* CorrespBiológica,Regional dCE, 63105-E-mail: hdm

PHYTOTHERAPY RESEARCHPhytother. Res. 27: 179–185 (2013)Published online 18 April 2012 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/ptr.4695

Copyright

Topical Antiinflammatory Activity of EssentialOil of Lippia sidoides Cham: Possible Mechanismof Action

Helenicy N. H. Veras,1 Mariana K. A. Araruna,2 José G. M. Costa,1 Henrique D. M. Coutinho,3*Marta R. Kerntopf,2 Marco A. Botelho1 and Irwin R. A. Menezes21Laboratório de Pesquisa em Produtos Naturais, Universidade Regional do Cariri–URCA, Rua Cel. Antonio Luis 1161, Pimenta,Crato, CE, 63105-000, Brazil2Laboratório de Farmacologia e Química Molecular, Universidade Regional do Cariri–URCA, Rua Cel. Antonio Luis 1161, Pimenta,Crato, CE, 63105-000, Brazil3Departamento de Química Biológica, Laboratório de Microbiologia e Biologia Molecular, Universidade Regional do Cariri–URCA,Rua Cel. Antonio Luis 1161, Pimenta, Crato, CE, 63105-000, Brazil

This work reports the chemical composition of the essential oil of Lippia sidoides (EOLS) and evaluation of thetopical effect of EOLS and thymol against different irritant agents in vivo. The essential oil was obtained byhydrodistillation, and gas chromatography/mass spectrometry analysis identified the main constituents: thymol(84.9%) and p-cymene (5.33%). The antiinflammatory activity was evaluated using the mouse models of acuteear inflammation induced by croton oil, arachidonic acid, phenol or histamine, and chronic inflammation inducedby croton oil. The topical application of EOLS or thymol at a dose of 2mg/ear significantly reduced ( p< 0.001)ear edema induced with arachidonic acid by 45.1% and 47.4% and reduced ear edema induced with phenol by33.2% ( p< 0.05) and 54.7% ( p< 0.01) in acute ear edema. However, a proinflammatory effect of EOLS andthymol was evidenced when it was applied for more than 1 day. There were no statistical differences inantiedematogenic activity between EOLS and thymol. In conclusion, the results indicate that thymol is theconstituent responsible for the topical antiinflammatory activity of EOLS. Thus, these findings could justify thepopular use of L. sidoides by alternative medicine, but chronic use has an inflammatory effect. Copyright ©2012 John Wiley & Sons, Ltd.

Keywords: Lippia sidoides; thymol; antiinflammatory activity; ear edema; essential oil.

INTRODUCTION

Medicinal plants have been traditionally used for woundhealing, fever, infection, edema, or rheumatic diseases,which indicates the presence of compounds with anti-inflammatory properties, and therefore, they should beinvestigated and their effectiveness determined. Theevolution of our understanding of the molecular aspectsof the physiopathology of inflammation has led to theestablishment of new test systems for the selection ofvarious substances that allow the identification of newantiinflammatory compounds (Carvalho, 2004).Antiinflammatory compounds are useful in the thera-

peutic treatment of diseases, such as non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids.Medicinal plants are used in the popular medicine ofmany countries to treat different inflammations of theskin. However, in many cases, the effectiveness, themechanism of action and the active principles for suchactivity are ignored (Falcão et al., 2005).Essential oils (also called ethereal oils or essences)

are complex mixtures of liquid volatile substances,

ondence to: HenriqueD.M. Coutinho, Departamento deQuímicaLaboratório de Microbiologia e Biologia Molecular, Universidadeo Cariri–URCA, Rua Cel. Antonio Luis 1161, Pimenta, Crato,000, Brazil.coutinho@gmail.com

© 2012 John Wiley & Sons, Ltd.

which are lipophilic and usually odoriferous and whichcan be obtained from several parts of the plant (flowers,leaves, branches, peels, fruits, roots, etc.). The chemicalcomposition of these oils from different species can bequite complex, including hydrocarbons, terpenes, phe-nols, esters, furans, or organic acids. In the mixture, suchcompounds are present at different concentrations, whereone of them comprises the majority, with others existingin smaller concentrations and some in trace amounts(Santos, 2007).

The species Lippia sidoides Cham. (Verbenaceae),popularly known as “alecrim pimento,” is a native bushof the semi-arid area of Northeast Brazil, widely used inpopular medicine as an antiseptic for local use on theskin and mucous membranes (Lorenzi and Matos,2002). The essential oil of the leaves is rich in thymol,a monoterpene phenolic with antimicrobial activity,larvicidal action, and antioxidant, antiinflammatory, andanalgesic properties, among others (Monteiro et al.,2007; Pavela, 2009; Rivas et al., 2010; Ozen et al., 2011).

The essential oil of L. sidoides (EOLS) showedpotential gastroprotective and topical antiinflammatoryactivities when used at different concentrations, probablydue to its antioxidant properties (Monteiro et al., 2007). Itis believed that the major representative, thymol, isresponsible for the biological activities of the EOLS, butthis had not yet been proven, as the essential oil cancontain minor substances that can also be responsiblefor the biological activities. For instance, the essential oil

Received 21 September 2011Revised 07 March 2012

Accepted 13 March 2012

180 H. N. H. VERAS ET AL.

ofCordia verbenaceae contains 4.64% a-humulene, whichaccounts for the antiinflammatory activity (Carvalhoet al., 2004; Fernandes et al., 2007).Therefore, the objective of this work was to evaluate

the topical antiinflammatory activity of the EOLS andthymol in animal models, as well as to elucidate thepossible mechanism of action.

MATERIALS AND METHODS

Plant material. Leaves of L. sidoides Cham. werecollected in August 2010 from the Small Aromatic andMedicinal Plants Garden of the Natural ProductsResearch Laboratory (LPPN) at University Regionaldo Cariri (URCA), Crato City, Ceará State, Brazil. Avoucher specimen was sent to the Herbarium CaririenseDárdano de Andrade, Lima, Department of BiologicalSciences (URCA), which was deposited under registra-tion no. 3038.

Chemicals and drugs. Croton oil, arachidonic acid(AA), capsaicin, histamine dihydrochloride, indometh-acin, and thymol were purchased from the Sigma Chem-ical Co. (St. Louis, MO, USA). Dexamethasone (DEX;DecadronW) was obtained from Aché (São Paulo,Brazil). Ketamine hydrochloride and xylazine hydro-chloride were purchased from Syntec (Brazil). Acetoneof analytical grade was from Dinâmica (Brazil).

Animals. Male and female Swiss mice (Mus musculus),weighing 25–35 g, were previously housed in standardpolypropylene cages under controlled conditions oftemperature (22� 2�C) and 12-h light/dark cycle, withfree access to water and rodent chow (LabinaW, Purina,Brazil). Mice were allowed to adapt to the laboratoryenvironment for at least 1 h before testing. All the pro-cedures were previously approved by the Research Eth-ics Committee of University of Fortaleza (Brazil), underno. 010/2010.

Essential oil. Samples of L. sidoides fresh leaves (140 g)were triturated and submitted to a hydrodistillationprocess in a Clevenger-type apparatus for 2 h. Thecollected essential oil was subsequently dried usinganhydrous sodium sulphate (Na2SO4) and stored underrefrigeration at <10 �C until analyzed and tested.

Analysis of essential oil. Analysis by CG/MS of theessential oil was carried out on a Hewlett-PackardModel 5971 GC/MS using a non-polar DB-1 fused silicacapillary column (30m� 0.25mm i.d., 0.25mm filmthickness), with helium as the carrier gas and flow rateof 0.8mL/min, using split mode (Palo Alto, California,USA). The injector temperature and detector tempera-ture were 250 and 200 �C, respectively. The column tem-perature was programmed from 35 to 180 �C at 4 �C/minand then 180 to 250 �C at 10 �C/min. Mass spectrometrywas by electron ionization, with ionization potential of70 eV, ion source temperature of 250 �C, ionizationcurrent of 1000mA, resolution of 1000, and mass rangeof 30–450m/z. Individual components were identified bycomputer matching against the library spectra (LibraryDatabase Wiley 275) and two other computer MS librarysearches by using retention indices as a pre-selection

Copyright © 2012 John Wiley & Sons, Ltd.

routine (Alencar, 1990); their retention indiceswere in refer-ence to an n-alkane series in a temperature-programmedrun, interpreting their fragmentation pattern and compari-son of the mass spectra with the literature data.

Croton oil-induced mouse ear edema: single application.Groups of seven mice were previously treated on theinner and outer surfaces of the right ear with 20 mL ofEOLS or thymol diluted in acetone at a concentrationof 100mg/mL (4mg/ear). The negative control receivedtopically 20 mL of vehicle (acetone) on the right ear.DEX (0.08mg/ear) was used in positive controls. After15min, edema was induced in the right ear by topicalapplication of 20 mL of croton oil (5%, v/v) in acetone,whereas the left ear received 20 mL of vehicle acetone.Ear edema was evaluated 6 h after croton oil application(Tubaro et al., 1985).

Croton oil-inducedmouse ear edema: multiple application.Inflammation was induced in mice (n=7/group) for9 days (days 0–8). Croton oil (5%, v/v) in acetone(20mL/ear) was applied on the right ear and acetoneon the left ear of mice (n=7/group) with a micropipetteon alternate days. Ear edema was evaluated daily bymeasuring ear thickness. On days 4–8, the mice weretreated on the inner and outer surfaces of the right earwith EOLS or thymol (20mL/ear), saline solution(0.9% NaCl, 20mL/ear), or DEX (0.08mg/ear) twice aday. On day 8, the mice were killed, and 6-mm-diameterear punch biopsies were collected and weighed (Stanleyet al., 1991).

Arachidonic acid-induced and phenol-induced mouseear edema. Inflammation was induced in mice (n=7/group) by applying on the inner and outer surfaces ofthe right ear 20mL of the following irritants: AA, 0.1mg/mLin acetone, and 10% (w/w) phenol in acetone. Fifteenminutes before the application of each irritant agent, theright ears were topically treated with EOLS or thymol(20mL/ear), saline solution (negative control, 20mL/ear),indomethacin (positive control for AA, 2mg/ear), orDEX (positive control for phenol, 0.08mg/ear). Earedemawas evaluated 1h afterAAand phenol application(Young et al., 1984; Gábor, 2000).

Subcutaneous histamine-induced mouse ear edema.Mice (n= 7/group) were previously anesthetized withketamine (20mg/kg i.p.) and xylazine (10mg/kg i.p.)Afterwards, their right ear was treated topically withsaline solution (20mL/ear), DEX (0.08mg/ear), or EOLSor thymol (20mL/ear). Fifteen minutes later, we inducededema in the right ear by intradermal application of5mL of histamine dihydrochloride (0.1mg/mL) dissolvedin phosphate-buffered saline (PBS, pH7.4) by using asyringe with a 29-G hypodermic needle, whereas the leftear received 5mLof PBSby using the same aforementionedprocedure (sham). Ear edema was evaluated 2h afterapplication of the histamine solution (Brand et al., 2002).

Ear edema measurement. Edema was expressed as thepercentage increase in ear weight (all models) or as earthickness variation (croton oil multiple application-inducedear edema) due to inflammatory challenge. Ear thicknesswas measured with a digital caliper (Jomarca). Thedigital caliper was applied near the tip of the ear justdistal to the cartilaginous ridges, and the thickness was

Phytother. Res. 27: 179–185 (2013)

181LIPPIA SIDOIDES

recorded in micrometers (mm). To evaluate the earweight, we previously anesthetized all animals withketamine (20mg/kg i.p.) and xylazine (10mg/kg i.p.),killed them by cervical dislocation, obtained 6-mm-diameter ear punch biopsies by using a metal punch,and individually weighed the biopsies on aMettler Toledo(AB204) balance. The extent of the edema was expressedas the percentage increase in ear punch biopsy weight(%), using the following formula: percentage of edemaweight (%)= [(wRE�wLE)� 100]/wLE, where wRE is thepunch biopsy weight obtained from the right ear(inflamed) and wLE is the punch biopsy weight obtainedfrom the left ear (non-inflamed). The mean of thepercentage (%) of inhibition of edema was calculated bycomparing it with the negative control group.

Statistical analysis. We expressed the results as mean�standard error of mean (SEM) by using the PrismaProgram. The comparison between groups was per-formed by one-way analysis of variance (ANOVA)followed by Student–Newmann–Keuls test. The timeeffect for ear edema volume was subjected to two-wayANOVA (treatment� time) followed by Bonferroni’spost hoc t test (to the replicated measures). Values ofp< 0.05 were considered as statistically significant.

A)

0 1 2 3 4 5 6 7 8 9 100

300

600

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1500

1800Acetone

DEX 0,08 mg/ear

EOLS 2 mg/ear

Thymol 2 mg/ear

*** *** *** ***

Topical treatment

******

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***

***

***

Time of evaluation (days)

Ear

Th

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ess

( m

)

B)

100

150

200

**

*****

of

edem

a w

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ht(

%)

RESULTS

Chemical composition of EOLS. The essential oilobtained by hydrodistillation of fresh leaves of L. sidoidesgave a yield of 1.06% (w/w). The major constituents of theEOLS were thymol (84.9%), p-cymene (5.33%), andethyl-methyl carvacrol (3.01%) (Table 1). Studies in theliterature have demonstrated that the thymol concentra-tion in this essential oil can vary between 34% and 95%(Girão et al., 2003; Fontenelle et al., 2007). These variationsare explained by climate conditions, crop developmentconditions, and the processing of the crop powder(Lavabre, 1992).

Topical antiinflammatory activity. The irritant agents(croton oil, AA, phenol, and histamine) caused an inflam-matory response with edema formation when comparedwith vehicle (acetone). The present study demonstratedthat EOLS and thymol reduced the edema formed by

Table 1. Chemical components of essential oil of Lippia sidoidesfresh leaves

Component RI (min)a IK Percentage

p-Cymene 4.2 1020 5.331,8-Cineol 4.4 1031 1.68g-Terpinene 5.0 1060 1.32Ethylmethyl carvacrol 9.7 1164 3.01Thymol 11.8 1288 84.9Carvacrol 12.9 1292 0.41b-Caryophyllene 15.1 1418 1.17Total identified 97.82

IK, Kovats index.aRetention index: n-alkanes were used as reference points in thecalculation of retention indices.

Copyright © 2012 John Wiley & Sons, Ltd.

the topical application of AA and phenol. There wereno statistical differences between EOLS and thymol withregard to antiedematogenic activity.

Croton oil-inducedmouse ear edema: multiple application.The analysis of the ear edema induced by multipleapplications of croton oil (a model of chronic inflammatoryresponse once daily) and by the application of EOLS orthymol (2mg/ear, twice daily for 4days) indicated a sig-nificant increase in the ear thickness after 5days (96h)after challenge with the croton oil (p< 0.001) (Fig. 1A).We observed necrosis of the treated ears when comparedwith the control group. Besides, DEX (0.08mg/ear, twicedaily for 4days) reduced the ear edema after 6, 7, 8, and9days, respectively (p< 0.001) (Fig. 1A). The weight ofthe edema showed a reduction when treated with DEX,showing a weight reduction of 46% (p< 0.01). However,when treated with EOLS or thymol, the treatmentshowed an increase of the edema of 59% and 62%,respectively (Fig. 1B).

Croton oil-induced mouse ear edema: single application.The essential oil of L. sidoides or thymol (2mg/ear)showed no antiedematogenic effect 6 h after topicalapplication of croton oil, when compared with the group

Contro DEX EOLS Thymol0

50

Croton oil multiple application

Per

cen

tag

e

Figure 1. Effect of EOLS and thymol on croton oil multiple applica-tion-induced ear edema. (A) The time–response curve of effect fromdays0 to 8. The croton oil in acetone was applied on alternate days(once daily). The thickness of the ear was measured daily, usinga digital caliper. On days4–7, the ear of the animals receivedsaline solution, dexamethasone (DEX; once daily), EOLS, or thymol(2mg/ear, twice daily in 12/12h—arrows indicate the days whenthe treatment occurred). The effect of the compoundswas examinedby varying the thickness of the ear, calculated as the differencebetween the initial and final thickness. Thepoints represent themeanof six animals and vertical bars means�SEM (two-way ANOVAfollowed by Bonferroni’s post hoc test). (B) The percentage of edemaweight of each group on day 8 (one-way ANOVA followed byStudent–Newman–Keuls post hoc test). *p<0.05, **p<0.01,and ***p<0.001 compared with saline-treated group.

Phytother. Res. 27: 179–185 (2013)

182 H. N. H. VERAS ET AL.

treated with acetone (negative control). In this model,only the positive control group, treated with the DEX(0.08mg/ear), showed a significant reduction in edemacompared with the negative control, with 76% inhibitionof inflammation (p< 0.001) (Fig. 2A).

Arachidonic acid-induced and phenol-induced mouseear edema. Arachidonic acid and phenol promoted theformation of edema 1h after topical application, in thesame way as described in the literature (Young et al.,1984; Crummey et al., 1987; Gábor, 2000). In these models,EOLS and thymol (2mg/ear) significantly reduced earedema 1h after application of the irritant, when compared

A)

Control DEX EOLS Thymol0

50

100

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Croton oil single application

nsns

B

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50

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D

Figure 2. Effect of OELS and thymol (2mg/ear/one time), dexamethasoone time) on mice ear edema induced by different irritant substan(AA—0.1mg/mL in acetone/one time); (C) phenol (10% (w/w) phenol ingroup received vehicle (acetone, saline, or PBS—20mL/ear/one time) as*p<0.05, **p<0.01, ***p<0.001, and ns=not significance comparedKeuls post hoc test).

Table 2. Effect of EOLS, thymol, and dexamethasone on ear edema in

Treatment

Phenol

Percentage of edema

Negative control 99.68�14.70Dexamethasone (0.08mg/ear) 47.27�6.83**EOLS 66.62�6.31*Thymol (2mg/ear) 45.13�9.60**

Data expressed as mean�SEM. AA, arachidonic acid. *p<0.05; **p<0ANOVA and Student–Newman–Keuls post hoc test).

Copyright © 2012 John Wiley & Sons, Ltd.

with the group control (Fig. 2B, C). In the edema inducedby AA, inhibition was 58%, 45%, and 47% (p< 0.001)for indomethacin (2mg/ear), EOLS, and thymol, respec-tively (Fig. 2B). Table 2 shows a mean inhibitory effect of33% (p< 005) for EOLS, 55% (p< 001) for thymol, and53% (p< 0.01) for DEX (0.08mg/ear) in the model ofear edema induced by phenol. There were no significantdifferences between the groups treatedwithDEX (positivecontrol), EOLS, and thymol.

Subcutaneous histamine-induced mouse ear edema. Thetreatment with EOLS or thymol administered topically(2mg/ear), 2 h after the intradermal application of

Per

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))

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ne (DEX—0.08mg/ear/one time) or indomethacin (IND—2mg/ear/ces. (A) Croton oil (20mL/ear/one time); (B) arachidonic acidacetone/one time); (D) histamine (0.1mg/mL/one time). The controltreatment. Each point represents the mean�SEM of seven mice.with control group (one-way ANOVA followed by Student–Newman–

duced by phenol and AA, single application

AA

IE (%) Percentage of edema IE (%)

– 89.97�10.10 –

52.6 37.42�4.70*** 58.433.2 49.42�3.16*** 45.154.7 47.30�8.64*** 47.4

.01; ***p<0.001 compared with negative control group (one-way

Phytother. Res. 27: 179–185 (2013)

183LIPPIA SIDOIDES

histamine (13mg/ear s.c.) solution, was not able toreduce ear edema significantly compared with the controlgroup. DEX (0.08mg/ear) inhibited edema (p< 0.001),with amean inhibitory effect of 46% (Fig. 2D). There wereno statistical differences between treatment with EOLSand thymol.

DISCUSSION

Several experimental pharmacological models can beused to evaluate the antiinflammatory activity of drugs.Models of ear edema induced by irritant agents allow usto identify compounds with an antiinflammatory activity,which can be potentially useful in the treatment ofinflammatory diseases of the skin, because they promoteconditions that resemble some types of dermatitisobserved in humans (Vane et al., 1998).The results show that EOLS and thymol did not cause

suppression of the edema induced by single (Fig. 2A)and multiple applications of croton oil (Figs. 1 and 2)at the dose tested. In the chronic experiment, it wasobserved that both EOLS and thymol were able to pro-mote the increase in ear thickness 24h after the beginningof the treatment (Fig. 1), increasing the degree of edemaconsiderably on the last day of the experiment (Fig. 2).EOLS and thymol were also not capable of significantlyreducing the edema induced by histamine, comparedwith the negative control (saline) (p> 0.05). This resultsuggests that EOLS and thymol are not involved in theinhibition of the histamine produced in the edematogenicaction of croton oil.In inflammation produced in the croton oil model,

corticosteroids and lipooxygenase (LOX) inhibitorsdemonstrate the best activity in reducing ear edema,whereas cyclooxygenase (COX) inhibitors and anti-histamines demonstrate little or no effect (Green andShuster, 1987). Our results suggest that EOLS and thymolare possibly unable to inhibit LOX or PLA2, but theyhave an important action on the COX enzymes.The topical application of AA generates a fast in-

flammatory response characterized by intense erythemaand edema with small neutrophil accumulation, whencompared with cell migration determined in thecroton oil model, whose main mediators are PGE2,LTC4, and LTD4 (Crummey et al., 1987; Humes et al.,1986; Young et al., 1984). Indomethacin, an NSAIDwhose antiinflammatory action is related to the non-selective inhibition of the isoforms of COX (COX1and COX2), clearly reverses edema induced by thetopical application of AA, as described in the literature(Gabor, 2000). In the same way, EOLS and thymol werealso shown to be effective in the inhibition of theformation of edema in this model. Our results agreewith those obtained by Monteiro et al. (2007), whodemonstrated the topical antiinflammatory activity ofEOLS (containing 66.67% thymol) in the acute earedema model induced by TPA in mice at doses 1 and10mg/ear, reducing edema by 45.93% and 35.26%,respectively.The model of ear edema induced by TPA and AA

is very useful in the in vivo detection of COX/LOXinhibitors (Carlson et al., 1985). However, the metabolitesof AA also promote the degranulation of mastocytescells, so that histamine release contributes partially to

Copyright © 2012 John Wiley & Sons, Ltd.

the formation of edema in this model (Camp, 1982). Simi-larly, it should be considered that the AA model is notspecific for the identification of compounds that inhibitCOX exclusively or LOX, because other antagonisticagents of histamine and antioxidants also reduce theedema induced byAA (Crummey et al., 1987). In general,corticoids do not show any effect against AA, whereassome monoterpenes inhibit AA metabolism (Juergenset al., 2003).

Thymol is capable of significantly inhibiting plateletaggregation induced by AA (Enomoto et al., 2001).Marsik et al. (2005) showed that thymol present in theseeds of Nigella sativa has an antiinflammatory activityas demonstrated by the inhibition of COX1, and theauthors suggested more studies related to this compoundfor possible use as an antiinflammatory. The essentialoil of Lippia gracilis, containing thymol as the majorcomponent (32.68%), was found to reduce inflammationin the paw induced by carrageenan and to have antinoci-ceptive effect in the acetic acid-induced writhing model(Mendes et al., 2010).

The inflammation induced by carrageenan involvescell migration and production of mediators includingnitric oxide, PGE2, IL-1, IL-6, and TNF-a (Loram et al.,2007). In the visceral pain model, there is release of AA,and through the COXs, there is the biosynthesis of prosta-glandins such as PGE2, which have an important role inthe nociceptive mechanism. Thus, in that study, thymolwas the component responsible for the observed pharma-cological activities, possibly involving the inhibition ofCOXs (Mendes et al., 2010).

The fact that EOLS and thymol inhibit edema inducedby an irritant agent such as AA and phenol demonstratesthe possible effectiveness of these in the treatment ofcontact dermatitis after challenge with irritant agents. Inresponse to an exogenous stimulus, such as phenol,keratinocytes produce important chemical mediators inprimary contact irritation, including cytokines associatedwith proinflammatory properties, such as IL-1, IL-8, andTNF-a (Lim et al., 2004; Wilmer et al., 1994).

Our results showed that in the chronic ear edemamodel, as observed in chronic inflammatory diseases,repeated use of EOLS and thymol treatment caused aproinflammatory effect and cutaneous damage. Apossible mechanism of action to account for this findinginvolves LOX enzymes. Chronic diseases, such asrheumatoid arthritis and psoriasis, involve an alterationin the release of proinflammatory cytokines (e.g., inter-leukins), growth factors, and proinflammatory enzymessuch as COX and LOX, which generate PGs and leuko-triene, respectively, resulting in leukocyte infiltration inskin (T cells, neutrophils and mastocytes) (Krueger andBawcock, 2005).

The study of Roschek et al. (2009) showed that SRB-AI extract (containing thymol) causes a dose-dependentinhibition of COX1 and COX2, selectively inhibitingCOX2 by an order of magnitude over COX1. No detect-able 5-LOX inhibition was found with SRB-AI. 5-LOXcatalyzes the biotransformation of AA into fatty acidhydroperoxides, lipoxines, and leukotrienes, such asB4, compounds implicated in allergy and suffering.Sahouo et al. (2003) showed that COX function ofPGHS was inhibited by only chemical constituents ofthe essential oil of Ocimum gratissimum, in whichthymol is the major component, with an IC50 value of125 mg/mL.

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184 H. N. H. VERAS ET AL.

Recently, Elhabazi et al. (2006) demonstrated thatthyme extracts increase the number of polymorpho-nuclear cells, total lymphocytes, and CD4+, CD8+, andNK cells. Neutrophils are important cells in host defenseagainst invading bacteria. These cells are also potentchemoattractants, and they are able to induce the forma-tion of reactive oxygen species (ROS) and the release oflysosome enzymes. However, these cells have also beenlinked to tissue destruction by stimulating the release ofeicosanoids (5-LOX derivatives) and other cytokines,such as IL-1 and TNF-a. IL-1 activates neutrophils andmacrophages, increasing the production and the releaseof reaction oxygen species and nitric oxide, which havebeen implicated in local tissue damage.Thus, our data suggest that EOLS and thymol exert an

antiinflammatory action (acute ear edema) involving theinhibition of COX enzymes, as is effectively demon-strated in the model of AA and phenol (Table 2).However, the proinflammatory effect observed inchronic ear edema can be explained by an increasedactivity of 5-LOX, a key enzyme in the biotransform-ation of AA into fatty acid hydroperoxides, lipoxines,and leukotrienes, with potent chemoattractant capacity,which induces the formation of ROS (Monteiro et al.,2007; Ozen et al., 2011).

Copyright © 2012 John Wiley & Sons, Ltd.

CONCLUSION

The antiinflammatory activity of the EOLS is explained, atleast in part, by the presence of the main constituent thy-mol. This work proves the efficacy of the topicalapplication of EOLS and thymol in models of skininflammation induced by AA and phenol, possiblyreducing the production of proinflammatory mediators inthe acute inflammation models. In repeated treatmentusing thymol or EOLS, there was an increase in inflamma-tion, which limits their use in chronic treatment. Theresults provide useful pharmacological informationrelated to the antiinflammatory activity of this species,suggesting its possible use as a source of active compoundswith antiedematogenic activity in acute treatment. Thus,this work could justify the popular use of L. sidoidesessential oil in alternative medicine and represents animportant contribution to the elucidation of the mecha-nism involved in the antiedematogenic effect of EOLS.

Conflict of Interest

The authors do not report any conflict of interest.

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