Odor-active constituents of Cedrus atlantica wood essential oil

Article abstract of DOI:10.1016/j.phytochem.2017.09.017

The main odorant constituents of Cedrus atlantica essential oil were characterized by GC-Olfactometry (GC-O), using the Aroma Extract Dilution Analysis (AEDA) methodology with 12 panelists. The two most potent odor-active constituents were vestitenone and 4-acetyl-1-methylcyclohexene. The identification of the odorants was realized by a detailed fractionation of the essential oil by liquid-liquid basic extraction, distillation and column chromatography, followed by the GC-MS and GC-O analyses of some fractions, and the synthesis of some non-commercial reference constituents.

Full text of DOI:10.1016/j.phytochem.2017.09.017

Phytochemistry 144 (2017) 208e215
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Odor-active constituents of Cedrus atlantica wood essential oil
Ayaka Uehara a, Basma Tommis b, Emilie Belhassen a, Badr Satrani b, Mohamed Ghanmi b,
,
*
Nicolas Baldovini a
a
ꢀ
^
Institut de Chimie de Nice, CNRS UMR 7272, Universite Cote d’Azur, Parc Valrose, F-06108, Nice, France
Laboratoire de Chimie des Plantes Aromatiques et Medicinales et de Microbiologie (LCPAMM), Centre de Recherche Forestiere, Haut-Commissariat aux
b
ꢀ
ꢁ
^
ꢁ
ꢀ
Eaux et Forets et a la Lutte Contre la Desertification, BP 763, 10000, Rabat, Morocco
a r t i c l e i n f o
a b s t r a c t
Article history:
The main odorant constituents of Cedrus atlantica essential oil were characterized by GC-Olfactometry
(GC-O), using the Aroma Extract Dilution Analysis (AEDA) methodology with 12 panelists. The two
most potent odor-active constituents were vestitenone and 4-acetyl-1-methylcyclohexene. The identi-
fication of the odorants was realized by a detailed fractionation of the essential oil by liquid-liquid basic
extraction, distillation and column chromatography, followed by the GC-MS and GC-O analyses of some
fractions, and the synthesis of some non-commercial reference constituents.
Received 23 May 2017
Received in revised form
20 September 2017
Accepted 21 September 2017
© 2017 Elsevier Ltd. All rights reserved.
Keywords:
Cedrus atlantica (Pinaceae)
Atlas Cedarwood
GC-O
AEDA
Key odorants
1. Introduction
reach a height up to 40 m and a trunk diameter of 2 m, and their
wood is still highly appreciated for construction. Indeed, C. atlantica
The Atlas cedar, native to the Atlas mountains of Algeria and
Morocco, is often considered as a unique species (Cedrus atlantica
is the main species of Moroccan forests used for timber production,
and the sawdust produced during wood processing is often valor-
ised by hydrodistillation to afford an essential oil displaying the
very typical odor of the wood. This oil is entirely different (chem-
ically and olfactorily) from the most common Texas and Virginia
cedarwood oils obtained from Juniperus species. Its very peculiar
odor has been described as slightly camphoraceous-cresylic, with a
sweet and tenacious, woody undertone, reminiscent of cassie and
mimosa (Arctander, 1960). The composition of Cedrus atlantica
ꢁ
(Endlicher) Carriere) separated from the two other main groups of
Mediterranean cedars located in Cyprus and Northeastern Medi-
terranea, since they show slight morphological differences (M'Hirit
ꢀ
and Blerot, 1999; M'Hirit, 1994). However, some authors prefer to
consider these three populations as subspecies of Cedrus libani:
C. libani subsp. atlantica (Endlicher) from North Africa, C. libani
subsp. brevifolia (J. Hooker) in Cyprus and C. libani A. Richard subsp.
libani (cedar of Lebanon) formerly widespread in Syria and
Lebanon, and now present mainly in Southern Turkey
(Eckenwalder, 2009). This latter subspecies was overexploited for
thousands of years by several ancient civilizations since the dura-
bility and the rot-resistant character of its aromatic wood was very
useful to build ships, houses and temples, the most famous
example being the King Solomon's temple in Jerusalem. Today,
C. atlantica is the largest remaining population, and the most
commonly cultivated subspecies. It covers about 160 000 ha,
mostly in the Rif and Atlas mountains of Morocco. The trees can
essential oil is generally dominated by a- and b-himachalenes 1 and
2 (Aberchane et al., 2004; Chalchat et al., 1994; Dahoun et al., 1993;
Derriche et al., 1996; Plattier and Teisseire, 1972) and/or himachalol
3 (Boudarene et al., 2004; Paoli et al., 2011; Satrani et al., 2006),
with significant amounts of bisabolane sesquiterpenic ketones such
as a-atlantone 4 (Aberchane et al., 2004; Boudarene et al., 2004;
Chalchat et al., 1994; Dahoun et al., 1993; Derriche et al., 1996;
Paoli et al., 2011; Plattier and Teisseire, 1972; Satrani et al., 2006;
Pfau and Plattner, 1934),
g-atlantone 5 (Aberchane et al., 2004;
Boudarene et al., 2004; Chalchat et al., 1994; Derriche et al., 1996;
Plattier and Teisseire, 1972; Pfau and Plattner, 1934), and deodar-
one 6 (Adams et al., 1974; Nam et al., 2015) which are besides
commonly found in the related himalayan Cedrus deodara species
(Adams et al., 1974).
* Corresponding author.
E-mail address: baldovin@unice.fr (N. Baldovini).
https://doi.org/10.1016/j.phytochem.2017.09.017
0031-9422/© 2017 Elsevier Ltd. All rights reserved.

A. Uehara et al. / Phytochemistry 144 (2017) 208e215
209
Despite the large number of publications dealing with the
composition of Atlas cedarwood oils, accurate descriptions of the
odor-active constituents of this material are scarce. As soon as 1902,
Grimal reported that the distillation of Atlas Cedarwood oil fur-
nished a fraction “which possessed exactly the odor of the original
essence” containing a ketone of the formula C9H14O (Grimal, 1902).
However, Pfau and Plattner claimed later that the mixture of
atlantones 4 and 5 is “the true aromatic principle of the oils” (Pfau
and Plattner, 1934). Thereafter, the odor of vestitenone 7 has been
described as characteristic of the wood (Shankaranarayan et al.,
1977), and recent secondary sources mentioned that the odor of
et al., 1993) to distinguish the olfactory contributors on the basis
of their relative importance. One of the drawbacks of the AEDA is
that it is time-consuming and not always compatible with the
participation of a large number of judges. However, Ferreira
mentioned that a good compromise between experimental time
and precision of the results can be achieved with a dilution rate of
4e20 (Ferreira et al., 2002), thus significantly lowering the number
of required experiments in comparison with AEDA studies based on
a dilution rate of 2. In our study, 12 panelists performed GC-O an-
alyses with a dilution rate of 4, corresponding then to a total of
about a dozen of 25 minutes sessions per panelist. They detected a
total of 44 different odor zones, but only 6 were perceived unani-
mously by all of the panelists. The individual olfactory descriptions
of the 20 zones perceived by more than half of the panelists are
reported in Table 2, together with each individual FD value. This
presentation of the results without any summarization of the data
is deliberate, as it illustrates the large differences of sensitivity
among the panelists and the complexity of the raw data obtained
with a large panel of judges of various cultural backgrounds and
olfactory educations. Indeed, our objective was to identify the most
characteristic cedarwood-like odorants of our sample, recognized
as such by an average population. Then, the only common training
to which was submitted our panel was the memorization of the
odor of the whole essential oil, for the panelists not yet familiar
with the odor of this material. The variability of the qualitative
descriptors used by the panelists in this study can then be due to
real differences in the perceptions, but is also probably the result of
the large variations in the olfactory cultures of non-trained panel-
ists, unable to define olfactory sensations with a common stan-
dardized vocabulary.
Atlas cedarwood was due to “materials such as
a-atlantone and
deodarone (4 and 6)” (Sell, 2003, 2006). However, the experimental
data supporting the olfactory importance of these components
were never accurately reported. Actually, the relative odor contri-
bution of 4e7 is still unknown, as well as the qualitative and
quantitative olfactory differences between each of the eight
and -atlantone isomers (4, 8, 5).
a-, b-
g
To better understand the olfactory contribution of the volatile
constituents of Atlas cedarwood, we performed a detailed investi-
gation on a sample of Moroccan essential oil. We first carried out a
series of fractionations by acido-basic liquid-liquid extraction,
distillation and flash chromatography, followed by GC-MS analysis.
Subsequently, GC-O analysis was applied to the whole essential oil
and its fractions to characterize the main odor-active constituents,
and to guide to other fractionations in order to identify additional
odor impact compounds.
2. Results and discussion
The chemical composition of the sample of Atlas Cedarwood
essential oil investigated in this work is reported in Table 1, and is
consistent with most of the studies published in the literature for
this material (Aberchane et al., 2004; Chalchat et al., 1994; Dahoun
et al., 1993; Derriche et al., 1996; Plattier and Teisseire, 1972). The
GC-O investigation of the whole essential oil permitted to charac-
terize several odorant zones, and a careful fractionation of the oil
combined with the GC-O analyses of the fractions and of pure
reference compounds helped us to attribute some odor-active
components to their corresponding zones. The AEDA methodol-
ogy (Ullrich and Grosch, 1987) was applied to characterize the most
important olfactory contributors of our sample. Although GC-O is
nowadays recognized as the most reliable tool for such task, it
should be kept in mind that it has some limitations as it does not
take into account the synergies between the odorants (Barkat et al.,
2012; Zou and Buck, 2006). Moreover, several authors have criti-
cized the Dilution to Threshold methodologies such as AEDA since
they are based on the false assumption that the odor intensity in-
creases linearly with the concentration. In fact, different odorants
are not necessarily characterized by the same slope in their psy-
chophysiological function (Neuner-Jehle and Etzweiler, 1991), and
for this reason a ranking of the odorants of a mixture cannot be
based solely on their evaluation in the mixture near their detection
threshold. Indeed, as pointed out by Ferreira (Ferreira et al., 2002),
it should be considered that initially, AEDA experiments were used
only as a rough guide for aroma reconstruction experiments. Hence,
the results reported in our study should be interpreted carefully, as
a simple attempt to bring some precisions to the apparent con-
troversies of the literature concerning the odor-active constituents
of Atlas Cedarwood. The AEDA method was selected since it does
not require well-trained panelists, on the contrary to the more
difficult Direct and Posterior Intensity methods (Casimir and
Whitfield, 1978; Delahunty et al., 1996; Etievant et al., 1999;
Guichard et al., 1995; Van Ruth and O'Connor, 2001). It is also
more informative than the Detection Frequency methods (Linssen
Despite such interindividual variability, interesting information
can be easily extracted from the whole set of data, in order to
characterize the main contributors of the typical odor of Atlas
Cedarwood oil. All of the panelists agreed that the contribution of
the atlantones and deodarone 4e6 and 8 was insignificant in
comparison with that of several more volatile constituents. Indeed,
only one odor zone at a Linear Retention Index (LRIGC-O) of 1748
seemed to match with the signal of one of these ketones, (Z)-4, and
was perceived by half of the panel with a rather low mean log4(FD).
Only one judge reported a cedarwood-like odor, while the 11 other
panelists described it as mushroom-like/woody, not particularly
typical of cedarwood. In contrast, two constituents showing the
highest mean FD value of all odorants were detected by all the
panelists at LRIGC-O ¼ 1152 and 1473. The former was almost
unanimously recognized as “typically cedarwood” (11 panelists out
of 12), and 7 panelists out of 12 described the latter as reminiscent
of cedarwood, while the other sniffers attributed less typical de-
scriptors, such as “lemon-like”. A first survey of our mass spectral
and LRI databases led us to the conclusion that these two constit-
uents could be respectively 4-acetyl-1-methylcyclohexene 9 and
vestitenone 7, contained in respective percentages of 1 and 0.45% in
the essential oil. 9 was probably the C9H14O ketone that Grimal had
mentioned earlier, without being able to determine the structure
(Grimal, 1902) and was besides described several times in the
following old phytochemical studies on Atlas Cedarwood essential
oil (Plattier and Teisseire, 1972; Pfau and Plattner, 1934) but sur-
prisingly much less in several more recent studies (see Fig. 1).
In view of the potential importance of 7 and 9 in our olfactory
study, we had to confirm unambiguously their identification and
their contribution to the odor of the essential oil. Even if the chro-
matograms of both constituents showed that their GC-O and GC-MS
signals matched perfectly, one cannot exclude the possibility that
the perceived odor is actually due to a coeluting minor constituent.
This situation is indeed one of the main difficulties of GC-O experi-
ments, which can be overcome only by the evaluation of pure

210
A. Uehara et al. / Phytochemistry 144 (2017) 208e215
Table 1
Constituents of Cedrus atlantica wood essential oil.
a
b
c
LRIHP-5
LRILitt
Identification
%
<800
831
963
936
992
771
828
957
932
988
1020
1024
1026
1044
1054
1071
e
toluene
furfural
5-methylfurfural
0.18
tr
tr
0.03
0.01
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.02
tr
1.02
tr
0.07
0.06
tr
a
-pinene
myrcene
1026
1030
1033
1048
1049
1073
1076
1079
1091
1092
1125
1134
1148
1150
1153
1166
1171
1181
1187
1194
1195
1213
1219
1221
1200
1207
1295
1298
1299
1359
1390
1395
1415
1436
1450
1461
1489
1492
1513
1523
1532
1536
1548
1553
1586
1610
1625
1632
1639
1661
1674
1677
1686
1700
1707
1712
1722
1783
p-cymene
limonene
1,8-cineole
(E)-b-ocimene
g
-terpinene
p-cresol 18d,e
6-methyl-3,5-heptadien-2-one
1086
1087
1089
1118
e
a
-terpinolene
guaiacold,e
dehydro-p-cymene
endo-fenchol
4-acetyl-1-methylcyclohexene 9e
1135
e
trans-pinocarveol
1-(4-methyl-3-cyclohexen-1-yl)ethanol 12 (diastereomer 1)e
e
1-(4-methyl-3-cyclohexen-1-yl)ethanol 12 (diastereomer 2)e
4-ethylphenold,e
borneole
1165
1174
1179
1186
1190
1204
e
tr
tr
terpineol-4
p-methylacetophenone 15e
0.15
0.02
0.01
0.01
tr
tr
tr
0.04
0.83
tr
a
-terpineole
decan-2-one
verbenone
1-(4-methyl-3-cyclohexen-1-yl)propanal 11 (diastereomer 1)e
1-(4-methyl-3-cyclohexen-1-yl)propanal 11 (diastereomer 2)e
dodecane
e
1200
1-(4-methylphenyl)ethanolf
1293
e
undecan-2-one 16e
1-(4-methyl-3-cyclohexen-1-yl)propanol 10 (diastereomer 1)e
e
1-(4-methyl-3-cyclohexen-1-yl)propanol 10 (diastereomer 2)e
tr
0.06
tr
1350
1383
e
a
b
-longipinene
-damascenone 17g
Unknownh
>1
1407
e
longifolenef
0.52
0.47
0.45
12.74
8.31
2.20
33.45
3.11
2.78
1.42
1.61
1.17
0.66
0.45
1.02
1.06
1.12
0.96
1.93
0.13
0.06
1.04
2.61
1.28
1.45
6.23
himachala-2,4-dienef
vestitenone 7e
1444
1449
1481
1485
1500
1516
1522
e
a
-himachalene 1
g-himachalene
himachala-1,4-diene
b-himachalene 2
a
-dehydro-ar-himachalene
d
-cadinene
(8,9)-dehydro-(neo)isolongifolenef
e
Unknowni
1544
1578
1599
1615
e
a-calacorene
himachalene epoxide
longiborneol
b
-himachalene oxide
Unknown (oxygenated sesquiterpenoid)j
1-epicubenol
1628
1652
1661
1668
1675
1694
1698
1706
1717
1777
himachalol 3
allo-himachalol
dihydroatlantone
cadalene
(Z)-g-atlantone (Z)-5
deodarone 6
(E)-
(Z)-
(E)-
g-atlantone (E)-5
a-atlantone (Z)-4
a-atlantone (E)-4
a
LRIHP-5: linear retention index in GC-MS on HP-5 column.
b
LRI Litt: linear retention index on HP-5 column reported in: Adams, R. P., 2007. Identification of Essential Oil Components by GC/MS, 4th ed., Allured Publishing Corp., Carol
Stream, USA.
c
Massic percentage calculated from predicted response factors according to Tissot et al. (2012). tr: trace (<0.01%).
Identification in the acidic fraction.
d
e
f
Identification confirmed by coinjection of commercial or synthesized reference compound.
Tentative identification.
Identification based on coincidence of the GC-O retention time with those of a reference compound.
m/z (%) ¼ 203 (5), 202 (31), 187 (6), 159 (18), 147 (7), 146 (41), 145 (16), 132 (13), 131 (100), 129 (11), 128 (6), 117 (5), 116 (5), 115 (10), 107 (5), 105 (8), 91 (17).
m/z (%) ¼ 202 (49), 188 (15), 187 (100), 159 (16), 146 (13), 145 (73), 143 (18), 133 (13), 132 (15), 131 (43), 129 (15), 128 (21), 121 (15), 119 (28), 115 (16), 109 (12), 105
g
h
i
(20), 93 (48), 91 (22), 79 (12).
j
m/z (%) ¼ 220 (50), 205 (17), 200 (16), 185 (24), 177 (17), 163 (15), 149 (15), 138 (100), 137 (77), 135 (24), 123 (37), 121 (32), 120 (46), 119 (15), 110 (71), 109 (48), 108
(24), 107 (56), 105 (37), 96 (20), 95 (28), 93 (43), 91 (52), 77 (31), 69 (22), 55 (24), 41(30).

A. Uehara et al. / Phytochemistry 144 (2017) 208e215
211
Table 2
GC-O Analysis of the Odorant Constituents of Cedrus atlantica wood essential oil.
LRIa
Constituentb
Olfactory Descriptionc
Individual log4(FD)d
AMe
1
2
2
3
0
4
1
5
0
6
7
8
9
10 11 12
1084 p-cresol 18
1112
Burnt plastic1, 9; wood4; woody, powdery, rancid6; phenolic7, 8; urine12
1
1
4
2
1
3
1
0
4
1
1
3
0
3
1
1
1
4
10
e
Olive6, 7; phenolic8, 10, 12; smoky, guaiacol-like
3
3
1152 4-acetyl-1-methylcyclohexene 9 Typically cedarwood1ꢀ5, 7ꢀ12; orange peel, buttery6; orange9; powdery,
sweet, dill10; cacao, floral, fruity12
4
1
3
1
2
1
3
0
2
0
3.1
0.7
1213 4-methylacetophenone 15
Almond1, 3, 5, 8; wet wood2; floral, jasmine6; fresh, woody7;
cedarwood9; cypress, soapy11; fatty, buttery, rancid10; floral, fresh
leaves12
1
1
1
1
0
1
2
2
1305 undecan-2-one 16
Fruity1; dry leaf, cedarwood2; almond5; capric, fatty6; stink bug8; mint,
citrus9; soap9ꢀ11; floral, fruity, fresh leaves12
0
1
1
0
0
0
0
2
3
0
1
4
0
1
0
2
1
0
0
4
3
1
1
1
4
1310
1392
1412
e
e
Honey, fatty6; anise, fennel7; fruity9; petrol, moldy11; rotten, fruity,
cyclopentanone-like, balsamic, valeric10
2
0
2
2
Almond1, 5, 8; incense6; hot, dust, milky7; coconut8; cinnamon9; sweet,
powdery cedarwood-like10; lemon11; fruity12
0
0
4
1
2
3
1
1
5
b-damascenone 17
Lemon, fresh fruit4; soapy, floral, violet, blackberry6; fruity,
damascenone-like8, 10; orange, fruity9; fresh, cedarwood11
Floral1, 10; cedarwood2ꢀ5, 7, 12; lemon4ꢀ5, 8ꢀ9, 11; lavender, pine,
Marseille soap6; liquorice7; powdery7, 10; eucalyptus9; slightly minty,
fruity10; pepper11
1
4
1473 vestitenone 7
5
2
1
4
0
0
0
3.1
2.0
1500
1560
e
e
Cedarwood2ꢀ4, 7ꢀ8, 10ꢀ12; citrus peel6; ginger, fruity9; dill10; apricot12
1
0
2
3
1
5
1
2
2
3
1
4
1
0
1
1
5
0
Herb1; unpleasant3; solvent, washing powder6; phenolic7; plastic8, 12
woody, dry, mushroom, mossy10; rust, mint12
;
1573
1623
e
e
Lemon1, 5; clove6; spicy, cinnamon7; eugenol8
0
0
1
0
2
Cedarwood, rose2; camphoraceous8; flowery, powdery, perfume10
;
1
1
0
0
1
1
0
1
4
3
minty12
1657
1703
e
e
Lemon1, 3, 8; cedarwood4; smoked saffron6; woody, fruity, vetiver,
1
1
1
1
2
3
1
2
vertofix-like10
Vegetal1; cedarwood2ꢀ3, 7, 8, 10; almond4; lemon5; rotten clementine6;
honey7; lactonic, coconut8; fir tree9; vetiver, buttery, fatty, fruity,
powdery10; curds, wood11; apricot, sweet12
0
1
1
2
2
2
1.3
1736
1748
1809
e
e
e
Soap, fruity4, 10; rosy, powdery7; floral, vetiver8; sweet9, 7; fruity,
damascenone10
1
2
1
1
0
0
0
Cedarwood2; dust, smoke, mushroom6; metallic, mushroom8; fruity,
woody, mushroom10; dry wood, pine, sauna11; fermented leaves12
Soap, almond4; cedarwood, damascenone9; fruity, plum-like10; lemon,
cypress11; floral12
2
1
1
0
0
1
4
5
1
2
0
1817
1828
e
e
Fruity1; fatty, soap6; lactonic8; spicy, cypress, tamarix11
Cedarwood2; citrus2, 9; fruity4, 8, 10; rose5, 2; almond4ꢀ5; violet, candy6;
cypress11; damascenone8, 10; fig7; grape12
1
0
1
2
1
3
0
3
0
2
0
4
0
0
2
4
3
1.8
a
Linear retention index in GC-O on HP5 column.
Odorants identified unambiguously by coinjection of reference compounds. -: Non identified. Additional constituents identified unambiguously by coinjection of reference
b
compounds, and detected by less than half of the panelists: Guaiacol (LRIGC-O ¼ 1105) and 4-ethylphenol (LRIGC-O ¼ 1172).
c
Individual olfactory descriptions (corresponding panelist letter in superscript).
Log4(FD) of each individual perceived odor zone.
Arithmetic mean of the log4(FD), for each odor zone unanimously perceived.
d
e
HO
O
O
1
2
3
(E)-4
(Z)-4
O
O
O
O
O
O
(E)-5
(Z)-5
6
7
8
Fig. 1. Typical constituents of Cedrus atlantica wood essential oil.

212
A. Uehara et al. / Phytochemistry 144 (2017) 208e215
reference compounds, obtained preferably from another origin than
the raw material itself. Therefore, we synthesized pure samples of 7
and 9 (Fig. 2), which we used in subsequent coinjection experiments
in GC-MS and GC-O. Several expeditious procedures are known for
the preparation of 9, either based on the Diels-Alder addition of
isoprene and methylvinylketone (Alder and Vogt, 1949) or the
oxidation of limonene (Baucherel et al., 2001). In our study, we fol-
lowed an original synthetic approach, which, albeit longer,
permitted to prepare three additional reference constituents
(10e12) to confirm their identification by coinjection, since pre-
liminary GC-MS investigations suggested that they could be actual
constituents of the essential oil. As our chiral GC investigations
showed that, not surprisingly, 9 was present in the essential oil as a
racemate, we decided to synthesize 9 in racemic form to ensure that
our synthetic reference compound would display the same olfactory
properties than the natural constituent. Hence, hydroboration of
( )-limonene by 9-BBN afforded the primary alcohol 10, which was
oxidized with PCC to aldehyde 11. The application of an original
oxidative decarbonylation procedure developed in our laboratory
(Baldovini et al., 2013) permitted to convert 11 in alcohol 12 con-
taining a small amount of 9. In these reactions, compounds 10e12
were obtained as ca. 1/1 diastereomeric pairs, and we noticed that
these three constituents were also contained in this form in the
essential oil. Eventually, the crude mixture of 9 and 12 led to 9 by PCC
oxidation. Vestitenone 7 was synthesized from 9 by a three-step
sequence (Wittig-Horner-Wadsworth-Emmons reaction, saponifi-
cation, and treatment by 2 equivalents of methyllithium). Both
synthetic samples were used in coinjection experiments which
confirmed unambiguously that 7 and 9 were the actual contributors
of the two main odor zones. Four other odorants (at LRIGC-O ¼ 1213,
1500, 1703, and 1828) were detected by all of the panelists, and also
reported to possess an Atlas cedarwood character by some of them,
though less unanimously than for 7 and 9. The characterization of
the first one as 4-methylacetophenone 15 was easily confirmed by
coinjection of a commercial reference sample. However, despite our
extensive analysis of the essential oil sample, the identification of
the three other odorants could not be achieved. These constituents
possess probably an extremely low detection threshold, since they
produced relatively strong olfactory stimuli for many panelists in
spite of their very low amount in the sample. It demonstrates that
the GC-O analyses of complex natural mixtures can be useful for the
discovery of new families of potent odorants, with many potential
applications in the fragrance industry. Three additional odorants
detected by almost all of the panelists could be identified unam-
biguously by coinjection of authentic reference samples: undecan-
constituents of the neutral part of the essential oil, and para-cresol
18 (animalic, leather) could be identified in the acidic extract of the
oil. In the context of an analysis devoted tothe identification of odor-
active compounds, such a preliminary acido-basic fractionation is
extremely useful since many naturally occurring carboxylic acids
and phenols have a low detection threshold and can contribute
significantly to the odor of the whole mixture (Boelens, 1996;
Cerutti-Delasalle et al., 2016). Indeed, in addition to 18, 4-
ethylphenol and guaiacol were also identified in this fraction and
detected by some panelists as odorant contributors bringing the
specific phenolic, animalic, sheepfold-like tonality of Cedarwood oil.
3. Conclusions
The determination of the most important olfactory contributors
of a natural mixture can be an extremely long and complex task,
which requires the combination of very efficient analytical tech-
niques with sensorial analyses involving a large number of panel-
ists. Consequently, the lack of accurate knowledge about the main
odoriferous constituents is rather common for many essential oils
and extracts obtained from fragrant plants. Even in modern studies,
the identification of the most important odor-active constituents is
often neglected, and this situation is paradoxical when it concerns
materials used for their odorant properties in the flavor and
fragrance industry.
In this study, we reported that 4-acetyl-1-methylcyclohexene 9
and vestitenone 7 are the two most important contributors to the
typical odor of Atlas cedarwood, together with some compounds of
minor importance like para-cresol 18, undecan-2-one 16 and 4-
methylacetophenone 15 (Fig. 3). Many other olfactorily important
unidentified constituents contribute to the overall odor of this
material, but in contrast with most of the previously published
data, we conclude that the atlantones play a negligible role in the
olfactory character of Atlas cedarwood. In such kind of studies on
O
OH
O
O
O
O
7
9
15
16
17
18
2-one 16 (soap, fatty, stink bug) and b-damascenone 17 (fruity) were
Fig. 3. Most important identified Cedrus atlantica wood essential oil odorants.
1) 9-BBN / THF
2) NaBO3
PCC
H2O2 / KOH
+
CH2Cl2
DMSO/MeOH
O
OH
O
OH
H
10
9
11
12
PCC
O
EtO
EtO
MeLi
Et2O
NaOH
EtOH
P
COOEt
CH2Cl2
NaH, THF
O
O
OH
O
OEt
7
14
13
Fig. 2. Synthesis of Cedrus atlantica wood essential oil constituents.

A. Uehara et al. / Phytochemistry 144 (2017) 208e215
213
the key odorants of natural raw materials, the main analytical issue
is due to the fact that the key contributors are often strongly potent
odorant constituents contained in low (or even trace) amounts.
Consequently, their identification requires an extensive analysis of
the whole mixture, involving several fractionation steps of a sig-
nificant amount of material. Moreover, the use of authentic stan-
dard compounds is often necessary to ensure the validity of the
identification of the odor active constituents. For instance, in this
study, the third most potent cedarwood-like odorant displayed a
The organic phases were then gathered, washed twice with brine,
dried with magnesium sulfate and evaporated to furnish 350 mg of
a dark brown thick oil. The ethereal phases containing the neutral
compounds were evaporated, and the resulting oil was distilled
under vacuum using a vacuum jacketed 50 cm column filled with
metallic packing. A total of eleven fractions (F1-F11) were collected,
and 13.5 g of residue (F12) was recovered in the boiler. On the basis
of the results of the GC-MS and GC-O experiments (see Tables 1 and
2), the characterization of several non-identified odorant constit-
uents required continuing the fractionations, and F1, F2, F9 and F12
were further fractionated by successive column chromatography on
silica gel using increasing amounts of diethyl ether in petroleum
ether for elution.
LRIGC-O of 1500, identical to the LRIGC-O of a-himachalene 1, but the
GC-O analysis of several fractions demonstrated that 1 was odor-
less, hence the olfactory perception was actually due to a coeluting
potent odorant. Finally, the large differences between all the pan-
elists illustrate how strongly can vary the olfactory representations
of complex mixtures from an individual to another. This last
observation justifies the use of large panels and the report of the
individual responses of each panelist, in order to give the best
possible representation of the actual identity of the key odorants of
a given raw material. In conclusion, this study is another demon-
stration of the difficulty to characterize the main odorant contrib-
utors of a mixture, because of the selectivity, the sensitivity and the
interindividual variability of the human olfactory system.
4.4. GC-MS analyses
Gas chromatography - Mass spectrometry (GC-MS) analyses
were carried out using an Agilent 6890N gas chromatograph
coupled to an Agilent 5973N mass selective detector working in
electron impact (EI) mode at 70 eV (scanning over 35e350 amu
range in SCAN mode). The gas chromatograph was equipped a
fused silica capillary column HP-5MS (60 m ꢂ 0.2 mm i.d., film
thickness: 0.3 mm). The analytical parameters were the following:
4. Experimental
The carrier gas was helium at a flow rate of 0.8 ml/min. The oven
temperature was programmed from 50 to 210 ꢁC at 2 ꢁC/min and
held isothermal for 10 min. The injector (split mode, ratio 1/100)
temperature was 240 ꢁC. The temperatures of the ion source and
transfer line were 230 and 250 ꢁC, respectively. LRI were deter-
mined from the retention times of a series of n-alkanes with linear
interpolation. The constituents were identified by comparison of
their mass spectra and LRI with those of pure compounds regis-
tered in commercial libraries and literature data, and with a
laboratory-made database built from authentic compounds.
4.1. General experimental procedures
All reagents and solvents were from Sigma Aldrich (L'Isle
d’Abeau, France). THF was dried by distillation over sodium/
benzophenone before use. Reference compounds 7, 9e12 were
synthesized according to the procedures described hereafter. NMR
Analyses were performed on a Bruker NanoBay Avance III HD
(400 MHz) spectrometer (Bruker, FR-Wissembourg) at 25 ꢁC in
CDCl3. All reference compounds, reagents and solvents were from
Sigma Aldrich (L'Isle d’Abeau, France) except sodium perborate
(Prolabo, France), undecan-2-one (Verley, France), and borneol
(Janssen, Belgium). THF and Diethylether were dried by distillation
over sodium/benzophenone before use.
4.5. GC-FID analyses
Gas chromatography e Flame Ionization Detector (FID) analyses
were performed in the same chromatographic conditions than
those described for the GC-MS, with a FID temperature set at
250 ꢁC. The quantification of the constituents was performed using
predicted response factors as described by Tissot et al. (2012) using
methyl octanoate as an internal standard.
4.2. Essential oil sample
ꢁ
The essential oil of Cedrus atlantica (Endlicher) Carriere (Pina-
ceae) was purchased from Aromaris Company (Taounate, Morocco).
It was obtained by distillation of Atlas Cedarwood sawdust gener-
ated by the sawing of the wood in an industrial sawmill processing
exclusively this species. The wood was collected in the Ain Leuh
4.6. GC-O analyses
GC-O analyses were performed on a Shimadzu GC-2010 Gas
forest
(area
of
Ifrane,
Morocco,
GPS
coordinates:
Chromatograph equipped with
a
HP-5 capillary column
33.209974, ꢀ5.424671). The distillation unit was a 1800 L inox
apparatus, in which a 500 kg charge of sawdust was hydrodistilled
during 7 h, to furnish the essential oil with a 8% yield. The chemical
composition and olfactory characteristics of the sample were
evaluated by trained experts who validated its conformity with
conventional Atlas cedarwood essential oil samples.
(50 m ꢂ 0.32 mm i.d.; 0.53
m
m film thickness), a FID detector and an
ATAS olfactory port OP275 mounted with a glass nasal cone. Sam-
ples were analyzed under the following conditions: injection vol-
ume: 1.0 m
L, in splitless mode. Injector temperature: 250 ꢁC. Oven
temperature program: 100 ꢁCe250 ꢁC at 5 ꢁC/min, then isothermal
for 5 min. Carrier gas (nitrogen) flow: 1.50 mL/min 60% of the flow
was directed to the FID while 40% was directed into the heated
sniffing port. FID temperature: 250 ꢁC.
4.3. Essential oil fractionation
The AEDA methodology (Ullrich and Grosch, 1987) was applied
to characterize the main odor active components: at first, a 2%
solution of the essential oil was analyzed (3e5 times) by each
panelist who was asked to describe freely her/his olfactory per-
ceptions, and to specify if the odor was reminiscent of the typical
smell of Atlas cedarwood. These experiments were then followed
by the injection of serial successive (1/4) dilutions until no more
olfactory perception was detected. Twelve evaluators participated
in this study. All of the panelists not familiar with the typical smell
of Atlas Cedarwood essential oil were asked to memorize the odor
A sample of Cedrus atlantica essential oil (659 g) was dissolved
in diethylether (1L) and extracted 3 times with 250 ml of a 1M
aqueous sodium hydroxide solution. The basic aqueous phases
were then gathered, washed 3 times with 250 ml of diethyl ether,
and poured in an Erlenmeyer flask with 250 ml of dichloromethane.
The content of the flask was cooled in an ice bath, and slowly
acidified by the dropwise addition of 37% hydrochloric acid (65 ml)
under vigorous stirring. The mixture was then decanted, and the
aqueous phase reextracted twice with dichloromethane (250 ml).

214
A. Uehara et al. / Phytochemistry 144 (2017) 208e215
of the sample studied in this work prior GC-O experiments.
(53), 91 (20), 93 (13), 94 (100), 95 (14), 105 (5), 119 (7), 134 (6), 152
(Mþ, 5).
4.7. Identification of the odorant constituents
4.8.3.
a,4-Dimethyl-3-cyclohexene-1-methanol 12
To identify the odorant constituents corresponding to the odor
zones detected in the GC-O experiments, the linear correlation
LRIGC-MS ¼ f(LRIGC-O) was determined by the measurement of the
retention indices of a series of typical EO constituents in GC-MS and
GC-O. This correlation helped to predict the LRIGC-MS of the pre-
sumed odorant corresponding to a given odor zone. GC-O experi-
ments on the various fractions and subfractions helped the search
for candidate odorants, by investigating chromatograms of lower
complexity. The identification of all odorants was confirmed by
coinjection of samples of reference compounds, either commer-
cially purchased or synthesized. In these coinjection experiments, a
panelist performed a GC-O experiment of the essential oil spiked
with the compound, and checked that a single odor perception was
detectable at the expected retention time, with a qualitative
description similar to those perceived in the non-spiked essential
oil analysis.
To a solution of 11 (1.98 g, 13.0 mmol, 1 eq) diluted in a mixture
of 7.6 ml dimethylsulfoxide and 18 ml isopropanol, a 10.2 M
aqueous solution of hydrogen peroxide (13 ml, 148 mmol, 11 eq)
was added at room temperature and stirred for 5 min. A 10.6 M
aqueous solution of potassium hydroxide (8.32 ml, 88 mmol, 6.8
eq) was then added dropwise for 90 min, and an exothermic re-
action occurred before the end of the addition. The mixture was
then heated at 80 ꢁC and stirred for 2 h. After cooling, 100 ml of
water and 50 ml diethyl ether were added, and the mixture was
decanted. The aqueous layer was extracted 3 times with 100 ml
diethyl ether, the organic phases were combined, washed with
brine, dried over MgSO4, and evaporated to give 1.67 g of a slightly
yellow oil which was purified by column chromatography on silica
gel with petroleum ether/diethyl ether (8/2) to furnish 840 mg
(46%) of 12 as a c.a. 6/4 mixture of diastereomers, together with
209 mg (12%) of 4-acetyl-1-methylcyclohexene 9. 12: 1H NMR
(400 MHz, CDCl3):
2.10e1.60 (m, 7H), 1.57 (s, 3H), 1.42 (s, large, 1H), 1.13e1.09 (m, 3H).
13C NMR (100 MHz, CDCl3):
133.16, 132.87, 119.25, 119.09, 70.64,
d
5.31 (s, 1H), 3.53 (dq, J ¼ 38.0, 6.1 Hz, 1H),
4.8. Synthesis
d
4.8.1.
b
,4-Dimethyl-3-cyclohexene-1-ethanol 10
70.50, 40.11, 39.93, 29.09, 26.87, 26.10, 24.32, 23.95, 22.43, 19.75,
19.56. MS (EI, 70 eV, m/z %): Diastereomer 1: 40 (17), 67 (23), 77
(22), 79 (36), 91 (38), 93 (100), 94 (19), 105 (17), 107 (39), 122 (49),
140 (Mþ, 8). Diastereomer 2: 67 (20), 77 (19), 79 (35), 81 (13), 91
(34), 93 (100), 94 (21), 105 (14), 107 (40), 122 (48), 140 (Mþ, 8).
A 0.5 M THF solution of 9-BBN (162 ml, 81 mmol, 1.1 eq) was
added dropwise to ( )-limonene (10.0 g, 73 mmol, 1 eq) at 0 ꢁC
under inert atmosphere. The cooling bath was removed 10 min
after the end of the addition, and the solution was stirred for 3 h at
room temperature. The reaction mixture was then cooled to 0 ꢁC
and a suspension of sodium perborate (56 g, 364 mmol, 5 eq) in
150 ml water was slowly added under stirring. After 2 h stirring at
0 ꢁC, 200 ml of saturated NH4Cl solution and 200 ml of diethyl ether
were added and the mixture was decanted. The aqueous layer was
extracted twice with 200 ml diethyl ether, the organic layers were
combined, washed with brine, dried over MgSO4 and concentrated
under vacuum to afford a yellow oil which was purified by column
chromatography on silica gel with petroleum ether/diethylether (8/
4.8.4. 4-Acetyl-1-methylcyclohexene 9
A solution of the crude mixture of 12 and 9 (1.48 g) in
dichloromethane (8 ml) was added dropwise to a suspension of PCC
(2.69 g, 7.77 mmol) and celite (2.67 g) in dichloromethane (40 mL)
at 0 ꢁC. The mixture was stirred at room temperature during 2 h
after the addition, and then filtered through a short pad of celite
and silica gel, using dichloromethane for rinse. After evaporation of
the solvent, the crude mixture was purified by filtration on silica gel
using petroleum ether/diethyl ether (8/2) for elution, to give
eventually 410 mg of 9 as a slightly yellow oil. When purified 12
(543 mg, 3.9 mmol) was used as a starting material, the same
2) to furnish 9.06 g (80%) of
10 (1/1 mixture of diastereomers) as a slightly yellow oil. 1H NMR
(400 MHz, CDCl3): 5.38 (s, 1H), 3.66e3.64 (m, 1H), 3.51e3.47 (m,
b,4-dimethyl-3-cyclohexene-1-ethanol
d
1H), 1.95 (s, 3H), 1.65 (s, 3H), 1.88e1.50 (m, 5H), 0.95e0.91 (dd,
protocol gave 500 mg (93%) of 9. 1H NMR (400 MHz, CDCl3):
(s, 1H), 2.50e2.39 (m, 1H), 2.16e1.84 (m, 6H), 2.10 (s, 3H), 1.58 (s,
3H). 13C NMR (100 MHz, CDCl3):
211.99, 133.77, 119.23, 47.20,
d 5.31
J ¼ 3.6, 17.0 Hz, 3H). 13C NMR (100 MHz, CDCl3):
d 134.01, 133.95,
120.71, 120.64, 66.35, 66.23, 40.13, 39.95, 35.27, 35.12, 30.73, 30.59,
29.80, 27.65, 27.22, 25.44, 23.44, 13.65, 13.23. MS (EI, 70 eV, m/z %):
Diastereomer 1: 40 (56), 67 (28), 77 (39), 79 (85), 91 (61), 92 (29),
93 (100), 94 (73), 107 (41), 121 (56), 136 (53), 154 (Mþ, 6). Diaste-
reomer 2: 40 (48), 67 (30), 77 (50), 79 (91), 91 (49), 93 (100), 94
(89), 95 (32), 107 (35), 121 (38), 136 (50), 154 (Mþ, 7).
d
29.46, 27.92, 27.02, 24.87, 23.36. MS (EI, 70 eV, m/z %): 43 (32), 67
(36), 77 (17), 79 (26), 93 (18), 95 (78), 105 (17), 123 (50), 138 (Mþ,
100).
4.8.5. 3-(4-Methylcyclohex-3-en-1-yl)-2-butenoic acid 14
A 60% dispersion of sodium hydride in mineral oil (167 mg,
4.2 mmol) was rinsed 3 times with dry petroleum ether under inert
atmosphere. Dry THF (8 ml) was then added, and the suspension
was cooled to 0 ꢁC. Ethyl 2-(diethylphosphoryl)acetate (988 mg,
4.4 mmol) was then added dropwise and stirred until the end of the
emission of hydrogen. 403 mg of crude 9 were then added drop-
wise, and the mixture thus obtained was then stirred for 30 min at
4.8.2.
a,4-Dimethyl-3-cyclohexene-1-acetaldehyde 11
To a suspension of PCC (22.5 g, 104.6 mmol, 2 eq) and celite
(22.8 g) in 345 mL dichloromethane at 0 ꢁC, a solution of 10 (7.87 g,
51 mmol, 1 eq) in 106 ml dichloromethane was added dropwise,
and the reaction mixture was stirred at room temperature during
2 h after the addition. The crude mixture was then filtered through
a short pad of celite and silica gel, rinsed with dichloromethane and
distilled on a Kugelrohr apparatus to give 5.70 g (74%) of 9 (1/1
mixture of diastereomers) as a colorless oil. 1H NMR (400 MHz,
CDCl3):
2.03e1.77 (m, 6H), 1.71e1.59 (m, 1H), 1.57 (s, 3H), 1.00 (t, J ¼ 6.9 Hz,
3H). 13C NMR (100 MHz, CDCl3):
205.48, 205.40, 134.04, 134.01,
119.95, 119.88, 50.96, 50.65, 34.32, 34.25, 30.13, 29.97, 29.65, 28.04,
27.29, 25.43, 23.39, 10.31, 10.22. MS (EI, 70 eV, m/z %): Diastereomer
1: 67 (16), 68 (10), 77 (12), 79 (59), 91 (16), 93 (12), 94 (100), 95 (17),
119 (7), 134 (5), 152 (Mþ, 5). Diastereomer 2: 67 (13), 77 (11), 79
0
ꢁC, and then for 6 h at room temperature. 40 ml of saturated
aqueous ammonium chloride solution was then added, and after
decantation the aqueous layer was extracted with 3 ꢂ 40 ml of
diethyl ether. The combined organic layers were dried on MgSO4
and evaporated to give 400 mg of a colorless oil which was purified
by column chromatography on silica gel with petroleum ether/
diethyl ether (95/5) for elution to furnish 177 mg of (E)-ethyl-3-(4-
methylcyclohex-3-en-1-yl)-2-butenoate 13 as a colorless oil: 1H
d 9.61e9.57 (m, 1H), 5.29 (s, 1H), 2.27e2.18 (m, 1H),
d
NMR (200 MHz, CDCl3):
d 5.65e5.58 (m, 1H), 5.39e5.27 (m, 1H),
4.08 (q, J ¼ 7.14 Hz, 2H), 2.23e2.06 (m, 1H), 2.09 (d, J ¼ 1.28 Hz, 3H),

A. Uehara et al. / Phytochemistry 144 (2017) 208e215
215
through the diene synthesis; diene syntheses with unsymmetrical addends.
Ann. 564, 109e120.
2.04e1.34 (m, 5H), 1.58 (s, 3H), 1.26e1.15 (m, 1H), 1.21 (t, J ¼ 7.13 Hz,
3H). 13C NMR (100 MHz, CDCl3):
167.18, 163.98, 133.86, 120.01,
d
Arctander, S., 1960. Perfume and flavor materials of natural origin. Allured Pub Corp.
139e140.
114.36, 59.49, 44.25, 30.29, 30.27, 27.32, 23.40, 17.14, 14.33. MS (EI,
70 eV, m/z %): 39 (17), 41 (66), 43 (13), 53 (12), 55 (47), 56 (25), 57
(10), 67 (65), 68 (28), 69 (100), 70 (17), 71 (28), 81 (70), 82 (64), 83
(15), 95 (66), 96 (13), 109 (28), 123 (44), 138 (22), 156 (10), 208 (Mþ,
0.1). To a solution of crude 13 (362 mg) in ethanol (2 ml), a solution
of sodium hydroxide (1.2 g, 30 mmol) in 9 ml water was slowly
added. The reaction mixture was heated at reflux during 6 h, and
eventually acidified by the addition of 6M hydrochloric acid (5 ml).
5 ml of ethyl acetate were then added, and after decantation, the
aqueous layer was extracted with 3 ꢂ 20 ml of ethyl acetate, and the
combined organic phases were washed with brine, dried over
MgSO4, and evaporated to give a light orange solid which was
purified by column chromatography on silica gel using dichloro-
methane/methanol (95/5) to give 144 mg of 14. 1H NMR (200 MHz,
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ꢀ
Baldovini, N., Belhassen, E., Filippi, J.-J., Brevard, H., 2013. Nouveau procede de
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decarbonylation oxydative des aldehydes, Patent FR3008089.
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Baucherel, X., Uziel, J., Juge, S., 2001. Unexpected catalyzed C: C bond cleavage by
molecular oxygen promoted by a thiyl radical. J. Org. Chem. 66, 4504e4510.
Boelens, M.H., 1996. Chemical and sensory evaluation of trace compounds in nat-
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Boudarene, L., Rahim, L., Baaliouamer, A., Meklati, B.Y., 2004. Analysis of Algerian
essential oils from twigs, needles and wood of Cedrus atlantica G. Manetti by
GC/MS. J. Essent. Oil Res. 16, 531e534.
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Cerutti-Delasalle, C., Mehiri, M., Cagliero, C., Rubiolo, P., Bicchi, C., Meierhenrich, U.J.,
Baldovini, N., 2016. The (þ)-cis- and (þ)-trans-Olibanic acids: key odorants of
frankincense. Angew. Chem. Int. Ed. 55, 13719e13723.
Chalchat, J.-C., Garry, R.-P., Michet, A., Benjilali, B., 1994. Essential oil components in
sawdust of Cedrus atlantica from Morocco. J. Essent. Oil Res. 6, 323e325.
Dahoun, A., Derriche, R., Belabbes, R., 1993. Influence of the extraction method on
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Delahunty, C.M., Piggott, J.R., Conner, J.M., Paterson, A., 1996. Flavor evaluation of
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CDCl3):
J ¼ 1.14 Hz, 3H), 2.04e1.37 (m, 5H), 1.59 (s, 3H), 1.32e1.11 (m, 1H).
13C NMR (100 MHz, CDCl3):
172.35, 167.31, 133.91, 119.87, 113.82,
d 5.65 (s, 1H), 5.33 (s, 1H), 2.28e2.04 (m, 1H), 2.10 (d,
d
44.55, 30.23, 30.19, 27.25, 23.40, 17.50. EIeMS m/z (%): 39 (14), 41
(15), 53 (14), 67 (52), 68 (87), 69 (12), 77 (18), 79 (29), 81 (10), 87
(14), 91 (24), 93 (31), 94 (100), 95 (18), 97 (10),105 (15),107 (14),111
(25), 119 (12), 120 (10), 121 (12), 135 (15), 147 (11), 180 (Mþ, 8).
4.8.6. Vestitenone 7
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intensity evaluation in gas chromatography-olfactometry by finger span
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Ferreira, V., Pet'ka, J., Aznar, M., 2002. Aroma extract dilution analysis. Precision and
optimal experimental design. J. Agric. Food Chem. 50, 1508e1514.
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Guichard, H., Guichard, E., Langlois, D., Issanchou, S., Abbott, N., 1995. GC sniffing
analysis: olfactive intensity measurement by two methods. Z. Lebensm.-Unters.
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Linssen, J.P.H., Janssens, J.L.G.M., Roozen, J.P., Posthumus, M.A., 1993. Combined gas
chromatography and sniffing port analysis of volatile compounds of mineral
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To a stirred solution of 14 (102 mg, 0.56 mmol, 1 eq) in 3 ml dry
diethylether, a 1.6 M solution of methyllithium in diethylether
(0.72 ml, 1.2 mmol, 2 eq) was added dropwise at 0 ꢁC and stirred for
90 min at this temperature, and then 3 h at room temperature. The
reaction mixture was then treated by the addition of 4 ml of a 0.5 M
solution of hydrochloric acid, followed by the addition of 4 ml
diethylether. After decantation, the aqueous layer was extracted
with 3 ꢂ 10 ml of diethylether. The combined organic phases were
washed with brine, dried over MgSO4, and evaporated to give a
colorless oil which was purified by column chromatography on
silica gel with petroleum ether/diethylether to give 55 mg (55%) of
ꢀ
ꢀ
ꢀ
ꢀ
M'Hirit, O., 1994. Le cedre de l'Atlas. Presentation generale et etat des connaissances
7 as a colorless oil. 1H NMR (200 MHz, CDCl3):
1H), 2.16e1.82 (m, 6H), 2.10 (s, 3H), 2.04 (s, 3H), 1.75e1.66 (m, 1H),
1.58 (s, 3H). 13C NMR (100 MHz, CDCl3):
199.03, 162.42, 133.74,
d 6.01 (s, 1H), 5.32 (s,
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a travers le reseau Silva mediterranea. "Le cedre". Ann. la Rech. For. Maroc 1,
4e5.
M'Hirit, O., Blerot, P., 1999. In: Mardaga, P. (Ed.), Le grand livre de la foret marocaine.
Sprimont, Belgium, pp. 74e76.
ꢀ
^
d
122.13, 119.93, 44.37, 31.80, 30.25, 30.20, 27.30, 23.34, 17.48. EI-MS
m/z (%): 43 (36), 67 (43), 68 (30), 77 (21), 79 (30), 91 (28), 93 (35),
94 (30), 95 (100), 105 (38), 107 (37), 109 (61), 120 (41), 121 (22), 135
(65), 145 (39), 163 (22), 178 (Mþ, 35).
Nam, A.M., Bighelli, A., Ghanmi, M., Satrani, B., Casanova, J., Tomi, F., 2015. Deo-
darone isomers in Cedrus atlantica essential oils and tar oils. Nat. Prod. Com-
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of the wood essential oil of Cedrus atlantica manetti from corsica. Chem. Biodiv.
8, 344e351.
Acknowledgement
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We gratefully acknowledge the Programme de Recherche
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Agronomique pour le Developpement (PRAD) N 12-01 for financial
support. AU is indebted to the French Ministry of Research for a
Grant. The authors wish tothank also Claire de March, Abderrahman
Aafi, Mariam Fatmi, Jean-Jacques Filippi, Najiba Mahmoud and Jean-
Guillaume Giraudon for their kind participation in the GC-O panel.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.phytochem.2017.09.017.
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