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Regioselective Glucuronidation of Andrographolide and Its Major Derivatives: Metabolite Identification, Isozyme Contribution, and Species Differences

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An Erratum to this article was published on 18 October 2014

Abstract

Andrographolide (AND) and two of its derivatives, deoxyandrographolide (DEO) and dehydroandrographolide (DEH), are widely used in clinical practice as anti-inflammatory agents. However, UDP-glucuronosyltransferase (UGT)-mediated phase II metabolism of these compounds is not fully understood. In this study, glucuronidation of AND, DEO, and DEH was characterized using liver microsomes and recombinant UGT enzymes. We isolated six glucuronides and identified them using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. We also systematically analyzed various kinetic parameters (K m, V max, and CLint) for glucuronidation of AND, DEO, and DEH. Among 12 commercially available UGT enzymes, UGT1A3, 1A4, 2B4, and 2B7 exhibited metabolic activities toward AND, DEO, and DEH. Further, UGT2B7 made the greatest contribution to glucuronidation of all three anti-inflammatory agents. Regioselective glucuronidation showed considerable species differences. 19-O-Glucuronides were present in liver microsomes from all species except rats. 3-O-Glucuronides were produced by pig and cynomolgus monkey liver microsomes for all compounds, and 3-O-glucuronide of DEH was detected in mouse and rat liver microsomes (RLM). Variations in K m values were 48.6-fold (1.93–93.6 μM) and 49.5-fold (2.01–99.1 μM) for 19-O-glucuronide and 3-O-glucuronide formation, respectively. Total intrinsic clearances (CLint) for 3-O- and 19-O-glucuronidation varied 4.8-fold (22.7–110 μL min−1 mg−1), 10.6-fold (94.2–991 μL min−1 mg−1), and 8.3-fold (122–1,010 μL min−1 mg−1), for AND, DEH, and DEO, respectively. Our results indicate that UGT2B7 is the major UGT enzyme involved in the metabolism of AND, DEO, and DEH. Metabolic pathways in the glucuronidation of AND, DEO, and DEH showed considerable species differences.

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Abbreviations

AND:

Andrographolide

DEH:

Dehydroandrographolide

DEO:

Deoxyandrographolide

HLM:

Human liver microsomes

PLM:

Pig liver microsomes

RLM:

SD rat liver microsomes

MLM:

Mouse liver microsomes

DLM:

Dog liver microsomes

CyLM:

Monkey liver microsomes

UDPGA:

Uridine diphosphate glucuronic acid

UGTs:

UDP-glucuronosyltransferases

AND-1:

Andrographolide-19-O-β-d-glucuronide

AND-2:

Andrographolide-3-O-β-d-glucuronide

DEH-1:

Dehydroandrographolide-19-O-β-d-glucuronide

DEH-2:

Dehydroandrographolide-3-O-β-d-glucuronide

DEO-1:

Deoxyandrographolide-19-O-β-d-glucuronide

DEO-2:

Deoxyandrographolide-3-O-β-d-glucuronide

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ACKNOWLEDGMENTS

We thank National Natural Science Foundation of China (No. 81073013,81473334, 81274047 and 81202589), Dalian Outstanding Youth Science and Technology Talent, Programs for Liaoning Excellent Talents and New Century Excellent Talents in University (NCET) for financial support.

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Authors and Affiliations

  1. College of Pharmacy, Academy of Integrative Medicine, Dalian Medical University, Dalian, 116044, China

    Xiangge Tian, Sicheng Liang, Chao Wang, Guangbo Ge, Sa Deng, Kexin Liu & Xiaochi Ma

  2. Joint Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences and Liaoning Medical University, Dalian, China

    Sicheng Liang, Ling Yang & Xiaochi Ma

  3. College of Pharmacy, Jinan University, Guangzhou, 510632, China

    Baojian Wu

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  1. Xiangge Tian
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Correspondence to Xiaochi Ma.

Additional information

Xiangge Tian, Sicheng Liang, and Chao Wang contributed equally to this work.

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Fig. S1

Representative HPLC chromatograms (left A, C, E) at 225 nm, 254 nm, and 210 nm and UV spectra (right B, D, F) spectra of Andrographolide, Dehydroandrographolide, and Deoxyandrographolide and their glucuronides among different species, respectively. The UV spectra of their glucuronides were similar with that of substrates. (DOC 217 kb)

Fig. S2

Chemical inhibition of Andrographolide (A), Dehydroandrographolide (B), Deoxyandrographolide (C) O-glucuronidation at different concentration of substrates by four potent inhibitors including diclofenac (400 μM), 20(S)-protopanaxatriol (100 μM), fluconazole (5 mM) and phenylbutazone (500 μM), in HLMs incubations. The incubations without inhibitors but the same volume of solvent were set as the solvent control in which the activity of three substrates O-glucuronidation was normalized to 100%. (DOC 137 kb)

Fig. S3

Correlation analysis between the formation rate of Andrographolide (A, 3 μM; B, 300 μM), Dehydroandrographolide (C, 20 μM; D, 200 μM) and Deoxyandrographolide (E, 2 μM; F, 100 μM) 19-O-glucuronides with UGT2B7-catalyzed AZT glucuronidation in HLMs from 11 individual. (DOC 158 kb)

Fig. S4

Eadie–Hofstee plots of glucuronidation profiles shown in Fig. 5 to determine the best-fit equation. (DOC 254 kb)

Fig. S5

Evaluation of DEO and DEH inhibition toward HLM catalyzed 19-O-glucuronide formation. A and C: Dixon plot of DEO and DEH’s inhibition toward AND 19-O-glucuronidation in HLMs; B and D: Lineweaver–Burk plots of DEO and DEH’s inhibition toward AND 19-O-glucuronidation in HLMs. The data point represents the mean of duplicate experiments. (DOC 165 kb)

Fig. S6

Kinetic profiles (A, B, C, D, E) of 19-O-β-glucuronidation derived from incubation of Andrographolide, Dehydroandrographolide, and Deoxyandrographolide with different species liver microsomes from Pig, Dog, Monkey, SDrat and Mouse, respectively. (DOC 298 kb)

Fig. S7

Kinetic profiles (A, B, C, D) of 3-O-β-glucuronidation derived from incubation of Andrographolide, Dehydroandrographolide, and Deoxyandrographolide with different species liver microsomes from Pig, Monkey, SDrat and Mouse, respectively. (DOC 228 kb)

Fig. S8

Kinetic profiles of 19-O-β-glucuronidation derived from incubation of 3-keton-dehydroandrographolide with UGT2B7. V max = 1.54 nmol/min/mg; K m = 22.1 μM. (DOC 322 kb)

Fig. S9

NMR spectra of in vitro metabolites of AND, DEH and DEO. (DOC 2,911 kb)

Table S1

1H NMR (600 MHz, MeOD) spectral data for metabolites of AND, DEH, and DEO. (DOC 63 kb)

Table S2

Kinetic parameters of 3-O-β-glucuronidation derived from the incubation of Andrographolide, Dehydroandrographolide, and Deoxyandrographolide with different species’ liver microsomes. Vm nmol/min/mg, K m μM, Ki μM. (n = 3) (DOC 37 kb)

Table S3

Inhibitory effects of diclofenac on AND, DEH and DEO glucuronidation in HLM and UGT2B7. The substrate concentrations were about K m values of AND, DEO and DEH. (n = 3) (DOC 30 kb)

Table S4

Inhibitory effects of AND, DEH, DEO and their metabolites against LPS- induced NO production in RAW264.7 macrophages. (n = 3) (DOC 32 kb)

Table S5

Clean of 3-O-β-glucuronidation in different species. CL μl/min · mg. (DOC 32 kb)

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Tian, X., Liang, S., Wang, C. et al. Regioselective Glucuronidation of Andrographolide and Its Major Derivatives: Metabolite Identification, Isozyme Contribution, and Species Differences. AAPS J 17, 156–166 (2015). https://doi.org/10.1208/s12248-014-9658-8

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