HPLC Analysis of Grapevine Phytoalexins Coupling Photodiode Array Detection and Fluorometry

Article abstract of DOI:10.1021/ac970582b

A reversed-phase HPLC method useful for the analysis of the grapevine phytoalexins resveratrol, its β-D-glucoside, ε-viniferin, and pterostilbene in leaf extracts was developed by coupling diode array detection and fluorometry. Phytoalexins were extracted

Full text of DOI:10.1021/ac970582b

Anal. Chem. 1997, 69, 5172-5177
Technical Notes
HP LC An a lys is o f Gra p e vin e P h yt o a le x in s Co u p lin g
P h o t o d io d e Arra y De t e c t io n a n d Flu o ro m e t ry
,
|
†
†
⊥
†
Philippe J e a nde t,* Anne C e´ line Bre uil, Ma rie lle Adria n, Le s lie A. We s ton, Sylva in De bord,
‡
§
†
Philippe Me unie r, Ga brie lle Ma um e , a nd Roge r Be s s is
Laboratoire d’Oenologie, UFR Sciences, Universit e´ de Reims, BP 1039, 51687 Reims, France, Laboratoire des Sciences de
la Vigne, Institut Jules Guyot, Laboratoire de Synth e` se et d’Electrosynth e` se Organom e´ talliques, UMR CNRS 5632, and
Laboratoire de Phytopharmacie et de Biochimie des Interactions Cellulaires, URA-INRA, Universit e´ de Bourgogne,
BP 138, 21004 Dijon, France, and Department of Horticulture and Landscape Architecture, N318 Agricultural Science
Building North, University of Kentucky, Lexington, Kentucky 40546
and pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene)4,5 (Fig-
ure 1). Stilbenes have provoked an intense interest due to their
antifungal properties, and their presence has been shown to be
A reversed-phase HP LC method useful for the analysis
of the grapevine phytoalexins resveratrol, its â- -gluco-
D
side, E-viniferin, and pterostilbene in leaf extracts was
developed by coupling diode array detection and fluorom-
etry. P hytoalexins were extracted from UV-irradiated
grapevine leaves with methanol/ water (8 0 :2 0 ) and pre-
purified on C1 8 solid phase extraction cartridges. Sepa-
ration by HP LC was achieved using a C1 8 column and a
gradient elution with acetonitrile and water (from 1 0 to
closely related to grape disease resistance, namely to gray mold
caused by Botrytis cinerea.6-8 Moreover, resveratrol is one of the
constituents of wine which is thought to confer protection against
9
10
artherosclerosis, coronary heart diseases, and cancer. In light
of these findings, recent papers have described analytical methods
to assay resveratrol in wines1
1-20
or in grapevines.15,21-23 In
8
5% acetonitrile). Analyses of grapevine leaf extracts were
contrast, there is only one work describing a method suitable for
the simultaneous analysis of the other stilbenic phytoalexins of
grapes,24 but the latter method did not allow for the identification
of the compounds analyzed, i.e., resveratrol, pterostilbene, and
performed by injecting the equivalent of 1 mg of leaf fresh
weight. Recovery was nearly 1 0 0 % for the three stilbenes
resveratrol, pterostilbene, and E-viniferin (ranging from
1
00.2 to 104.8%), and replicate analyses gave coefficients
of variation of 0 .6 -2 .5 %. Identification of each phyto-
alexin was accomplished by line spectral comparisons
with known standards, and E-viniferin was further char-
acterized by MS and GC/ MS. Simultaneously, stilbenes
were detected by fluorometry, allowing specific identifica-
tion of these compounds. This procedure provided excel-
lent separation and enabled quantitation of all grapevine
phytoalexins present in the extracts. The method can
easily be extended to the analysis of wine or biological
fluids.
(4) Langcake, P.; Pryce, R. J. Experientia 1 9 7 7 , 33, 151-152.
(
5) Langcake, P.; Cornford, C. A.; Pryce, R. J. Phytochemistry 1 9 7 9 , 18, 1025-
027.
6) Langcake, P. Physiol. Plant Pathol. 1 9 8 1 , 18, 213-216.
1
(
(7) Jeandet, P.; Bessis, R.; Sbaghi, M.; Meunier, P. J. Phytopathol. 1 9 9 5 , 143,
35-139.
8) Sbaghi, M.; Jeandet, P.; Faivre, B.; Bessis, R.; Fournioux, J. C. Euphytica
9 9 5 , 86, 41-47.
1
(
1
(9) Pace-Asciak, C. R.; Hahn, S.; Diamandis, E. P.; Soleas, G.; Goldberg, D. M.
Clin. Chim. Acta 1 9 9 5 , 235, 207-219.
(
10) Jang, M.; Cai, L.; Udeani, G. O.; Slowing, K. V.; Thomas, C. F.; Beecher, C.
W. W.; Fong, H. H. S.; Farnsworth, N. R.; Kinghorn, A. D.; Mehta, R. G.;
Moon, R. C.; Pezzuto, J. M. Science 1 9 9 7 , 275, 218-220.
(
(
11) Siemann, E.; Creasy, L. L. Am. J. Enol. Vitic. 1 9 9 2 , 43, 49-52.
12) Lamuela-Raventos, R. M.; Waterhouse, A. L. J. Agric. Food Chem. 1 9 9 3 ,
Phytoalexins are biologically active compounds that are
produced by plants in response to fungal infection or abiotic
stresses such as heavy metal ions or UV light.1 In grapevines,
such a response includes the synthesis of a simple stilbene,
resveratrol (trans-3,5,4′-trihydroxystilbene), and its glucoside,3
together with the biosynthetically related compounds ꢀ-viniferin
4
1, 521-523.
(13) Mattivi, F. Z. Lebensmit. Untersuch. Forsch. 1 9 9 3 , 196, 522-525.
(
(
14) Goldberg, D. M.; Yan, J.; Ng, E.; Diamandis, E. P.; Karumanchiri, A.; Soleas,
G. J.; Waterhouse, A. L. Anal. Chem. 1 9 9 4 , 66, 3959-3963.
15) Pezet, R.; Pont, V.; Cuenat, P. J. Chromatogr. A 1 9 9 4 , 663, 191-197.
2
(16) Goldberg, D. M.; Ng, E.; Karumanchiri, A.; Diamandis, E. P.; Soleas, G. J.
J. Chromatogr. A 1 9 9 5 , 708, 1245-1250.
*
To whom correspondence should be addressed. Fax: 11-33-326-05-33-40.
(17) Lamuela-Raventos, R. M.; Romero-Perez, A. I.; Waterhouse, A. L.; De la
Torre-Boronat, M. C. J. Agric. Food Chem. 1 9 9 5 , 43, 281-283.
(18) Jeandet, P.; Bessis, R.; Maume, B. F.; Meunier, P.; Peyron, D.; Trollat, P.
J. Agric. Food Chem. 1 9 9 5 , 43, 316-319.
E-mail: philippe.jeandet@univ-reims.fr.
|
Laboratoire d’Oenologie, Universit e´ de Reims.
Laboratoire des Sciences de la Vigne, Universit e´ de Bourgogne.
Laboratoire de Synth e` se et d’Electrosynth e` se Organom e´ talliques, Universit e´
†
‡
(19) Jeandet, P.; Bessis, R.; Sbaghi, M.; Meunier, P.; Trollat, P. Am. J. Enol.
de Bourgogne.
Vitic. 1 9 9 5 , 46, 1-4.
(20) Goldberg, D. M.; Tsang, E.; Karumanchiri, A.; Diamandis, E. P.; Soleas, G.
J.; Ng, E. Anal. Chem. 1 9 9 6 , 68, 1688-1694.
(21) Creasy, L. L.; Coffee, M. J. Am. Soc. Hortic. Sci. 1 9 8 8 , 113, 230-234.
(22) Jeandet, P.; Bessis, R.; Gautheron, B. Am. J. Enol. Vitic. 1 9 9 1 , 42, 41-46.
(23) Adrian, M.; Jeandet, P.; Bessis, R.; Joubert, J. M. J. Agric. Food Chem. 19 96 ,
44, 1979-1981.
§
Laboratoire de Phytopharmacie et de Biochimie des Interactions Cellulaires,
Universit e´ de Bourgogne.
⊥
University of Kentucky.
(
(
(
1) Bailey, J. A. Phytoalexins; Blackie: Glasgow and London, 1982; Chapter 9.
2) Langcake, P.; Pryce, R. J. Physiol. Plant Pathol. 1 9 7 6 , 9, 77-86.
3) Waterhouse, A. L.; Lamuela-Raventos, R. M. Phytochemistry 1 9 94 , 37, 571-
5
73.
(24) Langcake, P.; Pryce, R. J. Phytochemistry 1 9 7 7 , 16, 1193-1196.
5172 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
S0003-2700(97)00582-9 CCC: $14.00 © 1997 American Chemical Society

Fig u re 1 . Chemical structures of the stilbene phytoalexins measured by this method.
ꢀ-viniferin. Moreover, the resveratrol glucosides were not in-
cluded in this analysis.
We now describe an HPLC technique coupling diode array
detection and fluorometry that is useful for the analysis of the cis
and trans isomers of resveratrol and its â-D-glucoside, ꢀ-viniferin,
and pterostilbene in grapevine leaf extracts. Identification of each
phytoalexin was accomplished by line spectral comparisons with
known standards, and ꢀ-viniferin was further characterized by MS
and GC/ MS. Specific detection of all stilbenes was also obtained
using fluorometric analysis.
showed a trans-stilbene chromophore similar to trans-resveratrol,
but in contrast the spectrum showed no base shift of the long-
wavelength chromophore in the presence of diluted NaOH,
indicating the trans-stilbene lacking a phenolic group in the 2- or
4
-position of the stilbene moiety.27 Moreover, the mass spectrum
MATERIAL AND METHODS
of this compound (EI, M+ at m/ e 454, relative intensity ) 100)
Standards. A chemically pure standard of trans-resveratrol
+
and that of its trimethylsilyl ether (GC/ MS, M at m/ e 814, relative
was synthesized by a Wittig condensation between a phosphorus
ylide and a silylated hydroxybenzaldehyde, as previously de-
scribed.22,25 trans-Pterostilbene was obtained from Drs. R. Pezet
and V. Pont (Swiss Federal Agricultural Station of Changins).
intensity ) 100) suggested that it consists of a pentaphenol with
a dehydrodimeric structure.
1
The structure of 1 was also confirmed by H-NMR (500 MHz,
).27 Cis isomers of each stilbene were obtained by UV
acetone-d
6
trans-Piceid, the 3-â-D
-glucoside of resveratrol,3,16,26 was isolated
irradiation of diluted solutions (5 µg/ mL) of the corresponding
trans-stilbene. The standards were placed in a UV cuvette and
irradiated for 10 min at 366 nm.
P lant Material. Two Vitis species differing in susceptibility
to gray mold were used in this study: Vitis vinifera L. cv Pinot
Noir (susceptible species) and Vitis rupestris L. cv Rupestris du
Lot (tolerant species). Only leaves (from in vitro grown plantlets
or from plants growing in the fields) have been assayed for
from the dried roots of Polygonum cuspidatum as described by
Waterhouse and Lamuela-Raventos.3 The purity of the glucoside
obtained was compared to a standard furnished by Professor D.
M. Goldberg (University of Toronto, Canada). A resveratrol
dehydrodimer analogous to ꢀ-viniferin was obtained by dimeri-
zation of trans-resveratrol with horseradish peroxidase-hydrogen
peroxide utilizing a modification of the method of Langcake and
Pryce27 or Calderon et al.28 trans-Resveratrol (6 mg, 26.3 µM)
phytoalexin production. Stilbene synthesis was induced in grape
leaves by UV irradiation as previously described.22
was treated with 2 mL of H
2
O
2
and 0.1 mg of horseradish
peroxidase (type IX, Catalog No. P-1139, 275 units/ mg solid,
purchased from Sigma, St. Louis, MO) in 100 mL of citrate buffer
and 8% dioxane (to ensure solubility of resveratrol) at pH 6.0. The
mixture was stirred for 15 min in the dark and then extracted
twice with ethyl acetate. The dehydrodimer 1 obtained under
these conditions was found to be an isomeric form of the grapevine
dehydrodimer ꢀ-viniferin.27 Compound 1 was purified by prepara-
tive TLC on reversed-phase C18 material (RP 18 F254S, Merck,
Sample P reparation. UV-irradiated leaves were ground in
a mortar with sand and 30 mL of 8:2 (v/ v) MeOH/ water. After
centrifugation at 10000g for 15 min, the supernatant was prepu-
rified on a Sep-Pak C18 cartridge (Waters, Milford, MA). After
elution with 8:2 (v/ v) MeOH/ water (30 mL), the eluate was
evaporated to dryness (<40 °C). Leaf extracts were then redis-
solved in 10 mL of methanol/ g fresh weight and filtered. For
HPLC analysis, 10 µL of each sample (i.e., 1 mg fresh weight)
was injected. During sample preparation, extracts were constantly
Darmstadt, Germany) in 7:3 (v/ v) MeOH/ water (R
f
) 0.53),
leading to 3 mg (6.61 µM) of 1 (50% yield). Its UV spectrum
(
27) Langcake, P.; Pryce, R. J. J. Chem. Soc. Chem. Commun. 1 9 7 7 , 208-210.
(
(
25) Moreno-Manas, M.; Pleixats, R. Anal. Quim. 1 9 8 5 , 81, 157-161.
26) Jeandet, P.; Bessis, R.; Sbaghi, M.; Meunier, P. Vitis 1 9 9 4 , 33, 183-184.
(28) Calderon, A. A.; Pedreno, M. A.; Ros-Barcelo, A.; Munoz, R. J. Biochem.
Biophys. Methods 1 9 9 0 , 20, 171-180.
Analytical Chemistry, Vol. 69, No. 24, December 15, 1997 5173

protected from light to avoid photochemical isomerization of trans-
resveratrol to the cis form.
ties similar to those of cis-resveratrol. Concentrations of resvera-
trol, its glucosides, pterostilbene, and ꢀ-viniferin in grape leaf
extracts were measured using the external standard method.
Response factors (i.e., amounts of standard/ peak area) were
determined with data from the calibration curves. The detection
limit was measured as the concentration corresponding to the
lowest signal which differs significantly (p < 0.01) from the
baseline. This value was ∼0.1 µg/ g fresh weight for the four
stilbenes and was sufficient due to the high levels of each
compound found in the extracts.
To evaluate the efficiency of this extraction procedure, 100 µg
portions of each of the three stilbenes were added to 1 g of
uninduced fresh material and treated as described above. This
was done in triplicate. The recovery concentrations of the
extracted standards determined by HPLC were 103.6 ( 4.6% for
resveratrol, 104.8 ( 5.3% for pterostilbene, and 100.2 ( 4.5% for
the dehydrodimer 1 . The precision of the method was evaluated
by performing six replicate analyses of three different grape leaf
extracts. The coefficients of variation ranged from 0.6 to 2.5% for
all stilbenes.
Enzymatic Hydrolyses of Resveratrol Glucosides. Enzy-
matic hydrolyses of resveratrol glucosides with â-
(Catalog No. 49290, 6.76 units/ mg protein, Fluka, Buchs, Swit-
zerland) or R- -glucosidase (Catalog No. G-6136, 25 units/ mg
D-glucosidase
HP LC Analysis. Analyses were performed on a Lichrocart
Merck C18 (Merck-Clevenot Corp., Darmstadt, Germany) reversed-
phase column (250 mm × 4 mm, 5 µm) preceded by a guard
column of Lichrospher 100 RP-18 (4 mm × 4 mm, 5 µm, Merck).
A Waters system comprising a Model W 600 system controller, a
Model W 717 sample injector, a Model W 996 photodiode array
detector, and a Model W 474 fluorometer was used. For
fluorometric detection, maximum excitation wavelength was
D
protein, Sigma, St. Louis, MO) were conducted as follows:
methanolic extracts of grape leaves (see above) were diluted with
deionized water such that the concentration of alcohol does not
exceed 10% and adjusted to pH 6.0 with 0.1 N NaOH. Enzymes
were added in a concentration of 1 mg/ mL of diluted samples.
The mixtures were then incubated at 30 °C for 17 h (overnight).
Samples were concentrated and dissolved in methanol and filtered
before HPLC analysis.
MS and GC/ MS Analyses. MS analysis of the resveratrol
dehydrodimers (ꢀ-viniferin and its isomeric form) was carried out
using a mass spectrometer (Kratos, Concept IS, Great Britain)
with direct introduction of the samples. GC/ MS analysis of both
dimers was done in the form of their trimethylsilyl derivatives18,22
using a quadrupole mass spectrometer (Nermag R 10-10C). In
both cases, ionization was obtained by electron impact (electron
energy, 70 eV, and filament current, 0.19 mA).29 Samples were
injected into a Chrompack gas chromatograph (Chrompack Corp.,
The Netherlands) equipped with a de Ros injector. Analysis was
performed on a capillary column SE-30 (25 m × 0.32 mm)
operating at 300 °C. The pressure of the carrier gas (nitrogen)
was maintained at 50 kPa at a flow rate of 1 mL/ min. Under these
conditions, the retention times of both resveratrol dehydrodimers
were 17.0 min.
measured at 330 nm and emission at 374 nm.2
,15
Stilbenes were
eluted from the HPLC C18 column with a gradient comprising
acetonitrile (solvent A) and water (solvent B). Solvents were
delivered according the following program: linear gradient elution
from 10% A and 90% B to 85% A and 15% B within 18 min; 85% A
and 15% B for 5 min; linear gradient elution from 85% A and 15%
B to 10% A and 90% B within 7 min. This was followed by a 5 min
equilibrium period with 10% A and 90% B prior to injection of the
next sample. The flow rate was 1 mL/ min.
Identification and Quantification of Stilbenes. Identifica-
tion of resveratrol, its â-D-glucoside, pterostilbene, and the
dehydrodimer ꢀ-viniferin in extracts was carried out by comparison
of the retention time of each standard and that within the extracts.
They were also characterized by their UV spectra from 250 to
4
00 nm using the photodiode array detector and by line spectral
comparisons with the standards. Further identification of res-
veratrol glucosides was achieved by enzymatic hydrolysis (see
below). MS and GC/ MS were also used for the characterization
of ꢀ-viniferin (see below). Quantification of trans-resveratrol, trans-
pterostilbene, and trans-ꢀ-viniferin was performed using the
fluorometer only since these three stilbenes were found to have
RESULTS AND DISCUSSION
Analysis of Standards. Stilbenes are highly fluorescent
compounds that are very easily detected by fluorometry. Figure
2
A shows the HPLC profile corresponding to the four standards
the same fluorometric characteristics (λex ) 330 nm and λem
)
using fluorometric detection. Under our chromatographic condi-
tions, retention times were 12.63, 15.30, 17.38, and 21.28 min for
the trans form of the resveratrol glucoside, trans-resveratrol, the
trans-dehydrodimer 1 , and trans-pterostilbene, respectively. Si-
multaneously, these four compounds were analyzed by a photo-
diode array detector and characterized by their UV spectra from
374 nm). Standard calibration curves were established using peak
area vs different amounts of each stilbene (i.e., 2, 10, 20, 50, 100,
and 200 ng). We used the calibration curve obtained for the
dehydrodimer 1 to quantify ꢀ-viniferin in the extracts. Three
replicates were made for each concentration. Mean coefficients
of variation (CV) and linear correlations were calculated using
the Waters Millenium system. Linear correlations for fluorometric
250 to 400 nm. All compounds showed two bands corresponding
to a high absorbance from 308 to 336 nm (band I) and from 281
to 313 nm (band II), bands which are characteristic of the trans-
stilbenes30 (Figure 3A,B). UV maximum absorbances of trans-
detection were excellent (r2 ) 0.999 for trans-resveratrol, r2
0
)
.9995 for trans-pterostilbene, and r2 ) 0.9975 for the trans-
dehydrodimer 1 ). The trans form of the resveratrol glucoside
was quantified using calibration curves of trans-resveratrol, since
both compounds have the same fluorometric characteristics. The
cis forms of stilbenes are normally not present in grape leaf
extracts.2,19,22 Quantification of cis-stilbenes was thus not neces-
sary, except for the cis form of the resveratrol glucoside, which
was always present in the extracts. cis-Resveratrol glucoside was
quantified by UV at 285 nm by using the calibration curve obtained
resveratrol and trans-pterostilbene were both at 307.8 nm, values
which are consistent with those previously published.2,5,18,31 Unlike
resveratrol and pterostilbene, the synthetic trans-dehydrodimer
of resveratrol showed a maximum absorbance at 224 nm (data
not shown). This compound also contains a trans-stilbene moiety
(
29) Maume, B. F.; Millot, C.; Mesnier, D.; Patouraux, D.; Doumas, J.; Tomori,
E. J. Chromatogr. 1 9 7 9 , 186, 581-594.
(
30) Hillis, W. E.; Ishikura, N. J. Chromatogr. 1 9 6 8 , 32, 323-336.
2
for cis-resveratrol (r ) 0.995). The cis-glucoside has UV proper-
(31) Trela, B. C.; Waterhouse, A. L. J. Agric. Food Chem. 1 9 9 6 , 44, 1253-1257.
5174 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

trans-resveratrol by the following criteria: (1) its retention time
was identical to that of synthetic trans-resveratrol, and it cochro-
matographed with pure resveratrol; and (2) its UV spectrum was
identical to that of trans-resveratrol, and its purity was confirmed
as >99% by diode array detection of the spectrum. Peak D was
identified as trans-pterostilbene using the same criteria as those
used for resveratrol. The two peaks (noted E and F) found in
grape leaf extracts (Figure 4B,C) were identified as the â-
glucosides of cis- and trans-resveratrol, respectively, by the
following criteria: (1) treatment of samples with â- -glucosidase
D-
D
resulted in the disappearance of these peaks with a concomittant
increase of peak A (corresponding to the trans-resveratrol peak)
and the occurrence of a new compound (noted peak G), whose
UV spectrum and retention time were identical with those of cis-
resveratrol (Figure 3C); (2) treatment of samples by an R-D-
glucosidase had no effects on these peaks; (3) UV spectra of the
two peaks were very close to those of cis- and trans-resveratrol,
respectively, and their purities were confirmed as >99% by diode
array detection of the spectrum; and (4) the two compounds
cochromatographed with authentic cis- and trans-piceid. The
occurrence of the cis form of the glucoside of resveratrol is
surprising since extracts have constantly been protected from light
during sample preparation. Thus, the presence of cis-resveratrol
glucoside in the extracts is likely not attributable to the photo-
chemical isomerization of the trans form (see below).
Peak C was identified as being the resveratrol dehydrodimer,
ꢀ-viniferin, by the following criteria: (1) it cochromatographed
with the synthetic resveratrol dehydrodimer 1 ; (2) its UV
spectrum was identical to that of the pure dehydrodimer 1 ; and
(
3) photochemical isomerization of peak C gives a new peak
corresponding to a cis-stilbene, which cochromatographed with
the cis form of 1 . Further identification of C was achieved by
mass spectrometry. In this way, small quantities of C were
purified from grapevine leaf extracts (V. rupestris) by preparative
TLC using reversed-phase material RP 18 in 7:3 (v/ v) MeOH/
water. Observation of the TLC plates under long-wavelength UV
light revealed the presence of four fluorescent compounds, three
of which were identified as resveratrol (R
) 0.78), and pterostilbene (R ) 0.30), respectively. The fourth
compound cochromatographed with the synthetic dehydrodimer
1 (R ) 0.53). After TLC separation and extraction, HPLC analysis
f
) 0.66), its glucoside
Fig u re 2 . HPLC analysis of standards of phytoalexins (detection
by fluorometry). (A) Peak A, trans-resveratrol; peak C, trans-
resveratrol dehydrodimer 1; peak D, trans-pterostilbene; peak E,
trans-resveratrol glucoside. (B) HPLC profile obtained after partial
photochemical isomerization of the standards. Peaks A, C, D, and
E were the same as in part A; peak F, cis-resveratrol glucoside; peak
G, cis-resveratrol; peak H, cis-resveratrol dehydrodimer; peak I, cis-
pterostilbene.
(
R
f
f
f
confirmed the correspondence between peak C and the compound
collected from TLC plates. Compound C was then analyzed by
MS and GC/ MS (after derivatization with BSTFA) (see Materials
and Methods). The mass spectrum of the free phenol (EI, M+ at
m/ e 454, relative intensity ) 100) (Figure 5) and that of the
+
with a long-wavelength chromophore similar to that of resveratrol
trimethylsilyl ether obtained by GC/ MS (M at m/ e 814, relative
(
λ
max ) 312.5 nm). The UV spectrum of the resveratrol glucoside
intensity ) 100) confirm the dimeric and pentaphenolic nature of
this compound. With diluted NaOH, its UV spectrum showed a
base shift of the long-wavelength chromophore, indicating the
presence of a trans-stilbene unit with a free p-hydroxy group.27 It
can thus be assumed that peak C corresponds to the resveratrol
dehydrodimer, ꢀ-viniferin. However, further characterization by
NMR was not possible, due to the small quantities available.
The concentrations of stilbenes in grape leaf extracts are
presented in Table 1. It can be seen that the phytoalexin
production potential (determined by the amounts of free resvera-
trol, its glucosides, and ꢀ-viniferin) is higher for the tolerant
species, V. rupestris, than for the susceptible one, V. vinifera.
Pterostilbene can also be synthesized in relatively large amounts
is very close to that of resveratrol (λmax ) 320 nm). UV maximum
absorbances of the cis forms were, for the four stilbenes (Figure
3
C,D), at 285 nm. UV spectra of cis-stilbenes are characterized
by the lack of band I (see above). Light-induced isomerization
of the trans-stilbene moiety in the dehydrodimer 1 leads to the
cis isomer.
Analysis of Grape Leaf Extracts. Figure 4 shows the HPLC
profiles corresponding to the injection of UV-induced leaf extracts
of V. vinifera (A) and V. rupestris (B). Five major peaks appeared
within the extracts of V. rupestris (peaks A, C, D, E, and F), and
only two major and two minor peaks appeared within the extracts
of V. vinifera (peaks A, C, E, and F). Peak A was identified as
Analytical Chemistry, Vol. 69, No. 24, December 15, 1997 5175

Fig u re 3 . Spectra of trans- and cis-stilbenes obtained by diode array detection. (A) Spectra of trans-resveratrol (peak A) and its trans-
glucoside (peak E). (B) Spectra of the trans-dehydrodimer 1 (peak C) and trans-pterostilbene (peak D). (C) Spectra of cis-resveratrol (peak G)
and its cis-glucoside (peak F). (D) Spectra of the cis-dehydrodimer 1 (peak H) and cis-pterostilbene (peak I).
Ta b le 1 . P ro d u c t io n o f P h yt o a le x in s in Vit is s p p . As
a
In d u c e d b y UV Irra d ia t io n
V. vinifera
V. rupestris
trans-resveratrolb
trans-resveratrol glucoside
cis-resveratrol glucoside
trans-ꢀ-viniferin
102 ( 25
8 ( 2
216 ( 42
80 ( 20
48 ( 12
70 ( 18
14 ( 7
3 ( 1
25 ( 6
0.2-0.8
trans-pterostilbene
a
All leaves were placed in Petri dishes on moist filter paper and
then irradiated on their abaxial surfaces for 10 min with UV radiation
-
2
(
0.36 J cm ). After 24 h in darkness, phytoalexins were extracted as
described in the Materials and Methods section. The values represent
average phytoalexin production of six leaves of each species. b Ex-
pressed in micrograms per gram fresh weight. Quantification of all
stilbenes was done by using the fluorometer, except for the cis-
resveratrol glucoside which was quantified in UV at 285 nm (see
Materials and Methods section).
in the leaves of V. rupestris, but this was not consistently observed
in our experiments. Pterostilbene is a very biologically active
compound which is usually found in low quantities in grape-
vines.5,32
The fact that extracts of the variety Pinot Noir contained very
low concentrations of resveratrol glucosides confirmed other
previously published results,33 which showed that Pinot Noir wines
generally have a low resveratrol glucoside content. In contrast,
in V. rupestris leaf extracts, the sum of the glucosides (cis and
trans) can exceed the free resveratrol content. The existence of
free and bound forms of resveratrol in grapevine leaf extracts leads
one to formulate interesting hypotheses regarding the metabolism
of this stilbene within the plant. trans-Resveratrol is likely
synthesized in the free form by the action of stilbene synthase
and then rapidly glycosylated by a glycosyl transferase to trans-
Fig u re 4 . HPLC chromatograms of Vitis spp. extracts. (A) V.
vinifera extract. (B) V. rupestris extract. (C) The same extract as in
part B but obtained after treatment with â-D-glucosidase for 17 h,
showing disappearance of peaks E and F, increase in peak A, and
occurrence of a new peak (peak G). Peak A, trans-resveratrol; peak
C, trans-ꢀ-viniferin; peak D, trans-pterostilbene; peak E, trans-
resveratrol glucoside; peak F, cis-resveratrol glucoside; peak G, cis-
resveratrol.
(
(
32) Pezet, R.; Pont, V. Plant Physiol. Biochem. 1 9 8 8 , 26, 603-607.
33) Goldberg, D. M.; Ng, E.; Karumanchiri, A.; Diamandis, E. P.; Soleas, G. J.
Am. J. Enol. Vitic. 1 9 9 5 , 46, 400.
5176 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

resveratrol, its â-D-glucoside, pterostilbene, and ꢀ-viniferin. Given
the high level of peak resolution, both the cis and the trans
isomers of these compounds could be detected. Use of fluoro-
metric detection together with diode array detection permits the
unambigous characterization of stilbene compounds, and this is
due, first, to the specificity of the fluorometric parameter detection
linked to the particular structure of stilbenes and second, to the
characteristic absorbance of the stilbene skeleton between 260
and 350 nm.30 Simultaneous determination of pterostilbene and
ꢀ-viniferin together with that of resveratrol and its glucosides
allows an excellent assessment of the grapevine phytoalexin
response. In fact, although there are a number of works on the
relationship between resveratrol production and grape disease
resistance,6,8,35,36,37 the biological role of the other stilbenes in the
defense mechanisms of grapevines is poorly understood, largely
due to the lack of data concerning their chromatographic deter-
mination. Moreover, study of pterostilbene and ꢀ-viniferin is also
Fig u re 5 . Mass spectrum (electron impact, direct introduction) of
compound C obtained after purification by TLC.
of great interest since they are both considered to be more
fungitoxic than resveratrol itself.6,38 This analytical method thus
allows the determination of the major phytoalexins in grapevine
leaves with the aim of clarifying their role in the resistance of
this species against fungal attack.
resveratrol glucoside; after this, isomerization to the cis-glycosidic
form is possible by a cis-isomerase (which has not yet been
characterized to date). The occurrence of free resveratrol in the
extracts is linked to the presence of endogenous â-D-glucosidases,
which probably remain active during the extraction process.34
Interestingly, we observed a direct negative correlation between
the amounts of free resveratrol and those of glycosylated resvera-
trol in the plant extracts.
ACKNOWLEDGMENT
We thank Professor D. M. Goldberg (University of Toronto,
Canada) and Mr. G. Soleas (Andres Wines Limited, Grimsby,
Canada) for the generous gift of a purified sample of resveratrol
glucoside. We are also indebted to Drs. R. Pezet and V. Pont
In this paper, we have described a method suitable for the
analysis of the four major stilbene phytoalexins of grapevines,
(
Swiss Federal Agricultural Station of Changins) for providing us
(
(
34) Ayran, A.; Wilson, B.; Strauss, C.; Williams, P. Am. J. Enol. Vitic. 1 9 8 7 ,
8, 182-188.
35) Barlass, M.; Miller, R. M.; Douglas, T. J. Am. J. Enol. Vitic. 1 9 8 7 , 38, 65-
8.
36) Dercks, W.; Creasy, L. L. Physiol. Mol. Plant Pathol. 1 9 8 9 , 34, 189-202.
37) Dercks, W.; Creasy, L. L. Physiol. Mol. Plant Pathol. 1 9 8 9 , 34, 203-213.
38) Adrian, M.; Jeandet, P.; Veneau, J.; Weston, L. A.; Bessis, R. J. Chem. Ecol.,
in press.
a pure sample of pterostilbene.
3
Received for review June 5, 1997. Accepted September
6
X
25, 1997.
(
(
(
AC970582B
X
Abstract published in Advance ACS Abstracts, November 1, 1997.
Analytical Chemistry, Vol. 69, No. 24, December 15, 1997 5177