Food Chemistry 124 (2011) 672–678

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Antioxidant activity of flavone C-glucosides determined by updated

analytical strategies
Danuta Zielińska a,*, Henryk Zieliński b
a
Department of Chemistry, University of Warmia and Mazury in Olsztyn, Plac Lodzki 4, 10-727 Olsztyn, Poland
b
Division of Food Science, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10, P.O. Box 55, 10-747 Olsztyn, Poland

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

Article history: The antioxidant activity of flavone C-glucosides, namely orientin (OR), homoorientin (hOR), vitexin (VT)
Received 27 January 2010 and isovitexin (iVT) in comparison to quercetin (Q) was measured against 2,20 -azinobis-(3-ethylbenzo-
Received in revised form 29 March 2010 thiazoline-6-sulphonate) radical cation (ABTS+), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), superox-
Accepted 14 June 2010
ide anion radical O
2 whilst the reducing activity was determined by cyclic voltammetry (CV) method.
The order of the antioxidant activity determined against ABTS+ and DPPH radicals was as follows:
Q  hOR > ORoiVT > VT. Orientin (OR) and homoorientin (hOR) showed the highest ability to scavenge
Keywords:
superoxide anion radicals whilst about threefold lower was found for VT and iVT. The order of the anti-
Flavone C-glucosides
Quercetin
oxidant activity determined against superoxide anion radicals was hOR > OR > Q > iVT = VT. The reducing
Antioxidant activity activity of hOR and OR derived from CV experiments performed within the range from 100 to +1300 mV
Reducing activity showed twofold higher values than those determined for VT and iVT. In contrast, when CV tracing was
carried out from 100 to +800 mV, the order of the antioxidant activity was as follows: Q > hOR = OR,
and no antioxidant activity of VT and iVT was noted.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction venting the pro-oxidation process or biological oxidative damage

Flavonoids, a large group of plant polyphenol secondary metab-
olites, are widely distributed in medicinal plants, fruits, teas and
health beverages (Walle, 2004). Interest in the flavonoids is related
to their diversity, biological significance as secondary plant metab-
olites, and their impact on essential plant growth, development,
stress adaptation and defence. Besides the importance for the plant
itself, its metabolites determine the nutritional quality of food as
well as its physiological effects and industrial applications. In con-
sequence, a commercial interest in these compounds is consider-
able (Zhang et al., 2005). All of these aspects justify the intense
interest in flavonoids, which has been manifested over several dec-
ades (Manach, Scalbert, Morand, Remesy, & Jimenez, 2004).
The putative protective effects of antioxidants against the oxi-
dative-induced reactions in food products as well as the pathogen-
esis of several human diseases have received increasing attention
lately, especially within biological, medical, nutritional, and agro-
chemical fields. According to the well-known definition, an antiox-
idant is a substance which when present in a low concentration
significantly prevents or delays oxidation of an oxidizable sub-
strate (Halliwell, Murcia, Chirico, & Aruoma, 1995). In addition,
an antioxidant may be classified as a compound capable of pre-
* Corresponding author. Tel.: +48 89 5233935; fax: +48 89 5234801.
E-mail address: dziel@uwm.edu.pl (D. Zielińska).
(Kohen & Nyska, 2002). A great multiplicity of methods has been
used to evaluate the activity of polyphenols by using different
techniques of inducing and catalysing oxidation and measuring
the end point of oxidation for foods and biological systems
(Magalhaes, Segundo, Reis, & Lima, 2008). More recently, highly
attractive, convenient and sensitive voltammetric and photochem-
iluminescene approaches to study antioxidant activities have been
reported (Zielińska, Szawara-Nowak, Ornatowska, & Wiczkowski,
2007). Flavone C-glucosides, a kind of important constituents of
the flavonoid family present in foodstuffs and nutraceuticals, have
received much attention recently because of their suggested anti-
oxidant and anticancer properties (Zhang, Jiao, Liu, Wu, & Zhang,
2008; Zhang et al., 2005). Orientin (OR) and homoorientin (hOR),
a pair of isomeric compounds, and their 40 -deoxy analogues,
namely vitexin (VT) and isovitexin (iVT), are the main flavone
C-glucosides found in some plants, such as Pterocapus marsupium
tree (Abou-Zaid, Lombardo, Kite, Grayer, & Veitch, 2001), in fruits
of Cucurbitaceae (Yadaw & Singh, 1998), in common (Fagopyrum
esculentum Moench) and tartary (Fagopyrum tataricum Gaertn.)
buckwheat seeds and sprouts (Kim et al., 2007; Watanabe, 2007),
and in Atractylodes japonica leaves (Kim, Jun, Leong, & Chung,
2005). Moreover, VT and iVT are the main C-glucosylflavones in
mung bean (Peng et al., 2008), bamboo and pigeonpea leaves (Fu
et al., 2007; Zhang et al., 2005). Various biological and pharmaco-
logical activities have been attributed to these compounds, such as

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.06.051

hypotensive, anti-inflammatory, anti-spasmodic (Prabhakar, Bamo,
Kumar, Shamsi, & Khan, 1981), antimicrobial (Agnese, Perez, &
Cabrera, 2001), radioprotective effects (Hien, Huong, Hung, &
Duc, 2002) and anti-glycation activities (Gugliucci & Menini,
2002; Lee, Jang, Lee, Kim, & Kim, 2006; Yamaguchi, Ariga, Yoshim-
ura, & Nakazawa, 2000), however no information has been re-
ported on the systematical screening of the antioxidant activity
of flavone C-glucosides. Scare information has been reported on
antioxidant/free radical scavenging activities of these compounds
(Bramami, Aquilano, & Pietta, 2003; Picerno, Mencherini, Lauro,
Barbato, & Aquino, 2003), and on their inhibitory activities on hu-
man low-density lipoprotein (Kim et al., 2005).
Therefore, the aim of this study was to screen the antioxidant
activity of flavone C-glucosides (OR, hOR, VT, iVT) as free radical-
scavenging activity against specific reactive oxygen species (ROS),
against stable, non-biological relevant radicals, and as total reduc-
ing capacity estimated by means of the electrochemical method.
2. Materials and methods
2.1. Chemicals
2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diam-
monium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hy-
droxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and
quercetin (3,30 ,40 ,5,7-pentaxydroxyflavone) were purchased from
Sigma (Sigma Chemical Co., St. Louis, MO, USA). Orientin
(30 ,40 ,5,7-tetrahydroxyflavone-8-glucoside), homoorientin (30 ,40 ,5,
7-tetrahydroxyflavone-6-glucoside), vitexin (40 ,5,7-trihydroxyflav-
one-8-glucoside) and isovitexin (40 ,5,7-trihydroxyflavone-6-gluco-
side) standards (HPLC-grade) were obtained from Extrasynthese
Company Inc. (Lyon, France). Methanol, acetic acid (supra-gradi-
ent) and sodium acetate were from Merck KGaA, Darmstadt, Ger-
many. ACL (Antioxidant Capacity of Lipid-soluble substances) kit
(No. 400.801) for the PCL assay was from Analytik Jena AG (Jena,
Germany). All other reagents of reagent-grade quality were from
POCh, Gliwice, Poland. Water was purified with a Milli-Q-system
(Milipore, Bedford, USA).
2.2. Preparation of solutions of flavone C-glucosides
The chemical structures of the investigated flavone C-glucosides
and quercetin are shown in Fig. 1. Appropriate amounts of the
standards were dissolved in methanol and their concentration
was confirmed by a UV measurement according to Wiczkowski,
R4
1 B
8
HO 9 O 2
7
A C
3
R3 6 10
4 R2
5
OH O
Name R1 R2
quercetin OH OH
orientin OH H H
homoorientin OH H glu
vitexin H H
isovitexin H H
glu: glucose
Fig. 1. Structures of the investigated compounds.
D. Zielińska, H. Zieliński / Food Chemistry 124 (2011) 672–678 673
Nemeth, Buciński, and Piskuła (2003) and Franke, Custer, Arakaki,
and Murphy (2004). The UV spectra of flavone C-glucosides were
identical to those shown recently by Zhang et al. (2005). For the
measurement of the antioxidant activity against ABTS+ radical cat-
ion and for the determination of the reducing capacity by cyclic
voltammetry, exactly 500 lM concentration of each compound
was prepared, whilst for the determination of the antioxidant
activity against DPPH and O2 radicals solutions of 150 lM and
100 lM of each were used, respectively.
2.3. Determination of the antioxidant activity of flavone C-glucosides
against ABTS+ radical cation
The method described by Re et al. (1999) was used to determine
the antioxidant activity of flavone C-glucosides with a minor mod-
ification described below. For measurements, the ABTS+ solution
was diluted with 80% methanol, respectively, to the absorbance
of 0.70 ± 0.02 at 734 nm. For the spectrophotometric assay,
1.48 ml of the ABTS+ solution and 20 ll of the flavone C-glucoside
standard solution (500 lM) or Trolox solution were mixed and
absorbance was measured immediately after 6 min at 734 nm at
30 °C. Appropriate solvent blanks were used in each assay. The
standard curve based on the length of time of the lag phase vs.
Trolox concentration was constructed within the range of
0.1–2.5 mM of Trolox standard solutions in methanol. The mea-
surements were carried out using a temperature-controlled spec-
trophotometer UV-160 1PC with CPS-Controller (Shimadzu, Japan).
2.4. Determination of the antioxidant activity of flavone C-glucosides
against DPPH radical
DPPH scavenging activity was determined using flavone C-glu-
cosides standard solutions (150 lM) or a Trolox solution as de-
scribed previously in detail (Zielińska, Szawara-Nowak, &
Zieliński, 2007). The Trolox standard solutions (concentration
within the range of 0.1–2.5 mM) in methanol were assayed under
the same conditions and then DPPH scavenging activity of the fla-
vone C-glucosides was expressed in terms of Trolox equivalents on
the basis of a reduction in the absorbance of the DPPH solution by
standards at 515 nm. The measurements were carried out using a
temperature-controlled spectrophotometer UV-160 1PC with
CPS-Controller (Shimadzu, Japan).
2.5. Determination of the antioxidant activity of flavone C-glucosides
against superoxide anion radical ðO
2 Þ
R1 The photochemiluminescence (PCL) method was used to mea-
3'
4'
OH sure the antioxidant activity of flavone C-glucosides standard solu-
2'
tions (100 lM) or a Trolox solution against superoxide anion
1'
5' radicals ðO
2 Þ generated from luminol, a photosensitizer, when ex-
6'
posed to UV light. The light source was a mercury discharge lamp
phosphorus coated with the maximal excitation at k = 351 nm. The
antioxidant activity of flavone C-glucosides was determined using
a commercial kit. The detailed protocol was followed as described
previously by Zielińska et al. (2007). The measurements were car-
ried out with a PhotochemÒ apparatus (Analytik Jena, Leipzig,
Germany).
R3 R4
H H 2.6. Measurement of the reducing capacity of flavone C-glucosides in
glu voltammetric experiments
H
H glu A potentiostat/galvanostat G 750 (Gamry Ins., USA) was used for
glu H
voltammetric experiments. A conventional three-electrode sys-
tem: a 3 mm diameter glassy carbon working electrode (BAS MF-
2012), a Ag/AgCl electrode as a reference electrode, and a platinum
as counter electrode, was used in the study. Cyclic voltammetric
674 D. Zielińska, H. Zieliński / Food Chemistry 124 (2011) 672–678
experiments were performed with flavone C-glucosides standard
solutions (500 lM) or Trolox solutions (0.1–5.0 mM) mixed with
0.2 M sodium acetate-acetic buffer (pH 4.5 in 80% methanol) at a
ratio of 1:1 (v/v) according to Cosio, Buratti, Mannino, and Bened-
etti (2006). The sodium acetate-acetic buffer acted also as a sup-
porting electrolyte for the cyclic voltammetry measurements. The
voltammetric experiments were performed at room temperature
using an electrochemical cell (volume 200 ll), to which the ana-
lysed standard solution mixed previously with the buffer solution
was introduced. The cyclic voltammograms were firstly acquired in
the range of 100 to 800 mV and secondly in that of 100 to
1300 mV at a scanning rate of 100 mV s1. Prior to use, the surface
of the glassy carbon electrode was carefully polished with 0.05 lm
alumina paste and ultrasonically rinsed in deionized water, and
after that washed with methanol. This procedure was repeated
after each cycle. For the test purpose, the total charge below the
anodic wave curve of the voltammogram was calculated. The CV
method is actually based on the correlation between the total
charge below the anodic wave of cyclic voltammograms and the
antioxidant activity of the compound investigated. The cyclic vol-
tammograms of Trolox solutions over the concentration range of
0.1–2.5 mM was also acquired in the range of 100 to 800 mV
and that of 100 to 1300 mV, and then the antioxidant activity
of the tested compounds was expressed as Trolox equivalent anti-
oxidant capacity. The total charge under the anodic wave of the
background signal (solvent + supporting buffer) was subtracted
from the total charge under the anodic wave obtained for each
compound and Trolox which were recorded firstly over the range
of 100–800 mV and secondly over that of 100–1300 mV.
2.7. Statistical analysis
Results of the chemical analyses are given as mean values and
the standard deviation of nine independent measurements. The re-
sults were subjected to one-way analysis of variation ANOVA using
Fisher’s Least Significant Difference (LSD) test. The significant dif-
ferences (P < 0.05) were calculated. The correlation analysis was
performed and the Pearson correlation coefficient was calculated.
3. Results and discussion
3.1. Antioxidant activity of flavone C-glucosides determined against
ABTS+ and DPPH radicals
In this study, the antioxidant activity of OR, hOR, VT and iVT
determined as free radical-scavenging activity against stable,
non-biological relevant radicals was expressed as Trolox Equiva-
lents. As it was defined, the antioxidant activity is equal to the mil-
limolar concentration of a Trolox solution having the antioxidant
capacity equivalent to a 1.0 mM solution of the substance under
investigation. The analytical strategy was based on the reduction
in the absorbance of the ABTS+ solution at 734 nm (TEAC; Trolox
Equivalent Antioxidant Capacity assay), whilst the results of the
antioxidant activity provided by scavenging activity of DPPH solu-
tion were based on the reduction in the absorbance of the DPPH
solution at 515 nm (DPPH RSA; DPPH Radical-Scavenging Activity).
In this study, the antioxidant activity of C-glucosylflavones un-
der these spectrophotometric assays is reported for the first time
ever (Table 1). The hOR (luteolin-6-C-glucoside) showed the high-
est ability to scavenge ABTS+ and DPPH radicals followed by OR
(luteolin-8-C-glucoside), whilst VT (apigenin-8-C-glucoside) and
iVT (apigenin-6-C-glucoside) showed a very weak scavenging
activity against ABTS+ and no DPPH scavenging effect. The DPPH
RSA of hOR, OR and Q were higher than the ability to scavenge
ABTS+ radical cations, whilst the respective differences in the anti-
Table 1
The antioxidant activity of flavone C-glucosides in comparison to the antioxidant
activity of quercetin determined by TEAC, DPPH RSA and PCL assays.
Compound/assay Antioxidant activity (mM Trolox)
TEAC DPPH RSA PCL
Orientin 1.56 ± 0.02a 2.38 ± 0.04a 1.99 ± 0.07a
Homoorientin 1.88 ± 0.02b 2.88 ± 0.03b 2.59 ± 0.10b
Vitexin 0.02 ± 0.01c 0.006 ± 0.003c 0.71 ± 0.01c
Isovitexin 0.14 ± 0.01d 0.007 ± 0.004c 0.77 ± 0.03c
Quercetin 2.52 ± 0.01e 2.65 ± 0.03d 1.41 ± 0.06d
Data are expressed as means ± standard deviation (n = 9). Means in a column
related to a respective assay followed by the different letters are significantly dif-
ferent (P 6 0.05).
oxidant activity of VT and iVT were negligible. The antioxidant
activity of Q, used in this study as a representative flavonol, was
in an excellent agreement with that reported by Re et al. (1999)
but was lower in respect to that determined by Rice-Evans, Miller,
and Paganda (1996). The order of the antioxidant activity provided
by the TEAC assay was as follows: Q > hOR > ORoiVT > VT,
whilst that provided by the DPPH RSA assay was: hOR > Q >
ORoiVT ¼ VT. The antioxidant activities of these compounds pro-
vided by both assays were highly correlated (r = 0.96). The antiox-
idant activity of hOR, OR and Q was significantly higher than those
reported for VT and iVT, thus clearly indicating that the antioxidant
properties of these compounds are the most dependent on the free
hydroxyl group forming catecholic set in the B ring of these com-
pounds. This conclusion was supported by a recent study on a
structure–activity relationship (SAR) of flavonoids (Balasundram,
Sundram, & Samman, 2006).
It was previously reported that the antioxidant activity of lute-
olin (TEAC = 2.06) determined by a persulfate decolorisation assay
(Re et al., 1999) and by a myoglobin/ABTS decolorisation assay
(TEAC = 2.10) (Rice-Evans et al., 1996) was lower than those of api-
genin (TEAC = 1.45) and chrysin (TEAC = 1.43) (Rice-Evans et al.,
1996). The values of TEAC determined in this work indicated that
the substitution of glucose at the 6-C and 8-C positions of the A
ring of luteolin slightly decreased the antioxidant activity of OR
and hOR whilst, in contrast, the same substitution at the 6-C and
8-C positions of the A ring of apigenin caused a significant reduc-
tion in the antioxidant activity of VT and iVT. In spite of this find-
ing, it was noted that the substitution of glucose at the 6-C position
of the A ring subsequently increased the antioxidant activity whilst
the substitution of glucose at the C-8 position of the A ring de-
creased the antioxidant activity as it was shown for hOR and iVT
when compared with the TEAC of OR and VT. It can be suggested
that this effect was due to the torsion angle and loss of coplanarity
of luteolin and apigenin-8-glucosides (Pietta, 2000). This conclu-
sion was supported by the recently established structure–activity
relationship for the anti-inflammatory effect of luteolin and its de-
rived glycosides, including also homoorientin, which showed that
the relativities of these compounds against arachidonic acid syn-
thesis and hydrogen peroxide scavenging were dependent on their
molecular structures. It was concluded that the presence of ortho-
dihydroxy groups at the B ring and –OH substitution pattern at C-5
position of the A ring could significantly contribute to the anti-
inflammatory and antioxidant activities of flavonoids (Odontuya,
Hoult, & Houghton, 2005).
Our results are in accordance to the very early report on the
antioxidative activity of some C-glycosylflavones (Budzianowski,
Pakulski, & Robak, 1991). Amongst ten flavonoid C-glycosyl deri-
vates such as OR, hOR, VT, iVT, isovitexin 7,200 -di-O-glucosides, iso-
vitexin 7-O-galactoside-200 -O-glucoside, two different 6,8-di-C-
hexosylapigenins, and two different 6-C-hexosyl-8-C-pentosylapi-
genins, only OR and hOR were shown to display the antioxidant
activity. More recently, Mun’im, Negishi, and Ozawa (2003)
 D. Zielińska, H. Zieliński / Food Chemistry 124 (2011) 672–678
showed strong anti-peroxidative activities of OR and hOR towards
linoleic acid and the protective effect against the bacterial action of
tert-butyl peroxyl radicals. Their activities were nearly equal to
that of epigallocatechin gallate. Moreover, similar activities of OR
and hOR towards DPPH and the inhibition of lipid peroxidation
was shown by Cheel, Theoduloz, Rodriguez, & Schmeda-Hirsch-
mann, 2005.
Currently, it is well recognised that the structural features and
the nature of substitutions on rings B and C determine the antiox-
idant activity of flavonoids. It includes: (a) the degree of hydroxyl-
ation and the positions of the –OH groups in the B ring, (b) the
presence of hydroxyl groups at the 30 -, 40 - and 50 -positions of the
B ring which has been reported to enhance the antioxidant activity
of flavonoids as compared to those that have a single hydroxyl
group, (c) a double bond between C-2 and C-3, conjugated with
the 4-oxo group in the C ring which enhances the radical scaveng-
ing capacity of flavonoids, (d) a double bond between C-2 and C-3,
combined with 3-OH in the C ring which also enhances the radical
scavenging capacity of flavonoids, whilst additional hydroxyl
groups at positions 5 and 7 of the A ring appear to be less impor-
tant (Balasundram et al., 2006; Rice-Evans et al., 1996). However,
the presence of a single hydroxyl group in the B ring appears also
unimportant because the antioxidant activity of apigenin (40 ,5,7-
trihydroxyflavone) and chrysin (5,7-dihydroxyflavone) was exactly
the same (TEAC = 1.45) (Arts, Hallinga, Voss, Haenen, & Bast, 2003;
Rice-Evans et al., 1996). Therefore, it can be concluded that a weak
antioxidant activity of VT and iVT may originate from hydroxyl
groups at positions 5 and 7 of the A ring, as shown in our study
by means of the TEAC assay.
3.2. Antioxidant activity of flavone C-glucosides provided by the PCL
method
The antioxidant activity of OR, hOR, VT and iVT in relation to Q
was evaluated by using the PhotochemÒ device, and the commer-
cial kit supplied by Analytik Jena AG. The PhotochemÒ device is the
first system applicable to determine the antioxidant activity of
individual compounds. It combines the very fast photochemical
excitation of radical generation with the highly sensitive lumino-
metric detection. Because of the high sensitivity of the chemilumi-
nescence of luminol, only nanomolar concentrations of non-
enzymatic antioxidant substances are required to observe the
photochemiluminesce effect. The principles of the assay have been
described recently (Besco et al., 2007). In this study, the methanol
stock solution of the standards was diluted in methanol according
to the PCL measurement requirements. The antioxidant activity of
C-glucosylflavones represents their ability to scavenge O 2 radicals
generated from luminol, a photosensitizer, when exposed to UV
light (Popov & Lewin, 1999). The results are compiled in Table 1.
All tested C-glucosylflavones as well as Q exhibited an ability to
scavenge O 2 radicals. The highest scavenging activity was found
for hOR and OR, whilst about threefold lower one for VT and iVT.
The order of the antioxidant activity provided by the PCL assay
was as follows: hOR > OR > Q > iVT = VT. The higher antioxidant
activity of OR and hOR in comparison to the lower activity of Q
indicates that the substitution of glucose at the 6-C and 8-C posi-
tions of the A ring of apigenin may increase the ability to scavenge
O
2 . The results obtained for hOR and OR indicate also that the sub-
stitution of glucose at the 6-C position of the A ring subsequently
increased the ability to scavenge O
2 whilst the substitution of glu-
cose at the C-8 position of the A ring decreased the antioxidant
activity, as it was shown for OR and VT. This finding was in agree-
ment to that noted for free radical scavenging of these compounds
against ABTS+ and DPPH radicals. Moreover, the antioxidant activ-
ities of these compounds provided by PCL were correlated with
those determined by TEAC (r = 0.71) and DPPH RSA (r = 0.88). The
675
order of the antioxidant activity provided for these four flavone
C-glycosides was consistent with that reported by Watanabe
(2007) who showed that hOR and OR scavenged O 2 produced in
the xanthine/xanthine oxidase system in a dose-depend manner,
whilst the scavenging activities of VT and iVT were substantially
low. Our findings support also a very early investigation of O 2
scavenging properties of 38 flavonoids in a non-enzymatic system
isolated from Sideritis mugronensis, Sideritis javalambrensis and Cay-
aponia tayuya (Huguet, Manez, & Alcaraz, 1990). It was shown that
OR and hOR behaved as potent scavengers able to inhibit nitroblue
tetrazolium reduction.
Polyphenolic compounds such as flavonoids are of great interest
for their radical-scavenging activity. The O 2 generated in living
cells by a single-electron reduction of oxygen under physiological
conditions are of special interest because they play harmful roles
as precursors of more reactive oxygen species, contributing to
pathological diseases (Halliwell et al., 1995). The benefits of flavo-
noids as chemopreventive dietary or dietary supplemental agents
are still only ‘‘potential”. Much has been learnt about possible
mechanisms of action of these agents, but whether they can reach
their multiple intended sites of action, particularly in humans, is
largely unknown (Walle, 2004). Recently, poor absorption of these
four compounds has been shown based on a metabolic study
(Zhang, Tie, Bao, Wu, & Zhang, 2007). Such poor absorption charac-
teristic of flavone C-glucosides in vivo indicated that they may have
potentials on pharmacological activities. It may indeed be true
since almost 30 years ago VT, isolated from the flowers of Ochro-
carpus longifolius L. and Arnebia hispididdima Dc., exhibited potent
hypotensive, anti-inflammatory and anti-spasmodic (nonspecific)
properties (Prabhakar et al., 1981). The hypotensive effect of VT
was attributed to its ganglion-blocking properties and anti-inflam-
matory effects to its anti-histaminic, anti-bradykinin and anti-
serotonin properties. Therefore, it may be suggested that these ef-
fects as well those reported for anti-glycation activities of VT and
iVT may be associated with their antioxidant function correspond-
ing to a resorcinol group in the A ring, which is in agreement with
the suggestion made by Peng et al. (2008).
3.3. Antioxidant activity of flavone C-glucosides provided by the cyclic
voltammetry method
In this study, cyclic voltammograms of the compounds exam-
ined were recorded in the range of 100 to +800 mV and that of
100 to +1300 mV at a scanning rate of 100 mV s1. Cyclic voltam-
mograms of 250 lM solutions of OR, hOR, VT, iVT, and Q in 0.1 M
acetate-acetic buffer (pH 4.5) in 90% methanol are shown in
Fig. 2a–e. The selection of these two ranges of applied potentials
was due to the clear discrimination of the electrochemical oxida-
tion process corresponding to the oxidation of 30 ,40 -dihydroxyl
moiety at the B ring of OR, hOR and Q, and that noted at a higher
potential corresponded to the oxidation of 5,7-dihydroxyl moiety
at the A ring of OR, hOR, VT, iVT and Q. The cyclic voltammograms
of flavone C-glucosides standards recorded in the range of 100 to
+800 mV showed that OR, hOR and Q have well-defined reversible
waves with oxidation peak potentials of 0.43, 0.43 and 0.34 V (vs.
Ag/AgCl), whilst no oxidation peaks were observed in relation to
VT and iVT (Fig. 2a–e). Therefore, taking into account the values
of the first oxidation potential of the studied C-glucosylflavones,
OR and hOR can be ranged as compounds with a high (intermedi-
ate) antioxidant power (Ep < 0.8 V), whilst VT and iVT as com-
pounds with a low antioxidant power (0.8 V < Ep < 1.3 V). This
conclusion was withdrawn according to the recent work by Blasco,
Rogerio, Gonzalez, and Escarpa (2005) in which differentiation of
the antioxidant power of phenolic compounds was based on values
of their oxidation potentials. Our conclusion was supported when
cyclic voltammograms of the examined compounds were recorded
676 D. Zielińska, H. Zieliński / Food Chemistry 124 (2011) 672–678

25 25
(a) OR (b) hOR
20 20

15 15

10 10

I/µ A
I/ µ A
5 5
0 0

-5 -5
-10 -10
-0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 -0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3
E/V vs. Ag/AgCl E/V vs. Ag/AgCl

20 20
(c) VT (d) iVT
15 15

10 10
I/ µ A

I/µ A
5 5

0 0

-5 -5
-0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 -0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3
E/V vs. Ag/AgCl E/V vs. Ag/AgCl

30
(e) Q
25
20
15
I/µ A

10
5
0
-5
-10
-0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3
E/V vs. Ag/AgCl

Fig. 2. Cyclic voltammograms of 0.25 mM of standards solution (final concentration) of (a) OR, (b) hOR, (c) VT, (d) iVT and (e) Q in 0.1 M sodium acetate-acetic buffer (pH 4.5)
in 90% methanol recorded from 100 to +800 mV and from 100 to +1300 mV; scan rate 100 mV s1.

in the range of 100 to +1300 mV (Fig. 2a–e). In this case, OR, hOR, der of the antioxidant activity was as follows: Q > OR = hOR, and
VT, iVT and Q showed the second oxidation peak occurring at showed no antioxidant activity of VT and iVT under those exper-
potentials of 1.14, 1.07, 1.05, 1.04 and 1.14 V, respectively, corre-
sponding to an irreversible reaction which involves the 5,7-dihydr-
Table 2
oxyl group. These findings clearly indicate that the catechol group
The reducing activity of flavone C-glucosides in comparison to the reducing activity of
in the B ring was more easily oxidizable than the resorcinol group quercetin determined by cyclic voltammetric method.
in the A ring. The provided electrochemical behaviour of these
Compound Reducing activity (mM Trolox)
compounds was consistent with that observed recently for Q (Brett
& Ghica, 2003; Timbola, De Souza, Giacomelli, & Spineli, 2006; Zie- Range of applied potential Range of applied potential
100–800 mV 100–1300 mV
lińska, Nagels, & Piskuła, 2008) and chrysin (Janeiro, Corduneanu, &
Brett, 2005), and had an impact on the antioxidant activity of the Orientin 0.68 ± 0.07a 1.79 ± 0.06a
Homoorientin 0.67 ± 0.02a 2.01 ± 0.07b
investigated compounds.
Vitexin <LOD 1.13 ± 0.09c
When the calculation of the antioxidant activity was based on Isovitexin <LOD 1.18 ± 0.09c
the area under the anodic current waveform within the range Quercetin 1.38 ± 0.01b 2.65 ± 0.12d
from 100 to 800 mV for each compound and Trolox, then the anti-
Data are expressed as means ± standard deviation (n = 9). Means in a column fol-
oxidant activity reflect only the activity corresponding to the cat- lowed by the different letters are significantly different (P 6 0.05). Operative con-
echol group in the B ring and a double bond between C-2 and C-3, ditions: final concentration of each standard (250 lM); 0.1 M sodium acetate–
conjugated with the 4-oxo group in the C ring. In this case, the or- acetic buffer (pH 4.5); scan rate 100 mV s1.
 D. Zielińska, H. Zieliński / Food Chemistry 124 (2011) 672–678
80
2.50 mM
70
60 1.25 mM
50 0.50 mM
40
I / µA
30
20
10
0 ond oxidation potentials and was useful for the antioxidant activ-
-10
-0.1 0.1 0.3 0.5 0.7 0.9 1.1
E / V vs. Ag / AgCl
Fig. 3. Selected cyclic voltammograms of a Trolox solution within the range of
0.10–2.5 mM in 0.1 M sodium acetate–acetic buffer (pH 4.5) in 90% methanol
recorded from 100 to +1300 mV; scan rate 100 mV s1.
imental conditions (Table 2). This order, similar to that provided
by TEAC and DPPH RSA assays, provides evidences for understand-
ing the free radical-scavenging activity of OR and hOR and a lack
of such an activity shown for VT and iVT against ABTS+ and DPPH
radicals. In contrast, when calculation was made within the range
of 100–1300 mV and then compared to Trolox (Fig. 3), all these
four C-glucosylflavones showed the antioxidant activity within
the range of 1.13–2.01 TEAC, which were lower than TEAC of Q
(Table 2). This finding was in agreement to the quality data of
the antioxidant activity of these compounds provided by the PCL
assay. The antioxidant activity of OR and hOR derived from CV
experiments reflected the presence of the catechol group in the
B ring, a double bond between C-2 and C-3, conjugated with the
4-oxo group in the C ring and the resorcinol group in the A ring,
whilst the antioxidant activity of VT and iVT corresponded to
the single hydroxyl group in the B ring, a double bond between
C-2 and C-3, conjugated with the 4-oxo group in the C ring and
the resorcinol group in the A ring (Fig. 1). Since the first oxidation
peak at a lower potential (100–800 mV) was observed only for OR
and hOR (Fig. 2a and b) and the second oxidation peak was noted
for all the four C-glucosylflavones at higher potentials (800–
1300 mV) (Fig. 2a–d), the simple explanation of the antioxidant
activity of OR and hOR in comparison to VT and iVT cannot be
based only on the presence of a catechol and/or resorcinol group
of C-glucosylflavones, even if a simple flavone as chrysin is also
characterised by the first and second oxidation peaks only (Janeiro
et al., 2005). The results obtained for hOR and iVT indicate also
that the substitution of glucose at the 6-C position of the A ring
subsequently increased their antioxidant activity when compared
to the antioxidant activity of OR and VT, respectively. This finding
was in agreement to that noted for free radical scavenging of these
compounds against ABTS+ and O 2 radicals. The substitution of
glucose at the 6-C and 8-C positions of the A ring had no effect
on the antioxidant activity of OR and hOR when CV tracing was
done from 100 to 800 mV. Therefore, it may be suggested that
in order to evaluate the antioxidant activity of C-glucosylflavones
by the CV method, the cyclic voltammograms need to be recorded
up to 1300 mV.
The antioxidant activities of OR, hOR, Vt, iVT and Q provided by
CV were correlated with those determined by TEAC (r = 0.98) and
DPPH RSA (r = 0.88) but not with PCL results (r = 0.56). Therefore,
it should be taken into account that variation in media, substrates,
oxidants, evaluating indices and detecting methods may greatly
677
influence the behaviour of antioxidants and their evaluation in dif-
ferent systems, which may account for the data presented in this
study.
4. Conclusion
In this study, the order of the antioxidant activity provided by
the TEAC assay was as follows: Q > hOR > ORoiVT > VT, whilst
that determined by the DPPH RSA assay was hOR > Q > ORo
iVT ¼ VT. The tested by PCL C-glucosylflavones as well as Q exhib-
ited an ability to scavenge O 2 radicals according to the rank:
hOR > OR > Q > iVT = VT. The application of CV showed dependence
of the antioxidant activity of these compounds on the first and sec-
ity–structure relationship discussion of these compounds. This
study showed the importance of the free hydroxyl groups in the
1.3
B ring to the antioxidant properties of these compounds with min-
or significance of the substitution of glucose at the 6-C and C-8 po-
sition of the A ring .
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