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Antioxidant and radical scavenging properties

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ARTICLE in CHEMICO-BIOLOGICAL INTERACTIONS AUGUST 2008
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Chemico-Biological Interactions 174 (2008) 2737

Contents lists available at ScienceDirect

Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Antioxidant and radical scavenging properties of curcumin

in
Tuba Ak, I lhami Gulc
Faculty of Arts and Sciences, Department of Chemistry, Atat
urk University, TR-25240 Erzurum, Turkey

a r t i c l e

i n f o

Article history:
Received 21 January 2008
Received in revised form 30 April 2008
Accepted 1 May 2008
Available online 7 May 2008
Keywords:
Antioxidant activity
Curcumin
Metal chelating
Reducing power
Radical scavenging

a b s t r a c t
Curcumin (diferuoyl methane) is a phenolic compound and a major component of Curcuma longa L. In the present paper, we determined the antioxidant activity of curcumin by
employing various in vitro antioxidant assays such as 1,1-diphenyl-2-picryl-hydrazyl free
radical (DPPH ) scavenging, 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)
radical scavenging activity, N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD)
radical scavenging activity, total antioxidant activity determination by ferric thiocyanate,
total reducing ability determination by the Fe3+ Fe2+ transformation method, superoxide
anion radical scavenging by the riboavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe2+ ) chelating activities. Curcumin inhibited 97.3% lipid
peroxidation of linoleic acid emulsion at 15 g/mL concentration (20 mM). On the other
hand, butylated hydroxyanisole (BHA, 123 mM), butylated hydroxytoluene (BHT, 102 mM),
-tocopherol (51 mM) and trolox (90 mM) as standard antioxidants indicated inhibition
of 95.4, 99.7, 84.6 and 95.6% on peroxidation of linoleic acid emulsion at 45 g/mL concentration, respectively. In addition, curcumin had an effective DPPH scavenging, ABTS+
scavenging, DMPD+ scavenging, superoxide anion radical scavenging, hydrogen peroxide
scavenging, ferric ions (Fe3+ ) reducing power and ferrous ions (Fe2+ ) chelating activities.
Also, BHA, BHT, -tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. According to the present study, curcumin can be used in the
pharmacological and food industry because of these properties.
2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Oxygen consumption inherent in cell growth leads to
the generation of a series of reactive oxygen species (ROS)
[1]. They are continuously produced by the bodys normal
use of oxygen such as respiration and some cell-mediated
immune functions. ROS include free radicals such as superoxide anion radicals (O2 ), hydroxyl radicals (OH ) and
non-free radical species such as hydrogen peroxide (H2 O2 )
and singlet oxygen (1 O2 ) [2]. ROS are continuously produced during normal physiologic events and can easily
initiate the peroxidation of membrane lipids, leading to

Corresponding author. Tel.: +90 442 2314444; fax: +90 442 2360948.
E-mail addresses: igulcin@atauni.edu.tr,
in).
igulcin@yahoo.com (I . Gulc
0009-2797/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2008.05.003

the accumulation of lipid peroxides. ROS are also capable

of damaging crucial biomolecules such as nucleic acids,
lipids, proteins and carbohydrates and may cause DNA
damage that can lead to mutations. If ROS are not effectively
scavenged by cellular constituents, they lead to disease
conditions. ROS have been implicated in more than 100
diseases [3].
All aerobic organisms have antioxidant defences, including antioxidant enzymes and antioxidant food constituents,
to remove or repair the damaged molecules. Antioxidant
compounds can scavenge free radicals and increase shelf
life by retarding the process of lipid peroxidation, which
is one of the major reasons for deterioration of food and
pharmaceutical products during processing and storage [4].
Antioxidants can protect the human body from free radicals and ROS effects. They retard the progress of many
chronic diseases as well as lipid peroxidation [5,6]. Hence,

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28

T. Ak, I . G
ulcin / Chemico-Biological Interactions 174 (2008) 2737

a need for identifying alternative natural and safe sources

of food antioxidants has been created, and the search for
natural antioxidants, especially of plant origin, has notably
increased in recent years. Antioxidants have been widely
used as food additives to provide protection against oxidative degradation of foods. At the present time, the most
commonly used antioxidants are butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), propylgallate and
tert-butyl hydroquinone. However, BHA and BHT have been
suspected of being responsible for liver damage and carcinogenesis [7,8]. Therefore, there is a growing interest in
natural and safer antioxidants [9,10].
Curcuma longa L. has been used for hundreds of years as a
avor, color, and preservative. Commercially, it is traded as
a dye, spice, and source of industrial starch [11]. Curcumin is
a nutriceutical compound reported to possess therapeutic
properties against a variety of diseases ranging from cancer
to cystic brosis [12]. Recently, it has attracted much attention due to its signicant medicinal potential. The aim of
this study was to investigate the inhibition of lipid peroxidation, ferric ions (Fe3+ ) reducing antioxidant power assay,
1,1-diphenyl-2-picryl-hydrazyl (DPPH ) radical scavenging, 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid
(ABTS+ ) radical scavenging, superoxide anion radical scavenging in the riboavin/methionine/illuminate system,
hydrogen peroxide scavenging and ferrous ions (Fe3+ )
chelating activities of curcumin. In addition, an important
main goal of this study was to clarify the antioxidant and
radical scavenging and metal chelating mechanisms of curcumin.
2. Materials and methods
2.1. Chemicals
N,N-Dimethyl-p-phenylenediamine
dihydrochloride
(DMPD), riboavin, methionine, ABTS, BHA, BHT, nitroblue
tetrazolium (NBT), DPPH , 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine), linoleic acid,
-tocopherol,
polyoxyethylenesorbitan
monolaurate
(Tween-20) and trichloroacetic acid (TCA) were obtained
from Sigma (SigmaAldrich GmbH, Sternheim, Germany).
Curcumin and ammonium thiocyanate were purchased
from Merck. All other chemicals used were analytical grade
and obtained from either SigmaAldrich or Merck.
2.2. Total antioxidant activity determination by ferric
thiocyanate method
The antioxidant activity of curcumin and standards was
determined according to the ferric thiocyanate method as
in [2]. A stock solution contained 10 mg
described by Gulc
of curcumin dissolved in 10 mL ethanol. Different concentrations of curcumin (from 15 to 45 g/mL) were prepared
by diluting the stock solution in 2.5 mL of sodium phosphate buffer (0.04 M, pH 7.0) and these were added to
2.5 mL of linoleic acid emulsion in sodium phosphate buffer
(0.04 M, pH 7.0). The linoleic acid emulsion was prepared
by homogenising 15.5 L of linoleic acid, 17.5 mg of Tween20 as emulsier, and 5 mL phosphate buffer (pH 7.0). The
control was composed of 2.5 mL of linoleic acid emul-

sion and 2.5 mL, 0.04 M sodium phosphate buffer (pH 7.0).
The reaction mixtures (5 mL) were incubated at 37 C in
polyethylene asks. The peroxide levels were determined
by reading the absorbance at 500 nm. The peroxides formed
during linoleic acid peroxidation will oxidize Fe2+ to Fe3+ ,
which forms a complex with thiocyanate that has a maximum absorbance at 500 nm. The assay step was repeated
every 5 h until reaching a maximum. The percent inhibition was calculated at this point (30 h). Solutions without
curcumin were used as blank samples. The percent inhibition of lipid peroxidation in linoleic acid emulsion was
calculated by the following equation:
inhibition of lipid peroxidation (%) =

100

AS
AC

100

in which AC is the absorbance of the control reaction, which

contains only linoleic acid emulsion and sodium phosphate
buffer, and AS is the absorbance of the sample in the presence curcumin or other test compounds [13,14].
2.3. Ferric cyanide (Fe3+ ) reducing antioxidant power
assay
The ferric reducing antioxidant power method of Oyaizu
[15] with slight modication [13] was used to measure
the reducing capacity of curcumin. The FRAP method is
based on the reduction of (Fe3+ ) ferricyanide in stoichiometric excess relative to the antioxidants [16]. Different
concentrations of curcumin (1545 g/mL) in 1 mL of distilled water were mixed with 2.5 mL of 0.2 M, pH 6.6 sodium
phosphate buffer and potassium ferricyanide [K3 Fe(CN)6 ].
2.5 mL of a 1% mixture was incubated at 50 C for 20 min.
After 20 min incubation, the reaction mixture was acidied with 2.5 mL of trichloroacetic acid (10%). Then; 2.5 mL
of the acidied sample of this solution was mixed with
2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%) and the
absorbance was measured at 700 nm in a spectrophotometer. Increased absorbance of the reaction mixture indicates
greater reduction capability [17,18].
2.4. Ferrous ion (Fe2+ ) chelating activity
The chelating of ferrous ion by curcumin was estimated by the method of Dinis et al. [19], wherein the
Fe2+ -chelating ability of curcumin was monitored by
the absorbance of the ferrous ironferrozine complex at
562 nm. Briey, curcumin (15 g/mL, 20 mM) in 0.4 mL was
added to a solution of 2 mM FeCl2 (0.2 mL). The reaction
was initiated by the addition of 5 mM ferrozine (0.4 mL).
The total volume was adjusted to 4 mL with ethanol. Then,
the mixture was shaken vigorously and left at room temperature for 10 min. Absorbance of the solution was then
measured spectrophotometrically at 562 nm. The percentage of inhibition of ferrozineFe2+ complex formation was
calculated by using the equation given below:
ferrous ion (Fe2+ ) chelating effect (%) =

AS
AC

100

where AC is the absorbance of the control and AS is the

absorbance in the presence of curcumin or standards. The
control contains only FeCl2 and ferrozine [13,20].

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2.5. Hydrogen peroxide scavenging activity

The hydrogen peroxide scavenging assay was carried out
following the procedure of Ruch et al. [21]. The principle of
this method is that there is a decrease in absorbance of
H2 O2 upon oxidation of H2 O2 . A solution of 43 mM H2 O2
was prepared in 0.1 M phosphate buffer (pH 7.4). Curcumin
at 15 g/mL concentration in 3.4 mL phosphate buffer was
added to 0.6 mL of H2 O2 solution (43 mM) and absorbance
of the reaction mixture was recorded at 230 nm. A blank
solution contained the sodium phosphate buffer without
H2 O2 . The concentration of hydrogen peroxide (mM) in the
assay medium was determined using a standard curve (r2 :
0.9895):
absorbance = 0.038[H2 O2 ] + 0.4397
The percentage of H2 O2 scavenging by curcumin and
standard compounds was calculated using the following
equation:
H2 O2 scavenging effect (%) =

AS
AC

100

where AC is the absorbance of the control and AS is the

absorbance in the presence of curcumin or other scavengers
[14,22].
2.6. DPPH free radical scavenging activity
The total radical scavenging capacity of the tested compounds was determined and compared to that of BHA,
BHT, -tocopherol and trolox by using the DPPH , ABTS+ ,
DMPD+ and superoxide anion radical scavenging methods.
The hydrogen atom or electron donation abilities of
some pure compounds were measured by the bleaching
of a purple colored methanol solution of the stable DPPH
radical. This spectrophotometric assay uses the stable radical, 1,1-diphenyl-2-picryl-hydrazyl (DPPH ), as a reagent
[23]. The method of Blois [24] previously described by
in [25] was used with slight modications in order
Gulc
to assess the DPPH free radical scavenging capacity of
curcumin. The DPPH radical absorbs at 517 nm, but upon
reduction by an antioxidant or a radical species its absorption decreases. When a hydrogen atom or electron was
transferred to the odd electron in DPPH , the absorbance
at 517 nm decreased proportionally to the increases of
non-radical forms of DPPH [26]. Briey, a 0.1 mM solution of DPPH was prepared in ethanol and 0.5 mL of
this solution was added to 1.5 mL of curcumin solution in
ethanol at different concentrations (1545 g/mL). These
solutions were vortexed thoroughly and incubated in the
dark for 30 min. A half hour later, the absorbance was measured at 517 nm against blank samples lacking scavenger.
A standard curve was prepared using different concentrations of DPPH . The DPPH scavenging capacity was
expressed as mM in the reaction medium and calculated
from the calibration curve determined by linear regression
(r2 : 0.9845):
absorbance = 9.692[DPPH ] + 0.215

29

The capability to scavenge the DPPH radical was calculated using the following equation:
DPPH scavenging effect (%) =

AS
AC

100

where AC is the absorbance of the control (0.5 mL, containing DPPH solution without curcumin), and AS is the
absorbance in the presence of curcumin [27,28]. DPPH
decreases signicantly upon exposure to radical scavengers.
2.7. ABTS radical cation decolorization assay
ABTS also forms a relatively stable free radical, which
decolorizes in its non-radical form [29]. The spectrophotometric analysis of ABTS+ scavenging activity was
determined according to the method of Re et al. [30]. In
this method, an antioxidant is added to a pre-formed ABTS
radical solution and after a xed time period the remaining ABTS+ is quantied spectrophotometrically at 734 nm
[14]. ABTS+ was produced by reacting 2 mM ABTS in H2 O
with 2.45 mM potassium persulfate (K2 S2 O8 ), stored in the
dark at room temperature for 4 h. The ABTS+ solution was
diluted to give an absorbance of 0.750 0.025 at 734 nm
in 0.1 M sodium phosphate buffer (pH 7.4). Then, 1 mL
of ABTS+ solution was added to 3 mL of curcumin solution in ethanol at different concentrations (1545 g/mL).
The absorbance was recorded 30 min after mixing and the
percentage of radical scavenging was calculated for each
concentration relative to a blank containing no scavenger.
The extent of decolorization is calculated as percentage
reduction of absorbance. For preparation of a standard
curve, different concentrations of ABTS+ were used. The
ABTS+ concentration (mM) in the reaction medium was
calculated from the following calibration curve, determined by linear regression (r2 : 0.9841):
absorbance (734 nm ) = 4.6788[ABTS+ ] + 0.199
The scavenging capability of test compounds was calculated using the following equation:
ABTS+ scavenging (%) =

AS
AC

100

where AC is absorbance of a control (blank) lacking any radical scavenger and AS is absorbance of the remaining ABTS+
in the presence of scavenger [13,31].
2.8. Superoxide anion radical scavenging activity
Superoxide radicals were generated by the method of
Beauchamp and Fridovich [32] described by Zhishen et al.
[33] with slight modication. Superoxide radicals are generated in riboavin/methionine/illuminate and assayed by
the reduction of NBT to form blue formazan. All solutions
were prepared in 0.05 M phosphate buffer (pH 7.8). The
photo-induced reactions were performed using uorescent
lamps (20 W). The concentration of curcumin in the reaction mixture was 15 g/mL. The total volume of the reaction
mixture was 3 mL and the concentrations of the riboavin,
methionine and NBT were 1.33 105 , 4.46 105 and

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30

8.15 108 M, respectively. The reaction mixture was illuminated at 25 C for 40 min. The photochemically reduced
riboavin generated O2 which reduced NBT to form
blue formazan. The unilluminated reaction mixture was
used as a blank. The absorbance was measured at 560 nm.
Curcumin was added to the reaction mixture, in which
O2 was scavenged, thereby inhibiting the NBT reduction.
Decreased absorbance of the reaction mixture indicates
increased superoxide anion scavenging activity. The percentage of superoxide anion scavenged was calculated by
using the following equation:
O2 scavenging (%) =

AS
AC

100

where AC is the absorbance of the control and AS is

the absorbance in the presence of curcumin or standards
[34,35].
2.9. Measurement of DMPD+ scavenging ability
DMPD radical scavenging ability of curcumin was performed according to the method of Fogliano et al. [36].
DMPD (100 mM) was prepared by dissolving 209 mg of
DMPD in 10 mL of deionized water and 1 mL of this solution was added to 100 mL of 0.1 M acetate buffer (pH 5.3),
and the colored radical cation (DMPD+ ) was obtained
by adding 0.2 mL of a solution of 0.05 M ferric chloride
(FeCl3 ). The absorbance of this solution, which is freshly
prepared daily, is constant up to 12 h at room temperature. Different concentrations of standard antioxidants or
curcumin (1030 g/mL) were added in test tubes and the
total volume was adjusted with distilled water to 0.5 mL.
Ten minutes later, the absorbance was measured at 505 nm.
One millilitre of DMPD+ solution was directly added to
the reaction mixture and its absorbance at 505 nm was
measured. The buffer solution was used as a blank sample.
The scavenging capability of ABTS+ radical was calculated
using the following equation:
DMPD+ scavenging (%) =

AS
AC

100

where AC is the absorbance of the initial concentration of

DMPD+ and AS is absorbance of the remaining concentration of DMPD+ in the presence of curcumin [37].
2.10. Energy calculation
All calculations were performed by using SPARTAN04
software for Windows, version 1.0.3. The optimization
was performed at semi-empirical AM1 level for neutral
molecules. No symmetry constraints were imposed during
the optimization process.
2.11. Statistical analysis
The experiments were performed in triplicate. The data
were recorded as mean standard deviation and analysed by SPSS (version 11.5 for Windows 2000, SPSS Inc.).
One-way analysis of variance (ANOVA) was performed
by standard procedures. Signicant differences between
means were determined by Dunnetts multiple range tests,

and p < 0.05 was regarded as signicant and p < 0.01 was
very signicant.
3. Results
The ferric thiocyanate method measures the amount
of peroxide, which is the primary product of oxidation
produced during the initial stages of oxidation. Curcumin
exhibited effective antioxidant activity in the linoleic acid
emulsion system. The effects of different concentrations
(1545 g/mL) of curcumin on lipid peroxidation of linoleic
acid emulsion are shown in Fig. 1A and were found to
be 97.3, 98.8 and 99.2%. This activity was greater than
45 g/mL concentrations of BHA (95.5%), -tocopherol
(84.6%) and trolox (95.6%), but similar to BHT (99.7%). It was
reported that curcumin exhibits strong antioxidant activity in other models, comparable to vitamins C and E [37].
The auto-oxidation of linoleic acid emulsion without curcumin or standard compounds was accompanied by a rapid
increase of peroxides. Consequently, these results clearly
indicated that curcumin had effective and powerful antioxidant activity.
As can be seen from Fig. 1B, curcumin had effective
reducing power using the potassium ferricyanide reduction
method when compared to the standards. For the measurements of the reductive ability of curcumin, the Fe3+ Fe2+
transformation was investigated using the method of
Oyaizu [15]. At different concentrations (1545 g/mL),
curcumin demonstrated powerful reducing ability (r2 :
0.9937) and these differences were statistically very significant (p < 0.01). The reducing power of curcumin, BHA, BHT,
-tocopherol and trolox increased steadily with increasing
concentrations of samples. Reducing power of curcumin
and standard compounds exhibited the following order:
BHA BHT > curcumin > -tocopherol > trolox. The results
demonstrate the electron donor properties of curcumin
for neutralizing free radicals by forming stable products.
In vivo, the outcome of the reducing reaction is to terminate the radical chain reactions that may otherwise be very
damaging.
Curcumin had effective ferrous ions (Fe2+ ) chelating
capacity. The difference between the 15 g/mL concentration of curcumin and the control values was statistically
signicant (p < 0.01, Table 1). In addition, at 15 g/mL
concentration, curcumin (20 mM) exhibited 56.7 4.2%
chelation of ferrous ion. On the other hand, the ferrous
ion chelating capacities of the same concentrations of BHA
(41 mM), BHT (34 mM), -tocopherol (17 mM) and trolox
(30 mM) were found to be 69.9 7.5, 60.0 9.3, 31.3 5.5
and 45.2 6.2%, respectively. These results show that the
ferrous ion chelating effect of curcumin was statistically
similar to BHA (p > 0.05) and BHT (p > 0.05) but higher than
-tocopherol (p < 0.05) and trolox (p < 0.05).
The ability of curcumin to scavenge hydrogen peroxide
is shown in Table 1 and compared with that of BHA, BHT
-tocopherol and trolox as reference compounds. Hydrogen peroxide scavenging activity of curcumin at 15 g/mL
(20 mM) was found to be 28.4 3.9%. On the other hand,
BHA, BHT, -tocopherol and trolox exhibited 13.6 3.5,
16.7 4.1, 13.6 2.9 and 25.6 3.3% hydrogen peroxide
scavenging activity, respectively, at the same concentration.

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T. Ak, I . G
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31

Fig. 1. (A) Total antioxidant activities of different concentrations (1545 g/mL) of curcumin and standard antioxidant compounds such as BHA, BHT,
-tocopherol and trolox at the concentration of 45 g/mL. The antioxidant activity was determined according to the ferric thiocyanate method in linoleic
acid system. Five millilitre of linoleic acid emulsion consists of 15.5 L of linoleic acid, 17.5 mg of Tween-20 as emulsier, and 5 mL phosphate buffer (pH
7.0). (B) Total reductive potential of different concentrations (1545 g/mL) of curcumin (r2 : 0.9937) and reference antioxidants: BHA, BHT, -tocopherol
and trolox using spectrophotometric detection of the Fe3+ Fe2+ transformations. In the presence of reductants, Fe3+ /ferricyanide complex reduces to the
ferrous form (BHA: butylated hydroxyanisole, BHT: butylated hydroxytoluene; p < 0.05).

(102 mM) curcumin (60 mM) > trolox (90 mM) (67.8, 64.9,
62.5, 62.2 and 29.4%, respectively) at the concentration
of 45 g/mL. DPPH free radical scavenging activity of curcumin also increased with increasing concentrations (r2 :
0.9947). EC50 for curcumin was 34.86 g/mL. Lower EC50
value indicates a higher DPPH free radical scavenging activity.
All the tested compounds exhibited effective radical
cation scavenging activity. As seen in Fig. 2B, curcumin is
an effective ABTS+ radical scavenger in a concentrationdependent manner (1545 g/mL, r2 : 0.9250). EC50 for
curcumin in this assay was 18.07 g/mL. There was a
signicant decrease (p < 0.01) in the concentration of
ABTS+ due to the scavenging capacity at all curcumin
concentrations. The scavenging effect of curcumin and
standards on ABTS+ decreased in the order: BHA > BHT > tocopherol > curcumin > trolox (100, 97.8, 96.9, 86.3, 79.6
and 4.4%, respectively) at the concentration of 45 g/mL.
No signicant differences in ABTS+ scavenging potential
were found among curcumin, BHA and BHT.
The inhibition by curcumin of superoxide radical generation is higher than that by for -tocopherol and trolox but
lower than BHA and BHT. As seen in Table 1, the inhibition
of superoxide anion radical generation at the concentra-

These results show that curcumin has an effective hydrogen

peroxide scavenging activity. At the above concentration,
the hydrogen peroxide scavenging effect of curcumin and
four standard compounds decreased in the order of curcumin > trolox > BHT > BHA -tocopherol.
DPPH has been widely used to evaluate the free
radical scavenging effectiveness of various antioxidant substances. In the DPPH assay, the antioxidants were able
to reduce the stable radical DPPH to the yellow-colored
diphenyl-picrylhydrazine. The method is based on the
reduction of DPPH in alcoholic solution in the presence of
a hydrogen-donating antioxidant due to the formation of
the non-radical form DPPH-H in the reaction. DPPH is usually used as a reagent to evaluate free radical scavenging
activity of antioxidants. DPPH is a stable free radical and
accepts an electron or hydrogen radical to become a stable
diamagnetic molecule [15].
Fig. 2A illustrates a signicant decrease (p < 0.01) in
the concentration of DPPH radical due to the scavenging
ability of curcumin and the reference compounds. BHA,
BHT, -tocopherol and trolox were used as references for
radical scavenger activity. The scavenging effect of curcumin and standards on the DPPH radical decreased in
the order of BHA (123 mM) -tocopherol (51 mM) BHT

Table 1
Comparison of hydrogen peroxide (H2 O2 ) scavenging activity, ferrous ion (Fe2+ ) chelating activity, superoxide anion radical (O2 ) scavenging activity of
curcumin and standard antioxidant compounds such as BHA, BHT, -tocopherol and trolox at the concentration of 15 (g/mL (BHA: butylated hydroxyanisole,
BHT: butylated hydroxytoluene)
H2 O2 scavenging

BHA
BHT
Trolox
-Tocopherol
Curcumin

Ferrous ion chelating

Superoxide scavenging

Activity (%)

P-value

Activity (%)

P-value

Activity (%)

P-value

<0.002
<0.002
<0.0015
<0.0001
<0.0031

69.9
60.0
45.2
31.3
56.7

<0.0004
<0.0026
<0.0035
<0.002
<0.0003

75.3
70.2
16.0
22.2
42.7

<0.0004
<0.0004
<0.003
<0.014
<0.013

13.6
16.7
25.6
13.6
28.4

3.5
4.1
3.3
2.9
3.9

7.5
9.3
6.2
5.5
4.2

6.5
7.1
1.9
3.3
8.1

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T. Ak, I . G
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ide anion radical scavenging activity than -tocopherol and

trolox but lower than BHA and BHT.
As shown in Fig. 2C, curcumin was an effective DMPD+
radical scavenger in a concentration-dependent manner (1030 g/mL, r2 : 0.9974). EC50 for curcumin was
34.5 g/mL. There was a signicant decrease (p < 0.05) in
the concentration of DMPD+ due to the scavenging capacity at all curcumin concentrations. The scavenging effect of
curcumin and standards on DMPD+ decreased in the order:
trolox > BHA > curcumin, which was at the concentration of
30 g/mL, respectively.
4. Discussion

Fig. 2. Radical scavenging activity of different concentrations

(1545 g/mL) of curcumin and compared with BHA, BHT, -tocopherol
and trolox. *Means in row with different superscripts differ signicantly. (A) DPPH free radical scavenging activity (r2 : 0.9947; DPPH :
1,1-diphenyl-2-picryl-hydrazyl free radical). (B) ABTS radical scavenging
activity (r2 : 0.9250; ABTS + : 2,2 -azino-bis(3-ethylbenzthiazoline-6sulfonic acid). (C) DMPD radical scavenging activity (r2 : 0.9974; DMPD + :
N,N-dimethyl-p-phenylenediamine radical).

tion of curcumin was 42.7 8.1%. On the other hand, at the

same concentration, BHA, BHT and -tocopherol and trolox
exhibited 75.3 6.5, 70.2 7.1, 22.2 3.3 and 16.0 1.9%
superoxide anion radical scavenging activity, respectively.
According to these results, curcumin had higher superox-

Many studies have been performed on the in vivo and

in vitro properties of curcumin in different systems. Curcumin with its proven anti-inammatory and antioxidant
properties has been shown to have several therapeutic
effects. It was shown to be a potent scavenger of a variety of reactive oxygen species including hydroxyl radicals
[38] and nitrogen dioxide radicals [39]. It was also shown
to inhibit lipid peroxidation in different animal models
[38]. Curcumin is an extremely potent lipid-soluble antioxidant. It positions itself within the cell membrane, where
it intercepts lipid radicals and becomes a phenoxyl radical. Being more polar than curcumin, the phenoxyl radical
may move to the surface of the membrane, where it may be
repaired by any water-soluble antioxidant such as ascorbic
acid [40].
Antioxidant mechanisms of curcumin have been studied by laser ash photolysis and pulse radiolysis [41]. In
that study, it was found that the keto-enol-enolate equilibrium of the heptadienone moiety of curcumin determined
its physicochemical and antioxidant properties. In neutral
and acidic aqueous solutions, the keto form dominates,
and curcumin acts as an extraordinarily potent H-atom
donor. Curcumin reacts with the tert-butoxyl radical in
acetonitrile solutions. Phenolic antioxidants usually scavenge free radicals by an electron-transfer mechanism.
The electron-donating ability is determined by the oneelectron oxidation potential of the parent antioxidants,
expressed by denition as the reduction potential of the
corresponding phenoxyl radicals [41]. In another study,
the antioxidant activity of curcumin was determined by
inhibition of controlled initiation of styrene oxidation
[42].
The reduction of chronic diseases, DNA damage,
mutagenesis, carcinogenesis and inhibition of pathogenic
bacterial growth is often associated with the termination of
free radical propagation in biological systems [43]. Antioxidant capacity is widely used as a parameter for medicinal
bioactive components. In this study, the antioxidant activity of curcumin was compared to BHA, BHT, -tocopherol
and its water-soluble analogue trolox.
Lipid peroxidation consists of a series of free radicalmediated chain reaction processes and is associated with
several types of biological damage. The ferric thiocyanate
method measures the amount of peroxide, which is the
primary product of lipid oxidation, produced during the
initial stages of oxidation. In this assay, hydroperoxides
produced from linoleic acid, which autoxidized during the

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experimental period, added to the reaction mixture were

indirectly measured. Ferrous chloride and thiocyanate react
with each other to produce ferrous thiocyanate by means
of hydroperoxides [44].
It was suggested that the electron donating capacity,
reecting the reducing power of bioactive compounds, is
associated with antioxidant activity [45]. Antioxidants can
be reductants, and inactivation of oxidants by reductants
can be described as redox reactions in which one reaction species is reduced at the expense of the oxidation
of the other. The presence of reductants, such as antioxidant substances in the samples, causes the reduction of the
Fe3+ /ferricyanide complex to the ferrous form. Fe2+ formed
can be monitored by measuring the formation of Perls
Prussian blue at 700 nm [46]. There are a number of assays
designed to measure overall antioxidant activity, or reducing potential, as an indication of a hosts total capacity to
withstand free radical stress [47]. The ferric ion reducing
antioxidant power assay takes advantage of an electrontransfer reaction in which a ferric salt is used as an oxidant
[16]. In this assay, the yellow color of the test solution
changes to various shades of green and blue depending on
the reducing power of antioxidant samples. The reducing
capacity of a compound may serve as a signicant indicator
of its potential antioxidant activity.
Because elemental species, such as ferrous iron (Fe2+ ),
can facilitate the production of ROS, the ability of substances to chelate iron can be a valuable antioxidant
property. Iron, in nature, can be found as either ferrous
(Fe2+ ) or ferric ion (Fe3+ ), with the latter form predominating in foods. Ferrous chelation may render important
antioxidative effects by retarding metal-catalysed oxidation [11].
Ferrous ion chelating activities of curcumin, BHA, BHT,
-tocopherol and trolox are shown in Table 1. The chelation
of ferrous ions by curcumin and standards was determined
according to the method of Dinis et al. [19]. Among the transition metals, iron is known as the most important lipid
oxidation pro-oxidant due to its high reactivity. The effective ferrous ion chelators may also afford protection against
oxidative damage by removing iron that may otherwise
participate in HO generating Fenton type reactions.
Fe2+ + H2 O2 Fe3+ + OH + OH
Ferric ions also produce radicals from peroxides
although the rate is 10-fold less than that of ferrous ion [48]
and hence curcumin was assessed for its ability to compete
with ferrozine for ferrous ion in the solution.
The data shown in Table 1 reveal that curcumin has a
marked capacity for iron binding, suggesting that its main
action as a peroxidation inhibitor may be related to its
iron binding capacity. In this assay, curcumin interfered
with the formation of the ferrousferrozine complex, indicating that curcumin has chelating activity and is able to
capture ferrous ion with a higher binding afnity than ferrozine. As depicted in Fig. 3, curcumin may chelate the
ferrous ion with its hydroxyl and methoxyl groups. It was
reported that compounds with structures containing COH
and C O functional groups can chelate metal ions. Kazazica et al. demonstrated that avonoids, such as kaempferol,

33

Fig. 3. The proposed reaction for chelating of ferrous ions by curcumin.

chelated Cu2+ and Fe2+ through the functional carbonyl

groups [49]. The compounds with structures containing
two or more of the following functional groups: OH, SH,
COOH, PO3 H2 , C O, NR2 , S and O in a favorable
structure-function conguration, can show metal chelation
activity [2,50]. The structure of curcumin and its binding
sites for metal chelation is given in Fig. 3. Recently, Fiorucci
et al. demonstrated that quercetin-chelated metal ions in
the same way [51].
Biological systems can produce hydrogen peroxide [52].
It also is produced from polyphenol-rich beverages under
quasi-physiological conditions and it increases in amount
with the incubation time. Hydrogen peroxide can be
formed in vivo by several oxidizing enzymes such as superoxide dismutase. It can cross-membranes and may slowly
oxidize a number of compounds. It is used in the respiratory burst of activated phagocytes [52]. The hydrogen
peroxide scavenging capacity of curcumin was determined
according to the method of Ruch et al. [21] (Table 1). Curcumin has effective hydrogen peroxide scavenging activity.
It is known that H2 O2 is toxic and induces cell death in
vitro [53]. Hydrogen peroxide can attack many cellular
energy-producing systems. For instance, it deactivates the
glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase [54].
The free radical chain reaction is widely accepted
as a common mechanism of lipid peroxidation. Radical
scavengers may directly react with and quench peroxide
radicals to terminate the peroxidation chain reactions and
improve the quality and stability of food products [55].
Assays based upon the use of DPPH and ABTS+ radicals
are among the most popular spectrophotometric methods
for determination of the antioxidant capacity of foods, beverages and vegetable extracts. Both chromogens and radical
compounds can directly react with antioxidants. Additionally, DPPH and ABTS+ scavenging methods have been used
to evaluate the antioxidant activity of compounds due to
the simple, rapid, sensitive, and reproducible procedures
[56].

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34

ABTS+ or DPPH radical-scavenging methods are

common spectrophotometric procedures for determining
antioxidant capacities of components. When an antioxidant is added to the radicals, there is a degree of
decolorization owing to the presence of the antioxidants,
which reverses the formation of the DPPH radical and
ABTS+ cation:
DPPH + AH DPPH2 + A
ABTS+ + AH ABTS+ + A
DPPH and ABTS radical scavenging are easy to use, have a
high sensitivity, and allow for rapid analysis of the antioxidant activity of a large number of samples. These assays
have been applied to determine the antioxidant activity of
pure components [57]. In this study, three different assays
were used to assess the radical scavenging activities of curcumin.
With the DPPH method it was possible to determine
the antiradical power of an antioxidant by measuring
a decrease in the absorbance of DPPH at 517 nm. The
absorbance decreased when DPPH was scavenged by an
antioxidant through donation of hydrogen to form a stable DPPH molecule. In the radical form, this molecule had
an absorbance at 517 nm, which disappeared after acceptance of an electron or hydrogen radical from an antioxidant
compound to become a stable diamagnetic molecule [58].
As can be seen in Fig. 4, in the keto form of curcumin,
the heptadienone linkage between the two methoxyphenol
rings contains a highly activated carbon atom. Curcumin
can easily abstract a hydrogen atom from this carbon
atom. Hydrogen atom abstraction from phenolic ring is
very difcult since curcumins phenolic hydrogen atoms
are intramolecularly H-bonded to the adjacent methoxy
groups. Based on theoretical calculations shows that B
is the most stable one among the intermediates (AC)
(Fig. 4). While the calculated formation energy (H)
for B is 42.05 kcal/mol, this energy was calculated as

39.45 kcal/mol for A and 54.70 kcal/mol for C. DPPH radicals

easily abstract an H-atom from free hydroxyl group which
was responsible for the superb antioxidant properties of
curcumin. As a consequence, the reaction of DPPH radicals
diminishes by curcumin in alcoholic media, and the phenolic part of curcumin takes over as (electron donor) reaction
site [41]. The electron donating ability of curcumin is
assessed from the measurements of one-electron-transfer
to DPPH radicals. H-atom transfer reactions of curcumin
were also investigated using the tert-butoxyl [(CH3 )3 CO ]
radicals, with same results. Same mechanism observed in
these radicals scavenging. In addition, the H-atom donation
from the -diketone moiety to a lipid alkyl or a lipid peroxyl radical was described as a potentially more important
antioxidant action of curcumin [41].
ABTS+ radicals are more reactive than DPPH radicals
and unlike the reactions with DPPH radical, which involve
H-atom transfer, the reactions with ABTS+ radicals involve
an electron-transfer process. Generation of the ABTS radical cation forms the basis of one of the spectrophotometric
methods that have been applied to the measurement of
the total antioxidant activity of pure substances, aqueous
mixtures and beverages [59]. A more appropriate format
for the assay is a decolorization technique, in which the
radical is generated directly in a stable form prior to reaction with putative antioxidants. The improved technique
for the generation of ABTS+ described here involves the
direct production of the blue/green ABTS+ chromophore
through the reaction between ABTS and potassium persulfate. ABTS , the oxidant, was generated by potassium
persulfate oxidation of ABTS2 and the radical cation is
measured spectrophotometrically. This is a direct generation of a stable form of radical to create a blue-green ABTS+
chromophore prior to the reaction with antioxidants [52].
Bleaching of a preformed solution of the blue-green radical cation ABTS+ has been extensively used to evaluate
the antioxidant capacity of complex mixtures and individual compounds. The reaction of the preformed radical with
free radical scavengers can be easily monitored by follow-

Fig. 4. Proposed reaction of DPPH with curcumin (DPPH: 1,1-diphenyl-2-picryl-hydrazyl, DPPH : 1,1-diphenyl-2-picryl-hydrazyl free radical).

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T. Ak, I . G
ulcin / Chemico-Biological Interactions 174 (2008) 2737

ing the decrease of the sample absorbance at 734 nm. The

ABTS radical cation can be prepared employing different
oxidants. Results obtained using K2 S2 O8 as oxidant show
that the presence of peroxodisulphate increases the rate of
ABTS+ . ABTS+ were generated in the ABTS/K2 S2 O8 system:
S2 O8 2 + ABTS SO4 2 + SO4 + ABTS+
where the scission of the peroxodisulphate could take place
after the electron transfer. In the presence of excess ABTS,
the sulphate radical will react according to the following
equation:
SO4 + 2ABTS SO4 2 + + 2ABTS+
leading to the overall reaction represented by
S2 O8 2 + 2ABTS 2SO4 2 + + 2ABTS+
ABTS+ radicals are more reactive than DPPH radicals and,
unlike the reactions with DPPH radicals, which involve Hatom transfer, the reactions with ABTS+ radicals involve
electron-transfer [60].
Superoxide is an oxygen-centred radical with selective
reactivity. Although a relatively weak oxidant, superoxide
exhibits limited chemical reactivity, but can generate more
dangerous species, including singlet oxygen and hydroxyl
radicals, which cause the peroxidation of lipids [61]. These
species are produced by a number of enzyme systems.
Superoxide can also reduce certain iron complexes such
as cytochrome c. Superoxide anions are thus precursors
to active free radicals that have potential for reacting
with biological macromolecules and thereby inducing tissue damage [62]. Also, superoxide has been observed
to directly initiate lipid peroxidation. It has also been
reported that antioxidant properties of some avonoids are
effective mainly via scavenging of superoxide anion radical [63]. Superoxide radicals are normally formed rst,
and their effects can be magnied because they produce
other kinds of free radicals and oxidizing agents [12].
Superoxide anions derived from dissolved oxygen by the
riboavin/methionine/illuminate system will reduce NBT
in this system. In this method, superoxide anion reduces the
yellow dye (NBT2+ ) to produce the blue formazan, which
is measured spectrophotometrically at 560 nm. Antioxidants are able to inhibit the blue NBT formation [64]. The
decrease of absorbance at 560 nm with antioxidants indicates the consumption of superoxide anion in the reaction
mixture. Table 1 shows the inhibition of superoxide radical generation by 15 g/mL concentrations of curcumin and
standards.
The principle of the DMPD+ assay is that at acidic pH
and in the presence of a suitable oxidant solution, DMPD
can form a stable and colored radical cation (DMPD+ ).
The UVvisible spectrum of DMPD+ shows a maximum
absorbance at 505 nm. Antioxidant compounds which are
able to transfer a hydrogen atom to DMPD+ quench the
color and produce a decoloration of the solution. This reaction is rapid and the end point, which is stable, is taken
as a measure of the antioxidative efciency. Therefore, this
assay reects the ability of radical hydrogen-donors to scavenge the single electron from DMPD+ [36].

35

In contrast to the ABTS procedure, the DMPD+ method

guarantees a very stable end point. This is particularly
important when a large-scale screening is required. It was
reported that the main drawback of the DMPD+ method
is the fact that its sensitivity and reproducibility dramatically decreased when hydrophobic antioxidants such as
-tocopherol or BHT were used. Hence, these standard
antioxidant compounds were not used in this antiradical
assay.
5. Conclusion
Curcumin was found to be an effective antioxidant in
different in vitro assays including: reducing power, DPPH ,
ABTS+ , O2 and DMPD+ radical scavenging, hydrogen
peroxide scavenging and metal chelating activities when
compared to standard antioxidant compounds such as BHA,
BHT, -tocopherol, a natural antioxidant, and trolox. Fig. 1
shows the total antioxidant activity of curcumin, BHA, BHT,
-tocopherol and trolox as determined by the ferric thiocyanate method in the linoleic acid system, demonstrating
that curcumin had a marked antioxidant effect in linoleic
acid emulsion. Reactive radicals scavenging and antioxidant activity of curcumin was interpreted as originating
by H-atom abstraction from the free hydroxyl group. We
concluded that it was H-atom donation from phenolic
group which was responsible for the superb antioxidant properties of curcumin. Based on the discussion
above, it can be used for minimizing or preventing lipid
oxidation in pharmaceutical products, retarding the formation of toxic oxidation products, maintaining nutritional
quality and prolonging the shelf life of pharmaceuticals.
Acknowledgements
This study partially was supported by the Research Fund
University. The author is grateful to the Research
of Ataturk

Fund of Ataturk
University for nancial support (Project
no. 2001/35). The authors thank Prof. Glen Lawrence,
Department of Chemistry and Biochemistry, Long Island
University, Brooklyn, NY, USA for language correction of
this manuscript. Also, the authors thank Dr. Mustafa Ark,
Department of Chemistry, Faculty of Science and Arts,
University for theoretical calculations.
Ataturk
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