How curcumin works preferentially with water soluble antioxidants

Article abstract of DOI:10.1021/ja003823x

In this study we investigated physicochemical characteristics of the curcumin radical by pulse radiolysis and laser flash photolysis. Two methylated curcumin derivatives, methylcurcumin and trimethyl-curcumin, were synthesized to explore the role of phenol hydroxy and β-diketone moieties in the free radical chemistry of curcumin. Our results show that the initially generated β-oxo-alkyl transforms rapidly, probably via an intramolecular H-atom shift, into the phenoxyl-type curcumin radical. This phenoxyl does not react with oxygen, k < 10⁵ M^-1 s^-1, and can be repaired by any water-soluble antioxidant with appropriate redox potential, E₆ < 0.83 V, for example, with vitamin C, k = (6 ± 1) × 10⁶ M^-1 s^-1. A molecular mechanism of cancer chemoprevention by curcumin is proposed, with special emphasis on the synergism with water-soluble antioxidants.

Full text of DOI:10.1021/ja003823x

3064
J. Am. Chem. Soc. 2001, 123, 3064-3068
How Curcumin Works Preferentially with Water Soluble Antioxidants
Slobodan V. Jovanovic,*,† Charles W. Boone,‡ Steen Steenken,§ Manuela Trinoga,§ and
Robert B. Kaskey
Contribution from Helix International, 381 Viewmount DriVe, Nepean, ON, Canada K2E 7R9, Department
of Pathology, Georgetown UniVersity Medical School, Washington, DC, Max-Planck-Institut fu¨r
Strahlenchemie, 34-36 Stiftstrasse, Mu¨lheim, Germany, and Gene Print, Inc., Bala Cynwyd, PennsylVania
ReceiVed October 30, 2000. ReVised Manuscript ReceiVed February 2, 2001
Abstract: In this study we investigated physicochemical characteristics of the curcumin radical by pulse
radiolysis and laser flash photolysis. Two methylated curcumin derivatives, methylcurcumin and trimethyl-
curcumin, were synthesized to explore the role of phenol hydroxy and â-diketone moieties in the free radical
chemistry of curcumin. Our results show that the initially generated â-oxo-alkyl transforms rapidly, probably
via an intramolecular H-atom shift, into the phenoxyl-type curcumin radical. This phenoxyl does not react
with oxygen, k < 105 M-1 s-1, and can be repaired by any water-soluble antioxidant with appropriate redox
potential, E6 < 0.83 V, for example, with vitamin C, k ) (6 ( 1) × 106 M-1 s-1. A molecular mechanism
of cancer chemoprevention by curcumin is proposed, with special emphasis on the synergism with water-
soluble antioxidants.
Introduction
of protein kinases,8 and especially its neutralization of car-
cinogenic free radicals.9,15-25 It is possible that any one, more
than one, or all of these biological, biochemical, and chemical
mechanisms are responsible for the anticarcinogenic potential
of curcumin. While biological and biochemical mechanisms tend
to be tissue and organ specific, chemical anti-carcinogenesis,
e.g. free radical neutralization, works at all levels of a biological
system.
Numerous studies have demonstrated the remarkable cancer
preventive properties of curcumin.1-10 This natural antioxidant
inhibits cancerous growth in various cancer models alone and
in combinations with other “plant phenolics”. The chemopre-
ventive effects of curcumin have been attributed to various
properties, including its anti-angiogenesis action,11,12 which
limits the blood supply to rapidly growing malignant cells, its
stimulation of Phase I and Phase II detox systems, e.g. inhibition
of COX-1 and COX-2 enzymes, and stimulation of glutathione
S-transferase,13,14 its interference with cell growth by inhibition
Of particular interest is the ability of curcumin to intercept
and neutralize potent chemical carcinogens, such as ROS
(superoxide, peroxyl, hydroxy radicals) and NOS (nitric oxide,
peroxynitrite).9,15-25 The inactivation of carcinogenic free
radicals, which induce and propagate chemical and biochemical
processes involved in the inflammatory response, could be the
major chemical anti-carcinogenic mechanism of curcumin and
curcuminoids. It is usually assumed that the phenol moiety is
responsible for antioxidant properties of any plant phenolic
compound. Consequently, the free radical chemistry of curcumin
(an o-methoxyphenol derivative) has focused on its phenol
rings.15,17-19 Other studies have pointed to the possible involve-
ment of the â-diketone moiety in the antioxidant action of
curcumin and its derivatives.9,25,26 A recent report by some of
us24 describes the H-atom donation from the â-diketone moiety
† Helix International.
‡ Georgetown University Medical School.
§ Max-Planck-Institut fu¨r Strahlenchemie.
Gene Print, Inc.
(1) Cheng, A. L.; Lin, J. K.; Hsu, M. M.; Shen, T. S.; Ko, J. Y.; Lin, J.
T.; Lin, B. J.; Wu, M. S.; Yu, H. S.; Jee, S. H.; Chen, G. S.; Chen, T. M.;
Chen, C. A.; lai, M. K.; Pu, Y. S.; Pan, M. H.; Wang, Y. J.; Tsai, C. C.;
Hsieh, C. Y. American Society of Clinical Oncology, 1998 Annual Meeting;
Abstract 2140.
(2) Singh, S. V.; Hu, X.; Srivastava, S. K.; Singh, M.; Xia, H.; Orchard,
J. L.; Zaren, H. A. Carcinogenesis 1998, 19, 1357.
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Carcinogenesis 1998, 19, 419.
(4) Reddy, B. S.; Kawamori, T.; Rao, C. V.; Lubet, R. A.; Steele, V. E.;
Kelloff, G. J. Proc. Am. Assoc. Cancer Res. 1979, 39, 126.
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B.; Rao, C. V.; Reddy, B. S. Cancer Res. 1999, 59, 597.
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Cancer Res. 1997, 57, 1301.
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Yamaguchi, H. J. Agric. Food Chem. 1999, 47, 71.
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H. K.; Maheswari, R. K. Cell Growth Differ. 1998, 9, 305.
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T.; Fisher, C.; Flynn, E.; Byers, H. R. Mol. Med. 1998, 4, 376.
(13) Venkatesan, N. Br. J. Pharmacol. 1998, 124, 425.
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10.1021/ja003823x CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/09/2001

Physicochemical Characteristics of the Curcumin Radical
J. Am. Chem. Soc., Vol. 123, No. 13, 2001 3065
to a lipid alkyl or a lipid peroxyl radical as a potentially more
important antioxidant action of curcumin.
Chart 1
In the case of the H-atom donation to a bis-allylic radical
(e.g. the linoleic acid radical) the following reaction occurs:
It is possible that the resulting resonance-stabilized â-oxo-alkyl
curcumin radical,24 with unpaired electron density distributed
between three C and two O atoms, adds oxygen at the central
C atom to become a peroxyl radical. This would be an
undesirable reaction because peroxyl radicals propagate lipid
peroxidation, which is highly damaging of cell membranes.27
In comparison, when the addition of oxygen is inefficient, the
curcumin radicals may react with each other or with other free
radicals to yield stable products, such as vanillin, ferrulic acid,
and curcumin dimers.9 The chemical and antioxidant properties
of these stable products are considerably different from those
of curcumin.28,29
the curcumin radical. Our results indicate that the phenoxyl is
the predominant curcumin radical. This radical is formed either
by direct one-electron oxidation of a methoxyphenol moiety or
by an intramolecular H-atom transfer from any methoxyphenol
ring to the incipient â-oxo-alkyl radical. The implications of
these findings for the chemoprevention of cancer by naturally
occurring curcumin, demethoxycurcumin, and bisdemethoxy-
curcumin are discussed below. A model for the molecular basis
of the synergism between curcumin and water-soluble antioxi-
dants is presented.
The question arises, what is the molecular basis of the
reported synergism3 of curcumin with catechin and epigallo-
catechin gallate? The water-insoluble curcumin molecule resid-
ing in the central lipid layer of cell membranes is probably not
even close to the aqueous milieu adjacent to the membranes in
which the water-soluble catechins are located. Assuming a
mechanism similar to the well-known synergism between lipid-
soluble (water-insoluble) vitamin E and water-soluble ascorbate
(vitamin C),30 it may be postulated that the curcumin radical,
generated by antioxidant action, positions itself at the border
of the cell membrane adjacent to the aqueous milieu, in short
“pops out” of the membrane, to be repaired by the catechins.
However, for this mechanism to operate, the curcumin radical
must be more polar than curcumin itself and, in addition, be
able to oxidize catechins, a requirement that is satisfied by the
curcumin phenoxyl radical.
Materials and Methods
Chemicals used in this study were of the highest purity available.
Curcumin (1, 99.95% purity) was the product of R-Kane Inc., N.J. The
structures of curcumin and methylated curcumin derivatives are
presented in Chart 1. Acetonitrile, dimethyl sulfoxide, methyl iodide,
potassium carbonate, sodium hydrogenphosphate, and potassium
dihydrogenphosphate were the products of Merck. Bis(azo-methyl-
amidine) dihydrochloride and tert-butyl peroxide were obtained from
Aldrich. Water used to prepare all solutions was purified through a
Millipore Milli-Q system to a resistivity better than 18 MΩ/cm. Prior
to irradiation, the solutions were saturated with high-purity gases
(Ar, N2O, O2) by gentle bubbling for 30 min. The pH of aqueous
solutions was adjusted with a phosphate buffer.
Fully computerized laser flash photolysis at the Max-Planck-Institut
fu¨r Strahlenchemie31 was used for photochemical investigations. A 248
nm Lambda Physik excimer laser was used to irradiate the samples
contained in the 0.1 cm flow-cell. The flow rate of ∼1 mL/min, which
ensured a constant supply of fresh solution, was controlled by a
peristaltic pump. The absorbance of the solution to be irradiated was
adjusted so that less than 10% of the laser light is absorbed by curcumin
derivatives to minimize their photolysis.
In this study we used pulse radiolysis of aqueous and laser
flash photolysis of acetonitrile solutions of curcumin and two
methylated curcumin derivatives to determine the properties of
(26) Schaich, K. M.; Fisher, C.; King, R. In Food phytochemicals for
cancer preVention II. Teas, Spices, and Herbs; Ho, C.-T., Osawa, T., Huang,
M.-T., Rosen, T. R., Eds.; ACS Symp. Ser. No. 547; American Chemical
Society: Washington, DC, 1994.
(27) von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor
and Francis: London, 1987.
(28) Jovanovic, S. V.; Steenken, S.; Tosic, M.; Marjanovic, B.; Simic,
M. G. J. Am. Chem. Soc. 1994, 116, 4846.
(29) Jonsson, M.; Lind, J.; Reitberger, T.; Eriksen, T. E.; Mere´nyi, G. J.
Phys. Chem. 1993, 97, 11278.
The 3 MeV Van-de-Graaff pulse radiolysis equipment with optical
detection at the Max-Planck-Institute fu¨r Strahlenchemie32 was used
for the pulse radiolysis studies. A 2 cm supra-sil quartz cell with
temperature variation through a thermostatically controlled liquid jacket
was used for sample irradiation.
The rate constants were determined at ∼1-2 Gy/pulse to minimize
interference from radical-radical reactions. Thiocyanate dosimetry was
(30) Doba, T.; Burton, G. W.; Ingold, K. U. Biochim. Biophys. Acta 1985,
835, 298.
(31) Anklam, E.; Steenken, S. J. Photochem. Photobiol. 1988, 43A, 233.
(32) Jagannadham, V.; Steenken, S. J. Am. Chem. Soc. 1984, 106, 6542.

3066 J. Am. Chem. Soc., Vol. 123, No. 13, 2001
JoVanoVic et al.
used in dose determinations, assuming G[(SCN)2•-] ) 6.2 × 10-7 mol
J-1 in N2O-saturated 10 mM KSCN aqueous solutions.
Synthesis and Characterization of Methylcurcumin (2). Meth-
ylcurcumin, 1-(3,4-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-
1,6-heptadiene-3,5-dione, was synthesized by a mild methylation of
curcumin. Curcumin (0.1 M) was refluxed with 1.9 M methyl iodide
in the presence of 0.024 M sodium carbonate in acetone at 60 °C for
40 h. The resulting mixture contained ∼35% 2 (according to HPLC
analysis). The mixture was evaporated to dryness on a rotary evaporator.
2 was separated by a preparative reverse phase HPLC in acetonitrile/
water. Evaporation of the HPLC fraction yielded dark yellow viscous
oil. The compound was identified by the 500 MHz 2D NMR
spectroscopy. The mass spectrum confirmed the structure of 2, M+ at
382, and additional peaks at 364, 191, and 181. The purity of the
compound according to the HPLC and mass spectroscopy was higher
than 99%.
Synthesis and Characterization of Trimethylcurcumin (3). Tri-
methylcurcumin, 1,7-di(3,4-dimethoxyphenyl)-4-methyl-1,6-heptadiene-
3,5-dione (3), was synthesized by a prolonged mild methylation of
curcumin. Curcumin (0.05 M) was stirred with 1.9 M methyl iodide in
the presence of 0.024 M sodium carbonate in acetone at 20 °C for 80
h. The resulting mixture, which contained more than 90% of 3, was
evaporated to dryness on a rotary evaporator and dissolved in ethyl
acetate. The ethyl acetate solution was washed with three volumes of
water and evaporated to dryness. The orange powder was further
purified by a preparative reverse-phase HPLC. The less polar component
was collected and characterized by the 500 MHz 2D NMR and mass
spectroscopy. The mass spectrum of the powder confirmed the presence
of trimethylcurcumin, M+ at 410, and additional peaks at 219, 191,
etc. The purity of the compound according to the mass spectra was
higher than 99%.
Figure 1. Transient absorption spectra upon completion of the reaction
of (CH3)3CO• with curcumin derivatives obtained by 248 nm laser flash
photolysis of Ar-saturated acetonitrile solution containing 10% water,
0.8 M tert-butylperoxide, and 0.05 mM curcumin (1) and its methyl
derivatives (2 and 3).
Results and Discussion
The curcumin radical has been generated by the pulse
radiolysis of an N2O-saturated 40% aqueous DMSO at pH 5.4
by the following set of reactions,24,33
Figure 2. Transient absorption spectra of radicals from 3 generated
by laser flash photolysis of acetonitrile solutions containing 10% water,
0.8 M tert-butylperoxide, and 0.05 mM 3: (b) Ar-saturated solution;
(O) O2 -saturated solution. Laser power was kept at ∼15% to supress
second-order processes.
H2O ' H, •OH, e-aq, H3O+, etc.
(1)
(2)
e-aq + N2O + H3O+ f •OH + H2O + N2
Oxidation of ascorbate and the lack of reactivity with oxygen
suggest that the curcumin radical is an oxyl rather than a carbon-
centered radical. The oxyl curcumin radical(s) are very likely a
mixture of the phenoxyl, where the unpaired electron is
delocalized over the phenol ring and the adjacent allyl chain,
and the â-oxo-alkyl, where the unpaired electron is delocalized
within the â-diketone linkage. We used the tert-butoxyl and
methylamidino peroxyl radicals to selectively generate alkoxyl
radicals from 1, 2, and 3 and study their spectral and kinetic
characteristics.
The tert-butoxyl radical is well-known alkoxyl radical, which
reacts preferentially by an H-atom abstraction. The tert-butoxyl
radical was generated by photolysis of 0.8 M tert-butylperoxide
in Ar-saturated acetonitrile. It reacts rapidly with the curcumin
derivatives, k ∼ 5 × 109 M-1 s-1, as derived from the
“bleaching” of strongly yellow-colored solutions. The resulting
transient spectra are presented in Figure 1.
As seen from Figure 1, the absorption spectra of radicals from
1, 2, and 3 are similar, the slight differences in “bleaching”
originating from different UV-vis absorption spectra. The
disappointing lack of any significant difference between the
spectra of radicals from 1 and 2, where both phenoxyl and
â-oxo-alkyl radicals may be generated in different proportions,
and 3, where only â-oxo-alkyl radicals are possible, is a
consequence of an apparent overlap of strongly absorbing parent
compounds and weak spectral bands of the daughter radicals.
However, in the presence of oxygen (at 2 and 20 mM), the
decay of radicals from 1 and 2 (with typical τ1/2 ∼ 100 µs)
•OH + (CH3)2SO f •CH3 + CH3SO2- + H3O+ (3)
•CH3 + (1) f CH4 + •(1); k ) 3.5 × 109 M-1 s-1 (4)
At 0.65 mM curcumin (1), the curcumin radical is generated
within 2 µs. Upon addition of ascorbate in concentrations from
0.3 to 3.4 mM, the spectrum of the curcumin radical decayed
with an apparent pseudo-first-order kinetics, as monitored at
510 nm. From the pseudo-first-order rate, the second-order rate
of k ) (6 ( 1) × 106 M-1 s-1 is derived.
This rate is slower than k ) 2.5 × 107 M-1 s-1 reported17
for the same reaction in acetonitrile/aqueous buffer (1:4) at pH
7. The reason for this discrepancy may be our choice of lower
pH 5.4. The reaction may be slower because of higher reduction
potential of the ascorbate radical at this pH (E5.4 ) 0.40 V, vs
E7 ) 0.28 V).34
Regardless of the method of generation (pulse radiolysis or
laser flash photolysis), the decay of the curcumin radical was
not affected by oxygen in concentrations from 0.2 to 2 mM.
From the second-order rate of the radical decay the upper limit
of the rate can be set, k < 105 M-1 s-1, in full agreement with
the earlier study.17
(33) Veltwisch, D.; Janata, E.; Asmus, K.-D. J. Chem. Soc., Perkin Trans.
2 1980, 146.
(34) Steenken, S.; Neta, P. J. Phys. Chem. 1982, 86, 3661.

Physicochemical Characteristics of the Curcumin Radical
J. Am. Chem. Soc., Vol. 123, No. 13, 2001 3067
Scheme 1
Figure 3. Transient absorption spectra upon completion of the reaction
of the methylpropionamidine peroxyl radical with curcumin derivatives
obtained by 248 nm laser flash photolysis of an air-saturated solvent
mixture (1:1 water/acetonitrile) containing 50 mM 2,2′-azobis(2-
methylpropionamidine)‚2HCl and 0.05 mM curcumin (1) and its methyl
derivatives (2 and 3).
remains unaffected in the studied spectral range, whereas the
360 nm band in the spectrum of the radical derived from 3
rapidly decays. Such decay indicates the reaction of the â-oxo-
alkyl radical from 3 with oxygen, as shown below. From the
We further studied the reactions of the alkyl peroxyl radicals
with curcumin and its methylated derivatives in physiologically
more relevant aqueous solutions.
The methylpropionamidine radicals were generated by pho-
todecomposition of 50 mM 2,2′-azobis(2-methylpropionamidine)‚
2HCl in acetonitrile/water (1:1) at pH 5.0, i.e.
pseudo-first-order decay, the reaction rate constant of the radicals
from 3 with oxygen can be estimated as k ∼ 108 M-1 s-1 in
pure acetonitrile and k ∼ 107 M-1 s-1 in acetonitrile/water
(9:1). The spectrum of the radicals from 3 in Ar-saturated
acetonitrile and upon saturation of the same solution with oxygen
is shown in Figure 2.
The addition of oxygen to the â-oxo alkyl curcumin radical,
at k ∼ 107 M-1 s-1, is slow in comparison with the diffusion-
controlled reactions of alkyl radicals (k ∼ 109 M-1 s-1).27 The
slow reaction with oxygen is suggested to be caused by
resonance-stabilization of the â-oxo alkyl radical. Similar
behavior was reported for the linoleic acid allylic radical, with
k ) 1.8 × 108 M-1 s-1 35
.
(35) AlSheikhly, M.; Simic, M. G. J. Phys. Chem. 1989, 93, 3103.

3068 J. Am. Chem. Soc., Vol. 123, No. 13, 2001
JoVanoVic et al.
In air-saturated solutions ([O2] ∼ 0.2 mM), the initially
generated methyl propionamidine radicals add oxygen at close
to a diffusion controlled rate of k ∼ 109 M-1 s-1 to become
peroxyl radicals. These peroxyl radicals readily react with
curcumin and its methylated derivatives in concentrations from
0.1 to 0.6 mM at k ∼ 108 M-1 s-1. The spectra of the transients
from these reactions are presented in Figure 3.
As seen from Figure 3, the spectra of radicals derived from
curcumin and its methylated derivatives are similar to those
generated by the tert-butoxyl reaction, with the notable exception
of 3, where the 360 nm band is missing from the spectrum
generated in oxygenated aqueous solutions. The initially gener-
ated â-oxo-alkyl radical of 3 reacts with oxygen to generate
the corresponding peroxyl radical. The similarity of spectra of
the radicals from 3 generated by the butoxyl radical in the
presence of oxygen and the alkyl peroxyl radical (see Figures
2 and 3) is taken to support this conclusion.
Curcumin and its derivatives efficiently and rapidly react with
the methyl propionamidine peroxyl radicals, which are the model
for lipid peroxyl radicals. Such antioxidant action of curcumin
and methylcurcumin may be expected from the favorable
reduction potential of the phenol moiety, E6) 0.83 V, e.g., there
is a ∼0.23 V or E ∼ 5 kcal/mol driving force for the reduction
of methyl peroxyl radicals.36 Trimethylcurcumin, 3, reacts
rapidly with alkyl peroxyl radicals by an apparent H-atom
donation. However, the latter reaction generates a radical that
reacts with oxygen, leading to the possible propagation of the
peroxidation. Therefore, the â-diketone moiety alone does not
have antioxidant properties. Apparently, the presence of both
â-diketone and phenol is necessary for optimal antioxidant
function of curcumin.
Conclusions
Our results indicate that one of the curcumin oxyl radicals
generated by its antioxidant action undergoes “molecular
reorganization”, i.e., the initially generated curcumin â-oxo alkyl
is transformed into the phenoxyl radical. When the molecular
reorganization is not possible, as in trimethylcurcumin, the
curcumin â-oxo alkyl radical adds oxygen at k ) 107-108 M-1
s-1 in aqueous and acetonitrile solutions, respectively, to
generate potentially damaging peroxyl radical.
The reduction potential of the phenoxyl curcumin radical is
∼0.8 V (0.83 V at pH 6)24 at physiological pH 7. Consequently,
curcumin may be fully repaired by electron donors with
favorable oxidation potentials, that is with E7 lower than ∼0.8
V, such as epigallocatechin gallate (E7 ) 0.43 V), catechin
(E7 ) 0.55 V), or vitamin C (0.28 V). Such electron transfer
(with accompanying proton transfer) reaction will maintain
optimal concentration of curcumin in the cell membrane at the
expense of a water-soluble antioxidant. Therefore, highly
beneficial effects of curcumin, which are otherwise lost because
of its fast turnover and low physiological uptake, may be
successfully maintained with the more bioavailable and water-
soluble catechins.
If generated by one-electron oxidation, the phenoxyl radicals
from demethoxycurcumin and bisdemethoxycurcumin, which
are present with curcumin in natural extracts of Curcuma Longa,
will be more reactive and damaging than that of curcumin,
because of their considerably higher reduction potentials. The
reduction potential of the p-styryl-phenoxyl at pH 7 can be
estimated as 1.00 V37(the phenoxyl present in bis-demethoxy-
curcumin), which is ∼0.2 V higher than the corresponding
potential of the curcumin radical. Moreover, this is higher than
the 0.90 V needed to oxidize tyrosine,38 an ubiquitous amino
acid, often crucial for enzyme activity. Therefore, demethoxy-
and bisdemethoxycurcumin are considerably inferior physi-
ological antioxidants to curcumin.
We propose that as a result of higher resonance stabilization
afforded in the phenoxyl radical, the initially generated curcumin
alkoxyl radical undergoes rapid intramolecular H-shift, as
presented below.
Pure curcumin is an extremely potent lipid-soluble antioxi-
dant. It positions itself within the cell membrane, where it
intercepts lipid (peroxyl) radicals and becomes a phenoxyl
radical. Being more polar than curcumin, the phenoxyl radical
may “travel” to the surface of the membrane, where it may be
repaired by any water-soluble antioxidant with E7 < 0.80 V.
Scheme 1 illustrates the physiological antioxidant mechanism
of curcumin and synergism with a water-soluble antioxidant such
as epigallocatechin gallate (EGCG).
Acknowledgment. S.V.J. would like to thank Prof. Z.
Stojanac for helpful discussions on the synthesis of natural
curcuminoids.
JA003823X
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