1. Introduction
Pain is a displeasing sensation associated with tissue damage (Karimi, Monajemi, & Amjad, 2014). Pain is a physiologic protective function occurring via an external or internal harmful stimulus (Ashok & Upadhyaya, 2013). Activation of nociceptors in visceral structures leads to visceral pain including angina, colic, dyspepsia, pancreatitis, appendicitis, and dysmenorrhea (de Oliveira Júnior et al., 2017). Visceral tissue injury and inflammation can activate nociceptive primary afferent fibers, which results in central sensitization or hyperexcitability of nociceptive neurons in the spinal cord dorsal horn (Grace, Hutchinson, Maier, & Watkins, 2014). 
Recently, nonprescription analgesics like Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) and opioids are not prescribed because of their adverse effects (Zendehdel, Torabi, & Hassanpour, 2015). Instead, a variety of plant-derived pharmaceutical products are used in traditional medicine due to their positive properties (Yama et al., 2011). Plants are a rich source of a wide variety of secondary metabolites such as flavonoids, thymol, carvacrol, terpenoids, alkaloids (Hassanpour, Sadaghian, MaheriSis, Eshratkhah, & ChaichiSemsari, 2011). Worldwide interest has increased on traditional medicine. People are more interested in consuming medicinal plants because of their therapeutic properties (Yousif, Ashoush, Donia, & Hala Goma, 2013). 
Pharmaceutical research is highly focused on identifying bioactive compounds in plants. Such knowledge can be used for the treatment of different conditions, such as anxiety, pain, and inflammation (de Oliveira et al., 2012). The evaluation of pharmacological effects can be used as a strategy for discovering new plant-deprived pharmaceuticals (Zendehdel, Taati, Jadidoleslami, & Bashiri, 2011).
Medicinal and aromatic plants are traditionally used for the treatment of various illnesses (Khodaverdi-Samani, Pirbalouti, Shirmardi, & Malekpoor, 2015). Ziziphora Clinopodioides belongs to the Lamiaceae family. It is called “Kakuti-e-kuhi” or “Taramoshk” in Persian. This plant is spread worldwide and particularly in Iran, Afghanistan, Iraq and Turkey. Fresh leaves and stems are prescribed for wound healing and sedation. These herbs are presented in the forms of stomach tonic, antiseptic, expectorant, antifungal, antibacterial and antiseptic substances, in traditional Iranian medicine (Shahbazi, 2015). 
The Essential Oil of Ziziphora Clinopodioides (EOZC) contains a diversity of biologically active compounds like monoterpenes and sesquiterpenes (Shahbazi, 2015). Most studies on rats and or mice with experimental models of pain have demonstrated that some of these terpenes have analgesic effects (Almeida, Navarro, & Barbosa-Filho, 2001). Therefore, the current study aimed to determine the antinociceptive effect of EOZC on opioidergic system in male rats.
2. Methods
2.1. Preparation of essential oil
Fresh leaves of Ziziphora Clinopodioides were collected from Gilan-e-Gharb County (Kermanshah Province, Iran) from March to July 2016. Specimen identification was demonstrated in Faculty of Agriculture, Razi University, Kermanshah, Iran. Voucher specimen (No. 6816) of the plant was deposited in the herbaria of the Research Center of Natural Resources of Tehran, Iran.
2.2. Isolation of essential oil
Ziziphora Clinopodioides leaves (100 g) were shade dried at room temperature (25±2°C). Samples were hydrodistilled using a Clevenger-type apparatus for 3.5 h till full of essential oil. Then, supernatant was collected and dried with 0.5 g anhydrous sodium sulfate (Merck, Darmstadt, Germany). The essential oil was stored in a dark glass bottle, and covered with aluminum foil at 4±1°C (Shahbazi, 2015).

2.3. Analysis of the EOZC
The chemical compounds of EOZC was determined by Gas Chromatography–Mass Spectrometry (GC-MS) (Thermo Quest Finningan, UK), as presented in Table 1. The GC-MS instrument was 5% phenyl methyl silicone and 95% dimethylpolysiloxane and equipped with DB5 capillary column (30 m, 0.25 mm, film thickness 0.25 μm). An electron ionization mode with ionization energy of 70 eV was used to determine the EOZC constituents (Shahbazi, 2015). The carrier gas was helium at a constant flow rate of 1.2 mL/min, with linear velocity of 29.6 cm/s and split ratio of 1:20. The initial oven temperature was held at 50°C for 3 min, then raised to 265ºC at program ramp rate of 2.5°C/min. The final temperature was 265ºC and maintained for 6°C. The temperature of the injector was 250°C. To improve accuracy of the results, the GC-MS analysis was performed in triplicate. 
2.4. Drugs
Morphine (an opioid receptor agonist), and naloxone hydrochloride (an opioid receptor antagonist) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tween-80 and formaldehyde (37%) were purchased from Merck Co. (GERBU, Germany). All drugs were dissolved in normal saline. Various doses of the EOZC were prepared in Tween-80 (0.5%). Distilled water was used to dissolve Tween-80 into 1% (v/v) and diluted with the same vol­ume of normal saline. The control group were injected with vehicle. All drugs were prepared before use.
2.5. Animals
Sixty-four male Wistar rats (weighing: 200–220 g) were obtained from the Laboratory Animal Facility of the School of Veterinary Medicine, Razi University, Kermanshah, Iran. The animals were randomly divided into 8 groups (8 in each group). Rats were maintained under standard laboratory conditions according to European Guidelines for Environmental Control in Laboratory Animal Facilities (ambient temperature of 22±1ºC, 12:12 h light-dark cycle). 
All animals had access to chow pellets and fresh water ad libitum. In group 1, animals were Intraperitoneally (IP) injected with normal saline, 30 minutes before intraplantar injection of formalin. In group 2, rats were IP injected with vehicle (Tween-80, 0.5%), 30 minutes before intraplantar injection of formalin. In groups 3, 4 and 5, EOZC was injected IP at doses of 10, 20 and 40 mg/kg, respectively 30 min before induction of formalin pain. In groups 6 and 7, animals received IP injection of morphine (5 mg/kg) and naloxone (2 mg/kg), respectively, 30 minutes before intraplantar injection of formalin. In group 8, animals received naloxone (2 mg/kg), then 15 min later received EOZC (20 mg/kg) followed by formalin solution after 15 min. Drug solutions were injected (1 mL/kg IP) using a 25-gauge injection needle. 
To reduce the possible effect of circadian rhythm on the nociceptive susceptibility, all experiments were done from 9 AM to 12 AM (Borowicz, Kleinrok, & Czuczwar, 2003). All experimental procedures were carried out in accordance with the guidelines for the care and use of laboratory animals to investigate experimental pain in conscious animals (Zimmermann, 1983).
2.6. Estimation of acute toxicity
In order to identify the acute toxicity of the essential oil with few animals, a limit test was conducted according to OECD 425 guidelines. The animals were maintained in cages for at least 5 days prior to dosing to allow adaptation to the laboratory conditions. The EOZC (1000 mg/kg IP) was initially administered to one animal, followed by 24 hours observation. If the animal survived, 4 additional animals were sequentially administered with EOZC (1000 mg/kg, IP) under similar conditions. A total of 5 animals were tested. Observation was carried on for 14 days.
2.7. Formalin test
The formalin test is frequently used as a valid model of pain (Erami, Azhdari-Zarmehri, Imoto, & Furue, 2017). To minimize the possible effect of stress during the study, rats were placed inside a Plexiglas observation chamber (30×30×25 cm3) equipped with a mirror angled at 45° below the chamber for 30 minutes for 3 consecutive days (Abbott & Bonder, 1997). In the test day, a 30-minute adaptation period was applied on the animals, prior to administrating the test. Formalin (50 µL, 2%) was injected subcutaneously via a 30-gauge needle into the plantar surface of the right hind paw (Sofiabadi et al., 2014). 
Following the formalin injection, rats were immediately returned to the observation chamber. The time spent on licking and biting of the injected paw was determined as nociceptive behavior. The formalin-induced behavioral responses were biphasic, as follows: 0-5 minutes (first phase, neurogenic phase) and 15-45 minutes (second phase, inflammatory phase) (Tamaddonfard & Hamzeh-Gooshchi, 2010).
2.8. Statistical analysis
The obtained data were prepared in Excel and Analyzed by the Analysis of Variance (ANOVA) and Tukey’s HSD post-hoc test using SPSS. The Student t test was employed to determine the differences between the 2 control groups of formalin test. The results were expressed as Mean±SEM. P<0.05 was defined to set the significant differences between the groups.
3. Results
3.1. Analysis of the EOZC 
Table 1 lists the composition of the EOZC. In total, 24 components were identified, covering 99.65% of the total composition. Regarding the chemical constituents, carvacrol (65.22%), thymol (19.51%), p-cymene (4.86%) and γ-terpinene (4.63%) were the main components of the EOZC (Table 1).
3.2. Acute toxicity testing
Single dose acute toxicity of the EOZC was demonstrated through a limit test (1000 mg/kg. IP). EOZC caused no animal mortality in a period of 14 days. Therefore, LD50 of the EOZC was considered to be more than 1000 mg/kg.
3.3. Effect of the EOZC on formalin-induced pain behaviors 
The intraplantar injection of formalin 2% produced a biphasic pain-related behavior. Effects of normal saline and vehicle (Tween-80, 0.5%) on licking and biting time of the injected paw in male rats are presented in Figure 1. No significant differences were observed on the first phase of pain in the control (72.16±6.39 s) and Tween-80 (0.5%) (66.50±5.02 s) groups (P>0.05). In addition, no significant differences were observed on the second phase of pain in the control (208.50±15.21 s) and Tween-80 (0.5%) (196.66±14.43 s) groups (P>0.05) (Figure 1). Therefore, the data obtained from the experimental groups were compared with vehicle treated group. 
The EOZC at dose of 10 mg/kg did not show any significant effect on both first and second phases of formalin pain in comparison with vehicle treated group (P>0.05). However, 20 and 40 mg/kg of EOZC induced a significant reduction in the pain response compared to the vehicle group in a dose-dependent manner in both first (39.16±3.80 s and 31.33±3.76 s, respectively) and second (121.66±10.44 s and 108.50±11.87 s, respectively) phases (P<0.05). As expected, the standard drug morphine (5 mg/kg) significantly decreased the nociceptive response in both first (10.33±2.61 s) and second (87.00±8.05 s) phases of formalin test, compared to vehicle treated group (P<0.05) (Figure 2).
Based on the findings, naloxone (2 mg/kg) alone had no significant effects on both phases of formalin test (P>0.05). In addition, pretreatment with naloxone (2 mg/kg) significantly reversed antinociception by EOZC (20 mg/kg) in the first (58.50±3.77 s) and second (176.00±10.25 s) phases of formalin test (P<0.05) (Figure 3).
4. Discussion
The present study investigated the antinociceptive effect of EOZC and possible involvement of opioidergic system on EOZC-induced antinociception in male rats using formalin test. Herbs and plants are widely used in traditional medicine to treat numerous illnesses due to their potentially positive effects (Sofiabadi et al., 2014). Numerous experiments have explored analgesic effects of medicinal plants (Riedel, Marrassini, Anesini, & Gorzalczany, 2015). 
To our knowledge, this is the first report on the interaction of antinociceptive effect of the EOZC and opioidergic system by the formalin test in rats. Two phases of pain were evoked by formalin injection into the hind paw of the animals. Each phase of formalin test has different mechanisms of nociception. The first phase consists of neurogenic nociception, by direct stimulation of nociceptors (via C fibers) to the dorsal horn of the spinal cord after substance P is secreted and acts as a neurotransmitter. The second phase consists of inflammatory-induced 

 

 

 

malin test (Shibata, Ohkubo, Takahashi, & Inoki, 1989), while peripherally acting drugs exert an inhibition only on the second phase of formalin test (Elisabetsky, Amador, Albuquerque, Nunes, & Carvalho Ado, 1995). The IP injection of EOZC revealed a dose-dependent antinociceptive effect on both phases of formalin-induced nociception in rats. Considering the effectiveness of EOZC in suppression of paw licking time in both phases of the formalin test, it seems that the analgesic activity of Z. 
clinopodioides is mediated by both peripheral and central antinociceptive mechanisms. Based on the GC-MS results, EOZC contained high concentrations of phenolic compounds including carvacrol (65.22%), thymol (19.51%), p-cymene (4.86%) and γ-terpinene (4.63%). Several studies have reported the chemical composition of EOZC (Aghajani et al., 2008; Behravan et al., 2007; Ozturk & Ercisli, 2007).
It has been shown that flavonoids and polyphenolic compounds possess a great variety of pharmacological properties 

 

including antioxidant activity (Altiok, Altiok, & Tihminlioglu, 2010), immunomodulatory activity (Lima et al., 2012), inhibition of histamine release from mast cells (Amresh, Reddy, Rao, & Singh, 2007a), and suppression of prostaglandin synthesis (Amresh, Zeashan, Rao, & Singh, 2007b). Prior investigations suggested an antinociceptive activity for carvacrol (Guimarães et al., 2010), thymol (Beer, Lukanov, & Sagorchev, 2007), p-cymene (De Sousa, 2011), and γ-terpinene (Hajhashemi, Sajjadi, & Zomorodkia, 2011) in the model of formalin-induced licking. Furthermore, it is reported that thymol partially blocks voltage-operated Na+channels and directly activates Cl- currents via GABAA receptors (Haeseler et al., 2002; Mohammadi et al., 2001). 
It was also expressed that thymol reversibly inhibited prostaglandin synthesis, probably related to the analgesic effect of thymol in endodontic therapy (Sarmento-Neto, do Nascimento, Felipe, & de Sousa, 2016). There is also evidence that the antinociceptive effects of carvacrol are partly related to antioxidant activity and its scavenging activity on NO and other Reactive Oxygen Species (ROS) (Guimarães et al., 2010). Accordingly, based on the above-mentioned findings and given that these 4 compounds are among the predominant components of EOZC, it can be concluded that the antinociceptive property of EOZC might be at least in part due to the presence of these chemical compounds.
Morphine was recognized as an effective inhibitor of both phases of formalin pain. Morphine and other opioid analgesics are used for alleviating pain. The opioidergic system consists of 3 receptors including µ, δ and κ which are located in the central nervous system and throughout the peripheral tissues (Trescot, Datta, Lee, & Hansen, 2008). Studies report that the endogenous opioidergic system and its receptors take part in many functions, e.g. behavior, pain and analgesia, stress, tolerance and dependence, learning and memory, alcohol and substance abuse, respiratory control, locomotion, seizures, neurological disorders and neuroendocrine physiology (Bodnar, 2016).
Naloxone (an opioid receptor antagonist) was evaluated in the formalin test to explore the effect of endogenous opioidergic system on the antinociception mechanism exerted by EOZC. Naloxone is a competitive antagonist of µ, δ and κ receptors, with a high affinity for the µ receptor (Trescot et al., 2008). Our findings revealed that the antinociception caused by the EOZC was significantly attenuated by the pretreatment of rats with naloxone (2 mg/kg), thus it reverses the analgesic activity of EOZC, to some extent.
The obtained results suggest that the constituents in the EOZC may act through opioidergic pathway to produce antinociceptive activity. However, further investigation is required to elucidate the underlying cellular and molecular signaling pathways. 
Ethical Considerations
Compliance with ethical guidelines
Animal experiments used in this study were approved by the Animal Ethics Committee of Razi University and followed with the Guidelines for the Care and Use of Laboratory Animals in Research (Ethics code: 396-2-012).
Funding
This research was supported by a grant from the Research Council of the Faculty of Veterinary Medicine, Razi University, Iran.
Authors contributions
Authors contribution is as follows: Faezeh Mohammadifard operated the experimental procedure and animal handling. Samad Alimohammadi participated in acquisition and analysis of behavioral studies data, statistical analysis, project supervision and preparation of the manuscript.
Conflicts of interest
The authors certify that they have no affiliation with or involvement in any organization or entity with any financial interest, or non-financial interest in the subject matter or materials discussed in this manuscript.
Acknowledgements
Authors gratefully acknowledge the financial support of Razi University. We would like to express our appreciation to Dr. Shahin Hassanpour for his assistance.


References