Vitis Vinifera L.)

Annals of Agrarian Science xxx (2016) 1e4

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Annals of Agrarian Science

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Impact of wine technology on the variability of resveratrol and piceids in Saperavi (Vitis vinifera L.)

* M.A. Surguladze , M.G. Bezhuashvili

Agricultural University of Georgia, Institute of Viticulture and Oenology, 240, David Aghmashenebeli Alley, Tbilisi, 0159, Georgia article info abstract

Article history: The biologically active stilbenoids-resveratrol and its glucosides were identified in the dry bulk wines of Received 18 October 2016 different types made with red-grape vintage variety of Saperavi (Vitis vinifera L.): cis-resveratrol, trans- Accepted 18 October 2016 resveratrol, cis-piceid and trans-piceid. Red and pink wines were made by different technology: I e dry, Available online xxx pink, of a European type; alcoholic fermentation with natural microflora; II e dry, pink, of a European type; alcoholic fermentation with dry yeast “B2000”. III e red, dry; alcoholic fermentation with no-stem Keyword: pomace and aging on it for 5 months; IV e dry, red, of a Kakhetian type; alcoholic fermentation with Antitumor stem pomace with natural microflora and aging on it for 5 months; V e dry, red, of a Kakhetian type, with preliminary fermentation of cluster stems, then, alcoholic fermentation with natural microflora and aging on it for 5 months. The impact of the wine-making technology on the variability of the concentrations of these substances is proved. Kakhetian bulk wines (IV and V) also differ from one another. Bulk wine-V made by the fermentation of grappa fermented in advance contains little concentration of study stilbenoids as a result of the oxidation transformations caused by the preliminary treatment. The role of grape juice, stem and seed in the localization of resveratrols and piceids in the bulk wines is identified. © 2016 Agricultural University of Georgia. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction The biological activity of stilbenoids in various fields is proved with experiments in vitro and in vivo. For instance, trans-resveratrol The chemical content of red grapes and wine presents a rich is characterized by antioxidant [5] and inhibitory [6] activities, spectrum of the components of different classes. Among them, the cardio-protective [7], antitumor (skin and gastrointestinal cancers) diversified profile of phenol compounds with flavonoid (procya- [8], antidiabetic [9], skin protecting [10] and other effects. The nidins e oligomeric and polymeric, flavanols, flavonols, anthocya- biological activity of epsilon-viniferin is presented by antioxidant nins, flavonons, etc.) and non-flavonoid (stilbenoids, phenolic acids, [11], anti-inflammatory [12], antitumor [13] and neuroprotective phenol aldehydes, etc.) compounds is noteworthy. Intense research [6] effects, as well as Sirt.1 activating and 5-alphareductase inhib- of stilbenoids in red wines is associated with the “French paradox” iting properties [14]. Hopeaphenol has antitumor [15] and anti- phenomenon. The studies proved the high biological activity of the inflammatory [16] effects. said compounds [1]. Resveratrol, a monomer representative of The study of vine stilbenoids in Georgia was initiated by us in stilbenoids, in red wines is fixed as two isomers: cis-resveratrol and Institute of Horticulture, Viticulture and Winemaking of Georgia in trans-resveratrol [2]. These compounds form glycosidic and other 1991. Trans-resveratrol [17], ε-viniferin [18] and two tetramer stil- derivatives and are localized in wines as cis-piceid and trans-piceid, bens, including hopeaphenol [19] were isolated and identified in dimers: pallidol, delta- and epsilon-viniferins, trimer: alpha- one-year-old vine canes. Trans-resveratrol, ε-viniferin, trans-piceid viniferin, tetramer: hopeaphenol. In addition, red wines contain and cis-piceid [20e26] were isolated and quantified in red-grape astringin: piceatannol glucoside and astringinin: piceatannol, etc. vintage vines growing in Georgia and wines. Trans-resveratrol [3,4]. was found to have a full inhibiting power of Agrobacterium tume- faciens causing vine cancer [27]. Trans-resveratrol stimulates wine yeasts: Sacch. vini- Kahuri 42 and Sacch. chodati- Teliani 77 in the * Corresponding author. process of alcoholic fermentation what is evidenced by an inten- E-mail addresses: [email protected] (M.A. Surguladze), m. sified growth and development of yeasts [28]. The antioxidant ac- [email protected] (M.G. Bezhuashvili). tion of stilbenoids isolated from the vine evidenced by the Peer review under responsibility of Journal Annals of Agrarian Science. http://dx.doi.org/10.1016/j.aasci.2016.10.002 1512-1887/© 2016 Agricultural University of Georgia. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: M.A. Surguladze, M.G. Bezhuashvili, Impact of wine technology on the variability of resveratrol and piceids in Saperavi (Vitis vinifera L.), Annals of Agrarian Science (2016), http://dx.doi.org/10.1016/j.aasci.2016.10.002 2 M.A. Surguladze, M.G. Bezhuashvili / Annals of Agrarian Science xxx (2016) 1e4 inhibition degree of malondialdehyde formation in human blood Saperavi variety: I e dry, pink, of a European type; alcoholic serum with “in vitro” experiments, occurs in the following order: fermentation with natural microflora; II e dry, pink, of a European trans-resveratrol (105%) < ε-viniferin (118%) < tetrameric stilben type; alcoholic fermentation with dry yeast “B2000”. III e red, dry; (169%) < hopeaphenol (178%) [29]. Trans-resveratrol and ε-vin- alcoholic fermentation with natural microflora and with no-stem iferin influence the process of malic-and-lactic acid fermentation in pomace, aging on it for 5 months; IV e dry, red, of a Kakhetian Saperavi bulk wine evidenced by the inhibition of the formation of type; alcoholic fermentation with stem pomace with natural the intermediate product [30]. Resveratrol isomers and stilbene microflora and aging on it for 5 months; V e dry, red, of a Kakhetian derivatives are characterized by different actions depending on the type, with preliminary fermentation of cluster stems, then, alco- environment they occur. They limit the enzymes causing the holic fermentation with natural microflora and aging on it for 5 inflammation processes, obstruct the origination of thrombocytes months. In experiment was used grapes 2014 vintage from lowland and development of atherosclerosis. These conclusions were made vineyard. Wine analyses were done in June of 2015 year. We also as a result of the research of the wines produced in some European used hard grape parts, such as stems and seeds, as the study ob- countries, and the wine of a Kakhetian type from Georgia was jects. We isolated the stilbenoid-containing fractions from the bulk among the wines being studied [31]. wines and hard parts of Saperavi grapes with relevant treatment, as The concentration of stilbenoids in red wines is influenced by per plan 1. the methods of technological treatment of the bulk wine. The ε concentrations of trans-resveratrol and -viniferin in the bulk HPLC analysis wines treated with gelatin, fish glue, bentonite or cold, are reduced [32]. Out of the following bulk wines made of Saperavi variety: dry, We identified the stilbenoids by using the method of high- fi naturally sweet and forti ed ones, the concentration of trans- pressure liquid chromatography with the following conditions: fi resveratrol is naturally highest in the forti ed wines [33]. chromatograph “Varian”, column Supelcosil™ LC18, 250X4, 6 Following the above-mentioned, the study of vine and wine (mm); solvent A, 0.025%, trifluoroacetic acid; solvent B, acetonitrile stilbenoids, the biologically active substances, is topical. By (ACN)/A, 80/20, gradient regime 0e35 min. 20e50% B; 35e40 min. continuing the study of stilbenoids in Saperavi variety, this time, 50e100% B; 41e46 min. 100% B; 46e48 min. 100e20% B; the goal of the study is to determine the variability of cis- and trans- 48e53 min. 20% B. Eluent transfer velocity 1 ml/min, wavelength resveratrol and their glucosides: cis-piceid and trans-piceid 306 nm and 286 nm [4]. The samples for chromatography were depending on the wine technology. filtered with a membrane filter (0.45 mm) in advance. We should note that we carried out the analysis on both Objectives and methods 306 nm and 286 nm waves twice for each, and presented their mean values. The objects of the study were various bulk wines made of

Table 1 Contents of resveratrol and piceids (mg/l) in Saperavi bulk wines.

Wines Trans-resveratrol Cis-resveratrol Total resveratrol Trans-piceid Cis-piceid Total piceid

I 0.076 0500 0.576 4.9 12.2 17.1 II 0.176 0920 1096 4.3 11.5 15.8 III 0.35 1.28 1.63 5.9 14.7 20.6 IV 0.24 2.38 2.62 5.1 14.1 19.2 V 0.65 0.84 1.49 4.8 13.7 18.5

Results and discussion

The impact of the wine-making technology on the concentrations of cis- and trans-resveratols and piceids is clear (Table 1). The impact of dry yeasts on the piceids in the grape must of the European-type bulk wines is clearly seen during the alcoholic fermentation. The cis- and trans-resveratols formed as a result of glucosidal bond splitting are localized in the European-type bulk The data of Kakhetian bulk wines differ from those of the wine. We proved this fact by the alcoholic fermentation of the European-type bulk wines. Bulk wine-IV is distinguished for high grape must in Saperavi, as well as red-grape vintage varieties, such concentration of cis-resveratrol and a big quantitative difference as Aladasturi and Mujuretuli (2013 vintage) [24]. An increase of the from trans-resveratrol (2.38 mg/l and 0.24 mg/l). It should be noted total amount of resveratrol is followed by the decrease of the total that Kakhetian bulk wine is made by the alcoholic fermentation of þ þ þ amount of piceids. The concentration of cis-resveratrol exceeds that stem pomace (grape juice stem skin pip) and each of them of trans-resveratrol. Piceids contain bulk of cis-isomers. In this enriches the bulk wine with relevant extract substances, including fi connection, the results of our past experiments are worthwhile. In stilbenoids. We identi ed trans-resveratrol in the stems of red- particular, more quantity of trans-piceid was found in Saperavi grape vintage vine varieties growing in Georgia. These varieties grape juice than cis-piceid, while the quantity of cis-piceid in the are: Saperavi, Cabernet Sauvignon, Otskhanuri Sapere, Aleksan- grape skin was much more than that of trans-piceid Consequently, drouli, Mujuretuli, Shavkapito, Tavkveri, Aladasturi, Dzelshavi and the great quantity of cis-isomers in the study bulk wines is the Ojaleshi [25]. The contents of resveratrol and piceids in the hard fi result of the transformation of the trans-forms into the cis-form in grape parts xed by the given experiment are given in Table 2. the process of the bulk wine formation. The same variability of cis- The hard grape parts are different sources of resveratrol and and trans-resveratrol and piceids was fixed in bulk wine-III made piceid accumulation in Kakhetian bulk wines. Skin is the richest by the fermentation of Saperavi no-stem pomace fermentation. structure what is proved by relevant studies. Dominant in a stem is trans-resveratrol, while only traces of cis-resveratrol are seen in it. Stems contain cis- and trans-piceids with the amount of the former much exceeding that of the latter. Pips contain little trans- resveratrol and only traces of cis-resveratrol. The amount of cis- piceid exceeds that of trans-piceid. Kakhetian bulk wines (IV and V) differ from each other. Bulk wine-V made by the fermentation of grapes fermented in advance contains less than detection limit of study stilbenoids as a result of the oxidation transformations caused by the preliminary treatment.

Conclusion

So, the experiment evidenced the impact of wine-making technology on the total concentration of resveratrol and piceids. Unlike the European-type wines, in Saperavi bulk wines made with Kakhetian technology, the stilbenoids coming from the grape skin, stem and pips are localized. One should note that the liquid chro- matograms also showed other known and non-identiﬁed stilbenoids, and their study is the subject of the future studies. Maintenance of the biologically active stilbenoids in high-quality Saperavi wines without impairing the organoleptic properties of the wines is important for their functional purposes. Considering the gained results is an important precondition for reaching this goal.

References Table 2 Content of resveratrol and piceids in hard grape parts (mg/100 g dry wt.). [1] S. Renaud, M. De Lorgeril, Wine, alcohol, platelets, and the French paradox for coronary heart disease, Lancet 339 (8808) (1992) 1523e1526. Objects Trans-resveratrol Cis-resveratrol Trans-piceid Cis-piceid [2] A.I. Romero-Perez, R.M. Lamuela-Raventos, A.L. Waterhouse, M.C. de la Torre- Stems 24.18 A little amount 2.35 5.23 Boronat, Levels on cis- and trans-resveratrol and their glucosides in white and rose' Vitis vinifera wines from Spain, J. Agric. Food Chem. 44 (1996) Seed 1.24 A little amount 0.75 2.31 2124e2128.

[3] X. Vitrac, A. Bornet, R. Vanderlinde, J. Valls, T. Richard, J.C. Delaunay, [19] M. Bezhuashvili, L. Mujiri, A. Shashkov, O. Chzhov, A. Stomakhin, Stilben J.M. Merillon, P.L. Teissedre, Determination of Stilbenes (delta-viniferin, trans- tetramers of one-year vine shoots, Bioorg. Chem. 23 (1997) 979e987 (in astringin, trans-piceid, cis- and trans-resveratrol, ε-viniferin) in Brazilian Russian). Wines, J. Agric. Food Chem. 53 (2005) 5664e5669. [20] M. Kokhtashvili, M. Bezhuashvili, Identification of trans-resveratrol in some [4] H.A. Guebaila, K. Chira, T. Richard, T. Mabrouk, A. Furiga, X. Vitrac, J.P. Monti, red grape species, Georgian Eng. News 4 (1998) 104e106 (in Russian). J.C. Delaunay, J.M. Merillon, Hopeaphenol: the first resveratrol tetramer in [21] M. Kokhtashvili, M. Bezhuashvili, M. Pataraia, Study of Trans-resveratrol in wines from North Africa, J. Agric. Food Chem. 54 (2006) 9559e9564. Dry Table Red Wines, vol. 19, Collection of Works of Georgian Agrarian Uni- [5] Z. Ovesna, K. Kozics, Y. Bader, P. Saiko, N. Handler, T. Erker, T. Szekeres, versity, Tbilisi,, 2002, pp. 79e86 (in Georgian). Antioxidant activity of resveratrol, piceatannol and 3,30, 4,40, 5,50 hexahy- [22] M. Bezhuashvili, N. Vepkhishvili, T. Kobaidze, L. Shubladze, D. Okruashvili, droxytransstilbene in the eleukemiacellline, Oncol. Rep. 16 (2006) 617e624. Content of the biologically active trans-resveratrol and ε-viniferin in color [6] C. Riviere, T. Richard, L. Quentin, S. Krisa, J.M. Merillon, J.M. Monti, Inhibitory vine varieties growing in Georgia, Bull. Georgian Natl. Acad. Sci. 5 (2011) activity of stillbenes on alzheimer’s b-amyloid fibrils in vitro, Bioorg. Med. 61e64. Chem. 15 (2007) 1160e1167. [23] M. Bezhuashvili, L. Shubladze, D. Okruashvili, Trans-piceid stilbenoid in the [7] P.E. Szmitko, S. Verma, Red wine and your heart, cardiology patient pages, juice and skin of the red grape(Vitis vinifera L.) varieties growing in Georgia, Circulation 111 (2005) 10e11. Bull. Georgian Natl. Acad. Sci. 7 (2013) 74e79. [8] M. Athar, J.H. Back, X. Tang, K.H. Kim, L. Kopelovich, D.R. Bickers, A.L. Kim, [24] M.G. Bezhuashvili, M.A. Surguladze, P.N. Vashakidze, Biologically active stil- Resveratrol: a review of preclinical studies for human cancer prevention, benoids of saperavi grape (Vitis vinifera L.), in: 3rd International Conference on Toxicol. Appl. Pharmacol. 224 (2007) 274e283. Pharmaceutical Sciences. “looking towards the Future, Honoring the Past” [9] J.A. Baur, K.J. Pearson, N.L. Price, H.A. Jamieson, C. Lerin, A. Kalra, V.V. Prabhu, Tbilisi, May 29-31, 2015, pp. 66e67. Georgia. Abstract Book. J.S. Allard, G. Lopez-Lluch, K. Lewis, P.J. Pistell, S. Poosala, K.G. Becker, O. Boss, [25] M.G. Bezhuashvili, P.N. Vashakidze, N.G. Vepkhishvili, L.D. Elanidze, Trans- D. Gwinn, M. Wang, S. Ramaswamy, K.W. Fishbein, R.G. Spencer, E.G. Lakatta, formation of biologicaly active stilbenoids from grapevine to red wine, Food D. Le Couteur, R.J. Shaw, P. Navas, P. Puigserver, D.K. Ingram, R. de Cabo, Suppl. Beverage Ann. Agrar. Sci. 12 (2014) 63e70. D.A. Sinclair, Resveratrol improves health and survival of mice on a high- [26] M.A. Surguladze, M.G. Bezhuashvili, Change correlation of saperavi (Vitis calorie diet, Nature 444 (2006) 337e342. vinifera L.) stilbenoides to wine type, in: 7th Imternational Conference on [10] F. Afaq, H. Mukhtar, Botanical antioxidants in the prevention of photo- Polyphenols and Health, Congress Center Tours, France, 2015. October 27e30. carcinogenesis and photoaging, Exp. Dermatol. 15 (2006) 678e684. [27] M. Bezhuashvili, M. Kokhtashvili, Z. Lomtatidze, K. Mamulashvili, Impact of [11] C. Privat, J.P. Telo, V. Bernardes Genisson, A. Vieira, J.P. Souchard, F. Nepveu, trans-resveratrol on the growth and development of some microorganisms, Antioxidant properties of trans-epsilon-viniferin as compared to stilbene Collect. Sci. Works Postgrad. Stud. Seek. Deg. IV (1999) 210e213 (in Georgian). derivatives in aqueous and nonaqueousmadia, J. Agric. Food Chem. 50 (2002) [28] M. Bezhuashvili, T. Kobaidze, M. Kokhtashvili, M. Meskhi, Z. Sturua, Some 1213e1217. biological activities of vine stilbens and new georgian grape products with a [12] Q.T. Do, I.I. Renime, P. Andre, C. Lugnier, C.D. Muller, P. Bernard, Reverse- functional designation, in: 33 rd World Congress of Vine and Wine, Tbilisi, pharmacognosy: application of selnergy, a new tool for lead discovery. The 2010, 20e25 June. example of epsilon-viniferin, Curr. Drug Discov. Technol. 2 (2005) 161e167. [29] M. Bezhuashvili, M. Меskhi, M. Bostoganashvili, M. Malania, Anti-oxidant [13] B. Piver, F. Berthou, Y. Dreano, D. Lucas, Differential inhibition of human cy- activity of stilben-containing extract in experiments “in vitro”, Winemak. tochrome P450 enzymes by epsilon-viniferin, the dimer of resveratrol:com- Vitic. 3 (2005) 26e27. parison with resveratrol and polyphenols from alcoholized beverages, Life Sci. [30] M. Bezhuashvili, N. Vepkhishvili, D. Okruashvili, Influence of phenolic com- 73 (2003) 1199e1213. pounds over the malolactic fermentation in red wines, Biodiversity and [14] FR 2816843, Inhibiteursdel’enzyme5-alpha-reductase, ACTICHEM, 2000. Biotechnology, in: Conference of Georgian Agrarian University, 2011. [15] Sahidin, E.H. Hakim, L.D. Juliawaty, Y.M. Syah, L. BinDin, E.L. Ghisalberti, December 5e6. J. Latip, I.M. Said, S.A. Achmad, Cytotoxic properties of oligostilbenoids from [31] Z. Kutil, V. Temml, D. Maghradze, M. Pribylova, M. Dvorakova, D. Schuster, the tree barks of Hopea dryobalanoides, Z. Naturforsch. 60 (2005) 723e727. T. Vanek, P. Landa, Impact of wines and wine constituents on Cyclooxygenase- [16] K.S. Huang, M. Lin, G.F. Cheng, Antiinflammatory tetramers of resveratrol from 1, Cyclooxygenase-2, and 5-lipoxygenase catalytic activity, Mediat. Inflamm. the roots of Vitis amurensis and the conformations of the seven membered 2014 (2014). ID 178931 8. ring in some oligostilbenes, Phytochemistry 58 (2001) 357e362. [32] M. Мeskhi, M. Bezhuashvili, Effects of the Technological treatment in wines on [17] M. Bezhuashvili, L. Mujiri, V. Kurkin, G. Zapesochnaya, Resveratrol of vine, the contents of the stilbenoids compounds, Magarach Vitic. Winemak. 3 Timber Chem. 6 (1991) 75e76 (in Russian). (2003) 22e24 (in Russian). [18] M. Bezhuashvili, Development of Theoretical Basics for Vine and Oak Timber [33] M. Kokhtashvili, Studying of Resveratrol in Some Standard Red Vine Grapes Lignin and Identification of the Ways to Use the Gained Products, Doctoral and its Wines, Doctoral Thesis, Tbilisi, 2006, p. 25 (in Georgian). Dissertation, Tbilisi, 1994, p. 260 (in Reorgian).