Original Papers

Essential Oil from Myrcia ovata: Chemical Composition, Antinociceptive and Anti-Inflammatory Properties in Mice

Authors

Gabriela Carmelinda Martins dos Santos 1, Geovany Amorim Gomes 2, Gabriela Mastrangelo Gonçalves 1, Leôncio Mesquita de Sousa 3, Gilvandete Maria Pinheiro Santiago 3, 4, Mário Geraldo de Carvalho 2, Bruno Guimarães Marinho 1

Affiliations

The affiliations are listed at the end of the article

Key words " Myrtaceae l " Myrcia ovate l " acute pain l " essential oil l " mice l

Abstract !

The leaves of Myrcia ovata, popularly known as “laranjinha do mato”, are frequently used as an infusion in folk medicine. The essential oil obtained from these leaves is rich in citral, a mixture of neral and geranial isomers, known for its analgesic effect. Male Swiss mice (20–22 g) were tested in models of acute pain (acetic acid-induced abdominal writhing, tail flick, and formalin tests) and acute inflammation (paw oedema and air pouch tests) as well as in a model for evaluation of spontaneous motor performance (openfield test). The essential oil from M. ovata was administered orally at doses of 50–300 mg/kg. In addition, water, vehicle, morphine (5.01 mg/kg for

Introduction ! received revised accepted

Dec. 6, 2013 July 11, 2014 August 21, 2014

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383120 Published online October 8, 2014 Planta Med 2014; 80: 1588–1596 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Bruno Guimarães Marinho DVM, MSc, PhD Federal Rural University of Rio de Janeiro Department of Physiological Sciences Laboratory of Pharmacology BR465, Km07 23890–000 Seropédica, RJ Brazil Phone: + 55 21 26 82 32 22 [email protected]

Plant essential oils are complex mixtures of volatile, odourous, and lipophilic organic substances synthesised as secondary metabolites in plants. They are composed of phenylpropanoid or terpenoid derivatives, the latter being the most abundant [1]. The Myrtaceae family comprises about 130 genera with about 4000 species. It has predominantly a pantropical and subtropical distribution, being concentrated in the neotropic region and Australia [2]. Psidium pohlianum, Psidium guajava, Eugenia candolleana, and Myrcia pubiflora are examples of species of the Myrtaceae family whose essential oils show antinociceptive activity in rodents [3–6]. The leaves of Myrcia ovata Cambess., popularly known as “laranjinha do mato”, are frequently used as an infusion in folk medicine to treat gastric diseases and diarrhoea [6]. Notably, opioid receptors are widely distributed in the central and peripheral nervous system and peripheral tissues, mainly in the GI tract, and participate in many physiological processes, includ-

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evaluation of pain and motor performance), acetyl salicylic acid (200 mg/kg in the formalin test), and dexamethasone (2.25 mg/kg for evaluation of oedema formation, leukocyte extravasation, and quantification of cytokines) were administered. The essential oil showed a significant effect at doses of 200 and 300 mg/kg in the acute pain and acute inflammation tests. The effect of the essential oil was reduced by pretreatment with naloxone. The essential oil did not induce motor impairment. The extract was not toxic after oral administration (LD50 > 3000 mg/kg). These data provide initial evidence that the traditional use of M. ovata can be effective in reducing pain and inflammation.

ing alleviation of abdominal pain and inhibition of gastrointestinal motility [7]. The essential oil obtained from the leaves of M. ovata, rich in citral, shows antibacterial [8] and insecticidal activities [9]. Citral, a mixture of neral and geranial isomers, is known for its analgesic effect [10]. There is no scientific evidence for the effects of the essential oil from M. ovata (EOMO), especially those related to the control of pain and inflammation. Based on this, the aim of this study is to evaluate the antinociceptive and anti-inflammatory activities of EOMO in mice.

Results !

EOMO was obtained with a yield of 1.27 % (w/w). A chemical analysis of the essential oil showed that oxygenated monoterpenes are its most abundant class of compounds. Seven components, representing 93.55 % of the essential oil, were determined. Of these, we identified one unsaturated aliphatic ketone (0.47 %), four oxygenated mono-

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terpenes (91.78 %), one sesquiterpenic hydrocarbon (0.57 %), and one sesquiterpene oxygenated (0.73%). Intraperitoneal injection of acetic acid (1.2%) induced an average of 54.2 ± 6.1 writhes in a period of 30 min. Doses of 200 and 300 mg/kg inhibited writhing by 29 and 51 %, respectively. Morphine (5.01 mg/kg) inhibited the number of writhes by approxi" Fig. 1). mately 50% in relation to the control group (l Pretreatment with essential oil significantly reduced the time that the mice spent licking the injected paw after formalin injection. In the first phase, the inhibitory effect was observed only with the highest doses (200 and 300 mg/kg), whereas in the second phase, inhibition occurred at all doses except 50 mg/kg " Fig. 2 A). (l In the first phase, EOMO showed 58 and 59 % inhibition at doses of 200 and 300 mg/kg, respectively. In the second phase, the percent of inhibition at doses of 100, 200, and 300 mg/kg was 35, 52, " Fig. 2 A). Morphine (5.01 mg/kg) inhiband 54%, respectively (l ited the number of licks by approximately 50 % in relation to the control group in both the 1st and 2nd phases. Acetyl salicylic acid (200 mg/kg) inhibited the number of licks by approximately 60 % in relation to the control group in both the 1st and 2nd phases. " Fig. 2 B, isolated use of In both phases of the model shown in l the antagonist naloxone produced a result similar to that obtained with the control group, whereas concomitant use of this antagonist (at the two highest doses used – 1 and 3 mg/kg) with EOMO completely blocked the antinociceptive effect of EOMO in both phases.

Fig. 1 Effects of orally administered essential oil from M. ovata on acetic acid-induced writhing. The mice were pretreated with PBS, vehicle, morphine (5.01 mg/kg), or EOMO (50, 100, 200, and 300 mg/kg) 60 min before i. p. injection of acetic acid. The results are expressed as the mean ± SEM (n = 7–10). The statistical significance was calculated by one-way ANOVA followed by Bonferroniʼs test. * P < 0.05, ** p < 0.01, and *** p < 0.001 when comparing EOMO-, vehicle- and morphine-treated groups with the control group.

An effect of EOMO was observed only at higher doses (200 and 300 mg/kg) on the tail-flick test, with the maximal effect of 34 % " Fig. 3 A). These at a dose of 200 mg/kg and 91% at 300 mg/kg (l " Fig. 3 B. results can be confirmed by the observations in l In the paw oedema test, oral administration of the higher doses 200 and 300 mg/kg of EOMO reduced the paw oedema induced by carrageenan by 32% and 50%, respectively. Dexamethasone

Fig. 2 Effects of orally administered essential oil from M. ovata in the formalin test. In A, the mice were pretreated with PBS, vehicle, morphine (5.01 mg/kg), acetyl salicylic acid (ASA, 200 mg/ kg), or EOMO (50, 100, 200, and 300 mg/kg) 60 min before the formalin injection. In B, the mice were pretreated intraperitoneally with naloxone (0.01, 0.1, 1, and 3 mg/kg) 15 min before the administration of M. ovata (MO). The dose of EOMO used was 300 mg/kg. The results are expressed as the mean ± SEM (n = 7–10). The statistical significance was calculated by one-way ANOVA followed by Bonferroniʼs test. In A, * p < 0.05, ** p < 0.01, and *** p < 0.001 when comparing EOMO-, vehicle- and morphine-treated groups with the control group. In B, * p < 0.05, ** p < 0.01, and *** p < 0.001 when comparing the EOMO-, naloxone- and naloxone + EOMO-treated groups with the control group.

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Original Papers

Original Papers

Fig. 3 Effects of orally administered essential oil from M. ovata on the tail-flick test. In A, the mice were treated with PBS, vehicle, morphine (5.01 mg/ kg), or EOMO (50, 100, 200, and 300 mg/kg). In B, the graph represents the AUC calculated for each time-effect curve of the tail-flick test. The results are expressed as the mean ± SEM (n = 7–10). The statistical significance was calculated by two-way ANOVA (in A) and one-way ANOVA (in B) followed by Bonferroniʼs test. * P < 0.05, ** p < 0.01, and *** p < 0.001 when comparing EOMO-, vehicleand morphine-treated groups with the control group. # P < 0.05 when comparing the different times of measurement.

(2.25 mg/kg) inhibited the area under the curve by approximately " Fig. 4 A). These results can 50 % in relation to the control group (l " Fig. 4 B. be confirmed by the observations summaris ed in l Carrageenan applied to the air pouch produced a significant increase in leukocyte extravasation compared to application of PBS. Oral administration of EOMO reduced this leukocyte extravasation induced by carrageenan at doses of 200 and 300 mg/kg " Fig. 5). Subcutaneous administration of dexamethasone also (l reduced the leukocyte extravasation induced by carrageenan. Doses of 200 and 300 mg/kg inhibited the leukocyte extravasation by 30 % and 77 %, respectively. Pretreatment of mice with EOMO significantly suppressed TNF-α " Fig. 6). Doses of 200 and 300 mg/kg inand IL-1β production (l hibited TNF-α production by 50% and 69%, respectively, and a dose of 300 mg/kg inhibited IL-1β production by 47%. In the open-field test, EOMO had no significant effect on locomotor activity relative to the control and vehicle groups at a dose of 300 mg/kg or with another dose tested, whereas morphine significantly decreased locomotor activity (data not shown). The essential oil described in this paper was evaluated for acute toxicity in mice. No intoxication symptoms (disorientation, hyperactivity, piloerection, or hyperventilation) were observed in the animals. EOMO was not toxic after oral administration (LD50 > 3000 mg/kg).

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Discussion !

The present study aimed to evaluate the antinociceptive and anti-inflammatory activities of EOMO in models of acute pain and inflammation in mice. Furthermore, no reports were found in the literature regarding the biological actions of M. ovata, and although some pharmacological actions of the Myrcia species have been reported, no specific antinociceptive or anti-inflammatory actions of M. ovata have been reported in the literature. This study provides a pharmacological basis for M. ovata use in folk medicine and shows that this plant has potential for the development of safe phytomedicines with antinociceptive and antiinflammatory effects. The acetic acid-induced writhing reaction in mice has long been used as a screening tool to assess the analgesic or anti-inflammatory properties of new agents and is described as a typical model for visceral inflammatory pain [11]. The most important transmission pathways for inflammatory pain are those comprising peripheral polymodal nociceptors sensitive to protons, such as ASICs (acid sensitive ion channels), and to algogen substances, such as bradykinin, prostaglandin, and cytokines. These receptors signal the central nervous system (CNS) via sensory afferent C-fibres entering the dorsal horn [12, 13]. Moreover, it is well established that the nociceptive response caused by acetic acid is also dependent on the release of certain cytokines, such as TNFα, interleukin 1β, and interleukin 8, via modulation of macrophages and mast cells localised in the peritoneal cavity [14]. EOMO reduced the number of writhes, implying that it had a significant antinociceptive effect. However, this test was unable to

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ascertain whether the antinociception was related to inflammatory or noninflammatory effects. The formalin model of nociception can discriminate between inflammatory and noninflammatory components of pain. In this model, EOMO also produced antinociceptive effects in both phases of the formalin test in mice, but only at the highest doses (200 and 300 mg/kg). The formalin test is a valid and reliable model of nociception and inflammatory pain [15]. It is sensitive for various analgesic drugs. The formalin test involves a neurogenic response with the release of substance P, and inflammatory action with the release of prostaglandins (PGs), histamine, bradykinin, and serotonin [16, 17]. The formalin test consists of two time phases: the direct chemical stimulation of nociceptors is measured in the first phase, while the peripheral inflammation and changes in central processing are observed in the second phase [17, 18]. Some previous studies demonstrated that substance P and bradykinin participate in the first phase, whereas histamine, serotonin, PGs, NO (nitric oxide), and bradykinin are involved in the second phase of the formalin test [17, 18]. Drugs that act primarily on the central nervous system inhibit both phases equally, while peripherally acting drugs inhibit only the second phase [15]. The second phase is an inflammatory response with pain that can be inhibited by anti-inflammatory drugs [18, 19]. Thus, the reductions in the time spent licking in both phases of measurement indicate that the essential

oil exerts an antinociceptive effect on both inflammatory and noninflammatory pain [20]. To elucidate a possible mechanism responsible for the effect of EOMO in the formalin test, naloxone was administered prior to the administration of EOMO. Naloxone acts by competitively binding to opiate receptors and shows maximum affinity towards the µ receptor, but also antagonistic activity to κ and δ receptors [21]. Previous administration of naloxone reduced the effect produced by EOMO in the formalin test, indicating the participation of the opioid system. However, the results of the formalin test alone cannot ascertain whether the antinociceptive effect was peripheral or central. Painful thermal stimuli are known to be selective to centrally but not peripherally acting analgesic drugs [22]. In the tail-flick test, the thermal stimulation activates peripheral nociceptors, leading to reflexive removal of the tail [23]. In the present study, EOMO produced an inhibitory effect on the nociceptive response in the tail-flick test at the highest doses (200 and 300 mg/kg), confirming its central activity. In the tail-flick test, EOMO showed an effect earlier than morphine. This event may be related to the fact that the bioavailability of oral morphine is one-fourth to onesixth of that obtained with parenteral administration, owing to incomplete and irregular enteric absorption and hepatic firstpass metabolism [24]. To evaluate a possible anti-inflammatory action of EOMO, paw oedema was induced by carrageenan. Three phases have been

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Fig. 4 Effects of orally administered essential oil from M. ovata in the paw oedema test. In A, the mice were treated with PBS, vehicle, dexamethasone (2.25 mg/kg, s. c.), or EOMO (50, 100, 200, and 300 mg/kg). In B, the graph represents the AUC calculated for each time-effect curve in the paw oedema test. Carrageenan was applied in the paws of the animals in all groups. The results are expressed as the mean ± SEM (n = 7–10). The statistical significance was calculated by two-way ANOVA (in A) and one-way ANOVA (in B) followed by Bonferroniʼs test. * P < 0.05, ** p < 0.01, and *** p < 0.001 when comparing EOMO-, vehicleand dexamethasone-treated groups with the control group. # P < 0.05 when comparing the different times of measurement.

Original Papers

Fig. 5 Effects of orally administered essential oil from M. ovata in the air pouch test. The mice were treated with PBS, vehicle, dexamethasone (2.25 mg/kg, s. c.), or EOMO (100, 200, and 300 mg/kg). PBS or carrageenan was applied in the pouch of the animals. The results are expressed as the mean ± SEM (n = 7–10). The statistical significance was calculated by one-way ANOVA followed by Bonferroniʼs test. * P < 0.05, ** p < 0.01, and *** p < 0.001 when comparing EOMO-, vehicle- and dexamethasonetreated groups with the control group. # P < 0.05, ## p < 0.01, and ### p < 0.001 when comparing carrageenan-injected groups with the PBSinjected group.

postulated for carrageenan-induced oedema: histamine and serotonin release in the early phase (first hour), kinin release in the second phase (second hour), and prostaglandin release in the third phase (third and fourth hours) [25]. In this test, EOMO inhibited the induction of paw oedema in all phases, thus demonstrating its anti-oedematogenic activity. Leukocyte influx from blood into an inflammatory site represents a crucial step in inflammatory responses. Early inflammatory reactions are usually characterised by an influx of neutrophils into the inflamed tissues, whereas at late time points, an intense influx of eosinophils and mononuclear cells are usually observed. According to previous data, our results showed a significant leukocyte accumulation in the air pouch 4 h after carrageenan stimulation, as previously demonstrated [26]. This accumulation was significantly inhibited by the essential oil used in the present work. Treatment with EOMO resulted in a pronounced anti-inflammatory effect against acute carrageenan-induced inflammation. It has been shown that the carrageenan-induced mouse inflammatory response elicits the release of chemical mediators such as histamine, bradykinin, substance P, and PGs, followed by exudation and leukocyte infiltration into the inflammatory site; this peaks 4 h after induction of inflammation [27–29]. This acute inflammatory response is usually inhibited by NSAIDs (nonsteroi-

Fig. 6 Effects of orally administered essential oil from M. ovata on tumour necrosis factor α (A) and interleukin-1β (B) production in the air pouch test. The mice were treated with PBS, vehicle, dexamethasone (2.25 mg/kg, s. c.), or EOMO (100, 200, and 300 mg/kg). PBS or carrageenan was applied in the pouch of the animals. The results are expressed as the mean ± SEM (n = 7–10). The statistical significance was calculated by one-way ANOVA followed by Bonferroniʼs test. * P < 0.05, ** p < 0.01, and *** p < 0.001 when comparing EOMO-, vehicleand dexamethasone-treated groups with the control group. # P < 0.05, ## p < 0.01, and ### p < 0.001 when comparing carrageenan-injected groups with the PBS-injected group.

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Materials and Methods !

Plant material The leaves of M. ovata Cambess. (Myrtaceae) were collected in August 2012 in the city of Guaramiranga – Ceará, Brazil (4°16′ 17′′S, 38°56′ 46′′W). The plant was classified by Marcos Sobral (Faculty of Pharmacy, Federal University of Rio Grande do Sul, Brazil), and a voucher specimen was deposited in the Prisco Bezerra Herbarium of the Federal University of Ceará under the number 039 558.

Extraction of the essential oil For oil extraction, the leaves (580 g) were placed in a 5-L roundbottom beaker with 2 L of distilled water and submitted to hydrodistillation in a Clevenger apparatus for 2 h. The oil obtained was dried with anhydrous sodium sulphate, filtered, and then maintained in glass bottles under refrigeration until analysis.

Analysis of the essential oil (gas chromatography/mass spectrometry) The chemical composition of the essential oil was analysed in a gas chromatograph coupled to a mass spectrometer (GC/MS – Shimadzu QP-2010 Plus) equipped with a Factor Four/VF‑5 ms fused-silica capillary column (30 m × 0.25 mm × 0.25 µm film thickness), using helium as carrier gas at 1 mL/min. The initial oven temperature was 35 °C, which after being held constant for 2 min, was increased at a rate of 4 °C/min to 180 °C, followed by 10 °C/min to 250 °C, with a final isotherm (250 °C) for 20 min. The sample injection volume was 1 µL (1 : 50 split mode). The injector and detector temperatures were both 250 °C. The mass spectra were obtained in a range of m/z 10–300 by the electron impact technique at 70 eV.

Gas chromatography/flame ionisation detector The quantitative analysis of the oilʼs chemical composition was carried out in a gas chromatograph coupled to an HP 5890 Series II flame ionisation detector (FID), using the same type of column as in the GC/MS analysis. The injector and detector temperatures were 240 and 300 °C, respectively. The percentage of each constituent was calculated by the integral area under its respective peak in relation to the total area of all the sample constituents.

Identification of essential oil constituents The various chemical constituents of the essential oil were identified by visual comparisons of their mass spectra with those in the literature [37] and spectra supplied by the equipment database (NIST08), as well as by comparison of the retention indices with those in the literature [37]. A standard solution of n-alkanes (C8-C40) was injected under the same chromatographic conditions as the sample and used to obtain the retention indices as described by Van Den Dool and Kratz [38].

Animals Male Swiss mice (20–22 g) were obtained from our animal facility. The animals were maintained in a room with a controlled temperature (22 ± 2 °C) and a 12-h light/dark cycle, with free access to food and water. Twelve hours before each experiment, the animals received only water to avoid a possible interference of the food with absorption of the drug. The protocol for this study was approved by the ethics committee for Animal Research of the Federal Rural University of Rio de Janeiro on August 12, 2012 (COMEP – UFRRJ) under the number 23083.004724/2012–16. dos Santos GCM et al. Essential Oil from …

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dal anti-inflammatory) such as indomethacin or corticoids such as dexamethasone, and these effects have been attributed to the inhibition of inflammatory mediator release and tissue expression of inducible cyclooxygenase (COX-2) [30]. Our results revealed that EOMO decreased the concentrations of TNF-α and IL-1β in the inflammatory exudates. TNF-α is a cytokine that plays a key role in the innate immune response and is associated with cell migration and exudation [31]. IL-1β promotes the expression of adhesion molecules, leukocyte migration, and increased vascular permeability, indicating that it acts as an important proinflammatory mediator [32]. The inhibition of leukocyte migration observed in our study may be related to a decrease in IL-1β and TNF-α levels. These results suggest that EOMO can act as modulators of the immune system by decreasing cell migration, exudation, and the production of proinflammatory cytokines. Here, we confirm these observations and demonstrate that treatment with EOMO reduced the main features of acute inflammation, including exudation and leukocyte number. These findings suggest that EOMO has a critical role in controlling acute inflammatory events. To exclude the possibility that the antinociceptive action of EOMO could be related to nonspecific disturbances in the locomotor activity of the animals, the open-field test was used. We observed that at the doses that have antinociceptive action, EOMO did not alter the motor performance of the mice. The EOMO analysis revealed that the monoterpene geranial is the major constituent (52.60 %), followed by its isomer neral (37.14 %). The essential oil of M. ovata used in another study [8– 9] presented a yield of 0.9% (w/w) and also showed geranial (50.4 %) and neral (35.8%) as the most abundant components. It is therefore similar to ours with regard to chemical composition. This isomeric mixture of geranial and neral aldehydes is known as citral, which can be found among the constituents of essential oils of other species of Myrtaceae. In previous pharmacological trials, citral exhibited anticonvulsant [33], anti-inflammatory and antinociceptive activities [17] in rodents. Studies have also shown the synergistic effect between citral and naproxen [34, 35]. Some minor constituents, such as oxygenated sesquiterpenes, also have reports of antinociceptive and anti-inflammatory activities and therefore may also be responsible for the effect shown by the essential oil [36]. In conclusion, this study has demonstrated the antinociceptive and anti-inflammatory activities of EOMO in the models of chemical nociception induced by acetic acid and formalin, in models of nociception induced by thermal stimuli, as well as in models of inflammation and further suggests that this antinociceptive activity might involve the opioid system. It is important to state that there are no descriptions of adverse effects or intoxication in humans following the use of the M. ovata in folk medicine. This absence of adverse effects and intoxication is supported in our study by the lack of physiological complications in the animals that received EOMO orally. These data provide initial evidence that traditional use of M. ovata can be effective in reducing pain and inflammation, but more studies are required to further elucidate its mechanism of action.

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Chemicals The following substances were used: acetic acid (Vetec), formaldehyde (Merck), dexamethasone (purity – 97 %), λ-carrageenan, dimethyl sulphoxide, Tween 80 (Sigma–Aldrich), morphine (purity – 97 %), and naloxone (purity – 99 %) (Cristália).

Treatments Increasing doses of EOMO were administered orally (50, 100, 200, and 300 mg/kg, p. o.). Morphine, acetyl salicylic acid, and dexamethasone were used as positive controls. The doses of morphine [5.01 (2.47–8.68) mg/kg, p. o. – opioid analgesic drug] and dexamethasone [2.25 (1.82–2.79) mg/kg, subcutaneous administration (s. c.) – steroidal anti-inflammatory] were obtained by calculating the ED50 (confidence limits) in acetic acid-induced abdominal writhing and paw oedema tests that were performed beforehand. The ED50 values (the dose producing 50% of the maximal effect) for the antinociceptive and anti-inflammatory actions were obtained by fitting the data points representing the antinociceptive and anti-inflammatory effects demonstrated in these models by nonlinear regression (sigmoidal dose response) using GraphPad Prism software version 5.0 (data not shown). The dose of acetyl salicylic acid was 200 mg/kg, p. o., according to Guilhon et al. [39]. PBS solution mixed with Tween 80 was used as a vehicle, at a concentration of 2 %, for the preparation of different doses of EOMO. The control group consisted of mice that received only PBS. Each test was performed three times to confirm the results.

Acetic acid-induced abdominal writhing test In order to evaluate the antinociceptive effect of EOMO, different groups were treated orally with vehicle, PBS, morphine, or EOMO (50–300 mg/kg) 60 min before the i. p. injection of acetic acid. The behavioural protocol was performed as previously described by Koster et al. [40]. In brief, the total number of writhes after i. p. administration of 1.2 % (v/v) acetic acid (0.01 mL/g) was recorded over a period of 30 min, beginning immediately after the injection.

Formalin test In order to discriminate between inflammatory and noninflammatory activities of EOMO, different groups were treated orally with vehicle, PBS, morphine, or EOMO (50–300 mg/kg) 60 min before an i. p. injection of formalin. Formalin-induced behaviour was assessed as previously described by Hunskaar et al. [41]. Mice received a subplantar injection of 0.02 mL of formalin (2.5 % v/v) into the dorsal surface of the left hind paw. The mice were immediately placed into an individual observation chamber and the time (in seconds) the animal spent licking the injected paw was recorded. The nociceptive response includes two phases. The first phase is the neurogenic pain response, and it was recorded in the first 5 min after the formalin injection. The second phase is the inflammatory response, and it was recorded 15–30 min after the formalin injection. In order to evaluate the contribution of the endogenous opioid system to the antinociceptive activity of the EOMO, experimental groups were included that received increasing doses of naloxone (0.01, 0.1, 1, and 3 mg/ kg, i. p.) prior to the administration of the EOMO.

Tail-flick test In order to evaluate the central antinociceptive effect of EOMO, different groups were treated orally with vehicle, PBS, morphine, or EOMO (50–300 mg/kg). The test was performed as described

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by DʼAmour and Smith [42]. The mice were kept in an acrylic tube and then placed on the equipment to perform the tail-flick test. A light beam was focused approximately 4 cm from the tip of the tail and the tail withdrawal latency was recorded automatically. The light intensity was adjusted for baseline values between 4 and 6 s; this intensity was not changed and the animals that had baseline values outside these limits were excluded from the experiment. Measures of latency time were made at intervals of 20 min between these. The first two measures were made before drug administration. The average of these measures was called the ”baseline”. After drug administration, six measures of the latency time were performed. Antinociception was quantified as the percent of increase over the baseline (IBL) at each measurement time, calculated using the following formula: IBLð%Þ ¼

RT  100  100 BL

Results were also expressed by calculating the area under the curve (AUC) of responses from 20 to 120 min after drug administration, calculated according to the following formula based on the trapezoid rule: AUC = 20 × IBL [(20 min) + (40 min) + … + (120 min)/2]

Paw oedema test In order to evaluate the anti-oedematogenic effect of EOMO, different groups were treated orally with vehicle, PBS, dexamethasone (s. c.), or EOMO (p. o., 50–300 mg/kg) 60 min before the injection of carrageenan. Mouse paw oedema was induced by subplantar injection of 0.02 mL of carrageenan (1%) into one of the hind paws. As a control, 0.02 mL of distilled water was injected into the contralateral paw. Oedema was measured plethysmographically [43]. Paw oedema (PO) was measured at 1, 2, 3, and 4 hours after the injection of carrageenan, and the results expressed as the increase in carrageenan-injected paw volume (µL) minus the volume of the distilled water-injected paw. Results were also expressed by calculating the AUC of responses from 1 to 4 h after drug administration, calculated according to the following formula based on the trapezoid rule: AUC = 1 × PO [(1 h) + (2 h) + … + (4 h)/2]

Air pouch test In order to evaluate the effect of EOMO on the vascular permeability of leukocytes, different groups were treated orally with vehicle, PBS, dexamethasone (s.c) or EOMO (p. o., 100–300 mg/ kg) 60 min before the injection of carrageenan. Air pouches were generated as previously described by Vigil et al. [31]. An area of dorsal skin (3 cm × 2.5 cm) was disinfected with iodophor and shaved to provide the pouch site. Seven millilitres of sterile air was injected subcutaneously at a single site with a 16-gauge needle and 10-mL syringe. The air pouches were injected with sterile air on alternate days for three days. During this period, redness, swelling, exudation, and air leak were not observed, which suggested the air pouch model was successfully established. On the fourth day, the animals received carrageenan (Cg; 1 %) administered by the subcutaneous route, and 4 h later the animals were euthanized with an overdose of pentobarbital. The animals were fixed on a surgical table and an incision in the dorsal skin was made to perforate the air pouch. The cavity was then washed with 1.0 mL of sterile PBS [pH 7.6, containing NaCl (130 mM), Na2PO4 (5 mM), KH2PO4 (1 mM), and heparin (20 IU/mL) in dis-

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Affiliations 1

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IL-6 and TNF-α measurements Supernatants from exudates collected in the air pouch cavity were used to determine TNF-α and IL-1β measurements. TNF-α and IL-1β quantifications were determined by an enzyme-linked immunosorbent assay (ELISA), using the protocol supplied by the manufacturer (Peprotech).

Open-field test In order to evaluate the motor impairment induced by EOMO, different groups were treated orally with vehicle, PBS, morphine, or EOMO (50–300 mg/kg). Five days before behavioural testing, each animal was handled daily for a few minutes. The procedure followed was similar to the method described by Barros et al. [44]. The mice received the oral administration and were placed individually in an observation chamber (60 min after oral administration), the floor of which was divided into 50 squares (5 × 5 cm). The total number of squares covered by the animals in 5 min was counted.

In vivo toxicological evaluation An acute toxicity test was performed according to the WHO guidelines [45] and the Organization of Economic Co-operation and Development (OECD) guidelines for testing of chemicals [46]. EOMO was administered orally at increasing doses up to 3000 mg/kg (100, 300, 1000, 2000, and 3000 mg/kg). The mice received the oral administration and were placed individually in an observation chamber, where the animalʼs behaviour (disorientation, hyperactivity, piloerection, or hyperventilation) was observed starting 5 h after a single administration of the essential oil and monitored daily until the 14th day. Acute toxicity was expressed by the dose required in g/kg body weight to cause death in 50 % of the animals tested (LD50).

Statistical analysis All experimental groups consisted of 7–10 animals. The results are presented as the mean ± SEM. Statistical significance between groups was determined by one-way analysis of variance (ANOVA) followed by Bonferroniʼs test for the acetic acid-induced abdominal writhing, formalin, air pouch and open-field tests; and the statistical significance between groups was determined by a two-way analysis of variance (ANOVA) followed by Bonferroniʼs test for the tail-flick and paw oedema tests. P values less than 0.05, 0.01, and 0.001 were considered to be statistically significant.

Acknowledgements !

The authors acknowledge CNPq and FAPERJ for financial support.

Conflict of Interest !

The authors report no conflicts of interest.

Laboratory of Pharmacology, Department of Physiological Sciences, Institute of Biology, Federal Rural University of Rio de Janeiro, Seropédica, RJ, Brazil Department of Chemistry, Institute of Exact Sciences, Federal Rural University of Rio de Janeiro, Seropédica, RJ, Brazil Post Graduate Program in Chemistry, Federal University of Ceará, Fortaleza, CE, Brazil Department of Pharmacy, Federal University of Ceará, Fortaleza, CE, Brazil

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Essential oil from Myrcia ovata: chemical composition, antinociceptive and anti-inflammatory properties in mice.

The leaves of Myrcia ovata, popularly known as "laranjinha do mato", are frequently used as an infusion in folk medicine. The essential oil obtained f...
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