The Anti-Hepatocellular Activities And Mechanisms Of Phenylethanoid Glycosides From Cistanche Tubulosa

Traditional Chinese medicine（TCM）has been widely used to treat various diseases including cancer.A large number of antitumor components have been isolated and identified from TCM,which are potential candidates for the development of antitumor drugs.Cistanche tubulosa has a variety of biological activities including anti-oxidation,anti-aging,anti-osteoporosis and immunoregulation,and contains many components including phenylethanoid glycosides,iridoids,alditols,oligosaccharides,polysaccharides and volatile oils.Phenylethanoid glycosides are the main active ingredients of Cistanche.Echinacoside and acteoside are the two main components of phenylethanoid glycosides.Here,the anti-hepatocellular activity and mechanism of C.tubulosa phenylethanoid glycosides（CTPG）were investigated.The high performance liquid chromatography（HPLC）was used to quantify echinacoside and acteoside in CTPG.MTT assay was used to detect the viability of human hepatocellular carcinoma（HCC）cells Hep G2 and BEL-7404 and normal murine liver cells NCTC1469,and the proliferation of murine splenocytes.Ki-67 staining was used to test the proliferation of HCC cells.The morphology of HCC cells was observed by inverted microscope.Hoechst 33342 and propidium iodide（PI）staining was used to observe the chromatin condensation.The cell cycle changes of HCC cells were detected by PI staining.The apoptosis of HCC cells was detected by Annexin V/PI staining.The mitochondrial membrane potential of HCC cells was detected by JC-1 staining.The levels of apoptosis-related proteins in HCC cells were detected by Western blot.The role of caspase was analyzed by inhibitors in apoptosis induced by CTPG.Kunming mice were treated intragastricaly,subcutaneously and intraperitoneally with CTPG to evaluate the safety and immunoregulation of CTPG.The body weight was monitored and the organ indexes were calculated.The number and activation status of immune cells in spleen were detected by flow cytometry.H22 tumor mosue model was established using male Kunming mcie and treated with different doses of CTPG to evaluate the antitumor effect in vivo.The body weight and tumor sizes were measured,and the survival rates were calculated.The changes of liver,lung and kidney tissues of tumor mice were detected by HE staining.Finally,the antitumor components of CTPG were isolated by85%ethanol to remove polysaccharides,extraction with organic reagents,AB-8macroporous resin adsorption,silica gel column chromatography,ODS-C18 column chromatography and qualitified by HPLC.The following results were obtained.（1）CTPG inhibited the growth of HCC cells in vitro through mitochondria-depedent apoptosis.HPLC was used to quantify the contents of echinacoside and acteoside in CTPG,which are 28%and 9.9%respectively.CTPG dose-and time-dependently reduced the viability of Hep G2 and BEL-7404 cells,and the IC₅₀ values are（405±11.2）μg/m L and（420.3±10.5）μg/m L at 24 h,respectively,which is consistent with the result of Ki-67 staining.However,it promoted the proliferation of splenocytes and showed weak cytotoxicity on normal murine liver cells NCTC1469.It was observed that the number of BEL-7404 cells was reduced and cells became round and lost the intact morphology after CTPG treatment.CTPG also induced chromatin condensation and cell cycle arrest at S phase in HCC cells.The protein levels of Bax and Bcl-2 were up-regulated and down-regulated,respectively,which led to the reduction of mitochondrial membrane potential and the release of cytochrome c in HCC cells,then caspase 9,caspase 7 and caspase 3 were activated to degrade PARP.The inhibitors of caspase and caspase 3 partly decreased the apoptosis of HCC cells induced by CTPG.We further found that CTPG up-regulated the phosphorylation of JNK and p38,and down-regulated the phosphorylation of ERK.The results indicated that CTPG might inhibit the growth of HCC cells through mitochondria-depedent apoptosis and MAPK signaling pathway.（2）CTPG suppressed the growth of HCC cells in vivo without side effect.Kunming mice were intragastrically,subcutaneously,and intraperitoneally injected with CTPG（200 mg/kg and 400 mg/kg）.Compared with control group,the body weight and organ indexes including heart,liver,lung and kidney of mice treated with CTPG had no significant changes.Intragastrical and subcutaneous injection of CTPG did not change spleen index but intraperitoneal injection significantly increased spleen index.Flow cytometry results showed that CTPG increased the number of T cells,B cells,natural killer cells and effector T cells in the spleen,suggesting that the selected doses of CTPG were safe for mice.CTPG can inhibit the proliferation of H22 cells in vitro,arrest cell cycle at G0/G1 phase and induce apoptosis.Therefore,the antitumor effect of CTPG was further investigated using H22 tumor mouse model.The results showed that CTPG significantly inhibited the growth of tumor and improved the survival of tumor mice（P<0.05）.HE staining showed that tumor tissues from mice treated with CTPG had different degrees of necrotic areas,while liver,lung and kidney had no obvious pathological damage.（3）The separation of antitumor components from CTPG.The anti-hepatocarcinoma activity of the mixture of standard echinacoside and acteoside according to the contents in CTPG was compared with CTPG at the same concentrations.The results showed that the anti-hepatocarcinoma activity of CTPG was better than the standard mixture.We speculate that there are other antitumor components in CTPG except echinacoside and acteoside.Therefore,the active components in CTPG were separated with 85%ethanol to remove polysaccharides,extraction with organic reagents,AB-8 macroporous resin adsorption,silica gel column chromatography,ODS-C18 column chromatography and qualitified by HPLC.The main antitumor components in CTPG still are echinacoside and acteoside.We did not find new antitumor component.