Study On The Anti-tumor Activity Of Rhizoma Cyperi By Supercritical CO₂ Fluid Extraction In Vitro

Objective:This study was performed to investigate the inhibitory effect of XF,extracted from Cyperus rotundus with the technique of Supercritical CO₂ fluid,on a variety of tumor cells,and to explore the anticancer mechanism at the cellular and molecular level.Method:1.XF was extracted by supercritical CO₂ fluid extraction?SFE-CO₂?without entrainer.2.Cell viability,evaluated by MTT assay,are the main indicator?s?to assess the inhibitory and killing effect of XF on tumor cells.4 tumor cell lines?HepG2,A549,PC12,LNcap?and normal liver cell line?LO2?,which was as a control,were studied.3.HepG2 cells were used to study the molecular mechanisms of anti-tumor effect of XF.?1?Intracellular MDA,SOD Levels were measured to survey the oxidative damage induced XF on the cells.?2?Cell apoptosis and necrosis were evaluated by Hoechst 33342,Annexin V-EGFP/PI double labeling and flow cytometry methods.?3?Mitochondrial membrane potential????was detected by Rhodamine123?Rh 123?.?4?Bcl-2 phosphorylation relative to the total Bcl-2 expression was tested by the Muse?Bcl-2 Activation Dual Detection Kit.?5?Expression levels of apoptosis related protein Bcl-2 and Bax were detected by Western blot method.Results:1.The mean yield of XF was 1.073%?2.XF showed a significant dose dependent and time dependent killing effect on the 4tumor cell lines.The IC₅₀ for HepG2,A549,PC12,LNcap cells for 72 h respectively were 43.4,71.9,75.3 and 75.4?g/ml.Compared to the positive control drug DDP,which has a great toxicity on both tumor and normal cells,XF is less toxic to LO2 cells.3.The anti-rumor mechanism of XF was studied at molecular level.?1?XF increased intracellular MDA,decreased SOD activity of HepG2 cells significantly,which means that XF could induce oxidative damage.?2?Hochest33324 staining and Annexin V-EGFP/PI double staining showed that XF quickly and efficiently induced apoptosis of HepG2 cells in a concentration manner.Flow cytometry of Annexin V-EGFP/PI double staining showed that the late apoptosis rate of HepG2 cells were up to 97%after incubated with XF at 200?g/ml for 6 h,demonstrating that induction of apoptosis was the mechanism for the anti-tumor effects of XF.?3?Rh 123 staining indicated that XF could cause dose-dependent mitochondrial damage and loss of??.?4?XF could cause Bcl-2 protein dephosphorylation.?5?XF could raise the expression of Bax,lower the expression of Bcl-2,leading to cell apoptosis.Conclusion:1.XF had efficient anti-tumor effect on all of the 4 cell lines in vitro with the maximum inhibition/killing rate of 99.9%.2.Apoptosis played a critial role in anti-tumor effect of XF.XF induced apoptosis by triggering mitochondrial oxidative stress,inducing Bcl-2protein dephosphorylation and elevating the ratio of Bax/Bcl-2,then leaded to activation of endogenous apoptosis pathway.4.XF is of more selectivity for tumor cells and less selectivity for normal cells and more efficient and safer compared with DDP.Therefore,with great potential and good prospects,XF is expected to be developed as a novel anti-tumor drug for tumor treatment in clinic.