C H A P T E R
25
The Use of Oxygen Radical Absorbance
Capacity (ORAC) and Trolox Equivalent
Antioxidant Capacity (TEAC) Assays in
the Assessment of Beverages’ Antioxidant
Properties
Simona Carmen Litescu, Sandra A.V. Eremia, Andreia Tache, Ioana
Vasilescu, Gabriel-Lucian Radu
National Institute for Biological Sciences, Bucharest, Romania
CHAPTER POINTS
• T  he appropriate ascription of the antioxidant
efficiency of a compound can not be performed
solely based on an experiment consisting in
determination of radical scavenging properties;
it should be completed by the evaluation of the
compound efficacy with respect to biological
systems and relevant oxidative markers.
• In setting up an experimental design for
antioxidant assessment, it is necessary
to consider sample treatment specificity
and consequently the solvent influence on
final results values, antioxidant solubility
characteristics, and the reaction pathway
(type of antioxidant action).
• Comparison between obtained antioxidant
capacity values must be performed using
the same methods (assays), similar/identical
solvents, and identical experimental
conditions.
• At least two different assays have to be
performed in order to suitably ascribe the
antioxidant properties, of which one must be a
method involving an oxidizable substrate.
Processing and Impact on Antioxidants in Beverages
http://dx.doi.org/10.1016/B978-0-12-404738-9.00025-8 © 2014 Elsevier Inc. All rights reserved.
INTRODUCTION
Antioxidants represent living organisms’ front line of
defense against major health conditions such as neuro-
degenerative diseases, cardiovascular diseases, cancer,
and macular degeneration. There is scientific consensus
that the key factor initiating such pathologies is cellular
damage induced by free radical attack, generally by reac-
tive oxygen species (ROS) radicals, so-called ­‘oxidative
stress.’ According to Sies oxidative stress takes place
when an imbalance between ROS and antioxidants
exists (Sies, 1991).
The definition of an ‘antioxidant compound’ refers to
a substance able to end, suspend, or retard the damag-
ing reaction between an oxidative substrate and ROS.
But the reaction itself is not sufficient to properly define
an antioxidant, the concentration level of the presumed
antioxidant being the main element in deciding the
compound effectiveness, since according to Halliwell
­(Halliwell and Gutteridge, 2007), the proper definition of
an antioxidant is “a substance that, when present at low
concentrations compared to those of an oxidizable sub-
strate, significantly delays or prevents oxidation of that
substrate.” It has to be emphasized that a proper antioxi-
dant (AOxH) has to generate as result of a chain reaction
a corresponding radical (AOx•) less reactive than the
245
246 25.  ORAC AND TEAC ASSAYS IN BEVERAGES ASSESSMENT
free radical initiating the oxidative stress, otherwise the
radical reaction is not quenched.
When dealing with antioxidants assays, several issues
related to free radical structure, oxidative substrate, and
mechanism of action have to be considered to avoid both
misunderstandings of the real effect and experimental
misinterpretation. Accordingly, it has to be mentioned that
there are not overall applicable antioxidants; rather, their
effect depends on the type of ROS initiating oxidative stress
that generates specific damage to a certain substrate.
As Prior and co-workers emphasized in one review
(Huang, 2005), paradoxically, despite the huge interest
in antioxidants, a major problem has been the absence
of validated assay protocols able to perform a reliable
measure of the antioxidant capacity of foods and biologi-
cal samples. At the same time a lot of research dealing
with antioxidant assays from foods was published, but
opinions on the results are quite often significantly dif-
ferent (Niederlander et al., 2008; Litescu et al., 2010; Prior
and Cao, 1999; Sanchez-Moreno, 2002; Laguerre et al.,
2007). These paradoxical opinions are mainly due to the
fact that the antioxidant concept is very comprehensive,
frequently even covering other effects (e.g., anti-inflam-
matory). The lack of validated protocols and validated
methods devoted to antioxidant assessment either from
food, beverages, biological samples, or pharmaceutical
formulations is another factor contributing to the previ-
ously mentioned paradox of reported results variability.
The proper assessment of beverage antioxidant capac-
ity became essential for suitable labeling of a particular
beverage as a potential contributor to antioxidant intake,
considering the importance of beverages in ensuring
the daily intake of antioxidants. This is due not only to
increased use of such products in daily diet, but also to
the improved bioavailability that beverages supply for
the active antioxidant compounds compared with solid
products, which may need additional metabolic steps
prior to antioxidant release.
The present chapter, based on critical evaluation of a
few experimental key points, attempts to set out the argu-
ment for an overall evaluation of foods or beverages from
the point of view of their antioxidant capacity, oxygen rad-
ical absorbance capacity (ORAC) method being one of the
compulsory assays to be performed in order to provide
reliable information with Trolox equivalent antioxidant
capacity (TEAC) being the required complementary assay.
BACKGROUND OF EXPERIMENTAL
SET-UP FOR ANTIOXIDANT ASSAYS
Any experimental set-up of antioxidant assessment,
no matter what the nature of the sample, involves
three key points: (i) sample treatment—including the
extraction procedures for antioxidants, if necessary;
3.  SELECTIVE ASSAYS FOR ANTIOXIDANTS
(ii) antioxidant capacity measurement; and (iii) results
calculation and reporting (Perez-Jimenez et al., 2008).
Sample Treatment
When the sample matrix is a beverage, the sample
treatment is easier, generally consisting simply of dilution
of the sample in the appropriate solvent (water; mixture
of water and alcohol; ethyl acetate). At this point it should
be mentioned that the solvent polarity has a significant
influence on the values of measured antioxidant capac-
ity, of course depending on the method used. The highest
influence was noticed for the methods involving hydro-
gen atom transfer (HAT) mechanisms and polar antioxi-
dants, e.g., phenols, polyphenols, flavonoids, meaning
frequently occurring active principles in beverages and
known as efficient antioxidants. Addressed methods are
numerous, either the DPPH (­ 2,2-diphenylpicrylhydrazyl)
or ORAC methods or even the TEAC method, etc. An
example is the influence of alcoholic solvents on the
results obtained by the DPPH method ­(Litwinienko and
Ingold, 2003, 2005): the rate constants for hydrogen atom
abstraction for various hindered (thus powerful anti-
oxidants) and unhindered polyphenols is abnormally
increased in alcoholic solvents, the demonstrated expla-
nation being the occurrence of a slight ionization of the
phenols followed by their implication in a sequential pro-
ton loss electron transfer reaction involving the solvent.
When measurements of the values of antioxidant capacity
were performed for a mixture of standard antioxidants,
catechin and gallic acid, in different solvents (methanol,
water, and acetone/water 50 : 50, v/v), it was shown that
the reaction medium influences the obtained results; the
effect was greater in ORAC and TEAC assays (Perez-
Jimenez et al., 2008).
It should be concluded at this point that the sample
treatment is highly important; the solvent influence on
the reported results is significant and, accordingly, every
comparison performed between obtained antioxidant
capacity values has to use an identical reaction medium
and similar assays.
Moreover, antioxidant solubility characteristics have
to be considered when sample treatment is performed,
and assessment of antioxidant properties has to com-
prise both hydrophilic and lypophilic conditions for an
accurate result.
Antioxidant Capacity Measurement
In order to provide reliable results with respect to anti-
oxidant efficiency measurement in a manner applicable
to a wide range of presumed antioxidant compounds,
a four-step draft-protocol was proposed (Becker et al.,
2004) for the drawing up of an experiment of antioxidant
efficacy assessment.
 Background of Experimental Set-Up for Antioxidant Assays
This draft-protocol involves:
1. t he quantification and identification of potential
antioxidant compounds from the raw sample;
2. the quantification of the radical scavenging
properties and the determination of formal redox
potential;
3. the evaluation of the degree of lipid oxidation
inhibition or determination of the oxidation end-
point using biological systems; and
4. the study of the antioxidant effectiveness against
relevant oxidative markers.
The antioxidant effect is related to the compound
structure; accordingly, the identification of the com-
pounds with potential antioxidant effect (step 1) is
important to predict the outcome of food or beverage
intake with respect to certain free radicals. For example
one class of antioxidants mostly used as food and bev-
erage natural flavors and colorants are anthocyans and
anthocyanidins, which are highly effective in quench-
ing lipid peroxides, especially those resulted from low-
density lipoproteins peroxidation (Denev et al., 2010);
another example are carotenoids, which are very effi-
cient in scavenging hydroxyl radicals and hydrogen
peroxide (Parker et al., 2010). Consequently, it might be
presumed that a beverage enriched with anthocyans and
carotenoids will be effective in preventing degenerative
diseases (Kahkonen and Heinonen, 2003; Ghosh and
Konishi, 2007; Bell and Gochenaur 2006). At the same
time, it has to be noted that quantification of the anti-
oxidant amount (step 1) is important in order to comply
with the given definition of the antioxidant (Halliwell
and Gutteridge, 2007). However, when dealing with
sample antioxidant effectiveness it has to be considered
that the antioxidant efficiency is really not an additive
property, since a higher amount of antioxidants does
not necessarily lead to better efficiency against oxida-
tive stress; this behavior is due to either the synergetic or
antagonist effect that may occur when various antioxi-
dants are mixed.
As is clear from the above-presented step-sequence,
the appropriate ascription of the antioxidant efficiency
of a compound cannot be performed solely based on an
experiment consisting in determination of radical scav-
enging properties (step 2); it should be completed by the
evaluation of the compound efficacy with respect to bio-
logical systems (step 3) and relevant oxidative markers
(step 4). This experimental approach is highly important
when food and beverages have to be assessed as poten-
tial antioxidant sources, since it is supposed that a proper
diet, rich in antioxidants, might prevent oxidative stress
and further, the occurrence of the related pathologies.
A rationale for this assertion is given below, in order
to provide a basis for understanding the authors’ plea
for a complex assessment of a food or beverage sample
3.  SELECTIVE ASSAYS FOR ANTIOXIDANTS
247
Hydrogen Atom Transfer:
AOxH + ROO ROOH + AOx
AOxH + HO H2O + AOx
Electron Transfer:
- +
ROO + AOxH ROO + AOxH
+ +
AOxH + H2O AOx + H3O
- +
ROO + H3O ROOH + H2O
FIGURE 25.1  Examples of mechanism of antioxidants (AOxH)
action.
before making claims about its antioxidant properties.
Accomplishing experimental step 2 involves an assay
based on the use of a relatively stable reagent or a
long lifetime free radical [e.g. Folin–Ciocalteu; DPPH.,
2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) cation
radical (ABTS+•)]. The information provided by subse-
quent experiments concerns only the radical scaveng-
ing capability of the tested compound, which does not
automatically correlate with proper antioxidant activ-
ity. Not all samples presenting a high radical scavenger
effect demonstrate equally good antioxidant properties
with respect to an oxidizable substrate. This appar-
ent discrepancy has its origin in the reaction pathway,
which commonly is the same for both effects—radical
­scavenging effect and antioxidant effect. The reaction
pathway involves either hydrogen atom transfer (HAT)
or electron transfer (ET), or even both HAT and ET
­(Figure 25.1).
While the radical scavenging property means reach-
ing chemical stability by coupling hydrogen atoms, elec-
trons (or both) between antioxidant and free radicals,
without the participation of a substrate subjected to
oxidative damage, the antioxidant property means the
substrate protection as a result of a competitive reaction
gained by the antioxidant against the ROS damaging the
substrate.
Our assertion complies with Laguerre’s conclusion,
based on the Halliwell definition of antioxidant:
“by this definition, any method that does not involve such a
substrate could not measure antioxidant activity. These are
called indirect methods, which are generally used to measure
the capacity of a molecule to reduce a stable artificial free
radical, or a transition metal”
(Laguerre et al., 2008).
An experimental example supporting this rationale is
Katsube and co-workers, study on antioxidant activity
of several edible plant products, which proved to have a
higher efficiency in preventing LDL oxidation than esti-
mated by subsequent DPPH• assay and Folin–­Ciocalteu
analysis (Katsube et al., 2004).
248 25.  ORAC AND TEAC ASSAYS IN BEVERAGES ASSESSMENT

Results Calculation and Reporting In the initial method, a fluorescent hydrosoluble pro-
tein, β-phycoerythrin (β-PE), was used as a fluorescent
Another important contribution in the above-­ probe, with an excitation wavelength at 540 nm and an
mentioned paradox of the reported variability is given by emission at 565 nm. β-PE absorbs the visible light and pos-
the manner of expressing the obtained values after antiox- sesses high fluorescence yield, proving highly sensitive to
idant capacity measurement. The reports of the obtained ROS (Cao et al., 1993). Further, due to inconsistency of β-PE
results differ from method to method; e.g., in TEAC and ­fluorescent emission (it is photo-sensitive so that it loses its
ORAC methods the results obtained are reported as refer- fluorescence even in the absence of the free radicals) and
ence to a standard antioxidant, Trolox—the hydro-soluble in order to avoid the interaction between β-PE and the
correspondent of vitamin E, while total radical-trapping samples containing phenols and polyphenols, different
antioxidant parameter (TRAP) assay reports the results fluorescent probes were proposed. The new probes had
as associated lag-phase in radical quenching, and DPPH to have high molar absorption coefficients, considerable
assay reports the results as the equivalent concentration fluorescent yields, and photochemical stabilities, the most
able to quench 50% from initial DPPH radical. Trolox is frequently used being fluorescein and ­6-carboxyfluorescein
nowadays generally accepted as the reference compound (Naguib, 2000; Ou et al., 2001; Huang et al., 2002).
in an attempt to support a common value to be used to The ORAC assay is based on in situ production of
compare results from different laboratories for various peroxyl free radicals generated via an azo-compound,
samples having similar effects, despite all the associated 2,2,-azobis(2-methylpropionamidine) dihydrochloride
drawbacks of using it. (It is not the most efficient, has no (AAPH), according to the reaction presented in
physiological relevance, has unsuitable solubility charac- Figure 25.2. AAPH thermally generates C centred free
teristics for assessment of oils, etc.) radicals which, in the presence of oxygen generates the
­peroxy-free radicals interacting with fluorescent probe.
The peroxyl radical is able to react with the oxidizable
OXYGEN RADICAL ABSORPTION substrate/fluorescent probe, changing the fluorescence
CAPACITY ASSAY intensity and generally increasing the rate of fluores-
cence decay. In the presence of an antioxidant com-
Oxygen radical absorbance capacity assay (ORAC) pound (mixture of antioxidants) the fluorescence decay
is a method that was first developed by Cao, Sofic, and is inhibited.
Prior (Cao et al.,1993,1996) and relies on assessing the The result is obtained by calculating the area under
effect of presumed antioxidants by measuring fluores- the fluorescence curve, and it is expressed as equivalent
cence quenching. of micromoles of Trolox per mL (for beverages) or per
Fluorescence measurements were used because the g (for food products from plant), since prior to sample
technique is highly sensitive, allowing limits of detection assessment, the proper calibration is performed for
lower than nanomolar. Trolox.

FIGURE 25.2  ORAC assay measuring principle.

3.  SELECTIVE ASSAYS FOR ANTIOXIDANTS

 Trolox Equivalent Antioxidant Capacity (TEAC) Assay
The following formula is used for results expression:
AUCsample − AUCblank
ACFl = . CTrolox
AUCTrolox − AUCblank
where ACFl represents the antioxidant capacity; AUC-
sample represents the net area under the curve of the mix-
ture: sample, AAPH, fluorescent probe; the AUCblank is
the net area under the blank curve buffer, AAPH, fluo-
rescent probe; AUCTrolox is the net area under the curve
of Trolox, AAPH, fluorescent probe and CTrolox repre-
sents the molar concentration of Trolox.
Usually, when the fluorescent probe is fluorescein
(excitation at 490 nm, emission at 514 nm), the working
conditions involve buffer at pH 7.40; millimolar concen-
tration of AAPH and nanomolar concentration of fluo-
rescein in the measuring cell). The Trolox concentration
normally is 1 μmol/l.
The ORAC method is frequently used to evaluate
the antioxidant capacity of water-soluble antioxidants,
juices, wines, teas, etc., despite the fact that it has several
drawbacks: long time of measurement (30–80 min), and
reproducibility is highly influenced by pH values and
ionic strength (since fluorescent emission is affected).
Its large applicability is generally based on the fact that
ORAC assay deals with the ubiquitous free radical from
biological systems, peroxyl radical.
TROLOX EQUIVALENT ANTIOXIDANT
CAPACITY (TEAC) ASSAY
The TEAC assay was originally developed and opti-
mized by Miller and Rice Evans, and was based on pro-
duction of a colored cation radical, ABTS cation radical
(ABTS•+), as a result of the reaction between ABTS and
ferrylmyoglobin radical generated in situ by the meth-
myoglobin activation with hydrogen peroxide (Rice-
Evans and Miller, 1994). In this first approach the sample
to be tested had to be added before the formation of
ABTS•+ and consequently, the lag phase of radical for-
mation induced by tested samples was measured.
SO3
S
O3S
S N
N N
N C2H5
C2H5
ABTS (λmax = 734 nm)
Asample – Ablank
TEACsample =
ATrolox – Ablank
FIGURE 25.3  TEAC measuring principle and results expression.
3.  SELECTIVE ASSAYS FOR ANTIOXIDANTS
249
This assay proved to be tedious and moreover it was
affected by the drawback that the antioxidant cannot
discriminate between ABTS•+ and ferrylmyoglobin
radicals, the reaction being similar. As a consequence,
Re and Rice-Evans improved the method by develop-
ing a new technique for ABTS•+ radical cation genera-
tion, through the reaction between ABTS and potassium
persulfate (Re et al., 1999). In order to produce the blue–
green ABTS•+ cation radical chromophore, 7  mmol
of ABTS were dissolved in water then treated with
2.45 mmol of potassium persulfate and the mixture was
allowed to react for 12–16 h at room temperature, in the
dark, up to a stable blue–green solution. The so-formed
radical cation has maximum absorption wavelengths at
415 nm (the most commonly used), 645 nm, 734 nm, and
815 nm, and it is stable for more than two days when
stored in the dark, at room temperature. Further, this
last solution is diluted before experiments with ethanol
or buffer (pH 7.40) until the absorbance reaches 0.7 a.u.,
at 734 nm. The sample to be tested is added to the
ABTS+• solution (blue–green), which is reduced by the
antioxidants from the sample to ABTS (colorless) and
the decrease of the maximum absorbency is measured
spectrometrically. The decrease of the maximum absor-
bency is proportional with the antioxidant efficacy.
The result is expressed as equivalent of micromoles of
Trolox (Re et al, 1999). In Figure 25.3 the ABTS cation
radical reaction and the formulas to be applied for anti-
oxidant capacity calculation are given.
In the formula, ATrolox represents the maximum absor-
bency, measured at end-point addition after addition of
an established volume of Trolox (μl) in a cell containing
ABTS+•; Asample represents the maximum absorbency,
measured at end-point addition after addition of an
established volume of sample (μl, same as Trolox vol-
ume) in a cell containing ABTS+•; Ablank represents the
maximum absorbency, measured at end-point addition
after addition of an established volume of solvent (μl,
same as Trolox volume) in a cell containing ABTS+•; f is
the dilution factor of the sample; and CTrolox is the effec-
tive concentration of Trolox, in μmol/l.
S SO3
O3S S N
+ antioxidant
N N
- K2SO5 N C2H5
C2H5
ABTS2 (colorless)
. f . CTrolox
250 25.  ORAC AND TEAC ASSAYS IN BEVERAGES ASSESSMENT

TEAC is one of the assays widely applied to the CONSISTENCY OF ORAC AND TEAC
study of both water-soluble and lipid-soluble antioxi- RESULTS BEYOND THE PARADOX OF
dants, despite the existing drawback given mainly by VALUES VARIABILITY
the fact that measuring time—generally 3–6  min—does
not allow reaching the end-point (reaction completion). As previously mentioned the expression of results is
An automated TEAC assay, using a sequential injection of high importance in order to accurately ascribe the anti-
analysis system, was reported for the evaluation of the oxidant properties of a sample (beverage). The resulting
antioxidant capacity of white and red wines (Pinto et al., reports and, further, the product labeling with regard to
2005). antioxidant intake have to consider the obtained anti-
oxidant capacity values strictly related to the composi-
tion of the sample and to the solubility characteristics of
TABLE 25.1  Romanian Red Wine Antioxidant Capacity the presumed antioxidants (hydrophilic or lypophylic)
Assessment by TEAC and ORAC Methods since the antioxidant action is highly influenced by the
solvent, as formerly stated. All these precautions have
Wine Sample TEAC μmol/l Trolox ORAC μmol/l Trolox
to be taken in order to avoid considering the obtained
Feteasca Neagra 428.03 ± 1.09 90.07 ± 3.20 results as artifacts, if there are significantly different val-
Merlot 359.26 ± 0.92 88.28 ± 2.08
ues when two different methods are used for antioxi-
dant assessment. Further, we provide several examples
Pinot 282.77 ± 0.72 87.33 ± 2.02 supporting the results consistency beyond the apparent
Burgundy 364.50 ± 0.93 89.02 ± 3.20 paradoxical values obtained for antioxidant capacities.
Experiments performed in the authors’ laboratories
to assess antioxidant capacity of different Romanian red
wines of controlled origin led, apparently, to different
results, depending on the method used to evaluate the anti-
oxidant capacity, ORAC or TEAC assay (see Table 25.1).
At a glance, only seeing the values, result discharge
might be considered as an option. However, a deeper
analysis of the obtained value leads to accepting the
results. It is obvious that the ORAC equivalent values
are in a very narrow range, which is consistent with the
associated anthocyan and anthocyanidin effectiveness
against lipoperoxide formation and the composition pro-
file of the red wine (all having an equivalent content of
anthocyans of 6.70–8.18 μg/ml), while TEAC equivalent
values are higher and more widely spread. The TEAC
values are consistent to the sample wines chemical pro-
FIGURE 25.4  Comparison of TEAC and ORAC antioxidant capac-
ity values for red wines samples. file too, since ABTS does not discriminate between OH

FIGURE 25.5  Comparison between ORAC values

and TEAC values for essential oils from edible plants fre-
quently used in infusion, teas, and spice formulations.

3.  SELECTIVE ASSAYS FOR ANTIOXIDANTS

 References
phenolics providing a response related to total groups
able to quench the radical reaction.
The results consistency is proven even by the quite
accurate fitting between the results obtained for the
investigated wines by the two employed assays, since
the antioxidant capacities matching values is linear, with
a correlation coefficient of R = 0.9703 (see Figure 25.4).
The same paradox was noticed even for plant extracts
or essential oils from edible plants mainly used either as
teas, infusions, flavors in beverages, or even as spices.
One accessible example is that provided by the results
obtained for ORAC and TEAC analysis of five essential
oils from some of the most commonly used edible plants:
sage, Hypericum, mint, fennel, and Pistacia.
As shown in Figure 25.5, it seems that ORAC assay
was more appropriate for comprehensive antioxidant
assessment of essential oils while TEAC gave only par-
tial information, considering even solubility character-
istics. It is important to underline that TEAC clearly
provides the antioxidant capacity value associated to the
few compounds extracted in essential oil with structures
compatible to ABTS+• quenching.
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