Research In Anti-tumor Active Fraction Of Chrysanthemum Indicum In Vitro

Objective: Objective: Comfirmed the anti-tumor active fraction and constituents of wild chrysanthemum in vitro,and studied the anti-tumor mechanism of the fractions to provide experimental data for further exploitation and utilization of wild chrysanthemum.Methods: 1.Wild chrysanthemum was heated under 65℃ with70% ethanol as the solvent to get for Chrysanthemum indicum extract(CIE),followed by the use of petroleum ether,ethyl acetate and n-butanol to extract CIE to obtain Petroleum ether extract(PEE),Ethyl acetate extract(EAE)and n-butanol extract(NAE).All the products were weighed and calculate the yield,and a series of chromogenic reaction were used to identify the chemical compositions in different polar fractions.2.Morphological observation,CCK-8 were used to detected the influence in cell morphology and proliferation of CIE,PEE,EAE and NAE on tumor cells including human lung cancer cells(A549),human erythroleukemia cells(K562),human breast cancer cells(MCF-7),and human liver cancer cells(Hep G2).Hoechst nuclei staining,wound healing assay and transwell invasion assay were used to evaluate the influence of apoptosis,migration and invasion effect of PEE,EAE and NAE on MCF-7.3.PEE and EAE were isolated by silica gel column chromatography method combined with TLC according to the wild chrysanthemum anti-tumor activity screening results,and the MCF-7 cell proliferation inhibitory activity were used as an index in screening the most strongest active segmented products.Results: 392.2 g CIE was extracted from 1 Kg chrysanthemum indicum,the yield was 39.22%;56.1 g PEE,39.3 g EAE and 33.8 g NAE were gained from CIE which were extracted by a series of organic solvent,the yield were 5.61%,3.93% and 3.38%,respectively.The results of the chromogenic reaction showed that PEE showed positive in Fe Cl3(aqueous solution)reaction,Dragendorff reaction,Wager response,Mayer reaction,Molish reaction,liebermann-burchard reaction and Salkowski reaction;EAE showed positive in PAULY reaction,Fe Cl3(alcohol solution)reaction,hydrochloric acid-magnesium reaction,Fe Cl3(water solution)reaction,Molish reaction,Liebermann-Burchard reaction and Salkowski reaction.Shrinkage and fragmentation of cells could be seen obviously under the fluorescent inverted microscope when A549,K562 and MCF-7cells were treated with CIE、PEE、EAE、NAE when their concentra-tions were above 150 μg/m L.The proliferation of A549,K562 and MCF-7cells can be inhibited by CIE、PEE、EAE、NAE in a dose-dependent manner in the concentration from 150 μg/m L to 250 μg/m L,PEE showed the best inhibition effects in MCF-7;EAE showed the best effect to MCF-7 in apoptosis-inducing and cell migration and invasion resisting.9 component groups were isolated from PEE and 6 component groups from EAE.The component group E1 had the most obvious inhibitory effect on the proliferation on breast cancer MCF-7 cells.Conclusion: 1.Chrysanthemum indicum has antitumor activity and has a good performance in inhibiting the proliferation of MCF-7 cells in vitro;2.The anti-tumor activity sites of wild chrysanthemum were Petroleum ether extract(PEE)and Ethyl acetate extract(EAE),their mechanism might be associated with induction of apoptosis and inhibition of migration and invasion;3.The components with the strongest anti-tumor activity were presented in EAE,in the elution using 70% petroleum ether and 30%ethyl acetate as eluant.