Study On The Pharmacokinetics Of Triptolide And Its Effect To Ovary Tumor Cells

Tripterygium have been used widely to treat autoimmune diseases including rheumatoid arthritis, immune complex nephritis, and systemic lupus erythematosus in China for many years. Triptolide, a diterpene triepoxide, is an active component of extracts derived from the medicinal plant Tripterygium wilfordii Hook F., but it's also the poisonous component of extracts derived from the plant. At present, the research of triptolide is centralized on the effects of anti-tumor and immunosuppressive. But the report about it's pharmacokinetics and it's effect on ovarian caner have not been found until now. Therefore, we established the method to detect the concentration of triptolide in plasma and review the phannacokinetics parameters in rats. On the other hand, we adopted the ovarian cancer cell, A2780 lines and OVCAR-3 lines, in different p53 status to investigate the effect of triptolide on ovarian cancer, and use the flow cytometry to detect it's effect on cell Apoptosis and cell Cycle. The results are as follows:We adopted ethanol, the mixture of chloroform and aether (1:3), chloroform and Solid-Phase Extraction(SPE) to distill drug from the blood sample, and use HPLC method to compare the extraction rate. We see, the sample is very clean if use the SPE method, there is no impurity apex near the drug apex. The retention time is about 8 minutes, the absolute reclamation rate is about 80% and the RSD value is very little. So we give a dose 0.737 mg/kg of triptolide to rats by paunch injection, The condition of HPLC as follows: Hyperssil 0DS2 C₁₈ column (5μm, 200mm × 4.6mm), the mobile phase: 45%methanol, 55%water, flow rate: 1mL·min^-1, detective wave length: 218nm.The result shows that the absorption and distribution of triptolide in rats by ip. are rapid, but the elimination is very slow, the half life time of elimination is 19.15h.The ovarian cancer cell, A2780 lines and OVCAR-3 lines, were treated by different concentration of triptolide (0.5ng/mL, 1.5ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 120ng/mL, 160ng/mL, the final concentration) for 24, 48 and 72 hours respectively. Then, the efficacy to ovarian cancer cells was evaluated by means of the 3-(4,5-dimethylthiazol-2-yl) - (2,6-dimethyl-morpholino)-2,5-dipheny 1-tetrazolium bromide(MTT) assay. The cell viability of the tumor cell lines were restrained obviously, the effect is in dose and time dependent manner. Ovarian cancer cell lines A2780 was more sensitive to tnptolide, comparing with the cell lines OVCAR-3, it's IC50 concentration on A2780 was 2.17, 1.31 and 1.16 ng/mL after 2^ 48 and 72 hours, and on OVCAR-3 was 92.79 > 10.23, 7.34 ng/mL.The results of the cell cycle distribution after treatment with IC,so concentration of triptolide on A2780 (2.6, 1.8 ,1.5ng/mL for 24, 48, 72h, respectively) and OVCAR-3 (123,13.6, 8.3ng/mLfor 24, 48, 72h, respectively)cell lines were assessed by flow cytometry with propidium iodide (PI) coloring. The results were, after treatment with IC6o concentration of triptolide on A2780, the proportional in the S phase fraction is increased. And OVCAR-3 cells were arrested in the G2/M phase of the cell cycle. The effect is in time dependent manner and there were a gradual increase of the two cells in the sub-Gl phase.When the A2780 cell lines and OVCAR-3 cell lines were treated with IC3o> IQo and IC90 concentration of triptolide for 48 hours, the percentage of apoptotic cell were calculated with Armexin V-PI stain by FACS. The results showed treatment with various concentrations of triptolide can resulted induction of cell apoptosis in some content and this inducement is in a dose dependent manner. Treatment with the IQo concentration of triptolide, the A2780 cell lines is most in late apoptosis, but the OVCAR-3 cell lines is most in early apoptosis. And the conclusion could be drawn: Triptolide can inhibit the proliferation of human ovarian cancer cell, which is related to the cell cycle arrest and apoptotic, and more mechanisms of triptolide induces the apoptosis on ovarian cancer remain to be investigated.