© 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
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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.
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HYDROALCOHOLIC EXTRACT AND THE FLAVONOID RUTIN
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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.
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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. Finally, the antinociceptive action demonstrated
in the present study supports, at least part, the ethnomedical
uses of this plant.
Acknowledgements
This 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 Tecnológica do Estado de Santa Catarina (FAPESC) and
Financiadora de Estudos e Projetos [FINEP, Rede Instituto
Brasileiro de Neurociência (IBN-Net)], Brazil.
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