Topical continuous use of Lippia sidoides Cham. essential oil induces cutaneous inflammatory response, but does not delay wound healing process

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Topical continuous use of

Lippia sidoides

Cham. essential oil induces

cutaneous in

_ﬂ

ammatory response, but does not delay wound

healing process

Maria Liduína Maia de Oliveira

a

_{, Belise Maria Oliveira Bezerra}

a

_{, Luana Oliveira Leite}

a

_,

Virgínia Cláudia Carneiro Girão

b

, Diana Célia Sousa Nunes-Pinheiro

a,n

a_{Programa de Pós-Graduação em Ciências Veterinárias, Faculdade de Veterinária, Universidade Estadual do Ceará, Campus do Itaperi,} CEP 60740-903 Fortaleza, Ceará, Brazil

b_{Departamento de Morfologia, Faculdade de Medicina, Universidade Federal do Ceará, Campus do Porangabuçu, CEP 60430-170 Fortaleza, Ceará, Brazil}

a r t i c l e

i n f o

Article history:

Received 14 October 2013 Received in revised form 5 February 2014 Accepted 15 February 2014 Available online 25 February 2014

Keywords: Lippia sidoides

Verbenaceae Essential oil Inﬂammation Wound healing Skin

a b s t r a c t

Ethnopharmacological relevance: The essential oil ofLippia sidoides(EOLS) has been used in Brazilian folk medicine as a topical antiseptic agent in skin for treatment of wounds and superﬁcial infections of the body. The aim of this study was to investigate the effects of EOLS on intact and damaged skin, including its action on expression of mediators, COX-2 and VEGF, involved in healing full-thickness cutaneous lesions in vivo.

Material and methods: EOLS was analyzed chemically and used at different concentrations to dose-response experiments in skin mice. Skin irritation tests by one-dosage and multiple-dosages and irritation to damaged skin were assessed by macroscopy, morphometry and histological and immuno-histochemical analyses. To evaluate the effects of EOLS on wound healing, excision wounds were surgically created on the dorsum of rats, and the ointments at 6% and 12% were applied daily to the wound area. Cutaneous lesions were assessed by planimetric (wound contraction) and macroscopic parameters.

Results: Skin irritation tests showed that topical application of EOLS promoted cutaneous inflammation in varying degrees, which was demonstrated by increase of skin thickness and formation of cutaneous edema and erythema. Topical administration of EOLS in high concentrations presented an irritant response to skin, but this irritation is lighter when low concentrations this oil were used. Histological evaluation supported the outcome of these models, which revealed accentuated presence of infl amma-tory cells infiltration. In wound healing process, the lesions treated with EOLS showed intense edema and exsudation up to day 5, but there were not significant differences in the wound contraction on days 14 and 21. No immunohistochemical staining was verified to COX-2 and VEGF mediators in skin treated with EOLS 12%.

Conclusion: The continuous application of EOLS in adequate concentrations on cutaneous wounds increases inﬂammatory response without delay the lesions closure. The association of these results with antimicrobial action previously related to EOLS allows its indication as an alternative therapeutic modality for topical treatment of infected cutaneous wound. Nevertheless, further studies need to be performed to determine the mechanism of action and support its application in clinical practice.

1. Introduction

As the primary interface between the body and the

environ-ment, the skin provides a ﬁrst line of defense against infection,

trauma, or injury. Upon skin injury, a series of events take place aiming at the reconstruction of the wounded and cutaneous

homeostasis maintenance (Bangert et al., 2011). Wound repair is

a natural process of regenerating tissue with multiple pathways, which are immediately activated after an injurious stimulus and

can be divided into three overlapping phases: inﬂammation,

proliferation or granulation tissue formation, and tissue

remodel-ing (Velnar et al., 2009). During the inﬂammatory phase, leukocyte

cells play a key role in protecting the tissue against infections through phagocytosis, the antibacterial effects of oxygen radicals,

and the activation of complement (Rock et al., 2010). This response

is executed and regulated by an equally complex signaling network

Contents lists available atScienceDirect

journal homepage:www.elsevier.com/locate/jep

Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2014.02.030

n

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involving numerous enzymes, growth factors, cytokines and che-mokines. Of particular importance is the cyclooxygenase-2 (COX-2),

which is involved in the production of inﬂammatory mediators such

as prostaglandins (Rajakariar et al., 2006), and vascular endothelial

growth factor (VEGF), which regulated the angiogenesis during the

wound repair (Bao et al., 2009). In general, the inﬂammatory

response is a beneﬁcial event that leads to removal of the offending

factor, repair the damage, and then the recruited cells need to be

removed themselves with resolution of inﬂammation (Widgerow,

2011).

In inﬂammatory and wound healing processes, several

medic-inal plants and their diverse biological compounds such as terpenes, phenols, lignols and essential oils (EO) have been traditionally used to inhibit or accelerate these events, respectively (Monteiro et al., 2007; Cavalcanti et al., 2012; Riella et al., 2012; Veras et al., 2013).Lippia sidoidesCham. (Verbenaceae) is a native aromatic bush from semiarid areas of the northeast Brazil,

popu-larly known as “alecrim-pimenta” (Matos, 2007). Essential oil

obtained from its leaves presented high concentration of thymol and exhibits multiple biological activities, including antimicrobial (Bertini et al., 2005; Fontenelle et al., 2007; Veras et al., 2012),

antioxidant, gastroprotective and topical anti-inﬂammatory

(Monteiro et al., 2007; Veras et al., 2013), and oral antiseptic (Girão et al., 2003; Botelho et al., 2009). In northeast Brazil,

pharmaceutical formulations from the essential oil of Lippia

sidoides(EOLS) have been available by programs of herbal medi-cine in primary health care to treat cutaneous wounds and

super-ﬁcial infections in people served at regional hospitals (Matos,

2002), which make the skin a common target to pharmacologic

actions of this plant.

Despite topical acute anti-inﬂammatory (Monteiro et al., 2007;

Veras et al., 2013) and chronic inﬂammatory effects recently

attributed to EOLS (Veras et al., 2013), its safe and continuous

use on skin in different physiologic conditions, including the use in open cutaneous wound was not reported. Thus, the aim of this work was to evaluate the effects of topical application of EOLS on intact and damaged skin, including its action on the expression of mediators, COX-2 and VEGF, involved in the wound healing process in experimental models.

2. Material and methods

2.1. Plant material and chemical analysis

EOLS was purchased commercially from Technological Devel-opment Center (PADETEC) of the Federal University of Ceará. The chemical composition of EOLS was determined by gas chromato-graphy coupled to mass spectrometry, using a Shimadzu 5050 GCMS-QP instrument under the following conditions-column: W

Scientiﬁc DB-5MS fused silica capillary column (50 m0.25 mm);

carrier gas: He (1 mL/min); injector temperature: 2501C; detector

temperature: 2001C; column temperature: 35–1801C at 41C/min

and then 2501C/15 min; mass spectrum: electronic impact 70 eV.

The identiﬁcation of the constituents was performed by a

computer-based library search, retention indices and visual

inter-pretation of the mass spectra (Alencar et al., 1984; Adams, 1989).

2.2. Experimental animals

Male Swiss albino mice (25–30 g) and male Wistar rats (150– 180 g) were used in the study. Animals were individually housed in polypropylene cages under standard experimental conditions of

humidity (40–45%), temperature (23–251C), 12 h light/dark cycle

and fed on normal pellet diet and water ad libitum. All experi-mental protocols were approved by Ethics Committee for Use of

Animals of the State University of Ceará (protocol no 10340180-6/ 2010).

2.3. Skin irritation tests

Skin irritation tests were performed as previously described

procedure (Jia et al., 2008) with some modiﬁcations. Swiss mice

were shaved on the dorsal surface of the body, and then were left under close observation for 24 h in order to ascertain no abnormal skin responses. The shaved animals were randomly allocated into

ﬁfteen groups (n¼6/group), that received EOLS (10

μ

L) at 100% (in

natureEOLS), 50%, 25% and 12% (v/v) in mineral oil (control group),

applied topically in skin nude area of about 1 cm2_{, as following:}

2.3.1. One-dosage irritation to healthy skin

Mice were treated with EOLS at different concentrations in a single dose.

2.3.2. Multiple-dosages irritation to healthy skin

Mice were repeatedly treated with EOLS at different concen-trations, once per day, for consecutive 7 days.

2.3.3. Irritation to damaged skin

Mice skin abrasion was made using a scalpel blade until the

presence of the noticeable tissueﬂuid, but not blood. The animals

were treated with EOLS in a dose (10

μ

L) fractionated three times a

day for one day.

Dermal reactions to the skin challenge, including edema and erythema, were evaluated by macroscopic examination daily for 7 consecutive days. Scoring method for skin irritation was

per-formed as follows: 1–absent; 2–light; 3–moderate; 4–intense.

Skin thicknesses were measured before and after application of treatments, every 24 h, using a micrometer (MMD IP54). On day 7, the animals were euthanized and skin samples were removed for histological and immunohistochemical analyses.

2.4. Histological analysis

Skin sample wereﬁxed in 10% neutral buffered formalin and

were embedded in parafﬁn wax by usual histological processing.

Five-micrometer sections were cut and stained with hematoxylin-eosin. A representative area was selected for descriptive light

microscopic analysis (Nikon, Tokyo, Japan) at 400x magniﬁcation.

2.5. Immunohistochemical analysis

Immunohistochemical investigations were performed on parafﬁ

n-embedded sections from skin tissue on day 7 for COX-2 and

VEGF. Sections of 5

μ

m were mounted on positive-charged glass

slides and submitted to retrieval antigen process with Dako EnVision TM FLEX Target Retrieval Solutions High pH (Code

DM828) or Low pH (Code DM829) for 20 min at 971C using the

Dako pre-treatment (PT) link module (Dako, Glostrup, Denmark). Endogenous peroxidase activity was inhibited by Peroxidase Block (Dako) for 5 min, and slides were subjected either to mouse monoclonal anti-human COX-2 (clone CX-294; Dako) diluted 1:100; and mouse monoclonal anti-human VEGF (clone VG1; Dako) diluted 1:100 and incubated for 1 h at room temperature. The slides were then washed three times in phosphate-buffered

saline (PBS, pH¼7.2) and then incubated with EnVision polymer

reagent (EnVision TMþDual Link System/HRP; Dako) for 30 min at

room temperature andﬁnally diaminobenzidine (DAB; Dako) for

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endometrium were used as positive control for COX-2 and VEGF, respectively, according to the manufacturer's instructions (Dako Corporation, Carpinteria, CA, USA). The intensity of the staining was analyzed by light microscopy (Nikon, Tokyo, Japan) at 400x

magniﬁcation.

2.6. Wound healing evaluation

Excision wound model was used to evaluate the effects of EOLS on wound healing. Wistar rats were anesthetized with an intra-peritoneal injection of xylazine (5 mg/kg) and ketamine (80 mg/

kg) (Sadigh-Eteghad et al., 2013). The dorsal surface of each animal

was shaved and area was disinfected with povidone-iodine.

Then, an area approximately 4 cm2 _{was delimited on the dorsal}

medial line, and one full-thickness wound was created with sterile

surgical blade and scissors as previously described (Magalhães

et al., 2008).

The animals were randomly divided into ﬁve groups (n¼

6/group). Test group was treated with EOLS ointment 6% and 12% (v/w). Control groups received ointment vehicle (vaseline and

lanolin–1:2) or 0.9% saline. Reference group was treated with 5%

clostebol acetate and neomycin sulfate cream. The treatments were applied topically once a day, starting from the wound induction until complete healing in enough quantity to cover all wounds. The wounds were left undressed to the open

environ-ment and observed daily (Oliveira et al., 2010). Inﬂammatory

parameters, edema and exudation, were monitored by

macro-scopic examination and graded on a four-point scale: 1–absent; 2

–light; 3–moderate; 4–intense.

Planimetrical analysis was performed on days 0, 3, 7, 14 and 21 on anesthetized animals. The contraction rate was assessed by tracing the raw wound on each evaluation day using transparency paper and a permanent marker. The wound area and one piece of

millimeter paper with known area (1 cm2_{) were digitalized using a}

scanner (Hewlett–Packard, Palo Alto, CA, USA). The measuring wound area was obtained with images analyses as previously

described (Oliveira et al., 2010). Thus, the unhealed wound area

and the percentage of wound contraction were calculated as reduction of initial wound size and used for statistical analysis.

2.7. Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.0

software (San Diego, CA, USA). The comparison between groups was carried out by ANOVA followed by Student-Newmann-Keuls

test. The analysis of the inﬂammatory parameters was performed

by Kruskal–Wallis test, followed by Dunn test. Results were

expressed as mean7standard deviation (SD) and values of

po0.05 were considered as statistically signiﬁcant

3. Results

The chemical analysis of EOLS is displayed inTable 1. The main

constituent was thymol, but other minor constituents were also

identiﬁed.

The results of skin irritation tests showed that topical

applica-tion of EOLS promoted cutaneous inﬂammation in varying

degrees. In one-dosage irritation to healthy skin model, EOLS

100% signiﬁcantly increased skin thickness (po0.05) throughout

the study when compared to the control group (Fig. 1). Cutaneous

edema and erythema were more intense in mice treated with EOLS 100% in comparison to the others treatment groups on day 3 (Fig. 4). On day 7, it was observed epidermal discontinuity and

moderate polymorphonuclear cells inﬁltration (Fig. 5A).

In multiple-dosages irritation to healthy skin model, EOLS 100%

treated group revealed intense inﬂammatory parameters in

cuta-neous tissue on day 3 (Fig. 4). On day 5, this group showed a

Table 1

Percentage composition of EOLS obtained by gas chromatography/mass spectrometry.

Constituents Yield (%)

Myrcene 2.12

α-Terpinene 0.50

p-Cymene 7.51

γ-Terpinene 0.80

Thymol methyl ether 1.45

Thymol 70.97

Carvacrol 0.30

Eugenol 0.11

Caryophyllene 8.30 Caryophyllene oxide 1.59 Others constituents 6.35

400 800 1200 1600 2000 2400

0 1 3 5 7

Skin thickness (

m)

Time (days)

EOLS 100% EOLS 50% EOLS 25%

EOLS 12% Vehicle

a a

ab

b

c a

b

c c

b a

b b

b

b a

b b

b

Fig. 1. Effect of topical treatment with EOLS (100%, 50%, 25% and 12%) on one-dosage irritation to healthy skin model at different time points. Measurement skin thickness was performed at the times 0, 1, 3, 5, and 7 days after the single exposure to EOLS. When used in natura, EOLS signiﬁcantly increased skin thickness throughout the study when compared to the control group (po0.05). Different

small letters on the same time point indicate statistical signiﬁcance among groups (po0.05). Each value represents the means7S.D. (n¼6/group).

300 1000 1700 2400 3100

0 1 3 5 7

Skin

thickness

(

m)

Time (days)

EOLS 100% EOLS 50% EOLS 25%

b a

bc

c

bc b

a

b a

b

c a

b a

a b

a

c a

b

Fig. 2.Effect of topical treatment with EOLS (100, 50, 25 and 12%) on multiple-dosage irritation to healthy skin model at different time points. Measurement skin thickness was performed at the times 0, 1, 3, 5, and 7 days after the continuous exposure to EOLS. When usedin natura, EOLS showed a signiﬁcant decrease in skin thickness when compared to EOLS 12% treated group (po0.05) and control group

(po0.05). Different small letters on the same time point indicate statistical

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signiﬁcant decrease in skin thickness when compared to EOLS 12%

treated group (po0.05) and control group (po0.05). EOLS 50 and

25% induced higher skin thickness in relation to EOLS 100 and 12%

(po0.05) on day 7 (Fig. 2). In histological evaluation, the main

result veriﬁed was the presence of ulcer and moderate inﬂ

amma-tory cells inﬁltration in EOLS 100% treated group (Fig. 5B).

In irritation to damaged skin model, the cutaneous thickness

was signiﬁcantly higher in EOLS 25 and 12% treated groups in

relation to EOLS 100 and 50% treated group (po0.05) and control

group (po0.05) on day 3 (Fig. 3). On the other hand, skin

erythema and edema were more intense in EOLS 50% and 25% treated groups, respectively, as compared to control group

(po0.05) on day 3 (Fig. 4). This result was associated to the skin

peeling, dehydration and tissue shrinkage, which were more intense from day 3 post-treatment with EOLS 100% and increased

progressively during the study. On day 7, it was veriﬁed intense

inflammatory cells infiltration andfibroblast proliferation in EOLS

100% treated group (Fig. 5C), while the group that received EOLS

50% presented intense epidermal thickening with keratinocytes

proliferation (Fig. 5D). This can have contributed to the signiﬁcant

increase in skin thickness observed in EOLS 50% treated group

(po0.05) when compared to the other groups at the end of

experiment (Fig. 3).

When the wounds were induced experimentally, the lesions appeared clean and free of exudate throughout the study in all groups, except in groups treated with EOLS at 6 and 12%. In these groups, it was observed intense edema and exudation on wounds up to day 5. Despite the presence of edema in all groups on day 3,

except in saline 0.9% control group, this inﬂammatory parameter

was more intense and noticeable in animals treated with EOLS

(po0.05) (Fig. 6); however, EOLS ointments promoted healing.

Table 2shows the reduction in wound area in the different groups over the 21-day study period. The wound areas decreased in all

groups compared with the initial wound size, but no signiﬁcant

differences were veriﬁed in the wound contraction on days 14 and

21 (p40.05).

Additionally, immunohistochemical analysis in skin treated with EOLS 12% showed that the expression of COX-2 and VEGF

mediators in both inﬂammatory and epidermal cells were absent

on day 7. Differences in the staining intensity of COX-2 and VEGF

were not veriﬁed to EOLS 12% treatment in any skin irritation tests.

4. Discussion

In folk medicine of northeast Brazil, EOLS has been used as

antiseptic agent for local use in skin (Matos, 2002). This oil

presents thymol and others phenolic compounds, which are effective biological molecules when used in appropriate manner. Although the bioactive constituents of EOLS substantiate their use

as the wound healing agent (Cavalcanti et al., 2012; Riella et al.,

2012), they are nonselective in their action and can cause damage

to host cells. Consequently, inadequate use might cause skin

damage and interfere with or prevent healing (Chang et al.,

2000). In the present study, we investigated the effects of

continuous topical administration of EOLS on intact and damaged skin, in order to evaluate safety of EOLS on cutaneous tissues.

Data present here indicate that EOLS in high concentrations,

mainly when it was usedin nature, showed irritant effects on mice

skin, which were demonstrated by increase cutaneous thick-ness, edema and erythema formation, loss of skin hydration and elasticity. On the other hand, in low concentrations (12%), irritant effects of EOLS on the mice skin, after seven days of treatment, were limited to discrete erythema and edema, and the skin response was estimated as light irritation. Although not indicated using pure essential oils on the skin, it must be considered that

there may be improper use (Vigan, 2010). However, essential oils

600 1500 2400 3300 4200

0 1 3 5 7

Skin

thickness

(

m)

Time (days)

EOLS 100% EOLS 50% EOLS 25%

ab a ab ab b a a b b b a a b a a b a c b b

Fig. 3.Effect of topical treatment with EOLS (100%, 50%, 25% and 12%) on irritation to damaged skin model at different time points. Measurement skin thickness was performed at the times 0, 1, 3, 5, and 7 days after the skin abrasion and exposure to EOLS. On day 3, the cutaneous thickness was signiﬁcantly higher in EOLS 25 and 12% treated groups in relation to EOLS 100 and 50% treated group (po0.05) and

control group (po0.05). When used in natura, EOLS promoted skin peeling, dehydration and tissue shrinkage, which increased progressively during the study. Different small letters on the same time point indicate statistical signiﬁcance among groups (po0.05). Each value represents the means7S.D. (n¼6/group).

0 1 2 3 4

I II III

S

everi

ty

scores

Edema EOLS 100%

EOLS 50% EOLS 25% EOLS 12% Vehicle 0 1 2 3 4 5

I II III

S

everi

ty

scores

Erythema EOLS 100%

EOLS 50% EOLS 25% EOLS 12% Vehicle a b

b b a a a a a b b a c c b a a a a ab a a b a b b b c c b

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Fig. 5.Skin sections in the group treated with EOLS 100% (A, B and C) and EOLS 50% (D) on day 7 in different skin irritation models: (A) one-dosage irritation to healthy skin; (B) multiple-dosage irritation to healthy skin; (C) and (D) irritation to damaged skin. (a) epidermal discontinuity; (b) inflammatory cells infiltration; (c) ulcer; (d)fibroblast proliferation; (e) epidermal thickening with keratinocytes proliferation. Haematoxylin and eosin staining. Original magnification: 200x. Scale bar: 100μm.

0 1 2 3 4 5

Day 1 Day 3

S

e

veri

ty

scores

Edema _{EOLS 6%}

EOLS 12%

Vehicle

Reference

Saline 0.9%

0 1 2 3 4 5

Day1 Day 5

S

e

veri

ty

scores

Exudation EOLS 6%

EOLS 12%

Vehicle

Reference

Saline 0.9% a

a

b b

a

ab

b

a a

b b

b

b b

b a

a

Fig. 6.Wound severity scores for lesions treated with EOLS (12% and 6%) ointments in excision wound model. Reference group was treated with 5% clostebol acetate and neomycin sulfate. Two variables, edema (A) and exudation (B) were assessed on days 1–3 and days 1–5, respectively, and graded on a four-point scale. A larger score represents more intense reaction of the skin wound. Signiﬁcant differences in wound status were found among EOLS 12 and 6% groups and control group (po0.05) in both

edema and exudation on evaluation days. Different small letters indicate signiﬁcant difference of inﬂammatory parameters among groups per evaluation day (po0.05).

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are used in a 12% concentration to treat skin wounds, excoriations

and infections (Kerr, 2002). The skin is an immune-competent

organ capable of rapid response to chemical and physical injuries

by mounting an inﬂammatory response that prevents damage and

restore tissue function (Bangert et al., 2011). In this context, the

results of the current study show that an increased cellular

inﬁltration was observed on histological analysis in EOLS treated

skin, which may be due to increased induction of chemotactic

molecules, which might have attracted inﬂammatory cells towards

the wound site and intensiﬁed the inﬂammatory response.

Considering that relative skin irritancy in response to the topical treatment may play a role to delay or impair the wound

healing (Jia et al., 2008), an issue addressed in this study was

determining the effect of EOLS ointments on excision wound healing and evaluating their therapeutic action for topical applica-tion. Wound repair is a dynamic and complex process that requires

inﬂammatory reaction and formation of new tissue to heal the

lesion. In inﬂammatory phase, soluble mediators increase vascular

permeability, leading toﬂuid extravasations that result in edema,

and attract inﬂammatory cells, facilitating the adhesion to the

endothelium and transmigration, which result in tissue exudation (Velnar et al., 2009). A successful inﬂammatory response is characterized by clearance of injurious stimuli and restoration of tissue normal physiology with resolution of process. However,

when this event is not controlled, the exaggerated inﬂammation

is a common factor that contributes to matrix destruction, cellular

senescence, and tissue nonhealing (Rajakariar et al., 2006;

Widgerow, 2011). In our study, we found that EOLS at 6% and

12% accentuated the inﬂammatory response by edema and

exuda-tion formaexuda-tion, but does not delay wound closure and promoted

healing in rat skin. Despite topical anti-inﬂammatory activity of

EOLS in acute edema model, the repeated use of both EOLS and its

major constituent, thymol, induced pro-inﬂammatory effect and

cutaneous damage, suggesting that the use this oil must be limited

mainly in chronic treatment (Veras et al., 2013). On the other hand,

thymol has been related as a promising compound to be used in

treatment of inﬂammatory processes as well as wound healing,

once it reduced the edema and diminished the inﬂux of leukocytes

to the injured area (Riella et al., 2012).

To understand part of the molecular mechanisms through

which topical treatment with EOLS 12% ampliﬁes the inﬂ

amma-tory response, we investigated the immunohistochemical staining intensity of COX-2 and VEGF. The COX-2 pathway is responsible for the production of mediators as prostaglandins, which increase microvascular permeability, promote edema and act synergisti-cally with other mediators, such as VEGF, to stimulate local neovascularization and cellular migration during the wound repair (Laulederkind et al., 2002; Rajakariar et al., 2006). Previously, it

has been suggested that single application of EOLS and thymol

exert an anti-inﬂammatory action by inhibition of COX enzymes

(Veras et al., 2013). However, our data showed that the

pro-inﬂammatory effect promoted by multiples applications of EOLS

12% in skin did not involve the COX-2 and VEGF expression. This activity could be also explained by an increased activity of 5- lipooxygenase (5-LOX), a key enzyme in the production of lipoxines, and leukotrienes, mediators with potent

chemoattrac-tant capacity, which induces the formation of ROS (Veras et al.,

2013). Therefore, further understanding of others signaling

path-ways and mediators involved in this process should be evaluated in future studies.

We and other researchers believed that the anti-inﬂammation

is theﬁrst step in the wound healing and this effect can play a

direct role in facilitating the fast healing (Jia et al., 2008; Oliveira

et al., 2010; Riella et al., 2012). However, a prolonged inﬂammatory

response can be beneﬁcial when it does not cause tissues damage

and there is the need to eliminate excessive potential pathogens (Rock et al., 2010; Widgerow, 2011). In other words, the

remark-able inﬂammatory response and normal wound closure promoted

by EOLS in this study associated with its previous potential

antimicrobial activity (Bertini et al., 2005; Fontenelle et al., 2007;

Veras et al., 2012) can be responsible for the inhibition of

super-ﬁcial infections of the damaged skin. Moreover, considering that

wound infection is likely the most common cause for deﬁcient

wound healing, the efﬁciency of EOLS in eradiation of the potential

pathogenic microorganisms after injury can played an essential role in controlling the morbidity in patients suffering from skin wounds, such as diabetic foot ulcers in humans and traumatic ulcers in domestic animals.

5. Conclusion

In summary, we provided evidence that adequate use of EOLS has positive effects on dermal irritation response and wound healing. EOLS revealed an irritant response to skin when applied topically in high concentrations; however this irritation was light when EOLS was used in low concentrations, suggesting that the dose of EOLS should be controlled for external use. In relation to wound repair process, continuous use of EOLS in adjusted

con-centrations ampliﬁes the inﬂammatory response, but does not

delay the cutaneous lesions closure. Take together, the

antimicro-bial action previously related and exacerbation of inﬂammatory

response during wound healing veriﬁed in our study, we suggest

that the EOLS in adequate concentrations can be used topically as an alternative therapeutic modality for treatment of infected cutaneous wound. However, further cellular and molecular Table 2

Effects of essential oil ofL. sidoides(EOLS) on wound contraction by excision wound model.

Day Unhealed wound area (mm2

) and wound contraction (%)

EOLS 6% EOLS 12% Vehicle Referencen

Saline 0.9%

0 432.95739.53a _488.38₇_52.91a _422.08₇_76.47a _464.08₇_53.10a _449.15₇_67.23a 3 503.90758.39a _539.90₇_52.26a _456.08₇_68.45ab _511.93₇_42.48a _406.20₇_69.05b 7 308.52744.40ab _386.90₇_41.55a _237.70₇_46.84b _310.63₇_65.86ab _269.48₇_60.62b

(28.5079.75)AB _(20.36₇_8.83)A _(43.12₇_10.53)B _(33.02₇_13.75)AB _(39.44₇_13.61)AB 14 72.4778.73ab _89.14₇_26.24a _58.38₇_16.03b _86.02₇_19.30ab _58.52₇_12.30b

(82.9973.81)A _(81.42₇_6.12)A _(85.62₇_5.04)A _(81.45₇_3.65)A _(86.95₇_2.09)A 21 24.6775.75a _30.26₇_19.69a _9.84₇_13.95a _28.33₇_16.15a _17.10₇_8.50a

(94.1871.97)A _(93.88₇_3.89)A _(97.29₇_4.11)A _(93.84₇_3.48)A _(96.17₇_1.82)A

Results are expressed as means7S.D. (n¼6).

Different small letters within the same line indicate signiﬁcant difference of unhealed wound area among groups (po0.05). Different capital letters within the same line

indicate signiﬁcant difference of wound contraction among groups (po0.05).

n

(7)

investigations to explore the mechanism of action of EOLS should be carried out before to obtain conclusions more accurate

regard-ing these heath beneﬁts.

Acknowledgments

The authors are grateful to Fundação Cearense de Apoio ao

Desenvolvimento Cientíﬁco e Tecnológico (FUNCAP) for ﬁnancial support. They also extend their thanks to Maria do Socorro França Monte for help with histological processing, to Suzana Moreira de Souza for assistance with the PT link module, and to Conceição da Silva Martins for immunohistochemical techniques help.

References

Adams, R.P. (Ed.), 1989. Identiﬁcation of Essential Oils by Ion Trap Mass Spectro-scopy. Academic Press, London.

Alencar, W.J., Craveiro, A.A., Matos, F.J.A., 1984. Kovats indices as preselection routine in mass spectra library search of volatiles. J. Nat. Prod. 47, 890–892.

Bangert, C., Brunner, P.M., Stingl, G., 2011. Immune functions of the skin. Clin. Dermatol. 29, 360–376.

Bao, P., Kodra, A., Tomic-Canic, M., Golinko, M.S., Ehrlich, H.P., Brem, H., 2009. The role of vascular endothelial growth factor in wound healing. J. Surg. Res. 153, 347–358.

Bertini, L.M., Pereira, A.F., Oliveira, C.L.L., Menezes, E.A., Morais, S.M., Cunha, F.A., Cavalcanti, E.S.B., 2005. Perﬁl de sensibilidade de bactérias frente a óleos essenciais de algumas plantas do Nordeste do Brasil. Infarma 17, 80–83.

Botelho, M.A., Santos, R.A., Martins, J.G., Carvalho, C.O., Paz, M.C., Azenha, C., Ruela, R.S., Queiroz, D.B., Ruela, W.S., Marinho, G., Ruela, F.I., 2009. Comparative effect of an essential oil mouthrinse on plaque, gingivitis and salivary Streptococcus mutanslevels: a double blind randomized study. Phytother. Res. 23, 1214–1219.

Cavalcanti, J.M., Leal-Cardoso, J.H., Diniz, L.R.L., Portella, V.G., Costa, C.O., Linard, C.F. B.M., Alves, K., Rocha, M.V.A.P., Lima, C.C., Cecatto, V.M., Coelho-de-Souza, A.N., 2012. The essential oil of Croton zehntneri and trans-anethole improves cutaneous wound healing. J. Ethnopharmacol. 144, 240–247.

Chang, Y.C., Tai, K.W., Huang, F.M., Huang, M.F., 2000. Cytotoxic and nongenotoxic effects of phenolic compounds in human pulp cell cultures. J. Endod. 26, 440–443.

Fontenelle, R.O.S., Morais, S.M., Brito, E.H.S., Kerntopf, M.R., Brilhante, R.S.N., Cordeiro, R.A., Tomé, A.R., Queiroz, M.G.R., Nascimento, N.R.F., Sidrim, J.J.C., Rocha, M.F.G., 2007. Chemical composition, toxicological aspects and antifungal activity of essential oil fromLippia sidoidesCham. J. Antimicrob. Chemother. 59, 934–940.

Girão, V.C.C., Nunes-Pinheiro, D.C.S., Morais, S.M., Sequeira, J.L., Gioso, M.A., 2003. A clinical trial of the effect of a mouth-rinse prepared withLippia sidoidesCham essential oil in dogs with mild gingival disease. Prev. Vet. Med. 59, 95–102.

Jia, Y., Zhao, G., Jia, J., 2008. Preliminary evaluation: the effects ofAloe feroxMiller and Aloe arborescens Miller on wound healing. J. Ethnopharmacol. 120, 181–189.

Kerr, J., 2002. The use of essential oils in healing wounds. Int. J. Aromather. 12, 202–206.

Laulederkind, S.J., Thompson-Jaeger, S., Goorha, S., Chen, Q., Fu, A., Rho, J.Y., Ballou, L.R., Raghow, R., 2002. Both constitutive and inducible prostaglandin H synthase affect dermal wound healing in mice. Lab. Investig. 82, 919–927.

Magalhães, M.S.F., Fechine, F.V., Macedo, R.N., Monteiro, D.L.S., Oliveira, C.C., Brito, G.A.C., Moraes, M.E.A., Moraes, M.O., 2008. Effect of a combination of medium chain triglycerides, linoleic acid, soy lecithin and vitamins A and E on wound healing in rats. Acta Cirúrgica Bras. 23, 262–269.

Matos, F.J.A., 2002. Farmácias vivas: sistemas de utilização de plantas medicinais projetado para pequenas comunidades. Editora UFC, Fortaleza, Brazil, 267p.

Matos, F.J.A., 2007. Plantas medicinais: guia de seleção e emprego de plantas usadas em ﬁtoterapia no Nordeste do Brasil, 3ª edição Imprensa Universitária, Fortaleza, Brazil p. 394.

Monteiro, M.V.B., Leite, A.K.R.M., Bertini, L.M., Morais, S.M., Nunes-Pinheiro, D.C.S., 2007. Topical anti-inﬂammatory, gastroprotective and antioxidant effects of the essential oil ofLippia sidoidesCham. leaves. J. Ethnopharmacol. 111, 378–382.

Oliveira, M.L.M., Nunes-Pinheiro, D.C.S., Tomé, A.R., Mota, E.F., Lima-Verde, I.A., Pinheiro, F.G.M., Campello, C.C., Morais, S.M., 2010. In vivo topical anti-inﬂammatory and wound healing activities of the ﬁxed oil of Caryocar coriaceumWittm. seeds. J. Ethnopharmacol. 129, 214–219.

Rajakariar, R., Yaqoob, M.M., Gilroy, D.W., 2006. COX-2 in inﬂammation and resolution. Mol. Interv. 6, 199–207.

Riella, K.R., Marinho, R.R., Santos, J.S., Pereira-Filho, R.N., Cardoso, J.C., Albuquerque-Junior, R.L.C., Thomazzi, S.M., 2012. Anti-inﬂammatory and cicatrizing activities of thymol, a monoterpene of the essential oil fromLippia gracilis, in rodents. J. Ethnopharmacol. 143, 656–663.

Rock, K.L., Latz, E., Ontiveros, F., Kono, H., 2010. The sterile inﬂammatory response. Ann. Rev. Immunol. 28, 321–342.

Sadigh-Eteghad, S., Dehnad, A., Mahmodi, J., Hoseyni, H., Khalili, I., Razmarayii, N., 2013. Healing potential of aStreptomycessp. secondary metabolite, SEM-1-111, on experimental full-thickness excision cutaneous wounds in Wistar rats. Clin. Exp. Dermatol. 38, 178–184.

Veras, H.N.H., Araruna, M.K.A., Costa, J.G.M., Coutinho, H.D.M., Kerntopf, M.R., Botelho, M.A., Menezes, I.R.A., 2013. Topical antiinﬂammatory activity of essential oil of Lippia sidoides Cham: possible mechanism of action. Phytother. Res. 27, 179–185.

Veras, H.N.H., Rodrigues, F.F.G., Colares, A.V., Menezes, I.R.A., Coutinho, H.D.M., Botelho, M.A., Costa, J.G.M., 2012. Synergistic antibiotic activity of volatile compounds from the essential oil ofLippia sidoidesand thymol. Fitoterapia 83, 508–512.

Velnar, T., Bailey, T., Smrkoli, V., 2009. The wound healing process: an overview of the cellular and molecular mechanisms. J. Int. Med. Res. 37, 1528–1542.

Vigan, M., 2010. Essential oils: renewal of interest and toxicity. Eur. J. Dermatol. 20, 685–692.