PHYTOTHERAPY RESEARCH Phytother. Res. (2012) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.4695
Topical Antiinflammatory Activity of Essential Oil of Lippia sidoides Cham: Possible Mechanism of 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. Menezes2 1
Laboratório de Pesquisa em Produtos Naturais, Universidade Regional do Cariri–URCA, Rua Cel. Antonio Luis 1161, Pimenta, Crato, CE, 63105-000, Brazil Laboratório de Farmacologia e Química Molecular, Universidade Regional do Cariri–URCA, Rua Cel. Antonio Luis 1161, Pimenta, Crato, CE, 63105-000, Brazil 3 Departamento 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 2
This work reports the chemical composition of the essential oil of Lippia sidoides (EOLS) and evaluation of the topical effect of EOLS and thymol against different irritant agents in vivo. The essential oil was obtained by hydrodistillation, 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 acute ear inflammation induced by croton oil, arachidonic acid, phenol or histamine, and chronic inflammation induced by croton oil. The topical application of EOLS or thymol at a dose of 2 mg/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 by 33.2% ( p < 0.05) and 54.7% ( p < 0.01) in acute ear edema. However, a proinflammatory effect of EOLS and thymol was evidenced when it was applied for more than 1 day. There were no statistical differences in antiedematogenic activity between EOLS and thymol. In conclusion, the results indicate that thymol is the constituent responsible for the topical antiinflammatory activity of EOLS. Thus, these findings could justify the popular 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 wound healing, fever, infection, edema, or rheumatic diseases, which indicates the presence of compounds with antiinflammatory properties, and therefore, they should be investigated and their effectiveness determined. The evolution of our understanding of the molecular aspects of the physiopathology of inflammation has led to the establishment of new test systems for the selection of various substances that allow the identification of new antiinflammatory compounds (Carvalho, 2004). Antiinflammatory compounds are useful in the therapeutic treatment of diseases, such as non-steroidal antiinflammatory drugs (NSAIDs) and corticosteroids. Medicinal plants are used in the popular medicine of many countries to treat different inflammations of the skin. However, in many cases, the effectiveness, the mechanism of action and the active principles for such activity are ignored (Falcão et al., 2005). Essential oils (also called ethereal oils or essences) are complex mixtures of liquid volatile substances,
* Correspondence to: Henrique D. M. Coutinho, Departamento 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. E-mail:
[email protected]
Copyright © 2012 John Wiley & Sons, Ltd.
which are lipophilic and usually odoriferous and which can be obtained from several parts of the plant (flowers, leaves, branches, peels, fruits, roots, etc.). The chemical composition of these oils from different species can be quite complex, including hydrocarbons, terpenes, phenols, esters, furans, or organic acids. In the mixture, such compounds are present at different concentrations, where one of them comprises the majority, with others existing in smaller concentrations and some in trace amounts (Santos, 2007). The species Lippia sidoides Cham. (Verbenaceae), popularly known as “alecrim pimento,” is a native bush of the semi-arid area of Northeast Brazil, widely used in popular medicine as an antiseptic for local use on the skin 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, and analgesic properties, among others (Monteiro et al., 2007; Pavela, 2009; Rivas et al., 2010; Ozen et al., 2011). The essential oil of L. sidoides (EOLS) showed potential gastroprotective and topical antiinflammatory activities when used at different concentrations, probably due to its antioxidant properties (Monteiro et al., 2007). It is believed that the major representative, thymol, is responsible for the biological activities of the EOLS, but this had not yet been proven, as the essential oil can contain minor substances that can also be responsible for the biological activities. For instance, the essential oil Received 21 September 2011 Revised 07 March 2012 Accepted 13 March 2012
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of Cordia verbenaceae contains 4.64% a-humulene, which accounts for the antiinflammatory activity (Carvalho et al., 2004; Fernandes et al., 2007). Therefore, the objective of this work was to evaluate the topical antiinflammatory activity of the EOLS and thymol in animal models, as well as to elucidate the possible mechanism of action.
MATERIALS AND METHODS Plant material. Leaves of L. sidoides Cham. were collected in August 2010 from the Small Aromatic and Medicinal Plants Garden of the Natural Products Research Laboratory (LPPN) at University Regional do Cariri (URCA), Crato City, Ceará State, Brazil. A voucher specimen was sent to the Herbarium Caririense Dárdano de Andrade, Lima, Department of Biological Sciences (URCA), which was deposited under registration no. 3038. Chemicals and drugs. Croton oil, arachidonic acid (AA), capsaicin, histamine dihydrochloride, indomethacin, and thymol were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Dexamethasone (DEX; DecadronW) was obtained from Aché (São Paulo, Brazil). Ketamine hydrochloride and xylazine hydrochloride were purchased from Syntec (Brazil). Acetone of analytical grade was from Dinâmica (Brazil). Animals. Male and female Swiss mice (Mus musculus), weighing 25–35 g, were previously housed in standard polypropylene cages under controlled conditions of temperature (22 2 C) and 12-h light/dark cycle, with free access to water and rodent chow (LabinaW, Purina, Brazil). Mice were allowed to adapt to the laboratory environment for at least 1 h before testing. All the procedures were previously approved by the Research Ethics Committee of University of Fortaleza (Brazil), under no. 010/2010. Essential oil. Samples of L. sidoides fresh leaves (140 g) were triturated and submitted to a hydrodistillation process in a Clevenger-type apparatus for 2 h. The collected essential oil was subsequently dried using anhydrous sodium sulphate (Na2SO4) and stored under refrigeration at 0.05). This result suggests that EOLS and thymol are not involved in the inhibition of the histamine produced in the edematogenic action of croton oil. In inflammation produced in the croton oil model, corticosteroids and lipooxygenase (LOX) inhibitors demonstrate the best activity in reducing ear edema, whereas cyclooxygenase (COX) inhibitors and antihistamines demonstrate little or no effect (Green and Shuster, 1987). Our results suggest that EOLS and thymol are possibly unable to inhibit LOX or PLA2, but they have an important action on the COX enzymes. The topical application of AA generates a fast inflammatory response characterized by intense erythema and edema with small neutrophil accumulation, when compared with cell migration determined in the croton oil model, whose main mediators are PGE2, LTC4, and LTD4 (Crummey et al., 1987; Humes et al., 1986; Young et al., 1984). Indomethacin, an NSAID whose antiinflammatory action is related to the nonselective inhibition of the isoforms of COX (COX1 and COX2), clearly reverses edema induced by the topical application of AA, as described in the literature (Gabor, 2000). In the same way, EOLS and thymol were also shown to be effective in the inhibition of the formation of edema in this model. Our results agree with those obtained by Monteiro et al. (2007), who demonstrated the topical antiinflammatory activity of EOLS (containing 66.67% thymol) in the acute ear edema model induced by TPA in mice at doses 1 and 10 mg/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/LOX inhibitors (Carlson et al., 1985). However, the metabolites of AA also promote the degranulation of mastocytes cells, so that histamine release contributes partially to Copyright © 2012 John Wiley & Sons, Ltd.
the formation of edema in this model (Camp, 1982). Similarly, it should be considered that the AA model is not specific for the identification of compounds that inhibit COX exclusively or LOX, because other antagonistic agents of histamine and antioxidants also reduce the edema induced by AA (Crummey et al., 1987). In general, corticoids do not show any effect against AA, whereas some monoterpenes inhibit AA metabolism (Juergens et al., 2003). Thymol is capable of significantly inhibiting platelet aggregation induced by AA (Enomoto et al., 2001). Marsik et al. (2005) showed that thymol present in the seeds of Nigella sativa has an antiinflammatory activity as demonstrated by the inhibition of COX1, and the authors suggested more studies related to this compound for possible use as an antiinflammatory. The essential oil of Lippia gracilis, containing thymol as the major component (32.68%), was found to reduce inflammation in the paw induced by carrageenan and to have antinociceptive effect in the acetic acid-induced writhing model (Mendes et al., 2010). The inflammation induced by carrageenan involves cell migration and production of mediators including nitric 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 prostaglandins such as PGE2, which have an important role in the nociceptive mechanism. Thus, in that study, thymol was the component responsible for the observed pharmacological activities, possibly involving the inhibition of COXs (Mendes et al., 2010). The fact that EOLS and thymol inhibit edema induced by an irritant agent such as AA and phenol demonstrates the possible effectiveness of these in the treatment of contact dermatitis after challenge with irritant agents. In response to an exogenous stimulus, such as phenol, keratinocytes produce important chemical mediators in primary contact irritation, including cytokines associated with proinflammatory properties, such as IL-1, IL-8, and TNF-a (Lim et al., 2004; Wilmer et al., 1994). Our results showed that in the chronic ear edema model, as observed in chronic inflammatory diseases, repeated use of EOLS and thymol treatment caused a proinflammatory effect and cutaneous damage. A possible mechanism of action to account for this finding involves LOX enzymes. Chronic diseases, such as rheumatoid arthritis and psoriasis, involve an alteration in the release of proinflammatory cytokines (e.g., interleukins), growth factors, and proinflammatory enzymes such as COX and LOX, which generate PGs and leukotriene, respectively, resulting in leukocyte infiltration in skin (T cells, neutrophils and mastocytes) (Krueger and Bawcock, 2005). The study of Roschek et al. (2009) showed that SRBAI extract (containing thymol) causes a dose-dependent inhibition of COX1 and COX2, selectively inhibiting COX2 by an order of magnitude over COX1. No detectable 5-LOX inhibition was found with SRB-AI. 5-LOX catalyzes the biotransformation of AA into fatty acid hydroperoxides, lipoxines, and leukotrienes, such as B4, compounds implicated in allergy and suffering. Sahouo et al. (2003) showed that COX function of PGHS was inhibited by only chemical constituents of the essential oil of Ocimum gratissimum, in which thymol is the major component, with an IC50 value of 125 mg/mL. Phytother. Res. (2012)
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Recently, Elhabazi et al. (2006) demonstrated that thyme extracts increase the number of polymorphonuclear cells, total lymphocytes, and CD4+, CD8+, and NK cells. Neutrophils are important cells in host defense against invading bacteria. These cells are also potent chemoattractants, and they are able to induce the formation of reactive oxygen species (ROS) and the release of lysosome enzymes. However, these cells have also been linked to tissue destruction by stimulating the release of eicosanoids (5-LOX derivatives) and other cytokines, such as IL-1 and TNF-a. IL-1 activates neutrophils and macrophages, increasing the production and the release of reaction oxygen species and nitric oxide, which have been implicated in local tissue damage. Thus, our data suggest that EOLS and thymol exert an antiinflammatory action (acute ear edema) involving the inhibition of COX enzymes, as is effectively demonstrated in the model of AA and phenol (Table 2). However, the proinflammatory effect observed in chronic ear edema can be explained by an increased activity of 5-LOX, a key enzyme in the biotransformation 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).
CONCLUSION The antiinflammatory activity of the EOLS is explained, at least in part, by the presence of the main constituent thymol. This work proves the efficacy of the topical application of EOLS and thymol in models of skin inflammation induced by AA and phenol, possibly reducing the production of proinflammatory mediators in the acute inflammation models. In repeated treatment using thymol or EOLS, there was an increase in inflammation, which limits their use in chronic treatment. The results provide useful pharmacological information related to the antiinflammatory activity of this species, suggesting its possible use as a source of active compounds with antiedematogenic activity in acute treatment. Thus, this work could justify the popular use of L. sidoides essential oil in alternative medicine and represents an important contribution to the elucidation of the mechanism involved in the antiedematogenic effect of EOLS.
Conf lict of Interest The authors do not report any conflict of interest.
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