Objective: An in vivo cocktail probe drug method was conducted to evaluate the effect of luteolin on CYP450 enzyme activity. Methods: The rats were randomly divided into two groups, the control and the luteolin groups were respectively given gavage administration of CMC-Na (2.0 mL·kg-1) and luteolin (30.0 mg·kg-1) for 7 successive days. On the 7th day, all rats were injected intraperitoneally with the mixed solution of 3 probe drugs (10.0 mg·kg-1 of phenacetin and testosterone and 2.5 mg·kg-1 of tolbutamide), the plasma samples were collected at different time points and precipitated with isopropanol, then determined by LC-MS/MS. The chromatographic separation was achieved on a ZORBAX SB-C18(2.1 mm×30 mm, 3.5 μm) column by gradient elution of acetonitrile-0.1% formic acid in water, and the MS instrument was operated in multiple reaction monitoring (MRM) with positive and negative electrospray ionization (ESI) modes. The UHPLC-MS/MS method was established for the determination of the concentrations of three probe drugs in plasma, the acquired data were calculated for pharmacokinetic parameters by using DAS software subsequently. Further, the protein expression levels of the three CYP450 enzymes were tested by western blot. Results: Methodological results suggested that the linear range of phenacetin, testosterone, and tolbutamide were in the concentration of 2.0-2 000.0 ng·mL-1, 0.3-300.0 ng·mL-1, 1.7-1 700.0 ng·mL-1, and the lower limit of quantification (LLOQ) were 2.0 ng·mL-1, 0.3 ng·mL-1, and 1.7 ng·mL-1, respectively. For all analytes, the accuracy was in the range of 96.9%-108.0%, and the RSD of intra-day and inter-day precision were within 8.9%. The extraction recovery, matrix effect and stability all met the criteria. The pharmacokinetic results showed that luteolin could significantly reduce the AUC value of phenacetin, and increase the AUC value of tolbutamide. But there was few significant variation in pharmacokinetic parameters of testosterone. The western blot results showed that the expression of CYP1A2 was significantly induced by luteolin, while the levels of CYP2C9 and CYP3A4 were inhibited, which verified the LC-MS/MS results. Conclusion: The luteolin is a moderate inducer on CYP1A2 and a weak inhibitor on CYP2C9, but has no significant effect on CYP3A4 according to the FDA guidance document.
SONG Ke-xin, YI Jie, GAO Lin, REN Ji-ping, LI Qian
. Investigation on effect of luteolin on CYP450 enzymes in rats using cocktail method*[J]. Chinese Journal of Pharmaceutical Analysis, 2022
, 42(2)
: 279
-286
.
DOI: 10.16155/j.0254-1793.2022.02.12
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