Cardamonin attenuates hyperalgesia and allodynia in a mouse model of chronic constriction injury-induced neuropathic pain: Possible involvement of the opioid system

Author’s Accepted Manuscript Cardamonin attenuates hyperalgesia and allodynia in a mouse model of chronic constriction injuryinduced neuropathic pain: Possible involvement of the opioid system Yogesvari Sambasevam, Ahmad Akira Omar Farouk, Tengku Azam Shah Tengku Mohamad, Mohd Roslan Sulaiman, B.Hemabarathy Bharatham, Enoch Kumar Perimal

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S0014-2999(16)30791-9 http://dx.doi.org/10.1016/j.ejphar.2016.12.020 EJP70982

To appear in: European Journal of Pharmacology Received date: 11 November 2016 Revised date: 14 December 2016 Accepted date: 14 December 2016 Cite this article as: Yogesvari Sambasevam, Ahmad Akira Omar Farouk, Tengku Azam Shah Tengku Mohamad, Mohd Roslan Sulaiman, B.Hemabarathy Bharatham and Enoch Kumar Perimal, Cardamonin attenuates hyperalgesia and allodynia in a mouse model of chronic constriction injury-induced neuropathic pain: Possible involvement of the opioid system, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.12.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cardamonin attenuates hyperalgesia and allodynia in a mouse model of chronic constriction injury-induced neuropathic pain: Possible involvement of the opioid system Yogesvari Sambasevama, Ahmad Akira Omar Farouka, Tengku Azam Shah Tengku Mohamada, Mohd Roslan Sulaimana, B.Hemabarathy Bharathamb, Enoch Kumar Perimala* a

Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. b Biomedical Science Programme, School of Diagnostic and Applied Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia. *Corresponding author: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: 603-89472774; Fax:03-89472774. [email protected]

ABSTRACT Neuropathic pain arises from the injury of nervous system. The condition is extremely difficult to be treated due to the ineffectiveness and presence of various adverse effects of the currently available drugs. In the present study, we investigated the antiallodynic and antihyperlagesic properties of cardamonin, a naturally occurring chalcone in chronic constriction injury (CCI)-induced neuropathic pain mice model. Our findings showed that single and repeated dose of intra-peritoneal administration of cardamonin (3, 10, 30 mg/kg) significantly inhibited (P<0.001) the chronic constriction injuryinduced neuropathic pain using the Hargreaves plantar test, Randall-Selitto analgesiometer test, dynamic plantar anesthesiometer test and the cold plate test in comparison with the positive control drug used (amitriptyline hydrochloride, 20mg/kg, i.p.). Pre-treatment with naloxone hydrochloride (1mg/kg, i.p.) and naloxone methiodide (1mg/kg, s.c) significantly reversed the antiallodynic and antihyperalgesic effects of cardamonin in dynamic plantar anesthesiometer test and Hargreaves plantar test, respectively. In conclusion, the current findings demonstrated novel antiallodynic and antihyperalgesic effects of cardamonin through the activation of the opioidergic system both peripherally and centrally and may prove to be a potent lead compound for the development of neuropathic pain drugs in the future.

Keywords: Cardamonin, Hyperalgesia, Allodynia, CCI, Neuropathic pain

Chemical compounds studied in this article

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Cardamonin (PubChem CID: 10424762); Dimethyl sulphoxide (PubChem CID: 679); Iodine (PubChem CID: 410087); Naloxone hydrochloride (PubChem CID: 5464092); Naloxone methiodide (PubChem CID: 16219719); Morphine sulphate (PubChem CID: 6321225); Amitriptyline hydrochloride (PubChem CID: 11065), Tribromoethanol (PubChem CID: 6400)

1. Introduction International Association for the Study of Pain (IASP) defined pain related to injury of both peripheral and central nervous system as neuropathic pain (Campbell and Meyer, 2006; Moalem and Tracey, 2006; Treede et al., 2008). According to general population studies, 7-8% of adults are affected with neuropathic signs and symptoms arising from various diseases (van Hecke et al., 2014). Clinically, patients suffering from painful diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, multiple sclerosis and cancer are usually present with neuropathic pain characteristics (Baron et al., 2010). Some of the characteristics of neuropathic pain are allodynia and hyperalgesia (Jensen and Finnerup, 2014).These symptoms have a significant impact on the individual’s quality of life, social and economic wellbeing (Leung and Cahill, 2010).

To date, neuropathic pain remains very challenging to manage and cure due to the complex pathophysiological underlying mechanisms (Attal et al., 2010; Campbell and Meyer, 2006). Pharmacological treatments such as anticonvulsants (gabapentin, carbamazepine), antidepressants (amitriptyline, nortriptyline) and opioid analgesics (morphine, tramadol) are used as the first line treatment to provide relieve to patients diagnosed with neuropathic conditions (Baron et al., 2010; O'Connor and Dworkin, 2009). Despite their potential benefits, all these drugs often exhibit various side and adverse effects such as drowsiness, constipation, cognitive impairment and addiction (Attal et al., 2010). Therefore, there is an urge to search for compounds that could provide relief from neuropathic pain symptoms with minimal or no side effects. In line with that, one of the natural compounds that have been found to exhibit promising therapeutic effects is cardamonin.

Cardamonin (2’, 4’-dihydroxy-6’-methoxychalcone) (Fig. 1) is a naturally occurring chalcone, primarily isolated from the fruits or seeds of Alpinia species (Bheemasankara Rao et al., 1976) and rhizomes of other zingiberous plant species. Previous studies have shown that cardamonin has several medicinal

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benefits such as anti-inflammatory (Ahmad et al., 2006; Hatziieremia et al., 2006; Israf et al., 2007; Lee et al., 2006; Li et al., 2015), anti-mutagenic (Trakoontivakorn et al., 2001), anti-tumor activity (Murakami et al., 1993), in vitro anti-HIV (Tewtrakul et al., 2003), vasorelaxation effects via nitric-oxide mediated processes (Wang et al., 2001) and hypoglycemic activity (Yamamoto et al., 2011). Importantly, studies have postulated that cardamonin potentially suppress inflammatory mediators such as tumor necrosis factor-α and nitric oxide (Ahmad et al., 2006), in which these mediators play major role in neuropathic pain mechanisms (Tracey and Walker, 1995). However, the mechanisms of action of cardamonin remain unclear.

The current study was carried out with the aim to investigate the antiallodynic and antihyperalgesic effects of cardamonin in the chronic constriction injury (CCI)-induced neuropathic pain model. It is also aimed to investigate the involvement of the opiodergic system in the cardamonin induced analgesia. The chronic constriction injury (CCI)-induced neuropathic pain animal model is used to mimic peripheral nerve injury (Bennett and Xie, 1988). In this model, the placement of loose ligatures around the sciatic nerve is recognized as foreign materials by the immune system, which then develops inflammatory reaction and thus, leads to the development of neuropathic pain (Cui et al., 2000; Orhan et al., 2010).

Fig. 1. Structure of cardamonin (2′,4′-dihydroxy-6′-methoxychalcone)

2. Materials and methods

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2.1 Materials Cardamonin or 2′, 4′-dihydroxy-6′-methoxychalcone with the purity of ≥ 98%, tribromoethanol, dimethyl sulphoxide (DMSO), Tween 20, iodine solution, naloxone hydrochloride, naloxone methiodide and morphine sulphate was purchased from Sigma-Aldrich (St.Louis, MO, USA). The BRILON non-absorbable surgical suture was purchased from Vigilenz Medical Devices Sdn. Bhd. and silk surgical suture was purchased from DemeTech (USA).

2.2 Experimental animals Male ICR mice (25-35g) were used in this study. The mice were housed at 20-24°C under 12 hours light-dark cycles and were allowed free access to water and food. Experiments were conducted in accordance with Ethical Guidelines for Investigation of Experimental Pain in Conscious Animals as issued by the International Association for the Study of Pain (IASP). The protocol and procedure of the experiment were approved by the Institutional Animal Care and Use Committee (IACUC) UPM (Ref: UPM/IACUC/AUP-R012/2015).

2.3 Chronic constriction injury-induced neuropathic pain model Chronic constriction injury (CCI) surgical procedures were performed following the method described (Bennett and Xie, 1988) with minor modifications. Mice were first anesthetized with an intraperitoneal dose of tribromoethanol (250 mg/kg, i.p.). Under aseptic conditions, the left thigh was shaved and a small incision was made in the middle of the left thigh. The biceps femoris and the gluteus superficialis muscles were separated and the common sciatic nerve was exposed. The nerve was loosely ligated with a single ligature using a silk suture until the mouse elicited a brief twitch in the respective hind limb. The skin was sutured by the non-absorbable surgical suture and iodine was applied externally. In sham-operated animals, the nerve will be exposed but not ligated.

2.4 Preparation of compound and drugs Cardamonin was freshly prepared by dissolving in dimethylsulfoxide (DMSO), Tween 20 and distilled water at a ratio of 5:5:90 (v/v). Different dosage of cardamonin were prepared (3, 10 and 30mg/kg) and were administered intraperitoneally into the mice on day 14 post-surgery and

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continued for another 7 days daily. Amitriptyline hydrochloride (20mg/kg), morphine sulphate (5mg/kg), naloxone hydrochloride (1mg/kg) and naloxone methiodide (1mg/kg) were dissolved in normal saline (0.99% NaCl).

2.5 Experimental design The animals were randomly allocated into groups of 6 mice each. The experimental groups are shown in Table 1 while the experimental timeline is shown in Fig. 2.

Table 1: Experimental design. Experimental groups

Dose

Experimental conditions

Sham

-

Without nerve ligature and no treatment

Vehicle (mL/kg, i.p.)

10

Subjected to CCI and treated with vehicle

3 Cardamonin (mg/kg, i.p.)

10 30

Amitriptyline hydrochloride

20

Subjected to CCI and treated with the respective doses of cardamonin Subjected to CCI and treated with amitriptyline

(mg/kg, i.p.)

Fig. 2. Experimental timeline

2.6 Measurement of pain threshold The development of allodynia and hyperalgesia were evaluated by using dynamic plantar anesthesiometer (UgoBasile Italy), Hot/Cold plate (UgoBasile, Italy), Randall-Selitto

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analgesiometer (IITC, CA, USA) and Hargreaves plantar test (UgoBasile Italy). The presence of allodynia and hyperalgesia was assessed on day 0 (before CCI surgery), day 14 (after CCI surgery) and day 21 as shown in Fig. 2. The first administration of treatments was carried out on day 14 after pre-treatment behavioural assessments, and continued for 7 consecutive days till day 21. All behavioural assessments were carried out 30 min after receiving vehicle (10 mL/kg, i.p.),cardamonin (3, 10, 30mg/kg,i.p.) and amitriptyline hydrochloride (20mg/kg, i.p.). The shamoperated group did not receive any treatment.

2.7 Behavioural observation The behaviour of the mice in each group was observed in terms of gait, posture and mobility focusing on both the ipsilateral and contralateral paws. The behaviour of CCI-induced neuropathic pain mice was compared with the sham-operated mice.

2.8 Mechanical allodynia Mechanical allodynia was assessed by using dynamic plantar anesthesiometer (Orhan et al., 2010).The mice were placed in a Plexiglas chamber on top of a wire mesh grid. Tactile stimulus was applied three times to each paw with 5 min interval in between and the mean was calculated. The nociceptive response was defined as brisk paw withdrawal, flinching, lifting or licking of hind paw. Pain response was recorded in as gram force eliciting withdrawal response.

2.9 Cold allodynia Animals were placed on the hot/cold plate and the temperature was set at 5 °C (Bennett and Xie, 1988). The numbers of paw withdrawals of both contralateral and ipsilateral paws were recorded for 5 min. The withdrawal score was counted using the formula below (Zulazmi et al., 2015). Numbers of ipsilateral paw elevation – Number of contralateral paw elevation

2.10 Mechanical hyperalgesia Mechanical hyperalgesia was assessed by using the Randall-Selitto analgesiometer (Randall et al., 1957; Santos-Nogueira et al., 2012). The tested paws of mice were carefully held and immobilized with soft cotton cloth. The tip of the device was applied to the medial plantar surface

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and gradually increased the pressure. The nociceptive endpoint was characterized by first paw withdrawal. The cut-off of 200g was set to avoid skin damage and to prevent extreme pain.

2.11 Thermal hyperalgesia Thermal sensitivity of the hind paws was measured by using the plantar test (Hargreaves et al., 1988). Animals were placed in a Plexiglas chamber and allowed to acclimatize for about 10 min. The heat stimulus was applied from beneath, to the middle of the plantar surface by means of a radiant heat source using Hargreaves machine. Paw lifting or licking in response to the stimuli was considered positive. Thermal sensitivity was recorded once per trial as withdrawal latency in seconds.

2.12 Involvement of opioid system The involvement of the opioid system in cardamonin-induced antiallodynic and antihyperalgesic effects was investigated based on (Ming-Tatt et al., 2013) with slight modification. Mice (n=6) were pre-treated with non-selective opioid receptor antagonist, naloxone hydrochloride (1mg/kg, i.p.) or naloxone methiodide (1mg/kg, s.c) (Hervera et al., 2013) which was administered 15 min prior to administration of cardamonin (10mg/kg, i.p.) or morphine sulphate (5mg/kg, i.p.), and the pain response was evaluated using Hargreaves plantar test and dynamic plantar anesthesiometer.

2.13 Data analysis The data obtained were expressed as mean ± S.E.M. and statistically analysed by using GraphPad Prism v5.0 software (GraphPad San Diego, CA). Statistical differences between groups were analysed by One-way Analysis of Variance (ANOVA) followed by Tukey’s post-hoc test. The statistical difference between day 14 and day 21 for each group is assessed using Two-way Analysis of Variance (ANOVA) followed by Bonferroni’s test. The results were considered significant at P<0.05.

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3

Results 3.1 Behavioural observation CCI-induced neuropathic pain mice demonstrated impairment in gait and posture compared to sham-operated mice. It was observed that after surgery, the affected ipsilateral limb was lifted repetitively and the paw was remained close together compared to the contralateral paw in CCIinduced neuropathic pain mice. General behaviour and social interactions did not change compared with sham-operated mice. Autotomy condition did not occur in any of the limbs. Shamoperated mice did not show any changes on both ipsilateral and contralateral limbs. Body weight remained normal in all groups.

3.2 Effects of cardamonin on thermal and mechanical hyperalgesia induced by CCI 3.2.1 Hargreaves Plantar Test Fig. 3 shows the changes in response to painful heat stimuli in plantar test in sham operated mice and CCI-induced neuropathic pain mice administered with vehicle, cardamonin (3, 10, 30 mg/kg) and amitriptyline hydrochloride (20mg/kg) on day 14 and day 21 post-surgery. Baseline for thermal hyperalgesia was recorded before surgery and pharmacological testing. The marked decrease in the ipsilateral paw withdrawal latencies of CCI-induced neuropathic pain mice shows the development of thermal hyperalgesia in response to the heat stimuli two weeks after surgery when compared to sham-operated mice. The withdrawal latencies of the ipsilateral paw of shamoperated mice as well as contralateral paw of CCI-induced neuropathic pain and sham-operated mice were not modified (data not shown). Intraperitoneal injection of cardamonin in different dosages (3,10, 30 mg/kg) and amitriptyline hydrochloride (20mg/kg) on day 14 and followed by daily treatment until day 21 demonstrated significant and dose-dependent inhibition of thermal hyperalgesia in CCI-induced neuropathic pain mice. All the doses of cardamonin exhibited pronounced significant inhibition (P<0.001) of neuropathic pain when compared to vehicle group. Besides that, doses of 3mg/kg, 10mg/kg and 30mg/kg of cardamonin has significant difference in the withdrawal latencies (P<0.01) when compared between day 14 and day 21. Amitriptyline hydrochloride showed significant inhibition (P<0.001) of thermal hyperalgesia when compared to vehicle and between day 14 and day 21. The effective dose (ED50) calculated for Hargreaves plantar test was approximately 10.12 mg/kg.

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3.2.2 Randall-Selitto test Fig. 4 shows the changes in response to painful mechanical stimuli in Randall-Selitto test in sham operated mice and CCI-induced neuropathic pain mice administered with vehicle, cardamonin (3, 10, 30 mg/kg)and amitriptyline hydrochloride (20mg/kg) on day 14 and day 21 post-surgery. Baseline for mechanical hyperalgesia was recorded before surgery and pharmacological testing. The marked decrease in the ipsilateral paw withdrawal threshold of CCI-induced neuropathic pain mice shows the development of mechanical hyperalgesia in response to the mechanical stimuli two weeks after surgery when compared to sham-operated mice. The withdrawal threshold of the ipsilateral paw of sham-operated mice as well as contralateral paw of CCI-induced neuropathic pain and sham-operated mice were not modified (data not shown). Intraperitoneal injection of cardamonin different dosages (3, 10, 30mg/kg) and amitriptyline hydrochloride (20mg/kg) on day 14 and followed by daily treatment until day 21 exhibited significant antihyperalgesic effects (P<0.001) in CCI-induced neuropathic pain mice when compared to vehicle group. Amitriptyline hydrochloride produced significant inhibition of mechanical hyperalgesia when compared to the vehicle group (P<0.001). The effective dose (ED50) obtained for Randall-Selitto test was approximately 9.86mg/kg.

3.3 Effects of cardamonin on mechanical and thermal allodynia induced by CCI 3.3.1 Dynamic Plantar Anesthesiometer Test Fig. 5 shows the changes in response to painful mechanical stimuli in dynamic plantar test in sham operated mice and CCI-induced neuropathic pain mice administered with vehicle, cardamonin (3, 10, 30 mg/kg)and amitriptyline hydrochloride (20 mg/kg) on day 14 and day 21 post-surgery. Baseline for mechanical allodynia was recorded before surgery and pharmacological testing. The marked decrease in the ipsilateral paw withdrawal threshold of CCIinduced neuropathic pain mice shows the development of mechanical allodynia in response to the tactile stimuli two weeks after surgery when compared to sham-operated mice. The withdrawal threshold of the ipsilateral paw of sham-operated mice as well as contralateral paw of CCIinduced neuropathic pain and sham-operated mice were not modified (data not shown). Intraperitoneal injection of cardamonin in different dosages (3, 10, 30mg/kg) and amitriptyline hydrochloride (20mg/kg) on day 14 and followed by daily treatment until day 21 caused significant

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and dose-dependent inhibition of mechanical allodynia in CCI-induced neuropathic pain mice. All cardamonin doses produced significant antiallodynic (P<0.001) properties in CCI-induced neuropathic pain mice when compared to vehicle group. In addition, cardamonin doses at 3mg/kg and 10mg/kg show significant differences (P<0.01 and P<0.05, respectively) when compared between day 14 and day 21. Amitriptyline hydrochloride significantly attenuates mechanical allodynia when compared to the control group. The effective dose (ED50) obtained for dynamic plantar anesthesiometer test was approximately 10.22mg/kg.

3.3.2 Cold plate test

Fig. 6 shows the changes in response to painful cold stimuli in cold plate test in sham operated mice and CCI-induced neuropathic pain mice administered with vehicle, cardamonin (3, 10, 30mg/kg) and amitriptyline hydrochloride (20mg/kg) on day 14 and day 21 post-surgery. Baseline for cold allodynia was recorded before surgery and pharmacological testing. The marked increased number of paw elevation of the ipsilateral paw of CCI-induced neuropathic pain mice shows the development of cold allodynia in response to the cold stimuli two weeks after surgery when compared to sham-operated mice. There was no paw elevation observed in the ipsilateral and contralateral paw of sham-operated mice. Intraperitoneal injection of cardamonin in different dosages (3, 10, 30mg/kg) and amitriptyline hydrochloride (20mg/kg) on day 14 and followed by daily treatment until day 21 caused significant inhibition (P<0.001) of cold allodynia in CCI-induced neuropathic pain mice when compared to vehicle group. The statistical analysis also showed that there is significant difference between day 14 and day 21 in cardamonin dosage of 3mg/kg, 10mg/kg and 30mg/kg. Amitriptyline hydrochloride showed significant inhibition of cold allodynia when compared to the vehicle treated group. The effective dose (ED50) obtained for cold plate was approximately 9.67mg/kg.

3.4 Involvement of opioid system Fig. 7 (Panel A and Panel B) show the pre-treatment of naloxone hydrochloride, a non-selective opioid receptor antagonist significantly reversed the antihyperalgesic and antiallodynic effects of cardamonin and morphine in Hargreaves plantar test and dynamic plantar anesthesiometer test,

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respectively. Fig. 8 (Panel A and Panel B) shows that pre-treatment with naloxone methiodide, a peripherally acting non-selective opioid antagonist, reversing the antihyperalgesic and antiallodynic properties of cardamonin and morphine.

Fig. 3.: Paw withdrawal latency values measured using Hargreaves plantar test for the effect of cardamonin on CCI-induced neuropathic pain in mice on day 14 and day 21. Each column represents the mean ± S.E.M. obtained from six mice. ***P<0.001 when compared with the respective vehicle group; ##P <0.01, ###P<0.001 when compared between day 14 and day 21 in each group.

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Fig. 4.:Paw withdrawal threshold values measured using Randall-Selitto test for the effect of cardamonin on CCI-induced neuropathic pain in mice on day 14 and day 21. Each column represents the mean ± S.E.M. obtained from six mice. ***P<0.001 when compared with the respective vehicle group; ##P<0.01 when compared between day 14 and day 21 in each group.

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Fig. 5.: Paw withdrawal threshold values measured using dynamic plantar anesthesiometertest for the effect of cardamonin on CCI-induced neuropathic pain in mice on day 14 and day 21. Each column represents the mean ± S.E.M. obtained from six mice. ***P<0.001 when compared with the respective vehicle group; #P<0.05, ##P<0.01 when compared between day 14 and day 21 in each group.

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Fig. 6.: Number of paw lifting scores measured using cold plate test for the effect of cardamonin on CCI-induced neuropathic pain in mice on day 14 and day 21. Each column represents the mean ± S.E.M. obtained from six mice. ***P<0.001 when compared with the respective vehicle group; #P<0.05, ###P<0.001 when compared between day 14 and day 21 in each group.

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A)

B)

Fig. 7.: Paw withdrawal latency values measured using Hargreaves plantar test (Panel A) and paw withdrawal threshold values measured using Dynamic Plantar Anesthesiometer test (Panel B) for the effect of pre-treatment with naloxone hydrochloride on CCI-induced neuropathic pain in mice on day 14 post-surgery. Each column represents the mean ± S.E.M. obtained from six mice. ***P<0.001 when compared to vehicle; ###P<0.001 when compared to cardamonin or morphine treated group. VEH=vehicle; NLX=naloxone hydrochloride; CDN=cardamonin; MOR=morphine

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A)

B)

Fig. 8.: Paw withdrawal latency values measured using Hargreaves plantar test (Panel A) and paw withdrawal threshold values measured using Dynamic Plantar Anesthesiometer test (Panel B) for the effect of pre-treatment with naloxone methiodide on CCI-induced neuropathic pain in mice on day 14 post-surgery. Each column represents the mean ± S.E.M. obtained from six mice. ***P<0.001 when compared to vehicle; ###P<0.001 when compared to cardamonin or morphine treated group. VEH=vehicle; NLX MET=naloxone methiodide; CDN=cardamonin; MOR=morphine

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4

Discussion Chronic constriction injury-induced neuropathic pain animal model provide an experimental basis for preclinical studies in order to investigate and evaluate new lead compounds (Clark et al., 2013; Colleoni and Sacerdote, 2010). It is one of the most reliable neuropathic animal models used in preclinical testing (Bennett and Xie, 1988; Costa et al., 2005). This model mimics neuropathic pain symptoms observed in humans with nerve damage such as hyperalgesia and allodynia (Bennett and Xie, 1988; Campbell and Meyer, 2006). As commonly known, hyperalgesia is an increased response to painful stimuli, while allodynia is pain response to a stimulus that does not usually elicits pain (Moalem and Tracey, 2006). In this model, the sciatic nerve will be ligated with silk suture to develop nerve injury. The ligatures were recognised as foreign materials by the immune system setting the ground for the inflammatory process to take place in this model (George et al., 2005).

Nerve compression causes Wallerian degeneration, in which the axon and myelin sheath undergoes degradation and causes immune and inflammatory cells infiltration such as mast cells, neutrophils and macrophages (Moalem and Tracey, 2006; Scholz and Woolf, 2007). This in turn causes the pro-inflammatory cytokines, inflammatory mediators and neurotrophic factors to be secreted abnormally causing the increased excitability of nociceptive afferent fibers. This leads to peripheral sensitization (Cui et al., 2000; Nickel et al., 2012; Tracey and Walker, 1995). Neuropathic pain is also being maintained by the central sensitization, in which the ectopic activity of afferent fibres releases excitatory amino acids and neuropeptides in the dorsal horn of the spinal cord. This mechanism causes phosphorylation and upregulation of receptors and ion channels such as voltage-gated sodium channels (Hains et al., 2004). Briefly, the relapse of inflammatory mediators and cytokines, sensitization of both the peripheral and central systems are the main contributors to the development of neuropathic pain in the model used. It is nerve injury and the accompanying changes outlined above that promote the development of the neuropathic pain symptoms (Sommer and Kress, 2004) similar to the ones observed in clinical subjects.

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The present study provides the first ever evidence of the antiallodynic and antihyperalgesic properties of cardamonin administered via intraperitoneal route and evaluated using various pain stimuli. Single and repeated administration of cardamonin (3, 10, 30mg/kg) exhibited a significant dose-dependent inhibition of the CCI-induced neuropathic pain in ICR mice. The antiallodynic and antihyperalgesic effects of cardamonin were investigated using the Hargreaves plantar, RandallSelitto, dynamic plantar and cold plate tests on day 14 and day 21 after seven days daily treatment. A significant reduction in pain response towards heat, tactile and cold stimuli in the cardamonin-administered mice indicates that this compound exhibit significant antiallodynic and antihyperalgesic effects. The correlation between single and repeated administration of cardamonin was also investigated. Based on our findings, the significant differences towards pain stimuli observed between the cardamonin treated groups on day 14 and day 21 indicates better therapeutic outcomes on repeated dose. The anti-inflammatory and antinociceptive effects produced by cardamonin have been previously reported (Ahmad et al., 2006; Israf et al., 2007; Park et al., 2014). In accordance with these findings, cardamonin possibly attenuate neuropathic pain by inhibiting the inflammatory mediators and cytokines partly by inhibiting the downstream intracellular signalling pathway. This can be further supported by the fact that cardamonin attenuate the PBQ-induced writhing and carrageenan-induced hyperalgesia in animals (Li et al., 2015; Park et al., 2014) This study also reveals, for the first time that the analgesic effects of cardamonin in CCIinduced neuropathic pain are probably dependent of activation of opioidergic system. This was based on the pre-treatment with naloxone hydrochloride, preventing an increase in the paw withdrawal latency and paw withdrawal threshold in Hargreaves plantar test and dynamic plantar anesthesiometer test, respectively. These indicate that the antihyperalgesic and antiallodynic effects of cardamonin are mediated, at least in part, by an interaction with central opioid system. On the other hand, the pre-treatment of mice with naloxone methiodide reversed the analgesic effects of cardamonin in both behavioural assessments. Naloxone methiodide does not penetrate the blood brain barrier thereby suggesting that the antihyperalgesic and antiallodynic effects of cardamonin are mediated through the peripherally located opioid receptors. The current findings confirmed the participation of both central and peripheral opioid receptors in the cardamonin-

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induced antinociception, supported by similar outcome with morphine in the CCI-induced neuropathic pain mice. In consistent with the findings obtained, cardamonin has been suggested to be classified under the large family of flavonoids (Li et al., 2015; Wang et al., 2001). Surprisingly, flavonoids have been speculated to possess analgesic properties via activation of opioid receptors (Higgs et al., 2013). In line with this, it is proposed that cardamonin may possibly act as opioid agonist by activating the opioidergic pathway to attenuate chronic constriction injury-induced hyperalgesia and allodynia in mice. Three are types of opioid receptors known as mu-, kappa, and delta-opioid receptors widely distributed in central and peripheral nervous system (Kieffer and Gaveriaux-Ruff, 2002; Ossipov et al., 2004).Previous studies have shown that opioid agonists are able to elicit potent analgesic effect peripherally and centrally by activating opioid receptors of animals and humans (Mousa et al., 2007; Rau et al., 2005; Stein and Lang, 2009; Stein et al., 2003). Upon activation by opioid agonist at the receptor, conformational changes lead to coupling of G-protein to the receptors. This action subsequently leads to decrease of cyclic adenosine monophosphate (cAMP), which then alters the ion channels such as potassium channels and voltage-gated calcium channels. Consequently, the potassium current will be increased, giving rise to hyperpolarisation while the calcium current is inhibited (Stein et al., 2003). This in turn attenuate the excitability of nociceptive fibres and neurotransmitter release, hence leading to analgesic effects (Stein et al., 2001). The ability of cardamonin in halting the mechanical and thermal stimuli further indicates the involvement of both peripheral and central regions. The increased response towards heat stimuli is known as thermal hyperalgesia. This occurs at the site of nerve injury and thought to be partially mediated by peripheral sensitization which involved the primary afferent fibers and nociceptors. Other than that, the light-stroking stimuli give rise to allodynia, in which it is associated with uninjured territory surrounding the site of injury and then elicited due to the central sensitization (Campbell and Meyer, 2006). Further mechanisms in which cardamonin mediates antihyperalgesic and antiallodynic effects in neuropathic pain models are still unclear. However, based on our findings, the reversal effect of cardamonin by both the centrally and peripherally acting opioid antagonist, suggest the significant involvement of the opioid receptors located on the

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primary afferent nerve as well as spinal and supraspinal regions through the central nervous system.

5

Conclusion Based on our current findings, we conclude that cardamonin possesses antiallodynic and antihyperalgesic properties in the CCI-induced neuropathic pain animal model, with possible participation of opioid receptors at both the peripheral and central regions. Other underlying mechanisms of cardamonin in attenuating neuropathic pain are currently being investigated in our laboratory. Based on literature, the analgesic effects demonstrated by cardamonin in the CCIinduced neuropathic pain could be possibly attributed to the anti-inflammatory properties of cardamonin in suppressing the immune and inflammatory mechanisms caused by the ligation of sciatic nerve, and the inhibition of the abnormal excitability of injured and non-injured neurons and central sensitization.

Acknowledgments The authors thank the staffs at the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, and the Physiology and Pharmacology and Toxicology Laboratories for providing the necessary support for this study. This research was supported by the Universiti Putra Malaysia Grant (GP-IPM/2014/9433700).

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