Research On The Mechanism Of Incompatibility Between Veratrum Nigrum And Ginseng Based On Cytochrome P450

In traditional medicine theory, it is hypothesized that the toxity of veratrum nigrum will increase combinated with ginseng because of incompatibility. As the most important phase I drug-metabolizing enzymes, cytochrome P450 (CYP) is the base of drug-drug interaction, participate in the metabolism of drugs, carcinogen and environmental pollutants, attenuate and protect the liver. In addition to the role of xenobiotic metabolism, CYP has endogenous effect via transformation of physiological substances. In this study, we investigated the mechanism of incompatibility between veratrum nigrum and ginseng in exogenous and endogenous roles of CYPs.The effect of ginseng and veratrum on CYPs was investigated in rats and cell. We researched the mechanism in regulation of CYPs by ginseng Rb1 and Rg1 initially. Exposured to ginseng Rb1 and Rg1, the alterations of CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were monitored in HepG2 cells by RT-PCR. The results indicated that CYP1A1 mRNA was upregulated and CYP3A4 mRNA was downregulated as time- and concentration- dependent. Other CYPs did not change obviously. CYPs activity of live microsomes, prepared from rats exposed to ginseng and veratrum nigrum, was evaluated by measuring metabolism of specific substrates. The total amount of CYPs was decreased, but the cytochrome b5 did not changed obviously. The activity of CYP1A was increased, and that of CYP3A was decreased in a concentration-dependent manner.To evaluate the alteration of veratrum nigrum transformation via CYPs, the metabolism of veratridine, one of the two major alkaloids in veratrine extracted from the plant Veratrum officinale, and the effects of selective CYP inhibitors on the metabolism of veratridine were studied in rat liver microsomes. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MSn) and furtherly identified by comparison of their mass spectra and chromatographic behaviors with reference substances. Four CYP isoforms (CYP1A, CYP2B, CYP2E1, CYP3A) were involved in the metabolism of veratridine in vitro. Different metabolites, as a result of individual variation of CYPs, may cause the individual difference of veratridine toxic response. Seven metabolites of veratridine were detected incubating with rat liver microsomes, some of which were identified as potential mediates of neurotoxicity via protein binging. Assuming a potential role of this bioactivation mechanism in veratridine toxicity, the effects are mainly due to the generation of the veratridine catechol (and ortho-quinone).To evaluate the effect of CYP1A1 up-regulation on pharmacological action of veratrum nigrum, the expression plasmid of human CYP1A1 was constructed and transfected into human vein endothelial cells (HVEC304), meanwhile the intracellular Ca²⁺ level was monitored. Ca²⁺ basal level was decreased obviously in HVEC304 cells over-expressing CYP1A1. Veratridine, which can increase the intracellular calcium ion by preventing Na+ channels inactivation and activating Na⁺- Ca²⁺ exchange, reversed this alteration in a concentration-dependent manner. In HVEC304 cells over-expressing CYP1A1, veratridine stimulated the intracellular Ca2+ more effectively. It is hypothesized that CYP1A1 over-expression can enhance the metabolism of arachidonic acid (AA) via CYPs, increase the production of hydroxyeicosatetraenoic acid (HETEs), and cause the unbalance between HETEs and epoxyeicosatrienoic acids (EETs). So veratridine can evoke the intracellular Ca²⁺ dysfunction together with the alteration of arachidonic acid metabolites.As a whole, ginseng combinated with veratrum nigrum decreased the total count of CYPs and the activity of CYP3A, increased the activity of CYP1A. The gene expression of cyp1a1 is up-regulated by ginsenoside Rb1 and Rg1, inversely that of cyp3a4 is down-regulated. The alteration in expression and activity of CYPs modified the metabolism of veratrum nigrum. The descent of CYPs total count and CYP3A activity would cause accumulation of veratridine, a major toxic ingredient in veratrum. On the other hand, CYP1A1 up-regulation would increase the generation of veratridine active intermediate via phase I metabolism. Moreover, up-regulation of CYP1A1 altered the pharmacologic action of veratridine, which stimulated the intracellular Ca2+ more efficiently. So CYPs modification can be one of the mechanisms under the interaction incompatibility between veratrum nigrum and ginseng.