CHEMICAL CONSTITUENTS FROM VITIS VINIFERA (VITACEAE)

J.D. Felicio1, R. da S. Santos1, E. Gonçalez1

Laboratório de Química e Farmacologia de Produtos Naturais e Sintéticos, Centro de Sanidade Animal, Instituto
1

Biológico, CP 12.898, CEP 04010-970, São Paulo, SP, Brazil. E-mail: felicio@biologico.br

ABSTRACT

Optimal conditions for the simultaneous quantitative separation of resveratrol, ε - viniferins,

balanocarpol, and balanocarpol glycoside a new substance, isolated of Vitis vinifera, were
investigated in extracts from leaves and branches. The best results were obtained by the use of
reversed – phase high performance liquid chromatography (HPLC) using a mixture of 5% acetic
acid and methanol (65:35) as the mobile phase, detector wavelength of 290nm and flow rate of 1mL/
min. The quantification showed the presence of all these compounds in the ethanolic extract from
branches, but only the resveratrol was quantified in the ethanolic extracts from leaves.

KEY WORDS: Resveratrol, ε - viniferins, balanocarpol, HPLC, Vitis vinifera, Vitaceae.

RESUMO

COMPONENTES QUÍMICOS DE VITIS VINIFERA. Os compostos químicos resveratrol, e -

viniferina, balanocarpol e balanocarpol glicosídeo uma substância nova, foram isolados de Vitis
vinifera e quantificados por Cromatografia Líquida de alta eficiência (CLAE). Condições ótimas para
análise simultanea dos quatro compostos em extratos de galhos e folhas da planta foram determi-
nadas, usando como fase móvel ácido acético e metanol (65:35), detector de UV 290nm fluxo 1mL/
min. Todos os quatro compostos foram quantificados no extrato etanólico dos galhos; no extrato
etanólico das folhas, apenas o resveratrol foi quantificado.

PALAVRAS-CHAVE: Resveratrol, ε - viniferina, balanocarpol , Vitis vinifera, Vitaceae, CLAE.

INTRODUCTION besides its anti-inflammatory and anticancer

Vitis vinifera is a plant of economic importance
because it is utilized in the popular medicine of several
countries. Young branches of V. vinifera for example,
is a popular drug for skin diseases and for eye
inflammation. The leaves, due to their astringent and
haemostatic properties, are used in the treatment of
diarrhea, haemorrhage and varicose veins, and the
juice of leaves has been used for eye washing
(BOMBARDELLI & MORAZZONI, 1995).
Flavonoids, terpenes, organic acids, vitamins,
carbohydrats, lipids and enzymes (BOMBARDELLI &
MORAZZONI, 1995) have been isolated from fruits and
leaves of V. vinifera. The stilbene groups, as resveratrol
and viniferins, have also been isolated from leaves of
V. vinifera. Resveratrol and its derived exhibited high
antifungical activity (LANGCAKE & PRYCE, 1977 a, b, c).
Resveratrol contributes to the antioxidant potential
of red wine and may play a role in the prevention of
human cardiovascular diseases (FREMONT, 2000).
Resveratrol has also been shown to modulate the
metabolism of lipids, to inhibit the oxidation of low
density lipoproteins and the aggregation of platelets
properties have been reported (FREMONT, 2000).
The chemical study of the extracts of the branches
of Vitis vinifera led to the isolation of resveratrol (1),
and its three dimers : ε-viniferin (2), balanocarpol (3)
and the isolated new compound β-glucopyranosyl 8-
balanocarpol (4) (Fig 1). We report a reversed phase
HPLC method which has been developed for
quantification of these four substances, from ethanolic
extracts of leaves and branches of Vitis vinifera.
MATERIAL AND METHODS
Reagents and material
The reagents and material used were: acetic acid,
methanol HPLC grade, chloroform, methanol, ethanol
PA, ultra pure grade water, membrane filters (0.45µm
φ 20mm), silica gel 60 and 60G. Standard resveratrol
and its dimers were previously obtained by isolation
from ethanolic extract of branches by classic liquid
chromatography and high performace liquid
chromatography. These compounds were utilized for
the calibration curve.

Arq. Inst. Biol., São Paulo, v.68, n.1, p.47-50, jan./jun., 2001
48 J.D. Felicio et al.

Apparatus under vacuum and 2.5 mg of each were homogenized

Liquid Chromatograph LC6AD from Shimadzu
with UV/VIS SPC 6AV with variable wavelength
detector and Class VP 5.02 software (Shimadzu);
rotovapor model Buchi, Milli-Q water purifier, UV
from Hitachi model U2001 spectrophotometer,
1
HNMR (300MHz) and 13CNMR (75MHz) spectra in
CDCl3 and DMSO were record in a varian Gemini 300
spectrometer using TMS as interanl standard.
Isolation of compounds
Vitis vinifera was collected in the Biological Institute
of São Paulo (SP, Brazil). Air dried branches (380g)
were extracted with ethanol by Soxhlet for 4 hours.
The solvent evaporated under vacuum, yielding an
ethanolic extract (26 g). The ethanolic extract (12g)
was passed through a glass column packed with
silica gel and eluted with hexane-methanol in crescent
proportions. The (24) fractions of 250 mL each were
collected and analyzed by thin layer chromatography
(TLC) using silica gel 60 G CHCl3:MeOH as solvent
system. Nuclear Magnetic Ressonance (NMR)
indicated that only fractions 7, 8, 9, 10 and 19 contained
stilbene compounds. Fractions 7-10 (940mg), were
purified by Si-gel CC eluted with CHCl3:MeOH 9:1
and 8:2 yielding the compounds 1 (130 mg) and 2
(68mg). The fraction 19 (400mg) was submitted to
High Performace Liquid Chromatography (HPLC) in
the down described conditions , to afforded the
compounds 3 (78 mg) and 4 (123mg).
Chromatographic conditions
Reversed phase C18 columns Shim-pack, 250 x
4mm, 5µm (analytical) and 250 x 25mm, 5µm
(preparative); flow rate 1mL/min (analytical) and
9mL/min (preparative); detector wavelength 290nm;
mobile phase 5% acetic acid and methanol (65:35).
Preparation of extracts for analysis
Air dried branches and leaves were extracted with
ethanol by Soxhlet for 4 hours, the solvent evapored
with 10 mL of mobile phase, followed by filtration
through a 0.45mm membrane filter. Triplicate aliquot
of 20 µL were injected onto the HPLC system for
analysis.
Quantification by HPLC of compounds
The quantification of the stilbenes in the extract
samples (20µl) was done by the external standard
method using calibration curves, of the four
compounds in the concentration 0.005, 0.01, 0.02, 0.05
and 0.1mg/mL of the and Shimadzu Class VP 5.02
program. The mass of each stilbene was converted in
percentage of the compound in the total volume of the
extract.
RESULTS AND DISCUSSION
Structural identification of the compounds
The compounds were identified by mass and
NMR spectrometry. The 1H and 13C NMR data of
resveratrol (1) and e-viniferin (2), previously isolated
from leaves of Vitis vinifera, were compared with
literature (LANGCAKE & PRYCE, 1977 a, b, c). Compound
3 was identified with balanocarpol, previously
isolated from Balanocarpus zeylam (DIYASENA et. al,
1985), but now isolated in Vitaceae species for the first
time. The elucidation of the compound 4 was through
comparison with data of the compound 3 . The mass
spectrum of compound 3 showed na [M+] ions at m/
z 470, which together with the data for 13C and 1H
spectra (Table 1) and literature, allowed us to deduce
the molecular formula C28H22O7. The UV absorptions
of these compounds 3 and 4 are very similary 284nm
and 283 nm respectivly, indicated that they are
hydroxystilbene derivative.
The 13C NMR datum of 3 and 4 (Table 1) showed
both compounds were resveratroldimers, the
comparison of the chemical shift with ε-viniferin
showed much difference, but similarity with the
balanocarpol dimers. The 1H and 13 C NMR spectra of
3 and 4 also are similarity except the carbon 1, 8, 1’, 7’,

Table 1 - 1H, 13C NMR data of compounds 3 and 4 ( DMS-d6, 300MHz).

H 3 4 C 3 4 C 3 4

2,6 7.55 d (8,3) 7.13 d (8,3) 1 133.9 131.1 1’ 138.3 136.0

3,5 7.01 d (8.3) 6.92 d (8.3) 2,6 131.2 130.2 2’,6’ 129.9 129.8
7 5.81 5.84 3,5 115.1 115.5 3’5’ 116.7 116.3
8 5.33 5.06 4 158.4 158.8 4’ 155.2 155.5
10 6.20 6.22 7 94.2 93.9 7’ 44.1 41.6
12 6.33 6.38 8 53.2 50.5 8’ 73.2 71.8
2’6’ 6.77 d (8.3) 6.77 d (8.3) 9 141.1 141.2 9’ 142.3 142.5
6.58 d (8.3) 6.58 d (8.3) 10 114.6 115.5 10’ 118.8 119.5
7’ 4.90 4.88 11 159.2 159.4 11’ 159.2 159.3
8’ 5.40 5.74 12 102.4 101.1 12’ 95.2 95.4
10’ 6.33 6.58 13 157.0 157.0 13’ 160.7 157.3
12’ 5.60 5.78 14 107.1 106.3 14’ 110.3 111.8

Arq. Inst. Biol., São Paulo, v.68, n.1, p.47-50, jan./jun., 2001
 Chemical constituents from Vitis vinifera (Vitaceae). 49

8’and 13 of compound 4, that occur protection in Table 2 - Quantification of the compounds resveratrol (1),
relation compound 3. These fact were atributted the ε - viniferins (2), balanocarpol (3), and β- glucopyranosyl-
substitution of OH at 8 carbon by glycoside unit. The 8’-balanocarpol (4) in the ethanolic extracts from branches
signals in 101.1; 74.9; 78.0; 71.7; 77.6 and 62.8 referen- and leaves of Vitis vinifera (% in the extract).
te at 1, 2, 3, 4, 5, and 6 carbon of glycose confirm the
Extracts 1 2 3 4
substitution. The results analysis permitted conclude
that compound was β-glucopyranosyl-8-balanocarpol Branches 3.92 12.64 6.84 6.08
still relates in the literature. Leaves 1.88 0 0 0

Quantification by HPLC of compounds

The retention time of standard resveratrol, ε-
viniferins, balanocarpol, and β- glucopyranosyl-8’-
balanocarpol were respectively 11.00; 8.31; 6.66 and
5.48 min, which allowed the quantification of each
compound. Results showed that all the compounds
were present and quantified in large amounts in the
ethanolic extract of branches (Fig. 2 and Table 2). But
in the ethanolic extract from leaves, only resveratrol
was quantified (Table 2 and Fig. 3). The conditions
here reported are rapid and sensitive and may be used
for determination of these compounds in extracts of
the plants. The resveratrol and its dimers here
quantified are known to related with plant protection
(LANGCAKE & PRYCE, 1977 a, b, c; CICHEWICZ et. al, 2000),
but the branches hardness may also be a funtion of
these compounds, because of the high concentrations
found there, as compared with other plant parts.
ACKNOWLEDGEMENTS
The authors are grateful the Fundação de Amparo
à Pesquisa do Estado de São Paulo (FAPESP) for
supporting the research and the grant of R. S. Santos.

HO
OH
OH

OH HO

HO
1 OH 2

OH
4
HO H
O 12'
7
OH
HO 8
H
12 8'
OR
7' 3 R = H
H
HO H

4' 4 R = GLUCOSE

OH

Fig. 1- Compounds isolated of Vitis vinifera: resveratrol (1), e-viniferin (2), balanocarpol (3) and b-glucopyranosyl
8- balanocarpol (4).

Arq. Inst. Biol., São Paulo, v.68, n.1, p.47-50, jan./jun., 2001
50 J.D. Felicio et al.

Fig. 2 - HPLC chromatogram of the extracts ethanolic of branches (Retention time 11.11 min to resveratrol; 8.35
min to e-viniferins; 6.73 min to balanocarpol, and 5.35 min to b- glucopyranosyl-8’-balanocarpol).

Fig. 3- HPLC chromatogram of the ethanolic extracts of leaves (Retention time 10.57 min to resveratrol).

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