CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
295
Population Variability of Essential Oils of Pinus heldreichii from the Scardo-Pindic Mountains OÐljak and Galicˇica by Biljana Nikolic´* a ), Mihailo Ristic´ b ), Srdjan Bojovic´ c ), Zoran KrivoÐej d ), Vlado Matevski e ), and Petar D. Marin f ) a
) University of Belgrade, Institute of Forestry, Kneza ViÐeslava 3, 11000 Belgrade, Serbia (phone: þ 38-111-3553454; fax: þ 38-111-2545969; e-mail: [email protected]) b ) University of Belgrade, Institute for Medicinal Plant Research Dr Josif Pancˇic´, TadeuÐa KoÐc´uÐka 1, 11000 Belgrade, Serbia (e-mail: [email protected]) c ) University of Belgrade, Institute for Biological Research SiniÐa Stankovic´, Department of Ecology, Boulevard Despota Stefana 142, 11060 Belgrade, Serbia (e-mail: [email protected]) d ) University of PriÐtina, Department of Biology, Faculty of Natural Sciences, 38220 Kosovska Mitrovica, Serbia (e-mail: [email protected]) e ) Institute of Biology, Faculty of Natural Sciences and Mathematics, University Ss. Kiril and Metodij and Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia (e-mail: [email protected]) f ) University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden Jevremovac, Studentski trg 16, 11000 Belgrade, Serbia (e-mail: [email protected])
The needle-terpene profiles of two natural Pinus heldreichii populations from Mts. OÐljak and Galicˇica (Scardo-Pindic mountain system) were analyzed. Among the 68 detected compounds, 66 were identified. The dominant constituents were germacrene D (28.7%), limonene (27.1%), and a-pinene (16.2%). b-Caryophyllene (6.9%), b-pinene (5.2%), b-myrcene (2.3%), pimaric acid (2.0%), ahumulene (1.2%), and seven additional components were found to be present in medium-to-high amounts (0.5 – 10%). Although the general needle-terpene profile of the population from Galicˇica was similar to those of the populations from Lovc´en, Zeletin, Bjelasica, and Zlatibor-PeÐter (belonging to the Dinaric Alps), the principle-component analysis (PCA) of seven terpenes (b-myrcene, limonene, belemene, b-caryophyllene, a-humulene, d-cadinene, and germacrene D-4-ol) in 121 tree samples suggested a partial divergence in the needle-terpene profiles between the populations from the ScardoPindic mountain system and the Dinaric Alps. According to previously reported data, the P. heldreichii samples from the Balkan-Rhodope mountains lack b-caryophyllene and germacrene D, but contain gmuurolene in their terpene profile. Differences in the terpene composition between populations growing in the three above-mentioned mountain systems were compared and discussed.
Introduction. – Pinus heldreichii Christ (Bosnian pine) is a Balkan subendemic species and Tertiary relict [1]. This two-needle pine belongs to the subsection Sylvestres along with P. nigra, P. sylvestris, P. mugo, P. halepensis, P. brutia, P. resinosa, P. pinaster, etc., but its taxonomic position is still undefined [1] (and refs. cit. therein). The existence of four varieties of P. heldreichii, viz., var. typica Markgr., var. leucodermis (Antoine) Markgr., var. pancici Fukarek, and var. longiseminis Papaioannou [1] [2] (and refs. cit. therein), two interspecific hybrids, viz., Pinus mugodermis [3] and Pinus nigradermis [4], two intermediate forms, viz., P. heldreichii f. vivipara [5] and Pinus nigra f. leucodermoides [6], and several transitional forms between 2015 Verlag Helvetica Chimica Acta AG, Zrich
296
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
varieties typica and leucodermis [7] also contribute to the confusion in Bosnian pine taxonomy. The terpene composition of a large number of conifers, as well as other vascular plants, has been studied to date [8]. A lot of work was done in the field of taxonomy, confirming the lower taxonomic units. Moreover, terpenes were investigated for their contribution to plant resistance to diseases, pests, and external factors. The composition of Bosnian pine oleoresin was investigated by Mirov [8], Pejoski [9] [10], Iconomu and Valkanas [11], Karapandzˇic´ and Jovanovic´ [12] [13], Weißmann [14], Fengel and Wegner [15], Spanoudaki [16], and Lange et al. [17] (and other authors cit. therein). The main constituent of Bosnian pine turpentine was found to be limonene, while among resin acids, those of the abietane type were dominant, and, in the mixture of neutral diterpenes, tunbergol and cembrol were prevailing [17]. Differences in the terpene composition between P. heldreichii and P. nigra have long been observed (in 1961 by Mirov [8], then by Wang et al. [18]). Menkovic´ et al. [19] reported that young shoots of Bosnian pine contained 1.25% of essential oil and 41% of limonene, while the pine cones contained 0.85% of essential oil and 76% of limonene. The percentage of oil in the branches was found to vary between populations and decreases with the age of the needles (1.3 – 1.7% in young trees and 1.0 – 1.2% in adult trees) [20]. The terpene composition of Bosnian pine needles has been analyzed by numerous authors [20 – 27] (and refs. cit. therein), but mostly for a small number of samples. Limonene was generally the most copious component among the terpenes identified (contents of up to 67%), but, in rare cases, germacrene D was found to be dominant [20] [23]. The oil content in the needles varied depending on the population and developmental stage of the needles (0.28 – 0.45%), but the ratio of the major terpene components (germacrene D and limonene) was found to be stable [23]. The interpopulation variability and population divergence based on the terpene composition have only been investigated for Bosnian pine samples from Bulgaria [28 – 30], Serbia, and Montenegro [31]. In this work, a detailed study of the composition of the needle essential oils of two natural populations of P. heldreichii from Mts. OÐljak and Galicˇica (Scardo-Pindic mountain system) was presented. According to the available literature data, it seems that this research is the first investigation of the population variability of needle essential oil of P. heldreichii from this mountain system [32]. To compare the terpene composition of the needle oils of the Scardo-Pindic populations from OÐljak and Galicˇica (defined as Populations I and II, resp.) with our previously published results [31] for Populations III (Lovc´en), IV (Zeletin), V (Bjelasica), and VI (ZlatiborPeÐter), situated in the Dinaric Alps [32], multivariate statistical methods were used. Furthermore, the terpene compositions obtained for Populations I and II were compared and discussed in relation to those previously reported for P. heldreichii samples [20] [23] [24] [26] [30] [33]. Results and Discussion. – A map of the study area, indicating the location of the P. heldreichii populations, is shown in Fig. 1, and the corresponding geographic and geologic data are presented in Table 1.
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
297
Fig. 1. Distribution area of Pinus heldreichii on the Balkan Peninsula (according to Critchfield and Little, 1966, as stated by Vidakovic´ [1]). Populations: I, Mt. OÐljak; II, Mt. Galicˇica; III, Mt. Lovc´en; IV, Mt. Zeletin; V, Mt. Bjelasica; VI, Mt. Zlatibor – Mt. PeÐter. Mountain systems classified according to Stevanovic´ [32]: dark grey, Dinaric Alps; medium grey, Balkan-Rhodope mountains; light grey, ScardoPindic mountains. Table 1. Location and Habitat Description of the Studied Pinus heldreichii Populations I and II from the Scardo-Pindic Mountain System (see also Fig. 1)
Location Latitude ( N ) Longitude ( E) Altitude [m] Exposure Terrain inclination [8] Geologic substratum
Population I
Population II
Mt. OÐljak 42811’ 57’’ 20855’21’’ 1885 S < 60 Limestone
Mt. Galicˇica 40856’37’’ 20849’48’’ 1929 E < 40 Limestone schist
Terpene Composition of the P. heldreichii Populations from the Scardo-Pindic Mountains. The relative contents of the different terpene classes represented in the needle essential oils of Populations I and II from OÐljak and Pelister, respectively, are given in Table 2. In the overall terpene profile, mono- and sesquiterpenes were found to dominate, comprising in average 92.8% of the essential-oil composition, whereas diterpenes had an average relative content of 5.3%. A total of 66 individual components were identified out of the 68 compounds detected (Table 2). The dominant constituents were germacrene D (28) 1), limonene 1)
Italic numbers in parentheses refer to the entries in Table 2.
298
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 2. Chemical Composition of the Needle Essential Oils of Pinus heldreichii Populations I and II from OÐljak and Galicˇica Mountains, respectively Entry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
Compound name and class a )
(E )-Hex-2-enal (Z )-Hex-3-en-1-ol Hexan-1-ol Tricyclene a-Pinene Camphene Sabinene b-Pinene b-Myrcene Limonene trans-b-Ocimene g-Terpinene a-Terpinolene Bornyl acetate trans-Verbenyl acetate a-Terpinyl acetate a-Copaene b-Cubebene b-Elemene Longifolene b-Caryophyllene Calarene Guaia-6,9-diene a-Humulene trans-Muurola-3,5-diene trans-b-Farnesene g-Muurolene Germacrene D g-Amorphene a-Muurolene g-Cadinene d-Cadinene trans-Longipinocarveol Germacrene D-4-ol Caryophyllene oxide Biotol t-Cadinol a-Muurolol a-Cadinol Khusinol trans,trans-Farnesol Benzyl benzoate 14-Hydroxy-a-muurolene Farnesyl acetate Benzyl salicylate (5E,9E )-Farnesyl acetone Sandaracopimara-8(14),15-diene
Content [%] b )
Average content [%] c )
Population I
Population II
0.64 0.38 0.47 0.35 0.10 0.08 0.21 0.05 13.21 4.79 0.68 0.19 0.13 0.02 4.19 1.85 2.11 0.22 24.70 3.56 0.24 0.31 0.03 0.00 0.13 0.11 0.12 0.24 0.02 0.03 0.25 0.18 0.07 0.01 0.06 0.01 0.12 0.03 0.03 0.03 8.07 1.75 0.43 0.09 0.17 0.03 1.36 0.25 0.05 0.01 0.07 0.18 0.08 0.11 32.09 6.87 0.04 0.01 0.28 0.05 0.15 0.05 0.35 0.10 0.07 0.04 0.76 0.29 0.06 0.03 0.02 0.01 0.08 0.04 0.02 0.03 0.07 0.02 0.12 0.04 0.15 0.21 0.02 0.02 0.11 0.05 0.03 0.03 0.01 0.01 0.09 0.03 0.13 0.25
0.26 0.20 0.27 0.14 0.08 0.06 0.31 0.08 22.17 3.65 1.18 0.24 0.17 0.03 7.21 1.64 2.79 0.28 31.86 2.90 0.01 0.01 0.03 0.01 0.16 0.04 0.01 0.02 0.00 0.01 0.44 0.19 0.06 0.02 0.05 0.01 0.09 0.01 0.01 0.02 4.49 1.97 0.30 0.04 0.11 0.01 0.80 0.26 0.04 0.00 0.01 0.01 0.10 0.08 21.95 2.91 0.07 0.12 0.25 0.05 0.09 0.01 0.24 0.02 0.10 0.06 0.40 0.11 0.04 0.02 0.03 0.02 0.02 0.02 0.09 0.03 0.08 0.01 0.11 0.03 0.12 0.07 0.03 0.02 0.07 0.01 0.03 0.04 0.08 0.08 0.11 0.01 0.02 0.02
Populations I and II 0.52 0.38 0.41 0.31 0.10 0.07 0.25 0.08 16.20 6.13 0.85 0.32 0.14 0.03 5.20 2.27 2.34 0.41 27.09 4.77 0.17 0.27 0.03 0.01 0.14 0.10 0.08 0.20 0.02 0.03 0.31 0.20 0.06 0.02 0.05 0.01 0.11 0.03 0.02 0.03 6.88 2.48 0.38 0.10 0.15 0.04 1.17 0.36 0.05 0.01 0.05 0.15 0.09 0.10 28.71 7.56 0.05 0.07 0.27 0.05 0.13 0.05 0.31 0.10 0.08 0.05 0.64 0.30 0.05 0.03 0.03 0.01 0.06 0.05 0.04 0.04 0.07 0.02 0.12 0.03 0.14 0.17 0.02 0.02 0.10 0.04 0.03 0.03 0.04 0.06 0.09 0.03 0.10 0.21
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
299
Table 2 (cont.) Entry
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68
Compound name and class a )
(E,Z)-Geranyl linalool Kaur-15-ene (E,E)-Geranyl linalool Thunbergol Abietadiene Abienol Abieta-8(14),13(15)-diene Sandaracopimarinal Unknown 1 Sclareol 7a-Hydroxymanool Sandaracopimarinol Dehydroabietal Tricosane Isopimarol Torulosol Pimaric acid Kauran-18-oic acid Triacontane Unknown 2 Octacosan-1-ol Total Monoterpene hydrocarbons Oxygenated monoterpenes Total Monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Total Sesquiterpenes Diterpene hydrocarbons Oxygenated diterpenes Total Diterpenes Others d ) Unknown
Content [%] b )
Average content [%] c )
Population I
Population II
0.04 0.04 0.19 0.04 0.04 0.02 0.72 0.26 0.08 0.11 0.04 0.05 0.03 0.02 0.12 0.04 0.05 0.03 0.34 0.10 0.10 0.07 0.06 0.06 0.09 0.06 0.07 0.03 0.65 0.29 0.02 0.04 3.11 1.78 0.77 0.79 0.07 0.09 0.04 0.07 0.99 1.57
0.01 0.02 0.18 0.02 0.08 0.01 0.90 0.52 0.08 0.03 0.00 0.01 0.05 0.02 0.03 0.02 0.06 0.04 0.43 0.08 0.00 0.00 0.18 0.08 0.09 0.09 0.03 0.05 0.46 0.17 0.11 0.08 0.08 0.22 0.18 0.44 0.04 0.13 0.06 0.18 0.00 0.00
Populations I and II 0.03 0.03 0.19 0.03 0.05 0.02 0.78 0.37 0.08 0.09 0.03 0.04 0.04 0.02 0.09 0.06 0.05 0.03 0.37 0.10 0.07 0.08 0.10 0.09 0.09 0.07 0.06 0.04 0.58 0.27 0.05 0.07 2.10 2.05 0.57 0.74 0.06 0.10 0.05 0.12 0.66 1.35
100.00
100.00
100.00
45.63 0.39 46.02 43.41 1.59 45.00 6.15 0.08 6.51 2.39 0.08
65.90 0.45 66.35 28.64 1.19 29.83 2.65 0.13 2.90 0.79 0.13
52.39 0.41 52.80 38.49 1.46 39.95 4.98 0.10 5.30 1.85 0.10
a ) Literature (trivial) names rather than fully systematic names are given. b ) Contents are given as percentages (mean standard deviation) relative to the total essential-oil composition; for the population details, see Table 1. c ) Average contents of Populations I and II (mean standard deviation). d ) Aliphatic aldehydes and alcohols, aromatic acid esters, and n-alkanes.
(10), and a-pinene (5), comprising together in average 72.0% of the total essential-oil composition. b-Caryophyllene (21), b-pinene (8), b-myrcene (9), pimaric acid (64), ahumulene (24), camphene (6), thunbergol (51), octacosanol (68), germacrene D-4-ol (34), isopimarol (62), kauran-18-oic acid (65), and (E)-hex-2-enal (1) were found to be present in medium-to-high amounts (0.5 – 10%, according to [34]). The compounds identified in very low amounts were found to comprise in average 5.7% of the total essential oil composition (Table 2).
300
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
The average chemical profile of the five main terpene components in the needle oils of P. heldreichii Populations I and II (present results) was germacrene D > limonene >> a-pinene >> b-caryophyllene > b-pinene (Tables 2 and 3), where, according to Petrakis et al. [26], > and >> represent relative differences in the oil contents of 1.1 – 5.0 and 5.1 – 15.0%, respectively. Interpopulation Variability of the Terpene Composition of the Scardo-Pindic P. heldreichii Populations I and II. The needle oil of Population I (OÐljak) was found to have significantly higher contents of b-caryophyllene (21), a-humulene (24), germacrene D (28), pimaric acid (64), and 17 additional components (P 0.05, LSD test, results were not presented; Table 2) than that of Population II. Alternatively, the oil of Population II (Galicˇica) was found to contain significantly higher amounts of a-pinene (5), b-pinene (8), b-myrcene (9), limonene (10), and nine other components than that of Population I. As in previous studies [31], no correlations between the geological structures and the needle-terpene profiles were observed in these two populations either. Population I had a needle-terpene profile similar to the average profile presented above, i.e., germacrene D >> limonene >> a-pinene >> b-caryophyllene > b-pinene, whereas Population II showed a different sequence of the most abundant components in its terpene profile, i.e., limonene >> a-pinene ¼ germacrene D >> b-pinene > bcaryophyllene, where ¼ represents a relative difference in the oil contents of 0.1 – 1.0% [26]. Trimodal Frequency Distribution of the Scardo-Pindic P. heldreichii Populations I and II. When testing the normality of the distribution of each of the 68 components, it was observed that the needle-oil content of 33 components deviated from normal distribution (n ¼ 24, c2 values ranging from 8.50 – 97.50, d.f. ¼ 3, P < 0.05). The frequency distribution of 21 of them suggested a monogenic type of heredity (n ¼ 24, c2 values ranging from 1.00 – 7.50, d.f. ¼ 3, P > 0.05). Frequencies of putative alleles determining high and low concentrations of terpenes were estimated from the frequency histograms, assuming no dominance [35]. Based on a genetic model [31] [36] [37], a trimodal frequency distribution was ascertained for many compounds, e.g., bornyl acetate (14), a-copaene (17), b-cubebene (18), and longifolene (20; shown in Fig. 2), as well as others. There was a single locus with two alleles (L and H), leading to three genotypes, i.e., homozygous LL and HH, producing low and high amounts of terpene compound, respectively, and heterozygous LH, producing a medium amount of this terpene. For example, in the case of bornyl acetate, a trimodal frequency distribution was accounted by assigning distribution intervals of 0.1 – 0.3, 0.3 – 0.7, and > 0.7% to the LL, LH, and HH genotypes, respectively. Although the frequencies of the above-mentioned compounds corresponded to the distribution defined by the law of inheritance, at this stage, it was difficult to prove that these components were under genetic control of a single locus with two alleles [38 – 43], because their contents were very low. Comparison of the Terpene Profile between Scardo-Pindic and Dinaric Populations. The terpene profile of the Scardo-Pindic Population II (limonene >> a-pinene ¼ germacrene D >> b-pinene > b-caryophyllene) was similar to that of Populations III, IV, V, and VI from Lovc´en, Zeletin, Bjelasica, and Zlatibor-PeÐter, respectively [31], located in the Dinaric Alps [32] (limonene >> a-pinene > germacrene D >> b
Dinaric
Dinaric Dinaric
Dinaric Scardo-Pindic Scardo-Pindic Scardo-Pindic Scardo-Pindic BalkanRhodope
[20]
[27]
[31]
[24]
[23]
Present results
[26]
[46]
[30]
17.3 20.3
Bjelasica Lovc´en
Pirin Slavjanka
Pinds
Katara
OÐljak Galicˇica
ara
18.0 17.3
11.1
13.8
13.2 22.2
8.8
13.3
16.1
Zeletin
Prokletije
12.9
10.2
10.0 7.3
a-Pinene
6.2 5.4
3.6
4.2
4.2 7.2
–
4.0
6.5
6.0
4.8
4.5
3.0
3.0 2.4
b-Pinene
37.1 46.5
23.7
34.3
24.7 31.9
22.5
67.0
25.1
29.7
23.2
29.5
52.8
43.8 28.0
Limonene
– –
8.6
8.4
8.1 4.5
12.0
2.2
8.9
11.4
11.0
11.1
7.7
11.9 11.6
b-Caryophyllene
– –
21.3
12.8
32.1 22.0
43.5
tr
15.4
11.4
13.1
16.3
15.8
9.6 33.0
Germacrene D
Content of main terpene compounds [%] a )
ZlatiborPeÐter
Prenj ( RujiÐta)
Trebevic´ Prenj ( RujiÐta)
Mountains
22.0 15.1
–
b-Farnesene (7.1%), d-car-3-ene (3.7%) b-Farnesene (5.1%), d-car-3-ene (4.8%)
d-Car-3-ene (18.6%), myrcene 82.2%), terpinolene (2.1%)
Aristolene (6.0%), d-car-3-ene (2.8%), b-myrcene (2.5%), camphene (1.5%)
b-Myrcene (2.1%), pimaric acid (3.1%) b-Myrcene (2.8%) – – –
d-Cadinene (7.6%), sabinene (1.5%)
a-Terpineol (1.9%)
d-Cadinene (2.5%), a-muurolene (2.3%), a-humulene (2.2%), b-myrcene (2.2%), b-gurjunene (1.8%), g-muurolene (1.7%) ( E )-Hex-2-enal (2.6%), a-humulene (2.2%), d-cadinene (1.9%), a-muurolene (1.8%), b-myrcene (1.8%) b-Myrcene (2.5%), a-humulene (2.2%), ( E )-hex-2-enal (2.0%), a-muurolene (1.5%). b-Myrcene (2.2%), a-humulene (1.9%), d-cadinene (1.7%), b-gurjunene (1.6%), a-muurolene (1.6%)
b-Myrcene (1.5%), a-humulene (1.5%)
a-Humulene (1.9%) 1,10-Epoxygermacrene D (3.6%), g-cadinene (1.5%)
–
–
–
–
–
–
–
– –
g-Muurolene
Other terpenes b )
a ) Content of main terpenes present in the needle essential oils given as percentages relative to the total essential-oil composition; tr, trace amount; –, not detected. b ) Other terpenes present in the oils at contents 1.5%; contents [%] are given in parentheses.
Mountain System
Reference
Table 3. Terpene Composition of Pinus heldreichii Needle Oils: Comparison of the Present with Literature Results
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015) 301
302
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Fig. 2. Frequency distribution of a) bornyl acetate (14)1), b) a-copaene (17), c) b-cubebene (18), and d) longifolene (20) in two populations of Bosnian pine from the Scardo-Pindic mountains (Populations I and II; ca. 24 trees) suggesting trimodality. Single locus with two alleles leading to three genotypes: homozygous (LL and HH), producing low and high amounts of terpene, and heterozygous (LH), producing medium amounts of this terpene.
caryophyllene > b-pinene; Tables 2 and 3). In a recent study of the terpene composition of Macedonian pine (Pinus peuce), the terpene profile of the population from Mt. Pelister (Scardo-Pindic mountains) was also found to be similar to that of the populations from the Dinarides [44]. Multivariate Statistical Analyses. For the principle-component analysis (PCA) and cluster analysis (CA) of the terpene profiles of Populations I – VI, seven terpene compounds, whose distribution was normal (c2 test; P 0.05) and which did not display statistically important differences between the standard deviations of the populations (Levenes test; P 0.05), were selected, i.e., b-myrcene, limonene, b-elemene, bcaryophyllene, a-humulene, d-cadinene, and germacrene D-4-ol. The PCA was performed based on a correlation matrix computed for all 121 tree samples, with the contents of the individual compounds expressed as percentage of the total terpene fraction (Fig. 3). The first two principal axes represented 75% of the total variation. According to the contents of the seven selected compounds, the geographically most distant population, i.e., Population II from the Scardo-Pindic mountain system, was clearly separated from the Scardo-Pindic Population I and the four natural populations from the Dinaric Alps (Populations III, IV, V, and VI), a finding that was confirmed by CA (Fig. 4). Genetic Analysis of Compounds from Dinaric and Scardo-Pindic Populations. Genetic analysis was carried out using a-pinene, which showed the trimodal distribution most clearly. To check whether some other components of Bosnian pine needle oils from the investigated part of its habitat area (6 populations) show signs of
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
303
Fig. 3. Principle-component analysis of seven selected terpenes isolated from 121 Bosnian pine samples from six populations. Populations: *, Population I (Mt. OÐljak); ^, Population II (Mt. Galicˇica); ~, Population III (Mt. Lovc ´ en); ^, Populaton IV (Mt. Zeletin); *, Population V (Mt. Bjelasica); 1 n, Population VI (Mt. Zlatibor – Mt. PeÐter). Terpenes: b-myrcene (9) ), limonene (10), b-elemene (19), b-caryophyllene (21), a-humulene (24), d-cadinene (32), and germacrene D-4-ol (34).
Fig. 4. Dendrogram obtained by cluster analysis based on the nearest-neighbor method (squared Euclidean distances) of the contents of seven selected terpenes in the needle oils of P. heldreichii Populations I – VI. The numbers on the vertical axis refer to the distance level, calculated on the basis of the differences between population contents of the selected components. For the geographical location of Populations I – VI, see Fig. 1.
monohybrid inheritance of the terpene content, all the components with contents deviating from the normal distribution were analyzed. Among the components with a larger content range, those with histograms suggesting genetic control (existence of
304
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
trimodal distribution) were chosen. a-Pinene was the most suitable compound, because its contents ranged from small amounts to dominance (3.36 – 54.92%), its distribution deviated from the norm (n ¼ 121, c2 ¼ 13.02, P < 0.05), and it was the only component whose experimental chemotype (genotype) frequency in every population matched the frequencies predicted by the Hardy–Weinberg (HW) law of inheritance (Table 4). The experimental frequencies of the three modalities (chemotypes or genotypes: LL ¼ low concentration, LH ¼ medium concentration, and HH ¼ high concentration) were previously matched against the frequencies expected according to the HW law (using the c2 compatibility test with the HW law). Frequencies of putative alleles determining low (L) and high (H) concentrations of this terpene were estimated from frequency histograms, assuming no dominance [45]. Fig. 5 shows an example of a-pinenefrequency histograms for Population III (Lovc´en). The trimodal a-pinene-frequency distribution found in this population was accounted for by assigning percentage intervals of 0 – 20, 20 – 40, and more than 40% to the LL, LH and HH genotypes, respectively. A dendrogram based on UPGMA clustering of Rogers genetic distances (as modified by Wright) [46] is shown in Fig. 6. Cluster analysis showed a partial divergence of two Dinaric populations (Populations IV and V, form Zeletin and Bjelasica, resp.) from the other Dinaric and the Scardo-Pindic populations. According to a very recent study of the essential-oil composition and the genetic distance of Macedonian pine (Pinus peuce), partial divergence between populations from the Dinaric Alps and the Scardo-Pindic mountains was also found [44]. Comparison of the Present with Literature Results. Due to limonene as the dominant needle-oil terpene, the oil of Population II (Galicˇica) was similar to those of the populations from the Dinaric Alps [20] [27] [31] [24], Scardo-Pindic mountains [26] [47], and the Balkan-Rhodope massif [30] (Table 3). When regarding the complete terpene profile, the oil of Population II was most similar to that of a population from Katara (Greece) [26] and those of several populations from Montenegro [31], and the least similar to those of two populations from the Balkan-Rhodope mountain system, which lacked b-caryophyllene and germacrene D, but contained g-muurolene in their Table 4. Frequencies of Genotypes ( LL, LH, and HH ) and Alleles ( L and H ) Determined from the aPinene Content of the Needle Oils of Pinus heldreichii Populations I – VI Population a )
Number of trees sampled (n) Number of oils with genotype LL Number of oils with genotype LH Number of oils with genotype HH c2 P Frequency of allele L c ) Frequency of allele H d )
I
II
III b )
IV
V
VI
16 7 8 1 2.00 > 0.05 0.69 0.31
8 2 5 1 – > 0.05 0.56 0.44
30 20 8 2 14.80 > 0.05 0.80 0.20
30 1 10 19 6.00 > 0.05 0.20 0.80
30 4 14 12 2.00 > 0.05 0.36 0.64
7 3 3 1 – > 0.05 0.64 0.36
a ) Populations: I, OÐljak; II, Galicˇica; III, Lovc´en; IV, Zeletin; V, Bjelasica; VI, Zlatibor-PeÐter; for the location of the populations, see Fig. 1. b ) Results for Population III are also presented in Fig. 5. c ) Frequency of allele L ¼ (2LL þ LH)/2n. d ) Frequency of allele H ¼ (2 HH þ LH )/2n.
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
305
Fig. 5. Frequency distribution of the a-pinene content in Population III (30 trees), suggesting trimodality. Single locus with two alleles leading to three genotypes: homozygous (LL and HH) and heterozygous (LH), producing low, high, and medium amounts of a-pinene, respectively. The trimodal a-pinene frequency distribution found in this population was accounted for by assigning percentage intervals of 0 – 20, 20 – 40, and more than 40% to the LL, LH, and HH genotypes, respectively.
Fig. 6. Dendrogram obtained by UPGMA clustering based on Rogers genetic distances as modified by Wright of estimated allele frequencies (L and H) of a-pinene in Pinus heldreichii Populations I – VI. The numbers on the horizontal axis refer to the distance level. For the geographical location of Populations I – VI, see Fig. 1.
terpene profile [30]. By its dominant oil component germacrene D, the oil of Population I (OÐljak) was similar to that of Bosnian pines from Prenj [20] and Mt. ara [23]. To better understand whether the differences found in the terpene composition of the investigated natural populations of Bosnian pine growing on different mountain systems are caused predominantly by genetic or environmental factors, further phytochemical and molecular investigations are needed. The differences in the abundance of major oil components and the terpene profiles of P. heldreichii between the present and literature results might be attributed to differences in the needlesampling technique, time of collection, method of essential-oil extraction, chromatographic processing, and, especially, in the number of individuals analyzed. Tectonic collisions of the Prokletije massif with the Scardo-Pindic mountain system in the pliocene and glaciation in the pleistocene, which allowed the survival of many relict species [48], might also have influenced the divergence of the Bosnian pine populations [31] (and refs. cit. therein), which should be tested by further genetic analyses.
306
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Conclusions. – According to the general terpene profile and seven terpene components selected for the multivariate analyses, the needle oil of Population II from the Scardo-Pindic mountain system (present investigation) was clearly different from those of Populations III, IV, V, and VI from the Dinaric Alps and Population I from the Scardo-Pindic mountains (present investigation). A fact, which was not confirmed by genetic analysis of a-pinene. In future research, the genetic distance of other terpene components should also be examined. In addition, the combination of morphological, phytochemical, and molecular analyses of P. heldreichii populations from the whole area of limited natural distribution are necessary and planned, to highlight phylogeographic and historical relationships of more or less distant and fragmented populations of this very interesting pine species. This work is part of two research projects (173029 and 173021) supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia.
Experimental Part Plant Material. Twigs with needles from the lowest third of the tree crown were collected in late summer 2012 from 16 and 8 randomly selected trees growing on Mt. OÐljak (Population I) and Mt. Galicˇica (Population II), resp. The collected twigs were stored at 208, and voucher specimens have been deposited with the Institute of Forestry, Belgrade, Serbia. Isolation of Essential Oils. Two-year-old needles, stored in a freezer at 208 until extraction, were cut into pieces of 2 – 3 mm length and extracted with pentane (1 g needles/ml solvent). The extracts were kept at 4 – 68 for 24 h, then filtered, and stored in chromatography vials with solid caps in a refrigerator until further analysis. GC-FID Analysis. The GC-FID analyses were carried out with an Agilent Technologies 7890A apparatus equipped with a split/splitless injector, an automatic liquid sampler (ALS), a flame ionization detector (FID), and a HP-5 fused-silica cap. column (30 m 0.32 mm i.d., film thickness 0.25 mm). The oven temp. was programmed linearly rising from 40 to 2808 at 4 8/min; injector temp., 2508; detector temp., 2808; carrier gas, H2 (1 ml/min). Samples (1 ml) were injected in splitless mode (1 : 15) using the ALS. GC/MS Analysis. The GC/MS analyses were performed with a Hewlett Packard G1800C-GCD apparatus equipped with a split/splitless injector, ALS, a mass-selective detector, and a HP-5 MS fusedsilica cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm). The chromatographic conditions were the same as described above (cf. GC-FID Analysis), except for the carrier gas, which was He. Electronimpact mass spectra (EI-MS; 70 eV) were acquired over the m/z range 40 – 450 amu. Compound Quantification and Identification. For the quantification of the contents of the needle-oil components, area-percent reports obtained by GC-FID were used as a base. The identification of the oil components was based on the comparison of their retention indices (RIs) and mass spectra with those previously reported [49] or listed in the mass-spectral libraries Wiley-275 and NIST/NBS. Statistical Analysis. The calculation of arithmetic means and standard deviations (SD) of the populations, frequencies, histograms, test for normality (c2 test), LSD test, one-way analyses of variance (ANOVA), Levenes test, principal-component analyses (PCA), as a descriptive multivariate method capable of suggesting the structure and tendency of the data set, and cluster analysis were all carried out with the software Statgraphics Plus (version 5.0; Statistical Graphics Corporation, USA).
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
307
REFERENCES ˇ etinjacˇe: Morfologija i Varijabilnost, JAZU i SveucˇiliÐna naklada Liber, Zagreb, [1] M. Vidakovic´, in C 1982, p. 428. [2] P. Fukarek, umarski list (Zagreb) 1960, 5 – 6, 152. [3] P. Fukarek, umarski list (Zagreb) 1960, 11 – 12, 393. [4] P. Fukarek, M. Vidakovic´, Naucˇno druÐtvo Bosne i Hercegovine 1965, XXVIII, 68. [5] A. Tucovic´, S. Stilinovic´, Genetika (Belgrade) 1972, 4, 193. [6] P. Fukarek, M. Nikolic´, SANU and ANUBiH (Belgrade) 1974, I, 20. [7] T. Nikolovski, J. Matvejeva, MorfoloÐke karakteristike apofize munike (Pinus heldreichii Christ) sa podrucˇja Makedonije i Kosova, Zbornik radova, Simpozijum o munici Pinus heldreichii Christ, 4 – 7. IX 1972, Decˇani (Kosovo), Pec´, Belgrade, 1975, p. 258. [8] N. T. Mirov, Composition of Gum Turpentines of Pines, U. S. Department of Agriculture Technical Bulletin No. 1239’, U. S. Government Printing Office, Washington, 1961. [9] B. Pejoski, umarski list (Zagreb) 1951, 1 – 2, 85. [10] B. Pejoski, umarski list (Zagreb) 1953, 11, 450. [11] N. Iconomu, G. Valkanas, Pharm. Acta Helv. 1966, 41, 59. [12] D. Karapandzˇic´, N. Jovanovic´, Hemijski sastav munike (Pinus heldreichii), Simpozijum o munici Pinus heldreichii Christ, 4 – 7. IX 1972, Decˇani (Kosovo), Pec´, Zbornik radova, Belgrade, 1972, p. 439. [13] D. Karapandzˇic´, N. Jovanovic´, Glasnik umarskog fakulteta (Belgrade) 1975, 48 (B), 59. [14] G. Weißmann, Holz Roh-Werkstoff 1973, 31, 341. [15] D. Fengel, G. Wegener, in Wood: Chemistry, Ultrastructure, Reactions, Walter de Gruyter, Berlin, 1984, p. 26. [16] M. Spanoudaki, Diplomarbeit, Hamburg, 1993. [17] W. Lange, T. Stevanovic´-Janezˇic´, M. Spanoudaki, Phytochemistry 1994, 36, 1277. [18] X. R. Wang, Y. Tsumura, H. Yoshimary, K. Nagasaka, A. E. Szmidt, Am. J. Bot. 1999, 86, 1742. [19] N. R. Menkovic´, M. S. Ristic´, Z. J. Samardzˇic´, N. N. Kovacˇevic´, S. R. Tasic´, Acta Hort. 1993, 344, 578. [20] J. Chalchat, R. P. Garry, M. S. Gorunovic´, Pharmazie 1994, 49, 852. [21] D. Karapandzˇic´, Ph.D. Thesis, University of Belgrade, 1964. [22] D. Karapandzˇic´, J. Serb. Chem. Soc. 1966, 31, 473. [23] N. Simic´, R. Palic´, S. Andjelkovic´, V. Vajs, S. Milosavljevic´, J. Essent. Oil Res. 1996, 8, 1. [24] T. M. Stevanovic´-Janezˇic´, D. M. Vilotic´, Lekovite sirovine (Belgrade) 1998, 47, 109. [25] P. Petrakis, O. Ortiz, O. Tzakou, M. Couladis, C. Vagias, V. Roussis, Chemical variability of the foliar metabolites and chemotaxonomic studies on pine trees growing in Greece, First Conference on Medicinal and Aromatic Plants of Southeast European Countries & VI Meeting Days of Medicinal Plants, Book of Proceedings, Arandjelovac, Yugoslavia, 2000, L 15. [26] P. V. Petrakis, C. Tsitsimpikou, O. Tzakou, M. Couladis, C. Vagias, V. Roussis, Flavour Fragrance J. 2001, 16, 249. [27] S. Maric´, M. Jukic´, V. Katalinic´, M. MiloÐ, Molecules 2007, 12, 283. [28] K. D. Naidenov, G. G. Markov, in Population Genetics and Genetic Conservation of Forest Trees, Eds. P. Baradat, W. T. Adams, G. Mller-Starck, SPB Academic Publishing, Amsterdam, 1995, p. 165. [29] K. D. Naydenov, Co-relationship between terpenes in the essential oil of palebark Heldreichii pine (Pinus heldreichii Christ.) determined through CGC in different provenances in Bulgaria, 18th International Symposium on Capillary Chromatography, May 1996, Riva del Garda, Italy, Book of Proceedings, Eds. P. Sandra, G. Devos, Dr. Alfred Huethig Publishing GmbH, Heidelberg, 1996, Vol. II, p. 1247. [30] K. D. Naydenov, F. M. Tremblay, Y. Bergeron, A. Alexandrov, N. Fenton, Biochem. Syst. Ecol. 2005, 33, 133. [31] B. Nikolic´, M. Ristic´, S. Bojovic, P. D. Marin, Chem. Biodiversity 2007, 4, 905. [32] V. Stevanovic´, Bocconea 1996, 5, 77.
308
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
[33] P. K. Koukos, K. I. Papadopoulou, D. T. Patiaka, A. D. Papagiannopoulos, J. Agric. Food Chem. 2000, 48, 1266. [34] E. Von Rudloff, Biochem. Syst. Ecol. 1975, 2, 131. [35] S. Bojovic, M. Jurc, D. Drazic, P. Pavlovic, M. Mitrovic, L. Djurdjevic, R. S. Dodd, Z. Afzal-Rafii, M. Barbero, Trees 2005, 19, 531. [36] E. Zavarin, K. Snajberk, L. Cool, Biochem. Syst. Ecol. 1990, 18, 117. [37] L. G. Cool, E. Zavarin, Biochem. Syst. Ecol. 1992, 20, 133. [38] R. Yazdani, R. Rudin, T. Aldn, L. Lindgren, B. Harbom, K. Ljung, Hereditas 1982, 97, 261. [39] R. Yazdani, J. E. Nilsson, T. Ericsson, Silvae Genet. 1985, 34, 201. [40] S. H. Strauss, W. D. Critchfield, Forest Sci. 1982, 28, 687. [41] A. E. Squillace, O. O. Wells, O. Rockwood, Silvae Genet. 1980, 29, 141. [42] E. E. White, Phytochemistry 1983, 23, 1399. [43] C. Bernard-Dagan, Biosynthesis of Lower Terpenoids: Genetic and Physiological Controls in Woody Plants, Plenum Press, New York, 1988; J. W. Hanover, D. E. Keathley, in Genetic Manipulation of Woody Plants, Plenum Press, New York, 1988, p. 329. [44] B. Nikolic´, M. Ristic´, S. Bojovic´, V. Matevski, Z. KrivoÐej, P. D. Marin, Chem. Biodiversity 2014, 11, 934. [45] Z. A. Rafii, R. S. Dodd, E. Zavarin, Biochem. Syst. Ecol. 1996, 24, 325. [46] F. J. Rohlf, NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, Exter Software, New York, 1990. [47] E. Ioannou, A. Koutsaviti, O. Tzakou, V. Roussis, Phytochem. Rev. 2014, 13, 741. [48] P. D. Hughes, J. C. Woodward, P. L. Gibbard, Prog. Phys. Geogr. 2006, 30, 334. [49] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th edn., Allured Publishing Corporation, Carol Stream, IL, 2007. Received April 1, 2014
295
Population Variability of Essential Oils of Pinus heldreichii from the Scardo-Pindic Mountains OÐljak and Galicˇica by Biljana Nikolic´* a ), Mihailo Ristic´ b ), Srdjan Bojovic´ c ), Zoran KrivoÐej d ), Vlado Matevski e ), and Petar D. Marin f ) a
) University of Belgrade, Institute of Forestry, Kneza ViÐeslava 3, 11000 Belgrade, Serbia (phone: þ 38-111-3553454; fax: þ 38-111-2545969; e-mail: [email protected]) b ) University of Belgrade, Institute for Medicinal Plant Research Dr Josif Pancˇic´, TadeuÐa KoÐc´uÐka 1, 11000 Belgrade, Serbia (e-mail: [email protected]) c ) University of Belgrade, Institute for Biological Research SiniÐa Stankovic´, Department of Ecology, Boulevard Despota Stefana 142, 11060 Belgrade, Serbia (e-mail: [email protected]) d ) University of PriÐtina, Department of Biology, Faculty of Natural Sciences, 38220 Kosovska Mitrovica, Serbia (e-mail: [email protected]) e ) Institute of Biology, Faculty of Natural Sciences and Mathematics, University Ss. Kiril and Metodij and Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia (e-mail: [email protected]) f ) University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden Jevremovac, Studentski trg 16, 11000 Belgrade, Serbia (e-mail: [email protected])
The needle-terpene profiles of two natural Pinus heldreichii populations from Mts. OÐljak and Galicˇica (Scardo-Pindic mountain system) were analyzed. Among the 68 detected compounds, 66 were identified. The dominant constituents were germacrene D (28.7%), limonene (27.1%), and a-pinene (16.2%). b-Caryophyllene (6.9%), b-pinene (5.2%), b-myrcene (2.3%), pimaric acid (2.0%), ahumulene (1.2%), and seven additional components were found to be present in medium-to-high amounts (0.5 – 10%). Although the general needle-terpene profile of the population from Galicˇica was similar to those of the populations from Lovc´en, Zeletin, Bjelasica, and Zlatibor-PeÐter (belonging to the Dinaric Alps), the principle-component analysis (PCA) of seven terpenes (b-myrcene, limonene, belemene, b-caryophyllene, a-humulene, d-cadinene, and germacrene D-4-ol) in 121 tree samples suggested a partial divergence in the needle-terpene profiles between the populations from the ScardoPindic mountain system and the Dinaric Alps. According to previously reported data, the P. heldreichii samples from the Balkan-Rhodope mountains lack b-caryophyllene and germacrene D, but contain gmuurolene in their terpene profile. Differences in the terpene composition between populations growing in the three above-mentioned mountain systems were compared and discussed.
Introduction. – Pinus heldreichii Christ (Bosnian pine) is a Balkan subendemic species and Tertiary relict [1]. This two-needle pine belongs to the subsection Sylvestres along with P. nigra, P. sylvestris, P. mugo, P. halepensis, P. brutia, P. resinosa, P. pinaster, etc., but its taxonomic position is still undefined [1] (and refs. cit. therein). The existence of four varieties of P. heldreichii, viz., var. typica Markgr., var. leucodermis (Antoine) Markgr., var. pancici Fukarek, and var. longiseminis Papaioannou [1] [2] (and refs. cit. therein), two interspecific hybrids, viz., Pinus mugodermis [3] and Pinus nigradermis [4], two intermediate forms, viz., P. heldreichii f. vivipara [5] and Pinus nigra f. leucodermoides [6], and several transitional forms between 2015 Verlag Helvetica Chimica Acta AG, Zrich
296
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
varieties typica and leucodermis [7] also contribute to the confusion in Bosnian pine taxonomy. The terpene composition of a large number of conifers, as well as other vascular plants, has been studied to date [8]. A lot of work was done in the field of taxonomy, confirming the lower taxonomic units. Moreover, terpenes were investigated for their contribution to plant resistance to diseases, pests, and external factors. The composition of Bosnian pine oleoresin was investigated by Mirov [8], Pejoski [9] [10], Iconomu and Valkanas [11], Karapandzˇic´ and Jovanovic´ [12] [13], Weißmann [14], Fengel and Wegner [15], Spanoudaki [16], and Lange et al. [17] (and other authors cit. therein). The main constituent of Bosnian pine turpentine was found to be limonene, while among resin acids, those of the abietane type were dominant, and, in the mixture of neutral diterpenes, tunbergol and cembrol were prevailing [17]. Differences in the terpene composition between P. heldreichii and P. nigra have long been observed (in 1961 by Mirov [8], then by Wang et al. [18]). Menkovic´ et al. [19] reported that young shoots of Bosnian pine contained 1.25% of essential oil and 41% of limonene, while the pine cones contained 0.85% of essential oil and 76% of limonene. The percentage of oil in the branches was found to vary between populations and decreases with the age of the needles (1.3 – 1.7% in young trees and 1.0 – 1.2% in adult trees) [20]. The terpene composition of Bosnian pine needles has been analyzed by numerous authors [20 – 27] (and refs. cit. therein), but mostly for a small number of samples. Limonene was generally the most copious component among the terpenes identified (contents of up to 67%), but, in rare cases, germacrene D was found to be dominant [20] [23]. The oil content in the needles varied depending on the population and developmental stage of the needles (0.28 – 0.45%), but the ratio of the major terpene components (germacrene D and limonene) was found to be stable [23]. The interpopulation variability and population divergence based on the terpene composition have only been investigated for Bosnian pine samples from Bulgaria [28 – 30], Serbia, and Montenegro [31]. In this work, a detailed study of the composition of the needle essential oils of two natural populations of P. heldreichii from Mts. OÐljak and Galicˇica (Scardo-Pindic mountain system) was presented. According to the available literature data, it seems that this research is the first investigation of the population variability of needle essential oil of P. heldreichii from this mountain system [32]. To compare the terpene composition of the needle oils of the Scardo-Pindic populations from OÐljak and Galicˇica (defined as Populations I and II, resp.) with our previously published results [31] for Populations III (Lovc´en), IV (Zeletin), V (Bjelasica), and VI (ZlatiborPeÐter), situated in the Dinaric Alps [32], multivariate statistical methods were used. Furthermore, the terpene compositions obtained for Populations I and II were compared and discussed in relation to those previously reported for P. heldreichii samples [20] [23] [24] [26] [30] [33]. Results and Discussion. – A map of the study area, indicating the location of the P. heldreichii populations, is shown in Fig. 1, and the corresponding geographic and geologic data are presented in Table 1.
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
297
Fig. 1. Distribution area of Pinus heldreichii on the Balkan Peninsula (according to Critchfield and Little, 1966, as stated by Vidakovic´ [1]). Populations: I, Mt. OÐljak; II, Mt. Galicˇica; III, Mt. Lovc´en; IV, Mt. Zeletin; V, Mt. Bjelasica; VI, Mt. Zlatibor – Mt. PeÐter. Mountain systems classified according to Stevanovic´ [32]: dark grey, Dinaric Alps; medium grey, Balkan-Rhodope mountains; light grey, ScardoPindic mountains. Table 1. Location and Habitat Description of the Studied Pinus heldreichii Populations I and II from the Scardo-Pindic Mountain System (see also Fig. 1)
Location Latitude ( N ) Longitude ( E) Altitude [m] Exposure Terrain inclination [8] Geologic substratum
Population I
Population II
Mt. OÐljak 42811’ 57’’ 20855’21’’ 1885 S < 60 Limestone
Mt. Galicˇica 40856’37’’ 20849’48’’ 1929 E < 40 Limestone schist
Terpene Composition of the P. heldreichii Populations from the Scardo-Pindic Mountains. The relative contents of the different terpene classes represented in the needle essential oils of Populations I and II from OÐljak and Pelister, respectively, are given in Table 2. In the overall terpene profile, mono- and sesquiterpenes were found to dominate, comprising in average 92.8% of the essential-oil composition, whereas diterpenes had an average relative content of 5.3%. A total of 66 individual components were identified out of the 68 compounds detected (Table 2). The dominant constituents were germacrene D (28) 1), limonene 1)
Italic numbers in parentheses refer to the entries in Table 2.
298
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 2. Chemical Composition of the Needle Essential Oils of Pinus heldreichii Populations I and II from OÐljak and Galicˇica Mountains, respectively Entry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
Compound name and class a )
(E )-Hex-2-enal (Z )-Hex-3-en-1-ol Hexan-1-ol Tricyclene a-Pinene Camphene Sabinene b-Pinene b-Myrcene Limonene trans-b-Ocimene g-Terpinene a-Terpinolene Bornyl acetate trans-Verbenyl acetate a-Terpinyl acetate a-Copaene b-Cubebene b-Elemene Longifolene b-Caryophyllene Calarene Guaia-6,9-diene a-Humulene trans-Muurola-3,5-diene trans-b-Farnesene g-Muurolene Germacrene D g-Amorphene a-Muurolene g-Cadinene d-Cadinene trans-Longipinocarveol Germacrene D-4-ol Caryophyllene oxide Biotol t-Cadinol a-Muurolol a-Cadinol Khusinol trans,trans-Farnesol Benzyl benzoate 14-Hydroxy-a-muurolene Farnesyl acetate Benzyl salicylate (5E,9E )-Farnesyl acetone Sandaracopimara-8(14),15-diene
Content [%] b )
Average content [%] c )
Population I
Population II
0.64 0.38 0.47 0.35 0.10 0.08 0.21 0.05 13.21 4.79 0.68 0.19 0.13 0.02 4.19 1.85 2.11 0.22 24.70 3.56 0.24 0.31 0.03 0.00 0.13 0.11 0.12 0.24 0.02 0.03 0.25 0.18 0.07 0.01 0.06 0.01 0.12 0.03 0.03 0.03 8.07 1.75 0.43 0.09 0.17 0.03 1.36 0.25 0.05 0.01 0.07 0.18 0.08 0.11 32.09 6.87 0.04 0.01 0.28 0.05 0.15 0.05 0.35 0.10 0.07 0.04 0.76 0.29 0.06 0.03 0.02 0.01 0.08 0.04 0.02 0.03 0.07 0.02 0.12 0.04 0.15 0.21 0.02 0.02 0.11 0.05 0.03 0.03 0.01 0.01 0.09 0.03 0.13 0.25
0.26 0.20 0.27 0.14 0.08 0.06 0.31 0.08 22.17 3.65 1.18 0.24 0.17 0.03 7.21 1.64 2.79 0.28 31.86 2.90 0.01 0.01 0.03 0.01 0.16 0.04 0.01 0.02 0.00 0.01 0.44 0.19 0.06 0.02 0.05 0.01 0.09 0.01 0.01 0.02 4.49 1.97 0.30 0.04 0.11 0.01 0.80 0.26 0.04 0.00 0.01 0.01 0.10 0.08 21.95 2.91 0.07 0.12 0.25 0.05 0.09 0.01 0.24 0.02 0.10 0.06 0.40 0.11 0.04 0.02 0.03 0.02 0.02 0.02 0.09 0.03 0.08 0.01 0.11 0.03 0.12 0.07 0.03 0.02 0.07 0.01 0.03 0.04 0.08 0.08 0.11 0.01 0.02 0.02
Populations I and II 0.52 0.38 0.41 0.31 0.10 0.07 0.25 0.08 16.20 6.13 0.85 0.32 0.14 0.03 5.20 2.27 2.34 0.41 27.09 4.77 0.17 0.27 0.03 0.01 0.14 0.10 0.08 0.20 0.02 0.03 0.31 0.20 0.06 0.02 0.05 0.01 0.11 0.03 0.02 0.03 6.88 2.48 0.38 0.10 0.15 0.04 1.17 0.36 0.05 0.01 0.05 0.15 0.09 0.10 28.71 7.56 0.05 0.07 0.27 0.05 0.13 0.05 0.31 0.10 0.08 0.05 0.64 0.30 0.05 0.03 0.03 0.01 0.06 0.05 0.04 0.04 0.07 0.02 0.12 0.03 0.14 0.17 0.02 0.02 0.10 0.04 0.03 0.03 0.04 0.06 0.09 0.03 0.10 0.21
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
299
Table 2 (cont.) Entry
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68
Compound name and class a )
(E,Z)-Geranyl linalool Kaur-15-ene (E,E)-Geranyl linalool Thunbergol Abietadiene Abienol Abieta-8(14),13(15)-diene Sandaracopimarinal Unknown 1 Sclareol 7a-Hydroxymanool Sandaracopimarinol Dehydroabietal Tricosane Isopimarol Torulosol Pimaric acid Kauran-18-oic acid Triacontane Unknown 2 Octacosan-1-ol Total Monoterpene hydrocarbons Oxygenated monoterpenes Total Monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Total Sesquiterpenes Diterpene hydrocarbons Oxygenated diterpenes Total Diterpenes Others d ) Unknown
Content [%] b )
Average content [%] c )
Population I
Population II
0.04 0.04 0.19 0.04 0.04 0.02 0.72 0.26 0.08 0.11 0.04 0.05 0.03 0.02 0.12 0.04 0.05 0.03 0.34 0.10 0.10 0.07 0.06 0.06 0.09 0.06 0.07 0.03 0.65 0.29 0.02 0.04 3.11 1.78 0.77 0.79 0.07 0.09 0.04 0.07 0.99 1.57
0.01 0.02 0.18 0.02 0.08 0.01 0.90 0.52 0.08 0.03 0.00 0.01 0.05 0.02 0.03 0.02 0.06 0.04 0.43 0.08 0.00 0.00 0.18 0.08 0.09 0.09 0.03 0.05 0.46 0.17 0.11 0.08 0.08 0.22 0.18 0.44 0.04 0.13 0.06 0.18 0.00 0.00
Populations I and II 0.03 0.03 0.19 0.03 0.05 0.02 0.78 0.37 0.08 0.09 0.03 0.04 0.04 0.02 0.09 0.06 0.05 0.03 0.37 0.10 0.07 0.08 0.10 0.09 0.09 0.07 0.06 0.04 0.58 0.27 0.05 0.07 2.10 2.05 0.57 0.74 0.06 0.10 0.05 0.12 0.66 1.35
100.00
100.00
100.00
45.63 0.39 46.02 43.41 1.59 45.00 6.15 0.08 6.51 2.39 0.08
65.90 0.45 66.35 28.64 1.19 29.83 2.65 0.13 2.90 0.79 0.13
52.39 0.41 52.80 38.49 1.46 39.95 4.98 0.10 5.30 1.85 0.10
a ) Literature (trivial) names rather than fully systematic names are given. b ) Contents are given as percentages (mean standard deviation) relative to the total essential-oil composition; for the population details, see Table 1. c ) Average contents of Populations I and II (mean standard deviation). d ) Aliphatic aldehydes and alcohols, aromatic acid esters, and n-alkanes.
(10), and a-pinene (5), comprising together in average 72.0% of the total essential-oil composition. b-Caryophyllene (21), b-pinene (8), b-myrcene (9), pimaric acid (64), ahumulene (24), camphene (6), thunbergol (51), octacosanol (68), germacrene D-4-ol (34), isopimarol (62), kauran-18-oic acid (65), and (E)-hex-2-enal (1) were found to be present in medium-to-high amounts (0.5 – 10%, according to [34]). The compounds identified in very low amounts were found to comprise in average 5.7% of the total essential oil composition (Table 2).
300
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
The average chemical profile of the five main terpene components in the needle oils of P. heldreichii Populations I and II (present results) was germacrene D > limonene >> a-pinene >> b-caryophyllene > b-pinene (Tables 2 and 3), where, according to Petrakis et al. [26], > and >> represent relative differences in the oil contents of 1.1 – 5.0 and 5.1 – 15.0%, respectively. Interpopulation Variability of the Terpene Composition of the Scardo-Pindic P. heldreichii Populations I and II. The needle oil of Population I (OÐljak) was found to have significantly higher contents of b-caryophyllene (21), a-humulene (24), germacrene D (28), pimaric acid (64), and 17 additional components (P 0.05, LSD test, results were not presented; Table 2) than that of Population II. Alternatively, the oil of Population II (Galicˇica) was found to contain significantly higher amounts of a-pinene (5), b-pinene (8), b-myrcene (9), limonene (10), and nine other components than that of Population I. As in previous studies [31], no correlations between the geological structures and the needle-terpene profiles were observed in these two populations either. Population I had a needle-terpene profile similar to the average profile presented above, i.e., germacrene D >> limonene >> a-pinene >> b-caryophyllene > b-pinene, whereas Population II showed a different sequence of the most abundant components in its terpene profile, i.e., limonene >> a-pinene ¼ germacrene D >> b-pinene > bcaryophyllene, where ¼ represents a relative difference in the oil contents of 0.1 – 1.0% [26]. Trimodal Frequency Distribution of the Scardo-Pindic P. heldreichii Populations I and II. When testing the normality of the distribution of each of the 68 components, it was observed that the needle-oil content of 33 components deviated from normal distribution (n ¼ 24, c2 values ranging from 8.50 – 97.50, d.f. ¼ 3, P < 0.05). The frequency distribution of 21 of them suggested a monogenic type of heredity (n ¼ 24, c2 values ranging from 1.00 – 7.50, d.f. ¼ 3, P > 0.05). Frequencies of putative alleles determining high and low concentrations of terpenes were estimated from the frequency histograms, assuming no dominance [35]. Based on a genetic model [31] [36] [37], a trimodal frequency distribution was ascertained for many compounds, e.g., bornyl acetate (14), a-copaene (17), b-cubebene (18), and longifolene (20; shown in Fig. 2), as well as others. There was a single locus with two alleles (L and H), leading to three genotypes, i.e., homozygous LL and HH, producing low and high amounts of terpene compound, respectively, and heterozygous LH, producing a medium amount of this terpene. For example, in the case of bornyl acetate, a trimodal frequency distribution was accounted by assigning distribution intervals of 0.1 – 0.3, 0.3 – 0.7, and > 0.7% to the LL, LH, and HH genotypes, respectively. Although the frequencies of the above-mentioned compounds corresponded to the distribution defined by the law of inheritance, at this stage, it was difficult to prove that these components were under genetic control of a single locus with two alleles [38 – 43], because their contents were very low. Comparison of the Terpene Profile between Scardo-Pindic and Dinaric Populations. The terpene profile of the Scardo-Pindic Population II (limonene >> a-pinene ¼ germacrene D >> b-pinene > b-caryophyllene) was similar to that of Populations III, IV, V, and VI from Lovc´en, Zeletin, Bjelasica, and Zlatibor-PeÐter, respectively [31], located in the Dinaric Alps [32] (limonene >> a-pinene > germacrene D >> b
Dinaric
Dinaric Dinaric
Dinaric Scardo-Pindic Scardo-Pindic Scardo-Pindic Scardo-Pindic BalkanRhodope
[20]
[27]
[31]
[24]
[23]
Present results
[26]
[46]
[30]
17.3 20.3
Bjelasica Lovc´en
Pirin Slavjanka
Pinds
Katara
OÐljak Galicˇica
ara
18.0 17.3
11.1
13.8
13.2 22.2
8.8
13.3
16.1
Zeletin
Prokletije
12.9
10.2
10.0 7.3
a-Pinene
6.2 5.4
3.6
4.2
4.2 7.2
–
4.0
6.5
6.0
4.8
4.5
3.0
3.0 2.4
b-Pinene
37.1 46.5
23.7
34.3
24.7 31.9
22.5
67.0
25.1
29.7
23.2
29.5
52.8
43.8 28.0
Limonene
– –
8.6
8.4
8.1 4.5
12.0
2.2
8.9
11.4
11.0
11.1
7.7
11.9 11.6
b-Caryophyllene
– –
21.3
12.8
32.1 22.0
43.5
tr
15.4
11.4
13.1
16.3
15.8
9.6 33.0
Germacrene D
Content of main terpene compounds [%] a )
ZlatiborPeÐter
Prenj ( RujiÐta)
Trebevic´ Prenj ( RujiÐta)
Mountains
22.0 15.1
–
b-Farnesene (7.1%), d-car-3-ene (3.7%) b-Farnesene (5.1%), d-car-3-ene (4.8%)
d-Car-3-ene (18.6%), myrcene 82.2%), terpinolene (2.1%)
Aristolene (6.0%), d-car-3-ene (2.8%), b-myrcene (2.5%), camphene (1.5%)
b-Myrcene (2.1%), pimaric acid (3.1%) b-Myrcene (2.8%) – – –
d-Cadinene (7.6%), sabinene (1.5%)
a-Terpineol (1.9%)
d-Cadinene (2.5%), a-muurolene (2.3%), a-humulene (2.2%), b-myrcene (2.2%), b-gurjunene (1.8%), g-muurolene (1.7%) ( E )-Hex-2-enal (2.6%), a-humulene (2.2%), d-cadinene (1.9%), a-muurolene (1.8%), b-myrcene (1.8%) b-Myrcene (2.5%), a-humulene (2.2%), ( E )-hex-2-enal (2.0%), a-muurolene (1.5%). b-Myrcene (2.2%), a-humulene (1.9%), d-cadinene (1.7%), b-gurjunene (1.6%), a-muurolene (1.6%)
b-Myrcene (1.5%), a-humulene (1.5%)
a-Humulene (1.9%) 1,10-Epoxygermacrene D (3.6%), g-cadinene (1.5%)
–
–
–
–
–
–
–
– –
g-Muurolene
Other terpenes b )
a ) Content of main terpenes present in the needle essential oils given as percentages relative to the total essential-oil composition; tr, trace amount; –, not detected. b ) Other terpenes present in the oils at contents 1.5%; contents [%] are given in parentheses.
Mountain System
Reference
Table 3. Terpene Composition of Pinus heldreichii Needle Oils: Comparison of the Present with Literature Results
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015) 301
302
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Fig. 2. Frequency distribution of a) bornyl acetate (14)1), b) a-copaene (17), c) b-cubebene (18), and d) longifolene (20) in two populations of Bosnian pine from the Scardo-Pindic mountains (Populations I and II; ca. 24 trees) suggesting trimodality. Single locus with two alleles leading to three genotypes: homozygous (LL and HH), producing low and high amounts of terpene, and heterozygous (LH), producing medium amounts of this terpene.
caryophyllene > b-pinene; Tables 2 and 3). In a recent study of the terpene composition of Macedonian pine (Pinus peuce), the terpene profile of the population from Mt. Pelister (Scardo-Pindic mountains) was also found to be similar to that of the populations from the Dinarides [44]. Multivariate Statistical Analyses. For the principle-component analysis (PCA) and cluster analysis (CA) of the terpene profiles of Populations I – VI, seven terpene compounds, whose distribution was normal (c2 test; P 0.05) and which did not display statistically important differences between the standard deviations of the populations (Levenes test; P 0.05), were selected, i.e., b-myrcene, limonene, b-elemene, bcaryophyllene, a-humulene, d-cadinene, and germacrene D-4-ol. The PCA was performed based on a correlation matrix computed for all 121 tree samples, with the contents of the individual compounds expressed as percentage of the total terpene fraction (Fig. 3). The first two principal axes represented 75% of the total variation. According to the contents of the seven selected compounds, the geographically most distant population, i.e., Population II from the Scardo-Pindic mountain system, was clearly separated from the Scardo-Pindic Population I and the four natural populations from the Dinaric Alps (Populations III, IV, V, and VI), a finding that was confirmed by CA (Fig. 4). Genetic Analysis of Compounds from Dinaric and Scardo-Pindic Populations. Genetic analysis was carried out using a-pinene, which showed the trimodal distribution most clearly. To check whether some other components of Bosnian pine needle oils from the investigated part of its habitat area (6 populations) show signs of
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
303
Fig. 3. Principle-component analysis of seven selected terpenes isolated from 121 Bosnian pine samples from six populations. Populations: *, Population I (Mt. OÐljak); ^, Population II (Mt. Galicˇica); ~, Population III (Mt. Lovc ´ en); ^, Populaton IV (Mt. Zeletin); *, Population V (Mt. Bjelasica); 1 n, Population VI (Mt. Zlatibor – Mt. PeÐter). Terpenes: b-myrcene (9) ), limonene (10), b-elemene (19), b-caryophyllene (21), a-humulene (24), d-cadinene (32), and germacrene D-4-ol (34).
Fig. 4. Dendrogram obtained by cluster analysis based on the nearest-neighbor method (squared Euclidean distances) of the contents of seven selected terpenes in the needle oils of P. heldreichii Populations I – VI. The numbers on the vertical axis refer to the distance level, calculated on the basis of the differences between population contents of the selected components. For the geographical location of Populations I – VI, see Fig. 1.
monohybrid inheritance of the terpene content, all the components with contents deviating from the normal distribution were analyzed. Among the components with a larger content range, those with histograms suggesting genetic control (existence of
304
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
trimodal distribution) were chosen. a-Pinene was the most suitable compound, because its contents ranged from small amounts to dominance (3.36 – 54.92%), its distribution deviated from the norm (n ¼ 121, c2 ¼ 13.02, P < 0.05), and it was the only component whose experimental chemotype (genotype) frequency in every population matched the frequencies predicted by the Hardy–Weinberg (HW) law of inheritance (Table 4). The experimental frequencies of the three modalities (chemotypes or genotypes: LL ¼ low concentration, LH ¼ medium concentration, and HH ¼ high concentration) were previously matched against the frequencies expected according to the HW law (using the c2 compatibility test with the HW law). Frequencies of putative alleles determining low (L) and high (H) concentrations of this terpene were estimated from frequency histograms, assuming no dominance [45]. Fig. 5 shows an example of a-pinenefrequency histograms for Population III (Lovc´en). The trimodal a-pinene-frequency distribution found in this population was accounted for by assigning percentage intervals of 0 – 20, 20 – 40, and more than 40% to the LL, LH and HH genotypes, respectively. A dendrogram based on UPGMA clustering of Rogers genetic distances (as modified by Wright) [46] is shown in Fig. 6. Cluster analysis showed a partial divergence of two Dinaric populations (Populations IV and V, form Zeletin and Bjelasica, resp.) from the other Dinaric and the Scardo-Pindic populations. According to a very recent study of the essential-oil composition and the genetic distance of Macedonian pine (Pinus peuce), partial divergence between populations from the Dinaric Alps and the Scardo-Pindic mountains was also found [44]. Comparison of the Present with Literature Results. Due to limonene as the dominant needle-oil terpene, the oil of Population II (Galicˇica) was similar to those of the populations from the Dinaric Alps [20] [27] [31] [24], Scardo-Pindic mountains [26] [47], and the Balkan-Rhodope massif [30] (Table 3). When regarding the complete terpene profile, the oil of Population II was most similar to that of a population from Katara (Greece) [26] and those of several populations from Montenegro [31], and the least similar to those of two populations from the Balkan-Rhodope mountain system, which lacked b-caryophyllene and germacrene D, but contained g-muurolene in their Table 4. Frequencies of Genotypes ( LL, LH, and HH ) and Alleles ( L and H ) Determined from the aPinene Content of the Needle Oils of Pinus heldreichii Populations I – VI Population a )
Number of trees sampled (n) Number of oils with genotype LL Number of oils with genotype LH Number of oils with genotype HH c2 P Frequency of allele L c ) Frequency of allele H d )
I
II
III b )
IV
V
VI
16 7 8 1 2.00 > 0.05 0.69 0.31
8 2 5 1 – > 0.05 0.56 0.44
30 20 8 2 14.80 > 0.05 0.80 0.20
30 1 10 19 6.00 > 0.05 0.20 0.80
30 4 14 12 2.00 > 0.05 0.36 0.64
7 3 3 1 – > 0.05 0.64 0.36
a ) Populations: I, OÐljak; II, Galicˇica; III, Lovc´en; IV, Zeletin; V, Bjelasica; VI, Zlatibor-PeÐter; for the location of the populations, see Fig. 1. b ) Results for Population III are also presented in Fig. 5. c ) Frequency of allele L ¼ (2LL þ LH)/2n. d ) Frequency of allele H ¼ (2 HH þ LH )/2n.
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
305
Fig. 5. Frequency distribution of the a-pinene content in Population III (30 trees), suggesting trimodality. Single locus with two alleles leading to three genotypes: homozygous (LL and HH) and heterozygous (LH), producing low, high, and medium amounts of a-pinene, respectively. The trimodal a-pinene frequency distribution found in this population was accounted for by assigning percentage intervals of 0 – 20, 20 – 40, and more than 40% to the LL, LH, and HH genotypes, respectively.
Fig. 6. Dendrogram obtained by UPGMA clustering based on Rogers genetic distances as modified by Wright of estimated allele frequencies (L and H) of a-pinene in Pinus heldreichii Populations I – VI. The numbers on the horizontal axis refer to the distance level. For the geographical location of Populations I – VI, see Fig. 1.
terpene profile [30]. By its dominant oil component germacrene D, the oil of Population I (OÐljak) was similar to that of Bosnian pines from Prenj [20] and Mt. ara [23]. To better understand whether the differences found in the terpene composition of the investigated natural populations of Bosnian pine growing on different mountain systems are caused predominantly by genetic or environmental factors, further phytochemical and molecular investigations are needed. The differences in the abundance of major oil components and the terpene profiles of P. heldreichii between the present and literature results might be attributed to differences in the needlesampling technique, time of collection, method of essential-oil extraction, chromatographic processing, and, especially, in the number of individuals analyzed. Tectonic collisions of the Prokletije massif with the Scardo-Pindic mountain system in the pliocene and glaciation in the pleistocene, which allowed the survival of many relict species [48], might also have influenced the divergence of the Bosnian pine populations [31] (and refs. cit. therein), which should be tested by further genetic analyses.
306
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Conclusions. – According to the general terpene profile and seven terpene components selected for the multivariate analyses, the needle oil of Population II from the Scardo-Pindic mountain system (present investigation) was clearly different from those of Populations III, IV, V, and VI from the Dinaric Alps and Population I from the Scardo-Pindic mountains (present investigation). A fact, which was not confirmed by genetic analysis of a-pinene. In future research, the genetic distance of other terpene components should also be examined. In addition, the combination of morphological, phytochemical, and molecular analyses of P. heldreichii populations from the whole area of limited natural distribution are necessary and planned, to highlight phylogeographic and historical relationships of more or less distant and fragmented populations of this very interesting pine species. This work is part of two research projects (173029 and 173021) supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia.
Experimental Part Plant Material. Twigs with needles from the lowest third of the tree crown were collected in late summer 2012 from 16 and 8 randomly selected trees growing on Mt. OÐljak (Population I) and Mt. Galicˇica (Population II), resp. The collected twigs were stored at 208, and voucher specimens have been deposited with the Institute of Forestry, Belgrade, Serbia. Isolation of Essential Oils. Two-year-old needles, stored in a freezer at 208 until extraction, were cut into pieces of 2 – 3 mm length and extracted with pentane (1 g needles/ml solvent). The extracts were kept at 4 – 68 for 24 h, then filtered, and stored in chromatography vials with solid caps in a refrigerator until further analysis. GC-FID Analysis. The GC-FID analyses were carried out with an Agilent Technologies 7890A apparatus equipped with a split/splitless injector, an automatic liquid sampler (ALS), a flame ionization detector (FID), and a HP-5 fused-silica cap. column (30 m 0.32 mm i.d., film thickness 0.25 mm). The oven temp. was programmed linearly rising from 40 to 2808 at 4 8/min; injector temp., 2508; detector temp., 2808; carrier gas, H2 (1 ml/min). Samples (1 ml) were injected in splitless mode (1 : 15) using the ALS. GC/MS Analysis. The GC/MS analyses were performed with a Hewlett Packard G1800C-GCD apparatus equipped with a split/splitless injector, ALS, a mass-selective detector, and a HP-5 MS fusedsilica cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm). The chromatographic conditions were the same as described above (cf. GC-FID Analysis), except for the carrier gas, which was He. Electronimpact mass spectra (EI-MS; 70 eV) were acquired over the m/z range 40 – 450 amu. Compound Quantification and Identification. For the quantification of the contents of the needle-oil components, area-percent reports obtained by GC-FID were used as a base. The identification of the oil components was based on the comparison of their retention indices (RIs) and mass spectra with those previously reported [49] or listed in the mass-spectral libraries Wiley-275 and NIST/NBS. Statistical Analysis. The calculation of arithmetic means and standard deviations (SD) of the populations, frequencies, histograms, test for normality (c2 test), LSD test, one-way analyses of variance (ANOVA), Levenes test, principal-component analyses (PCA), as a descriptive multivariate method capable of suggesting the structure and tendency of the data set, and cluster analysis were all carried out with the software Statgraphics Plus (version 5.0; Statistical Graphics Corporation, USA).
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
307
REFERENCES ˇ etinjacˇe: Morfologija i Varijabilnost, JAZU i SveucˇiliÐna naklada Liber, Zagreb, [1] M. Vidakovic´, in C 1982, p. 428. [2] P. Fukarek, umarski list (Zagreb) 1960, 5 – 6, 152. [3] P. Fukarek, umarski list (Zagreb) 1960, 11 – 12, 393. [4] P. Fukarek, M. Vidakovic´, Naucˇno druÐtvo Bosne i Hercegovine 1965, XXVIII, 68. [5] A. Tucovic´, S. Stilinovic´, Genetika (Belgrade) 1972, 4, 193. [6] P. Fukarek, M. Nikolic´, SANU and ANUBiH (Belgrade) 1974, I, 20. [7] T. Nikolovski, J. Matvejeva, MorfoloÐke karakteristike apofize munike (Pinus heldreichii Christ) sa podrucˇja Makedonije i Kosova, Zbornik radova, Simpozijum o munici Pinus heldreichii Christ, 4 – 7. IX 1972, Decˇani (Kosovo), Pec´, Belgrade, 1975, p. 258. [8] N. T. Mirov, Composition of Gum Turpentines of Pines, U. S. Department of Agriculture Technical Bulletin No. 1239’, U. S. Government Printing Office, Washington, 1961. [9] B. Pejoski, umarski list (Zagreb) 1951, 1 – 2, 85. [10] B. Pejoski, umarski list (Zagreb) 1953, 11, 450. [11] N. Iconomu, G. Valkanas, Pharm. Acta Helv. 1966, 41, 59. [12] D. Karapandzˇic´, N. Jovanovic´, Hemijski sastav munike (Pinus heldreichii), Simpozijum o munici Pinus heldreichii Christ, 4 – 7. IX 1972, Decˇani (Kosovo), Pec´, Zbornik radova, Belgrade, 1972, p. 439. [13] D. Karapandzˇic´, N. Jovanovic´, Glasnik umarskog fakulteta (Belgrade) 1975, 48 (B), 59. [14] G. Weißmann, Holz Roh-Werkstoff 1973, 31, 341. [15] D. Fengel, G. Wegener, in Wood: Chemistry, Ultrastructure, Reactions, Walter de Gruyter, Berlin, 1984, p. 26. [16] M. Spanoudaki, Diplomarbeit, Hamburg, 1993. [17] W. Lange, T. Stevanovic´-Janezˇic´, M. Spanoudaki, Phytochemistry 1994, 36, 1277. [18] X. R. Wang, Y. Tsumura, H. Yoshimary, K. Nagasaka, A. E. Szmidt, Am. J. Bot. 1999, 86, 1742. [19] N. R. Menkovic´, M. S. Ristic´, Z. J. Samardzˇic´, N. N. Kovacˇevic´, S. R. Tasic´, Acta Hort. 1993, 344, 578. [20] J. Chalchat, R. P. Garry, M. S. Gorunovic´, Pharmazie 1994, 49, 852. [21] D. Karapandzˇic´, Ph.D. Thesis, University of Belgrade, 1964. [22] D. Karapandzˇic´, J. Serb. Chem. Soc. 1966, 31, 473. [23] N. Simic´, R. Palic´, S. Andjelkovic´, V. Vajs, S. Milosavljevic´, J. Essent. Oil Res. 1996, 8, 1. [24] T. M. Stevanovic´-Janezˇic´, D. M. Vilotic´, Lekovite sirovine (Belgrade) 1998, 47, 109. [25] P. Petrakis, O. Ortiz, O. Tzakou, M. Couladis, C. Vagias, V. Roussis, Chemical variability of the foliar metabolites and chemotaxonomic studies on pine trees growing in Greece, First Conference on Medicinal and Aromatic Plants of Southeast European Countries & VI Meeting Days of Medicinal Plants, Book of Proceedings, Arandjelovac, Yugoslavia, 2000, L 15. [26] P. V. Petrakis, C. Tsitsimpikou, O. Tzakou, M. Couladis, C. Vagias, V. Roussis, Flavour Fragrance J. 2001, 16, 249. [27] S. Maric´, M. Jukic´, V. Katalinic´, M. MiloÐ, Molecules 2007, 12, 283. [28] K. D. Naidenov, G. G. Markov, in Population Genetics and Genetic Conservation of Forest Trees, Eds. P. Baradat, W. T. Adams, G. Mller-Starck, SPB Academic Publishing, Amsterdam, 1995, p. 165. [29] K. D. Naydenov, Co-relationship between terpenes in the essential oil of palebark Heldreichii pine (Pinus heldreichii Christ.) determined through CGC in different provenances in Bulgaria, 18th International Symposium on Capillary Chromatography, May 1996, Riva del Garda, Italy, Book of Proceedings, Eds. P. Sandra, G. Devos, Dr. Alfred Huethig Publishing GmbH, Heidelberg, 1996, Vol. II, p. 1247. [30] K. D. Naydenov, F. M. Tremblay, Y. Bergeron, A. Alexandrov, N. Fenton, Biochem. Syst. Ecol. 2005, 33, 133. [31] B. Nikolic´, M. Ristic´, S. Bojovic, P. D. Marin, Chem. Biodiversity 2007, 4, 905. [32] V. Stevanovic´, Bocconea 1996, 5, 77.
308
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
[33] P. K. Koukos, K. I. Papadopoulou, D. T. Patiaka, A. D. Papagiannopoulos, J. Agric. Food Chem. 2000, 48, 1266. [34] E. Von Rudloff, Biochem. Syst. Ecol. 1975, 2, 131. [35] S. Bojovic, M. Jurc, D. Drazic, P. Pavlovic, M. Mitrovic, L. Djurdjevic, R. S. Dodd, Z. Afzal-Rafii, M. Barbero, Trees 2005, 19, 531. [36] E. Zavarin, K. Snajberk, L. Cool, Biochem. Syst. Ecol. 1990, 18, 117. [37] L. G. Cool, E. Zavarin, Biochem. Syst. Ecol. 1992, 20, 133. [38] R. Yazdani, R. Rudin, T. Aldn, L. Lindgren, B. Harbom, K. Ljung, Hereditas 1982, 97, 261. [39] R. Yazdani, J. E. Nilsson, T. Ericsson, Silvae Genet. 1985, 34, 201. [40] S. H. Strauss, W. D. Critchfield, Forest Sci. 1982, 28, 687. [41] A. E. Squillace, O. O. Wells, O. Rockwood, Silvae Genet. 1980, 29, 141. [42] E. E. White, Phytochemistry 1983, 23, 1399. [43] C. Bernard-Dagan, Biosynthesis of Lower Terpenoids: Genetic and Physiological Controls in Woody Plants, Plenum Press, New York, 1988; J. W. Hanover, D. E. Keathley, in Genetic Manipulation of Woody Plants, Plenum Press, New York, 1988, p. 329. [44] B. Nikolic´, M. Ristic´, S. Bojovic´, V. Matevski, Z. KrivoÐej, P. D. Marin, Chem. Biodiversity 2014, 11, 934. [45] Z. A. Rafii, R. S. Dodd, E. Zavarin, Biochem. Syst. Ecol. 1996, 24, 325. [46] F. J. Rohlf, NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, Exter Software, New York, 1990. [47] E. Ioannou, A. Koutsaviti, O. Tzakou, V. Roussis, Phytochem. Rev. 2014, 13, 741. [48] P. D. Hughes, J. C. Woodward, P. L. Gibbard, Prog. Phys. Geogr. 2006, 30, 334. [49] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th edn., Allured Publishing Corporation, Carol Stream, IL, 2007. Received April 1, 2014
