Analytica Chimica Acta 763 (2013) 110

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Analytica Chimica Acta
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Review
Evaluating the antioxidant capacity of natural products: A review on
chemical and cellular-based assays
Camilo Lpez-Alarcn
a
, Ana Denicola
b,
a
Departamento de Farmacia, Facultad de Qumica, Ponticia Universidad Catlica de Chile, Av. Vicuna Mackenna 4860, 7820436, Santiago, Chile
b
Instituto de Qumica Biolgica, Facultad de Ciencias, Center for Free Radical and Biomedical Research, Universidad de la Repblica, Igu 4225, 11300
Montevideo, Uruguay
h i g h l i g h t s
There is high interest on determi-
nation of antioxidant capacity of
natural products.
Several chemical in vitro methods
should be used.
Cellular antioxidant activity assays
should also be performed.
No strong correlation between
chemical and cellular assays.
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
Article history:
Received 1 July 2012
Received in revised form
24 November 2012
Accepted 26 November 2012
Available online 5 December 2012
Keywords:
Antioxidant capacity
Chemical assays
Nanoparticle assays
Cellular assays
Reactive oxygen species
Reactive nitrogen species
a b s t r a c t
Oxidative stress is associated with several pathologies like cardiovascular, neurodegenerative, cancer and
even aging. It has been suggested that a diet rich in antioxidants would be benecial to human health
and a lot of interest is focused on the determination of antioxidant capacity of natural products. Different
chemical methods have been developed including the popular ORAC that evaluates the potential of a
sample as inhibitor of a target molecule oxidation. Chemical-based methods are useful for screening,
they are low cost, high-throughput and yield an index value (expressed as equivalents of Trolox) that
allows comparing and ordering different products. More recently, nanoparticles-based assays have been
developed to sense the antioxidant power of natural products. However, the antioxidant capacity indexes
obtained by chemical assays cannot extrapolate the performance of the sample in vivo. Considering that
antioxidant action is not limited to scavenging free radicals but includes upregulation of antioxidant and
detoxifying enzymes, modulation of redox cell signaling and gene expression, it is necessary to move to
cellular assays in order to evaluate the potential antioxidant activity of a compound or extract. Animal
models and human studies are more appropriate but also more expensive and time-consuming, mak-
ing the cell culture assays very attractive as intermediate testing methods. Cellular antioxidant activity
(CAA) assays, activation of redox transcription factors, inhibition of oxidases or activation of antioxidant
enzymes are reviewed and compared with the classical in vitro chemical-based assays for evaluation of
antioxidant capacity of natural products.
2012 Elsevier B.V. All rights reserved.
Abbreviations: ROS, reactive oxygen species; RNS, reactive nitrogen species; SOD, superoxide dismutase; DPPH

, 2,2-diphenyl-1-picrylhydrazyl radical; ABTS

+, 2,2

-
azinobis-(3-ethylbenzothiazole-6-sulphonate) radical cation; FRAP, ferric reducing antioxidant power; CUPRAC, cupric ion reducing antioxidant capacity; ORAC, oxygen
radical absorbance capacity; TRAP, total radical-trapping antioxidant parameter; LDL, lowdensity lipoprotein; ESR, electronspinresonance; HepG2, humanhepatocarcinoma;
DCFH
2
, dihydrodichlorouorescein; DCFH
2
-DA, dihydrodichlorouorescein diacetate; CAA, cellular antioxidant activity; RBC, red blood cells; NOx, NADPH oxidases; NOS,
nitric oxide synthase; Nrf-2, nuclear factor E2-related protein 2; NF-B, nuclear factor kappa B.

Corresponding author. Tel.: +598 2 5250749; fax: +598 2 5250749.

E-mail addresses: clopezr@uc.cl (C. Lpez-Alarcn), denicola@fcien.edu.uy (A. Denicola).
0003-2670/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aca.2012.11.051
2 C. Lpez-Alarcn, A. Denicola / Analytica Chimica Acta 763 (2013) 110
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1. Mechanisms of antioxidant actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.1. The classic (traditional) antioxidant mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.2. New mechanisms based on redox signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Methodologies to evaluate antioxidant capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1. Chemical-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.1. Scavenging activity toward stable free radicals (DPPH

, ABTS

+) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.2. Reduction of metal ions (FRAP and CUPRAC assays) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.3. Competitive methods (ORAC and TRAP assays) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.4. Oxidation of low density lipoprotein (LDL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1.5. Nanoparticles-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1.6. Other chemical assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2. Evaluation of the antioxidant activity at cellular level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2.1. Cellular antioxidant activity (CAA) assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2.2. Expression of antioxidant enzymes vs inhibition of pro-oxidant enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.2.3. Activation vs repression of redox transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3. Correlation between chemical-based antioxidant capacity indexes and cellular-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Ana Denicola is Head of the Physical Biochemistry
laboratory at the Chemical Biology Institute of Fac-
ulty of Science, University of the Republic (UdelaR),
Uruguay. She graduated inChemistry/Pharmaceutical
Chemistry at the UdelaR, Uruguay, and received her
PhD in Biochemistry at Virginia Tech, Va, USA. She is
nowFull Professor of Physical Biochemistryat the Fac-
ulty of Science, UdelaR. Her major research interests
concern mechanisms of bioproduction and reactivity
of oxygen and nitrogen species, structural and func-
tional characterization of oxidative modications of
proteins, synthetic and natural antioxidants.
Camilo Lpez Alarcn received his Doctorate in
Chemistry in 2004 fromthe University of Chile, Chile.
He then gained a posdoctoral position at Dr. Eduardo
Lissis laboratory at University of Santiago of Chile,
where he focused on free radicals and antioxidants
chemistry. At present, he is Associate Professor at
Ponticial Catholic University of Chile, where he is
studying new methodologies to evaluate in vitro
antioxidant capacity of foods, beverages and human
uids. His current research focuses on the interac-
tionof biologically-relevant compounds withreactive
species such as nitrous acid, peroxyl radicals, super-
oxide, hypochlorite and nitrogen dioxide.
1. Introduction
During the last decades it has been proposed that oxidative
stress, denedas theunbalancebetweenreactiveoxygenandnitro-
gen species (ROS/RNS) production and the antioxidant defense,
plays a pivotal role in different pathophysiological conditions [1,2].
Oxidative stress, originated from an increase in ROS/RNS produc-
tion or froma decrease in the antioxidant network, is characterized
bytheinabilityof endogenous antioxidants tocounteract theoxida-
tive damage on biological targets [3]. In this context, it has been
suggested that an intake of a rich antioxidant diet is inversely
associated with the risk to develop some pathologies like car-
diovascular diseases [46]. Thus, attention has been paid on the
antioxidant capacity of natural products, with particular interest
on those that are frequently (or potentially) consumed by peo-
ple. Different in vitro chemical-based assays have been developed
to determine the antioxidant capacity of natural products, includ-
ing the popular ORAC, DPPH

scavenging method, ferric reducing

capacity, etc. and more recently the use of nanoprobes to evaluate
the metal-reducing capacity of antioxidants [714]. These assays
are based on different strategies and endow different informa-
tion about the ROS/RNS-sample interaction. However, to study the
potential antioxidant health-protecting effects of natural products,
considering the complexity involved in their in vivo mechanisms
of action, a single in vitro chemical method is not enough to eval-
uate and compare their antioxidant properties. In addition, these
chemical assays do not consider relevant parameters involved in
biological environments such as lipophilicity and bioavailability;
therefore, the obtained antioxidant capacity indexes not necessary
reect the antioxidant effects that would be associated with a par-
ticular sample in vivo. The ability of natural products to induce
an antioxidant response at the cellular level also has to be eval-
uated. A good antioxidant is not just a good radical scavenger and
reducing compound but a molecule that can exert its antioxidant
activity by activating transcription factors that induce the expres-
sion of antioxidant enzymes, thus, ameliorating oxidative stress
[15]. To evaluate a newproduct as antioxidant, as a compound able
to change the redox cellular state, we must use a cellular antiox-
idant assay since participation of different components of the cell
are critical to nally develop an antioxidant response.
In the present work we review the chemical-based method-
ologies employed for screening antioxidant capacity of natural
products discussing the classical and non-classical mechanisms
of action of antioxidant-containing natural products. In particu-
lar, we highlight the advantages and drawbacks of each method;
introduce the importance of pursuing the evaluation of antioxidant
capacity at the cellular level and compare reported results between
chemical- and cellular-based assays.
1.1. Mechanisms of antioxidant actions
1.1.1. The classic (traditional) antioxidant mechanism
The classical denition of antioxidant as any substance that
delays, prevents or removes oxidative damage toa target molecule
[2] is usually understood as the ability of these compounds to
C. Lpez-Alarcn, A. Denicola / Analytica Chimica Acta 763 (2013) 110 3
neutralize free radicals, acting for example, as chain-breaking
derivatives in agreement with Reaction (1).
AH+FR

+FRH (1)
where AH and FR

represent an antioxidant and a free radical,

respectively.
Thus, Reaction (1) is considered the base of the classical mecha-
nismof actionof antioxidants andexplains their abilitytoinhibit (or
delay) many damaging processes inducedby FR

onlipids, proteins,
or DNA. However, Reaction (1) does not represent all the factors
affecting the antioxidant activity of a compound or an antioxidant-
containingmixture. Amongthese factors, the most relevant are: the
reactivity of antioxidants toward FR

, the number of FR

molecules
neutralizedby eachantioxidant molecule (stoichiometric factor, n),
the liposolubility of the antioxidant, and the presence of secondary
reactions.
1.1.1.1. Reactivity and stoichiometric factor. Taking into account
Reaction (1) as a bimolecular process, the rate of the reaction, r,
expressed as:
r = k
1
[AH] [FR

]
ss
(1)
depends on the kinetic rate constant k
1
, the antioxidant concen-
tration [AH], and the steady state concentration of FR

([FR

]
ss
).
Accordingly, froma kinetic point of view, a compound with a high
k
1
value wouldbe a goodantioxidant. Nonetheless, for antioxidants
expected to act in biological environments (for example human
blood plasma) the latter is relevant only if their reactions with FR

are faster or comparable with the rate of endogenous antioxidants

(for example human serumalbumin or uric acid) reacting with FR

.
In that case, the stoichiometry of Reaction (1), dened as the num-
ber of FR

molecules that each antioxidant is able to neutralize (n),

could also be a relevant factor in a food or beverage preservative.
1.1.1.2. Liposolubility. The capacity of a particular antioxidant to
reach the place where FR

are being generated is also a critical

aspect. In this context, antioxidants with high in vitro antioxidant
capacity are not necessarily efcient neutralizers of FR

incompart-
mentalized systems. This is a pivotal point when trying to inhibit
lipid peroxidation processes in cell membranes, where antioxi-
dants should have an appropriate liposolubility to be incorporated
into the membrane and to react with FR

through chain-breaking
reactions [16]. For instance, -tocopherol is liposoluble and is
the most important biological antioxidant in membranes, but its
solubility in water is very low. One particular case is ascorbic
acid, which in spite of its well-known hydrophilic character is
able to work additively with membrane-immersed -tocopherol
to prevent lipid peroxidation, by reducing the surface-exposed -
tocopheroxyl radical back to -tocopherol [17].
1.1.1.3. Secondary reactions. Other aspect toconsider inthe antiox-
idant behavior of a particular sample is the ability of secondary free
radicals (A

) generated in Reaction (1) to damage biological targets

(BTH in Reaction (2)).
A

+BTH AH+BT

(2)
It has been observed that secondary phenoxyl radicals can, in
some conditions, trigger oxidative modications on proteins, DNA,
as well as on cell membrane lipids [18]. Furthermore, depending on
the antioxidant compound and/or the place where A

is generated,
it can react with O
2
to forma secondary peroxyl radical or superox-
ide anion (Reaction (3) and Reaction (4), respectively). In the latter
case, dismutation of superoxide (usually catalyzed by superoxide
dismutase, SOD) yields hydrogen peroxide (H
2
O
2
), which in the
presenceof metals generates hydroxyl radical (Fentonmechanism),
a strongly oxidant free radical.
A

+O
2
AOO

(3)
A

+O
2
A
ox
+O

2
(4)
1.1.2. New mechanisms based on redox signaling
In 1985 Helmut Sies introduced the concept of oxidative stress
as a disturbance in the prooxidant-antioxidant balance in favor
of the former [3]. Experimental evidence supported the idea that
oxidative stress contributes to the development of several patholo-
gies including cardiovascular disease, neurodegenerative diseases,
cancer andalsoaging. ROS suchas superoxide, singlet oxygen, H
2
O
2
and hydroxyl radical were mainly considered responsible for these
damaging oxidative reactions. More recently, new radical species
were identied, now centered on nitrogen, thus termed reactive
nitrogen species RNS, derived fromthe biologically produced rad-
ical, nitric oxide (

NO). The concept of ROS/RNS as just biologically

damaging species has begun to change. Enzyme systems produce
reactive species not only for chemical defense or detoxication, but
also for cell signaling and biosynthetic reactions. The presence of
both, toxic and benecial effects of ROS/RNS precludes a simple
denition of oxidative stress. At the same time, the intervention
with antioxidant supplements did not show the results expected
[1921], in essence because most pathologies are multifactorial
and oxidative stress is just one of many contributing factors. A
new concept of oxidative stress was emerging, not limited to free
radical damage of the macromolecular machinery but to pertur-
bation of cellular redox status. Based on new accumulating data
on redox signaling pathways, antioxidant intervention trials and
oxidative stress markers, Dean Jones in 2006 re-dened oxidative
stress as a disruption in redox signaling and control [15]. It is
clear today that the mechanism of action of antioxidants is more
complex than just intercepting reactive free radicals [15,22]. Free
radicals can be deleterious to life depending on the type of radical
produced and the level and site of production, but at the same time,
low concentrations of these reactive species are essential to per-
formnormal physiological functions like gene expression, cellular
growth and defense against infection. The redox cellular network
is nely regulated and its perturbation provokes oxidative stress.
The idea behind antioxidant supplementation is to restore redox
cellular status. Some studies have indicated that antioxidants could
alsohave deleterious effects onhumanhealthdepending ondosage
and bioavailability [19,20]. It is therefore necessary to explore the
mechanismof action of a potential antioxidant at the cellular level
in order to extrapolate its therapeutic potential (Fig. 1).
1.1.2.1. Discrete and compartmentalized redox signaling pathways.
Evidence has accumulated showing that endogenous oxidants like
H
2
O
2
can act as second messengers and trigger a cascade of intra-
cellular responses resulting in the expression of antioxidant and
detoxifying enzymes in order to control the cellular redox status
[23]. Much research is still needed in this area but some redox
signaling pathways have already beenidentied. Two transcription
factors associated with redox control are: Nrf-2 (nuclear factor E2-
related protein 2) and NF-B (nuclear factor kappa B) with dened
redox control compartimentalized in cytosol and nucleus.
Nrf-2 is a redox-sensitive transcription factor that is activated
by an oxidative signal in the cytoplasm that causes its transloca-
tion to the nucleus where it binds to DNA ARE-regions (antioxidant
response elements) inducing the expression of cytoprotective
enzymes like glutathione S-transferase GST, superoxide dismu-
tase SOD, heme oxigenase-1 HO-1, NADPH-quinone oxidase NQO
(ARE-regulated genes) [24]. In the cytoplasm, Nrf-2 is associated
with Keap1 (Kelch-like ECH associated protein) that facilitates its
ubiquitination and degradation, thus, keeping down the levels of
4 C. Lpez-Alarcn, A. Denicola / Analytica Chimica Acta 763 (2013) 110
Fig. 1. Different mechanisms of action of antioxidants: (1) inhibition of oxidant
enzymes, that translate ina decrease inthe ROS/RNS cellular production, (2) interac-
tion with redox signaling pathways that translate in a cellular antioxidant response
(less classical viewof an antioxidant compound or mixture), (3) direct reaction with
ROS/RNS yielding a less toxic/reactive product (the most popular viewof an antiox-
idant, as a free radical scavenger). Enzymes producing ROS/RNS: NOS (nitric oxide
synthase), NOx (NADPH oxidase), COX (cyclooxygenase), MPO (myeloperoxidase).
Antioxidant enzymes: CAT (catalase), SOD (superoxide dismutase), Prx (peroxire-
doxin), GPx (glutathione peroxidase), Trx (thioredoxin), TR (thioredoxin reductase),
GR (glutathione reductase).
Nrf-2. Activation of Nrf-2 by ARE-inducers provokes dissociation of
Nrf-2/Keap1 complex, less ubiquitination and the corresponding
accumulation of Nrf-2 that reach the nucleus leading to increase
transcription of genes under control of ARE. Not only Keap1 has
critical cysteine residues that are modied under oxidative stress,
but also Nrf-2 needs a reduced cysteine to bind to DNA.
Compounds that increase the expression of Nrf-2 and/or
facilitate the dissociation of Nrf-2/Keap1 and translocate Nrf-2
to the nucleus, provoke an antioxidant response since ARE-
genes are induced, i.e. the expression of antioxidant enzymes
increased.
The NF-B family consists of a group of inducible transcrip-
tion factors which regulate immune and inammatory responses
and protect cells from undergoing apoptosis in response to cel-
lular stress (including oxidative stress). NF-B is kept inactive
in the cytoplasm by association with inhibitors IB proteins. In
response to inammatory stimulus, such as oxidants, IB proteins
are rapidly degraded by the proteasome liberating NF-B protein
to the nucleus where it binds to specic DNA sequences, activat-
ing the expression of specic pro-inammatory and anti-apoptotic
genes [25].
Compounds that decrease the expression of NF-B and/or
inhibit its activation prevent its translocation to the nucleus and
the induction of pro-inammatory/pro-oxidant genes.
2. Methodologies to evaluate antioxidant capacity
2.1. Chemical-based assays
Taking into account the complexity involvedinthe invivo action
of antioxidants, different in vitro methodologies have been devel-
oped to estimate, in a simple experimental way, the capacity of
antioxidants and their complex mixtures to interact with (neutral-
ize) ROS/RNS. The assays are based on diverse strategies aimed to
evaluate:
The consumption of stable free radicals by antioxidants.
The capacity of antioxidants to reduce cupric or ferric ions.
The ability of antioxidants to protect a target molecule exposed
to a free radical source.
The capacity of antioxidants to inhibit the oxidation of low-
density lipoprotein LDL.
2.1.1. Scavenging activity toward stable free radicals (DPPH

,
ABTS

+)
DPPH

(2,2-Diphenyl-1-picrylhydrazyl) and ABTS (2,2

-
Azinobis-(3-ethylbenzothiazole-6-sulphonate) radical cation,
ABTS

+) are two stable and colored free radicals that have been
widely employed to determine antioxidant capacity [26,27]. DPPH

is commercially available, whereas ABTS

+ must be generated
from the oxidation of ABTS by oxidants such as K
2
S
2
O
8
, MnO
2
or peroxyl radicals [28]. DPPH

is soluble in organic solvents and

presents a typical absorption band at 517nm (in ethanol); while
ABTS

+ is soluble in aqueous as well as in alcoholic media and its

absorption at 734nm is used. Usually, the decrease in absorption
intensity in the presence of antioxidant-containing samples is
registered after a xed incubation time (30min or less), thus no
kinetic data are considered. In this context, Snchez-Moreno et al.
[29] introduced the antiradical efciency (AE) parameter which
includes kinetic factors to characterize the reaction between
DPPH

and polyphenols.
The shortcomings of these assays are the complexity of the
mechanisms of reaction, the dependence of the index with the
experimental conditions, and the poor correlation between DPPH

and ABTS

+ chemical structures with free radicals produced in bio-

logical systems [30].
2.1.2. Reduction of metal ions (FRAP and CUPRAC assays)
These methods aim to evaluate the capacity of the sample to
reduce ferric or cupric ions in aqueous media. The FRAP (ferric
reducing antioxidant power) assay, originally developed to eval-
uate the ability of human plasma as antioxidant, uses the reaction
of tripyridyl triazine-ferric complex (Fe
III
-TPTZ) with antioxidants
[31], while the CUPRAC (Cupric ion-Reducing Antioxidant Capac-
ity) method, developed by Apak et al. [32], determines the ability
of a sample to reduce the neocuproine-cupric complex (Cu
II
Nc).
The basis of both methodologies is that the complexes of TPTZ or
Nc with the reduced formof the metals present characteristic visi-
ble absorption bands with maximal intensity at 593 and 450nmfor
FRAP and CUPRAC assay, respectively. Thus, the capacity of a sam-
ple to reduce the metal complexes generating the corresponding
visible absorption bands in a xed incubation time, is employed to
determine FRAP or CUPRAC values. FRAP assay requires an acidic
pH (3.6), that is far fromthe physiological pH. In contrast, CUPRAC
assay, performed at pH 7.0, better simulate physiological condi-
tions. In addition, CUPRAC assay has shown to differentiate the
reducing power of thiol-type antioxidants [33].
The main advantage of these methods is the simplicity of the
experimental conditions. However, as DPPH

and ABTS

+ -based
assays, FRAP and CUPRAC indexes depend on the reaction time.
Depending on the sample under study, different endpoints of the
reaction can be registered. In this context, it has recently been pro-
posed a kinetic matching approach to express antioxidant capacity
in a more standardized way [34].
2.1.3. Competitive methods (ORAC and TRAP assays)
These methodologies evaluate the ability of a particular sam-
ple to inhibit the consumption of a target molecule (usually
followed by UVvisible absorption or uorescence spectroscopy)
mediated by peroxyl radicals. Usually, AAPH (2,2

-Azobis (2-
methylpropionamidine) hydrochloride, also abbreviated ABAP) is
employed as peroxyl radical (ROO

) source, that generates ROO

C. Lpez-Alarcn, A. Denicola / Analytica Chimica Acta 763 (2013) 110 5

Fig. 2. Protection of uorescein oxidation (induced by ROO

) elicited by Peumus-
boldus extract. Control experiment (in the absence of extract, ), Peumusboldus
0.1 L/mL (); 0.3L/mL (); 0.5L/mL (). Data taken from[40].
at a known rate in the rst hours of incubation in aqueous media,
according to Reaction (5) [35]
AAPH+O
2
2ROO

+N
2
(5)
The incubation of AAPH with a particular target molecule (TM)
leads to a change in the UVvisible absorption or uorescence
intensity of that TM. Thus, the co-incubation of TM, AAPHand pure
antioxidants or their complex mixtures (XH) usually leads to the
inhibition of TM consumption. The minimal reactions involved in
these assays are:
ROO

+TMconsumption (6)
ROO

+XH X

+ROOH (7)
2ROO

nonradicals products (8)

2.1.3.1. ORAC (oxygen radical absorbance capacity) assay. Among
the methodologies developed to estimate antioxidant capacity,
this assay is one of the most employed. In fact, this method has
been used for estimating the antioxidant capacity of the mainly
consumed foods and beverages in USA [36]. ORAC assay is an
integrative method based on Reactions (5)(8). In this assay the
antioxidant capacity is evaluated from the area under the curve
(AUC) of the kinetic proles of the TM consumption (Fig. 2).
AUC values are commonly compared with gallic acid or Trolox (a
hydrosoluble vitamin E analog) allowing determine an ORAC index
in terms of these reference compounds.
The ORAC assay, proposed by Cao et al. in 1993 was originally
applied employing beta-phycoerythrin as TM [37]. Nonetheless,
in 2001 Priors group proposed an improved ORAC assay employ-
ing uorescein as probe which is photostable, cheap, and does not
interact with polyphenols [38].
Considering Reactions (5)(8), the ORAC index is expected to
be independent fromthe TMemployed. However, reports employ-
ing pyrogallol red and other compounds as TM, have shown that
the selection of the TM can inuence signicantly this index [39].
Furthermore, in some cases, depending on the selected TM, contra-
dictory results were obtained [40].
The ORAC assay has the advantage to be a simple and stan-
dardized method, however, secondary reactions or even reactions
associated with repair mechanisms can be present [41]. In addi-
tion, it has recently been reported a re-examination of the ORAC
assay that indicates that antioxidant-metal reactions could result
in a lower concentration of antioxidants and therefore to an under-
estimation of the ORAC value [42].
2.1.3.2. TRAP (total radical-trapping antioxidant parameter) assay.
This method is based on the oxygen uptake associated to the per-
oxidation process of human plasma [43]. In particular, the method
evaluates the induction period (lag time ), generated in the kinetic
proles of oxygen uptake during the incubation of human plasma
with AAPH (Eq. (2)).
TRAP = R
i
x
plasma
(2)
where R
i
is the rate of peroxyl radicals formation and
plasma
is
the lag time in oxygen consumption generated by the presence of
human plasma.
Thus, taking into account Eq. (2), TRAP index is dened as
the number of moles of ROO

trapped per liter of uid (plasma)

[43,44]. Different variations have been proposed to assess the TRAP
index of natural products. For example, the use of luminol (o-
aminophthaloylhydrazide) or pyranine (8-hydroxy-1,3,6-pyrene
trisulfonic acid) as probes to determine this index. The reaction
of luminol with ROO

emits photons that can be measured in a

luminometer, while pyranine oxidation by ROO

can be followed
by uorescence [44,45]. It is important to consider that TRAP
index reects mainly the number of ROO

trapped per antioxidant

molecule, thus, this is anindexexclusively relatedtothe stoichiom-
etry of the ROO

-antioxidant reaction.
2.1.4. Oxidation of low density lipoprotein (LDL)
The oxidation of LDL mediated by ROS/RNS has been studied
since many years ago. At present, it is well known that ROS play a
pivotal role inthe initiation, propagationandterminationreactions
of the LDL lipid peroxidation processes [2]. In vitro assays usually
employ cupric sulfate as initiator of LDL oxidationandthe lipidper-
oxidation processes are easily followed by UV spectroscopy and/or
chemiluminiscence techniques [46]. In the rst case, the formation
of diene conjugates at 234nmis followed, while in the second one,
the emission of photons related to the formation of nal oxida-
tive products is measured. When cupric sulfate is added to a LDL
solution, the kinetic proles are characterized by the presence of a
lag time associated with the presence of endogenous antioxidants
(mainly vitamin E and coenzyme Q) in the LDL particle. After that
period, the peroxidation of lipids is evidenced as an increase in
the absorbance at 234nm that eventually reaches a plateau, after
minutes or hours. In the presence of antioxidants, this lag time is
increased, implying an additional protection of LDL given by the
sample. Thus, measurement of the lag time has been frequently
used to evaluate antioxidant capacity.
The main advantage of this in vitro assay is the use of a biologi-
cal relevant target. One of the most important shortcomings of this
methodis the variabilitybetweenlot tolot of the LDL sample. This is
a consequence of the complexity of the LDL particle, whichincludes
proteins (apoB), lipids (phospholipids, saturated and unsaturated
lipids, triglycerides, free and esteried cholesterol), and endoge-
nous antioxidants (vitamin E, coenzyme-Q), that can vary from
donor to donor. Also, when cupric sulfate is employed as initia-
tor of oxidation, it should be considered that the antioxidant effect
can also derive fromthe copper-chelating capacity of the sample.
2.1.5. Nanoparticles-based assays
More recently, novel chemical assays based on nanotechnology,
in particular the use of nanoparticles, have been proposed to evalu-
ate antioxidant capacity. Inpioneeringwork, Scampicchioet al. [13]
estimated the antioxidant power of several phenolic acids fromthe
generation and growth of gold nanoparticles (AuNPs) from a Au
III
solution (HAuCl
4
) by the appearance of a sharp plasmon absorp-
tion band at 555nm. The optical properties of the generated AuNPs
correlated well with the reduction potential of the phenolic acids
as measured by cyclic voltametry, and it was proposed as a form
6 C. Lpez-Alarcn, A. Denicola / Analytica Chimica Acta 763 (2013) 110
to evaluate the antioxidant capacity of pure compounds and com-
plex mixtures. For example, this experimental approach has been
recently employed by Liu et al. [47] to evaluate the antioxidant
capacity of chrysanthemum extracts and tea beverages. In addi-
tion, Vilela andcollaborators [14], following the generationof AuNP
at 540nm (A
540
) described a sigmoidal behavior as a function of
polyphenol concentration (X) that t the equation:
A
540
=
A max
1 +e
KAuNPs

XX
50
C
(3)
where Xc
50
represents the concentration of polyphenol that gives
half maximum plasmon resonance absorption and K
AuNPs
the
number of AuNPs produced per polyphenol concentration unit.
The authors proposed to use K
AuNPs
as a parameter to estimate
antioxidant capacity. The assay has been applied to evaluate the
antioxidant activity of natural products such as tea, apples, pears,
red wines and honey. Interestingly, a good correlation between
K
AuNPs
and total phenolic content has been observed [14].
Inadditiontothe latter studies, zyreket al. [12] have reported
a novel methodology employing silver nanoparticles to assess
antioxidant capacity. The method, namedSNPAC(Silver NanoParti-
cle Antioxidant Capacity) employs Trolox as standard antioxidant,
andreects thetotal antioxidant capacity(TAC) of thesampleunder
study. The assay is based on the ability of polyphenols to reduce
Ag
+
ions in the presence of citrate-stabilized silver seeds, and eval-
uates the intensity of the plasmon visible-absorbance at 423nmto
quantitatively determine antioxidant capacity. In comparison with
previous reports the novelty of this assay is to employ a rst step
reductionof Ag
+
ions by citrate to formsilver seeds. Afterwards, the
addition of the polyphenol-containing sample increase the plas-
mon absorption intensity. The role of polyphenols as secondary
reducing agents would imply a more robust and reproducible
methodology than the assays employing a direct reduction of
metal ions by antioxidants. Interestingly, the authors describe that
growth but not nucleation of silver nanoparticles showed a linear
concentration-dependent response. The SNPAC method has been
applied to a wide variety of pure antioxidants and complex mix-
tures suchas fruit juices andherbal teas. As advantages, the method
has shown good linearity with polyphenol concentration and has
not been affected by the presence of reducing sugars, fruits acids,
nor amino acids present in the extracts.
These newnanoparticles-basedassays todetermine antioxidant
capacity of natural products comprise a novel and promising area
combining nanoscience with food and health research.
2.1.6. Other chemical assays
In addition to the above discussed assays, other methodologies
have also been proposed to evaluate antioxidant capacity. These
methods aim to evaluate scavenging activity toward ROS/RNS
formed in vivo like superoxide anion, hydroxyl radical, peroxyni-
trite and hypochlorite.
The classical approach to determine antioxidant capacity
toward superoxide employs the xanthine-xanthine oxidase sys-
temas the superoxide source and probes such as ferricytochrome
c or nitrobluetetrazolium (NBT) [2,7,9]. The reduction of both
probes, mediated by superoxide, is followed by visible absorbance,
assessing the formation of ferrocytochrome c or formazan, respec-
tively. Alternatively or in conjunction, the reactivity toward
superoxideis determinedbyelectronspinresonance(ESR) employ-
ing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin-trap [2,7].
In the presence of an antioxidant-containing sample, the reduction
of ferricytochrome c, NBT or the formation of DMPO-OH adduct
is inhibited. These assays have been widely employed, however,
it is important to consider that some antioxidants, particularly
avonoids, can inhibit xanthine oxidase leading to overestimation
of the antioxidant capacity index [48]. For instance, polyphenols
have been observed to inhibit xanthine oxidase activity using a
parallel CUPRAC assay [49].
Hydroxyl radical (

OH) is one of the most reactive free radicals

and the strongest oxidant, thus in vivo, its toxicity depends strongly
on its site of formation. Its high reactivity results in a very short
diffusion range that may prevent reaction with a critical biological
target. In a general context, the proposed methods employ a

OH-
sensitive molecule such as 2-deoxy-D-ribose, uorescein, or DMPO
and a source of

OH, usually a system containing ferric sulfate-
H
2
O
2
-ascorbic acid or Fenton-like reactions [7,9]. In the case of
uorescein, the consumption of this probe in the absence and pres-
ence of the sample under study is estimated by uorescence, while
in the case of deoxyribose as a probe, the formation of colored
adducts with thiobarbituric acid are determined. In a similar way
to superoxide, the inhibition of the DMPO-OHadduct formation by
ESR has also been used. It is worth to point that, considering the
extraordinary high reactivity of

OH toward biological targets and
many organic compounds, it is somehowirrelevant to evaluate the
capacityof a sample as

OHscavenger invitro. Onlyif the compound

is present at a high concentration in the site of cellular formation
of

OH, will it efciently compete for

OH.
Peroxynitrite (ONOO

), the product of the diffusional reac-

tion between superoxide anion and nitric oxide, is implicated in
different pathological conditions [50]. As in the other cases, the
reaction of antioxidants with ONOO

(or the free radicals derived

fromhomolysis of peroxynitrous acid, ONOOH) has been assessed
employing probe molecules. Dihydrorhodamine, dihydrouores-
cein, pyrogallol red and tyrosine have been used. Oxidation of
the rst two probes is followed by uorescence increase [51],
while pyrogallol red oxidation is monitored by decrease in absorp-
tion at 540nm. 3-Nitrotyrosine formation as a footprint of ONOO-
(although other

NO
2
-dependent biological systems can also per-
form this modication) can be followed by HPLC-MS, UVvis
spectroscopy, or even immunochemistry [52,53]. It is worth to
point that the biological nitration of tyrosine residues is a radical
process initiated by NO-derived species and not by oxygen radicals
[53] that it was not considered (nor quantied) in the classical free
radical scavenging methods.
Hypochlorite ion (ClO

) and its protonated form, hypochlor-

ous acid (HOCl), are oxidative species generated by the enzyme
myeloperoxidase in activated neutrophils. Hypochlorite is a
powerful oxidizing agent able to damage biomolecules and/or
cellular structures during processes associated with inamma-
tion or degenerative diseases [2]. Different approaches have been
employed to evaluate the scavenging activity of antioxidants or
their complex samples toward hypochlorite, being the use of
proteins or low-molecular weight targets the strategy most fre-
quently employed. For instance, 1-antiproteinase, myoglobin
and serum albumin have been used as protein targets, while
5-thio-2-nitrobenzoic acid (TNB), 5-aminosalycilic acid, uores-
cein and luminol have been employed as hypochlorite-oxidizable
low-molecular weight target molecules [9]. The inhibition given
by antioxidants, on the consumption of such targets, is followed
evaluating chloramines formation, by changes in both UVvisible
absorption and/or uorescence as well as chemiluminescence.
2.2. Evaluation of the antioxidant activity at cellular level
Going beyond the in vitro chemical-based assays for testing
potential antioxidants is more expensive because it requires com-
plex cellular testing systems or full clinical trials analyzing blood
samples before and after consuming the product. Nevertheless,
it is very important to proceed to cellular assays after screening
antioxidant activity with an in vitro chemical method in order
to approach some aspects of the bioavailability of the potential
C. Lpez-Alarcn, A. Denicola / Analytica Chimica Acta 763 (2013) 110 7
Fig. 3. Schematic representation of the basis of the cellular antioxidant activity
(CAA) assay. Cells are loaded with the redox-sensitive probe DCFH
2
(the ester form
DCFH
2
-DA is supplied so that to permeate the membrane and once inside the cell is
hydrolyzed by endogenous esterases). Cells are incubated with the natural product
extract or just medium(control) and exposed to AAPH that generates a ux of per-
oxyl radicals (ROO

). The antioxidant compounds can protect DCFH

2
fromoxidation
(thus diminish uorescence) by different mechanisms: (1) scavenge peroxyl radi-
cals in the membrane diminishing lipoperoxidation, (2) react with AAPH avoiding
intracellular ROO

formation, (3) compete with DCFH

2
for oxidants ROS/RNS, (4)
react with ROO

preventing other radicals formation, (5) inhibit a redox pathway

toward formation of ROS/RNS that oxidize DCFH
2
.
antioxidant like uptake, metabolism, partitioning in membranes,
that are crucial to the effectiveness of the antioxidant in vivo.
Animal models and human studies are more appropriate but more
expensive and time-consuming, making the cell culture assays
very attractive as intermediate testing methods.
Accordingtothe newdenitionof anantioxidant, a redox-active
compound or mixture able to modulate the redox status of the cell,
it is critical to use cellular assays in order to evaluate the potential
antioxidant activity of a compound or extract. Antioxidant action
is not limited to ROS/RNS scavenging but includes upregulation
of antioxidant and detoxifying enzymes, modulation of redox cell
signaling and gene expression (Fig. 1).
2.2.1. Cellular antioxidant activity (CAA) assay
Wolfe and Liu developed a cellular antioxidant activity (CAA)
assay to evaluate the antioxidant activity of food extracts and
dietary supplements [54]. They use human hepatocarcinoma
HepG2 cells loaded with the redox sensor dihydrodichlorouores-
cein DCFH
2
that easily oxides to uorescent dichlorouorescein
DCF by ROO

(from AAPH decomposition). The method measures

the ability of compounds to prevent the oxidation of intracellu-
lar DCFH
2
that can be followed by uorescence (
exc
=485nm,

em
=535nm) and the results are expressed in moles of quercetin
equivalents. The CAA assay is an important tool for screening
antioxidant activity in natural products extracts that evaluates its
potential to exert an antioxidant response at the cellular level,
not just its capacity as a reducing agent. The principle of the
method is depicted in Fig. 3. ROO

generated from temperature-

decomposition of the azo compound AAPH can be produced at the
cell membrane or intracellularly and either directly oxidize DCFH
2
or produce other oxidants (ROS/RNS) that nally oxidize the probe.
It is worthtomentionthat DCFH
2
is oxidizedby ROO

but alsoother
biologically-produced ROS/RNS are capable to performthis oxida-
tion, like H
2
O
2
in the presence of peroxidases (not H
2
O
2
alone),
peroxynitrite or hydroxyl radical [51]. The antioxidant compound
can prevent DCHF
2
oxidation, thus reduce uorescence levels in
different ways, either directly reacting with ROO

or secondary
oxidants ROS/RNS, or indirectly, inducing an antioxidant cellular
response that in sum reduce the levels of oxidant cellular status.
The CAA assay protocol is detailed in [54]. Briey, HepG2 cells are
seededonamicroplateat adensityof 610
4
cell/100L/well. After
24h of growth, cells are incubated with the natural product extract
and the probe dichloro-dihydrouorescein diacetate (DCFH
2
-DA,
25M) for 1h at 37

C. The ester is used so that it permeates

the cellular membrane and once inside the cell is hydrolyzed by
endogenous esterases to the more polar DCFH
2
. Then, 600M
AAPHis addedtoeachwell anduorescenceis recordedevery5min
for 1h (
exc
=485nm,
em
=535nm). CAA values are calculated as:
CAA = 1

SA

CA

(4)
where SA is the integrated area under the curve sample uores-
cence vs time and CA is the integrated area fromthe control curve.
Quercetin is used as standard and CAA values are expressed as
mol equivalents of quercetin per 100mol of compound (pure
avonoid) or 100g product (fruit, vegetable) or per 100mol of
total phenolics (considering all phenol content as expressed as gal-
lic acid equivalents).
Among the pure compounds examined in the CAA assay,
quercetinshowedthehighest CAAactivity, followedbykaempferol,
myricetin, epigallocatechin gallate and luteolin. In contrast, caffeic
acid, gallic acid and ascorbic acid showed less than 1% CAA activity
[54].
Cellular uptake of DCFH
2
-DA is rapid and relatively stable
although leakage is observed after 1h, thus long runs should be
avoided [55]. Exposure of loaded cells to light should be minimized
to avoid artifactual generation of superoxide and oxidation of the
probe[56]. Theuseof darkmicroplates is recommended. We should
not forget that working with cells is not like isolated chemical reac-
tions. The cell response is quite complex, thus, more variable results
are expected depending on the assay conditions like growth status
of the cell line, endogenous antioxidant levels and initial ROS/RNS
production. Several replicas should be performed in order to mini-
mize these variables.
Since the publication of the CAA assay by Wolfe and Liu in
2007 [54], this method has been applied to several natural prod-
uct extracts, food and dietary supplements. Among fruits analyzed,
the ones with higher phenolics content were the ones display-
ing higher CAA values; berries (blueberry, cranberry, etc.) with
>50mol quercetin/100g followed by apples, and grapes with less
than 10mol quercetin/100g [57].
Different cell types have been used for the CAA assay besides
HepG2, including Caco-2 matured differentiated intestinal cells
[58], human gastric adenocarcinoma cell line AGS with rapid pro-
liferation properties [59], vascular endothelial cells EA.hy926 [60],
human macrophage cell line U937 [61], human lung broblasts
(WI38, IMR-90) [62]. An interesting one is erythrocytes, since it
is easily available and testing the potential protection of red blood
cells (RBC) is biologically relevant per se as RBC play a critical role
in reducing oxidative stress in the vasculature [63,64]. The same
principle is used, i.e., cells are loaded with the redox probe DCFH
2
,
incubated with the antioxidant to be tested and exposed to oxida-
tive stress via generation of ROO

with AAPH. Cellular oxidative

stress can also be generated by cell culture exposure to H
2
O
2
in the
mMrange(althoughplasmamembraneis permeabletoH
2
O
2
, there
is a signicant drop on H
2
O
2
concentration achieved intracellu-
larly). Again, the oxidation of the probe is followed by uorescence
[65,66]. RBCdonot have nucleus nor mitochondria, thus the antiox-
idant cellular response in this type of cells will not involve gene
expression regulation but will consider the membrane permeation
8 C. Lpez-Alarcn, A. Denicola / Analytica Chimica Acta 763 (2013) 110
of the compounds as well as interaction with cytosolic antioxidant
systems.
The properties of the cell line used in the CAA assay could
affect the result. The use of transformed cell lines has been criti-
cized because they have altered antioxidant enzymes expression
(for example increased catalase mRNA expression in HepG2) and
performed asymmetrical cell divisions [65]. In addition, some cell
lines (like MCF-7) contain estrogen receptors that could be acti-
vated by natural products with phytoestrogen activity.
Another cellular-based assay uses Saccharomyces cerevisiae
as a simple model to evaluate antioxidant capacity of natural
products in living systems [6769]. Exponentially growing yeast
(OD
600
=0.50.7) is exposed to H
2
O
2
to induce oxidative stress,
in the absence and presence of serial dilutions of the extract
(non-toxic nal concentrations) and cell viability is determined by
plating in solid YPDmediumand counting the colonies. S. cerevisiae
is sensitive to H
2
O
2
insult (only 45% survived after 1h incubation
at 28

C/160rpmwith 0.75mM H
2
O
2
). Pretreatment with isolated
avonoids [6769] or complex polyphenolic mixtures like wine
[70,71] partially suppressed the damage triggered by H
2
O
2
.
2.2.2. Expression of antioxidant enzymes vs inhibition of
pro-oxidant enzymes
The previous CAA assay evaluates the capacity of individual
compounds or complex mixtures to effectively reduce the intracel-
lular oxidative state (the levels of the internal biomarker oxidized
uorescein are lower) but it does not say about the mechanismthe
antioxidant compound(s) used to reduce the oxidative stress: is it
reacting directly with a radical oxidant?, which one?, inhibiting the
production of more radical oxidants?, repairing the biomolecules
oxidized by a radical oxidant?. Nevertheless, it is an index that bet-
ter reects the new concept of an antioxidant: a compound (or
mixture) that modulates the cellular redox state. But we can go
further and investigate if the compound displaying a good CAA
is in fact inhibiting an oxidase (for example NADPH oxidase that
produces superoxide) or inducing the expression of an antioxidant
enzyme (for example SOD), resulting in both cases in the reduction
of superoxide/H
2
O
2
levels.
NADPHoxidases are heme-avoenzymes that produce ROS [72].
Theclassical Nox(gp91
phox
) alsotermedNox2, is foundinthemem-
brane of phagocytes and forms an active complex with the other
membrane subunit p22
phox
as well as soluble regulatory proteins,
to catalyze the production of superoxide anion that plays a crucial
role in host defense. We now know that there is an entire family
of NADPH oxidases (Nox) that mediate diverse functions including
cell growth, apoptosis, innate immunity, angiogenesis, regulation
of the extracellular matrix and thyroid hormone biosynthesis. Nox
15 produce superoxide from molecular oxygen at the expense
of NAD(P)H, whereas Duox enzymes are known to release H
2
O
2
without forming detectable amounts of superoxide.
Certain polyphenols and metabolites are able to inhibit Nox
activity reducing the endogenous production of ROS [73,74]. In
addition, complex mixtures from plant extracts or natural prod-
ucts like wine, cocoa, propolis, have been demonstrated to inhibit
this enzyme, in particular the Nox1 and/or Nox 4 isoforms, respon-
sible for the constant production of superoxide at the endothelium
[7579].
Further examples of inhibition of key pro-oxidant enzymes by
polyphenols have been given, e.g., with 5-lipoxygenase and xan-
thine oxidase [49,77,79].
Nitric oxide synthases (NOS) are a family of heme-avoenzymes
that produce the radical

NO from l-arginine using NADPH and
molecular oxygen (also requires tetrahydrobiopterine BH
4
and
Ca
2+
/calmodulin) [80]. Different isoforms have been identied:
NOS1 (neuronal nNOS), NOS2 (inducible iNOS present in phago-
cytes) and NOS3 (endothelial eNOS). A prolonged high production
of

NO from iNOS should be avoided to diminish the inamma-
tory state, while active production fromeNOS at the endothelium
is desirable to promote a healthy vascular function.
The benecial effect of some polyphenols to increase eNOS
activity and at the same time inhibit endothelial Nox, translates
into an effective increase of

NO bioavailability, since not only
more

NO is been produced by eNOS but also the formation of
peroxynitrite from the reaction of

NO with superoxide is being
prevented [78,81,82]. Increased

NObioavailability at the endothe-

lium is likely responsible for the benecial effect observed at the
cardiovascular level by a diet rich in polyphenols [5,83].
Enzymes involved in the metabolism of glutathione (GSH) are
important to maintain cellular redox status. In particular, glu-
tathione reductase (GR) regulates the balance between reduced
(GSH) and oxidized (GSSG) glutathione at the expense of NADPH.
The GSH/GSSG ratio is used as an index of oxidative stress. Acti-
vation of GR raises the level of GSH, thus reduces oxidative stress
and in addition recycles GPx, glutathione peroxidase, an important
H
2
O
2
-detoxifying enzyme. Baroni et al. observed that GR and GPx
activities were increased in yeast cells when exposed to wine in
the presence of H
2
O
2
that could explain the observed increased
survival rates [70].
The thioredoxin system (thioredoxin Trx, thioredoxin reductase
TR and peroxiredoxins Prx) is tightly connected to the GSH sys-
tem and also contributes to maintain the redox status of the cell.
Increased Trx activity have been found in S. cerevisiae treated with
green tea extract due to activation of Yap1 transcription factor by
catechins present in the extract [84].
It is interesting to point that among polyphenols, curcumin and
some avonoids such as myricetin, quercetin and catechin, have
been identied as potential anticancer agents with a mechanism
of action that involves strong specic inhibition of TR activity in
cancer cells and it is currently a focus of attention [8587].
2.2.3. Activation vs repression of redox transcription factors
If the antioxidant is able to interact with a redox transcription
factor, up-regulating the expression of antioxidant enzymes, it is
effectively reducing the oxidative status of the cell, and the inter-
esting point of this mechanism is that the antioxidant response
is amplied. We need just a few molecules of the transcription
factor modied in order to express an antioxidant enzyme that
will catalytically reduce the reactive oxidant. Instead, the amount
of antioxidant compound needed to obtain the same response by
directly reducing the radical oxidant is much higher, even with
a strong reducing compound. Nevertheless, the free radical scav-
enging capacity of an antioxidant is a rapid pathway to protect
biological targets fromoxidation while the effect mediated by up-
regulating antioxidant enzymes is much slower, depending on the
biosynthesis of newproteins.
Isolated polyphenols (like curcumin and quercetin) as well as
natural products extracts have been found to exert an antioxidant
response via activation of Nrf-2 [8891].
In addition, inhibition of Nf-kB activation rendering an
anti-inammatory/anti-oxidant response has been obtained by
incubation of cell cultures to phenolic compounds like curcumin
[92] or fruit extracts like blueberries [93]. Even more, amelioration
of inammatory processes were observed in mice supplemented
with a diet rich in blueberries extracts associated with inhibition
of Nf-kB activation [93].
3. Correlation between chemical-based antioxidant
capacity indexes and cellular-based assays
As mentioned above, Lius group proposed an interesting assay
to evaluate antioxidant capacity in cell cultures [54,57,94]. This
C. Lpez-Alarcn, A. Denicola / Analytica Chimica Acta 763 (2013) 110 9
Fig. 4. (A) CAA values vs ORAC indexes for isolated avonoids (quercetin,
kaempferol, myricetin, morin, luteolin, epicathechin, catechin, galangin). (B) CAA
values vs logP dened as the octanol-water partition coefcient for the selected
avonoids. Data taken from [94]. () represents CAA values using a protocol that
includes washing of the cells before peroxyl radical exposure. () represents CAA
data without previous wash. See [94] for experimental details.
method, named cellular antioxidant activity (CAA) assay, was born
from the need for developing standard methods that allowed
establish the antioxidant capacity of samples in biologically more
relevant conditions. CAA index reects the capacity of antioxidants
to reduce intracellular oxidative stress and evaluates more than
the reduction potential of an antioxidant compound and its free
radical scavenging ability but also takes into account other fac-
tors like membrane permeability, cell uptake, distribution and
metabolism. Therefore, unlike the classical antioxidant capacity
assays which mainly focus this issue froma chemical point of view,
CAA methodology has a more biological approach, in agreement
with the new concept of antioxidant, as a compound able to mod-
ulate the redox state of the cell.
This does not meanthat chemical methods shouldbe considered
useless when compared to cell-based assays. On the contrary, they
should be understood as complementary methodologies and it is
highly recommended to analyze the antioxidant capacity of natural
products byusingmorethanonechemical-basedas well as cellular-
basedassay. Inthis context, Honzel et al. [65] proposeda sequential
strategy to determine antioxidant capacity, including ORAC assay,
evaluation of the protection given by the sample to erythrocytes
(CAP-e method) and response of polymorphonuclear cells (PMN)
exposed to ROS.
Considering that chemical and cell-based assays are inuenced
by very different factors, it is not surprising that the antioxidant
capacity of a sample determined by cell-based assays does not
correlate well with their chemical indexes. Moreover, the results
obtained from chemical-based assays depend on the method
employed since they explore different chemical aspects of the
potential antioxidant compound(s). For example, a particular sam-
ple could yield a high FRAP index because it contains a good metal
reducing compound but it could be a poor scavenger of peroxyl
radicals, thus showing a lowORACvalue. Inaddition, different com-
ponents of the natural product extract may have synergistic effects
and the antioxidant response will depend on the particular phe-
nolic prole. As an example, in Fig. 4 we analyze data previously
reported by Wolfe et al. [94]. As is depicted in Fig. 4A, ORAC values
of pure avonoids do not correlate well with their ability to inhibit
the peroxyl radical-mediated damage on HepG2 cells (CAA index).
Some structures displaying high ORAC values did not showthe cor-
responding highCAAindexes. Similarly, some compounds withlow
ORAC index showed surprisingly high response in the CAA assay.
In addition, CAA values reported so far for isolated avonoids, did
not correlate well withtheir partitioncoefcients (Fig. 4B), suggest-
ing that membrane/water partition of the antioxidant compound is
not the only factor affecting the antioxidant capacity in cell-based
systems.
4. Conclusions
Considering the diversity of mechanisms an antioxidant com-
pound or mixture can exert in vivo, it is not possible to nd a single
analytical method to evaluate its antioxidant capacity. It is nec-
essary to apply more than one in vitro chemical-based assay that
evaluates different aspects of the reactivity of the compound(s)
toward ROS/RNS. It is very important to standardize the analytical
methods used (indexes change depending on the assay conditions)
and always express the results as equivalents of a standard in order
to make possible the comparison between samples. It is essential
to add a cellular-based assay to evaluate the potential of the sam-
ple to elicit a cellular antioxidant response besides its potential
as a good scavenger of ROS/RNS. For testing natural products as
antioxidants, a sequential multifaceted approach is highly recom-
mended, includingfor exampledeterminationof total polyphenolic
content withFolinreagent, a competitive chemical assay like ORAC,
metal-reducing activity like FRAP or SNPAC, inhibition of LDL per-
oxidation, inhibition of tyrosine nitration, and importantly, a CAA
assay. The antioxidant capacity of a natural product extract will
essentially depend on the bioavailability of the specic mixture of
compounds present, their synergistic interactions to yield the nal
antioxidant response at the cellular level.
Acknowledgements
The authors thank Dr. Matas N. Mller for critical reading of the
manuscript and assistance on Figures. The work was supported by
CSIC, UdelaR, Uruguay and FONDECYT (grant no. 1100659), Chile.
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