Mutation Research 629 (2007) 100–110

Investigation of the mutagenic and antimutagenic effects of

Origanum compactum essential oil and some of its constituents
N. Mezzoug a , A. Elhadri a , A. Dallouh a , S. Amkiss a , N.S. Skali a , J. Abrini a ,
A. Zhiri b , D. Baudoux b , B. Diallo c,d , M. El Jaziri c,d , M. Idaomar a,∗
a Laboratory of Biology and Health, Faculté des Sciences, Université Abdelmalek Essaâdi BP 2121,
93002 Tétouan, Morocco
b S.A. PRANAROM International, 37 Avenue des Artisans, B-7822 Ghislenghien, Belgium
c Laboratory of Plant Biotechnology, Université Libre de Bruxelles, Belgium
d BIOVALLEE, Rue Adrienne Bolland 8,

B-6041 Gosselies, Belgium

Received 22 April 2006; received in revised form 4 December 2006; accepted 25 January 2007
Available online 1 March 2007

Abstract
In the present study, the chemical composition of Origanum compactum essential oil was determined by gas chromatography and
mass spectrometry, and its mutagenic and antimutagenic activities were investigated by the somatic mutation and recombination
test (SMART) in Drosophila melanogaster. No significant increase in the number of somatic mutations was observed with the
essential oil tested using both the standard (ST) and high bio-activation (HB) cross. In order to investigate the antimutagenic effect
of the essential oil, we have tested the effect on the indirect-acting mutagen urethane (URE), as well as the direct-acting mutagen
methyl methanesulfonate (MMS). O. compactum essential oil showed a strong inhibitory effect against URE-induced mutagenicity,
especially with the HB cross. However, only a weak inhibitory effect on the mutagenicity induced by MMS was observed. These
results suggest that the detected antimutagenicity could be mediated by an inhibitory effect on metabolic activation. The essential
oil was fractionated to identify the components responsible of the suppressing effect detected. Seven fractions were obtained: two of
them showed the most potent inhibitory effect against URE-induced mutagenicity and were further fractionated. The sub-fractions
obtained from the second chromatographic fractionation were tested for their antimutagenic activity, together with carvacrol and
thymol. The highest antimutagenic effect obtained with the sub-fractions was similar to the effect of the crude essential oil, as well
as to the effect of carvacrol alone. These results suggest the absence of a synergic antimutagenic effect between the components of
O. compactum essential oil and indicate that carvacrol was the most active oil component.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Antimutagenicity; Mutagenicity; Origanum compactum essential oil; SMART assay; Chromatographic fractionation

1. Introduction

Recently, cancer chemoprevention has become a

∗
promising alternative to control cancer, now that a large
Corresponding author at: Département de Biologie, Faculté des
Sciences, Université Abdelmalek Essaâdi BP 2121, 93002 Tétouan,
number of modulating factors including antimutagens
Morocco. Tel.: +212 39 97 24 23; fax: +212 39 99 45 00. and anticarcinogens have been widely studied to assess
E-mail address: idaomar@gmail.com (M. Idaomar). their ability to suppress or prevent the process of car-

1383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2007.01.011
 N. Mezzoug et al. / Mutation Research 629 (2007) 100–110
cinogenesis. It has been accepted that plants and their
products represent one of the main sources for com-
pounds with chemopreventive potential and, indeed,
several secondary plant metabolites have demonstrated
chemopreventive activity [1].
Essential oils are isolated from many plants by
mechanical pressing or hydro- and steam-distillation.
They are complex mixtures of odorous and volatile
compounds from secondary plant metabolism: monoter-
penes, sesquiterpenes, alcohols, ethers, aldehydes, esters
and ketones are the main constituents of these mixtures.
The essential oils are often responsible for a plant’s dis-
tinctive scent or taste and are widely used as flavour
enhancers in many food products and as odorants in
fragrances [2]. Moreover, the essential oils and their
ingredients are lipophilic compounds that are able to
cross cell membranes and therefore absorbed through the
skin [3], which is the reason why they have a long his-
tory of use for many medical applications [4]. Nowadays,
the essential oils have been shown to possess a range of
biological activities including antibacterial, antioxidant,
antifungal and insecticidal activity [5–7]. However, just
a few of the essential oils have been studied for their
antimutagenic activity [8].
All over the world, the Origanum genus was the
most commonly found among oregano species. Forty-
nine taxa divided into 10 sections belong to this genus,
most of them having a very local distribution around
the Mediterranean [9]. Origanum compactum with very
compact pinkish flowers is the endemic oregano in
Morocco. In general, the oregano spices are widely
used to enhance the flavour of many different foods,
but O. compactum for its part is used as herbal rem-
edy in folk medicine, especially as infusion for many
purposes [10]. No published data are available on the
mutagenicity and antimutagenicity of O. compactum oil
and only a few studies with some of its components
have been performed. Carvacrol was suggested to be
antimutagenic against 4-nitro-o-phenylenediamine and
2-aminofluorene using the Ames test [11]. Carvacrol is
also able to prevent mutagenicity of some known muta-
gens in human lymphocytes by inhibiting chromatid
exchange [12]. The thymol together with the carvacrol
␥-terpinene were reported for their protective effect
against 2-amino-3-methylimidazo[4,5-f]-quinoline and
mitomycin C using the comet assay [13].
In this study, we first evaluated the mutagenic activity
of O. compactum oil and then investigated the antimu-
tagenic potential of the crude essential oil against a
promutagen and a direct alkylating agent using the
somatic mutation and recombination test in Drosophila
melanogaster. To identify the components responsible
101
of the detected antimutagenic activity, the Origanum
essential oil was separated into a series of fractions and
each fraction was evaluate for its antimutagenic activ-
ity. A second fractionation was performed for fractions
that had demonstrated the highest level of antimutagenic
activity. Determination of the chemical composition of
the essential oil as well as the series of the fractions
obtained was elaborated by gas chromatography and gas
chromatography–mass spectrometry. Finally, we evalu-
ated the antimutagenic activity of the major constituents
of the most potent fraction, mainly carvacrol and thy-
mol, to compare the antimutagenic activity of the crude
Origanum essential oil to that of its constituents.
2. Materials and methods
2.1. Compounds and plant material
The Origanum was collected from the north of Morocco,
only flowering tops of the plant were used. Oil of O.
compactum was produced by PRANAROM International
Company, Belgium. Urethane (URE) [CAS no. 51-79-6] and
methyl methanesulfonate (MMS) [66-27-3] as well as car-
vacrol [499-75-2], thymol [89-83-8], pentane, HPLC grade
[109-66-0] and diethyl ether [60-29-7] were purchased from
Sigma Aldrich. Acetone [67-64-1] was from Riedel-deHaën.
2.2. Extraction of the essential oil and identification of its
constituents
The essential oil was extracted by hydro-distillation. This
was performed at low pressure without chemical descalers.
The essential oil analyses were carried out by GC–MS using a
Hewlett-Packard GCD system. An HP-INNOWAX capillary
column (60 m × 0.25 mm, 0.5 ␮m film thickness) was used
with helium as carrier gas with flow 22–25 psi. The GC oven
temperature was held at 50 ◦ C for 6 min, and programmed to
increase by 2 ◦ C/min till 250 ◦ C and then held at this temper-
ature for 20 min. The injector and detector temperatures were
250 and 280 ◦ C, respectively, injection was in split mode, and
the volume injected was 1 ␮l of a 5/100 solution of the oil in
hexane. Automatic calibration of the masses by auto-tuning
was used in MS. The mass range was from m/z 30 to 350. A
library search was carried out using the combination of the
NBS library with 75,000 spectra provided with the software
of interpretation of the spectra from NIST SCIENTIFIC and a
personal aromatic library.
2.3. Fractionation of O. compactum essential oil
Column chromatography was used for the fractionation of
the essential oil. Ten gram of the O. compactum essential oil
was fractionated using a column (1.5 cm× 500 cm) packed with
100 g of silica gel 60, equilibrated with pentane. The col-
umn was eluted successively with 250 ml each of pentane,
102 N. Mezzoug et al. / Mutation Research 629 (2007) 100–110
pentane–diethyl ether (75/25), pentane–diethyl ether (50/50),
pentane–diethyl ether (25/75), diethyl ether (1/1), acetone and
methanol. The organic solvents were removed from the eluates
by evaporation under reduced pressure to get seven fractions
(F1–F7). Two of these fractions (F2 and F6) showed a sig-
nificant antimutagenic activity and were further fractionated.
The first fraction was subjected to column chromatography
under the same conditions as described above. Seventy sub-
fractions were collected and pooled into seven after analysis
by thin-layer chromatography (TLC). This TLC analysis was
achieved using silica gel plates with pentane–diethyl ether
(95/05) as the mobile phase. The chromatogram was revealed
by spraying with vanillin/sulfuric acid reagent and subse-
quent heating at 105 ◦ C. The second fraction was fractionated
by preparative TLC, six sub-fractions were obtained using
pentane–diethylether (60/40) as the mobile phase.
2.4. Mutagenicity and antimutagenicity assay
2.4.1. Drosophila stocks and crosses
The following two crosses of flies were used for the
study of the mutagenic and antimutagenic activity of the
Origanum essential oil: (1) standard cross (ST): mwh/mwh
females mated to flr3/In (3LR) TM3, ri pp sep l(3)89Aa
bx34e e Bds (flr3/TM3) males, (2) high bio-activation (HB)
cross: NORR/NORR; flr3/In (3LR) TM3, ri pp sep l(3)89Aa
bx34e e Bds (flr3/TM3) females mated to NORR/NORR;
NORR/NORR; mwh/mwh males. NORR strains (New ORR)
have chromosomes 1 and 2 from a DDT-resistant strain, which
are responsible for a high constitutive level of cytochrome P450
[14]. The mwh strain is homozygous for the wing-cell marker
multiple wing hairs (mwh, 3–0.3). The flr3/TM3 strain con-
tains the wing-cell marker allele flare3 (flr3 , 3–38.8) and the
balancer chromosomes TM. For the antimutagenic evaluation
of the fractions obtained from the essential oil, only the ST
cross was used.
2.4.2. Experimental procedures
Test compounds were administered to Drosophila larvae
in food. The essential oil of O. compactum was dissolved in
Tween 80. The oil was administered to larvae at three concen-
trations, 0.005, 0.1 and 0.2%. Each of the different fractions
was administered at two concentrations. F1, F2, F3, F6 and F7
were dissolved in 2% Tween 80. However, 2% Tween 80 + 10%
ethanol was used as solvent for fractions F4 and F5, as well
as the fractions obtained from F2 and F6. The same solvent
was used for carvacrol and thymol. The solutions were always
freshly prepared, immediately before use. The solvent alone
was used as negative control. Urethane and MMS were tested
as positive controls and were dissolved each time in the solvent
used in the treated series.
Eggs from the crosses were collected for 8 h in culture bot-
tles containing live fermenting yeast. After 72 ± 4 h, the larvae
were collected off the food with a 20% NaCl solution and
transferred to individual vials containing 1.5 g of food pre-
pared from Drosophila Instant Medium (Carolina Biological
Supply) hydrated with 5 ml of the test solutions at different
concentrations.
Essential oil solution, mutagen and a combination of the
mutagen with each of the Origanum oil, each of the differ-
ent fractions obtained as well as with carvacrol and thymol
were used for chronic treatment. The larvae were fed on this
medium for the rest of their development, during approximately
48 h. Negative solvent controls were included in all treatments.
All experiments were conducted at 25 ◦ C and 65% relative
humidity. The adult flies were collected and flies of the trans-
heterozygous (mwh flr + /mwh + flr) genotype were stored in
70% ethanol. The wings of adult flies were mounted on slides
and scored under 400× magnification for the presence of spots.
The spots were recorded according to standard procedures [15].
2.5. Data evaluation and statistical analysis
For the evaluation of the mutagenic activity of O. com-
pactum essential oil, the frequencies of spots per wing obtained
with different concentrations of the essential oil were com-
pared with the negative control. The treated series were
compared with the concurrent positive control for the evalu-
ation of the mutagenic activity. These statistical comparisons
were done with a programme that employs the chi-square
test. Accordingly, we distinguished small single spots (one
or two cells affected), large single spots (more than two cells
affected) and twin spots. The percentage of genotoxicity inhi-
bition was calculated based on the spot frequencies according
to the formula: [(mutagen alone − (essential oil or oil frac-
tion))/mutagen alone] × 100 [16].
3. Results
The different constituents of the O. compactum essen-
tial oil were identified and quantified by GC and GC–MS.
The chemical composition of the essential oil is pre-
sented in Table 1. The essential oil was particularly rich
in carvacrol 22%, ␥-terpinene 22%, thymol 19% and
p-cymene 13%.
The Origanum essential oil was first analyzed for the
evaluation of its genotoxic/mutagenic activity. The num-
bers of small spots, large spots and twin spots together
with the total number of spots are given in Table 2.
Three concentrations of the Origanum oil were tested.
The concentrations chosen are not toxic according to
progeny mortality relative to the control. Negative con-
trols showed that the number of spontaneous spots per
wing is significantly different between the ST and HB
cross. The frequency of spots obtained with ST was
higher when compared with the HB cross because the
solvent used for ST cross was Tween 80 at 2%, which
was responsible of the increase of the frequency of the
spontaneous mutations, while only 0.1% of Tween 80
was used for the HB cross to reduce the number of muta-
 N. Mezzoug et al. / Mutation Research 629 (2007) 100–110 103

Table 1
Chemical composition of Origanum compactum essential oil
Constituentsa % Constituentsa %

Methyl butanal 0.08 Linalol 2.91

␣-Pinene 1.15 cis-Thuyanol 0.12
␣-Thuyene 1.51 Terpinene-4-ol 0.91
Camphene 0.20 ␤-Caryophyllene 3.47
␤-Pinene 0.25 cis-Dihydrocarvone 0.20
3-carene 0.16 Pulegone 0.45
␤-Myrcene 2.58 ␣-Humulene 0.22
␣-Phellandrene 0.40 Neral 0.14
␣-Terpinene 3.29 ␣-Terpineol 0.56
Limonene 0.51 Borneol 0.38
1.8-Cineole 0.25 Geranial 0.12
␤-Phellandrene 0.36 ␦-Cadinene 0.14
␥-Terpinene 22.90 p-Cymene-8-ol 0.16
3-Octanone 0.25 Piperitenone 0.05
p-Cymene 13.26 Caryophyllene oxide 0.14
Terpinolene 0.20 Thymol isomer 0.11
␣,p-Dimethylstyrene 0.54 Thymol 19.36
trans-Thuyanol 0.21 Carvacrol isomer 0.23 Fig. 1. Inhibitory effect of the Origanum compactum essential oil on
Camphre 0.23 Carvacrol 22.00 the somatic mutations induced by MMS at 0.5 mM and URE at 10 mM
using the ST cross in addition of URE at 5 mM using HB cross.
a The constituents of the Origanum compactum essential oil were
identified and quantified by GC and GC–MS.
a direct-acting genotoxicant. Under our experimental
tions induced in the absence of the essential oil. Both conditions, both URE and MMS showed to be clearly
with the ST and the HB crosses, O. compactum essential mutagenic (Table 3 and Fig. 1). URE increased the fre-
oil, at the concentrations tested, did not show any effect quency of induced spots from 0.84 spots per wing in the
on the frequencies of somatic mutations when compared control to 2.25 spots per wing when it was administered
with their respective negative controls. A slight increase at 10 mM using the larvae from the ST cross. Approxi-
in the total number of spots was noticed with 0.05% of mately the same frequency of mutations per wing (2.29)
the Origanum essential oil in HB cross, but this was not was obtained with only 5 mM using the HB cross. For its
significant. part, MMS at 0.5 mM in the ST cross showed a highly
The evaluation of the antimutagenic potential of the significant increase in induced somatic mutations (8.15
essential oil from O. compactum was established by spots per wing) compared with the negative control (0.84
means of a cotreatment protocol in which the essential spot per wing).
oil was administered simultaneously with the mutagens The essential oil from O. compactum displayed sig-
used: URE, an indirect-acting genotoxicant and MMS, nificant antimutagenic effect (Table 3) on the somatic

Table 2
Mutagenicity of Origanum compactum essential oil in the Drosophila wing spot test
Compound concentration No. of wings Spots per wing (no. of spots), statistical diagnosis

Small single Large single Twin Total

ST cross
Solvent (2% Tween) 77 0.83 (64) 0.08 (6) 0.00 (0) 0.91 (70)
0.05 (v/v) 71 0.93 (66) 0.03 (2) 0.00 (0) 0.96 (68)
0.1 (v/v) 75 0.64 (48) 0.03 (2) 0.00 (0) 0.67 (50)
0.2 (v/v) 73 0.47 (34) b 0.05 (4) 0.00 (0) 0.52 (38)a
HB cross
Solvent (0.1% Tween) 82 0.46 (38) 0.01 (1) 0.00 (00) 0.47 (39)
0.05% (v/v) 32 0.47 (15) 0.06 (2) 0.00 (00) 0.53 (17)
0.1% (v/v) 40 0.22 (9) 0.08 (3) 0.03 (1) 0.33 (13)
0.2% (v/v) 34 0.26 (9) 0.00 (0) 0.00 (0) 0.26 (9)
a Significantly different from control at p < 0.01.
104 N. Mezzoug et al. / Mutation Research 629 (2007) 100–110

Table 3
Effect of Origanum Compactum essential oil on URE and MMS induced mutagenicity in the Drosophila wing spot test
Compound concentration No. of wings Spots per wing (no. of spots), statistical diagnosis % inhibition

Small single Large single Twin Total

ST cross
Solvent (2% Tween) 38 0.76 (29) 0.08 (3) 0.00 (0) 0.84 (32)
URE 10 mM 40 2 (80) 0.25 (10) 0.00 (0) 2.25 (90) ***
URE and 0.05% (v/v) 40 1.32 (53) a 0.05 (2) a 0.00 (0) 1.37 (55) b 39.11
URE and 0.1% (v/v) 40 1 (40) c 0.10 (4) 0.00 (0) 1.1 (44) c 51.11
URE and 0.2% (v/v) 40 0.77 (31) c 0.10 (4) 0.00 (0) 0.87 (35) c 61.33
MMS 0.5 mM 40 2.2 (88) 3.43 (137) 2.52 (101) 8.15 (326) ***
MMS and 0.05% (v/v) 40 2.9 (116) 3.2 (128) 1.87 (75) 7.97 (319) 2.21
MMS and 0.1% (v/v) 37 3.05 (113) a 2.68 (99) 0.97 (36) c 6.70 (248) a 17.79
MMS and 0.2% (v/v) 40 1.72 (69) 2.4 (96) b 2.35 (94) 6.47 (259) b 20.61
HB cross
Solvent (0.1% Tween) 40 0.37 (15) 0.05 (2) 0.00 (0) 0.42 (17)
URE 5 mM (41) 1.90 (78) 0.34 (14) 0.05 (2) 2.29 (94) ***
URE and 0.05% (v/v) (36) 1 (36) b 0.08 (3) a 0.00 (0) 1.08 (39) c 52.83
URE and 0.1% (v/v) (41) 0.61 (25) c 0.10 (4) a 0.00 (0) 0.71 (29) c 68.99

URE: urethane; MMS: methyl methanesulfonate; Different letters (a, b, c) denote significantly different from positive control at p < 0.05, p < 0.01
and p < 0.001, respectively; three asterisks (***) denote % inhibition not calculated.

mutations induced by URE and MMS. The suppressing fractions were co-administered to larvae with URE at
effect detected against the promutagen URE was more 10 mM. Each fraction was tested in three concentrations
important than the effect detected against the direct- except F4 and F5, which were tested at two concen-
alkylating agent MMS (Table 3, Fig. 1). At 0.2%, the trations as shown in Table 4. None of the fractions
essential oil reduced significantly (p < 0.01) the muta- was toxic at the doses tested. All fractions demon-
genicity induced by MMS, but by just 20%. However, strated antimutagenic activity (Fig. 3). A weak effect
Origanum at 0.05% caused a 39% inhibition of the muta- of 23–29% was observed with F1, F3, F4 and F7.
genicity induced by URE. This inhibition reached 61%
against the same promutagen with 0.2% of the essential
oil co-administered to larvae of the ST cross. Using lar-
vae of the HB cross, 0.1% of the essential oil reduced by
69% the mutagenicity induced by the promutagen.
The crude essential oil was fractionated using column
chromatography. Seven fractions were isolated and des-
ignated F1–F7 (Fig. 2). Using the ST cross, the seven

Fig. 3. The inhibition of the mutagenicity induced by URE at 10 mM

Fig. 2. TLC analysis of Origanum compactum essential oil. The left
hand lane corresponds to the crude essential oil and lines F1–F7
correspond to the fractions obtained after separation by column chro-
matography on silica gel.
obtained with the seven fractions isolated from the crude Origanum
compactum essential oil (F1–F7), the sub-fractions isolated from F2
and F6, carvacrol, thymol and HMP. The F1, F2, F3 and F7 were
tested at 0.2%; F4, F5 and F6 were tested at 0.05‰, 0.01% and 0.1%,
respectively. The sub-fractions isolated from F2 were tested at 0.027‰
(F2-1), 1.5‰ (F2-2), 5‰ (F2-3), 2‰ (F2-4), 0.1‰ (F2-5), 0.04‰ (F2-
6) and 0.03‰ (F2-7). The sub-fractions obtained from F6 were tested at
0.055‰ (F6-1), 0.045‰ (F6-2), 0.1‰ (F6-3), 0.02‰ (F6-4), 0.14‰
(F6-5), 0.06‰ (F6-6). Carvacrol was tested at 0.2% and thymol at
0.15%.
 N. Mezzoug et al. / Mutation Research 629 (2007) 100–110 105

Table 4
The effect of Origanum compactum essential oil fractions on repressing the induction of mutation by URE at 10 mM
Compound concentration No. of wings Spots per wing (no. of spots), statistical diagnosis % inhibition

Small single Large single Twin Total

Fraction 1
URE 10 mM 41 0.97 (40) 0.17 (7) 0.05 (2) 1.19 (49) ***
URE and 0.05% (v/v) 40 0.97 (39) 0.1 (4) 0.05 (2) 1.12 (45) 5.88
URE and 0.1% (v/v) 40 0.93 (37) 0.07 (3) 0.00 (0) 1.00 (40) 15.97
URE and 0.2% (v/v) 38 0.81 (31) 0.05 (2) 0.03 (1) 0.89 (34) 25.21
Fraction 2
URE 10 mM 40 2.27 (91) 0.23 (9) 0.05 (2) 2.55 (102) ***
URE and 0.05% (v/v) 40 1.47 (59) a 0.13 (5) 0.00 (0) 1.60 (64) b 37.25
URE and 0.1% (v/v) 40 1.17 (47) c 0.25 (10) 0.13 (5) 1.55 (62) b 39.21
URE and 0.2% (v/v) 40 0.75 (30) c 0.08 (3) 0.12 (5) 0.95 (38) c 62.74
Fraction 3
URE 10 mM 40 1.07 (43) 0.23 (9) 0.00 (0) 1.3 (52) ***
URE and 0.05% (v/v) 40 1.03 (41) 0.05 (2) 0.07 (3) 1.15 (46) 11.54
URE and 0.1% (v/v) 40 1.05 (42) 0.15 (6) 0.03 (1) 1.23 (49) 5.38
URE and 0.2% (v/v) 40 0.77 (31) 0.15 (6) 0.00 (0) 0.92 (37) 29.23
Fraction 4
URE 10 mM 40 0.97 (39) 0.10 (4) 0.075 (3) 1.15 (46) ***
URE and 0.05‰ (w/v) 40 0.67 (27) 0.15 (6) 0.00 (0) 0.82 (33) 28.69
URE and 0.1‰ (w/v) 40 1.17 (47) 0.05 (2) 0.05 (2) 1.27 (51) ***
Fraction 5
URE 10 mM 40 1.22 (49) 0.075 (3) 0.075 (3) 1.37 (55) ***
URE and 0.01% (w/v) 40 0.63 (25) b 0.12 (5) 0.00 (0) 0.75 (30) b 45.25
URE and 0.02% (w/v) 40 1.05 (42) 0.1 (4) 0.025 (1) 1.175 (47) 14.23
Fraction 6
URE 10 mM 40 2.67 (107) 0.42 (17) 0.13 (5) 3.22 (129) ***
URE and 0.025% (v/v) 40 1.47 (59) c 0.35 (14) 0.10 (4) 1.92 (77) c 40.37
URE and 0.05% (v/v) 40 1.35 (54) c 0.12 (5) a 0.18 (7) 1.65 (66) c 48.75
URE and 0.1% (v/v) 40 1.12 (45) c 0.10 (4) b 0.10 (4) 1.32 (53) c 59.01
Fraction 7
URE 10 mM 40 2.07 (83) 0.2 (8) 0.00 (0) 2.27 (91) ***
URE and 0.05% (v/v) 40 1.65 (66) 0.12 (5) 0.15 (6) a 1.92 (77) 15.42
URE and 0.1% (v/v) 40 1.53 (61) 0.22 (9) 0.02 (1) 1.77 (71) 22.03
URE and 0.2% (v/v) 40 1.45 (58) a 0.15 (6) 0.15 (6) a 1.75 (70) 22.91

URE: urethane; different letters (a, b, c) denote significantly different from URE control at p < 0.05, p < 0.01 and p < 0.001, respectively; three
asterisks (***) denote % inhibition not calculated.

The antimutagenic activity detected for those fractions
was not statistically significant. However, a significant
reduction (50%, p < 0.01) of the number of somatic muta-
tions induced by URE was detected with F5, and the
strongest antimutagenic effect was exhibited by F6 and
F2 when inhibition rates reached 59 and 63%, respec-
tively.
F2 and F6, which showed the potent antimutagenic
activity, were further fractionated. Seven fractions were
isolated from F2 and six from F6, which were designated
as F2-1–F2-7 and F6-1–F6-6, respectively. The results
of the study of the antimutagenic effect of the fractions
obtained from F2 and F6 are presented in Tables 5 and 6,
respectively. All the fractions obtained from F2 demon-
strated a suppressing effect against the mutagenicity of
URE tested at 10 mM (Fig. 3). In particular, F2-3 and
F2-5 reduced the mutagenicity by more than 50%. In
addition, F2-3 at 2‰ exhibited a marked decrease in the
total spots by 63%, which was equal to the inhibition
rate of the unfractionated F2. For their part, all the frac-
tions obtained from F6 demonstrated an antimutagenic
effect that was less than the effect detected for the entire
fraction. The strongest effect was obtained with F6-4
where the inhibition rate was about 38%. However, 59%
was the inhibition rate of the URE-induced mutagenicity
provoked by unfractionated F6.
106 N. Mezzoug et al. / Mutation Research 629 (2007) 100–110

Table 5
Summary of results obtained in the Drosophila wing spot test after cotreatment of the F2 sub-fractions with URE
Compound concentration No. of wings Spots per wing (no. of spots), statistical diagnosis % inhibition

Small single Large single Twin Total

Fraction 2-1
URE 10 mM 56 2.35 (132) 0.25 (14) 0.04 (2) 2.64 (148) ***
URE and 0.027‰ (w/v) 58 1.67 (97) a 0.12 (7) 0.07 (4) 1.86 (108) b 29.54
URE and 0.055‰ (w/v) 60 1.77 (106) a 0.28 (17) 0.06 (4) 2.11 (127) a 20.07
Fraction 2-2
URE 10 mM 64 2.09 (134) 0.22 (14) 0.09 (6) 2.41 (154) ***
URE and 1.5‰ (v/v) 55 0.85 (47) c 0.16 (9) 0.02 (1) 1.03 (57) c 57.26
URE and 2.5‰ (v/v) 56 1.02 (57) c 0.14 (8) 0.00 (0) 1.16 (65) c 51.87
Fraction 2-3
URE 10 mM 64 1.59 (102) 0.31 (20) 0.09 (6) 2 (128) ***
URE and1‰ (v/v) 55 1.69 (93) 0.13 (7) 0.05 (3) 1.87 (103) 6.5
URE and 5‰ (v/v) 63 1.32 (83) 0.19 (12) 0.05 (3) 1.56 (98) 22
Fraction 2-4
URE 10 mM 63 2.03 (128) 0.35 (22) 0.08 (5) 2.46 (155) ***
URE and 1‰ (v/v) 64 0.92 (59) c 0.05 (3) c 0.05 (3) 1.02 (65) c 58.54
URE and 2‰ (v/v) 61 0.80 (49) c 0.08 (5) b 0.02 (1) 0.90 (55) c 63.41
Fraction 2-5
URE 10 mM 60 1.83 (110) 0.33 (20) 0.03 (2) 2.20 (132) ***
URE and 0.05‰ (w/v) 60 1.25 (75) a 0.12 (7) a 0.03 (2) 1.40 (84) b 36.36
URE and 0.1‰ (w/v) 60 0.90 (54) c 0.06 (4) b 0.02 (1) 0.98 (59) c 55.45
Fraction 2-6
URE 10 mM 60 2.68 (161) 0.32 (19) 0.08 (5) 3.08 (185) ***
URE and 0.04‰ (w/v) 54 1.61 (87) c 0.18 (10) 0.04 (2) 1.83 (99) c 40.58
URE and 0.08‰ (w/v) 59 2 (118) a 0.27 (16) 0.08 (5) 2.35 (139) a 23.70
Fraction 2-7
URE 10 mM 58 1.64 (95) 0.35 (20) 0.10 (6) 2.09 (121) ***
URE and 0.015‰ (w/v) 60 1.48 (89) 0.18 (11) 0.06 (4) 1.73 (104) 17.22
URE and 0.03‰ (w/v) 57 1.44 (82) 0.07 (4) b 0.05 (3) 1.56 (89) a 25.36

URE: urethane; different letters (a, b, c) denote significantly different from URE control at p < 0.05, p < 0.01 and p < 0.001, respectively; three
asterisks (***) denote % inhibition not calculated.

Carvacrol and thymol, the major components of the
fraction F2 (Table 7) were tested at three concentrations
against URE (Table 8, Fig. 3). Carvacrol at 0.2% was
more powerful than thymol tested at 0.24%. Carvacrol
suppressed the URE-induced spots by 63%, while thy-
mol was able to suppress the induced mutations by just
43%.
4. Discussion
The somatic mutation and recombination test in D.
melanogaster was performed to study the mutagenic-
ity and antimutagenicity of O. compactum essential
oil, using the ST and the HB cross. The larvae from
the HB cross are characterized by a high level of
cytochrome P450 [17]. P450 plays the main role
in the bio-activation of numerous classes of com-
pounds, including drugs, carcinogens and pesticides,
as well as natural products and plant constituents
[18–20].
Data from the wing SMART assay in D. melanogaster
demonstrated that Origanum essential oil was not muta-
genic. The negative results were obtained by the ST cross
as well as the HB cross, though the larvae from the HB
cross exhibited a slight but not significant increase of
the somatic mutations compared with the control. To
our knowledge, no previous study on the genotoxicity
of the essential oil from O. compactum was performed.
Another study was performed on essential oil extracted
from Origanum onites [11] using the Ames assay. In
this study, different concentrations of the oregano essen-
tial oil did not show any mutagenic effect on TA98 and
TA100 strains. However, in the presence of S9 microso-
mal fraction, a single dose of the oil caused statistically
significant (p < 0.05) mutagenic activity in both strains
[11].
 N. Mezzoug et al. / Mutation Research 629 (2007) 100–110 107

Table 6
Summary of results obtained in the Drosophila wing spot test after co-treatment of the F6 sub-fractions with URE
Compound concentration No. of wings Spots per wing (no. of spots), statistical diagnosis % inhibition

Small single Large single Twin Total

Fraction 6-1
URE 10 mM 58 1.74 (101) 0.27 (16) 0.09 (5) 2.10 (122) ***
URE and 0.027‰ (w/v) 60 1.7 (102) 0.30 (18) 0.08 (5) 2.08 (125) 0.95
URE and 0.055‰ (w/v) 60 1.77 (106) 0.23 (14) 0.05 (3) 2.05 (123) 2.38
Fraction 6-2
URE 10 mM 59 1.69 (100) 0.25 (15) 0.08 (5) 2.03 (120) ***
URE and 0.022‰ (w/v) 60 1.53 (92) 0.23 (14) 0.01 (1) 1.78 (107) 12.31
URE and 0.045‰ (w/v) 60 1.47 (88) 0.15 (9) 0.03 (2) 1.65 (99) 18.71
Fraction 6-3
URE 10 mM 60 1.65 (99) 0.22 (13) 0.08 (5) 1.95 (117) ***
URE and 0.05‰ (w/v) 60 1.67 (100) 0.23 (14) 0.15 (9) 2.05 (123) ***
URE and 0.1‰ (w/v) 60 1.45 (87) 0.18 (11) 0.06 (4) 1.7 (102) 12.82
Fraction 6-4
URE 10 mM 60 1.65 (99) 0.22 (13) 0.08 (5) 1.95 (117) ***
URE and 0.01‰ (w/v) 60 1.50 (90) 0.10 (6) 0.05 (3) 1.65 (99) 15.38
URE and 0.02‰ (w/v) 60 1.12 (67) a 0.07 (4) 0.01 (1) 1.20 (72) b 38.46
Fraction 6-5
URE 10 mM 60 1.87 (112) 0.15 (9) 0.06 (4) 2.08 (125) ***
URE and 0.07‰ (w/v) 60 1.73 (104) 0.22 (13) 0.02 (1) 1.97 (118) 5.29
URE and 0.14‰ (w/v) 60 1.80 (108) 0.11 (7) 0.11 (7) 2.03 (122) 2.40
Fraction 6-6
URE 10 mM 62 1.71 (106) 0.22 (14) 0.11 (7) 2.04 (127) ***
URE and 0.03‰ (w/v) 58 2.14 (124) 0.19 (11) 0.09 (5) 2.41 (140) ***
URE and 0.06‰ (w/v) 59 1.71 (101) 0.11 (7) 0.07 (4) 1.89 (112) 7.35

URE: urethane; different letters (a, b) denote significantly different from URE control at p < 0.05 and p < 0.01, respectively; three asterisks (***)
denote % inhibition not calculated.

The study of the antimutagenic activity of the essen-
tial oil was performed against URE and MMS. The
mutagenic activity of URE depends on metabolic activa-
tion to highly reactive electrophilic metabolites, which is
catalyzed by the cytochrome P450-dependent monooxy-
genase system [21]. MMS for its part, a direct SN2
alkylating agent, does not require metabolic activation
[22]. Therefore, with the ST cross, MMS induced a
higher frequency of somatic mutations when compared
with URE. The mutagenic of URE was amplified using
the HB cross. Nevertheless, its should be noted that lar-
vae derived not only from the HB cross but also from
the ST cross have sufficient bio-activation capacity to
produce the ultimate metabolites of URE that led to the
detection of mutagenic effects when the ST cross was
used.
The O. compactum essential oil has demonstrated an
antimutagenic activity against URE and MMS. Since the
mutagens used were co-administered with the essential
oil, desmutagenic mechanisms resulting in direct inac-
tivation by scavenging of radicals or reducing activity
of the mutagens can be responsible for the suppressing
effect observed. However, there is an evident differ-
ence in the protective activity of essential oil against
a direct and an indirect mutagen. The antimutagenic
activity against MMS investigated in the present study
was very low. This observation indicates that this essen-
tial oil was unable to interact and neutralize effectively
this direct-alkylating agent. However, O. compactum oil
was more effective against the mutagenicity induced by
URE and particularly using the HB cross. This suggests
that the inhibition of cytochrome P450 activity is the
major desmutagenic mechanism by which the essen-
tial oil exerts its modulating effect. In addition, it was
reported that some essential oils were able to inhibit the
activity of the cytochrome P450 enzymes [23], which is
in line with the results of the present study.
On the other hand, it is known that antioxidants can
inhibit the mutagenicity induced by chemicals through
different mechanisms [24]. Many of the oregano essen-
tial oils were reported for their antioxidant activity [25].
Therefore, the mechanism of the antimutagenic activity
108 N. Mezzoug et al. / Mutation Research 629 (2007) 100–110

Table 7 may be due to the antioxidant capacity of the Origanum

Results of GC analysis of the F2 and F6 obtained from chromatographic essential oil. The possibility that the essential oil may
fractionation of the Origanum compactum essential oil
reduce mutagenicity by blocking oxidation processes is
Constituents % not excluded [24].
F2
Only two doses are used in the evaluation of the
␤-Myrcene 0.29 antimutagenicity of the Origanum essential oil using the
␣-Terpinene 0.35 HB cross. The dose 0.02% was not tested because this
1,8-Cineole 0.71 concentration was toxic for the larvae from HB cross
␥-Terpinene 2.22 when it is co-administered with the mutagen. The high
3-Octanone 0.50
p-Cymene 3.47
toxicity observed in larvae of the HB cross is most prob-
1-Octen-3-ol + ␣,p-dimethylstyrene 0.23 ably due to the constitutively high levels of cytochromes
Camphre 0.52 P450 in these larvae, which may lead to increased pro-
Linalol 3.23 duction of toxic metabolites [26]. For the same reason,
Terpinene-4-ol 1.22 the study of the antimutagenic activity of the fractions
Methyl carvacrol ether 0.86
cis-Dihydrocarvone 0.24
obtained from the essential oil was performed using the
Pulegone 0.68 ST crosses, since the fractions isolated demonstrated to
Caryophyllene oxide 0.23 be highly toxic compared with the crude essential oil.
Thymol isomer 0.28 The fractionation of the crude essential oil had
Thymol 40.84 demonstrated that the F2 fraction, together with F6,
Carvacrol 44.13
presents the powerful antimutagenic activity against
F6 URE. The inhibition rates detected with the fractions
4-Hydroxy-4-methyl-2-pentanonea 76.21
isolated from F6 were lower than the effect of the entire
Methyl propanoic acid 1.65
Isovalerianic acid 1.66 fraction. These data suggest the presence of a synergic
cis-3-Hexenyle butyrate 1.38 effect between the constituents of F6 responsible for the
Menthadienyle esterb 3.89 increase of the inhibitory effect detected from the entire
Menthadienyle isomer esterb 6.38 F6. For its part, F2 is composed mainly by the carvacrol
Oxo-cetonic compoundb 2.91
at 44% and thymol at 40%. The study of the antimuta-
Oxo-cetonic unsaturated compoundb 3.66
Hydroxy-cetonic compoundb 2.26 genicity of the fractions of F2 demonstrated that there is
a 4-Hydroxy-4-methyl-2-pentanone is the polluting synthetic chem-
no synergic or additional effect between the constituents
of F2, because the maximum inhibition obtained with
ical.
b Identification need to be confirmed. the F2 fractions was equal to the rate of inhibition of the
entire F2. Moreover, the evaluation of the antimutagenic
effect of carvacrol and thymol, the major components
of F2, demonstrated that carvacrol has a stronger effect
on the mutagenic compound compared with thymol. The

Table 8
Antimutagenic effect of carvacrol, thymol and HMP on URE-induced mutagenicity in Drosophila wing spot
Compound concentration No. of wings Spots per wing (no. of spots), statistical diagnosis % inhibition

Small single Large single Twin Total

Carvacrol
URE 10 mM 60 1.80 (108) 0.20 (12) 0.07 (4) 2.07 (124) ***
URE and 0.05% (v/v) 60 1.20 (72) b 0.11 (7) 0.00 (0) 1.31 (79) a 36.71
URE and 0.1% (v/v) 63 0.88 (56) c 0.06 (4) 0.02 (1) 0.96 (61) c 53.62
URE and 0.2% (v/v) 62 0.74 (46) c 0.03 (2) a 0.00 (0) 0.77 (48) c 62.80
Thymol
URE 10 mM 123 1.89 (232) 0.15 (19) 0.06 (8) 2.10 (259) ***
URE and 0.05% (v/v) 62 1.40 (87) a 0.06 (4) 0.08 (5) 1.54 (96) a 26.67
URE and 0.15% (v/v) 60 0.91 (55) c 0.15 (9) 0.02 (1) 1.08 (65) c 48.57
URE and 0.24% (v/v) 62 1.1 (68) c 0.06 (4) 0.02 (1) 1.18 (73) c 43.81

URE: urethane; different letters (a, b, c) denote significantly different from URE control at p < 0.05, p < 0.01 and p < 0.001, respectively; three
asterisks (***) denote % inhibition not calculated.
 N. Mezzoug et al. / Mutation Research 629 (2007) 100–110
antimutagenic activity obtained with carvacrol was equal
to the activity of the entire F2, as well as that of the crude
Origanum essential oil. This excludes the possibility
of a synergic antimutagenic effect between the differ-
ent fractions and components of F2 and provided strong
evidence that the suppressing effect of the somatic muta-
tions by the O. compactum essential oil is due mainly to
carvacrol.
Carvacrol and thymol were reported for their
antimutagenic effect. Both carvacrol and thymol had
demonstrated a protective effect on lymphocytes using
the comet assay [13]. Carvacrol for its part was reported
to exhibit a significant antigenotoxic activity in mam-
malian cells by inhibiting the rate of sister chromatid
exchange [12]. A strong suppressing effect of carvacrol
was also observed using the Salmonella/microsome
mutagenicity test [11]. This monoterpene was also
reported for its anti-tumour proprieties. Inhibitory effects
of carvacrol on tumorigenesis induced in rats and on the
growth of melanomas in vitro as well as of myoblast
cells were reported [27–29]. On the other hand, car-
vacrol and its isomer thymol were extensively reported
for their antioxidant activity [30–32] that contributes to
their antimutagenic potential.
In conclusion, this investigation demonstrated that
the essential oil from O. compactum does not show
any mutagenic activity using the somatic mutation and
recombination test in D. melanogaster. However, an
important antimutagenic potential was detected. The
Origanum essential oil was most effective against a
promutagen compared with a direct-acting mutagen.
Moreover, the effect detected with the high bio-activation
system was most pronounced, which suggests that
inhibition of the bio-activating enzymes is the major
mechanism behind the detected antimutagenic effect.
The inhibitory effect can be attributed to some major
component, mainly carvacrol. Finally, the antimutagenic
property of O. compactum essential oil and its con-
stituents reported in this study could make this essential
oil a promising candidate for future applications in can-
cer chemoprevention and therapy.
Acknowledgements
This work was supported by PRANAROM Interna-
tional Company and the Ministry of Wallone region,
Belgium (Direction générale des technologies, de la
recherche et de l’énergie, Promotion de l’innovation
technique, Jambe-Namur). Acknowledgements to the
Manuscript Review Committee at Biomatecus and Dr
B. Edderkaoui for revision of manuscript and language.
109
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