Journal of Food Composition and Analysis 23 (2010) 689698

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Article

A novel hydrogen peroxide scavenging assay of phenolics and avonoids using

cupric reducing antioxidant capacity (CUPRAC) methodology
zyurek, Burcu Bektasoglu, Kubilay Guclu, Nilay Gungor, Resat Apak *
Mustafa O
Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 7 December 2009
Received in revised form 16 February 2010
Accepted 22 February 2010

Hydrogen peroxide (H2O2), a biologically relevant, non-radical oxidizing species, may be formed in
tissues through oxidative processes, but there has been limited information regarding its scavenging by
polyphenolic antioxidants. The present study was undertaken to investigate the hydrogen peroxide
scavenging (HPS) activity of several polyphenolic compounds, some fruit juices and green tea extract.
HPS activity has usually been determined by following the rate of H2O2 consumption in an incubation
system (H2O2 + scavenger) using the classical UV-method at 230 nm. Since some polyphenols have
strong absorption in the UV-region, their HPS activity was alternatively determined without interference
by directly measuring the concentration of H2O2 using the HPSCUPRAC (cupric reducing antioxidant
capacity) method at 450 nm in the presence of a Cu(II) salt (since H2O2 is relatively stable, and not
scavenged unless transition metal compounds are present as catalysts). Comparison between two
groups of phenolics revealed that benzoate derivatives are much stronger H2O2 scavengers than
cinnamic acids. The ndings of the developed method for polyphenolics were statistically alike with
those of the reference GSH-Px-DTNB (glutathione peroxidase-5,50 -dithio-bis(2-nitrobenzoic acid)) and
HPLCECD methods. The proposed spectrophotometric method was rapid, practical, low-cost, and could
reliably assay H2O2 in the presence of polyphenolic scavengers, without being affected from UVabsorbing substances.
2010 Elsevier Inc. All rights reserved.

Keywords:
Hydrogen peroxide scavenging
CUPRAC assay
Phenolics
Flavonoids
Glutathione peroxidase
HPLCECD
HPS activity of polyphenolic compounds
and method of detection
Food analysis
Food composition

1. Introduction
Among reactive oxygen species (ROS), hydrogen peroxide
(H2O2) is a relatively stable, non-radical oxidant, which can diffuse
across biological membranes. It is produced by 2-electron
reduction of molecular oxygen or by dismutation of the superoxide
anion radical (enzymatic (SOD) or non-enzymatic):
O2 2e 2H ! H2 O2

(1.1)

O2  e 2H ! H2 O2

(1.2)

SOD

2O2  2H !H2 O2 O2

(1.3)

A biological system (including humans and bacteria) generating

O2 from molecular oxygen will inevitably produce H2O2 by the
dismutation reaction, the rate of which will depend on pH and O2
concentration (Halliwell and Gutteridge, 1984). Hydrogen peroxide causes toxicity via two main mechanisms: it leads to depletion
of adenosine triphosphate (ATP), reduced glutathione (GSH), and
reduced nicotinamide-adenine dinucleotide phosphate (NADPH),

* Corresponding author. Tel.: +90 212 473 7028; fax: +90 212 473 7180.
E-mail address: rapak@istanbul.edu.tr (R. Apak).
0889-1575/$ see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2010.02.013

and produces strand breaks in DNA that trigger energy-consuming

DNA repair mechanisms and activate the nuclear enzyme
poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)
activity leading to cell death by the so-called enzyme-suicide
mechanism (Szabo and Dawson, 1998). In another way, it induces
the oxidative degradation of most biological macromolecules such
as lipids, proteins or enzymes, carbohydrates and nucleic acids,
through generation of the hydroxyl radical (OH) as being the most
potent and reactive ROS species (Jonas et al., 1989). If both H2O2
and Cu(I) or Fe(II) salts are available in vivo, then OH radicals will
form, since transition metal ion-catalyzed Fenton and Haber
Weiss reactions of hydrogen peroxide produce the hydroxyl radical
(Halliwell and Gutteridge, 1984). Therefore, the measurement of
hydrogen peroxide scavenging (HPS) activity in biological uids
and food extracts is important.
A large number of assays developed for the determination of
HPS activity depend on the oxidation of a spectrophotometric,
uorogenic or chemiluminogenic probe (detector molecule) by
H2O2 using horseradish peroxidase (HRP) as the oxidation catalyst.
The most extensively studied uorogenic probes are p-hydroxyphenylacetic acid (p-HPA), homovanilic acid (HVA), scopoletin
(7-hydroxy-6-methoxy-coumarin),
2,7-dichlorodihydrouorescein (DCFH), and amplex red (N-acetyl-3,7-dihydroxyphenoxazine) (Gomes et al., 2005). In these systems, the amount of H2O2

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M. Ozyurek et al. / Journal of Food Composition and Analysis 23 (2010) 689698

present is estimated by following the decrease (i.e. scopoletin) or

increase (i.e. HVA) of uorescence of the initial probes. Spectrophotometric probes used in HRP-coupled reactions with optical
properties that change upon reaction with H2O2 are guaiacol, and
phenol red. In the most common chemiluminescence (CL)-based
methods, luminol and peroxyoxalates have been used as chemiluminogenic probes, the emissions of which are affected by pH
changes (Bartosz, 2006). The HRP-linked assays have some
disadvantages. Halliwell stated that peroxidase also oxidizes
certain substrates without it being necessary to add H2O2
(Halliwell, 1978). Several biological compounds including thiols
and ascorbate can serve as substrate for HRP and thus compete
with the probe for oxidation, leading to underestimation of H2O2
formation. As a result, these assays are not suitable for HPS activity
of plasma or serum because of the presence of reducing agents in
extracellular uid (e.g., GSH, ascorbate) which interfere with the
assay (Tarpey et al., 2004). The nature of the inhibition is
ambiguous because of the existence of several potential inhibition
pathways. Antioxidants can inhibit the reaction by (a) reacting
directly with H2O2, (b) reacting with intermediates formed from
enzyme and H2O2, or (c) inhibiting the horseradish peroxidase
from binding to H2O2. Therefore, it is difcult to explain the actual
chemical meaning of the collected data (Martnez-Tome et al.,
2001). Moreover, HRP is expensive and unstable in solution, and
has strict requirements for experimental conditions (Lu et al.,
2006). In the HPS activity measurement of some nonsteroidal
antiinammatory drugs, it was concluded that the lack of
specicity of horseradish peroxidase for its donor substrate may
lead to erroneous results for drugs like 5-aminosalicylic acid
capable of both scavenging hydrogen peroxide and interacting
with horseradish peroxidase, and therefore a more specic assay
system based on the absolute specicity of the enzyme glutathione
peroxidase for glutathione was subsequently used (Parij and Ne`ve,
1996).
In the most common UV-measurement method for detection of
H2O2, the scavenging capacity depends on the change of the
absorbance at 230 nm when H2O2 is consumed by scavenger
compounds. Naturally, there might be possible interferences from
other organic materials that also absorb at the same wavelength,
and thus, a blank measurement should be performed (Magalhaes
et al., 2008). Moreover, this UV assay does not use any catalyst for
following HPS (which is a prerequisite for efcient H2O2
degradation), and as a result, many literature works (e.g., Chinese
green tea, Ruch et al., 1989, curcumin, Ak and Gulcin, 2008, various
tea extracts, Yen and Chen, 1995, garlic, Bozin et al., 2008) using
this method have reported quite close scavenging activities for a
diverse series of scavengers that should normally be expected to
cover a wide range of HPS values.
In practice, hydrogen peroxide is not very labile and survives
reasonably well at moderate temperatures unless traces of
transition metal catalysts are present (Shriver et al., 1995).
Effective (transition metal ion) catalysts for its decomposition
into oxygen and water should have standard potentials within the
range of 1.76 V and 0.70 V, as E0 (H2O2, H2O) = 1.76 V and E0 (O2,
H2O2) = 0.70 V.
There has been limited information in literature regarding the
HPS activity of polyphenolic compounds. Thus, the aim of this
study is to precisely measure the HPS activity of several
polyphenolic compounds, including simple phenolic acids, hydroxycinnamic acids, avonoids, and other antioxidants (i.e. ascorbic
acid, trolox), using a HPSCUPRAC method in comparison with the
GSH-Px-DTNB (glutathione peroxidase-5,50 -dithio-bis(2-nitrobenzoic acid)) reference method claimed to produce more reliable
results (Parij and Ne`ve, 1996; Miles and Grisham, 1994). The
CUPRAC method of antioxidant measurement, introduced by our
research group to world literature (Apak et al., 2004, 2007), is

based on the absorbance measurement of the CUPRAC chromophore, Cu(I)-neocuproine (Nc) chelate, formed as a result of the
redox reaction of antioxidants with the CUPRAC reagent, Cu(II)neocuproine (Nc: 2,9-dimethyl-1,10-phenanthroline), where
absorbance is recorded at the maximal light absorption wavelength of 450 nm. In this respect, Campos et al. (2009) developed a
similar spectrophotometric method using bathocuproinedisulfonic acid disodium salt (BCS) as the chelating agent for Cu(I)
emerging from the reaction of Cu(II) with antioxidants (i.e. the
cupric ion reducing capacity BCS assay, not competent with
conventional CUPRAC in regard to kinetics and response to
lipophilic antioxidants) for the total antioxidant capacity (TAC)
assessment in human plasma and urine. To give a few examples
for the use of the conventional CUPRAC procedure in the
antioxidant assay of plant foods, Gorinsteins research group
has used the method in several occasions; namely garlic extract
(Gorinstein et al., 2006); kiwifruit (Park et al., 2006), ethylenetreated kiwifruit (Park et al., 2008), and cereal and pseudo-cereal
methanol extracts (Gorinstein et al., 2008). The original CUPRAC
method was modied so as to serve diverse purposes in
antioxidant research such as measuring the hydroxyl radical
scavenging activity of water soluble antioxidants and polyphe zyurek et al., 2008a),
nolics (Bektasoglu et al., 2006, 2008; O
simultaneously determining lipophilic and hydrophilic antioxidants (e.g., b-carotene, a-tocopherol, ascorbic acid, quercetin,
etc.) in the same solvent medium of acetonewater with the aid of
their inclusion complexes formed with methyl-b-cyclodextrin
zyurek et al., 2008b), determining the xanthine oxidase (XO)
(O
zyurek et al., 2009), nding
inhibition activity of polyphenolics (O
the antioxidant capacity of protein thiols (Demirci Cekic et al.,
2009), and nally measuring the Cu(II)-catalyzed hydrogen
peroxide scavenging activity of polyphenols subject to this study.
2. Materials and methods
2.1. Reagents, materials and apparatus
The following chemical substances of analytical reagent grade
were supplied from the corresponding sources: neocuproine (2,9dimethyl-1,10-phenanthroline),
()epigallocatechin
gallate,
()epicatechin gallate, ()epigallocatechin, ()epicatechin, quercetin, naringin, gallic acid (GA), chlorogenic acid, glutathione
(GSH), catalase from bovine liver (1340 U mg1 solid), methanol
(MeOH): Sigma (Steinheim, Germany); caffeic acid, p-coumaric
acid, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid), and L-ascorbic acid: Aldrich (Steinheim, Germany); copper(II) chloride dihydrate, ammonium acetate, hydrogen peroxide
(30%, by wt.), Na2HPO42H2O, NaH2PO42H2O, and Tris (tris
(hydroxymethyl) aminomethane): Merck (Darmstadt, Germany);
glutathione peroxidase (GSH-Px) (from bovine erythrocytes,
100 U mg1 solid), 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB)
(Ellmans reagent), pyrogallol, rosmarinic acid, (+)catechin (CAT),
kaempferol: Fluka (Buchs, Switzerland); HCl (37%), absolute
ethanol (EtOH): Riedel-de Haen (Steinheim, Germany).
Lipton green tea (Camellia sinensis) was purchased from
Unilever San. Tic. Turk AS. (Istanbul, Turkey), fruit juices (orange,
apple, and sour cherry) from Tamek Gida Kons. San. Tic. AS (Bursa,
Turkey).
The visible spectra and absorption measurements were
recorded in matched quartz cuvettes using a Varian CARY Bio
100 UVVis spectrophotometer (Mulgrave, Victoria, Australia).
Other related apparatus and accessories were a J.P. Selecta water
bath (Barcelona, Spain), E521 model Metrohm pH-meter equipped
with glass electrodes (Herisau, Switzerland), Ultra-Turrax CAT X620 model homogenizer apparatus (Staufen, Germany), and
Elektromag vortex stirrer (Istanbul, Turkey).

M. Ozyurek et al. / Journal of Food Composition and Analysis 23 (2010) 689698

A Waters BreezeTM 2 Model HPLC system (Milford, MA, USA)

equipped with a 1525 binary pump, a column thermostat, a 2465
Electrochemical Detector (ECD) (Chelmsford, MA, USA), a Hamilton
25 mL-syringe (Reno, NV, USA), and ACE C18 column
(4.6 mm  250 mm, 5 mm particle size) (Milford, MA, USA) was
used for chromatographic measurements. Data acquisition was
accomplished using Empower PRO (Waters Associates, Milford,
MA).
2.2. Preparation of solutions
Hydrogen peroxide at 1.0 mM concentration was prepared from
a 0.5 M intermediary stock solution, the latter being prepared from
30% H2O2 and standardized with permanganate titration. The
NaH2PO4Na2HPO4 buffer solution (pH 7.4) at 0.2 M total
phosphate concentration was prepared in water. Copper(II) at
1.0  102 M was prepared by dissolving 0.4262 g of CuCl22H2O in
water, and diluting to 250 mL. Ammonium acetate (NH4Ac; 1.0 M)
aqueous buffer solution at pH 7.0 contained 19.27 g of the salt in
250 mL. Neocuproine (Nc) at 7.5  103 M was freshly prepared by
dissolving 0.078 g of the free base in EtOH, and diluting to 50 mL
with ethanol. The standard solutions at 1.0  104 M concentration of polyphenolics (avonoids and phenolic acids); at
1.0  103 M concentration of naringin were all prepared in EtOH,
and ascorbic acid at 1.0  104 M concentration in water prior to
measurement. The original catalase solution of initial activity
1340 U mg1 solid was diluted with 0.2 M phosphate buffer (pH
7.4) to a concentration of 268 U mL1.
DTNB at 4 mg mL1 was prepared in 50 mM TrisHCl buffer
solution (pH 8.5). Glutathione at 1.0 mM, and TrisHCl buffer
solution (pH 8.5) at 50 mM were prepared in water. The original
GSH-Px solution of initial activity 100 U mg1 solid was diluted
with 50 mM TrisHCl buffer (pH 8.5) to a concentration of
0.25 U mL1.
The mobile phase for HPLC analysis with isocratic elution was
prepared from 50 mM ammonium acetate. This solution was
prepared by dissolving 0.9635 g of ammonium acetate in 250 mL of
bidistilled water.
2.3. Preparation of plant sample and fruit juices for analysis
The air-dried plant specimens were crushed in a mill, and 2-g
samples were taken for each plant species. These samples were
soaked in 80% MeOH overnight, and homogenized in an UltraTurrax apparatus by gradually increasing the number of cycles per
unit time. The obtained extracts were transferred to centrifuge
tubes and centrifuged for 10 min (5000 rpm), and subsequently
ltered through a Whatman quantitative lter paper into 100-mL
asks. The same procedure was repeated three times with 25 mL
portions of 80% MeOH on the remaining parts of the plants. All
ltered extracts were combined, and diluted to 100 mL using the
same solvent. Each extraction procedure was run three times in
parallel (Guclu et al., 2006). The obtained extracts were analyzed
for their HPS activity on the next day after preserving the N2bubbled and stoppered extracts in a refrigerator at +4 8C. During
the measurement of HPS activity of green tea extract with the
proposed (HPSCUPRAC) procedure, 0.2 mL of the diluted (at 1:20
ratio with H2O) extract was used. Among the fruit juices, sour
cherry was diluted with H2O (1:10), and others were analyzed
without dilution.
2.4. Recommended procedure (HPSCUPRAC method)
To a test tube were added 0.7 mL of phosphate buffer (pH 7.4),
0.4 mL of 1 mM H2O2, 0.4 mL of 0.1 mM CuCl22H2O in this order
(hydrogen peroxide incubation solution, used as reference). To

691

other two test tubes were added 0.5 mL of phosphate buffer (pH 7.4),
0.4 mL of 1.0 mM H2O2, 0.2 mL scavenger sample solution, and
0.4 mL of 0.1 mM CuCl22H2O solution rapidly in this order
(scavenger solutions-I and -II, identical at this stage). The mixtures
in a total volume of 1.5 mL were incubated for 30 min in a water bath
kept at 37 8C. At the end of this period, to reference and scavenger
solution-I was added 0.4 mL H2O and to scavenger solution-II was
added 0.4 mL of 268 U mL1 catalase solution (the contents of
scavenger solution-I and -II became different at this stage), and
vortexed for 30 s. To 1.0 mL of the nal incubation solutions, the
HPSCUPRAC method was applied in the following manner:
1 mL CuII 1 mL Nc 2 mL NH4 Ac buffer
1:0 mL final incubation solution
The absorbance at 450 nm of the nal solution at 5.0 mL total
volume was recorded 30 min later against a reagent blank.
The colors of the (H2O2 + CuCl2) mixtures (initial concentration
of H2O2 = 1.0 mM) in the absence of scavenger were compared
with respect to their stabilities, the corresponding CUPRAC
absorbances being recorded within 06 h (in order to optimize
the incubation period).
The HPS activity (%) of polyphenols and real samples were
calculated using the following formula:


A0  A1  A2
HPS % 100
(2.1)
A0
where A0 is the CUPRAC absorbance of reference hydrogen
peroxide incubation solution, A1 and A2 the CUPRAC absorbances
of scavenger solutions-I and -II, respectively.
2.4.1. Testing incubation reaction products with the HPSCUPRAC
method
Potential incubation reaction systems containing various combinations of catalyst, H2O2, and scavenger were prepared as; (a)
GA + H2O2 + CuCl2 (scavenger solution-II); (b) GA + CuCl2; (c) GA; (d)
GA + H2O2 + CuCl2 (scavenger solution-I); (e) H2O2 + CuCl2; (f) H2O2;
(g) GA + H2O2. Phosphate buffer at pH 7.4 was added to each mixture,
plus water to adjust the nal incubation volume to 1.5 mL. The tubes
bearing the reaction mixtures were stoppered, and incubated for
30 min in a 37 8C water bath. At the end of the reaction, to (a) mixture
solution was added 0.4 mL of catalase prepared in phosphate buffer,
and to the other mixture solutions were added 0.4 mL of H2O, and the
nal mixtures were subjected to the HPSCUPRAC assay.
2.5. GSH-Px-DTNB method
The colorimetric GSH-Px-DTNB method was applied as the
reference method of comparison for determining the HPS activity
of polyphenols. The reacting mixture for the DTNB method (Miles
and Grisham, 1994) contained in a nal volume of 1.5 mL the
following constituents: (0.7  x) mL of phosphate buffer (pH 7.4),
0.4 mL of 1 mM H2O2, x mL (x = 0 or 0.2 mL) of scavenger sample
solution, and 0.4 mL of 0.1 mM CuCl22H2O solution. Reaction
mixtures were incubated at 37 8C for 30 min. At the end of the
incubation period, 0.5 mL of 1.0 mM GSH, and 0.5 mL GSH-Px
enzyme solution (0.25 U mL1) was added to each mixture and
incubated at 37 8C for 5 min again.
At the end of this period, 50 mL DTNB (4 mg mL1) was added to
each mixture and incubated at 25 8C for 5 min to develop the
yellow colored TNB, and the absorbances of the resulting solutions
(total volume = 2.55 mL) were measured at 412 nm:

1:5 mL incubation solution 0:5 mL GSH

5 min

0:5 mL GSH-Px ! 50 mL DTNB

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M. Ozyurek et al. / Journal of Food Composition and Analysis 23 (2010) 689698

The HPS activity of polyphenolics and real samples (%) were

calculated using the following formula:


A  A0
HPS % 100
A0

(2.2)

where A0 and A are the absorbances of incubation mixtures in the

absence and presence of scavengers, respectively.
2.6. HPLCECD method
A suitable aliquot of the incubation mixture solution was
injected (injection volume: 20 mL) without derivatization (i.e.
without conversion into a uorophore or a chromophore, enabling
post-column detection) into the HPLC column (5 mm ODS) at
ambient temperature, and isocratic elution was employed for
detection of the hydrogen peroxide. The HPLCECD method
developed by Yue et al. (2009) was adapted with slight
modications, namely the use of an ACE C18 column
(4.6 mm  250 mm, 5 mm particle size) (Milford, MA, USA) in
conjunction with an ECD system comprising Ag/AgCl reference and
glassy carbon working electrodes. The mobile phase consisting of
50 mM ammonium acetate in water was delivered at a rate of
1 mL min1. The column efuent was pumped to an ECD detector
(as recommended by Takahashi et al., 1999) equipped with a Ag/
AgCl reference electrode and a glassy carbon working electrode.
The detector was operated in direct current (DC) mode. Each run
was 7 min long. Quantication of hydrogen peroxide was
performed using the data generated at 500 mV potential (Yue
et al., 2009).
2.7. Testing of HPS abilities of catechin and green tea extract with the
UV-method
Synthetic incubation reaction systems containing various
combinations of catechin (or green tea extract) and H2O2 were
prepared as: (a) 0.4 mL 5.0  103 M H2O2 + 0.2 mL 104 M CAT (or
0.2 mL of 1:20 diluted green tea extract); (b) 0.2 mL 104 M CAT (or
0.2 mL of 1:20 diluted green tea extract); (c) 0.4 mL 5.0  103 M
H2O2. Phosphate buffer at pH 7.4 (0.5 mL) was added to each
mixture, plus distilled water to adjust the nal incubation volume
to 1.9 mL. The tubes bearing the reaction mixtures were incubated
for 30 min in a 37 8C water bath. At the end of the reaction, the UV
spectra of the mixtures of 1.9 mL total volume were recorded
against distilled water (reagent blank).
2.8. Statistical analysis
Descriptive statistical analyses were performed using Excel
software (Microsoft Ofce 2002) for calculating the means and the
standard error of the mean. Results were expressed as the
mean  standard deviation (SD). Using SPSS software for Windows
(version 13), the data were evaluated by two-way ANalysis Of
VAriance (ANOVA) (Miller and Miller, 1993).
3. Results and discussion
3.1. Optimization of the recommended method
A modied CUPRAC method was applied to assess the HPS
activity of polyphenols and other antioxidant compounds (e.g.,
ascorbic acid). CUPRAC absorbance of the tested solutions arises
from the reduction of the Cu(II)-Nc reagent to the Cu(I)-chelate
(Apak et al., 2004) by the products of the incubation system
including scavenger + H2O2 or H2O2 alone (reference solution),
where hydrogen peroxide is oxidized to molecular oxygen. Although

H2O2 is known to be a strong oxidant, it may act as a reductant under

certain conditions, e.g., toward Cu(II)-neocuproine. The molar
absorptivity for H2O2 was: e = 1.42  104 L mol1 cm1, and the
linear concentration range: 2.11  106 to 9.91  105 M (linear
correlation coefcient: r = 0.9992). The relative standard deviation
(RSD) within this concentration range was approximately 1.9%. The
limit of detection (LOD) and limit of quantication (LOQ) were
calculated using the equations; LOD = 3sbl/m and LOQ = 10sbl/m,
respectively, where sbl is the standard deviation of a blank and m is
the slope of the calibration line. The LOD and LOQ for H2O2 were
found to be 6.3  104 mM and 2.1  103 mM, respectively.
A phosphate buffer was used in the recommended HPSCUPRAC
procedure, but copper-phosphate complexes are about nine orders
of magnitude less stable than that of neocuproine which signicantly stabilizes the lower valency of copper (Hawkins and Perin,
1962), and therefore this would not make any difference in the
determination. On the other hand, site-specic DNA cleavage by
Cu(II) + H2O2 via reactive species emerging from copper-peroxide
complexation was not hindered in phosphate buffer medium at pH
7.9 (Yamamoto and Kawanishi, 1989), conrming that the redox
activity of copper(II) can be experimentally followed in phosphatebuffered media. Moreover, Zhu et al. (2002) followed the redox
activity of copper (in an investigation associated with Cu(II)-induced
oxidative damage on ascorbate by a Cu-thiourea complex) in 0.1 M
phosphate buffer at pH 7.4.
An HPLCECD system was used in method validation. An
electrochemical detector may be more analyte-specic due to the
different redox potentials of electroactive species. Takahashi et al.
(1999) have reported that in the HPLC detection of H2O2, ECD is
more popular than the chemiluminescent (CL) detector, and the
electrolyte for electrochemical reaction might also be less
expensive than microperoxidase, which is used for the postcolumn reaction of luminol CL-detection. Another advantage of an
ECD system is that electrochemical detector responses are linear
over several orders of magnitude (Coudray et al., 1995), and that
the detection technique does not need derivatization of the
analyte. The chromatograms used for hydrogen peroxide determination in the absence and presence of the phenolic scavenger
are shown in Figs. 1 and 2. These chromatograms demonstrate that
H2O2 remains intact upon incubation with either Cu(II) or phenolic
scavenger (GA or CAT) alone within the protocol time period of the
assay, but is effectively degraded in admixture with Cu(II) catalyst
and phenolic scavenger together (Figs. 1 and 2). Another point
observable from these chromatograms is that the widely used UVmethod of HPS assay without using any catalyst (Magalhaes et al.,
2008) is incapable of effectively degrading the analyte within a
reasonable time period.
Although hydrogen peroxide is a redox-active compound with a
high oxidation potential, it is relatively stable and its reaction with
most substances occurs slowly unless catalyzed by transition
metal salts (Halliwell et al., 2000). For example, Cu(II)-catalyzed
chemiluminescence (CL) techniques (e.g., metal ion-catalyzed
oxidation of luminol) were employed for detection of HPS activity
(Lu et al., 2006), but Co(II) was generally preferred as the
alternative catalyst for the luminol-H2O2 reaction due to its higher
sensitivity (Yuan and Shiller, 1999).
It has been assumed in literature that H2O2 reduces Cu(II) to
Cu(I), followed by the reaction of Cu(I) with H2O2 and the
formation of hydroxyl free radicals (Gutteridge and Wilkins, 1983;
Yamamoto and Kawanishi, 1989) as shown in Eqs. (3.1) and (3.2).
CuII H2 O2 ! CuI HO2 H

(3.1)

CuI H2 O2 !  OH OH CuII

(3.2)

However, the results obtained by Yamamoto and Kawanishi

(1989) suggest that the main active species causing site-specic

[(Fig._1)TD$IG]

M. Ozyurek et al. / Journal of Food Composition and Analysis 23 (2010) 689698

693

Fig. 1. The HPLCECD chromatogram for hydrogen peroxide in the presence and absence of a scavenger (gallic acid): (a) 0.4 mL of 1 mM H2O2 + 0.4 mL of 0.1 mM Cu(II); (b)
0.4 mL of 1 mM H2O2 + 0.2 mL of 0.1 mM GA and (c) 0.4 mL of 1 mM H2O2 + 0.4 mL of 0.1 mM Cu(II) + 0.2 mL of 0.1 mM GA.

DNA damage in the presence of Cu(II) + H2O2 are more likely

copper-peroxide complexes, with similar reactivity to singlet
oxygen and/or hydroxyl radical rather than hydroxyl free radical.
During copper-catalyzed scavenging of H2O2 by phenolic antioxidants, the phenolic-reduced Cu(I) may be expected to be the
main active species leading to H2O2 degradation (essentially by
Eq. (3.2) through the generation of hydroxyl radicals or via a
copper-bound oxygen intermediate, Zhu et al., 2002) due to faster
reaction rates, which was aimed to be measured by the proposed
assay.
In the reaction systems comprised of (a) GA + H2O2 + CuCl2 + catalase (scavenger solution-II); (b) GA + CuCl2; (c) GA; (d)
GA + H2O2 + CuCl2 (scavenger solution-I); (e) H2O2 + CuCl2 (hydrogen peroxide incubation solution, used as reference); (f) H2O2; (g)
GA + H2O2, 30 min incubation in phosphate-buffered medium
followed by reaction with the CUPRAC reagent gave the spectra
depicted in Fig. 3. When catalase was absent, hydrogen peroxide
produced the CUPRAC chromophore (Fig. 3f), whereas catalase,
being an effective H2O2 scavenger, completely annihilated the
CUPRAC
signal due to hydrogen peroxide (Fig. 3a). The highest
[(Fig._2)TD$IG]

Fig. 2. The HPLCECD chromatogram for hydrogen peroxide in the presence and
absence of a scavenger (catechin): (a) 0.4 mL of 1 mM H2O2 + 0.2 mL of 0.1 mM CAT
and (b) 0.4 mL of 1 mM H2O2 + 0.4 mL of 0.1 mM Cu(II) + 0.2 mL of 0.1 mM CAT.

CUPRAC signal was obtained from a mixture of H2O2 and GA

(Fig. 3g). Hydrogen peroxide was neither degraded with GA
scavenger (Fig. 3g) nor with Cu(II) catalyst (Fig. 3e) alone, but was
effectively degraded when both scavenger and catalyst were
present in admixture (Fig. 3d). It is apparent that H2O2 was not
scavenged by a polyphenol such as GA unless a transition metal ion
(Cu(II)) was present as catalyst (Fig. 3g). Since gallic acid could only
scavenge hydrogen peroxide in the presence of Cu(II) catalyst
within the protocol time period of the assay, CUPRAC absorbance
diminished (Fig. 3d), enabling the measurement of HPS activity of
GA from the difference (3d!3a).
CUPRAC absorbances of the (H2O2 + CuCl2) incubation system
were measured as a function of time in the absence of a phenolic
scavenger to show any change in the initial absorbance of the
H2O2 solution (Fig. 4), and an optimal incubation time of 30 min
was chosen. As a result, there were no appreciable chemical

[(Fig._3)TD$IG]

Fig. 3. Visible spectra of Cu(I)-Nc chelate produced as a result of CUPRAC reaction

with (a) GA + H2O2 + CuCl2 + catalase; (b) GA + CuCl2; (c) GA; (d) GA + H2O2 + CuCl2;
(e) H2O2 + CuCl2; (f) H2O2 and (g) GA + H2O2 (all mixtures were incubated in
phosphate-buffered medium, followed by addition of CUPRAC reagent).

[(Fig._4)TD$IG]

694

M. Ozyurek et al. / Journal of Food Composition and Analysis 23 (2010) 689698

Table 1
Analysis of hydrogen peroxide standard solution before and after incubation with
phenolic scavengers (GA and CAT) in admixture with Cu(II) catalyst using HPS
CUPRAC and HPLCECD methods (N = 3).

Fig. 4. Absorbance vs incubation time curves of H2O2 (1.0 mM) in the presence of
CuCl2 (1.0  104 M) using the HPSCUPRAC method.

interactions among the constituents of the incubation system

during 30 min. Although, in principle, a number of transition metal
ions may equally catalyze the polyphenolics scavenging of H2O2,
Cu(II) was the preferred catalyst in this work, because Fe(II) may
cause undesired Fenton reactions with H2O2 which would arise
uncertainty whether the actual oxidant for the tested polyphenol
was H2O2 or OH (produced from the Fenton reaction).
The effect of hydrogen peroxide that may possibly be present in
plant samples (Orozco-Cardenas and Ryan, 1999), e.g., in 80%
methanolic green tea extract, as a potential interferent to the
proposed method was experimentally investigated. Mixture-1 was
composed of 0.2 mL of 1:20 diluted green tea extract + 1.7 mL of
H2O, and mixture-2 of 0.2 mL of 1:20 diluted green tea
extract + 0.4 mL of catalase + 1.3 mL of H2O. To 1.0 mL of the nal
mixture solutions, the HPSCUPRAC method was applied, and the
absorbances (A450) recorded with mixture-1 and mixture-2 were
0.4054 and 0.3929, respectively, conrming that the H2O2 content
of the original (diluted) green tea sample extract yielded a
difference of 3.1% in measurements. Cheeseman (2006) reported
the existence of 0.73.6 mmol H2O2 per gram of fresh weight in
various plant leaves. Adapting these data to our case and
calculating the contributions of H2O2 coming from the plant and
the test medium reveal that the percentage contribution of plantderived H2O2 to the total H2O2 budget would make only 3%
difference in measurements, which is well within the relative error
of spectrophotometric determinations.
3.2. Comparison of the ndings with HPSCUPRAC and HPLCECD
methods
In the chromatograms for hydrogen peroxide assay (Figs. 1 and
2), the retention time for H2O2 varied between 2.65 and 2.68 min.
The amounts of hydrogen peroxide in the incubation mixtures
were calculated using the calibration curve drawn as peak area vs
concentration. The percentage HPS activities of gallic acid using the
HPSCUPRAC and HPLCECD methods were (58.6  1.5) and
(62.8  0.9), respectively, and both methods reected in agreement
the relative decrease in hydrogen peroxide quantities in the presence
of phenolic inhibitors (Table 1). In the presence of potent inhibitors
such as phenolic compounds (e.g., gallic acid (Fig. 1) and catechin
(Fig. 2)), there was considerable decrease in hydrogen peroxide
concentration upon incubation with scavenger and catalyst, as is

Mixtures

Conc. of H2O2
with respect to
HPLCECD
method (mM)

H2O2 (initial)
H2O2 + Cu(II)
Gallic acid
(GA) + H2O2
Gallic acid
(GA) + H2O2 + Cu(II)
Catechin
(CAT) + H2O2
Catechin
(CAT) + H2O2 + Cu(II)

0.266
0.271
0.263
0.110
0.280
0.221

HPS (%) value

with respect
to HPLCECD
method

58.6  1.5

16.9  0.5

HPS (%) value

with respect to
HPSCUPRAC
method

62.8  0.9

14.6  1.9

apparent from the signicant lowering of the peak heights of

hydrogen peroxide, e.g., the initial peak area for hydrogen peroxide as
3.09  104 decreased to 1.28  104 with GA (Fig. 1c) and to 2.57  104
with CAT (Fig. 2b). Degradation of H2O2 was not observed by merely
incubating the analyte with the polyphenol, and Cu(II) catalyst was
necessary (Table 1). The data depicted in Table 1 validates the ndings
of the proposed HPSCUPRAC assay against HPLC, and shows that
gallic acid is a more efcient scavenger of H2O2 than catechin. Sroka
and Cisowski (2003) state that the anti-radical activity of phenolic
acids correlated positively with the number of OH groups
(preferably in ortho-position to one another) bonded to the aromatic
ring. Campos et al. (2009) also found that there is signicant positive
linear correlation between the number of phenolic hydroxyl and TAC
of individual antioxidants. Among the benzoic acid derivatives tested,
HPS activity (90%) was maximal for 3,4,5-trihydroxybenzoic acid,
whereas the corresponding value for 2,3-dihydroxybenzoic acid (47%)
was signicantly lower (Sroka and Cisowski, 2003). The lower activity
observed for catechin (Table 1) may be related to the lack of 2,3double bond in catechin (normally in conjugation with the 4-oxo
function in avonols) enhancing both electron transfer and ROS
scavenging ability through electron delocalization (Apak et al., 2007).
The superior antioxidant ability of quercetin results from the
formation of a stable aryloxy radical, due to C25
5C3-double bond
and the resulting planar geometry (Rice-Evans et al., 1996) which
delocalizes the radical throughout the entire molecule, whereas A and
B rings are perpendicular to each other in catechin (Fukuhara et al.,
2002). When the aryloxy radical produced from 1  e oxidation of a
avonoid is stabilized by conjugation, the redox potential of the
avonoid is lowered, increasing radical scavenging and antioxidant
activity. Flavanonols and avanones, due to the lack of conjugation
are weak antioxidants (Pietta, 2000). The ABTS-assayed troloxequivalent antioxidant capacity (TEAC) of quercetin (4.7) is almost
twice that of (+)-catechin (2.4), illustrating the signicance of both 2
3 unsaturation and a carbonyl at position 4 (Heim et al., 2002).
3.3. Comparison of the ndings with modied CUPRAC and GSH-PxDTNB methods
According to the reaction sequence of the GSH-Px-DTNB
method (Miles and Grisham, 1994) shown by Eqs. (3.3)(3.5),
when H2O2 is not scavenged (blank or reference solution), GSH-Pxcatalyzed reduction of H2O2 to water at the expense of reduced
glutathione (GSH) will be more, and therefore the DTNB method
response (A412nm) to the remaining GSH will be less. Ellmans thiol
reagent (DTNB) responded to unreacted GSH (Schimmel and Bauer,
2002). On the other hand, when there are H2O2 scavengers (sample
solution), less GSH conversion to GSSG (according to reaction (3.3))
will occur, and the remaining GSH will produce a higher DTNB

M. Ozyurek et al. / Journal of Food Composition and Analysis 23 (2010) 689698

response (according to reaction (3.4)). As a result, the sample

reading will be greater than the blank reading in Eq. (2.2), i.e.
A > A0, in the GSH-Px-DTNB method of H2O2 assay.
GSHPx

H2 O2 2GSH ! 2H2 O GSSG

(3.3)

GSH DTNB ! TNB GSTNB

(3.4)

H2 O2 Scavenger-1 ! 2H2 O Product-1

(3.5)

On the other hand, the modied HPSCUPRAC method

proposed in this work uses Cu(II)-neocuproine colorimetric
measurement of H2O2 according to the reaction:
H2 O2 2CuNc2 2 ! O2 2CuNc2 2H

(3.6)

as mentioned in Section 3.1. Therefore, the greatest absorbance

will come from the blank, as its initial H2O2 content will not have
been scavenged, i.e. A1, A2 < A0 (described in Eq. (2.1)). To
summarize, the relative magnitudes of the blank (A0) and sample
absorbance (A) readings are interchanged in the reference and
proposed methods.
Comparison between benzoic acids and hydroxycinnamic acids
indicated that benzoic acid derivatives (i.e. simple phenolic acids)
appeared to be the strongest hydrogen peroxide quenchers in the
HPSCUPRAC method (Table 2). Similarly, Mansouri et al. (2005)
have found that benzoate derivatives (e.g., gallic acid) are stronger
scavengers than their hydroxycinnamic acid analogues (e.g., caffeic
acid). The same researchers have also reported that caffeic acid has
a lower IC50 value (IC50 = 12.21  0.48 mM) than p-coumaric acid
(IC50 = 435.00  35.35 mM) for HPS activity (Mansouri et al., 2005).
This is in accordance with the authors earlier ndings on the CUPRAC
antioxidant capacities of hydroxycinnamic acids (Apak et al., 2004,
2007), because structural requirements of these compounds would
Table 2
HPS activities (%) of various antioxidant compounds found by the HPSCUPRAC
method in comparison to the GSH-Px-DTNB reference method.
HPS value (%)
with respect to
HPSCUPRAC
method

HPS value (%)

with respect to
GSH-Px-DTNB
method

Simple phenolic acids

Gallic acid
Pyrogallol

62.8  0.9
54.9  1.2

63.8  4.0
57.8  2.7

Hydroxycinnamic acids
Rosmarinic acid
Caffeic acid
p-Coumaric acid
Chlorogenic acid

26.0  0.5
17.3  1.6
10.6  0.3
14.6  0.8

15.5  1.6
17.0  0.6
N.D.a
N.D.a

Flavan-3-ols
()Epigallocatechin gallate (EGCG)
()Epicatechin gallate (ECG)
()Epigallocatechin (EGC)
()Epicatechin (EC)
(+)Catechin (CAT)

42.9  0.5
38.6  1.1
35.2  1.3
30.3  0.1
14.6  1.9

53.7  4.3
42.1  2.8
37.0  2.2
30.0  3.8
14.3  1.2

Flavons
Apigenin

2.5  0.1

N.D.a

Flavonols
Quercetin
Kaempferol

47.1  2.0
31.7  0.4

48.4  4.9
21.9  1.9

Flavanons
Naringin

N.D.a

N.D.a

Others
Ascorbic acid
Trolox

17.5  0.5
17.0  1.2

17.2  1.2
11.0  0.9

Polyphenolics

P = 0.05, Fexp = 0.093, Fcrit(table) = 4.747, Fexp < Fcrit(table).

Data presented as (mean  SD), N = 3.
a
N.D.: HPS activity at the level of studied conc. could not be detected.

695

normally dictate that the twoOH bearing caffeic and chlorogenic

acids should exhibit higher antioxidant activity than the oneOH
bearing p-coumaric acid (Apak et al., 2007), which is the case in this
work (Table 2). Moreover, rosmarinic acid would be expected to show
the highest antioxidant activity among the tested hydroxycinnamic
acids (Yldz et al., 2008), which was also conrmed in this work in
regard to HPS activity (Table 2). Rosmarinic acid is the dimer of caffeic
acid, possessing four phenolic hydroxyl groups correlating with
antioxidant power (Rice-Evans et al., 1996). Moreover, it has an
excellent conjugated structure, further stabilizing the aryloxy radicals
produced during the course of its oxidation. In the Rancimat test of oil
oxidation when the lipid substrate was changed to corn oil,
rosmarinic acid proved to be the strongest antioxidant among
hydroxycinnamic acids in inhibition of oil degradation (Chen and Ho,
1997). In the same study, the DPPH. radical scavenging activity order
of the tested antioxidants was: rosmarinic acid  caffeic acid
phenethyl ester > caffeic acid > chlorogenic acid > a-tocopherol > ferulic acid > ferulic acid phenethyl ester > BHT (Chen and Ho,
1997).
The data for the avan-3-ol group of antioxidant polyphenolic
compounds listed in Table 2 demonstrate that the highest HPS
activities in the HPSCUPRAC method were observed for epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin,
and catechin in this order. This is in accordance with theoretical
expectations, because the number and position of the hydroxyl
groups as well as the degree of conjugation of the whole molecule
are important (Rice-Evans et al., 1996). The antioxidant potency of
avonoids of similar conjugation level is roughly proportional to
the total number ofOH groups and is positively affected by the
presence of an o-dihydroxy moiety in the B-ring (Robards et al.,
1999). Likewise, the decreasing order of HPS activity (Table 2)
using the HPSCUPRAC method for epigallocatechin gallate [8 OH;
42.9  0.5], epicatechin gallate [7 OH; 38.6  1.1], epigallocatechin [6
OH; 35.2  1.3], epicatechin [5 OH; 30.3  0.1], catechin [5OH;
14.6  1.9], the integer values ofOH in brackets showing the number
of hydroxyl groups in the molecules, is again in accordance with the
authors previous experiences with the parent CUPRAC assay (of total
antioxidant capacity measurement, Apak et al., 2004, 2007). The HPS
activities for this class of avonoids found with the proposed CUPRAC
and reference GSH-Px-DTNB methods were almost equal (Table 2),
validating the HPSCUPRAC assay.
The HPS activities of avonols were relatively high with an
activity order of quercetin > kaempferol (Table 2). Quercetin,
possessing all the structural requirements of a avonol antioxidant, namely the 5-hydroxy-4-keto substitution in the A-ring, the
2,3-double bond promoting conjugation between the rings, and
the 30 ,40 -dihydroxy catechol substitution in the B-ring, is a typical
avonoid that is expected to show relatively high antioxidant
(Apak et al., 2007) and anti-radical activity (Seyoum et al., 2006).
The plot of HPS activity (percentage scavenging) of gallic acid
calculated with respect to Eq. (2.1) as a function of concentration
of scavenger using the HPSCUPRAC method is given in Fig. 5.
There was a non-linear increase in HPS activity with increasing
concentration of GA. Pyrogallol gave a similar non-linear curve of
HPS activity vs antioxidant concentration (gure not shown).
Actually in a complex reaction where hydrogen peroxide is
catalytically scavenged with phenolic antioxidants, the rate of the
disappearance of H2O2 (HPS activity measured in a xed-time
assay) is related to H2O2, polyphenol, and copper concentrations,
and cannot simply be a linear function of antioxidant concentration. Likewise, it has been reported in literature that the DPPH.
radical scavenging capacities of selected plant extracts varied nonlinearly (i.e. showing sigmoid curves) with the amount of extract
(Amarowicz et al., 2004; Maisuthisakul et al., 2007).
The two-way ANalysis Of VAriance (ANOVA) comparison by the
aid of F-test of the mean-squares of between-treatments (i.e. HPS

[(Fig._5)TD$IG]

696

[(Fig._6)TD$IG]

M. Ozyurek et al. / Journal of Food Composition and Analysis 23 (2010) 689698

Fig. 6. UV spectra of possible mixtures comprised of (a) catechin + H2O2, (b)

catechin and (c) H2O2.

forming a given mixture (i.e. no chemical deviation from Beers

law).
Fig. 5. Effect of concentration on the hydrogen peroxide scavenging (HPS) action of
gallic acid (2.0  105 M to 2.0  104 M) using the HPSCUPRAC method.

values of different samples with respect to HPSCUPRAC method

and GSH-Px-DTNB reference method depicted in Table 2) and of
residuals (Miller and Miller, 1993) for a number of real samples
(consisting of fteen polyphenols) enabled to conclude that there
was no signicant difference between-treatments. In other words,
the results obtained with the proposed CUPRAC and reference
GSH-Px-DTNB methods were alike at 95% condence level
(Fexp = 0.093, Fcrit = 4.747, Fexp < Fcrit at P = 0.05). Thus, the
proposed methodology was validated against the GSH-Px-DTNB
reference method.
3.4. Analysis of a scavenger (catechin) and a real matrix (green tea
extract) solution using the direct UV-method
Catechin and green tea extract solutions were tested for their
ability to scavenge hydrogen peroxide by using the classical UVmethod (Magalhaes et al., 2008) (Figs. 6 and 7, respectively). The
following observations were made:

[(Fig._7)TD$IG]

(i) Both catechin and green tea extract gave background

absorption at 230 nm (working wavelength of classical UVmethod).
(ii) The absorbances of catechin (or green tea extract) and
hydrogen peroxide were approximately additive at 230 nm.
(iii) Both catechin and green tea extract at the working concentration level could not scavenge H2O2, as observed from the
additivity of absorbances of the corresponding constituents

In comparison with the direct UV-method, the HPSCUPRAC

method eliminates the interference from UV-absorbing materials
such as antioxidant compounds present in plant extracts. Unlike
the UV-method, the proposed method utilizing a catalyst enables
effective H2O2 elimination within a reasonable time period of the
test protocol.
3.5. Application of the method to green tea extract and some fruit
juices
The hydrogen peroxide scavenging activity of green tea extracts
and some fruit juices were determined by both the proposed and
reference methods as HPS (%) values of the incubation reaction
mixture (Table 3). The green tea extract was shown to exhibit the
higher HPS activity (HPS, % = 41.9) with the HPSCUPRAC method,
in agreement with many literature reports. The HPS activities
found with HPSCUPRAC were almost equal to those found with
the GSH-Px-DTNB method measured at the same concentrations of
plant extracts.
Comparison of the means of the two assays with a two-way
ANOVA without interaction test applied to HPS activities of plant
extracts tabulated in Table 3 yielded the following results: The null
hypothesis: H0, stating that all the rows effects are zero was
rejected, meaning that the plant samples were different. The null
hypothesis: H00 , stating that all the columns effects are zero was
accepted, meaning that the same plant extracts analyzed with the
proposed and reference methods essentially yielded the same results.
The same test was performed on the data in Table 2, yielding the same
results, thereby validating the proposed method in comparison to the
reference method in regard to both precision and accuracy.

Fig. 7. UV spectra of possible mixtures comprised of (a) green tea extract + H2O2, (b) green tea extract and (c) H2O2.

M. Ozyurek et al. / Journal of Food Composition and Analysis 23 (2010) 689698

Table 3
HPS activities (%) of green tea extract and some fruit juices using HPSCUPRAC
method in comparison with the GSH-Px-DTNB method (N = 3).
Samples

HPS (%) value

with respect to
HPSCUPRAC
method

HPS (%) value with

respect to GSH-Px-DTNB
method

Green tea extract

Sour cherry juice
Orange juice
Apple juice

41.9  0.8
12.7  0.6
23.1  0.8
26.2  1.3

47.1  3.7
14.2  1.0
20.18  1.9
21.99  1.7

P = 0.05, Fexp = 0.0021, Fcrit(table) = 10.13, Fexp < Fcrit(table); data as (mean  SD), N = 3.

4. Conclusion
The idea in the present study is to measure the HPS activity of
polyphenols (i.e. avonoids, simple phenolic acids, and hydroxycinnamic acids) and other antioxidant compounds (i.e. ascorbic
acid) with a simple, low-cost and versatile colorimetric procedure.
In the most common UV-method for determination of HPS activity,
scavenging ability depends on the change of the absorbance value
at 230 nm when H2O2 concentration is decreased by scavenger
compounds. Since the UV-method suffers from both the interference of some phenolics in real samples having strong absorption in
the UV-region and from inefcient degradation of H2O2 with
polyphenols in the absence of Cu(II), HPS activity of polyphenols
was alternatively determined without interference by directly
measuring the concentration of undegraded H2O2 using the HPS
CUPRAC method in the presence of a Cu(II) catalyst. The proposed
methodology is also superior to the rather non-specic horseradish
peroxidase-based assays. Another contribution of the developed
method to antioxidant research is its integration to the CUPRAC
train of antioxidant measurements, starting from total antioxidant
capacity (TAC) measurement of plant extracts and biological uids
and extending up to the simultaneous assay of both lipophilic and
hydrophilic antioxidants, determination of hydroxyl radical
scavenging and xanthine oxidase inhibition of polyphenols, and
TAC measurement of protein thiols (Apak et al., 2004, 2007;
zyurek et al., 2008a,b, 2009;
Bektasoglu et al., 2006, 2008; O
Demirci Cekic et al., 2009; Guclu et al., 2006). In conclusion, the
proposed method can be considered as a precise and accurate
alternative for measuring the HPS activity of polyphenols and plant
extracts.
Acknowledgements
zyurek would like to thank Istanbul University
Author Mustafa O
Research Fund, Bilimsel Arastirma Projeleri (BAP) Yurutucu
Sekreterligi (Project T-1428/11092007), and to Istanbul University,
Institute of Pure and Applied Sciences (I.U. Fen Bilimleri Enstitusu),
for the support given to his Ph.D. thesis entitled Development of
Modied CUPRAC Methods for Reactive Oxygen Species Scavenging
Antioxidant Activity Measurement. The authors also express their
gratitude to Istanbul University Research Fund for the support of two
projects (BAP-2724 and UDP-7344), the latter enabling author Resat
Apak to attend the 240th ACS National Meeting & Exposition in
Boston, Massachusetts, U.S.A., on 2226 August 2009, for an oral
presentation of the Main and modied CUPRAC methods of
antioxidant characterization.
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