Volatiles of Geranium purpureum Vill. and Geranium phaeum L.: Chemotaxonomy of Balkan Geranium and Erodium Species (Geraniaceae)

2042 Volatiles of Geranium purpureum Vill. and Geranium phaeum L.: Chemotaxonomy of Balkan Geranium and Erodium Species (Geraniaceae) by Niko S. Radulović* a ) and Milan S. Dekić a ) b ) a ) Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia (phone: þ381-628049210; fax: þ381-18533014; e-mail: nikoradulovic@yahoo.com) b ) Department of Chemical and Technological Sciences, State University of Novi Pazar, Vuka Karadžića bb, 36300 Novi Pazar, Serbia The essential oils obtained by hydrodistillation of Geranium purpureum and G. phaeum were characterized by GC-FID and GC/MS analyses (the former for the first time in general). In total, 154 constituents were identified, accounting for 89.0 95.8% of the detected GC peak areas. The investigated essential oils consisted mainly of fatty acids and fatty-acid-derived compounds (45.4 81.3%), with hexadecanoic acid and (E)-phytol as the major components. The chemotaxonomic significance of the variations in the essential-oil composition/production of the presently and previously investigated Geranium and highly related Erodium taxa from Serbia and Macedonia was assessed by multivariate statistical analyses. The main conclusions drawn from the high chemical similarity of the two genera, visible from the obtained dendrograms and biplots, confirm the close phylogenetic relationship between the investigated Geranium and Erodium taxa, i.e., that there is no great intergeneric oil-composition variability. Changes in the composition and production of essential oils of the herein investigated taxa and 60 other randomly chosen species belonging to different plant genera were also statistically analyzed. The results put forward pro arguments for the oil-yield oil-composition correlation hypothesis. Introduction. The genus Geranium L. (Geraniaceae) comprises ca. 250 species, distributed mainly in the temperate region of the northern hemisphere. According to Janković [1], only 19 Geranium species can be found in the Serbian flora. G. purpureum Vill. (syn. G. robertianum var. purpuereum (Vill.) DC.), commonly known as little robin, is a herbaceous annual plant growing spontaneously in the Mediterranean and Submediterranean area. It occupies open and dry areas and hillsides. G. phaeum L., or dusky cranesbill (zdravinjak, in Serbian), is also a European herbaceous, perennial plant. It can usually be found in open woods, shrub communities, forest glades, and meadows of foothills in subalpine areas in southern and eastern European mountains. In general, the essential-oil constituents of Geranium taxa have been poorly investigated, except for the renowned G. macrorrhizum [2]. Previous reports on the phytochemistry of G. purpureum mainly dealt with the analysis and/or isolation of flavonoids, phenolic acids, and other phenolic compounds [3a] [3b], the investigation of their antimicrobial and antioxidant activity [3a] [3c][3d], and the isolation of a new disaccharide, a derivative of neohesperidoside [3e]. Only two previous investigations concerned the volatiles chemistry of G. phaeum, conducted by Chalchat et al. [2a] and Fodorea et al. [2b]. Motivated by the complete lack of chemical data on the essential oil of G. purpureum and the scarce data in the case of G. phaeum, as well as in continuation of 2013 Verlag Helvetica Chimica Acta AG, Zürich

2043 our previous investigations of Geranium taxa [2c] [4], we set, as the first goal of this work, to provide detailed compositional data on G. purpureum and G. phaeum volatiles. The second aim of this study was to evaluate the chemotaxonomic significance of such compositional data at an intergeneric level. Erodium Aiton and Geranium L. are two phylogenetically closely related plant genera of the Geraniaceae family (with difficult taxonomy). Hence, we decided to statistically compare the differences in the essential-oil composition/production of the two Geranium species characterized here and other previously investigated taxa belonging to the genera Geranium (G. macrorrhizum, G. robertianum, G. sanguineum, G. columbinum, and G. lucidum) and Erodium (E. cicutarium, E. ciconium, and E. absinthoides) from Serbia and Macedonia. This was done by multivariate statistical analyses (agglomerative hierarchical clustering analysis and principal component analysis). In addition, changes in the composition and production of essential oils of the presently investigated taxa and 60 other randomly chosen species belonging to different plant genera (characterized by a wide range of essential-oil yields) were also mutually correlated, with the aim to test a hypothesis claiming a possible link between the oil-yield and the chemical oil composition [5]. Results and Discussion. Chemistry. Hydrodistillation of the entire plants of G. purpureum (Sample A) and the aerial (Samples B and C 1 ) and underground (Sample C 2 ) parts of G. phaeum gave yellow, semisolid essential oils in low yields (Table 1). In total, the GC/MS analyses allowed the identification of 154 volatiles, accounting for 89.0 95.8% of the detected GC peak areas. The list of the identified volatile constituents as well as their attribution to four compound classes according to their likely biogenetic origin, viz., fatty acids and fatty-acid-derived compounds (FAD), carotenoid-derived compounds (CDC), terpenoids (TER), and the class of the other (unclassified) constituents (OTH), is given in Table 2. FAD predominated in all oils (45.4 81.3%). The essential oil isolated from G. purpureum almost entirely consisted of FAD (81.3%), whereas the content of Table 1. Collection Data and Essential-Oil Yield of the Geranium Species Characterized in This Study Geranium species G. purpureum Sample code A Plant parts Collection site Collection date Entire plants (flowering stage) G. phaeum B Aerial parts (flowering stage) C 1 Aerial parts (flowering stage) C 2 Underground parts Close to the village Dolac, Bela Palanka, alongside the railway line, on rocky places Ploče, north slopes of the Suva Planina mountain, forest area Ploče, north slopes of the Suva Planina mountain, forest area Ploče, north slopes of the Suva Planina mountain, forest area Yield [% (w/w)] Voucher No. 05.06.2006 0.024 MD0406 12.06.2006 0.010 MD0506 09.06.2007 0.014 MD0407 09.06.2007 0.006 MD0407

2044 Table 2. Composition of the Essential Oils Isolated from Geranium purpureum (Oil Sample A) and G. phaeum (Samples B, C 1, and C 2 ) Compound Class a ) RI b ) Content [%] c ) Identification d ) A e ) B C 1 C 2 Pyridine OTH 752 tr tr RI, MS, CoI (Z)-Pent-2-enol FAD 768 0.1 RI, MS Toluene OTH 773 tr tr tr RI, MS, CoI Hexanal FAD 802 tr tr RI, MS, CoI 2-Methylpyridine OTH 820 tr RI, MS Furfural OTH 834 0.1 0.1 RI, MS, CoI Dimethylsulfoxide OTH 842 0.1 tr RI, MS, CoI (Z)-Hex-3-en-1-ol FAD 852 0.3 1.1 RI, MS 3-Methylhexan-2one FAD 853 tr RI, MS Hexan-1-ol FAD 862 0.3 RI, MS, CoI Ethylbenzene OTH 867 0.1 RI, MS 2,6-Dimethylpyridine OTH 885 tr RI, MS 2-Butylfuran FAD 887 tr RI, MS (Z)-Hept-4-enal FAD 901 0.4 RI, MS 2,5-Dimethylpyridine OTH 931 tr RI, MS 5,5-Dimethyl-2(5H)-furanone OTH 952 tr RI, MS Benzaldehyde OTH 963 tr 1.2 0.9 RI, MS, CoI Hexanoic acid FAD 968 tr tr RI, MS, CoI Phenol OTH 976 tr RI, MS, CoI Oct-1-en-3-one FAD 977 tr RI, MS Oct-1-en-3-ol FAD 977 0.2 0.5 RI, MS b-pinene TER 983 tr RI, MS, CoI 2-Pentylfuran FAD 993 tr RI, MS 2,4,6-Trimethylpyridine OTH 995 tr 0.5 RI, MS Octanal FAD 1004 tr RI, MS, CoI (E,E)-Hepta-2,4-dienal FAD 1012 0.1 RI, MS 3-Ethyl-4-mehylpentan-1-ol OTH 1020 0.5 RI, MS Benzyl alcohol OTH 1035 tr 0.3 3.6 RI, MS, CoI Phenylacetaldehyde OTH 1046 tr 2.2 4.8 RI, MS Heptanoic acid FAD 1068 tr tr RI, MS, CoI Octan-1-ol FAD 1069 tr RI, MS, CoI Acetophenone OTH 1069 tr 0.4 0.3 RI, MS trans-linalool oxide (furanoid) TER 1092 3.2 RI, MS, CoI p-cymenene TER 1094 tr RI, MS Guaiacol OTH 1095 tr RI, MS Linalool TER/CDC 1101 0.5 1.2 RI, MS, CoI 5-Hydroxy-4,5-dimethyl-2(5H)- TER/CDC 1104 tr tr RI, MS furanone Nonanal FAD 1105 tr RI, MS, CoI Hotrienol TER 1106 tr RI, MS 2,6-Dimethylcyclohexanol CDC 1114 0.2 RI, MS b-phenylethyl alcohol OTH 1117 0.3 1.9 1.2 RI, MS Isophorone CDC 1125 0.3 RI, MS Nopinone TER 1144 0.1 0.3 RI, MS 4-Oxoisophorone CDC 1146 0.1 RI, MS Camphor TER 1150 0.2 tr RI, MS, CoI Benzoic acid FAD 1165 0.5 4.3 0.7 RI, MS, CoI Borneol TER 1170 1.6 RI, MS, CoI

2045 Table 2 (cont.) Compound Class a ) RI b ) Content [%] c ) Identification d ) A e ) B C 1 C 2 Nonanol FAD 1171 0.3 RI, MS, CoI Octanoic acid FAD 1171 0.4 0.2 RI, MS 3,5-Dimethylphenol OTH 1171 0.6 RI, MS p-cymen-8-ol TER 1189 tr RI, MS, CoI a-terpineol TER 1196 0.2 1.1 RI, MS Decanal FAD 1207 tr tr RI, MS 4-Vinylphenol OTH 1222 0.4 RI, MS Phenylacetic acid OTH 1250 tr tr RI, MS (E)-Geraniol TER/CDC 1255 1.3 RI, MS, CoI Nonanoic acid FAD 1270 0.3 tr RI, MS, CoI 2,6-Dimethylocta-1,7-diene- TER 1276 0.4 RI, MS 3,6-diol f ) (2Z)-2-Phenylbut-2-enal OTH 1277 tr 0.3 RI, MS Tridecane FAD 1300 tr RI, MS, CoI Indole OTH 1300 0.1 RI, MS Eugenol OTH 1362 0.1 2.8 RI, MS, CoI Decanoic acid FAD 1366 0.4 0.9 RI, MS, CoI Undecan-1-ol FAD 1374 tr RI, MS Tetradecane FAD 1400 0.1 tr RI, MS, CoI Tetrahydrogeranylacetone CDC 1407 0.1 tr RI, MS Dodecanal FAD 1411 tr RI, MS b-caryophyllene TER 1427 0.1 RI, MS, CoI (E)-Geranyl acetone CDC/TER 1455 0.3 RI, MS a-humulene TER 1461 0.4 RI, MS, CoI Homofarnesane f ) TER 1463 0.1 tr RI, MS Undecanoic acid FAD 1465 0.1 RI, MS, CoI Dodecan-1-ol FAD 1476 tr 0.6 RI, MS, CoI Germacrene D TER 1488 1.3 RI, MS, CoI (E)-b-Ionone CDC 1491 1.4 RI, MS Tridecan-2-one FAD 1497 tr tr 0.2 RI, MS Pentadecane FAD 1500 0.3 tr RI, MS, CoI Tridecanal FAD 1512 tr tr RI, MS Dibenzofuran OTH 1519 tr RI, MS 3,4-Dimethyl-5-pentyl-furan- FAD 1523 0.1 0.5 0.8 RI, MS 2(5H)-one (¼dihydrobovolide) f ) trans-calamenene TER 1530 tr RI, MS Dihydroactinidiolide CDC 1538 0.2 1.0 0.1 RI, MS a-calacorene TER 1550 tr RI, MS 5,5-Dimethyl-4-(3-oxobutyl)dihydro-2(3H)-furanone OTH 1556 tr MS Dodecanoic acid FAD 1563 1.4 1.7 RI, MS, CoI 11-Norbourbonan-1-one TER 1570 tr RI, MS Tridecan-1-ol FAD 1578 tr RI, MS (E,E)-4,8,12-Trimethyltrideca- TER 1582 0.1 RI, MS 1,3,7,11-tetraene Germacrene D-4-ol TER 1583 1.1 RI, MS Spathulenol TER 1586 tr RI, MS, CoI Caryophyllene oxide TER 1592 0.3 RI, MS, CoI Hexadecane FAD 1600 0.3 RI, MS, CoI

2046 Table 2 (cont.) Compound Class a ) RI b ) Content [%] c ) Identification d ) A e ) B C 1 C 2 Globulol TER 1610 tr RI, MS Tetradecanal FAD 1614 tr 0.1 RI, MS, CoI b-oplopenone TER 1618 0.6 RI, MS Benzophenone OTH 1633 tr RI, MS Hexahydrofarnesol (isomer II) f ) TER 1644 tr RI, MS Norpristane f ) CDC/TER 1650 0.2 RI, MS epi-a-muurolol TER 1651 tr RI, MS Tridecanoic acid FAD 1662 0.5 tr RI, MS a-cadinol TER 1665 0.4 RI, MS 3-Methylhexadecane FAD 1671 tr RI, MS Unidentified component g ) UIC 1676 0.4 Tetradecanol FAD 1678 0.1 0.4 0.3 RI, MS, CoI b-sinensal CDC/TER 1680 0.1 RI, MS Cadalene TER 1683 tr RI, MS Pentadecan-2-one FAD 1700 0.3 RI, MS Heptadecane FAD 1700 0.7 0.1 RI, MS, CoI Pristane f ) CDC/TER 1706 0.5 RI, MS 10-Norcalamenen-10-one TER 1711 tr RI, MS Pentadecanal FAD 1718 tr 0.5 0.6 0.9 RI, MS Methyl tetradecanoate FAD 1726 tr tr RI, MS, CoI Oplopanone TER 1748 tr RI, MS Tetradecanoic acid FAD 1766 3.8 1.9 2.6 RI, MS, CoI Benzyl benzoate OTH 1771 0.6 0.2 RI, MS, CoI 3-Methylheptadecane FAD 1772 tr RI, MS Pentadecan-1-ol FAD 1779 0.8 0.3 RI, MS Phenanthrene OTH 1784 0.7 tr RI, MS, CoI g-tridecalactone FAD 1788 0.5 RI, MS Octadecane FAD 1800 1.1 0.1 0.2 RI, MS, CoI Phytane f ) CDC/TER 1810 1.0 tr RI, MS Hexadecanal FAD 1818 0.5 tr RI, MS Methyl pentadecanoate FAD 1826 0.1 RI, MS Neophytadiene (isomer I) CDC/TER 1841 tr 0.2 RI, MS Hexahydrofarnesyl acetone CDC/TER 1848 2.9 10.4 3.7 RI, MS Pentadecanoic acid FAD 1865 1.7 1.1 1.1 RI, MS, CoI Benzyl salicylate OTH 1877 0.3 tr RI, MS Hexadecan-1-ol FAD 1883 0.6 1.7 RI, MS, CoI g-tetradecalactone FAD 1895 0.5 RI, MS Nonadecane FAD 1900 1.8 0.3 RI, MS, CoI Heptadecan-2-one FAD 1905 tr RI, MS (E,E)-Farnesyl acetone CDC/TER 1923 0.7 RI, MS Methyl hexadecanoate FAD 1928 1.0 RI, MS, CoI Isophytol h ) CDC/TER 1951 1.6 0.7 RI, MS Hexadecanoic acid FAD 1969 33.5 36.1 3.2 55.2 RI, MS 3-(4,8,12-Trimethyltridecyl)furan CDC 1971 tr RI, MS ( ¼ Phytofuran) f ) Icosane FAD 2000 2.2 0.4 0.1 RI, MS Octadecanal FAD 2023 tr RI, MS (E,E)-Geranyllinalool TER 2034 0.7 RI, MS Heptadecanoic acid FAD 2063 1.9 RI, MS, CoI

2047 Table 2 (cont.) Compound Class a ) RI b ) Content [%] c ) Identification d ) A e ) B C 1 C 2 3-Methylicosane FAD 2073 0.6 RI, MS Octadecan-1-ol FAD 2086 0.7 1.3 RI, MS, CoI Heneicosane FAD 2100 4.8 1.3 0.1 RI, MS, CoI Isooctyl dodecanoate FAD 2104 1.1 MS g-hexadecalactone FAD 2107 1.3 tr RI, MS (E)-Phytol h ) TER/CDC 2117 25.9 RI, MS. CoI Unidentified component i ) UIC 2140 3.2 Oleic acid FAD 2140 8.6 RI, MS, CoI Octadecanoic acid FAD 2164 1.7 0.7 RI, MS, CoI Docosane FAD 2200 2.6 0.8 0.8 RI, MS, CoI Tricosane FAD 2300 3.8 3.4 1.4 RI, MS, CoI 4,5-Dihydro-5-methyl-5-(4,8,12- OTH 2354 0.8 1.2 RI, MS trimethyl-tridecyl)furan-2(3h)-one Tetracosane FAD 2400 1.9 2.8 3.1 RI, MS, CoI Pentacosane FAD 2500 8.5 11.5 8.3 RI, MS, CoI Hexacosane FAD 2600 4.3 6.4 15.9 0.8 RI, MS, CoI Heptacosane FAD 2700 4.1 RI, MS, CoI Total 92.2 96.2 92.5 94.6 Fatty acids and fatty-acid-derived compounds (FAD) 81.3 80.1 45.4 71.7 Carotenoid-derived compounds (CDC) 4.9 11.4 6.9 Terpenoids (TER) 0.3 2.6 31.8 8.7 Monoterpenoids 0.2 0.4 0.8 8.7 Monoterpene hydrocarbons tr tr Oxygenated monoterpenes 0.2 0.4 0.8 8.7 Sesquiterpenoids 0.1 0.6 4.4 Sesquiterpene hydrocarbons 0.1 tr 1.9 Oxygenated sesquiterpenes 0.6 2.5 Diterpenoids 1.6 26.6 Diterpene hydrocarbons Oxygenated diterpenes 1.6 26.6 Others (OTH) k ) 2.5 1.7 8.4 14.2 Unidentified constituents (UIC, >0.1%) 3.2 0.4 a ) The abbreviations of the compound classes are given at the end of the table. b ) RI: Linear retention indices determined experimentally on the HP-5MS column relative to a series of n-alkanes (C 8 C 27 ). c ) Values are means of three individual analyses; tr, trace amounts ( <<0.05%). d ) Identification method: RI, retention indices matching with literature data [6]; MS, mass spectra matching with those listed in the Wiley 6, NIST05, MassFinder 2.3, and a homemade mass spectral library; CoI, coinjection with pure reference compound. e ) Sample codes: A, oil isolated from entire plants of G. purpureum; B and C 1, oils isolated from aerial parts of G. phaeum; C 2, oil isolated from underground parts of G. phaeum; for further details of samples, cf. Table 1. f ) Correct isomer not defined. g ) EI-MS: 85 (45), 81 (42), 71 (62), 70 (42), 69 (49), 57 (100), 56 (48), 55 (53), 43 (73), 41 (55). h ) Phytol isomers possibly formed by chlorophyll degradation during hydrodistillation. i ) EI-MS: 99 (13), 75 (53), 71 (74), 70 (11), 69 (13), 57 (100), 56 (13), 55 (22), 43 (62), 41 (26). k ) Unclassified constituents, compounds of possible anthropogenic origin.

2048 terpenoids was very low (0.3%). Hexadecanoic acid (33.5%) and the n-alkanes pentacosane (8.5%), heneicosane (4.8%), and hexacosane (4.3%) were the major constituents of G. purpureum essential oil. As mentioned above, hydrodistillation of the aerial parts of G. phaeum yielded also oils rich in FAD compounds (80.1 and 45.4% for Samples B and C 1, resp.). In contrast to the oil of Sample B, the terpenoid fraction of that of Sample C 1 was relatively high (31.8%), with the diterpene alcohol (E)-phytol (25.9%) as the most abundant component, possibly formed by chlorophyll degradation during hydrodistillation and/or senescing of the plant material. Hexadecanoic (55.2%) and oleic acids (8.6%) were the main compounds of the oil isolated from the underground parts of G. phaeum (Sample C 2 ). There were noticeable differences in the essential-oil composition of G. phaeum found this study, when compared to those previously reported [2a][2b]. Piperitone (12.3%), which was the most abundant component of the oil from aerial parts of G. phaeum (also originating from Serbia) reported by Chalchat et al. [2a], was not even detected in the oils of Samples B or C 1. The most abundant constituent of the oil isolated from the aerial parts of the same species by Fodorea et al. [2b], germacrene D, was present in the oil of Sample C 1, but only as a minor compound. Essential-Oil-Composition Essential-Oil-Yield Hypothesis. The proposed hypothesis considered the existence of a possible link between the oil yield and the oil composition, i.e., it was thought that essential-oil-rich species have a pronounced biosynthesis of TER compounds and/or phenylpropanoids, whereas essential-oil-poor species mostly produce FAD and CDC volatile constituents [5b]. The variations in the volatile profiles of the taxa analyzed in this study and 60 other randomly chosen species belonging to different plant genera (characterized by a wide range of essential-oil yields) [5c] were correlated with the oil yields, with the aim to test this compoundclass yield hypothesis [5b]. The class distribution of the essential-oil components, i.e., the summed-up contents ( percentages) of the constituents belonging to the FAD and CDC classes were plotted vs. the essential-oil yields (Fig. 1). The diagram in Fig. 1 reveals a good Boltzmann fit and nicely demonstrates that the essential oils of oil-poor species with oil yields much lower than 0.1% are mostly dominated by FAD and/or CDC volatiles. The predominance of these compounds might be explained by the lack of an elaborate biosynthetic apparatus responsible for the production of volatile terpenes and/or phenylpropanoids, which dominate in aromatic species with relative abundance of essential oils (yields of 0.1 1.0%). FAD and CDC compounds are generally connected with rather non-specific, omnipresent plant biosynthetic pathways (e.g., green-leaf volatiles as oxylipin products, higher alkanes as wax constituents, etc.), so that these compounds represent major oilcompound classes of non-aromatic, oil-poor species (relative yields of 10 3 10 2 %). Multivariate Statistics. The chemotaxonomic significance of the variation in the essential-oil profiles among Geranium and Erodium species originating from the Balkan Peninsula (investigated here or previously studied under the same experimental conditions in our laboratory [2c][4]), i.e., G. macrorrhizum, G. robertianum, G. sanguineum, G. columbinum, G. lucidum, E. cicutarium, E. ciconium, and E. absinthoides, was inferred from multivariate statistical analyses (MVA), i.e., agglomerative hierarchical clustering (AHC) analysis and principal component analysis (PCA; for observations included in the MVA, cf. Table 3). The dendrogram obtained

2049 Fig. 1. Correlation diagram between the essential-oil yields and the class distribution of the essential-oil components, i.e., the summed-up contents (percentages) of the constituents belonging to the fatty acids and fatty-acid-derived compounds (FAD) and carotenoid-derived compounds (CDC), of the taxa characterized in this study and other 60 randomly chosen species belonging to different plant genera by AHC analysis, performed using the relative contents (original variables) of the identified oil constituents, depicted in Fig. 2, revealed four groups of essential oils (Groups I IV). The oil-sample codes, i.e., the italic capital letters A L, used as designations for the oil samples, are listed in Table 3. Group I was composed of one single oil (Sample D), isolated from G. macrorrhizum and distinguished from the other oils mainly by the predomination of germacrone, i.e., by the production and accumulation of this sesquiterpenoid in this plant species. Whereas the G. macrorrhizum oil was characterized by the predomination of germacrone, the remaining essential oils were very similar to each other and different from the former, which was also evident from the biplot obtained as a result of the PCA, which was based on the same individual component variable set (original variables) than the AHC (Fig. 3). Another interesting point that arises from the biplot was that the PCA distinguished the group of Erodium species (Samples I L) from the cluster containing the investigated Geranium taxa, although the differentiation at both the infrageneric and intergeneric level was ca. at the same level. The main conclusion, based on the dendrogram and biplot obtained as the results of the performed analyses, using relative abundances of the identified oil constituents, consists in that there was no great intergeneric variability observed in the oil compositions, i.e., the chemical composition of the investigated Geranium and Erodium taxa was generally very similar. These results confirm the close phylogenetic relationship between the two genera.

2050 Table 3. Balkan Geranium and Erodium Species (characterized in the present study or investigated previously in our laboratory under the same experimental conditions) Included in the Multivariate Statistical Analyses Plant species Sample code Plant parts Reference G. purpureum Vill. A Entire plant Present study G. phaeum L. B Aerial parts Present study G. phaeum L. C 1 Aerial parts Present study G. macrorrhizum L. D Aerial parts [2c] G. columbinum L. E Aerial parts [4b] G. lucidum L. F Entire plant [4b] G. sanguineum L. G Entire plant [4a] G. robertianum L. H Aerial parts [4a] E. cicutarium (L.) L He r. I Entire plant [4c] E. cicutarium (L.) L He r. J Aerial parts [4d] E. ciconium (Jusl.) Aiton K Entire plant [4d] E. absinthoides Willd. L Entire plant [4d] Fig. 2. Dendrogram (AHC analysis) representing the dissimilarity relationship of the chemical composition (original variables, i.e., relative compound contents that exceed 7.5% of the total oil composition in at least one observation) of the twelve essential-oil samples (observations) of seven Geranium and three Erodium species obtained by Euclidean distance dissimilarity (dissimilarity within interval [0, 2900]), using Wards method as aggregation criterion. Four groups of essential oils (I IV, from bottom to top) were distinguished. For the oil codes (Oils A L), cf. Table 3. The authors acknowledge the Ministry of Education, Science, and Technological Development of Serbia for financial support of this work (Project No. 172061).

2051 Fig. 3. Principal component analysis (original variables, i.e., the relative compound contents that exceed 7.5% of the total oil composition in at least one observation) ordination of twelve essential-oil samples (observations) of seven Geranium and three Erodium species. The axes (F1 and F2 factors, i.e., the first and second principal components) refer to the ordination scores obtained from the samples. Axes F1 and F2 account for ca. 34.1 and 23.7% of the total variance, respectively. For the oil codes (Oils A L), cf. Table 3. Experimental Part Plant Material. The plant samples investigated in this work were collected from natural populations. The complete details on the collected taxa are summarized in Table 1. The plant material subjected to analyses was dried at r.t. for two weeks. Voucher specimens have been deposited with the Herbarium collection of the Faculty of Science and Mathematics, University of Niš, under the acquisition numbers given in Table 1. Isolation of the Essential Oils. Air-dried (to constant weight) plant material (two times three batches of ca. 500 g of each taxon) was subjected to hydrodistillation with ca. 2 l of dist. H 2 O for 2.5 h using an original Clevenger-type apparatus [4c]. The obtained oils were separated by extraction with freshly dist. Et 2 O(Merck, Germany), dried (anh. Na 2 SO 4, Aldrich, USA), and immediately analyzed. The oil yields are given in Table 1. GC-FID and GC/MS Analyses. The chemical composition of the oils was determined by GC-FID and GC/MS analyses (three repetitions for each sample). The GC/MS analyses were performed on a Hewlett-Packard 6890N gas chromatograph equipped with a fused-silica cap. column HP-5MS (5% phenylmethylsiloxane, 30 m 0.25 mm i.d., film thickness 0.25 mm, Agilent Technologies, USA) and coupled with a 5975B mass selective detector (MSD) from the same company. The injector and interface were operated at 250 and 2808, resp. The oven temp. was raised from 70 to 2258 at 58/min and then isothermally held at 2258 for 10 min; carrier gas, He (1.0 ml/min). The samples, 1 ml of the oil solns. in Et 2 O (1 : 100), were injected in a pulsed split mode (flow of 1.5 ml/min for the first 0.5 min and then 1.0 ml/min throughout the remainder of the analysis; split ratio, 40 : 1). The MSD (EI) was operated at the ionization energy of 70 ev, over the mass range 35 500 amu, at a scan speed of 0.34 s 1. The GC-FID analyses were carried out under the same experimental conditions and using the same column as described for the GC/MS analyses.

2052 Identification and Quantification of Constituents. The identification of the essential-oil constituents was based on the comparison of their linear retention indices (RIs, determined rel. to the retention times (t R ) of a series of n-alkanes (C 8 C 27 ) on the HP-5MS column [7]) to those reported in the literature [6] and their mass spectra to those of authentic standards as well as to those listed in the Wiley 6, NIST05, and MassFinder 2.3 mass spectral libraries. Moreover, a homemade mass spectral library built with the spectra of pure substances and components of known essential oils was used, and, finally, whenever possible, the identification was achieved by coinjection with an authentic sample (alkanes, some of the terpenoids, and aromatics). The content (percentage composition) of the components was computed from the GC-FID peak areas without the use of correction factors. Data Analyses. The multivariate statistical analyses (MVA), i.e., the principal components analysis (PCA) and the agglomerative hierarchical clustering (AHC) were performed using the Excel program plug-in XLSTAT (version 2009.3.02, Addinsoft, France). Both methods were applied to examine the inter-relationships between the investigated species and their chemical constituents utilizing the original variables (compound contents that exceeded 7.5% of the total oil composition in at least one of the oils). The AHC was determined using the Pearson dissimilarity (the aggregation criteria were simple linkage, unweighted pair-group average, and complete linkage) and the Euclidian distance (the aggregation criteria were weighted pair-group average, unweighted pair-group average, and Wards method). The definition of the oil groups was based on the Pearson correlation, using the complete linkage and unweighted pair-group average method. The data set used in the correlation diagram (essential-oil yields and the class distribution of the essential-oil components) was analyzed using Origin 6.1 (Originlab Corporation, USA). REFERENCES [1] M. M. Janković, in Flora of Serbia, Ed. M. Josifović, Srpska Akademija Nauka i Umetnosti, Belgrade, 1973, Vol. 5, p. 136. [2] a) J.-C. Chalchat, S. D. Petrović, Z. A. Maksimović, M. S. Gorunović, J. Essent. Oil Res. 2002, 14, 333; b) C. Ş. Fodorea, R. Oprean, M. Tămaş, Contrib. Bot. 2005, 40, 243; c) N. S. Radulović, M.S. Dekić, Z. Z. Stojanović-Radić, S. K. Zoranić, Chem. Biodiversity 2010, 7, 2783. [3] a) C. Proestos, I. S. Boziaris, G.-J. E. Nychas, M. Komaitis, Food Chem. 2006, 95, 664; b) D. Sohretoglu, M. K. Sakar, O. Sterner, Turk. J. Chem. 2011, 35, 809; c) O. Ertürk, Fresenius Environ. Bull. 2010, 19, 3113; d) D. Sohretoglu, M. Ekizoglu, M. Ozalp, M. K. Sakar, Hacettepe Univ. J. Fac. Pharm. 2008, 28, 115; e) D. Sohretoglu, T. Liptaj, M. K. Sakar, O. Sterner, Nat. Prod. Commun. 2011, 6, 1321. [4] a) N. S. Radulović, M. S. Dekić, Z. Z. Stojanović-Radić, Med. Chem. Res. 2012, 21, 601; b) N. Radulović, M. Dekić, Z. Stojanović-Radić, R. Palić, Turk. J. Chem. 2011, 35, 499; c) N. Radulović, M. Dekić, Z. Stojanović-Radić, R. Palić, Cent. Eur. J. Biol. 2009, 4, 404; d) Z. Z. Stojanović-Radić, L. R. Čomić, N. S. Radulović, M. S. Dekić, V. N. Randjelović, O. Stefanović, Chem. Pap. 2010, 64, 368. [5] a) B. M. Lawrence, Essential Oils 1988 1991, Allured Publishing Corporation, Carol Stream, IL, 1992; b) N. S. Radulović, P. D. Blagojević, R. M. Palić, Nat. Prod. Commun. 2009, 4, 405; c) N. Radulović, P. Blagojević, in Book of Abstracts, 41st International Symposium on Essential Oils, Wroclaw, Poland, September 5 8, 2010, p. 42 (OP-12). [6] a) R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th edn., Allured Publishing Corp., Carol Stream, IL, 2007; b) N. S. Radulović, P. D. Blagojević, Chem. Biodiversity 2010, 7, 2856; c) N. Radulović, M. Dekić, M. Joksović, R. Vukićević, Chem. Biodiversity 2012, 9, 106; d)n.s. Radulović, M.Z. Mladenović, N.D. or ević, Chem. Biodiversity 2012, 9, 1320; e) N. S. Radulović, M.S. Dekić, P. J. Ran elović, N. M. Stojanović, A.R. Zarubica, Z. Z. Stojanović-Radić, Food Chem. Toxicol. 2012, 50, 2016; f) N. S. Radulović, M. S. Denić, Chem. Biodiversity 2013, 10, 658; g)n. S. Radulović, P. D. Blagojević, Chem. Biodiversity 2012, 9, 2324; h) N. S. Radulović, M. Z. Mladenović, P. D. Blagojević, Chem. Biodiversity 2013, 10, 1202. [7] H. Van den Dool, P. D. Kratz, J. Chromatogr., A 1963, 11, 463. Received June 20, 2013