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Journal of the Science of Food and Agriculture
J Sci Food Agric 79:1011±1014 (1999)
Electron spin resonance (ESR) spectroscopic
assessment of the contribution of quercetin and
other flavonols to the antioxidant capacity of red
wines
Peter T Gardner,1 Donald B McPhail,2 Alan Crozier3 and Garry G Duthie1*
1
Rowett Research Institute, Aberdeen, AB21 9SB, Scotland, UK
Macaulay Land Use Research Institute, Aberdeen, AB15 8QH, Scotland, UK
3
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ,
Scotland, UK
2
Abstract: Health bene®ts of red wine have been ascribed in part to the antioxidant properties of
quercetin and other ¯avonols. Red wines, however, contain many other polyphenolic compounds with
antioxidant potential. The present study has assessed the antioxidant capacity of seven red wines using
electron spin resonance spectrometry to measure their ability to donate hydrogen atoms to a stable
Fremy's radical. Antioxidant capacity of the wines was strongly related to the total phenol content
(r = 0.872, P < 0.01) but less so to total ¯avonol content (r = 0.651, P < 0.10). The only ¯avonols detected
in the wines by HPLC with post-column derivatisation were the aglycones and conjugated forms of
quercetin and myricetin. These accounted for less than 2% of total phenolic content when expressed as
gallic acid equivalents. Although these ¯avonols showed marked ability to quench Fremy's radical,
they contributed less than 1.5% to the total antioxidant capacity of the wines. Consequently, quercetin
is not a major antioxidant in red wine. The antioxidant activity of other polyphenols may be of greater
importance in contributing to the reputed health bene®ts of moderate wine consumption.
# 1999 Society of Chemical Industry
Keywords: ESR; wine; ¯avonols; quercetin; myricetin; polyphenols; antioxidant capacity; Fremy's salt; free
radical; coronary heart disease
INTRODUCTION
Moderate consumption of red wine is reported to
reduce the incidence of coronary heart disease
(CHD).1±3 This health bene®t has been ascribed in
part to the antioxidant properties of the polyphenolic
compounds in red wine whose extensive conjugated pelectron systems allow ready donation of electrons or
hydrogen atoms from the hydroxyl moieties to free
radicals.4 Such polyphenols may reduce CHD risk by
preventing the oxidation of low density lipoprotein
(LDL) to an atherogenic form, thus inhibiting the
development of the occlusive arterial plaque.5,6
A major source of the antioxidant potential of red
wine is thought to be due to the ¯avonoids (reviewed
by Maxwell7). Attention has focused on quercetin
(2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1benzopyran-4-one; 3,3',4',5,7-pentahydroxy ¯avone)
which possesses strong antioxidant activity in chemical
systems,8 prevents the oxidation of LDL in vitro,9 may
be absorbed from the diet10 and low dietary intakes of
which are associated with increased CHD risk.11 As
well as quercetin, however, there are many other
phenolic compounds in red wine with potential
antioxidant activity, including anthocyanidins, resveratrol, hydroxycinnamic acids and catechin derivatives,
which may have health bene®ts.12±14
Consequently, in order to ascertain whether quercetin was a major determinant of the antioxidant
capacity in wine, the hydrogen/electron donating
ability of seven red wines has been assessed using
electron spin resonance (ESR) spectroscopy. The
ability of the wines to quench Fremy's salt (potassium
nitrosodisulphonate), a semi-stable radical which
responds to good hydrogen donors,14 was related to
the concentrations of total phenols and the antioxidant
activity of individual ¯avonols in the wines.
MATERIALS AND METHODS
Materials
The selected wines (Corbieres, Cabernet Sauvignon,
Minervois, Chianti, CoÃtes du Rhone, Pinot Noir,
* Correspondence to: Garry G Duthie, Rowett Research Institute, Aberdeen, AB21 9SB, Scotland, UK
Contract/grant sponsor: EC; contract/grant number: FAIR-CT95
Contract/grant sponsor: Scottish Office Agriculture, Environment and Fisheries Department
(Received 3 April 1998; revised version 1 September 1998; accepted 9 December 1998)
# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50
1011
PT Gardner et al
Valpolicella) were purchased from a local supermarket. Ethanol (analar grade) was obtained from
BDH (Poole, Dorset, UK). Fremy's salt, quercetin
(dihydrate form), rutin and myricetin were from
Sigma-Aldrich (Poole, UK). Myricitrin was obtained
in the dihydrate form from INDOFINE Chemical
Company Inc (Somerville, New Jersey, USA).
Methods
Determination of radical-scavenging ability of wines
Each of the seven red wines were diluted to 50 ml lÿ1 in
aqueous EtOH, 125 ml lÿ1. Aliquots (3 ml) were
reacted with an equal volume of a solution of Fremy's
radical (1 mM in aqueous EtOH, 125 ml lÿ1). The ESR
spectrum of the low ®eld resonance of the Fremy's
radical tm plot was obtained after 20 min by which
time the reaction was complete. Signal intensity was
obtained by double integration and the concentration
calculated by comparison with a control reaction using
aqueous EtOH, 125 ml lÿ1 instead of wine.
Determination of radical-scavenging ability of individual
¯avonols
Ethanolic solutions of quercetin, rutin, myricetin and
myricitrin were made up to a concentration of 0.2 mM.
Aliquots (3 ml) of each were added to an equal volume
of aqueous EtOH solution 125 ml lÿ1, of Fremy's salt
(1.0 mM.) The ESR spectrum was obtained after
20 min and the signal intensity calculated as above.
The percentage reduction of the radical was calculated
by comparing the signal intensities to that obtained
when 3 ml of ethanol were added to the Fremy's
solution.
Spectra were obtained at 21 °C on a Bruker ECS
106 spectrometer working at ca 9.5 GHz (X-band
frequency) and equipped with a cylindrical (TM110
mode) cavity. The microwave power and modulation
amplitude were set at 2 mW and 0.01 mT, respectively.
All measurements were made in ¯at, quartz cells
(Wilmad Glass, NJ, USA) speci®cally designed for use
with aqueous solutions. Sample volume was 275 ml.
Analysis of ¯avonol content and total phenols
Concentrations of ¯avonols in the wines were
Table 1. ESR-determined antioxidant potential of the red wines, as
determined by the ability of a 5% wine solution to quench the Fremy’s
radical and the total phenol contents of the wines
Wine
Table 2. Myricetin and quercetin
contents (mg lÿ1) as the free aglycone a
or the conjugated glycoside and the
total flavonol content (mg lÿ1) of the red
wines
1012
Pinot Noir
Chianti
Valpolicella
Minervois
Cotes du Rhone
Corbieres
Cabernet Sauvignon
Phenols
(mg gallic acid
equiv lÿ1)
6.78 0.19
7.91 0.19
6.59 0.06
8.53 0.13
7.26 0.05
8.55 0.14
7.94 0.06
1560 14
1666 3.2
1222 18
1780 24
1440 51
1987 19
1894 78
Pinot Noir
Chianti
Valpolicella
Minervois
Cotes du Rhone
Corbieres
Cabernet Sauvignon
a
Expressed as the number of radicals quenched by 1 litre of wine (at 100%
concentration).
measured by HPLC in conjunction with post-column
derivatisation with aluminium nitrate to form ¯uorescent ¯avonol complexes.15,16 The total phenol
contents of the red wines were determined using the
Folin±Ciocalteu method.17
All measurements for wines and individual compounds were made in triplicate and presented as
mean SD. Pearsons regression analysis was performed where appropriate in the estimation of the
contribution of individual compounds to the antioxidant capacity of the whole wine.
RESULTS
All the red wines had marked hydrogen donating
abilities; greatest quenching of Fremy's radicals was by
Corbieres and Minervois (8.5 1021) and least by
Valpolicella (6.6 1021) (Table 1). This ESR-determined antioxidant capacity was strongly associated
with the total phenol content of the wines (r = 0.872,
P < 0.01) but less so with total ¯avonol content of the
wines (r = 0.651, P < 0.10) (Table 2).
The only ¯avonols detected in the wines were
myricetin and quercetin which were present to varying
degrees as both free aglycones and conjugated glycosides (Table 2). The free ¯avonols contributed 22±
60% of the total ¯avonol contents, depending on wine
Myricetin
Wine
Number of Fremy's
radicals reduced
( 1021) a
Quercetin
Free
Conjugated
Free
Conjugated
Total ¯avonols
1.5 0.1
1.6 0.3
0.9 0.1
5.0 0.1
4.0 0.04
7.0 0.01
4.1 0.3
0.8 0.6
2.2 0.2
0.6 0.1
2.4 0.2
1.3 0.3
7.3 0.1
17.9 0.1
1.5 0.1
4.7 0.1
0.5 0.2
4.8 0.3
2.1 0.1
9.9 0.1
7.6 0.4
1.5 0.04
9.6 0.3
3.6 0.1
7.9 0.1
6.4 0.3
12.4 0.3
24.6 1.0
5.3 0.1
18.1 0.3
5.6 0.1
20.1 0.2
13.8 0.7
36.62 0.01
54.2 0.3
a
The free ¯avonols as a % of the total ¯avonols (ie free quercetin ‡ free myricetin/total 100) is as follows: Pinot
Noir 60%; Chianti 35%; Valpolicella 25%; Minervois 49%; Cotes du Rhone 44%; Corbieres 46%; Cabernet
Sauvignon 22%.
J Sci Food Agric 79:1011±1014 (1999)
Antioxidant properties of ¯avonols in red wine
Table 3. Antioxidant potential of individual flavonols, expressed as the number
of Fremy’s radicals reduced by one
molecule of the respective compounds
Flavonoid
Antioxidant potential
Quercetin
Rutin
Myricetin
Myricitrin
1.18 0.12
0.89 0.10
2.24 0.07
1.88 0.06
type (Table 2). Although the precise identity of
¯avonol conjugates in grapes and red wines is unclear,
direct injection of ®ltered samples onto chromatographic columns indicates primarily the presence of
rutin and myricitrin.13 Consequently, these compounds were also used in the assessment of the
antioxidant capacity of the ¯avonol components of
the wines. Cabernet Sauvignon had markedly more
conjugated ¯avonols than the others, whereas Corbieres had the greatest concentration of free ¯avonols.
The ¯avonols, however, accounted for less than 2% of
the total amount of phenolic material in the wines
when expressed as gallic acid equivalents.
The free, aglycone forms of quercetin and myricetin
and their respective glycones (rutin and myricitrin)
were all capable of markedly reducing the Fremy's
radical (Table 3) although substitution of the
3-hydroxyl group for a sugar group did cause a
10±15% decrease in antioxidant ability. From the
compositional data and the antioxidant potential of the
individual compounds, the calculated amount of
radicals reduced that could be ascribed to the ¯avonols
in the wines ranged from 9 1018±100 1018. This
accounts for no greater than 1.5% of the number of
radicals reduced by each of the wines (Table 4).
DISCUSSION
The ability of the red wines to quench the Fremy's
radical was closely related to total phenol content. The
superoxide and nitric oxide radical scavenging ability
and the Trolox equivalent antioxidant capacity
(TEAC) of wines also correlates closely with the total
phenol content18,19 suggesting that hydrogen/electron
donation from the extensive conjugated p-electron
systems of the phenolic constituents is the major
antioxidant mechanism.
Quercetin and other ¯avonols in wines and foods
have marked antioxidant properties20 in a range of
model systems and their ability to inhibit the oxidation
of LDL in vitro has been suggested as a mechanistic
explanation for epidemiological associations relating
moderate red wine consumption to lowered CHD
risk.9,21±24 The only ¯avonols detected in the wines
used in the present study were the aglycone and
conjugated forms of quercetin and myricetin, all of
which were capable of reducing Fremy's radicals.
These accounted for less than 2% of the total phenolic
material in the wines, however. Therefore the number
of radicals each ¯avonol would reduce at concentrations equivalent to that in the wines is correspondingly
small, accounting for less than 1.5% of the total
antioxidant capacity of the wines. Moreover, the
relatively poor correlation between total ¯avonol
content and antioxidant capacity also suggests that
¯avonols are minor antioxidants in red wine.
The results of the present study suggest that while
the ¯avonols in red wine exhibit good antioxidant
potential, they are not present in suf®cient amounts to
be considered the main group of antioxidants. This is
an important observation as there is the general
perception that quercetin and related ¯avonols may
be major nutritional antioxidants. The results of this
study suggest that any cardio-protective effects of
moderate wine consumption are unlikely to be
ascribed solely to the antioxidant effects of quercetin
and myricetin and their conjugates. Other polyphenolic products of the phenylpropanoid biosynthetic
pathway, such as anthocyanidins, catechins, resveratrol and hydroxycinnamic and hydroxybenzoic acids,
may contribute to a greater degree to the antioxidant
properties of the wines.12,14,25 Their signi®cance as
biological antioxidants, however, will remain unclear
until the bioavailability, mechanism of uptake and
consequences of biotransformation by intestinal ¯ora
are established.
ACKNOWLEDGEMENTS
This study was funded by the EC (FAIR-CT95) and
the Scottish Of®ce Agriculture, Environment and
Fisheries Department (SOAEFD).
Table 4. Calculated number of radicals ( 1018) reduced by each of the flavonols in wine, and the percentage contribution of the total flavonols to the antioxidant
potential of the wines
Wine
Pinot Noir
Chianti
Valpolicella
Minervois
Cotes du Rhone
Corbieres
Cabernet Sauvignon
Quercetin
Quercetin conjugates
Myricetin
6.15 0.15
6.87 1.06
3.69 0.51
21.06 0.34
16.80 0.17
29.79 0.04
17.54 1.10
1.83 1.34
5.44 0.39
1.56 0.20
5.90 0.44
3.24 0.76
17.80 0.34
43.60 0.12
3.41 0.19
11.10 0.21
1.13 0.52
11.30 0.75
4.94 0.31
23.30 0.28
17.80 0.94
J Sci Food Agric 79:1011±1014 (1999)
Myricetin conjugates Total ¯avonoids % contribution
1.29 0.04
8.45 0.24
3.18 0.11
6.89 0.5
5.65 0.22
10.86 0.25
21.60 0.90
12.67 1.90
31.78 1.90
9.55 1.34
45.12 1.58
30.66 1.45
81.76 0.91
100.56 3.07
0.19
0.40
0.14
0.53
0.42
0.96
1.27
1013
PT Gardner et al
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