IN VITRO HEPATIC METABOLISM OF THE PHYTOBIOTIC 1,8- CINEOLE IN DOMESTIC FOWL
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Date
2018
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD
Abstract
Chickens reared under intensive systems are likely to be exposed to feed
additives and various xenobiotics. Phytobiotics, especially essential oils (EOs), are
a new class of feed additives that have attracted attention due to their attributed
antimicrobial and growth promoter properties. 1,8- cineole is a naturally occurring
monocyclic monoterpene ether with an aromatic and camphor like odour and is one
of the EO components used in poultry feed as phytobiotic. Even though the
metabolism of 1,8-cineole is studied in rats, rabbits, koala and brushtail possum and
humans, its fate in poultry is not yet reported. The present study was conducted in
vitro to identify the cytochrome P450 enzyme orthologs involved in the
biotransformation of 1,8-cineole in chicken hepatic S9 and microsomal fractions.
The approaches used included the use of specific cytochrome P450 (CYP3A4)
inhibitors and the correlation of prototype substrate activities with the formation of
the hydroxylated metabolite of 1,8-cineole. Nifedipine and phenacetin were used as
specific substrates for the CYP3A and CYP1A isoforms and ketoconazole was used
as an inhibitor. A high performance liquid chromatography method was validated
and applied for the determination of nifedipine, phenacetin and their formed
metabolites nifedipine oxide and acetaminophen in hepatic microsomes and S9
fractions. Analysis of 1,8-cineole and its metabolite was done by gas
chromatography mass spectrophotometry(GC-MS). No significant difference in
metabolic activity of specific probes and 1,8-cineole was noted between the male
and female birds. Incubation of 1,8-cineole in the hepatic S9 fraction with a protein
concentration of 7 mg/ml up to 180 min did not exhibit any significant decrease in
the concentration of the parent compound compared to samples at ‘zero’ minutes.
On the contrary, 1,8 cineole was significantly metabolized by the microsomal
fraction with a linear decrease in the concentration of the drug. The formation of
nifedipine oxide at different incubation times indicated the existence of chicken
CYP ortholog of the human enzyme studied. Phenacetin was not metabolized by
the hepatic cytosolic enzymes till 180 minutes. However, both phenacetin and 1,8-
cineole was metabolised in the S9 fraction 6 hours post incubation. The study
indicated that CYP3A isoform catalyzed the hydroxylation of 1,8-cineole in
chicken liver microsomes with the formation of 2α-hydroxy-1,8-cineole. The
human isoenzyme CYP3A specific inhibitor ketoconazole significantly inhibited
the metabolisms of nifedipine and 1,8-cineole in chicken liver microsomes. Since
ketoconazole is also reported as a nonspecific inhibitor of CYP 1A in chickens, the
role of CYP 1A2 in the metabolism of 1,8-cineole in chicken in the present study
cannot be ruled out.