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V.

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

Volatiles of the Grape Hybrid Cultivar Othello (Vitis


vinifera x (Vitis labrusca x Vitis riparia)) cultivated in
Serbia
N. Radulovic*,
P. Blagojevic and R. Palic
Department of Chemistry, Faculty of Science and Mathematics, University of Ni, Viegradska 33, 18000 Ni, 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, hentriacontane, ethyl oleate, nonacosane.

Plant Name, Source and Part

Present work

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.

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% phenylmethylsiloxane, 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 temperature was raised from 70290oC 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, acquisition mass range 35500, scan time 0.32 s. Extract constituents

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, using a different methodology (8).

Rec: August 2008

*Address for correspondence

Acc: November 2008


1041-2905/10/0006-01$14.00/0 2010 Allured Business Media
Vol. 22, November/December 2010

Journal of Essential Oil Research/1

Radulovic et al.

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 washings 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 compound classes (green leaf volatiles (1.8%), free acids (0.2%),
methyl esters (0.5%) and alcohols (3.2%)), have not exceeded
1%. -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 insectattractants. 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 previously 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
2/Journal of Essential Oil Research

Table I. Percentage composition of diethyl ether washings of


the grape hybrid variety Othello berries
RI1

Class

Method

780
2,3-butanediol
O
785
meso-2,3-butanediol
O
A
800
octane2
855
(E)-2-hexenal
GL
859
methyl 3-hydroxybutyrate
E
866
(Z)-2-hexenol
GL
867
hexanol
GL
936
ethyl 3-hydroxybutyrate
E
A
1100
undecane2
1106
nonanal
GL/AL
1119
2-phenylethanol
O
1277
nonanoic acid
AC
1353
methyl anthranilate
E
1384
ethyl (E)-4-decenoate
E
1397
ethyl decanoate
E
E
1471
ethyl (E,Z)-2,4-decadienoate2
1500
pentadecane
A
2173
ethyl (Z,Z)-9,12-octadecadienoate

(syn. ethyl linoleate)
E
2179
ethyl (Z)-9-octadecenoate

(syn. ethyl oleate)
E
2185
ethyl (E)-9-octadecenoate

(syn. ethyl elaidate)
E
2205
ethyl octadecanoate

(syn. ethyl stearate)
E
2300
tricosane
A
2398
ethyl eicosanoate

(syn. ethyl arachinoate)
E
2400
tetracosane
A
2500
pentacosane
A
2597
ethyl docosanoate

(syn. ethyl behenate)
E
2600
hexacosane
A
2635
tetracosanal
AL
2700
heptacosane
A
2796
ethyl tetracosanoate

(syn. ethyl lignocerate)
E
2800
octacosane
A
2839
hexacosanal
AL

2867
unknown3
2900
nonacosane
A
2993
ethyl hexacosanoate

(syn. ethyl cerotate)
E
3000
triacontane
A
3038
octacosanal
AL
-tocopherol
O
3058
3100
hentriacontane
A
-tocopherol
O
3120
Total
Esters (E)
Ethyl
Methyl
Alkanes (A)
Aldehydes (AL)
Green leaf volatiles (GL)
Acids (AC)
Others (O)

Compound

0.5
0.3
0.3
0.1
0.3
0.8
tr
1.0
0.1
0.9
0.9
0.2
0.2
0.6
0.1
1.3
0.2

a, b
a, b
a, b, c
a, b
a, b
a, b
a, b, c
a, b
a, b, c
a, b, c
a, b, c
a, b, c
a, b, c
a, b
a, b
a, b
a, b, c

3.8

a, b

12.4

a, b, c

0.8

a, b

2.2
1.0

a, b, c
a, b, c

3.2
tr
8.3

a, b
a, b, c
a, b, c

2.4
tr
2.6
15.0

a, b
a, b, c
a, b
a, b, c

3.9
tr
3.9
2.4
11.0

a, b
a, b, c
a, b
a, b
a, b, c

0.5
tr
1.2
0.8
15.0
0.7
98.9
32.7
32.2
0.5
50.9
7.7
1.8
0.2
3.2

a, b
a, b, c
a, b
a, b
a, b, c
a, b, c

Compounds 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 coinjection of an authentic sample.

Vol. 22, November/December 2010

V. vinifera

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

OT, ppm

methyl anthranilate
0.003
2,3-butanediol
150
meso-2,3-butanediol
150
(E)-2-hexenal
0.017
hexanol
2.5
ethyl 3-hydroxybutyrate
1
nonanal
0.001
2-phenylethanol
1.1
nonanoic acid
3
ethyl decanoate
0.2
ethyl octadecanoate (syn. ethyl stearate) 15000

OAV

Odor description

222.22
foxy, peachy
0.01
fruity
0.01
fruity
19.61
green, fruity, pungent vegetable-like
<0.05
herbaceous, woody
3.33
grapesque
3000.00
sweet/flowery
2.73
rosy
0.22
fatty, dry
1.67
pleasant, soapy
0.00

isomerization product of ethyl oleate. The terpenoids, repeatedly 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 Saccharomyces cerevisiae can produce monoterpenoids even in
the absence of grape derived precursors (17).
The aroma of any particular wine depends on many factors, 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).

Vol. 22, November/December 2010

20, 28
21
21
24
24
23
24
21
24
25
22

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Acknowledgments

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

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Vol. 22, November/December 2010