Essential oil composition of Hypericum L. species from Southeastern Serbia and their chemotaxonomy

Biochemical Systematics and Ecology 35 (2007) 99e113 www.elsevier.com/locate/biochemsyseco

Essential oil composition of Hypericum L. species from Southeastern Serbia and their chemotaxonomy Andrija Smelcerovic a, Michael Spiteller a,*, Axel Patrick Ligon a, Zaklina Smelcerovic a, Nils Raabe b a

Institute of Environmental Research, University of Dortmund, Otto-Hahn-Str. 6, 44221 Dortmund, Germany b Department of Statistics, University of Dortmund, Vogelpothsweg 87, 44221 Dortmund, Germany Received 25 March 2005; accepted 16 September 2006

Abstract The essential oils of the aerial parts of nine species of Hypericum (Hypericum barbatum, Hypericum hirsutum, Hypericum linarioides, Hypericum maculatum, Hypericum olympicum, Hypericum perforatum, Hypericum richeri, Hypericum rumeliacum and Hypericum tetrapterum), collected from different locations in Southeast Serbia, were obtained by steam distillation and analyzed by GC and GCeMS. The essential oils investigated were characterized by a high content of non-terpene compounds and a low content of monoterpenes. The contents of non-terpenes, monoterpenes and sesquiterpenes in oils of the species H. barbatum, H. richeri and H. rumeliacum (section Drosocaprium) were similar and these oils were characterized by high contents of fatty acids. The oils of H. hirsutum and H. linarioides (section Taeniocarpium) contained a high percentage of n-nonane. There were similarities in contents of non-terpenes and sesquiterpenes in oils of species that belong to the section Hypericum (H. maculatum, H. perforatum and H. tetrapterum). The oil of H. olympicum differed from others by higher terpene content. A comparison was also carried out of the chemical composition of the essential oils from flower, leaf and stem of H. perforatum and it revealed that the highest concentration of non-terpene compounds was found in the flower and stem oil, while a high concentration of sesquiterpenes was characteristic for leaf oil. There were significant differences in the concentrations of the same compounds in the essential oils of H. maculatum, H. olympicum and H. perforatum, collected in different years from the same location which could be explained by seasonal differences. All data were statistically processed with principal component analysis and cluster analysis. The main conclusion from the above data is that genetic and environmental factors both play a role in determining the composition of essential oils of the Hypericum species studied. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Hypericum barbatum; Hypericum hirsutum; Hypericum linarioides; Hypericum maculatum; Hypericum olympicum; Hypericum perforatum; Hypericum richeri; Hypericum rumeliacum; Hypericum tetrapterum; Essential oil composition

* Corresponding author. Tel.: þ49 2317554080; fax: þ49 2317554085. E-mail address: [email protected] (M. Spiteller). 0305-1978/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2006.09.012

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1. Introduction Hypericum L. is a genus of about 400 species, widespread in warm-temperature areas throughout the world and well represented in the Mediterranean area (Robson and Strid, 1986). Plants of the genus Hypericum have traditionally been used as medicinal plants in various parts of the world (Yazaki and Okada, 1994). Hypericum perforatum occupies a special position among the species of Hypericum. The chemical composition of H. perforatum oil has been the subject of many publications (Cakir et al., 1997; Baser et al., 2002; Osinska, 2002; Schwob et al., 2002a; Mockute et al., 2003; Smelcerovic et al., 2004). The content of the oil depends on the origin of the plant. Thus, a-pinene was the most abundant component of the oil of H. perforatum from Turkey (61.7 %) (Cakir et al., 1997) and b-caryophyllene of the oil from Uzbekistan (11.7%) (Baser et al., 2002). The two monoterpenes (a- and b-pinene) made up to 70% of the leaf essential oil of H. perforatum from India (Weyerstahl et al., 1995). The H. perforatum oils from Lithuania have been classified into three chemotypes: b-caryophyllene, caryophyllene oxide and germacrene D (Mockute et al., 2003). Considerable variation has already been reported in oil composition among different populations of H. perforatum from Serbia (Mimica-Dukic et al., 1997). The essential oil content of many other Hypericum species has been described: Hypericum androsaemum (Guedes et al., 2003), Hypericum brasiliense (Abreu et al., 2004), Hypericum coris (Schwob et al., 2002b), Hypericum dogonbadanicum (Sajjadi et al., 2001), Hypericum foliosum (Santos et al., 1999), Hypericum heterophyllum (Cakir et al., 2004), Hypericum hircinum (Bertoli et al., 2000), Hypericum hyssopifolium (Cakir et al., 2004), Hypericum lanceolatum (Vera et al., 1996), Hypericum linarioides (Cakir et al., 2005), Hypericum maculatum (Vasilieva et al., 2003), Hypericum perfoliatum (Couladis et al., 2001), Hypericum richeri (Ferretti et al., 2005), Hypericum rumeliacum (Couladis et al., 2003), Hypericum scabrum (Cakir et al., 1997; Baser et al., 2002), Hypericum triquetrifolium (Bertoli et al., 2003). The flora of Serbia lists 19 species of Hypericum (Josifovic, 1972). Recently, the chemical composition has been determined of the essential oils of Hypericum atomarium (Gudzic et al., 2004), H. maculatum (Gudzic et al., 2002), Hypericum olympicum (Gudzic et al., 2001) and H. perforatum (Gudzic et al., 2001; Smelcerovic et al., 2004), all originating from Southeastern Serbia. The objective of this study was to determine the essential oil composition of nine wild-growing species of Hypericum (H. barbatum, H. hirsutum, H. linarioides, H. maculatum, H. olympicum, H. perforatum, H. richeri, H. rumeliacum and Hypericum tetrapterum) from the Southeastern region of Serbia and to examine their potential chemotaxonomic significance. The chemical composition of oils obtained from flower, leaf and stem of H. perforatum and of the oils of H. maculatum, H. olympicum and H. perforatum collected in years 1998, 2001 and 2003 are also discussed. 2. Materials and methods 2.1. Plant material Table 1 contains information concerning the species of Hypericum studied, the voucher numbers of the specimens deposited in the herbarium (Herbarium Moesicum Doljevac, Serbia and Montenegro), the site and date of collection, together with their taxonomic placement within sections of the genus Hypericum (Robson, 1977). All the plant samples were collected at bloom stage. Dried and ground drug was steam distilled for 2.5 h using a Clevenger apparatus. 2.2. Identification procedure The oils were analyzed by analytical GC and GCeMS. Constituents were identified by comparison of their retention times with standards and/or their mass spectra with those from the NIST MS library (Version 2.0a), Wiley MS library (Version 6) and the literature (Adams, 1989, 1995). The fatty acids and their methyl esters were identified by methylation of the essential oils. The solutions of essential oils in 2-propanol were derivatized with trimethylsulfonium hydroxide to yield the methyl esters of the fatty acids (Vosman et al., 1998; Ishida et al., 1999). The coherence of the retention indexes of the analyzed compounds with the retention indexes obtained with a DB-5 column (Adams, 1989, 1995) constituted an additional criterion in the confirmation of each compound.

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Table 1 Species of Hypericum from Southeast Serbia studied Sectiona

Codeb

Plant species

Voucher number(HMDc)

Collection period

Locality

Drosocarpium Spach.

A2003 B2003 C2003 D2003 D2001 D1998 EF2004 EL2004 ES2004 E2003 E2001 E1998 F2003 G2003 H2003 I2003 I2001 I1998

H. barbatum Jacq. H. richeri Vill. H. rumeliacum Boiss. H. maculatum Crantz H. maculatum Crantz. H. maculatum Crantz. Flower of H. perforatum L. Leaf of H. perforatum L. Stem of H. perforatum L. H. perforatum L. H. perforatum L. H. perforatum L. H. tetrapterum Fries. H. hirsutum L. H. linarioides Bosse. H. olympicum L. H. olympicum L. H. olympicum L.

705 715 717 711 701 e 728 728 728 714 703 e 722 708 709 713 702 e

July 2003 August 2003 July 2003 July 2003 June 2001 July 1998 June 2004 June 2004 June 2004 July 2003 June 2001 July 1998 August 2003 August 2003 August 2003 July 2003 June 2001 July 1998

Rtanj Suva planina Rujan planina Stara planina Vlasina Vlasina Pasina cesma Pasina cesma Pasina cesma Rujan planina Rujan planina Rujan planina Beljanica Suva planina Suva planina Rujan planina Rujan planina Rujan planina

Hypericum

Taeniocarpium Jaub. et Spach. Olympia (Spach.) Nyman

a b c

Taxonomic classification according to Robson (1977). This is the code used for identifying samples studied in Tables 2e4. Herbarium Moesicum Doljevac (Serbia and Montenegro).

2.2.1. Analytical GC A Thermo Finnigan Trace Gas Chromatograph, equipped with a fused silica capillary column (DB-5 30 m  0.25 mm  0.25 mm) and FID was used. The operating conditions were: temperature program, 60e320  C at 10  C/min and 320  C (4 min); injector temperature, 310  C; detector temperature, 320  C; carrier gas helium (1 mL/min); and split mode (1:25). 2.2.2. GCeMS Analyses were performed on a Thermo Finnigan Trace Gas Chromatograph and Trace MSPLUS detector, equipped with a fused silica column (DB-5 30 m  0.25 mm  0.25 mm); carrier gas helium (1 mL/min) with the same temperature program as for the analytical GC. Ionization was performed at 70 eV. Oil solutions were injected in two split modes (1:10 and 1:20). 2.3. Data analyses All data were statistically analyzed using statistical Software R (Foundation for Statistical Computing, Vienna, Austria, 2005, ISBN 3-900051-07-0). Analyses included principal component analysis and cluster analysis. 2.3.1. Principal component analysis A two-dimensional visualization of the position of the exemplars relative to each other was created by depicting the values of the first two principal components. The principal components are the axes of that orthogonal projection for which the values of the first axis have the highest possible variance, and those of the second have the second highest and so on (Hartung and Baerbel, 1999). 2.3.2. Cluster analysis For a clearer arrangement the compounds measured were grouped in a manner that assigned similar behaving, i.e. highly correlated compounds to the same groups. The hierarchical cluster analysis method ’’average linkage’’ has been applied to achieve such a grouping (Buttler, 2000).

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3. Results and discussion 3.1. Essential oil composition of nine species of Hypericum The compositions of the oils isolated from nine species of Hypericum are reproduced in Table 2. The oils were complex mixtures of non-terpenes, monoterpenes and sesquiterpenes: 98 components were identified in nine essential oils under study. The non-terpene compounds made up the higher contribution (43.5%) in the essential oil of H. barbatum (Table 2, code A2003). The content of two fatty acids (hexadecanoic and octadecadienoic) amounted to 18.0%. The sesquiterpenes amounted to 28.3% with hydrocarbons dominating (19.7%). The monoterpenes amounted to 12.7% and the content of oxygenated monoterpenes was relatively low amounting to 2.3%. The sesquiterpenes made up the highest contribution (41.2%) in the essential oil of H. richeri (Table 2, code B2003), and the oxygenated sesquiterpenes amounted to 24.9%. The monoterpenes amounted to 7.3%. The content of three fatty acids (dodecanoic, tetradecanoic and hexadecanoic) amounted to 27.0% in the essential oil of H. rumeliacum (Table 2, code C2003). The sesquiterpene hydrocarbons amounted to 26.5% and the content of oxygenated sesquiterpenes was 7.7%. The monoterpene fraction was relatively small (4.2%). The content of non-terpene compounds amounted to 48.2% in the essential oil of H. maculatum (Table 2, code D2003). The content of sesquiterpene hydrocarbons amounted to 9.4% and the content of oxygenated sesquiterpenes was 20.9%. In the essential oil of H. perforatum (Table 2, code E2003) the contents of non-terpenes, monoterpenes and sesquiterpenes compounds amounted to 44.1, 19.8 and 23.4%, respectively. The oxygenated monoterpenes amounted to 1.9%. The sesquiterpenoid fraction contained the highest proportion of oxygenated compounds (17.8%). In the essential oil of H. tetrapterum (Table 2, code F2003) the contents of non-terpenes, monoterpenes and sesquiterpenes amount to 32.3, 4.0 and 26.4%, respectively. The content of the hydrocarbons (11.6%) and oxygenated compounds (14.8%) was similar in the sesquiterpenoid fraction; while monoterpene hydrocarbons were not detected. The major components in the essential oil of H. hirsutum (Table 2, code G2003) were n-nonane and n-undecane amounting to 52.3%. The sesquiterpene hydrocarbons amounted to 7.9% and the oxygenated sesquiterpenes amounted to 5.1%. A low content of monoterpenes (0.5%) and a high content of n-nonane (40.5%) were the characteristics of this sample. In the essential oil of H. linarioides (Table 2, code H2003) the content of non-terpenes, monoterpenes and sesquiterpenes in the oil sample amounted to 40.1%, 18.2% and 18.1%, respectively. Hydrocarbons (10.6%) and oxygenated compounds (7.5%) in the sesquiterpenoid fraction did not differ much. Hydrocarbons predominated in the monoterpenoid fraction (15.6%). The sesquiterpenes formed the main fraction in the essential oil of H. olympicum (Table 2, code I2003) that amounted to 47.7% and the content of hydrocarbons (23.9%) and oxygenated compounds (23.8%) was almost identical. The content of monoterpenes amounted to 10.7%. The content of the non-terpene and terpene compounds was similar in the majority of oils particularly those from H. barbatum, H. linarioides, H. perforatum, H. rumeliacum and H. tetrapetrum. Terpenes made up the main fraction (58.4%) in the oil of H. olympicum. The oil of H. hirsutum contained the lowest amount of terpene compounds (13.5%). All oil samples exhibited a relative low content of monoterpenes. Their content ranged from 0.5% (H. hirsutum) to 19.8% (H. perforatum). This was in agreement with previous data on the content of monoterpenes in Hypericum oils of Serbian origin (Mimica-Dukic et al., 1997; Gudzic et al., 2001, 2002; Smelcerovic et al., 2004). Diterpenes were found in only three oil samples in this study: H. richeri (1.8%) and H. rumeliacum (1.1%), and H. tetrapterum (0.1%) which contrasts to earlier results of the investigation of oils of Hypericum species from Southeastern region of Serbia that did not reveal the presence of any diterpenes at all (Gudzic et al., 2001, 2002, 2004; Smelcerovic et al., 2004). A great variability was found in the oil composition of species investigated in this work. Thus, n-nonane was the major component in H. hirsutum and H. maculatum oils, a-pinene in H. linarioides oil, germacrene D in H. olympicum oil, 2-methyl-octane in H. perforatum oil and (E)-anethol in H. richeri oil. On the other hand, n-nonane was not found in H. olympicum and H. rumeliacum oils and a-pinene in H. tetrapterum oil. Germacrene D was found in

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Table 2 Compositions of the essential oils of nine species of Hypericum (value given in % weight fraction) Constituents

2-Methyl-octane n-Nonane a-Pinene Camphene 3-Methyl-nonane b-Pinene 6-Methyl-5-hepten-2-one cis-Meta-mentha-2,8-diene n-Decane n-Octanal Unknown Limonene (Z)-b-ocimene g-Terpinene 2-Methyl-decane 2,6-Dimethyl-3,5-heptanedione 2-Nonanone Methyl benzoate 2-Nonanol n-Undecane Linalool n-Nonanal a-Campholenal 2,2,6-Trimethyl-hepta-3,5-diene n-Octanoic acid 4-Terpineol a-Terpineol Dihydro carveol Geraniol 2-Methyl-dodecane n-Nonanoic acid 3-Undecanone (E)-anethole 2-Undecanone n-Tridecane (E,E)-2,4-decadienal a-Cubebene a-Longipinene n-Decanoic acid n-Octadecanal a-Yalangene a-Copaene n-Dodecanal Unknown b-Cedrene b-Caryophyllene b-Copaene (Z)-b-farnesene a-Himachalene allo-Aromadrene 10-epi-Italicene g-Muurolene g-Himachalene Germacrene D Unknown a-Muurolene

RI

858 897 941 953 967 982 984 993 996 999 1026 1034 1045 1062 1068 1076 1090 1094 1097 1103 1105 1107 1133 1161 1166 1187 1202 1205 1256 1261 1269 1283 1285 1288 1293 1314 1350 1355 1358 1361 1372 1375 1392 1417 1423 1424 1435 1442 1455 1460 1462 1473 1476 1479 1493 1496

Identified by

Drosocarpium

a, b a, b a, b a a a, b a a a, b a, b e a, b a a a a a, b a, b a, b a, b a a, b a a a, c a a a a a a, c a a, b a, b a, b a a a a, c a a a a, b e a a, b a a a a a a a a e a

e 3.0 4.7 0.2 e 4.6 e e e e e 0.9 e e e e e e e 2.9 1.0 e e e e 0.4 0.9 e e 1.0 0.3 e e e 1.1 e e 1.7 e e e 1.0 e e e 3.8 0.7 2.8 e 0.8 e 3.7 e e e e

Hypericum

Taeniocarpium

A2003 B2003 C2003 D2003 E2003 F2003 G2003 1.4 7.9 4.4 e 0.7 1.1 e e e e 0.6 e e 0.6 e e e 0.4 e 2.0 0.4 e e e e 0.4 0.2 0.2 e e 0.4 e 9.5 e e e e 0.4 e e e 0.4 e e e 1.8 e 0.5 0.6 1.0 3.1 1.2 e e 0.8

e e 0.8 e e 1.5 e e e e 0.5 e e e e e e e e 0.9 0.4 e e e 0.5 0.5 1.0 e e 0.6 0.7 e e 0.2 e e 0.2 1.9 0.4 e e 0.6 2.1 1.1 e 5.8 e 2.7 e 1.1 0.9 3.0 e 4.8 e 1.4

1.2 14.9 2.2 e 1.0 0.4 e e e e 0.5 e e 0.3 e e e e e 5.9 e e e e e 0.6 0.4 e e e e e e 0.5 e e e e 0.3 e 0.2 0.3 0.6 e 0.4 1.4 e 0.7 e e e 1.6 e 0.7 e 0.2

20.5 1.6 13.7 e 2.0 3.5 e e e e 1.3 0.4 0.3 e 1.7 e e e e 0.8 1.0 e 0.9 e e e e e e 0.4 e e e e e e 0.2 e e e 0.2 e e e 3.0 e 0.2 e e e 0.9 e 0.4 e e

e 0.6 e e e e 1.5 e e e e e e e e 7.6 e e e 1.5 3.4 0.1 e 0.9 e 0.2 0.4 e e e 1.2 e 6.0 e e e e e 1.1 e 0.7 e 0.2 15.5 1.6 0.7 e e e e 1.0 1.9 1.3 e 6.4 e

e 40.5 0.2 e e e e e 0.7 0.3 e e e e e e 1.0 e 1.7 11.8 e 0.6 e e e e 0.3 e e e 1.1 0.5 e 1.6 e 0.9 e 1.4 e 0.1 e 0.4 e e e e e e e 3.8 e 0.9 e e e e

Olympia

H2003

I2003

e 10.6 11.5 0.2 0.6 1.4 2.3 0.6 e e e 1.5 0.4 e e e e e e 3.8 2.6 e e e e e e e e e e e e e e e e e e e e e e e e 0.6 e 2.5 e 0.6 e 1.2 e e e 0.3

6.1 e 4.6 e 0.1 2.1 e e e e 0.5 1.1 e e 1.1 e e e e 0.9 1.1 e e e e 0.3 0.6 e 0.9 e e e e e e e 0.1 e 0.2 e 0.1 0.5 e e 1.4 1.3 e 2.6 e 1.5 e 0.9 e 7.6 e 1.7

(continued on next page)

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Table 2 (continued ) Constituents

d-Cadinene Cadina-3,9-diene Cadina-1,3,5-triene a-Bisabolene Nerolidol n-Dodecanoic acid 3-Hexenyl-benzoate Spathulenol Caryophyllene oxide Sesquiterpene C15H24O Globulol 12-Methyl-tridecanol 1,10-di-epi-Cubenol epi-a-Cubenol epi-a-Cadinol epi-a-Muurolol 3,6-Pentadecadienal a-Cadinol Caryophylla-3(15),7-dienol (6) I n-Tetradecanol Caryophylla-3(15),7-dienol (6) II a-Bisabolol Unknown n-Tetradecanoic acid Benzyl benzoate Hexahydrofarnesyl aceton n-Pentadecanoic acid n-Hexadecanol n-Nonadecane n-Hexadecanoic acid, methyl ester n-Hexadecanoic acid Ethyl hexadecanonate n-Eicosane Kaurene (Z,Z)-9,12-octadecadienoic acid, methyl ester n-Heneicosane (E)-phytol (Z,Z)-9,12-octadecadienoic acid (Z,Z,Z)-9,12,15-octadecatrienoic acid, methyl ester n-Docosane n-Tricosane n-Tetracosane n-Pentacosane n-Hexacosane n-Heptacosane n-Octacosane n-Nonacosane Total

Identified by

Drosocarpium

A2003 B2003 C2003 D2003 E2003 F2003 G2003

H2003

I2003

1514 1520 1528 1536 1556 1562 1570 1575 1582 1592 1599 1625 1633 1635 1644 1647 1651 1657 1664 1670 1682 1684 1699 1748 1769 1842 1856 1873 1888 1907

a a a a a a, c a a a, b a a, b a a a a a a a a a, b a a e a, b, c a, b a a, b, c a, b a, b a, c

0.5 1.1 2.8 0.8 e 1.4 0.7 3.7 2.1 e e e e 0.3 0.5 0.2 e 1.3 0.5 e e e e 0.3 2.7 2.7 e e 0.3 e

2.1 3.8 0.6 e 1.3 e e 1.1 7.1 0.6 9.4 e 0.3 e 0.2 1.3 e 3.6 e e e e 2.0 0.8 e 0.7 e e 0.8 0.2

1.3 2.8 e e e 8.0 e 1.9 2.4 e 0.6 2.3 e e 0.8 0.4 e 1.6 e 0.3 e e e 7.3 e 1.5 e e 0.5 0.4

1.1 0.9 1.9 e e 1.8 e 7.0 1.5 e 8.0 e e e 0.7 0.7 e 3.0 e e e e e 1.4 1.1 0.8 e e 0.4 0.3

0.3 0.4 e e 1.8 e 0.6 9.8 2.9 e 0.3 3.7 e e e e e e 1.7 2.8 1.3 e e 0.4 0.2 0.9 e 1.2 0.2 e

1.2 3.1 0.1 e e 1.3 e 2.6 3.2 1.3 6.1 e e 0.5 0.3 e 0.5 0.8 e e e e e 1.1 e 0.9 0.1 e 0.8 e

0.7 e 0.7 e e 1.3 e 2.9 2.0 e e e e e 0.2 e e e e e e e e 1.3 e 0.6 e e e e

0.8 1.9 e 2.7 e 1.3 1.0 2.5 1.7 e e e e 0.2 0.9 0.3 e 0.5 0.5 e e 0.9 e e 2.2 3.9 e e 0.2 e

2.3 3.9 e e e 1.6 0.5 6.9 1.0 e 5.5 e e 0.9 0.4 0.3 2.5 6.1 e e e 2.7 e 0.5 1.5 0.7 e e e e

1968 1981 1993 2071 2091

a, b, c a a, b a a, c

8.0 e e e e

6.2 e 0.1 e e

11.7 e 0.3 e e

9.2 e 0.1 e e

4.0 e e e e

4.1 e 0.2 0.1 0.1

3.1 e e e e

2.6 e 0.1 e e

6.5 e e e e

2099 2111 2146 2158

a a, b a, c a

1.0 e 10.0 1.9

0.5 1.8 2.4 1.0

1.2 1.1 1.3 2.8

0.6 e 1.1 3.5

0.6 e e e

0.7 e 0.7 0.7

0.1 e 0.8 0.1

0.8 e 3.3 0.3

0.2 e 1.7 0.7

2199 2304 2399 2504 2597 2706 2797 2902

a, a a, a a, a a, a

0.5 0.6

e 0.4 e 0.4 e 0.3 e 0.7 89.7

0.2 0.6 0.1 0.5 0.1 e e 0.7 86.3

0.1 0.6 0.1 0.5 e 0.6 e 1.6 82.9

e 0.4 e 0.3 e 0.4 t 1.4 88.6

t

e 0.2 0.2 0.2 e 0.2 0.3 0.4 83.1

0.1 0.7 0.1 1.0 0.2 2.4 0.4 2.2 76.4

t

RI

RI ¼ calculated retention indices. a ¼ identification based on comparison of mass spectra. b ¼ retention time identical to authentic compounds. c ¼ identification based on methylation. t ¼ less than 0.1%. The codes are given in Table 1.

b b b b

t 0.6 0.1 1.6 0.2 2.6 84.5

Hypericum

Taeniocarpium

0.2 e 0.1 e t e 0.1 84.7

Olympia

0.6 0.1 0.4 e 0.5 e 1.9 87.2

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H. maculatum, H. olympicum, H. perforatum and H. rumeliacum oils, 2-methyl-octane in H. maculatum, H. olympicum, H. perforatum and H. richeri oils and (E)-anethol in H. richeri and H. tetrapterum oils. The oils studied had 11 common components. All nine oil samples contained spathulenol and caryophyllene oxide; this is in agreement with the literature report on essential oils of H. perforatum growing wild in ten habitats in Lithuania (Mockute et al., 2003). It is interesting that there were also significant differences in the oil composition of samples collected from the same location (Tables 1 and 2; locality Suva planina, codes B2003, G2003 and H2003; and locality Rujan planina, codes C2003, E2003 and I2003), which suggest that genetic factors play a role in determining the oil composition. Our results are in agreement with the previous study of H. olympicum and H. perforatum oils (Gudzic et al., 2001). 3.2. Compositions of the flower, leaf and stem oils of Hypericum perforatum The composition of oils isolated from flowers (code FF2004), leaves (code FL2004) and stems (code FS2004) of H. perforatum, are presented in Table 3. Significant differences were observed in the chemical composition of oils of various parts of the plant. The non-terpene compounds were the major fraction of the oil obtained from flowers and they amounted to 44.4%. A high content of sesquiterpenes (73.8%) and low content of non-terpenes (10.6%) characterized the leaf oil. The content of non-terpene in the stem oil amounted to 50.4%. The chemical composition of the leaf essential oil of H. perforatum investigated in this study differed from the leaf essential oil of H. perforatum originating from India (Weyerstahl et al., 1995). a-Pinene was the major component of the leaf oil from India (67.3%), while its content in the leaf oil from Southeastern Serbia was considerably lower (1.2%). Germacrene D was the main component in the leaf oil from Southeastern Serbia, which was not the case of the leaf oil from India. On the other hand, there were similarities in the composition of leaf oil of H. perforatum investigated in this study and the oils of H. perforatum from Lithuania (Mockute et al., 2003). All these oils were characterized by high contents of b-caryophyllene, germacrene D and caryophyllene oxide. 3.3. Variability of chemical compositions of oils of Hypericum maculatum, Hypericum olympicum and Hypericum perforatum collected in years 1998, 2001 and 2003 Published data are available on the essential oil compositions of H. maculatum, H. olympicum and H. perforatum, collected in 1998 (Gudzic et al., 2001, 2002) and in this study the composition of oils of the same species from the same locations collected in 2001 and 2003 were determined so that a comparison was possible over a longer period of time. The results are summarized in Table 4 and the significant differences in the amounts of the same compound in samples from different years are evident. A high content of the sesquiterpenes (61.0%) characterized the H. maculatum oil of the year 1998 (Gudzic et al., 2002). The content of non-terpenes compounds were relatively high in the H. maculatum oils of years 2001 (56.0%) and 2003 (48.2%). There were 11 common components in H. maculatum oils collected in different years. Sesquiterpenes were the main fraction of the H. olympicum oils and amounted to 47.7%, 74.5% and 56.0% in the oils collected in 2003, 2001 and 1998, respectively. The major component in the H. olympicum oil of the year 1998 was (E)-anethole (30.7%) (Gudzic et al., 2001). It was not found in the H. olympicum oils of years 2001 and 2003. These samples contained 18 common components. 2-Methyl-octane was the main constituent of the H. perforatum samples collected in years 2003 (20.5%) and 1998 (13.1%), while its content in the H. perforatum sample of the year 2001 was lower (3.1%). a-Pinene and b-pinene amounted to 17.2% in the oil of the year 2003, while their content in oils of the years 2001 and 1998 was 3.8% and 2.2%, respectively. There were 18 common components in H. perforatum oils. The differences in the oil composition of samples collected from the same species and the same location, in different years, might be explained by seasonal differences because the samples were collected at different dates. 3.4. Chemotaxonomic significance of the oils obtained from Hypericum species An examination of Table 2 reveals definite chemotaxonomic similarities and differences among the nine species. The contents of non-terpenes, monoterpenes and sesquiterpenes in oils of the species H. barbatum, H. richeri and

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Table 3 Compositions of the flower, leaf and stem essential oils of Hypericum perforatum (value given in % weight fraction) Constituents

RI

Identified by

EF2004

EL2004

ES2004

2-Methyl-octane n-Nonane a-Pinene 3-Methyl-nonane b-Pinene a-Terpinene Unknown b-Phellandrene (Z)-b-ocimene g-Terpinene 2-Methyl-decane 2,6-Dimethyl-3,5-heptanedione n-Undecane Linalool 4-Terpineol a-Terpineol Dihydro carveol Carvone (Z)-anethole 2-Methyl-dodecane (E)-anethole d-Elemene a-Cubebene n-Octadecanal a-Yalangene a-Copaene b-Bourbonene Unknown b-Caryophyllene b-Copaene Aromadendrene (Z)-b-farnesene allo-Aromadrene 10-epi-Italicene n-Dodecanol g-Muurolene a-Bergamotene g-Himachalene Germacrene D Unknown a-Muurolene d-Cadinene Cadina-3,9-diene Cadina-1,3,5-triene Nerolidol 3-Hexenyl-benzoate Spathulenol Caryophyllene oxide Sesquiterpene C15H24O Globulol Humulene epoxide II 12-Methyl-tridecanol epi-a-Cubenol epi-a-Cadinol 3,6-Pentadecadienal a-Cadinol n-Tetradecanol a-Bisabolol

858 897 941 967 982 1019 1026 1034 1045 1062 1068 1076 1103 1105 1187 1202 1205 1245 1250 1261 1285 1342 1350 1361 1372 1375 1383 1417 1424 1435 1439 1442 1460 1462 1470 1473 1475 1476 1479 1493 1496 1514 1520 1528 1556 1570 1575 1582 1592 1599 1619 1625 1635 1644 1651 1657 1670 1684

a, b a, b a, b a a, b a e a a a a a a, b a a a a a a a a, b a a a a a a e a, b a a a a a a, b a a a a e a a a a a a a a, b a a, b a a a a a a a, b a

27.3 1.3 15.2 0.9 3.4 e e e 2.7 e 1.5 e 0.6 0.1 0.5 0.3 e e e 0.6 e e 0.1 0.2 0.1 0.4 e e 5.0 e e 3.2 e e e 1.6 e 0.9 1.7 e e 0.8 1.4 0.2 1.5 1.0 3.8 1.6 e 0.3 e 4.3 e 0.7 e 1.1 2.1 e

1.8 e 1.2 0.3 1.0 0.2 0.5 0.3 0.3 0.6 e e e 0.9 0.9 0.4 0.4 e e e e 1.7 0.5 e 0.1 1.1 1.1 e 5.5 1.6 2.8 3.5 2.0 e 1.4 4.3 11.5 e 10.0 e 0.9 3.2 4.7 0.3 1.9 0.7 3.4 6.8 0.7 0.9 1.2 e 2.6 0.2 e 1.1 5.1 0.2

e 0.1 0.1 e 0.4 e 0.3 e e e e 2.4 1.2 1.0 e e e 1.2 0.2 e 25.0 e 0.2 e e 0.5 e 5.2 2.4 e e 0.6 0.4 0.5 e 2.1 e 2.3 e 2.7 e 1.0 1.9 0.1 1.5 e 3.5 2.9 1.2 3.5 e 2.1 0.3 e 0.2 1.9 e e

A. Smelcerovic et al. / Biochemical Systematics and Ecology 35 (2007) 99e113

107

Table 3 (continued ) Constituents

RI

Identified by

EF2004

EL2004

ES2004

n-Tetradecanoic acid Benzyl benzoate n-Pentanoic acid, eugenyl ester Hexahydrofarnesyl aceton n-Pentadecanoic acid n-Hexadecanol Butanoic acid, 2-methyl-,4-methoxy-2(3-methyloxiranyl) phenyl ester n-Nonadecane n-Hexadecanoic acid Kaurene (Z,Z)-9,12-octadecadienoic acid, methyl ester n-Heneicosane (Z,Z)-9,12-octadecadienoic acid (Z,Z,Z)-9,12,15-octadecatrienoic acid, methyl ester n-Tricosane n-Pentacosane n-Heptacosane n-Nonacosane Total

1748 1769 1832 1842 1856 1873 1882

a, a, a a a, a, a

b, c b

0.2 0.6 e 0.4 e 0.3 e

e 0.3 e 0.2 e e e

0.8 e 3.4 0.6 0.5 e 0.3

1888 1968 2071 2091 2099 2146 2158 2304 2504 2706 2902

a, a, a a, a a, a a a a a

b b, c

e 1.7 e e 0.1 0.3 0.3 t 0.1 0.2 0.4 91.0

e 0.5 e e e e e t 0.1 e 0.2 91.1

0.2 8.1 0.3 0.3 0.9 2.1 1.6 0.1 0.2 e 0.1 88.4

b, c b

c c

RI ¼ calculated retention indices. a ¼ identification based on comparison of mass spectra. b ¼ retention time identical to authentic compounds. c ¼ identification based on methylation. t ¼ less than 0.1%. The codes are given in Table 1.

H. rumeliacum (section Drosocaprium) were similar and they were characterized with high contents of fatty acids. The oils of H. hirsutum and H. linarioides (section Taeniocarpium) contained a high percentage of n-nonane. There were similarities in contents of non-terpenes and sesquiterpenes in oils of species that belong to the section Hypericum (H. maculatum, H. perforatum and H. tetrapterum). The oil of H. olympicum (section Olympia) differed from others by higher terpene content. Principal component analysis revealed that there were significant differences among the compositions of the essential oils of the nine species of Hypericum under study (Fig. 1a), the compositions of the essential oils from flower, leaf and stem of H. perforatum (Fig. 1b), and the compositions of the essential oils of H. maculatum, H. olympicum and H. perforatum, collected in different years from the same location (Fig. 1c). The greatest similarity between the essential oil content and the sectional botanical classification (Robson, 1977) is observed for the Drosocaprium section (oils of H. barbatum (code A2003), H. richeri (code B2003) and H. rumeliacum (code C2003)). The oils of H. hirsutum (code G2003) and H. perforatum (code E2003) are clearly distant from the oils of other seven species investigated (Fig. 1a). The greatest similarity between the essential oils contents for different years between the three species investigated (H. maculatum, H. olympicum and H. perforatum) is observed by H. perforatum (codes E2003, E2001, E1998) (Fig. 1c). The dendrogram obtained by cluster analysis of the volatile metabolites of the nine species of Hypericum under study (Fig. 2) suggest the existence of eight clusters (clusters IeVIII), representing principally compounds from oils of eight species (I e H. perforatum; II e H. linarioides; III e H. tetrapterum; IV e H. barbatum; V e H. rumeliacum; VI e H. hirsutum; VII e H. richeri; VIII e H. olympicum). The oil of H. maculatum alone does not form a separate cluster because it contains only two compounds whose content is highest in this species (4-terpineol and (Z,Z,Z)-9,12,15-octadecatrienoic acid, methyl ester). The dendrogram contains a relative range between 0.50 and 0.65 where no clusters are merged which suggests that the groups of characteristic compounds from oils of eight species were clearly distinguished. The species H. barbatum and H. rumeliacum belong to the section Drosocaprium, and their clusters (IV and V) are merged in the dendrogram. No other relationship can be established between the clustermerging and sectional botanical classification (Robson, 1977).

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Table 4 Variability of the essential oils compositions of Hypericum maculatum, Hypericum olympicum and Hypericum perforatum, collected in different years (value given in % weight fraction) Constituents

RI

Identified by

H. maculatum

H. perforatum

D2003

D2001

D1998

E2003

E2001

E1998

I2003

I2001

I1998

2-Methyl-octane n-Nonane Tricyclene a-Pinene 2,5,6-Trimethyl-hepta-1,3,6triene 3-Methyl-nonane b-Pinene Myrcene n-Decane a-Phellandrene a-Terpinene Unknown p-Cymene Limonene 1,8-Cineole (Z)-b-ocimene (E)-b-ocimene g-Terpinene 2-Methyl-decane Fenchone Terpinolene p-Cymenene n-Undecane Linalool a-Thujone a-Campholenal 2-Nonanol (E)-Pinocarveol Pinocarvone g-Terpineol 4-Terpineol p-Cumene-8-ol a-Terpineol Myrtenal Myrtenol Estragole Cuminyl aldehyde Carvone Geraniol 2-Methyl-dodecane (E)-anethole 2-Undecanone n-Tridecane a-Cubebene a-Longipinene n-Decanoic acid a-Yalangene a-Copaene b-Bourbonene b-Cubebene n-Dodecanal a-Gurjunene b-Cedrene b-Caryophyllene b-Copaene

858 897 e 941 e

a, b a, b e a, b e

1.2 14.9 e 2.2 e

29.9 3.2 e 12.1 e

e 1.1 1.3 e e

20.5 1.6 e 13.7 e

3.1 0.4 e 2.8 e

13.1 1.4 e e e

6.1 e e 4.6 e

2.7 e e 1.2 e

e e 0.7 e 0.2

967 982 e e e e 1026 e 1034 1036 1045 e 1062 1068 e e e 1103 1105 e 1133 e e e e 1187 e 1202 e e e e e 1256 1261 e 1288 1293 1350 1355 1358 1372 1375 e 1388 1392 e 1423 1424 1435

a a, b e e e e e e a, b a a e a a e e e a, b a e a e e e e a e a e e e e e a a e a, b a, b a a a, c a a e a a, b e a a, b a

1.0 0.4 e e e e 0.5 e e e e e 0.3 e e e e 5.9 e e e e e e e 0.6 e 0.4 e e e e e e e e 0.5 e e e 0.3 0.2 0.3 e e 0.6 e 0.4 1.4 e

3.8 2.7 e e e e e e 0.5 e e e e 5.3 e e e 3.3 0.2 e 0.3 e e e e 0.1 e 0.3 e e e e e e 2.8 e e 1.0 e 0.4 e e 0.3 e e e e e 6.9 0.2

e 0.5 e

2.0 3.5 e e e e 1.3 e 0.4 e 0.3 e e 1.7 e e e 0.8 1.0 e 0.9 e e e e e e e e e e e e e 0.4 e e e e 0.2 e e 0.2 e e e e e 3.0 e

0.9 1.0 e e e e 1.0 e e 0.5 e e e 2.9 e e e 1.7 e e e e e e e e e e e e e e e e 5.0 e e 1.3 e 0.3 e e 0.8 e e e e 1.0 2.7 e

e 2.2 0.3 0.2 e e e 0.8 e e e 0.3 e 7.9 e e 0.2 3.9 e e 0.1 0.4 0.4 0.1 e 0.5 0.2 0.3 e t e e 0.1 t 4.5 0.1 e 1.3 e 0.4 e e 0.2 0.2 e e e e 14.2 e

0.1 2.1 e e e e 0.5 e 1.1 e e e e 1.1 e e e 0.9 1.1 e e e e e e 0.3 e 0.6 e e e e e 0.9 e e e e 0.1 e 0.2 0.1 0.5 e e e e 1.4 1.3 e

0.8 1.4 e e e e 0.3 e 0.5 e 0.2 e e 1.0 e e e 0.5 e e e e e e e e e e e e e e e e 0.1 e e e 2.1 e e 0.3 2.8 e 1.2 e e e 2.4 3.8

e 0.3 0.1 e t t e t 0.3 e e 0.2 e e 0.5 e e 0.3 e e e e e e t e e e e e 0.5 e 4.2 e e 30.7 e e 1.3 e e 0.6 2.7 0.1 0.7 e 0.1 e 2.1 e

e e e 0.8 0.2 0.5 e e 0.4 e e 0.2 e 8.2 e 0.4 e e 0.2 0.2 e 1.9 e 0.4 0.6 0.3 e 0.2 e e e 0.1 e 0.6 1.4 e e 0.8 2.7 1.0 0.1 e 1.6 e 7.6 e

H. olympicum

A. Smelcerovic et al. / Biochemical Systematics and Ecology 35 (2007) 99e113

109

Table 4 (continued ) Constituents

RI

Identified by

H. maculatum D2003

D2001

D1998

E2003

E2001

E1998

I2003

I2001

I1998

b-Gurjunene Aromadendrene (Z)-b-farnesene a-Himachalene a-Patchoulene Bicyclosesquiphellandrene a-Caryophyllene b-Farnesene allo-Aromadrene a-Acoradiene g-Muurolene g-Himachalene Germacrene D Eremophyllene a-Muurolene a-Farnesene g-Cadinene g-Bisabolene Calamenene d-Cadinene Cadina-3,9-diene Cadina-1,4-diene Cadina-1,3,5-triene Muurola-4,9-diene a-Calacorene Germacrene B b-Calacorene Nerolidol n-Dodecanoic acid 3-Hexenyl-benzoate Spathulenol Caryophyllene oxide Globulol Viridiflorol Humulene epoxide II 12-Methyl-tridecanol epi-a-Cubenol a-Elemene epi-a-Cadinol epi-a-Muurolol 3,6-Pentadecadienal a-Cadinol Caryophylla-3(15),7-dienol (6) I n-Tetradecanol Caryophylla-3(15),7-dienol (6) II a-Bisabolol n-Tetradecanoic acid Benzyl benzoate Hexahydrofarnesyl aceton n-Hexadecanol n-Nonadecane n-Hexadecanoic acid, methyl ester n-Hexadecanoic acid n-Eicosane n-Heneicosane (Z,Z)-9,12-octadecadienoic acid

e e 1442 1455 e e 1458 e 1460 e 1473 1476 1479 e 1496 e e e e 1514 1520 e 1528 e e e e 1556 1562 1570 1575 1582 1599 e e 1625 1635 e 1644 1647 1651 1657 1664 1670 1682 1684 1748 1769 1842 1873 1888 1907

e e a a e e a e a e a a a e a e e e e a a e a e e e e a a, c a a a, b a, b e e a a e a a a a a a, b a a a, b, c a, b a a, b a, b a, c

e e 0.7 e e e e e e e 1.6 e 0.7 e 0.2 e e e e 1.1 0.9 e 1.9 e e e e e 1.8 e 7.0 1.5 8.0 e e e e e 0.7 0.7 e 3.0 e e e e 1.4 1.1 0.8 e 0.4 0.3

e e 2.2 0.4 e e e e e e 0.6 0.5 e e e e e e e e 0.5 e e e e e e 1.2 e e 3.1 3.1 0.1 e e 2.1 e e e e e e e 1.4 e e e e 0.9 0.4 0.2 e

1.7 0.7 e 0.4 e e 1.3 10.0 e 0.3 5.2 e e 1.9 1.4 e 4.2 e 1.2 7.2 e 0.2 e e 0.6 e 0.1 0.8 e e 1.4 2.5 3.4 0.3 0.2 e e e 0.4 e e 0.2 e e e e e e e e e e

e e 0.2 e e e e e e e 0.9 e 0.4 e e e e e e 0.3 0.4 e e e e e e 1.8 e 0.6 9.8 2.9 0.3 e e 3.7 e e e e e e 1.7 2.8 1.3 e 0.4 0.2 0.9 1.2 0.2 e

e e 5.8 e e e e e e e 5.6 e e e e e e e e 2.7 e e 2.0 e e e e 3.8 e 0.6 8.7 2.2 0.5 e e 7.5 e e 0.5 0.2 e 1.5 e 7.5 0.2 e e 0.4 1.9 1.1 0.2 e

0.2 e e 0.4 e e 0.4 3.2 e e 3.5 e e e e 0.1 0.5 e e 0.5 e e e e e e e 0.8 e 0.5 4.5 e e e 4.2 e 0.7 e e e e e e 4.1 e e e 0.1 e 0.4 0.2 e

e e 2.6 e e e e e 1.5 e 0.9 e 7.6 e 1.7 e e e e 2.3 3.9 e e e e e e e 1.6 0.5 6.9 1.0 5.5 e e e 0.9 e 0.4 0.3 2.5 6.1 e e e 2.7 0.5 1.5 0.7 e e e

e e 2.6 e e e 2.0 e 2.9 e 16.1 e 1.3 e 3.4 e e e e 12.6 10.0 e 1.5 e e e e e e 0.9 1.5 0.5 0.4 e e e 0.5 e 2.0 0.5 e 3.5 e e e 0.6 e e 0.5 0.4 e e

1.1 e e e 0.5 1.8 0.3 12.4 e e 7.5 e 4.3 0.7 1.5 0.5 4.2 t 0.4 8.7 0.7 0.7 e 0.7 0.3 0.7 t e e e 0.4 e e e e e 0.1 t 0.2 t e 0.5 e e e 0.2 e e e e e e

1968 1993 2099 2146

a, b, c a, b a a, c

9.2 0.1 0.6 1.1

e e e e

4.0 e 0.6 e

2.7 0.1 0.5 e

e e e e

1.1 t 0.4 e

H. perforatum

H. olympicum

6.5 e 0.2 1.7 (continued

e e t e on next

e e e e page)

110

A. Smelcerovic et al. / Biochemical Systematics and Ecology 35 (2007) 99e113

Table 4 (continued) Constituents

RI

Identified by

(Z,Z,Z)-9,12,15-octadecatrienoic acid, methyl ester n-Docosane n-Tricosane n-Tetracosane n-Pentacosane n-Heptacosane n-Octacosane n-Nonacosane Total

2158

a

2199 2304 2399 2504 2706 2797 2902

a, b a a, b a a a, b a

H. maculatum D2003

H. perforatum

H. olympicum

D2001

D1998

E2003

E2001

E1998

3.5

e

e

e

e

e

0.1 0.6 0.1 0.5 0.6 e 1.6 82.9

t

e e e e e e e 78.9

e 0.4 e 0.3 0.4 t 1.4 88.6

t

e e e e e e e 77.6

0.1 e t t e 0.1 91.7

0.3 e 0.2 0.2 e 0.6 82.9

I2003 0.7 t 0.6 0.1 0.4 0.5 e 1.9 87.2

I2001

I1998

e

e

e 0.2 t t 0.2 e 1.1 86.5

e e e e e e e 94.0

RI ¼ calculated retention indices. a ¼ identification based on comparison of mass spectra. b ¼ retention time identical to authentic compounds. c ¼ identification based on methylation. t ¼ less than 0.1%. The codes are given in Table 1.

Fig. 1. Principal component analysis of the volatile metabolites of nine species of Hypericum under study (a), the volatile metabolites from flower, leaf and stem of H. perforatum (b), and the volatile metabolites of H. maculatum, H. olympicum and H. perforatum, collected in different years from the same location (c). The codes are given in Table 1.

A. Smelcerovic et al. / Biochemical Systematics and Ecology 35 (2007) 99e113

Fig. 2. Dendrogram obtained by cluster analysis of the volatile metabolites of nine species of Hypericum under study.

111

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A. Smelcerovic et al. / Biochemical Systematics and Ecology 35 (2007) 99e113

The dendrogram obtained by cluster analysis of the volatile metabolites from flower, leaf and stem of H. perforatum revealed that clusters representing the volatile metabolites from leaf and stem are first merged each other and then with clusters representing the volatile metabolites from flowers. Dendrogram obtained by cluster analysis of the volatile metabolites of H. maculatum, H. olympicum and H. perforatum, collected in different years from the same location, revealed that many merged clusters and individual samples are not obviously separated. This result could be explained by the postulate, which indeed could be also the main conclusion of this present study that both genetic and environmental factors play a role in determining the composition of essential oils of the Hypericum species studied. A detailed study has been performed on the secondary metabolite contents of the nine species of Hypericum under study, originating from Serbia and the F.Y.R. Macedonia (Smelcerovic and Spiteller, 2006). The conclusion from the data above is that a stronger correlation exists between Robson’s sectional classification (Robson, 1977) and the hypericin and hyperforin contents of the nine Hypericum species (Smelcerovic and Spiteller, 2006) in contrast to a rather low correlation with essential oil composition. Naphthodianthrones and phloroglucinol derivatives are characteristic for Hypericum species and they represent excellent chemotaxonomical markers. A phytochemical analysis and genetic characterisation of H. barbatum, H. hirsutum, H. linarioides, H. maculatum, H. rumeliacum and H. tetrapterum has been performed recently, showing that a correlation exists between secondary metabolite contents (hypericin, pseudohypericin, hyperforin, hyperoside and quercitrin), RAPD (random amplification of polymorphic DNA) data and Robson’s sectional classification among the six Hypericum species from Serbia (Smelcerovic et al., 2006). Acknowledgements We thank Prof. Dr. N. Randjelovic, Faculty of Occupation Safety Nis, Serbia, for taxonomic identification of the plant material and Prof. Dr. J. Jovanovic for critical reading of the manuscript. The Alexander von Humboldt Foundation, Bonn, Germany, supported the work through a fellowship to A. Smelcerovic. References Abreu, I.N., Reis, M.G., Marsaioli, A.J., Mazzaefera, P., 2004. Essential oil composition of Hypericum brasiliens choise. Flavour Fragrance J. 19, 80e82. Adams, R.P., 1989. Identification of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press, San Diego. Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. Allured Publ. Corp., Carol Stream. Baser, K.H.C., Oze, T., Nuriddinov, H.R., Demirci, A.B., 2002. Essential oils of two Hypericum species from Uzbekistan. Khim. Prir. Soedin. 38, 54e57. Bertoli, A., Pistelli, L., Morelli, I., Spinelli, G., Manichini, F., 2000. Constituens of Hypericum hircinum oils. J. Essent. Oil Res. 12, 617e620. Bertoli, A., Menichini, F., Mazzetti, M., Spinelli, G., Morelli, I., 2003. Volatile constituents of the leaves and flowers of Hypericum triqetrifolium Tuura. Flavour Fragrance J. 18, 91e94. Buttler, G., 2000. Cluster analyse. In: Voss, W. (Ed.), Taschenbuch der Statistik. Fachbuchverlag Leipzig, Leipzig. Cakir, A., Duru, M.E., Harmandar, M., Ciriminna, R., Passannanti, S., Piozzi, F., 1997. Comparison of the volatile oils of Hypericum scabrum L. and Hypericum perforatum L. from Turkey. Flavour Fragrance J. 12, 285e287. Cakir, A., Kordali, S., Zengin, H., Hirata, T., 2004. Composition and antifungal activity of essential oils isolated from Hypericum hyssofilium and Hypericum heterophyllum. Flavour Fragrance J. 19, 62e68. Cakir, A., Kordali, S., Kilic, H., Kaya, E., 2005. Antifungal properties of essential oils and crude extracts of Hypericum linarioides. Biochem. Syst. Ecol. 33, 245e256. Couladis, M., Baziou, P., Petrakis, P.V., Harvala, C., 2001. Essential oil of Hypericum perfoliatum L. growing in different locations in Greece. Flavour Fragrance J. 16, 204e206. Couladis, M., Chinou, I.B., Tzakou, O., Petrakis, P.V., 2003. Composition and antimicrobial activity of the essential oil of Hypericum rumeliacum subsp. apollinis (Boiss. & Heldr.). Phytother. Res. 17, 152e154. Ferretti, G., Maggi, F., Tirillini, B., 2005. Essential oil composition of Hypericum richeri Vill. from Italy. Flavour Fragrance J. 20, 295e298. Gudzic, B., Dordevic, S., Palic, R., Stojanovic, G., 2001. Essential oils of Hypericum olympicum L. and Hypericum perforatum L. Flavour Fragrance J. 16, 201e203. Gudzic, B., Djokovic, D., Vajs, V., Palic, R., Stojanovic, G., 2002. Composition and antimicrobial activity of the essential oil of Hypericum maculatum Crantz. Flavour Fragrance J. 17, 392e394. Gudzic, B., Dordevic, S., Nedeljkovic, J., Smelcerovic, A., 2004. Essential oil composition of Hypericum atomarium Boiss. Chem. Ind. 58, 413e 415. Guedes, A.P., Amorim, L.R., Vicente, A.M.S., Ramos, G., Fernandes- Ferriera, M., 2003. Essential oils from plants and in vitro shoots of Hypericum androsaemum L. J. Agric. Food Chem. 51, 1399e1404.

A. Smelcerovic et al. / Biochemical Systematics and Ecology 35 (2007) 99e113

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Hartung, J., Elpelt, B., 1999. Multivariate Statistik. Oldenbourg Verlag, Mu¨nchen. Ishida, Y., Wakamatsu, S., Yokoi, H., Ohtani, H., Tsuge, S., 1999. Compositional analysis of polyunsaturated fatty acid oil by one-step thermally assisted hydrolysis and methylation in the presence of trimethylsulfonium hydroxide. J. Anal. Appl. Pyrol. 49, 367e376. Josifovic, M. (Ed.), 1972, Flora of Serbia, vol. 3. SANU, Belgrade. Mimica-Dukic, N., Ivancev-Tumbas, I., Igic, R., Popovic, M., Gasic, O., 1997. The content and composition of essential oil of Hypericum perforatum L. from Serbia. Pharm. Pharmacol. Lett. 7, 26e28. Mockute, D., Bernotiene, G., Juzdentiene, A., 2003. Volatile compounds of the aerial parts of wild St. John’s wort (Hypericum perforatum L.) plants. Chemija 14, 108e111. Osinska, E., 2002. Comparative study on essential oil content and its composition of different ecotypes of St. Johns wort (Hypericum perforatum L.). Herba Pol. 48, 174e177. Robson, N.K.B., 1977. Studies in the genus Hypericum L. (Guttiferae) I. Infrageneric classification. Bull. Br. Mus. Nat. Hist. (Bot.) 5, 293e355. Robson, N.K.B., Strid, A., 1986. Hypericum L. In: Strid, A. (Ed.), Mountain Flora of Greece, vol. 1 Cambridge University Press, Cambridge, pp. 594e608. Sajjadi, S.E., Rahiminezhad, M.R., Mehregan, I., Poorassar, A., 2001. Constituents of essential oil of Hypericum dogonbadanicum Assadi. J. Essent. Oil Res. 13, 43e44. Santos, P.A.G., Figueiredo, A.C., Barroso, J.G., Pedro, L.G., Schefer, J.J.C., 1999. Composition of the essential oil of Hypericum foliosum Aiton from five Azorean islands. Flavour Fragrance J. 14, 283e286. Schwob, I., Bessiere, J.M., Viano, J., 2002a. Composition of the essential oils of Hypericum perforatum L. from southeastern France. C. R. Biologies 325, 781e785. Schwob, I., Bessiere, J.M., Dherbomez, M., Viano, J., 2002b. Composition and antimicrobial activity of the essential oil of Hypericum coris. Fitoterapia 73, 511e513. Smelcerovic, A., Mimica-Dukic, N., Djordjevic, S., 2004. Essential oil composition of Hypericum perforatum L. ssp angustifolium from South Serbia. J. Essent. Oil Bear. Plants 7, 275e278. Smelcerovic, A., Spiteller, M., 2006. Phytochemical analysis of nine Hypericum L. species from Serbia and the F.Y.R. Macedonia. Pharmazie 61, 251e252. Smelcerovic, A., Verma, V., Spiteller, M., Ahmad, S.M., Puri, S.C., Qazi, G.N., 2006. Phytochemical analysis and genetic characterisation of six Hypericum species from Serbia. Phytochemistry 67, 171e177. Vasilieva, R., Georgieva, V., Vojnova, J., Milkova, Ts, 2003. GCeMS study on the essential oils of Bulgarian Hypericum maculatum Crantz and Hypericum perforatum L. C. R. Acad. Bulg. Sci. 56, 71e74. Vera, R.R., Chane-Ming, J., Fraisse, D.J., 1996. Essential oils of Hypericum lanceolatum Lam. from Reunion. Riv. Ital. EPPOS 7, 639e644. Vosman, K., Klein, E., Weber, N., 1998. S-methyl derivatives from thiol compounds by the pyrolytic reaction with trimethylsulfonium hydroxide. Lipids 33, 1037e1041. Weyerstahl, P., Splittgerber, U., Marschall, H., Kaul, V.K., 1995. Constituents of the leaf essential oil of Hypericum perforatum L. from India. Flavour Fragrance J. 10, 365e370. Yazaki, K., Okada, T., 1994. Medicinal and Aromatic Plants VI. In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry, vol. 26. Springer-Verlag, Berlin, pp. 167e178.