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Accepted in revised form 6 October 2009

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L. origanoides EO chemical composition was determined by gas chromatographymass

e) isf Cen

L. origanoides EO inhibits in vitro replication of yellow fever

Based on the major constituents found in L. origanoides

Fitoterapia 81 (2010) 343349

Contents lists available at ScienceDirect

Fitoter

j ourna l homepage: www.e lsnorthern South America. It is used as seasoning and intraditional medicine for treatments of gastrointestinal andrespiratory diseases [1]. In Colombia, L. origanoides iscommonly known as Organo de monte (mountain oreg-ano) and habits in semiarid zones of Guajira, Magdalena,Cauca, Cundinamarca, Santander and North of Santanderstates.

Different antimicrobial activities have been reported forL. origanoides EOs, supporting their use in traditional medi-

EOs, different chemotypes have been reported in Brazil,Venezuela and Colombia [2,3,5,6]. Although it has beenshown that extractionmethods [7] and environmental factors[5] can affect secondary metabolite production in Lippiaspecies; at least three different chemotypes can be differen-tiated for L. origanoides, two chemotypes whose major com-pounds are carvacrol and thymol respectively and a rarechemotype characterized by the absence or very low contentof these compounds. Carvacrol and thymol also showed anti-Lippia origanoidesHBK (Vervenaceaor small tree (up to 3 m tall) native ocine. The EO of this plant shows antimicrobibacteria involved in respiratory diseases a

Corresponding author. Microbiology and EnviroLaboratory, Biology School, Faculty of Sciences, IndSantander, A.A. 678, Bucaramanga, Colombia. Tel.: +5

E-mail addresses: jfuentes@uis.edu.co, jlfuentes200(J.L. Fuentes).

0367-326X/$ see front matter 2009 Elsevier B.V.doi:10.1016/j.tote.2009.10.008an aromatic shrubtral America and

virus [4].1. Introductioncompound (epigallocatechin gallate) were assayed in co-incubation procedures using theSOS chromotest in Escherichia coli. Both EOs and their major compounds protected bacterialcells against bleomycin-induced genotoxicity indicating that these two compounds wereprincipally responsible for the antigenotoxicity detected in the oils. Thymol and carvacrolantigenotoxicity was lower than those observed with epigallocatechin gallate. The resultswere discussed in relation to the chemopreventive potential of L. origanoides EOs and theirmajor components, carvacrol and thymol.

2009 Elsevier B.V. All rights reserved.

opathogens [2,3]. Recently, it has been demonstrated thatKeywords:Lippia origanoidesEssential oilThymolCarvacrolAntigenotoxicityBleomycinSOS chromotestAvailable online 27 October 2009 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 standardChemical composition of the Lippia oantigenotoxicity against bleomycin-i

Gloria Carolina Vicua a, Elena E. Stashenko b,a Microbiology and Environmental Mutagenesis Laboratory, Biology School, Facb Research Center for Biomolecules (CIBIMOL), Research Center of Excellence (C

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 August 2009

The present work evLippia origanoides aal activity againstnd against enter-

nmental Mutagenesisustrial University of7 7 6344000x2354.5@yahoo.es

All rights reserved.anoides essential oils and theiruced DNA damage

e Luis Fuentes a,b,Sciences, Industrial University of Santander, A.A. 678, Bucaramanga, ColombiaM), Industrial University of Santander, A.A. 678, Bucaramanga, Colombia

d the chemical composition of the essential oils (EO) obtained fromeir DNA protective effect against bleomycin-induced genotoxicity.

apia

ev ie r.com/ locate / f i to temicrobial [816], antiparasitic [17] and insecticide [18,19]activities, supporting the application of these compounds indisease and pest management.

Recent works emphasize the EO use as a source ofantitumor, 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

344 G.C. Vicua et al. / Fitoterapia 81 (2010) 343349carvacrol and/or thymol contents, have showed antigeno-toxic properties against known mutagens [2226]. Addition-ally, carvacrol inhibited DMBA-induced tumorigenesis in rat[27] and the growth of murine B16 melanomas [28] and ofhuman A549 non-small lung cancer [29] cell lines, whilethymol signicantly increased antioxidant enzyme activitiessuch as superoxide dismutase and glutathione peroxidase inrats [30]. Carvacrol and thymol also showed antigenotoxicactivity against H2O2-induced genotoxicity in human colonicCaco-2, hepatoma HepG2 and leukemic K562 cell lines[31,32].

Consequently, the L. origanoides EO is considered as apotential source of antigenotoxic compounds. In this study,we determined the chemical composition of L. origanoides EOby GCMS and then evaluated antigenotoxic activity of theseoils against the clastogenic mutagen bleomycin by means ofthe SOS chromotest [33]. The antigenotoxic activity wasrelated to the major constituents of the EO (carvacrol andthymol). Our work provides evidence about the chemopre-ventive potential of L. origanoides EO and its major com-pounds, carvacrol and thymol.

2. Materials and methods

2.1. Chemicals

Sodiumsulfate anddichloromethanewere purchased fromAldrich Chemical Co. Inc. (Milwaukee, WI, USA). High puritygases (helium, nitrogen, hydrogen, and air) for chromatogra-phy were obtained from AGA-Fano S.A. (Bucaramanga,Colombia). Different standard compounds (n-tetradecane,n-alkanes, C8C25, epigallocatechin gallate (EGCG), thymoland carvacrol), LuriaBertani (LB) media, and antibiotics(ampicillin, bleomycin and tetracycline) were obtained fromSigma-Aldrich Co. Inc. (Milwaukee, WI, USA). The substratefor -galactosidase (o-nitrophenyl--D-galactopyranoside)and alkaline phosphatase (p-nitrophenylphosphate) werepurchased from Merck (Darmstadt, Germany).

2.2. Plant material

L. origanoides plants were collected from the Chicamochariver canyon (Santander, Colombia). The taxonomic identi-cation was performed by Dr. Jos Luis Fernndez Alonso(National University, Bogot, Colombia). Two L. origanoidesspecimens (COL519799 and COL516290) were stored at theColombian National Herbarium (Bogot). Propagation cut-tings from these specimens were used in establishing ex-perimental gardens at CENIVAMAgroindustrial Pilot Complexlocated at the Universidad Industrial de Santander campus(Bucaramanga, Colombia). Plant growing conditions were asindicated by Stashenko et al. [6].

2.3. EO extraction and chromatographic analysis

Fresh leaves and owers from L. origanoides plants wereemployed for EO extraction. Microwave-assisted hydrodis-tillation was used as described by Stashenko et al. [7].

Compound identication was based on chromatographic(retention times, retention indices, standard compounds) andmass spectroscopic (spectral interpretation, comparison withdatabases and standard) criteria [34]. Gas chromatography(GC) analyses were performed with a 6890 Plus gaschromatograph (Agilent Technologies, Palo Alto, CA, USA)equipped with a mass selective detector MSD 5975 (Electronimpact ionization, EI, 70 eV, Agilent Technologies, Palo Alto,CA, USA), a split/splitless injector (1:30 split ratio), a 7863automatic injector and a MS-ChemStation G1701-DA datasystem, that included the spectral libraries WILEY, NIST andQUADLIB 2007. A fused-silica capillary column DB-5MS (J&WScientic, Folsom, CA, USA) of 60 m0.25 mm i.d., coatedwith 5%-phenyl poly (methysiloxane) (0.25 m lm thick-ness) was used. Samples were prepared mixing aliquots ofdehydrated EO (25 L) with n-tetradecane (25 L) anddichloromethane (974 L). Chromatographic conditionswere as follows: The GC oven temperature was programmedfrom 45 C (5 min) to 150 C (2 min) at 4 C/min, then to250 C (5 min) at 5 C/min, and nally, to 275 C (15 min) at10 C/min. The temperatures of the injection port, ionizationchamber and of the transfer line were set at 250, 230 and285 C, respectively. Helium (99.99%, AGA-Fano, Bucara-manga, Colombia) was used as carrier gas, with 155 kPacolumn head pressure and 27 cm s1 linear velocity(1 mL min1, at constant ow). A standard solution of n-alkanes (C8C25) was used to obtain the retention indices.Mass spectra and reconstructed (total) ion chromatogramswere obtained by automatic scanning in the mass range m/z30300 at 5.1 scan 1. Chromatographic peaks were checkedfor homogeneity with the aid of the mass chromatograms forthe characteristic fragment ions and with the help of the peakpurity program.

2.4. Bacterial strains and culture

Escherichia coli PQ37 strain [F thr leu his-4 pyrD thi galEgalK 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 onthe chromosome as a reporter of the primary DNA damageinduced during SOS response. The cells were grown overnightat 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 chro-motest as described by Quillardet et al. [33]. Briey, overnightcultures were grown in fresh LB medium as described aboveuntil anoptical densityOD600nm=0.4, diluted10-fold in doubleforce LB medium (20 g tryptone/L, 10 g yeast extract/L, 20 gsodiumchloride/L, pH7.4), andmixed (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 andthen cultured during 2 h at 37 C. To assay -galactosidaseactivity, cell membranes were disrupted mixing 1.42 mL of Zbuffer (Na2HPO4 60 mM, NaH2PO4 40 mM, KCl 10 mM,Mg2SO41 mM, SDS 0.1%, -mercaptoethanol 40 mM, pH 7.0) with0.15 ml of cell culture for 20 min at room temperature. Theenzyme reaction was started by the addition of 0.3 mL of

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 byGCMS are listed in Table 1. The L. origanoides EO specimensshowed high percentage of monoterpenes (36.139.4%)followed by oxygenated monoterpenes (27.330.6%), andsesquiterpenes (22.227.2%). The major compound of EOfrom COL519799 was thymol (60%), while the specimenCOL516290 was characterized by high proportion of thymol(34%) and carvacrol (26%) as is shown in Fig. 1. Based on

Table 1Chemical composition of the Lippia origanoides EO obtained by microwave-assisted hydrodistillation.

Compounds IK Relative amount (%)

COL519799 COL516290

1 -Thujene 928 1.2 1.32 -Pinene 936 0.4 0.33 Camphene 954 b0.1 b0.14 Sabinene 976 b0.1 0.15 -Pinene 982 0.2 0.26 Myrcene 990 3.6 3.07 -Phellandrene 1009 0.2 0.28 -Terpinene 1017 2.2 2.49 -Cymene 1027 8.4 9.310 Limonene 1030 1.0 0.811 1.8-Cineole 1035 1.2 12 (E)--ocimene 1044 0.2 0.213 -Terpinene 1060 7.3 9.114 cis-sabinene hydrate 1071 0.6 0.515 Terpinolene 1085 0.3 0.116 -Cymenene 1090 0.1 0.117 Linalool 1096 0.3 0.318 trans-Sabinene hydrate 1102 0.2 0.119 Umbellunone 1177 0.120 Terpinen-4-ol 1184 1.1 0.821 Thymol methyl ether 1228 2.7 2.322 Carvacrol methyl ether 1238 0.123 Thymol 1296 59.6 34.524 Carvacrol 1301 25.825 Thymyl acetate 1347 1.6 0.726 (E)-caryophyllene 1432 4.3 3.427 (E)--bergamotene 1439 0.9 0.528 -Humulene 1468 2.3 1.929 -Muurclene 1484 0.2

345G.C. Vicua et al. / Fitoterapia 81 (2010) 343349o-nitrophenyl--D-galactopyranoside at a concentration of4 mg/mL in T buffer (TRISHCl 1 M, pH 8.8). After 40 min,the enzymatic reaction was stopped by adding 0.5 mL of1 M Na2CO3. After 5 min, 0.5 mL of 2 M TRIS was added torestore 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% ofsodium 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 mLof p-nitrophenyl phosphate solution (4 mg/mL in T buffer).After 40 min, the enzymatic reaction was stopped byadding 0.5 mL of 2 M HCl. After 5 min, 0.5 mL of 2 M TRISwas added to restore the color. The absorbance both for -galactosidase and alkaline phosphatase assays was mea-sured at 420 nm using an HACH DR/2000 spectrophotom-eter (Loveland, CO, USA).

The genotoxicity criterion was the Induction Factor (IF)that represents the fold induction of the sulA gene in eachtreatment (EO, mutagen, etc) and this was thereforeconsidered to be an indirect measure of the primary DNAdamage (genotoxicity) induced by the treatment. The IF wascalculated as: IF=(-galactosidase/alkaline phosphatase)t/(-galactosidase/alkaline phosphatase)nt, where t and nt arethe treated and non-treated cells, respectively. A substance isclassied as not genotoxic if IFb1.5, not conclusive, if IF isbetween 1.5 and 2.0, and genotoxic if IF exceeds 2.0 and adoseresponse relationship is observed.

2.6. Antigenotoxicity assay

The antigenotoxicity was assayed using a co-incubationprocedure as indicated by Fuentes et al. [35]. The antigeno-toxicity procedure was performed basically as the genotoxi-city protocol but the cells were co-treated with differentconcentrations of the assayed substances (EO, thymol,carvacrol and EGCG) and mutagen (1 g/mL of bleomycin),simultaneously. Antigenotoxicity, i.e., the capacity for DNAprotection by the tested substance, was measured as asignicant reduction of the IF in the combined treatments(substance+bleomycin) and was expressed as percentage ofgenotoxicity inhibition:

%GI = 1 IFcoIFbasalIFbleoIFbasal

100

where, IFco is the SOS induction factor in co-treated cells(substance+bleomycin), IFbasal is the basal SOS inductionfactor and IFbleo is the SOS induction factor in bleomycin-treated cells.

2.7. Statistical analysis

The average IF values and the corresponding standarderrors were calculated. The normality of the data was testedusing the KolmogorovSmirnov test. Variance homogeneityand analysis of variance (ANOVA) tests were also conducted.Mean values were compared using Student's t-test. Product-moment (Pearson) correlation analysis was used to examinedoseresponse relationship in genotoxicity study. For allstatistical analyses, pb0.05 was considered signicant. The30 Germacrene D 1492 0.131 -Bisabolene 1512 0.3 0.332 -Alaskene 1521 0.133 -Alaskene 1522 0.1 34 -Cadinene 1523 b0.1 35 -Cadinene 1526 0.1 b 0.136 (E)--bisabolene 1532 0.1 0.1

Monoterpene hydrocarbons 39.4 36.1Oxygen containingmonoterpenes

27.3 30.6

Sesquiterpene hydrocarbons 27.2 22.2Oxygen containingsesquiterpenes

0.0 0.0

Other not identied 6.0 11.1Total 100 100

Order of elution is given on DB-5MS column. IK. Values of retentionindexes (Kovats [36]) calculated from a minimum of three independentchromatograms.

and 912.0 mM showing complete inhibition at doses between228.0 and 912.0 mM. Percentages of genotoxicity inhibition(% GI) increased with carvacrol and thymol doses suggestinga direct mode of action for antigenotoxicity of these com-

ere numbered according to elution order on DB-5MS column as indicated in Table 1.

346 G.C. Vicua et al. / Fitoterapia 81 (2010) 343349EO densities and the percentage of chromatogram area formajor compounds, the thymol and carvacrol concentrationsinto 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 antigenotoxicityassay, the genotoxicity of bleomycin on PQ37 E. coli strainwas studied for a range between 62 and 2000 ng/mL. TheSOSIF values increased with bleomycin dose and were sig-nicant beginning at 250 ng/mL (data not shown). Based onthese results, a bleomycin dose of 1 g/mL that yields sixtimes the basal IF value (6.00.7 vs. 1.10.4), was chosenfor the antigenotoxicity study. In addition, genotoxicity ofL. origanoides EO was assayed before antigenotoxic effect wasinvestigated. Oils that had not increased the IF values in PQ37E. coli strain were considered as not genotoxic in E. coli cells(data not shown).

The antimutagenic properties of L. origanoidesEO are shownin Table 2. L. origanoides EO (COL519799 and COL516290)

Fig. 1. Typical GCMS proles of L. origanoides EO. Major constituents of EO wproduced a signicant decrease in the bleomycin-inducedgenotoxicity (IF values) at doses between 7.4 and 477 mg/mL,but not at lowerdoses. Percentagesof genotoxicity inhibition (%GI) increased with EO doses. This suggested a direct mode ofaction of antigenotoxic compounds present in the EO obtainedfrom L. origanoides. A complete inhibition of bleomycin-induced genotoxicity was observed at doses between 29.7and 475.0 mg/mL of EO from specimen COL516290, while EOfrom COL519799 showed total inhibition only between 180.7and 475.0 mg/mL. This result suggests that antigenotoxicity ofthe EO from specimen COL516290 originates from the synergicaction of its constituents.

3.3. Carvacrol and thymol antigenotoxic effect

Results of antigenotoxicity of carvacrol and thymol areshown in Table 3. Carvacrol produced signicant reduction ofbleomycin-induced genotoxicity at a dose between 50.7 and812.0 mM, while thymol produced a signicant decrease inthe bleomycin-induced genotoxicity at a dose between 28.5pounds and supporting the antigenotoxicity results observedwith the EO.

Data on antigenotoxicity of positive standard EGCG werepresented for comparison with carvacrol and thymol(Table 3). Assayed doses were selected considering previousantigenotoxicity reports of this standard compound [37,38].As previously reported [37], EGCG produced a signicantdecrease in the bleomycin-induced genotoxicity at dosesbetween 0.5 and 4.0 mM showing complete genotoxicityinhibition at 4.0 mM. This dose was near 57 times lower thanthe thymol dose (228 mM) for complete genotoxicityinhibition.

Table 2Antigenotoxic effect of L. origanoides EO against bleomycin-induced DNAdamage in PQ37 Escherichia coli cells.

Cell treatments IF (% GI)COL519799 COL516290

Distilled water(negative control)

1.10.3 1.10.3

Bleomycin (positive control) 8.51.6 5.41.7EO (475.0 mg/mL) 0.30.1 1.20.3EO (475.0 mg/mL)+bleomycin 0.30.1 (100%) 0.60.1 (100%)EO (237.5 mg/mL)+bleomycin 0.60.3 (100%) 0.60.2 (100%)EO (118.7 mg/mL)+bleomycin 0.60.2 (100%) 0.60.3 (100%)EO (59.3 mg/mL)+bleomycin 1.40.5 (96%) 0.70.2 (100%)EO (29.7 mg/mL)+bleomycin 2.01.0 (88%) 1.10.3 (100%)EO (14.8 mg/mL)+bleomycin 2.30.9 (84%) 1.50.8 (91%)EO (7.4 mg/mL)+bleomycin 4.41.7 (55%) 2.41.3 (70%)EO (3.7 mg/mL)+bleomycin 7.01.6 (20%) n.s 5.02.5 (9%) n.sEO (1.8 mg/mL)+bleomycin 9.21.2 (0%) n.s 5.83.2 (0%) n.s

Bleomycin was used at dose of 1 g/mL. Densities of essential oils wereestimated at 0.95 g/ml using a BRAND picnometer of 9.814 mL (Wertheim.Germany).Average IF values from a minimum of three independentexperiments with three replicates each and the corresponding standarderror are given. Percentage of genotoxicity inhibition (% GI) was calculated asindicated in Materials and methods. Signicant reduction (pb0.05) withrespect to positive control was found using Student's t-test. n.s. Nosignicant reduction was found.

microwave-assisted hydrodistillation. Thymol, carvacrol,-cymene and -terpinene were identied as the principalcomponents of the oils, with thymol as the major constituentin both studied specimens (COL519799 and COL516290),although there were variations in the relative amounts (%).

A summary about chemical composition of L. origanoides EOavailable in the literature is presented in Table 4. Based on dataabout proportion of major EO constituents, we have proposedthree chemotypes for L. origanoides species. A rst chemotype(A) whose major components are -cymene and 1,8-cineole, asecond chemotype (B) with a high fraction of carvacrol, and athird chemotype (C) with a high fraction of thymol. Accordingto this classication, L. origanoides specimens COL519799 andCOL516290 correspond to chemotype C. It has been suggested

Table 3Antigenotoxic effect of thymol, carvacrol and EGCG against bleomycin-induced DNA damage in PQ37 Escherichia coli cells.

Cell treatments IF (% GI)

Distilled water (negative control) 1.00.2Bleomycin (positive control) 7.41.0Carvacrol (812.0 mM) 0.50.2Carvacrol (812.0 mM)+bleomycin 1.10.5 (98%)Carvacrol (406.0 mM)+bleomycin 1.20.7 (97%)Carvacrol (203.0 mM)+bleomycin 1.30.7 (95%)Carvacrol (101.5 mM)+bleomycin 1.70.7 (89%)Carvacrol (50.7 mM)+bleomycin 4.20.9 (50%)Carvacrol (25.4 mM)+bleomycin 6.62.0 (12%) n.sCarvacrol (12.7 mM)+bleomycin 7.61.1 (0%) n.sCarvacrol (6.3 mM)+bleomycin 7.51.9 (0%) n.sDistilled water (negative control) 1.00.2

347G.C. Vicua et al. / Fitoterapia 81 (2010) 343349Bleomycin (positive control) 7.41.0Thymol (912.0 mM) 0.90.4Thymol (912.0 mM)+bleomycin 0.50.2 (100%)Thymol (456.0 mM)+bleomycin 0.80.2 (100%)Thymol (228.0 mM)+bleomycin 1.00.3 (100%)Thymol (114.0 mM)+bleomycin 1.40.4 (94%)4. Discussion

This study focused on chemical composition and anti-genotoxic properties of L. origanoides EO obtained by

Thymol (57.0 mM)+bleomycin 1.80.8 (87%)Thymol (28.5 mM)+bleomycin 3.91.3 (55%)Thymol (14.2 mM)+bleomycin 7.31.6 (2%) n.sThymol (7.1 mM)+bleomycin 7.71.1 (0%) n.sDistilled water (negative control) 1.00.1Bleomycin (positive control) 4.20.6EGCG (4.00 mM) 0.70.4EGCG (4.00 mM)+bleomycin 0.70.2 (100%)EGCG (2.00 mM)+bleomycin 1.40.4 (87%)EGCG (1.00 mM)+bleomycin 2.00.6 (67%)EGCG (0.50 mM)+bleomycin 2.60.8 (47%)EGCG (0.25 mM)+bleomycin 3.10.9 (30%) n.sEGCG (0.12 mM)+bleomycin 3.20.7 (27%) n.sEGCG (0.06 mM)+bleomycin 3.30.6 (24%) n.sEGCG (0.03 mM)+bleomycin 4.21.0 (0%) n.s

Bleomycin was used at dose of 1 g/m. Carvacrol and thymol densities were0.966 and 0.965 g/mL, respectively; according to technical product data(Sigma-Aldrich Co. St. Louis. Missouri. USA). Carvacrol and thymol doseranges were estimated based on their amount (%) in the chromatogram andthe oil density as indicated in Table 2. Average IF values from a minimum ofthree independent experiments with four replicate each and thecorresponding standard error are given. Percentage of genotoxicityinhibition (% GI) was calculated as indicated in Materials and methods.Signicant reduction (pb0.05) with respect to positive control was foundusing Student's t-test. n.s. No signicant reduction was found.

Table 4Lippia origanoides chemotypes according to the major constituent amount (%) in th

Chemotypes Major constituents (%)

Carvacrol Thymol -Cymene

A 1116B 3343 58

39 18 104452 714 910

C 817 4862 60 826 034 9

Only compounds with percentage higher than 5% were considered. Order A to C wacarvacrol and thymol content.that L. origanoides chemotypes can be inuenced by differencesin soils and climatic conditions [2]. At this study, we founddifferences in theproportionof carvacrol studyingL. origanoidesspecimens with similar origin (Chicamocha river canyon,Santander, Colombia) and that were propagated under similarclimatic conditions at the university campus (UIS). This resultsuggests that additional factors can inuence the quantity andproportion of constituents into EO.

L. origanoides is widely distributed in Colombia and it canbe founded in several Andean states and in the northernpeninsula of Guajira. Since L. origanoides can be easily adaptedto grow in commercial gardens this specie is very promissoryas commercial seasoning. As previously indicated for Oreganospices [39], the carvacrol content is the prerequisite key forselecting the adequate plant specimen for the preparation ofOregano condiment. High carvacrol content determines theplant's odor and therefore, spices quality. Considering this,the specimen COL516290 appears as the best candidate forseasoning preparation. A study that compares the quality ofthe Colombian L. origanoides specimens as spices is now inprogress in our laboratory.

This work presents the rst report on antigenotoxicproperties of L. origanoides EO. The oils signicantly reducedbleomycin-induced genotoxicity in a dose dependent man-ner; an effect that was directly related with carvacrol andthymol constituents (Table 3). Antioxidant effects fromL. origanoides EO and from other oregano species have beenpreviously related with the presence of carvacrol and thymolin the oils [6,4042]. In addition, carvacrol and thymolresulted to be antigenotoxic against H2O2-induced genotoxi-city in human colonic Caco-2, hepatoma HepG2 and leukemicK562 cell lines [31,32]. Thymol also inhibited antioxidant

e essential oils reported in the literature.

References

1.8-Cineole -Terpinene

711 Stashenko et al. [6] 810 Dos Santos et al. [2] Oliveira et al. [3]

710 Stashenko et al. [6] Rojas et al. [5] 7 Present work 9 Present work

s assigned considering potential use of oregano as condiment based on high

348 G.C. Vicua et al. / Fitoterapia 81 (2010) 343349activity against malonaldehyde formation from blood plasmaoxidation [43].

Since bleomycin genotoxicity involves generation ofradicals on the DNA molecule which induce DNA strandbreakages [44] and both carvacrol and thymol can interactwith DNA [45], it can be expected that the antigenotoxic effectof L. origanoides EO and/or carvacrol and thymol againstbleomycin occurs through radical scavenging mechanisms onthe DNAmolecule. Thymol also increases antioxidant enzymeactivities in rat [30], therefore reduction of DNA radicalscaused by thymol-increased antioxidant enzymes could notbe discarded as a potential antigenotoxic mechanism. How-ever, these hypotheses could be examined in further studies.

Based on literature data [24,6], L. origanoides clearly hasbroad-ranging therapeutic potential. The antigenotoxic prop-erties against bleomycin widen its potential as a source ofcompoundswith application in cancer chemoprevention.Otheroregano species whose EO have high fraction of carvacrol andthymol, such as Lippia graveolens, Origanum compactum, Ori-ganum onites, Thymus spicata and Thymus vulgaris, have shownantigenotoxic properties against known mutagens [2226].Carvacrol and thymol have also exhibited protective, antitumorand anti-carcinogenic properties in different experimentalmodels [2732,4648]. All these works highlight the potentialbenet of L. origanoides EO and its major components carvacroland thymol asdietary supplementswith chemopreventive and/or antioxidant properties.

Several reasons support the use of L. origanoides EO aschemopreventive agent. First, the L. origanoides species canbe easily established from wild to cultivated conditions withadequate EO yield levels (between 1.4 and 2.0%). Second, theL. origanoides EO with high thymol content (between 326 and566 mg/mL) supports direct preparation of therapeuticformulations. Third, previous work [49] has demonstratedsystemic thymol availability in human plasma (limit ofquantitation of 8.1 ng/mL) after intake of oral therapeuticdoses of thyme preparation, which indicates clinical usefeasibility. Four, carvacrol and thymol are natural enhancersof transdermal drug delivery by increasing percutaneouspermeation [50], supporting the simultaneous use of thesecompounds in gel preparation for skin chemoprotection.

In conclusion, this study showed the antigenotoxicproperties of L. origanoides EO and carvacrol and thymolagainst the drug bleomycin, supporting the potential of theoils and compounds in chemoprevention and cancer therapy.Since the role of chemopreventive agents in the etiology ofcancer is very complex and involves several modes of actionand our results concern only in vitro experiments with abacterial assay, additional animal and human studies involv-ing different endpoints should be addressed in order to clarifythe antimutagenic potential of L. origanoides EO and its majorconstituents carvacrol and thymol. In addition, harmonizedstudies on genotoxicity of carvacrol and thymol using abattery of in vivo assays that evaluates different levels of DNAdamage expression will be required for practical use of thesecompounds in chemoprevention.

Acknowledgments

The authors wish to thank Dr. Jos Luis Fernndez Alonsofor botanical identication of specimens and Dr. MontserratLlagostera Casal from Universidad Autnoma de Barcelonafor gently supplies the PQ37 E. coli strain. This work wassupported by the Vice-Rectory for Science Research atUniversidad Industrial de Santander (UIS, Grant 5154), andby the Colombian Institute of Science and Technology,CENIVAM-COLCIENCIAS (Grant RC-432-2004).

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349G.C. Vicua et al. / Fitoterapia 81 (2010) 343349

Chemical composition of the Lippia origanoides essential oils and their antigenotoxicity agains.....IntroductionMaterials and methodsChemicalsPlant materialEO extraction and chromatographic analysisBacterial strains and cultureGenotoxicity assayAntigenotoxicity assayStatistical analysis

ResultsEO analysisAntigenotoxic effect of L. origanoides EOCarvacrol and thymol antigenotoxic effect

DiscussionAcknowledgmentsReferences