Antinociceptive activity of the Psidium brownianum Mart ex DC. leaf essential oil in mice

Food and Chemical Toxicology 135 (2020) 111053

Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Antinociceptive activity of the Psidium brownianum Mart ex DC. leaf essential oil in mice

T

Renata de Souza Sampaioa,b, Emmily Petícia do Nascimentoc, Irwin Rose Alencar de Menezesa,∗, Valterlúcio dos Santos Salesd, Anita Oliveira Brito Pereirae, Giovana Mendes de Lacerdaa,∗∗, Enaide Soares Santosa, Maria Janice Pereira Lopesa, Luanna Gomes da Silvaa, Gyllyandeson de Araújo Delmondesa,∗∗∗∗, Nélio Barreto Vieiraf, Victor Mantoani Zaiaf, Daniel Souza Bezerraa, José Galberto Martins da Costaa, Cícero Francisco Bezerra Felipeb, Marta Regina Kerntopfa,∗∗∗ a

Regional University of Cariri, Crato, Ceará, Brazil Federal University of Paraiba, João Pessoa, Paraíba, Brazil c Postgraduate Program in Health Sciences of the Faculty of Medical Sciences of Santa Casa de São Paulo, Brazil d Federal University of São Paulo, São Paulo, Brazil e Federal University of Pernambuco, Recife, Pernambuco, Brazil f Programa de Pós-graduação em Ciência da Saúde, Centro Universitário Saúde ABC, Santo André, São Paulo, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Psidium brownianum Antinociceptive activity Essential oil Natural products

Chronic pain management has several adverse effects and research looking for new and effective pain management drugs posing lower undesirable effects is necessary. Given the above, the pharmacological investigation of medicinal plants significantly contributes to the dissemination of plant-derived therapeutics. The aim of this study was to evaluate the antinociceptive activity of the Psidium brownianum Mart ex DC. leaf essential oil (PBEO) and the participation of the opioid pathway in this effect in mice. Swiss Mus musculus male mice were tested using acute nociception models (acetic acid induced abdominal contortions, formalin, capsaicin and hot plate tests). The possible myorelaxant action of the PBEO was tested using the rotarod test. The essential oil reduced animal nociception in chemical and heat models, with this action being devoid of a myorelaxant effect. Naloxone (2 mg/kg, intraperitoneally – i.p.) partially antagonized the PBEO activity, possibly acting via opioid receptors. The results obtained provide evidence that the traditional Psidium brownianum use may be effective for pain treatment.

1. Introduction Pain is an unpleasant sensory and emotional experience, which causes a loss in quality of life regardless of age, race or social group (Brunelli et al., 2014; Von Korff et al., 2016). However, pain has a physiological role acting as an organism's threat signal, for both potential and real threats, thus, preserving the organism's integrity (Van Hecke et al., 2013). Studies have shown that 50% of individuals seeking medical attention have pain-related complaints which burden the health systems (Andrew et al., 2014; Gaskin and Richard, 2012; Linde

et al., 2012) in countries such as Brazil, the United States of America (Johannes et al., 2010) and European countries such as France, England, Germany and Spain (Liedgens et al., 2016). Although drugs used for pain management have a high efficacy level (Pereira et al., 2015), they have also shown considerable amounts of adverse effects (Batlouni, 2010; Castel-Branco et al., 2013; Coluzzi et al., 2016; Michellin et al., 2006; Monteiro et al., 2008). Due to the numerous adverse effects associated with pain management, the search for new and effective drugs with no or at least low undesirable effects is necessary (Guilhermino et al., 2012). The use of medicinal plants and

∗

Corresponding author. Rua Cel. Antônio Luis, 1161 - 63105-000, Pimenta, Crato, CE, Brazil. Corresponding author. Rua Cel. Antônio Luis, 1161 - 63105-000, Pimenta, Crato, CE, Brazil. Corresponding author. Rua Cel. Antônio Luis, 1161 - 63105-000, Pimenta, Crato, CE, Brazil. ∗∗∗∗ Corresponding author. Rua Cel. Antônio Luis, 1161 - 63105-000, Pimenta, Crato, CE, Brazil. E-mail addresses: [email protected] (I.R. Alencar de Menezes), [email protected] (G. Mendes de Lacerda), [email protected] (G. de Araújo Delmondes), [email protected] (M.R. Kerntopf). ∗∗

∗∗∗

https://doi.org/10.1016/j.fct.2019.111053 Received 5 December 2018; Received in revised form 3 November 2019; Accepted 10 December 2019 Available online 17 December 2019 0278-6915/ © 2019 Elsevier Ltd. All rights reserved.

Food and Chemical Toxicology 135 (2020) 111053

R. de Souza Sampaio, et al.

QP5050A model), equipped with a DB-5HT fused silica capillary column (30 m long, with 0.25 mm internal diameter and 0.25 μm film thickness). Helium was used as a carrier gas at 0.8 mL/min. Injector and detector (or interface) temperatures were held at 250 °C and 200 °C, respectively. Column temperatures were raised from 35 to 180 °C at a rate of 4 °C/min and then heated from 180 to 250 °C/min at a rate of 10 °C/min. The mass detector was operated at the ionization energy of 70 eV and in the 30–450 amu range. The identity of each compound was determined by comparison of its retention index relative to those of C8–C20 n-alkanes (Fluka Analytical, 1.0 mL Alkane Standard Solution) with retention indices reported in the literature, as well as by visual comparison of their mass spectra with those from the spectrometer database and those reported in the literature(Adams, 2007; Alencar et al., 1984, 1990).

their efficacy significantly contributes to the dissemination of plantderived therapies (Pimentel et al., 2015). Psidium brownianum Mart ex DC. (Myrtaceae), known as “araçá-deveado” and “murtinha do mato”, is widely distributed across the vegetation of the interior of Brazil (Albuquerque et al., 1991) and in some communities, Psidium brownianum is used for flu and fever treatment (Freitas et al., 2015). While cytoprotective effects have been reported by (Coutinho et al., 2018), other species from the Psidium genus have been shown to possess antimicrobial (Alvarenda et al., 2015; Nair and Chanda, 2007), antifungal (de Macêdo et al., 2018; Morais et al., 2017; Morais et al., 2016), antioxidant (Sobral-Souza et al., 2014), anti-inflammatory (Siani et al., 2013) and antinociceptive activity, such as Psidium cattleyanum Sabine (Alvarenda et al., 2015; Alvarenga et al., 2013), Psidium guajava Linnaeus (Denny et al., 2013; Sekhar et al., 2014) and Psidium pohlianum Berg (Santos et al., 1996). In terms of chemical compounds, the following terpenes have been identified in different species from the Psidium genus: δ-cadinene, αpinene, (E)-β-caryophyllene, α-terpineol, β-pinene, 1,8-cineole, myrcene, limonene, terpinen-4-ol, β-pinene, ρ-cymene, α-humulene, linalool and β-eudesmol. These compounds are responsible for the biological and pharmacological activity attributed to species from this genus (Pereira, 2010), where some of these compounds are also present in the P. brownianum essential oil. Therefore, the aim of this study was to evaluate the essential oil chemical composition and antinociceptive activity of P. brownianum leaves.

2.5. Acute toxicity and median lethal dose (LD50) study The animals were divided into groups (n = 4) and were orally treated with the following PBEO doses: 19, 61, 195, 625 and 2,000 mg/ kg. Following treatment, the animals were observed for fourteen days, with their weight and ingested ration noted daily, as well as any possible alteration signs such as tremors, seizures, salivation, piloerection, bleeding, among other toxicity indicators. The LD50 was calculated according to the method established by the Organization for Economic Co-operation and Development (OECD, 2008), defined by the number of death occurrences and calculated with the aid of the LC50 Modem System program.

2. Methods 2.1. Botanic material

2.6. Acetic acid-induced contortion test

Fresh Psidium brownianum Mart ex DC. leaves were collected from the Barreiro Grande site, in Crato-CE, Brazil, during July 2011. Prof. Dr. Maria Arlene Pessoa da Silva performed botanical identification and a plant specimen was deposited in the Herbarium Caririense Dárdano de Andrade Lima – HCDAL of the Regional University of Cariri - URCA, cataloged under registration number #10,671.

The animals were divided into groups (n = 8) and were pretreated with vehicle (2% Tween 80 dissolved in distilled water, p.o.), PBEO (100 and 200 mg/kg; p.o.) or diclofenac (10 mg/kg; p.o.). In order to observe the potentiating effect of the oil, one group was treated with the PBEO (100 mg/kg, p.o.) in association with diclofenac (10 mg/kg, p.o.). After 60 min, the animals were injected with 0.6% acetic acid (10 mL/kg), intraperitoneally. The intensity of nociception was quantified by the number of contortions during 30 min of observation after acetic acid administration (Koster et al., 1959).

2.2. Drugs The following substances were used for experimental assays: distilled water (Farmace, Brazil), capsaicin (Sigma, USA), capsazepine (Sigma, USA), diclofenac (Aché, Brazil), formalin (Sigma-Aldrich, USA), morphine (Sigma-Aldrich, USA), naloxone (Cristalia, Brazil) and Tween 80 (Sigma-Aldrich, USA).

2.7. Formalin test Animals were divided into groups (n = 8) and treated with the PBEO (100 and 200 mg/kg, p.o.) or vehicle (2% Tween 80 dissolved in distilled water, p.o.) 60 min prior to 1% formalin (20 μL/paw) injection. Morphine (7.5 mg/kg, i.p.) was administered 30 min prior to the formalin injection. To verify the PBEO antinociceptive action on the opioid system, naloxone (1 mg/kg, subcuteneous – s.c.) was injected 15 min prior to the PBEO (100 mg/kg, p.o.) and morphine (7.5 mg/kg, i.p.) groups treatment. 1% formalin (20 μL/paw) was injected into the mice's right hind paw. The paw licking time (PLT) was recorded, in seconds, from 0 to 5 min (acute phase) and from 15 to 30 min (latent phase) after formalin administration (Hunskaar and Hole, 1987).

2.3. Animals Adult male Swiss (Mus musculus) mice, weighing between 28 ± 5 g were used. Animals were acclimated in polypropylene cages at room temperature between 23 ± 2 °C, following a of 12 h light/dark cycle with ad libitum access to food (Labina, Purina) and water. The animals were monitored in accordance with biosafety norms and procedures for vivariums (Cardoso, 1998–2001) and bioethics (Bazzano, 2006). The project containing the protocols in this study was submitted and approved by the Animal Experimentation and Use Commission of the Regional University of Cariri (URCA), under number #0045/2015.2.

2.8. Capsaicin-induced nociception in mice 2.4. Extraction and GC-MS analysis of the essential oil Animals were divided into groups (n = 8) and treated with the PBEO (100 and 200 mg/kg, p.o.) or vehicle (2% Tween 80 dissolved in distilled water, p.o.) 60 min prior to injection of the nociceptive agent, while capsazepine (2 mg/kg, i.p.) was administered 30 min prior to the nociceptive agent injection. Capsaicin (1.6 μg/paw intraplantar, i.pl.) was injected in the mice's right hind paw. Then, licking or biting time in the injected paw, a measure indicative of pain (Santos and Calixto, 1997), was recorded for 5 min.

The collected plant material (523 g) was subjected to hydrodistillation with 3 L of distilled water for 4 h using a Clevenger-type apparatus. The oil obtained was separated by extraction, dried over anhydrous sodium sulphate and stored in a refrigerator until analysis. The yield of the obtained oil was 0.79%. Chemical analysis of the essential oil was performed using a gas chromatograph coupled to a mass spectrometer (Shimadzu GC/MS, 2

Food and Chemical Toxicology 135 (2020) 111053

R. de Souza Sampaio, et al.

2.9. Hot plate test

Table 1 Chemical composition of the Psidium brownianum Mart ex DC. leaf essential oil.

Animals were divided into groups (n = 8) and individually placed on a hot plate with constant temperature (55 ± 0.5 °C) for up to 30 s (cut off time) to avoid paw lesions (LAPA et al., 2008). The latency of reaction to the thermal stimulus (time taken to lick, shake the hind paw or jump) was registered. The animals were selected 24 h before the experiment in order to exclude tolerant individuals with a reaction time > 15 s. The animals were orally treated with vehicle (2% Tween 80 dissolved in distilled water, p.o.), PBEO (100 and 200 mg/kg p.o.) or morphine (7.5 mg/kg i.p.). To observe the PBEO antinociception activity on the opioid system, separate groups were pretreated with naloxone (1 mg/kg, s.c.) 15 min prior to PBEO (100 mg/kg, p.o.) and morphine (7.5 mg/kg, i.p.) administration. Reaction latency measurements were performed at 30, 60 and 90 min following drug administration.

Compounds

Retention Time (min)

Retention Indexa

Kovats Retention Indexb

Percentage (%)

α-pinene β-pinene p-cineol α-terpineol α-elemol Guaiol α-cadinol β-eudesmol

3.17 3.59 4.15 6.19 11.03 12.12 12.20 12.39

944 994 1183 1209 1534 1576 1640 1654

939 1002 1185 1198 1529 1589 1652 1649

11.35 8.35 24.68 3.30 11.78 9.13 4.13 27.09

TOTAL

99.81

RIa: Retention indices calculated from retention times in relation to those of a C8–C20 series of n-alkanes in a DB-5HT column; RIb: Retention indices from the literature (Adams R.P. 2007, Alencar et al., 1984, and Alencar et al., 1990).

2.10. Rotarod test Motor incoordination and myorelaxant effects were evaluated in the rotarod test (Carlini and Burgos, 1979). In order to select the best animals, mice were submitted to a training session 24 h before the experiment. This step consisted of placing each animal on a rotating bar (16 rpm), 2.5 cm in diameter, raised 25 cm above ground. After this procedure, mice that remained 180 s in the rotating bar with a limit of up to three falls, were selected. On the following day, the animals were divided into four groups (n = 8) and treated with vehicle (2% Tween 80 dissolved in distilled water, p.o.), diazepam (2 mg/kg, i.p.) or PBEO (100 and 200 mg/kg, p.o.). After 30 min (for intraperitoneally doses) or 60 min (for oral doses), each animal was placed in the device and parameters such as time spent in the rotating bar and the number of falls were evaluated (Dunham and Miya, 1957). 2.11. Statistical analysis The results were represented as mean ± standard deviation (SD) and analyzed using an analysis of variance (ANOVA) followed by Dunnett's (one-way) or Bonferroni's (two-way) multiple comparisons post-hoc test. The results were considered significant when p < 0.05. Analyzes were performed using the GraphPad Prism 7.0 software.

Fig. 1. Effect of the Psidium brownianum Mart ex DC. leaf essential oil on acetic acid-induced contortions. All results were expressed as the mean ± standard deviation (SD), analyzed by ANOVA followed by Dunnett's multiple comparisons test. **** = Significant values when compared vs Vehicle when p < 0.05. Diclofen = Diclofenac 10 mg/kg; PBEO100 = Psidium brownianum Mart ex DC. essential oil 100 mg/kg; PBEO200 = Psidium brownianum Mart ex DC. essential oil 200 mg/kg, Diclofen + PBEO100 = Diclofenac 10 mg/ kg + Psidium brownianum Mart ex DC. essential oil 100 mg/kg.

3. Results 3.1. Chemical composition Chemical analysis (GC/MS) of the PBEO allowed the identification of 8 chemical constituents representing 99.81% of the total GC peak areas detected, with β-eudesmol (27.09%) and p-cineol (24.68%) as the major components, Table 1.

3.4. Effects on formalin-induced nociception The PBEO at 100 and 200 mg/kg doses was able to significantly reduce paw licking time in both phases of the test (49.02 and 44.94%, 68.04 and 46.35%, respectively, p < 0.001) when compared to the vehicle group. As expected, Morphine (7.5 mg/kg, i.p) significantly reduced paw licking time in both phases (80.47% and 87.59%, respectively). Naloxone (1 mg/kg, s.c.) significantly reversed the inhibition of the antinociceptive effect induced by morphine and the PBEO in both phases. This result may indicate the participation of the opioid system in the antinociceptive action promote by the PBEO. Fig. 2A and B demonstrate these results.

3.2. Acute toxicity and LD50 PBEO-treated animals showed no signs of acute toxicity at any of the oral doses administered, according to the OECD protocol, thus presenting an LD50 above ≥2,000 mg/kg (OECD, 2008). The dosage used in the pharmacological activity tests were based on the LD50 value. 3.3. Nociceptive contortions induced by acetic acid Diclofenac (10 mg/kg) and the PBEO at 100 and 200 mg/kg doses significantly reduced the number of abdominal contortions by 63.67, 40.62 and 41.79%, respectively, when compared to the vehicle group. Moreover, when combined with diclofenac, the PBEO promoted a significant reduction in abdominal contortions (86.32% vs 40.62%) indicating a possible synergistic effect (Fig. 1).

3.5. Hot plate test The PBEO at 100 and 200 mg/kg doses significantly prolonged the mice's reaction time on the hot plate test (30 min: 68.54 and 76.16%, 3

Food and Chemical Toxicology 135 (2020) 111053

R. de Souza Sampaio, et al.

Fig. 3. Effect of the Psidium brownianum Mart ex DC. leaf essential oil in the hot plate test. All results were expressed as the mean ± standard deviation (SD) analyzed by an ANOVA followed by Dunnett's multiple comparisons test. **** = Significant values when compared vs Vehicle when p < 0.05. Morphine = Morphine 7.5 mg/kg; PBEO100 = Psidium brownianum Mart ex DC. essential oil 100 mg/kg; PBEO200 = Psidium brownianum Mart ex DC. essential oil 200 mg/kg, Naloxone = Naloxone 1 mg/kg, Naloxone + Morphine = Naloxone 1 mg/kg + Morphine 7.5 mg/kg, Naloxone + PBEO100 = Naloxone 1 mg/kg + Psidium brownianum Mart ex DC. essential oil 100 mg/kg.

3.6. Effects on capsaicin-induced nociception The orally administered PBEO at 100 and 200 mg/kg doses significantly reduced the animals paw licking time (PLT) (45.36 and 42.26%, respectively, p < 0.001) after capsaicin (20 μL/paw) administration, in comparison to the vehicle group. Capsazepine (5 mg/ kg, s.c.) also inhibited PLT by 96.90% when compared to the vehicle group (Fig. 4). However, when associated with Capsazepine, no modification of the PBEO effect was observed, which suggests no participation of the TPRV system in the antinociceptive effect.

Fig. 2. Effect of the Psidium brownianum Mart ex DC. leaf essential oil in the first phase of formalin-induced nociception. All results were expressed as the mean ± standard deviation (SD), analyzed by ANOVA followed by Dunnett's multiple comparisons test. **** = Significant values when compared vs Vehicle when p < 0.05. Morphine = Morphine 7.5 mg/kg; PBEO100 = Psidium brownianum Mart ex DC. essential oil 100 mg/kg; PBEO200 = Psidium brownianum Mart ex DC. essential oil 200 mg/kg, Naloxone = Naloxone 1 mg/kg, Naloxone + Morphine = Naloxone 1 mg/kg + Morphine 7.5 mg/kg, Naloxone + PBEO100 = Naloxone 1 mg/kg + Psidium brownianum Mart ex DC. essential oil 100 mg/kg.

60 min: 105.47 and 106.52%, 90 min: 105.57 and 96.58%, respectively, p < 0.001) when compared to the vehicle group. As expected, morphine (7.5 mg/kg, i.p.) also significantly prolonged the thermal stimulus response latency from 30 min up to 90 min (Fig. 3). As observed in the previous test, when associated with naloxone, the effect of the PBEO was significantly reduced after 30 min with a duration of up to 90 min, thus corroborating with a participation of the opioid system in its antinociceptive effect.

Fig. 4. Effect of Psidium brownianum Mart ex DC. leaf essential oil on capsaicininduced nociception in mice. All results were expressed as the mean ± standard deviation (SD), analyzed by ANOVA followed by Dunnett's multiple comparisons test. **** = Significant values when compared vs Vehicle when p < 0.05. Morphine = Morphine 7.5 mg/kg, Capsazepine = Capsazepine 5 mg/kg; PBEO100 = Psidium brownianum Mart ex DC. essential oil 100 mg/kg; PBEO200 = Psidium brownianum Mart ex DC. essential oil 200 mg/kg. Capsazepine + PBEO100 = Capsazepine 5 mg/kg + Psidium brownianum Mart ex DC. essential oil 100 mg/kg. 4

Food and Chemical Toxicology 135 (2020) 111053

R. de Souza Sampaio, et al.

et al., 2011), which possibly modulates GABAA receptor inhibitory transmission (Watt et al., 2008) or which may act as a nerve excitability depressant (Moreira et al., 2001). As for β-pinene, in addition to increasing latency time in the same experimental model, by exerting supraspinal antinociceptive actions, β-pinene had its effect antagonized by naloxone, suggesting an opioid mechanism of action (Liapi et al., 2007). According to Santos and Rao (2000), cineole, one of the major PBEO compounds, displays an important inhibitory effect on the formation of prostaglandins and cytokines, which may contribute to the PBEO antinociceptive action. Similar to what was observed in the capsaicin test, formalin sensitizes nociceptive fibers by stimulating the release of excitatory amino acids and pro-inflammatory substances. The formalin-induced nociception test exhibits two distinct phases, the first arises from direct substance stimulation of nociceptors and the second arises from the action of inflammatory products (Hunskaar and Hole, 1987) such as macrophages and cytokines (Deftu et al., 2016). Moreover, formalin interaction with NMDA receptors is associated with the onset of nociceptive effect (Davidson et al., 1997) and TRPA1 (McNamara et al., 2007) receptor activation. The results from this study suggest the PBEO possibly reduces inflammatory products and potentiates opioid signaling, minimizing licking time in the two test phases. In other studies, P. pohlianum (Santos et al., 1996), P. guajava (Santos et al., 1998; Victor et al., 2005), α-pinene, one of the monoterpenes present in the PBEO (Santos et al., 1998), and α-terpineol (Quintans-Júnior et al., 2011), showed equivalent effects. The aforementioned effects were also found to be partially reversed with naloxone administration, suggesting a possible interaction between the PBEO and opioid receptors, an important pathway in downward pain modulation (Ghelardini et al., 2015; Heinricher, 2013; Lumb, 2014; Ren and Dubner, 2002). However, other species from this genus, such as P. guajava (Somchit et al., 2004), P. pohlianum (Santos et al., 1996) and P. guianense (Santos et al., 1997) do not present this mechanism of action. On the other hand, β-pinene had its effect reversed when administered alongside naloxone and, when combined with morphine, it antagonized the drug effect, suggesting that this monoterpene is probably a partial μ opioid receptor agonist (Bourgou et al., 2010). The opioid-like antinociceptive effect of the PBEO does not seem to be associated with motor deficits, an effect commonly exhibited by opioid drugs (Cunha-Oliveira et al., 2008), since it did not present a myorelaxant effect in the rotarod test (Hamm et al., 1994). This was similarly observed with α-pinene (Him et al., 2008) and α-terpineol (Oliveira et al., 2016), unlike P. guajava which presented a significant myorelaxant action (Verma et al., 2010). The major PBEO compound, β-eudesmol, is not described in the literature with an analgesic action. However, evidences indicate its association with an analgesic effect. It is known that β-eudesmol presents an important noncompetitive nicotinic acetylcholine receptor antagonism (Kimura et al., 1987, 1991, 1995), which is useful in the treatment of inflammatory allodynia pain (Bennett and Xie, 1988), hyperalgesia (Livett et al., 2005, 2008; McIntosh et al., 2005; Nevin et al., 2007; Satkunanathan et al., 2005; Vincler et al., 2006; Vincler and McIntosh, 2007) and neuropathic pain (Seltzer et al., 1990), also presenting lower toxicity when compared to nicotinic agonists (Wala et al., 2012). Many nicotinic receptor antagonists also act by inhibiting glutamate NMDA receptors (Amador and Dani, 1991), which may justify the PBEO action in the tests above.

Table 2 Effect of the Psidium brownianum Mart ex DC. leaf essential oil on the rotarod test. Groups

Number of falls

Permanence Time (seconds)

Vehicle Diazepam 5 mg/kg PBEO 100 mg/kg PBEO 200 mg/kg

0.2 ± 0.44 1.8 ± 0.44 0 ± 0 0 ± 0

59.25 49.63 60 ± 60 ±

± 1.4 ± 6.14*** 0 0

All results were expressed as the mean ± standard deviation (SD), analyzed by an ANOVA followed by Dunnett's multiple comparisons test. **** = Significant values when compared vs Vehicle when p < 0.05. PBEO = Psidium brownianum Mart ex DC. leaf essential oil.

3.7. Rotarod test The PBEO (100 and 200 mg/kg) failed to produce any significant effects on motor coordination (time spent on rotating bar and number of falls) in the tested animals. Diazepam (2 mg/kg, i.p.) reduced the rotating bar permanence time by 17.1% (49.7 ± 2.4) and increased the number of falls by 466.6% (Table 2). 4. Discussion In this study, the results indicate the PBEO produces peripheral and central antinociceptive action when assessed using different chemical and thermal models of nociception. The PBEO also presents low acute oral toxicity (OECD, 2008). The acetic acid-induced contortion test has been used largely as a screening tool in order to assess the analgesic properties of a diverse range of substances (Blumberg et al., 1965; Hokanson, 1978; Koster et al., 1959; Siegmund et al., 1957). This test is used to cause peripheral sensitization through the indirect release of nociceptive endogenous mediators in the animal (Duarte et al., 1988). The PBEO significantly reduced the number of abdominal contortions at all tested doses, with a maximum activity when PBEO100 was associated with diclofenac (10 mg/kg), showing a significant synergistic effect. Since acetic acid is responsible for promoting the release of bradykinin, prostaglandins, serotonin, histamine, cytokines and noradrenaline, among other nociceptive mediators, the aforementioned effect may be due to the inhibition of the release of these nociceptive mediators (Duarte et al., 1988; Ribeiro et al., 2000). In the literature, studies corroborating with the present study, such as Psidium guajava (Jayakumari et al., 2012; Ojewole, 2006; Olajide et al., 1999; Santos et al., 1998; Sarkar et al., 2011; Sekhar et al., 2014; Somchit et al., 2004), P. cattleianum (Alvarenda et al., 2015; Alvarenga et al., 2013) and P. guianense Pers. (Santos et al., 1997), have already been described reporting similar antinociceptive effects. α-terpineol, a monoterpene identified in the PBEO chemical profile, was shown to be an effective antinociceptive compound at 25, 50 and 100 mg/kg doses (Quintans-Júnior et al., 2011). Kimura and Sumiyoshi (2012) described the antagonistic action of β-eudesmol on serotonin 5HT3 receptors, which are involved in animal nociceptive effects in the formalin test (Garcia et al., 2013), thus suggesting that this compound's action possibly contributes to the PBEO antinociceptive action. The hot plate test, employed to evaluate central antinociceptive drugs, is sensitive to opioid and non-opioid compounds such as nicotinic receptor agonists, N-methyl-d-aspartate (NMDA) receptor antagonists and tricyclic antidepressants (Bannon and Malmberg, 2007). In this test, the PBEO prolonged the latency time at all doses suggesting an important participation of supraspinal mechanisms (Dickenson and Besson, 1997), also seen in P. guajava (Ojewole, 2006) and Psidium pohlianum O.Berg (Santos et al., 1996). Other studies using monoterpenes present in the PBEO reinforce the findings in this study. Such is the case for α-terpineol (Quintas-Júnior

5. Conclusions The results obtained in this study provide pharmacological evidence for the use of the P. brownianum leaf essential oil in pain treatment and shows their potential for new analgesic drug development. It is noteworthy that new studies aiming to evaluate the relationship between 5

Food and Chemical Toxicology 135 (2020) 111053

R. de Souza Sampaio, et al.

the P. brownianum chemical constituents and their antinociceptive activity need to be performed.

Bourgou, S., Pichette, A., Marzouk, B., Legault, J., 2010. Bioactivities of black cumin essential oil and its main terpenes from Tunisia. South Afr. J. Bot. 76 (2), 210–216. Brunelli, C., Bennett, M.I., Kaasa, S., Fainsinger, R., Sjøgren, P., Mercadante, S., 2014. European Association for Palliative Care (EAPC) Research Network. Classification of neuropathic pain in cancer patients: a Delphi expert survey report and EAPC/IASP proposal of an algorithm for diagnostic criteria. Pain 155 (12), 2707–2713. Cardoso, T.A.O., 1998-2001. Considerações sobre a biossegurança em arquitetura de biotérios. Boletim Central. Panamaense Fiebre Aftosa 64–67, 3–17. Carlini, E.A., Burgos, V., 1979. Screening farmacológico de ansiolíticos: metodologia laboratorial e comparação entre o diazepam e o clorobenzapam. Rev Assoc Bra Psiq 1, 25–31. Castel-Branco, M.M., Santos, A.T., Carvalho, R.M., Caramona, M.M., Santiago, L.M., Fernandez-Llimos, F., Figueiredo, I.V., 2013. As bases farmacológicas dos cuidados farmacêuticos: o caso dos AINEs. Acta Farm Port 2 (2), 19–27. Coluzzi, F., Taylor, R., Pergolizzi, J.V., Mattia, C., Raffa, R.B., 2016. Orientação para boa prática clínica para opioides no tratamento da dor: os três “Ts”–titulação (teste), ajustes (individualização), transição (redução gradual). Br. J. Anaesth. 66 (3), 310–317. Coutinho, H.D.M., Souza, C.E.S., Leite, Nadghia F., Costa, JOSÉ Galberto M., Cunha, Francisco A.B., Rolim, L.A., Silva, A.R.P., Menezes, I.R.A., 2018. LC-MS analysis and cytoprotective effect against the mercurium and aluminium toxicity by bioactive products of Psidium brownianum Mart. ex DS. J. Hazard Mater. 370, 54–62. Cunha-Oliveira, T., Rego, A.C., Oliveira, C.R., 2008. Cellular and molecular mechanisms involved in the neurotoxicity of opioid and psychostimulant drugs. Brain Res. Rev. 58 (1), 192–208. Davidson, E.M., Coggeshall, R.E., Carlton, S.M., 1997. Peripheral NMDA and non-NMDA glutamate receptors contribute to nociceptive behaviors in the rat formalin test. Neuroreport 8 (4), 941–946. De Macêdo, D.G., Souza, Marta M.A., Morais-braga, M.F.B., Coutinho, H.D.M., dos Santos, A.T.L., da Cruz, R.P., da Costa, José, G.M., Rodrigues, Fábio F.G., Quintans-Junior, L.J., da Silva Almeida, J.R.G., de Menezes, I.R.A., 2018. Effect of seasonality on chemical profile and antifungal activity of essential oil isolated from leaves Psidium salutare (Kunth). O. Berg. PeerJ 6, e5476. Deftu, A.F., Fiorenzani, P., Ceccarelli, I., Pinassi, J., Gambaretto, M., Ristoiu, V., Aloisi, A.M., 2016. Macrophage migration inhibitory factor modulates formalin induced behaviors in rats. Anim. Biol. Leiden 66, 249–258. Denny, C., Melo, P.S., Franchin, M., Massarioli, A.P., Bergamaschi, K.B., de Alencar, S.M., Rosalen, P.L., 2013. Guava pomace: a new source of anti-inflammatory and analgesic bioactives. BMC Complement Altern. Med. 13 (1), 1. Dickenson, A., Besson, J.M., 1997. The Pharmacology of Pain. Springer, Berlin. Duarte, J.D.G., Nakamura, M., Ferreira, S.H., 1988. Participation of the sympathetic system in acetic acid induced writhing in mice. J Med Biol Res 21, 341–343. Dunham, N.W., Miya, T.S., 1957. A Note on a single apparatus for detecting neurological déficits in rats and mice. J. Am. Pharm. Assoc. 46 (3), 208–209. Freitas, A.V.L., Coelho, M.F.B., Pereira, Y.B., Freitas Neto, E.C., Azevedo, R.A.B., 2015. Diversidade e usos de plantas medicinais nos quintais da comunidade de São João da Várzea em Mossoró. RN. Rev Bras Plantas Med. 17 (4), 845–856. Garcia, T.A., Nunes, X.A., Teixeira, D.G., Ferreira, C.M.R., 2013. Influência dos Receptores 5-HT3 no Processamento Nociceptivo de Ratos Submetidos ao Teste da Formalina. Rev Neurocienc 20 (4), 527–533. Gaskin, D.J., Richard, P., 2012. The economic costs of pain in the United States. J. Pain 13 (8), 715–724. Ghelardini, C., Mannelli, L.D.C., Bianchi, E., 2015. The pharmacological basis of opioids. Clin Cases Miner Bone Metab 12 (3), 219. Guilhermino, J.F., Siani, A.C., Quental, C., Bomtempo, J.V., 2012. Desafios e Complexidade para Inovação a partir da Biodiversidade Brasileira. Rev Pesq Inov Farm 4 (1), 18–30. Hamm, R.J., Pike, B.R., O'Dell, D.M., Lyeth, B.G., Jenkins, L.W., 1994. The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury. 11 (2), 187–196. Heinricher, M.M., 2013. Opiates, rostral ventromedial medulla, and descending control. In: Encyclopedia of Pain. Springer Berlin Heidelberg, pp. 2399–2405. Him, A., Ozbek, H., Turel, I., Oner, A.C., 2008. Antinociceptive activity OF alpha-pinene and FENCHONE. Pharmacologyonline 3, 363–369. Hokanson, G.C., 1978. Acetic acid for analgesic screening. J. Nat. Prod. 41, 497–498. Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between inflammatory and non inflammatory pain. Pain 30, 103–114. Jayakumari, S., Anbu, J., Ravichandiran, V., Nithya, S., Anjana, A., Sudharani, D., 2012. Evaluation of toothache activity of methanolic extract and its various fractions from the leaves of Psidium guajava Linn. Int. J. Pharm. Biol. Sci. 3, 238–249. Johannes, C.B., Le, T.K., Zhou, X., Johnston, J.A., Dworkin, R.H., 2010. The prevalence of chronic pain in United States adults: results of an Internet-based survey. J. Pain 11 (11), 1230–1239. Kimura, M., Diwan, P.V., Yanagi, S., Kon-no, Y., Nojima, H., Kimura, I., 1995. Potentiating effects of. BETA.-Eudesmol-Related cyclohexylidene derivatives on succinylcholine-induced neuromuscular block in isolated phrenic nerve-diaphragm muscles of normal and alloxan-DiabeticMice. Biol. Pharm. Bull. 18 (3), 407–410. Kimura, M., Kimura, I., Muroi, M., Yoshizaki, M., Hikino, H., 1987. Pharmacological evidence for an interaction between constituents (blend effect) of the Japanese‐Sino medicine “Keishi‐ka‐zyutubu‐tō” in neuromuscular blockade in diabetic mice. Phytother Res. 1 (3), 107–113. Kimura, M., Nojima, H., Muroi, M., Kimura, I., 1991. Mechanism of the blocking action of β-eudesmol on the nicotinic acetylcholine receptor channel in mouse skeletal muscles. Neuropharmacology 30 (8), 835–841. Kimura, Y., Sumiyoshi, M.M., 2012. Effects of an Atractylodeslancea rhizome extract and a volatile component β-eudesmol on gastrointestinal motility in mice. J.

Competing interests and funding statements The authors declare there are no conflicts of interest. This study was funded in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) Finance Code BPI, the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - Finance Code 304291/2017–0 and the Financiadora de Estudos e Projetos - Brasil (FINEP). Authors’ contributions GML, RSS, EPN, VSS, AOBP, LGS, GAD, JGMC, CFBF, IRAM and MRK contributed to the conception or design of the study. RSS, EPN, VSS and AOBP contributed to the data collection. RSS, EPN, VSS, AOBP, JGMC, CFBF, IRAM and MRK contributed to the data analysis and interpretation. DSB has contributed with the article revision. GML, GAD, LGS, and MRK drafted the article. All authors critically revised and approved the article. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors thank the Regional University of Cariri and the Natural Products Pharmacology Laboratory. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fct.2019.111053. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. Allured publishing corporation. Albuquerque, E.X., Costa, A.C.S., Alkondon, M., Shaw, K.P., Ramoa, A.S., Aracava, Y., 1991. Functional properties of the nicotinic and glutamatergic receptors. J. Recept. Res. 11 (1–4), 603–625. Alencar, J.W., Craveiro, A.A., Matos, F.J.A., 1984. Kovats indices as a preselection routine in mass spectra library search of volatiles. J. Nat. Prod. 47, 890–892. Alencar, J.W., Craveiro, A.A., Matos, F.J.A., Machado, M.I.L., 1990. Kovats indices simulation in essential oils analysis. Quím. Nova 13, 282–284. Alvarenda, F., Royo, V., Mota, B., Laurentiz, R., Menezes, E., Melo Junior, A.F., Oliveira, D., 2015. Atividade Antinociceptiva e Antimicrobiana da Casca do Caule de Psidium Cattleyanum Sabine. Rev. Bras. Plantas Med. 17 (4), 1125–1133. Alvarenga, F.Q., Mota, B.C., Leite, M.N., Fonseca, J.M., Oliveira, D.A., de Andrade Royo, V., Laurentiz, R.S., 2013. In vivo analgesic activity, toxicity and phytochemical screening of the hydroalcoholic extract from the leaves of Psidium cattleianum Sabine. J. Ethnopharmacol. 150, 280–284. Amador, M., Dani, J.A., 1991. MK‐801 inhibition of nicotinic acetylcholine receptor channels. Synapse 7 (3), 207–215. Andrew, R., Derry, S., Taylor, R.S., Straube, S., Phillips, C.J., 2014. The costs and consequences of adequately managed chronic non‐cancer pain and chronic neuropathic pain. Pain Pract. 14 (1), 79–94. Bannon, A.W., Malmberg, A.B., 2007. Models of nociception: hot-plate, tail-flick, and formalin tests in rodents. Curr Protoc Neurosci 8 (8–9), 1–8. Batlouni, M., 2010. Anti-inflamatórios não esteroides: efeitos cardiovasculares, cérebrovasculares e renais. Arq. Bras. Cardiol. 94 (4), 556–563. Bazzano, F.C.O., 2006. Aspectos Éticos da Pesquisa Científica, p. 149-180. In: SILVA, José Vitor da (Org.) et al. Bioética: meio ambiente, saúde e pesquisa. 1, (São Paulo: Iátria). Bennett, G.J., Xie, Y.K., 1988. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33 (1), 87–107. Blumberg, H., Wolf, P.S., Dayton, H.B., 1965. Use of the writhing test for evaluating analgesic activity of narcotic antagonists. Proc. Soc. Exp. Biol. Med. 118, 763–766.

6

Food and Chemical Toxicology 135 (2020) 111053

R. de Souza Sampaio, et al.

Rev. BNDES (Banco Nac. Desenvolv. Econ. Soc.) 43, 41–89. Quintas-Júnior, L.J., Oliveira, M.G., Santana, M.F., Santana, M.T., Guimarães, A.G., Siqueira, J.S., Almeida, R.N., 2011. α-Terpineol reducesnociceptivebehavior in mice. Pharm. Biol. 49 (6), 583–586. Ren, K., Dubner, R., 2002. Descending modulation in persistent pain: an update. Pain 100 (1–2), 1–6. Ribeiro, R.A., Vale, M.L., Thomazzi, S.M., Paschoalato, A.B., Poole, S., Ferreira, S.H., Cunha, F.Q., 2000. Involvement of resident macrophages and mast cells in the writhing nociceptive response induced by zymosan and acetic acid in mice. Eur. J. Pharmacol. 387 (1), 111–118. Santos, A.R., Calixto, J.B., 1997. Further evidence for the involvement of tachykinin receptor subtypes in formalin and capsaicin models of pain in mice. Neuropeptides 37, 381–389. Santos, F.A., Rao, V.S.N., 2000. Antiinflammatory and antinociceptive effects of 1, 8‐cineole a terpenoid oxide present in many plant essential oils. Phytother Res.: Int. J. Devoted to Pharmacol. Toxicol. Eval. Nat. Prod. Deriv. 14 (4), 240–244. Santos, F.A., Rao, V.S.N., Silveira, E.R., 1996. Naloxone-resistant antinociceptive activity in the essential oil of Psidium pohlianum Berg. Phytomedicine 3 (2), 197–201. https:// doi.org/10.1016/S0944-7113(96)197-201. Santos, F.A., Rao, V.S.N., Silveira, E.R., 1997. Anti-inflammatory and analgesic activities of the essential oil of Psidium guianense. Fitoterapia 68 (1), 65–68. Santos, F.A., Rao, V.S.N., Silveira, E.R., 1998. Investigations on the antinociceptive effect of Psidium guajava leaf essential oil and its major constituents. Phytother Res. 12 (1), 24–27. Sarkar, J., Pal, S., Bhattacharya, S., Biswas, M., 2011. Thin layer chromatographic profiling and evaluation of analgesic activity of Psidium guajava leaf extracts in mice. J. Adv. Pharm. Educ. Res. 2 (2249–3379), 177–183. Satkunanathan, N., Livett, B., Gayler, K., Sandall, D., Down, J., Khalil, Z., 2005. Alphaconotoxin Vc1. 1 alleviates neuropathic pain and accelerates functional recovery of injured neurones. Brain Res. 1059 (2), 149–158. Sekhar, N.C., Jayasree, T., Ubedulla, S., Dixit, R., 2014. Evaluation of antinociceptive activity of aqueous extract of bark of Psidium guajava in albino rats and albino mice. J. Clin. Diagn. Res. 8 (9), 1–4. Seltzer, Z.E., Dubner, R., Shir, Y., 1990. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 43 (2), 205–218. Siani, A.C., Souza, M.C., Henriques, M.G., Ramos, M.F., 2013. Anti-inflammatory activity of essential oils from Syzygium cumini and Psidium guajava. Pharm. Biol. 51 (7), 881–887. Siegmund, E., Cadmus, R., Lu, G., 1957. A method for evaluating both non-narcotic and narcotic analgesics. Proc. Soc. Exp. Biol. Med. 95 (4), 729–931. Sobral-Souza, C.E., Leite, N.F., Cunha, F.A., Pinho, A.I., Costa, J.G., Coutinho, H.D., 2014. Evaluation of the Cytoprotective and Antioxidant Activity of the Extracts of Eugenia Uniflora Lineau e Psidium Sobraleanum Proença Landrum Against Heavy Metals. Rev Cienc de la Salud 12 (3), 401–409. Somchit, M.N., Sulaiman, M.R., Ahmad, Z., Israf, D.A., Hosni, H., 2004. Non-Opioid antinociceptive effect of Psidium guajava leaves extract. J. Nat. Remedies 4 (2), 174–178. Van Hecke, O., Torrance, N., Smith, B.H., 2013. Chronic pain epidemiology and its clinical relevance. Br. J. Anaesth. 111 (1), 13–18. Verma, S., Jain, S.K., Gautam, O.P., 2010. Anticonvulsivant and myorelaxation activity of Psdium guajava Linn. leaf extract. Pharmacology 2, 575–578. Victor, B.O., Timothy, O.J., Ayodele, O.S., 2005. Analgesics and antipyretic activities of ethanolic extract of Psidium guajava in rats. Recent Progress Medicinal Plants 13, 473–480. Vincler, M., McIntosh, J.M., 2007. Targeting the α9α10 nicotinic acetylcholine receptor to treat severe pain. Expert Opin. Ther. Targets 11 (7), 891–897. Vincler, M., Wittenauer, S., Parker, R., Ellison, M., Olivera, B.M., McIntosh, J.M., 2006. Molecular mechanism for analgesia involving specific antagonism of α9α10 nicotinic acetylcholine receptors. Proc. Natl. Acad. Sci. 103 (47), 17880–17884. Von Korff, M., Scher, A.I., Helmick, C., Carter-Pokras, O., Dodick, D., Goulet, J., HamillRuth, R., LeResche, L., Porter, L., Tait, R., Terman, G., Veasley, C., Mackey, S., 2016. United States national pain strategy for population research: concepts, definitions and pilot data. J. Pain 17 (10), 1068–1080. Wala, E.P., Crooks, P.A., McIntosh, J.M., Holtman Jr., J.R., 2012. Receptor antagonist prevents and reverses. Chemotherapy-evoked neuropathic pain in rats. Anesth. Analg. 115 (3), 713. Watt, E.E., Betts, B.A., Kotey, F.O., Humbert, D.J., Griffith, T.N., Kelly, E.W., Veneskey, K.C., Gill, N., Rowan, K.C., Jenkins, A., Hall, A.C., 2008. O mentol compartilha a atividade anestésica geral e os locais de ação no receptor GABA (A) com o agente intravenoso propofol. Eur. J. Pharmacol. 590 (1–3), 120–126.

Ethnopharmacol. 141 (1), 530–536. Koster, R., Anderson, M., De Beer, E.J., 1959. Acetic acid-induced analgesic screening. Fed. Proc. LAPA, A.J., et al., 2008. Métodos de avaliação da atividade farmacológica de plantas medicinais. Sociedade Brasileira de Plantas Medicinais. Liapi, C., Anifantis, G., Chinou, I., Kourounakis, A.P., Theodosopoulos, S., Galanopoulou, P., 2007. Antinociceptive properties of 1, 8-cineole and β-pinene, from the essential oil of Eucalyptus camaldulensis leaves, in rodents. Planta Med. 73 (12), 1247–1254. Liedgens, H., Obradovic, M., De Courcy, J., Holbrook, T., Jakubanis, R., 2016. A burden of illness study for neuropathic pain in Europe. Clinicoecon Outcomes Res 8 (113). Linde, M., Gustavsson, A., Stovner, L.J., Steiner, T.J., Barré, J., Katsarava, Z., Ruiz De La Torre, E., 2012. The cost of headache disorders in Europe: the Eurolight project. Eur. J. Neurol. 19 (5), 703–711. Livett, B.G., Khalil, Z., Gayler, K.R., Down, J.G., Sandall, D.W., Keays, D.A., 2008. Alpha Conotoxin Peptides with Analgesic Properties, vol 212 Patent Application n. 12/054. Livett, B.G., Khalil, Z., Gayler, R.K., Down, J.G., Sandall, D.W., Keays, D.A., 2005. Alpha Conotoxin Peptides with Analgesic Properties. Patent Application n. 2005/0215480 A1. . Lumb, B.M., 2014. Descending controls: how to harness for the relief of pain? J. Physiol. 592 (19) 4097-4097. McIntosh, J.M., Plazas, P.V., Watkins, M., Gomez-Casati, M.E., Olivera, B.M., Elgoyhen, A.B., 2005. A novel α-conotoxin, PeIA, cloned from Conuspergrandis, discriminates between rat α9α10 and α7 nicotinic cholinergic receptors. J. Biol. Chem. 280 (34), 30107–30112. McNamara, C.R., Mandel-Brehm, J., Bautista, D.M., Siemens, J., Deranian, K.L., Zhao, M., Fanger, C.M., 2007. TRPA1 mediates formalin-induced pain. Proc. Natl. Acad. Sci. 104 (33), 13525–13530. Michellin, A.F., Ferreira, A.A.P., Bitar, V.G., Lopes, L.C., 2006. Toxicidade renal de inibidores seletivos da ciclooxigenase-2: celecoxib e rofecoxib. Rev. Cienc. Med. (Lourenco Marques) 15 (4), 321–332. Monteiro, E.C.A., Trindade, J.M.D.F., Duarte, A.L.B.P., Chahade, W.H., 2008. Os antiinflamatórios não esteroidais (AINEs). T Reumatol Clín. 9 (2), 53–63. Morais-Braga, Maria, F.B., Carneiro, J.N.P., Machado, A.J.T., Sales, D.L., dos Santos, A.T.L., Boligon, A.A., Athayde, M.L., Menezes, I.R.A., Souza, D.S.L., Costa, J.G.M., Coutinho, H.M.D., 2017. Phenolic composition and medicinal usage of Psidium guajava Linn.: antifungal activity or inhibition of virulence? Saudi J. Biol. Sci. 24, 302–313. Morais-Braga, M.F.B., Sales, Carneiro, J.N.P., Machado, A.J.T., Santos, A.T.L., Freitas, M.A., Martins, G.M.A.B., Leite, N.F., de Matos, Y.M.L.S., Tintino, S.R., Souza, D.S.L., de Menezes, I.R.A., Ribeiro-Filho, J., Coutinho, H.D.M., Costa, J.G.M., 2016. Psidium guajava L. and Psidium brownianum Mart ex DC.: chemical composition and anti Candida effect in association with fluconazole. Microb. Pathog. 95, 200–207. Moreira, M.R., Cruz, G.M.P., Lopes, M.S., Albuquerque, A.A.C., Leal-Cardoso, J.H., 2001. Effects of terpineol on the compound action potential of therat sciatic nerve. Braz. J. Med. Biol. Res. 34 (10), 1337–1340. Nair, R., Chanda, S., 2007. In-vitro antimicrobial activity of Psidium guajava L. leaf extracts against clinically important pathogenic microbial strains. Braz. J. Microbiol. 38 (3), 452–458. Nevin, S.T., Clark, R.J., Klimis, H., Christie, M.J., Craik, D.J., Adams, D.J., 2007. Are alpha9alpha10 nicotinic acetylcholine receptors a pain target for alpha-conotoxins? Mol. Pharmacol. 72 (6), 1406–1410. OECD - Organization for Economic Co-operation and Development, 2008. Guideline 425: Acute Oral Toxicity: Modified Up-And-Down Procedure. Ojewole, J.A.O., 2006. Antiinflammatory and analgesic effects of Psidium guajava Linn. (Myrtaceae) leaf aqueous extract in rats and mice. Methods Find. Exp. Clin. Pharmacol. 28 (7), 441–446. Olajide, O.A., Awe, S.O., Makinde, J.M., 1999. Pharmacological studies on the leaf of Psidium guajava. Fitoterapia 70 (1), 25–31. Oliveira, M.G., Brito, R.G., Santos, P.L., Araújo-Filho, H.G., Quintans, J.S., Menezes, P.P., Scotti, L., 2016. α-Terpineol, a monoterpene alcohol, complexed with β-cyclodextrin exerts antihyperalgesic effect in animal model for fibromyalgia aided with docking study. Chem. Biol. Interact. 254, 54–62. Pereira, C.K.B., 2010. Estudo químico e atividades microbiológicas de espécies do gênero Psidium (Myrtaceae). Dissertação (Mestrado em Bioprospecção Molecular). Universidade Regional do Cariri, Crato, Brazil 108pp. Pereira, D.T.D.S., Andrade, L.L.D., Agra, G., Costa, M.M.L., 2015. Condutas terapêuticas utilizadas no manejo da dor em oncologia. Rev Pesq Cuid Fundam (Online) 7 (1), 1883–1890. Pimentel, V.P., Vieira, V.A.M., Mitidieri, T.L., Oliveira, F.F.S., Pieroni, J.P., 2015. Biodiversidade brasileira como fonte da inovação farmacêutica: uma nova esperança?

7