Spectral analysis allows using the DPPH* UV–Vis assay to estimate antioxidant activity of colored compounds

Article abstract of DOI:10.1007/s11696-020-01110-8

Abstract: An accurate measurement of antiradical activity to samples, such as herbal extract and natural or synthetic pure compounds, is essential to estimate its scope as antioxidant, for example, in functional foods. The 2,2-diphenyl-1-picrylhydrazyl (DPPH*) UV–Vis assay is the most widely used method for this aim. Nevertheless, it can drive to erroneous results if the spectroscopic properties of each substance, in the reaction medium, are not considered. To overcome this limitation, we present a new methodology based on spectral analysis which, instead of reading at fixed wavelength, uses the UV–Vis spectra of selected compounds, as well as simple algebraic operations and fitting by residual sum of squares. The proposed methodology was assayed successfully to determine the antiradical activity of three compounds: naringin, which does not present absorbance in the absorption band of DPPH*, naringin-Cu(II) complex, with moderate absorbance at this range, and naringin-Fe(III) complex, which shows intense absorption at the studied range. The proposed methodology is simple with a broad scope, since it allows to study the free radical scavenging activity on samples with high color that could not be analyzed, by UV–Vis spectrophotometry, up to the moment. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11696-020-01110-8

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https://doi.org/10.1007/s11696-020-01110-8
ORIGINAL PAPER
Spectral analysis allows using the DPPH* UV–Vis assay to estimate
antioxidant activity of colored compounds
Gustavo Celiz1,2 · Melisa Renfge2 · Mariela Finetti2
Received: 1 November 2019 / Accepted: 18 February 2020
© Institute of Chemistry, Slovak Academy of Sciences 2020
Abstract
An accurate measurement of antiradical activity to samples, such as herbal extract and natural or synthetic pure compounds,
is essential to estimate its scope as antioxidant, for example, in functional foods. The 2,2-diphenyl-1-picrylhydrazyl (DPPH*)
UV–Vis assay is the most widely used method for this aim. Nevertheless, it can drive to erroneous results if the spectroscopic
properties of each substance, in the reaction medium, are not considered. To overcome this limitation, we present a new
methodology based on spectral analysis which, instead of reading at fxed wavelength, uses the UV–Vis spectra of selected
compounds, as well as simple algebraic operations and ftting by residual sum of squares. The proposed methodology was
assayed successfully to determine the antiradical activity of three compounds: naringin, which does not present absorbance
in the absorption band of DPPH*, naringin-Cu(II) complex, with moderate absorbance at this range, and naringin-Fe(III)
complex, which shows intense absorption at the studied range. The proposed methodology is simple with a broad scope,
since it allows to study the free radical scavenging activity on samples with high color that could not be analyzed, by UV–Vis
spectrophotometry, up to the moment.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11696-020-01110-8) contains
supplementary material, which is available to authorized users.
* Gustavo Celiz
gceliz@unsa.edu.ar
1
Instituto de Investigaciones para la Industria Química
(INIQUI, CONICET), Universidad Nacional de Salta,
Avenida Bolivia 5150, A4408FVY Salta, Argentina
2
Facultad de Ciencias Exactas, Universidad Nacional de Salta,
Avenida Bolivia 5150, A4408FVY Salta, Argentina
Vol.:(0123456789)
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Graphic abstract
Keywords DPPH* assay · Free radical scavenging activity · Antioxidant activity · Naringin · Citrus favonoids · Metal
favonoid complex
rich in antioxidants so as to minimize the harmful efects
of the free radicals in the organism. To address this issue
properly, it is essential to count with reliable methodolo-
gies to compare the efectiveness of diferent substances
in reducing the generation of radicals or to increase the
passivation of the previously formed free radicals.
Introduction
Free radicals of oxygen and nitrogen species (ROS and
RNS, respectively) are present in the atmosphere and in
living organisms, since they are generated from stable
molecules by solar radiation and by respiration. Unpaired
electrons in free radicals give them a great reactivity,
which may cause benefcial and harmful efects in liv-
ing systems. At low concentrations, the benefcial efects
involve responses in several cellular signaling systems
such as defense against infectious agents (Pham-Huy et al.
2008). Conversely, at high level they may damage lipids,
proteins and nucleic acids and thus the cell structures
(Kedare and Singh 2011). High levels of free radicals are
diminished by the antioxidant action of enzymatic systems
or by antioxidant substances; if they are not, they can lead
to the development of diseases such as arteriosclerosis,
arthritis, Parkinson’s disease, heart disease and cancer
(Klein and Ackerman 2003). It is necessary to consume
food, medicines, nutraceuticals or dietary supplements
The 2,2-diphenyl-1-picrylhydrazyl (DPPH*) assay is the
most widespread method to quantify the radical scavenging
activity of pure substances, mixtures or extracts. By search-
ing for “DPPH” just in www.sciencedirect.com, it can be
verifed that since 1995 up to the present there was an expo-
nential increase in published papers concerning the DPPH*
assay (Supplementary Fig. 1). It was used to determine anti-
oxidant activities of pure substances or mixtures; to study
the reaction itself in theoretical and/or experimental form;
to develop new methodologies; and to fnd it new uses, such
as following the loss of antioxidant substances in various
matrices after drying them (Branisa et al. 2017).
The DPPH* free radical, in alcoholic solution, has a deep
violet color resulting from the molecular planarity. When
antiradicals with phenolic group are present in the medium,
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/
the main reaction is the transfer of a hydrogen atom from an
–OH group to the nitrogen atom that supports the unpaired
electron (Fig. 1). There are others mechanisms through
an antioxidant can reacts with the DPPH* radical, and the
extention of each one depends of the antioxidant molecule
and the medium in which the reaction is carried out (Foti
2015). For example, a polyhydroxylated favonoid such
as quercetin in methanol/water reacts through a complex
mechanism with secondary reactions that ends up obeying
a kinetics of a fractional order with respect to the antioxi-
dant (Foti et al. 2011). Despite the mechanism by which the
reaction is carried out, it is caused by the loss of DPPH* pla-
narity and the consequent disappearance of its violet color.
Therefore, the consumption of the radical can be followed
spectrophotometrically by measuring the absorbance of the
remnant DPPH* (λmax around 517–520 nm). The diferent
procedures to determine the antiradical activity measure the
initial DPPH* concentration and the remaining DPPH* in
the reaction medium after an incubation period (% DPPHR).
If Eq. 1 is applied, the percentage of remaining DPPH* can
be determined.
At
[DPPH ∗]
ꢀDPPH∗
b
b
A
.100 = t .100
A0
%DPPHR =
t .100 =
/
A0
[DPPH ∗]0
ꢀDPPH∗
(1)
In Eq. 1, At is the absorbance of the sample after incuba-
tion, A0 is the absorbance of the control with solvent instead
of antioxidant at the same incubation time, ε is the molar
absorptivity, and b is the optical path length. This equa-
tion does not take into account the spectroscopic properties
of all the compounds in the system, and thus, it may yield
incorrect results, since the reduced analog (DPPH-H), the
antioxidant and the product of the antioxidant might have
non-negligible absorptivities and therefore absorb part of
the radiation. The limitation of the traditional methodology,
which uses reading at fxed wavelength, was clearly demon-
strated in a recent publication (Olszowy and Dawidowicz
2018). Diferent methodological alternatives have been pro-
posed to overcome this interference. However, most of these
alternatives require specialized and expensive instruments
such as HPLC with DAD and/or MS/MS detectors (Kedare
Fig. 1 Structures of a
2,2-diphenyl-1-picrylhydrazyl
(DPPH*), b its reduced analog
(DPPH-H), c naringin and d
naringin complexes (M+n can be
Cu+2 or Fe+3
)
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and Singh 2011) or electron paramagnetic resonance (EPR)
spectroscopy (Yeo and Shahidi 2019).
Germany, respectively). All other reagents were of analytical
grade.
In this work, we propose a variant of the DPPH* assay
that corrects this underestimation, based on UV–Vis spectral
analysis. The method involves minimizing the residual sum
of squares using software such as Excel datasheet or simi-
lar. The developed methodology was applied to measure the
antioxidant activity of three compounds whose spectral fea-
tures present diferent degrees of complexity: naringin that
does not present absorbance at DPPH* band wavelength, and
two naringin complexes that show absorption at this range:
naringin-Cu(II) with moderate absorbance and naringin-
Fe(III) with intense absorbance.
Experimental for the proposed DPPH* assay
The foundation of the presented method comes from Brand-
Williams et al. (1995). An DPPH* stock solution was prepared
by dissolving the radical in methanol at a concentration of
6.3×10−5 M. A methanol solution of reduced DPPH* (DPPH-
H) at the same concentration (6.3×10−5 M) was obtained from
the previous one by the addition of a small amount of BHA
which does not absorb at the spectral range studied. The anti-
oxidants tested were dissolved at a concentration of 20 mM in
dimethyl sulfoxide (DMSO).
The goal of the experimental methodology and the data
processing presented is to determine the antiradical activity
of samples that present high absorbance in the same range
of the DPPH* absorption band.
Absorbance spectra were obtained from the individual
species dissolved in DMSO/methanol (1:19, v/v): DPPH*;
DPPH-H; and antioxidants (from 0.025 to 1.00 mM). From
these spectra, the molar absorptions for each compound were
determined.
The reaction time needed to reach a state where the concen-
tration does not change against time (steady state) was deter-
mined as follows: solutions at a concentration of 1 mM of the
antioxidants were prepared in DMSO. Then 1900 μL of the
DPPH* solution was mixed with 100 μL of antioxidant solu-
tion (fnal concentration 0.05 mM) in several capped tubes.
The solutions were kept under dark conditions at 20 °C. At
every hour, one tube was removed and its absorbance was read
at 515 nm.
Experimental
Reagents
Naringin was produced from agro-industrial grapefruit waste
by a simple and economic process (Geronazzo et al. 2000).
It briefy consists in grinding the solid discarded parts to an
average size of 2–4 mm and then performing an extraction
in a fxed bed column with distilled water at 80 °C. The
obtained extract is cooled, resulting in crystallization of the
favanone. The precipitate is fltered, washed and, fnally,
dried at 50 °C. This naringin is re-crystallized in water. The
naringin obtained had a purity by HPLC (UV detection at
280 nm) over 98%.
To determine the antiradical activity of the tested com-
pounds, 1900 μL of DPPH* solution and 100 μL of antioxidant
solution in DMSO at diferent concentrations (0.5–20 mM)
were mixed. When the steady state was reached (8 h), the spec-
tra of the solutions between 400 and 600 nm were recorded in
a spectrophotometer Cintra 101 (GBC, Australia).
Naringin-copper (II) complex was obtained by putting
25 mL of 25 mM copper (II) acetate in water (Anedra,
Argentina) in a glass and adding dropwise 50 mL of 25 mM
naringin in water/ ethanol (1:1, v/v) solution. The pH was
adjusted to 7, and the reaction medium was kept under stir-
ring at 60 °C for 1 h. Next, the reaction medium was cooled
at 4 °C overnight. The solid was recovered by centrifugation.
Then, it was washed with acetone to eliminate any naringin
rest (naringin is soluble and the complex not). Afterward, it
was dried in an oven at 50 °C. The complex obtained was a
light green color.
Calculation methodology
When DPPH* reacts with an antioxidant, one must consider
that DPPH*, DPPH-H, the antioxidant and the product (or
products) derived from the antioxidant can be present in the
medium. Therefore, it was suggested that each experimental
spectrum could be constructed by a linear combination of the
individual spectra of the species involved. This means that the
total absorbance (measured in unities of absorbance, UA) at
each wavelength will be given by the following expression:
Naringin-Fe(III) complex: It was obtained similarly to
the naringin-copper (II) complex replacing Cu (II) salt by
Fe(III) chloride (Biopack, Argentina). This complex did not
precipitate in the reaction medium, and thus, the solvent was
evaporated. The resulting solid was purifed as the Cu (II)
complex. The complex obtained was burgundy color.
Butylhydroxyanisole (BHA) and 2,2-diphenyl-1-picryl-
hydrazyl (DPPH*) were purchased from Sigma (USA and
A(UA) = ADPPH∗ + ADPPHH + Aantioxidant + Aproduct
(2)
where, according to the Lambert–Beer law,
ADPPH∗ = b ∗ ꢀDPPH∗ ∗ CDPPH∗ = b ∗ ꢀDPPH∗ ∗ x1 ∗ CD0 PPH∗
(2a)
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