Study Of Effect Of Procyanidins Angelica Dahurica Alkaloids On Cervical Cancer And Its Mechanism

Behind the breast cancer, cervical cancer is the second malignant tumor which threatens the health of women. But the current use of chemical synthetic drugs such as cyclophosphamide, not only kills tumor cells, but also damages blood cells, resulting in decreased immune function, or even died from complications caused serious side-effects. To be safe, effective and with little adverse effect, natural medicine has been paid many attention to treat the cervical cancer, with overwhelming advantages. Pinus koraiensis Bark Procyanidins, main active component of the Pinus koraiensis, is effective in cancer treatment, antimutation and antioxidants. So far, Pinus koraiensis Bark Procyanidins extract (PKBPE) used in cervical cancer treatment hasn't been reported systematically. Though it is valuable as medicine in prevention of sunshine, ultraviolet and inhibition of tyrosinase, Angelica dahurica alkaloids (AAD) haven't been reported in cervical cancer treatment.In this study, PKBPE was extracted with macroporous resin, Angelica dahurica alkaloids were extracted with the help of ultrasound wave, and they were then seperated, purified and identified. Mice bearing U14 cervical cancer were divided into control group, CTX positive group, PKBPE low dose group, PKBPE high dose group, AAD low dose group and AAD high dose group, and operated following the HE method, immunohistochemistry SP method, transmission electron microscopy(TEM), colorimetric analysis antigenic, enzyme linked immunosorbent(ELISA) method and reverse transcription-polymerase chain reaction(RT-PCR), then the effects of PKBPE on mice bearing U14 cervical cancer were researched in vivo. HeLa cells which were inhibited and induced apoptosis by PKBPE and AAD were studied by MTT method, TRAP argentation, TEM, flow cytometry, agarose gel electrophoresis and western blot method in vitro. The results were as following.Marron powder were extracted from Pinus koraiensis Bark with the rate of 2.3%, which was identified as Procyanidins and the procyanidins content was 22.84mg/g. White crystalline was extracted from angelica dahurica with the rate of 2%. The angelica dahurica alkaloids content was 0.16mg/g, identified as total alkaloids. In vivo experiments, PKBPE and AAD could significantly extend the survival time of U14 ascites mice, inhibit the tumor growth of tumor-bearing mice, arrest tumor cells in G0/G1 period, and decrease the positive numbers of mutant P53, Ki-67, PCNA and Bcl-2 protein expression in tumor cells without liver and kidney injury.In immunity and anti-oxygenation experiments of mice bearing U14 cervical cancer,PKBPE and AAD significantly increased SOD activity and decreased MDA content of mice plasma in free radicals testing;in immune function tests, PKBPE and AAD significantly increased the thymus index and the spleen index of U14 mice, significantly lowed ear swelling, raised mice macrophage phagocytosis function, increased serum TNF-α(P<0.01) and lowed IL-8 levels (P<0.01), increased IL-2 activity significantly.In HeLa cell experiments, the IC50 values of PKBPE and AAD were 196.38μg/ml and 387.45μg/ml respectively. Not only cell growth could be inhibited, but also Caspase-3 activities were significantly increased by PKBPE and AAD, thus apoptosis were induced significantly.PKBPE and AAD could make HeLa cell appeared the typical ultrastructure characteristics of apoptosis by TEM observation. In HeLa cell cycle experiments, PKBPE and AAD could make the agarose gel electrophoresis of HeLa cells appear"ladder"zone with a dose-dependent, inhibit tumor cell replication and DNA synthesis, inhibit normal cell division, arrest cell to S phase, so that cells were accumulated in G1 period with G2/M block, further leading to cell mitosis prevented and apoptosis.PKBPE and AAD could increase the expression of Bax and reduce Bcl-2 and Survivin protein expressions in line with the anti-tumor effects. These results showed that the molecular basis of PKBPE and AAD inducing HeLa cells apoptosis.Mice bearing U14 cervical cancer and HeLa cell were acted as experimental material in vitro and in vivo, by the methods of immunohistochemistry SP, ELISA, MTT, TEM, western blot, flow cytometry, RT-PCR, et al, the effects and mechanisms of PKBPE and AAD anti-cervical was systematically researched to provide important references for the treatment of cervical cancer and utilization of natural medicines in cancer treatment.