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Composition of the essential oil of the Rhododendron tomentosum Harmaja from Estonia a
b
b
Ain Raal , Anne Orav & Tatjana Gretchushnikova a
Department of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, Estonia b
Institute of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia Published online: 09 Apr 2014.
To cite this article: Ain Raal, Anne Orav & Tatjana Gretchushnikova (2014): Composition of the essential oil of the Rhododendron tomentosum Harmaja from Estonia, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.907287 To link to this article: http://dx.doi.org/10.1080/14786419.2014.907287
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.907287
Composition of the essential oil of the Rhododendron tomentosum Harmaja from Estonia Ain Raala*, Anne Oravb and Tatjana Gretchushnikovab Department of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, Estonia; bInstitute of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia
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a
(Received 27 December 2013; final version received 16 March 2014) Wild Rhododendron tomentosum Harmaja shoots were collected from four localities of Estonia. Essential oils, isolated from dried samples by simultaneous distillation and extraction, were analysed using GC-FID and Gas chromatography – mass spectrometry methods. The yields of oils were in the range 0.14 – 0.87%. A total of 72 constituents, accounting for over 95% of the total oil yield, were identified. The major components in the all four oils studied were palustrol (15.9– 53.5%), ledol (11.8 – 18.3%), g-terpineol (0 – 31.2%), p-cymene (0.1 – 13.9%), lepalone (0.7 – 6.5%), lepalol (1.0 – 6.5%) and cyclocolorenone (1.0 – 6.4%). Two different chemotypes of R. tomentosum were found in Estonia and one of them was rich in palustrol (41.0– 53.5%) and ledol (14.6– 18.3%). The second chemotype, for the first time, was rich in g-terpineol (24.7– 31.2%) and contained less of palustrol (15.9– 16.7%) and ledol (11.8– 12.8%), but more p-cymene (12.5 – 13.9%). Also, g-terpineol was identified for the first time in the oils of R. tomentosum. Keywords: Ledum palustre L.; Rhododendrum tomentosum Harmaja; marsh rosemary; essential oil; chemotypes; ledol; palustrol
1. Introduction Wild rosemary or Marsh tea has long been referred to as Ledum palustre L. Recently, it has been discovered that L. palustre L. belongs to the Rhododendron family, its Latin nomenclature has changed to Rhododendron tomentosum Harmaja. It is a low shrub with evergreen leaves growing wild in North and Central Europe, North Asia and America. The tiny white flowers are very fragrant and sticky. It is in leaf all year and flowers from April to August, depending on the area. R. tomentosum prefers acidic soils and can grow with various sun exposures. As its name suggests, it is most appropriate to grow in marshy and swampy areas. The leaves and flowers of R. tomentosum have strong smell and may cause headache to some people. All the parts of the plant contain poisonous terpenes that affect the central nervous system, causing aggressive behaviour. This plant is widely used in folk medicine and homeopathy for the treatment of rheumatism, arthrosis and insect bites. The expectorant and antitussive effect of marsh tea is due to the content of ledol in its essential oil (Dampc & Luczkiewicz 2013). The composition of the essential oils from R. tomentosum varied in a wide range in different localities (Schantz et al. 1973; Mikhailova et al. 1978; Tettje & Bos 1981; Klokova et al. 1983; Belousova & Khan 1990; Belousova et al. 1990, 1991; Ylipahkala & Jalonen 1992; Jaenson et al. 2005, 2006; Butkiene et al. 2008; Gretsˇusˇnikova et al. 2010; Butkiene & Mockute 2011; Judzentiene, Butkiene, et al. 2012, Judzentiene, Budiene, et al. 2012). The major components in
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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its oils were mostly palustrol, ledol and myrcene. Cyclocolorenone, sabinene, limonene, pcymene, cis-p-mentha-1(7) and 8-dien-2-ol were found in high amounts (. 5%) besides palustrol and ledol in R. tomentosum oils from different localities (Belousova et al. 1990; Jaenson et al. 2005; Dampc & Luczkiewicz 2013). Ledol, as toxic compound, can affect the central nervous system, leading finally to paralysis, breathing problems and even death (Dampc & Luczkiewicz 2013). Therefore, the potential chemotypes poor in ledol can be important from the perspective of herbal drug safety. In this work, the variations of the essential oil amounts and compositions were determined in four samples of R. tomentosum grown wild in different localities of Estonia.
2. Results and discussion R. tomentosum shoots were separated from wild plants collected in four localities. The amounts of essential oils from dried Samples 1 and 4 (0.78 – 0.87%) exceeded four times those from Samples 2 and 3 (0.14 – 0.24%). In our previous studies, much more significant differences (10to 17-fold) were found between the minimum and the maximum yields of essential oils of Coriandrum sativum, Levisticum officinale, Thymus serpyllum and Chamomilla recutita from various countries (Paaver et al. 2008; Raal et al. 2008; Orav et al. 2010, 2011). A 14-fold annual variance was detected in the yield of the essential oil in Juniperus communis branches collected once in month (Raal et al. 2010), but only about a 2-fold variance in the yield of essential oils was observed in the aerial parts of Origanum vulgare from different countries (Ivask et al. 2005). Thus, a moderate variance in the yield of essential oil has been found in R. tomentosum samples we studied. A total of 72 compounds were identified in the studied R. tomentosum samples, accounting for over 95% of the total oil yield. In Samples 1, 3 and 4 correspondingly, 65, 61 and 62 constituents were identified but in Sample 2, only 33 compounds were found. Mass spectral data of unidentified compounds are presented in Table 2 ([Mþ] value and characteristic peaks m/z values). The major components in the all four shoot oils of Estonian R. tomentosum, as evident in Table 1, were palustrol (15.9 – 53.5%), ledol (11.8 – 18.3%), g-terpineol (0 –31.2%), p-cymene (0.1 – 13.9%), lepalone (0.7 – 6.5%), lepalol (1.0 – 6.5%) and cyclocolorenone (1.0 – 6.4%). The composition of the essential oils was based on the locality. Two different chemotypes of R. tomentosum were found in Estonia. The dominant constituent in Samples 2 and 3 was palustrol (41.0 –53.5%), followed by ledol (14.6 – 18.3%). These samples were also rich in cyclocolorene (4.3 – 6.4%) and compounds with furyl structure: lepalone (5.0 –6.5%) and lepalol (3.5 – 6.5%). As evident in Table 1, the content of compounds with menthane skeleton (0.9 – 5.8%) and compounds with aromatic structure (0.4 – 2.9%) in these samples was low. The second chemotype was rich in g-terpineol (24.7 – 31.2%). Samples 1 and 4 contained less of palustrol (15.9 – 16.7%) and ledol (11.8 –12.8%), but with more p-cymene (12.5 – 13.9%). As it was demonstrated by Gretsˇusˇnikova et al. (2010), the dominant compound of stem oil from R. tomentosum growing in Estonia was b-myrcene followed by palustrol and ledol, still the leaves and shoots contained three – four times more the essential oil than the stems. The main carbon skeletons of R. tomentosum oil constituents were aromadendrene, menthane, furane and 2,6-dimethyloctane as reported by Butkiene et al. (2008). In Samples 1 and 4, compound with menthane skeleton 1-methyl-4-(1-methylethylidene) cyclohexanol (g-terpineol) dominated (24.7 – 31.2%), followed by palustrol (15.9 –16.7%), ledol (11.8 – 14.6%) and p-cymene (12.5 – 13.9%). g-Terpineol was not found in the earlier investigated above-mentioned oils. Isoascaridol (2.7%), geranyl acetate (0.2 – 3.5%) and (Z)-nerolidol acetate (1.7 – 2.1%) were also found in these oils in remarkable amounts.
a-Pinene Camphene Sabinene b-Pinene b-Myrcene a-Phellandrene (E,E)-2,4-heptadienal Bicyclo[3.3.1]nonane a-Terpinene p-Cymene Limonene 1,8-Cineole (Z)-b-ocimene (E)-b-ocimene g-Terpinene Terpinolene p-Cymenene Linalool Isothujol n-Nonanal 2-Methyl-6-methylene-3,7-octadien-2-ol 1,3,8-Menthatriene Dihydrosabinaketone (lebakon) (E)-p-mentha-2-en-1-ol (Z)-p-mentha-2,8-dien-1-ol (E)-Pinocarveol 5-(3-Furyl)-2-methyl-1-pentene (lepalin) 2-Methyl-6-methylene-1,7-octadien-3-one (E)-3(10)-caren-4-ol Pinocarvone
Compound 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3
Identification method
930 945 970 972 990 1002 1009 – 1018 1022 1026 1030 1040 1050 1054 1086 1090 1098 – 1102 – – – – – 1137 – – – 1162
NIST
Table 1. The composition of the essential oil of L. palustre L. from Estonia (%).
930 943 969 971 990 1002 1006 1008 1013 1023 1025 1028 1035 1045 1054 1084 1085 1100 1100 1102 1102 1107 1118 1121 1129 1132 1132 1138 1147 1155
SPB-5
RI
1182 1277 1203 1211 1240 1256 1247 1280 1426 1558 1560 1400 2077 1395 1395 1627 1620 1654 1554 1554 1662 1563
1024 1067 1120 1111 1168 1168 1329
SW-10
0.7 0.1 tr 0.5 1.3 0.1 0.2 – 0.1 12.5 0.3 0.1 0.1 tr 0.1 0.1 0.5 0.3 – 0.2 – 0.2 0.2 0.1 0.5 0.7 0.7 0.1 0.1 0.8
1
– tr – tr 2.8 – – 0.1 – 0.1 – – – – – – – – – – 1.1 – – – – – 0.6 0.6 0.2 –
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0.1 tr – 0.1 0.8 – – 0.3 0.2 1.9 0.1 tr tr tr 0.1 0.1 tr – 0.4 – 0.9 0.1 0.1 0.1 0.1 0.2 1.9 0.8 0.2 0.2
3
0.7 0.3 tr 0.6 1.0 0.1 0.1 – 0.1 13.9 0.3 0.1 0.2 0.1 0.2 0.2 0.5 0.3 – 0.2 – 0.3 0.2 0.1 0.6 0.4 0.3 – 0.2 0.5
SDE 2 h
Sample number
0.6 0.2 – 0.4 0.8 tr 0.1 – 0.2 11.7 0.3 – 0.1 – 0.2 – 0.7 0.3 – 0.2 – 0.5 0.2 tr 0.6 0.8 0.3 – 0.1 0.5
SDE 4 h
(Continued)
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2-Methyl-6-methylene-1,7-octadien-3-ol Terpinen-4-ol a-Thujenal p-Cymen-8-ol a-Terpineol Myrtenal (E)-p-Mentha-1(7),8-dien-1-ol (Z)-p-Mentha-1(7),8-dien-1-ol p-Cuminal 5-(3-Furyl)-2-methyl-3-penten-2-ol m-Isopropylbenzaldehyde g-Terpineol Piperitone 5-(3-Furyl)-2-methyl-1-penten-3-one (lepalone) NI (1) Isopiperitenone þ lepalol isomer Perillaldehyde Vitispirane Isogeraniol 5-(3-Furyl)-2-methyl-1-penten-3-ol (lepalol) Bornyl acetate NI (2) Carvacrol Isoascaridol 5-Isopropenyl-2-methylcyclo-pent-1-ene carboxaldehyde 2-Methyl-5(fur-3-yl)-pent-1-en-3-ol (lepalol isomer) Neryl acetate Geranyl acetate 2-Ethyl-5-propylphenol Aromadendrene Alloaromadendrene g-Muurolene
Compound
Table 1. (Continued)
1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 3 1, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2
1, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2 1, 2, 3
Identification method
– 1176 – 1183 1190 1193 – – – – – – 1255 – – – 1272 – – 1285 – 1299 – – – 1365 1379 – 1438 1456 1477
NIST
1155 1176 1183 1187 1190 1190 1193 1220 1224 1232 1236 1238 1251 1256 1263 1263 1269 1270 1276 1282 1291 1296 1300 1329 1353 1367 1382 1393 1439 1456 1477
SPB-5
RI
1730 1761 2320 1598 1632 1685
1720 1726 1853 1855 1777 1700 1840 2051 1577 1902 2226 1860
1604 1651 1855 1702 1623 1794 1636 1756 2077
SW-10
2.6 0.1 0.6 0.3 2.7 0.1 0.1 tr 0.2 0.1 tr 0.6 tr
– 0.2 0.4 0.7 – 0.8 0.3 0.1 0.2 0.6 0.1 31.2 0.7 1.3 0.4 tr 0.1
1
3.5 – 0.1 – – 0.2 0.1 – 0.9 – tr 0.8 tr
0.6 tr 0.1 – 0.2 – 0.1 0.7 0.3 tr – – – 5.0 0.4 – tr
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6.5 0.3 0.4 tr 0.3 0.2 – 0.3 0.2 0.1 0.1 0.8 0.2
1.0 0.1 0.3 tr 0.5 0.1 0.4 1.0 0.8 0.2 0.1 2.4 1.0 6.5 1.1 – 0.3
3
1.0 0.7 0.5 0.7 2.7 – 0.3 – 3.5 0.1 – 0.6 0.1
– 0.3 – 0.5 – 0.5 0.3 – tr 0.4 0.1 24.7 0.3 0.7 1.7 0.2 0.2
SDE 2 h
Sample number 4
1.0 0.4 0.3 0.2 2.3 – 0.2 – 2.2 0.1 0.1 0.9 0.1
– 0.1 0.2 0.6 – 0.4 – – – 0.2 – 18.7 0.7 1.4 0.2 – 0.2
SDE 4 h
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1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3
– – – – – 1580 – 1559 – – – 1653 – – 98.8 0.78
1486 1506 1522 1545 1557 1573 1584 1594 1600 1626 1655 1655 1715 1746 99.4 0.14
1683 1890 1904 2100 1920 1983 2060 2022 1983 2108 2174 2216 2350 2351 97.5 0.24
0.4 0.2 0.2 0.1 15.9 – 0.7 11.8 0.4 0.1 2.1 0.1 0.2 1.6 96.7 0.87
0.5 – – – 53.5 tr 1.5 18.3 0.6 0.1 – – – 6.4 96.1 1.11
0.8 0.1 – – 41.0 0.3 1.6 14.6 0.7 0.1 0.1 – – 4.3
0.4 0.4 0.1 0.4 16.7 0.2 0.8 12.8 0.1 0.3 1.7 tr 0.2 1.0
0.5 0.3 0.2 0.2 23.9 0.2 1.3 16.5 – 0.1 2.0 tr 0.2 1.8
Notes: tr, trace (,0.05%); identification methods: 1, RISPB-5, 2, RISW-10, 3, mass spectra. Main components (.1%) are indicated in bold, NIST, NIST Chemistry WebBook http://webbook. nist.gov/chemistry/.
Ledene Shyobunone a-Calacorene NI (3) Palustrol Caryophyllene oxide Epiglobulol Ledol b-Oplopenone NI (4) b-Elemenone (Z)-nerolidol acetate a-Cadinol Isocalamendiol Cyclocolorenone Total Oil yield, %
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Table 2. Mass spectral data of unidentified (NI) compounds. NI
RISPB-5
MW
1 2 3
1263 1291 1545
150
4
1626
m/z (relative intensity) 85 (100), 82 (50), 41 (25), 53 (18), 57 (15) 109 (100), 43 (70), 81 (55), 127 (40) 205 (100), 121 (90), 81 (85), 41 (82), 95 (80), 109 (75), 53 (70), 55 (65), 105 ((60), 107 (60), 135 (60), 149 (60) 41 (100), 55 (95), 69 (83), 105 (83), 93 (80), 107 (80), 79 (75), 81 (75), 95 (75)
The extension of distillation time from 2 –4 h increased the oil yield from 0.87% to 1.11% (Sample 4), but did not significantly change the composition of the oils (Table 1). The content of monoterpene compounds decreased from 60.1% to 47.6% and the content of oxygenated sesquiterpenes increased from 34.6% to 46.5%. According to the Pharmacopoeia of U.S.S.R. (1990), the distillation time for Cormus Ledi palustris should be 4 h, which may be considered when the monograph of European Pharmacopoeia will be composed. It is also important that young shoots of R. tomentosum biosynthesised three – four times more of essential oil and its principal compounds compared with aged shoots (von Schantz & Hiltunen 1971). The essential oils of R. tomentosum from Lithuania were rich in palustrol (31.4 – 42.8%) and ledol (23.6 – 30.8%) (Butkiene et al. 2008) or palustrol (1.2 – 31.8%), ledol (5.2 –23.4%), isoascaridol (16.7 – 24.1%) and p-cymene (6.2 – 20.3%). Although the herbs were collected from limited area, significant compositional variability was observed in the essential oils (Judzentiene, Butkiene, et al. 2012). Also, palustrol (38%) and ledol (27%) were predominant constituents of the seed essential oil of R. tomentosum growing in Lithuania (Judzentiene, Budiene, et al. 2012), the inflorescence oil contained considerable amounts of myrcene (Butkiene & Mockute 2011). The content of R. tomentosum oils separated from shoots, leaves and stems was rather similar (Gretsˇusˇnikova et al. 2010). In Lithuanian samples (Butkiene et al. 2008), the content of oxygenated monotepenes (6.3 –16.7%) was lower than it was in Estonian R. tomentosum oils (14.6 –46.9%). In general, the content of palustrol and ledol in the R. tomentosum oils of Russian origin was low, instead, the Russian plants were characterised by high sabinene content (Dampc & Luczkiewicz 2013). The R. tomentosum oils from East Siberia (Belousova et al. 1990) contained small amounts of ledol (0 – 3.9%) and palustrol (0.7 – 7.2%); the main compounds in the samples were limonene, sabinene and p-cymene. In Sweden, p-cymene, sabinene and terpinyl acetate from the leaves of R. tomentosum were found as the principal compounds (Jaenson et al. 2006). Thus, the content of ledol (up to 16.5%) was not so high in our Samples 1– 4 compared with R. tomentosum oils from Lithuania. Myrtenal, besides palustrol and ledol, predominated the oils from Netherlands; R. tomentosum oil from China contained much a-thujenal and cyclocolorenone was mentioned among the main constituents in the essential oil of R. tomentosum collected near Leningrad (Russia). Dampc and Luczkiewicz (2013) concluded that changes in composition of the R. tomentosum essential oil depend on the systematic inhomogeneity of the species, influence of ecological factors, geographical location and on the phases of vegetation. 3. Experimental 3.1 Plant material Four R. tomentosum samples were collected in August 2007 (Sample 3: Raplamaa, VanaVigala), in September 2007 (Sample 1: Harjumaa, Kuusalu, Pudisoo), in October 2007 (Sample 2: Po˜lvamaa, Ahja) and in October 2007 (Sample 4: Ida-Virumaa, Iisaku, Jo˜uga lakes) from
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West-, North-, East- and South-Estonia. Voucher specimens (No Ericaceae/Led Nos 1 – 4) have been deposited at the Institute of Pharmacy, University of Tartu, Estonia.
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3.2 Isolation of essential oil Essential oil was isolated from the dried R. tomentosum shoots (10 g) by simultaneous distillation and extraction (SDE) with n-hexane (Fluka, . 99%) as a solvent (0.5 mL) using a Marcusson type micro-apparatus (Jaenson et al. 2006). The SDE process was carried out for 2 h (Samples 1– 4) and 4 h (Sample 4). The oil amount (%) was determined using n-tetradecane (Reachim, . 99%) as internal standard (2 mL). The reproducibility of three parallel SDE procedures with a single sample indicated the variation coefficient to be below 20%. 3.3 Capillary gas chromatography (GC-FID) GC analysis was carried out using a Chrom 5 chromatograph with FID on two fused silica capillary columns with bonded stationary phases (30 m £ 0.25 mm, Supelco, Bellefonte, PA, USA): SPB-5(poly (5%-diphenyl-95%-dimethylsiloxane)) and SW-10 (polyethylene glycol). The film thickness of both stationary phases was 0.25 mm. The carrier gas was helium with the split ratio of 1:150, and a flow rate of 30 –35 cm/s applied. The temperature program was within 50 – 2508C at 28C/min, and the injector temperature was at 2508C. A Clarity Lite chromatography station (DataApex Ltd, Prague, Czech Republic) was used for data processing. The identification of oil components was accomplished by comparing their retention indices (RIs) on two columns with the RI values of reference standards, our RI data and literature data (Bicchi et al. 1990; Davies 1990; Zenkevich 1996, 1997, 1999; Butkiene et al. 2008). The results obtained were confirmed by gas chromatography – mass spectrometry. The percentage composition of the oils was calculated using normalisation method without correction factors. The relative standard deviation of the percentages of oil components of threerepeated GC analysis of the single oil sample did not exceed 5%. 3.4 Gas chromatography– mass spectrometry GC –MS analysis was carried out using a GCMS-QP2010 (Shimadzu, Kyoto, Japan) on a fused silica capillary column (30 m £ 0.32 mm) with a bonded stationary phase: poly (5%-diphenyl95%-dimethylsiloxane) (ZB-5, Zebron, Torrance, CA, USA). The film thickness of the stationary phase was 0.25 mm. The carrier gas was helium with the split ratio of 1:17, and the flow rate of 1.8 mL/min was applied. The temperature program was 2 min at 608C, and then from 60 to 2808C at 128C/min, the injector temperature was at 2808C. The MS detector was operated in the EI mode of 70 eV, and at a scan rate of 2 scans/s with an acquisition mass range of 40– 500 u. 4. Conclusion The essential oil of R. tomentosum plants grown in Estonia contained high amount of palustrol (15.9 – 53.5%) and ledol (11.8 – 18.3%). g-Terpineol (24.7 – 31.2%), which dominated in two samples studied, was not earlier identified in the oils of R. tomentosum. Two other samples were also rich in cyclocolorene and compounds with furyl structure: lepalone and lepalol (3.5 – 6.5%). Two chemotypes of R. tomentosum plants grow in Estonia; they are rich in both palustrol and ledol, and rich in g-terpineol. Acknowledgement The authors gratefully acknowledge Mati Mu¨u¨risepp, Ph.D., for performing GC– MS analysis.
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References Belousova NI, Khan VA. 1990. Bicyclic monoterpenoids of the essential oil of Ledum palustre. Chem Nat Compd. 5:627–629. Belousova NI, Khan VA, Berezovskaya TP. 1990. Intraspecies chemical variability of the essential oil of Ledum palustre. Chem Nat Compd. 4:472–480. Belousova NI, Tkachev AV, Shakirov MM, Khan VA. 1991. New terpenoids of the essential oil of Ledum palustre. Chem Nat Compd. 27:20–24. Bicchi C, D’Amato A, Nano M, Frattini C. 1990. Improved method for the analysis of small amounts of essential oils by microdistillation followed by capillary gas chromatography. J Chromatogr A. 279:409–416. Butkiene R, Mockute D. 2011. The variability of the essential oil composition of wild Ledum palustre L. shoots during vegetation period. J Essent Oil Res. 23:9–13. Butkiene R, Sˇakociut V, Latvenaite D, Mockute D. 2008. Composition of young and aged shoot essential oils of the wild Ledum palustre L. Chimija. 19:19–24. Dampc A, Luczkiewicz M. 2013. Rhondodendron tomentosum (Ledum palustre). A review of traditional use based on current research. Fitoterapia. 85:130–143. Davies NW. 1990. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. J Chromatogr. 503:1– 25. Gretsˇusˇnikova T, Ja¨rvan K, Orav A, Koel M. 2010. Comparative analysis of the composition of the essential oil from the shoots, leaves and stems the wild Ledum palustre L. from Estonia. Procedia Chem. 2:168–173. Ivask K, Orav A, Kailas T, Raal A, Arak E, Paaver U. 2005. Composition of the essential oil from wild marjoram (Origanum vulgare L. ssp. vulgare) cultivated in Estonia. J Essent Oil Res. 17:384–387. Jaenson TG, Palsson K, Borg-Karlson AK. 2005. Evaluation of extracts and oils of tick-repellent plants from Sweden. Med Vet Entomol. 19:345–352. Jaenson TG, Palsson K, Borg-Karlson AK. 2006. Evaluation of extracts and oils of mosquito (Diptera: Culiciae) repellent plants from Sweden and Guinea-Bissau. J Med Entomol. 43:113–119. Judzentiene A, Budiene J, Misiunas A, Butkiene R. 2012. Variation in essential oil composition of Rhododendron tomentosum gathered in limited population (in Eastern Lithuania). Chemija. 23:131–135. Judzentiene A, Butkiene R, Budiene J, Tomi F, Casanova J. 2012. Composition of seed essential oils of Rhondrodendron tomentosum. Nat Prod Commun. 7:227–230. Klokova MV, Khan VA, Dubovenko ZV, Pentegova VA, Berezovskaya TP, Serykh EA. 1983. Terpenoids of the essential oil of Ledum palustre. Chem Nat Compd. 19:278–281. Mikhailova NS, Konovalova OA, Rybalko KS. 1978. Chemical composition of Ledum palustre. Chem Nat Compd. 14:103–105. Orav A, Arak E, Raal A. 2011. Essential oil composition of Coriandrum sativum L. fruits from different countries. J Essent Oil Bear Plant. 14:118– 123. Orav A, Raal A, Arak E. 2010. Content and composition of the essential oil of Chamomilla recutita (L.) Rauschert from some European countries. Nat Prod Res. 24:48–55. Paaver U, Orav A, Arak E, Ma¨eorg U, Raal A. 2008. Phytochemical analysis of the essential oil of Thymus serpyllum L. growing wild in Estonia. Nat Prod Res. 22:108–115. Pharmacopoeia of U.S.S.R. 1990. 11th ed. Moscow: Meditsina. Raal A, Arak E, Orav A, Kailas T, Mu¨u¨risepp M. 2008. Composition of the essential oil of Levisticum officinale W.D.J. Koch from some European countries. J Essent Oil Res. 20:318–322. Raal A, Kanut M, Orav A. 2010. Annual variation of yield and composition of the essential oil of common juniper (Juniperus communis L.) branches from Estonia. Balt For. 16:50–56. Schantz M, Widen KG, Hiltunen R. 1973. Structures of some aliphatic monoterpenoids isolated from the essential oil of Ledum palustre L. Acta Chem Scand. 27:551–555. Tattje DH, Bos R. 1981. Composition of essential oil of Ledum palustre. Planta Med. 41:303–307. von Schantz M, Hiltunen R. 1971. Composition of essential oils from Ledum palustre including the geographic races, L. groenlandicum and decumbens. Sci Pharm. 39:137– 146. Ylipahkala TM, Jalonen JE. 1992. Isolation of very volatile compounds from the leaves of Ledum palustre using the purge and trap technique. Chromatography. 34:159–162. Zenkevich IG. 1996. Analytical parameters of components of essential oils for their GC and GC–MS identification. Mono- and sesquiterpenes. Rastit Res. 32:48–58. Zenkevich IG. 1997. Analytical parameters of essential oils for their GC and GC–MS identification. Oxygen containing derivatives of monoterpene and sesquiterpene hydrocarbons. Rastit Res. 33:16–28. Zenkevich IG. 1999. Analytical parameters of components of essential oils for their GC and GC–MS identification. Acetates of terpenic alcohols. Rastit Res. 35:30–37.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Composition of the essential oil of the Rhododendron tomentosum Harmaja from Estonia a
b
b
Ain Raal , Anne Orav & Tatjana Gretchushnikova a
Department of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, Estonia b
Institute of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia Published online: 09 Apr 2014.
To cite this article: Ain Raal, Anne Orav & Tatjana Gretchushnikova (2014): Composition of the essential oil of the Rhododendron tomentosum Harmaja from Estonia, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.907287 To link to this article: http://dx.doi.org/10.1080/14786419.2014.907287
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.907287
Composition of the essential oil of the Rhododendron tomentosum Harmaja from Estonia Ain Raala*, Anne Oravb and Tatjana Gretchushnikovab Department of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, Estonia; bInstitute of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia
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a
(Received 27 December 2013; final version received 16 March 2014) Wild Rhododendron tomentosum Harmaja shoots were collected from four localities of Estonia. Essential oils, isolated from dried samples by simultaneous distillation and extraction, were analysed using GC-FID and Gas chromatography – mass spectrometry methods. The yields of oils were in the range 0.14 – 0.87%. A total of 72 constituents, accounting for over 95% of the total oil yield, were identified. The major components in the all four oils studied were palustrol (15.9– 53.5%), ledol (11.8 – 18.3%), g-terpineol (0 – 31.2%), p-cymene (0.1 – 13.9%), lepalone (0.7 – 6.5%), lepalol (1.0 – 6.5%) and cyclocolorenone (1.0 – 6.4%). Two different chemotypes of R. tomentosum were found in Estonia and one of them was rich in palustrol (41.0– 53.5%) and ledol (14.6– 18.3%). The second chemotype, for the first time, was rich in g-terpineol (24.7– 31.2%) and contained less of palustrol (15.9– 16.7%) and ledol (11.8– 12.8%), but more p-cymene (12.5 – 13.9%). Also, g-terpineol was identified for the first time in the oils of R. tomentosum. Keywords: Ledum palustre L.; Rhododendrum tomentosum Harmaja; marsh rosemary; essential oil; chemotypes; ledol; palustrol
1. Introduction Wild rosemary or Marsh tea has long been referred to as Ledum palustre L. Recently, it has been discovered that L. palustre L. belongs to the Rhododendron family, its Latin nomenclature has changed to Rhododendron tomentosum Harmaja. It is a low shrub with evergreen leaves growing wild in North and Central Europe, North Asia and America. The tiny white flowers are very fragrant and sticky. It is in leaf all year and flowers from April to August, depending on the area. R. tomentosum prefers acidic soils and can grow with various sun exposures. As its name suggests, it is most appropriate to grow in marshy and swampy areas. The leaves and flowers of R. tomentosum have strong smell and may cause headache to some people. All the parts of the plant contain poisonous terpenes that affect the central nervous system, causing aggressive behaviour. This plant is widely used in folk medicine and homeopathy for the treatment of rheumatism, arthrosis and insect bites. The expectorant and antitussive effect of marsh tea is due to the content of ledol in its essential oil (Dampc & Luczkiewicz 2013). The composition of the essential oils from R. tomentosum varied in a wide range in different localities (Schantz et al. 1973; Mikhailova et al. 1978; Tettje & Bos 1981; Klokova et al. 1983; Belousova & Khan 1990; Belousova et al. 1990, 1991; Ylipahkala & Jalonen 1992; Jaenson et al. 2005, 2006; Butkiene et al. 2008; Gretsˇusˇnikova et al. 2010; Butkiene & Mockute 2011; Judzentiene, Butkiene, et al. 2012, Judzentiene, Budiene, et al. 2012). The major components in
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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its oils were mostly palustrol, ledol and myrcene. Cyclocolorenone, sabinene, limonene, pcymene, cis-p-mentha-1(7) and 8-dien-2-ol were found in high amounts (. 5%) besides palustrol and ledol in R. tomentosum oils from different localities (Belousova et al. 1990; Jaenson et al. 2005; Dampc & Luczkiewicz 2013). Ledol, as toxic compound, can affect the central nervous system, leading finally to paralysis, breathing problems and even death (Dampc & Luczkiewicz 2013). Therefore, the potential chemotypes poor in ledol can be important from the perspective of herbal drug safety. In this work, the variations of the essential oil amounts and compositions were determined in four samples of R. tomentosum grown wild in different localities of Estonia.
2. Results and discussion R. tomentosum shoots were separated from wild plants collected in four localities. The amounts of essential oils from dried Samples 1 and 4 (0.78 – 0.87%) exceeded four times those from Samples 2 and 3 (0.14 – 0.24%). In our previous studies, much more significant differences (10to 17-fold) were found between the minimum and the maximum yields of essential oils of Coriandrum sativum, Levisticum officinale, Thymus serpyllum and Chamomilla recutita from various countries (Paaver et al. 2008; Raal et al. 2008; Orav et al. 2010, 2011). A 14-fold annual variance was detected in the yield of the essential oil in Juniperus communis branches collected once in month (Raal et al. 2010), but only about a 2-fold variance in the yield of essential oils was observed in the aerial parts of Origanum vulgare from different countries (Ivask et al. 2005). Thus, a moderate variance in the yield of essential oil has been found in R. tomentosum samples we studied. A total of 72 compounds were identified in the studied R. tomentosum samples, accounting for over 95% of the total oil yield. In Samples 1, 3 and 4 correspondingly, 65, 61 and 62 constituents were identified but in Sample 2, only 33 compounds were found. Mass spectral data of unidentified compounds are presented in Table 2 ([Mþ] value and characteristic peaks m/z values). The major components in the all four shoot oils of Estonian R. tomentosum, as evident in Table 1, were palustrol (15.9 – 53.5%), ledol (11.8 – 18.3%), g-terpineol (0 –31.2%), p-cymene (0.1 – 13.9%), lepalone (0.7 – 6.5%), lepalol (1.0 – 6.5%) and cyclocolorenone (1.0 – 6.4%). The composition of the essential oils was based on the locality. Two different chemotypes of R. tomentosum were found in Estonia. The dominant constituent in Samples 2 and 3 was palustrol (41.0 –53.5%), followed by ledol (14.6 – 18.3%). These samples were also rich in cyclocolorene (4.3 – 6.4%) and compounds with furyl structure: lepalone (5.0 –6.5%) and lepalol (3.5 – 6.5%). As evident in Table 1, the content of compounds with menthane skeleton (0.9 – 5.8%) and compounds with aromatic structure (0.4 – 2.9%) in these samples was low. The second chemotype was rich in g-terpineol (24.7 – 31.2%). Samples 1 and 4 contained less of palustrol (15.9 – 16.7%) and ledol (11.8 –12.8%), but with more p-cymene (12.5 – 13.9%). As it was demonstrated by Gretsˇusˇnikova et al. (2010), the dominant compound of stem oil from R. tomentosum growing in Estonia was b-myrcene followed by palustrol and ledol, still the leaves and shoots contained three – four times more the essential oil than the stems. The main carbon skeletons of R. tomentosum oil constituents were aromadendrene, menthane, furane and 2,6-dimethyloctane as reported by Butkiene et al. (2008). In Samples 1 and 4, compound with menthane skeleton 1-methyl-4-(1-methylethylidene) cyclohexanol (g-terpineol) dominated (24.7 – 31.2%), followed by palustrol (15.9 –16.7%), ledol (11.8 – 14.6%) and p-cymene (12.5 – 13.9%). g-Terpineol was not found in the earlier investigated above-mentioned oils. Isoascaridol (2.7%), geranyl acetate (0.2 – 3.5%) and (Z)-nerolidol acetate (1.7 – 2.1%) were also found in these oils in remarkable amounts.
a-Pinene Camphene Sabinene b-Pinene b-Myrcene a-Phellandrene (E,E)-2,4-heptadienal Bicyclo[3.3.1]nonane a-Terpinene p-Cymene Limonene 1,8-Cineole (Z)-b-ocimene (E)-b-ocimene g-Terpinene Terpinolene p-Cymenene Linalool Isothujol n-Nonanal 2-Methyl-6-methylene-3,7-octadien-2-ol 1,3,8-Menthatriene Dihydrosabinaketone (lebakon) (E)-p-mentha-2-en-1-ol (Z)-p-mentha-2,8-dien-1-ol (E)-Pinocarveol 5-(3-Furyl)-2-methyl-1-pentene (lepalin) 2-Methyl-6-methylene-1,7-octadien-3-one (E)-3(10)-caren-4-ol Pinocarvone
Compound 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3
Identification method
930 945 970 972 990 1002 1009 – 1018 1022 1026 1030 1040 1050 1054 1086 1090 1098 – 1102 – – – – – 1137 – – – 1162
NIST
Table 1. The composition of the essential oil of L. palustre L. from Estonia (%).
930 943 969 971 990 1002 1006 1008 1013 1023 1025 1028 1035 1045 1054 1084 1085 1100 1100 1102 1102 1107 1118 1121 1129 1132 1132 1138 1147 1155
SPB-5
RI
1182 1277 1203 1211 1240 1256 1247 1280 1426 1558 1560 1400 2077 1395 1395 1627 1620 1654 1554 1554 1662 1563
1024 1067 1120 1111 1168 1168 1329
SW-10
0.7 0.1 tr 0.5 1.3 0.1 0.2 – 0.1 12.5 0.3 0.1 0.1 tr 0.1 0.1 0.5 0.3 – 0.2 – 0.2 0.2 0.1 0.5 0.7 0.7 0.1 0.1 0.8
1
– tr – tr 2.8 – – 0.1 – 0.1 – – – – – – – – – – 1.1 – – – – – 0.6 0.6 0.2 –
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0.1 tr – 0.1 0.8 – – 0.3 0.2 1.9 0.1 tr tr tr 0.1 0.1 tr – 0.4 – 0.9 0.1 0.1 0.1 0.1 0.2 1.9 0.8 0.2 0.2
3
0.7 0.3 tr 0.6 1.0 0.1 0.1 – 0.1 13.9 0.3 0.1 0.2 0.1 0.2 0.2 0.5 0.3 – 0.2 – 0.3 0.2 0.1 0.6 0.4 0.3 – 0.2 0.5
SDE 2 h
Sample number
0.6 0.2 – 0.4 0.8 tr 0.1 – 0.2 11.7 0.3 – 0.1 – 0.2 – 0.7 0.3 – 0.2 – 0.5 0.2 tr 0.6 0.8 0.3 – 0.1 0.5
SDE 4 h
(Continued)
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2-Methyl-6-methylene-1,7-octadien-3-ol Terpinen-4-ol a-Thujenal p-Cymen-8-ol a-Terpineol Myrtenal (E)-p-Mentha-1(7),8-dien-1-ol (Z)-p-Mentha-1(7),8-dien-1-ol p-Cuminal 5-(3-Furyl)-2-methyl-3-penten-2-ol m-Isopropylbenzaldehyde g-Terpineol Piperitone 5-(3-Furyl)-2-methyl-1-penten-3-one (lepalone) NI (1) Isopiperitenone þ lepalol isomer Perillaldehyde Vitispirane Isogeraniol 5-(3-Furyl)-2-methyl-1-penten-3-ol (lepalol) Bornyl acetate NI (2) Carvacrol Isoascaridol 5-Isopropenyl-2-methylcyclo-pent-1-ene carboxaldehyde 2-Methyl-5(fur-3-yl)-pent-1-en-3-ol (lepalol isomer) Neryl acetate Geranyl acetate 2-Ethyl-5-propylphenol Aromadendrene Alloaromadendrene g-Muurolene
Compound
Table 1. (Continued)
1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 3 1, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2
1, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2 1, 2, 3
Identification method
– 1176 – 1183 1190 1193 – – – – – – 1255 – – – 1272 – – 1285 – 1299 – – – 1365 1379 – 1438 1456 1477
NIST
1155 1176 1183 1187 1190 1190 1193 1220 1224 1232 1236 1238 1251 1256 1263 1263 1269 1270 1276 1282 1291 1296 1300 1329 1353 1367 1382 1393 1439 1456 1477
SPB-5
RI
1730 1761 2320 1598 1632 1685
1720 1726 1853 1855 1777 1700 1840 2051 1577 1902 2226 1860
1604 1651 1855 1702 1623 1794 1636 1756 2077
SW-10
2.6 0.1 0.6 0.3 2.7 0.1 0.1 tr 0.2 0.1 tr 0.6 tr
– 0.2 0.4 0.7 – 0.8 0.3 0.1 0.2 0.6 0.1 31.2 0.7 1.3 0.4 tr 0.1
1
3.5 – 0.1 – – 0.2 0.1 – 0.9 – tr 0.8 tr
0.6 tr 0.1 – 0.2 – 0.1 0.7 0.3 tr – – – 5.0 0.4 – tr
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6.5 0.3 0.4 tr 0.3 0.2 – 0.3 0.2 0.1 0.1 0.8 0.2
1.0 0.1 0.3 tr 0.5 0.1 0.4 1.0 0.8 0.2 0.1 2.4 1.0 6.5 1.1 – 0.3
3
1.0 0.7 0.5 0.7 2.7 – 0.3 – 3.5 0.1 – 0.6 0.1
– 0.3 – 0.5 – 0.5 0.3 – tr 0.4 0.1 24.7 0.3 0.7 1.7 0.2 0.2
SDE 2 h
Sample number 4
1.0 0.4 0.3 0.2 2.3 – 0.2 – 2.2 0.1 0.1 0.9 0.1
– 0.1 0.2 0.6 – 0.4 – – – 0.2 – 18.7 0.7 1.4 0.2 – 0.2
SDE 4 h
4 A. Raal et al.
1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3
– – – – – 1580 – 1559 – – – 1653 – – 98.8 0.78
1486 1506 1522 1545 1557 1573 1584 1594 1600 1626 1655 1655 1715 1746 99.4 0.14
1683 1890 1904 2100 1920 1983 2060 2022 1983 2108 2174 2216 2350 2351 97.5 0.24
0.4 0.2 0.2 0.1 15.9 – 0.7 11.8 0.4 0.1 2.1 0.1 0.2 1.6 96.7 0.87
0.5 – – – 53.5 tr 1.5 18.3 0.6 0.1 – – – 6.4 96.1 1.11
0.8 0.1 – – 41.0 0.3 1.6 14.6 0.7 0.1 0.1 – – 4.3
0.4 0.4 0.1 0.4 16.7 0.2 0.8 12.8 0.1 0.3 1.7 tr 0.2 1.0
0.5 0.3 0.2 0.2 23.9 0.2 1.3 16.5 – 0.1 2.0 tr 0.2 1.8
Notes: tr, trace (,0.05%); identification methods: 1, RISPB-5, 2, RISW-10, 3, mass spectra. Main components (.1%) are indicated in bold, NIST, NIST Chemistry WebBook http://webbook. nist.gov/chemistry/.
Ledene Shyobunone a-Calacorene NI (3) Palustrol Caryophyllene oxide Epiglobulol Ledol b-Oplopenone NI (4) b-Elemenone (Z)-nerolidol acetate a-Cadinol Isocalamendiol Cyclocolorenone Total Oil yield, %
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Table 2. Mass spectral data of unidentified (NI) compounds. NI
RISPB-5
MW
1 2 3
1263 1291 1545
150
4
1626
m/z (relative intensity) 85 (100), 82 (50), 41 (25), 53 (18), 57 (15) 109 (100), 43 (70), 81 (55), 127 (40) 205 (100), 121 (90), 81 (85), 41 (82), 95 (80), 109 (75), 53 (70), 55 (65), 105 ((60), 107 (60), 135 (60), 149 (60) 41 (100), 55 (95), 69 (83), 105 (83), 93 (80), 107 (80), 79 (75), 81 (75), 95 (75)
The extension of distillation time from 2 –4 h increased the oil yield from 0.87% to 1.11% (Sample 4), but did not significantly change the composition of the oils (Table 1). The content of monoterpene compounds decreased from 60.1% to 47.6% and the content of oxygenated sesquiterpenes increased from 34.6% to 46.5%. According to the Pharmacopoeia of U.S.S.R. (1990), the distillation time for Cormus Ledi palustris should be 4 h, which may be considered when the monograph of European Pharmacopoeia will be composed. It is also important that young shoots of R. tomentosum biosynthesised three – four times more of essential oil and its principal compounds compared with aged shoots (von Schantz & Hiltunen 1971). The essential oils of R. tomentosum from Lithuania were rich in palustrol (31.4 – 42.8%) and ledol (23.6 – 30.8%) (Butkiene et al. 2008) or palustrol (1.2 – 31.8%), ledol (5.2 –23.4%), isoascaridol (16.7 – 24.1%) and p-cymene (6.2 – 20.3%). Although the herbs were collected from limited area, significant compositional variability was observed in the essential oils (Judzentiene, Butkiene, et al. 2012). Also, palustrol (38%) and ledol (27%) were predominant constituents of the seed essential oil of R. tomentosum growing in Lithuania (Judzentiene, Budiene, et al. 2012), the inflorescence oil contained considerable amounts of myrcene (Butkiene & Mockute 2011). The content of R. tomentosum oils separated from shoots, leaves and stems was rather similar (Gretsˇusˇnikova et al. 2010). In Lithuanian samples (Butkiene et al. 2008), the content of oxygenated monotepenes (6.3 –16.7%) was lower than it was in Estonian R. tomentosum oils (14.6 –46.9%). In general, the content of palustrol and ledol in the R. tomentosum oils of Russian origin was low, instead, the Russian plants were characterised by high sabinene content (Dampc & Luczkiewicz 2013). The R. tomentosum oils from East Siberia (Belousova et al. 1990) contained small amounts of ledol (0 – 3.9%) and palustrol (0.7 – 7.2%); the main compounds in the samples were limonene, sabinene and p-cymene. In Sweden, p-cymene, sabinene and terpinyl acetate from the leaves of R. tomentosum were found as the principal compounds (Jaenson et al. 2006). Thus, the content of ledol (up to 16.5%) was not so high in our Samples 1– 4 compared with R. tomentosum oils from Lithuania. Myrtenal, besides palustrol and ledol, predominated the oils from Netherlands; R. tomentosum oil from China contained much a-thujenal and cyclocolorenone was mentioned among the main constituents in the essential oil of R. tomentosum collected near Leningrad (Russia). Dampc and Luczkiewicz (2013) concluded that changes in composition of the R. tomentosum essential oil depend on the systematic inhomogeneity of the species, influence of ecological factors, geographical location and on the phases of vegetation. 3. Experimental 3.1 Plant material Four R. tomentosum samples were collected in August 2007 (Sample 3: Raplamaa, VanaVigala), in September 2007 (Sample 1: Harjumaa, Kuusalu, Pudisoo), in October 2007 (Sample 2: Po˜lvamaa, Ahja) and in October 2007 (Sample 4: Ida-Virumaa, Iisaku, Jo˜uga lakes) from
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West-, North-, East- and South-Estonia. Voucher specimens (No Ericaceae/Led Nos 1 – 4) have been deposited at the Institute of Pharmacy, University of Tartu, Estonia.
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3.2 Isolation of essential oil Essential oil was isolated from the dried R. tomentosum shoots (10 g) by simultaneous distillation and extraction (SDE) with n-hexane (Fluka, . 99%) as a solvent (0.5 mL) using a Marcusson type micro-apparatus (Jaenson et al. 2006). The SDE process was carried out for 2 h (Samples 1– 4) and 4 h (Sample 4). The oil amount (%) was determined using n-tetradecane (Reachim, . 99%) as internal standard (2 mL). The reproducibility of three parallel SDE procedures with a single sample indicated the variation coefficient to be below 20%. 3.3 Capillary gas chromatography (GC-FID) GC analysis was carried out using a Chrom 5 chromatograph with FID on two fused silica capillary columns with bonded stationary phases (30 m £ 0.25 mm, Supelco, Bellefonte, PA, USA): SPB-5(poly (5%-diphenyl-95%-dimethylsiloxane)) and SW-10 (polyethylene glycol). The film thickness of both stationary phases was 0.25 mm. The carrier gas was helium with the split ratio of 1:150, and a flow rate of 30 –35 cm/s applied. The temperature program was within 50 – 2508C at 28C/min, and the injector temperature was at 2508C. A Clarity Lite chromatography station (DataApex Ltd, Prague, Czech Republic) was used for data processing. The identification of oil components was accomplished by comparing their retention indices (RIs) on two columns with the RI values of reference standards, our RI data and literature data (Bicchi et al. 1990; Davies 1990; Zenkevich 1996, 1997, 1999; Butkiene et al. 2008). The results obtained were confirmed by gas chromatography – mass spectrometry. The percentage composition of the oils was calculated using normalisation method without correction factors. The relative standard deviation of the percentages of oil components of threerepeated GC analysis of the single oil sample did not exceed 5%. 3.4 Gas chromatography– mass spectrometry GC –MS analysis was carried out using a GCMS-QP2010 (Shimadzu, Kyoto, Japan) on a fused silica capillary column (30 m £ 0.32 mm) with a bonded stationary phase: poly (5%-diphenyl95%-dimethylsiloxane) (ZB-5, Zebron, Torrance, CA, USA). The film thickness of the stationary phase was 0.25 mm. The carrier gas was helium with the split ratio of 1:17, and the flow rate of 1.8 mL/min was applied. The temperature program was 2 min at 608C, and then from 60 to 2808C at 128C/min, the injector temperature was at 2808C. The MS detector was operated in the EI mode of 70 eV, and at a scan rate of 2 scans/s with an acquisition mass range of 40– 500 u. 4. Conclusion The essential oil of R. tomentosum plants grown in Estonia contained high amount of palustrol (15.9 – 53.5%) and ledol (11.8 – 18.3%). g-Terpineol (24.7 – 31.2%), which dominated in two samples studied, was not earlier identified in the oils of R. tomentosum. Two other samples were also rich in cyclocolorene and compounds with furyl structure: lepalone and lepalol (3.5 – 6.5%). Two chemotypes of R. tomentosum plants grow in Estonia; they are rich in both palustrol and ledol, and rich in g-terpineol. Acknowledgement The authors gratefully acknowledge Mati Mu¨u¨risepp, Ph.D., for performing GC– MS analysis.
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