Some Biological Activities of Vine Stilbens and New ... - Oiv2010.ge

Trans-resveratrol and ε-viniferin in red Georgian wines
M. Bezhuashvili, M. Kokhtashvili, T. Kobaidze, N. Vepkhishvili
Institute of Horticulture, Viticulture and Oenology of Georgia
#6, Marshal Gelovani St., 0159, Tbilisi, Georgia
mbezhuashvili@yahoo.com
ABSTRACT
At present, out of the rich spectrum of the phenol substances of Georgian red wines, stilbens
are not thoroughly studied. In 1991-1994, we isolated and identified from Rkatsiteli vine
species trans-resveratrol, ε-viniferin and two Tetramers of resveratrol. The dependence of
trans-resveratrol concentration in the red Georgian wines on the vine species and wine type
has been identified. Of naturally semi-sweet, semi-sweet, dry and naturally cleared fortified
wines, trans-resveratrol was found in the greatest concentrations in the fortified wines. 0,78-
3,20 mg/l (in Saperavi); 0,69-2,62 mg/l (Otskhanuri Sapere); 0,65-2,87mg/l (Cabernet
Sauvignon); 0,47-1,92 mg/l (Tavkveri). ε-viniferin in self-cleared dry bulk wine of Saperavi
and Cabernet Sauvignon amounts to 0,98 mg/l and 0,40 mg/l, respectively.
Key words: Red wine, Trans-resveratrol, ε-viniferin.
RESUME
A nos jours parmi le spectre riche de produits phénoliques des vins rouges géorgiens les
stilbènes ne sont pas encore étudiés complètement. En 1991-1994 il a été distingué et identifié
du cépage Rkatsitéli le trans-resvératrol, la ε-viniférine et deux tetramètres de resvératrol. Il
est découvert que dans les vins rouges géorgiens le taux de concentration de trans-resvératrol
dépend des cépages de vignes et des types de vins. Le taux de concentration est élevé dans les
vins naturellement demi-sucré, demi-sucré, sec et les vins fortifiés filtrés naturellement.
Sapéravi 0,78 – 3,20 mg/L ; Otskhanouri Sapéré 0,69 – 2,62 mg/L ; Le taux de ε-viniférine
dans les vins fins de Sapéravi et Cabernet Sauvignon filtrés naturellement, secs est : 0,98
mg/L et 0,40 mg/L.
Mots clés : Vin rouge, Trans-resvératrol, ε-viniférine.
INTRODUCTION. Georgia is the homeland of vine and wine. The gene pool of Georgian
vines including approximately 525 unique species of white and versicolored vine has been
tried by the Georgians for centuries. The diversified technologies to produce different types of
wine developed as a result of the rich experience, have been shifted to one generation to
another and as time went, they were perfected and new technologies were developed. In this
respect, the merit of Georgian scientists as the foundation of Georgian viticulture and
winemaking is worth mentioning. The curative properties of grape and wine always attracted
a great deal of attention of different scientific branches. This is particularly true with red
Georgian wines known for their curative properties from the ancient times and are a panacea
in Georgian folk medicine, particularly, the bread soaked in red wine. The scientific progress
allowed thoroughly explaining the close connection between the useful properties of red wine
and its rich chemical content.
1

The search for the fundamentals of the phenomenon of the “French paradox” was followed by
the intense study of the content of red wines on an international scale. Numerous experiments
have demonstrated the leading role of biologically active phenol substances in the formation
of the curative and nutritive value of red wines. Identification of stilbens and particularly
resveratrol, which is their monomer representative, and their determination in red and pink
wines made of different vine species in a range of countries is associated with these studies.
(Waterhouse,1993; Roggero, Archier, 1994; .Lamuela-Raventos et al.,1995; Romero-
Perez et al.,1996; Lamuela-Rawentos, Waterhouse, 1993; Lamikarna, et al.,1996).The
further studies, besides resveratrol, identified its derivatives: piceids, dimmers: ε-viniferin and
δ-viniferin; trimer α-viniferin, etc. as well as other stilbens. (Ribeiro de Lima et al.,1999;
Guebailia et al., 2006). Out of resveratrol isomers, it is red wines with the higher content of
trans-resveratrol if compared to cis-resveratrol. At the same time, trans-resveratrol is
biologically more active than cis-resveratrol. The established diversified biological activities
of stilbens greatly make for the positive effect of red wines against cardiovascular, cancerous,
thrombotic and other diseases( Blond et al., 1995; Klatsky et al.,1997; Yang et al., 1997;
Szmitko, Verma, 2005; Balestrieri et al., 2008).
.
We started to study stilbens in 1991-1994, and individually isolated and identified transresveratrol,
ε-viniferin and two tetramers of resveratrol from 1-year-old Rkatsiteli (Vitis
vinifera L.) vine shoot (Bezhuashvili et al., 1991; Bezhuashvili et al., 1997; Bezhuashvili,
1994).
Trans-resveratrol
ε-viniferin
2

Tetramer-I
Tetramer-II
Then, we continued studying them in red Georgian wines. It should be noted that a
Georgian scientist Durmishidze S. and his colleagues have made a great contribution to the
study of the diversified spectrum of phenol compounds of the vine species spread in Georgia
(Durmishidze, Khachidze, 1979; 1985). Out of phenol compounds, proantocyanidines
(oligomeric and polymeric), phenolic acids, anthocyanins, catechines, flavonols, etc. have
been identified. Saperavi species growing in different regions of Kakheti have been studied
by us, and we identified the differences between the chemical compositions of bulk wines.
The property of an early (9-month-long) stabilization of Saperavi growing in Khashmi has
been identified in Kakheti Region. This property is demonstrated by the wine changing for the
coloured complex of anthocyanins by preserving its intense ruby color, with the colouring
intensity coefficient T

engaged in an intense study in the direction of “Wine and health” to identify specifically the
stilben spectrum in red Georgian wines. At present, we have identified and determined
resveratrol dimmer ε-viniferin.
MATERIALS AND METHODS. We used the different types of naturally cleared red
wines made with Saperavi, Otskhanuri Sapere, Cabernet Sauvignon and Tavkveri species as
the objects of the study, in particular, dry table, naturally semi-sweet, semi-sweet and fortified
wines. For qualitative and quantitative analyses of trans-resveratrol, we extracted the wines in
advance with ethyl acetate and used the gained fraction for analyses. We used thin-layer
chromatography with silufol plates (20 cm x 20 cm), system: chloroform : methanol (80:20)
and denitrated sulfanilic acid as a developer. We defined the quantity of trans-resveratrol by
high-efficiency liquid chromatography in terms of gradient. The column of Nucleosil C 18 ,
eluent A: water + H3PO4; eluent B: acetonitrile+H3PO4, pH=3,5-4,0. Out of naturally cleared
dry table bulk wines made with the 2008 harvest of Saperavi and Cabernet Sauvignon, we
isolated the total stilbens with further treatment of their ethyl acetate fractions (Ribeiro de
Lima et al., 1999). We analyzed the gained fraction by high-efficiency liquid chromatography
in terms of gradient (the chromatograph made by “Varian”); UV detector/visible spectrum,
column – Microsorb 100 C18, 250X4,6 LxId (mm); 5µm – Particle Size. Eluent A: TF
(trifluoroacetic acid) 0,025% water solution; eluent B: AСN/AA, 80/20 (v/v); (Guebailia, et
al., 2006).
RESULTS AND DISCUSSION. The gained results demonstrate that the concentration of
trans-resveratrol in bulk wines depends on different factors, one of which is the generic factor.
In this respect, Saperavi and Otskhanuri Sapere are the most obvious examples. The
concentration of trans-resveratrol in bulk wines also changes according to the sugar content of
the must. In particular, within the range of 19,1-26,0%, the content of trans-resveratrol in the
red wines made of Saperavi with different sugar contents changes from 1,69 mg/l to 2,87
mg/l; for Otskhanuri Sapere (with the sugar content of 19,2%-22,4%), it is 1,25 mg/l 1,95
mg/l and for Cabernet Sauvignon (with the sugar content of 19,3%-22,7%), it is 1,15 mg/l to
2,25 mg/l. At this point, in addition to the generic factor, the factor of alcohol-content of the
pomace fermented is obviously seen. As for the wine type, it is seen as the factor making the
wines made with the same grape species significantly different from one another with their
concentration of trans-resveratrol. This is proved by the diagram in “Fig. 1”.
“Fig. 1”. Change of the concentration of trans-resveratrol according to the types of wine.
I – naturally semi-sweet, II – semi-sweet, III – dry, IV – fortified.
Among the naturally semi-sweet, semi-sweet, dry and fortified wines, the concentration of
trans-resveratrol is the highest in the fortified wine and is the least in the naturally semi-sweet
wine. Besides the above-mentioned factors, temperature and squeezing and mixing of
4

fermented pomace were also identified as the factors affecting the quantity of trans-resveratrol
in bulk wines (Kokhtashvili, Bezhuashvili; 1998, Kokhtashvili et al. 2002; Kokhtashvili,
2006).
The data of thin-layer and liquid chromatographs of the total stilben fraction gained from
dry table bulk wines made of Saperavi and Cabernet Sauvignon prove their variety. The
stilben spectrum, besides trans-resveratrol and ε-viniferin, is presented by some other stilbens
(“Fig. 2”). The concentration of ε-viniferin in Saperavi bulk wines is 0.98 mg/l and is 0.40
mg/l in Cabernet Sauvignon. At present, an intense research is carried out to study the stilben
spectrum in red Georgian wines.
“Fig. 2”. Liquid chromatogram of stilben fractions gained from dry table bulk wines of
Saperavi.
CONCLUSIONS. The content of biologically active high-oxidant trans-resveratrol and ε-
viniferin in red Georgian wines rich in phenol substances is a certain proof of the useful
properties of the given wines. Thoroughly studied stilben spectrum in red Georgian wines as
that of the product of a functional designation, is an important bio-chemical marker.
ACKNOWLEDGMENTS. We thank the following scientists for their assistance in liquid
chromatography:
Shubladze L., the head of the central laboratory and Sikharulidze T., the chemical engineer
of instrumental analysis of the Institute of Horticulture, Viticulture and Oenology of Georgia.
Butkova O. of the Institute of Beer and Soft Drinks in Moscow and Larionov O. of the
Institute of Physical Chemistry.
5

Bibliography
1. Balestrieri M., Schiano C., Felice F., Casamassimi A., Balestrieri A., Milone L., Sarvillo
L., Napoli Cl. 2008. Effect of low doses of red Wine and Pure Resveratrol on Circulating
Endothelial Progenitor Cells. Journal of Biochemistry, 143 (2): pp. 179-186.
2. Bezhuashvili M., Mujiri L., Kurkin V., Zapesochnaya G. 1991. Resveratrol of vine.
Timber chemistry, №6: pp. 75-76.
3. Bezhuashvili M. 1994. Development of theoretical basics for vine and oak timber lignin
and identification of the ways to use the gained products. Doctoral thesis, Tbilisi, 260 p.
4. Bezhuashvili M., Mujiri L., Shashkov A., Chzhov O., Stomakhin A. 1997. Stlben
tetramers of one-year vine shoots. Bioorganic chemistry, vol. 23, №12: pp. 979-987.
5. Bezhuashvili M., Deisadze I., Kobaidze T. 2008. Correlation of proanthocyanins and
selected sorts of red bulk wines made of technical sorts and hybrids – immediate
producers. Winemaking and Viticulture, №3.: pp. 20-22.
6. Bezhuashvili M., Deisadze I., Shubladze L., Sikharulidze T. 2009. Chromatographic
profile of anthocyanins of the grape of red technical sorts and wines made of them.
Magarach Winemaking and Viticulture, №3: pp.27-29.
7. Bezhuashvili M., Vepkhishvili N., Kobaidze T. 2010. Identification and determination of
biologically active ε-viniferin in grapes and bulk wines of Saperavi and Cabernet
Savignon sorts. Collection of Scientific Works of the National Institute of Grape and
Wine “Magarach”, Yalta,pp.71-75.
8. Bezhuashvili M., Vepkhishvili N., Kobaidze T. 2010. Identification of resveratrol dimmer
ε-viniferin in versicolored vine species. Georgian National Academy of Sciences,
Republican Scientific Conference “Biologically active natural and synthetic substances”.
Tbilisi, March 30. The Theses. pp. 25-26.
9. Blond Y., Denis M. Bezard I. 1995. Antioxidant action of resveratrol in Lipid
peroxidation. Sciences Aliments, 15,: pp. 347-358.
10. Durmishidze S., Khachidze O. 1979. Chemical composition of grape. Metsniereba.
Tbilisi: Editor Lashkhi A.
11. Durmishidze S., Khachidze O. 1985. Biochemistry of vine. Metsniereba. Tbilisi: . Editor
Kezeli T.
12. Durmishidze S. 1955, Tannin and anthocyanins of the vine and wine. Publishing house of
the Academy of Sciences of the USSR, Moscow: Editor Sisakian H.
13. Guebailia H., Chilra K., Richard T., Mabrouk T., Furiga A., Vitrac X., Monthi Jean-
Pierre, delaunay Yean-Claude, Merillon Yean-Michael. 2006. Hopeaphenol: The first
resveratrol Tetramer in Wines from North Africa. J. Agric. Food Chem., 54: pp. 9559-
9564.
14. Klatsky A., Armstrong M., Friedman G. 1997. Red wine, white wine, liquor, beer and risk
for coronary artery disease hospitalization. American Journal of Cardiology., 80: pp. 416-
420.
15. Kokhtashvili М., Bezhuashvili М. Identification of trans-resveratrol in some red grape
species. Georgian Engineering News. 1998, №4: pp. 104-106.
16. Kokhtashvili М., Bezhuashvili М., Pataraia М. Study of trans-resveratrol in dry table red
wines. Collection of Works of Georgian Agrarian University, Tbilisi, 2002, vol. 19: pp.
79-86
17. Kvlividze D., Bezhuashvili M. 2005-a. Phenol compounds in the wine of Saperavi sort
and its table bulk wines for wines controlled by origination. Winemaking and Viticulture,
№2: pp. 21-22.
6

18. Kvlividze D., Bezhuashvili M. 2005-b.Study of anthocyanins of the Saperavi sort grape
and making dry table bulk wines of it according to the place of origination. Magarach
Winemaking and Viticulture, №1: pp. 25-27.
19. Kvlividze D., Bezhuashvili M. 2005-c. Economic and technological properties of Saperavi
in Khashmi microzone. The Bulletin of the Academy of Agricultural Sciences of Georgia.
№14: pp. 47-55.
20. Lamikarma O., Grimm C., Rodin B., Lnyang I. 1996. Hydroxylated stilbenes in Selected
American Wines. j.Agric.food Chem., 44: pp. 1111-1115.
21. Lamuela-Raventos, R., romero-Perez, A., Waterhouse, A., Torre-Bornat, M. 1995.Direct
HPLC Analysis of cis- and trans-resveratrol and piceid isomers in Spanish red vitis
vinifera wines. J. Agric. Food Chem., 43: pp.281-283.
22. Lamuela-Rawentos R., Waterhouse A. 1993. Occurrence of Resveratrol and California
wines by a new HPLQ method. J. Agric. Food Chem. 41: pp. 521-524.
23. Ribeiro de Lima M., Waffo Heguo, P., Tessedre, P., Pujolas, A., Vercauteren, I., Cabanis,
I. C., Merillon, I. 1999. determination of stilbenes (trans-astrigin, cis- and trans-piceid,
and cis- and trans-resveratrol) in Portuguese wines. J. Agric. Food Chem., 47: pp.2665-
2670.
24. Roggero, I., Archier, P. 1994. Quantitave determination of resveratrol and one of its
glycosides in wines. Sci. Alimests, 14: pp. 99-107.
25. Romero-Perez A., Lamuela – Raventos R., Waterhouse A., de la Torre-Boronat M. 1996.
Levels of cis- and trans- rezveratrol and their Glucosides in white and Rose Vitis vinifera
Wines from Spain. J. Agric. Food Chem., 44: pp. 2124-2128.
26. Szmitko P., Verma S. 2005. Antiatherogenic potential of red wine: clinican update. Am
Journal Physiol Heart Circ. Physiol.,288.:pp. 2023-2030.
27. Yang M., Gai L., Udeani G., Slowing K., Thomas C., Beecher C., Fong H., Fansworth N.,
Kinghorn A., Metha R., Moon R., Perruto Y. 1997. Cancer chemopreventive Activity of
Resveratrol, a natural Product Derived from Grapes. Science, 275: pp. 218-220.
7