CHEMICAL CONSTITUENTS FROM VITIS VINIFERA (VITACEAE)

COMPONENTES QUÍMICOS DE VITIS VINIFERA

ABSTRACT

Optimal conditions for the simultaneous quantitative separation of resveratrol, ɛ - 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; ɛ; - viniferins; balanocarpol; HPLC; Vitis vinifera; Vitaceae.

RESUMO

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 determinadas, 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; ɛ; - viniferina; balanocarpol; Vitis vinifera; Vitaceae; CLAE.

INTRODUCTION

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 besides its anti-inflammatory and anticancer 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 : ɛ-viniferin (2), balanocarpol (3) and the isolated new compound β-glucopyranosyl 8balanocarpol (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.

Apparatus

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, ¹HNMR (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 under vacuum and 2.5 mg of each were homogenized 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 ɛ-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’, 8’and 13 of compound 4, that occur protection in relation compound 3. These fact were atributted the substitution of OH at 8 carbon by glycoside unit. The signals in 101.1; 74.9; 78.0; 71.7; 77.6 and 62.8 referente at 1, 2, 3, 4, 5, and 6 carbon of glycose confirm the substitution. The results analysis permitted conclude that compound was β-glucopyranosyl-8-balanocarpol still relates in the literature.

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

Quantification by HPLC of compounds

The retention time of standard resveratrol, ɛviniferins, balanocarpol, and βglucopyranosyl-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.

Table 2
Quantification of the compounds resveratrol (1), ɛ - viniferins (2), balanocarpol (3), and βglucopyranosyl8’-balanocarpol (4) in the ethanolic extracts from branches and leaves of Vitis vinifera (% in the extract).

Extracts	1	2	3	4
Branches	3.92	12.64	6.84	6.08
Leaves	1.88	0	0	0

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

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 bglucopyranosyl-8’-balanocarpol).

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

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.

REFERENCES

• BOMBARDELLI, E. & MORAZZONI, I. Vitis vinifera L. Fitoterapia, v.6LXVL, p.291-317, 1995.
• LANGCAKE, P. & PRYCE, R.P. The production of resveratrol and the viniferins by gravepines in response to ultraviolet irradiation. Phytochemistry, v.16, p.11931196, 1977a.
• LANGKACE, P. & PRYCE, R.P. α- Viniferin: an antifungigal resveratrol trimer from grapevines. Phytochemistry, V.16, p.1452-1454, 1977a.
• LANGCAKE, P. & PRYCE, R.P. A new class of phytoalexins from gravepines. Experientia Basel, v.33, p.151-152, 1977c.
• FREMONT, L. Biological effects of resveratrol . Life Sci., v.66, p.663-673, 2000.
• DIYASENA, M. N. C.; SOTHEESWARAN, S.; BALASUBRAMANIAN, S.; BOKEL, M.E.; KRAUS, W. Balanocarpae, a new polyphenol from Balanocarpus zeylanias (Trimen) and Hopea jucunda (Thw). J. Chem. Soc. Perkin Trans I, p.1807-1809, 1985.
• CICHEWICZ, R . H.; KOUZI, S. A. ; HAMANN, M. T. Dimerization of resveratrol by the grapevine pathogen Botrytis cinerea. J. Nat. Prod., v.63, p.29-33, 2000.

Publication Dates

• Publication in this collection
  20 Sept 2024
• Date of issue
  Jan-Jun 2001

History

• Received
  09 Oct 2000