Delta- and mu-opioid pathways are involved in the analgesic effect of Ocimum basilicum L in mice

Journal of Ethnopharmacology 250 (2020) 112471

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Delta- and mu-opioid pathways are involved in the analgesic effect of Ocimum basilicum L in mice

T

Ah Hyun Baea, Gyuna Kima, Geun Hee Seolb, Seon Bong Leec, Jeong Min Leec, Wonseok Changa, Sun Seek Mina,∗ a

Department of Physiology and Biophysics, School of Medicine, Eulji University, Daejeon, Republic of Korea Department of Basic Nursing Science, Korea University School of Nursing, Seoul, 136-713, South Korea c KT&G Research Institute, Daejeon, 34337, South Korea b

A R T I C LE I N FO

A B S T R A C T

Keywords: Ocimum basilicum L. Pain Formalin test Acetic-acid test Opioid pathway

Ethnopharmacological relevance: Ocimum basilicum L. is a perennial herb that has been used in traditional Asian Indian medicine for thousands of years as a natural anti-inflammatory, antibiotic, diuretic, and analgesic. Aim of the study: The present study was conducted to investigate the analgesic effects of basil essential oil (BEO) in inflammatory pain models and identify underlying mechanisms. We further investigated whether BEO affects physiological pain and motor coordination. Materials and methods: The analgesic effects of BEO were assessed in various mouse experimental pain models using formalin, acetic acid, heat, and carrageenan as stimuli. BEO was administered by intraperitoneal injection or inhalation. The involvement of various pathways in the analgesic effect of BEO was assessed by pretreating mice with selective pharmacological inhibitors, administered intraperitoneally. Opioid pathways were tested using the κ-opioid antagonist 5′-guanidinonaltrindole (GNTI; 0.3 mg/kg), δ-opioid antagonist naltrindole (NTD; 5 mg/kg) and μ-opioid antagonist naloxone (NAL; 8 mg/kg); nitric oxide (NO) pathways were tested using the NO synthase inhibitor N-nitro l-arginine methyl ester (L-NAME; 37.5 mg/kg) and NO precursor L-arginine (LArg; 600 mg/kg); and KATP channel pathways were tested using the ATP-sensitive K+ channel blocker, glibenclamide-hippuric acid (GHA, 2 mg/kg). Potential effects of BEO on motor coordination were assessed using a rotarod test. Results: BEO exerted analgesic effects in all pain models. Notably, pretreatment with naltrindole, naloxone, or Larginine significantly reduced the analgesic effects of BEO in the formalin test. BEO increased mean withdrawal latencies in a thermal plantar test at a high dose, but not at lower doses. BEO had no effect on motor coordination. Conclusions: Our findings indicate that the analgesic effects of BEO are primarily mediated by delta- and muopioid pathways and further suggest that BEO has potential for development as an analgesic agent for the relief of inflammatory pain.

1. Introduction Pain, defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage (1979), is a common health problem for many people. According to the International Association for the Study of Pain, 20% of adults worldwide suffer from pain and 10% are newly diagnosed with chronic pain each year (Goldberg and McGee, 2011). Pain does not only causes physical discomfort, but it is also a contributor to many other problems, including mental health issues and economic impacts related to medical expenses and reduced productivity. Therefore, controlling ∗

pain is an important task. Numerous drugs are clinically prescribed for controlling pain. Unfortunately, most of these drugs have a wide range of side effects ranging from mild to deadly, including drug addiction and respiratory depression. Therefore, continuing efforts to develop new therapeutic agents with higher safety profiles are important from a clinical standpoint. In this context, among the more notable potential sources of such agents are essential oils extracted from plants. Although essential oils, which consist of a number of different chemical compounds, have been used to effectively treat many diseases for thousands of years, they are not readily incorporated into the concept and development process of

Corresponding author. E-mail address: [email protected] (S.S. Min).

https://doi.org/10.1016/j.jep.2019.112471 Received 27 February 2019; Received in revised form 8 December 2019; Accepted 9 December 2019 Available online 16 December 2019 0378-8741/ © 2019 Published by Elsevier B.V.

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2.3. Injection and inhalation of essential oils

synthetic medicines, which are designed to target a single mechanism of action. As a result, these natural products have been excluded from the “medicine” category. However, the field of synthetic medicine has increasingly come to recognize that a multi-target/multi-component approach is effective for treating chronic diseases with complex pathogenic mechanisms, such as cancer, immune diseases, mental diseases, circulatory diseases and diseases associated with aging, among others (Oh, 2011). Thus, although essential oils have not yet been proven scientifically, their long history of clinical efficacy suggests that that they are a potential starting point for the development of new painkillers. Ocimum basilicum L. (basil) is an annual plant belonging to Lamiaceae family, which includes more than 150 species of various botanical varieties and forms of basil distributed around the world (Pushpangadan, 1995). In traditional Asian Indian medicine (Rakshit and Ramalingam, 2010), basil is used as a natural anti-inflammatory, antibiotic (Srivastava et al., 2014), diuretic and analgesic (Al Abbasy et al., 2015). Also, leaves and flowering parts of O. basilicum are used as antispasmodic, aromatic, carminative, digestive, galactogogue, stomachic, and tonic agents in traditional medicine (Adigüzel, 2005). Notably, essential oil, steam-distilled from basil leaves, stems and flowers, has beneficial properties like high levels of antioxidants (Chenni, 2016). The main components of basil essential oil (BEO) are linalool, methyl cinnamate, methyleugenol, squalene, 2-methyltriacontane, 9-methyldotriacontane, β-sitosterol (El-Ghffar, 2018) and eugenol (Akgül, 1989). Among these, linalool and eugenol are known analgesic (Peana, 2004) as well as anti-inflammatory properties (Daniel, 2009) (Peana, 2002). This suggests that BEO is biologically active and can be used effectively in the context of pain control. The present study was designed to investigate the analgesic effect of BEO through various mouse experimental pain models using formalin, acetic acid, heat, and carrageenan as noxious stimuli. In addition, we examined the involvement of opioid, KATP channel and nitric oxide (NO) pathways in the analgesic effects of BEO. Our findings demonstrate that the BEO exerts analgesic effects in pain models that are associated with delta- and mu-opioid pathways. We further found that BEO does not affect physiological pain or motor coordination.

BEO (Aromarant Co. Ltd., batch no. 103369, Rottingen, Germany) dissolved in almond oil, was injected intraperitoneally (i.p.) into mice at different doses (11.25, 11.5, 22.5, 45, 90 and 180 mg/kg) in an injection volume of 0.1 mL/100 g body weight. Mice in the control group were administered 0.9% saline; a separate vehicle group was injected with almond oil. Saline, vehicle or essential oil was injected 30 min before formalin tests, carrageenan-induced paw edema and pyrexia tests, thermal plantar tests, and rotarod tests (see below). In the acetic acid-induced writhing test, control, vehicle or essential oil was administered 35 min before the start of the experiment. Experiments employing inhalation of essential oils utilized a holeboard apparatus consisting of a square transparent Plexiglas cage (50 cm × 50 cm × 30 cm) with four holes in the bottom (Aloisi et al., 2002). BEO was efficiently vaporized by mixing it with an equal volume of distilled water, followed by soaking the mixture into cotton balls, and then placing the oil-soaked cotton balls on the upper side of the inhalation box. For the BEO group, 1 mL of BEO was vaporized in a Petri dish (90 × 15 mm); the control group received only distilled water. Animals were placed in the transparent Plexiglas cage and exposed for 1, 2, 4, 8 and 24 h before the formalin test. 2.4. Drugs The following pharmacological agents were used at the indicated doses: morphine (4 mg/kg), indomethacin (10 mg/kg), naloxone (8 mg/kg), 5′-guanidinonaltrindole (0.3 mg/kg), naltrindole (5 mg/kg), N-nitro l-arginine methyl ester (L-NAME; 37.5 mg/kg), L-arginine (LArg; 600 mg/kg) and glibenclamide-hippuric acid (GHA; 2 mg/kg). Except for inhalation tests, all drugs and vehicle were injected intraperitoneally 15 min prior to BEO injection; the injection volume was 0.1 mL/100 g body weight. All drugs were obtained from Sigma-Aldrich (Sigma, St. Louis, MO, USA). 2.5. Formalin-induced licking behavior The formalin test procedure was similar to the method described by Dubuisson et al. (1977). Animals (n = 7–12) received an injection of 20 μl of formalin (2% v/v) into the dorsal surface of the left hind paw. The time that the animals spent licking their injected paws was immediately recorded. The nociceptive response develops in two phases: 0–5 min after formalin injection (first phase, neurogenic pain response), and 20–25 min after formalin injection (second phase, inflammatory pain response) (Raymundo, 2011). The times established for the first and second phases of the formalin test are based on a previous publication (Parker et al., 2007).

2. Materials and methods 2.1. Experimental animals Male 8-week-old C57BL/6 mice (Samtaco Inc., Osan City, Korea) were received in the experimental facility 1 week prior to experiments. All animals were maintained under a 12-h light-dark cycle (lights on from 7:00 a.m. to 07:00 p.m.) at a constant temperature of 21°C ± 2°C. Mice were housed in groups of four with ad libitum access to sterilized food and tap water. All experimental procedures were conducted in accordance with guidelines relevant to the care of experimental animals, as approved by the Institutional Animal Research Ethics Committee of Korea University (approval no. KUIACUC-2011-84).

2.6. Acetic acid-induced abdominal writhing test Abdominal writhing was assessed in mice (n = 8–10) according to the method of Koster et al. (1959). Acetic acid was injected 5 min before the start of the experiment. After intraperitoneal administration of a 0.5% acetic acid solution (10 mL/kg), the total number of writhing events was recorded over a period of 30 min, starting 5 min after acetic acid injection.

2.2. Chemical analysis of the essential oils The components of basil essential oil (BEO) were determined by gas chromatography coupled to mass spectrometry (GC-MS) on a HPINNOWAX equipped with a 60 m × 0.25 mm ID, 0.5 μm film thickness capillary column. The analyses were conducted under the following conditions: oven temperature program, 40°C (3 min), increase at a rate of 3°C/min up to 230°C, and keeping of 230°C (30 min). Sample injections (1 μL) were performed in the split mode (50 : 1) at 230°C using helium as the carrier gas (1 mL/min). The solvent delay time of mass spectrometer was 3 min with m/z 40–350 of scan range and temperatures of 230°C (ion source) and 150°C (analyzer).

2.7. Carrageenan-induced paw edema and pyrexia Paw edema was induced according to the method of Winter et al. (1962). Animals were randomly divided in four groups (n = 10/group). Edema was induced injecting 25 μL of 2% carrageenan into the left hind paw. Inflammation was quantified by measuring the paw edema volume (mL) using a model 7140 plethysmometer (Ugo Basile, Varese, Italy) at 1 d before carrageenan injection, and 6 and 8 h and 1, 3, 5 and 7 d, after carrageenan injection. 2

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Changes in paw temperature induced by carrageenan were recorded using an AR872D infrared thermometer (Shenzhen Graigar Technology Co., Ltd., Shenzhen, Guangdong, China) 1 d before carrageenan injection, and 4, 6 and 8 h and 1, 3, 5 and 7 d after carrageenan injection.

Table 1 Chemical components of the BEO.

2.8. Thermal plantar test The Hargreaves method (Hargreaves et al., 1988) was used to assess paw withdrawal latency to a thermal nociceptive stimulus. Animals (n = 9/group) were tested after intraperitoneal administration of 0.9% saline (control group), vehicle, or BEO (45, 90 mg/kg). The center of a focused beam of radiant heat was applied to the plantar surface of the hind paw. The cut-off time was adjusted 20 s to avoid tissue damage. Four measurements were performed on each hind paw at intervals of 1 min. The mean paw withdrawal latency for each group was then determined using the values obtained from each hind paw. 2.9. Rota-rod test A rotarod test was used to assess potential effects of BEO on motor coordination. Animals (n = 10/group) were placed on a rotating rod (lane width, 50 mm; diameter, 30 mm; Scitechkorea, RRM, Seoul, Korea), spinning at 4 rpm. After mice had stabilized, the rotation speed was gradually increased at a rate of 1 rpm per 8 s. Each animal was subjected to three trials. The latency to fall and the speed at which the rod was rotating when mice fell off the apparatus were scored automatically using infrared sensors (Ayton et al., 2013). The average of three trials was used for further analysis.

Compounds

RT (min)

Area

% Area

α-Pinene Camphene β-Pinene Sabinene β-Myrcene α-Phellandrene α-Terpinene Limonene 1,8-Cineole (E)-Ocimene (Z)-Ocimene 3-Octanone Terpinolene 1-Hexanol cis-β-Terpineol α-Copaene Linalool Linalyl acetate α-Bergamotene α-Guaiene β-Caryophyllene Estragol α-Terpineol Borneol γ-Cadinene trans-Geraniol Eugenol Total

19.26 21.68 23.92 24.50 26.39 26.85 27.59 28.66 29.13 30.03 30.97 31.22 32.93 35.47 41.02 43.08 44.23 44.95 46.12 47.48 48.00 50.49 51.10 51.56 54.26 56.23 68.19 –

12,317,645 2,036,002 23,641,499 12,932,067 17,973,515 298,781 908,392 6,015,122 89,181,089 1,033,582 7,226,034 289,311 1,162,195 85,427 1,252,634 4,119,974 729,049,188 1,378,116 1,583,580 11,233,560 2,121,185 15,970,311 17,392,345 7,941,386 82,812,600 4,410,645 233,345,851 1,038,395,874

0.96 0.16 1.84 1.00 1.40 0.02 0.07 0.47 6.93 0.08 0.56 0.02 0.09 0.01 0.10 0.32 56.6 0.11 0.12 0.87 0.16 1.24 1.35 0.62 6.43 0.34 18.1 100

2.10. Statistical analysis Data are expressed at means ± SEMs. Differences among treatment groups, except for rotarod tests, were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Rotarod tests were evaluated using Student's t-test. In all analyses, a P-value < 0.05 was considered to be statistically significant. 3. Results 3.1. Chemical components of the BEO identified by GC-MS analysis The GC-MS analyses were conducted to evaluate the chemical composition of the BEO. Twenty-seven components were identified out of the GC-MS chromatograms (Table 1). The BEO was characterized mainly by linalool, eugenol, 1,8-cineole, γ-Cadinene, and β-Pinene, representing 89.9%.

Fig. 1. Analgesic effects of BEO and morphine in the formalin test. Graph shows paw-licking time in mice injected with control (0.9% saline, i.p.), vehicle (almond oil, i.p.), basil essential oil (BEO, 11.25, 22.5 and 45 mg/kg, i.p.) or morphine (4 mg/kg, i.p.) 30 min before the injection of 20 μL of formalin (2% v/v) into the dorsal surface of the left hind paw. In the first phase (0–5 min), only morphine, used as a positive control, significantly reduced paw-licking time. In the second phase (20–25 min), BEO (45 mg/kg) in addition to morphine significantly reduced licking time. Values represent means ± S.E.M. (*p < 0.05 compared with vehicle control; one-way ANOVA followed by Tukey's post hoc test; n = 12 for controls and 8 for other groups).

3.2. Analgesic effect of injected and inhaled BEO in the formalin test To determine whether BEO has analgesic effects in the formalin test, we recorded licking time during the first phase (0–5 min) and the second phase (20–25 min) of the test (Fig. 1) after pretreating intraperitoneal cavity of mice with BEO. BEO (45 mg/kg) did not reduce licking time during the first phase, but did reduce licking time in the second phase by 74.32% (12.3 s for basil vs. 47.9 s for vehicle; p = 0.007). As expected, morphine used as positive control caused inhibition in both phases, reducing licking in the first phase by 79.96% (9.6 s for morphine vs. 47.9 s for vehicle; p = 0.007) and in the second phase by 47.99% (59.5 s for morphine vs. 114.4 s for vehicle; p = 0.003) (Fig. 1). A comparison of the effect of BEO with that of the morphine positive control in the second phase showed no significant between-group difference (p = 1.000). Next, we investigated whether inhalation of BEO also affected licking time in the formalin test (Fig. 2). In the first phase, inhalation of BEO reduced licking time compared with that in the control group, but the difference was statistically significant only after 2 h (p = 0.022) and

24 h (p = 0.016) of inhalation. Inhalation of BEO for more than 8 h caused significantly shorter licking time compared with the control group in the second phase (p = 0.042 and 0.008 for 8 and 24 h, respectively). Taken together, the results of injection and inhalation of BEO indicate that BEO exerts analgesic effects in the formalin test in mice. 3.3. Association between the analgesic effect of BEO and opioid pathways in the formalin test We next investigated whether the analgesic effect of BEO is related to opioid pathways by pretreating mice with antagonists of κ-, δ- and μopioid receptors 15 min before treatment with BEO (45 mg/kg, i.p.) and 3

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then assessing analgesic effects in first and second phases of the formalin test (Fig. 3). In these experiments, antagonists were injected 45 min before the injection of 20 μL of formalin (2% v/v) into the dorsal surface of the left hind paw of experimental mice. In response to pretreatment with 5′-guanidinonaltrindole (0.3 mg/kg, i.p.), a κ-opioid antagonist, licking time in the second phase trended higher compared with that in the essential oil-only group, but this difference did not reach statistical significance. Notably, licking time in the second phase was substantially and significantly increased by pretreatment with either the δ-opioid antagonist naltrindole (5 mg/kg; p < 0.001 or μopioid antagonist naloxone (8 mg/kg; p = 0.011) compared with that in the BEO-only group. As was the case for BEO treatment alone, none of these antagonists significantly affected licking time in the first phase. Collectively, these results suggest that BEO acts primarily through delta- and mu-opioid pathways to exert its analgesic effects in the formalin test.

Fig. 2. Inhalation of BEO reduces paw-licking time in the formalin test. Graph shows paw-licking time following inhalation of control solution (distilled water) or BEO mixed with the same volume of distilled water for 1, 2, 4, 8, and 24 h before the formalin test. In the first phase (0–5 min), BEO exerted analgesic effects at 2 and 24 h. In the second phase (20–25 min), inhalation of BEO for more than 8 h significantly reduced paw-licking time. Values represent means ± S.E.M. (*p < 0.05 compared with vehicle control; one-way ANOVA followed by Tukey's post hoc test; n = 7 for control and 24-h groups and 8 for other groups).

3.4. Involvement of nitric oxide and KATP channel pathways in the analgesic effects of BEO Possible involvement of NO and KATP channel pathways in the analgesic effects of BEO in the formalin test was investigated using the same basic experimental paradigm as that used to assess the role of opioid signaling (Fig. 4). We first assessed the role of the NO pathway by treating with the NO synthase (NOS) inhibitor N-nitro l-arginine methyl ester (L-NAME; 37.5 mg/kg, i.p.) or the NO precursor L-arginine (600 mg/kg, i.p.) 45 min before the injection of 20 μL of formalin (2% v/v). Almond oil (i.p.) was used as a vehicle control. Licking time in the first phase was significantly reduced by L-NAME (p = 0.007) and Larginine (p = 0.015) alone, as well as L-NAME + basil (p = 0.017) or L-arginine + basil (p = 0.001) compared with vehicle controls. The combination of L-arginine pretreatment and BEO treatment also exerted a greater analgesic effect in the first phase compared with BEO alone (p = 0.012), which had no significant effect on its own, but produced an analgesic effect less than that of BEO alone in the second phase of the formalin test (p = 0.015). In contrast to the effects of NO pathway, pretreatment with the ATP-sensitive K+ channel blocker glibenclamide (2 mg/kg) had no effect on nociception in first or second phases of the formalin test. Taken together, our results suggest that the NO pathway may be associated with formalin-induced pain responses but that the KATP channel pathway is unrelated to the analgesic effects of BEO.

Fig. 3. Basil essential oil acts through delta- and mu-opioid pathways to exert analgesic effects in the formalin test. Graph shows paw-licking time after pretreatment with an opioid receptor antagonist 15 min before the injection of BEO (45 mg/kg, i.p.). Mice in each group were administered the κopioid antagonist 5′-guanidinonaltrindole (GNTI; 0.3 mg/kg, i.p.), δ-opioid antagonist naltrindole (NTD; 5 mg/kg, i.p.) or μ-opioid antagonist naloxone (NAL; 8 mg/kg, i.p.). In the second phase (20–25 min), BEO alone (Saline) significantly decreased licking time compared with vehicle control, an effect that was significantly attenuated by pretreatment with NTD and NAL. Values represent means ± S.E.M. (*p < 0.05 for BEO alone compared with vehicle control; #p < 0.05, ##p < 0.001 for BEO alone compared with BEO plus opioid antagonist; one-way ANOVA followed by Tukey's post hoc test; n = 8 for NAL plus BEO group and 7 for all other groups).

3.5. Analgesic effects of BEO in the acetic acid-induced abdominal writhing test The acetic acid-induced abdominal writhing test is a visceral pain model that causes abdominal contraction. Acetic acid induces pain by liberating endogenous substances (Khan et al., 2010) as well as by promoting the biosynthesis of arachidonic acid-derived pain mediators via cyclooxygenase and prostaglandin pathways (Lu et al., 2007). This visceral pain model differs from the formalin test in that it is a somatic

Fig. 4. Association between analgesic effects of BEO and the NO pathway in the formalin test. Graph shows paw-licking time after pretreatment with L-NAME (37.5 mg/kg, i.p.), L-arginine (L-Arg; 600 mg/kg, i.p.) or the ATP-sensitive K+ channel blocker glibenclamide-hippuric acid (GHA; 2 mg/ kg, i.p.) 45 min before the injection of 20 μL of formalin (2% v/v) into the dorsal surface of the left hind paw. Vehicle (almond oil) or BEO (45 mg/kg) was injected i.p. in mice 15 min after the injection of drug. Paw-licking time in the first phase (0–5 min) was significantly decreased compared with the vehicle group in all groups except the GHA + BEO group. In addition, paw-licking time in the second phase (20–25 min) was significantly increased in the LArg + BEO group. Values represent means ± S.E.M. (*p < 0.05 compared with vehicle control; #p < 0.05 for the BEO group compared with drug groups; oneway ANOVA followed by Tukey's post hoc test; n = 8 for all groups). 4

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Treatment with BEO produced no significant difference in mean withdrawal latencies at a dose of 45 mg/kg (p = 0.792) or 90 mg/kg (p = 0.539) compared with the vehicle group, but did cause a significant difference at a higher dose (180 mg/kg; p < 0.001, ANOVA with Tukey post-hoc test). These findings indicate that BEO is capable of inhibiting physiological pain associated with thermal stimuli at high doses, but not at low doses. 3.8. Altered latency to fall in a rotarod test in mice treated with BEO Finally, a rotarod test was conducted to determine whether BEO at the highest dose tested in the various nociception models (180 mg/kg) influenced motor coordination in mice. As shown in Fig. 8, there was no significant difference in latency to fall between mice treated with 180 mg/kg BEO and mice in the vehicle control group. Thus, even at high doses, BEO does not affect motor coordination.

Fig. 5. Effects of BEO on abdominal writhing events induced by acetic acid. Graph shows number of writhing events in mice injected with control (0.9% saline, i.p.), vehicle (almond oil, i.p.), BEO (11.5, 22.5 and 45 mg/kg, i.p.) or indomethacin (10 mg/kg, i.p.) 30 min before intraperitoneal administration of a 0.5% acetic acid solution (10 mL/kg). The total number of writhing was recorded over a period of 30 min, starting 5 min after acetic acid injection. Only indomethacin and 45 mg/kg BEO decreased the number of writhing. Values represent means ± S.E.M. (*p < 0.05 with vehicle control; one-way ANOVA followed by Tukey's post hoc test; n = 10 for the vehicle control group and 8 for all other groups).

4. Discussion O. basilicum L. (basil), a perennial plant with yellow, white and violet flowers, and sharp-tipped oval leaves (Darrah, 1974), has been used for thousands of years as a natural anti-inflammatory, antibiotic, diuretic, and analgesic in traditional Asian Indian medicine. In this study, we investigated the analgesic effects of BEO using various pain models and assessed the underlying mechanism to establish the benefits of BEO in the context of pain control. In the formalin test, formalin was injected into a hind paw, and nociceptive behavior was recorded. Two phases of the response were observed: the first phase reflects acute pain in the injured tissue caused by transient activation of primary afferent fibers (A δ and C fibers) by injected formalin (neurogenic pain response). The second-phase reaction is a consistently strong pain state reflecting peripheral and central sensitization produced by inflammation of the peripheral tissues (inflammatory pain response) (Shibata, 1989). The formalin test is a good pain model for assessing both acute pain and persistent pain in a single experiment. In the formalin test, paw-licking time in the first phase was not affected by BEO injected intraperitoneally, but was reduced by BEO administered by inhalation. These results contrast with those of a previous study (Venancio et al., 2011) in which injected BEO was reported to exert analgesic effects in both first and second phases. However, in the previous study, 200 mg/kg of BEO was subcutaneously injected, whereas a much lower dose (45 mg/kg) of BEO was intraperitoneally administrated in the present study, a difference that may account for the apparent discrepancies. It is also possible that these discrepancies are due to differences in BEO composition. Differences in time and route of administration (inhalation vs. i.p. injection) is also a consideration in these experiments. Because inhaled BEO was administered at least 1 h before the start of the formalin test, there was enough time for the BEO to reach the formalin injection site and exert an analgesic effect. In addition, inhaled BEO is rapidly absorbed into the blood owing to the large surface area of the alveolar epithelium, and thus could immediately enter the brain through the nose to affect the limbic system. This, in turn, could activate descending pain control pathways. In contrast, intraperitoneally administered BEO was injected just 30 min before the formalin test; thus, there may not have been sufficient time for BEO to enter the systemic circulation and exert analgesic effects at the site of formalin injection. However, since various mechanisms are involved in formalin test results, further research on this difference is needed. In the second phase, both intraperitoneal injection and inhalation of BEO reduced paw-licking time. Considering both the results of first and second phases, this suggests that BEO is effective against direct effects of formalin on nociceptors and also significantly controls inflammatory pain mediated by the combination of peripheral input and spinal cord sensitization.

pain model the reflects pain conduction. We therefore used the acetic acid-induced abdominal writhing test to investigate whether different doses of BEO (11.5, 22.5 and 45 mg/kg) have analgesic effects on visceral pain (Fig. 5). BEO (45 mg/kg) and indomethacin (10 mg/kg), used as a positive control, inhibited writhing, reducing the number of events by 33.15% (p = 0.014) and 47.66% (p = 0.003), respectively, compared with the vehicle control group (basil 45 mg/kg, 12.3 events; indomethacin, 9.63 events; vehicle, 18.4 events). Although the number of writhing events trended lower in mice treated with BEO at a dose of 22.5 mg/kg, this difference did not reach statistical significance (Fig. 5). 3.6. Anti-inflammatory activity of BEO in the carrageenan-induced paw edema and pyrexia tests To examine the anti-inflammation activity of BEO in acute-phase inflammation in vivo, we assessed paw edema and pyrexia induced by carrageenan (2%; i.p.) as a function of time (Fig. 6A and B). At a dose of 45 mg/kg, BEO tended to reduce edema at all time points measured compared with vehicle controls, reaching statistical significance at 6 h (p < 0.001), 3 d (p < 0.001),8 h (p = 0.004) and 7 d (p = 0.001). Interestingly, at a higher dose (90 mg/kg), basal essential oil significantly reduced edema compared to vehicle only 6 h after carrageenan injection (p < 0.001) (Fig. 6A). In carrageenan-induced paw pyrexia experiments, BEO exerted a remarkable inhibitory effect 6 h after carrageenan injection, reducing pyrexia by 15.47% and 13.21% at doses of 45 mg/kg (26.17°C for basil vs. 30.96°C for vehicle; p < 0.001) and 90 mg/kg (26.87°C for basil vs. 30.96°C for vehicle; p < 0.001) respectively, compared with the vehicle control group (Fig. 6B). BEO also inhibited pyrexia compared with vehicle by 5.89% at a dose of 45 mg/kg (27.63°C for basil vs. 29.36°C for vehicle; p = 0.007) and 4.73% at a dose of 90 mg/kg (27.97°C for basil vs. 29.36°C for vehicle; p = 0.039) on day 5. On day 7, BEO inhibited pyrexia compared with vehicle by 3.95% at a dose of 45 mg/kg (28.17°C for basil vs. 29.33°C for vehicle; p = 0.017) and 4.84% at a dose of 90 mg/kg (27.91°C for basil vs. 29.33°C for vehicle; p = 0.003). Collectively, the results of carrageenan tests suggest that BEO possesses anti-inflammatory activity. 3.7. The effect of BEO in the thermal plantar test To determine whether BEO affects physiological pain associated with a thermal stimulus, we performed a thermal plantar test (Fig. 7). 5

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Fig. 6. Anti-inflammatory activity of BEO in carrageenan-induced paw edema and pyrexia tests. Graphs show paw volume (edema) (A) and paw temperature (pyrexia) (B) over time after injection of a 2% carrageenan solution into the left hind paw, with or without treatment with 45 or 90 mg/kg BEO (i.p.). BEO significantly inhibited carrageenan-induced paw edema and pyrexia at multiple time points compared with the vehicle control group. #p < 0.05, ##p < 0.001 for 45 mg/kg BEO compared with vehicle control; *p < 0.05, **p < 0.001 for 90 mg/kg BEO compared with vehicle control; one-way ANOVA followed by Tukey's post hoc test; n = 10 for all groups.

Fig. 7. Basil essential oil exerts dose-dependent effects in the thermal plantar test. Graph shows latency to paw withdrawal in mice pre-treated with BEO (45, 90 and 180 mg/kg, i.p.). Control and vehicle groups were administered 0.9% saline (i.p.) or almond oil (i.p.). After 30 min, the center of a focused beam of radiant heat was applied to the plantar surface of the hind paw. BEO significantly increased mean withdrawal latencies only at the highest dose tested (180 mg/kg). Values represent means ± S.E.M. (**p < 0.001 compared with vehicle control; one-way ANOVA followed by Tukey's post hoc test; n = 9 for all groups).

Fig. 8. Basil essential oil does not affect motor coordination in the rotarod test. Graph shows elapsed time on a rotarod apparatus for mice pretreated with BEO (180 mg/kg) or vehicle (almond oil). BEO had no significant effect on latency to fall. Values represent means ± S.E.M. (n = 10/group). Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test.

and Hole, 1987). In addition, a variety of drugs that exhibit analgesic effects in the formalin test, such as a lectin from Canavalia brasiliensis (de Freitas Pires et al., 2013), also act through opioid receptors. We therefore investigated whether opioid pathways are involved in the

Previous studies have reported that opioid analgesics such as morphine exert analgesic effects in both phases of the formalin test, although these drugs are more effective in the second phase (Hunskaar 6

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It is also possible to consider the mechanism of the analgesic effect of BEO in the formalin test in relation to the action on the opioid receptor, N-methyl-D-aspartate (NMDA). In the formalin test, the NMDA receptor is activated by repeated stimulation of afferent C-fibers, which releases the intracellular Mg2+ block that maintains stable NMDA receptor activity. Ca2+ influx into the secondary neuronal cytoplasm through the NMDA receptor pathway (MacDermott, 1986) results in the production of NO, prostaglandin E2 (PGE2), and other metabolites, and increases the secretion of neurotransmitters in the synapse membrane through positive feedback and upregulation of the α-amino-3-hydroxyl5-methyl-4-isoxazole propionic acid (AMPA) receptor. It also induces transcription of immediate early genes, causing a vicious cycle of pain in the central nervous system and potentiating long-term pain (Woolf, 1991). Therefore, inhibition of the NMDA receptor produces antinociception (Peana et al., 2004). Previous studies investigating the chemical composition of essential oil from O. basilicum L. have shown that linalool is the predominant component of BEO, accounting for 61.6%–69.5% of the total (Venancio et al., 2011). It is known that linalool acts as a competitive antagonist at the NMDA receptor (Elisabetsky et al., 1999). Considering the results of the present study in the context of previous studies, it could be that the analgesic effect of BEO in the formalin test is associated with inhibition of the NMDA receptor. To investigate whether BEO also affected visceral pain, we conducted acetic acid tests. Previous studies indicate that pain induction in the acetic acid-induced writhing test is elicited by the production of a localized inflammatory response caused by the release of free arachidonic acid from tissue phospholipids and the production of prostaglandins, specifically PGE2 and PGF2α, via cyclooxygenase. These products cause inflammation and pain by increasing capillary permeability (Muhammad et al., 2012). Our results showed that BEO induces a dose-dependent reduction in the number of writhing events, a result consistent with a previous study by Antônio Medeiros Venâncio et al. (2011). Thus, it is possible that BEO exerts analgesic effects, at least in part, through inhibition of prostaglandin synthesis via cyclooxygenase and release of free arachidonic acid from tissue phospholipids. To assess the anti-inflammatory activity of BEO in vivo, we performed carrageenan-induced paw edema and pyrexia tests. Previous studies have reported that carrageenan strongly stimulates the release of inflammatory and proinflammatory mediators, including bradykinin, histamine, tachykinins, and reactive oxygen and nitrogen species (Morris, 2003). Carrageenan-induced edemas form owing to exudation of fluid and plasma proteins and accumulation of mainly neutrophilic granulocytes at the inflammatory site (Du et al., 2014), whereas pyrexia results from local vasodilation and increased blood flow to the injured area induced by histamine. In addition, damaged tissue initiates the secretion and enhanced formation of pro-inflammatory chemical mediators (including the cytokines, interleukin-1α and -β, and TNF-α), which induce an upsurge in the synthesis of PGE2 near the pre-optic hypothalamic area, thereby triggering an increase in hypothalamic activity that elevates normal body temperature (Raju et al., 2014). In this experimental series, we found that BEO reduced carrageenan-induced edema and pyrexia in the acute phase as well as in the chronic phase. Accordingly, it suggests that BEO likely suppresses the release of inflammatory mediators and consequently reduces capillary permeability and inhibits extravasation of plasma proteins. Our results are supported by a recent study showing that oral administration of essential oil obtained from O. basilicum complexed with β–cyclodextrin prevents paw edema formation induced by carrageenan, histamine or arachidonic acid, thereby decreasing vascular permeability in vivo and inhibiting leukocyte recruitment into the peritoneal cavity (Rodrigues et al., 2017). To determine whether BEO affects physiological pain associated with a thermal stimulus, we performed a thermal plantar test. We found that, although lower doses of BEO (45 and 90 mg/kg) did not affect latency to paw withdrawal compared with vehicle, at a higher dose

analgesic effects of BEO observed in the formalin test. We found that the analgesic effect of BEO was reversed by treatment with either a delta opioid antagonist or a mu-opioid antagonist, suggesting that the analgesic effect of BEO is related to the delta and mu opioid pathways. This finding is supported by previous reports that the analgesic effect of BEO may be associated with the mu opioid pathway, as evidenced by inhibition of the analgesic effect of BEO in the hot plate test by the mu opioid antagonist, naloxone (Venancio et al., 2011). We also investigated the possible involvement of NO and KATP channel pathways in the analgesic effect of BEO in the formalin test. According to a previous study (Kawabata et al., 1994), the role NO plays in formalin-induced pain is complicated, since peripheral NO plays a dual role in nociceptive modulation, depending on its level in tissue. Increasing NO levels through regional activation of NOS by injection of formalin into the hind paw induces nociceptive responses in the second phase, whereas NO is also able to increase the concentration of cGMP, which leads to activation of KATP channels. Activation of these channels, in turn, induces hyperpolarization of primary afferent neurons, thereby inducing analgesia (Nguelefack et al., 2010). In the current study, we showed that treatment with L-arginine or L-NAME before formalin injection exerted analgesic effects in the first phase. We further found that pretreatment with L-arginine altered the analgesic effect of BEO in the second phase of the formalin test. This is similar to previous reports that intraplantar injection of a large dose of L-arginine induces antinociception in the second phase of the formalin test (Kawabata et al., 1994). However, this previous study differs from ours in reporting an analgesic effect of L-arginine in the second phase, but not the first phase. However, this difference can be explained by the fact that the interval between drug injection and formalin test start time in the current study was longer than that in previous studies. Interestingly, L-arginine inhibited the analgesic effect of BEO in the second phase of the formalin test, suggesting that L-arginine prevents the analgesic effect of BEO by increasing NO concentration. Our interpretation of these results is that the analgesic effect of BEO in the second phase of formalin test is related to NO inhibition. Further research is needed to explain why L-arginine exerts an analgesic effect in the first phase of the formalin, and prevents the analgesic effect of BEO in the second phase. The analgesic effect of BEO in the second phase of the formalin test was reduced in groups that received L-NAME and BEO together. This apparently contradicts our conjecture that BEO would exert analgesic effects in the second phase of the formalin test by inhibiting NO production. However, these results can be explained by previous studies showing that a high dose of L-NAME decreases the analgesic effect of ellagic acid—a phenolic compound in a methanol extract of basil (MHM et al., 2015)—in the second phase of the formalin test (Ghorbanzadeh et al., 2014). Thus, it is possible that L-NAME decreased the analgesic effect of BEO through effects on ellagic acid in the second phase of the formalin test. However, since ellagic acid is not a major component of BEO, additional studies will be needed to assess the effects of L-NAME on major components of BEO. In addition, a previous study in which LNAME was administered intraperitoneally, as was done here, recorded results 15–45 min after injection of the drug (Moore et al., 1991), whereas we observed licking behavior 45–70 min after drug injection, highlighting the importance of recording pain behavior after a longer time period in obtaining an accurate second-phase record. The ATP-sensitive K+ channel blocker, glibenclamide had no effect on the analgesic effect of BEO in either phase of the formalin test. This observation is at odds with a previous study (Venancio et al., 2011) that could be interpreted as predicting that BEO would exert analgesic effects through K+-ATP channels because linalool, a major component of BEO, acts through this mechanism. Although our data suggest that the analgesic effect of BEO is not related to the KATP channel pathway, it is possible that the dose of glibenclamide used in this experiment (2 mg/ kg) is insufficient to suppress hyperpolarization induced by activation of KATP channels. 7

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(180 mg/kg), BEO did increase latency. These results are not entirely congruent with a previous study showing that BEO suppresses the response to heat-related physiological pain in a hot plate test (Venancio et al., 2011). Differences between our study and this previous study may reflect differences in the mechanism by which the thermal plantar test and hot plate test cause physiological pain. Finally, to investigate whether BEO influenced motor coordination in mice, we conducted a rotarod test. We found no difference in latency to fall between mice treated with BEO (180 mg/kg) and those treated with vehicle. Thus, given the high dose used in these experiments, we conclude that BEO does not affect motor coordination. This finding contrasts with a previous report that oral supplementation with O. basilicum L. (100 mg/mL of solvent/kg body weight) improved motor performance in the rotarod test (Zahra et al., 2015). A recent study has shown that oral treatment with BEO resulted in a significant reduction in mechanical hyperalgesia in an animal model of fibromyalgia, and significantly increased Fos protein expression in the central nervous system (Nascimento et al., 2014). In addition, more recently published studies have reported that essential oil obtained from cultivated O. basilicum L. inhibits excitability in the peripheral nervous system (Medeiros Venancio et al., 2016). Taken together with the results of this study, this suggests that BEO has the potential for development as an analgesic.

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5. Conclusions Our results indicate that BEO exerts analgesic effects in formalin, acetic acid-induced writhing, and carrageenan-induced paw edema and pyrexia tests. Furthermore, we found an association between the deltaand mu-opioid pathways and the analgesic effect of BEO in the formalin test. In this study, BEO did not affect motor coordination or physiological pain related to thermal stimulation, despite having an analgesic effect. These findings indicate that BEO could be developed as an analgesic agent to relieve inflammatory pain. By reducing the required medication dose, the BEO may also help prevent side effects associated with anti-inflammatory and analgesic therapeutics. Improving our understanding of the pharmacology of BEO and applying BEO as an effective analgesic agent will require further research into the mechanisms and side effects of BEO. Additional studies on the NMDA receptor, as well as GABA and cholinergic pathways, could also provide insight into the mechanisms underlying the analgesic effects of BEO. Authors’ contributions AHB and WSC participated in data processing and analysis, and organizing and drafting the manuscript. MSK conducted behavioral experiments. SBL and JML analyzed the chemical composition of BEO. GHS and SSM conceived the study and participated in the design of the study, data processing and analysis, and organizing and drafting the manuscript. Acknowledgement This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) & funded by the Korean government (MSIP&MOHW) (No. 2016M3A9B6904244) and NRF(2015R1D1A1A01061326). References Adigüzel, A., Güllüce, M., ŞENGÜL, M., Öğütcü, H., ŞAHİN, F., Karaman, İ., 2005. Antimicrobial effects of Ocimum basilicum (Labiatae) extract. Turkish J. Biol. 29 (3), 155–160. Akgül, A., 1989. Volatile oil composition of sweet basil (Ocimum basilicum L.) cultivating in Turkey. Food/Nahrung 33 (1), 87–88 1979. Pain terms: a list with definitions and notes on usage. Recommended by the IASP Subcommittee on Taxonomy. Pain 6(3), 249.

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Venancio, A.M., Onofre, A.S., Lira, A.F., Alves, P.B., Blank, A.F., Antoniolli, A.R., Marchioro, M., Estevam Cdos, S., de Araujo, B.S., 2011. Chemical composition, acute toxicity, and antinociceptive activity of the essential oil of a plant breeding cultivar of basil (Ocimum basilicum L.). Planta Med. 77 (8), 825–829.

Zahra, K., Khan, M.A., Iqbal, F., 2015. Oral supplementation of Ocimum basilicum has the potential to improves the locomotory, exploratory, anxiolytic behavior and learning in adult male albino mice. Neurol. Sci. : Off. J. Ital. Neurol. Soc. Ital. Soc. Clin. Neurophysiol. 36 (1), 73–78.

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