Online - 2455-3891
Print - 0974-2441Vol 9, Suppl. 2, 2016
A REVIEW ON ANTIOXIDANT METHODS
SUNITHA DONTHA*
Department of Pharmaceutical Chemistry, Malla Reddy College of Pharmacy, Maisammaguda, Secunderabad, Telangana, India.
Email: chmrcp@gmail.com
Received: 26 May 2016, Revised and Accepted: 28 June 2016
ABSTRACT
To provide an outlook of the various available methods of antioxidant activity. Various available in vitro and in vivo methods are listed and the
procedure to perform the method, its mechanism is also explained in brief. 1,1-diphenyl-2-picrylhydrazyl method was found to be used mostly for the
in vitro antioxidant activity evaluation purpose while lipid peroxidation was found as mostly used in vivo antioxidant assay. An ethanol was with the
highest frequency as a solvent for extraction purpose. Summarized information on the various methods available provides with reliable information
to confirm the benefits of antioxidant effects.
Keywords: Antioxidant activity, Reactive oxygen species, Free radical, 1,1-diphenyl-2-picrylhydrazyl, Flavonoid.
INTRODUCTION
Antioxidants became a vital part of our lives today since antioxidants
neutralizes or destroys “reactive oxygen species” (ROS) or free radicals
before they damage cells. The oxidation induced by ROS results in
cell membrane disintegration, membrane protein damage, and DNA
mutations, which results in aging and further initiates or propagates
the development of many diseases such as arteriosclerosis, cancer,
diabetes mellitus, liver injury, inflammation, skin damages, coronary
heart diseases, and arthritis.
The chemical compounds, which decrease the rate of lipid oxidation
reaction in food systems, are called antioxidants. By definition, a
substance that opposes oxidation or inhibits reactions promoted
by oxygen or peroxides; many of these substances being used as
preservatives in various products are antioxidants. Biologically
antioxidants are defined as synthetic or natural substances added to
products to prevent or delay their deterioration by the action of oxygen
in air. For example, enzymes or other organic substances such as
Antioxidants are chemical compounds which bind to free oxygen
radicals and prevents these radicals from damaging healthy cells.
This review focuses mainly on the types of damaging free radicals
generated in metabolic processes and also gives an insight of
mechanistic aspect of various in vitro and in vivo methods for the
evaluation of antioxidant capacity (Fig. 1).
By the normal use of oxygen [1], free radicals are produced continuously
by the body. Oxygen is an element indispensable for life. When cells
use oxygen to generate energy, free radicals are produced by the
mitochondria. These by-products are generally ROS as well as reactive
nitrogen species (RNS) that result from the cellular redox process. The
free radicals have a special affinity for lipids, proteins, carbohydrates,
and nucleic acids [2].
A free radical is a chemical species, capable of independent existence
possessing one or more unpaired electron. The free radicals are less
stable than non-radicals and are capable of reacting indiscriminately
with molecules. Once radicals are formed, they can either react with
another radical or with another non-radical molecule by various
interactions. When two radicals collide with their unpaired electron,
forms a covalent bond. The most molecules found in vivo are non-
radicals. A radical donates its unpaired electron to the other molecules,
or takes one electron from it, thus transforming its radical character.
At the same time, a new radical is formed [3,4]. ROS/RNS are present
in the atmosphere as pollutants and can be generated (i) during ultra-
violet (UV) light irradiation, by X-rays and gamma rays; (ii) during metal
catalyzed reactions; (iii) by neutrophils, esinophils and macrophages
during inflammatory cell activation [5,6]; (iv) as by-products of
mitochondrial catalyzed electron transport reactions; (v) by cytochrome
P450 metabolism and the enzyme xanthine oxidase, which catalyzes the
reaction of hypoxanthine to xanthine and xanthine to uric acid [7].
Depending on the environment and concentration of ROS, it is both
harmful and beneficial in biological systems [8,9]. For example, the
physiological roles in cellular responses to noxia such as defense against
infectious agents, and in the function of a number of cellular signaling
systems and gene expression. In contrast, at high concentrations, ROS
mediates damage to cell structures including lipids and membranes,
proteins, and nucleic acids; which is known as “oxidative stress” [10].
Oxidative stress is defined as an imbalance between the production of
free radicals and reactive metabolites, so-called oxidants or ROS, and
their elimination by protective mechanisms referred to as antioxidants.
This imbalance leads to damage of important biomolecules and cells, with
potential impact on the whole organism [11]. The harmful effects of ROS
are balanced by the action of antioxidants, example like enzymes present
in the body [12]. Despite the presence of the cell’s antioxidant defense
system to counteract oxidative damage from ROS, oxidative damage
accumulates during the life cycle and has been implicated in diseases,
aging and age-dependent diseases such as cardiovascular disease, cancer,
neurodegenerative disorders, and other chronic conditions [13].
ROS is classified into oxygen-centered radicals and oxygen-centered
non-radicals.
2
–
), hydroxyl
reactive species are nitrogen species such as nitric oxide (NO·), nitric
dioxide (NO
2
2
O
2
) and
singlet oxygen (
1
O
2
), hypochlorous acid and ozone [14,15].
ROS, which consist of free radicals such as superoxide anion (O
2
)
2
O
2
and singled oxygen (O
2
), are different forms of activated oxygen.
Review Article
© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2016.v9s2.13092
Asian J Pharm Clin Res, Vol 9, Suppl. 2, 2016, 14-32
Dontha
15
ROS are produced by all aerobic organisms and can easily react with
most biological molecules including proteins, lipids, lipoproteins,
and DNA. Thus, the generation of ROS proceeds to a variety of
pathophysiological disorders such as arthritis, diabetes, inflammation,
cancer, and genotoxicity. Therefore, living organisms possess a number
of protective mechanisms against the oxidative stress and toxic effects
of ROS. Antioxidants regulate various oxidative reactions naturally
occurring in tissues. Furthermore, terminates or retards the oxidation
process by scavenging free radicals, chelating free catalytic metals and
also by acting as electron donors.
A diet high in foods of animal origin and saturated fats increases the
risk of cardiovascular diseases and some cancers [16], which has
generated interest in promoting the consumption of plant-derived
proteins [17,18]. Legumes such as cereals, fruits, and vegetables
have health-promoting compounds and nutritional value [19]. The
nutritional quality and nutraceutical content associated with the
antioxidant activity of legumes such as common bean are important
sources of nutritional components (proteins, carbohydrates, fiber,
vitamins, and some minerals) [20,21].
presence of wide variety of phytochemicals such as phenolic
compounds, flavonoids, tannins, and unsaturated fatty acids.
Nutraceutical foods are preferred because they prevent degenerative
diseases and maintain good health [22]. From the epidemiological and
pharmacological evidence, it was found that nutraceutical properties
of active compounds in edible plants have increased, contribution for
the prevention and reduction of heart disease, diabetes, hypertension,
Alzheimer’s disease and arteriosclerosis, etc. [23-26].
ROLE OF ANTIOXIDANTS
An antioxidant is a molecule capable of inhibiting the oxidation of
another molecule. It breaks the free radical chain of reactions by
sacrificing their own electrons to feed free radicals, without becoming
free radicals themselves (Fig. 2).
ANTIOXIDANTS PREVENTS AGAINST FREE RADICAL DAMAGE
Antioxidants are nature’s way of defending cells against attack by ROS.
Our body naturally circulates a variety of nutrients for their antioxidant
properties and manufactures antioxidant enzymes to control these
destructive chain reactions. Forexample, vitamin C, vitamin E, carotenes,
and lipoic acid.
Oxidative stress is defined as the state in which the free radicals in
the body outnumber our antioxidant defenses. They also decrease the
telomere length of the chromosome (Fig. 3).
Oxidation is a chemical reaction that transfers electrons from a
substance to an oxidizing agent. Free radicals produced by these
oxidation reactions, start chain reactions that damage cells.
Antioxidants terminate these chain reactions by removing free radical
intermediates and inhibit other oxidation reactions by being oxidized
polyphenols.
Oxidation reactions are important for life, but they are also damaging
as well as enzymes such as catalase (CAT), superoxide dismutase (SOD),
and various peroxidases. Low levels of antioxidants, or inhibition of
antioxidant enzymes, causes oxidative stress and damages or kill cells.
These oxidants damage cells by chain reactions such as lipid
peroxidation (LPO), or by oxidizing DNA or proteins. Damage to DNA
causes mutations and possibly cancer, if not reversed by DNA repair
mechanisms, while damage to proteins causes enzyme inhibition,
denaturation and protein degradation. The brain is vulnerable to
oxidative injury, by LPO due to its high metabolic rate and elevated
levels of polyunsaturated lipids. Antioxidants prevent oxidative stress
in neurons and prevent apoptosis and neurological damage [27-29].
CLASSIFICATION OF ANTIOXIDANTS
Antioxidants can be categorized into two types.
Non-enzymatic antioxidants
Non-enzymatic antioxidants interrupt free radical chain reactions. For
example, vitamin E interrupts a chain of free radical activity after only
five reactions. Other examples include vitamin C, plant polyphenols,
antioxidant” and is found in every single cell of your body, maximizing
gamma peptide linkage between the amine group of cysteine (which
is attached by a normal peptide linkage to a glycine) and the carboxyl
group of the glutamate side-chain [30].
reduced state, the thiol group of cysteine is able to donate a reducing
++
e
) to other unstable molecules such as ROS. In donating
the liver).
Enzymatic antioxidants
Enzymatic antioxidants work by breaking down and removing free
radicals. In general, these antioxidant enzymes flush out dangerous
oxidative products by converting them into hydrogen peroxide, then
Fig. 1: Oxidation and reduction process
Fig. 2: Electrons in the outer shell
Asian J Pharm Clin Res, Vol 9, Suppl. 2, 2016, 14-32
Dontha
16
into water, in a multi-step process that requires a number of trace
metal cofactors (copper, zinc, manganese, and iron). These enzymatic
antioxidants cannot be supplemented orally but must be produced in
our body.
The principle enzymatic antioxidants are the following.
SOD
Assisted by copper, zinc, manganese and iron, SOD breaks down
superoxide (which plays a major role in lipid peroxidation) into oxygen
and hydrogen peroxide. SOD is present in nearly all aerobic cells and
extracellular fluids.
CAT
Converts hydrogen peroxide into water and oxygen (using iron and
manganese cofactors), hence finishing up the detoxification process
that SOD started.
Selenoproteins
These selenium-containing enzymes help break down hydrogen
peroxide and organic peroxides into alcohols and are particularly
abundant in liver. Selenium is an essential trace element having
fundamental importance to human health as it is a constituent of the
small group of selenocysteine-containing selenoproteins (over 25
different proteins) which are important for structural and enzymatic
functions. Selenoproteins include several forms of the enzymes
deiodinase.
GSHpx
Catalyzes the elimination of hydrogen peroxide as well as organic
GSR
maintaining the reducing environment of the cell.
WATER-SOLUBLE (HYDROPHILIC) AND LIPID-SOLUBLE
(LIPOPHILIC) ANTIOXIDANTS
Another categorization of antioxidants is based on whether they are
soluble in water (hydrophilic) or in lipids (hydrophobic). The interior
of our cells and the fluid between them are composed mainly of water,
but cell membranes are made largely made of lipids [32].
The lipid-soluble antioxidants (such as vitamins E and A, carotenoids,
and lipoic acid) are primarily located in the cell membranes, whereas the
are present in aqueous body fluids such as blood and the fluids within
and around the cells (the cytosol, or cytoplasmic matrix). Free radicals
can strike the watery cell contents or the fatty cellular membrane, so
the cell needs defenses for both. The lipid-soluble antioxidants are the
ones that protect the cell membranes from LPO.
Natural and artificial antioxidants
Antioxidants are divided into two groups according to their origin
synthetic antioxidants are of the phenolic type. The differences in their
antioxidant activities are related to their chemical structures, which
also influence their physical properties such as volatility, solubility, and
thermal stability.
Natural phenolic compounds are widely distributed in plants and
are the main contributors to the antioxidant activities of food [33].
atherosclerosis or heart failure are connected with oxidative stress.
Therefore, the increasing interest in elucidating the antioxidant activity
of different natural compounds [34,35].
The commercially available and currently used synthetic antioxidants
In recent years, there is an increasing interest in natural antioxidants
and subsequently looking through the literature; it is recognized that
the replacement of synthetic antioxidants by natural ones may have
several benefits and much of the research on natural antioxidants has
focused on phenolic compounds, in particular, flavonoids as potential
sources of natural antioxidants [36-38].
Numbers of naturally existing antioxidant compounds present in fruits,
phenolic acids (benzoic acid, trans-cinnamic acid, and hydroxycinnamic
acid), coumarins, lignans, stilbenes (in glycosylated form), flavonoids,
isoflavonoids, and phenolic polymers (tannins) [39].
Flavonoids as antioxidants
Flavonoids are secondary plant products recognized as the characteristic
red, blue and purple anthocyanin pigments of plant tissues. Apart
from their physiological roles in the plants, flavonoids as important
components in human diet but never considered as nutrient [40]. The
basic structure of flavonoid is a phenylated benzopyrone consists of
3 rings A, B and C (Fig. 5).
The various classes of flavonoids differ in the level of oxidation and
pattern of substitution of the C ring. Among the various classes of
flavonoids, the important ones are flavones, flavanones, isoflavones,
flavonols, flavanol (catechin), flavanonols, flavan-3-ols, and
anthocyanidins. Flavonoids are polyphenolic compounds representing
the majority of plant secondary metabolites and have shown to possess
remarkable health promoting effects including antioxidant activity [41].
Fig. 3: Role of antioxidants
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Aluminum chloride colorimetrilac estimation is commonly used
to quantify flavonoid content of plant extracts [42]. Total flavonoid
contents can be determined by reaction with sodium nitrite, followed
by the development of colored flavonoid-aluminum complex formation
using aluminum chloride in alkaline condition which can be monitored
spectrophotometrically at a maximum wavelength of 510 nm [43].
CLASSIFICATION OF ANTIOXIDANT METHODS [44]
In vitro antioxidant methods
Antioxidant activity is not concluded based on a single antioxidant
test model. There are several in vitro test procedures for evaluating
antioxidant activities with the samples of interest. Another aspect is
that antioxidant test models vary in different respects. Therefore, it is
difficult to compare fully one method to another one. In general, in vitro
antioxidant tests using free radical traps are relatively straightforward
to perform. Among free radical scavenging methods, 1,1-diphenyl-2-
involved with many steps and reagents) and inexpensive in comparison
to other test models. On the other hand, 2, 2-azinobis (3-ethyl
benzothiazoline-6-sulfonic acid) diamonium salt (ABTS) decolorization
assay is applicable for both hydrophilic and lipophilic antioxidants. In
this article, all in vitro methods are described and it is important to note
that no one method is absolute in nature rather than an example. All in
vitro antioxidant methods are listed in Table 1.
Based on the chemical reaction involved between the antioxidant
compounds and the free radicals, antioxidant capacity assays are
broadly classified into two types.
2. Electron transfer (ET) reaction based assays.
ET-based assays
These assays measure the reducing capacity of the antioxidant
compounds. It is based on the simple redox reaction, where antioxidant
compounds reduce the free radicals and get themselves oxidized.
Reduction by antioxidant compounds results in the color change of
the reagent, which correlates with the antioxidant capacity, which is
measured by the change in absorbance.
X
2
3
O
+
X
3
O
+
2
O
HAT-based assays
These assays measures/quantify the hydrogen atom donating ability of
the antioxidant compounds by a proton-coupled ET reaction, where it
measures the chain breaking antioxidant capacity. These assays based
on the reaction between synthetic free radical generator, oxidisable
molecular probe, and an oxidant where reaction kinetics is derived
from the kinetic curve.
X
ET-based assays
2. Superoxide anion radical scavenging assay
3. Ferric ion reducing antioxidant power (FRAP)
4. Trolox equivalence antioxidant capacity (TEAC), using ABTS
5. Cupric ion reducing antioxidant capacity (CUPRAC) assay
Fig. 4: Synthetic antioxidants
Fig. 5: Flavonoid
Table 1: List of in vitro anti-oxidant methods
Serial number Name of the antioxidant method
1. In vitro antioxidant methods
1.1. ET based assays
1.1.1.
1.1.2. Superoxide anion radical scavenging assay
1.1.3. FRAP
1.1.4. TEAC, using ABTS
1.1.5. CUPRAC assay
1.1.6. FCR, the total phenols assay
1.1.7. Reducing power assay
1.1.8.
1.1.9. Nitric oxide radical inhibition activity
1.1.10. TBARS assay
1.2.
1.2.1. ORAC
1.2.2. ABTS radical scavenging method
1.2.3. Crocin Bleaching Assays
1.2.4. TRAP
1.2.5.
1.2.6.
1.2.7. LPIC assay
1.2.8.
2
O
2
radicals
1.2.9. IOC
1.2.10. PCL Assay
1.2.11.
1.3. Other in vitro antioxidant methods
1.3.1. Ascorbic acid content assay
1.3.2. CAA
1.3.3. EPR spectroscopy investigations
1.3.4. Phosphomolybdenum assay
1.3.5. Xanthine oxidase method
1.3.6.
1,1-diphenyl-2-picrylhydrazyl, FRAP: Ferric ion reducing antioxidant power,
TEAC: Trolox equivalence antioxidant capacity, ABTS: 2, 2-azinobis (3-ethyl
benzothiazoline-6-sulfonic acid) diamonium salt, CUPRAC: Cupric ion
N-dimethyl-p-Phenylenediamine, TBARS: Thiobarbituric acid reactive
substances, ORAC: Oxygen radical absorbance capacity, TRAP: Total radical
LPIC: Lipid peroxidation inhibition capacity, IOC: Inhibited oxygen uptake,
PCL: Photochemiluminescence, CAA: Cellular antioxidant activity, EPR: Electron
paramagnetic resonance
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6. Folin-Ciocalteu reagent (FCR), the total phenols assay
7. Reducing power assay
9. NO radical inhibition activity
10. Thiobarbituric acid reactive substances (TBARS) assay.
HAT-based assays
1. Oxygen radical absorbance capacity (ORAC),
2. ABTS radical scavenging method
3. Crocin bleaching assays (CBA),
4. Total radical-trapping antioxidant parameter (TRAP),
7. LPO inhibition capacity (LPIC) assay
2
O
2
radicals
9. Inhibited oxygen uptake (IOC)
10. Photochemiluminescence (PCL) assay
Antioxidant testing of natural products has increasing interest in
recent years, mainly due to the fact that antioxidants can neutralize the
harmful free radicals in vitro, thus suggesting that an antioxidant-rich
diet, provides health benefits.
ET-based assays
DPPH radical scavenging activity (Fig. 6)
assay method is very simple and is also quick for manual analysis of
antioxidant contents. This method can be used for solid or liquid
samples and is not only specific to any particular antioxidant but also
applies to the overall antioxidant capacity of the sample.
picrylhydrazyl free radical to react with hydrogen donors [46,47].
the stable free radical becomes paired off in the presence of a hydrogen
donor (e.g., a free radical-scavenging antioxidant) and is reduced to the
more is the reducing ability. This test has been the most accepted model
for evaluating the free radical scavenging activity of any new drug.
that can donate a hydrogen atom, then this gives rise to the reduced
form (diphenyl picryl hydrazine; nonradical) with the loss of violet
(pale yellow of the picryl group present) [48].
In this test, a solution of radical is decolorized after reduction with
equation [49]:
(Purple) (Yellow)
from light by covering the test tubes with aluminum foil. 150 ml
taken immediately at 517 nm for control reading. 50 ml of various
concentrations of compounds as well as standard compound (e.g.,
ascorbic acid) were taken, and the volume was made uniformly to
150 ml using methanol. Each of the samples was then further diluted
absorbance was taken after 15 minutes at 517 nm using methanol as
blank on UV-vis spectrometer Shimadzu, UV-1601. The IC
50
values for
each drug compounds as well as standard preparation were calculated.
The free radical scavenging activity was calculated using the following
formula:
Absorbance of control]×100
radical by 50% (IC
50
value) was obtained by linear regression analysis of
dose-response curve plotting between % inhibition and concentrations.
The better way of comparison of antioxidant activity between the
samples is using IC
50
values. Inhibition concentration (IC
50
) values
defined as the concentration of sample required for 50% inhibition of
free radicals. IC
50
is determined from the plot between the remaining
absorbance of free radical and concentration with each analysis in
triplicates. In this test, quercetin, 6-hydroxy-2,5,7,8-tetramethyl
chroman-2-carboxylic acid (trolox), tocopherol, and ascorbic acid are
used as positive controls [50,51].
result. Furthermore, it requires only a UV-Vis spectrophotometer to
perform, which explains its widespread use in screening antioxidant
reactive and transient peroxyl radicals involved in LPO.
a. Conventional cuvette assay of radical scavenging activity is
replaced by 96-well plate titer assay from past couple of years.
Cuvette assay method uses UV-Vis spectrophotometer to see the
absorbance, whereas 96-well plate method uses ELISA plate reader
method, allows only 1 sample to read a time and requires a high
quantity of reagent, whereas the second method is time saving and
it reads about 96 samples at a time, with a small amount of reagent.
b. Thin layer chromatography (TLC) autography technique
The antiradical screening by TLC autography technique provides an
easy, effective and rapid way to study plant extract profiles. No sample
purification is needed as this technique provided a simultaneous
separation and radical scavenging activity measurement of antioxidative
analysis of antioxidants can be done by this technique.
Qualitative analysis
To detect the antioxidant activity, a method based on the reduction
is a free radical stable at room temperature, which produces a violet
solution in methanol. When the free radical reacts to an antioxidant, its
free radical property is lost due to chain breakage and its color changes
to light yellow. In this assay by TLC, the extracts that produced yellow or
white spots in the purple background were considered as antioxidants.
Fig. 6: 1, 1-diphenyl-2-picrylhydrazyl