Journal of Ethnopharmacology 148 (2013) 205–209

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Journal of Ethnopharmacology

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Evaluation of mechanisms involved in the antinociception of theethanol extract from the inner bark of Caesalpinia pyramidalis in mice

Cliomar A. Santos, Dayanne S. Santos, Danielle G. Santana, Sara M. Thomazzi n

Pharmacology of Inflammation and Pain Laboratory, Department of Physiology, Federal University of Sergipe, CEP 49100-000, São Cristóvão, Sergipe, Brazil

a r t i c l e i n f o

Article history:Received 17 November 2012Accepted 31 March 2013Available online 17 April 2013

Keywords:Caesalpinia pyramidalisFabaceaeGlutamatergic systemL-arginine–nitric oxide pathwayNociception

41/$ - see front matter & 2013 Elsevier Irelanx.doi.org/10.1016/j.jep.2013.03.081

esponding author. Tel.: +55 79 21056640; faxail address: sarathomazzi@ufs.br (S.M. Thoma

a b s t r a c t

Ethnopharmacological relevance: Caesalpinia pyramidalis Tul. (Fabaceae) is an endemic tree of theNortheast region of Brazil, mainly in the Caatinga region. More commonly, inner bark or flowers aretraditionally used to treat many painful and inflammatory processes. A common use of this plant is madeby macerating a handful of its stem bark in a liter of wine or sugarcane brandy. It is drunk againststomachache, dysenteries, and diarrheas.Materials and methods: The ethanol extract of Caesalpinia pyramidalis inner bark was used in mice via oralroute, at the doses of 10, 30, and 100 mg/kg, in behavioral models of nociception and investigates some of themechanisms underlying this effect.Results: The ethanol extract (30 and 100 mg/kg, Po0.001), given orally, produced dose dependent inhibition ofacetic acid-induced visceral pain. The ethanol extract also caused significant and dose-dependent inhibition ofcapsaicin-(100 mg/kg, Po0.001) and glutamate-(10, 30, and 100 mg/kg, Po0.01) induced pain. The anti-nociception caused by the ethanol extract (30 mg/kg) in the abdominal constriction test was significantlyattenuated (Po0.001) by intraperitoneal treatment of mice with L-arginine (600 mg/kg).Conclusions: Collectively, the present results suggest that the ethanol extract of Caesalpinia pyramidalisproduced dose-related antinociception in several models of pain through mechanisms that involved bothglutamatergic system and/or the L-arginine–nitric oxide pathway, supporting the folkloric usage of the plant totreat various painful processes.

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1. Introduction

The use of natural therapy constitutes an attractive approach forthe treatment of several painful disorders. Different products of naturalorigin have been reported to possess significant antinociceptiveactivity (Amorim et al., 2009; Nguelefack et al., 2010). Many speciesfrom Caesalpinia genus (Fabaceae) have been used in traditionalmedicine and its analgesic activity demonstrated (Shukla et al., 2010;Kannur et al., 2012; Lima et al., 2012; Ochieng et al., 2012).

Caesalpinia pyramidalis Tul. is an endemic tree of the Northeastregion of Brazil, mainly in the Caatinga region, which is knownpopularly as “catingueira” (Albuquerque et al., 2007; Agra et al.,2007, 2008). More commonly, inner bark or flowers are tradition-ally used to treat colic, stomachache, collision, cough, bronchitis,asthma, respiratory infection, influenza, gastritis, diarrhea, dia-betes, dysenteries, and fever (Albuquerque et al., 2007; Agra et al.,2007, 2008). A common use of this plant is made by macerating ahandful of its stem bark in a liter of wine or sugarcane brandy. It isdrunk before the meals two times a day, against stomachache,dysenteries, and diarrheas (Agra et al., 2007, 2008).

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: +55 79 21056474.zzi).

Our previous study showed that the inner bark ethanol extract ofCaesalpinia pyramidalis possessed both central and peripheral anti-nociceptive effect when tested using chemical and thermal models ofnociception in mice (Santos et al., 2011). In the same study, we alsodemonstrated that the antinociceptive effect of the ethanol extractwas significantly reversed by the non-selective opioid receptorantagonist, naloxone (Santos et al., 2011). Although this result maysuggest that of activation of opioid receptors are involved in theanalgesic mechanism of extract, the actual mechanism of actions bywhich the extract exerts its antinociceptive activity remains to beelucidated. In another study, we recently showed that the ethanolextract of Caesalpinia pyramidalis reduced the abdominal hyperalgesiain rats with acute pancreatitis (Santana et al., 2012). The pre-treatmentwith the ethanol extract did not produce any significant motorperformance alterations (Santos et al., 2011; Santana et al., 2012).

Therefore, the present study was conducted to further investigatepossible mechanisms of action that participate in the ethanol extract-induced antinociception.

2. Materials and methods

2.1. Plant collection and extraction of the ethanol extract

The inner bark of Caesalpinia pyramidalis was collected in themunicipality of Canindé de São Francisco-SE, Brazil, in September

C.A. Santos et al. / Journal of Ethnopharmacology 148 (2013) 205–209206

2008 (09166′00″S, 37178′94″W). The plant was authenticatedby Professor Ana Paula Prata, Department of Biology, FederalUniversity of Sergipe. A voucher specimen has been deposited atthe Herbarium of the Federal University of Sergipe (number ASE13,164). The inner bark was dried at 40 1C in a forced air oven fortwo days and subsequently powdered (2.840 g) and extracted bymaceration at room temperature with 90% ethanol for five days.The extract was filtered in vacuum, and the solvent was removedusing a rotary evaporator (45 1C). The percentage of yield of theethanol extract (EE) was 2.6% (73.8 g).

The EE of Caesalpinia pyramidalis was analyzed using high perfor-mance liquid chromatography (Shimadzu, Prominence model, Kyoto,Japan) consisting of a vacuum degasser DGU-20A3 model, SIL-10 Aautosampler, two high pressure pumps LC-6 A, and an SPDM20Avpphotodiode array detector system coupled with a CBM 20 A interface.Analysis was performed in an analytical Phenomenex LUNAs C18column (250�4.6 mm i.d., 5 mm of particle diameter, Torrance, CA,USA). Separation of compounds was done by reverse mode gradientelution as previously described (Santana et al., 2012).

2.2. Drugs

The following substances were used: acetic acid (Merck, Darm-stadt, Germany); acetylsalicylic acid (ASA), Nω-nitro-L-arginine(L-NOARG), L-arginine hydrochloride, capsaicin, L-glutamic acid hydro-chloride, morphine hydrochloride (Sigma Chemical Co., St. Louis, MO).All substances used were dissolved in 0.2% Tween 80 in 0.9% NaClsolution.

2.3. Animals

Young adults Swiss mice (20–30 g) of both sexes were obtainedfrom the Central Biotery of the Federal University of Sergipe (SãoCristóvão, Brazil). Animals were maintained at controlled roomtemperature (2172 1C) with free access to food (Purinas) andwater, under a 12 h light/dark cycle. The experiments wereperformed after approval of the protocol by the Institutional EthicsCommittee (CEPA/UFS 05/09) and were carried out in accordancewith the current guidelines for the care of laboratory animals andthe ethical guidelines for investigations of experimental pain inconscious animals (Zimmermann, 1983).

2.4. Acetic acid-induced abdominal constriction test

The procedure used was similar to that previously described(Santos et al., 2011). Abdominal writhes were induced by intraper-itoneal (i.p.) injection of acetic acid (0.6%, 0.1 mL/10 g) inmice. Animalswere pre-treated orally (p.o.) with the EE (10–100 mg/kg), acetylsa-licylic acid (ASA, 300mg/kg), or vehicle (0.2% Tween 80, 0.1 mL/10 g)60 min before initiating the algesic stimulation (n¼6/group). Theabdominal writhes were observed for a period of 20 min and began5min after the injection of the nociceptive agent.

2.5. Analysis of possible involvement of L-arginine–nitric oxide (NO)pathway

To investigate the role played by the L-arginine–NO pathway inthe antinociception caused by the EE, the mice (n¼8/group) werepre-treated with L-arginine (600 mg/kg, i.p., a nitric oxide precur-sor) and after 15 min they received the EE (30 mg/kg, p.o.),Nω-nitro L-arginine (L-NOARG, 75 mg/kg, i.p., a nitric oxide synthaseinhibitor), or vehicle (p.o.). The algesic responses to acetic acid wererecorded 60 min after the administration of the EE, L-NOARG, orvehicle. Another group of animals (n¼8/group) was pre-treatedwith vehicle and after 15 min received the EE, L-NOARG, or vehicle,60 min before acetic acid injection. The abdominal writhes were

observed for a period of 20 min and began 5 min after the injectionof the nociceptive agent.

2.6. Capsaicin-induced nociception

In order to provide more direct evidence concerning theparticipation of vanilloid receptor-1 (TRPV1) in the effect of theEE, we investigated its antinociceptive effect in capsaicin-(a TRPV1agonist) induced licking in the mouse paw. The procedure usedwas similar to that described previously (Santos and Calixto, 1997).The animals (n¼8/group) were treated with the EE (10–100 mg/kg,p.o., 60 min before), morphine (3 mg/kg, i.p., 30 min before), orvehicle (0.2% Tween 80, p.o., 60 min before). After 60 min, 20 μL ofcapsaicin (1.6 μg/paw prepared in saline) were injected intraplan-tarly in the ventral surface of the right hind paw. Animals wereobserved individually for 5 min following capsaicin injection. Theamount of time spent licking the injected paw was recorded with achronometer and was considered as indicative of nociception.

2.7. Glutamate-induced nociception

In an attempt to provide more direct evidence concerning theinteraction of the EE with the glutamatergic system, we investi-gated whether the EE was able to antagonize glutamate inducedlicking of the mouse paw. The procedure used was similar to thatdescribed previously (Beirith et al., 2002). After, a volume of 20 μLof glutamate (20 μmol/paw) was injected intraplantarly in theventral surface of the right hind paw. Animals were observedindividually for 15 min after glutamate injection. The amount oftime spent licking the injected paw was recorded with a chron-ometer and was considered as indicative of nociception. Theanimals (n¼8/group) were treated with the EE (10–100 mg/kg,p.o., 60 min before), morphine (3 mg/kg, i.p., 30 min before), orvehicle (0.2% Tween 80, p.o., 60 min before).

2.8. Statistical analysis

The results are presented as the means7SEM of n animals pergroup. Statistical evaluation of the data was performed using one-way analysis of variance (ANOVA) followed by Bonferroni's test.P values lower than 0.05 were considered significant.

3. Results

3.1. Abdominal constriction response caused by intraperitonealinjection of acetic acid

The writhes evoked by injection of acetic acid in the abdominalcavity were markedly reduced, dose-related manner, by the pre-treatment with the EE at 30 and 100 mg/kg (61.9 and 77.6%,respectively, Po0.001, Fig. 1). ASA (300 mg/kg) also significantinhibited (80.9%, Po0.001) the writhes induced by acetic acid(Fig. 1).

3.2. Involvement of L-arginine–NO pathway

The systemic pre-treatment of mice with the nitric oxideprecursor L-arginine (600 mg/kg, i.p.), given 15 min earlier, sig-nificantly reversed (Po0.001) the antinociception caused by bothL-NOARG (75 mg/kg, i.p., a nitric oxide synthase inhibitor) and EE(30 mg/kg, p.o.), when analyzed against acetic acid-induced noci-ception (Fig. 2).

Fig. 1. The effect of the EE on acetic acid-induced visceral nociception. Mice werepre-treated with vehicle, EE (10–100 mg/kg), or acetylsalicylic acid (ASA, 300 mg/kg) before of acetic acid injection. nPo0.001 vs. the vehicle group (n¼6/group).

Fig. 2. Effect of pre-treatment of animals with L-arginine (600 mg/kg) on theantinociceptive profile of L-NOARG (75 mg/kg, i.p.) and EE (30 mg/kg, p.o.) againstthe acetic acid-induced visceral nociception. nPo0.05, nnPo0.001 vs. the vehiclegroup (animals injected with the vehicle alone, n¼8/group). #Po0.001 vs.L-NOARG or EE plus vehicle.

Fig. 3. The effect of the EE on capsaicin-induced nociception. Mice were pre-treated with vehicle, EE (10–100 mg/kg), or morphine (Morph, 3 mg/kg) before acapsaicin injection. nPo0.001 vs. the vehicle group (n¼8/group).

Fig. 4. The effect of the EE on glutamate-induced nociception. Mice were pre-treated with vehicle, EE (10–100 mg/kg), or morphine (Morph, 3 mg/kg) before aglutamate injection. nPo0.01 and nnPo0.001 vs. the vehicle group (n¼8/group).

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3.3. Capsaicin-induced nociception

The neurogenic pain evoked by injection of the capsaicin wasmarkedly reduced, dose-related manner, by the pre-treatmentwith the EE at 100 mg/kg (40.1%, Po0.001, Fig. 3). Morphine(3 mg/kg) also significant inhibited (58.0%, Po0.001) the nocicep-tion induced by capsaicin (Fig. 3).

3.4. Glutamate-induced nociception

The EE, given orally, at 10, 30, and 100 mg/kg produced markedand dose-dependent attenuation of the glutamate-induced noci-ception (23.8, 49.2, and 72.9%, respectively, Po0.01, Fig. 4). Mor-phine (3 mg/kg) also inhibited (77.4%, Po0.001) the nociceptioninduced by glutamate (Fig. 4).

4. Discussion

The present study demonstrates that the oral administration ofthe EE of Caesalpinia pyramidalis elicits a potent and dose-dependent inhibition of the nociceptive behavioral response in

mice, and provides some evidence on the mechanisms implicatedin this action.

We recently demonstrated the antinociceptive action of the EEfrom Caesalpinia pyramidalis in the acetic acid-induced abdominalwrithing test (Santos et al., 2011). The acetic acid-induced writhingreaction in mice, described as a typical model for inflammatorypain, has long been used as a screening tool for the assessment ofanalgesic and/or anti-inflammatory properties of new compounds(Tjølsen and Hole, 1997). Acetic acid, in the abdominal constrictionassay, acts causing the release of different mediators that excitethe nociceptors (Ikeda et al., 2001; Ribeiro et al., 2000; Feng et al.,2003). Of particular interest, the results reported here againshowed that p.o. administration of the EE, using doses belowthose used in the previous article, produced significant dosedependent antinociception when assessed in acetic acid-inducedvisceral nociception. The results showed here are consistent withour previous study (Santos et al., 2011).

Other than the role of opioid receptors, which has beendiscussed in another study (Santos et al., 2011), further studieswere also carried out to study the effect of the EE against vanilloidreceptors induced nociceptive transmission and to explore the roleof glutamatergic system and L-arginine/NO pathway in the mod-ulation of the EE-induced antinociception.

Capsaicin, an active ingredient in hot chili peppers, stimulates atransient receptor potential (TRP), the vanilloid receptor-1 (TRPV1), a

C.A. Santos et al. / Journal of Ethnopharmacology 148 (2013) 205–209208

ligand-gated non-selective cation channel in primary sensory neu-rons (Caterina et al., 1997). Activation of TRPV1 receptor by capsaicin,results in an increase in synaptic release of both glutamate andneuropeptides (Ferrini et al., 2007). Besides the involvement of TRPchannels in acute nociceptive pain, they are also important players ininflammatory pain. The TRP activation also sends an efferent signal atperipheral terminal via secretion of inflammatory agents, causinglocal neurogenic inflammation (Julius and Basbaum, 2001). Ourresults show that oral administration of the EE of Caesalpiniapyramidalis produced a partial, but significant, reduction of thenociceptive response caused by intraplantar injection of capsaicininto the mouse hindpaw. The antinociceptive effect of the EE couldbe by regulating the TRP receptor activation, which in turn reducesthe neurogenic inflammation and the glutamate release, contributingto the modulation of nociceptive transmission.

The EE from Caesalpinia pyramidalis also produced a dose-dependent antinociceptive effect on the glutamate induced pawlicking response. Glutamate is a major excitatory neurotransmitterin the central nervous system (Coderre, 1993), and various studieshave shown that the nociceptive response induced by glutamate ismediated by glutamatergic receptors [both N-methyl-D-aspartate(NMDA) and non-NMDA receptors] as well as by the release of NOand NO-related substances in both central and peripheral nervoussystems (Fundytus, 2001; Beirith et al., 2002). The EE from Caesalpi-nia pyramidalis produced a dose-dependent antinociceptive effect onthe glutamate induced paw licking response. Therefore, the anti-nociception of Caesalpinia pyramidalis could be dependent on eitherperipheral or central sites of action.

Nitric oxide is an essential bioregulatory molecule required forseveral physiological processes (Hierholzer et al., 1998). NO increasesthe level of cyclic guanosine monophosphate (GMP) through theactivation of soluble guanylyl cyclase (sGC), which influences a widerange of physiological functions including pain and analgesia(Abacioglu et al., 2000). Cyclic GMP acts on the ion channels directlyor through the activation of protein kinases and phosphodiesterases(Xu et al., 1995).

Based on our findings, it was observed that the pre-treatment ofanimals with L-arginine (a nitric oxide precursor) at a dose that didnot produce any significant changes to the acetic acid-inducednociception, significantly reversed antinociceptive effect caused bythe EE of Caesalpinia pyramidalis. Furthermore, under same condi-tions, the pre-treatment with L-arginine reversed the antinocicep-tion caused by L-NOARG (a nitric oxide synthase inhibitor). Thesefindings may possibly suggest the involvement of the L-arginine/nitric oxide pathway in the antinociceptive activity of the EE.Previous studies have reported that NO synthase inhibitors reducednociception caused by acetic acid (Larson et al., 2000; Meotti et al.,2006). Therefore, the inhibitory effect of L-NOARG observed in thepresent study are in line with the notion that NO is one of themediators of acetic acid induced nociception, justifying the use ofthis model.

5. Conclusion

In summary, the present results provide convincing evidencethat the EE from Caesalpinia pyramidalis presents a pronouncedsystemic antinociception in several models of nociception in themouse, supporting the folkloric usage of the plant to treat variouspainful processes. The antinociceptive effect of the EE involves aninteraction with glutamatergic (through NMDA receptors) systemand/or L-arginine–NO pathway. This ethanol extract is of greatinterest as a source of novel molecules for developing strategiesmore appropriate in treating pain. In order to identify the activecompounds present in the EE of Caesalpinia pyramidalis, pharma-cological and chemical studies are in progress.

Acknowledgements

Authors are grateful to Coordenação de Aperfeiçoamento dePessoal de Nível Superior (CAPES) and Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq). SMT is recipientof CNPq productivity grants.

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Glossary

ASA,: acetylsalicylic acid;EE,: ethanol extract;GMP,: guanosine monophosphate;L-NOARG,: Nω-nitro-L-arginine;Morph,: morphine;NMDA,: N-methyl-D-aspartate receptor;NO,: nitric oxide;sGC,: soluble guanylyl cyclase;TRP,: transient receptor potential;TRPV1,: vanilloid receptor-1.