Oral treatment with essential oil of Hyptis spicigera Lam. (Lamiaceae) reduces acute pain and inflammation in mice: Potential interactions with transient receptor potential (TRP) ion channels

Journal of Ethnopharmacology 200 (2017) 8–15

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Oral treatment with essential oil of Hyptis spicigera Lam. (Lamiaceae) reduces acute pain and inflammation in mice: Potential interactions with transient receptor potential (TRP) ion channels

MARK

Róli Rodrigues Simõesa,b, Igor dos Santos Coelhoa,b, Stella Célio Junqueiraa,b, Glauce ⁎ Regina Pigattob, Marcos José Salvadorc, Adair Roberto Soares Santosa,b, , Felipe Meira de Fariac,⁎⁎ Programa de Pós-Graduação em Neurociências, Universidade Federal de Santa Catarina – UFSC, Florianópolis, Santa Catarina, Brazil Laboratório de Neurobiologia da Dor e Inflamação, Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina – UFSC, Florianópolis, Santa Catarina, Brazil c Departamento de Biologia Vegetal, Universidade Estadual de Campinas – UNICAMP, Campinas, São Paulo, Brazil a

b

A BS T RAC T Ethnopharmacological relevance: The genus Hyptis comprehends almost 400 species widespread in tropical and temperate regions of America. The use of Hyptis spicigera Lam. (Lamiaceae) is reported in traditional medicine due to its gastroprotective, anti-inflammatory and analgesic properties. Aim of the study: The rationale of this study was to investigate the potential use of the essential oil of H. spicigera (EOHs) as analgesic. Material and methods: The antinociceptive effect of EOHs was verified analyzing acute nocifensive behavior of mice induced by chemical noxious stimuli [i.e., formalin and transient receptor potential (TRP) channels agonists]. We also verified the effects of EOHs on locomotor activity and motor performance in mice. Finally, we investigate the involvement of central afferent C-fibers with EOHs analgesic effect. Results: EOHs presented antinociceptive effect at 300 and 1000 mg/kg on formalin-induced pain behavior model, presenting 50% and 72% of inhibition during the first phase (ED50 =292 mg/kg), and 85% and 100% during de second phase (ED50 =205 mg/kg), respectively. Temperature of the hind paw was reduced by EOHs treatment in a dose-dependent manner; oedema was diminished only by EOHs 1000 mg/kg. EOHs does not impaired locomotor activity or motor performance. For mice injected with capsaicin, a TRPV1 activator, EOHs (1000 mg/kg, ED50 =660 mg/kg) showed decreased (63%) nociceptive behavior. When injected with cinnamaldehyde (TRPA1 activator), mice treated with EOHs showed 23%, 43% and 66% inhibition on nociceptive behavior (100, 300 and 1000 mg/kg, respectively; ED50 402 mg/kg). When mice were injected with menthol (TRPM8 activator), EOHs showed 29%, 59% and 98% inhibition of nociceptive behavior (100, 300 and 1000 mg/kg, respectively; with ED50 =198 mg/kg. Finally, when desensitized mice were injected with menthol, EOHs (300 mg/kg) does not show antinociceptive effect. Conclusions: This study demonstrated the efficacy of EOHs on experimental models of nociception. We have found the involvement of TRP channels V1, A1 and M8 with EOHs activity, which was remarkably potent and efficient in inhibiting pain evoked by menthol, a TRPM8 channel activator. TRPM8 channels from TRPV1+ Cfibers, but not TRPM8+ C-fibers nor TRPM8+ Aδ mechanosensory fibers, mediate EOHs analgesic effects.

Abbreviation: ANOVA, analysis of variance; COX-2, cyclooxygenase 2; ED50, median effective dose; EOHs, essential oil of Hyptis spicigera; GC-MS, gas chromatography-mass spectrometry; i.pl., intraplanter; i.t., intrathecal; IL-1β, interleukin 1; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MAPK, mitogen activated protein kinase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NSAIDs, non-steroidal anti-inflammatory drugs; S.E.M., standard error of the mean; TNF-α, tumor necrosis factor alpha; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPM8, transient receptor potential cation channel melastatin 8; TRPV1, transient receptor potential cation channel subfamily V member 1 ⁎ Correspondence to: Departamento de Ciências Fisiológicas, Universidade Federal de Santa Catarina, Florianópolis, 88040-90 Santa Catarina, Brazil. ⁎⁎ Corresponding author. Current address: Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, 13084-153 SP, Brazil. E-mail addresses: [email protected] (A.R.S. Santos), [email protected] (F.M. de Faria). http://dx.doi.org/10.1016/j.jep.2017.02.025 Received 26 August 2016; Received in revised form 20 January 2017; Accepted 14 February 2017 Available online 16 February 2017 0378-8741/ © 2017 Elsevier B.V. All rights reserved.

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Sigma-Aldrich (St. Louis, MO). EOHs and cinnamaldehyde were dissolved in Tween 80-saline (0.9% NaCl) solution (vehicle), capsaicin was dissolved in 10% ethanol in saline, and menthol was dissolved in 1.6% ethanol/0.01% Tween 80 in saline. The final solutions containing ethanol failed to cause any nociceptive effects per se when administered alone (control groups).

1. Introduction Despite the huge amount of pain relievers available to treat a number of pain-related diseases, their large range of side effects and low efficacy lead patients to switch to alternative medicines, such as herbal medicines (Quintans et al., 2014; Zareba, 2009). In the therapeutic of pain, non-steroidal anti-inflammatory drugs (NSAIDs) block the pain stimuli to sensory tissue (Fernandez-Duenas et al., 2008), on the other hand, the NSAIDs induce several negative effects, mainly damaging gastrointestinal mucosa. Natural products are commonly associated with a wide range of biological activities, often presenting additional properties to their main effect. For instance, recently Nishijima et al. (2014) have demonstrated antinociceptive and gastroprotective activities of the monoterpene citral. Indeed, there are several data from literature describing the antinociceptive properties of essential oils (de Souza et al., 2009). The Hyptis genus (Lamiaceae) comprehends almost 400 species widespread in tropical and temperate regions of America (Pinheiro et al., 2015), where the Brazilian Cerrado presents their major diversity (Takayama et al., 2011). Within this genus, several plants have been found on traditional medicine reports and recently have been addressed to the scientific literature with a number of uses including, but not restricted to, gastro-protective, anti-inflammatory and analgesic properties (Caldas et al., 2011; Diniz et al., 2013; Jesus et al., 2013; Menezes et al., 2007; Raymundo et al., 2011; Takayama et al., 2011; Violante et al., 2012). The usage of H. spicigera has been popularly reported as reinvigorate, embalming and repellent; to treat skin diseases, cough reliever, bronchitis, gastric ulcer, migraine, headaches, pain, colds and catarrh (Onayade et al., 1990; Takayama et al., 2011). Accordantly to McNeil et al. (2011), oils obtained from Hyptis plants contain mainly mono- and sesquiterpenes. The following constituents were common among some species: 1,8-cineole; β-caryophyllene, eugenol, gamma-cadinene; p-cymene; α-pinene. The sesquiterpenes (mainly, β-caryophyllene and caryophyllene oxide) from H. pectinata essential oil exhibited antinociceptive effects mediated by opioid and cholinergic receptors (Raymundo et al., 2011). Menezes et al. (2007) showed bicyclogermacrene (12.32%), 1,8-cineole (16.86%), α-pinene (11.32%), and β-caryophyllene (8.82%) as major components of Hyptis fruticosa essential oil; the authors reported diminished writhing behavior in animals challenged with acetic acid. Franco et al. (2011) have also demonstrated the antinociceptive effects of essential oils from H. fruticosa in mice. In this study, they found an inhibitory effect on the neurogenic phase of the formalin assay, which was correlated to the concentrations of 1.8 cineole (18.70%), α-pinene (20.51%) and β-pinene (13.64%). Based on the amount of data raising the potential role of Hyptis species in the treatment of several conditions and the previous literature suggesting its traditional use (Onayade et al., 1990) we decided to investigate whether the essential oil of H. spicigera (EOHs) presents antinociceptive effect on experimental models of acute inflammatory pain in mice. Previously, we had shown the composition of EOHs and its gastroprotective effect (Takayama et al., 2011). In the present study, EOHs composition has been confirmed, highlighting three monoterpenes as major compounds: α-pinene (50.8%), cineole (20.3%) and βpinene (18.3%). We have extended the knowledge on the usage of EOHs as an antinociceptive agent and provided some mechanisms of action supporting this property.

2.2. Plant material The essential oil of H. spicigera (EOHs) was donated by Mr. Adriano Galvão de Carvalho, who also collected the plants in Distrito de Caatinga (João Pinheiro, MG, Brazil), a Cerrado region. A flowered “voucher” was identified by Professor Jorge Yoshio Tamashiro at the State University of Campinas (UNICAMP) and deposited under the number 150422 at UEC herbarium (Campinas, SP, Brazil). 2.2.1. Isolation of the essential oil The essential oil was obtained by steam distillation in Clevenger apparatus from the aerial parts (inflorescences, leaves and stems) of the fresh H. spicigera plant (500 g), yielding 0.8% w/w of the oil. The EOHs was then kept at 4 °C until usage. 2.2.2. Identification of essential oil constituents The EOHs samples were analyzed in a gas chromatographer (GC2010/QP2010 Plus, Shimadzu, Japan) coupled to an lectronic mass spectrometer equipped with a capillary column of fused silica (DB-5; 5.30 m x 0.32 mm x 0.25 µm), helium as carrier gas (1.52 mL/min, White Martins 99.9%), injector at 250 °C, detector at 250 °C and split injection mode. Mass spectrum acquisition was performed with a voltage of 70 eV at the mass range from 40 to 600 m/z. The EOHs (10 µL) was diluted in chloroform to produce a 1 mL of chromatographic grade solvent, 1 µL of which was injected as sample at the split ratio of 1:30. The column temperature was heated to 60 °C and programmed at 5 °C/min to 220 °C. The identification of substances was realized by comparing its mass spectra with the GC-MS system database (NIST 62 lib.), the literature and with the Kovats retention time indices (Adams, 1995). 2.3. Animals The experiments were performed using 2.5-months-old female Swiss mice (30 – 40 g) obtained from the animal facility of the Federal University of de Santa Catarina (Florianópolis, SC, Brazil) and housed in groups of 5 per cages at 22 ± 2 °C and humidity (60– 80%) under a 12-h light/dark cycle (lights on at 06:00 h), with access to standard laboratory diet and water ad libitum. The total number of animals used in this study was recorded at 359 individuals. Animals were habituated to laboratory conditions for at least 1 h before testing, and all experiments were performed during the light phase of the cycle. The animals were randomly distributed between the experimental groups (n=6 – 18 animals per group – each experiment was repeated 3 times in order to complete the number of animals). All experiments reported in this study were carried out in accordance with current guidelines for the care of laboratory and ethical guidelines for investigation of experimental pain in conscious animals (Zimmermann, 1983) and were approved by the Ethics Committee for Animal Research of Federal University of Santa Catarina (protocol number PP00745). The number of animals used and the intensity of the noxious stimuli were the minimum necessary to obtain reliable data.

2. Methods and materials

2.4. Pharmacological assay

2.1. Drugs and reagents

2.4.1. Formalin-induced nociception The procedure used was similar to that described previously (Santos and Calixto, 1997). After an adaptation period, mice received

Capsaicin, cinnamaldehyde, menthol, and Tween 80 were from 9

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2.4.4. Involvement of the central afferent fibers sensitive to capsaicin (TRPV1+) on the antinociceptive effect of EOHs High doses of capsaicin destroy TRPV1+ C-fibers. We explored this fact to investigate the role of the TRPM8 channels found in the central afferents fibers sensitive to capsaicin (TRPV1+) in the antinociceptive effect of EOHs. To this end, mice were anesthetized with isoflurane 1 – 2% and treated intrathecally (i.t.) with capsaicin (10 μg/site, i.t.; in a volume of 5.0 µL with a 30-gauge needle attached to a Hamilton syringe) as described previously (Cavanaugh et al., 2009), with minor modifications. Control animals received, by the same route and same volume the vehicle used to dissolve capsaicin (10% ethanol, 10% Tween 80%, and 80% saline). The efficiency of the ablation of the central afferent fibers sensitive to capsaicin was confirmed by paw withdrawal latency in the hot plate test (50 °C, cut-off 60 s). Forty-eight (48) hours after i.t. treatment with capsaicin or vehicle, animals (n =10) were pretreated with EOHs (300 mg/kg, p.o.) or vehicle prior to nocifensive behavior induced by menthol (1.2 μmol/paw), then, animals were analyzed as described above.

an intraplantar (i.pl.) injection of 20 µL of a 2.5% formalin solution (0.92% formaldehyde in saline) in the ventral surface of the right hind paw. To determine the effects of EOHs on nociception, mice were pretreated with EOHs (100, 300 and 1000 mg/kg, p.o.) 1 h before formalin injection in the right hind paw. Control animals were pretreated with vehicle (5% tween 80 in saline, 10 mL/kg, p.o.) 1 h before formalin injection. After the formalin injection, the animals were immediately individually placed in an acrylic chamber, and the time spent licking the injected paw was recorded with a chronometer for both the early neurogenic phase (0–5 min) and late inflammatory phase (15 – 30 min) of this model. These values were considered measures of nociception. In addition, oedema and local temperature of the paw were verified after the observation time of the nociceptive response induced by formalin. Paw oedema was measured from the central region of the right paw before and after the injection of formalin using digital micrometer (MT-045B; Shanghai Metal Great Tools Co., Shanghai, China) and was expressed as the difference between paw thickness before and after formalin challenge (Δ paw thickness, in millimeters). The temperature difference (°C) between the ventral surface of the right paw, before and after formalin injection, was regarded as an index of the local paw temperature of the animals using a Mallory-Pro Thermosensor (10–50 °C).

2.5. Statistical analysis Results are presented as mean ± S.E.M. and the data were analyzed by one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc test or two-way ANOVA followed by Bonferroni post-test; p values less than 0.05 were considered significant. The median effective dose (ED50) values (i.e., dose capable of reducing nociceptive response by 50% relative to the control value) were determined by non-linear regression analysis of each experiment and reported as geometric mean. All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA).

2.4.2. Measurement of locomotor activity and motor coordination In order to exclude the possibility that the observed antinociceptive action could be related to non-specific effects in the locomotor activity, the open field test was carried out as previously described (Broadhurst, 1957). Mice were placed in a wooden box (40×60×50 cm) with the floor divided in 12 identical squares, and the number of squares crossed with all paws was counted during 6 min. Animals (n=6 – 12) were pre-treated with EOHs (100 – 1000 mg/kg, p.o.) or vehicle 1 h prior to the test, while control animals received the same volume of vehicle. We have also performed the rota-rod test (Scheidt et al., 2002) to exclude the possible nonspecific muscle relaxant or sedative effects of EOHs. The apparatus (model-DS 37; Ugo Basile) consisted of a bar with a diameter of 2.5 cm, subdivided into six compartments by disks, 25 cm in diameter (Dunham and Miya, 1957). The bar rotated at a constant speed of 22 rpm. The animals (n =8) were selected 24 h previously by eliminating those mice that did not remain on the bar for two consecutive periods of 60 s. Animals (n =8 per group) were treated with EOHs (100 – 1000 mg/kg, p.o.) or with vehicle, and were retested. The time they remained on the rotating bar (maximum of 60 s) was recorded and the number of falls was counted.

3. Results 3.1. Chemical analysis of the essential oil The GC-MS analysis of EOHs (Table 1) revealed fourteen compounds, mainly terpenes. Among these compounds three monoterpenes were found as major compounds: α-pinene (50.8%), 1,8-cineole (20.3%) and β-pinene (18.3%). 3.2. Formalin-induced nociception The results showed in Fig. 1 (A–D) demonstrate the effects of EOHs in the formalin test. EOHs caused a significant dose-related inhibition of the neurogenic (phase 1, 0–5 min) and the inflammatory phase (phase 2, 15–30 min) of the formalin-induced nocifencive behavior. The inhibition values were 11, 50% and 72% for the phase 1 (Fig. 1A) and 0, 85% and 100% for the phase 2 (Fig. 1B) at doses of 100, 300 and

2.4.3. Effects of EOHs on pain behavior induced by the injection (i.pl.) of TRPV1, TRPA1 and TRPM8 agonists in mice To evaluate the possible involvement of transient receptor potential cation channels (TRP), subfamily V member 1 (TRPV1), subfamily A member 1 (TRPA1) and melastatin 8 (TRPM8), on the antinociceptive effect of EOHs, mice were submitted to a test using either capsaicin, cinnamaldehyde or menthol, all activators of these channels respectively, as previously described by Cordova et al. (2011). Briefly, the mice were pretreated with EOHs (100 – 1000 mg/kg, p.o.) or vehicle (10 mL/kg, p.o.), 1 h prior the injection of 20 µL of capsaicin (an activator of the TRPV1 channel, 5.2 nmol/paw), cinnamaldehyde (an activator of the TRPA1 channel, 10 nmol/paw), menthol (an activator of the TRPM8 channel, 1.2 μmol/paw) or corresponding vehicle into the ventral surface of the right hind paw. The animals (n =10 – 24) were individually placed in an acrylic chamber (9×11×13 cm), and paw licking or biting was recorded with a chronometer for 5 min (capsaicin and cinnamaldehyde), 15 min (acidified saline) or 20 min (menthol). The time spent licking/biting the injected paw was considered indicative of nociception.

Table 1 Chemical composition of the essential oil of Hyptis spicigera.

10

Peak

Compound

Composition %

1 2 3 4 5 6 7 8 9 10 11 12 13 14

α-thujene α-pinene Sabinene β-pinene β-myrcene α-phellandrene β-cymene 1,8-cineole Copaene β-bourbonene β-caryophyllene Humulene Germacrene Caryphyllene oxide

0.18 50.78 1.10 18.30 0.13 0.30 0.23 20.31 0.24 0.29 7.01 0.38 0.16 0.59

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Fig. 1. Effects of the oral administration of EOHs (100 – 1000 mg/kg) on formalin-induced pain behavior in mice (n=6 – 13). (A) Shows first phase (neurogenic) of licking behavior provoked by formalin injection, (B) Shows the second phase (inflammatory), (C) Indicates the temperature changes between beginning and ending of the procedure, (D) Represents the changes in paw oedema between beginning and ending of the procedure. Columns represents the mean ± S.E.M. ANOVA followed by Newman-Keuls test, *p < 0.05, **p < 0.01 and ***p < 0.001. Percentage values indicate the inhibition of licking behavior compared to control. ED50 are presented as geometrical mean determined by non-linear regression.

crossings (Fig. 2A). In addition, motor performance was not significantly influenced by EOHs (Fig. 2B).

1000 mg/kg, respectively. Moreover, the calculated ED50 values for the first and second phase were 292 mg/kg and 205 mg/kg, respectively. However, EOHs was more effective in relation to the phase 2 of the formalin test. As expected the temperature (Δt°1-t°0) of the hind paw was also reduced by EOHs, in a dose-dependent manner, with ED50 of 285 mg/kg and inhibition of 27%, 53% and 74% at the dose of 100, 300 and 1000 mg/kg, respectively (Fig. 1C). Surprisingly, EOHs at dose of 1000 mg/kg, but not 100 or 300 mg/kg, prevented the oedema caused by the formalin injection, with inhibition of 43% compared with control group (Fig. 1D).

3.4. Effects of EOHs on pain behavior induced by the intraplantar injection of TRPV1, TRPA1 and TRPM8 agonists in mice As illustrated in Fig. 3, injection (i.pl.) of capsaicin (an activator of the TRPV1 channel, 5.2 nmol/paw), cinnamaldehyde (an activator of the TRPA1 channel, 10 nmol/paw) or menthol (an activator of the TRPM8 channel, 1.2 μmol/paw) produced marked nocifensive behavior in mice. Interestingly, EOHs treatment at the highest dose (1000 mg/kg, p.o.) significantly reduced the nocifensive behavior induced by capsaicin, with ED50 of 660 mg/kg (Fig. 3A). Furthermore, EOHs treatment inhibited the nociceptive behavior responses induced by cinnamaldehyde or menthol injection (i.pl.)

3.3. Measurement of locomotor activity and motor coordination The locomotor activity of mice was not altered by EOHs administration presenting non-statistical differences values in the number of

Fig. 2. Effects of the oral administration of EOHs (100 – 1000 mg/kg) on A: locomotor activity (n =6 – 12, animals were observed for 6 min and the number of crossings recorded) and B: motor coordination (n =8 per group, the time they remained on the rotating bar, max 60 s, was recorded). Columns represents the mean ± S.E.M. ANOVA followed by Newman-Keuls test, p > 0.05.

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Fig. 3. Effects of the oral administration of EOHs (100 – 1000 mg/kg) on pain behavior induced in mice by intraplantar administration of capsaicin (n =10–15), a TRPV1 activator (panel A), cinnamaldehyde (n =15–24), a TRPA1 activator (panel B) and menthol, a TRPM8 (n =12–19) activator (panel C). Columns represents the mean ± S.E.M. ANOVA followed by Newman-Keuls test, *p < 0.05, **p < 0.01 and ***p < 0.001. Percentage values indicate the inhibition of licking behavior compared to control. ED50 presented as geometrical mean determined by non-linear regression.

usage of natural occurring essential oils (Bakkali et al., 2008; de Sousa, 2011). Among those compounds, the Hyptis genus is a well-known source of essential oil and has recently been investigated on several experimental models, including gastrointestinal diseases, inflammation and pain (Diniz et al., 2013; Grassi et al., 2006; Menezes Pdos et al., 2015; Sanchez Miranda et al., 2013; Takayama et al., 2011). Previously, we had investigated the effects of EOHs on gastric ulcer experimental models in which we demonstrated its gastroprotective and healing effects; moreover, no sign of toxicity of EOHs (100 mg/kg) oral administration was found (Takayama et al., 2011). Considering that: (1) usually pain relievers (e.g. non-steroidal anti-inflammatories) impair gastric mucosa leading to ulceration, (2) EOHs is gastroprotective and presented no sign of toxicity, and (3) there is ethnopharmacological data suggesting both, gastroprotective and analgesic effects of EOHs, thus we have decided to investigate the role of EOHs (100 – 1000) on experimental models of acute pain. Considering the experimental models for acute pain study, the formalin test is the most useful tool to verify whether a compound has antinociceptive activity or not. We have employed this experimental model to test to possible antinociceptive effect of EOHs. Injection of formalin into the hind paw induces a biphasic pain response, the first phase is thought to result from direct activation of primary afferent sensory neurons, whereas the second phase has been proposed to reflect the combined effects of afferent input and central sensitization in the dorsal horn (McNamara et al., 2007); thus, this test provides information from two phases of pain generation, neurogenic and inflammatory. In the present study, we have found antinociceptive effects of EOHs on both, neurogenic and inflammatory, phases in the formalin test (Fig. 1A – B). However, EOHs was significantly more effective against the nocifensive behavior evoked by formalin in the inflammatory phase.

when compared to the control group. The inhibition values for the paw cinnamaldehyde test were 26%, 43% and 66% and for the paw menthol of 29%, 59% and 98% at doses of 100, 300 and 1000 mg/kg, with ED50 values of 402 and 198 mg/kg, respectively (Fig. 3B and C). At the ED level, EOHs was about 2.0–3.3-fold more potent and effective in inhibiting the nocifensive behavior induced by menthol that that caused by caused by capsaicin and cinnamaldehyde, respectively. 3.5. Involvement of the central afferent fibers sensitive to capsaicin (TRPV1+) on the antinociceptive effect of EOHs Intrathecal administration of capsaicin (acting on TRPV1 receptor) results in a rapid inactivation of C-fibers at the peripheral terminals, which likely results from direct actions on the spinal cord leading to desensitization. Moreover, the desensitization of TRPV1+ C-fibers in the spinal cord inhibits this nociceptive pathway. Here, we confirmed previous findings and demonstrated that an i.t. injection of capsaicin (25.5 ± 1.4 s) significantly (p < 0.001) increased the pain latency in the hot plate test compared with the vehicle (13.3 ± 0.7 s) group (Fig. 4A). Depletion of TRPV1+ C-fibers by capsaicin decreases the nociceptive behavior induced by menthol (in control and EOHs-treated mice) and abolishes the analgesia produced by EOHs. The result presented in Fig. 4B shows that TRPM8 localized in TRPV1+ C-fibers are required for the antinociceptive effect of EOHs (300 mg/kg). TRPM8+ C-fibers and TRPM8+ Aδ fibers (AM) do not contribute to the antinociceptive action of EOHs, suggesting that EOHs acts on TRPM8 channels from TRPV1+ C-fibers. 4. Discussion There is a strong body of evidences supporting the pharmacological

Fig. 4. A: latency to lick on 50 °C hot plate one day after i.t. injection of vehicle or capsaicin. B: effects of the oral administration of EOHs (300 mg/kg) on pain behavior induced by intra-plantar administration of menthol (a TRPM8 activator) two days after i.t. injection of vehicle or capsaicin. Columns represent the mean ± S.E.M. two-way ANOVA followed by Bonferroni's test, **p < 0.01 compared with the appropriate control group, # indicates non-statistical difference (NS).

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releasing inhibited when exposed to cineole (Juergens et al., 2004). Bastos et al. (2011) showed that inhaled 1,8-cineole prevented the hyperresponsiveness of guinea pig airways challenged with ovalbumin and inflammatory response as measured by pro-inflammatory cytokines tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) levels and decreased neutrophil infiltration. Liapi et al. (2007) have demonstrated the antinociceptive properties of 1,8-cineole and β-pinene in mice and rats, employing hot plate and tail flick tests. The authors showed that 1,8-cineole has a non-opioid antinociceptive effect on both, rats and mice. Beta-pinene, however, showed opioid-like effects reversing the effects of morphine, reason by which the authors suggested it may act as partial agonist. Because EOHs showed prominent antinociceptive effect on formalin-test and, because one of the major compounds, 1,8-cineole, was demonstrated to promote relaxation and sedation in certain contexts, we verified the possible influence of myorelaxant or sedative effects of EOHs on behavioral tests. In order to investigate this possibility, we submitted mice the open field and rotarod tests. Neither locomotor activity (Fig. 2A) nor motor performances (Fig. 2B) were significantly influenced by EOHs oral administration to mice. Differently than reported by Santos and Rao (2000), our data suggest that the compounds in EOHs, markedly 1,8-cineole, did not impair the locomotor activity neither motor performance of mice. Maybe this effect has not been found because of the differences on the concentration and dosage used in our study (1,8-cineole 20.31% within a mixture of substances, 100 – 1000 mg/kg) and their report (1,8cineole 100%, 100 – 400 mg/kg), or, perhaps, the gender employed on these studies. After determining the antinociceptive activity of EOHs, we have speculated the involvement of TRP in the antinociceptive activity of EOHs. Numerous functional studies have shown that these TRP channels mediate hyperalgesia following tissue and/or nerve injury and therefore may represent potential targets for novel analgesic drugs (Basbaum et al., 2009). Activation of these peptidergic C-fibers likely contributes to acute and chronic pain associated with a wide range of pathophysiological conditions involving inflammation, such as arthritis, irritable bowel syndrome, migraine headache, and cancer pain (Julius, 2013). TRP channels are a large family of non-selective cation channels. Several TRP channel family members, including TRPV1, TRPA1 and TRPM8, are expressed in distinct populations of primary afferent neurons (Huang et al., 2012). It is known that the nociceptive response begins when primary sensory fibers are activated by noxious stimulus, which may be chemical, thermal or mechanical. For instance, the TRP channels, especially TRPV1 and TRPA1, are highly involved in the transduction and sensitization in primary afferent somatosensory neurons (Basbaum et al., 2009). Besides activated by irritant chemicals, these ion channels are transducers of both thermal and mechanical stimuli, acting as molecular integrators for a range of diverse noxious stimuli (Montrucchio et al., 2013). Taken this into consideration, there are some reports indicating that plant essential oil components are active towards ion channels and receptors (de Araujo et al., 2011). Calixto et al. (2005) provided important aspects from the contribution of natural products over the characterization of TRP channels, especially the natural occurring essential oils constituents (i.e. monoterpenes). Plant essential oils are typically composed of volatile aromatic terpenes and phenylpropanoids. These lipophilic substances are classified as monoterpenes and sesquiterpenes based on the number of isoprene units (two and three respectively), besides the phenylpropanoids, which are made up of C6C3 units. These molecules freely cross cellular membranes and may serve various signaling roles inside the cell (de Araujo et al., 2011). Although monoterpenes usually activate TRP channels, there are several data suggesting the role of these substances in decreasing nociception-induced behavior through the

Even though EOHs (300 and 1000 mg/kg) has shown antinociceptive effects during both phases of this test, especially during inflammatory phase (2), EOHs 300 mg/kg failed in preventing oedema formation (Fig. 1D). In addition to the antinociceptive action, EOHs 1000 mg/kg markedly decreased oedema and temperature change. These results suggest that EOHs may have anti-inflammatory effects since it blocked three (pain, oedema, heat) out of five signs of inflammation (redness, heat, swelling, pain and loss of function). Although oedema and heat account for inflammatory settings, the presence of oedema in mice treated with EOHs 300 mg/kg did not influence its antinociceptive action, which was statistically indistinguishable from EOHs 1000 mg/kg (Fig. 1A – D). Based on the chemical analysis of the EOHs (Table 1), three monoterpenes were found as major constituents: α-pinene, 1,8-cineole and β-pinene. These compounds (and the other minor compounds) are commonly found in a variety of essential oils and are frequently speculated to possess medicinal properties. We focused our literature search on the major compounds cited above. Using a carrageenan-induced paw inflammation model Popovic et al. (2014) showed the antinociceptive and anti-edematous activity of essential oils from two Laserpitium species they both presenting more than 30% α-pinene in their composition. Their results suggest that αpinene may be responsible for these effects. Indeed, α-pinene had already been shown to possess antinociceptive effect. A report (Him, 2008) showed that α-pinene prevented thermal pain in the tail-flick test and did not impair motor coordination in mice. Quintao et al. (2010) reported that inflammatory (carrageenan or complete's Freud adjuvant-induced paw inflammation) and neuropathic pain (partial ligation of the sciatic nerve) were abolished in mice treated with essential oil of Ugni myricoides or αpinene (its major compound – 52.1%), suggesting that the effects could be attributed to the presence of α-pinene. More recently, α-pinene was also shown to prevent the inflammatory activation of mouse peritoneal macrophages by lypopolysaccharides (LPS) (Kim et al., 2015). The authors suggested that α-pinene suppress mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB) and therefore, inhibits several down-streams inflammatory enzymes such as inducible nitric oxide (iNOS) synthase and cyclooxygenase-2 (COX-2). Franco et al. (2011) have shown differences in α-pinene and βpinene concentration between essential oils from different parts of H. fruticosa and their corresponding activity on formalin test. Interestingly, they showed that the higher is the concentration of αpinene 5.27–20.51%) and β-pinene 6.59–13.64%) the stronger is the effect in the first phase of formalin test. Our data show that EOHs has more potent effect in the first phase of formalin test compared to their finding for H. fruticosa. Despite the different dosage between these studies, the concentration of α-pinene and β-pinene in EOHs are higher (50.78% and 18.30%, respectively) than those indicated by Franco et al. (2011), which, perhaps, seems to account for the antinociceptive action of EOHs. The literature exploring the effects of α-pinene strongly suggests a role for α-pinene on inflammatory and painful contexts. Likewise, our data suggest that α-pinene is, reasonably, contributing for the antinociceptive effects of EOHs. Another major compound in EOHs – cineole – had also been studied for its antinociceptive effect before. Santos and Rao (2000) demonstrated the antinociceptive and anti-inflammatory activities of 1,8-cineole in the formalin test, including suppression of oedema formation. The authors have reported, however, the impact of 1,8cineole on locomotor activity. Conversely, 1,8-cineole presented myorelaxant effect on humans prior to electromyography (Gobel et al., 1994). Eucolyptus oil, which is ~ 90% 1,8-cineole, showed analgesic and anti-inflammatory effects, presenting oedema inhibition (Silva et al., 2003). Cultured human lymphocytes and monocytes had cytokines 13

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desensitized mice (TRV1+ depleted) injected with menthol (i.pl). We tested the thermal responsiveness of these animals employing a hot plate, assuring that TRPV1+ C-fibers were effectively abolished. Normal and desensitized mice were treated with EOHs (300 mg/kg, p.o.) or vehicle and injected with menthol (i.pl.). The result presented in Fig. 4B shows both, nocifensive effect of menthol is diminished by TPRV1+C-fiber ablation, and TRPM8 localized in TRPV1+ C-fibers are necessary to the antinociceptive effect of EOHs (300 mg/kg). Desensitized mice presented diminished (~60%) nociceptive behavior, suggesting that the unique TRPM8+ afferents and/or myelinated Aδ fibers (AM subset) accounts for the remaining behavior evoked by menthol. It is intriguing, however, the fact that EOHs show no effect on residual TRPM8+ afferents, at least on the C-fibers subset. Although we cannot rule out how EOHs interacts with TRPM8, it is more likely that compounds in EOHs are TRPM8 partial agonist thus, impeding the activation of TRPM8 by menthol. This result confirms our finding (Fig. 3C) and shows that TRPM8 channel from TRPV1+ fibers, but not from TRPM8+ C-fibers or AM fibers are targeted by EOHs.

administration of TRP agonists (Amorim et al., 2009; Nishijima et al., 2014; Rios et al., 2013). Our findings shown that EOHs produces significant inhibition from capsaicin-induced pain behavior when administered at the highest dose (1000 mg/kg), as demonstrated in Fig. 3A. The ED50 for this assay was calculated at 660 mg/kg, suggesting the EOHs low efficacy of EOHs in diminishing pain stimuli when elicited selectively via TRPV1 nociceptors. Almost 50% of the somatosensory neurons within the rodent ganglia are unmyelinated TRPV1+ C-fiber. Initially determined as the capsaicin receptor, TRPV1 represents the detector of noxious heat (threshold of ~43 °C), neurogenic inflammation, and thermal hyperalgesia (Szolcsanyi and Sandor, 2012). Tissue acidification and lipid metabolites can activate TRPV1 and many intracellular signaling triggered under inflammatory responses can sensitize this cation channel (Le Pichon and Chesler, 2014). Our data suggests then, that EOHs effects are might due to a mechanism other than specific TRPV1 interaction. Current evidence suggests that TRPA1 plays little role in acute cold sensation but more likely contributes to injury-evoked cold hypersensitivity (Bautista et al., 2006). There is widespread agreement that TRPA1 plays an important role in chemo nociception by serving as a detector of chemical irritants that elicit acute and inflammatory pain, especially stinging pain (Basbaum et al., 2009). TRPA1 is activated by isothiocyanates and thiosulfonates that constitute pungent agents from mustard and allium plants, respectively (Julius, 2013). EOHs exhibits antinociceptive effect for all doses tested (100, 300 and 1000 mg/kg) against the nocifensive behavior caused by cinnamaldehyde. In addition, ED50 was found at 402 mg/kg, suggesting a preference of EOHs compounds for TRPA1 rather than TPRV1. Importantly, TRPA1 has been demonstrated to mediate formalininduced nociception compared to that evoked by TRPA1 agonists, indicating that TRPA1 activation by formalin fires this subset of C-fiber (McNamara et al., 2007). EOHs might have its prominent antinociceptive effect on formalin (Fig. 1A – B) because of the blocking of TRPA1 signaling, as suggested by cinnamaldehyde evoked nociception (Fig. 3B). Finally, our investigation among EOHs effects on TRP channels has found the highest efficacy and potency when nocifensive behavior was evaluated after injection (i.pl.) of menthol. In this set of experiment (showed in Fig. 3C), we have found that EOHs inhibits menthol evoked nociceptive response dose-dependently in 29%, 59% and 98% (100, 300 and 1000 mg/kg, respectively), with ED50 value of 198 mg/kg. TRPM8 channels are expressed in about 15% of all somatosensory neurons, mostly unmyelinated C-fibers, as well as a cohort of myelinated Aδ fibers in a subset of mechanoreceptors (AM-fibers) (Bautista et al., 2007). Despite the difference in numbers, a significant proportion of TRPM8+ C-fibers co-express TRPV1, according to Takashima et al. (2007) 38%, while Dhaka et al. (2008) reported 18%. Several data have provided the knowledge from interactions of monoterpenes and TRP channels, including menthol as a TRPM8 agonist. There are many other natural occurring drugs such as essential oils compounds interacting with TRPM8 afferents as both, activators and inactivators (Bautista et al., 2007; Calixto et al., 2005; Julius, 2013). Taken together, these results clearly towards TRPM8 as principal target in the context of our research, indicating that compounds found in EOHs may act on TRPM8 afferents. However, despite our findings, the sensory neurons responsible for antinociceptive effects of EOHs remain unclear. To address this question, we have ablated TRPV1+C fibers by i.t. injection of capsaicin. Ablation of TRPV1+ afferents abolishes responsiveness of superficial and deep dorsal horn neurons to noxious thermal, with no change in response to noxious mechanical stimulation (Cavanaugh et al., 2009). Since a subset of C-fibers are TRPM8+ and TRPV1+, we asked whether EOHs would have antinociceptive effect in

5. Conclusions In the present work, we have demonstrated the efficacy of EOHs in experimental models of nociception, including the attenuation of temperature and oedema – two elements of the inflammatory response in the formalin test. We have also found the involvement of TRP channels V1 (ED50 660 mg/kg), A1 (ED50 402 mg/kg) and M8 (ED50 198 mg/kg) with EOHs, which was verified by the inhibition of pain behavior evoked by specific activators of TRPV1, TRPA1 and TRPM8 (V1 < A1 < M8). The inhibition was more potent on TRPM8 in a dosedependent manner. Indeed, we have demonstrated that this effect depends on TRPM8 channels from TRPV1+ C-fibers. The present study shows for the first time evidences for some of the mechanisms implicated in the antinociceptive effects of EOHs. Considering these findings and those from a previous gastro-protective study (Takayama et al., 2011), EOHs arises as an interesting natural occurring drug for pain treatment being safe and protective for the gastrointestinal tract. Author contributions FMF, RRS, ISC, SCJ and GRP conducted the experiments; FMF, ISC and ARSS analyzed the data; FMF, MJS, ARSS design and the study; ARSS and FMF oversaw and wrote the study. Acknowledgements The authors would like to acknowledge Mr. Adriano Galvão de Carvalho (for donating plant material and essential oil), and Prof. Jorge Yoshio Tamashiro (for botanical identification). This research was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC). FMF was recipient of a PNDP/CAPES grant. References Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. Allured Publishing Corporation, Carol Stream, Ill, USA. Amorim, A.C., Lima, C.K., Hovell, A.M., Miranda, A.L., Rezende, C.M., 2009. Antinociceptive and hypothermic evaluation of the leaf essential oil and isolated terpenoids from Eugenia uniflora L. (Brazilian Pitanga). Phytomedicine 16, 923–928. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils–a review. Food Chem. Toxicol. 46, 446–475. Basbaum, A.I., Bautista, D.M., Scherrer, G., Julius, D., 2009. Cellular and molecular mechanisms of pain. Cell 139, 267–284. Bastos, V.P., Gomes, A.S., Lima, F.J., Brito, T.S., Soares, P.M., Pinho, J.P., Silva, C.S., Santos, A.A., Souza, M.H., Magalhaes, P.J., 2011. Inhaled 1,8-cineole reduces

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