Measurement of Antioxidant Capacity by Electron Spin Resonance Spectroscopy Based on Copper(II) Reduction

Article abstract of DOI:10.1021/acs.analchem.6b00049

A new method is proposed for measuring the antioxidant capacity by electron spin resonance spectroscopy based on the loss of electron spin resonance signal after Cu²⁺ is reduced to Cu⁺ with antioxidant. Cu⁺ was removed by precipitation in the presence of SCN^-. The remaining Cu²⁺ was coordinated with diethyldithiocarbamate, extracted into n-butanol and determined by electron spin resonance spectrometry. Eight standards widely used in antioxidant capacity determination, including Trolox, ascorbic acid, ferulic acid, rutin, caffeic acid, quercetin, chlorogenic acid, and gallic acid were investigated. The standard curves for determining the eight standards were plotted, and results showed that the linear regression correlation coefficients were all high enough (r > 0.99). Trolox equivalent antioxidant capacity values for the antioxidant standards were calculated, and a good correlation (r > 0.94) between the values obtained by the present method and cupric reducing antioxidant capacity method was observed. The present method was applied to the analysis of real fruit samples and the evaluation of the antioxidant capacity of these fruits. (Graph Presented).

Full text of DOI:10.1021/acs.analchem.6b00049

Article
pubs.acs.org/ac
Measurement of Antioxidant Capacity by Electron Spin Resonance
Spectroscopy Based on Copper(II) Reduction
Dan Li,† Jia Jiang,† Dandan Han,‡ Xinyu Yu,‡ Kun Wang,† Shuang Zang,† Dayong Lu,‡ Aimin Yu,†
and Ziwei Zhang*,†
†College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, People’s Republic of China
‡Department of Materials Science and Engineering, Jilin Institute of Chemical Technology, Jilin 132022, People’s Republic of China
S
* Supporting Information
ABSTRACT: A new method is proposed for measuring the antioxidant capacity
by electron spin resonance spectroscopy based on the loss of electron spin
resonance signal after Cu2+ is reduced to Cu+ with antioxidant. Cu+ was removed
by precipitation in the presence of SCN−. The remaining Cu2+ was coordinated
with diethyldithiocarbamate, extracted into n-butanol and determined by electron
spin resonance spectrometry. Eight standards widely used in antioxidant capacity
determination, including Trolox, ascorbic acid, ferulic acid, rutin, caffeic acid,
quercetin, chlorogenic acid, and gallic acid were investigated. The standard
curves for determining the eight standards were plotted, and results showed that
the linear regression correlation coefficients were all high enough (r > 0.99).
Trolox equivalent antioxidant capacity values for the antioxidant standards were
calculated, and a good correlation (r > 0.94) between the values obtained by the
present method and cupric reducing antioxidant capacity method was observed.
The present method was applied to the analysis of real fruit samples and the evaluation of the antioxidant capacity of these fruits.
Electron spin resonance (ESR) spectroscopy, also called
electron paramagnetic resonance (EPR) spectroscopy, is a
magnetic resonance technique for detecting chemicals with
unpaired electrons including free radicals and paramagnetic
transition metal ions (e.g., Cu2+).17 ESR has been applied in
many fields, such as physics, chemistry, and biological
science.18−20 So far, the measurment of antioxidant capacity
by ESR technique mainly focus on two aspects: (1) measure
the decrease of signal intensity of stable artificial free radicals
(e.g., DPPH); and (2) measure the efficiency of antioxidant in
scavenging short-lived radicals (e.g., hydroxyl radical) yielded
from radical generation system by ESR spin-trapping
technique.17 In the past decade, ESR technique was applied
to the determination of antioxidant capacity of wine,21 beer,22
white tea,23 fruits and vegetables,24 wheat extracts,25 and herbal
extracts.26
In this work, a new method was developed for measuring
antioxidant capacity by detecting ESR signal from the
remaining Cu2+ after Cu2+ was reduced to Cu+ with various
antioxidant standards and real samples. Cu+ was removed by
precipitation with SCN−. The remaining Cu2+ was chelated
with diethyldithiocarbamate (DDC) in aqueous phase,
extracted into n-butanol phase and then determined by ESR.
The present method was applied to the measurement of
antioxidant capacity of eight antioxidant standards and six fruit
ree radicals and reactive oxygen species generated from
Foxidation reactions in the human body may cause damage
or death to the cell by starting chain reactions, resulting in
health problems such as cancer, aging, and heart disease.1−3
Antioxidants can terminate these chain reactions by scavenging
free radicals or reacting with reactive oxygen species.2,4,5 Most
antioxidants are reducing agents and inhibit oxidation reactions
by being oxidized themselves.2 Natural antioxidants are divided
into several categories based on their common chemical
structures, including phenolic acids, flavonoids, vitamins, and
phenolic diterpenes.2,6 Fruits and vegetables contain a variety of
antioxidants, such as vitamin C, polyphenols, and carotenoids.7
Trolox equivalent antioxidant capacity (TEAC) or vitamin C
equivalent antioxidant capacity (VCEAC) is frequently used to
quantify antioxidant capacity of a given substance.8,9
A number of analytical methods, especially the spectrophoto-
metric method, have been developed to measure antioxidant
capacity of antioxidants in either food/plant extracts or
biological samples. The most popular methods include 1,1-
diphenyl-2-picrylhydrazyl (DPPH),3 2,2′-azino-bis-3-ethylbenz-
thiazoline-6-sulfonic acid (ABTS),10 ferric reducing ability of
plasma (FRAP),11 and oxygen radical absorbance capacity
(ORAC).12 A new spectrophotometric method was proposed
by Apak et al. in 2004 and named as cupric reducing
antioxidant capacity (CUPRAC) method.13−15 In the CU-
PRAC assay, the Cu2+-neocuproine (Nc) complex is reduced
with antioxidant to Cu+-Nc, which has an orange−yellow color
and gives a maximum UV absorption at 450 nm.16
Received: January 6, 2016
Accepted: February 29, 2016
© XXXX American Chemical Society
A
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juice samples. TEAC values of eight standards and six fruit
juices were compared with those obtained by CUPRAC, ABTS,
DPPH, and FRAP methods. The results obtained by the
present method are proved to be highly correlated with those
obtained by CUPRAC method.
solution in a tube. The resulting Cu2+−DDC complex was then
extracted into 750 μL of n-butanol. The resulting solution was
referred to as analytical solution and measured with both
electron spin resonance spectrometer and UV spectrometer.
Electron Spin Resonance Measurement. The ESR
spectra were collected at room temperature on a commercial
Bruker A300 X-band spectrometer at a frequency of 9.84 GHz.
A 40 μL aliquot of analytical solution sealed in a 1.5 mm i.d.
glass capillary tube (Kimble Chase) was placed into a quartz
tube (Wilmad LabGlass), and the quartz tube was put in the
resonator cavity. The modulation amplitude was 2.0 G, the
microwave power was 2.3 mW, the center field was 3450 G, and
the sweep width was 500 G. The experiments for the
measurement of the standards and the samples were carried
out in two replicates and triplicate, respectively.
UV Spectrometric Measurement. The UV spectra were
obtained at room temperature on a Shimadzu UV-1700
PharmaSpec spectrophotometer. A 200 μL aliquot of the
analytical solution was diluted four times with n-butanol, and
400 μL of the resulting solution was injected into a 1 mm
quartz cuvette. Absorbance at 430 nm was recorded against a
blank of n-butanol. The experiments were carried out in
triplicate.
DPPH Method. DPPH (0.1 mmol/L) was prepared daily by
dissolving 9.86 mg DPPH in 250 mL of methanol. A 3 mL
aliquot of 0.1 mmol/L DPPH solution and 100 μL of sample
solution were added into a test tube and blended immediately,
reacting in the dark for 60 min. The resulting solution was
measured with UV spectrometer at 515 nm. The experiments
were carried out in triplicate.
CUPRAC Method. Nc solution (7.5 mmol/L) was prepared
by dissolving 39.00 mg neocuproine in 25 mL of ethanol.
Ammonium acetate (0.1 mol/L, NH4Ac) aqueous buffer was
prepared and adjusted to pH 7.0 with 50% HAc. A 1 mL aliquot
of 10 mmol/L CuCl2 solution, 1 mL of NH4Ac buffer solution,
1 mL of Nc solution, 100 μL of sample solution, and 1 mL of
H2O were added into a test tube in sequence, and the mixture
stood for 60 min. The resulting solution was measured with UV
spectrometer at 450 nm. The experiments were carried out in
triplicate.
EXPERIMENTAL SECTION
■
Chemicals and Materials. Eight antioxidant standards,
including Trolox (a water-soluble derivative of vitamin E), one
vitamin (ascorbic acid), two flavonoids (rutin and quercetin),
and four phenolic acids (ferulic acid, caffeic acid, chlorogenic
acid, and gallic acid) were used. Trolox was purchased from
J&K Chemicals. Ascorbic acid, rutin, quercetin, and gallic acid
were purchased from Aladdin Industrial Corporation. Ferulic
acid and caffeic acid were obtained from National Institute for
The Control of Pharmaceutical and Biological Products.
Chlorogenic acid was obtained from Shanghai Yuanye Bio-
Technology Co., Ltd. n-Butanol, methanol, and acetic acid were
purchased from Beijing Chemical Works. Sodium diethyldi-
thiocarbamate and potassium thiocyanate were obtained from
Aladdin Industrial Corporation. Copper(II) chloride and
ethanol were purchased from Sinopharm Chemical Reagents
Co., Ltd. Sodium acetate anhydrous was purchased from
Tianjin Fengchuan Chemical Reagent Co., Ltd. DPPH was
purchased from Alfa Aesar. Nc was purchased from J&K
Chemicals.
Preparation of Standard Solution. A stock solution of 10
mmol/L copper(II) chloride (CuCl2) was prepared by
dissolving 170.48 mg CuCl2·2H2O in distilled water and
diluted to 1.8 mmol/L when used. Sodium acetate (0.1 mol/L,
NaAc) buffer solution was prepared in water and adjusted to
pH 6.0 with 50% acetic acid (HAc). Potassium thiocyanate (50
mmol/L, KSCN) was prepared by dissolving 242.95 mg KSCN
in 50 mL of water. Sodium diethyldithiocarbamate (50 mmol/
L, DDC) was prepared by dissolving 536.25 mg of DDC in 50
mL water. Stock solutions of eight standards in a concentration
of 2−5 mmol/L were prepared in 100% ethanol except for
ascorbic acid, which was prepared in water and quercetin, which
was prepared in 100% methanol. Each stock solution, except for
quercetin, which was diluted by methanol, was diluted by water
to a series of concentrations in order to obtain the standard
curve and time curve for each standard.
Preparation of Sample Solution. Six fruits, including
lemon, orange, grapefruit, mandarin orange, watermelon, and
strawberry, were obtained from local supermarkets. The eaten
part of each fruit was cut into pieces and pressed for juice.
Milliliters of juice were filtered once with filter paper and
centrifuged at 10000 rpm twice. The juices from grapefruit,
mandarin orange, and watermelon were used as the sample
solution without dilution. Juices from lemon, orange, and
strawberry were diluted four times with water before analysis,
and the resulting solution was referred to as sample solution. A
total of 100 μL of sample solution were used to prepare the
analytical solution. Three parallel sample solutions for each fruit
were prepared and used.
RESULTS AND DISCUSSION
■
Optimization of Experimental Parameters. To optimize
the experimental conditions, the effects of pH value of the
buffer solution, concentration of KSCN, volume of DDC
solution, and volume of n-butanol were investigated. These
experiments were performed using Trolox as the antioxidant.
NaAc buffer solutions with pH values ranging from 4.5 to 6.5
were prepared. The experimental results indicated that there
was not an obvious difference in antioxidant capacity when pH
was changed. pH 6.0, which is close to physiological pH, was
selected for further experiments. Aliquots of 200 μL of KSCN
solution at concentrations ranging from 0 to 100 mmol/L were
used, and we found that the scavenging capacity of Trolox
reached maximum once KSCN concentration was over 40
mmol/L. Thus, 50 mmol/L was selected as the concentration
of KSCN in order to fully precipitate Cu+. The effect of volume
ranging from 0 to 140 μL of 50 mmol/L DDC solution was
investigated. The antioxidant capacity increased with volume of
DDC and reached a plateau when the volume was over 40 μL.
Thus, 80 μL of DDC solution were used. The volume of n-
butanol was set to be 750 μL to achieve the highest extraction
efficiency for Cu2+−DDC complex.
Preparation of Analytical Solution. A total of 200 μL of
CuCl2 working solution, 200 μL of NaAc buffer solution, and
200 μL of KSCN solution were in sequence added into a test
tube. Then 100 μL of antioxidant standard or sample solution
were added into the tube. The resulting mixture stood for a
fixed period of time (reaction time) and then was filtered with a
0.22 μm syringe filter to remove CuSCN precipitate. After
filtration, 450 μL of the filtrate were mixed with 80 μL of DDC
B
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Standard Curves. The ESR spectra for Cu2+−DDC in n-
butanol are displayed in Figure 1. As seen in the figure, all
Time Curves. The effect of the reaction time was
investigated to determine the optimal period in the redox
reaction for each standard. The curves of inhibition versus time
obtained by ESR and UV methods are shown in Figures 2 and
3, respectively. The experimental results show that, for Trolox,
ascorbic acid, ferulic acid, and rutin, the reaction is fast and
completes mostly within 10 min. The time of 40 min was
selected for the construction of their standard curves. However,
for caffeic acid, quercetin, chlorogenic acid, and gallic acid, the
redox process is slow, and it took about 40−90 min to reach
equilibrium after the antioxidant was mixed with CuCl2
solution. Thus, 110, 120, 90, and 80 min were selected for
the construction of the standard curves of caffeic acid,
quercetin, chlorogenic acid, and gallic acid, respectively.
Correlation with CUPRAC Assay. TEAC value (defined as
mmol Trolox/L)29 for each standard was obtained by dividing
slope of the standard curve by that of Trolox curve, and results
are listed in Table 1. As shown in Table 1, the increasing order
of antioxidant capacity determined by the present ESR method
is ferulic acid < Trolox < ascorbic acid < gallic acid < rutin <
caffeic acid < chlorogenic acid < quercetin. These results are
mostly consistent with those from theoretical evaluation. In
theory, antioxidant capacity of either phenolic acids or
flavonoids depends largely on the number and position of
their hydroxyl (−OH) groups.30 For example, free radical
scavenging capability of flavonoids is highly dependent on the
presence of a free 3-OH in the C ring.31,32 In this study,
quercetin has a TEAC of 4.7, whereas rutin has a TEAC of only
2.9 because of the substitution of the free 3-OH with rutinose.
Gallic acid has a TEAC of 2.6, which is close to its expectation
value of 3.0 mmol/L Trolox corresponding to the three
hydroxyl groups.30 The antioxidant abilities of hydroxycinnamic
acids in increasing the resistance of low density lipoproteins
(LDL) to peroxidation were investigated by Castelluccio et al.,
and the order of antioxidant effectiveness was chlorogenic acid
∼ caffeic acid > ferulic acid.33 The scavenging of ferulic acid was
weakened by the methoxylation of the ortho-hydroxyl group
when compared with the two diphenolics, chlorogenic, and
caffeic acids.33 The results are consistent with ours, except for
chlorogenic acid, which has a slightly higher TEAC than caffeic
acid. We found that the linearity of the standard curve of
chlorogenic acid becomes poor when the concentration is
above 0.3 mmol/L. The highest concentration to construct the
standard curve was 0.25 mmol/L, which may result in the
slightly higher TEAC of chlorogenic acid.
Figure 1. ESR spectra of Trolox at concentrations of 0.0, 0.8, and 1.6
mmol/L.
spectra obtained with different concentrations (0.0, 0.8, and 1.6
mmol/L) of Trolox have the same line shape. Thus, we
quantified the signal simply by measuring the peak to peak
amplitude of the third line among the four lines, without taking
efforts to double-integrate the signals. The inhibition (%) of
antioxidant is represented as the ratio of the height reduction in
the presence of antioxidant to the height in the absence of
antioxidant. In Figure 1, we can find a split of two peaks in the
rightmost line of the ESR signal of Cu2+. We suspect that such
extra splitting of two peaks comes from the two isotopes 63Cu
and 65Cu existing in the CuCl2 reagent.27,28 Even with this extra
signal, the quantitative analysis was not affected, especially
when the third line was used for the analysis.
Standard curves were constructed for each antioxidant
standard by plotting the inhibition (%) versus antioxidant
concentration. In order to prove the reliability of the present
ESR method, the UV experiments on the same antioxidant
standards were carried out. The standard curves for the eight
standards obtained by ESR and UV spectrometry are displayed
respectively in Figures S1 and S2. All these standard curves
were simulated with linear equation. The regression equations
and correlation coefficients (r) for the eight antioxidant
standards are listed in Table 1. From Figures S1 and S2, as
well as Table 1, it is found that the linearity for all curves is
good enough and the values of r are higher than 0.994. The
results obtained by ESR and UV assay are quite consistent.
The TEAC values of these eight standards obtained by the
present method were compared with the values obtained by
some popular assays, including CUPRAC,14 ABTS,34,35
DPPH,36−38 and FRAP.39,40 To obtain the correlation of
Table 1. Regression Equations and TEAC Values (mmol Trolox/L) of Antioxidant Standards Determined by ESR and UV Assay
regression equation
r
TEAC
std
Trolox
ESR
UV
ESR
UV
ESR
UV
y = 0.502c + 0.013
y = 0.561c + 0.008
y = 0.429c − 0.023
y = 1.44c + 0.024
y = 1.51c + 0.037
y = 2.44c − 0.008
y = 1.86c − 0.002
y = 1.28c + 0.015
y = 0.500c + 0.001
y = 0.568c − 0.009
y = 0.406c − 0.026
y = 1.45c + 0.018
y = 1.46c + 0.015
y = 2.23c + 0.010
y = 1.69c + 0.010
y = 1.28c − 0.004
0.997
0.999
0.994
0.996
0.994
0.997
0.997
0.995
0.998
0.999
0.997
0.996
0.999
0.998
0.997
0.998
1.00
1.12
0.85
2.87
3.01
4.86
3.71
2.55
1.00
1.14
0.81
2.90
2.92
4.46
3.38
2.56
ascorbic acid
ferulic acid
rutin
caffeic acid
quercetin
chlorogenic acid
gallic acid
C
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Figure 2. Change of inhibition with time for the antioxidants obtained by ESR spectroscopy: (A) Trolox, (B) ascorbic acid, (C) ferulic acid, (D)
rutin, (E) caffeic acid, (F) quercetin, (G) chlorogenic acid, and (H) gallic acid.
Figure 3. Change of inhibition with time for the antioxidants obtained by UV spectrometry: (A) Trolox, (B) ascorbic acid, (C) ferulic acid, (D)
rutin, (E) caffeic acid, (F) quercetin, (G) chlorogenic acid, and (H) gallic acid.
TEAC values between any two methods, the calibration curves
were constructed by plotting TEAC values obtained by first
method versus those obtained by second method. The slope of
the calibration curve is taken as the correlation coefficient (r).
As seen in Table 2 and Figure 4, TEAC values obtained by the
present method has a high correlation with those obtained by
CUPRAC method (r = 0.94). However, the correlations of
TEAC values obtained by the ESR method with those obtained
by ABTS (r = 0.59), DPPH (r = 0.66), and FRAP (r = 0.73) are
relatively low.
Analysis of Real Fruit Juice Samples. The present
method was applied to the analysis of six real fruit juice
samples, including lemon, orange, grapefruit, mandarin orange,
watermelon, and strawberry juices. The reaction time is 60 min.
The values of TEAC for these juice samples are calculated as
mmol TEAC per liter of juice, and listed in Table 3. From these
D
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Table 2. Comparison of TEAC Values (mmol Trolox/L) of Antioxidant Standards Obtained by ESR with Other Methods
14
std
Trolox
ESR
CUPRACN
ABTS34,35
DPPH36−38
FRAP39,40
1.00
1.12
0.85
2.87
3.01
4.86
3.71
2.55
1.00
0.96
1.20
2.56
2.89
4.38
2.47
2.62
1.00
0.99
1.90
2.42
1.26
4.72
1.24
3.01
1.00
0.91
0.60
2.77
1.11
2.55
4.7
1.00
1.04
1.01
1.99
2.32
3.50
1.05
2.27
ascorbic acid
ferulic acid
rutin
caffeic acid
quercetin
chlorogenic acid
gallic acid
2.66
The TEAC values for the standards and juice samples
obtained by the present method have respectively low (r =
0.66) and high (r = 0.97) correlation with those obtained by
DPPH method. The results should be due to the difference of
the matrices for standard and sample. Some similar
experimental results were found in some publications.1,14,43
Method Evaluation. The advantages of the present ESR
method are as follows: (1) The agents used, including CuCl2,
KSCN, and DDC, are low cost, stable, and insensitive to heat or
light; (2) When UV spectroscopy was applied too high
absorbance (such as >1.0) is not suitable. However, when the
analyte concentration in the final analytical solution is too high,
no dilution is needed before measurement for ESR signal
collection; (3) In ESR spectroscopy, the resonance signal from
Cu2+ was measured, and color from any sample itself (e.g., color
of juices) will not influence the quantification; (4) Complex of
Cu2+−DDC was finally extracted into the n-butanol phase so
that interference of sample matrix was eliminated; (5) The
redox reaction is performed at pH 6.0, which is close to
physiology pH in human body; (6) Samples were filtered after
the redox reaction completes and Cu2+−DDC complex was
extracted into the organic phase for the final analysis. Thus, it is
not necessary for the sample to be measured immediately after
the reaction. We measured the sample within several days and
experimental results were repeatable. However, compared with
CUPRAC method, when the present method was applied,
more operation steps were required.
Figure 4. Correlation of TEAC values obtained by ESR method with
those obtained by CUPRAC method (r = 0.94).
Table 3. TEAC Values (mmol Trolox/L juice) of Six Fruit
Juices Obtained by ESR, DPPH, and CUPRAC
fruit
ESR
DPPH
CUPRAC
lemon
1.92 0.25
0.79 0.33
1.45 0.01
1.21 0.06
0.31 0.06
3.99 0.49
2.21 0.02
1.82 0.03
1.49 0.03
1.80 0.02
0.38 0.00
6.36 0.31
1.38 0.03
2.01 0.04
1.27 0.02
2.29 0.02
0.39 0.01
7.06 0.16
orange
grapefruit
mandarin orange
watermelon
strawberry
TEAC values, we could find that the increasing order of
antioxidant capacity determined by the present ESR method is
watermelon < grapefruit < mandarin orange < orange < lemon
< strawberry. We also measured each fruit juice sample by
DPPH and CUPRAC assays, and the TEAC values are listed in
Table 3. As seen in Table 3, the TEAC values obtained by the
present method has a high correlation with those obtained by
either DPPH (r = 0.97) or CUPRAC method (r = 0.91).
The DPPH method involves an electron transfer of the free
radical, whereas CUPRAC method and the present method
involve measurement of the reducing potential of juices.41 The
present method is based on the reaction: Cu2+ + e + SCN− →
CuSCN, and CUPRAC method is based on the reaction:
CONCLUSIONS
■
A new ESR method was proposed for measuring antioxidant
capacity taking advantage of the reduction of Cu2+ into Cu+. In
order to evaluate the method, the present method was applied
to the measurement of both antioxidant standards and real fruit
juice samples. Standard curves for the eight antioxidant
standards show good linearity, and the TEAC values obtained
by the present method has a high correlation with those
obtained by CUPRAC method. The present method could be
applied to the determination of thiols in medicine and
biological samples by changing the experimental conditions.
+
Cu(Nc)22+ + e → Cu(Nc)2 . The same sample matrix may have
significantly different effect on the redox potentials of the two
reactions. The difference in the redox potentials may result in
the difference in the TEAC values. The matrices of juices are
much more complicated than those of the eight standards that
we studied. The complicated juice sample matrices may have
very different effect on the potentials of the two reactions.
Various antioxidants may interact with each other, as might
affect the total antioxidant capacity.42 So, for some juice
samples there is no good agreement of the TEAC values
between CUPRAC and ESR methods. Although different
methods gave different TEAC values for specific sample (e.g.,
orange), the correlations between methods were generally
good.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.6b00049.
Standard curves for the antioxidants obtained by ESR
spectroscopy and by UV spectrometry (PDF).
E
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(31) Heim, K. E.; Tagliaferro, A. R.; Bobilya, D. J. J. Nutr. Biochem.
2002, 13, 572−584.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +86 13610709861. Fax: +86 431 85112355. E-mail:
zzw@jlu.edu.cn.
■
(32) Rice-Evans, C.; Miller, N.; Paganga, G. Trends Plant Sci. 1997, 2,
152−159.
(33) Castelluccio, C.; Paganga, G.; Melikian, N.; Bolwell, G. P.;
Pridham, J.; Sampson, J.; Rice-Evans, C. FEBS Lett. 1995, 368, 188−
192.
Notes
The authors declare no competing financial interest.
(34) Miller, N. J.; Diplock, A. T.; Rice-Evans, C. A. J. Agric. Food
Chem. 1995, 43, 1794−1801.
ACKNOWLEDGMENTS
■
(35) Salah, N.; Miller, N. J.; Paganga, G.; Tijburg, L.; Bolwell, G. P.;
Rice-Evans, C. Arch. Biochem. Biophys. 1995, 322, 339−346.
(36) Villano, D.; Fernandez-Pachon, M. S.; Troncoso, A. M.; Garcia-
Parrilla, M. C. Anal. Chim. Acta 2005, 538, 391−398.
(37) Ozgen, M.; Reese, R. N.; Tulio, A. Z.; Scheerens, J. C.; Miller, A.
R. J. Agric. Food Chem. 2006, 54, 1151−1157.
This project is supported by the Research Startup Foundation
of Jilin University (No. 450060521178).
REFERENCES
■
(1) Celik, S. E.; Ozyurek, M.; Gucl
̧
u, K.; Apak, R. Talanta 2010, 81,
̈
̈
̈
(38) Campos, C.; Guzman
Biochem. 2009, 392, 37−44.
(39) Pulido, R.; Bravo, L.; Saura-Calixto, F. J. Agric. Food Chem. 2000,
48, 3396−3402.
́ ́ ́
, R.; Lopez-Fernandez, E.; Casado, A. Anal.
1300−1309.
(2) Hamid, A.; Aiyelaagbe, O.; Usman, L.; Ameen, O.; Lawal, A. Afr.
J. Pure Appl. Chem. 2010, 4, 142−151.
(3) Mishra, K.; Ojha, H.; Chaudhury, N. K. Food Chem. 2012, 130,
1036−1043.
(40) Jiao, J.; Gai, Q. Y.; Luo, M.; Wang, W.; Gu, C. B.; Zhao, C. J.;
Zu, Y. G.; Wei, F. Y.; Fu, Y. J. Food Res. Int. 2013, 53, 857−863.
(41) Granato, D.; Karnopp, A. R.; Ruth, S. M. J. Sci. Food Agric. 2015,
95, 1997−2006.
(42) Hidalgo, M.; Sanchez-Moreno, C.; Pascual-Teresa, S. Food
Chem. 2010, 121, 691−696.
(43) Alpinar, K.; Ozyurek, M.; Kolak, U.; Guclu, K.; Aras, Q.; Altun,
M.; Celik, S. E.; Berker, K. I. J.; Bektasoglu, B.; Apak, R. Food Sci.
Technol. Res. 2009, 15, 59−64.
(4) Yildiz, L.; Bask
304−313.
(5) Lim, Y. Y.; Lim, T. T.; Tee, J. J. Food Chem. 2007, 103, 1003−
1008.
̧ an, K. S.; Tutem, E.; Apak, R. Talanta 2008, 77,
̈
(6) Brewer, M. S. Compr. Rev. Food Sci. Food Saf. 2011, 10, 221−247.
(7) Pisoschi, A. M.; Cheregi, M. C.; Danet, A. F. Molecules 2009, 14,
480−493.
(8) Miller, N. J.; Rice-Evans, C.; Davies, M. J.; Gopinathan, V.;
Milner, A. Clin. Sci. 1993, 84, 407−412.
(9) Floegel, A.; Kim, D. O.; Chung, S. J.; Koo, S. I.; Chun, O. K. J.
Food Compos. Anal. 2011, 24, 1043−1048.
(10) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.;
Rice-Evans, C. Free Radical Biol. Med. 1999, 26, 1231−1237.
(11) Benzie, I. F. F.; Strain, J. J. Anal. Biochem. 1996, 239, 70−76.
(12) Cao, G.; Alessio, H. M.; Cutler, R. G. Free Radical Biol. Med.
1993, 14, 303−311.
(13) Apak, R.; Guclu, K.; Ozyurek, M.; Karademir, S. E. J. Agric. Food
Chem. 2004, 52, 7970−7981.
̈
(14) Apak, R.; Gucl
̧
u, K.; Ozyurek, M.; C
̧
elik, S. E. Microchim. Acta
̧ u, K.; Apak, R. TrAC, Trends Anal. Chem.
̈
̈
̈
̈
2008, 160, 413−419.
̈
(15) Ozyurek, M.; Gucl
̈
̈
2011, 30, 652−664.
(16) Ozyurek, M.; Gucl
̈
̧
u, K.; Tutem, E.; Bask
elik, S.; Baki, S.; Yıldız, L.; Karaman, S
̧
̆
an, K. S.; Erca̧ g, E.; Esin
̈
̈
̈
̈
Ç
̧.; Apak, R. Anal. Methods
2011, 3, 2439−2439.
(17) Yu, L. L.; Cheng, Z. Mol. Nutr. Food Res. 2008, 52, 62−78.
(18) Weil, J. Phys. Chem. Miner. 1984, 10, 149−165.
(19) Borbat, P. P.; Costa-Filho, A. J.; Earle, K. A.; Moscicki, J. K.;
Freed, J. H. Science 2001, 291, 266−269.
(20) Burns, D. T.; Flockhart, B. D. Philos. Trans. R. Soc., A 1990, 333,
37−48.
(21) Bartoszek, M.; Polak, J. Food Chem. 2012, 132, 2089−2093.
(22) Polak, J.; Bartoszek, M.; Stanimirova, I. Food Chem. 2013, 141,
3042−3049.
(23) Azman, N. A.; Peiro, S.; Fajari, L.; Julia, L.; Almajano, M. P. J.
Agric. Food Chem. 2014, 62, 5743−5748.
(24) Tzika, E. D.; Papadimitriou, V.; Sotiroudis, T. G.; Xenakis, A.
Food Biophys. 2008, 3, 48−53.
(25) Yu, L.; Haley, S.; Perret, J.; Harris, M.; Wilson, J.; Qian, M. J.
Agric. Food Chem. 2002, 50, 1619−1624.
(26) Orlowska, M.; Kowalska, T.; Sajewicz, M.; Pytlakowska, K.;
Bartoszek, M.; Polak, J.; Waksmundzka-Hajnos, M. J. AOAC Int. 2015,
98, 876−882.
(27) Gersmann, H. R.; Swalen, J. D. J. Chem. Phys. 1962, 36, 3221.
(28) Rieger, P. H.; Corden, B. J. Inorg. Chem. 1971, 10, 263−272.
(29) Karaman, S.; Tutem, E.; Basķ an, K. S.; Apak, R. J. Sci. Food Agric.
̈
2013, 93, 867−875.
(30) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Free Radical Biol.
Med. 1996, 20, 933−956.
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DOI: 10.1021/acs.analchem.6b00049
Anal. Chem. XXXX, XXX, XXX−XXX