Microsoft Word - 0404_Cavar_et_al BIOLOGICA NYSSANA 1 (1-2) December 2010: 99-103 Čavar, S. et al. Comparison of essential oil composition of Stachys … 31 Original Article ! Comparison of essential oil composition of Stachys menthifolia Vis. from two natural habitats in Croatia Sanja Ćavar1,2*, Milka Maksimović1, Marija Edita Šolić3 1University of Sarajevo, Faculty of Science, Department of Chemistry, Zmaja od Bosne 33-35, 71000 Sarajevo, Bosnia and Herzegovina 2University of Ljubljana, Faculty of Chemistry and Chemical Technology, Aškerčeva 5, 1000 Ljubljana, Slovenia 3Institute „Mountain and Sea“, Franjevački put 1, 21300 Makarska, Croatia E-mail: sanja.cavar@pmf.unsa.ba Abstract: Ćavar, S., Maksimović, M., Šolić, M. E.: Comparison of Essential Oil Composition of Stachys menthifolia Vis. from Two Natural Habitats in Croatia. Biologica Nyssana, 1 (1-2), December 2010: 99-103. Stachys menthifolia Vis. is an endemic species from the Balkan Peninsula. Aerial parts of the plant were collected from its natural habitat near Dubrovnik. Hydrodistilled volatile oil obtained from the plant material of S. menthifolia was subjected to gas chromatographic analysis coupled to mass spectrometry. More than 70 compounds were identified, representing 94.5% of the total oil. The major constituents of the oil were diterpenoid abietatriene (11.7%), and sesquiterpene hydrocarbons α-bisabolene (8.4%), and β-caryophyllene (7.4%). Presented results are comparable to our previous findings on essential oil composition of the same species from Biokovo Mountain, with small differences in quantitative and qualitative constitution of the oil. Although plants belonging to the Stachys genus show significant variability in their chemical compositions depending on the location and stage of plant development, this work indicates that chemical polymorphism of endemic S. menthifolia does not manifest in the region of Croatian Mediterranean area. Key words: abietatriene, essential oil, GC-MS, Stachys menthifolia Vis. Introduction ! The genus Stachys is one of the largest representative genera of the Lamiaceae family, and includes about 300 species in the subtropical and tropical regions of both hemispheres (M a b b e r l e y , 1997). Like most of species belonging to the family Lamiaceae, Stachys species produce essential oils, but the composition of volatile compounds is known only in a small number of species (M a l y , 1985; C a k i r et al., 1997; M a r i o t t i et al., 1997; P é l i s s i e r at al., 1999; C h a l c h a t et al., 2001; K u k i ć et al., 2006; P a l i ć et al., 2006; R a d u l o v i c et al., 2007). Available literature data indicate the existence of a chemical polymorphism of essential oil composition among Stachys taxa. Stachys menthifolia Vis. is an endemic species of the West Balkan region. Š i l i ć & Š o l i ć (2001) discovered its natural habitat in the region of Biokovo Mountain in Croatia. The first location in Croatia was near Dubrovnik, discovered by Roberto Visiani, who was the first who described this species. Continuing our phytochemical research on S. menthifolia, this paper presents the essential oil composition of the plant collected from its natural habitat near Dubrovnik. 10th SFSES • 17-20 June 2010, Vlasina lake1 (1-2) • December 2010: 99-103 BIOLOGICA NYSSANA 1 (1-2) December 2010: 99-103 Ćavar, S. et al. Comparison of essential oil composition of Stachys … 32 Material and methods Plant material was collected in May 2008 from the natural habitat near Dubrovnik. Voucher specimens have been deposited at the Department of Chemistry, Faculty of Science, University of Sarajevo, Bosnia and Herzegovina. All applied reagents were of the highest purity available and purchased from the Sigma- Aldrich Chemical Company. Essential oil from the air-dried parts of S. menthifolia was isolated by hydrodistillation according to European Pharmacopoeia for 2 h. The oil was extracted with dichloromethane and dried over anhydrous sodium sulphate and stored at 4°C in the dark until the analysis. Gas chromatography-mass spectrometry analysis was carried out on a Hewlett-Packard 6890 Series II gas chromatograph fitted with a fused silica HP-5 (5% phenyl methyl siloxane) capillary column (30 m × 0.252 mm, 0.25 μm film thickness), coupled to a HP 6890 Series II mass selective detector (MSD). Column temperature was programmed from 60°C to 240°C at 3°C min-1, and helium was used as carrier gas. Other operating conditions were as follows: inlet pressure 9.43 psi, injector temperature 250°C, detector temperature 280°C, split ratio 1:25, injection volume 1 μL. Ionization of the sample components was performed in the EI mode, (70 eV), with scan range 20-555 amu, and scan time 1.60 s. The linear retention indices, RI, for all compounds were determined by injection of the hexane solution containing the homologous series of C8-C26 n-alkanes (V a n D e n D o o l & K r a t z , 1963). The identification of the essential oil constituents was accomplished by the visual interpretation, comparing their retention indices and mass spectra with literature data (A d a m s , 2007) by computer library search (HP Chemstation computer library NBS75K.L, NIST/EPA/NIH Mass Spectral Library 2.0 and Mass Finder 4 Computer Software and Terpenoids Library), and in the laboratory own database. Semi-quantitative analysis was carried out directly from peak areas in the GC profile. Results and discussion The yield of S. menthifolia hydrodistilled oil was 0.09% based on the dry weight of the plant material. Exactly seventy-six compounds were identified in the essential oil, representing 94.5% of the total oil. The components identified in investigated sample of S. menthifolia, their retention indices, and percentage composition is summarized in Table 1. The major constituents of the oil were diterpenoid abietatriene (11.7%), and sesquiterpene hydrocarbons α-bisabolene (8.4%), and β- caryophyllene (7.4%). In general, sesquiterpenoid compounds predominate in the oil with 59.1%, where 35.1% are sesquiterpene hydrocarbons. Presented results can be compared to essential oil composition of S. menthifolia from Biokovo Mountain (Ć a v a r et al., 2010), with some differences in quantitative and qualitative constitution of the oil (Figure 1). Oxygenated sesquiterpenes were the most abundant volatiles in S. menthifolia from Biokovo (Table 1), followed by diterpene hydrocarbons. This sample was characterized as the oil of abietatriene– 8−α−acetoxyelemol chemotype. Significant differences between these samples were in the content of aromatic compounds, particularly 4’- methoxyacetophenone, which was not found in the population from Dubrovnik. Moreover, the content of sesquiterpene hydrocarbons was much higher in the sample from Dubrovnik. The observed differences might be explained by the fact that this plant material was harvested one month earlier than the sample from Biokovo. Figure 1. GC chromatograms of essential oil of S. menthifolia from Dubrovnik (1) and Biokovo (2) Literature survey on studies related to the essential oils obtained from the plants belonging to Stachys genus from the Balkan Peninsula, showed significant variability in their chemical composition depending on location and stages of plant development. Essential oil obtained from S. milanii Petrović (P a l i ć et al., 2006) had borneol and terpinen-4-ol as the most abundant compounds, while the major constituents of essential oil from Bosnian S. alpina L. spp. dinarica Murb. were β- BIOLOGICA NYSSANA 1 (1-2) December 2010: 99-103 Čavar, S. et al. Comparison of essential oil composition of Stachys … 33 caryophyllene and germacrene D (K u k i ć et al., 2006). Dehydroabietane is the predominant compound in the essential oil of S. plumosa Griseb. (P e t r o v i ć et al., 2006). Sesquiterpenoids were the main class of the constituents in Croatian Stachys species as follows: (E)-nerolidol in S. alpina; germacrene D in S. officinalis, S. recta subsp. subcrenata, S. salviifolia, and S. sylvatica, caryophyllene oxide, along with 1-octen-3-ol, in S. palustris, while S. recta subsp. recta contained β- ionone as the dominant volatile constituent (B i l u š i ć V u n d a c et al., 2006). Table 1. Comparison of essential oil composition of S. menthifolia from Croatia # RI Compound Dubrovnik RA† (%) Biokovo* RA (%) 1. 801 Hexanal 0.3 tr‡-0.2 2. 847 (2E)-Hexenal 0.6 tr-0.2 3. 861 n-Hexanol 0.3 - 4. 874 2-Methyl butyl acetate 0.2 - 5. 930 α-Pinene 0.3 tr-1.0 6. 975 1-Octen-3-ol 0.9 tr-0.5 7. 1026 Limonene 1.0 0.1-3.4 8. 1041 Benzene acetaldehyde 0.1 tr 9. 1056 γ-Terpinene 0.2 tr 10. 1099 Linalool 3.6 0.7-1.7 11. 1103 Nonanal 0.2 tr 12. 1107 2-Methyl butyl isovalerate 0.5 - 13. 1111 1-Octen-3-yl acetate 1.9 - 14. 1122 3-Octanol acetate 0.4 - 15. 1189 α-Terpineol 0.3 tr-0.3 16. 1190 Methyl salycilate tr tr 17. 1255 Linalool acetate 0.3 - 18. 1286 trans-Linalool oxide acetate (pyranoid) 0.2 - 19. 1373 α-Copaene 0.5 tr-0.3 20. 1381 β-Bourbonene 0.3 tr 21. 1416 β-Caryophyllene 7.4 0.5-2.8 22. 1422 β-Maaliene 0.2 - 23. 1434 β-Guaiene 0.1 tr 24. 1450 α-Humulene 3.4 tr-0.2 25. 1456 (E)-β-Farnesene 1.4 - 26. 1478 γ-Curcumene 0.3 - 27. 1481 ar-Curcumene 0.3 tr-0.2 28. 1492 α-Muurolene 0.6 tr-0.2 29. 1499 (E,E)-α-Farnesene 1.3 - 30. 1503 α-Zingiberene 3.8 - 31. 1508 α-Bisabolene 8.4 tr-0.1 32. 1512 γ-Cadinene 1.7 tr-0.4 33. 1522 δ-Cadinene 3.3 - 34. 1530 trans-Cadina-1,4-diene 0.3 - 35. 1535 α-Cadinene 0.9 tr-0.2 36. 1547 Elemol 1.4 2.2-3.6 37. 1564 2-epi-(E)-β-Caryophyllene 0.3 - 38. 1574 Spathulenol 0.7 0.7-1.7 39. 1579 Caryophyllene oxide tr 2.4-5.2 40. 1581 Clovenol 1.1 - 41. 1588 Globulol 1.9 1.5-2.4 42. 1600 Guaiol tr tr-0.3 43. 1606 β-Oplopenone 0.3 tr 44. 1627 trans-Isolongifolanone 0.9 1.2-1.6 45. 1629 γ-Eudesmol 0.9 1.1-1.9 BIOLOGICA NYSSANA 1 (1-2) December 2010: 99-103 Ćavar, S. et al. Comparison of essential oil composition of Stachys … 34 R a d u l o v i c et al., (2007) suggested that high degree of variation in the main volatiles of the species of Stachys genus, especially of germacrene D and its congeners, was correlated with possible rearrangements under hydrodistillation conditions. According to the presence and quantity of dominant compounds, the essential oil of investigated populations of S. menthifolia significantly differs from the previously published data concerning this species (S k a l t s a et al., 2003). Greek S. menthifolia essential oil had abietatriene (13.7%), kaurene (9.0%) and 13-epi- manoyl oxide (7.5%) as the most abundant compounds. The observed differences in qualitative and quantitative composition of the essential oils between these two geographically isolated populations of S. menthifolia, confirm the influence of the environmental conditions on the volatiles # RI Compound Dubrovnik RA† (%) Biokovo* RA (%) 46. 1633 Caryophylla-4(15),8(13)-dien-5-α-ol 0.4 tr-0.4 47. 1640 epi-α-Murrolol 1.7 1.8-2.6 48. 1647 β-Eudesmol 2.3 2.4-5.0 49. 1650 α-Eudesmol 1.5 1.4-3.3 50. 1652 α-Cadinol 1.5 2.0-3.1 51. 1655 allo-Aromadendrene epoxide 1.3 - 52. 1669 Valeranone 4.5 - 53. 1676 n-Tetradecanol 0.5 0.5-0.6 54. 1682 α-Bisabolol 0.4 - 55. 1695 Isolongifolol 2.7 - 56. 1761 Benzyl benzoate tr - 57. 1788 8-α-Acetoxyelemol 2.1 6.9-21.3 58. 1807 Cryptomeridiol 0.3 0.5-6.7 59. 1844 6,10,14-Trimethylpentadecan-2-one 0.2 - 60. 1903 Isopimara-9(11),15-diene 0.6 0-5-1.1 61. 1954 Pimaradiene 0.5 0.2-0.8 62. 1963 n-Hexadecanoic acid 0.7 - 63. 1983 Manool oxide 1.9 0.7-2.3 64. 2002 (E)-Labda-7,12,14-triene 0.3 0.5-2.7 65. 2006 13-epi-Dolabradien tr 0.3-0.6 66. 2050 Abietatriene 11.7 3.5-21.1 67. 2073 Abietadiene 0.6 tr-0.8 68. 2141 Abienol 0.4 0.7-0.9 69. 2206 2-Keto manool oxide 0.7 0.7-1.2 70. 2226 7-α-Hydroxy manool 0.4 0.2-0.3 71. 2258 Larixol 0.5 0.4-1.1 72. 2288 Dehydroabietal 1.9 1.5 73. 2298 n-Tricosane tr tr 74. 2499 n-Pentacosane 0.3 tr-0.2 75. 2599 n-Hexacosane tr tr-0.2 76. 2698 n-Heptacosane 0.6 - Aliphatic compounds 7.4 0.5-1.4 Aromatic compounds 2.2 4.5-17.0 Monoterpene hydrocarbons 1.5 0.1-4.6 Oxygenated monoterpenes 4.4 1.7-2.9 Sesquiterpene hydrocarbons 35.1 1.4-6.4 Oxygenated sesquiterpenes 24.0 48.4-58.9 Diterpene hydrocarbons 13.1 3.5-25.2 Oxygenated diterpenes 6.8 1.5-5.8 Total identified 94.5 86.8-90.8 *Data taken from Ć a v a r et al., (2010); †RA-Relative area; ‡tr-trace (<0.1%). BIOLOGICA NYSSANA 1 (1-2) December 2010: 99-103 Čavar, S. et al. Comparison of essential oil composition of Stachys … 35 found in plants. It is essential to notify that these prominent variations were not retrieved in the populations from Croatia. Conclusion In conclusion, GC-MS analysis on the essential oil of endemic S. menthifolia populations from Croatian Mediterranean area indicated similarities in qualitative, but small differences in quantitative composition of their essential oils. Although plants belonging to Stachys genus show significant variability in their chemical compositions depending on the location and stage of plant development, this work indicates that chemical polymorphism of endemic S. menthifolia does not significantly manifest in the region of Croatian Mediterranean area. References Adams, R. P. 2007: Identification of essential oil components by gas chromatography/mass spectrometry, 4th Ed. Allured Publ., Carol Stream, IL. Bilušić Vundac,V., Pfeifhofer, H. 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