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Antinociceptive effect of Lecythis pisonis Camb. (Lecythidaceae) in models of acute pain in mice
Journal of Ethnopharmacology 146 (2013) 180–186
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Antinociceptive effect of Lecythis pisonis Camb. (Lecythidaceae) in models of acute pain in mice M.S. Branda~ o a, S.S. Pereira a, D.F. Lima a, J.P.C. Oliveira b, E.L.F. Ferreira b, M.H. Chaves b, F.R.C. Almeida a,n a b
´tima s/n, 64049-550 Teresina, Brazil Medicinal Plants Research Center, Federal University of Piauı´, Av. Nossa Senhora de Fa Department of Chemistry, Federal University of Piauı´, Teresina, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2012 Received in revised form 14 December 2012 Accepted 20 December 2012 Available online 28 December 2012
Ethnopharmacological relevance: Lecythis pisonis Camb., also known in Brazil as sapucaia, is used in folk medicine against pruritus, muscle pain and gastric ulcer. Aim of the study: To investigate the antinociceptive effect of ethanol extract from Lecythis pisonis leaves (LPEE), fractions (hexane-LPHF, ether-LPEF and ethyl acetate-LPEAF) and mixture of triterpenes [ursolic and oleanolic acids (MT)] in mice. Materials and methods: LPEE and LPEF were evaluated on the acetic acid induced writhings and formalin, capsaicin and glutamate tests. In addition, MT was investigated on the writhings induced by acetic acid, capsaicin and glutamate tests. In the study of some possible mechanisms involved on the þ channels and antinociceptive effect of LPEF, it was investigated the participation of opioid system, KATP L-arginine-nitric oxide pathway. Results: LPEE (12.5 and 25 mg/kg, p.o.), LPEF and MT (6.25, 12.5 and 25 mg/kg, p.o.) reduced the writhings in comparison to saline. LPEE (100 mg/kg, p.o.) and LPEF (50 mg/kg, p.o.) were effective in inhibiting both phases of formalin test. In capsaicin test, LPEE (100 and 200 mg/kg, p.o.), LPEF (12.5– 50 mg/kg, p.o) and MT (6.25–25 mg/kg, p.o.) showed a significant antinociceptive effect compared to the control. LPEE (25 and 50 mg/kg, p.o.), LPEF (50 and 100 mg/kg, p.o.) and MT (12.5 and 25 mg/kg, p.o.) reduced the glutamate-evoked nociceptive response. Treatment with naloxone, L-arginine and glibenclamide reversed the effect of LPEF in glutamate test. Conclusions: These results indicate the antinociceptive effect of Lecythis pisonis leaves and suggest that þ channels, and L-arginine-nitric oxide modulation. this effect may be related to opioid pathway, KATP Furthermore, these data support the ethnomedical use of this plant. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Lecythis pisonis Lecythidaceae Antinociceptive effect Ursolic/oleanolic acid
1. Introduction Lecythis pisonis Camb. belongs to the Lecythidaceae botanic family and is popularly known in Brazil as ‘‘sapucaia’’ or ‘‘cumbuca de macaco’’. It is found mainly in the states of Piauı´, from Pernambuco to Sa~ o Paulo and in the Amazon region. The seeds of this species are a valuable source of essential amino acids, fatty acids and minerals, being a functional and nutritious food for human consumption (Vallilo et al., 1999). In traditional medicine, it is used for the treatment of itching (pruritus) in the body (Franco and Barros, 2006) and as an emollient, reducing muscle pain (Agra et al., 2007).
Abbreviations: IL-1b, interleukin-1beta; IL-8, interleukin-8; LD50, lethal dosis 50%; L-NOARG, L-NG-nitro arginine; NO, nitric oxide; NMDA, N-methyl-D-aspartate; PGE2, prostaglandin E2; SP, substance P; TNFa, tumor necrosis factor-alfa; TRPV1, transient receptor potential cation channel subfamily V member 1 n Corresponding author. Tel.: þ55 86 3215 58 72; fax: þ 55 86 3237 18 50. E-mail address: [email protected] (F.R.C. Almeida). 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.12.028
Phytochemical investigation of the ethanolic extract of Lecythis pisonis leaves has led to the identification of different fractions such as hexane, ether and ethyl acetate. However, the chromatographic fractioning of the ethereal fraction and its subfractions spectrometric analysis resulted in the identification of 10 isoprenic origin substances, distributed in three binary mixtures of triterpenoid skeletons and ursano oleanano, a mixture of steroid and triterpenoid skeleton called friedelan-3-ol. The presence of triterpenes in the species was confirmed by the isolation and identification of the mixture of ursolic and oleanolic acids (Oliveira et al., 2012). Plants rich in triterpenoids are widely used in medicine because of its anti-inflammatory, protecting of the vascular system, antiallergic, analgesic and antipyretic properties (Andrade et al., 2007). Several species have triterpenoids in its composition and show antinociceptive activity, for example Achyranthes aspera Linn. (Barua et al., 2010), Muehlenbeckia platyclada F. Muell. (Fagundes et al., 2010) and Aegiceras corniculatum L. Blanco (Roome et al., 2011). The species Emilia sonchifolia L. has demonstrated antinociceptive
~ et al. / Journal of Ethnopharmacology 146 (2013) 180–186 M.S. Brandao
activity and chemical composition similar to Lecythis pisonis, including the presence of ursolic acid, sitosterol and stigmasterol (Couto et al., 2011). There are reports showing that ursolic acid is able to produce a significant reduction in acetic acid-induced writhings, indicating that it is effective against inflammatory pain (Taviano et al., 2007). In addition, the oleanolic acid inhibits visceral nociception in the mustard oil-induced colonic nociception in mice through an opioid mechanism, and it may have a modulatory influence on vanilloid receptors (Maia et al., 2006). A previous study provided experimental evidence that justifies the popular use of Lecythis pisonis leaves in the treatment of pruritus and suggests that this effect may be related to a stabilizing action on mast cell membrane (Silva et al., 2012). The precise relationship between itch and pain remains controversial. Itch seems to share numerous features with pain, including induction by noxious chemical stimuli, arousal of attention, negative affective valence and initiation of motor responses (Drzezga et al., 2001). By virtue of aforementioned properties, it is expected that Lecythis pisonis have antinociceptive activity associated with its antipruriginous effect. So, this study aims to evaluate Lecythis pisonis Camb. antinociceptive effect in animal models of chemically induced acute pain and some of the possible mechanisms involved in this effect.
2. Materials and methods 2.1. Plant material and identification Leaves of Lecythis pisonis Camb. (Lecythidaceae) were collected in July 2008 at the Centre for Agrarian Sciences, Federal University of Piauı´ (CCA-UFPI) in the city of Teresina, Piauı´ state, Brazil (51 020 53.200 S, 421 470 16.800 O to 68 m sea level). After collection, a voucher specimen has been identified by Gardene M. Sousa and deposited in the Graziela Barroso Herbarium of Federal University of Piauı´ (TEPB 26488). 2.2. Extraction and isolation Dried and powdered leaves of Lecythis pisonis (2 kg) were extracted at room temperature, consecutively six times with ethanol 98%. The solvent was removed by evaporation under reduced pressure using Hedolph Rotary Evaporator to yield the ethanol extract (LPEE, 272.0 g, 13.6%). Part of the LPEE (200.0 g) was fractionated by serial extraction with hexane, ethyl ether, EtOAc and H2O to yield hexane (LPHF, 60.0 g, 34.0%), ethyl ether (LPEF, 24.0 g, 12.0%), EtOAc (LPEAF, 21.0 g, 10.5%) and H2O (LPAF, 70.0 g, 37.5%) fractions and a precipitate formed in the EtOAc phase which was separated by simple filtration (ppt-EtOAc, 10 g, 5%). The ethereal fraction (LPEF, 10 g) was fractionated on a silica gel column with elution gradient from CHCl3 to MeOH to yield 103 fractions collected as follows: 1–31 (CHCl3, 100%), 32–54 (CHCl3-MeOH, 98:2), 55–62 (CHCl3-MeOH, 95:5), 63–72 (CHCl3MeOH, 9:1), 73–89 (CHCl3-MeOH, 8:2), 90–96 (CHCl3-MeOH, 7:3), 97–103 (MeOH 100%). The fraction B (17–23, 603 mg) was suspended in MeOH to form an amorphous precipitate (F17-ppt, 475 mg, 4.75%) composed of a mixture of ursolic and oleanolic acid triterpenes (Fig. 1). The structural characterization of this mixture was performed using spectroscopic methods such as nuclear magnetic resonance (1H and 13C NMR) and compared with literature data (Mahato and Kundu, 1994; Junges et al., 2000). The ratio of ursolic acid and oleanolic acid in this mixture was 59:41, calculated by 1H NMR, by dividing the signal area of olefinic hydrogens d ¼5.15 (ursolic acid) and d ¼5.19 (oleanolic
181
29
R1 19
1
25
14 10
3
5
HO
H 24
26
23
H 7
20
21
17
13
11
R2
H
COOH 16
28
27
1: R1 = CH3 ; R2 = H 2: R1= H ; R2 = CH3
Fig. 1. Structure of ursolic (1) and oleanolic (2) acids.
acid) with the signal area of d ¼ 3.11 (dd, J¼7.0 and 9.0 Hz) attributed to H-3 in the two triterpenes and multiplying by 100 (Oliveira et al., 2012). The 1H and 13C NMR spectra were recorded on a Varian Inova 500 spectrometer, in CDCl3, at 500 and 125 MHz, respectively, using TMS as internal standard. The chemical shifts values are on d scale and the coupling constants (J) values are in Hertz. Column chromatography was carried out using silica gel 60 (0.063–0.200 mm). Thin layer chromatography (TLC) was carried out on silica gel 60 G (Merck) plates (0.25 mm layer thickness). Extract, fractions and mixture of triterpenes of Lecythis pisonis leaves were dissolved in or diluted with physiological saline (0.9%) and prepared immediately before each experiment. The triterpenes concentration of the extract, fractions and mixture were adjusted to a volume of 10 mL/kg. 2.3. Chemicals and reagents Capsaicin, glibenclamide, glutamate, L-arginine, L-NOARG, Dizocilpine (MK 801), naloxone and Tween 80 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Morphine was purchased from Crista´lia Produtos Quı´micos e Farmacˆeuticos Ltda. (SP, Brazil). Formaldehyde was obtained from Dinˆamica Quı´mica Contemporˆanea Ltda. (SP, Brazil). Acetic acid and sodium chloride were from Vetec Quı´mica Fina Ltda. (RJ, Brazil). 2.4. Animals Male Swiss mice 20–30 g (n ¼6–11) were used, 10 weeks old from the Sector Animal House for Research on Medicinal Plants of the Federal University of Piauı´. The animals were kept at 2471 1C with a light/dark cycle of 12 h, with free access to food and water. They were fasted for a period of 18 h and were accustomed to the test environment for 1 h before each experiment. All experimental protocols were approved by the Ethics Committee for Animal Experimentation at the Federal University of Piauı´ (No. 52/2010). 2.5. Antinociceptive tests 2.5.1. Acetic acid-induced writhing The procedure was similar to a previously described method (Collier et al., 1968). Swiss mice (n ¼6–8) were pretreated with saline vehicle (0.1 mL/10 g), LPEE, LPEF and MT (6.25, 12.5 and 25 mg/kg, p.o.) 60 min before the intraperitoneal administration of 0.75% acetic acid, and then, the total number of writhings was counted over a period of 20 min. The strength of the elicited antinociceptive effect was compared with that of an effective dose
~ et al. / Journal of Ethnopharmacology 146 (2013) 180–186 M.S. Brandao
182
of morphine (2.5 mg/kg, s.c.) administered 30 min before the acetic acid injection. 2.5.2. Formalin test Mice (n ¼6–8) were given orally LPEE (25, 50, 100 and 200 mg/ kg), LPEF (25, 50 and 100 mg/kg) or vehicle (10 mL/kg) 1 h before the test. Morphine (5 mg/kg) was administered subcutaneously 30 min before the test and used as positive control. The right hind foot pad was injected with formalin (20 mL, 2%) in the intraplantar region. Nociception was evaluated by quantifying paw licking time during the first 5 min (first phase) and at 15–30 min (second phase) (Hunskaar and Hole, 1987). 2.5.3. Capsaicin test The animals were individually placed in the observation chamber for an adjustment period of 20 min. After this period, mice (n ¼6–8) were given orally LPEE (50, 100 and 200 mg/kg), LPEF (12.5, 25 and 50 mg/kg), MT (6.25, 12.5 and 25 mg/kg) or vehicle (10 mL/kg). Morphine (5 mg/kg) was administered subcutaneously 30 min before the test and used as a positive control. One hour after these treatments, the right hind paw was injected with capsaicin (2 mg/paw) prepared in 5% Tween solution and 2% ethanol. Nociception was evaluated immediately after injection and quantified by paw licking time during a 5 min period (Sakurada et al., 1992). Table 1 Antinociceptive effect of the ethanol extract, ethereal fraction and the mixture of ursolic and oleanolic acids from leaves of Lecythis pisonis on acetic acid-induced writhings in mice. Treatment
Dose (mg/kg)
Number or writhings
Inhibition (%)
Vehicle Morphine LPEE
– 2.50 6.25 12.50 25.00 6.25 12.50 25.00 6.25 12.50 25.00
73.25 7 2.37 6.17 7 0.91 60.43 7 4.80 31.17 7 2.31nnn 42.17 7 6.01nnn 48.71 7 7.63nnn 34.00 7 3.39nnn 38.67 7 1.91nnn 58.29 7 5.55n 42.00 7 4.57nnn 39.57 7 2.83nnn
– 91.58 17.50 57.45 42.43 33.49 53.58 47.21 20.43 42.66 49.98
LPEF MT
Mice were treated with extract (LPEE), fraction (LPEF) or mixture of triterpenic acids (MT) 60 min (p.o.) before writhing test. Data represent the mean 7S.E.M. of 6–8 animals. The symbols report significance level. npo 0.05, nnnpo 0.001.
2.5.4. Glutamate test The procedure used was similar to the previously described by Beirith et al. (2002). Mice (6–11) received an intraplantar injection of glutamate (20 mmol/paw) 60 min after oral administration of LPEE (12.5, 25 and 50 mg/kg), LPEF (25, 50 and 100 mg/kg), MT (6.25, 12.5 and 25 mg/kg) or vehicle (0.1 mL/10 g), and 30 min after the administration of MK 801 (0.03 mg/kg, i.p.) as a positive control. Animals were observed individually for 15 min following glutamate injection. The amount of time spent licking the injected paw was taken to indicate nociception. 2.6. Evaluation of possible mechanisms of action of the LPEF in mice To address some of the mechanisms by which LPEF of Lecythis pisonis causes antinociception in the glutamate-induced nociception, animals (6–11) were treated with different drugs. The doses of the drugs used were selected on the basis of the literature (Santos et al., 2005) data and also based on previous results from our laboratory. 2.6.1. Participation of opioid system Mice (6–11) were pretreated with naloxone (2 mg/kg, i.p., a non-selective opioid receptor antagonist), and after 20 min the animals received an injection of LPEF (50 mg/kg, p.o.), morphine (5 mg/kg, s.c.) or vehicle (10 ml/kg, p.o.). One hour after administration of LPEF or 30 min after administration of morphine, it was injected 20 mL of glutamate solution (20 mmol/paw, i.pl.) and evaluated the nociceptive response like in glutamate test. 2.6.2. Participation of K þ ATP channels Mice (n ¼6–11) were previously treated with glibenclamide (3 mg/kg, i.p.) and 20 min latter, they received LPEF (50 mg/kg, p.o.) or vehicle (0.1 mL/10 g, p.o.). After 60 min, the animals were assessed for nociception induced by intraplantar injection of 20 mL solution of glutamate (20 mmol/paw). 2.6.3. Participation of L-arginine-nitric oxide Mice (7–11) were pre-treated with L-arginine (600 mg/kg, i.p., a nitric oxide precursor) and after 20 min, they received LPEF (50 mg/kg, p.o.), N$-nitro-L-arginine (L-NOARG, 75 mg/kg, i.p., a nitric oxide inhibitor) or vehicle (10 ml/kg, p.o.). The nociceptive responses to glutamate were recorded 1, 0.5 and 1 h after the administration of LPEF, L-NOARG, or vehicle, respectively.
Table 2 Antinociceptive effect of the ethanol extract and ethereal fraction of leaves from Lecythis pisonis in the formalin induced nociceptive response in mice. Treatment
Vehicle 1 LPEE
Morphine 1 Vehicle 2 LPEF
Morphine 2
Dosage (mg/ kg)
Licking time (s) 0–5 min
Inhibition (%)
15–30 min
Inhibition (%)
– 25 50 100 200 5 – 25 50 100 5
78.69 7 8.27 72.86 7 3.05 74.92 7 6.95 45.82 7 3.08nnn 46.097 4.19nnn 8.887 1.79nnn 82.78 7 8.50 57.22 7 9.27 53.707 4.26n 52.037 4.58n 8.067 1.65nnn
– 7.41 4.79 41.77 41.43 88.94 – 30.88 35.13 37.15 93.64
115.627 7.38 96.69 7 1.83 74.14 7 6.89nnn 81.08 7 9.36nn 104.49 7 10.62 8.70 7 2.67nnn 124.60 7 6.07 102.00 7 12.45 49.37 7 6.85nnn 82.42 7 11.56n 5.26 7 1.95nnn
– 16.37 35.88 29.87 9.63 92.47 – 18.14 60.37 33.86 95.78
Mice were treated with extract (LPEE) or fraction (LPEF) 60 min (p.o.) before formalin test. Data represent the mean 7 S.E.M. of 6–8 animals. The LPEE was compared to vehicle 1 and morphine 1, while LPEF to vehicle 2 and morphine 2. The symbols report significance level. np o 0.05, nnpo 0.01, nnnpo 0.001.
~ et al. / Journal of Ethnopharmacology 146 (2013) 180–186 M.S. Brandao
2.7. Statistical analysis
3. Results
75
Licking time (s)
The results were expressed as the mean 7S.E.M. and analyzed by one-way ANOVA followed by post hoc Bonferroni test. Differences between groups were considered significant when p o0.05 (GraphPad Prism software version 3.0).
183
50
** ***
25
3.1. Acetic acid-induced writhing LPEE (12.5 and 25 mg/kg, p.o.), LPEF and MT (6.25, 12.5 and 25 mg/kg, p.o.) reduced the number of writhings when compared to vehicle. The positive control (morphine 2.5 mg/kg s.c.) was effective when compared to vehicle (Table 1).
0
*** C
50
200
5 Morphine (mg/kg, s.c.)
LPEE (mg/kg, p.o.)
3.2. Formalin test
75
Licking time (s)
LPEE (100 mg/kg p.o.) significantly reduced the time (seconds) the animal stayed licking its stimulated paw in both phases of testing when compared with vehicle, while LPEE (50 mg/kg p.o.) was effective only in the second phase and LPEE (200 mg/kg p.o.) showed significant result only in the first phase. LPEF (50 and 100 mg/kg p.o.) was effective in two phases of response compared with vehicle. Morphine was used as positive control (5 mg/kg s.c.) and decreased significantly response time of the animals when compared to vehicle in both phases (Table 2).
100
50
**
*** ***
25
3.3. Capsaicin test
*** 0
C
12.5
25
50
75
50
* ***
25
***
3.4. Glutamate test Fig. 3A shows that LPEE (25 and 50 mg/kg p.o.) presented a significant reduction of glutamate-induced nociception (64.247 8.29, 47.7175.00 seconds) relative to vehicle (92.5472.06), with corresponds inhibition of 30.58% and 48.43%, respectively; the positive control (MK801 0.03 mg/kg i.p.) significantly reduced the time the animal stayed licking its stimulated paw (20.4573.28) when compared to vehicle, presenting an inhibition of 77.90%. Fig. 3B shows that LPEF (50 and 100 mg/kg p.o.) produced attenuation of the glutamate-induced nociception (53.8475.41, 67.37 710.45) compared to the vehicle (100.86 76.46), leading to an inhibition of 46.62% and 33.20%, respectively; MK801 (0.03 mg/kg i.p.) showed a decrease of the response (20.457 3.28) when compared to vehicle, corresponding to inhibition of 79.72%. MT decreased significantly and dose-dependent response time of the animal at doses of 12.5 and 25 mg/kg p.o. (88.9677.86, 40.50 75.65) compared to vehicle (121.3375.38),
5 Morphine (mg/kg, s.c.)
LPEF (mg/kg, p.o.)
Licking time (s)
The results depicted in Fig. 2A show that LPEE (100 and 200 mg/kg p.o.) reduced the time (seconds) of paw licking of the animal (37.23 74.45 and 24.3472.36) after administration of capsaicin in relation to vehicle (62.8178.32), presenting an inhibition of 40.73% and 61.24%, respectively. The results presented in Fig. 2B show that LPEF orally produced marked inhibition of the capsaicin-induced neurogenic pain in mice in all doses tested (12.5, 25 and 50 mg/kg) (32.42 74.00, 25.4774.11, 35.41 74.31) compared to vehicle (62.81 78.32), corresponding to inhibition of 48.38%, 59.45% and 43.62%, respectively. MT was effective at the doses of 6.25, 12.5 and 25 mg/kg (30.4075.33, 27.37 72.50, 39.38 76.64) compared to vehicle (62.8178.32) (Fig. 2C), with inhibition of 51.60%, 56.43% and 37.30%, respectively. Morphine (5 mg/kg s.c.) caused a reduction in the response (5.40 70.36) when compared to vehicle, presenting an inhibition of 91.40%.
0
*** C
6.25
12.5 MT (mg/kg, p.o.)
25
5 Morphine (mg/kg, s.c.)
Fig. 2. Effect of ethanol extract from Lecythis pisonis—LPEE (Panel A), ethereal fraction—LPEF (Panel B) and the mixture of ursolic and oleanolic acids—MT (Panel C) administered orally against capsaicin induced nociception in mice. Each column represents the mean 7 S.E.M of 6–8 animals. Control value (C) indicates the animals treated with vehicle and the asterisks denote the significance levels, when compared with control groups (one-way ANOVA followed by Bonferroni test) (*p o 0.05, **p o 0.01, ***p o 0.001).
corresponding to losses of 26.68% and 66.62%, respectively (Fig. 3C); MK801 (0.03 mg/kg i.p.) produced marked inhibition of the nociception (20.4573.28) when compared to vehicle, corresponding to decreasing of 83.14%.
~ et al. / Journal of Ethnopharmacology 146 (2013) 180–186 M.S. Brandao
184
150
75
Licking time (s)
Licking time (s)
100
* ***
50
b
a
100
*** 50
***
25
***
0
0
-
-
-
-
-
-
+ -
-
+ -
-
Morphine Glutamate +
-
+
+ + -
+
+
+ +
+ +
+
C
12.5
25
50
LPEE (mg/kg, p.o.)
0.03 MK801 (mg/kg, i.p.)
Naloxone LPEF
150
150
Licking time (s)
Licking time (s)
+
Vehicle
100
** ***
50
b
100
*** 50
*** 0
C
25
50
100
LPEF (mg/kg, p.o.)
0.03 MK801 (mg/kg, i.p.)
150
0
+
-
-
-
Glibenclamide -
Vehicle
+
-
+ -
-
LPEF
+
+
Glutamate
+
+
+
+
100
** a
50
*** ***
0
C
6.25
12.5 MT (mg/kg, p.o.)
25
100
MK 801 (mg/kg, i.p.)
3.5. Evaluation of possible mechanisms of action of the LPEF in mice Naloxone (2 mg/kg i.p.), glibenclamide (3 mg/kg i.p.) and (600 mg/kg i.p.) significantly reversed the effect of LPEF (50 mg/kg p.o.) (Fig. 4).
b
a ***
50
***
0.03
Fig. 3. Effect of ethanolic extract from Lecythis pisonis—LPEE (Panel A), ethereal fraction—LPEF (Panel B) and the mixture of ursolic and oleanolic acids—MT (Panel C) administered orally against glutamate induced nociception in mice. Each column represents the mean 7 S.E.M of 6–11 animals. Control value (C) indicates the animals treated with vehicle; the asterisks denote the significance levels, when compared with control groups; and a when compared to MT 12.5 mg/kg group (one-way ANOVA followed by Bonferroni test) (*po 0.05, **p o 0.01, ***p o 0.001, apo 0.001).
L-arginine
Licking time (s)
Licking time (s)
150
0
Vehicle
+
-
-
-
-
-
L-ARG
-
+ -
+
+ +
-
L-NOARG
-
+ -
LPEF
-
-
-
-
+
+
Glutamate
+
+
+
+
+
+
Fig. 4. Effect of LPEF (50 mg/kg, p.o.) against the action of naloxone (2 mg/kg, i.p.), glibenclamide (3 mg/kg, i.p.), L-arginine (600 mg/kg, i.p.) and vehicle on glutamate-induced nociception (20 mL, 20 mmol/paw) in mice. Data represent mean 7 S.E.M of 6–11 animals. The symbols indicate the level of significance (***po 0.001 compared with vehicle (C), ap o 0.001 compared with the morphine or L-NOARG groups, bpo 0.001 compared with group LPEF; þ, treatment present; , missing treatment) (one-way ANOVA followed by Bonferroni test).
~ et al. / Journal of Ethnopharmacology 146 (2013) 180–186 M.S. Brandao
4. Discussion The results of this study validate the use of Lecythis pisonis leaves in the traditional medicine and show for the first time that the ethanol extract (LPEE) and ethereal fraction (LPEF) have an antinociceptive activity when administered orally in different models of chemical nociception in mice. These data also confirm and extend the literature regarding the antinociceptive action of ursolic and oleanolic acids (main constituents identified in LPEF) (Maia et al., 2006; Oliveira et al., 2012). Oral administration of LPEE from Lecythis pisonis leaves, up to the dose of 2 g/kg, showed no signs of acute toxicity within 14 days of observation (Silva et al., 2012). The absence of toxicity presented by LPEE enabled the establishment of the doses used in this study. We started the investigation of the antinociceptive activity of Lecythis pisonis using acetic acid-induced writhing reaction in mice. LPEE, LPEF and MT (mixture of triterpenes–ursolic and oleanolic acids) produced marked suppression of writhing response at dose levels tested. This response has been described as a typical model of inflammatory pain and has long been used as a screening tool for the assessment of analgesic or antiinflammatory properties of new agents (Collier et al., 1968). Protons depolarize sensory neurones by directly activating a non-selective cationic channel localized on cutaneous, visceral and other types of peripheral afferent C fibers (Julius and Basbaum, 2001). Local irritation also induces the release of endogenous mediators such as bradykinin, prostaglandins and cytokines (TNF-a, IL-1b and IL-8), which stimulate the nociceptive neurons. This method shows good sensitivity but poor specificity because the abdominal writhing response may be suppressed by muscle relaxants and other drugs, leaving scope for the misinterpretation of results (Le Bars et al., 2001). This can be avoided by complementing the test with other models of nociception. Our results also show that LPEE and LPEF were effective in both phases of the formalin test. The intraplantar injection of formalin promotes two phases of pain sensitivity. The first phase (neurogenic pain) occurs through direct chemical stimulation promoted by formalin on nociceptors, in type C and part of the Ad afferent fibers, and it is associated with the release of excitatory amino acids, nitric oxide and substance P, among others. The second phase (inflammatory pain) is characterized as inflammatory pain, associated with the release of chemical mediators such as histamine, serotonin, bradykinin, prostaglandins and excitatory amino acids (Hunskaar and Hole, 1987; Corrˆea and Calixto, 1993). In addition, LPEE, LPEF and MT were able to inhibit the neurogenic nociception caused by capsaicin, an alkaloid extracted from red pepper Capsicum, which stimulates nociceptive and thermal nerve endings causing intense pain. Capsaicin acts specifically in unmyelinated type C fibers and poorly in myelinated and thin Ad fibers through the vanilloid receptor (TRPV-1) in the peripheral nervous system, by opening a nonselective cation channel, allowing the influx of cations, mainly Ca2 þ and Na þ , causing depolarization and initiation of action potentials. Capsaicin determines the release of neuropeptides, especially tachykinins (substance P, neurokinin A and neurokinin B), which operate in the transmission of pain sensation, in the nociceptive pathway and in inflammatory processes (Palazzo et al., 2008). In this study, we also observed that oral administration of LPEE, LPEF and MT produced a significant inhibition of the nociceptive response caused by glutamate injection into the mouse hind paw, at doses that did not produce any statistically significant motor dysfunction (Silva et al., 2012). It is known that glutamate is the major excitatory neurotransmitter involved in nociceptive signal transmission. Moreover, the intraplantar injection of glutamate releases excitatory amino acids, PGE2
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(Prostaglandina E2), NO (Nitric oxide), kinins, protons, SP (Substance P) and more glutamate in the dorsal horn. Glutamate induces a nociceptive response through its action on glutamate receptors that are present in peripheral, spinal and supraspinal sites of action and is largely mediated by both NMDA (N-methylD-aspartate) and non-NMDA receptors as well as by the release of NO or by some NO-related substance (Beirith et al., 2002; Sakurada et al., 2003). It is known that drugs with sedative activity can also inhibit scratching behavior and motor response (Watanable et al., 1999). However, in a previous study LPEE did not alter the locomotor activity of animals with the highest doses that presented antinociceptive effect (Silva et al., 2012), suggesting that the sedative action of LPEE may not be involved in the antinociceptive activity observed here. Therefore, the suppression of the capsaicin, formalin and glutamate induced licking response and acetic acid-induced abdominal writhing, caused by treatment with LPEE and LPEF, are complementary indications that the antinociceptive action of this plant could be associated with its ability to inhibit NO production or through interaction with the glutamatergic system. This fact has been corroborated with the reversion of LPEF antinociceptive effect by the pretreatment with L-arginine (substrate for NO formation) in this model. In the present study, we attempted to characterize some of the mechanisms through which LPEF exerts its antinociceptive action on glutamate-induced nociception in mice. We observed clearly that naloxone (nonselective antagonist of opioid receptors), at a dose that produced no significant effect on glutamate nociception, completely reversed the antinociception induced by both morphine and LPEF. The LPEF antinociception was also antagonized þ by pretreatment with glibenclamide (a blocker of KATP channels), þ suggesting that opioid system via KATP channels is likely to be involved in LPEF antinociception. These results can be related to the popular use of this species like antipruriginous (Silva et al., 2012). Some studies show that itch-sensing neurons (pruriceptors) can respond to pain-inducing (algogenic) stimuli. The ‘‘spatial contrast’’ theory proposes that itch is encoded when a minority of nociceptive fibers are activated in a receptive field, and pain is encoded when a large number of nociceptor fibers are activated. This theory argues that pain and itch can be coded in the absence of pain-specific and itch-specific labeled lines (Sikand et al., 2009). However, this theory appears to conflict with increasing evidence supporting the existence of itch-specific primary and relay sensory neurons (Sun and Chen, 2007; Sun et al., 2009). Various pruritic chemicals produce itch and scratching when injected or applied to the skin and also play a pivotal role in pain and hyperalgesia (increased sensitivity to pain) in inflamed tissues by exciting or sensitizing C-fiber nociceptors. These compounds include most proinflammatory molecules – such as histamine, serotonin, and prostaglandins – for which there are corresponding well-characterized G-protein-coupled receptors (GPCRs). Based on the marked specificity of interaction between GPCRs and ligands, each pruritic chemical has been assumed to transmit itch and pain messages through the activation of the same cognate GPCR. Thus, it was thought that differentiation between itch and pain messages was likely depending on the cell types that are specialized as parts of neural circuits segregated for mediating itch or pain responses. Alternatively, it has been suggested that pruritic compounds may produce complex response patterns in the skin, and the resultant ‘‘codes’’ may be processed and deciphered in the central nervous system to generate the sensations of itch or pain (Han and Simon, 2011). Increasing evidence also points to the fact that many GPCRs important for itch also regulate PLC/Ca2 þ signaling in nociceptive
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processing. Thus, multiple intracellular signal transduction pathways required for pruriception are likely to converge, cross talk, and even compete with nociceptive signaling in various settings (Jeffry et al., 2011). In conclusion, the results presented in this study revealed that the LPEE, LPEF and MT from leaves of Lecythis pisonis exhibited antinociceptive activity in animal models of acute nociception, þ what can be related to its medicinal use. The opioid pathway, KATP channels and negative modulation of L-arginine-nitric oxide are involved in this effect and the presence of triterpenoids like ursolic and oleanolic acids may contribute to the antinociceptive response observed. So, the multiplicity of mechanisms demonstrated by this vegetable preparation is a great opportunity to develop novel antagonists, ligands, and ion channel–active drugs that will provide relief of pain and itch. References Agra, M.F., Freitas, P.F., Barbosa Filho, J.M., 2007. Synopsis of the plants known as medicinal and poisonous in Northeast of Brazil. Brazilian Journal of Pharmacognosy 17, 116–155. Andrade, S.F., Cardoso, L.G.V., Carvalho, J.C.T., Bastos, J.K., 2007. Anti-inflammatory and antinociceptive activities of extract, fractions and populnoic acid from bark wood of Austroplenckia populnea. Journal of Ethnopharmacology 109, 464–471. Barua, C.C., Talukdar, A., Begum, S.A., Lahon, L.C., Sarma, D.K., Pathak, D.C., Borah, P., 2010. Antinociceptive activity of methanolic extract of leaves of Achyranthes aspera Linn. (Amaranthaceae) in animal models of nociception. Indian Journal of Experimental Biology 48, 817–821. Beirith, A., Santos, A.R.S., Calixto, J.B., 2002. Mechanisms underlying the nociception and paw oedema caused by injection of glutamate into the mouse paw. Brain Research 924, 219–228. Collier, H.O.J., Dinneen, L.C., Johnson, C.A., Schneider, C., 1968. The abdominal constriction response and its suppression by analgesic drugs in the mouse. British Journal of Pharmacology Chemotherapy 32, 295–310. Corrˆea, C.R., Calixto, J.B., 1993. Evidence for participation of B1and B2 kinin receptor in formalin-induced nociceptive response in mouse. British Journal of Pharmacology 110, 193–198. Couto, V.M., Vilela, F.C., Dias, D.F., Santos, M.H., Soncini, R., Nascimento, C.G.O., Giusti-Paiva, A., 2011. Antinociceptive effect of extract of Emilia sonchifolia in mice. Journal of Ethnopharmacology 134, 348–353. Drzezga, A., Darsowb, U., Treedec, R., Siebnerd, H., Frischb, M., Munza, F., Weilked, F., Ringb, J., Schwaigera, M., Bartensteina, P., 2001. Central activation by histamineinduced itch: analogies to pain processing: a correlational analysis of O-15 H2O positron emission tomography studies. Pain 92, 295–305. Fagundes, L.L., Vieira, G.D.V., Pinho, J.J.R.G., Yamamoto, C.H., Alves, M.S., Stringheta, P.C., Sousa, O.V., 2010. Pharmacological proprieties of ethanol extract of Muehlenbeckia platyclada (F. Muell.) Meisn. leaves. International Journal of Molecular Sciences 11, 3942–3953. Franco, E.A.P., Barros, R.F.M., 2006. Uso e diversificac- a~ o de plantas medicinais no quilombo olho d‘a´gua dos Pires, Esperantina, Piauı´. Revista Brasileira de Plantas Medicinais, Botucatu 8, 78–88.
Han, S.-K., Simon, M.I., 2011. Intracellular signaling and the origins of the sensations of itch and pain. Science Signaling 4, 1–3. Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30, 103–114. Jeffry, J., Kim, S., Chen, Z., 2011. Itch signaling in the nervous system. Physiology 26, 286–292. Julius, D., Basbaum, A.I., 2001. Molecular mechanisms of nociception. Nature 413, 203–210. Junges, M.J., Fernandes, J.B., Vieira, P.C., Fernandes, M.F.G.S., Rodrigues Filho, E., ¨ ˜ ano, A.G., 2000. Triterpenos ursˆanicos e oleanˆanicos isolados Fruhauf, M., Baran do caule de Eugenia florida DC. Revista de Pesquisa e Po´s-Graduac- a~ o da Universidade Regional Integrada 1, 13–30. Le Bars, D., Gozariu, M., Cadden, S.W., 2001. Animal models of nociception. Pharmacological Reviews 53, 597–652. Mahato, S.B., Kundu, A.P., 1994. 13C NMR spectra of pentacyclic triterpenoids—a compilation and some salient features. Phytochemistry 37, 1517–1575. Maia, J.L., Lima-Ju´nior, R.C.P., David, J.P., David, J.M., Santos, F.A., Rao, V.S., 2006. Oleanolic acid, a pentacyclic triterpene attenuates the mustard oil-induced colonic nociception in mice. Biological & Pharmaceutical Bulletin 29, 82–85. Oliveira, J.P., Ferreira, E.L.F., Chaves, M.H., Milita~ o, G.C.G., Junior, G.M.V., Costa, A. ´ ., Moraes, M. O. de, Costa-Lotufo, L.V., 2012. Chemical de M., Pessoa, C. do O constituents of Lecythis pisonis and cytotoxic activity. Brazilian Journal of Pharmacognosy 22, 1140–1144. Palazzo, E., Rossi, F., Malone, S., 2008. Role of TRPV1 receptors in descending modulation of pain. Molecular and Cellular Endocrinology 286, 79–83. Roome, T., Dar, A., Naqvi, S., Choudhary, M.I., 2011. Evaluation of antinociceptive effect of Aegiceras corniculatum stems extracts and its possible mechanism of action in rodents. Journal of Ethnopharmacology 135, 351–358. Sakurada, T., Katsumata, K., Tanno, K., Sakurada, S., Kisara, K., 1992. The capsaicin test in mice for evaluating tachykinin antagonists in the spinal cord. Neuropharmacology 31, 1279–1285. Sakurada, T., Matsumura, T., Moriyama, T., Sakurada, C., Ueno, S., Sakurada, S., 2003. Differential effects of intraplantar capsazepine and ruthenium red on capsaicin-induced desensitization in mice. Pharmacology Biochemistry and Behavior. 75, 115–121. Santos, A.R.S., Gadotti, V.M., Oliveira, G.L., Tibola, D., Paszcuk, A.F., Neto, A., 2005. Mechanisms involved in the antinociception caused by agmatine in mice. Neuropharmacology 48, 21–34. Sikand, P., Shimada, S.G., Green, B.G., LaMotte, R.H., 2009. Similar itch and nociceptive sensations evoked by punctuate cutaneous application of capsaicin, histamine and cowhage. Pain 144, 66–75. Silva, L.L., Gomes, B.S., Sousa-Neto, B.P., Oliveira, J.P.C., Ferreira, E.L.F., Chaves, M.H., Oliveira, F.A., 2012. Effects of Lecythis pisonis Camb. (Lecythidaceae) in a mouse model of pruritus. Journal of Ethnopharmacology 139, 90–97. Sun, Y.G., Chen, Z.F., 2007. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord. Nature 448, 700–703. Sun, Y.G., Zhao, Z.Q., Meng, X.L., Yin, J., Liu, X.Y., Chen, Z.F., 2009. Cellular basis of itch sensation. Science 325, 1531–1534. Taviano, N.F., Miceli, N., Monforte, M.T., Tzakou, O., Galati, E.M., 2007. Ursolic acid plays a role in Nepeta sibthorpii Bentham CNS depressing effects. Phytotherapy Research 21, 382–385. Vallilo, M.I., Tavares, M., Pimentel, S.A., Campos, N.C., Moita Neto, J.M., 1999. Lecythis pisonis Camb. nuts: oil characterization, fatty acids and minerals. Food Chemistry. 66, 197–200. Watanable, C., Satoh, T., Tahara, E., Murakami, K., Hayashi, K., Hase, K., 1999. Inhibitory mechanism of glycoprotein fraction derived from Miscanthus sinensis for the immediate phase response of an IgE-mediated cutaneous reaction. Biological & Pharmaceutical Bulletin 22, 26–30.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Antinociceptive effect of Lecythis pisonis Camb. (Lecythidaceae) in models of acute pain in mice M.S. Branda~ o a, S.S. Pereira a, D.F. Lima a, J.P.C. Oliveira b, E.L.F. Ferreira b, M.H. Chaves b, F.R.C. Almeida a,n a b
´tima s/n, 64049-550 Teresina, Brazil Medicinal Plants Research Center, Federal University of Piauı´, Av. Nossa Senhora de Fa Department of Chemistry, Federal University of Piauı´, Teresina, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2012 Received in revised form 14 December 2012 Accepted 20 December 2012 Available online 28 December 2012
Ethnopharmacological relevance: Lecythis pisonis Camb., also known in Brazil as sapucaia, is used in folk medicine against pruritus, muscle pain and gastric ulcer. Aim of the study: To investigate the antinociceptive effect of ethanol extract from Lecythis pisonis leaves (LPEE), fractions (hexane-LPHF, ether-LPEF and ethyl acetate-LPEAF) and mixture of triterpenes [ursolic and oleanolic acids (MT)] in mice. Materials and methods: LPEE and LPEF were evaluated on the acetic acid induced writhings and formalin, capsaicin and glutamate tests. In addition, MT was investigated on the writhings induced by acetic acid, capsaicin and glutamate tests. In the study of some possible mechanisms involved on the þ channels and antinociceptive effect of LPEF, it was investigated the participation of opioid system, KATP L-arginine-nitric oxide pathway. Results: LPEE (12.5 and 25 mg/kg, p.o.), LPEF and MT (6.25, 12.5 and 25 mg/kg, p.o.) reduced the writhings in comparison to saline. LPEE (100 mg/kg, p.o.) and LPEF (50 mg/kg, p.o.) were effective in inhibiting both phases of formalin test. In capsaicin test, LPEE (100 and 200 mg/kg, p.o.), LPEF (12.5– 50 mg/kg, p.o) and MT (6.25–25 mg/kg, p.o.) showed a significant antinociceptive effect compared to the control. LPEE (25 and 50 mg/kg, p.o.), LPEF (50 and 100 mg/kg, p.o.) and MT (12.5 and 25 mg/kg, p.o.) reduced the glutamate-evoked nociceptive response. Treatment with naloxone, L-arginine and glibenclamide reversed the effect of LPEF in glutamate test. Conclusions: These results indicate the antinociceptive effect of Lecythis pisonis leaves and suggest that þ channels, and L-arginine-nitric oxide modulation. this effect may be related to opioid pathway, KATP Furthermore, these data support the ethnomedical use of this plant. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Lecythis pisonis Lecythidaceae Antinociceptive effect Ursolic/oleanolic acid
1. Introduction Lecythis pisonis Camb. belongs to the Lecythidaceae botanic family and is popularly known in Brazil as ‘‘sapucaia’’ or ‘‘cumbuca de macaco’’. It is found mainly in the states of Piauı´, from Pernambuco to Sa~ o Paulo and in the Amazon region. The seeds of this species are a valuable source of essential amino acids, fatty acids and minerals, being a functional and nutritious food for human consumption (Vallilo et al., 1999). In traditional medicine, it is used for the treatment of itching (pruritus) in the body (Franco and Barros, 2006) and as an emollient, reducing muscle pain (Agra et al., 2007).
Abbreviations: IL-1b, interleukin-1beta; IL-8, interleukin-8; LD50, lethal dosis 50%; L-NOARG, L-NG-nitro arginine; NO, nitric oxide; NMDA, N-methyl-D-aspartate; PGE2, prostaglandin E2; SP, substance P; TNFa, tumor necrosis factor-alfa; TRPV1, transient receptor potential cation channel subfamily V member 1 n Corresponding author. Tel.: þ55 86 3215 58 72; fax: þ 55 86 3237 18 50. E-mail address: [email protected] (F.R.C. Almeida). 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.12.028
Phytochemical investigation of the ethanolic extract of Lecythis pisonis leaves has led to the identification of different fractions such as hexane, ether and ethyl acetate. However, the chromatographic fractioning of the ethereal fraction and its subfractions spectrometric analysis resulted in the identification of 10 isoprenic origin substances, distributed in three binary mixtures of triterpenoid skeletons and ursano oleanano, a mixture of steroid and triterpenoid skeleton called friedelan-3-ol. The presence of triterpenes in the species was confirmed by the isolation and identification of the mixture of ursolic and oleanolic acids (Oliveira et al., 2012). Plants rich in triterpenoids are widely used in medicine because of its anti-inflammatory, protecting of the vascular system, antiallergic, analgesic and antipyretic properties (Andrade et al., 2007). Several species have triterpenoids in its composition and show antinociceptive activity, for example Achyranthes aspera Linn. (Barua et al., 2010), Muehlenbeckia platyclada F. Muell. (Fagundes et al., 2010) and Aegiceras corniculatum L. Blanco (Roome et al., 2011). The species Emilia sonchifolia L. has demonstrated antinociceptive
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activity and chemical composition similar to Lecythis pisonis, including the presence of ursolic acid, sitosterol and stigmasterol (Couto et al., 2011). There are reports showing that ursolic acid is able to produce a significant reduction in acetic acid-induced writhings, indicating that it is effective against inflammatory pain (Taviano et al., 2007). In addition, the oleanolic acid inhibits visceral nociception in the mustard oil-induced colonic nociception in mice through an opioid mechanism, and it may have a modulatory influence on vanilloid receptors (Maia et al., 2006). A previous study provided experimental evidence that justifies the popular use of Lecythis pisonis leaves in the treatment of pruritus and suggests that this effect may be related to a stabilizing action on mast cell membrane (Silva et al., 2012). The precise relationship between itch and pain remains controversial. Itch seems to share numerous features with pain, including induction by noxious chemical stimuli, arousal of attention, negative affective valence and initiation of motor responses (Drzezga et al., 2001). By virtue of aforementioned properties, it is expected that Lecythis pisonis have antinociceptive activity associated with its antipruriginous effect. So, this study aims to evaluate Lecythis pisonis Camb. antinociceptive effect in animal models of chemically induced acute pain and some of the possible mechanisms involved in this effect.
2. Materials and methods 2.1. Plant material and identification Leaves of Lecythis pisonis Camb. (Lecythidaceae) were collected in July 2008 at the Centre for Agrarian Sciences, Federal University of Piauı´ (CCA-UFPI) in the city of Teresina, Piauı´ state, Brazil (51 020 53.200 S, 421 470 16.800 O to 68 m sea level). After collection, a voucher specimen has been identified by Gardene M. Sousa and deposited in the Graziela Barroso Herbarium of Federal University of Piauı´ (TEPB 26488). 2.2. Extraction and isolation Dried and powdered leaves of Lecythis pisonis (2 kg) were extracted at room temperature, consecutively six times with ethanol 98%. The solvent was removed by evaporation under reduced pressure using Hedolph Rotary Evaporator to yield the ethanol extract (LPEE, 272.0 g, 13.6%). Part of the LPEE (200.0 g) was fractionated by serial extraction with hexane, ethyl ether, EtOAc and H2O to yield hexane (LPHF, 60.0 g, 34.0%), ethyl ether (LPEF, 24.0 g, 12.0%), EtOAc (LPEAF, 21.0 g, 10.5%) and H2O (LPAF, 70.0 g, 37.5%) fractions and a precipitate formed in the EtOAc phase which was separated by simple filtration (ppt-EtOAc, 10 g, 5%). The ethereal fraction (LPEF, 10 g) was fractionated on a silica gel column with elution gradient from CHCl3 to MeOH to yield 103 fractions collected as follows: 1–31 (CHCl3, 100%), 32–54 (CHCl3-MeOH, 98:2), 55–62 (CHCl3-MeOH, 95:5), 63–72 (CHCl3MeOH, 9:1), 73–89 (CHCl3-MeOH, 8:2), 90–96 (CHCl3-MeOH, 7:3), 97–103 (MeOH 100%). The fraction B (17–23, 603 mg) was suspended in MeOH to form an amorphous precipitate (F17-ppt, 475 mg, 4.75%) composed of a mixture of ursolic and oleanolic acid triterpenes (Fig. 1). The structural characterization of this mixture was performed using spectroscopic methods such as nuclear magnetic resonance (1H and 13C NMR) and compared with literature data (Mahato and Kundu, 1994; Junges et al., 2000). The ratio of ursolic acid and oleanolic acid in this mixture was 59:41, calculated by 1H NMR, by dividing the signal area of olefinic hydrogens d ¼5.15 (ursolic acid) and d ¼5.19 (oleanolic
181
29
R1 19
1
25
14 10
3
5
HO
H 24
26
23
H 7
20
21
17
13
11
R2
H
COOH 16
28
27
1: R1 = CH3 ; R2 = H 2: R1= H ; R2 = CH3
Fig. 1. Structure of ursolic (1) and oleanolic (2) acids.
acid) with the signal area of d ¼ 3.11 (dd, J¼7.0 and 9.0 Hz) attributed to H-3 in the two triterpenes and multiplying by 100 (Oliveira et al., 2012). The 1H and 13C NMR spectra were recorded on a Varian Inova 500 spectrometer, in CDCl3, at 500 and 125 MHz, respectively, using TMS as internal standard. The chemical shifts values are on d scale and the coupling constants (J) values are in Hertz. Column chromatography was carried out using silica gel 60 (0.063–0.200 mm). Thin layer chromatography (TLC) was carried out on silica gel 60 G (Merck) plates (0.25 mm layer thickness). Extract, fractions and mixture of triterpenes of Lecythis pisonis leaves were dissolved in or diluted with physiological saline (0.9%) and prepared immediately before each experiment. The triterpenes concentration of the extract, fractions and mixture were adjusted to a volume of 10 mL/kg. 2.3. Chemicals and reagents Capsaicin, glibenclamide, glutamate, L-arginine, L-NOARG, Dizocilpine (MK 801), naloxone and Tween 80 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Morphine was purchased from Crista´lia Produtos Quı´micos e Farmacˆeuticos Ltda. (SP, Brazil). Formaldehyde was obtained from Dinˆamica Quı´mica Contemporˆanea Ltda. (SP, Brazil). Acetic acid and sodium chloride were from Vetec Quı´mica Fina Ltda. (RJ, Brazil). 2.4. Animals Male Swiss mice 20–30 g (n ¼6–11) were used, 10 weeks old from the Sector Animal House for Research on Medicinal Plants of the Federal University of Piauı´. The animals were kept at 2471 1C with a light/dark cycle of 12 h, with free access to food and water. They were fasted for a period of 18 h and were accustomed to the test environment for 1 h before each experiment. All experimental protocols were approved by the Ethics Committee for Animal Experimentation at the Federal University of Piauı´ (No. 52/2010). 2.5. Antinociceptive tests 2.5.1. Acetic acid-induced writhing The procedure was similar to a previously described method (Collier et al., 1968). Swiss mice (n ¼6–8) were pretreated with saline vehicle (0.1 mL/10 g), LPEE, LPEF and MT (6.25, 12.5 and 25 mg/kg, p.o.) 60 min before the intraperitoneal administration of 0.75% acetic acid, and then, the total number of writhings was counted over a period of 20 min. The strength of the elicited antinociceptive effect was compared with that of an effective dose
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182
of morphine (2.5 mg/kg, s.c.) administered 30 min before the acetic acid injection. 2.5.2. Formalin test Mice (n ¼6–8) were given orally LPEE (25, 50, 100 and 200 mg/ kg), LPEF (25, 50 and 100 mg/kg) or vehicle (10 mL/kg) 1 h before the test. Morphine (5 mg/kg) was administered subcutaneously 30 min before the test and used as positive control. The right hind foot pad was injected with formalin (20 mL, 2%) in the intraplantar region. Nociception was evaluated by quantifying paw licking time during the first 5 min (first phase) and at 15–30 min (second phase) (Hunskaar and Hole, 1987). 2.5.3. Capsaicin test The animals were individually placed in the observation chamber for an adjustment period of 20 min. After this period, mice (n ¼6–8) were given orally LPEE (50, 100 and 200 mg/kg), LPEF (12.5, 25 and 50 mg/kg), MT (6.25, 12.5 and 25 mg/kg) or vehicle (10 mL/kg). Morphine (5 mg/kg) was administered subcutaneously 30 min before the test and used as a positive control. One hour after these treatments, the right hind paw was injected with capsaicin (2 mg/paw) prepared in 5% Tween solution and 2% ethanol. Nociception was evaluated immediately after injection and quantified by paw licking time during a 5 min period (Sakurada et al., 1992). Table 1 Antinociceptive effect of the ethanol extract, ethereal fraction and the mixture of ursolic and oleanolic acids from leaves of Lecythis pisonis on acetic acid-induced writhings in mice. Treatment
Dose (mg/kg)
Number or writhings
Inhibition (%)
Vehicle Morphine LPEE
– 2.50 6.25 12.50 25.00 6.25 12.50 25.00 6.25 12.50 25.00
73.25 7 2.37 6.17 7 0.91 60.43 7 4.80 31.17 7 2.31nnn 42.17 7 6.01nnn 48.71 7 7.63nnn 34.00 7 3.39nnn 38.67 7 1.91nnn 58.29 7 5.55n 42.00 7 4.57nnn 39.57 7 2.83nnn
– 91.58 17.50 57.45 42.43 33.49 53.58 47.21 20.43 42.66 49.98
LPEF MT
Mice were treated with extract (LPEE), fraction (LPEF) or mixture of triterpenic acids (MT) 60 min (p.o.) before writhing test. Data represent the mean 7S.E.M. of 6–8 animals. The symbols report significance level. npo 0.05, nnnpo 0.001.
2.5.4. Glutamate test The procedure used was similar to the previously described by Beirith et al. (2002). Mice (6–11) received an intraplantar injection of glutamate (20 mmol/paw) 60 min after oral administration of LPEE (12.5, 25 and 50 mg/kg), LPEF (25, 50 and 100 mg/kg), MT (6.25, 12.5 and 25 mg/kg) or vehicle (0.1 mL/10 g), and 30 min after the administration of MK 801 (0.03 mg/kg, i.p.) as a positive control. Animals were observed individually for 15 min following glutamate injection. The amount of time spent licking the injected paw was taken to indicate nociception. 2.6. Evaluation of possible mechanisms of action of the LPEF in mice To address some of the mechanisms by which LPEF of Lecythis pisonis causes antinociception in the glutamate-induced nociception, animals (6–11) were treated with different drugs. The doses of the drugs used were selected on the basis of the literature (Santos et al., 2005) data and also based on previous results from our laboratory. 2.6.1. Participation of opioid system Mice (6–11) were pretreated with naloxone (2 mg/kg, i.p., a non-selective opioid receptor antagonist), and after 20 min the animals received an injection of LPEF (50 mg/kg, p.o.), morphine (5 mg/kg, s.c.) or vehicle (10 ml/kg, p.o.). One hour after administration of LPEF or 30 min after administration of morphine, it was injected 20 mL of glutamate solution (20 mmol/paw, i.pl.) and evaluated the nociceptive response like in glutamate test. 2.6.2. Participation of K þ ATP channels Mice (n ¼6–11) were previously treated with glibenclamide (3 mg/kg, i.p.) and 20 min latter, they received LPEF (50 mg/kg, p.o.) or vehicle (0.1 mL/10 g, p.o.). After 60 min, the animals were assessed for nociception induced by intraplantar injection of 20 mL solution of glutamate (20 mmol/paw). 2.6.3. Participation of L-arginine-nitric oxide Mice (7–11) were pre-treated with L-arginine (600 mg/kg, i.p., a nitric oxide precursor) and after 20 min, they received LPEF (50 mg/kg, p.o.), N$-nitro-L-arginine (L-NOARG, 75 mg/kg, i.p., a nitric oxide inhibitor) or vehicle (10 ml/kg, p.o.). The nociceptive responses to glutamate were recorded 1, 0.5 and 1 h after the administration of LPEF, L-NOARG, or vehicle, respectively.
Table 2 Antinociceptive effect of the ethanol extract and ethereal fraction of leaves from Lecythis pisonis in the formalin induced nociceptive response in mice. Treatment
Vehicle 1 LPEE
Morphine 1 Vehicle 2 LPEF
Morphine 2
Dosage (mg/ kg)
Licking time (s) 0–5 min
Inhibition (%)
15–30 min
Inhibition (%)
– 25 50 100 200 5 – 25 50 100 5
78.69 7 8.27 72.86 7 3.05 74.92 7 6.95 45.82 7 3.08nnn 46.097 4.19nnn 8.887 1.79nnn 82.78 7 8.50 57.22 7 9.27 53.707 4.26n 52.037 4.58n 8.067 1.65nnn
– 7.41 4.79 41.77 41.43 88.94 – 30.88 35.13 37.15 93.64
115.627 7.38 96.69 7 1.83 74.14 7 6.89nnn 81.08 7 9.36nn 104.49 7 10.62 8.70 7 2.67nnn 124.60 7 6.07 102.00 7 12.45 49.37 7 6.85nnn 82.42 7 11.56n 5.26 7 1.95nnn
– 16.37 35.88 29.87 9.63 92.47 – 18.14 60.37 33.86 95.78
Mice were treated with extract (LPEE) or fraction (LPEF) 60 min (p.o.) before formalin test. Data represent the mean 7 S.E.M. of 6–8 animals. The LPEE was compared to vehicle 1 and morphine 1, while LPEF to vehicle 2 and morphine 2. The symbols report significance level. np o 0.05, nnpo 0.01, nnnpo 0.001.
~ et al. / Journal of Ethnopharmacology 146 (2013) 180–186 M.S. Brandao
2.7. Statistical analysis
3. Results
75
Licking time (s)
The results were expressed as the mean 7S.E.M. and analyzed by one-way ANOVA followed by post hoc Bonferroni test. Differences between groups were considered significant when p o0.05 (GraphPad Prism software version 3.0).
183
50
** ***
25
3.1. Acetic acid-induced writhing LPEE (12.5 and 25 mg/kg, p.o.), LPEF and MT (6.25, 12.5 and 25 mg/kg, p.o.) reduced the number of writhings when compared to vehicle. The positive control (morphine 2.5 mg/kg s.c.) was effective when compared to vehicle (Table 1).
0
*** C
50
200
5 Morphine (mg/kg, s.c.)
LPEE (mg/kg, p.o.)
3.2. Formalin test
75
Licking time (s)
LPEE (100 mg/kg p.o.) significantly reduced the time (seconds) the animal stayed licking its stimulated paw in both phases of testing when compared with vehicle, while LPEE (50 mg/kg p.o.) was effective only in the second phase and LPEE (200 mg/kg p.o.) showed significant result only in the first phase. LPEF (50 and 100 mg/kg p.o.) was effective in two phases of response compared with vehicle. Morphine was used as positive control (5 mg/kg s.c.) and decreased significantly response time of the animals when compared to vehicle in both phases (Table 2).
100
50
**
*** ***
25
3.3. Capsaicin test
*** 0
C
12.5
25
50
75
50
* ***
25
***
3.4. Glutamate test Fig. 3A shows that LPEE (25 and 50 mg/kg p.o.) presented a significant reduction of glutamate-induced nociception (64.247 8.29, 47.7175.00 seconds) relative to vehicle (92.5472.06), with corresponds inhibition of 30.58% and 48.43%, respectively; the positive control (MK801 0.03 mg/kg i.p.) significantly reduced the time the animal stayed licking its stimulated paw (20.4573.28) when compared to vehicle, presenting an inhibition of 77.90%. Fig. 3B shows that LPEF (50 and 100 mg/kg p.o.) produced attenuation of the glutamate-induced nociception (53.8475.41, 67.37 710.45) compared to the vehicle (100.86 76.46), leading to an inhibition of 46.62% and 33.20%, respectively; MK801 (0.03 mg/kg i.p.) showed a decrease of the response (20.457 3.28) when compared to vehicle, corresponding to inhibition of 79.72%. MT decreased significantly and dose-dependent response time of the animal at doses of 12.5 and 25 mg/kg p.o. (88.9677.86, 40.50 75.65) compared to vehicle (121.3375.38),
5 Morphine (mg/kg, s.c.)
LPEF (mg/kg, p.o.)
Licking time (s)
The results depicted in Fig. 2A show that LPEE (100 and 200 mg/kg p.o.) reduced the time (seconds) of paw licking of the animal (37.23 74.45 and 24.3472.36) after administration of capsaicin in relation to vehicle (62.8178.32), presenting an inhibition of 40.73% and 61.24%, respectively. The results presented in Fig. 2B show that LPEF orally produced marked inhibition of the capsaicin-induced neurogenic pain in mice in all doses tested (12.5, 25 and 50 mg/kg) (32.42 74.00, 25.4774.11, 35.41 74.31) compared to vehicle (62.81 78.32), corresponding to inhibition of 48.38%, 59.45% and 43.62%, respectively. MT was effective at the doses of 6.25, 12.5 and 25 mg/kg (30.4075.33, 27.37 72.50, 39.38 76.64) compared to vehicle (62.8178.32) (Fig. 2C), with inhibition of 51.60%, 56.43% and 37.30%, respectively. Morphine (5 mg/kg s.c.) caused a reduction in the response (5.40 70.36) when compared to vehicle, presenting an inhibition of 91.40%.
0
*** C
6.25
12.5 MT (mg/kg, p.o.)
25
5 Morphine (mg/kg, s.c.)
Fig. 2. Effect of ethanol extract from Lecythis pisonis—LPEE (Panel A), ethereal fraction—LPEF (Panel B) and the mixture of ursolic and oleanolic acids—MT (Panel C) administered orally against capsaicin induced nociception in mice. Each column represents the mean 7 S.E.M of 6–8 animals. Control value (C) indicates the animals treated with vehicle and the asterisks denote the significance levels, when compared with control groups (one-way ANOVA followed by Bonferroni test) (*p o 0.05, **p o 0.01, ***p o 0.001).
corresponding to losses of 26.68% and 66.62%, respectively (Fig. 3C); MK801 (0.03 mg/kg i.p.) produced marked inhibition of the nociception (20.4573.28) when compared to vehicle, corresponding to decreasing of 83.14%.
~ et al. / Journal of Ethnopharmacology 146 (2013) 180–186 M.S. Brandao
184
150
75
Licking time (s)
Licking time (s)
100
* ***
50
b
a
100
*** 50
***
25
***
0
0
-
-
-
-
-
-
+ -
-
+ -
-
Morphine Glutamate +
-
+
+ + -
+
+
+ +
+ +
+
C
12.5
25
50
LPEE (mg/kg, p.o.)
0.03 MK801 (mg/kg, i.p.)
Naloxone LPEF
150
150
Licking time (s)
Licking time (s)
+
Vehicle
100
** ***
50
b
100
*** 50
*** 0
C
25
50
100
LPEF (mg/kg, p.o.)
0.03 MK801 (mg/kg, i.p.)
150
0
+
-
-
-
Glibenclamide -
Vehicle
+
-
+ -
-
LPEF
+
+
Glutamate
+
+
+
+
100
** a
50
*** ***
0
C
6.25
12.5 MT (mg/kg, p.o.)
25
100
MK 801 (mg/kg, i.p.)
3.5. Evaluation of possible mechanisms of action of the LPEF in mice Naloxone (2 mg/kg i.p.), glibenclamide (3 mg/kg i.p.) and (600 mg/kg i.p.) significantly reversed the effect of LPEF (50 mg/kg p.o.) (Fig. 4).
b
a ***
50
***
0.03
Fig. 3. Effect of ethanolic extract from Lecythis pisonis—LPEE (Panel A), ethereal fraction—LPEF (Panel B) and the mixture of ursolic and oleanolic acids—MT (Panel C) administered orally against glutamate induced nociception in mice. Each column represents the mean 7 S.E.M of 6–11 animals. Control value (C) indicates the animals treated with vehicle; the asterisks denote the significance levels, when compared with control groups; and a when compared to MT 12.5 mg/kg group (one-way ANOVA followed by Bonferroni test) (*po 0.05, **p o 0.01, ***p o 0.001, apo 0.001).
L-arginine
Licking time (s)
Licking time (s)
150
0
Vehicle
+
-
-
-
-
-
L-ARG
-
+ -
+
+ +
-
L-NOARG
-
+ -
LPEF
-
-
-
-
+
+
Glutamate
+
+
+
+
+
+
Fig. 4. Effect of LPEF (50 mg/kg, p.o.) against the action of naloxone (2 mg/kg, i.p.), glibenclamide (3 mg/kg, i.p.), L-arginine (600 mg/kg, i.p.) and vehicle on glutamate-induced nociception (20 mL, 20 mmol/paw) in mice. Data represent mean 7 S.E.M of 6–11 animals. The symbols indicate the level of significance (***po 0.001 compared with vehicle (C), ap o 0.001 compared with the morphine or L-NOARG groups, bpo 0.001 compared with group LPEF; þ, treatment present; , missing treatment) (one-way ANOVA followed by Bonferroni test).
~ et al. / Journal of Ethnopharmacology 146 (2013) 180–186 M.S. Brandao
4. Discussion The results of this study validate the use of Lecythis pisonis leaves in the traditional medicine and show for the first time that the ethanol extract (LPEE) and ethereal fraction (LPEF) have an antinociceptive activity when administered orally in different models of chemical nociception in mice. These data also confirm and extend the literature regarding the antinociceptive action of ursolic and oleanolic acids (main constituents identified in LPEF) (Maia et al., 2006; Oliveira et al., 2012). Oral administration of LPEE from Lecythis pisonis leaves, up to the dose of 2 g/kg, showed no signs of acute toxicity within 14 days of observation (Silva et al., 2012). The absence of toxicity presented by LPEE enabled the establishment of the doses used in this study. We started the investigation of the antinociceptive activity of Lecythis pisonis using acetic acid-induced writhing reaction in mice. LPEE, LPEF and MT (mixture of triterpenes–ursolic and oleanolic acids) produced marked suppression of writhing response at dose levels tested. This response has been described as a typical model of inflammatory pain and has long been used as a screening tool for the assessment of analgesic or antiinflammatory properties of new agents (Collier et al., 1968). Protons depolarize sensory neurones by directly activating a non-selective cationic channel localized on cutaneous, visceral and other types of peripheral afferent C fibers (Julius and Basbaum, 2001). Local irritation also induces the release of endogenous mediators such as bradykinin, prostaglandins and cytokines (TNF-a, IL-1b and IL-8), which stimulate the nociceptive neurons. This method shows good sensitivity but poor specificity because the abdominal writhing response may be suppressed by muscle relaxants and other drugs, leaving scope for the misinterpretation of results (Le Bars et al., 2001). This can be avoided by complementing the test with other models of nociception. Our results also show that LPEE and LPEF were effective in both phases of the formalin test. The intraplantar injection of formalin promotes two phases of pain sensitivity. The first phase (neurogenic pain) occurs through direct chemical stimulation promoted by formalin on nociceptors, in type C and part of the Ad afferent fibers, and it is associated with the release of excitatory amino acids, nitric oxide and substance P, among others. The second phase (inflammatory pain) is characterized as inflammatory pain, associated with the release of chemical mediators such as histamine, serotonin, bradykinin, prostaglandins and excitatory amino acids (Hunskaar and Hole, 1987; Corrˆea and Calixto, 1993). In addition, LPEE, LPEF and MT were able to inhibit the neurogenic nociception caused by capsaicin, an alkaloid extracted from red pepper Capsicum, which stimulates nociceptive and thermal nerve endings causing intense pain. Capsaicin acts specifically in unmyelinated type C fibers and poorly in myelinated and thin Ad fibers through the vanilloid receptor (TRPV-1) in the peripheral nervous system, by opening a nonselective cation channel, allowing the influx of cations, mainly Ca2 þ and Na þ , causing depolarization and initiation of action potentials. Capsaicin determines the release of neuropeptides, especially tachykinins (substance P, neurokinin A and neurokinin B), which operate in the transmission of pain sensation, in the nociceptive pathway and in inflammatory processes (Palazzo et al., 2008). In this study, we also observed that oral administration of LPEE, LPEF and MT produced a significant inhibition of the nociceptive response caused by glutamate injection into the mouse hind paw, at doses that did not produce any statistically significant motor dysfunction (Silva et al., 2012). It is known that glutamate is the major excitatory neurotransmitter involved in nociceptive signal transmission. Moreover, the intraplantar injection of glutamate releases excitatory amino acids, PGE2
185
(Prostaglandina E2), NO (Nitric oxide), kinins, protons, SP (Substance P) and more glutamate in the dorsal horn. Glutamate induces a nociceptive response through its action on glutamate receptors that are present in peripheral, spinal and supraspinal sites of action and is largely mediated by both NMDA (N-methylD-aspartate) and non-NMDA receptors as well as by the release of NO or by some NO-related substance (Beirith et al., 2002; Sakurada et al., 2003). It is known that drugs with sedative activity can also inhibit scratching behavior and motor response (Watanable et al., 1999). However, in a previous study LPEE did not alter the locomotor activity of animals with the highest doses that presented antinociceptive effect (Silva et al., 2012), suggesting that the sedative action of LPEE may not be involved in the antinociceptive activity observed here. Therefore, the suppression of the capsaicin, formalin and glutamate induced licking response and acetic acid-induced abdominal writhing, caused by treatment with LPEE and LPEF, are complementary indications that the antinociceptive action of this plant could be associated with its ability to inhibit NO production or through interaction with the glutamatergic system. This fact has been corroborated with the reversion of LPEF antinociceptive effect by the pretreatment with L-arginine (substrate for NO formation) in this model. In the present study, we attempted to characterize some of the mechanisms through which LPEF exerts its antinociceptive action on glutamate-induced nociception in mice. We observed clearly that naloxone (nonselective antagonist of opioid receptors), at a dose that produced no significant effect on glutamate nociception, completely reversed the antinociception induced by both morphine and LPEF. The LPEF antinociception was also antagonized þ by pretreatment with glibenclamide (a blocker of KATP channels), þ suggesting that opioid system via KATP channels is likely to be involved in LPEF antinociception. These results can be related to the popular use of this species like antipruriginous (Silva et al., 2012). Some studies show that itch-sensing neurons (pruriceptors) can respond to pain-inducing (algogenic) stimuli. The ‘‘spatial contrast’’ theory proposes that itch is encoded when a minority of nociceptive fibers are activated in a receptive field, and pain is encoded when a large number of nociceptor fibers are activated. This theory argues that pain and itch can be coded in the absence of pain-specific and itch-specific labeled lines (Sikand et al., 2009). However, this theory appears to conflict with increasing evidence supporting the existence of itch-specific primary and relay sensory neurons (Sun and Chen, 2007; Sun et al., 2009). Various pruritic chemicals produce itch and scratching when injected or applied to the skin and also play a pivotal role in pain and hyperalgesia (increased sensitivity to pain) in inflamed tissues by exciting or sensitizing C-fiber nociceptors. These compounds include most proinflammatory molecules – such as histamine, serotonin, and prostaglandins – for which there are corresponding well-characterized G-protein-coupled receptors (GPCRs). Based on the marked specificity of interaction between GPCRs and ligands, each pruritic chemical has been assumed to transmit itch and pain messages through the activation of the same cognate GPCR. Thus, it was thought that differentiation between itch and pain messages was likely depending on the cell types that are specialized as parts of neural circuits segregated for mediating itch or pain responses. Alternatively, it has been suggested that pruritic compounds may produce complex response patterns in the skin, and the resultant ‘‘codes’’ may be processed and deciphered in the central nervous system to generate the sensations of itch or pain (Han and Simon, 2011). Increasing evidence also points to the fact that many GPCRs important for itch also regulate PLC/Ca2 þ signaling in nociceptive
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processing. Thus, multiple intracellular signal transduction pathways required for pruriception are likely to converge, cross talk, and even compete with nociceptive signaling in various settings (Jeffry et al., 2011). In conclusion, the results presented in this study revealed that the LPEE, LPEF and MT from leaves of Lecythis pisonis exhibited antinociceptive activity in animal models of acute nociception, þ what can be related to its medicinal use. The opioid pathway, KATP channels and negative modulation of L-arginine-nitric oxide are involved in this effect and the presence of triterpenoids like ursolic and oleanolic acids may contribute to the antinociceptive response observed. So, the multiplicity of mechanisms demonstrated by this vegetable preparation is a great opportunity to develop novel antagonists, ligands, and ion channel–active drugs that will provide relief of pain and itch. References Agra, M.F., Freitas, P.F., Barbosa Filho, J.M., 2007. Synopsis of the plants known as medicinal and poisonous in Northeast of Brazil. Brazilian Journal of Pharmacognosy 17, 116–155. Andrade, S.F., Cardoso, L.G.V., Carvalho, J.C.T., Bastos, J.K., 2007. Anti-inflammatory and antinociceptive activities of extract, fractions and populnoic acid from bark wood of Austroplenckia populnea. Journal of Ethnopharmacology 109, 464–471. Barua, C.C., Talukdar, A., Begum, S.A., Lahon, L.C., Sarma, D.K., Pathak, D.C., Borah, P., 2010. Antinociceptive activity of methanolic extract of leaves of Achyranthes aspera Linn. (Amaranthaceae) in animal models of nociception. Indian Journal of Experimental Biology 48, 817–821. Beirith, A., Santos, A.R.S., Calixto, J.B., 2002. Mechanisms underlying the nociception and paw oedema caused by injection of glutamate into the mouse paw. Brain Research 924, 219–228. Collier, H.O.J., Dinneen, L.C., Johnson, C.A., Schneider, C., 1968. The abdominal constriction response and its suppression by analgesic drugs in the mouse. British Journal of Pharmacology Chemotherapy 32, 295–310. Corrˆea, C.R., Calixto, J.B., 1993. Evidence for participation of B1and B2 kinin receptor in formalin-induced nociceptive response in mouse. British Journal of Pharmacology 110, 193–198. Couto, V.M., Vilela, F.C., Dias, D.F., Santos, M.H., Soncini, R., Nascimento, C.G.O., Giusti-Paiva, A., 2011. Antinociceptive effect of extract of Emilia sonchifolia in mice. Journal of Ethnopharmacology 134, 348–353. Drzezga, A., Darsowb, U., Treedec, R., Siebnerd, H., Frischb, M., Munza, F., Weilked, F., Ringb, J., Schwaigera, M., Bartensteina, P., 2001. Central activation by histamineinduced itch: analogies to pain processing: a correlational analysis of O-15 H2O positron emission tomography studies. Pain 92, 295–305. Fagundes, L.L., Vieira, G.D.V., Pinho, J.J.R.G., Yamamoto, C.H., Alves, M.S., Stringheta, P.C., Sousa, O.V., 2010. Pharmacological proprieties of ethanol extract of Muehlenbeckia platyclada (F. Muell.) Meisn. leaves. International Journal of Molecular Sciences 11, 3942–3953. Franco, E.A.P., Barros, R.F.M., 2006. Uso e diversificac- a~ o de plantas medicinais no quilombo olho d‘a´gua dos Pires, Esperantina, Piauı´. Revista Brasileira de Plantas Medicinais, Botucatu 8, 78–88.
Han, S.-K., Simon, M.I., 2011. Intracellular signaling and the origins of the sensations of itch and pain. Science Signaling 4, 1–3. Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30, 103–114. Jeffry, J., Kim, S., Chen, Z., 2011. Itch signaling in the nervous system. Physiology 26, 286–292. Julius, D., Basbaum, A.I., 2001. Molecular mechanisms of nociception. Nature 413, 203–210. Junges, M.J., Fernandes, J.B., Vieira, P.C., Fernandes, M.F.G.S., Rodrigues Filho, E., ¨ ˜ ano, A.G., 2000. Triterpenos ursˆanicos e oleanˆanicos isolados Fruhauf, M., Baran do caule de Eugenia florida DC. Revista de Pesquisa e Po´s-Graduac- a~ o da Universidade Regional Integrada 1, 13–30. Le Bars, D., Gozariu, M., Cadden, S.W., 2001. Animal models of nociception. Pharmacological Reviews 53, 597–652. Mahato, S.B., Kundu, A.P., 1994. 13C NMR spectra of pentacyclic triterpenoids—a compilation and some salient features. Phytochemistry 37, 1517–1575. Maia, J.L., Lima-Ju´nior, R.C.P., David, J.P., David, J.M., Santos, F.A., Rao, V.S., 2006. Oleanolic acid, a pentacyclic triterpene attenuates the mustard oil-induced colonic nociception in mice. Biological & Pharmaceutical Bulletin 29, 82–85. Oliveira, J.P., Ferreira, E.L.F., Chaves, M.H., Milita~ o, G.C.G., Junior, G.M.V., Costa, A. ´ ., Moraes, M. O. de, Costa-Lotufo, L.V., 2012. Chemical de M., Pessoa, C. do O constituents of Lecythis pisonis and cytotoxic activity. Brazilian Journal of Pharmacognosy 22, 1140–1144. Palazzo, E., Rossi, F., Malone, S., 2008. Role of TRPV1 receptors in descending modulation of pain. Molecular and Cellular Endocrinology 286, 79–83. Roome, T., Dar, A., Naqvi, S., Choudhary, M.I., 2011. Evaluation of antinociceptive effect of Aegiceras corniculatum stems extracts and its possible mechanism of action in rodents. Journal of Ethnopharmacology 135, 351–358. Sakurada, T., Katsumata, K., Tanno, K., Sakurada, S., Kisara, K., 1992. The capsaicin test in mice for evaluating tachykinin antagonists in the spinal cord. Neuropharmacology 31, 1279–1285. Sakurada, T., Matsumura, T., Moriyama, T., Sakurada, C., Ueno, S., Sakurada, S., 2003. Differential effects of intraplantar capsazepine and ruthenium red on capsaicin-induced desensitization in mice. Pharmacology Biochemistry and Behavior. 75, 115–121. Santos, A.R.S., Gadotti, V.M., Oliveira, G.L., Tibola, D., Paszcuk, A.F., Neto, A., 2005. Mechanisms involved in the antinociception caused by agmatine in mice. Neuropharmacology 48, 21–34. Sikand, P., Shimada, S.G., Green, B.G., LaMotte, R.H., 2009. Similar itch and nociceptive sensations evoked by punctuate cutaneous application of capsaicin, histamine and cowhage. Pain 144, 66–75. Silva, L.L., Gomes, B.S., Sousa-Neto, B.P., Oliveira, J.P.C., Ferreira, E.L.F., Chaves, M.H., Oliveira, F.A., 2012. Effects of Lecythis pisonis Camb. (Lecythidaceae) in a mouse model of pruritus. Journal of Ethnopharmacology 139, 90–97. Sun, Y.G., Chen, Z.F., 2007. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord. Nature 448, 700–703. Sun, Y.G., Zhao, Z.Q., Meng, X.L., Yin, J., Liu, X.Y., Chen, Z.F., 2009. Cellular basis of itch sensation. Science 325, 1531–1534. Taviano, N.F., Miceli, N., Monforte, M.T., Tzakou, O., Galati, E.M., 2007. Ursolic acid plays a role in Nepeta sibthorpii Bentham CNS depressing effects. Phytotherapy Research 21, 382–385. Vallilo, M.I., Tavares, M., Pimentel, S.A., Campos, N.C., Moita Neto, J.M., 1999. Lecythis pisonis Camb. nuts: oil characterization, fatty acids and minerals. Food Chemistry. 66, 197–200. Watanable, C., Satoh, T., Tahara, E., Murakami, K., Hayashi, K., Hase, K., 1999. Inhibitory mechanism of glycoprotein fraction derived from Miscanthus sinensis for the immediate phase response of an IgE-mediated cutaneous reaction. Biological & Pharmaceutical Bulletin 22, 26–30.