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Journal of Ethnopharmacology 137 (2011) 345– 351

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l h o me page: www.elsev ier .com/ locate / je thpharm

ntinociceptive effect of the essential oil of Zingiber zerumbet in mice: Possibleechanisms

ohamed Hanief Khalida, Muhammad Nadeem Akhtarc, Azam Shah Mohamadb,noch Kumar Perimalb, Ahmad Akirab, Daud Ahmad Israfb,c, Nordin Lajis c,d, Mohd Roslan Sulaimanb,c,∗

Department of Human Anatomy, Universiti Putra Malaysia, Serdang, MalaysiaDepartment of Biomedical Science, Universiti Putra Malaysia, Serdang, MalaysiaInstitute of Bioscience, Universiti Putra Malaysia, Serdang, MalaysiaDepartment of Chemistry, Universiti Putra Malaysia, Serdang, Malaysia

r t i c l e i n f o

rticle history:eceived 12 February 2011eceived in revised form 12 May 2011ccepted 26 May 2011vailable online 6 June 2011

eywords:ingiber zerumbet (L.) Smithssential oilingiberaceaentinociceptiveitric oxideyclic GMPotassium channels

a b s t r a c t

Ethnopharmacological relevance: Zingiber zerumbet (L.) Smith, a wild edible ginger species or locallyknown as “lempoyang”, commonly used in the Malays traditional medicine as an appetizer or to treatstomachache, toothache, muscle sprain and as a cure for swelling sores and cuts.Aim: The present study was conducted to investigate the possible mechanism of actions underlying thesystemic antinociception activity of the essential oil of Zingiber zerumbet (EOZZ) in chemical-inducednociception tests in mice.Materials and methods: Acetic acid-induced abdominal constriction, capsaicin-, glutamate- and phorbol12-myristate 13-acetate-induced paw licking tests in mice were employed in the study. In all experi-ments, EOZZ was administered systemically at the doses of 50, 100, 200 and 300 mg/kg.Results: It was shown that EOZZ given to mice via intraperitoneal and oral routes at 50, 100, 200and 300 mg/kg produced significant dose dependent antinociception when assessed using acetic acid-induced abdominal writing test with calculated mean ID50 values of 88.84 mg/kg (80.88–97.57 mg/kg)and 118.8 mg/kg (102.5–137.8 mg/kg), respectively. Likewise, intraperitoneal administration of EOZZat similar doses produced significant dose dependent inhibition of neurogenic pain induced byintraplantar injection of capsaicin (1.6 �g/paw), glutamate (10 �mol/paw) and phorbol 12-myristate13-acetate (1.6 �g/paw) with calculated mean ID50 of 128.8 mg/kg (118.6–139.9 mg/kg), 124.8 mg/kg(111.4–139.7 mg/kg) and 40.29 (35.39–45.86) mg/kg, respectively. It was also demonstrated that pre-treatment with l-arginine (100 mg/kg, i.p.), a nitric oxide precursor significantly reversed antinociceptionproduced by EOZZ suggesting the involvement of l-arginine/nitric oxide pathway. In addition, methylene

blue (20 mg/kg, i.p.) significantly enhanced antinociception produced by EOZZ. Administration of gliben-clamide (10 mg/kg, i.p.), an ATP-sensitive K+ channel antagonist significantly reversed antinociceptiveactivity induced by EOZZ.Conclusion: Together, the present results suggested that EOZZ-induced antinociceptive activity was pos-sibly related to its ability to inhibit glutamatergic system, TRPV1 receptors as well as through activationof l-arginine/nitric oxide/cGMP/protein kinase C/ATP-sensitive K+ channel pathway.

. Introduction

The Zingiberaceae family is among the most prolific plants inhe tropical rainforests. In Peninsular Malaysia, approximately 160pecies from 18 genera are found, mostly growing naturally in

∗ Corresponding author at: Department of Biomedical Science, Faculty ofedicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor,alaysia. Tel.: +60 38947 2603; fax: +60 38947 2585.

E-mail addresses: mrs4969@gmail.com, mrs@medic.upm.edu.myM.R. Sulaiman).

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.05.043

© 2011 Elsevier Ireland Ltd. All rights reserved.

damp, shaded parts of the lowland or hill slopes, as scattered plantsor thickets (Larsen et al., 1999). In the Southeast Asian countries,several species are normally used as spices, medicines, flavouringagents, as well as the source of certain dyes (Burkill, 1966). Zingiberzerumbet (L.) Smith, locally known in Malaysia as “lempoyang” isone of the commonly used wild ginger species in Malays traditionalmedicine. The concoction of Zingiber zerumbet rhizomes is normallydrunk to treat indigestion, stomachache, fever and worm infesta-

tion. The young stems, rhizomes and inflorescence are also usedas a poultice for topical application to treat muscle sprain and asa cure for swelling sores. The juice extracted from the rhizomesor the cooked rhizomes are usually taken by post partum women

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r post surgical patients to improve appetite, enhance recovery orealing as well as to alleviate pain (Grant and Lutz, 2000; Habsaht al., 2000).

Our previous study showed that the rhizome essential oil ofingiber zerumbet (EOZZ) possessed both central and peripheralntinociceptive activity when tested using chemical and thermalodels of nociception (Sulaiman et al., 2010). In the same study,e also demonstrated that the antinociceptive effect of the EOZZ

t doses that did not produce any toxic and sedative effects, was sig-ificantly reversed by the non-selective opioid receptor antagonist,aloxone (Sulaiman et al., 2010). Although this result may suggesthat activation of opioid receptors were involved in the analgesic

echanism of EOZZ induced antinociception, however, the actualechanism of actions by which the EOZZ exerts its antinociceptive

ctivity remains to be elucidated. Moreover, we recently showedhat zerumbone, one of the major compounds found in the EOZZ,roduced its antinociception through the activation of nitric oxideNO)/cyclic guanosine monophosphate (cGMP)/protein kinase CPKC)/K+ channel pathways (Perimal et al., 2011). This has led tohe assumption that the mechanism of EOZZ-induced antinocicep-ion at least in part, involved the interaction of EOZZ with similarathways. Therefore, the present study was conducted to further

nvestigate possible mechanism(s) of action that participate in theOZZ-induced antinociception.

. Materials and methods

.1. Plant material and essential oil extraction

The rhizomes of Zingiber zerumbet were purchased from thehow Kit’s wet market, Kuala Lumpur, Malaysia in September 2007nd were subsequently identified by Mr. Shamsul Khamis, a resi-ent botanist at the Institute of Bioscience (IBS), Universiti Putraalaysia, (UPM), Serdang, Selangor, Malaysia. A voucher specimen

SK 622/07) was deposited at the Herbarium of the Laboratoryf Natural Products, IBS, UPM, Malaysia. A small part of the rhi-omes were cultivated in the Medicinal Plant Garden, Institutef Bioscience, Universiti Putra Malaysia for future reference. Thessential oil from the rhizomes of Zingiber zerumbet (EOZZ) wasbtained from the fresh rhizomes by hydrodistillation and wasnalyzed using a Shimadzu GC-17A gas chromatograph interfacedith a Shimadzu GCMS-QP5050A quadrupole mass spectrometer.ualitative analysis was based on a comparison of retention timesnd mass spectra with corresponding data in the literature andomputer mass spectra libraries (NIST and Wiley) as previouslyescribed (Sulaiman et al., 2010).

.2. Animals

Experiments were conducted using adult male ICR mice25–30 g) purchased from the Institute for Medical Research, Kualaumpur. They were housed at 22 ± 2 ◦C under 12 h light/dark cycleith access to food and water ad libitum. The mice were divided into

roup of 10 mice each, acclimatized and habituated to the labora-ory environment for at least one week prior to the experimentsnd were used only once throughout the experiments. Experi-ents reported in this study were performed in accordance with

he current guidelines for the care of laboratory animals and thethical guidelines for investigations of experimental pain in con-cious animals (Zimmermann, 1983), approved by the Animal Carenit Committee, Faculty of Medicine and Health Sciences, Univer-

iti Putra Malaysia. The number of animals and intensity of noxioustimuli used were minimum and just at necessary to demonstratehe consistent effects of the treatments. In all experiments, dataere collected by a blinded, randomized and controlled design.

rmacology 137 (2011) 345– 351

2.3. Drugs and chemicals

N�-nitro-l-arginine (l-NOARG), l-arginine hydrochloride (l-Arginine), l-glutamic acid, capsaicin, methylene blue, phorbol 12-myristate 13-acetate, glibenclamide, dimethyl sulfoxide (DMSO),acetylsalicylic acid (ASA) and Tween 20 were purchased from SigmaChemical Co. (USA). All drugs were dissolved in physiological saline(0.9% NaCl) and buffered to a pH of 7, while EOZZ and ASA weredissolved in DMSO, Tween 20 and saline in 5:5:90 (v/v) fractions,respectively. Respective controls received only solvent vehicle. Alldrugs, chemicals and the EOZZ solutions were freshly prepared andadministered intraperitoneally (i.p.) in volumes of 10 mL/kg unlessotherwise stated in the method. The vehicle alone had no effects perse on the nociceptive responses. The doses of EOZZ in the presentstudy were selected based on our previous study, whereby at thesedoses there were no toxic and sedative effects observed (Sulaimanet al., 2010).

2.4. Antinociceptive analysis

2.4.1. Acetic acid-induced abdominal writhing testThe procedure used was similar to that previously described

(Sulaiman et al., 2010). Mice were intraperitoneally or orally pre-treated with EOZZ (50, 100, 200, 300 mg/kg, i.p. or p.o.), 30 minprior to i.p. injection of 0.6% acetic acid (v/v). The control groupreceived a similar volume of vehicle (10 mL/kg, i.p.). Acetylsalicylicacid (ASA, 100 mg/kg, i.p. or p.o.) was used as the reference drug andadministered 30 min before the nociceptive agent. Following thei.p. injection of acetic acid, the animals were immediately placedinto a perspex chamber and the number of writhing was recordedfor 30 min, starting from 5 min post injection.

2.4.2. Capsaicin-induced nociceptionThe procedure used was similar to that previously described

(Meotti et al., 2006). Mice were treated with EOZZ (50, 100, 200, and300 mg/kg, i.p.), ASA (100 mg/kg, i.p.), capsazepine (0.17 mmol/kg,i.p.), or vehicle (10 mL/kg, i.p.) 30 min before intraplantar injectionof 20 �L of capsaicin (1.6 �g/paw) into the right hind paw. Themice were then observed individually for 5 min following capsaicininjection. The amount of time each mice spent licking its injectedpaw was timed with a chronometer as indication of nociception.

2.4.3. Glutamate-induced nociceptionThe experiment was carried out according to the method as

previously described (Beirith et al., 2002). 20 �L of glutamate(10 �mol/paw) was injected into the ventral surface of the righthind paw of the mice. The mice were observed for 15 min followingglutamate injection. The amount of time each mice spent licking itsinjected paw was timed with a chronometer as indicative of noci-ception. Mice were treated with EOZZ (50, 100, 200, and 300 mg/kg,i.p.), ASA (100 mg/kg, i.p.) or vehicle (10 mL/kg, i.p.) 30 min beforeglutamate injection.

2.4.4. Phorbol 12-myristate 13-acetate (PMA)-inducednociception

The experiment was conducted as previously described (Perimalet al., 2011; Siebel et al., 2004). 20 �L of PMA (0.03 �g/paw) wasinjected i.pl. into the ventral surface of the right hind paw of themice. The mice were observed individually from 15 to 45 min fol-lowing PMA injection. The amount of time each mice spent licking

the injected paw was timed with a chronometer as indicative ofnociception. The mice were treated with EOZZ (50, 100, 200, and300 mg/kg, i.p.), ASA (100 mg/kg, i.p.) or vehicle (10 mL/kg, i.p.)30 min before PMA injection.

nopharmacology 137 (2011) 345– 351 347

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Fig. 1. Effects of EOZZ (50, 100, 200, 300 mg/kg) administered intraperitoneally (A)and orally (B) against acetic acid-induced nociception. Each column represents themean ± S.E.M. of 10 mice. Control group (C) receives only the vehicle (DMSO: Tween

M.H. Khalid et al. / Journal of Eth

.5. Analysis of the possible mechanism of action of EOZZ

.5.1. Involvement of l-arginine/NO pathwayThe method used was according to previously described

Abacioglu et al., 2000; Perimal et al., 2011). The mice were pre-reated with l-arginine (100 mg/kg, i.p.), and after 15 min, the miceeceived EOZZ (200 mg/kg, i.p.), N�-nitro-l-arginine (l-NOARG,0 mg/kg, i.p.) or vehicle (10 mL/kg, i.p.). Nociceptive responsesgainst acetic acid injection were observed for 30 min. The numbersf abdominal writhing were counted as indication of pain behavior.

.5.2. Involvement of cyclic guanosine monophosphate (cGMP)athway

To verify the possible involvement of cGMP in the antinocicep-ive action caused by EOZZ, similar procedure previously describedas employed (Abacioglu et al., 2000; Perimal et al., 2011). The miceere pre-treated with methylene blue (a non specific inhibitor ofO/guanylyl cyclase, 20 mg/kg, i.p.) 15 min before administrationf EOZZ (200 mg/kg, i.p.). Nociceptive responses against acetic acidnjection were observed for 30 min. The numbers of abdominal

rithing were counted as indication of pain behavior.

.5.3. Involvement of ATP-sensitive K+ channel pathwayPossible contribution of K+ channel in the antinociceptive effect

f EOZZ was evaluated using the method previously describedMohamad et al., 2011; Perimal et al., 2011). The mice were pre-reated with glibenclamide (an ATP-sensitive K+ channel inhibitor,0 mg/kg, i.p.) 15 min before injection of either vehicle (10 mL/kg,

.p.) or EOZZ (200 mg/kg, i.p.). The mice were challenged with i.p.njection of 0.6% acetic acid, 30 min post-treatment. Following thenjection of acetic acid, the animals were immediately placed in

Perspex chamber and the number of writhing was recorded for0 min, starting from 5 min post injection.

.6. Statistical analysis

Results were expressed as mean ± standard error mean (S.E.M.)f 10 mice and analyzed by one-way ANOVA followed by Dunnett’sost hoc test unless otherwise stated. P < 0.05 was considered sig-ificant. The ID50 (dose that produced 50% inhibition in total timef paw licking) and 95% confidence intervals (CI) values were deter-ined by using linear regression and graphs were drawn by usingraphPad Prism 5 software.

. Results

.1. Acetic acid-induced abdominal writhing test

The result in Fig. 1 showed the effect of systemic adminis-ration of EOZZ (50, 100, 200, 300 mg/kg, i.p. or p.o.) in aceticcid-induced abdominal writhing test. EOZZ exhibited signifi-ant dose-dependent inhibition on abdominal writhing whendministered intraperitoneally with percentage of inhibition of3.02, 53.89, 83.63 and 98.57%. Similar dose dependent inhi-ition of EOZZ was also observed in mice administered orallyith percentage of inhibition of 13.04, 28.30, 54.69 and 75.68%.SA (100 mg/kg, i.p. or p.o.) also produced similar inhibition.he calculated mean ID50 for i.p. and p.o. administration ofOZZ were 88.8 mg/kg (80.88–97.57 mg/kg) and 118.8 mg/kg102.5–137.8 mg/kg), respectively. Since, the administration of

OZZ by i.p. route was more potent than p.o. route in preventingociception caused by acetic acid, thus the i.p. administration ofOZZ was selected as a route of choice for all further studies withndependent groups of animals.

20: saline in 5:5:90, v/v) used to dilute the oil. Acetylsalicylic acid (ASA) was used asthe positive control. The asterisks denote the significance levels compared with thecontrol group (one-way ANOVA, followed by Dunnett’s post hoc test): ***p < 0.001.

3.2. Capsaicin-induced nociception

The result from the capsaicin-induced nociception (Fig. 2)showed that administration of EOZZ (50, 100, 200, 300 mg/kg) viaintraperitoneal route produced dose-dependent inhibition of thecapsaicin-induced neurogenic nociception with percentage of inhi-bition of 8.75, 33.00, 73.40, and 97.64%, respectively. Only 50 mg/kgEOZZ treatment showed no significant difference compared withthe vehicle group, while the rest showed significant antinocicep-tive activity. Administration of capsazepine (TRPV1 antagonist)at 0.17 mmol/kg was comparable with 200 mg/kg EOZZ. The ID50value for EOZZ in capsaicin-induced nociception is 128.8 mg/kg(118.6–139.9 mg/kg). ASA and capsazepine (0.17 mmol/kg) wereused as positive control drugs showed similar significant inhibi-tion as compared to the control group with inhibition of 40.07 and62.29%, respectively.

3.3. Glutamate-induced nociception

The result from the glutamate-induced nociception (Fig. 3)showed that i.p. administration of EOZZ (50, 100, 200, 300 mg/kg)

produced dose-dependent inhibition of the glutamate-inducednociception with the percentage of inhibition observed of 11.27,41.70, 64.81, and 99.30%. ASA (100 mg/kg) was used as positivecontrol drug showed similar significant inhibition as compared

348 M.H. Khalid et al. / Journal of Ethnopharmacology 137 (2011) 345– 351

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Fig. 2. Effects of EOZZ administered intraperitoneally against capsaicin inducednociception. Each column represents the mean ± S.E.M. of 10 mice. Control group (C)receives only the vehicle used to dilute the oil. Capsazepine (CAP) is used as the posi-tive control for capsaicin induced nociception. Acetylsalicylic acid (ASA) was used astc

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Fig. 4. Effects of EOZZ administered intraperitoneally against PMA-induced noci-ception. Each column represents the mean ± S.E.M. of 10 mice. Control group (C)

and methylene blue (20 mg/kg) treatments (Fig. 6). EOZZ andmethylene blue administration alone significantly inhibited aceticacid induced abdominal writhing test. Given together, methylene

he positive control. The asterisks denote the significance levels compared with theontrol group (one-way ANOVA, followed by Dunnett’s post hoc test): ***p < 0.001.

o the control group at 63.88%. All treatments showed significantntinociceptive activity compared with the control group. The ID50alue for EOZZ in glutamate-induced nociception was 124.8 mg/kg111.4–139.7 mg/kg).

.4. Phorbol 12-myristate 13-acetate (PMA)-induced nociception

The i.p. administration of EOZZ (50, 100, 200, 300 mg/kg)lso produced a marked and dose-dependent inhibition of PMA-nduced paw licking in the mice (Fig. 4). The percentage ofnhibition observed for EOZZ treatments were 59.94, 80.42, 94.58,nd 100%. ASA (100 mg/kg) was used as positive control drughowed similar significant inhibition as compared to the controlroup at 56.93%. The mean ID50 value obtained was 40.29 mg/kg35.39–45.86 mg/kg) and the inhibition was 100% when adminis-

rated at 300 mg/kg.

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ig. 3. Effects of EOZZ administered intraperitoneally against glutamate inducedociception. Each column represents the mean ± S.E.M. of 10 mice. Control groupC) receives only the vehicle used to dilute the oil. Acetylsalicylic acid (ASA) was useds the positive control. The asterisks denote the significance levels compared withhe control group (one-way ANOVA, followed by Dunnett’s post hoc test): *p < 0.05,**p < 0.001.

receives only the vehicle used to dilute the oil. Acetylsalicylic acid (ASA) was used asthe positive control. The asterisks denote the significance levels compared with thecontrol group (one-way ANOVA, followed by Dunnett’s post hoc test): ***p < 0.001.

3.5. Involvement of l-arginine/NO pathway

The involvement of l-arginine/NO pathway (Fig. 5) was ana-lyzed with the pretreatment of nitric oxide precursor, l-arginine(100 mg/kg). l-arginine which was administered at a dose thatwas not significantly different to the control group was able tosignificantly reverse the antinociceptive activity exhibited by NOsynthase (NOS) inhibitor, l-NOARG (20 mg/kg), and 200 mg/kgEOZZ in acetic acid induced abdominal writhing test.

3.6. Involvement of cyclic guanosine monophosphate (cGMP)pathway

The present study also looked at the effects of 200 mg/kg EOZZ

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Fig. 5. Effects of pretreatment with l-arginine (100 mg/kg) on antinociceptive pro-files of EOZZ (200 mg/kg) and l-NOARG (20 mg/kg) against acetic acid-inducedabdominal writhing test in mice. Each column represents the mean ± S.E.M. of 10mice. ***p < 0.001 when compared to control group; ###p < 0.001 when comparedto l-NOARG or ##p < 0.01 when compared to EOZZ-treated group (one-way ANOVAfollowed by Dunnett’s post hoc test).

M.H. Khalid et al. / Journal of Ethnopha

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lue significantly enhanced EOZZ induced antinociception com-ared to the treatment of both EOZZ and methylene blue alone.

.7. Involvement of ATP-sensitive K+ channel pathway

It was also found that glibenclamide (10 mg/kg) administra-ion alone did not alter abdominal writhing count when assessedhrough the injection of 0.6% acetic acid (Fig. 7). Given together,ntinociceptive activity of EOZZ was significantly reversed bylibenclamide.

. Discussion

In the present study, we demonstrated that systemic admin-stration of EOZZ at the doses of 50, 100, 200 and 300 mg/kgvoked significant dose-dependent inhibition against chemical-nduced nociception test models. Another important additionalnding of the present study was the demonstration of the possible

nvolvement of the glutamatergic system, TRPV1 receptor and l-rginine/nitric oxide/cGMP/PKC/ATP-sensitive K+ channel pathwayn the EOZZ-induced antinociception in mice.

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ig. 7. Effects of glibenclamide (10 mg/kg) pretreatment on EOZZ (200 mg/kg)nduced antinociceptive activity in acetic acid induced abdominal writhing test.ach column represents the mean ± S.E.M. of 10 mice. Comparing glibenclamidend EOZZ treatment versus EOZZ treatment alone. ***p < 0.001 when compared toontrol group and ###p < 0.001 when compared to EOZZ-treated group (one-wayNOVA followed by Dunnett’s post hoc test).

rmacology 137 (2011) 345– 351 349

We recently described the antinociceptive effect of EOZZ inthe acetic acid-induced abdominal writhing test (Sulaiman et al.,2010). It was suggested that acetic acid, which was used to induceabdominal writhing, causes algesia by liberation of various endoge-nous substances that excite the peripheral nociceptors (Collieret al., 1968). Of particular interest, the results reported here againdemonstrated that i.p. administration of EOZZ produced significantdose dependent reduction in the number of abdominal writhinginduced by acetic acid. In addition, it was also demonstratedthat at the ID50 level, administration of EOZZ via i.p. route wasabout 1.3-fold more potent than that of p.o. route. These find-ings were consistent with our previous study (Sulaiman et al.,2010).

In the present study, the possible involvement of TRPV1receptor inhibition in the EOZZ-induced antinociception was inves-tigated. Our findings showed that EOZZ significantly inhibitedcapsaicin-induced nociceptive behavior in a dose-dependent man-ner. Furthermore, the effect of capsaicin was antagonized with aTRPV1 receptor antagonist, capsazepin. These findings suggest thatthe antinociceptive activity of EOZZ possibly involves the inhibitionof TRPV1 receptors. It is believed that capsaicin directly activates anon-selective ionotropic channel in primary sensory neurons, thecapsaicin receptor, also known as the transient receptor poten-tial vanilloid 1 (TRPV1) (Calixto et al., 2005; Julius and Basbaum,2001; Szallasi and Blumberg, 1999). It has been reported else-where that capsaicin also evokes the release of neuropepetides,excitatory amino acids (glutamate and aspartate), nitric oxide andpro-inflammatory mediators in the periphery and transmits noci-ceptive information to the spinal cord (Sakurada et al., 2003; Santosand Calixto, 1997).

In relation to the above, we also demonstrated the involvementof PKC in the antinociceptive activity of EOZZ. The i.p. administra-tion of EOZZ dose-dependently inhibited the nociception causedby intraplantar PMA injection (a PKC activator). PKC activation isan essential step for the nociceptive effects of numerous stimuli,including those that are caused by inflammatory mediators. PKCphosphorylates many cellular components, including membrane-bound receptors, ion channels and enzymes, which are knownto regulate the excitation of nociceptors (Ferreira et al., 2005).Peripheral introduction of PMA produces nociception, thermalhyperalgesia and mechanical allodynia in mice and rats. PMA, act-ing on PKC can directly stimulate TRPV1 channels leading to thepropagation of nociceptive impulses (Numazaki et al., 2002). Thisfinding can be linked to our earlier findings in which EOZZ wasfound to inhibit the activation of TRPV1 receptors by reducing thenociceptive response induced by intraplantar injection of capsaicin.We therefore suggest that EOZZ exerts its antinociceptive effect byinhibiting TRPV1 receptor activation through the inhibition of PKCphosphorylation.

Another interesting finding of the present study was the factthat i.p. administration of EOZZ produced a dose-dependent inhi-bition of the nociceptive response caused by i.pl. injection ofglutamate into the mouse hindpaw. It was reported that this noci-ceptive response caused by glutamate involves peripheral, spinaland supraspinal sites of action with glutamate receptors (AMPA,kainate and NMDA receptors) play an important role modulat-ing this nociceptive response (Beirith et al., 2002; Neugebauer,2001a,b). In addition, it has been previously reported that PKC isdirectly involved in the phosphorylation of AMPA receptors andindirectly involved in NMDA receptors (Ji and Woolf, 2001). Thisfurther suggests that the glutamatergic system is also affected bythe PKC activation which is in line with our earlier findings showing

the ability of EOZZ in inhibiting PMA-induced nociception. Thus,such results suggest that the antinociception induced by EOZZis associated with its interaction with the glutamatergic system.However, these findings are preliminary, and further studies are

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eing conducted to identify types of glutamate receptors that arenvolved in the EOZZ-induced antinociception.

Further experiments were conducted to investigate the partici-ation of l-arginine/NO/cyclic GMP pathway on the antinociceptiveroperties of EOZZ. NO is a diffusible gas that permeates cell mem-ranes and is not stored in vesicles. NMDA receptors activation

ncreases intracellular calcium, which in turn stimulates NOS toatalyze the substrate l-arginine to NO and l-citrulline (Mellert al., 1992; Schmidtko et al., 2009). NO seems to be involved inll three levels of pain pathway, which are the peripheral, dorsalorn of spinal cord and cerebral cortex where perception of pain isrocessed (Anbar and Gratt, 1997).

In the present study, it was found that the pretreatment with-arginine (a nitric oxide precursor) at a dose that did not pro-uce any significant changes to the acetic acid-induced nociception,ignificantly reversed antinociceptive activity caused by EOZZ.urthermore, treatment with l-NOARG (a nitric oxide synthasenhibitor) produced significant inhibition of the effect observedollowing l-arginine pretreatment but was significantly higherompared to l-NOARG treatment alone. These findings may possi-ly suggest the involvement of the l-arginine/nitric oxide pathway

n the antinociceptive activity of EOZZ. Previous studies haveeported that NOS inhibitors reduced nociception caused by aceticcid (Larson et al., 2000; Meotti et al., 2006; Moore et al., 1993).herefore, the inhibitory effect of l-NOARG observed in the presenttudy are in line with the notion that NO is one of the mediators ofcetic acid induced nociception, justifying the use of this model. Inddition, our findings are also coherent with previous studies whicheported that zerumbone, one of the major compounds present inOZZ demonstrated potent inhibition of NO (Chien et al., 2008;erimal et al., 2011).

In addition to the above findings, NO also increases the levelf cyclic guanosine monophosphate (cGMP) through the activationf soluble guanylyl cyclase (sGC), which influences a wide rangef physiological functions including pain and analgesia (Abacioglut al., 2000). cGMP acts on the ion channels directly or throughhe activation of protein kinases and phosphodiesterases (Xu et al.,995). In order to further prove the involvement/inhibition of cGMP

n EOZZ-induced antinociception, methylene blue (MB), a guanylylyclase and/or NOS inhibitor, was administered prior to inducingociception with i.p. injection of acetic acid. It was demonstratedhat the pretreatment of methylene blue significantly enhanced thentinociceptive effect exerted by EOZZ. It has been suggested thatB promotes antinociception by sequentially inhibiting periph-

ral NOS and GC, resulting in NO interference (Abacioglu et al.,000). The activation or deactivation of nociresponsive neuronsre dependent on the availability of cGMP. Intracellular cGMPoncentrations are regulated by the action of GCs and also byhe rate of degradation by cGMP-specific phosphodiesterases (Jaint al., 2001). Therefore, cGMP is very important for the func-ioning of nociceptors. Nitric oxide activates the sGC, which inurn catalyses the formation of cGMP from guanosine triphos-hate, whereas cyclic GMP-specific phosphodiesterase catalyseshe hydrolysis of cGMP to GMP, thus consequently ending the signalransduction (Pyne et al., 1996). Our finding that MB reduces l-rginine-induced antinociception is consistent with the inhibitoryffect of MB on GC and/or NOS. On the other hand, the combina-ion of EOZZ’s potent analgesic activity and its ability to inhibit the-arginine-induced NO and cGMP increase, may partially explainhe significant increase in the analgesic effect of MB (Ehab and ELesoky, 2005).

The present study also shows the participation of ATP-sensitive

+ channel inhibition in EOZZ-induced antinociception. Admin-

stration of ATP-sensitive K+ channel antagonist, glibenclamideignificantly reversed the antinociceptive activity evoked by EOZZ.here have been many reports on the fact that glibenclamide specif-

rmacology 137 (2011) 345– 351

ically blocked ATP-sensitive K+ channel, with no effects on othertypes of K+ channel such as Ca2+ activated K+ channel and voltage-gated potassium channels (Alves and Duarte, 2002; Jesse et al.,2007). Therefore, the present findings might suggest that EOZZexerted its antinociceptive activity through the opening of ATP-sensitive K+ channel that allows the efflux of K+ ion, thus leadingto membrane repolarization and/or hyperpolarization state whichreduces the membrane excitability (Lawson, 1996). Altogether, thepresent findings provide strong evidence on the participation ofl-arginine/NO/cGMP/ATP-sensitive K+ channel pathway in EOZZ-induced antinociceptive activity.

As a conclusion, EOZZ at doses that did produce any toxic andsedative effects provide conclusive evidence on the activation ofl-arginine/NO/cGMP/ATP-sensitive K+ channel pathway in EOZZ-induced antinociceptive activity, apart from its ability to interactand inactivate the TRPV1 receptor, inactivation of protein kinase Cand also the inhibition of the glutamatergic system. Further studiesinvolving the mechanism of actions of EOZZ are under way in ourlaboratory to fully comprehend the role of other receptors possiblyinvolved in the antinociceptive activity of EOZZ.

Acknowledgements

The authors thanked the Faculty of Medicine and Health Sci-ences and the Institute of Bioscience, Universiti Putra Malaysia,for providing necessary support for the study. This research wasfunded by Research University Grant Scheme 2009 (04-01-09-0780RU/F1). Special acknowledgement to Mr. Ramli Suhaimi forhis assistance in animal handling and management.

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