Analytica Chimica Acta 763 (2013) 1–10

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Analytica Chimica Acta

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

Review

Evaluating the antioxidant capacity of natural products: A review on

chemical and cellular-based assays
Camilo López-Alarcón a , Ana Denicola b,∗
a
Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, 7820436, Santiago, Chile
b
Instituto de Química Biológica, Facultad de Ciencias, Center for Free Radical and Biomedical Research, Universidad de la República, Iguá 4225, 11300
Montevideo, Uruguay

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 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.

a r t i c l e i n f o a b s t r a c t

Article history: Oxidative stress is associated with several pathologies like cardiovascular, neurodegenerative, cancer and
Received 1 July 2012 even aging. It has been suggested that a diet rich in antioxidants would be beneficial to human health
Received in revised form and a lot of interest is focused on the determination of antioxidant capacity of natural products. Different
24 November 2012
chemical methods have been developed including the popular ORAC that evaluates the potential of a
Accepted 26 November 2012
sample as inhibitor of a target molecule oxidation. Chemical-based methods are useful for screening,
Available online 5 December 2012
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
Keywords:
Antioxidant capacity
developed to sense the antioxidant power of natural products. However, the antioxidant capacity indexes
Chemical assays obtained by chemical assays cannot extrapolate the performance of the sample in vivo. Considering that
Nanoparticle assays antioxidant action is not limited to scavenging free radicals but includes upregulation of antioxidant and
Cellular assays detoxifying enzymes, modulation of redox cell signaling and gene expression, it is necessary to move to
Reactive oxygen species cellular assays in order to evaluate the potential antioxidant activity of a compound or extract. Animal
Reactive nitrogen species 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, low density lipoprotein; ESR, electron spin resonance; HepG2, human hepatocarcinoma;
DCFH2 , dihydrodichlorofluorescein; DCFH2 -DA, dihydrodichlorofluorescein 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. López-Alarcón), 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. López-Alarcón, A. Denicola / Analytica Chimica Acta 763 (2013) 1–10

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 Camilo López Alarcón received his Doctorate in
laboratory at the Chemical Biology Institute of Fac- Chemistry in 2004 from the University of Chile, Chile.
ulty of Science, University of the Republic (UdelaR), He then gained a posdoctoral position at Dr. Eduardo
Uruguay. She graduated in Chemistry/Pharmaceutical Lissi‘s laboratory at University of Santiago of Chile,
Chemistry at the UdelaR, Uruguay, and received her where he focused on free radicals and antioxidants
PhD in Biochemistry at Virginia Tech, Va, USA. She is chemistry. At present, he is Associate Professor at
now Full Professor of Physical Biochemistry at the Fac- Pontificial Catholic University of Chile, where he is
ulty of Science, UdelaR. Her major research interests studying new methodologies to evaluate in vitro
concern mechanisms of bioproduction and reactivity antioxidant capacity of foods, beverages and human
of oxygen and nitrogen species, structural and func- fluids. His current research focuses on the interac-
tional characterization of oxidative modifications of tion of biologically-relevant compounds with reactive
proteins, synthetic and natural antioxidants. species such as nitrous acid, peroxyl radicals, super-
oxide, hypochlorite and nitrogen dioxide.

1. Introduction therefore, the obtained antioxidant capacity indexes not necessary

reflect the antioxidant effects that would be associated with a par-
During the last decades it has been proposed that oxidative ticular sample in vivo. The ability of natural products to induce
stress, defined as the unbalance between reactive oxygen and nitro- an antioxidant response at the cellular level also has to be eval-
gen species (ROS/RNS) production and the antioxidant defense, uated. A good antioxidant is not just a good radical scavenger and
plays a pivotal role in different pathophysiological conditions [1,2]. reducing compound but a molecule that can exert its antioxidant
Oxidative stress, originated from an increase in ROS/RNS produc- activity by activating transcription factors that induce the expres-
tion or from a decrease in the antioxidant network, is characterized sion of antioxidant enzymes, thus, ameliorating oxidative stress
by the inability of endogenous antioxidants to counteract the oxida- [15]. To evaluate a new product as antioxidant, as a compound able
tive damage on biological targets [3]. In this context, it has been to change the redox cellular state, we must use a cellular antiox-
suggested that an intake of a rich antioxidant diet is inversely idant assay since participation of different components of the cell
associated with the risk to develop some pathologies like car- are critical to finally develop an antioxidant response.
diovascular diseases [4–6]. Thus, attention has been paid on the In the present work we review the chemical-based method-
antioxidant capacity of natural products, with particular interest ologies employed for screening antioxidant capacity of natural
on those that are frequently (or potentially) consumed by peo- products discussing the classical and non-classical mechanisms
ple. Different in vitro chemical-based assays have been developed of action of antioxidant-containing natural products. In particu-
to determine the antioxidant capacity of natural products, includ- lar, we highlight the advantages and drawbacks of each method;
ing the popular ORAC, DPPH• scavenging method, ferric reducing introduce the importance of pursuing the evaluation of antioxidant
capacity, etc. and more recently the use of nanoprobes to evaluate capacity at the cellular level and compare reported results between
the metal-reducing capacity of antioxidants [7–14]. These assays chemical- and cellular-based 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, 1.1. Mechanisms of antioxidant actions
considering the complexity involved in their in vivo mechanisms
of action, a single in vitro chemical method is not enough to eval- 1.1.1. The classic (traditional) antioxidant mechanism
uate and compare their antioxidant properties. In addition, these The classical definition of antioxidant as “any substance that
chemical assays do not consider relevant parameters involved in delays, prevents or removes oxidative damage to a target molecule”
biological environments such as lipophilicity and bioavailability; [2] is usually understood as the ability of these compounds to
 C. López-Alarcón, A. Denicola / Analytica Chimica Acta 763 (2013) 1–10
neutralize free radicals, acting for example, as chain-breaking
derivatives in agreement with Reaction (1).
AH + FR• → A• + FRH
where AH and FR• represent an antioxidant and a free radical,
respectively.
Thus, Reaction (1) is considered the base of the classical mecha-
nism of action of antioxidants and explains their ability to inhibit (or
delay) many damaging processes induced by FR• on lipids, proteins,
or DNA. However, Reaction (1) does not represent all the factors
affecting the antioxidant activity of a compound or an antioxidant-
containing mixture. Among these factors, the most relevant are: the
reactivity of antioxidants toward FR• , the number of FR• molecules
neutralized by each antioxidant 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 = k1 [AH] [FR• ]ss
depends on the kinetic rate constant k1 , the antioxidant concen-
tration [AH], and the steady state concentration of FR• ([FR• ]ss ).
Accordingly, from a kinetic point of view, a compound with a high
k1 value would be a good antioxidant. 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 serum albumin or uric acid) reacting with FR• .
In that case, the stoichiometry of Reaction (1), defined 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 efficient neutralizers of FR• in compart-
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 to consider in the 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•
It has been observed that secondary phenoxyl radicals can, in
some conditions, trigger oxidative modifications 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 O2 to form a 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 (H2 O2 ), which in the
3
presence of metals generates hydroxyl radical (Fenton mechanism),
a strongly oxidant free radical.
(1) A• + O2 → AOO• (3)
•−
A• + O2 → Aox + O2 (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 and also aging. ROS such as superoxide, singlet oxygen, H2 O2
and hydroxyl radical were mainly considered responsible for these
damaging oxidative reactions. More recently, new radical species
were identified, now centered on nitrogen, thus termed reactive
nitrogen species RNS, derived from the 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 detoxification, but
also for cell signaling and biosynthetic reactions. The presence of
(1) both, toxic and beneficial effects of ROS/RNS precludes a simple
definition of oxidative stress. At the same time, the intervention
with antioxidant supplements did not show the results expected
[19–21], 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-defined 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-
form normal physiological functions like gene expression, cellular
growth and defense against infection. The redox cellular network
is finely 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
also have deleterious effects on human health depending on dosage
and bioavailability [19,20]. It is therefore necessary to explore the
mechanism of 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
H2 O2 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 been identified. Two transcription
factors associated with redox control are: Nrf-2 (nuclear factor E2-
related protein 2) and NF-␬B (nuclear factor kappa B) with defined
(2) 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. López-Alarcón, A. Denicola / Analytica Chimica Acta 763 (2013) 1–10
Fig. 1. Different mechanisms of action of antioxidants: (1) inhibition of oxidant
enzymes, that translate in a decrease in the ROS/RNS cellular production, (2) interac-
tion with redox signaling pathways that translate in a cellular antioxidant response
(less classical view of an antioxidant compound or mixture), (3) direct reaction with
ROS/RNS yielding a less toxic/reactive product (the most popular view of 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 modified 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 inflammatory 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 I␬B proteins. In
response to inflammatory stimulus, such as oxidants, I␬B proteins
are rapidly degraded by the proteasome liberating NF-␬B protein
to the nucleus where it binds to specific DNA sequences, activat-
ing the expression of specific pro-inflammatory 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-inflammatory/pro-oxidant genes.
2. Methodologies to evaluate antioxidant capacity
2.1. Chemical-based assays
Taking into account the complexity involved in the in vivo 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 K2 S2 O8 , MnO2
or peroxyl radicals [28]. DPPH• is soluble in organic solvents and
presents a typical absorption band at 517 nm (in ethanol); while
ABTS• + is soluble in aqueous as well as in alcoholic media and its
absorption at 734 nm is used. Usually, the decrease in absorption
intensity in the presence of antioxidant-containing samples is
registered after a fixed incubation time (30 min or less), thus no
kinetic data are considered. In this context, Sánchez-Moreno et al.
[29] introduced the antiradical efficiency (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 (FeIII -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 (CuII –Nc).
The basis of both methodologies is that the complexes of TPTZ or
Nc with the reduced form of the metals present characteristic visi-
ble absorption bands with maximal intensity at 593 and 450 nm for
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 fixed incubation time, is employed to
determine FRAP or CUPRAC values. FRAP assay requires an acidic
pH (3.6), that is far from the 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 UV–visible absorption or fluorescence 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. López-Alarcón, A. Denicola / Analytica Chimica Acta 763 (2013) 1–10
Fig. 2. Protection of fluorescein oxidation (induced by ROO• ) elicited by Peumus-
boldus extract. Control experiment (in the absence of extract, ), Peumusboldus
0.1 ␮L/mL (); 0.3 ␮L/mL (); 0.5 ␮L/mL (♦). Data taken from [40].
at a known rate in the first hours of incubation in aqueous media,
according to Reaction (5) [35]
AAPH + O2 → 2ROO• + N2
The incubation of AAPH with a particular target molecule (TM)
leads to a change in the UV–visible absorption or fluorescence
intensity of that TM. Thus, the co-incubation of TM, AAPH and pure
antioxidants or their complex mixtures (XH) usually leads to the
inhibition of TM consumption. The minimal reactions involved in
these assays are:
ROO• + TM → consumption
ROO• + XH → X• + ROOH
2ROO• → non radicals products
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 profiles 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 Prior’s group proposed an improved ORAC assay employ-
ing fluorescein 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 from the TM employed. However, reports employ-
ing pyrogallol red and other compounds as TM, have shown that
the selection of the TM can influence significantly 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].
5
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
profiles of oxygen uptake during the incubation of human plasma
with AAPH (Eq. (2)).
TRAP = Ri xplasma (2)
where Ri 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 defined as
the number of moles of ROO• trapped per liter of fluid (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 fluorescence [44,45]. It is important to consider that TRAP
index reflects mainly the number of ROO• trapped per antioxidant
molecule, thus, this is an index exclusively related to the stoichiom-
etry of the ROO• -antioxidant reaction.
(5)
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 in the initiation, propagation and termination reactions
of the LDL lipid peroxidation processes [2]. In vitro assays usually
employ cupric sulfate as initiator of LDL oxidation and the lipid per-
oxidation processes are easily followed by UV spectroscopy and/or
(6)
chemiluminiscence techniques [46]. In the first case, the formation
(7) of diene conjugates at 234 nm is followed, while in the second one,
the emission of photons related to the formation of final oxida-
(8) tive products is measured. When cupric sulfate is added to a LDL
solution, the kinetic profiles 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 234 nm 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
method is the variability between lot to lot of the LDL sample. This is
a consequence of the complexity of the LDL particle, which includes
proteins (apoB), lipids (phospholipids, saturated and unsaturated
lipids, triglycerides, free and esterified 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 from the 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. In pioneering work, Scampicchio et al. [13]
estimated the antioxidant power of several phenolic acids from the
generation and growth of gold nanoparticles (AuNPs) from a AuIII
solution (HAuCl4 ) by the appearance of a sharp plasmon absorp-
tion band at 555 nm. 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. López-Alarcón, A. Denicola / Analytica Chimica Acta 763 (2013) 1–10
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 and collaborators [14], following the generation of AuNP
at 540 nm (A540 ) described a sigmoidal behavior as a function of
polyphenol concentration (X) that fit the equation:
A max
A540 =   (3)
−KAuNPs X−X 50
1+e C and a source of • OH, usually a system containing ferric sulfate-
where Xc50 represents the concentration of polyphenol that gives
half maximum plasmon resonance absorption and KAuNPs the
number of AuNPs produced per polyphenol concentration unit.
The authors proposed to use KAuNPs 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
KAuNPs and total phenolic content has been observed [14].
In addition to the latter studies, Özyürek et al. [12] have reported
a novel methodology employing silver nanoparticles to assess
antioxidant capacity. The method, named SNPAC (Silver NanoParti-
cle Antioxidant Capacity) employs Trolox as standard antioxidant,
and reflects the total antioxidant capacity (TAC) of the sample under
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 423 nm to
quantitatively determine antioxidant capacity. In comparison with
previous reports the novelty of this assay is to employ a first step
reduction of Ag+ ions by citrate to form silver 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 such as fruit juices and herbal 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 new nanoparticles-based assays to determine 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-
tem as 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
superoxide is determined by electron spin resonance (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
flavonoids, 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, fluorescein, or DMPO
H2 O2 -ascorbic acid or Fenton-like reactions [7,9]. In the case of
fluorescein, the consumption of this probe in the absence and pres-
ence of the sample under study is estimated by fluorescence, 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-OH adduct 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 somehow irrelevant to evaluate the
capacity of a sample as • OH scavenger in vitro. Only if the compound
is present at a high concentration in the site of cellular formation
of • OH, will it efficiently 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
from homolysis of peroxynitrous acid, ONOOH) has been assessed
employing probe molecules. Dihydrorhodamine, dihydrofluores-
cein, pyrogallol red and tyrosine have been used. Oxidation of
the first two probes is followed by fluorescence increase [51],
while pyrogallol red oxidation is monitored by decrease in absorp-
tion at 540 nm. 3-Nitrotyrosine formation as a footprint of ONOO-
(although other • NO2 -dependent biological systems can also per-
form this modification) can be followed by HPLC-MS, UV–vis
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 quantified) 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 inflamma-
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, fluores-
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 UV–visible
absorption and/or fluorescence 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. López-Alarcón, A. Denicola / Analytica Chimica Acta 763 (2013) 1–10
Fig. 3. Schematic representation of the basis of the cellular antioxidant activity
(CAA) assay. Cells are loaded with the redox-sensitive probe DCFH2 (the ester form
DCFH2 -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 flux of per-
oxyl radicals (ROO• ). The antioxidant compounds can protect DCFH2 from oxidation
(thus diminish fluorescence) by different mechanisms: (1) scavenge peroxyl radi-
cals in the membrane diminishing lipoperoxidation, (2) react with AAPH avoiding
intracellular ROO• formation, (3) compete with DCFH2 for oxidants ROS/RNS, (4)
react with ROO• preventing other radicals formation, (5) inhibit a redox pathway
toward formation of ROS/RNS that oxidize DCFH2 .
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.
According to the new definition of an antioxidant, 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 dihydrodichlorofluores-
cein DCFH2 that easily oxides to fluorescent dichlorofluorescein
DCF by ROO• (from AAPH decomposition). The method measures
the ability of compounds to prevent the oxidation of intracellu-
lar DCFH2 that can be followed by fluorescence (exc = 485 nm,
em = 535 nm) 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 DCFH2
or produce other oxidants (ROS/RNS) that finally oxidize the probe.
It is worth to mention that DCFH2 is oxidized by ROO• but also other
biologically-produced ROS/RNS are capable to perform this oxida-
tion, like H2 O2 in the presence of peroxidases (not H2 O2 alone),
peroxynitrite or hydroxyl radical [51]. The antioxidant compound
can prevent DCHF2 oxidation, thus reduce fluorescence levels in
7
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]. Briefly, HepG2 cells are
seeded on a microplate at a density of 6 × 104 cell/100 ␮L/well. After
24 h of growth, cells are incubated with the natural product extract
and the probe dichloro-dihydrofluorescein diacetate (DCFH2 -DA,
25 ␮M) for 1 h 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 DCFH2 . Then, 600 ␮M
AAPH is added to each well and fluorescence is recorded every 5 min
for 1 h (exc = 485 nm, em = 535 nm). CAA values are calculated as:
 
SA
CAA = 1 −  (4)
CA
where ʃSA is the integrated area under the curve sample fluores-
cence vs time and ʃCA is the integrated area from the control curve.
Quercetin is used as standard and CAA values are expressed as
␮mol equivalents of quercetin per 100 ␮mol of compound (pure
flavonoid) or 100 g product (fruit, vegetable) or per 100 ␮mol of
total phenolics (considering all phenol content as expressed as gal-
lic acid equivalents).
Among the pure compounds examined in the CAA assay,
quercetin showed the highest CAA activity, followed by kaempferol,
myricetin, epigallocatechin gallate and luteolin. In contrast, caffeic
acid, gallic acid and ascorbic acid showed less than 1% CAA activity
[54].
Cellular uptake of DCFH2 -DA is rapid and relatively stable
although leakage is observed after 1 h, 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]. The use of dark microplates 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
>50 ␮mol quercetin/100 g followed by apples, and grapes with less
than 10 ␮mol quercetin/100 g [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 fibroblasts
(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 DCFH2 ,
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 H2 O2 in the
mM range (although plasma membrane is permeable to H2 O2 , there
is a significant drop on H2 O2 concentration achieved intracellu-
larly). Again, the oxidation of the probe is followed by fluorescence
[65,66]. RBC do not 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. López-Alarcón, A. Denicola / Analytica Chimica Acta 763 (2013) 1–10
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 [67–69]. Exponentially growing yeast
(OD600 = 0.5–0.7) is exposed to H2 O2 to induce oxidative stress,
in the absence and presence of serial dilutions of the extract
(non-toxic final concentrations) and cell viability is determined by
plating in solid YPD medium and counting the colonies. S. cerevisiae
is sensitive to H2 O2 insult (only 45% survived after 1 h incubation
at 28 ◦ C/160 rpm with 0.75 mM H2 O2 ). Pretreatment with isolated
flavonoids [67–69] or complex polyphenolic mixtures like wine
[70,71] partially suppressed the damage triggered by H2 O2 .
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
fluorescein are lower) but it does not say about the mechanism the
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 reflects 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/H2 O2 levels.
NADPH oxidases are heme-flavoenzymes that produce ROS [72].
The classical Nox (gp91phox ) also termed Nox2, is found in the mem-
brane of phagocytes and forms an active complex with the other
membrane subunit p22phox 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
1–5 produce superoxide from molecular oxygen at the expense
of NAD(P)H, whereas Duox enzymes are known to release H2 O2
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
[75–79].
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-flavoenzymes
that produce the radical • NO from l-arginine using NADPH and
molecular oxygen (also requires tetrahydrobiopterine BH4 and
Ca2+ /calmodulin) [80]. Different isoforms have been identified:
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 inflamma-
tory state, while active production from eNOS at the endothelium
is desirable to promote a healthy vascular function.
The beneficial 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 • NO bioavailability at the endothe-
lium is likely responsible for the beneficial 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
H2 O2 -detoxifying enzyme. Baroni et al. observed that GR and GPx
activities were increased in yeast cells when exposed to wine in
the presence of H2 O2 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 flavonoids such as myricetin, quercetin and catechin, have
been identified as potential anticancer agents with a mechanism
of action that involves strong specific inhibition of TR activity in
cancer cells and it is currently a focus of attention [85–87].
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 amplified. We need just a few molecules of the transcription
factor modified 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 from oxidation while the effect mediated by up-
regulating antioxidant enzymes is much slower, depending on the
biosynthesis of new proteins.
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 [88–91].
In addition, inhibition of Nf-kB activation rendering an
anti-inflammatory/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 inflammatory 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, Liu’s group proposed an interesting assay
to evaluate antioxidant capacity in cell cultures [54,57,94]. This
 C. López-Alarcón, A. Denicola / Analytica Chimica Acta 763 (2013) 1–10
Fig. 4. (A) CAA values vs ORAC indexes for isolated flavonoids (quercetin,
kaempferol, myricetin, morin, luteolin, epicathechin, catechin, galangin). (B) CAA
values vs logP defined as the octanol-water partition coefficient for the selected
flavonoids. Data taken from [94]. (䊉) 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 reflects 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 from a 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 mean that chemical methods should be 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 by using more than one chemical-based as well as cellular-
based assay. In this context, Honzel et al. [65] proposed a 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.
9
Considering that chemical and cell-based assays are influenced
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 low ORAC value. In addition, different com-
ponents of the natural product extract may have synergistic effects
and the antioxidant response will depend on the particular phe-
nolic profile. 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 flavonoids 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 show the cor-
responding high CAA indexes. Similarly, some compounds with low
ORAC index showed surprisingly high response in the CAA assay.
In addition, CAA values reported so far for isolated flavonoids, did
not correlate well with their partition coefficients (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 find 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, including for example determination of total polyphenolic
content with Folin reagent, 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 specific mixture of
compounds present, their synergistic interactions to yield the final
antioxidant response at the cellular level.
Acknowledgements
The authors thank Dr. Matías N. Möller 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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