research article topical antinociceptive effect of ...in order to verify the possible involvement of...

Research ArticleTopical Antinociceptive Effect of Vanillosmopsis arboreaBaker on Acute Corneal Pain in Mice

Laura Heacutevila Inocecircncio Leite1 Gerlacircnia de Oliveira Leite1

Thales Silva Coutinho1 Severino Deniacutecio Gonccedilalves de Sousa1

Renata Souza Sampaio1 Joseacute Galberto Martins da Costa1

Irwin Rose Alencar de Menezes1 and Adriana Rolim Campos2

1 Programa de Pos-Graduacao em Bioprospeccao Molecular Universidade Regional do Cariri 63105-000 Crato CE Brazil2 Universidade de Fortaleza Avenida Washington Soares 1321 60811-905 Fortaleza CE Brazil

Correspondence should be addressed to Adriana Rolim Campos adrirolimuniforbr

Received 25 October 2013 Revised 7 December 2013 Accepted 19 December 2013 Published 10 February 2014

Academic Editor Olumayokun A Olajide

Copyright copy 2014 Laura Hevila Inocencio Leite et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

This study aimed to assess the possible topical antinociceptive activity of Vanillosmopsis arborea Baker essential oil (EOVA)and to clarify the underlying mechanism using the acute model of chemical (eye wiping) nociception in mice EOVA (25 to200mgkg po and topical) evidenced significant antinociception against chemogenic pain in the test model of formalin-inducedneuroinflammatory pain Local application of 5MNaCl solution on the corneal surface of the eye produced a significant nociceptivebehavior characterized by eye wipingThe number of eye wipes was counted during the first 30 s EOVA (25 50 100 and 200mgkgpo and topical) significantly decreased the number of eye wipes Naloxone yohimbine L-NAME theophylline glibenclamideand ruthenium red had no effect on the antinociceptive effect of EOVA However ondansetron p-chlorophenylalanine methylester (PCPA) capsazepine prazosin and atropine prevented the antinociception induced by EOVA These results indicate thetopical antinociceptive effect of EOVA and showed that 5-HT 1205721 TRPV1 and central muscarinic receptors might be involved inthe antinociceptive effect of EOVA in the acute corneal model of pain in mice

1 Introduction

The small size of the cornea and the extensive branching ofthe peripheral axons of corneal neurons make this structurethe most densely innervated tissue of the body [1] Themajority of corneal sensory fibers are polymodal nociceptorswhich are activated by noxious mechanical thermal andchemical stimuli [2]

Safe long-lasting pain relief following corneal abrasionscorneal ulcers or ophthalmic surgery is difficult to achievewith current analgesics The use of topical NSAIDs andacetominophen is constrained by their gradual onset andlimited efficacy and the adverse side effects of systemicopioids are well known [3]

Vanillosmopsis arborea Baker is native to the AraripeNational Forest in the Northeast of Brazil in the state ofCearaThere are few studies concerning the traditional use of

this plant However biological and pharmacological studieshave shown that its essential oil presents antimicrobialantiinflammatory and gastroprotective activities [4]

Topically applied Vanillosmopsis arborea essential oilproduced antinociception in acetic acid cyclophosphamidemustard oil capsaicin or formalin-induced visceral pain [5]The essential oil also suppressed the ear edema induced byCroton oil and phenol but no that one induced by histamineor capsaicin [6]

Ocular pain has not been adequately controlled in manypatients and there is a lack of studies that examine theeffect of Vanillosmopsis arborea on the perception of cornealpainTherefore the present study was designed to investigatethe effects of acute topical administration of Vanillosmopsisarborea on acute corneal pain that was induced by hypertonicsaline applied locally on the corneal surface in mice

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 708636 6 pageshttpdxdoiorg1011552014708636

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Pharmacologic agents used for test in order to verify the possible action mechanism of EOVA antinociceptive effect

Drug Target Dose(mgkg)

Administrationroute

Naloxone Opioid receptors 2 (11) IntraperitonealPrazosin 120572

1-receptors 015 (12) Intraperitoneal

Yohimbine 1205722-receptors 2 (11) Intraperitoneal

Theophylline Adenosine receptors 5 (13) IntraperitonealL-NAME NO synthesis 2 (11) IntraperitonealOndansetron 5-HT3 receptors 05 (14) IntraperitonealAtropine Muscarinic receptors 01 (15) IntraperitonealGlibenclamide K+ATP channels 2 (16) IntraperitonealRuthenium red TRPV1 receptors 5 (13) Intraperitoneal(noncompetitive antagonist) 3 (11 17 18) SubcutaneousCapsazepine (competitiveantagonist) 5 (16) Intraperitoneal

2 Material and Methods

21 Essential Oil The essential oil from Vanillosmopsisarborea Baker bark (EOVA) was obtained from the Nat-ural Products Research Laboratory of Regional Universityof Cariri The composition (ww) of EOVA revealed thepresence of 120572-bisabolol to the extent of 70 Other identifiedcompounds were 120572-cadinol (84) elemicin (621) 120573-bisabolene (446) 120575-guaiene (231) 120573-cubebene (176)and estragole (108)

22 Animals Male Swiss albino mice (20ndash25 g) obtainedfrom the Central Animal House of Regional University ofCariri were used They were housed in environmentallycontrolled conditions (22∘C 12 h light-dark cycle) with freeaccess to standard pellet diet (Purina Sao Paulo Brazil) andwater Animalswere kept in cageswith raised floors to preventcoprophagy The experimental protocols were in accordancewith the ethical guidelines of National Institute of HealthBethesda USA

23 Formalin-Induced Paw Licking The formalin-inducednociception was performed as described previously by Hun-skaar and Hole [7] Groups of mice (119899 = 8) were treatedwith vehicle (10mLkg po) EOVA (25 50 100 or 200mgkgpo or topical) or morphine (75mgkg sc) 30 or 60minbefore the administration of 20 120583L of 1 formalin (in 09saline) into the plantar surface of the right hind paw Theduration of paw licking (s) as an index of painful responsewas determined at 0ndash5min (early phase neurogenic) and 20ndash25min (late phase inflammatory) after formalin injection

24 Eye Wiping Test Corneal nociception was induced by alocal application of hypertonic saline to the corneal surface[8 9] One drop (40120583L) of 5M NaCl solution was appliedlocally on the corneal surface of mice using a fine dropper[10] The number of eye wipes performed with the ipsilateralforepaw was counted for a period of 30 s Topical (20120583Leye)or orally (10mLkg) EOVA (25 50 100 or 200mgkg 119899 =

8group) or vehicle were given 1 h before the noxiou agentMorphine 7mgkg (sc 119899 = 8) was used as positive controlA normal control group (119899 = 8) received one drop of 015MNaCl (09)

In order to verify the possible involvement of opioidnoradrenergic nitrergic 5-HT

3 muscarinic K+ATP adenosin-

ergic and TRPV1systems in the effect of EOVA the animals

(119899 = 8group) were treated with the respective antagonist(Table 1) 30min before the topical administration of EOVA(50mgkg) The doses of antagonists were chosen based onprevious studies

To assess the possible contribution of endogenous sero-tonin animals (119899 = 8group) were pretreated with 120588-chlo-rophenylalanine methyl esther (PCPA 100mgkg ip aninhibitor of serotonin synthesis) or with vehicle once a dayfor 4 consecutive days Then 24 hours after the last PCPA orvehicle injection animals received EOVA (50mgkg) and 1 hlater they were tested in the eye wiping test

25 Statistical Analysis All data are presented as meanplusmn sem The data were evaluated by one-way analysis ofvariance with Student-Newman-Keuls post hoc test usingthe GraphPad Prism 40 statistical program The level ofsignificance was set at 119875 lt 005

3 Results and Discussion

In formalin test pretreatment with EOVA (oral and top-ical) caused significant diminutions of both first phase(neurogenic) and second phase (inflammatory) nocicep-tion responses (Tables 2(a) and 2(b)) Morphine (5mgkg)the reference standard also significantly suppressed theformalin-response at both phases

Topically administered EOVA (25 50 100 or 200mgkg)respectively (119875 lt 0001 119875 lt 001 119875 lt 001 and 119875 lt00001 resp) decreased the number of eye wipes inducedby the local application of 5M NaCl solution on the cornealsurface (Table 3(a)) Oral treatment with EOVA also reducedthe number of eye wipes (Table 3(b))

Evidence-Based Complementary and Alternative Medicine 3

Table 2 (a) Effect of topical EOVA on formalin test in mice (b)Effect of oral EOVA on formalin test in mice

(a)

Group Dose (mgkg) Paw licking time (s)1st phase 2nd phase

Control mdash 9963 plusmn 595 4175 plusmn 807

EOVA

25 1068 plusmn 1003 1967 plusmn 883lowastlowastlowastlowast

50 8183 plusmn 653lowastlowastlowastlowast

466 plusmn 427lowastlowastlowastlowast

100 4217 plusmn 863lowastlowastlowastlowast1833 plusmn 683

lowastlowastlowastlowast



Morphine 75 1983 plusmn 433lowastlowastlowastlowast1383 plusmn 456


Data are expressed as mean plusmn sem lowastlowastlowastlowast119875 lt 00001 compared to controlgroup ANOVA followed by Student-Newman-Keuls test

(b)



EOVA












EOVA was evaluated for topical antinociceptive activityin mice using experimental models of chemogenic nocicep-tion EOVA (oral and topical) was effective in attenuatingacute neurogenic and tonic inflammatory phases of theformalin response The essential oil given topically eliciteda dose-unrelated antinociceptive effect on the paw-lickingresponse just as observed in the eye wiping test

The local application of a 5MNaCl solution to the cornealsurface produced corneal nociception Previous work hasshown that the application of hypertonic saline to the tongueand cornea transiently activates nociceptive neurons withwide dynamic range property in the trigeminal subnucleuscaudalis [11] Moreover infusion of hypertonic saline into themasseter muscle produces hind paw shaking in addition toactivating c-Fos positive neurons in the ipsilateral trigeminalsubnucleus caudalis [12] The results presented here are inagreement with previous findings [8 9]

Study of trigeminal pain and analgesic effects on trigem-inal acute pains such as headache muscle spasms dentalproblems or postsurgery pain seems to bemore problematicThe cornea is used for nociception studies in trigeminalsystem [13] since corneal nociceptive receptors have a largerepresentation in the trigeminal ganglion through the oph-thalmic branch of trigeminal nerve [14] Thin myelinatedfibres [15] as well as unmyelinated fibers in cornea respondto chemical mechanical and thermal noxious stimuli [16]In rat wiping the eye with forelimb is an obvious withdrawalresponse to corneal chemical stimuli Some researchers haveused eye wiping test for investigating the chemical pungency[17] or the presence of C-fiber activity [18 19] Eye wiping test

Table 3 (a) Effect of topically applied EOVA on the cornealnociception induced by a local application of a 5M NaCl solutionin mice (b) Effect of orally administered EOVA on the cornealnociception induced by a local application of a 5M NaCl solutionin mice

(a)

Group Dose (mgkg) Number of eye wipes (30 s)Control mdash 2033 plusmn 066

EOVA

25 1533 plusmn 091lowastlowastlowast

50 1667 plusmn 084lowastlowast



Morphine 75 1217 plusmn 083lowastlowastlowastlowast

Data are expressed as mean plusmn sem lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 andlowastlowastlowastlowast119875 lt 00001 compared to control group ANOVA followed by Student-

Newman-Keuls test(b)


EOVA





Morphine 75 1188 plusmn 083lowastlowastlowast


Newman-Keuls test

is a phasic analgesic test and is sensitive to centrally actinganalgesics Hypertonic saline-induced corneal pain has beenintroduced as a model of acute pain for study of mechanismsof pain in the trigeminal system in rats [8]

The results of this investigation provide evidence thatthe essential oil from V arborea bark is topically activein the attenuation of corneal pain induced by 5M NaClPrevious study showed the local antiinflammatory effect ofthe essential oil from Vanillosmopsis arborea It was observedthat EOVA effect can be related to release of leukotrienesdecrease the production of inflammatory eicosanoids andinfluence on the production of AA metabolites [6]

This antinociceptive effect may be related to the high 120572-bisabolol content in EOVA since that 120572-bisabolol possess vis-ceral antinociceptive activity [20] and it is able to reduce theneuronal excitability in a concentration-dependent manner[21]

In the present study we attempted to characterize furthersome of the mechanisms through which EOVA exerts itsantinociceptive action in chemical model of corneal pain inmice

The antinociceptive effect induced by the EOVA(50mgkg) was significantly inhibited by ondansetronPCPA prazosin atropine and capsazepine (Tables 45 6 7 and 8) On the other hand the administrationof glibenclamide naloxone ruthenium red yohimbineLNAME or theophylline did not prevent the EOVA-inducedantinociception (Tables 7 9 10 11 12 and 13)


Table 4 Effect of pretreatment with ondansetron on the EOVA-induced corneal antinociception in mice


OEVA 50 833 plusmn 105lowastlowastlowast

Ondansetron 05 500 plusmn 051lowastlowastlowastlowast

+OEVA 50 1250 plusmn 191

Data are expressed as mean plusmn sem lowastlowastlowast119875 lt 0001 and lowastlowastlowastlowast119875 lt 00001compared to control group ANOVA followed by Student-Newman-Keulstest

Table 5 Effect of pretreatment with 120588-chlorophenylalanine methylesther (PCPA) on the EOVA-induced corneal antinociception inmice



PCPA 100 912 plusmn 083

+OEVA 50 1413 plusmn 035lowastlowastlowast

Data are expressed as mean plusmn sem lowastlowastlowast119875 lt 0001 compared to controlgroup ANOVA followed by Student-Newman-Keuls test

Serotoninergic neurons also play a crucial role in thecontrol of pain [22] and the diversity of subtype receptors forserotonin makes this system able to exert either facilitatoryor inhibitory function [23] Spinal 5-HT

3receptors have been

shown tomediate antinociception possibly via GABA release[24 25] Concerning the mechanism through which EOVAexerts its antinociceptive action the present study shows thatthe 5-HT

3receptor is likely involvedThis conclusion derives

from the fact that pretreatment of animals with the 5-HT3

antagonist ondansetron reversed the antinociception causedby EOVA

In addition the treatment of mice with tryptophanhydroxylase inhibitor (PCPA) at a dose known to decreasethe cortical content of serotonin and to significantly reversemorphine antinociception [26 27] attenuated the effect ofEOVA indicating the involvement of 5-HT in the antinoci-ceptive effect of EOVA

In the present study the involvement of 1205721-receptors in

the antinociceptive action of EOVA is suggested by the resultsshowing that pretreatment of animals with prazosin (an 120572

1-

receptor antagonist) significantly blocked the antinociceptioncaused by EOVA Prazosin a former selective 120572

1-receptor

antagonist has high affinity for1205722B-receptors [28] It has been

suggested that the effects of prazosin against spinal 120572 agonistsappear through 120572

2B-receptors although yohimbine interactswith 120572

2A-receptor site as well as 1205722B-receptor site [29]The roles of acetylcholine cholinergic agonists and

cholinesterase inhibitors collectively termed cholinomimet-ics have been established in the modulation of pain andanalgesia [30] Here we show that the pretreatment of animalswith atropine prevented the antinociceptive effect inducedby EOVA This result indicates that the muscarinic receptorsare involved in EOVA-induced antinociceptive effect It isalready well established that the analgesic effect of systemicmorphine is mediated by a descending cholinergic pathway

Table 6 Effect of pretreatment with prazosin on the EOVA-inducedcorneal antinociception in mice


EOVA 50 833 plusmn 105lowastlowastlowast

Prazosin 015 1233 plusmn 080

+EOVA 50 1333 plusmn 095


Table 7 Effect of pretreatment with atropine or glibenclamide onthe EOVA-induced corneal antinociception in mice



Atropine 01 1083 plusmn 087lowastlowastlowastlowast


Glibenclamide 2 666 plusmn 095lowastlowastlowastlowast

+EOVA 50 1150 plusmn 084lowastlowastlowast


Table 8 Effect of pretreatment with capsazepine on the EOVA-induced corneal antinociception in mice


EOVA 50 1100 plusmn 057lowast

Capsazepine 5 1067 plusmn 076lowast


Data are expressed as mean plusmn sem lowast119875 lt 005 compared to control groupANOVA followed by Student-Newman-Keuls test

Table 9 Effect of pretreatment with naloxone on the EOVA-induced corneal antinociception in mice


EOVA 50 1317 plusmn 079lowastlowastlowastlowast

+Naloxone 2 1150 plusmn 088lowastlowastlowastlowast


Table 10 Effect of pretreatment with ruthenium red on the EOVA-induced corneal antinociception in mice



Ruthenium red 3 583 plusmn 233lowastlowast

+EOVA 50 516 plusmn 030lowastlowast

Data are expressed as mean plusmn sem lowast119875 lt 005 and lowastlowast119875 lt 001 compared tocontrol group ANOVA followed by Student-Newman-Keuls test

[31] aswell as spinal endogenous acetylcholine acting throughmuscarinic receptors [32ndash35]


Table 11 Effect of pretreatment with yohimbine on the EOVA-induced corneal antinociception in mice


EOVA 50 1229 plusmn 077lowastlowast

Yohimbine 2 800 plusmn 166lowastlowastlowast


Data are expressed asmeanplusmn sem lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 comparedto control group ANOVA followed by Student-Newman-Keuls test

Table 12 Effect of pretreatment with L-NAME on the EOVA-induced corneal antinociception in mice



L-NAME 10 1750 plusmn 084

+EOVA 50 1283 plusmn 065lowastlowastlowastlowast


Table 13 Effect of pretreatment with theophylline on the EOVA-induced corneal antinociception in mice



Theophylline 5 733 plusmn 206lowastlowastlowastlowast



Ruthenium red (a noncompetitive TRPV1antagonist) did

not affect the antinociceptive effect of EOVA However cap-sazepine (a competitive TRPV

1channel antagonist) inhibited

this response indicating that EOVA interacts directly withTRPV

1receptors may contribute to this antinociception

In summary we have demonstrated that topical EOVAreduces the nociceptive behavior in models of formalin-induced paw licking and eye wiping in mice In the cornealpain model the observed antinociception is possibly medi-ated by 5-HT 120572

1 muscarinic and TRPV

1receptors

Conflict of Interests

There is no conflict of interests

Acknowledgments

This research was supported by the grants and fellowshipsfrom CNPq CAPES and FUNCAP Brazil

References

[1] L J Muller C F Marfurt F Kruse and TM T Tervo ldquoCornealnerves structure contents and functionrdquo Experimental EyeResearch vol 76 no 5 pp 521ndash542 2003

[2] C Belmonte M C Acosta and J Gallar ldquoNeural basis of sen-sation in intact and injured corneasrdquoExperimental Eye Researchvol 78 no 3 pp 513ndash525 2004

[3] K Wishaw D Billington D OrsquoBrien and P Davies ldquoThe useof orbital morphine for postoperative analgesia in pterygiumsurgeryrdquoAnaesthesia and Intensive Care vol 28 no 1 pp 43ndash452000

[4] A V Colares F Almeida-Souza N N Taniwaki et al ldquoInvitro antileishmanial activity of essential oil of Vanillosmopsisarborea (Asteraceae) bakerrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 727042 7 pages2013

[5] G D O Leite R S Sampaio L H I Leite et al ldquoAttenuationof visceral pain in mice by the essential oil from Vanillosmopsisarborea barkrdquo Revista Dor vol 12 no 1 pp 46ndash49 2011

[6] G D O Leite L H I Leite R D S Sampaio et al ldquoMod-ulation of topical inflammation and visceral nociception byVanillosmopsis arborea essential oil in micerdquo Biomedicine andPreventive Nutrition vol 1 no 3 pp 216ndash222 2011

[7] S Hunskaar and K Hole ldquoThe formalin test in mice dissocia-tion between inflammatory and non-inflammatory painrdquo Painvol 30 no 1 pp 103ndash114 1987

[8] R Farazifard F Safarpour V Sheibani and M Javan ldquoEye-wiping test a sensitive animal model for acute trigeminal painstudiesrdquo Brain Research Protocols vol 16 no 1ndash3 pp 44ndash492005

[9] E Tamaddonfard E Khalilzadeh N Hamzeh-Gooshchi and SSeiednejhad-Yamchi ldquoCentral effect of histamine in a rat modelof acute trigeminal painrdquo Pharmacological Reports vol 60 no2 pp 219ndash224 2008

[10] S Kasture and S Ingale ldquoEvaluation of analgesic activity ofthe leaves of Passiflora incarnata Linnrdquo International Journal ofGreen Pharmacy vol 6 no 1 pp 36ndash39 2012

[11] E Carstens N Kuenzler and H O Handwerker ldquoActivationof neurons in rat trigeminal subnucleus caudalis by differentirritant chemicals applied to oral or ocular mucosardquo Journal ofNeurophysiology vol 80 no 2 pp 465ndash492 1998

[12] J Y Ro N F Capra J S Lee R Masri and Y H ChunldquoHypertonic saline-induced muscle nociception and c-fos acti-vation are partially mediated by peripheral NMDA receptorsrdquoEuropean Journal of Pain vol 11 no 4 pp 398ndash405 2007

[13] I DMeng JWHu A P Benetti andD A Bereiter ldquoEncodingof corneal input in two distinct regions of the spinal trigem-inal nucleus in the rat cutaneous receptive field propertiesresponses to thermal and chemical stimulation modulationby diffuse noxious inhibitory controls and projections to theparabrachial areardquo Journal of Neurophysiology vol 77 no 1 pp43ndash56 1997

[14] C de Felipe G G Gonzalez J Gallar and C BelmonteldquoQuantification and immunocytochemical characteristics oftrigeminal ganglion neurons projecting to the cornea effect ofcorneal woundingrdquo European Journal of Pain vol 3 no 1 pp31ndash39 1999

[15] C Belmonte J Gallar M A Pozo and I Rebollo ldquoExcitationby irritant chemical substances of sensory afferent units in thecatrsquos corneardquo Journal of Physiology vol 437 pp 709ndash725 1991

[16] J Gallar M A Pozo R P Tuckett and C Belmonte ldquoResponseof sensory units with unmyelinated fibres to mechanical ther-mal and chemical stimulation of the catrsquos corneardquo Journal ofPhysiology vol 468 pp 609ndash622 1993

[17] J Lee J Lee J Kim et al ldquoN-(3-acyloxy-2-benzylpropyl)-N1015840-(4-hydroxy-3-methoxybenzyl)thiourea derivatives as potent


vanilloid receptor agonists and analgesicsrdquo Bioorganic andMedicinal Chemistry vol 9 no 1 pp 19ndash32 2001

[18] R Farazifard R Kiani M Noorbakhsh and H EstekyldquoEffects of neonatal C-fiber depletion on the integration ofpaired-whisker inputs in rat barrel cortexrdquo Experimental BrainResearch vol 162 no 1 pp 115ndash121 2005

[19] L Karai D C Brown A J Mannes et al ldquoDeletion ofvanilloid receptor 1-expressing primary afferent neurons forpain controlrdquo Journal of Clinical Investigation vol 113 no 9 pp1344ndash1352 2004

[20] G D O Leite L H I Leite R D S Sampaio et al ldquo(minus)-120572-bisabolol attenuates visceral nociception and inflammation inmicerdquo Fitoterapia vol 82 no 2 pp 208ndash211 2011

[21] M A Ade J C R Goncalves J S Cruz and D A M AraujoldquoEvaluation of the sesquiterpene (minus)-120572-bisabolol as a novelperipheral nervous blockerrdquo Neuroscience Letters vol 472 no1 pp 11ndash15 2010

[22] H L Fields M M Heinricher and P Mason ldquoNeurotrans-mitters in nociceptive modulatory circuitsrdquo Annual Review ofNeuroscience vol 14 pp 219ndash245 1991

[23] L Bardin J Lavarenne and A Eschalier ldquoSerotonin receptorsubtypes involved in the spinal antinociceptive effect of 5-HTin ratsrdquo Pain vol 86 no 1-2 pp 11ndash18 2000

[24] A A Alhaider S Z Lei and G L Wilcox ldquoSpinal 5-HT3receptor-mediated antinociception possible release of GABArdquoJournal of Neuroscience vol 11 no 7 pp 1881ndash1888 1991

[25] M J Millan ldquoDescending control of painrdquo Progress in Neurobi-ology vol 66 no 6 pp 355ndash474 2002

[26] L A Pini M Sandrini and G Vitale ldquoThe antinocicep-tive action of paracetamol is associated with changes in theserotonergic system in the rat brainrdquo European Journal ofPharmacology vol 308 no 1 pp 31ndash40 1996

[27] A R S Santos R O P de Campos O G Miguel V Cechinel-Filho R A Yunes and J B Calixto ldquoThe involvement of K+channels and G(io) protein in the antinociceptive action of thegallic acid ethyl esterrdquo European Journal of Pharmacology vol379 no 1 pp 7ndash17 1999

[28] D B Bylund C Ray-Prenger and T J Murphy ldquoAlpha-2A andalpha-2B adrenergic receptor subtypes antagonist binding intissues and cell lines containing only one subtyperdquo Journal ofPharmacology and Experimental Therapeutics vol 245 no 2pp 600ndash607 1988

[29] Y Takano and T L Yaksh ldquoCharacterization of the phar-macology of intrathecally administered alpha-2 agonists andantagonists in ratsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 261 no 2 pp 764ndash772 1992

[30] P G Jones and J Dunlop ldquoTargeting the cholinergic system asa therapeutic strategy for the treatment of painrdquoNeuropharma-cology vol 53 no 2 pp 197ndash206 2007

[31] C Y Chiang and M Zhuo ldquoEvidence for the involvementof a descending cholinergic pathway in systemic morphineanalgesiardquo Brain Research vol 478 no 2 pp 293ndash300 1989

[32] S R Chen and H L Pan ldquoSpinal endogenous acetylcholinecontributes to the analgesic effect of systemic morphine in ratsrdquoAnesthesiology vol 95 no 2 pp 525ndash530 2001

[33] R Radhakrishnan andKA Sluka ldquoSpinalmuscarinic receptorsare activated during low or high frequency TENS-inducedantihyperalgesia in ratsrdquo Neuropharmacology vol 45 no 8 pp1111ndash1119 2003

[34] K Honda S Ando K Koga and Y Takano ldquoThe spinalmuscarinic receptor subtypes contribute to the morphine-induced antinociceptive effects in thermal stimulation in micerdquoNeuroscience Letters vol 371 no 2-3 pp 235ndash238 2004

[35] Y P Chen S R Chen and H L Pan ldquoSystemic morphineinhibits dorsal horn projection neurons through spinal cholin-ergic system independent of descending pathwaysrdquo Journal ofPharmacology and ExperimentalTherapeutics vol 314 no 2 pp611ndash617 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Pharmacologic agents used for test in order to verify the possible action mechanism of EOVA antinociceptive effect

Drug Target Dose(mgkg)

Administrationroute

Naloxone Opioid receptors 2 (11) IntraperitonealPrazosin 120572

1-receptors 015 (12) Intraperitoneal

Yohimbine 1205722-receptors 2 (11) Intraperitoneal

Theophylline Adenosine receptors 5 (13) IntraperitonealL-NAME NO synthesis 2 (11) IntraperitonealOndansetron 5-HT3 receptors 05 (14) IntraperitonealAtropine Muscarinic receptors 01 (15) IntraperitonealGlibenclamide K+ATP channels 2 (16) IntraperitonealRuthenium red TRPV1 receptors 5 (13) Intraperitoneal(noncompetitive antagonist) 3 (11 17 18) SubcutaneousCapsazepine (competitiveantagonist) 5 (16) Intraperitoneal

2 Material and Methods

21 Essential Oil The essential oil from Vanillosmopsisarborea Baker bark (EOVA) was obtained from the Nat-ural Products Research Laboratory of Regional Universityof Cariri The composition (ww) of EOVA revealed thepresence of 120572-bisabolol to the extent of 70 Other identifiedcompounds were 120572-cadinol (84) elemicin (621) 120573-bisabolene (446) 120575-guaiene (231) 120573-cubebene (176)and estragole (108)

22 Animals Male Swiss albino mice (20ndash25 g) obtainedfrom the Central Animal House of Regional University ofCariri were used They were housed in environmentallycontrolled conditions (22∘C 12 h light-dark cycle) with freeaccess to standard pellet diet (Purina Sao Paulo Brazil) andwater Animalswere kept in cageswith raised floors to preventcoprophagy The experimental protocols were in accordancewith the ethical guidelines of National Institute of HealthBethesda USA

23 Formalin-Induced Paw Licking The formalin-inducednociception was performed as described previously by Hun-skaar and Hole [7] Groups of mice (119899 = 8) were treatedwith vehicle (10mLkg po) EOVA (25 50 100 or 200mgkgpo or topical) or morphine (75mgkg sc) 30 or 60minbefore the administration of 20 120583L of 1 formalin (in 09saline) into the plantar surface of the right hind paw Theduration of paw licking (s) as an index of painful responsewas determined at 0ndash5min (early phase neurogenic) and 20ndash25min (late phase inflammatory) after formalin injection

24 Eye Wiping Test Corneal nociception was induced by alocal application of hypertonic saline to the corneal surface[8 9] One drop (40120583L) of 5M NaCl solution was appliedlocally on the corneal surface of mice using a fine dropper[10] The number of eye wipes performed with the ipsilateralforepaw was counted for a period of 30 s Topical (20120583Leye)or orally (10mLkg) EOVA (25 50 100 or 200mgkg 119899 =

8group) or vehicle were given 1 h before the noxiou agentMorphine 7mgkg (sc 119899 = 8) was used as positive controlA normal control group (119899 = 8) received one drop of 015MNaCl (09)

In order to verify the possible involvement of opioidnoradrenergic nitrergic 5-HT

3 muscarinic K+ATP adenosin-

ergic and TRPV1systems in the effect of EOVA the animals

(119899 = 8group) were treated with the respective antagonist(Table 1) 30min before the topical administration of EOVA(50mgkg) The doses of antagonists were chosen based onprevious studies

To assess the possible contribution of endogenous sero-tonin animals (119899 = 8group) were pretreated with 120588-chlo-rophenylalanine methyl esther (PCPA 100mgkg ip aninhibitor of serotonin synthesis) or with vehicle once a dayfor 4 consecutive days Then 24 hours after the last PCPA orvehicle injection animals received EOVA (50mgkg) and 1 hlater they were tested in the eye wiping test

25 Statistical Analysis All data are presented as meanplusmn sem The data were evaluated by one-way analysis ofvariance with Student-Newman-Keuls post hoc test usingthe GraphPad Prism 40 statistical program The level ofsignificance was set at 119875 lt 005

3 Results and Discussion

In formalin test pretreatment with EOVA (oral and top-ical) caused significant diminutions of both first phase(neurogenic) and second phase (inflammatory) nocicep-tion responses (Tables 2(a) and 2(b)) Morphine (5mgkg)the reference standard also significantly suppressed theformalin-response at both phases

Topically administered EOVA (25 50 100 or 200mgkg)respectively (119875 lt 0001 119875 lt 001 119875 lt 001 and 119875 lt00001 resp) decreased the number of eye wipes inducedby the local application of 5M NaCl solution on the cornealsurface (Table 3(a)) Oral treatment with EOVA also reducedthe number of eye wipes (Table 3(b))



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