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. 2018 Aug;39(8):1393-1404.
doi: 10.1038/aps.2017.144. Epub 2018 Feb 8.

Charactering the metabolism of cryptotanshinone by human P450 enzymes and uridine diphosphate glucuronosyltransferases in vitro

Jin Zeng  1 , Yu-Juan Fan  1 , Bo Tan  1 , Hui-Zong Su  1 , Yue Li  1 , Lin-Lin Zhang  1 , Jian Jiang  2 , Fu-Rong Qiu  3
Affiliations

Charactering the metabolism of cryptotanshinone by human P450 enzymes and uridine diphosphate glucuronosyltransferases in vitro

Jin Zeng et al. Acta Pharmacol Sin. 2018 Aug.

Abstract

Cryptotanshinone (CT) is the main active component in the root of Salvia miltiorrhiza Bunge (SMB) that displays antibacterial, anti-inflammatory and anticancer activities. In this study, we characterized phase I and phase II metabolism of CT in human liver microsomes in vitro and identified the metabolic enzymes (CYPs and UGTs) involved. The metabolites of CT generated by CYPs were detected using LC-MS/MS and the CYP subtypes involved in the metabolic reactions were identified using chemical inhibitors of CYP enzymes and recombinant human CYP enzymes (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Glucuronidation of CT was also examined, and the UGT subtypes involved in the metabolic reactions were identified using recombinant human UGT enzymes (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). After adding NADPH to the human liver microsomes incubation system, CT was transformed into 6 main dehydrogenation and hydroxylation metabolites. CYP2A6, CYP3A4 and CYP2C19 were the major contributors to the transformation of its hydroxylation metabolites. CYP2C19, CYP1A2 and CYP3A4 were the major contributors to the transformation of its hydrogenation metabolites in human liver microsomes. This study showed that the metabolites at m/z of 473 were mediated by UGT1A9 and that the metabolites at m/z of 489 were mediated by UGT2B7 and UGT2B4. CT was extensively metabolized by UGTs following metabolism by CYPs in the liver.

Keywords: P450 enzymes; cryptotanshinone; human liver microsomes; metabolic characteristics; uridine diphosphate glucuronosyltransferases.

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Figures

Figure 1
Figure 1
MRM chromatograms of CT and its metabolites in HLMs. These chromatograms show the formation of (A) unchanged, (B) dehydrogenated, (C) hydroxylated, (D) glucuronidation forms, and (E) glucuronidation forms of CT in HLMs.
Figure 2
Figure 2
Time course of degradation of cryptotanshinone during a 60-min incubation in HLMs (A) and the generation of its main metabolites, including M1-1, M1-2, M1-3, M2-1, M2-2, and M2-3 in NADPH (B), M1-1, M1-2, M1-3, M2-1, M2-2, and M2-3 (C), M3-1, M3-2, M3-3, M4-1 and M4-2 (D) in NADPH+UDPGA.
Figure 3
Figure 3
(A) Effects of various chemical inhibitors of CYP enzymes on CTS metabolism in HLM (KTC: ketoconazole, QND: quinidine, FUR: Furafylline, MOS: methoxsalen, QUE: quercetin, SUL: sulfaphenazole, TIC: ticlopidine). (B) The metabolism of cryptotanshinone by CYP recombinant enzymes. * P<0.05 vs control group.
Figure 4
Figure 4
(A) Formation of the dehydrogenation metabolites (M1-1, M1-2 and M1-3) from cryptotanshinone using human cDNA-expressed CYP isoforms. Cryptotanshinone at 10 μmol/L was incubated with each recombinant CYP isoform (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6 and 3A4). (B) Formation of hydroxylation metabolites from cryptotanshinone using human cDNA-expressed CYP isoforms. Cryptotanshinone at 10 μmol/L was incubated with each recombinant CYP isoform (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6 and 3A4). (C) Formation of cryptotanshinone glucuronide from cryptotanshinone using human cDNA-expressed UGT isoforms. Cryptotanshinone at 10 μmol/L was incubated with each recombinant UGT isoform (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). Each bar represents the mean±standard deviation (SD) of triplicate determinations. (D) Formation of cryptotanshinone glucuronide (m/z of 489) from cryptotanshinone using human cDNA-expressed UGT isoforms. Cryptotanshinone at 10 μmol/L was incubated with each recombinant UGT isoform (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). Each bar represents the mean±standard deviation (SD) of triplicate determinations.
Figure 5
Figure 5
The proposed metabolic pathway of cryptotanshinone in HLMs.
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