Phytochemistry 57 (2001) 71±76

www.elsevier.com/locate/phytochem

Variability of essential oils of Satureja montana L. and

Satureja kitaibelii Wierzb. ex Heu€. from the central
part of the Balkan peninsula
Violeta Slavkovska a, Radisa Jancic a, Srdjan Bojovic b,*, Slobodan Milosavljevic c,
Dejan Djokovic c
a
Department of Botany, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Yugoslavia
b
Institute of Forestry, Kneza Viseslava 3, 11030 Belgrade, Yugoslavia
c
Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11001 Belgrade, Yugoslavia

Received 21 March 2000; received in revised form 3 November 2000

Abstract
The results of the essential oil composition con®rm that there are di€erences between populations within the same subspecies
(Satureja montana ssp. montana), between the subspecies (Satureja montana ssp. montana and Satureja montana ssp. pisidica) and
the species (Satureja montana and S. kitaibelii). The essential oil composition of S. montana ssp. montana is more related to the
Satureja montana ssp. pisidica than S. kitaibelii which separates as a whole species. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Satureja montana ssp. montana; S. montana ssp. pisidica; S. kitaibelii; Lamiaceae; Chemotaxonomy; Essential oils

1. Introduction morphologic characteristics such as the form and size of

The genus Satureja L. embraces over 30 species whose
center of distribution is located in the eastern part of the
Mediterranean (Silic, 1979). These are annual or per-
ennial semi-bushy aromatic plants that inhabit the arid,
sunny, stony and rocky habitats.
The genus Satureja belongs to the family Lamiaceae,
sub-family Nepetoideae, and the tribe Mentheae (Can-
tino et al., 1992). One of the diagnostic characteristics of
the sub-family Nepetoideae is that its representatives
contain more than 0.5% of essential oil (El-Gazzar and
Watson, 1970). The essential oils have certain biological
functions such as physiological function (in plant meta-
bolism) and ecological function (in interactions of the
plants with their environment) (Jancic et al., 1995). The
essential oils are also signi®cant for an explanation of
phylogenetic relations.
The species Satureja montana L. is confusing from the
taxonomic and, therefore, chorological point of view. Its
high variability is evident, even within a single popula-
tion polymorphism, and especially in populations com-
ing from distant habitats. The variability is re¯ected in
* Corresponding author. Fax: +381-11-545-969.
E-mail address: bojos@ptt.yu (S. Bojovic).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00458-1
the calyx and corolla, calycal dents, size, form and color
of the nutlets, and particularly in the form, size and
pubescence of the leaves and in a more or less pro-
nounced pubescence of the stems (Silic, 1979). The taxa
S. montana contains numerous subspecies.
Satureja kitaibelii Wierzb. ex Heu€. has been shown by
the older authors as a member of one of the infraspecies
systematic categories within an extremely polymorphic
species S. montana (Briquet, 1893; Hayek, 1931; Stojanov
and Stefanov, 1948; Ball and Getli€e, 1972; Diklic, 1974).
Silic (1979) thinks that the pronounced morphologic dif-
ferences and clearly separated area, in regard to the related
taxons, con®rm the view of those authors that consider S.
kitaibelii as a separate species (Ravarut, 1961).
The essential oil composition of the species S. montana
and S. kitaibelii is very variable. According to some
authors the dominant components in the oil of S. montana
are caryophyllene and geraniol (Sevarda et al., 1986),
and according to others it is carvacrol (Palic and Gasic,
1993; Kustrak et al., 1996). The dominant components
in the oil of S. kitaibelii are r-cymene, geraniol and b-
elemene (Sevarda et al., 1989; Palic and Gasic, 1993;
Chalchat et al., 1999).
The aim of the work is investigating the essential oil
composition variability on di€erent levels: within a
72 V. Slavkovska et al. / Phytochemistry 57 (2001) 71±76
population, between populations of the same subspecies
(two populations of S. montana ssp. montana), between
the subspecies (S. montana ssp. montana and S. montana
ssp. pisidica) and between the species S. montana and S.
kitaibelii. The results could aid in solving the position of
these taxons whose place within the clasi®cation system
is unclear.
2. Results and discussion
The composition and content of essential oils in the
aerial parts of the plants of the studied populations are
given in Table 2. Monoterpene hydrocarbons and their
derivatives dominate in the chemical composition of
essential oils of the studied populations. The sesqui-
terpene compounds are present in small quantities.
In the population S. montana ssp. montana from Beli
Rzav gorge in the essental oil of the 33% of plants the
acyclic monoterpene alcohol linalool is found as the
dominating component, while in 43% of individuals it was
aromatic monoterpene, -cymene. In 10% of plants the
essential oil is dominated by bicyclic alcohol of bornan
class, borneol, while in 13% of individuals dominates
bicyclic alcohol of thujanic class, trans-sabinen hydrate.
The population of S. montana ssp. montana from the
surroundings of Zvornik is characterized by the pre-
Cymene as the dominant component of the essential oil
of the species S. kitaibelii had been also determined in
earlier investigations (Sevarda et al., 1989; Palic and
Gasic, 1993; Chalchat et al., 1999).
The principal component analysis was done on the
basis of the quantitative content of 19 analyzed com-
pounds in 120 individuals (Fig. 2). The PCA revealed that
the ®rst three axes represent 47.5% of total information.
The ®rst axe explains the most variabilities (22.7%) and
possesses the highest biological signi®cance, since it is
capable to clearly separates the species S. montana ssp.
pisidica from the species S. kitaibelii according to the
limonene, a-pinene and camphene percentages (Fig. 2).
By comparing the studied populations (based on the
qualitative and quantitative composition of essential
oils) a conclusion could be made that the most similar
populations are the ones of S. montana ssp. montana.
The essential oil of S. montana ssp. pisidica di€ers
from the essential oil of S. montana ssp. montana from
the gorge of the river Beli Rzav and the surroundings of
Zvornik in domination of carvacrol which in other
populations is found in irrelevant quantities (Table 2).
The small quantity of trans-sabinene hydrate and dom-
ination of carvacrol in the essential oils of some indivi-
duals could be a ``reason plus'' to di€erentiate it from
the subspecies montana.
In the plants of S. kitaibelii, linalool appears in small
sence of the same components in essential oils, except - quantities, while in the essential oils of some individuals
caryophyllene, as the previous population (from the there is a clear domination of limonene (Fig. 2). The S.
gorge of the river Beli Rzav). Linalool as a dominant kitaibelii population is speci®c for not having some com-
component was detected in the essential oils of 23% of pounds such as z-()-ocymene, -terpinene, -pinene and
individuals, -cimene also in 23%, trans-sabinen hydrate carvacrol (Table 2). The qualitative di€erence in the
in 27% and borneole in 3% of individuals. In this popu- composition of essential oils in S. kitaibelii (absence of
lation 23% of the plants are dominated by phenol of the some compounds) might be a result of a speci®c genetic
menthane class (-cymen-8-ol).
According to the presence and quantity of dominant
compounds, the essential oil composition of the investi-
gated populations of S. montana ssp. montana di€ers
from the results published earlier (Sevarda et al., 1986;
Kustrak et al., 1996).
In 33% of the plants of S. montana ssp. pisidica from
the mountain Galicica dominates linalool, in 37% of
individuals -cymene, in 3% borneole and in 27% Ð
monoterpene phenol of the menthane class Ð carvacrol.
These results agree with those from previous investiga-
tions of S. montana ssp. pisidica (Palic and Gasic, 1993).
In the essential oils of the plants of population S.
kitaibelii coming from the gorge of the river Jelasnica,
14 components were identi®ed (Table 2). The com-
pounds which are absent in the essential oils with regard
to the essential oils from populations of the species S.
montana are: -pinene, -terpinene, z-()-ocymene and
carvacrol. In 27% of the plants the dominant compo-
nent in essential oils is monoterpene of the menthane
class Ð limonene, in 63% is -cymene and in 10% the
dominant component is trans-sabinene hydrate. -
Fig. 1. Location of the analyzed populations. *, Satureja montana
ssp. montana; Beli Rzav; *, Satureja montana ssp. montana; Zvornik;
&, Satureja montana ssp. pisidica; Galicica; &, Satureja kitaibelii,
Jelasnica.
 V. Slavkovska et al. / Phytochemistry 57 (2001) 71±76 73

basis of this species. Since there are clear morphologic and
chorologic di€erences, including even the pronounced
chemical di€erences, it is to be accepted that the view of
the authors that this taxon classify into a separate species
S. kitaibelii (Ravarut, 1961; Silic, 1979).
The multivariate analysis reveals that S. montana ssp.
montana represents a heterogeneous group while S. mon-
tana ssp. pisidica and S. kitaibelii are clearly separated
between themselves. Cluster analysis using three methods
(UPGMA, single linkage and complete linkage) revealed
that essential oil composition of S. montana ssp. montana
is more related to the one of S. montana ssp. pisidica than
of S. kitaibelii (Fig. 3).
Our results contribute to the better knowledge
of the chemotaxonomic structure of this polymor-
phic genus from the central part of the Balkan
peninsula Ð and particularly the speci®city of the
species S. kitaibelii.
3. Experimental
3.1. Plant material
The plant material was collected from the following
localities: S. montana ssp. pisidica (Wettst.) Silic from the

Table 1
Basic characteristic of localities

Locality Ha (m) Nb ( ) Ec ( ) Exposition Substrate Climate Studied species

Beli Rzav gorge 600 43.7 19.2 S Limestone Sub-mediterranean S. montana ssp. montana
Zvornik area 200 44.2 19.0 SE Limestone Sub-mediterranean S. montana ssp. montana
Galicica mountain 1800 40.9 20.9 E Limestone Sub-med. montane S. montana ssp. pisidica
Jelasnica gorge 400 43.2 22.0 SW Limestone Sub-mediterranean S. kitaibelii
a
H=height above sea level.
b
N=north geographical latitude.
c
E=east geographical longitude.

Table 2
Composition of essential oils of studied populations of Satureja montana and Satureja kitaibelii

Satureja montana ssp. montana Satureja montana ssp. pisidica Satureja kitaibelii

Compound Beli Rzav gorge Zvornik area Galicica mountain Jelasnica gorge

1. -Pinene 1.5 (0.9)a 3.6 (2.7) 0.9 (0.5) 6.0 (2.5)

2. Camphene 2.2 (1.3) 1.9 (1.2) 1.1 (0.9) 3.7 (1.2)
3. -Pinene 0.8 (0.5) 1.3 (1.1) 1.1 (1.4) ±
4. Myrcene 1.2 (0.6) 1.9 (1.6) 1.3 (0.6) 1.0 (0.5)
5. -Terpinene 0.9 (0.5) 2.5 (3.0) 0.8 (0.4) ±
6. Limonene 2.0 (1.7) 4.5 (2.8) 0.6 (0.5) 16.0 (7.5)
7. (z)--Ocimene 3.0 (2.6) 2.8 (2.1) 1.9 (1.6) ±
8. -Terpinene 4.9 (3.6) 5.0 (3.6) 6.2 (3.3) 2.0 (1.4)
9. -Cymene 18.8 (12.2) 13.3 (12.2) 29.3 (15.8) 20.9 (8.2)
10. t-Sabinene hyd. 7.2 (7.5) 9.3 (9.7) 1.6 (2.0) 8.2 (8.7)
11. Linalool 22.8 (23.8) 8.1 (10.7) 24.0 (17.7) 1.6 (1.0)
12. Terpinen-4-ol 2.6 (2.4) 5.9 (4.3) 2.4 (2.9) 3.8 (3.0)
13. -Caryophyllene 0.1 (0.2) ± 1.1 (0.9) ±
14. Borneol 10.6 (7.5) 7.1 (3.5) 4.6 (8.3) 9.8 (3.3)
15. -Cymen-8-ol 3.7 (5.7) 8.6 (8.0) 1.3 (2.3) 0.7 (0.5)
16. C15H26O 2.0 (0.9) 3.0 (1.6) 1.8 (0.8) 3.9 (2.0)
17. C15H26O 1.9 (0.9) 1.3 (0.8) 1.0 (0.7) 2.4 (1.5)
18. Thymol 0.7 (1.0) 1.9 (3.0) 1.5 (1.4) 0.9 (0.6)
19. Carvacrol 0.4 (0.2) 1.1 (0.9) 18.3 (16.1) ±
Monoterpene comp. 84.3 78.8 95.9 74.6
Monoter. hydrocarbons 35.3 36.8 42.2 49.6
Oxygenated monoterp. 49.0 42.0 53.7 25.0
Alcohols 43.2 30.4 32.6 23.4
Phenols 5.8 11.6 21.1 1.6
Sesquiterpene comp. 4.3 4.3 3.9 6.3
Sesquit. hydrocarbons 0.4 ± 1.1 ±
Oxygenated sesquit. 3.9 4.3 2.8 6.3
Total 88.6 83.1 99.8 80.9
a
Average value in bold; standard deviation in parentheses.
74 V. Slavkovska et al. / Phytochemistry 57 (2001) 71±76

mountain Galicica Ð the south-western Macedonia; S. 3.2. Steam distillation

montana ssp. montana from the surroundings of Zvor-
nik and the gorge of the river Beli Rzav Ð western
Serbia; S. kitaibelii Wierzb. ex Heu€. from the gorge of
the river Jelasnica Ð eastern Serbia (Fig. 1). Basic
characteristics of the mentioned localities are given in
Table 1. From each locality 30 plants were collected,
picked in the period of blooming.
The dried aerial part of each individual was submitted
to the steam distillation with simultaneous extraction of
the distillate by methylene-chloride (CH2Cl2), which
enabled the separation of the essential oil. For this pro-
cedure the Likens±Nickerson apparatus was used
(Maarse and Belz, 1981).

Fig. 2. PCA Ð graphical review of observations grouped in characters and individuals in the plan of the ®rst two axes. *, Satureja montana ssp.
montana; Beli Rzav; *, Satureja montana ssp. montana; Zvornik; &, Satureja montana ssp. pisidica; Galicica; &, Satureja kitaibelii, Jelasnica. The
space containing all the individuals of one populations is approximately bordered with a line.
 V. Slavkovska et al. / Phytochemistry 57 (2001) 71±76
Fig. 3. Cluster analyses of Satureja montana and Satureja kitaibelii
based on population means of essential oils. *, Satureja montana ssp.
montana; Beli Rzav; *, Satureja montana ssp. montana; Zvornik; &,
Satureja montana ssp. pisidica; Galicica; &, Satureja kitaibelii, Jelasnica.
3.2.1. Combined GC±MS
The qualitative and quantitative analysis of the
essential oils were done by GC and GC/MS under the
following conditions: Analytical GC-A Varian model
3400 gas chromatograph equipped with a split/splitless
injector (250 C), and a 30 m0.32 mm DB-Wax silica
capillary column and FID (300 C), were used for GC
and GC/MS masurements. The column was tempera-
ture programmed from 50 C (3 min) to 200 C at 10 C/
min; carrier gas 3 ml H2/min. Peak areas were calcu-
lated electronically by a Varian DS-604 data station.
GC/MS Ð the gas chromatograph was connected via
an open split interface, and a fused silica capillary (at
250 C) to the ion source of a Finnigan MAT 8230 mass
spectrometer, equipped with a PDP 11/74 computer.
Working conditions: carrier gas 2 ml He/min; other GC
conditions as above. MS: ion source (electron impact),
170 C, 70 eV. Constituents were identi®ed by co-injec-
tion with authentic compounds, and by comparison of
their mass spectra to those from the MS library (Wiley,
1984) taking into account the relative retention times.
75
3.3. Statistical analysis
In data processing, the multivariatiate statistic analy-
sis was applied (De Lagarde, 1983). The principal com-
ponent analysis (PCA) was used as a statistical method
capable to suggest the structure of the set and analysis
of the variables in relation to the studied characteristics.
In total, 19 characteristics (chemical compounds) were
analyzed in 120 individuals.
The program Statgraphics 5.0 (Statistical Graphics
Corporation, USA, 1991) was used. Agglomerative clus-
ter analyses on population means for all essential oils used
the UPGMA (unweighted pair-group method, arith-
metic average), single-link and complete-link clustering
methods of the SAHN procedure of the NTSYS-pc
computer program (Rohlf, 1990).
Acknowledgements
Our special thanks go to Dr. Bruno Fady for his use-
ful comments and suggestions, and to Danica Bajic,
Institute of Chemistry, Technology and Metallurgy
from Belgrade for the GC analysis.
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