Study Of The Metabolism And Toxicity Of Ginkgolic Acid (15:1)

Ginkgo biloba has long been used in traditional Chinese medicine to treat circulatory disorders and enhance memory. The pharmacological activity and toxicity of Ginkgo biloba is attributed to different constituents present in it. Ginkgolic acids and related alkylphenols (e.g. cardanols and cardols) have been recognized as hazardous compounds with suspected cytotoxic, allergenic, mutagenic and carcinogenic properties.The question arises whether the metabolism of ginkgolic acids in the liver could decrease or increase their toxicity. In this study, the in vitro metabolism of ginkgolic acid (15:1), one component of ginkgo acids, was investigated as a model compound in Sprague-Dawley rat liver microsomes. The metabolites were analyzed by ultra-performance liquid chromatography equipped with photodiode array detector /negative-ion electrospray ionization tandem mass spectrometry (UPLC-PDA-ESI-MS/MS) and hydrogen/deuterium (H/D) exchange. The result showed that the benzene ring remained unchanged and the oxidations occurred at the side alkyl chain in rat liver microsomes. At least eight metabolites were found. Among them, six phase I metabolites were tentatively identified. This study might be useful for the investigation of toxicological mechanism of ginkgolic acids.To determine whether the phase I metabolism could contribute to their cytotoxicity, we investigated the cytotoxicity of ginkgolic acid (15:1), using in vitro bioassay systems. In the first step, cytochrome P450 enzymes involved in ginkgolic acid (15:1) metabolism were investigated in rat liver microsomes; then, two in vitro cell-based assay systems, primary rat hepatocytes and HepG2 cells, were used and the measurement of MTT reduction was used to assess cell viability. Results indicated that the cytotoxicity of ginkgolic acid in primary rat hepatocytes was lower than in HepG2 cells. Ginkgolic acid (15:1) demonstrated less cytotoxicity in four-day-cultured primary rat hepatocytes than that in 20-hour-cultured ones. Co-incubation with selective CYP inhibitors, a-naphthoflavone and ketoconazole, could decrease the cytotoxicity of ginkgolic acid in primary rat hepatocytes. In agreement, pretreatment with selective CYP inducers,β-naphthoflavone and rifampin, could increase the cytotoxicity of ginkgolic acid (15:1) in HepG2 cells. These findings suggest that HepG2 cells were more sensitive to the cytotoxicity of ginkgolic acid than primary rat hepatocytes, and CYP1A and CYP3A could metabolize ginkgolic acid to more toxic compounds.little information is available about the direct effects of P-gp on ginkgolic acids toxicity in kidney and there is no report on the mechanisms of how ginkgolic acids induce renal tubular epithelial cell toxicity. The cytotoxic potential of ginkgolic acids and their effect on the rhesus monkey kidney was investigated (Hecker et al.,2002). Instead of mixture, the present study investigated GA renal cytoxicity using MDCK cells. The investigation was divided into two phases: in the first phase, the effect of GA on cell viability and its interaction with P-gp were studied in MDCK cells and MDCK-MDR1 cells, and in the second phase, especially by DNA agarose gel electrophoresis and flow cytometry, the mechanism of renal toxicity in MDCK cells was studied.The cellular uptake and cytotoxicity of GA in MDCK and MDCK-MDRlcells displayed a time and concentration dependent manner. Pre-incubation with a typical P-glycoprotein inhibitor such as verapamil or cyclosporin A before the incubation with GA significantly increased GA uptake in MDCK-MDR1 cells. GA also inhibited Rhodamine 123 accumulation in MDCK-MDR1 cells. These results suggested that P-gp expressed in the renal epithelial cells could exclude GA to reduce its toxicity.Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions. Using flow cytometry, further study of the cytotoxic mechanism demonstrated that GA could induce cell cycle arrest at the G0/G1 and G2/M phases in a time and concentration dependent manner, and GA could decrease the mitochondrial trans-membrane potential..The results of this study might provide some useful information to understand the toxicity of GA(15:1).