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Essential Oils of Micromeria dalmatica Benth., a Balkan Endemic Species of Section Pseudomelissa by Regina Karousou* a ), Effie Hanlidou a ), and Diamanto Lazari b ) a
) Laboratory of Systematic Botany and Phytogeography, School of Biology, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki (phone: þ 30-2310-998282; fax: þ 30-2310-998295; e-mail:
[email protected]) b ) Department of Pharmacognosy-Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki
The essential oils of 13 Greek populations of Micromeria dalmatica, a Balkan endemic species and member of the section Pseudomelissa, were examined for the first time. Among the studied populations, two main oil types could be distinguished. Type I was found to be rich in b-pinene, limonene, and germacrene D (accounting for 55.6 – 70.2% of the total oil), and Type II was characterized by the preponderance of p-menthane compounds (accounting for 64.2 – 89.9% of the oil). The latter oil type could be further divided into two subtypes, one comprising oils with predominance of piperitenone and piperitenone oxide and another composed of oils containing high proportions of pulegone, menthone, and isomenthone. The abundance of p-menthane compounds is a common feature of the oils of all members of the section Pseudomelissa studied to date. However, the existence of oils of Type I has not been previously reported for M. dalmatica, neither for other members of the section Pseudomelissa.
Introduction. – Micromeria dalmatica Benth. [syn. Clinopodium dalmaticum (Benth.) Bruchler & Heubl., Satureja dalmatica (Benth.) Nyman] is a Balkan endemic species occurring in Montenegro, Bulgaria, and Greece [1]. It is a member of the old-world section Pseudomelissa Benth., comprising 20 taxa, among which nine are endemic to the Balkan Peninsula and Anatolia [1] [2]. In Greece, M. dalmatica is confined in the northeastern part of the mainland (East Macedonia and Thrace). Several taxa of the section Pseudomelissa are used in the traditional medicine as mint alternatives, due to their prominent mint-like odor [3 – 7]. The potential economic value of the plants attracted the researchers interest, resulting in several publications on their essential-oil composition. It has been found that, besides the fluctuations in the quantitative composition, in all cases, the essential oils of the examined taxa were characterized by the preponderance of C(3)-oxygenated p-menthane compounds [3 – 5] [8 – 23]. Moreover, it has been proposed that the presence of p-menthane compounds might have a taxonomic significance, since it might allow the distinction of section Pseudomelissa from section Eumicromeria, respectively [18]. Publications regarding M. dalmatica oils are scarce, and only four wild growing populations (three from Montenegro and one from Bulgaria) have been studied to date [16] [18] [20] [23]. Thus, this study presents, for the first time, the chemical profile of 13 Greek M. dalmatica populations, aiming to increase the knowledge of the species essential-oil diversity. The obtained results were further compared to the published 2012 Verlag Helvetica Chimica Acta AG, Zrich
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data concerning the non-Greek populations of M. dalmatica and other taxa of the section Pseudomelissa studied. Results and Discussion. – Thirteen M. dalmatica populations were examined, representing the species range in the country. The collected plants were growing at altitudes between 180 and 1400 m in the following habitat types: pseudomaquis (mixed deciduous and evergreen scrub formations, dominated by Quercus coccifera L.), Ostrya, Carpinus, and mixed thermophilous forests (mixed deciduous scrub formations, dominated by Carpinus orientalis Miller), and openings of Greek Pinus sylvestris forests. The collection localities and the corresponding altitudes and habitat types are presented in Table 1 and Fig. 1.
Table 1. Collection Locality and Essential-Oil Yield of the Micromeria dalmatica Populations Examined Population
Collection locality a )
Habitat type b )
Oil yield [%] c ) 0.4
800
Ostrya, Carpinus, and mixed thermophilous forests (925A) d ) Pseudomaquis (5350) e ) Ostrya, Carpinus, and mixed thermophilous forests (925A) d ) Ostrya, Carpinus, and mixed thermophilous forests (925A) d ) Openings of Greek Pinus sylvestris forest (9440) Openings of Greek P. sylvestris forest (9440) Pseudomaquis (5350) e )
900
Pseudomaquis (5350) e )
0.5
Ostrya, Carpinus, and mixed thermophilous forests (925A) d ) Ostrya, Carpinus, and mixed thermophilous forests (925A) d ) Ostrya, Carpinus, and mixed thermophilous forests (925A) d ) Ostrya, Carpinus, and mixed thermophilous forests (925A) d ) Ostrya, Carpinus, and mixed thermophilous forests (925A) d )
0.6
Altitude [m]
2 3
Prefecture of Drama Mt Orvilos, near the village of Katafyto Mt Falakro Mt Falakro
4
Mt Falakro
1010
5
Mt Falakro, near the ski center Mt Falakro, near the ski center Mt Falakro, between ski center and village Volakas Mt Falakro, between ski center and village Volakas Prefecture of Xanthi Near the town Stavroupoli
1300
1
6 7 8
9
830 435 900
1400
180m
10
Outside the village Sminthi 180m
11
Outside the village Echinos 300m
12
Near the junction to the village Dimario Outside the village Thermes
13 a
520 760
0.2 0.7 0.7 0.4 1.4 0.5
1.3 1.4 1.2 1.5
) The geographical location of the collection localities is shown in Fig. 1. b ) The classification of the habitat type, with habitat codes given in parentheses, is according to [24]. c ) The essential-oil yield is given in % (v/w) based on the dry weight of the plant material, i.e., ml/100 g dry weight. d ) Mixed deciduous scrub formations, dominated by Carpinus orientalis Miller. e ) Mixed deciduous and evergreen scrub formations, dominated by Quercus coccifera L.
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Fig. 1. Geographical location and main essential-oil components of the 13 Micromeria dalmatica populations studied. Oils rich in b-pinene, limonene, and germacrene D are indicated by open circles, oils rich in piperitenone oxide and piperitenone by open squares, and oils rich in pulegone, menthone, and isomenthone by solid squares.
Essential-Oil Content and Composition. The essential-oil content of the studied populations ranged from 0.2% (v/w), based on the dry weight of the plant material, for Population 2 up to 1.5% for Population 13 (Table 1). Plants collected in localities with the habitat type Ostrya, Carpinus, and mixed thermophilous forests had higher contents of essential oil (1.0 0.4%) than those growing in localities with the habitat types pseudomaquis and Greek Pinus sylvestris forests (0.6 0.5%). In total, 57 compounds have been identified, accounting for 97.2 – 99.4% of the oil composition (Table 2). Among them, nine components, viz., the monoterpene hydrocarbons b-pinene and limonene, the C(3)-oxygenated p-menthane compounds menthone, isomenthone, pulegone, piperitone, piperitenone, and piperitenone oxide, and the sesquiterpene germacrene D, were identified as the main oil constituents, i.e., they were detected at a content of at least 10.0% in at least one of the analyzed oils (Table 2). They constituted the bulk of the oil, with the sum of their contents ranging from 69.9 to 98.6%. However, a considerable variation in their amounts was observed among the different populations. Willing to understand the interpopulation relationships with respect to their essential-oil composition, a principal component analysis (PCA) was applied using as variables the contents of the nine main components. In Fig. 2, the eigenvalues of the 13 populations are presented along the first two components, accounting for 72.3% of the total variance. As can be seen, along the principal component 1 (PC1), accounting for 47.6% of the total variance, except piperitenone oxide, all the p-menthane compounds were negatively related to b-pinene, limonene, and germacrene D. As a result, two groups of populations were distinguished. Group A comprised four populations (Populations 1, 2, 8, and 9) having oils rich in b-pinene (29.4 6.9%) and limonene (22.6 3.4%), containing also considerable amounts of germacrene D (11.8 2.1%), and low contents of p-menthane compounds (mean of their sum ¼ 9.2 6.7%). Group B was composed of nine populations (Populations 3 – 7 and 10 – 13) with essential oils characterized by the preponderance of C(3)-oxygenated p-menthane
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Table 2. Chemical Composition of the Essential Oils Obtained from the Aerial Parts of Micromeria dalmatica Populations 1 – 13, Growing Wild in Different Localities of Northeastern Greece Compound a )
AI b ) Content [%] c ) e
a-Thujene a-Pinene Camphene Sabinene b-Pinene Oct-1-en-3-ol Myrcene Octan-3-ol a-Phellandrene a-Terpinene p-Cymene Limonene 1,8-Cineole cis-Ocimene trans-Ocimene g-Terpinene cis-Sabinene hydrate Terpinolene Octan-3-ol acetate Menthone Isomenthone Benzyl acetate d-Terpineol Terpinen-4-ol a-Terpineol Myrtenal 2,3-Dimethylbenzofuran Coahuilensol methyl ether ( Z )-Hex-3-enyl 3-methylbutanoate Pulegone Carvone Carvacrol methyl ether Piperitone cis-Piperitone oxide Dec-9-en-1-ol Neomenthyl acetate Thymol Carvacrol Myrtenyl acetate Piperitenone Piperitenone oxide a-Copaene
925 930 945 970 972 980 991 997 1000 1017 1024 1028 1026 1038 1048 1058 1067 1086 1125 1152 1163 1163 1166 1176 1190 1195 1210
Identification d )
1 )
2
3
4
5
6
7
8
9
10
11
12
13
0.2 2.9 0.1 4.9 34.1 0.2 1.0 0.2 0.3 tr n.d 25.5 3.2 0.2 0.3 0.1 0.1 0.1 0.1 0.7 0.3 n.d. n.d 0.1 tr 0.1 n.d
0.2 2.8 n.d. 9.8 33.9 n.d. 1.6 n.d. 0.3 n.d. 0.4 25.0 5.1 0.3 0.5 0.8 n.d. n.d. 0.4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
0.1 0.9 n.d. 1.2 10.0 0.2 0.6 0.2 0.1 n.d. n.d. 11.3 0.9 n.d. n.d. 0.1 n.d. n.d. n.d. 15.7 7.7 n.d. n.d. n.d. n.d. n.d. n.d.
0.1 0.9 n.d. 1.3 8.4 0.3 0.5 0.1 tr n.d. tr 12.0 0.5 n.d. 0.1 0.1 tr tr tr 18.2 17.4 n.d. n.d. n.d. n.d. n.d. n.d.
tr 0.8 n.d. 0.7 9.0 n.d. 0.4 n.d. tr n.d. n.d. 9.6 0.8 0.1 0.2 0.1 tr tr n.d. 21.0 0.6 tr n.d. n.d. n.d. n.d. n.d.
0.3 n.d. n.d. 0.3 2.9 n.d. 0.3 n.d. n.d. n.d. n.d. 4.1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 33.3 24.7 n.d. n.d. n.d. n.d. n.d. n.d.
n.d. 0.6 n.d. 1.5 9.7 n.d. 0.6 n.d. n.d. n.d. n.d. 10.5 0.7 n.d. n.d. n.d. n.d. n.d. n.d. 0.3 24.6 n.d. n.d. n.d. n.d. n.d. n.d.
0.2 1.8 tr 5.0 19.4 0.1 1.0 0.1 0.4 0.1 0.2 21.6 5.3 0.2 0.2 0.5 0.1 0.1 n.d. n.d. 0.4 n.d. n.d. 0.1 n.d. n.d. n.d.
0.2 2.7 0.1 3.2 30.2 0.2 1.1 0.1 0.2 tr 0.1 18.1 3.2 0.2 0.2 0.2 0.1 0.1 0.1 n.d. n.d. n.d. 0.1 0.1 0.1 n.d. n.d.
tr 0.7 n.d. 0.3 0.9 0.2 0.5 n.d. n.d. tr 0.3 3.4 n.d. n.d. n.d. 0.2 n.d. n.d. n.d. 5.1 0.5 tr n.d. n.d. n.d. n.d. n.d.
n.d. 0.8 n.d. 0.5 1.9 0.1 0.6 n.d. n.d. n.d. n.d. 12.1 n.d. tr tr n.d. n.d. n.d. n.d. 14.1 0.4 n.d. n.d. n.d. n.d. n.d. n.d.
tr 1.0 n.d. 0.5 2.2 0.2 0.7 0.1 tr n.d. tr 11.8 0.1 tr tr tr tr tr n.d. 1.4 n.d. n.d. n.d. n.d. n.d. n.d. tr
n.d. 0.7 n.d. 0.3 0.9 0.2 0.5 tr n.d. n.d. n.d. 6.0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 16.8 4.4 n.d. n.d. n.d. n.d. n.d. n.d.
0.1
0.2
0.3
n.d. tr
1236 n.d
n.d. n.d. n.d. n.d. n.d. n.d. 0.1 0.1
1238 0.8 1241 n.d 1243 n.d
n.d. 41.0 29.1 23.7 24.8 37.8 0.4 n.d. 73.5 46.4 12.0 45.3 AI, MS, Co-GC n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. AI, MS, Co-GC n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.2 n.d. n.d. n.d. AI, MS
2.4 0.4 1.2 n.d. n.d n.d. n.d n.d. n.d n.d. n.d n.d. n.d n.d. 1.8 tr 1.0 0.3 0.2 0.2
1.5 0.5 n.d. n.d. n.d. n.d. n.d. 1.4 0.2 0.1
2.6 n.d. n.d. n.d. n.d. n.d. n.d. 0.6 1.0 0.1
0.6 tr
8.6 2.2 n.d. 0.1 0.1 tr 0.1 6.0 2.1 0.3
6.5 n.d. n.d. n.d. n.d. n.d. n.d. 0.6 n.d. n.d.
n.d. 0.1 tr
MS MS, Co-GC MS MS MS, Co-GC MS MS, Co-GC MS MS MS MS, Co-GC MS, Co-GC MS, Co-GC MS MS MS, Co-GC MS MS MS MS, Co-GC MS MS MS MS, Co-GC MS MS, Co-GC MS
1214 0.1
1250 1250 1263 1275 1291 1300 1325 1340 1366 1375
0.1
AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI, AI,
3.4 n.d. n.d. n.d. n.d. n.d. n.d. 0.9 n.d. n.d.
10.0 2.5 n.d. n.d. tr n.d. n.d. 1.6 1.9 0.4
2.6 5.1 n.d. n.d. tr n.d. n.d. 1.5 10.8 0.3
0.1 AI, MS
n.d. n.d. n.d. n.d. AI, MS
0.6 n.d. n.d. n.d. n.d. 3.6 n.d. 8.5 0.1 n.d.
4.7 n.d. n.d. n.d. n.d. n.d. n.d. 16.3 0.4 n.d.
8.2 8.1 0.2 n.d. 0.1 n.d. n.d. 21.9 27.4 0.1
10.5 n.d. n.d. n.d. n.d. n.d. n.d. 11.4 1.2 n.d.
AI, AI, AI, AI, AI, AI, AI, AI, AI, AI,
MS MS MS MS MS, Co-GC MS MS MS MS MS
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Table 2 (cont.) Compound a )
AI b ) Content [%] c ) e
1 ) 2 b-Bourbonene b-Elemene b-Caryophyllene b-Copaene Guaia-6,9-diene ( E )-b-Farnesene cis-Muurola-4(14), 5-diene Germacrene D Bicyclogermacrene b-Bisabolene g-Cadinene d-Cadinene Spathulenol a-Cadinol Germacra-4(15),5, 10(14)-trien-1a-ol Total identified
3
Identification d ) 4
5
6
7
8
1384 1.8 2.1 1391 0.3 n.d. 1419 0.4 0.3 1429 0.3 tr 1444 n.d n.d. 1456 0.4 tr 1467 n.d n.d.
0.4 0.1 0.1 0.1 n.d. 0.1 n.d.
0.5 0.1 0.1 0.1 n.d. tr n.d.
2.8 0.2 0.3 0.3 0.1 0.1 0.1
0.3 n.d. n.d. n.d. n.d. n.d. n.d.
0.5 n.d. n.d. n.d. n.d. n.d. n.d.
2.7 0.4 0.5 0.4 n.d. 0.2 n.d.
1481 9.4 11.3 1496 1.5 2.4 1505 n.d n.d. 1514 0.1 tr 1524 0.3 0.2 1578 0.5 0.5 1656 0.1 n.d. 1687 0.2 n.d.
4.5 0.2 n.d. tr 0.1 0.1 n.d. n.d.
4.0 0.2 n.d. tr 0.1 tr n.d. n.d.
5.6 1.1 0.1 tr 0.2 0.4 n.d. n.d.
1.7 tr n.d. n.d. n.d. n.d. n.d. n.d.
5.3 0.8 n.d. n.d. n.d. n.d. n.d. n.d.
14.6 3.7 n.d. 0.1 0.3 0.5 n.d. n.d.
9
10
11
12
13
1.4 tr 0.3 n.d. 0.4 n.d. 0.2 n.d. n.d. n.d. 0.1 n.d. n.d. n.d.
0.1 n.d. n.d. n.d. n.d. n.d. n.d.
0.1 tr tr n.d. n.d. n.d. n.d.
0.1 n.d. n.d. n.d. n.d. n.d. n.d.
AI, MS AI, MS AI, MS, Co-GC AI, MS AI, MS AI, MS AI, MS
11.9 2.4 n.d. 0.1 0.2 0.1 n.d. n.d.
0.6 tr n.d. n.d. n.d. n.d. n.d. n.d.
1.5 0.6 n.d. n.d. tr 0.1 n.d. n.d.
0.8 0.1 n.d. n.d. n.d. 0.1 n.d. n.d.
AI, MS AI, MS AI, MS AI, MS AI, MS AI, MS AI, MS AI, MS
0.2 n.d. n.d. n.d. n.d. 0.2 n.d. n.d.
97.5 98.8 99.4 98.5 98.4 99.8 97.2 97.2 98.1 99.4 99.2 98.6 99.4
a
) Compounds are listed in the order of elution from an HP-5 MS capillary column. b ) AI: Arithmetic index determined relative to a homologous series of n-alkanes (C9 – C25 ) on a HP-5 MS capillary column. c ) The content of the essential oil components is given as percentage of the total oil composition; tr: trace ( < 0.05%); n.d.: not detected. d ) Identification method: AI, arithmetic index; MS, mass spectrum; Co-GC, coinjection with authentic compound. e ) For the collection localities and the habitat conditions of the Micromeria dalmatica Populations 1 – 13, cf. Table 1 and Fig. 1.
compounds (mean of their sum ¼ 76.3 11.1%) and by low contents of b-pinene, limonene, and germacrene D (5.1 4.0, 9.0 3.5, and 2.7 2.1%, resp.). No obvious relation between the two groups formed and the geographic origin of the examined populations was observed (Fig. 1). However, our results showed that the plants of Group A presented a significantly lower essential-oil content than those belonging to Group B (independent sample t-test with p ¼ 0.002). The differences in the essential-oil contents as well as in the oil compositions resulted in an evident diversity in the plant odors. Thus, plants belonging to Group A were almost scentless, while the plants of Group B emitted a prominent mint-like odor. Distinction of Oil Types within M. dalmatica. Aiming to compare our findings with the essential-oil composition of the other, non-Greek M. dalmatica populations previously studied [16] [18] [20] [23], a hierarchical cluster analysis was carried out (Fig. 3). The chemical profiles of the three main population clusters formed can be described as follows: Cluster I comprised the four Greek populations belonging to Group A described above for the PCA (Fig. 2), i.e., Populations 1, 2, 8, and 9 (Table 1 and Fig. 1), with oils characterized by the preponderance of b-pinene, limonene, and germacrene D (sum of the three compounds ¼ 55.6 – 70.2%) and by low contents of the p-menthane
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Fig. 2. Ordination scores obtained by principal component analysis of the essential oils of the 13 Greek Micromeria dalmatica populations studied. The variables used are represented as vectors from the origin. For the collection localities and the habitat conditions of the Micromeria dalmatica Populations 1 – 13, cf. Table 1 and Fig. 1.
compounds (sum of b-pinene, limonene, and germacrene D contents/sum of contents of p-menthane compounds > 3). Cluster II was composed of twelve populations having as main oil components the p-menthane compounds (sum ¼ 64.2 – 89.9%), while the oil content of the monoterpene hydrocarbons and germacrene D was very low (sum of b-pinene, limonene, and germacrene D contents/sum of contents of p-menthane compounds 0.4). The fluctuations in the relative amounts of the individual p-menthane constituents allowed a further distinction of two subgroups of populations. Cluster IIa included one Greek population (Population 12; Fig. 1) and two populations from Montenegro (Mng1 [16] and Mng2 [23]), with oils rich in piperitenone oxide (31.5 8.9), followed by high amounts of piperitenone (17.9 6.7) and pulegone (18.2 7.6). The sum of the three components ranged from 61.3 up to 73.9%, while the other p-menthane compounds were detected in small amounts ( < 10%). Oils with a similar composition have also been reported from other taxa of the section Pseudomelissa, viz., M. fruticosa (L.) Druce subsp. fruticosa [14] and M. thymifolia (Scop.) Fritsch [13] [15]. Cluster IIb comprised eight of the Greek populations examined in the present study (Populations 3 – 7, 10, 11, and 13; Fig. 1) and one Bulgarian population (Bu) [20], having oils rich in pulegone (15.2 – 73.5%) and menthone (not detected – 33.3%) and/or
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Fig. 3. Hierarchical cluster analysis of the essential oils of wild growing Micromeria dalmatica populations. The nearest neighbor method based on the squared Euclidian distance was used. The main compounds of the essential oils for the population clusters were identified as follows: Cluster I, bpinene, limonene, and germacrene D; Cluster IIa, piperitenone oxide and piperitenone; Cluster IIb, pulegone, menthone, and isomenthone; Cluster III, piperitenone. Populations: Populations 1 – 13, data from the present study; Mng1, Mng2, and Mng3, populations from Montenegro ([16], [23], and [18], resp.); Bu, Bulgarian population [20].
isomenthone (not detected – 24.7%). In addition, high amounts of piperitone and piperitenone were found in Population 13 (10.5 and 21.9%, resp.), while a high percentage of trans-p-menthane-3-one (15.8%) was found in the Bulgarian population. A similar essential-oil composition has been frequently encountered in other taxa of the section Pseudomelissa, namely in M. abyssinica Hochst [8], M. cilicica Hausskn. [4], M. dolichodonta P.H.Davis [3], M. fruticosa ssp. brachycalyx P.H.Davis [9], M. fruticosa ssp. fruticosa [14] [19] [22], M. fruticosa ssp. giresunica P.H.Davis [12], and M. thymifolia [11] [15] [16] [18] [23]. However, pulegone contents higher than 70%, as those found in the present study for Population 10, were reported only twice, viz., for M. fruticosa ssp. barbata P.H.Davis from Turkey [3] and for M. thymifolia from Montenegro [18]. Cluster III consisted of only one population from Montenegro, Mng3 [18], having oils rich in piperitenone (56.7%), followed by pulegone (12.1%). The amounts of the other p-menthane compounds and those of the monoterpene hydrocarbons b-pinene and limonene as well as of germacrene D were low ( < 11.0%). A similar oil composition has been reported for M. congesta Boiss. & Hausskn. ex Boiss. [3], M. fruticosa ssp. fruticosa [5], and M. fruticosa ssp. serpyllifolia (M.Bieb.) P.H.Davis [10]. Conclusions. – Our results bring into light new evidence for the essential-oil composition of M. dalmatica, contributing to a better circumscription of its chemical
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diversity. Two main oil types were distinguished. Type I was characterized by the preponderance of the monoterpene hydrocarbons b-pinene and limonene and of the sesquiterpene germacrene D, while Type II was found to be rich in p-menthane compounds. The abundance of p-menthane compounds is common to the oils of all Micromeria taxa of section Pseudomelissa studied to date, while the occurrence of an oil type as Type I was reported for the first time here for M. dalmatica as well as for all taxa of the section Pseudomelissa. Although our findings are not in disagreement with the suggestion that taxa of the sections Pseudomelissa and Eumicromeria might be distinguished by the presence or absence, respectively, of p-menthane compounds, they showed that a more complicated variation pattern exists in section Pseudomelissa. Further study is needed to reveal whether an oil type as Type I is species specific of M. dalmatica or whether it is also found in other members of the section Pseudomelissa. Experimental Part Plant Material. The aerial parts of Micromeria dalmatica plants were collected from 13 wild growing populations in the northeast of Greece (Table 1 and Fig. 1). The sample of each population consisted of at least ten randomly collected individuals. The plants were collected during the flowering stage in August, 2011. The habitat-type classification was done according to [24]. Voucher specimens have been deposited with the Herbarium of the Institute of Systematic Botany and Phytogeography, Aristotle University of Thessaloniki (TAU). Essential-Oil Isolation. The collected plant material was air-dried at r.t. for 10 d. Then it was grossly pulverized and subjected to hydrodistillation for 2 h using a Clevenger-type apparatus. The oil yield was expressed as ml/100g of dry weight of the plant material. GC/MS Analysis. The GC/MS analyses of the essential oils were performed with a Shimadzu GC2010-GCMS-QP2010 system equipped with a split/splitless injector and a fused silica HP-5 MS cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm). The oven temp. was programmed rising from 50 to 2908 at 48/min; injector temp., 2308; carrier gas, He (1.0 ml/min); ionization voltage, 70 eV; injection volume, 1.0 ml. Arithmetic indices (AIs) for all compounds were determined using a homologous series of n-alkanes (C9 – C25 ) as standards [25]. The contents (relative percentages) of the separated compounds were calculated from the total ion chromatogram by a computerized integrator. The identification of the components was based on the comparison of their mass spectra with those of the NIST21 and NIST107 mass spectral libraries [26] and of their AIs with literature data [27]. When possible, the essential oils were subjected to cochromatography with authentic compounds (Fluka, Sigma). Statistical Analysis. After applying the KolmogorovSmirnov test for normality testing of the data, significant differences between different data subsets were traced by the independent samples t-test. Principal component analysis (PCA) was applied using as variables the essential-oil contents of b-pinene, limonene, menthone, isomenthone, pulegone, piperitone, cis-piperitone oxide, piperitenone, piperitenone oxide, and germacrene D. Hierarchical cluster analysis (HCA) was performed with the nearest neighbor method, based on the squared Euclidian distance, using as variables the essential-oil contents of trans-p-menthan-3-one and the variables used in the PCA. All analyses were performed with the PASW Statistics18.0 software package for Windows.
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