ANTIOPHIDIAN ACTIVITY OF BROSIMUM GUIANENSE (AUBL) HUBER

Snakebites envenomations are a problem public healt h in worldwide due to the high rates of morbidity a nd mortality. The Bothrops venom causes local tissue damage and inflammation is e of the most important events that occur. At present, effective treatment for snakebites is serum therapy with antivenom, whi ch neutralizes systemic alterations but does not preve nt local damage that can cause disabilities. Many p lants are used in popular medicine to treat these acciden ts but few attempts have been made to investigate t he scientific validity of these assertions. In Amazon region, indigenous and local people use the macerat ed bark of Brosimum guinanensis applied in the form of cataplasm, on the site of s nakebite. This study aimed to analyze the ability of the Brosimum guianensis aqueous extract in the neutralization several effe cts induced by Bothrops atrox snake venom to investigate the scientific validity of folk medicine informations by means of controlled experiments. Our results sho wed that Brosimum guianensis aqueous extract was not effective to inhibit oedema, peritonitis, coagulant , myotoxic, phospholipase A2 activity (indirect hem olytic method) induced by B. atrox venom, but was able to inhibited significantly hemorrhag ic and nociceptive activities. These results support a potential effec t of this extract as a compounds source for biotechonological application and synthesis of new drugs with therapeutic purpose.


INTRODUCTION
In Brazil, significant snakebites numbers occurs annually and are considered a neglected tropical disease of high impact in the rural areas (Gutiérrez et al., 2006;WHO, 2007a). Bothrops atrox is ophidian most frequently found in northern Brazil and envenoming caused by this snake are associated with a variety of pathophysiological manifestations, frequently including a severe local tissue damage (Gutiérrez, 2002). In addition, an unknow number of cases end up with permanent sequelar secondary to the tissue-damaging effects of the venom (WHO, 2007b;Warrell, 2010). The use of plants in traditional medicine systems of many cultures has been extensively documented. These plantbased systems continue to play an essential role in health care and the World Health Organization estimates that 80% of the world inhabitants continue to rely mainly on traditional medicine systems for their health care (Gurib-Fakim, 2006;WHO, 2002).
Several vegetal species are popularly known as antiophidian, but only a few species have been Science Publications AJPT scientifically investigated and still less have had their active principles isolated and characterized (Veronese et al., 2005;Coea and Anderson, 2005;Da Silva et al., 2005). The use of plants to treatment snakebites is spread out among Amazon population, that use the macerated bark of Brosimum guinanensis, applied in the form of cataplasm, on the site of snakebite.
In the present study was evaluated the ability of the Brosimum guinanensis aqueous extract of in the neutralization oedema, peritonitis, nociceptive, coagulant, myotoxic, hemorrhagic and phospholipase A2 activity (indirect hemolytic method) induced by Bothrops atrox venom.

Venom and Antivenom
B. atrox venom was supplied from the Serpentarium at the Toxicology Laboratory, Federal University of Amapá, Brazil. The venom was lyophilized and kept under refrigeration at 4°C. The venom was diluted in Phosphate Buffered Saline (PBS) immediately prior to its use.

Preparation of Plant Extract
Dry and worn-out stem leaves were extracted with distilled water, maintained in infusion for 24 h at room temperature and then vacuum filtered. The aqueous Extract (CEE) was lyophilized and stored at-18°C. Before use, it was weighed and dissolved in PBS.

Animals
All animal care was performed in accordance with the guidelines of the Brazilian College for Animal Experimentation. Male Swiss webster mice weighing 20-25 g were used for the experiments and were randomly divided into groups of five animals each. The mice were kept in plastic cages with access to water and food ad libitum and were maintained under controlled temperatures (18-20°C) on a 12 h light/dark cycle.

Groups and Experimental Protocols
The experimental groups consisted of five male mice each administered B.Atrox Venom (BAV) alone, Brosimum Guinanensis Extract alone (BGE), B. atrox venom+Brosimum guinanensis extract in concentrations different (BAVBGE), or PBS alone. The venom doses used were selected from previous dose-response experiments, in which it was observed that the venom induced a minimum response for all activities evaluated.

Nociception Activity
The method used was a modification of the method previously described by Hunskaar and Hole (1987) modified by (Soares et al., 2009;Sousa, 2012). Sample containing 0.05, 0.10 or 0.20 mg kg −1 of venom in 50 µL of PBS were injected subcutaneously into the right hind paw for evaluated venom nociceptive effect. The minimum dose was defined as the lowest venom dose required for a statistically significant increase (p<0.05) at time (in seconds) spent in licking and biting responses of the injected paw. For inhibition studies, a fixed amount of Bothrops atrox venom (0,20 mg/25 µL PBS) was mixed with Brosimum guinanensis extract concentrations different (1,25; 2,5; 5,0; 7,5 and 10 mg de BGE/Kg/25 µL PBS). Then, the mixture was administered subcutaneously into the right hind paw of mice. Controls animals received only PBS (50 µ L), venom (0, 20 mg/Kg/50 µ L PBS) or B. guinanensis extract (10 mg/Kg/50 µ L PBS). Mice were then put back individually under glass funnel with mirrors behind and also to the side to facilitate observation.
Distinct periods of intensive licking activity were identified and scored separately unless otherwise Science Publications AJPT stated. The first period (early phase) was recorded 0-5 min and the second period (late phase) was recorded 20-30 min after the injection in the corresponding groups. The time (in seconds) spent in licking and biting responses of the injected paw were taken as an indicator of pain response. The test was performed at ambient temperature of 22-26°C and care was taken to exclude environmental disturbances (high temperature, noise and excessive movement) that might interfere in the study. The animals were individually assessed by only one observer, responsible for all tests. Due to number of animals, tests were performed during two consecutive days.
After four hours, the animals were euthanized in a CO 2 chamber and the peritoneal exudates were collected with a plastic Pasteur pipette by abdominal laparoscopy. To facilitate collection, all the animals received an injection of 2.0 mL of heparinized PBS (1 mL/1000 mL de PBS) and their abdomens were massaged to release any cells that had stuck to them. A sample of the peritoneal wash was diluted 1:20 in Türk's solution and the cells were counted in a Neubauer chamber. Peritoneal fluid part was centrifuged at 1000 rpm for 10 min and the supernatant was suspended in 0.4 mL of a solution of albumin in PBS 3%. Differential leukocytes were stained in Instant-Prov and counted under a light microscope, using oil immersion objective. The results were expressed as the total number of cells per peritoneal cavity.

Hemorrhagic Activity
Hemorrhage was performed as previously described by Kondo et al. (1960). Firstly, were administered Intradermally (ID) venom concentrations different on the back of mice to determine a Minimal Hemorrhagic Dose (MHD) defined as that concentration of venom resulting in a 10 mm hemorrhagic spot. After 2 h, the animals were euthanized in a CO 2 chamber. The skin near the injection site was removed and hemorrhagic halo formed was measured in millimeters (mm). Two diameters were achieved for the spot of hemorrhage by measuring the longest diameter of the spot and the diameter perpendicular to the first measurement. In the inhibition assays, a fixed amount of Bothrops atrox venom (0, 20 mg/25 µL PBS) was mixed with Brosimum guinanensis extract concentrations different (1, 25; 2, 5; 5, 0; 7, 5 and 10 mg de BGE/Kg/25 µL PBS). Then, the mixture was administered by ID injection in mice. Controls animals received only PBS (50 µL), venom (0,20 mg/Kg/50 µL PBS) or B. guinanensis extract (10 mg/Kg/50µL PBS).

Myotoxic Activity
Myotoxic activity was determined using the method of quantification of creatine kinase enzyme (Kaplan and Pesce, 1986). The principle of this method consists in the reaction of creatine phosphate and Adenosine Phosphate (ADP), catalyzed by creatine kinase to form creatine and Adenosine Triphosphate (ATP). Activity was expressed in units/L, one unit corresponding to the production of one micromole of NADH per min. Mice were injected in the gastrocnemius muscle with several concentrations Bothrops atrox venom to determined minimum myotoxic dose. 3 h after injection, mice were bled from the orbital plexus with heparinized Pasteur pipettes. After centrifugation, plasma was separated and the CK activity was determinate by using Liquiform CK-NAC Kit (Labtest Diagnostica). In the inhibition assays, a fixed amount of Bothrops atrox venom (0, 20 mg/25 µL PBS) was mixed with Brosimum guinanensis extract concentrations different (1, 25; 2, 5; 5, 0; 7, 5 and 10 mg de BGE/Kg/25 µL PBS). Then, the mixture was administered by intramuscular injection in mice. Controls animals received only PBS (50 µL), venom (0, 20 mg/Kg/50 µL PBS) or B. guinanensis extract (10 mg/Kg/50 µL PBS) by intramuscular route.

Statistical Analysis
The results are presented as the mean±S.E.M. Differences among groups were Analyzed by Oneway Analysis of Variance (ANOVA) followed by Tukey-Kramer test. Differences with an associated probability (P value) of less than 5% (p<0.05) were considered significant.

RESULTS
The venom dose used for oedema induction was 0.20 mg PBBa/25 µL PBS, measured at times 0, 1, 2, 3 and 4 h after treatments. Figure 1 shows the effect of venom and BGE in the edema formation in mice. At the end 4 h, the results in all test groups were not significantly different from BAV control group. Therefore, BGE was not effective in paw edema reducing.
In the inhibition assays, was used Bothrops atrox venom (0.20 mg/25 µ L PBS) and BGE concentrations different. The influx of leukocytes was analyzed in the peritoneal wash observing cells total count and differential leukocytes. . The responses were measured at 5 min (first phase -A) and 20-30 min (second phase -B) after venom or saline solution administration. The results are presentedthe mean ± S.E.M. of five animals. Differences among groups were analysed by one-way Analysis of Variance (ANOVA), followed by the Tukey-Kramer test. Differences with an associated probability (p value) of less than 5% (p<0.05) were considered significant. *p<0.05, **p<0.01, ***p<0.001 compared with BAV group The results showed that all BGE concentrations used in the present study were not effective on the migration of inflammatory cells induced by venom administration (Fig. 3).
Intramuscular injections of B. atrox venom (0.20 mg/25 µL PBS) induced a significant increase on plasma CK activity when compared to PBS control group. The results also showed that PBE was not able to inhibit increase on plasma activity when compared to BAV control group (Fig.  6). Indirect hemolytic method, showed that BGE concentrations used in the present study did not inhibit the Phospholipase A2 (PLA2) activity, as shown in Fig. 7.

DISCUSSION
In many countries, plants have been used for snakebite treatments. In Amazon region, local people use the macerated bark of B. guinanensis applied in the form of cataplasm, on the site of snakebite. However, scientific validation of the antiophidian properties is needed. In the present study we analyzed for the first time the ability of the B. guianensis aqueous extract in the neutralization several effects induced by B. atrox venom.
Our results showed that B. guianensis aqueous extract was not effective to inhibit oedema, peritonitis coagulant, myotoxic, indirect hemolytic activities induced by B. atrox venom, but was able to inhibited significantly hemorrhagic and nociceptive activities. Soares et al. (2005) and coworkers summarized 850 species from 138 families of plants used ethnobotanically and ethnopharmacologically or confirmed by biological assays. Several plant species used by Brazilian folk medicine had been studied against snake venom activities (Melo et al., 1994;Da Silva et al., 2005;Nishijima et al., 2009). De Paula et al. (2010 and coworkers evaluated antiophidian properties of 12 Brazilian plants extracts against the hemolytic, coagulant, hemorrhagic and proteolytic effects of Lachesis muta venom. Data revealed that most of these aqueous products were capable of inhibiting those activities at different levels, except for Sapindus saponaria extract. In contrast, Stryphnodendron barbatiman extract completely neutralized all the analyzed biological activities.

CONCLUSION
The results of this study indicated that Brosimum guianensis extract reduced nociception and hemorrhage produced by Bothrops atrox venom in mice, however, was not effective to inhibit oedema, peritonitis, coagulant, myotoxic, phospholipase A2 activity (indirect hemolytic method). Further studies are necessary to isolated and identified yours active components, opening the possibility of synthesis of new compounds and application for therapeutic purpose as supplements to conventional serum therapy in reducing nociception and hemorrhage induced by B. atrox venom.
These results also support a potential effect of this extract as a compounds source for biotechonological application and synthesis of new drugs with therapeutic purpose.

ACKNOWLEDGEMENT
The researchers wish to thank the Santander Bank for their financial support, Instituto Evandro Chagas, Instituto de Pesquisas Científicas e Tecnológicas do Amapá, Neotrópica Tecnologia Ambiental LTDA, Dra. Camila Moreira Barreto Gomes and Dra. Wegliane Campelo da Silva Aparício for their contributions.