Food Anal. Methods (2014) 7:1474–1480
DOI 10.1007/s12161-013-9776-4
Comparative Analysis of Antioxidative Activity of Flavonoids
Using HPLC–ED and Photometric Assays
Paweł Piszcz & Magdalena Woźniak &
Monika Asztemborska & Bronisław K. Głód
Received: 9 August 2013 / Accepted: 25 November 2013 / Published online: 11 December 2013
# The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract The present investigation reports on the application
of a new antioxidant activity assay for the examination of
flavonoids. It has been shown that the high-performance liquid
chromatography with electrochemical detection (HPLC–ED)
measurements allow to obtain additional information about
the antioxidative properties of pure compounds by measuring
their half-wave potential, the chromatographic peak height, and
the product of the peak height and exponent of potential. In
comparison to the classical electrochemical measurements, the
HPLC–ED is characterized by a much smaller detection limit.
The results were compared with the standard photometric measurement based on 1,1-diphenyl-2-picrylhydrazyl radicals. The
possible antioxidant activity forecasting is also discussed.
Keywords Antioxidants . DPPH . Flavonoids . Free
radicals . HPLC–ED
Introduction
Flavonoids are the main bioactive components of vegetables
and fruits. They have antioxidant, antimicrobial, antiallergenic, anti-inflammatory, insecticidal, and light-screening activities and act as photoreceptors, visual attractors, and feeding
repellants. Therefore, they are predominant in the human diet,
and intake may reach up to 1 g/day (Pietta 2000). Their
P. Piszcz : M. Woźniak : B. K. Głód (*)
Department of Analytical Chemistry, Institute of Chemistry, Faculty
of Science, University of Natural Sciences and Humanities, 3 Maja
54, 08-110 Siedlce, Poland
e-mail: bkg@onet.eu
M. Asztemborska
Institute of Physical Chemistry, Polish Academy of Sciences,
Kasprzaka 44/52, 01-224 Warsaw, Poland
antioxidant activity is regarded as scavenging free radicals
by donation of electrons.
Many methods of antioxidant activity evaluation have been
developed (Wantusiak et al. 2011; Głód et al. 2012), such as
scavenging activity determination using 1,1-diphenyl-2picrylhydrazyl (DPPH) or Folin–Ciocalteu (FCR) reagent. Photometric (Uma Devi et al. 2008) and chromatographic (Głód
et al. 2000, 2012) assays are, generally, based on competition
between the sample and the sensor for reaction with the free
radicals. Often, flavonoids show inconsistent antioxidant activities, depending on the assay used (Zhang et al. 2011).
A good measure of the antioxidant activity of pure compounds, like flavonoids, is their oxidation potential. It can be
determined potentiometrically (Brainina et al. 2007) or using
the voltammetric measurements (Alonso et al. 2003). These
measurements cannot be applied to real samples with complex
biological (food, blood, etc.) matrix, containing many various
antioxidants at different concentrations. Recently, we have
proposed an original assay of the total antioxidant potential
(TAP) measurement, based on the reverse-phase highperformance liquid chromatography (RP-HPLC) antioxidant
separation followed by electrochemical (amperometric) detection (Wantusiak et al. 2012). This assay was used to the TAP
estimation of various food samples. In the anodic potential
range, only oxidation current is observed. It means that chromatographic peaks originate from the antioxidants. The TAP
measure is the total surface area of all chromatographic peaks.
It is dependent on the potential of the working electrode.
The main purpose of this work was to study the antioxidative
properties of 15 flavonoids (Fig. 1) by using high-performance
liquid chromatography with electrochemical detection (HPLC–
ED) (involving measurement of chromatographic peak height);
moreover, the structural parameters (energy of the highest occupied molecular orbital (HOMO) orbital) were calculated. The
results (obtained using photometric assay) were compared to the
antioxidant activity related to the DPPH radicals.
Food Anal. Methods (2014) 7:1474–1480
Materials and Methods
Instrumentation
Chromatographic measurements were performed by means of
a chromatograph comprising an Interface Box, four-channel
Smartline Manager 5000 with Degasser K-5004, Solvent Organizer K-1500, Dynamic Mixing Chamber, HPLC Pump
Smartline 1000, ClinLab Digital Amperometric Detector
EC3000 (Recipe, Munich, Germany) with a glassy carbon
working electrode (reference electrode—Ag/AgCl, auxiliary
electrode—Pt), Smartline Diode Array Detector 2800,
Autosampler Smartline 3900 (all from Knauer GmbH, Berlin,
Germany), and Smartline 4000 Column Thermostat (Industrial Electronics, Langenzersdorf, Austria). Samples were separated on a Cosmosil RP-C18-MS-II column (5 μm, 250×
4.6 mm I.D.; Nacalai, USA). The system was controlled,
and data acquisition was performed on an IBM PC-type
computer with ClarityChrom V 2.6 2007 software.
Photometric measurements were performed on a Helios
Epsilon spectrophotometer (Thermo Scientific, USA).
The application HyperChem™ Release 8.0.7 (molecular
modeling system; Hypercube, Gainesville, FL, USA) for Windows was used for the structural parameter (energy of the
HOMO orbital) evaluation.
pH was measured using an OP-208/1 pH meter (Radelkis,
Budapest, Hungary) with an OSH 10-10 electrode (MetronWCF, Czekanów, Poland).
Reagents
Gallic acid, sodium perchlorate, DPPH, and HPLC-grade
methanol were obtained from Sigma (St. Louis, MO, USA).
All other reagents (Fluka, Buchs, Switzerland; Alchem, Inform, and POCh, Poland) were of analytical reagent grade and
were used without further purification. Water was distilled
three times from quartz. Mobile phases were filtered through
a 0.22-μm membrane filter (Millipore, Bedford, USA).
Various flavonoid standards including (+)-catechin,
(−)-epicatechin, hesperetin, hesperidin, neohesperidin,
naringin, naringenin, flavanone, 7,8-benzoflavanone, 6methoxyflavanone, 6-hydroxyflavanone, 4′-hydroxyflavanone,
2′-hydroxyflavanone, quercetin, and rutin were purchased from
Sigma-Aldrich, USA (Fig. 1).
Chromatographic/Amperometric Measurements
of Antioxidant Activity
Chromatographic experiments were performed at a flow rate
of 1.0 mL min−1. The column was stabilized at 20 (±0.1)°C by
passage of mobile phase for 0.5 h prior to the chromatographic
measurements. Methanol + sodium perchlorate (80+20 %,
v/v) in water (0.1 mol L−1) was used as a mobile phase; 1.0-
1475
mmol L−1 stock solutions of the analyzed flavonoids were
prepared in methanol and diluted to 0.1 mmol L−1 before use.
Using an autosampler, 20-μL samples were injected. Output
signal from the electrochemical detector was continuously
displayed on the computer (Wantusiak et al. 2012).
Photometric DPPH Assay
The antioxidative activity of the flavonoid samples was
assessed on the basis of the scavenging activity of the stable
DPPH free radical according to previous reports (Brand-Williams et al. 1995; Sharma and Bhat 2009; Huang et al. 2012)
with slight modifications. To 700 μL of 0.1 mmol L−1 DPPH
in methanol, 1.4 mL of a 0.01-mmol L−1 flavonoid solution in
methanol was added. The mixture was shaken vigorously and
left to stand for 20 min at room temperature in the dark. The
change of absorbance at 517 (±0.2)nm was measured. The
antioxidant activity was expressed as percentage of DPPH
radical concentration (AA = [(A blank − A sample ) / A blank ] ×
100[%]), where A blank is the absorbance of the DPPH radical
solution and A sample is the absorbance of the DPPH radical
solution after the addition of 10 μmol L−1 of sample solution.
Results and Discussion
TAP can be measured using RP-HPLC separation of antioxidants followed by their amperometric measurements
(Wantusiak et al. 2012). At anodic potentials, only antioxidants are recorded on chromatograms. In this paper, it will be
discussed whether this assay can be used to the antioxidant
activity (AA) estimations of pure compounds. Although in
this case AA can be simply expressed as a red/ox potential, the
disadvantage of these methods is very small sensitivity. In
voltammetry, current is measured as a function of potential.
However, much higher signals (than those in traditional
voltammetric measurements) are observed in the flowing
(among them HPLC) systems. This is due to (1) neglected
capacity current (measurements are performed at a constant
potential) and (2) huge and stable convectional current (current is directly proportional to the flow rate of depolarizer to
the electrode). A very small volume of the electrochemical
detector cell means that the mobile phase flows through it at a
very high linear flow rate. This, in turn, causes a large convection current and, consequently, lowers detection limit and
yields higher sensitivity of chromatographic measurements
with electrochemical detection than that of voltammetric measurements. So, 0.1-mmol L−1 flavonoid solutions are undetectable using voltammetry, hence the advantage of hydrodynamically recorded voltammograms. Changes of convection
current are responsible for the current (chromatographic signal) changes observed at the dead column volume (Fig. 2).
The current flowing through the system depends also on the
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Food Anal. Methods (2014) 7:1474–1480
Fig. 1 Structures of the
investigated flavonoids
O
O
O
O
O
O
7,8-benzoflavanone
6-hydroxyflavanone
HO
flavanone
OH
HO
O
O
O
O
CH3
O
O
O
2’-hydroxyflavanone
6-methoxyflavanone
4’-hydroxyflavanone
OH
OH
OH
CH3
OH
O
HO
O
HO
HO
O
O
OH
OH
O
OH
OH
O
hesperetin
naringenin
(-)epicatechin
OH
OH
OH
OH
OH
O
OH
O
O
HO
HO
O
HO
O
O
HO
H3C
OH
OH
O
OH
O
OH
quercetin
(+)catechin
O
OH
HO
OH
OH
naringin
OH
CH 3
HO
OH
O
HO
CH 3
OH
O
OH
H 3C
O
O
O
O
O
O
O
HO
HO
HO
HO
O
O
OH
OH
HO
O
OH
OH
O
OH
O
H3C
O
HO
HO
OH
HO
O
OH
O
OH
O
O
OH
H3C
O
HO
HO
OH
hesperidin
changes of the solvent dielectric constant and on the concentration of the supporting electrolyte. Usually, polar solvents
and strong electrolytes are used as mobile phase in RP-HPLC.
They are eluted near the dead column volume. Hence, when a
sample is not dissolved in the mobile phase, then, often, the
additional peak is observed in the dead column volume.
Therefore, the described measurements cannot be obtained
using flow injection systems, without a column. The column
is necessary to separate the solute peak from the dead volume.
The reported method is based on isocratic elution, and for the
given mobile phase conditions, the solutes were eluted at 2.8±
0.1 min, while the dead time was approximately 2 min
(Fig. 2).
The hydrodynamic voltammograms of five different flavonoids are presented in Fig. 3. It is easy to see their characteristic wave shapes. They differ significantly in comparison to
neohesperidin
rutin
the classical voltammograms. These waves are more similar to
waves obtained polarographically, using a rotating disc electrode or microelectrode. It turned out (Table 1) that among the
investigated flavonoids, quercetin is the strongest antioxidant,
which is consistent with the available literature data (RiceEvans et al. 1996). Quercetin is characterized by the smallest
half-wave potential (i.e., potential at which the wave current is
equal to one half of its maximum current) and the highest
chromatographic peaks, particularly the peak obtained at
0.1 V. At higher potentials, the additional waves are observed,
similarly to the voltammetric results (Brett and Ghica 2003).
Quercetin is also the best electron donor from all investigated
flavonoids. It contains a catechol moiety, namely, the 3′,4′dihydroxyl electron-donating group in ring B. The oxidation
mechanism proceeds in sequential steps, related to the catechol moiety and 3-hydroxyl group. The oxidation of the
Food Anal. Methods (2014) 7:1474–1480
Fig. 2 RP-HPLC–ED chromatogram of 6-hydroxyflavanone (2), separated from the dead column volume (1). Chromatographic conditions:
column—Cosmosil RP-C18-MS-II (Nacalai; 5 μm, 250×4.6 mm); mobile phase—0.1 mol L−1 NaClO4/MeOH 80:20 % (v/v); flow rate—
1.0 mL min−1, electrochemical detector with the glassy carbon used as
a working electrode; potential—0.9 V vs. Ag/AgCl
catechol electron-donating group occurs first at a low positive
potential and involves a two-electron–two-proton reversible
Fig. 3 Hydrodynamic
voltammograms of quercetin
(circle), (+)-catechin (square), 4′hydroxyflavanone (diamond),
hesperedin (triangle), and
flavanone (asterisk).
Chromatographic conditions are
as in Fig. 2 except the working
electrode range of potentials—
0.0–1.2 V vs. Ag/AgCl. The
points represent the results from
three independent measurements.
In all cases, RSD≤5 %
1477
reaction and forms o-quinone. The following steps are oxidation of the 3-hydroxyl group located in ring C and the resorcinol group (5,7-dihydroxyl) in ring A undergoing an irreversible reaction (Pierożyński and Zielińska 2011). Quercetin is
also characterized by the highest antioxidant activity measured in relation to the DPPH radical and the smallest energy
of the HOMO orbital (Table 1).
Catechin (E 1/2 =0.55 V) is structurally similar to quercetin;
it has also a catechol group in ring B, resorcinol moiety in ring
A, and hydroxyl group at position 3 in ring C. However, the
lack of a carbonyl group as well as double bond in ring C
causes the catechin to be unconjugated and characterized by
less mesomeric structures, and therefore, it is a weaker antioxidant (Janeiro and Brett 2004). Even weaker antioxidative
properties are observed for those compounds in which one
hydroxyl group is changed to a methoxyl one, as it is observed
for hesperitin (E 1/2 = 0.73 V), or removed (4′hydroxyflavanone, E 1/2 =0.55 V). Also, glycosylation increases the half-wave potential (hesperidin, E 1/2 =0.55 V).
Glycosylation additionally decreases the height of the chromatographic peak because of a decrease of the diffusion
c o e f f i c i e n t . F i n a l l y, 6 - m e t h o x y f l a v a n o n e , 7 , 8 benzoflavanone, and flavanone, where the B ring is
unsubstituted phenyl, are not oxidized electrochemically
(Table 1).
A good correlation between peak heights and E 1/2 was
found (Table 1), but only for peaks recorded at the smallest
potentials. Due to the nonlinearity of hydrodynamic voltammograms (for example, see Fig. 3), such correlation is not
observed for peak heights recorded at higher potentials. Comparison of the peak heights obtained at the various potentials
gives deeper insight into the antioxidative sample properties.
According to the Randles–Sevcik equation, the oxidation
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Food Anal. Methods (2014) 7:1474–1480
Table 1 Antioxidant activity of flavonoids
Sample
E 1/2 (V)
AADPPH (%)
e (2.08−E) h 0.4 (nA)
E HOMO (eV)
h 0.4 (nA)a
h 0.7 (nA)a
h 0.9 (nA)a
Quercetin
(−)-Epicatechin
Rutin
(+)-Catechin
Hesperetin
6-Hydroxyflavanone
4′-Hydroxyflavanone
2′-Hydroxyflavanone
Neohesperidin
Hesperidin
Naringin
Naringenin
6-Methoxyflavanone
7,8-Benzoflavanone
Flavanone
0.12
0.35
0.52
0.55
0.73
0.73
0.74
0.75
0.76
0.80
0.82
0.83
–
–
–
75.3
52.8
65.2
43.9
10.6
1.3
0.7
0.8
3.0
9.5
1.5
0.9
0.5
0.4
0.4
28.98
24.25
11.2
14.11
11.51
8.06
12.04
11.02
11.07
6.27
7.43
5.71
0
0
0
−8.55
−8.67
−8.86
−8.85
−8.81
−8.96
−9.17
−9.15
−8.87
−8.82
−9.25
−9.14
−8.87
−8.69
−9.36
381.1
263.8
121.6
60.8
3.0
0.9
1.4
19.4
3.7
4.3
3.8
0.9
0.3
0.1
0.3
811.4
864.6
322.4
508.6
336.1
218.7
285.7
256.6
287.8
336.1
91.3
25.3
1.3
1.2
1.99
930.6
1,060.7
356.6
688.8
764.1
593.5
884.1
760.0
825.6
423.5
555.4
416.7
2.9
2.0
3.6
E 1/2 (volts) is the half-wave potential of the first oxidation step; AADPPH (percent) is the antioxidant activity related to DPPH; E HOMO (volts) is the
energy of the highest occupied molecular orbital; h 0.4,0.7,0.9 (nanoamperes) are the heights of the chromatographic peaks obtained at various potentials of
the working electrode; and e (2.08−E) h 0.4 (nanoamperes) is the product of the chromatographic peak height obtained at 0.4 V by exponent from the
potential related to the reduction potential of hydroxyl radicals
a
RSD<4 %, for all experiments
current is directly proportional to the solute concentration and
to the square root of the depolarizer diffusion coefficient.
Hence, smaller peaks are observed for glycosides (as an
example, compare hesperitin and hesperedin or quercetin
and rutin).
From the above discussion, it turned out that the antioxidative properties of flavonoids can be expressed as half-wave
potential and or height of the chromatographic peak. The
former value describes the sample antioxidative activity. The
latter depends on the sample concentration and, indirectly, on
Fig. 4 Correlation between
(circle) E OH–E 1/2 and TAPHPLC–
ED
as well as between (square)
h 0.4 and TAPHPLC–ED
its antioxidative properties. The aim of this paper was to find a
more universal assay of AA measurements. It should enable
the AA measurement of single compounds as well as any
biological or food sample. Therefore, it is impossible to derive
proper equation because it is impossible to take any assumption, like that the reaction is reversible or that it is in equilibrium. It is expected that AA will be proportional to the electrode potential at which the chromatographic measurements of
the peak height are performed. Therefore, we propose to
measure the potential in relation to the redox potential of
Food Anal. Methods (2014) 7:1474–1480
1479
hydroxyl radical (E OH–E ). This new potential value is
directly proportional to the antioxidant activity. In the
next step, the product of peak height and potential
should be used as an AA measure. The strong antioxidants are oxidized at low potential and low reaction
current (the chromatographic peak height). Therefore,
we purpose to use the exponential function. Finally, a
new assay of the total antioxidant potential (named
TAP HPLC–ED ) can be performed using the equation
which is expressed as follows:
TAPHPLC–ED ¼
Xn
AAi ¼
i¼1
Xn
i¼1
eð E
OH
–E Þ
⋅hEi
characterized by similar HOMO energy values because
of their structural similarity.
Conclusions
1. HPLC–ED assay can be used for the estimations of the
antioxidant activity of pure compounds as it was shown
for flavonoids.
2. As an antioxidant activity measure, the first half-wave
potential and/or height of the chromatographic peak can
be used.
3. The new TAP measure is proposed:
OH
TAPHPLC–ED ¼ ∑n AA ¼ ∑n eðE –EÞ ⋅hE .
i¼1
It turned out (Table 1) that TAP values are directly correlated with the E 1/2 and h 0.4 values, as it is presented in Fig. 4.
In this case, only qualitative correlation is expected. TAP
measured using the proposed equation is less sensitive to the
experimental inaccuracies.
In the literature, many methods of the antioxidative
measurements are described (Głód et al. 2000). One of
the most popular is assay based on the photometric
measurements of DPPH radicals (Brand-Williams
et al. 1995; Huang et al. 2012). The main mechanism
of the reaction between flavonoid and DPPH is the
formation of the phenoxyl radical, characterized by
many resonance structures. Also, in this case, quercetin
is characterized by the strongest antioxidant activity
(Table 1), and flavanone does not react with DPPH.
Generally, a good correlation is obtained between
TAPHPLC–ED and AA measured using DPPH and peak
heights recorded at 0.4 V. Because DPPH is a weak
radical and oxidant, therefore, AA DPPH is correlated
with the electrochemical oxidation at relatively low
potentials.
The antioxidant activity of flavonoids has been the
subject of several studies in the past years, and important structure–activity relationships of the antioxidant activity have been established. It was interesting
to check if there are any correlations between AA and
HOMO (highest occupied orbital) energies. The HOMO orbitals and map of the electrostatic potential
were calculated using semiempirical AM1 method
(HyperChem™ Release 8.0.7) using optimization conditions described in the literature (Lien et al. 1999;
Seyoum et al. 2006). The results are presented in
Table 1. When the energy difference between HOMO
and LUMO orbitals is small, then the electron can be
easily triggered. In the case when this difference
increases, the greater energy is needed to excite an
electron. In summary, the lower the HOMO energy,
the slower is the reaction observed. Flavonoids are
i
i¼1
i
4. The proposed assay is much more sensitive than the
electrochemical measurements because of the lack of
capacity current and very big convection current.
5. The results were confirmed with those obtained using
DPPH assay as well as energy of HOMO orbitals.
Conflict of Interest Paweł Piszcz declares that he has no conflict of
interest. Magdalena Woźniak declares that she has no conflict of interest.
Monika Asztemborska declares that she has no conflict of interest.
Bronisław Głód declares that he has no conflict of interest. This article
does not contain any studies with human or animal subjects.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
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