Fitoterapia 71 Ž2000.

162]175

Characterization of proanthocyanidins from

grape seeds
Bruno Gabetta, Nicola Fuzzati, Alberto Griffini,
Enrico Lolla, Roberto Pace, Tiziano Ruffilli,
Federico PeterlongoU
Indena SpA, Laboratori Ricerca e S¨ iluppo, Via Don Minzoni 6, 20090, Settala, Milano, Italy

Received 15 November 1999; accepted 29 November 1999

Abstract

Leucoselect TM Žgrape seed selected proanthocyanidins. was analyzed. HPLC thermospray

mass spectrometry ŽTSP-MS. allowed the detection of monomeric flavan-3-ols and dimeric
proanthocyanidins. Fractionation over Sephadex W LH-20 resin and analysis of the isolated
fractions by gel permeation chromatography ŽGPC. and electrospray mass spectrometry
ŽESI-MS. led to the complete characterization of the proanthocyanidin constituents of
Leucoselect TM. The analysis revealed the presence of approximately 15% of Žq.-catechin
(1) and Žy.-epicatechin (2), 80% of Žy.-epicatechin 3-O-gallate (3), dimers, trimers,
tetramers and their gallates and 5% of pentamers, hexamers, heptamers and their gallates.
Q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Vitis ¨ inifera; Grape seeds; Proanthocyanidins; GPC; HPLC TSP-MS; ESI-MS

1. Introduction

Proanthocyanidins, also known as condensed tannins, are polymeric compounds,

the basic structural elements of which are polyhydroxyflavan-3-ol units linked
together by carbon]carbon bonds. They are widely distributed in the plant king-

U
Corresponding author. Fax:q 39-0295-413-695.
E-mail address: indenard@tin.it ŽF. Peterlongo.

0367-326Xr00r$ - see front matter Q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 6 7 - 3 2 6 X Ž 9 9 . 0 0 1 6 1 - 6
 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175 163

dom and represent one of the most abundant groups of higher plant secondary
metabolites. It has been shown that polymeric proanthocyanidins from grape w Vitis

Scheme 1. Grape seed polymeric proanthocyanidins.

164 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175

¨ inifera L. ŽVitaceae.x seeds are procyanidins, consisting of the flavan-3-ols Žq.-

catechin (1), Žy.-epicatechin (2) and Žy.-epicatechin 3-O-gallate (3), linked by
C-4]C-8 or C-4]C-6 bonds, as shown in Scheme 1 w1]4x.
Extracts containing proanthocyanidins obtained from Vitis ¨ inifera seeds are used
as active ingredients of medicinal products for the treatment of circulatory dis-
orders such as capillary fragility, peripheral chronic venous insufficiency and
microangiopathy of the retina. Their pharmacological properties are related to an
increase of tonicity and resistance of capillary walls Žmaking them more resistant to
the degradative action of elastase and collagene. and to radical scavenging and
inhibition of superoxide ion formation w5,6x. The sparingrrecycling effect of
Leucoselect TM Žgrape seed selected proanthocyanidins. on a-tocopherol in differ-
ent in vitro models has been reported w7x. The efficacy of proanthocyanidin
constituents of Leucoselect TM on cardiac mechanics following ischemiarre-perfu-
sion stunning in the rat has been studied. Significant increases of the total
antioxidant plasma capacity Ž40% in young and 30% in aged rats. and of the
ascorbic acid plasma level have been observed, showing that proanthocyanidin
supplementation in the rat makes the heart less susceptible to the ischemiar
reperfusion damage and that this is positively associated with an increase of the
plasma antioxidant activity w8x.
Following in vitro investigation for an antimutagenic effect, proanthocyanidins
were shown to strongly counteract spontaneous mutation at both mitochondrial
and nuclear level w9x. Recently, the evaluation of the antioxidant activity toward
human low density lipoprotein ŽLDL. oxidation in vitro of phenolic compounds
from 12 different varieties of grapes Žred and white wines. has been reported w10x.
To date, investigation of the structures of grape proanthocyanidins has been
performed in different ways depending on the extension of the polymeric chain. In
fact, only low molecular weight tannins Žup to tetramers . can be isolated and
characterized as pure compounds. An improved procedure for the isolation and
purification of dimeric and trimeric procyanidins has been recently described w11x.
Structure elucidation of oligomers has been performed using two-dimensional
NMR w12,13x and FAB mass spectrometry w14x, while reversed phase ŽRP. HPLC
has been applied for the analysis of lower molecular weight oligomeric constituents
w1]4,15x. Isolation of pure polymeric tannins is difficult. In most cases, only
fractions corresponding to mixtures of several polymers can be obtained. To clarify
the nature of the constituents the obtained fractions are subjected to acid degrada-
tion in the presence of a nucleophilic agent such as phloroglucinol or benzylmer-
captan w2,4,16x. It is thus possible to identify the terminal units and to gather
information on the average degree of polymerization.
Various methods have been proposed for the estimation of proanthocyanidin
contents: vanillin assay w17]19x; depolymerization in HClrbutanol w20x; p-dimethyl-
aminocinnamaldehyde reaction w21,22x; and Folin]Ciocalteau reaction w23x. These
methods, however, lack satisfactory specificity and reproducibility w24x.
Gel permeation chromatography ŽGPC. could directly provide information on
both degree of polymerization and average molecular weight. GPC analysis of
proanthocyanidins has traditionally been carried out on acetylated derivatives w25x.
 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175 165

Derivatization was considered necessary because proanthocyanidins were too polar

to be separated on currently available GPC columns. However, molecular weights
determined by GPC were slightly higher compared to the ones obtained by
degradation, due to the lack of suitable calibrating standards w4x.
Mass spectrometry was used as a fast and direct method for elucidating the
polyphenol constituents of grape seed extracts. In particular, the composition of
various commercial extracts and of oligomeric and polymeric procyanidin fractions
from grape seeds were determined by liquid secondary ion mass spectrometry
ŽLSIMS., in negative ion mode w26,27x. Electrospray mass spectrometry ŽESI-MS.
was also used, directly or combined with liquid chromatography, for studying
oligomeric and polymeric tannins contained in grape seeds; the negative ion ESI
mass spectra showed the presence of a series of non-galloylated and galloylated
oligomeric procyanidins up to trigalloylated octamer w27x.
The present study was aimed at characterizing the constituents of Leucoselect TM ,
combining Sephadex W LH-20 fractionation, thermospray and electrospray mass
spectrometry results of fractions, HPLC and GPC analyses. The Leucoselect TM
commercial batch submitted to this investigation can then be used as a reference
standard for proanthocyanidin assay in the routine analysis of both Leucoselect TM
industrial batches and other commercial grape seed extracts ŽGSEs..

2. Experimental

2.1. Samples and standards

Leucoselect TM Žgrape seed selected proanthocyanidins. from V. ¨ inifera seeds

w29x. 2,6-Di-tert-butyl-4-methylphenol ŽBHT, HPLC potency 99.89%. was obtained
from Merck ŽDarmstadt, Germany.. Žq.-Catechin hydrate ŽHPLC potency 93.36%.
and Žy.-epicatechin ŽHPLC potency 97.70%. were purchased from Aldrich-Fluka
`
ŽMilan, Italy.. Gallic acid ŽHPLC potency 89.97%. was obtained from Extrasynthese
ŽGenay, France.. Žy.-Epicatechin gallate ŽHPLC potency 97.79%. was isolated and
characterized in Indena laboratories.

2.2. Sol¨ ents and reagents

Acetonitrile ŽMeCN. and methanol ŽMeOH. HPLC grade were from J.T. Baker
ŽDeventer, The Netherlands.. Water was purified by a Milli-Q plus system from
Millipore ŽMilford, MA, USA.. Tetrahydrofuran ŽTHF. for HPLC ŽBDH Labora-
tory Supplies, England., 85% phosphoric acid reagent grade from Ashland ŽMilan,
Italy., trifluoroacetic acid ŽTFA. and lithium bromide ŽLiBr. extra pure from
Merck ŽDarmstadt, Germany., were used.

2.3. Sample fractionation

Leucoselect TM Ž20 g. was dissolved in 90% EtOH and CC on Sephadex W LH-20

166 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175

resin Ž160 g. in a 50 = 4.5 cm i.d. column. The elution, under pressure, started
using 90% EtOH, followed by 20%, 40% and 70% acetone, at a flow rate of 1 lrh.
The obtained fractions Ž500 ml each. were analyzed by GPC and ESI mass
spectrometry according to Sections 2.4.1 and 2.4.4, respectively, then collected on
the basis of their constituent pattern. Ten fractions were obtained as detailed in
Table 1.

Table 1
Summary of Leucoselect TM Sephadex W LH-20 fractionation

Fraction Yield Analytical Main

no. Ž%. characterization constituents a

1 15.0 HPLC; GPC C, E,

2 8.9 GPC; HPLC-MS ŽTSP. EG, D, DG
3 9.6 GPC; MS ŽESI. D, DG, T
4 9.2 GPC; MS ŽESI. DG, DG2, T, TG, Te
5 50.7 GPC; MS ŽESI. T, TG, Te, TeG, P
6 3.9 GPC; MS ŽESI. TG, Te, TG2, TeG, P, TeG2, PG
7 0.92 GPC; MS ŽESI. TeG, P, TeG2, PG, Hx, PG2
8 0.22 GPC; MS ŽESI. PG, TeG3, Hx, PG2, HxG
9 0.38 GPC; MS ŽESI. PG2, HxG, PG3, HxG2
10 0.22 GPC; MS ŽESI. PG2, HxG, PG3, HxG2, Hp, HpG
a
C, Žq.-catechin; E, Žy.-epicatechin; EG, Žy.-epicatechin 3-O-gallate; D, dimer; DG, dimer
gallate; DG2, dimer digallate; T, trimer; TG, trimer gallate; TG2, trimer digallate; Te, tetramer; TeG,
tetramer gallate; TeG2, tetramer digallate; P, pentamer; PG, pentamer gallate; PG2, pentamer
digallate; PG3, pentamer trigallate; Hx, hexamer; HxG, hexamer gallate; HxG2, hexamer digallate; Hp,
heptamer; HpG, heptamer gallate.

2.4. Analytical procedures

2.4.1. Gel permeation chromatography (GPC)

2.4.1.1. GPC conditions. GPC separations were carried out at room temperature
on a PL Gel column Ž300 = 7.6 mm i.d.., particle size 5 mm, pore type 500 A
ŽHewlett Packard, part no. 1110-6525., connected to a pre-column 0.5-mm filter
ŽSupelco.. The elution was isocratic using THFraqueous LiBr 1.2 = 10y4 M 95:5
at a flow rate of 1.0 mlrmin. Detection was at 280 nm. Injection volume, 10 ml.
BHT was used as an internal standard. Acquisition time, 15 min.
2.4.1.2. GPC assay, proanthocyanidins. For the quantitative analysis three solu-
tions containing Leucoselect TM Ž0.8, 1.0 and 1.2 mgrml. and BHT Ž0.3 mgrml.
reference standards, dissolved in the eluting solvent, were prepared Ž Reference
solutions.. In double, solutions of the examined GSE Ž1.0 mgrml. containing BHT
reference standard Ž0.3 mgrml., in the same eluting solvent, were also prepared
Ž Sample solutions.. After verification of the suitability of the system, the reference
and sample solutions were injected, two injections for each solution, following the
 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175 167

preparation order. The proanthocyanidins content ŽT . in the examined GSE,

compared to Leucoselect TM reference standard, is calculated using the following
equation:

Ž A GSE rA BHT . sample y a 100

T Ž%. s =
b Csample

where:

A GSE s GSE peak area in the sample solution;

A BH T s BHT peak area in the sample solution;
a s calibration curve wŽ A LeucoselectrA BHT . RST vs. C RST x intercept;
A Leucoselect RST s Leucoselect TM peak area in the reference solution;
A BH T RST s BHT peak area in the reference solution;
C RST s Leucoselect TM reference standard concentration Žmgrml.;
b s calibration curve wŽ A LeucoselectrA BHT . RST vs. C RST x slope; and
Csample s GSE sample solution concentration Žmgrml..

2.4.2. High performance liquid chromatography (HPLC-UV)

2.4.2.1. HPLC-UV conditions. HPLC separations were obtained at room temp on
a SupelcoSil LC-18 column Ž250 = 4.6 mm i.d.., particle size 5 mm ŽSupelco.. The
solvent system was a linear gradient using MeCN Žsolvent A. and 0.3% phosphoric
acid Žsolvent B. from 10% A to 20% A in 45 min, then to 60% A in 20 min. The
flow-rate was 0.7 mlrmin, the UV detector was set at 278 nm and the injection
volume was 10 ml.
2.4.2.2. HPLC-UV assay, (q)-catechin and (y)-epicatechin. In order to quantitate
Žq.-catechin (1) in grape seed extracts ŽGSEs., linearity was determined for the
reference standard. Linearity of responses was determined on five levels of
concentration with three injections for each level. Response was linear from 0.05 to
0.8 mgrml and the curve had a coefficient of linear correlation G 0.999 with
y-intercept close to zero. GSE Ž50 mg. was dissolved in 10 ml of MeCN]0.3%
phosphoric acid 1:9 Žvrv.. Reference standard 1 Ž10 mg. was dissolved in 20 ml of
the same solvent mixture. After verification of the suitability of the system, all
standards and samples Žtwo preparations for each of them. were injected alterna-
tively. The content of 1 was calculated using the following equation:

A GSE = 100
Content Ž % . s
RFstd = CGSE

where:

A GSE s peak area of 1 in the examined GSE;

RFstd s mean response factor of 1 in the reference standard solutions
 response factor s 100 = arearwconc. Žmgrml.x = potency4 ; and
168 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175

CGSE s concentration of the solution of the examined GSE Žmgrml..

The same equation was used to obtain the content of Žy.-epicatechin (2),
expressed as 1.

2.4.3. High performance liquid chromatography thermospray-mass spectrometry (HPLC

TSP-MS)
For the HPLC TSP-MS analyses, the conditions described in Section 2.4.2 were
used, substituting 0.3% phosphoric acid with 0.3% TFA, in order to avoid precipi-
tation inside the vaporizer. The liquid chromatography system was connected to a
Finnigan-MAT TSQ700 triple quadrupole mass spectrometer equipped with a
TSP-2 thermospray interface and a 5100 DEC Station with ICIS data system. Mass
spectrometer conditions were optimized in order to achieve maximum sensitivity as
follows: source block temperature 2308C, vaporizer temperature 1008C, repeller
voltage 150 V, discharge off-mode, filament on-mode Želectron energy 400 eV,
emission current 350 mA.. The electron multiplier and dynode voltages were set to
2000 V and 15 kV, respectively. Pre-amplifier sensitivity was 10y8 ArV. Positive
thermospray mass spectra from mrz 160 to 1200 Žscan time, 1 s. were obtained
scanning the Q3 analyzer and acquired in centroid mode.

2.4.4. Electrospray mass spectrometry (ESI-MS)

An API III q triple quadrupole mass spectrometer ŽPE-Sciex, Thornhill, ON,
Canada. equipped with an articulated ion spray interface was employed. Tuning
and calibration were performed on both the first ŽQ1. and the third quadrupole
using a solution of polypropylene glycols ŽPPGs. in 3 mM ammonium acetate. All
the samples, diluted in MeOH added with 1% TFA, were injected via a 20-ml loop
Rheodyne valve at 50 mlrmin using as eluent a 40:60 Žvrv. mixture of 0.1% TFA
in waterrMeCN. A Waters mod. 626 HPLC pump controlled the delivery of the
solvent. Zero grade compressed air was used as nebulizer gas Žpressure set at 60
psi.. A curtain gas Ž99.999% UHP nitrogen. flow of 0.8 lrmin was employed. The
interface heater was set at 608C. The ion spray tip which was held at a potential of
q5.3 kV. Mass spectra were obtained at a dwell time of 1.00 ms ŽQ1 scan range:
300 % 2300 u, 10 scans averaged. and a step size of 0.10 u. The orifice voltage was
maintained at 70 V.

3. Results and discussion

HPLC-UV analysis of Leucoselect TM showed the presence of 9.2% of Žq.-

catechin (1) and 6.8% of Žy.-epicatechin (2). The chromatographic profile ob-
tained at 278 nm ŽFig. 1. showed, besides well-separated low molecular weight
constituents, a hump at approximately 60 min retention time due to the co-eluted
polymeric polyphenols Žpart of polyphenols might also be retained and not eluted
from the column..
 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175 169

Fig. 1. HPLC-UV profile of Leucoselect TM .

GPC analysis of Leucoselect TM displayed a distribution of the polyphenols as

shown in Fig. 2.
Leucoselect TM was submitted to fractionation over Sephadex W LH-20 and

Fig. 2. GPC profile of Leucoselect TM . Region A: Žy.-epecatechin. Region B: Žy.-epicatechin 3-O-gal-

late, dimers, and their gallates. Region C: trimers, tetramers, pentamers, hexamers, heptamers and their
gallates.
170 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175

identification of the polyphenolic constituents was obtained by HPLC, GPC, TSP

and ESI mass spectrometry analyses. Fraction yields, relevant analytical characteri-
zation and constituents are summarized in Table 1.
Characterization of the proanthocyanidins with a limited number of flavan-3-ol
units Žfractions 1 and 2. was obtained by HPLC-UV analysis combined with TSP
mass spectrometry. TSP interface allows the introduction of aqueous eluents into
the mass spectrometer using flow-rates compatible with those used in RP-HPLC.
The latter technique is currently a powerful detection tool for on-line identification
of plant constituents in crude extracts w30x. Analysis of fractions 1]2 allowed the
identification of Žq.-catechin (1), Žy.-epicatechin (2), Žy.-epicatechin 3-O-gallate
(3), gallic acid, four dimers and a dimer gallate. The HPLC TSP-MS profile of
fraction 2 is reported in Fig. 3. Positive TSP mass spectra contain only few ions, but
they are useful for the identification. In the case of 1 and 2 only the protonated
molecule wM q Hxq at mrz 291 is present. Dimers give the protonated molecule
wM q Hxq at mrz 579, base peak being the flavan-3-ol protonated unit Ž mrz 291..
The presence of a peak at mrz 601, corresponding to the wM q Naxq ion, strongly
supports the molecular weight attribution. The dimer gallate gives a positive TSP
mass spectrum showing, besides the protonated molecule wM q Hxq at mrz 731,
the ions corresponding to the loss of gallic acid residue at mrz 579 and to the loss
of a flavan-3-ol unit at mrz 443. Žy.-Epicatechin 3-O-gallate (3) shows a mass
spectrum containing the protonated molecule wM q Hxq at mrz 443 as the base
peak. Compounds 1–3 and gallic acid were thus identified on the basis of their
chromatographic retention times and positive TSP mass spectra by comparison
with reference substances. The four dimers and the dimer gallate were identified
on the basis of the corresponding positive TSP mass spectra and by comparison
with literature data w1x. This technique was not suitable for the analysis of fractions
3]10 since in the reported conditions thermal decomposition of polymeric proan-
thocyanidins occurs.
Fractions 3]10 were analyzed by GPC and by ESI-MS. Comparison of the GPC
profiles with that of Leucoselect TM showed that polymers with decreasing molecu-
lar weights are eluted in succession. A typical example Žfraction 6. is shown in Fig.
4. Even if suitable calibrating standards for GPC analysis were lacking, molecular
weight determinations were obtained by ESI mass spectrometry, a soft ionization
technique applicable to the analysis of large and unstable polar molecules w31x.
Recently, application of this technique to the analysis of proanthocyanidin oligomers
and polymers extracted from cider apple, grape seeds and litchi pericarp allowed
the determination of the nature of units of polymeric proanthocyanidins, their
degree of polymerization, type of interflavanoid bonds and presence of gallate
derivatives; negative double charge wM]2Hx 2y ions allowed to detect the presence
up to a trigallate octamer constituent w28x.
ESI positive mass spectra of fractions 3]10 showed that oligomeric proantho-
cyanidins can be readily distinguished. In fact, each fraction was characterized by a
mass spectrum containing peaks corresponding to the protonated molecule wM q
Hxq for each polymeric constituent. Fractions 3]6 showed ions at mrz 579, 867
and 1155 due to dimers, trimers and tetramers and ions at mrz 731, 1019 and 1307
 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175 171

Fig. 3. HPLC TSP-MS profile of fraction 2. wD, dimer; C, Žq.-catechin; E, Žy.-epicatechin; DG, dimer
gallate; EG, Žy.-epicatechin 3-O-gallatex.
 172
B. Gabetta et al. r Fitoterapia 71 (2000) 162]175
Fig. 4. Positive ESI mass spectrum and GPC profile Žcompared to Leucoselect TM . of fraction 6.
 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175 173

corresponding to their monogallates. Ions at mrz 883, 1171 and 1459 correspond-
ing to digallate derivatives of dimers, trimers and tetramers, respectively, and ions
at mrz 1323 and 1611 corresponding to trigallate derivatives of trimers and
tetramers, were also present. Fractions 7]10 showed ions at mrz 1443, 1731 and
2019 corresponding to pentamers, hexamers and heptamers; ions at mrz 1595,
1883 and 2171 corresponding to their monogallate derivatives; ions at mrz 1747
and 2035 corresponding to pentamer and hexamer digallate derivatives; and ions at
m r z 1899 and 2187 corresponding to pentamer and hexamer trigallate derivatives.
The last one was the largest polymeric proanthocyanidin detected in the mass
spectra. No ions were detected above mrz 2200, or double charge peaks over the
whole mass range. Ions corresponding to the loss of flavan-3-ol units were also
present, showing that a certain degree of fragmentation took place even in ESI-MS
conditions. ESI mass spectrometric analysis, without any degradation step, pro-
vided evidence of the existence of a complete series of oligomeric proanthocyani-
dins Žup to heptamers. in Leucoselect TM .
This was further confirmed by direct positive ESI-MS analysis of unfractionated
Leucoselect TM . Fig. 5 shows a characteristic distribution of proanthocyanidin
molecular weights, each proanthocyanidin constituent giving the corresponding
wM q Hxq ion. The highest detected molecular weight is due to a monogallate
pentamer. Hexamers, heptamers and their gallates were not detected, probably due
to their low content. The mass spectrum was acquired above 350 Da to avoid the
suppression effect exerted by monomer signal on the other ions.
In conclusion, analysis of Leucoselect TM by HPLC-UV, according to the proce-
dure described in Section 2.4.2.2, allowed to evaluate the assays of monomers 1 and

Fig. 5. Positive ESI mass spectrum of Leucoselect TM .

174 B. Gabetta et al. r Fitoterapia 71 (2000) 162]175

2 and to provide a chromatographic profile of oligomeric constituents. Fractiona-

tion over Sephadex W LH-20 and combined GPC and ESI-MS analysis of the
obtained fractions, led to the identification of Žy.-epicatechin 3-O-gallate (3),
dimers, trimers, tetramers and their gallates Žapprox. 80% wrw., pentamers,
hexamers, heptamers and their gallates Žapprox. 5% wrw..
Several grape seeds extracts ŽGSEs. are commercially available, the composition
of which depends on the used starting plant materials, solvents and production
methods. Some of them contain a higher degree of polymeric proanthocyanidins,
other ones a higher degree of oligomers, in comparison to Leucoselect TM . The
coupling of the above discussed HPLC and GPC analytical methods appears to be
presently the most efficient tool to inspect the composition of a GSE. In particular,
GPC analysis, performed as described in Section 2.4.1.2, is able not only to provide
information on the composition of the extract, by evaluating the shape of the
chromatographic profile, but also to allow a quantitative evaluation of the poly-
phenols in comparison to Leucoselect TM reference standard, thus ensuring con-
stancy and reproducibility of biological effects.

Acknowledgements

We are grateful to Dr Luigi Colombo, Biosearch Italia, for the measurement of

the ESI-MS spectra.

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