Journal of Functional Foods 18 (2015) 782796

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Reprint of Hurdles and pitfalls in measuring

antioxidant efficacy: A critical evaluation of
ABTS, DPPH, and ORAC assays

K.M. Schaich *, X. Tian, J. Xie

Department of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901-8520, USA

A R T I C L E I N F O A B S T R A C T

Article history: Assays developed to measure radical scavenging ability of natural compounds have been
Available online 12 June 2015 used as a basis for ranking and recommending best foods for consumption. However, assays
often were adapted for screening assays with inadequate consideration of reaction chem-
Keywords: istry, particularly kinetics. Recent research results raise serious questions about the chemistry,
Antioxidant efficacy assays execution, and application of these assays. This paper critically evaluates conceptual and
Limitations technical issues that limit use and compromise validity of three commonly-used assays
TEAC trolox equivalent TEAC/ABTS+, DPPH, and ORAC. Recommendations are made for discontinuing use of ABTS+
antioxidant capacity assay and DPPH radicals for measuring radical quenching, redirecting them instead to distin-
ABTS+ 2,2-azino-bis(3- guishing electron transfer reaction mechanisms. Conditions required for accurate results
ethylbenzothiazoline-6-sulfonic in ORAC are reviewed, and recommendations are made for redirecting this assay to distin-
acid) assay guishing compounds that quench radicals by hydrogen atom transfer. The mechanistic
ORAC oxygen radical absorbance information so gained can be then applied to understand how natural antioxidants can be
capacity assay used most effectively in foods.
Radical quenching kinetics 2015 Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...................................................................................................................................................................................... 783

2. Hurdles and pitfalls in three common antioxidant assays ....................................................................................................... 784
2.1. TEAC/ABTS+ trolox equivalent antioxidant capacity/2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay . 784
2.1.1. TEAC I ........................................................................................................................................................................... 785
2.1.2. TEAC II .......................................................................................................................................................................... 785
2.1.3. Limitations of the TEAC/ABTS+ assay .................................................................................................................... 785
2.1.4. Summary and recommendations TEAC/ABTS+ assay ...................................................................................... 787
2.2. DPPH (2,2-diphenylpicryl hydrazyl free radical) assay ................................................................................................... 787
2.2.1. Limitations of the DPPH assay ................................................................................................................................. 787
2.2.2. Summary and recommendations DPPH assay ................................................................................................... 789

* Corresponding author. Department of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901-8520, USA. Tel.: +1 848
932 5454; fax: +1 732 932 6776.
E-mail address: schaich@aesop.rutgers.edu (K.M. Schaich).
Abbreviations: SET, single electron transfer; HAT, hydrogen atom transfer; TEAC, Trolox equivalent antioxidant capacity; ABTS+, 2,2-
azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); ORAC, Oxygen Radical Absorbance Capacity; AAPH, 2,2-azobis(2-amidinopropane)
dihydrochloride; IU, International Units; CuPRAC, Cupric ion reducing antioxidant capacity; FRAP, Ferric reducing antioxidant power
http://dx.doi.org/10.1016/j.jff.2015.05.024
1756-4646/ 2015 Elsevier Ltd. All rights reserved.
 Journal of Functional Foods 18 (2015) 782796 783

2.3. ORAC oxygen radical absorbance capacity .................................................................................................................... 790

2.3.1. Advantages of the ORAC assay ................................................................................................................................ 790
2.3.2. Limitations of the ORAC assay ................................................................................................................................. 790
2.3.3. Summary and recommendations ORAC assay ................................................................................................... 793
3. Summary and recommendations ................................................................................................................................................. 794
References ......................................................................................................................................................................................... 794

vivo. Most antioxidants are poorly absorbed or are rapidly con-

1. Introduction
Recognition of potential health effects of antioxidants more
than twenty years ago stimulated what could be called the An-
tioxidant Bandwagon of research seeking to determine which
natural materials contain the highest levels of the most active
antioxidants, addition of antioxidants to beverages and all forms
of foods, prophylactic and therapeutic medical applications,
and hyper marketing. At the same time, numerous in vitro an-
tioxidant assays were developed to measure radical scavenging
and to compare and rank antioxidants in different foods and
natural materials. Antioxidant assays have measured three
values:
antioxidant capacity the total number of electrons donated
or target molecules converted per mol of antioxidant at full
reaction under given conditions. This usually approxi-
mates the number of phenolic OH groups, or two electrons
per OH group, though not always. Unfortunately, the re-
quirement for full reaction ignores reaction rate and creates
a kind of tortoise and hare situation where slow react-
ing antioxidants with many phenol groups receive highest
rankings while fast reacting antioxidants with few phenol
groups can be greatly underestimated or even overlooked.
antioxidant activity the antioxidant concentration re-
quired to provide a specified rate or extent of reaction
antioxidant potential a nebulous general term used to de-
scribe the expectation that an antioxidant can quench
radicals under specific conditions. This term can easily be
confused with thermodynamic potential, so its applica-
tion to antioxidant assays has been controversial.
Assays based on these measures have been applied so ex-
tensively to evaluation of fruits, vegetables, leaves and stems,
herbs and spices that TEAC, DPPH, ORAC, CuPRAC, and FRAP
are now household words in scientific publications, health food
publications, and internet websites. Thousands of research
papers have been published comparing values of different foods,
and these results have provided the basis for recommending
the best foods to eat, for labeling Super Foods, and for mar-
keting products.
Despite extensive use of in vitro antioxidant assays to dis-
tinguish which antioxidants and foods are most efficacious,
time has shown that the assays have both conceptual and tech-
nical limitations (Apak et al., 2013). One major conceptual
limitation is that, although antioxidant assays were designed
to identify which antioxidants should be expected to provide
greatest protective effects against free radicals in vivo, the radical
scavenging observed in test tubes probably does not occur in
jugated and eliminated in the urine, so circulating phenol
concentrations reach trace levels at best. It is unlikely that such
minute amounts can mediate physiological actions of anti-
oxidants merely by scavenging radicals. Reviews of the
antioxidant literature suggest that microbial or physiological
metabolites of antioxidants may actually be more active in vivo
(Frankel & Finley, 2008), but such metabolites remain largely
unidentified and they are not the compounds tested in in vitro
assays. Cell culture assays attempt at least in part to include
effects of metabolites, but most cell types used in assay cul-
tures never see antioxidants in vivo. Phenols bind readily to
proteins in cells and foods, so they may be inactivated for clas-
sical radical quenching or activated for some other physiological
action not tested in the assays. Finally, original assumptions
regarding radical scavenging being the mode of antioxidant
action in vivo may well be erroneous in light of new evidence
regarding signal transduction and regulation of gene expres-
sion of redox controlling and detoxifying enzymes (Frankel,
2007; Halliwell, Rafter, & Jenner, 2005). Indeed, the U.S. De-
partment of Agriculture recently withdrew the compiled tables
of ORAC values of many foods, herbs, and spices which had
been posted on their website for some time, leaving behind
the following statement (USDA, 2010):
There is no evidence that the beneficial effects of
polyphenol-rich foods can be attributed to the antioxi-
dant properties of these foods. The data for antioxidant
capacity of foods generated by in vitro (test-tube) methods
cannot be extrapolated to in vivo (human) effects and the
clinical trials to test benefits of dietary antioxidants have
produced mixed results. We know now that antioxidant mol-
ecules in food have a wide range of functions, many of which
are unrelated to the ability to absorb free radicals. For these
reasons the ORAC table, previously available on this web site
has been withdrawn.
A second conceptual limitation is that the chemistry and
molecular targets of most in vitro assays are not relevant to in
vivo conditions. Antioxidant concentrations used in these assays
are orders of magnitude higher than would ever be found in
vivo, the assays use sterically-hindered stable radicals (e.g.
ABTS+ or DPPH) as targets rather than small, readily acces-
sible but short-lived radicals such as HO, O2, or lipid oxyl
radicals that are active in vivo, and they follow reaction times
much longer than radical lifetimes in vivo (ranging from 10 s
for peroxyl radicals to 109 s for hydroxyl radicals). If an anti-
oxidant requires many minutes to hours to quench radicals,
its action as a radical scavenger must be irrelevant in vivo in
cells or even in situ in foods.
784 Journal of Functional Foods 18 (2015) 782796
A third conceptual limitation is that the assays in common
use do not address radical reactions in lipids, when lipids are
clearly involved in oxidation of both membranes and foods.
A few lipophilic versions of assays have been developed (Alcolea,
Cano, Acosta, & Arnao, 2003; Arnao, Cano, & Acosta, 2001;
Huang, Ou, Hampsch-Woodill, Flanagan, & Deemer, 2002;
Jimenez-Alvarez et al., 2008; Prior et al., 2003), but these ap-
proaches mostly solubilize lipophilic antioxidants for reaction
in aqueous phase rather than testing reactions in lipid phases.
As a result, relatively little information is available about
how natural antioxidants partition into and interact with
lipids.
Technical limitations are no less problematic. First and fore-
most, the assays have unclear chemistry for quantitative
analyses. They were developed for rapid screening without de-
tailed investigations of underlying initiators, targets, antioxidant
interactions, kinetics, solvent effects, concentration effects, etc.
Lacking full understanding of the chemistry, control issues such
as oxygen (dissolved and atmospheric), light, temperature,
reagent concentrations, solvent, pH, and running blanks are
routinely ignored. Most assays measure stoichiometry (capac-
ity) rather than kinetics of antioxidant reactions, i.e. how much
reacts rather than how fast. Ignoring the chemistry leads to
incorrect assessment of antioxidant activity, both quantita-
tively and qualitatively. In addition, each laboratory has adapted
procedures to work with instrumentation and capabilities avail-
able. As a result, there is currently almost total lack of
standardization in experimental procedures in the assays, par-
ticularly in expressing results where methods are quite varied
and often meaningless in terms of action in situ. Finally, most
screening assays and natural antioxidants are water-based
and ignore lipid phases.
After more than twenty years of antioxidant assays, it is clear
that fruits and vegetables, herbs, and spices have antioxi-
dants and that antioxidant activity generally correlates with
content of total phenols in natural products. What remains
unclear is which fruits and vegetables are best, whether and
how antioxidants interact with each other in assays and with
other compounds in vivo and in foods, and how in vitro assay
results correlate with multiple in situ actions of antioxidants
in foods and biological tissues. Thus, when antioxidant assays
are used for screening, results must be taken with a prover-
bial grain of salt.
Recent attempts to standardize assays have raised new ques-
tions about reliability and appropriate applications of
antioxidant assays:
1. Do the assays accurately reflect antioxidant chemistry
occurring in vivo? Increasing evidence suggests that radical
scavenging may occur in the gastro-intestinal tract but
other actions of antioxidants may be even more impor-
tant there and systemically. Thus, there are critical
needs to move beyond these simplistic assays to find ac-
curate models that test signaling processes of antioxidants,
to conduct detailed pharmacokinetic analyses in animals
where do antioxidants go, what do they do, and how do
they break down? and to identify other actions of
antioxidants.
2. Do the assays accurately reflect radical quenching capabil-
ity of antioxidants with different structures or in mixtures?
3. How can the assays be used to predict effectiveness or op-
timize usage of natural antioxidants in foods?
This critical evaluation focuses on Question 2, with em-
phasis on ABTS+, DPPH, and ORAC assays.
2. Hurdles and pitfalls in three common
antioxidant assays
Radicals are most commonly quenched by two mechanisms,
transfer of either a hydrogen atom or an electron to convert
the radical to a stable species:
HAT hydrogen atom transfer (H atom transferred to
target radical, possible secondary quenching by radical
recombinations)
ROO + AH PheOH ROOH + A Phe-O (1)
ROO + A ROOA (2)
SET single electron transfer (one or more electrons trans-
ferred to reduce target compounds)
ROO + AH PheOH
+ H2O (3)
ROO + AH+ PheOH+ A Phe-O + H3O+
ROO + H3O+ ROOH + H2O (4)
Ascorbic acid ( AH2 ) O2
O2
+ AH A + O2
H2O2 2 HO
O2
(5)
AH + O2 O2
where AH = any antioxidant with donatable H, PheOH = phenol
or polyphenol, M = redox-active metal.
While the end result of the two mechanisms may be the
same, kinetics and dependence on system conditions, espe-
cially solvent and pH, vary tremendously. Electron transfer is
very fast (femtoseconds) (Ganapathi, Hermann, Naumov, &
Brede, 2000), it is not diffusion-controlled, and it increases with
pH as ionization increases availability of electrons. Hydrogen
atom transfer, in contrast, is considerably slowed by diffu-
sion; it is independent of pH but strongly enhanced by water
and inhibited by hydrogen bonding solvents such as alco-
hols. These differences actually have critical impacts on how
antioxidants act in complex, multiphase systems.
Effects of electron versus hydrogen atom transfer can be
seen in the three most commonly-used assays.
2.1. TEAC/ABTS+ trolox equivalent antioxidant
capacity/2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) assay
TEAC measures antioxidant capacity as the ability of test an-
tioxidants (AH) to decrease ABTS+ color by (a) intercepting initial
oxidation and preventing ABTS+ production, or (b) reacting di-
rectly with the ABTS+. Two versions of the assay are in use,
based on these two approaches.
 Journal of Functional Foods 18 (2015) 782796
2.1.1. TEAC I
TEAC 1 utilizes metmyoglobin-H2O2 to generate hydroxyl radi-
cals, which oxidize ABTS to its colored free radical form, ABTS+
(Rxn 1a); subsequent reaction with antioxidants (AH) results
in loss of the green color (Rxn 1b) (Miller & Rice-Evans, 1997;
Miller, Rice-Evans, Davies, Gopinathan, & Milner, 1993).
This version is available in commercial kits. However, the
reaction is ambiguous because antioxidants (AH) can react with
the original HO radical oxidant, the metmyoglobin, and the
ABTS+, causing an overestimation of antioxidant activity
(Strube, Haenen, van den Berg, & Bast, 1997). Furthermore, the
antioxidant reaction mechanism cannot be determined. Are
different mechanisms active with each radical, e.g. HAT with
HO and SET with ABTS+? Do active mechanisms vary with
phenol structure? It is not possible to accurately compare an-
tioxidants without this information.
2.1.2. TEAC II
To circumvent the problems of double reaction, ABTS+ can be
generated directly in high yield using potassium persulfate as
the oxidizing agent (Re et al., 1999). Antioxidants then react
only with ABTS+ and color is diminished by only one reac-
tion (Rxn 7):
For reaction, aliquots of ABTS+ solution are diluted to an
absorbance of ~1.0 at 734 nm, which is recorded as the start-
ing point. The antioxidant is added and mixed, and the drop
in absorbance (A0Af) is typically measured after reaction periods
varying from minutes to hours. Antioxidant action is re-
ported as Trolox equivalents by comparing A0Af of the test
antioxidant to A0Af of Trolox standards, or the antioxidant con-
centration that gives the same response as 1 mM Trolox (Rxn
8):
(A 0 A f )sample blank
TEAC ( ABTS) = (8)
(A 0 A f )1 mM Trolox blank
2.1.3. Limitations of the TEAC/ABTS+ assay
Despite the extensive use of the ABTS+ reaction to screen an-
tioxidant activity in a wide range of materials, serious flaws in
design and performance compromise the usefulness and even
the validity of this assay. First, the most important chemistry
in this reaction the antioxidant reaction rate and kinetic pattern
785
is totally ignored. The absorbance drop (A0Af) upon which
TEAC calculations are based provides only reaction stoichi-
ometry (total mols ABTS+ reduced per mol antioxidant after
full reaction). Usually two electrons can be donated per phenol
OH group, so antioxidant capacity as measured in TEAC should
be closely predictable from antioxidant structure. Indeed, under
these conditions, polyphenols are highly favored and other struc-
tural effects on antioxidant reactivity are obscured. TEAC
correlation with number of phenol groups have been claimed,
but we found the relation held primarily within common struc-
tural groups and that other factors have stronger control over
antioxidant reaction with ABTS+ (Tian & Schaich, 2013).
Recording the ABTS+ absorbance continuously during reac-
tion rather than at the beginning and the end generates curves
from which reaction kinetics can be calculated (Fig. 1). These
curves also reveal marked differences between antioxidant be-
haviors that reflect whether SET or HAT reaction mechanisms
are dominant. Some antioxidants react completely in less than
mixing time, some react slowly and gradually, and others have
mixed fast and slow initial reactions (Tian & Schaich, 2013).These
differences in antioxidant reaction rates are critical because they
reflect ability to quench short-lived reactive radicals that are
present in biological systems and in foods. Such capability is
missed entirely in normal assay procedures.
What molecular properties account for differences in initial
reaction rates and patterns? Only key points will be reviewed
here; further details are available in Tian and Schaich (2013).
Two factors control reaction rates and shapes of response
curves: SET vs HAT mechanisms and steric access to the hin-
dered ABTS+ radical site. Antioxidants acting by electron
transfer and having full access to ABTS+ radical site react within
milliseconds. Reactions are slowed by the presence of mul-
tiple OH groups and rings, bulky ring adducts, high
antioxidant concentrations, and hydrogen atom transfer. Thus,
almost instantaneous initial reaction (Group 1, Fig. 1) arises from
electron transfer. The reaction is so fast that accurate results
can be obtained only with rapid mixing methods such as
autodispensing plate readers or stopped-flow mixers, which
take the assay out of routine laboratory capabilities. Contin-
ued reaction after rapid initial absorbance drop (Group 2, Fig. 1)
results from phenol structure (ring adducts) that interferes with
access to ABTS+. Smaller initial absorbance drop with contin-
ued reaction afterwards (Group 3, Fig. 1) is still mostly electron
transfer, but now the phenol has bulky side groups or mul-
tiple rings that impede diffusion and orientation toward ABTS+;
these compounds may also act partially by slower hydrogen
atom transfer. No initial fast reaction but gradual drop from
starting absorbance (Group 4, Fig. 1) arises from dominant HAT.
Critical controlling effects of steric accessibility are shown
quite dramatically in effects of antioxidant concentration on
reaction rate (Fig. 2). Small molecules and single phenols reduce
~1:1 ABTS+ per phenolic OH and reaction response remains
linear as antioxidant concentration increases (Group 1). However,
the reaction becomes increasingly impeded at higher antioxi-
dant concentrations as the structural complexity and size of
the phenol increases because the additional ring adducts (Group
2, Group 3-simple phenols) and especially secondary rings (Group
3-polyphenols) interfere with phenol access to the hindered
ABTS+. As a result, rates slow and mols ABTS+ reduced/mol
antioxidant and per OH group decrease, particularly for
786 Journal of Functional Foods 18 (2015) 782796

1.4 Group 1, inst & complete, SET mM 1.4 Group 2, very fast, SET 1.4 Group 3, fast SET, some steric
AOX hindrance or HAT
1.2 0 1.2 1.2
Absorbance, 734 nm

0.001
1.0 0.005 1.0 1.0
0.05
0.8 0.10 0.8 0.8
0.6 0.25 0.6 0.6
0.4 0.50 0.4 0.4
0.2 0.2 0.2
0.0 0.0 0.0
0 2 4 6 0 2 4 6 0 2 4 6
Reaction time (min) Reaction time (min) Reaction time (min)

1.4 Group 4, hindered SET, HAT, or mixed 1.4 Group 5, HAT at low conc, SET at high Group 6, unreactive
1.4
1.2 1.2 1.2
Absorbance, 734 nm

1.0 1.0 1.0

0.8 0.8 0.8
0.6 0.6 0.6
0.4 0.4 0.4
0.2 0.2 0.2
0.0 0.0 0.0
0 2 4 6 0 2 4 6 0 2 4 6
Reaction time (min) Reaction time (min) Reaction time (min)

Fig. 1 Kinetic response curves showing different types of reactivity of phenolic compounds with ABTS+. Adapted from
Tian and Schaich (2013), used with permission.

polyphenols where reaction efficiency decreases with each ad- Rate dependence on steric accessibility is further sup-
ditional OH. Such marked differences in rate patterns mean ported by lack of correlation with initial reaction rate and
that this assay must be conducted with over a range of known stoichiometry or antioxidant properties such as redox poten-
phenol concentrations to obtain accurate activity comparisons tials, with only weak correlations (p = 0.75.86) between TEAC
of compounds or extracts with different phenol compositions. values and reaction rates with number of phenol OH groups

Group 1 0.8 Group 2 1.0 Group 3 - simple phenols

0.8
Hydroquinone Pyrogallol Gallic acid
Ascorbic
acid 3-Methylcatechol
0.8 2-Propyl gallate
0.6 Chlorogenic 0.6
Coniferyl alcohol
Initial A

acid Catechol
0.6 Caffeic acid
Trolox
0.4 0.4
Protocatechuic
0.4 Ferulic acid
acid

0.2 0.2
0.2

0.0 0.0 0.0

0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5
AOX Concentration (mM) AOX Concentration (mM) AOX Concentration (mM)

1.4 Group 3 - polyphenols 0.8 Group 4 0.8 Group 5

Resorcinol
Gallocatechin gallate
1.2
Catechin gallate p-Coumaric acid
Quercetin
Final A

1.0 0.6 0.6

Gallocatechin
0.8 Glutathione
Catechin 0.4 0.4
0.6
0.4 Curcumin
0.2 0.2
0.2 Rutin

0.0 0.0 0.0

0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5
AOX Concentration (mM) AOX Concentration (mM) AOX Concentration (mM)

Fig. 2 Effects of antioxidant concentrations on reaction rates with ABTS+. Adapted from Tian and Schaich (2013), used
with permission.
 Journal of Functional Foods 18 (2015) 782796
(Tian & Schaich, 2013). TEAC stoichiometry generally in-
creased while the rate per OH decreased with the number
of phenol OH groups.
2.1.4. Summary and recommendations TEAC/ABTS+ assay
As a large nitrogen-centered and sterically-hindered radical,
ABTS+ is not a good model for small highly-reactive radicals
[e.g. OH, NO, O2, LO(O)] that are active in biological tissues center highly protected (Foti, Daquino, Mackie, DiLabio, & Ingold,
and foods. That steric accessibility rather than chemical prop-
erties of antioxidants controls the reaction invalidates the
chemical accuracy of this assay and its ability to predict
which antioxidants will be active in real situations. Negli-
gible hydrogen atom transfer rates with ABTS+ further
compromise the usefulness of this assay because it totally
misses activity of compounds such as glutathione, thiols,
and proteins that are HAT-dominant and very important an-
tioxidants in vivo.
Van den Berg recognized problems with the TEAC fifteen
years ago, stating that Quantitative evaluation of antioxi-
dant capacity using the TEAC assay can be troublesome or even
impossible (van den Berg, Haenen, van den Berg, & Bast, 1999).
We concur with his conclusion and, further, consider the limi-
tations of the TEAC/ABTS+ assay fatal. We thus recommend
that the use of this assay to screen and compare antioxi-
dants of different structure be discontinued. This assay does
not provide information that is sufficiently accurate on a chemi-
cal basis to be used for ranking of antioxidants or for
construction of SARS (structureactivity relationships).
Nevertheless, the TEAC/ABTS+ assay can be used to assess
whether antioxidants are HAT or SET dominant in their reac-
tions, and it can be used to compare changes in the same
antioxidant during processing or storage. For example, ABTS+
has been used to monitor changes in tocopherol activity after
heat exposure in frying oils and in extrusion of packaging
films (Lang, 2009) and to determine loss of antioxidant activ-
ity in strawberries dried with different methods (Wojdyo,
Figiel, & Oszmianski, 2009). In such applications, limitations
of the assay remain but antioxidant components are con-
stant, and variations in ABTS+ accessibility are not the prime
determinant of reactivity. Rather, alterations in a single anti-
oxidant structure modify radical quenching capabilities.
2.2. DPPH (2,2-diphenylpicryl hydrazyl free radical)
assay
DPPH is a stable radical with a deep purple color. Reaction with
other radicals, electrons, or hydrogen atoms leads to loss of color
at 515 nm and loss of the EPR free radical signal (Rxns 9ac).
NO2 NO2
O2N
.
N N O2 N
H
N
. N
NO2 NO2
Purple Colorless
787
DPPH + Phe-OH DPPH (H) + Phe-O (9a)
DPPH + Phe-O DPPH + Phe-O (9b)
DPPH + Phe-OH DPPH + Phe-O+ (9c)
Like ABTS+, DPPH is sterically-hindered with the radical
2008; Williams, 1966). In contrast to water-soluble ABTS+, DPPH
is hydrophobic so its reactions must be run in organic sol-
vents. Although literature reports attribute DPPH reactions
mostly attributed to HAT, reactions in strong hydrogen-
bonding solvents such as methanol interfere with release of
hydrogen atoms and thus strongly enhance SET over HAT
(Barclay, Edwards, & Vinqvist, 1999; Foti, Daquino, & Geraci, 2004;
Foti et al., 2008). Results outlined in the Limitations discus-
sion suggest that electron transfer dominates in the organic
solvents required to dissolve DPPH.
DPPH assays are performed very much like ABTS+ reac-
tions. A stock solution of concentrated DPPH in methanol is
diluted to an absorbance of ~1 at 515 nm, the starting absor-
bance (A0) is measured, and the antioxidant is added to start
the reaction. Traditional methodology then measures final ab-
sorbance (Af) at 515 nm after reaction times that range from 4
minutes to many hours, or even days. The quantity A0Af is used
to generate a value for ranking tested materials, e.g. EC50 (an-
tioxidant concentration required to reduce DPPH 50%) (Bondet,
Brand-Williams, & Berset, 1997; Brand-Williams, Cuvelier, &
Berset, 1995), %DPPH loss or %DPPH remaining (Burda & Oleszek,
2001; Stratil, Klejdus, & Kuban, 2006), or Antiradical Efficiency
[1/[EC50(Sample) EC50(Trolox)] (Awika, Rooney, Wu, Prior, &
Cisneros-Zevallos, 2003; Butkovic, Klasinc, & Bors, 2004).
2.2.1. Limitations of the DPPH assay
The DPPH reaction has been used as if it is a simplistic chemi-
cal black box reagents are mixed and a number is generated,
and the chemistry occurring between is ignored. The calcula-
tion methods noted in the previous paragraph provide relative
rather than absolute values, and carry no direct chemical in-
formation unless absorbance change is converted to mols DPPH
lost using Beers Law and extinction coefficient values of
10,90012,500 (Lebeau et al., 2000). Even more importantly, this
assay does not measure reaction rates and it misses impor-
tant information contained in reaction curves. Hence, deleting
reaction rates can lead to erroneous assessment of antioxi-
dant efficacy.
That antioxidant reactions with DPPH are actually rather
complex becomes obvious when absorbance changes are fol-
lowed continuously. Reaction curves show multiple reactivity
patterns (Fig. 3) that are similar to ABTS+ but comparisons of
28 phenolic compounds showed some key differences (Xie &
Schaich, 2014). Antioxidants react with DPPH by very rapid
electron transfer and by slow hydrogen atom transfer. Initial
reactions (electron transfer) are quite rapid but slower than
ABTS+ reactions due to difficult phenol access to the hin-
dered DPPH radical site. Hindered access impedes all reactions
but especially hydrogen atom transfer for which formation
of a hydrogen bonded complex between the radical -CH
and the nitrogen lone pair is required (Salamone, DiLabio, &
Bietti, 2011; Salamone, Martella, & Bietti, 2012). Compounds
788 Journal of Functional Foods 18 (2015) 782796

Group 1 - Extremely fast (SET) mM AOX Group 2 - Fast (hindered SET)

1.0 Blank 1.0
1

Absorbance 515 nm
0.8 2.5
0.8
5

0.6 0.6

0.4 0.4
10

0.2 25 0.2

1000
0.0 0.0
0 2 4 6 8 10 0 2 4 6 8 10
Reaction time (min) Reaction time (min)

Group 3 - Moderate, delayed (mixed) Group 4 - Slow (HAT)

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0 2 4 6 8 10 0 2 4 6 8 10
Reaction time (min) Reaction time (min)

Fig. 3 Kinetic patterns of phenol reactions with DPPH. From Xie and Schaich (2014), used with permission. Examples
for each group are shown. Antioxidants showing similar reaction patterns: Group 1 (instantaneous initial absorbance drop)
n-propyl gallate, pyrogallol, and ascorbic acid; Group 2 (rapid initial absorbance drop, then reaction continues more
slowly) catechol, 3-methylcatechol, -tocopherol, caffeic acid, gallic acid, quercetin, epicatechin; Group 3 (moderate,
delayed initial reaction) hydroquinone, protocatechuic acid, rosmarinic acid, Trolox, ferulic acid, chlorogenic acid; Group 4
(absorbance drop very slow and almost linear over time) glutathione, resorcinol, and vanillic acid.

reacting rapidly (dominant electron transfer) are ascorbic acid
and simple phenols with no ring adducts; reactions slow with
addition of acid groups or other side chains to aromatic rings,
and there are some inductive effects of ring adducts within
phenol classes.
Effects of steric hindrance to DPPH are exhibited quite dis-
tinctively in plots of initial rates as a function of antioxidant
concentration (Fig. 4) (Xie & Schaich, 2014). Similar to ABTS+,
particularly evident in variability of DPPH reactions with solvent.
Methanol, the solvent most commonly used for the DPPH assay,
strongly binds hydrogen atoms and inhibits HAT processes
(Barclay et al., 1999; Foti et al., 2004; Hogg, Lohmann, & Russell,
1
Pyrogallol
Initial rate, nmol DPPH/sec

small compounds with fast initial rates (Group 1, e.g. pyrogal-

lol) show a linear increase at low concentrations, but the linear 0.8
Catechol
reaction range shrinks and saturation concentrations de-
crease with addition of bulky ring adducts and multiple rings.
0.6
Molecules with complex structures more readily get in the way
of each other and impede access to DPPH at low concentra-
tions and they strongly block reaction at high concentrations. 0.4
These results show that single antioxidant concentrations are Hydroquinone
clearly inadequate to quantitate and compare antioxidant re-
0.2
activity with DPPH, that phenol concentrations of extracts must
be known for accurate comparisons, and that the patterns of Resorcinol
antioxidant and extract responses over a reasonable phenol 0
concentration range must be determined to understand what 0 0.2 0.4 0.6 0.8 1
is happening in the reactions, especially when phenol struc- [AOX], mM
tures are different or unknown.
DPPH reactions are highly sensitive to reaction environ- Fig. 4 Effects of phenol concentrations on rates of phenol
ment, i.e. water and solvent (even more than ABTS+), pH, reactions with DPPH. Only samples from each group are
oxygen, light exposure (Xie & Schaich, 2014). The presence of shown. Full details for all tested compounds are available
both electron and hydrogen atom transfer mechanisms is in Xie and Schaich (2014).
 Journal of Functional Foods 18 (2015) 782796
1961). However, water disrupts the binding and facilitates H
atom transfer, so when it is added to the reaction medium, any
test compounds with H atom transfer capabilities increase their
reaction rates. Similarly, electron transfer is pH-dependent, in-
creasing in rate with pH and degree of ionization, while HAT
is pH-independent. The dominant mechanism of a test com-
pound can thus be assessed by reacting it with DPPH in
methanol and in 50% methanol in which the aqueous phase
is buffered to a range of pH from acid to strongly alkaline (Xie
& Schaich, 2014). Reaction rates of compounds that are HAT-
dominant (e.g. glutathione) increase markedly in 50% water but
not with pH. In contrast, SET-dominant compounds such as
hydroquinone will react extremely fast in methanol, show small
change in 50% water, then increase markedly with pH of the
water phase. The strength of SET processes for individual an-
tioxidants can thus be evaluated by plotting DPPH initial
reaction rates as a function of pH. High slopes indicate domi-
nant electron transfer (Xie & Schaich, 2014).
DPPH reactions also vary with dissolved oxygen in the re-
action mixtures (Xie & Schaich, 2014). Compounds that
autoxidize, e.g. ascorbic acid (with metal catalysts) and some
phenols, reduce oxygen to O2 which then enters the reac-
tion mix:
Ascorbic acid ( AH2 )
O2
O2 + AH O2
H2O2 2 HO
AH + O2 O2
(10)
O2 reacts instantaneously with ABTS+, leading to aberrantly
high antioxidant activity, while it reacts poorly with DPPH, re-
sulting in aberrantly low antioxidant reactivity because
oxidation of phenols to less reactive quinones or unreactive
products competitively removes them from the reaction and
reduces detected reactivity. Thus, a good working rule of thumb
to eliminate potential artifacts is to sparge all solutions with
argon before running the DPPH and ABTS+ reactions.
An additional issue with DPPH assays is what happens in
mixtures of compounds. Given problems with steric inacces-
sibility of the DPPH radical site, do DPPH reactions accurately
reflect radical quenching capabilities of mixtures of antioxi-
dants, such as would be found in fruit and vegetable extracts?
Can they detect synergisms that have been reported from in
vivo feeding studies, or do multiple compounds in mixtures in-
terfere with each other in reaching the DPPH radical? Studies
in our laboratory show that reaction of phenol mixtures with
DPPH is nearly always less than the sum of individual phenol
components because steric factors control the reactions (G. Wu
and K.M. Schaich, unpublished data). The response shifts with
the proportion of components and balance between HAT and
SET, synergisms and antagonisms are difficult to detect, and
ascorbic acid, when present, dominates reactions. This is a criti-
cal limitation when comparing mixtures of compounds in
extracts and infusions.
Finally, for any quantitative assay to be valid, reactivity must
be directly related to chemical properties of the test com-
pounds, and the reaction mechanism must be constant.
Unfortunately, our results suggest that neither of these re-
quirements can be met in the DPPH assay. Steric accessibility
to the DPPH radical site is the overall most important driver
of reaction rate, with secondary contributions from balance
between electron and hydrogen atom transfer quenching
789
mechanisms. In contrast, both initial reaction rate and final
stoichiometry are poorly correlated with molecular structure
and chemical characteristics, including number of phenolic
OH groups and redox potential (Xie & Schaich, 2014). These
observations raise serious questions about using the DPPH
assay to rank antioxidant efficacy of foods and extracts.
2.2.2. Summary and recommendations DPPH assay
Like ABTS+, DPPH is a poor model for radical quenching in vivo
and in foods because it is nitrogen-centered and the radical
site is highly hindered. Questions must be raised regarding use
of DPPH in antioxidant screening and quantitation of radical
quenching in extracts of natural materials for the following
reasons (Xie & Schaich, 2014):
1. The main reaction for fast reactors is complete in <10 s. Long
reaction times to allow for full reaction merely count the
phenolic groups, overestimate polyphenols and underes-
timate small phenols, and are irrelevant to fast radical
reactions in situ.
2. Recording initial fast reaction rates accurately requires use
of fast mixing techniques such as dispensing plate readers
and continuous or stopped flow mixers. Normal hand mixing
can miss much of the early reaction.
3. Rate calculations are not straightforward because the re-
sponse curves lack linear regions, change with antioxidant
concentration and reaction conditions, and do not exhibit
a constant reaction order.
4. DPPH reaction rates are more closely related to steric ac-
cessibility than chemical characteristics of test antioxidants.
5. DPPH reacts by mixed SET and HAT mechanisms, the pro-
portion of which changes with the antioxidant.
6. DPPH reaction mechanisms and quantitative responses are
altered by many environmental factors.
7. Reaction of antioxidant mixtures with DPPH is less than the
total reactivity of individual components, suggesting that
radical quenching by mixed extracts is actually depressed
in the DPPH assay. The assay thus may greatly underesti-
mate potential radical quenching in cells or foods. Steric
interferences do not account for all antagonisms or syner-
gisms in the assay.
All these factors invalidate the chemical accuracy of this
assay and its ability to predict which antioxidants will be active
in real situations. We thus recommend that the DPPH assay
no longer be used to compare activity of antioxidants with
different or unknown structures, especially as occurs in ex-
tracts of natural materials. At the same time, although there
is still much to be elucidated about the fundamental chem-
istry of this reaction, we feel the DPPH assay may be usefully
redirected to gain information about active quenching mecha-
nisms of antioxidants being tested.
Such redirection requires a nearly total revamping of the
assay. Fast mixing methods must be used to capture early re-
actions. Reactions must be recorded continuously from the point
of mixing to determine initial reaction rate and shape of re-
action response curves. Kinetics (rate constants) of initial fast
response (mixing to point of diversion from linear, usually a
few seconds to about thirty seconds) must be calculated.
790 Journal of Functional Foods 18 (2015) 782796
When initial fast absorbance drop is absent, rates should be
calculated from the slope of the first thirty seconds of re-
sponse curves. Reaction rates must be calculated over a range
of antioxidant concentrations, then plotted versus concentra-
tion to calculate second order rate constants and determine
concentration effects on reactivity. For comparability between
laboratories, standard protocols for calculating rates and first
and second order rate constants must be adopted. Currently,
nearly every study that reports kinetics uses a different pro-
cedure. Antioxidant reactions with DPPH must be tested in
different solvents and pHs over a range of antioxidant or phenol
concentrations to determine dominant radical quenching
mechanisms. Extracts, juices, infusions, etc. should be nor-
malized to identical phenol and ascorbic acid concentrations
before assays. Finally, DPPH blanks must always be sub-
tracted from test samples. This step seems to be ignored in
many current research reports.
2.3. ORAC oxygen radical absorbance capacity
The ORAC assay originally developed by Glazer and Ghiselli
(Ghiselli, Serafini, Maiani, Azzini, & Ferro-Luzzi, 1995; Glazer,
1990) was refined and applied to extensive analyses of hun-
dreds of foods by Prior and colleagues in U.S. Dept Agriculture
research laboratories (Cao, Sofic, & Prior, 1996; Huang,
Ou, Hampsch-Woodill, Flanagan, & Prior, 2002; Ou,
Hampsch-Woodill, & Prior, 2001; Prior et al., 2003; Wu et al.,
2004) and commercialized by Brunswick Laboratories (Wareham,
MA, USA). Perhaps most widely recognized of all the antioxi-
dant assays, the reaction is simple in concept but complex in
practice. Radicals are generated by heating an azide com-
pound. The azide decomposes, eliminating nitrogen gas and
leaving behind two carbon-centered radicals, R (Rxn 11). In the
presence of oxygen, R are converted almost instantaneously
to reactive peroxyl radicals, ROO (Rxn 11) which can either
attack target molecules that have color or fluorescence (Rxn.
12) or react with antioxidants (Rxns. 13,14).
R N = N-R
Heat
N2 + 2 R
O2
ROO
(11)
ROO + target ROOH + oxidized target (product ) (12)
ROO + AH ROOH + A (13)
ROO + A ROO-A (14)
Competition between reaction of targets and antioxidants
with the ROO forms the basis of the assay. Fluorescein, the
most common target in current use, is intensely fluorescent
in its native form. When attacked by peroxyl radicals, the fluo-
rescence is lost. An antioxidant slows the loss of fluorescence
by quenching the peroxyl radicals via hydrogen atom trans-
fer (Rxn 13) or radical addition (Rxn 14) (Prior, Wu, & Schaich,
2005). The reaction is followed by recording fluorescence over
time (Prior et al., 2005). Results are calculated as the total area
under the reaction curves (AUC) for each antioxidant sample
minus the reaction blank area (no antioxidant) to account for
increased induction period (lag time) and total extent of in-
hibition, including secondary reactions, induced by antioxidants
(Fig. 5A). To convert these unitless areas into chemically-
meaningful values then requires that AUCs from test samples
be compared to AUCs from Trolox standards, and results are
reported as Trolox equivalents. This practice, however, does not
measure rates and it can underestimate fast reactors.
2.3.1. Advantages of the ORAC assay
The ORAC assay offers several advantages over ABTS+ and DPPH
assays. It uses peroxyl radicals that are better models of an-
tioxidant reactions with oxidizing lipids and reactive oxygen
species (ROS) in foods and in vivo, and it provides continuous
generation of radicals on a realistic time scale (more like actual
reactions in situ). The completely hydrogen atom transfer radical
quenching mechanism presents a contrast and comparison to
electron transfer in ABTS+ and DPPH assays. The assay can be
adapted to detect both hydrophilic and hydrophobic antioxi-
dants generally or specifically by altering the radical source,
solvent, and target molecule, and has been routinely automated.
2.3.2. Limitations of the ORAC assay
Nonetheless, the complexity of the ORAC reaction means there
are many points to cause difficulties for researchers using this
assay. The issues outlined below pose significant limitations
and problems for ORAC and can compromise the accuracy and
use of this assay. The purpose in raising these issues is not to
argue that one set of concentrations is correct or optimum, but
to call attention to problems in the assay and the critical need
for more systematic investigations of all ORAC reaction con-
ditions. More detailed studies of the chemistry are essential
to ensure optimization, understand the reactions better for
general use, and guide decisions about standardization of pro-
tocols. More details may be found in Apak et al. (2013).
Very careful control of ORAC reaction temperature is critical! Con-
sistent generation of radicals in the azide reaction requires
accurate and reproducible temperature control to ensure timely
and complete azide decomposition. The temperature required
depends on the azide used, e.g. for AAPH (2,2-azobis-2-
amidinopropane dihydrochloride), it is 37 C. When required
temperatures are not reached, reactions are slow and incom-
plete, and results are poorly reproducible. Even more problematic,
slow reaction can be misinterpreted as increased radical scav-
enging, leading to an overestimation of antioxidant activity.
Never assume that a reaction temperature is achieved just because
it is set on an instrument. Temperatures must be measured. The most
accurate control is obtained by reactions in single cells in heated
holding blocks and fluorescence sample blocks but this elimi-
nates rapid throughput. Obtaining required temperatures with
plate readers is difficult. Plastic plates (e.g. 96 wells) are good
insulators, so temperatures of most ovens must be set at some
higher point to ensure that azide decomposition tempera-
ture in the wells is reached. We tested a number of different
plate readers and found that each unit had to be evaluated in-
dependently to determine the required set temperature. We also
found that plate readers in which the oven and optical reader
were separate gave highly irreproducible results across the plate
and from plate to plate because the temperature varied too
much as the plate was moved back and forth between oven
and reader. Most accurate, stable, and consistent tempera-
tures are obtained in plate readers that are heated from above
and below the plate, not from the sides, and the optical system
 Journal of Functional Foods 18 (2015) 782796 791

Fig. 5 Standard ORAC response curves and some aberrations that compromise accuracy of results. (A) Standard ORAC
reaction curves showing area under curve used to quantitate reactions. Fluorescence quenching at high fluorescein
concentrations can be seen in temporary increases in fluorescence immediately after mixing with antioxidants (B) and in
maintenance of fluorescence levels even after dilution (C). Non-radical antioxidant interactions with fluorescein can prevent
fluorescence decay (D). Samples must be diluted until reactions run in reasonable time. Dilution required for each extract
must be determined independently. (E) Effects of non-radical antioxidant interactions with fluorescein can be seen in ORAC
values (area under curve) that increase as antioxidant concentrations decrease. Fluorescein and azide concentrations are
different from D. (E) Overlap of fluorescein and antioxidant fluorescence spectra can dissipate fluorescence in absence of
azide. Fluoresceinantioxidant extract mixture has same reagent concentrations as for individual spectra. Possibility of
multiple effects means that each antioxidant effect must be tested independently with fluorescein alone. Y-axis was
fluorescence intensity in counts per second for AE; Absorbance or fluorescence units in F. All graphs: K.M. Schaich,
unpublished data.

rotates over the plates to take a reading. In this configura-
tion, the plates never move and the temperature is maintained
more accurately.
Very careful control of oxygen is critical! Full and reproducible
oxygenation is required for the azide reaction to efficiently gen-
erate radicals. However, solubility of oxygen declines as
temperature increases, so oxygen becomes depleted when
solutions are pre-warmed before runs, during thermal equili-
bration in ovens, and when reaction must be heated over long
times due to strong antioxidant inhibition. Variations in han-
dling and heating times between samples cause considerable
variability in dissolved pO2, hence inconsistent results. With
insufficient oxygen, reactions are slow, variable, and do not run
to completion. In our laboratory, most robust reactions and most
reproducible results are obtained when all solutions are sparged
thoroughly with oxygen just before mixing in both manual and
plate reader testing.
Very careful control of reagent concentrations is critical! Abso-
lute and relative concentrations of azide, fluorescein, and
samples control the reaction. Because fluorescein emission
intensity is so intense, only nM or lower concentrations are
needed to follow the reaction. However, the reaction is diffu-
sion controlled. At typical nM concentrations, fluorescein
molecules are greatly scattered in the medium. At the same
time, the peroxyl radicals generated from the azide are very
short-lived and must encounter a fluorescein molecule before
decaying. Thus, very high concentrations of peroxyl radicals
are needed to surround the fluorescein and ensure efficient re-
action. Prior and colleagues investigated azide levels up to 2000
greater than fluorescein concentrations (Prior et al., 2003) yet
adopted 1.6 mol AAPH and 7.5 nmols fluorescein per reac-
tion in their standard procedure (Wu, 2005). We find that azide
concentrations in their higher range provide more consistent
reactions and are necessary to keep reaction times for con-
trols at about twenty minutes.
Imbalance in reagent proportions e.g. fluorescein too high
(leading to quenching) and azide too low (leading to insuffi-
cient ROO for rapid reaction) results in very slow reactions that
do not always go to completion. We find consistent reactions
running to completion in about 15 minutes for controls when
792 Journal of Functional Foods 18 (2015) 782796
half Priors fluorescein concentration (1.9 nM) and 210 times
his AAPH concentration (1.6 M) are used. High fluorescein con-
centrations are a problem because extensive H bonding between
the phenol groups and pi stacking of the rings decrease net fluo-
rescence, a process called self-quenching (Schauenstein,
Schauenstein, & Wick, 1978). When fluorescence quenching
occurs, emissions do not diminish with dilution they stay the
same or even increase, as can be seen as a rise at the begin-
ning of some reaction curves when consumption of fluorescein
in reaction releases fluorescence quenching (Fig. 5B) or when
emission intensity does not decrease with dilution (Fig. 5C). Delay
in fluorescence decay leads to loss of resolution between samples
and overestimation of antioxidant action. We find fluores-
cence quenching at the 1.9 nM fluorescein concentrations
recommended in USDA and Brunswick Labs protocols (Prior et al.,
2003; Wu, 2005); using half this fluorescein concentration or less
eliminates the initial fluorescence increases and speeds reactions.
Sample concentrations are no less important. When working
with isolated compounds or Trolox standards, the recom-
mended M solutions can be prepared with certainty. However,
phenol identity and concentrations in extracts from foods and
natural materials are seldom measured before analysis. Most
commonly, a single standard aliquot (e.g. 20 l) of extract is
analyzed without standardization to phenol or ascorbic acid
content and without testing a range of dilutions. This prac-
tice can lead to distorted if not outright incorrect results, due
in part to varying concentration response patterns with dif-
ferent antioxidants as well as to non-radical interactions with
fluorescein. Phenolic, quinone, and aromatic rings in the fluo-
rescein structure provide multiple sites and modes for non-
radical associations that block normal reactions with
antioxidants. Polyphenol binding may block reactive OH
groups and stabilize fluorescein during reaction. Such inter-
actions between antioxidants and fluorescein mostly increase
apparent radical quenching activity, as can be seen in excep-
tionally long reaction times and implausibly high ORAC values
(Fig. 5D) or higher ORAC values with more dilute antioxi-
dants (Fig. 5E). Alternatively, if antioxidant and fluorescein
excitation or emission manifolds overlap, fluorescein
antioxidant binding can either stabilize or reduce fluorescence
emissions (Fig. 5F). Interaction interferences must be tested
for using appropriate blanks with extracts plus fluorescein
(no azide) and analyzing overlaps of fluorescein and phenol
excitation and emission manifolds in fluorescence spectra.
Thus, considering all these concentration effects, as with
the ABTS+ and DPPH assays, it is necessary to assay a range
of antioxidant or extract concentrations to determine linear
response ranges or concentration ranges for valid measure-
ments, and also the cross-over concentration at which anti-
oxidants may become pro-oxidant. To eliminate non-radical
interferences, extracts should be diluted to a concentration that
gives complete reaction in less than one hour. Longer times
increase complicating side reactions and likelihood that the
system will become oxygen limited. Furthermore, the occur-
rence of very long reaction times without substantial fluorescein
decay often signals extract interactions with the fluorescein.
Variations in calculation methods as well as reporting and
use of results lead to huge inconsistencies in ORAC values re-
ported by different laboratories and on internet websites. This
is perhaps the greatest problem with ORAC. Simply calculating
the area under the curve leads to differences and inaccura-
cies imposed by timing of data recording and equations used
for integration. One equation proposed for calculating the AUCs
integrates successive slices of the curve determined by inter-
val sampling time when point values are recorded (Cao, Alessio,
& Cutler, 1993) or a selected time interval when absorbance
is recorded continuously:
AUC = ( 0.5 + f 5 f 4 + f 6 f 4 + f 7 f 4 + + fi f 4) CT (15)
where fi = fluorescence reading at cycle i (i.e. f4 = initial fluo-
rescence reading at cycle 4), and CT = cycle time in minutes.
It is easy to see from this equation that the time interval
matters. The smaller the time interval in data recording, the
more closely the calculated AUC approaches actual area, or con-
versely, the longer the sampling interval, the more the
calculated AUC underestimates actual area. Variations in cal-
culation method contribute to inconsistent quantitative results
between laboratories.
Converting AUC to Trolox equivalents (ORAC units) is a
source of additional vagaries and inconsistencies. Early methods
recommended the following equation for calculating ORAC units
(Ou et al., 2001):
ORAC units =
( AUCsample AUCblank ) M Trolox Msample (16)
(AUCTrolox AUCblank )
This equation includes a factor (circled in equation) for nor-
malizing to a known Trolox standard concentration (usually
1 mM) that takes the place of a standard curve. Unfortu-
nately, phenol concentrations in extract samples are seldom
determined before analyses, and the equation is often applied
without sample normalization, or with just comparison to 1 mM
Trolox. This practice can lead to huge errors in ORAC values.
Taking problems of ORAC normalization one step further,
the practice of reporting ORAC units as micromolar equiva-
lents of Trolox generates very large numbers which give the
erroneous impression that extracts of foods and other natural
materials have components that are more reactive than to-
copherols by many orders of magnitude. In fact, ORAC values
of 1,000,000 micromolar equivalents are needed to provide the
same protection as 1 M -tocopherol. Thus, care needs to be
taken so that expression of ORAC values do not psychologi-
cally or actually overestimate antioxidant action.
Perhaps the greatest problem in calculating ORAC values
is what to do with extracts and edible materials. Calculations
are straightforward for solutions of pure phenols where con-
centrations are known, but phenol concentrations in extracts
are seldom known or measured. What normalization base
should be used in these conditions mmol Trolox equiva-
lents per ml extract? L of extract? Liter of starting material (e.g.
fruit juice)? g or kg fresh wt? g or kg dried extract? mol phenol?
per 100 g? per serving? unspecified? All of these bases are in
common use, and the variable units and normalization bases
for expressing ORAC values lead to considerable confusion, un-
certainty, and obfuscation of results as researchers and
marketers alike try to enlarge the numbers to amplify psy-
chological response to results. So which reporting basis is
preferable?
 Journal of Functional Foods 18 (2015) 782796 793

It cannot be overstressed that ORAC units must be speci- had much higher antioxidant power than fruits and veg-
fied and reported on a common and reasonable basis for useful etables. However, with further consideration several factors that
comparisons. Reporting without normalization to a concen- strongly distort interpretation of the ORAC values immedi-
tration standard and without units, as has occurred often in ately become apparent. First is moisture content when the
reports on the internet, is meaningless and often reflects intent comparison basis is weight. The spices listed are dry, but the
to deceive or over-exaggerate results. However, even in valid foods have moisture contents of typically 90% or greater, which
research, determining a basis that is reasonable for practical markedly dilute the antioxidants. The second issue is, what
understanding and still not misleading is not straightfor- does 100 g mean in terms of how much of a food is normally
ward. Compare the ORAC values presented in Table 1, for consumed? 100 g cinnamon, for example, is about 1 cup, a
example, where ORAC values were determined for 100 g of ma- volume very far from consumable. In contrast, 100 g straw-
terial. ORAC values varied by several orders of magnitude, and berries is only half a normal serving, and a medium-size apple
taking the numbers at face value might lead one to think spices is about 145 g rather than 100 g. Thus, for the two fruits, and
indeed most foods in the list, normalizing to 100 g underes-
timates to variable degrees the antioxidant potential that would
actually be consumed. Reporting on a fresh weight basis that
Table 1 ORAC values for foods, selected to illustrate ignores moisture contents distorts and underreports antioxi-
effects of food moisture content and serving size on dant potential even more. This example demonstrates that
numerical values reported.
ORAC values cannot be accepted as just numbers for com-
Serving ORAC parison, but must be expressed on bases that are meaningful
sizea (mol
to consumption and include normalization to dry solids.
TE/100 g)b
In addition, to ascertain whether an extract has higher
Spices phenol content or higher phenol activity, normalization at least
Cloves, ground 1 cup 290,283
to total phenols should be included.
Sumac, bran, raw 312,400
One further limitation in using and interpreting ORAC
Cinnamon, ground 0.8 cup 131,420
Sorghum, bran, hi-tannin 240,000 values is understanding what they mean chemically, physi-
Oregano, dried 2 cups ologically, or nutritionally. Chemically, although phenols are
Acai berry, freeze-dried 102,700 assumed to be the key reactants, correlations of phenol con-
Turmeric, ground 0.7 cup 127,068 tents to ORAC values have not been established and there is
Sorghum, bran, black 100,800 little to no other documentation of the compounds respon-
Sumac, grain, raw 86,800
sible for extract response in ORAC assays. Since some phenols
Cocoa, dry powder, 1.2 cup 55,653
unsweetened
have known toxicity (Bermdez-Saldaa, Escuder-Gilabert,
Cumin seed 50,372 Medina-Hernndez, Villanueva-Camaas, & Sagrado, 2007;
Maqui berry, 75,000 Hansch, McKarns, Smith, & Doolittle, 2000; Moridani, Siraki,
concentrated powder & OBrien, 2003; Selassie, DeSoyza, Rosario, Gao, & Hansch,
Parsley, dried 4 cups 73,670 1998), especially at higher concentrations, using such a blind
Sorghum, bran, red 71,000
assay to make recommendations or decisions about best foods
Basil, dried 3 cups 61,063
to eat is dangerous. Physiologically, lack of substantive docu-
Foods
Aronia black chokeberry 100 g 16,062 mentation of radical quenching by phenols in vivo has led to
Small red bean 1/2 c. dried beans (112 g) 8606 removal of the ORAC Values of Foods list from the USDA website,
Wild blueberry 1 cup (145 g) 9621 as noted in the Introduction (USDA, 2010).
Red kidney bean 1/2 c. dried beans 8606 Nutritionally, ORAC values are very confusing. At one point,
Pinto bean 1/2 cup 8033 the USDA recommended that 30005000 ORAC units be con-
Blueberry 1 cup berries (145 g) 4669
sumed per day for health. 5000 ORAC units can be obtained from
Cranberry 1 cup berries (120 g) 9090
one Granny Smith apple or one ounce of pecans, but few people
Artichoke hearts 1 cup, cooked 6552
Blackberry 1 cup berries (145 g) 5905 would consider that either of these food quantities contains suf-
Prune 1/2 cup (75 g) 8059 ficient antioxidant levels for health. Taking a different approach,
Raspberry 1 cup (123 g) 5065 for ORAC expressed as moles Trolox equivalent, 5000 ORAC units
Strawberry 1 cup (200 g) 4302 5 mmol Vit E = 2153 mg. If it is assumed that tocopherol re-
Red Delicious apple 1 apple (154 g, medium) 4275 action with fluorescein is comparable to that of Trolox, 5000 ORAC
Granny Smith apple 1 apple (154 g, medium) 3898
units is equivalent to about 3230 IU of tocopherol daily. Com-
Pecan 1 oz. 17,940
Sweet cherry 1 cup (140 g) 3747
pared to the 30 IU Toc needed to prevent deficiency and the 400 IU
Black plum 1 plum (78 g) 7581 maximum recommended supplement level, 5000 ORAC units
Russet potato 1, cooked (148 g) 1680 totally absorbed could be a potentially dangerous overdose.
Black bean 1/2 cup dried beans 8040
Plum 1 plum (78 g) 6100 2.3.3. Summary and recommendations ORAC assay
Gala apple 1 apple (154 g, medium) 2828
The ORAC assay provides a means of evaluating ability of an
a
Serving sizes for spices and herbs is the approximate volume for antioxidant to quench radicals by hydrogen atom transfer in-
100 g, to provide perspective. Weights taken from Aqua-Calc (2015). dependently of electron transfer. It also provides a means of
Cups are U.S. volumes.
b
testing reactivity with a number of different types of radicals,
(Haytowitz & Bhagwat, 2010).
e.g. by changing initiators (Ou et al., 2002). Nevertheless, the
794 Journal of Functional Foods 18 (2015) 782796
radical reactions employed in the ORAC assay are complex, even
with non-azide initiators, they require rigid control over re-
action conditions to ensure reproducible and accurate results,
and they are subject to numerous complications and limita-
tions that can compromise use of this assay.
We recognize that assays such as ORAC provide impor-
tant tools for preliminary evaluation and screening of
antioxidants. However, ORAC values have been used more as
political and marketing tools than as chemical tools. Despite
the extensive use of ORAC assays to analyze a very wide range
of materials and current focus on standardizing this assay as
an official method, many glitches remain in the way the assay
is handled. ORAC reaction chemistry is not well enough defined
in either reaction progress or connection to specific molecu-
lar properties to warrant standardization of any existing
protocols. Key problem areas in chemistry include:
Fluorescein needs to be replaced with a target that has no
side reactions and does not interact with itself or phenols
by non-radical mechanisms.
Detailed investigations of the effects of absolute and rela-
tive reagent concentrations on reaction rates and extent are
urgently needed before standardization.
Non-radical target self-associations and interactions with
phenols in the absence of radicals are insufficiently docu-
mented and may contribute to erroneously high ORAC
values.
Alternate methods for following reactions that will clearly
determine kinetics as well as stoichiometry need to be
developed.
To provide chemical logic relating ORAC reactivity to phenol
structures, structural features that control reactivity of an-
tioxidants with fluorescein or other target molecules must
be identified and ORAC values need to be connected di-
rectly to levels and types of antioxidants in extracts rather
than analyzing extracts blindly.
Critical problems exist also with use and applications of
ORAC values. Although correlations between ORAC values of
consumed foods and elevated plasma antioxidant capacity or
beneficial anti-disease actions have been claimed, the appear-
ance of components of tested materials in the bloodstream have
not been demonstrated. Phenols are poorly absorbed and rapidly
conjugated and excreted so actual intake from consumed foods
cannot be predicted from ORAC values, and toxicity of high
levels of phenols in general and some specific phenols is well
documented (Bermdez-Saldaa et al., 2007; Hansch et al., 2000;
Moridani et al., 2003; Selassie et al., 1998; Yen, Duh, & Tsai, 2002).
Thus, high ORAC values should not be used to rank foods, sell
foods, or determine food choices. Much more research is needed
to establish clear causal connections between molecules de-
tected in ORAC assays and physiological actions.
3. Summary and recommendations
Twenty five years of antioxidant screening in natural materi-
als have shown that polyphenols are good antioxidants; that
most plant materials have high contents of phenols which
exhibit excellent antioxidant activity; that within classes of an-
tioxidants, the number and position of phenolic OH groups
affect antioxidant efficacy, and that non-phenol antioxidant
structures are poorly detected in most existing antioxidant
assays. Twenty five years of antioxidant screening have NOT
resolved issues of assay chemistry, standardization, and re-
porting; provided significant insight into chemical mechanisms
and factors controlling antioxidant action; clearly connected
in vitro assay chemistry to in vivo actions; established rate con-
stants for reaction of antioxidants with radicals that are relevant
in foods and biological tissues; shown how antioxidants with
negligible absorption exert systemic effects; or addressed
counter issues of (poly)phenol toxicity.
The early need to merely identify which natural materials
had strong antioxidant efficacy has been replaced by the need
to elucidate accurately how natural antioxidants act. The re-
action patterns discussed in this paper demonstrate the
chemical inaccuracies of three common in vitro antioxidant
assays and argue for redirecting their orientation and empha-
sis. ABTS+, DPPH, and ORAC assays conducted with more
detailed and revised protocols can still be useful for reveal-
ing dominant quenching mechanisms. However, for quantitative
antioxidant analysis of natural materials, these assays should
be replaced with new antioxidant assays, including lipid oxi-
dation assays, with clearly identifiable reaction mechanisms,
fully tested reaction conditions, and kinetic as well as stoi-
chiometric analyses.
Finally, it is essential that all assays used to investigate an-
tioxidant efficacy emphasize fundamental chemistry rather
than easy or rapid analysis in order to elucidate radical quench-
ing mechanisms for different classes of phenols, determine rate
constants and specificity for reactions of antioxidants with dif-
ferent radicals (will require rapid-mix techniques), identify
synergisms and antagonisms among classes of antioxidant
compounds, and then learn how to exploit this information
for more effective application of natural antioxidants in medical
therapies and food stabilization.
REFERENCES
Alcolea, J. F., Cano, A., Acosta, M., & Arnao, M. B. (2003).
Determination of the hydrophilic and lipophilic antioxidant
activity of white and red wines during the wine-making
process. Italian Journal of Food Science, 15, 207214.
Apak, R., Gorinstein, S., Bhm, V., Schaich, K. M., zyrek, M., &
Gl, K. (2013). Methods of measurement and evaluation of
natural antioxidant capacity/activity (IUPAC Technical
Report). Pure and Applied Chemistry, 85, 957998.
Aqua-Calc. Conversions and calculations. (2015). <http://www.aqua
-calc.com/calculate/food-weight-to-volume> Accessed
06.07.15.
Arnao, M. B., Cano, A., & Acosta, M. (2001). The hydrophilic and
lipophilic contribution to total antioxidant activity. Food
Chemistry, 73, 239244.
Awika, J. M., Rooney, L. W., Wu, X., Prior, R. L., & Cisneros-
Zevallos, L. (2003). Screening methods to measure
antioxidant activity of sorghum (Sorghum bicolour) and
sorghum products. Journal of Agricultural and Food Chemistry,
51, 66576662.
Barclay, L. R. C., Edwards, C. E., & Vinqvist, M. R. (1999).
Media effects on antioxidant activities of phenols and
 Journal of Functional Foods 18 (2015) 782796
catechols. Journal of the American Chemical Society, 121,
62266231.
Bermdez-Saldaa, J. M., Escuder-Gilabert, L., Medina-
Hernndez, M. J., Villanueva-Camaas, R. M., & Sagrado, S.
(2007). Chromatographic retentionactivity relationships for
prediction of the toxicity pH-dependence of phenols.
Chemosphere, 69, 108117.
Bondet, V., Brand-Williams, W., & Berset, C. (1997). Kinetics and
mechanisms of antioxidant activity using the DPPH free
radical method. LWT Food Science and Technology, 30, 609615.
Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a
free radical method to evaluate antioxidant activity. LWT
Food Science and Technology, 28, 2530.
Burda, S., & Oleszek, W. (2001). Antioxidant and antiradical
activities of flavonoids. Journal of Agricultural and Food
Chemistry, 49, 27742779.
Butkovic, V., Klasinc, L., & Bors, W. (2004). Kinetic study of
flavonoid reactions with stable radicals. Journal of Agricultural
and Food Chemistry, 52, 28162820.
Cao, G., Alessio, H. M., & Cutler, R. G. (1993). Oxygen-radical
absorbance capacity assay for antioxidants. Free Radical
Biology and Medicine, 14, 303311.
Cao, G., Sofic, E., & Prior, R. L. (1996). Antioxidant capacity of tea
and common vegetables. Journal of Agricultural and Food
Chemistry, 44, 34263431.
Foti, M. C., Daquino, C., & Geraci, C. (2004). Electron-transfer
reaction of cinnamic acids and their methyl esters with the
DPPH. radical in alcoholic solutions. The Journal of Organic
Chemistry, 69, 23092314.
Foti, M. C., Daquino, C., Mackie, I. D., DiLabio, G. A., & Ingold, K. U.
(2008). Reaction of phenols with the 2,2-diphenyl-1-
picrylhydrazyl radical. Kinetics and DFT calculations applied
to determine ArO-H bond dissociation enthalpies and
reaction mechanism. The Journal of Organic Chemistry, 73, 9270
9282.
Frankel, E. N. (2007). Antioxidants in food and biology: Facts and
fiction. Bridgwater, England: The Oily Press, PJ Barnes &
Associates.
Frankel, E. N., & Finley, J. (2008). How to standardize the
multiplicity of methods to evaluate natural antioxidants.
Journal of Agricultural and Food Chemistry, 56, 49014908.
Ganapathi, M. R., Hermann, R., Naumov, S., & Brede, O. (2000).
Free electron transfer from several phenols to radical cations
of non-polar solvents. Physical Chemistry Chemical Physics, 2,
49474955.
Ghiselli, A., Serafini, M., Maiani, G., Azzini, E., & Ferro-Luzzi, A.
(1995). A fluorescence-based method for measuring total
plasma antioxidant capability. Free Radical Biology and
Medicine, 18, 2936.
Glazer, A. N. (1990). Phycoerythrin fluorescence-based assay for
reactive oxygen species. Methods in Enzymology, 186, 161168.
Halliwell, B., Rafter, J., & Jenner, A. (2005). Antioxidant or not.
Health promotion by flavonoids, tocopherols, tocotrienols,
and other phenols: Direct or indirect effects? The American
Journal of Clinical Nutrition, 81, 268S276S.
Hansch, C., McKarns, S. C., Smith, C. J., & Doolittle, D. J. (2000).
Comparative QSAR evidence for a free-radical mechanism of
phenol-induced toxicity. Chemico-Biological Interactions, 127,
6172.
Haytowitz, D. B., & Bhagwat, S. USDA database for the oxygen
radical absorbance capacity (orac) of selected foods. (2010)
<http://www.orac-info-portal.de/download/ORAC_R2.pdf>
Accessed 06.04.15. Release 2 (2010)
Hogg, J. S., Lohmann, D. H., & Russell, K. S. (1961). The kinetics of
reaction of 2,2-diphenyl-1-picrylhydrazyl with phenols.
Canadian Journal of Chemistry, 39, 15881594.
Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J. A., &
Deemer, E. K. (2002). Development and validation of oxygen
795
radical absorbance capacity assay for lipophilic antioxidants
using randomly methylated -cyclodextrin as the solubility
enhancer. Journal of Agricultural and Food Chemistry, 50, 1815
1821.
Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J. A., & Prior, R.
L. (2002). High-throughput assay of oxygen radical absorbance
capacity (ORAC) using a multichannel liquid handling system
coupled with a microplate fluorescence reader in 96-well
format. Journal of Agricultural and Food Chemistry, 50, 4437
4444.
Jimenez-Alvarez, D., Giuffrida, F., Vanrobaeys, F., Golay, P. A.,
Cotting, C., Lardeau, A., & Keely, B. J. (2008). High-throughput
methods to assess lipophilic and hydrophilic antioxidant
capacity of food extracts in vitro. Journal of Agricultural and
Food Chemistry, 56, 34703477.
Lang, J. (2009). Tocopherol stability in controlled release packaging
films. M.S. thesis, Rutgers University, New Brunswick, NJ.
Lebeau, L., Furman, C., Bernier, J.-L., Duriez, P., Teissier, E., &
Cotelle, N. (2000). Antioxidant properties of di-tert-
butylhydroxylated flavonoids. Free Radical Biology and Medicine,
29, 900912.
Miller, N. J., & Rice-Evans, C. A. (1997). Factors influencing the
antioxidant activity determined by the ABTS+. radical cation
assay. Free Radical Research, 26, 195199.
Miller, N. J., Rice-Evans, C. A., Davies, M. J., Gopinathan, V., &
Milner, A. (1993). A novel method for measuring antioxidant
capacity and its application to monitoring the antioxidant
status in premature neonates. Clinical Science, 84, 407412.
Moridani, M. Y., Siraki, A., & OBrien, P. J. (2003). Quantitative
structure toxicity relationships for phenols in isolated rat
hepatocytes. Chemico-Biological Interactions, 145, 213223.
Ou, B., Hampsch-Woodill, M., Flanagan, J. A., Deemer, E. K., Prior,
R. L., & Huang, D. (2002). Novel fluorometric assay for
hydroxyl radical prevention capacity using fluorescein as
the probe. Journal of Agricultural and Food Chemistry, 50, 2772
2777.
Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development
and validation of an improved oxygen radical absorbance
capacity assay using fluorescein as the fluorescent probe.
Journal of Agricultural and Food Chemistry, 49, 46194626.
Prior, R. L., Hoang, A., Gu, L., Wu, X., Bacchiocca, M., Howard, L.,
Hampsch-Woodill, M., Huang, D., Ou, B., & Jacob, R. (2003).
Assays for hydrophilic and lipophilic antioxidant capacity
(oxygen radical absorbance capacity (ORACFL) of plasma and
other biological and food samples. Journal of Agricultural and
Food Chemistry, 51, 32733279.
Prior, R. L., Wu, X., & Schaich, K. M. (2005). Standard methods for
the determination of antioxidant capacity and phenolics in
foods and dietary supplements. Journal of Agricultural and Food
Chemistry, 53, 42904302.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-
Evans, C. A. (1999). Antioxidant activity applying an improved
ABTS radical cation decolourization assay. Free Radical Biology
and Medicine, 26, 12311237.
Salamone, M., DiLabio, G. A., & Bietti, M. (2011). Hydrogen atom
abstraction reactions from tertiary amines by benzyloxyl and
cumyloxyl radicals: Influence of structure on the rate-
determining formation of a hydrogen-bonded prereaction
complex. The Journal of Organic Chemistry, 76, 62646270.
Salamone, M., Martella, R., & Bietti, M. (2012). Hydrogen
abstraction from cyclic amines by the cumyloxyl and
benzyloxyl radicals. The role of stereoelectronic effects and of
substrate/radical hydrogen bonding. The Journal of Organic
Chemistry, 77, 85568561.
Schauenstein, K., Schauenstein, E., & Wick, G. (1978).
Fluorescence properties of free and protein bound fluorescein
dyes. I. Macrospectrofluorometric measurements. The Journal
of Histochemistry and Cytochemistry, 26, 277283.
796 Journal of Functional Foods 18 (2015) 782796
Selassie, C. D., DeSoyza, T. V., Rosario, M., Gao, H., & Hansch, C.
(1998). Phenol toxicity in leukemia cells: A radical process?
Chemical-Biological Interactions, 113, 175190.
Stratil, P., Klejdus, B., & Kuban, V. (2006). Determination of total
content of phenolic compounds and their antioxidant activity
in vegetables Evaluation of spectrophotometric methods.
Journal of Agricultural and Food Chemistry, 54, 607616.
Strube, M., Haenen, G. R. M. M., van den Berg, H., & Bast, A. (1997).
Pitfalls in a method for assessment of total antioxidant
capacity. Free Radical Research, 26, 515521.
Tian, X., & Schaich, K. M. (2013). Effects of molecular structure on
kinetics and dynamics of the Trolox equivalent antioxidant
capacity assay with ABTS+. Journal of Agricultural and Food
Chemistry, 61, 55115519.
USDA. Oxygen Radical Absorbance Capacity (ORAC) of selected foods.
(2010). <http://www.ars.usda.gov/Services/docs.htm?docid
=15866> Accessed 06.05.15. Release 2 (2010).
van den Berg, R., Haenen, G. R. M. M., van den Berg, H., & Bast, A.
(1999). Applicability of an improved Trolox equivalent
antioxidant capacity (TEAC) assay for evaluation of
antioxidant capacity measurements of mixtures. Food
Chemistry, 66, 511517.
Williams, D. E. (1966). Structure of 2,2-diphenyl-1-picrylhydrazyl
free radical. Journal of the American Chemical Society, 88, 5665
5666.
Wojdyo, A., Figiel, A., & Oszmianski, J. (2009). Effect of drying
methods with the application of vacuum microwaves on the
bioactive compounds, colour, and antioxidant activity of
strawberry fruits. Journal of Agricultural and Food Chemistry, 57,
13371343.
Wu, X. (2005). Standard operating procedure of oxygen radical
absorbance capacity (ORACFL). Provided by R. Prior, Univ.
Arkansas.
Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S.
E., & Prior, R. L. (2004). Lipophilic and hydrophilic antioxidant
capacities of common foods in the United States. Journal of
Agricultural and Food Chemistry, 52, 40264037.
Xie, J., & Schaich, K. M. (2014). Re-evaluation of the 2,2-diphenyl-
1-picrylhydrazyl free radical (DPPH) assay for antioxidant
activity. Journal of Agricultural and Food Chemistry, 62, 4251
4260.
Yen, G.-C., Duh, P.-D., & Tsai, H.-L. (2002). Antioxidant and pro-
oxidant properties of ascorbic acid and gallic acid. Food
Chemistry, 79, 307313.