Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Research Paper

Tabebuia aurea decreases inflammatory, myotoxic and hemorrhagic activities induced by the venom of Bothrops neuwiedi Fernanda Paes Reis a, Iluska Moslaves Senna Bonfa a, Rafael Bezerra Cavalcante a, Denis Okoba a, Simone Bertozi de Souza Vasconcelos a, Luciane Candeloro a, Wander Fernando de Oliveira Filiu a, Antônio Carlos Duenhas Monreal a, Valter Jose da Silva a, Paula Helena Santa Rita b, Carlos Alexandre Carollo a, Mônica Cristina Toffoli-Kadri a,n a b

Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, 79070-900 Campo Grande, Mato Grosso do Sul, Brazil Biotério Central, Universidade Católica Dom Bosco, 79117-900 Campo Grande, Mato Grosso do Sul, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 11 August 2014 Received in revised form 16 October 2014 Accepted 21 October 2014

Ethnopharmacological relevance: Ethnobotanical studies show that Tabebuia aurea has been used as antiinflammatory and for snake bite. Evaluate the effect of treatment with the hydroethanolic extract of Tabebuia aurea (HETa) on inflammatory, hemorrhagic and myotoxic activities induced by Bothrops neuwiedi (BnV) in mice. Materials and methods: The anti-inflammatory, antihemorragic and antimyotoxic properties of the HETa 100, 200 and 400 mg/kg or BnV neutralized with HETa (1:50) were evaluated using the following animal models: BnV-induced paw edema, BnV-induced recruitment of polymorphonuclear cells into the peritoneal cavity, hemorrhagic activity, myotoxic activity and hydrogen peroxide production by peritoneal macrophages in vitro. Results: HETa inhibited the paw edema and polymorphonuclear cell recruitment into the peritoneal cavity. BnV neutralized with HETa reduced the hemorrhagic activity and histopathological analysis of skeletal muscle tissue showed that the hemorrhagic area was smaller and multifocal. The leukocyte infiltrate was less intense and muscle necrosis discrete. BnV induced hydrogen peroxide production and BnV neutralized reduced this production. In addition, the HETa was nontoxic to macrophages. Conclusions: The activities of the HETa presented herein justify the popular use of Tabebuia aurea in inflammatory situations from snake bite. & 2014 Published by Elsevier Ireland Ltd.

Keywords: Paratudo Bothrops Anti-inflammatory Antihemorragic Antimyotoxic Snake bite

1. Introduction Bothrops family is responsible for most of the snake bites in Brazil. Snake bites are a public health problem in tropical regions and a significant cause of morbidity and mortality; they are included in the World Health Organization (WHO) list of neglected diseases (Harrison et al., 2009). Venoms from snakes, which include the Bothrops genus, trigger systemic effects such as coagulopathy, hypotension, acute renal failure and local effects including pain, hemorrhage, and inflammation with predominance of edema and leukocyte influx to the bite site. In most cases, the disease progresses to tissue necrosis

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Corresponding author. Tel.: þ 55 67 3345 3534. E-mail address: [email protected] (M.C. Toffoli-Kadri).

and may lead to serious consequences such as amputation of the affected limb (Gutierrez and Lomonte, 1989). The conventional treatment for snake bites is antivenom; it reverses the systemic effects; however, it has not been effective for local effects (Gutierrez et al., 1981). The use of plants as an antidote for snake bites is old and common in native populations who use them orally or topically on the bite site (Rizzini et al., 1988; Ruppelt et al., 1991; Martz, 1992). Tabebuia genus (Bignoniaceae) has a wide distribution in tropical and subtropical regions of the American continent, including Pantanal, the Amazon and Caatinga (Lorenzi, 1992). Ethnobotanical studies have shown that Tabebuia aurea, known as “paratudo”, is widely used in traditional medicine, as infusion or maceration with alcohol or chewing of stem bark during the day, to treat snake bites (Pott and Pott, 1994; Agra et al., 2007; Hadju and Hohmann, 2012) and as an anti-inflammatory (Nunes et al., 2003).

http://dx.doi.org/10.1016/j.jep.2014.10.045 0378-8741/& 2014 Published by Elsevier Ireland Ltd.

Please cite this article as: Reis, F.P., et al., Tabebuia aurea decreases inflammatory, myotoxic and hemorrhagic activities induced by the venom of Bothrops neuwiedi. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.10.045i

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The aim of this study was to evaluate the effect of hydroethanolic extract of Tabebuia aurea (HETa) on inflammatory, hemorrhagic, and myotoxic activities induced by Bothrops neuwiedi venom (BnV) in mice.

of the edema was monitored by measuring the volume of footpad swelling at 0, 30, 60, 120 and 240 min after BnV injection using a digital plethysmometer (Insights). 2.6. Polymorphonuclear recruitment into the peritoneal cavity

2. Materials and methods 2.1. Plant material and preparation of the hydroethanolic extract of Tabebuia aurea (HETa) The stem bark of Tabebuia aurea was collected from the “Paratudal”, 19134″37″S;57100″42″W located in the region of Abobral, Passo do Lontra, Miranda, MS, Brazil in July 2009 and were identified by Dr. Arnildo Pott. Voucher specimens of the plants were deposited in the CGMS herbarium, Campo Grande, Mato Grosso do Sul, Brazil (Number 29578). This study was approved for the acquisition and shipment of samples of genetic heritage components – 010273/2013. HETa was obtained by extracting the stem bark (200 g) with percolation (EtOH:H2O – 7:3, v/v) followed by concentration under reduced pressure.

After four hours of the administration of BnV (10 μg/0.5 mL, i.p.), the mice were killed in a CO2 chamber, and the cells from the peritoneal cavity were harvested by injecting 3 mL of phosphatebuffered saline (PBS) containing 10 U/mL of heparin (Basel, Switzerland) into the peritoneal cavity. The peritoneal exudates was aspirated with a syringe and placed into plastic tubes. Total cell counts were performed immediately in a Neubauer chamber after dilution (1:20, v/v) in Turk's solution (0.2% crystal violet dye in 30% acetic acid). For differential cell analyses and determination of number of polymorphonuclear, the preparations were stained with Instant-Prov (Newprov). 2.7. Hemorrhagic activity

Male Swiss mice weighing 18–25 g were obtained from the Federal University of Mato Grosso do Sul (UFMS), Brazil and maintained in a room with controlled temperature (22 7 2 1C) and a 12 h light/dark cycle with free access to food and water. Six hours before each experiment, the animals received only water to avoid food interference with substance absorption. All experiments were approved by the Ethics Committee on Animal Experimentation of the UFMS (Protocol 303/2011) and were conducted in accordance with the National Institutes of Health regulations on the use and care of animals for scientific purposes. Snake venom was extracted from adult specimens of Bothrops neuwiedi (BnV) from the State of Mato Grosso do Sul and supplied by Catholic University Dom Bosco (UCDB). Only one batch of venom was used. The venom was lyophilized and kept at  20 1C until use and the concentration was expressed as dry weight.

Assay was performed according to the method described by Nikai et al. (1984). The hemorrhagic activity of BnV was assessed by measuring the formation and hemorrhagic halo in millimeters into shaved dorsal skin. The Minimum Hemorrhagic Dose (MHD) was determined as the minimum dose of venom (mg) to induce a hemorrhagic halo of 10 mm diameter in the dorsal region and the concentration was established at 10 μg/50 μL. Animals were distributed into groups and received intradermally (i.d), saline (50 mL), HETa (500 μg/50 μL), BnV (10 μg/50 μL) or neutralized BnV (50 μL). After three hours, animals were killed in a CO2 chamber and the dorsal skin removed delimiting the hemorrhagic area. The image of hemorrhagic lesions was photographed and transferred to ImageJs software to determine the area. Quantifying hemoglobin (Hb) was performed as Rucavado and Lomonte (2000). The area limited by hemorrhagic skin was fragmented and transferred to tubes containing 1500 μL solution Drabkins. The material was kept in refrigerator (2–8 1C) for 12 h and then centrifuged at 700 rpm/15 min. The supernatant was collected for hemoglobin assay with spectrophotometer at 540 nm Microchems Analyzer.

2.3. Neutralization of snake venom

2.8. Myotoxic activity

Experimental approach to assess neutralization has been previously described by Gutierrez et al. (1990). BnV (10 mg) was incubated with HETa to obtain venom:extract ratios (1:50; w/w) and carried out at 37 1C for 30 min. Control was performed with aliquots containing BnV without HETa.

Myotoxic activity was assessed by the quantification of the activity of the enzyme creatine kinase (CK) in plasma and histopathological analysis of the gastrocnemius muscle, according to Gutierrez et al. (1980). Animals were injected intramuscularly (i.m.), in the right gastrocnemius muscle, with saline (50 mL), HETa (500 mg/50 mL), BnV (20 mg/50 mL) or neutralized BnV (50 mL). After three hours, mice were bled from the orbital plexus with heparinized Pasteur pipettes. After centrifugation, plasma was separated and the CK activity was determined by using the kit CK-NAC (Gold Analisas, Brazil). After, animals were killed in a CO2 chamber and gastrocnemius muscle was removed and placed in 10% formalin for fixation for 48 h. The sections were stained with hematoxylin and eosin (HE).

2.2. Animals and snake venom

2.4. Treatments For the edema paw and leukocyte recruitment into the peritoneal cavity assay, the animals received per os (p.o.; gavage, 10 mL/ kg), water (vehicle), HETa 100, 200 and 400 mg/kg, or indomethacin (15 mg/kg, Sigma-Aldrich, St. Louis, USA) 60 min before the BnV administration (10 mg/0.5 mL, intraperitoneally, i.p.). Animals that received neutralized BnV were injected at the time of administration of BnV for the other groups.

2.9. Hydrogen peroxide production by peritoneal macrophages in vitro

2.5. Paw edema BnV-induced paw edema assay was adapted from the method described by Winter et al. (1962). Paw edema was induced in the hind paw of the mice by intraplantar injection (i.pl.) of BnV (10 μg/ 40 μL). The contralateral paw was used as a control, which was injected with the same volume of the saline (vehicle). The course

A hydrogen peroxide assay was performed according to the method described by Pick and Mizel (1981) with the modifications proposed by Russo et al. (1989). Mice were pretreated (i.p.) with 3% thioglycollate (1 mL, Oxoid, Hampshire, U.K.) for 96 h before cell harvesting. Subsequently, the mice were killed in a CO2 chamber, and macrophages were harvested by washing the

Please cite this article as: Reis, F.P., et al., Tabebuia aurea decreases inflammatory, myotoxic and hemorrhagic activities induced by the venom of Bothrops neuwiedi. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.10.045i

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cavities with 3 mL of buffered saline (PBS) containing heparin (10 U/mL). The cells (2  105 macrophages) were resuspended in RPMI 1640 medium supplemented and incubated for 60 min in a 96-well plate to allow for adhesion. After, the non-adherent cells were removed, and 100 mL of a phenol red solution (140 mM NaCl, 10 mM potassium-phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red) containing 8.5 U/mL of horseradish peroxidase (Sigma-Aldrich, St. Louis, USA) and HETa (500 mg/mL, 10 mL), BnV (10 mg/mL, 10 mL) or neutralized BnV (10 mL) were added, and the cells were incubated for 60 min at 37 1C in a 5% CO2 atmosphere. The reaction was stopped by the addition of 10 mL of 1 N NaOH. The absorbance was determined using an ELISA reader at a wavelength of 620 nm. Hydrogen peroxide concentration values were extrapolated from a standard calibration curve and expressed as mM H2O2. Cell viability was determined after one hour of incubation of macrophages with different stimuli under the same conditions described. The supernatants were collected and 100 mL of 0.1% Trypan blue was added . Viable cells was determined using an inverted microscope (OptiPhase, MecLab, São Paulo, Brazil) counting a total of 100 cells. 2.10. Statistical analysis Data are shown as the mean 7S.E.M. Statistical differences were analyzed by one- or two-way ANOVA followed by the Bonferroni test. P values less than 0.05 were considered statistically significant (GraphPad Prism 5, GraphPad Software Inc., USA).

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400 mg/kg (43.8%; 465 745 cells/mm3). Neutralized BnV reduced this cells influx in 52.0% (389 785 cells/mm³). 3.3. Effect of BnV neutralized with HETa on hemorrhagic activity Fig. 2 shows the effect of neutralized BnV on hemorrhagic activity. There was no significant difference between BnV and neutralized BnV. BnV induced a hemorrhagic area of 172570.265 cm², whereas the area induced by neutralized BnV was 162570302 cm². Saline or HETa alone (controls) did not induce hemorrhagic area in the animals evaluated (data not shown). However, hemoglobin assay showed that treatment with the neutralized BnV decreased hemoglobin concentration by 40.6% (8.8470.89 g/dL) compared to the BnV group (14.8870.64 g/dL). 3.4. Effect of BnV neutralized with HETa on myotoxic activity Quantification of serum CK showed that neutralized BnV decreased the release of CK in 22.0% (1711 7272 U/L) compared to the group receiving BnV only (2203 7355 U/L), but was not statistically significant. Serum CK concentration in the group of animals that received HETa was only 902 7 164 U/L (Fig. 3). Histopathological analysis of skeletal muscle tissue after three hours shows that HETa preserved the muscle fibers (data not shown). Animals injected with BnV showed marked leukocyte infiltrate between muscle fibers and hemorrhage (Fig. 4). In

3. Results 3.1. Effect of treatment with HETA on BnV-induced paw edema Treatment with the HETa inhibited paw edema in mice (Table 1). The maximum inflammatory response induced by BnV occurred between 30 and 60 min after administration. Indomethacin inhibited paw edema throughout the evaluated period. HETa (200 mg/kg) effectively reduced edema from 60 min after administration, and significantly neutralized BnV paw edema from 120 min after administration. 3.2. Effect of treatment with HETA on BnV-induced polymorphonuclear recruitment into the peritoneal cavity Fig. 1 shows the effect of HETa on BnV-induced polymorphonuclear recruitment (811 766 cells/mm³) into the peritoneal cavity when compared to saline (120732 cells∕mm3). Positive control, indomethacin reduced this influx in 39.5% (4917 42 cells/ mm³). Pre-treatment with HETa at 100 mg/kg inhibited in 32.2% (551 760 cells/mm³), 200 mg/kg (43.9%; 455 794 cells/mm3) and

Fig. 1. Effect of the hydroethanolic extract of Tabebuia aurea (HETa) on polymorphonuclear (PMN) influx into the peritoneal cavity in mice injected with BnV (10 mg/0.5 mL, i.p.). The control group was injected i.p. with sterile saline solution. Indomethacin (15 mg/kg, p.o, positive control) or water (10 mL/kg, p.o., vehicle) was administered 60 min before injection of the BnV. Cells numbers are expressed as the mean7 S.E.M (n ¼7). #Po 0.05 compared to the saline group. nPo 0.05 compared to BnV. ANOVA and Bonferroni test.

Table 1 Effects of the hydroethanolic extract of Tabebuia aurea (HETa) on BnV-induced mice paw edema. Group

Control Indomethacin HETa HETa HETa BnV neutralized

Dose (mg/kg)

15 100 200 400 1:50

Paw edema (mL) and inhibition (%) 30 min 0.1527 0.006 0.104 7 0.006 0.1337 0.009 0.1407 0.014 0.1247 0.008 0.1187 0.008

(%) 31.6n 12.5 7.9 18.4 22.4

60 min 0.1277 0.006 0.053 7 0.009 0.0977 0.008 0.084 7 0.007 0.095 7 0.009 0.1027 0.004

(%) 58.3n 23.7 33.9n 25.2 19.7

120 min 0.1087 0.003 0.036 7 0.009 0.090 7 0.008 0.0637 0.007 0.095 7 0.009 0.0727 0.005

(%) 66.7n 16.7 41.7n 12.0 33.3n

240 min 0.088 7 0.007 0.025 7 0.006 0.0777 0.008 0.0427 0.009 0.0487 0.006 0.053 7 0.006

(%) 71.6n 12.5 52.3n 45.5n 39.8n

Values are expressed as the mean 7 S.E.M. (n ¼7). Data were analyzed by two-way ANOVA followed by Bonferroni's test for comparisons between groups. n

Statistical significance: Po 0.05 when compared with the control.

Please cite this article as: Reis, F.P., et al., Tabebuia aurea decreases inflammatory, myotoxic and hemorrhagic activities induced by the venom of Bothrops neuwiedi. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.10.045i

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Fig. 2. Effect of hydroethanolic Tabebuia aurea extract (HETa) on hemorrhage induced by Bothrops neuwiedi venom (BnV). (A) Hemorrhagic area, and (B) hemoglobin concentration. Three hours after intradermal injection of BnV in the dorsal region of mice (n¼ 6). Results expressed as mean7 EPM. Student t test. Po 0.05.

Fig. 3. Effect of hydroethanolic Tabebuia aurea extract (HETa) on myotoxicity induced by Bothrops neuwiedi venom (BnV). Determination of serum creatine kinase (CK, U/L) three hours after BnV injection into the gastrocnemius muscle (n¼10). Results expressed as mean 7 EPM. ANOVA and Bonferroni test. P40.05.

neutralized BnV group the hemorrhagic area was smaller and multifocal. Leukocyte infiltrate was less intense and muscle necrosis discrete (Fig. 5). 3.5. Effect of BnV neutralized with HETa on hydrogen peroxide production by peritoneal macrophages Fig. 6 shows that incubating macrophages with BnV induced hydrogen peroxide production (11.37 70.14 mM H2O2) compared to unstimulated macrophages (1.63 70.28 mM H2O2). Neutralized BnV reduced this production by 63.2% (4.19 70.44 mM H2O2). HETa did not induce production (2.74 70.30 mM H2O2). Cell viability was greater than 95% for all experiments.

4. Discussion Our results demonstrate that the anti-inflammatory, antihemorragic and antimyotoxic activities of Tabebuia aurea supports the ethnomedicinal data available regarding this species (Nunes et al., 2003; Agra et al., 2007; Hadju and Hohmann, 2012). HETa was able to inhibit polymorphonuclear cell influx to the peritoneal cavity of the mice. The leukocyte profile observed with BnV corroborates the results found by Zamuner and Teixeira (2002) showing that Bothrops venoms increased leukocytes, predominantly polymorphonuclear, in the lesion focus. These cells, in specific activation situations, can contribute to the evolution of tissue and/or vascular lesions caused by different venom components (Zamuner et al., 2001). Intraplantar-injected BnV triggered edema formation which was inhibited by HETa. Previous studies have shown the ability

of other extracts to inhibit edema induced by Bothrops venoms if administered orally before envenomation (Pereira et al., 1994). The mediators liberated by venoms which contribute to vascular and cellular inflammatory reaction development are the eicosanoids, vasoactive amines, adrenergic agonists, and cytokines (Chaves et al., 1995; Barros et al., 1998; Petricevich et al., 2000; Moreira et al., 2009). In this context, the phospholipases A2 (PLA2) of Bothrops venoms hydrolyze cell membrane glycerophospholipids, generating lysophospholipids and fatty acids which are precursors for the formation of eicosanoids (Burke and Dennis, 2009). This enzyme is also responsible for mastocyte degranulation which potentializes edema (Lloret and Moreno, 1993). Our results show that HETa blocked one or production pathway and/or activation of these mediators. In hemorrhagic activity evaluation, HETa did not reduce lesion area, but inhibited the quantity of hemoglobin in the area. One possible explanation is the fact that the metalloproteinases are dependent on zinc (Zn2 þ ) for their catalytic action (Schenkel et al., 2004) and have suffered a complex forming reaction during the venom extract incubation period reducing their activity. From this, we can assume that HETa does not interfere in metalloproteinase distribution in tissue, or that it does not affect the non-catalytic sites responsible for the adhesive properties of this enzyme, but does affect its lytic activity. In relation to myotoxicity, we observed that HETa did not significantly inhibit CK liberation, but histopathological results showed a reduction in the myotoxic event with smaller multifocal hemorrhagic area, less intense leukocyte infiltration, and discrete muscle necrosis. Myotoxins are proteins with a PLA2 structure with specific action on skeletal muscle provoking irreversible damage in muscle fibers at the bite site (Lomonte et al., 2003). These are classified as ASP49-PLA2 with catalytic activity and LYS49-PLA2, which does not have enzymatic activity; however both can cause muscle lesion and are present in Bothrops venoms. ASP49-PLA2 was not identified in Bothrops neuwiedi venom, indicating that catalytic activity does not seem to be a condition for myotoxicity (Leite et al., 2001). Our results showed that BnV induced hydrogen peroxide production and that venon neutralization inhibited it. In phagocytosis, macrophages produce reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide (Babior, 2004). Accentuated ROS production and liberation can damage the tissue involved in bothrops venom triggered inflammatory response (Setubal et al., 2011). It is possible that the HETa action mechanism, by direct action, is inhibiting signal pathways, such as the protein C kinase pathway, not permitting NADPH oxidase activation and impeding respiratory burst (Babior, 2004) or indirectly, chelating bivalent ions, as ROS can also be generated independent mechanisms from NADPH oxidase, through calcium-activated potassium conductance channels (Fay et al., 2006).

Please cite this article as: Reis, F.P., et al., Tabebuia aurea decreases inflammatory, myotoxic and hemorrhagic activities induced by the venom of Bothrops neuwiedi. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.10.045i

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Fig. 4. Histological section of mouse gastrocnemius muscle three hours after intramuscular BnV administration. Intense hemorrhagic (H) and accentuated leukocyte infiltrated (I) tumefied muscle. H/E staining.

Fig. 5. Histological section of mouse gastrocnemius muscle three hours after intramuscular neutralized BnV administration. Hemorrhage (H) and moderate leukocyte infiltrated (I) tumefied muscle (T). H/E staining.

5. Conclusion HETa inhibited leukocyte infiltration and paw edema, displaying its anti-inflammatory action; it also reduced hemorrhage, myotoxicity, and hydrogen peroxide production induced by Bothrops neuwiedi venom. The results confirmed the potential of ethnobotanically-sellected plant, Tabebuia aurea, as an alternative for neutralization of venom-induced local effect.

Uncited references Silva and Filho (2002); Zuliani et al. (2005). Fig. 6. Effect of the hydroethanolic extract of Tabebuia aurea (HETa) on hydrogen peroxide (H2O2) production by peritoneal macrophages (MF). The cells were collected 96 h after ip injection of 3% thioglycolate. Macrophages (2  105 cells/ well) were resuspended in phenol red solution containing horseradish peroxidase. H2O2 production was determined one hour after incubation with Bothrops neuwiedi venom (BnV, 10 mg/mL, 10 mL), HETa (250 mg/mL, 10 mL) or neutralized BnV (1:50, 10 mL). Values represent the mean 7S.E.M. of three experiments performed in triplicate. #Po 0.05 compared to control. nPo 0.05 compared to BnV. ANOVA and Bonferroni test.

Members of the Tabebuia genus produce iridoids, saponins, cumarins, and flavonoids, among its other secondary metabolites. Studies with Tabebuia aurea have shown the presence of iridoids, flavonoids, terpenes and steroids (Guerbas-Neto, 2003). Some of these secondary metabolites, alone or in combination, should be responsible for the anti-inflammatory, antimyotoxic and antihemorrhagic activities, and future studies are necessary for clarifying the activate compound(s) of HETa.

Acknowledgments The authors acknowledge CNPq and FUNDECT for providing research grants. The authors are also grateful to Helder Antônio de Souza – UNIDERP for support with snake venom extraction. References Agra, M.F., Bracho, G.S., Nurit, K., Basílio, I.J.L.D., Coelho, V.P.M., 2007. Medicinal and poisonous diversity of the flora of Cariri Paraibano, Brazil. Journal of Ethnopharmacology 111, 383–395. Babior, B.M., 2004. NADPH oxidase. Current Opinion Immunology 16, 42–47. Barros, S.F., Friedlanskaia, I., Petricevich, V.L., Kipnis, T.L., 1998. Local inflammation, lethality and cytokine release in mice injected with Bothrops atrox venom. Mediators of Inflammation 7, 339–346. Burke, J.E., Dennis, E.A., 2009. Phospholipases A2 structure/function, mechanism, and signaling. Journal of Lipid Research 50, 237–242. Chaves, F., Barboza, M., Gutierrez, J.M., 1995. Pharmacological study of edema induced by venom of the snake Bothrops asper (terciopelo) in mice. Toxicon 33, 31–39.

Please cite this article as: Reis, F.P., et al., Tabebuia aurea decreases inflammatory, myotoxic and hemorrhagic activities induced by the venom of Bothrops neuwiedi. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.10.045i

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Please cite this article as: Reis, F.P., et al., Tabebuia aurea decreases inflammatory, myotoxic and hemorrhagic activities induced by the venom of Bothrops neuwiedi. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.10.045i

Tabebuia aurea decreases inflammatory, myotoxic and hemorrhagic activities induced by the venom of Bothrops neuwiedi.

Ethnobotanical studies show that Tabebuia aurea has been used as anti-inflammatory and for snake bite. Evaluate the effect of treatment with the hydro...
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