VARIABILITY IN THE CHEMICAL COMPOSITION OF WILD Thymus vulgaris
L.
Cases, A.1; Pérez, B.1; Navarrete, P.1; Mora, E.3; Peña, B. 3; Peluzzo, A.2; Calvo, R.4; Sánchez de
Ron, D.5; Varela, F.1
1
MA-INIA. Apdo. 8111. 28040 Madrid
Apdo. 1045. 28800 Alcalá de Henares
3 JCCM- CIA-. Ctra. Cuenca-Tarancón km7. 16194 Cuenca
4 INIA-Servicio de Biometría. Apdo. 8111. 28040 Madrid
5 INIA-CIFOR. Apdo. 8111. 28040 Madrid
2 INIA-CRF.
Introduction
The genus Thymus is composed of approximately 150 spices, distributed throughout Asia, Africa and
Greenland. It is widely extended in the Iberian Peninsula, most species being endemic. Thymus L.
(Lamiaceae) is a polymorphic genus with some taxonomical problems. This natural resource is
cultivated for commercial production of thyme essential oil.
Thyme essential oils rich in phenols have antiseptic and fungicidal activity and are also used in the
food flavour industry (Leung, A. Y., 1980.). Such oils are preferred with total phenol contents of over
40% with thymol constitute 90% of these phenols (Arctander, S., 1961). Not all thyme populations
meet these criteria, since several infraspecifyc chemotype of T. vulgaris are know and only one
chemotype contain thymol as the major component (Morgan, R. K., 1989). From Thymus vulgaris L.
growing in France and other countries (Granger and Passet, 1973; Piccaglia and Marotti, 1973) six
chemotypes have been described whose principal components are: geraniol, linalol, α- terpineol,
carvacrol, thymol and trans tujan-4-ol/terpinen-4-ol. From Thymus vulgaris L. growing in Spain another
chemotype with 1,8-cineol as its main component has been described (Adzet, T.; Genager, R.; Passet,
J. and San Martin, R., 1977). The composition of the spice thyme can be very different from one
location to another and the essential oil of Thymus vulgaris L. can correspond to different blends of
components with different activities and organoleptic properties (Guillén, M.D. and Manzanos, M.J.,
1998). Studies on the essential oils can be useful as taxonomic aids in the characterization of the taxa,
in the detection of hybridization in natural population and in the detection of infraespecific variability
(Salgueiro, L.R, Vila, R., Tomás, X., Cañigueral, S., Proença Da Cunha, A., Adzet, T., 1997).
The aim of this study is to show the composition and variability of the essential oils of T. vulgaris from
Spain. With this purpose, 63 samples localities of T. vulgaris were chosen, and the qualitative and
quantitative analysis of the volatile oil of a representative population sample of each one was
performed by GC. The results obtained were submitted to statistical analysis for to detect some pattern
distribution of localities and to identify who constituents can differentiate the different groups.
Material and method
Plant material: we collected 63 representative population samples from Castilla - La Mancha (Spain).
Flowering branches of the 50 plants of each population were collected. A minimum distance of 30km
between each population collected was taken in order to avoid sampling siblings.
Essential Oil obtaining: the essential oils were obtained by hydrodistillation of the dried ground material
in a Clevenger-like apparatus for 2 h. at atmospheric pressure on about 100g of sample. Time was
measured from the falling of the first drop of distillate. For each sample two replications of each
extraction were done. The essential oil yield was evaluated gravimetrically
Essential Oil Analysis: the Gas Chromatography analysis was accomplished with an Hewlett Packard
6890 Series instrument equipped with a FID and HP-5 capillary columns (30m x 0.25mm, 0.25μm film
thickness), working with the following temperature program: 70ºC for ten minutes and then ramp of
3ºC/min to 220ºC; injector and detector temperature, 250ºC; carrier gas nitrogen was adjusted to a
flow of 2ml/min. The sample were injected using the split mode (split ratio 1:30) and the injection
volume 0.2μL. The identification of the components was performed by comparison of their retention
times with those of pure authentic samples and by means of their linear retention indices (LRI) relative
to the series of n-hydrocarbons.
Results
The samples have been grouped in four classes according to the edafo-climatic characteristics of the
collection sites: Z1 Mancha, Z2 Guadalajara, Z3 Mountains of Cuenca and Z4 Mountains of Albacete
(Figure 1).
Figure 1. Eco-regions of Castilla – La Mancha
The chromatographic analysis of essential oils of Thymus vulgaris L. obtained of populations from
different colleted sites of Castilla La-Mancha, allowed knowing the great diversity of the oils
composition. Table 1 shows individual oil yield of each sample and average, maximum and minimum
values of each one group.
Table 1. Essential oil yield
Num.
1
2
11
13
16
17
21
23
24
32
42
44
45
54
401
402
403
404
406
407
409
420
426
514
537
M
MAX
MIN
Z1
1.25
2.95
2.50
2.00
2.89
1.88
0.94
1.38
0.97
0.75
1.38
1.99
1.77
1.30
1.76
1.62
1.94
1.55
1.21
1.42
2.44
1.48
1.76
1.87
1.06
1.68
2.95
0.75
% essential oil
Num.
Z2
58
1.22
61
1.24
62
1.45
67
0.69
437
1.25
439
0.53
441
0.91
443
0.17
445
0.68
447
0.35
449
0.17
458
0.90
460
0.13
0.69
1.45
0.13
yield
Num.
19
20
22
26
27
28
29
30
31
40
41
57
64
66
69
Z3
0.61
1.27
1.26
1.36
1.13
1.06
0.52
0.38
0.94
1.18
1.37
1.22
0.81
1.02
0.82
0.93
1,37
0.38
Num.
48
405
408
412
413
416
417
418
419
430
432
Z4
2.36
1.94
0.22
2.22
1.66
1.17
1.68
2.03
1.52
1.67
1.93
1.67
2.36
0.22
The best average oil yield were for samples from areas Z1 (1.68%) and Z4 (1.67%), whereas the
lowest were area Z2 (0.69%). The populations from Campo de Criptana (CR), Ossa de Montiel (CR),
Solana (CR) and Bonillo (AB), showed the highest levels of yield, with values of: 2.95%, 2.89%, 2.50%
and 2.44% respectively. In the 65 populations recollected, eight exceeded 2%, 35 are within 1.0% and
2.0% and the rest of populations had values close to 1.0%.
Related to the essential oil composition, the chromatographic analysis of the volatile profile of Thymus
vulgaris L. essential oil allowed the identification of a total of 40 volatile components whose
concentration surpassed 0.1%. The comparative study of the four groups the components with
concentration higher than 1.0% represented the major volatile components of this thyme oil: myrceno,
β-pineno, p-cymene, limonene, 1,8-cineole, linalol, camphor and borneol. Thymol and carvacrol also
were considered because are characteristic components of these essential oil, though in occasions
they not surpass the concentration mentioned.
The samples from the area Z1 (Table 2) were rich in 1,8-cineole and linalol with the average values of
36,98% and 11.34% respectively, more than the average values of β-pineno (7.54%) and p-cymene
(7.25%). The sample 420 from Lezuza (AB), had high concentration of 1,8-cineole (57.33%) and the
sample 32 from Caudete (CU) had high level of linalol (22.44%). Concentration of thymol in this group
in general was very low, with average value of 0.81%. The population 44 from Tarazona de la Mancha
with 2.19% of this component is the more rich.
Majority of samples had content in carvacrol less than 0.1%, except 514 from Manzanares (CR) with a
concentration of 0.95%. Concentration of camphor was appreciable in all populations with average
concentration of 4.37%. Sample 17 from San Clemente (CU) had the maximum value (10.90%).
Table 2. Essential oil components group Z1
NUM
1
2
11
13
16
17
21
23
24
32
42
44
45
54
401
402
403
404
406
407
409
420
426
514
537
M
MAX
MIN
β-pineno
2.84
3.53
5.96
4.67
7.03
8.00
2.65
5.53
8.00
12.72
9.52
8.68
7.89
11.10
7.01
10.68
11.44
11.19
14.04
10.17
0.85
1.95
9.02
5.19
8.98
7.54
14.04
0.85
p-cymene
6.18
7.22
6.57
6.18
5.40
3.36
6.85
5.88
5.95
5.15
8.84
5.44
8.26
9.17
11.16
9.14
7.08
7.57
6.03
7.69
13.93
9.93
9.47
8.71
8.09
7.25
13.93
3.36
limonene
0.15
0.27
0.10
0.11
0.34
0.48
1.36
1.37
0.36
1.87
0.30
0.61
1.87
0.10
1,8 cineol
19.40
41.11
39.39
38.30
30.60
15.10
29.15
34.22
34.52
20.21
32.80
32.11
34.54
28.90
42.42
41.52
36.34
36.24
37.23
42.86
57.05
57.33
39.89
56.00
47.33
36.98
57.33
15.10
linalol
3.84
5.87
10.69
10.40
12.30
20.20
3.40
10.63
13.40
22.44
5.54
14.21
13.20
17.20
10.26
11.91
13.37
17.25
16.04
12.93
1.84
3.09
11.77
7.62
14.07
11.34
22.44
0.98
camphor
0.73
1.32
1.38
1.37
10.90
2.87
1.47
1.55
9.81
5.74
4.40
5.87
3.88
8.59
6.61
5.52
8.15
5.61
1.34
4.38
3.71
4.59
4.37
10.90
1.84
borneol
2.35
3.33
3.12
4.54
3.31
1.70
1.06
3.30
2.60
1.92
1.57
1.00
1.26
1.18
1.24
1.23
1.33
1.30
0.98
1.96
1.13
1.97
3.33
0.73
thymol
1.14
0.62
1.39
0.77
1.24
1.17
0.43
0.22
0.16
0.59
1.09
2.19
0.80
0.69
0.47
0.59
0.97
0.67
0.81
0.65
0.28
0.81
2.19
0.22
carvacrol
0.11
0.10
0.95
0.64
0.45
0.95
0.10
Content of 1,8-cineole of the populations from Z2 (Table 3), was similar to the Z1, with medium value
of 34,69%. Sample 443 from Corduente (46.84%), had the highest value. Samples from Z2 had lower
values for linalol than samples from Z1, but they were richer in camphor, thymol and carvacrol. Sample
458 from Illana (GU) had the higest level of carvacrol (3.52%), and with sample 460 from Pastrana
(GU) had the highest level of camphor (18.52%, 11.93%) and borneol (6.00%, 5.85%). Sample 447
from Anguita(GU) had high level of thymol and limonene (9,87% and 4.52%).
Table 3. Essential oil components group Z2
NUM
58
61
62
67
437
439
441
443
445
447
449
458
460
M
MAX
MIN
β-pineno
7.62
6.60
9.09
2.67
5.62
10,88
6.80
6.43
5.39
6.42
6.08
10.30
7.20
7.00
10.88
2.67
p-cymene
8.52
8.54
8.84
10.00
8.78
7,14
8.50
9.12
9.43
6.41
7.10
6.75
3.80
7.92
10.00
3.80
limonene
0.31
0.11
0.37
0.30
2,64 -4.52
0.32
0.40
3.91
1.39
3.91
0.11
1,8 cineole
33.90
41.42
42.15
42.56
40.78
1,38
34,67
42.20
46.84
38.02
20.80
22.93
30.51
14.20
34.69
46.84
14.20
linalol
12.50
9.32
15.60
2.22
12,98
11.26
10.23
7.30
8.23
6.60
9.62
12.98
2.22
camphor
4.01
6.63
5.24
2.22
8.57
4,66
4.41
3.77
3.51
4.44
2.49
18.52
11.93
5.94
18.52
2.22
borneol
1.17
1.64
1.35
3.22
2.50
1.44
1.73
0.94
0.85
6.00
5.85
2.37
6.00
0.85
thymol
3.42
1.32
1.02
0.32
5.48
0,75
0.63
0.71
0.75
9.87
3.84
4.46
2.71
9.87
0.32
carvacrol
0.91
0.35
3.52
1.57
3.52
0.35
Samples from Z3 (Table 4) were rich in borneol and carvacrol, whit medium value of 2.51% and 1.45%
respectively, however had low content of β-pineno (5.16%), limonene (0.32%), 1,8-cineole (34.68%),
linalol (8.99%), camphor (3.37%) and thymol (0.78%). Sample 22 from La Almarcha (CU) had high
content of carvacrol (6.00%) and very low concentration β-pineno (1.93%). Sample 57 from Villar de
Domingo García (CU) had the lowest content of 1,8-cineole (3.83%) of all the samples analysed in this
work and the highest value of β-pineno (8.30%) and linalol (14.65%) of this group.
Table 4. Essential oil components group Z3
NUM
22
26
27
28
29
30
31
40
41
57
64
66
69
M
MAX
MIN
β-pineno
1.93
7.26
3.65
7.10
3.08
1.40
8.83
3.72
8.00
8.30
4.76
4.40
4.66
5.16
8.83
1.40
p-cymene
0.94
6.54
8.82
6.37
10.10
6.77
6.23
8.14
8.83
7.63
9.02
8.47
9.72
7.50
10.10
6.23
limonene
0.38
0.16
0.45
0.17
0.27
0.36
0.34
0.34
0.36
0.39
0.44
0.43
0.32
0.45
0,16
1,8 cineole
28.21
30.75
44.43
36.40
44.36
42.69
29.23
27.50
38.70
3,83
42.95
35.46
46.41
34,68
46.41
27,50
linalol
9.67
10.22
6.50
12.51
3.91
5.78
13.43
6.52
13.40
14.65
7.39
5.81
7.10
8.99
14.65
3.91
camphor
6.07
1.49
1.50
1.93
1.16
2.35
1.69
5.64
5.26
3.05
7.14
3.28
3.37
7.14
1.16
borneol
2.87
2.33
3.86
3.13
2.46
4.79
2.24
1.33
1.66
1.41
2.57
1.52
2,51
4.79
1.33
thymol
0.99
0.74
0.17
0.75
0.26
0.40
0.48
0.10
2.83
1.30
1.23
0.28
0.64
0.78
1,30
0.10
carvacrol
6.00
0.11
0.28
0.13
0.23
0.39
4.00
0.53
1.45
6.00
0.11
borneol
2,26
1.29
1.16
1.29
1.35
1,43
1.34
1.16
1.42
1.34
1.62
1,38
1,42
2,26
1,16
thymol
0.64
2,83
1.21
0.55
0.91
0,99
1.06
0,75
1,12
2,83
0,64
carvacrol
0,12
0.64
1.00
0.85
0.82
0,69
1,00
0,12
Table 5. Essential oil components group Z4
NUM
19
41
48
405
408
412
413
416
417
418
419
430
432
M
MAX
MIN
β-pineno
8,19
8,00
10.45
5.99
10.22
10.58
10,71
8.88
9.92
12.57
11.71
0.17
10,88
9,10
12,57
0,17
p-cymene
9,57
8,83
7.93
9.80
7.76
7.33
6,95
7.14
6.82
6.39
7.05
3.29
7,14
6,84
9,80
3,29
limonene
0,10
0,34
1,81
2,64
1,22
2,64
0,10
1,8 cineole
37,34
38,70
34.53
54.12
43.11
42.38
40,12
40.20
36.20
35.02
35.34
57.06
34,67
40,67
54,12
34,53
linalol
8,17
13,40
14.18
7.56
12.84
12.47
13,55
12.35
13.92
15.22
15.82
0.41
12,98
11,75
15,82
0,41
camphor
1,10
5,64
6.69
3.42
5.57
5.70
7,00
5.75
5.81
8.26
7.52
4,66
5,59
8,26
1,10
Samples from Z4 (Table 5) had a concentration of limonene less than 0.1% in majority of cases,
however were rich in linalol. Sample 430 from Yeste (AB) was an exception, with a content of linalol of
0.41% and level very low of β-pineno and p-cymene. This population from Yeste is a chemotype 1,8cineole with 57.06% of this component. Sample 405 from Fuente Álamo (AB) also had a high level of
1,8-cineole (54.12%) and 7,56% of linalol. The rest of populations from Z4 were 1,8-cineol/linalol
chemotype. Sample 418 from Elche de la Sierra (AB) and sample 419 from Ayna (AB), they had high
concentration of linalol (15.22% and 15.82%) respectively.
The Table 6 show medium value of majority components:
Table 6. Average values
Medium value
Z1
Z2
Essential oil yield
1.68
0.69
β-pineno
7.54
7.00
p-cymene
7.25
7.92
limonene
0.61
1.39
1,8 cineole
36.98 34.69
linalol
11.34
9.62
camphor
4.37
5.94
borneol
1.97
2.37
thymol
0.81
2.71
carvacrol
0.45
1.57
Z3
0.93
5.16
7.50
0.32
37.26
8.99
3.37
2.51
0.78
1.45
Z4
1.67
9.10
6.84
1.22
40.67
11.75
5.59
1.42
1.12
0.69
Statistical analysis showed significance differences between altitude, longitude and latitude face to
yield of essential oil with R2=0.5. The biggest difference was for the latitude with 41% and 6% for the
altitude and 3% for longitude. The influence of these variables over greater part of main components of
essential oils was not significant. Individually, when the altitude increase, also increase the yield
essential oil and content of 1,8-cineole decrease.
Conclusions
 Samples from Z1 and Z4 had high content of essential oil and linalol, however their content of
borneol, thymol and camphor was very low.
 Samples from Z2 had very high content of thymol and camphor, however yield of essential oil
and content of linalol were low.
 Samples from Z3 yield essential oil were low, in this way like concentration borneol and
carvacrol, while that content of thymol and camphor were low.
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