Antiox Fenolici

Phenol-Based Antioxidants and the In Vitro
Methods Used for Their Assessment

Brian D. Craft, Adrian L. Kerrihard, Ryszard Amarowicz, and Ronald B. Pegg
Abstract: In recent years, much interest has been observed in the eld of phenol-based antioxidants. As a result of
this, many analytical methods have been developed for the purpose of the quantication of phenolic and polyphenolic
antioxidant capacities in biological materials. Many of these methods have been altered for application toward the in vitro
assessment of antioxidant activities in animal and human model systems as well as in vivo. Due to the varied applicability
and usage, methods for the assessment of phenol antioxidant capacities have become so widespread that they are often
misused. It is the intent of this work to review the chemistry behind the antioxidant activity of phenolics as well as
summarize the many methods applicable for the measurement of in vitro phenolic antioxidant capacity.
Introduction
Due to the growing popularity of phenolic antioxidants over
the past 2 decades, new scientic methods have been developed
to measure either the content or antioxidant capacity of phenolics
present in plants, foods, and food components (Naczk and Shahidi
2004; Stratil and others 2006; Moon and Shibamoto 2009; Deng
and others 2011). Methods have been established to determine
phenolics antioxidant efcacy in lipid and food model systems
(Becker and others 2004; Decker and others 2005; Laguerre and
others 2007), as well as gauge relative antioxidative activities of
phenolic compounds in vitro (Llesuy and others 2001; Schlesier
and others 2002; Apak and others 2007; Yoo and others 2007)
and in vivo (Halliwell 2008; Jensen and others 2008; Larrosa and
others 2008). Perhaps of particular interest is research directed to-
ward the elucidation of structure-activity relationships of different
classes of phenolic antioxidants (Rice-Evans and others 1996; Lien
and others 1999; Lema nska and others 2001; Nijveldt and others
2001; Kim and Lee 2004; Hoelz and others 2010). Given the pres-
ence of many different antioxidants in biological systems, methods
involving the quantication of total antioxidant capacity (TAC)
are commonplace in todays scientic and medical laboratories.
Antioxidant capacity corresponds to the total radical-scavenging
capability of a test solution, independent of individual antioxidant
activity constants (Ghiselli and others 2000).
Many publications have involved the use of very few antiox-
idant evaluation methods per source, and there exist notable
MS 20111047 Submitted 8/30/2011, Accepted 10/11/2011. Authors Craft,
Kerrihard, and Pegg are with Dept. of Food Science and Technology, The Univ.
of Georgia, 100 Cedar Street, Athens, GA 306022610, U.S.A. Author Amarow-
icz is with Division of Food Science, Inst. of Animal Reproduction and Food Research
of the Polish Academy of Sciences, ul. Tuwima 10, 10747 Olsztyn, Poland. Current
address for author Craft: Department of Food Science & Technology, Nestl e Research
Center, Vers-chez-les-Blanc, Case Postale 44, 1000 Lausanne 26, Switzerland.
Direct inquiries to author Pegg (E-mail: rpegg@uga.edu).
inconsistencies in the methods employed. The lack of uniformity
in the standards used for calibration, assay modications employed,
and the basis for the expression of determinations contributes to
inconsistent data and impairs the ability to compare the results re-
ported in the literature. This concern was illustrated in a publica-
tion by Bhagwat and others (2007) who showed that the ORAC
values of common foods from two different laboratories varied
wildly (in reference to the published values of Ou and others
2002; and Wu and others 2004a).
The wide variety of modications employed in assays mea-
suring antioxidants is undoubtedly due to these assays inherent
versatility. Many in vitro antioxidant assays can be modied for the
production/quenching of several radical species based on the use
of a variety of azo-initiator compounds, metal-ion catalysts, and
thermal or photodegradative processes. For example, the ORAC
assay (Huang and others 2002b; Prior and others 2003) typically
measures peroxyl-radical scavenging; but it has been successfully
modied for the screening of hydroxyl-radical; a radical species
of much greater reaction rate (Ou and others 2002). Given the
different chemistries involved in each group of antioxidants and
the different rates of reactions in the varying radical-scavenging
reactions, the choice of assay can have great effect upon the results
obtained. Not all methods and antioxidant sources are compati-
ble, and the same antioxidant species can yield dissimilar results in
different assays. This suggests a need to complete a multitude of
antioxidant assessment assays on potential phenolic sources, bear-
ing in mind the chemistry involved and the important factors
regarding the assays.
Another concern of many of the assays employed is that of
external relevance. Though some assays can be quick and easy to
perform, they may yield data with little biological signicance. For
instance, despite being two of the most widely used antioxidant
methods, the mixed-mode assays of DPPH
(S anchez-Moreno
and others 1998) and TEAC (Re and others 1999) do not involve
reactive oxygen species (ROS), but instead organic nitro-radicals.
Furthermore, the radical substrates ABTS
+
and DPPH
are very
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2012 Institute of Food Technologists
doi: 10.1111/j.1541-4337.2011.00173.x
Phenolic antioxidants and their assessment . . .
large compounds and steric issues may apply when considering
the potential docking of incoming reactants.
When chosen and performed properly, antioxidant capacity
measurements can produce valuable in vitro data involving the
potential capabilities of antioxidant compounds in vivo (although
still only in the case of screening; bioavailability, and bioactiv-
ity are other issues entirely). Two excellent reviews discussing
the strengths and weaknesses of antioxidant methods/techniques
have recently been published (Schaich 2006; Frankel and Finley
2008), as well as a review specically examining the evidences of
the in vivo relevance of in vitro phenolic antioxidant assessments
(Fernandez-Panchon and others 2008). Though standardization
of methods has been suggested (Prior and others 2005), no of-
cial antioxidant capacity assays exist to date. However, a review of
the scientic literature reveals that certain in vitro methods have
emerged as being commonly implemented for the measurement
of phenolic antioxidant content and efcacy. The purpose of this
work is to discuss these assays, summarize the chemistry of their
processes, and address the benets and drawbacks of each.
Background
Prior to an examination of phenolics and the common method-
ologies of their assessment, this review will provide a brief back-
ground of oxidation, antioxidants, and their relevance to the hu-
man body and to the food industry.
Oxidation
Oxidation can be dened as a chemical reaction involving the
transfer of electrons between molecules or an electron-rich species
to an oxidizing agent (which undergoes a simultaneous reduction).
This transfer of electrons between entities can give rise to radicals.
In nature, molecules are made of protons, neutrons, and elec-
trons. While protons and neutrons comprise the nucleus of atoms,
electrons are left to occupy regions of space outside of the nu-
cleus known as orbitals. In compounds, each molecular orbital
can contain a maximum of two paired electrons with opposite
spins. Orbital shape (s-1, p-3, d-5, f -7) and orientation (x, y, and
z dimensions) will differ from molecule to molecule depending
on its composition. A free radical is an atom or molecule that
can exist independently with one or more unpaired electrons in
its outermost shell. These shells can be atomic or compounded.
Given that molecules are most stable in the ground state, radicals
are highly reactive species that often do not last long in a given
form.
Among the most important oxidation mechanisms in food sys-
tems is that of autoxidation of lipids, which will be discussed here as
a descriptive example of biological oxidative reactions. These free-
radical reactions involve the following mechanisms: (1) initiation
reactions in which the number of free radicals increases; (2) prop-
agation reactions in which the total number of radicals remains
constant (although the number of radical species may change);
and (3) termination reactions in which the number of free radicals
decreases. The following reaction schemes (15) illustrate these
processes:
RH + Initiator R
+ H
(Initiation) (1)
R
+ O
2
ROO
(Propagation) (2)
ROO
+ R
H ROOH + R
(Propagation) (3)
ROO
+ R
ROOR(Termination) (4)
R
+ R
RR(Termination) (5)
The generated radical (1) can interact with molecular oxygen
(
3
O
2
) (2) and undergo many subsequent propagation reactions (3)
with endogenous or exogenous substrates resulting in a variety of
ROS. It is important to note that R
(1) is relatively unreactive;

however, once it propagates with
3
O
2
to form ROO
, it becomes
highly reactive.
The primary products of lipid autoxidation are lipid hydroper-
oxides (LOOHs), which are not sensorially active. LOOHs are
very unstable and degrade to secondary oxidation or scission
products, such as aldehydes, ketones, alcohols, and hydrocarbons
that can impact food quality. Though many of the in vitro radi-
cal generation reactions discussed herein are initiated by chemical
(metal-ion catalyst), thermal (heat), and electromagnetic (light)
means; there are also important enzymatic radical-generation sys-
tems (Hodgson and Fridovich 1976).
Antioxidants
Although the term antioxidant originally referred to molecules
that prevent the consumption of oxygen by human tissues, it has
evolved to refer to the prevention of oxidative systems as a whole.
An antioxidant is a molecule or species that slows or prevents the
oxidation of another molecule, and therefore can be considered
as a reductant. It is important to note, however, that not all re-
ductants are necessarily antioxidants. In explanation, reductant
and oxidant are chemical (redox) terms, while antioxidant
and pro-oxidant hold a specic reference to biological systems
(Prior and Cao 1999).
Antioxidants can be classied either as primary antioxidants
(those which actively inhibit oxidation reactions) or secondary
antioxidants (those which inhibit oxidation indirectly, by mech-
anisms such as oxygen-scavenging, binding pro-oxidants, etc.)
(Shahidi and Wanasundara 1992). Much of primary antioxi-
dant chemistry reactions can be grouped into the categories of
hydrogen-atom transfer (HAT) and single-electron transfer (SET),
both of which are applicable to the discussion of phenolic antiox-
idant action. Phenolics are also considered to operate as secondary
oxidants due to their ability to bind with potentially pro-oxidative
metal ions (Rice-Evans and others 1997).
Hydrogen-Atom Transfer (HAT) Mechanism
The HAT mechanism occurs when an antioxidant compound
quenches free-radical species by donating hydrogen atoms. Reac-
tion scheme (6) below illustrates HAT chemistry: an antioxidant
component (abbreviated as an aromatic component [Ar] and a
hydroxy component [OH]) donates an H-atom to an unstable
free radical and in this process becomes a more stable free-radical
species. This more stable species is then less likely to propagate fur-
ther radical reactions with initiation substrates (Wright and others
2001):
(n)RO

2
+ ArOH (n)ROOH + ArO
(HAT) (6)
Figure 1 is an illustration of the conjugated resonance stabiliza-
tion of phenoxyl radicals. Although the phenoxyl-radical electron
(6) initially exists on the highly electronegative oxygen atom, it is
likely that the electron is delocalized and shared throughout the
aromatic ring. In reaction (6), n represents the stoichiometric factor
for the reactant free radical and the resultant phenolic compound.
Vitamin E has been shown to react with two peroxyl radicals per
molecule (Burton and Ingold 1981). The weaker the hydrogen
atom is held to the reactant hydroxy substituent of the antioxidant
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Comprehensive Reviews in Food Science and Food Safety 149
O O O O
Stable phenoxyl radical
O
H
Unstable peroxyl radical
Phenol-hydroxybenzene
Figure 1A mechanism of phenolic antioxidant efcacy; conjugative resonance stabilization.
compound in reaction (6), the more likely and faster it will par-
ticipate in HAT reactions with free-radical substrates. Therefore,
the bond dissociation enthalpy (BDE) of an antioxidant species is
a parameter when studying the capacity of a phenolic compound
to undergo a HAT in free-radical reactions (Wright and others
2001). That is, the greater the BDE required, the less active a phe-
nolic compound will be in participating in free-radical quenching
reactions via the HAT mechanism.
Antioxidant size, chemistry, and polarity play a role in their ca-
pacity and speed in HAT reactions (Silva and others 2000). HAT
reactions may be hindered by the presence of electron withdraw-
ing groups in the 3- and 5-positions (meta) via deactivation of the
aromatic ring (Streitwieser and Heathcock 1981). HAT reactions
increase with the presence of t-butyl groups at the 2- and 6-
positions (ortho), and methoxy constituents in the 4-position (para)
by inductive donation of electron density to help in the reso-
nance stabilization of the generated phenoxyl radical (Howard and
Ingold 1963). The p-type lone pair orbital of an oxygen-containing
substituent located in the 4-position on a phenolic ring is thought
to overlap with the semi-occupied molecular orbital (SOMO)
of the generated radical species upon hydrogen abstraction
(Burton and Ingold 1986). If, however, the 4-methoxy substituent
is forced out of the plane by neighboring alkyl groups, as in the
case of TMMP (4-methoxy-2,3,5,6-tetramethylphenol), its p-type
lone pair electrons are no longer available to participate in reso-
nance structures with the aromatic ring (Burton and Ingold 1981).
Generally, the presence of large substituents on the aromatic ring
reduces the capability of free radicals to dimerize with the phe-
nolic hydroxy group by steric crowding (Mahoney 1969), thereby
increasing the likelihood of HAT. These parameters give a possi-
ble explanation for the strong antioxidant activity observed for the
food preservatives butylated hydroxytoluene (BHT) and butylated
hydroxyanisole (BHA).
Figure 2 is an example of two sequential hydrogen abstrac-
tions incurred by peroxyl radicals resulting in the conversion of
l-ascorbic acid (vitamin C) to dehydroascorbic acid, and the re-
sultant creation of two hydroperoxides. Although ascorbic acid
lacks phenol chemistry, it does contain two hydroxy functional
groups located on a conjugated furan ring, which renders it
sufciently stable to participate in free-radical redox chemistry
(Brand-Williams and others 1995). Although generated phe-
noxyl radicals (6) could terminate with each other (dimerization)
or with substrate-radical initiators (complexation), the generated
phenoxyl radicals are sufciently stable to readily react with fur-
ther substrate stoichiometrically, until fully oxidized (Blois 1958).
Rice-Evans and others (1996) offer excellent insight into the
structure-activity relationships of phenolic acid and avonoid HAT
reactions. Furthermore, Dangles and others (2000) have assessed
the phenomenon exhibited by DPPH
HAT mechanisms with

3
,4
,7-trihydroxyavylium cation and (+)-catechin (as models for

anthocyanins and proanthocyanidins [PACs], respectively).
Single Electron Transfer (SET) Mechanism
The SET mechanism describes the cases where an antioxidant
transfers a single electron to aid in the reduction of potential target
compounds. The following reaction schemes (79) illustrate a SET
mechanism, in which an antioxidant transfers a single electron to
a ROS. The resultant radical-cationic antioxidant compound is
then deprotonated through interaction with water.
(n)RO
2
+ ArOH (n)RO
2
+ [ArOH]
+
(SET) (7)
[ArOH]
+
+H
2
OArO
+H
3
O
+
(Deprotonation Equilibrium)
(8)
RO
2
+ H
3
O
+
ROOH + H
2
O(Hydroperoxide Formation)
(9)
The nality of a SET reaction (8) is the same as a HAT reaction
(6) in terms of radical scavenging; however, the SET reaction
(7) can be subject to further radical-propagation reactions with
the extended life of [ArOH]
+
(Wright and others 2001). The
resultant antioxidant species from reaction (7) [ArOH]
+
illustrates
that even though the radical electron and formal charge do initially
exist on the oxygen atom, it is likely that the electron is delocalized
and distributed throughout the aromatic ring.
Given that reaction (7) involves the creation of ionic species,
the ionization potential (IP) of an antioxidant compound be-
comes a parameter for predicting the capability of a phenolic
species to scavenge free radicals via SET. The greater the ioniza-
tion energy required, the more reluctant an antioxidant molecule
will be to donate an electron (Wright and others 2001). IP de-
creases with increasing pH, so SET reactions are favored in alkaline
environments.
Mixed HAT and SET Mechanisms
Leopoldini and others (2004) and Wright and others (2001)
assert that although many antioxidant reactions are characterized
as following either HAT or SET chemical processes, these reac-
tion mechanisms can, and do, simultaneously occur. Migliavacca
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O
OH HO
O
HO
HO
RO
2
ROOH
L-Ascorbic acid
(Vitamin C)
O
OH O
O
HO
HO
RO
2
ROOH
O
O O
O
HO
HO
.
O
O O
O
HO
HO
Dehydroascorbic acid
Figure 2HAT conversion of L-ascorbic acid (vitamin C) to dehydroascorbic acid.
and others (1997) assert that -tocopherol undergoes simultane-
ous HAT and SET mechanisms with radical substrates, and that
these processes are interrelated. Zhang and Ji (2006) corroborate
this assertion through studies of the interaction of vitamin E with
DPPH
in polar protic media, in which both HAT and sequen-

tial proton-loss electron transfer (SPLET), also termed proton-
coupled electron transfer (PCET) by Huang and others (2005),
were found to be thermodynamically favorable reactions. Reac-
tion schemes (1012) illustrate a SPLET mechanism (Klein and
Lukes 2006).
ArOH ArO
+ H
+
(10)
ArO
+ ROO
ArO
+ ROO
(11)
ROO
+ H
+
ROOH (12)
SPLET reactions represent one of the main mechanistic sources
of error in falsely denoting SET reactions as HAT, because they
can occur rapidly in certain environs. SET reactions are often
slower than HAT ones; therefore, if the reaction kinetics between
an antioxidant substrate and free radical are expeditious in a given
system, HAT is often assumed to be the predominant mechanism.
Figure 3 is an example of a SET mechanism between -tocopherol
and 4-methoxybenzoyloxyl radical, as suggested by Evans and oth-
ers (1992). Although -tocopherol can undergo a SETwith radical
substrates, its radical-scavenging behavior is still thought to be pre-
dominantly HAT (Burton and Ingold 1981; Nakanishi and others
2002; Zhang and Ji 2006).
The most prevalent mechanismin any systemwill depend on an-
tioxidant structure, properties, and medium of interaction (Huang
and others 2005; Prior and others 2005). If bulky constituents
are located adjacent to phenolic hydroxy groups, steric issues may
hinder HAT or SET reactions. Similarly, if antioxidant reactions
are carried out in hydrogen bond-accepting environments, HAT
efciency will be greatly reduced (Evans and others 1992; Barclay
and others 1999).
Secondary Antioxidative Action by Metal-Ion Chela-
tion
The presence of ionic metals such as copper and iron in a
system can accelerate the rate of oxidation in that system. This
action can occur either by the promotion of the decomposition
of hydroperoxides, or through the production of hydroxyl radicals
by the Fenton reaction (Koppenol 1993; Fernandez and others
2002). The Fenton reaction is shown in reaction schemes (13) and
(14).
Fe
2+
+ H
2
O
2
Fe
3+
+

OH +

OH (13)
Fe
3+
+H
2
O
2
Fe
2+
+

OOH + H (14)
Phenolic compounds have been shown to inhibit the pro-
oxidative action of metals by a chelation process, in which the
phenolics bind with the metal ions to form a complex incapable
of promoting oxidation (Mira and others 2002). By this chelation
process, phenolics operate as secondary or preventive antiox-
idants; effectively inhibiting oxidation without directly interact-
ing with oxidative species. In phenolic compounds, the 5-OH
and/or 3-OH moiety with a 4-oxo group in the A/C ring struc-
ture or a large number of hydroxyl groups are important for the
binding/chelation of metal ions (Khokhar and Apenton 2003). In
avonoids, the presence of 3
-4
and/or 7
-8
o-dihydroxyphenyl
groups on the B- and A-rings also inuences the chelation of
metal ions, as does a double bond between the C2 and C3 of
avones (Mira and others 2002). Hydroxyl groups in conjugation
with methyl groups or a carbohydrate moiety are not involved in
the complexation of metal ions (Andjelkovi c and others 2006).
The propensity of transition metal ions to be chelated follows the
order: Cu
2+
> Fe
2+
> Fe
3+
, which is to be expected according
to their relative stabilization energies (Mira and others 2002).
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O
CH
3
HO
H
3
C
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
4-CH
3
OC
6
H
4
CO
2
4-CH
3
OC
6
H
4
CO
2
O
CH
3
HO
H
3
C
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
.
H
2
O
H
3
O
+
O
CH
3
O
H
3
C
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
Figure 3A SET mechanism between -tocopherol (vitamin E) and 4-methoxybenzoyloxyl radical; adapted from Evans and others (1992). J Am Chem
Soc 114:458993.
The ability of phenolics to enact chelation in vivo is as of yet
uncertain, but the use of phenolics has been suggested as treat-
ment for metal-overload diseases such as hemochromatosis (iron
overload) and Wilsons disease (copper overload) (Afanasev and
others 1995). The extent to which the chelation process explains
the observed antioxidative action of phenolics (be it in synergy
with HAT and SET mechanisms or in the place of ) is an area of
ongoing research.
Reactive Oxygen Species (ROS) and the Human Body
Oxygen is required for life; yet, its reaction products with bio-
logical substrates can generate compounds (such as reactive oxy-
gen species or ROS) that can damage health if left unchecked.
It has been stated that oxygen is the greatest paradox in biologi-
cal science. ROS are of interest in biology and biological chem-
istry due to strong evidence relating them to the pathogenesis of
many degenerative diseases and aging in humans (Halliwell and
others 1992; Barber and Harris 1994; Halliwell 1996; Hiramatsu
and others 1997). ROS have even been implicated in the dis-
ruption of cellular signaling pathways, and thus can affect gene
expression (Palmer and Paulson 1997). Perhaps the best-known
forms of ROS include certain oxygen radicals like the superoxide
radical anion (O
2
), hydroxyl radical (HO
), as well as alkoxyl
and peroxyl radicals (RO
& RO
2
, respectively). In addition to
these, there are non-radical oxidizing agents such as hydrogen per-
oxide (H
2
O
2
), hypochlorous acid (HOCl), singlet oxygen (
1
O
2
),
and ozone (O
3
) (Halliwell and others 1995). Reactive nitrogen
species (RNS) such as nitroxyl radicals (NO
, NO
2
) and per-
oxynitrite (ONOO
) also exist and can have adverse effects on

human health and disease (Halliwell and others 1992; Halliwell
1996).
Though ROS are often viewed as exogenous and deleterious to
mankind, these compounds are naturally present within humans
and held in check by multiple defense systems found within the
body. These defenses including the following: endogenous antiox-
idant enzymes (such as catalase, glutathione reductase, glutathione
peroxidase, superoxide dismutase), endogenous factors (such as
glutathione, co-enzyme Q), metal-ion sequestration systems, and
endogenously-generated primary and secondary antioxidants (in-
cluding vitamin E, vitamin C, and carotenoids) (Machlin and
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Bendich 1987; Sies 1993; Halliwell 1996). In fact, all vascular
cell types produce ROS enzymatically via membrane-associated
NAD(P)H oxidase. ROS are involved in many stages of vascular
function, including cell contraction/dilation, cell growth, pro-
grammed cell death, and inammation (Touyz 2005; Park and
others 2011), various stages of cellular respiration, including mo-
bilization of the electron transport system, oxidative phospho-
rylation, and, consequently, in various redox signaling pathways
(Adam-Vizi 2005).
ROS can become dangerous when present in excess in an an-
imal body. They result in the overproduction of free radicals that
can damage multiple components of cells including deoxyribonu-
cleic acid (DNA), ribonucleic acid (RNA), lipids, proteins, car-
bohydrates, and enzymes (Machlin and Bendich 1987; Aruoma
1994). Furthermore, cellular damage incurred by free radicals can
cause further radical production, as well as an increased risk of
inammation, cardiovascular disease, cancer, diabetes, Alzheimers
disease, and age-related functional decline; even though it is per-
haps not the primary cause of these diseases (Temple 2000). Of
recent interest is the role of redox modulation in insulin signaling
as it pertains to vascular endothelial function and possible links
to diabetes (Christon and others 2005; Stevens 2005; Estivalet
and others 2011). Even though some may think that free-radical
research in biological systems is fairly recent, signicant break-
throughs in free-radical theory have occurred since the work of
D. Harman in the mid 1950s (Niki 1997).
As a recognized contributing factor to the overproduction of
ROS, oxidative stress is thought to be linked to many degen-
erative diseases. For example, oxidative processes are involved in
the formation of atherosclerotic plaques within arterial walls and,
therefore, can lead to an increased incidence of cardiovascular dis-
ease in humans (Steinberg 1991). Oxidized nucleic acids in DNA
can be mutagenic and lead to carcinogenesis (Nakabeppu and oth-
ers 2006). Many inammatory processes can lead to an increased
incidence of oxidative stress; this often results from an overpro-
duction of ROS, which the bodys natural defense systems cannot
overcome (Sies 1993). Greater nutritional intake of pro-oxidant
food sources or prolonged nutrient deciency can also result in
oxidative overload within the body (Sies and others 2005). Al-
though chromatographic and spectrophotometric assays are able
to measure the extent of nucleic acid oxidation (Collins 2005),
studies in oxidative/antioxidative research have tended toward in
vitro antioxidant and radical-scavenging capacity assays, lipid oxida-
tion model systems in foods, and in vivo assays of human biological
uids.
The Role of Antioxidants in Humans
Antioxidants are important to humans because of the multiple
benecial interactions they may be able to have within our bodies.
Often this protection is dependent upon case, type, and location.
For example, an antioxidant generated to help protect against lipid
peroxidation in human tissues may or may not be able to prevent
oxidative stress caused to DNA, proteins, or other compounds.
In some cases, they may in fact cause more damage than good
(Halliwell 1996). Antioxidant effectiveness in vitro may not corre-
late with effectiveness in vivo. The human digestive tract can de-
grade or alter the chemical form of antioxidant compounds as they
pass through the stomach, preventing them from being absorbed
in the lower intestines, and, therefore, rendering them ineffec-
tive at preventing oxidation in the body (Scalbert and Williamson
2000). As a screening process, it is reasonable to assume that if an
antioxidant has a poor capability of scavenging free radicals or pre-
venting oxidative reactions in vitro, then it likely will also have poor
efcacy in vivo. Given the cost of animal model systems and human
intervention studies, cell culture models to directly assess antiox-
idant effectiveness in vivo have been attempted and hold promise
(Liu and Finley 2005; Wolfe and Liu 2007; Ara ujo and others
2011).
Assessment of antioxidant proles in human plasma (Polidori
and others 2001) and other biological uids is commonplace as an
index of oxidative stress. Past research suggests that exogenously
supplemented antioxidants may provide relief from multiple ox-
idative reactions within humans and act as anti-inammatory, an-
ticarcinogenic, anticancer, and antiradical agents (Rice-Evans and
Diplock 1993; Diplock 1994, 1996). However, the role of such
supplementation in eliciting a favorable response in the body is
still under debate (Halliwell and others 2005). Some in vivo studies
have suggested that supplementing the human diet with antioxi-
dants may not be warranted given the possibility of pro-oxidative
reactions. A pro-oxidant effect of supplemented vitamins C and
E was observed in in vivo dietary trials (Kontush and others 1996;
Paolini and others 1999; Abudu and others 2004). The possibility
for pro-oxidative effect by phenolics has been demonstrated in vitro,
but this effect been shown to lessen dramatically with decreases in
copper ions present (Cao and others 1997; Fukumoto and Mazza
2000; Ru an-Henares and others 2006). To date, neither the pro-
oxidative or antioxidative effect of phenol consumption has been
adequately substantiated in vivo (Halliwell 2008).
The Role of Antioxidants in Foods
Another important application of antioxidants is their inclusion
in food products as preservatives to extend shelf-life and to main-
tain quality. This has led to the attempted correlation of in vitro
antioxidant capacity data with projected capabilities of antioxidants
to perform in food systems. Even though antioxidant capacity as-
says can gauge the relative capabilities of antioxidant components,
antioxidant activity in food systems depends on many factors in-
cluding the antioxidants physical location in the food, interac-
tion(s) with other food constituents, and the overall conditions of
the food environment (pH, ionic strength, hydrophilic/lipophilic
balance, and so on) (Decker and others 2005). An antioxidants
efcacy at scavenging free radicals in the aqueous phase depends
on its solubility in both the aqueous and lipid phases of a food
or beverage. To this end, antioxidant model systems in vitro need
to take into account the complex nature of foods if they are to
achieve high relevance and accuracy.
Phenolic and Polyphenolic Antioxidants
One of the most well-known groups of antioxidant compounds
in the scientic literature is the phenolics. Any compound that
contains a hydroxy-substituted aromatic ring is a phenolic com-
pound. Phenolics and polyphenolics (polymeric phenolics) can
provide relief from certain physical ailments and degenerative dis-
eases in humans, including the reduction of cardiovascular disease
and certain cancers (Scalbert and others 2002, 2005; Arts and
Hollman 2005). Therefore, it is not surprising that the extrac-
tion and analysis of phenolics from plants and other food sources
have been extensively studied (Naczk and Shahidi 2004; Dai and
Mumper 2010).
Occurrence of Phenolics in the Plant Kingdom
In plants, phenolic compounds are metabolized from the amino
acid l-phenylalanine and, in some cases, l-tyrosine (Shahidi 2000,
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Phenylalanine
Phenylalanine
lyase
Cinammic acid
Phenyl propanoid
C
6
n
n
n
Lignan
Lignin
Suberin, Cutin
+ Fatty acids
+ Fatty alcohols
+ Hydroxyf atty acids
+ Dicarboxylic acids
3 Malonyl
CoA
3 Malonyl
CoA
Chalcone
synthase
Stilbene
synthase
Flavanoid
- Flavone
- Flavonol
- Flavonone
- Flavononol
- Flavanol
- Anthocyanidin
n
Tannin
C
3
C
6
C
6 C
3
C
6 C
3
C
6
C
6
C
6 C
3
C
3
C
6
C
6
C
2
C
6
C
6
C
3
C
6
Stilbene
Chalcone
C
6
C
3
C
6
NH
2
O
OH
O
OH
Figure 4Synthesis of phenylpropanoids from phenylalanine, the origin of phenolics.
2002). Figure 4 is an illustration of the pathways of production
of phenylpropanoids including stilbenes, lignans, suberins, cutins,
avonoids, and tannins. Figures 5 and 6 are illustrations of the
enzymatic reactions undergone in the synthesis of phenolic acids
(trans-cinnamic and benzoic acids) and avonoids from phenylala-
nine. Phenolic compounds exist as a monomeric aglycone or in
various bound forms. They are also the building blocks of large
polymeric compounds such as tannins (Shahidi and Naczk 2004;
Cheynier 2005). Figure 7 is a summary of the current classi-
cation of dietary phenolics, including examples. Many phenolic
compounds and mixtures thereof are prevalent in a wide vari-
ety of fruits, vegetables, grains, and other plant products (Madsen
and Bertelsen 1995; Pietta and others 1998; Paganga and others
1999; Adom and Liu 2002; Chu and others 2002; Sun and others
2002; Shan and others 2005; Stratil and others 2006). Research has
shown that diets rich in fruits, vegetables, whole grains, and other
sources of phenolics can lead to an increased quantity of antioxi-
dants in the human body (Cao and others 1998). Also, phenolics
may work together synergistically to improve ones total health
status (Liu 2004).
Phenolic acids
As depicted in Figure 5, phenolic acids of the benzoic and
trans-cinnamic acid families are synthesized from l-phenylalanine
(and l-tyrosine) in plants. This process is commonly referred to
as phenylpropanoid metabolism. Hydroxycinnamic acids are most
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c

HOOC
HOOC
HOOC
HOOC
HOOC
HOOC HOH
2
C
OH
OCH
3
OH
OCH
3
OH
OH
OH
OH OH
HOOC
HOH
2
C
HOH
2
C
-C
2
Benzoic acid
p-Hydroxybenzoic acid
Phenol
-CO
2
Coniferyl alcohol
-C
2
P
450
Monooxygenase
trans-Cinnamic acid
p-Coumaric acid
Caffeic acid
Ferulic acid
OH
OCH
3
OH
OCH
3
OCH
3
OH
OCH
3
Sinapyl alcohol
Coumaryl alcohol
Sinapic acid
Hydroxylase
O-Methyl transferase
Phenylalanine
Tyrosine
Phenylalanine
lyase
Tyrosine
lyase
-NH
3
-NH
3
Figure 5Formation of phenylpropanoids from phenylalanine and tyrosine; adapted from Shahidi (2000). Nahrung 44:15863.
widely distributed in plant tissues. They are often found in the
form of hydroxyacid esters with quinic, shikimic, or tartaric acid
residues (Herrmann and Nagel 1989).
Phenolic acids have been associated with many aspects of food
quality including color, avor properties, and nutrition (Maga and
Katz 1978). Of the many methods available for their selective sep-
aration, RP-HPLC methods with spectrophotometric detection
are the overwhelming majority, with gas chromatography (GC)
along with derivatization steps being employed to a lesser extent
(Robbins 2003). Figures 8 and 9 are UV-spectral scans of phe-
nolic acids of the benzoic acid and trans-cinnamic acid families.
While benzoic acids typically yield their primary UV-maximum
near 260 nm (especially p-hydroxybenzoic, vanillic, and proto-
catechuic acids), most trans-cinnamic acids absorb UV-radiation
nearer to 320 nm. The inherent differences in UV-spectra exhib-
ited by the two phenolic acid families provide for their selective
chromatographic identication.
Lignans
Lignans consist of two phenylpropane units, which are joined by
oxidative dimerization (Ignat and others 2011). Lignans typically
exist in nature in their free form, with the glycoside derivatives
occurring only in small quantities. Once consumed by humans,
lignans are metabolized to enterodiol and enterolactone by the
intestinal microora (El Gharras 2009). It is speculated that there
are additional precursors to these products that have not yet been
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p-Coumaryl CoA + 3 Malonyl CoA
HO
OH
HO
OH
OH
O
OH
OH
HO
OH
O
O
OH
Chalcone
synthase
Stilbene
synthase
Stilbene
(Resveratrol)
Chalcone
(Tetrahydrochalcone)
Chalcone
isomerase
Flavonoid
(Flavonone)
Flavone
Flavonol
Flavononol
Flavanol
Isoflavone
Anthocyanidin
Figure 6Production of avonoids and stilbenes from phenylpropanoid (p-coumaryl CoA) and malonyl CoA; adapted from Shahidi (2000). Nahrung
44:15863.
identied (Heinonen and others 2001). Flax seed is considered
to be by far the greatest dietary source of lignans, but lignans are
also found in appreciable quantities in sesame seed and, to a lesser
degree, in a variety of grains, seeds, fruits, and vegetables (Milder
and others 2005). Table 1 shows a portion of the prominent di-
etary sources of lignans and their relative concentration levels. In
addition to serving as antioxidants, lignans have been shown to be
capable of inhibiting the cancer-promoting effects of estrogen on
breast tissue by binding to estrogen receptors (Pianjing and oth-
ers 2011). The potential effect of lignans upon a variety of cancer
types has become a subject of ongoing research (Saleem and others
2005).
Stilbenes
Stilbenes are a family of hydrocarbons consisting of two
phenyl groups joined via an ethene double bond (Leopoldini
and others 2011). This double bond may occur naturally in ei-
ther a cis or a trans conguration, and the stilbenes are gen-
erally present in glycosylated forms (Delmas and others 2006).
The primary representatives of this family of compounds are
pterostilbene, piceatannol, and resveratrol (Leopoldini and oth-
ers 2011). Resveratrol has received particular attention for its po-
tential health-promoting effects, including reports of possessing
cardio-protective, neuro-protective, anticancer, antidiabetic, and
antiaging capabilities (Pandey and Rizvi 2011). Resveratrol occurs
in more than 70 plant species, including berries and peanuts, but
is most commonly associated with red wine, in which resveratrol
concentrations have been measured to be 0.3 to 7 mg aglycones/L
and 15 mg glycosides/L (El Gharras 2009). The prevalence of
resveratrol in red wine is often considered to play a major role in
the so-called French Paradox, the observation that the people
of France experience relatively low occurrences of cardiovascu-
lar disease despite high consumption of fats, high occurrence of
cigarette smoking, and little exercise (Orallo 2006). Studies have
suggested that this observed protection against heart disease could
be a result of a combination of antioxidant activity, modulation of
lipoprotein metabolism, and vasodilatory and platelet antiaggrega-
tory properties.
Flavonoids
Flavonoids are the most common and widely distributed group
of phenolic compounds in plants. As seen in Figure 6, their ba-
sic makeup is a diphenylpropane core structure that consists of
two outer aromatic rings with a three-carbon bridge, which can
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Phenolic
acids
p-Hydroxybenzoic
Protocatechuic
Gallic
Vanillic
Syringic
(+)-Catechin, (-)-Epicatechin
Epigallocatechin (EGC)
Epigallocatechingallate (EGCG)
Pelargonidin
Cyanidin
Delphinidin
CLASIFICATION OF PHENOLICS
Flavonols
Hydroxy-
trans-cinammic
acids
Flavones Flavanols Flavanones Flavanonols Anthocyanidins
Stilbenes Flavonoids Isoflavonoids Lignans
Phenolic
polymers
p-Coumaric
Caffeic
Ferulic
Kaempferol
Quercetin
Myricetin
Chrysin
Apigenin
Luteolin
Naringenin
Eriodictyol
Hesperitin
Taxifolin
Isoflavones Coumestans
Daidzein
Genistein
Glycitein
Coumestrol
Coumarins
Proanthocyanidins
(Condensed tannins)
Procyanidin
Prodelphinidin
Propelargonidin
Gallotannins
Ellagitannins
Hydroxy-
benzoic
acids
Hydrolyzable
Tannins
Figure 7 Classication of dietary phenolics; adapted from Liu (2004). J Nutr 134:3479S85S.
Figure 8UV-spectra of phenolic acids in the benzoic acid family.
be closed (as in avones, avanols, and anthocyanidins) or open
(chalcones). Flavonoids most commonly occur as glycosides in
plants, with some classes consisting of up to 380 variations in
their chemical structure (Bravo 1998). In the case of avonoids,
their altered substitution and saturation patterns can result in
the production of avones, avonols, avanones, avanonols, a-
vanols, and anthocyanidins. The chemical backbones of various
avonoids/isoavonoids commonly found in plants are depicted
in Figure 10.
Phenolic polymers
Phenolic polymers, or tannins, were named because of their ca-
pacity to bind to proteins in the transformation of animal hides to
leather. Tannins can be subdivided into two classes based on their
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Wavelength (nm)
220 240 260 280 300 320 340 360 380
A
b
s
o
r
b
a
n
c
e
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Caffeic acid
p-Coumaric acid
Ferulic acid
Isoferulic acid
Sinapic acid
Figure 9UV-spectra of phenolic acids in the trans-cinnamic acid family.
Table 1Lignan content of solid foods.
a
Total lignan
Product concentration (g/100 g)
Oilseeds and nuts
Flaxseed 301,129
Sesame seed 39,348
Sunower seed 891
Cashew 629
Peanut 94
Poppy seed 10
Grain products
Multi-grain bread 6,744
Muesli 764
Rye bread (dark) 320
Vegetables
Curly kale 2,321
Broccoli 1,325
Garlic 536
French bean 273
Fruits
Apricot 450
Strawberry 334
Peach 293
a
Milder and others (2005).
inherent chemical make-up: hydrolyzable and condensed tannins.
Hydrolyzable tannins can be further segregated into gallotannins
and ellagitannins. Gallotannins consist of gallic acid subunits ester-
ied to glucose. Ellagitannins are simply polymers of ellagic acid
and gallic acid. Figures 11 and 12 are examples of gallo- and ellag-
itannins, respectively. Hydrolyzable tannins are so named because
they easily hydrolyze in weak acid or alkali to their individual
monomeric units (Bravo 1998).
Condensed tannins, often called proanthocyanidins or PACs,
release anthocyanidin monomers when heated in the presence of
acid (Cheynier and others 1999). In foods, PACs are usually classi-
ed as procyanidins or prodelphinidins according to the chemistry
of their avan-3-ol subunits. Procyanidins are comprised of (-)-
epicatechin monomers, whereas prodelphinidins are comprised
of epigallocatechin subunits. PACs are subdivided into A- and
B-types according to their interavonoid linkages. B-type PACs
are most common and have a C4D8 or C4D6 interavonoid
linkage; whereas, A-type PACs have an additional ether linkage
from C2D7 (Ferreira and Li 2000) as seen in Figure 13. PACs
can range from dimeric to oligomeric species with many subunits.
In fact, decamers with a molecular mass greater than 30 kDa have
been reported in cocoa and sorghum (Gu and others 2002).
Extraction of Phenolics from Plants
In order for proper chromatographic analysis, phenolics must
rst be extracted from their respective plant or food matrices. Ex-
traction efciency is inuenced by analyte particle size, extraction
solvent(s), pH, time, temperature, and agitation, as well as the pres-
ence of potential interfering substances such as sugars (Naczk and
Shahidi 2004). Solubility of targeted phenolic compounds in the
selected extraction solvent is largely dependent on their relative
polarities. If one is attempting to extract a wide variety of phenolic
and polyphenolic constituents from a single plant or food source,
the conditions for extraction should take into account the com-
plex nature of the selected compounds. Often this is accomplished
through the use of multiple extraction solvents and sequential
liquid partitioning followed by the chromatographic analysis of
the components in each fraction. Nevertheless, there is no assess-
ment that exhaustive extraction of the analytes-of-interest has
been achieved. The most common methods of phenolic extrac-
tion employed involve pH-buffered aqueous/organic mixtures of
methanol, ethanol, acetone, and ethyl acetate (Naczk and Shahidi
2004).
Phenolics in Food
Along with providing possible health benets, ingredients rich
in phenolics are employed as antioxidants in a variety of food sys-
tems (Andersen and others 2005; Brewer 2011). More recently,
polyphenolics have been added to functional foods and nutraceu-
ticals to bestow targeted health benets to consumers. However,
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O
O
O
O
OH
O
O
O
O
OH
O
OH
O
+
O
O
O O
O
Flavonone
Flavononol
Flavone
Flavonol
n i d i n a y c o h t n A ) n i h c e t a C ( l o n a v a l F
Isoflavone
Coumestan
Figure 10Chemical backbone of selected avonoids and isoavonoids found in plants.
the inclusion of phenol-rich components in nutritive foods and
beverages needs to be intelligently employed to ensure that the
phenolics do not adversely affect sensory attributes of the food
(Lesschaeve and Noble 2005) and that they are not signicantly
biodegraded before reaching their point of absorption in the hu-
man body. This is often accomplished by microencapsulation and
other stabilization techniques.
Phenolic Bioavailability/Bioactivity after Ingestion
As discussed, the human body contains a very complex sys-
tem of chemical and enzymatic defense mechanisms. Once an-
tioxidants enter the body, they do not necessarily pass through
unaltered or reach their intended absorption site in the gastroin-
testinal (GI) tract; hence, bioavailability and bioactivity must be
considered. The bioavailability of phenolics and polyphenolics
has been studied extensively over the past 2 decades, whether by
examining the kinetic patterns of polyphenol absorption in the
bodily uids of healthy volunteers (Manach and others 2005) or
by epidemiological intervention studies in hospitals (Williamson
and Manach 2005). These studies have, however, yielded
conicting results. Though much knowledge has been acquired
involving the absorption of phenolic acids and avonoids in the GI
tract (Scalbert and Williamson 2000), more targeted investigations
are warranted.
Dietary origins of polyphenolics have been established including
PACs in dark chocolate and ellagitannins in pomegranate, but
methods for screening daily intake of these compounds have only
recently been developed (Prior and Gu 2005). PACs have gained
considerable attention as of late and are quickly becoming the
most popular ingredient for natural in vivo antioxidant therapy
(Dixon and others 2005). Much of this attention is due to their
capability of binding to proteins and surviving passage through the
human GI tract. Tannins can also survive certain thermal processes
and greatly retard lipid oxidation in foods (Pegg and Amarowicz
2004; Amarowicz 2007). Whether or not the large-scale addition
of phenolics to the human diet in the form of supplements or
formulated foods is safe and/or of potential benet is still a matter
of debate (Pokorn y 2007; Martin and Appel 2010).
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OH
HO
HO
O
O
OH
OH
O O
O
O
O
O
O
O
HO
HO
O O
HO
OH
OH
O
HO
OH
O
O
OH
OH
OH
O
OH
OH
O
O
OH
OH
O
O
O
HO
OH
OH
OH
OH
OH
Gallic acid monomer
Figure 11A hydrolyzable gallotannin (tannic acid).
HO
OH
OH
OH
HO
HO
O
O
H
O
O
O
OH
OH
O
O
HO
O
O
O
O
HO
HO OH
HO
OH
O
OH
OH
O
OH
Ellagic acid monomer
Figure 12A hydrolyzable ellagitannin (punicalagin).
Relationship between Phenolic Structure and
Antioxidant Activity
Another key point of study regarding the antioxidant activity
of phenolic compounds is that of identifying and dening the
relationships between the structures of phenolic compounds and
their relative ability to performas antioxidants. Within real systems,
this relationship will be highly dependent upon the conditions of
the system such as substrates, temperature, light, oxygen pressure,
relative physical characteristics, polarity, and metals (Chen and
Ho 1997; Chaiyasit and others 2007; Shahidi and Zhong 2011).
Still, studies have successfully determined generalized relationships
between phenolic structures and their relative antioxidant activities
when assessed independent of real systems.
In a recent study, Hoelz and others (2010) compared the struc-
tural characteristics of 15 common phenolic antioxidants and re-
lated those characteristics to their determined antioxidant activity;
the comparison of which was used for the creation of a pre-
dictive model. In this case, antioxidant potential was measured
according to their relative capacity to inhibit peroxide formation
in accelerated conditions (Zhiyong and others 2003). They found
the structural variables OH bond homolytic dissociation enthalpy
(BDEOH) and ionization potential (IP) to be sufcient to form
a successful model, while lipophilicity and relative lipophilicity re-
quired no consideration. The best determined equation [pIC
50
=
6.682 0.023(BDEOH) 0.0036(IP)] attained an adjusted R
2
value of 0.866 signifying a strong predictive power. The result
shows that increases in either OH bond homolytic dissociation
enthalpy or ionization potential negatively affect a phenolic com-
pounds antioxidant activity, both of which are in agreement with
our discussion of HAT and SET mechanisms above. The authors
assert their results suggest that the best phenolic antioxidants are
compounds, which contain electron donor groups directly at-
tached to an aromatic ring. A summary of their results is shown in
Table 2.
Another study (Kim and Lee 2004) examined the antioxi-
dant potential of a representative variety of phenolic antioxidants
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c

O
OH
HO
OH
OH
HO O
OH
OH
OH
OH
OH
4
8
HO
OH
O
O
OH
OH O
OH
4
8
2
7
OH
OH
HO
OH
B-type A-type
Figure 13Condensed tannins, B-type (48) and A-type (48 27) procyanidin dimers (B
2
and A
2
).
Table 2Comparison of physical attributes of certain phenolic compounds with their observed and predicted antioxidant activities.
a
OH bond homolytic
Compound dissociation enthalpy Ionization potential pIC
50Calc
b
pIC
50Exp
c
% Error
o-Coumaric acid 84.4 188.8 4.10 4.14 0.98
p-Coumaric acid 84.9 184.9 4.10 4.10 0.00
Ferulic acid 84.5 177.6 4.13 4.15 0.48
Caffeic acid 74.9 181.6 4.34 4.21 3.09
Catechol 76.4 184.5 4.29 4.29 0.00
Pyrogallol 77.7 183.2 4.27 4.31 0.94
Phloroglucinol 87.7 188.2 4.02 3.98 1.00
Resorcinol 86.1 186.9 4.06 4.02 1.00
Hydroquinone 80.6 178.4 4.22 4.28 1.42
p-Aminophenol 76.8 163.2 4.36 4.42 1.38
Protocatechuic acid 79.6 228.2 4.06 4.09 0.74
Gallic acid 79.8 228.6 4.06 4.08 0.49
Salicylic acid 93.0 196.6 3.88 3.94 1.55
m-Hydroxybenzoic acid 88.9 199.0 3.96 3.92 1.02
p-Hydroxybenzoic acid 89.2 200.3 3.95 3.90 1.28
a
Hoelz and others (2010);
b
pIC
50
= 6.682 0.023(BDE-OH)0.0036(IP);
c
Zhiyong and others (2003), measured by lipid peroxide inhibition assay.
using the Vitamin C Equivalent Antioxidant Capacity (VCEAC)
assay (a method conceptually similar to the TEAC assay), and
compared the results to structural characteristics. The study was
able to determine key emerging patterns. Specically, antioxi-
dant activity generally increased with increasing number of phe-
nolic rings (meaning polyphenolics are generally more effective
than monophenolics), cinnamic acid derivatives generally showed
greater antioxidant activity than benzoic acid derivatives, the sub-
stitution of sugars into avonoids resulted in impaired antioxidant
activity (speculated to be due to steric hindrance), and antioxidant
activity of avonoids increased in a linear fashion with an increase
in free OH groups around the avonoid framework. The obser-
vation of positive correlation with free OH groups was also made
by Lien and others (1999), who measured antioxidant potential
according to TEAC.
Additional studies investigating such ndings, implementing
different analytical techniques, and incorporating different assay
conditions may still be required for more comprehensive con-
clusions regarding the structure-activity relationships of phenolic
compounds.
Correlation between Phenolics Content and Antioxi-
dant Activity
Total phenolics content can have a strong association with the
antioxidant activity observed within a system, but this will cer-
tainly not always be the case. As addressed previously, the mode
of action of antioxidants is complex and may be highly dependent
upon a wide range of variables within a system. Many studies have
been conducted with specic food sources evaluating the level of
correlation between total phenolics content and observed antiox-
idant activity; the results of which have shown great variation (for
example, Di Majo and others 2008; Hu and others 2010; Yose
and others 2010; Sulaiman and others 2011).
In sources in which a strong correlation is observed, it is typically
concluded that phenolics are largely responsible for the antioxidant
activities seen within the samples. In sources in which strong cor-
relations are not observed, it is commonly concluded that there are
signicant amounts of antioxidants other than the measured phe-
nolics present in the system, or that the specic phenolic species
present in the system cannot be quantied properly through the
total phenol assay. Another key point of consideration here may
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Table 3Examples of antioxidant assessment assays.
Descriptive reference
Assay Radical/ion measured (listed in order of relevance for each assay)
HAT assays
Azo-initiated chemiluminescence (CL) RO
2
Alho and Leinonen (1999)

Photochemiluminescence (PCL) O
2
Popov and Lewin (1999a); Pegg and others (2007)

Total antioxidant reactivity (TAR) RO
2
Campos and others (1996); Lissi and others (1995)

ORAC
FL
RO
2
Huang and others (2002b); Prior and others (2003); Wu and

other (2004b)
TRAP RO
2
Wayner and others (1985); Wayner (1987); Lussignoli and

others (1999)
Crocin or -carotene bleaching assays RO
2
UV-Vis Miller (1971); Kampa and others (2002); Tanizawa and others
(1983); Tubaro and others (1998)
Microtiter plate Mikami and others (2009)
Total oxyradical-scavenging capacity (TOSC) RO
2
and HO
Regoli and Winston (1999); Winston and others (1998)

Liposome model systems RO
2
and HO
Roberts and Gordon (2003)

Low-density lipoprotein (LDL) oxidation models RO
2
and HO
Esterbauer and others (1992); Frankel and others (1995)

SET assays
Cupric reducing antioxidant capacity (CUPRAC) Cu
2+
Cu
+
[complexed]
UV-Vis Apak and others (2004); Moffet and others (1985)
Microtiter plate Ribeiro and others (2011)
Ferric reducing antioxidant power (FRAP) Fe
3+
TPTZ Fe
2+
TPTZ
UV-Vis Benzie and Strain (1996); Pulido and others (2000); Amarowicz
and others (2004)
Microtiter plate Firuzi and others (2005)
Mixed-mode assays
TEAC ABTS
+
UV-Vis Miller and others (1993); Re and others (1999)
Microtiter plate Kambayashi and others (2009)
DPPH
DPPH
UV-Vis Hatano and others (1988), S anchez-Moreno and others (1998)

Microtiter plate Fukumoto and Mazza (2000)
Chelation assays
Ferrozine Divalent metal cations Dinis and others (1994)
Tetramethylmurexide (TMM) Divalent metal cations Shimada and others (1992)
Quantication assay
Total phenolics content (TPC) Mo
6+
[yellow] Mo
5+
[blue])
UV-Vis Singleton and Rossi (1965); Folin and Ciocalteu (1927);
Singleton and others (1999)
Microtiter plate Zhang and others (2006)
be the possible synergistic and antagonistic effects that can oc-
cur within the system based on additional components, as well as
interactions between the phenolic compounds and their physical
environment of the food matrix.
Although the phenolics content assay may in many cases provide
indication of the potential antioxidant capacity of an extract, it
should not be confused with an accurate assessment thereof. This
must be evaluated by more direct and specic means.
Quantication of Antioxidant Content and Capacity
Most phenolic antioxidant assessment assays can be grouped
according to the chemistry of the reactions involved; meaning it
may specically pertain to the mechanisms of either HAT or SET,
or may be a mixed-mode method pertaining to both (Schaich
2006). Also of importance is the TPC, which is commonly used
to directly quantify inherent phenol content, as well as methods
that evaluate chelation activity. Examples of antioxidant assays are
shown according to their categorization in Table 3, and Figure 14
demonstrates the changes in the respective rates of citation for
some of the most commonly used assays. A more comprehensive
list of in vitro antioxidant assays is beyond the scope of this work.
One should not expect the results of antioxidant contents or
capacity assessed by a HAT assay to necessarily be compatible
(either quantitatively or qualitatively) to that obtained by a SET
assay. In fact, it has been reported that they do not directly com-
pare (Bhagwat and others 2007); Table 4 serves to illustrate this
phenomenon. One possible explanation of these discrepancies is
that different mechanisms of measurement may present different
determinations of antioxidant activity according to the particular
antioxidant composition of the sample. For example, the antioxi-
dant capacities of foods rich in ascorbic acid have been shown to
be underrepresented by ORAC (Ou and others 2001). Another
explanation is that different foods, when analyzed, may enact inter-
ferences, which have different magnitudes of effect upon different
assays. Prior and others (2005) suggest that assessment of SET
reactions may be more sensitive to potential interferences than
those of HAT reactions. SET reactions often take long periods
of time to reach completion, and interfering substances (such as
trace components, metal contaminants, and uric acid) can exert a
great effect on their measurement. Nevertheless, each assaybe
it HAT, SET, or mixedinvolves the correlation of an antioxi-
dants capability to perform in relation to a standard antioxidant
compound.
Also of relevance to this review are assays, which monitor the
effect upon oxidation rates within a system (peroxide value, con-
jugated dienes, and so on), and these will be discussed.
HAT Assays
Oxygen radical absorbance capacity (ORAC
FL
) assay
The ORAC assay was developed by Dr. Alexander N. Glazer
in the early 1990s for the determination of ROS in biologi-
cal systems. The original assay was based on the uorescence of
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Vol. 11, 2012
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Figure 14Frequency of citation of popular antioxidant assays.
a
Determined according to queries within SciFinder
on August 16, 2011 (see http://cas.org/products/scindr/index.html). All searches used full

names of assays (no acronyms or abbreviations), and results were ltered according to the keyword antioxidant. Results were rened to timeframes
of 5 years (or 1 year and 7.5 months in the case of 20102011) and the number of hits was then divided according to the number of years within the
search (1.625 in the case of 20102011).
Table 4Comparison of the total phenolics content and antioxidant capacities of fruits and vegetables as measured by the TPC, ORAC
FL
, TEAC, and
FRAP assays.
TPC
a,b
ORAC
FL
c,d,e
TEAC
a,f
FRAP
a,f
(mg GAE/100 g FW) SD (mol TE/100 g FW) SD (mol TE/100 g FW) SD (mol Fe
2+
/100 g FW) SD
Fruit/vegetable [Column rank] [Column rank] [Column rank] [Column rank]
Lettuce 14 1[14] 1550 [10] 17112[14] 124 7[14]
Red cabbage 158 4[4] 300 30 [14] 1377 49[4] 1870 18[4]
Spinach 72 1[9] 2640 [7] 75754[6] 1009 35[7]
Broccoli 128 4[6] 1590 [9] 64825[8] 833 16[8]
Onion 881[8] 1029 [11] 53229[9] 369 13[10]
Tomato 30 1[13] 460 [13] 25514[12] 344 7[11]
Apple 48 1[11] 2936 [6] 34313[10] 394 8[9]
Pear 60 3[10] 5235 [2] 28219[11] 315 24[12]
Orange 126 6[7] 1814 [8] 84925[5] 1181 6[6]
Banana 384[12] 879 [12] 18139[13] 164 32[13]
Red plum 32012[2] 6239 [1] 1825 28[3] 2057 25[3]
Blueberry 151 19[5] 4848 [4] 743 [7] 1861 [5]
Strawberry 330 4[1] 3577 [5] 2591 68[1] 3352 38[1]
Raspberry 228 6[3] 4925 [3] 1846 10[2] 2325 53[2]
a
Proteggente and others (2002).
b
Vinson and others (2001).
c
Wu and others (2004a).
d
Bhagwat and others (2007).
e
Cao and others (1996).
f
Pellegrini and others (2003).
photosynthetic phycobiliproteins from cyanobacteria (blue-green
algae) and two groups of eukaryotic algae (red algae and cryp-
tomonads) (Glazer 1990). The ORAC assay was adapted by Cao
and others (1993) for the assessment of antioxidant species in
human plasma. It was later automated on the Cobas Fara II cen-
trifugal analyzer (Cao and others 1995) and used to determine the
TRAP of human plasma (Ghiselli and others 1995). After the ap-
plication of the phycoerythrin-based assay to tea, vegetables, and
biological uids (Cao and others 1996; Cao and others 1998), Dr.
Ronald L. Prior and his colleagues modied the method using u-
orescein (FL) (3
-dihydroxy-spiro[isobenzofuran-1[3H],9
[9H]-
xanthen]-3-one) as a more stable and reproducible uorescent
probe (namely, the ORAC
FL
assay) (Ou and others 2001). Over
the following years, the ORAC
FL
assay was adapted to a multi-
channel liquid handling system coupled with a microplate u-
orescence reader (Huang and others 2002b) and applied to both
hydrophilic and lipophilic systems (Huang and others 2002a; Prior
and others 2003). Dr. Priors laboratory further modied the
ORAC
FL
assay for the controlled generation and scavenging of
HO
(Ou and others 2002). More recently, a derivative of u-

orescein (namely, dichlorouorescein) (Adom and Liu 2005) has
been applied as the uorescent probe in the ORAC assay, but
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uorescein still remains the probe of choice for the majority of
applications.
Despite the series of modications discussed, the principles of
the initial assay remain the same and include the following: azo-
initiation of RO
2
via thermal degradation of AAPH followed by

the competitive HAT reaction between antioxidant samples (or
standard Trolox) and the generated peroxyl radicals with the uo-
rescent probe. Fluorescence gives off a real-time signal registered
by the plate reader at an excitation/emission wavelength pair of
493/515 nm and declines rapidly as it undergoes a HAT reaction
with the azide-generated peroxyl radicals. The following reaction
scheme (15) illustrates this process:
2RO

2
+(FL)OH(Fluorescence at 515 nm) 2ROOH
+(FL)O
(HAT)
(15)
Any antioxidant species present in the reaction mixture will un-
dergo HAT with the peroxyl radicals (15) and delay the reduction
of the uorescent signal. Figure 15 is a proposed mechanism by
which FL (pictured in its free acid form) interacts with peroxyl
radicals resulting in the loss of uorescence at
Em
= 515 nm.
Photochemiluminescent (PCL) detection of water- and lipid-
soluble antioxidants
The capabilities of water- and lipid-soluble antioxidants to scav-
enge O
2
can be assessed using a Photochem
unit from Ana-

lytik Jena USA (The Woodlands, TX). The initial protocol and
system upon which Photochem
was developed is the work of

Drs. Igor Popov and Gudrun Lewin from 1987 to 1999. The span
of their research covers the photochemiluminescent quantication
of ascorbic acid and superoxide dismutase (SOD) in human plasma
(Analytik Jena sells kits for these assays, PCL
ASC
and PCL
SOD
, re-
spectively) (Popov and others 1987, 2001; Lewin and Popov 1994;
Popov and Lewin 1999b), as well as the measurement of antiox-
idant capacities of water- and lipid-soluble antioxidants (sold as
PCL
ACW
and PCL
ACL
kits, respectively) (Popov and Lewin 1994,
1996, 1999a). Each assay involves the photodegradation of lu-
minol (5-amino-2,3-dihydro-1,4-phthalazinedione) and results in
the production/quenching of O
2
. From this work, Analytik Jena

developed their testing kits for photochemiluminescent measure-
ments as well as the Photochem
system. The simplied radical-

generation reaction scheme (16) is as follows (Pegg and others
2007):
Luminol + hv
1
(UV) L
+
3
O
2
[L
O
2
] L
+
+ O

2
(16)
In reaction (16), L
is an intermediate product of the photo-

induced luminol and
3
O
2
is triplet oxygen (no
1
O
2
is involved in
the reaction). Once the O
2
and luminol radicals are generated,

they proceed through a series of reactions resulting in the pro-
duction of blue luminescence (Mer enyi and others 1986; Popov
and Lewin 1994; Schneider 1970). Though all the steps in the
detection reaction are not known, an example of possible chemical
intermediates in the chemiluminescence of luminol is illustrated
in reaction scheme (17) (Pegg and others 2007):
L
+
+ O

2
N
2
+ AP
2
AP
2
+ hv
2
(Blue at 360 nm)
(17)
In reaction (17), AP
2
is an excited aminophthalate anion,
and AP
2
is the aminophthalate anion at the ground state. The
O
O
O
HO
OH
Fluorescin (emission at 515 nm)
2ROOH
O
O
O
O
O
Dehydrofluorescin (no signal at 515 nm)
O
O
O
O
O
2ROO
Figure 15Proposed mechanism for the ORAC HAT FL(H) FL (loss of
signal).
chemical structure of luminol and the aminophthalate anion are
discussed by Schneider (1970).
Once O
2
radicals are generated, any exogenous antioxidant

species present in the reaction mixture will out-compete the
luminol radical in the action of donating a hydrogen atom
(a HAT reaction). This will halt the production of blue lumines-
cence until the concentration is exhausted. The resultant lag/log
relationships of antioxidant compounds performing in this closed
system are then compared to the effectiveness of standards (ascor-
bic acid in ACW, and Trolox in ACL). The antioxidant capacity
of compounds in the ACW and ACL assays gauge their relative
antioxidant capacities in hydrophilic and lipophilic media.
SET Assays
Ferric reducing antioxidant power (FRAP) assay
Benzie and Strain (1996) developed an assay to measure the
ferric reducing power of human plasma. This method was later
adapted to the quantication of ferric reducing antioxidant power
(FRAP) of plant extracts (Pulido and others 2000). Recently, the
FRAP assay was adapted to a microtiter plate reader in 96-well
format (Dragsted and others 2004). The assay reaction involves the
reduction of Fe
3+
TPTZ (iron[III]-2,4,6-tripyridyl-s-triazine) to
Fe
2+
TPTZ through SET with an antioxidant compound. The
result of this reaction is an intense blue color at
max
= 550 nm,
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AROH
N
N
N
N
N
N
N
N N
N
N
N
N
N
N
N
N
N
N
N N
N
N
N
Fe
2+
-TPTZ (blue at 595 nm)
Fe
3+
-TPTZ (colorless)
AROH
Fe
3+
Fe
2+
Figure 16FRAP color production reaction: SET reduction of iron(III)TPTZ to iron(II)TPTZ (blue at
max
= 595 nm).
as seen in reaction scheme (18):
Fe
3+
TPTZ + ArOH Fe
2+
TPTZ(Blue at 595 nm)
+[ArOH]
+
(SET)
(18)
Figure 16 is a structural representation of the conversion of
iron(III)TPTZto iron(II)TPTZ. Reaction (18) can occur with
antioxidant compounds with redox potentials lower than 0.7 V
(the E
of Fe
3+
TPTZ) and is, thus, comparable to ABTS
+
(E
= 0.68 V)-based assays (namely, TEAC) (Prior and others

2005). Furthermore, reducing power appears to be related to the
extent of conjugation in phenols as well as the number of hydroxy
constituents (Schaich 2006).
Because the FRAP assay solely measures reducing power, the
activity of compounds that follow traditional hydrogen abstraction
mechanisms may go unmeasured. This can in some instances serve
as a shortcoming, but when used in conjunction with other assays
this trait provides unique opportunities for determining the dom-
inant mechanisms of antioxidant compounds. It is also important
to note that the results of FRAP assays have been shown to pro-
duce considerably different results depending on the analysis time
and the reaction medium used (Prior and others 2005). Further,
the assay reaction must be carried out at acidic pH in order to
maintain iron solubility, but this can lower the IP of the reactants
and reduce the redox potential of the system.
Cupric reducing antioxidant capacity (CUPRAC) assay
In comparison with iron, copper has a greater potential to
undergo redox reactions with antioxidant components (E
of
copper[I] and [II] spectrophotometric-complexation reactions are
generally lower than iron[II] and [III]) (Schaich 2006). Redox
reactions with copper are often faster than with iron, thus reduc-
ing time constraints in the laboratory. Further, copper has more
3d-electrons than iron which may lend to its greater capability
to coordinate with the -electrons of incoming ligands during
metal-ion chelation (Chatterjee and others 1983). Just like iron,
copper ions coordinate with nitrogen-containing chelating agents
such as 2,2
-bipyridine or 1,10-phenanthroline and its derivatives

(Pilipenko and Falendysh 1972).
It follows from the reaction of iron(II) with 1,10 phenanthro-
line (the ferroin reaction) that the related copper(I) complexes
with 1,10 phenanthroline derivatives began to carry the sufx
cuproine (Smith and Wilkins 1953). T utem and others (1991)
sought to improve an existing bathocuproine (BC) (2,9-dimethyl-
4,7-diphenyl-1,10-phenanthroline) method for the selective spec-
trophotometric determination of copper(I) in the presence of
copper(II) (Moffett and others 1985), by introducing the use of
neocuproine (NC) (2,9-dimethyl-1,10-phenanthroline) as an al-
ternative chelating agent. Later, Apak and others (2004) revised
their copper(I)-NC method, applied it to the analysis of dietary
polyphenols, and created the CUPRAC assay. The CUPRAC
method involves the reduction of free copper(II) to copper(I) in
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Cu
2+
AROH
AROH
N
N
N
N
H
3
C CH
3
CH
3
H
3
C
Cu
+
N
N
N
N
CH
3
H
3
C
H
3
C CH
3
Cu
+
Bathocuproine (2:1) (
max
= 490 nm)
Neocuproine (2:1) (
max
= 450 nm)
AROH
AROH
Figure 17CuPRAC color production reaction: SET reduction of copper(II)

to copper(I)-BC/NC (blue at
max
= 490 or 450 nm).
the presence of NC, which results in the coordinated complex
Cu(I)-NC at a ratio of 2:1 according to the following reaction
scheme (19):
Cu
2+
+ ArOH + 2NC Cu
+
(NC)
2
(Blue at 450 nm)
+[ArOH]
+
(SET) (19)
Figure 17 is a structural representation of reaction (19), includ-
ing NC and BC complexes. Reagents for the CUPRAC assay
include a 0.1 M solution of copper(II) chloride (for free Cu
2+
), a
7.5 mM NC solution prepared in 95% (v/v) ethanol, ammonium
acetate buffer (pH 7) for the reaction medium and diluent of sam-
ples, and a standard (usually uric acid). A variation of the old
BC (copper[I]-BC,
max
= 490 nm) method for copper is sold as
a Bioxytech
AOP-490
TM
assay kit by OXIS International, Inc.
(Portland, OR). This kit consists of a ready-made dilution buffer,
cupric sulfate solution, uric acid standard, and stop solution to halt
the reaction.
Mixed-Mode Assays
Trolox equivalent antioxidant capacity (TEAC) assay
The Trolox equivalent antioxidant capacity (TEAC) assay is a
spectrophotometric method based on the capability of an antiox-
idant to scavenge the free-radical cation ABTS
+
. The TEAC
assay was originally developed by Miller and others (1993) for
the measurement of the antioxidant capacity of human plasma in
infants. Re and others (1999) modied the assay for the direct
generation of ABTS
+
without radical intermediates and applied
it to hydrophilic and lipophilic antioxidants. Dragsted and others
(2004) adapted the assay to a microplate reader for high through-
put. Though the TEAC assay is generally accepted as a SET assay,
ABTS
+
can be neutralized by SET and HAT mechanisms. The
HAT and SET assay reaction schemes (2021) are as follows:
ABTS
+
(Green at 734 nm) + ArOH ABTS(Colorless)
+[ArOH]
+
(SET)
(20)
ABTS
+
(Green at 734 nm) + ArOH ABTS(H)(Colorless)
+ArO
(HAT)
(21)
Figure 18 serves as a structural representation of the ABTS
+
decolorization reaction (21). It is important to note that there are
many points for careful consideration in the TEAC assay including
the controlled generation of ABTS
+
, pH, and temperature of the
assay medium. Also, ABTS
+
is a nitro-radical; therefore, it may
not correlate well with other antioxidant capacity assays that mea-
sure oxyl-radical scavenging. As with the FRAP and CUPRAC
assays, the complex nature of ABTS
+
may render interaction
with polyphenolics time-dependent, so time-curves are often pre-
pared. Given the prevalence of both HAT and SET reactions with
ABTS
+
, the TEAC assay should be considered a mixed-mode
assay.
DPPH
(2,2
-diphenyl-1-picrylhydrazyl radical cation)

Aassay
DPPH
has been examined for its use as an organic colorimetric

reagent since the 1950s (Blois 1958). Braude and others (1954)
made the observation that DPPH
undergoes a HAT mechanism

with antioxidant compounds according to the following reaction
scheme (22):
DPPH
(Violet at 515 nm) + ArOH DPPH(H)(Colorless)

+ArO
(HAT)
(22)
Figure 19 is a structural representation of the reaction (22). Blois
(1958) determined that if the phenolic compound under analysis
contains more than one phenolic hydroxy functional group, the
resultant ArO
formed is sufciently stable to undergo a second

simultaneous HAT reaction with another molecule of DPPH
,
thereby preserving the stoichiometry of the reaction.
Over the past two decades the DPPH
assay has resurfaced as a

method for the analysis of phenols in plants and plant-derived food
products (S anchez-Moreno and others 1998). The current version
of the assay involves adaptation to a high-throughput 96-well mi-
crotiter plate system (Fukumoto and Mazza 2000). The assay is
often run in-tube due to relative inexpensiveness. This renewed
interest in the DPPH
assay has resulted in a re-examination of the

kinetics of its reaction with phenolics (Brand-Williams and oth-
ers 1995; Bondet and others 1997; Silva and others 2000) and
possible mechanisms of interaction, whether HAT (Brand-
Williams and others 1995; Dangles and others 2000; Litwinienko
and Ingold 2003), SET (Foti and others 2004; Huang and others
2005), or mixed (Schaich 2006). The following reaction scheme
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S
N
N N
N
S
CH
2
CH
3
HO
3
S
SO
3
H
CH
2
CH
3
AROH
S
N
N N
NH
S
CH
2
CH
3
HO
3
S
SO
3
H
CH
2
CH
3
ABTS (green at 734 nm)
ABTS(H) (colorless)
ARO
Figure 18Conversion of ABTS
+
(green at
max
= 734 nm) to a colorless species ABTS(H) through a HAT mechanism with antioxidant compound
ArOH.
(23) is an example of a SET mechanism between DPPH
and a
phenolic antioxidant:
DPPH
(Violetat515nm) + ArOH DPPH
(Colorless)
+[ArOH]
+
(SET)
(23)
As with the TEAC assay, the medium of interaction, size, polar-
ity, and acidity of phenolic hydroxy groups play a role in whether
SET or HAT mechanisms dominate. DPPH
is known to react
with a variety of compounds including aromatic amino acids, glu-
tathione, -tocopherol, ascorbic acid, and polyhydroxy aromatics
(phenolics); therefore, the amount of potential interferences in the
assays progress is great.
Evaluation of Chelation Activity
The most commonly employed methods to determine the
metal-ion chelating activity of phenolic compounds are the
tetramethylmurexide (TMM) and the ferrozine assays, for fer-
rous and cuprous ions, respectively (Lee and others 2007). Both
of these assays are spectrophotometric and easy to use. In both
methods, a reagent (TMM or ferrozine) is used to form com-
plexes with divalent metal cations; the result of which produces
increases in absorbance readings at the measured wavelengths (485
and 562 nm, respectively). Absorbancies increase in a linear fash-
ion with concentration according to Beers Law, thereby allowing
for the formation of a standard curve by linear regression. The
standard curve can then be used for the comparative evaluation of
the chelating properties of other compounds relative to reference
compounds.
Although both assays have become commonplace, Karama c and
Pegg (2009) reported the TMM assay to present limitations in the
assessment of phenolic preparations rich in tannin constituents.
Specically, control samples not containing TMM produced high
absorbance values, suggesting interference in the measurement.
The authors suggest the use of the ferrozine method as the pre-
ferred one in such cases.
Methods to Evaluate Lipid Oxidation
Another strategy that may be employed in assessing antioxidants
is to incorporate the antioxidant into a system and then monitor
the affected oxidative stability (that is, the resistance to oxidation).
This approach may offer a practical advantage in that it can suc-
cessfully incorporate any multitude of variables (which may or
may not be well understood) to give a relatively telling account of
an antioxidants capabilities within a particular system.
There are many methods used to evaluate oxidation rates within
a system, most of which require the repeated performance of an
assay over a period of time. The period of time may be abbreviated
by use of radical initiators (Alsante and others 2007) or acceler-
ated storage techniques like the Schaal oven storage stability test
(Frankel 1993). Below is a very brief summary of the methods
commonly used for the evaluation of oxidation.
Peroxide value
Given that the primary products of lipid oxidation are hydroper-
oxides (commonly referred to as peroxides), their quantication
can provide a suitable measurement of the extent of oxidation
present in a lipid sample. Protocols for the quantication of per-
oxide values (PVs) in foods can be iodometric or colorimetric
methods, each with its strong and weak points (for example, iodo-
metric titration endpoint) (Pegg 2005). Because the extent of
oxidation of a lipid is related to its PV, the capability of an antiox-
idant compound to perform in a closed system can be gauged by
the prevention of peroxide formation over time with respect to a
control.
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r
AROH
NO
2
NO
2
N N O
2
N
NO
2
NO
2
H
N N O
2
N
DPPH (purple at 517 nm)
DPPH(H) (colorless)
ARO
Figure 19HAT conversion of DPPH
(purple at
max
= 517 nm) to
colorless species DPPH(H).
Conjugated dienes
The majority of polyunsaturated fatty acids (PUFAs) in na-
ture have a 1,4-diene structure (their points of unsaturation are
methylene-interrupted), so the occurrence/detection of conju-
gated dienes (CD) is an indication that fatty acids have undergone
autoxidation (Corongiu and Banni 1994). The number of posi-
tional isomeric peroxides that can result from autoxidation of a
lipid depends on the number of double bonds (n) contained and
is equal to 2n-2 (Esterbauer 1993).
The premise of the CD assay, which has been in use since
before the 1950s (Farmer and Sutton 1943), is the strong UV
absorbance of the CD moiety at
max
= 234 nm. Conjugated
dienes are the rst indicator of oxidation in model lipid systems
and are often retained in many secondary products, even after PVs
decrease in later stages of oxidation. Given the lack of complicated
reagents and preparative work required, the assay is a very attractive
option for a quick assessment of lipid oxidation or the capability of
an exogenous antioxidant to inhibit autoxidation. In some cases;
however, absorbance of the diene moiety of an oxidized lipid is not
easily related to the full extent of oxidation in a sample. The effects
of autoxidation on lipids can vary (Gray 1978), and results are best
explained if the composition of the lipid is known (Holman and
Burr 1946).
Anisidine test
The p-anisidine value (PAV) measures secondary lipid oxidation
products by an assessment of aldehyde concentration. Aldehyde
carbonyl bonds react with the amine group of the p-anisidine
reagent to forma Schiff base which absorbs radiation at 350 nmand
can therefore be quantied using spectrophotometry (Laguerre
and others 2007). The colorimetric response is affected by the level
of aldehyde unsaturation (greater reactivity with higher degree
of unsaturation), and therefore the results of the assay must be
interpreted with a level of caution. The PAV is often combined
with peroxide values to form a totox value; a simple summary
of lipid oxidation that incorporates measures of both primary and
secondary oxidation products (Nielsen 2010).
Thiobarbituric acid-reactive substances test
The thiobarbituric acid-reactive substances (TBARS) test quan-
ties malonaldehyde and malonaldehyde-type products (such as
trans,trans-2,4-heptadienal, trans-2-heptenal, trans-2-hexenal, and
hexanal), as well as secondary oxidation decomposition products of
polyunsaturated fatty acids (Dahle and others 1962). These prod-
ucts react with 2-thiobarbituric acid (TBA) to form a stable pink
chromophore, with a
max
of 532 nm, and can therefore be quanti-
ed spectrophotometrically (Dahle and others 1962; Frankel 1993;
Nielsen 2010). Because the malonaldehyde-type compounds are
highly reactive, the TBARS test only measures products temporar-
ily occurring in the steps of oxidation.
Volatile organic compounds
Volatile organic compounds (hexanal, pentane, trans,trans-2,4-
decadienal, and others) are common secondary oxidation products
of foods containing linoleic acid, and their quantication with gas
chromatography (GC) can be useful in the assessment of oxidation.
This method can be of particular value in the case of foods because
it allows not only for a strict assessment of oxidation, but also for
the assessment of oxidation as it relates to the products quality
(Panseri and others 2011). The proliferation of volatile organic
compounds is often the causative agent in the development of
oxidized off-avors. The introduction of solid-phase microextrac-
tion (SPME) has advanced the method and now allows for greater
sensitivity (Nielsen 2010; Panseri and others 2011). Recent work
has established successful methods by which the use of SPME/GC
accurately assesses hexanal concentrations in systems at concentra-
tions in the range of g/g, thus allowing for detection of volatiles
at early stages of formation (Giuffrida and others 2005).
Total Phenolics Content (TPC) with Folin and
Ciocalteus Phenol Reagent
Another assay of relevance is the total phenolics content or TPC
assay with Folin and Ciocalteus phenol reagent, which quanties
the phenolic content of a sample. In addition to providing the ob-
vious benet of the assessment of quantities within food sources,
this method may also be used in tandem with antioxidant assess-
ment assays to determine antioxidant potential relative to quantity
and/or concentration.
Phosphotungstic and phosphomolybdic heteropolyacids have
been used as colorimetric reagents since the early 1900s (Folin
and Macallum 1912). In 1912, a breakthrough in colorimetry oc-
curred with the creation of a sensitive chromophoric complexing
reagent for the quantication of tyrosine residues in protein hy-
drolysates (Folin and Denis 1912a, b). The Folin-Denis phenol
reagent (as it was named then) was again reformulated, in 1927
by Folin and Ciocalteu (1927), with a greater incorporation of
molybdenum for increased redox sensitivity. Singleton and Rossi
(1965) applied Folin & Ciocalteus phenol reagent to the assess-
ment of antioxidant contents in wine, resulting in the well-known
TPC assay. With the increased interest in phenolics over the past 2
decades, this assay has become a mainstay in laboratories the world
over.
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The TPCassay is often modied based on the antioxidant source
under investigation. While the most up-to-date method involves
automation on a 96-well microtiter plate reader (Zhang and oth-
ers 2006), the original in-tube assay is most prevalent. Despite
modications, the bulk of the total phenol protocol has remained
the same since Singleton and Rossi (1965). Briey, the assay in-
volves the mixture of excess phenol reagent and a diluted sample
or standard (gallic acid). The mixture is then treated with alkali
to a nal pH of 10 to 11. The resultant color from interaction of
phenolate anions with the phenol reagent complex is allowed to
develop over 30 to 60 min and yields a
max
in the range of 745
to 765 nm, depending on the standard employed.
The mechanism behind the TPC assay involves reduc-
tion of the molybdenum component in the phosphotungstic-
phosphomolybdic complexing reagent according to the following
reaction scheme (24):
Mo
6+
(yellow) + ArOH Mo
5+
(blue at 750 nm)
+[ArOH]
+
(SET) (24)
Reaction (24) is subject to a great many interferences, partic-
ularly any readily reducible component present within the assay
mixture. Ascorbic acid is the major interference in the case of wine
analysis (Singleton and others 1999) and most fruits.
Although the phenol reagents of Folin-Denis and Folin-
Ciocalteu have been around for 75+ years, the chemistry of the
reagents and the phenol-reagent reactions are still not well under-
stood. It is possible that the reaction product between phenolate
anions and Folin and Ciocalteus phenol reagent is a group of
Keggin clusters: a common formof heteropolyacids comprised of a
cage structure of oxygen-containing phosphomolybdic and phos-
photungstic repeats bearing the common formula [XM
12
O
40
]
n
,
where X is the heteroatom (phosphorus) and M is an added metal
atom (molybdenum or tungsten in this case) (Pope 1983). The
inherent stability of Keggin clusters promotes the reduction of the
metal ion contained and, thus, facilitates the utilization of such
reagents for colorimetry. It is generally accepted that in alkaline
media three competitive reactions are proceeding simultaneously,
including the destruction of the yellow Folin-Denis/Ciocalteu
phenol reagent, the reduction of the reagent by phenolate anions to
produce the characteristic molybdenum blue, and the destruc-
tion of the blue pigment by alkali (fading). Increasing the alkalinity
or temperature of the assay promotes the development of the
color complex in a shorter period of time. However, precipitation
of the phenol reagent can occur. The precipitate is a dense, white,
crystalline material that can be formed by excessive heat (above
60

C), alkalinity (above pH 1011), or the quantity of reagent in
the assay (above 5 mL/100 mL) (Rosenblatt and Peluso 1941).
Conclusions
During the dawn of the functional food and nutraceutical era,
new antioxidant assessment methods are constantly being created
for the fast and cost-efcient screening of extracts and food prod-
ucts with biological activities. Each of these assays includes a variety
of critical steps that must be followed in order to maintain accuracy
and precision. Furthermore, the chemistry behind the assays must
be assessed throughout, in order to run the assays and troubleshoot
in an effective manner. There has been a great need for the stan-
dardization of antioxidant methods to promote uniformity in the
scientic literature for the better comparison between sources.
Yet, even with the development of standard protocols for antiox-
idant evaluation, the complex nature of these methods and their
compatibility with certain antioxidant sources must still be of con-
cern for future research and, thus, great care should be exercised
when formulating ones battery of methods for the measurement
of antioxidant capabilities.
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Phenol-Based Antioxidants and the In Vitro

Methods Used for Their Assessment

(1) is relatively unreactive;

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HAT mechanisms with

,7-trihydroxyavylium cation and (+)-catechin (as models for

Phenolic antioxidants and their assessment . . .

in polar protic media, in which both HAT and sequen-

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), hydroxyl radical (HO

) also exist and can have adverse effects on

Phenolic antioxidants and their assessment . . .

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Phenolic antioxidants and their assessment . . .

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Phenolic antioxidants and their assessment . . .

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Phenolic antioxidants and their assessment . . .

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Phenolic antioxidants and their assessment . . .

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Alho and Leinonen (1999)

Popov and Lewin (1999a); Pegg and others (2007)

Campos and others (1996); Lissi and others (1995)

Huang and others (2002b); Prior and others (2003); Wu and

Wayner and others (1985); Wayner (1987); Lussignoli and

Regoli and Winston (1999); Winston and others (1998)

Roberts and Gordon (2003)

Esterbauer and others (1992); Frankel and others (1995)

UV-Vis Hatano and others (1988), S anchez-Moreno and others (1998)

Phenolic antioxidants and their assessment . . .

on August 16, 2011 (see http://cas.org/products/scindr/index.html). All searches used full

(Ou and others 2002). More recently, a derivative of u-

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via thermal degradation of AAPH followed by

can be assessed using a Photochem

unit from Ana-

was developed is the work of

. From this work, Analytik Jena

system. The simplied radical-

is an intermediate product of the photo-

and luminol radicals are generated,

radicals are generated, any exogenous antioxidant

Phenolic antioxidants and their assessment . . .

= 0.68 V)-based assays (namely, TEAC) (Prior and others

-bipyridine or 1,10-phenanthroline and its derivatives

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Figure 17CuPRAC color production reaction: SET reduction of copper(II)

-diphenyl-1-picrylhydrazyl radical cation)

has been examined for its use as an organic colorimetric

undergoes a HAT mechanism

(Violet at 515 nm) + ArOH DPPH(H)(Colorless)

formed is sufciently stable to undergo a second

assay has resurfaced as a

assay has resulted in a re-examination of the

Phenolic antioxidants and their assessment . . .

(Violetat515nm) + ArOH DPPH

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Phenolic antioxidants and their assessment . . .

free radical method. Lebensm-Wiss u

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,7-trihydroxyavylium ion. J Chem Soc Perkin Trans 2:165363.