Inhaled 1,8-Cineole Reduces Inflammatory Parameters in Airways of Ovalbumin-Challenged Guinea Pigs

Basic & Clinical Pharmacology & Toxicology, 108, 34 39 Doi: 10.1111/j.1742-7843.2010.00622.x Inhaled 1,8-Cineole Reduces Inflammatory Parameters in Airways of Ovalbumin-Challenged Guinea Pigs Vasco P.D. Bastos 1,2,AntoniellaS.Gomes 1, Francisco J.B. Lima 1, Teresinha S. Brito 1, Pedro M.G. Soares 3,Jo¼oP.M.Pinho 1, Claudijane S. Silva 2, ArmÞnio A. Santos 1, Marcellus H.L.P. Souza 1 and Pedro J.C. Magalh¼es 1 1 Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Cearµ, Fortaleza, CE, Brazil, 2 Faculdade Integrada do Cearµ, Fortaleza, CE, Brazil, and 3 Department of Morphology, Faculty of Medicine, Federal University of Cearµ, Fortaleza, CE, Brazil (Received 29 March 2010; Accepted 9 June 2010) Abstract: Eucalyptol, also known as 1,8-cineole, is a monoterpene traditionally used to treat respiratory disorders due to its secretolytic properties. In addition to its myorelaxant effects, it also has anti-inflammatory actions in vitro. Inthisstudy, we aimed to evaluate the efficacy of acute treatment with 1,8-cineole on reducing airway inflammatory parameters. Ovalbumin (OVA)-sensitized guinea pigs were submitted to antigenic challenge (OVA) with or without pre-treatment with a single dose of 1,8-cineole administered by inhalation. Airway inflammatory parameters were reduced or absent in 1,8-cineole-treated animals as compared with untreated guinea pigs. Acute treatment with 1,8-cineole impaired the development of airway hyperresponsiveness to carbachol in isolated tracheal rings. Levels of the pro-inflammatory cytokines TNFa and IL-1b was lower in bronchoalveolar lavage fluid (BALF) of 1,8-cineol-treated guinea pigs than in untreated animals. 1,8-Cineole impaired the OVA-induced increase of the myeloperoxidase activity in BALF. 1,8-Cineole also prevented the reduction of the mucociliary clearance induced by the antigen presentation. The present investigation provides evidence that inhaled 1,8-cineole prevents hyperresponsiveness and inhibits inflammation in airways of ovalbumin-challenged guinea pigs. Author for correspondence: Pedro Magalh¼es, Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Cearµ, R. Cel. Nunes de Melo 1127, Rodolfo Teófilo, 60430-270, Fortaleza, CE, Brazil (fax +55 85 3366 8333, e-mail pjcmagal@ ufc.br). Eucalyptol is a terpenoid oxide found in several essential oils, mainly those extracted from Eucalyptus species such as E. globulus Labill. and E. tereticornis Sm. [1]. Also known as 1,8-cineole, it possesses myorelaxant properties on intestinal and vascular smooth muscle [2,3]. Even in cardiac muscle, 1,8-cineole has inhibitory actions, which are attributed to its ability in reducing transmembrane Ca 2+ influx in papillary muscle [4]. As a matter of fact, myorelaxant properties on airway smooth muscle were already reported [5,6] and a preferential action of this terpene on contractions elicited by electromechanical events seems to be involved in the underlying mechanisms of its relaxant effects [7]. Actions of 1,8- cineole on airways are particularly interesting because they are traditionally used to treat respiratory disorders due to their well-known secretolytic properties [8]. In recent years, there has been an increasing interest in the beneficial effects of 1,8-cineole in asthma, a condition defined as a bronchoconstrictive inflammatory disease characterized by infiltration of eosinophils, lymphocytes and mast cells in airways [9]. Among several key events, airway hyperresponsiveness is a central feature of this clinical syndrome and inflammation of the mucosa is often associated with its onset, which involves multiple cytokines and inflammatory mediators [10]. In vitro, eucalyptol inhibited the development of increased contractions in airway smooth muscle of guinea pigs subjected to antigen challenge [7]. In addition to its myorelaxant effects, 1,8-cineole also shows anti-inflammatory actions probably due to its ability to reduce the production of cytokines such as the arachidonic acid metabolites LTB 4 and PGE 2 as demonstrated in vitro by Juergens et al. [8] in human monocytes obtained from patients with bronchial asthma or from healthy volunteers. Additionally, decreased production of TNFa, interleukin 1 (IL-1), leukotriene B 4 and thromboxane B 2 was observed ex vivo in unselected lymphocytes and monocytes stimulated with LPS-and IL-1 [11,12]. It has been reported that a long-term systemic therapy with 1,8-cineole has a significant steroid-saving effect by reducing the daily prednisolone dosage in steroid-depending asthmatic patients, which corroborates its anti-inflammatory activity [13]. In a recent paper, Worth et al. [14] showed that 1,8-cineole reduced exacerbations as well as dyspnoea and improved lung function and health status in patients with stable chronic obstructive pulmonary disease. Notwithstanding, up to date, there are no scientific demonstrations that 1,8-cineole administered by inhalation decreases production of the inflammatory process in airways. Thus, in this study, we aimed to evaluate the efficacy of acute treatment with 1,8-cineole on reducing inflammatory parameters, especially cytokines levels on bronchoalveolar lavage fluid of ovalbumin (OVA)-sensitized guinea pigs submitted to antigenic challenge with OVA. Basic & Clinical Pharmacology & Toxicology Ó 2010 Nordic Pharmacological Society

ANTI-INFLAMMATORY EFFECTS OF 1,8-CINEOLE ON GUINEA PIG AIRWAYS 35 Materials and Methods Animals. Male guinea pigs (400 500 g) were housed under standard conditions with free access to food and water in the Central Housing Station of the Federal University of Cearµ. All animals were handled in accordance with the Ethical Principles for Care and Use of Laboratory Animals, published by the Brazilian College for Animal Experimentation (COBEA). The study protocol was approved by the Animal Ethics Committee of the Federal University of Cearµ (protocol n/ 34-06). Treatment with 1,8-cineole and antigen challenge. The guinea pigs were actively sensitized by means of 3 intraperitoneal injections of a solution containing OVA (10 mg kg), which was applied every other day. After at least 21 days (and no more than 50 days), OVA-sensitized conscious guinea pigs were placed in a plastic box (21 20 30 cm) for inhalation of a single dose of 1,8-cineole aerosolized by 15 min. from a solution of 1 mg ml via an ultrasonic nebulizer (RespiraMax; NS Indfflstria de Aparelhos MØdicos, S¼o Paulo, Brazil). Control animals inhaled saline instead of 1,8-cineole. Immediately (3 min.) after treatment with 1,8-cineole or saline, the animals were challenged by inhalation with OVA (first challenge 1 mg ml; second challenge 5 mg ml; 15 min. each) or saline again (for 30 min.). Thus, the animals were divided into the following experimental groups, all sensitized to OVA: Group I 1,8-cineole-untreated, challenged with saline; Group II 1,8-cineole-untreated, challenged with OVA; Group III 1,8-cineole-treated, challenged with saline; Group IV 1,8-cineole-treated, challenged with OVA. Each guinea pig was euthanized 24 hr later under chloral hydrate (400 mg kg, i.p.) sedation followed by exsanguination. Inflammatory parameters such as tracheal responsiveness to carbachol, cytokine levels and myeloperoxidase activity on bronchoalveolar lavage fluid, as well as mucociliary clearance were evaluated. Tracheal responsiveness to carbachol. The trachea was dissected out and placed into a dish containing physiological salt solution for removal of adhering fat and connective tissue. Next, it was cut transversely as cylindrical rings (with 3 4 cartilage rings), which were suspended in 5 ml organ baths containing physiological solution continuously aerated at 37 C with ph adjusted to 7.4. Tracheal rings were stretched with a passive tension of 1 g. One side of the tracheal ring was attached to a fixed pin in the bath and the other to a force transducer (Grass Model FTO3, Quincy, MA, USA) connected to a digital recording device (Dataq, PM-1000, Akron, OH, USA) to record tension isometrically in a micro-computer. Time to equilibration was 1 hr. Airway smooth muscle reactivity was evaluated in tracheal rings obtained from OVA-sensitized guinea pigs, 24 hr after they were submitted to challenge by inhalation with either the sensitizing antigen or saline, with or without pre-treatment with 1,8-cineole as described above. Concentration-effect curves were calculated using the cumulative method of Van Rossum [15]. Briefly, they were obtained by exposing the preparation to cumulatively increasing concentrations of carbachol added to the bath and maintained at a given concentration during 3 min. A maximum response (E max ) was obtained when the increase in agonist concentration did not induce a significant additional response. The contractile amplitude was measured at the peak deflection. Bronchoalveolar lavage fluid. After 24 hr antigen or saline challenge, the guinea pigs were deeply sedated and euthanized by exsanguination via carotid artery sectioning. Bronchoalveolar lavage fluid (BALF) was obtained by two repeated washes using 5 ml of warmed (37 C) deoxygenated saline, which was introduced into the lungs via tracheal cannula connected to a 5 ml syringe. The recovered lavage fluid was divided in two samples, one for leukocyte count (8 ml) and the other for enzymatic evaluation (myeloperoxidase assay) as well as cytokine levels determination (2 ml). The former was centrifuged (at 4 C, 10 min., 200 g) and the cells were resuspended in 2 ml of heparinized saline (1:1000). Pelleted cells were counted in Turk (1:20) stained samples which were placed in a haemocytometer (Neubauer counting chamber, Inlab, Ribeir¼o Preto, Brazil). The remaining aliquot was centrifuged again (400 g, 10 min.) and cells were stained with haematoxylin and eosin (H&E) for examination by light microscopy (magnification 100) to determine cell differentials. Myeloperoxidase assay. The myeloperoxidase (MPO) activity in BALF collected 24 hr after antigen or saline challenge was determined as a measurement of neutrophil accumulation. Enzymatic activity was evaluated spectrophotometrically according to the method described by Bradley et al. [16]. In brief, BALF was obtained as described above and stored at )70 C. The material was suspended in 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mm potassium phosphate buffer, ph 6.0, to solubilize MPO. After homogenization in an ice bath (15 sec.), the samples were freeze thawed twice. Additional buffer was added to the test tube to reach 400 ll of buffer per 15 mg of tissue for 12 min. After centrifugation (1000 g 12 min.), 0.1 ml of the supernatant was added to 2 ml phosphate buffer 50 mm, ph 6.0, containing 0.167 mg ml o-dianisidine dihydrochloride, distillated water and 0.0005% hydrogen peroxide to give a final volume of 2.1 ml per tube. The absorbance was measured spectrophotometrically (460 nm). One unit of activity was defined as that degrading 1 lmol of peroxide min. at 25 C. Results are expressed in MPO units (U) ml. Detection of cytokines (TNFa, IL-1b, IL-10) in BALF. Cytokine levels were determined in BALF collected at 24 hr after antigen or saline challenge. The BALF was stored at )70 C until required for assay. The detection of TNFa, IL-1b and IL-10 concentrations was determined by ELISA, as described previously [17]. Briefly, microtitre plates were coated overnight at 4 C with antibody against mice TNFa, IL-1b and IL-10 (2 lg ml). After blocking the plates, the samples and standard at various dilutions were added in duplicate and incubated at 4 C for 24 hr. The plates were washed three times with buffer. After washing the plates, biotinylated sheep polyclonal anti-tnfa or anti-il-1b was added to the wells. After further incubation at room temperature for 1 hr, the plates were washed and 50 ll of avidin-hrp diluted 1:5000 were added. The colour reagent o-phenylenediamine (OPD; 50 ll) was added 15 min. later and the plates were incubated in the dark at 37 C for 15 20 min. The enzyme reaction was stopped with H 2 SO 4 and absorbance was measured at 490 nm. Values were expressed as pg ml. Measurement of tracheal mucociliary transport. Airway mucociliary transport was evaluated according to the method described by Kimoto et al. [18]. In brief, male guinea pigs were anaesthetized with urethane (1.2 g kg, i.p.) and fixed in the dorsal position on a fixation board (inclined 10 from the horizontal with the animal s head up). After careful exposition, the trachea was punctured with a needle, and 2 ll of a 0.3 g ml gelatin solution containing 0.5% Evans blue dye was injected into the trachea using a microsyringe. After 2 min., the trachea was opened and the distance the dye was transported from the injection point was measured with caliper rule to determine mucociliary transport distance. Statistical analysis. Data are expressed as mean standard error of the mean (number of experiments). EC 50 values, i.e. concentration that can be expected to cause a defined effect on 50%, were shown as geometric mean (95% confidence interval). Significance of the results was determined using unpaired Student s t-test, one- or twoway analysis of variance (ANOVA). When ANOVA was significant, it was followed by the Holm Sidak multiple comparison test. Statistical significance was accepted when p < 0.05.

36 VASCO P.D. BASTOS ET AL. Results Effects of inhaled 1,8-cineole on tracheal responsiveness to carbamylcholine. Full concentration response curves (fig. 1) obtained under carbachol (CCh; 10-9 to 3 10-5 M) treatment showed typical tracheal contractions, in a concentration-dependent manner (p < 0.001, ANOVA). Tracheal rings of 1,8-cineoleuntreated animals contracted more effectively (p <0.05, two-way ANOVA) in response to CCh when preparations were obtained from OVA-challenged animals (group II; E max of 1.21 0.11 g; n = 6) than when tissues were obtained from guinea pigs that inhaled just saline (group I; E max of 0.75 0.08 g; n = 6), but without significant difference (p > 0.05, ANOVA) in their EC 50 values [0.92 (0.27 1.56) lm in OVA-challenged animals versus 0.62 (0.06 1.18) lm insal- ine-treated guinea pigs]. On the other hand, when guinea pigs were submitted to inhalation of 1,8-cineole before the challenge with OVA (group IV), their tracheal rings did not develop tracheal contractions higher than those obtained from guinea pigs that inhaled 1,8-cineole before challenge with saline (group III). Indeed, E max values were 0.74 0.08 g (n =6)and0.86 0.09g(n = 7) for 1,8-cineole-treated guinea pigs challenged with OVA- or saline, respectively, and they did not differ significantly (p > 0.05, ANOVA) from those values observed in tracheal preparations from group I. Fig. 1. Inhibitory effect of 1,8-cineole treatment on the development of airway hyperresponsiveness on OVA-sensitized guinea pigs challenged with sensitizing antigen. Full concentration-effect curves for carbachol (CCh; 10 )9 to 3 10-5 M) obtained in tracheal rings from 1,8-cineole-untreated guinea pigs submitted to challenge with saline (group I; empty square; n = 6) or OVA (group II; empty circle; n = 6), as well as in tracheal rings from 1,8-cineol-treated guinea pigs challenged with saline (Group III; full square; n = 7) or OVA(group IV;fullcircle;n = 6). *, p < 0.05, two-way anova followed by the Holm Sidak test, compared with group I. Effects of 1,8-cineole on antigen-induced changes of airway inflammatory cells in BALF. Table 1 shows the major findings of the inhibitory effects induced by 1,8-cineole on the development of inflammatory parameters such as presence of leukocytes, MPO activity and level of cytokines in BALF of guinea pigs. In each group, recovery of lavage fluid was consistently 80 90% of that instilled. In 1,8-cineole-untreated guinea pigs, after 24 hr OVA inhalation (group II), antigen increased the number of inflammatory cells in BALF, reaching values that were significantly higher than those in group I (p <0.05,Holm Sidak). In particular, OVA caused a significant increase of eosinophils (from 0.70 0.18 10 3 cells mm 3 (n = 12) to 4.35 0.42 10 3 cells mm 3 (n = 8)) and neutrophils (from 0.20 0.05 10 3 cells mm 3 to 1.85 0.38 10 3 cells mm 3 ). When compared with tissues obtained from group II, group IV showed that 1,8-cineole inhalation significantly prevented (p < 0.05, Holm Sidak) the increase in leucocytes seen after antigen challenge. In OVA-challenged guinea pigs, the number of inflammatory cells was always smaller in BALF of 1,8-cineole-treated guinea pigs than in BALF obtained from animals that did not receive 1,8- cineole. Effects of 1,8-cineole on antigen-induced changes in the MPO activity in BALF. Neutrophil infiltration in airways of guinea pigs was evaluated by means of the MPO assay in BALF samples (table 1). Antigen challenge caused a statistically significant (p < 0.05, Holm Sidak) increase in MPO activity in 1,8-cineoleuntreated animals (group II) compared with the 1,8-cineoleuntreated group I (6.28 0.97 versus 0.56 0.22 U ml, respectively; n = 4). Pre-treatment with 1,8-cineole markedly impaired the increase in the MPO activity (p < 0.05, Holm Sidak) induced by the antigen inhalation, which corresponded to 1.38 0.52 U ml (n = 5) in animals challenged with saline (group III) versus 3.38 0.56 U ml (n =8)in OVA-challenged guinea pigs (group IV), a value significantly lower than that observed in BALF of the 1,8-cineoleuntreated group that inhaled just saline (group I; 6.28 0.97 U ml; p < 0.05, Holm Sidak). Effects of 1,8-cineole on antigen-induced changes in the cytokine levels in BALF. Measured by ELISA, cytokine levels were evaluated in BALF obtained 24 hr after bronchoprovocation of guinea pigs by inhalation of either OVA or saline. TNFa and IL-1 levels in BALF of 1,8-cineole-untreated guinea pigs challenged with OVA (324.1 11.7 and 540.7 55.1 pg ml, respectively) were significantly increased (p < 0.05, Holm Sidak) compared to those levels in group I that inhaled just saline (172.1 16.0 and 250.6 35.6 pg ml, respectively; table 1). Conversely, IL-10 levels were significantly decreased in BALF of guinea pigs from group II. Pre-treatment of the animals with 1,8-cineole completely impaired the OVAinduced increase in both TNFa and IL-1 levels after antigen challenge. Additionally, pre-treatment with 1,8-cineole also

ANTI-INFLAMMATORY EFFECTS OF 1,8-CINEOLE ON GUINEA PIG AIRWAYS 37 Table 1. Effects of 1,8-cineole on inflammatory parameters measured in BALF obtained from OVA-challenged guinea pigs. Group I II III IV Cell counts ( 10 3 mm 3 ) Leukocytes 3.23 0.34 Eosinophils 0.70 0.18 Neutrophils 0.20 0.05 Lymphocytes 0.82 0,12 Macrophages 1.53 0.17 Cytokine levels (pg ml) IL-1 280.30 50.31 TNFa 188.06 19.69 IL-10 1976.87 20.91 (4) Enzyme activity (U ml) MPO 0.55 0.22 (4) 10.94 1.45 a 4.35 0.42 a 1.85 0.38 a 1.37 0.22 a 3.38 0.69 a 540.73 55.07 a 324.13 11.75 a 665.07 189.51 a (7) 6.27 0.97 a (3) Values are mean S.E.M. with the number of experiments in parenthesis; a p < 0.05 compared with group I, Holm Sidak test; b p < 0.05 compared with group III, Holm Sidak test; c p < 0.05 compared with group II, Holm Sidak test. 4.11 0.29 1.00 0.29 0.58 0.11 0.91 0.09 1.63 0.08 322.70 60.20 206.05 35.12 1507.66 70.45 (5) 1.38 0.51 (5) 6.82 0.65 b,c 2.30 0.31 b,c 0.91 0.14 b,c 1.18 0.15 2.42 0.33 b 338.64 46.69 (10) 223.27 17.61 (10) 1314.04 59.54 (11) 3.38 0.55 c impaired the decreased levels for IL-10 after antigen challenge as observed in 1,8-cineole-untreated guinea pigs. Effects of 1,8-cineole on antigen-induced changes in the tracheal mucociliary transport. The Evans blue-containing gelatin solution (0.3 g ml) used in this study as a distance marker moved within the limits of the length of trachea in all the animals. In the 1,8-cineoleuntreated guinea pigs that inhaled saline (group I), the traversed distance of the marker on the luminal surface of the guinea pig trachea was 0.80 0.11 cm (n = 6; fig. 2), a value significantly higher (p < 0.05, unpaired Student s t-test) than that observed in the guinea pigs of group II (0.32 0.04 cm; n = 5). In the guinea pigs that previously inhaled 1,8-cineole before bronchoprovocation with OVA (group IV), the distance traversed by the marker was 0.58 0.14 cm (n =6),a value that did not reach statistical significance (p >0.05, unpaired Student s t-test) compared to the 1,8-cineole-treated guinea pigs that inhaled saline (0.83 0.07; n =6). Discussion The major findings of this study are that 1,8-cineole effectively inhibits airway inflammatory signs induced by antigen challenge in guinea pigs. Although it is used commonly as a decongestant and expectorant for upper respiratory tract infections or inflammations [19], there is yet little objective evidence of its biological effects, especially related to airway Fig. 2. Effects of 1,8-cineole on antigen-induced changes in tracheal mucociliary transport of guinea pigs submitted to antigen challenge. Mucociliary transport (MCT) was measured as the distance traversed by a marker (Evans blue in a gelatin solution; 0.3 g ml) added on the luminal surface of the guinea pig trachea. In 1,8-cineole-untreated guinea pigs that inhaled saline (group I; n = 6), the traversed distance of the marker was significantly higher than in guinea pigs of group II (1,8-cineole-untreated challenged with OVA; n = 5). Previous inhalation of 1,8-cineole prevented the slowing of the MCT in guinea pigs of group IV (OVA-challenged; n = 6). Values are mean and S.E.M. *, p < 0.05, unpaired Student s t-test, compared with group I. physiology. Thus, in this study, we report that airway inflammatory response induced by a sensitizing antigen may be inhibited by a single dose of 1,8-cineole administered by inhalation to antigen-sensitized guinea pigs.

38 VASCO P.D. BASTOS ET AL. The animal model for asthma used in the present work is reliable to reproduce many characteristic features of human asthma such as airway hyperresponsiveness [20]. As a matter of fact, we have recently demonstrated that, in vitro, 1,8-cineole reduced the contractions of tracheal rings obtained from OVA-challenged guinea pigs, which showed airway hyperresponsiveness to CCh and KCl [7]. In this study, CCh-induced contractions of tracheal rings from OVA-challenged 1,8-cineole-treated guinea pigs showed amplitude similar to those contractions obtained with tracheal preparations of guinea pigs challenged with saline, indicating that the airway hyperresponsiveness phenotype was prevented, or not fully developed, in animals pre-treated with 1,8-cineole. Because there is evidence that the degree of inflammation is loosely related to airway hyperresponsiveness [21], we hypothesized that, in vivo, 1,8-cineole acts as an anti-inflammatory agent. It has been proposed that 1,8-cineole is a strong in vitro inhibitor of the release of cytokines involved in airway inflammation. It suppressed arachidonic acid metabolism and production of TNFa, IL-1b, LTB 4 and TXB 2 in human blood monocytes [8,11]. Juergens et al. [12] also reported that this monoterpene is inhibitory on stimulated cytokine production by human unselected lymphocytes and LPS-stimulated monocytes. Notwithstanding, no study addressed hitherto the in vivo pharmacological efficacy of 1,8-cineole to produce inhibition of the cytokine release on airways submitted to antigen challenge. Indeed, just recently, a study further suggested that 1,8-cineole may control airway inflammation in patients with chronic obstructive pulmonary disease by intervening in its pathophysiology [14]. Here, we demonstrate that 1,8-cineole pre-treatment reduced the levels of inflammatory cytokines, such as TNFa and IL-1b, in BALF samples collected 24 hr after antigen presentation to sensitized guinea pigs. Conversely, the immunomodulatory interleukin IL-10, which had reduced their levels in BALF of OVA-challenged animals after the antigenic challenge, was partially recovered with the treatment with 1,8-cineole. Since it is well known that IL-10 exerts anti-inflammatory effects [22], we can conclude that 1,8-cineole-induced effects are due to a balance between reducing the release of pro-inflammatory cytokines and maintaining the physiological production of immunomodulatory substances, achieving desirable beneficial effects. It is well known that synthesis and release of a variety of cytokines occur in the course of an inflammatory process mediated by antigen presentation, involving airway structural cells such as epithelial cells and endothelial cells, as well as inflammatory cells such as macrophages, mast cells, eosinophils and lymphocytes [23]. Guinea pigs pre-treated with 1,8-cineole showed reduced counts for inflammatory cells in their BALF samples as compared with 1,8-cineole-untreated animals. Pre-treatment with 1,8-cineole also markedly inhibited the antigen-induced increase in the MPO activity, an enzyme considered an index of neutrophil infiltration [24], corroborating with the hypothesis that 1,8-cineole really acts as an anti-inflammatory agent. After antigen challenge, a deficient mucociliary clearance, an important physiological function of the ciliated epithelial cells [25], is often seen. Dorow et al. [26] first showed that a mixture of volatile substances, including 1,8-cineole, improved mucociliary clearance of patients with chronic obstructive pulmonary disease. In the present study, we observed that OVA-challenged guinea pigs showed a reduced distance traversed by the marker added to their tracheal lumen, and that under 1,8-cineole pre-treatment such reduction was prevented. This is indicative that the inhibitory actions of 1,8-cineole on cytokine release may also prevent mucus accumulation in airways as a result of the increased secretory response due to inflammatory mediators acting on submucosal glands [27]. Thus, our study corroborates early in vitro data and helps to increase evidence of an important role of 1,8-cineole, a monoterpene found in numerous over-the-counter cough and cold lozenges as well as in inhalation vapours or topical ointments to control airway mucus hypersecretion and other airway diseases, but with yet scarce scientific confirmation of its efficacy. Acknowledgements This work is part of a thesis submitted by V.P. Bastos in partial fulfilment of the requirements for the degree of Doctor in Pharmacology. It was supported by CAPES and INCT-IBISAB from the Brazilian research agency CNPq. We thank Ms Maria Silvandira FranÅa Pinheiro for technical assistance. References 1 Barton A, Tjandra J, Nicholas P. Chemical evaluation of volatile oils in Eucalyptus species. J Agric Food Chem 1989;37: 1253 7. 2 Magalh¼es PJC, Criddle DN, Tavares RA, Melo EM, Mota TL, Leal-Cardoso JH. Intestinal myorelaxant and anti-spasmodic effects of the essential oil of Croton nepetaefolius, and its constituents cineole, methyl-eugenol and terpineol. Phytother Res 1998;12:172 7. 3 Lahlou S, Figueiredo AF, Magalh¼es PJ, Leal-Cardoso JH. Cardiovascular effects of 1,8-cineole, a terpenoid oxide present in many plant essential oils, in normotensive rats. Can J Physiol Pharmacol 2002;80:1125 31. 4 Soares MC, Damiani CE, Moreira CM, Stefanon I, Vassallo DV. 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