ORIGINAL ARTICLE Simplified Methods for Microtiter Based

Analysis of In Vitro Antioxidant Activity

Somanjana Khatua, Sandipta Ghosh, Krishnendu Acharya*
Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, Centre of Advanced
Study, University of Calcutta, Kolkata, West Bengal, India

Abstract

Context: The research in exploring the antioxidant activity of pharmaceuticals has recently been increased
considerably and to determine such property a variety of testing methods are available. However, the major
drawback of these conventional tests is their large reaction volumes varying from 2 to 6 ml that in turn demand
high quantity of reagents and biological resources. Aims: Thus, this work was focused for optimization of routinely
used five antioxidant experiments such as superoxide radical (O2.− ) inhibition, 2, 2-diphenyl-1-picrylhydrazyl
(DPPH) quenching, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS.+) scavenging, chelating ability
of ferrous ion and reducing power by means of 96-well plates in minimal reaction volume ranging from 60
to 200 µl. Subjects and Methods: To authenticate the processes some renowned standards such as ascorbic
acid, butylated hydroxyanisole, Trolox as well as ethylenediaminetetraacetic acid were used, and their calibration
curves were prepared. Results: Analysis depicted similar activities of all references as reported by traditional
protocols suggesting validation of standardized procedures. Conclusion: Thus, the recommended systems clearly
improve original one in a number of ways. First, the methods provide concurrent multi-sample investigation
with automatic data storage. Second, the approach is time-saving with the use of a multichannel pipette. Third,
assays are inexpensive as the use of chemicals is reduced by 10 times. Fourth, the investigating pure compounds
or extracts are required in low quantity. Fifth, except O2.− quenching assay, all the methods are applicable to
lipophilic and aqueous components both.

Key words: 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical, chelating ability, 2, 2-diphenyl-1-

picrylhydrazyl radical, reducing power, scatter plots, superoxide radical, 96-well plates

INTRODUCTION through diets are required to neutralize excess radicals,

A
protect cells against toxic effects and prevent ailments.
ntioxidant activity is a broadly used Nowadays, several synthetic antioxidants such as butylated
term to characterize substances with the hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
ability of scavenging or neutralizing and tert-butylhydroquinone (TBHQ) are routinely used in
free radicals.[1] These radicals can be produced foods and medicines.[5] However, carcinogenic nature, strict
inside human body by both exogenous and legislation on the use of synthetic food additives and consumer
endogenous sources such as inflammation, preferences have shifted the attention of industrialists from
immune cell activation, excessive exercise,
synthetic to natural antioxidants.[6] Thus, there has been
mental stress, infection, ischemia, cancer,
ageing, water pollution, alcohol, cigarette
Address for correspondence:
smoke, heavy metals, industrial solvents, certain
Krishnendu Acharya, Molecular and Applied Mycology
drugs, radiation, and cooking.[2] The presence of
and Plant Pathology Laboratory, Department of Botany,
these reactive components in excess amount
Centre of Advanced Study, University of Calcutta,
can generate a phenomenon known as oxidative
35, Ballygunge Circular Road, Kolkata - 700 019,
stress, a deleterious process that can alter the
West Bengal, India.
structure of proteins, lipids, lipoproteins, and
E-mail: krish_paper@yahoo.com
DNA.[3] Consequently, a number of disorders
may propagate that can lead to degenerative,
Received: 06-04-2017
cardiovascular, renal, neurological, liver, and
Revised: 27-04-2017
autoimmune diseases.[4] In this backdrop,
Accepted: 05-05-2017
antioxidant components externally supplied

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 Khatua, et al.: Antioxidant methods by microplate technology

a worldwide trend toward the use of natural substances as sodium persulfate, ascorbic acid, ethylenediaminetetraacetic
therapeutic antioxidants to reduce risk factors of human from acid (EDTA), BHA, and Trolox were purchased from Sigma
deadly diseases.[7] Chemicals Co. (St. Louis, MO, USA). All chemicals were of
analytical reagent grade.
To date, a great number of in vitro methodologies have been
established to measure the efficacy of natural antioxidants
either as pure compound or extract. However, there are several Instrument
impediments that limited the applicability of these techniques
and handling samples with different concentrations.[8] For In this study, Bio-Rad iMarkTM Microplate Reader (USA)
instance, an enormous volume of reaction mixture ranging was used for absorbance reading and spectra recording. The
from 3 to 6 ml is required for conducting conventional machine is comprised an eight-channel, vertical path length
superoxide radical (O2.−) scavenging activity and reducing photometer that measures the absorbance of contents in
power assay. As a consequence, not only reagents are utilized 96-well microtitration plate at specific wavelengths such as
in high quantity but the biological materials are also needed 415, 450, 490, 595, 655, and 750 nm. Before every read, the
in significant amount. This becomes of major concern in plate can be shaken at low, medium, or high speed for 0-999
the case of pure components or complex samples that have s to mix the reaction mixture.
been isolated in low extent. Besides, both these techniques
offer robust and time-consuming processes demanding
their modification in a simplified way. While, methods Superoxide radical scavenging assay
of 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS.+) as well as 2, 2-diphenyl-1-picrylhydrazyl (DPPH.) Original method
radical quenching activity are operationally simple and have Each 3 ml reaction mixture sequentially contained 750 µl
been used in many research laboratories; however high price sodium phosphate buffer (50 mM, pH 7.8), 390 µl methionine
of these chemicals is the foremost and unavoidable limitation (13 mM), 300 µl EDTA (100 nM), 1300 µl of standards or
from the routine analysis.[9] antioxidant compounds, 250 µl NBT (75 mM), and 6 µl
riboflavin (2 mM). The reaction was started by turning on a
Therefore, the development of new, easy to perform, rapid
fluorescent lamp (15 W), and illumination was run for 10 min.
and inexpensive methods are an urgent call to determine
The production of violet color was monitored by measuring
radical scavenging potentiality of pharmaceuticals,
absorbance at 560 nm. Tubes with identical reaction mixture
nutraceuticals, and foods. In this context, microplate
technology can be considered as an effective step toward were kept in the dark and served as blanks.[15]
more sensitive and fast measurement of spectral absorption
values.[10] Eventually, some antioxidant experiments such Proposed method
as DPPH, ABTS, and reducing power tests have been Methionine solution was prepared by dissolving 0.074 g in
modified by adopting 96 as well as 48-well plates in a 5 ml water (2 nM). 0.004 g EDTA was dissolved in 10 ml
reaction volume of 220-1500 µl.[10-14] However, to the best water to make 0.1 nM solution. NBT and riboflavin were
of our knowledge, no previous study has been performed to prepared daily by dissolving 0.004 g in 5 ml (0.1 nM)
simplify O2.− inhibition and chelating ability of ferrous ion and 100 ml (1 nM) water, respectively. In 200 µl reaction
processes. In this backdrop, here we describe rapid, small-
mixture, x µl of sample was added followed by serial
scale, high-throughput microplate-based methodologies for
inclusion of water (83-x µl), 50 µl buffer, 26 µl methionine,
those aforementioned antioxidant assay using 60-200 µl
20 µl EDTA, 17 µl NBT, and 4 µl riboflavin. The plate was
reaction mixture. The modified approach is more precise,
shaken for 10 seconds in medium speed by microplate reader
sample-saving, rapid, and eco-friendly as well which would
be more suitable for substituting the traditional laborious instrument, and initial absorbance was measured at 595 nm.
techniques. Overall, the proposed assays are easy to execute Further, the 96-well plates were placed under 15 W lights
for manual screening of antioxidant capacity of a large and incubated for 10 min at room temperature to initiate
number of samples in a day. the reaction. Finally, change in absorbance was recorded at
the same wavelength, and initial absorbance was deducted
to nullify background color. Ascorbic acid at different
SUBJECTS AND METHODS concentrations (10-150 µg/ml) was considered as a reference
in this technique.
Chemicals
The degree of scavenging was calculated by the following
Sodium dihydrogen phosphate, disodium hydrogen equation:
phosphate, L-methionine, nitroblue tetrazolium (NBT),
riboflavin, ferrous chloride, ferrozine, trichloroacetic acid Scavenging effect (%) = {(Absorbance of control−
(TCA), potassium ferricyanide, ferric chloride, DPPH, ABTS, Absorbance of sample)/Absorbance of control} × 100

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 Khatua, et al.: Antioxidant methods by microplate technology

DPPH radical scavenging assay The degree of scavenging was calculated by the following
equation:
Original method
Scavenging effect (%) = {(Absorbance of control−
About 5 ml of standard or antioxidant compound solution was
Absorbance of sample)/Absorbance of control} × 100
mixed with 5 ml of 0.008% DPPH prepared in 50% ethanol.
The reaction was incubated for 30 min at room temperature
in the dark. Decolorization of DPPH solution was measured Chelating ability of ferrous ion
at 528 nm.[16]
Original method
Proposed method About 1850 µl of standards or antioxidant compounds were
About 0.004 g DPPH reagent was dissolved in 100 ml incubated with 50 µl Fe2+ (20 µM) (ammonium ferrous
methanol or 4:1 aqueous methanol solution depending sulfate) in 5% ammonium acetate, pH 6.9. Reaction was
on nature of investigating component. In 96-well plates, initiated by addition of 100 µl of ferrozine (100 µM) and after
x µl sample and 200-x µl DPPH solution were added. The 10 min incubation absorbance was read at 562 nm.[18]
plate was incubated for 30 min at room temperature in
the dark, and finally, absorbance was recorded at 595 nm Proposed method
wavelength. To authenticate the process, three synthetic About 10 mg ferrous chloride was dissolved in 40 ml
antioxidants including ascorbic acid, BHA, and Trolox at water to prepare 3 nM solutions. While 0.012 g ferrozine
different concentrations ranging from 1 to 50 µg/ml were was dissolved in 5 ml water (0.12 nM). In 200 µl reaction
used. mixture, x µl sample was added to 96-well plates along with
5 µl of ferrous chloride and mixed well. Then, 10 µl ferrozine
The degree of scavenging was calculated by the following was included followed by addition of 185-x µl of water
equation: or methanol depending on investigating component. The
system was incubated at room temperature for 10 min, and
Scavenging effect (%) = {(Absorbance of control− absorbance was measured at 595 nm. EDTA at concentrations
Absorbance of sample)/Absorbance of control} × 100 of 5, 10, 15, and 20 µg/ml was considered as a reference.

ABTS radical scavenging assay The degree of scavenging was calculated by the following
equation:
Original method
Scavenging effect (%) = {(Absorbance of control−
At first, ABTS was dissolved in water to prepare 7 mM Absorbance of sample)/Absorbance of control} × 100
concentrated solutions. Further, ABTS radical cation
(ABTS•+) was generated by reacting the stock solution with
2.45 mM of potassium persulfate followed by incubation in Reducing power
the dark at room temperature before use. After 12-16 h, the
ABTS•+ solution was diluted with ethanol or PBS (pH 7.4) Original method
to an absorbance of 0.7 at 734 nm. 1 ml of diluted ABTS•+ About 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml
solution was mixed with 10 ml of standards or antioxidant of K3 [Fe(CN)6] (1% w/v) were added to 1 ml of sample or
compounds and absorbance was measured against the antioxidant compounds dissolved in distilled water. The
appropriate solvent blank at 734 nm.[17] resulting mixture was incubated at 50°C for 20 min, followed
by addition of 2.5 ml of TCA (10% w/v). The mixture was
Proposed method centrifuged at 3000 rpm for 10 min to collect an upper layer
ABTS radicals were prepared freshly in each time according of solution (2.5 ml), mixed with distilled water (2.5 ml)
to the original method. After 12-16 h, ABTS•+ solution and 0.5 ml of FeCl3 (0.1%, w/v). The absorbance was then
was diluted to an absorbance of 0.7 ± 0.02 at 750 nm. In measured at 700 nm against blank sample.[19]
200 µl reaction mixture, x µl standard was added followed
Proposed method
by addition of 200-x µl reagent solution. The plate was
shaken for 10 seconds at medium speed, and absorbance Reagents were prepared at per the original method. In
was measured at 750 nm following 5 min incubation in the 96-well plates, 10 µl of sample solution, 25 µl of buffer and
dark. Herein, three references such as ascorbic acid, BHA, 25 µl of K3[Fe(CN)6] were added sequentially. The mixture
and Trolox were used at different concentrations (1, 3, 5, and was incubated for 20 min at room temperature, and reaction
10 µg/ml). was stopped by adding 25 µl of TCA solution. Further,

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 Khatua, et al.: Antioxidant methods by microplate technology

85 µl of water and 8.5 µl of FeCl3 were added to each well.

The contents were mixed, incubated for another 15 min at
room temperature and absorbance was measured at 750 nm.
Ascorbic acid, BHA, and Trolox at concentrations of 10,
30, 50, and 70 µg/ml were considered as standards in this
technique.
a b

Data analysis

All data were expressed as mean ± standard deviation of

three independent experiments. The sample concentrations
providing 0.5 of absorbance or 50% of antioxidant activity
were calculated from graphs of antioxidant activity c
percentages and regarded as EC50 value. Linear regression Figure 1: Superoxide radical scavenging activity (a) Photograph
and correlation analyses were performed using Microsoft of 96-well plates presenting dose-dependent radical quenching
Excel® (USA). potentiality of ascorbic acid with respect to control (Lane
A1-3: Blank, B1-3: 10 µg/ml, B4-6: 50 µg/ml, B7-9: 100 µg/ml,
B10-12: 150 µg/ml), (b) graphical presentation of the effect
of ascorbic acid on radicals in terms of percent inhibition,
RESULTS AND DISCUSSION (c) scatter plot of the standard showing Pearson’s correlation
coefficient (R2). Experiments were triplicated, and values are
Superoxide radical scavenging assay presented as mean ± standard deviation

Superoxide anion (O2−•) is generated by one-electron reduction

DPPH radical scavenging assay
of oxygen and acts as the precursor of most reactive oxygen
species. Dismutation of this primary radical can produce Use of DPPH. to analysis antioxidant potentiality dates back
hydrogen peroxide that in turn may be partially reduced to to 1950s, when Brand-Williams et al. published the first
one of the strongest oxidants in nature, hydroxyl radical. In technique to quantify scavenging property of components
addition, the anion may react with other radicals such as nitric using a spectrophotometer.[21] Presently, this commercially
acid resulting a more powerful oxidant, peroxynitrite.[20] These available stable nitrogen-centered free radical is the most
harmful components can initiate cellular damage that may extensively used reagent to determine radical scavenging
lead to different pathophysiological conditions.[3] Therefore, activity of nutraceuticals. The violet colored radical
evaluation of superoxide anion scavenging is extremely possesses a characteristic absorption and readily accepts
important for determining antioxidant activities. To assess electron from antioxidant compounds. Consequently, it is
such potentiality of pharmaceuticals, Martinez et al. proposed reduced to yellow colored diphenylpicrylhydrazine that
an in vitro method based on the generation of O2−• by auto- can be measured through colorimeter.[22] However, a large
oxidation of riboflavin in the presence of light. The radical volume of freshly prepared DPPH solution (1-5 ml) is
in turn reduced NBT to a blue colored formazan that can be required each time to conduct the conventional method. To
measured photometrically. However, decrease in absorbance overcome the limitation, protocol has been later modified
indicated consumption of O2−• by antioxidants present in by adopting microwell-based technique to make the assay
resulting mixture.[15] simpler as well as faster.[23] So far, few researchers have
validated and described the assay by applying 96-well
This original method has been revised herein by adopting plates although reaction mixture was in the range of
96-well plates to reduce the reaction volume from 3000 220-300 µl.[12]
to 200 µl at minimal expense of samples. In addition, less
concentration of all reacting reagents was used as compared For further simplification, miniaturization of volume and
to the traditional one. Besides, the maximum absorption reduction in expenditure of radicals, the original method
wavelength (λmax) was changed from 560 nm to 595 nm has been improved herein. In this context, the stock solution
due to the limitation of the automated analyzer. The was prepared with the low concentration of DPPH. and
proposed technique was standardized by using ascorbic acid reaction system was standardized to only 200 µl signifying
as a reference which showed excellent radical scavenging less use of the costly reagent. Besides, λmax was changed
activity [Figure 1]. At the concentration of 10, 50, and from 528 nm to 595 nm due to limitation of the automated
100 µg/ml, the reference was able to inhibit 11.751 ± 2.732%, analyzer. To authenticate the technique, three standards were
25.547 ± 1.838%, and 41.217 ± 0.285% radicals, respectively, used, and results have been summarized in Table 1. Ascorbic
which reached to the level of 59.6 ± 3.085% at the dose of acid exhibited a dose-dependent response by inhibiting
150 µg/ml. 30.183 ± 4.255%, 65.463 ± 1.766%, 89.506 ± 1.138%, and

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 Khatua, et al.: Antioxidant methods by microplate technology

Table 1: Antioxidant activity of standards

Antioxidant assays Ascorbic acid BHA Trolox EDTA
Superoxide radical scavenging assay 123.147±15.628 NT NT NT
DPPH radical scavenging assay 7.695±0.023 7.447±0.024 9.613±0.022 NT
ABTS radical scavenging assay 3.18±0.001 3.654±0.017 8.618±0.226 NT
Chelating ability of ferrous ion NT NT NT 11.814±0.585
Reducing power 18.739±0.007 32.215±6.907 36.095±0.001 NT
The results are presented in EC50 values (µg/ml) corresponding to effective concentration at which 50% radicals are scavenged or
absorbance is 0.5. Experiments were triplicated, and values are presented as mean±SD. SD: Standard deviation, NT: Not tested,
DPPH: 2, 2‑diphenyl‑1‑picrylhydrazyl, ABTS: 2, 2′‑azinobis (3‑ethylbenzothiazoline‑6‑sulfonic acid), BHA: Butylated hydroxyanisole,
EDTA: Ethylenediaminetetraacetic acid, EC50: Effective concentration 50%

91.716 ± 0.189% of radicals at the level of 5, 10, 25, and 50 respectively, depicting strong antioxidant potential. Whereas,
µg/ml, respectively. While BHA showed 36.832 ± 0.576%, BHA presented 15.37 ± 7.246%, 39.97 ± 1.923% of
68.829 ± 3.029%, 86.314 ± 0.383%, and 91.144 ± 0.197% quenching ability at the levels of 1 and 3 µg/ml that gradually
scavenging ability of DPPH• at those above-mentioned elevated to 69.02 ± 7.333% and 91.42 ± 1.931% at the dose
concentrations. However, Trolox presented lower potentiality of 5 and 10 µg/ml. Conversely, inhibition activities of Trolox
by quenching 31.134 ± 1.267%, 52.457 ± 3.168%, 92.617 ± were 8.21 ± 7.451%, 19.34 ± 8.542%, 29.43 ± 7.597%, and
0.198%, and 95.654 ± 0.31% radicals at the level of 5, 10, 58.81 ± 3.661% at those concentrations ranges demonstrating
25, and 50 µg/ml, respectively [Figure 2]. Nevertheless, our moderate activity. Thus, it can be said that ascorbic acid and
findings were found to be in agreement with the previous BHA presented extremely powerful potential while Trolox
reports.[24-26] exhibited lower effects [Table 1]. However, properties of
Trolox were detected to be in accordance with the previously
published reports.[26,29]
ABTS radical scavenging assay

Original ABTS•+ scavenging assay was proposed by Chelating ability of ferrous ion
Wolfenden and Wilson where the radical cation was generated
by activation of metmyoglobin with hydrogen peroxide in Some transition metals including Fe2+, Cu2+, and Pb2+
the presence of ABTS.[27] Later the method was modified by as well as Co2+ can trigger oxidative stress and magnify
direct production of blue-green ABTS•+ chromophore through cellular damage. Among them, Fe2+ has ability to react with
reaction between ABTS salt and a strong oxidizing agent H2O2 and produces highly reactive hydroxyl radical via
such as potassium persulfate or potassium permanganate.[17] Fenton’s reaction. Thus, antioxidants with metal chelating
However, the blue colored ABTS•+ is converted back to its ability may be of beneficial use in the treatment of several
colorless neutral form in the presence of hydrogen-donating pathophysiological disorders.[30] To determine such activity,
antioxidant compounds measured by decrease of its Dinis et al. proposed a method where ferrozine competitively
characteristic absorption spectrum.[28] As a consequence, the reacts with Fe2+ forming red complexes. When other chelators
assay becomes operationally simple, and nowadays it has been are present in reaction mixture, this complex formation
routinely used in many research laboratories to determine is disrupted, and red color of solution decreases. Thus
radical scavenging activity of components. However, with reduction in absorbance signifies Fe2+ trapping potentiality
sophistication and advancement in instrumental techniques, of investigating component.[18] The assay is a quite popular
the protocol has experienced several modifications, while technique for determination of antioxidant property of
basic principle remained unique. Recently, microplate samples, though the method has not being modified so far for
adaptation of ABTS radical quenching method has been higher sample throughput and better reproducibility.
described by some investigators in a reaction volume of not
<255 µl.[10] In this background, this study was conducted in an effort to
improve the traditional method. As shown in Figure 4, EDTA,
In an aim to reduce the quantity of reaction mixture and use of the reference, revealed exceptional dose-dependent chelating
expensive reagent, the assay was revised herein. The system pattern at the tested concentrations. The chelating activity
was optimized in resulting solution of only 200 µl, and of 18.18 ± 3.04% and 38.82 ± 0.961% was achieved at the
three references were considered for validation [Figure 3]. concentration of 5 and 10 µg/ml of EDTA, respectively. While
Analysis showed that all standards possessed strong radical it increased steadily to 65.99 ± 2.333% and 89.23 ± 0.209%
scavenging capacity that incremented in a dose-dependent at the level of 15 and 20 µg/ml of standard. In a recent study,
manner. At the concentration of 1, 3, 5, and 10 µg/ml, radical Chai et al. reported ferrous ion chelating potentiality of
scavenging abilities of ascorbic acid were 15.16 ± 0.647%, EDTA following classical method which was found to be in
47.07 ± 3.38%, 79.15 ± 5.586%, and 89.82 ± 2.387%, agreement with our data.[31]

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 Khatua, et al.: Antioxidant methods by microplate technology

a b

c d e
Figure 2: 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (a) Photograph of 96-well plates where Lane B
represents negative control; Lane C, E, and G depicts DPPH. quenching potentialities of ascorbic acid, butylated hydroxyanisole
(BHA), and Trolox, respectively. The standards were investigated at different concentrations such as 5, 10, 25, and 50 µg/ml
in triplicates, (b) graphical presentation of the effect of three references on radicals in terms of percent inhibition. Scatter plot
of ascorbic acid (c), BHA (d) and Trolox (e) was prepared to calculate Pearson’s correlation coefficient (R2). Experiments were
triplicated, and values are presented as mean ± standard deviation

a b

c d e
Figure 3: 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity (a) Photograph of 96-well
plates where Lane B represents negative control; Lane C, E, and G depicts ABTS•+ quenching potentialities of ascorbic acid,
butylated hydroxyanisole (BHA), and Trolox, respectively. The standards were investigated at variable concentrations of 1, 3, 5,
and 10 µg/ml in triplicates, (b) graphical presentation of the effect of three references on radicals in terms of percent inhibition.
Scatter plot of ascorbic acid (c), BHA (d), and Trolox (e) was prepared to calculate Pearson’s correlation coefficient (R2).
Experiments were triplicated, and values are presented as mean ± standard deviation

Reducing power of reducing the power of a substance may serve as a

significant indication for its antioxidant potentiality. In that
It is well-known that free radicals are molecules, atoms or view, Oyaizu described a process based on reduction of Fe3+
ions that contain unpaired electrons; as a result, they are to Fe2+ depending on the activity of investigating compounds
highly reactive in nature. Antioxidants are substances that which in turn generates KFe [Fe(CN)6] (Prussian blue)
are stable enough to donate their own electrons to stabilize appropriate for spectrophotometric measurement.[19,32] Thus
radicals and inhibit further damages.[5] Thus, determination increase in absorbance denotes an increment in reducing

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 Khatua, et al.: Antioxidant methods by microplate technology

power as well as antioxidant effects. However, originally To perform the test in 96-well plates, the assay needs to be
the assay was designed for plasma, but after realizing re-designed. In this context, the protocol has been modified
its applicability, it is now used in an enormous number herein and to fulfill requirement resultant solution was
of matrixes.[4] Subsequently, the methodology has been deducted to only 60 µl. Moreover, the centrifugation step as
modified using 48 well microtitration plates in a reaction described in original technique has been removed to simplify
mixture of 1500 µl.[13] the method. Besides, λmax was changed from 700 to 750 nm
due to limitation of automated analyzer. To authenticate
the revised technique, three standards were used, and their
activities have been presented in Figure 5. All the references
were capable of reducing Fe3+ to Fe2+ in a linear dose-dependent
manner. Ascorbic acid exhibited a dose-dependent response
by increasing absorbance 0.218 ± 0.011, 0.909 ± 0.025, 1.2 ±
0.0618, and 1.2 ± 0.017 at the level of 10, 30, 50, and 70 µg/ml,
a b respectively. While BHA showed 0.145 ± 0.015, 0.526 ±
0.074, 0.731 ± 0.018, and 0.922 ± 0.014 reducing power at
those above-mentioned concentrations. However, Trolox
presented lower potentiality by increasing absorbance of 0.07
± 0.011, 0.445 ± 0.041, 0.698 ± 0.08, and 0.933 ± 0.003 at the
level of 10, 30, 50, and 70 µg/ml, respectively. Thus, it can be
observed that ascorbic acid possesses the strongest reducing
c effect followed by BHA and Trolox [Table 1].
Figure 4: Chelating ability of ferrous ion (a) Photograph
of 96-well plates presenting dose-dependent activity of
ethylenediaminetetraacetic acid (EDTA) in respect to negative
CONCLUSION
control (Lane B1-3: Blank, C1-3: 5 µg/ml, B4-6: 10 µg/ml,
B7-9: 15 µg/ml, and B10-12: 20 µg/ml), (b) graphical presentation
of the effect of EDTA in terms of percent chelation, (c) scatter The work presents improved version of routinely used
plot of the standard showing Pearson’s correlation coefficient antioxidant assays based on microplate reader technique.
(R2). Experiments were triplicated, and values are presented as Major advantage of the proposed methods is a reduction
mean ± standard deviation in reaction volume ranging from 200 µl for DPPH and

a b

c d e
Figure 5: Reducing power (a) Photograph of 96-well plates where Lane B represents negative control; Lane C, E, and G
depicts reducing power activity of ascorbic acid, butylated hydroxyanisole (BHA), and Trolox, respectively. The standards were
investigated at variable concentrations of 10, 30, 50, and 70 µg/ml in triplicates, (b) Graphical presentation of the reducing ability
of three references in terms of increase in absorbance. Scatter plot of ascorbic acid (c), BHA (d) and Trolox (e) was prepared
to determine Pearson’s correlation coefficient (R2). Experiments were triplicated, and values are presented as mean ± standard
deviation

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 Khatua, et al.: Antioxidant methods by microplate technology

ABTS radical scavenging protocols to 60 µl in the case of et al. Phenolic compounds and antioxidant activities of
reducing power method. In addition, this study provides the Liriope muscari. Molecules 2012;17:1797-808.
first time attempt for optimization of O2.− quenching as well 15. Martinez CA, Loureiro ME, Oliva MA, Maestri M.
as chelating ability of ferrous ion assays in 96-well plates Differential responses of superoxide dismutase in
at per our knowledge. The modified techniques may be freezing resistant Solanum curtilobum and freezing
considered as economically inexpensive, time-saving and sensitive Solanum tuberosum subjected to oxidative and
most importantly useful for analysis of a large number of water stress. Plant Sci 2001;160:505-15.
samples in a day. 16. Shimada K, Fujikawa K, Yahara K, Nakamura T.
Antioxidative properties of xanthan on the autoxidation
of soybean oil in cyclodextrin emulsion. J Agric Food
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