© 2009 The Authors

Doi: 10.1111/j.1742-7843.2008.00365.x

Journal compilation

© 2009 Nordic Pharmacological Society

. Basic & Clinical Pharmacology & Toxicology

,

104

, 306–315

Blackwell Publishing Ltd

Antinociceptive Properties of the Hydroalcoholic Extract and the Flavonoid Rutin Obtained from

Polygala paniculata

L. in Mice

Fernanda da R. Lapa

1

, Vinicius M. Gadotti

2

, Fabiana C. Missau

3

, Moacir G. Pizzolatti

3

, Maria Consuelo A. Marques

1

, Alcir L. Dafré

2

, Marcelo Farina

4

, Ana Lúcia S. Rodrigues

4

and Adair R. S. Santos

2

1

Department of Pharmacology, Center of Biological Sciences, Federal University of Paraná, Curitiba, 88015-420, PR, Brazil,

2

Department of Physiological Sciences, Center of Biological Sciences, Federal University of Santa Catarina, Florianópolis, 88040-900, SC, Brazil,

3

Department of Chemistry, Center of Physical and Mathematical Sciences, Federal University of Santa Catarina, Florianópolis, 88040-900, SC, Brazil, and

4

Department of Biochemistry, Center of Biological Sciences, Federal University of Santa Catarina, Florianópolis, 88040-900, SC, Brazil

(Received 4 December 2007; Accepted 30 June 2008)

Abstract:

The present study examined the antinociceptive effects of a hydroalcoholic extract of

Polygala paniculata

inchemical and thermal behavioural models of pain in mice. The antinociceptive effects of hydroalcoholic extract was evaluatedin chemical (acetic-acid, formalin, capsaicin, cinnamaldehyde and glutamate tests) and thermal (tail-flick and hot-platetest) models of pain or by biting behaviour following intratecal administration of both ionotropic and metabotropicagonists of excitatory amino acids receptors glutamate and cytokines such as interleukin-1

β

(IL-1

β

) and tumour necrosisfactor-

α

(TNF-

α

) in mice. When given orally, hydroalcoholic extract (0.001–10 mg/kg), produced potent and dose-dependentinhibition of acetic acid-induced visceral pain. In the formalin test, the hydroalcoholic extract (0.0001–0.1 mg/kgorally) also caused significant inhibition of both the early (neurogenic pain) and the late (inflammatory pain) phases offormalin-induced licking. However, it was more potent and efficacious in relation to the late phase of the formalin test. Thecapsaicin-induced nociception was also reduced at a dose of only 1.0 mg/kg orally. The hydroalcoholic extract significantlyreduced the cinnamaldehyde-induced nociception at doses of 0.01, 0.1 and 1.0 mg/kg orally. Moreover, the hydroalcoholicextract (0.001–1.0 mg/kg orally) caused significant and dose-dependent inhibition of glutamate-induced pain. However,only rutin, but not phebalosin or aurapten, isolated from

P. paniculata

, administered intraperitoneally to mice, produceddose-related inhibition of glutamate-induced pain. Furthermore, the hydroalcoholic extract (0.1–100 mg/kg orally) had noeffect in the tail-flick test. On the other hand, the hydroalcoholic extract caused a significant increase in the latency toresponse at a dose of 10 mg/kg orally, in the hot-plate test. The hydroalcoholic extract (0.1 mg/kg orally) antinociception,in the glutamate test, was neither affected by intraperitoenal treatment of animals with

l

-arginine (precursor of nitric oxide,600 mg/kg) and naloxone (opioid receptor antagonist, 1 mg/kg.) nor associated with non-specific effects such as musclerelaxation or sedation. In addition, oral administration of hydroalcoholic extract produced a great inhibition of the pain-related behaviours induced by intrathecal injection of glutamate,

N

-methyl-

d

-aspartate (NMDA), IL-1

β

and TNF-

α

, but notby

α

-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), kainate or trans-1-amino-1.3-cyclopentanediocarboxylicacid (trans-ACPD). Together, our results suggest that inhibition of glutamatergic ionotropic receptors, may account forthe antinociceptive action reported for the hydroalcoholic extract from

P. paniculata

in models of chemical pain used in

this study.

The plants of the

Polygalaceae

family are widespread intropical regions. Among the members of this family, is thegenus

Polygala

, with approximately 500 species [1]. In addi-tion,

Polygala paniculata

Linneu, a plant of the genus

Polygala

,is a native small arbust, that grows in Brazil’s Atlanticcoast, known by the popular names of ‘barba-de-são-joão’,‘bromil’, ‘vassourinha branca’ and ‘mimosa’. Furthermore,

P. paniculata

L. is used in the folk medicine as a tonic remedyand for the treatment of different inflammatory diseases,such as asthma, bronchitis, arthritis and other pathologies,including disorders of the kidney [2]. Apart from thesemedicinal uses, there are reports showing antipsychotic [3],

anti-tumoural [4], anti-inflammatory [5], antinociceptive [6,7],anti-spasmodic [8] activity of some

Polygala

species. Inaddition, preliminary data from our group have demon-strated that hydroalcoholic extract from

P. paniculata

L.,produced

in vivo

protective effects against methylmercury-induced neurotoxicity [9].

Phytochemical studies carried out with plant of the genus

Polygala

revealed an abundant amount of several com-pounds, including cytotoxic lignans [4], saponins [3], xan-thones, coumarins and flavonoids [10]. More recently, it hasbeen reported the isolation of two xanthones, 1-hydroxy-5-methoxy-2.3-methylenedioxyxanthone and 1.5-dihydroxy-2.3-dimethoxyxanthone, together with coumarin murragatinand flavonol rutin and two sterol, spinasterol and

δ

25-spinasterol from

P. paniculata

L. [11].Recently, we have demonstrated that the hydroalcoholic

extract from

P. paniculata

markedly inhibited gastricmucosal lesions induced by ethanol 70%. According to our

Author for correspondence: Adair R. Santos, Department of Physio-logical Sciences, Center of Biological Sciences, Federal Universityof Santa Catarina, 88040-900, Florianópolis, SC, Brazil (fax+55 48 37219672, e-mail arssantos@ccb.ufsc.br).

HYDROALCOHOLIC EXTRACT AND THE FLAVONOID RUTIN

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results, the protective effect of hydroalcoholic extract mayhave been related to a slight increase or maintenance of gastricmucus secretion, an effect that may involve prostaglandinsand the antioxidant activity of some constituents found inthe extract [12].

In addition, preliminary studies have shown that thehydroalcoholic extract of

P. cyparissias

, another plant of thegenus

Polygala

, and an isolated xanthone (dihydroxy-2.3-dimethoxyxanthone) produced a dose-related and markedantinociception [6]. Recently, we have demonstrated that thehydroalcoholic extract from

Polygala sabulosa

(A. W. Bennett)produced antinociceptive action against the acetic acid-induced visceral nociception. The presence of styryl-pyrones,coumarin (scopoletin) and steroid (

α

-spinasterol) gives theplant a potent antinociceptive effect [7].

However, to date, no pharmacological study on the anti-nociceptive activity of

P. paniculata

has been carried out. Inthe present study we have attempted to examine the possibleantinociceptive action of the hydroalcoholic extract of

P. paniculata

L., in chemical and thermal models of nocicep-tion in mice. In addition, we also analysed the possible anti-nociceptive effect of the phebalosin, aurapten and rutinisolated from this plant.

Material and Methods

Animals.

Experiments were conducted with male Swiss mice (25–35 g),housed at 22 ± 2

°

under a 12-hr light/12-hr dark cycle (lights onat 6 a.m.) and with access to food and water

ad libitum

. Animalswere acclimatised to the laboratory for at least 1 hr before testingand were used only once throughout the experiments. The experi-ments were performed after approval of the protocol by the Institu-tional Ethics Committee and were carried out in accordance withthe current guidelines for the care of laboratory animals and theethical guidelines for investigations of experimental pain in con-scious animals [13]. The numbers of animals (n = 6–12) and inten-sities of noxious stimuli used were the minimum necessary todemonstrate the consistent effects of the drug treatments.

Preparation of ethanolic extract, isolation and chemical identificationof the isolated compound.Polygala paniculata

L. was collected in Daniela Beach (Florianópolis,Santa Catarina State, Brazil) and was classified by M.Sc. OlavoAraújo Guimarães (Federal University of Paraná, Curitiba, Brazil).A voucher specimen of this plant was deposited in the herbarium ofthe Botanical Department of Federal University of Paraná. Thedried whole plant (3500 g) was minced and submitted to exhaustiveextraction by maceration with ethanol: water (80 : 20) in closedrecipient. After maceration, the extract was filtered through a paperfilter and the solvent was evaporated under reduced pressure (50

°

)in a rotative evaporator. The respective crude hydroalcoholicextract (yield 50 g) was obtained.

After the solvent removal of the hydroalcoholic extract, theextract was concentrated with reduced pressure and successivelypartitioned with EtOH and dichloromethane CH

2

Cl

2

. The flavo-noid quercetin-3-rutinoside (rutin) yielded (430 mg), the majorcompound of hydroalcoholic extract, was found in the ethanol frac-tion. The fraction eluated with CH

2

Cl

2

was subjected to a chromato

-

graphy over silica gel with a mixture of

n

-hexano–EtOAc–EtOHwith increasing polarity. Elution with

n

-hexane–EtOAc–EtOH yield7- genariloxi cumarin (87 mg) also known as aurapten. The edicu-ticular (cuticle) wax was obtained from the hexanic extract of

P. paniculata

. This wax was successively partitioned with

n

-hexane

and phebalosin was yielded (2 g). The antinociceptive activity ofhexanic extract was not investigated in this work.

Drugs.

The following substances were used: acetic acid, formalin and mor-phine hydrochloride (Merck, Darmstadt, Germany); N

ω

-nitro-

l

-arginine,

l

-arginine hydrochloride, capsaicin, cinnamaldehyde,

l

-glutamic acid hydrochloride, naloxone hydrochloride, morphinehydrochloride (Sigma Chemical Co., St. Louis, MO). All drugswere dissolved in saline, with the exception of capsaicin, phebalosinand aurapten that were dissolved in tween 80 plus saline and absoluteethanol. The final concentration of tween 80 and ethanol did notexceed 10% and did not cause any

per se

effect.

Assessment of antinociceptive effect of HE from P. paniculata andisolated compound.Abdominal constriction response caused by intraperitoneal injectionof acetic acid.

The abdominal constrictions were induced accord-ing to procedures described previously [14] and resulted in contrac-tion of the abdominal muscle together with a stretching of the hindlimbs in response to an intraperitoneal injection of acetic acid(0.6%) at the time of the test. Mice were pre-treated with hydro

-

alcoholic extract (0.001–10 mg/kg orally), 1 hr before injection ofthe irritant. Control animals received a similar volume of vehicle(10 ml/kg). After the challenge, the mice were individually placedinto glass cylinders of 20 cm diameter, and the abdominal constric-tions were counted cumulatively over a period of 20 min. Antinoci-ceptive activity was expressed as the reduction in the number ofabdominal constrictions, that is, the difference between controlanimals (mice pre-treated with vehicle) and animals pre-treatedwith hydroalcoholic extract.

Formalin-induced nociception.

The procedure used was essentiallythe same as that described previously [14,15]. Animals received20

μ

l of a 2.5% formalin solution (0.92% formaldehyde) made up insaline, injected intraplantarly in the ventral surface of the right hindpaw. Animals were observed from 0 to 5 min. (neurogenic phase)and 15–30 min. (inflammatory phase) and the time spent licking theinjected paw was recorded with a chronometer and considered asindicative of nociception. The animals received hydroalcoholicextract of

P. paniculata

(0.0001–0.1 mg/kg orally) 1 hr before, withbasis of a previous time-response curve. Control animals receivedvehicle (10 ml/kg orally).

Capsaicin- and cinnamaldehyde-induced nociception.

In order to pro-vide more direct evidence concerning the participation of TRPV1and TRPA1 in the effect of the hydroalcoholic extract of

P. panicu-lata

, we investigated its antinociceptive effect in capsaicin- (aTRPV1 receptor agonist) and cinnamadehyde- (a TRPA1 receptoragonist) induced licking in the mouse paw. The procedure used wassimilar to that described previously [15,16]. After an adaptationperiod (20 min.), 20

μ

l of capsaicin (1.6

μ

g/paw prepared in saline)or 20

μ

l of cinnamaldehyde (10 nmol/paw prepared in saline) wereinjected intraplantarly in the ventral surface of the right hind paw.Animals were observed individually for 5 min. following capsaicinor cinnamaldehyde injection. The amount of time spent licking theinjected paw was recorded with a chronometer and was consideredas indicative of nociception. The animals were treated with hydro

-

alcoholic extract of

P. paniculata

in doses of 0.01–10 mg/kg orally,1 hr before capsaicin injection or in doses of 0.01–1.0 mg/kg orally,1 hr before cinnamaldehyde injection. Control animals receivedvehicle (10 ml/kg orally).

Glutamate-induced nociception.

In an attempt to provide moredirect evidence concerning the interaction of the hydroalcoholicextract of

P. paniculata

or isolated compounds with the glutamatergicsystem, we separately investigated whether or not hydroalcoholicextract or isolated compounds was able to antagonize glutamate-induced licking of the mouse paw. The procedure used was similarto that described previously [17]. A volume of 20

μ

l of glutamate

308

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ET AL.

© 2009 The AuthorsJournal compilation

© 2009 Nordic Pharmacological Society.

Basic & Clinical Pharmacology & Toxicology

,

104

, 306–315

(10

μ

mol/paw prepared in saline) was injected intraplantarly in theventral surface of the right hind paw. Animals were observed indi-vidually for 15 min. after glutamate injection. The amount of timespent licking the injected paw was recorded with a chronometer andwas considered as indicative of nociception. The animals weretreated with hydroalcoholic extract (0.001–1.0 mg/kg orally) 1 hrbefore and with the isolated compounds, phebalosin (1–30 mg/kgintraperitoneally), aurapten (3–100 mg/kg intraperitoneally) andrutin (1–100 mg/kg intraperitoneally) 0.5 hr before glutamate injec-tion. Control animals received a similar volume of vehicle (10 ml/kg,orally or intraperitoneally).

In separate series of experiments, the hydroalcoholic extract from

P. paniculata

(0.1 mg/kg) was given orally and the time-course ofthe hydroalcoholic extract antinociceptive effect was evaluated atthe time points of 1, 2, 4, 6, 8 and 12 hr after the administration, inthe glutamate-induced nociception. Control animals received asimilar volume of vehicle orally (10 ml/kg) and were observed at thesame intervals of time.

Intrathecal injection of excitatory amino acids and pro-inflammatorycytokine-induced pain behaviour in mice.

In another set of experi-ments, the interaction of the hydroalcoholic extract from

P. panicu-lata

with the glutamatergic system was investigated by using bothionotropic and metabotropic agonists of excitatory aminoacidsreceptors, which were administered by the intrathecal route causingbiting behaviour in mice [18]. In addition, we investigated

P. panicu

-

lata

effect upon pro-inflammatory cytokines IL-1

β

- and TNF-

α

-induced biting behaviour in mice [19,20]. The animals receivedhydroalcoholic extract of

P. paniculata

(1 mg/kg orally) 1 hr beforeintrathecal injection of 5

μ

l of the drugs. Injections were given tofully conscious mice using the method described by Hylden andWilcox [21]. Briefly, the animals were manually restrained and a 30-gauge needle, attached to a 50-

μ

l microsyringe, was insertedthrough the skin and between the vertebrae into the subdural spaceof the L5–L6 spinal segments. The biting behaviour was defined asa single head movement directed at the flanks or hind limbs, result-ing in contact of the animal’s snout with the target organ. Intrathecalinjections were given over a period of 5 sec. The nociceptiveresponse was elicited by glutamate (an excitatory amino acid,30

μ

g/site), NMDA (a selective agonist of NMDA-subtype of gluta-matergic ionotropic receptors, 25 ng/site), AMPA (a selective agonistof AMPA-subtype of glutamatergic ionotropic receptors, 25 ng/site),kainate (a selective agonist of kainate-subtype of glutamatergicionotropic receptors, 23.5 ng/site), trans-ACPD (an agonist ofmetabotropic glutamate receptors, 8.6

μ

g/site, i.t.), IL-1

β

(1 pg/site)and TNF-

α

(0.1 pg/site) [19–21]. As control, a group of micereceived vehicle (saline) intrathecally, the amount of biting behav-iour was quantified and discounted from all groups. The amount oftime the mice spent biting or licking was evaluated following localpost-injections of one of the following agonists: glutamate 3 min.;NMDA 5 min.; AMPA 1 min.; kainate 4 min.; t-ACPD 15 min.;IL-1

β

and TNF-

α

15 min.

Tail-flick test.

A radiant heat tail-flick analgesimeter (Ugo-Basile,Italy) was used to measure response latencies according to themethod described previously by D’Amour & Smith [22], with minormodifications. Animals responded to a focused heat stimulus byflicking or removing their tail, when exposed to a photocell in theapparatus immediately below it. The reaction time was recorded forcontrol mice (vehicle, 10 ml/kg orally) and for animals pre-treated1 hr before with hydroalcoholic extract (0.1–10 mg/kg orally) orwith morphine (5.0 mg/kg subcutaneously). An automatic 20-sec.cut-off was used to minimize tissue damage. Animals thatremained on the apparatus for an average of 12 sec. were selected2 hr previously on the basis of their reactivity in the test. To deter-mine the baseline, each animal was tested before administration ofdrugs, using a moderate radiant heat stimulus. The maximal per-centage of the effect (MPE) of drug-induced antinociception wascalculated as follows: %MPE = [(post-drug

−

pre-drug)/(20

−

pre-drug)]

×

100.

Hot-plate test.

The hot-plate test was used to measure the responselatencies according to the method described previously by Eddyand Leimbach [23]. In these experiments, the hot-plate (Ugo-Basile,Socrel, model-DS 37) was maintained at 50 ± 1

°

. Animals wereplaced into a glass cylinder and the time (sec.) between placementand shaking or licking of the paws or jumping was recorded as theindex of response latency. The reaction time was recorded foranimals pre-treated with the of

P. paniculata

(0.1–10 mg/kg orally., 1 hrbefore) or with morphine (5.0 mg/kg subcutaneously, 0.5 hr before),which was used as a positive control. Animals that remained on theapparatus for an average of 16 sec. were selected 2 hr previously onthe basis of their reactivity in the model. A latency period (cut-off)of 30 sec. was defined as complete antinociception. Control animalsreceived the vehicle used to dilute these drugs. To determine thebaseline, each animal was tested before administration of drugs,using the same heat stimulus. The MPE of drugs-induced antinoci-ception was calculated as follows: %MPE = [(post-drug

−

pre-drug)/(30

−

pre-drug)]

×

100.

Analysis of possible mechanism of action of hydroalcoholic extract.

To address some of the mechanisms by which hydroalcoholicextract causes antinociception, the glutamate-induced nociceptiontest was chosen, in which hydroalcoholic extract was shown to bemore potent and efficacious as compared to other nociceptive tests.The doses of the drugs used were selected on the basis of literaturedata [14] and also based in previous results from our laboratory.

Involvement of opioid system.

To assess the possible participationof the opioid system in the antinociceptive effect of hydroalcoholicextract, mice were pre-treated with naloxone (1 mg/kg intraperito-neally, a non-selective opioid receptor antagonist), and after20 min. the animals received an injection of hydroalcoholic extract(0.1 mg/kg orally), morphine (5 mg/kg subcutaneously) or vehicle(10 ml/kg orally). The algesic responses to glutamate were recorded1, 0.5 or 1 hr after the administration of hydroalcoholic extract,morphine or vehicle, respectively. Another group of animals waspre-treated with vehicle and after 20 min. received hydroalcoholicextract, morphine or vehicle, 1, 0.5 or 1 hr before glutamate injec-tion, respectively.

Involvement of nitric oxide-l-arginine pathway.

To investigate therole played by the nitric oxide-

l

-arginine pathway in the antinocic-eption caused by hydroalcoholic extract, the mice were pre-treatedwith

l

-arginine (600 mg/kg intraperitoneally, a nitric oxide precursor)and after 20 min. they received hydroalcoholic extract (0.1 mg/kgorally),

N

ω

-nitro-

l

-arginine (

l

-NOARG, 100 mg/kg intraperito-neally, a nitric oxide synthase inhibitor) or vehicle (10 ml/kg orally).The algesic responses to glutamate were recorded 1, 0.5 or 1 hrafter the administration of hydroalcoholic extract,

l

-NOARG, orvehicle, respectively. Another group of animals was pre-treated withvehicle and after 20 min. received hydroalcoholic extract,

l

-NOARG or vehicle, 1, 0.5 or 1 hr before glutamate injection,respectively.

Measurement of locomotor activity.

To evaluate some non-specific muscle-relaxant or sedative effects ofhydroalcoholic extract from

P. paniculata

, mice were submitted tothe open-field test. The ambulatory behaviour was assessed in theopen-field test as described previously [24]. The apparatus consistedof a wooden box measuring 40

×

60

×

50 cm. The floor of the arenawas divided into 12 equal squares, and the number of squarescrossed with all paws (crossings) was counted in a 6-min. ses-sion. Mice were treated with hydroalcoholic extract from

P. panicu-lata

(0.1 or 10 mg/kg orally) or vehicle (10 ml/kg orally) 1 hr previously.

Statistical analysis.

The results are presented as mean ± S.E.M., except the ID

50

values(i.e., the dose of hydroalcoholic extract reducing the nociceptiveresponse by 50% relative to the control value), which are reportedas geometric means accompanied by their respective 95% confidence

HYDROALCOHOLIC EXTRACT AND THE FLAVONOID RUTIN

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limits. The ID

50

value was determined by linear regression fromindividual experiments using linear regression GraphPad software(GraphPad software, San Diego, CA). The statistical significance ofdifferences between groups was detected by Student’s unpairedt

-

test and

anova

followed by Newman-Keuls’ test when indicated.P-values less than 0.05 (P < 0.05) were considered as indicative ofsignificance.

Results

Abdominal constriction response caused by intraperitoneal injection of acetic acid.

Figure 1 shows that hydroalcoholic extract (0.001–10 mg/kg),given orally 1 hr earlier than, produced dose-related inhibition

of the acetic acid-induced visceral nociception in mice, withmean ID50 values (and their respective 95% confidence limits)of 0.22 (0.13–0.38) mg/kg and inhibition of 76 ± 7% at adose of 10 mg/kg.

Formalin-induced nociception.

The results depicted in fig. 2A and B shows that thehydroalcoholic extract from P. paniculata (0.0001–0.1 mg/kgorally) caused a significant inhibition of both the neurogenic(0–5 min.) and inflammatory (15–30 min.) phases of formalin-induced licking. However, its antinociceptive effects weresignificantly more pronounced against the second phase ofthis model of pain. The calculated mean ID50 value (and itsrespective 95% confidence limits) for these effects were: >0.1and 0.0042 (0.0035–0.0049) mg/kg and the inhibitionsobserved were 24 ± 6 and 69 ± 4% at a dose of 0.01 mg/kg,for first and second phase, respectively.

Capsaicin- and cinnamaldehyde-induced nociception.

Oral administration of hydroalcoholic extract of P. pani-culata inhibited the capsaicin-induced neurogenic pain,only at a dose of 1.0 mg/kg orally, with inhibition of50 ± 3% (fig. 3A). The cinnamaldehyde-induced pain wasalso inhibited significantly at doses of 0.01–1.0 mg/kgorally, the inhibitions observed were 57 ± 5, 47 ± 7 and67 ± 8% at doses of 0.01, 0.1 and 1.0 mg/kg respectively andthe calculated mean ID50 value was 0.27 (0.13–0.57) mg/kg(fig. 3B).

Glutamate-induced nociception.

The hydroalcoholic extract of P. paniculata (0.001–1 mg/kg),given orally, produced marked and dose-dependent attenu-ation of the glutamate-induced nociception. The ID50 valuewas 0.0084 (0.0077–0.0092) mg/kg. The peak of inhibitionwas 72 ± 3% at 1 mg/kg (fig. 4A). A time-course analysis ofthe antinociceptive effect of hydroalcoholic extract given

Fig. 1. Effect of the hydroalcoholic extract of P. paniculataadministered orally against acetic acid-induced writhing movementsin mice. Each column represents the mean of the values obtained in6–12 animals and the error bars indicate the S.E.M. The closedcolumn indicates the control value (C) (animals injected withvehicle) and the open columns correspond to animals treated withextract, the asterisks denote the significance levels, when comparedwith control group, (one-way anova followed by Newman–Keulstest) **P < 0.01 and ***P < 0.001.

Fig. 2. Effect of the hydroalcoholic extract of P. paniculata administered orally against formalin-induced licking (first phase, panel A, andsecond phase, panel B) in mice. Each column represents the mean of the values obtained in 6–12 animals and the error bars indicate theS.E.M. The closed columns indicates the control value (C) (animals injected with vehicle) and the open columns correspond to animalstreated with extract, the asterisks denote the significance levels, when compared with control group, (one-way anova followed by Newman–Keuls test) *P < 0.05; **P < 0.01 and ***P < 0.001.

310 FERNANDA DA R. LAPA ET AL.

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orally is shown in fig. 4B. Hydroalcoholic extract producedmarked antinociception as early as 1 hr after oral adminis-tration, an action that remained significant up to 8 hr afterthe administration (fig. 4B). Thus, the time-point of 1 hrwas chosen for all further studies with independent groupsof animals.

Interestingly, when rutin, isolated from P. paniculata, wasadministered intraperitoneally to mice, it produced dose-related inhibition of glutamate-induced pain, with a meanID50 value of 10.9 (7.8–15.4) mg/kg and the peak of inhibi-tion observed was 82 ± 7% (fig. 4C). However, phebalosinor aurapten, administered intraperitoneally to mice, produced

Fig. 3. Effect of the hydroalcoholic extract obtained from P. paniculata administered orally against capsaicin (A)- and cinnamaldehyde(B)-induced nociception in mice. Each column represents the mean of the values obtained in 6–12 animals and the error bars indicate theS.E.M. The closed columns indicates the control value (C) (animals injected with vehicle) and the open columns correspond to animalstreated with extract, the asterisks denote the significance levels, when compared with control group, (one-way anova followed by Newman–Keuls test) **P < 0.01, ***P < 0.001.

Fig. 4. Effect of the hydroalcoholic extract (A), rutin (C), phebalosin (D) and aurapten (E) obtained from P. paniculata against glutamate-induced licking in mice. Panel (B) Time-course of the antinociceptive effect of hydroalcoholic extract (HE) on glutamate-induced licking inmice. Each column represents the mean of the values obtained in 6–12 animals and the error bars indicate the S.E.M. The closed columnsindicates the control value (C) (animals injected with vehicle) and the open columns correspond to animals treated with extract orcompounds, the asterisks denote the significance levels, when compared with control group, (one-way anova followed by Newman–Keulstest) *P < 0.05, **P < 0.05 and ***P < 0.001.

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inhibition of 64 ± 12% and 71 ± 7% of glutamate-inducedlicking at doses of 10 and 100 mg/kg, respectively(fig. 4D,F).

Intrathecal injection of excitatory amino acids and pro-inflammatory cytokine-induced pain behaviour in mice.

The results depicted in fig. 5 shows that hydroalcoholicextract of P. paniculata (1 mg/kg orally) inhibited the noci-ceptive responses induced by spinal injections of glutamate,NMDA, IL-1β and TNF-α in mice. The inhibition valueswere 48 ± 9%, 81 ± 5%, 68 ± 13% and 62 ± 15%, respec-tively. In contrast, hydroalcoholic extract had no effectagainst AMPA, kainate and trans-ACPD-mediated bitingresponses (fig. 5).

Hot-plate test and tail-flick test.

The hydroalcoholic extract from P. paniculata (1.0–10 mg/kgorally) did not alter the latency response to the tail-flick

test (fig. 6B). The tail-flick test basal latency values (in sec.)were 10.4 ± 0.9; 10.4 ± 0.8; 9.2 ± 1; 9.7 ± 0.8; 11.0 ± 1.1 forthe groups of animals that were afterwards treated withsaline, morphine, hydroalcoholic extract at doses of 0.1, 1.0and 10 mg/kg, respectively. In the tail-flick test was used abasal cut-off of 12 sec. In contrast, the hydroalcoholicextract (10 mg/kg orally) given 1 hr previously, caused a sig-nificant increase of the latency response in the hot-plate test(fig. 6A). Under similar conditions morphine (5.0 mg/kgsubcutaneously), used as reference drug, caused a significantand marked analgesic effect in both models.

Analysis of possible mechanism of action of hydroalcoholic extract.

Involvement of the opioid system. The results in fig. 7A showsthat the pre-treatment of mice with naloxone (1 mg/kg intra-peritoneally, a non-selective opioid receptor antagonist),given 20 min. before, largely reversed the antinociception

Fig. 5. Effect of the hydroalcoholic extract obtained from P. paniculata (1.0 mg/kg) administered orally on biting response caused byintrathecal injection of glutamate (30 μg/site), NMDA (25 ng/site), AMPA (25 ng/site), kainate (23.5 ng/site), trans-ACPD (8.6 μg/site), IL-1β (1 pg/site) and TNF-α (0.1 pg/site) in mice. Each column represents the mean of the values obtained in 6-12 animals and the error barsindicate the S.E.M. The asterisks denote the significance levels, when compared with untreated groups, ***P < 0.001 by Student’s unpaired t-test.

Fig. 6. Effect of the hydroalcoholic extract obtained from P. paniculata administered orally in hot plate (A) and tail flick (B) tests in mice.Each column represents the mean of the values obtained in 6–12 animals and the error bars indicate the S.E.M. The closed columns indicatesthe control value (C) (animals injected with vehicle), hatched columns indicates the control group treated with morphine and the opencolumns correspond to animals treated with extract, the asterisks denote the significance levels, when compared with control group (C), (one-wayanova followed by Newman–Keuls test) **P < 0.01.

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caused by injection of morphine, but did not significantlychange the antinociceptive action caused by hydroalcoholicextract, when assessed against glutamate test.

Involvement of nitric oxide–l-arginine pathway.

The systemic pre-treatment of mice with the nitric oxideprecursor l-arginine (600 mg/kg intraperitoneally), given20 min. earlier, significantly reversed the antinociceptioncaused by l-NOARG (100 mg/kg intraperitoneally, a nitricoxide synthase inhibitor) when analysed against glutamate-induced nociception (fig. 7B). Under the same conditions,l-arginine did not significantly modify the antinociceptioncaused by hydroalcoholic extract in the glutamate test (fig. 7B).

Evaluation of locomotor activity.

The hydroalcoholic extract from P. paniculata (0.1, 1 and10 mg/kg orally) did not affect the locomotor activity ofmice submitted to the open-field test when compared toanimals that received vehicle (control group). In the locomotoractivity the means ± S.E.M. of crossings number were99.3 ± 6.3; 95.8 ± 6.8; 89.8 ± 6.7 and 97.3 ± 4.9 for the con-trol, 0.1, 1 and 10 mg/kg group, respectively.

Discussion

The present study demonstrates that systemic (oral) admin-istration of hydroalcoholic extract from P. paniculata elicitsa potent and dose-dependent inhibition of the nociceptivebehavioural response in mice submitted to chemicalpain-inducing stimuli. The most relevant findings in thework are that (1) oral administration of hydroalcoholicextract caused significant inhibition of acetic acid-inducedvisceral pain response; (2) oral administration of hydroalco-holic extract also caused significant inhibition against bothphases of the pain response to the intraplantar injection of

formalin, and against the capsaicin- and cinnamaldehyde-induced nociception; (3) the algesic response caused byintraplantar injection of glutamate was also greatly inhibitedby hydroalcoholic extract; (4) the antinociceptive action ofhydroalcoholic extract in the glutamate test was not significantlyreversed by intraperitoneal pre-treatment of animals withnaloxone and l-arginine; (5) oral administration ofhydroalcoholic extract significantly reduced the glutamate,NMDA-, IL-1β- and TNF-α-induced biting, although, itdid not cause any significant reduction on trans-ACPD,AMPA and kainate nociception response; (6) hydroalco-holic extract did not change the response latency of animalsin the tail-flick test, but increased the response latency in thehot-plate test and (7) the dose of hydroalcoholic extract thatcaused significant antinociception did not produce anystatistically significant motor dysfunction or any detectableside-effect.

The acetic acid-induced writhing reaction in mice,described as a typical model for inflammatory pain, haslong been used as a screening tool for the assessment ofanalgesic or anti-inflammatory properties of new agents[25,26]. At the cellular level, protons depolarize sensoryneurones by directly activating a non-selective cationicchannel localized on cutaneous, visceral and other types ofnocisponsive peripheral afferent C-fibres [27,28]. The resultsreported here indicate, that oral administration of hydro-alcoholic extract produced marked and dose-related anti-nociception when assessed in acetic acid-induced visceralnociception, at doses that did not produce any statisticallysignificant motor dysfunction. To our knowledge this is thefirst report of its kind in the literature.

Also of interest are the results showing that hydro-alcoholic extract of P. paniculata caused significant and dose-related antinociception when administered orally againstboth neurogenic (early phase) and inflammatory (late phase)

Fig. 7. Effect of pre-treatment of animals with naloxone (1 mg/kg, A) or l-arginine (600 mg/kg, B) on the antinociceptive profiles ofhydroalcoholic extract of P. paniculata (HE – 0.1 mg/kg orally), morphine (5 mg/kg, A) or l-NOARG (100 mg/kg, intraperitoneally, B)against the glutamate-induced licking in mice. Each column represent the mean of the values obtained in 6–12 animals and the error barsindicate the S.E.M. #P < 0.001 comparing agonist (P. paniculata, morphine or l-NOARG) plus antagonists (naloxone or l-arginine) versusagonist plus vehicle (control); ***P < 0.01 compared with corresponding control values (animals injected with the vehicle alone).

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pain responses caused by formalin injection in mice. Theformalin-induced nociception is a well-described model ofnociception and can be consistently inhibited by typicalanalgesic and anti-inflammatory drugs, including morphine,indomethacin and dexamethasone [29,30]. Considering theinhibitory property of P. paniculata on the second phase offormalin, we might suggest an anti-inflammatory action ofthe plant extract.

In addition, recent studies have shown that formalin acti-vates primary afferent sensory neurons through a specificand direct action on TRPA1, a member of the TransientReceptor Potential family (TRP) of cation channels that ishighly expressed by a subset of C-fibre nociceptors [31].To investigate the further participation of TRPA1 receptorin the antinociceptive effect of hydroalcoholic extract ofP. paniculata, we assessed the cinnamaldehyde-induced noci-ception. Currently, it was demonstrated that intraplantaradministration of cinnamaldehyde, a TRPA1 agonist recep-tor, to mice produced a dose-dependent spontaneous noci-ception [16]. Our results show that a hydroalcoholic extractsignificantly reduced the cinnamaldehyde-induced pain.This result is in line with that obtained in formalin modeland indicates that hydroalcoholic extract at low doses probablyinteracts with TRPA1 receptor located in C-fibres reducingthe formalin-induced nociception.

It has been proposed that the capsaicin-induced noci-ception is brought about by activation of another TRPreceptor, the vanilloid receptor (TRPV), termed TRPV1,a ligand-gated non-selective cation channel in primarysensory neurons [32–34]. Our results also show that oraladministration of hydroalcoholic extract of P. paniculataproduced a partial, but significant, reduction of the noci-ceptive response caused by intraplantar. injection of capsaicininto the mouse hindpaw.

Of note, the licking response induced by formalin, capsaicinand glutamate results from a combination of peripheralinput and spinal cord sensitization [15,17,30,35]. The intra-plantar injection of formalin, capsaicin or glutamatereleases excitatory amino acids, PGE2, NO, neuropeptidesand kinins in the spinal cord [15,17,30,31,35,36]. Taking thisinto account, the antinociception of P. paniculata could bedependent on either peripheral or central sites of action.

In this study, we observed in formalin and capsaicin modelsthat the antinociceptive effect of hydroalcoholic extractwas higher at low doses. These finding suggested that theconstituents from hydroalcoholic extract at lower dosesmight be interacted peripherally with nociceptors or servedas scavengers. We have recently shown the antioxidant activityof hydroalcoholic extract and its isolated flavonoid rutin,verified in the DPPH free-radical scavenging assay [12].Hence, it is possible that an antioxidant activity of hydro-alcoholic extract might have been related to the inhibitoryeffect against the inflammatory component and generationof free radicals observed in these models and consequentlyreducing the nociceptive behaviour.

The hydroalcoholic extract from P. paniculata produced adose-dependent antinociceptive effect on the glutamate

induced paw licking response. Recently, Beirith et al. [17]found that the nociceptive response induced by glutamateappears to involve peripheral, spinal and supraspinal sitesof action and is greatly mediated by both NMDA and non-NMDA receptors as well as by the release of nitric oxide orby some nitric oxide-related substance. Hence, an effect ofthe plant extract directly on the receptors or second messen-gers related to these transmitters could avoid the nociceptiveresponse. Interestingly, the P. paniculata antinociception wasextended up to 8 hr after the treatment, an effect that ishardly reached for clinically used analgesics.

The effect of P. paniculata against nociception induced byglutamate is of great interest since glutamate plays a signifi-cant role in nociceptive processing in both central andperipheral nervous systems [17,37,38]. Indeed, drugs capableof blocking either iGluRs (ionotropic glutamate recep-tors) or mGluRs (metabotropic glutamate receptors) exhibitantinociceptive effects in several mammalian species includ-ing human beings [39]. Therefore, it is supposed that sub-stances that block GluRs may have clinical potential inthe management of some painful states. On the other hand, theuse of these substances as analgesics is hampered due to theunaccepted side-effects displayed by these drugs [37,40].Here, we have verified that the highest dose of P. paniculata(10 mg/kg) did not cause any disturbance on the locomotoractivity when assessed in the open field test. In addition,hydroalcoholic extract of P. paniculata administered at adose of 1.0 mg/kg, inhibited efficaciously the biting behav-iour induced by iGluRs, mainly those induced by NMDA,but not AMPA and kainate agonists. However, P. paniculatatreatment did not reduce biting behaviour induced bymGluR (trans-ACPD). Therefore, it is plausible that someconstituents of P. paniculata, when the extract was adminis-tered at a higher dose (1.0 mg/kg orally), may reach thecentral nervous system and interact with the pathwaysdepending on the activation of iGluRs (NMDA receptor),instead of observed on formalin-induced pain where a onehundred time lower dose of hydroalcoholic extract might beresponsible for a local effect probably related to interactionof active compounds from hydroalcoholic extract with localTRPA1 receptors on afferent fibres.

The intrathecal injection of cytokines, such as TNF-α,IL-1β, IL-1α and IFN-γ, induces nociceptive behaviour thatis partly mediated through the activation of central nerveterminals and subsequent release of glutamate [40,41]. Thisstudy shows that hydroalcoholic extract was able to inhibitthe nociceptive behaviour induced by intrathecal injectionof IL-1β and TNF-α. There are two ways of interpretingthis result. One possibility is that the constituents present inthe hydroalcoholic extract directly inhibit the action ofcytokines, preventing them from depolarizing projectionneurons and primary afferents. Another possibility is thatthe hydroalcoholic extract inhibits the further activation ofprojection neurons by glutamate, through the inhibition ofNMDA receptors. The second hypothesis seems more plau-sible, since hydroalcoholic extract was capable of inhibitingnociceptive behaviour induced by NMDA. Therefore, it is

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suggested that the inhibition of the cytokines-induced bitingbehaviour is due to the antagonism of glutamatergic trans-mission at the spinal level.

Further experiments were undertaken to elucidate thepossible involvement of opioid and l-arginine–nitric oxidesystems on the antinociceptive properties of the hydro-alcoholic extract from P. paniculata. The results obtaineddemonstrated that the activation of the opioid naloxone-sensitive pathway is unlikely to be involved in the antinocic-eption caused by hydroalcoholic extract as naloxone, underconditions where it fully reversed morphine-induced anti-nociception, had no effect against the hydroalcoholic extractaction.

In addition, the results of the present study also suggestthat the antinociception caused by hydroalcoholic extractdid not involve any interaction with l-arginine–nitric oxidepathway, since the treatment of animals with l-arginine (aprecursor of nitric oxide), under conditions in which it con-sistently reversed the antinociception caused by l-NOARG(a known nitric oxide synthase inhibitor), failed to interferewith hydroalcoholic extract-induced antinociception.

The hydroalcoholic extract of P. paniculata was devoid ofantinociceptive action when assessed in a thermal model ofnociception, the tail-flick test, under conditions that mor-phine has a marked antinociceptive effect. Interestingly, ourresults showed that the hydroalcoholic extract at the higherdose tested (10 mg/kg orally) had increased the latencyresponse in the hot-plate test. These results may suggests acentral analgesic action for hydroalcoholic extract since thehot-plate test is known to involve the activation of suprasp-inal structures and the tail-flick response is more related tospinal reflex triggered by C fibres when it is elicited by heat[42].

Finally, the chemical studies carried out with thishydroalcoholic extract allowed us to isolate and identifyrutin, phebalosin and aurapten in P. paniculata, which seemnot to be responsible, at least in part, for the antinociceptiveproperties reported for the hydroalcoholic extract of P.paniculata, considering that the necessary doses to producesignificant inhibition of the nociception caused by glutamatewas about 3–100 times larger than the one of hydroalcoholicextract. On the basis of this finding, we can speculate thatthese compounds as well as others found in P. paniculata,might act synergically contributing to the potent antinocice-ptive action of P. paniculata. Additional studies are inprogress to address this hypothesis.

In summary, the present results provide convincing evi-dence that hydroalcoholic extract from P. paniculata exerts arapid onset, relatively long-lasting and pronounced systemicantinociception in several chemical models of nociceptionin the mouse, at a dose that does not produce any statisticallysignificant motor dysfunction or any detectable side-effect. Inaddition, the antinociceptive effect of hydroalcoholicextract involves an interaction with glutamatergic(through NMDA receptors) system or pro-inflammatorycytokines (IL-1β and TNF-α), but not with nitric oxide l-arginine pathway or opioid receptors sensitive to naloxone.

Pharmacological and chemical studies are in progress, inorder to characterize the precise mechanism(s) responsiblefor the antinociceptive action, and also to identify otheractive compounds present in hydroalcoholic extract of P.paniculata. Finally, the antinociceptive action demonstratedin the present study supports, at least part, the ethnomedicaluses of this plant.

AcknowledgementsThis work was supported by grants from Conselho

Nacional de Desenvolvimento Científico e Tecnológico(CNPq), Programa de Apoio aos Núcleos de Excelência(PRONEX), Fundação de Apoio à Pesquisa Científica Tec-nológica do Estado de Santa Catarina (FAPESC) andFinanciadora de Estudos e Projetos [FINEP, Rede InstitutoBrasileiro de Neurociência (IBN-Net)], Brazil.

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