Journal of the Science of Food and Agriculture

J Sci Food Agric 86:20462056 (2006)

Review
Methodology for the determination
of biological antioxidant capacity in vitro:
a review
Lesley K MacDonald-Wicks,1 Lisa G Wood2 and Manohar L Garg3
1 School

of Health Sciences, University of Newcastle, Callaghan, NSW 2308, Australia

Medical Research Institute, Respiratory & Sleep Medicine, John Hunter Hospital, NSW, 2305, Australia
3 School of Biomedical Sciences, University of Newcastle, Callaghan, NSW, 2308, Australia
2 Hunter

Abstract: One of the effective means of adding value to agricultural produce is the development of functional foods
or dietary supplements enriched with antioxidants. This process involves extraction of antioxidant rich fractions
from their sources and then testing for antioxidant activity prior to adding these back to the foods or packing into
pills or capsules. Ideally, the antioxidant activity should be tested using both in vitro and in vivo techniques but
due to the high cost of conducting animal and human feeding trials, many products undergo only in vitro testing.
This review describes popular methods commonly used for testing antioxidant activity in vitro, with particular
reference to their reliability, efficiency, accessibility and biological relevance.
2006 Society of Chemical Industry

Keywords: antioxidant capacity; in-vitro assays; oxidative stress; antioxidants

INTRODUCTION
There is increasing evidence that oxidative stress,
in particular reactive oxygen species (ROS) and
reactive nitrogen species (RNS), are involved in
several inflammatory and degenerative diseases. In
humans, oxidative damage is usually not involved in
the initiation of chronic disease although it can be
a promoter of disease.1 Therefore there is escalating
interest in the efficacy of antioxidant activity of the
many naturally occurring molecules in food and
biological systems.
In foods, antioxidants have been defined as
substances that in small quantities are able to prevent
or greatly retard the oxidation of easily oxidisable
materials such as fats,2 therefore in food science
antioxidants are usually equated with chain-breaking
inhibitors of lipid peroxidation, but not exclusively
so. Many antioxidants are used and studied in a
wide range of foods including beverages. Therefore for
foods and beverages, antioxidants are molecules that
can be equated with the protection of macromolecules
from oxidation.3
In biological systems the definition has been
accepted to be any substance that, when present at
low concentrations compared to those of an oxidisable
substrate, significantly delays or prevents oxidation
of that substrate.4,5 This is a broader definition
encompassing many vulnerable macromolecules (e.g.
DNA, lipids and proteins) that can be affected by
oxidation. In biological terms, it is accepted that any

molecule that can retard or prevent the action of

oxidants could be considered to be an antioxidant.6
Such a broad definition means that compounds that
inhibit specific oxidising enzymes, react with oxidants
before they damage molecules, sequester dangerous
metal ions or even repair systems such as iron
transport proteins, can fit the definition. A definition
as broad as this, however, loses its meaning, and this
can be demonstrated when, for example, mutagenic
or anti-allergic compounds that are described as
having antioxidant actions, may, in fact, have little
antioxidant activity.
None of the above definitions defines antioxidant
activity, and there is no accepted international
definition for antioxidant or antioxidant capacity.
Mechanistic definitions of antioxidants are usually
focused on the ability to be a hydrogen donor or
an electron donor. Many of the frequently cited assays
of antioxidant capacity can be broadly categorised
as either hydrogen transfer assays or single electron
transfer reaction based assays. These assays measure
the radical scavenging capacity or the reducing ability,
respectively, not the preventative antioxidant capacity
of the sample.
Antioxidant activity and antioxidant capacity are
terms that are often used interchangeably but it should
be recognised that they have different meanings.
Activity refers to the rate constant of a reaction between
a specific antioxidant and a specific oxidant. Capacity
is a measure of the amount (in moles) of a given

Correspondence to: Manohar L Garg, Room 305C, Medical Sciences Building, University of Newcastle, Callaghan, NSW 2308, Australia
E-mail: manohar.garg@newcastle.edu.au
(Received 28 September 2005; revised version received 17 February 2006; accepted 25 May 2006)
Published online 23 August 2006; DOI: 10.1002/jsfa.2603

2006 Society of Chemical Industry. J Sci Food Agric 00225142/2006/$30.00

Methods for assessing in vitro antioxidant activity

free radical scavenged by a sample. Measurements

of antioxidant capacity yield the amount of a
heterogeneous mixture of antioxidants that react
together to produce the total or net scavenging ability
of the sample. The antioxidant capacity of each
individual component is not measured.
It must also be recognised that the antioxidant
capacity of a sample will differ with the different
oxidants it is measured against. Reactions between
different antioxidants and oxidants have different
rate constants, and therefore the overall antioxidant
capacity varies.7 It should also be realised that the
analytical methods of measurement and the conditions
under which the analysis has occurred, can lead to
variable results for the same food types.
Due to the increasing interest in these biological
molecules for consumers, food scientists and the
medical fraternity, a quick and easy method for
determining antioxidant capacity would be most
useful. This review will show that a simple onedimensional test of antioxidant capacity is not possible,
and would not accurately reflect the complexity of
the interactions of antioxidants in vivo.8,9 Therefore
it is important that a variety of tests is used on a
food to investigate its antioxidant potential. Becker
et al.3 recognise the need for a variety of approaches
in their three-step procedure. Becker and colleagues
propose that there be a series of tests performed on
a food, thereby capturing the complexity of the issue
of antioxidant capacity. The food would then go on
to either storage studies, to look at stability and the
relevance to a food product, or to human intervention
studies.3
The aim of this review is to provide evidence of
the strengths and limitations of antioxidant capacity
assays, therefore demonstrating the need to combine
assays to test the relevance of any single aspect of
a food as having antioxidant potential. Any claims
that a food has potential antioxidant action must be
supported by in vitro tests before it is involved in
human intervention trials. As a result the valuable
work done on the quantification of antioxidants, the
antioxidant capacity of a number of specific food
compounds (such as Maillard reaction products), the
effect of storage on antioxidant capacity and studies
testing the incorporation of antioxidants in foods are
beyond the scope of this review.
SCAVENGING REACTIVE OXYGEN SPECIES
AND REACTIVE NITROGEN SPECIES
There are six major ROS and RNS that regularly
interact and damage the major macromolecules
in physiological and food-related systems: (1) the

superoxide anion (O2 ), (2) hydrogen peroxide

(H2 O2 ), (3) the peroxyl radical (ROO ), (4) the

hydroxyl radical (OH ), (5) singlet oxygen (1 O2 ), and

(6) peroxynitrite (ONOO ).
Sophisticated antioxidant protection systems for
these compounds have been developed. For example, the superoxide anion is converted to oxygen and
J Sci Food Agric 86:20462056 (2006)
DOI: 10.1002/jsfa

hydrogen peroxide by the enzyme superoxide dismutase, and hydrogen peroxide is converted to water and
oxygen by the enzyme catalase. For those radicals
without endogenous enzymatic antioxidant protection, exogenous non-enzymatic dietary compounds
are used as scavengers. The peroxyl radical has been
the most frequently used compound in assays to measure antioxidant capacity as it is a key component
of autoxidation and can be easily produced by the
decomposition of azo compounds.
Measuring superoxide scavenging

The superoxide radical (O2 ) is easily formed by

radiolysis of water in the presence of oxygen and
formate, which allows accurate reaction rate constants
to be measured.5 However, this method is unsuitable
for reactions with slow rate constants, and requires the
presence of appropriate equipment.
In normal tissue, xanthine oxidase is a dehydrogenase enzyme that transfers electrons to nicotinamide
adenine dinucleotide (NAD). During times of stress
this enzyme is converted to an oxidase enzyme and
produces superoxide and hydrogen peroxide. Thus,
xanthine oxidase plus hypoxanthine (or xanthine) at
pH 7.4, in the test tube, can be used to generate the
superoxide anion. This reaction can be measured by
the ability of the superoxide anion to reduce nitro blue
tetrazolium (NBT)10 to formazan, and the formation
of formazan can be measured spectrophotometrically
at 560 nm.11 To improve the throughput and ease of
this assay, it has recently been adapted to micro-plate
format using cytochrome c instead of NBT and read
at 550 nm (the Amax for cytochrome c).12
The appropriate ratio of substrate (hypoxanthine) to
enzyme is essential to ensure that optimum amounts
of superoxide radical are formed. If there is too
much substrate a two-electron transfer occurs which
causes the formation of hydroperoxide and thence in
undesirable side reactions that could potentially cloud
the results. The addition of EDTA to the assay, as
originally outlined by Bull et al.10 was not as efficient
as the addition of catalase in sequestering hydrogen
peroxide production.12
In general, problems arise if controls are not
established for substances that may directly interfere
with the action of the enzyme, or if the antioxidant
itself directly reduces cytochrome c or NBT. It could
also be that the radical produced by the reduced
antioxidant also could interact with cytochrome c
or NBT.5 Therefore this method is not suitable for
non-enzymatic antioxidants.
The scavenging ability of superoxide can also be
measured by reaction of the anion with -ketomethiolbutyric acid (KMB) to produce ethane, which
can be measured by using gas chromatography. The
scavenging ability can also be measured by using
electron spin resonance (ESR) spectrometry. Here,
the anion is trapped with 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) and the DMPOOH adduct is
2047

LK MacDonald, LG Wood, ML Garg

detected by ESR using manganese oxide as the internal

standard.
Interest in the scavenging ability of the superoxide
anion is largely due to its role in the production
of the highly reactive hydroxyl radical in the
presence of metal ions. However, this is not
the only mechanism of oxidising lipids, and the
ability to scavenge the superoxide anion is not
necessarily effective in preventing lipid oxidation. In
addition to the limitations of this assay mentioned
above, measurement of the scavenging ability of the
superoxide anion must be interpreted with care as
no equilibrium can be reached when superoxide
radicals are generated constantly throughout the assay.
Therefore, measurement of the superoxide radical is
sufficiently problematic that the uses of these assays
are not yet at a standard to recommend its reliability
and utility.
Measuring hydrogen peroxide scavenging
Hydrogen peroxide (H2 O2 ) can cross membranes
easily but it is unreactive at low concentrations
and reacts quite slowly with most compounds (the
exceptions being oxygen, nitric oxide and some
ironsulfur proteins). It is used in the respiratory
burst of activated phagocytes, and generated in vivo
by several oxidase enzymes.
Peroxide-based assays are sensitive measures of
hydrogen peroxide. For example, the most common
assay uses horseradish peroxidase and hydrogen
peroxide to oxidise scopoletin into a non-fluorescent
product, which can be measured in the presence of
a scavenging molecule by inhibiting the oxidation of
scopoletin.11
Similar to the superoxide radical scavenging, it
is important that the antioxidant being measured
is not a substrate for the peroxidase enzyme. It is
also possible that the superoxide radical is being
produced as well, which will interfere with the
measurement of hydrogen peroxide, and it may be
necessary to add superoxide dismutase to counteract
the superoxide radical.5 From this assay it is difficult
to determine whether the antioxidant is acting with
the hydrogen peroxide, reacting with intermediates
formed from enzyme and hydrogen peroxide, or
inhibiting the horseradish peroxidase from binding
hydrogen peroxide; therefore it is difficult to interpret
the results.13 Thus, similarly to the superoxide
scavenging assays the reliability and utility of the assays
is questionable.
Mueller14 describes in detail a non-enzymatic
and sensitive hydrogen peroxide assay based on
the chemiluminescence reaction of luminal with
hypochlorite. The assay is based on the oxidation
of luminol by sodium hypochlorite (NaOCl) to
diazaquinone in a two-electron oxidation, which is
then specifically converted by H2 O2 to an excited
aminophthalate. The reaction has a very short
luminescence signal (2 s) at a maximum wavelength
of 431 nm, and is linearly dependent on hydrogen
2048

peroxide down to a concentration of 109 mol L1 .

It therefore measures the concentration of hydrogen
peroxide not the generation rate of hydrogen peroxide,
and may be used to describe/assess fast enzyme kinetics
at very low concentrations of H2 O2 .
The assay requires a chemiluminometer with a flow
cell installed, a photomultiplier and a perfusion pump
for the substrates. The strength of this assay is that
it bypasses the one-electron transfers inherent in the
peroxidase assays, which may cause redox cycling and
possibly reduction of oxygen to the superoxide anion.
Therefore this assay has potential as one of a group of
assays designed to interpret the antioxidant activity of
compounds in vitro.
Measuring hydroxyl radical scavenging
Pulse radiolysis15 is the most often used technique for

measuring the reaction of the hydroxyl radical (OH )

and antioxidants.4 Again, this requires the specialised
equipment and could be a costly assay. However,
approximations of rate constants can be more easily
obtained, and are not significantly less accurate. The
hydroxyl radical is formed by the combination of Fe(II)
and hydrogen peroxide, which is a Fenton reaction.
Since the spin trap DMPO reacts quickly with the
hydroxyl radical adding a hydroxyl radical scavenger
will compete for the hydroxyl radical and decrease

the DMPOOH ESR signal. By using an ESR

spectrometer with appropriate data systems the rate
constant can be obtained from a competition plot.16
2-Deoxy-D-ribose is also a hydroxyl radical detector.
The hydroxyl radical is formed by mixing ascorbic
acid, hydrogen peroxide and Fe(III)-EDTA. The
radical reacts with deoxyribose, causing it to fragment
and combine with thiobarbituric acid (TBA) to
form a chromogen when heated, scavengers of
the hydroxyl radical prevent the formation of
this chromogen by competing for the radical.17
Recently Zhu and co-workers18 reported a mixture
of tetrachlorohydroquinone (TCHQ) and H2 O2
hydroxylates salicylic acid to yield 2,3- and 2,5dihydroxybenzoic acid (DHBA), a process inhibited
by hydroxyl radical scavenging agents.18 It may be that
this new assay can provide a direct, or more direct,
way of measuring hydroxy radical scavenging ability,
as it may be that this assay is a direct producer of the
hydroxyl radical.
The hydroxyl radical is extremely reactive in vivo
with rate constants greater than 109 mol L1 s1 .5
Therefore the hydroxyl scavenging ability of a
molecule would be irrelevant in vivo. Of more use
is the ability to sequester metal ions and therefore
prevent the reaction that leads to the hydroxyl radical
formation. A dietary molecule that acts in this way
would behave as a preventative antioxidant. Ou
et al.19 developed an assay for measuring the metalchelating ability of compounds, termed HORAC:
hydroxyl (OH) radical (R) averting (A) capacity (C).
The hydroxyl radical is generated by a Co(II)mediated Fenton-like reaction, and radical production
J Sci Food Agric 86:20462056 (2006)
DOI: 10.1002/jsfa

Methods for assessing in vitro antioxidant activity

is confirmed indirectly by the hydroxylation of phydroxybenzoic acid (measured by high-performance

liquid chromatographymass spectrometry (HPLCMS)). The scavenging ability is measured by using a
fluorescein (FL) probe, and the fluorescence decay
curve of FL is measured with and without an
antioxidant (in this case a metal-ion sequester). Ou
et al.19 used a COBAS FARA II programmed to
automate the reaction and to measure the decay curve
initially after 0.5 s and then every minute for 35 min,
at an excitation wavelength of 493 nm and with an
emission filter at 515 nm. The quantification of this
assay is the same as the ORAC assay (discussed later
in this review), it uses area-under-the-curve (AUC)
calculations where the sample AUC is subtracted
from the blank AUC and the assay uses gallic acid
as the reference standard. The HORAC value is
related directly to the ability of the antioxidant to
chelate metals, and the ability to form a stable complex
between Co(II) and the antioxidant measured. This
method has been rigorously validated for linearity,
precision, accuracy and ruggedness. For details see
reference 19. However, it must be noted that although
the quantification processes are the same in the ORAC
and HORAC assays there is no correlation between
the ORAC and HORAC values. Ou and co-workers
state that to accurately evaluate the antioxidant action
of food constituents a variety of validated assays are
required, and suggest the HORAC assay could be part
of this battery of tests.19
Singlet oxygen scavenging
Normally, singlet oxygen (1 O2 ) is believed to be
formed in the presence of light or photosensitisers.
The formation of singlet oxygen, in vivo, without the
presence of light is thought to be the result of the
spontaneous dismutation of the superoxide anion.
Singlet oxygen can also be physically quenched by
transferring its excitation energy to another molecule.
Singlet oxygen emits a characteristic phosphorescence
at 1270 nm; the decay rates of the light intensity can be
used to measure the singlet oxygen quenching ability
of compounds. Wilkinson et al.20 have calculated the
rate constants of singlet oxygen reactions with various
compounds.
Fu and co-workers21 reported a more sensitive
method by monitoring the quenching of singlet
oxygen sensitised delayed fluorescence of tetra-tbutylphthalocyanine at 703 nm. This method uses
10-nm band-pass filters to focus the laser to the
required nanometre range and a cryogenic germanium
photodetector to measure the output as photons. This
data are transferred to a digital storage oscilloscope
and the decay rates are calculated. This method is not
widely used but may provide a measure of quenching
ability in systems where the 1270 nm luminescence is
difficult to detect. Specialised equipment is required
and the 10-nm filters increase the cost of this
method. Further use of the method will enable the
determination of the reliability and utility.
J Sci Food Agric 86:20462056 (2006)
DOI: 10.1002/jsfa

Peroxynitrite scavenging
Peroxynitrite (ONOO ) and nitric oxide are not strong
oxidising agents. Both can be protonated to form
peroxynitrous acid (ONOOH) at a physiological pH
(pKa 6.8), which decays rapidly to a series of strong
oxidising agents.22 The main route of damage is the
nitration or hydroxylation of aromatic compounds,
particularly tyrosine. Physiologically, peroxynitrite
forms an adduct with carbon dioxide and this could
be responsible for the oxidative damage to proteins.
There are few reports on this method. The main
process requires HPLC separation and quantification
of the product of nitration of proteins, particularly
tyrosine and nitrotyrosine, a process which is time
consuming and requires the appropriate equipment.
Methods for measuring the scavenging of peroxynitrite usually depend on either the inhibition of tyrosine
nitration or the inhibition of dihydro-rhodamine 123
(DHR) oxidation to rhodamine 123. Pannala et al.23
measured the antioxidant capacity of catechin and
other polyphenolics by determining the changes in
concentration of tyrosine at 275 nm and of 3-nitrotyrosine (the main product of nitration of tyrosine) at
430 nm in the presence of varying concentrations of
the antioxidant. This was then confirmed by HPLC
separation and quantification of nitro-tyrosine.
In the method by Kooy et al.,24 fluorescent intensity
was measured by using a spectrophotometer with the
excitation wavelength set at 500 nm and an emission
wavelength of 536 nm. The oxidation of dihydrorhodamine by peroxynitrite is rapid. The reaction
is linear and concentration dependent and does
not require a metal-ion catalyst. The final product
is stable. The initial rate approach was used to
quantify the peroxynitrite scavenging capacity; this
assay shows that dihydro-rhodamine is a potentially
useful in vitro probe for measuring concentrations of
peroxynitrite production. This method has since been
used with a micro-plate fluorescent spectrophotometer
with excitation and emission wavelengths of 485 and
530 nm, respectively.25

METHODS THAT MEASURE THE UPTAKE OF

OXYGEN
These methods require the measurement of the
rate of O2 consumption or the rate of conjugated
diene peroxide formation in order to interpret the
antioxidants rate constant for inhibition using a
pressure transducer system under one atmospheric
pressure of oxygen. This method has not found great
use due to a number of factors. Firstly, the data
need to be collected under very high O2 pressure.
Secondly, there are questions about the difficulty and
accuracy of measuring the uptake of O2 , especially
during the inhibition phase when the uptake is very
low. Thirdly, in foods, antioxidant concentrations are
usually lower than the levels needed to perform this
assay, therefore the sensitivity may not be sufficient.
And lastly, the transition between inhibition and
2049

LK MacDonald, LG Wood, ML Garg

uninhibited O2 uptake may not be distinct. Burton and

Ingold used this method to successfully determine the
inhibition rate constant of the tocopherol isomers26
but it is thought that ambiguous induction period
values would result from using this method.

METHODS THAT MEASURE THE INHIBITION OF

INDUCED LIPID AUTOXIDATION
These methods induce autoxidation of linoleic acid or
low-density lipoproteins (LDL) by Cu(II) or an azo
initiator and measures the UV absorbance at 234 nm
(the Amax of conjugated diene peroxides from linoleic
acid oxidation).27 Problems arise because (1) it is
difficult to measure the small lag times that occur, and
(2) many substances in foods also absorb at 234 nm.
The reaction can occur in micelles or in organic
solvents, but measuring the absorbance in micelles
is not straightforward. Additionally, linoleic acid will
form micelles in the presence of water. This is a critical
issue, as the way an antioxidant performs between the
two phases (aqueous and lipid) is important to its
in vivo behaviour. The problem of micelle formation
by linoleic acid can be overcome by using methyl
esters, although products of methyl esters can show
inter-batch differences. Similarly, conjugated dienes
can only be formed by polyunsaturated fatty acids;
therefore it is only applicable if comparing to PUFA.
Meyer and co-workers28 used the Cu(II) induction
method to measure oxidation of LDL by the increase
in hexanal in the headspace of the reaction vessel
used for gas chromatography. Hexanal was chosen
as it is one of the major secondary by-products of
the peroxidation of n-6 fatty acids. The per cent
inhibition of hexanal production was used as a measure
of antioxidant capacity and, from this, the IC50 can
be used to compare antioxidants. Problems with this
method are due to the measurement of hexanal. It
is only one of the secondary peroxidation products,
and therefore may not be a good marker of oxidation.
Also, it has a relatively high boiling point and thus
much of the product will be in the liquid phase and
therefore not measured using this methodology. It
was found that Cu(II) is not a good initiator of LDL
autoxidation, and the reaction needed to be initiated
by the tocopherol (acting as a proxidant) present in
the LDL donating an electron to the Cu(II) and then
autoxidation proceeded.

HYDROGEN ATOM TRANSFER ASSAYS USING

MOLECULAR PROBES
Experimentally, it is difficult to directly measure the
reaction kinetics of the methods discussed so far.
Therefore more convenient methods are required to
measure antioxidant capacity. Many of the current colorimetric or fluorometric antioxidant capacity assays
provide a radical reaction without a chain propagation
step. As this step is essential for lipid autoxidation,
it is debatable whether these assays are relevant for
2050

measuring chain-breaking antioxidant capacity.29 In

general, these assays provide a steady flux of peroxyl radicals (from a radical initiator usually 2,2 azobis(2-amidinopropane)hydrochloride (AAPH)) in
air saturated solutions in the presence of a molecular
probe that fluoresces when oxidised. Added antioxidants compete with the probes for oxidation. The
difference between the following methods lies mostly
in the approach to quantification of antioxidant capacity. The ORAC assay applies the AUC, the TRAP
assay relies on lag time, and the crocin-bleaching assay
utilises initial reaction rate. The following methods
are based on the antioxidant scavenging principle of
hydrogen atom transfer.
The oxygen radical absorbance capacity
The oxygen radical absorbance capacity (ORAC) assay
has been used widely in measuring the net resultant
antioxidant capacity (or peroxyl radical absorbance
capacity) of botanical and other biological samples.
Initially, this method was developed by Cao et al.30
using B-phycoerthrin (B-PE) as the probe and the
results were expressed with reference to a known
amount of an antioxidant, Trolox (a water soluble
analogue of vitamin E). B-PE was chosen because of its
excitation and emission wavelengths, high fluorescent
yield, sensitivity to ROS, and water solubility. A
criticism of the ORAC method was the need to use
a FARA COBAS II analyser, which are not widely
available.
Eventually B-PE was replaced with fluorescein
(FL) (3 ,6 -dihydroxyspiroisobenzofuran-13H,9 ,9Hxanthen-3-one) by Ou and co-workers31 because
B-PE photobleached under plate-reader conditions,
had large inter-batch differences and interacted with
polyphenols due to non-specific protein binding and
therefore lost fluorescence even without the added
radical generator. FL overcame these problems and
was tested for precision and accuracy, ruggedness,
specificity to antioxidant action and linearity of the
relationship between the net AUC and antioxidant
concentration.28 However, FL is pH sensitive and this
must be carefully monitored.
In general, sample, control and standards (Trolox of
four or five different concentrations used to construct
a standard curve) are mixed with the FL solution and
incubated at 37 C before AAPH initiates the reaction.
The reaction is measured at 485 nm(ex)/525 nm(em)
for changes to fluorescence every minute for 35 min.
As the reaction progresses FL is consumed and
the fluorescence diminishes. In the presence of an
antioxidant the decay of FL is prevented. The data
from the assay obtained by calculating the AUC and
net AUC (AUCsample AUCblank ) and by calculating
the standard curve by plotting the concentration of
Trolox and the AUC, and also by calculating the
Trolox equivalents of a sample using the standard
curve.
The antioxidant in the sample can have a different
curve of concentration to the AUC of the Trolox
J Sci Food Agric 86:20462056 (2006)
DOI: 10.1002/jsfa

Methods for assessing in vitro antioxidant activity

standard. Forcing the samples AUC to the standard

equation of Trolox can cause the ORAC values to
become scattered; this is normally due to interference
from the sample. This is found especially when
the antioxidant capacity is low and therefore the
concentration of the sample has to be high to fit
into the range of the standard curve.
Using AUC to measure the antioxidant capacity
is advantageous as it applies both to an antioxidant
that has a lag phase and one that does not. This
allows the unification of the lag time method and the
initiator method of analysis. This is particularly useful
in foods, as there is often a mix of activities that can be
accounted for by using this method. The FL probe is
inexpensive and the use of a micro-plate reader means
the method is readily accessible.
Most of the antioxidant capacity assays, including
the ORACFL are conducted in aqueous conditions,
but many antioxidants are lipophilic, and require
lipophilic assay conditions. Therefore an ORAC based
on an organic solvent would be useful. Naguib,
cited in Huang et al.,9,32 applied 4,4-difluoro-3,5bis(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-sindacene (BO-DIPY 665/667) as a fluorescent probe
and 2,2 -azobis(2,4-dimethylvaleronitrile) (AMVN)
as the radical generator. This provided the equivalent to an ORAC in either a liposome or an
octane and butyronitrile mixture. This has allowed
the antioxidant capacity of many carotenoids to be
characterised, although it is a 100-fold less sensitive than the ORAC assay. Huang et al.32 used
cyclodextrins to increase the solubility of lipophilic
antioxidants and used this in the ORACFL assay
to determine their antioxidant capacity. This assay
was validated and found to be robust, reliable and
sensitive. It uses the FARA COBAS II and this
can be problematic as this equipment has limited
availability.
Total peroxyl radical-trapping antioxidant
parameter assay
The total peroxyl radical-trapping antioxidant parameter (TRAP) assay was introduced to measure the total
antioxidant status of human plasma. Using an azo initiator (e.g. 2,2 -diazobis-(2-amidinopropane)dihydrochloride (ABAP)), peroxyl radicals are produced at
a controlled rate. The oxidation rate is measured by
oxygen consumption during the reaction, which is
problematic as accurate measurement of oxygen consumption is difficult. The induction period, i.e. where
the oxidation is prevented by the antioxidant measured, is compared to the induction period of Trolox.33
This assay was modified to allow the peroxyl radical to
react with lipids,33 but this proved physically difficult
due to the high dilution of the plasma. A small amount
of linoleic acid was introduced to overcome the dilution problem but this introduces another source of
error.7 DeLange and Glazer34 replaced the lipid substrate with R-phycoerythrin (R-PE) as a fluorescent
probe. This method directly measures the attack of
J Sci Food Agric 86:20462056 (2006)
DOI: 10.1002/jsfa

peroxyl radical on the target molecule, not the oxygen

consumption, and is therefore less problematical.
Ghiselli et al.7 propose an assay based on the original
TRAP assay, where the ability of plasma to trap a flow
of peroxyl radicals at a constant rate is measured via
the decomposition of AAPH. The reaction progress
of R-PE and AAPH is measured fluorometrically
(ex = 495 nm and em = 575 nm). The antioxidant
capacity is expressed as Trolox equivalents (X) by the
equation
Ctrolox
X
=
Ttrolox
Tplasma
where CTrolox is Trolox concentration; TTrolox is the
lag time of the kinetic curve of R-PE in the presence
of Trolox, X is the antioxidant capacity of plasma
and Tplasma is the lag time of the kinetic curve in the
presence of plasma. X is then multiplied by 2.0 as
this is the stoichiometric factor of Trolox, and by the
dilution factor of the sample (mol L1 ).
There are some issues with this assay, primarily
when used in biological fluid. The use of plasma
versus serum is an issue, due to the instability of
antioxidants during treatment of the blood. Storage
of the sample is an issue, again due to the instability
of some of the antioxidants present in the sample.
This is true in biological and food-based systems.
Finally, the presence of proteins and uric acid can
have a significant effect. The method accounts for the
noise of proteins by having the standards measured
in the presence of the proteins as well. The authors
suggest that the concentration of uric acid should also
be measured and used as an accompanied reference
measurement.7
Crocin-bleaching assay
This assay measures the level of protection provided
to the naturally occurring carotenoid derivative crocin
from bleaching by the radical generator AAPH. The
assay is performed by preparing a 2.0 mL phosphate
buffer (0.1 mol L1 , pH 7.0) containing 10 mol L1
crocin and a known amount of antioxidant. The
reaction is initiated by the addition of AAPH (50 L;
0.5 mol L1 ) and the bleaching of the crocin becomes
linear after approximately 1 min at the wavelength
of 443 nm. Antioxidants prevent the bleaching. The
sample is monitored for 10 min. To eliminate possible
interference by the sample, blanks without crocin
should also be measured.35
This assay has only found limited use in food systems. Some of the reaction rates of the phytochemicals
are similar to crocin and therefore there is no lag phase,
whereas other chemicals will have a lag phase. At times
the reduction in bleaching is very small and the assay is
not sensitive to concentration changes in antioxidants.
Crocin absorbs at 450 nm and many food pigments
also absorb at this wavelength, although the inclusion
of a blank measurement may control for this. Crocin
is a mixture of natural food pigments from saffron and
therefore has the potential for inter-batch variation.
2051

LK MacDonald, LG Wood, ML Garg

These problems limit the reliability and utility of this

assay.

ELECTRON TRANSFER ASSAYS USING

MOLECULAR PROBES
The following assays are characterised by their ability
to undergo single electron transfer. They include the
total phenol assay, the Trolox equivalent antioxidant
capacity (TEAC) assay, the ferric ion reducing
antioxidant power (FRAP) assay, the N,N-dimethylp-phenylenediamine (DMPD) assay, and the Cu(II)
reduction capacity assay. They are based on the
following reaction and electron transfer
Probe(oxidant) + e (from antioxidant)
reduced probe + oxidised antioxidant
The probe is an oxidant, which extracts an electron
from the antioxidant, causing the probe to change
colour. The degree of colour change is proportional
to the antioxidant capacity; the change of absorbance
(A) is plotted against the antioxidant concentration,
giving a linear curve where the slope of the curve
reflects the reducing capacity (expressed as Trolox
equivalent (TE) or gallic acid equivalent (GAE)). As
there is no oxygen radical in this equation, relating
it to antioxidant capacity is very questionable in
peroxidating food or in vivo situations;3 it is only
relevant if the antioxidant action of the compound
of interest equates to reducing ability. One of the key
strengths to these assays is the speed of the reaction
and the ease of the assay.
Total-phenol assay by using the FolinCiocalteu
reagent
This assay was originally intended to analyse protein,
taking advantage of the phenol group in tyrosine.
Singleton and co-workers36 extended this assay to
measure total phenols in wine. This assay, the
Folin-Ciocalteu reagent (FCR)-based assay has since
become known as the total-phenol (phenolic) assay,
but it actually measures the reducing capacity of the
sample.
The FC reagent is not specific for phenolic
compounds as it can also be reduced by many
non-phenolic compounds. The phenolic compounds
can only react with FCR under basic conditions,
where dissociation of the phenolic proton leads to
a phenolate anion, which is capable of reducing
FCR. Thus, the assay uses an electron transfer
reaction. The stoichiometry is not always simple,
and needs to been corrected for ascorbate content.3
It is simple, reproducible and convenient, however,
and therefore has been widely used when studying
phenolic antioxidants. It is usually more correct to use
HPLC analysis to determine phenolic concentrations,
however, and this limits the quantification to one class
of phenolic compounds.3
2052

Trolox equivalent antioxidant capacity

The Trolox equivalent antioxidant capacity (TEAC)
assay was developed by Miller et al.37 and Re et al.38
and has been used widely in testing antioxidant
capacity in food samples. The original method was
based on the activation of metmyoglobin by hydrogen peroxide in the presence of 2,2 -azinobis(3-ethylbenzothiazolline-6-sulfonic acid) (ABTS). The criticism of this assay was that fast reacting antioxidants
may also reduce the ferryl myoglobin radical. In the

improved version38 ABTS , the oxidant, was generated by potassium persulfate oxidation of ABTS2 and
the radical cation is measured spectrophotometrically.
This is a direct generation of a stable form of radical prior to the reaction with antioxidants, to create

a bluegreen ABTS + chromophore. The extent of

decolourisation as percentage inhibition of ABTS + is

determined as a function of concentration and time
and calculated relative to the standard Trolox, determined under the same conditions. The solution is
then diluted until the absorbance reads 0.7 at 734 nm.
One millilitre of this solution is then mixed with the
sample, the absorbance is read at 30 C, 1, 4 and
6 min after mixing. The differences in absorbance are
plotted against the antioxidant concentrations to give
a straight line.
Due to the simplicity of this assay it has been
frequently used for studying antioxidant capacity, but
there is little correlation between the TEAC number
and the number of electrons an antioxidant can give
away. For example, the TEAC value for ascorbic
acid (1.05), -tocopherol (0.97), glutathione (1.28)
and uric acid (1.01) are quite similar even though
glutathione can normally only donate one electron,
and the others are two-electron reductants.9 The fact

that ABTS + is not found in biological systems and is

not similar to radicals found in those systems is also a
problem. A favourable point, though, is that this assay
has been used in aqueous and lipid phases and thus
can be used to determine the antioxidant capacity in
both.
Another assay that is very similar to the use of the

ABTS + , is the N,N-dimethyl-p-phenylenediamine,

or the DMPD assay. In the presence of an oxidant
or an acidic pH the DMPD is converted to a

stable and coloured DMPD radical cation (DMPD + ).

Antioxidants capable of transferring a hydrogen atom
to the radical cause the decolourisation of the solution,
which is measured spectrophotmetrically at 505 nm for
approximately 10 min. The reaction is stable and the
endpoint is taken to be the measure of antioxidant
efficiency, which is measured in Trolox equivalents
from a calibrated Trolox concentration curve. This
assay is used on hydrophilic compounds, although
organic acids may cause interference.11
Ferric ion reducing antioxidant power assay
The Ferric ion reducing antioxidant power (FRAP)
assay takes advantage of an electron transfer reaction39
in which a ferric salt is used as an oxidant. The redox
J Sci Food Agric 86:20462056 (2006)
DOI: 10.1002/jsfa

Methods for assessing in vitro antioxidant activity

potential of the Fe(III) salt (approximately 0.70 V)

is similar to ABTS and therefore there is little

difference between the FRAP and TEAC assay. The
difference is that TEAC occurs at a neutral pH whereas
FRAP needs acidic conditions (pH 3.6). This assay
was originally designed to measure the antioxidant
capacity of plasma, but has subsequently been used
with tea and wine.40 It is a measure of reducing ability,
therefore antioxidants not acting in this way will not
have FRAP values. This was demonstrated by Pulido
et al.40 when they could not determine FRAP values
for carotenoids.
Briefly, in this method 900 L of FRAP reagent
was mixed with 90 L of distilled water and 30 mL
of sample or blank (distilled water or methanol). The
final dilution of the test sample was 1/34 and readings
were at 595 nm on a spectrophotometer. Temperature
was maintained at 37 C and the sample was measured
every 15 s for up to 30 min.
Pulido et al.40 noted that there is a potential
for bias introduced by the dissolvent, there were
different values for the intercepts and the slopes on
the regression curves for the aqueous and alcoholic
Fe(II) solutions. This shows the influence of the
dissolvent redox potential. This resulted in differences
in the FRAP values for the polyphenols measured
when they were dissolved in distilled water or
methanol. Therefore care should be taken when nonaqueous samples are analysed, and comparison to the
corresponding Fe(II) solutions should be made.
Potential problems occur as the mixture contains
other Fe(III) species, which can bind to chelators
in the food extract and these complexes are capable
of reacting with the antioxidants. Results show that,
similar to TEAC, there is no relation between the
FRAP value and the number of electrons that an
antioxidant can donate.3 The FRAP value for bilirubin
is one-fold higher than vitamin C, indicating that 1 mol
of ascorbic acid can reduce 2 mol of Fe(III) and 1 mol
bilirubin can reduce 4 mol of Fe(III), although both
these antioxidants are two-electron donors. When used
to determine the antioxidant potential of polyphenols
in water and methanol,40 the change in absorbance
continued after 4 min, therefore the FRAP values for
these compounds cannot be accurately determined in
4 min.
Total antioxidant potential assay using Cu(II) as
an oxidant
This method has not been widely used or reported.
This assay is based on the reduction of Cu(II)
to Cu(I) by the antioxidants present in the sample. Cu(I) is complexed with a chromogenic
reagent, bathocuproine (2,9-dimethyl-4,7-diphenyl1,10-phenanthroline) with a maximum absorbance
at 490 nm. One mole of -tocopherol was found to
reduce 2 mol of Cu(II) to Cu(I).41
Similarly, the reducing power of herbal products was investigated using solid-phase spectrophotometry and the tetrabenzo-[b,f ,j,n][1,5,9,13]tetraazacyclohexadecineCu(II) complex immobilised
J Sci Food Agric 86:20462056 (2006)
DOI: 10.1002/jsfa

on silica gel (CuTAAB-SG),42 in a redox reaction.

This assay has one great advantage: it is very quick,
needing only 10 min per sample, and there are no
complex stages that require prolonged periods of time.
It is known that test methods using analytical reagents
immobilised on matrixes are easy to handle, and
require little cost or expertise. Studies by Zaporozhets
et al.42 have shown that this complex may be a specific
reagent for organic antioxidants.
The absorbance is measured on a UVvisible
spectrophotometer. Briefly, the method is as follows.
A portion (0.070 0.001 g) of Cu-TAAB-SG is put
in a cell in combination with one drop (40 L) of
carbonate buffer (pH 10.5 0.1) and one drop (40 L)
of antioxidant sample. The absorbance is measured at
712 and 870 nm (where 712 = Amax and 870 = Amin ).
Quercetin was used as the standard.
The results from Zaporozhets et al.42 show that this
assay has the potential to provide accurate results with
only the need to measure time and absorbance. It does
not require great expertise or the use of expensive
equipment.
2,2-diphenyl-1-picrylhydrazyl radical scavenging
capacity assay
Sanchez-Moreno11 considered that this assay is an
easy and accurate method for use in fruit and
vegetable juice extracts. It is one of the few stable and
commercially available organic nitrogen radical assays.
Originally, it was monitored by ESR spectroscopy and
relied on the signal intensity of the 2,2-diphenyl-1
picrylhydrazyl radical (DPPH ) being inversely related
to the antioxidant concentration and the reaction time.
More recently, this reaction has been measured by the
decoloration assay where the decrease in absorbance
at 515528 nm produced by the addition of the

antioxidant to the DPPH in methanol or ethanol

is measured. This assay is not suitable for measuring
the antioxidant capacity of plasma as proteins are
precipitated in the presence of the ethanol/methanol
solvent used.11
Using this method the efficiency and the EC50
of an antioxidant can be measured. The method is
technically simple; for example, the DPPH solution
(3.9 mL, 25 mg L1 ) in methanol is mixed with
sample solution (0.1 ml), and then monitored at
515 nm for 30 min or until the colour change is
stable. Despite the simplicity of the method, there
are some disadvantages: the DPPH molecule is a
long-lived nitrogen radical and has little similarity
to the reactive peroxyl radicals involved in lipid
peroxidation. Also, antioxidants that may react
quickly to the in vivo peroxyl radicals may react
slowly with DPPH. The reaction kinetics between
DPPH and antioxidants is not linear to DPPH
concentration, therefore measurement of the EC50
is problematic.
Originally, it was believed that the DPPH assay
was a hydrogen transfer reaction but recent work
by Foti and co-workers43 suggests that it is, in fact,
2053

LK MacDonald, LG Wood, ML Garg

Table 1. Methods for assessing antioxidant capacity compared by key criteria

Principle
ROS/RNS scavenging

Hydrogen atom transfer

Electron transfer

Examples

Superoxide
Hydrogen peroxide
Hydroxyl
Singlet oxygen, Peroxynitrite

ORAC
TRAP
Crocin
LDL oxidation

Biologically relevant?
Simple to measure?
Instrumentation readily available?
Reproducible?
Suitable for hydrophilic and lipophilic antioxidants?

No
No
No
Undetermined
No

Yes
No (except ORAC)
Yes (except TRAP and ORAC)
Undetermined
No (except ORAC)

Total phenols
TEAC
FRAP
DMPD
Cu(II) reduction
DPPH
No
Yes
Yes
Undetermined
No (except TEAC)

an electron transfer reaction. The initial electron

transfer occurs very quickly, and the subsequent
hydrogen transfer occurs more slowly and depends on
the hydrogen-bond accepting solvent. For example,
methanol and ethanol, which many assays use as
solvents for antioxidant capacity assays, are strong
hydrogen-bond accepting solvents and therefore the
hydrogen abstracting reaction occurs very slowly.
Similarly, the acids or bases present in the solvent
may influence the ionisation equilibrium of phenols
and therefore cause a reduction or enhancement of
the measured rate constants. This means that the
rate of the reaction can be dramatically increased or
decreased by basic and acidic impurities, respectively,
in the solvent, therefore suggesting that these aspects
affect whether a reaction involves electron transfer or
hydrogen atom transfer.43

SUMMARY
The various methods of assessing antioxidant capacity
of foods are summarised in Table 1. Electron transfer
assays measure the reducing ability of the substrate
(antioxidant); hydrogen transfer assays measure the
hydrogen donating ability of the substrate. It is
clear that hydrogen atom donation is essential in
the radical chain reaction stage of lipid peroxidation,
therefore hydrogen transfer assays are relevant to the
measurement of chain-breaking antioxidant capacity.
It is clear that in many cases the antioxidant capacity,
or the ability to trap radicals, of a compound is related
to the ease of hydrogen atom donation, not necessarily
the redox potential of the compound.
Therefore, electron transfer, or reducing power
assays, are not as relevant to antioxidant capacity
in vivo and are more difficult to justify when looking for
the antioxidant potential of new or novel compounds
found in foods, although in the case of some oxidants
such as peroxynitrite and hypochlorite, reduction
can render these compounds harmless and therefore
reducing power has relevance in a few isolated cases.
In general, assays that measure hydrogen atom transfer
would be preferable to assays that measure electron
transfer reactions.
2054

The use of multiple electron transfer assays is

redundant. When papers cite the use of multiple
methods the correlations between them is excellent,
largely because they rely on the same redox reactions.
When deciding to use an assay to measure reducing
ability it is important to choose one that is widely
reported and validated.
Similarly, for assays that measure hydrogen atom
transfer Huang et al.9 recommend the use of the
ORAC assay. This is largely due to the number of
laboratories using this method and the comparable
data available. This assay has been extensively
validated, and there can be confidence that it is
measuring the hydrogen atom transfer ability of the
sample and will provide useful information about the
radical chain-breaking capacity of the sample. Also, the
ORAC assay has been modified for lipophilic sample
testing, which increases its usefulness. However,
corroborating evidence of the ability of the assay to
predict the efficacy of the antioxidant action in food
and biological samples is needed.9
Experimentally, it is difficult to distinguish between
hydrogen atom transfer and electron transfer, and
often the two are mistaken. The pH of the solvent
is particularly important in electron transfer assays
under acidic conditions the reducing capacity may be
suppressed; under basic conditions proton dissociation
of phenolic compounds would enhance the reducing
capacity of a compound.9 Solvent effects should always
be monitored, as the type of solvent can affect the
scavenging rate constants which are dependent on
the solvent and through this affect the measure of an
antioxidants efficiency.3 Therefore careful monitoring
of all aspects of the assay conditions is essential for
the successful use of these assays, and comparisons
of antioxidant capacity of foods tested under varying
conditions is not appropriate.
The homogenous nature of the in vitro assays
also present challenges to the relevance of these
molecules as antioxidants. It remains to be proven
that the antioxidant capacity in a heterogenous food
or biological system reflects the results shown in
an in vitro assay. While appropriate in vitro data is
undeniably valid and can be used to inform in vivo
J Sci Food Agric 86:20462056 (2006)
DOI: 10.1002/jsfa

Methods for assessing in vitro antioxidant activity

and clinical studies, it is difficult to extrapolate from

an in vitro assay to an in vivo situation, and supporting
evidence of in vivo antioxidant capacity should also be
provided.7 9,11
It is important to use samples, antioxidants or
oxidants in physiologically relevant concentrations. In
the hydrogen atom transfer assays using fluorescent
probes the process is similar to lipid peroxidation,
but the concentration of substrate (i.e. probe) is often
lower than the concentration of antioxidant. This is
not the case in food and biological systems: in food
systems the antioxidant is in much lower concentration
that the oxidisable substrate. In order to reflect the
antioxidant potential of a molecule, it must be tested
using relevant concentrations.
The assays described in this review are based on
chemical reactions in vitro and therefore to extrapolate
to biological systems is impossible. These assays do not
take into account the bioavailability, in vivo stability,
in vivo storage patterns by tissues and the reactivity
of the sample matrix in vivo, nor do they reflect the
well documented synergistic effects of antioxidants.
Therefore unless the in vitro evidence is supported
by in vivo evidence it is questionable whether there
will be in vivo efficacy based on these methodologies.
It is clear from the existing data that some in vitro
antioxidants are powerful in vivo pro-oxidants. Aside
from this there are some very complex chemical
matrices in food and biological systems that cannot
be predicted, especially when, in vivo, the antioxidants
will need to perform in both aqueous and lipid phases.
When using assays to measure antioxidant capacity,
several parameters are manipulated to accelerate
oxidation. These oxidative stress conditions induced
by various agonists used in the in vitro assays do
not reflect the oxidative stress present in the body
(in vivo) which is a combination of not only the six
ROS described in this paper but others as well. For
example, accelerated testing by increasing temperature
presents various problems, not least of which is the
invalid extrapolation of reaction kinetics between the
temperature changes.8 Similar problems arise when
using transition metal ion catalysts and azo initiators,
resulting in misinterpretation of the data. Therefore a
battery of relevant tests, measuring different aspects
of the behaviour of antioxidants is required to truly
assess the relevance of a compound in vivo.
Lastly, it may be necessary to use a variety of
methods and a variety of conditions in order to
correctly describe the in vitro antioxidant capacity
of a compound. It is necessary to support in vitro
data with in vivo assay data, to truly describe the
likelihood of in vivo antioxidant capacity if this is the
desirable endpoint. Similarly, it is difficult to compare
the results from studies that all use separate and noncomparable methods. Therefore it is advantageous
to select methods that are validated, standardised
and widely reported. Misunderstanding what these
methods are measuring has led to misrepresentation
and misinterpretation of data, which has complicated
J Sci Food Agric 86:20462056 (2006)
DOI: 10.1002/jsfa

the measurement of antioxidant capacity in vitro.

Finally, it is important to use concentrations of
reactants in assays that are physiologically relevant.
While it is difficult to accurately mimic in vivo
heterogenous conditions, one-dimensional and overly
simplistic homogenous in vitro assays do not aid the
search for compounds in the food supply that will act
as antioxidants in vivo. Antioxidant action in foods is
important as the availability of toxic material in the
food supply is reduced, but the role in food and the
role in vivo are separate issues and the researcher needs
to be aware of the aim of the research when choosing
assays to measure antioxidant capacity of foods.

REFERENCES
1 Rice-Evans CA and Diplock AT, Current status of antioxidant
therapy. Free Rad Biol Med 15:7797 (1992).
2 Chipault JR, Antioxidants for Use in Foods, vol II, ed. by
Lundberg W. New York: Interscience (1962).
3 Becker EM, Nissen LR and Skibsted LH, Antioxidant evaluation protocols: Food quality or health effects. Eur Food Res
Technol 219:561571 (2004).
4 Halliwell B, How to characterize a biological antioxidant. Free
Rad Res Commun 9:132 (1990).
5 Halliwell B, Antioxidants: The basics what they are and how
to evaluate them. Adv Pharmacol 38:320 (1997).
6 Scott G, Antioxidants in Science, Technology, Medicine and
Nutrition. Chichester: Albion Publishing (1997).
7 Ghiselli A, Serafini M, Natella F and Saccini C, Total antioxidant capacity as a tool to assess redox status: Critical view and
experimental data. Free Rad Biol Med 29:11061114 (2000).
8 Frankel EN and Meyer AS, Review: The problems of using
one-dimensional methods to evaluate multifunctional food
and biological antioxidants. J Sci Food Agric 80:19251941
(2000).
9 Huang D, Ou B and Prior RL, The chemistry behind antioxidant capacity assays. J Agric Food Chem 53:18411856
(2005).
10 Bull C, McClune GJ and Free JA, The mechanism of FeEDTA catalyzed superoxide dismutation. J Am Chem Soc
105:52905300 (1983).
11 Sanchez-Mareno C, Review: Methods used to evaluate the free
radical scavenging activity in foods and biological systems.
Food Sci Technol Intern 8:121137 (2002).
12 Quick KL, Hardt JI and Dugan LL, Rapid microplate assay
for superoxide scavenging efficiency. J Neurosci Methods
97:138144 (2000).
13 Martinez-Tome M, Carcia-Carmona F and Murcia MA, Comparison of the antioxidants and pro-oxidants activities of
broccoli amino acids with those of common food additives.
J Sci Food Agric 81:10191026 (2001).
14 Mueller S, Sensitive and nonenzymatic measurement of hydrogen peroxide in biological systems. Free Rad Biol Med
29:410415 (2000).
15 Bielski BHJ, Reactivity of HO2 /O2 radicals in aqueous
solution. J Phys Chem Reference Data 14:10411100 (1985).
16 Finkelstein E, Rosen GM and Rauckman EJ, Spin trapping:
Kinetics of the reaction of superoxide and hydroxyl radicals
with nitrones. J Am Chem Soc 102:49944999 (1980).
17 Halliwell B, Gutteridge JM and Aruoma OI, The deoxyribose
method: a simple test tube assay for determination of rate
constants for reactions of hydroxyl radicals. Anal Biochem
165:215219 (1987).
18 Zhu BZ, Kitrosky N and Chevion M, Evidence of production of hydroxyl radicals by pentachlorophenol metabolites
and hydrogen peroxide. A metal-independent organic Fenton reaction. Biochem Biophys Res Commun 270:942946
(2000).
2055

LK MacDonald, LG Wood, ML Garg

19 Ou B, Hampsch-Woodhill M, Flanagan J, Deemer EK, Prior RL
and Huang D, Novel fluorometric assay for hydroxyl radical
prevention capacity using fluorescein as the probe. J Agric
Food Chem 50:27722777 (2002).
20 Wilkinson F, Helman WP and Ross AB, Rate constants for the
decay and reactions of the lowest electronically excited singlet
state of molecular oxygen in solution. An expanded and
revised compilation. J Phys Chem Reference Data 24:6631021
(1995).
21 Fu YL, Krasnovsky AA and Foote CS, Quenching of singlet
oxygen and sensitized delayed phthalocyanine fluorescence.
J Phys Chem 101:25522554 (1997).
22 Pryor WA and Sqadrito GL, The chemistry of peroxynitrite: a
product from the reaction of NO and O2 . Am J Physiol
268:L699 (1995).
23 Pannala A, Razaq R, Halliwell B, Singh S and Rice-Evans CA,
Inhibition of peroxynitrite dependent typrosine nitration by
hydroxycinnamates: Nitration or electron donation? Free Rad
Biol Med 24:594606 (1998).
24 Kooy NW, Royall JA, Ischiropoulos H and Beckman JS,
Peroxynitrite-mediated oxidation of dehydrorhodamine 123.
Free Rad Biol Med 16:149156 (1994).
25 Chung HY, Choi HR, Park HJ, Choi JS and Choi WC,
Peroxynitrite scavenging and cytoprotective activity of
2,3,6-tribromo-4,5-dihydroxybenzylmethylether from the
marine alga Symphyocladia latiuscula. J Agric Food Chem
46:36143621 (2001).
26 Burton GW and Ingold KU, Autoxidation of biological
molecules. 1. The autoxidation of vitamin E and related chain
breaking antioxidants in vitro. J Am Chem Soc 103:64726477
(1981).
27 Pryor WA, Cornicelli JA, Devall LJ, Tait B, Trivedi BK,
Witiak DT, et al, A rapid screening test to determine the
antioxidants potencies of natural and synthetic antioxidants.
J Org Chem 58:35213532 (1993).
28 Meyer AS, Yi OS, Pearson DS, Waterhouse AL and Frankel EN,
Inhibition of low density lipoprotein oxidation in relation to
composition of phenolic antioxidants in grape (Vitis vinifera).
J Agric Food Chem 45:16381643 (1997).
29 Antunes F, Barclay RC, Ingold KU, King M, Norris J, Scaiano JC, et al, On the antioxidant activity of melatonin. Free
Rad Biol Med 26:117128 (1999).
30 Cao GH, Alessio H and Cutler RG, Oxygen-radical absorbency
capacity assay for antioxidants. Free Rad Biol Med 14:303311
(1993).
31 Ou B, Hampsch-Woodhill M and Prior RL, Development and
validation of an improved oxygen radical absorbance capacity
assay using fluorescein as the fluorescent probe. J Agric Food
Chem 49:46194626 (2001).

2056

32 Huang D, Ou B, Hampsch-Woodhill M, Flanagan J and

Deemer EK, Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using
randomly methylated cyclodextrin as the solubility enhancer.
J Agric Food Chem 50:18151821 (2002).
33 Wayner DDM, Burton GW, Ingold KU and Locke S, Quantitative measurement of the total peroxyl radical-trapping
antioxidant capability of human blood plasma by controlled
peroxidation. FEBS 187:3337 (1985).
34 DeLange RJ and Glazer AN, Phycoerythrin fluorescencebased assay for peroxyl radicals: a screen for biologically
relevant protective agents. Anal Biochem 177:300306
(1989).
35 Bors W, Michel C and Saran M, Inhibition of bleaching of the
carotenoid crotin, a rapid test for quantifying antioxidant
activity. Biochim Biophys Acta 796:312319 (1984).
36 Singleton VL, Orthofer R and Lamuela-Ravenos RM, Analysis
of total phenols and other oxdiation substrates and
antioxidants by means of FolinCiocalteu reagent. Methods
Enzymol 299:1527 (1999).
37 Miller NJ, Rice-Evans CA, Davies MJ, Gopinathan V and
Milner AA, A novel method for measuring antioxidant
capacity and its application to monitoring antioxidant status
in premature neonates. Clin Sci 84:407412 (1993).
38 Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and
Rice-Evans CA, Antioxidant activity applying an improved
ABTS radical cation decolorization assay. Free Rad Biol Med
26:12311237 (1999).
39 Benzie IFF and Strain JJ, The ferric reducing ability of plasma
as a measure of antioxidant power: the FRAP assay. Anal
Biochem 239:7076 (1996).
40 Pulido R, Bravo L and Saura-Calixo F, Antioxidant activity
of dietary polyphenols as determined by a modified ferric
reducing/antioxidant power assay. J Agric Food Chem
48:33963402 (2000).
41 Yamashita N, Murata M, Inoue S, Burkitt MJ, Milne L and
Kawanishi S, -tocopherol induces oxidative damage to
DNA in the presence of copper(II) ions. Chem Res Toxicol
11:855862 (1998).
42 Zaporozhets OA, Krushynska OA, Lipkovska NA and
Barvinchenko VN, A new test method for the evaluation
of total antioxidant activity of herbal products. J Agric Food
Chem 52:2125 (2004).
43 Foti MC, Daquino C and Geraci C, Electron-transfer reaction
of cinnamic acids and their methyl esters with the DPPH
radical in alcoholic solutions. J Org Chem 69:23092314
(2004).

J Sci Food Agric 86:20462056 (2006)

DOI: 10.1002/jsfa