Resistance factors to grey mould in grape berries: Identification of some phenolics inhibitors of Botrytis cinerea stilbene oxidase

Article abstract of DOI:10.1016/S0031-9422(99)00351-9

Grey mould caused by Botrytis cinerea is one of the most important diseases of grapes. Between bloom and veraison, grape berries are resistant to B. cinerea, although they can harbour the pathogen without any visible signs of disease development. After veraison, B. cinerea can produce disease in susceptible grape varieties (e.g. Gamay), but remains quiescent in resistant varieties (e.g. Gamaret). Pathogen resistance in the quiescent stage is not yet fully understood, but is thought to involve multiple parameters including chemical and mechanical factors. The pathogenesis of B. cinerea is essentially linked to excretion of lyric enzymes such as polyphenoloxidases or laccases. One lytic enzyme, stilbene oxidase, can detoxify grape stilbenic phytoalexins, destroying the grapes' defence mechanisms and allowing the fungus to grow. Some constitutive grape berry phenolic compounds, however, strongly inhibit stilbene oxidase activity. Constitutive berry phenolic compounds were isolated from Gamay and Gamaret varieties and their biological activities, concentrations and chemical structures were comparatively analysed. Catechin, epicatechin-3-O-gallate, trans-caftaric, trans- and cis-coutaric and trans-coumaric acids, taxifoline- 3-O-rhamnoside and quercetine-3-O-glucuronide were identified as potent stilbene oxidase inhibitors. High concentrations of some of those compounds could be closely involved in the persistence of the quiescent stage of B. cinerea, between bloom and veraison in all grape varieties and after veraison in resistant varieties.

Full text of DOI:10.1016/S0031-9422(99)00351-9

Phytochemistry 52 (1999) 759±767
Resistance factors to grey mould in grape berries: identi®cation
of some phenolics inhibitors of Botrytis cinerea stilbene oxidase
Gilles Goetza, Abdellatif Fkyerata, Nadine Metaisa, Manuela Kunza,
Ra€aele Tabacchia,*, Roger Pezetb, Vincent Pontb
a
Â
Ã
Ã
Institut de Chimie de l'Universite de Neuchatel, Avenue de Bellevaux 51, CH-2000, Neuchatel, Switzerland
b
 Â
Station Federale de Recherches Agronomiques de Changins, C.P. 254, CH-1260, Nyon, Switzerland
Received 26 March 1999; received in revised form 14 June 1999; accepted 18 June 1999
Abstract
Grey mould caused by Botrytis cinerea is one of the most important diseases of grapes. Between bloom and veraison, grape
berries are resistant to B. cinerea, although they can harbour the pathogen without any visible signs of disease development.
After veraison, B. cinerea can produce disease in susceptible grape varieties (e.g. Gamay ), but remains quiescent in resistant
varieties (e.g. Gamaret ). Pathogen resistance in the quiescent stage is not yet fully understood, but is thought to involve multiple
parameters including chemical and mechanical factors. The pathogenesis of B. cinerea is essentially linked to excretion of lytic
enzymes such as polyphenoloxidases or laccases. One lytic enzyme, stilbene oxidase, can detoxify grape stilbenic phytoalexins,
destroying the grapes' defence mechanisms and allowing the fungus to grow. Some constitutive grape berry phenolic
compounds, however, strongly inhibit stilbene oxidase activity. Constitutive berry phenolic compounds were isolated from
Gamay and Gamaret varieties and their biological activities, concentrations and chemical structures were comparatively analysed.
Catechin, epicatechin-3-O-gallate, trans-caftaric, trans- and cis-coutaric and trans-coumaric acids, taxifoline-3-O-rhamnoside and
quercetine-3-O-glucuronide were identi®ed as potent stilbene oxidase inhibitors. High concentrations of some of those
compounds could be closely involved in the persistence of the quiescent stage of B. cinerea, between bloom and veraison in all
grape varieties and after veraison in resistant varieties. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Vitis vinifera; Grapevine; Botrytis cinerea; Stilben oxydase; Caftaric acid; Coutaric trans acid; Coutaric cis acid; p trans Coumaric
acid; Taxifolin-3-O-rhamnoside; Quercetin-3-O-glucuronide; Catechin; Epicatechin-3-O-gallate
1. Introduction
healthy berries collected during this stage are able to
strongly inhibit germination of B. cinerea conidia. The
fungicidal activity of these extracts can be explained
by the presence of low concentrations of pterostilbene
(trans-3,5-dimethoxy-4-hydroxystilbene) (Pezet & Pont,
1988a), a known vitaceae phytoalexin (Langcake,
Cornford & Ryce, 1979), and glycolic acid, which
enhances the biocidal activity of pterostilbene (Pezet &
Pont, 1988b). However, other chemical and mechanical
processes could explain the resistance of the immature
berries: polygalacturonase inhibiting protein (PGIP)
(Grassin, 1987), pathogenesis-related protein (b-1,3-
glucanase) (Renault, Deloire & Bierne, 1996), antho-
cyanins and phenolic compounds (Jersch, Scherer,
Huth & Schlosser, 1989), as well as the cuticle mechan-
Grey mould is one of the most important diseases of
grapes. Before bloom and after veraison, grape clusters
can be infected and destroyed by Botrytis cinerea,
depending on climatic conditions and the sensitivity of
the grape variety. Between these two developmental
stages, young clusters are resistant to B. cinerea. This
natural resistance period is called the quiescent stage
(McClellan & Hewitt, 1973; Pezet & Pont, 1986). Pezet
and Pont (1984) have shown that crude extracts of
* Corresponding author. Tel.: +41-32-7182429; fax: +41-32-
7182511.
E-mail address: raphael.tabacchi@ich.unine.ch (R. Tabacchi)
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00351-9

760
G. Goetz et al. / Phytochemistry 52 (1999) 759±767
Fig. 1. Structures of isolated compounds.
ical defence (Kolattukudy, 1985). Although it can be
concluded that the resistance of grape berries to B.
cinerea is the result of several conjugated processes,
these processes are not yet completely understood.
Exoenzymes excreted by B. cinerea are strongly
implicated in the pathogenesis of this parasite
(Grassin, 1987). Compounds from grape berries that
inhibit the activities of these enzymes may be impli-
cated in the defence against grey mould. The aim of
this research project was to isolate metabolites from
the crude extracts of young berries. In addition to the
mechanisms described previously, these metabolites
could be implicated in maintaining B. cinerea quies-
cence and inhibiting laccases. One of the laccases
excreted by B. cinerea oxidizes stilbenes (Pezet, 1998),
detoxifying the compound (Pezet, Pont & Hoang-Van,
1991) and breaking the grapes' induced defence. In
this study, free phenolic compounds were isolated
from young grape berries. Their potent inhibition of
B. cinerea stilbene oxidase may contribute to the pro-
tection of stilbenic phytoalexins and their fungicidal
properties.
2. Results and discussion
Immature grape berries, variety Pinot Noir, were
harvested from June to September in vineyards situ-
ated in Epernay (France). Free phenolic compounds
were extracted from the berries and puri®ed by semi-
preparative HPLC. Eight peaks, numbered 1 to 8
according to their retention time, were identi®ed by
means of UV, DCI-NH3 or electrospray-mass spec-
1
troscopy, or H and 13C NMR (Fig. 1). All the result-
ing compounds were very polar, soluble only in water
or methanol, with high molecular weights. In further
biological studies we avoided, when possible, chemical

G. Goetz et al. / Phytochemistry 52 (1999) 759±767
761
Scheme 1. Synthesis of ( ) epicatechin-3-O-gallate.
modi®cations and use of solvents such as THF or
DMSO.
1985; Weber, Hoesch & Rast, 1995). The data com-
parison indicated compounds 4±8 were catechin, epica-
techin-3-O-gallate, p-coumaric acid, taxifolin-3-
rhamnosyl and quercetin-3-glucuronid, respectively.
13C-NMR of quercetin-3-glucuronid (8) had to be
recorded at 408C to enable visualization of the sig-
nals corresponding to the glucuronic acid moiety. Acid
hydrolysis and TLC analysis con®rmed the sugar moi-
ety to be glucuronic acid by comparison with a pure
sample. Although all the compounds identi®ed in this
study have been previously described in grapes
(Ricardo da Silva, Rigaud, Cheynier, Cheminat &
Moutounet, 1991; Singleton, Zaya & Trousdale, 1986;
Souquet, Cheynier, Brossaud & Moutounet, 1996;
Trousdale & Singleton, 1983; Weber et al., 1995),
some of the published data is incomplete or outdated.
Therefore, we chose to describe compounds 1±8 exten-
sively in this experiment.
Except for di€erent retention times, the UV spec-
trum of 1 was identical to the spectrum of ca€eic acid
and the spectrums of 2 and 3 were identical to couma-
ric acid. At 408C, 1, 2 and 3 showed 1H and 13C
NMR spectra similar to those of ca€eic or coumaric
acid (Satake, Murakami, Saiki & Chen, 1980). The
1
extra signals observed at 5.65 and 4.85 ppm in the H-
NMR, and four more signals in the 13C-NMR, could
be assigned to a tartaric acid moiety. 1H-13C short-
and long-range correlations at 408C, as well as MS,
con®rmed compounds 1, 2, and 3 to be E-caftaric
acid, Z-coutaric acid and E-coutaric acid, respectively
(Cheminat, Zawatzky, Becker & Brouillard, 1988;
Strack, Hartfeld, Austenfeld, Grotjahn & Wray, 1985;
Weber, Hoesch & Rast, 1995).
The 1H, 13C-NMR data for compounds 4±8 was
compared with previously published data for
4
Compound 5 was synthesized to obtain sucient
amounts of the sample biological testing. This syn-
thesis is illustrated in Scheme 1. The key step of this
synthesis was the esteri®cation of the hindered alcohol
using dicyclohexyl carbodiimid (DCC). The hydroxyl
groups of ( ) epicatechin (9) and methyl gallate were
(Mendez, Bilia & Morelli, 1995; Seto, Nakamura,
Nanjo & Hara, 1997), 5 (Sakar, Petereit & Nahrstedt,
1993), 6 (Satake et al., 1980), 7 (De Britto, Manickam,
Gopalakrishnan, Ushioda & Tanaka, 1995; Trousdale
& Singleton, 1983) and 8 (Mohle, Heller & Wellmann,

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G. Goetz et al. / Phytochemistry 52 (1999) 759±767
Table 1
Stilbene oxidase inhibition (and anity) of Botrytis cinerea laccase
Inhibitors
nmoles of pterostilbene oxidized/mina
IC50 (mM)b
Km (mM)
Inhibitor concentrations (mg/ml)
0.66
6.6
20
3.23
33
7.89
(E) Ca€eoyl tartaric acid (1)
(Z) Coumaroyl tartaric acid (2)
(E) Coumaroyl tartaric acid (3)
Catechin (4)
40.24
45.38
38.07
41.84
31.57
40.99
38.64
47.55
20.00
18.98
43.21
40.19
15.63
2.49
0.0150
0.1110
0.0537
0.0114
0.0022
0.0540
0.0222
0.0297
0.0021
0.11
ND
0.64
0.58
ND
0.25
ND
ND
ND
24.47
18.61
4.86
30.93
15.10
2.07
Epicatechin-3-O-gallate (5)
(E) p-Coumaric acid (6)
Taxifoline-3-O-rhamnoside (7)
Quercetine-3-O-glucuronide (8)
Epicatechin (9)
1.82
1.24
25.62
27.99
41.42
4.45
21.30
16.54
15.21
3.41
21.75
0.00
7.03
0.77
a Without inhibitor pterostilbene is degraded at the rate of 50.84 nmoles/min; Km=0.66. Activity is determined in 0.1 M phosphate bu€er
pH 5.2 with 0.054 mM of pterostilbene as substrate.
b Calculated with a single exponential decay function (Gra®t Erithacus software).
protected with benzyl groups, using standard proto-
cols, to generate 10 and 11, respectively. The ester (11)
was hydrolysed into the acid derivative, 12. Then, con-
densation of 10 and 12 yielded the fully protected ( )
epicatechin-3-O-gallate (13). After esteri®cation the
proton in position 3 shifted from 4.18 to 5.7 ppm.
Catalytic hydrogenation of 13 on Pd-charcoal to
remove the protecting benzyl groups yielded 5. The
spectral data for the synthetic compound 5 were identi-
cal to those of the natural compound, con®rming its
structure. This synthetic route could be used as a gen-
eral method for the preparation of other epicatechin
or catechin ester derivatives.
All isolated phenols and catechols inhibited B.
cinerea stilbene oxidase activity in vitro using pterostil-
bene as substrate, with IC50 values ranging from
0.0021 to 0.111 mM (Table 1). The highest inhibition
was observed with epicatechin-3-O-gallate (5). This
compound gave the same level of inhibition as the
positive control, epicatechin. (Z ) and (E ) coumaroyl
tartaric acid (2 and 3) and (E )-p-coumaric acid (6)
were the least active phenols of the extracts. With B.
cinerea laccase, (E )-coumaroyl tartaric acid (3) has a
Km of 0.64 mM, close to that of pterostilbene
(Km=0.66 mM), while (E)-ca€eoyl tartaric acid (1),
one of the most active inhibitors, has a Km of 0.11 mM
(Gunata, Sapis & Moutounet, 1987; Pezet & Pont,
1992). These results suggest that these phenolic com-
pounds competitively inhibit stilbene oxidase activity.
However, further studies would be necessary to con-
®rm this hypothesis.
ditions are favourable. The latter variety is considered
to be resistant to B. cinerea in all conditions (Pezet &
Pont, 1992). Young clusters and berries of those two
varieties were harvested in the vineyards of the Swiss
Federal Agricultural Research Station of Changins in
1991. Similar HPLC peaks were detected in the two
varieties and the phenolic compounds identi®ed in
Pinot Noir were identical to those separated in Gamay
and Gamaret; only quantitative di€erences were
detected.
Fig. 2a and b shows the concentrations of each com-
pound (determined by analytical HPLC) from grapes
collected (two or three times per month from 19 June
to 25 September) at every stage of development of the
two varieties. Both pro®les were very similar. Before
blooming, when the grapes are most sensitive, p-cou-
maric acid (6) was the major phenol detected in both
varieties; at the very start of blooming (27 June) it rep-
resented more than 90% of the extract, and almost dis-
appeared after blooming (9 July). This can be
explained by the fact that 6 (under its coenzyme A
form) is the biosynthetic precursor of other shikimic
acid metabolism components of the extract (Rupprich
& Kindl, 1978; Smith & Banks, 1986). As the level of
6 decreased, the level of catechin (4) increased, up to a
maximum observed on 23 July. Within 10 days (2
August), it dropped to a minimum, corresponding to
the bunch closure period during which many berries
dehydrate. This normal process sheds from the cluster
the nascent fruit for which fertilization did not occur
or the embryo aborted (Jackson, 1994). The catechin
level then rose again to a maximum immediately prior
to veraison (4 September), and a ®nal setting level of 4
accompanied the later sensitive stage. The level of cate-
chin (4) in the resistant Gamaret was twice as high as
the level in the sensitive Gamay variety. At a lower
To compare these compounds quantitatively, we
analysed the phenolic extracts from Gamay and
Gamaret; two grape varieties with di€erent sensitivities
to grey mould. The former variety develops a high
level of grey mould after veraison, if the climatic con-

G. Goetz et al. / Phytochemistry 52 (1999) 759±767
763
Fig. 2. Evolution over a growing season of the concentration of compounds extracted from (a) Gamaret Grapes and (b) Gamay grapes.
order of magnitude, quiescence for both varieties
seems to be correlated with increased levels of E cafta-
ric acid (1) and E coutaric acid (3), with epicatechin-3-
O-gallate (5) produced only by Gamaret. Here again,
levels of E caftaric acid (1) in Gamaret are twice as
high as in Gamay. Z Coutaric acid (2), in the Gamay
variety, shows two maxima, one during bloom, the
other during bunch closure.
ate the Botrytis defence, leaving `safer ground' for the
classical phythoalexins, resveratrol and pterostilbene.
The resistance of young berries results from several
conjugated processes; inhibition of the stilbene oxidase
activity of B. cinerea laccases is one of them. Along
with glycolic acid, low concentration of pterostilbene,
a polygalacturonase inhibiting glycoprotein, a patho-
genesis-related protein, the mechanical barrier of the
epidermal complex, etc., high levels of the catechin (4)
contribute to the quiescent stage. The fact that the
catechin level in the resistant Gamaret is twice as high
as in the sensitive Gamay variety can help to explain,
but is not sucient proof by itself, why di€erences in
sensitivity to B. cinerea are observed in the two var-
ieties.
3. Conclusion
This study showed all isolated phenols and catechols
were active against B. cinerea laccases. One can assume
that by inhibiting the stilbene oxidase they may attenu-

764
G. Goetz et al. / Phytochemistry 52 (1999) 759±767
4. Experimental
4.1. Extraction
photometer, with a kinetic program (lag period 10 s,
absorbance recorded every 30 s over a 3 min period).
The reaction mixture for the enzyme assay contained
2.940 ml of phosphate bu€er (0.1 M, pH 5.2), with
30 ml of ethanolic stock solution of pterostilbene (®nal
conc. 0.054 mM) and 30 ml of protein solution. In
each case 2, 20, 60 and 100 mg of inhibitor were added
in the 3 ml quartz cuvettes.
Free phenolic acids were extracted according to
Southerton & Deverall (1990). Grapes (Vitis vinifera
L. ) were harvested at di€erent growth stages. Freeze-
dried and ®nely powdered plant materiel (10 g) was
extracted with MeOH (800 ml) at room temperature in
the dark for 45 min. The solution was centrifuged at
4500 rpm for 45 min. The supernatant was kept; the
residue was dissolved in MeOH (400 ml) and centri-
fuged at 4500 rpm for 10 min. The supernatants were
combined and MeOH was evaporated at 408 under
vacuum. The residue was dissolved in H2O (800 ml)
and the aq. soln was extracted with Et2O (4 Â 200 ml).
Organic layers were combined, dried over Na2SO4, ®l-
tered and Et2O evaporated. The residue was dissolved
in MeOH±H2O (60:40). The soluble part was pre-puri-
®ed by elution over RP-18 column (length 4 cm  id
4 cm) using MeOH±H2O (60:40). Extracts were then
concentrated and ready for HPLC.
4.4. NMR and MS
1H±NMR spectra (400 MHz), 13C (100.6 MHz),
COSY, DEPT, 1H-13C inverse correlation (125 Hz,
10 Hz and 5 Hz) were measured on a Bruker AMX400
with CD3OD as solvent. Chemical shifts (d) are given
in ppm using TMS as reference. Mass spectra obtained
by electronic impact (EIMS) and chemical ionization
(CIMS) with ammonia (positive) were measured on a
Delsi±Nermag R-30-10. Electrospray mass spectra
(ESMS positive) were performed on
Packard 59987A.
a Hewlett
4.4.1. (E )-Ca€eoyl tartaric acid (1)
UV: lmax (MeOH+formic acid 50 mM) nm: 218,
235, 242, 297, 326. H NMR (400 MHz, CD3OD, 258):
4.2. Isolation
1
Analytical HPLC experiments (RP 18, 10 mm, length
250 mm, id 4.6 mm) were performed with a Perkin±
Elmer Liquid Chromatograph (series 3B) connected to
a DAD detector coupled to an HP 9000 series 300
computer. The chromatographic elution condition was
a linear gradient MeOH±aq.HCOOH 50 mM from
(20:80) to (60:40) within 40 min, then from (60:40) to
(100:0) within 10 min. 10 ml of extract were injected at
a 1.5 ml/min ¯ow rate and detected at 254, 300 and
360 nm. The detector used for preparative HPLC (RP
18, 5 mm, length 250 mm, id 8 mm) was a Perkin±
Elmer LC-75 Spectrophotometric Detector. Elution
conditions: a non-linear gradient (curve 2) MeOH±
aq.HCOOH 50 mM from (20:80) to (40:60) within
20 min, followed by a linear gradient from (40:60) to
(60:40) for 20 min and ®nally isocratically at (60:40)
for 10 min. 100 ml of extract were injected at a 2.5 ml/
min ¯ow rate and detected at 300 nm. Each compound
was puri®ed once again using the same conditions but
without formic acid in order to avoid traces of acid.
Isolated compounds were dried and stored at +48. All
the analysis of peaks 1, 2, 3 and 4 were carried out
with compounds extracted from grapes collected
during both periods (cf Fig. 2a and b) that proved to
be identical.
d 7.83 (1H, d, J = 15.9 Hz, H-7'), 7.18 (1H, d,
J = 1.8 Hz, H-2'), 7.06 (1H, dd, J = 1.8 Hz and
8.17 Hz, H-6'), 6.88 (1 H, d, J = 8.17 Hz, H-5'), 6.39
(1H, d, J = 15.9 Hz, H-8'), 5.65 (1H, broad, H-2),
4.85 (1H, broad, H-3). 13C NMR (100 MHz, CD3OD,
258): d 174.4 (C-4), 171.0 (C-1), 168.3 (C-9'), 150.1 (C-
4'), 148.5 (C-7'), 146.5 (C-3'), 128.0 (C-1'), 123.5 (C-
6'), 116.4 (C-5'), 115.6 (C-2'), 114.4 (C-8'), 74.9 (C-2),
71.6 (C-3). ESMS (positive) m/z: 335 [M+Na+], 351
[M+K+].
4.4.2. (Z )-p-Coumaroyl tartaric acid (2)
UV: lmax (MeOH+formic acid 50 mM) nm: 210,
1
226, 308. H NMR (400 MHz, CD3OD, 258): d 7.77
(2H, d, J = 8.0 Hz, H-2', 6'), 6.99 (1H, d, J = 12.0 Hz,
H-7'), 6.86 (2H, d, J = 8.2 Hz, H-3', 5'), 5.95 (1H, d,
J = 12.0 Hz, H-8'), 5.62 (1H, broad, H-2), 4.82 (1H,
broad, H-3). 13C NMR (100 MHz, CD3OD, 258): d
174.6 (C-4), 171.2 (C-1), 167.2 (C-9') 160.6 (C-7'),
146.9 (C-4'), 134.3 (C-1'), 127.7 (C-2', 6'), 116.1 (C-3',
5'), 115.6 (C-8'), 74.8 (C-2), 71.8 (C-3). ESMS (posi-
tive): m/z 319 [M+Na+], 335 [M+K+].
4.4.3. (E )-p-Coumaroyl tartaric acid (3)
UV: lmax (MeOH+formic acid 50 mM) nm: 210,
1
226, 311. H NMR (400 MHz, CD3OD, 258): d 7.83
4.3. Enzyme assays
(1H, d, J = 15.9 Hz, H-7'), 7.57 (2H, d, J = 8.5 Hz,
H-2', 6'), 6.90 (2H, d, J = 8.5 Hz, H-3', 5'), 6.47 (1H,
d, J = 15.9 Hz, H-8'), 5.65 (1H, broad, H-2), 4.86
(1H, broad, H-3). 13C NMR (100 MHz, CD3OD, 258):
d 174.0 (C-4), 170.7 (C-1), 169.3 (C-9'), 162.7 (C-7'),
Enzymatic degradation of pterostilbene was assayed
by measuring the change in absorbance of the reaction
mixture at 300 nm using a Shimadzu UV-160 spectro-

G. Goetz et al. / Phytochemistry 52 (1999) 759±767
765
149.0 (C-4'), 132.4 (C-1'), 128.2 (C-2', 6'), 117.8 (C-3',
5'), 115.1 (C-8'), 75.0 (C-2), 71.6 (C-3). ESMS (posi-
tive) m/z: 319 [M+Na+], 335 [M+K+].
95.1, 77.9, 75.6, 73.2. ESMS (positive) m/z: 479
[M+H+], 501 [M+Na+], 979 [2 M+Na+].
4.4.8. Tetrabenzyloxy-5,7,3',4'-epicatechin (10)
A mixture of epicatechin (9) (116 mg, 0.4 mmol),
K2CO3 (414 mg, 3 mmol) and benzyl bromide (240 ml,
2 mmol) in DMF (5 ml) was stirred at room tempera-
ture for 18 h. Water was added and extracted with
Et2O. The organic layer was washed with aq. HCl
10%, NaCl satd and dried with MgSO4. The solvent
was evaporated under reduced pressure and the oily
residue was puri®ed by column chromatography on
silica gel (hexane±EtOAc, 9:1) to a€ord the desired
product 10 (240 mg, 90%). 1H NMR (400 MHz,
CDCl3): d 7.40 (20H, m, Ar±H), 7.16 (1H, d, H-2'),
6.99 (2H, d, H-5', H-6'), 6.28 (2H, d, H-6, H-8), 5.20
(2H, s, CH2), 5.18 (2H, s, CH2), 5.03 (2H, s, CH2),
5.02 (2H, s, CH2), 4.92 (1H, s, H-5), 4.20 (1H, m, H-
3), 2.98 (2H, m, H-4). 13C NMR (100 MHz, CDCl3): d
159.4, 159.0, 155.9, 149.7, 149.5, 137.9, 137.8, 137.6,
137.6, 132.1, 129.2, 129.2, 129.1, 129.1, 129.1, 128.7,
128.6, 128.5, 128.5, 128.4, 128.4, 128.2, 128.2, 127.9,
127.8, 127.8, 127.6, 120.2, 115.7, 114.2, 101.7, 95.4,
94.7, 79.0, 72.0, 72.0, 70.8, 70.6, 67.0, 28.9.
4.4.4. Catechin (4)
1H NMR (400 MHz, CD3OD, 258): d 6.93 (1H, d,
J = 1.6 Hz, C-2'H), 6.80±6.88 (2H, m, H-6', H-5'),
6.03 (1H, d, J = 2.2 Hz, H-6), 5.96 (1H, d, J = 2.2 Hz,
H-8), 4.66 (1H, d, J = 7.5 Hz, H-2), 4.07 (1H, m, H-
3), 2.94 (1H, dd, J = 5.4 Hz, J = 16.1 Hz, H-4ax.),
2.60 (1H, dd, J = 8.1 Hz, J = 16.1 Hz, H-4eq.). 13C
NMR (100 MHz, CD3OD, 258): d 158.1, 157.8, 157.2,
146.5, 146.5, 132.5, 120.3, 116.4, 115.5, 101.1, 96.6,
95.8, 83.1, 69.1, 28.7.
4.4.5. (E )-p-Coumaric acid (6)
UV: lmax (MeOH+formic acid 50 mM) nm: 227,
1
310. H NMR (400 MHz, CD3OD, 258): d 7.69 (1H, d,
J = 15.9 Hz, H-7), 7.54 (2H, d, J = 8.6 Hz, H-2, 6),
6.89 (2H, d, J = 8.6 Hz, H-3, 5), 6.37 (1H, d,
J = 15.9 Hz, H-8). 13C NMR (100 MHz, CD3OD,
258): d 171.3 (C-9), 161.2 (C-7), 146.9 (C-4), 131.3 (C-
1), 127.5 (C-2, 6), 117.1 (C-3, 5), 115.8 (C-8). CIMS
(NH3, positive) m/z: 182 [M+NH+4 ], 165 [M+H+],
147 [(M OH)+].
4.4.9. Methyl tribenzyloxy-3,4,5-benzoate (11)
The reaction was performed in the same conditions
as above. The compound was obtained as a white
4.4.6. Taxifolin-3-O-rhamnoside (7)
UV: lmax (MeOH+formic acid 50 mM) nm: 230,
1
solid (74% yield). H NMR (400 MHz, CDCl3): d 7.40
1
290. H NMR (400 MHz, CD3OD, 258): d 7.06 (1H, d,
(15H, m, Ar±H), 7.27 (2H, m, Ar±H), 5.15 (4H, s, 2
CH2), 5.12 (2H, s, CH2), 3.90 (3H, s, CH3). 13C-NMR
(CDCl3), 167.3, 153.2, 143.1, 138.1, 137.3, 129.2, 129.2,
128.8, 128.7, 128.6, 128.2, 125.9, 109.8, 75.8, 71.9,
52.9.
J = 1.81 Hz, H-2'), 6.92 (1H, dd, J = 8.2 Hz,
J = 1.86 Hz, H-6'), 6.89 (1H, d, J = 8.1 Hz, H-5'),
6.01 (1H, d, J = 2.2 Hz, H-6), 5.99 (1H, d, J = 2.2 Hz,
H-8), 5.16 (1H, d, J = 10.7 Hz, H-2), 4.67 (1H, d,
J = 10.7 Hz, H-3), 4.14 (1H, d, J = 1.27 Hz, H-10),
4.34 (1H, dq, J = 9.6 Hz, J = 6.0 Hz, H-50), 3.74 (1H,
dd, J = 9.6 Hz, J = 3.2 Hz, H-40), 3.62 (1H, dd,
J = 3.0 Hz, J = 1.5 Hz, H-30), 3.4 (under the MeOH,
H-20), 1.28 (3H, d, J = 6.14 Hz, 3H-60). 13C NMR
(100 MHz, CD3OD, 258): d 196.2 (C-4), 168.9 (C-9),
165.8 (C-5), 164.4 (C-7), 147.7 (C-3'), 146.9 (C-4'),
129.5 (C-1'), 120.6 (C-6'), 116.2 (C-5'), 115.5 (C-2),
102.8 (C-10), 102.5 (C-10), 97.8 (C-6), 96.2 (C-8), 84.0
(C-2), 79.5 (C-3), 74.3 (C-20), 72.5 (C-40) 71.8 (C-30),
71.1 (C-50), 18.1 (C-60). ESMS (positive) m/z: 473
[M+Na+], 923 [2 M+Na+].
4.4.10. Tribenzyloxy-3,4,5-benzoic acid (12)
Methyl tribenzyloxy-3, 4, 5-benzoate 11 (2 g,
4.4 mmol), KOH (3.15 g, 40 mmol) in dioxan (50 ml)
and MeOH (50 ml) were heated at re¯ux for 18 h. The
solvent was removed under reduced pressure; the resi-
due was diluted in water and extracted with EtOAc.
The organic layer was washed with aq. HCl 10%,
NaCl satd and dried with MgSO4. The solvent was
evaporated under reduced pressure to obtain the
desired compound 12 as a white solid (1.7 g, 89%).1H
NMR (400 MHz, CDCl3): d 7.44 (15H, m, Ar±H),
7.27 (2H, m, Ar±H), 5.17 (6H, m, 3 CH2).
4.4.7. Quercetin-3-O-glucuronide (8)
UV: lmax (MeOH+formic acid 50 mm) nm: 205,
256, 354. H NMR (400 MHz, CD3OD, 258): d 7.72
4.4.11. Tribenzyloxy-3,4,5-benzoate-3-tetrabenzyloxy-
5,7,3',4'-epicatechin (13)
1
(1H, d, J = 8.4 Hz), 7.71 (1H, s), 6.94 (1H, d,
J = 8.2 Hz), 6.47 (1H, s), 6.28 (1H, s), 5.4 (1H, d,
J = 7.3 Hz), 3.85 (1H, m ), 3.62 (3H, m ), 3.43 (1H,
under the MeOH). 13C NMR (100 MHz, CD3OD,
258): d 179.5, 166.3, 163.3, 159.3, 158.7, 150.2, 146.2,
135.7, 123.8, 123.1, 117.5, 116.3, 105.9, 104.6, 100.2,
The acid 12 (63 mg, 0.13 mmol) and DCC (36 mg,
0.17 mmol) were dissolved in dry toluene (1 ml) under
Argon and stirred for 5 min. The alcohol 10 (32 mg,
0.05 mmol) and DMAP (cat.) were added and the mix-
ture was heated at 728 for 3 days. The solvent was
evaporated under reduced pressure and the residue

766
G. Goetz et al. / Phytochemistry 52 (1999) 759±767
Jackson, R. S. (1994). Wine science, principles and applications. San
Diego, CA: Academic press.
was diluted with water and extracted with EtOAc. The
organic layer was washed with aq. NaOH 1N, HCl
10%, NaCl satd and dried with MgSO4. The solvent
was evaporated under reduced pressure; the residue
was puri®ed by column chromatography on silica gel
(hexane±EtOAc, 95:5) to a€ord the ester 13 as a white
Jersch, S., Scherer, C., Huth, G.,
& Schlosser, E. (1989).
Proanthocyanidins as basis for quiescence of Botrytis cinerea in
immature strawberry fruits. Journal of Plant Diseases and
Protection, 96(4), 365±378.
Kolattukudy, P. E. (1985). Enzymatic penetration of the plant cuticle
by fungal pathogens. Ann. Rev. Phytopathol., 23, 223±250.
Langcake, P., Cornford, C. A., & Ryce, R. J. (1979). Identi®cation
1
solid (48 mg, 88%). H NMR (400 MHz, CDCl3): d
7.40 (32H, m, Ar±H), 7.04 (1H, d, H-2'), 6.85 (2H, m,
H-5', H-6'), 6.35 (2H, 2 d, H-6, H-8), 5.62 (1H, m, H-
3), 5.05 (12H, m, 6CH2), 4.97 (1H, d, H-2), 4.74 (2H,
2 d, CH2), 3.10 (2H, m, H-4). 13C NMR (100 MHz,
CDCl3): d 165.6, 159.5, 158.7, 156.4, 153.0, 153.0,
149.6, 149.6, 143.2, 138.1, 137.8, 137.7, 137.5, 137.5,
137.2, 131.7, 129.3, 129.2, 129.2, 129.1, 129.0, 129.0,
128.8, 128.8, 128.7, 128.6, 128.6, 128.5, 128.3, 128.1,
128.0, 128.0, 127.8, 125.6, 120.7, 115.4, 114.3, 109.7,
101.6, 95.3, 94.6, 78.3, 77.9, 75.7, 71.8, 71.8, 71.8, 71.7,
70.8, 70.6, 69.2, 26.8.
of pterostilbene as
Phytochemistry, 18, 1025±1027.
a phytoalexin from Vitis vinifera leaves.
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4.4.12. Epicatechin-3-O-gallate (5)
Pezet, R., & Pont, V. (1984). Botrytis cinerea: activite antifongique
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A suspension of tribenzyloxy-3,4,5-benzoate-3-tetra-
benzyloxy-5,7,3',4'-epicatechin 13 (2.1 mg, 0.002 mmol)
and 10% Pd±charcoal in EtOAc (2 ml) were stirred
under pressure of H2 (40 bar) for 5 h. The catalyst was
®ltered o€, washed with MeOH and the solvent was
evaporated under reduced pressure. The residue was
puri®ed by prep. TLC (silica gel) to a€ord the desired
compound 5. 1H NMR (400 MHz, CD3OD): d 7.04
(2H, s, H-20, H-60), 7.02 (1H, d, J = 2 Hz, H-2'), 6.91
(1H, dd, J = 8.5 Hz, J = 2 Hz, H-6'), 6.78 (1H, d,
J = 8 Hz, H-5'), 6.05 (2H, 2 d, J = 2.5 Hz, H-6, H-8),
5.61 (1H, m, H-3), 5.12 (1H, s, H-2), 3.08 (1H, dd,
J = 4.7 Hz, J = 17 Hz, H-4ax.), 2.93 (1H, dd,
J = 2.4 Hz, J = 17 Hz, H-4eq.). 13C NMR (100 MHz,
CD3OD): d 167.9 (CO), 158.2, 157.6 (C-5,7,9), 146.6
(C-30,50), 146.3 (C-3'), 146.2 (C-4'), 140.1 (C-6'), 131.7
(C-1'), 121.8 (C-10), 119.7 (C-6'), 116.3 (C-5'), 115.4
(C-2'), 110.5 (C-20,60), 99.7 (C-10), 96.8 (C-6), 96.2 (C-
8), 78.9 (C-2), 70.3 (C-3), 27.2 (C-4). EIMS m/z: 442
[M+], 273 [M-gallate+], 170 [gallate+]
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dans les grappes Vitis vinifera (var. Gamay et Pinot). Plant
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studies of two grape cultivars in the view of their respective sus-
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