2211

J. Sep. Sci. 2010, 33, 22112218

Clara Grosso1
Ana Cristina Figueiredo2
Jesus Burillo3
Ana M. Mainar4
Jose S. Urieta4
Jose G. Barroso2
Jose A. Coelho5
Antonio M. F. Palavra1
1

Departamento de Engenharia
Qumica e Biologica, IST, Lisboa,
Portugal
2
Universidade de Lisboa,
Faculdade de Ciencias de
Lisboa, DBV, IBB, Centro de
Biotecnologia Vegetal, Lisboa,
Portugal
3
Centro de Investigacion y
Tecnologa Agroalimentaria,
Departamento de Ciencia,
Tecnologa y Universidad,
Gobierno de Aragon, Zaragoza,
Spain
4
Qumica Organica y Qumica
Fsica, Universidad Zaragoza,
Pedro Cerbuna, Zaragoza, Spain
5
CIEQB/DEQ, ISEL, Lisboa,
Portugal

Received March 21, 2010

Revised April 28, 2010
Accepted May 2, 2010

Research Article

Composition and antioxidant activity of

Thymus vulgaris volatiles: Comparison
between supercritical fluid extraction and
hydrodistillation
Supercritical fluid extraction (SFE) of the volatile oil from Thymus vulgaris L. aerial
flowering parts was performed under different conditions of pressure, temperature, mean
particle size and CO2 flow rate and the correspondent yield and composition were
compared with those of the essential oil isolated by hydrodistillation (HD). Both the oils
were analyzed by GC and GC-MS and 52 components were identified. The main volatile
components obtained were p-cymene (10.042.6% for SFE and 28.934.8% for HD),
g-terpinene (0.86.9% for SFE and 5.17.0% for HD), linalool (2.35.3% for SFE and
2.83.1% for HD), thymol (19.540.8% for SFE and 35.441.6% for HD), and carvacrol
(1.43.1% for SFE and 2.63.1% for HD). The main difference was found to be the relative
percentage of thymoquinone (not found in the essential oil) and carvacryl methyl ether
(1.01.2% for HD versus t 0.4 for SFE) which can explain the higher antioxidant activity,
assessed by Rancimat test, of the SFE volatiles when compared with HD. Thymoquinone
is considered a strong antioxidant compound.
Keywords: Antioxidant activity / Hydrodistillation / Rancimat test / Supercritical
fluid extraction / Thymus vulgaris L.
DOI 10.1002/jssc.201000192

1 Introduction
Plant extracts, especially essential oils, have been employed
in pharmaceutical, agronomic, food, cosmetic, and perfume
industries due to several reported biological properties.
According to the Portuguese [1] and the European [2]
Pharmacopoeias, an essential oil is the plant extract
obtained just by distillation processes, like hydrodistillation
(HD) and steam distillation (SD), with the exception of the
Citrus sp. peel oil, which is isolated by cold expression.
When other isolation techniques are employed, other
designations, such as volatiles or volatile oil, must be used.
The quality of the extract containing the volatile
components can be improved by alternative techniques,
such as supercritical fluid extraction (SFE). With this technique the plant has no contact with water and therefore,
interconversion of monoterpenes as well as hydrolysis
reactions are avoided, preventing the changes in the original

Correspondence: Dr. Clara Grosso, Av. Rovisco Pais, 1, 1049-001

Lisboa, Portugal
E-mail: clara.f.grosso@ist.utl.pt
Fax: 135-121-846-4455

Abbreviations: d.w., dry weight; HD, hydrodistillation; IP,

induction period; PF, protector factor; SD, steam distillation;
SFE, supercritical fluid extraction

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

volatiles composition. Moreover, since the most used

supercritical fluid is carbon dioxide, which has moderate
critical pressure and temperature (Pc 5 73.8 bar and Tc 5
31.11C), the extraction of volatile components is usually
performed in the range of 40501C and 80120 bar, which
means that thermodegradation is avoided [3].
Thymus vulgaris L., commonly known as thyme, is a
perennial aromatic and medicinal plant from the Mediterranean region. It belongs to the Lamiaceae family and as
it occurs within several species of this family, different
varieties based on different chemotypes have been reported
in the literature. Therefore, until now, seven chemotypes
were described: 1,8-cineole, linalool, a-terpineol, geraniol,
trans-thujan-4-ol, thymol, and carvacrol [46]. The composition of the volatiles from thyme has already been assessed
using different isolation techniques: HD [710], microwaveassisted HD [9], SD [11], SD under reduced pressure [12],
simultaneous distillation-extraction [5], ultrasound-assisted
extraction with dichloromethane [13], CO2-SFE [5, 11], and
pressurized liquid extraction with hexane [11].
The demand for the volatile components from this plant
is due to the medicinal properties attributed to thyme.
Studies indicated that thyme possesses antispasmodic,
antiseptic, expectorant, antitussive, antibroncholitic, carminative, and diuretic properties [7, 14]. Moreover, the essential oil obtained by HD and SD from T. vulgaris showed to
have antibacterial [10, 1417], antifungal [16, 18, 19] and

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2212

J. Sep. Sci. 2010, 33, 22112218

C. Grosso et al.

antioxidant activities [12, 15, 20] which led to its incorporation in skin care [21] and food products [18, 22].
The aim of this work was to test the effect of
pressure, temperature, particle size, and CO2 flow rate
conditions and to evaluate how these process parameters
influence the SFE extraction yield and composition of the
volatile oil from T. vulgaris. As reported above, SFE has
already been performed for this species. However, except for
Daz-Maroto et al. [5], who used the conditions of pressure
and temperature appropriate for the extraction of a pure
volatile oil without contamination with heavier compounds
(120 bar and 401C), the other extractions were performed
at higher CO2 densities corresponding to pressure and
temperature of 300 bar/401C [20], 400 bar/601C [23] and
500 bar/801C [11].
Moreover, the antioxidant activities of the supercritical
fluid volatile oil, as well as the HD essential oil were studied
using the Rancimat test at 1201C, an assay that measures
the ability to inhibit lipid oxidation [24]. The aim of this
test is to simulate the storage of fat and oils, at room
temperature, but in a expedite way. Therefore, it is used
to predict, more quickly, the shelf life of fats and oils, by
using conditions of oxidation which promote the forced
aging of fatty oils (high temperatures in the range of
1201401C and a stream of dry air). This test was already
applied at 1001C for this plant by Schwarz et al. [22] and
by Simandi et al. [23] to evaluate the antioxidant activity of
the n-hexane extract and SFE extract obtained at 400 bar/
601C, respectively.

regulator and measured with a Bourdon-type manometer

and the preset temperature is reached with the aid of a water
jacket. The total volume of CO2 used in the extraction is
determined with a dry test meter. The CO2 (99.995% purity)
was supplied by Air Liquide (Lisbon, Portugal).

2.3 Extraction of essential and volatile oils

The essential oil was obtained by HD, during 4 h, with a
Clevenger-type apparatus, according to the European
Pharmacopoeia method [2]. The extractions were performed
with about 40 g dry weight (d.w.) of plant material with a
mean particle size of 0.4, 0.6, and 0.8 mm.
The volatile oil was isolated by SFE. The extractions
were carried out with about 100 g (d.w.). Different conditions of pressure (90 and 100 bar), temperature (40 and
501C), mean particle size (0.4, 0.6 and 0.8 mm), and
flow rate (0.7, 1.1, and 1.3 kg/h) were tested. To obtain
a pure volatile oil with a composition near to that
of the essential oil, a fractional separation was conducted
at 80 bar and 81C (first separator), and 20 bar and
151C (second separator). The amount of the waxes
and volatile oil collected in the first and second separators, respectively, was determined gravimetrically w/w,
although only the composition of the volatile oils was
analyzed by GC.

2.4 Extraction of the nonvolatile extracts

2 Materials and methods

2.1 Plant material
Dried aerial flowering parts of T. vulgaris (from Centro de
n y Tecnologa Agroalimentaria, cultivated at
Investigacio
Ejea de los Caballeros, Spain) were ground with a
commercial mill to obtain particles in the range from 0.1
to 1.0 mm. The mill was operated after freezing the plant
material with liquid N2 to avoid the loss and thermal
degradation of the volatile compounds. After being separated by a series of sieves, the fractions were kept at 201C
and mean particle sizes of about 0.4, 0.6, and 0.8 mm were
used for the subsequent extractions.
To calculate the initial moisture content of the plant
material, a sample was oven dried at 1051C until a constant
weight (72 h). A value of 10.9% was obtained.

2.2 SFE apparatus

The SFE apparatus was described in detail in a previous
work [25]. The apparatus is composed of a diaphragm
pump, an extraction vessel (1 L) and two separators (0.27 L),
operating in series and allowing a fractional separation. The
pressure of the extractor is regulated by a back-pressure
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

After the HD, the plant residue was dried and about 5 g was
subjected to a Soxhlet extraction with pentane p.a. (Prolabo,
Paris, France), for 5 h, to obtain the nonvolatile components.
With the aim of comparing with HD followed by Soxhlet
extraction, after the SFE at 90 bar, the same plant matrix
(100 g) was submitted to an extraction at 250 bar, 401C, and
1.1 kg/h of CO2 flow rate, for 4 h.

2.5 GC
Quantitative analyses of the volatile and essential oils were
performed in a Hewlett-Packard 5890 gas chromatograph
(HP, Waldbronn, Germany), equipped with a flame ionization detector and a fused-silica DB-5 capillary column (J&W
Scientific; 30 m  0.25 mm id, film thickness 0.25 mm;
Folsom, CA, USA). Oven temperature was programmed
isothermally to 401C, during 2 min, subsequently at 31C/
min to 2301C, and finally increased at 51C/min to 3101C and
held isothermally for 15 min; injector and detector temperatures, 3101C; carrier gas, helium, adjusted to a linear
velocity of 24 cm/s. The samples were injected using a split
ratio 1:50. The volume of injection was 0.1 mL. The
percentage composition of the oils was computed by the
normalization method from the GC peak areas without
using response factors.
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Other Techniques

J. Sep. Sci. 2010, 33, 22112218

2.6 GC-MS
The GC-MS unit consisted of a Perkin Elmer Autosystem
XL gas chromatograph (Perkin Elmer, Shelton, CT, USA)
equipped with a DB-1 fused-silica column (30 m  0.25 mm
id, film thickness 0.25 mm; J&W Scientific, Agilent Technologies, Santa Clara, CA, USA), interfaced with a PerkinElmer Turbomass mass spectrometer (software version 4.1;
Perkin-Elmer). Oven temperature was programmed from 45
to 1751C, at 31C/min, subsequently at 151C/min up to
3001C, and then held isothermal for 10 min; injector
temperature, 2801C; transfer line temperature, 2801C; ion
trap temperature, 2201C; carrier gas, helium, adjusted to a
linear velocity of 30 cm/s; split ratio, 1:40; ionization energy,
70 eV; ionization current, 60 mA; scan range, 40300 u; and
scan time, 1 s.
The identity of the components was assigned by
comparison of their retention indices, relative to C9C36
n-alkanes indices (Supelco, Bellefonte, PA, USA) and
GC-MS spectra from a homemade library, constructed
based on the analyses of reference oils, laboratory-synthesized components, and commercially available standards.

2213

determined according to NP EN ISO 660 [29] and the

peroxide value was obtained with AOAC 965.33 [30].
The fatty acid composition, the percentages of free fatty
acids, of saturated fatty acids and of unsaturated fatty acids
were determined, respectively, according to the methods
described in NP EN ISO 660 [29], EN ISO 5508 [31] and EN
ISO 5509 [32]. For the GC analysis, a Varian 3300 chromatograph (Walnut Creek, CA, USA) equipped with a flame
ionization detector and a Supelcowax 10 column
(30 m  0.32 mm id; film thickness 0.25 mm) was used.
Helium was used as the carrier gas and the injector and
detector temperatures were kept at 200 and 2501C, respectively. The oven temperature was maintained at 2001C.

2.9 Statistical analysis

All experiments were carried out in triplicate. Mean values
of PF were compared with the respective controls and
separated according to t-test at the 0.05 probability level.

3 Results and discussion

2.7 Antioxidant activity

3.1 Composition of the essential and the volatile oils

The antioxidative property of T. vulgaris volatiles was

assessed by the Rancimat test. The measurements were
performed with a homebuilt Rancimat and to validate the
obtained results, the control assay was also done with a
commercial Rancimat (Rancimat 679 apparatus, Metrohm
AG, Schweiz, Switzerland).
After the validation, the protective effect of the essential
and the volatile oil on sunflower oil (Diasol, Madrid, Spain)
was performed using the following conditions. The vessel
containing the sample (9.9 g of oil 1 0.1 g of extract) was
heated at 1201C and compressed air (Air Liquide) with a
flow rate of 20 L/h was bubbled through it. The volatile
compounds formed were collected in the water vessel and
the increase of water conductivity was measured. The time
necessary to achieve the conductivity value that corresponds
to the inflection point of the oxidation curve was considered
as the induction period (IP, h). The protector factor (PF) is
defined as the ratio between the IP of each sample and the
IP of the control (sample without plant volatiles) [26].
To predict the shelf life at 201C of the sunflower oil
used, assays at different temperatures (from 110 to 1601C)
were performed. The IP(201C) was calculated from the plot of
the log10(IP) versus temperature [27].

The compositions of the volatile oils (SFE) and essential oils

(HD) isolated from T. vulgaris, obtained under the different
extraction conditions, are summarized in Table 1 considering the order of the components elution on a DB-5 column.
Fifty-two components were identified and both extracts
were richer in p-cymene (10.042.6% for SFE and
28.934.8% for HD), g-terpinene (0.86.9% for SFE and
5.17.0% for HD), linalool (2.35.3% for SFE and 2.83.1%
for HD), thymol (19.540.8% for SFE and 35.441.6% for
HD), and carvacrol (1.43.1% for SFE and 2.63.1% for
HD). The broader range of relative percentages observed for
SFE is due to different conditions of pressure (90 bar versus
100 bar) and temperature (401C versus 501C).
The main difference between the essential and the
volatile oil composition is the amount of two components.
Only the volatile oil contains thymoquinone (2.06.2%) and
the essential oil is richer in carvacryl methyl ether (1.01.2%
for HD versus t0.4 for SFE).

2.8 Chemical composition of the sunflower oil

Different tests were performed to characterize the sunflower
oil used in this study.
The iodine value was obtained using the method
described by the EN 14111 [28]; the acidity value was
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3.2 Influence of the pressure and the temperature

The first step in SFE is to choose the working pressure and
temperature that allow obtaining the highest recoveries of
volatile oils, compared with HD or SD, while, at the
same time, the coextraction of unwanted high molecular
mass compounds, such as fatty acids, is avoided. Several
plants belonging to Lamiaceae family have already been
submitted to SFE, being the best conditions of pressure in
the range of 90100 bar and temperatures from 40 to 501C
[25, 3339].
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RIa)
1009
1016
1027
1059
1063
1081
1081
1093
1100
1108
1117
1122
1122
1124
1146
1157
1166
1173
1174
1201
1202
1210
1254
1279
1292
1299
1305
1371
1371
1381
1407
1412

Components

a-Thujene
a-Pinene
Camphene
1-Octen-3-ol
b-Pinene
3-Octanol
b-Myrcene
a-Phellandrene
D-3-Carene
a-Terpinene
p-Cymene
1,8-Cineole
b-Phellandrene
Limonene
trans-b-Ocimene
g-Terpinene
trans-Sabinene hydrate
cis-Linalool oxide
trans-Linalool oxide
cis-Sabinene hydrate
Nonanol
Linalool
Camphor
Borneol
Terpinen-4-ol
cis-Dihydrocarvone
a-Terpineol
Thymoquinone
Thymyl methyl ether
Carvacryl methyl ether
Geraniol
Thymyl formate

Flow rate (kg/h)

0.5
0.8
0.7
0.2
0.9
0.4
0.4
0.1
0.1
0.9
34.7
0.3
0.3
0.7
t
7.0
0.5
0.1
0.1
0.2
t
2.9
0.7
1.1
0.8
0.2
0.2
0.2
1.0
0.1
0.3

0.4
0.7
0.6
0.2
0.7
0.3
0.3
0.1
0.1
0.7
28.9
0.3
0.3
0.6
t
5.1
0.5
0.1
0.1
t
t
3.1
0.8
1.2
0.8
0.3
0.2
0.2
1.2
0.1
0.3

0.2
0.3
0.3
t
0.9
0.2
0.2
t
0.2
0.5
24.4
0.3
0.3
0.7
t
2.5
1.1
0.2
0.2
0.3
t
4.0
0.9
1.2
0.7
0.3
0.2
6.2
t
t
0.2
t

0.1
0.3
0.3
0.1
0.8
0.2
0.2
0.1
t
0.4
22.8
0.2
0.2
0.5
t
2.9
1.2
0.2
t
0.4
t
4.0
0.9
1.4
0.8
0.3
0.3
5.2
t
0.2
0.2
0.3

0.8

0.6

40

90

0.2
0.4
0.4
0.2
0.7
0.2
0.2
0.1
t
0.5
28.6
0.2
0.2
0.6
t
4.1
1.2
0.1
0.1
0.3
t
3.6
0.9
1.2
0.7
0.3
0.2
3.5
t
t
t
0.2

1.1

0.6

40

90

0.2
0.4
0.4
0.2
0.9
0.3
0.3
0.2
t
0.3
26.2
0.2
0.2
0.6
t
3.9
0.7
0.2
0.1
t
t
3.7
t
1.3
0.8
0.1
0.2
3.2
t
t
t
0.1

1.3

0.6

40

90

SFE

0.2
0.4
0.4
0.1
0.8
0.2
0.2
0.2
0.1
0.4
29.3
0.2
0.2
0.6
0.1
3.6
0.8
0.2
0.1
t
t
3.8
0.4
1.2
0.8
0.3
0.3
3.4
t
t
t
0.2

1.1

0.8

40

90

0.3
0.4
0.4
0.3
1.4
0.4
0.4
0.2
0.1
0.7
42.6
0.4
0.4
0.9
0.1
6.9
0.8
0.3
t
0.2
t
5.3
t
1.2
0.7
0.7
0.1
3.2
t
0.4
t
0.1

1.1

0.6

50

90

tb)
t
t
0.2
t
0.1
0.1
0.1
0.1
0.4
10.0
1.9
1.9
0.3
t
0.8
0.6
0.3
0.3
0.1
t
2.3
0.5
0.7
0.6
0.3
0.4
2.6
t
t
t
0.2

1.1

0.6

40

100

C. Grosso et al.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0.2
1.2
t
0.3

0.5
0.9
0.8
0.2
0.9
0.4
0.4
0.1
0.1
0.7
34.8
0.3
0.3
0.6
t
5.5
0.5
0.1
0.1
0.2
t
2.8
0.8
1.1
0.8
0.2
0.2

1.1

0.4

0.8

Particle size (mm)

0.6

40

Temperature (1C)
0.4

90

HD

Pressure (bar)

Extraction method

size (0.4, 0.6, and 0.8 mm), and flow rate (0.8, 1.1, and 1.3 kg/h)

Table 1. Yield and percentage composition of T. vulgaris volatiles obtained by HD and SFE at different conditions of pressure (90 and 100 bar), temperature (40 and 501C), mean particle

2214
J. Sep. Sci. 2010, 33, 22112218

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& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1419
1431
1483
1507
1551
1551
1561
1570
1585
1606
1616
1642
1647
1654
1666
1684
1707
1750
1767
1792

Thymol
Carvacrol
Bornyl proprionate
b-Bourbonene
2-Methoxy-4-ethyl-6-methylphenolc)
trans-b-Caryophyllene
b-Copaene
a-Humulene
g-Muurolene
Germacrene D
a-Muurolene
g-Cadinene
trans-Calamenene
D-Cadinene
a-Calacorene
Geranyl butirate
b-Caryophyllene oxide
10-epi-g-Eudesmol
epi-a-Cadinol
a-Cadinol
Identified components (%)
Monoterpene hydrocarbons
Oxygen-containing monoterpenes
Sesquiterpene hydrocarbons
Oxygen-containing sesquiterpenes
Others
Yield % w/w

35.4
2.6
t
0.1
1.0
1.0
0.2
0.1
0.2
t
t
0.1
0.3
0.3
t
0.8
t
0.1
0.1
98.7
47.8
47.2
2.3
1.0
0.4
0.9

41.6
3.1
t
0.1
1.2
1.2
0.2
0.1
0.3
0.1
0.1
0.1
0.3
0.4
t
1.3
t
0.1
t
98.4
39.0
54.8
2.9
1.4
0.3
1.1

a) RI retention indices relative to C9C18 n-alkanes on DB-5 column.

b) t trace (o0.05%).
c) Identification based on mass-spectra only.
Values represent means of three samples.

RIa)

Components

Flow rate (kg/h)

t
1.4
0.1
0.3
0.1
98.7
47.5
46.2
2.6
1.9
0.4
0.8

35.4
2.8
t
0.1
1.0
1.0
0.2
0.1
0.2
0.1
0.1
0.1
0.3
0.4

36.3
2.6
t
0.2
1.6
1.6
2.0
t
0.3
0.2
0.2
0.2
0.3
0.5
t
t
1.6
0.2
0.3
t
94.6
31.9
54.9
5.5
2.1
0.2
1.1

1.1

0.4

0.8

Particle size (mm)

0.6

40

Temperature (1C)
0.4

90

HD

Pressure (bar)

Extraction method

Table 1. Continued

38.7
2.6
0.2
0.2
1.4
1.4
2.3
t
0.3
t
t
0.1
0.4
0.5
t
t
1.4
0.1
0.3
0.1
94.5
30.2
57.0
5.2
1.9
0.2
0.8

0.8

0.6

40

90

36.8
2.6
0.1
0.2
1.4
1.4
1.4
0.1
0.4
0.1
0.1
0.1
0.4
0.5
t
t
1.5
0.1
0.3
0.1
96.5
37.5
52.1
4.7
2.0
0.2
0.8

1.1

0.6

40

90

39.7
3.0
0.1
0.2
1.5
1.5
2.6
0.2
0.3
0.1
0.1
0.1
0.3
0.6
t
t
1.7
0.1
0.5
0.1
97.4
34.3
54.4
6.0
2.4
0.3
0.8

1.3

0.6

40

90

SFE

36
3.3
t
0.1
1.4
1.4
2.5
0.2
0.4
0.1
0.2
0.2
0.5
0.6
t
t
1.9
0.1
0.4
0.1
97.9
37.3
51.7
6.2
2.5
0.2
0.7

1.1

0.8

40

90

19.5
1.4
t
0.2
1.2
1.2
1.8
0.1
0.1
0.1
0.1
0.1
0.2
0.3
t
t
0.5
t
0.1
t
95.8
55.8
34.8
4.2
0.6
0.4
0.4

1.1

0.6

50

90

40.8
3.1
t
0.1
1.1
1.1
3.0
0.1
0.4
0.1
0.2
0.1
0.4
0.5
0.1
t
1.4
t
0.4
0.2
77.9
14.4
55.3
6.1
2.1
0.1
1.1

1.1

0.6

40

100

J. Sep. Sci. 2010, 33, 22112218

Other Techniques
2215

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2216

J. Sep. Sci. 2010, 33, 22112218

C. Grosso et al.

The effect of pressure and temperature and therefore

of density of CO2 influenced the yield and composition of
T. vulgaris SFE volatile oil. However, the effect of temperature was more evident than that of the pressure. When the
pressure was increased by 10 bar (from 90 to 100 bar, at
401C), the extraction yield slightly increased from 0.8 to
1.1% (kg/kg d.w.), while the change of temperature from 40
to 501C (at 90 bar), which corresponded to a density decrease
from 485.5 to 285.0 kg/m3, induced a decline from 0.8 to
0.4% (Fig. 1). Moreover, the volatiles obtained at 100 bar/
401C are richer in oxygen-containing monoterpenes, while
those isolated at 90 bar/501C are dominated by monoterpene hydrocarbons (Table 1).
Dapkevicius et al. [20] extracted the volatiles from aerial
flowering parts at higher densities than in the present study
and the extraction yield was close to that observed for HD
(5.5% for SFE and 6.3% for HD). As mentioned above, at
pressures of 300 bar, the volatile fraction is usually
contaminated with heavier compounds and therefore, it was
expected that SFE yield was higher for SFE than for HD.
However, the volatiles were collected in methanol and the
subsequent evaporation step could lead to the loss of some
components, and consequently, to the decrease of the
extraction yield. On the other hand, Simandi et al. [23]
obtained higher extraction yields for Soxhlet extraction with
ethanol (12.3%) and SFE at 400 bar and 601C, r 5 890.1 kg/
m3 (4.9%), than for HD (2.2%). These results showed that
apart from the effect of the density, the solvent polarity is
also of great importance. In fact, since ethanol is a polar
solvent, the polar components from T. vulgaris can also be
extracted.

3.3 Influence of the particle size

For the lowest particle size (0.4 mm), the extraction yield
was higher (1.1%) (Fig. 2), because during the milling

Figure 1. Yield of the SFE volatile oil extracted as a function of

time under different conditions of pressure and temperature (~
90 bar/401C; m 90 bar/501C; & 100 bar/401C). Mean particle
size and flow rate were 0.6 mm and 1.1 kg/h, respectively.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

process the cuticle of the peltate trichomes of T. vulgaris [40]

is ruptured and the oil is released into the surroundings,
facilitating its solubility in supercritical CO2. However, for
practical reasons, the mean particle size of 0.6 mm was
chosen for the subsequent extractions.

3.4 Influence of the flow rate

Three different flow rates were tested for the mean particle
size of 0.6 mm: 0.8, 1.1, and 1.3 kg/h at 90 bar and 401C
(Fig. 3). Within this range, the maximum yield is achieved
(about 0.8%), showing that no deviation from the plug flow
pattern occurs. This means that the equilibrium was
achieved in the first part of the extraction, for all the flow
rates, and the contact time between the CO2 and the plant
matrix, in the second part, was enough to dissolve all the
available solute.

3.5 Antioxidant activity

From the studies on the influence of pressure, temperature,
mean particle size, and CO2 flow rate, the extraction
conditions of 90 bar, 401C, 0.6 mm, and 1.1 kg/h were
chosen. The comparative study of the antioxidant activity,
assessed by Rancimat test, between the volatile oil obtained
using the above-mentioned conditions and the essential oil
extracted by HD, using the same mean particle size, was
performed. The validation of the in-homebuilt Rancimat
was performed by comparing the IP of the control
(sunflower oil), using the commercial and the in-homebuilt
apparatuses. The obtained values were, respectively,
4.06 7 0.16 and 3.94 7 0.23. As our method was validated,
all the subsequent measurements were performed using the
homebuilt apparatus.

Figure 2. Yield of the SFE volatile oil isolated as a function of

time under different mean particle sizes (& 0.4 mm; ~
0.6 mm; m 0.8 mm). Pressure, temperature, and flow rate were
90 bar, 401C, and 1.1 kg/h, respectively.

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Other Techniques

J. Sep. Sci. 2010, 33, 22112218

Figure 3. Yield of the SFE volatile oil extracted as a function of

time under different conditions of flow rate (& 0.8 kg/h; ~
1.1 kg/h; m 1.3 kg/h) and using a pressure of 90 bar, a
temperature of 401C, and a mean particle size of 0.6 mm.

Figure 4. IP of the sunflower oil, with and without plant volatiles,

measured by Rancimat test. SO sunflower oil (control); A HD;
B 90 bar/401C. The difference between B and SO is statistically
significant (po0.001).

From Fig. 4, it can be seen that the SFE extract has

the highest IP and protector factor (IP 5 6.66 7 0.26 h;
PF 5 1.63 versus IP 5 4.1070.16 h; PF 5 1 of the control;
po0.001), whereas the essential oil presented a weak antioxidant activity (IP 5 4.4370.18; PF 5 1.08; difference not
statistically significant). Using the b-carotene bleaching test,
Dapkevicius et al. [20] also observed a higher antioxidant
activity for the extract obtained by SFE at 300 bar and 401C
and for the acetone oleoresin than for the hydrodistilled
essential oil. However, as pointed out above, with the
conditions employed by these authors, not only the volatile
fraction is isolated.
To assess whether the nonvolatile fraction also showed
some degree of activity, in the current study, the deodorized
fractions were also tested. The corresponding IP of the
deodorized SFE extracts isolated at 250 bar and 401C were
2.7570.11 h (first separator) and 3.5970.14 h (second
separator) and for Soxhlet extract, 2.9670.12 h. These
results strengthen the idea that it is the volatile fraction that

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2217

contains the active compounds. In fact, Lee et al. [12]

isolated the volatiles by SD at reduced pressure from thyme
leaves and observed that thymol and carvacrol, amounting to
72 and 5.7% in the essential oil, inhibited the oxidation of
hexanal to hexanoic acid in a similar way than a-tocopherol
and butylated hydroxytoluene. Moreover, Schwarz et al. [22]
tested some isolated compounds by Rancimat test at 1001C,
with an air flow of 15 L/h using lard and found protector
factors between 1.7 (carvacrol and thymol) and 10.6
(p-cymene-2,3-diol).
Some authors have suggested that one of the disadvantages of Rancimat test and other accelerated tests that
use high temperatures is that they can lead to the loss of the
more volatile components [41]. If it is true, it can be
speculated that at room temperature the protector factor of
the volatile fraction is even higher. However, in this particular case, the values obtained for 1201C and those reported
by Schwarz et al. [22] and Simandi et al. [23] at 1001C are
similar, showing that the degree of thermodegradation is
low.
The predicted shelf life of the sunflower oil used, at
room temperature (201C), corresponds to about 35 days.
Regarding its composition (Table 2), it can be seen that the
percentage of linoleic acid is higher than that of oleic acid,
therefore justifying the lowest IP (4.1070.16 h at 1201C).
rquez-Ruiz et al. [42] tested two different sunflower oils,
Ma
one conventional high linoleic oil (21.0% of C18:1 and
67.7% of C18:2) and one genetically modified high oleic oil
(72.4% of C18:1 and 16.8% of C18:2) and they observed that
the higher the oleic acid percentage, the longer was the IP
(7.4 against 20.1 h, at 1001C) [42].

Table 2. Chemical composition of the sunflower oil

Sunflower oil
Acid value (mg KOH/g)
Peroxide value, POV (mEq peroxide/kg)
Iodine value (g I2/100 g)
Free fatty acid value (as oleic acid %)
Fatty acid composition (% w/w)
Saturated fatty acids
Unsaturated fatty acids
C14:0
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
C20:0
C20:1
C22:0
C24:0
Others
MUFA/PUFAb) ratio

0.073
11.2
127
0.037
9.66
89.81
0.01
5.75
0.10
3.03
31.01
58.58
0.12
0.20
n.d.a)
0.67
n.d.
0.53
0.5:1

a) n.d. not determined.

b) Monounsaturated fatty acids/polyunsaturated fatty acids.

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2218

C. Grosso et al.

J. Sep. Sci. 2010, 33, 22112218

4 Concluding remarks

[16] Hammer, K. A., Carson, C. F., Riley, T. V., J. Appl.

Microbiol. 1999, 86, 985990.

Different conditions of pressure, temperature, mean particle

size, and CO2 flow rate were tested to choose the best SFE
conditions that allowed obtaining a volatile oil with a yield
and composition similar to that isolated by HD, which was
at 90 bar, 401C, 0.40.6 mm of mean particle size, and
1.1 kg/h of CO2 flow rate. Both volatile and essential oils are
dominated by p-cymene (8.842.6% for SFE and 28.934.8%
for HD), g-terpinene (0.56.9% for SFE and 5.17.0% for
HD), linalool (2.75.3% for SFE and 2.83.1% for HD),
thymol (19.546.1% for SFE and 35.441.6% for HD), and
carvacrol (1.43.4% for SFE and 2.63.1% for HD). The
major differences between both the extract compositions,
which can explain the different antioxidant activities
observed, are the amounts of thymoquinone (only present
in SFE 2.06.2%) and carvacryl methyl ether (1.01.2% for
HD versus t 0.4 for SFE).

[17] Dorman, H. J. D., Deans, S. G., J. Appl. Microbiol. 2000,

88, 308316.

The authors have declared no conflict of interest.

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