The Study Of The Anti-tumor Activity And Mechanism Of The Anthriscus Sylvestris(L.)Hoffm’ Extracts And Monomers

Objective To investigate the effect of the extracts of Anthriscus sylvestris (L.)Hoffm on inhibiting proliferation and inducing apoptosis HEPG2, MG-63, B16, HELA cells in vitro; To explore the acute toxicity of the Anthriscus lactone and the anti-tumor activity and molecular mechanism in H22tumor-bearing mice in vivo.Methods To detect the inhibitive effect of different concentration of the extracts of Anthriscus sylvestris (L.)Hoffm on proliferation of HEPG2, MG-63, B16, HELA cells in vitro after48h by MTT Assay method. And to calculate the IC50value of the petrol extract of the extracts of Anthriscus sylvestris (L.)Hoffm. To detect the cell apoptosis character of HEPG2, MG-63, B16, HELA cells by treating with different doses of the petrol extract of Anthriscus sylvestris (L.)Hoffm by the DAPI method and DNA ladder method. And to calculated the IC50value of the Anthriscus lactone; To establish the anmimal model by inoculating the H22cell line in the left oxter asepticly, and then devided the mice into7groups randomly. took the Anthriscus lactone high-dose, medium-dose and low-dose group, Anthriscus polysaccharide group and Tegafur group as the treatment group, model group and blank group were treated with0.5%CMC-Na as the control, then gave the mice drugs daily and weighed once a day respectively, after12days,7mice in each group were killed. Then to take out the tumor, thymus and spleen, and weighed them to calculate the mortality, inhibition rate, thymus index, spleen index, and the rate elongation of life. To detect the necrosis of tumor and expression of bax、bcl-2、P53by HE and immunohistochemistry method.Results1、The extracts of chloroform, petroleum of Anthriscus sylvestris (L.)Hoffm all had the better inhibitive effect on the proliferation of HEPG2、HELA cells, the IC50value were36.53μg/ml and18.25μg/ml on HEPG2、HELA cells by The extracts of petroleum. The IC50value were40.62μg/ml and45.66μg/ml on HEPG2、HELA cells by The extracts of chloroform. Anthriscus ethyl acetate extract and Anthriscus polysaccharide almost have no anti-tumor activity in vitro. The Anthriscus lactone had better inhibitive effect on the proliferation of the four tumor cells. The IC50value were12.24μg/ml、14.37μg/ml、43.25μg/ml and18.72μg/ml for HEPG2、MG-63、B16、HELA cells respectively. The rate of inhibitive effect was related with the concentration, and the rate of inhibitiv effect reached70%at the80μg/ml.The isoan-thricin had significantly inhibitive effect or the proliferation of B16cells and MG-63cells, and the IC50value were34.68μg/ml and65.72μg/ml for B16cells and MG-63cells respectively.2、By DAPI stained, then to observe the impact of different concentrations of Anthriscu lactone on HEPG2, MG-63, B16, HELA cells by gel electrophoresis method. These fou tumor cells’growth was suppressed, including the cell collapse, fragmentation of cel nucleus, pyknosis and so on. And higher concentrations, more obvious. The cells in the control group were intact. At different concentrations treated, The DNA apopsis electrophoresis lines were observed by DNA ladder method.3、The acute toxicity test of Anthriscus lactone used Kunming mice. The test showed the Anthriscus lactone althougth had higher anti-tumor activity, but also had higher acute toxicity. the LD50value was151.52mg/kg, the95%confidence interval was93.28mg/kg-252.94mg/kg.4、The inhibitive rate of high-dose group, medium-dose group, low-dose group, and the Tegafur group were54.39%、63.74%、56.14%、53.80%respectively. To detect the bax,bcl-2and P53expression in different group’s tumor tissues by immunohistochemical method. The preliminary study showed Anthriscus lactone can significantly improve the lives of mice and can upregulate the expression of bax and reduce the ratio of bcl-2and bax So Anthriscus lactone had the effect of inducing the H22cells apoptosis. Although each group’s p53had certain expression, but there was no significant differences.Conclusions The Anthriscus lactone had significant anti-tumor activity on HEPG2、 MG-63、B16、HELA cells in vivo. And can significantly improve the lives of mice which have H22tumor cells. Its one of the ways to promote apoptosis may be related with bax expression raised and the ratio of bcl-2and bax reduced.