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Journal of Food Composition and Analysis 24 (2011) 516–522

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Research Article

Rapid and effective evaluation of the antioxidant capacity of propolis extracts

using DPPH bleaching kinetic profiles, FT-IR and UV–vis spectroscopic data
Augustin Cătălin Moţ *, Radu Silaghi-Dumitrescu, Costel Sârbu
Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, 11 Arany Janos Str., 400028 Cluj-Napoca, Romania

A R T I C L E I N F O A B S T R A C T

Article history: An informative and effective measure of antioxidant capacity based on DPPH (2,2-diphenyl-1-
Received 28 September 2009 picrylhydrazyl) bleaching kinetic profiles has been developed using principal component analysis (PCA).
Received in revised form 9 August 2010 The activity score and a related parameter, called quercetin factor (QF), were used to estimate antioxidant
Accepted 12 November 2010
capacity for 39 propolis extracts based on the first principal component (which explains 98% of the total
Available online 8 December 2010
variance). Determination of the QF parameter requires less time and reagents than previous DPPH-based
antioxidant capacity parameters, but does require additional equipment. Additionally, UV–vis and FT-IR
Keywords:
spectroscopic features of propolis extracts have been identified, which have been correlated to
Propolis extract
Antioxidant capacity
antioxidant capacity, and offer a spectroscopic and reagent-less rapid evaluation method of the
DPPH kinetic profile antioxidant activity of biological samples. Together the QF scores based on DPPH bleaching and FT-IR and
Principal component analysis UV–vis spectra are analyzed in terms of antioxidant capacity, floral origin, and geographic location.
FT-IR spectroscopy ß 2010 Elsevier Inc. All rights reserved.
UV–vis spectroscopy
Floral origin
Food analysis
Food composition

1. Introduction consuming since several trials are required at different extract

Propolis is a resinous material collected by bees from the buds of
conifers, poplar trees and other botanical sources. Propolis is well
known for its antibacterial, antifungal, antitrypanosomal, antimi-
crobial (Katircioglu and Mercan, 2006; Kartal et al., 2003; Prytzyk
et al., 2003), antioxidant (Jasprica et al., 2007; Sun et al., 2000), anti-
inflammatory (Burdock, 1998; Miyataka et al., 1997), and anti-
hepatotoxic (Banskota et al., 2001) activities. Propolis is used in a
number of commercial dietary supplements, as a food preservative
(Han and Park, 1995; Tosi et al., 2007), and as a germicide and
insecticide (Mizuno, 1989). The chemical composition and biological
activities of propolis depend mainly upon the local flora (Bankova,
2005a; Markham et al., 1996; Pena, 2008; Salatino et al., 2005), the
geographic region, and the climate. Thus, development of analytical
methods to evaluate the antioxidant capacity and to discriminate
the floral origin of propolis is necessary (Bankova, 2005b).
There are numerous methods for determining the antioxidant
capacity of soluble natural extracts (Ayse et al., 2009; Thaipong et al.,
2006) as well as for insoluble food components (Serpen et al., 2007).
Measurements of the radical scavenging activity (EC50, TEC50) values
and related parameters, such as antiradical efficiency (AE), and
radical scavenging efficiency (RSE) (Villano et al., 2007) can be time
* Corresponding author. Tel.: +40 264 593877; fax: +40 264 590818.
E-mail address: augustinmot@chem.ubbcluj.ro (A.C. Moţ).
concentrations. The DPPH radical scavenging capacity assay is a
commonly used assay to evaluate antioxidant capacity of natural
extracts. This simple assay monitors the percentage of the DPPH
remaining after a given time (Brand-Williams et al., 1995). However,
the reaction kinetics between DPPH and a given antioxidant are non-
linear with respect to DPPH concentration, which affects the EC50
accuracy (Ayse et al., 2009). Secondly, this assay is sensitive to light,
oxygen, and impurities (Ozcelik et al., 2003; Apak et al., 2007).
Furthermore, the rate-limiting step is a proton transfer step, which is
dependent on the solvent system (Huang et al., 2005), and influenced
by the ionization equilibrium of phenol and phenolate compounds in
solution (Ayse et al., 2009; MacDonald-Wicks et al., 2006).
Calculation of the DPPH concentration remaining at a given reaction
time, as performed in the traditional DPPH radical scavenging
capacity assay, is indeed informative of the antioxidant capacity of
that sample, but does not take into account the whole kinetic profile.
Herein, we discuss a new antioxidant capacity score and related
quercetin factor (QF), which can be obtained by applying PCA to the
antioxidant data matrix resulting from the whole DPPH bleaching
kinetic profiles. Since the chemical composition of a natural extract
determines its antioxidant capacity, it is expected that related
spectroscopic data in the infrared (FT-IR) and UV–vis regions would
carry useful information concerning the antioxidant capacity. FT-IR
was successfully employed to determine the antioxidant capacity of
fruit extracts (Lam et al., 2005). However, to our knowledge, there

0889-1575/$ – see front matter ß 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2010.11.006
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A.C. Moţ et al. / Journal of Food Composition and Analysis 24 (2011) 516–522 517

are no literature precedents established as markers of antioxidant
capacity for propolis extracts or in general for botanical extracts. In
this study, absorbance at specific wavelengths (for UV–vis) and wave
numbers (for FT-IR) are found to be convenient spectroscopic tools
that can be used as a rapid screen for antioxidant capacity of natural
extracts. Moreover, using the DPPH kinetic profiles along with the
UV–vis and FT-IR spectroscopic data, a connection can be drawn
between the antioxidant capacity of a sample, its floral origin, and
the geographic region where it was collected.
2. Materials and methods
2.1. Reagents and instrumentation
UV–vis spectra and the absorption time-courses for DPPH
bleaching were recorded on a UV–vis spectrophotometer (Agilent
8453, Waldbronn, Germany) equipped with a diode array detector.
The IR spectra were recorded using a Bruker Vector 22 FT-IR
spectrophotometer equipped with a full-sized sample compart-
ment and a kinematic base plate. The spectroscopic range analyzed
for each sample was 4000–400 cm1 and generally 20 scans were
collected per sample. The ethanol (Reactivul, Bucharest, Romania)
and DPPH (Alfa-Aesar, Karlsruhe, Germany) used within this study
were analytical grade.
2.2. Sampling and sample preparation
A total of 39 propolis samples of different floral origins were
collected from several geographical regions of Romania. These
samples were collected by professional beekeepers with assistance
from a specialized chemist. Freshly collected samples were frozen
and stored at 20 8C until further use. Specific details on the origin,
color, and texture of the propolis samples are listed in Table 1.
Frozen propolis samples were ground to a fine powder using a
mortar and pestle. Then 95% ethanol was added to this
homogenate, to a final concentration of 166 mg solid sample/
mL. This suspension was then ultrasonicated for 1.5 h and then
incubated anaerobically for 48 h at 37 8C in a shaker at 200 rpm.
These samples were then spun at 1600 rpm for 2 h in a centrifuge.
The clear supernatant was collected and used as described below.
extract samples which were diluted 100-times in absolute
ethanol. Each UV–vis spectrum was recorded between 200 and
1000 nm.
A stock solution of 3.8 mM DPPH (2,2-diphenyl-1-picrylhy-
drazyl) in ethanol was prepared for each sample. Twenty
microliters of this stock solution was added into 950 mL ethanol
found in a quartz UV–vis cuvette resulting in a DPPH solution with
absorbance value 0.936  0.017 at 520 nm (molar absorptivity
12,350 cm1 M1 (Yordanov and Christova, 1997)). After approxi-
mately 150 s, 30 mL of 100-times diluted ethanol extract of propolis
was added to the DPPH solution. A reference sample was also
generated, which contains all the components described above except
the 30 mL ethanol extract was replaced by 30 mL of ethanol. The
bleaching of DPPH by propolis and other reference antioxidants was
monitored spectrophotometrically at 520 nm for 400 s.
2.4. Multivariate data analysis
Multivariate data analysis was performed using PCA incorpo-
rated in Statistica software, version 7.0 (StatSoft, 2010). The main
purpose of PCA is to conveniently represent the location of the
objects (samples) in a reduced coordinate system where, instead of
m-axes (corresponding to m variables), only p (p < m) are used to
describe the data set with the maximum possible information
(Mackiewicz and Ratajczak, 1993; Moţ et al., 2010; Sârbu and Pop,
2005). The new variables are called principal components, and
represented by a linear combination of the n raw variables (in our
case absorbances). The most important results obtained are
loadings and scores. Loadings indicate the relative importance of
the corresponding raw variable in the principal component, and
scores represent the new coordinates corresponding to each
principal component for every sample. Moreover, it may well turn
out that two or three principal components provide a good
summary of the complex propolis matrix. Scores plots are very
useful as a display tool for examining the relationships between
objects, looking for trends, and sorting out outliers.
3. Results and discussions
3.1. DPPH kinetics

2.3. Procedures Two processes are occurring in the kinetic profiles associated
with DPPH bleaching, a fast process in the first seconds followed by
IR spectra were collected on the propolis extract between a significantly slower one at longer reaction times. Attempts to fit
4000 and 400 cm1. UV–vis spectra were collected on propolis the experimental data to a single-exponential decay function have

Table 1
Floral, geographical origins and description of the studied propolis samples.

Sample No. of Origin place of samples Origin Flora Color and

codes samples (place, county) coordinates type texture

T1 T11 4 Nadaselu, CJ 468500 N/238270 E Meadow, low content mixture Brown, rigid, powder
T12 3 Sarmasu, MS 46845N/248120 E of deciduous forest Brown, rigid, powder
T13 4 Gledin, BN 468570 N/248330 E Yellowish brown, waxy
T2 T21 1 Pestera, CT 448110 N/288070 E Grassy meadow Brownish yellow, waxy
T22 2 Livada, AR 468130 N/218230 E Yellowish brown, waxy
T23 1 Sanleani, AR 468120 N/218230 E Dark greenish brown, rigid,
waxy
T24 2 Carei, SM 478400 N/228250 E Brownish yellow, rigid, waxy
T3 T31 2 Cristian, SB 458480 N/248020 E Meadow, high content of mixture Brown, waxy
T32 4 Orlat, SB 458450 N/238570 E of deciduous forests Brown, waxy
T4 T41 2 Brebi, SJ 478130 N/238100 E Mixture of deciduous and Reddish brown, rigid
T42 2 Romuli, BN 478320 N/248240 E resinous forests Reddish brown, rigid powder
T43 2 Fagaras, BN 458470 N/248580 E brown
T5 T51 7 Romuli, BN 478320 N/248250 E Mixture of deciduous forests Light reddish brown, rigid,
waxy
T52 2 Gura Raului, SB 458430 N/238580 E Light brown, rigid, waxy
Pastorala 1 Pastoral Pastoral Very complex Dark brown, rigid, waxy
a
Pastoral – refers to the fact that the beehive was moved to various places during a season.
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518 A.C. Moţ et al. / Journal of Food Composition and Analysis 24 (2011) 516–522

Fig. 1. Kinetic profiles of DPPH consumption by two ethanol extracts of propolis Fig. 2. Linear domain of the normalized PCA scores vs. quercetin concentration for
samples with two different antioxidant capacities,sample1-T21 and sample2-T42 quercetin in DPPH bleaching assays.
(see Table 1). The double-exponential model (R2 = 0.9997, F = 319,146; R2 = 0.9996,
F = 165,476) is a better model with respect to the mono-exponential model
(R2 = 0.98825, F = 7953; R2 = 0.9837, F = 6466). Relative Abs refers to Abst/Abs0,
where Abs0 is the initial absorbance (at time zero) at 520 nm of the DPPH solution quercetin samples corresponding to the first component are
and the Abst is the absorbance of the DPPH solution at a reaction time t. linearly correlated with quercetin concentration (R = 0.9985)
between 0.1 and 11 mg/L (Fig. 2).
For propolis samples, a comparison between the PCA-derived
scores and the more traditional %DPPH parameter (derived from
failed, while a double-exponential function (Eq. (1)) fits to the the DPPH concentration at a single time point) are informative in
bleaching data (Fig. 1). This suggests that two major processes are terms of efficiently establishing antioxidant capacity. A good
occurring, which can be characterized by the two kinetic constants correlation between PCA-derived scores and the %DPPH parameter
a1 and a2 in Eq. (1). These two processes are presumably due to the was found as expected (data not shown). However, it was also
two main groups of antioxidants present in the samples that can be found that samples with identical values for %DPPH250 s (% of DPPH
classified as either ‘‘fast-acting’’ or ‘‘slow-acting’’ antioxidants consumed in the first 250 s) can have different PCA-derived score
(Espin et al., 2000; Schauss et al., 2006). The fast-acting values. This illustrates how the PCA-derived antioxidant scoring
antioxidants are responsible for the DPPH consumption at short system offers more information on antioxidant behavior than the
reaction times (30–50 s) while the slow-acting antioxidants traditional %DPPH250 s. Since the PCA-derived score values are
dominate at longer reaction times, where the kinetic profile generated from the analysis of a given set of samples, such analysis
becomes linear. When y0 is zero (González-Forero and Alvarez, would not allow for comparisons between specific samples
2005) A + B = 1, in this case A is the fraction of the fast-acting belonging to different data sets. Therefore, the DPPH bleaching
antioxidants while B indicates the fraction of the slow-acting kinetic profile of a pure antioxidant (quercetin) was chosen as
antioxidants. reference and the PCA-derived scores were converted into
‘‘quercetin equivalents’’, i.e., the quercetin concentration whose
   
t t score value is identical with the score of the tested sample
Abst ¼ y0 þ A exp þ B exp (1)
a1 a2 according to the calibration curve from Fig. 2. Working in the linear
part of the calibration curve, the quercetin equivalents in mg/L for
The fast-acting and slow-acting antioxidant are typically each sample were predicted, and a non-dimensional parameter as
considered to be two different types of molecules (Espin et al.,
2000; Schauss et al., 2006) with different antioxidant capacity.
However, they can also refer to two different antioxidant moieties
within the same molecule that have different radical scavenging
capacity (Bernardi et al., 2007). Either way, the analysis of the
entire DPPH bleaching kinetic profile brings useful information
about types of antioxidants, their relative proportions, as well as
measures the total antioxidant capacity of a sample. The
disadvantages associated with the traditional DPPH antioxidant
assay have led us to propose a new assay and antioxidant
parameter, which are based on the entire kinetic profiles of DPPH
bleaching rather than single point measurements.
The PCA was applied to the data collected on the 39 propolis
samples and eight quercetin standards (0.1–17 mg/L and 80
absorbances). It was found that the first principal component
explains 98.08% of total variance. Quercetin, a well-known
member of the polyphenol family of antioxidants, was chosen as
an antioxidant reference, which account for most of the antioxi-
dant properties of propolis (Moţ et al., 2009). Furthermore, the
DPPH kinetic profiles observed with quercetin references resemble Fig. 3. Correlation of the proposed QFs and the %DPPH250 s for the studied propolis
well those obtained with propolis samples. The scores for samples.
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A.C. Moţ et al. / Journal of Food Composition and Analysis 24 (2011) 516–522
Fig. 4. Averages of the QFs for the studied propolis samples, for number of samples
and codes see Table 1.
associated with the antioxidant capacity was generated. This
parameter, termed the quercetin factor (QF), defines the ratio
between quercetin equivalent in mg/L of the assayed propolis
sample and the corresponding propolis concentration in mg/L.
By plotting the QFs corresponding to the first principal
component vs. the %DPPH consumed at a given reaction time
for all propolis samples, a linear function with highly significant
correlation coefficient (R = 0.9852) is obtained (Fig. 3). For all the
studied samples the antioxidant capacity was calculated in
terms of QF (Fig. 4). Most of the samples from meadow regions
present higher antioxidant activities than those from forest
regions (Table 1).
Based on the kinetic curves of the DPPH bleaching assays,
some well known parameters were calculated for each propolis
sample. These parameters were %DPPH250 s, A250 s (area under
the kinetic curve for the first 250 s), Slope0% (the slope of the
tangent to the kinetic curve in the zero reaction time point),
Slope50% (the slope of the tangent to the kinetic curve in reaction
time point where 50% of the DPPH was consumed), and they
were highly correlated with the QF (Table 2, first row). It can be
observed that the parameter which takes into account the whole
kinetic curve (A250 s) has a higher correlation coefficient with the
QF than the traditional parameters based on DPPH concentration
at a single time point. Furthermore, the antioxidant activity is
mainly due to the fast-acting, rather than the slow-acting,
antioxidants according to the correlation coefficient (last two
columns from Table 2).
To conclude, the PCA-derived antioxidant scores and the
related QF parameter offers advantages over the classical
antioxidant assay. It offers more information associated with
the nature of the antioxidant. It also takes less time and uses less
reagents to evaluate the antioxidant capacity of propolis samples.
In addition, similar PCA-derived protocols can be applied to
antioxidant assays systems aimed at many other types of
biological extracts.
Table 2
Correlation coefficients between several calculated parameters of the kinetic profile of DPPH bleaching assays and some relevant IR and UV–vis absorbances.
Correl. coeff. (R) %DPPH250 s A250 s
QF 0.9852 0.9988
A1515 cm1 0.2380 0.2166
A1630 cm1 0.7520 0.7436
A290 nm 0.4645 0.4161
A353 nm 0.7691 0.7399
a
The factors from Eq. (1).
519
3.2. FT-IR spectra
Noticeable differences of the FT-IR spectra of propolis extracts
can be seen in the 1100–1800 cm1 region (Fig. 5). Prominent IR
absorption bands within this region and their corresponding
functional groups are listed in Table 3, which are consistent with IR
data collected on other extracts from natural products (Tarantilisa
et al., 2008; Wu et al., 2008). Clearly, the 1515 cm1 band is specific
to the propolis extracts. This feature is most likely due to stretching
and bending modes associated with the aromatic rings of the
polyphenols (Manthey, 2006). The 1168 cm1 band is also present
in all the collected spectra, but its intensity differs from sample to
sample.
The 1515 cm1, 1633 cm1 and 1683 cm1 bands correspond
to compounds specific to propolis extracts (Table 3). The
antioxidant capacity of the samples is well correlated at
1630 cm1 signal. The intensity of other bands have poor
correlation to the measured antioxidant activity, as exemplified
by the 1515 cm1 band (Table 2).
It is clear that the antioxidant activity does not only depend on
the amount of compounds in solution (Teixeira et al., 2008), but
rather on their chemical structures. Thus, the 1515 cm1 band
corresponds to an aromatic vibrational mode, but has little
relationship to the antioxidant behavior, while the 1630 cm1
band correlates to some antioxidant compounds (most likely a
flavonoid compound). Therefore, this IR band can be used as
parameter for measure of antioxidant capacity.
3.3. UV–vis spectra
Absorbances in UV–vis spectra of the extracts indicate the
degree to which the propolis samples contain inert material which
cannot be extracted into the solvent, since all the samples have the
same concentration in mg solid propolis/mL. The UV–vis spectra of
the ethanol extracts are similar to typical polyphenol spectra. The
primary feature of these spectra is a broad band centered around
280–330 nm (Fig. 6). The best correlation between antioxidant
capacity and absorbance of UV–visible spectra is found at 353 nm.
At other wavelengths, such as 290 nm, the correlation to
antioxidant activity is poor (Table 2). Generally samples with
both a 1630 cm1 band in the IR region and a 353 nm absorbance
band in the UV–vis region have antioxidant capacity (Fig. 6).
Together these two spectroscopic ‘‘hand-holds’’ can be effectively
used for rapid screening of the antioxidant capacity of ethanol
extracts of propolis and other biological samples.
3.4. PCA-based mapping
The score plot associated with the first two principal
components of the DPPH bleaching profiles shows a polarization
of samples into distinctive groups (i.e. the T1/T2 group, the T4/
T5 group, and a transitional T3 group (see Fig. 7c)), which are in
good agreement with the sample’s floral origin (Table 1). The
floral origin of each sample can be discriminated by the second
principal component. Most of the propolis samples collected
from meadow regions present higher antioxidant activity than
Slope0% Slope50% QF Aa Ba
0.8924 0.9637 1 0.9439 0.9592
0.3357 0.3003 0.2345 0.1158 0.1171
0.5754 0.5634 0.7375 0.6875 0.7181
0.2425 0.2720 0.4010 0.4243 0.4430
0.5540 0.5711 0.7285 0.6684 0.6912
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520 A.C. Moţ et al. / Journal of Food Composition and Analysis 24 (2011) 516–522

Fig. 5. FT-IR spectra of several propolis extracts. The main spectroscopic differences are within 1500–1800 cm1 region. The sample codes and the description of the samples
are found in Table 1.

Table 3 those collected from forest regions. The score plots resulting
Distinct bands in FT-IR spectra found in propolis extracts. from the PCA of the DPPH bleaching profiles, and the FT-IR and
UV–vis spectroscopic data, form a pattern with distinctive
Bands (cm1) Functional group Possible compounds
vibration modea subgroups (Fig. 7a and b). The grouping of samples obtained
using IR data tend to be more condensed, which suggests the
1160s n(C–O), d(C–OH) Lipid, alcohol groups
1450s d(C–H), n(aromatic) CH3, CH2, flavonoids, aromatic ring information provided by this technique is more reliable. The
1515 n(C5
5C) (aromatic) Flavonoids, aromatic ring data above suggest when FT-IR or UV–vis spectroscopic data
1630s n(C5
5O), n(C5
5C), das (N–H) Flavonoids, amino acids (Fig. 7a and b), and activity assay data are compiled, they can be
1690s n(C5
5O) Lipids, flavonoids used to identify both the geographic and botanical origins of
a
n – stretching; d – bending; as-asymmetrical. propolis samples.
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A.C. Moţ et al. / Journal of Food Composition and Analysis 24 (2011) 516–522 521

Fig. 6. UV–vis electronic spectra of some propolis extracts (a). The 353 nm absorbance correlates with the antioxidant capacity and with 1630 cm1 absorbance (R = 0.889,
p < 0.0001) (b).

Fig. 7. Grouping of the propolis samples according to their origin using the scores of the principal components obtained after the PCA was applied on the 240–410 nm UV–vis
spectroscopic range (a) and on the 1454–1780 cm1 IR spectroscopic range (b), DPPH kinetic profile (c).

4. Conclusions antioxidant capacity marker, since can be estimated to be 98%

A new, more informative and effective scale of antioxidant
capacity is obtained by applying PCA on DPPH (2,2-diphenyl-1-
picrylhydrazyl) bleaching kinetic profiles. This technique has been
developed and used to characterize several propolis extracts from
various regions of Romania. The activity score corresponding to the
first principal component was successfully assigned as an
of the sample by PCA. In order to obtain comparable antioxidant
activities, a non-dimensional parameter was generated which is
termed the quercetin factor (QF), which defines the ratio between
quercetin equivalent in mg/L of the assayed propolis sample and
the corresponding propolis concentration in mg/L. Further
application of this methodology to other botanical extracts will
confirm this new method for assessing antioxidant activity.
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522 A.C. Moţ et al. / Journal of Food Composition and Analysis 24 (2011) 516–522
Furthermore, the UV–vis and FT-IR spectra were also shown to bear
relevant information regarding antioxidant activity of the extracts
and can be used as a tool to predict both the floral origin and
geographic location in which samples are collected.
Acknowledgements
Funding from 41/1.10.2008 Scientific Research Scholarship
granted by Babes-Bolyai University (2008–2009) and the Ph.D.
scholarship, Contract No. POSDRU/88/1.5/S/60185 – ‘‘Innovative
doctoral studies in a Knowledge Based Society’’ (2009–2010) is
gratefully acknowledged by ACM. We would like to thank Dr
Joseph P. Emerson (Mississippi State University) for helpful
discussions.
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