V. vinifera

Vol. 22, November/December 2010 Journal of Essential Oil Research/1

Rec: August 2008

Acc: November 2008

Volatiles of the Grape Hybrid Cultivar Othello (Vitis vinifera x (Vitis labrusca x Vitis riparia)) cultivated in

Serbia

N. Radulovic*, P. Blagojevic and R. PalicDepartment of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia;

vangelis0703@yahoo.com

Abstract

GC and GC/MS analyses of the diethyl ether washings of fresh grapes of Vitis vinifera x (Vitis labrusca x Vitis riparia) interspecific hybrid Othello from Serbia enabled the identification of 39 constituents that accounted for 96.5% of the total GC peak areas. Five major contributors were the following: heptacosane (15.0%), hentriacontane (15.0%), ethyl oleate (12.4%), nonacosane (11.0%) and pentacosane (8.3%). n-Alkanes (50.9%), fatty acid ethyl esters (32.2%) and higher aldehydes (7.7%) alone constituted ~90% of the extract. Ethyl (E,Z)-2,4-decadienoate, ethyl hexacosanoate, ethyl tetracosanoate, ethyl docosanoate, octane and undecane are reported for the first time as grape constituents.

Key Word Index

Vitis vinifera x (Vitis labrusca x Vitis riparia), Vitaceae, Othello cultivar, extract volatiles, heptacosane, hentria-contane, ethyl oleate, nonacosane.

1041-2905/10/0006-01$14.00/0 —© 2010 Allured Business Media

J. Essent. Oil Res., 22 (November/December 2010)

*Address for correspondence

Plant Name, Source and Part

The cultivar Othello is a highly fragrant hybrid of two North American grape species Vitis labrusca L. and Vitis riparia L. and European Vitis vinifera L. (Vitaceae) (1). Fresh intact grapes of the interspecific hybrid Othello, grown in SE Serbia, harvested in the beginning of October 2007, were obtained from the local market.

Previous work

Vitis vinifera L. and related taxa have been used for various utilizations since ancient times. Today, they are of worldwide interest for pharmaceutical applications, nutritional purposes, including raw and dried consummation, but, most of all, for wine production (2). From the first application of the GC/MS for the study of grape and wine chemistry, in the early 1980s, this technique has been readily used for the investigation of different grape volatile metabolites: sulphur compounds, methoxypyrazines, polyphenols, etc. (3). Many studies concerning volatile and semivolatile compounds of V. vinifera point out to the significant influence of the climatic and geographical origin of the specific grape varieties on their aroma profiles (4-7). To the best of the authors’ knowledge, aroma compounds of the highly fragrant grapes of V. vinifera x (V. labrusca x V. riparia) interspecific hybrid Othello were previously analyzed only once, more than thirty years ago, us-ing a different methodology (8).

Present work

The whole intact fresh berries of Vitis sp. hybrid Othello (150 g) were immersed into vessels with 100 mL of diethyl ether in an ultrasonic bath (Bandelin electronic, GmbH & Co. KG, Germany) for 15 min at room temperature. The obtained ether washings were gravity filtered through small columns packed with 1 g of Celite (Merck, Germany) in order to remove all the insoluble material and then concentrated to 10 mL at room temperature using a steam of N2 before GC and GC/MS analyses. Yield of the dry residue, obtained by completely evaporating the ether, was 50 mg.

The GC/MS analyses (three repetitions) were carried out using a Hewlett-Packard 6890N gas chromatograph equipped with a fused silica capillary column HP-5MS (5% phenylmeth-ylsiloxane, 30 m ´ 0.25 mm, film thickness 0.25 mm, Agilent Technologies, USA) and coupled with a 5975B mass selective detector from the same company. The injector and interface were operated at 250oC and 300oC, respectively. Oven tem-perature was raised from 70–290oC at a heating rate of 5oC/min and then isothermally held for 10 min. As a carrier gas He at 1.0 mL/min was used. The sample, prepared as previously mentioned, was injected in a pulsed split mode (the flow was 1.5 mL/min for the first 0.5 min and then set to 1.0 mL/min throughout the remainder of the analysis; split ratio 40:1). MS conditions were as follows: ionization voltage of 70 eV, acquisi-tion mass range 35–500, scan time 0.32 s. Extract constituents

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2/Journal of Essential Oil Research Vol. 22, November/December 2010

were identified by comparison of their linear retention indices (relative to C7-C32 alkane (9) on the HP-5MS column) with literature values (10) and their mass spectra with those of authentic standards, as well as those from Wiley 6, NIST02, MassFinder 2.3, and a homemade MS library with the spectra corresponding to pure substances, and wherever possible, by co-injection with an authentic sample. GC-FID analysis was carried out under the same experimental conditions using the same column as described for the GC/MS. The percentage composition of the extract was computed from the GC peak areas without any corrections.

Results and Discussion

GC and GC/MS analyses of the diethyl ether grape wash-ings of hybrid Othello cultivated in SE Serbia enabled the identification of overall 39 components representing 96.5% of the total GC peak areas. Major contributors of the extract were hentriacontane (15.0%), heptacosane (15.0%), ethyl oleate (12.4%), nonacosane (11.0%) and pentacosane (8.3%). Alongside the n-alkanes (50.9%) and ethyl esters (32.2%), which alone constituted more than 80% of the total extract, higher aldehydes were also present in a significant amount (7.7%). These are grape surface wax constituents (11). The relative percentage of all additionally identified constituents, belonging to other com-pound classes (“green leaf” volatiles (1.8%), free acids (0.2%), methyl esters (0.5%) and alcohols (3.2%)), have not exceeded 1%. α-Tocopherol (0.7%) and g-tocopherol (0.8%), well known antioxidants, were among the extract constituents.

Certain components listed in Table I are probably products of the post-harvesting biochemical transformations of native plant metabolites. The isomeric 2,3-butanediols, for example, could be the products of anaerobic intracellular fermentation (12). “Green leaf” volatiles, on the other hand, are most probably produced by enzymatic degradation of unsaturated fatty acids, as the plants’ injury-induced response to stress, emitted during the collection and preparation of Othello grape sample (13).

Some of the Othello extract volatiles are known insect-attractants. For example, a recent study concerning antennal and behavioral responses of the grapevine moth Lobesia botrana females to volatiles from grapevine (14) showed that among the compounds, which were consistently eliciting a strong antennal response, were pentadecane and nonanal, also identified as Othello volatiles. It could mean that these, and perhaps some other compounds listed in Table I are involved in the host plant signaling that mediate female moth reproductive behavior. Another constituent found in the extract, potentially possessing insect-attractant properties, was 2-phenylethanol (15).

Although most of the identified compounds were previ-ously found in V. vinifera, this is the first known report of some fatty acid ethyl esters occurring as Vitis sp. constituents: ethyl (E,Z)-2,4-decadienoate (1.3%), ethyl docosanoate (2.4%), ethyl hexacosanoate (0.5%), ethyl tetracosanoate (3.9%) and ethyl (E)-9-octadecenoate (0.8%), as well as the ubiquitous octane (0.3%) and undecane (0.1%). It should be noted, however, that methyl tetracosanoate, along with some other fatty acid methyl esters, was previously reported for Vitis sp. (16). As for the ethyl elaidate, there is a possibility that this compound is not a native grape metabolite, but an artifact, formed as the

Table I. Percentage composition of diethyl ether washings of the grape hybrid variety Othello berries

RI1 Compound Class % Method

780 2,3-butanediol O 0.5 a, b785 meso-2,3-butanediol O 0.3 a, b800 octane2 A 0.3 a, b, c855 (E)-2-hexenal GL 0.1 a, b859 methyl 3-hydroxybutyrate E 0.3 a, b866 (Z)-2-hexenol GL 0.8 a, b867 hexanol GL tr a, b, c936 ethyl 3-hydroxybutyrate E 1.0 a, b1100 undecane2 A 0.1 a, b, c1106 nonanal GL/AL 0.9 a, b, c1119 2-phenylethanol O 0.9 a, b, c1277 nonanoic acid AC 0.2 a, b, c1353 methyl anthranilate E 0.2 a, b, c1384 ethyl (E)-4-decenoate E 0.6 a, b1397 ethyl decanoate E 0.1 a, b1471 ethyl (E,Z)-2,4-decadienoate2 E 1.3 a, b1500 pentadecane A 0.2 a, b, c2173 ethyl (Z,Z)-9,12-octadecadienoate

(syn. ethyl linoleate) E 3.8 a, b2179 ethyl (Z)-9-octadecenoate

(syn. ethyl oleate) E 12.4 a, b, c2185 ethyl (E)-9-octadecenoate

(syn. ethyl elaidate) E 0.8 a, b2205 ethyl octadecanoate

(syn. ethyl stearate) E 2.2 a, b, c2300 tricosane A 1.0 a, b, c2398 ethyl eicosanoate

(syn. ethyl arachinoate) E 3.2 a, b2400 tetracosane A tr a, b, c2500 pentacosane A 8.3 a, b, c2597 ethyl docosanoate

(syn. ethyl behenate) E 2.4 a, b2600 hexacosane A tr a, b, c2635 tetracosanal AL 2.6 a, b2700 heptacosane A 15.0 a, b, c2796 ethyl tetracosanoate

(syn. ethyl lignocerate) E 3.9 a, b2800 octacosane A tr a, b, c2839 hexacosanal AL 3.9 a, b2867 unknown3 2.4 a, b2900 nonacosane A 11.0 a, b, c2993 ethyl hexacosanoate

(syn. ethyl cerotate) E 0.5 a, b3000 triacontane A tr a, b, c3038 octacosanal AL 1.2 a, b3058 γ-tocopherol O 0.8 a, b3100 hentriacontane A 15.0 a, b, c3120 γ-tocopherol O 0.7 a, b, cTotal 98.9 Esters (E) 32.7 Ethyl 32.2 Methyl 0.5 Alkanes (A) 50.9 Aldehydes (AL) 7.7 “Green leaf” volatiles (GL) 1.8 Acids (AC) 0.2 Others (O) 3.2

1Compounds listed in order of elution on HP-5MS column (RI- experimentally determined retention indices on the mentioned column by co-injection of a homologous series of n-alkanes C7-C32);

2Previously not reported as a V. vinifera constituent; 3MS, m/z (EI, 70 eV): 281(2), 106(8), 105(100), 104(68), 96(1), 95(3), 91(2), 83(4), 81(4), 67(5), 57(2), 55(7), 43(4), 41(9); tr- trace (<0.05%); syn. – synonym; a- constituent identified by mass spectra comparison; b- constituent identified by retention index matching; c- constituent identity confirmed by co-injection of an authentic sample.

V. vinifera

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isomerization product of ethyl oleate. The terpenoids, repeat-edly identified as wine volatiles, have not been found in the analyzed extract. One explanation could be that the analyzed grape samples contained only glycosylated conjugates (4,17), or that these compounds simply and understandably accumulate in some other part of the plant and not on the surface of grapes, i.e. marcs (used for the preparation of the extract). Otherwise, the origin of isoprenoids in wine could be connected to the grape fermentation process. It was shown that the yeast Sac-charomyces cerevisiae can produce monoterpenoids even in the absence of grape derived precursors (17).

The aroma of any particular wine depends on many fac-tors, but one of the most important is the volatile profile of the grapes employed for its production. This is particularly true for wine made from the grapes of “aromatic” Vitis sp. (18). In general, the aroma compounds are considered to be present in much higher concentrations in the marc or skins than in the pulp (19). In that sense, one could speculate that those components responsible for the specific aroma of the Othello hybrid would be also significant contributors to the flavor of wine made from that grape variety. Table II lists the literature values for odor thresholds (OT), calculated odor activity values (OAV) and odor descriptions of some aroma compounds identified in the extract. According to the OAV values, it seems that the significant contributors to the Othello fragrance, present on the grape surface in the concentration higher than their thresholds, were methyl anthranilate, nonanal, (E)-2-hexenol, ethyl 3-hydroxybutanoate, ethyl decanoate and 2-phenylethanol, giving it a peach-green-grape-sweet aroma. This is in general agreement with the results of Ivanov et al. (8). Methyl anthranilate (0.2% of the extract of the hybrid Othello ((V. riparia x V. labrusca) x V. vinifera)) was considered to be responsible for the characteristic aroma of the North American V. labrusca as well. Unfortunately, this renowned odor-active compound with a high ecological and economical value is, because of the “foxy” aroma, generally undesirable in wines (20). It is worth mentioning that the European species, opposite to the North American ones, seems to totally lack the ability to produce this ester (20).

Acknowledgments

This work was funded by the Ministry of Science and Technologi-cal Development of Serbia (Project 142054 B).

Table II. Odor thresholds (OT), odor activity values (OAV) and odor descriptions for some extract constituents

Compound OT, ppm OAV Odor description Reference

methyl anthranilate 0.003 222.22 foxy, peachy 20, 282,3-butanediol 150 0.01 fruity 21meso-2,3-butanediol 150 0.01 fruity 21(E)-2-hexenal 0.017 19.61 green, fruity, pungent vegetable-like 24hexanol 2.5 <0.05 herbaceous, woody 24ethyl 3-hydroxybutyrate 1 3.33 grapesque 23nonanal 0.001 3000.00 sweet/flowery 242-phenylethanol 1.1 2.73 rosy 21nonanoic acid 3 0.22 fatty, dry 24ethyl decanoate 0.2 1.67 pleasant, soapy 25ethyl octadecanoate (syn. ethyl stearate) 15000 0.00 22

References

B. Jankovic, M. Rogatkin and J. Radosevic, Fluorometric 1. determination of the content of the diglucoside malvoside in black hybrid wine of different ages. Jug. Vinograd. Vinar., 12, 11-16 (1979).

S. Bail, G. Stuebiger, S. Krist, H. Unterweger and G. Buchbauer, 2. Characterization of various grape seed oils by volatile compounds, triacylglycerol composition, total phenols and antioxidant capacity, Food Chem., 108, 1122–1132 (2008).

R. Flamini and A. Panighel, Mass spectrometry in grape and wine 3. chemistry. Part II: the consumer protection, Mass Spectrom. Review, 25, 741-774 (2006).

A. Nasi, P. Ferranti, S. Amato and L. Chianse, Identification of free and 4. bound volatile compounds as typicalness and authenticity markers of non-aromatic grapes and wines through a combined use of mass spectrometric techniques. Food Chem., 110, 762-768 (2008).

M. J. Cabrita, A. M. Costa Freitas, O. Laureano, D. Borsa and R. D. 5. Stefano, Aroma compounds in varietal wines from Alentejo, Portugal. J. Food Compos. Anal., 20, 375-390 (2007).

G. Buchbauer, L. Jirovetz, M. Wasicky, and A. Nikiforov, Headspace 6. analysis of Vitis vinifera (Vitaceae) flowers. J. Essent. Oil Res., 6, 311-314 (1994).

Sh. A. Abramov and K. Vlasova, Essential oils in early-ripening grape. 7. Prikl. Biokhim. Mikrobiol., 35, 90-95 (1999).

I. Ivanov, V. Vulchev and V. Dikov, Composition of aromatic compounds 8. in grapes of the Vitis vinifera variety and some interspecies hybrids. Gradin. Lozar. Nauka, 12, 63-73 (1975).

H. Van Den Dool and P. D. Kratz, A generalization of the retention 9. index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr., 11, 463-471 (1963).

R. P. Adams, 10. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th Edition. Allured Publ. Corp., Carol Stream, IL (2007).

D. Borsa and R. Di Stefano, Analysis of grape lipids, 11. Riv. Viticolt. Enol., 46, 3-21 (1993).

D. Y. Yang, Y. Kakuda and R. E. Subden, Higher alcohols, diacetyl, 12. acetoin and 2,3-butanediol biosynthesis in grapes undergoing carbonic maceration. Food Res. Int., 39, 112–116 (2006).

A. Hatanaka, T. Kajiwara and K. Matsui, The biogeneration of green 13. odor by green leaves and its physiological functions: Past, present and future. Z. Naturforsch. C, 50, 467-472 (1995).

M. Tasin, G. Anfora, C. Ioriatti, S. Carlin, A. De Cristofaro, S. Schmidt, 14. M. Bengtsson, G. Versini and P. Witzgall, Antennal and behavioral responses of grapevine moth Lobesia botrana females to volatiles from grapevine, J. Chem. Ecol., 31, 77-87 (2005).

B. A. Roy and R. A. Raguso, Olfactory versus visual cues in a floral 15. mimicry system. Oecologia, 109, 414-426 (1997).

L. dos Santos Freitas, J. V. de Oliviera, C. Dariva, R. A. Jacques 16. and E. B. Caramao, Extraction of grape seed oil using compressed carbon dioxide and propane: extraction yields and characterization of free glycerol compounds. J. Agric. Food. Chem., 56, 2558-2564 (2008).

F. M. Carrau, E. Boido and E. Dellacassa, Terpenoids in grapes and 17. wines: origin and micrometabolism during the vinification process.

Radulovic et al.

4/Journal of Essential Oil Research Vol. 22, November/December 2010

Nat. Prod. Commun., 3, 577-592 (2008).

D. J. Caven-Quantrill and A. J. Buglass, Comparison of micro-scale 18. simultaneous distillation-extraction and stir sorptive extraction for the determination of volatile organic constituents of grape juice, J. Chromatogr., 1117, 121-131 (2006).

Z. Pineiro, R. Natera, R. Castro, B. Puertas and C. G. Barroso, 19. Characterization of volatile fraction of monovarietal wines: influence of winemaking practices. Anal. Chim. Acta, 563, 165-172 (2006).

J. Wang and V. De Luca, The biosynthesis and regulation of 20. biosynthesis of Concord grape fruit esters, including ‘foxy’ methyl anthranilate. Plant J., 44, 606-619 (2005).

R. A. Peinado, J. Moreno, M. Medina and J. C. Mauricio, Changes 21. in volatile compounds and aromatic series in sherry wine with high gluconic acid levels subjected to aging by submerged flor yeast cultures. Biotechnol. Lett., 26, 757-762 (2004).

S. Boonbumrung, H. Tamura, J. Mookdasant, H. Nakamoto, M. 22. Ishihara, T. Yoshizawa and W. Varanyanond, Characteristic aroma components of the volatile oil of yellow kea mango fruits determined by limited odor unit method. Food Sci. Technol. Res., 7, 200-206 (2001).

L. Moyano, Z. Luis, J. Moreno and M. Medina, Analytical study of 23. aromatic series in sherry wines subjected to biological aging. J. Agric. Food Chem. 50, 7356-7361 (2002).

R. G. Buttery, J. G. Turnbaugh and L. C. Ling, Contribution of volatiles 24. to rice aroma. J. Agric. Food Chem., 36, 1006-1009 (1988).

V. Ferreira, N. Ortin, A. Escudero, R. Lopez and J. Cacho, Chemical 25. characterization of the aroma of Grenache rose wines: aroma extract dilution analysis, quantitative determination and sensory reconstitution studies. J. Agric. Food Chem., 50, 4048-4054 (2002).