Stability and anti-inflammatory activity of the reduction-resistant curcumin analog, 2,6-dimethyl-curcumin

Article abstract of DOI:10.1039/c8ob00639c

The efficacy of the curry spice compound curcumin as a natural anti-inflammatory agent is limited by its rapid reductive metabolism in vivo. A recent report described a novel synthetic derivative, 2,6-dimethyl-curcumin, with increased stability against reduction in vitro and in vivo. It is also known that curcumin is unstable at physiological pH in vitro and undergoes rapid autoxidative transformation. Since the oxidation products may contribute to the biological effects of curcumin, we tested oxidative stability of 2,6-dimethyl-curcumin in buffer (pH 7.5). The rate of degradation was similar to curcumin. The degradation products were identified as a one-carbon chain-shortened alcohol, vanillin, and two isomeric epoxides that underwent cleavage to vanillin and a corresponding hydroxylated cleavage product. 2,6-Dimethyl-curcumin was more potent than curcumin in inhibiting NF-κB activity but less potent in inhibiting expression of cyclooxygenase-2 in LPS-activated RAW264.7 cells. 2,6-Dimethyl-curcumin and some of its degradation products covalently bound to a peptide that contains the redox-sensitive cysteine of IKKβ kinase, the activating kinase upstream of NF-κB, providing a mechanism for the anti-inflammatory activity. In RAW264.7 cells vanillin, the chain-shortened alcohol, and reduced 2,6-dimethyl-curcumin were detected as major metabolites. These studies provide new insight into the oxidative transformation mechanism of curcumin and related compounds. The products resulting from oxidative transformation contribute to the anti-inflammatory activity of 2,6-dimethyl-curcumin in addition to its enhanced resistance against enzymatic reduction.

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DOI: 10.1039/C8OB00639C
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ARTICLE
Stability and anti-inflammatory activity of the reduction-resistant
curcumin analog, 2,6-dimethyl-curcumin
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
b
Akil I. Joseph, Rebecca L. Edwards , Paula B. Luis , Sai Han Presley , Ned A. Porter and Claus
a
Schneider *
DOI: 10.1039/x0xx00000x
The efficacy of the curry spice compound curcumin as a natural anti-inflammatory agent is limited by its rapid reductive
metabolism in vivo. A recent report described a novel synthetic derivative, 2,6-dimethyl-curcumin, with increased stability
against reduction in vitro and in vivo. It is also known that curcumin is unstable at physiological pH in vitro and undergoes
rapid autoxidative transformation. Since the oxidation products may contribute to the biological effects of curcumin, we
tested oxidative stability of 2,6-dimethyl-curcumin in buffer (pH 7.5). The rate of degradation was similar to curcumin. The
degradation products were identified as a one-carbon chain-shortened alcohol, vanillin, and two isomeric epoxides that
underwent cleavage to vanillin and a corresponding hydroxylated cleavage product. 2,6-Dimethyl-curcumin was more
potent than curcumin in inhibiting NF-κB activity but less potent in inhibiting expression of cyclooxygenase-2 in LPS-
activated RAW264.7 cells. 2,6-Dimethyl-curcumin and some of its degradation products covalently bound to a peptide that
contains the redox-sensitive cysteine of IKKβ kinase, the activating kinase upstream of NF-κB, providing a mechanism for
the anti-inflammatory activity. In RAW264.7 cells vanillin, the chain-shortened alcohol, and reduced 2,6-dimethyl-
curcumin were detected as major metabolites. These studies provide new insight into the oxidative transformation
mechanism of curcumin and related compounds. The products resulting from oxidative transformation contribute to the
anti-inflammatory activity of 2,6-dimethyl-curcumin in addition to its enhanced resistance against enzymatic reduction.
www.rsc.org/
1
6
curcumin (Fig. 1) . Introduction of steric bulk around the 1,2-
and 6,7-double bonds was proposed to hinder enzymatic
reduction (saturation) to di- and tetrahydro metabolites. The
novel analog, 2,6-dimethyl-curcumin, showed enhanced anti-
angiogenic activity in vitro and in an animal model compared
to curcumin. It was less efficiently reduced by recombinant
alcohol dehydrogenase, and there appeared to be less
reductive metabolism in mice injected intraperitoneally with
Introduction
The biological activities described for the spice compound
curcumin include antioxidant, anti-inflammatory, anti-
1, 2
microbial, and anti-tumorigenic effects
. The majority of
these effects have been established using cultured cells and
other in vitro models, and some have been replicated in
3
-5
animal models . As a consequence of successful preclinical
evaluation, a large and yet increasing number of clinical trials
with curcumin has been registered at clinicaltrials.gov.
1
6
2,6-dimethyl-curcumin than with curcumin . The authors
concluded that inhibition of reductive metabolism accounted
for the increased biological activity of 2,6-dimethyl-curcumin
6
,
Successful trials with curcumin or turmeric have been sparse
7
1
6
.
although some recent controlled trials report improvements
in cognitive function, joint arthritis, and inflammatory bowel
8
-11
disease
.
A key reason suggested for the lack of successful application in
1
2, 13
humans is the low bioavailability of curcumin
, and rapid
metabolism via reduction and conjugation of what little is
1
4, 15
absorbed is another
. With the aim of impeding reductive
metabolism, Koo and coworkers have placed methyl groups at
double bond carbons 2 and 6 of the heptadienedione chain of
Figure 1 Chemical structures of curcumin, 2,6-dimethyl-
curcumin, and the curcumin metabolites tetrahydrocurcumin
and bicyclopentadione.
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Curcumin is chemically unstable at physiological pH and transformation. After 5 h reaction time
1
,
4
, and
absent or decreased, respectively
autoxidation involving reaction with oxygen to yield a (Fig. 2d). Consistent with the UV/Vis assay, unreacted 2,6-
5 became the
1
7
undergoes rapid degradation . The degradation is an major products with
2 and 3
1
8, 19
bicyclopentadione as the final stable product (Fig. 1)
initiating step of autoxidation is loss of a hydrogen atom from analyses, chromatographing as two peaks representing the
one of the phenolic hydroxyls followed by delocalization of the diketo and enol forms ( max 331 and 395 nm, respectively,
. The dimethyl-curcumin was the most abundant compound in the
λ
resulting radical into the heptadienedione chain. The radical recorded in RP-HPLC solvent). Large scale reactions for
facilitates cyclization and oxygenation reactions that transform identification of the degradation products were conducted for
the linear 7-carbon chain to a bicyclic cyclopentadione- 1 h or 24 h, respectively, in order to produce the intermediate
2
0
tetrahydrofuran moiety . The initial H-abstraction can be and final products. Products were isolated by RP-HPLC and
1
8, 21
catalyzed by peroxidases
, suggesting that oxidative identified using UV/Vis, LC-MS, and homo- and heteronuclear
transformation may occur in vivo although this has not been 1D- and 2D-NMR analyses.
established yet. Oxidative activation of curcumin as well as the
minor curcuminoids, demethoxy- and bisdemethoxycurcumin,
is required for the inhibition of human topoisomerase II
α
and
. Inhibition
was mediated by reaction intermediates
adducting to active site cysteine residues since neither
2
2-24
contributes to their anti-inflammatory effects
of topoisomerase II
α
2
2
curcumin nor the final bicyclopentadione were active . For
the anti-inflammatory activity, a strong correlation between
the rate of oxidative transformation and inhibition of NF-κB
was observed when synthetic analogs of curcumin were
2
4
analyzed
.
Considering the potential role of oxidative transformation
1
9
products in the biological activity of curcumin we prepared
,6-dimethyl-curcumin and tested its chemical stability. We
2
found it to be unstable similar to curcumin, identified
degradation products in buffer, and analyzed their possible
role in mediating the anti-inflammatory effect of 2,6-dimethyl-
curcumin.
Results and discussion
Stability of 2,6-dimethyl-curcumin
Chemical stability of 2,6-dimethyl-curcumin was tested by
incubation in pH 7.5 buffer. UV/Vis analysis indicated presence
of both the enol tautomer (λmax 410 nm) and the diketo
tautomer with absorbance around 350 nm. Repetitive
scanning using a UV/Vis spectrophotometer showed rapid
disappearance of the absorbance at 410 nm of 2,6-dimethyl-
curcumin in the first 2 min and then a decrease in rate until an Fig. 2. Degradation of 2,6-dimethyl-curcumin. (a) Stability of
apparent halt after about half of the absorbance had 2,6-dimethyl-curcumin at pH 7.5. 2,6-Dimethyl-curcumin (25
disappeared (Fig. 2a). There was a concomitant increase in
absorbance around 340 nm. The rate of degradation during sample was analyzed by repetitive scanning every 1 min or (b)
the initial 2 min of reaction was calculated as 2.8 ± 0.1 M/min in the time drive mode at 410 nm in
UV/Vis
Fig. 2b), slightly less than the initial rate of degradation of spectrophotometer. (c) Incubation reactions of 2,6-dimethyl-
ꢀM) was diluted in 20 mM phosphate buffer pH 7.5, and the
ꢀ
a
(
curcumin (4.0 ꢀM/min). The change in absorbance was not a curcumin in 20 mM phosphate buffer pH 8 were extracted
mere slow shift in the equilibrium of the diketo and enol after 1 h or (d) 5 h and analyzed using RP-HPLC with diode
tautomers as was evident from analysis of the reaction array detection. The chromatograms were recorded at UV 205
products by HPLC.
nm. (e) Isolated and identified products of degradation of 2,6-
dimethyl-curcumin.
Identification of degradation products
Product
1 eluting at 7.5 min retention time was identified as
Autoxidation of 2,6-dimethyl-curcumin showed formation of 5
vanillin based on its UV/Vis spectrum and positive ion LC-ESI-
products (
1-
5) when the reaction was terminated after 1 h (Fig.
2c). Products 2 and 3 were unstable and underwent further
2
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MS analysis (m/z 153.1) in comparison to an authentic Thus, the immediate products of autoxidative degradation of
standard. 2,6-dimethyl-curcumin are alcohol , the 6,7(or 1,2)-epoxide
Product appeared to be isomeric products. Due to isomers and , and vanillin (Fig. 2e). The epoxides undergo
differences in stability we were able to obtain clean NMR secondary cleavage, forming vanillin and alcohol
spectra only for more stable isomer , and its identification is
presented in detail. Product gave a molecular ion of m/z Time course of degradation
13.2 in positive ion LC-MS analysis, indicating incorporation of
5
2
and
3
2
3
1
4.
3
3
4
A time course for the degradation of 2,6-dimethyl-curcumin
was established by analyzing reaction aliquots every 1 h for a
total of 10 h (Fig. 3a). 2,6-Dimethyl-curcumin was decreased to
one atom of oxygen. The oxygen was present as a C-6/C-7
epoxide‡, based on diagnostic chemical shifts of H-7, C-6, and
1
C-7 in the H and heteronuclear NMR spectra. Consistent with
≈
40% within the first h of reaction and remained stable from
the lack of a direct neighbor, H-7 was a singlet, and thus, it was
not possible to determine cis or trans configuration of the
epoxide. Absence of the 6,7 double bond was consistent with
the UV/Vis spectrum with a λmax at 360 nm, a hypsochromic
shift compared to max 420 nm of 2,6-dimethyl-curcumin.
λ
Based on these data product
2
was identified as 6,7-epoxy-2,6-
dimethyl-curcumin.
1
Product
NMR data as
,6-dimethyl-curcumin, likely with changed configuration of
the epoxide group.
Product showed a
chromophore as in
2
(
λ
max 359 nm) had similar UV/Vis, LC-MS, and H
3
and was identified as an isomer of 6,7-epoxy-
2
4
λmax at 355 nm indicating a shortened
and 3. The H NMR spectrum showed the
1
2
presence of only one methoxyphenol ring. The signals for H-1
and H-4 of the original heptadienedione chain were present as
well as both original methyl groups at C-2 and C-6. One of the
methyl groups, however, was shifted upfield to 1.47 ppm,
indicating shielding by a hydroxyl. A C-6 hydroxyl was evident
from the carbon chemical shift (82.3 ppm) and confirmed
absence of the 6,7-double bond. The expected hydrogen signal
(
H-6), however, was not observed due to exchange with
deuterated solvent. The NMR data identified as 2,5-
dihydroxy-7-(4-hydroxy-3-methoxyphenyl)-6-methylhepta-
Z,6E-dien-3-one. LC-MS in positive ion mode gave an
4
4
abundant ion at m/z 261.1, which was 18 amu less than the
predicted ion and indicated facile loss of water during the
ionization process.
Product 5 shared features in the UV/Vis (λmax at 358 nm) and
NMR analyses with the other products indicating that one
methoxyphenol ring and the attached carbon chain through C-
5
were unchanged from the starting material. HMBC analyses
indicated lack of a signal for C-7 of the original carbon chain. In
order to provide further support for loss of this carbon,
Fig. 3 Time course of the degradation of 2,6-dimethyl-
curcumin. (a) The disappearance of 2,6-dimethyl-curcumin is
plotted as % remaining of the starting amount on the right
axis. Formation of products in % of total products is plotted on
1
3
sufficient material was prepared for direct C-NMR analysis
which confirmed absence of a signal for C-7. The upfield shift
of the methyl group attached at C-6 was explained by a
geminal hydroxyl, compatible with the absence of a hydrogen
at this carbon. Additional evidence for the loss of C-7 came
from the HMBC cross-peaks of H-2” and H-6” of the ring with
C-6, placing this carbon one bond closer to the methoxyphenol
the left axis. 2,6-Dimethyl-curcumin (25
ꢀ
0 mM phosphate buffer pH 8 in an HPLC autosampler vial.
M) was dissolved in
2
Aliquots (10 ꢀl) were injected every 1 h for 10 h. The mean ±
SD of three independent replicates is shown. (b) RP-HPLC
analysis of the incubation of isolated epoxide in buffer pH 7.5
for 1 h. (c) RP-HPLC analysis of the incubation of isolated
epoxide . The chromatograms were recorded at UV 205 nm.
2
ring than in 2,6-dimethyl-curcumin. Loss of a carbon from
5
was supported by LC-MS data. A prominent ion was obtained
at m/z 383.2 which resulted from loss of water from the
molecular ion which was not detected, similar to the facile
3
there on. Product
and likewise did not change in abundance during the
remainder of the experiment. Epoxides and peaked at 1 h
5 was formed in parallel within the first h
dehydration observed with
the chain-shortened alcohol, 2,5-dihydroxy-2,7-bis(4-hydroxy-
-methoxyphenyl)-6-methylhepta-4Z,6E-dien-3-one.
4. These analyses identified 5 as
2
3
3
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and disappeared within the next 2-5 h to undergo further >20
transformation. Disappearance of and was paralleled by results showed that 2,6-dimethyl-curcumin has in vitro anti-
the formation of cleavage products and vanillin . Vanillin, inflammatory activity similar to curcumin.
however, was already present in the first aliquot analyzed (t =
) and showed an earlier increase than . This indicated that Peptide adduction
there was an alternative route of formation of vanillin that did
ꢀM whereas curcumin was effective at 10 ꢀM. These
2
3
4
1
0
4
Inhibition of NF-
κ
B activity by curcumin involves covalent
, the
. To test whether 2,6-
dimethyl-curcumin and/or its degradation products adducted
to IKK , we used a 27-amino acid peptide that mimics the
activation domain of IKK
not include epoxides
When isolated and
to vanillin
2
3
and
were incubated in buffer both gave rise
(Fig. 3b,c). Several additional
3.
adduction of oxidative metabolites of curcumin to IKK
β
2
24
upstream kinase that activates NF-κB
1
and product
4
products were formed but these were not sufficiently
abundant for isolation and structural identification.
Transformation of the purified epoxides required longer
incubation times than during the degradation reactions. After
β
24, 25
β
. The peptide contains a redox-
active cysteine (Cys179) that has been well characterized as a
target of covalent modification by small molecule electrophiles
24 h incubation in buffer about half of
2 and 3
remained that 26, 27
.
had not converted to 1, 4, and the less abundant by-products.
Anti-inflammatory activity
We analyzed the anti-inflammatory activity of 2,6-dimethyl-
curcumin by quantifying the inhibition of NF- B in activated
RAW264.7 mouse macrophage-like cells that stably express
luciferase under the control of an NF- B response element.
κ
κ
Treatment of the cells with LPS increased luciferase activity,
and pretreatment with 2,6-dimethyl-curcumin for 45 min prior
to LPS resulted in dose-dependent inhibition (Fig. 4a). The IC50
for inhibition of NF-
κB by 2,6-dimethyl-curcumin was 7 ꢀM,
about half of the value observed for curcumin (IC = 18
5
ꢀ
M
0
2
4
).
Fig. 5 Peptide adduction by 2,6-dimethyl-curcumin and its
degradation products. A 27-amino acid peptide containing
redox-regulated Cys179 of Inhibitor of nuclear factor
kinase subunit (IKK ) (10 M) was reacted with 2,6-
dimethyl-curcumin (50 M) at 37 C for 1 h. An aliquot of the
κ-B
β
β
ꢀ
ꢀ
°
reaction was analyzed using LC-MS. (a) The first panel shows
the total ion chromatogram (TIC) and the following panels
show extracted ion chromatograms for peptide adducts with
2,6-dimethyl-curcumin (+396; m/z 1703), epoxides
2 and 3
(
+412; m/z 1711), methoxyphenol (+122; m/z 1567), 1-
Fig. 4 Anti-inflammatory activity of 2,6-dimethyl-curcumin. (a)
hydroxy-ethyl-methoxyphenol (+166; m/z 1589), and for the
unreacted peptide and its dimer, respectively, (m/z 1506). (b)
RAW264.7 cells stably expressing luciferase downstream of an
NF-κ
dimethyl-curcumin (1-50
B response element were treated with vehicle or with 2,6-
M) 1 h prior to LPS (100 ng/mL).
Total ion chromatogram for analysis of the IKK
incubation with 2,6-dimethyl-curcumin and of a Cys179Ala
mutant IKK peptide (10 M) reacted with 2,6-dimethyl-
curcumin (50 M) for 1 h.
β peptide before
ꢀ
Cells were harvested 4 h after LPS addition, and luciferase
activity was measured in the cell supernatant. (b) Western blot
detection of COX-2 in RAW264.7 cells treated with 2,6-
dimethyl-curcumin or curcumin 1 h prior to stimulation with
LPS (100 ng/mL). The cells were harvested after 5 h, and
protein was resolved by SDS-PAGE. The image shown is
representative of three replicates with identical results.
β
ꢀ
ꢀ
The peptide was added to autoxidation reactions of 2,6-
dimethyl-curcumin, and peptide adducts were analyzed using
LC-MS (Fig. 5a). The most prominent adduct was formed by
addition of 122 amu, indicating adduction of the
methoxyphenol terminal group of 2,6-dimethyl-curcumin. An
identical adduct was observed when the same peptide was
Conversely, 2,6-dimethyl-curcumin was less potent than
curcumin in inhibiting the expression of COX-2 in LPS-
stimulated RAW264.7 cells (Fig. 4b). Decrease of COX-2
expression required 2,6-dimethyl-curcumin at concentration
24
incubated with curcumin . Less abundant adducts were
formed by the addition of 396 amu, representing the relative
4
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mass of 2,6-dimethyl-curcumin, and 412 amu, the relative Instability of 2,6-dimethyl-curcumin
ARTICLE
mass of epoxides
addition of 166 amu, likely representing a hydroxy-ethyl-
methoxyphenol cleavage product. The latter may result by
fragmentation of an intermediate C-6 alkoxyl radical during the
2 and 3. A fourth adduct was formed by the
Introduction of methyl groups at the 2- and 6-position of the
heptadienedione chain of curcumin increases stability against
16
β
-
enzymatic reduction . The gain in metabolic stability was
suggested to account for the increased anti-angiogenic
16
potency of 2,6-dimethyl-curcumin compared to curcumin .
Reduction is not the only possible metabolic transformation of
formation of chain-shortened alcohol
mechanism in Scheme 1).
5
(see a proposed
curcumin. Curcumin is chemically unstable at physiological pH
19, 20
Using a 5-fold molar excess of 2,6-dimethyl-curcumin over
peptide, there was no unreacted peptide left, although some
peptide had dimerized under these conditions (Fig 5a, b).
When a Cys179Ala mutant peptide was used, no adducts with
and undergoes rapid autoxidative degradation
. Here we
show that the addition of methyl groups to curcumin at C-2
and C-6 did not increase chemical stability, in contrast to the
gain in stability against enzymatic reduction. Similar to
curcumin 2,6-dimethyl-curcumin was subject to spontaneous
autoxidative degradation. The chemical transformation
showed differences but also similarities with the autoxidation
of curcumin.
2,6-dimethyl-curcumin were detectable (Fig. 5b). This
suggested that the mechanism of adduct formation was by a
Michael-type addition of electrophilic degradation products
with the nucleophilic cysteine (Cys179) of the peptide.
Metabolism of 2,6-dimethyl-curcumin in RAW264.7 cells
Differences in degradation to curcumin
We analyzed whether the degradation products and reduced
metabolites were present in the supernatant of RAW264.7
cells incubated with 2,6-dimethyl-curcumin. LC-ESI-SRM
analyses were developed based on characteristic MS2
fragment ions of the major degradation products identified
above. A standard of the reduced metabolite (tetrahydro-2,6-
dimethyl-curcumin) was generated by catalytic hydrogenation
of 2,6-dimethyl-curcumin. RAW264.7 cells transformed 2,6-
The major outcome of oxidative transformation of curcumin is
cyclization of C-2 and C-6 of the heptadienedione chain to
form a cyclopentadione ring and incorporation of two atoms of
20
oxygen into the final products . With 2,6-dimethyl-curcumin
only one atom of oxygen is incorporated in the final and
intermediate products (Fig. 2e). Cyclopentadione formation
was not observed, and instead, cleavage of the methyl-
substituted double bond was a prevalent outcome. As with
curcumin, the oxidation reaction of 2,6-dimethyl-curcumin is
initiated by H-abstraction from the phenolic hydroxyl. H-
abstraction occurs as a sequential proton loss-electron transfer
dimethyl-curcumin to vanillin
whereas the other oxidative products, epoxides
as cleavage product were not detected (Fig. 6). The reduced
1
and chain shortened alcohol
5
2
and as well
3
4
metabolite tetrahydro-2,6-dimethyl-curcumin was also
detected. This indicated that 2,6-dimethyl-curcumin
underwent oxidative as well as reductive metabolic
transformation in the presence of RAW264.7 cells.
28
process (SPLET) , and the methyl groups at C-2 and C-6 that
stabilize the diketo form may influence the kinetics of the
29
initial oxidation reaction . H-abstraction yields a phenoxyl
radical that is stabilized at C-2 (C-6) by the electron-donating
effect of the methyl group. Radical cyclization of the tertiary
radical C-2 to C-6 is disfavored, and instead O is added to form
2
a peroxyl radical (ROO•). Two possibilities for further reaction
of the peroxyl radical appear plausible based on the products
we have identified.
Proposed mechanisms of degradation
The first proposed reaction of the peroxyl radical is
intramolecular addition to the exocyclic double bond of the
quinone methide (Scheme 1a). This gives a 1,2-dioxetane with
the radical transferred to the ring.
aldehydic fragments, one of which is vanillin
carbonyl equivalent of . The latter was not among the
products identified, possibly due to the reactive nature of the
,2,4-tricarbonyl moiety. Dioxetane formation and
β
-Cleavage yields two
1
, the other is the
4
1
Fig. 6. Metabolism of 2,6-dimethyl-curcumin in RAW264.7
fragmentation provides a direct pathway to vanillin in the
degradation of 2,6-dimethyl-curcumin.
cells. RAW264.7 cells were incubated with 2,6-dimethyl-
curcumin (10
analysis was performed in the positive ion mode. The ion
chromatograms for detection of vanillin, product
ꢀM) for 30 min and extracted. LC-SRM-MS
Alternatively, the peroxyl radical can add to the 6,7 (1,2)
double bond of a second molecule of 2,6-dimethyl-curcumin
5,
tetrahydro-2,6-dimethyl-curcumin, and 2,6-dimethyl-curcumin
are shown.
(
Scheme 1b). This results in the formation of a peroxide-linked
dimer. The radical can add to either C-6 or C-7 (C-1, C-2)
forming two isomeric dimers that will eventually yield the
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same products, namely epoxides
2
and
3. Epoxide formation is new bond between C-6 and the methoxyphenol ring is
achieved (1) by intramolecular homolytic substitution (S
the carbon-centered radical at the dimer peroxide bond and peroxide-linked dimer (Scheme 1c) . Following migration of
2) by the resulting alkoxyl radical (RO ) reacting with the the methoxyphenol ring, breaking of the C-6/C-7 bond to form
exocyclic double bond of the quinone methide (Scheme 1b). one-carbon chain shortened alcohol proceeds through a
Thus, both parts of the dimer give the same epoxides ( and mechanism that was not further probed but likely involves
H
i) of provided by radical 1,2-migration of the phenolic ring in the
3
7
(
•
5
2
3)
when the peroxide bond breaks, regardless of whether the additional reaction with oxygen.
peroxyl radical added initially to C-6 or C-7. Equivalent
dimerization pathways to epoxide formation are well Formation of vanillin
precedented in radical reactions during non-enzymatic and
Vanillin is formed by two mechanisms during autoxidative
degradation of 2,6-dimethyl-curcumin (Fig. 2). One is a direct
pathway through a dioxetane radical intermediate. The
predicted complementary cleavage product, a keto analog of
3
0-35
enzymatic lipid oxidation
.
4, has not been identified. The other, indirect pathway
involves epoxides 2 and 3 as reaction intermediates. Vanillin is
the most abundant product of degradation of 2,6-dimethyl-
curcumin, whereas in the autoxidation of curcumin it is only a
3
8
very minor product . The mechanism of how vanillin is
formed from curcumin has not been established although
3
cleavage of a dioxetane intermediate as well as retro Claisen
9
4
0
condensation have been suggested. It is conceivable that the
increased stability of the tertiary radical at C-6 or C-2,
respectively, formed upon H-abstraction, favors reaction with
oxygen in the case of 2,6-dimethyl-curcumin. At the same time
cyclization of the carbon-centered radical with the double
bond is disfavored due to steric bulk provided by the methyl
groups. In the degradation of 2,6-dimethyl-curcumin formation
of the peroxyl radical is a key step toward subsequent carbon
bond cleavage to vanillin, through either the direct (dioxetane)
or indirect (epoxide) pathway. With curcumin, there is no
evidence for formation of a peroxyl radical at C-2 (C-6)
immediately following the initial hydrogen abstraction and
2
0
prior to 5-exo cyclization . This may explain why vanillin is not
an abundant product of the degradation of curcumin.
Scheme 1. Proposed mechanisms of oxidative transformation
of 2,6-dimethyl-curcumin. (a) Direct pathway of formation of
vanillin
b) Formation of epoxides
dimerization of 2,6-dimethyl-curcumin. The first step is
formation of a peroxyl radical (ROO ). In the second step the
1
via
β
-cleavage of a dioxetane radical intermediate. Conclusions
and via peroxide mediated
(
2
3
The experiments with peptide adduction and metabolism in
RAW264.7 cells suggested a key role of oxidative degradation
products in mediating the anti-inflammatory activity of 2,6-
dimethyl-curcumin. The cell incubations showed that 2,6-
dimethyl-curcumin undergoes reductive as well as oxidative
metabolic transformation. Based on LC-MS ion intensities (a
non-quantitative measure of abundance) vanillin was the most
•
peroxyl radical adds to the double bond of a second molecule
of 2,6-dimethyl-curcumin (blue). Addition can occur at either
3
6
C-6 or C-7 and results in the formation of the same epoxides,
and via homolytic intramolecular substitution S i or
addition of the alkoxyl radical to the double bond, respectively.
c) Formation of chain-shortened alcohol by phenyl 1,2-
2
3
H
prominent product, about 10-fold higher than
tetrahydro-2,6-dimethyl-curcumin, respectively.
5 and reduced
(
5
migration rearrangement of a peroxide-linked dimer of 2,6-
dimethyl-curcumin.
A recent analysis of the anti-inflammatory activity of curcumin
suggested that inhibition of NF- B may be mediated by the
κ
degradation products in addition to or possibly even instead of
Epoxides
and further incubation, they both transformed to vanillin and
product (Fig. 2e). This reaction likely involves the addition of
water to introduce a hydroxyl in although a detailed reaction
mechanism could not be proposed.
2 and 3 are unstable intermediates. Upon isolation
24
the parent compound . The same may be the case for 2,6-
4
dimethyl-curcumin. This was supported by the peptide binding
assay that showed more prominent adduction with
degradation products, especially methoxyphenol, than with
4
2
,6-dimethyl-curcumin (Fig. 7). Even for 2,6-dimethyl-curcumin
Product
5 was an unusual product due to the loss of C-7 from
it was not apparent whether adduction occurred via the enone
the molecule. A satisfactory explanation for formation of a
6
| J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
of the parent molecule or the quinone methide oxidation For the UV/Vis assay, 2,6-dimethyl-curcumin (25
product (Fig. 7). concentration) was dissolved in 20 mM NH OAc buffer (pH 7.5)
in a 500 L-cuvette. The sample was analyzed in time-drive
ꢀM final
4
ꢀ
mode following the chromophore at 410 nm and by repetitive
scanning from 700 – 220 nm every 1 min using a Perkin-Elmer
Lambda 35 spectrophotometer. The time-course analysis was
performed using 2,6-dimethyl-curcumin (25
0 mM NH OAc buffer (500 L) in an autosampler vial. The
autosampler was maintained at 25 C, and aliquots were
injected every 1 h over a period of 10 h.
HPLC analysis of product formation was performed using an
Agilent Eclipse XDB-C18 3.5 m column (3.0 × 100 mm) eluted
a flow rate of 0.6 mL/min with a gradient of acetonitrile/water,
0:80 (by volume) to 80:20 (by volume), containing 0.01%
acetic acid, within 20 min. Elution of products was monitored
Fig. 7. Proposed structures of 2,6-dimethyl-curcumin and its using an Agilent 1200 diode array detector.
degradation products adducting to the IKK peptide. The red Large-scale reactions were conducted by dissolving 2,6-
arrow indicates predicted sites of reaction with Cys179 of the dimethyl-curcumin (25
M) in 2 L of 20 mM NH OAc buffer (pH
ꢀM) dissolved in
2
4
ꢀ
°
ꢀ
2
β
ꢀ
4
2
7-amino acid peptide of IKK
β
.
8). The reaction was left at 25 °C for 16 h and extracted with
CH Cl (4 × 100 mL). The organic extracts were combined and
2
2
The anti-inflammatory activity of curcumin involves covalent dried with anhydrous MgSO4 (s). The solvent was evaporated
2
4, 41
adduction to Cys179 of IKK
electrophiles to Cys179 prevents phosphorylation of mixture of methanol/water (50:50) and isolated by RP-HPLC
neighboring serines 177 and 181 and inhibits kinase activity using a Waters Symmetry C18, 5
m column (4.6 × 250 mm)
that is required for the phosphorylation of I and activation with a gradient of 20:80 (by volume) to 80:20 (by volume)
. For curcumin, there is evidence to support the containing 0.01% acetic acid, within 20 min. The flow rate was
hypothesis that adduction to Cys179 occurs by oxidative 1 mL/min. The products were monitored using an Agilent 1200
β
. Adduction of small molecule under a stream of N . The crude products were dissolved in a
2
ꢀ
κBα
4
2
of NF-κB
4
3
metabolites as well as parent curcumin . This implies that the diode array detector. Products were recovered from the
enones in curcumin and 2,6-dimethyl-curcumin are not the solvent by lyophilization.
(
only) functionally relevant electrophilic moiety. The methyl
groups at the 2- and 6-positions of 2,6-dimethyl-curcumin NF-
likely further decrease the ability to form a Michael-type RAW264.7 cells stably expressing luciferase under the control
κ
B inhibition
24
adduct due to increased steric bulk and decreased electrophilic of an NF-
character at C-1 (C-7) by the electron-donating effect of the at 37
C in 5% CO in DMEM containing glucose (4.5 g/L) and
methyl group at the adjacent carbon. Thus, it is unlikely that fetal bovine serum (10%, v/v) in the presence of geneticin (1
,6-dimethyl-curcumin is more potent than curcumin in mg/mL). Cells were treated with 2,6-dimethyl-curcumin at the
inhibiting NF- B if the 1,2-enone were the electrophile concentrations indicated from a stock solution in DMSO (10
reacting with Cys179 of IKK . This supports the hypothesis in mM) for 45 min prior to stimulation with LPS (100 ng/mL). The
which not the parent molecule but its degradation products medium was removed after 4 h, and 50
l were analyzed using
Ready-to-glow Secreted Luciferase Reporter assay
κB response element were used . Cells were grown
°
2
2
κ
β
ꢀ
are the electrophilic agents and thus the ultimate anti-
inflammatory agents.
a
(Clontech). Statistical analysis was performed using GraphPad
Prism 7,00 software using an unpaired non-parametric t-test
(Mann-Whitney) with significance of p < 0.05.
Experimental
COX-2 expression
Materials
RAW264.7 cells were treated with 2,6-dimethyl-curcumin or
Curcumin and 2,6-dimethyl-curcumin were synthesized
1
6, 44
curcumin (1-50
ꢀ
ng/mL). Cells were harvested after 5 h, and protein (5
M) for 1 h prior to treatment with LPS (100
g each)
according to published procedures
. The IKK
LDQGSLCTSFVGTLQYLAPELLEQQK ; M 3012.43 g/mol,
obtained by in silico tryptic digest of human IKK ) was
synthesized by United Biosystems Inc. (Herndon, VA).
RAW264.7 cells were from ATCC. For quantification of NF-
activity RAW264.7 cell stably transfected with a pNFkB-TA-
β peptide
1
72
198
ꢀ
(E
r
was resolved on a 10% SDS-PAGE gel. Gels were blotted onto
nitrocellulose membranes and probed with antibodies for
COX-2 (mouse polyclonal, catalog-# 160126, Cayman Chemical)
β
κ
B
and β-actin (mouse monoclonal, 8H10D10, catalog-# 3700, Cell
Signaling Technology) as loading control.
2
4
MetLuc with TB vector (Clontech) were used
.
Peptide adduction and analysis
Degradation reactions and HPLC analysis
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ARTICLE
Journal Name
IKKβ peptide (10 μM) was incubated with 2,6-
dimethylcurcumin (50 μM) at 37°C in 20 mM NH OAc buffer,
Acknowledgements
4
This work was supported by award R01AT006896 from the
National Center for Complementary and Integrative Health
pH 8, for 1 h. Aliquots were analyzed directly using a Waters
Synapt HDMS Q-TOF instrument in positive ionization mode
following separation of the sample using a Waters XSelect C18
(NCCIH) and the Office of Dietary Supplements (ODS) of the
National Institutes of Health (NIH). NAP acknowledges support
by award R01ES024133 from the National Institute of
Environmental Health Sciences of NIH. PBL is supported by
postdoctoral fellowship award 16POST27250138 from the
American Heart Association. Mass spectrometric analyses
were in part performed through Vanderbilt University Medical
Center’s Digestive Disease Research Center supported by NIH
grant P30DK058404 Core Scholarship. The content is solely the
responsibility of the authors and does not necessarily
represent the official views of the NIH.
3
.5 ꢀm column (2.1 ×50 mm) with a gradient of
acetonitrile/water, 15:85 (by volume) to 65:35 (by volume),
containing 0.1% formic acid, within 10 min at a flow rate of
0.25 mL/min.
Metabolism in RAW264.7 cells
RAW264.7 cells were seeded in 6-well plates using DMEM
containing 10% fetal bovine serum. After 24 h 2,6-dimethyl-
curcumin (10
added to the cells and incubated for 30 min. Media (800
were removed, acidified (pH 4-5), and extracted using 30-mg
Waters HLB cartridges. The cartridges were eluted with Notes and references
ethylacetate (350 L) and methanol (700 L), and the
combined eluates were evaporated under a stream of
ꢀ
M; from a 10 mM stock solution in DMSO) was
ꢀ
l)
ꢀ
ꢀ
‡
2,6-Dimethyl-curcumin is symmetric, and thus C-1 through C-3
are identical to C-7 through C-4. We decided to describe the
nitrogen. The residue was dissolved in 700
L acetonitrile and reactions as occurring at C-6/C-7 because that avoided changing
numbers of the carbon atoms in the 7-carbon linker chain of
00 l water, and 10 ꢀl were analyzed by LC-SRM-MS.
ꢀ
1
ꢀ
product
5.
LC-MS
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. T. Esatbeyoglu, P. Huebbe, I. M. Ernst, D. Chin, A. E. Wagner
A TSQ Vantage Triple Quadrupole LC-MS instrument with and G. Rimbach, Angew. Chem. Int. Ed. Engl., 2012, 51, 5308-
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curcumin: m/z 397.2
92.9 (10 eV); for
61.1 (-H O) 137.1 (20 eV); for
15 eV); for tetrahydro-2,6-dimethyl-curcumin: m/z 401.5
37.0 (15 eV).
→
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: m/z 413.2 137.1 (15 eV); for
: m/z 383.2 (-H O)
1
: m/z 153.1 5. J. Yu, Y. Peng, L. C. Wu, Z. Xie, Y. Deng, T. Hughes, S. He, X.
: m/z Mo, M. Chiu, Q. E. Wang, X. He, S. Liu, M. R. Grever, K. K. Chan
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,
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(
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. B. Chandran and A. Goel, Phytotherapy research : PTR, 2012,
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. G. W. Small, P. Siddarth, Z. Li, K. J. Miller, L. Ercoli, N. D.
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8
Samples were dissolved in 150
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L of methanol-d
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O in 3-
Emerson, J. Martinez, K. P. Wong, J. Liu, D. A. Merrill, S. T. Chen,
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residual non-deuterated solvent signals (δ 3.31 for methanol-
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Degradation products are linked to anti-inflammatory activity of 2,6-dimethyl-
curcumin, a synthetic analog of curcumin stable against metabolic reduction in
vivo.