Abstract!

The present work describes the chemical charac-terization of a chloroform fraction (CF) obtainedfrom an extract of Ocotea puberula (Lauraceae)fruits, and preliminary antinociceptive analysisof CF and the alkaloid dicentrine, isolated fromthis fraction. CF (30–300mg/kg, p.o.) causeddose-related inhibition of abdominal constric-tions caused by acetic acid and also inhibited bothphases of formalin-induced nociception. Howev-er, hexane or ethyl acetate fractions did not pro-duce any effect. Antinociception caused by CF(100mg/kg, p.o.) in the acetic acid test was not af-fected either by caffeine, an adenosine receptorantagonist, or by naloxone, an opioid receptor an-tagonist, and neither was associatedwith nonspe-

cific effects such as muscle relaxation or sedation.Furthermore, dicentrine (30–300mg/kg, p.o.)produced dose-related inhibition of acetic acid-induced pain without causing changes in the mo-tor performance of mice. The results show, for thefirst time, that CF from Ocotea puberula fruits pro-duced marked antinociception in different mod-els of chemical pain, and this effect appears to be,at least in part, due to the presence of dicentrine.Themechanism by which CF and the alkaloid pro-duced antinociception still remains unclear, butthe adenosinergic or opioid system seems un-likely to be involved in this action.

Supporting information available online athttp://www.thieme-connect.de/ejournals/toc/plantamedica

Antinociceptive Effects of a Chloroform Extractand the Alkaloid Dicentrine Isolated from Fruitsof Ocotea puberula

Authors Deise Prehs Montrucchio1,2, Obdulio Gomes Miguel1, Sandra Maria Warumby Zanin1, Gabriel Araujo da Silva3,Alcíbia Maia Cardozo4, Adair Roberto Soares Santos2,5

Affiliations The affiliations are listed at the end of the article

Key wordsl" Ocotea puberulal" Lauraceael" alkaloidl" dicentrinel" antinociception

received May 7, 2012revised June 5, 2012accepted June 8, 2012

BibliographyDOI http://dx.doi.org/10.1055/s-0032-1315026Published onlinePlanta Med © Georg ThiemeVerlag KG Stuttgart · New York ·ISSN 0032‑0943

CorrespondenceProf. Dr. Adair R. S. SantosDepartamento de CiênciasFisiológicasUniversidade Federal deSanta CatarinaR. Roberto SampaioGonzaga, s/nFlorianópolis, 88040–900, SCBrazilPhone: + 554837219352(206)Fax: + 554837219672arssantos@ccb.ufsc.br

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Introduction!

Ocotea puberula (Rich.) Nees, Lauraceae is a Bra-zilian native tree whose phytochemical composi-tion includes several classes of chemicals, such asalkaloids, tannins, steroids, and triterpenes [1,2].Among the alkaloids, the aporphinic types are themost abundant in this species, including ocoteine,dehydroocotein, didehydroocotein, dicentrine,predicentrine, isodomesticine, and leucoxine [1,3–7]. Several biological properties have been de-scribed for many aporphinic alkaloids, includingdicentrine. This alkaloid was already reported ashaving a cytostatic effect against some tumor celllines frommice and humans [8–11], inhibition ef-fects on platelet aggregation induced by arachi-donic acid [12,13], and vasorelaxing and antihy-pertensive actions by antagonism of α1-adreno-ceptors [14–16]. In an ethnobotanical study withthe indigenous populations from Parana and San-ta Catarina states, Brazil, it was reported that O.puberula is used for the treatment of tumors andcutaneous affections [17]. Moreover, it has been

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reported that several species of plants belongingto the genus Ocotea are endowed with antinoci-ceptive and anti-inflammatory activity [18,19].Taking into account the biological activities ofthe Ocotea genus and the popular uses of O. pu-berula, it is surprising that no pharmacologicalstudy has been carried out on the possible antino-ciceptive effects of O. puberula up to now. Fur-thermore, in the state of Parana, south Brazil,some trees of O. puberula were found to producelarger fruits than those described in the literatureby Souza and Moscheta [20], and here we at-tempted to examine: (i) the possible antinocicep-tive action of the chloroform (CF), hexane (HF), orethyl acetate (EAF) fractions obtained from theselargerO. puberula fruits in chemical models of no-ciception in mice; (ii) the isolation, chemical iden-tification, and preliminary pharmacological eval-uation of the alkaloid dicentrine isolated fromthis plant; and (iii) the possible involvement ofadenosinergic and opioidergic systems in theantinociceptive action of the CF fraction.

ontrucchio DP et al. Antinociceptive Effects of… Planta Med

Fig. 1 Chemical struc-ture of dicentrine.

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Materials and Methods!

Plant material, chemical extraction and identificationOcotea puberula fruits were collected in Curitiba, state of Parana,Brazil in February 2008, from trees exhibiting larger fruits, andvoucher specimens were identified by Dr. Marcelo Brotto and de-posited in the Botanical Museum of Curitiba, under the numbers342996, 342997, and 342998. Fresh fruits (1.0 kg, equivalent to0.6 kg in dried basis) were triturated with ethanol, placed in aSoxhlet apparatus, and extracted with a mixture of ethanol andacetone (1:1) under reflux for 24 hours.The crude extract (approximately 1000mL) was concentratedunder reduced pressure to approximately 300mL, filtered, andfractioned in a modified Soxhlet apparatus [21] for 6–8 hourswith solvents of increasing polarity. The modification of Soxhletequipment consists of a lateral extension of the siphon from theupper curve, preventing the reflux of the solvent, and allowingthe extraction from the liquid material. From this process, fourfractions were obtained and then concentrated under reducedpressure until total removal of the solvent, yielding dried resi-dues of the n-hexanic fraction (HF, 1.54 g), chloroform fraction(CF, 10.69 g), ethyl acetate fraction (EAF, 1.90 g), and residual al-coholic fraction (RES, 1.93 g). The CF fraction was tested by sim-ple reaction with Dragendorff reagent (bismuth subnitrate, nitricacid, and potassium iodide) and revealed to contain alkaloids.CF was adsorbed in silica gel 60 (70–230 mesh; Merck™) andsubmitted to a column chromatography (30 × 6 cm, with a baseof G3 sintered glass), eluted with solvents of increasing polarityin portions of 100mL, ranging from n-hexane/chloroform(70:30), increasing polarity at 5% until pure chloroform, andthen addingmethanol at 5% increases until chloroform/methanol(70:30). Fractions were collected in 10mL portions and moni-tored by TLC using a mobile phase of toluene/ethyl acetate/dieth-ylamine (8:90:2) and were revealed with Dragendorff reagent.Fractions with the same TLC profile were united and the solventwas left to spontaneously evaporate, allowing crystallization. Thewhite crystals were filtered in a sintered glass filter and washedwith cold methanol, yielding a pure substance, which was sub-mitted to 1H and 13C NMR analysis in CDCl3 (NMR Bruker DPX200, 200 and 50MHz, respectively) and identified as being theaporphinic alkaloid S-(+)-dicentrine (DCTN, l" Fig. 1), based oncomparisonwith literature data [6]. Themelting point was deter-mined as being 168–169°C (PF 1000 Gehaka) and the optical ro-tation was measured on a Polartronic E (Schmidt + HaenschGmbH & Co.), [α]D20: + 62.0° (c = 1.0, chloroform).HPLC of DCTN and CF was performed on a Finnigan Surveyor(Thermo Fisher Scientific) equipped with a C18–5 µm ACE col-umn (250 × 4.6mm) and a mobile phase composed of 15% aceto-nitrile in acidified water (H2SO4 0.02N and H3PO4 2%), with aflow rate of 1mL/min.

AnimalsExperiments were conducted using adult male Swiss mice (25–35 g) obtained from the animal facility of Universidade Federalde Santa Catarina. Animals were housed at 22 ± 2°C under a 12-h dark/12-h light cycle with free access to food andwater, and ac-climatized to the laboratory for at least one hour prior to testing.All experiments were previously approved by the Ethics Commit-tee for Animal Research of Universidade Federal de Santa Catari-na (Protocol number PP00462, approved on December 9, 2010)and were conducted in accordance to the current guidelines for

Montrucchio DP et al. Antinociceptive Effects of… Planta Med

the care of laboratory animals and the ethical guidelines for in-vestigations of experimental pain in conscious animals [22]. Thenumber of animals and intensity of noxious stimuli used werethe minimum necessary to demonstrate consistent effects of thedrug treatment.

Formalin-induced nociceptionThe formalin test was carried out as described previously [23].Animals received an intraplantar injection of 20 µL of a 2.5% for-malin solution (0.92% formaldehyde), in the ventral surface ofthe right hind paw, and were observed during the first 5 minutes(neurogenic phase) and during the 15th to 30th minute (inflam-matory phase). Mice were pretreated with HF, CF, or EAF (30–300mg/kg, p.o.), aspirin (400mg/kg, p.o., used as a positive con-trol), or morphine (5mg/kg, s. c., used as a positive control) 1 or0.5 h before formalin injection [24], and control animals receivedthe same volume of vehicle. After the formalin injection, animalswere immediately placed into glass cylinders of 20 cm diameter,and the time spent licking the injected paw was recorded with achronometer, as indicative of nociception.

Abdominal constriction response caused byintraperitoneal injection of acetic acidNociception induced by acetic acid was evaluated as describedpreviously [23], where animals received a single intraperitonealinjection of a 0.6% acetic acid solution (450 µL) and the nocicep-tive response, observed as contractions of the abdominal muscletogether with a hind limb stretching, were cumulatively countedduring a period of 20minutes. Micewere pretreatedwith CF (30–300mg/kg, p.o.) or aspirin (400mg/kg, p.o., used as a positivecontrol) or with DCTN (30–300mg/kg, p.o.) 1 h before acetic acidinjection, and control animals received the same volume of ve-hicle. After the irritant injection, mice were placed into 20-cmdiameter glass cylinders and the abdominal constrictions werecumulatively counted during a 20-minute period.

Evaluation of a possible involvement of opioidergicsystemTo evaluate the possible involvement of the opioid system, micewere pretreatedwith the nonselective opioid receptor antagonistnaloxone (1mg/kg, i.p.) 20 minutes before morphine (2.5mg/kg,s. c., used as a positive control), CF (100mg/kg, p.o.), or vehicle(10mL/kg, p.o.) and the nociceptive response to an intraperito-neal injection of acetic acid was recorded 0.5 or 1 h later, respec-tively [18].

Fig. 2 Effects of the chloroform fraction of O. pu-berula fruits extract (CF, 30–300mg/kg, p.o.), as-pirin (Asp, 400mg/kg, p.o.), and morphine (Morph,5mg/kg, s. c.) in the first (panel A) and second(panel B) phases of formalin-induced nociception inmice. Each bar represents the mean ± S.E.M. of 6–8animals, column C being indicative of control val-ues. Significance levels are indicated by ** p < 0.01and *** p < 0.001 when compared to the controlgroup (ANOVA followed by the Newman-Keulstest).

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Evaluation of a possible involvement of adenosinergicsystemIn order to evaluate the involvement of the adenosinergic systemas a possible mechanism of action for the antinociceptive prop-erty of CF, mice were pretreated with the nonselective adenosinereceptor antagonist caffeine (3mg/kg, i.p.), as described previ-ously [25]. After 20 minutes, mice received N6-cyclohexyladeno-sine (CHA; a selective adenosine A1 receptor agonist, 0.03mg/kg,i.p., used as a positive control), CF (100mg/kg, p.o.), or vehicle(10mL/kg, p.o.) and the nociceptive response to an intraperito-neal injection of acetic acid was recorded 0.5 or 1 h later, respec-tively.

Measurement of locomotor activityIn order to exclude the possibility that the observed antinocicep-tive action could be related to nonspecific effects in the locomo-tor activity, the open-field test was carried out as previously de-scribed [26]. Mice were placed in a wooden box (40 × 60 × 50 cm)with the floor divided into 12 identical squares, and the numberof squares crossed with all paws was counted during 6 minutes.Animals were treated with CF (100mg/kg, p.o.) or DCTN(100mg/kg, p.o.) 1 h prior to the test, while control animals re-ceived the same volume of vehicle.

Reagents and drugsThe following substances were used: acetonitrile (Tedia Brasil),ethanol, acetone, n-hexane, chloroform, ethyl acetate, silicagel60, acetic acid and formalin (Merck), morphine sulphate (inject-able solution, 10mg/mL; União Química), naloxone hydrochlor-ide (Cristália), aspirin (99% purity), and CHA (Sigma Aldrich).The drugs were dissolved in saline (0.9% NaCl). The organic frac-tions of O. puberula extract were dissolved in saline with 5% ofTween 80 (CRQ). Aspirin and DCTN were dissolved in saline with1% DMSO (Merck) and 5% Tween 80 (CRQ). The final concentra-tion of Tween 80 or DMSO did not exceed 5 and 1%, respectively,and did not cause any effect per se. All other chemicals were ofanalytical grade and obtained from standard commercial suppli-ers.

Statistical analysisResults are presented as mean ± S.E.M. and the data were ana-lyzed by one-way analysis of variance (ANOVA) followed by New-man-Keuls post hoc test. P values less than 0.05 were consideredsignificant. ID50 values (dose capable of reducing nociceptive re-sponse by 50% relative to the control value) were determined bynonlinear regression analysis and reported as geometric meanwith 95% confidence limits. All statistical analyses were per-formed using GraphPad Prism 5.0 (GraphPad Software).

Supporting informationHPLC chromatograms for DCTN and CF, results of HF and EAF inthe formalin test, and 13C NMR of DCTN are available as Support-ing Information.

Results!

When submitted to HPLC, the CF fraction showed a major com-pound with RT = 17.06min, which was isolated with 97% of pu-rity (Fig. 1S, available as Supporting Information) and identifiedas DCTN (13C NMR available as Supporting Information, Fig. 2S).This alkaloid was used as the standard in a calibration curve,made by 6 points, with concentrations ranging from 5–100 µg/mL. When comparing the peak areas, DCTN was shown to bepresent as 577mg/g of CF, corresponding to 57% of CF (Table 1S,available as Supporting Information).For initial screening of antinociceptive properties, micewere pre-treated with different doses of HF, EAF, and CF obtained from O.puberula fruits, morphine or aspirin as positive controls, andthen submitted to the formalin test. The administration of HF orEAF (30–300mg/kg, p.o., 1 h prior) did not cause any antinoci-ceptive effect against formalin-induced licking (Table 2S, avail-able as Supporting Information). However, CF (30–300mg/kg,p.o., 1 h prior) caused a significant inhibition of both neurogenic(0–5min) and inflammatory (15–30min) phases of formalin-in-duced licking. The calculated mean ID50 values (and their respec-tive 95% confidence limits) for these effects were: 190.0 (127.6–282.8) mg/kg and 120.3 (88.6–163.3) mg/kg and the inhibitionsobserved were 65 ± 8 and 91 ± 7% at a dose of 300mg/kg, for firstand second phases, respectively (l" Fig. 2A and B).The reference opioid analgesic, morphine (5mg/kg, s. c., 0.5 h pri-or), produced a significant inhibition of neurogenic (72 ± 6%) andinflammatory (94 ± 10%) phases of formalin-induced licking. Incontrast, the nonsteroidal analgesic, aspirin (400mg/kg, p.o., 1 hprior) was able to significantly reduce only the inflammatory(66 ± 7%) phase of formalin-induced pain (l" Fig. 2A and B).In order to confirm the antinociceptive property of CF, mice werepretreated with the same doses used in the formalin test (30–300mg/kg, p.o., 1 h prior) and then submitted to the acetic acidmodel, a typical model of visceral inflammatory pain. The CF frac-tion produced a dose-related inhibition of acetic acid-inducedabdominal constrictions in mice when compared to the controlgroup, with a mean ID50 value of 89.0 (52.5–150.9) mg/kg and in-hibition of 92 ± 6% at a dose of 300mg/kg (l" Fig. 3A). The DCTN(30–300mg/kg, p.o., 1 h prior), the main alkaloid present in CF,also produced a dose-related inhibition of acetic acid-inducedabdominal constrictions, with a mean ID50 of 75.7 (55.0–104.1)

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Fig. 3 Effects of the chloroform fraction of O. pu-berula fruits extract (CF, 30–300mg/kg, p.o., panelA) and aspirin (Asp, 400mg/kg, p.o., panel A) oralkaloid dicentrine (DCTN, 30–300mg/kg, p.o.,panel B) on the acetic acid-induced nociception inmice. Each bar represents the mean ± S.E.M. of 6animals, column C being indicative of control val-ues. Significance levels are indicated by ** p < 0.01and *** p < 0.001 when compared to the controlgroup (ANOVA followed by the Newman-Keulstest).

Fig. 4 Effects of pretreatment with naloxone(1mg/kg, i. p., panel A) or caffeine (3mg/kg, i. p.,panel B) on the antinociceptive effects of thechloroform fraction of O. puberula fruits extract (CF,100mg/kg, p.o.), morphine (2.5mg/kg, s. c.), orCHA (0.03mg/kg, i. p.) in the acetic acid-inducednociception in mice. Each bar represents the mean± S.E.M. of 6–8 animals. Significance levels are in-dicated by * p < 0.05, ** p < 0.01, and *** p < 0.001when compared to the control group and # p < 0.05and ## p < 0.01 when compared to the agonists(morphine or CHA) (ANOVA followed by the New-man-Keuls test).

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mg/kg and an inhibition of 87 ± 3% at a dose of 300mg/kg(l" Fig. 3B). The positive control aspirin also inhibited acetic ac-id-induced abdominal constrictions in 84 ± 4% at the dose of400mg/kg.The results presented in l" Fig. 4A and B show that the antinoci-ceptive effect of morphine (2.5mg/kg, s. c., a nonselective opioidreceptor agonist) or CHA (0.03mg/kg, i.p., a selective adenosineA1 receptor agonist), but not that of CF, were significantly re-versed by the previous treatment of mice with naloxone (1mg/kg, i.p., a nonselective opioid receptor antagonist) or caffeine(3mg/kg, a nonselective adenosine receptor antagonist), respec-tively, when analyzed against acetic acid-induced pain.In order to discard a nonspecific action on motor performance,the active dose of CF (100mg/kg) and the alkaloid DCTN(100mg/kg) were orally given 1 h prior to the test, and neitherCF nor DCTN affected the locomotor activity of mice in the open-field test compared with control animals (treated with vehiclealone). The means ± S.E.M. of crossing numbers 1 h after admin-istration were 112.7 ± 8.1 for the control and 105.4 ± 8.5 for theCF, and 106.5 ± 7.9 for DCTN and 113.0 ± 3.8 for the control.

Discussion!

The present results show the occurrence of the aporphinic alka-loid dicentrine as the major compound found in O. puberulafruits. This alkaloid is known to occur in a small amount in sev-eral different plant species, mainly from the Lauraceae family, in-cluding O. puberula leaves, bark, and roots [1,7]; however, thisappears to be the first report of a phytochemical evaluation ofthe atypical larger fruits of this species, showing dicentrine asthe major alkaloid compound. The confirmation of alkaloid iden-tity was made by comparison of 1H and 13C NMR data with those

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obtained in the literature [6], and HPLC quantification showedthat dicentrine occurs as 57% of CF, being the majority alkaloid.The reasons why some subjects from this species develop thesefruits were not investigated, but considering the fact that theirphytochemical composition is based on a single type of a majorsubstance, it may be suggested that this atypical developmentmay be due to some specific injury suffered by the plant, leadingto an overproduction of secondary metabolites, in this case, thealkaloid dicentrine [27].Furthermore, no reports were found in the literature regardingan antinociceptive action of this plant and, although some biolog-ical actions of dicentrine have been reported, no specific antino-ciceptive effect of this alkaloid was found in any database. Thepresent results show that the chloroform, but not hexane or ethylacetate fractions obtained from the fractioning of O. puberulafruits extract, when given orally to mice, was able to reduce thenociceptive behavior in both phases of formalin-induced noci-ception, an effect that was confirmed in the acetic acid model.Surprisingly, when DCTN was administered, it also showed anantinociceptive effect that was similar in potency to CF (meanID50 of 75.7mg/kg and 89.2mg/kg, respectively), suggesting thatthis alkaloid greatly contributes to the antinociceptive effect of O.puberula fruits extract.When compared to a well-known nonsteroidal analgesic such asaspirin, administered by the oral route, the CF was shown to bemore effective, since it reduced both phases of formalin-inducedpain, while aspirin was effective only on the inflammatory phase.At the dose of 300mg/kg, however, CF antinociceptive activitywas similar to that produced by morphine. In the acetic acidmodel, on the other hand, both CF and DCTN displayed a potencythat was similar to that produced by aspirin. Furthermore, themean ID50 of DCTN in the acetic acid model (75.7mg/kg) is com-parable to the mean ID50 of aspirin (117.0mg/kg) in the same

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model [28]. Given the potency similarity with NSAIDs, it may beinteresting to further investigate other similarities in the mecha-nism of action, such as the possible role of O. puberula as an anti-inflammatory agent. Indeed, several other species from the Oco-tea genus have already been reported to have anti-inflammatoryproperties, mainly by inhibition of cyclooxygenases (COX-1 andCOX-2) and 5-lypoxygenase (5-LOX) [19,29–31]. Antinociceptiveactivity of theOcotea genus, on the other hand, has been reportedto a lesser extent and its mechanism of action has not been wellestablished. However, data from our group has shown that theantinociceptive effect of O. suaveolens, for instance, does not in-volve the activation of opioid, nitric oxide, or serotonergic sys-tems [18], common endogenous pathways of pain control. Withthis in mind, we attempted here to verify if opioid and adeno-sinergic systems would be involved in the mechanism of actionof CF from O. puberula fruits. Thus, our data show that the antino-ciceptive effect of CF was not reversed by the pretreatment ofmice either with naloxone, an opioid receptor antagonist, or withcaffeine, an adenosine receptor antagonist, suggesting that its ac-tion is unlikely to occur via opioidergic or adenosinergic systems.Finally, neither CF nor DCTN, at a dose that causes antinocicep-tion, produced any impairment of locomotor activity in theopen-field test, which may be interpreted that the antinocicep-tive effect is not derived from a nonspecific effect on motor per-formance or sedation.Of note, the doses used to assess the antinociceptive effects ofDCTN differ from other reports in the literature, where the hypo-tensive effect on anesthetized rats was found with doses of 2mg/kg by the intravenous route [12]. Moreover, DCTN (at doses of 5to 10mg/kg) was able to reduce plasma lipid levels and mean ar-terial pressure in hypertensive rats with a high-fat cholesterol di-et [15]. These latter effects, however, besides being achieved bythe oral route, were found with a continuous treatment of ani-mals, twice a day during 4 weeks. For the antinociceptive effects,mice received a single oral administration of DCTN previous tothe nociceptive stimulus. Those differences on the administrationroute and duration of treatment, besides the type of investiga-tion, may account for the increased doses necessary for the anti-nociceptive effect of DCTN, in comparisonwith other reported ef-fects.In addition, it is well known that xenobiotics are metabolized inorder to make their elimination from the body easier. In the caseof DCTN, a few reports on pharmacokinetics have shown that itshalf-life of distribution and elimination is very short (4.3 and 45.2minutes, respectively) after intravenous administration in rats[32], and at least 24 metabolites were found in miniature pigsurine 24 h after oral administration of DCTN [33]. These datacould suggest that the antinociceptive effect of DCTN may bedue to one or more of its metabolites. However, among the DCTNdescribed metabolites [33], only actinodaphnine has a single re-port in the literature regarding an antinociceptive effect [34].Thus, additional studies can be done to define whether this anti-nociceptive effect of DCTN is due to the alkaloid itself or its me-tabolites.Taken together, these results show for the first time thatO. puber-ula fruits have an important antinociceptive effect that seems tobe derived from the presence of the alkaloid dicentrine, andalthough not established, themechanism of this action is unlikelyto occur via the adenosinergic or opioid system, or sedative ef-fects. The mechanism by which CF and dicentrine produce anti-nociception still remains unclear, but we are still carrying outpharmacological studies for the characterization of the mecha-

nism(s) responsible for the antinociceptive action. These findingssupport, at least partially, the use of O. puberula in traditionalmedicine.

Acknowledgements!

This work was supported by grants from Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq), Coordenaçãode Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fun-dação de Apoio à Pesquisa Científica Tecnológica do Estado deSanta Catarina (FAPESC), and Programa de Apoio a Núcleos de Ex-celência (PRONEX-CNPq/FAPESC), Brazil. The authors kindlythank Pedro Zanin for all the help in the phytochemical studies,Cristina Mayumi Myiazaki for the NMR spectral data, and Profes-sor Maria da Graça Nascimento for the optical rotation data.

Conflict of Interest!

None.

Affiliations1 Departamento de Farmácia, Setor de Ciências da Saúde, Universidade Federaldo Paraná, Curitiba, PR, Brazil

2 Programa de Pós-Graduação em Farmacologia, Universidade Federal deSanta Maria, Santa Maria, RS, Brazil

3 Programa de Pós-Graduação em Ciências Farmacêuticas, UniversidadeFederal do Rio Grande do Norte, Natal, RN, Brazil

4 Departamento de Patologia, Centro de Ciências da Saúde, UniversidadeFederal de Santa Catarina, Florianópolis, SC, Brazil

5 Laboratório de Neurobiologia da Dor e Inflamação, Departamento deCiências Fisiológicas, Universidade Federal de Santa Catarina, Florianópolis,SC, Brazil

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