Archives of Toxicology

https://doi.org/10.1007/s00204-020-02689-3

REVIEW ARTICLE

Antioxidants and antioxidant methods: an updated overview

İlhami Gulcin1

Received: 17 January 2020 / Accepted: 24 February 2020

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Antioxidants had a growing interest owing to their protective roles in food and pharmaceutical products against oxidative
deterioration and in the body and against oxidative stress-mediated pathological processes. Screening of antioxidant prop-
erties of plants and plant-derived compounds requires appropriate methods, which address the mechanism of antioxidant
activity and focus on the kinetics of the reactions including the antioxidants. Many studies evaluating the antioxidant activ-
ity of various samples of research interest using different methods in food and human health have been conducted. These
methods are classified, described, and discussed in this review. Methods based on inhibited autoxidation are the most suited
for termination-enhancing antioxidants and for chain-breaking antioxidants, while different specific studies are needed for
preventive antioxidants. For this purpose, the most common methods used in vitro determination of antioxidant capacity of
food constituents were examined. Also, a selection of chemical testing methods was critically reviewed and highlighted. In
addition, their advantages, disadvantages, limitations and usefulness were discussed and investigated for pure molecules and
raw extracts. The effect and influence of the reaction medium on the performance of antioxidants are also addressed. Hence,
this overview provides a basis and rationale for developing standardized antioxidant methods for the food, nutraceuticals, and
dietary supplement industries. In addition, the most important advantages and shortcomings of each method were detected
and highlighted. The chemical principles of these methods are outlined and critically discussed. The chemical principles of
methods of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical (­ ABTS·+) scavenging, 1,1-diphenyl-2-picrylhydrazyl
(DPPH·) radical scavenging, F ­ e3+–Fe2+ transformation assay, ferric reducing antioxidant power (FRAP) assay, cupric ions
2+
­(Cu ) reducing power assay (Cuprac), Folin–Ciocalteu reducing capacity (FCR assay), peroxyl radical (ROO·), superoxide
radical anion (­ O2·−), hydrogen peroxide (­ H2O2) scavenging assay, hydroxyl radical (OH·) scavenging assay, singlet oxygen
(1O2) quenching assay, nitric oxide radical (NO·) scavenging assay and chemiluminescence assay are outlined and critically
discussed. Also, the general antioxidant aspects of main food components were discussed by a number of methods, which
are currently used for the detection of antioxidant properties of food components. This review consists of two main sections.
The first section is devoted to the main components in the food and pharmaceutical applications. The second general section
comprises some definitions of the main antioxidant methods commonly used for the determination of the antioxidant activity
of components. In addition, some chemical, mechanistic and kinetic basis, and technical details of the used methods are given.

Keywords  Antioxidants · Antioxidant activity · Antioxidant methods · Reactive oxygen species

Introduction as with other compounds. It is present in the atmosphere

in its ground state as a stable triplet biradical (3O2), under-
Reactive oxygen species and oxidative stress goes a stepwise reduction process (Taslimi and Gulçin
2018; Rezai et al. 2018). Molecular oxygen, in the ground
Oxygen is a highly reactive nonmetal and an oxidizing state, has two unpaired electrons with parallel spins in its
agent that readily forms oxides with most elements as well two separate anti-bonding orbitals. Due to this spin restric-
tion, it can accept a pair of electrons from an electron
donor. On the other hand, redox is an essential metabolic
* İlhami Gulcin reaction in the living system, wherein the electrons are
igulcin@atauni.edu.tr; igulcin@yahoo.com
transferred from one species to another. These processes
1
Department of Chemistry, Faculty of Sciences, Atatürk are the main reactions in biological systems, whereby the
University, 25240 Erzurum, Turkey

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chain of chemical reactions takes place in the living organ- protection (Huyut et al. 2017; Oztaskin et al. 2017). ROS
ism that uses oxygen from the air for oxidation and to occur in living organisms during normal cellular metabolism
provide energy in the form of ATP (Gulcin 2012). Oxygen and can be harmful decisive biomolecules including lipids,
is a highly integrated series of oxidation–reduction and carbohydrates, nucleic acids, and proteins (Cetin Cakmak
enzymatic process in living organisms. Also, it can trans- and Gulcin 2019). Living organisms are constantly exposed
fer electrons from one atom to another and represents an to ROS, which occur as by-products of metabolism, normal
essential part of aerobic life and our metabolism since oxy- respiration, and the autoxidation of xenobiotics, or as the
gen is the ultimate electron acceptor in the electron flow result of stress that accompanies a range of diseases (Anr-
system that produces energy in the form of ATP (Davies aku et al. 2018). Oxidative stress is a result of an imbal-
1995; Gülçin et  al. 2005; Elmastas et  al. 2018). How- ance between ROS and antioxidant defenses. This oxidative
ever, problems may arise when the electron flow becomes stress deregulates a series of cellular functions and leads to
uncoupled (transfer of unpaired single electrons), gener- various pathological conditions in which ROS overwhelm
ating free radicals. Free radicals are atoms, molecules or antioxidative defenses of the organism, leading to oxidative
ions with unpaired electrons that are highly unstable and modification of the above-indicated biological macromol-
active towards chemical reactions with other molecules. ecules, tissue injury, and accelerated cellular death as the
They derive from three elements: oxygen, nitrogen and sul- foundation of many diseases (Sindhi et al. 2013; Apak et al.
fur. For example, oxygen-centered free radicals are known 2016). The putative ROS, reactive nitrogen species (RNS),
as reactive oxygen species (ROS) and include superoxide and non-free-radical species are summarized in Table 1.
­(O2·−), hydroxyl (HO·), peroxyl (ROO·), alkoxyl (RO·) and Free radicals, also known as ROS and RNS, which con-
nitric oxide (NO·). The hydroxyl (half-life of ­10–9 s) and tain one or more unpaired electrons in the outermost orbital
the alkoxyl (half-life of seconds) free radicals are very is an unstable species that exists independently. A contribut-
reactive and rapidly attack the molecules in nearby cells, ing factor for this instability is the lack of octet configura-
and probably the damage caused by them is unavoidable tion. ROS and RNS have characteristics such as the gen-
and is dealt with by repair processes. On the other hand, eration of new vulnerable ROS, short life, and damage to
­O2·−, NO· and lipid hydroperoxides are less reactive (Ames various tissues. The four-step sequential univalent reduction
et al. 1993; Han et al. 2018; Bulut et al. 2018). In addi- of molecular oxygen to water can be given as follows (Gul-
tion to these ROS radicals, in living organisms, there are cin 2012):
other nonradicalic ROS, such as the singlet oxygen (1O2), e− e− e− e−
hydrogen peroxide (­ H2O2), and hypochlorous acid (HOCl) O2 → O⋅−
2
→ O2−
2
→ OH ⋅ → H2 O.
(Pietta 2000). Regarding ROS, the reactions leading to
the production of reactive species are displayed in Fig. 1. In this electron flow, O ­ 2 is reduced from a variety of
The formation of these ROS is crucial for the mainte- sources, including either the formation of O ­ 2·− or conver-
1
nance of cell homeostasis and living organisms achieve sion to O2 or the formation of H ­ 2O2. The vast majority
this by a system of antioxidant defense, allowing them to of oxygen consumed in the body is fully reduced to ­H2O
maintain a balance between oxidative stress and antioxidant in a single stage at the terminal cytochrome of the oxygen

NADPH GSSG H2O +O2

GR GP
GP
CAT

NADP+ 2GSH H2O2

α-Tocopheryl Asc Lipoic acid NADP+

LOOH 2GSH
DNA
Damage

LOO. AscH Dehydrolipoic acid NADPH

α-Tocopherol GSSG
O2.-
Hypoxanthine Xanthine OOH
Fe2+ Fe3+ H
OO
. O O
LH O2
O2.- L.
NAD(P)H
O2 Oksidaz SOD H2O2 .OH Cyclization O O
(O2/RH) H
H
Xanthine Uric acid MDA

Fenton reaction
C5H11 O . Cyclization
H
O
H OH
LO. C5H11 R O
R .
4-Hydroxynonenal

Fig. 1  Overview of the reactions leading to the formation of ROS and their effects. SOD superoxide dismutase enzyme, CAT​ catalase enzyme,
GR Glutathione reductase, GP glutathione peroxidase

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Table 1  Reactive oxygen species (ROS), reactive nitrogen species ROS are either radicals that contain at least one unpaired
(ROS) and non free-radical species electron or reactive nonradical compounds, capable of oxi-
Reactive oxygen species Non free-radical species dizing biomolecules. Therefore, these intermediates are
also called oxidants or prooxidants (Sies 1991; Koksal et al.
Hydroxyl radical HO· Hydrogen peroxide H2O2
2017a). ROS are continuously produced during normal
Superoxide radical O2·− Singlet oxygen 1
O2
physiologic events and can easily initiate the peroxidation
Hydroperoxyl radical HOO· Ozone O3
of membrane lipids, leading to the accumulation of lipid per-
Lipid radical L· Lipid hydroperoxide LOOH
oxides (Gulçin 2010; Ekinci Akdemir et al. 2017; Kandemir
Lipid peroxyl radical LOO· Hypochlorite HOCl
et al. 2017a). ROS at physiological concentrations may be
Peroxyl radical ROO· Peroxynitrite ONOO−
required for normal cell function. However, excessive ROS
Lipid alkoxyl radical LO· Dinitrogen trioxide N2O3
production can cause gradual oxidative damage and eventu-
Nitrogen dioxide NO2· Nitrous acid HNO2
ally disruption of cell function and consequently cell death
radical
via damaging DNA, RNA, proteins, and lipids (Isik et al.
Nitric oxide radical NO· Nitryl chloride NO2Cl
2017; Krawczyk 2019). They are also capable of damaging
Nitrosyl cation NO+ Nitroxyl anion NO−
crucial biomolecules such as nucleic acids, lipids, proteins,
Thiyl radical RS· Peroxynitrous acid ONOOH
polyunsaturated fatty acids and carbohydrates (Tohma et al.
Protein radical P· Nitrous oxide N2O
2017; Koksal et al. 2017b). Also, they may cause DNA dam-
age that can lead to mutations. If cellular constituents cannot
effectively scavenge ROS, they can stimulate free radical
transport chain in the mitochondria. The damage by ROS chain reactions subsequently damaging the cellular biomol-
is created by the partial reduction of oxygen. In fact, most ecules such as proteins, lipids, carbohydrates and nucleic
ROS are formed at low levels by normal aerobic metabo- acids and finally they lead to disease conditions (Craft et al.
lism and play an important role in the redox-dependent 2012). Another important destructive effect of ROS is on
regulation of many signaling processes in living organ- mitochondria. The role of mitochondria, the powerhouse of
isms (Shivakumar and Kumar 2018). However, ROS are the cell, is to produce ATP via oxidative phosphorylation
continuously produced by the body’s normal use of oxy- via the electron transport chain. ROS and oxidative stress
gen such as respiration and some cell-mediated immune can also result from mitochondrial dysfunction, or a com-
functions (Gülçin 2006a). A free radical is defined as bination of any of these factors (Gulcin et al. 2004a; Kraw-
chemical species capable of independent existence, pos- czyk 2019). Living organisms including the human body
sessing one or more unpaired electrons. Free radicals are can protect themselves by scavenging ROS and producing
highly unstable molecules. They are internally produced endogenous or exogenous antioxidant compounds that scav-
as a normal part of metabolism within the mitochondria, enge free radicals (Hamad et al. 2017; Anraku et al. 2018).
through xanthine oxidase (XO), peroxisomes, phagocyto- The human body has a complex system of natural enzymatic
sis, inflammation processes, ischemia, arachidonate path- and non-enzymatic antioxidant defenses, which counteract
ways, and physical exercise. On the other hand, external the harmful effects of free radicals, which are responsible
factors that help to promote the production of free radicals for causing a large number of diseases and other oxidiz-
are environmental pollutants, smoking, radiation, drugs, ing agents (Ekinci Akdemir et al. 2016a, b). Scavenging of
ozone, pesticides, and industrial solvents. It is ironic that ROS from the human body can help to prevent or reduce
these elements which are essential to life have deleterious the incidence of these and other diseases, thus contributing
effects on the human body through these reactive species to a better quality of life (Gülçin 2012; Aksu et al. 2016).
(Gulcin et al. 2006b, c; Lobo et al. 2010). Free radicals Protection against free radicals can be enhanced by intake
have different reaction mechanisms. They can react with of dietary antioxidants (Alam et al. 2013; Ekinci Akdemir
surrounding molecules by electron donation, electron et al. 2016a).
acceptance, reducing radicals, and oxidizing radicals (a), ROS are also produced in the living organism as a part of
hydrogen abstraction (b), self-annihilation reactions (c), the primary immune defense. Phagocytic cells such as neu-
addition reactions (d) and disproportionation (e) (Carocho trophils, monocytes, or macrophages defend against foreign
and Ferreira 2013). organisms by synthesizing large amounts of ­O2·− or NO·
as a part of their killing mechanism. Several diseases are
a. OH⋅ + RS− → OH− + RS⋅ accompanied by excessive phagocyte activation resulting in
b. CCl⋅3 + RH → CHCl3 + R⋅ tissue damage, which is at least in part due to the activity
c. CCl⋅3 + CCl⋅3 → CH2 Cl6 of ROS (Diplock et al. 1998; Bae et al. 2016; Tohma et al.
d. CCl⋅3 + CH2 = CH2 → CH2 (CCl3 ) − CH2 2016). In addition, ROS induce some oxidative damage to
e. CH3 CH⋅2 + CH3 CH⋅2 → CH2 = CH2 + CH3 − CH3

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biomolecules like lipids, nucleic acids, proteins, and car- in blood-vessel walls resulting in lowered blood pressure.
bohydrates. Their damage causes aging, cancer, and many It is an important cellular messenger molecule involved in
other diseases (Aruoma 1994). It is known that ROS have many physiological and pathological processes. It is also
been implicated in more than 100 diseases, including produced by activated macrophages contributing to the
malaria, acquired immunodeficiency syndrome, heart dis- primary immune defense. An excess of NO· is cytotoxic.
ease, stroke, arteriosclerosis, diabetes, and cancer (Tanizawa It might react directly with biomolecules or combine with
et al. 1992; Duh 1998). Also, the biological pathways for ­O2·− to form peroxynitrite (ONOO–) (Hou et al. 1999).
endogenous ROS formation are examples of a whole class of Peroxynitrite (ONOO–) is not only capable of inducing
reactive intermediates and their ways of generation. It should lipid peroxidation in lipoproteins but might also interfere
further be noted that the organism is also exposed to ROS with cellular signaling by nitrating tyrosine residues in pro-
from external sources. In living organisms, various ROS can teins (Packer 1996). Peroxynitrite is an oxidant and nitrating
form in different ways. Normal aerobic respiration stimulates agent. Because of its oxidizing properties, ­ONOO− can dam-
polymorphonuclear leukocytes and macrophages, and per- age a wide array of molecules in cells, including proteins
oxisomes appear to be the main endogenous sources of most and DNA. The reaction of NO with ­O2·− to form the much
of the oxidants produced by cells. Exogenous sources of more powerful oxidant O ­ NOO− is a key element in resolv-
ROS include tobacco smoke, certain pollutants, organic sol- ing the contrasting roles of NO in physiology and pathol-
vents, and pesticides (Halliwell and Gutteridge 1989; Topal ­ NOO− is a strong oxidant and can react directly
ogy. Also, O
et al. 2016a). With the diet, many compounds of prooxidant with electron-rich groups like sulfhydryls, zinc-thiolates,
nature, such as quinones capable of redox cycling, are deliv- iron–sulphur centres and the active site sulfhydryl in tyros-
ered to the organism. Also, an array of radicals is inhaled ine phosphatases (Pacher et al. 2007).
with cigarette smoke; ozone, which increasing levels are Peroxyl radical (ROO·) is relatively long lived with a
reported due to air pollution, is an ROS, which can oxidize considerable diffusion path length in biological systems. It
lipids (Diplock et al. 1998; Polat Kose et al. 2015). There can be generated in the process of lipid peroxidation, which
are various sources for specific ROS in the living organism. is initiated by the abstraction of an H atom from polyun-
However, the O ­ 2·− appears to play a central role since other saturated fatty acids (PUFA). OH· is capable of starting
reactive intermediates are formed in reaction sequences this reaction sequence (Reaven and Witzum 1996). Further
starting with O ­ 2·−. It has been estimated that about 1–3% products generated in lipid peroxidation are alkoxyl radi-
of the ­O2 we utilize is converted to ­O2·− (Halliwell, 1996; cals (RO·) and organic hydroperoxides (ROOH). The latter
Gulcin 2012). might rearrange to endoperoxide intermediates, which are
Hydroxyl radicals (OH·) are the neutral form of the cleaved to yield aldehydes. The reaction of aldehydes with
hydroxide ions (­ OH−). These radicals are highly reactive, amine groups of proteins has been discussed as a mechanism
easily becoming hydroxyl groups and consequently short- involved in the modification of the protein part of lipopro-
lived. It is reported that every day a human cell is targeted teins (Diplock et al. 1998).
by the hydroxyl radical (OH·) and other radical species Hydrogen peroxide (H2O2) is the simplest peroxide
and average of 1­ 05 times inducing oxidative stress (Valko with oxygen–oxygen single bond. It is unstable and slowly
et al. 2004; Carocho and Ferreira 2013). Hydroxyl radicals decomposes in the presence of light. ­H2O2 is found in bio-
can occasionally be produced as a byproduct of immune logical systems including the human body. The enzymes
action. Microglia and macrophages most frequently gener- that use or decompose H ­ 2O2 are classified as peroxidases.
ate this compound when exposed to very specific patho- It is formed by the two-electron reduction of ­O2 is not a
gens and certain bacteria. The destructive action of OH· has free radical, but is an oxidizing agent. In the presence of O
­ 2
been implicated in several neurological autoimmune when and transition metal ions, ­H2O2 can generate OH· via the
immune cells become over-activated and toxic to neighbor- Fenton reaction (Halliwell and Gutteridge 1989). Also, the
ing healthy cells (Gulcin 2012). This radical is known as the Haber–Weiss reaction generates OH· from ­H2O2 and super-
most reactive species with an estimated half-life of about oxide ­O2·− catalyzed by iron ions. This rection was first pro-
­10–9 s. It might be formed in vivo on high-energy irradiation posed by Fritz Haber and his student Joseph Joshua Weiss.
by hemolytic cleavage of body water or from endogenous Then, it was proven that the Haber–Weiss and Fenton reac-
­H2O2 in metal-catalyzed processes. UV light is insufficiently tions together were the main sources of radicals responsible
energetic to split water but it can cleave ­H2O2 to yield two for oxidative stress and cellular damages.
molecules of the hydroxyl radical. The high reactivity of this
radical implies immediate reaction at the place where it is Fe2+ + H2 O2 → Fe3+ + OH− + OH∙ (Fenton reaction).
generated (Diplock et al. 1998).
Nitric oxide (NO·) is a signaling compound formed O∙−
2
+ H2 O2 → O2 + H2 O + OH∙ (Haber−Weiss reaction).
enzymatically from arginine and relaxes smooth muscles

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The formed short-lived radical of OH· unspecifically All aerobic organisms have antioxidant defenses, includ-
attacks the biomolecules in a diffusion-limited reaction and ing antioxidant enzymes and antioxidant constituents to
thus is able to crack proteins, polysaccharides, and nucleic remove or repair the damaged molecules. As with the chemi-
acids located within a few nanometers from its site of gener- cal antioxidants, cells are protected against oxidative stress
ation (Shivakumar and Kumar 2018). On the other hand, the by an interacting network of antioxidant enzymes (Davies
­H2O2, as a substrate of Fenton reaction, is naturally produced 1995). Also, enzymes have been evaluated as new types of
in organisms as a by-product of oxidative metabolism. It is a natural antioxidants in some food applications. They can be
non-radical reactive species and can easily diffuse between used beneficially to remove oxygen and ROS and to reduce
living cells. It is efficiently converted to water by the catalase lipid hydroperoxides (Frankel 1998; Isik et al. 2015).
enzyme, a process which determines its half-life. Some evi-
dence suggests that ­H2O2 is involved in signal transduction Antioxidants
regulating the expression of genes through the nuclear factor
and apoprotein-1 pathways (Sen and Packer 1996). Antioxidants play a vital role in both food systems as well as
Singlet oxygen (lO2) is not a radical but a highly ROS in the human body to reduce oxidative processes and harm-
and is responsible for skin damage induced by ultraviolet ful effects of ROS (Cakmakci et al. 2015; Gocer et al. 2013).
irradiation and for the cytotoxic anti-cancer effect in photo- In food systems, retarding lipid peroxidation and formation
dynamic therapy. It causes a cytotoxic process in tumor cells of secondary lipid peroxidation product can be prevented by
in photodynamic therapy and skin photoaging. Despite its the use of nutritional antioxidant molecules thereby helping
significant roles, the biological effects of lO2 are not fully to maintain the flavor, color and texture of the food product
understood (Homma et  al. 2019). Singlet oxygen is the during storage (Bursal et al. 2013; Cakmakci et al. 2015).
common name used for the diamagnetic form of molecu- Also, antioxidants are helpful in reducing amino acids,
lar oxygen ­(O2), which is less stable than the normal triplet protein oxidation as well as the interaction of lipid-derived
oxygen. It is another non-radical ROS, which suggested to carbonyls with proteins that leads to an alteration of protein
be formed in vivo in light-exposed tissue. Its half-life has function (Sindhi et al. 2013). An antioxidant is a molecule
been estimated to be ­10–6 s depending on the nature of the capable of inhibiting the oxidation of other molecules. In
surrounding matrix. Singlet oxygen can react with multiple terms of food, an antioxidant has been defined as “any sub-
cellular components, its preference for reacting with conju- stance that when present in low concentrations compared to
gated double bonds is very high, and hence it preferentially that of an oxidizable substrate significantly delays or inhibits
attacks polyunsaturated fatty acids (PUFA) or guanine in the oxidation of that substrate” but later defined them as “any
DNA bases in cells (Di Mascio et al. 2019). Also, it can substance that delays, prevents or removes oxidative damage
interact with other molecules either by transferring its exci- to a target molecule” (Halliwell and Gutteridge 1989; Sies
tation energy or by combining chemically (Stahl and Sies 1993; Halliwell 1995). In another definition, an antioxidant
1993). is defined as a substance that directly scavenges ROS or
In a normal cell, there is an appropriate prooxi- indirectly acts to up-regulate antioxidant defenses or inhibit
dant–antioxidant balance. However, this balance can be ROS production”. Antioxidant compounds can scavenge free
shifted towards the prooxidants when the production of radicals and increase shelf-life by retarding the process of
oxygen species is increased greatly or when levels of anti- lipid peroxidation, which is one of the major reasons for the
oxidants are diminished. This state is called oxidative stress. deterioration of food and pharmaceutical products during
Sies (1997) introduced the concept of oxidative stress, that processing and storage (Halliwell 1997). Antioxidants can
is, the dissolution of the prooxidant–antioxidant equilibrium. also protect the human body from free radicals and ROS
Another definition, oxidative stress represents an imbalance effects. They retard the progress of many chronic diseases
between the production and manifestation of reactive oxygen as well as lipid peroxidation. In recent years, there is great
species and a biological system’s ability to readily detoxify interest in identifying alternative natural and safe sources of
the reactive intermediates or to repair the resulting damage food antioxidants, and the search for natural antioxidants,
(Kalin et al. 2015; Oztaskin et al. 2015). Oxidative stress is especially of plant origin. Antioxidants are often added to
basically caused by two main mechanisms. The concentra- foods to prevent the radical chain reactions of oxidation,
tion of antioxidants is reduced due to mutated antioxidant and they act by inhibiting the initiation and propagation
enzymes, toxins, or the reduced intake of natural antioxi- step leading to the termination of the reaction and delay the
dants. The number of oxygen, nitrogen or carbon-based reac- oxidation process (Shahidi et al. 1992; Gülçin 2006a). Anti-
tive species derived from activated phagocytes is increased oxidants have become an indispensable group of food addi-
in the case of chronic inflammation (Somogyi et al. 2007; tives mainly because of their unique properties of extending
Gulcin 2012). the shelf-life of food products without any adverse effect on
their sensory or nutritional qualities. However, antioxidants

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for use in food system must be inexpensive, effective and color, and texture. The simpler phenolic substances include
nontoxic at low concentrations; highly stable and capable monophenols with a single benzene ring, such as 3-ethyl-
of surviving processing; have no odor, taste or color of their phenol and 3,4-dimethylphenol found in fruits and seeds,
own; easy to incorporate and have good solubility in the the hydroxycinnamic acid group which contains caffeic and
product (Shahidi and Ambigaipalan 2015). For the determi- ferulic acid, and the flavonoids and their glycosides which
nation of antioxidant ability of food components, the terms include catechins, proanthocyanins, anthocyanidins, and fla-
of antioxidant activity and antioxidant capacity are often vonols. The tannins are a complex and poorly defined group
used interchangeably but it should be recognized that they of water-soluble phenolics with high molecular weights. The
have different meanings. Activity refers to the rate constant daily intake of phenolic substances may be as high as 1 g per
of a reaction between a specific antioxidant and a specific day, but the quantity of defined flavonoids in the diet prob-
oxidant. Capacity is a measure of the amount of a given ably amounts to no more than a few tens of milligrams per
free radical scavenged by a sample (MacDonald-Wicks et al. day (Pokorny et al. 2000).
2006). Phenolic compounds in foods originate from one of
Consumption of fruits and vegetables has been associated the main classes of secondary metabolites in plants. They
with reduced risk of some chronic diseases including the act as an antioxidant and protect food from oxidative ran-
most dangerous coronary atherosclerosis (Rimm et al. 1996). cidity at a low concentration (Karakaya 2004). The anti-
Epidemiological studies have demonstrated an inverse asso- oxidant potential of phenolic compounds depends on the
ciation between intake of fruits and vegetables and mortality number and arrangement of the hydroxyl groups in the
from age-related diseases, such as coronary heart disease molecules of interest. Phenolics are not active antioxidants
and cancer, which may be attributed to their antioxidant unless substitution at either the ortho- or para-position
activity (Ganesan et al. 2011). The major bioactive com- has increased the electron density at the hydroxyl group
pounds of these natural sources are especially phenolics and and lowered the oxygen–hydrogen bond energy, in effect
flavonoids, which are responsible for their health benefits increasing the reactivity towards the lipid free radicals.
(Bocco et al. 1998). The antioxidant properties of phenolics Substitution in phenolic compounds at the meta-position
are responsible for the inhibition of oxidation of low-density has a rather limited effect. Steric and electronic effects
lipoprotein cholesterol (Eberhardt et al. 2000). As a conse- are responsible for the antioxidant activities and stoichio-
quence, the consumption of fruits and vegetables is inversely metric factors of the chain-breaking phenolic antioxidants
related to coronary atherosclerosis (Rimm et al. 1996). (Barclay et  al. 1993). For elucidation of the hydrogen
The term bioavailability is used for the indication of abstraction mechanism of phenolic antioxidants in the
the nutrient that is digested, absorbed, and metabolized chain process of autoxidation, molecular orbital theory
through normal pathways. It is well known that after the has been applied (Tomiyama et al. 1993; Gulcin 2012).
consumption of polyphenol-rich foods, indirect evidence Recently, it was demonstrated that bromophenols possess
of the absorption of polyphenols from the intestinal bar- many medicinal properties such as antioxidant activity
rier is given by the increase in the antioxidant capacity of (Oztaskin et al. 2015; Boztas et al. 2019), antimicrobial
plasma. The absorption and speed limit of polyphenols in activity, anticancer activity, anti-inflammatory, and anti-
the intestine are related to their chemical feature and struc- diabetic activities (Cherian et al. 2019) and carbonic anhy-
ture. In dead, the aglycones are absorbed from the small drase (Boztas et al. 2015; Taslimi et al. 2016), acetylcho-
intestine; however, the polyphenols present in the form of linesterase (Oztaskin et al. 2015), butyrylcholinesterase
esters, glycosides, or polymers cannot be absorbed in the (Bayrak et al. 2017, 2019), aldose reductase (Demir et al.
native form. Therefore, they are hydrolyzed by intestinal 2018), α-amylase, α-glycosidase (Taslimi et al. 2018b)
enzymes like lactase and β-glycosidases or by the colonic and paraoxonase (Cherian et al. 2019) inhibition proper-
microflora and then adsorbed (Nemeth et al. 2003). So, ties. These enzymes were linked to some global health
polyphenol forms that are present in the human blood and diseases.
tissues are different from those present in foods, making A number of scientific studies are going about address-
the identification of all the metabolites and the evaluation ing the varied health benefits of antioxidants in processes
of their biological activity difficult. Actually, chemical like stress, pathogen infestation, aging, apoptosis, and
structure of polyphenols is more important than that of neurological diseases. Antioxidants reduce cell-damaging
their concentration, as it determines the rate and extent of effects of free radicals. Humans take antioxidants directly
absorption and the nature of metabolites circulating in the from fresh and dried fruits and vegetables, which contain
plasma (Cipolletti et al. 2018). a large amount of flavonoids and antioxidant supplements
A huge variety of biologically active phenolic compounds that contribute to protection against different types of dis-
containing one or more aromatic rings are found naturally eases including cancers and cardiovascular health problems
in plant foods, where they provide much of the flavor, (Sindhi et al. 2013).

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Synthetic antioxidants the flavor or odor of the material to which it is added (Kasha-
nian and Dolatabadi 2009). It was reported that amongst the
Synthetic antioxidants had been developed to have a standard synthetic antioxidants, TBHQ was more effective than BHA
antioxidant activity measurement system to compare with and BHT and served as the strongest antioxidant in borage
natural antioxidants and to be incorporated into food. They and evening primrose oil. The two para-hydroxyl groups in
are added to food so it can withstand various treatments and TBHQ are responsible for its antioxidant activity (Nanditha
conditions as well as to prolong shelf life. Almost all pro- and Prabhasankar 2009; Shahidi and Ambigaipalan 2015).
cessed food products have synthetic antioxidants incorpo- PG is generally recognized as a safe antioxidant to protect
rated that are reported to be safe, although some studies indi- oils, fats, and fat-containing food from rancidity that results
cate otherwise (Carocho and Ferreira 2013). These synthetic from the formation of peroxides. It has been used since 1948
antioxidants are deliberately added to products to prevent for stabilizing food and cosmetic-packaging materials, foods
or delay the onset of lipid oxidation during processing and containing fats, and as an additive in edible fats, oils, mayon-
storage of fats, oils, and lipid-containing foods. They have naise, shortening, pressure-sensitive adhesives, baked prod-
been used by the food industry for some 70 years. It was ucts, lubricating oil additives and transforming oils. It was
reported that the food and beverage market for antioxidants reported that PG and its metabolites have demonstrated liver
is a $500 billion industry and is growing at 5–7% per year, toxicity and enhanced carcinogenesis (Shahidi and Ambi-
and is expected to grow at that rate through the year 2017 gaipalan 2015).
(Shahidi and Ambigaipalan 2015). As seen in Fig. 2, the OG is the ester of 1-octanol and gallic acid and used as an
more popular synthetic antioxidants used are phenolic com- antioxidant and food preservative. However, BHT and BHA
pounds such as butylated hydroxyanisole (BHA), butylated are the most widely used synthetic antioxidants, which are
hydroxytoluene (BHT) tert-butylhydroquinone (TBHQ), used for food and pharmacological applications and author-
propyl gallate (PG) and octyl gallate (OG) (Gulcin 2012). ized for use by several national authorities as food additives
BHA is particularly effective in controlling the oxidation of (Koudelka et al. 2015). Both synthetic antioxidants have
short-chain fatty acids. Especially, BHT is not as effective been suspected of being responsible for liver damage and
as BHA mainly due to the presence of two tert-butyl groups carcinogenesis when used at high levels in laboratory ani-
that show greater steric hindrance than BHA to the molecule mals Synthetic antioxidants are always substituted by alkyls
(Nanditha and Prabhasankar 2009). However, it was referred to improve their solubility in fats and oils (Hudson 1990).
to as a prooxidant, an antioxidant, an anticarcinogen, a car- Safety concerns have been raised about synthetic antioxi-
cinogen, and a tumor promoter. Furthermore, BHA exhibits dants. Governments, due to their potential toxicity effects,
anticarcinogenic properties at concentrations close to food strictly regulate the usages of synthetic antioxidants in food-
additive levels (Shahidi and Wanasundara 1992). stuffs. Because of safety concerns, synthetic antioxidants are
TBHQ is a highly effective preservative for many edible limited to be used as food preservatives (Biparva et al. 2012;
animal fats, unsaturated vegetable oils, and meat products. It Shahidi and Ambigaipalan 2015). It has been reported that
preserves and stabilizes the freshness, nutritive value, color amongst the various food additives BHA, TBHQ, and PG
and flavor of animal food products. It does not cause discol- have the potential to form molecular complexes with nucleic
oration even in the presence of iron. Also, it does not change acid structure and cause damage to double helical structure
of DNA (Dolatabadi and Kashanian 2010). However, their
acceptability for consumers is diminished, because they are
OH OH OH
synthetic compounds, raising concerns about their safety
related to their metabolism and possible absorption and
accumulation in cells, tissues, organs and body (Kulawik
OCH3 CH3 OH et al. 2013; Anraku et al. 2018). BHA and BHT have been
TBHQ
restricted by legislative rules due to doubts over their toxic
BHA BHT
and carcinogenic effects (Wichi 1988; Sherwin 1990). How-
ever, it was reported that BHA is slightly better than BHT
O O
HO CH3 HO in its carry-through properties (Shahidi and Ambigaipalan
O O CH3
2015). Because of these reasons, it is widely held that many
HO
HO
OH OH
of these objections can be overcome by replacing synthetic
Octyl gallate
antioxidants with effective natural antioxidants for general
Propyl gallate
use in the food and medicinal fields (Anraku et al. 2018).
The usage of synthetic antioxidants is limited and regulated
Fig. 2  The chemical structures of most putative and popular synthetic by the Food and Drug Administration (FDA), necessitating
antioxidants the search for effective antioxidants from natural sources.

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Therefore, there is a growing interest in natural and safer (Shahidi and Ambigaipalan 2015). These metabolites play
antioxidants for food applications, and a growing trend in a major role in protecting plants from undesired effects.
consumer preferences towards natural antioxidants, all of The human diet contains an array of different compounds
which have given impetus to the attempts to explore natural that possess antioxidant activities or have been suggested
sources of antioxidants (Gülçin 2006b, 2007). to scavenge ROS based on their structural properties. The
The four major synthetic antioxidants in use are subjected most prominent representatives of dietary antioxidants are
to a good manufacturing practice limit of 0.02% of the fat vitamin C, tocopherols, carotenoids, and flavonoids. Apart
or oil content of the food (Simic 1981). The most suitable from vitamin C, each group of these antioxidants consists
antioxidant for vegetable oils is TBHQ. BHA and BHT are of a number of structurally different compounds, e.g.,
fairly stable to heat and are often used for the stabilization more than 600 different carotenoids have been identified
of fats in baked and fried products. Antioxidants that are to date and about fifty of them might occur in the human
heat stable have the property referred to as carry-through. diet (Sies and Stahl 1995; Rice-Evans and Miller 1996). In
The disadvantages of gallates such as PG lie in their ten- the diet, there may be synergistic effects of these various
dency to form dark participate with the iron ions and their dietary compounds, which are difficult to assess at present.
heat sensitivity. Some antioxidants, such as BHA and BHT, Indeed, the diet may be considered as an orchestra where
are used in combination with resulting synergistic effects interactions between constituents may bring about effects,
(Omura 1995). BHA is also synergistic with PG (St Angelo which are not the necessary properties of the individual
1996). PG has been shown to exhibit liver toxicity and constituents (Diplock et al. 1998).
enhance carcinogenesis (Shahidi and Ambigaipalan 2015). Phenolic compounds as natural antioxidants have great
The synthetic antioxidants have been very thoroughly tested structural diversity and variations in chemical composition
for their toxicological behaviors, but some of them are com- among plant-derived substances. Also, they are found in
ing, after a long period of use, under heavy pressure as new almost all plants, microorganisms, fungi, and even in ani-
toxicological data impose some caution in their use (Thomp- mal tissues (Pokorny 1999). Phenolic compounds known
son and Moldeus 1988). In this context, natural products to serve as multipurpose bioactive compounds are widely
appear as healthier as and safer than synthetic antioxidants. spread throughout the plant kingdom. Most of the phenolic
Since about 1980, natural antioxidants have appeared as an compounds are an integral part of the human diet and are
alternative to synthetic antioxidants. So, antioxidants can also consumed as medicinal preparations. Many of the
be classified as synthetic and natural based on their origin health-protective effects of phenolic compounds have been
(Gulcin 2012). ascribed to their antioxidant, anticarcinogenic, antimuta-
genic, antimicrobial, anti-inflammatory and other biological
Natural antioxidants properties The most widely occurring plant phenolic com-
pounds include phenolic acids, flavonoids, tannins, lignans,
Recently, the possible toxicity and undesired effects of and terpenes (Nazck and Shahidi 2006; Shahidi and Ambi-
synthetic antioxidants have been considered. Also, the gaipalan 2015). Phenolic compounds are secondary plant
potential of plant products to serve as antioxidants to pro- metabolites naturally present in almost all plant materials,
tect against ROS and various diseases induced by free rad- including food products of plant origin. These compounds
icals has been explored (Hou et al. 2003). Therefore, the are thought to be an integral part of both human and animal
increased popularity of natural food additives may prompt diets (Gülçin 2006b). The majority of natural antioxidants
more food manufacturers to replace synthetic antioxidants are phenolic compounds and the most important groups of
with ingredients containing natural antioxidative com- natural antioxidants are the tocopherols, flavonoids and phe-
pounds. Also, the research on natural additives has gained nolic acids (Fig. 3).
acceleration. They are perceived as posing no health risk Phenolic acids are present in almost all plants and plant-
for consumers (Shahidi and Wanasundara 1995; Shahidi derived foods, representing a significant portion of the
and Ambigaipalan 2015). Sources of natural antioxidants human diet. The average phenolic acid intake in human diet
are primarily plant phenolics that may occur in all parts has been reported to be in the order of 200 mg/day depend-
of plants including fruits, vegetables, seeds, nuts, leaves, ing on preferences and diet habits (Clifford and Scalbert
roots flours, and barks. Plants produce a vast repertoire 2000). The recent focus of interest on phenolic acids stems
of secondary metabolites such as flavonoids, essential from their potential protective role, through the ingestion
oils, alkaloids, lignans, terpenes, terpenoids, tocopherols, of fruits and vegetables, against oxidative damage diseases
phenolic acids, phenolics, peptides, polyfunctional organic such as coronary heart disease, stroke and cancers (Gülçin
acids, amongst others in their normal metabolic pathways. et al. 2010a) and antiglaucoma (Çoban et al. 2007; Öztürk
However, the main sources of natural antioxidants are pri- Sarıkaya et al. 2010, 2011; Innocenti et al. 2010a, b; Şenturk
marily plant phenolics that may occur in all parts of plants et al. 2011). Phenolic acids constitute approximately 30%

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Fig. 3  Classification of phenolic
antioxidants Phenolic antioxidants

Natural antioxidants Synthetic antioxidants

Hydroxybenzoic acid derivtives

Phenolic acids BHA
Hydroxycynamic acid derivtives
BHT
Flavonoids

TBHQ
Carotenoids Flavonols

PG
Stilbens Flavononols
OG
Coumarins Flavones

Lignans Flavanols

Tannins Flavanones

Antocyanidins

Isoflavonoids

of the dietary phenolic present in plants in free and bound higher antioxidant activity than the hydroxybenzoic acids.
forms (Robbins 2003). The latter is found more frequently In relation to the phenolic acids, it is possible to observe
and occurs in the form of esters, glycosides, and insoluble- that hydroxycinnamic acids are more effective antioxidants
bound complexes (Ross et al. 2009). Phenolic acids are than that of hydroxybenzoic acids. This situation is due to
hydroxy derivatives of aromatic carboxylic acids, which the conjugation through the double bonds of the ring to the
arise from either the benzoic acid group or the cinnamic −CH=CH–COOH of the cinnamic acid structure, which
acid group. Phenolic acids, such as p-hydroxybenzoic acid, enhances the ability to stabilize free radicals. The pres-
3,4-dihydroxybenzoic acid, vanillic acid, syringic acid, ence of –CH2=CH–COOH group in cinnamic acids ensures
p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, chlo- greater antioxidant capacity than –COOH group in benzoic
rogenic acid, and rosmarinic acid, are widely distributed in acid (Cuppett et al. 1997). The presence of different sub-
the plant kingdom (Öztürk Sarikaya et al. 2010). Rosmarinic stituents in the phenol backbone structure modulates their
acid, which possessed a potent some metabolic enzymes antioxidant property, in particular their hydrogen-donating
including carbonic anhydrase glutathione S-transferase, lac- capacity. In general, unsubstituted phenol is inactive as
toperoxidase, acetylcholinesterase, and butyrylcholinesterase hydrogen donor and monophenol is a less efficient antioxi-
enzymes, is an ester of caffeic acid and 3,4-dihydroxyphenyl dant than polyphenol. The introduction of electron-donating
lactic acid (Topal and Gülçin 2014; Gulcin et al. 2016). The group such as hydroxyl group in the ortho- or para-position
derivatives of cinnamic acid are more active antioxidants increases the antioxidant activity of phenol or phenolic acid
than the derivatives of benzoic acid derivatives (Chen and (Chimi et al. 1991). Catechin, which presented the same
Ho 1997). number of –OH groups in the molecule as quercetin, pre-
The antioxidant activity of phenolic acids and their sented significantly lower antioxidant activity. This is due to
derivatives depends on the number and position of the the fact that the structure of catechin does not have unsatu-
hydroxyl groups bound to the aromatic ring, the binding rated bonds at the C ­ 2–C3 position in conjunction with the
site and mutual position of hydroxyl groups in the aromatic oxo (−C=O) on C ring, which comparatively gives quercetin
ring, and the type of substituents (Rice-Evans et al. 1996; a higher antioxidant activity. With the addition of an –OH
Sroka and Cisowski 2003; Gulcin 2012). As can be seen group on the B ring of catechin molecule, this compound is
in Table 2, there are two main groups of phenolic acids: called epigallocatechin and, with this new structure, there
hydroxybenzoic acids and hydroxycinnamic acids. The is an increase in the antioxidant activity, but not equiva-
hydroxycinnamic acids have been found to have significantly lent to quercetin (Rice-Evans et al. 1996). In addition, the

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Table 2  Chemical structures of naturally occurring phenolic acids In the literature, the position and the degree of hydroxyla-
and related compounds as benzoic acid and cinnamic acid derivatives tion are of primary importance in determining antioxidant
Phenolic acids activity (Dziedzic and Hudson 1984; Chen and Ho 1997).
The relationship between antioxidant activity and the struc-
O O
ture of many phenolic acids and their antioxidant activity
OH OH was established (Chen and Ho 1997; Goçer and Gülçin
2011). Monophenols were less efficient than diphenols or
polyphenols. Recently, in a relationship between structure
Benzoic acid Cinnamic acid and activity study, it was demonstrated that l-dopa had
higher antioxidant and radical scavenging activities than that
Benzoic acid derivatives Cinnamic acid derivatives
of l-tyrosine. On the basis of this analysis, it was confirmed
Gallic acid Caffeic acid that the radical scavenging and antioxidant activities of the
p-Hydrobenzoic acid Sinapic acid studied antioxidants were highly controlled by the number
3,4-Dihydrobenzoic acid Ferulic acid of phenolic hydroxyl groups (Gülçin 2007). The introduction
Vanillic acid Rosmarinic acid of a second hydroxy group in the ortho- or para-position
Syringic acid Chlorogenic acid increased the antioxidative activity. The inhibiting effective-
Protocatechuic acid Caffeoylquinic acid ness of monophenols was increased substantially by one or
Veratric acid O-Coumaric acid two methoxy substitutions. The combination of two acid
Gentisic acid P-Coumaric acid phenols increased the efficiency, e.g., rosmarinic acid is a
better antioxidant than caffeic acid. Caffeic acid is found
in the form of esters, chlorogenic acid being the most fre-
presence of a carbonyl group, such as aromatic acid, ester, quently encountered. Recently, the antioxidant activity of
or lactone enhanced its antioxidant activity. The antioxidant caffeic acid was well demonstrated and its possible anti-
activity of a molecule also increases when its carbonyl group oxidant mechanism clarified (Gülçin 2006b). Esterification
is separated from the aromatic ring. So, cinnamic acids are of caffeic acid by sugar moiety decreased its activity, e.g.,
more effective antioxidants than the corresponding benzoic chlorogenic acid was less effective than caffeic acid (Chen
acids. Steric hindrance of the phenolic hydroxyl groups by a and Ho 1997). Caffeic acid is among the major hydroxycin-
neighboring inert group, such as methoxy groups, enhanced namic acids. Also, Pekkarinen et al. (1999b) have examined
its antioxidant activity (Dziedzic and Hudson 1984; Gocer the effect of selected hydroxybenzoic and hydroxycinnamic
and Gülçin 2011). Also, it is well known that antioxidant acids on both bulk and emulsified methyl linoleate oxidation
activity of the phenolic compounds depends on their struc- in the dark. It has been demonstrated that specific interac-
ture, particularly on the position and number of the –OH tions of the antioxidant with other compounds, for exam-
groups and the nature of the substitutions on the aromatic ple, the emulsifier, and intramolecular hydrogen bonds may
rings (Cosme et al. 2018). It must be emphasized that gallic play an important role in reducing and antioxidant activi-
acid has more antioxidant activity than catechin, which has ties. Oleuropein, tyrosol and hydroxytyrosol are among the
five –OH groups in its structure (Rice-Evans et al. 1996). most important phenolic substances in olive oil (Gülçin et al.
Cinnamic acids exhibit a wide range of biological activi- 2006a; Gulcin 2012). Also, it was found that hydroxytyrosol
ties including free radicals scavenging, antioxidants, pro- and caffeic acid had greater protection factors than BHT
tecting of ultraviolet radiation, antiviral and antibacterial (Papadopoulos and Boskou 1991; Gülçin 2006b). Sesame
effects. Cinnamic acids and their derivatives are bioactive
plant food ingredients. They are widely distributed in cere-
ROO ROOH
als, legumes, oilseeds, fruits, and vegetables. The most HO HO
common hydroxycinnamic acids include coumaric, caffeic, R R
HAT -O
ferulic, and sinapic acids. They exhibit in vitro antioxidant HO

activity, which might have a beneficial health impact in vivo

(Kroon and Williamson 1999; Gulcin 2012). The fact that H+

the antioxidant activity of phenolic acids follows an inverse ROO ROO ROO-
ROOH
dependence on the magnitude of their O–H bond dissocia- -O HO HO

tion enthalpies values provides evidence that in weakly polar R

HAT
R
ET
R
O -O O
organic media. The key mechanism of the chain-breaking
action is the hydrogen atom transfer (HAT) from the phe-
nolic OH to ROO· (Fig. 4). Fig. 4  Purposed mechanism for reaction of catechol (1,2-dihydroxy-
benzene) with peroxyl radicals (ROO·)

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Archives of Toxicology

oil contains several natural antioxidants, such as sesamol of oxidation and pattern of substitution of the C ring, while
(Budowski, Menezes and Dollear 1950). Sesame oil is an individual compounds within a class differ in the pattern
edible vegetable oil derived from sesame seeds. Besides of substitution of the A and B rings. The basic structure
being used as cooking oil and flavor enhancer in South India, of these compounds consists of two aromatic rings linked
Chinese, Korean, Japanese and to a lesser extent Southeast by a three-carbon aliphatic chain, which normally has been
Asian cuisine. It is popularly used in alternative medicine condensed to form a pyran or less commonly, a furan ring
for traditional massages and treatments to modern-day fads (Harborne 1986; Gulcin 2012).
(Gülçin 2012). Flavonoids are a large group of polyphenolic natural
Antioxidants may respond in a different manner to differ- compounds, extensively distributed in plant-based foods. A
ent radical or oxidant sources. For example, carotenoids are multitude of substitution patterns in the two benzene rings
not particularly good quenchers of ROO· relative to pheno- (A and B) of the basic structure occur in nature (Ghosh et al.
lics and other antioxidants but are exceptional in quenching 2015). Flavonoids are very effective antioxidants and it has
singlet oxygen, at which most other phenolics and antioxi- been proposed that they protect against cardiovascular dis-
dants are relatively ineffective (Prior et al. 2005; Gulcin ease by reducing the oxidation of low-density lipoproteins.
et al. 2012a, b). Also flavonoids are among the major antioxidant constitu-
Flavonoids are cyclized diphenylpropanes that com- ents of our diet. Flavonoids are mostly present in human
monly occur in plants and particularly plant foods. They diet, such as fruits, vegetables and plant-derived beverages
are polyphenolic compounds, which are very effective anti- such as tea. Daily intake of flavonoids is a few hundreds of
oxidants that serve against chronicle diseases. More than milligrams per day. Up to now, over 4000 different natu-
8000 polyphenolic compounds, including over 4000 flavo- rally occurring flavonoids have been described (Ghosh et al.
noids have been identified in various plant species, and the 2015). Flavonoids are generally poorly absorbed from food
number is still growing (Harborne et al. 1999), have been and their effects on the overall antioxidant capacity of the
isolated from almost all parts of the plant such as leaves, plasma remain to be established (Formica and Regelson
stems, roots, fruits, or seeds. Flavonoids are formed in plants 1995). The most significant profits of flavonoids are the pro-
from the aromatic amino acids phenylalanine and tyrosine, tection against oxidative diseases, ability to modulate the
and malonate (Harborne 1986). Flavonoids, which include activity of various enzymes and interactions with specific
flavones, flavanols, isoflavones, flavanones, and chalcones, receptors (Williams and Spencer 2004). In general, the effec-
are seen in higher plant tissues. Flavanones undergo a series tive antioxidant ability of flavonoids depends on some fac-
of transformations affecting the heterocyclic carbon ring to tors: the metal-chelating potential that is strongly dependent
give rise to anthocyanins and catechins. Flavonoid deriva- on the arrangement of hydroxyls and carbonyl group around
tives vary in their structure around the heterocyclic oxy- the molecule, the presence of hydrogen or electron-donating
gen ring, but all have the characteristic ­C6–C3–C6 carbon substituents able to reduce free radicals and the ability of
skeleton. All flavonoids are generally derivatives from the the flavonoid to delocalize the unpaired electron leading to
2-phenylchromone-parent compound composed of three formation of a stable phenoxy radical (Gulcin et al. 2011a;
phenolic rings (A, B and C), which exhibit various levels Gulcin 2012).
of methoxylation and hydroxylation (Shahidi and Ambi- Flavonols with varied hydroxyl substitution can act as
gaipalan 2015). The basic flavonoid structure is the flavan strong antioxidants and belong to flavonoids. They are a
nucleus, which consists of 15 carbon atoms arranged in three group of polyphenolic natural compounds widely distrib-
rings ­(C6–C3–C6), which are labeled A, B, and C (Table 3). uted in the plant kingdom. For this reason, they are present
The general structure of these compounds containing three in vegetables, fruits, and foods from plant origin (Samsono-
rings designated A, B and C is depicted in Table 3. The wicz and Regulska 2017). About 200 flavonols have been
presence of a double bond, a carbonyl and a hydroxyl group identified in vegetables and plants so far. They accumulate
in the pyranyl ring C serves as a basis for their classifica- mainly in the outer and aerial tissues, therefore, skin and
tion into several classes and subclasses. The substitution of leaves of fruits, plants, and vegetables since their biosynthe-
A and B rings by hydroxyl groups distinguishes individual sis is stimulated by light. Structurally, like flavonoids, fla-
members of each class (Musialik et al. 2009). Flavonoids vonols consist of two aromatic rings (A and B rings) linked
constitute a large group of naturally occurring plant phe- by a 3-carbon chain that forms an oxygenated heterocyclic
nolics. All these sub-groups of compounds share the same ring (C ring) and hydroxyl group substituted in 3 positions.
diphenylpropane ­(C6–C3–C6) skeleton. Also, the biochemi- A number of functional groups mainly hydroxyl can be
cal profiles of flavonoids and their derivatives depend on attached to ring structures of flavonols. Due to the presence
their chemical structures and the relative orientation of vari- of carbonyl (–C=O) and hydroxyl groups (–OH), they can
ous moieties in the molecules (Shahidi and Ambigaipalan coordinate metal ions forming complexes (Samsonowicz and
2015). The various classes of flavonoids differ in the level Regulska 2017).

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Table 3  Chemical structure of naturally occurring flavonoids

R3
3
R2 2 4
R4
8
B 5
HO O 2
R5
7
A C 6
6
5 4
3 R1
OH O

R1 R2 R3 R4 R5

Falavones Apigenin H H H OH H
Chrysin H H H H H
Luteolin H H OH OH H
Flavonols Datiscetin OH H OH OH H
Quercetin OH H OH OH H
Myricetin OH H OH OH OH
Morin OH OH H OH H
Kaemperol OH H H OH H
R3
3
R2 2 4 R4
8 B 5
HO O 2
R5
7
A C 6
6 3
5 4 R1
OH O

Flavanones Hesperetin H H OH OCH3 H

Naringenin H H H OH H
R3
3
R2 2 4 R4
8 B 5
HO O 2
R5
7
A C 6
6 3
5 4 R1
OH O

Flavanonol Taxifolin OH H OH OH H
8
A7 O 2
7
A C
6 3
4
5
B
A5 O
R4

Isoflavones A5 A7 R4

Genistein OH OH OH
Genistin OH Oglc OH
Daidzein H OH OH
Daidzin H Oglc OH
Biochanin A OH OH OCH3
Formononetin H OH OCH3

Metal chelation is widely considered as another mechanism ion; because of its ability to act as a free radical acceptor
of the antioxidant activity of flavonoids. The interaction of (Gulcin et al. 2010b; Ghosh et al. 2015). As can be seen in
metal ions with flavonoids may also change the antioxidant Fig. 5, the proposed binding sites for metals to taxifolin as
properties and also the biological effects of the flavonoids. a kind of flavanonol are the catechol moiety in ring B, the
It is suggested that the biological activity of a molecule can 3-hydroxyl, 4-oxo groups in the heterocyclic ring, and the
be increased when coordinated or mixed with suitable metal 4-oxo, 5-hydroxyl groups between the heterocyclic and the A

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rings. Figure 5 displays that taxifolin has a strong capability to with distinct boundaries (Apak et al. 2016). HAT-based meth-
bind ­Fe2+. It is submitting that its main action as a peroxidation ods measure the classical ability of an antioxidant to quench
inhibitor may be involved in its F ­ e2+-linking capacity. Taxifo- free radicals by hydrogen donation. On the other hand, SET-
lin prohibited the formation of the ferrous ion–ferrozine com- based methods detect the ability of a potential antioxidant to
plex. This flavanonol is able to catch ferrous ion before ferro- transfer one electron (­ e−) to reduce any compound, including
zine. It can convert ­Fe2+ ions into insoluble metal complexes metals, carbonyls, and radicals (Shahidi and Ambigaipalan
or generate sterically hindrance, which can prevent the interac- 2015).
tions between metals and lipid intermediates. It may probably
ArOH → ArO∙ + H∙ (HAT),
chelate three ferrous ions by the agency of its hydroxyl and
carbonyl groups (Topal et al. 2016b). Metallic center stabilized
semiquinone–metal complex (Pieta 2000). However, the major ArOH → ArO∙+ + e− (SET − PT),
contribution to metal chelation is due to the catechol moiety,
as exemplified by the more pronounced bathochromic shift ArO∙+ → ArO∙ + H+ .
produced by the chelation of copper to quercetin compared to
that of kaempferol (van Acker et al. 1996). Flavonoids play a Recently, another mechanism has been developed, named
crucial role in the bioavailability for metal ions present in low sequential proton loss electron transfer (SPLET) mechanism.
amounts in the body like aluminum as well as in the detoxifi- ArOH → ArO− + H+ ,
cation of heavy metals including Cr, Cd, Sn, and Pb. Chelat-
ing agents strongly bind to toxic metal ions forming complex
ArO− + ROO∙ → ArO∙ + e− .
structures, which are easily evacuated from the body (Dehghan
and Khoshkam 2012; Ghosh et al. 2015). The 7-OH group in flavonoids plays an important role as the
The stability of the radical generated from flavonoid moiety site of ionization and of electron transfer according to sequen-
relies on the hydrogen bond formed between the hydroxyl and tial proton loss electron transfer (SPLET). This mechanism has
oxygen possessing unpaired electron. The presence of a C=C been discovered recently (Litwinienko and Ingold 2004; Foti
double bond in ring C conjugated with a 4-oxo group is also of et al. 2004a, b) and takes place in two steps:
great importance for the delocalization of an unpaired electron.
Additional antioxidant activity is assigned to the presence of ArOH → ArO− + H+ ,
a hydroxyl group at the 3- and 5-positions. An internal hydro-
gen bond to a 4-oxo group makes these positions kinetically ArOH + ROO∙ → ArO∙ + ROO− .
equivalent (Bors et al. 1990). There are numerous methods The reaction enthalpy of the first step corresponds to the
for measuring antioxidant activity. These may be classified proton affinity of the phenoxide anion (­ ArO−) (Vianello and
into two categories. The first category measures the ability Maksic 2006). In the second step, electron transfer from
of antioxidants in inhibiting oxidation in a model system by phenoxide anion to ROO· occurs and the phenoxy radical
monitoring the associated changes using physical, chemical or is formed. The reaction enthalpy of this step is denoted as
instrumental means. Radical scavenging assays include meth- electron transfer enthalpy. From the antioxidant action view-
ods based on hydrogen atom transfer (HAT) or single electron point, the net result of SPLET is the same as in mechanisms
transfer (SET) mechanisms (Shahidi and Ambigaipalan 2015) HAT to the free radicals. For example, quercetin is a biofla-
besides the two generally accepted mechanisms of phenolic vonoid and well known for its good antioxidant and radical
antioxidants (generally Ar–OH) action (Wright et al. 2001), scavenging activity. The possible mechanism for querce-
namely hydrogen atom transfer (HAT) and single-electron tin and taxifolin reactions with DPPH· radicals is given in
transfer followed by proton transfer (SET-PT). However, in Figs. 6 and 7. Both flavonoids have the ability to convert
some cases, these two mechanisms may not be differentiated the steady radical DPPH to the yellow-colored DPPH-H.
Also, it was reported that Taxifolin, as a naturally bioac-
tive flavonoid, had marked inhibition effects against some
OH
H
O metabolic enzymes including human carbonic anhydrase I,
HO O HO O
Fe2+ and II isoenzymes and acetylcholinesterase enzyme (Gocer
O
OH
3Fe2+ H et al. 2016).
OH
OH
HO O
In the same manner, the structure of the usnic acid leads
OH O Fe2+
Fe2+ to interference in the DPPH·. After the interaction of usnic
Taxifolin Taxifolin-Fe2+ Complex
acid and radicals, DPPH· disappears after accepted an elec-
tron or hydrogen radical from usnic acid to become ­DPPH2.
Fig. 5  Schematic representation of metal binding sites for taxifolin as The featured reaction between DPPH· and usnic acid is
flavonoids

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 Archives of Toxicology

shown in Fig. 8. In the usnic acid molecule, phenolic group and Ingold 2004). In addition, the occurrence of SPLET in
has also two hydroxyl units. Withdrawal of hydrogen atoms methanol and ethanol has also been clearly demonstrated
from phenolic hydroxyl groups may occur easily. Usnic acid by Foti et al. (2004a, b). In the study, it was shown that the
can be found in the triradical structures by removing three reactions of DPPH· with some phenolic acids such as caf-
DPPH molecules using resonance structures (Cetin Cakmak feic acid, p-coumaric acid, ferulic acid, and sinapic acid.
and Gulcin 2019). Rate constants for reactions for the methyl esters of these
These reactions are strongly accelerated by increased acids were a few times greater than for the free acids as a
electron density in rings A and C. Furthermore; the alter- consequence of the suppression of ionization of the phenolic
native route is a fast electron transfer (ET) from phenolate –OH group by the free carboxylic acid. These experiments
anion to DPPH radicals. Ring A is strongly electron with- nicely confirm the role that phenol ionization can play in
drawing, and as an effect of conjugation, the catechol moiety the reactions of phenols with DPPH· in solvents that can
in ring B is the most probable site of deprotonation. As a support ionization.
result of the presence of –OH groups, many flavonoids are Flavonoids including flavones, flavonols, isoflavones,
mainly located in the water phase of the biological systems. flavonones, and chalcones occur in all types of higher plant
Reactions of flavonoids with electron-deficient radicals can tissues (White and Xing 1997). Flavones and flavonols
be accelerated by the SPLET mechanism to effectively mini- are found in almost every plant, particularly in the leaves
mize the accumulation of the reactive oxygen species in the and petals, with flavonols occurring more frequently than
cell (Musialik et al. 2009). Because SET-PT and SPLET flavones (Table 3). Some of the common flavonoids are
mechanisms are of importance in solvated media, it is inter- apigenin, chrysin, luteolin, datiscetin, myricetin, querce-
esting to ascertain how the water alters the reaction enthalp- tin, kaemferol and morin. Also, most of the flavonoids
ies of individual steps of the three mechanisms (Klein et al. in plants occur as glycosides (Macheix et al. 1990). The
2007). Also, Litwinienko and Ingold proposed another ability of flavonoids to inhibit lipid peroxidation is well
mechanism for SPLET (2004). This mechanism is favored documented and well known, both for natural lipid prod-
for reactions of phenols having low pKas with electron- ucts and for model lipids (Bors et al. 1996). It was reported
deficient radicals having relatively low HAT activities and that flavonoids may act as antioxidants by scavenging
yielding product molecules having low pKas (Litwinienko radicals such as lipid peroxyl radicals (Takahama 1985),

Fig. 6  Possible mechanisms for DPHPH DPPH-H

OH
OH
quercetin reaction as a flavonoid OH
OH
with DPPH radicals -O O
HO O

OH
OH
OH O
OH O
Quercetin

ET
t)

(V
n
lve

ery
o
T
ar s

fas
HA

t)
pol
on -
(N

O H
OH
O OH
T
HA

HO O O O

OH OH
OH O OH O

DPHPH

H+ DPPH-H

O H
O O H

O O O-
O O
OH
OH O OH
OH O

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Archives of Toxicology

3 O O O
2 4 OH
8 B DPPH HO O H HO O H HO O H
HO O 2
5 DPPH-H
O O O
OH
7
A C 6

6 OH OH OH
5 4
3 OH
OH O OH O OH O
OH O

O O
H O O
HO O HO O OH HO O
OH O HO O OH
OH
OH OH OH
OH
O O OH O OH O
H OH O

O O O
O

HO O HO O HO O
HO O OH OH OH
OH

OH OH OH
OH
O OH O OH O OH
O O
H

Fig. 7  The proposed reaction scheme between DPPH free radicals and taxifolin (dihydroquercetin)

Fig. 8  Purposed radical scav-

enging mechanism between O O O
usnic acid and DPPH radicals DPPH. DPPH2
HO O O O O O O O O

OH O O OH O O OH O O
DPPH.

DPPH2
O O

HO O O O O O

O O O OH O O

O O O

HO O O HO O O O O O

O O O O O O O O O

O O
O
O O O O O O
HO O O

O O O O O O
O O O

superoxide anion radicals (Hu et al. 1995) and hydroxyl and Das 1993). It is concluded that for maximal radical
radicals (Husain et al. 1987), singlet oxygen quenchers scavenging activity, a flavonoid molecule needs to meet
(Takahama 1984), and metal ion chelators (Ramanathan the following criteria; 3′,4′-dihydroxy structure in the

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 Archives of Toxicology

B-ring, 2,3-double bond in conjunction with a 4-oxo group growth factors, provitamin A effect, stimulation of immune
in the C-ring, presence of a 3-hydroxyl group in the C-ring response, cell differentiation, regulation of cell cycle, and
and a 5-hydroxyl group in the A-ring (Hu and Ding 1996). important role as antioxidant agents. Because of their anti-
Flavonoids with free hydroxyl groups act as free-radical oxidant properties, a diet rich in carotenoids is associated
scavengers and multiple hydroxyl groups, especially in the with a reduced risk of development of several disorders
B-ring, enhance their antioxidant activity. The hydroxyls caused by oxidative stress, such as various types of cancer,
in ring B are the primary active sites in interrupting the ophthalmologic and cardiovascular diseases (Monego et al.
oxidation chain (Jovanovic et al. 1994). 2017). It was reported that there are more than 700 carot-
Flavonoids are the most common and popular group enoids, from which 40 are ingested in the human diet, which
of polyphenolic compounds in human diet and are found obtained from vegetables and fruits. Their chemical proper-
ubiquitously in plants. They can prevent coronary heart dis- ties are closely related to the presence of an extended sys-
ease and have antioxidant properties (Gülçin et al. 2011a, tem of conjugated double bonds, which is substituted with
2011b). The antioxidant and biological activities of flavo- various end groups. They are one class of major food micro-
noids depend on their chemical structure. There are three nutrients in the human diet. In plants, they have potential
structure groups responsible for the determination of free antioxidant properties due to their chemical structure (Gul-
radical scavenging and antioxidant activities of flavonoids: cin 2012). In the human organism, carotenoids are part of
a catechol moiety of the B-ring, the 2,3-double bond in con- the antioxidant defense system, too. They can be separated
jugation with a 4-oxo function of a carbonyl group in the into two vast groups: the carotenoid hydrocarbons known
C-ring and the presence of hydroxyl groups at the 3 and 5 as the carotenes which contain specific end groups like
positions. Quercetin possesses all the three structure groups lycopene and b-carotene; and the oxygenated carotenoids
and thus usually gives a higher antioxidant potential than known as xanthophylls, like zeaxanthin and lutein (Caro-
kaempferol, which has no catechol moiety in the B-ring. The cho and Ferreira 2013). They can be classified as carotenes
presence of a hydroxyl group at 5′ in the B-ring increases the and xanthophylls, based on their chemical structure. As can
antioxidant potential significantly. be seen in Fig. 9, α-, β-carotene, and lycopene are exam-
Quercetin and kaempferol are flavonoids widely distrib- ples of carotenes, i.e., non-oxygenated carotenoids. Lutein,
uted in nature. Also, it was described that quercetin was zeaxanthin, astaxanthin, and canthaxanthin are examples
much more effective than kaemferol in retarding autoxida- of xanthophylls, i.e., oxygenated derivatives of carotenes.
tion of lard (Yanishlieva et al. 1984) and of methyl linoleate The quantitative most important carotenoids in the human
(Pekkarinen et al. 1999a). The relative reactivities, as well diet are β-carotene, lycopene, lutein, β-cryptoxanthin, zeax-
as the stoichiometric coefficients for a number of flavo- anthin, and astaxanthin (Riccioni 2009). Numerous obser-
noids, catechols and standard phenolic antioxidants, have vational studies have supported the hypothesis that anti-
been determined (Roginsky et al. 1996). In addition, it was oxidants like carotenoids and vitamin E or metabolites of
analyzed the kinetics of oxygen consumption in organic and these nutrients are associated with cardiovascular diseases
micellar systems, with peroxidation initiated by lipid- or (Lichtenstein 2009). Carotenoids could be used as an inex-
water-soluble initiators. Roginsky et al. (1996) demonstrated pensive means of prevention and as a possible treatment,
that the flavonoids did not behave as classic phenolic antioxi- even though human intervention trials showed controversial
dants such as α-tocopherol, but showed only moderate chain- results, with some positive findings, many null findings,
breaking activities. The antioxidant activity of flavonoids is and some suggestion of harm in certain high-risk popula-
greatly affected by the system used as a substrate and the tions. Carotenoids are the principal pigments responsible
conditions used to catalyze oxidation. The structure–activ- for the colors of vegetables and fruits including ß-carotene,
ity relationship for the antioxidant activities of flavonoids lutein, zeaxanthin and lycopene, which are responsible for
was well argued and well known (Foti et al. 1996; Chen the red color of red tomatoes and other fruits, it is found
et al. 1996). in. Its color is due to its many conjugated carbon double
Carotenoids are natural colorants and lipid-soluble plant bonds. Each double bond reduces the energy required for
pigments widely distributed in nature with pronounced electrons to transition to higher energy states, allowing the
antioxidant activity. They are a class of natural pigments molecule to absorb visible lengths of progressively longer
derived from a basic structure of at least 40 carbons with an wavelengths (Riccioni 2009). Due to this antioxidant poten-
extensive conjugated double bond system. They are synthe- tial, carotenoids are widely employed in the food industry
sized by plants and microorganisms but not by animals. In by their strong coloration. Recent smaller interventional
plants, they can be found esterified to fatty acids or unest- studies with carefully chosen populations, such as those
erified. In the human diet, carotenoids and derivatives like under high levels of oxidative stress, have yielded largely
retinol had a large spectrum of biological effects including positive results (Lichtenstein 2009). The main antioxidant
modulation of gap junction communication, modulation of effect of carotenoids is due to 1O2 quenching. It results

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Archives of Toxicology

Fig. 9  The chemical struc-

tures of common carotenoids
including lycopene, α-carotene,
β-carotene, β-cryptoxanthin,
lutein, zeaxanthin and astax-
anthin

in excited carotenoids that dissipate the newly acquired is distinguished by having beta-rings at both ends of the
energy through a series of rotational and vibrational inter- molecule (Susan and Van Arnum 1998).
actions with the solvent, thus returning to the unexcited β-Cryptoxanthin is a natural carotenoid pigment, which
state and allowing them to quench more radical species. isolated from a variety of sources including the petals and
This situation can occur while the carotenoids have con- flowers of plants in the genus Physalis, orange rind, papaya,
jugated double bonds within. The only free radicals that egg yolk, butter, apples, and bovine blood serum (Lorenzo
completely destroy these pigments are ROO·. Carotenoids et al. 2008). Also, lutein is a xanthophyll and one of the
are relatively unreactive but may also decay and form non- most known naturally occurring carotenoids. It was found in
radical compounds that may terminate free radical attacks green leafy vegetables such as kale and spinach. Organisms
by binding to the indicated radicals (Carocho and Ferreira employed lutein as an antioxidant for blue light absorption.
2013). They are a class of natural fat-soluble compounds Lutein is also found in egg yolks, animal fats, and the retina
in which their chemical properties are closely related to (Johnson et al. 2000). Zeaxanthin is one of the two primary
the presence of extended conjugated double bonds. In the xanthophyll carotenoids contained within the retina of the
human system, such carotenoids as β-carotene, lycopene, eye. Within the central macula, zeaxanthin is the dominant
lutein, β-cryptoxanthin, zeaxanthin, and astaxanthin are component, whereas in the peripheral retina, lutein predomi-
greatly associated with mitigating cardiovascular diseases nates. The name is derived from Zea mays in which zeax-
(Lichtenstein 2009). anthin provides the primary yellow pigment (Krishnadev
Lycopene is a carotenoid pigment, found in tomatoes et al. 2010). Like many carotenoids, astaxanthin is a color-
and other red fruits, like watermelon, papaya, pink grape- ful, lipid-soluble pigment. It is found in microalgae, yeast,
fruit and pink guava. Its name is derived from the tomato’s salmon, trout, krill, shrimp, crayfish, crustaceans, and the
species classification, Solanum lycopersicum. Lycopene feathers of some birds. It provides the red color of salmon
absorbs most of the visible spectrum, so it appears red. The meat and cooked shellfish. Astaxanthin, unlike some carot-
chemical structures of some carotenoid pigments such as enoids, is not converted to retinol (vitamin A) in the human
β-carotene, lycopene, lutein, β-cryptoxanthin, zeaxanthin, body. Too much vitamin A is toxic for a human, but astax-
and astaxanthin are shown in Fig. 9. β-Carotene is a strongly anthin has lower toxicity. It is an antioxidant with a slightly
colored red-orange pigment abundant in plants and fruits. lower antioxidant activity than other carotenoids (Mortensen
It is an organic compound and chemically is classified as and Skibsted 1997; Gulcin 2012).
a hydrocarbon and specifically as an isoprenoid, reflecting Retinol (Vitamin A) is a carotenoid produced in the
its derivation from isoprene units. β-Carotene is biosynthe- liver and results from the breakdown of β-carotene. It was
sized from geranylgeranyl pyrophosphate. It is a member reported a dozen forms of vitamin A, which can be iso-
of the carotenes, which are tetraterpenes, synthesized bio- lated. It had a beneficial effect on the skin, eyes and internal
chemically from eight isoprene units and thus having 40 organs. What confers the antioxidant activity is the ability
carbons. Among this general class of carotenes, β-carotene to combine with ROO· before they propagate peroxidation

13
 Archives of Toxicology

to lipids (Jee et al. 2006; Carocho and Ferreira 2013). They that, despite also being radicals, are unreactive and unable
play a role in protecting plants against photooxidative pro- to continue the oxidative chain reaction. It is the only major
cesses. They are efficient antioxidants, e.g., in scavenging lipid-soluble, chain-breaking antioxidant found in plasma,
singlet oxygen (Di Mascio et al. 1989) and ROO· (Stahl and red cells and tissues, allowing it to protect the integrity of
Sies 2003). lipid structures, mainly membranes (Carocho and Ferreira
Tocopherols (Vitamin E) are fat-soluble vitamins, and- 2013). α-Tocopherol is a hydrophobic antioxidant, which is
possess antioxidant properties to protect living organism only soluble in inorganic solvents and membranes, and is
especially body cells from the damage caused by free radi- difficult to handle in buffered reaction media. This situation
cals and ROS. Vitamin E is the generic name for tocophe- creates difficulties for experimental studies. This revives
rols, which are a class of chemical phenolic compounds the use of α-tocopherol analogues that enables the ability
(ArasHisar et al. 2004; Gülçin et al. 2005a). They are the to work in homogeneous, aqueous solutions, besides hav-
best-known and most widely used antioxidants (Pokorny ing a significant antioxidant activity. Among them, trolox,
1987; Koksal et al. 2011; Cetinkaya et al. 2012). Tocophe- in which the polyisoprenoid tail of α-tocopherol has been
rols naturally are present in a wide range of foods, including replaced by a carboxyl moiety, has the precedence of being
green leafy vegetables and fatty foods such as vegetable moderately water-soluble. In this molecule, the carboxyl
oils, seeds, nuts, and egg yolk. Vitamin E is composed of group provides the water solubility property, while antioxi-
eight isoforms with four tocopherols and four tocotrienols. dant effect is supplied by the chromanol moiety (Aksu et al.
Tocopherols and tocotrienols are collectively known as 2015; Çelik et al. 2018).
tocols. They are monophenolic compounds and comprise They are present, at least in traces, in nearly all food
a group of eight chromanol homologs that possess vitamin materials. In general, food sources with the highest con-
E activity in the diet They can be classified as tocophe- centrations of vitamin E are vegetable oils, followed by
rols and tocotrienols and within each of these two classes, nuts and seeds including whole grains. The most important
there are four isomers (α-, β-, γ- and δ-tocopherols) mak- antioxidant of this group is α-tocopherol, which has lower
ing a total of eight tocopherol isomers (Table 4). However, antioxidant activity in edible oils than other tocopherols.
α-tocopherol is the most potent and plentiful isoform in bio- α-Tocopherol is a lipid-soluble antioxidant. In addition to
logical systems. Tocopherols includes a 6-chromanol group decreasing lipid peroxidation, α-tocopherol may exert intra-
and an apolar phytal chain, having the prefix α-, β-, γ-, or cellular effects. α-Tocopherol is a lipid-soluble vitamin,
δ-, depending on the number and position of methyl groups present in the outer membrane of cells and cell organelles.
attached to the chroman rings (Dziezak 1986; Shahidi and It breaks the chain and protects membranes from further
Ambigaipalan 2015). The chroman head group confers the oxidation (Godbout et al. 2004). Among the vitamin E iso-
antioxidant activity to tocopherols, but the phytyl tail has no forms, α- and γ-tocopherol are the most abundant in diet
influence. All feature a chromanol ring, with a –OH group and tissues. However, α-tocopherol is the most biologically
that can donate a hydrogen atom to reduce free radicals active and clinically relevant. Like α-tocopherol isoform,
and a hydrophobic side chain which allows for penetra- γ-tocopherol also reacts with ROS and RNS and thus may
tion into biological membranes (Burton and Ingold 1981). be beneficial for inflammation. α-Tocopherol is the main
α-Tocopherol halts lipid peroxidation by donating its phe- source found in supplements and in the European diet while
nolic hydrogen to the ROO· forming tocopheroxyl radicals γ-tocopherol is the most common form in the American diet

Table 4  The classification of tocopherols and tocotrienols

R5 R4 R5 R4
HO HO

R7 O R7 O
R8 R8

Tocopherols Tocotrienols

Tocopherols R4 R5 R7 R8 Tocotrienols R4 R5 R7 R8

α-Tocopherol H CH3 CH3 CH3 α-Tocotrienol H CH3 CH3 CH3

β-Tocopherol H CH3 H CH3 β-Tocotrienol H CH3 H CH3
γ-Tocopherol H H CH3 CH3 γ-Tocotrienol H H CH3 CH3
δ-Tocopherol H H H CH3 δ-Tocotrienol H H H CH3

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Archives of Toxicology

(Jiang et al. 2001). The compound α-tocopherol, a common Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-car-

form of tocopherol added to food products. Tocopherols act boxylic acid) or water-soluble analogue of tocopherol,
as antioxidants by donating the hydrogen of the hydroxyl serves as a positive control, which inhibits the formation
group to the lipid ROO·. One radical formation from a of radical cation. It is not a naturally occurring antioxidant
tocopherol is stabilized through the delocalization of the (Bursal et al. 2019; Gulcin et al. 2019a). The antioxidant
solitary electron over the aromatic ring structure (Fig. 10). activity present in the biological fluids, cells, tissues, and
This group of compounds is highly lipophilic, and operative natural extracts is optimized by trolox as a standard. Trolox
in membranes or lipoproteins. Their most important antioxi- is a water-soluble analog of α-tocopherol. It has antioxidant
dant function appears to be the inhibition of lipid peroxida- activity like α-tocopherol. Trolox has been widely used as a
tion, scavenging lipid peroxyl radicals to yield lipid hydrop- model compound of α-tocopherol in biological, biochemical
eroxides and a tocopheroxyl radical (Diplock et al. 1998). and pharmaceutical applications to scavenge free radicals
This radical forms non-radical products, including sta- and reduce oxidative stress damage (Garibov et al. 2016;
ble peroxides, which can be reduced to tocoquinones and to Çelik et al. 2018). Trolox equivalent antioxidant capacity
tocopherol dimers. α-Tocopherol has also been associated measures the concentration of trolox solution with reference
with retarding the decomposition of hydroperoxides (Fran- to the concentration of standard antioxidant solution under
kel 1996). Etminan et al. (2005) reported that α-tocopherol investigation (Shivakumar and Kumar 2018; Aras et  al.
had a protective effect against Parkinson’s disease. Tocoph- 2019; Gulcin et al. 2019b, 2020).
erol deficiency causes neurological problems such as Ascorbic acid (Vitamin C) Ascorbic acid includes
spinocerebellar ataxia and myopathies (Brigelius-Flohé two forms with antioxidant activity: l-ascorbic acid and
and Traber 1999). The hydrogen-donating power of toco- l-dehydroascorbic acid, which are both absorbed through
pherols in fats, oils, and lipoproteins is in the order δ > β the gastrointestinal tract and can be interchanged enzymati-
≈ γ > α-tocopherol. It has been found that the antioxidant cally in vivo. It was reported that ascorbic acid is effective
strength of α-tocopherol in butter oil triacylglycerol was less scavenging effects against ­O2·−, ­H2O2, OH·, lO2 and reac-
than that of γ-tocopherol (Lampi and Piironen 1998). tive ­NO2· (Barros et al. 2011). It is considered to be one of

Fig. 10  Radical scavenging LOOH

LOO
mechanism of α-tocopherol CH3
CH3
O
OH

H3C O R
H3C O R
CH3
CH3
α-Tocopherol

CH3
O CH2
HO
H 3C O R
OL
CH3 H3C O R
Tocoquinone CH3

e-
CH3
CH3
O
O

H3C O R
O H 3C O R
CH3
OL CH3
Tocopheril peroxide

LOO

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 Archives of Toxicology

the most powerful, least toxic natural antioxidants (Weber NADP+ Reduced Dehidro H2O
et al. 1996). It is water-soluble vitamin and is found in high Glutathione ascorbic acid

concentrations in many dietary food or plants. Typically NADPH Oxidase Dehydroascorbate

oxidase
Ascorbic acid
oxidase

it reacts with oxidants. Ascorbic acid can terminate chain Oxidized

NADP + H+ Ascorbic acid X2O2
radical reactions by electron transfer. It is special because it Glutathione

can transfer a single electron. Ascorbic acid is an effective

reductones. It behaves as a vinylogous carboxylic acid where
Fig. 12  The enzymatic conversion dehydroascorbate to ascorbic acid
the electrons in the double bond, hydroxyl group lone pair, by dehydroascorbate oxidase
and the carbonyl double bond form a conjugated system.
Because the two major resonance structures stabilize the
deprotonated conjugate base of ascorbic acid, the hydroxyl it can also become a prooxidant. This happens when it
group in ascorbic acid is much more acidic than typical combines with Fe and Cu reducing ­Fe3+ to ­Fe2+ or ­Cu3+
hydroxyl groups. In other words, ascorbic acid can be con- to ­Cu2+, which in turn reduces ­H2O2 to OH· (Duarte and
sidered an enol where the deprotonated form is a stabilized Lunec 2005). Also, it was reported that some phenolic
enolate (Fig. 11). antioxidants lose their activity at high concentrations and
Ascorbic acid was recorded to regenerate α-tocopherol behave as prooxidants by involvement in initiation reac-
from its tocopheroxyl radical in  vivo and in  vitro and tions (Gorden 1990).
thereby restoring its antioxidant activity (Kamal-Eldin ROO∙ + Toc − OH → ROOH + Toc − O∙ ,
and Budilarto 2015). Also, human plasma contains about
60 µmol ascorbate. On interaction with ROS, it is oxi-
Toc − O∙ + Ascorbic acid → Toc − OH + Ascorbate.
dized to dehydroascorbate via the intermediate ascorbyl
free radical. As can be seen in Fig. 12, dehydroascorbate This process would allow for the transport of a radical
is recycled back to ascorbic acid by the enzyme dehy- load from a lipophilic compartment to an aqueous com-
droascorbate reductase. Thus, dehydroascorbate is found partment where it is taken care of by efficient enzymatic
in only very low levels compared with ascorbate. defense systems. It should be noted, however, that ascor-
As a scavenger of ROS, ascorbic acid has been shown bate might also act as a prooxidant in vivo. In the presence
to be effective against the ­O2·–, ­H2O2, the OH· and 1O2. In of free transition metal ions and ascorbate, the OH· can be
aqueous solutions, ascorbic acid also scavenges reactive generated and initiation of lipid peroxidation may occur.
nitrogen oxide species efficiently. The major sources of However, the amounts of free transition metals in vivo
ascorbic acid in the diet are fruits, especially citrus fruits, are very small because they are efficiently bound to pro-
kiwi fruit, cherries, melons, and vegetables such as toma- teins. Vitamin C has additional well-established biological
toes, leafy greens, broccoli, cauliflower, Brussels sprouts, functions including cofactor-activity for several important
and cabbage; its content might exceed g ascorbate/kg enzymes such as hydroxylases (Levine et al. 1996).
fresh weight. Recently, Bursal and Gülçin (2011) recorded Stilbenes are a small group of phenylpropanoids charac-
approximately 1000  mg vitamin C per kg lyophilized terized by a 1,2-diphenylethylene backbone. They are a fam-
kiwifruit (Actinidia deliciosa) extract. At low dose levels ily of secondary metabolites derived via phenylpropanoid
(100 mg), the bioavailability values for vitamin C from pathway or via the polyketide pathway, either of which con-
synthetic and food sources are very similar (Mangels et al. sists of a cis- or trans-ethene double bond substituted with a
1993). The efficacy of absorption decreases with increas- phenyl on both carbon atoms of the double bond. Naturally
ing dose levels. There is evidence from studies in vitro occurring nucleosides modified by stilbene derivatives may
that vitamin C is capable of regenerating tocopherol from constitute one of the components of such a system. Stilbenes
the tocopheroxyl radical, which is formed on inhibition of in particular trans-resveratrol and its glycoside are beneficial
lipid peroxidation by vitamin E (Niki et al. 1985). Vitamin to health, having antioxidative, anticarcinogenic, and antitu-
C is considered an effective antioxidant and intervenes in mor properties (Krawczyk 2019). Most of stilbenes are their
many physiological and biochemical reactions. However, derivatives are found in particular plant families (Table 5).
Some examples of common stilbenes isolated from diverse
HO HO HO plant families contain grape, pine, peanut, and sorghum.
HO HO HO
In recent years, plant stilbenes have received considerable
H
O
O -H
O
O
O interest, due to their biological activities and possible phar-
H H O
macological applications. Since resveratrol was postulated
HO OH O OH O OH to be involved in the health benefits. It is one of the most
extensively studied natural products (Gulcin 2010).
Fig. 11  Mechanistic steps on antioxidant action ascorbic acid

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Archives of Toxicology

Resveratrol has gained significant worldwide attention and synthetic sources are useful in a number of fields and are
because of its ability to inhibit or retard a wide variety of commonly used in medicine, often in the form of glycosides.
animal diseases, including cardiovascular disease and can- Coumarin is highly important O-heterocyclic that consists
cer. Also, it has been speculated that resveratrol acts as an of the fusion of benzene and α-pyrone rings. Coumarin is
antioxidant, inhibit platelet aggregation, promotes nitric also a member of flavonoid family, which is associated with
oxide production, and increases high-density lipoprotein low toxicity. Recently, it was shown that biochemical and
cholesterol, thereby serving as a cardio-protective agent. In pharmacological properties of coumarins depend on the pat-
addition, resveratrol was shown to function as a chemo-pre- tern of substitutions, containing the therapeutic applications,
ventive agent, and there has been a great deal of experimen- and can beneficially affect toxicity. It is well known that the
tal effort directed toward defining this effect. Furthermore, presence of a hydroxyl and amine groups is important for
resveratrol exhibits anti-inflammatory, neuroprotective, and many biological active coumarins. The chemical structure of
antiviral properties (Wolter and Stein 2002; Gülçin 2010). coumarin and its putative and simple derivatives were shown
The inhibition effects of resveratrol against some metabolic in Table 6 (Skalicka-Wozniak et al. 2016).
enzymes including human carbonic anhydrase, linked to Recently, the synthesis of a large spectrum of coumarins
some diseases were well known and well established (Inno- and their derivatives has attracted attention in research and
centi et al. 2010b; Senturk et al. 2011; Koksal et al. 2020). development to both medicinal and organic chemists due to
It was reported that resveratrol has an effective antioxidant their various biological activities like antitumor, antimicro-
effect in different in vitro assays including total antioxidant bial, anti-HIV, serine protease inhibition, anti-cancer, anti-
activity, reducing power, DPPH·, ­ABTS·+, ­DMPD·+ and oxidant activity and vasorelaxant effect (Koleva et al. 2019).
­O2·− scavenging, ­H2O2 scavenging, and metal chelating Different pharmacological activities of coumarins mainly
activities, when compared to standard antioxidant com- depend on the type of coumarin nucleus. The most important
pounds such as BHA, BHT, and α-tocopherol (Gülçin 2010). group of commonly used drugs based on coumarin deriva-
Coumarins (2H-chromen-2-one) and its related ana- tives is vitamin K antagonists such as warfarin, phenpro-
logues occur naturally in higher plants and also in microor- coumon or acenocoumarol used as anticoagulants (Salvo
ganisms as secondary metabolites (Jayashree et al. 2014). It et al. 2017). Coumarins are already of clinical significance.
plays a major role in the proper functioning of the individual They first acquired interest when the anticoagulant activity
plant parts. It is an important group of compounds from of dicumarol was established and the synthetic coumarin
synthetic and natural sources, which are useful in a number Warfarin (Coumadin) became a commercial drug in 1954.
of fields. It can be described as a benzene molecule with two Warfarin is the most widely prescribed oral anticoagulant
adjacent hydrogen atoms replaced by a lactone-like chain, drug with established efficacy for the prevention of thrombo-
forming a second six-membered heterocycle that shares two embolic events (Orhan and Gulcan 2015; Skalicka-Wozniak
carbons with the benzene ring. Its derivatives from natural et al. 2016).

Table 5  The chemical structure of common plant stilbenes (O-Gly:

O-β-D-glycopyranoside) Table 6  The chemical structure of coumarin and its putative and sim-
ple derivatives
R5
R1

R2
‘
R4
R3 R3 O O
R3
‘

R4

Stilbens R3 R5 R3’ R4’ Coumarins R1 R2 R3 R4

trans-Resveratrol OH OH H OH Umbelliferone H H H H
trans-Piceid O-Gly OH H OH Esculetin H OH H H
trans-Pterostilbene OCH3 OCH3 H OH Scopletine H OCH3 H H
Pinosylvin OH OH H – Scoparone H OCH3 OCH3 H
Pinosylvin monoethylether OCH3 OH H OH Osthole H H OCH3 CH2–
Piceatannol OH OH OH OH CH=C(CH3)2
Astringin O-Gly OH OH OH Fraxetin H OCH3 OH OH
Rhapontin O-Gly OH OH OCH3 Daphnetin H H OH OH

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Lignans are a large group of naturally occurring poly- Lignans are generally found as dimers, but some of were
phenols that are derived from the shikimic acid biosynthetic found as trimers or tetramers. In plants, most of the lignans
pathway with a wide spectrum of biological functions found are in a free state, while some of them can combine with
abundantly in the plant kingdom and human food sources. glycon and form glycosides and other derivatives.
Lignans are widely distributed in the plant kingdom, and When the molecular linkage of monomers occurs between
they exist in plant roots, stems, rhizomes, leaves, fruits, β-β′ positions, lignan is designated as “classical lignans”.
flowers, seeds, resins, and xylem. Until now, lignans are However, if the main structural units are combined in
found in over 70 plant families, and more than 100 neol- another manner without the β-β′ linkage, the compounds are
ignans and 200 classical lignans have been characterized. grouped as "neolignan". Figure 14 shows the monomers and
Structurally, they contain a basic scaffold of two or more the classification of lignans. As seen in this figure, neolignan
phenylpropanoid units and the monomers forming lignans has more different structures than classical lignans (Cui et al.
are cinnamic acid, propenyl benzene, allyl benzene, and 2020). The pest example of neolignan is silymarin, which
cinnamyl alcohol. Lignans are structurally a very diverse possessed therapeutic effects of silymarin and naringin on
group. Up to now, almost two thousands of lignans have methotrexate-induced nephrotoxicity, anti-inflammatory,
been described (Markulin et al. 2019). They are polyphe- antiapoptotic and anti-autophagic properties (Kandemir et al
nolic substances derived from phenylalanine via dimeriza- 2017b). Silymarin is a plant-derived flavonoid, which is clas-
tion of substituted cinnamic alcohols to a dibenzylbutane sified as benzopyranone. It is isolated from the fruits and
skeleton (2,3-dimethylbutane-1,4-diyl)dibenzene) catalyzed seed of the milk thistle (Silymarin marianum) and widely
by oxidative enzymes and is often controlled by dirigent pro- used for the treatment of several diseases. This putative
teins. Neolignan is formed by joining the two-propylbenzene lignan has been possessed antioxidant and mammalian car-
residues at other than the β-carbon atom of the propyl side bonic anhydrase isoforms I–XV inhibition properties (Kok-
chain. When a part of the human diet, some plant lignans sal et al. 2009; Innocenti et al. 2010b).
are metabolized by intestinal bacteria to mammalian lig- They are dimers of two phenylpropanoid units with a dif-
nans enterodiol (1,4-bis(3-hydroxyphenyl)butane-2,3-diol) ferent degree of oxidation and substitution patterns of their
and enterolactone (3,4-bis(3-hydroxybenzyl)dihydrofuran- aromatic moieties. Lignans had phytoestrogenic effects.
2(3H)-one) (Fig. 13). In mammals, lignans can easily metab- Phytoestrogens are plant-derived compounds that have
olized to mammalian lignans including pinoresinol, secoi- estrogenic properties. The term “phytoestrogens” includes
solariciresinol, lariciresinol, matairesinol, syringaresinol, two main groups of compounds including isoflavones and
hydroxymatairesinol, and sesamin (Heinonen et al. 2001). lignans, which present in significant amounts in the human

Fig. 13  Some mammalian
lignans including enterodiol and
enterolactone

Fig. 14  The classification and

chemical structures of lignans

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diet (Lampe 2003). It was reported that lignans have been anticarcinogenic activities. Also, it inhibited skin, lung and
possessed potent biological activities including antimalarial, forestomach tumors induced by polycyclic aromatic hydro-
antiparasitic, antioxidant, antibacterial, immunosuppressive, carbon carcinogens and N-methyl-N-nitrosourea in mice. It
antifungal, antiasthmatic effects (Neuhaus et al. 2019; Cui was proven that, in addition, tannic acid had an effective
et al. 2010), and carbonic anhydrase, acetylcholinesterase superoxide, DPPH and ABTS radical scavenging activity,
and butyrylcholinesterase enzyme properties (Polat Köse ­H2O2 scavenging activity, ­Fe3+ reducing power and metal
and Gulcin 2017). There is a growing interest in promoting chelation on ferrous ion activities (Gulcin et al. 2010a, b,
the inclusion of lignan-rich foods in humans’ diets. Lignan c, d).
rich foods had advantageous for human health, breast cancer Natural antioxidants are generally preferred by consum-
patients with higher lignan intake through food showed bet- ers, and may gain legislative approval more easily than syn-
ter survival chance and reduction in tumor growth (Ezzat thetic additives do. However, the fact that a substance is
et al. 2018). commonly found in a food is no guarantee that it is entirely
Tannins are defined as condensed or hydrolyzable nontoxic (Matsuo and Kaneko 1999). Synthetic antioxidants
proanthocyanidins depending on their chemical structures are tested for carcinogenic or mutagenic effects, but many
(Fig. 15). They are widely distributed in many plant spe- natural food compounds have not yet been tested. The advan-
cies, where they play a role in protection from predation tages and disadvantages of synthetic and natural antioxi-
including as pesticides and might help in regulating plant dants are summarized in Table 7 (Ames 1996; Valenzuela
growth. Likewise, the destruction or modification of tan- and Nieto 1996; Gülçin 2012). However, the benefits of
nins with time plays an important role when determining using antioxidants outweigh the risk. Without antioxidants
harvesting times. The tannins are oligomers and polymers in foods, oxidation products are formed, and these cause a
of flavonoids, specifically flavan-3-ols, whilst hydrolyzable greater risk to health than the possible hazardous effects of
tannins are glycosylated gallic acid. The phenolic groups of antioxidants (Gulcin 2012).
tannins bind very tightly with the –NH groups of peptides
and proteins; they prevent their hydrolysis and digestion in
the stomach and known as anti-nutritional in nature. Proan- Antioxidant effects
thocyanidins are principally found in fruits, especially cocoa
and tea. However, the major sources of hydrolyzable of tan- In the past years, the importance of antioxidants in the
nins are legumes, berries, and leafy vegetables (Shahidi and protection of organisms or tissues, or of nonliving systems
Naczk 2004; Shahidi and Ambigaipalan 2015). against oxidative stress has become evident. This statement
Tannic acid is the most putative type of tannin. It has is supported by studies performed in a variety of areas,
a structure consisting of a central glucose and ten galloyl including physiology, pharmacology, nutrition and even
groups (Fig. 15). They is a type of water-soluble polyphe- food processing (Magalhaes et al. 2009). In terms of food,
nol that is present in the bark and fruits of many plants and antioxidants may be defined as any substances, which are
fruits including bananas, raisins, grapes, sorghum, spinach, capable of delaying, retarding or preventing the develop-
coffee, persimmons, chocolate, and tea. It is always used as a ment in food of rancidity or other flavor deterioration due
food additive and has safe dosage ranges from 10 to 400 μg, to oxidation. Antioxidants delay the development of off-
depending on the type of food to which it is added. Also, it flavors by extending the induction period. Addition of anti-
was demonstrated that tannic acid had antimutagenic and oxidants after the end of this period tends to be ineffective
in retarding rancidity development (Taslimi et al. 2018a, b).
Also, they can inhibit or retard oxidation in two ways: either
by scavenging free radicals, in which case the compound
is described as a primary antioxidant, or by a mechanism
that does not involve direct scavenging of free radicals.
Primary antioxidants include phenolic compounds such as
α-tocopherol (Altay et al. 2019). These compounds are con-
sumed during the induction period. Secondary antioxidants
operate by a variety of mechanisms including binding of
metal ions, scavenging reactive oxygen species, converting
hydroperoxides to non-radicalic species, absorbing UV radi-
ation or deactivating singlet oxygen. Normally, secondary
Tannin Tannic acid antioxidants only show antioxidant activity when a second
minor component is present. This can be seen in the case of
Fig. 15  Chemical structure of tannin and tannic acid sequestering agents such as citric acid which are effective

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Table 7  Advantages and Synthetic antioxidants Natural antioxidants

disadvantages of natural
and synthetic antioxidants Inexpensive Expensive
commonly used for food
Widely applied Usage of some products restricted
protections
Medium to high antioxidant activity Wide ranging antioxidant activity
Increasing safety concern Perceived as innocuous substances
Usage of some of them banned Increasing usage and expanding applications
Low water-solubility Broad range of solubility
Decreasing interest Increasing interest
Some of them stored in adipose tissues Completely metabolized

only in the presence of metal ions, and reducing agents such are generated during irradiation by UV light, by X-rays and
as ascorbic acid which are effective in the presence of toco- by gamma rays; are products of metal-catalyzed reactions;
pherols or other primary antioxidants (Gülçin et al. 2008a). are present as pollutants in the atmosphere; are produced
The activities of antioxidants depend not only on their by neutrophils and macrophages during inflammation; are
structural features, for instance, on their chemical reactivi- by-products of mitochondria-catalyzed electron transport
ties towards peroxyl and other active species, but also on reactions and other mechanisms (Cadenas 1989). It was
many other factors, such as concentration, temperature, light reported that ROS might contribute to oxidative damage of
level, type of substrate, physical state of the system, as well lipids, protein, nucleic acids and polyunsaturated fatty acids
as on the numerous microcomponents acting as prooxidants in living cells. It is well known that free radicals or ROS
or synergists. It was reported that the participation of an play important roles in the development of many chronic
antioxidant in the side reactions of chain initiation and prop- diseases, such as the aging process, heart disease and can-
agation might also result in decreasing its effectiveness. For cer (Gülçin et al. 2006a). During the past years, the forma-
example, peroxydienones (Fig. 16a), resulting from reaction, tion of ROS and RNS have been implicated in the oxidative
limit the usefulness of BHT at high temperature or under UV deterioration of food products as well as in the pathogenesis
light, because they produce new free radicals that continue of several human diseases such as atherosclerosis, diabetes
the kinetic chain reaction (Gulcin 2012). Also, the resonance mellitus, chronic inflammation, neurodegenerative disor-
stabilization of phenoxy radical was illustrated in Fig. 16b. ders and certain types of cancer (Valko et al. 2006). It is
To the best of our knowledge, ROS and RNS such as significant for health to look for efficient ways to decrease
superoxide, hydrogen peroxide, and singlet oxygen and other or depress yielding of free radicals in the body. Oxidative
free radicals such as nitrogen free radicals will be generated stress was defined as an imbalance between the production
in the human body because of various inside sources or the of ROS and antioxidant defense (Buldurun et al. 2020).
outside source factor. Accumulated evidence indicates that Due to the disturbance in the equilibrium state of prooxi-
ROS, such as ROO·, HO·, O ­ 2−·, and 1O2 are involved in the dant–antioxidant reaction, ROS are overproduced to induce
pathophysiology of aging and a multitude of diseases such oxidative stress that inhibits normal functions of cellular
as cancer, Alzheimer’s and Parkinson’s diseases (Finkel and lipids, proteins, DNA and RNA (Fig. 16b). Thus, increas-
Holbrook 2000; Turkan et al. 2020). ROS and other free ing attention has been directed toward finding some natural
radicals are by-products of normal cellular metabolism in antioxidant compounds that could be isolated from herbal
aerobic life where molecular oxygen is ubiquitous. ROS medicine and efficiently clear free radicals. The protective

Fig. 16  Reaction products from

a hindered phenol (BHT) during OH
LOO LOOH O O
autoxidation (a), resonance sta- A LOO
bilization of phenoxy radical (b)

CH3 H3C OOL

CH3

O O O O

CH3 CH3 CH3 CH3

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effects of antioxidants against these deleterious oxidative- oxygen quenching activity, hydrogen peroxide decomposi-
induced reactions have received increasing attention lately, tion activity. Antioxidants serve as a defensive factor against
especially within biological, medical, nutritional, and agro- free radicals’ effects in the body. The antioxidants not only to
chemical fields (Magalhaes et al. 2008). While oxidants and eliminate ROS, but also to adjust the cellular redox state and
reductants are chemical terms, in biological environments enable redox signal transduction. They act by inhibiting the
they are usually termed as pro-oxidants and antioxidants, initiation and propagation steps, leading to the termination
respectively (Cao and Prior 1998). Prooxidants are the sub- of the reaction and delaying the oxidation process (Gülçin
stances that can induce oxidative damage to various biologi- 2006a). At present, a variety of synthetic antioxidants are
cal targets such as carbohydrates, nucleic acids, lipids and commonly used. However, the use of these compounds has
proteins (Fig. 17). been restricted by legislation due to doubts over their toxic
On the other hand, antioxidants are substances that can and carcinogenic effects. Plant foods are potential sources of
efficiently reduce a pro-oxidant with concomitant formation natural antioxidants, such as vitamin C, α-tocopherol, carot-
of products having no or low toxicity. However, up to now, enoids, flavonoid, and phenolic acids, which prevent free
Halliwell and coworkers purposed the best antioxidant defi- radical damage. They can provide phenolic hydroxyl group
nition (1995). According to these investigators, antioxidant to react with free radicals. Consequently, they will inhibit the
is a substance that when present at low concentrations, com- oxidative mechanisms, which degenerate diseases. Hence,
pared to those of an oxidizable substrate significantly delays there is a growing interest in natural and safer antioxidants
or prevents oxidation of that substrate. This protection may for food applications, and a growing trend in consumer pref-
be based on different mechanisms of action, namely: inhibi- erences towards natural antioxidants, all of which have given
tion of generation and scavenging capacity against ROS or impetus to the attempts to explore natural sources of antioxi-
RNS; reducing capacity; metal chelating capacity; singled dants (Gülçin 2006b, 2007, 2010).

-Base modification
-Base deleteion
-Frame chifts
-DNA-Protein cross linking
-Chromosomal arrangements

Nucleic
acids

-Lipid peroxidations -Sugar nonenzymatic

-Cyclic endoperoxide Lipids fragmantations
Free Radicals Carbohydrates
formation -Chain reactions leading
-MDA formation mutagenesis

Proteins

-Amino acids modification

-Peptide clavage
-Cros linking with lipid
peroxidation products

Fig. 17  Summarised biological targets of free radicals

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In recent years, a wide range of spectrophotometric assays attempting in fulfilling the gap between the reaction mecha-
has been adopted for measurement of antioxidant capacity nism of antioxidants and methodology involved.
of foods and pharmaceuticals. Therefore, it is desirable to
establish and standardize methods that can measure the
antioxidant level directly from plant-based materials, food Antioxidant methods
extracts and biological samples. The agreement on standard-
ized antioxidant methods requires (Apak et al. 2016): Different antioxidant methods have been introduced to meas-
ure and investigate the antioxidant property and capacity of
• General basis for the application of antioxidant assays commercial antioxidants, foods, medicinal, pharmaceuticals
should include the test conditions, exact description, and biological samples. The concept of antioxidant capac-
experimental apparatus, and stability of experimental ity first originated from chemistry and was later adapted
reagents. to biology, medicine, epidemiology and nutrition (Floegel
• All antioxidant methods should be validated within their et al. 2011). One of the objectives of this review article is
framework, associated with validation parameters includ- to accumulate all probable methods, used for the evaluation
ing the production of repeatability, reproducibility, and of the antioxidant property of various samples. In the last
recovery data, internal quality control, standardization, decades, there is a growing interest in the studies of the anti-
proficiency testing, and analytical quality assurance. oxidant activity of foods and diets due to the known impli-
• The results should be a reasonable comparison of the cations of oxygen free radicals in the progress and develop-
antioxidant content of pharmaceuticals, foods, and other ment of cardiovascular and neurodegenerative disease, aging
commercial products. and cancer (Garcia-Parrilla 2008). On this purpose, many
• The measurements should be improved and provided to different procedures have been developed to test the total
meet the requirement of quality standards for legal issues antioxidant capacity of foodstuffs (Perez-Jimenez and Saura-
and health demands. Calixto 2005). It was reported that the reliable methods for
antioxidant assessment are needed (Magalhaes et al. 2008).
However, up to now, the most commonly used methods A standardized method for antioxidant activity of a food
for in vitro determination of antioxidant capacity of food component should meet the following ideal requirements
constituents are inhibition of lipid peroxidation in linoleic (Prior et al. 2005):
acid system, total radical-trapping antioxidant parameter
(TRAP assay), oxygen radical absorbance capacity (ORAC • It measures chemistry actually occurring in potential
assay), ferric ion ­(Fe3+) reducing antioxidant power assay applications.
(FRAP), cupric ion (­Cu2+) reducing antioxidant power • It utilizes a biologically relevant radical source.
assay (CUPRAC assay), ­Fe3+–Fe2+ transformation assay, • It should be simple.
Folin–Ciocalteu reducing capacity (FCR assay), ­DPPH· radi- • It uses a method with a defined endpoint and chemical
cal scavenging, A­ BTS·+ radical scavenging, D­ MPD·+ radical mechanism.
scavenging, superoxide anion radical scavenging and fer- • Its chemicals and instrumentation is readily available.
rous ions (­ Fe2+) chelating activities. Most of the methods • It has good within-run and between-day reproducibility.
employ the same principle: a synthetic colored radical or • It should adaptable for assay of both hydrophilic and lipo-
redox-active compound is generated; and the ability of a philic antioxidants and use of different radical sources.
biological sample to scavenge the radical or to reduce the • It should adaptable to high-throughput analysis for rou-
redox-active compound is monitored by spectrophotometer, tine quality control analyses.
applying an appropriate standard to quantify antioxidant
capacity (Floegel et al. 2011). These multiple methods are Also, antioxidant activity should not be concluded based
recommended to measure antioxidant properties of food on a single antioxidant test model. Several in vitro antioxi-
components that better reflect their potential protective dant procedures should be carried out to evaluate the anti-
effects. A main objective of this overview is to review the oxidant activities with the samples of interest (Alam et al.
chemical principles, some of its variants, recent applications 2013). Another important point is that antioxidant tests vary
as well as the advantages and shortcomings. Also, another in different respects. Therefore, it is difficult to compare fully
aim of this review is to provide comprehensive chemical one method to another one. For this purpose, researchers
investigation methods available for the antioxidant assays. have to critically verify methods of analysis before adopting
There are quite number of research articles pertaining to that one for research purpose. However, the ideal method
the assay of antioxidant methodology and analytical reac- for determination of antioxidant properties should assess
tion mechanistic steps. In this route, I have made a modest the effect of a food compounds in reaction conditions that
mimic those found when oxidative stress is induced in vivo

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by ROS and RNS. However, this kind of assessment may be transfer one electron to reduce any compound, including
considered and exaggerated for screening purposes, consid- metals, carbonyl groups and radicals:
ering the individual testing against numerous ROS or RNS
(Magalhaes et al. 2009). On the other hand, methods can be AH + X ⋅ → AH⋅+ + X− ,
divided according to reaction mechanisms in hydrogen atom
transfer (HAT) and single electron transfer (SET) methods. AH⋅+ + H2 O → A⋅ + H3 O+ ,
In general, the methods for the determination of the
antioxidant capacity of food components can deactivate X− + H3 O+ → XH + H2 O,
radicals by two major mechanisms and were divided into
two major groups: assays based on the SET reaction, and
assays based on a HAT. The end result is the same, regard- AH + M3+ → AH+ + M2+ .
less of mechanism, but kinetics and potential for side reac- Relative reactivity in SET methods is based primarily
tions are different. SET-based methods detect the ability of on deprotonation (Lemanska et al. 2001) and ionization
a potential antioxidant to transfer one electron to reduce any potential of the reactive functional group (Wright et al.
compound, including metals, carbonyls, and radicals. SET 2001). So SET reactions are pH dependent. In general,
displayed through a change in color as the oxidant is reduced ionization potential values decrease with increasing pH,
(Huang et al. 2005; Apak et al. 2016). HAT-based methods reflecting increased electron donating capacity with
measure the classical ability of an antioxidant to quench free deprotonation. A correlation between redox potential and
radicals by hydrogen donation. HAT reactions are solvent SET methods has been suggested (Ou et al. 2005), but
and pH independent and are usually quite rapid, typically not consistently demonstrated. SET reactions are usually
completed in seconds to minutes. The presence of reducing slow and can require long times to reach completion, so
agents, including metals, is a complication in HAT assays antioxidant capacity calculations are based on percent
and can lead to erroneously high apparent reactivity (Prior decrease in product rather than kinetics. When ­AH+ has a
et al. 2005). In the HAT reaction-based methods, the anti- sufficient lifetime, secondary reactions become a signifi-
oxidant is able to quench free radicals by hydrogen donation: cant interference in assays and can even lead to toxicity
AH + X ⋅ → A ⋅ + XH. or mutagenicity in vivo (Sartor et al. 1999). SET methods
are very sensitive to ascorbic acid and uric acid, which are
The relative reactivity of these methods is determined important in maintaining plasma redox tone, and reducing
by the bond dissociation energy of the H-donating group polyphenols are also detected. Importantly, trace compo-
of the antioxidant, which is characteristic for compounds nents and contaminants such as metals interfere with SET
with an interval of bond dissociation energy (≈ − 10 kcal/ methods and can account for high variability and poor
mol) and ionization potential (< − 36 kcal/mol). These reproducibility and consistency of results (Ou et al. 2005).
reactions are pH and solvent independent and are very fast Relative reactivity of the SET method is based on depro-
and usually completed in seconds to minutes. The presence tonation and in ionization potential of the reactive functional
of reducing agents like metal ions in such methods is not group. Therefore, SET methods are pH dependent. Gener-
recommended because it can originate an apparently high ally, ionization potential decreases with increasing pH val-
reactivity (Prior et al. 2005). Methods based on the HAT ues, which reflects a higher electron-donating capacity with
reaction include the following (Huang et al. 2005): deprotonation. The antioxidant mechanism is predominantly
of the electron transfer type when the ionization potential
• Oxygen radical absorbance capacity (ORAC). values are superior to − 45 kcal/mol. The reactions based
• Total radical trapping antioxidant parameter (TRAP). on the electron transfer are usually slow and calculations are
• Inhibition of induced LDL oxidation. based on product percentage decrease more than in kinetic
• Total radical scavenging capacity assay (TOSCA). terms (Prior et al. 2005). The SET-based methods include
• β-Carotene bleaching assays. the following assays:
• Chemiluminescent assay.
• Total phenolics assay by Folin–Ciocalteu reagent assay.
SET-based methods measure the ability of a poten- • Trolox equivalence antioxidant capacity assay (TEAC).
tial antioxidant. These methods transfer one electron to • Ferric ion reducing antioxidant power assay (FRAP).
reduce any compound, including metal ions, carbonyls • Total antioxidant potential assay, using a ­Cu2+-complex
and radicals (Wright et al. 2001). SET and HAT mecha- as an oxidant.
nisms almost always occur together in all samples, with • 2,2-Diphenyl-1-picrylhydrazyl radical scavenging assay
the balance determined by antioxidant structure and pH. (DPPH·).
SET-based methods detect the ability of an antioxidant to

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• 2,2-Azinobis 3-ethylbenzthiazoline-6-sulfonic acid radi- (Yanishlieva and Marinova 2001). Lipid peroxidation in bio-
cal scavenging assay ­(ABTS·+). logical systems has long been thought to be a toxicological
• N,N-Dimethyl-p-phenylenediamine radical scavenging phenomenon that can lead to various pathological conse-
assay ­(DMPD·+). quences (Hochstein and Atallah 1988). The resulting lipid
• Cupric ions ­(Cu2+) reducing antioxidant power assay hydroperoxides can affect membrane fluidity and the func-
(CUPRAC). tion of membrane proteins. In addition, lipid hydroperox-
ides can undergo iron-mediated, one-electron reduction and
It was reported that ABTS methods used both HAT and oxygenation to form epoxyallylic ROO· that triggers a chain
SET mechanisms (Prior et al. 2005). There are also other reaction of free radical-mediated lipid peroxidation. The end
assays that measure the sample’s scavenging ability for oxi- products of lipid peroxidation are reactive aldehydes, such as
dants, which interact and damage the major macromolecules 4-hydroxyl nonenal and malondialdehyde, many of which are
either in biological systems or in foodstuffs (Huang et al. highly toxic to cells (Yu and Yang 1996). In addition, reactive
2005; MacDonald-Wicks et al. 2006; Miguel, 2010): aldehydes generated by lipid peroxidation can attack other
cellular targets, such as proteins and DNA; thereby propagate
• Superoxide anion radical ­(O2·−) scavenging assays. the initial damage in cellular membranes to other macromol-
• Hydroxyl radical (HO·) scavenging assays. ecules. Because lipid hydroperoxides formed in membranes
• Singlet oxygen (1O2) quenching assays. are important components of ROS generation in vivo. Their
• Peroxynitrite ­(ONOO−) scavenging assays. detoxification appears to be critical for the survival of an
• Hydrogen peroxide ­(H2O2) scavenging assays. organism in oxidative stress (Dargel 1992). Therefore, anti-
oxidants play a vital role in the inhibition of lipid peroxidation
Total antioxidant capacities or in protection against cellular damage by free radicals. Vari-
ous antioxidant assay methods have been developed for food
Thiocyanate methods and biological samples. Hydroperoxides formed from polyun-
saturated fatty acids such as linoleic acid may undergo further
Total antioxidant capacity is widely used as a parameter oxidation reactions to form dihydroperoxides and molecules,
for food and medicinal bioactive components. This assay which have oxygen-containing rings including hydroperoxy
is defined as the ability of a compound to inhibit oxida- epidioxides and bicycloendoperoxides. The otooxidation
tive degradation like lipid peroxidation (Roginsky and Lissi mechanism of a polyunsaturated fatty acid is shown in Fig. 18.
2005). Lipid peroxidation can be defined as the oxidative Lipid peroxidation consists of a series of free radical-
deterioration of lipids containing any number of carbon–car- mediated chain reaction processes, and is associated with
bon double bonds. Generally, for determination of possible several types of biological damages. The thiocyanate method
effect of a bioactive compound, its affinity to restrict the measures the amount of peroxide produced during the initial
peroxidation of a linoleic acid emulsion was tested. Linoleic stages of oxidation, which is the primary product of lipid
acid oxidation generates linoleic acid hydroperoxides, which oxidation. In this assay, indirect measurement was per-
decomposes to secondary oxidation products. The thiocy- formed for the amount of hydroperoxides produced from
anate method was used to measure the peroxide quantity dur- linoleic acid emulsion by auto-oxidation during the experi-
ing the initial level of lipid peroxidation. Peroxides formed ment period. Then ferrous chloride and thiocyanate react
during the linoleic acid oxidation, which react with F ­ e2+ to with each other to produce ferrous thiocyanate by means of
3+
form ­Fe . The latter ions form a complex with thiocyanate hydroperoxides (Inatani et al. 1983; Gulcin 2012). Lipid per-
­(SCN−) and this complex has a maximum absorbance at oxidation occurs during harvesting, storage and processing
500 nm. In the presence of antioxidants, oxidation of linoleic of foods, and causes chemical spoilage, resulting in rancidity
acid will be slow. Hence, the color development, because of and deterioration of the nutritional value, flavor, color, safety
the formation of thiocyanate, will be slow (Gülçin 2002). and texture of the food and pharmaceutical products. Also,
Lipid peroxidation is initiated by an attack towards a it is responsible for quality deterioration of dairy products,
fatty acid’s side chain by a radical to abstract a hydrogen meat, vegetable, and fruits crops. Furthermore, it oxidizes
atom from a methylene carbon. The more the double bonds the membrane lipids, cellular proteins and enzymes, DNA
present in the fatty acid the easier it is to remove hydrogen and disrupts the cells. Food manufacturers use antioxidants
atoms and consequently form a radical, making monounsatu- for stabilization of food lipids and prevent quality deterio-
rated and saturated fatty acids more resistant to radicals than ration of the food products. They use the antioxidants as
polyunsaturated fatty acids (Carocho and Ferreira 2013). the most effective method for controlling of lipid oxidation
Lipid peroxidation has long been classified as the major (Shahidi and Zhong 2015).
deterioration process affecting both the sensory quality
and nutritional of foods, especially lipid-based products

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antioxidant capacity can be expressed as α-tocopherol or

trolox equivalents.

Polyunsaturated fatty acid

β‑Carotene bleaching assay
H
β-Carotene bleaching assay is one of the most common,
practiced and oldest techniques for the determination of
Lipid radical
lipid peroxidation in oil-in-water emulsion. This assay is
one of the rapid and simple methods to screen antioxidant
effects, which is mainly based on the principle that linoleic
H
Conjugated diene
acid, which is an unsaturated fatty acid that gets oxidized by
O2 ROS produced by oxygenated water. In this assay, β-carotene
dissolved in chloroform is added to linoleic acid emulsion.
H Then chloroform is evaporated at 40 °C, the emulsion of
O O
distilled water saturated with oxygen is slowly added to the
Lipid peroxy radical
residue and the solution is vigorously agitated to form a
H stable emulsion. Then an aliquot of this mixture is added
into the test tubes containing sample prepared in methanol at
final concentrations. The oxidative level of emulsion, which
includes β-carotene, linoleic acid, and water, is recorded
HOO H
O O spectrophotometrically in the absence and presence of the
Hydroperoxide
Endoperoxide antioxidant samples. The products formed will initiate the
β-carotene oxidation, which will lead to discoloration. As
soon as the emulsified solution is added to the tubes, the
H H
end products, formed followed by β-carotene oxidation, were
O O spectrophotometrically monitored at 470 nm. The tubes are
Malondialdehyde incubated for 2 h at 50 °C. Thus, radicals generated by the
spontaneous oxidation of linoleic acid are promoted by ther-
mal induction at 50 °C. Radicals were generated in autoxi-
Fig. 18  Reaction of 12-hydroperoxide from α-linolenic acid to form dation of linoleic acid as a lipid model system and cause
9-hydroperoxy endoperoxide β-carotene bleaching, which is retarded by antioxidants. The
reaction is performed in an aqueous emulsion of β-carotene
Phosphomolybdenum method and linoleic acid using Tween 40 surfactant. The addition
of an antioxidant compound retards β-carotene bleaching.
Another total antioxidant capacity assay is phosphomolyb- However, the incorrect quantification, low reproducibility,
denum method. This method was another developed assay complexity of the reagent preparation, and certain interfer-
for the quantitative determination of antioxidant capacity, ence parameters including pH, temperature, and solvents are
through the formation of phosphomolybdenum complex. the main limitations of this antioxidant assay (Alam et al.
­ o6+ to M
The assay is based on the reduction of M ­ o5+ by the 2013; Apak et al. 2016). The mechanism involves the follow-
sample analyte and subsequent formation of a green phos- ing steps. The ROO· is formed by the unimolecular thermal
phate ­Mo5+ complex at acidic pH (Alam et al. 2013). In this degradation of an initiator in the presence of oxygen in air.
method, the sample, which contained antioxidant solution, The ROO· formed can either bleach the abstract a proton
is combined with phosphomolybdenum reagent (0.6 M sul- from radical scavengers. The radical derived from radical
furic acid, 28 mM sodium phosphate and 4 mM ammonium scavengers can also bleach β-carotene. The bleaching of
molybdate). The tube is capped and incubated in a boiling β-carotene can either be abstraction of hydrogen atom or
water bath at 95 °C for 90 min. After cooling the sample addition of radical to polyene system. The hydroxyl and azyl
to room temperature, the absorbance of the aqueous solu- radicals cannot be assayed because of their high reactivity
tion is measured at 695 nm against blank in UV spectropho- toward organic compounds. Superoxide and alkyl radicals
tometer. A typical blank solution contained reagent solu- are not found suitable due to least reactivity (Shivakumar
tion and the appropriate volume of the same solvent used and Kumar 2018).
for the sample and it is incubated under same conditions as
rest of the sample. For samples of unknown composition,

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Oxygen radical absorbance capacity (ORAC assay) • β-phycoerythrin has large variability in reactivity to
ROO·, leading to inconsistency in assay results,
Oxygen radical absorbance capacity (ORAC) is an exciting • β-phycoerythrin becomes photobleached after exposure
and revolutionary new test tube analysis that can be utilized to excitation light interaction with polyphenols by non-
to test antioxidant power of foods and other chemicals. This specific protein binding,
assay measures the radical chain breaking ability of antioxi- • Particularly proanthocyanidins bind to β-phycoerythrin
dants by monitoring the inhibition of ROO·-induced oxida- via nonspecific protein binding.
tion. This method uses an area-under-curve technique and
thus combines both inhibition time and inhibition degree of Both of these latter factors cause false low ORAC values.
free radical action by an antioxidant into a single quantity, To overcome these limitations, the synthetic, non-protein
while other similar methods use either the inhibition time at fluorescein has been used as the fluorescent probe, instead
a fixed inhibition degree or the inhibition degree at a fixed of the original β-phycoerythrin. The oxidized fluorescein
time as the basis for quantifying the results (Miller et al. products induced by ROO· have been identified by LC–MS,
1993; Whitehead et al. 1992). and the reaction mechanism has been verified as a clas-
ORAC measures antioxidant inhibition of ROO·-induced sic HAT mechanism (Ou et al. 2001). Probe reaction with
oxidations and thus reflects classical radical chain breaking ROO· is followed by loss of fluorescence over time. Anti-
antioxidant activity by HAT (Ou et al. 2001). In the basic oxidant analyses followed extension of the lag phase only,
assay, the ROO· reacts with a fluorescent probe to form a but antioxidant effects often extend well beyond early stages
nonfluorescent product, which can be quantitated easily of oxidation (Ou et al. 2005). Fluorescent markers, although
by fluorescence. Antioxidant capacity is determined by a sensitive, require detection by fluorometers, which may not
decreased rate and amount of product formed over time: be routinely available in analytical laboratories although
this instrument is used routinely in many cell culture lab-
O2
R − N = N = R → N2 + 2ROO∙ , oratories. The long analysis time has also been a major
criticism, but this limitation has been partially overcome
ROO∙ + Probe(Fluorescent) → ROOH + Probe(Non−fluorescent) , by development of high-throughput assays (Huang et al.
2002a).
The ORAC assay supplies a controllable source of ROO·
AH + ROO ⋅ → ROOH + A⋅, that model reactions of antioxidants with lipids in both food
and physiological systems. It can be adapted to detect both
A ⋅ + ROO ⋅ → ROO − A. hydrophilic and hydrophobic antioxidants by altering the
The principle of this assay is based on the intensity of radical source and solvent (Ou et al. 2002a, b). The applica-
fluorescent molecule such as β-phycoerythrin or fluorescein tion to both lipophilic and hydrophilic chain-breaking anti-
decrease of the target along time under reproducible and oxidants was carried out using a mixture of acetone or water
constant flux of ROO·, generated from the thermal decom- containing 7% of randomly methylated β-cyclodextrin as a
position of 2,2ʹ-azobis(2-amidino-propane) dihydrochloride water solubility enhancer (Huang et al. 2002b). Lipophilic
(AAPH) in aqueous buffer. This assay is based on generation compounds were also quantified by ORAC assay using either
of free radical using AAPH and measurement of decrease organic media or liposomes (Naguib 1998). To improve the
in fluorescence in the presence of free radical scavengers this assay, Huang et al. developed a high-throughput assay
(Alam et al. 2013). So far, AAPH is the sole free-radical using a multichannel liquid handling system coupled with a
generator used for ORAC assay determination. A wide microplate fluorescence reader in 96-well format (2002b).
variety of foods and food constituents has been tested using Small temperature differences in the external wells of the
this methodology. In the presence of a sample that contains microplate can decrease the reproducibility of the assay. This
chain-breaking antioxidants, the decay of fluorescence is is not unique to the ORAC assay, but will be true for any
inhibited. Initially, the protein isolated from Porphyridium assay that is highly temperature sensitive that uses micro-
cruentum, β-phycoerythrin was used as the fluorescent plates and microplate readers in the assay (Ou et al. 2001).
probe, which react with ROO· to form a non-fluorescent Also, one benefit of using the ORAC method to evaluate
product (Cao et al. 1993). The fluorescent probes that are antioxidant capacity of food substances is that it takes into
currently preferred fluorescein (Ou et al. 2001) or dichloro- account samples with and without lag phases of their antiox-
fluorescein are more stable and less reactive. However, the idant capacities. This is especially beneficial when measur-
use of β-phycoerythrin in antioxidant assays has shortcom- ing foods and supplements that contain complex ingredients
ings for some reasons: with various slow- and fast-acting antioxidants, as well as
ingredients with combined effects that cannot be pre-calcu-
lated. Several modified ORAC methods have been proposed.

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Most of them employ the same principle; however, electron dichlorofluorescein-diacetate as the fluorescent oxidisable
paramagnetic resonance-based ORAC (ORAC-EPR) method substrate (1997). In both these studies, they evaluated the
directly measures the decrease of AAPH radical level by contribution and synergistic effects of main antioxidant
the scavenging action of the antioxidant substance (Kohri compounds, and the effect of plasma storage in the TRAP
et al. 2009). values. The oxidation of dichlorofluorescein-diacetate by
As a conclusion, this assay measures the oxidative deg- ROO· yields the formation of highly fluorescent dichloro-
radation of the fluorescent molecule such as β-cyclodextrin fluorescein product. In this case, the presence of antioxidant
or fluorescein after being mixed with free radical generators compounds competitively inhibits the increase of fluores-
such as azo-initiator compounds. Azo-initiators are consid- cence signal (Gulcin 2012).
ered to produce the ROO· by heating, which damages the The TRAP assay involves the initiation of lipid peroxida-
fluorescent molecule, resulting in the loss of fluorescence. tion by generating water-soluble ROO· and is sensitive to
The fluorescent intensity decreases as the oxidative degen- all known chain-breaking antioxidants, but it is relatively
eration proceeds, and this intensity is typically recorded for complex and time-consuming to perform, requiring a high
half hour after the addition of the azo-initiator as free radical degree of expertise and experience (Prior et al. 2005). Also,
generator. Antioxidants are considered to protect the fluores- this method permits the quantification of the absorbance
cent molecule from the oxidative degeneration. The degree capacity of antioxidants specifically toward three potent oxi-
of protection is quantified using a fluorometer. Fluorescein dants, which is hydroxyl radicals, peroxyl radicals, and per-
is currently used most as a fluorescent probe. Equipment oxynitrite (Regoli and Winston 1999). An earlier detection
that can automatically measure and calculate the capacity is method used the principle that peroxyl radicals produced
commercially available. from AAPH oxidize luminol, which led to the formation of
luminol radicals that emitted light. The emitted light used to
Total radical‑trapping antioxidant parameter (TRAP be detected by a luminometer. Under normal circumstances,
assay) this reaction produces low-intensity light emission that may
decay rapidly. The characteristics of the reaction can be
TRAP assay is based on the protection provided by anti- altered substantially by the addition of the enhancer para-
oxidants on the fluorescence decay of R-phycoerythrin dur- iodophenol, giving a more intense, prolonged, and stable
ing a controlled peroxidation reaction. The fluorescence of light emission. The light emission is sensitive to interfer-
R-Phycoerythrin is quenched by AAPH as a radical gen- ence by antioxidants. The length of the induction period
erator. This quenching reaction is measured in the pres- is compared to that of an internal standard such as trolox
ence of antioxidants. The antioxidative potential is evalu- and then quantitatively related to the antioxidant capacity
ated by measuring the decay in decoloration. TRAP values food constituents (Somogyi et al. 2007). One of the major
are calculated from the length of the lag-phase due to the problems with the original TRAP assay lies in the utiliza-
sample compared with standard. This method monitors the tion of the oxygen electrode as detector, since it may not
ability of antioxidant compounds to interfere with the reac- maintain its stability over the period of time required (Rice-
tion between ROO· generated by AAPH and a target probe Evans and Miller 1994). Then this assay was improved using
(Prior et al. 2005). Different variations of the method have β-phycoerythrin as the fluorescent probe. The ability of the
used oxygen uptake (Wayner et al. 1985), fluorescence of plasma to protect β-phycoerythrin from ROO· oxidation
R-phycoerythrin (Ghiselli et al. 1995), or absorbance of was fluorimetrically monitored (DeLange and Glazer 1989).
ABTS as the reaction probe (Bartosz et al. 1998). Their test Disregarding the different variations discussed above, the
is based on the measure of oxygen consumption during a quantification is based on the lag phase duration, in which
controlled lipid peroxidation reaction induced by thermal oxidation is inhibited by the antioxidants, compared to the
decomposition of an azo-compound. It was reported that lag phase of trolox. The antioxidant capacity expressed as
this method was the most widely used method for measuring trolox equivalents (TE) is calculated as
total antioxidant capacity of plasma or serum during the last
TE = (ATrolox ∕BTrolox ) x AAO ,
decade (Leinonen et al. 1998). The TRAP assay uses ROO·
generated from AAPH and peroxidizable materials. After where ­ATrolox is the trolox concentration, whilst ­BTrolox is
adding AAPH to the plasma, the oxidation of the oxidisable the lag time of the kinetic curve of target oxidation in the
materials is monitored by measuring the oxygen consumed presence of trolox. ­AAO is the lag time of the kinetic curve
during the reaction. During an induction period, this oxida- of target oxidation in the presence of antioxidant molecules
tion is inhibited by the antioxidants in the plasma (Prior and such as food constituents. Also, it was reported that the
Cao 1999). Ghiselli and co-workers proposed some modi- main shortcoming of TRAP assay was the use of the lag
fications to circumvent interferences from plasma proteins, phase for quantifying antioxidant capacity since not every
lipids and metal ions (1995). Valkonen and Kuusi applied

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Fig. 19  Chemiluminescent mechanism lucigenin in the assay of superoxide (a), and luminol (b)

antioxidant possesses an obvious lag phase and also the anti- including lucigenin (Fig. 19a), luminol (Fig. 19b), oxalates,
oxidant capacity profile after the lag phase is totally ignored Cypridina luciferin or pyrogallol. Among them, luminol is
(Somogyi et al. 2007). Also, Mulholland and Strain (1991) the most commonly used aqueous chemiluminescent reagent.
reported that serum TRAP experimental values were sig- Luminol reacts with the potent oxidizing agent H ­ 2O2 to yield
nificantly lower in patients with acute myocardial infarc- 3-aminophthalate in an excited electronic state, which is a
tion when compared to the sex- and age-matched controls. light-producing emitter. Antioxidant molecules can quench
Additionally, the plasma TRAP experimental values were ­H2O2 by hydrogen atom donation and inhibit ­H2O2-induced
also reported to decline significantly by about 40% during chemiluminescence. However, chemiluminescence assay is
chemotherapy in patients with various hematologic malig- based on the competition between antioxidants and luminol
nancies (Durken et al. 1995). for ­H2O2 and presence of antioxidants results in a decrease
in light emission intensity. Sometimes, a catalyst is added
to magnify the chemiluminescence signal, by converting
Chemiluminescence assay the relatively weak oxidant ­H2O2 into stronger ­O2·− and
­OH· (Shahidi and Zhong 2015). In alkaline DMSO, the
Chemiluminescence is a phenomenon observed when a luminol can be univalently oxidized by different oxidants,
chemical reaction produces an electronically excited spe- in the presence of O ­ 2, and the luminol radical generated
cies that either direct chemiluminescence (luminesces) or can add O ­ 2·−, yielding an unstable endoperoxide. Chemi-
transfers the energy to a fluorophore that generates indirect luminescence assay takes place by the transfer of energy to
chemiluminescence (emission). The main chemilumines- chemicals with compatible energy background, which then
cence method for antioxidant activity is direct chemilumi- becomes an emitter of the light, either by direct or sensitized
nescence based and generally includes a chemiluminescent procedure. This assay can be accepted in the quantitative
species, an oxidant ­(H2O2) in the absence or presence of a feature of the concentration in terms of emission intensity
metal or enzymatic catalyst, and an antioxidant molecule or measurement. Chemiluminescence assays are based on the
plant extract. There are a lot of chemiluminescent reagents

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Archives of Toxicology

attack of ROS with chemiluminescence active compounds. ­ e3+. An antioxidant is a reduct-

be able to efficiently reduce F
Formation of ROS was determined by chemiluminescence ant, but a reductant is not necessarily an antioxidant (Prior
assay using luminol, which is sensitive toward hypochlor- and Cao 1999). As reported in many studies, the activities
ous (HOCl), while lucigenin is oxidized by superoxide. It of natural antioxidants in influencing diseases are closely
is known that O ­ 2·− may oxidize both luminol and
­ H· or O related to their ability to reduce DNA damage, mutagen-
lucigenin (Shivakumar and Kumar 2018). The mechanism of esis, carcinogenesis, and inhibition of pathogenic bacterial
the chemiluminescence of luminol is shown in Fig. 19b. In growth (Roginsky and Lissi 2005).
this reaction, luminol is converted in basic solution into the
resonance-stabilized dianion (1), which is oxidized by ­H2O2 Ferric reducing antioxidant power (FRAP assay)
into the dicarboxylate ion (2) along with the loss of molecu-
lar nitrogen. The dicarboxylate ion is in an excited electronic FRAP assay is a typical ET-based method, which meas-
state and sheds its extra energy by emitting a photon of light ures the reduction of ferric ions ­(Fe3+)–ligand complex to
(hν), allowing the molecule to go to its ground state form (3) the intensely blue-colored ferrous ions ­(Fe2+) complex by
(White et al. 1964). Antioxidant properties of many biologi- antioxidants in acidic media. In another word, this method
cally active compounds were determined by the formation measure is based on antioxidants to reduce the ferric
high luminescent ­O2·− from potassium superoxide ­(KO2) 2,4,6-tripyridyl-s-triazine complex [­ Fe3+-(TPTZ)2]3+ to the
and 18-crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane) intensely blue colored ferrous complex ­[Fe2+-(TPTZ)2]2+
in dimethyl sulfoxide (DMSO). Antioxidants have received in acidic medium. Measuring the increasing absorption at
greater attention by permangnometric chemiluminescence 593 nm using a spectrophotometer monitors this reduction
assay (Shivakumar and Kumar 2018). and results are expressed as micromolar ­Fe2+ equivalents
Potassium permanganate (­ KMnO4) shows characteristic or relative to an antioxidant standard (Benzie and Strain
red chemiluminescence emission property at of 689 nm due 1999). FRAP, originally applied to assay of plasma later
to the relaxation of ­Mn2+ species from an excited species on extended to other biological fluids, foods, plant extracts,
(Adcock et al. 2007). A radical intermediate generates by the beverages, and food, tea, vegetables, juice (Gulcin 2012),
oxidation of antioxidant by M ­ n4+. This permanent radical spices (Gülçin et al. 2004a; Gülçin 2005b) and fruits (Gülçin
3+
species reacts with M ­ n previously present in the solution et al. 2005a, 2011b, c, 2012c; Bursal and Gülçin 2011). As
to produce ­Mn2+-emission source. The enhancement in the can seen in Fig. 20, the reaction measures reduction of fer-
chemiluminescence intensity is due to the prevention of dis- ric 2,4,6-tripyridyl-s-triazine (TPTZ) to a colored product
proportionate ­Mn3+ intermediate in which polyphosphates (Gulcin 2012).
form a cage-like structure around ­Mn2+, limiting nonradio- The FRAP assay is conducted at acidic pH 3.6 to maintain
active pathways. iron solubility. Reaction at low pH decreases the ionization
potential that drives electron transfer and increases the redox
Reducing antioxidant power potential, causing a shift in the dominant reaction mecha-
nism (Hagerman et al. 1998). It has been argued that the
Reducing antioxidant power is based on the principle of ability to reduce iron has little relationship to the radical
increase in the absorbance of the reaction mixtures. Increase quenching processes (H transfer) mediated by most antioxi-
in the absorbance indicates an increase in the antioxidant dants. However, oxidation or reduction of radicals to ions
activity. Reduction of a chemical is defined as a gain of elec- still stops radical chains, and reducing power reflects the
trons. Oxidation is defined as a loss of electrons. A reductant ability of compounds to modulate redox tone in plasma and
or a reducing agent is a substance that donates electrons and, tissues. The mechanism of FRAP assay is totally electron
thereby, causes another reactant to be reduced. An oxidant transfer rather than mixed SET and HAT, so in combina-
or an oxidizing agent is a substance that accepts electrons tion with other methods can be very useful in distinguish-
and causes another reactant to be oxidized. An oxidation ing dominant mechanisms with different antioxidants (Prior
is impossible without a reduction elsewhere in the system. et al. 2005). In addition, because reduced metals are active
When reduction and oxidation characterize a chemical reac- propagators of radical chains via hydroperoxide reduction to
tion, it is called a redox reaction. Redox reactions are the RO·, it would be interesting to evaluate whether high FRAP
main reaction of biological oxidation, the chain of chemi- values correlate with the tendency of polyphenols to become
cal reactions whereby we use oxygen from air to oxidize pro-oxidants under some conditions. This has been shown
chemicals from the breakdown of food to provide energy for some flavones and flavanones, which also have marked
for living system (Gulcin 2012). Reductant and oxidant are FRAP values (Cao et al. 1993).
chemical terms, whereas antioxidant and pro-oxidant have FRAP values are calculated by measuring the absorbance
meaning in the context of a biological system. In addition, an increase at 593 nm and relating it to a ferrous ion standard
antioxidant that can effectively reduce pro-oxidants may not solution or to an antioxidant standard solution. The change

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does not require specialized equipment. The FRAP assay can

be performed using automated, semiautomatic or manual
N N
methods (Prior et al. 2005). In addition, the FRAP assay
N N N N has several drawbacks. Since there are no free radicals intro-
duced into the system, there is no way of comparing the
N N
N N N N
antioxidant capacity towards different kinds of radicals. As
the FRAP assay measures the reducing capacity based upon
Fe3+ Fe2+ reduction of ferric ions (­ Fe3+). Antioxidants act by radical
N N quenching (H transfer). The FRAP assay cannot measure

N
N

N N the antioxidant capacity of certain antioxidants accurately,

N N N N which can react with ferrous ion (­Fe2+) and SH group-
containing antioxidants (Somogyi et al. 2007). One FRAP
N N unit is arbitrarily defined as the reduction of 1 M ferric ions
­(Fe3+) to ferrous ions (­ Fe2+) (MacDonald-Wicks et al. 2006).
Fe3+-TPTZ Fe2+-TPTZ In this method, the reducing ability of thiols and carotenoids
will not be determined (Pulido et al. 2000; Ou et al. 2002a,
b). Another point to take into consideration is the concomi-
Fig. 20  [Fe3+-(TPTZ)2]3+–[Fe3+-(TPTZ)2]2+ reduction reaction of tant production of ­Fe2+, which is a well-known prooxidant
FRAP assay and may result in the generation of additional radicals in
the reaction medium, as OH· from ­H2O2. Finally, com-
pounds that absorb at the wavelength of the determination
in absorbance is proportional to the combined FRAP value may interfere, causing overestimation of the FRAP value.
of the antioxidants in the sample (Ou et al. 2002a, b). The For instance, Benzie and Strain reported an unusually high
absorption (λ593) slowly increased for polyphenols such as FRAP value for bilirubin (1999) because it was oxidized
caffeic acid, tannic acid, ferulic acid, ascorbic acid, and to biliverdin, which absorbs considerably at 593 nm. Also,
quercetin, even after several hours of reaction time. Thus, a the low pH (3.6) necessary for this assay may lead to some
single-point absorption endpoint may not represent a com- protein precipitation, as casein in milk samples (Chen et al.
pleted reaction (Prior et al. 2005). Concerning its limita- 2003). Despite the low pH value applied when compared to
tions, any compound with redox potential lower than that of physiological conditions (pH 7.4), the FRAP may indirectly
the redox pair F­ e3+/Fe2+, can theoretically reduce F
­ e3+–Fe2+, reflect the antioxidant capacity, although the results should
contributing to the FRAP value and inducing falsely high be compared with that obtained by an HAT-based assay.
results. On the other hand, not all antioxidants reduce ­Fe3+ This lack of agreement may be due to the chemical variabil-
at a rate fast enough to allow its measurement within the ity found when such a large variety of samples is analyzed,
observation time. FRAP assay evolve from assay that rely especially considering the differences in rate of reactivity
on the hypothesis that the redox reaction proceeds so rapidly attained in the FRAP assay by different types of compounds.
that all reactions are complete within 4 and 6 min, respec- In summary, the FRAP assay is simple, inexpensive, and
tively, but, in fact, this is not always true. FRAP results can may offer a putative index of antioxidant capacity (Magal-
vary tremendously depending on the timescale of analysis. haes et al. 2008). In addition, Ou and co-workers (2002a,
Fast-reacting phenols that bind the iron or break down to b) analyzed a lot of freeze-dried vegetable samples using
compounds with lower or different reactivity are best ana- ORAC and FRAP methods. The obtained data show that the
lyzed with short reaction times. However, some polyphe- ORAC and FRAP values of vegetable are not only depend-
nols react more slowly and require longer reaction times for ent on species, but also highly dependent on geographical
detection. Pulido et al. observed that dietary polyphenols origin and harvest time. Both the antioxidant assays also
react more slowly and require longer reaction times for total give different antioxidant activity trends. They concluded
quantification and depending on the analysis time the order that the ORAC method is chemically more relevant to chain
of their reactivity is changed (2003). The polyphenols with breaking antioxidant activity, while the FRAP assay has
such behavior include caffeic acid, ferulic acid, quercetin, some drawbacks such as interference, reaction kinetics and
and tannic acid. Also, the FRAP assay does not measure quantitation methods.
thiol antioxidants, such as glutathione. FRAP actually meas-
ures only the reducing capability based upon the ferric ion,
which is not relevant to antioxidant activity mechanistically
and physiologically. However, in contrast to other tests, the
FRAP assay is simple, speedy, inexpensive and robust and

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Cupric ions ­(Cu2+) reducing antioxidant power (CUPRAC of antioxidants. Cuprac chromogenic redox reaction is car-
assay) ried out at a pH (7.0) close to the physiological pH, and
the method is capable of measuring thiol-type antioxidants
This assay was first developed and used by Apak’s group such as glutathione and non-protein thiols unlike the widely
(2006). It has also been applied to various matrices con- applied FRAP test, which is non-responsive to –SH group
taining both lipophilic and hydrophilic antioxidants. These antioxidants (Gülçin and Daştan 2007; Karaman et al. 2009).
assays are based on the reduction of ­Cu2+ to ­Cu+ by the The CUPRAC reagent is selective, because it has a lower
combined action of all antioxidants or reducing in aqueous- redox potential than those of Folin or ferric ion-based oxida-
ethanolic medium (pH 7.0) in the presence of neocuproine tive reagents, close to that of A­ BTS+/ABTS redox couple.
(2,9-dimethyl-1,10-phenanthroline), by polyphenols, yield- Also, simple sugars and citric acid, which are not classified
ing a ­Cu+ complexes with maximum absorption peak at as true antioxidants, are not oxidized with the CUPRAC rea-
450 nm (Fig. 21) (Gülçin 2008). This method can be used gent, whereas most phenolic antioxidants are easily oxidized
for the determination of the antioxidant capacity of food due to their favorable redox potentials. Some antioxidants
constituent by the C ­ u2+-neocuproine reagent as the chro- remaining inert toward F ­ e3+-based reagents like FRAP (such
mogenic oxidizing agent. The reduction of C ­ u2+ in the as thiols) and ­Fe –Fe2+ transformation methods are eas-
3+

presence of neocuproine by a reducing agent yields a ­Cu+ ily oxidized with the CUPRAC reagent (Apak et al. 2005).
complex with maximum absorption peak at 450 nm (Tutem The optimum pH for the CUPRAC assay is 7.0 and close to
et al. 1991). The CUPRAC reagent is much more stable and physiological pH (7.4), simulating antioxidant action under
readily accessible than the chromogenic radical reagents. It real conditions. This method is capable of measuring both
gives perfectly linear absorbance–concentration curves. In hydrophilic and lipophilic antioxidants (Apak et al. 2008).
this reducing assay, the liberated protons may be buffered
with the relatively concentrated acetate buffer. In this reac- Ferric ion (­ Fe3+) reducing antioxidant power ­(Fe3+–Fe2+
tion, the reactive Ar-OH groups of polyphenols and other transformation assay)
antioxidants are oxidized to the corresponding quinones
and ­Cu2+–neocuproine is reduced to the highly colored Reducing power of bioactive compounds or food compo-
­C u +–neocuproine complex, which showing maximum nents reflects the electron donating capacity and is associ-
absorption at 450 nm against a reagent blank by spectrom- ated with antioxidant activity. Bioactive compounds with
eter. Neocuproine is a heterocyclic organic compound and antioxidant effects can be reductants and inactivate oxidants
chelating agent. Cuprac assay is based on reduction of C ­ u2+ (Koksal and Gulcin 2008). The reducing capacity of a bio-
+
to ­Cu by antioxidants. This method is simultaneously cost- active compound can be measured by the direct reduction
effective, rapid, stable, selective, and suitable for a variety of Fe[(CN)6]3 to Fe[(CN)6]2. Addition of free F ­ e3+ to the
of antioxidants regardless of chemical type or hydrophobic- reduced product leads to the formation of the intense Perl’s
ity. Moreover, it was reported that the results obtained from Prussian blue complex, ­Fe4[Fe(CN−)6]3, which has a strong
in vitro cupric ion (­ Cu2+) reducing measurements might be absorbance at 700 nm (Bursal and Gülçin 2011).
more efficiently extended to the possible in vivo reactions

Fig. 21  Putative CUPRAC
mechanism by an antioxidant. 2+ +
The ammonium acetate buffer
neutralizes protons liberated in
the reaction. HA an antioxidant
molecule, A+ an oxidized anti-
HA A+
oxidant molecule N N N N
+ H+
Cu Cu
N N N N

CUPRAC Reagent Reduced CUPRAC Reagent

(Light blue) (Yellow-orange)

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Fe(CN)3− 4− . + sense that they can act as reducing agents, hydrogen atom
6 + AH → Fe(CN)6 + A + H ,
donators, and singlet oxygen scavengers. Certain polyphe-
+
nols are also effective as antioxidants capable of chelating
Fe(CN)4−
6 + Fe
3+
→ Fe4 [Fe(CN)4− .
6 ]3 + A + H . transition metal ions, which may otherwise induce Fenton-
type oxidation reactions in their free states (Rice-Evans et al.
An increase in the absorbance of the reaction mixture
1996; Karaman et al. 2009; Gulcin 2012).
would indicate an increase in the reducing capacity due to
The Folin–Ciocalteu assay is the well-known method
an increase in the formation of the complex. There are a
to determine the total phenolic contents. It was initially
number of assays designed to measure overall antioxidant
designed for the analysis of proteins, which takes advan-
activity, or reducing potential, as an indication of a host’s
tage of tyrosine as phenolic amino acid in proteins (Folin
total capacity to withstand free radical stress. The ferric ion
and Ciocalteu 1927). Then, it was later adopted to analyze
­(Fe3+) reducing antioxidant power assay takes advantage of
phenolic content in food and plant extracts by (Singleton
an SET in which a ferric salt is used as an oxidant (Gulcin
et  al. 1999). This method was frequently used for total
2009). In this assay, the yellow color of the test solution
phenolic compounds determination in plants (Gülçin et al.
changes to various shades of green and blue depending on
2003a; Oktay et al. 2003). Folin–Ciocalteu reducing (FCR)
the reducing power of antioxidant samples. The reducing
assay is the modification of Folin–Denis reducing (FDR)
capacity of a compound may serve as a significant indicator
capacity, which was applied to indirect total determina-
of its potential antioxidant activity (Gülçin et al. 2010c).
tion of tyrosine and tryptophan amino acids with higher
Antioxidant compounds reduce F ­ e3+–ferricyanide com-
2+ sensitivity and good reproducibility. The basic principle
plexes to the ferrous ­(Fe ) form. The Prussian’s blue-
of both methods relies on the reaction between FCR and
colored complex was formed by adding ­FeCl3 to the fer-
an oxidizing agent with either amino acids resulting in the
rous form ­(Fe2+). Therefore, the amount of reduction can be
formation of reduced molybdenum blue proportional to the
determined by measuring the formation of Perl’s Prussian
concentration of the protein. The basic difference between
blue at 700 nm. In this assay, the yellow color of the test
FCR and FDR lies in the molybdate concentration used in
solution changes to green or blue depending on the reduc-
the preparation. The amount of molybdate added is more in
ing power of the antioxidant. A higher absorbance indicates
FCR to avoid the white precipitate formed during the assay
higher ferric reducing power (Gülçin et al. 2002a). However,
involving FDR. The FCR assay is based on the reduction of
it was reported that the results might vary depending on the
the FCR by phenolic compounds under alkaline condition
analysis time as observed for the reaction between antioxi-
(Shahidi and Zhong 2015). In this assay, the FCR can be
dants and F­ e3+, which ranged from several minutes to several
prepared by dissolving 0.1 g of sodium tungstate with 25 g
hours (Pulido et al. 2000).
of sodium molybdate in 700 mL distilled water followed
by acidification with of concentrated HCl (50 mL) and of
Folin–Ciocalteu reducing capacity (FCR assay)
85% orthophosphoric acid (­ H3PO4, 50 mL). The acidified
solution is boiled for 10 h and 150 g of lithium sulphate
Total phenolic content (TPC) is actually not an antioxidant
­(Li2SO4) is added for intense yellow solution called FCR.
method. In this method, total phenolic content of plant and
However, the exact chemical nature of the FCR is not clearly
food materials are determined as gallic acid or another puta-
defined, but it is believed that it contain phosphomolybdic/
tive phenolic compound equivalent. Also, TPC are some-
phosphotungstic acid complexes that are reduced to yield
times expressed as caffeic acid, catechin, chlorogenic acid,
a blue-colored chromophore with maximum absorbance at
or ferulic acid equivalents. However, the high quantity of
765 nm (Shahidi and Zhong 2015).
phenolic content was associated with high antioxidant abil-
The FCR assay has for many years been used as a
ity. Hence, this assay is important parameter for the deter-
measure of total phenolics in natural products (Prior
mination of total antioxidant capacity. It is widely used for
et al. 2005). The total phenolic contents in foods were
evaluation of antioxidant extracts, including extracts from
estimated using the Folin–Ciocalteu method, which relied
herbs, spices and fruits, cereals and legumes, among others.
on the transfer of electrons from phenolic compounds to
This assay has several drawbacks including reaction time
the FCR in alkaline medium, and is a simple and widely
sensitive to pH and temperature (Shahidi and Zhong 2015).
used method (Singleton and Rossi 1965). FCR or Folin’s
Phenolics share the same general structure composed of an
phenol reagent or Folin–Denis reagent, also called the
aromatic hydroxyl nucleus and exist in an approximated
gallic acid equivalence method (GAE), is a mixture of
number of 8000 in nature (Ozturk Sarikaya et al. 2011). So
phosphomolybdate and phosphotungstate used for the
far, plant phenolics constitute one of the major groups of
colorimetric assay of phenolic and polyphenolic antioxi-
compounds acting as primary antioxidants or free radical
dants (Singleton et al. 1999). It works by measuring the
terminators. Plant polyphenols are multifunctional in the
amount of the substance being tested needed to inhibit the

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oxidation of the reagent. The exact chemical nature of the in which the metals have lower valence (Bray and Thorpe
FCR is not known, but it is accepted that it contains phos- 1954).
phomolybdic/phosphotungstic acid complexes (Singleton Generally, gallic acid is extensively used as the reference
and Rossi, 1965). The testing system is the mixture of standard compound and results are expressed as gallic acid
tungstate and molybdate in highly basic medium (5–10% equivalents (Gülçin et al. 2002b, 2003b, 2004b). In addition,
aqueous ­Na2CO3). Phenolics are energetically oxidized in in the literature, a number of papers have presented the rec-
basic medium resulting in the formation of O ­ 2·−, which ommended gallic acid reference standard with pyrocatechol
in turn, reacts with molybdate with formation of molyb- equivalents (Gulcin et al. 2002b), tannic acid equivalents,
denum oxide, ­MoO4+ having a very intensive absorbance catechin equivalents, chlorogenic acid equivalents, caffeic
near 750  nm. The original FCR method developed in acid equivalents, protocatechuic acid equivalents, vanillic
1927 originated from chemical reagents used for tyrosine acid equivalents and ferulic acid equivalents (Gulcin 2012).
analysis (Folin and Ciocalteu 1927) in which oxidation of It should be stressed that, the blue complexes formed are
phenols by a molybdotungstate reagent yields a colored independent of the structure of phenolic compounds, there-
product with λmax at 745–750 nm. The molybdenum center fore ruling out the possibility of coordination complexes
in the complexes is generally accepted as the reduction formed between the metal and the phenolic compounds
site, where the Mo ion by accepting an electron donated (Singleton et al. 1999; Gülçin et al. 2004c; Elmastas et al.
by the phenolic antioxidant (Gulcin 2012). 2006a). The FCR is non-specific to phenolic compounds.
)4− Many non-phenolic compounds such as aromatic amines,
sulfur dioxide, ascorbic acid, some ions including ­Cu+, and
(
Na2 WO4 ∕ Na2 MoO4 + Phenol → Phenol − MoW11 O40 ,
­Fe2+ can reduce FCR. Additional non-phenolic organic sub-
Mo5+ + e− → Mo4+ . stances that react with the FCR included adenine, adenosine,
(Yellow) (Blue)
alanine, aniline, aminobenzoic acid, ascorbic acid, benza-
The chemistry behind the FCR assay relies on the SET ldehyde, creatinine, cysteine, cytidine, cytosine, dimethy-
in alkaline medium from phenolic compounds and other aniline, diphenylamine, EDTA, fructose, guanine, guano-
reducing species to molybdenum, forming blue complexes sine, glycine, histamine, histidine, indole, methylamine,
that can be detected spectrophotometrically at 750–765 nm nitriloacetic acid, oleic acid, phenylthiourea, proteins,
(Singleton et al. 1999). This method is precise, sensitive, pyridoxine, sucrose, sulfanilic acid, thiourea, thymine, thy-
and simple. It can be useful in characterizing and stand- midine, trimethylamine, tryptophan, uracil, uric acid and
ardizing botanical samples provided that some of the limi- xanthine. Also, some inorganic substances such as hydra-
tations and variations mentioned previously are properly zine, hydroxyammonium chloride, iron ammonium sulfate,
controlled. However, the reaction is slow at acid pH. It iron sulfate, manganese sulfate, potassium nitrite, sodium
lacks specificity. The improved method outlined by Single- cyanide, sodium metabisulfite, sodium phosphate, sodium
ton and Rossi (1965) specified the conditions to minimize sulfite, and tin chloride may also react with the FCR to give
variability and eliminate erratic results. This method was elevated apparent phenolic concentrations (Prior et al. 2005).
improved with a molybdotungstophosphoric heteropoly- For that reason, it is not suitable for determination of “total
anion reagent ­(3H2O–P2O5–13WO3–5MoO3–10H2O) and phenolic content”, unless interfering species are considered
­(3H2O–P2O5–14WO3–4MoO3–10H2O) that reduced phe- or removed (Prior et al. 2005; Singleton et al. 1999). This
nols more specifically; the λmax for the product is 765 nm. reagent should not be confused with Folin’s reagent, which
They also imposed mandatory steps and conditions to is used to detect amines and sulfur-containing compounds.
obtain reliable and predictable data: proper volume ratio Therefore, the FCR assay was recently proposed for the
of alkali and FCR; optimal reaction time and temperature measurement of total reducing capacity of samples (Huang
for color development; monitoring of optical density at et al. 2005). Also, it was known that the excellent linear cor-
765 nm; and use of gallic acid as the reference standard relations were found between FCR assay and other ET-based
phenol. antioxidant assays such as TEAC and DPPH· (Roginsky and
FCR does not contain phenol. Rather, the reagent will Lissi 2005; Gülçin 2005). Also, the contribution from other
react with phenols and nonphenolic reducing substances to dietary antioxidant compounds with non-ET mechanism of
form chromogens that can be detected spectrophotometri- action may not be assessed by FCR assay. Despite that, the
cally. It can also be used as a spray reagent in chromato- relationship between the FCR assay and HAT based anti-
graphic procedures. The color development is due to the oxidant assays such as ORAC is usually good (Prior et al.
transfer of electrons at basic pH to reduce the phosphomo- 2005). These correlations confirm the value, usefulness of
lybdic–phosphotungstic acid complexes to form chromogens FCR reducing capacity for the assessment of antioxidant
capacity of food constituents or samples. In addition, the

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FCR assay is operationally simple, reproducible and con- ­ PPH· radicals, A

the formation of the D ­ BTS·+ and D
­ MPD·+
venient for assessment of dietary antioxidant capacity since cation (Gulcin 2012):
the reagent is commercially available. This procedure is
DPPH∙ + AH → DPPH2 + A∙ ,
rather standardized, and the absorption of the product at a
long-wavelength minimizes interferences from the sample
matrix. Additionally, it is performed in aqueous phase, thus ABTS∙+ + AH → ABTS+ + A∙ ,
it is not applicable for lipophilic food components.
On the other hand, the most common usage of FCR is DMPD∙+ + AH → DMPD+ + A∙ .
in the Lowry method for determining protein concentration
(Lowry et al. 1951). In this method, protein is pre-treated These methods are rapid; a sample analysis takes 15 min
with ­Cu2+ in a modified biuret reagent. Alkaline copper solu- in total and little manpower, no expensive reagents or
tion is stabilized with sodium potassium tartrate. Addition of sophisticated instrumentation is required. These chromo-
FCR generates chromogens that give increasing absorbance gens are easy to use, have a high sensitivity, and allow for
between 550 and 750 nm. rapid analysis of the antioxidant activity of a large number
of samples. These assays have been applied to determine the
Radical scavenging assays antioxidant activity of food components (Köksal et al. 2009).

The free radical chain reaction is widely accepted as a com- DPPH· scavenging assay
mon mechanism of lipid peroxidation. Radical scavengers
may directly react with and scavenge peroxide radicals to 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
terminate the peroxidation chain reactions, and improve the assay is one of the most frequently used methods and offers
quality and stability of food products (Soares et al. 1997). the first approach for evaluating antioxidant activity. It has
Free radical scavenging is one of the known mechanisms deep blue color and it is a long-lived nitrogen radical spe-
by which antioxidants inhibit lipid oxidation. This test is a cies, due to its inability to undergo dimerization (Gulcin
standard assay in antioxidant activity studies, and offers a et al. 2002a, b). It is characterized as a stable free radical
rapid technique for screening the radical scavenging activ- by virtue of the delocalization of the spare electron over the
ity of specific compounds. Assays based upon the use of molecule as a whole, so that the molecule does not dimerize,
DPPH·, ­ABTS·+, ­DMPD·+ and ­O2·− radicals are among the as would be the case with most other free radicals. Thus,
most popular spectrophotometric methods for determination the delocalization of electron gives rise to the deep violet
of the antioxidant capacity of foods, beverages and vegeta- color, characterized by an absorption band in organic solu-
ble extracts. Both chromogens and radical compounds can tion centered at about 517 nm. When the DPPH radical
directly react with antioxidants. Additionally, DPPH· and solution is mixed with an antioxidant molecule, which can
­ABTS·+ scavenging methods have been used to evaluate the donate a hydrogen atom, it gives rise to the reduced form
antioxidant activity of compounds due to the simple, rapid, with the loss of this violet color (Alam et al. 2013). The
sensitive and reproducible procedures (Gülçin et al. 2005c). DPPH radical scavenging method a simple, easy, economic,
Antioxidants are believed to intercept the free radical and rapid and efficient assay commonly employed for meas-
chain of oxidation and donate hydrogen from the phenolic uring antioxidant activity and the evaluation of the radical
hydroxyl groups, thereby form a stable end product that does scavenging activity of non-enzymatic antioxidants. DPPH·
not initiate or propagate further oxidation of lipid (Amaro- scavenging assay is the oldest indirect method for determin-
wicz et al. 2004). ing of antioxidant activity and was firstly used to determine
Radical scavenging activity is very important, due to the the antioxidant potential of phenolic compounds. It was
deleterious role of free radicals in foods and in biological firstly suggested in 1950s originally to discover H-donors in
systems. Diverse methods are currently used to assess the natural materials. Later the test was quantified to determine
antioxidant activity of plant phenolic compounds. Chemi- the antioxidant potential of both individual phenolics and
cal assays are based on the ability to scavenge synthetic free food as well as of biologically relevant samples (Roginsky
radicals, using a variety of radical-generating systems and and Lissi 2005). The DPPH radical is one of the few stable
methods for detection of the oxidation end-point. D ­ PPH·, organic nitrogen radicals, which bears a deep purple color.
·+ ·+ ·−
­ABTS , ­DMPD or ­O2 radical scavenging methods are It is commercially available and does not have to be gener-
common spectrophotometric procedures for determining ated before assay like A­ BTS·+. In DPPH assay, PPH assay is
antioxidant capacities of components. When an antioxidant based on the reduction of the purple DPPH· to 1,1-diphenyl-
is added to the radicals, there is a degree of decolorization 2-picryl hydrazine. This method is based on the reduction of
owing to the presence of the antioxidants, which reverses DPPH in alcoholic solution in the presence of a hydrogen-
donating antioxidant due to the formation of the non-radical

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Archives of Toxicology

form DPPH-H in the reaction. This assay is based on the The stable free DPPH radical is a useful reagent to inves-
measurement of the reducing ability of antioxidants toward tigate the scavenger properties of phenols, catechols, and
DPPH·. The ability can be evaluated by ESR or by measur- anilines. It is now widely accepted that the reaction between
ing the decrease of its absorbance. This assay is convenient phenols and DPPH proceeds through two different mech-
in their application and thus most popular; nevertheless, they anisms: the direct HAT and SPLET (Foti et al. 2004a, b;
are limited as they use non-physiological radical. The widely Musialik and Litwinienko 2004).
used decoloration assay was first reported by Blois (1958).
ArOH + DPPH∙ → ArOH∙ + DPPH2 (HAT),
DPPH is usually used as a reagent to evaluate free radical
scavenging activity of antioxidants (Elmasatas et al. 2006b).
It is a stable free radical showing a maximum absorbance ArOH ⇆ArO− + H+ (SPLET),
at 517 nm. When DPPH radicals encounter a proton-donor
substrate such as an antioxidant, the radicals would be scav- ArO− + DPPH∙ → ArO∙ + DPPH− ,
enged and the absorbance is reduced (Blois 1958; Gülçin
et al. 2009). This assay is simple and does not require special DPPH− + H+ → ArO∙ + DPPH2 .
sample treatment. Its sensitivity may be affected by a num-
ber of factors, such as the type and amount of solvent used, A freshly prepared DPPH solution exhibits a deep pur-
presence and concentration of hydrogen and metal ion and ple color with absorption maximum at 517 nm. This purple
freshness of DPPH reagent (Shahidi and Zhong 2015). This color generally disappears when an antioxidant is present
assay has one major limitation. Some compounds absorb in in the medium (Gülçin et al. 2004d, 2006a). Thus, antioxi-
the same wavelength range as DPPH. For example, antho- dant molecules can quench DPPH free radicals by provid-
cyanins have strong absorption in the similar wavelength ing hydrogen atoms or by electron donation, conceivably
range (500–550 nm) as DPPH, and thus may introduce inter- via a free-radical attack on the DPPH molecule and convert
ference with the results and their interpretation (Shahidi and them to colorless or bleached product. In this assay, it was
Zhong 2015). measured the DPPH initial absorbance and the absorbance
In this assay, as seen in Fig. 22, the purple chromogen once the potential antioxidant had been added. The reduc-
radical (DPPH·) is reduced by antioxidants (AH) to the cor- tion of absorbance is a measure of the free DPPH due to the
responding pale yellow hydrazine (DPPH-H) (Blois 1958; action of the antioxidant (Gülçin et al. 2007b; Samadi et al.
Elmastas et al. 2006c). The test is simple and rapid and needs 2011). The blank is the reaction mixture without the test
only a UV–Vis spectrophotometer to perform, which prob- compounds. Also, define half maximal scavenging concen-
ably explains its widespread use in antioxidant screening. tration ­(IC50) value was defined as the concentration of the
DPPH· radical is one of the few stable organic nitrogen sample that causes fifty percent of DPPH radicals neutraliza-
radicals, which has a deep purple color. It is commercially tion. These values were calculated from the plot of inhibition
available and unlike ABTS, it does not have to be generated percentages in function of concentrations (Cakmakcı et al.
before assay. The DPPH· assay is considered mainly based 2019; Oztaskin et al. 2019; Eruygur et al. 2019).
on an electron transfer reaction, and hydrogen-atom abstrac- DPPH scavenging capacity is generally evaluated in
tion. This assay is based on the measurement of the reducing organic media such as ethanol or methanol by monitoring
ability of antioxidants toward DPPH· (Prior et al. 2005). the absorbance decrease at 515–528 nm until the absorbance
remains constant (Brand-Williams et al. 1995; Gulcin et al.
2018; Maharramova et al. 2018) or by electron paramagnetic
NO2 NO2
resonance (EPR) (Calliste et al. 2001). EPR spectroscopy
can directly measures the DPPH· concentration at submi-
cromolecular levels. This resonance technic has advantages
over the classic spectrophotometric detection, not only for
O2N NO2 AH A O2 N NO2 highly colored samples, but also for samples appearing tur-
N NH
bid in the chosen solvent (Gardner et al. 1998; Shahidi and
N
Zhong 2015). The usage of methanol as organic media is
N
not preferred due to its toxic properties. Recently, Milardo-
vic et al. (2006) determined the radical scavenging capacity
based on the amperometric reduction of DPPH· at the glassy
DPPH DPPH-H carbon electrode. The resulting current on a glassy carbon
electrode polarized at fixed potential was proportional to
the residual concentration of DPPH· after reaction with the
Fig. 22  DPPH· scavenging mechanisms by an antioxidant (AH) radical scavenger. A biamperometric method using DPPH/

13
 Archives of Toxicology

DPPH· redox couple and two identical glassy carbon disc spectrophotometric measurements can be affected by com-
electrodes was also presented (Milardovic et al. 2006). The pounds, such as carotenoids, that absorb at the wavelength
reaction mechanism is based on an electron transfer reaction of determination as well as by the turbidity of the sample. In
whilst the hydrogen atom abstraction is a marginal reaction this way, the electrochemical detection proposed by Milar-
pathway, because it occurs slowly in strong hydrogen-bond dovic et al. (2006) may be a valid alternative to analyze
accepting solvents, such as ethanol and methanol (Foti et al. colored or turbid samples with low content of antioxidant
2004a, b). As occurs in other electron transfer based assays, compounds. The DPPH· assay is not suitable for measur-
the scavenging capacity against DPPH· radical is strongly ing the antioxidant capacity of plasma, because proteins
influenced by the solvent and the pH of reaction (Magal- are precipitated in the alcoholic reaction medium. Finally,
haes et al. 2008). It was studied the suitable conditions and the DPPH· scavenging reaction is time-consuming and it
limits for water as a component of a mixed water–ethanol may take 20 min up to 6 h (Brand-Williams et al. 1995).
solvent of DPPH· radical assay (Stasko et al. 2007). It was Despite the limitations above mentioned, the DPPH radical
concluded that the aqueous/ethanol (v/v:50/50) solutions are is stable and commercially available. Also, it does not have
a suitable choice for lipophilic and hydrophilic antioxidants. to be generated before assay like ­ABTS·+. Therefore, it is
Also, the reaction rate between DPPH· and the radical scav- considered an easy and useful spectrophotometric method
enger compounds may increase considerably with increasing with regard to screening/measuring the radical scavenging
water ratios. However, at water content over the aqueous/ capacity of pure compounds (Gülçin et al. 2007a, 2008b),
ethanol (v/v:60/40) solutions the radical scavenging activ- food constituents (Gülçin et al. 2006c, 2011c; Ak and Gülçin
ity decreased, since a part of the DPPH· coagulates. It is not 2008), plant extracts (Elmastas et al. 2006c; Buyukokuroglu
easily accessible to the reaction with radical scavenger. Gen- and Gulcin 2009; Serbetci Tohma and Gülçin 2010) and the
erally, the results are reported as the efficient concentration other samples such as synthesized compounds (Talaz et al.
­(IC50). ­IC50 value is the amount of antioxidant necessary to 2009; Balaydın et al. 2010).
decrease by 50% the initial DPPH· concentration (Brand- In a recent study, the interaction between DPPH· and
Williams et al. 1995). Percentage of inhibition and ­IC50 are usnic acid was documented (Sehitoglu et al. 2015; Cetin
used very frequently as parameters characterizing the radi- Cakmak and Gulcin 2019). The structure of the usnic acid
cal scavenging activity. Originally, this parameter has been leads to interference in the DPPH·. After the interaction of
derived from biochemistry to characterize the capability of usnic acid and radicals, DPPH· disappears after accepted
some substrates to inhibit enzyme activity. In some cases, an electron or hydrogen radical from usnic acid to become
this approach remains acceptable when we determine the ­DPPH2. The featured reaction between DPPH· and usnic
inhibition of non-chain processes, such as the oxidative deg- acid is shown in Fig. 8. DPPH· scavenging mechanism of
radation of DNA, reactions with stable free radicals (Rogin- usnic acid has not been reported, so far. However, the best
sky and Lissi 2005). The lower ­IC50 indicates the higher knowledge is that a phenolic group stabilizes radicals formed
radical scavenging efficiency. The main limitation of ­IC50 on phenolic carbon with their resonance structure. In usnic
determination is that the percentage of radical scavenged acid molecule, phenolic group has also two hydroxyl units.
is dependent of the initial concentration of DPPH· radical A withdrawing of hydrogen atoms from phenolic hydroxyl
(Magalhaes et al. 2008; Askin et al. 2018). For this reason, groups may occur easily. Usnic acid can be found in the
it is more accurate to use the absorbance variation rather triradical structures by removing three DPPH molecules
than the percentage of the radical consumed. For this pur- using resonance structures. Different resonance structures
pose, DPPH· concentration (­ 10–3 M) seems the most effec- for these triradical structures are shown in Fig. 8.
tive concentration and this concentration was frequently It was reported that food constituents have a powerful
used (Büyükokuroğlu et al. 2001; Gulcin et al. 2006a, b; DPPH· scavenging capacity. Resveratrol is the main constitu-
Büyükokuroğlu and Gülçin 2009). This absorbance value ent in the skin and leaves of grape. Also, it is well known that
is further interpolated in a dose–response curve of a stand- resveratrol is a naturally occurring phytoalexin belonging to
ard radical scavenging compound such as ascorbic acid or the stilbene family of phenolic compounds, present in a lot
Trolox. The results can be expressed as equivalent concen- of medical and edible plants. It has been reported to exhibit
tration. The steric accessibility of DPPH· radical is a major antioxidant, cardioprotective, antidiabetic, anticancer, and
determinant of the reaction. Because small molecules that antiaging properties. The antioxidant potency was recently
have better access to the radical site have relatively higher evaluated with different methods (Rodriguez-Bonilla et al.
antioxidant capacity (Huang et al. 2005). On the other hand, 2017). Recently, Gülçin demonstrated that Resveratrol had
many large antioxidant compounds that react quickly with a marked DPPH radical scavenging activity (2010). It was
ROO· may react slowly or may even be inert in this assay. well-known that the ability of the polyphenolic compounds
The inexistence of DPPH· or similar radicals in biological to act as antioxidants depends on the redox properties of
and food systems is also a shortcoming. In addition, the their phenolic hydroxyl groups and the potential for electron

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very difficult since curcumin’s phenolic hydrogen atoms are

HO HO
DPPH DPPH-H intramolecular H-bonded to the adjacent methoxy groups.
OH
Based on theoretical calculations shows that B is the most
O
HO HO stable one among the intermediates (A, B and C) (Gulcin
Resveratrol Resveratrol phenoxyl radical 2012).
While the calculated formation energy (ΔH) for B is
− 42.05 kcal/mol, this energy was calculated as 39.45 kcal/
Fig. 23  DPPH radical scavenging mechanism of resveratrol mol for A and 54.70 kcal/mol for C. DPPH radicals eas-
ily abstract an H-atom from free hydroxyl group which was
responsible for the “superb antioxidant” properties of cur-
delocalization across the chemical structure. The structure of cumin. As a consequence, the reaction of DPPH radicals
Resveratrol provides a chromophoric system, which leads to diminishes by curcumin in alcoholic media, and the phenolic
interference in DPPH·. DPPH radical capacity of Resvera- part of curcumin takes over as (electron donor) reaction site
trol molecule is summarized in Fig. 23. It is well known (Jovanovic et al. 1999). The electron donating ability of
that phenolic groups stabilize a radical formed on phenolic curcumin is assessed from the measurements of one-elec-
carbon with their resonance structure. tron-transfer to DPPH radicals. HAT reactions of curcumin
Resveratrol has two phenolic rings: monophenol and were also investigated using the tert-butoxyl [(CH3)3CO·]
diphenol. An abstraction of a hydrogen atom from monophe- radicals, with same results. Same mechanism observed in
nolic hydroxyl group may occur easily (Gulcin 2010). It was these radicals scavenging. In addition, the H-atom donation
reported that a different mechanism had been observed in from the β-diketone moiety to a lipid alkyl or a lipid peroxyl
curcumin. Curcumin is a yellow-orange hydrophobic com- radical was described as a potentially more important anti-
pound extracted from Curcuma longa and widely used by oxidant action of curcumin (Jovanovic et al. 1999; Ak and
oriental cultures. Curcumin displays good tolerability and Gülçin 2008).
safety profiles and discloses numerous biological properties On the other hand, Kawabata et al. (2002) reported the
including antimicrobial antioxidant and anti-inflammatory formation of dimers from Gallic and protocatechuic acids,
activities (Ak and Gülçin 2008; Arshad et al. 2017). As can after reaction with DPPH radical. Also, Brand-Williams
be seen in Fig. 24, in the keto form of curcumin, the hepta- et al. (1995) and Bondet et al. (1997) studying the possi-
dienone linkage between the two methoxyphenol rings con- ble mechanism of DPPH with BHT, eugenol and isoeuge-
tains a highly activated carbon atom. At the same manner, nol, proposed the intervention of a more complex reaction
curcumin can easily abstract a hydrogen atom from this car- mechanism, eventually involving dimeric species. Dehy-
bon atom. Hydrogen atom abstraction from phenolic ring is drodiisoeugenol occurred by oxidative coupling of eugenol

OH OH OH OH

OCH3 OCH3 OCH3 OCH3

DPPH DPPH-H
O O O O
CH CH CH
HO O O O

OCH3 OCH3 OCH3 OCH3

OH OH OH OH

Curcumin A B C

Fig. 24  Proposed reaction between DPPH radicals and curcumin

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(Bortolomeazzi et al. 2010; Gulcin 2011). To confirm obser- compounds provide a chromophoric system, which leads to
vations made on the role of the side chain, a group of three interference in DPPH·. It is well known that phenolic groups
monophenols, namely isoeugenol, dihydroeugenol, and stabilize a radical formed on phenolic carbon with their reso-
eugenol, differing in one double bond and/or its position nance structure (Gulcin 2011).
in the chain, was also known. Also, conjugation seemed to
enhance the antioxidant and radical scavenging activity of ABTS·+ scavenging assay
eugenol (Nenadis et al. 2003). It is accepted that the scav-
enging mechanism of reaction is abstracting a hydrogen In this assay, ABTS is oxidized by oxidants to its radical
atom from a phenol donor to give DPPH-H and a phenoxy cation, ­ABTS·+, which is intensely colored, and antioxidant
radical (Mastelic et al. 2008). It was reported that Eugenol capacity is measured as the ability of test compounds to
reduce two or more DPPH radicals, despite the availability decrease the color reacting directly with the ABTS radical.
of hydrogen from a hydroxyl group, and there are different ­ABTS·+ is applicable for both lipophilic and hydrophilic
suggested hypotheses to explain the antiradical efficiencies compounds. ­ABTS·+ radicals are more reactive than DPPH
of the different monophenolic compounds (Brand-Williams radicals, and unlike the reactions with DPPH radical, which
et al. 1995; Mastelic et al. 2008). In another study, co-anti- involve HAT, the reactions with A ­ BTS·+ radicals involve
oxidant behavior of some relevant phenols such as eugenol, both HAT and SET. Generation of the ABTS radical cation
isoeugenol was studied. Synergism, implying regeneration forms the basis of one of the spectrophotometric methods
of α-tocopherol by the co-antioxidant, was observed with that have been applied to measure the total antioxidant activ-
combination of α-tocopherol/eugenol with peroxyl radicals ity of pure substances, aqueous mixtures, and beverages
(Kadoma et al. 2006; Gülçin 2011). As hypothesized in (Gulcin 2009). A more appropriate format for the assay is a
Fig. 25, the dimers with two phenolic hydroxyl groups of decolorization technique in which the radical is generated
A, B and C were present at a very low amount compared to directly in a stable form prior to the reaction with putative
C. All these compounds can be originated from the C ­ 8–C8 antioxidants. ­ABTS·+ has absorption maxima in aqueous
and ­C5–C5 coupling processes (Bortolomeazzi et al. 2010). media (414, 734 and 815 nm) and in ethanolic media (414,
Eugenol has an aromatic ring. This phenolic group sta- 730 and 873). The original ­ABTS·+ scavenging assay devel-
bilized a radical formed on a carbon with conjugation in oped by Miller et al. (1993). This method was based on the
eugenol molecule. Eugenol scavenged radical on this aro- activation of metmyoglobin, acting as peroxidase, with ­H2O2
matic ring. Similarly, the structures of the other aromatic

OH O O O O
DPHPH DPPH-H
OCH3 OCH3 OCH3 OCH3 OCH3

2 2

Eugenol A B C D

Eugenyl intermediate radicals

OH OH
H3CO OCH3 H3CO OH

O
HO OCH3
OCH3
HO
OCH3

1 2 3
Dehydrodieugenol

Fig. 25  Possible DPPH· scavenging mechanism of eugenol and formation of dehydrodieugenol by coupling reaction

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to generate ferrylmyoglobin radical, which then reacted with and have shown that the reaction can be studied over a wide
ABTS to form the ­ABTS·+ radical cation. range of pH values. However, the reaction mechanism may
Originally, this assay used metmyoglobin and ­H2O2 to shift with pH; for example, electron transfer is facilitated
generate ferrylmyoglobin, which then reacted with ABTS to at acid pH (8.0). This variation has been adapted also to
form ­ABTS·+ (Miller et al. 1993). In terms of assay condi- measure selectively hydrophilic and lipophilic antioxidants
tions, different strategies have been implemented for ­ABTS·+ by running the assay in buffered media and organic solvents,
generation, applied reaction time, detection of wavelength, respectively (Alcolea et al. 2002) or by partitioning antioxi-
and the reference antioxidant chosen. ABTS is oxidized dants in mixtures between hexane and aqueous solvents (Wu
by oxidants to its A ­ BTS·+ radical cation. For this purpose, et al. 2004). However, water-soluble reactions appear to be
·+
­ABTS radical cation can be generated by chemical reac- favored (Pulido et al. 2003).
tion using M ­ nO2. The important reactions of M ­ nO2 are This spectrophotometric assay is technically simple,
associated with its redox, oxidation and reduction (Miller which accounts for its application for screening and routine
et al. 1996), AAPH (Van den Berg et al. 1999), or potas- determinations. Generally, ­ABTS·+ was generated by oxida-
sium persulfate ­(K2S2O8) (Miller et al. 1996), by enzymatic tion of ABTS with K ­ 2S2O8. This assay is based on the inhi-
reaction using metmyoglobin (Miller et al. 1993) or horse- bition of the absorbance of the radical cation ­ABTS·+, which
radish peroxidase (Cano et al. 1998), and by electrochemical has a characteristic long-wavelength absorption spectrum
generation (Alonso et al. 2002). As seen in Fig. 26, ­ABTS·+ showing absorption at 734 nm (Gulcin 2012).
radical cation is generally generated by chemical reaction It should also be noted that the reaction with ­ABTS·+
of ­K2S2O8 (Gülçin et al. 2010a, 2011c). The generation of was quite fast and almost in all cases was completed in
ABTS radical cation by persulfate system involves the scis- 0.25–0.5 min. Bleaching of a preformed solution of the
sion of the K­ 2S2O8 after the transfer of electron. Concerning blue-green radical cation A ­ BTS·+ has been extensively
the wavelength of detection, the determination at 734 nm used to evaluate the antioxidant capacity of complex mix-
is preferred because the interference from other absorb- tures and individual compounds. The reaction of preformed
ing components and from sample turbidity is minimized radical with free-radical scavengers can easily visualize by
(Arnao 2000). In terms of quantification, the absorbance following the decay of the sample absorbance at 734 nm
value, proportional to the remaining A ­ BTS·+ concentration, (Gülçin et al. 2009). The ABTS radical cation can be pre-
is measured after a fixed reaction time (Magalhaes et al. pared employing different oxidants. Results obtained using
2008). Generally, chemical generation requires a long time ­K2S2O8 as oxidant show that the presence of peroxodisul-
(up to 16 h for ­K2S2O8 generation) or high temperatures phate increases the rate of ­ABTS·+. ­ABTS·+ were generated
(60 °C for ABAP generation), whereas enzyme generation in the ABTS/K2S2O8 system.
is faster and the reaction conditions are milder. Cano et al.
(1998) utilized horseradish peroxidase to generate A ­ BTS·+ S2 O2−
8
+ ABTS → SO2−
4
+ SO∙−
4
+ ABTS∙+

where the scission of the K­ 2S2O8 could take place after the
electron transfer. In the presence of excess ABTS, the sul-
-O S phate radical will react according to
+

3 -O S
3

SO∙−
4
+ 2ABTS → SO2−
4
+ 2ABTS∙+

leading to the overall reaction represented by

C2H5
C2H5 S2 O2− + 3ABTS → 2SO2− + 3ABTS∙+ .
K2S2O8 8 4
N
N
N ABTS·+ radicals are more reactive than DPPH radicals
N
and, unlike the reactions with DPPH radicals, which involve
C2H5
S N
S N
C2H5 HAT; the reactions with ­ABTS·+ radicals generally involve
SET (Kaviarasan et al. 2007; Köksal et al. 2009).
Also, ABTS radical scavenging method can be evaluated
over a wide pH range, which is useful to study the effect of
-O S
3 -O
3S
pH on antioxidant mechanisms for food components. Fur-
thermore, the ABTS radical is soluble in water and organic
ABTS
ABT S
solvents, enabling the determination of antioxidant capacity
of both hydrophilic and lipophilic compounds. However, as
the results obtained for samples are related to an antioxidant
­ BTS·+
Fig. 26  Oxidation of ABTS with ­K2S2O8 and generation of A

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standard compound that shows different kinetic behavior,

the results provided by this assay are dependent of time of NH2 NH2 NH
analysis. Reaction times ranging from 1 to 30 min have been
adopted throughout the protocols described in the literature.
e /H e /H
Moreover, this assay has been criticized, as the ABTS radi-
cal is not representative of biomolecules and not even found
in any biological and food system. Thermodynamically,
a compound that has a redox potential lower than that of N N N
­ABTS·+ may react with the radical (Magalhaes et al. 2008).
DMPD DMPD DMPD
As a conclusion, ­ABTS·+ reacts rapidly with antioxidants
in food components, typically within 30 min. It can be used
over a wide pH range and can be used to study effects of Fig. 27  The formation of DMPD radicals (­ DMPD·+)
pH on antioxidant mechanisms. Also, ­ABTS·+ is soluble in
both aqueous and organic solvents and is not affected by
ionic strength. So it can be used in multiple media to deter- of antioxidant efficiency (Fogliano et al. 1999; Ak and Gul-
mine both hydrophilic and lipophilic antioxidant capacities cin 2008).
of extracts and body fluids (Awika et al. 2003). Preliminary experiments show that the choice of oxidant
solution and the ratio between the concentration of D ­ MPD·+
DMPD·+ scavenging assay and the concentration of the oxidative compound are crucial
for the effectiveness of the method. In fact, formation of radi-
DMPD radical cation is an unstable species that can be cal cation is very slow and results in a continuous increase of
generated in situ by oxidation with potassium persulfate the absorbance. The best results were obtained with F ­ eCl3,
­(K2S2O8) and ferric chloride ­(FeCl3). In the presence of fer- which gives a stable colored solution up to a final concentra-
ric ions ­(Fe3+), N,N-dimethyl-p-phenylenediamine dihydro- tion of 0.1 mM. Moreover, this method ensures low cost and
chloride (DMPD) is converted to the colored DMPD radical highly reproducible analysis (Gülçin 2008). DMPD assay
cation ­(DMPD·+). Antioxidant molecules present in test sam- is particularly suitable for hydrophilic antioxidants, but is
ples easily scavenge DMPD radicals, forming the principle less sensitive to hydrophobic bioactive compounds, while
of the ­DMPD·+ assay. Another assay that is very similar to the opposite case applies for the other two tests. In contrast
the use of the A­ BTS·+ is the DMPD, or the D ­ MPD·+ assay. to the ABTS procedure, the ­DMPD·+ method guarantees a
Fogliano et al. (1999) suggested a new version of the ABTS very stable end point. This is particularly important when
test where ­ABTS·+ was changed for the stable ­DMPD·+ radi- a large-scale screening is required. It was reported that the
cal cation derived from N,N-dimethylphenylenediamine. As main drawback of the ­DMPD·+ method is that its sensitivity
Fogliano et al. (1999) and Schleisier et al. (2002) reported and reproducibility dramatically decreased when hydropho-
that this method is simpler, more productive, and less expen- bic antioxidants such as α-tocopherol or BHT were used.
sive as compared with the traditional ABTS test. In the pres- It was reported that organic acids may cause interference
ence of an oxidant or an acidic pH the DMPD is converted (Sanchez-Mareno 2002). Hence, the both standard antioxi-
to a stable and colored DMPD radical cation (­ DMPD·+). dant compounds were not used in this antiradical assay.
Antioxidant molecules that are able to transfer a hydrogen
atom (or an electron) to ­DMPD·+ cause rapid decolorization Superoxide anion radical scavenging assay
of the solution and measured by an absorbance decrease at
505 nm with a stable end-point. However, no data of its stoi- Superoxide anions (­ O2·−) are weak oxidants; however, they
chiometry with antioxidant standard and radical stability are ultimately produces powerful and dangerous hydroxyl radi-
available. A further problem is that DMPD is only soluble cals ­(OH·) as well as singlet oxygen (1O2), both of which con-
in water and cannot be used with hydrophobic antioxidants tribute to oxidative stress (Gulcin et al. 2006a). These radi-
(Fogliano et al. 1999). As can seen in Fig. 27, antioxidant cals are biologically quite toxic and deployed by the immune
compounds, which are able to transfer a hydrogen atom to system to kill invading microorganisms. In phagocytes, it is
­DMPD·+, quench the color and produce a decoloration of produced in large quantities by the enzyme NADPH oxidase
the solution. This reaction is rapid, and the end point, which for use in oxygen-dependent killing mechanisms of invading
is stable, is taken as a measure of antioxidative efficiency. pathogens. ­O2·− is an oxygen-cantered radical with selective
Therefore, this assay reflects the ability of radical hydrogen- reactivity. It is produced as a result of the donation of one
donors to scavenge the single electron from ­DMPD·+. The electron to oxygen in vivo. This radical arises either from
reaction is stable and the endpoint is taken to be the measure several metabolic processes or following oxygen activation
by irradiation (Halliwell 2006). Superoxide anion radical is

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Archives of Toxicology

converted to ­H2O2 by enzymatic and nonenzymatic mecha- adapted to micro-plate format using cytochrome c instead
nisms. ­H2O2 is further converted to a more reactive HO· by of NBT and read at 550 nm (MacDonald-Wicks et al. 2006).
the Fenton reaction (Gardner and Fridovich 1992), which Also, the scavenging capacity towards ­O2·−, using xan-
requires reduced iron or copper (Halliwell and Gutteridge thine-xanthine oxidase generating system, has also been
1990). Although a relatively weak oxidant, superoxide measured by reaction with α-ketomethiolbutyric acid to
exhibits limited chemical reactivity, but can generate more produce ethylene, which is measured by gas chromatogra-
dangerous species, including 1O2 and HO·, which cause the phy (Lavelli et al. 1999). The scavenging capacity against
peroxidation of lipids (Halliwell and Chirico 1993). Super- this radical can also be measured by using ESR spectrom-
oxide anions are precursors to active free radicals that have etry (Calliste et al. 2001). Similarly to xanthine-xanthine
potential for reacting with biological macromolecules, and system, in another method (Fig. 29), ­O2·− is also generated
thereby, inducing tissue damage (Halliwell and Gutteridge using a non-enzymatic reaction of phenazine methosulphate
1984). Superoxide is easily formed by radiolysis of water (PMS) in the presence of nicotinamide adenine dinucleotide
in the presence of oxygen, which allows accurate reaction (NADH). In both generation systems, O ­ 2·− may reduce NBT
rate constants to be measured (Gülçin and Daştan 2007). into formazan, which is spectrophotometrically monitored
It has been implicated in several pathophysiological pro- at 560 nm (Aruoma et al. 1993).
cesses due to its transformation into more reactive species Another common O ­ 2·− production assay is riboflavin—
such as HO·. Also, superoxide has been observed to directly methionine—illuminate system. Superoxide anions derived
initiate lipid peroxidation (Wickens 2001). It has also been from dissolved oxygen by the riboflavin-methionine-illumi-
reported that antioxidant properties of some flavonoids are nate system will reduce NBT in this system. In this method,
effective mainly via scavenging of superoxide anion radi- superoxide anion reduces the yellow dye (­ NBT2+) to pro-
cal. Superoxide anion is the precursor of H ­ 2O2, HO·, and duce the blue formazan, which is measured spectrophoto-
1
O2, which induce oxidative damage in lipids, proteins and metrically at 560 nm. Antioxidants inhibit the blue NBT
DNA. ­O2·− is normally formed first, and their effects can be formation. The decrease in absorbance at 560 nm with anti-
magnified because they produce other kinds of free radicals oxidants indicates the consumption of superoxide anion in
and oxidizing agents (Pietta 2000). However, detection and the reaction mixture. Antioxidants inhibit the formation of
measurement of ­O2·− levels within cells is as difficult as it is blue NBT (Parejo et al. 2002). The two principal reactions
desirable, because of the instability of this ROS in aqueous are involved in this assay are (Liochev and Fridovich 1995):
solutions. The methodologies that are applied for determina-
2NBTH∙ → NBT + NBTH2 (a),
tion of ­O2·−, which generation in biological systems involve
the reaction of superoxide with an indicator that forms a sta-
ble product by oxidation, reduction, or binding of superoxide NBTH⋅ + O2 ↔ NBT + O⋅−
2
(b).
to the indicator. The efficiency, sensitivity and specificity
When riboflavin is photochemically activated, it reacts
of detection of O ­ 2·− vary greatly depending on the different
with NBT to generate NBTH∙ (Beauchamp and Fridovich
reaction pathways involved (Fridovich 1997). The bioana-
1971; Gulcin 2002), which leads to formazan according to
lytical methods for determination of ­O2·− scavenging activity
reaction (a). In the presence of oxygen, radical species are
make use of the xanthine-xanthine oxidase system at pH 7.4
controlled by quasi-equilibrium (b). Thus, superoxide anion
to generate superoxide anion radical. As can see in Fig. 28,
radicals appear indirectly when the assay is performed under
­O2·− may reduce nitroblue tetrazolium (NBT) into formazan,
aerobic conditions. In the presence of an antioxidant mol-
which is spectrophotometrically monitored at 560 nm in this
ecule that can donate an electron to NBT, the typical purple
system (Aruoma et al. 1993).
color of formazan decays, which can be followed spectro-
In normal tissue, xanthine oxidase is a dehydrogenase
photometrically at 560 nm (Gulcin et al. 2003c, 2005b).
enzyme that transfers electrons to nicotinamide adenine
Antioxidants are able to inhibit the formation of NBT and
dinucleotide (NAD). During times of stress, this enzyme
scavenge superoxide anion radicals. The decrease in absorb-
is converted to an oxidase enzyme and produces O ­ 2·− and
ance at 560 nm in the presence of antioxidants indicates
­H2O2. Thus, xanthine oxidase plus hypoxanthine (or xan-
the scavenging of superoxide anions in the reaction mixture
thine) at pH 7.4, in the test tube, can be used to generate the
(Bursal and Gülçin 2011).
­O2·−. Xanthine oxidase reduces oxygen while catalyzing the
Antioxidant compounds compete with NBT for O ­ 2·− and
oxidation of its substrates, thus producing O ­ 2·− and H
­ 2O 2.
·− decrease the rate of reaction. Another widely used probe for
Then, ­O2 reduces NBT to formazan (Bull et al. 1983) and
­O2·− is cytochrome c. The kinetic analysis of reduction of
the formation of formazan can be measured spectrophoto-
ferricytochrome c to ferrocytochrome c was monitored at
metrically at 560 nm (Sanchez-Mareno 2002). To improve
550 nm (Aruoma et al. 1993; Quick et al. 2000; Gulcin et al.
the throughput and ease of this assay, it has recently been
2004d). It was observed that inhibition of NBT reduction

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Fig. 28  The formation of
superoxide anion radicals ­(O2·−) OH H OH
H
in xanthine-xanthine oxidase N N N
N
and reducing effect of nitroblue
­NBT2+ into formazan N N O N N
H
Xanthyne
Hypoxanthyne

Xanthine
oxidase

2O2 2O2
.

NO2 O2N NO2 O2N

NN NN HN N
N NH
NN NN
NN NN

H3CO OCH3
H3CO OCH3

NBT2+ Formazan

was generally greater than that of cytochrome c reduction Nitric oxide is an important cell-signaling molecule in
(Aruoma et al. 1993). This is because ­O2·− reacts much faster mammals, including humans (Hou et al. 1999). Low lev-
with cytochrome c than it does with NBT, so a given concen- els of NO production are important in protecting an organ
tration of added ­O2·− scavenger competes less efficiently in such as liver from ischemic damage. In metabolism, NO is
the cytochrome c system and exerts less inhibition. biosynthesized endogenously from l-arginine, oxygen and
NADPH by various nitric oxide synthase enzymes. Nitric
Nitric oxide radical (NO·) scavenging assays oxide is highly reactive and has a few seconds lifetime. It dif-
fuses easily across membranes. These attributes make nitric
Nitric oxide radicals (NO·) are generated in biological sys- oxide ideal for a transient paracrine and autocrine-signaling
tems by specific nitric oxide synthases, which metabolizes molecule (Stryer 1995).
arginine to citrulline with the formation of NO· via a five Vriesman et al. (1997) developed a relatively simple
electron oxidative reaction. It has crucial role in the regula- method for the quantification of NO· scavenging capacity
tion of different physiological and pathophysiological pro- of sulfur-containing compounds in aqueous solution using
cesses (Pacher et al. 2007; Gulcin 2012). an amperometric NO· sensor. The natural logarithm of
the NO· concentration and time are linearly related. After

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Fig. 29  The formation of
superoxide anion radicals ­(O2·−)
in NADH-PMS and reducing N A
­ BT2+ into
effect of nitroblue N
N N
formazan CH3 CH3
Oxidised PMS Reduced PMS

NADH NAD+ + H+

2e-

2O2 2O2
.

NO2 O2N NO2 O2N

NN NN HN N
N NH
NN NN
NN NN

H3CO OCH3
H3CO OCH3

NBT2+ Formazan

correction for the spontaneous degradation of NO·, second- detection technique is not easily available and has long reac-
order rate kinetics of the scavenging reaction was deter- tion time approximately 2 h.
mined. They observed that only those compounds, which Sodium nitroprusside is known to decompose in aqueous
contained a thiol group, displayed a considerable NO· scav- solution at physiological pH producing NO·. Under aero-
enging capacity. In this method, there is a non-competitive bic conditions, NO· reacts with oxygen to produce stable
reaction mechanism, since in the reaction medium is only products including nitrate and nitrite, which their quanti-
present the reactive species such as NO· and the scaven- ties can be determined using Griess reagent (Marcocci et al.
ger molecules. Also, the assessment of NO· scavenging 1994). When sulfanilamide is added the nitrite ion reacts
capacity has also been performed using ESR spectrometry to form a diazonium salt. When the azo dye agent (N-α-
(Asanuma et al. 2001). The NO· was generated from the naphthyl-ethylenediamine) is formed a pink color develops.
donor 3-(2-hydroxy-1-methyl-ethyl-2-nitrosohydrazino)- This diamine is used in place of the simpler and cheaper
N-methyl-1-propanamine was oxidized to N ­ O2 by the tar- α-naphthylamine because this latter is a potent carcinogen
get 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline- and moreover the diamine forms a more polar and hence a
1-oxyl-3-oxide, named as carboxy-PTIO with the formation much more soluble dye in acidic aqueous medium.
of carboxy-PTI spin adduct, that was measured by ESR. The Griess reaction is frequently used for assessment
The method was applied to non-steroidal anti-inflammatory of NO· production by whole cells or enzymes (Krol et al.
drugs. The main limitation of this method, considering the 1995). Briefly, the Griess reaction is a two-step diazotization

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Fig. 30  Assessment of NO· production by Griess reagent

reaction in which the NO-derived nitrosating agent (e.g., will interact with N-(1-naphthyl)ethylenediamine to yield
­N2O3), generated from the acid-catalyzed formation of a colored diazo product that absorbs strongly at 548 nm
nitrous acid from nitrite, reacts with sulfanilic acid to pro- (Tarpey et al. 2004).
duce a diazonium ion that is then coupled to N-(1-naphthyl) When this method compared to other methods, this
ethylenediamine to form a chromophoric azo product that methodology is not straightforward, requiring the addi-
absorbs strongly at 548 nm (Grisham et al. 1996). Its appli- tion of several enzymatic reagents. The fluorescent target
cation to in vitro determination of NO· scavenging capac- 4,5-diaminofluorescein widely used for in vivo NO· detec-
ity is also frequent. In this case, remaining amount of the tion and imaging, has also been applied for screening of NO·
nitric oxide after reaction with the test sample is measured scavenging capacity (Nagata et al. 1999). The nitrosation
as nitrite. It is important to emphasize that nitrate may of the weak fluorescent target 4,5-diaminofluorescein by
also be formed, thus it should be reduced to nitrite prior to derivative NO· species yielded the formation of the strong
determination. For this purpose, NADH-dependent nitrate green-fluorescent triazolofluorescein product. The NO· scav-
reductase was used for elimination of interference of NADH enging capacity was determined by the ability of compounds
by addition of lactate dehydrogenase and pyruvate (Perez to prevent the NO·-induced nitration of 4,5-diaminofluores-
et al. 2007). The chromophoric azo derivative formed from cein. The results are expressed as the percentage inhibition
nitrite after Griess reaction is then measured spectrophoto- of the 4,5-diaminofluorescein oxidation as a function of
metrically at 548 nm. Standard curves were generated using concentration of NO· scavenging compound. Nevertheless,
sodium nitrite and results were expressed as percentage the results obtained using 4,5-diaminofluorescein as a target
change from control response (Magalhaes et al. 2008). In for NO· quantification should be interpreted with caution,
this method, nitrite is detected and analyzed by formation since some antioxidant compounds such as ascorbic acid
of a red pink color upon treatment of a N ­ O2−-containing and dehydroascorbic may react directly with 4,5-diamino-
sample with the Griess reagent. When sulphanilic acid is fluorescein and form fluorescent compounds with emission
added, the nitrites form a diazonium salt. When the azo dye spectra similar to that of green-fluorescent triazolofluores-
agent (α-naphthylamine) is added a pink colour develops. cein product (Magalhaes et al. 2008).
A typical commercial Griess reagent contains naphthylen-
ediamine dihydrochloride (0.2%) and sulfanilamide (2%) in Peroxynitrite radical (ONOO·) scavenging assays
phosphoric acid (5%).
Griess reagent was used to detect nitrite photometrically. Peroxynitrite (ONOO) is one of the major reactive oxidants
The reagent contains two chemicals, sulfanilic acid and and nitrating species and can stable to cause neuronal cell
N-(1-naphthyl)ethylenediamine. Under acidic condition sul- injury, and has been suggested to contribute to the pathogen-
fanilic acid is converted by nitrite to a diazonium salt, which esis of neurodegeneration (Chen et al. 2012). On the other
readily couples with N-(1-naphthyl)ethylenediamine to form hand, peroxynitrite anion ­(ONOO−) is a potent and rela-
a highly colored azo dye that can be detected at 548 nm tively short-lived oxidant with a half-life of approximately
(Fig. 30). Under physiological condition, NO is unstable and 1 s under physiological conditions and it is highly diffusible
is rapidly oxidized to a mixture of nitrite and nitrate. To across cell membranes (Schieke et al. 1999). It is a potent
measure NO level indirectly from nitrite, nitrate is reduced oxidizing and nitrating species that can be generated from
to nitrite enzymatically via nitrate reductase so that the total the bi-radical reaction of nitric oxide and superoxide at a
amount of nitrite can be measured (Gulcin 2012). diffusion-limited rate. Peroxynitrite radical (ONOO·) is a
Also, the nitrosating agent dinitrogen trioxide ­(N2O3) cytotoxic effect with strong oxidizing properties toward
generated from the autoxidation of nitric oxide (NO) or from various food and cellular constituents, including lipids,
the acidification of nitrite ­(NO2) reacts with sulfanilamide sulfhydryls, amino acids and nucleotides. They can cause
to yield a diazonium derivative. This reactive intermediate

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Archives of Toxicology

cell death, lipid peroxidation, carcinogenesis and aging. as a by-product of oxygen metabolism. Electron reduction
These radicals are in vivo generated by endothelial cells, of ­O2 initially forms the ­O2·−, which is then spontaneously
neutrophils and macrophages. It is a relatively stable species or enzymatically converted to ­H2O2, which can induce the
compared with other free radicals but once protonated gives degradation of biological macromolecules such as proteins,
highly reactive peroxynitrous acid (ONOOH), decomposing enzymes, lipids, carbohydrates, and nucleic acids through
with a very short half-life (1.9 s) at 37 °C to form various generation of ­OH ·. In the presence of transition metal
cytotoxicants. It can induce the lipid peroxidation, oxidation ions, ­H2O2 can easily converted to ­OH· by Fenton (1984)
of thiol groups (–SH) on proteins, nitration of tyrosine, and or Haber–Weiss reactions (1934). Almost all living things
also nitrosation reactions, which affecting cell metabolism possess enzymes known as peroxidases, which harmlessly
and signal transduction. It can ultimately contribute to tissue and catalytically decompose low concentrations of H ­ 2O 2
and cellular injury with DNA strand breakage and apoptotic to water and oxygen (Gülçin et al. 2003c). It is generated
cell death. Its excessive formation may also be involved in in vivo, under physiological conditions by peroxisomes, by
several human diseases such as Alzheimer’s disease, cancer a variety of oxidative enzymes including glucose oxidase
rheumatoid arthritis, and atherosclerosis. Because of the lack and d-amino acid oxidase, and by dismutation of superox-
of endogenous enzymes responsible for ONOO· inactivation, ide radical, catalyzed by superoxide dismutase. It can cross
developing specific ONOO radical scavengers is of consid- membranes easily but it is unreactive at low concentrations
erable importance. The described method by involves the and reacts quite slowly with most compounds. It is used in
use of a stock solution of dihydroxyrhodamine (5 mM) in the respiratory burst of activated phagocytes, and generated
dimethylformamide, which purged with nitrogen and stored in vivo by several oxidase enzymes.
at − 80  °C. Working solution with dihydroxyrhodamine It is also produced from polyphenol-rich beverages under
(5 μM) is diluted from the stock solution and is placed on quasi-physiological conditions, and it increases in amount
ice in the dark immediately prior to the experiment (Kooy with longer incubation time. The generation of H ­ 2O2 by
et al. 1994). Phosphate buffer solution, (50 mM, pH 7.4), activated phagocytes is known to play an important part in
including NaCl (90 mM) and KCl (5 mM) with diethylen- killing several bacterial and fungal strains. ­H2O2 is most
etriaminepentaacetic acid (0.1 mM) are purged with nitrogen generally considered as a powerful oxidizing agent. There
and placed on ice before use. Scavenging activity of ONOO is increasing evidence that ­H2O2, either directly or indirectly
radicals by the oxidation of dihydroxyrhodamine is meas- via its reduction product ­OH−, acts as a messenger molecule
ured on a microplate fluorescence spectrophotometer with in the synthesis and activation of inflammatory mediators. It
excitation and emission wavelengths of 485 nm and 530 nm can cross membranes and may slowly oxidize a number of
at room temperature, respectively. The background and final compounds. It is used in the respiratory burst of activated
fluorescent intensities are measured 5 min after treatment phagocytes (Gülçin et al. 2003c). One of the most common
without 3-morpholino-sydnonimine or authentic ONOO·. methods for assessing the scavenging capacity against this
Oxidation of dihydroxyrhodamine by decomposition of molecule is based on the intrinsic absorption of H ­ 2O2 in
3-morpholino-sydnonimine gradually increased whereas the UV region at 230 nm (Santocono et al. 2006; Berges
authentic ONOO· rapidly oxidized dihydroxyrhodamine et al. 2007). The measurement of ­H2O2 scavenging activity
with its final fluorescent intensity being stable over time in foods and biological fluids is important. A lot of assays
(Alam et al. 2103). were developed for the determination of ­H2O2 scavenging
activity depending on the oxidation of a spectrophotomet-
Nonradical reactive oxygen species scavenging ric, fluorogenic, or chemiluminogenic probe by H ­ 2O2 using
assay horseradish peroxidase or transition metal ions as oxidation
catalysts (Ozyurek et al. 2010). A common assay for assess-
Hydrogen peroxide ­(H2O2) scavenging ing the scavenging capacity against H ­ 2O2 is based on the
intrinsic absorption of this molecule in the UV region at
Human beings are exposed to ­H 2O 2 indirectly via the 230 nm (Gülçin et al. 2003c). A solution of H ­ 2O2 (40 mM)
environment nearly about 0.28 mg/kg per day with intake is prepared in phosphate buffer (pH 7.4, 50 mM). In this
mostly from leaf crops. ­H2O2 may enter into the human body method, the remaining concentration of H ­ 2O2 is spectro-
through inhalation of vapor or mist and through eye or skin photometrically determined by absorption at 230 nm after
contact. It is rapidly decomposed into molecular oxygen and incubation (10 min) against a blank solution, which includ-
water and this may produce hydroxyl radicals (OH·), which ing phosphate buffer (pH 7.4, 50 mM) without ­H2O2. As
can initiate lipid peroxidation and cause DNA damage in the ­H2O2 concentration is decreased in the presence of
the body. It is a biologically relevant and non-radical-oxi- scavenger compounds, the absorbance value at 230 nm is
dizing species and may be formed in tissues through oxida- decreased. Nevertheless, it is quite usual that samples also
tive processes. Also, it is naturally produced in organisms absorb at this wavelength, requiring the performance of a

13
 Archives of Toxicology

“blank” measurement (Magalhaes et al. 2008). This situ- (Wilkinson et al. 1995). Nevertheless, the intensity of lumi-
ation can compromise both the precision and accuracy of nescence based on the self-emission of 1O2 is often insuf-
this method. Magalhaes and co-workers report that this ficient to provide reproducible quantitative information, even
situation can compromise both the precision and the accu- in aqueous medium. A more sensitivity method based on
racy of this method (2008). This method has some disad- monitoring the scavenging of 1O2 fluorescence of tetratert-
vantage and undesired results. Firstly, it may be difficult butylphthalocyanine was developed (Fu et al. 1997). Singlet
to distinguish between small changes when there is much oxygen is usually formed in the presence of light and pho-
larger background absorption. Secondly, the absorption of tosensitizers. It is thought that singlet oxygen is responsi-
samples may change after reaction with ­H2O2 and the blank ble for UV radiation skin damage, cataract formation in the
measurement would not be valid. Another common assay lenses of the eyes, macular degeneration and photosensitiv-
employs horseradish peroxidase, which uses ­H2O2 to oxidize ity derived from absorption or ingestion of phytochemicals,
scopoletin into a non-fluorescent product. In the presence pharmaceuticals and pesticides that act as photosensitizers
of putative H ­ 2O2 decomposer compounds, the oxidation (Zigman 2000). The assay was applied to compounds, which
of scopoletin is inhibited and the decomposition reaction are well documented to be ­1O2 quenchers such as β-carotene,
can be fluorimetrically monitored (Corbett 1989). Also, it α-tocopherol and lauric acid. This method is not widely
is known that ­H2O2 is toxic and induces cell death in vitro. applied but it is useful for measurement of rate constants
­H2O2 can attack many cellular energy-producing systems. for singled oxygen quenching, requiring commonly avail-
For instance, it deactivates the glycolytic enzyme glyceral- able equipment and also applicable to systems where the
dehyde-3-phosphate dehydrogenase (Ak and Gülçin 2008). 1270 nm luminescence is difficult to detect. The decay rates
Hydrogen peroxide itself is not very reactive; however, it of light intensity have been used to measure 1O2 quenching
can sometimes be toxic to cells because it may give rise to ability by a given compound (Huang et al. 2005). The 1O2,
HO· within the cells. Its toxicity derives from its conversion selectively generated by the thermal decomposition of the
to HO· (Gülçin et al. 2003c). ­H2O2 to cells in culture can endoperoxide disodium 3,3′-(1,4-naphthalene) bispropion-
lead to transition metal ion-dependent OH radicals medi- ate, oxidize the highly sensitive target dihydrorhodamine to
ating oxidative DNA damage. Levels of ­H2O2 at or below the fluorescent form rhodamine 123. The assay was success-
about 20–50 μg/cell seem to have limited cytotoxicity to fully applied for screening scavenging activity of several
many cell types. It is therefore widely thought that ­H2O2 is recognized antioxidant compounds against 1O2 (Magalhaes
very toxic in vivo and must be rapidly eliminated, employing et al. 2008). The in vivo formation of 1O2, without the pres-
enzymes such as catalases, peroxidases, and thioredoxin- ence of light, seems to be result of the spontaneous dismuta-
linked systems (Halliwell et al. 2000). Thus, removing H ­ 2O2 tion of the ­O2·−. Chemically, the 1O2 can be generated from
as well as superoxide anion is very important for protec- the non-photochemical decomposition of ­H2O2 by metals or
tion of pharmaceuticals and food systems (Chai et al. 2003). hypochlorite (MacDonald-Wicks et al. 2006; Huang et al.
The danger of ­H2O2 largely comes from its ready conversion 2005).
to the indiscriminately reactive HO·, either by exposure to
ultraviolet light or by interaction with a range of transition Metal chelating assay
metal ions, of which the most important is probably iron
(Halliwell et al. 2000). Metal chelation capacity is determined by measuring the
chelating effect of antioxidants for metal ions including
Singlet oxygen (1O2) quenching assays ferrous iron (­ Fe2+). Elemental species, such as F ­ e2+ can
facilitate the production of ROS within animal and human
Singlet oxygen (1O2) may behave as a more selective oxi- systems, and the ability of substances to chelate iron can be
dant than other ROS and can give rise to specific damage. valuable for antioxidant property. Iron is an essential min-
It is not a radical and does not react via radical mechanisms eral for normal physiology, but excess can result in cellular
but reacts mostly by the addition to double bonds, form- injury. Among the transition metals, iron is known as the
ing endoperoxides that can be reduced to alkoxyl radicals most important lipid oxidation pro-oxidant due to its high
that initiate radical chain reactions (Prior et al. 2005). It is reactivity (Gulcin 2012). Transition metal ions are known to
an excited state of molecular oxygen that has no unpaired stimulate lipid peroxidation via Fenton reaction and also by
electrons and it is known to be a powerful oxidizing agent, decomposing lipid hydroperoxides into more reactive per-
reacting directly with a wide range of biomolecules. Due oxyl and alkoxyl radicals. The effective ­Fe2+ chelators may
to its decay to the lower energy ground state, singled oxy- also afford protection against oxidative damage by removing
gen absorbs characteristic phosphorescence at 1270 nm. iron that may otherwise participate in HO· generating Fenton
Therefore, the 1O2 scavenging ability of several compounds type reactions. If they undergo the Fenton reaction, these
was measured through the decay rates of the light intensity reduced metals may form highly reactive hydroxyl radicals,

13
Archives of Toxicology

­ e3+ also produce

and thereby, contribute to oxidative stress. F (Siah et al. 2005). Iron-mediated formation of ROS leading
radicals from peroxides although the rate is tenfold less than to DNA and lipid damage appears to result from an exag-
that of ­Fe2+ (Kehrer 2000). geration of the normal function of iron, which is to transport
oxygen to tissues. Iron-induced free radical damage to DNA
Fe2+ + H2 O2 → Fe3+ + OH− + OH⋅ appears to be important for the development of cancer and
cancer cells are known to grow rapidly in response to iron
The name “Fenton reagent” refers to a mixture of H ­ 2O 2
(Valko et al. 2006).
and ferrous salts, which is an effective oxidant of a large
Transition metals, which possess two or more valence
variety of organic substrates. Fenton discovered that in the
states with a suitable oxidation–reduction potential affect
presence of a low concentration of ferrous salts and H ­ 2O 2,
both the speed of autoxidation and the direction of hydrop-
tartaric acid is oxidized to dihydroxymaleic acid in (1894).
eroxide breakdown to volatile compounds. Among the tran-
Then, Haber and Weiss, the authors suggested that in the
sition metals, iron is known to be the most important lipid
decomposition of ­H2O2 catalyzed by iron salts, OH· is
oxidation prooxidant due to its high reactivity. Iron is an
formed as an active intermediate (1934). Superoxide ion
essential element for all living cells. It plays a vital role
­(O2·−) can also be detoxified to ­H2O2 through a dismuta-
as component of heme-containing proteins, and as a metal
tion reaction with the SOD (through the Haber–Weiss reac-
cofactor of non-heme iron-containing enzymes (Wood and
tion) and finally to water by the CAT. If ­H2O2 reacts with
Ronnenberg 2006). Despite its essential nature, iron can be
an iron catalyst like ferrous ions (­ Fe2+), the Fenton reaction
potentially toxic when present as free ion. Its toxicity may
can take place forming the hydroxyl radical HO· (Carocho
arise from its capacity to participate, via the Fenton reaction,
and Ferreira 2013). The involvement of HO· as a reactive
as a catalyst in the formation of hydroxyl radical, a species
intermediate in the Fenton reagent was suggested by the fact
capable of inducing the oxidation of lipids, proteins, and
that it is an efficient hydroxylation agent. It was found later
DNA (Halliwell and Gutteridge 1984; Valko et al. 2006). An
that numerous metal ions and their complexes in their lower
effective ferrous ion chelator affords protection against oxi-
oxidation states such as ­Cu+, ­Ti3+, ­Cr2+, and ­Co2+ react with
dative damage by removing iron that may otherwise partici-
­H2O2 in a similar pattern as F ­ e2+, and the mixtures of these
pate in HO· generation via the Fenton type reactions. Ferric
metals with ­H2O2 were thus christened “Fenton-like” rea-
ions ­(Fe3+) also produce radicals from peroxides although
gents (Ou et al. 2002a, b). Studies have demonstrated that
the rate is tenfold less than that of ferrous ion. Ferrous ions
the electron transfer reaction between a transition metal and
­(Fe2+) are the most powerful prooxidants among the various
­H2O2 does not follow an outer-sphere electron transfer mech-
species of metal ions (Halliwell and Gutteridge 1984; Gülçin
anism. Instead, the reaction occurs through an inner-sphere
et al. 2004d). As a form to secure its availability and to pro-
electron transfer process, in which ­H2O2 forms a complex
tect cells from undergoing the toxic effects of free iron ions,
with transition metals before electron transfer takes place
the metal is bound to various peptides and proteins, among
(Goldstein et al. 1993). Therefore, if the transition metal is
which ferritin is recognized as the single most important
coordinately saturated, it does not react with H ­ 2O2 to give
iron-storing protein. In fact, ferritin has been estimated to
reactive oxygen species. Indeed, Halliwell et al. have shown
be able to sequester up to 4500 iron atoms per molecule;
that iron chelators inhibit the F ­ e2+-mediated Fenton reaction
storage of such atoms occurs under the form of a ferrihydrite
and therefore prevent the oxidative damage caused by ­Fe2+/
mineral core (Arosio et al. 2009).
H2O2 (Ou et al. 2002a, b).
Minimizing ferrous ion ­(Fe2+) may afford protection
The resulting oxy radicals cause damage to cellular lipids,
against oxidative damage by inhibiting production of ROS
nucleic acids, proteins, and carbohydrates, leading to cel-
and molecular damage. Ferrozine can form a complex with
lular impairment. Since ­Fe2+ are the most effective pro-
a red color by forming chelates with ferrous ions ­(Fe2+).
oxidants in food systems, the good chelating effect would
This reaction is restricted in the presence of other chelating
be beneficial, and removal of free iron ion from circulation
agents and results in a decrease of the red color of the fer-
could be a promising approach to prevent oxidative stress-
rozine–Fe2+ complexes (Fig. 31). Measurement of the color
induced diseases. When iron ion is chelated, it may lose its
change determines the chelating activity to compete with
pro-oxidant properties. Iron, in nature, can be found as either
ferrozine for the ­Fe2+ ions. The antioxidant effects of metal
­Fe2+ or F ­ e3+, with the latter form predominant in foods. Fer-
chelators is assessed when a complex is formed between
rous chelation may render important antioxidative effects by
the antioxidant and the metal, in such a way that metal ions
retarding metal-catalyzed oxidation (Gulcin 2012). From a
can no longer act as an initiator of lipid oxidation. The fer-
compilation of biochemical, animal and human data, links
rozine method can detect iron up to nanomolar concentra-
have been proposed between increased levels of iron in the
tions after preconcentration on resins (King et al. 1991).
body and an enhanced risk of a variety of diseases such as
In the presence of chelating agents, complex formation is
vascular disease, cancer and certain neurological conditions

13
 Archives of Toxicology

)2+
HO3S 3Ferrozine + Fe H2 O 6 → Fe − (Ferrozine)4−
(
3
+ 6H2 O.

The metal chelating capacity was significant since it

N reduced the concentration of the catalyzing transition metal
NaO3S in lipid peroxidation. It was reported that chelating agents
N N N are effective as secondary antioxidants because they reduce
the redox potential, thereby, stabilizing the oxidized form of
the metal ions (Gulcin 2012).
Fig. 31  The chemical structure of ferrozine
It was reported that food constituent has a marked capac-
ity for iron binding, suggesting that its main action as a per-
oxidation inhibitor may be related to its iron binding capacity
disrupted, resulting in a reduction in the red color of the (Ak and Gülçin 2008; Gülçin 2010). In this assay, curcumin,
complex. Measurement of color reduction therefore allows as a food constituent, interfered with the formation of the
estimation of the metal chelating activity of the coexisting ferrous–ferrozine complex, has chelating activity and is able
chelator. Lower absorbance indicates higher metal chelating to capture ferrous ion with a higher binding affinity than
activity (Kehrer 2000). Hence, metal chelation ability is also ferrozine. For example, as depicted in Fig. 32, curcumin
used as an indicator of antioxidant activity, usually in com- may chelate the ferrous ion with its –OH and –OCH3. It was
bination with other antioxidant assays (Gülçin et al. 2003c). reported that compounds with structures containing C–OH
Ferrous sulphate (­ FeSO4) and ferrous chloride (­ FeCl2) and C=O functional groups can chelate metal ions. Kazazica
are the most commonly used sources of ferrous ion (Gulcin and co-workers (2006) demonstrated that flavonoids, such as
et al. 2004e). A loss of absorbance at 485 nm (2,2′-bipyri- kaempferol, chelated ­Cu2+ and ­Fe2+ through the functional
dine) or 562 nm (for ferrozine) after the addition of antioxi- carbonyl groups. The compounds with structures containing
dants represents the formation of metal-antioxidant complex, two or more of the following functional groups: –OH, –SH,
and metal chelation capacity of the added antioxidant can –COOH, –PO3H2, C=O, –NR2, –S– and –O– in a favorable
be quantified spectrophotometrically (Sujayev et al. 2016; structure–function configuration can show metal chelation
Turan et al. 2016). One measurement of the metal-chelating activity (Yuan et al. 2005; Gülçin 2006a). The structure of
activity of an antioxidant is based on the absorbance meas- curcumin and its binding sites for metal chelation is given
urement of ­Fe2+–ferrozine complex after prior treatment of in Fig. 32. Recently, Fiorucci and co-workers (2007) dem-
a ferrous ion solution with test material. Ferrozine forms a onstrated that quercetin chelated metal ions in the same way.
complex with free ­Fe2+, but not with ­Fe2+ bound to other In another study, it was shown that l-carnitine chelated
chelators; thus, a decrease in the amount of ferrozine–Fe2+ ferrous ions ­(Fe2+) through the carbonyl and hydroxyl func-
complex formed after treatment indicates the presence of tional groups. In the same way, it was indicated that cur-
antioxidant chelators. The ferrozine–Fe2+ complex produced cumin bounded ferrous ions ­(Fe2+) through the carbonyl and
a red chromophore with absorbance that can be measured at
λ562 nm. Ethylenediaminetetraacetic acid (EDTA) is gener-
ally used as a standard metal chelator in food and pharma- OH
O Fe2+
ceutical applications. In most cases, metal chelation ability
of antioxidant compounds or plant extracts is expressed as O
OCH3
H3 C
EDTA equivalents (Ozbey et al. 2016). A significant draw-
back of this complexation reaction in measuring the pres-
ence of antioxidant chelator is that the reaction is affected 3 Fe2+ O
O
by both the antioxidant-Fe2+ and ferrozine-Fe2+ complex Fe2+
formation constants, and the competition between the two HO
O
H
chelators for binding to iron (Taslimi et al. 2017; Sarı et al.
2018). Thus, a weak antioxidant iron chelator would be seri- CH3
ously underestimated in quantitative determination. From a O
nutritional point of view, it is not yet possible to assess the OCH3
Fe2+

role of a weak antioxidant iron chelator in preventing the O

OH
Fenton reaction in vivo. Nonetheless, this reaction serves H
as a convenient assay to assess iron-chelating activity of Curcumin Curcumin-Fe2+ complex
antioxidant.
Fig. 32  The purposed ferrous ion ­(Fe2+) chelating mechanism of cur-
cumin

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Archives of Toxicology

H H
OH O O
HO HO OH
2 + Fe2+ Fe2+

OH O O
H H
Resveratrol Resveratrol-Fe2+ complex

Fig. 33  Two resveratrol molecules chelate activity one ferrous ion before ferrozine

hydroxyl functional groups (Ak and Gülçin 2008). Simi- spectrophotometrically at 532 nm (Gulcin 2012). MDA
larly, L-Adrenaline bounded ferrous ions ­(Fe2+) on amine is the organic compound and this reactive species occurs
and hydroxyl groups (Gülçin 2009). At the same manner, naturally and is a marker for oxidative stress. ROS degrade
two resveratrol molecules bonded ferrous ions ­(Fe2+) on polyunsaturated lipids, forming MDA (Pryor and Stanley
hydroxyl groups. Resveratrol demonstrates a marked capac- 1975). It is a reactive aldehyde and is one of the many
ity for iron binding, suggesting that its main action as a per- reactive electrophile species that cause toxic stress in cells
oxidation inhibitor may be related to its iron binding capac- and form covalent protein adducts referred to as advanced
ity. In this assay, resveratrol interfered with the formation of lipooxidation end products in analogy to advanced glyca-
the ferrous-ferrozine complex. This suggests that resvera- tion end products. The production of this aldehyde is used
trol has chelating activity and is able to capture ferrous ion as a biomarker to measure the level of oxidative stress in
before ferrozine. The structure of resveratrol and its binding an organism (Farmer and Davoine 2007). MDA may be
sites for metal chelation are given in Fig. 33. formed from PUFA with at least three double bonds. The
In a recent study, the possible ­Fe2+ chelating mechanism of concentration of this product may be assessed by reaction
usnic acid was studied. In this study, usnic acid interfered with with TBA, which reacts with MDA to form red condensa-
the formation of the ferrozine–Fe2+ complex. Usnic acid had tion products (Fig. 35) that absorb at 532–535 nm with
­Fe2+ chelating effect and was able to capture F ­ e2+ ions before molar absorptivity of 27.5 absorbance U/mmol. MDA and
ferrozine as a metal biding agent. The structure of usnic acid other TBARS condense with two equivalents of TBA to
and its binding sites for metal chelation is shown in Fig. 34. give a fluorescent red derivative that can be assayed spec-
It may chelate the ­Fe2+ with its hydroxyl and carboxyl groups trophotometrically (Nair et al. 2008). The MDA equiva-
bounded phenolic ring (Çetin Çakmak and Gulcin 2019). lents (μmol) of the samples being tested are calculated
with the use of the molar extinction coefficient of chromo-
Thiobarbituric acid value (TBA) genic product (1.56 × 105 M−1 cm−1). The TBARS assay
measures the amount of MDA formed from lipid peroxida-
The thiobarbituric reactive substances (TBARS) method tion, but other aldehydes simultaneously generated during
has been commonly employed to screen and monitor lipid lipid peroxidation may react with TBA and also absorb
peroxidation, for its ease of operation and low cost. In this at 532 nm. This assay is not a specific method for lipid
assay, malondialdehyde (MDA) emerging as an advanced peroxidation products. TBA reacts with different forms
product of unsaturated lipid degradation reacts with thio- of aldehydes, not just those formed as a result of lipid
barbituric acid (TBA) under acidic conditions to give peroxidation (Apak et al. 2016).
a characteristic chromogenic product [MDA-(TBA) 2], However, the reaction is not specific, and reaction with
which produced at high temperature (100 °C) is measured a wide variety of other products may contribute to the

Fig. 34  Possible ferrous ions

­(Fe2+) chelating mechanism of 2+Fe O
O
usnic acid
O O O
HO O O 2Fe2+

OH O O
OH O O Fe2+

Usnic acid Usnic acid-Fe2+ complex

13
 Archives of Toxicology

O OH
occurring antioxidant compounds requires reliable methods
OH
O O
HCl / H2O
of antioxidant activity evaluation. On the other hand, at the
HN NH
N +
H
CH2
H
present study, a large diversity of bioanalytical methods for
S N OH O N S
SH N OH H H determination of antioxidant capacity of food component is
TBA MDA (TBA)2-MDA
available as reported here. These bioanalytical assays dif-
fer from each other in terms of antioxidant reaction mecha-
Fig. 35  Formation of condensation reaction between thiobarbituric nisms, substrate type, oxidant and target species, reaction
acid (TBA) and malondialdehyde (MDA) conditions and oxidation initiator, result expression and ease
of operation. These methods vary in terms of mechanism.
Also, total antioxidant capacity is dependent of a multitude
absorbance. 2,4-alkadienals such as 2,4-decadienal also react of factors, however the behavior of antioxidants is strongly
with TBA to show strong absorption at 532 nm. Saturated recommended to generate a complete antioxidant profile.
aldehydes normally absorb at lower wavelengths after reac- The comparison of data from different studies is also diffi-
tion with TBA. Several food components including proteins, cult. In this context, when a method is selected, the primary
Maillard browning products and sugar degradation products factor of reaction mechanism should be considered. For the
affect the determination. To emphasize the lack of specific- determination of in vitro analytical assays should be know
ity, the values obtained in the test are commonly described about some information such as oxidation sources, concen-
as TBARS or TBA reactive substances. The TBA test has trations, the interactions with other oxidant species and bio-
recently been reviewed (Guillen-Sans et al. 1998). MDA logical targets, their significance to oxidative stress, and the
reacts with deoxyadenosine and deoxyguanosine in DNA, surrounding environment. The choice of appropriate method
forming DNA adducts, the primary one being M1G, which or combination of methods is important for the valid assess-
is mutagenic. The guanidine group of arginine residues ment of antioxidant activity and ultimately for the potential
condenses with MDA to give 2-aminopyrimidines (Marnett of antioxidant as food preservatives and pharmaceuticals or
1999). The TBARS method cannot distinguish between the health-promoting agents.
stoichiometry of the oxidation reaction and the kinetics. It
has some limitations because of oxidation reaction of TBA
with other substances not associated with lipid peroxida-
tion and the formation of Schiff bases between MDA and References
amines, leading to misestimating of the antioxidant protec-
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ganate as a chemiluminescence reagent—a review. Anal Chim
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inhibitory properties of novel ureas derived from phenethyl-
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