MethodsX 2 (2015) 283291

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MethodsX
journal homepage: www.elsevier.com/locate/mex

Optimized enzymatic colorimetric assay for

determination of hydrogen peroxide (H2O2)
scavenging activity of plant extracts
Chamira Dilanka Fernando * , Preethi Soysa 1
Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Colombo, Kynsey Road,
Colombo 08, Sri Lanka

G R A P H I C A L A B S T R A C T

A B S T R A C T

The classical method to determine hydrogen peroxide (H2O2) scavenging activity of plant extracts is evaluated by
measuring the disappearance of H2O2 at a wavelength of 230 nm. Since this method suffers from the interference
of phenolics having strong absorption in the UV region, a simple and rapid colorimetric assay was developed
where plant extracts are introduced to H2O2, phenol and 4-aminoantipyrine reaction system in the presence of
horseradish peroxidase (HRP). This reaction yields a quinoneimine chromogen which can be measured at 504 nm.
Decrease in the colour intensity reects the H2O2 scavenged by the plant material.

Optimum conditions determined for this assay were 30 min reaction time, 37  C, pH 7, enzyme concentration of
1 U/ml and H2O2 concentration of 0.7 mM. The limit of detection (LOD) and limit of quantitation (LOQ) were
136 mM and 411 mM, respectively.

Half maximal effective concentration required to scavenge 50% of H2O2 in the system (EC50 value) calculated for
several plant extracts and standard antioxidants resulted in coefcient of variance (CV%) of the EC50 values less
than 3.0% and correlation coefcient values (R2) > 0.95 for all dose response curves obtained.

* Corresponding author. Present address: College of Chemical Sciences, Institute of Chemistry Ceylon, 341/22, Kotte Road,
Welikada, Rajagiriya, Sri Lanka. Tel.: +94 723546610.
E-mail addresses: dilankafdo86@gmail.com (C.D. Fernando), indunilsree@gmail.com (P. Soysa).
1
Tel.: +94 771825814.

http://dx.doi.org/10.1016/j.mex.2015.05.001
2215-0161/ 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
284 C.D. Fernando, P. Soysa / MethodsX 2 (2015) 283291

This method is convenient and very precise which is suitable for the rapid quantication of H2O2 scavenging
ability of standard antioxidants and natural antioxidants present in plant extracts.
2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).

A R T I C L E I N F O
Method name: Enzymatic colorimetric assay for H2O2 scavenging activity
Keywords: Colorimetric assay, Hydrogen peroxide, Scavenging activity, Plant extracts
Article history: Received 17 December 2014; Accepted 11 May 2015; Available online 18 May 2015

Method details

Background information

Hydrogen peroxide (H2O2) scavenging activity of natural antioxidants present in plant extracts has
been determined widely [15] by measuring decrement of H2O2 in an incubation system containing
H2O2 and the scavenger using the classical UV-method at 230 nm [6]. The main disadvantage of this
method is the possible interference from secondary metabolites present in plants which absorb in UV
region [7]. Therefore, a simple and rapid colorimetric assay was developed to determine H2O2
scavenging activity of plant extracts and standard antioxidants based on the reaction system where
H2O2 rapidly reacts with phenol and 4-aminoantipyrine in the presence of horseradish peroxidase
(HRP) to produce a pink coloured quinoneimine dye (Fig. 1) [8]. H2O2 scavengers will eventually result
in decreased production of this particular chromophore. This method was applied to standard
antioxidants ascorbic acid, gallic acid and tannic acid in addition to selected plant extracts to
determine their hydrogen peroxide scavenging abilities.

Chemicals and equipment

The chemicals gallic acid, 4-aminoantipyrine and horse radish peroxidase (HRP) were purchased
from Sigma Chemicals Co. (P.O. Box 14508, St. Louis, MO 63178, USA). L-Ascorbic acid and hydrogen
peroxide were purchased from BDH Chemicals (BDH Chemicals Ltd Poole, England). Tannic acid was
purchased from Riedel De Haen Ag, Wunstorfer Strasse 40, SEELZE1, D3016, Germany. Phenol was
purchased from Fluka (Fluka chemie GmbH, CH-9471, Buchs, Switzerland). Plant extracts were freeze
dried using LFT 600EC freeze dryer. SHIMADZU UV 1601 UV Visible spectrophotometer (Shimadzu
Corporation, Kyoto, Japan) was used to measure the absorbance.

Fig. 1. The chemical reaction catalyzed by HRP [8].

 C.D. Fernando, P. Soysa / MethodsX 2 (2015) 283291 285

Plant materials

Atalantia ceylanica (Yaki-naran), Eriocaulon quinquangulare (Heen kokmota) and Semecarpus

parvifolia (Heen badulla) were collected from Anuradhapura, Kalutara and Colombo districts,
respectively. Mollugo cerviana (Pathpadagam) total plant was purchased from a traditional medicinal
drug store. Camellia sinensis Crush-Tear-Curl (CTC) black tea powder was purchased from the local
market. Plants were identied and conrmed by Department of Botany, Bandaranaike Memorial
Ayurvedic Research Institute, Nawinna, Sri Lanka.

Preparation of the plant extracts

All the plant materials A. ceylanica (leaves), E. quinquangulare (whole plant), M. cerviana (whole
plant), S. parvifolia (leaves) were prepared separately as decoctions according to the proportions
followed by Ayurvedic practitioners. The plant materials described above were washed separately
with tap water followed by distilled water and de-ionized water, dried to achieve a constant weight.
Each plant material was cut into small pieces and ground to a ne powder using a clean kitchen
blender. Powdered samples (30 g) except S. parvifolia were boiled with 800 ml of deionized water until
the total volume reduced to 100 ml (1/8th of the original volume) using a beaker. Powdered leaves of S.
parvifolia (30 g) was reuxed with 800 ml of deionized water to prepare the aqueous extract of the
plant material. The decoctions were sonicated and ltered through a cotton wool plug and then using
lter paper (Whatman No. 1). The ltrates were centrifuged at 2000 rpm for 10 min. The supernatants
were freeze dried. The freeze dried samples were weighed, and stored at 20  C in sterile tubes until
further use. A weight of 2.0 g of C. sinensis (black tea powder) was added into 200 ml of boiling water
and allowed to stand in a closed beaker to prepare black tea infusion. The solution was allowed to cool
to room temperature, ltered through lter paper (Whatman No. 1) and the ltrate was used for the
experiments.

Determination of incubation period, enzyme concentration and H2O2 concentration

A mixture containing phenol (12 mM, 350 ml), 4-aminoantipyrene (0.5 mM, 100 ml), H2O2 (0.7 mM,
160 ml) and phosphate buffer at pH 7 (84 mM, 350 ml) was prepared in separate tubes for each
incubation period (560 min) to optimize the incubation time needed for the completion of the
reaction. HRP (0.1 U/ml, 40 ml) was added to each tube and incubated at 37  C. At the end of the each
incubation period the absorbance was measured at 504 nm against reagent blank consisting of
phosphate buffer instead of phenol.
Varying concentrations of HRP (0.011 U/ml) were incubated with substrates at pH 7 for 30 min. at
37  C as described above to optimize the enzyme concentration needed for the assay.
Similarly different concentrations of H2O2 (0.1750.70 mM), phenol, 4-aminoantipyrene,
phosphate buffer (pH 7) were incubated with HRP (1 U/ml) as described above and the absorbance
was read to study the best H2O2 concentration to be used for the assay.
In the present colorimetric method to determine H2O2 scavenging activity, maximum wavelength
of absorbance (lmax) of the quinoneimine dye at pH 7 was observed at 504 nm (Fig. 2A). At 37  C, pH 7,
H2O2 concentration of 0.7 mM and horse radish peroxidase enzyme concentration of 0.1 U/ml,
maximum intensity of the quinoneimine dye was resulted at 30 min and the colour was stable until
60 min (Fig. 2B). When solutions having enzyme concentrations of 0.011 U/ml (maintained at 37  C,
pH 7 with H2O2 concentration of 0.7 mM) were incubated for 30 min, the colour increased drastically
from enzyme concentration of 0.010.08 U/ml but very slight increment of the colour was observed
from 0.08 to 1 U/ml (Fig. 2C). Although enzyme concentration of 0.1 U/ml turned out to be suitable for
the experiment, further studies were carried out keeping enzyme concentration 10 folds higher
(1 U/ml) to compensate for possible inhibition of enzyme that can be caused by phytochemicals
present in plant extracts. When varying the H2O2 concentration from 0.175 to 0.70 mM of the solutions
having enzyme concentration of 1 U/ml (maintained at pH 7, 37  C for an incubation period of 30 min),
it was observed that the enzyme is being saturated at H2O2 concentration of 0.70 mM (Fig. 2D) and
therefore this concentration was used for the assay.
 286 C.D. Fernando, P. Soysa / MethodsX 2 (2015) 283291

Fig. 2. The UVvis spectrum for the chromogen formed at wave length range from 200 to 800 nm (A), variation of absorbance
with time (B), variation of absorbance with enzyme concentration (C), dependance of absorbance on H2O2 concentration (D), pH
stability of the chromogen formed (E) and variation of absorbance with temperature (F). The results are presented as mean + SD
of three independent experiments.

Determination of optimum pH and temperature

Mixtures containing phenol (12 mM, 350 ml), 4-aminoantipyrene (0.5 mM, 100 ml), H2O2 (0.7 mM,
160 ml) and phosphate buffer at pH 7 (84 mM, 350 ml) were prepared in separate tubes and pH was
varied from 1 to 11. HRP (1 U/ml, 40 ml) was added to each tube and was incubated at 37  C for 30 min.
The absorbances of the resulting solutions were measured at 504 nm against the reagent blank
consisting of phosphate buffer at pH 7 instead of phenol.
Substrates were incubated with HRP (1 U/ml) at different temperatures (1854  C) at pH 7 for
30 min and absorbance was measured at 504 nm as described above.
When pH was varied from 1 to 11 of the solutions having H2O2 and peroxidase concentrations of
0.7 mM and 1 U/ml, respectively, maintained at 37  C and incubated for 30 min, maximum intensity of
colour of the resultant dye was yielded at pH 7 (Fig. 2E). When temperature was varied from 18 to 54  C
of the solutions maintained at similar conditions as above and at pH 7, maximum intensity of the
colour of the dye was observed at 37  C (Fig. 2F).
 C.D. Fernando, P. Soysa / MethodsX 2 (2015) 283291 287

Table 1
Selected optimum conditions for the reaction catalyzed by
horse radish peroxidase enzyme.

Parameter Optimum value

Incubation time 30 min
Enzyme concentration 1 U/ml
H2O2 concentration 0.7 mM
Temperature 37  C
pH 7

Determination of limit of detection (LOD) and limit of quantitation (LOQ)

The LOD and LOQ values were determined according to the ICH guidelines provided [9]. Microsoft
Excel was used to perform regression analysis of the calibration curve constructed using diluted
samples of H2O2. The standard deviation of the y-intercepts (d) of the regression line and the slope of
the calibration curve (S) were estimated. LOD and LOQ were calculated as 3.3  d/S and 10  d/S,
respectively [9]. The calculated values were 136 mM and 411 mM for LOD and LOQ, respectively.

Determination of H2O2 scavenging activity

According to the above experiments the optimum conditions selected for the reaction catalyzed by
horse radish peroxidase is stated in Table 1. These conditions were maintained when plant extracts
were introduced into these systems to assess their scavenging ability of H2O2 molecules. Phenol
(12 mM) and 4-aminoantipyrene (0.5 mM) were chosen and used for all the above tests as these
concentrations led to maximum intensity of the resultant chromophore. The percentage inhibition
(% I) of H2O2 caused by plant extracts and standard antioxidants was calculated as follows. Reaction
mixture comprising of test sample (plant extract/standard antioxidant; 350 ml), phenol solution
(12 mM, 350 ml), 4-aminoantipyrene (0.5 mM, 100 ml), H2O2 (0.7 mM, 160 ml) and HRP (1 U/ml)
prepared in phosphate buffer (84 mM, pH 7) was incubated at 37  C for 30 min. The absorbances of the
resulting solutions were measured at 504 nm against reagent blank consisting of phosphate buffer
instead of plant extract/standard antioxidant and phenol. The control was made out of same reagents
except plant extract replaced by phosphate buffer. Interference for the assay from the plant extracts
was minimized as follows. For each concentration of plant extract, samples for background
subtraction were made using the plant extract with other reagents replacing phenol by phosphate
buffer. Each resulting absorbance value was subtracted from the relevant original absorbance reading.
Five types of plant extracts known for their antioxidant properties were tested for their H2O2
scavenging activities. L-Ascorbic acid, gallic acid and tannic acid were used as reference standard
antioxidants. The percentage inhibition of hydrogen peroxide was calculated by the equation as
described as for many antioxidant assays [35]:

Abs: of control  Abs: of sample

%Inhibition  100%
Abs: of control

The effective concentration required to scavenge 50% of H2O2 in the system (EC50 value) was
calculated from either linear or logarithmic dose response curves plotted between % Inhibition of
hydrogen peroxide versus concentration of test samples/standards. EC50 values were presented as
mean  standard deviation (Mean  SD) of six independent experiments. Students t-test was used to
compare mean EC50 values of standard antioxidants/plant extracts and p value <0.05 was considered
as signicant. Coefcient of variance (CV %) was computed for the EC50 values obtained for plant
extracts/standard antioxidants. All regression and statistical analyses were performed using Microsoft
Excel software.
In this study, various plant extracts and standard antioxidants were investigated for their hydrogen
peroxide scavenging ability utilizing the developed method by comparing the EC50 (half maximal
effective concentration) values obtained from the corresponding dose response curves via linear or
 288 C.D. Fernando, P. Soysa / MethodsX 2 (2015) 283291

Fig. 3. The dose response curves for percentage inhibition (% I) of hydrogen peroxide by L-ascorbic acid (A), Gallic acid (B), tannic
acid (C) standard antioxidants, A. ceylanica (D), E. quinquangulare (E) decoctions, S. parvifolia aqueous extract (F) M. cerviana
decoction (G) and C. sinensis infusion (H). The results are presented as mean + SD of six independent experiments. Correlation
coefcient values (R2) exceeded 0.95.
 C.D. Fernando, P. Soysa / MethodsX 2 (2015) 283291 289

Table 2
The EC50 values, coefcient of variance (CV %), regression equations and correlation coefcients (R2 values) obtained for dose
response curves of various plant extracts and reference standard antioxidants using the developed H2O2 scavenging activity
test.

Sample (n = 6) EC50 value (mg/ml) CV % Regression equation R2 value

mean  SD
A. ceylanica (Yakinaran) 388.11  4.11 1.1 y = 14.75 ln(x)  37.97 0.994
E. quinquangulare (Heen kokmota) 381.98  1.83 0.5 y = 0.130x  0.689 0.996
S. parvifolia (Heen badulla) 156.25  2.85 1.8 y = 35.67 ln(x)  130.1 0.956
M. cerviana (Pathpadagam) 1480.3  43.1 2.9 y = 0.026x + 12.40 0.990
C. sinensis (black tea) 91.96  2.51 2.7 y = 33.88 ln(x)  102.0 0.995
L-Ascorbic acid 10.0  0.14 1.4 y = 10.65x  38.68 0.999
Gallic acid 7.82  0.19 2.4 y = 8.094x  13.35 0.992
Tannic acid 8.17  0.10 1.2 y = 40.33 ln(x)  35.68 0.992

EC50 = half maximal effective concentration, SD = standard deviation, y = percentage inhibition (% I) of H2O2,x = concentration
(mg/ml), CV % = coefcient of variance %, R2 = correlation coefcient.

logarithmic regression analyses (Fig. 3AH). The amount of chromogen formed in the reaction
between H2O2, phenol and 4-aminoantipyrine (catalyzed by HRP) decreased in a dose dependant
manner of the plant extracts/standard antioxidants due to their scavenging ability of H2O2 molecules.
C. sinensis black tea infusion had the highest ability to scavenge H2O2 molecules followed by
S. parvifolia. There was no signicant difference in H2O2 scavenging ability between A. ceylanica and
E. quinquangulare (p > 0.05) but both of these extracts had lesser H2O2 scavenging ability than
S. parvifolia. M. cerviana had the least ability to scavenge hydrogen peroxide molecules. However, the
scavenging ability of hydrogen peroxide was superior in the standard antioxidants (i.e., L-ascorbic acid,
gallic acid and tannic acid) than all the plant extracts studied. H2O2 scavenging ability of L-ascorbic
acid was signicantly lower (p < 0.001) than gallic acid and tannic acid. There was no signicant
difference in H2O2 scavenging ability observed between gallic acid and tannic acid (p > 0.05).
In addition, we have conducted most widely used antioxidant assay i.e.; DPPH test [10] for some of
the above mentioned plant extracts and antioxidants. With respect to this assay, the antioxidant
potential for these substances varied according to L-ascorbic acid > C. sinensis > S. parvifolia > A.
ceylanica > M. cerviana [1013]. Similar pattern of variation in the antioxidant potential of the same
substances was observed in the method developed for the determination of H2O2 scavenging activity.
For the current method for analysis of H2O2 scavenging activity, coefcient of variance (CV%) for the
EC50 values obtained were less than 3.0% for all the plant extracts and standard antioxidants studied.
The correlation coefcient values (R2) of the dose response curves were greater than 0.95 (Table 2).
When considering other methods used for the determination of H2O2 scavenging activity, the
classical UV-method is widely used where the decrement of H2O2 in an incubation system containing
H2O2 and the scavenger is measured at 230 nm. According to this method described by Ruch et al. [6],
we experienced uctuations in absorbance. This encountered with less reproducible and reliable
results. The interference from secondary metabolites present in plants which absorb in UV region
affects the results [7]. The present method involves background correction for endogenous interfering
substances which improves the reproducibility. Czochra and Widnska [14] have developed
uorescence spectroscopic method using homovanillic acid (4-hydroxy-3-methoxyphenylacetic
acid) and peroxidase for the determination of H2O2 scavenging activity of plant extracts and standard
antioxidants. Although it is a highly selective and sensitive method, uorescence generated can be lost
by quenching, resonance energy transfer and inner lter effect in the presence of various endogenous
phytochemicals apart from the fact that uorescence spectrometry being a costly method [15]. UVvis
spectrophotometers are widely used in most of the laboratories in low-income countries, and the
current method can be used without additional burden. Zhang [16], has estimated hydrogen peroxide
scavenging activity of plant extracts by replacement titration method. This method described based on
iodide oxidation by hydrogen peroxide is a time consuming macro method where a titration is
involved and may not be feasible with higher number of analytes [17]. Many different systems too
have been described and are commercially available for quantication of H2O2 in experimental
290 C.D. Fernando, P. Soysa / MethodsX 2 (2015) 283291

systems, for instance, chemiluminescent, uorogenic substrates like Amplex Red [18], chromogenic
substrates like tetramethylbenzidine (TMB) [19] and 2,20 -azino-bis(3-ethylbenzothiazoline-6-
sulphonicacid) (ABTS) [20]. The reaction of peroxidase catalyzed conversion of H2O2, phenol and
4-aminoantipyrine to a chromogenic substance is utilized in many commercially available assay kits
designed for bio-analytical tests which include determination of blood glucose [21] and cholesterol
[22]. However this reaction is not involved so far for the determination of H2O2 scavenging activity of
various antioxidants. Therefore use of the same reagents for determination of H2O2 scavenging activity
gives a dual purpose for the reagents which in turn reduces costs for the purchase of special chemicals
like Amplex red, TMB and ABTS.
In conclusion, the colorimetric method developed and optimized in the current study for the
quantication of H2O2 scavenging activity of standard antioxidants as well as natural antioxidants
present in plant extracts is less expensive, precise, rapid and yields reproducible results. Therefore this
method is ideal for routine laboratory analyses.

Additional information

During aerobic metabolism as well as in the process of drug biotransformation, reactive oxygen
species (ROS) are produced as by-products. These include radicals such as superoxide anion (O2
),
hydroxyl radical (HO
), alkoxyl radical (RO
), peroxyl radical (ROO
) and non radicals such as hydrogen
peroxide (H2O2) and singlet oxygen (1O2) [23]. ROS can cause lipid oxidation, protein oxidation, DNA
strand breaks, and modulation of gene expression. Experimental evidences show that these ROS are
involved in liver diseases and also lead to atherosclerosis, cancer, stroke, asthma, arthritis and other
age related diseases [24]. In order to combat ROS, living organisms have developed defense
mechanisms consisting of variety of antioxidant enzymes such as superoxide dismutase, catalase,
glutathione peroxidase, glutathione reductase [25] as well as non-enzymatic antioxidants such as
ascorbic acid, a-tocopherol, b-carotene, avonoids and many phenolic compounds [26]. Antioxidants
are compounds that when present in low concentration in relation to the oxidant prevent or delay the
oxidation of a particular oxidizable-substrate [27]. Since natural antioxidants are capable of
scavenging various ROS, many methods have been developed for the estimation of these properties.
Hydrogen peroxide can be formed in vivo by various oxidizing enzymes such as superoxide
dismutase. It can permeate through biological membranes slowly oxidizing number of compounds.
Hydrogen peroxide is used in the respiratory burst of activated phagocytes [28]. Although hydrogen
peroxide itself is not very reactive [29], it can generate the highly reactive hydroxyl radical (HO
)
through the Fenton reaction [30] and is found to be main reason for toxicity associated with hydrogen
peroxide. Hydrogen peroxide can deactivate enzymes involved in cellular energy production such as
glyceraldehyde-3-phosphate dehydrogenase found in glycolytic pathway [31] as well as aconitase and
a-ketoglutarate dehydrogenase found in Krebs cycle [32] by oxidation of essential thiol (SH) groups.
Therefore, scavenging of hydrogen peroxide is considered as an important feature of antioxidants [33].
Accepting electrons in the presence of electron donors, hydrogen peroxide is decomposed into water
[34]. Hydrogen peroxide scavenging activity especially of phenolic compounds is assigned to their
electron-donating ability [35].

Acknowledgements

Freeze-dried samples of S. parvifolia and M. cerviana were donated by Ms. D.D.P. Jayarathna and Dr.
D.N. Jayasinghe. We acknowledge nancial assistance by Department of Biochemistry & Molecular
Biology, Faculty of Medicine, University of Colombo and National Science Foundation, Sri Lanka.
Authors like to thank Ms. Sudeepa Sugathadasa and Ms. Pushpa Jeewandara, Department of Botany,
Bandaranayake Memorial Ayurveda Research Institute, Nawinna, Colombo, Sri Lanka, for the
identication of the plant material used in this study. The technical assistance offered by Mr. Thisira
Andrahennadi, Mr. Jayantha Weerasinghe, Mr. Saman Kolombage and Ms. Nilusha Rajapakse,
Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Colombo, is
gratefully acknowledged. MethodsX thanks the reviewers of this article for taking the time to provide
valuable feedback.
 C.D. Fernando, P. Soysa / MethodsX 2 (2015) 283291 291

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