Study of the Metabolism and Toxicity of Ginkgolic Acid (15:1)
|Keywords||Ginkgo acid (15) Rat liver microsomes UPLC-PDA-MS/MS Metabolites H / D exchange Rat primary hepatocytes HepG2 cells MDCK MDR1 cells MDCK cells P-glycoprotein Cell cycle arrest|
Ginkgo used in traditional Chinese medicine for several thousand years of history, is mainly used to treat blood circulation disorders and enhance memory capacity. Ginkgo biloba containing a variety of active chemical ingredient, resulting in Ginkgo biloba having a wide range of pharmacological activity and toxic effects. Ginkgo acid is a derivative of salicylic acid, is widely present in ginkgo leaves, fruits and testa of bioactive components outside the seed coat content is the highest. Ginkgo acid is recognized as harmful compounds, has cytotoxicity, sensitization, mutagenic and carcinogenic properties. In recent years, the international ginkgo phenolic acids as Ginkgo pharmaceutical intermediates quality standard control indicators, Ginkgo phenolic content (mass fraction) control the following five millionths. Ginkgo acid more concentrated in the pharmacological and toxicological aspects, and for in vitro and in vivo metabolism and its metabolites have not been reported. In the present study, the extract from Ginkgo biloba exocarp, separation and preparation of GA (15:1). In vitro metabolism studies, the application of SD rat liver microsomes investigated its metabolism. Phase I metabolites were analyzed by ultra performance liquid chromatography / diode array detector / negative ion electrospray ionization tandem mass spectrometry (UPLC-PDA-ESI-MS/MS) and hydrogen / deuterium (H / D) exchange. The results showed that, in the metabolic process, GA (15:1) on the benzene ring structure did not change, the oxidation occurs mainly on the alkyl side chains in the C6 position. Compared with control group, in PDA FIG least 8 metabolites, wherein the six metabolites structure has been confirmed, and new metabolites. This study in vitro cytotoxicity assays to detect the phase Ⅰ metabolic whether enough increase GA (15:1) that liver toxicity. In primary rat hepatocytes of the phase I metabolic enzymes CYP1A, and CYP3A chemical inhibition of HepG2 cells-phase I metabolic enzymes CYP1A, and CYP3A chemically induced cell survival MTT assay to assess given GA (15:1) rate. The results showed that in two cells, the cytotoxic effects of GA (15:1) were tested time concentration-dependent relationship in which toxicity was significantly less than the toxicity of HepG2 cells in primary rat hepatocytes. Rat primary hepatocytes cultured for 4 days, the sensitivity of the cells to GA (15:1) than cultured for 20h primary hepatocytes is much smaller. While giving GA (15:1) and the selective CYPP450 inhibitors a-naphthoflavone (CYP1A) or ketoconazole (CYP3A), rat primary hepatocytes on GA (15:1) toxic effects of sensitivity was significantly reduced. The the selective CYPP450 enzyme inducer beta-naphthoflavone (CYP1A) or rifampicin (CYP3A), pretreatment of HepG2 cells after 3 days to give the Ga (15:1), can significantly improve on HepG2 cells GA (15:1) toxicity. These results indicate that, compared with rat primary hepatocytes, GA (15:1) HepG2 cells have greater cytotoxicity and CYP1A and CYP3A metabolism can increase the cytotoxicity of GA (15:1) effect. Ginkgo acid has been shown to have induced allergic, genotoxic and cytotoxic activity. However, P-glycoprotein (P-gp) efflux is able to reduce the toxic effects of ginkgo acid in the kidney; ginkgo acid renal tubular epithelial cell toxicity mechanisms leading to cell necrosis or apoptosis has not reported. In the present study, the MDCK cells ScS GA (15:1) renal toxicity. It was found that the toxicity of GA (15:1) in MDCK cells and MDCK-MDR1 cells have time concentration dependencies, and MDCK-MDR1 cells are less toxic than the MDCK cells, and GA (15:1) in MDCK-MDR1 The accumulation amount intracellular accumulation amount may be less than in MDCK cells have a certain relationship. Application of P-glycoprotein inhibitors such as verapamil or cyclosporin A pre-incubated for 15 min, can increase the accumulation of GA (15:1) in MDCK-MDR1 cells, and significantly increase GA (15:1) MDCK -MDR1 cell toxicity, which indirectly kidney P-gp efflux GA (15:1), to reduce its renal toxicity. In order to study GA (15:1) cytotoxicity leading to cell necrosis or induction of apoptosis, a clear mechanism of toxic reaction in MDCK cells, DNA agarose gel electrophoresis and flow cytometry experiments study. The results show that the short period of time at a high concentration, GA (15:1) can directly cause cell necrosis; GA (15:1) induced apoptosis generated in a low concentration for a long time, have not found. MDCK cell cycle analysis found that different concentrations of GA (15:1) MDCK cells arrest in G1 and G2 phase, so that in G1 and G2 phase cells increased, S and M phase cells decreased further study found, GA ( 15:1) is also possible to reduce the MDCK cells in the mitochondrial membrane potential. These results provide a reference for the understanding of the toxicity of ginkgo acid.