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We examined the in vitro metabolism of (+)-terpinen-4-ol by human liver microsomes and recombinant enzymes. The biotransformation of (+)-terpinen-4-ol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Terpinen-4-ol was found to be oxidized to (+)-(1R,2S,4S)-1,2-epoxy-p-menthan-4-ol, (+)-(1S,2R,4S)-1,2-epoxy-p-menthan-4-ol, and (4S)-p-menth-1-en-4,8-diol by human liver microsomal P450 enzymes. The identities of (+)-terpinen-4-ol metabolites were determined through the relative abundance of mass fragments and retention times on GC-MS. Of 11 recombinant human P450 enzymes tested, CYP1A2, CYP2A6, and CYP3A4 were found to catalyze the oxidation of (+)-terpinen-4-ol. Based on several lines of evidence, CYP2A6 and CYP3A4 were determined to be major enzymes involved in the oxidation of (+)-terpinen-4-ol by human liver microsomes. First, of the 11 recombinant human P450 enzymes tested, CYP1A2, CYP2A6 and CYP3A4 catalyzed oxidation of (+)-terpinen-4-ol. Second, oxidation of (+)-terpinen-4-ol was inhibited by (+)-menthofuran and ketoconazole, inhibitors known to be specific for these enzymes. Finally, there was a good correlation between CYP2A6 and CYP3A4 activities and (+)-terpinen-4-ol oxidation activities in the 10 human liver microsomes.

The in vitro metabolism of (-)-terpinen-4-ol was examined in human liver microsomes and recombinant enzymes. The biotransformation of (-)-terpinen-4-ol was investigated by gas chromatography-mass spectrometry. (-)-Terpinen-4-ol was found to be oxidized to (-)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol, major metabolic product by human liver microsomal P450 enzymes. The formation of metabolites of (-)-terpinen-4-ol was determined by relative abundance of mass fragments and retention times on GC. CYP2A6 in human liver microsomes was a major enzyme involved in the oxidation of (-)-terpinen-4-ol by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 had the highest activity for oxidation of (-)-terpinen-4-ol. Second, oxidation of (-)-terpinen-4-ol was inhibited by (+)-menthofuran. Finally, there was a good correlation between CYP2A6 maker activity and (-)-terpinen-4-ol oxidation activities in liver microsomes of 10 human samples. Kinetic analysis showed that the V(max)/K(m) values for (-)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol catalysed by liver microsomes of human sample HH-18 was 2.49 μL/min/nmol. Human recombinant CYP2A6 catalysed (-)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol with V(max) values of 13.9 nmol/min/nmol P450 and apparent K(m) values of 91 μM.

The in vitro metabolism of (+)-(1S,3S,4R) and (-)-(1R,3R,4S)-menthol enantiomers was examined by incubation with human liver microsomes, and the oxidative metabolites thus formed were analyzed using gas chromatography-mass spectrometry (GC-MS). The (+)- and (-)-menthols were found to be oxidized to the respective (+)-(1S,3S,4S)- and (-)-(1R,3R,4R)-trans-p-menthane-3,8-diol derivatives by human liver microsomal P450 enzymes. Cytochrome P450 (CYP) 2A6 was determined to be the major enzyme involved in the hydroxylation of (+)- and (-)-menthols by human liver microsomes on the basis of the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 catalyzed the oxidation of (+)- and (-)-menthols. Second, oxidation of (+)- and (-)-menthols was inhibited by (+)-menthofuran and anti-CYP2A6 antibody. Finally, (+)- and (-)-menthol activities were found to correlate with contents of CYP2A6 in liver microsomes of 9 human samples.