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The present work evaluated the chemical composition and the DNA protective effect of the essential oils (EOs) from Lippia alba against bleomycin-induced genotoxicity. EO constituents were determined by Gas Chromatography/Mass Spectrometric (GC-MS) analysis. The major compounds encountered being citral (33% geranial and 25% neral), geraniol (7%) and trans-β-caryophyllene (7%) for L. alba specimen COL512077, and carvone (38%), limonene (33%) and bicyclosesquiphellandrene (8%) for the other, COL512078. The genotoxicity and antigenotoxicity of EO and the compounds citral, carvone and limonene, were assayed using the SOS Chromotest in Escherichia coli. The EOs were not genotoxic in the SOS chromotest, but one of the major compound (limonene) showed genotoxicity at doses between 97 and 1549 mM. Both EOs protected bacterial cells against bleomycin-induced genotoxicity. Antigenotoxicity in the two L. alba chemotypes was related to the major compounds, citral and carvone, respectively. The results were discussed in relation to the chemopreventive potential of L. alba EOs and its major compounds.
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Chemical composition and antigenotoxic properties
of
Lippia alba
essential oils
Molkary Andrea López1, Elena E. Stashenko2and Jorge Luis Fuentes1,2
1Microbiology and Environmental Mutagenesis Laboratory, Biology School, Faculty of Sciences,
Industrial University of Santander, Bucaramanga, Colombia.
2Research Center for Biomolecules, Research Center of Excellence, Industrial University of Santander,
Bucaramanga, Colombia.
Abstract
The present work evaluated the chemical composition and the DNA protective effect of the essential oils (EOs) from
Lippia alba
against bleomycin-induced genotoxicity. EO constituents were determined by Gas Chromatogra-
phy/Mass Spectrometric (GC-MS) analysis. The major compounds encountered being citral (33% geranial and 25%
neral), geraniol (7%) and
trans
-b-caryophyllene (7%) for
L. alba
specimen COL512077, and carvone (38%),
limonene (33%) and bicyclosesquiphellandrene (8%) for the other, COL512078. The genotoxicity and antigenotoxi-
city of EO and the compounds citral, carvone and limonene, were assayed using the SOS Chromotest in
Escherichia
coli
. The EOs were not genotoxic in the SOS chromotest, but one of the major compound (limonene) showed
genotoxicity at doses between 97 and 1549 mM. Both EOsprotected bacterial cells against bleomycin-induced
genotoxicity. Antigenotoxicity in the two
L. alba
chemotypes was related to the major compounds, citral and carvone,
respectively. The results were discussed in relation to the chemopreventive potential of
L. alba
EOs and its major
compounds.
Key words: Lippia alba, essential oil, antigenotoxicity, bleomycin, SOS chromotest.
Received: September 30, 2010; Accepted: March 23, 2011.
Introduction
Lippia alba (Mill.) N.E. Brown (Verbenaceae), an ar-
omatic shrub reaching 1.7 m high, is distributed throughout
the Caribbean, South and Central America and Tropical Af-
rica. The species is mainly used in folk medicine against di-
gestive and respiratory ailments, but also as a sedative,
analgesic, anti-inflammatory, antipyretic and antihy-
pertensive remedy (Pascual et al., 2001a; Hennebelle et al.,
2008a). In Colombia it is popularly known as “Orégano de
cerro” (Hill oregano), “Pronto alivio” (ready-relief) and
“Curatodo” (all-round cure) depending on the region
(Stashenko et al., 2003).
The species L. alba is characterized by variability in
the chemical composition of the essential oils, depending
on the origin of plant material, as well as the stage of the
plant and the part selected for distillation of the oil (Zoghbi
et al., 1998). Various chemotypes have been proposed
(Hennebelle et al., 2006; Oliveira et al., 2006). Based on
both the composition and the possible common bio-
synthetic pathways among the different oils, the existence
of at least seven has been indicated (Hennebelle et al.,
2008a). These are: chemotype I (citral, linalool and b-ca-
ryophyllene, as the main constituents), chemotype II (tage-
tenone), chemotype III (limonene and carvone or related
monoterpenic ketones), chemotype IV (myrcene), chemo-
type V (g-terpinene), chemotype VI (camphor-1,8-cineole)
and chemotype VII (estragole). In Colombia, L. alba
chemotypes I and III, and a combined (I/III) form, not pre-
viously reported, have been found.
Various studies on L. alba bioactivities bolster their
use in traditional medicine. The essential oils (EOs), either
extracts or their constituents, have revealed antiviral, anti-
bacterial, antifungal and antiparasitic activities (Pino-Alea
et al., 1996; Abad et al., 1997; Andrighetti-Fröhner et al.,
2005; Teixeira-Duarte et al., 2005; de Carvalho and da
Fonseca, 2006; Sena-Filho et al., 2006; Paduch et al., 2007;
Ara et al., 2009; Arruda et al., 2009; Mesa-Arango et al.,
2009; Shukla et al., 2009), thus sustaining their use in the
treatment of diseases of microbial origin. Currently, several
major compounds from L. alba EOs are used to control
food pathogens (Burt, 2004; Rojas-Graü et al., 2007; Tei-
xeira-Duarte et al., 2007; du Plooy et al., 2009; Linde et al.,
2009). Moreover, analgesic, anti-inflammatory and seda-
tive effects in mammalian models have been related to cer-
Genetics and Molecular Biology, 34, 3, 479-488 (2011)
Copyright © 2011, Sociedade Brasileira de Genética. Printed in Brazil
www.sbg.org.br
Send correspondence to Jorge Luis Fuentes. Microbiology and En-
vironmental Mutagenesis Laboratory, Biology School, Faculty of
Sciences, Industrial University of Santander, A.A. 678, Bucara-
manga, Colombia. E-mail: jfuentes@uis.edu.co.
Research Article
tain EO major constituents, such as citral, myrcene and
limonene (Viana et al., 1998, 2000; Vale et al., 1999). Sed-
ative effects, attributed to non-volatile flavonoids and
iridoids (Zétola et al., 2002, Hennebelle et al., 2008b), were
also encountered with L. alba ethanol extracts.Further-
more, aqueous extracts also reduced cardiac rate and gastric
ulceration induced by indomethacin in rats (Pascual et al.,
2001b; Gazola et al., 2004).
In a previous work (Vicuña et al., 2010), the impor-
tance of EOs as sources of antitumor, anti-carcinogenic and
chemopreventive agents, was emphasized. Although the
chemopreventive properties of L. alba terpenoids, such as
carvone, geraniol, limonene, and perillyl alcohol have been
well-documented (He et al., 1997; Crowell, 1999; Uedo et
al., 1999; de Carvalho and da Fonseca, 2006; Paduch et al.,
2007; Patil et al., 2009; Rabi and Bishayee, 2009), little is
known about the chemopreventive potential of other L.
alba EO constituents, for example, citral (Connor, 1991;
Nakamura et al., 2003; Seo et al., 2008).
After determining the EOs composition of the two L.
alba specimens by GC-MS analysis, their specific anti-
genotoxic activity against the clastogenic mutagen, bleo-
mycin, was evaluated by using the SOS Chromotest
(Quillardet et al., 1982). The antigenotoxic properties of
the major EO constituents (citral, carvone and limonene)
were also studied and their activity compared with the
antigenotoxic standard compound Trolox. Our work pro-
vides new insights into chemoprevention by L. alba EO
major compounds.
Materials and Methods
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 (C8-C25), citral (40:60 neral:ge-
ranial), S(+)-carvone and S(–)-limonene), Luria-Bertani
(LB) media, and antibiotics (ampicillin, bleomycin and tet-
racycline) were obtained from Sigma-Aldrich Co. Inc.
(Milwaukee, WI, USA). The standard antioxidant com-
pound 6-hydroxy-2,5,7,8-tetramethylchromane-2-carbo-
xylic acid (Trolox) was purchased from Fluka (Steinheim,
Germany). The substrates for b-galactosidase (ortho-nitro-
phenyl-b-d-galactopyranoside) and alkaline phosphatase
(p-nitrophenylphosphate) were purchased from Merck
(Darmstadt, Germany).
Plant material
L. alba plants were collected from the experimental
gardens at CENIVAM Agroindustrial Pilot Complex, lo-
cated at the Universidad Industrial de Santander campus
(Bucaramanga, Colombia). Plant growing conditions were
as indicated by Stashenko et al., (2008). Taxonomic identi-
fication was undertaken by Dr. José Luis Fernández Alonso
(National University, Bogotá, Colombia). The two L. alba
specimens (COL512077 and COL512078) were stored at
the Colombian National Herbarium.
EO extraction and chromatographic analysis
Fresh leaves and flowers from L. alba plants were
used for EO extraction using the microwave-assisted
hydrodistillation method, as described by Stashenko et al.,
(2004). Briefly, a Clevenger-type hydro-distillation appa-
ratus was placed inside a domestic microwave oven (LG,
1100 W, 2.45 GHz) with a side orifice, through which an
external glass condenser linked the 2 l-round flask with the
plant material (ca. 300 g) and water (ca. 0.5 l) inside the
oven. The oven was operated for 40 min (4 x 10 min) at full
power, which caused water to boil vigorously and reflux.
Essential oil was decanted from the condensate, and then
dried with anhydrous sodium sulfate. For chromatographic
analysis, neat essential oil (50 mL) and n-tetradecane
(0.5 mL) were dissolved in 1 mL of dichloromethane (Chro-
matography-grade reagent, Merck, Darmstadt, Germany).
EO compound identification was based on chromatogra-
phic/spectroscopic analysis, as previously indicated by Vi-
cuña et al., (2010).
Bacterial strains and culture
The Escherichia coli PQ37 strain, as proposed by
Quillardet et al. (1982) for detecting genotoxic carcino-
gens, was used. The cells, grown overnight at 37 °C, were
stired at 100 rpm in Luria-Bertani (LB) medium (10 g
tryptone/L, 5 g yeast extract/L, 10 g sodium chloride/L, pH
7.4), supplemented with 50 mg/mL ampicillin and
17 mg/mL tetracycline.
Genotoxicity assay
The SOS Chromotest, as indicated by Quillardet et
al., (1982), was used for genotoxicity assaying. Briefly,
overnight-cultures were grown in fresh LB medium (indi-
cated above) until reaching an optical density of
OD600nm = 0.4. They were then diluted 10-fold in double-
strength LB medium, and mixed (v/v) with a specific sub-
stance for identification (EO, citral, carvone and limonene).
Pure EOs (density of 900 mg/mL determined with a
BRAND picnometer, Wertheim, Germany) were diluted in
distilled water by vigorously stirring to a concentration
ranging between 1.7 and 450.0 mg/mL, this including the
antioxidant dose as previously indicated (Stashenko et al.,
2004). Negative (distilled water) and positive (1 mg/mL of
bleomycin) controls were always included in each assay.
Cells were exposed to substances during 30 min at 8 °C,
and then cultured during2hat3C.Theassays for
b-galactosidase and alkaline phosphatase activities were
according to Vicuña et al. (2010).
480 López et al.
The genotoxicity criterion applied was the Induction
Factor (IF), which, by representing fold induction of the
sulA gene in each treatment (EO, mutagen, etc), could be
considered as an indirect measure of induced primary DNA
damage. The IF was calculated as: IF = (b-galactosidase/al-
kaline phosphatase)t/(b-galactosidase/alkaline phospha-
tase)nt, where tand nt are the treated and non-treated cells,
respectively.
Antigenotoxicity assay
Antigenotoxicity was assayed using the co-incuba-
tion procedure, as indicated by Fuentes et al., (2006). Al-
though the procedure was basically the same as that of the
genotoxicity protocol, the cells were simultaneously co-
treated with different concentrations of the tested substan-
ces (EO, citral, carvone and limonene) and the mutagen
(1 mg/mL of bleomycin). Antigenotoxicity, i.e., the DNA-
protective capacity of the tested substance, was measured
as a significant reduction in IF in the combined treatments
(substance + bleomycin), and expressed as a percentage of
genotoxicity inhibition:
%GI 1 IF IF
IF IF 100
co basal
bleo basal
=- -
-´
where IFco is the SOS induction factor in co-treated cells
(substance + bleomycin), IFbasal the basal SOS induction
factor, and IFbleo the SOS induction factor in bleomycin-
treated cells.
Statistical analysis
The average values of alkaline phosphatase and IF
and the corresponding standard errors were calculated.
Normality of the data was tested using the Kolmogorov-
Smirnov test. Variance homogeneity and analysis of vari-
ance (ANOVA) tests were also conducted. Mean values
were compared using Student’s t-test. Product-moment
(Pearson) correlation analysis was applied for examining
dose-response relationships in genotoxicity studies. In all
statistical analyses, p < 0.05 was considered significant.
The STATISTICA software package (Version 6.0, StatSoft
Inc (2003), Tulsa, OK, USA) was used for all analyses.
Results
EO chemical analysis
L. alba EO compounds, as defined by GC-MS analy-
sis, are listed in Table 1. Essential oil chemical composition
in the two L. alba specimens was different. In specimen
COL512077, oxygenated monoterpenes (70.5%) were pre-
dominant, followed by sesquiterpenes (13.6%) and mono-
terpenes (3.5%). In specimen COL512078, there were high
percentages of oxygenated monoterpenes (49.4%) and mo-
noterpenes (36.0%), followed by sesquiterpenes (13.6%).
The major compounds in specimen COL512077 were citral
(geranial 33% and neral 25%), geraniol (7%) and trans-b-
caryophyllene (7%), whereas specimen COL512078 was
characterized by a high proportion of carvone (38%), li-
monene (33%) and bicyclosesquiphellandrene (8%) (Fig-
ure 1). Based on EO densities and the percentage of chro-
matogram area for major compounds, compound
concentrations were thus estimated: neral (231 mg/mL),
geranial (302 mg/mL), geraniol (65 mg/mL), trans-b-ca-
ryophyllene (59 mg/mL), carvone (345 mg/mL), limonene
(301 mg/mL) and bicyclosesquiphellandrene (70 mg/mL).
The L. alba EOs studied here were classified as citral
(COL512077) and carvone/limonene (COL512078) che-
motypes.
Genotoxic and antigenotoxic effects of
L. alba
EOs
The genotoxicity of L. alba EO was assayed before
the antigenotoxic effect was investigated. Oils did not in-
creased the IF values in PQ37 Escherichia coli strain indi-
cating that they do not induce the SOS response in E. coli
cells (Table 2). Interestingly, a stimulating effect on protein
synthesis, measured as alkaline phosphatase activity, was
observed with increased EO concentration in the case of
Lippia alba essential oils 481
Figure 1 - GC-MS profiles of EO from L. alba specimens, COL512077
(A) and COL512078 (B). Major EO constituents were numbered accord-
ing to elution order on DB-5MS column indicated in Table 1.
482 López et al.
Table 1 - Chemical composition of the Lippia alba essential oils obtained by microwave-assisted hydrodistillation of each specimen or chemotype.
Relative amount (%)
No. Compounds IKCOL512077 (citral) COL512078 (carvone/limonene)
1a-pinene 937 0.1 0.1
2 Camphene 954 - 0.4
3 Verbenene 967 - 0.2
4 1-octen-3-ol 981 0.3 -
5b-pinene-1-octen-3-ol 982 - 0.1
6 6-methyl-5-hepten-2-one 986 2.2 -
7b-Myrcene 990 0.2 1.0
8a-phellandrene 1010 0.2 -
9r-cymene 1028 0.2 -
10 Limonene 1035 2.5 33.2
11 trans-b-ocimene 1048 0.4 1.0
12 Linalool 1101 1.8 0.7
13 trans-p-mentha-2,8-diene-1-ol 1127 - 0.3
14 cis-limonene oxide 1138 - 0.1
15 Hexenyl cis-3-isobutanoate 1140 0.1 -
16 cis-p-mentha-2,8-diene-1-ol 1141 - 0.2
17 Citronellal 1153 1.0 -
18 Rosefuran epoxide 1171 0.1 -
19 Borneol 1180 - 0.6
20 cis-dihydrocarvone 1203 - 0.3
21 trans-dihydrocarvone 1210 - 0.2
22 trans-carveol 1227 - 0.3
23 Nerol 1228 2.0 -
24 Neral 1246 25.4 -
25 Geraniol 1254 7.1 -
26 Carvone 1257 - 38.1
27 Piperitone 1263 - 4.4
28 Geranial 1276 33.1 -
29 Piperitenone 1349 - 4.3
30 Neryl acetate 1357 0.2 -
31 Geranyl acetate 1376 2.7 -
32 a-copaene 1384 - 0.1
33 b-bourbonene + b-Elemene 1394 - 2.2
34 b-elemene 1397 1.9 1.2
35 b-ylangene 1429 - 0.3
36 trans-b-caryophyllene 1432 6.6 0.2
37 b-gurjunene 1441 - 0.3
38 a-guaiene 1444 1.2 -
39 trans-b-farnesene 1456 0.2 0.8
40 a-humulene 1469 1.3 -
41 Caryophyllene-9-epi-E 1473 0.1 0.3
42 Bicyclosesquiphellandrene 1493 1.2 7.7
43 Bicyclogermacrene 1507 - 0.5
44 a-bulnesene 1511 0.6 -
45 cis-a-bisabolene 1547 0.6 -
46 Caryophyllene oxide 1599 0.5 -
Monoterpene hydrocarbons 3.5 36.0
Oxygen containing monoterpenes 70.5 49.4
Sesquiterpene hydrocarbons 13.6 13.6
Oxygen containing sesquiterpenes 0.5 0.0
Other not identified 5.4 0.0
Total 93.5 99.1
No., Order of elution is given in DB-5MScolumn, IK, Values of retention index (Kovats, 1965) calculated from a minimum of three independent
chromatograms.
citral chemotype. Since this did not occur with a water solu-
ble EO fraction (data not shown), apolar compounds in the
EO mix are possibly involved.
The antigenotoxic properties of L. alba EO are shown
in Table 3. As previously indicated (Vicuña et al., 2010), a
dose of 1 mg/mL bleomycin was used for antigenotoxicity
assaying. EOs produced a significant decrease in bleomy-
cin-induced genotoxicity (IF values) at doses between 28.1
and 450 mg/mL, though insignificant at those lower. Com-
plete inhibition occurred with both citral and carvone/li-
monene chemotypes at doses higher than 56.2 mg/mL.
Genotoxic and antigenotoxic effects of
L. alba
EO
major constituents
Genotoxicity of the major EO constituents (citral,
carvone and limonene) was also assayed (Table 4). Citral
Lippia alba essential oils 483
Table 2 - Genotoxicity study of the L. alba EO measured by the SOS chromotest.
COL512077 (citral) COL512078 (carvone/limonene)
Treatments AP IF AP IF
Distilled water (negative control) 0.015 ±0.006 1.0 ±0.3 0.032 ±0.008 0.9 ±0.6
Bleomycin (positive control) 0.011 ±0.004 n.s 6.2 ±2.7 * 0.03 ±0.012 n.s 7.5 ±0.4 *
EO (0.9 mg/mL) 0.015 ±0.005 n.s 0.8 ±0.6 n.s 0.022 ±0.009 n.s 0.7 ±0.5 n.s
EO (1.7 mg/mL) 0.05 ±0.016 * 0.4 ±0.3 n.s 0.05 ±0.013 * 0.3 ±0.2 n.s
EO (3.5 mg/mL) 0.16 ±0.039 * 0.2 ±0.1 n.s 0.11 ±0.043 * 0.4 ±0.2 n.s
EO (7.0 mg/mL) 0.17 ±0.052 * 0.3 ±0.1 n.s 0.22 ±0.080 * 0.5 ±0.3 n.s
EO (14.1 mg/mL) 0.14 ±0.023 * 0.4 ±0.1 n.s 0.33 ±0.059 * 0.7 ±0.3 n.s
EO (28.1 mg/mL) 0.14 ±0.034 * 0.6 ±0.2 n.s 0.29 ±0.055 * 0.7 ±0.2 n.s
EO (56.2 mg/mL) 0.22 ±0.052 * 0.3 ±0.1 n.s 0.25 ±0.073 * 0.6 ±0.2 n.s
EO (112.5 mg/mL) 0.27 ±0.060 * 0.2 ±0.1 n.s 0.18 ±0.062 * 0.6 ±0.3 n.s
EO (225.0 mg/mL) 0.228 ±0.073 * 0.5 ±0.3 n.s 0.10 ±0.043 * 1.0 ±0.7 n.s
EO (450.0 mg/mL) 0.141 ±0.030 * 0.5 ±0.1 n.s 0.07 ±0.027 * 0.7 ±0.3 n.s
Bleomycin dosage was 1 mg/mL. Densities of essential oils were estimated in 900 mg/mL using a 9.814 mL BRAND picnometer (Wertheim, Germany).†,
Average values for direct absorbance measurement of alkaline phosphatase (AP) activity and SOS Induction Factor (IF), from a minimum of three inde-
pendent experiments with three replicates each, as well as the corresponding standard error, are given. *, The significant increase (p < 0.05) in negative
control was found by Student t-testing. n.s., no significant differences were found.
Table 3 - Antigenotoxic effect of L. alba EO against bleomycin-induced DNA damage in PQ37 Escherichia coli cells.
IF(%GI)
Cell treatments COL512077 (citral) COL512078 (carvone/limonene)
Distilled water (negative control) 1.0 ±0.1 1.0 ±0.0
Bleomycin (positive control) 5.8 ±1.2 5.4 ±1.4
EO (450.0 mg/mL) 0.5 ±0.1 0.7 ±0.3
EO (450.0 mg/mL) + Bleomycin 0.4 ±0.1 (100%) * 0.4 ±0.1 (100%) *
EO (225.0 mg/mL) + Bleomycin 0.5 ±0.1 (100%) * 0.6 ±0.2 (100%) *
EO (112.5 mg/mL) + Bleomycin 0.9 ±0.3 (100%) * 0.5 ±0.2 (100%) *
EO (56.2 mg/mL) + Bleomycin 0.9 ±0.5 (100%) * 0.9 ±0.6 (100%) *
EO (28.1 mg/mL) + Bleomycin 1.9 ±0.5 (81%) * 2.4 ±1.6 (68%) *
EO (14.1 mg/mL) + Bleomycin 5.0 ±1.2 (17%) n.s 5.0 ±2.3 (9%) n.s
EO (7.0 mg/mL) + Bleomycin 5.0 ±1.2 (17%) n.s 6.5 ±2.2 (0%) n.s
EO (3.5 mg/mL) + Bleomycin 5.1 ±1.2 (15%) n.s 6.6 ±1.1 (0%) n.s
EO (1.7 mg/mL) + Bleomycin 5.5 ±2.1 (6%) n.s 5.6 ±0.8 (0%) n.s
Bleomycin dosage was1 mg/mL. The densities of essential oils were estimated in 900 mg/mL using a 9.814 mL BRAND picnometer (Wertheim, Ger-
many).†, SOS Induction Factor (IF) averages from a minimum of three independent experiments with three replicates each, as well as the corresponding
standard errors, are given. Percentages of genotoxicity inhibition (%GI) were calculated as indicated in Materials and Methods. *, significant reduction
(p < 0.05) in positive control was found by Student t-testing. n.s., no significant reduction was found.
and carvone did not increase the IF values in PQ37 Esche-
richia coli strain, indicating that these compounds do not
induce the SOS response in E. coli cells. Limonene signifi-
cantly increased IF values for a dose range between 97 and
1549 mM, but no response association was observed by
means of Product-moment correlation analysis (R = -0,06,
n.s) and, therefore, results were considered as non conclu-
sive.
Citral induced a significant reduction in bleomycin-
induced genotoxicity at a dose of 182 mM. Percentages of
genotoxicity inhibition (% GI) increased with citral doses
suggesting a direct mode of action for antigenotoxicity of
this compound mixture and supporting the results observed
with the EO. Carvone and limonene were also antigeno-
toxic. S(+)-carvone was significantly active only from a
dose of 798 mM on, as also very similarly its isomer
(R(-)-carvone) (data not shown). Limonene was antige-
notoxic from a relatively lower dose (97 mM) on, although
GI percentages were always consistently lower than those
observed with citral (Table 4). Thus, citral was considered
of higher antigenotoxic potential.
Data on antigenotoxicity of positive standard Trolox
were also presented for comparison with citral, carvone and
limonene. Assayed doses were determined experimentally,
since no previous reports on this standard compound and
SOS Chromotest were available in the literature. Trolox
produced a significant decrease in bleomycin-induced ge-
notoxicity from a dose of 586 mM, onwards, thus compara-
tively nearly 90, 398 and 1548 times lower than those of
citral, carvone and limonene, respectively.
Discussion
The chemical composition and genotoxic and anti-
genotoxic properties of L. alba EOs obtained by micro-
wave-assisted hydrodistillation, were evaluated. Citral,
geraniol, trans-b-caryophyllene, carvone, limonene and
bycyclosesquiphellandrene were identified as the principal
components. According to Hennebelle et al. (2008a), there
are at least seven chemotypes: I (Citral, linalool and b-ca-
ryophyllene, as the main constituents), II (tagetenone), III
(limonene and carvone or related monoterpenic ketones),
IV (myrcene), V (g-terpinene), VI (camphor-1,8-cineole)
and VII (estragole). So, the two EOs studied here were clas-
sified as citral and carvone/limonene chemotypes, thus cor-
responding to chemotypes I and III, respectively.
Apparently, this is the first report on the genotoxic
and antigenotoxic properties of L. alba EOs. Under the ex-
perimental conditions assayed here (absence of exogenous
metabolic activation), the L. alba chemotypes (citral and
carvone/limonene) did not induce DNA primary damage in
the SOS Chromotest. In addition, EO major constituents as
citral and carvone were not genotoxic in the SOS Chro-
motest. This was in accordance with previous studies using
SOS Chromotest, Salmonella/microsome and Drosophila
melanogaster SMART assays (Franzios et al., 1997; Go-
mes-Carneiro et al., 1998; Stammati et al., 1999). For
limonene, IF increased at doses between 194 and 774 mM,
thereby contrasting with the results obtained with EO of the
carvone/limonene chemotype. This limonene-effect was
possibly masked in the EO by interaction with other con-
stituents, perhaps even carvone itself. Nevertheless, this
presumption needs to be tested. A previous study (Vu-
kovic-Gacic et al., 2006) indicated non-mutagenic effects
for limonene using Salmonella/microsome assay. As the re-
sults so far have been inconclusive, harmonized studies on
the genotoxicity of these compounds are now underway in
our laboratory.
The antigenotoxic potential of L. alba EO was also
shown. Although both the citral and carvone/limonene che-
motypes were antigenotoxic against the clastogen bleomy-
cin, citral appears as the most promising source of chemo-
preventive compounds, apparent by the antigenotoxicity
observed in the major constituents (Table 4). The order of
antigenotoxic activity for these compounds was found to be
citral > carvone > limonene, indicating that citral was the
most active compound. Although the chemopreventive
properties of L. alba terpenoids, as carvone, geraniol, limo-
nene and perillyl alcohol, have already been well-docu-
mented (He et al., 1997; Crowell, 1999; Uedo et al., 1999;
de Carvalho and da Fonseca, 2006; Paduch et al., 2007;
Patil et al., 2009; Rabi and Bishayee, 2009), little is really
known as regards citral. Connor (1991) was the first to indi-
cate citral chemopreventive potentiality against skin chem-
ical carcinogenesis in mice. Further experimental evidence
lent supported that citral has an ability to suppress oxidative
stress, possibly through the induction of endogenous anti-
oxidant proteins, such as phase II xenobiotic metabolizing
enzymes, as well as glutatione S-transferase (Nakamura et
al., 2003). In addition, it has been recently demonstrated
that citral strongly inhibited the CYP2B60 hydroxylase ac-
tivity (Seo et al., 2008) involved, not only in xenobiotic ac-
tivation of a wide variety of pro-mutagens, but also in the
synthesis of Aflatoxin B1mycotoxin (Shukla et al., 2009),
involved in gastric carcinogenesis. The present work pro-
vides new insights into citral and carvone chemopreven-
tion. Since bleomycin genotoxicity involves the generation
of radicals in the DNA molecule, which thus induce DNA-
strand breakages (Claussen and Long, 1999), it can be ex-
pected that the antigenotoxic effect of citral and carvone
against bleomycin occurs through radical scavenging
mechanisms within the molecule. In fact, Stashenko et al.
(2004) have previously demonstrated antioxidant proper-
ties for the L. alba carvone/limonene chemotype.
On considering the importance of oxidative damage
in carcinogenesis, the antioxidant effect of citral and car-
vone can be explored as cancer chemopreventive agents
against inflammation-related disorders, such as skin and
colon cancers. Since carvone and limonene are natural
enhancers of transdermal drug delivery by increasing per-
484 López et al.
Lippia alba essential oils 485
Table 4 - The genotoxiceffects of citral, carvone and limonene, and respective antigenotoxicity†† against bleomycin-induced DNA damage in PQ37
Escherichia coli cells. Antigenotoxic data on standard compound Trolox are also shown.
Genotoxicity Antigenotoxicity
Cell treatments IFCell treatments IF†† (% GI)
Distilled water (negative control) 1.0 ±0.1 Distilled water (negative control) 1.0 ±0.3
Bleomycin (positive control) 7.7 ±1.6 * Bleomycin (positive control) 12.5 ±4.4
Citral (2915 mM) 0.6 ±0.1 n.s Citral (2915 mM) + Bleomycin 0.5 ±0.1 (100%) **
Citral (1457 mM) 0.6 ±0.1 n.s Citral (1457 mM) + Bleomycin 0.6 ±0.2 (100%) **
Citral (729 mM) 0.8 ±0.2 n.s Citral (729 mM) + Bleomycin 1.0 ±0.2 (100%) **
Citral (364 mM) 1.3 ±0.2 n.s Citral (364 mM) + Bleomycin 1.5 ±0.3 (96%) **
Citral (182 mM) 1.2 ±0.2 n.s Citral (182 mM) + Bleomycin 1.5 ±0.3 (96%) **
Citral (91 mM) 1.2 ±0.2 n.s Citral (91 mM) + Bleomycin 6.9 ±2.4 (49%) n.s
Citral (45 mM) 1.2 ±0.2 n.s Citral (45 mM) + Bleomycin 17.1 ±5.6 (0%) n.s
Citral (23 mM) 1.2 ±0.2 n.s Citral (23 mM) + Bleomycin 17.2 ±6.8 (0%) n.s
Citral (12 mM) 1.0 ±0.3 n.s Citral (12 mM) + Bleomycin 13.9 ±6.0 (0%) n.s
Distilled water (negative control) 1.0 ±0.1 Distilled water (negative control) 0.9 ±0.1
Bleomycin (positive control) 5.8 ±0.9 * Bleomycin (positive control) 9.5 ±2.8
Carvone (3192 mM) 1.2 ±0.3 n.s Carvone (3192 mM) + Bleomycin 2.2 ±0.5 (85%) **
Carvone (1596 mM) 1.0 ±0.2 n.s Carvone (1596 mM) + Bleomycin 1.7 ±0.6 (84%) **
Carvone (798 mM) 1.3 ±0.4 n.s Carvone (798 mM) + Bleomycin 5.2 ±1.5 (50%) **
Carvone (399 mM) 1.4 ±0.3 n.s Carvone (399 mM) + Bleomycin 7.6 ±3.1 (50%) n.s
Carvone (199 mM) 1.3 ±0.1 n.s Carvone (199 mM) + Bleomycin 11.4 ±2.5 (22%) n.s
Carvone (100 mM) 1.2 ±0.2 n.s Carvone (100 mM) + Bleomycin 11.0 ±4.3 (0%) n.s
Carvone (50 mM) 1.3 ±0.3 n.s Carvone (50 mM) + Bleomycin 9.6 ±1.8 (0%) n.s
Carvone (25 mM) 1.1 ±0.2 n.s Carvone (25 mM) + Bleomycin 9.7 ±2.9 (0%) n.s
Carvone (12 mM) 0.9 ±0.2 n.s Carvone (12 mM) + Bleomycin 9.8 ±3.6 (0%) n.s
Distilled water (negative control) 1.0 ±0.1 Distilled water (negative control) 0.9 ±0.2
Bleomycin (positive control) 8.9 ±1.1 * Bleomycin (positive control) 7.2 ±1.4
Limonene (3098 mM) 0.9 ±0.3 n.s Limonene (3098 mM) + Bleomycin 2.0 ±0.9 (82%) **
Limonene (1549 mM) 2.0 ±0.9 * Limonene (1549 mM) + Bleomycin 2.0 ±0.6 (82%) **
Limonene (774 mM) 5.7 ±1.2 * Limonene (774 mM) + Bleomycin 4.6 ±1.5 (41%) **
Limonene (387 mM) 4.7 ±1.0 * Limonene (387 mM) + Bleomycin 4.6 ±1.5 (41%) **
Limonene (194 mM) 2.0 ±0.3 * Limonene (194 mM) + Bleomycin 4.7 ±1.6 (40%) **
Limonene (97 mM) 1.6 ±0.2 * Limonene (97 mM) + Bleomycin 4.8 ±1.5 (38%) **
Limonene (48 mM) 1.4 ±0.3 n.s Limonene (48 mM) + Bleomycin 5.0 ±1.6 (35%) n.s
Limonene (24 mM) 1.4 ±0.2 n.s Limonene (24 mM) + Bleomycin 5.6 ±1.4 (25%) n.s
Limonene (12 mM) 1.3 ±0.3 n.s Limonene (12 mM) + Bleomycin 6.5 ±1.2 (11%) n.s
Distilled water (negative control) - Distilled water (negative control) 1.1 ±0.1
Bleomycin (positive control) - Bleomycin (positive control) 9.9 ±2.1
Trolox (4687 mM) -Trolox (4687 mM) + Bleomycin 0.9 ±0.3 (100%) **
Trolox (2344 mM) -Trolox (2344 mM) + Bleomycin 1.1 ±0.3 (100%) **
Trolox (1172 mM) -Trolox (1172 mM) + Bleomycin 0.8 ±0.4 (100%) **
Trolox (586 mM) -Trolox (586 mM) + Bleomycin 2.0 ±1.0 (90%) **
Trolox (293 mM) -Trolox (293 mM) + Bleomycin 7.7 ±2.1 (25%) n.s
Trolox (146 mM) -Trolox (146 mM) + Bleomycin 12.9 ±2.9 (0%) n.s
Trolox (73 mM) -Trolox (73 mM) + Bleomycin 14.3 ±3.0 (0%) n.s
Trolox (37 mM) -Trolox (37 mM) + Bleomycin 10.8 ±2.6 (0%) n.s
Trolox (18 mM) -Trolox (18 mM) + Bleomycin 10.8 ±2.1 (0%) n.s
Bleomycin was always used at a dose of 1 mg/mL. According to technical product data (Sigma-Aldrich Co, St. Louis, Missouri, USA), citral, carvone and
limonene densities were 0.888, 0.959 and 0.844 g/mL, respectively. Major compound dose ranges were estimated based on their amount (%) in the
chromatogram and oil density, as indicated in Tables 1 and 2. Average IF values for genotoxicityand antigenotoxicity†† from a minimum of three inde-
pendent experiments with four replicates each, and the corresponding standard error, are given. A substance is classified as nongenotoxic if IF re-
mains < 1.5, nonconclusive if IF is between 1.5 and 2.0, and genotoxic if IF exceeds 2.0 and a dose-response relationship is observed. Percentage of
genotoxicity inhibition (% GI) was calculated as indicated in Materials and Methods. *, a significant increase (p < 0.05) in negative control was foundus
-
ing the Student t-test. **, a significant reduction (p < 0.05) in positive control was found using Student t-test. n.s., no significant differences were found.
cutaneous permeation (Aqil et al., 2007; Sapra et al., 2008),
the simultaneous use of either of these compounds together
with citral in gel preparation, should be an effective ap-
proach for skin chemoprotection. However, cytotoxicity in
human fibroblast cells has been reported at concentrations
higher than 1% of citral (Hayes and Marcovic, 2002).
Moreover, lemongrass (Cymbopogon citratus) EO with a
high citral content (70%-90%) induced phototoxic effects
in murine fibroblastic cell-line 3T3 and rabbit cornea de-
rived cell-line SIRC (Dijoux et al., 2006). Thus, further
studies on skin sensitization and phototoxicity with ter-
penoids are required.
Based on data from the literature (Pino-Alea et al.,
1996; Abad et al., 1997; Viana et al., 1998, 2000; Vale et
al., 1999; Pascual et al., 2001b; Zétola et al., 2002, Gazola
et al., 2004; Andrighetti-Fröhner et al., 2005; Teixeira-
Duarte et al., 2005; de Carvalho and da Fonseca, 2006;
Sena-Filho et al., 2006; Paduch et al., 2007; Hennebelle et
al., 2008a,b; Ara et al., 2009; Arruda et al., 2009; Mesa-
Arango et al., 2009), L. alba clearly has a wide-ranging
therapeutic potential, even further amplified by the anti-
genotoxic properties against bleomycin, as a source of
compounds with application in cancer chemoprevention.
As indicated above, citral, carvone and limonene have
shown protective properties in vitro and in vivo (Connor,
1991; He et al., 1997; Crowell, 1999; Uedo et al., 1999;
Nakamura et al., 2003; de Carvalho and da Fonseca, 2006;
Paduch et al., 2007; Seo et al., 2008; Patil et al., 2009; Rabi
and Bishayee, 2009; Shukla et al., 2009), all of which high-
light the potential benefit of L. alba and its major compo-
nents, citral, carvone and limonene, as dietary supplements
with chemopreventive and/or antioxidant properties.
In conclusion, this study showed the antigenotoxic
properties of L. alba EO, citral, carvone and limonene
against the drug bleomycin, lending support to 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 ex-
periments with a bacterial assay, additional animal and
human studies involving different endpoints should be ad-
dressed in order to clarify the antimutagenic potential of L.
alba EOs and their major constituents. In addition, harmo-
nized studies on the genotoxicity of citral, carvone and
limonene, using a battery of in vivo assays that evaluate dif-
ferent levels of DNA damage expression, are required,
prior to the practical use of these compounds in chemo-
prevention.
Acknowledgments
The authors wish to thank Dr. José Luis Fernández
Alonso for botanical identification of specimens and Dr.
Montserrat Llagostera Casal from the Universidad Autó-
noma de Barcelona for gently supplying the PQ37 E. coli
strain. This work was supported by the Vice-Rectory for
Scientific Research at the Universidad Industrial de San-
tander (UIS, Grant 5154), and by the Colombian Institute of
Science and Technology, CENIVAM-COLCIENCIAS
(Grant RC-432-2004).
References
Abad MJ, Bermejo P, Villar A, Sanchez-Palomino S and Carrasco
L (1997) Antiviral activity of medicinal plant extracts. Phy-
tother Res 11:198-202.
Andrighetti-Fröhner CR, Sincero TCM, da Silva AC, Savi LA,
Gaido CM, Bettega JMR, Mancini M, de Almeida MTR,
Barbosa RA, Farias MR, et al. (2005) Antiviral evaluation of
plants from Brazilian Atlantic Tropical Forest. Fitoterapia
76:374-378.
Aqil M, Ahad A, Sultana Y and Ali A (2007) Status of terpenes as
skin penetration enhancers. Drug Discov Today 12:1061-
1067.
Ara N, Nur MH, Amran MS, Wahid MII and Ahmed M (2009) In
vitro antimicrobial and cytotoxic activities of leaves and
flowers extracts from Lippia alba. Pak J Biol Sci 12:87-90.
Arruda DC, Miguel DC, Yokoyama-Yusunaka JKU, Katzin AM
and Uliana SRB (2009) Inhibitory activity of limonene
against Leishmania parasites in vitro and in vivo. Biomed
Pharmacother 63:643-649.
Burt S (2004) Essential oils: Their antibacterial properties and po-
tential applications in foods - A review. Int J Food Microbiol
94:223-253.
Connor MJ (1991) Modulation of tumor promotion in mouse skin
by the food additive citral (3, 7-dimethyl-2, 6-octadienal).
Cancer Lett 56:25-28.
Crowell PL (1999) Prevention and therapy of cancer by dietary
monoterpenes. J Nutr 129:775S-778S.
Claussen CA and Long EC (1999) Nucleic acid recognition by
metal complexes of bleomycin. Chem Rev99:2797-2816.
de Carvalho CCCR and da Fonseca MMR (2006) Carvone: Why
and how should one bother to produce this terpene. Food
Chem 95:413-422.
Dijoux N, Guingand Y, Bourgeois C, Durand S, Fromageot C,
Combe C and Ferret PJ (2006) Assessment of the phototoxic
hazard of some essential oils using modified 3T3 neutral red
uptake assay. Toxicol in Vitro 20:480-489.
du Plooy W, Regnier T and Combrinck S (2009) Essential oil
amended coatings as alternatives to synthetic fungicides in
citrus postharvest management. Postharvest Biol Tech
53:117-122.
Franzios G, Mirotsou M, Hatziapostolou E, Kral J, Scouras ZG
and Mavragani-Tsipidou P (1997) Insecticidal and geno-
toxic activities of mint essential oils. J Agr Food Chem
45:2690-2694.
Fuentes JL, Vernhe M, Cuetara EB, Sánchez-Lamar A, Santana
JL and Llagostera M (2006) Tannins from barks of Pinus
caribeae Morelet protect Escherichia coli cells against DNA
damage induced by g-rays. Fitoterapia 77:116-120.
Gazola R, Machado D, Ruggiero C, Singi G and Alexandre MM
(2004) Lippia alba, Melissa officinalis and Cymbopogon
citratus: Effects of the aqueous extracts on the isolated
hearts of rats. Pharmacol Res 50:477-480.
Gomes-Carneiro MR, Viana MES, Felzenszwalb I and Paum-
gartten FJR (1998) Mutagenicity testing of (±)-camphor,
486 López et al.
1,8-cineole, citral, citronellal, (-)-menthol and terpineol with
the Salmonella/microsome assay. Mutat Res 416:129-136.
Hayes AJ and Marcovic B (2002) Toxicity of Australian essential
oil Backhousia citriodora (Lemon myrtle). Part 1. Antimi-
crobial activity and in vitro cytotoxicity. Food Chem To-
xicol 40:535-543.
He L, Mo H, Hadisusilo S, Qureshi AA and Elson CE (1997)
Isoprenoids suppress the growth of murine B16 melanomas
in vitro and in vivo. J Nutr 127:668-674.
Hennebelle T, Sahpaz S, Dermont C, Joseph H and Bailleul F
(2006) The essential oil of Lippia alba: Analysis of samples
from French overseas departments and review of previous
works. Chem Biodiv 3:1116-1125.
Hennebelle T, Sahpaz S, Joseph H and Bailleul F (2008a) Ethno-
pharmacology of Lippia alba. J Ethnopharmacol 116:211-
222.
Hennebelle T, Sahpaz S, Gressier B, Joseph H and Bailleul F
(2008b). Antioxidant and neurosedative properties of poly-
phenols and iridoids from Lippia alba. Phytother Res
22:256-258.
Kovats E (1965) Gas chromatographic characterization of organic
substances in the retention index system. Adv Chrom
1:229-247.
Linde JH, Combrinck S, Regnier TJC and Virijevic RS (2009)
Chemical composition and antifungal activity of the essen-
tial oils of Lippia rehmannii from South Africa. S Afr J Bot
76:37-42.
Mesa-Arango AC, Montiel-Ramos J, Zapata B, Duran C, Betan-
cur-Galvis L and Stashenko E (2009) Citral and carvone
chemotypes from the essential oils of Colombian Lippia
alba (Mill.) N.E. Brown: Composition, cytotoxicity and
antifungal activity. Mem Inst Oswaldo Cruz 104:878-884.
Nakamura Y, Miyamoto M, Murakami A, Ohigashi H, Osawa T
and Uchida K (2003) A phase II detoxification enzymes in-
ducer from lemongrass: Identification of citral and involve-
ment of electrophilic reaction in the enzyme induction. Bio-
chem Biophys Res Commun 302:593-600.
Oliveira DR, Leitão GG, Santos SS, Bizzo HR, Lopes D, Alvino
CS, Alvino DS and Leitão SG (2006) Ethnopharmacological
study of two Lippia species from Oriximiná, Brazil. J Ethno-
pharmacol 108:103-108.
Paduch R, Kandefer-Szerszen K, Trytek M and Fiedurek J (2007)
Terpenes: Substances useful in human healthcare. Arch Im-
munol Ther Exp 55:315-327.
Pascual ME, Slowing K, Carretero E, Sánchez-Mata D and Villar
A (2001a) Lippia: Traditional uses, chemistry and pharma-
cology: A review. J Ethnopharmacol 76:201-214.
Pascual ME, Slowing K, Carretero ME and Villar A (2001b).
Antiulcerogenic activity of Lippia alba (Mill.) N.E. Brown
(Verbenaceae). IL Farmaco 56:501-504.
Patil JR, Jayaprakasha GK, Chindambara-Murthy KN, Tichy SE,
Chetti MB and Patil BS (2009) Apoptosis-mediated prolifer-
ation inhibition of human colon cancer cells by volatile prin-
ciples of Citrus aurantifolia. Food Chem 114:1351-1358.
Pino-Alea JA, Ortega-Luis AG, Rosado-Pérez A, Rodriguez-Jor-
ge M and Baluja R (1996) Composición y propiedades
antibacterianas del aceite esencial de Lippia alba (Mill.)
N.E. Brown. Rev Cubana Farm 30:29-35.
Quillardet P, Huisman O, D’Ari R and Hofnung M (1982) SOS
Chromotest, a direct assay of induction of an SOS function
in Escherichia coli K-12 to measure genotoxicity. Proc Natl
Acad Sci USA 79:5971-5975.
Rabi T and Bishayee A (2009) Terpenoids and breast cancer
chemoprevention. Breast Cancer Res Treat 115:223-239.
Rojas-Graü MA, Avena-Bustillos RJ, Olsen C, Friedman M,
Henika PR, Martí-Belloso O, Pan Z and McHugh TH (2007)
Effect of plant essential oils and oil compounds on mechani-
cal barrier and antimicrobial properties of alginate-apple pu-
ree edible films. J Food Eng 81:634-641.
Sapra B, Jain S and Tiwary AK (2008) Percutaneous permeation
enhancement by terpenes: Mechanistic view. AAPS J
10:120-132.
Sena-Filho JG, Melo JGS, Saraiva AM, Goncalves AM, Cae-
tano-Psiottano MN and Xavier HS (2006) Antimicrobial ac-
tivity and phytochemical profile from the roots of Lippia
alba (Mill.) N.E. Brown. Rev Bras Farmacogn 16:506-509.
Seo KA, Kim H, Ku HY, Ahn HJ, Park SJ, Bae SK, Shin JG and
Liu KH (2008) The monoterpenoids citral and geraniol are
moderate inhibitors of CYP2B6 hydroxylase activity. Chem
Biol Interact 174:141-146.
Shukla R, Kumar A, Singh P and Dubey NK (2009) Efficacy of
Lippia alba (Mill.) N.E. Brown essential oil and its mono-
terpenes aldehyde constituents against fungi isolated from
some edible legume seeds and aflatoxin B1production. Int J
Food Microbiol 135:165-170.
Stammati A, Bonsi P, Zucco F, Moezelaar R, Alakomi HL and
von Wright A (1999) Toxicity of selected plant volatiles in
microbial and mammalian short-term assays. Food Chem
Toxicol 37:813-823.
Stashenko EE, Jaramillo BE and Martínez JR (2003) Compara-
ción de la composición química y de la actividad antioxi-
dante in vitro de los metabolitos secundarios volátiles de
plantas de la familia Verbenaceae. Rev Acad Col Cien
27:579-597.
Stashenko EE, Jaramillo BE and Martínez JR (2004) Comparison
of different extraction methods for the analysis of volatile
secondary metabolites of Lippia alba (Mill.) N.E. Brown,
grown in Colombia and evaluation of its in vitro antioxidant
activity. J Chromatogr A 1025:93-103.
Stashenko EE, Ruiz C, Muñoz A, Castañeda M and Martínez J
(2008) Composition and antioxidant activity of essential oils
of Lippia origanoides HBK grown in Colombia. Nat Prod
Commun 3:563-566.
Teixeira-Duarte MC, Figueira GM, Sartoratto A, García-Rehder
VL and Delarmelina C (2005) Anti-Candida activity of Bra-
zilian medicinal plants. J Ethnopharmacol 97:305-311.
Teixeira-Duarte MC, Leme EE, Delarmelina C, Soarces AA,
Figueira GM and Sartoratto A (2007) Activity of essential
oils from Brazilian medicinal plants on Escherichia coli.J
Ethnopharmacol 111:197-201.
Uedo N, Tasuta M, Iishi H, Baba M, Sakai N, Yano H and Otani T
(1999) Inhibition by D-limonene of gastric carcinogenesis
induced by N-methyl-N-nitro-N-nitrosoguanidine in Wistar
rats. Cancer Lett 137:131-136.
Vale TG, Matos FJA, de Lima TCM and Viana GSB (1999) Be-
havioral effects of essential oils from Lippia alba (Mill.)
N.E. Brown chemotypes. J Ethnopharmacol 167:127-133.
Viana GSB, Vale TG, Rao VSN and Matos FJA (1998) Analgesic
and anti-inflammatory effects of two chemotypes of Lippia
alba: A comparative study. Pharm Biol 36:347-351.
Lippia alba essential oils 487
Viana GSB, Vale TG, Silva CMM and Matos FJA (2000) Anti-
convulsant activity of essential oils and active principles
from chemotypes of Lippia alba (Mill.) N.E. Brown. Biol
Pharm Bull 23:1314-1317.
Vicuña GC, Stashenko EE and Fuentes JL (2010) Chemical com-
position of the Lippia origanoides essential oils and their
antigenotoxicity against bleomycin-induced DNA damage.
Fitoterapia 81:343-349.
Vukovic-Gacic B, Nikcevic S, Beric-Bjedov T, Knezevic-Vu-
kcevic J and Simic D (2006) Antimutagenic effect of essen-
tial oil of sage (Salvia officinalis L.) and its monoterpenes
against UV-induced mutations in Escherichia coli and
Saccharomyces cerevisiae. Food Chem Toxicol 44:1730-
1738.
Zétola M, de Lima TCM, Sonaglio D, González-Ortega G, Lim-
berger RP, Petrovick PR and Bassani VL (2002) CNS activi-
ties of liquid and spray-dried extracts from Lippia alba -
Verbenaceae (Brazilian false melissa). J Ethnopharmacol
82:207-215.
Zoghbi MGB, Andrade EHA, Santos AS, Silva MHL and Maia
JGS (1998). Essential oils of Lippia alba (Mill.) N.E. Brown
growing wild in Brazilian Amazon. Flavour Fragr J 14:411-
414.
Associate Editor: Catarina S. Takahashi
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Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
488 López et al.
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Background Parasite persistence, exacerbated and sustained immune response, and continuous oxidative stress have been described to contribute to the development of the cardiac manifestations in Chronic Chagas Disease. Nevertheless, there are no efficient therapies to resolve the Trypanosoma cruzi infection and prevent the disease progression. Interestingly, trypanocide, antioxidant, and immunodulatory properties have been reported separately for some major terpenes, as citral (neral plus geranial), limonene, and caryophyllene oxide, presents in essential oils (EO) extracted from two chemotypes (Citral and Carvone) of Lippia alba . The aim of this study was to obtain L. alba essential oil fractions enriched with the aforementioned bioactive terpenes and to evaluate the impact of these therapies on trypanocide, oxidative stress, mitochondrial bioenergetics, genotoxicity, and inflammatory markers on T. cruzi- infected macrophages . Methods T. cruzi -infected J774A.1 macrophage were treated with limonene-enriched (ACT1) and citral/caryophyllene oxide-enriched (ACT2) essential oils fractions derived from Carvone and Citral- L. alba chemotypes, respectively. Results ACT1 (IC 50 = 45 ± 1.7 μg/mL) and ACT2 (IC 50 = 80 ± 1.9 μg/mL) exhibit similar trypanocidal effects to Benznidazole (BZN) (IC 50 = 48 ± 2.5 μg/mL), against amastigotes. Synergistic antiparasitic activity was observed when ACT1 was combined with BZN (∑FIC = 0.52 ± 0.13 μg/mL) or ACT2 (∑FIC = 0.46 ± 1.7 μg/mL). ACT1 also decreased the oxidative stress, mitochondrial metabolism, and genotoxicity of the therapies. The ACT1 + ACT2 and ACT1 + BZN experimental treatments reduced the pro-inflammatory cytokines (IFN-γ, IL-2, and TNF-α) and increased the anti-inflammatory cytokines (IL-4 and IL-10). Conclusion Due to its highly trypanocidal and immunomodulatory properties, ACT1 (whether alone or in combination with BZN or ACT2) represents a promising L. alba essential oil fraction for further studies in drug development towards the Chagas disease control.
... Similar results were indicated by Glamočlija et al. (2011) who reported geranial (50.94%) and neral (33.32%) as major components in L. alba essential oil in Brazil. However, López et al. (2011) reported carvone (38%), citral (33%), and limonene (33%) as the major compounds in essential oils of different specimens of L. alba that was collected from different habitats of Bucaramanga, Colombia. In the same series, Mesa-Arango et al. (2009) reported two chemotypes, citral and carvone, and Tavares et al. (2005) reported three chemotypes, carvonem, citral, and linalool in L. alba oils. ...
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The red spider mite, Oligonychus coffeae Nietner (Acari: Tetranychidae), is a prevalent mite pest damages tea crops. The present study evaluated the efficacy of L. alba essential oil against O. coffeae. GC and GC-MS analysis of L. alba essential oil revealed geraniol (52.19%) and neral (34.13%) as major compounds. The L. alba essential oil applied at 1500 ppm exhibited 100% mortality of treated mites 48 h after exposure through fumigant toxicity method, while 99% ovicidal activity of essential oil was achieved at 1000 ppm. Similarly, in fumigant bioassay LC50 value against O. coffeae was 87.75 ppm for geraniol and 103.49 ppm for neral at 72 h after exposure. The findings suggest that L. alba essential oil and its major constituents geraniol and neral could be used as eco-friendly acaricide, and can be incorporated as one of the components in IPM after multi-location field trials.
... Interestingly, the inclusion of ACT1 in the Mixture 10, signi cantly decreased the DNA damage caused by BZN (41% of cells without any damage and 54% with minimal or type 1 damage). In this regard, López [27] referenced data on the genotoxicity of Lippia alba and its enriched fraction, concluding that the major compounds of this plant did not show genotoxic effects when tested on Escherichia coli. Likewise, limonene has also been found to reduce the toxicity of certain substances by interaction when mixed. ...
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Background: Parasite persistence, exacerbated and sustained immune response, and continuous oxidative stress have been described to contribute to the development of the cardiac manifestations in chronic Chagas disease. Nevertheless, there are no efficient therapies to resolve the Trypanosoma cruzi infection and prevent the disease progression. Interestingly, trypanocide, antioxidant, and immunodulatory properties have been reported separately for some major terpenes (citral, limonene, and caryophyllene oxide) presents in essential oils extracted from two chemotypes (Citral and Carvone) of Lippia alba. The aim of this study was to obtain L. alba essential oil fractions enriched with the aforementioned bioactive terpenes and to evaluate the impact of these therapies on trypanocide, oxidative stress, mitochondrial bioenergetics, genotoxicity, and inflammatory markers on T. cruzi-infected macrophages. Methods: T. cruzi-infected J774A.1 macrophage were treated with limonene-enriched (ACT1) and citral/caryophyllene oxide-enriched (ACT2) essential oil fractions derived from Carvone and Citral-L. alba chemotypes, respectively. Results: ACT1 and ACT2 exhibit similar trypanocidal effects to Benznidazole (BZN), against amastigotes. Synergistic antiparasitic activity was observed when ACT1 was combined with BZN or ACT2. This compound also decreased the oxidative stress, mitochondrial metabolism, and genotoxicity of the therapies. The experimental treatments (ACT1+ACT2 and ACT1+BZN) reduced the pro-inflammatory cytokines (IFN-γ, IL-2, and TNF-α), while increased the anti-inflammatories (IL-4 and IL-10). Conclusion: Due to its highly trypanocidal and immunomodulatory properties, ACT1 (whether alone or in combination with BZN or ACT2) represents a promising compound for further studies in drug development towards the Chagas disease control.
... Different biological activities, such as antibacterial (Porfírio et al., 2017), antifungal (Mesa-Arango et al., 2009), antiviral (Ocazionez et al., 2010), antigenotoxic (López, Stashenko and Fuentes, 2011), antiprotozoal (Escobar et al., 2010), antioxidant (Stashenko, Jaramillo and Martínez, 2004) and vasorelaxant , have been identified in L. alba essential oils. ...
... Different biological activities, such as antibacterial (Porfírio et al., 2017), antifungal (Mesa-Arango et al., 2009), antiviral (Ocazionez et al., 2010), antigenotoxic (López, Stashenko andFuentes, 2011), antiprotozoal (Escobar et al., 2010), antioxidant (Stashenko, Jaramillo and Martínez, 2004) and vasorelaxant , have been identified in L. alba essential oils. ...
... Different biological activities, such as antibacterial (Porfírio et al., 2017), antifungal (Mesa-Arango et al., 2009), antiviral (Ocazionez et al., 2010), antigenotoxic (López, Stashenko andFuentes, 2011), antiprotozoal (Escobar et al., 2010), antioxidant (Stashenko, Jaramillo and Martínez, 2004) and vasorelaxant , have been identified in L. alba essential oils. ...
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