Syntheses of chiral 1,8-cineole metabolites and determination of their enantiomeric composition in human urine after ingestion of 1,8-cineole- containing capsules

Article abstract of DOI:10.1002/cplu.201200253

The chiral metabolites in human urine were investigated after ingestion of a 1,8-cineole (eucalyptol)-containing enterocoated capsule (Soledum). For identification of the various enantiomers the enantiomerically pure (-/+)-α2-hydroxy-1,8- cineole, (-/+)-β2-hydroxy-1,8-cineole, (-/+)-9-hydroxy-1,8-cineole, and (-/+)-2-oxo-1,8-cineole were prepared. To achievethis aim, after acetylation of the synthesized racemic 2-and 9-hydroxy-1,8-cineoles, pig liver esterase- or yeast-mediated hydrolysis provided the (-)-alcohols with their antipodal(+)-acetates with enantiomeric excess of 33-100 %. Dess-Martin periodinane oxidation of the alcohol (+)-α2-hydroxy-1,8-cineole, obtained by hydrolysis of the resolved acetate, provided the corresponding (+)-2-oxo-1,8-cineole, meanwhile the oxidation of (-)-α2-hydroxy-1,8-cineole gave (-)-2-oxo-1,8-cineole. Using these standards seven metabolites (+/-)-α2-hydroxy-1,8-cineole, (+/-)-β2-hydroxy-1,8-cineole, (+/-)-α3-hydroxycineole,(+/-)-3-oxo-1, 8-cineole, 4-hydroxy-1,8-cineole, 7-hydroxy-1,8-cineole, and (+/-)-9-hydroxy-1,8-cineole, all liberated from their glucuronides, were identified in urine by GCMS on a chiral stationary phase after consumption of 10 mg of 1,8-cineole. Metabolite screening using ²H₃-1,8- cineol as the internal standard revealed (+/-)-α2-hydroxy-1,8-cineole as the predominant metabolite followed by (+/-)-9-hydroxy-1,8-cineole. Furthermore, the results showed that one enantiomer is always formed preferentially.

Full text of DOI:10.1002/cplu.201200253

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DOI: 10.1002/cplu.201200253
Syntheses of Chiral 1,8-Cineole Metabolites and
Determination of Their Enantiomeric Composition in
Human Urine After Ingestion of 1,8-Cineole-Containing
Capsules
Monika Schaffarczyk,[a, b] Teodor Silviu Balaban,[c] Michael Rychlik,[b, e] and
Andrea Buettner*[a, d]
The chiral metabolites in human urine were investigated after
ingestion of 1,8-cineole (eucalyptol)-containing entero-
the oxidation of (À)-a2-hydroxy-1,8-cineole gave (À)-2-oxo-1,8-
cineole. Using these standards seven metabolites (+/À)-a2-hy-
droxy-1,8-cineole, (+/À)-b2-hydroxy-1,8-cineole, (+/À)-a3-hy-
droxycineole, (+/À)-3-oxo-1,8-cineole, 4-hydroxy-1,8-cineole, 7-
hydroxy-1,8-cineole, and (+/À)-9-hydroxy-1,8-cineole, all liber-
ated from their glucuronides, were identified in urine by GC-
MS on a chiral stationary phase after consumption of 100 mg
a
coated capsule (Soledum). For identification of the various
enantiomers the enantiomerically pure (À/+)-a2-hydroxy-1,8-
cineole, (À/+)-b2-hydroxy-1,8-cineole, (À/+)-9-hydroxy-1,8-
cineole, and (À/+)-2-oxo-1,8-cineole were prepared. To ach-
ieve this aim, after acetylation of the synthesized racemic 2-
and 9-hydroxy-1,8-cineoles, pig liver esterase- or yeast-mediat-
ed hydrolysis provided the (À)-alcohols with their antipodal
(+)-acetates with enantiomeric excess of 33–100%. Dess–
Martin periodinane oxidation of the alcohol (+)-a2-hydroxy-
1,8-cineole, obtained by hydrolysis of the resolved acetate,
provided the corresponding (+)-2-oxo-1,8-cineole, meanwhile
2
of 1,8-cineole. Metabolite screening using H3-1,8-cineol as the
internal standard revealed (+/À)-a2-hydroxy-1,8-cineole as the
predominant metabolite followed by (+/À)-9-hydroxy-1,8-cin-
eole. Furthermore, the results showed that one enantiomer is
always formed preferentially.
Introduction
The monoterpenoid 1,8-cineole (1,3,3-trimethyl-2-oxabicyclo-
[2.2.2]octane), commonly known as eucalyptol, is the main
component of eucalyptus essential oil and is abundant in sev-
eral plant species. Because of its fresh and menthol-like odor it
is used for flavoring foods and cosmetics. Furthermore, 1,8-cin-
eole is applied in pharmaceutical preparations to treat cough,
bronchitis, asthma, and muscular pain.[1]
The biotransformation of 1,8-cineole has been studied ex-
tensively in brushtail possum (Trichosurus vulpecula) and koala.
The main metabolites in these species were the hydroxycineol-
ic acids, cineolic acids, and diols with preferred location for the
oxidation in position-9 and -7. In minor concentrations also
some hydroxycineoles were found: a2-hydroxy-1,8-cineole, b2-
hydroxy-1,8-cineole, a3-hydroxy-1,8-cineole, b3-hydroxy-1,8-
cineole, 7-hydroxy-1,8-cineole, 9-hydroxy-1,8-cineole.[2,3,4] After
administration of 1,8-cineole to rabbits, which normally do not
consume eucalyptus in their regular diet, a/b2-hydroxy-1,8-cin-
eole and a/b3-hydroxy-1,8-cineole were identified. Higher oxi-
dized products were not found.
[a] M. Schaffarczyk, Prof. A. Buettner
Fraunhofer Institute for Process Engineering and Packaging (IVV)
85354 Freising (Germany)
Fax: (+49)9131-8522587
E-mail: andrea.buettner@lmchemie.uni-erlangen.de
The metabolism of 1,8-cineole was also investigated in in vi-
tro assays involving human liver microsomes. Mainly the en-
zymes CYP3A4 and CYP3A5 catalyzed the oxidation to a2-
and a3-hydroxy-1,8-cineole.[5]
[b] M. Schaffarczyk, Prof. M. Rychlik
BIOANALYTIK Weihenstephan
Z I E L Research Center for Nutrition and Food Sciences
Technical University Munich, 85350 Freising (Germany)
[c] Prof. T. S. Balaban
Only recently the metabolites of 1,8-cineole have also been
studied in humans. Horst and Rychlik[6] identified the four me-
tabolites a2-hydroxy-1,8-cineole, a3-hydroxy-1,8-cineole, 7-hy-
droxy-1,8-cineole, and 9-hydroxy-1,8-cineole in blood and urine
after ingestion of sage tea. The metabolites were quantified
after liberation from their glucuronides using stable isotope di-
lution assays. Also, for the first time, the metabolites of cineole
in human milk were examined in another study; thereby, milk
samples were analyzed after lactating mothers ingested the
non-prescription pharmaceutical Soledum (Klosterfrau Health-
Aix Marseille Universitꢀ, iSm2, CNRS, UMR 7313
13397 Marseille (France)
[d] Prof. A. Buettner
Department of Chemistry and Pharmacy, Food Chemistry, Emil Fischer
Center, University of Erlangen-Nuremberg
91052 Erlangen (Germany)
[e] Prof. M. Rychlik
Chair of Analytical Food Chemistry
Technical University of Munich, 85350 Freising (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201200253.
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Results and Discussion
Synthesis of (À)- and (+)-a2-hydroxy-1,8-cineole
The synthetic sequence commences with epoxidation of the
commercially available a-terpineol mediated by m-chloroper-
benzoic acid as shown in Scheme 1. After a reaction time of
24 hours, upon acid catalysis and ring closure to the [2,2,2]-
care Group, Cologne, Germany) containing 100 mg of 1,8-cin-
eole. In addition to the previously known human hydroxylated
metabolites a2-hydroxy-1,8-cineole, b2-hydroxy-1,8-cineole a3-
hydroxy-1,8-cineole, 7-hydroxy-1,8-cineole, and 9-hydroxy-1,8-
cineole, five additional new human metabolites could be iden-
tified: 4-hydroxy-1,8-cineole, 2-oxo-1,8-cineole, 3-oxo-1,8-cin-
eole, 2,3-dehydro-1,8-cineole and 2,3-a-epoxy-1,8-cineole.
Nevertheless, in all previous studies until today, no distinc-
tion was made between the respective enantiomeric forms of
the metabolites. Accordingly, on the basis of the synthesized
racemic reference substances, the results were represented as
the sum of both enantiomers in each case.
Scheme 1. Synthesis of (À)-a2-hydroxy-1,8-cineole: a) mCPBA, CH2Cl2, 08C,
2 h; b) p-toluenesulfonic acid, CH2Cl2, RT, 24 h; c) Acetyl chloride, DMAP,
CH2Cl2, 0–58C; d) PLE, pH 7, 12 h, 378C.
However, for determining the enantiomeric ratio of the me-
tabolites the enantiopure references are required. As these ref-
erences are not commercially available, this involves extensive
synthetic and preparative work. Previously, Luzzio and
Duveau[7] prepared for the first time (1R,3R,4S)-(À)-3-hydroxy-
1,8-cineole by pig liver esterase (PLE)-mediated hydrolysis of
the racemic acetate (+/À)-b3-acetyloxy-1,8-cineole in an enan-
tiomeric purity exceeding 99%. After hydrolysis of the antipo-
dal (1S,3S,4R)-(+)-3-acetyloxy-1,8-cineole the two enantiopure
b3-hydroxy-1,8-cineoles were oxidized using pyridinium chloro-
chromate (PCC) to give (1R,4R)-(+)-3-oxo-1,8-cineole and
(1S,4S)-(À)-3-oxo-1,8-cineole.[8]
oxabicyclooctane skeleton, the racemic (À/+)-a2-hydroxy-1,8-
cineole was obtained in 15% yield after purification by chro-
matography on silica gel. Treatment of this alcohol with acetyl
chloride in the presence of 4-dimethylaminopyridine (DMAP) in
dichloromethane provided the corresponding racemic acetate
(À/+)-a2-acetyloxy-1,8-cineole in 69% yield after purification
by flash chromatography on silica gel. Inspired by the stereo-
specific synthesis of (À)- and (+)-b3-hydroxy-1,8-cineole by
Luzzio and Duveau[7] we selected a porcine liver esterase (PLE)-
mediated desymmetrization of the secondary acetate. After in-
cubation of (À/+)-a2-acetyloxy-1,8-cineole with the commer-
cially available porcine liver esterase (EC 3.1.1.1) for 16 hours at
378C afforded a chromatographically separable mixture of (À)-
a2-hydroxy-1,8-cineole (43%) and (+)-a2-acetyloxy-1,8-cineole
(45%). After separation by flash chromatography the remain-
ing (+)-acetate was saponified with lithium hydroxide at room
temperature and provided (+)-a2-hydroxy-1,8-cineole (44%).
To determine the enantiomeric purity the products were ana-
lyzed on two gas chromatography capillary columns with
chiral stationary phases of bDEX-sm and gDEX-sa. The enantio-
meric excess of 98% for (À)-a2-hydroxy-1,8-cineole indicated
that the PLE is selective for the (À)-ester according to many
similar types of substrates.[7] However, the substrate was not
fully hydrolyzed showing an enantiomeric excess of just 78%
for (+)-a2-hydroxy-1,8-cineole. Nevertheless, the PLE-mediated
Nevertheless, no other previous study was targeted at deter-
mining the natural ratios of cineole metabolites being formed
in humans. Accordingly, in the present study, the enantiopure
references of a2-hydroxy1,8-cineole, b2-hydroxy1,8-cineole, 2-
oxo-cineole, and 9-hydroxy-1,8-cineole were synthesized by re-
ferring to or by adapting and optimizing previously described
methods.
Using these references, an initial screen was performed to
characterize the metabolism profile of 1,8-cineole and the
enantiomeric ratios of its metabolites in human urine after in-
gestion of one Soledum capsule by application of gas chroma-
tography-mass spectrometry on a chiral stationary phase.
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enantioselective hydrolysis proved to be an effective possibility
PCC. In conclusion, an environmentally friendlier oxidation to
the enantiopure 2-oxo-1,8-cineole could be developed in addi-
tion to the original PCC oxidation.
for the synthesis of enantiopure a2-hydroxy-1,8-cineole.
Synthesis of (À)- and (+)-2-oxo-1,8-cineole
Synthesis of (À)- and (+)-b2-hydroxy-1,8-cineole
Oxidation of (À)-a2-hydroxy-1,8-cineole using Dess–Martin pe-
riodinane cleanly provided the ketone (À)-2-oxo-1,8-cineole
with 70% yield (96% ee), while the oxidation of (+)-a2-hy-
droxy-1,8-cineole gave the corresponding (+)-2-oxo-1,8-cineole
(77% ee) with 71% yield after purification by a short pad of
silica gel (Scheme 2). The enantiomeric excess corresponded to
As shown in Scheme 3 the synthesis of b2-hydroxycineole
starts from its diastereomer a2-hydroxy-1,8-cineole (see
above). After Dess–Martin oxidation the received 2-oxo-1,8-cin-
eole (70% yield) was reduced with sodium borohydride to
a mixture of a/b2-hydroxy-1,8-cineoles. After separation on an
alumina column with an elution gradient from apolar to polar,
pure b2-hydroxy-1,8-cineole was obtained with 10% yield.
Analogous to the stereospecific synthesis of a2-hydroxy-1,8-
cineole described above the alcohol was esterified with acetyl
chloride to b2-acetyloxy-1,8-cineole (73%). Incubation with PLE
(EC 3.1.1.1) for 18 hours at 378C afforded a chromatographically
separable mixture of (À)-b2-hydroxy-1,8-cineole (34%) and
(+)-b2-acetyloxy-1,8-cineole (53%). After separation by flash
chromatography the remaining (+)-acetate was saponified to
provide (+)-b2-hydroxy-1,8-cineole (48%).
The determination of the enantiomeric purity by GC-MS on
a chiral stationary phase revealed a 100% optical purity for
(À)-b2-hydroxy-1,8-cineole corresponding to the expected se-
lectivity of PLE. Despite an incubation time of 18 hours the hy-
drolysis was not complete, so the resulting enantiomeric
excess of (+)-b2-hydroxy-1,8-cineole was only 33%. The poorer
results, in comparison to the corresponding diastereomer
(+)-a2-hydroxy-1,8-cineole, were presumably due to the exo-
position of the hydroxyl group and a probably sterically re-
strained binding in the active center of PLE.
Scheme 2. Synthesis of (+)-2-oxo-1,8-cineole and (À)-2-oxo-1,8-cineole:
a) Dess–Martin periodinane, CH2Cl2, RT, 24 h.
the enantiomeric purities of the stereospecifically synthesized
educts. For comparison, the described pyridinium chlorochro-
mate oxidation was performed with the enantiopure (À)- and
(+)-a2-hydroxy-1,8-cineole. Our results showed that similar
yields of the desired products were obtained. However, the de-
veloped Dess–Martin periodinane oxidation is comparatively
easier to perform because it is less laborious. Further, the
Dess–Martin periodinane is less toxic than the cancerogenic
Scheme 3. Synthesis of (À)-b2-hydroxy-1,8-cineole: a) Dess–Martin periodinane, CH2Cl2, RT, 24 h; b) 2m NaOH, 2 h, 258C; c) Acetyl chloride, DMAP, CH2Cl2, 0–
58C; d) PLE, pH 7, 18 h, 378C.
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maining acetate with lithium hydroxide, enantiomeric analysis
by GC-MS on a chiral stationary phase was performed. The re-
sults showed that the hydrolysis of the racemic primary ace-
tate by the esterases present in S. cerevisiae, is not totally ste-
reospecific. (À)-9-Hydroxy-1,8-cineole was obtained with an
enantiomeric excess of 40%, whereas the enantiomeric excess
of its enantiomer (+)-9-hydroxy-1,8-cineole was 73%. In con-
clusion, the stereospecific hydrolysis of the primary acetate 9-
acetyloxy-1,8-cineole was only possible with the esterases pres-
ent in yeast yielding moderate results. To achieve an improved
enantioselective hydrolysis the reaction conditions have to be
optimized, or further esterases have to be tested with regard
to their ability of enantiospecific conversion of 9-acetyloxy-1,8-
cineole.
Determination of the enantiomeric composition of chiral
1,8-cineole-metabolites in human urine
A further goal of the present study was to investigate the
enantiomeric composition of chiral 1,8-cineole metabolites in
human urine after ingestion of 1,8-cineole using the synthe-
sized enantiopure references. After ingestion of an enterocoat-
ed Soledum capsule containing 100 mg of 1,8-cineole, samples
were collected at regular time intervals (2 h). Accordingly, char-
acterization of each metabolite or enantiomer, respectively,
was accomplished based on the confirmation of the structural
identities of the compounds in the human urine samples in
comparison to the respective mass spectral and chromato-
graphic data (retention indices, compare with Table 1) of the
reference substances.
Scheme 4. Synthesis of (À)-9-hydroxy-1,8-cineole: a) Pb(Ac)4, THF, 4 h, 658C;
b) KOH (2%), EtOH, 45 min, 608C; c) Hg(Ac)2, THF, 24 h, 558C; d) NaBH4,
NaOH, 16 h, RT; e) Acetyl chloride, DMAP, CH2Cl2, 0–58C; f) Saccharomyces
cerevisiae, sucrose, 24 h. THF=tetrahydrofuran.
Synthesis of (À)- and (+)-9-hydroxy-1,8-cineole
The racemic 9-hydroxy-1,8-cineole (Scheme 4) was obtained by
dihydroxylation of limonene to uroterpenol and subsequent
ring closure with mercuric acetate and sodium borohydride as
described by Horst and Rychlik[6] After acetylation 9-acetyloxy-
1,8-cineole (62%) was incubated with PLE. Enantiomeric analy-
sis showed that PLE did not discriminate the racemate of the
primary acetate, which was completely hydrolyzed to the cor-
responding racemic alcohol 9-hydroxy-1,8-cineole. The same
observation was already described by Loandos et al.[8] Thus, we
have confirmed that PLE does not show selectivity for primary
cineolyl acetates. This outcome corresponds with the de-
scribed syntheses, which showed high stereospecifity of PLE
only for primary meso-diols and secondary or tertiary alco-
hols.[9]
Overall, the metabolites (+/À)-a2-hydroxy-1,8-cineole, (+
/À)-b2-hydroxy-1,8-cineole, (+/À)-a3-hydroxy-1,8-cineole, (+
/À)-3-oxo-1,8-cineole, 4-hydroxy-1,8-cineole, 7-hydroxy-1,8-cin-
eole, and (+/À)-9-hydroxy-1,8-cineole along with traces of
their parent compound were detectable in urine of four volun-
teers (two men and two women) after liberation from their
glucuronides. Not every metabolite could be found in every
sample at each time point after ingestion, but in the samples
with the highest excretion rate each metabolite could be iden-
On the other hand, under the
same conditions the synthesized
racemic 9-acetyloxy-1,8-cineole
was not a substrate for the re-
combinant g-isoenzyme of LPE
expressed in E. coli so that no
conversion occurred. Thus, after
purification, the unchanged ace-
tate was obtained.
Table 1. Retention indices (RI).
Substance
CAS number
RI
[FFAP]
RI
RI
RI
[TG-SQC]
[b-DEX-sm][a]
[gDEX-sa][a]
(+/À)-a2-hydroxy-1,8-cineole
(+/À)-a2-acetyloxy-1,8-cineole
(+/À)-b2-hydroxy-1,8-cineole
(+/À)-b2-acetyloxy-1,8-cineole
(+/À)-2-oxo-1,8-cineole
18679-48-6
–
92999-78-5
57709-95-2
70222-88-7
98920-24-2
–
–
1890
1714
1707
1774
1675
n.d.
n.d.
n.d.
n.d.
1534
1752
n.d.
n.d.
1628
n.d.
n.d.
n.d.
n.d.
1389/1389
1375/1390
1328/1332
1408/1428
1307/1340
1423/1425
1290/1312
1351
1488/1492
1484/1500
1452/1459
1520/1528
1507/1512
1515/1521
1429/1453
1457
Finally the incubation of the
primary ester 9-acetyloxy-1,8-cin-
eole with yeast (Saccharomyces
cerevisiae) and sucrose provided
(À)-9-hydroxy-1,8-cineole in 13%
yield and (+)-9-acetyloxy-1,8-cin-
eole in 19% yield after separa-
tion by flash chromatography.
After saponification of the re-
(+/À)-a3-hydroxy-1,8-cineole
(+/À)-3-oxo-1,8-cineole
4-hydroxy-1,8-cineole
7-hydroxy-1,8-cineole
–
–
–
1390
1473
(+/À)-9-hydroxy-1,8-cineole
(+/À)-9-acetyloxy-1,8-cineole
(+/À)-2,3-dehydro-1,8-cineole
(+/À)-2,3-epoxy-1,8-cineole
1860
1816
n.d.
1673
1811
n.d.
1401/1417
1456/1456
1052/1061
1258/1269
1504/1506
1554/1558
1091/1091
1339/1343
92760-25-3
–
n.d.
n.d.
[a] First (+) enantiomer, second value (À) enantiomer. n.d.=not determined.
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tified for each volunteer. All seven metabolites found in
human urine have been previously reported in humans: 9-hy-
droxy-1,8-cineole, 7-hydroxy-1,8-cineole, a2-hydroxy-1,8-cin-
eole, b2-hydroxy-1,8-cineole, and a3-hydroxy-1,8-cineole were
already known as animal metabolites since long.[4] 7-Hydroxy-
1,8-cineole was first described by Horst and Rychlik as human
metabolite.[6] Recently 4-hydroxy-1,8-cineole and 3-oxo-1,8-cin-
eole were identified in human milk after ingestion of a Soledum
capsule by Kirsch et al.[10] In this study the authors proved that
the Soledum capsules themselves contained traces of all me-
tabolites, but the amounts were much lower than the concen-
trations observed in human milk samples. Thus, it can be ex-
cluded that the metabolites in milk, as well as in the present
case in human urine, cannot be derived from the capsule con-
tent but have to be built presumably inside the body.
1,8-cineole in the exhaled breath of volunteer panelists even
25 hours after consumption of one Soledum capsule.
Also, as can be seen from the error bars in Figure 1, metabo-
lism profiles of the three samples were significantly different
from subject to subject and the ratio of metabolites between
each individual varied drastically. This variation could be ex-
plained by the different metabolic activities of each person.
Higher concentrations in one sample could be due to especial-
ly intensive metabolic processes of the respective donor. Fur-
thermore, it has to be considered that the amount of 1,8-cin-
eole, which was absorbed, was apparently different for each
person. In addition, the different metabolism profiles could be
caused by different times of sampling under the assumption
that the time–concentration curves of the metabolites do not
run in parallel. Therefore, the timing of the sampling can signif-
icantly influence the concentrations of the metabolites. This
phenomenon has also been described previously by several
other authors.[12,13] Studies thereby showed that the expressed
amounts of metabolic enzymes may differ by a factor of up to
50 between each individual, being related with the respective
differences in biotransformatory conversion rates.[14] According-
ly, the inter-individual differences of the elimination rate of 1,8-
cineole and its metabolites are most likely high, as can be de-
duced both from our studies on the elimination of 1,8-cineole
via breath as well as the urine studies.[15]
Metabolism of 1,8-cineole in humans has been studied ex-
tensively in human liver microsomes. According to present
knowledge, the cytochrome P450 enzymes CYP3A4 and
CYP3A5 are mainly responsible for the first oxidative steps in
1,8-cineole metabolism leading to 2- and 3-hydroxy-1,8-cin-
eole.[5,11]
Furthermore, a semiquantitative assessment was carried out
in relation to the concentration of the internal standard (la-
beled 1,8-cineole), still, without consideration of the presuma-
bly different responses. Thus, these ratios have to be treated
with caution, but they can be used to compare the different
samples to each other and to discuss inter- and intra-individual
differences. In addition, considering the different extent of con-
centration of the urine samples, the metabolite concentration
was expressed as a ratio relative to mmol of creatinine.
Summation of the excretion rates of all metabolites in urine
during the time interval up to 8 hours shows that the predomi-
nant metabolite in all samples was (+/À)-a2-hydroxy-1,8-cin-
eole followed by (+/À)-9-hydroxy-1,8-cineole, (+/À)-3-hy-
droxy-1,8-cineole, and 7-hydroxy-1,8-cineole. The same result
were obtained by Horst and Rychlik after consumption of sage
tea (1.02 mg of 1,8-cineole).[6] However, the authors did not
detect the less abundant metabolites (+/À)-b2-hydroxy-1,8-
cineole, 3-oxo-1,8-cineole, and 4-hydroxy-1,8-cineole. It is likely
that the concentrations of these compounds were below the
limit of detection as the ingested dose of 1,8-cineole was 100-
fold lower than in the present study. Moreover, higher concen-
trations of xenobiotica can significantly change the metabo-
lism owing to enzyme induction or saturation, so that other
metabolism pathways might be involved than in case of low
doses.
The first sample was collected no earlier than 30 minutes
after the absorption of 1,8-cineole. The successful transfer of
1,8-cineole into the blood could be detected by the eucalyp-
tol-like odor of the exhaled breath.[12] As already observed in
previous investigations of our group, the absorption of the
capsule content varied from subject to subject between 0.5
and 1.5 hours. These variations are presumably influenced by
differences in the underlying digestion processes spanning dif-
ferent time periods after capsule ingestion.
Figure 1 shows the average relative concentrations of each
metabolite in the three samples. It can be clearly seen that the
first samples (0.5 h–1 h after absorption) contained the lowest
concentrations of all metabolites. (+/À)-b2-Hydroxy-1,8-cin-
eole, (+/À)-3-oxo-1,8-cineole, and 4-hydroxy-1,8-cineole were
not detectable or only traces of them. Generally these substan-
ces were the metabolites with the lowest concentrations. The
highly concentrated metabolites showed their maximum in the
second sample. It is interesting to note that this is divergent to
the metabolites with low concentrations as these substances
showed their maximum in the last samples, indicating differen-
ces in the underlying biotransformation or their pharmacoki-
netic processes.
In previous studies, the metabolites of 1,8-cineole in urine
and plasma were found to be mainly composed of their corre-
sponding glucuronides and were virtually not detected in their
free form. Accordingly, in our study, the metabolite profile of
one urine sample was compared once with and once without
glucuronidase treatment. Thereby, we could confirm that only
traces of free metabolites were detectable. Therefore, the
major part of the functionalized metabolites (phase I reaction)
were conjugated to glucuronic acid by phase II reaction.
Table 2 shows the overall enantiomeric ratios of the chiral
metabolites (results of all time points are integrated into com-
bined data). The temporally resolved results are additionally
provided in Table S1 in the Supporting Information. It can be
clearly seen that each enantiomeric pair displayed one pre-
ferred enantiomer, that means either the (+)- or the (À)-form
predominated.
Generally, decrease of the detected metabolites in the series
of urine samples after reaching their maximum was rather
slow as even more than 8 hours after absorption the concen-
trations of some metabolites were remarkably high. Similar ob-
servations were made by Beauchamp et al.[12] who detected
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2b-hydroxy-1,8-cineole-dehydro-
genase from Citrobacter braakii
after incubation with 1,8-cin-
eole.[16] The authors described
(1R)-2b-hydroxy-1,8-cineole to be
the predominantly formed enan-
tiomer with cytochrome P450cin.
Furthermore, only (1R)-2b-hy-
droxy-1,8-cineole was oxidized
to (1R)-2-keto-1,8-cineole by
(1R)-2b-hydroxy-1,8-cineole-de-
hydrogenase, whereas the re-
maining three isomers of 2-hy-
droxy-1,8-cineole were not con-
verted.
Overall, the question arises if
in any case the same CYP mono-
oxigenase produces both enan-
tiomers or if different enzymes
are involved in catalyzing these
reactions. The latter might be
more likely in view of results de-
scribed by Garcia et al. for the
diasteromers a/b2-hydroxy-1,8-
cineole and a/b3-hydroxy-1,8-
cineole.[17] These authors report-
ed that biotransformation of 1,8-
cineole by a strain of Aspergillus
terreus isolated from Eucalyptus
provided four oxygenated com-
pounds. The inhibition of
CYP450 using ketoconazole or
metronidazole showed that only
the endo position a2-hydroxy-
1,8-cineole and a3-hydroxy-1,8-
cineole were inhibited, mean-
while the exo position was not
affected by the inhibitors. Conse-
quently, these authors conclud-
ed that the b-isomers could be
built by another oxidase or
could be generated from the
corresponding
cineole.
a-hydroxy-1,8-
In our study a shift of the
ratios of the respective enantio-
mers with time was observed, as
well as major variations from
person to person (metabolite
concentrations of each volun-
teer, compare Table S3–S6). Thus,
different reaction pathways may
be responsible for the formation
of the respective enantiomers.
Accordingly, further studies are
Figure 1. Average profile of a) high and b) low concentrated metabolites of 1,8-cineole in human urine after in-
gestion of a Soledum capsule. Error bars show the range of values measured for the individual volunteers.
Similar results were reported by Slessor et al., who deter-
needed to address the mechanistic aspects of the bioconver-
sion of 1,8-cineole.
mined the stereo and enantioselectivity of CYP450cin and (1R)-
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HP-2, 100000 unitsmLÀ1), aluminium oxide (neutral), lead tetraace-
tate, Dess–Martin periodinane, acetyl chloride 98%, lithium hydrox-
ide, m-chloroperbenzoic acid 77%, sodium azide, sodium borohy-
dride>96%, phosphate buffer (pH 7), p-toluenesulfonic acid, yeast
(Saccharomyces cerivisiae), pig liver esterase (EC 3.1.1.1,ꢀ
150 unitsmgÀ1 protein), g-isoenzyme of porcine liver esterase ex-
pressed in E. coli (ꢀ1.2 unitsmgÀ1; Sigma Aldrich, Steinheim, Ger-
many); dichloromethane, acetic acid 99.5%, diethyl ether 99.5%,
hexane 99.5%, (R)-limonene, sodium sulfate 98.5%, sodium thiosul-
fate 99%, tetrahydrofuran 99.9% (Th. Geyer, Renningen, Germany),
Table 2. Percentages of the enantiomeric ratios of 1,8-cineole metabo-
lites in human urine.
1,8-Cineole
derivatives
Range
Ø Total content
Enantiomeric ratio
[%] in urine[a]
[mgmmolÀ1
creatinine]
x
[mgmmolÀ1
x
creatinine]
(+/À)-3-oxo
(À/+)-3-oxo
(+)-b2-OH
(À)-b2-OH
(+)-a2-OH
(À)-a2-OH
(+)-9-OH
0.21–0.36
0.0005–0.003
0.2–0.79
0.32
0.0016
0.36
99.5
0.5
72
28
38
62
15
85
86
14
0.06–0.25
0.14
2
15.36–66.72
34.44–95.41
1.24–30.87
38.49–91.83
6.68–24.13
0.72–4.17
37.58
60.30
11.68
65.32
16.84
2.66
and H3-1,8-cineol (aromaLAB AG, Munich, Germany). 2,3-Dehydro-
1,8-cineole, 2,3-epoxy-1,8-cineole, a3-hydroxycineole, 3-oxo-1,8-cin-
eole, 4-hydroxy-1,8-cineole and 7-hydroxy-1,8-cineole were a gener-
ous gift from Frauke Kirsch (Friedrich-Alexander-University of Erlan-
gen-Nuremberg, Germany).
(À)-9-OH
(+/À)-a3-OH
(À/+)-a3-OH
[a] [100%=amount of both enantiomers].
Syntheses
Detailed experimental procedures are given in the Supporting In-
formation. Some of the experimental procedures were adapted
from literature methods.[18,19,20]
Conclusion
Pig liver esterase (PLE) can be efficiently employed for the
enantioselective hydrolysis of secondary racemic cineolyl ace-
tates. Using this enzyme (À)-a2-hydroxy-1,8-cineole and (À)-
b2-hydroxy-1,8-cineole could be stereospecifically synthesized
with high enantiomeric excess and chemical yield. For the pri-
mary 9-acetyloxy-1,8-cineol PLE showed no selectivity, as this
substance was completely hydrolyzed to the corresponding
racemic 9-hydroxy-1,8-cineole. In contrast to this, for the re-
combinant g-isoenzyme of PLE the primary acetate was shown
not to be a substrate. Only after incubation with Saccharomy-
ces cerevisiae an enantioselective hydrolysis was observed with
moderate enantiomeric excess and chemical yield. Further, an
environmentally friendlier oxidation to the enantiopure (À/
+)-2-oxo-1,8-cineole with Dess–Martin-periodinane was devel-
oped in addition to the PCC oxidation used in former studies.
Another objective of this study was to investigate the enan-
tiomeric composition of the chiral metabolites in human urine
after consumption of 100 mg of 1,8-cineole using the newly
synthesized enantiopure reference compounds. Seven metabo-
lites could be clearly identified after liberation from their glu-
curonides. The individual metabolite profiles varied widely
from person to person. Enantiomeric analyses showed that
one enantiomer always predominated for each metabolite.
On the whole, in future studies these results can be com-
pared with the enantiomeric composition of 1,8-cineole me-
tabolites in blood and human milk for better understanding of
1,8-cineole metabolism. Such investigations are currently
under way.
Methods
Column chromatography
Column chromatography was carried out using silica gel 60
(0.063–0.200 mm) or neutral aluminium oxide (water-cooled glass
column; 500 mmꢁ20 mm i.d.). Flash column chromatography was
performed with silica gel 60 (0.040–0.063 mm; water-cooled glass
column; 400 mmꢁ20 mm i.d.) and nitrogen pressure of 0.2 bar.
Gas chromatography–mass spectrometry
GC-MS analysis was performed with a Finnigan Trace DSQ Single
Quadrupole GC/MS (Thermo Fisher Scientific) with GERSTEL CIS 4C
injection system and GERSTEL MPS 2 autosampler (GERSTEL GmbH
& Co. KG, Duisburg) using the following capillary columns: DB-
FFAP (30 mꢁ0.32 mm, film thickness 0.25 mm; J&W Scientific,
Fisons Instruments, Mainz-Kastel, Germany) and GOLD TG-SQC
(30 mꢁ0.25 mm I.D. film thickness 0.25 mm, Thermo Scientific,
Schwerte, Germany). For chiral analysis were used the two capillary
columns: gDEX-sa and bDEX-sm (30 mꢁ0.32 mm i.d., film thickness
0.25 mm, Restek Chromatography Products, Kriftel, Germany).
For all analyses the following temperature program was used: The
samples were applied by the cold-on-column injection technique
at 408C. After 2 min, the temperature of the oven was raised at
48CminÀ1 to 1708C, then raised at 408CminÀ1 to 2308C and held
for 5 minutes. The flow rate of the helium carrier gas was
2.5 mLminÀ1
.
EI-mass spectra were generated in full scan mode (m/z range 40 to
250) at 70 eV ionization energy.
Experimental Section
Chemicals
The following reagents were purchased from the sources given in
parentheses: potassium hydroxide, silica gel 60 (0.040–0.063 mm),
silica gel (0.063–0.200 mm), sodium acetate, sodium carbonate
99%, sodium hydrogen carbonate, sodium hydroxide, pentane, tol-
uene (Merck, Darmstadt, Germany); 4-dimethylamino pyridine, a-
terpineole, b-glucuronidase from Helix pomatia (EC 3.2.1.31, Type
Nuclear magnetic resonance spectroscopy
1H NMR and 13C spectra were recorded on a Bruker AMX400
(Bruker, Karlsruhe, Germany) at 297 K in CDCl3 with TMS as internal
standard (d=0 ppm). Optical rotations were measured on a polar-
imeter P 3000 A. KRꢂSS Optronic GmbH (Germany).
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Study design
Isolation of the volatile fraction of human urine without enzy-
matic hydrolysis
The study design was in concordance with the requirements of the
declaration of Helsinki, and was conducted after consultation of
the local ethical committee of the University of Erlangen-Nurem-
berg for approval of the study.
To estimate the concentrations of free metabolites, one probe
were subjected directly to SAFE distillation without glucuronidase
treatment.
Therefore, 2.25 mg 2H3-1,8-cineole and 30 mL dichloromethane
were added to 50 mL urine and stirred 30 minutes at room tem-
perature. Subsequently, the untreated sample was worked up by
SAFE distillation analogous to the outline described above for the
glucuronidase treated samples.
Donors
Donors were volunteers (non-smokers, Germans of Caucasian eth-
nicity, 2 females and 2 males, age range 23–33, mean age 28,
body-mass-index: 21.7–22.6) exhibiting no known illnesses at the
time of examination, and consuming their freely chosen meals
without any specified dietary protocol.
The sample was analyzed on the DSQ system using the two chiral
capillaries bDEX-sm and gDEX-sa.
Prior to sample collection and analysis written consent was ob-
tained from all participants providing urine after a full explanation
of the purpose and nature of the study. Withdrawing from the
study was possible at any time.
Photometric creatinine determination in urine (Jaffꢀ reac-
tion)
Creatinine concentration in urine is a marker for concentration of
urine. It was used as reference level of the excretion of the ana-
lytes. Photometrical creatinine determination was performed using
the Jaffꢃ reaction.[22] After dilution of the urine (1+49, v+v) with
distilled water, 100 mL of the diluted urine was mixed with 500 mL
Samples
Urine was collected in sterile 500 mL amber glass bottles. The
urine samples were either processed and analyzed directly after
donation as described below or frozen at À208C until analysis.
Frozen samples were thawed at room temperature and then ana-
lyzed according to the same procedure described below.
sodium hydroxide (300 mmolLÀ1
)
and 500 mL picric acid
(8.7 mmolLÀ1). The orange-red complex formed was measured
spectrophotometrically at 492 nm in relation to an internal stan-
dard (177 mmolLÀ1 creatinine).
One blank sample from urine was collected as control before in-
gestion of one Soledum capsule (100 mg 1,8-cineole). After per-
ceiving a eucalyptus-like odor in the breath the urine was collected
three times during the workday (up to total duration of 8 h).
Semiquantification of the metabolites in urine
2
Triply deuterated H3-1,8-cineole was used as internal standard and
the concentration of each metabolite was calculated as percentage
2
of H3-1,8-cineole added to the samples prior to sample prepara-
Solvent Extraction and Solvent Assisted Flavor Evaporation
of Urine Volatiles
tion. Therefore, the peak area of the individual metabolite was di-
vided trough the peak area of labeled 1,8-cineole and multiplied
Solvent assisted flavor evaporation (SAFE)[21] was applied for the
fast and careful isolation of the urinary volatiles. The urine was
either subjected to the SAFE directly for isolation of the volatile
fraction, or immediately subjected to the hydrolysis procedure
with b-glucuronidase prior to SAFE as described below, and subse-
quently subjected to volatile isolation via SAFE.
2
with the absolute concentration of H3-1,8-cineole (2.25 mg). After
normalizing to the sample amount the concentration was ex-
pressed in relation to the molar creatinine concentration in urine.
Acknowledgements
We thank Oliver Frank, Chair of Food Chemistry and Molecular
Sensory Science of the Technical University of Munich for record-
ing the NMR spectra. This study was supported by the German
Federal Ministry of Education and Research (BMBF). The study
sponsor had no involvement in the design and accomplishment
of the study, nor in the process of publication. We are exclusively
responsible for the contents of this publication.
Isolation of the volatile fraction of human urine with enzy-
matic hydrolysis (b-glucuronidase assays)
Acetic acid-sodium acetate buffer (10 mL) adjusted to pH=5 were
added to freshly collected urine (10 mL). Then 0.1 mL of a sodium
azide solution at a concentration of 200 mgmLÀ1, and 0.2 mL of the
2
b-glucuronidase-solution were added. H3-1,8-Cineol (2.25 mg) was
used as internal standard. The mixture was stirred for 15 hours at
378C. Afterwards, 10 mL of purified dichloromethane were added
and the mixture was stirred for 30 minutes. After distillation of the
mixture additional aliquots of 5 mL of dichloromethane were ad-
ministered and distillation was re-performed thrice to achieve com-
plete transfer of the respective odor compounds. The obtained
aqueous distillate phase was additionally extracted thrice with
20 mL of dichloromethane. Then all combined dichloromethane
phases were dried over anhydrous Na2SO4, and finally concentrated
to a total volume of 100–200 mL at 508C by means of Vigreux-dis-
tillation and micro-distillation.
Keywords: cineoles · enantioselectivity · enzyme catalysis ·
hydrolysis · metabolism
[1] F. A. Santos, V. S. Rao, Phytother. Res. 2000, 14, 240–244.
[2] R. M. Carman, W. T. Robinson, C. J. Wallis, Aust. J. Chem. 2005, 58, 785–
791.
[3] R. Boyle, S. McLean, N. W. Davies, Xenobiotica 2000, 30, 915–932.
[4] R. M. Carman, K. D. Klika, Aust. J. Chem. 1992, 45, 651–657.
[5] M. Duisken, F. Sandner, B. Blomeke, J. Hollender, Biochim. Biophys. Acta-
Gen. Subj. 2005, 1722, 304–311.
The samples were analyzed on the DSQ system using the two
chiral capillaries bDEX-sm and gDEX-sa.
[6] K. Horst, M. Rychlik, Mol. Nutr. Food Res. 2010, 54, 1515–1529.
[7] F. A. Luzzio, D. Y. Duveau, Tetrahedron: Asymmetry 2002, 13, 1173–1180.
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPlusChem 2013, 78, 77 – 85 84

CHEMPLUSCHEM
FULL PAPERS
www.chempluschem.org
[8] M. d. H. Loandos, M. B. Villecco, E. Burgueno-Tapia, P. Joseph-Nathan,
C. A. N. Catalan, Nat. Prod. Commun. 2009, 4, 1537–1545.
[9] U. T. Bornscheuer, R. J. Kazlauskas, Hydrolases In Organic Synthesis:
Regio- And Stereoselective Biotransformations, Vol. 2, Wiley-VCH, Wein-
heim, 2006.
[10] F. H. Kirsch, K. Beauchamp, J. Rychlik, M. Buettner, Wartburg Symp.
Flavor Chem. Biol. 2010.
[11] M. Miyazawa, M. Shindo, Nat. Prod. Lett. 2001, 15, 49–53.
[12] J. Beauchamp, F. Kirsch, A. Buettner, J. Breath Res. 2010, 4, 026006.
[13] M. Miyazawa, H. Kameoka, K. Morinaga, K. Negoro, N. Mura, J. Agric.
Food Chem. 1989, 37, 222–226.
[16] K. E. Slessor, J. E. Stok, S. M. Cavaignac, D. B. Hawkes, Y. Ghasemi, J. J.
De Voss, Bioorg. Chem. 2010, 38, 81–86.
[17] C. Garcꢄa, P. Rodrꢄguez, E. Dꢄas, H. Heinzen, P. Menꢃndez, J. Mol. Catal. B
2009, 59, 173–176.
[18] H. L. Kopperman, R. C. Hallcher, Sr. , A. Riehl, R. M. Carlson, R. Caple, Tet-
rahedron 1976, 32, 1621–1626.
[19] R. M. Carman, M. T. Fletcher, Aust. J. Chem. 1984, 37, 1117–1122.
[20] I. C. Macrae, V. Alberts, R. M. Carman, I. M. Shaw, Aust. J. Chem. 1979, 32,
917–922.
[21] W. Engel, W. Bahr, P. Schieberle, Eur. Food Res. Technol. 1999, 209, 237–
241.
[14] K. Aktories, W. Forth, U. Fçrstermann, Allgemeine und spezielle Pharma-
kologie und Toxikologie: fꢁr Studenten der Medizin, Veterinꢂrmedizin,
Pharmazie, Chemie und Biologie sowie fꢁr ꢃrzte, Tierꢂrzte und Apotheker,
mit 303 Tabellen, Vol. 9, Elsevier, Urban & Fischer, Amsterdam, New York,
2005.
[22] H. H. Taussky, Clin. Chim. Acta 1956, 1, 210–224.
Received: September 25, 2012
[15] F. Kirsch, J. Beauchamp, A. Buettner, Clin. Nutr. 2012, 31, 682.
Published online on December 18, 2012
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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