FLAVOUR AND FRAGRANCE JOURNAL, VOL.

8,53-57 (1993)

The Essential Oils of Two Endemic Portuguese Thyme Species:

Thymus capitellatus Hoffmanns. & Link and T. lotocephalus
G. L6pez & R. Morales
A. Cristina Figueiredo, JoSe G. Barrom, Luis G. Pedro and M. Salomb S. Pais
Departamenlo de Biologia Vegetal, Faculhde de Ciincias & Lisboa, Bloc0 C2, Camp0 Gran&, I700 Lisbon, Portugal

Johannes J. C. Sche6er
Division of Pharmacognosy, Center for Bio-Pharmaceutical Sciences, Leiden University, Gorlaeus Laboratories, PO Box 9502,
2300 RA Leiden, The Netherlandr

The composition of the essential oils of Thymus capitellatus Hoffmanns. & Link and T . lotocephalus G. L6pez & R
Morales was studied by GC and GC-MS. The essential oils of T . capitellatus, isolated either from flowers or fron
leaves of the plant during its vegetative phase, showed a more or less similar composition, but the corresponding oil
from T . lotocephulus showed marked differences. The oils of both species consisted mainly of oxygen-containin
monoterpenes (55-69 % of the total oils). 1,8-Cineole (50-56 %) was the main component of the oils of T . capitellatw
both in the oil from its flowers and in that from its leaves.
The oil isolated from the flowers of T . lotocephalus was dominated by linalyl acetate (23%); the other major
components were linalol (1 1%) and 1,8-cineole (10 %). However, in the oil from the leaves of this species 1.8-cineolt
(24%) was again the major component, while linalyl acetate and linalol amounted to 5 % and 6 % respectivelj
Although present in relatively small amounts, sesquiterpenes were more important in the oils of 7: lotocephalu
(15-17%) than in those of T . capitellatus (3-473. The presence of l,8-cineole, in relatively large amounts, in the oil
analysed was in agreement with the earlier finding of this compound as major component of other Iberian thyme oils
However, the oil isolated from flowers of T. lotocephalus must be considered as being characterized by linalyl acetate,
linalol instead of by 1,8-cineole.

KEY WORDS Thymus capitellatus Hoffmanns. & Link Thymus lotocephalus G. Lopez & R. Morales
Thymus cephalotos Hoffmanns. & Link Lamiaceae Essential oil l,&Cineole Linalyl acetate
Linalol Abietatriene

INTRODUCTION there were more similarities between the oils of

Thyme is the common name of more than three
hundred species and many hybrids, varieties and
ecotypes belonging to the genus Thymus.' Thyme
produces an essential oil which is used for flavour-
ing foods and in the manufacture of perfumes and
cosmetics. It is also used for medicinal purposes,
among others as an antispasmodic, a carminative,
an emmenagogue and a tonic.'
The essential oils of different Thymus species
have been extensively studied by several
authors,2-8 as is also clearly shown by a recent
review article by Stahl-Bi~kup.~ According to
Morales Valverde,' a general relationship could be
seen for all the oils he analysed, but in some cases
0882-5734/93/010053-05$07.50
0 1993 by John Wiley & Sons, Ltd.
different species than between the oils of different
populations of one certain species. Such variations
seem to be related to the age of the plant, and to
genetic and environmental condition^.^^
Several chemotypes have been defined for thyme
oils. Granger and Passetlo described six chemo-
types of T. vulgaris using the dominant terpene in
the oil as criterion: geraniol, linalol, a-terpineol
carvacrol, thymol and the complex trans-thujan-4
ol/terpinen-4-ol. Subsequent studies led to the iden.
tification of a seventh chemotype, observed ir.
populations of the Iberian peninsula, which was
characterized by high levels of 1,8-cineole.'' Ac-
cording to Simon et d.,' the thyme oils can be
grouped into three main types: thyme oil, contain-
Received 4 June 199.
Accepted 10 July 199.
54 A. C. FIGUEIREDO ET AL.

ing 42-60% of phenols, thymol being the main (Seixal, Portugal). Two batches of plant material
component; origanum-type oil containing 63-74 % were collected: flowers during full flowering (June)
of phenols, carvacrol being the main component; and leaves during the vegetative phase of the plants
lemon thyme oil, containing citral. In the review (May).
mentioned above,g the most frequently occurring Aerial parts of T . lotocephalus G. L6pez & R.
constituents of the essential oils of the 84 Thymus Morales ( = T. cephalotos sensu Hoffmanns. &
species studied are, listed in decreasing order of Link) were collected from plants growing in the
occurrence, thymol and carvacrol, linalol, p-cy- Botanical Garden of Lisbon: flowers were collected
mene, y-terpinene, borneol, 1,8-cineole,terpinen-4- , during full flowering (May) and leaves during the
01, a-terpineol, /I-caryophyllene,linalyl acetate, and vegetative phase (October).
camphor. Voucher specimens of T . capitellatus and 7'.
Studies carried out on Portuguese thyme oils led lotocephalus have been deposited in Herbarium of
to the characterization of four main groups: thy- the Instituto BotAnico da Faculdade de Cibncias de
mol-type oil for T. vulgaris and T . zygis; citral-type Lisboa (LISU: 160057 and LISU: P 31296, respec
oil for T . hirtus; cineole and linalol-type oil for T . tively).
mastichina;and an oil type with alcohols and esters
for T . caespititiu~.'~*'~ Later, another Portuguese
thyme oil, that of T . camphoratus, was ~tudied;'~Isolation Procedure
the composition of this oil allows its inclusion in The oils were isolated from fresh plant materia
the last group. by distillation-extraction, for 3 h, using a Likens
T. capitellatus and T. lotocephalus are two vul- Nickerson-type apparatus, and by hydrodistilla
nerable endemic Portuguese thyme species with tion, for 3 h, using a Clevenger-type apparatus. Thb
special ecological niche^.'^ In the first reports on oil samples isolated by hydrodistillation were usec
the composition of their essential oils Fernandes to sstimate the oil yields, and those isolated by
C o ~ t a ' ~ .found
' ~ 30% cineole and 30% alcohols distillation-extraction to determine the percentage
for T . capitellatus, and 60% cineole and 12% composition of the oils.
alcohols for T. lotocephalus. According to
Palhinha," the latter species contained linalyl ace-
tate in addition to cineole (probably 1,8-cineole is Gas Chromatography
meant). GC analyses were performed using a Perkic
Recently, Salgueiro described three chemotypes Elmer 8600 gas chromatograph equipped with ar
of Portuguese T . capitellatus,two of which contain- FID, a data handling system and a DB-1 fused-
ing large amounts of 1,8-cineole." In the oils silica column (30 m x 0.25 mm id., film thicknes!
isolated from a number of plants of this species, the 0.25 pm; J & W Scientific Inc., Rancho Cordova
percentage of 1,8-cineolevaried from 25 % to 59 %. CA, USA). Oven temperature was programmed
In the same paper," the essential oil of T . lotoce- 45-175"C, at 3"C/min, subsequently at 15"C/mir
phalus was described as consisting of large amounts up to 280°C, and then held isothermal for 10 min
of 1,8-cineole,camphor, linalol, linalyl acetate and injector and detector temperatures, 280°C; carriei
a-pinene, but more precise data on this species were gas, hydrogen, adjusted to a linear velocity 01
not given. 30 cm/s. The samples were injected using the split
In the study described in this paper, we analysed sampling technique, ratio 1:50. The percentage
the essential oils from flowers of T . capitellatus and composition of the oils was computed from the GC
T . lotocephalus using GC and GC-MS. In addition peak areas without using correction factors. The
the oils from the leaves collected during the vegeta- data shown are mean values of two injections.
tive phase of these species were investigated.
Gas Chromatography-Mass Spectrometry
EXPERIMENTAL The GC-MS unit consisted of a Carlo Erba 600(
Vega gas chromatograph, equipped with a DB-I
Plant Material fused-silica column (30 m x 0.25 mm i.d., filrr
thickness 0.25 pm; J & W Scientific Inc.), anc
Aerial parts of Thymus capitellatus Hoffmanns. interfaced with a Finnigan MAT 800 Ion TraF
& Link were collected randomly at Verdizela Detector (ITD; software version 4.1). Oven tem-
 ESSENTIAL OIL OF PORTUGUESE THYME SPECIES 55

perature was as above; transfer line temperature, Table 1. Percentage composition of the essential oils of Thy-
280°C; ion trap temperature, 220°C; carrier gas, MIF capitellatus Hoffmanns. & Link and T. lotocephalus G.
helium, adjusted to a linear velocity of 30cm/s; Upez & R. Moraks isolated from the flowers, and from the
leaves collected during the vegetative phase of the plants
splitting ratio, 1:40;ionization energy, 70 eV; ion-
ization current, 60 FA; scan range, 40-300 u; scan T. capitellatus T. lotocephalus
time, 1 s. Component. Flowers Leaves Flowers Leaves
The identity of the components was assigned by ~

comparison of their retention times and mass spec- Tricyclene t t t 0.2

tra with corresponding data of components of a-Thujene
a-Pinene
t 0.1 0.1 0.1
5.3 7.8 7.4 9.0
reference oils. Camphene 2.6 0.7 0.7 1.9
Sabinene 3.9 4.1 0.7 0.6
8-Pinene 2.8 2.1 0.9 0.7
8-Myrcene 1.0 1.o 1.7 0.9
a-Terpinene 0.2 0.2 0.1 t
RESULTS AND DISCUSSION pCymene t t 0.1 0.4
1.8-Cineok 50.4 55.5 10.3 24.1
The essential oils isolated from the flowers of T. Limonene 0.7 0.8 1.4 t
capitellatus and of T . lotocephalus were obtained in cis-8-Ocimene t 0.1 0.5 0.3
trans-8-Ocimcne 2.3 2.1 2.4 1.2
almost similar yields (1.7 % and 1.5 %, v/w, respec- y-Terpinene 0.3 0.3 0.4 0.1
tively). Although the yield of the oils isolated from trans-Sabinene hydrate 0.7 0.4 0.3 0.8
the leaves, collected during the vegetative phase of cis-Linalol oxide t 0.2 0.6 1.6
both species, was again more or less similar, it was trans-Linalol oxide 0.5 1.o
Terpinolene t 0.1 0.4 0.3
much lower, being 0.8% for T. capitellatus and cis-Sabinene hydrate 0.3 0.2 0.4 0.7
0.5 % for T. lotocephalus. Linalol 2.4 3.8 11.3 6.0
The essential oils isolated from the flowers and a-Campholenal 0.1 0.2 0.3 0.7
from the leaves of T. capitellatus were complex trans-pMenth-2en- 1-01 t t
mixtures consisting mainly of monoterpenes (88 % Camphor 0.5 0.5 1.8 3.4
trans-Pinocarveol 0.3 0.3 t 0.4
in both cases); the oxygen-containing compounds allo-Ocimene 0.3
formed the major fraction of the oils (69% and trans-Verbenol 0.5 0.7 t 3.1
68 %, respectively). In both cases l,8-cineole was Pinocarvone 0.3 0.2 0.1 0.5
the most abundant component (50 % in the oil from Nerol oxide 0.1
Borneol 6.9 t 0.9
flowers and 56% in the leaf oil). Borneol was the 6-Terpineol t 1.2 t 0.4
second most important component in the oil from Terpinen401 0.9 0.7 0.5
the flowers (7%) and a-pinene in that from the Myrtenal 0.3 0.2 0.1 0.3
leaves (8 %). Apart from borneol, which was only a-Terpineol 1.6 1.3 5.3 2.6
Myrtenol 0.4 0.3 t t
found in the oil isolated from the flowers, the cis-Verbenone 1.3
composition of both oils was quite similar, both n-Decanal 0.1
qualitatively and quantitatively. The identified Carvone t 0.2 t
components and their percentages, attaining 92 % Geraniol 0.9
of the total oil from the flowers and 91 % of that Linalyl acetate 22.8 5.0
Bornyl acetate 1.o 0.8 0.3 1.1
from the leaves, are given in Table 1, where the a-Terpenyl acetate 2.3 1.5 t
components are listed in order of their elution on Geranyl acetate t 0.1 2.0 1.1
the DB-1 column. a-Copaene t
The sesquiterpenefraction represented 4% of the /3-Bourbonene t t 0.1 t
8-Elemene 0.4 0.3 0.1 0.2
oil isolated from the flowers and 3 % of the leaf oil. a-Gujunene 0.1 0.5 0.3
The relative amounts of sesquiterpene hydrocar- 8-Caryophyllene 0.3 0.5 1.6 0.1
bons and oxygen-containing sesquiterpenes were Aromadendrene 0.1
almost similar in the oil isolated from the leaves a-Cadinene’ 0.7
(1.4% and 1.6%, respectively), whereas in the oil allo-Aromadendrene 1.2 0.9
Germacrene-D 0.1 3.9 1.1
from the flowers, the oxygen-containing sesquiter- Valencene 0.1 t
penes represented the major part of the sesquiter- a-blinene 0.1 t
pene fraction (3%). In both oils elemol was the Bicyclogermacreng 3.5 1.1
dominant sesquiterpene (1.2 % and 2.5 %, respec- y-Cadinene 0.2
tively). l:ontinuel
56 A. C. FIGUEIREDO ET RL.

Table 1. Continued important monoterpenes were a-pinene (9 %) lina-

lo1 (6 %) and linalyl acetate (5 %). The other mono-
T . capitellatus T . lotocephalus terpenes were each found in a concentration less
Component" Flowers Leaves Flowers Leaves than 4 %. The sesquiterpene fraction of the leaf oil
Calamenene 0.4 0.2 0.3 0.3
amounted to 17 %, the oxygen-containing sesqui-
6-Cadinene t 0.1 0.3 terpenes representing the major part of this fraction
Elemol 2.5 1.2 t (12%).
Ledol 0.2 It is noteworthy that linalyl acetate and linalol
Spathulenol 1.2 1.8 were the major components of the oil isolated from
8-Caryophyllene epoxide 0.3 0.3 0.9 1.3
Globulol 0.6 the flowers of T. lotocephalus. Schmaus and
ocs 5.1 Kubeczka" have shown that in oils with linalyl
ocs 1.7 acetate as major component, high amounts ol
8-Eudesmol t 0.1 linalol can be found as a distillation artefact. Ir,
T-Cadinol 0.1
or-Cadino] 0.6 0.2 order to investigated the influence of the isolatior
ocs 0.7 procedure on the formation of linalol, the influores
cences of T. lotocephalus were extracted with n
Identified 91.9 90.7 89.9 79.7 pentane-diethyl ether (1: 1). The analyses per
formed showed that only small amounts of h a l o
Grouped components
MHs 19.1 19.4 17.1 15.7 could be found in this extract (0.5%), wherea
OCMs 68.9 68.3 57.6 55.0 linalyl acetate attained 25% of the total oil. Ou
SHs 1.1 1.4 12.3 4.9 results thus confirm the finding reported b
ocss 2.8 1.6 2.8 11.6 Schmaus and Kubeczka."
Others 0.1
The essential oil isolated from the flowers of 7
lotocephalus contained two compounds with GC
'MH = monoterpene hydrocarbon; OCM = oxygen-
containing monoterpene; SH = sesquiterpene hydrocarbon; retention times longer than those of sesquiterpenes
OCS = oxygencontaining sesquiterpene; t = trace (< 0.05%). The mass spectra of these compounds showec
'Identification based on mass spectra only. fragmentation patterns which were quite similar tc
those reported in the literat~re,'~ for rimuene ant
abietatriene, respectively. A comparison of the re
tention times and mass spectra of the two compo.
The identified components of the corresponding nents with those of authentic samples confirmec
oils of T. lotocephalus, and their percentages are the presence of abietatriene (0.1%), but the othe:
also given in Table 1. Three oil components with a compound (0.2%) although having a mass spec
relative amount of more than 0.1 % which were trum similar to that of rimuene (Figure l), had i
recognized as oxygen-containingsesquiterpenes by different retention time.
means of their mass spectra, but could not be Although the essential oils of T. capitellatus anc
identified until now, have also been included in the T . lotocephalus showed different qualitative anc
table. The components identified in the oils isolated quantitative compositions, they had in commor
from the flowers and from the leaves amounted to 1,8-cineole as one of their major components. Ir
90% and 80%, respectively. Both oils consisted that way our results agree with those reported ir
mainly of oxygen-containing monoterpenes (58 % the literature, namely that Iberian thyme oils arc
and 55 %, respectively). In the oil from the flowers, characterized by high levels of 1,8-cineole.'' How-
the main components were linalyl acetate (23 %), ever, the oil isolated from the flowers of T. lotoce
linalol (1 1 %), l,&cineole (10 %), a-pinene (7 %) and phalus can better be considered as beinl
a-terpineol (5%). The percentage of each of the characterized by linalyl acetate/linalol. Since thc
remaining components was less than 4%. The oils isolated from the leaves and from the flowers o
sesquiterpene fraction of this oil amounted to 15 %; T. lotocephalus were markedly different, the oi
hydrocarbons formed the major part of this frac- composition of this species will depend on the stag0
tion 12 %. Germacrene-D (3.9 %) and bicycloger- of development of the plants and on the amount o
macrene (3.5 %) were the dominant sesquiterpenes. flowers present in the plant material used to isolatc
The leaf oil, which was again dominated by 1,8- the essential oil. This should be kept in mind, wher
cineole (24 %), was markedly different from that studies on the occurrence of chemotypes of thc
obtained from the flowers. The other quantitatively species in question are carried out.
 ESSENTIAL OIL OF PORTUGUESE THYME SPECIES 57

100% 257

41
50-

50 100 150 200 250 m/z

Fig. 1. Mass spectrum of an unidentified diterpene obtained by GC-MS (ion trap detector) anal@ of the essential oil isolated from
the flowers of Thymus lotocephulus G. Mpez & R. Morales; the spectrum, which is similar to the mas spectrum published To,
rimuen~,'~ was obtained by background subtraction.

Acknowledgements-The authors gratefully acknowledge Mr
Belo-Correia and Ms Fdtima Costa for identification of the
plant material, Professor Adams (Baylor University, Texas,
USA) for authentic samples of abietatrieneand rimuene, and the
Instituto Nacional de Investip@o Cientilica (INK), Ccntro de
Engenharia Biolbgica, Lisbon, for financial support.
REFERENCES
1. J. E. Simon, A. F. Chadwick and L. E. Craker, Herbs: An
I d x e d Bibliogruphy 1971-1980, p. 92,Elsevier, Amsterdam
(1984).
2. R. Morales Valverde, Taxonomiu de 10s g h r o s Thymus
(excluido la seccibn Serpyllum) y Thymbro en la P e n h u h
Ibhicu, Monogra6a del Real Jardin Bothico, vol. 3, Mad-
rid (1986).
3. M.E. Crespo. E. Gomis, J. Jimena and C. Navarro. Plonto
Med., 161 (1988).
4. J. Cabo. M.E. Crespo, J. Jimencz, C. Navarro and S . Risco,
Planta Med., 380 (1987).
5. T. Adzet, R. Vila, S. Cailigueral and C. IMiiez, Flowur
Fragr. J., 4, 133 (1989).
6. T. Adzct, R Vila,X Badori and C. IWa, Fluwur Fragr
I , 4,63 (1989).
7. R Piccaglia and M. Marotti, Flooour Fragr. J , 6,241(1991)
8. J. Iglesias, R. Vila,S. Caiiigwral, J. Bellakhdar and A. I
Idrisai, I. E s m t . Oil Ru, 3.43 (1991).
9. E. Stahi-Biskup, 1.Essmt. 011 Ru.,3.61 (1991).
10 R Granger and J. Panset, P h y r o c k m b y , 12,1683(1973).
11. T.Adzet, R. Granger, J. Panset and R. San Martin, Biochen
Syst. Ecol, 5.269 (1977).
12. A. Fernandm h t a , EIacntos da Flora Aromdtica, Junk
dc Inveatigqiks Cihtificos no Ultramar, Lisbon (1975).
13. A. Fernandm Costa, BoL Soc. h t , 19,687 (1945).
14. T.Adzet. R. Vila. C. -1 and S. Coainueral
- . Planto Med.
369 (1988).
15. A. M. Dray. Plantus o Roteaa em Portuaol Continenta
Scrvip N & o d de Parqw; Rer#rvas e Conservaglo di
Naturaa, Lisbon (1985).
16. R. T.Palhinha, B r o t h 1%97 (1Wa).
17. L. M. R. ~ @ C i r O ,Fluuour Fragr. I.,7,159 (1992).
18. G. schmaus pnd K.-H. Ku- in Essarthl Oils d
Aromutic P h s , cd. A. Bacrheim Svcndaen and J. J. C
Scheffer, p. 127, Martinus Nijhoff/Dr W.Junk Publishers
Dordrecht (1985).
19. R. P. Adam8, ldentifcatwn of Essential Oils by Ion TraL
M a w Spectroscopy, Acpdemic Press, San Diego (1989).