Anti-hyperalgesic activity of corilagin, a tannin isolated from Phyllanthus niruri L. (Euphorbiaceae)

Journal of Ethnopharmacology 146 (2013) 318–323

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Anti-hyperalgesic activity of corilagin, a tannin isolated from Phyllanthus niruri L. (Euphorbiaceae) Jeverson Moreira a, Luiz Carlos Klein-Ju´nior a,b, Valdir Cechinel Filho a,b, Fa´tima de Campos Buzzi a,b,n a b

´ cleo de Investigac- o~ es Quı´mico-Farmacˆeuticas—NIQFAR, Universidade do Vale do Itajaı´-UNIVALI, 88.302-202, Itajaı´, Santa Catarina, Brazil Nu ´s-Graduac- a~ o em Ciˆencias Farmacˆeuticas, Universidade do Vale do Itajaı´-UNIVALI, 88.302-202, Itajaı´, Santa Catarina, Brazil Programa de Po

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 August 2012 Received in revised form 17 December 2012 Accepted 27 December 2012 Available online 17 January 2013

Ethnopharmacological relevance: Corilagin (b-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose) is a tannin isolated from Phyllanthus niruri (Euphorbiaceae). This plant is well known for their therapeutic purposes to treat several diseases associated with dolorous process and are used in several ethnomedicines in tropical and subtropical countries. Aim of the study: This study was designed to evaluate the anti-hyperalgesic activity of corilagin using chemically and thermally based nociception models in mice. Materials and methods: Corilagin was isolated from Phyllanthus niruri (Euphorbiaceae) by extraction and chromatographic procedures and the anti-hyperalgesic activity was evaluated by using writhing, formalin, capsaicin, glutamate and hot plate tests in mice. Results: Corilagin presented activity in acetic acid model with the ID50 calculated value of 6.46 (3.09– 13.51) being about 20.6 fold more potent than acetylsalicylic acid. It also exhibited activity against the first phase of formalin test with ID50 value of 18.38 (15.15–22.59) mmol/kg. In the capsaicin and glutamate models, corilagin demonstrated significant activity at the 3 mg/kg. Conclusion: The experimental data demonstrated that corilagin exhibits anti-hyperalgesic activity that may be due to interaction with the glutamatergic system. & 2013 Elsevier Ireland Ltd. Open access under the Elsevier OA license.

Keywords: Corilagin Antinociception Phyllanthus niruru L.

1. Introduction Phyllanthus niruri Linn., Euphorbiaceae, known in Brazil as ‘‘quebra-pedra’’ and in other Latinoamerican countries as ‘‘chancapiedra’’ occurs widely in the tropical and subtropical countries. Infusion of different parts of this plant and other from the genus Phyllanthus is frequently used with therapeutic purposes to treat several diseases particularly disturbances of kidney and urinary bladder associated with the dolorous process (Calixto et al., 1998; Khanna et al., 2002; Ranilla et al., 2010; Shanbhag et al., 2010). Experimental studies have confirmed different pharmacological properties such as hepatoprotective (Harish and Shivanandappa, 2006), antiplasmodial (Cimanga et al., 2004), antihyperlipemic (Khanna et al., 2002), antihyperuricemic (Murugaiyah and Chan, 2009) and antioxidant activities (Sarkar et al., 2009). Moreover, there are quite a few studies reporting the antinociceptive effect of Phyllanthus niruri (Santos et al., 1994, 1995a, 1995b; Obidike et al., 2010). The medicinal effects are attributed to the active

n Corresponding author at: Programa de Po´s-Graduac- a~ o em Ciˆencias Farm~ Quı´mico-Farmacˆeuticas—NIQFAR, Rua Uruguai acˆeuticas, Nu´cleo de Investigac- oes 458, Universidade do Vale do Itajaı´—UNIVALI, Itajaı´, Santa Catarina, Brazil. Tel.: þ55 4733417855. E-mail address: [email protected] (F.d.C. Buzzi).

0378-8741 & 2013 Elsevier Ireland Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.jep.2012.12.052

components present in Phyllanthus niruri, such as lignans (Murugaiyah and Chan, 2007), flavonoids (Shakil et al., 2008), terpenoids (Singh et al., 1989) and in particular, tannins (Markom et al., 2007). Corilagin (b-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose) (Fig. 1), a tannin isolated from several plants, including Phyllanthus niruri, has also been used as a phytochemical marker for qualitative and quantitative studies of this plant (Colombo et al., 2009). Some pharmacological activities of corilagin have already been described, such as antiatherogenic (Duan et al., 2005), antioxidant (Chen and Chen, 2011), hepatoprotective (Kinoshita et al., 2007) and anti-tumor (Hau et al., 2010). Furthermore, corilagin was also able to inhibit the release of cytokines such as TNF-a, IL-1b and IL-6 as well as the production of nitric oxide, both of which are mediators of inflammation and pain (Okabe et al., 2001; Zhao et al., 2008; Dong et al., 2010). Considering that Phyllanthus niruri has already demonstrated antinociceptive activity in vivo and, that one of the main constituents of the species is corilagin, which was able to inhibit mediators related to inflammatory pain; and since there is no study evaluating the antinociceptive properties of the tannin, this study assesses the effects of corilagin treatment on chemically and thermally based nociception models in mice.

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319

Table 1 13 C NMR chemical shifts for corilagin in CD3ODa. Sudjaroen et al., 2012

Fig. 1. Chemical structure of corilagin (b-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl -D-glucose).

2. Material and methods 2.1. Plant material Phyllanthus niruri L. (Euphorbiaceae) was collected in January 2011 at Praia da Esplanada-Jaguaruna, Santa Catarina, Brazil. The plant was authenticated by Professor Oscar Iza (UNIVALI) and a voucher specimen was deposited at the Barbosa Rodrigues Herbarium (HBR, Itajaı´) under number VC Filho 075. 2.2. Extraction and isolation procedures The whole plant (337 g, dry weight) was cut into small pieces and submitted to maceration with methanol at room temperature for 5 days, giving 30.98 g of the crude extract. 25 g of the extract was solubilized in methanol/water (2:5 v/v) and extracted with dichloromethane (3 times, 2:1 v/v) and ethyl acetate (3 times, 2:1 v/v), to give the dichloromethane (4.05 g), ethyl acetate (5.58 g) and methanol (10.74 g) fractions after evaporation. 1 g of the ethyl acetate fraction was submitted to chromatography using silica gel eluted with a dichloromethane/ethyl acetate/ methanol gradient (40:55:5-0:0:100 v/v/v), giving six main fractions. The third fraction (272.9 mg) was chromatographed using isocratic flash chromatography (dichloromethane/ethyl acetate/ methanol, 30:50:20 v/v/v), to give pure corilagin (88.4 mg). The compound with purity grade 495% was identified by 13C-NMR spectral data by comparison with those previously reported (Table 1). 2.3. Pharmacological procedures 2.3.1. Animals Male Swiss mice weighting 25–30 g obtained from the Animal Facility of the Universidade do Itajaı´ (UNIVALI, Itajaı´, Brazil) were used in this study. The animals were housed in plastic cages, under a 12 h light/dark cycle (lights on 7:00 a.m.) at constant room temperature (23 1C72 1C) and humidity (60–80%) with water and food ad libitum. The animals were acclimated to the experimental room at least 1 h before each experiment. The allocation of animals to the different groups was randomized, and the experiments were carried out in blind conditions. Each group was constituted of 6–8 mice; each animal was used in one experiment only, and sacrificed immediately at the end of each experiment. All the experimental procedures were performed in accordance with NIH publication no. 85–23—‘‘Principles of Laboratory Animal Care’’ and with the approval of the UNIVALI Ethics Committee, under protocol no. 599/2007.

Glucose 1 2 3 4 5 6 Galloyl 1 2 3 4 5 6 Ring A 1 2 3 4 5 6 7 Ring B 1 2 3 4 5 6 7 a

Experimental

95.1 69.4 71.6 62.5 76.2 65.0

95.1 71.3 71.8 61.7 77.1 65.2

120.7 111.0 146.4 140.4 146.4 111.0

120.4 111.0 146.3 140.9 146.6 111.0

117.3 125.5 110.3 145.7 138.3 145.5 168.7

117.3 125.5 109.0 145.7 138.3 145.4 168.6

116.8 125.6 108.4 146.0 137.8 145.4 170.2

116.4 125.7 108.3 146.3 137.7 145.4 170.1

Obtained in ppm rel. TMS; 1H NMR 300 MHz.

Control animals received a similar volume of vehicle (10 mL/kg, 0.9% NaCl solution). Acetic acid-induced writhing test (Collier et al., 1968) consisting of an i.p. injection of 10 mL/kg acetic acid (0.6% in vehicle) 30 min after treatment of the animals, to cause abdominal constrictions. Pairs of mice were placed in separate boxes and the number of abdominal constrictions was cumulatively counted over a period of 20 min. Antinociception was expressed as the reduction in the number of abdominal constrictions between the control animals and the mice pretreated with corilagin. 2.3.2.2. Formalin-induced paw licking test. The procedure was similar to that described previously (Hunskaar and Hole, 1987; Sakurada et al., 1993). Animals were treated with corilagin intraperitoneally at 3, 6, 10 mg/kg or vehicle (10 mL/kg, 0.9% NaCl solution), 30 min before formalin injection. The observation chamber was a glass cylinder of 20 cm in diameter, with a mirror at a 451 angle to allow clear observation of the animal’s paws. The 0.92% aqueous formalin solution was prepared as a 2.5% dilution of analytical 37% formaldehyde solution in water (Hunskaar and Hole, 1987). This solution was injected (20 mL) under the surface of the right hind paw. Two mice (control and treated) were observed simultaneously from 0 to 30 min following formalin injection. The amount of time spent licking the injected paw was recorded and expressed as the total licking time in the early phase (0–5 min) and late phase (15–30 min) after formalin injection, representing the tonic and inflammatory pain responses, respectively.

2.3.2. Evaluation of antinociceptive activity in mice 2.3.2.1. Acetic acid-induced writhing test. Groups of mice (8/group) were treated with different doses of corilagin (1, 3, 6 and 10 mg/kg) by intraperitonial injection (i.p.) or by oral gavage (50 mg/kg).

2.3.2.3. Capsaicin-induced nociception. The procedure used was similar to that described previously (Sakurada et al., 1993). Animals were placed individually in transparent glass cylinders. Following the adaptation period, 20 mL of capsaicin solution

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(1.6 mg/paw) was injected under the skin of the plantar surface of the right hind paw, using a micro-syringe. The animals were observed individually for 5 min following capsaicin injection. The amount of time spent licking the injected paw was timed with a chronometer and was considered as indicative of nociception. The animals were treated intraperitoneally with corilagin at 3 mg/kg or vehicle (10 mL/kg, 0.9% NaCl solution) 30 min before administration of capsaicin. 2.3.2.4. Glutamate-induced nociception. Similarly to the procedure described by Beirith et al. (2002), in this test animals were treated with corilagin i.p. at 3 mg/kg or vehicle (10 mL/kg, 0.9% NaCl solution) 30 min before glutamate injection. A volume of 20 mL glutamate solution (30 mmol/paw), made up in phosphate buffered saline (PBS), was injected intraplantarly under the surface of the right hind paw as described previously. After injection with glutamate, the animals were individually placed into glass cylinders of 20 cm in diameter and observed for 0 to 15 min. The time spent licking and biting the injected paw was timed with a chronometer and considered as indicative of pain. 2.3.2.5. Hot-plate test. The hot-plate test was used to estimate the latency of responses according to the method described by Eddy and Leimback (1953) with minor modifications. The temperature of the hot-plate was maintained at 55 71 1C. The animals were placed on glass funnels on a heated surface, and the time between placing the animals and the beginning of licking paws or jumping was recorded as the response latency index. Reaction time was recorded before and 30 min after i.p. administration of corilagin (3 mg/kg i.p.) or the same volume of vehicle (saline 10 mL/kg). A cut-off time of 20 s was chosen to indicate complete analgesia and avoid tissue injury. 2.3.3. Measurement of motor performance 2.3.3.1. Rota-rod test. The interference of corilagin on motor coordination was also evaluated (Duhnam and Miya, 1957). This test was performed using a horizontal rota-rod device (Letica Scientific Instrument, Barcelona, Spain) set to rotate at a constant speed of 15 rpm. The animals were trained and then selected 24 h before the test by eliminating those mice that did not remain on the bar for two consecutive periods of 60 s. The animals were treated with compound (3 mg/kg, i.p.) or with the same volume of 0.9% NaCl solution (10 mL/kg, i.p.) 30 min before being tested. The results are expressed as the time (s) the animals remained on the rota-rod. The cut-off time used was 60 s. 2.3.3.2. Open field test. To exclude possible nonspecific effects of corilagin on locomotor activity, mice were treated with compound (3 mg/kg, i.p.) and after 30 min, the locomotor activity was assessed in the open-field test, as described previously (Bortolanza et al., 2002). The number of squares crossed with all paws (‘‘crossings’’) in a 6 min session was counted. The control mice received vehicle (10 mL/kg, i.p. 0.9% NaCl). 2.4. Statistical analysis The results are presented as mean7SEM, except the mean ID50 values (i.e. the dose of compound reducing the algesic responses by 50% compared with the control value) which are reported as geometric means accompanied by their respective 95% confidence limits. The statistical significance between groups was analyzed by one-way ANOVA followed by Dunnett’s posttest, unless otherwise stated. P-values of less than 0.05 were

considered as indicative of significance. ID50 values were determined by graphical interpolation from individual experiments. 2.5. Drugs The following drugs were used: capsaicin and glutamate (Sigma Chemical Company, St. Louis, U.S.A); formalin, acetic acid (Vetec, Rio de Janeiro, Brazil). The compounds studied, as well as the reference drugs, were dissolved in 0.9% of NaCl solution, with the exception of capsaicin, which was dissolved in ethanol, plus 0.9% of NaCl solution. The final concentration of ethanol did not exceed 5% and did not cause any effect ‘‘per se’’.

3. Results 3.1. Acetic acid-induced writhing test The administration of corilagin 1, 3, 6 and 10 mg/kg i.p. reduced the number of writhing episodes induced by acetic acid by 22.2, 49.0, 53.5 and 57.4%, respectively, compared to the control group (Table 2). ID50 calculated value (and its 95% confidence limits) was 6.46 (3.09–13.51) mmol/kg or 4.10 (1.96– 8.57) mg/kg As oral administration of 50 mg/kg of corilagin did not reduce acetic acid writhing responses, intraperitoneal administration was preferred (Data not shown). 3.2. Formalin-induced paw licking test In the formalin test, intraperitoneal pre-administration (30 min) of corilagin in different doses (3, 6 and 10 mg/kg) significantly reduced the formalin-induced licking in the early phase (po0.01), in a dose-dependent way, but not in the late phase (Fig. 2). The calculated ID50 (and its 95% confidence limits) was 18.38 (15.15–22.59) mmol/kg or 11.66 mg/kg (9.61–14.33). The 3 mg/kg corilagin was the lowest dose used in this study capable of causing a marked inhibition in both, acetic acid and formalin tests. Therefore, this dose was chosen to be used in the subsequent tests. 3.3. Capsaicin-induced pain The effect of corilagin against capsaicin-induced nociception in mice is shown in Fig. 3A. Significant reduction of 32.6% was observed in the time spent on licking the paw in mice receiving corilagin at the dose of 3 mg/kg compared to the control group (p o0.01). 3.4. Glutamate-induced nociception The corilagin dose of 3 mg/kg caused a significant inhibition, 66.2% (p o0.01), in the licking and biting behavior in

Table 2 Effects of different doses of corilagin against acetic acid-induced writhing in mice. Compound dose (mg/kg i.p.)

Inhibition (%)

1 3 6 10

22.2 49.0 53.5 57.4

( 74.85)n ( 71.59)nn ( 73.35)nn ( 72.46)nn

ID50 4.10 (1.96–8.57) mg/kg or 6.46 (3.09–13.51) mmol/kg

Each group represents the mean 7 SEM of 6–8 experimental values. n

p o0.05. po 0.01 compared to the respective control values.

nn

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321

Fig. 2. Effects of corilagin administered intraperitoneally against formalin induced licking in mice (Panel A: early phase; Panel B: late phase). Each column represents the mean 7 SEM (n¼ 6–8/group). Asterisks denote the significance levels, when compared to the control group, *p o 0.05; **po 0.01. ANOVA followed by Dunnett’s test.

Fig. 3. Panel A: Effect of the intraperitoneal administration of corilagin (3 mg/kg) against capsaicin-induced nociception in mice. Panel B: Effect of the intraperitoneal administration of corilagin (3 mg/kg) against glutamate-induced nociception in mice. Panel C: Effect of the intraperitoneal administration of corilagin (3 mg/kg) on the hotplate test in mice. Each column represents the mean 7 SEM (n ¼6–8/group). Asterisks denote the significance levels, when compared to the control group, *p o0.05; ** p o 0.01. ANOVA followed by Dunnett’s test.

Fig. 4. Panel A: Effects of corilagin administered intraperitoneally on the rota-rod in mice. Each column represents the mean 7 SEM (n¼ 6–8/group). Panel B: Effects of corilagin administered intraperitoneally on the open field test. Each column represents the mean 7SEM (n¼ 6–8/group). Asterisks denote the significance levels, when compared to the control group, *po 0.05, **po 0.01. ANOVA followed by Dunnett’s test.

glutamate-induced nociception in mice, when compared to the control group (Fig. 3B). 3.5. Hot-plate test The i.p. administration of 3 mg/kg of corilagin did not change the latency time to the nociceptive response of mice in the hot plate test (Fig. 3C). 3.6. Rota-rod test As shown in Fig. 4A, corilagin (3 mg/kg i.p.) did not cause significant alterations in the motor performance of mice in the rota-rod test.

3.7. Open field test Mice treated with corilagin (3 mg/kg i.p.) did not demonstrate a statistical reduction in the number of crossings and rearings when compared to control group (Fig. 4B).

4. Discussion Considering that corilagin is one of the major constituents of Phyllanthus niruri and has a wide spectra of biological activities (Zhao et al., 2008; Chen and Chen, 2011), including antiinflammatory activity, we investigated, in this work, the antihyperalgesic properties of this tannin using animals models of pain behavior.

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The acetic acid-induced writhing test is a classic visceral pain model commonly used for assessment of potential antinociceptive and anti-inflamatory agents (Collier et al., 1968). In this test, pain is caused by acetic acid directly through the activation of non-selective cation channels present at primary afferent pathways (Julius and Basbaum, 2001) and indirectly via release of endogenous mediators, including bradykinin, substance P, prostaglandins, histamine and others, which are able to increase vascular permeability, and to stimulate both peripheral nociceptive neurons and nerve terminal of nociceptive fibers (Derardt et al., 1980; Martinez et al., 1999; Ikeda et al., 2001). Our results show that corilagin significantly reduced acetic acid writhing responses in a dose-dependent manner, which may suggest that this tannin attenuates the release of inflammatory endogenous mediators or blocks inflammation-promoting receptors, resulting in peripheral anti-hyperalgesic activity. However, even if acetic acid is a sensitive model for screening analgesic substances, it constitutes a non-specific model (Takahashi and Paz, 1987) and thus other tests such as formalin, capsaicin and glutamate were conducted to better characterize the antihyperalgesic pharmacology of corilagin. Comparison with previous results (Santos et al., 1995a, 1995b) indicated that corilagin was more potent than the hydroalcoholic extract obtained from this same plant. The oral administration of corilagin was not able to inhibit the nociception induced by acetic acid. This result may be due to the rapid metabolization of corilagin by intestinal microflora. It was demonstrated that the anaerobic incubation of the tannin with rat fecal suspension led to hydrolysis to several different metabolites, like gallic acid, ellagic acid and other derivatives. This hydrolysis seems to not occur by i.p. administration (Ito, 2011). Two distinct phases constitute the formalin test. The first, called the neurogenic phase, starts immediately after formalin injection and seems to be elicited by direct activation of C-fibers (McNamara et al., 2007). The second (or inflammatory) phase is a combination of a peripheral inflammatory reaction response induced by release of nociceptive mediators, and changes in central processing of pain (Hunskaar and Hole, 1987; Shibata et al., 1989; Tjølsen et al., 1992). Corilagin produced significant dose-dependent anti-hyperalgesic properties only in the neurogenic phase of the formalin test. These findings strongly suggest that corilagin could induce changes both in the direct activation of nociceptors and in the neurogenic pain response induced by the release of mediators such as bradykinin, 5-hydroxytryptamine, histamine and adenosine triphosphate (Tjølsen et al., 1992; Sawynok and Liu, 2003). The lack of significant activity on the second phase of formalin test could be due to the hidrophilicity or high polar surface area of corilagin, which could selectively limit its passage to the brain (Kelder et al., 1999; Pardridge, 2007). Nociception evoked by capsaicin occurs by activation of a ligand-gated cation channel TRPV1 (or VR1 channel) (Caterina et al., 1997). As a consequence, the release of several chemical mediators, such as substance P, excitatory amino acids (aspartic and glutamic acids), neurokinin A, calcitonin gene-related peptide, nitric oxide (NO) and pro-inflammatory mediators also contribute to increase nociception (Sakurada et al., 1993; Teoh et al., 1996; Mohamad et al., 2010). Our results show that corilagin significantly reduced capsaicin-induced nociception, suggesting that the tannin may be involved in the antagonism of the TRPV1 receptor. Another finding of our study is that corilagin also inhibited the nociceptive pain response induced by intraplantar injection of glutamate into mouse hind paw. Glutamate can act in peripheral, spinal and supra-spinal areas that are involved in the processing of nociception. The glutamate peripheral induced-pain response appears to be mediated through the activation of ionotropic

receptors by N-methyl-D-aspartate (NMDA), a-amino-3hydroxy-5-methylisoxazole-4-propionic acid (non-NMDA ligand), as well as the release of NO or other NO-derived substances. NO release contributes to enhancing the inflammatory reaction through the induction of the synthesis of pro-inflammatory mediators such as cytokines (Fundytus, 2001; Beirith et al., 2002; Jesse et al., 2009; Mohamad et al., 2010). Thus, the antihyperalgesic activity of corilagin in the glutamate test may be due to inhibition of NO production or to the interaction of corilagin with the glutamatergic system. Concerning the hot plate test, administration of corilagin 3 mg/kg i.p. in mice did not exert any change in response latency time to the thermal nociceptive stimulus when compared to the control group. Considering that the supraspinal and spinal opioid receptors play a key role in the hot plate assay, our results suggest that this compound does not act on the central opioid receptors, nor does it produce a release of endogenous opioid peptides (Eddy and Leimback, 1953). Importantly, corilagin at 3 mg/kg i.p. was devoid of significant effects on locomotor activity and motor coordination in mice (i.e. open field and rotarod test, respectively), eliminating the possibility of non-specific muscle relaxation and sedative effects in corilagin-induced antinociception. This observation strongly suggests that the antinociceptive activities of corilagin measured in this study are pharmacologically specific. Several studies have reported that NMDA and non-NMDA receptor antagonists, as well as NO inhibitors, were able to inhibit nociception induced by acetic acid, formalin, and glutamate (Fundytus, 2001; Jesse et al., 2009; Mohamad et al., 2010). In addition, Zhao et al. (2008) showed that corilagin was able to suppress the release of proinflammatory cytokines in vitro and reduced the production of NO and other pro-inflammatory mediators. Our study demonstrates for the first time that intraperitoneal administration of corilagin produced marked and significant dose-dependent suppression of nociceptive responses in all chemical nociceptive test models used and importantly, that no alteration in motor performance contributed to the observed behaviors of the animals. Exact mechanism underlying the anti-hyperalgesic effects of corilagin remains unclear, but our results suggest that its antihyperalgesic properties could be due in part to the inhibition of NO production and release of pro-inflammatory mediators such as cytokines as well as the potential interaction with the glutamatergic system. Several studies from our and other research groups have evidenced the antinociceptive activity of Phyllanthus niruri and other species of the genus, sometimes attributing this effect to both polar and non-polar compounds (Calixto et al., 1998). However, this is the first time that the antinociceptive potential of corilagin is demonstrated. Our findings presented herein are of interest because they support the idea that corilagin, a tannin isolated from Phyllanthus niruri, may be used for the development of potentially novel and effective peripheral analgesics devoted to the control of pain.

Acknowledgment We are grateful to Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Universidade do Vale do Itajaı´ (UNIVALI) for their financial support. The authors also wish to thank Professor Oscar Iza for the assistance with plant authentication. References Beirith, A., Santos, A.R.S., Calixto, J.B., 2002. Mechanisms underlying the nociception and paw oedema caused by injection of glutamate into mouse paw. Brain Research 924, 219–228.

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