J Sci Food Agric 1996,70, 55-61

Inhibition of In Vitro Human LDL Oxidation by

Phenolic Antioxidants from Grapes and Wines
Pierre L Teissedre,"" Edwin N Frankel,b Andrew L Waterhouse,"? Hanna Peleg" and
J Bruce Germanb
a Departments of Viticulture and Enology, and Food Science & Technology, University of California,
Davis, CA 95616, USA
(Received 8 March 1995; revised version received 17 J uly 1995; accepted 8 August 1995)
Abstract: Current research suggests that wine contains substances that may
reduce the mortality rate from coronary diseases. The oxidation of low-density
lipoprotein (LDL) is thought to be a key step in the development of atheroscle-
rosis. Phenolic fractions of a Petite Syrah wine were evaluated for their anti-
oxidant activity in inhibiting LDL oxidation in vitro. The more active fractions
contained components of the catechin family. The catechin oligomers and the
procyanidin dimers (B,, B, , B,, B, , B8) and trimers ( C, , C,) were extracted,
isolated and purified from grapes seeds. These compounds were tested for their
inhibition of LDL oxidation, along with other monomeric wine phenolics. The
procyanidin dimers B, and B, , and trimer C,, and the monomers catechin, epi-
catechin and myricetin had the highest antioxidant activity. The procyanidin
dimers B, , B, and C, and the monomers gallic acid, quercetin, caffeic acid, and
rutin, and a group of compounds that included the dimer B,, ellagic acid,
sinapic acid, cyanidin had lower antioxidant activity and a-tocopherol had the
least activity. Thus, the numerous phenolic compounds found in wine are potent
antioxidants in inhibiting LDL oxidation in vitro.
Key words : antioxidant, phenolic, LDL, catechin, wine, grape, flavonoid, pro-
cyanidin.
INTRODUCTION
Chronic diseases have been associated with oxidative
damage (Ames 1989). Diets high in fruits and veget-
ables are associated with lower chronic disease rates,
especially coronary diseases and cancer, and the anti-
oxidants vitamin E, p-carotene, and vitamin C have
been investigated as potential constituents that reduce
the mortality (Willett 1994). However, one recent study
correlates increased dietary levels of phenolic com-
pounds and reduced coronary heart disease (CHD)
mortality (Hertog et a1 1992), suggesting that this class
of materials may have some nutritional benefit. High
consumption rates of vitamin E, have also been corre-
lated with reduced mortality from CHD (Rimm et a1
1993). In addition, epidemiological studies have shown
that the consumption of wine correlates with reduced
* Permanent address: Universite de Montpellier I, Faculte de
Pharmacie, 34060 Montpellier Cedex 1, France.
i To whom correspondence should be addressed.
CHD mortality, and this effect has been described as
the 'French paradox' (Renaud and De Lorgeril 1992;
Criqui and Ringel 1994).
A mechanism to explain the potential beneficial
effects of red wine was suggested in a study showing
that the oxidation of human low-density lipoproteins
(LDL) was significantly inhibited by the addition of
wine diluted 1000-fold (Frankel et a1 1993a). Based on
these results we hypothesised that the non-alcoholic
phenolic substances of wine may be responsible for the
'French paradox'. We subsequently demonstrated that
three phenolic compounds known to occur in wine were
potent antioxidants for LDL oxidation (Frankel et a1
1993b), and these substances were more powerful anti-
oxidants than a-tocopherol (vitamin E). The non-
alcoholic components of different wines as well as white
and red grapes also had powerful antioxidant proper-
ties (Kanner et a1 1994).
Free radical oxidation of the polyunsaturated fatty
acid components of LDL appears to play a major role
in the process of atherosclerosis (Esterbauer et a1 1992;
55
J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain
56
P L Teissedre et a1
Steinberg 1992a). Oxidative modification of LDL is pro-
moted by arterial endothelial cells, smooth muscle cells
and macrophages. Oxidation proceeds by interaction of
polyunsaturated fatty acid with active oxygen species
generated by catalytic metal radical initiators. Some
studies indicate that this modification of LDL occurs
primarily through oxidative modification of the apo-
protein, and it is thought that this protein-modified
LDL is the atherogenic form (Steinberg 1992a).
To reduce the chance of arterial damage, current
clinical practice focuses dietary treatment strategies on
modification of dietary fat intake (American Dietetic
Association 1983). However, reduction of fat intake
may not be the sole means of reducing risk of athe-
rogenesis and coronary disease. Several researchers
have demonstrated that oxidised LDL particles in vitro
can be decreased by the presence of antioxidants either
in serum or dissolved in the lipoproteins themselves
(Esterbauer et a1 1991, 1992; J alial and Grundy 1992;
Steinberg 1992b). Therefore, dietary phenolic com-
pounds may protect circulating LDL from oxidation if
they act as antioxidants, providing an explanation for
the reduced rates of cardiac disease in those popu-
lations consuming diets rich in antioxidants (Frankel et
a1 1993a; Kinsella et a1 1993).
In evaluating a large number of commercial wines,
significant variations were found in antioxidant activ-
ities (Frankel et a1 1995). Analysis of the mixtures of
phenolic compounds in commercial wines was too
complex to allow us to interpret the effects of specific
compounds on antioxidant activity. Much work has
been published on the antioxidant activity of mono-
meric phenolic compounds that also occur in wine
(Pratt and Hudson 1990; Frankel et a1 1993b).
However, there is limited information on the anti-
oxidant activity of the oligomeric and polymeric phe-
nolic compounds present in wine. (Ricardo Da Silva et
a1 1991a).
This paper presents a study of antioxidant activity of
selected pure phenolic compounds found in commercial
wines and grapes, using an assay based on the inhibi-
tion of in vitro oxidation of LDL particles. The specific
aim was to obtain a better understanding of the anti-
oxidant effectiveness of individual polyphenolic com-
pounds, and to compare their activity with structures of
these wine and grape phenolic compounds.
EXPERIMENTAL
Materials
Gallic acid, caffeic acid, catechin, epicatechin, sinapic
acid, rutin, ellagic acid (found in Vitis rotundifolia
wine), rutin and quercetin were purchased from Aldrich
(Milwaukee, WI, USA) and Sigma (St Louis, MO,
USA). Cyanidin was provided by V L Singleton
(University of California, Davis, CA, USA). Pro-
cyanidins and dimers B,, B, , B, , B, , B, and pro-
cyanidins trimers C, and C, were obtained from grape
seeds as detailed below.
Wine fractions
A Petit Syrah wine from the 1992 vintage was selected
from the Department of Viticulture and Enology, Uni-
versity of California, Davis cellar. The wine was con-
centrated 10-fold by rotary evaporation at 25C at
0.13 Pa. This concentrate (25 pl) was injected into the
high-performance liquid chromatograph (HPLC)
system described below, except that Solvent A was a
25 mM phosphate buffer adjusted to pH 2.5. Fractions
were collected at 6 min intervals, yielding 10 fractions
of 3 ml each. The methanol in each fraction was evapo-
rated by rotary evaporation at 5C and the final volume
adjusted to 3 ml with water. The total phenol content
of each fraction was determined according to the pro-
cedure of Singleton and Rossi (1965), which uses the
Folin-Ciocalteau reagent to develop an absorption at
765 nm, and the results are expressed in gallic acid
equivalents (GAE).
Extraction and isolation of crude procyanidins
Grape seeds (Vitis vinifera), 150 g, were extracted with
methanol as described by Bourzeix et a1 (1986) and by
Weinges and Piretti (1971). The extract (3 ml, 300 mg)
was chromatographed on Fractogel TSK HW-40(s)
(25-40 pm) (450 x 25 mm id) with methanol as eluant,
using an ISCO (Lincoln, NE, USA) model UA-5
absorbance monitor set at 280 nm, a peristaltic
Miniplus2 pump (Gilson Inc, Middletin, WI, USA)
and an ISCO 328 fraction collector. Ten fractions con-
taining procyanidins were collected.
Isolation of purified procyanidins
Semi-preparative HPLC was performed with a Waters
510 pump (Waters, Miford, MA, USA) a U6K injector,
and a Hewlett-Packard (Palo Alto, CA, USA) model
1050 UV-Vis detector set at 280 nm. The column was a
Waters RCM Novapak C18 25 x 100 mm, 4 pm parti-
cle size. Elution was carried out by a linear gradient of
0-500 ml litre- methanol with the solvents described
below at 2 ml min ~ .
TLC analysis
Silica plates (DC Alufolien-Kieselgel 60, 0.2 mm thick,
Merck, EM Separations Technology, Gibbstown, NJ ,
USA), were developed with toluene/acetone/formic acid
(3:3:1 v/v/v) as described by Lea et a1 (1979). The
Phenolic antioxidants from grapes and wines 57
plates were visualised by spraying with a solution of
vanillin (100 g litre- ') in concentrated HCl.
HPLC analysis
A Hewlett-Packard Model 1090 with three low-pressure
pumps and a diode array UV-Vis detector coupled to
an Hewlett-Packard Chem Station was used for solvent
delivery and detection. A Waters Novapack Cl 8
column, 3.9 x 150 mm, 4 pm particle size, ther-
mostated at 40C was used with a solvent flow of
0.5 ml min-'. The solvents used for the separation
were: solvent A, 30 mM dichloroacetic acid adjusted to
pH 1.5 with sulphuric acid; solvent B, methanol. The
solvent gradient consisted of increasing the concentra-
tion of methanol from 0 at the start to 100 at 7 min,
123 at 19 min, 250 at 27.5 min, 395 at 42 min, 505 at
48 min, 700 at 53 min and 800 ml litre-' at 55 min
with a 3 min hold. Detection was at 280 nm, 313 nm,
520 nm and 620 nm. The retention time of each com-
pound is shown in Table 1.
LDL antioxidant activity
The same assay was used as described previously
(Frankel et a1 1992) to determine the inhibition of
Cut '-catalysed oxidation of freshly prepared human
LDL by monitoring the production of hexanal by head-
space gas chromatography (GC). Blood was collected
by venepuncture in EDTA from two normolipidaemic
volunteers and the plasma was obtained by centrifu-
gation at 1500 x g at 4C. Plasma LDL was prepared
by sequential density ultracentrifugation (Orr et a1
TABLE 1
Analytical HPLC retention times (average R, values)
-~ ~
Phenolic compounds R, (min)
Gallic acid
Procyanidin B3
Catechin
Procyanidin trimer C2
Procyanidin B8
Procyanidin B4
Procyanidin B6
Caffeic acid
Procyanidin B2
Epicatechin
Procyanidin trimer C I
Sinapic acid
Cyanidin
Rutin
Myricetin
Quercetin
4.13
8.91
9.81
10.02
10.4 1
11.11
11.86
12.12
13.77
17.71
20.3 1
28.24
30.47
34.93
36.08
41.64
1991) in the presence of 0.1 g litre-' EDTA. Prior to
oxidation, LDL was thoroughly dialysed with deoxy-
genated phosphate-buffered (10 mM, pH 7.4) saline
(100 mM) for 24 h. The final concentration of each
LDL sample was diluted with phosphate-buffered saline
(10 mM) to the same protein concentration
(1.0 mg m1-I). The antioxidant effect of phenolic com-
pounds dissolved in dimethyl suphoxide was investi-
gated by GC measurement of the hexanal produced.
Duplicate samples of freshly dialysed LDL in
phosphate-buffered saline in the presence of 80 p~
CuSO, were sealed in special 6 ml headspace bottles
and incubated for 2 h at 37C. The headspace bottles
were then transferred into a headspace sampler (Perkin-
Elmer Sigma 3B GC with an H-6 headspace sampler,
Norwalk, CT, USA), pressurised with helium carrier
gas for 30 s and an aliquot of the gas phase (headspace)
was automatically injected directly into the GC.
Analyses were standardized daily with solutions of
10 p~ hexanal. Relative inhibition obtained by different
phenolic compounds and fractions were expressed as
% inhibition =((C - S)/C) x 100
where C is the hexanal formed in control and S is the
hexanal formed in sample. Differences in antioxidant
activity were tested statistically by one-way analysis of
variance (Minitab Statistical software).
RESULTS
The phenolic components of a new Petit Syrah wine
concentrate were separated into 10 fractions, which
were tested for their activity to inhibit LDL oxidation
after dilution to the same phenolic concentrations of
5 p~ GAE. The first eight fractions inhibited LDL oxi-
dation by between 31.1 and 70.6%, and the last two
fractions had no antioxidant activity. The first fraction
contained largely benzoic acids, such as gallic acid, syr-
ingic acid, gentisic acid and p-hydroxybenzoic acid, as
well as cinnamic acids such as caftaric acid, caffeic acid,
coumaric acid and ferulic acid. This fraction inhibited
LDL oxidation by only 43.1%, and fractions 5-8 inhib-
ited oxidation by 31.1-45.1%. Fraction 5 contained a
few of the procyanidin oligomers, while fraction 6 con-
tained flavonols such as rutin, myrecitin and the antho-
cyanins, including cyanidin and malvidin-3-glucoside,
and fraction 7 contained flavonols such as quercetin.
The highest antioxidant activity was observed with
fraction 2 (70.6%), fraction 3 (64.8%) and fraction 4
(60.9%). Fraction 2 contained the flavan-3-01s includ-
ing catechin, procyanidin dimers B,, B, , B, , B, , B,
and trimer C,. Fraction 3 contained epicatechin and
procyanidin B, , and fraction 4 procyanidin C,. Given
the high antioxidant activity of these fractions, the
activity of the phenolics components in these fractions
58
P L Teissedre et a1
Hoyqo$R' &OH : : @3 H
R,
0.
OH
Caffeic Acid. R1, R2 =H
Sinaplc Acld. R1 =CH3. Rp =OCH3
Catechin. R = *OH
Epicatechin. R= ' ' I OH
H o d o H / / >
HO$ HO / OH
OH
?
OH glucose
Cyanidin Gallic Acid
HO. dH / R*
?
OH 0 R,
Quercetin. Rt, R2 =H
Myricetin. R1 =H. Rp =OH
Fig 1. Monomeric wine phenolics
were investigated and compared to a selection of the
other major wine phenolics.
Proanthocyanidins (often referred to as procyanidins)
are oligomers and polymers of polyhydroxy flavan-3-01
units, ( +)-catechin and ( - )-epicatechin, and their
gallate esters. The simplest procyanidins are dimers and
the most common of these in grapes are the four 4 -+ 8
HO
OH R4
8-2: R2. R4 =OH, epi(4-8)epi
OH
8-3. Rt, R3 =OH. cat(4-8)cat
8-4: R1. R4 =OH. cat(4-8)epi
OH
C-1: R2, R4 =OH B - 6 R2 =OH
C-2: R2. R3 =OH B-8: R1 =OH
Unlabe!led R groups =H
Fig 2. Oligomeric wine phenolics
TABLE 2
Inhibition of LDL oxidation by phenolic compounds in wine"
Phenolic Average content Inhibition
compounds (mg litre- l ) b (%)c
Red White 5 PM 10 pM 20 pM
wines wines (GA-Qd ( GAOd
Catechin 191.3 34.9 74.9 k 0.3bc
Myricetin 8. 5 0.0 68.1 f 8.7bc
Epicatechin 82.0 21.2 67.6 k 7.6bc
Rutin 9.1 0.0 67.6 f l5bc
Gallic acid 95.0 6.8 63.3 f 3.4bc
Quercetin 7.7 0.0 61.4 f 1.4b
Caffeic acid 7.1 2.8 58.5 f 12b
Ellagic acid ND ND 36.6 rf: 0.2a
Sinapic acid 1.8 0.1 35.1 It: 0.9a
Cyanidin 2.8 0.0 27.0 f- 4.6a
a-Tocopherol ND ND 32.6 k 1.8a
98.2 k 1. 1~
97.4 f 1. 3~
96.4 1. 5~
98.2 f 0. 2~
71.8 & 3.0b
97.7 f 0. 4~
98.1 f 0. 1~
ND
ND
54.6 & 7.la
54.7 * 7.1 a
98.9 & 0. 2~
97.6 f 0. 9~
ND'
99.1 k 0. 1~
97.8 f 3. 0~
98.7 f 0. 1~
98.5 f 0. 5~
ND
ND
89.4 f 4.2b
73.8 f 3.1a
~~~ ~~ ~~ ~ ~
' Mean values SD in the same columns followed by the same letter are not significantly
different at P <0.05.
Average contents in 14 California red wines and 6 white wines (Frankel r t al 1995).
Inhibition of hexanal formation in human LDL oxidised in the presence of CuZf
Molar concentrations in gallic acid molecular weight equivalents.
ND, not determined.
(Frankel et a1 1992).
Phenolic antioxidants from grapes and wines
59
TABLE 3
Inhibition of LDL oxidation by catechin, epicatechin and
their dimeric and trirneric compounds in wine
-
Phenolic Inhibition
compounds (%y
HPLC fractions
5 I1M 10 /AM
Dimer B2
Dimer B3
Dimer B4
Dimer B6
Dimer B8
Trimer C2
Trimer Cul
Catechin
Epicatechin
79.5 f 0.7cd
67.5 f 2. 1~
51.0 & l l b
36.5 f 3.5a
81.0 f Od
66.0 f 5. 7~
85.5 f 0.7d
82.5 f 0.7d
83.0 f Od
98.0 & 1.4b
98.0 f Ob
95.5 f 0.7b
82.5 f 6.4a
99.0 f Ob
97.0 f 1.4b
99.0 f Ob
98.0 & Ob
98.0 f 1.4b
See footnote c in Table 2.
See footnote di n Table 2.
linked compounds (B1-B,) (Lea et a1 1979). These
4 .+8 linked dimers are typically accompanied by lower
concentrations of the corresponding 4 + 6 linked
isomers (B,-B,) as described by Thompson et a1 (1972),
and Boukharta et al(1988). The procyanidin trimers C,
and C, which have the 4 -+ 8 linkage and those which
have the 4 -+6 linkage (B3-B6) have also been identi-
fied in grapes recently (Ricardo Da Silva et a1 1991b).
The catechin oligomers (procyanidins) from a grape
seed extract were separated by column chromatography
on Fractogel TSK HW-40(s) into 10 fractions that were
further purified by semi-preparative HPLC. The iden-
tity of the procyanidin dimer and trimer fractions were
confirmed by chromatography with authentic reference
samples on TLC silica plates and analytical HPLC. All
samples used were 95-99% pure as determined by
HPLC analysis.
The antioxidant activities of 10 pure phenolic com-
pounds (Figs 1 and 2) in inhibiting LDL oxidation were
compared at different concentrations with that of a-
tocopherol. The most significant differences among
phenolic compounds were found when they were tested
at 5 /AM concentration (Table 2). When compared at
this concentration, the relative activities of the phenolic
compounds decreased in the order given in Table 2.
Catechin was the most active antioxidant of the phe-
nolic compounds tested. The other phenolic compounds
can be grouped by analysis of variance in order of
decreasing antioxidant activity as follows :
catechin >myrecitin x epicatechin x gallic
acid >quercitin z caffeic acid
x cyanidin x sinapic acid x a-tocopherol.
The antioxidant activity of the procyanidin dimers
and trimers are compared with that of catechin and epi-
catechin in Table 3. At 5 p ~ , dimers B, and B, and
acid z rutin >ellagic
trimer C, had the same activity as the monomeric cate-
chins, ranging between 79.5 and 85.5%. However, the
dimers B3 and B, and trimer C, were less active (51.0-
67.5% inhibition) than the monomeric catechins.
DISCUSSION
Wine made from grapes contains numerous phenolic
compounds (Lamuela-Raventos and Waterhouse 1994),
including flavonoids and non-flavonoids. Previous
studies have shown that these types of substances
exhibit antioxidant activity (Pratt and Hudson 1990).
Others have shown the antioxidant activity of mono-
meric phenolics compounds and polymers (Mangiapane
et a1 1992). However, in these studies antioxidant activ-
ity was determined by employing non-specific tests of
oxidation that have a limited ability to measure the oxi-
dation of LDL lipids. In the present study, oxidation of
LDL was determined by analysing hexanal, a specific
volatile oxidation product of n-6 polyunsaturated lipids
(Frankel et a1 1992).
There is a considerable body of literature relating to
the structure of flavonoids and antioxidant activity.
The dihydroxylation in both rings and in the 3-position
in catechin, myricetin and quercetin are required for
antioxidant activity reported in various lipid systems
(Pratt and Hudson 1990). Hydroxylation of the 3,5,7-
positions of the A-ring and 3,4-dihydroxylation of the
B-ring flavone were considered important to anti-
oxidant activity. In a lard system oxidised at 60C the
antioxidant activity of the hexahydroxy myricetin was
greater than that of the pentahydroxy quercetin, fol-
lowed by the pentahydroxy catechin (Mehta and
Seshadi 1959). Using, the bleaching time of j-carotene
in a lard solution, the antioxidant activity of myricetin
was greater than that of quercetin, and the 3-
glycosylation of the 3-hydroxy flavones did not alter
antioxidant activity (Pratt and Watts 1964). Others
have confirmed a relationship between increased
hydroxylation and increased antioxidant activity of
phenolic compounds (Darmon et a1 1990). By alkyl-
ating quercetin in different positions, Letan (1966) con-
cluded that flavonoid antioxidant activity is enhanced
markedly by having free hydroxyl groups at the 5- and
7-positions of the A ring, at the 3- and 4-positions of
the B ring and at the 3-position of the C ring.
To explain the observed differences in inhibition of
LDL oxidation described here, these same structural
features were considered : the number of phenolic
hydroxyl or methoxyl groups, flavone hydroxyl and
keto groups, glycoslylation and other structural fea-
tures. In the LDL oxidation system used, catechin
without a keto group in the 4-position was more inhibi-
tory than the flavonols. Among the flavonols, myricetin
with 3, 4, 5-trihydroxylation of the B ring was more
active than quercetin with dihydroxylation in the B
60
P L Tei ssedre et a1
ring. The greater antioxidant activity of caffeic acid
than sinapic acid confirms that methoxylation of ortho-
dihydroxy groups decreases antioxidant activity. Gly-
cosylation of the 3-hydroxyl group of flavonols did not
change the antioxidant activity, as rutin and had similar
activity at 5 ~ L M as quercetin (Table 2) , which has a free
hydroxyl group in the 3-position.
To explain the difference in antioxidant activity of
the other phenolic compounds in Table 2, other factors
may be important, including their solubilities in and
partitioning between the aqueous and lipid phases of
LDL. Based on previous studies, both the physical and
chemical properties of individual phenolic compounds
strongly affect their antioxidant potency (Frankel
1993).
However, these factors cannot explain the activity
differences between the procyanidins which have very
similar physical properties. Ricardo Da'Silva (1992) has
demonstrated no variation between trapping capacity
for the hydroxyl radical (*OH) and the degree of
flavan-3-01 polymerization. Thus, in a simple chemical
system sensitive largely to chemical reduction potential,
these compounds have little difference in activity.
Because their chromatographic behavior is very similar,
their differences in antioxidant activity cannot be
entirely explained by altered lipophilicity. One possible
explanation for the large variation in the activity of
procyanidins in the LDL system used in the present
study may be related to their ability to bind proteins.
The procyanidins bind with proteins by hydrophobic
interactions, hydrogen bonds and covalent bonds
(Loomis 1974). Hydrogen bonding has been proposed
to arise between the amide protein linkages and the aro-
matic phenolic hydroxyls on the polyphenolic com-
pounds. Hydrophobic bonding can also occur
between the aromatic rings on the procyanidins and
aromatic amino acids. The relative importance of
hydrogen versus hydrophobic bonding depends on a
number of factors, including solvent polarity and pH,
which will affect the extent of the ionisation of acidic,
including phenols, and basic functionality. Changes in
pH will drastically affect the hydrogen bonding inter-
actions, but hydrophobic interactions will be largely
affected by solvent polarity (Oh et a1 1980).
The relative importance of hydrophobic and hydro-
gen bonding in procyanidin interactions has been
studied by several investigators. Haslam (1974) and
Hagerman and Butler (1981) have suggested that the
binding was stabilized by hydrogens bonding. Oh et a1
(1980) studied the effects that a series of substances
exerted on tannin-protein complex and concluded that
the major binding factor was hydrophobic. Artz et a1
(1987) came to a similar conclusion by analysing the
equilibrium binding constants between procyanidins
and bovine serum albumin.
Thus, the protein binding of these substances may
explain in part the different antioxidant activity of these
procyanidins in the LDL oxidation assay. Since the
ApoB protein constitutes 21% of LDL particle mass
(Gurr and Harwood 1991), it is possible that differen-
tial protein binding to this portion of the particle may
affect the antioxidant efficiency of the phenolic constit-
uent. Further studies on binding of these compounds
will be necessary to clarify its impact on the mechanism
of inhibition of LDL oxidation.
CONCLUSIONS
Wine contains many phenolic compounds that are
effective antioxidants. The fractionation of wine phe-
nolics revealed that the flavan-3-01s are the flavonoid
class with the highest activity, but some members of
this class were not as active. Of the structural features
that could be compared, there are some similarities
between factors that affect antioxidant activity. These
factors include the flavonoid class, the number of phe-
nolic hydroxyl groups and methylation of those
hydroxyl groups. A more comprehensive and systematic
structural analysis will be required to elucidate the con-
tribution of each factor. However, another factor
involving protein binding may affect antioxidant activ-
ity towards LDL, a factor that would not be present in
other oxidations systems. Future studies should be
directed to the binding of procyanidins with proteins
such as ApoB of LDL. It will also be important to
study the synergistic effects of combinations of phenolic
compounds such as those present in wine.
ACKNOWLEDGEMENT
This work was supported by the American Vineyard
Foundation. PLT was supported by the Region
Languedoc-Roussillon and the Comite Inter-
professionnel des vins d'AOC. C6tes du Rh6ne et
Vallee du RhBne, France, and HP was supported by
Research Fellow Grant No FL-0124-90 from BARD-
the United States-Israel Binational Agricultural
Research and development fund. Thanks to A C Noble
for her assistance.
REFERENCES
Ames B N 1989 Endogenous oxidative DNA damage, aging
and cancer. Free Radical Res Commun 7 121-127.
Anon 1981 American Dietetic Associution Handbook of Clini-
cul Dietetics. Yale University Press, New Haven, USA, pp
E3-E64.
Artz W E, Bishop P D, Dunker A K, Schanus E G, Swanson
B G 1987 Interaction of synthetic proanthocyanidin dimer
and trimer with bovine serum albumin and purified bean
globulin fraction G-1. JAgri c Food Chem 35 417-421.
Phenolic antioxidants from grapes and wines 61
Boukharta M, Girardin M, Metche M 1988 Procyanidines
galloylees du sarment de vignes (Vitis pingera) Separation
et identification par chromatographie liquide haute per-
formance et chromatographie en phase gazeuse. J Chro-
matogr 455 406-409.
Bourzeix M, Weyland D, Heredia N 1986 A study of cate-
chins and procyanidins of grape clusters, the wine and
other by-products of the wine. Bull OW59 1171-1254.
Criqui M H, Ringel B L 1994 Does diet or alcohol explain the
French paradox? Lancet 344 1719-1723.
Darmon N, Fernandez Y, Cambon-Gros C, Mitjavila S 1990
Quantification of the Scavenger capacity of different flavo-
noids in regards to the superoxide ion. Foods Addit Contam
7 560-563.
Esterbauer H, Puhl H, Dieber-Rotheneder M 1991 Effects of
antioxidants on oxidative modification of LDL. Free
Radical Biol Med 23 573-581.
Esterbauer H, Gebicki J , Puhl H, J urgens G 1992 The role of
lipid peroxidation and antioxidants in oxidative modifi-
cation of low density lipoproteins. Free Radical Biol Med
13 341-390.
Frankel E N 1993 I n search of better methods to evaluate
natural antioxidants and oxidative stability in food lipids.
Trends Food Sci Technol 4 220-225.
Frankel E N, German J B, Davis P A 1992 Headspace gas
chromatography to determine human low density lipopro-
tein oxidation. Lipids 27 1047-1051.
Frankel E N, Kanner J , German J B, Parks E, Kinsella J E
1993a Inhibition of oxidation of human low-density lipo-
protein by phenolic substances in red wine. Lancet 341
454-457.
Frankel E N, Waterhouse A L, Kinsella J E 1993b Inhibition
of human LDL oxidation by resveratrol. Lancet 341 1103-
1 104.
Frankel E N, Waterhouse A L, Teissedre P L 1995 Principal
phenolic phytochemicals in selected California wines and
their antioxidant activity in inhibiting oxidation of human
low-density lipoprotein. J Agric Food Chem 43 890-894.
Gurr M I, Harwood J L 1991 Lipid Biochemistry, An Intro-
duction (4th edn). Chapman and Hall, London, UK, p 200.
Hagerman A E, Butler L G 1981 The specificity of
proanthocyanidin-protein interactions. J Biol Chem 256
4494-4497.
Haslam E 1974 Polyphenols-protein interactions. Biochem J
139 285-288.
Hennekens C H, Buring J E, Pet0 R 1994 Antioxidant
vitamins-benefits not yet proved. New Engl J Med 330
1080-108 1.
Hertog M G, Hollman P C H, Katan M 1992 Content of
potentially anticarcinogenic flavonoids of 28 vegetables of 9
fruits commonly consumed in the Netherlands J Agric Food
Chem 40 2379-2383.
J alial I , Grundy S M 1992 Effects of dietary supplementation
with cc-tocopherol on the oxidative modification of low-
density lipoprotein. J Lipid Res 33 899-906.
Kanner J , Frankel E, Granit R, German B, Kinsella J E 1994
Natural antioxidants in grapes and wine. J Agric Food
Chem 42 64-69.
Kinsella J E, Frankel E N, German J B, Kanner J 1993 Pos-
sible mechanisms for the protective role of antioxidants in
wine and plant foods. Food Technol 47 85-89.
Lamuela-Raventos R M, Waterhouse A L 1994 A direct
HPLC separation of wine phenolics. Am J En01 Vit 45 1-5.
Lea A G H, Bridle P, Timberlake C F, Singleton V L 1979
The procyanidins of white grapes and wines. Am J En01 Vit
Letan A 1966 The relation of structure to antioxidant activity
of quercetin and some of its derivatives. Primary activity. J
Food Sci 31 5 18-523.
Loomis W D 1974 Overcoming problems of phenolics and
quinones in the isolation of plants enzymes and organelles.
Meth Enzymol31 528-544.
Mangiapane H, Thomson J , Salter A, Brown S, Bell G D,
White D A I992 The inhibition of the oxidative of low
density lipoproteins by ( +)-catechin, naturally occuring
flavonoid. Biochem Pharmacol43 445-450.
Mehta A C, Seshadi T R 1959 Flavonoids as antioxidants. J
Sci Industr Res 18B 24-28.
Oh H I, Hoff J E, Armstrong G F, Haff L A 1980 Hydropho-
bic interaction in tannin-protein complexes. J Agric Food
Chem 28 394-398.
Orr J R, Adamson G L, Lindgren F T 1991 Preparative ultra-
centrifugation and analytic ultracentrifugation of plasma
lipoproteins. In : Analyses of fats, oils, and lipoproteins, ed
Perkins E G. American Oil Chemists Society, Champaign,
ILL, USA, pp 524-554.
Pratt D E, Hudson B J F 1990 Natural antioxidants not
exploited commercially. In : Food Antioxidants, ed Hudson
B J F. Elsevier, London, UK, pp 171-191.
Pratt D E, Watts B M 1964 The antioxidant activity of
vegetable extracts. I. Flavone aglycones. J Food Sci 29
27-33.
Renaud S, De Lorgeril M 1992 Wine, alcohol, platelets, and
the French paradox for coronary heart disease. Lancet 339
1523-1526.
Ricardo Da Silva J M 1992 Procyanidines du raisin et du vin
structure et proprietes chimiques. These de Doctorat Spe-
cialite: Sciences Agro-Alimentaires. ENSAM, Montpellier,
France.
Ricardo Da Silva J M, Darmon N, Fernandez Y, Mitjavila S
1991a Oxygen free radical scanvenger capacity in aqueous
models of different procyanidins from grape seeds. J Agric
Food Chem 39 1549-1 552.
Ricardo Da Silva J M, Rigaud J , Cheynier V, Cheminat A,
Moutounet M 1991b Procyanidin dimers and trimers from
grape seeds. Phytochemistry 30 1259-1 264.
Rimm E B, Stampfer M J , Ascherio A, Giovannucci E,
Colditz G A, Willet W C 1993 Vitamin E consumption and
the risk of coronary heart disease in men. New Engl J Med
328 1450-1456.
Singleton C P, Rossi J A 1965 Colorimetry of total phenolics
with phosphomolybdic-phosphotungstic acid reagents. J
En01 Vitic 16 144-158.
Steinberg D 1992a Metabolism of lipoproteins and their role
in pathogenesis of artherosclerosis. Atherosclerosis Rev 18
1-6.
Steinberg D 1992b Antioxidants in the prevention of humans
atherosclerosis. Circulation 85 2337-2344.
Thompson R S, J acques D, Haslam E, Tanner R J N 1972
Plant proanthocyanidins. Part 1 Introduction : The iso-
lation, structure and distribution in nature of plant pro-
cyanidins. J Chem Soc Perkin Trans I 138771399,
Weinges K, Piretti M V 1971 Isolierung des procyanidins B1
Aus Weintrauben. Liebigs Ann Chem 748 218-220.
Willett W C 1994 Diet and health-what should we eat?
Science 264 532-537.
30 289-300.