Kinetic Characteristics of Curcumin and Germacrone in Rat and Human Liver Microsomes: Involvement of CYP Enzymes

Abstract

Curcumin and germacrone, natural products present in the Zingiberaceae family of plants, have several biological properties. Among these properties, the anti-NSCLC cancer action is noteworthy. In this paper, kinetics of the two compounds in rat liver microsomes (RLMs), human liver microsomes (HLMs), and cytochrome P450 (CYP) enzymes (CYP3A4, 1A2, 2E1, and 2C19) in an NADPH-generating system in vitro were evaluated by UP-HPLC-MS/MS (ultrahigh-pressure liquid chromatography-tandem mass spectrometry). The contents of four cytochrome P450 (CYP) enzymes, adjusting by the compounds were detected using Western blotting in vitro and in vivo. The t_1/2 of curcumin was 22.35 min in RLMs and 173.28 min in HLMs, while 18.02 and 16.37 min were gained for germacrone. The V_max of curcumin in RLMs was about 4-fold in HLMs, meanwhile, the V_max of germacrone in RLMs was similar to that of HLMs. The single enzyme t1/2 of curcumin was 38.51 min in CYP3A4, 301.4 min in 1A2, 69.31 min in 2E1, 63.01 min in 2C19; besides, as to the same enzymes, t1/2 of germacrone was 36.48 min, 86.64 min, 69.31 min, and 57.76 min. The dynamic curves were obtained by reasonable experimental design and the metabolism of curcumin and germacrone were selected in RLMs/HLMs. The selectivities in the two liver microsomes differed in degradation performance. These results meant that we should pay more attention to drugs in clinical medication-drug and drug-enzyme interactions.

Keywords: UPLC–MS/MS; curcumin; cytochrome P450 enzymes; drug–drug interaction; germacrone.

Figure 1

Chemical structures of curcumin and germacrone.

Figure 2

Representative HPLC–MS/MS chromatograms. (A,F): blank RLMs; (B,G): blank HLMs; (C,H): standard curcumin and germacrone (50 and 60 μM); (D,I): extract from RLMs with 50 μM curcumin and 60 μM germacrone after incubation for 120 min with NADPH; (E,J): extract from HLMs with 50 μM curcumin and 60 μM germacrone after incubation for 120 min with NADPH.

Figure 3

Degradation of curcumin (A), 50 μM and germacrone (B), 60 μM in RLMs and HLMs (n = 3).

Figure 4

Enzyme kinetics of curcumin (A) and germacrone (B) in RLMs and HLMs (n = 3).

Figure 5

Degradation of curcumin (A), 5 μM and germacrone (B), 5 μM in CYP3A4, 1A2, 2E1 and 2C19 in vitro (n = 3).

Figure 6

The effects of curcumin (A) or germacrone (B) on CYP3A4, 1A2, 2E1, and 2C19 activity in vitro (n = 3).

Figure 7

Plasma concentration-time curves of midazolam (A), phenacetin (B), chlorzoxazone (C), omeprazole (D) after i.g. administrated with curcumin (C: control, L: 10, M: 40, H: 80 mg/kg/d), and midazolam (E), phenacetin (F), chlorzoxazone (G), omeprazole (H) after i.g. administrated with germacrone (C: control, L: 1, M: 4, H: 8 mg/kg/d) in rats (mean ± SD, n = 6).

Figure 8

The expression levels of CYP3A4 (A), 1A2 (B), 2E1 (C) and 2C19 (D) were decreased in curcumin groups and CYP3A4 (E), 1A2 (F), 2E1 (G) and 2C19 (H) were increased in germacrone groups in vitro (mean ± SD, n = 6, * p < 0.05 vs. control rats).

Figure 9

The expression levels of CYP3A4 (A), 1A2 (B), 2E1 (C) and 2C19 (D) were decreased in curcumin groups and CYP3A4 (E), 1A2 (F), 2E1 (G) and 2C19 (H) were increased in germacrone groups in vivo (mean ± SD, n = 6, * p < 0.05 vs. control rats).