Autoxidative and cyclooxygenase-2 catalyzed transformation of the dietary chemopreventive agent curcumin

Article abstract of DOI:10.1074/jbc.M110.178806

The efficacy of the diphenol curcumin as a cancer chemopreventive agent is limited by its chemical and metabolic instability. Non-enzymatic degradation has been described to yield vanillin, ferulic acid, and feruloylmethane through cleavage of the heptadienone chain connecting the phenolic rings. Here we provide evidence for an alternative mechanism, resulting in autoxidative cyclization of the heptadienone moiety as a major pathway of degradation. Autoxidative transformation of curcumin was pH-dependent with the highest rate at pH 8 (2.2 μM/min) and associated with stoichiometric uptake of O ₂. Oxidation was also catalyzed by recombinant cyclooxygenase-2 (COX-2) (50 nM; 7.5 μM/min), and the rate was increased ≈10-fold by the addition of 300 μM H₂O₂. The COX-2 catalyzed transformation was inhibited by acetaminophen but not indomethacin, suggesting catalysis occurred by the peroxidase activity. We propose a mechanism of enzymatic or autoxidative hydrogen abstraction from a phenolic hydroxyl to give a quinone methide and a delocalized radical in the heptadienone chain that undergoes 5-exo cyclization and oxygenation. Hydration of the quinone methide (measured by the incorporation of O-18 from H₂¹⁸O) and rearrangement under loss of water gives the final dioxygenated bicyclopentadione product. When curcumin was added to RAW264.7 cells, the bicyclopentadione was increased 1.8-fold in cells activated by LPS; vanillin and other putative cleavage products were negligible. Oxidation to a reactive quinone methide is the mechanistic basis of many phenolic anti-cancer drugs. It is possible, therefore, that oxidative transformation of curcumin, a prominent but previously unrecognized reaction, contributes to its cancer chemopreventive activity.

Full text of DOI:10.1074/jbc.M110.178806

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 2, pp. 1114–1124, January 14, 2011
2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
©
Autoxidative and Cyclooxygenase-2 Catalyzed
Transformation of the Dietary Chemopreventive Agent
□
S
*
Curcumin
Received for publication, August 25, 2010, and in revised form, November 10, 2010 Published, JBC Papers in Press, November 11, 2010, DOI 10.1074/jbc.M110.178806
‡2
‡
1
‡
‡
‡
§
Markus Griesser , Valentina Pistis , Takashi Suzuki , Noemi Tejera , Derek A. Pratt , and Claus Schneider
‡
From the Department of Pharmacology and Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232 and the Department of Chemistry, Queen’s University, Kingston, Ontario KL7 3N6, Canada
§
The efficacy of the diphenol curcumin as a cancer chemo-
Altogether more than one hundred molecular targets of cur-
preventive agent is limited by its chemical and metabolic insta- cumin have been identified using in vitro cell culture-based
bility. Non-enzymatic degradation has been described to yield
vanillin, ferulic acid, and feruloylmethane through cleavage of
the heptadienone chain connecting the phenolic rings. Here
assays (2). The diversity of biological effects of curcumin has
been attributed to its ability to act as an antioxidant, anti-in-
flammatory, and anti-viral agent (3). As a consequence of
we provide evidence for an alternative mechanism, resulting in promising in vitro results, several clinical trials have been ini-
autoxidative cyclization of the heptadienone moiety as a major tiated to investigate the effect of dietary curcumin in the pre-
pathway of degradation. Autoxidative transformation of cur-
cumin was pH-dependent with the highest rate at pH 8 (2.2
vention of inflammatory bowel disease, colon, and pancreatic
cancer, and Alzheimer Disease, among others (3).
␮
M/min) and associated with stoichiometric uptake of O . Oxi-
Prior to the more recent interest in its chemopreventive
properties, curcumin was being considered as a food coloring
agent but its chemical and photochemical instability pre-
vented widespread application. Light-induced degradation of
curcumin in organic solvents results in cleavage of the hepta-
dienone chain, and the most abundant products have been
identified as vanillin, ferulic aldehyde, ferulic acid, and feru-
loylmethane (4, 5). The same products have been observed as
degradation products of curcumin in aqueous buffer solutions
(6, 7). The identification of the products, however, was partly
based on HPLC retention times and monitoring using fixed
wavelength UV detection. Besides the chain cleavage products
there is evidence of additional, prominent products in the
literature but these were left uncharacterized (8, 9). Degrada-
tion of curcumin is also a prominent reaction upon addition
to cultured cells (7), and therefore, defining the products and
kinetics of degradation of curcumin is relevant to the question
whether metabolites could be mediating some of the biologi-
cal activities, an issue that has been raised frequently in recent
2
dation was also catalyzed by recombinant cyclooxygenase-2
COX-2) (50 nM; 7.5 ␮M/min), and the rate was increased ≈10-
fold by the addition of 300 ␮M H O . The COX-2 catalyzed
(
2
2
transformation was inhibited by acetaminophen but not indo-
methacin, suggesting catalysis occurred by the peroxidase ac-
tivity. We propose a mechanism of enzymatic or autoxidative
hydrogen abstraction from a phenolic hydroxyl to give a qui-
none methide and a delocalized radical in the heptadienone
chain that undergoes 5-exo cyclization and oxygenation. Hy-
dration of the quinone methide (measured by the incorpora-
18
tion of O-18 from H O) and rearrangement under loss of
2
water gives the final dioxygenated bicyclopentadione product.
When curcumin was added to RAW264.7 cells, the bicyclo-
pentadione was increased 1.8-fold in cells activated by LPS;
vanillin and other putative cleavage products were negligible.
Oxidation to a reactive quinone methide is the mechanistic
basis of many phenolic anti-cancer drugs. It is possible, there-
fore, that oxidative transformation of curcumin, a prominent
but previously unrecognized reaction, contributes to its cancer years, e.g. Refs. 3, 10–12.
chemopreventive activity.
One of the suggested chemopreventive mechanisms of cur-
cumin is the suppression of prostaglandin formation by the
3
cyclooxygenase-2 (COX-2) enzyme (13, 14). Induction of
The yellow plant phenolic pigment curcumin shows a re-
markable ability to affect a wide variety of signaling pathways
that are dysregulated during tumorigenesis, including prolif-
eration, invasion, apoptosis, and cell cycle checkpoints (1).
COX-2 and the formation of PGE are hallmarks in the devel-
2
opment of many cancers, best studied in the case of colon
carcinogenesis. Suppression of COX-2 activity by selective or
non-selective inhibitors of the cyclooxygenase enzymes leads
to a significant reduction of colon cancer in animal models
and in human populations with a genetically elevated cancer
risk (15). Adverse cardiovascular side effects due to inhibition
*
This work was supported, in whole or in part, by National Institutes of
Health Pilot and Feasibility Grant P30 ES000267 from the Center of Mo-
lecular Toxicology and Grant R01GM076592 from the NIGMS.
S
The on-line version of this article (available at http://www.jbc.org) con-
tains supplemental Figs. S1–S3.
□
3
The abbreviations used are: COX, cyclooxygenase; ABTS, [2,2Ј-azinobis-(3-
1
Present address: Consumer Safety, Crop Protection, BASF SE, Germany.
To whom correspondence should be addressed: Dept. of Pharmacology,
Vanderbilt University School of Medicine, 23rd Ave. S. at Pierce, Nashville,
TN 37232-6602. Tel.: 615-343-9539; Fax: 615-322-4707; E-mail: claus.
schneider@vanderbilt.edu.
ethyl-benzothiazoline-6-sulphonic acid; ApAP, acetaminophen (N-acetyl-
p-aminophenol); CID, collision-induced dissociation; ESI, electrospray
ionization; HRMS, high resolution mass spectrometry; LC-MS, liquid chro-
matography-mass spectrometry; LOX, lipoxygenase; LPS, lipopolysaccha-
ride; PG, prostaglandin; SPLET, sequential proton loss electron transfer.
2
1
114 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidative Transformation of Curcumin
of COX-2 in the vascular endothelium substantially limit the
dimethoxycurcumin. The product was filtered from solvent
usefulness of these drugs for cancer treatment. Dietary curcu- and allowed to air dry (93% pure by HPLC-DAD analysis; m.p.
ϩ
min, in contrast, with its low systemic bioavailability but high
concentration in the small intestine and colon (16), may pro-
vide suitable pharmacokinetic properties to target COX-2
specifically in the gastrointestinal tract while sparing other
organs (17).
128 °C) HRMS (ES ) calcd for C H O H 397.1646, found
2
3
24
6
1
397.1648. H NMR, 600 MHz, CD CN (␦ 1.93): ␦ 7.58 (d, J ϭ
3
15.8 Hz, H1, 2H), ␦ 7.24 (d, J ϭ 1.7 Hz, ar-H2, 2H), ␦ 7.20 (dd,
J ϭ 8.3/1.8 Hz, ar-H6, 2H), ␦ 6.96 (d, J ϭ 8.3 Hz, ar-H5, 2H), ␦
6.70 (d, J ϭ 15.8 Hz, H2, 2H), ␦ 5.93 (s, H4, 1H), ␦ 3.85 (s,
The interactions of curcumin with arachidonic acid metab- -OCH , 3H), ␦ 3.84 (s, -OCH , 3H).
3
3
olism and prostaglandin biosynthesis are remarkably com-
plex: curcumin has been shown to down-regulate the expres-
Synthesis of 4Ј-Methoxycurcumin—The same procedure as
for synthesis of 4Ј,4Љ-dimethoxycurcumin was used. Instead of
sion of COX-2 mRNA and protein (18, 19) and also to directly 3 g of 3,4-dimethoxybenzaldehyde, 1.5 g (10 mmol) each of
inhibit the enzymatic activities of COX-2 (20) and of
mPGES-1, one of the terminal prostaglandin synthases that
converts the COX-derived unstable PGH to PGE (21). In
vanillin and 3,4-dimethoxybenzaldehyde were used. After
extraction of the reaction mixture with ethyl acetate the crude
products were purified using preparative thin layer chroma-
tography developed with a solvent of chloroform/EtOH (25:1,
by vol). Regions of the plate containing 4Ј-methoxycurcumin
were scraped off and extracted with ethyl acetate. A 5 mM
2
2
addition, curcumin has been shown to inhibit the enzymatic
activity of 5-lipoxygenase, the enzyme that initiates biosyn-
thesis of leukotrienes from arachidonic acid (22–24), and,
unexpectedly, to be a substrate for the lipoxygenase from soy- stock solution was prepared in MeOH and stored at Ϫ20 °C
ϩ
bean seeds (25). Here we analyzed the chemical mechanism
underlying a previously unrecognized oxidative transforma-
tion of curcumin that is in stark contrast to the published lit-
erature describing cleavage into smaller fragments as the ma-
jor non-enzymatic transformation of curcumin.
(90% pure by HPLC-DAD analysis; HRMS (ES ) calculated
for C H O H 383.1489, found 383.1482). ESI-MS: m/z
2
2
22
6
Ϫ
1
381.25 ([M-H] ). H NMR, 600 MHz, CD COCD (␦ 2.04): ␦
3
3
7.59 (d, J ϭ 15.8 Hz, H1&H7, 2H), ␦ 7.32 (d, J ϭ 1.6 Hz, H2Љ,
1H), ␦ 7.31 (d, J ϭ 1.6 Hz, H2Ј, 1H), ␦ 7.22 (dd, J ϭ 8.3/1.7 Hz,
H6Ј, 1H), ␦ 7.17 (dd, J ϭ 8.2/6.1 Hz, H6Љ, 1H) ␦ 7.00 (d, J ϭ 8.3
Hz, H5Ј, 1H), ␦ 6.88 (d, J ϭ 8.1 Hz, H5, 1H), ␦ 6.73 (d, J ϭ
15.8, H2, 1H), ␦ 6.69, (d, J ϭ 15.8, H6, 1H), ␦ 5.98 (s, H4, 1H),
␦ 3.91 (s, 3Љ-OCH , 3H), ␦ 3.87 (s, 3Ј-OCH , 3H), ␦ 3.86 (s,
EXPERIMENTAL PROCEDURES
Materials—Curcumin was synthesized according to the
method of Pabon (26) and purified by crystallization from
ethyl acetate/MeOH at Ϫ20 °C (90% pure by HPLC-DAD
3
3
4Ј-OCH , 3H).
3
ϩ
analysis; HRMS (ES ) calcd for C H O H 369.1333, found
Autoxidative Transformation of Curcumin—Curcumin was
diluted to a concentration of 70 ␮M in 500 ␮l of 100 mM Tris-
21
20
6
3
69.1324). A 5 mM stock solution in ethanol was prepared
fresh daily. Hexahydrocurcumin was prepared by reduction of HCl buffer pH 8 in a cuvette. The sample was scanned repeti-
curcumin with H in the presence of Pd/Al in ethanol. Vanil-
tively from 700 to 220 nm in 2-min intervals using a Perkin
Elmer PE35 UV/Vis spectrophotometer. When Ͼ70% of the
absorbance at 430 nm had disappeared the sample was acidi-
fied to pH 4 using 1 N HCl and loaded on a preconditioned 30
mg of Waters HLB cartridge. After washing with water the
2
lin, ferulic acid, indomethacin, and acetaminophen were ob-
tained from Sigma. Feruloylmethane was from ICI. Oxy-
18
gen-18 labeled water (97 atom-% O) was obtained from
Isotec. Recombinant His -tagged human COX-2 was ex-
6
pressed in the baculovirus/Sf9 insect cells system and purified cartridge was eluted with 2 ϫ 500 ␮l of methanol. The solvent
by Ni-NTA affinity chromatography as described (27). The
was evaporated under a stream of nitrogen, and the residue
concentration of the purified COX-2 stock solution was 5 ␮M. was dissolved in 50 ␮l of MeOH for RP-HPLC analysis. For
Ovine COX-1 was obtained from Cayman Chemical.
RAW264.7 were obtained from ATCC.
product identification, 20 separate 2-ml reactions were con-
ducted in parallel using 100 ␮M curcumin in the same buffer
and a reaction time of 30 min. Extraction was performed as
described above.
Synthesis of 4Ј,4Љ-Dimethoxycurcumin—The procedure fol-
lowed the method of Pabon for the synthesis of curcumin
(
(
26). Boron oxide (B O ), 0.5 g (7 mmol), was mixed with 1 g
COX-2 Catalyzed Transformation of Curcumin—COX-2, 5
␮l (50 nM final), was diluted in a cuvette using 500 ␮l of 100
mM K-phosphate buffer pH 9 containing 1 ␮M hematin for
reconstitution of the holoenzyme. Curcumin (100 ␮M) was
added, and the reaction was followed at 430 nm using the
spectrophotometer in the time drive mode. In some of the
reactions, 5 nM COX-2 and 300 ␮M of H O were added. For
2
3
10 mmol) of acetylacetone and stirred for 3 h to give a thick
white suspension. The compound was added to a mixture of
3
g (20 mmol) of 3,4-dimethoxybenzaldehyde and 9.2 g (40
mmol) tributylborate dissolved in 10 ml of ethyl acetate. The
entire mixture was stirred for 5 min, and then 4 aliquots of 50
␮
l of butylamine were added over the course of 30 min (total
2
2
of 200 ␮l; 2 mmol), followed by stirring for 4 h and standing
overnight. The next day, 15 ml of 0.4 M HCl (heated to 60 °C)
were added, and the mixture was stirred for 1 h. The organic
layer was collected, and the aqueous phase was extracted
twice with ethyl acetate. The combined organic phases were
washed with water and dried over sodium sulfate. The organic
phase was reduced to 10 ml under a stream of nitrogen, and
reactions in the presence of the inhibitors (indomethacin and
acetaminophen) the enzyme, hematin, and inhibitors were
preincubated for 1 min before addition of curcumin to start
the reaction. All enzymatic transformations were conducted
in the absence of arachidonic acid and phenol.
Oxygen Electrode Assay—A Clark-type oxygen electrode
(Hansatech Instruments, obtained through PP Systems,
Amesbury, MA) was maintained at 30 °C using a circulating
1
0 ml of MeOH were added to induce crystallization of 4Ј,4Љ-
JANUARY 14, 2011•VOLUME 286•NUMBER 2
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Oxidative Transformation of Curcumin
water bath. The calibration was performed using air-saturated ucts were analyzed by RP-HPLC with diode array detection as
distilled water and sodium dithionite assuming 235 ␮M for the described below.
maximum concentration of oxygen. 1 ml of Na-phosphate/
HPLC and LC-MS Analyses—The transformation reactions
citrate buffer pH 8.0 was added to the chamber and allowed to were analyzed using a Waters Symmetry C18 5-␮m column
equilibrate for 5 min before addition of 12 ␮l of curcumin
(4.6 ϫ 250 mm) eluted with a gradient of 20% acetonitrile to
80% acetonitrile in 0.01% aqueous acetic acid over 20 min
followed by 10 min of isocratic elution at a flow rate of 1 ml/
min. Elution of the products was monitored using an Agilent
1200 diode array detector.
from a 5 mM stock in ethanol or the same amount of ethanol.
18
Reaction in H O—Autoxidative transformation of curcu-
2
min was conducted using 100 ␮l of buffer. 5 ␮l of a 1 M stock
solution of Tris-HCl buffer pH 8 in water were diluted with
1
8
18
9
5 ␮l of H O (97 atom-% O). Curcumin, 3 ␮l from a 5 mM
For LC-MS analyses a Finnigan TSQ Quantum triple qua-
drupole MS instrument equipped with an electrospray inter-
face was used. The instrument was operated in the negative
ion mode, and mass spectra were acquired at a rate of 2
s/scan. The heated capillary was set at 300 °C, and the spray
voltage was set at 4.4 kV. For LC-MS analysis of oxygen incor-
2
stock solution in ethanol, was added, and the reaction was
allowed to proceed for 30 min followed by acidification to pH
4
using 1 N HCl and extraction using a HLB cartridge as de-
scribed above. A reaction using unlabeled H O was con-
2
ducted in parallel. In a control reaction curcumin was autoxi-
18
18
n
dized in 100 ␮l of buffered H O, extracted, and then
poration from O-buffer (MS analyses), a Thermo Finnigan
2
incubated in unlabeled water for 60 min followed by acidifica- LTQ ion trap instrument was used. The settings for the
18
tion and extraction. Only a minimal exchange of O content
of the main oxidized product was observed.
heated capillary (300 °C), spray voltage (4.0 kV), spray current
(0.22 ␮A), auxiliary (37 mTorr) and sheath gas (16 mTorr)
were optimized using direct infusion of a solution of curcu-
min (20 ng/␮l) in acetonitrile/water 95/5, by vol., containing
1
8
Reaction of 4Ј-Methoxycurcumin in H O—The transfor-
2
mation of 4Ј-methoxycurcumin was conducted in the pres-
ence of COX-2. 5 ␮l of a 1 M Tris-HCl pH 8 stock solution in
10 mM NH OAc. Samples were introduced into both instru-
4
18
18
water were diluted with 95 ␮l of H O (97 atom-% O) and
ments using a Waters Symmetry Shield C18 3.5-␮m column
2
5
␮l of COX-2 and 1 ␮M heme (final) were added. After 2
(2.1 ϫ 100 mm) eluted with a gradient of acetonitrile/water
min, 100 ␮M 4Ј-methoxycurcumin and 300 ␮M H O were
(5/95, by vol., containing 10 mM NH OAc) to acetonitrile/
2
2
4
added. After 10 min of reaction time, a second 5 ␮l aliquot of
COX-2 was added, and the reaction was allowed to proceed
for additional 60 min before acidification and extraction as
described above. After elution from the HLB cartridge and
evaporation of MeOH, the sample was dissolved in 50 ␮l of
water (95/5, by vol., containing 10 mM NH OAc) over 10 min
4
followed by 3 min of isocratic elution and re-equilibration in
the starting solvent.
For HRMS a Waters Synapt hybrid quadrupole orthogonal
access time-of-flight mass spectrometer was used. Samples
MeOH, and analyzed by LC-ESI-MS using a Thermo Finnigan were analyzed in the positive mode using a dual ESI/CI ion
LTQ ion trap instrument.
source with lock spray channel.
Reaction of 4Ј,4Љ-dimethoxycurcumin—4Ј,4Љ-dimethoxycur-
cumin (50 ␮M) was dissolved in 500 ␮l of 100 mM potassium
phosphate buffer, pH 8, in a cuvette and the absorbance at
NMR—Samples were dissolved in 150 ␮l of acetone-d or
6
acetonitrile-d in a 3 mm tube, and the NMR spectra were
3
recorded using a Bruker DRX 600 MHz spectrometer
equipped with a cryoprobe. Chemical shifts are reported in
ppm relative to residual non-deuterated solvent (␦ 2.04 for
acetone-d and ␦ 1.93 for acetonitrile-d ). The pulse frequen-
4
30 nm was monitored using the time-drive mode of the
spectrophotometer. COX-2 (50 nM final) and/or H O (300
2
2
␮
M final) were added as indicated.
6
3
Determination of Superoxide—The amount of superoxide
cies for the H,H-COSY, HSQC, and HMBC experiments were
taken form the Bruker library. The spectra were acquired at
285 K.
formed during transformation of curcumin was measured as
the reduction of cytochrome c (from bovine heart). Cyto-
chrome c, 12 ␮M, was dissolved in 500 ␮l of 100 mM potas-
sium phosphate buffer pH 8 in a cuvette and 50 ␮M curcumin
were added. The solution was scanned form 700–200 nm in
the spectrophotometer in 2 min intervals. Reduction of cyto-
chrome c was measured as the increase in absorbance at 550
RESULTS
Autoxidative Transformation of Curcumin—The degrada-
tion of curcumin (Fig. 1A) in aqueous buffers of pH values
ranging from 5 to 9 was monitored by the loss of the absor-
Ϫ1
Ϫ1
nm using ⑀ 29.5 mM cm (28). In some of the reactions,
superoxide dismutase (450 units) or catalase (50 units) was
included.
bance at the ␭
of 430 nm using a UV/Vis spectrophotome-
max
ter. Curcumin was unstable at basic pH (7.5–9) but its stabil-
ity was increased at acidic pH (Fig. 1B), similar to findings
Cell Culture—RAW264.7 cells were cultured in DMEM and reported by (7). Curcumin was most unstable at pH 8, and,
grown in 6-well plates at 37 °C in an atmosphere of 5% CO .
therefore, most of the following assays were performed at pH
8. Repetitive scanning from 700–220 nm confirmed that the
chromophore at 430 nm was lost during the transformation
indicating that the conjugated double bond system between
the phenolic rings of curcumin was disrupted (Fig. 1C).
The reaction observed in the spectrophotometer was com-
pared with the amount and rate of oxygen consumed during
the transformation of curcumin. Addition of 60 ␮M curcumin
2
Cells of passages 5 to 8 were used. Some of the cells were
stimulated by treatment with 100 ng/ml LPS for 5 h in
DMEM adjusted to pH 7.6 using 20 mM sodium phosphate
buffer and in the absence of FBS. Curcumin (20 ␮M) was
added from a stock solution in DMSO. After 2 h of incubation
the culture medium was removed, acidified to pH 4, and ex-
tracted using ethyl acetate/isopropanol (90/10, by vol). Prod-
1
116 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidative Transformation of Curcumin
FIGURE 2. Oxygen consumption during the transformation of curcumin.
A, 1 ml of Na-phosphate/citrate buffer pH 8.0 was placed in a Clark-type
oxygen electrode and allowed to adjust to 30 °C. Curcumin (60 ␮M) or vehi-
cle (ethanol) was added after 2 min (arrow), and the oxygen concentration
was monitored for 10 min. B, curcumin (60 ␮M) was added to 1 ml of Na-
phosphate/citrate buffer pH 8.0 in a spectrophotometer cuvette, and the
absorbance at 430 nm was recorded for 10 min.
FIGURE 1. Effect of pH on the transformation of curcumin. A, extended
conjugation of the seven-carbon heptadienone chain connecting the two
phenolic rings results in the yellow-orange color of curcumin. In 4Ј-me-
thoxy- and 4Ј,4Ј-dimethoxycurcumin the phenolic hydroxyl group is re-
placed by a methoxy group. B, curcumin was added to buffer of the pH indi-
cated and the decrease in absorbance at 430 nm at room temperature was
monitored for 20 min using a UV/Vis spectrophotometer. For all reactions
4
30 nm was similar to the rate of oxygen consumption meas-
ured in the oxygen electrode (Fig. 2B). The amount of curcu-
min degraded in the spectrophotometric assay (about 75% or
4
5 ␮M) was equivalent to the amount of oxygen consumed
(Ϸ38 ␮M) implicating that curcumin reacted with an equimo-
lar amount of oxygen during degradation.
7
0 ␮M curcumin was added except for pH 9.0 where 50 ␮M curcumin was
added. The buffers pH 5 to 8 were generated using mixtures of 200 mM
Na HPO and 100 mM citric acid, pH 8.5 and 9.0 were 100 mM K HPO ad-
HPLC Analysis of the Reaction Products—LC-ESI-MS anal-
ysis with online diode array detection and recording of the
absorbance at 205 nm showed a major transformation prod-
uct (1) of curcumin eluting at 3.5 min and two less abundant
products 2 and 3 eluting as a double peak at 4.2 min (Fig. 3A).
Concomitant ESI-MS analysis with detection in the negative
ion mode gave a molecular ion of m/z 399 for the three prod-
ucts 1, 2, and 3, equivalent to a molecular weight of 400 (Fig.
2
4
2
4
justed with NaOH. C, repetitive scans in 2-min intervals of a solution of 70
M curcumin in 100 mM Tris-HCl buffer, pH 8.
␮
(
dissolved in 5 ␮l of ethanol) to 1 ml of Na-phosphate/citrate
buffer pH 8.0 in a Clark-type oxygen electrode resulted in
immediate and rapid uptake of oxygen (Fig. 2A). The total
reduction of the oxygen concentration in the buffer was cal-
culated as 43 ␮M. Addition of the ethanol vehicle resulted in a 3B). This is an increase of 32 amu over curcumin (7.4 min
drift of about 5 ␮M oxygen over the same time. When the
transformation of 60 ␮M curcumin was monitored in the
spectrophotometer, the rate of disappearance of the signal at
retention time; m/z 367; MW 368) suggesting that the prod-
ucts had been formed by reaction with molecular oxygen
(Fig. 3C).
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Oxidative Transformation of Curcumin
6
00
A
B
standards
feruloylmethane
mAU
vanillin &
ferulic acid
4
2
00
00
0
0
5
10
15
20
5
00
curcumin
autoxidation
mAU
1
3
2
1
75
50
FIGURE 3. RP-HPLC analysis of the transformation products of curcu-
min. The transformation reaction of curcumin as shown in Fig. 1B was ex-
tracted and analyzed by RP-HPLC with detection by (A) UV 205 nm and (B
and C) LC-ESI-MS. Three products 1, 2, and 3 were detected in the UV chro-
matogram and gave a signal in the ion trace m/z 399 (panel B), equivalent to
a molecular weight of 400 for all three products. C, unreacted curcumin was
vanillin and/or
ferulic acid
curcumin
2
,3
25
0
Ϫ
detected as the [M-H] molecular ion in the ion trace m/z 367.
Products 1-3 were compared with authentic standards of
three of the four reported major degradation products of cur-
cumin (4, 5, 7) using RP-HPLC analysis with diode array de-
tection (Fig. 4). A standard of the fourth putative product,
trans-6-(4-hydroxy-3-methoxyphenyl)-2,4-dioxo-hexenal (7),
was not available. The standards of vanillin and ferulic acid
co-eluted at 7.4 min, feruloylmethane eluted at 9.4 min (Fig.
0
5
10
15
20
8
00
co-chromatography
C
mAU
00
vanillin &
ferulic acid
6
1
feruloylmethane
curcumin
400
4
A). The main oxygenation product of curcumin (1) eluted at
.2 min (Fig. 4B), about 0.2 min earlier than vanillin and feru-
7
200
lic acid. Co-chromatographic analysis of autoxidized curcu-
min with the standards confirmed the difference in retention
times of 1 and vanillin/ferulic acid (Fig. 4C). Based on the UV
spectrum, a minor peak eluting at the corresponding reten-
tion time in the autoxidation reaction of curcumin is vanillin
0
0
5
10
15
20
Retention time (min)
FIGURE 4. RP-HPLC comparison of the products of curcumin autoxida-
tion with the putative degradation products, vanillin, ferulic acid, and
feruloylmethane. A, 500 ng each of vanillin and ferulic acid and 1 ␮g of
feruloylmethane were injected on a Waters Symmetry 5-␮m column (4.6 ϫ
50 mm) eluted with a linear gradient of acetonitrile/water/acetic acid (20:
0:0.01, by vol) to (80:20:0.01, by vol) in 20 min at a flow rate of 1 ml/min
and diode array detection. B, analysis of autoxidized curcumin showing the
elution of products 1, 2, and 3. C, co-chromatographic analysis of a mixture
of the sample in B and 500 ng each of vanillin, ferulic acid, and feruloyl-
methane. The chromatograms shown were at recorded at UV 205 nm.
(
see Fig. 4B) whereas ferulic acid and feruloylmethane were
not detected. These analyses provided evidence that the re-
ported cleavage of the heptadienone chain of curcumin into
vanillin, ferulic acid, or feruloylmethane is not an abundant
reaction (7) and that, in contrast, the almost exclusive trans-
formation of curcumin in aqueous buffer at physiological pH
is an autoxidation reaction.
2
8
Identification of the Transformation Products—The main
product 1 was prepared by autoxidation of 500 ␮g of curcu-
min and isolated using RP-HPLC. HRMS analysis gave an ex-
act mass of 401.1225 for the protonated adduct resulting in a
molecular formula of C H O for 1 (calculated: 401.1231)
carbon 1 and the phenolic ring (supplemental Fig. S1). Chro-
matographic, mass-spectrometric, and NMR data were used
to identify 2 and 3 as configurational isomers of 1 by compar-
ison to published data (29).
2
1
20
8
and compatible with the addition of molecular oxygen to cur-
COX-2 Catalyzed Oxidative Transformation of Curcumin—
1
cumin. The H NMR and H,H-COSY spectra of 1 recorded in The formation of 1 by autoxidation and by lipoxygenase-cata-
d -acetone are shown Fig. 5. The proton chemical shifts of
lyzed oxygenation of curcumin (25) prompted the hypothesis
that a similar transformation would also be catalyzed by the
cyclooxygenase enzymes, COX-1 and COX-2. Reactions with
6
product 1 were identical with data reported for the transfor-
mation product of curcumin by soybean lipoxygenase (25).
Therefore, the main autoxidation product 1 is a bicyclopenta- COX-2 were carried out using the purified recombinant hu-
dione formed by cyclization and oxygenation of the heptadi-
enone carbon chain of curcumin. Carbons 2 and 6 form the
bridge of the two fused five membered rings, and carbons 1
man enzyme in the presence of hematin (1 ␮M) to reconsti-
tute the COX-2 holoenzyme but in the absence of arachidonic
acid or a co-substrate for the peroxidase activity (e.g. phenol).
and 7 are connected through an oxygen bridge to form a furan The rate of degradation of curcumin increased about 3.5-fold
ring. The second atom of oxygen has been inserted between
when 50 nM COX-2 was added, and there was a further 10-
1
118 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidative Transformation of Curcumin
1
FIGURE 5. H and H,H-COSY NMR spectra of product 1 (bicyclopentadi-
1
one) isolated from autoxidation of curcumin. The H spectrum at the top
shows five signals corresponding to the hydrogens at carbons 1, 2, 4, 6, and
7
of the original heptadienone chain of curcumin and the signals of the aro-
matic protons. The H,H-COSY spectrum (below) shows a cross peak be-
tween H6 and H7 as well as between H6 and H2. Note the lack of a cross-
peak between the neighboring H1 and H2 due to unfavorable arrangement
of the two spins at the ring structures. In addition, H1 shows weak coupling
(J ϭ 3.5 Hz) with the hydroxyl group at C3 (␦ 5.76). H2Ј of the aromatic ring
showed a cross-peak with C1Ј at 148.4 ppm in the HMBC experiments (not
shown) that allowed assignment of all six aromatic protons to the corre-
sponding phenolic ring.
fold increase by the addition of 300 ␮M H O (Fig. 6A and
2
2
Table 1). In the absence of enzyme, addition of 300 ␮M H O
2
2
led only to a 2-fold increase of the rate of autoxidative trans-
formation. Product analysis using RP-HPLC with diode array
detection or LC-MS confirmed that the same products were
formed in the COX-2 catalyzed transformation as in the au-
toxidative transformation of curcumin. The COX-1 isozyme
was active in the presence of H O (300 ␮M) but inactive
2
2
when H O was absent.
2
2
The effect of inhibitors of the cyclooxygenase active site
(
indomethacin) and peroxidase active site (acetaminophen,
ApAP) was tested to establish which of the two COX-2 active
sites was involved in the transformation of curcumin. Indo-
methacin at 10 ␮M concentration had no effect on the COX-2
catalyzed transformation of curcumin (Fig. 6B) whereas acet-
aminophen dose-dependently decreased the rate of COX-2
catalyzed degradation of curcumin (Fig. 6C). Acetaminophen
also had a small inhibitory effect on the autoxidative transfor-
mation of curcumin. Neither inhibitor showed an effect on
the COX-2-catalyzed transformation of curcumin in the pres-
ence of 300 ␮M H O .
FIGURE 6. Spectrophotometric analysis of the COX-2-catalyzed transfor-
mation of curcumin. A, transformation of curcumin was accelerated by the
presence of 50 nM COX-2 compared with autoxidation. Addition of 300 ␮M
H O further accelerated the rate of COX-2-catalyzed transformation
2 2
whereas it had only a small effect on autoxidation. B, addition of 10 ␮M in-
domethacin did not change the rate of COX-2 (50 nM) catalyzed transforma-
tion of curcumin. C, addition of acetaminophen (ApAP) dose-dependently
inhibited the COX-2 (50 nM) catalyzed transformation of curcumin. All reac-
tions were conducted using 100 ␮M curcumin and 1 ␮M hematin in 100 mM
potassium phosphate buffer pH 9 in the absence or presence of 50 nM
COX-2 as indicated. The absorbance at 430 nm was recorded in the time-
drive mode using a UV/Vis spectrophotometer.
2
2
JANUARY 14, 2011•VOLUME 286•NUMBER 2
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Oxidative Transformation of Curcumin
TABLE 1
Rates of autoxidative and COX-2-catalyzed transformation of curcumin and curcumin derivatives
Rates were determined using 50 ␮M substrate in 500 ␮l of 100 mM potassium phosphate buffer, pH 8.0, in a 1-cm cuvette, and in the absence or presence of COX-2, 1
Ϫ1
Ϫ1
␮
M hematin, and 300 ␮M H O . Curcumin and its derivatives were monitored at 430 nm (⑀ ϭ 25,000 M cm ); the reaction with ABTS was monitored at 417 nm (⑀ ϭ
2 2
Ϫ1 Ϫ1
36,000 M cm ).
Initial rate ؎ S.D. (␮M/min)
Autoxidation (hematin ؉ H O )
COX-2a
COX-2 turnover number
b
Substrate
Autoxidation
COX-2 ؉ H O
(at 50 ␮M substrate)
2
2
2
2
3
3
2
2
Curcumin
2.2 Ϯ 0.4
4.4 Ϯ 0.4
7.5 Ϯ 0.6
88.0 Ϯ 7.0
1.76 ϫ 10 Ϯ 1.4 ϫ 10
c
4
Ј-Methoxycurcumin
Ј,4Љ-Dimethoxycurcumin
1.0 Ϯ 0.2
3.9 Ϯ 1.0
3.9 Ϯ 1.0
86.2 Ϯ 5.0
1.72 ϫ 10 Ϯ 1.0 ϫ 10
0
.1 Ϯ 0.05
d
d
0.06 Ϯ 0.02a
7.2 Ϯ 0.6
e
4
–
0.07 Ϯ 0.01
–
ND
ABTS
ND
ND
ND
144 Ϯ 12
a
b
c
d
e
The concentration of COX-2 was 50 nM, 1 ␮M hematin.
The concentration of COX-2 was 5 nM, 1 ␮M hematin. The calculated rate is corrected for the dilution of enzyme.
The reaction proceeded at an initial, faster rate during the first 1–2 min followed by a slower rate over the next 30 min.
No reaction.
Not determined.
Reactions in H218O-Buffer—The origin of oxygen in the
the phenolic rings can be used to assign the mass spectromet-
ric fragment ions (supplemental Fig. S3). The reaction of
COX-2 with 4Ј-methoxycurcumin proceeded at similar initial
rate but less total conversion was observed (Table 1). As ex-
pected, the oxygenated product had a MW of 414 or 416, re-
bicyclopentadione 1 was established by incubations in buffer
18
with H O or unlabeled water. The reactions were analyzed
2
by LC-ESI-MS using an ion trap mass detector. The calcu-
18
lated concentration of H O in the labeled buffer was 92%.
2
1
8
Product 1 showed an increase of 2 amu from m/z 399 to m/z
spectively, when formed in H O-enriched buffer. The incor-
2
18
18
4
01 when the reaction was carried out in H O-buffer (Fig. 7, poration of O into the product was calculated to be about
2
18
A and B). The incorporation of H O into 1 was 85%, impli-
25%. MS2 analyses of the molecular ions at m/z 413 and 415,
respectively, gave m/z 247 or 249 as the base peak (supple-
mental Fig. S3). This indicated that the additional 4Ј-methyl
group was not part of the bicyclic ring structure that con-
2
cating that one of the two oxygen atoms incorporated during
autoxidative transformation of curcumin was derived from
water. Control experiments showed that 1 did not exchange
18
18
O during incubation (pH 8), extraction (pH 4), or LC-MS
tained the O but was located on the phenolic ring (ring A)
analysis.
that is attached to carbon 1 through the ether bridge.
The site of incorporation of oxygen from water into 1 was
Additional Mechanistic Experiments—We hypothesized
that 4Ј,4Љ-dimethoxycurcumin would be protected from au-
toxidative as well as COX-2 catalyzed transformation due to
blocking of both phenolic hydroxyl groups. 4Ј,4Љ-Dimethoxy-
curcumin at 50 ␮M was stable in potassium phosphate buffer
pH 8 showing no measurable decrease of the chromophore at
430 nm over the course of 15 min. Addition of 300 ␮M H O
Ϫ
determined by CID of the [M-H] molecular ions at m/z 399
n
and 401, and by subsequent MS of the respective fragment
ions. The MS2 spectra of the molecular ion showed a frag-
ment ion at m/z 247 or 249, respectively, as the base peak
(
supplemental Fig. S2). In addition, both spectra contained
fragments at m/z 151 and 353, implicating that these ions
2
2
were derived by loss of a fragment containing the labeled oxy- and/or 50 nM COX-2 induced only a minimal loss of the chro-
gen, the latter m/z 353 likely being derived from the loss of
mophore of 4Ј,4Љ-dimethoxycurcumin (Table 1).
18
water and CO or C O, respectively. Further fragmentation
Catalysis by peroxidase and lack of reaction of 4Ј,4Љ-dime-
thoxycurcumin implicated formation of a phenoxyl radical as
(
MS3) of m/z 247 or 249 showed consecutive loss of 15 amu
(
-CH ) to give m/z 232 or 234, respectively, followed by loss of a likely initial step of the transformation of curcumin. We
3
18
CO or C O, respectively, to result in m/z 204 in both cases
hypothesized that the phenoxyl radical is formed by oxidation
of the phenolate anion with molecular oxygen serving as the
oxidant (electron acceptor) that would be reduced to superox-
ide. The formation of superoxide was determined as the re-
duction of cyctochrome c (12.5 ␮M) measured at 550 nm, and
its inhibition by superoxide dismutase (28). The rate of super-
oxide formed was 0.1 ␮M/min over the course of autoxidation
of 50 ␮M curcumin (20 min). Addition of catalase in order to
(
Fig. 7, C and D). Based on these results, the major fragment
ions m/z 247 or 249, respectively, were found to represent the
bicyclic ring moiety with one of the phenolic rings attached
(
ring B) but lacking carbon 1 of the bicyclic ring (Fig. 7E).
Carbon 1 was part of the fragment m/z 151 formed by CID of
the molecular ions (m/z 399 and 401). Together, these data
18
indicated that the O derived from water was incorporated
into the substituted furan ring of 1, i.e. between carbons 1 and prevent the possibility of re-oxidation of reduced cytochrome
7
.
c by H O (derived from dismutation of superoxide) did not
2
2
To provide additional evidence for the assignment of the
change the rate of reduction of cytochrome c.
n
fragments observed in the MS experiments, the asymmetric
Attempts to trap a putative hydroperoxide intermediate of
oxidative transformation by reduction were not successful.
When the autoxidative and COX-2 catalyzed transformation
curcumin analog 4Ј-methoxycurcumin was reacted with
18
COX-2 in unlabeled and in H O-buffer. The proposed
2
mechanism of autoxidative and COX-2 catalyzed transforma- of curcumin were extracted and treated immediately with an
tion of curcumin implies that only one of two isomeric oxy-
genation products will be formed, and that, therefore, the in-
crease in 14 amu due to the additional methyl group at one of
excess of triphenylphosphine no change in the product profile
was observed upon analysis by RP-HPLC with diode array
detection. Similarly, inclusion of 1 mM SnCl in the reaction
2
1
120 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidative Transformation of Curcumin
MS3: 399.0@CID15.0; 247.0@CID25.0
C
232
A
H2O
399
204
1
00
100
80
60
40
20
0
247
124
8
6
4
2
0
0
0
0
-CH3
-CO
123
400
109
FIGURE 8. RP-HPLC analysis of the incubation of curcumin with LPS-acti-
vated RAW264.7 cells. RAW264.7 cells were activated with 100 ng/ml LPS
for 5 h in DMEM adjusted to pH 7.6 using 20 mM sodium phosphate buffer
in the absence of FBS. Curcumin (20 ␮M) was added to the RAW264.7 cells
and incubated for additional 2 h. The medium was removed, acidified to pH
0
390
400
m/z
410
80
120
160
200
240
280
m/z
4
, and extracted twice with 2.5 ml of ethyl acetate/isopropanol (90:10, by
H218O
01
MS3: 401.0@CID15.0; 249.0@CID25.0
D
249
B
vol). An aliquot was injected on RP-HPLC with diode array detection using
the same conditions as in Fig. 4. The peak identified as hexahydrocurcumin
4
100
100
(
structure sown) contained an unidentified product that eluted as a front
shoulder. The chromatogram was recorded at 205 nm.
204
234
8
6
4
2
0
0
0
0
8
6
4
2
0
0
0
0
1
26
2 h. RP-HPLC analyses with UV detection showed that 1 was
one of two major transformation products of curcumin de-
tectable (Fig. 8). A second prominent peak at 11.4 min reten-
tion time consisted of hexahydrocurcumin and an unidenti-
fied co-eluting product. Hexahydrocurcumin was identified
by comparison of its retention time and UV spectrum to an
authentic standard prepared by partial hydrogenation of
curcumin.
-CH3
18
-
C O
4
02
125
3
99
1
11
0
0
3
90
400
m/z
410
80
120
160
200
240
2
80
The metabolic profile of curcumin was similar in
m/z
RAW264.7 cells activated with LPS and in unactivated cells.
In unstimulated cells, however, 83.0 Ϯ 0.9% of curcumin was
degraded after 2 h incubation whereas in activated cells
92.7 Ϯ 0.4% of curcumin was degraded. The peak area of the
bicyclopentadione product was increased 1.8-fold in LPS-
activated RAW264.7 cells compared with unactivated cells.
E
151
OH
B
H CO
O
O*
3
OCH3
1
7
A
1
23; -14(-CH ) = 109
2
HO
O
HO
DISCUSSION
2
2
2
2
47-123 = 124
2
47 or 249*
47-15(-CH ) = 232 ; 232-109 = 123
The obvious structural differences of curcumin and the
bicyclopentadione product prevented a ready explanation of
the mechanism of oxidative transformation. The net increase
of 32 amu of the bicyclopentadione initially suggested that
both oxygen atoms were derived from incorporation of mo-
3
49*-123 = 126*
49*-15 = 234*; 234-109 = 125*
FIGURE 7. LC-ESI-MS/MS analysis of the bicyclopentadione 1 obtained
by autoxidation of curcumin in regular and H2 O-buffer. MS1 spectrum
of 1 obtained form the incubation of curcumin in (A) regular buffer and (B)
buffer containing 92% H2 O. The MS2 spectra obtained by CID of the mo-
lecular ions m/z 399 and 401 are shown in supplemental Fig. S2. MS3 spec-
tra obtained by CID of (C) m/z 247 and (D) m/z 249. The solid arrows indicate
fragments derived from m/z 247 and m/z 249, respectively. The dashed ar-
1
8
1
8
lecular oxygen, similar to the addition of O to a polyunsatu-
2
rated fatty acid during autoxidation, and likewise, the incor-
poration of O into a fatty acid substrate in COX-2 and
2
rows indicate the fragments obtained by CID of m/z 232 (in panel C) and m/z lipoxygenase catalysis (30, 31). Important clues about the
2
34 (in panel D), respectively. E, proposed fragmentation of 1 in the CID ex-
18
mechanism came from analysis of the reaction catalyzed by
COX-2. In the case of enzymatic transformation of curcumin,
binding in the fatty acid binding site and catalytic reaction
periments. The asterisk denotes the oxygen atom derived from H O and
2
1
8
n
the fragments that contain O. The MS analyses were performed using an
ion trap instrument as described under “Experimental Procedures.”
with O appeared a straightforward possibility. Certainly for
2
of curcumin with COX-2 did not change the product profile
but reduced the extent of transformation.
COX-2 catalysis this is not the case since indomethacin, an
inhibitor that blocks access of substrate to the oxygenase ac-
tive site, did not inhibit COX-2 catalyzed transformation of
Transformation of Curcumin in RAW264.7 Cells—Oxida-
tive transformation of curcumin was also analyzed upon addi- curcumin. Acetaminophen, on the other hand, which acts at
tion to RAW264.7 murine macrophage cells. Cells were
treated for 5 h without or with LPS (100 ng/ml) to induce ex-
pression of COX-2 and then treated with 20 ␮M curcumin for
the peroxidase active site of COX-2 (32) inhibited the reac-
tion, implicating that the peroxidase rather than the oxygen-
ase activity of the enzyme was crucial.
JANUARY 14, 2011•VOLUME 286•NUMBER 2
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Oxidative Transformation of Curcumin
FIGURE 9. Proposed mechanism of autoxidative and COX-2 catalyzed transformation of curcumin. The reaction steps are explained in the “Discus-
sion.” Products 2 and 3 are isomers of 1 differing in the configuration of carbons 1 and 7, respectively. R ϭ -OCH3.
A mechanism for autoxidative and COX-2 catalyzed trans-
formation of curcumin that is consistent with the experimen-
electrophilic oxygen of the hydroperoxide to form a spiro cy-
clohexadienone epoxide intermediate (step 7). The unstable
tal observations is proposed in Fig. 9. In both cases, reaction is epoxide is opened by the neighboring alcohol to yield the bi-
initiated by the formal hydrogen atom abstraction from one
of the phenolic hydroxyl groups (step 1 in Fig. 9). Given the
cyclopentadione 1 (step 8).
Incorporation of water during the transformation was evi-
1
8
n
pH of the medium (pH 8) and the first pK of curcumin
dent from reactions carried out in H O-buffer. MS analy-
a
2
(
Ϸ8.5), it is likely that the reaction is initiated by an electron
ses proved that the oxygen derived from water is the one
forming the ether bridge linking carbons 1 and 7 of the tetra-
hydrofuran ring. This was confirmed by analysis of the main
reaction product formed from the asymmetrical analog 4Ј-
methoxycurcumin. The second oxygen connecting the distal
aromatic ring with the newly formed bicyclic moiety is de-
transfer from the phenolate anion, akin to the concept of “se-
quential proton loss electron transfer (SPLET)” advanced by
Litwinienko and Ingold (33). The peroxidase active site of the
COX enzyme is promiscuous with regard to the H-atom do-
nor and accepts a large variety of phenols, aromatic amines,
␤
-dicarbonyls, and naturally occurring compounds (e.g. ascor- rived from molecular oxygen.
bic acid, uric acid, glutathione) (34). As expected for catalysis
The antioxidant mechanism of curcumin has been the sub-
by the peroxidase active site, addition of H O greatly acceler- ject of a controversial debate for more than a decade, e.g. Ref.
2
2
ated the rate of reaction (Table 1). The initial oxidation is pre- 33, 36–38. What is clear is that the antioxidant reaction of
sumably the only true enzymatic reaction in the COX-2 cata-
lyzed transformation of curcumin, all of the following steps
being non-enzymatic. The resulting phenoxyl radical has sig-
nificant radical character at C6 of the heptadienone chain,
which allows it to undergo 5-exo cyclization onto C2 to yield
the cyclopenta-1,3-dione moiety of 1 upon rotation about the
curcumin (as with other phenols) is the donation of a hydro-
gen atom to a lipid peroxyl radical, thereby forming a lipid
hydroperoxide and interrupting the radical chain reaction.
What is less clear is at which of the two possible sites hydro-
gen abstraction takes place, i.e. at the phenolic OH or the cen-
tral CH in the ␤-diketo form of curcumin, and also whether a
2
C4-C5 bond (steps 2–4). Addition of O to the newly formed
classical hydrogen atom transfer (“HAT”) takes place or
rather what has been described as a sequential proton loss
electron transfer (SPLET) reaction (33, 38). The essence of the
SPLET reaction is that first loss of a proton occurs and then
an electron is donated from the phenolate anion. This is the
most likely mechanism in polar solvents such as water as we
have used here. Nevertheless, our results contribute little to
this discussion since they are concerned with what happens to
curcumin after the antioxidant reaction, i.e. after the hydro-
2
C1 radical is presumably diffusion controlled (step 5), and the
resultant peroxyl radical can serve as the autoxidation chain-
carrying radical through reaction with another molecule of
curcumin. It is not clear whether hydration of the quinone
methide moiety or reduction of the peroxyl radical to the hy-
droperoxide by curcumin would occur next (step 6). While
the rate constants for hydration of analogous o-methoxy p-
quinone methides have been determined (pseudo first order
Ϫ1
rate constant of Ϸ0.12 s ) (35), the rate constant for the cur- gen atom has been abstracted from the phenolic hydroxyl.
cumin peroxyl reaction has only been determined in chloro-
Leaving detailed mechanistic considerations aside, there is
the unexpected finding that in the COX-2 catalyzed transfor-
5
Ϫ1 Ϫ1
benzene (3.4 ϫ 10
M
s
) (36) where the mechanism is
presumably H-atom transfer rather than a SPLET mechanism, mation of curcumin the peroxidase rather than the cyclooxy-
making comparison of the rates difficult. The tetrahydrofuran genase active site is involved in the oxygenation reaction. The
ring of the bicyclic moiety is formed when the C1-hydroper-
oxide dehydrates to undergo a 1,2-carbon-to-oxygen aryl mi-
gration whereby the electron rich phenolic ring attacks the
insertion of oxygen, however, is non-enzymatic and depend-
ent on the particular structure of curcumin that enables a rad-
ical intermediate to undergo reaction with molecular oxygen
1
122 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidative Transformation of Curcumin
and to further rearrange the peroxide into a stable product.
Other natural phenolic compounds that react at the peroxi-
dase site of the COX enzymes, for example eugenol and res-
veratrol, undergo a similar initial hydrogen abstraction to
yield a phenoxyl radical but fail to incorporate molecular oxy-
gen as part of the transformation since phenoxyl radicals are
Acknowledgment—We thank Dr. M. Wade Calcutt for help with
HRMS.
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The degradation of curcumin in buffer at alkaline pH and
upon addition to cultured cells is well documented (7). Our
studies implicate that oxidative transformation could also
contribute to the degradation of curcumin in cultured cells.
Using RAW264.7 mouse macrophage-like cells we found that
the cyclopentadione metabolite is almost equally abundant to
hexahydrocurcumin, the main reductive metabolite found in
animals and humans (43). Furthermore, since LPS-activated
RAW264.7 cells showed greater degradation of curcumin and
almost 2-fold increased formation of the bicyclopentadione
product there is the possibility of enzymatic contribution to the
oxidative transformation of curcumin. Enzymatic oxidation of
curcumin is likely to occur not only by COX-2 but also by other
peroxidases like myeloperoxidase and NADPH oxidase.
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