antioxidants

Review
Oxidative Stress and Antioxidants—A Critical Review on In
Vitro Antioxidant Assays
Raghavendhar R. Kotha 1 , Fakir Shahidullah Tareq 1 , Elif Yildiz 2 and Devanand L. Luthria 1, *

1 Methods and Application of Food Composition Laboratory, Beltsville Human Nutrition Research Center,
Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705, USA
2 Keles Vocational School, Bursa Uludag University, 16740 Bursa, Turkey
* Correspondence: [email protected]; Tel.: +1-301-504-7247; Fax: +1-301-504-8314

Abstract: Antioxidants have been widely studied in the fields of biology, medicine, food, and
nutrition sciences. There has been extensive work on developing assays for foods and biological
systems. The scientific communities have well-accepted the effectiveness of endogenous antioxidants
generated in the body. However, the health efficacy and the possible action of exogenous dietary
antioxidants are still questionable. This may be attributed to several factors, including a lack of basic
understanding of the interaction of exogenous antioxidants in the body, the lack of agreement of the
different antioxidant assays, and the lack of specificity of the assays, which leads to an inability to
relate specific dietary antioxidants to health outcomes. Hence, there is significant doubt regarding
the relationship between dietary antioxidants to human health. In this review, we documented the
variations in the current methodologies, their mechanisms, and the highly varying values for six
common food substrates (fruits, vegetables, processed foods, grains, legumes, milk, and dairy-related
products). Finally, we discuss the strengths and weaknesses of the antioxidant assays and examine
the challenges in correlating the antioxidant activity of foods to human health.

Citation: Kotha, R.R.; Tareq, F.S.;

Keywords: oxidative stress; antioxidants; foods; antioxidant assays limitations; challenges in correlating
Yildiz, E.; Luthria, D.L. Oxidative
with health benefits
Stress and Antioxidants—A Critical
Review on In Vitro Antioxidant
Assays. Antioxidants 2022, 11, 2388.
https://doi.org/10.3390/
1. Introduction
antiox11122388
By definition, antioxidants prevent/inhibit/reduce oxidation processes [1–11]. Histor-
Academic Editor: Mohammad
ically, the industry has used antioxidants to prevent metal corrosion and rubber vulcan-
Hossain
ization [2]. More recently, antioxidants have been used as food preservatives, lubricants,
Received: 4 November 2022 and stabilizers [2]. Allied market research shows the worldwide industrial market value
Accepted: 19 November 2022 for natural and synthetic antioxidants in 2015 was USD 2.9 billion and forecasted more
Published: 1 December 2022 than 50% overall growth to over USD 4.5 billion by 2022. In another study, Grand View
Research forecasted that the global market for natural antioxidants is expected to be USD
Publisher’s Note: MDPI stays neutral
4.1 billion by 2022 [12]. In each case, the functions of these antioxidants meet the strict
with regard to jurisdictional claims in
published maps and institutional affil-
chemical definition and are not associated with bodily functions.
iations.
In recent years, there has been considerable interest in the impact of naturally occurring
phytochemicals in foods that may function as antioxidants in the human body. This interest
has arisen from popular literature that defines oxidants as harmful and antioxidants as
the antithesis and, therefore, healthful. There have been many reviews on all aspects of
Copyright: © 2022 by the authors. antioxidant assays and the relationship of antioxidants to health, including their activity,
Licensee MDPI, Basel, Switzerland. classification, and applications [2,3,5,10,13–18]. Apak et al. [15–17] published reviews
This article is an open access article on various antioxidant assays, their mechanisms, advantages, and limitations. Recently,
distributed under the terms and Gulcin et al. [18] published a detailed review on in vitro antioxidant methods used for the
conditions of the Creative Commons determination of the antioxidant capacity of food constituents. Similarly, Shahidi et al. [3]
Attribution (CC BY) license (https:// and Alam et al. [13] published review articles on in vitro and in vivo antioxidant assays
creativecommons.org/licenses/by/ and their experimental procedures.
4.0/).

Antioxidants 2022, 11, 2388. https://doi.org/10.3390/antiox11122388 https://www.mdpi.com/journal/antioxidants

Antioxidants 2022, 11, 2388 2 of 30

There have also been numerous papers questioning the health benefits of the many
compounds that are in vitro antioxidants and whether they exhibit similar in vivo activ-
ity [19–24]. The U.S. Food and Drug Administration (FDA) and the European Food Safety
Authority (EFSA) allow health claims for vitamin antioxidants (i.e., vitamins A, C, and E,
those with a recommended daily intake (RDI) [25,26]. However, even for these compounds,
there is some controversy with respect to their performance as antioxidants in vivo [22].
In the current review, we briefly describe oxidative stress, types of antioxidants,
and in vitro assays, their mechanisms, their strengths and weaknesses, bioaccessibility,
and bioavailability. We have also compared the analytical variations between the reported
methodologies and activities for six commonly consumed food substrates (fruits, vegetables,
processed foods, grains, legumes, milk, and dairy-related products). It is important for
consumers, nutritionists, and other healthcare professionals to understand the health
benefits gained from the consumption of fruits and vegetables and to distinguish facts from
commercial hype regarding antioxidants.

2. Oxidative Stress
Oxidative stress is defined as the imbalance between the occurrence of reactive oxy-
gen/nitrogen species (ROS/RNS) and cellular antioxidant defenses [1,4]. Oxidative stress
is a result of excess ROS/RNS, which occurs due to a lack of counteraction by cellular
antioxidant systems [1,5]. Increased oxidative stress can have severe consequences in bio-
logical systems, including molecular damage (such as nucleic acids, lipids, and proteins),
which can severely impact health, as shown in Figure 1A [1,5]. Damage to biomolecules
or the induction of several secondary reactive species due to oxidative stress ultimately
leads to cell death (apoptosis or necrosis). It has been assessed that oxidative stress is asso-
ciated with more than 100 diseases, including cardiovascular disease, cancer, hypertension,
diabetes, neurogenerative diseases, aging, etc. [1,5].
Contrary to their harmful effects on health, ROS/RNS can have beneficial effects de-
pending on their function, location, and amount. For instance, superoxide (O2 −• ) and nitric
oxide (• NO) radicals at low or medium concentrations are involved in cellular responses
and participate in signaling pathways [1]. H2 O2 , formed by various oxidase enzymes, and
the action of superoxide dismutase (SOD), allows its use as an important signaling molecule,
also it is substrate for generating further reactive species such as HOCl [27,28]. ROS are
also involved in immunological responses, degrading xeno compounds and organisms
through phagocytosis.
ROS are oxygen-containing molecules, including radicals (like the superoxide anion)
and non-radicals (like H2 O2 ) that greatly vary in their chemical abilities, such as diffusion
in living cells and chemical reactivity with biomolecules. ROS examples include singlet
oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals (Figure 1B). Singlet oxygen
is the highest energy spin state of molecular oxygen. In contrast to molecular oxygen in
ground state, the two valence electrons are paired in an anti-bonding orbital. Singlet oxygen
is therefore only generated, when molecular oxygen is energized via radiation. Importantly,
and in contrast to other ROS subspecies, no electron transfer does occur during this process.
Singlet oxygen is very reactive towards organic compounds and plays a deleterious role in
biological systems, for instance, by involving in the oxidation of LDL cholesterol, which can
lead to cardiovascular diseases. Moreover, increased ROS can trigger mtDNA mutations as
well as promote uncontrolled proliferation and carcinogenesis [29]. The delicate balance of
harmful and beneficial effects of free radicals is crucial for life processes, and antioxidants
play an essential role in achieving this balance.
 logical systems, including molecular damage (such as nucleic acids, lipids, and proteins),
which can severely impact health, as shown in Figure 1A [1,5]. Damage to biomolecules
or the induction of several secondary reactive species due to oxidative stress ultimately
leads to cell death (apoptosis or necrosis). It has been assessed that oxidative stress is as-
Antioxidants 2022, 11, 2388 sociated with more than 100 diseases, including cardiovascular disease, cancer, hyperten- 3 of 30
sion, diabetes, neurogenerative diseases, aging, etc. [1,5].

Antioxidants 2022, 11, x FOR PEER REVIEW 3 of 28

Figure Figure
1. Schematic representation
1. Schematic of (A) radicals’
representation impact impact
of (A) radicals’ on human health, health,
on human and (B)and
the (B)
generation
the generation
of various radicals in vivo.
of various radicals in vivo.

Contrary to their harmful effects on health, ROS/RNS can have beneficial effects de-
3. Antioxidants
pending onAntioxidants
their function,can location, and amount.
be broadly categorized For in
instance, superoxide
many different (O2−•
ways: (i)) and nitric
natural and syn-
•NO) radicals
oxide (thetic; (ii) polaratand
lownon-polar;
or medium(iii)concentrations
enzymatic and arenon-enzymatic;
involved in cellular responses and
(iv) endogenous
and participate
exogenous; in signaling
and (v) bypathways [1]. H2Oin
the mechanisms 2, formed by various
which they oxidase
are involved enzymes,
[30]. and pri-
Antioxidants
the action of superoxide dismutase (SOD), allows its use as an important signaling
marily exhibit activities based on three mechanisms, hydrogen atom transfer, single electron mole-
cule, also it is substrate
transfer, and metal forchelation
generating further
[10]. Theyreactive
show theirspecies suchthrough
activity as HOClthree
[27,28]. ROS path-
different
are also involved in immunological responses, degrading xeno compounds
ways: (i) preventive: prevention of free radical formation and derivatives; (ii) interruption:and organ-
isms through
interrupt phagocytosis.
radical oxidation reactions; and (iii) inactivation: inactivate free radical/radical
ROS are oxygen-containing
derivative reaction products molecules, includingantioxidants
[30]. Endogenous radicals (like arethe superoxide
primarily anion)such as
enzymes,
and non-radicals
superoxide(like H2O2) that
dismutase greatly
(SOD), vary(CAT),
catalase in their chemical abilities,
glutathione reductase such
(GR),as and
diffusion
glutathione
in living cells and(GPx).
peroxidase chemicalOnreactivity
the other with
hand,biomolecules.
non-enzymatic ROS examples include
endogenous antioxidants,singletsuch as
oxygen, superoxide,
glutathione andhydrogen peroxide,
lipoic acid, and hydroxyl
are products radicalsmetabolism
of the body’s (Figure 1B).[2,31].
Singlet Theoxy-
first-line
gen is the highest
defense energy spin
antioxidants state of molecular
(enzymatic) oxygen.superoxide
convert reactive In contrastand to molecular
hydrogenoxygenperoxide into
in ground
waterstate, the two The
and oxygen. valence electrons are
non-enzymatic paired in an
antioxidants cananti-bonding orbital.defense
act as a second-line Singletagainst
oxygenROS by rapidly
is therefore inactivating
only generated, radicals
when and oxidants.
molecular The enzymatic
oxygen is energized antioxidants
via radiation.further act
as the third-line
Importantly, defense
and in contrast involved
to other ROSin the detoxifying
subspecies, and removal.
no electron Dietary
transfer does occurantioxidants,
dur-
ing this process. Singlet oxygen is very reactive towards organic compounds and plays a
deleterious role in biological systems, for instance, by involving in the oxidation of LDL
cholesterol, which can lead to cardiovascular diseases. Moreover, increased ROS can trig-
ger mtDNA mutations as well as promote uncontrolled proliferation and carcinogenesis
[29]. The delicate balance of harmful and beneficial effects of free radicals is crucial for life
Antioxidants 2022, 11, x FOR PEER REVIEW 4 of 28

Antioxidants 2022, 11, 2388 peroxide into water and oxygen. The non-enzymatic antioxidants can act as a second-line
4 of 30
defense against ROS by rapidly inactivating radicals and oxidants. The enzymatic antiox-
idants further act as the third-line defense involved in the detoxifying and removal. Die-
tary antioxidants, such as vitamins, carotenoids, polyphenols, flavonoids, and bioflavo-
such as vitamins, carotenoids, polyphenols, flavonoids, and bioflavonoids (Figure 2), are
noids (Figure 2), are exogenous antioxidants that have in vivo activity [30].
exogenous antioxidants that have in vivo activity [30].

Figure 2.2.
Figure Different approaches
Different approachesfor
forthe
theclassification
classificationofofantioxidants.
antioxidants.

4.4.Antioxidant
AntioxidantAssays Assays
Various analytical
Various analytical methods methods have
have been
been developed
developed to evaluate
to evaluate the the antioxidant
antioxidant prop-
proper-
erties
ties of plant-based
of plant-based phytochemicals
phytochemicals [11,13,32,33].
[11,13,32,33]. The antioxidant
The antioxidant activityactivity
depends depends
on theiron
their chemical structure; specifically, it depends on their ability to donate hydrogen with
chemical structure; specifically, it depends on their ability to donate hydrogen with elec-
electron, metal chelation, and their ability to delocalize the unpaired electron within the
tron, metal chelation, and their ability to delocalize the unpaired electron within the aro-
aromatic structure. Numerous analytical methods for evaluating each aspect of their an-
matic structure. Numerous analytical methods for evaluating each aspect of their antiox-
tioxidant action, including either in vitro or in vivo, have been reported and discussed in
idant action, including either in vitro or in vivo, have been reported and discussed in the
the literature [34].
literature [34].
Antioxidant assays can be categorized into five mechanistic pathways, as summarized
Antioxidant assays can be categorized into five mechanistic pathways, as summa-
in Figure 3.
rized in Figure 3.
(i) Electron transfer-based assays: In these assays, a single electron transfer occurs be-
(i) Electron transfer-based assays: In these assays, a single electron transfer occurs
tween the antioxidant and substrate, which is measured to assess the potential of the plant’s sec-
between the antioxidant and substrate, which is measured to assess the potential of the
ondary metabolites. Assays like cupric ion reducing antioxidant capacity (CUPRAC) [35–37],
plant’s secondary metabolites. Assays like cupric ion reducing antioxidant capacity (CU-
N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) [38], ferric reducing-antioxidant
PRAC)
power[35–37],
(FRAP) N,N-dimethyl-p-phenylenediamine dihydrochloride
[39,40], Folin-Ciocalteu (FC), Trolox equivalent (DMPD)
antioxidant [38], (TEAC)
capacity ferric
reducing-antioxidant power (FRAP) [39,40], Folin-Ciocalteu (FC),
method/ABTS radical cation decolorization assay [41] come under this category. Trolox equivalent anti-
oxidant capacity (TEAC) method/ABTS radical cation decolorization assay
(ii) Hydrogen atom transfer-based assays: In these assays, a hydrogen atom transfers [41] come un-
der
fromthisthecategory.
antioxidant to the substrate. Assays such as oxygen radical absorbance capacity
(ORAC) [42] and atom
(ii) Hydrogen transfer-based assays:
total radical-trapping In theseparameter
antioxidant assays, a hydrogen
(TRAP) [43] atom transfers
methods fall
from the antioxidant
under this category. to the substrate. Assays such as oxygen radical absorbance capacity
(ORAC) (iii)[42] and total radical-trapping
Electron/hydrogen antioxidant
atom transfer (mixed) parameter (TRAP)In[43]
based assays: methods
these assays,fall
the
under this category.
hydrogen atom transfer occurs via two-step mechanisms (Figure 3(iii)). Assays like DPPH
(iii) Electron/hydrogen
scavenging activity [39,44,45] atomandtransfer (mixed)
TEAC follow based
this assays: In these assays, the hy-
mechanism.
drogen(iv) atom transfer
Metal occurs viaassays:
chelation-based two-step mechanisms
In these (Figure 3(iii)).
assays, antioxidants Assays
chelate liketransition
with DPPH
scavenging activity [39,44,45] and TEAC follow this mechanism.
metals like Fe(II) and Cu(II). Ferrous ion and cuprous ion chelating activity are examples of
this(iv) Metal chelation-based assays: In these assays, antioxidants chelate with transition
category.
metals(v). likeLipid
Fe(II)oxidation
and Cu(II). andFerrous
ROS/RNS ion and cuprousactivity
scavenging ion chelating
assays: activity are examples
These assays are based
ofonthis
thecategory.
ability of antioxidants in reducing/preventing lipid oxidation and scavenging ROS
and(v).RNS.Lipid oxidation
β-carotene and ROS/RNS
linoleic scavenging activity
acid method/conjugated assays:
diene assayThese
[45], assays are based
ferric thiocyanate
onmethod
the ability of [39,46],
(FTC) antioxidants in reducing/preventing
thiobarbituric acid method (TBA), lipid oxidation
hydrogen and scavenging
peroxide (H2 O2 )ROS
scav-
enging assay [39,47], hydroxyl radical averting capacity method (HORAC) [42], nitric
oxide scavenging activity [48], peroxynitrile radical scavenging activity, superoxide radical
scavenging activity (SRSA/SOD), and xanthine oxidase methods come under this category.
 and RNS. β-carotene linoleic acid method/conjugated diene assay [45], ferric thiocyanate
method (FTC) [39,46], thiobarbituric acid method (TBA), hydrogen peroxide (H2O2) scav-
enging assay [39,47], hydroxyl radical averting capacity method (HORAC) [42], nitric ox-
Antioxidants 2022, 11, 2388 ide scavenging activity [48], peroxynitrile radical scavenging activity, superoxide radical 5 of 30
scavenging activity (SRSA/SOD), and xanthine oxidase methods come under this cate-
gory.
(vi). Enzymatic antioxidant assays: Antioxidant enzyme systems that catalyze reac-
(vi). Enzymatic antioxidant assays: Antioxidant enzyme systems that catalyze reac-
tionstotocounterbalance
tions counterbalancefree
freeradicals
radicals and
and reactive
reactive oxygen
oxygen species
species include
include superoxide
superoxide dis-
dismu-
mutase and catalase. Catalase [49,50], ferric reducing ability of plasma [40,51,52],
tase and catalase. Catalase [49,50], ferric reducing ability of plasma [40,51,52], γ-glutamyl γ-glu-
tamyl transpeptidase
transpeptidase [53], glutathione
[53], glutathione peroxidase peroxidase
estimationestimation [49,54], glutathione
[49,54], glutathione (reduced) GSH (re-
estimation [55], glutathione-S-transferase [56,57], LDL assay [58], lipid peroxidation pe-
duced) GSH estimation [55], glutathione-S-transferase [56,57], LDL assay [58], lipid as-
roxidation
say [59], andassay [59], anddismutase
superoxide superoxidemethod
dismutase
[60] method [60] can be categorized
can be categorized under
under enzymatic
enzymatic antioxidant
antioxidant assays. assays.

(i) Single electron transfer (SET)

ArOH + X ArO + X

(ii) Hydrogen atom transfer (HAT)

ArOH + X ArO + XH

(iii) Hydrogen atom transfer via

(a) Proton-coupled electron transfer (PCET)

ArOH ArO + H

ArO H + X ArO + XH

(b) Single electron transfer-proton transfer (SET-PT)

ArOH + X ArOH + X

ArOH3 ArO + H

(iv) Metal chelation

OH 2+
pH 6, Fe O
R Fe + 2H
OH O

(v) ROS/RNS Scavenging activity/ Lipid oxidation

Figure3.3.Mechanistic
Figure Mechanisticaspects
aspectsof
ofantioxidant
antioxidantassays
assaysboth
bothin
invitro
vitroand
andininvivo.
vivo.

Table
Table11summarizes
summarizesthe thevarious
variousmethods
methodsusedusedtotomeasure
measureantioxidant
antioxidantactivity.
activity.The
The
principles, advantages, and limitations associated with each method, along
principles, advantages, and limitations associated with each method, along with recent with recent
references,
references,are
arealso
alsopresented
presented[13,14,18,36,46,51,61–88].
[13,14,18,36,46,51,61–88].The advantages
The advantagesof of
electron transfer
electron trans-
assays include
fer assays faster
include reaction
faster reaction rates, ease
rates, of of
ease experimentation,
experimentation,sample
samplethroughput,
throughput,and and
reproducibility. main advantages
reproducibility. The main advantagesassociated
associatedwith
withhydrogen
hydrogen atom
atom transfer
transfer assays
assays in-
include close
clude close physiological
physiological resemblance,
resemblance, taking
taking initiation
initiation andand propagation
propagation intointo account,
account, and
and uses of physiologically relevant radicals. Moreover, ORAC assay can be performed for
antioxidants with a wide range of polarities, from lipophilic to hydrophilic [64]. Similarly,
ROS/RNS scavenging activity/lipid oxidation assays, ET/HAT mixed, metal chelation,
and lipid peroxidation inhibition assays have the advantages listed in Table 1.
Antioxidants 2022, 11, 2388 6 of 30

Table 1. Various in vitro and in vivo antioxidant assays, their principles, advantages, and limitations.

Mechanism (Category) Assay Technique/Principle Advantages Limitations Ref.

In vitro
In this assay, phenolic groups in the
CUPRAC (Cupric ion (+) Copper reaction rates are faster
polyphenols are oxidized to quinones, (−) it ignores the
reducing antioxidant than that of ferric ions, and it is [36]
whereas Cu(II) is reduced to Cu(I), reaction kinetics
capacity method) more specific for antioxidants
which is measured at 450 nm.
DMPD (−) it ignores the reaction
In the presence TROLOX, reduction of
(N,N-dimethyl-p-phenylene kinetics, and the DMPD
DMPD radical cation by antioxidants, (+) easy, cheaper, and reproducible [62]
diamine dihydrochloride) radical is a
the absorbance at 505 nm is decreased.
method non-physiological radical
(+) a good representation of
electron transfer mechanism
FRAP (Ferric Antioxidants at low pH reduce (−) it ignores the reaction
(+) the is inexpensive, easy to
reducing-antioxidant ferric-tripyridyltriazine (FeIII -TPTZ)to kinetics and non-specific [51]
Electron transfer (Total prepare reagents, reproducibility,
power assay) FeII form which is measured at 593 nm. to antioxidants
Antioxidant Capacity) and speedy and a straight
forward procedure
In this assay, phenols are oxidized in a
basic medium by a mixture of
(−) it ignores the reaction
Follin-Ciocalteu tungstate and molybdate
(+) easy and reproducible kinetics and non-specific [14]
reducing capacity (Folin-Ciocalteu reagent), with the
to antioxidants
consequent formation of colored
molybdenum ions, MoO4 + (750 nm).
Upon reaction with an antioxidant
(−) it ignores the reaction
(Trolox), ABTS (2,2-azo-bis(3-
Trolox equivalent antioxidant kinetics, and the ABTS is a
ethylbenz-thiozoline-6-sulfonic acid)) (+) it can screen both hydrophilic
capacity (TEAC) method non-physiological radical
radical cation, which is a blue-green and lipophilic antioxidants, easy [63]
(ABTS radical cation (−) The assay is not suitable
chromophore, reduces and decolorized. and reproducible
decolorization assay) for the determination of
This assay uses a diode-array
proteins antioxidant activity
spectrophotometer at 750 nm.
Antioxidants 2022, 11, 2388 7 of 30

Table 1. Cont.

Mechanism (Category) Assay Technique/Principle Advantages Limitations Ref.

AAPH (2,2-azobis-2-aminopropane
(−) lack of consistency and
dihydrochloride) decomposition
the possible underestimation
induces peroxyl radicals, and radical
of antioxidant activity as
scavengers are used to measure the
(+) physiologically resemble B-PE can interact with
ORAC (Oxygen radical decrease in fluorescence. AAPH is
method, and it takes initiation and phenolic acids [64]
absorbance capacity) method used as a radical generator and Trolox
propagation into account (−) The method has been
as the antioxidant control. 485 nm is
reported to fail determining
used as the excitation wavelength, and
Hydrogen Atom Transfer both hydrophilic and
520 nm is used as the
(Antioxidant Activity) lipophilic antioxidants
emission wavelength.
In this method, the antioxidant
potential is assessed by measuring the
TRAP (Total radical-trapping
decay in decoloration. ABAP (+) peroxyl radical is a common (−) detection probe(oxygen)
antioxidant parameter
(2,20 -azo-bis(2-ami-dino- and physiologically that is not stable and may [65,67,68]
method), both in vivo and
propane)hydrochloride) is a radical representative radical cause issues in measurements
in vitro
initiator that quenches the fluorescence
of R-Phycoerythrin (R-PE).
The ROS oxidizes linoleic acid, and the
resulting products initiate β-carotene
β-carotene linoleic acid (+) shows a strong correlation with
oxidation, which leads to discoloration. (−) lack of reproducibility
method/conjugated the total phenolics measured by the [14,68,69]
In the presence of antioxidants, the and crude kinetic treatment
diene assay F-C method.
discoloration will be delayed and
measured at 434 nm.
During linoleic acid peroxidation,
peroxides were formed, which oxidize (+) used to measure peroxide
ROS/RNS scavenging Ferric thiocyanate
Fe(II) to Fe(III). The Fe(III) reacts with amount at the starting phase (−) lack of specificity [46,70,71]
activity/lipid oxidation (FTC) method
thiocyanate to form a red color of peroxidation
complex, which is measured at 500 nm.
In this assay, TBA and trichloroacetic
acid are mixed with the sample (+) used to measure the
Thiobarbituric acid solution, placed in the hot water bath concentration of free radicals
(−) Not specific [14]
(TBA) method for 10 min, centrifuged in the solution, present at the end of
and supernatant absorbance activity is peroxide oxidation
measured at 552 nm.
Antioxidants 2022, 11, 2388 8 of 30

Table 1. Cont.

Mechanism (Category) Assay Technique/Principle Advantages Limitations Ref.

(−) most plant and food
Antioxidants reduce hydrogen samples also absorb at this
Hydrogen peroxide peroxide concentration, which is wavelength, which can
[72]
scavenging (H2 O2 ) assay measured at 230 nm using a compromise both the
spectrophotometer. precision and accuracy of
the method
In the presence of antioxidant, the
Hydroxyl radical degraded product of deoxyribose (+) Useful for ketone (−) Higher concentration of
[73]
scavenging activity (TBARS) measured colorimetrically at containing antioxidants antioxidants required
532 nm.
Under aerobic conditions, nitric oxide
reacts with oxygen to form nitrate and (+) relatively simple (−) detection technique is not
Nitric oxide
nitrite, which can be quantified using experimentation and easily available and has a [18,74]
scavenging activity
Griess reagent, and the absorbance is physiologically relevant long reaction time
measured at 546 nm.
(−) Under anaerobic
ONOO.scavenging activity is
conditions, nitric oxide did
measured by the oxidation of
(+) Peroxynitrile is a good not oxidize
Peroxynitrile radical dihydroxyrhodamine to rhodamine
oxidizing agent for dihydrorhodamine and [75]
scavenging activity fluorescence spectrophotometer with
dihydroxyrhodamine inhibited spontaneous
an excitation wavelength of 485 nm
oxidation of
and emission wavelength of 530 nm.
dihydrorhodamine
This assay is based on the removal rate
(−) irreproducibility due to
Superoxide radical of superoxide radical (O2 − ) using (+) peroxyl radical is a common
the water insolubility issue of
scavenging activity antioxidants, which is measured by and physiologically [76,77]
diformazan, the end product
(SRSA/SOD) nitro blue tetrazolium (NBT) at representative radical
of NBT reduction
560 nm.
Xanthine is a substrate in XOD-
catalyzed reaction, which yields uric
Xanthine oxidase (−) Enzyme collection
acid as a product. Allopurinol is used (+) Possible to get kinetics [8,78]
inhibition assay is tricky
as a xanthine oxidase inhibitor,
measured at 293 nm.
Antioxidants 2022, 11, 2388 9 of 30

Table 1. Cont.

Mechanism (Category) Assay Technique/Principle Advantages Limitations Ref.

1-diphenyl-2-picrylhydrazyl
(α,α-diphenyl-β-picrylhydrazyl;
DPPH) is a stable free radical due to (−), difficult to get the
electron delocalization, which prevents reaction kinetics, and the
ET/HAT_mixed DPPH scavenging activity (+) easy and reproducible [66]
its dimerization. DPPH reacts with DPPH radical is a
antioxidants, which diminishes its non-physiological radical
deep violet color, which is measured at
517 nm (515–518 nm).
(+) High sensitivity, correlated with
structure-activity relationships,
TAC assay obtained
higher molar absorptivity,
via reduction of Fe(III) to Fe(II), and
Ferrous ion chelating activity relatively lower interference (−) Not correlated with
formed Fe(II) is determined with [87,88]
assay/Ferrozine assay-Fe(II) from foreign FRAP, DPPH, and TPC
ferrozine using spectrophotometric
ions, wide pH tolerance, complex
absorbance measurement at 562 nm.
stability constant, water solubility,
Metal chelation and low viscosity
(+) good repeatability and
Cuprous ion chelating Free Cu(II) that is not complexed with reproducibility, Cu2+ chelating
activity/Pyrocatechol antioxidants is bound to Pyrocatechol, ability is significantly and [88]
violet-Cu(II) which is assessed at 632 nm. positively correlated to DPPH,
FRAP, and total phenolic content
In vivo
The catalase activity is measured in an
erythrocyte lysate as the difference in
absorbance (λ240 ) per unit as the H2 O2 (+) a good representation of
Hydrogen atom transfer Catalase (CAT) [78]
maximum absorption wavelength is physiological conditions
240 nm. Catalase activity is used both
in vivo and in vitro.
Antioxidants 2022, 11, 2388 10 of 30

Table 1. Cont.

Mechanism (Category) Assay Technique/Principle Advantages Limitations Ref.

This assay is primarily based on the
principle that, at low pH,
(+) most simple, rapid, inexpensive
Electron transfer/reducing ferric-tripyridyltriazine (FeIII -TPTZ) is (−) it ignores the reaction
Ferric reducing ability of tests and very useful for routine
power (Total reduced to Fe(II). The antioxidant kinetics and non-specific [51,79]
plasma (FRAP) analysis, a good representation of
Antioxidant Capacity) capacity is measured using the to antioxidants
electron transfer mechanism
increased FeII , which is measured
spectrophotometrically at 593 nm.
GGT transfers the γ-glutamyl group
from the L-γ-Glutamyl-p-nitroanilide
γ-glutamyl transpeptidase (+) a good representation of
and liberates the chromogen [80]
(GGT) physiological conditions
p-nitroanilide (pNA, 418 nm)
proportional to the GGT present.
GSHPx is a seleno-enzyme that
catalyzes the reaction of
Glutathione peroxidase (+) a good representation of
Lipid peroxidation inhibition hydroperoxides with GSH to form [13,85]
(GSHPx) estimation physiological conditions
GSSG and reduction of
hydrogen peroxide.
GR catalyzes the reduction of GSSG to
GSH. GR activity is determined at 340
nm and 412 nm.
Glutathione reductase One may expect a decrease of activity (+) a good representation of
[13,81]
(GR) assay at 340 nm as a result of the oxidation of physiological conditions
NADPH or an increase at 412 nm
caused by the reduction of dithiobis
(2-nitrobenzoic acid) DTNB.
This assay utilizes
1-Chloro-2,4-dinitrobenzene (CDNB).
Potassium phosphate, GSt, and CDNB
Glutathion-S-transferase (+) a good representation of
mixture are incubated at 37 C, pH 6.5 [13]
(GSt) physiological conditions
for 5 min, followed by adding
substrate. 340 nm absorbance is used
for monitoring the assay.
Antioxidants 2022, 11, 2388 11 of 30

Table 1. Cont.

Mechanism (Category) Assay Technique/Principle Advantages Limitations Ref.

The extent of low-density lipoprotein
(LDL) oxidation is determined by the (−) limitations in the
amount of lipid peroxides, also by (+) LDL is a true representation isolation of LDL from the
LDL assay [13,83–85]
using a thiobarbituric acid reactive of physiologically blood, and it is difficult to
substances (TBARS) assay determined monitor the lag phase
at 532 nm.
Malondialdehyde (MDA) is one of the
Lipid peroxidation end products of lipid peroxidation, (+) a good representation of
Lipid peroxidation inhibition [84,85]
(LPO) assay which is used for the LPO assay physiological conditions
measured at 586 nm.
The SOD assay works based on the
Superoxide dismutase (+) a good representation of
absorbance change at 420 nm related [86]
(SOD) method physiological conditions
to pyrogallol.
Antioxidants 2022, 11, 2388 12 of 30

The major limitation associated with all these assays is their lack of specificity. How-
ever, specific limitations include the solubility of antioxidants in the extraction solvent,
interferences with coloring substances and other reducing phytochemicals, ignoring reac-
tion kinetics, and not representing the physiological radicals used in these assays.
A second major limitation is the lack of equivalence of the methods. It is not possible
to convert ORAC to FRAP values with a simple proportionality factor. As shown in the next
section, some foods high in FRAP values may be low in ORAC values, and the opposite can
be true. This situation is best summed up by stating that the FRAP assay generates FRAP
values, ORAC generates ORAC values, DPPH generates DPPH values, etc., and there are
no equivalency factors.

5. Antioxidant Activity of Selected Prominent Foods

We have summarized (Table 2) [89–121] peer-reviewed literature reports on the an-
tioxidant activity/capacity data for six prominent groups of food and related products
that are consumed globally: fruits (apples and berries), vegetables (spinach and olives),
processed products (wine, coffee, and tea), dairy products (milk and yogurt), legumes
(soybeans, beans), and grains (wheat and corn), documented by different researchers using
various assay procedures. Results from each group are separately presented below. In
each case, antioxidant assay methods are used to document changes in composition and
emphasize the impact of genetics and processing on composition. However, it must be
remembered that a specific antioxidant activity can be achieved in literally multiple ways
by different possible combinations of components. Without data for specific components, it
is impossible to relate antioxidant values to composition or health outcomes.

Table 2. Variations in the in vitro antioxidant activity for six prominent groups of food and related
products that are consumed globally: fruits (apple and berries), vegetables (spinach and olives),
processed products (wine, coffee, and tea), dairy products (milk and yogurt), legumes (soybeans,
beans), and grains (wheat and corn), as documented in peer-reviewed published literature.

Samples Matrix Assay Results Ref.

6.82 mg GAE/g fw for Benoni cultivars
TPC
from the location Mukhwa

Fresh apple DPPH 10.87 mmol AAE/kg fw [89]

ABTS 24.57 mmol AAE/kg fw
FRAP 24.05 mmol AAE/kg fw
TPC 4.18 ± 0.1 mg GAE/g dw
DPPH 22.14 ± 1.2 µmol TE/g dw
Fresh apple [96]
FRAP 26.98 ± 0.9 µmol TE/g dw
ABTS 32.85 ± 1.5 µmol TE/g dw
Fruits
TPC 0.48 g GAE/kg
Apple
Apple peel DPPH 121 mol TEAC/kg [102]
ABTS 13 mol TEAC/kg
8.00 mg GAE/g fw in peel
TPC
6.64 mg GAE/g fw in pulp

Wild apples peel and pulp IC50 : 240.00 ± 6.00 µg/mL peel
DPPH [97]
(ultra-sonic extract) IC50 : 286.00 ± 7.00 µg/mL pulp
IC50 : 134.00 ± 3.00 µg/mL peel
ABTS
IC50 : 167.00 ± 4.00 µg/mL pulp
Antioxidants 2022, 11, 2388 13 of 30

Table 2. Cont.

Samples Matrix Assay Results Ref.

3.48 ± 0.12 mg GAE/g apple pomace
TPC
for MeOH extract

Apple pomace DPPH 72.6 ± 1.6% (Inhibition) [98]

FRAP 65.8 ± 1.8% (Inhibition)
ABTS 84.3 ± 1.6% (Inhibition)
TPC 143.84 ± 37.79 mg GAE/g
DPPH 259.68 ± 46.91 µmol TE/g
Apple leaves [99]
ABTS 625.26 ± 141.31 µmol TE/g
FRAP 328.02 ± 130.38 µmol TE/g
TPC 443.60 ± 17.00 mg GAE/g
87.90 ± 0.20% inhibition (100 µg/mL);
DPPH
Blueberry IC50 1.40 ± 0.10 µg/mL
23.10 ± 0.60% inhibition (100 µg/mL);
ABTS
IC50 14.00 ± 0.50 µg/mL
TPC 269.5 ± 16 mg GAE/g
77.80 ± 2.00% inhibition (100 µg/mL);
DPPH
Blackberry IC50 1.30 ± 0.10 µg/mL
25.30 ± 1.10% inhibition (100 µg/mL);
ABTS
IC50 23.00 ± 5.00 µg/mL
TPC 965.60 ± 2.90 mg GAE/g
89.03 ± 0.040% inhibition (100 µg/mL); [100]
DPPH
Black raspberry IC50 3.40 ± 0.40 µg/mL
Berries ABTS 21.3 ± 1% (per 100 µg/mL);
IC50 79.00 ± 18.07 µg/mL
TPC 434.3 ± 6.3 mg GAE g−1
87 ± 1.2% inhibition (100 µg/mL);
DPPH
Red raspberry IC50 1.40 ± 0.10 µg/mL
31.1 ± 0.6% inhibition (100 µg/mL);
ABTS
IC50 15.00 ± 0.90 µg/mL
TPC 250.10 ± 17.10 mg GAE/g
70.20 ± 1.00% inhibition (100 µg/mL);
DPPH
Strawberry IC50 3.1 ± 0.02 µg/mL
26.20 ± 0.70% inhibition (100 µg/mL);
ABTS
IC50 9.9 ± 0.40 µg/mL
TPC 24.50 ± 0.69 mg GAE/g
ABTS 127.00 ± 5.30 µmol TE/g
Lowbush blueberry [103]
389.00 ± 19.40 µmol FeSO4
FRAP
equivalent/g
DPPH 36.71% inhibition (180 µg sample/mL)
Dried, powdered ABTS 68.34% inhibition (180 µg sample/mL) [91]
FRAP 0.14% inhibition (180 µg sample/mL)
Vegetables
Spinach DPPH EC50 42–50% inhibition
Gamma irradiated (above
0.75 kGy) FRAP EC50 0.48–0.7% inhibition [104]
samples TPC 208.9–216.2 mg GAE/g
Antioxidants 2022, 11, 2388 14 of 30

Table 2. Cont.

Samples Matrix Assay Results Ref.

DPPH 68.51 ± 0.89% inhibition
ABTS 70.12 ± 0.04% inhibition
Polysaccharides [90]
1590 ± 53.98 µmol/L at 10 mg/mL BHT
FRAP
and AA
TPC 31.52 mg GAE/g
Lyophilized table Olive; ABTS 308.68 µmol TE/g [92]
methanol extract
DPPH 228.46 µmol TE/g
DPPH 69.15 ± 0.06% Inh
β-carotene
54.98 ± 0.03%
bleaching
Leaves; ethanol extract [105]
Olives
TPC 82.63 ± 0.02 mg AAE/g extract
FRAP 07.53 ± 0.06 mol Fe2+ /g extract
TPC ~4.50 mg GAE/g dw
ABTS ~12 µmol TE/g dw
Sprouted olive seeds [106]
DPPH ~11 µmol TE/g dw
FRAP ~9 µmol TE/g dw
TPC 317.62 ± 18.75 mg/mL
DPPH 3.16 ± 0.15 mg GAE/mL
Red wine [107]
ABTS 7.10 ± 0.75 mg TE/mL
FRAP 8.20 ± 0.76 mg TE/mL
FRAP 336.70 ± 15.20 µmol TE
DPPH 2103.30 ± 115.60 µmol TE
Processed food
Wine Standard white wine ABTS 3037.50 ± 333.30 µmol TE [108]
ORAC 4756.70 ± 41.20 µmol TE
TPC 305.30 ± 3.40 mg GAE/L
FRAP 0.33 ± 0.01 µmol TE/g dry residue
Merlot wines from Serbia
and Spain DPPH 0.16 ± 0.01 µmol TE/g dry residue [109]
* Red wine ABTS 0.35 ± 0.03 µmol TE/g dry residue
DPPH ~13.00% RSA at 0.5 mg/mL sample
Green coffee- light roasted
ABTS ~90.00% RSA at 0.5 mg/mL sample
Green coffee- DPPH ~10.00% RSA at 0.5 mg/mL sample
medium roasted [110]
ABTS ~90.00% RSA at 0.5 mg/mL sample
Green coffee- DPPH ~6.50% RSA at 0.5 mg/mL sample
French roasted ABTS ~90.00% RSA at 0.5 mg/mL sample
Coffee
TPC 13.94 ± 0.2 mg GAE/g dm
Filtered coffee,
DPPH 82.40 ± 2.86 µmolTE/g dm
water extract
ABTS 140.31 ± 2.80 µmolTE/g dm
[111]
TPC 23.43 ± 0.06 mg GAE g−1 dm
Defatted coffee DPPH 110.33 ± 1.97 µmol TE/g dm
ABTS 218.38 ± 0.55 µmol TE/g dm
Antioxidants 2022, 11, 2388 15 of 30

Table 2. Cont.

Samples Matrix Assay Results Ref.

FRAP 2670.13 ± 34.02 µmol Fe2+ /g dw
Black Tea
TEAC 994.56 ± 12.64 µmol Trolox/g dw
(Dianhong Congou)
TPC 101.29 ± 1.58 mg GAE/g dw
[112]
FRAP 4647.47 ± 57.87 µmol Fe2+ /g dw
Green Tea (Dianqing Tea) TEAC 2532.41 ± 50.18 µmol Trolox/g dw
TPC 252.65 ± 4.74 mg GAE/g dw
TPC 0.37 ± 0.02 mg GAE/mL at 90 ◦ C temp
Tea Green Tea leaves
DPPH 42.4 ± 2.6% RSA at 90 ◦ C temp
TPC 0.64 ± 0.02 mg GAE/mL at 90 ◦ C temp
Green Teabags
DPPH 70.3 ± 3.4% RSA at 90 ◦ C temp
[113]
TPC 0.19 ± 0.00 mg GAE/mL at 90 ◦ C temp
Black Tea leaves
DPPH 20.7 ± 1.5% RSA at 90 ◦ C temp
TPC 0.50 ± 0.02 mg GAE/mL at 90 ◦ C temp
Black Teabags
DPPH 36.0 ± 2.0% RSA at 90 ◦ C temp
TPC 21.9 ± 2.8 mg GAE/g
TAC 648 ± 18 (U/g)
Chickpea—60%
OH scavenging [101]
ethanol extract 66.22 ± 0.09%
capacity
Legumes DPPH ~15% RSA
Beans
TPC 60.09 ± 4.17 mg GAE/100 g
ORAC 52.73± 0.96 mg TE/g dry base
Chickpea aqueous extract [114]
OH scavenging
56.36 ± 1.54%
capacity
42.2 ± 2.23–50.40 ± 1.00 mg CE/g
extract
TPC
1.40 ± 0.04 to 1.95 ± 0.00 mg CE/g fw
for seven growth stages
177.00 ± 11.00–245.00 ± 21.00 µmol
TE/g extract
The aerial part of TEAC
6.26 ± 0.41–8.43 ± 1.28 µmol TE/g fw [115]
the soybean for seven growth stages
623.00 ± 3.00–780.00 ± 0.700 µmol
Fe2+ /g extract
FRAP
21.4 ± 2.6–28.5 ± 0.7 µmol Fe2+ /g fw
for seven growth stages
Soybeans
DPPH EC50 : 0.125–0.22 mg/mL
TPC 2.20 ± 0.03 mg GAE/g
Fermented (by ABTS 59.61 ± 6.68 µmol TE/g
M. purpureus) defatted [93]
FRAP 14.26 ± 0.44 µmol TE/g
soybean flour
DPPH 0.74 ± 0.02 µmol TE/g
TPC 3.71–6.83 mg GAE/g
Water-soluble black ABTS IC50 : 1.72–3.48 mg/mL
soybean polysaccharide [116]
from sprouted seeds DPPH IC50 : 4.45–8.00 mg/mL
Reducing power IC50 : 3.42–5.84 ± 0.12 mg/mL
Antioxidants 2022, 11, 2388 16 of 30

Table 2. Cont.

Samples Matrix Assay Results Ref.

TPC 9.06 ± 0.07 GAE/kg
Grounded purple corn DPPH IC50 : 66.3 ± 0.80 µg/mL
extracted with acidified [94]
Grains 80:20 methanol: water ABTS IC50 : 250 ± 0.40 µg/mL
Corn
FRAP 26.10 ± 0.04 µmol TE/g
TPC ~1230–1410 µg GAE/g dm
Corn [95]
DPPH 37–45% RSA
TPC 1556.11 ± 20.42 µg FAE/g
Whole fresh flour DPPH 4.68 ± 0.45 µmol TE/g [117]
FRAP 42.09 ± 2.82 µmol Fe2+ /g
TPC 26.01 ± 0.40 mg GAE/g

Wheat aleurone- water DPPH 147.85 ± 8.54 µmol TE/g WEAX

extract (WA-f50) ABTS 355.26 ± 0.01 µmol TE/g WEAX
ORAC 527.47 ± 13.21 µmol TE/g WEAX
[118]
TPC 16.78 ± 0.35 mg GAE/g

Wheat bran- water extract DPPH 106.29± 12.13 µmol TE/g WEAX
Wheat
(WA-f50) ABTS 320.40 ± 21.06 µmol TE/g WEAX
ORAC 484.91 ± 34.15 µmol TE/g WEAX
DPPH 3.1 µmol TE/g
TEAC 1.3 µmol TE/g
Whole grain flour Peroxyl
scavenging 0.55 mmol TE/g
capacity [119]

DPPH 6.7 µmol TE/g

Wheat bran TEAC 2.6 µmol TE/g
ORAC 1.05 µmol TE/g
Whole milk (UHT):
14,481± 328 µmol TE
Deproteinized Milk (UHT):
129 ± 5.9 µmol TE
Whole milk (Pasteurized):
14,216 ± 1051 µmol TE
Deproteinized (Pasteurized):
Milk and Dairy 464 ± 21.4 µmol TE
Milk ORACFL [120]
products Lowfat milk (UHT):
13,874 ± 312 µmol TE
Lowfat Deproteinized Milk (UHT):
35 ± 2.2 µmol TE
Lowfat milk (Pasteurized):
13,748 ± 397 µmol TE
Deproteinized milk (Pasteurized):
610± 16.9 µmol TE
Yoghurt; 0.14 ± 0.01 mg GAE/g
(control)
Yoghurt + 0.25% FSE;
Yoghurt TPC [121]
0.43 ± 0.02 mg GAE/g
Yoghurt + 0.5% FSE;
0.65 ±0.02 mg GAE/g
Antioxidants 2022, 11, 2388 17 of 30

Table 2. Cont.

Samples Matrix Assay Results Ref.

Yoghurt; 0.40 ± 0.03 µmol TE/g dw
Yoghurt + 0.25% FSE;
FRAP 2.57 ± 0.09 µmol TE/g dw
Yoghurt + 0.5% FSE;
4.19 ± 0.05 µmol TE/g dw
Yoghurt; 0.40 ± 0.04 µmol TE/g dw
Yoghurt + 0.25% FSE;
ABTS 3.63 ± 0.08 µmol TE/g dw
Yoghurt + 0.5% FSE;
5.34 ± 0.23 µmol TE/g dw
* fw: fresh weight; dw: dry weight; TE: Trolox equivalent; GAE: gallic acid equivalent; CE: Catechin equivalent
Inh: inhibition; AAE: ascorbic acid equivalent; FAE: ferulic acid equivalent; RSA: radical scavenging activity;
FDS: fortified with stevia extract; WEAX: water extractable arabinoxylan.

5.1. Fruits-Apples and Berries

Apples provide a rich source of phytochemicals, and epidemiological studies have
shown that the consumption of apples reduces the risk of certain cancer types, cardiovascu-
lar diseases, asthma and diabetes. The antioxidant activity of apples has been attributed
to various phytochemicals, particularly quercetin, catechin, phloridzin, and chlorogenic
acid [122]. Antioxidant properties of different apple matrices (leaves, fresh fruit, pulp and
peel, and pomace) have been investigated using various colorimetric assays (FC, DPPH,
ABTS, and FRAP). The results from different matrices were expressed as gallic acid equiv-
alents (GAE), ascorbic acid equivalents (AAE), Trolox equivalents (TE), and IC50 (50%
inhibition concentration) (Table 2). For instance, Bahukhandi et al. [89] investigated the
antioxidant activity of apples after pulverizing them to a fine texture. They studied the
antioxidant activity of apples in terms of total phenolic contents using an FC reagent, and
the results were expressed in GAE (10.87 mmol/kg). The antioxidant capacity for DPPH,
ABTS, and FRAP was reported in AAE as 10.87, 24.57 mmol/kg, and 24.05 mmol/kg
(of fresh apple), respectively. They also evaluated the correlation between total phenolic
content, flavonoid, flavonol, TEACABTS , TEACFRAP and determined positive correlation
values as 0.895, 0.843, 0.812, 0.856, and 0.830, respectively [89]. As noted, depending on the
type of assay used, the reported values were significantly different. Additionally, signifi-
cant variations in antioxidant activity were observed in different sample matrices (leaves,
fresh fruit, pulp and peel, and pomace) even when the same assay was used. In a recent
systematic review by Antonic et al. [123] on apple pomace, the authors showed that the
high antioxidant content and dietary fibers present in apple pomace play an essential role
as a food fortification ingredient in the food industry. The review highlights that fortified
apple pomace increased the antioxidant activity and dietary fiber content in various food
products. In a recent study, Li et al. [124] reported that red-fleshed apples showed greater
antioxidant activities, phenolics, and flavonoid content than regular fuji apples. Particularly,
one of the red-fleshed varieties, ‘A38’, showed about 3-fold higher FRAP, DPPH, and ABTS
activities than the fuji apple [124]. The above results illustrate that proper documentation
of genotypes is important. Unfortunately, none of these studies documented the difference
in specific chemical components.
Berries, like apples, are considered to have several health benefits as they contain
phenolic acids, flavonoids, and anthocyanins, which are localized mainly in berries, seeds,
skins, and leaves [125]. For instance, blueberry anthocyanins are nature’s most potent
antioxidants [125]. Similarly, blackberries show high antioxidant activity as they have
highly abundant phenolic compounds, such as gallic acid, ellagic acid, ellagitannins,
tannins, quercetin, cyanidins, and anthocyanins [126,127]. The antioxidant activities of
berries are presented in Table 2. The data clearly shows significant variations in the
results obtained from different assays. In another blueberry study, Liović et al. [128]
Antioxidants 2022, 11, 2388 18 of 30

studied the influence of freeze-drying, high-intensity ultra-sound, and pasteurization on

gastrointestinal stability and antioxidant activity. The authors found that both total phenolic
content and antioxidant capacities were improved for the freeze-dried and simulated gastric
digested samples as compared to control untreated samples investigated in that study.
These results suggested that external factors, such as drying and digestion, can also have
a significant impact on bioactivity. Similarly, Dalmau and coworkers hypothesized that
the drying process might alter the microstructures of vegetables as they found that both
convective drying (CD) and freeze-drying (FD) decreased the TPC and antioxidant activity
of beetroot samples [129]. The authors, however, claimed that these drying processes
facilitate the better release of bioactive compounds during the digestion process and, in
turn, lead to higher TPC and antioxidant activities [129].

5.2. Vegetables-Spinach and Olives

Spinach (Spinacia oleracea) is a vegetable with a wide array of phytoconstituents such
as polyphenols, flavonoids, tocopherols, carotenoids, ascorbates, p-coumarins, vitamins,
and polysaccharides, which are responsible for its nutritional properties [130,131]. The
prominent antioxidants from spinach identified by different researchers include chlorogenic
acid and spinacetins, and their analogs [132]. However, Mzoughi et al. reported the antiox-
idant activity of polysaccharides from spinach using DPPH, ABTS, and FRAP assays [90].
The water-soluble polysaccharides from Spinacia oleracea were extracted and characterized
using FT-IR, UV–vis, 1 H-NMR, SEC (Size Exclusion Chromatography)/MALS (multi-
angle light scattering), and DRI (differential refractive index) techniques. The average
molecular mass of the polysaccharide was 408 kDa composed of monosaccharides like
arabinose, glucose, galactose, mannose, and rhamnose. Spinacia polysaccharide significantly
prevented oxidation-induced Cd damage on HEK293 and HCT116 cells [90]. The results
from both DPPH and ABTS assay were presented as percent inhibition (68.51 ± 0.89% and
70.12 ± 0.04%, respectively), whereas FRAP results were shown as reducing capacity in
µmol/L (1590 ± 53.98 µmol/L at 10 mg/mL). On the other hand, Galla et al. [91] reported
the antioxidant activity of the methanolic extract of spinach leaves assayed by DPPH, ABTS,
and FRAP. In these two cases, one assay used water extraction to measure polysaccharides
activity, and the other used methanol extraction to measure the activity of hydrophobic
analytes. Hence, it becomes extremely challenging to identify the true antioxidant activity
of foods using a single assay or extraction methodology. Another challenge involving these
methods is the units used to report the activity. For instance, for the same assay (FRAP),
Galla et al. [91] reported results in terms of percent inhibition, Hussain et al. [100] reported
EC50 , and Mzoughi et al. [90] reported reducing capacity in µmol/L [91]. Additional
details on the antioxidant, antimicrobial activities, and clinical efficacy of Spinacia oleracea
were presented in a recent review by Salehi et al. [131]. Recently, in 2020, Kamiloglu [133]
reported the industrial freezing effects on the phenolic content and related antioxidant
capacity of spinach. The results of both TPC and TAC (CUPRAC, ABTS, DPPH, and FRAP
assays) showed that the freezing process increased both the TPC and TAC of spinach.
Interestingly, undigested frozen samples have shown higher TPC and TAC results than
digested (oral, gastric, and intestinal) samples [133]. These observations further illustrate
that it is challenging to compare health claims just based on colorimetric assays commonly
used for reporting antioxidant activities in foods.
Olives and olive oil have several health benefits due to the presence of phytochemi-
cals [134,135]. Olives contain phenolic acids (caffeic acid, gallic acid) and their derivatives,
phenolic alcohols (tyrosol, hydroxytyrosol), secoiridoids (oleuropein, oleocanthal), lignans
(pinoresinol), and flavones (luteolin), which account for their antioxidant activity [134,135].
Hydroxytyrosol (HyT) is one of the main polyphenols found in virgin olive oil and olive
mill waste [136] and has been shown to have strong ROS scavenging properties. HyT is the
only phenolic compound that has received health claim approval from the European Food
Safety Authority (EFSA). According to the EFSA, the consumption of olive oil polyphenols
Antioxidants 2022, 11, 2388 19 of 30

such as HyT and its derivatives play a protective role in preventing oxidative damage of
blood lipids [136]. However, the antioxidant assays are not specific for HyT.
Recently, Fernández-Poyatos et al. [92] studied the antioxidant potential of table olives
from Olea europaea L. They determined activity using conventional spectrophotometric
methods ABTS and DPPH to be 308.68 µM and 228.46 µM Trolox equivalent per gram
of dried extracts, respectively. Cheurfa et al. [105] investigated the antioxidant potential
of the extract of olive leaves using water and aqueous ethanol separately. The ethanol
extract of O. europeae leaves showed significant antioxidant activity (IC50 69.15 mg/mL,
% inhibition 54.98, % inhibition 49.71, 82.63 mg ascorbic acid equivalent/g extract, and
7.53 mol of Fe2+ /g extract for the DPPH, β-carotene bleaching, ferric thiocyanate, TAC,
and FRAP assays, respectively) [105]. As noted above, there are significant variations in
the antioxidant capacity values for olives depending on the assay used and the units for
the results.

5.3. Processed Products-Wine, Coffee, and Tea

Wine is primarily made from grapes but is also made from several other fruits, including
apple, cherry, pear, peach, plum, banana, mango, strawberry, blueberry, blackberry, and rasp-
berry [137]. These fruits are widely known for their antioxidant activity. Similarly, coffee and tea
are also considered to have significant antioxidant activity [138]. The coffee-brewed drink is pre-
pared from coffee beans, and tea is prepared from fresh tea leaf extracts. Antioxidant activities
of these three processed food products (wine, coffee, and tea) have been widely studied using
different assays, including FRAP, ABTS, DPPH, ORAC, and TPC [110–113,139–142]. However,
the data in Table 2 showed significant variations in the outcomes from different assays, despite
using the same sample for analysis [110–113,139–142]. Table 2 presents DPPH and ABTS studies
by Jung et al. [110] reported in percent radical scavenging activity (RSA), whereas, for the same
assays, Bravo et al. [111] reported results in µM TE/g. Moreover, the results from either study
(DPPH vs. ABTS) were distinctly different, which can be attributed to the mechanisms involved
in the assays. Similar variations were observed for tea and wine antioxidant activities (Table 2).

5.4. Legumes-Bean, Soybean

Soybean (Glycine max (L.) Merr.) is one of the most important plant protein sources
consumed by humans and animals and receives growing interest as a source of high-protein
crops in Europe, North America, South Asia, and Japan [115]. The antioxidant activities of
different matrices of soybeans have been studied, as shown in Table 2. However, for each
assay, different units were reported, which makes it difficult to compare the antioxidant
potential of different foods. For instance, Peiretti et al. [115] reported the TPC activity in
catechin eq/g, whereas Handa et al. [93] reported in GA eq/g.
Common beans (Phaseolus vulgaris L.) are one of the most important legumes con-
sumed globally and are used for nutritional and medical purposes [125]. Beans are rich
in polyphenols, flavonoids, anthocyanidins, and procyanidins, which may explain their
antioxidant activities. Zhao et al. [101] and Cid-Gallegos et al. [114] reported antioxidant
activities for chickpeas. The results presented by the authors are not comparable as they
used different solvents for extractions and expressed their results in different units (Table 2).

5.5. Grains-Corn, Wheat

Epidemiological studies have shown that the consumption of whole grains and grain-
based products is associated with a reduced risk of oxidative stress-related chronic diseases
and age-related disorders, such as cardiovascular diseases, carcinogenesis, type II diabetes,
and obesity [143]. The health benefits of whole-grain flours are attributed to the presence
of antioxidants, such as tocopherols and tocotrienols, vitamin E, carotenoids, phenolic
acids, and flavonoids [144]. In 2019, Ranjbar et al. [117] studied the antioxidant potential of
wheat flour with iron enrichment using DPPH and FRAP assays. However, the authors
reported phenolic acid content as µg equivalent of ferulic acid/g, whereas, usually, the
Antioxidants 2022, 11, 2388 20 of 30

TAC is reported as either gallic acid or ascorbic acid equivalents/g. Hence, the comparison
of antioxidant results becomes complicated for researchers and consumers.
Corn is among the largest produced staple foods across the globe. Corn is well known
for its antioxidant properties, which may be attributed to the presence of high amounts of
anthocyanins [145]. Antioxidant studies by Ramos-Escudero et al. [94] and Horvat et al. [95]
showed no significant relationship between TPC and DPPH radical scavenging in corn
samples (r = 0.202) [95]. This lack of correlation may be due to several reasons, such as
experimental conditions, mechanisms, interferences, and types of the analytes assayed [95].

5.6. Dairy Products-Milk, Yogurt, and Others

Dairy products constitute about 25–30% of the average diet of an individual [146]. Milk
and milk products are rich in essential nutrients such as vitamins (tocopherol, retinol, and
carotenoids), minerals, oleic acid, omega-3 fatty acids, conjugated linoleic acid, antioxidants
like milk caseins, ascorbate, low molecular weight thiols, and whey proteins, and other
bioactive compounds [146,147].
Zulueta et al. [120] reported the antioxidant capacity of multiple commercial samples of
pasteurized and ultra-high temperature (UHT) treated whole milk, whey, and deproteinized
milk using ORACFL assay [120]. According to the study, the TAC of whole milk was
attributed mainly to casein fractions, albumin, and whey protein, whereas hydrophilic
antioxidant compounds, such as ascorbic acid and uric acid, were the main contributors
to the total TAC of the deproteinized milk. A significant correlation was found between
the fat% and the TAC of milk samples. In addition, pasteurized milk was found to have
significantly higher TAC than UHT-treated milk for both whey and deproteinized milk
samples. In contrast, the TAC values of pasteurized and UHT whole milk were not
significantly different [120]. The reported antioxidant results were entirely based on a
single ORACFL assay, which is the main limitation of this report, as no single assay is
expected to provide an accurate measurement of total antioxidant capacity. In addition,
this report also suggested that sample processing plays a very important role in the overall
antioxidant activity/capacity of the food substrate.
In a recent study by de Carvalho et al. [121], yogurt samples were fortified with 0.25% and
0.5% freeze-dried stevia extract (FSE). The control and stevia-fortified yogurts were evaluated
and compared for the TPC and antioxidant activity (using FRAP and ABTS). Table 2 shows the
TPC, FRAP, and ABTS results for the yoghurt 0.14 ± 0.01 mg GAE/g, 0.40 ± 0.03 µmol Trolox/g,
0.40 ± 0.04 µmol Trolox/g, respectively. Upon adding 0.25% FSE, the antioxidant activity of the
yoghurt significantly increased (TPC- 0.43 ± 0.02 mg GAE/g, FRAP- 2.57 ± 0.09 µmol Trolox/g,
ABTS- 3.63 ± 0.08 µmol Trolox/g. Moreover, upon addition of 0.5% FSE, the antioxidant activity
further increased TPC- 0.65 ± 0.02, FRAP- 4.19 ± 0.05 µmol Trolox/g, ABTS- 5.34 ± 0.23 µmol
Trolox/g. Apart from FSE, the simulated digestion also increased the antioxidant activity of the
fortified yogurts compared to the undigested fractions [121].

6. Strengths and Weaknesses of Antioxidant Assays

6.1. Strengths
In general, antioxidant assays provide rapid and inexpensive means of measuring
the status of a sample. Changes in the composition of a food or supplement arising from
genetics, environment, management, and processing are readily detected. Antioxidant
assays are sensitive to the metadata associated with a sample. The assays cannot specify
which components are changing, but changes are readily detected.

6.2. Weaknesses
Questions have been raised about the relationship between dietary/exogenous antiox-
idants and health. Lack of specificity, lack of harmonization, and the inability to correlate
in vitro measurements with in vivo activity have been major factors. The U.S. Food and
Drug Administration (FDA) and the European Food Safety Authority (EFSA) do allow
health claims for vitamins (A, C, and E), which have shown ambiguous results in vivo.
Antioxidants 2022, 11, 2388 21 of 30

Several journals have banned papers whose primary measurements are antioxidant activity.
This suggests the complexity of the issue [148]. The major limitations of currently used
antioxidant assays are summarized below.
(a) There is a huge divergence in the results for food and dietary antioxidants because
the assays are non-specific and are influenced by every component in a sample matrix.
A change in an assay value cannot be correlated with a change in a specific component.
Consequently, antioxidant assays cannot be correlated with health outcomes.
(b) Antioxidant assays cannot be compared or inter-converted. Antioxidant assays
measure activities. A detailed explanation for these limitations has been described by Apak
et al. [15]. Both AOAC International (Association of Analytical Communities) and the
IUPAC (International Union of Pure and Applied Chemistry) reported that the results from
different antioxidant methods could not be compared as each method employs different
mechanisms, pH, temperature, and sample matrix [148]. Hence, the IUPAC concluded
that there is no single universally accepted antioxidant assay available that accurately
determines the antioxidant activity or the total antioxidant capacity [10,60,148]. In 2012, the
U.S. Department of Agriculture (USDA) removed the ORAC database from online ‘because
of increasing evidence that the data infers there is no relevance between antioxidant activity
and the effects of bioactive compounds, including polyphenols on human health’ [10,148].
(c) Reporting results in multiple units for the same assay complicates data compari-
son [149]. For example, results for TPC have been reported as equivalents of gallic acid,
ascorbic acid, or ferulic acid, which complicates the understanding of the results.
(d) The measurements in vitro experiments cannot be directly correlated with in vivo
activity. This can be attributed to the complex physiological environment in vivo assays
as compared with the controlled environments in vitro assays [149,150]. The activity of
plant-based secondary metabolites such as polyphenols, phenolic acids, flavonoids, and
proanthocyanidins, is little known [148].
(e) The interpretation of antioxidant assay results mainly focuses on the antioxidant-
oxidant reactions and related kinetics; however, they often ignore the chromophore/
luminescent interactions with the probe, which can cause interferences in the results [151].

7. Other Factors Influencing Antioxidant Activity

Unlike the in vitro assays, the measurements based on in vivo antioxidant assays are
impacted by several factors relevant to physiological conditions. For instance, the fate of the
antioxidant in vivo is determined by the pharmacokinetic phenomena ADME (absorption,
distribution, metabolism, excretion) profiles, which are not evaluated in vitro [152]. Unlike
in vitro, the actual concentrations of antioxidants at tissue levels are essentially depend-
ing on ADME profiles. Below are some of the critical limitations of in vivo antioxidant
assays [15,152,153].

7.1. Bioaccessibility and Bioavailability of Antioxidants

Bioaccessibility and bioavailability are closely related, but they have different defini-
tions. Bioaccessibility is the fraction of bioactive compounds that are released from a matrix
during the digestion process and become available for absorption, whereas bioavailability is
the fraction of compounds that are absorbed into the bloodstream, distributed by systemic
circulation, and exert their effect after being metabolized then eliminated [154,155]. For
phytochemicals, as with any food component, to exert their biological activity, they must be
released from the matrix in an absorbable form into the stomach/intestine/colon (bioacces-
sible), followed by absorption into the bloodstream (bioavailable) [154,155]. Factors such
as matrix interactions and chemical structures influence the bioaccessibility of phytochem-
icals, whereas bioavailability can be affected by multiple parameters, such as biological
membranes (GI wall), the physicochemical properties of the phytochemicals, biological
environment inside the GI, etc., The optimum properties of phytochemicals/drugs for GI
absorption have been defined by Lipinski’s Rule [156], including appropriate molecular
weight, hydrogen bonding capability, and partition coefficient (LogP) [156]. In addition,
Antioxidants 2022, 11, 2388 22 of 30

the presence of adjuvants, food processing techniques, sample preparation, and extraction
methods can also significantly influence the bioavailability of the compounds by influencing
their bioaccessibility [154,155].
The bioaccessibility and bioavailability of phytochemicals can be enhanced using pro-
cessing techniques that induce physical or chemical modifications in the food [154]. These
modifications include: (a) chemical modifications, such as cleavage of hydrophobic forces,
hydrogen bonds, and bonds that attach phenolic compounds to matrix macromolecules,
conjugation, and derivatization; (b) physical modifications, such as grinding, drying by
different approaches; (c) disrupting the cell wall barriers so that phytochemicals can be
released from the matrix; and (d) using encapsulation techniques/solubilization using
nanotechnologies that protect phytochemicals until they are absorbed. One should note
that these techniques can cause the degradation of phytochemicals; however, it is possible
to reduce it by altering the operating conditions, which can facilitate increased bioaccessi-
bility and bioavailability [154]. Techniques such as drying, freezing, thermal processing,
sterilization and pasteurization, ultrasounds treatment, milling and grinding, chemical and
enzymatic treatments, and encapsulation, which are commonly used for food processing
and supplements, can influence the overall phytochemical availability [154]. The positive
or adverse effects of processing techniques on bioaccessibility and bioavailability depend
on compounds’ stability, matrix protective effects, and existing interactions.
Bioaccessibility, bioavailability, the metabolism of antioxidants, and their consequences,
must be taken into consideration. For instance, flavonoids are structurally altered in vivo.
Hence, the nutritional application of flavonoids requires extensive studies on their metabolism
and controlled comparison of antioxidant activity of their structural isoforms. Additionally,
the evidence shows that the removal of glycosidic substituents (sugar moiety) by enzymes
or bacteria is likely to increase the antioxidant activity of flavonoids in vivo. In contrast,
the methylation and glycosylation of OH groups in flavonoids reduce their prooxidant
behavior by catechol-o-methyltransferase (COMT) and other enzymes. The impact of sugar
moiety on the bioactivities of flavonoids has been discussed in recent reviews by different
researchers [153,157,158].

7.2. Chelation
The efficacy of antioxidants’ function based on metal chelation mechanism, such as
polyphenols, need to be investigated thoroughly in vivo. Though ascorbate is an antioxi-
dant, it can also act as a pro-oxidant, especially in the presence of transition metals like iron
and copper [159]. Hence, it is important to understand the actual factors contributing to the
antioxidant activity in the presence of metals, ascorbate, and other possible interferences,
such as uric acid, which is elevated upon consumption of polyphenol-rich foods and may
cause increased plasma total antioxidant capacity in vivo. Finally, it has been suggested
that the large increase in plasma total antioxidant capacity observed after the consumption
of polyphenol-rich foods is not caused by the polyphenols themselves but is likely the
consequence of increased uric acid levels [160].

7.3. In Vivo Assays

Over the years, researchers have discussed the validity of applying various assays
to in vivo conditions. All of them provide an estimate of the antioxidant capacity of
plasma/serum without distinguishing the contribution by exogenous molecules of dietary
origin (i.e., ascorbic acid, vitamin E, polyphenols, and other phytochemicals) compared to
endogenously-derived molecules such as enzymatic components (i.e., SOD, GSH-Px, and
catalase, CAT) and small macromolecules (i.e., albumin, bilirubin, ceruloplasmin, ferritin,
glutathione) [161]. In this regard, the EFSA remarked on the inappropriateness of the
methods used to determine antioxidant capacity in humans and extrapolate possible effects
on human health [162].
Antioxidants 2022, 11, 2388 23 of 30

7.4. Sample Matrix

Sample/substrate matrix always has a great influence on the outcomes of antioxidant
studies, both in vitro and in vivo. Along with matrix effects and multiple variable experi-
mental conditions, performing the assays (radical generator, time of measure, expression of
results, etc.) makes it difficult to compare the reported data.

7.5. Experimental Parameters

Most assays lack a detailed evaluation of important factors that affect the antioxidant
activity measurements, such as concentration, pH, localization, distribution, metabolism,
reactivity toward other non-targeted molecules, interaction with other antioxidants, and
the fate of the radical that is derived from the antioxidant.

8. Perspectives/Recommendations
To achieve absolute efficacy of food antioxidants acceptable to various communities
such as nutrition and clinical professionals and consumers, it is essential to consider the
following factors:
(a) Matrix information must be presented in detail in the experimental section of the
in vitro and in vivo antioxidant assays. The matrix effects should also be accounted for.
(b) Sample preparation (grinding, drying, processing, etc.) and the extraction proce-
dures for antioxidants have a direct impact on the results. Hence, one should use optimized
sample preparation and extraction methods to extract antioxidants with a wide range
of polarities.
(c) Since there is no universal standard available currently, the development of uni-
versal multi-component standards is essential to address the variances associated with
antioxidant capacity measurements.
(d) Both antioxidant activity and antioxidant capacity must be distinguished as these
two terms are used interchangeably even though they are both measured completely
differently, i.e., antioxidant activity is measured kinetically, and antioxidant capacity is
measured thermodynamically. An ideal method for an antioxidant activity measurement
requires: (i) usage of a biologically relevant radical source; (ii) determines actual chemistry
that occurs in the assays; (iii) the use of a method with a defined endpoint and chemical
mechanism involved in a particular assay; (iv) a suitable method for both hydrophilic
and lipophilic antioxidants; and (v) instrumentation, which is readily available, simple,
economical, rugged, user friendly, and facilitates high-throughput for routine analyses.
(e) The harmonization of methodology is required, which includes detailed standard
operating procedures for different assays, test conditions, good internal/external stan-
dards, proper method validation, including intra- and inter-lab validation (reproducibility,
accuracy, precision, and recovery), quality control, and quality assurance.
(f) All antioxidant results should be presented in a single international system of units
(SI). Correlations tables between various assays should be established to promote easy
comparison of results between different assay procedures.
(g) A better understanding of the bioaccessibility and bioavailability of antioxidants
in vivo is essential. To achieve maximum efficacy from the antioxidants in vivo, their
bioaccessibility and bioavailability must be increased by using appropriate delivery systems,
such as liposomes, nanoparticles, etc.
Overall, it is not easy to accomplish the above recommendations altogether. However,
focusing on each of the above steps will improve the credibility of antioxidants. This
will enable nutrition professionals, and researchers to correlate accurately the role of
antioxidants as it relates to nutrition and health.

9. Conclusions
The present review defines antioxidants and describes antioxidant assays, their mech-
anisms, and their strengths and weaknesses. These methods have been used to evaluate
the “health benefits” of phytochemicals, as documented in the literature. Unfortunately,
Antioxidants 2022, 11, 2388 24 of 30

with antioxidant assays, it is not certain what has been measured. The lack of specificity of
the assays creates confusion and ambiguity for consumers, healthcare professionals, and
researchers. To have a wide acceptance and clear understanding of the health benefits of
phytochemicals, there is a critical need to develop multi-omic approaches which measure
specific food and supplement components. This will allow health outcomes to be correlated
with specific food components.

Author Contributions: Conceptualization, D.L.L.; methodology, D.L.L., R.R.K., F.S.T. and E.Y.; formal
analysis, D.L.L., R.R.K., F.S.T. and E.Y.; investigation, D.L.L., R.R.K., F.S.T. and E.Y.; resources, D.L.L.,
R.R.K., F.S.T. and E.Y.; data curation, D.L.L., R.R.K., F.S.T. and E.Y.; writing—original draft preparation,
D.L.L., R.R.K., F.S.T. and E.Y.; writing—review and editing, D.L.L., R.R.K., F.S.T. and E.Y.; visual-
ization, D.L.L., R.R.K., F.S.T. and E.Y.; supervision, D.L.L.; project administration, D.L.L.; funding
acquisition, D.L.L. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Agricultural Research Service, US Department of Agricul-
ture, Project # 1235-52000-066-00D.
Acknowledgments: We would like to thank James Harnly for his feedback on the preparation of
this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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