Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 546–549

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Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy
journal homepage: www.elsevier.com/locate/saa

A comparison of antioxidative capacities of fruit juices, drinks and

nectars, as determined by EPR and UV–vis spectroscopies
Mariola Bartoszek ⁎, Justyna Polak
Department of Materials Chemistry and Chemical Technology, Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland

a r t i c l e i n f o a b s t r a c t

Article history: The differences in the Trolox Equivalent Antioxidant Capacity (TEAC) values at the same incubation time obtain-
Received 1 March 2015 ed by two different techniques: electron paramagnetic resonance (EPR) spectroscopy and ultraviolet visible (UV–
Received in revised form 13 August 2015 vis) spectroscopy, which use the same antioxidant-free radical reaction mechanism, were determined for fruit
Accepted 26 September 2015
juices, nectars and drinks. For this study, the stable free radical 1,1-Diphenyl-2-picryl-hydrazyl (DPPH•) was
Available online 28 September 2015
used. The antioxidant capacity was presented in Trolox Equivalents, e.g., μM trolox per 100 ml of sample.
Keywords:
All of the studied fruit juices, drinks and nectars showed antioxidative properties. Dependencies between TEAC
EPR spectroscopy values and the percent fruit content and sample color were observed for the studied beverages. It was found
UV–vis spectroscopy that EPR spectroscopy is the more adequate method for determining TEAC values for these kinds of samples.
Antioxidant capacity © 2015 Elsevier B.V. All rights reserved.
Fruit juices
Drinks
Nectars

1. Introduction
Fruit juices are a good source of many biologically active antioxidant
compounds, particularly ascorbic acid and phenols. Ascorbic acid is well
known for its antioxidant and anti-inflammatory activity, while pheno-
lic compounds are known for their anticancer and cardio-protective
properties, and their effect on age-related diseases [1]. Hence dietary
recommendations for healthy nutrition include the consumption of
fruit juices [2]. Citrus fruit juices consistently rank first among the
most consumed fruit juices in the US. On average, citrus fruit juice con-
sumption is 2.5 times greater than that of apple juice, the second most
popular juice [3]. The European Fruit Juice Association (AIJN) published
a market report concerning the European fruit and vegetable juice and
nectar market, which indicates that the most popular nectar and juice
flavor is orange (38.5%), then mixed (19.9%) and then apple (13.3%)
[4]. Orange juice is not only the most widely consumed juice but also
it's one of the most studied with respect to its antioxidative properties,
content and its beneficial health effects on consumers [1,3,5,6].
Studies of antioxidant properties of fruit juices are conducted with
the use of a variety of methods. These methods are basically classified
into two groups, depending on the reaction mechanism: methods
based on hydrogen atom transfer (HAT) and methods based on electron
⁎ Corresponding author.
E-mail address: mariola.bartoszek@us.edu.pl (M. Bartoszek).
transfer (ET) [6,7]. The majority of HAT-based assays apply a competi-
tive scheme, in which antioxidant and substrate compete for thermally
generated peroxyl radicals through the decomposition of azo-com-
pounds. ET-based assays measure the capacity of an antioxidant in re-
ducing an oxidant, which changes color when reduced. The degree of
color change is correlated with the sample's antioxidant concentrations.
One of the most popular methods is the method employing the stable,
2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), called the DPPH meth-
od. DPPH• is a stable radical and appears purple in color absorbing at
515 nm in ethanol solution. This assay is based on the principle that
DPPH•, on accepting a hydrogen (H) atom from the scavenger molecule
i.e. antioxidant, reduces to DPPH2, and the purple color changes to yel-
low with concomitant decrease in absorbance at 515 nm. This color
change is monitored spectrophotometrically in order to determine anti-
oxidant properties.
The antioxidant concentration necessary to decrease the initial
DPPH• concentration by 50% is used for the comparison of antioxidant
activities of different compounds. A refinement of this method is the
use of trolox as the standard and determining the total antioxidative po-
tential TEAC in the units of μmol trolox per 100 ml, allowing for the com-
parison of the obtained values with TEAC values obtained by other
methods. So far, the TEAC values obtained with the use of the same rad-
ical (DPPH•) determined by UV–vis spectroscopy and EPR spectroscopy
have not been compared. In the case of UV–vis spectroscopy, a decrease
in absorbance is observed, while in the case of EPR, the decrease in free
radical concentration is proportional to the radical quenching capacity

http://dx.doi.org/10.1016/j.saa.2015.09.022
1386-1425/© 2015 Elsevier B.V. All rights reserved.
 M. Bartoszek, J. Polak / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 546–549
of the tested antioxidant. Both methods are based on the reduction of
the DPPH• radical in reaction with an antioxidant (AH) species accord-
ing to the formula:
AH + DPPH• → A• + DPPH − H
DPPH• + A• → DPPH − A.
Earlier studies have shown that TEAC values obtained with the UV–
vis method with the use of DPPH• depend on the type of solvent used for
antioxidant dissolution, water content in the measuring system, metal
and hydrogen ion concentrations, the concentration of DPPH•, incuba-
tion time (5 min–1 h), reaction solvent and the pH of the reaction mix-
ture [8].
The goal of our study is to compare the values of TEAC obtained for
the same samples with two different techniques at the same incubation
time: EPR spectroscopy and UV–vis, which make use of the same antiox-
idant-radical reaction mechanism.
2. Materials and methods
2.1. Samples and chemicals
12 samples were purchased from local markets and 3 samples were
squeezed from fresh fruit. The juices differed in the type of fruit that was
used for their production, color, % fruit content and turbidity. The fol-
lowing beverages were included in the study: 6 juices, 5 drinks and 4
nectars; S. No. 1 orange juice (50% fruit by volume), S. No. 2 apple
juice (50% fruit by volume), S. No. 3 fresh grapefruit juice (100% fruit
by volume), S. No. 4 fresh orange juice (100% fruit by volume), S. No. 5
fresh citron juice (100% fruit by volume), S. No. 6 fresh raspberry juice
(100% fruit by volume), S. No. 7 blackcurrant drink (20% fruit by vol-
ume), S. No. 8 red grape drink (50% fruit by volume), S. No. 9 grape
drink (30% fruit by volume), S. No. 10 cherry drink (30% fruit by vol-
ume), S. No. 11 plum drink (30% fruit by volume), S. No. 12 apple nectar
(50% fruit by volume), S. No. 13 blackcurrant nectar (25% fruit by vol-
ume), S. No. 14 cherry nectar (25% fruit by volume), and S. No. 15
plum nectar (30% fruit by volume).
All the commercial juices, drinks and nectars were used as received
and kept refrigerated (2–8 °C). Fresh orange, grapefruit and lemon
juices were obtained by squeezing. Fresh juices were measured imme-
diately after preparation.
1,1-diphenyl-2-picrylhydrazyl (DPPH•) (Sigma-Aldrich, Poznań, Po-
land) was used as the source of free radicals. To quantify the antioxidant
activity of the tinctures, trolox (Acros Organics, Geel, Belgium) was
used. Trolox (molecular formula C14H18O4) is the water-soluble deriva-
tive of vitamin E. All other chemicals and solvents were of analytical
grade and used without further purification.
2.2. Determination of antioxidant capacity by the EPR spectroscopy
Antioxidant capacity was determined using the method described
previously [9]. Electron paramagnetic resonance spectra were obtained
with a Bruker EMX EPR spectrometer (Bruker-Biospin, Germany) oper-
ating at the X-band frequency at room temperature. The typical instru-
ment parameters were: central field, 3480 G; modulation amplitude,
2.0 G; time constant, 40.96; gain, 1 ∗ 104 G; microwave power,
20.12 mW.
The regression equation for the linear relationship between the per-
cent inhibition of EPR signal intensity and the mol number of trolox was
assessed as: y = 987.60x + 16.36, where: y is the inhibition [%] and x is
the volume of the sample [ml]. This equation was used to calculate the
antioxidant activity of the studied samples in μmol trolox per 100 ml
of the studied samples.
547
A typical reaction mixture contained 1 ml of 200 μmol/dm3 DPPH•
solution in ethanol together with 0.008 ml to 0.6 ml of sample, depend-
ing on the manifested antioxidant properties. For all samples, regression
equation of the linear relationship between the percent inhibition (%I)
of the EPR signal intensity and the volume of sample (V) was deter-
mined. Based on this equation the %I corresponding to 100 ml of the
studied sample was calculated. Then, from the standard curve obtained
for trolox, the antioxidant activity given in μmol trolox per 100 ml of
sample was defined. The data presented here are the result of three
trials.
2.3. Determination of antioxidant capacity by UV–vis spectroscopy
Antioxidant capacity was determined using the DPPH method [17].
The method was modified to use trolox as the standard. On increasing
the concentration of trolox, the intensity of the corresponding value of
absorbance decreased and consequently the percent inhibition (%I) in-
creased. The regression equation of the linear relationship of the percent
inhibition of absorbance to mol number of trolox was used to calculate
the antioxidation activity of the studied samples in units called TEAC,
e.g., μmol TE/100 ml of the studied sample. The regression equation
for the linear relationship between the percent inhibition of absorbance
and the mol number of trolox was assessed as: y = 876.55x − 2.7,
where: y is the inhibition [%] and x is the volume of the sample [ml].
This equation was used to calculate the antioxidant activity of the stud-
ied samples in μmol trolox per 100 ml of the studied samples.
The percent inhibition of the decrease in absorption at 515 nm calcu-
lated according to the following equation: % Inhibition = [(A0 − A)/
A0] ∗ 100%, where A0 is the absorbance of DPPH• (control sample), and
A is the absorbance of DPPH• with a sample.
A typical reaction mixture contained 3 ml of the 200 μM/l DPPH• so-
lution in ethanol together with 0.024 ml to 1.8 ml of sample of commer-
cially available juices, drinks and nectars. For all samples regression
equation of linear relationship of the percent inhibition (%I) of the ab-
sorbance to the volume of sample (V) was determined. On the basis of
this equation %I corresponding to 100 ml of the studied sample was cal-
culated. Then from the standard curve the antioxidant activity given in
TEAC values in μM trolox per 100 ml of sample was defined. The pre-
sented data are the means of three determinations.
UV–vis spectrophotometric measurements were performed at
515 nm using a Lambda Bio 40 spectrometer (Perkin Elmer, USA).
2.4. Statistical analysis
The presented data are the mean values whereas errors were calcu-
lated as standard deviation (SD). Correlation coefficients (r): TEACEPR
(Trolox equivalent antioxidant capacity obtained by EPR spectroscopy),
TEACUV–vis (Trolox equivalent antioxidant capacity obtained by UV–vis
spectroscopy) and fruit content were calculated by the Pearson test
with a level of significance of p = 0.05.
3. Results and discussion
The antioxidative capacity TEAC of fruit juices, nectars and drinks
that was determined with the use of DPPH• and by EPR spectroscopy
ranges from 41.37 to 412.33 μM/100 ml of sample. The same samples
as analyzed by UV–vis spectroscopy yield results from 26.43 to 382.78
μM/100 ml of sample (Table 1).
A linear relationship was observed between the TEAC values obtain-
ed by EPR and UV–vis spectroscopies, which can be expressed by the
equation y = 1.08x + 13.2 (r = 0.99), where y is the TEAC value obtain-
ed from EPR spectroscopy (TEACEPR), and x is the value obtained from
UV–vis spectroscopy (TEACUV–vis).
The slope of the resulting line is close to 1, indicating a constant dif-
ference between TEAC values obtained with these two methods. Both
methods make use of the same free radical (DPPH•) and have the
548 M. Bartoszek, J. Polak / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 546–549

Table 1 compounds (anthocyanins, carotenoids, etc.) in the samples or due to

Names of the samples and TEAC values obtained for the studied samples by EPR and UV– the appearance of secondary reaction products between the chromogen
vis spectroscopies.
and the samples being analyzed [12]. In the case of sample S11 a greater
Sample Fruit TEACEPR TEACUV–vis TEAC value was obtained with the UV–vis method as compared to that
[μM/100 ml] [μM/100 ml] obtained by the EPR method. This can be due to the turbidity of this
S1 Orange juice 412.33 ± 7.30 381.46 ± 13.21 sample. It is known that the UV–vis method can be easily affected by
S2 Apple juice 175.62 ± 4.08 158.06 ± 11.86 the color and turbidity of a sample [8,12,13]. In addition, it must be stat-
S3 Fresh grapefruit juice 263.67 ± 5.99 232.37 ± 8.55
ed that a large turbidity may even make it impossible to measure the
S4 Fresh orange juice 318.35 ± 6.04 282.92 ± 21.93
S5 Fresh citron juice 405.87 ± 7.83 382.78 ± 13.10 TEAC total antioxidative capacity (e.g., for sample S15 it was impossible
S6 Fresh raspberry juice 239.27 ± 4.34 198,42 ± 8.19 to determine the TEAC value using UV–vis spectroscopy). Due to the
S7 Blackcurrant drink 314.8 ± 3.38 250.01 ± 38.00 above, the EPR spectroscopy method seems to be the more adequate
S8 Red grape drink 239.34 ± 7.80 207.91 ± 71.28 one for determining antioxidative capacity using the DPPH• free radical.
S9 Grape drink 41.37 ± 4.18 26.43 ± 3.79
S10 Cherry drink 148,77 ± 10.11 108,27 ± 7.27
It is suited for studying turbid samples and samples of a color close to
S11 Plum drink 50,69 ± 2.71 78.31 ± 9.02 that of the free radical.
S12 Apple nectar 225.39 ± 39.90 188.22 ± 27.24 High TEAC values by both methods were obtained for samples S1, S3,
S13 Blackcurrant nectar 407.32 ± 14.51 357.59 ± 21.45 S4, and S5 (Table 1). All of these samples are citrus juices, which are the
S14 Cherry nectar 204.61 ± 21.23 162.69 ± 13.78
most popular juices among consumers [3]. The flavinoids, beta-caro-
S15 Plum nectar 53,2 ± 4.14 Not determined
tenes and large amounts of ascorbic acid that these juices contain are re-
Abbreviations: TEACEPR: trolox equivalent antioxidant capacity obtained by EPR sponsible for their antioxidative capacity. It must be stated that the
spectroscopy.
TEACUV–vis: trolox equivalent antioxidant capacity obtained by UV–vis spectroscopy.
vitamin C content in these types of products has a wide range, and it de-
Each value of TEACEPR and TEACUV–vis is presented as mean ± SD (n = 3). pends to a large degree on fruit type, ripeness, and losses due to process-
ing and storage [1,3,5,6,14].
Among the studied antioxidants, the blackcurrant nectar and
same reaction mechanism of the radical with the oxidant. However, EPR blackcurrant drink are worthy of attention (S7 and S13) (Table 1). It
spectroscopy is a method that is unique to free radicals, and other sub- must be stated that despite a low fruit content in the blackcurrant nectar
stances have no effect on the registered signal, i.e. it reflects the true an- and blackcurrant drink, their TEAC values are very high (Table 1). It is
tioxidative properties of the studied material. In the case of UV–vis known that blackcurrants have very high value of total antioxidative ca-
spectroscopy, the change in the absorbance of the DPPH free radical so- pacity, which amounts to 200.3 μmol TE/g dried fruit mass. This results
lution is measured before and after the reaction with the antioxidant, from high polyphenol, anthocyanin and vitamin C contents [15,16]. The
and therefore the antioxidative capacity determined with this method total anthocyanin (cyanidin-3-rutinoside + cyanidin-3-glucoside +
may contain some interference. Earlier studies of the correlation be- delphinidin-3-rutinoside + delphinidin-3-glucoside) content is very
tween the optical and paramagnetic properties of DPPH• solutions high in fresh fruits and amounts to 897.2 given as mg/100 g of fresh
show that the decrease of the absorption maximum in the UV–vis spec- weight. The four main pigments: delphinidin 3-O-glucoside,
trum does not always correlate with a decrease in the concentration of delphinidin 3-O-rutinoside, cyanidin-3-O-glucoside and cyanidin-3-O-
the free radical [10,11]. rutinoside contribute up to 97% of the total anthocyanin in
It was observed that in the case of samples that were of a light color blackcurrants [15,17]. However, it was shown that the retention of an-
(S1–S5, S9, S11–S12, S15) a closer agreement (described by the equa- thocyanins in the blackcurrant beverages appears to be rather poor [18].
tion y = 1.09x + 0.87, r = 0.99) between the values obtained from The next group of studied fruit antioxidants were apple juice and
both techniques can be found than for dark juices (S6–S8, S10, S13– nectar S2 and S12 (Table 1). Apples are especially rich in chlorogenic
S14) where a greater difference was observed (described by the equa- acid, caffeic acid, p-coumaric acid, ferulic acid, catechin, epicatechin,
tion y = 1.06x + 31.94, r = 0.99) (Table 1, Fig. 1). The greater difference procyanidines (B1, B2, trimer C1), rutin and phloridzin [19], which are
in the TEAC values obtained by UV–vis and EPR spectroscopies for prod- responsible for relatively high values of TEAC for apple juices. Relatively
ucts of a dark color is caused by the fact that the more color there is in a high TEAC values were also obtained for cherry nectar and the cherry
sample, the smaller the absorbance decrease and the less antioxidant drink (S10 and S14). Cherries are a flavinoid, organic acid and cyanide
activity is measured, even when working with minimal sample vol- source. They also contain numerous vitamins, including vitamins A, C
umes. The interference arises because of the presence of colored and B-group vitamins, which affects their antioxidative properties. On
the other hand, low TEAC values were obtained for plum nectar and
plum drink (S11 and S15), despite the fact that plums contain bioactive
compounds such as carotenoid, vitamins A, C and E, anthocyanins and
other phenolic compounds [20,21].
Another group of studied antioxidants were grape drinks (S8 and
S9). The value of TEAC for the red grape drink was higher than that of
the white grape drink (Table 1). The difference in TEAC values between
white and red grapes is caused by the higher polyphenol and flavonoid
content in red grapes [22]. It was generally observed that for darker
samples, TEAC values are higher than for lighter-colored samples
(Fig. 2). Similar dependencies were observed in comparing the antioxi-
dative properties of red and white wines [22,23]. Only the citrus juice
group distinguishes itself by having high TEAC values with a light
color. It must be noted that all of the studied fruit juices had a high per-
cent fruit content (50–100%) and were characterized by a high vitamin
C content.
Differences were observed between the antioxidative capacities for
juices, nectars and drinks (Fig. 2). The mean value for the studied juices
Fig. 1. Linear dependence of TEAC value obtained by the UV–vis and EPR spectroscopy (301.34 μM/100 ml) is the highest as compared to that of drinks (228.43
techniques. μM/100 ml) and nectars (146.59 μM/100 ml). A juice defined as a
 M. Bartoszek, J. Polak / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 546–549
Fig. 2. A comparison of TEAC values of fruit juices, drinks and nectars, as determined by EPR and UV–vis spectroscopies for dark and light samples.
beverage that is capable of fermentation but is unfermented, and is pro-
duced from healthy, ripe, fresh or chilled fruits, while nectar is water-di-
luted juice, fruit paste, or mixture of the two. Drinks have the lowest
percent fruit content, and therefore the lowest nutrient content, since
they contain even less juice than nectars. The TEAC values for juices
are the highest because they contain the highest percent fruit content.
Only in the case of two juices (orange and apple) the fruit content
amounted to 50% — in the other juices it was 100%. For all the studied
samples, there is a moderate correlation between TEAC values and per-
cent fruit content (r = 0.4). This moderate correlation between TEAC
values and percent fruit content is highly lowered by samples S7 and
S13, for which, despite a low fruit content, a high TEAC value was ob-
tained. These samples were produced from blackcurrants, which, as de-
scribed earlier, have very strong antioxidative properties. For all of the
studied samples, ignoring samples S7 and S13, the correlation coeffi-
cient between TEACEPR and TEACUV–vis values and the percent fruit con-
tent is r = 0.7 and 0.6, which indicates a correlation and confirms a
dependence between antioxidative properties and percent fruit
content.
4. Conclusions
All of the studied fruit juices, drinks and nectars showed antioxida-
tive properties. A dependence was found between TEAC and the percent
fruit content in the studied samples.
Based on the conducted research of antioxidant properties using EPR
and UV–vis spectroscopy techniques, it was determined that EPR spec-
troscopy is the more adequate method for determining antioxidative
capacity with the use of the DPPH• free radical. It is a method unique
to free radicals, and other substances that are present in the studied ma-
terial do not participate in the registered signal. Furthermore, unlike in
549
UV–vis spectroscopy, the sample color has no influence on the results,
and therefore it is possible to study unclear and turbid samples.
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