Fitoterapia 81 (2010) 343349

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Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f i t o t e

Chemical composition of the Lippia origanoides essential oils and their

antigenotoxicity against bleomycin-induced DNA damage
Gloria Carolina Vicua a, Elena E. Stashenko b, Jorge Luis Fuentes a,b,
a
b

Microbiology and Environmental Mutagenesis Laboratory, Biology School, Faculty of Sciences, Industrial University of Santander, A.A. 678, Bucaramanga, Colombia
Research Center for Biomolecules (CIBIMOL), Research Center of Excellence (CENIVAM), Industrial University of Santander, A.A. 678, Bucaramanga, Colombia

a r t i c l e

i n f o

Article history:
Received 1 August 2009
Accepted in revised form 6 October 2009
Available online 27 October 2009
Keywords:
Lippia origanoides
Essential oil
Thymol
Carvacrol
Antigenotoxicity
Bleomycin
SOS chromotest

a b s t r a c t
The present work evaluated the chemical composition of the essential oils (EO) obtained from
Lippia origanoides and their DNA protective effect against bleomycin-induced genotoxicity.
L. origanoides EO chemical composition was determined by gas chromatographymass
spectrometry (GCMS). The major compounds of the L. origanoides EOs were thymol (3458%)
and carvacrol (26%). The antigenotoxic effects of the EOs, major compounds and standard
compound (epigallocatechin gallate) were assayed in co-incubation procedures using the
SOS chromotest in Escherichia coli. Both EOs and their major compounds protected bacterial
cells against bleomycin-induced genotoxicity indicating that these two compounds were
principally responsible for the antigenotoxicity detected in the oils. Thymol and carvacrol
antigenotoxicity was lower than those observed with epigallocatechin gallate. The results
were discussed in relation to the chemopreventive potential of L. origanoides EOs and their
major components, carvacrol and thymol.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Lippia origanoides HBK (Vervenaceae) is an aromatic shrub
or small tree (up to 3 m tall) native of Central America and
northern South America. It is used as seasoning and in
traditional medicine for treatments of gastrointestinal and
respiratory diseases [1]. In Colombia, L. origanoides is
commonly known as Organo de monte (mountain oregano) and habits in semiarid zones of Guajira, Magdalena,
Cauca, Cundinamarca, Santander and North of Santander
states.
Different antimicrobial activities have been reported for
L. origanoides EOs, supporting their use in traditional medicine. The EO of this plant shows antimicrobial activity against
bacteria involved in respiratory diseases and against enter-

Corresponding author. Microbiology and Environmental Mutagenesis

Laboratory, Biology School, Faculty of Sciences, Industrial University of
Santander, A.A. 678, Bucaramanga, Colombia. Tel.: +57 7 6344000x2354.
E-mail addresses: jfuentes@uis.edu.co, jlfuentes2005@yahoo.es
(J.L. Fuentes).
0367-326X/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.tote.2009.10.008

opathogens [2,3]. Recently, it has been demonstrated that

L. origanoides EO inhibits in vitro replication of yellow fever
virus [4].
Based on the major constituents found in L. origanoides
EOs, different chemotypes have been reported in Brazil,
Venezuela and Colombia [2,3,5,6]. Although it has been
shown that extraction methods [7] and environmental factors
[5] can affect secondary metabolite production in Lippia
species; at least three different chemotypes can be differentiated for L. origanoides, two chemotypes whose major compounds are carvacrol and thymol respectively and a rare
chemotype characterized by the absence or very low content
of these compounds. Carvacrol and thymol also showed antimicrobial [816], antiparasitic [17] and insecticide [18,19]
activities, supporting the application of these compounds in
disease and pest management.
Recent works emphasize the EO use as a source of
antitumor, anti-carcinogenic and chemopreventive agents
[20,21]. Aromatic plants considered as Oregano spices
(Lippia graveolens, Origanum compactum, Origanum onites,
Thymus spicata, and Thymus vulgaris) since their EO have high

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G.C. Vicua et al. / Fitoterapia 81 (2010) 343349

carvacrol and/or thymol contents, have showed antigenotoxic properties against known mutagens [2226]. Additionally, carvacrol inhibited DMBA-induced tumorigenesis in rat
[27] and the growth of murine B16 melanomas [28] and of
human A549 non-small lung cancer [29] cell lines, while
thymol signicantly increased antioxidant enzyme activities
such as superoxide dismutase and glutathione peroxidase in
rats [30]. Carvacrol and thymol also showed antigenotoxic
activity against H2O2-induced genotoxicity in human colonic
Caco-2, hepatoma HepG2 and leukemic K562 cell lines
[31,32].
Consequently, the L. origanoides EO is considered as a
potential source of antigenotoxic compounds. In this study,
we determined the chemical composition of L. origanoides EO
by GCMS and then evaluated antigenotoxic activity of these
oils against the clastogenic mutagen bleomycin by means of
the SOS chromotest [33]. The antigenotoxic activity was
related to the major constituents of the EO (carvacrol and
thymol). Our work provides evidence about the chemopreventive potential of L. origanoides EO and its major compounds, carvacrol and thymol.
2. Materials and methods
2.1. Chemicals
Sodium sulfate and dichloromethane were purchased from
Aldrich Chemical Co. Inc. (Milwaukee, WI, USA). High purity
gases (helium, nitrogen, hydrogen, and air) for chromatography were obtained from AGA-Fano S.A. (Bucaramanga,
Colombia). Different standard compounds (n-tetradecane,
n-alkanes, C8C25, epigallocatechin gallate (EGCG), thymol
and carvacrol), LuriaBertani (LB) media, and antibiotics
(ampicillin, bleomycin and tetracycline) were obtained from
Sigma-Aldrich Co. Inc. (Milwaukee, WI, USA). The substrate
for -galactosidase (o-nitrophenyl--D-galactopyranoside)
and alkaline phosphatase (p-nitrophenylphosphate) were
purchased from Merck (Darmstadt, Germany).
2.2. Plant material
L. origanoides plants were collected from the Chicamocha
river canyon (Santander, Colombia). The taxonomic identication was performed by Dr. Jos Luis Fernndez Alonso
(National University, Bogot, Colombia). Two L. origanoides
specimens (COL519799 and COL516290) were stored at the
Colombian National Herbarium (Bogot). Propagation cuttings from these specimens were used in establishing experimental gardens at CENIVAM Agroindustrial Pilot Complex
located at the Universidad Industrial de Santander campus
(Bucaramanga, Colombia). Plant growing conditions were as
indicated by Stashenko et al. [6].
2.3. EO extraction and chromatographic analysis
Fresh leaves and owers from L. origanoides plants were
employed for EO extraction. Microwave-assisted hydrodistillation was used as described by Stashenko et al. [7].
Compound identication was based on chromatographic
(retention times, retention indices, standard compounds) and
mass spectroscopic (spectral interpretation, comparison with

databases and standard) criteria [34]. Gas chromatography

(GC) analyses were performed with a 6890 Plus gas
chromatograph (Agilent Technologies, Palo Alto, CA, USA)
equipped with a mass selective detector MSD 5975 (Electron
impact ionization, EI, 70 eV, Agilent Technologies, Palo Alto,
CA, USA), a split/splitless injector (1:30 split ratio), a 7863
automatic injector and a MS-ChemStation G1701-DA data
system, that included the spectral libraries WILEY, NIST and
QUADLIB 2007. A fused-silica capillary column DB-5MS (J&W
Scientic, Folsom, CA, USA) of 60 m 0.25 mm i.d., coated
with 5%-phenyl poly (methysiloxane) (0.25 m lm thickness) was used. Samples were prepared mixing aliquots of
dehydrated EO (25 L) with n-tetradecane (25 L) and
dichloromethane (974 L). Chromatographic conditions
were as follows: The GC oven temperature was programmed
from 45 C (5 min) to 150 C (2 min) at 4 C/min, then to
250 C (5 min) at 5 C/min, and nally, to 275 C (15 min) at
10 C/min. The temperatures of the injection port, ionization
chamber and of the transfer line were set at 250, 230 and
285 C, respectively. Helium (99.99%, AGA-Fano, Bucaramanga, Colombia) was used as carrier gas, with 155 kPa
column head pressure and 27 cm s 1 linear velocity
(1 mL min1, at constant ow). A standard solution of nalkanes (C8C25) was used to obtain the retention indices.
Mass spectra and reconstructed (total) ion chromatograms
were obtained by automatic scanning in the mass range m/z
30300 at 5.1 scan 1. Chromatographic peaks were checked
for homogeneity with the aid of the mass chromatograms for
the characteristic fragment ions and with the help of the peak
purity program.
2.4. Bacterial strains and culture
Escherichia coli PQ37 strain [F thr leu his-4 pyrD thi galE
galK or galT lacU169 srl300::Tn10 rpoB rpsL uvrA rfa trp::Muc+
sA::Mud(Ap,lac)ts] proposed to detect genotoxic carcinogens
[33] was used. This strain carried the sulA::lacZ gene fusion on
the chromosome as a reporter of the primary DNA damage
induced during SOS response. The cells were grown overnight
at 37 C and shaken at 100 rpm in LuriaBertani (LB) media
(10 g tryptone/L, 5 g yeast extract/L, 10 g sodium chloride/L,
pH 7.4) supplemented with 50 g/mL ampicillin and 17 g/
mL tetracycline.
2.5. Genotoxicity assay
Genotoxicity assays were performed using the SOS chromotest as described by Quillardet et al. [33]. Briey, overnight
cultures were grown in fresh LB medium as described above
until an optical density OD600nm = 0.4, diluted 10-fold in double
force LB medium (20 g tryptone/L, 10 g yeast extract/L, 20 g
sodium chloride/L, pH 7.4), and mixed (v/v) with test substance
(essential oils). Negative (distilled water) and positive (1 g/
mL of Bleomycin) controls were always included in each assay.
Cells were exposed to substances during 30 min at 8 C and
then cultured during 2 h at 37 C. To assay -galactosidase
activity, cell membranes were disrupted mixing 1.42 mL of Z
buffer (Na2HPO4 60 mM, NaH2PO4 40 mM, KCl 10 mM, Mg2SO4
1 mM, SDS 0.1%, -mercaptoethanol 40 mM, pH 7.0) with
0.15 ml of cell culture for 20 min at room temperature. The
enzyme reaction was started by the addition of 0.3 mL of

G.C. Vicua et al. / Fitoterapia 81 (2010) 343349

o-nitrophenyl--D-galactopyranoside at a concentration of
4 mg/mL in T buffer (TRISHCl 1 M, pH 8.8). After 40 min,
the enzymatic reaction was stopped by adding 0.5 mL of
1 M Na2CO3. After 5 min, 0.5 mL of 2 M TRIS was added to
restore the color. To assay alkaline phosphatase activity,
cell membranes were disrupted by addition of T buffer
(1.3 mL), chloroform (0.07 mL) and 0.05 mL of 0.1% of
sodium dodecyl sulfate solution to 0.15 mL of cell culture,
mixing them vigorously for 20 min at room temperature.
The enzyme reaction was started by the addition of 0.3 mL
of p-nitrophenyl phosphate solution (4 mg/mL in T buffer).
After 40 min, the enzymatic reaction was stopped by
adding 0.5 mL of 2 M HCl. After 5 min, 0.5 mL of 2 M TRIS
was added to restore the color. The absorbance both for galactosidase and alkaline phosphatase assays was measured at 420 nm using an HACH DR/2000 spectrophotometer (Loveland, CO, USA).
The genotoxicity criterion was the Induction Factor (IF)
that represents the fold induction of the sulA gene in each
treatment (EO, mutagen, etc) and this was therefore
considered to be an indirect measure of the primary DNA
damage (genotoxicity) induced by the treatment. The IF was
calculated as: IF = (-galactosidase/alkaline phosphatase)t/
(-galactosidase/alkaline phosphatase)nt, where t and nt are
the treated and non-treated cells, respectively. A substance is
classied as not genotoxic if IF b 1.5, not conclusive, if IF is
between 1.5 and 2.0, and genotoxic if IF exceeds 2.0 and a
doseresponse relationship is observed.
2.6. Antigenotoxicity assay
The antigenotoxicity was assayed using a co-incubation
procedure as indicated by Fuentes et al. [35]. The antigenotoxicity procedure was performed basically as the genotoxicity protocol but the cells were co-treated with different
concentrations of the assayed substances (EO, thymol,
carvacrol and EGCG) and mutagen (1 g/mL of bleomycin),
simultaneously. Antigenotoxicity, i.e., the capacity for DNA
protection by the tested substance, was measured as a
signicant reduction of the IF in the combined treatments
(substance + bleomycin) and was expressed as percentage of
genotoxicity inhibition:

%GI = 1

IFco IFbasal
100
IFbleo IFbasal

where, IFco is the SOS induction factor in co-treated cells

(substance + bleomycin), IFbasal is the basal SOS induction
factor and IFbleo is the SOS induction factor in bleomycintreated cells.
2.7. Statistical analysis
The average IF values and the corresponding standard
errors were calculated. The normality of the data was tested
using the KolmogorovSmirnov test. Variance homogeneity
and analysis of variance (ANOVA) tests were also conducted.
Mean values were compared using Student's t-test. Productmoment (Pearson) correlation analysis was used to examine
doseresponse relationship in genotoxicity study. For all
statistical analyses, p b 0.05 was considered signicant. The

345

STATISTICA software package (Version 6.0, StatSoft Inc, 2003,

Tulsa, OK, USA) was used for all analyses.
3. Results
3.1. EO analysis
The main compounds of L. origanoides EO determined by
GCMS are listed in Table 1. The L. origanoides EO specimens
showed high percentage of monoterpenes (36.139.4%)
followed by oxygenated monoterpenes (27.330.6%), and
sesquiterpenes (22.227.2%). The major compound of EO
from COL519799 was thymol ( 60%), while the specimen
COL516290 was characterized by high proportion of thymol
(34%) and carvacrol ( 26%) as is shown in Fig. 1. Based on
Table 1
Chemical composition of the Lippia origanoides EO obtained by microwaveassisted hydrodistillation.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36

Compounds

IK

-Thujene
-Pinene
Camphene
Sabinene
-Pinene
Myrcene
-Phellandrene
-Terpinene
-Cymene
Limonene
1.8-Cineole
(E)--ocimene
-Terpinene
cis-sabinene hydrate
Terpinolene
-Cymenene
Linalool
trans-Sabinene hydrate
Umbellunone
Terpinen-4-ol
Thymol methyl ether
Carvacrol methyl ether
Thymol
Carvacrol
Thymyl acetate
(E)-caryophyllene
(E)--bergamotene
-Humulene
-Muurclene
Germacrene D
-Bisabolene
-Alaskene
-Alaskene
-Cadinene
-Cadinene
(E)--bisabolene
Monoterpene hydrocarbons
Oxygen containing
monoterpenes
Sesquiterpene hydrocarbons
Oxygen containing
sesquiterpenes
Other not identied
Total

928
936
954
976
982
990
1009
1017
1027
1030
1035
1044
1060
1071
1085
1090
1096
1102
1177
1184
1228
1238
1296
1301
1347
1432
1439
1468
1484
1492
1512
1521
1522
1523
1526
1532

Relative amount (%)

COL519799

COL516290

1.2
0.4
b 0.1
b 0.1
0.2
3.6
0.2
2.2
8.4
1.0
1.2
0.2
7.3
0.6
0.3
0.1
0.3
0.2

1.1
2.7

59.6

1.6
4.3
0.9
2.3
0.2

0.3

0.1
b 0.1
0.1
0.1
39.4
27.3

1.3
0.3
b 0.1
0.1
0.2
3.0
0.2
2.4
9.3
0.8

0.2
9.1
0.5
0.1
0.1
0.3
0.1
0.1
0.8
2.3
0.1
34.5
25.8
0.7
3.4
0.5
1.9

0.1
0.3
0.1

b 0.1
0.1
36.1
30.6

27.2
0.0

22.2
0.0

6.0
100

11.1
100

Order of elution is given on DB-5MS column. IK. Values of retention

indexes (Kovats [36]) calculated from a minimum of three independent
chromatograms.

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G.C. Vicua et al. / Fitoterapia 81 (2010) 343349

Fig. 1. Typical GCMS proles of L. origanoides EO. Major constituents of EO were numbered according to elution order on DB-5MS column as indicated in Table 1.

EO densities and the percentage of chromatogram area for

major compounds, the thymol and carvacrol concentrations
into the oils were estimated as: thymol (326566 mg/mL)
and carvacrol (244 mg/mL).
3.2. Antigenotoxic effect of L. origanoides EO
In order to select the optimal dose for antigenotoxicity
assay, the genotoxicity of bleomycin on PQ37 E. coli strain
was studied for a range between 62 and 2000 ng/mL. The
SOSIF values increased with bleomycin dose and were signicant beginning at 250 ng/mL (data not shown). Based on
these results, a bleomycin dose of 1 g/mL that yields six
times the basal IF value (6.0 0.7 vs. 1.1 0.4), was chosen
for the antigenotoxicity study. In addition, genotoxicity of
L. origanoides EO was assayed before antigenotoxic effect was
investigated. Oils that had not increased the IF values in PQ37
E. coli strain were considered as not genotoxic in E. coli cells
(data not shown).
The antimutagenic properties of L. origanoides EO are shown
in Table 2. L. origanoides EO (COL519799 and COL516290)
produced a signicant decrease in the bleomycin-induced
genotoxicity (IF values) at doses between 7.4 and 477 mg/mL,
but not at lower doses. Percentages of genotoxicity inhibition (%
GI) increased with EO doses. This suggested a direct mode of
action of antigenotoxic compounds present in the EO obtained
from L. origanoides. A complete inhibition of bleomycininduced genotoxicity was observed at doses between 29.7
and 475.0 mg/mL of EO from specimen COL516290, while EO
from COL519799 showed total inhibition only between 180.7
and 475.0 mg/mL. This result suggests that antigenotoxicity of
the EO from specimen COL516290 originates from the synergic
action of its constituents.
3.3. Carvacrol and thymol antigenotoxic effect
Results of antigenotoxicity of carvacrol and thymol are
shown in Table 3. Carvacrol produced signicant reduction of
bleomycin-induced genotoxicity at a dose between 50.7 and
812.0 mM, while thymol produced a signicant decrease in
the bleomycin-induced genotoxicity at a dose between 28.5

and 912.0 mM showing complete inhibition at doses between

228.0 and 912.0 mM. Percentages of genotoxicity inhibition
(% GI) increased with carvacrol and thymol doses suggesting
a direct mode of action for antigenotoxicity of these compounds and supporting the antigenotoxicity results observed
with the EO.
Data on antigenotoxicity of positive standard EGCG were
presented for comparison with carvacrol and thymol
(Table 3). Assayed doses were selected considering previous
antigenotoxicity reports of this standard compound [37,38].
As previously reported [37], EGCG produced a signicant
decrease in the bleomycin-induced genotoxicity at doses
between 0.5 and 4.0 mM showing complete genotoxicity
inhibition at 4.0 mM. This dose was near 57 times lower than
the thymol dose (228 mM) for complete genotoxicity
inhibition.
Table 2
Antigenotoxic effect of L. origanoides EO against bleomycin-induced DNA
damage in PQ37 Escherichia coli cells.
Cell treatments

Distilled water
(negative control)
Bleomycin (positive control)
EO (475.0 mg/mL)
EO (475.0 mg/mL) + bleomycin
EO (237.5 mg/mL) + bleomycin
EO (118.7 mg/mL) + bleomycin
EO (59.3 mg/mL) + bleomycin
EO (29.7 mg/mL) + bleomycin
EO (14.8 mg/mL) + bleomycin
EO (7.4 mg/mL) + bleomycin
EO (3.7 mg/mL) + bleomycin
EO (1.8 mg/mL) + bleomycin

IF (% GI)
COL519799

COL516290

1.1 0.3

1.1 0.3

8.5 1.6
0.3 0.1
0.3 0.1 (100%)
0.6 0.3 (100%)
0.6 0.2 (100%)
1.4 0.5 (96%)
2.0 1.0 (88%)
2.3 0.9 (84%)
4.4 1.7 (55%)
7.0 1.6 (20%) n.s
9.2 1.2 (0%) n.s

5.4 1.7
1.2 0.3
0.6 0.1 (100%)
0.6 0.2 (100%)
0.6 0.3 (100%)
0.7 0.2 (100%)
1.1 0.3 (100%)
1.5 0.8 (91%)
2.4 1.3 (70%)
5.0 2.5 (9%) n.s
5.8 3.2 (0%) n.s

Bleomycin was used at dose of 1 g/mL. Densities of essential oils were

estimated at 0.95 g/ml using a BRAND picnometer of 9.814 mL (Wertheim.
Germany).Average IF values from a minimum of three independent
experiments with three replicates each and the corresponding standard
error are given. Percentage of genotoxicity inhibition (% GI) was calculated as
indicated in Materials and methods. Signicant reduction (p b 0.05) with
respect to positive control was found using Student's t-test. n.s. No
signicant reduction was found.

G.C. Vicua et al. / Fitoterapia 81 (2010) 343349

Table 3
Antigenotoxic effect of thymol, carvacrol and EGCG against bleomycininduced DNA damage in PQ37 Escherichia coli cells.
Cell treatments

IF (% GI)

Distilled water (negative control)

Bleomycin (positive control)
Carvacrol (812.0 mM)
Carvacrol (812.0 mM) + bleomycin
Carvacrol (406.0 mM) + bleomycin
Carvacrol (203.0 mM) + bleomycin
Carvacrol (101.5 mM) + bleomycin
Carvacrol (50.7 mM) + bleomycin
Carvacrol (25.4 mM) + bleomycin
Carvacrol (12.7 mM) + bleomycin
Carvacrol (6.3 mM) + bleomycin
Distilled water (negative control)
Bleomycin (positive control)
Thymol (912.0 mM)
Thymol (912.0 mM) + bleomycin
Thymol (456.0 mM) + bleomycin
Thymol (228.0 mM) + bleomycin
Thymol (114.0 mM) + bleomycin
Thymol (57.0 mM) + bleomycin
Thymol (28.5 mM) + bleomycin
Thymol (14.2 mM) + bleomycin
Thymol (7.1 mM) + bleomycin
Distilled water (negative control)
Bleomycin (positive control)
EGCG (4.00 mM)
EGCG (4.00 mM) + bleomycin
EGCG (2.00 mM) + bleomycin
EGCG (1.00 mM) + bleomycin
EGCG (0.50 mM) + bleomycin
EGCG (0.25 mM) + bleomycin
EGCG (0.12 mM) + bleomycin
EGCG (0.06 mM) + bleomycin
EGCG (0.03 mM) + bleomycin

1.0 0.2
7.4 1.0
0.5 0.2
1.1 0.5 (98%)
1.2 0.7 (97%)
1.3 0.7 (95%)
1.7 0.7 (89%)
4.2 0.9 (50%)
6.6 2.0 (12%) n.s
7.6 1.1 (0%) n.s
7.5 1.9 (0%) n.s
1.0 0.2
7.4 1.0
0.9 0.4
0.5 0.2 (100%)
0.8 0.2 (100%)
1.0 0.3 (100%)
1.4 0.4 (94%)
1.8 0.8 (87%)
3.9 1.3 (55%)
7.3 1.6 (2%) n.s
7.7 1.1 (0%) n.s
1.0 0.1
4.2 0.6
0.7 0.4
0.7 0.2 (100%)
1.4 0.4 (87%)
2.0 0.6 (67%)
2.6 0.8 (47%)
3.1 0.9 (30%) n.s
3.2 0.7 (27%) n.s
3.3 0.6 (24%) n.s
4.2 1.0 (0%) n.s

Bleomycin was used at dose of 1 g/m. Carvacrol and thymol densities were
0.966 and 0.965 g/mL, respectively; according to technical product data
(Sigma-Aldrich Co. St. Louis. Missouri. USA). Carvacrol and thymol dose
ranges were estimated based on their amount (%) in the chromatogram and
the oil density as indicated in Table 2. Average IF values from a minimum of
three independent experiments with four replicate each and the
corresponding standard error are given. Percentage of genotoxicity
inhibition (% GI) was calculated as indicated in Materials and methods.
Signicant reduction (p b 0.05) with respect to positive control was found
using Student's t-test. n.s. No signicant reduction was found.

4. Discussion
This study focused on chemical composition and antigenotoxic properties of L. origanoides EO obtained by

347

microwave-assisted hydrodistillation. Thymol, carvacrol,

-cymene and -terpinene were identied as the principal
components of the oils, with thymol as the major constituent
in both studied specimens (COL519799 and COL516290),
although there were variations in the relative amounts (%).
A summary about chemical composition of L. origanoides EO
available in the literature is presented in Table 4. Based on data
about proportion of major EO constituents, we have proposed
three chemotypes for L. origanoides species. A rst chemotype
(A) whose major components are -cymene and 1,8-cineole, a
second chemotype (B) with a high fraction of carvacrol, and a
third chemotype (C) with a high fraction of thymol. According
to this classication, L. origanoides specimens COL519799 and
COL516290 correspond to chemotype C. It has been suggested
that L. origanoides chemotypes can be inuenced by differences
in soils and climatic conditions [2]. At this study, we found
differences in the proportion of carvacrol studying L. origanoides
specimens with similar origin (Chicamocha river canyon,
Santander, Colombia) and that were propagated under similar
climatic conditions at the university campus (UIS). This result
suggests that additional factors can inuence the quantity and
proportion of constituents into EO.
L. origanoides is widely distributed in Colombia and it can
be founded in several Andean states and in the northern
peninsula of Guajira. Since L. origanoides can be easily adapted
to grow in commercial gardens this specie is very promissory
as commercial seasoning. As previously indicated for Oregano
spices [39], the carvacrol content is the prerequisite key for
selecting the adequate plant specimen for the preparation of
Oregano condiment. High carvacrol content determines the
plant's odor and therefore, spices quality. Considering this,
the specimen COL516290 appears as the best candidate for
seasoning preparation. A study that compares the quality of
the Colombian L. origanoides specimens as spices is now in
progress in our laboratory.
This work presents the rst report on antigenotoxic
properties of L. origanoides EO. The oils signicantly reduced
bleomycin-induced genotoxicity in a dose dependent manner; an effect that was directly related with carvacrol and
thymol constituents (Table 3). Antioxidant effects from
L. origanoides EO and from other oregano species have been
previously related with the presence of carvacrol and thymol
in the oils [6,4042]. In addition, carvacrol and thymol
resulted to be antigenotoxic against H2O2-induced genotoxicity in human colonic Caco-2, hepatoma HepG2 and leukemic
K562 cell lines [31,32]. Thymol also inhibited antioxidant

Table 4
Lippia origanoides chemotypes according to the major constituent amount (%) in the essential oils reported in the literature.
Chemotypes

A
B

Major constituents (%)

References

Carvacrol

Thymol

-Cymene

1.8-Cineole

-Terpinene

3343
39
4452
817

26

58
18
714
4862
60
034

1116

10
910

8
9

711

810

710

7
9

Stashenko et al. [6]

Dos Santos et al. [2]
Oliveira et al. [3]
Stashenko et al. [6]
Rojas et al. [5]
Present work
Present work

Only compounds with percentage higher than 5% were considered. Order A to C was assigned considering potential use of oregano as condiment based on high
carvacrol and thymol content.

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G.C. Vicua et al. / Fitoterapia 81 (2010) 343349

activity against malonaldehyde formation from blood plasma

oxidation [43].
Since bleomycin genotoxicity involves generation of
radicals on the DNA molecule which induce DNA strand
breakages [44] and both carvacrol and thymol can interact
with DNA [45], it can be expected that the antigenotoxic effect
of L. origanoides EO and/or carvacrol and thymol against
bleomycin occurs through radical scavenging mechanisms on
the DNA molecule. Thymol also increases antioxidant enzyme
activities in rat [30], therefore reduction of DNA radicals
caused by thymol-increased antioxidant enzymes could not
be discarded as a potential antigenotoxic mechanism. However, these hypotheses could be examined in further studies.
Based on literature data [24,6], L. origanoides clearly has
broad-ranging therapeutic potential. The antigenotoxic properties against bleomycin widen its potential as a source of
compounds with application in cancer chemoprevention. Other
oregano species whose EO have high fraction of carvacrol and
thymol, such as Lippia graveolens, Origanum compactum, Origanum onites, Thymus spicata and Thymus vulgaris, have shown
antigenotoxic properties against known mutagens [2226].
Carvacrol and thymol have also exhibited protective, antitumor
and anti-carcinogenic properties in different experimental
models [2732,4648]. All these works highlight the potential
benet of L. origanoides EO and its major components carvacrol
and thymol as dietary supplements with chemopreventive and/
or antioxidant properties.
Several reasons support the use of L. origanoides EO as
chemopreventive agent. First, the L. origanoides species can
be easily established from wild to cultivated conditions with
adequate EO yield levels (between 1.4 and 2.0%). Second, the
L. origanoides EO with high thymol content (between 326 and
566 mg/mL) supports direct preparation of therapeutic
formulations. Third, previous work [49] has demonstrated
systemic thymol availability in human plasma (limit of
quantitation of 8.1 ng/mL) after intake of oral therapeutic
doses of thyme preparation, which indicates clinical use
feasibility. Four, carvacrol and thymol are natural enhancers
of transdermal drug delivery by increasing percutaneous
permeation [50], supporting the simultaneous use of these
compounds in gel preparation for skin chemoprotection.
In conclusion, this study showed the antigenotoxic
properties of L. origanoides EO and carvacrol and thymol
against the drug bleomycin, supporting the potential of the
oils and compounds in chemoprevention and cancer therapy.
Since the role of chemopreventive agents in the etiology of
cancer is very complex and involves several modes of action
and our results concern only in vitro experiments with a
bacterial assay, additional animal and human studies involving different endpoints should be addressed in order to clarify
the antimutagenic potential of L. origanoides EO and its major
constituents carvacrol and thymol. In addition, harmonized
studies on genotoxicity of carvacrol and thymol using a
battery of in vivo assays that evaluates different levels of DNA
damage expression will be required for practical use of these
compounds in chemoprevention.
Acknowledgments
The authors wish to thank Dr. Jos Luis Fernndez Alonso
for botanical identication of specimens and Dr. Montserrat

Llagostera Casal from Universidad Autnoma de Barcelona

for gently supplies the PQ37 E. coli strain. This work was
supported by the Vice-Rectory for Science Research at
Universidad Industrial de Santander (UIS, Grant 5154), and
by the Colombian Institute of Science and Technology,
CENIVAM-COLCIENCIAS (Grant RC-432-2004).
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