Identification of the condensed tannins content in grape and Bordeaux wine

by means of standards of synthesis

S. FABRE (1, 2), E. FOUQUET (1), I. PIANET (1) and P-L.
TEISSEDRE*(2)
1) Institut des Sciences Molculaires, CNRS-UMR 5255, Universit Bordeaux 1, Laboratoire
Synthse-Molcules Bioactives, 351 cours de la Libration, Talence 33405 cedex, FRANCE
2) Facult dnologie, UMR nologie 1219, Universit Victor Segalen Bordeaux 2,
Laboratoire de Chimie Applique, 351 Cours de la Libration, Talence, 33405 cedex, France
*Corresponding author Tel: +33 (0)5 40 00 64 56; Fax: +33 (0)5 40 00 64 68; E-mail:
p.teissedre@u-bordeaux2.fr

Keywords: Procyanidins; High Performance Liquid Chromatography; wine; grape seeds;

grape skins; grape ripening; wine ageing;

Our study consists in evaluating the concentration and compositional changes of procyanidins
(condensed tannins) in grape seeds and skins during fruit ripening, winemaking and ageing
process of the resulting wine. Grapes from vinis vinifera varieties have been sampled over four
periods of the 2006 harvest season in Medoc area. After some extraction steps, an
analysis of samples by HPLC-UV allows to obtain phenolic concentrations of catechin,
epicatechin, procyanidins dimers (B1, B2, B3 and B4), trimers (C2 and cat-cat-epi) and one
tetramer (cat-cat-cat-cat). Subsequently, the various wine samples taken during winemaking
and ageing process will be also analyzed with the same HPLC methodology.

Introduction:
Procyanidins (condensed tannins) are a widely recognized, fundamental quality component of
grapes and wines. Present in skin, seed and stem tissues of the fruit, they are extracted during
red wine making. They are responsible for many important properties of wine
including colour, bitterness, astringency and antioxidant capacity. Because these attributes
contribute so much to red wine quality 1, a knowledge and understanding of their evolution
during grape
ripening, during winemaking and wine ageing is important. Wine is a complex matrix with a
lot of different component, so it is difficult to analyse it. Our aim is precisely to quantify the
procyanidins of the grapes and wine with a HPLC method, by direct injection for the wine and
after the minimum of preparation for the skins and skins.
MATERIALS AND METHODS:
Samples:
Grapes of Merlot and Cabernet Sauvignon varieties were manually harvested in at least two
different plots for each variety, in a Medoc vineyard during the 2006 season. The berries were
sampled randomly at four different periods of grape ripening: veraison, middle of maturity,
maturity and at harvest. In the end, we have eighteen samples of grapes.
Preparation of extracts:

Grape seeds and skins were carefully separated before to be freeze-dried and reduced
in
powder. Then, each sample was extracted in two steps. First, 50 ml of a solution of water and
acetone (30/70 v/v) was added to five gram of the powder and leave for four hour
under nitrogen with magnetic stirring at 20C. The resulting mixture was centrifuged for 15
min (5000g). The solid residue was re-extracted with a solution of water and methanol (40/60
v/v) for three hours. The crude extracts obtained are typically complex, and need to be purified
before HPLC analyses. A liquid-liquid extraction with chloroform allows to eliminate
chlorophylls, lipids and other undesirable compounds: then an extraction with ethyl acetate
allows to eliminate polymers tannins and to preserve only the procyanidins of interest i.e.
oligomers and monomers. The both upper layer were combined, the acetone and methanol
were evaporated and the resulting aqueous solution was diluted with water to a find volume of
250 mL. This solution was extracted three times with 250 ml of chloroform and three times
with 250 ml ethyl acetate. The ethyl acetate was evaporated from the organic phase and the
resulting extract was lyophilised. For the seed extracts the preparation is finished and they can
be directly analysed by HPLC. For the skin extract, it is necessary to have a free
anthocyanidin extract if we want to quantify the condensed tannins. We will use a cationic
exchange resin (Dowex, Sigma)) in batch with our extracts. The anthocyanidin will be fixed
by the resin and after filtration, we will have a free anthocyanidin extract.
HPLC standards and analysis:
All chromatographic solvent are HPLC grade and were purchased from Prolabo
(France).
Catechin and epicatechin was purchased from extrasynthese (Genay, France). B1, B2, B3 and
B4 dimers, C2 and cat-cat-epi trimers and cat-cat-cat-cat were synthesized in the laboratory of
ISM.
A modification of the reversed-phase HPLC method of Lamuela-Raventos and al.2 was used
for the analyses of the procyanidins. This procedure use a ternary solvent system
which allows the separation of phenolic compounds,by direct injection of wine.
The standards and extracts were analysed by a Thermo apparatus including a diode array UVvisible detector model UV6000, an autosampler model AS100and a four pump system model
P1500.
A Agilent Nucleosil (250*4mm, 5 m particule size) C18 column was used with a flow of 0,5
ml/min. The solvent used for the separation were: Solvent A= 50 mM dihydrogen ammonium
phosphate adjusted to pH 2,- with orthophosphoric acid; Solvent B= 20%A with 80%
acetonitrile and Solvent C: 0,2 M orthophosphoric acid adjusted with ammonia to pH 1,5.
RESULTAT AND DISCUSSION
The extraction effectiveness between aqueous methanolic, acetonic and ethanolic solvents
was initially compared. It is generally accepted that aqueous acetone is the most efficient
overall extraction solvent for proanthocyanidins. According to our essays, even if this
hypothesis is generally verified, some procyanidins are better extract with an alcoholic
extraction (methanol more effective than ethanol). As a result, a two step extraction
was chosen: a first extraction with acetone follows by a second extraction with methanol
For the quantification by HPLC, an external standard calibration is used. For this, we need to
have standards well characterized with a high purity in sufficient quantity. The new efficient
iterative methodology developed in the ISM laboratory3, which permits a perfect control of the
interflavan regio- and stereoselectivity as well as the degree of oligomerization, allows us to
have easily access to high purity standards (going dimer to tetramer)

Figure 1: Synthesis pathway to dimer, trimer and tetramer of condensed tannins

The modified HPLC method allows to distinguish the different standards, but for a
good
quantification, we need to optimize a little more the separation, in particular concerning the
C2 trimer and the tetramer.
When this will be finished, we could do the external standard calibration and analyze our
extracts and the different samples of wine by direct injection.
Time

Solvent A

Solvent B

Solvent C

100

100

10

96

12

96

15

12

88

20

18

82

25

20

80

30

22

78

40

24,5

75,5

50

25

75

70

40

60

75

60

40

80

100

Figure 2: Chromatogram of standards synthetic solution and gradient used

(1)Singleton V.L. In Plant Polyphenols; Hemingway, R. W.; Laks P.E., Plenum Press, 1992
(2) Lamuela-Raventos R.M. and Waterhouse A.L. Am.J.Enol.Vitic., 1994, 45:1-5.
(3) Tarascou I.; Barathieu K.; Andr, Y., Pianet, I., Dufourc E.J., Fouquet E., Eur. J. Org.
Chem. 2006, 5367-5377.