Food Research International 40 (2007) 714

www.elsevier.com/locate/foodres

Biomimetic oxidation of quercetin: Isolation of a naturally

occurring quercetin heterodimer and evaluation of its
in vitro antioxidant properties
Aytac Gulsen, Dimitris P. Makris *, Panagiotis Kefalas
Department of Food Quality Management & Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania (M.A.I.Ch.),
P.O. Box 85, 73100 Chania, Greece
Received 28 March 2006; accepted 14 July 2006

Abstract
Quercetin was oxidized with potassium ferricyanide under alkaline conditions, a reaction that has been previously shown to generate
the same set of products deriving from oxidation by mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD). The major
oxidation product was isolated and characterized by means of 1H NMR and LCMS, and it was found to be a quercetin heterodimer [1,3,11a-trihydroxy-9-(3,5,7-trihydroxy-4H-1-benzopyran-4-on-2-yl)-5a-(3,4-dihydroxyphenyl)-5,6,11-hexahydro-5,6,11-trioxanapthacene-12-one], occurring in onion skins. Its in vitro antioxidant properties were evaluated in comparison with the parent molecule
and other natural polyphenols, employing three representative tests and the alterations in the activity observed were discussed on the
basis of structureactivity relationships. Biomimetic chemical oxidations of natural polyphenols may help understand oxidation pathways in plant food commodities and evaluate the consequences on their nutritional value.
 2006 Elsevier Ltd. All rights reserved.
Keywords: Antiradical activity; Hydrogen peroxide scavenging activity; Hydroxyl free radical scavenging activity; Onions; Oxidation; Polyphenols;
Potassium ferricyanide; Quercetin

1. Introduction
Over the past 10 years, researchers and food manufacturers have become increasingly interested in polyphenols,
as they comprise a wide area of natural substances of plant
origin, almost all of them exhibiting a marked antioxidant
activity (Scalbert, Manach, Morand, Remesy, & Jimenez,
2005). The main reason for this interest is the recognition
of their great abundance in human diet, and their probable
role in the prevention of various diseases associated with
oxidative stress, such as cancer, cardiovascular disorders
and other degenerative processes.
Improper post-harvest processing and storage of plant
tissues and products may lead, in some instances, to oxida*

Corresponding author. Tel.: +30 28210 35056; fax: +30 28210 35001.
E-mail address: dimitris@maich.gr (D.P. Makris).

0963-9969/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2006.07.009

tion of certain components which can aect the overall

quality of foods. Browning, which is a result of polyphenol
oxidation occurring in plant foods, is generally regarded as
a detrimental process. However, in certain cases, the most
characteristic being black tea, native plant polyphenols
(avanols) are extensively transformed into oxidation
products (theaavins, thearubigins, etc.) that largely dene
the quality of the commodity. The oxidation reactions that
take place during tea fermentation alter substantially the
polyphenolic prole by decreasing the content of avanols
(Tanaka & Kouno, 2003), which are well characterized
antioxidants, yet black tea appears to exhibit antioxidant
properties that are comparable to those of green tea (Frei
& Higdon, 2003; Hashimoto et al., 2003). Thus polyphenol
oxidation in plant foods, although may bring about undesirable organoleptic features, merits further investigation
with regard to the consequences on the nutritional value.

A. Gulsen et al. / Food Research International 40 (2007) 714

Quercetin is a avonol that is ubiquitous in edible plant

material, and has been a subject of detailed examination
with respect to its antioxidant and other biological properties (Hollman & Katan, 1999; Rajnarayana, Reddy,
Chaluvadi, & Krishna, 2001). Oxidative enzymes that
occur in plant tissues, such as polyphenol oxidase (PPO)
and peroxidase (POD), have been shown to act on quercetin, yielding three major oxidation products, which were
also possible to generate by oxidation with potassium ferricyanide under alkaline conditions (Makris & Rossiter,
2002a). As a further step for elucidating the reaction pathway(s), the investigation presented herein aimed at characterizing one of the major quercetin oxidation products,
generated by potassium ferricyanide oxidation, with the
view to exploiting similar reactions for generating specic
polyphenol oxidation products.
2. Materials and methods
2.1. Chemicals
All solvents used for HPLC and LCMS analyses were of
HPLC grade. Caeic acid, gallic acid, catechin, quercetin,
butylated hydroxytoluene (BHT), rutin (quercetin 3-Orutinoside), EDTA, luminol (3-aminophthalhydazine),
and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, 9,10dimethylanthracene (9,10-DMA), imidazole, and bis(2,3,6trichlorophenyl) oxalate (TCPO) were from Sigma Chemical
Co. (St. Louis, MO, USA). Hydrogen peroxide and cobalt(II) chloride hexahydrate were from (Merck Germany).
2.2. Quercetin oxidation with potassium ferricyanide
Three hundred and forty milligrams quercetin was dissolved in 80 mL acetonitrile in a 200 mL conical ask
and 20 mL of a solution containing 50 mM potassium ferricyanide and 50 mM sodium carbonate was added dropwise over 30 min time period at room temperature, under
continuous stirring. The nal concentrations of potassium
ferricyanide, sodium carbonate and quercetin in the reaction mixture were 10 mM. After addition of the oxidizing
agent the ask containing the reaction mixture was
wrapped with aluminium foil to avoid any possible light
induced eect and left under stirring for 3 h. Following
this, the pH of the reaction mixture was brought to 2 with
concentrated HCl and concentrated under vacuum
(T 6 40 C) until all acetonitrile was removed. The remaining aqueous solution was extracted with ethyl acetate/
diethyl ether (8/2 v/v) (4 40 mL), and the extracts were
pooled and dried over magnesium sulphate. The dried
extract was concentrated to dryness in vacuo and dissolved
in MeOH.
2.3. Isolation of the oxidation product
An appropriate aliquot of the methanolic solution
prepared as described above, was applied onto cellulose

column (40 180 mm) and eluted successively with 15%

aqueous acetic acid and 15% aqueous acetic acid/isopropanol (3:1 v/v). The elution was monitored by cellulose
TLC using aqueous 15% acetic acid/isopropanol (5:1, v/v)
as developing solvent. The fractions containing the
product were collected, concentrated to dryness under
vacuum (T 6 40 C), and further dried over magnesium
sulphate.
2.4. HPLC analysis
The equipment utilized was an HP 1090, series II liquid
chromatograph, coupled with an HP 1090 diode array
detector and controlled by Agilent ChemStation software.
The column was a LiChrosphere RP18, 5 lm, 250 4 mm
(Merck), protected by a guard volume packed with the same
material. Both columns were maintained at 40 C. Eluent
(A) and eluent (B) were 2.5% aqueous acetic acid solution
and MeOH, respectively. The ow rate was 1 mL min1,
and the elution programme used was as follows; 05 min,
80% A; 525 min, 80% B; 2530 min, 80% B. Monitoring
of the eluate was performed at 278 nm.
2.5. Liquid chromatographymass spectrometry
A Finnigan MAT Spectra System P4000 pump was
used coupled with a UV6000LP diode array detector
and a Finnigan AQA mass spectrometer. Analyses were
carried out on a Superspher RP-18, 125 2 mm, 4 lm,
column (Macherey-Nagel, Germany), protected by a
guard column packed with the same material, and maintained at 40 C. Analyses were carried out employing
electrospray ionization (ESI) at the positive ion mode,
with acquisition set at 12 and 80 eV, capillary voltage
3.5 kV, source voltage 4.9 kV, detector voltage 650 V
and probe temperature 450 C. All other conditions were
as for HPLC analyses, except that ow rate was set at
0.33 mL min1.
2.6. 1H NMR
The spectra were recorded on a Bruker DRX, 400 MHz,
using DMSO-d6 and CD3OD as solvents.
2.7. Evaluation of the in vitro antioxidant properties
2.7.1. Antiradical activity (AAR)
A method previously described by Brand-Williams,
Cuvelier, and Berset (1995) was used. Dierent dilutions
of each component tested were prepared in MeOH, and
0.05 mL of the diluted sample was mixed with 1.95 mL
DPPH solution (7 105 M in MeOH). The decrease
in the absorbance was determined at 515 nm when the
reaction reached a plateau. The exact initial DPPH concentration in the medium was calculated from the
equation:

A. Gulsen et al. / Food Research International 40 (2007) 714

A515 27:153  C DPPH mg mL1 0:0002

2.8. Statistics

as determined by linear regression. For each concentration,

the percentage of DPPH remaining at the steady state was
calculated according to the following equation:

All measurements were carried out at least in triplicate,

unless otherwise specied, and values averaged and given
along with the standard deviation (SD). Calibration curves
for the antioxidant tests were established using simple linear regression analysis. All statistics were performed with
MicrosoftTM Excel.

%DPPHrem

DPPH T
 100;
DPPH T 0

where T is the time to reach the steady state. These

values were plotted vs moles antioxidant/moles DPPH,
to obtain the amount of sample necessary to decrease
the initial DPPH concentration by 50% (EC50 value).
Antiradical eciency (AE) of samples was calculated as
1
.
EC50
2.7.2. Hydroxyl free radical scavenging activity (SAHFR)
A highly sensitive, luminol chemiluminescence method
was used, as described by Parejo, Codina, Petrakis, and
Kefalas (2000), with some minor modications. One mL
of borate buer solution (50 mM, pH = 9) containing
Co2+ (0.4 mg mL1) and EDTA (2 mg mL1) was rst
mixed with 0.1 mL of luminol solution (100 lg L1,
0.56 mM in borate buer) and vortexed for 15 s. An aliquot
of 0.025 mL of sample and 0.05 mL of H2O2 solution
(5.4 mM) were then added into the test tube, the solution
was thoroughly mixed and rapidly transferred into 10 mm
glass cuvette, and the CL measurements were recorded.
The instantaneous reduction in the CL intensity, elicited
by the addition of the sample, was recorded as I, and the
CL intensity in the absence of the sample was recorded
as I0. For all experiments, the ratio I0/I was calculated
and plotted vs sample concentration (lM). From the
equation established after linear regression analysis, the
concentration of sample, which is required to decrease
the CL intensity by 50% (IC50), was calculated. The hydroxyl free radical scavenging activity (SAHFR) was termed
as IC150 .
2.7.3. Hydrogen peroxide scavenging activity (SAHP)
A previously established methodology (Arnous, Petrakis, Makris, & Kefalas, 2003) based on peroxyoxalate
chemiluminescence, was employed. An aliquot of 1.8 mL
9,10-dimethylanthracene (9,10-DMA) [0.5 mM in EtOAc/
acetonitrile (9:1)] was mixed with 0.2 mL imidazole
solution [4.5 mM in EtOAc:MeCN (9:1)] and 0.025 mL
H2O2 (2.25 mM in MeCN). This mixture was called as
mixture A. In a 1 cm path length cuvette, 0.2 mL of
bis(2,3,6-trichlorophenyl) oxalate (TCPO) (0.45 mM in
MeCN) and 0.05 mL of test compound were placed,
and mixture A was immediately added and vortexed for
5 s. Chemiluminescence intensity was continuously
monitored until it reached a plateau. Reference samples
were tested by adding 0.05 mL of solvent instead of antioxidant solution. SAHP was determined as described for
SAHFR.

3. Results
3.1. Generation and characterisation of quercetin
heterodimer
Quercetin oxidation by potassium ferricyanide was
performed under conditions previously suggested (Makris
& Rossiter, 2002a), which favour the generation of
relatively higher amounts of an oxidation product termed
as Q-DP2 (Fig. 1). Liquid chromatographyelectrospray
ionization mass spectrometry analysis of the isolated
product, performed at positive ion mode, showed a
molecular ion at m/z 603 [M + H]+ and further fragmentation induced by increased source voltage resulted in
major daughter ions of m/z 303 [M  300 + H]+ and
273 [M  330 + H]+, reported in order of abundance
(Fig. 2). The ion m/z 303 was assigned to quercetin. The
ion m/z 273 could result from quercetin quinone cleavage,
formed through autoxidation and/or disproportionation,
after a further step of CO removal, yielding the benzofuran derivative. On the basis of these observations the
structure was tentatively assigned to a quercetin dimer.
The 1H NMR data obtained using both DMSO-d6 and
DMSO-d6/CD3OD as solvents were consistent with those
previously reported (Hirose, Fujita, & Nakayama, 1999;
Krishnamachari, Levine, & Pare, 2002) on a doubly
linked quercetin heterodimer (QHD), suggesting that the
component is actually a mixture of two regioisomers
(Fig. 3).
3.2. Assessment of in vitro antioxidant characteristics
The antioxidant potential of the QHD was assessed
employing three representative tests: the antiradical eciency (AE), the hydroxyl free radical scavenging activity
(SAHFR) and the hydrogen peroxide scavenging activity
(SAHP). For a more descriptive evaluation, the tests
were carried out on a comparative basis with other, well
studied antioxidants (Table 1). QHD exhibited the weakest
AE, which was 6.2 times lower than that of the parent molecule (quercetin). Based on this test, the order of activity
was
Quercetin > gallic acid > rutin > catechin > caffeic acid
> BHT > QHD:

10

A. Gulsen et al. / Food Research International 40 (2007) 714

Fig. 1. Chromatograms showing similarities in the prole of a quercetin solution treated with (a) mushroom tyrosinase, (b) horseradish peroxidase, and
(c) with potassium ferricyanide/sodium carbonate reagent (after Makris and Rossiter, 2002a, 2002b). Q, quercetin; Q-DP 1, 2, 3 and 4, quercetin
degradation products.

Likewise, QHD also possessed the weakest SAHFR of all

antioxidants tested, the overall rank being as follows:
Gallic acid > caffeic acid > quercetin > catechin > rutin
> BHT > QHD:

Contrary to these ndings, QHD was proven more

potent than catechin and caeic acid in scavenging
hydrogen peroxide, although less so compared with
quercetin and rutin. In this test, the order of ecacy
was

A. Gulsen et al. / Food Research International 40 (2007) 714

11

Fig. 2. Fragmentation pathway proposed for the quercetin heterodimer, employing electrospray ionization mass spectrometry at positive ion mode.

Quercetin > gallic acid > rutin > QHD > catechin
> caffeic acid:
4. Discussion
Potassium ferricyanide has been used for model oxidations of mixtures of gallic acid and epicatechin-3-O-gallate
(ECG) to produce a benzotropolone derivative similar to
those occurring in black (fermented) tea (Bailey, Nursten,
& McDowell, 1993). The structural elucidation of such a

derivative, termed theaavate A, generated from ECG oxidation by potassium ferricyanide and isolated from tea
(Wan et al., 1997), provided sound evidence that potassium
ferricyanide mimics oxidations performed during tea fermentation, which are catalysed by PPO and POD (Finger,
1994).
Quercetin oxidation by potassium ferricyanide was
shown to mimic the action of mushroom PPO and horseradish POD yielding the same set of products (Makris &
Rossiter, 2002a). One of these products was isolated
and identied as a doubly linked quercetin heterodimer

12

A. Gulsen et al. / Food Research International 40 (2007) 714

OH

HO

O
OH

5'

4'

HO

HO
6'

3'
2'

4*

OH

OH

6*

5''

HO

5*

8*

OH

OH

OH

7*

OH

OH

O
HO

1''
4''

7
6

3''

HO

3*

6''
2*

1'

2''

OH

Regioisomer 1:

Regioisomer 2:

HO

OH

Fig. 3. Structures of the two regioisomers of the quercetin heterodimer.

Table 1
Antioxidant properties of the quercetin heterodimer, as compared with the
parent molecule (quercetin) and other, well established polyphenolic
antioxidants
Compound

AEa

SAHFRb

SAHPc

Quercetin
Rutin
Catechin
Quercetin heterodimer
Gallic acid
Caeic acid
BHT

19.64 0.99
11.45 0.15
8.64 0.04
3.17 0.23
12.82 1.53
7.72 0.37
3.96 0.23

0.67 0.01
0.58 0.02
0.66 0.01
0.32 0.04
0.91 0.04
0.77 0.03
0.41 0.02

2.21 0.02
0.48 0.01
0.20 0.00
0.46 0.00
1.80 0.12
0.08 0.00
NT

OH

HO
O
O

HO
O

OH

O
HO

OH

NT, not tested.

a
Antiradical eciency.
b
Hydroxyl free radical scavenging activity.
c
Hydrogen peroxide scavenging activity.

Fig. 4. Proposed mechanism of quercetin oxidation leading in the

formation of the heterodimer (after Krishnamachari et al., 2002).

(QHD) (Fig. 3). Early studies indicated that a strict

requirement for oxidation of some chalcone derivatives
by potassium ferricyanide is the presence of a hydroxyl
group in the B-ring (Dean & Podimuang, 1965). Oxidation
of various avonoids with potassium ferricyanide (Pelter,
Bradshaw, & Warren, 1971) further conrmed that only
the B-ring is acted upon, and no reaction takes place if
hydroxyl groups on the B-ring are methylated. These
observations strongly indicate that modications in the
B-ring of the avonoid skeleton are primarily involved in
the oxidation mechanism. QHD has been proven to derive
from radical oxidation of quercetin (Krishnamachari et al.,
2002), and a possible route of generation has been proposed (Fig. 4). In a following study it was indeed suggested
that the presence of a free C-3 hydroxyl, along with a
B-ring ortho hydroxy unit appear essential for dimer formation (Krishnamachari et al., 2002). Therefore it can be
supported that potassium ferricyanide oxidation of quercetin proceeds via the same pathway, which is also obeyed in
the case of enzyme-catalysed (either PPO or POD) oxidation. Quercetin oxidations performed with POD have also
been demonstrated to result in cleavage of the avonol
skeleton, giving protocatechuic and phloroglucinol carboxylic acids, as well as dimers and trimers (Schreier & Miller,
1985). These products were also detected in hydroxyl free

radical oxidation of quercetin (Makris & Rossiter,

2002b). The presence of QHD was demonstrated in onion
peels together with an array of other quercetin oxidation
products (Furusawa et al., 2003; Ly et al., 2005), including
protocatechuic acid, a quercetin trimer, and other quercetin conjugates.
Antioxidant testing performed using autoxidation of
methyl linoleate showed that the antioxidant activity of
QHD is inferior to that of parent molecule (Ly et al.,
2005). In the tests performed in this study (Table 1) it
was shown that QHD possesses inferior antiradical eciency and hydroxyl free radical scavenging activity compared with quercetin, its rutinoside rutin and several
other polyphenolic antioxidants. The obvious explanation
for this low ability is that the component moieties of
QHD have abolished part of conjugation (the C2C3 double bond) and the ortho dihydroxy function at C300 C400
(Fig. 3). Both these structural features are considered very
important for the expression of antioxidant activity,
because they contribute to radical scavenging, metal chelating and stabilisation of the phenoxyl radical produced by
hydrogen donation (Arora, Nair, & Strasburg, 1998; Chen,
Zhu, Hu, & Zhu, 2002; Rice-Evans, Miller, & Paganga,
1997; Silva et al., 2002). As a result, QHD appears less
ecient than quercetin. Since both AE and SAHFR actually

Dimer

A. Gulsen et al. / Food Research International 40 (2007) 714

measure the ability of an antioxidant for hydrogen atom

donation, the fact that QHD was ranked as the least eective provides an evidence for its radical quenching potential.
Regarding the hydrogen peroxide scavenging, QHD was
also less active than quercetin and rutin but its activity
surpassed those of catechin and caeic acid. Structure
activity relationships of simple benzoic and cinnamic acid
derivatives (Mansouri, Makris, & Kefalas, 2005) indicated
that the structural requirements for ecient radical scavenging may not be favourable for hydrogen peroxide scavenging. Thus hydrocaeic acid was demonstrated a better
hydrogen peroxide scavenger than its unsaturated analogue, caeic acid. Furthermore, vanillic acid (one phenolic
hydroxyl) was much more eective than protocatechuic
(two ortho phenolic hydroxyls) and gallic acid (three ortho
phenolic hydroxyls). On the basis of these considerations,
the activity of QHD should not be interpreted taking into
account only criteria that hold true for radical scavenging.
Hotta et al. (2002) demonstrated that the high antioxidant potency observed for several polyphenols may be
ascribed to chemical reactions following their oxidation.
In this regard, polyphenol oxidation products may merit
further investigation as biological antioxidants. Furthermore, as the biological activity does not always depend
on or relate with the antioxidant characteristics, assays
involving in vitro cell cultures and in vivo examinations
for absorption and bioavailability should be carried out,
to fully assess the biological properties and functionality
of polyphenol oxidation products.
5. Conclusions
The isolation and structural elucidation of a quercetin
oxidation product further conrmed that potassium ferricyanide oxidation mimics the pathway followed by PPOand POD-catalysed oxidation. The oxidation product was
found to be a quercetin heterodimer (QHD), which can
also be generated by radical oxidation, and represents a
natural product occurring in the outer dry layers of onion
bulb. The detailed examination of its in vitro antioxidant
characteristics showed that QHD is less active than its parent molecule. Similar observations may help understand
the role of oxidative enzymes in the quality of processed
foods, as this may be illustrated by the transformations
of bioactive constituents.
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