© 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. Lapa1, Vinicius M. Gadotti2, Fabiana C. Missau3, Moacir G. Pizzolatti3, Maria Consuelo A. Marques1, Alcir L. Dafré 2, Marcelo Farina4, Ana Lúcia S. Rodrigues4 and Adair R. S. Santos2 1 Department of Pharmacology, Center of Biological Sciences, Federal University of Paraná, Curitiba, 88015-420, PR, Brazil, 2Department 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 4Department 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 in chemical and thermal behavioural models of pain in mice. The antinociceptive effects of hydroalcoholic extract was evaluated in chemical (acetic-acid, formalin, capsaicin, cinnamaldehyde and glutamate tests) and thermal (tail-flick and hot-plate test) models of pain or by biting behaviour following intratecal administration of both ionotropic and metabotropic agonists of excitatory amino acids receptors glutamate and cytokines such as interleukin-1 β (IL-1β) and tumour necrosis factor-α (TNF-α) in mice. When given orally, hydroalcoholic extract (0.001–10 mg/kg), produced potent and dose-dependent inhibition of acetic acid-induced visceral pain. In the formalin test, the hydroalcoholic extract (0.0001–0.1 mg/kg orally) also caused significant inhibition of both the early (neurogenic pain) and the late (inflammatory pain) phases of formalin-induced licking. However, it was more potent and efficacious in relation to the late phase of the formalin test. The capsaicin-induced nociception was also reduced at a dose of only 1.0 mg/kg orally. The hydroalcoholic extract significantly reduced the cinnamaldehyde-induced nociception at doses of 0.01, 0.1 and 1.0 mg/kg orally. Moreover, the hydroalcoholic extract (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, produced dose-related inhibition of glutamate-induced pain. Furthermore, the hydroalcoholic extract (0.1–100 mg/kg orally) had no effect in the tail-flick test. On the other hand, the hydroalcoholic extract caused a significant increase in the latency to response 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 -arginine (precursor of nitric oxide, 600 mg/kg) and naloxone (opioid receptor antagonist, 1 mg/kg.) nor associated with non-specific effects such as muscle relaxation or sedation. In addition, oral administration of hydroalcoholic extract produced a great inhibition of the painrelated behaviours induced by intrathecal injection of glutamate, N-methyl--aspartate (NMDA), IL-1β and TNF-α, but not by α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), kainate or trans-1-amino-1.3-cyclopentanediocarboxylic acid (trans-ACPD). Together, our results suggest that inhibition of glutamatergic ionotropic receptors, may account for the 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 in tropical regions. Among the members of this family, is the genus Polygala, with approximately 500 species [1]. In addition, Polygala paniculata Linneu, a plant of the genus Polygala, is a native small arbust, that grows in Brazil’s Atlantic coast, 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 remedy and for the treatment of different inflammatory diseases, such as asthma, bronchitis, arthritis and other pathologies, including disorders of the kidney [2]. Apart from these medicinal uses, there are reports showing antipsychotic [3], Author for correspondence: Adair R. Santos, Department of Physiological Sciences, Center of Biological Sciences, Federal University of Santa Catarina, 88040-900, Florianópolis, SC, Brazil (fax +55 48 37219672, e-mail arssantos@ccb.ufsc.br). anti-tumoural [4], anti-inflammatory [5], antinociceptive [6,7], anti-spasmodic [8] activity of some Polygala species. In addition, preliminary data from our group have demonstrated that hydroalcoholic extract from P. paniculata L., produced in vivo protective effects against methylmercuryinduced neurotoxicity [9]. Phytochemical studies carried out with plant of the genus Polygala revealed an abundant amount of several compounds, including cytotoxic lignans [4], saponins [3], xanthones, coumarins and flavonoids [10]. More recently, it has been reported the isolation of two xanthones, 1-hydroxy-5methoxy-2.3-methylenedioxyxanthone and 1.5-dihydroxy2.3-dimethoxyxanthone, together with coumarin murragatin and flavonol rutin and two sterol, spinasterol and δ25spinasterol from P. paniculata L. [11]. Recently, we have demonstrated that the hydroalcoholic extract from P. paniculata markedly inhibited gastric mucosal lesions induced by ethanol 70%. According to our HYDROALCOHOLIC EXTRACT AND THE FLAVONOID RUTIN results, the protective effect of hydroalcoholic extract may have been related to a slight increase or maintenance of gastric mucus secretion, an effect that may involve prostaglandins and the antioxidant activity of some constituents found in the extract [12]. In addition, preliminary studies have shown that the hydroalcoholic extract of P. cyparissias, another plant of the genus Polygala, and an isolated xanthone (dihydroxy-2.3dimethoxyxanthone) produced a dose-related and marked antinociception [6]. Recently, we have demonstrated that the hydroalcoholic extract from Polygala sabulosa (A. W. Bennett) produced antinociceptive action against the acetic acidinduced visceral nociception. The presence of styryl-pyrones, coumarin (scopoletin) and steroid (α-spinasterol) gives the plant a potent antinociceptive effect [7]. However, to date, no pharmacological study on the antinociceptive activity of P. paniculata has been carried out. In the present study we have attempted to examine the possible antinociceptive action of the hydroalcoholic extract of P. paniculata L., in chemical and thermal models of nociception in mice. In addition, we also analysed the possible antinociceptive effect of the phebalosin, aurapten and rutin isolated 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 on at 6 a.m.) and with access to food and water ad libitum. Animals were acclimatised to the laboratory for at least 1 hr before testing and were used only once throughout the experiments. The experiments were performed after approval of the protocol by the Institutional Ethics Committee and were carried out in accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals [13]. The numbers of animals (n = 6 –12) and intensities of noxious stimuli used were the minimum necessary to demonstrate the consistent effects of the drug treatments. Preparation of ethanolic extract, isolation and chemical identification of the isolated compound. Polygala paniculata L. was collected in Daniela Beach (Florianópolis, Santa Catarina State, Brazil) and was classified by M.Sc. Olavo Araújo Guimarães (Federal University of Paraná, Curitiba, Brazil). A voucher specimen of this plant was deposited in the herbarium of the Botanical Department of Federal University of Paraná. The dried whole plant (3500 g) was minced and submitted to exhaustive extraction by maceration with ethanol: water (80 : 20) in closed recipient. After maceration, the extract was filtered through a paper filter and the solvent was evaporated under reduced pressure (50°) in a rotative evaporator. The respective crude hydroalcoholic extract (yield 50 g) was obtained. After the solvent removal of the hydroalcoholic extract, the extract was concentrated with reduced pressure and successively partitioned with EtOH and dichloromethane CH2Cl2. The flavonoid quercetin-3-rutinoside (rutin) yielded (430 mg), the major compound of hydroalcoholic extract, was found in the ethanol fraction. The fraction eluated with CH2Cl2 was subjected to a chromatography over silica gel with a mixture of n-hexano–EtOAc–EtOH with increasing polarity. Elution with n-hexane–EtOAc–EtOH yield 7- genariloxi cumarin (87 mg) also known as aurapten. The edicuticular (cuticle) wax was obtained from the hexanic extract of P. paniculata. This wax was successively partitioned with n-hexane 307 and phebalosin was yielded (2 g). The antinociceptive activity of hexanic extract was not investigated in this work. Drugs. The following substances were used: acetic acid, formalin and morphine hydrochloride (Merck, Darmstadt, Germany); Nω-nitro-arginine, -arginine hydrochloride, capsaicin, cinnamaldehyde, glutamic acid hydrochloride, naloxone hydrochloride, morphine hydrochloride (Sigma Chemical Co., St. Louis, MO). All drugs were dissolved in saline, with the exception of capsaicin, phebalosin and aurapten that were dissolved in tween 80 plus saline and absolute ethanol. The final concentration of tween 80 and ethanol did not exceed 10% and did not cause any per se effect. Assessment of antinociceptive effect of HE from P. paniculata and isolated compound. Abdominal constriction response caused by intraperitoneal injection of acetic acid. The abdominal constrictions were induced according to procedures described previously [14] and resulted in contraction of the abdominal muscle together with a stretching of the hind limbs 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 of the irritant. Control animals received a similar volume of vehicle (10 ml/kg). After the challenge, the mice were individually placed into glass cylinders of 20 cm diameter, and the abdominal constrictions were counted cumulatively over a period of 20 min. Antinociceptive activity was expressed as the reduction in the number of abdominal constrictions, that is, the difference between control animals (mice pre-treated with vehicle) and animals pre-treated with hydroalcoholic extract. Formalin-induced nociception. The procedure used was essentially the same as that described previously [14,15]. Animals received 20 µl of a 2.5% formalin solution (0.92% formaldehyde) made up in saline, injected intraplantarly in the ventral surface of the right hind paw. Animals were observed from 0 to 5 min. (neurogenic phase) and 15–30 min. (inflammatory phase) and the time spent licking the injected paw was recorded with a chronometer and considered as indicative of nociception. The animals received hydroalcoholic extract of P. paniculata (0.0001–0.1 mg/kg orally) 1 hr before, with basis of a previous time-response curve. Control animals received vehicle (10 ml/kg orally). Capsaicin- and cinnamaldehyde-induced nociception. In order to provide more direct evidence concerning the participation of TRPV1 and TRPA1 in the effect of the hydroalcoholic extract of P. paniculata, we investigated its antinociceptive effect in capsaicin- (a TRPV1 receptor agonist) and cinnamadehyde- (a TRPA1 receptor agonist) induced licking in the mouse paw. The procedure used was similar to that described previously [15,16]. After an adaptation period (20 min.), 20 µl of capsaicin (1.6 µg/paw prepared in saline) or 20 µl of cinnamaldehyde (10 nmol/paw prepared in saline) were injected intraplantarly in the ventral surface of the right hind paw. Animals were observed individually for 5 min. following capsaicin or cinnamaldehyde injection. The amount of time spent licking the injected paw was recorded with a chronometer and was considered as indicative of nociception. The animals were treated with hydroalcoholic 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 received vehicle (10 ml/kg orally). Glutamate-induced nociception. In an attempt to provide more direct evidence concerning the interaction of the hydroalcoholic extract of P. paniculata or isolated compounds with the glutamatergic system, we separately investigated whether or not hydroalcoholic extract or isolated compounds was able to antagonize glutamateinduced licking of the mouse paw. The procedure used was similar to that described previously [17]. A volume of 20 µl of glutamate © 2009 The Authors Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 306–315 308 FERNANDA DA R. LAPA ET AL. (10 µmol/paw prepared in saline) was injected intraplantarly in the ventral surface of the right hind paw. Animals were observed individually for 15 min. after glutamate injection. The amount of time spent licking the injected paw was recorded with a chronometer and was considered as indicative of nociception. The animals were treated with hydroalcoholic extract (0.001–1.0 mg/kg orally) 1 hr before and with the isolated compounds, phebalosin (1–30 mg/kg intraperitoneally), aurapten (3–100 mg/kg intraperitoneally) and rutin (1–100 mg/kg intraperitoneally) 0.5 hr before glutamate injection. 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 of the hydroalcoholic extract antinociceptive effect was evaluated at the time points of 1, 2, 4, 6, 8 and 12 hr after the administration, in the glutamate-induced nociception. Control animals received a similar volume of vehicle orally (10 ml/kg) and were observed at the same intervals of time. Hot-plate test. The hot-plate test was used to measure the response latencies according to the method described previously by Eddy and Leimbach [23]. In these experiments, the hot-plate (Ugo-Basile, Socrel, model-DS 37) was maintained at 50 ± 1°. Animals were placed into a glass cylinder and the time (sec.) between placement and shaking or licking of the paws or jumping was recorded as the index of response latency. The reaction time was recorded for animals pre-treated with the of P. paniculata (0.1–10 mg/kg orally., 1 hr before) or with morphine (5.0 mg/kg subcutaneously, 0.5 hr before), which was used as a positive control. Animals that remained on the apparatus for an average of 16 sec. were selected 2 hr previously on the basis of their reactivity in the model. A latency period (cut-off ) of 30 sec. was defined as complete antinociception. Control animals received the vehicle used to dilute these drugs. To determine the baseline, each animal was tested before administration of drugs, using the same heat stimulus. The MPE of drugs-induced antinociception was calculated as follows: %MPE = [(post-drug − pre-drug)/ (30 − pre-drug)] × 100. Intrathecal injection of excitatory amino acids and pro-inflammatory cytokine-induced pain behaviour in mice. In another set of experiments, the interaction of the hydroalcoholic extract from P. paniculata with the glutamatergic system was investigated by using both ionotropic and metabotropic agonists of excitatory aminoacids receptors, which were administered by the intrathecal route causing biting behaviour in mice [18]. In addition, we investigated P. paniculata effect upon pro-inflammatory cytokines IL-1β- and TNF-αinduced biting behaviour in mice [19,20]. The animals received hydroalcoholic extract of P. paniculata (1 mg/kg orally) 1 hr before intrathecal injection of 5 µl of the drugs. Injections were given to fully conscious mice using the method described by Hylden and Wilcox [21]. Briefly, the animals were manually restrained and a 30gauge needle, attached to a 50-µl microsyringe, was inserted through the skin and between the vertebrae into the subdural space of the L5–L6 spinal segments. The biting behaviour was defined as a single head movement directed at the flanks or hind limbs, resulting in contact of the animal’s snout with the target organ. Intrathecal injections were given over a period of 5 sec. The nociceptive response was elicited by glutamate (an excitatory amino acid, 30 µg/site), NMDA (a selective agonist of NMDA-subtype of glutamatergic ionotropic receptors, 25 ng/site), AMPA (a selective agonist of AMPA-subtype of glutamatergic ionotropic receptors, 25 ng/site), kainate (a selective agonist of kainate-subtype of glutamatergic ionotropic receptors, 23.5 ng/site), trans-ACPD (an agonist of metabotropic glutamate receptors, 8.6 µg/site, i.t.), IL-1β (1 pg/site) and TNF-α (0.1 pg/site) [19–21]. As control, a group of mice received vehicle (saline) intrathecally, the amount of biting behaviour was quantified and discounted from all groups. The amount of time the mice spent biting or licking was evaluated following local post-injections of one of the following agonists: glutamate 3 min.; NMDA 5 min.; AMPA 1 min.; kainate 4 min.; t-ACPD 15 min.; IL-1β and TNF-α 15 min. Analysis of possible mechanism of action of hydroalcoholic extract. To address some of the mechanisms by which hydroalcoholic extract causes antinociception, the glutamate-induced nociception test was chosen, in which hydroalcoholic extract was shown to be more potent and efficacious as compared to other nociceptive tests. The doses of the drugs used were selected on the basis of literature data [14] and also based in previous results from our laboratory. Tail-flick test. A radiant heat tail-flick analgesimeter (Ugo-Basile, Italy) was used to measure response latencies according to the method described previously by D’Amour & Smith [22], with minor modifications. Animals responded to a focused heat stimulus by flicking or removing their tail, when exposed to a photocell in the apparatus immediately below it. The reaction time was recorded for control mice (vehicle, 10 ml/kg orally) and for animals pre-treated 1 hr before with hydroalcoholic extract (0.1–10 mg/kg orally) or with morphine (5.0 mg/kg subcutaneously). An automatic 20-sec. cut-off was used to minimize tissue damage. Animals that remained on the apparatus for an average of 12 sec. were selected 2 hr previously on the basis of their reactivity in the test. To determine the baseline, each animal was tested before administration of drugs, using a moderate radiant heat stimulus. The maximal percentage of the effect (MPE) of drug-induced antinociception was calculated as follows: %MPE = [(post-drug − pre-drug)/(20 − pre-drug)] × 100. Involvement of opioid system. To assess the possible participation of the opioid system in the antinociceptive effect of hydroalcoholic extract, mice were pre-treated with naloxone (1 mg/kg intraperitoneally, a non-selective opioid receptor antagonist), and after 20 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 recorded 1, 0.5 or 1 hr after the administration of hydroalcoholic extract, morphine or vehicle, respectively. Another group of animals was pre-treated with vehicle and after 20 min. received hydroalcoholic extract, morphine or vehicle, 1, 0.5 or 1 hr before glutamate injection, respectively. Involvement of nitric oxide-l-arginine pathway. To investigate the role played by the nitric oxide--arginine pathway in the antinociception caused by hydroalcoholic extract, the mice were pre-treated with -arginine (600 mg/kg intraperitoneally, a nitric oxide precursor) and after 20 min. they received hydroalcoholic extract (0.1 mg/kg orally), Nω-nitro--arginine (-NOARG, 100 mg/kg intraperitoneally, a nitric oxide synthase inhibitor) or vehicle (10 ml/kg orally). The algesic responses to glutamate were recorded 1, 0.5 or 1 hr after the administration of hydroalcoholic extract, -NOARG, or vehicle, respectively. Another group of animals was pre-treated with vehicle and after 20 min. received hydroalcoholic extract, 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 of hydroalcoholic extract from P. paniculata, mice were submitted to the open-field test. The ambulatory behaviour was assessed in the open-field test as described previously [24]. The apparatus consisted of a wooden box measuring 40 × 60 × 50 cm. The floor of the arena was divided into 12 equal squares, and the number of squares crossed with all paws (crossings) was counted in a 6-min. session. Mice were treated with hydroalcoholic extract from P. paniculata (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 ID50 values (i.e., the dose of hydroalcoholic extract reducing the nociceptive response by 50% relative to the control value), which are reported as geometric means accompanied by their respective 95% confidence © 2009 The Authors Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 306–315 HYDROALCOHOLIC EXTRACT AND THE FLAVONOID RUTIN 309 of the acetic acid-induced visceral nociception in mice, with mean ID50 values (and their respective 95% confidence limits) of 0.22 (0.13–0.38) mg/kg and inhibition of 76 ± 7% at a dose of 10 mg/kg. Formalin-induced nociception. Fig. 1. Effect of the hydroalcoholic extract of P. paniculata administered orally against acetic acid-induced writhing movements 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 column indicates the control value (C) (animals injected with vehicle) and the open columns correspond to animals treated with extract, the asterisks denote the significance levels, when compared with control group, (one-way  followed by Newman–Keuls test) **P < 0.01 and ***P < 0.001. limits. The ID50 value was determined by linear regression from individual experiments using linear regression GraphPad software (GraphPad software, San Diego, CA). The statistical significance of differences between groups was detected by Student’s unpaired t-test and  followed by Newman-Keuls’ test when indicated. P-values less than 0.05 (P < 0.05) were considered as indicative of significance. The results depicted in fig. 2A and B shows that the hydroalcoholic extract from P. paniculata (0.0001–0.1 mg/kg orally) caused a significant inhibition of both the neurogenic (0–5 min.) and inflammatory (15–30 min.) phases of formalininduced licking. However, its antinociceptive effects were significantly more pronounced against the second phase of this model of pain. The calculated mean ID50 value (and its respective 95% confidence limits) for these effects were: >0.1 and 0.0042 (0.0035–0.0049) mg/kg and the inhibitions observed 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. paniculata inhibited the capsaicin-induced neurogenic pain, only at a dose of 1.0 mg/kg orally, with inhibition of 50 ± 3% (fig. 3A). The cinnamaldehyde-induced pain was also inhibited significantly at doses of 0.01–1.0 mg/kg orally, the inhibitions observed were 57 ± 5, 47 ± 7 and 67 ± 8% at doses of 0.01, 0.1 and 1.0 mg/kg respectively and the calculated mean ID50 value was 0.27 (0.13–0.57) mg/kg (fig. 3B). Glutamate-induced nociception. 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 The hydroalcoholic extract of P. paniculata (0.001–1 mg/kg), given orally, produced marked and dose-dependent attenuation of the glutamate-induced nociception. The ID50 value was 0.0084 (0.0077–0.0092) mg/kg. The peak of inhibition was 72 ± 3% at 1 mg/kg (fig. 4A). A time-course analysis of the antinociceptive effect of hydroalcoholic extract given Fig. 2. Effect of the hydroalcoholic extract of P. paniculata administered orally against formalin-induced licking (first phase, panel A, and second phase, panel B) 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 indicates the control value (C) (animals injected with vehicle) and the open columns correspond to animals treated with extract, the asterisks denote the significance levels, when compared with control group, (one-way  followed by Newman– Keuls test) *P < 0.05; **P < 0.01 and ***P < 0.001. © 2009 The Authors Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 306–315 310 FERNANDA DA R. LAPA ET AL. 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 the S.E.M. The closed columns indicates the control value (C) (animals injected with vehicle) and the open columns correspond to animals treated with extract, the asterisks denote the significance levels, when compared with control group, (one-way  followed by Newman– Keuls test) **P < 0.01, ***P < 0.001. orally is shown in fig. 4B. Hydroalcoholic extract produced marked antinociception as early as 1 hr after oral administration, an action that remained significant up to 8 hr after the administration (fig. 4B). Thus, the time-point of 1 hr was chosen for all further studies with independent groups of animals. Interestingly, when rutin, isolated from P. paniculata, was administered intraperitoneally to mice, it produced doserelated inhibition of glutamate-induced pain, with a mean ID50 value of 10.9 (7.8–15.4) mg/kg and the peak of inhibition observed was 82 ± 7% (fig. 4C). However, phebalosin or aurapten, administered intraperitoneally to mice, produced Fig. 4. Effect of the hydroalcoholic extract (A), rutin (C), phebalosin (D) and aurapten (E) obtained from P. paniculata against glutamateinduced licking in mice. Panel (B) Time-course of the antinociceptive effect of hydroalcoholic extract (HE) on glutamate-induced licking 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 indicates the control value (C) (animals injected with vehicle) and the open columns correspond to animals treated with extract or compounds, the asterisks denote the significance levels, when compared with control group, (one-way  followed by Newman–Keuls test) *P < 0.05, **P < 0.05 and ***P < 0.001. © 2009 The Authors Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 306–315 HYDROALCOHOLIC EXTRACT AND THE FLAVONOID RUTIN 311 Fig. 5. Effect of the hydroalcoholic extract obtained from P. paniculata (1.0 mg/kg) administered orally on biting response caused by intrathecal 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), IL1β (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 bars indicate the S.E.M. The asterisks denote the significance levels, when compared with untreated groups, ***P < 0.001 by Student’s unpaired t-test. inhibition of 64 ± 12% and 71 ± 7% of glutamate-induced licking 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 hydroalcoholic extract of P. paniculata (1 mg/kg orally) inhibited the nociceptive responses induced by spinal injections of glutamate, NMDA, IL-1β and TNF-α in mice. The inhibition values were 48 ± 9%, 81 ± 5%, 68 ± 13% and 62 ± 15%, respectively. In contrast, hydroalcoholic extract had no effect against AMPA, kainate and trans-ACPD-mediated biting responses (fig. 5). Hot-plate test and tail-flick test. The hydroalcoholic extract from P. paniculata (1.0–10 mg/kg orally) 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 for the groups of animals that were afterwards treated with saline, morphine, hydroalcoholic extract at doses of 0.1, 1.0 and 10 mg/kg, respectively. In the tail-flick test was used a basal cut-off of 12 sec. In contrast, the hydroalcoholic extract (10 mg/kg orally) given 1 hr previously, caused a significant increase of the latency response in the hot-plate test (fig. 6A). Under similar conditions morphine (5.0 mg/kg subcutaneously), used as reference drug, caused a significant and 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 shows that the pre-treatment of mice with naloxone (1 mg/kg intraperitoneally, a non-selective opioid receptor antagonist), given 20 min. before, largely reversed the antinociception 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 indicates the control value (C) (animals injected with vehicle), hatched columns indicates the control group treated with morphine and the open columns correspond to animals treated with extract, the asterisks denote the significance levels, when compared with control group (C), (one-way  followed by Newman–Keuls test) **P < 0.01. © 2009 The Authors Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 306–315 312 FERNANDA DA R. LAPA ET AL. Fig. 7. Effect of pre-treatment of animals with naloxone (1 mg/kg, A) or -arginine (600 mg/kg, B) on the antinociceptive profiles of hydroalcoholic extract of P. paniculata (HE – 0.1 mg/kg orally), morphine (5 mg/kg, A) or -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 bars indicate the S.E.M. #P < 0.001 comparing agonist (P. paniculata, morphine or -NOARG) plus antagonists (naloxone or -arginine) versus agonist plus vehicle (control); ***P < 0.01 compared with corresponding control values (animals injected with the vehicle alone). caused by injection of morphine, but did not significantly change the antinociceptive action caused by hydroalcoholic extract, when assessed against glutamate test. Involvement of nitric oxide–-arginine pathway. The systemic pre-treatment of mice with the nitric oxide precursor -arginine (600 mg/kg intraperitoneally), given 20 min. earlier, significantly reversed the antinociception caused by -NOARG (100 mg/kg intraperitoneally, a nitric oxide synthase inhibitor) when analysed against glutamateinduced nociception (fig. 7B). Under the same conditions, -arginine did not significantly modify the antinociception caused by hydroalcoholic extract in the glutamate test (fig. 7B). Evaluation of locomotor activity. The hydroalcoholic extract from P. paniculata (0.1, 1 and 10 mg/kg orally) did not affect the locomotor activity of mice submitted to the open-field test when compared to animals that received vehicle (control group). In the locomotor activity the means ± S.E.M. of crossings number were 99.3 ± 6.3; 95.8 ± 6.8; 89.8 ± 6.7 and 97.3 ± 4.9 for the control, 0.1, 1 and 10 mg/kg group, respectively. Discussion The present study demonstrates that systemic (oral) administration of hydroalcoholic extract from P. paniculata elicits a potent and dose-dependent inhibition of the nociceptive behavioural response in mice submitted to chemical pain-inducing stimuli. The most relevant findings in the work are that (1) oral administration of hydroalcoholic extract caused significant inhibition of acetic acid-induced visceral pain response; (2) oral administration of hydroalcoholic extract also caused significant inhibition against both phases of the pain response to the intraplantar injection of formalin, and against the capsaicin- and cinnamaldehydeinduced nociception; (3) the algesic response caused by intraplantar injection of glutamate was also greatly inhibited by hydroalcoholic extract; (4) the antinociceptive action of hydroalcoholic extract in the glutamate test was not significantly reversed by intraperitoneal pre-treatment of animals with naloxone and -arginine; (5) oral administration of hydroalcoholic extract significantly reduced the glutamate, NMDA-, IL-1β- and TNF-α-induced biting, although, it did not cause any significant reduction on trans-ACPD, AMPA and kainate nociception response; (6) hydroalcoholic extract did not change the response latency of animals in the tail-flick test, but increased the response latency in the hot-plate test and (7) the dose of hydroalcoholic extract that caused significant antinociception did not produce any statistically significant motor dysfunction or any detectable side-effect. The acetic acid-induced writhing reaction in mice, described as a typical model for inflammatory pain, has long been used as a screening tool for the assessment of analgesic or anti-inflammatory properties of new agents [25,26]. At the cellular level, protons depolarize sensory neurones by directly activating a non-selective cationic channel localized on cutaneous, visceral and other types of nocisponsive peripheral afferent C-fibres [27,28]. The results reported here indicate, that oral administration of hydroalcoholic extract produced marked and dose-related antinociception when assessed in acetic acid-induced visceral nociception, at doses that did not produce any statistically significant motor dysfunction. To our knowledge this is the first report of its kind in the literature. Also of interest are the results showing that hydroalcoholic extract of P. paniculata caused significant and doserelated antinociception when administered orally against both neurogenic (early phase) and inflammatory (late phase) © 2009 The Authors Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 306–315 HYDROALCOHOLIC EXTRACT AND THE FLAVONOID RUTIN pain responses caused by formalin injection in mice. The formalin-induced nociception is a well-described model of nociception and can be consistently inhibited by typical analgesic and anti-inflammatory drugs, including morphine, indomethacin and dexamethasone [29,30]. Considering the inhibitory property of P. paniculata on the second phase of formalin, we might suggest an anti-inflammatory action of the plant extract. In addition, recent studies have shown that formalin activates primary afferent sensory neurons through a specific and direct action on TRPA1, a member of the Transient Receptor Potential family (TRP) of cation channels that is highly expressed by a subset of C-fibre nociceptors [31]. To investigate the further participation of TRPA1 receptor in the antinociceptive effect of hydroalcoholic extract of P. paniculata, we assessed the cinnamaldehyde-induced nociception. Currently, it was demonstrated that intraplantar administration of cinnamaldehyde, a TRPA1 agonist receptor, to mice produced a dose-dependent spontaneous nociception [16]. Our results show that a hydroalcoholic extract significantly reduced the cinnamaldehyde-induced pain. This result is in line with that obtained in formalin model and indicates that hydroalcoholic extract at low doses probably interacts with TRPA1 receptor located in C-fibres reducing the formalin-induced nociception. It has been proposed that the capsaicin-induced nociception is brought about by activation of another TRP receptor, the vanilloid receptor (TRPV), termed TRPV1, a ligand-gated non-selective cation channel in primary sensory neurons [32–34]. Our results also show that oral administration of hydroalcoholic extract of P. paniculata produced a partial, but significant, reduction of the nociceptive response caused by intraplantar. injection of capsaicin into the mouse hindpaw. Of note, the licking response induced by formalin, capsaicin and glutamate results from a combination of peripheral input and spinal cord sensitization [15,17,30,35]. The intraplantar injection of formalin, capsaicin or glutamate releases excitatory amino acids, PGE2, NO, neuropeptides and kinins in the spinal cord [15,17,30,31,35,36]. Taking this into account, the antinociception of P. paniculata could be dependent on either peripheral or central sites of action. In this study, we observed in formalin and capsaicin models that the antinociceptive effect of hydroalcoholic extract was higher at low doses. These finding suggested that the constituents from hydroalcoholic extract at lower doses might be interacted peripherally with nociceptors or served as scavengers. We have recently shown the antioxidant activity of 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 hydroalcoholic extract might have been related to the inhibitory effect against the inflammatory component and generation of free radicals observed in these models and consequently reducing the nociceptive behaviour. The hydroalcoholic extract from P. paniculata produced a dose-dependent antinociceptive effect on the glutamate 313 induced paw licking response. Recently, Beirith et al. [17] found that the nociceptive response induced by glutamate appears to involve peripheral, spinal and supraspinal sites of action and is greatly mediated by both NMDA and nonNMDA receptors as well as by the release of nitric oxide or by some nitric oxide-related substance. Hence, an effect of the plant extract directly on the receptors or second messengers related to these transmitters could avoid the nociceptive response. Interestingly, the P. paniculata antinociception was extended up to 8 hr after the treatment, an effect that is hardly reached for clinically used analgesics. The effect of P. paniculata against nociception induced by glutamate is of great interest since glutamate plays a significant role in nociceptive processing in both central and peripheral nervous systems [17,37,38]. Indeed, drugs capable of blocking either iGluRs (ionotropic glutamate receptors) or mGluRs (metabotropic glutamate receptors) exhibit antinociceptive effects in several mammalian species including human beings [39]. Therefore, it is supposed that substances that block GluRs may have clinical potential in the management of some painful states. On the other hand, the use of these substances as analgesics is hampered due to the unaccepted 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 locomotor activity when assessed in the open field test. In addition, hydroalcoholic extract of P. paniculata administered at a dose of 1.0 mg/kg, inhibited efficaciously the biting behaviour induced by iGluRs, mainly those induced by NMDA, but not AMPA and kainate agonists. However, P. paniculata treatment did not reduce biting behaviour induced by mGluR (trans-ACPD). Therefore, it is plausible that some constituents of P. paniculata, when the extract was administered at a higher dose (1.0 mg/kg orally), may reach the central nervous system and interact with the pathways depending on the activation of iGluRs (NMDA receptor), instead of observed on formalin-induced pain where a one hundred time lower dose of hydroalcoholic extract might be responsible for a local effect probably related to interaction of active compounds from hydroalcoholic extract with local TRPA1 receptors on afferent fibres. The intrathecal injection of cytokines, such as TNF-α, IL-1β, IL-1α and IFN-γ, induces nociceptive behaviour that is partly mediated through the activation of central nerve terminals and subsequent release of glutamate [40,41]. This study shows that hydroalcoholic extract was able to inhibit the nociceptive behaviour induced by intrathecal injection of IL-1β and TNF-α. There are two ways of interpreting this result. One possibility is that the constituents present in the hydroalcoholic extract directly inhibit the action of cytokines, preventing them from depolarizing projection neurons and primary afferents. Another possibility is that the hydroalcoholic extract inhibits the further activation of projection neurons by glutamate, through the inhibition of NMDA receptors. The second hypothesis seems more plausible, since hydroalcoholic extract was capable of inhibiting nociceptive behaviour induced by NMDA. Therefore, it is © 2009 The Authors Journal compilation © 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 306–315 314 FERNANDA DA R. LAPA ET AL. suggested that the inhibition of the cytokines-induced biting behaviour is due to the antagonism of glutamatergic transmission at the spinal level. Further experiments were undertaken to elucidate the possible involvement of opioid and -arginine–nitric oxide systems on the antinociceptive properties of the hydroalcoholic extract from P. paniculata. The results obtained demonstrated that the activation of the opioid naloxonesensitive pathway is unlikely to be involved in the antinociception caused by hydroalcoholic extract as naloxone, under conditions where it fully reversed morphine-induced antinociception, had no effect against the hydroalcoholic extract action. In addition, the results of the present study also suggest that the antinociception caused by hydroalcoholic extract did not involve any interaction with -arginine–nitric oxide pathway, since the treatment of animals with -arginine (a precursor of nitric oxide), under conditions in which it consistently reversed the antinociception caused by -NOARG (a known nitric oxide synthase inhibitor), failed to interfere with hydroalcoholic extract-induced antinociception. The hydroalcoholic extract of P. paniculata was devoid of antinociceptive action when assessed in a thermal model of nociception, the tail-flick test, under conditions that morphine has a marked antinociceptive effect. Interestingly, our results showed that the hydroalcoholic extract at the higher dose tested (10 mg/kg orally) had increased the latency response in the hot-plate test. These results may suggests a central analgesic action for hydroalcoholic extract since the hot-plate test is known to involve the activation of supraspinal structures and the tail-flick response is more related to spinal reflex triggered by C fibres when it is elicited by heat [42]. Finally, the chemical studies carried out with this hydroalcoholic extract allowed us to isolate and identify rutin, phebalosin and aurapten in P. paniculata, which seem not to be responsible, at least in part, for the antinociceptive properties reported for the hydroalcoholic extract of P. paniculata, considering that the necessary doses to produce significant inhibition of the nociception caused by glutamate was about 3–100 times larger than the one of hydroalcoholic extract. On the basis of this finding, we can speculate that these compounds as well as others found in P. paniculata, might act synergically contributing to the potent antinociceptive action of P. paniculata. Additional studies are in progress to address this hypothesis. In summary, the present results provide convincing evidence that hydroalcoholic extract from P. paniculata exerts a rapid onset, relatively long-lasting and pronounced systemic antinociception in several chemical models of nociception in the mouse, at a dose that does not produce any statistically significant motor dysfunction or any detectable side-effect. In addition, the antinociceptive effect of hydroalcoholic extract involves an interaction with glutamatergic (through NMDA receptors) system or pro-inflammatory cytokines (IL-1β and TNF-α), but not with nitric oxide arginine pathway or opioid receptors sensitive to naloxone. Pharmacological and chemical studies are in progress, in order to characterize the precise mechanism(s) responsible for the antinociceptive action, and also to identify other active compounds present in hydroalcoholic extract of P. paniculata. 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