Essential oil and its systematic significance in species of Micromeria Bentham from Serbia & Montenegro

Abstract

The composition and quantity of the essential oil of Micromeria allowed us to clearly distinguish between sections Pseudomelissa and Eumicromeria. According to our results the species of section Pseudomelissa (M. thymifolia, M. albanica, M. dalmatica and M. pulegium) contained a large quantity of oil (>0.5%) dominated by oxygenated monoterpenes of the menthane type, while the species of
section Eumicromeria (M. croatica, M. juliana, M. cristata and M. parviflora) had a small quantity of essential oil (<0.5%) dominated by various terpene compounds. 0.5% of essential oil is defined like
boundary value on the basis of the lowest quantity of essential oil measured in populations of species of section Pseudomelissa.

Full text

Essential oil and its systematic significance in species of Micromeria
Bentham from Serbia & Montenegro
V. Slavkovska
1
, M. Couladis
2
, S. Bojovic
3
, O. Tzakou
2
, M. Pavlovic
4
, B. Lakusic
1
,
and R. Jancic
1
1
Department of Botany, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia and Montenegro
2
Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of
Athens, Athens, Greece
3
Institute for Biological Research ‘‘Sinisa Stankovic’’, Belgrade, Serbia and Motenegro
4
Department of Pharmacognosy, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia and
Montenegro
Received July 7, 2003; accepted July 23, 2004
Published online: July 25, 2005
Springer-Verlag 2005
Abstract. The composition and quantity of the
essential oil of Micromeria allowed us to clearly
distinguish between sections Pseudomelissa and
Eumicromeria. According to our results the species
of section Pseudomelissa (M. thymifolia,
M. albanica,M. dalmatica and M. pulegium)
contained a large quantity of oil (>0.5%) domi-
nated by oxygenated monoterpenes of the men-
thane type, while the species of section
Eumicromeria (M. croatica,M. juliana,M. cristata
and M. parviflora) had a small quantity of essential
oil (<0.5 %) dominated by various terpene
compounds. 0.5% of essential oil is defined like
boundary value on the basis of the lowest quantity
of essential oil measured in populations of species
of section Pseudomelissa.
Key words: Micromeria,Pseudomelissa,Eumicromeria,
Lamiaceae, chemotaxonomy, essential oils.
The genus Micromeria Benth. includes about
130 species widespread in the Mediterranean
region (Diklic 1974). In the flora of Serbia and
Montenegro this genus is represented by ten
species, seven of which are endemic (Silic
1979).
On the basis of their morphological char-
acteristics and phylogenetic relationships, the
species of the genus Micromeria are grouped
in three sections (Boissier 1879): Cymularia,
Eumicromeria and Pseudomelissa. Boissier
used the term section for classification of
species in the genus Micromeria (Flora Orien-
talis, 1879, pp. 568–575). The species of
Micromeria inhabiting the territory of Serbia
and Montenegro belong to sections Pseudome-
lissa and Eumicromeria.
The species of Micromeria are well known
as aromatic species because they contain con-
siderable quantities of essential oil. The quan-
tity of essential oil (‡0.5%) is one of the
characteristics on the basis of which the species
of this genus have been classified into subfamily
Nepetoideae (El-Gazzar and Watson 1970). It is
well known that the following species possess
this characteristic: Micromeria thymifolia
(Scop.) Fritsch (Savarda et al. 1979; Pavlovic
Pl. Syst. Evol. 255: 1–15 (2005)
DOI 10.1007/s00606-005-0303-y

et al. 1983; Kalodjera et al. 1990, 1994; Mar-
inkovic et al. 2001; Vladimir-Knezevic et al.
2001), M. dalmatica Benth. (Savarda et al.
1979, Pavlovic et al. 1983, Karuza-Stojakovic
et al. 1989), M. pulegium (Rochel.) Benth.
(Pavlovic et al. 1983), M. albanica (Griseb. ex
K. Maly) Silic (Stojanovic et al. 1999, Mar-
inkovic et al. 2001), Micromeria fruticosa (L.)
Druce (Fleisher and Fleisher 1991; Kirimer
1992; Kirimer et al. 1993a, 1993b; Putievsky
et al. 1995; Baser et al. 1996, 1998) and M.
dolichodonta P. H. Davis (Baser et al. 1997a).
However, by examining the chemical composi-
tion of the species of the genus Micromeria as
well as according to data gathered from liter-
ature (Pavlovic et al. 1983; Stanic et al. 1988;
O
¨zek et al. 1992; Baser et al. 1995, 1997b;
Pe
´rez-Alonso et al. 1996; Mastelic et al. 1998)
we have established that certain species contain
only small quantities or even merely traces of
essential oils. These differences in quantity
prompted us to make a detailed examination
of the essential oil characteristics of chosen
populations of different species of the genus
Micromeria on the territory of Serbia and
Montenegro and compare them to the existing
literature data. The number of examined pop-
ulations is directly commensurate to areal size
and width of ecological species valence, in the
examined area. The results will be the basis for
determining the interpopulation, interspecies
and intersection variability of quantity and
composition of essential oil which will point to
the adaptive, and possibly, systematic impor-
tance of these characters.
Material and methods
Plant material. We have investigated the follow-
ing plant material: M. thymifolia from five
different localities, Derventa canyon, Beli Rzav
gorge, Moraca canyon, Semolj and mount Orjen;
M. dalmatica from the area of Kotor; M.
pulegium from Beli Rzav gorge, M. albanica from
Prizren area, M. croatica from Beli Rzav gorge,
M. juliana from three localities, Moraca canyon,
Cijevna canyon and mount Orjen; M. cristata
from Jerma gorge and M. parviflora from three
localities, Moraca canyon, Cijevna canyon and
Rijeka Crnojevica (Fig. 1). The basic characteris-
tics of the 16 mentioned localities are given in
Table 1. The samples were gathered in the
flowering period.
Voucher specimens are kept at the Herbarium
of the Institute of Botany, Faculty of Pharmacy,
University of Belgrade, Serbia and Montenegro.
Steam distillation. Air-dried aerial parts of the
plant were subjected to hydrodistillation for 3 h
using a Clevenger-type apparatus.
Combined GC-MS. GC-MS analysis was car-
ried out using the Hewlett Packard 5973–6890 GC-
MS system operating in EI mode at 70 eV,
equipped with a HP 5MS capillary column
(30 m ·0.25 mm; film thickness 0.25 lm). The
initial temperature of the column was 60
o
C and
was heated to 280
o
C with a rate of 3
o
C/min.
Carrier gas He; flow rate 1 ml/min; split ratio, 1:10.
The injection volume was 1 ll. Relative percentage
amounts were calculated from total ion chromato-
grams (TICs).
The identification of the compounds was based
on the comparison of their Kovats indices (KI),
their retention times (RT) and mass spectra with
those obtained from authentic samples and/or the
MS library (Adams 1995).
Statistical analysis. Cluster analysis represents
the most appropriate method to illustrate the
similarities and dissimilarities of the data matrix.
The dendrogram acquired by Optimal clustering
(Chord distance) emphasizes intergroup hetero-
geneity which is, according to Orloci (1966) and
Pielou (1984), primary characteristic of this clus-
tering method. Cluster analyses were carried out
using FLORA software (Karadzic et al. 1998). In
total, 120 characteristics (quantity of essential oils
and chemical compounds) were analyzed in 16
populations.
Results
The composition and content of essential oils
of the studied species are given in Table 2. The
qualitative composition of the essential oils of
M. thymifolia populations was always similar.
Oxygenated terpenes of the menthane type
dominated in all the oils (>73%). However,
the contribution of certain major components
2 V. Slavkovska et al.: Essential oil and its systematic significance in Micromeria

was varying. In the essential oil from the
Derventa canyon population there was a
considerable presence of piperitone oxide
(63.8%). The proportion of piperitenone oxide
was 9.2%. Piperitenone was present in a very
small quantity (0.4%), while pulegone was
Fig. 1. Locations of the analyzed populations of species of Micromeria. For explanation of numbers compare
Table 1
Table 1. Basic characteristics of the studied populations of Micromeria spp.
Species Code Locality H
a
(m) Coordinates Substrate Climate
M. thymifolia 1 Derventa canyon 500 439N 194E limestone sub-mediterranean
M. thymifolia 2 Beli Rzav gorge 600 437N 193E limestone sub-mediterranean
M. thymifolia 3 Moraca canyon 500 425N 194E limestone sub-mediterranean
M. thymifolia 4 Semolj 700 426N 192E limestone sub-mediterranean
M. thymifolia 5 Mt Orjen 1540 424N 185E limestone sub-med. montane
M. dalmatica Kotor area 800 423N 188E limestone sub-mediterranean
M. pulegium Beli Rzav gorge 600 437N 193E limestone sub-mediterranean
M. albanica Prizren area 500 421N 207E limestone sub-med. montane
M. croatica Beli Rzav gorge 600 437N 193E limestone sub-mediterranean
M. juliana 1 Moraca canyon 300 425N 194E limestone sub-mediterranean
M. juliana 2 Cijevna canyon 45 423N 195E limestone sub-mediterranean
M. juliana 3 Mt Orjen 900 424N 185E limestone sub-med. montane
M. cristata Jerma gorge 900 429N 226E limestone sub-mediterranean
M. parviflora 1 Moraca canyon 300 425N 194E limestone sub-mediterranean
M. parviflora 2 Cijevna canyon 45 423N 195E limestone sub-mediterranean
M. parviflora 3 Rijeka Crnojevica 15 423N 191E limestone sub-mediterranean
a
H = height above sea level
V. Slavkovska et al.: Essential oil and its systematic significance in Micromerica 3

Table 2. The composition (%) of essential oils of the studied populations of Micromeria spp
Populations M. thymifolia M.
dalmatica
M.
pulegium
M.
albanica
M.
croatica
M. juliana M.
cristata
M. parviflora
12345 123 1 2 3
(%) 0.5 0.9 0.5 1.3 0.6 1.7 1.0 0.9 0.12 0.08 0.09 0.04 0.13 <0.05<0.05<0.05
Compounds
MONO-
TERPENES
KI
Hydrocarbons
1. Tricyclene 922 t
c
t t –t t t t – –––– – – –
2. a-Thujene 923 – – – t – – – – – – – – – – – –
3. a-Pinene 929 1.7 1.9 1.0 1.0 1.2 1.8 1.2 1.4 0.6 t t t t t t t
4. Camphene 944 t t t – – t t t 1.4 – – – t t t t
5. Verbenene 960 – – – – – – – – – – – – t – – –
6. Sabinene 967 0.6 t t 0.4 0.1 0.4 t t – – – – – – – –
7. b-Pinene 973 3.0 2.7 4.3 2.2 1.7 2.7 2.4 2.6 1.3 t 1.9 t t t t t
8. Myrcene 988 0.9 0.4 0.9 0.4 0.3 0.8 0.9 0.5 – – – – – – – –
9. p-Mentha-1(7),
8-diene
999–––t –– – – – –––– – – –
10. d-3-carene 1011 – – – – – – – – t – – – – – – –
11. a-terpinene 1015 t – – t – t t t t – – – – – – –
12. o-Cymene 1020 – – – – – – – – 0.6 10.8 ––– –––
13. q-Cymene 1021 t t t t t t t t 1.6 5.3 2.1 1.6 1.1 11.5 t14.6
14. Limonene 1029 11.7 4.5 8.9 3.0 4.4 8.3 6.8 7.0 0.9 – t 0.8 – 1.3 t t
15. (Z)-b-Ocimene 1035 t – t t t t 0.7 t – – – – – – – –
16. (E)-b-Ocimene 1044 t – – t t t t t – – – – – – – –
17. c-terpinene 1056 t t t t t t 1.2 t 0.6 t t – – t t t
18. Terpinolene 1089 – – – t – t t t t – – – – – – –
Oxygenated
monoterpenes
ALDEHYDES
19. a-Campholenal 1126 – – – – – – – – 0.6 t t – 0.7 – – –
20. Myrtenal 1195 – – – – – – – – 0.9 t 4.3 – 1.1 – – –
ALCOHOLS
21. cis-Sabinene
hydrate
1068 t – t – t t 6.4 t – – – – – – – –
22. trans-Sabinene
hydrate
1098 – – – – – t t t – – – – – – – –
4 V. Slavkovska et al.: Essential oil and its systematic significance in Micromeria

23. Linalool 1099 t – – – t – – – t 1.4 1.9 t t 2.0 14.3 t
24. endo-Fenchol 1114 – – – – – – – – – – – – t – – –
25. trans-Pino-
carveol
1139 – – – – – – – – t t 2.6 t – – – –
26. trans-Verbenol 1146 – – – – – – – – – t 4.8 – – – – –
27. Borneol 1165 – – – – – – – – 10.8 3.8 6.3 t 5.7 2.3 t –
28. Terpinen-4-ol 1183 t – 0.5 – – – 2.5 – 0.8 1.5 t t t 0.7 t –
29. q-Cymen-8-ol 1187 – – – – – – – – – t t t t – – –
30. Neo-iso-men-
thol
1189 – – – – – – 2.6 – – – – – – – – –
31. a-terpineol 1194 0.2 t t – 0.3 0.6 0.2 – t – – – 0.8 0.9 3.0 t
32. Myrtenol 1197 – – – – – – – – t – – – – – – –
33. trans-Carveol 1220 – – – – – – – – – – – – t – – –
34. Geraniol 1260 – – – – – – – – – – – – – – t –
35. Thymol 1294 0.8 0.6 0.2 – 0.2 – – – – 3.4 t 7.3 – – t t
36. Carvacrol 1303 – – – – – – 3.5 – – 18.1 ttt – 10.6 t
ETHERS
37. 1,8 Cineole 1031 – – – – – – – – – – – – 5.0 t t t
38. methyl-ether
Carvacrol
1245 – – – – – – – – – t – t – 0.7 t –
OXIDES
39. cis-Linalool
oxide
1072 – – – – – – – – – – – – – t t –
40. trans-Linalool
oxide
1088 – – – – – – – – – – – – – t t –
41. Piperitone
oxide *
1259 63.8 20.0 1.8 – t – 7.4 36.9 –––––1.2–t
42. Piperitenone
oxide
1366 9.2 9.8 16.0 2.4 2.8 0.9 3.6 21.9 0.7 – – 1.2 t – – –
KETONES
43. cis-Thujone 1105 – – – – – – – – – – – – 3.1 – – –
44. trans-Thujone 1117 – – – – – – – – – – – – t – – –
45. Camphor 1145 – – – – – – – – 2.2 – – – 7.5 – – –
46. Menthone 1156 t 0.5 2.7 1.1 8.7 t 4.3 4.5 – t t 1.3 – t t t
12345 123 1 2 3
47. Pinocarvone 1161 – – – – – – – – – t 1.8 t t – – –
48. Isomenthone 1164 t 6.9 t 5.0 8.3 0.6 27.2 t–tt10.1 – – – 7.9
49. cis-Isopulegone 1173 – t t 2.1 2.2 0.5 – – – – – – – – – –
V. Slavkovska et al.: Essential oil and its systematic significance in Micromerica 5

Table 2. (Continued)
Populations M. thymifolia M.
dalmatica
M.
pulegium
M.
albanica
M.
croatica
M. juliana M.
cristata
M. parviflora
12345 123 1 2 3
50. Verbenone 1209 – – – – – – – – – – – – 0.6 – – –
51. Pulegone 1240 t 37.2 6.2 72.3 48.0 12.1 4.0 7.8 t t t 8.1 t t t t
52. Carvone 1244 – – – – – – – – – – – – t – – –
53. Piperitone 1257 – 1.7 24.0 0.9 2.8 3.3 – – – – – – – – – –
54. Piperitenone 1340 0.4 6.3 28.7 4.3 13.9 56.7 1.2 10.0 1.0 t t 0.8 t – – –
ESTERS
55. Bornyl acetate 1285 – – – – – – – – – – – – 4.1 1.1 – t
56. Isobornyl
acetate
1285 – – – – – – – – 2.9 – – – – – – –
57. Isomenthyl
acetate
1318 – – – – – – 3.0 – – – – – – – – –
58. Neo-iso-Iso-
pulegol acetate
1321 – – – – – – 1.1 – – – – – – – – –
59. Isobornyl
formate
1233 – – – – – – – – 1.1 – – – t – – –
SESQUITERPENES
Hydrocarbons
60. a-Cubebene 1351 – – – – – – – – 0.5 – – – – – – –
61. a-Copaene 1373 0.2 – – 0.1 0.3 – t – 0.5 t t – – – – –
62. b-Bourbonene 1382 0.5 – 0.9 0.2 0.3 – 1.2 – 2.4 1.5 2.1 1.1 – 4.9 t t
63. Isolongifolene 1386 – – – – – – – – – – – – 1.3 – – –
64. b-Cubebene 1391 – – – – – – – – t – – – – – – –
65. b-Elemene 1392 t t t t – t t – – t – t t t – –
66. 1,7-di-epi-a-Ce-
drene
1399 – – – – – – – – 0.5 – – – – – – –
67. b-Longipinene 1400 – – – – – – – – – – – – t – – –
68. (Z)-Car- yo-
phyllene
1405 – – – – – – – – – – – – t – – –
69. a-Cedrene 1408 – – – – – – – – t – – – – – – –
70. a-Gurjunene 1414 – – – – – – – – – – – – 1.2 – – –
71. b-Cedrene 1417 – – – – – – – – 0.5 – – – – – – –
72. (E)-Caryo-
phyllene
1423 – – – – – – 4.7 t – 3.7 7.1 5.6 1.9 1.5 t t
73. b-Gurjunene 1427 t t t – – 0.2 t t t t t t – t – t
6 V. Slavkovska et al.: Essential oil and its systematic significance in Micromeria

74. a-Guaiene 1440 – – – – – – – – – – – – t – – –
75. a-Humulene 1452 – – – – – – 1.2 – – t t 0.8 t – – –
76. (E)-b-Farne-
sene
1458 – – – – – – 0.6 – – – – – – – – –
77. allo-Aromaden-
drene
1460 – t t – t – – – – 1.1 3.5 5.3 3.7 – – –
78. cis-Muurola-
4(15)5-diene
1462 – – – – – – – – 1.1 – – – – – – –
79. b-Acoradiene 1465 – – – – – – – – t – – – – – – –
80. c-Muurolene 1475 t t t – t – – – t t t t t – –
81. Germacrene D 1480 1.1 0.5 t 1.0 0.2 3.0 2.7 2.1 0.7 1.1 2.9 5.2 – 2.5 t t
82. ar-Curcumene 1483 – – – – – – – – 1.3 t t 1.3 – – – –
83. cis-b-Guaiene 1487 – – – – – – – – t – – – – – – –
84. Bicyclogerma-
crene
1489 0.4 – – 0.5 – 0.6 0.5 0.5 – – – – – 2.6 t t
85. a-Muurolene 1496 – – – – – – – – – t t t – – – –
86. Germacrene A 1501 – – – – – – – – t – – – – – – –
87. b-Bisabolene 1502 – – – – – – – – – – – – – t t t
88. b-Curcumene 1506 – – – – – – – – – t t t – – – –
89. c-Cadinene 1518 – – – – – t t t 10.9 ttt1.3 – – –
90. d-Cadinene 1523 0.1 0.2 t t t 0.3 t 0.4 – 3.8 3.0 3.7 t 1.8 t t
91. trans-Calame-
nene
1526 – – – – – – – – 3.9 – – – – – – –
92. a-Cadinene 1536 – – – – – – – – 0.4 t t t – – – –
93. Unknown 1554 – – – – – – – – – – – – 17.7 –––
Oxygenated sesquiterpenes
ALCOHOLS
94. endo-1-Bourbo-
nanol
1522 – – – – – – – – 4.3 2.9 2.3 0.9 – – – –
95. (E)-Nerolidol 1565 – – – – – – – – – – – – 3.9 – – –
96. Spathulenol 1572 1.9 2.3 2.0 1.1 1.9 1.3 0.9 2.3 – t t 1.5 11.7 46.7 12.7 19.0
97. Germacrene
D-4-ol
1574 – – – – – – – – – 4.1 4.8 1.4 – – – –
98. Globulol 1591 – – – – – – – – – – – – 6.0 – – –
99. Viridiflorol 1595 – – – – – – – – – – – – t – – –
100. 1,10-di-epi-
Cubenol
1616 – – – – – – – – 1.1 – – – 2.9 – – –
101. Caryophylla-
3,8(13)-dien-
5-b-ol
1635 – – – – – – – – – – – – 1.0 – – –
V. Slavkovska et al.: Essential oil and its systematic significance in Micromerica 7

Table 2. (Continued)
Populations M. thymifolia M.
dalmatica
M.
pulegium
M.
albanica
M.
croatica
M. juliana M.
cristata
M. parviflora
12345 123 1 2 3
102. Isospathulenol 1641 0.1 0.6 0.5 – 0.2 0.5 t t – – – – – – – –
103. epi-a-Muralol 1642 – – – – – – – – – 3.9 3.4 1.6 – – – –
104. a-Muurolol 1647 – – – – – – – – 8.1 – – – – – – –
105. a-Cadinol 1658 t 0.5 t – t t t t 1.6 4.4 6.6 1.9 4.3 – – –
106. Caryophyllenol
II
1674 – – – – – – – – 1.4 – – – 0.6 – – –
OXIDES
107 Caryophyllene
oxide
1583 – – – – – – 1.5 t 24.4 18.1 20.4 15.9 – –––
KETONES
108. 1-nor
Bourbonanone
1560 – – – – – – – – – 1.4 1.9 2.2 – – – –
109. b-Oplopenone 1611 – – – – – – – – 2.9 – – – – – – –
110. Hexahydro-
farnesyl
acetone
1846 – – – – – – – – 0.4 1.6 1.2 2.6 0.5 1.9 5.0 4.4
FATTY ACIDS
111. Hexadecanoic
acid
– – – – – – – – – – t t 1.0 – t 3.9 t
ESTERS
112. Ethyl linoleate – – – – – – – – – – – – – – – 7.9 t
HYDROCARBONS
ALDEHYDES
113. (E)-2-Hexenal – – – – 0.2 – – – – – – – – – – – –
114. (E,E)-2,4-hepta-
dienal
1006 – – – t – – – – – – – – – – – –
ALCOHOLS
115. 3-Octanol 993 t t 0.4 0.3 0.6 0.3 1.6 t – – – – – – – –
PHENYLPRO-
PANOIDS
116. Eugenol 1362 – – – – – – t – – – – – – – – –
8 V. Slavkovska et al.: Essential oil and its systematic significance in Micromeria

OTHERS
117. Benzaldehyde 956 – – – – – t t – – – – – – – – –
118. Dihydroedullan
II
1285 – – – – – – – – – 2.9 4.2 1.3 – – – –
119. b-(E)-Ionone 1485 t t t t – – t t – – – – t – – –
Monoterpene
compounds
92.3 92.5 95.2 95.1 94.9 88.7 80.2 92.6 28.0 44.3 25.7 31.2 29.7 21.7 27.9 22.5
Monoterpene
hydrocarbons
17.9 9.5 15.1 7.0 7.7 14.0 13.2 11.5 7.0 16.1 4.0 2.4 1.1 12.8 t 14.6
Oxygenated
monoterpenes
74.4 83.0 80.1 88.1 87.2 74.7 67.0 81.1 21.0 28.2 21.7 28.8 28.6 8.9 27.9 7.9
Oxygenated
monoterpenes of
the menthane type
73.4 82.4 79.4 88.1 86.7 74.1 51.8 81.1 1.7 t t 21.5 t 1.2 t 7.9
Sesquiterpene
compounds
4.3 4.1 3.4 2.9 2.9 5.9 13.3 5.3 66.9 47.6 59.2 52.0 58.0 61.9 17.7 23.4
Sesquiterpene
hydrocarbons
2.3 0.7 0.9 1.8 0.8 4.1 10.9 3.0 22.7 11.2 18.6 23.0 27.1 13.3 t t
Oxygenated
sesquiterpenes
2.0 3.4 2.5 1.1 2.1 1.8 2.4 2.3 44.2 36.4 40.6 29.0 30.9 48.6 17.7 23.4
Other
compounds
t t 0.4 0.5 0.6 0.3 1.6 t – 2.9 4.2 1.3 – t 11.8 t
Total 96.6 96.6 99.0 98.5 98.4 94.9 95.1 97.9 94.9 94.8 89.1 84.5 87.7 83.6 57.4 45.9
% percentage calculated from TIC data
t – traces
*
correct isomer not identified
V. Slavkovska et al.: Essential oil and its systematic significance in Micromerica 9

found in traces. Pulegone (37.2%) and piperi-
tone oxide (20.0%) were the main compounds
in the essential oil of M. thymifolia from Beli
Rzav gorge. The quantities of piperitenone
oxide and piperitenone corresponded to 9.8%
and 6.3% of total content of essential oil,
respectively. The population from Moraca
canyon had a high content of piperitenone
(28.7%), piperitone (24.0%) and piperitenone
oxide (16.0%), while the concentration of
pulegone amounted to 6.2%. The essential oil
of the Semolj populations contained 72.3% of
pulegone, 4.3% piperitenone, 12.4% piperite-
none oxide and 0.9% of piperitone. There was
no piperitone oxide. The mount Orjen popu-
lation contained essential oil rich in pulegone
(48.0%) and piperitenone (13.9%). Piperitone
and piperitenone oxide were ascertained in
smaller quantities (2.8%). Piperitone oxide
was found in traces. The proportion of sesqui-
terpenes in all M. thymifolia populations
varied between 2.9 and 4.3 %.
Oxygenated terpenes of the menthane type
were dominant (74.1%) in the oil of M. dalma-
atica. The oil was rich in piperitenone (56.7%)
and pulegone (12.1%). The proportion of
sesquiterpenes was 5.9%.
Oxygenated terpenes of the menthane type
were dominant in the oils of M. pulegium
(51.8%). The main compound in the essential
oil was isomenthone (27.2%). Sesquiterpene
compounds were found to contribute 13.3% to
the essential oil content.
The oil of M. albanica contained 81.1% of
oxygenated terpenes of the menthane type.
Piperitone oxide (36.9%), piperitenone oxide
(21.9%) and piperitenone (10.0%) were dom-
inant in the oil. The proportion of sesquiter-
penes was 5.3%.
All four species of sect. Pseudomelissa
were characterized by a higher quantity of
essential oils (0.8%–1.7%) in which monoter-
pene compounds dominated (even higher than
80.2%). Among the monoterpenes oxygenated
monoterpenes of the menthane type had the
highest presence (51.8%–82.0%). They shared
an increased piperitenone, pulegone, piperite-
none oxide content, as well as the absence of
borneol. Sesquiterpene compounds appeared
in a smaller quantity (3.5%–13.3%).
The main components of the M. croatica
essential oil were sesquiterpenes (66.9%).
Caryophyllene oxide (24.4%) and c-cadinene
(10.9%) were dominant among them. The
bicyclic monoterpene, borneol, was also one
of the dominant components (10.8%). Oxy-
genated terpenes of the menthane type were
ascertained in the concentration of 1.7%.
The content of monoterpene (44.3%) and
sesquiterpene (47.6%) compounds in the oil of
the M. juliana population from Moraca canyon
was approximately the same, while sesquiterp-
enes dominated in the oils of the other popu-
lations (59.2% - Cijevna canyon population;
52.0% - Mt Orjen population). The largest
quantity of oxygenated terpenes of the men-
thane type was present in the oil of the Orjen
population (21.5%), while in the other popu-
lations these components were present in
traces. Caryophyllene oxide was the main
component of all the oils. The Moraca canyon
population had a high content of caryophyllene
oxide (18.1%), carvacrol (18.1%) and o-cym-
ene (10.8%). Traces of isomenthone were
found. In the essential oil from the Cijevna
canyon population there was a considerable
presence of caryophyllene oxide (20.4%).
Isomenthone and carvacrol were found in
traces, while no o-cymene was detected in the
essential oil of this population. The Mt. Orjen
population contained essential oil rich in
caryophyllene oxide (15.9%) and isomenthone
(10.1%). There were traces of carvacrol, while
there was no presence of o-cymene.
An unidentified sesquiterpene (KI 1554)
was dominant (17.7%) in the oil of M. cristata.
Spathulenol was also found in a large quantity
(11.7%). A considerable quantity of sesqui-
terpenes (58.0%) was also found in this oil,
while oxygenated menthane monoterpenes
were found in traces.
The essential oil of M. parviflora from
Rijeka Crnojevica had approximately the same
content of monoterpene (22.5%) and sesqui-
terpene (23.4%) compounds, while sesquiter-
penes dominated in the oil of the Moraca
10 V. Slavkovska et al.: Essential oil and its systematic significance in Micromeria

canyon populations (61.9%), and monoter-
penes dominated (27.9%) in the oil of the
Cijevna canyon population. In the essential oil
from the Cijevna canyon population there was
a considerable presence (11.8%) of other
compounds (fatty acids and fatty acid esters).
These compounds were found in traces in the
Rijeka Crnojevica and Moraca canyon popu-
lations.
High content of spathulenol (46.7%) and
p-cymene (11.5%) was found in the oil of the
Moraca canyon population. Linalool (2.0%)
was ascertained in a smaller quantity. Carva-
crol was not present. Linalool (14.3%), spath-
ulenol (12.7%), and carvacrol (10.6%) were
the dominant compounds of the oil from the
Cijevna canyon population. p-Cymene was
found in traces. The Rijeka Crnojevica popu-
lation contained essential oil rich in spathule-
nol (19.0%) and p-cymene (14.6%). Linalool
and carvacrol were found in traces.
The higher content of borneol, p-cymene,
hexahydrofarnesyl acetone and lower content
of piperitone oxide was what the species of the
Eumicromeria section had in common. Also,
they were characterised by a small quantity of
essential oil (0.05–0.13%) in which sesquiter-
penes predominated (34.3%–66.9%), while the
proportion of monoterpene compounds of the
menthane type varied from 0.1%–7.2%.
The optimal clustering (Chord distance)
dendrogram based on a matrix of total char-
acter differences is shown in Fig. 2. The
dendogram Fig. 2a represents the interrelation
of all examined populations. The mountain
populations of the M. thymifolia species from
Orjen and Semolj were the most alike, and the
Beli Rzav gorge population was the one closest
to them. All three populations were character-
ized by pulegone dominance in the oil. The
Derventa Canyon M. thymifolia was the most
similar to the M. albanica species because of
the piperitone oxide domination, while the
Moraca Canyon M. thymifolia was similar to
the M. dalmatica species because of the piperi-
tenone predominance. The Rijeka Crnojevica
and Moraca canyon M. parviflora populations
were closer to the M. cristata species according
to their quantity of spathulenol. The Cijevna
canyon M. parviflora stood apart from the
previous group because of its quantity of
linalool and carvacrol. According to its b-
pinene and myrtenal content, M. juliana from
the Cijevna canyon was similar to M. croatica.
M. juliana from the Moraca canyon was
prominent for its o-cimene and carvacrol
domination, and the Orjen population for its
quantity of isomenthone and pulegone.
The dendogram (Fig. 2b) shows a distinct
division of the species into three groups, the
first of which is clearly separated from the
others. The first group consists of represen-
tatives of section Pseudomelissa (M. thymi-
folia,M. albanica,M. dalmatica and M.
pulegium). The M. thymifolia populations,
although heterogeneous, were closest to M.
albanica because of their domination of
piperitone oxide. M. dalmatica contained
somewhat more a-pinene and the largest
quantity of piperitenone. M. pulegium espe-
cially distinguished itself from the other
species of the section Pseudomelissa by its
higher content of isomenthone, cis-sabinene
hydrate, (E) caryophyllene, carvacrol and
isomenthyl acetate.
The representatives of section Eumicro-
meria are divided in two groups. Because of
the domination of caryophyllene oxide and
presence and content of endo-1-bourbonanol
the species M. croatica and M. juliana are the
most similar. M. cristata and M. parviflora
are between the species of section Pseudome-
lissa section and M. croatica and M. juliana,
and they are characterized by the domination
of spathulenol.
Discussion
The essential oil of Micromeria thymifolia has
been analyzed quite frequently. Our results
referring to the quantity of essential oil agree
with previously published results (Savarda
et al. 1979, Pavlovic et al. 1983, Kalodjera
et al. 1990, Marinkovic et al. 2001, Vladimir-
Knezevic et al. 2001). In works published so
far, in the essential oil of M. thymifolia,
V. Slavkovska et al.: Essential oil and its systematic significance in Micromerica 11

pulegone was found in the largest quantity
(Kalodjera et al. 1993, 1994; Vladimir et al.
1994; Duletic-Lausevic et al. 2001; Marinkovic
et al. 2001). However, in our researches pule-
gone appeared as dominant in the Beli Rzav
gorge, Semolj and Orjen populations, while
piperitone, piperitenone and their epoxides
were the main components in the rest of the
populations.
M. dalmatica had the highest quantity of
essential oil among all the Micromeria species
investigated in this work. Previously published
works confirm the large quantity of oil,
although piperitenone oxide, established as
dominant in earlier researches (Savarda et al.
1979, Karuza-Stojakovic et al. 1989), was
discovered in a very small quantity (0.9%) in
our examination.
In earlier works concerning the quantity of
essential oils of the species M. pulegium, the
same approximate value was found as in our
researches (Pavlovic et al. 1983). In our work,
for the first time the qualitative composition of
the oil of this species was determined as well.
In the works of Stojanovic et al. (1999),
and Marinkovic et al. (2001) epoxyketones
were determined as dominant components in
the M. albanica oil, which was also confirmed
by our research. The antibacterial activity of
the oil is ascribed to the presence of these
compounds, too. However, the first of the
listed studies points out the absence of sesqui-
terpenes which has not been confirmed by our
research.
Earlier publications point out a very small
quantity of oil in M. croatica, in which
sesquiterpenes were dominant while the quan-
tity of terpene hydrocarbons and ketones was
very small (Stanic et al. 1988), which agrees
with our results.
If we compare our results about the oil of
M. juliana with those published previously
(Mastelic et al. 1998) we can conclude
that they are similar in the quantities of
Fig. 2. Dendrogram based on Optimal cluster analysis (Chord distance) of 16 populations (a) and 8 species (b)
of Micromeria sp. essential oils
12 V. Slavkovska et al.: Essential oil and its systematic significance in Micromeria

caryophyllene and borneol and that they differ
in the presence of caryophyllene oxide and the
quantity of a-pinene, b-pinene, limonene, b-
gurjunene and ar-curcumene. Considerable
quantities of a- and b-pinene as well as
bisabolol were determined in the oil of M.
juliana from Greece (Skaltsa et al. 1998).
Previously published results about the
essential oil of M. cristata (Tabanca et al.
2001) differ from ours in the presence of
caryophyllene oxide and trans-verbenol, as
well as in the quantity of dominant compo-
nents. In the work of Tabanca et al. (2001)
borneol was found in the quantity of 27.0%–
29.0%, whereas its quantity in our research
was 5.7%.
The small quantity of essential oil in the
species M. parviflora was also established by
Pavlovic et al. (1983). It is in our work that the
qualitative composition of the oil of this
endemic plant species was determined for the
first time.
The species of section Pseudomelissa that
cannot be found on the territory of Serbia and
Montenegro, such as Micromeria fruticosa
(Fleisher and Fleisher 1991; Kirimer 1992;
Kirimer et al. 1993a, 1993b; Baser et al. 1996),
M. dolichodonta (Baser et al. 1997a) and
M. capitellata Benth. (Baser et al. 1998) were
characterized by a large quantity of oil (0.6%–
4%) and domination of oxygenated terpenes
of the menthane type (78%–91%), especially
pulegone (15%–80%).
Representatives of section Eumicromeria
that do not inhabit our territory contained a
small quantity of oil (0.02%–0.4%), however,
the oils differed in the quantity of dominant
components. M. biflora (D. Don.) Benth. was
rich in the oxygenated monoterpenes neral and
geranial (25.3%–41.3%) (Mallavarapu et al.
1997). The species M. carminea P. H. Davis
(Baser et al. 1995), M. varia Benth., M.
herpyllomorpha Webb. and Berth., M. lachno-
phylla Webb. and Berth. (Pedro et al. 1995,
Pe
´rez-Alonso et al. 1996) also abounded in
oxygenated monoterpenes but of the bornane
(5.0%–26.0%) and pinane type (8.3%–13.9%).
M. myrtifolia Boiss.et Hohen. (O
¨zek et al.
1992), M. cremnophila Boiss. et Heldr. (Baser
et al. 1997b) and M. graeca (L.) Benth. et
Rchb. (Tzakou and Couladis 2001) were the
most similar to the representatives of our flora
of this section in their content of sesquiterpene
compounds, b-caryophyllene (22.6%–42.6%),
caryophyllene oxide (9.9%–17.9%) and ger-
macrene D (7.0%–24.0%).
If one takes into consideration the results of
our research, literature data about the oil of the
selected species and the species that do not live
on the territory of Serbia and Montenegro it is
clear that the variability in quantity and quality
is less among populations of the same species
and that it increases with the taxon rank. It is
higher among the species of the same section
and it is highest among different sections.
The oil composition showed a greater
variability than the quantity, noting that the
variability is less prominent within section
Pseudomelissa. In the oils of the representa-
tives of this section different oxygenated mon-
oterpenes dominated, but they were all of the
menthane type. Heterogeneity was more pro-
nounced in section Eumicromeria, since several
different chemical types were observed within
it.
The quantity of oil is a less variable
characteristic. All representatives of section
Pseudomelissa had a large quantity of essential
oil (>0.5%), and section Eumicromeria had
less than 0.5%.
The relative stability of the quantity and
quality of essential oil within each taxon of
section Pseudomelissa means that these char-
acteristics are probably under a relatively strict
control of natural selection and that they are
of adaptive importance. Consequently we can
say that the characteristics of the essential oils
can also be used as additional characters when
defining the species of this section.
The species of section Eumicromeria as
well as their undoubtable relatives from the
same genus, contain essential oil, but in a
considerably lesser quantity. The composition
of their essential oil is variable, so the
adaptive value of this characteristic is small
as well. Therefore, in the case of the Eumi-
V. Slavkovska et al.: Essential oil and its systematic significance in Micromerica 13

cromeria species, only the small quantity of
essential oil can be taken into consideration as
a taxonomic character.
This work forms part of the research projects
(Project Code 1568 and 1932) sponsored by The
Ministry of Science, Technology and Development
of Republic Serbia.
References
Adams R. P. (1995) Identification of essential oil
components by gas chromatography/mass spec-
troscopy. Allured Publishing Co, Illinois, USA.
Baser K. H. C., Kirimer N., O
¨zek T., Tu
¨men G.
(1995) Essential oil of Micromeria carminea
P. H. Davis. Journal of Essential Oil Research
7: 457–458.
Baser K. H. C., Kirimer N., O
¨zek T., Tu
¨men G.,
Karaer F. (1996) Essential oil composition of
three Labiatae endemic to Turkey (Micromeria
fruticosa (L.) Druce subsp. giresunica P.H.Davis,
Sideritis lycia Boiss. et Heldr. and S. arguta
Boiss. et Heldr.). Journal of Essential Oil
Research 8: 699–701.
Baser K. H. C., Kirimer N., Duman H. (1997a)
Composition of the essential oil of Micromeria
dolichodonta P. H. Davis. Flavour and Fra-
grance Journal 12: 289–291.
Baser K. H. C., Demirc¸ akmak B., Duman H.
(1997b) Composition of the essential oil of
Micromeria cremnophila Boiss. et Heldr. subsp.
amana (Rech. fil) P. H. Davis. Journal of
Essential Oil Research 9: 725–726.
Baser K. H. C., Kirimer N., Tu
¨men G. (1998)
Pulegone-rich essential oils of Turkey. Journal of
Essential Oil Research 10: 1–8.
Boissier E. (1879) Flora Orientalis. Basileae, Gene
`-
vae & Lugduni, pp. 568–575.
Diklic N. (1974) Micromeria Benth.In: Josifovic
M. (ed.) Flore de la Re
´publique Socialiste de
Serbie, Vol. 6. Acade
´mie Serbe des Sciences et
des Arts, Belgrade, pp. 458–462.
Duletic-Lausevic S., Marinkovic B., Sokovic M.,
Marin P., Vukojevic J. (2001) Antifungal effect
of essential oil of two Micromeria species. X
OPTIMA Meeting, 13–19 September, Palermo,
Italy. Book of Abstracts, D/3.
El-Gazzar A., Watson L. (1970) A taxonomic study
of Labiatae and related genera. New Phytologist
69: 451–486.
Fleisher Z., Fleisher A. (1991) The essential oil of
Micromeria fruticosa (L.) Druce subsp. barbata
(Boiss. et Kotschy) P.H.Davis. Aromatic plants
of the Holy Land and the Sinai, Part VII.
Journal of Essential Oil Research 3: 477–479.
Kalodjera Z., Vladimir S., Petricic J. (1990) Chem-
ical investigation of Micromeria thymifolia
(Scop.) Fritsch. Lekovite sirovine 9: 59–65.
Kalodjera Z., Pepeljnjak S., Vladimir S. (1993)
Antibacterial and antifungal activity of Micro-
meria thymifolia. Pharmazie 48: 311–313.
Kalodjera Z., Pepeljnjak S. V., Blazevic N. (1994)
Antimicrobial activity of essential oil from
Micromeria thymifolia (Scop.) Fritsch. Pharma-
zie 49 (5): 376–377.
Karadzic B., Saco-Jovanovic V., Jovanovic Z.,
Popovic R. (1998) ‘‘FLORA’’ a database and
software for floristic and vegetation analyzes. In:
Tsekos I., Moustakas M. (eds.) Progress in
Botanical Research. Kluwer Academic Press,
Dordrecht, pp. 69–72.
Karuza-Stojakovic Lj., Pavlovic S., Zivanovic P.,
Todorovic B. (1989) Parallel study of the essential
oil configurations of populations of Micromeria
dalmatica Benth. species from the mountain
Orjen. Archive of Pharmacy 39(3): 59–63.
Kirimer N. (1992) The essential oil of Micromeria
fruticosa (L.) Druce ssp. brachycalyx P.H.Davis.
Journal of Essential Oil Research 4: 521–522.
Kirimer N., Tu
¨men G., O
¨zek T., Baser K. H. C.
(1993a) The essential oil of Micromeria fruticosa
(L.) Druce subsp. barbata (Boiss. & Kotschy) P.
H. Davis of Turkish Origin. Journal of Essential
Oil Research 5: 79–80.
Kirimer N., Tu
¨men G., O
¨zek T., Baser K. H. C.,
Harmandar M. (1993b) The essential oil of
Micromeria fruticosa (L.) Druce subsp. serpylli-
folia (Bieb.) P. H. Davis. Journal of Essential Oil
Research 5: 199–200.
Mallavarapu G. R., Srinivasaiyer R., Karna S.
(1997) Composition of the essential oil of
Micromeria biflora. Journal of Essential Oil
Research 9: 23–26.
Marinkovic B., Knezevic-Vukcevic J., Marin D. P.,
Sokovic M., Duletic-Lausevic S. (2001) Antibac-
terial activity of essential oil of Micromeria
thymifolia and M. albanica (Lamiaceae). X
OPTIMA Meeting, 13–19 September, Palermo,
Italy. Book of Abstracts, D/11.
Mastelic J., Kustrak D., Milos M. (1998) Essential
oil composition of Micromeria juliana (L.)
14 V. Slavkovska et al.: Essential oil and its systematic significance in Micromeria

Benth. ex Rchb. 29
th
International symposium
on Essential Oils, 6–9 September, Frankfurt am
Main, Germany. Abstracts, P5-3.
Orloci L. (1966) Geometric models in ecology. I.
The theory and application of some ordination
methods. Journal of Ecology 54: 193–215.
O
¨zek T., Kirimer N., Baser K. H. C. (1992)
Composition of the essential oil of Micromeria
myrtifolia Boiss. et Hohen. Journal of Essential
Oil Research 4: 79–80.
Pavlovic S., Ivanic R., Savin K., Zivanovic P.,
Jancic R., Milinkovic D., Vujcic S. (1983)
Essential oil, tanins and flavonoids from wild
species of genus Micromeria,Satureja,Calamin-
tha,Teucrium and Acinos occuring in Yugosla-
via. Archive of Pharmacy 33 (6): 287–291.
Pedro L. G., Figueiredo A. C., Barroso J. G.,
Fontinha S. S., Looman A., Scheffer J. J. C.
(1995) Composition of the essential oil of
Micromeria varia Benth. ssp. thymoides (Sol. ex
Lowe) Pe
´rez var. thymoides, an endemic species
of the Madeira archipelago. Flavour and Fra-
grance Journal 10(3): 199–202.
Pe
´rez-Alonso M. J., Velasco-Neguerela A.,
Gil-Pinilla M., Pe
´rez De Paz P. L., Vallejo C.
G., Esteban J. L. (1996) The volatiles of five
Micromeria species endemic to the Canary
Islands. Biochem. Syst. Ecol. 24(6): 571–
576.
Pielou E. C. (1984) Interpretation of ecological
data. Primer of ordination and classification.
Wiley Interscience, New York.
Putievsky E., Dudai N., Ravid U., Katzir I.,
Michaelovich Y., Zuabi E., Saadi D. (1995)
Morphology, phenology and essential oil of
Micromeria fruticosa (L.) Druce in different
seasons. Jornal of Herbs, Species & Medicinal
Plants 3 (3): 27–34.
Savarda L. A., Pavlovic S., Jancic R., Kuznjecova
A. G. (1979) Uporedna proucavanja ekofiziolo-
skih osobina vrsta Micromeria thymifolia (Scop.)
Fritsch i Micromeria dalmatica Benth. Matica
Srpska 56: 179–202.
Silic C. (1979) Monographie der Gattungen Satureja
L., Calamintha Miller, Micromeria Benth., Acinos
Miller und Clinopodium L. in der Flora Jugoslaw-
iens, Zemaljski Muzej BiH, Sarajevo, pp. 172–
262.
Skaltsa H. D., Lazaris D. M., Loukis A. E. (1998)
Composition of the essential oil of Satureja
juliana (L.) Benth. ex Rchb. from Greece.
Journal of Essential Oil Research 10(6): 641–641.
Stanic G., Kalodjera Z., Petricic J., Todoric A.,
Blazevic N. (1988) Essential oil content and
composition of Micromeria croatica (Pers.)
Schott and Micromeria thymifolia (Scop.) Fritsch.
Acta Pharmaceutica Jugoslavica 38: 251–254.
Stojanovic G., Palic I., Ursic-Jankovic J., Vajs V.,
Djokovic D. (1999) Chemical composition of the
essential oil of Micromeria albanica (Griseb. ex
K. Maly) Silic. Journal of Essential Oil Research
11: 785–787.
Tabanca N., Kirimer N., Demirci B., Demirci F.,
Baser K. H. C. (2001) Composition and antimi-
crobial activity of the essential oil of Micromeria
cristata subsp. phrygia and the enantiomeric
distribution of borneol. J. Agric. Food Chem. 49
(9): 4300–4303.
Tzakou O., Couladis M. (2001) The essential oil of
Micromeria graeca (L.) Benth. et Rchb. growing
in Greece. Flavour and Fragrance Journal 16(2):
107–109.
Vladimir S., Kalodjera Z., Pepeljnjak S., Blazevic
N., Hazler K. (1994) Activity of essential oil and
ethanolic extract isolated from Micromeria
thymifolia (Scop.) Fritsch. Periodicum Biologo-
rum 4: 383–385.
Vladimir-Knezevic S., Blazevic N., Kalodjera Z.
(2001) Seasonal variations in the content and
composition of the essential oil of Micromeria
thymifolia (Scop.) Fritsch. Acta Pharm. 51:
147–151.
Addresses of the authors: Violeta Slavkovska,
Branislava Lakusic, Radisa Jancic, Department of
Botany, Faculty of Pharmacy, University of Bel-
grade Vojvode Stepe 450, 11000 Belgrade, Serbia
and Montenegro. Maria Couladis, Olga Tzakou,
Department of Pharmacognosy and Chemistry of
Natural Products, School of Pharmacy, University
of Athens, Panepistimiopolis Zografou, 15771
Athens, Greece. Srdjan Bojovic (correspondence)
(e-mail: bojos@ibiss.bg.ac.yu), Institute for Bio-
logical Research ‘‘Sinisa Stankovic’’ 29 Novembra
142, 11060 Belgrade, Serbia and Montenegro.
Milica Pavlovic, Department of Pharmacognosy,
Faculty of Pharmacy, University of Belgrade,
Vojvode Stepe 450, 11000 Belgrade, Serbia and
Montenegro.
V. Slavkovska et al.: Essential oil and its systematic significance in Micromerica 15