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Characterization of cardamonin metabolism by P450 in different species via HPLC-ESI-ion trap and UPLC-ESI-quadrupole mass spectrometry.

He YQ, Yang L, Liu Y, Zhang JW, Tang J, Su J, Li YY, Lu YL, Wang CH, Yang L, Wang ZT - Acta Pharmacol. Sin. (2009)

Bottom Line: Furafylline and clomethiazole significantly inhibited cardamonin hydroxylation.CYP 1A2 and 2E1 were identified as the P450 isozymes involved in the metabolism of cardamonin in human liver microsomes.Furthermore, our research suggests that guinea pigs could be used in the advanced pharmacokinetic studies of cardamonin in vivo.

View Article: PubMed Central - PubMed

Affiliation: The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China.

ABSTRACT

Aim: To characterize the metabolism of cardamonin by the P450 enzymes in human and animal liver microsomes.

Methods: Cardamonin was incubated with both human and animal liver microsomal incubation systems containing P450 reaction factors. High performance liquid chromatography coupled with ion trap mass spectrometry was used to identify the metabolites. Serial cardamonin dilutions were used to perform a kinetic study in human liver microsomes. Selective inhibitors of 7 of the major P450 isozymes were used to inhibit cardamonin hydroxylation to identify the isozymes involved in cardamonin metabolism. The cardamonin hydroxylation metabolic capacities of human and various other animals were investigated using the liver microsomal incubation system.

Results: Two metabolites generated by the liver microsome system were detected and identified as hydroxylated cardamonin. The Km and Vmax values for cardamonin hydroxylation were calculated as 32 micromol/L and 35 pmol x min(-1) x mg(-1), respectively. Furafylline and clomethiazole significantly inhibited cardamonin hydroxylation. Guinea pigs showed the highest similarity to humans with respect to the metabolism of cardamonin.

Conclusion: CYP 1A2 and 2E1 were identified as the P450 isozymes involved in the metabolism of cardamonin in human liver microsomes. Furthermore, our research suggests that guinea pigs could be used in the advanced pharmacokinetic studies of cardamonin in vivo.

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MS2 spectrums of cardamonin (A), M1 (B), and M2 (C).
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fig2: MS2 spectrums of cardamonin (A), M1 (B), and M2 (C).

Mentions: To elucidate the structures of the metabolites, the MS2 fragmentation of the parent compound was first obtained as the reference. The fragment ions and MS2 spectrum of cardamonin are shown in Table 1 and Figure 2A, respectively. In the MS2 spectrum of the [M-H]− ion at m/z 269, three representative ions at m/z 124, 139, and 165 were observed (Figure 2A). The ion at m/z 165 was generated by cleavage between C8 and C9. Serial neutral loss from the ion at m/z 165 produced another two fragment ions (Table 1) at m/z 139 (165-CO) and 124 (165-CO-CH3·), respectively. All the three ions could be valuable to identify the structure of ring A. The fragment ions that represent Part I (Table 1) of the cardamonin structure were also observed at m/z 177 and 179 (Figure 2A).


Characterization of cardamonin metabolism by P450 in different species via HPLC-ESI-ion trap and UPLC-ESI-quadrupole mass spectrometry.

He YQ, Yang L, Liu Y, Zhang JW, Tang J, Su J, Li YY, Lu YL, Wang CH, Yang L, Wang ZT - Acta Pharmacol. Sin. (2009)

MS2 spectrums of cardamonin (A), M1 (B), and M2 (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4007323&req=5

fig2: MS2 spectrums of cardamonin (A), M1 (B), and M2 (C).
Mentions: To elucidate the structures of the metabolites, the MS2 fragmentation of the parent compound was first obtained as the reference. The fragment ions and MS2 spectrum of cardamonin are shown in Table 1 and Figure 2A, respectively. In the MS2 spectrum of the [M-H]− ion at m/z 269, three representative ions at m/z 124, 139, and 165 were observed (Figure 2A). The ion at m/z 165 was generated by cleavage between C8 and C9. Serial neutral loss from the ion at m/z 165 produced another two fragment ions (Table 1) at m/z 139 (165-CO) and 124 (165-CO-CH3·), respectively. All the three ions could be valuable to identify the structure of ring A. The fragment ions that represent Part I (Table 1) of the cardamonin structure were also observed at m/z 177 and 179 (Figure 2A).

Bottom Line: Furafylline and clomethiazole significantly inhibited cardamonin hydroxylation.CYP 1A2 and 2E1 were identified as the P450 isozymes involved in the metabolism of cardamonin in human liver microsomes.Furthermore, our research suggests that guinea pigs could be used in the advanced pharmacokinetic studies of cardamonin in vivo.

View Article: PubMed Central - PubMed

Affiliation: The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China.

ABSTRACT

Aim: To characterize the metabolism of cardamonin by the P450 enzymes in human and animal liver microsomes.

Methods: Cardamonin was incubated with both human and animal liver microsomal incubation systems containing P450 reaction factors. High performance liquid chromatography coupled with ion trap mass spectrometry was used to identify the metabolites. Serial cardamonin dilutions were used to perform a kinetic study in human liver microsomes. Selective inhibitors of 7 of the major P450 isozymes were used to inhibit cardamonin hydroxylation to identify the isozymes involved in cardamonin metabolism. The cardamonin hydroxylation metabolic capacities of human and various other animals were investigated using the liver microsomal incubation system.

Results: Two metabolites generated by the liver microsome system were detected and identified as hydroxylated cardamonin. The Km and Vmax values for cardamonin hydroxylation were calculated as 32 micromol/L and 35 pmol x min(-1) x mg(-1), respectively. Furafylline and clomethiazole significantly inhibited cardamonin hydroxylation. Guinea pigs showed the highest similarity to humans with respect to the metabolism of cardamonin.

Conclusion: CYP 1A2 and 2E1 were identified as the P450 isozymes involved in the metabolism of cardamonin in human liver microsomes. Furthermore, our research suggests that guinea pigs could be used in the advanced pharmacokinetic studies of cardamonin in vivo.

Show MeSH