2005_FFJ_20_Plagiochila

ESSENTIAL OIL OF PLAGIOCHILA SPP. 703 FLAVOUR AND FRAGRANCE JOURNAL Flavour Fragr. J. 2005; 20: 703–709 Published online 29 June 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1627

Comparison of the essential oil composition of four Plagiochila species: P. bifaria, P. maderensis, P. retrorsa and P. stricta A. Cristina Figueiredo,1* Manuela Sim-Sim,2 Monya M. Costa,1 José G. Barroso,1 Luis G. Pedro,1 M. Glória Esquível,3 Francisco Gutierres,2 Carlos Lobo4 and Susana Fontinha4 1

2

3 4

Universidade de Lisboa, Faculdade de Ciências de Lisboa, DBV, Centro de Biotecnologia Vegetal, C2, Campo Grande, 1749-016 Lisbon, Portugal Universidade de Lisboa, Faculdade de Ciências de Lisboa, DBV, Centro de Ecologia e Biologia Vegetal, C2, Campo Grande, 1749-016 Lisbon, Portugal Centro de Botânica Aplicada à Agricultura, Dep. Botânica e Engenharia Biológica, ISA, UTL, 1399 Lisboa, Codex, Portugal Serviço do Parque Natural da Madeira, Caminho do Meio, Quinta do Bom Sucesso, 9050-251 Funchal, Madeira, Portugal

Received 12 October 2004; Revised 30 March 2005; Accepted 17 April 2005

ABSTRACT: Essential oils isolated by distillation–extraction from P. bifaria, P. maderensis, P. retrorsa and P. stricta, collected on Madeira, were analysed by GC and GC–MS. Methyl everninate (1–35%), peculiaroxide (13–16%) and enteudesm-4(15)-ene-6-one (9–19%) were the main components in all P. bifaria specimens analysed. Terpinolene (34–60%) dominated the oils isolated from P. maderensis specimens. β -Phellandrene (16–46%) was the main component of two of the three specimens of P. retrorsa, allo-ocimene (15%), terpinolene (13%), peculiaroxide (12%) and neo-allo-ocimene (10%) being the main components of the third specimen. P. stricta oils were dominated by peculiaroxide (11–21%), allo-ocimene (7–19%), bicyclogermacrene (4–17%), neo-allo-ocimene (4–11%) and spathulenol (2–14%). Essential oil cluster analysis showed a high degree of similarity between three of the four species studied, the least correlated being P. maderensis oils.

Copyright © 2005 John Wiley & Sons, Ltd. KEY WORDS: Plagiochila bifaria (Sw.) Lindenb.; Plagiochila maderensis Gottsche ex Steph.; Plagiochila retrorsa Gottsche; Plagiochila stricta Lindenb.; Plagiochilaceae; liverworts; essential oil; GC; GC–MS

Introduction The genus Plagiochilla comprises over 1600 species, being one of the largest of the liverwort genera.1 Madeiran Plagiochila species constitute more than 50% of the total species referred to Europe. According to Schumacker and Vá@a,2 Söderström et al.,3 Rycroft et al.4 and Sim-Sim et al.5 (2004), nine Plagiochila spp. are presently referred to Madeira: P. bifaria (Sw.) Lindenb., P. exigua (Taylor) Taylor, P. maderensis Gottsche ex Steph., P. porelloides (Torrey ex Nees) Lindenb., P. punctata (Taylor) Taylor, P. retrorsa Gottsche, P. spinulosa (Dicks.) Dumort., P. stricta Lindenb. and P. virginica A. Evans.

P. bifaria is the most frequently found of the Plagiochila spp. in Madeira’s native laurel forest (‘laurissilva’).

It can be found in sheltered or exposed habitats along water courses, forming loose to dense patches on rocks, boulders and stone walls.6 Morphological evidence of the

* Correspondence to: A. C. Figueiredo, Universidade de Lisboa, Faculdade de Ciências de Lisboa, DBV, Centro de Biotecnologia Vegetal, C2, Campo Grande, 1749-016 Lisbon, Portugal.

E-mail: acsf@fc.ul.pt Contract/grant sponsor: Fundação para a Ciência e Tecnologia, Portugal; Contract/grant number: POCTI/AGR/42501/2001.

Copyright © 2005 John Wiley & Sons, Ltd.

conspecificity between this Neotropical species and the European P. killarniensis Pearson. was presented by Heinrichs et al.7 and recent molecular, morphological and phytochemical evidence support a broad species concept of P. bifaria.8 P. maderensis is a Madeiran endemic species that was formerly synonymized with P. spinulosa (Dicks.) Dumort.4 It grows on the north side of the Madeiran ‘laurissilva’.4 P. retrorsa occurs in Central America and in the Southern Appalachian Mountains of the eastern USA. It is also known from the Azores and Madeira archipelagos in Macaronesia.9 On the island of Madeira P. retrorsa is a common species, especially in the less humid parts of the ‘laurissilva’, where it can be found on rocks and slopes.6 P. stricta is a Neotropical and Macaronesian liverwort widespread on the Madeiran ‘laurissilva’, where it grows forming scattered or pure mats in shaded slopes located along water rivulets.5,6 With the exception of a recent study on the volatiles of P. bifaria,10 previous phytochemical studies have examined only the solvent extracts of the four species.4,7–9,11,12 As part of our studies on the essential oil-bearing plants of Portugal and with the aim of determining the potential of essential oils as chemical markers, we report on the comparison of the essential oil composition of four Plagiochila spp. collected on Madeira.

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Table 1. Sites of collection and voucher numbers of Plagiochila species studied on the Madeira Islands Species

Collection site

Year

Voucher No.

Abbreviation

P. bifaria

Levada do Norte Montado dos Pessegueiros Balcões, vereda para Ribeiro Frio Fajã da Nogueira Levada do Ribeiro Bonito Fajã da Nogueira, Levadinha João de Deus Levada do Norte Montado dos Pessegueiros Fanal, Lagoa do Fanal Levada do Ribeiro Bonito Levada do Ribeiro Bonito Montado dos Pessegueiros Montado dos Pessegueiros Montado dos Pessegueiros Fajã da Nogueira, Levadinha João de Deus Fajã da Nogueira, Levadinha João de Deus

2002 2003 2003 2003 2002 2003 2002 2003 2003 2002 2002 2002 2002 2003 2003 2003

LISU190979 LISU190983 LISU190984 LISU190981 LISU190980 LISU190985 LISU182272 LISU190986 LISU182245 LISU182235 LISU182239 LISU182228 LISU182229 LISU190982 LISU190988 LISU190987

P.bif37/02 P.bif41/03 P.bif50/03 P.bif62/03 P.mad29/02 P.mad62/03 P.ret36/02 P.ret42/02 P.ret59/03 P.str32/02 P.str33/02 P.str40/02 P.str41/02 P.str42/03 P.str60/03 P.str62/03

P. maderensis P. retrorsa

P. stricta

Materials and Methods Plant Material Plagiochila bifaria, P. maderensis, P. retrorsa and P. stricta samples were collected on Madeira at different locations and collection dates (Table 1). Voucher specimens have been deposited in the Herbarium of the Botanical Garden of Lisbon (LISU) and in the Herbarium of the Madeira Botanical Garden (MADJ) (Table 1).

Essential Oil Isolation Procedure Each essential oil sample was isolated from deep-frozen (−20 °C) fresh plant material (ca. 50 g fresh weight), by distillation–extraction for 3 h using a Likens–Nickersontype apparatus13 with distilled n-pentane (50 ml) as the organic solvent. The isolation procedure was run at a distillation rate of 3 ml/min. The oil recovered in pentane was concentrated, at room temperature and under reduced pressure, on a rotary evaporator, collected in a vial and concentrated to a minimum volume, again at room temperature, under nitrogen flux.

Gas Chromatography (GC) GC analyses were performed using a Perkin-Elmer 8700 gas chromatograph equipped with two FIDs, a datahandling system and a vaporizing injector 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 µm; J&W Scientific Inc., Rancho Cordova, CA, USA) and a DB-17HT fused-silica column (30 m × 0.25 mm i.d., film thickness 0.15 µm; J&W Scientific Inc.). Oven temperature was programmed to 45–175 °C at 3 °C/min, rising at 15 °C/min to 300 °C, then held

Copyright © 2005 John Wiley & Sons, Ltd.

isothermal for 10 min; injector and detector temperatures, 280 °C and 290 °C, respectively; carrier gas, hydrogen, adjusted to a linear velocity of 30 cm/s. The samples were injected using the split sampling technique, ratio 1:50. The percentage composition of the oil samples was computed from the GC peak areas using the normalization method; the data were calculated as mean values of two injections from each oil sample without using response factors.

Gas Chromatography–Mass Spectrometry (GC–MS) The GC–MS units were equipped with DB-1 fused-silica columns (30 m × 0.25 mm i.d., film thickness 0.25 µm; J&W Scientific, Inc.) each interfaced either with an ion trap detector (ITD; software version 4.1) or a PerkinElmer Turbomass mass spectrometer (software version 4.1). Oven temperature was as above; transfer line temperature, 280 °C; ion trap temperature, 220 °C; carrier gas, helium, adjusted to a linear velocity of 30 cm/s; split ratio, 1:40; ionization energy, 70 eV; ionization current, 60 µA; scan range, 40–300 u; scan time, 1 s.

The identity of the components was assigned by comparison of their retention indices, relative to C8–C17 n-alkanes, and GC–MS spectra with corresponding data of components of reference oils, literature data,10,14–18 laboratory-synthesized components and commercially available standards from a home-made library.

Statistical analysis The percentage composition of the essential oil samples was used to determine the relationship between the different samples of Plagiochila spp. by cluster analysis using the NTSYS software.19 Correlation was selected as

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ESSENTIAL OIL OF PLAGIOCHILA SPP.

a measure of similarity, and the unweighted pair-group method with arithmetic average (UPGMA) was used for cluster definition.

Results and Discussion Seventy-two components were identified in the lightyellow essential oil samples isolated from the different Plagiochila spp., amounting to 66–92% of the total oils.

The identified oil components are listed in Table 2 in order of their elution on the DB-1 column. A limited number of components, with relative amounts of 0.5–3% each, and some trace components could not be identified; these are not included in Table 2.

The sesquiterpene (63–82%) and monoterpene (47– 78%) fractions were the most representative in P. bifaria and P. maderensis, respectively. In P. retrorsa, one of the three specimens studied was dominated by the monoterpene fraction (58%), while in the other the mono- (46%) and sesquiterpenes (43%) occurred in comparable amounts, the third specimen being sesquiterpenedominated (52%). Sesquiterpenes (43–56%) were the main components of six of the seven P. stricta specimens studied, whereas one specimen had mono- (41%) and sesquiterpenes (46%) in approximately equal amounts.

P. bifaria Despite the qualitative and quantitative variability of the monoterpenes, the P. bifaria specimens studied showed resemblances in the types of sesquiterpenes and aromatic compounds present in their oils (Table 2).

The sesquiterpene fraction was the most representative (63–82%) in all P. bifaria specimens, methyl everninate (1–35%), peculiaroxide (13–16%) and ent-eudesm-4(15)ene-6-one (9–19%) being the main components. ent-7Hydroxy-eudesm-4-en-6-one also occurred in appreciable amount (11%) in the sample, in which methyl everninate attained the lowest relative amount (1%).

Although no quantitative data were reported, the GC profile of the essential oil of P. bifaria isolated by hydrodistillation by Hackl et al.10 showed major resemblance with the present reported data. This is the only previous study on the volatiles of this species, since preceding studies reported only on the phytochemical profiles of its solvent extracts.8,15,20 According to Rycroft,20 the first attempts to find a chemical resemblance between P. bifaria and P. killarniensis were unsuccessful. Nevertheless, later studies on other P. bifaria specimens showed the presence in P. killarniensis of characteristic compounds, such as methyl everninate and methyl 6-methoxy-2-methyl-3,4-methylenedioxybenzoate.20 Both compounds were detected in the present study in high amounts in the Madeiran specimens.

Copyright © 2005 John Wiley & Sons, Ltd.

705

Based on solvent extracts studies, Heinrichs et al.8 consider that the European P. bifaria populations investigated so far, none of which were from Madeira, belong to a single methyl everninate chemotype, which has considerable variation in the proportions of the minor constituents. Of the four populations presently studied, one had only 1% methyl everninate, ent-eudesm-4(15)ene-6-one and ent-7-hydroxy-eudesm-4-en-6-one being the dominant components, thus suggesting the existence of other chemotypes.

P. maderensis Terpinolene (34–60%) was the main component from the oil isolated from P. maderensis specimens (Table 2).

Although terpinolene could also be found in all other Plagiochila spp. studied, only P. maderensis attained this high relative amount. This is also the only oil in which the monoterpene fraction is clearly dominant (47–78%), the usual sesquiterpenes of Plagiochila, peculiaroxide, methyl everninate and ent-eudesm-4(15)-ene-6one, attaining only relative amounts <3%.

Rycroft et al.4 have analysed the solvent extracts of two P. maderensis specimens and detected 4-hydroxy-3′methoxybibenzyl and terpinolene as the major aromatic and terpenoid compounds, respectively. 4-Hydroxy-3′methoxybibenzyl, which did not show major quantitative variation in the Rycroft et al.4 study, was not detected in the essential analyses. Terpinolene, bicyclogermacrene and spathulenol showed major quantitative variations in the two specimens studied by those authors. The two latter sesquiterpenes were also detected in the present study in variable amounts.

It is noteworthy that, according to Toyota et al.21 spathulenol is an artefact produced from the oxidation of bicyclogermacrene, encountered particularly in old liverwort extracts. Nevertheless, in the present study, the essential oil isolation procedure was run within 2–3 weeks after liverwort collection and thus the spathulenol relative amount should not be considered age-related.

P. retrorsa The essential oils isolated from P. retrorsa samples showed a major qualitative and quantitative variability, being dominated by either the monoterpene fraction (58%) or by the sesquiterpenes (52%) or both appearing in approximately equal amounts (46% and 43%, respectively). β-Phellandrene (46%) was the main component of the monoterpene dominated oil. Peculiaroxide (9%), bicyclogermacrene (6%) and allo-ocimene (5%) were the other main components (≥5%) of this oil. β-Phellandrene (16%) was also the main component of the sesquiterpene-

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RI 900 924 930 958 961 961 975 995 1002 1003 1005 1009 1017 1027 1035 1037 1059 1064 1074 1066 1073 1074 1086 1095 1110 1110 1117 1148 1148 1148 1159 1264 1332 1310 1375 1379 1388

Components

n-Nonane α-Thujene α-Pinene Sabinene Octen-3-ol 3-Octanone Myrcene α-Phellandrene α-Terpinene p-Cymene β -Phellandrene Limonene cis-β-Ocimene trans-β-Ocimene γ-Terpinene trans-Sabinene hydrate 2,5-Dimethyl styrene Terpinolene 1,3,8-Menthatriene cis-Sabinene hydrate Nonanal Linalol 1-Octen-3-yl acetate trans-p-Menthen-1-ol cis-p-Menthen-1-ol allo-Ocimene neo-allo-Ocimene Terpinen-4-ol m-Cymen-8-ol p-Cymen-8-ol α-Terpineol cis-Verbenyl acetate δ -Elemene Geranyl acetate α-Copaene β-Bourbonene β-Elemene

P.bif37/02 t 0.2 0.1

0.7

t

t t t t t

t

t 0.1 t t 2.9 0.1

t 0.1 0.1

P.bif41/03 1.3 0.4

3.2

0.3

t t 7.7

t

t

0.6 5.0

t

t t

5.3

P.bif50/03

t t t

P.bif62/03

t

P.mad29/02 2.2

1.4 t

3.6 33.5

2.5 t 1.8 t

2.2

0.7 0.6

4.8 60.1 0.7

0.5 t t 3.3 t 2.8 0.4

t t

1.5

0.3 1.3 2.0

t 0.2

2.4

t t t

P.mad62/03

P. maderensis P.ret36/02 t t t t 0.6

t 0.3 t 4.9 4.2 t

t

t 0.3 0.3 t 46.0 0.5 t 1.5 t

t

t t

P. retrorsa

t

0.9 t 1.7 t t t

15.0 9.6

t

2.0 12.9

0.8 t t t 0.5 1.2 t 1.6 t

1.2

t t

P.ret42/02

P. bifaria P.ret59/03 t 1.4

t t

t t t 15.5

P.str32/02 t

1.2 0.7

18.2 7.6

0.6

1.8

t 1.2

0.8

1.3

t

P.str33/02 t

1.4 0.9

15.4 6.7

0.1

1.0

t 0.7

0.5

0.9

t

t

6.7 3.7

t

t t t

t

t

t

P.str40/02

P. stricta

0.6 t 0.1 t 0.3

t

8.9 3.9 0.4

1.1

t

0.3

0.3 t t t 3.9 0.6 t 0.5 t t

t t t 0.8

1.5 2.5

t 0.4 t 1.9 t

0.6 0.6 t

t t

P.str42/03 1.4

t t

16.9 11.4

Table 2. Percentage composition of the essential oil samples isolated by distillation–extraction from Plagiochila species collected on Madeira

P.str41/02

P.str60/03 t

t

t

13.0 7.4

t 8.1

t t 1.7 t 1.8 t

t t 0.6

t t

P.str62/03 0.8

t t

19.1 10.5

0.5

1.6 6.0

t 1.1 t 2.3

0.4 0.4 t

t t

706 A. C. FIGUEIREDO ET AL.

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8.0 19.3 0.6 1.1 2.1 9.3

4.0

3.6

0.8 22.8 1.4 2.2 1.0 10.3

3.7

4.2 82.2 11.2 t 8.7 57.7 4.6 t

t 35.4 1.4 1.4 0.5 2.8 11.1 t t 0.5 2.7 0.3 8.8 88.1 3.2 t 4.1 77.7 3.0 0.1

1644 1672

0.9

5.3 t 11.4 51.2 12.9 t

5.5 80.8

0.9

4.6

3.5

3.3

1.6

0.9 0.9

0.8

0.9

0.6

13.3 t

0.6

15.2

1.1

14.7

1413 1414 1414 1427 1428 1434 1454 1473 1468 1468 1474 1476 1487 1494 1495 1487 1500 1500 1500 1505 1505 1536 1550 1551 1566 1569 1582 1583 1616 1618 1626 1626

a RI = Retention index relative to C8–C17 n-alkanes on the DB-1 column. * Based on mass spectra only. ** Based on mass spectra from literature data.7,10–14 t = trace (<0.05%).

Peculiaroxide β-Caryophyllene α-Himachalene β-Chamigrene* Aromadendrene trans-α-Bergamotene allo-Aromadendrene Zierene (=deoxysaccogynol) Dodecanol γ-Himachalene Germacrene-D β-Selinene Bicyclogermacrene α-Muurolene β-Bisabolene Viridiflorene α-trans,trans-Farnesene γ-Cadinene β-Curcumene trans-Calamenene δ-Cadinene 2,4,5-Trimethoxy-allylbenzene Methyl everninate Spathulenol Globulol Viridiflorol ent-Eudesm-4-ene-6-one** ent-Eudesm-4(15)-ene-6-one** T-Cadinol δ -Cadinol α-Cadinol Methyl 6-hydroxy-2-methyl-3,4-methylene dioxybenzoate** Methyl 6-methoxy-2-methyl-3,4-methylene dioxybenzoate** ent-7-Hydroxy-eudesm-4-en-6-one** % Identification Grouped components Monoterpene hydrocarbons Oxygen-containing monoterpenes Sesquiterpene hydrocarbons Oxygen-containing sesquiterpenes Aromatic Others 5.6 t 9.9 53.6 2.7 t

10.6 71.8

0.5

2.2

3.1 18.7

0.6 2.0

t

5.1

2.7

2.4 2.4

15.9

44.3 2.2 12.5 14.8 t 2.4

1.3 76.2

3.4 2.5 1.4 1.4 2.3

1.9

5.3

1.0

1.7 3.6

1.5

76.1 2.2 4.5 6.8 0.4 1.6

0.5 91.6

0.4

t

1.4 2.6

1.1 0.5

t

t

3.2

1.3

0.7

57.7 0.3 10.5 23.1 0.8 t

1.0 92.4

t

4.3 1.5 3.1 1.1 t 1.7 t t t 0.8

t

t

6.0 1.0

0.8

1.5

0.9

1.2

8.9

43.6 2.6 9.9 32.6 t 1.2

0.6 89.9

3.7 1.1 7.2 1.1 1.9 1.8 t t 0.7

0.3

t t t

4.8

2.6

4.8

11.9

15.5 t 12.1 40.0 t t

8.5 67.6

t

t

t 4.5 t t 2.7 12.7

1.7 0.8

t

t

3.0 3.6

t t t

0.8 0.8

11.6

29.6 0.7 19.5 23.3 t 1.9

75.0

9.9 0.9 1.4

1.2

1.3

10.0

2.7

1.4 1.7

11.1

24.3 0.9 22.6 24.6 t 1.0

73.4

10.0 1.4 2.0

1.6

2.5

10.9

2.9

1.9 1.4

11.2

10.4 t 17.7 38.3 t t

66.4

14.2 0.8 2.3 t t

3.7

3.8

3.7

2.8 3.7

21.0

18.4 0.4 12.1 43.6 0.8 1.9

1.7 77.2

0.5

0.3

9.6 5.6 4.6 0.6 1.6 2.3

0.3

0.3

3.6

0.3

1.2

5.4

17.6

34.6 t 20.2 26.3 t 1.2

t 82.3

t

t

1.7 1.4

1.3 2.7 2.0

1.2

t

12.7

2.2

t

1.4 1.3

17.2

32.6 t 14.8 31.4 t t

1.8 78.8

t

t

2.0 4.7

2.1 2.1 2.2

1.4

1.3 t 8.9

t

1.6 1.6

16.5

40.6 t 27.1 19.3 t 1.3

t 88.3

t

t

0.8 t

t 2.6

2.1

t

17.3

2.8

1.6

0.8 1.7

15.9

ESSENTIAL OIL OF PLAGIOCHILA SPP. 707

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dominated oil, and ent-eudesm-4(15)-ene-6-one (13%) was the other main component. In the oil with balanced amounts of mono- and sesquiterpenes, β-phellandrene attained only 1%, allo-ocimene (15%), terpinolene (13%), peculiaroxide (12%) and neo-allo-ocimene (10%) being the most representative compounds. β-Phellandrene was also the dominant monoterpene identified by Rycroft et al.9 on the solvent extracts of 12 collections of this species, three of which from Madeira.

Among the other main components these authors also detected peculiaroxide and bicyclogermacrene. According to Rycroft et al.,9 P. retrorsa belongs to the 9,10dihydrophenantrene chemotype, due to the dominant components, 3,5-dimethoxy-9,10-dihydrophenanthren-2-ol and 4-hydroxy-4′-O-methyllunularate. However, these two compounds were not extracted on the essential oil from this species in the present study.

P. stricta Peculiaroxide (11–21%), allo-ocimene (7–19%), bicyclogermacrene (4–17%), neo-allo-ocimene (4–11%) and spathulenol (2–14%) were the main components of

the oils of the P. stricta specimens studied. These oils showed some monoterpene qualitative and quantitative variability.

Rycroft et al.,11 detected aromatic and terpenoid compounds on the solvent extracts of two specimens from Tenerife and two from Ecuador. The aromatic profile was not uniform between the four specimens, although similar for the Tenerife specimens. None of these compounds were detected in the Madeiran essential oils. Concerning the terpenoid fraction, Rycroft et al.11 also detected alloocimene, neo-allo-ocimene, peculiaroxide and bicyclogermacrene as the main terpenoid compounds.

Cluster analysis (Figure 1), showed a high degree of similarity between the oils from each of the Plagiochila spp. studied. With the exception of the oils from P. retrorsa, which intermingled with P. bifaria and P. stricta, each of the remaining Plagiochila oils showed a high correlation within their own populations. Noteworthy is the fact that the oils from P. maderensis showed not only a correlation higher than 0.95 among themselves but also were the ones that were less correlated with the other Plagiochila spp. oils. The lower similarity of the P. maderensis oils seems to be related to the high content of terpinolene (34–60%), while its amount was less than

Figure 1. Dendrogram obtained by cluster analysis of the percentage composition of essential oils from the Plagiochila species examined, based on correlation and using the unweighted pair-group method with arithmetic average (UPGMA). For abbreviations, see Table 1.

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ESSENTIAL OIL OF PLAGIOCHILA SPP.

13% in all other species. Interestingly, the molecular phylogeny of Plagiochila spp., based on ITS1-, 5.8Sand ITS2-nrDNA sequence comparisons4 includes P. maderensis in the section Rutilantes, whereas the remaining P. bifaria, P. retrorsa and P. stricta are included in the section Arrectae.

In conclusion, (a) the fact that previous phytochemical studies on these species were performed on solvent extracts does not allow a direct comparison and correlation between the preceding and the present new essential oil data; (b) the ubiquity of some mono- and sesquiterpenes [terpinolene, allo-ocimene, neo-allo-ocimene, peculiaroxide, methyl everninate, ent-eudesm-4(15)-ene6-one] as well as aromatic compounds in the genus Plagiochila does not allow clear species identification solely based on these compounds; (c) nevertheless, although volatiles have not been used as chemotype markers or in the establishment of taxonomic entities in liverworts, if used in conjunction with morphological, enzymatic and molecular studies,22–24 the essential oils analyses provide an helpful tool for Plagiochila spp. identification.

Acknowledgements—This study was partially funded by the Fundação para a Ciência e Tecnologia (FCT) under research contract POCTI/ AGR/42501/2001.

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4.

5.

6.

7.

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20.

21.

References 1. Asakawa Y. Phytochemistry 2004; 65: 623–669.

2. Schumacker R, Váña J. Identification Keys to the Liverworts and Hornworts of Europe and Macaronesia. Documents de la Station Scientifique des Hautes-Fagnes, No. 31, 2000; 160 pp.

Copyright © 2005 John Wiley & Sons, Ltd.

22.

23.

24.

709

Söderström L, Urmi E, Vá@a J. Lindbergia 2002; 27: 3–47.

Rycroft DS, Groth H, Heinrichs J. J. Bryol. 2004; 26: 37–45.

Sim-Sim M, Esquível MG, Fontinha S, Carvalho S. Nova Hedwgia 2004; 79 (in press).

Sim-Sim M, Carvalho S, Figueiredo AC et al. Bocagiana 2003; 210: 1–24.

Heinrichs J, Grolle R, Drehwald U. J. Bryol. 1998; 20: 495– 497.

Heinrichs J, Groth H, Linder M, Feldberg K, Rycroft DS. Bryologist 2004; 107: 28–40.

Rycroft DS, Heinrichs J, Cole WJ, Anton H. J. Bryol. 2001; 23: 23–34.

Hackl T, König WA, Muhle H. 34th International Symposium on Essential Oils, 2003 (Abstract); 77.

Rycroft DS, Cole WJ, Heinrichs J et al. Bryologist 2002; 105: 363–372.

Asakawa Y. Chemical constituents of the bryophytes. In Progress in the Chemistry of Organic Natural Products, vol 65, Hertz W, Kirby GW, Moore RE, Steglich W, Tamm C (eds).

Springer-Verlag: Berlin, 1995.

Likens ST, Nickerson GB. Proc. Am. Soc. Brewing Chemists 1964; 5–13.

Wu C-L, Huang C-D, Shih T-L. Tetrahedr. Lett. 1993; 34: 4855– 4856.

Rycroft DS, Cole WJ, Aslam N, Lamont YM, Gabriel R. Phytochemistry 1999; 50: 1167–1173.

Asakawa Y, Campbell EO. Phytochemistry 1982; 21: 2663–2667.

Kawabata J, Fukushi Y, Tahara S, Mizutani J. Agric. Biol. Chem.

1985; 49: 1479–1485.

Niwa M, Nishiyama A, Iguchi M, Yamamura S. Bull. Chem. Soc. Japan 1975; 48: 2930–2934.

Rohlf JF. NTSYS-pc, Numerical Taxonomy and Multivariate Analysis System. Applied Biostatistics: New York, 1992.

Rycroft DS. Chemosystematics and the liverwort genus Plagiochila. J. Hattori Bot. Lab. 2003; 93: 331–342.

Toyota M, Koyama H, Mizutani M, Asakawa Y. Phytochemistry 1996; 41: 1347–1350.

Sim-Sim M, Carvalho S, Figueiredo AC et al. Bocagiana 2003; 210: 1–24.

Esquível MG, Carvalho S, Figueiredo AC, Fontinha S, Sim-Sim M. An. Inst. Sup. Agronom. 2003; 49: 41–50.

Sim-Sim M, Stech M, Esquível MG et al. Hattori Bot. Lab. 2005 (in press).

Flavour Fragr. J. 2005; 20: 703–709