were isolated from ethyl acetic acid ethyl acetate.quercetin,orthotwobenzoicacid,palmiticacid,gallategallate,3,4-dihydroxybenzo ic acid,5,4 '-dihydroxy-6,7,3'-trimethoxy flavonoids,oleic acid,isochrin,gallic acid,caffeic acid;2 compounds were isolated from the acetone.apigenin,luteolin,corlijing,3-O-methyl ellagic acid-4 '-rat liposide.3.The expression of cmyc ? cyclin D1 ? ER? in MCF-10 A cells was down-regulated by 5,4 '-2 hydroxy-6,7,3'-trimethoxyflavone and apigenin obtained from the treatment of the active site of breast hyperplasia. |
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