In Vitro Assessment Of Increasing Cytotoxicity Of Veratrum Nigrum Induced By Panax Gingseng And Quantification Of Hepatic Drug-metabolizing Enzymes

Objective To compare the potential cytotoxicity induced by Veratrum nigrum and coadministered with Panax gingseng, to provide experimental evidences to the mode of herb-herb interaction based on human liver drug metabolizing enzymes.Methods The effects of Veratrum nigrum and coadministration on cultured human hepatoma (HepG2) cells were investigated by detecting morphological changes, cell viability, cytomembrane integrity, and apoptosis after the cells were treated for24h. The mRNA expression levels of drug metabolizing enzymes influenced by Panax gingseng were determined by real-time quantitative reverse-transcriptase polymerase chain reaction. Four QconCATs corresponding to41proteins were designed and built to encode two or three analyte peptides per protein, and after stable isotope labeling of the standard in vivo, protein levels were determined by liquid chromatography tandem mass spectrometry.Results Veratrum nigrum coadministered with Panax gingseng had a more inhibitive effects on growth of HepG2cells with the IC50value of (15.18±1.03)mg/mL compared with the value of IC50(21.46±1.10)mg/mL of Veratrum nigrum; Coadministration could significantly raise the LDH level in cell culture medium compared with the same dose of Veratrum nigrum; Moreover, in coadministration group (Cveratrum nigrum=10mg/mL20mg/mL), compared with the same dose of Veratrum nigrum,the total apoptosis and necrosis rate of HepG2cells was significantly increased (P<0.05).Panax gingseng had effect on the expression of CYP3A4, CYP1A1, CYP1A2, CYP2B6and CYP2R1mRNA. We demonstrate successful application of the QconCAT technology to the absolute quantification of Cytochromes P450and UDP-Glucuronosyltransferases in human liver microsomes, The nature of the technical variance and the biological variance were assessed and the CV<25%. As a result, the determination coefficients (r2) of all targets were greater than0.98and the overall procedure showed a wide dynamic range (≥100-fold). We were able to determine absolute protein concentrations between161.66and2.84pmol/mg microsome.Conclusion The result indicated that compatibility of medicines in a prescription also have herb-herb interaction based on drug metabolizing enzymes. The interaction mode could express as Panax gingseng inhibit and induce the CYPs and the modulated CYP isozymes would further have an impact on the metabolism of constitutes in coadmistrated herbs Veratrum nigrum and cause herb-herb interaction. The QconCAT approach proved to be reproducible, cost-effective and readily suitable for high-throughput assays.