Metabolism of curcuminoids in tissue slices and subcellular fractions from rat liver

Article abstract of DOI:10.1021/jf058146a

Curcumin and its natural congeners are of current interest because of their putative anti-inflammatory and anticarcinogenic activities, but knowledge about their metabolic fate is scant. In the present study conducted with precision-cut liver slices from

Full text of DOI:10.1021/jf058146a

756 J. Agric. Food Chem. 2006, 54, 756−764
Metabolism of Curcuminoids in Tissue Slices and Subcellular
Fractions from Rat Liver
SIMONE I. HOEHLE,† ERIKA PFEIFFER,† ANIKOÄ M. SOÄ LYOM,§ AND
MANFRED METZLER*,†
Institute of Applied Biosciences, Section of Food Chemistry and Toxicology, University of Karlsruhe,
P.O. Box 6980, D-76128 Karlsruhe, Germany, and College of Pharmacy, University of Arizona,
1703 East Mabel Street, Tucson, Arizona 85721-0207
Curcumin and its natural congeners are of current interest because of their putative anti-inflammatory
and anticarcinogenic activities, but knowledge about their metabolic fate is scant. In the present study
conducted with precision-cut liver slices from male and female Sprague-Dawley rats, five reductive
but no oxidative metabolites of curcumin and its demethoxy and bis-demethoxy analogues were
observed and identified by HPLC and GC-MS analysis, mostly by comparison with authentic reference
compounds. The major reductive metabolites were the hexahydrocurcuminoids in both male and
female rat liver slices, whereas male rats formed more octahydro than tetrahydro metabolites and
female rats more tetrahydro- than octahydrocurcuminoids. Tetrahydro, hexahydro, and octahydro
metabolites were predominantly present as glucuronides, but a significant proportion of sulfate
conjugates was also observed. The lack of formation of oxidative metabolites of curcumin and the
ready generation of reductive metabolites were confirmed using rat liver microsomes and cytosol,
respectively. Results of enzymatic hydrolysis studies conducted under various conditions revealed
that curcumin and demethoxycurcumin are chemically less stable than bis-demethoxycurcumin,
whereas the reductive metabolites of all three curcuminoids are stable compounds. This is the first
report on the metabolism of demethoxycurcumin and bis-demethoxycurcumin. In view of the chemical
instability of the parent curcuminoids, it is proposed to use their major phase I metabolites, that is,
the stable hexahydro products, as biomarkers for exposure in clinical studies.
KEYWORDS: Curcumin; liver slices; curcuminoids; reductive metabolites; instability
INTRODUCTION
Curcuminoids are natural yellow-orange pigments present in
the rhizomes of the Asian plant Curcuma longa and other
Curcuma species, for example, C. xanthorriza. They are
commonly isolated from the spice and food-coloring agent
turmeric. Curcuma extracts contain three diarylheptanoids,
namely, curcumin as the major component together with smaller
amounts of demethoxycurcumin and bis-demethoxycurcumin
(1). As depicted in Figure 1, curcuminoids have a unique
conjugated structure including a â-diketone moiety due to which
they undergo keto-enol tautomerization and exist entirely in
the enol form in solution (2, 3).
In traditional Oriental medicine, turmeric has been used as a
medicinal herb for thousands of years. Curcumin is recognized
for its broad spectrum of biological activities including chola-
gogic and choleretic, anti-inflammatory, and antioxidant activi-
ties, the most important being its putative anticarcinogenic effect
* Author to whom correspondence should be addressed (telephone
+49-721-6082132; fax +49-721-6087255; e-mail manfred.metzler@
chemie.uni-karlsruhe.de).
Figure 1. Chemical structures of the curcuminoids from turmeric.
(4-6). Curcumin acts as a scavenger of reactive oxygen species
and interferes with lipid peroxidation (7, 8). The antioxidant
† University of Karlsruhe.
§ University of Arizona.
10.1021/jf058146a CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/13/2006

Curcuminoid Metabolism in Rat Liver
J. Agric. Food Chem., Vol. 54, No. 3, 2006 757
Figure 2. Reductive metabolism of curcumin.
properties of curcumin are assumed to be imparted by its
polyphenolic structure, the â-diketone moiety, and the formation
of relatively stable free radicals due to its extended conjugated
double-bond system (5, 9). The anti-inflammatory and cancer-
preventive properties have been linked to numerous biological
activities including effects on arachidonate metabolism through
inhibition of both cyclooxygenase and lipoxygenase pathways
(5, 10, 11). Curcumin is also a potent inhibitor of mutagenesis
and chemically induced carcinogenesis (5, 12-14).
for biotransformation studies because the physiological liver
microarchitecture, that is, cell-to-cell contacts as well as cell-
matrix interactions, is maintained. Thus, the in vivo situation
concerning phase I and phase II metabolism is reflected very
well in tissue slices (26, 27).
MATERIALS AND METHODS
Chemicals and Animals. Curcumin, demethoxycurcumin, bis-
demethoxycurcumin, and tetrahydrocurcumin were provided by the
Arizona Center for Phytomedicine Research (Tucson, AZ): tetrahy-
drocurcumin had been chemically synthesized by Sabinsa Corp.
(Payson, UT), whereas the other three curcuminoids were isolated from
turmeric and purified by column chromatography and recrystallization.
All four compounds had a purity of >99% according to HPLC analysis.
Their HPLC retention times and UV-vis spectra are given in Table
1. Pure curcumin and bis-demethoxycurcumin were also chemically
synthesized as described below. Commercial curcumin obtained from
turmeric and containing 75% curcumin, 18% demethoxycurcumin, and
7% bis-demethoxycurcumin, sulfatase (type VI from Aerobacter
aerogenes), â-glucuronidase (type B-1 from bovine liver), alcohol
dehydrogenase (from equine liver), and all other chemicals and reagents
were obtained from Sigma-Aldrich Chemical Co. (Steinheim, Germany).
â-Glucuronidase/arylsulfatase from Helix pomatia was purchased from
Roche Diagnostic Co. (Mannheim, Germany). HPLC grade acetonitrile
was from Carl Roth Co. (Karlsruhe, Germany).
Male and female Sprague-Dawley rats were purchased from Harlan
Winkelmann GmbH (Borchen, Germany). Animals were kept under a
12-h light/dark cycle and received water and commercial laboratory
chow ad libitum. Rats of 200-300 g weight were used for the
preparation of liver slices and hepatic microsomes and cytosol.
Chemical Syntheses. Curcumin and bis-demethoxycurcumin were
synthesized according to the method of Pabon et al. (28). Briefly, 2.5
mL of acetylacetone (25 mmol) and 1.25 g of boric acid anhydride
(17 mmol) were stirred for 0.5 h to form a thick paste, to which 10
mL of dry ethyl acetate was added. This mixture was then added to a
solution of 50 mmol of the respective substituted benzaldehyde (7.6 g
of vanillin for curcumin, 6.1 g of 4-hydroxybenzaldehyde for bis-
demethoxycurcumin) and 26.2 mL of tributylborate (100 mmol) in 15
mL of dry ethyl acetate. After 10 min of stirring, 0.5 mL of
n-butylamine (5 mmol) was added dropwise over a period of 10 min,
and stirring was continued for 4 h. The mixture was then kept without
stirring overnight, followed by the addition of 37.5 mL of 0.6 N aqueous
HCl at 60 °C and stirring for 1 h to hydrolyze the reaction product.
The organic layer was separated and the aqueous layer extracted three
times with ethyl acetate. The combined organic phases were washed
acid-free and dried over Na2SO4, and solvent was evaporated under
reduced pressure. The synthetic curcuminoids were recrystallized from
methanol at 4 °C and washed several times with cold methanol and
Although numerous aspects of the pharmacology of curcumin,
in particular its activity as a chemopreventive agent, have been
studied, the absorption, pharmacokinetics, and metabolism in
humans and experimental animals have not been fully character-
ized. The metabolism of curcumin, both phase I and phase II,
has mostly been studied in rats in vivo and in vitro (15-21).
More recently, information on the metabolism of curcumin in
humans has been obtained from in vitro studies with hepatic
and intestinal cells and subcellular fractions (20, 21), as well
as from clinical studies with cancer patients (22-25). The
present knowledge can be summarized as follows: phase I
metabolism comprises the successive reduction of the four
double bonds of the heptadiene-3,5-dione system (Figure 2).
Tetrahydrocurcumin and hexahydrocurcumin are the major
products observed in most studies, whereas dihydrocurcumin
and octahydrocurcumin (also named hexahydrocurcuminol)
usually represent minor products or are not detected at all. The
enzymes responsible for the bioreduction have been found to
reside in the cytosol of liver and intestine and include alcohol
dehydrogenase. Curcumin and its reduced metabolites appear
to be easily conjugated in vivo and in vitro. Reported conjugates
of curcumin include a monoglucuronide, a monosulfate, and a
mixed sulfate/glucuronide. Although mass spectra of these phase
II metabolites have been published, their exact chemical
structures have not yet been established. Glucuronidation and
sulfation of curcumin are believed to occur in the liver and
intestine of rats and humans. No data have yet been published
on the metabolism of demethoxycurcumin and bis-demethoxy-
curcumin.
The aim of the present study was to compare the phase I and
phase II metabolism of curcumin, demethoxycurcumin, and bis-
demethoxycurcumin in precision-cut tissue slices prepared from
the liver of male and female rats. Of particular interest was the
question of whether oxidative phase I metabolites would be
formed, for example, through hydroxylation or demethylation
of the curcuminoids. Liver slices represent a valuable model

758 J. Agric. Food Chem., Vol. 54, No. 3, 2006
Hoehle et al.
Table 1. Chromatographic and Spectrometric Properties of Curcuminoids and Their Reductive Metabolites.
HPLC
(min)
GC-MS after trimethylsilylation retention time in min;
m/z (% relative intensity)
compound
UV−vis spectrum λmax nm (ꢀ)
curcumina
25.4
428 (58530) in MeOH
na
na
dihydrocurcuminb
24.6, 26.0c
23.3
376 (32000) and 285 (7500) in EtOHb
281 (15100) in MeOH
tetrahydrocurcumina
23.4; 588 (M , 0.4), 573 (10), 365 (15), 323 (5), 223 (5),
+
209 (100), 193 (8), 179 (13), 73 (19)
21.5; 590 (M , 2), 500 (18), 291 (19), 278 (9), 251 (6),
+
hexahydrocurcumina
octahydrocurcumina
11.4
282 (7400) in MeOH
249 (8), 235 (17), 209 (100), 195 (9), 193 (7),
179 (18), 73 (24)
+
8.8, 9.1c
282 (∼
7400) in HPLC eluent
20.6, 21.3; 664 (M , 0.1), 574 (15), 484 (23), 324 (19),
275 (18), 262 (98), 247 (23), 235 (29), 209 (100),
179 (27), 73 (53)
na
20.5; 560 (M , 5), 470 (43), 291 (15), 278 (8), 263 (11), 249
demethoxycurcumind
24.7
11.1
424 (54800) in MeOH
+
hexahydro-demethoxy-curcumine
282 (∼
7100) in HPLC eluent
35, 235 (44), 209 (84), 195 (10), 193 (18), 179 (100), 73 (57)
+
octahydro-demethoxy-curcumina
8.6, 8.9c
282 (∼
7100) in HPLC eluent
20.0, 20.3; 634 (M , 0.1), 544 (10), 454 (23), 386 (5),
324 (14), 275 (11), 262 (100), 258 (8), 247 (17),
235 (15), 232 (27), 209 (44), 192 (10), 179 (60),
73 (44)
na
bis-demethoxycurcumina
24.0
10.8
416 (46400) in MeOH
280 (6000) in MeOH
+
hexahydro-bis-demethoxy-curcumina
19.2; 530 (M , 0.01), 440 (19), 295 (8), 261 (18), 248 (43),
219 (13), 205 (11), 192 (15), 179 (100), 73 (51)
18.4, 19.0; 604 (M , 0.03), 514 (16), 424 (25), 356 (11),
294 (11), 258 (11), 232 (100), 205 (28), 192 (15),
179 (83), 73 (53)
+
octahydro-bis-demethoxy-curcumina
8.3, 8.7c
280 (∼
6000) in HPLC eluent
a Obtained through chemical synthesis. b According to Uehara et al. (29). c Two isomers. d Isolated from turmeric. e Obtained through enzymatic reaction; MeOH, methanol;
EtOH, ethanol; na, not applicable because curcumin, dihydrocurcumin, demethoxycurcumin, and bis-demethoxycurcumin could not be gas chromatographed even after
trimethylsilylation.
deionized water to remove traces of unreacted aldehyde. Melting points
were 179-181 °C for curcumin [lit. (2) 182-183 °C] and 224-225
°C for bis-demethoxycurcumin [lit. (2) 223-224 °C]. UV-vis spectra
and HPLC retention times were identical with those of the authentic
compounds isolated from turmeric (Table 1).
(solvent B) in deionized water (solvent A, adjusted to pH 3.0 with
formic acid) in 35 min was used. The flow rate was 1 mL/min. The
curcuminoids were recorded at 420 nm and the reductive metabolites
at 280 nm. 3,5,3′,5′-Tetramethyl-bisphenol A (TMBPA) was used as
internal standard and monitored at 280 nm. Peaks were identified by
comparison of HPLC retention times and absorption spectra with those
of authentic standards (Table 1). Quantification was based on the peak
area ratio (test compound to internal standard), applying different
absorption coefficients for curcuminoids and their reductive metabolites
(Table 1). The absorbance of hexahydrocurcumin and hexahydro-bis-
demethoxycurcumin did not change upon reduction to octahydrocur-
cumin and octahydro-bis-demethoxycurcumin, respectively. Therefore,
octahydrocurcuminoids were quantified by using the same absorption
coefficient as for hexahydrocurcuminoids and by adding the peak areas
of both diastereomers. The limits of quantification were 3 pmol for
curcuminoids and 15 pmol for hexahydro- and octahydrocurcuminoids.
Hexahydrocurcumin was prepared by hydrogenation of tetrahy-
drocurcumin according to the method of Uehara et al. (29). A solution
of 20 mg of tetrahydrocurcumin (54 mmol) in 20 mL of methanol was
stirred with 10 mg of a Pd catalyst (5% Pd on charcoal) under a H2
atmosphere at 20 °C for 5 h and filtered, and the filtrate was evaporated
in vacuo. The colorless oily product contained one major peak (>95%)
according to HPLC analysis with UV detection at 280 nm. The chemical
structure of the synthetic hexahydrocurcumin was confirmed by GC-
MS analysis of the trimethylsilylated product (Table 1). The same
hydrogenation procedure was used to synthesize hexahydro-bis-
demethoxycurcumin from bis-demethoxycurcumin, whereas hexahy-
drodemethoxycurcumin was obtained from demethoxycurcumin by
enzymatic reduction with cytosol (see below) and purified by HPLC.
The structures of both hexahydro compounds were also confirmed by
GC-MS analysis (Table 1).
GC-MS Analysis. A Finnigan GCQ capillary gas chromatograph
equipped with a 30 m × 0.25 mm i.d., 0.25 µm, 5% phenylmethyl
MDN-5S fused-silica column (Supelco, Bellefonte, PA) and coupled
to an ion-trap detector was operated with electron impact (EI) ionization
at 70 eV (Thermo Finnigan, Austin, TX). Oven temperature was
programmed from 150 °C (1-min hold) to 295 °C (10-min hold) at a
rate of 10 °C/min. Temperatures of injector, transfer line, and ion source
were 275, 275, and 250 °C, respectively. Samples (1 µL) dissolved in
N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were splitlessly
injected with helium as carrier gas with a column pressure of 83 kPa.
Mass spectra were scanned from m/z 50 to 650 at a rate of 0.5 s/scan.
Metabolites of curcuminoids from the incubations were identified by
comparison of their HPLC and GC retention times and their mass
spectra with those of authentic reference substances.
Octahydrocurcumin was obtained from 1 mL of a 2.5 mM solution
of hexahydrocurcumin in methanol by adding a 10-fold molar excess
of sodium borohydride. After 2 h at ambient temperature, the methanol
was evaporated in vacuo and the residue dissolved in 1 mL of deionized
water adjusted to pH 4.5. The product was extracted with 3 × 0.5 mL
of ethyl acetate, the combined extract evaporated in vacuo, and the
residue dissolved in 50 µL of methanol. Analysis by HPLC at 280 nm
and by GC-MS after trimethylsilylation showed the presence of two
products, which gave identical mass spectra consistent with the structure
of octahydrocurcumin (Table 1). The two octahydrocurcumins represent
diastereomers due to the presence of the two chiral carbon atoms
carrying the aliphatic hydroxyl groups (Figure 2).
Incubation of Liver Slices. Precision-cut slices were prepared from
the livers of two male and two female untreated Sprague-Dawley rats
as previously described (30). Two independent experiments were carried
out for each of the three curcuminoids and both genders, using slices
from different rats. For the reductive metabolites tetrahydrocurcumin
and hexahydrocurcumin, two independent experiments with slices from
one male and one female rat were conducted. For each experiment,
three liver slices were separately incubated according to the method of
Fisher et al. (31). The final concentration of the test compounds was
HPLC Analysis. A HP 1100 system equipped with a binary pump,
a photodiode array detector, and HP Chemstation software for data
collection and analysis (Agilent Technologies, Waldbronn, Germany)
was used, and separation was carried out on a 250 × 4.6 mm i.d., 5
µm, reversed-phase Prodigy 5ODS2 column (Phenomenex, Torrance,
CA). Samples were dissolved in methanol, and the injection volume
was 10-20 µL. A linear gradient changing from 30 to 70% acetonitrile

Curcuminoid Metabolism in Rat Liver
J. Agric. Food Chem., Vol. 54, No. 3, 2006 759
50 or 200 µM. Two types of control cultures, that is, one without liver
slice and the other without test compound but with DMSO, were
performed. Slices were incubated for 2, 4, 6, and 24 h. After incubation,
the media of the three slices used in each experiment were combined
and stored at -80 °C until analyzed.
Enzymatic Hydrolysis. Prior to workup, 10 µL of a 5 mM solution
of TMBPA in DMSO was added as internal standard to each milliliter
of the combined media. Two different methods of enzymatic hydrolysis
were used.
the microsomes, control incubations were also conducted with 100 µM
testosterone and analyzed as described by Pfeiffer et al. (30).
For reductive in vitro metabolism, incubations were conducted in a
final volume of 0.5 mL containing a 100 µM mixture of curcuminoids
from turmeric (75% curcumin, 18% demethoxycurcumin, and 7% bis-
demethoxycurcumin) dissolved in DMSO (final concentration ) 1%),
equine alcohol dehydrogenase (5 U) or rat hepatic cytosol (1 mg of
cytosolic protein/mL), and 10 mM NADH in 0.1 M phosphate buffer
(pH 7.4). After a preincubation period of 5 min at 37 °C, NADH was
added and the mixture incubated for 1 h at 37 °C. The reaction was
terminated by extraction with 3 × 0.5 mL of ethyl acetate, the extract
evaporated to dryness in vacuo, the residue dissolved in 50 µL of
methanol, and an aliquot used for HPLC analysis. In control experi-
ments, the curcuminoid mixture was incubated with (i) the cytosol but
without NADH and (ii) NADH without equine alcohol dehydrogenase.
In vitro reduction and glucuronidation of curcumin were studied
separately and in combination, using liver cell fractions from male
Sprague-Dawley rats. Incubations were performed in 0.1 M phosphate
buffer (pH 7.4) containing 100 µM curcumin dissolved in DMSO (final
concentration ) 1%) and 10 mM magnesium chloride in a total volume
of 1 mL. For reduction, incubation mixtures included 1 mg of cytosolic
protein/mL and 10 mM NADH. For glucuronidation, 1 mg of
microsomal protein/mL and 4 mM UDPGA were added. For simulta-
neous reduction and glucuronidation, cytosol and microsomes were
combined and their cofactors added. After 5 min of preincubation of
cell fractions and substrates at 37 °C, reactions were started by the
addition of the cofactor(s) and incubations continued for 1 h at 37 °C.
Control incubations were carried out with cytosol and microsomes but
without NADH or UDPGA or both cofactors. Reactions were terminated
by adding 25 µL of 20% aqueous trichloroacetic acid. Subsequently,
the incubation mixture was neutralized with 25 µL of 1 M aqueous
sodium hydroxide, and the precipitated proteins were removed by
centrifugation (5 min at 1000g). The resulting supernatant was analyzed
by HPLC directly and after enzymatic hydrolysis in a 0.2-mL aliquot
with a â-glucuronidase/arylsulfatase preparation from H. pomatia in
0.2 mL of 0.15 M acetate buffer (pH 5.0) as described for method I
above.
Method I. For the determination of unconjugated metabolites (value
IA), a 0.5-mL aliquot of the medium was extracted three times with
0.5 mL each of ethyl acetate. The combined extracts were evaporated
in vacuo, the residue was dissolved in 50 µL of methanol,and a 10-
20-µL aliquot was analyzed by HPLC. For measuring the sum of
unconjugated and conjugated material (value IB), another 0.5-mL
aliquot of the medium was mixed with 0.2 mL of 0.15 M acetate buffer
(pH 5.0), incubated with 10 µL of â-glucuronidase/arylsulfatase
preparation from H. pomatia at 37 °C for 16 h, extracted with ethyl
acetate, and analyzed by HPLC as described above. The amount of
conjugates was calculated as the difference between values IB and IA.
Method II of the enzymatic hydrolysis provided information about
the type of conjugate by separate treatment of the incubation medium
with â-glucuronidase and sulfatase. Unconjugated material (value IIA)
was first determined in a 0.2-mL aliquot mixed with 0.2 mL of 0.15
M acetate buffer (pH 5.0) but no enzyme prior to extraction with ethyl
acetate. Another 0.2-mL aliquot of the medium was mixed with 0.2
mL of 0.15 M acetate buffer (pH 5.0) and incubated with 5000 Fishman
units (U) of â-glucuronidase type B-1 from bovine liver prior to
extraction with ethyl acetate (value IIB). For the analysis of sulfates,
0.2 mL of medium was mixed with 0.2 mL of 0.1 M phosphate buffer
(pH 7.1) containing 0.1 U of sulfatase type VI from Aerobacter
aerogenes prior to incubation and extraction (value IIC). For total
metabolites, that is, unconjugated, glucuronides, and sulfates, a 0.2-
mL aliquot of the medium was mixed with 5000 Fishman U of
â-glucuronidase type B-1 and 0.1 U of sulfatase type VI in 0.2 mL of
0.15 M acetate buffer (pH 5.0) (value IID). All enzymatic hydrolyses
were carried out at 37 °C for 2 h, followed by extraction with ethyl
acetate and analysis by HPLC as described above for method I. The
difference between value IIB and value IIA gave the amount of
glucuronides and the difference between value IIC and value IIA the
amount of sulfates. The sum of unconjugated material, glucuronides,
and sulfates derived from these calculations corresponded well with
value IID, which was also used for comparison with value IB.
RESULTS AND DISCUSSION
Metabolism of Curcuminoids in Liver Slices. Freshly
prepared liver slices from male and female Sprague-Dawley
rats were incubated with 200 µM curcumin for 24 h and the
culture media analyzed for curcumin and its phase I and phase
II metabolites. Authentic reference compounds were available
for the reductive curcumin metabolites tetrahydrocurcumin,
hexahydrocurcumin, and octahydrocurcumin, which differed in
their HPLC retention times (Table 1).
For confirmation by GC-MS analysis, the reductive metabolites
tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin gen-
erated from curcumin in liver slices were extracted with ethyl acetate
from the HPLC fractions obtained after enzymatic hydrolysis using
method I. The extract was then evaporated in vacuo and the residue
dissolved in methanol. Solvent was removed from an aliquot of the
sample under a stream of nitrogen, and trimethylsilyl (TMS) ether
derivatives were prepared by treatment with 25 µL of BSTFA at room
temperature for at least 16 h.
A representative HPLC profile of the phase I metabolites of
curcumin extracted from the incubation medium of a male liver
slice after hydrolysis of the conjugates is depicted in Figure 3.
The absorbance at 280 nm was selected because the known
reductive metabolites of curcumin do not absorb at 420 nm
(Table 1). In addition to curcumin, two major peaks were
observed, which eluted after 11.4 and 9.1 min. The peak at 11.4
min coeluted with synthetic hexahydrocurcumin and had an
identical UV-vis spectrum. When the HPLC peak was collected
and the metabolite extracted and analyzed by GC-MS, the GC
retention time and mass spectrum were the same as for authentic
hexahydrocurcumin. Likewise, the peak at 9.1 min coeluted with
one of the diastereomers of synthetic octahydrocurcumin,
whereas the other authentic diastereomer had a HPLC retention
time of 8.8 min and cochromatographed with the small peak
preceding the peak at 9.1 min in Figure 3. Collection of the
HPLC eluate from 8.5 to 9.5 min and analysis by GC-MS
confirmed the presence of the two diastereomers of octahy-
drocurcumin in a ratio of 1:3. The HPLC peaks at 11.4 and 9.1
min were not detectable in control incubations containing no
Incubation of Liver Subcellular Fractions. Microsomes and cytosol
were prepared from the livers of untreated male Sprague-Dawley rats
as described previously by Lake (32). Protein concentrations were
measured according to the method of Bradford (33) with bovine serum
albumin as standard. The concentration of active cytochrome P450
(CYP) was determined by using the method of Omura and Sato (34).
Oxidative in vitro metabolism of curcuminoids was studied by
incubating rat hepatic microsomes (1 mg of microsomal protein/mL)
with 100 µM substrate dissolved in DMSO (final concentration ) 1%)
and a NADPH-generating system (0.9 U of isocitrate dehydrogenase,
9.4 mM isocitrate, 1.21 mM NADP+, and 4.3 mM magnesium chloride)
in a final volume of 1 mL in 0.1 M phosphate buffer (pH 7.4). After
preincubation for 5 min at 37 °C, the NADPH-generating system was
added and the mixture incubated for 40 min at 37 °C. Subsequently,
the incubation mixture was extracted with 3 × 0.5 mL pf ethyl acetate
and the extract evaporated to dryness. The residue was dissolved in 50
µL of methanol for HPLC analysis. Control incubations contained
substrate but no NADPH-generating system. To ensure the activity of

760 J. Agric. Food Chem., Vol. 54, No. 3, 2006
Hoehle et al.
Figure 3. HPLC profile of the phase I metabolites of curcumin extracted
from the medium of a 24-h incubation of 200
of a male Sprague Dawley rat liver after conjugate hydrolysis with
-glucuronidase/arylsulfatase from H. pomatia. TMBPA, internal standard.
µM curcumin with the slice
−
â
curcumin or no liver slice. However, the incubation of curcumin
in the absence of a liver slice gave rise to the formation of
vanillin and ferulic acid, which are known products of the
chemical decomposition of curcumin (35) and which were also
observed in the HPLC profile of the complete incubation
(Figure 3).
Of the other minor peaks visible in Figure 3, only those
eluting after 23.3, 24.6, and 26.0 min were not present in the
HPLC profile of control incubations. The peak at 23.3 min
cochromatographed with synthetic tetrahydrocurcumin and had
the same UV-vis spectrum and mass spectrum upon GC-MS
analysis. The peaks at 24.6 and 26.0 min were tentatively
identified as isomers of dihydrocurcumin on the basis of their
UV-vis spectra, which were identical and corresponded to the
published spectrum for dihydrocurcumin in ethanol (29). The
formation of two isomers is probably due to the reduction of
either the allylic or the enolic double bond of curcumin (see
Figure 1).
No hints for oxidative metabolites of curcumin, for example,
products of aromatic hydroxylation or demethylation, were
obtained from the HPLC profiles, even when the detector was
set to 420 nm, which should be near the maximum of absorption
of oxidative metabolites due to the presence of the curcumin
chromophore. On the basis of the limit of detection (LOD) at
this wavelength, it is estimated that oxidative curcumin me-
tabolites would have been observed if at least 0.01% of the
curcumin were converted to such products.
When demethoxycurcumin and bis-demethoxycurcumin were
incubated with rat liver slices under the same conditions as
curcumin and the incubation media analyzed by HPLC, the
pattern of their phase I metabolites was very similar to that of
curcumin: the major metabolites were the hexahydro and
octahydro products, which were unambiguously identified by
cochromatography with the respective reference compounds in
HPLC and GC-MS analysis (Table 1). The other phase I
metabolites of demethoxycurcumin and bis-demethoxycurcumin
were tentatively identified as two dihydro and one tetrahydro
product, on the basis of their HPLC retention times and UV-
vis spectra. No evidence for the formation of oxidative
metabolites of the demethoxylated curcuminoids was provided
by HPLC analysis.
Figure 4. Pattern of reductive metabolites of curcumin, demethoxycur-
cumin, and bis-demethoxycurcumin in incubation media of liver slices from
male (top) and female (bottom) Sprague−Dawley rats after 24 h of
incubation with curcuminoids. For each metabolite, the proportion of
unconjugated (darker part of bar) and conjugated (lighter part) material
was determined using hydrolysis with
â-glucuronidase/arylsulfatase from
H. pomatia (method I). Data are expressed as percent of the sum of
recovered curcuminoid and metabolites and represent the mean of two
rats for each gender and three independently incubated slices per rat
liver.
used, the proportion of conjugates indicated in each column of
Figure 4 represents the sum of glucuronides and sulfates. The
conjugated hexahydro product was the predominating metabolite
of all three curcuminoids in liver slices from both genders,
whereas the two isomers of the dihydro metabolites were formed
only to a small extent. Interestingly, one isomer was completely
conjugated, whereas the other isomer appeared to be a poor
substrate for conjugation. The phase I metabolism of the three
curcuminoids suggested a slight gender difference: male rats
generated more octahydro metabolites but fewer tetrahydro
metabolites than female rats; all of these metabolites were
present as conjugates (Figure 4).
In addition to the metabolites, the amounts of parent cur-
cuminoids were quantified in the 24-h incubations with liver
slices (Table 2). Most notably, the parent compounds were
present only in unconjugated form, and the extent of metabolic
conversion of parent curcuminoids to reductive and conjugated
metabolites was in the range of 30-50%. When the sums of
recovered parent curcuminoids and their reductive metabolites
were compared to the amounts of curcuminoids added to the
incubations, it became apparent that major portions of the
incubated curcuminoids could not be accounted for. The
recoveries were only ∼50% for curcumin and demethoxycur-
cumin but ∼75% for bis-demethoxycurcumin with both male
A quantitative account of the phase I metabolites and their
extent of conjugation in liver slices from male and female rats
is given in Figure 4 for all three curcuminoids. As enzymatic
hydrolysis with â-glucuronidase/arylsulfatase (method I) was

Curcuminoid Metabolism in Rat Liver
J. Agric. Food Chem., Vol. 54, No. 3, 2006 761
Table 2. Recoveries of Parent Curcuminoids and Their Reductive Metabolites (Unconjugated and Conjugated Forms) from Incubations with Male
and Female Rat Liver Slices after Conjugate Hydrolysis with
rat
â-Glucuronidase/Arylsulfatase from H. pomatia
parent
compound
reductive
metabolites
total
recovery
metabolic
conversiona
compound
gender
curcumin
male
female
27.9
29.7
±
±
3.2
11.6
25.8
12.1
±
±
2.9
1.1
53.7
41.8
±
±
6.1
10.5
48.0
31.6
±
±
0.1
10.7
demethoxycurcumin
male
female
30.5
26.1
±
±
7.9
6.6
22.2
20.1
±
±
1.3
0.5
52.7
46.2
±
±
9.2
7.1
43.1
44.3
±
±
5.1
5.7
bis-demethoxycurcumin
male
female
48.8
46.4
±
±
16.0
9.7
27.4
28.2
±
±
1.1
4.1
76.3
74.6
±
±
17.1
13.8
37.5
38.1
±
±
7.0
1.5
a Metabolic conversion: percent of total recovered material present as reductive and conjugated metabolites. Data are expressed as percent of the administered amount
of curcuminoid and represent the mean range of two independent experiments.
±
Table 3. Pattern of Conjugates in the Medium of Liver Slices from Male and Female Rats after 24 h of Incubation with Curcumin,
Tetrahydocurcumin, and Hexahydrocurcumin
curcumin (200
male
µ
M)
tetrahydrocurcumin (200
µ
M)
hexahydrocurcumin (50 µM)
compound
curcumin
female
male
female
male
female
unconjugated
glucuronide
sulfate
26.3
16.5
9.2
±
±
±
5.7
1.2
2.5
50.6
13.2
6.7
±
±
±
14.0
1.3
0.6
na
na
na
na
na
na
na
na
na
na
na
na
tetrahydrocurcumin
hexahydrocurcumin
octahydrocurcumin
unconjugated
glucuronide
sulfate
nd
0.9
nd
nd
5.5
nd
4.4
2.0
nd
±
±
0.4
0.1
5.9
9.5
nd
±
±
1.0
0.8
na
na
na
na
na
na
±
0.1
±
0.1
unconjugated
glucuronide
sulfate
0.9
31.9
4.1
±
±
±
0.9
6.7
0.4
2.0
15.2
2.2
±
±
±
1.0
7.9
1.6
8.3
70.8
3.5
±
±
±
0.4
1.8
0.6
3.8
74.9
4.3
±
±
±
0.2
0.5
1.8
nd
42.2
14.6
nd
76.8
19.6
±
±
2.7
1.7
±
±
1.2
2.5
unconjugated
glucuronide
sulfate
nd
6.5
nd
nd
0.7
nd
2.0
9.0
nd
±
±
0.4
2.7
nd
1.7
nd
8.6
34.6
nd
±
±
2.0
1.0
2.1
1.6
nd
±
±
2.1
1.6
±
2.1
±
0.7
±
1.7
a na, not applicable; nd, not detectable, <0.03% for tetrahydrocurcumin and <0.25% for hexahydrocurcumin and octahydrocurcumin. The limit of quantification was the
3-fold value of the LOD. Data are expressed as percent of all detected compounds (parent compound plus metabolites) and represent the mean
determination of three independently incubated slices per rat.
± range of a duplicate
and female liver slices. Thus, about half of the curcumin and
demethoxycurcumin, but only 25% of the bis-demethoxycur-
cumin, administered to the liver slices had “disappeared”. About
the same losses were noted when the individual curcuminoids
were incubated for 24 h with the medium in the absence of
liver slices. Disappearance of curcumin and demethoxycurcumin
and, to a lesser extent, bis-demethoxycurcumin was also
observed when the curcuminoid mixture from turmeric, consist-
ing of 75% curcumin, 18% demethoxycurcumin, and 7% bis-
demethoxycurcumin, was incubated for 24 h without liver slices,
resulting in a shift of the composition from curcumin toward
bis-demethoxycurcumin. The observed loss of curcuminoids is
in agreement with previous studies showing that these com-
pounds are unstable in aqueous solution, with curcumin being
the least and bis-demethoxycurcumin the most stable curcumi-
noid (35, 36). Indeed, when the 24-h incubations of curcumin
were analyzed by HPLC for the presence of known degradation
products, vanillin and ferulic acid were detected both in the
absence and presence of liver slices (Figure 3).
tions of curcumin of <200 µM resulted in lower recoveries of
metabolites and parent compound.
Detailed Investigation of Curcumin Metabolism in Liver
Slices. To further study the reductive metabolites and conjugates
of curcumin in rat liver slices, 24-h incubations of curcumin,
tetrahydrocurcumin, and hexahydrocurcumin were conducted
in liver slices of male and female Sprague-Dawley rats. A
modification of the enzymatic hydrolysis was used, involving
separate hydrolysis with â-glucuronidase and sulfatase and a
hydrolysis time of only 2 h (method II). The results of these
experiments are summarized in Table 3.
With 200 µM curcumin, the major phase I metabolites again
were hexahydrocurcumin, tetrahydrocurcumin, and octahy-
drocurcumin. As in the earlier study (Figure 4), liver slices from
male rats generated more octahydrocurcumin and liver slices
from female rats more tetrahydrocurcumin. The preferred
pathway for conjugation of all three major reductive curcumin
metabolites was glucuronidation, but sulfates were also formed
in significant amounts (Table 3). Large amounts of the
glucuronide and sulfate were also found for curcumin itself,
which is in clear contrast to the earlier study in which the parent
curcuminoids were present in the 24-h incubations only in
unconjugated form.
The issue of chemical instability of curcuminoids raises
questions about the incubation conditions, in particular, with
respect to the incubation time. Pilot studies with slices from
male rat liver showed that short-term incubations of 2, 4, and
6 h gave rise to the same pattern of phase I and II metabolites
as observed after 24 h, but the sum of metabolites was smaller
and the proportion of unmetabolized curcumin higher, as was
the total recovery. For example, the metabolic conversion rate
was 17% and the recovery 69% in 6-h incubations as compared
with 48 and 54%, respectively, in 24-h incubations. Concentra-
When 200 µM tetrahydrocurcumin was incubated for 24 h,
little parent compound but large amounts (>80%) of hexahy-
drocurcumin together with small quantities of octahydrocur-
cumin were found in the incubation media of male and female
slices (Table 3). Again, male rat liver formed more octahydro
metabolites than female liver, and glucuronides were predomi-

762 J. Agric. Food Chem., Vol. 54, No. 3, 2006
Hoehle et al.
−Dawley Rats and with Alcohol Dehydrogenase from
Table 4. Metabolic Studies with Microsomes and Cytosol from the Liver of Male Sprague
Equine Liver
metabolic system
microsomes/NADPHa
substrate
workup
result of HPLC analysisd
curcumin from turmericb
extractionc
extractionc
extractionc
parent curcuminoids, no oxidative metabolites, traces
of hexahydrocurcuminoids
reductive metabolites of curcuminoids (hexahydro >
alcohol dehydrogenase/NADH
cytosol/NADH
curcumin from turmericb
curcumin from turmericb
tetrahydro
reductive metabolites of curcuminoids (hexahydro >
tetrahydro octahydro dihydro-1 and dihydro-2)
= octahydro . dihydro-1 and dihydro-2)
=
.
microsomes/UDPGA
microsomes/UDPGA
pure curcumin
pure curcumin
precipitation of proteins
large peaks of curcumin and of curcumin-glucuronide
(at 16.6 min)
large peak of curcumin, but less than the sum of
curcumin and curcumin-glucuronide
precipitation of proteins, then
hydrolysis by method I
detected prior to hydrolysis
cytosol/NADH plus microsomes/UDPGA
cytosol/NADPH plus microsomes/UDPGA
pure curcumin
pure curcumin
precipitation of proteins
large peaks of curcumin and curcuminglucuronide,
small peak of unconjugated hexahydrocurcumin
large peaks of curcumin and of hexahydro-, tetrahydro-,
and octahydrocurcumin
precipitation of proteins, then
hydrolysis by method I
a NADPH-generating system. b Consisting of 75% curcumin, 18% demethoxycurcumin, and 7% bis-demethoxycurcumin. c With ethyl acetate. d LOD was 1 pmol for
curcuminoids, 3 pmol for tetrahydrocurcuminoids, and 5 pmol for hexahydro- and octahydrocurcuminoids.
nating. The metabolic conversion rate and total recovery of
parent compound plus metabolites were much higher for
tetrahydrocurcumin (>90%) than for curcumin (∼50%).
The higher capacity of the male versus the female rat liver
for the reduction of hexahydrocurcumin to octahydrocurcumin
was again obvious when 50 µM hexahydrocurcumin was
incubated for 24 h (Table 3): 90% of hexahydrocurcumin,
although mostly glucuronidated, was still present in slices from
female rat liver, but only ∼50% was found for males. Total
recoveries were near 100% for both genders. The comparison
of the metabolism of tetrahydrocurcumin and hexahydrocur-
cumin emphasizes the fact that tetrahydrocurcumin is avidly
reduced to hexahydrocurcumin by both male and female rat
liver, although female liver is somewhat less efficient. The
further reduction to octahydrocurcumin is less efficient in both
genders, especially in females. Glucuronidation is preferred over
sulfation for the conjugation of the reductive metabolites.
Furthermore, hints for a sulfoglucuronide of hexahydrocurcumin
were obtained: when the amounts of hexahydrocurcumin
released by separate hydrolysis with â-glucuronidase and
sulfatase were added, the sum was always less than the amount
of hexahydrocurcumin liberated by hydrolysis with a mixture
of the two enzymes (data not shown).
Metabolism of Curcuminoids in Subcellular Fractions.
Because no products of aromatic hydroxylation or demethylation
were observed in liver slices, the ability of rat liver to form
oxidative metabolites of curcuminoids was probed with rat
hepatic microsomes. Furthermore, because of the observed
discrepancy in the amount of curcumin conjugates found after
enzymatic cleavage with â-glucuronidase/arylsulfatase from H.
pomatia (hydrolysis method I: no conjugated curcumin ob-
served) and with pure â-glucuronidase and sulfatase (hydrolysis
method II: glucuronides and sulfates of curcumin observed),
the glucuronidation of curcumin and its reductive metabolites
was studied with rat hepatic microsomes. The experiments and
corresponding results are listed in Table 4.
of curcumin were converted to an oxidative metabolite, assum-
ing the same absorbance as for curcumin. For reductive
metabolites, the LOD would have indicated a conversion of
0.05% of the administered curcumin. Thus, neither oxidative
nor reductive curcumin metabolites were formed to any
significant extent by rat liver microsomes. The monooxygenese
activity of the microsomes was ensured by incubations with
testosterone, which gave rise to the expected pattern of oxidative
testosterone metabolites as described previously by Pfeiffer et
al. (30).
In contrast to the failure to observe oxidative or reductive
curcumin metabolites in microsomal incubations, the full
spectrum of reductive phase I metabolites was formed when
curcuminoids were incubated with an alcohol dehydrogenase
preparation or with rat liver cytosol in the presence of NADH
(Table 4).
For the experiments on microsomal glucuronidation, pure
curcumin was used, and the incubation medium after precipita-
tion of proteins was analyzed by HPLC without extraction to
avoid the loss of glucuronides. Incubation of microsomes with
curcumin in the presence of UDPGA clearly gave rise to a novel
product eluting earlier than curcumin from the HPLC column
and most likely representing a glucuronide of curcumin, because
it disappeared after hydrolysis with â-glucuronidase/arylsulfatase
from H. pomatia (Table 4). However, the area of the curcumin
peak observed after hydrolysis was clearly smaller than the sum
of the areas for the curcumin peak and curcumin-glucuronide
peak determined prior to hydrolysis. When curcumin was
incubated with cytosol plus microsomes and the respective
cofactors, curcumin-glucuronide together with small amounts
of unconjugated reductive curcumin metabolites were detected
by HPLC prior to hydrolysis; after cleavage of conjugates, the
peaks of the reductive metabolites increased markedly. It is
assumed that the reductive metabolites are extensively glucu-
ronidated but that these glucuronides eluted with the solvent
front under the HPLC conditions used.
When microsomes were incubated with a mixture of the three
curcuminoids in the presence of a NADPH-generating system
for 40 min, only unchanged curcuminoids and traces of the
hexahydro metabolites were detectable in the ethyl acetate
extract by HPLC with diode array detection, using detector
wavelengths suitable for both curcumin and its reductive
metabolites. Our HPLC method would have detected if 0.01%
Conclusion. Our study on the metabolism of curcumin in
precision-cut liver slices from male and female Sprague-
Dawley rats and with hepatic microsomes and cytosol has
confirmed earlier reports from other laboratories (16, 20, 21,
37) that reduction of the aliphatic moiety is the only pathway
in phase I metabolism. No evidence for the formation of
hydroxylation or demethylation products of curcumin was

Curcuminoid Metabolism in Rat Liver
J. Agric. Food Chem., Vol. 54, No. 3, 2006 763
obtained in our experiments. Although no reference substances
for such oxidative curcumin metabolites were at hand, they
should have been detectable in our HPLC system due to their
putative similarity of absorbance with curcumin and their
expected retention times, which should be somewhat shorter
than that of curcumin on the reversed-phase column. The major
reductive curcumin metabolite formed in rat liver slices and in
hepatic cytosol was hexahydrocurcumin, together with smaller
amounts of tetrahydrocurcumin and octahydrocurcumin. This
is consistent with the report by Holder et al. (16) that
glucuronides of hexahydrocurcumin and tetrahydrocurcumin are
the major biliary metabolites after intraveneous injection of
tritium-labeled curcumin to bile duct-cannulated male Sprague-
Dawley rats. After intraperitoneal injection of curcumin into
female BALB/c mice, glucuronides of tetrahydrocurcumin and
dihydrocurcumin were the major phase I metabolites in plasma
(37). Ireson et al. (20) reported that curcumin is metabolized to
hexahydrocurcumin and octahydrocurcumin in hepatocytes from
male and female Fischer 344 rats. This laboratory also studied
the reductive metabolism of curcumin in more detail and
observed that cytosol or alcohol dehydrogenase is required for
the formation of tetrahydrocurcumin and hexahydrocurcumin,
but microsomes are needed for the reduction of hexahydrocur-
cumin to octahydrocurcumin (21).
separately hydrolyzed by â-glucuronidase and sulfatase, respec-
tively, for 2 h only (Table 3), in agreement with the formation
of curcumin-glucuronide and curcumin-sulfate reported by
others (19-21, 37). This discrepancy is most likely due to the
chemical instability of curcumin. The spontaneous time- and
pH-dependent degradation of curcumin in aqueous systems has
been previously described by Wang et al. (35). It has also been
reported that curcumin was more stable in aqueous media in
the presence of fetal calf serum and in blood than in pure buffer
(35, 36). Obviously, the curcumin released from its glucuronide
and sulfate in our study had completely decomposed after 16 h
of hydrolysis but was still present to a significant extent after
2 h of hydrolysis. In contrast to curcumin, a large proportion
of the major reductive metabolites was found in the 16-h
incubation, indicating their higher chemical stability as compared
to curcumin. It has previously been reported by Pan et al. (37)
that tetrahydrocurcumin is much more stable in aqueous buffer
than curcumin.
The instability of free curcumin may be one of the reasons
for the poor recovery observed in our incubations of the
curcuminoids, which were about 50% for curcumin and
demethoxycurcumin and 75% for bis-demethoxycurcumin. The
better recovery for bis-demethoxycurcumin implies that this
curcuminoid has a higher chemical stability, which has also been
reported earlier (36).
No reports have been published in the literature to date on
the phase I metabolism of demethoxycurcumin and bis-
demethoxycurcumin. Our study has demonstrated for the first
time that these curcuminoids follow the same pathways as
curcumin: no oxidative metabolites were observed, and the
pattern of the reductive products was about the same as obtained
with curcumin. For the major reductive metabolites of demethoxy-
curcumin and bis-demethoxycurcumin, the EI mass spectra
obtained by GC-MS analysis of the trimethylsilylated metabo-
lites provided characteristic features suitable for unambiguous
identification (Table 1). For example, the mass spectra of the
hexahydro metabolites of all three curcuminoids exhibit very
small molecular ions and the loss of 90 mass units from the
molecular ions, which is due to the elimination of trimethylsi-
lanol. Other characteristic and abundant ions in the mass spectra
arise from cleavage of the benzylic bonds. The mass spectra of
the octahydrocurcuminoids indicate two successive eliminations
of trimethylsilanol, together with fragmentations at the benzylic
positions. Another prominent fragmentation of the octahy-
drocurcuminoids, leading to m/z 262 and 232, occurs in the
aliphatic chain after the loss of the two trimethylsilanol
molecules.
The chemical instability of curcumin may also explain, at
least in part, the failure of clinical studies to detect significant
concentrations of curcumin in the plasma and urine of patients
even after heroic doses of curcumin. For example, maximum
plasma levels of only 1.8 µM curcumin were observed after an
oral dose of 8 g of curcumin, and no curcumin was detectable
in urine (22). Following oral doses of 180 mg of curcumin for
up to 4 months, no curcumin was detectable in plasma or urine
(23). In a more recent study by Sharma et al. (25) with colorectal
cancer patients receiving daily oral doses of 3.6 g of curcumin
for up to 4 months, maximum blood plasma levels in the range
of 11, 16, and 9 nmol/L were measured for curcumin, curcumin-
glucuronide, and curcumin-sulfate, respectively. In the urine of
the same patients, maximum concentrations of curcumin,
curcumin-glucuronide, and curcumin-sulfate were on the order
of 1, 500, and 40 nmol/L. From the very low levels of curcumin
and its conjugates in blood and urine, it is currently concluded
that curcumin possesses poor systemic bioavailability after oral
dosing. To corroborate this assumption, it is proposed to use
the urinary excretion of the glucuronides of hexahydrocurcumin
and octahydrocurcumin as markers for systemic bioavailability
in future clinical studies. These major metabolites of curcumin
have the distinct advantage of being much more chemically
stable than curcumin and will also account for the extensive
metabolic bioreduction of this important curcuminoid.
In addition to bioreduction, curcumin and its reductive
metabolites undergo conjugation with glucuronic acid and sulfate
in liver slices. The formation of such conjugates has been
reported by others before, for example, by Holder et al. (16) in
the bile of bile duct-cannulated rats, by Pan et al. (37) in the
blood plasma of mice, and by Ireson et al. (20) in rats in vivo
and rat hepatocytes in vitro. The evidence for the type of
conjugate was mostly based on cleavage with either â-glucu-
ronidase or sulfatase, but in some cases mass spectra of the
glucuronide or sulfate were obtained by LC-MS/MS (20, 21,
37).
ABBREVIATIONS USED
BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; NADH,
nicotinamide dinucleotide, reduced form; NADPH, nicotinamide
dinucleotidephosphate, reduced form; TMS, trimethylsilyl; U,
enzyme unit; UDPGA, uridine-5′-diphosphoglucuronic acid;
LOD, limit of detection.
In our study, no conjugates of the parent curcuminoids but
large amounts of conjugates of the reductive metabolites were
observed when the hydrolysis of conjugates was conducted with
a â-glucuronidase/arylsulfatase preparation from H. pomatia for
16 h (Figure 4). However, significant amounts of glucuronides
and sulfates were found both of the reductive metabolites and
of parent curcumin when glucuronides and sulfates were
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Received for review September 6, 2005. Revised manuscript received
November 17, 2005. Accepted November 17, 2005. This study was, in
part, supported by NIH Grant P50 AT00474 to the Arizona Center
for Phytomedicine Research.
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JF058146A