Flavonoid oxidation by the radical generator AIBN: a unified mechanism for quercetin radical scavenging.

Article abstract of DOI:10.1021/jf020045e

Four oxidized flavonoid derivatives generated from reacting quercetin (a pentahydroxylated flavone) with the peroxyl radical generator 2,2'-azobis-isobutyronitrile (AIBN) were isolated by chromatographic methods and identified by NMR and MS analyses. Comp

Full text of DOI:10.1021/jf020045e

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J. Agric. Food Chem. 2002, 50, 4357−4363 4357
Flavonoid Oxidation by the Radical Generator AIBN: A Unified
Mechanism for Quercetin Radical Scavenging
VENKAT KRISHNAMACHARI, LANFANG H. LEVINE,† AND PAUL W. PAREÄ *
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409
Four oxidized flavonoid derivatives generated from reacting quercetin (a pentahydroxylated flavone)
with the peroxyl radical generator 2,2′-azobis-isobutyronitrile (AIBN) were isolated by chromatographic
methods and identified by NMR and MS analyses. Compounds included 2-(3,4-dihydroxybenzoyl)-
2,4,6-trihydroxy-3(2H)-benzofuranone (2); 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-trioxanaphthacene-12-one (3); 2-(3,4-
dihydroxybenzoyloxy)-4,6-dihydroxybenzoic acid (4); and methyl 3,4-dihydroxyphenylglyoxylate (5).
Product ratios under different hydrogen ion concentrations and external nucleophiles revealed that
two of the products, namely the substituted benzofuranone (2) and the depside (4), are generated
from a common carbocation intermediate. Indirect evidence for the operation of a cyclic concerted
mechanism in the formation of the dimeric product (3) is provided. The identification of these products
supports the model that the principal site of scavenging reactive oxygen species (ROS) in quercetin
is the o-dihydroxyl substituent in the B-ring, as well as the C-ring olefinic linkage.
KEYWORDS: Quercetin; peroxyl radicals; AIBN; oxidizing agents; flavonols; antioxidant
INTRODUCTION
By chemical or enzymatic oxidation, several reaction products
have been reported which provide direct evidence as to reactive
site(s) of flavonoid antioxidant activity (4).
Flavonoids are naturally occurring polyphenolics present in
fruits and vegetables and are an integral part of the human diet
(1). Consumption of flavonoid-rich foods is inversely correlated
with the risk of coronary heart disease, and the antioxidant or
free-radical-scavenging property of flavonoids has been pro-
posed to contribute to this chemopreventive effect (2). Radical-
absorbing capacities of flavonoids have been estimated by
measuring the loss of fluorescence of â-phycoerythrin, an
indicator of oxidative damage; flavonoids compete with the
fluorescent indicator to quench peroxyl radical species, and
reduce oxidation of â-phycoerythrin. On the basis of such
studies, flavonoid oxygen-radical absorbing capacity increases
with the number of hydroxyl substitutions in the skeleton.
Using fast reaction kinetics techniques, spectral changes for
a variety of hydroxylated flavonoids and the corresponding
aroxyl radical species allow identification of the position of
primary radical attack (i.e., formation of the aroxyl radical) and
the stability of the radical species formed (i.e., decay of the
aroxyl radical) (3). Three reactive centers have been identified
by rate constant measurements as important for flavonoid radical
scavenging potential: the o-dihydroxyl structure in the B-ring;
the 2,3 double bond in conjugation with the 4-oxo function in
the C-ring; and the 3- and 5-hydroxyl groups with hydrogen
bonding to the keto group (for flavonoid numbering see Figure
1).
The picture emerging from these studies is that the oxidation
of flavonoids is complex, resulting in a wide variety of, in many
cases, unidentified reaction products. The objectives of this study
were to identify products generated from the oxidation of
quercetin with the peroxyl radical generating agent AIBN, and
to compare the levels of products formed under different reaction
conditions to provide insight into the mechanism of quercetin
radical scavenging.
MATERIALS AND METHODS
Chemicals. Quercetin (dihydrate, 99%) was obtained from Acros
Organics (Fischer Scientific; Pittsburgh, PA). AIBN (2,2′-azobis-
isobutyronitrile) was purchased from Sigma-Aldrich Chemical Co (St.
Louis, MO). Acetonitrile and methanol were HPLC grade and other
chemicals were reagent grade.
Oxidation with AIBN (Condition 1). AIBN (1.97 g, 12.0 mmol,
20 equiv) was added to a solution of quercetin (200 mg, 0.6 mmol) in
CH3CN (250 mL). The temperature of the reaction mixture was
maintained at 60 °C for 3 h. Acetonitrile was then removed under
reduced pressure, and the resulting solid was washed with hexane to
remove the unreacted AIBN.
Purification of Oxidized Products. The quercetin oxidation reaction
was run 10 times, and the pooled products were passed through a silica
gel (70-230 mesh) column (55 cm × 2 cm) eluting first with CHCl3
and then CHCl3 with increasing concentrations of MeOH. Fractions
with common TLC profiles were pooled and subsequently run through
sephadex LH-20 columns (55 cm × 2 cm) eluted with MeOH. Repeated
column chromatography over sephadex LH 20 yielded compounds 2
(60 mg), 3 (200 mg), 4 (52 mg), and 5 (16 mg).
* Corresponding author. Tel: 806 742-3062. Fax: 806 742 1289.
E-mail: Paul.Pare@TTU.edu.
† Current address: Dynamac Corporation, Kennedy Space Center, FL
32899.
10.1021/jf020045e CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/18/2002

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4358 J. Agric. Food Chem., Vol. 50, No. 15, 2002
Krishnamachari et al.
and acidic fractions. Preparative TLC of the neutral fraction yielded
the heptamethyl derivative.
Methylation of Depside (4). To a solution of 4 (25 mg, 82 µmol)
in acetone, K2CO3 (46 mg, 4 equiv) and dimethyl sulfate (5 equiv, 38
µL) were added, and the resulting mixture was refluxed for 8 h. The
crude pentamethyl derivative obtained was purified by Sephadex LH-
20 (40 cm × 2 cm) eluted with methanol.
1H and 13C NMR Spectroscopy. 1H, 13C, and 2D 1H-13C correlated
NMR spectra were obtained in DMSO-d6/CD3CN/CD3OD using a
Bruker DRX 500 MHz instrument, operating at 500 MHz (1H) and
125 MHz (13C).
LC-MS. Analyses were performed on a Thermo Separation
Products HPLC system coupled with a photodiode array (PDA) detector
and a Thermo Finnigan (San Jose, CA) LCQ deca mass spectrometer
in sequence. A 250 × 2.1 mm i.d., 5 µm Altima C18 column (Alltech)
was used for separation using the same mobile phase gradient program
described in the product quantification section. Trifluoroacetic acid
(0.1%) or formic acid (1%) was used as a mobile phase modifier to
facilitate ionization. A flow rate of 0.2 mL/min was used and directly
directed to a PDA detector, then to a quadrupole ion trap mass
spectrometer via an electron spray ionization or atmospheric pressure
chemical ionization (APCI) interface. The mass spectrometer was
operated in a negative mode; MS/MS was performed as needed.
Compound 2. 1H NMR (DMSO-d6, 500 MHz): 5.89 (d, J ) 2 Hz,
1H, H-6), 5.94 (d, J ) 2 Hz, 1H, H-8), 6.79 (d, J ) 8 Hz, 1H, H-5′),
7.53-7.55 (m, 2H, H-2′ and H-6′), 8.62 (s, 1H, OH), 9.37 (s, 1H, OH),
9.98 (s, 1H, OH), 10.79 (s, 1H, OH), 10.85 (s, 1H, OH).
13C NMR (DMSO-d6, 125 MHz): 90.34 (C-8), 96.56 (C-6), 100.47
(C-4a), 104.57 (C-3), 114.89 (C-5′), 117.33 (C-2′), 123.82 (C-6′), 124.99
(C-1′), 144.73 (C-3′), 151.36 (C-4′), 158.53 (C-8a), 168.46 (C-5), 171.86
(C-7), 189.87 (C-4), 190.24 (C-2).
Compound 2b (water adduct). 1H NMR (CD3OD, 500 MHz): 0.96
(t, J ) 7 Hz, 3H, CH3), 3.25-3.31 (m, 1H, CH2), 3.37-3.42 (m, 1H,
CH2), 5.96 (d, J ) 2.5 Hz, 1H, H-6/H-8), 5.97 (d, J ) 2.5 Hz, H-8/
H-6), 6.78 (d, J ) 8.5 Hz, 1H, H-5′), 7.02 (dd, J ) 8.5 Hz, 2.5 Hz,
1H, H-6′), 7.14 (d, J ) 2.5 Hz, 1H, H-2′).
Figure 1. Quercetin (1) and oxidized products generated from the reaction
of 1 with the radical generator AIBN.
13C NMR (CD3OD, 125 MHz): 15.31 (CH3), 59.92 (CH2), 95.08
(C-3), 97.06 (C-6/C-8), 97.44 (C-8/C-6), 101.31 (C-4a), 107.56 (C-2),
115.24 (C-5′), 117.49 (C-2′), 121.84 (C-6′), 127.05 (C-1′), 145.41 (C-
3′), 147.18 (C-4′), 160.45 (C-8a), 165.16 (C-5), 168.70 (C-7), 194.14
(C-4).
The TLC conditions were CHCl3/MeOH/HOAc in the ratio of 75:
25:0.1 or 80:20:0.1.
Modified Oxidation Conditions. Quercetin (100 mg, 0.3 mmol)
was dissolved in a fixed volume of 250 mL and varied ratios of
acetonitrile/methanol/ethanol/concentrated HCl. To this solution AIBN
(984 mg, 6.0 mmol, 20 equiv) was added, and the reaction mixture
was stirred at 60 °C for 3 h. Differences in the reaction mixtures are
noted as follows. For condition 2 (low pH) 100 µL of concentrated
HCl was added to a solution of quercetin in acetonitrile (250 mL). For
condition 3 (external nucleophile) 10 mL of MeOH was added to the
solution of quercetin in acetonitrile (240 mL). For condition 4 (low
pH with external nucleophile) 100 µL of concentrated HCl was added
to a solution of quercetin in acetonitrile (240 mL) and methanol (10
mL). For condition 5 (low pH with external nucleophile) 100 µL of
concentrated HCl was added to a solution of quercetin in acetonitrile
(240 mL) and ethanol (10 mL).
Product Quantification and Data Analysis. The oxidized products
were quantified by a Hewlett-Packard 1100 series HPLC system
equipped with a 250 × 4.6 mm i.d., 5 µm (Alltech) Econosphere C18
column and variable wavelength UV detector. A 2-mg aliquot of
4-hydroxybenzoic acid as an internal standard (ISTD) was added to
reaction mixtures at the end of the reaction. The components of the
reaction mixture (10 µL) were eluted with water containing 0.1% CF3-
COOH/MeOH (v/v) gradient and detected at 254 nm. The mobile phase
gradient program started with 40% methanol was maintained at 40%
for 15 min, increased linearly to 80% within 25 min, and kept at 80%
for another 10 min. The flow rate was set to 0.5 mL/min. Analysis of
variance procedure was run using SAS statistical software (5). Means
were separated using Tukey’s mean separation method at a P value
less than 0.05.
Compound 3. 1H NMR (DMSO-d6, 500 MHz): 2.06 (s, OH), 5.97
(bs, 2H, H-6, H-8), 6.19 (d, 1H, J ) 2 Hz, H-8/H-6), 6.46 (d, 1H, J )
2 Hz, H-6/H-8), 6.68 (dd, J ) 8.5 Hz, 2.5 Hz, 1H, H-6′), 6.92 (d, J )
8.5 Hz, 1H, H-5′), 7.14 (bs, 1H, H-2′), 7.26 (d, J ) 9 Hz, 1H, H-5′),
7.79 (d, J ) 2 Hz, 1H, H-2′), 7.85 (dd, J ) 8.5 Hz, 2 Hz, 1H, H-6′),
8.92 (s, OH), 9.16 (s, OH), 9.29 (s, OH), 9.70 (s, OH), 10.85 (s, OH),
11.14 (s, OH), 12.36 (s, OH). (Tentative assignments based on 1H-1H
COSY, HMQC, HMBC, and ROESY spectra in comparison with
quercetin.)
13C NMR (CD3CN, 125 MHz) (tentative assignments based on 1H-
1H COSY, HMQC, HMBC, and ROESY spectra in comparison with
quercetin.): 91.16 (C-3), 94.82 (C-8), 97.50 (C-8*), 98.28 (C-6*), 99.25
(C-6), 100.88 (C-4a), 101.43 (C-2), 104.39 (C-4a*), 115.80 (C-5′),
116.20 (C-2′), 117.60 (C-2′*), 118.30 (C-5′*), 121.50 (C-6′), 123.70
(C-6′*), 126.26 (C-1′), 127.09 (C-1′*), 137.43 (C-3), 141.46 (C-3′*),
142.65 (C-4′*), 144.97 (C-3′), 145.22 (C-2), 147.31 (C-4′), 157.79 (C-
8a), 160.59 (C-8a*), 162.01 (C-5), 164.73 (C-7), 165.06 (C-5), 168.90
(C-7*), 176.51 (C-4), 188.62 (C-4*).
1
Heptamethyl Derivative of 3. H NMR (CDCl3, 500 MHz): 3.78
(bs, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.86 (s, 6H,
2 OCH3), 3.87 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.11 (d, J ) 2 Hz,
1H, H-6), 6.16 (bs, 1H, H-8), 6.35 (d, J ) 2.5 Hz, 1H, H-6), 6.43 (d,
J ) 2.5 Hz, 1H, H-8), 6.79 (d, J ) 8.5 Hz, 1H, H-5′), 7.19 (d, J ) 8
Hz, 1H, H-5′*), 7.28 (dd, J ) 8.5 Hz, 2 Hz, 1H, H-6′), 7.35 (d, J )
2.5 Hz, 1H, H-2′), 7.85-7.87 (m, 2H, H-2′*, H-6′*).
Compound 4. 13C NMR (DMSO-d6, 125 MHz): 100.99 (C-4a),
101.73 (C-8), 104.62 (C-6), 116.19 (C-5′), 118.06 (C-2′), 122.39 (C-
1′), 124.39 (C-6′), 145.56 (C-3′), 151.33 (C-4′), 154.64 (C-8a), 164.15
(C-7), 166.07 (C-2), 166.11 (C-5), 172.79 (C-4). ESI-MS (negative
mode): 304.9 (M+ - 1), 168.9 (M+ - C6H5(OH)2CO).
Methylation of Dimer (3). To a solution of dimer (3) (50 mg, 83
µmol) in methanol, an excess of diazomethane (ca. 16 mmol) in ether
was added, and the reaction mixture was allowed to stir overnight. The
crude reaction mixture was then fractionated using NaOH into neutral

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Quercetin Radical Scavenging
J. Agric. Food Chem., Vol. 50, No. 15, 2002 4359
Figure 2. Partial dimer structure with dioxane linkage for the AM1 optimized regioisomers of an octamethyl dimer derivative of quercetin.
1
Pentamethyl Derivative of 4. H NMR (CDCl3, 500 MHz): 3.67
(s, 3H, COOCH3), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.93 (s,
3H, OCH3), 3.95 (s, 3H, OCH3), 6.39 (Aps, 2H, H-6, H-8), 6.92 (d, J
) 8.5 Hz, 1H, H-5′), 7.61 (d, J ) 2 Hz, 1H, H-2′), 7.79 (dd, J ) 8.5
Hz, 2 Hz, 1H, H-6′). 13C NMR (CDCl3, 125 MHz): 52.14 (COOCH3),
55.65 (ArOCH3), 56.09 (2 ArOCH3), 56.25 (ArOCH3), 96.89 (C-6/8),
100.13 (C-8/6), 109.39 (C-4a), 110.39 (C-5′), 112.35 (C-2′), 121.48
(C-1′), 124.52 (C-6′), 148.78 (C-3′), 151.06 (C-8a), 153.63 (C-4′),
159.43 (C-5), 162.38 (C-7), 164.38 (C-2/C-4), 165.05 (C-4/C-2).
Compound 5. 1H NMR (CD3OD, 500 MHz): 3.92 (s, 3H,
COOCH3), 6.86 (d, J ) 8.5 Hz, 1H, H-5′), 7.36 (dd, J ) 8.5 Hz, 2 Hz,
1H, H-6′), 7.38 (d, J ) 2 Hz, 1H, H-2′). 13C NMR (CD3OD, 125
MHz): 52.96 (COOMe), 116.28 (C-5′), 116.56 (C-2′), 125.64 (C-6′),
125.77 (C-1′), 147.11 (C-3′), 154.49 (C-4′), 166.72 (COOMe), 186.84
(CdO). CI-MS (positive): 197 (M+ + 1), 137 (M+ - COOMe). CI-
HRMS (positive): observed 197.044602; C9H9O5 requires 197.044999.
RESULTS AND DISCUSSION
Quercetin (1) was oxidized by thermolysis of AIBN in
oxygenated acetonitrile (condition 1). To ensure that all oxidized
quercetin derivatives were produced from AIBN generated
radicals, reactions devoid of AIBN or heating were also
analyzed. Without the full compliment of reagents, neither the
formation of products nor loss of quercetin was observed. Time-
course studies carried out on the reaction revealed the presence
of quercetin at the end of 1-h and 2-h intervals and its complete
disappearance at the end of 3 h. The disappearance of quercetin
was accompanied by the formation of a complex mixture of
products, of which, at least four prominent UV absorbing
products could be readily detected by TLC and HPLC. These
oxidized products, isolated pure (TLC/HPLC) by column
chromatography, were identified on the basis of various spectral
analyses. The fused heterocyclic ring of quercetin served as the
Figure 3. Atmospheric pressure chemical ionization mass spectra of the
reactive center for all the oxidation products detected with an
substituted 3(2H)benzofuranone (2), and the methoxylated (2a) and
accompanying loss or gain of a substituent to the original C-ring
ethoxylated adducts (2b).
unit (Figure 1).
In the least-modified of the products generated from the
reaction, the pyranone C-ring of quercetin was converted to a
furanone-carbonyl derivative. This benzofuranone derivative (2,
Figure 1) has been observed previously in a two-electron
electrochemical (6) and a metal ion mediated oxidation (7) of
quercetin. Our NMR and mass spectral data for 2 (Figure 3)
were in agreement with the published values (6). Furthermore,
our long-range C-H correlation data (HMBC) were consistent
with those reported by Jungbluth et al. (7), showing that the
compound formed has in fact a 3(2H)benzofuranone skeleton
and not a benzopyranone skeleton.
The other major product formed from the oxidation of
quercetin was 3 (Figure 1), a dimer that has been observed
previously during the autoxidation of methyl linoleate solutions
containing quercetin (8). Our spectral data are in agreement with
those reported by Hirose et al. (8). However, because the
oxidative coupling of two quercetin units (the o-dihydroxyl
group of the B-ring of one quercetin unit and the conjugated
olefinic linkage (C-2, C-3) of the C-ring of the other) potentially
gives rise to two regioisomers with either a C2-C3′*/C3-C4′*
or a C2-C4′*/C3-C3′* dioxane linkage (see Figure 2 for the
dioxane linkage), we confirmed the structure by a series of NOE
experiments. As the two quercetin subunits present in 3 are
bridged through insulating oxygen atoms, the nature of this
linkage could not be directly assigned. The absence of protons
associated with the linking carbons of the two subunits also
precluded any long-range carbon-proton NMR analyses of 3.
The difficulty in assigning all of the hydroxyl groups in 3 also
made ROESY studies unfeasible. The structure of 3 was earlier
assigned on the basis of a differential NOE observed for its
methylated derivative (8). However, uncertainty in the spatial
orientation of the B/B* ring protons relative to the tertiary
methoxyl at C-3 ruled out a definitive interpretation of the NOE
interactions. To circumvent the uncertainty we carried out AM1
calculations (9), using Gaussian 94 (10) on theoretical regioi-
someric octamethyl derivatives of 3 (shown as 6A and 6B in

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4360 J. Agric. Food Chem., Vol. 50, No. 15, 2002
Krishnamachari et al.
Figure 2). AM1 is the most successful semiempirical model
for predicting molecular geometry. This method parametrizes
for a list of atoms including H, C, and O and partly reproduces
intermolecular hydrogen bonds which is critical for biological
molecules (11, 12). These Gaussian calculations provided
optimized structures (Figure 2) from which the spatial distances
between protons present in the two quercetin subunits were
obtained. These distances, in turn, were used to unambiguously
assign the NOE interactions that were observed in the ROESY
spectrum of the heptamethyl derivative of 3, prepared by
methylating 3 using diazomethane. The calculations revealed
that for the tertiary methoxyl group at C-3 in 6A, the spatially
closest B* ring proton was H-2′* at 4.5 Å (the spatial distance
for H-5′* being 6.5 Å), whereas in 6B, H-5′* (the B* ring
proton) was found to be the closest to the tertiary methoxyl
group at 4.6 Å; H-2′* was 6.6 Å away from the tertiary methoxyl
group. These calculated distances, in conjunction with the cross-
peak observed in the ROESY spectrum of the heptamethyl
derivative of 3 between the C-3 methoxyl group and H-2′*,
indicated the presence of a C2-C4′*/C3-C3′* regioisomer and
in turn confirmed the nature of the dioxane linkage in 3.
Furthermore, the cross-peak observed in the ROESY spectrum
of the heptamethyl derivative, between the protons of the C-3
methoxyl and H2′/H6′ of the same quercetin subunit, also
established the presence of an R,R and S,S pair of stereoisomers.
On the basis of the molecular modeling studies and spectral
data, the dimer (3) was assigned the stereochemistry as shown
(3, Figure 1).
Table 1. HPLC Peak Areas for the Oxidized Products of Quercetin
under Four Different Conditionsa
+
+
CH CN
3
CH CN/H
CH CN/MeOH CH CN/MeOH/H
3
3
3
product (condition 1) (condition 2)
(condition 3)
(condition 4)
2
2a
3
1.83 ± 0.08b
ND
0.95 ± 0.21
4.86 ± 0.24
1.49 ± 0.09
ND
0.60 ± 0.09
4.47 ± 0.35
3.40 ± 0.02
ND
3.39 ± 0.41
3.43 ± 0.17
0.35 ± 0.04
2.68 ± 0.25
1.99 ± 0.42
1.53 ± 0.13
c
4
a Values (× e4) represent mean peak areas from four replications. b Represents
standard error. c ND, not detected.
methoxylated and the ethoxylated adducts of quercetin were
observed by LC-MS (Figure 3) and MS-MS analyses. The
quercetin-methanol adduct was not purified in sufficient
quantities for NMR analyses. However, the structure of the
quercetin-ethanol adduct was confirmed by a series of NMR
experiments. Long-range C-H correlations observed for the
carbon signal at δ107 with the CH2 protons of the ethoxy moiety
and with those of the B ring protons, established the presence
of a C-2 ethoxy adduct. The signal at δ 95, for C-3, in the 13
NMR spectrum of 2b indicated the addition of water to the
carbonyl at C-3. The other signals showed the expected 1H and
13C chemical shifts.
C
The level of substituted 3(2H)benzofuranone (2) and depside
(4) decreased significantly in the presence of an external
nucleophile at reduced pH (condition 4) compared to that of
acetonitrile alone (p < 0.0001). This decrease in accumulation
of products 2 and 4 was accompanied by the appearance of the
methoxylated adduct (2a). Interestingly there was no significant
difference in dimer (3) formation with an external nucleophile
at reduced pH or simply at a reduced pH compared to that of
acetonitrile alone. It was also observed that acidification of
reaction mixture (condition 2) did not result in any significant
changes in the accumulation of products 2 and 4 (p < 0.0001).
In contrast, the addition of the external nucleophile methanol
(condition 3) enhanced the accumulation of products 2 and 3
(p ) 0.0003), and reduced accumulation of compound 4.
Oxidation of flavon-3-ols has been studied under several
oxidizing conditions, including peroxidase oxidation (4), two-
electron copper ion mediated oxidation (7, 16, 17), and DPPH/
CAN treatment (18). Oxidation in nonpolar systems usually
occurs via H-atom donation, and in aqueous media occurs by
electron donation (19). Quercetin probably acts a chain-breaking
antioxidant, which traps peroxyl radicals and thus suppresses
radical chain oxidation. Oxidation mechanisms are thought to
vary with the oxidizing agent, and the mechanism described
for a specific oxidizing agent may not directly apply to
biological antioxidant systems (20). In addition, under some
metal ion conditions it can be difficult to control chemical
oxidation because these oxidizing agents are not specific and
can react with already oxidized products. Advantages of using
azo radical generators such as AIBN for probing oxidation
mechanisms include the selectivity with which the radical
generator yields peroxyl radicals, the ability to control oxidation
rates by changing temperature and/or radical generator concen-
trations, and the stability of the oxidized product (21). Because
azo compounds decompose thermally without biotransformation
they are well suited as radical sources for in vivo systems or to
mimic peroxyl radical reactions that occur in biological systems
in vitro (22).
The first indication of a cleaved C-ring structure for 4 (Figure
1), generated from the oxidation of quercetin (condition 1), was
the absence of signals corresponding to the C2 and C3 carbons
in the 13C NMR spectrum. The methylation of 4 with dimethyl
sulfate generated a pentamethoxy derivative with the 1H NMR
chemical shifts of the methyl groups indicating the presence of
four phenolic hydroxyls and a carboxylic acid. The mass spectral
data for 4 and its methoxyl derivative were consistent and
confirmed the structural assignments. This phenolic carboxylic
acid ester, a depside, has been reported under different oxidation
conditions such as singlet oxygen and superoxide anion radical
(13, 14), as well as in the enzyme system using quercetinase
(15). However, the absence of 13C NMR data in the earlier
reports has raised doubts as to its correct identification (6).
The 1H NMR spectrum of 5, the fourth product of quercetin
oxidation (condition 1), showed the absence of signals corre-
sponding to the A-ring protons and the presence of the methyl
signal of a carbomethoxyl functionality. This evidence combined
with the appearance of two carbonyl functionalities in the 13C
NMR spectrum indicated the presence of an R-keto ester moiety.
The molecular ion peak, [M + H]+, at m/z 197 in the CI-MS
(positive mode) confirmed the structural assignment. The
o-dihydroxy benzoyl unit of this quercetin fragment presumably
corresponds to the B-ring of the parent flavanoid quercetin.
Product 5 to the best of our knowledge has not been observed
earlier, under this or other reported oxidizing conditions. This
may, at least in part, be due to its low abundance and close
association with other polar reaction products.
To elucidate the reaction mechanism for quercetin radical
scavenging, the abundance of the major oxidized products 2,
3, and 4 formed under different hydrogen ion concentration and
external nucleophile were measured (Table 1). The values in
the table represent peak HPLC areas for compounds 2, 2a
(methoxylated adduct), 3, and 4 under conditions 1-4. When
ethanol was used as the external nucleophile, formation of a
quercetin-ethanol adduct was observed. The formation of the
The formation of depsides and substituted 3(2H)benzofura-
nones have been reported in several oxidation systems (6, 7,
13, 14). Electrochemical oxidation of quercetin and kaempferol