Plant Science 169 (2005) 1112–1117 www.elsevier.com/locate/plantsci
Chemical polymorphism of populations of Thymus caespititius grown on the islands Corvo, Flores, Sa˜o Miguel and Terceira (Azores) and on Madeira, assessed by analysis of their essential oils Pedro A.G. Santos a, Jose´ G. Barroso a,*, A. Cristina Figueiredo a, Luis G. Pedro a, Lı´gia R. Salgueiro b, Susana S. Fontinha c, Stanley G. Deans d, Johannes J.C. Scheffer e a
Universidade de Lisboa, Faculdade de Cieˆncias de Lisboa, Dep. de Biologia Vegetal, Centro de Biotecnologia Vegetal, zC2, Campo Grande, 1749-016 Lisbon, Portugal b Laborato´rio de Farmacognosia, Faculdade de Farma´cia, Universidade de Coimbra, Rua do Norte, 3000 Coimbra, Portugal c Jardim Botaˆnico da Madeira, Caminho do Meio, Bom Sucesso, 9050-244 Funchal, Madeira, Portugal d Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow G4 0NR, Scotland, UK e LACDR, Leiden University, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands Received 29 April 2005; received in revised form 13 July 2005; accepted 14 July 2005 Available online 8 August 2005
Abstract The composition of the essential oils isolated from 24 populations of Thymus caespititius collected on Corvo, Flores, Sa˜o Miguel and Terceira (Azores) and on Madeira were studied by GC and GC–MS. All the oil samples analysed were dominated by their monoterpene fraction (66–89%). In the Azorean populations, the proportion of the oxygenated monoterpenes (51–79%) was higher than that of the monoterpene hydrocarbons (8–27%). In contrast, the monoterpene hydrocarbons and the oxygenated monoterpenes represented 35–44 and 42–43%, respectively, of the total oils from the populations grown on Madeira. Cluster analysis of the identified components with a concentration 1% grouped the oils into three main clusters that corresponded with their main components: carvacrol (41–65%), thymol (35– 51%) and a-terpineol (33–37%). Although the populations collected on Madeira were grouped in the same cluster, the chiral analysis of sabinene, terpinen-4-ol and a-terpineol showed that there was a clear chemical polymorphism. Actually, in the oils from two populations ( )-sabinene, ( )-terpinen-4-ol and (+)-a-terpineol were the predominant enantiomers while in that from the third population an opposite ratio was found. The chemical polymorphism of the essential oils from T. caespititius may result either from the genetic variability of the populations or from the influence of edaphic factors. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Thymus caespititius; Lamiaceae; Essential oil; Chemotypes; Azores; Madeira; Thymol; Carvacrol; a-Terpineol; Chiral analysis
1. Introduction Thymus caespititius Brot. is a taxon of the section Micantes; it is endemic of the NW Iberian peninsula, and grows wild also in the Madeiran and Azorean archipelagos [1–3]. The essential oils obtained from populations of this * Corresponding author. Fax: +351 21 7500048. E-mail address: jgbarroso@fc.ul.pt (J.G. Barroso).
species collected in the Portuguese mainland [1,4–6] were all dominated by a-terpineol, whereas those isolated from populations grown on Azorean islands showed a remarkable chemical polymorphism [1–3]. Continuing our studies on T. caespititius from Macaronesia, we report in this paper on the compositions of the essential oils from 24 populations (Table 1): two grown on Corvo, eight on Flores, six on Sa˜o Miguel, five on Terceira (Azores) and three on Madeira.
0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.07.004
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Table 1 Sites of collection of 24 populations of Thymus caespititius growing on Corvo, Flores, Sa˜o Miguel and Terceira (Azores) and on Madeira Island
Collection site
Population
Corvo
Cabaceiras Montinho do Queijo
CO1 CO2
Flores
Ladeiras Ladeiras Lajedo Pedra Alta Tapada da Forcada Matosa Miradouro da Rocha dos Bordo˜es Caldeira Comprida
Sa˜o Miguel
Oil type
Voucher number
300 600
Carvacrol Carvacrol
LISU:173789 LISU:173788
FL1 FL2 FL3 FL4 FL5 FL6 FL7 FL8
200 400 280 300 400 350 380 550
Carvacrol Carvacrol Carvacrol Carvacrol Carvacrol Carvacrol Carvacrol Carvacrol
LISU:173796 LISU:173797 LISU:173795 LISU:173791 LISU:173792 LISU:173794 LISU:173793 LISU:173790
Miradouro do Carva˜o Serra Devassa—Sete Cidades ´ gua de Pau Serra da A Cumeeiras Pico da Barrosa Pico Bartolomeu
SM1 SM2 SM3 SM4 SM5 SM6
600 700 650 450 900 800
Carvacrol Carvacrol Carvacrol Carvacrol Carvacrol Carvacrol
LISU:173786 LISU:173785 LISU:173783 LISU:173787 LISU:173784 LISU:173781
Terceira
Cerrado dos Sete Serra do Cume Pico Rachado Tombo Miste´rio Negro
TE1 TE2 TE3 TE4 TE5
530 550 550 300 650
Thymol Thymol Thymol Thymol Thymol
LISU:173800 LISU:173798 LISU:173799 LISU:173801 LISU:173782
Madeira
Fonte Pau´l da Serra Fonte Pau´l da Serra Pico Ruivo
MA1 MA2 MA3
1185 1170 1800
a-Terpineol a-Terpineol a-Terpineol
MADJ:08395 MADJ:08395 MADJ:08893
2. Experimental 2.1. Plant material On each site of collection a collective representative sample (120 g) of the aerial parts of T. caespititius were collected during the full flowering period of the plant from 24 populations growing on the islands Corvo, Flores, Sa˜o Miguel and Terceira (Azores) and on Madeira. The plant material from the Azorean populations was collected in July 2000 and that from Madeira in June 2000. A voucher specimen of each population has been deposited in the Herbarium of the Museu, Laborato´rio e Jardim Botaˆnico de Lisboa and Jardim Botaˆnico da Madeira (Table 1). 2.2. Isolation procedure The essential oil samples were isolated from deep-frozen ( 20 8C) plant material by distillation-extraction for 3 h, using a Likens–Nickerson-type apparatus with n-pentane as organic solvent, and by hydrodistillation for 3 h, using a Clevenger-type apparatus [7]. The oil samples isolated by hydrodistillation were used to estimate the oil yields, and those isolated by distillation-extraction to determine their percentage composition. 2.3. Gas chromatography GC analyses were performed using a twin FID instrument, a data handling system and a vaporizing injector
Altitude (m)
port into which two columns of different polarities were installed: a DB-1 fused-silica column (30 m 0.25 mm i.d., film thickness 0.25 mm) and a DB-17HT fused-silica column (30 m 0.25 mm i.d., film thickness 0.15 mm). The oven temperature was programmed, 45–175 8C, at 3 8C min 1, subsequently at 15 8C min 1 up to 300 8C and then held isothermally for 10 min; injector and detector temperatures were 280 and 290 8C, respectively; carrier gas, H2 at 30 cm s 1. Chiral GC analyses were performed using an FID instrument, a data handling system and a CyclodexB or a CyclosilB fused-silica column (30 m 0.25 mm i.d., film thickness 0.25 mm). The oven temperature was programmed, 40–230 8C, at 2 8C min 1, for sabinene, and 75–230 8C, at 2 8C min 1, for the oxygenated monoterpenes; injector and detector temps were 280 and 290 8C, respectively; carrier gas, H2 at 30 cm s 1. Samples were injected using the split-sampling technique with a ratio of 1:50. Percentage composition of oils was computed using the normalization method from the GC peak areas without correction factors. Percentage data shown are mean values of two injections of each oil sample. 2.4. Gas chromatography–mass spectrometry The GC–MS unit was equipped with a DB-1 fused-silica column (30 m 0.25 mm i.d., film thickness 0.25 mm) and interfaced with an ion trap detector (ITD; software version 4.1). Injector and oven temperature were as described above; transfer line temperature, 280 8C; ion trap temperature,
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220 8C; carrier gas, He at 30 cm s 1; split ratio, 1:40; ionization energy, 70 eV; ionization current, 60 mA; scan range, 40–300 U; scan time, 1 s. The identity of the components was assigned by comparison of their retention indices, relative to C9–C17 n-alkanes, and GC–MS data with corresponding data of components of reference oils, laboratory-synthesized components and commercially available standards from a home-made library. For chiral GC–MS analyses the chromatograph was equipped with a CyclodexB or a CyclosilB fused-silica column (30 m 0.25 mm i.d., film thickness 0.25 mm) and interfaced with a Turbomass quadrupole mass spectrometer. Injector, oven and transfer line temperature, carrier gas, ionization energy, scan range, and scan time were as described above; ion source, 220 8C; split ratio, 1:50. 2.5. Cluster analysis The percentage composition of the essential oil samples was used to determine the relationship between the different populations of T. caespititius by cluster analysis using the NTSYS software [8]. Euclidean distance was selected as a measure of similarity, and the unweighed pair-group method with arithmetic average (UPGMA) was used for cluster definition.
3. Results and discussion The essential oil samples isolated from the populations of T. caespititius, collected during the flowering phase,
possessed a strong odour and had a light-yellow to a dark-yellow colour. The oils were obtained in yields ranging from 0.1 to 1.2% (v/w) (Table 2). The 24 oil samples were complex mixtures in which 36– 54 components were identified, amounting to a total percentage of 93–99%. The identified components and their percentage limits in each oil type are given in Table 2, where the components are listed in order of their elution from the DB-1 column. The monoterpene fraction was dominant in all the oils analysed (66–89%). With the exception of the populations CO1 from Corvo, FL2 from Flores and MA3 from Madeira, this fraction represented more than 80% of total oils. In the oils from the Azorean populations, the amount of oxygenated monoterpenes (51–79%) was higher than that of monoterpene hydrocarbons (8–27%). In contrast, the proportion of these two groups of components in the oils from the populations grown on Madeira was almost similar (35–44% for the monoterpene hydrocarbons and 42–43% for oxygenated monoterpenes). For population MA2, the amount of monoterpene hydrocarbons was even higher than that of oxygenated monoterpenes. The sesquiterpene fraction was rather small (7–28%), being always dominated by the oxygenated compounds (6–22%). Non-terpenoid compounds occurred in relative amounts from traces to 1%. The populations grown on Corvo, Flores and Sa˜o Miguel yielded carvacrol-rich oils (41–65%), but they differed in their second main components: T-cadinol (15%) and pcymene (14%) in the oils from the populations CO1 and CO2, respectively, p-cymene (10–19%) or carvacryl acetate (8–11%) in those from the Flores populations, and carvacryl
Table 2 Percentage limits, in each oil type, of components detected in the essential oils isolated from the aerial parts, collected during the flowering phase, from 24 populations of T. caespititius grown on Corvo, Flores, Sa˜o Miguel and Terceira (Azores) and on Madeira Components
Tricyclene a-Thujene a-Pinene Camphene Sabinene 1-Octen-3-ol b-Pinene b-Myrcene a-Phellandrene d-3-Carene a-Terpinene p-Cymene b-Phellandrene Limonene (E)-b-Ocimene (Z)-b-Ocimene g-Terpinene trans-Sabinene hydrate Terpinolene cis-Sabinene hydrate n-Nonanal Linalool
RIa
921 924 930 938 958 961 963 975 995 1000 1002 1003 1005 1009 1017 1027 1035 1037 1064 1066 1073 1074
Azorean populations
Madeiran populations
Carvacrol-rich oil [Corvo (CO), Flores (FL), Sa˜o Miguel (SM)]
Thymol-rich oil [Terceira (TE)]
a-Terpineol-rich oil [Madeira (MA)]
n.d.–t 1.1–2.3 0.4–1.0 0.1 t–0.2 n.d.–0.1 0.2–0.6 n.d.–0.5 0.1 t–0.1 0.5–1.1 4.0–19.0 0.1–0.2 0.1–1.2 n.d. n.d. 0.9–5.7 0.1–0.4 0.1 n.d.–0.1 n.d.–t n.d.–t
n.d. 1.6–2.2 0.5–1.4 0.1–1.8 t–0.4 0.1–0.4 0.5–0.7 t–0.3 t–0.2 t–0.1 0.9–1.4 10.2–14.3 0.2–0.3 0.3–0.6 n.d. n.d. 2.1–5.2 0.2–0.4 0.1–0.2 t–0.1 n.d. n.d.
0.1 0.7–1.0 0.7–0.8 1.3–1.5 16.3–7.7 t 0.3–0.6 7.3–10.0 0.1–0.2 n.d. 1.3–1.8 1.5–4.1 0.5–1.2 2.1–2.7 t–0.2 0.8–1.6 4.2–5.5 0.2 0.7–0.9 0.1–0.2 n.d. 0.1–0.2
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Table 2 (Continued ) Components
RI a
Azorean populations
Madeiran populations
Carvacrol-rich oil [Corvo (CO), Flores (FL), Sa˜o Miguel (SM)]
Thymol-rich oil [Terceira (TE)]
a-Terpineol-rich oil [Madeira (MA)]
n.d.–0.4 n.d. n.d.–0.1 n.d.–t t–0.1 0.1–1.3 n.d.–0.6 0.1–3.9 n.d. n.d. t–0.5 n.d.–0.3 t–0.2 n.d.–0.4 n.d. 0.1–0.4 41.0–64.8 n.d. 2.3–23.8 n.d. n.d. n.d.–0.2 n.d.–0.2 n.d. n.d.–0.1 n.d. n.d.–t n.d. n.d. 1.2–2.8 n.d. n.d. n.d.–4.9 n.d.–1.1 n.d.–0.3 0.6–1.3 n.d.–0.2 n.d.–0.1 n.d.–0.1 n.d. n.d.–0.1 n.d. 0.8–3.4 n.d.–1.2 n.d.–15.0 n.d. n.d.–1.2 n.d.–2.5
0.4–0.8 n.d. n.d. n.d. 0.1–1.9 0.4–1.1 t–0.5 0.8–7.6 n.d. n.d. t–0.8 n.d. n.d. n.d. n.d. 34.9–51.1 2.6–3.5 10.1–18.9 0.5–0.8 n.d. n.d. 0.1–0.3 n.d. n.d. 0.2–0.4 n.d. n.d. t–0.1 0.1 0.6–1.3 n.d. n.d. 0.9–1.5 0.2–0.3 t–0.3 0.2–0.5 n.d. n.d. n.d. n.d. n.d. n.d. 0.7–1.0 t–0.4 t–3.3 n.d. t–0.7 1.2–3.3
t–0.1 t–0.2 0.2 n.d. 1.2–1.8 3.3–5.2 t 32.8–36.7 t–0.1 t n.d. n.d. t n.d. 0.8–1.6 n.d. t–0.4 n.d. n.d. t t–0.1 t–0.3 0.1–0.3 t–0.1 0.2–0.3 0.1 0.1–0.2 n.d. n.d. 2.0–3.0 0.1–0.3 0.3–0.5 0.4–0.5 n.d. 0.6–1.2 0.9–1.5 n.d. n.d. t–0.1 t–0.1 n.d. n.d.–0.4 3.1–4.8 n.d. 1.5–1.6 0.1–0.2 0.7–1.3 n.d.–1.0
Identified components (%)
92.5–97.0
95.6–99.0
95.1–97.2
Grouped components Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others
8.4–26.6 51.3–79.1 0.3–6.2 6.1–22.2 t–0.5
17.3–26.9 58.6–71.4 2.0–2.8 6.1–7.1 0.5–1.1
35.4–43.7 41.5–43.4 2.5–3.5 9.4–12.8 t–0.1
0.1–0.8
0.4–0.8
0.5–1.2
1-Octen-3-yl acetate Camphor trans-p-Menth-2-en-1-ol trans-Pinocarveol Borneol Terpinen-4-ol p-Cymen-8-ol a-Terpineol trans-Carveol Nerol Carvone Thymoquinone Carvacrol methyl ether Geraniol Bornyl acetate Thymol Carvacrol Thymyl acetate Carvacryl acetate a-Copaene b-Bourbonene b-Elemene b-Caryophyllene a-Humulene allo-Aromadendrene g-Muurolene Germacrene-D Valencene a-Selinene trans-Dihydroagarofuran a-Muurolene b-Bisabolene g-Cadinene Calameneneb d-Cadinene Kessanec a-Calacorene a-Cadinene Elemol Germacrene-D-4-ol Tridecanol Viridiflorol epi-Guaiol epi-Cubenol T-Cadinol a-Muurolol a-Cadinol Intermedeol
1086 1095 1095 1106 1134 1148 1148 1159 1189 1206 1206 1210 1224 1236 1265 1275 1286 1330 1348 1375 1379 1388 1414 1447 1454 1469 1474 1477 1486 1489 1494 1495 1496 1505 1505 1517 1525 1529 1530 1557 1565 1569 1593 1600 1616 1618 1626 1629
Oil yield (%, v/w) n.d. = not detected t = trace (< 0.05%). a Relative to C9–C17 n-alkanes on a DB-1 column. b Correct isomer not determined. c Identification based on mass spectra only.
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acetate (10–24%) or p-cymene (6%) in the oils from the Sa˜o Miguel populations. As stated above, the sesquiterpene fraction varied considerably, but T-cadinol was the dominant (5–15%) component of this fraction in all but the Sa˜o Miguel oils, where its presence was not detected. In the latter oils, epi-guaiol (3%), trans-dihydroagarofuran (2–3%) and intermedeol (not detected–3%) where the dominant sesquiterpenes. The populations collected on Terceira (TE1–TE5) yielded also phenol-rich oils, but the main component was thymol (35–51%) followed by thymyl acetate (10–19%) and p-cymene (10–14%). The sesquiterpene fraction (9%) was mainly composed by oxygenated compounds (6–7%), intermedeol (1–3%) and T-cadinol (t–3%) being the major components. The Azorean populations under study show a clear chemical polymorphism. However, regarding their main components, they are more or less homogeneous within each island, with exception of those from Corvo. These results contrast with those reported for oil samples obtained from populations collected on four other Azorean islands, namely Sa˜o Jorge [2], Pico, Faial and Graciosa [3]. For these islands, the chemical polymorphism of T. caespititius was, in some cases, more evident among populations from the same island than among those from different islands. In contrast with the Azorean populations, those grown on Madeira yielded a-terpineol-rich oils (33–37%), sabinene (8–16%) and b-myrcene (7–10%) being their second and third main components, respectively. Of the phenols detected as major components in the oil samples from the populations grown on the Azorean archipelago, only carvacrol (t–0.4%) could be detected in these oil samples. As in the oils from the other populations under study, the oxygenated compounds (9–13%) dominated the sesquiterpene fraction (12–16%), epi-guaiol (3–5%), trans-dihydroagarofuran (2–3%) and T-cadinol (2%) being again the main components of this fraction. Although the essential oils obtained from the populations collected on Madeira possessed a high proportion of aterpineol like those grown either in the Portuguese mainland [1] or on Azorean islands, namely the populations SJ5, SJ6, SJ8 and SJ10 from Sa˜o Jorge, [2] and the populations G1– G4 from Graciosa [3], their chemical composition differed markedly. In fact, the Portuguese mainland populations showed both higher levels of sesquiterpenes (25–30%) and of T-cadinol (6–9%). The same held true for the population G4 (28% of sesquiterpenes and 11% of T-cadinol). For all the other above-mentioned populations, a-terpineol was detected along with important amounts of thymol and/or carvacrol. Moreover, sabinene (8–16%) and b-myrcene (7– 10%), two important components of the oils from the Madeiran populations, were only detected in small amounts (<0.5%) in the oils from the other populations. Cluster analysis of the identified components with a concentration 1%, at least in one oil sample, grouped the oils from the 24 populations into three main groups (Fig. 1),
Fig. 1. Dendrogram obtained by cluster analysis of the percentage composition of essential oils from 24 populations of Thymus caespititius, based on Euclidean distance and using the unweighed pair-group method with arithmetic average (UPGMA).
corresponding with their main components: carvacrol (populations CO1, CO2, FL1–FL8 and SM1–SM6), thymol (populations TE1–TE5) and a-terpineol (populations MA1– MA3). Although the populations producing carvacrol-rich oils, i.e. those grown on Corvo, Flores and Sa˜o Miguel, are grouped in the same cluster, the populations SM1–SM5 and the populations CO2 and FL1–FL8 formed two distinct smaller clusters. This can be explained by the absence of Tcadinol in the oils from the populations SM1–SM5 and by the higher relative amount of p-cymene in those from the populations CO2 and FL1–FL8. In addition, the populations CO1 and SM6 are clearly individualized: the essential oil of the former population possessed a higher proportion of Tcadinol, and that of the latter a larger amount of carvacryl acetate. According to a recent review of Stahl-Biskup [9], thymol and carvacrol are the two top 10 terpenes present in the oils from the genus Thymus, followed by linalool, p-cymene, gterpinene, borneol, 1,8-cineole, geraniol, a-terpinyl acetate and a-terpineol, respectively. The presence of a low percentage of sesquiterpenes, found in the present study, is, according Stahl-Biskup [9], a characteristic of the Thymus taxa, T-cadinol being widely distributed within the Thymus species of northern Europe. In common with this review, the non-terpenoid compounds were found in very low percentages. Since the oils isolated from T. caespititius showed chemical polymorphism, the study of the enantiomeric distribution of the chiral compounds in the oils may provide important additional information. Nevertheless, although the variability in essential oil composition is widespread in the genus Thymus, the enantiomeric composition of these oils has not been particularly studied, mostly because thymol and carvacrol, that dominate Thymus taxa oils, are both achiral terpenes [9]. Of the chiral compounds present in the oils from T. caespititius, only sabinene, terpinen-4-ol and aterpineol were investigated for their enantiomeric ratio, due to their high concentration in the oil samples from the
P.A.G. Santos et al. / Plant Science 169 (2005) 1112–1117
populations grown on Madeira. The results obtained showed that, although the oils were dominated by the same major components, they possessed in fact a clear chemical polymorphism. ( )-Sabinene was the predominant enantiomer in the oils from the populations MA1 (75%) and MA2 (77%), whereas the (+) enantiomer (83%) was dominant in that from population MA3. Although with lower enantiomeric purity, terpinen-4-ol and a-terpineol behaved in the same way as sabinene: ( )-terpinen-4-ol and (+)-aterpineol were the predominant enantiomers in the oils from the populations MA1 (60 and 64%, respectively) and MA2 (61 and 63%, respectively), while (+)-terpinen-4-ol (61%) and ( )-a-terpineol (53%) were the predominant enantiomers in that from population MA3. In essential oil-bearing plants, the yield of oil and its chemical composition vary considerably due to both intrinsic (sexual, seasonal, ontogenetic and genetic variations) and extrinsic (ecological and environmental aspects) factors [9,10]. Since it was not possible to find any correlation between the chemical composition of the oils and the altitudes of the collection sites, or the longitude and the climate of the islands, the polymorphism recorded in the present study may result either from the genetic variability of the populations or from the influence of edaphic factors.
Acknowledgement This study was funded by the Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT), Lisbon, under research contract no. Praxis/P/BIA/11054/98.
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