Abstract
Ebracteolata compound B (ECB) is one major active component of both Euphorbia ebracteolata and Euphorbia fischeriana, which have been extensively used as a tuberculocide in the Asian countries. The aim of our present study was to characterize ECB metabolism in human liver microsomes, HepG2 cells, and recombinant human enzymes. One monohydroxylation metabolite, determined by mass spectrometry to be 1-(2,4-dihydroxy-6-methoxy-3-methylphenyl)-2-hydroxyethanone, and one monoglucuronide, isolated and determined by hydrolysis with β-glucuronidase, mass spectrometry, and 1H NMR to be 2-hydroxy-6-methoxy-3-methyl-acetophenone-4-O-β-glucuronide, were observed in human liver microsomal incubates in the presence of NADPH or UDP-glucuronic acid (UDPGA), respectively. However, the mixed incubation of ECB with human liver microsomes in the presence of both NADPH and UDPGA showed the monoglucuronide to be the most major metabolite, indicating that glucuronidation was probably the major clearance pathway of ECB in humans. No glucuronide and only trace monohydroxylation metabolite were observed in HepG2 cells. The cytochrome P450 and UDP-glucuronosyltransferase (UGT) isoenzymes were identified by using selective chemical inhibition and recombinant human enzymes. The results indicated that CYP3A4 was probably involved in ECB oxidative metabolism and UGT1A6 and UGT1A9 were important catalytic enzymes in ECB glucuronidation. The results from enzymatic kinetic analysis showed the oxidative metabolism in human liver microsomes; the glucuronidation in human liver microsomes and recombinant UGT1A6 exhibited a typical Michaelis-Menten pattern, but the glucuronidation in UGT1A9 exhibited a substrate inhibition pattern. UGT1A6 had the highest affinity compared with human liver microsomes and UGT1A9, indicating its important role in ECB glucuronidation.
Footnotes
This work was supported by the National Major Special Project for Science and Technology Development of Ministry of Science and Technology of China [2009ZX09304-003]; and the National Science Foundation for Post-doctoral Scientists of China [20090461391].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.034496.
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ABBREVIATIONS:
- XDR
- drug-resistant
- TB
- tuberculosis
- ECB
- ebracteolata compound B
- P450
- cytochrome P450
- UGT
- UGP glucuronosyltransferase
- HPLC
- high-performance liquid chromatography
- UPLC
- ultraperformance liquid chromatography
- MS
- mass spectrometry
- ESI
- electrospray ionization
- PCR
- polymerase chain reaction
- UDPGA
- UCP-glucuronic acid
- MTT
- 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
- PBS
- phosphate-buffered saline
- DMSO
- dimethyl sulfoxide
- SIR
- selected ion recording
- MS/MS
- tandem mass spectrometry.
- Received May 14, 2010.
- Accepted September 13, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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