The dried roots of Euphorbia kansui L.(Euphorbiaceae)are known as “Gan Sui” in Traditional Chinese Medicine.It is abundantly distributed in northwestern China.“Gan Sui” was firstly recorded in Sheng Nung's Herbal as a low grade drug and it is bitter,cold and poisonous.Modern researches indicated that it had numbers of activities,such as purgative,abortive,analgesic,immunosuppressive and anti-tumor activity.Previous investigations have shown that the ethyl acetate extract of Euphorbia kansui showed strongest anti-tumor activity in all kinds of commonly used solvent extracts.However the research of monomers was still in blank.In this study,a new strategy of the convergence of two-dimensional preparative high performance liquid chromatography(HPLC)and real-time cell analysis(RTCA)was developed for bioassay-guided separation of anti-tumor components from the ethyl acetate extract of Euphorbia kansui.The method might lay the material foundation for the anti-tumor mechanism of this medicine.The first-dimensional separation was conducted on silica column in industrial preparative high performance liquid chromatography and seven fractions were gatherd.After anti-tumor screening by iCELLigence real-time cell analysis system,fraction 4 and 6 were selected to sepatare by the second-dimensional preparation.Fraction 4 was prepared by 2D-NPLC/RPLC chromatography system with silica column and C18 column and fraction 6 was prepared by 2D-NPLC/NPLC chromatography preparation system with silica column and amide column.In this method,eight compounds esulone A(1),kansuinin A(2),(3?,11?)-3,11-dihydroxylanosta-8,24-dien-7-one(3),kansuinin E(4),kansuinin B(5),isoscopoletin(6),kansuinin D(7)and kansuinin G(8)were efficiently isolated in purity above 97%.Of all the compounds,esulone A has been isolated from this plant for the first time.The structures were identified by NMR spectroscopy and comparison with the previous literatures.The bioassay revealed that compounds 1,2,3,7 and 8 almost entirely inhibited the proliferation of A549 cells and that compound 8 was also thought to be the most active compound against Hep-G2 cells.The result of primary pharmacodynamics revealed that kansuinin A and kansuinin B showed obvious concentration-dependent effect on lewis lung cancer mice.The inhibitory ratios of kansuinin A and kansuinin B were similar to cyclophosphamide with the concentrations of 11.11mg/kg and 99.99mg/kg,respectively.The experiment might provide the reference for further research of its action mechanism.The convergence of 2D-HPLC and RTCA system had distinctly shorten the experiental period.It also prepared considerable assist for separation and anti-tumor screening of complicated compounds of Traditional Chinise Medicine. |