NEUTRALIZATION OF THE NEUROMUSCULAR ACTIVITY OF BOTHROPSTOXIN-I, A MYOTOXIN FROM Bothrops jararacussu SNAKE VENOM, BY A HYDROALCOHOLIC EXTRACT OF Casearia sylvestris Sw. (GUAÇATONGA)

Numerous plants are used as snakebite antidotes in Brazilian folk medicine, including Casearia sylvestris Swartz, popularly known as guaçatonga. In this study, we examined the action of a hydroalcoholic extract from C. sylvestris on the neuromuscular blockade caused by bothropstoxin-I (BthTX-I), a myotoxin from Bothrops jararacussu venom, in mouse isolated phrenic nerve-diaphragm (PND) preparations. Aqueous (8 and 12 mg/ml, n=4 and 5, respectively) and hydroalcoholic (12 mg/ml, n=12) extracts of the leaves of C. sylvestris caused facilitation in PND preparations followed by partial neuromuscular blockade. BthTX-I (20 μg/ml, n=4) caused 50% paralysis after 65±15 min (mean ± S.E.M). Preincubation (30 min at 37°C) of BthTX-I (20 μg/ml, n=4) with a concentration of the hydroalcoholic extract (4 mg/ml) that had no neuromuscular activity, such as the control (n=5), prevented the neuromuscular blockade caused by the toxin. This protection may be mediated by compounds such as flavonoids and phenols identified by thin-layer chromatography and colorimetric assays.


INTRODUCTION
Currently, the standard treatment for snakebites in humans is the administration of antivenom (5).However, numerous species of plants have also been used to treat snakebites in animals and humans.Thus, in Trinidad, the standard treatment for snakebites and scorpion stings in dogs involves the use of steroids, antibiotics, and the enzyme ananase (from the pineapple Ananas comosus).This enzyme, the administration of which must occur within 2 hours of the bite (15), reduces the inflammatory response and helps to degrade necrotic tissue.
Casearia sylvestris Sw. (Flacourtiaceae), popularly known as guaçatonga, grows wild throughout the tropics, where it easily adapts to forests and plains (28).This plant is widely used in folk medicine as an antiseptic, wound healer, topical anesthetic, and antitumor, anti-ulcer and anti-snakebite agent (1,2,3,14).In this study, we examined the neuromuscular activity of aqueous and hydroalcoholic extracts of C. sylvestris, and assessed the activity of the hydroalcoholic extract to neutralize the neuromuscular blockade caused by BthTX-I, a myotoxin from Bothrops jararacussu venom, that causes muscle damage (12,24) and blockade of directly and indirectly evoked contractions in mouse phrenic nerve-diaphragm preparations (11).

Plant material and extraction
The leaves of C. sylvestris were collected in February 2003 from adult plants growing in an orchard at the University of Sorocaba (UNISO).A voucher specimen was deposited in the UNISO herbarium after identification by the Botanical Institute (Instituto de Botânica) in São Paulo, Brazil.The leaves were dried and powdered, and aqueous and hydroalcoholic (70%) extracts were obtained by maceration, concentration, and lyophilization.The lyophilized material was stored at room temperature and protected from light and humidity until assayed.Partial physicochemical characterization of the extracts was done as described in the sections 2 and 3 below.

Thin-layer chromatography
Aliquots of the hydroalcoholic (HA) extract were spotted onto thin-layer (0.3 mm thick) silica gel plates (Merck ® ), along with appropriate standards.All solvents were purchased from Merck ® (Darmstadt, Germany).The solvent system for running the hydroalcoholic extract consisted of ethyl acetate : formic acid : glacial acetic acid : water (100:11:11:26, v/v).The following phytochemical groups were used as standards (all from Sigma-Aldrich ® , St. Louis, MO, USA): apigenin, quercetin, rutin, caffeic acid, and chlorogenic acid.The separated spots were visualized with diphenylboric acid 2-aminoethyl ester (NP) and polyethylene glycol 4000 (PEG) and observed with ultraviolet light at 365 nm.The retention factor (Rf) of each spot was compared to the standards indicated above.

Flavonoids content
The content of flavonoids was determined as described by other authors (9,17), based on the UV absorption of Al-Cl 3 -flavonoid complexes, and was expressed as the total content of quercetin.Briefly, 2 g of the extract were extracted under reflux for 30 min with 50 ml of 50% methanol and 5 ml of this extract was then completed to 50 ml with 80% methanol.Aliquots (2 ml) of this solution (n=4) were mixed with 2 ml of methanol containing 5% anhydrous aluminum chloride (AlCl 3 ; complexing agent) and completed to 10 ml with 80% methanol.After 30 min, the absorbances were read at 420 nm against a blank containing 80% methanol (8 ml) and 5% AlCl 3 (2 ml).The percentage of flavonoids (%FL) was calculated from a standard curve of quercetin (Sigma-Aldrich ® ) prepared in methanol (0, 4, 8, 12, and 16 µg/ml).

Polyphenol content
The content of polyphenols was determined as described by other authors (13,22), using an adaptation by Reicher et al. (22).Briefly, 2 g of the extract were extracted under reflux with 50 ml of 50% ethanol, and 5-ml aliquots of this solution (n=3) were completed to 50 ml with 50% ethanol.From this solution, 5-ml aliquots were withdrawn and completed to 25 ml using distilled water.Five hundred microliters of phosphomolybdotungstic reagent was added to 1 ml of this solution and the volume was completed to 10 ml with 15% sodium carbonate solution.After 30 min, the absorbance at 720 nm was read against a blank of 15% sodium carbonate solution.

Animals
Male Swiss white mice (26-32 g) were supplied by the Animal Services Unit of the State University of Campinas (UNICAMP).The animals were housed at 25 ± 3°C on a 12-h light/dark cycle and had access to food and water ad libitum.This project (protocol number 644-1) was approved by the institutional Committee for Ethics in Animal Experimentation (CEEA-IB, UNICAMP), and the experiments were done within the guidelines of the Brazilian College for Animal Experimentation (COBEA).

Statistical analysis
Each experimental protocol was repeated at least three times.The results were expressed as the mean + S.E.M., as appropriate.Student's t-test was used for statistical comparison of the data.A value of p<0.05 indicated significance.

Thin-layer chromatography
Figure 1 shows the chromatographic profile of the hydroalcoholic extract of C. sylvestris leaves.Comparison with standards of quercetin, rutin, caffeic acid, and chlorogenic acid run, simultaneously, showed that the phenolic constituents of the extract had Rf values that were most similar to those of rutin and chlorogenic acid.

Content of flavonoids and polyphenolic compounds
Figure 2A shows the standard curve for flavonoids (quercetin).Using the equation Y = 0.0563X -0.041 (r = 0.99939) for this curve, the concentration of quercetin in the hydroalcoholic extract of C. sylvestris was calculated to be 5.12 µg/ml and the content of flavonoids (%FL) was 0.640 g%.The standard curve for polyphenols (pyrogallol) (Figure 2B) resulted in the equation Y = 0.135X + 0.0356 (r = 0.99749).
From this, the concentration of pyrogallol in the extract was calculated to be 1.77 µg/ml and the content of polyphenols (%PP) was 2.22 g%.Both extracts caused significant facilitation (p<0.05,compared to the Tyrode control) of an "all or nothing" type, starting with 8 mg/ml for the aqueous extract and 12 mg/ml for the hydroalcoholic extract.High concentrations of the extracts caused neuromuscular blockade.The facilitation and neuromuscular blockade were more intense with the hydroalcoholic extract.For this reason, this extract was chosen for the neutralization experiments with BthTX-I.A concentration of 4 mg/ml was considered ideal because it produced no facilitation or significant neuromuscular blockade.(8,25).Bothrops jararacussu venom produces these effects mainly through bothropstoxin-I (BthTX-I), the major myotoxin of this venom (12).

Neutralization assays
The use of plant extracts as antidotes for envenomings represents an alternative for communities that do not have access to antivenom therapy, and may be useful against the local effects of venoms (3,16,21,23,26).Plant products also have applications against snakebites and scorpion stings (15).
The efficacy of C. sylvestris against snake venom has been assessed in some studies (2,3).As shown here, alcohol was a better solvent for extraction than water.
The presence of active compounds such as flavonoids and phenols was initially demonstrated by thin-layer chromatography followed by direct quantification.Many of these plant substances structurally resemble biologically active compounds, and this similarity may be the basis of their physiological action (10).
In plants, flavonoids most likely serve to filter out UV light (adstringency), scavenge free radicals in situ, and exert an antipredatory effect.Flavonoids can also act as free radical scavengers in vitro and may modulate the redox properties and mechanisms of radical interactions (4).As shown here, the aqueous and hydroalcoholic extracts caused facilitation followed by partial inhibition of the twitch tension.Similar results have been reported for green tea (Camellia sinensis) extracts in rat diaphragm and were reproduced by a crude polyphenol preparation (7).The hydroalcoholic extract of guaçatonga contains flavonoids and phenolic compounds.The role of the phenolic compounds in the neuromuscular blockade caused by this extract is unclear.
In addition to the extracts tested here, other substances with a facilitatory action on neuromuscular transmission, such as heparin (20) and manganese (a neuromuscular blocker that shows spontaneous reversion and post-wash facilitation) (18,19,27), also protect against neuromuscular blockade.
The results reported here extend the findings of Borges et al. (2,3) regarding the ability of extracts of C. sylvestris to protect against the hemorrhagic and phospholipase A 2 activities of snake venom.However, the mechanism by which the hydroalcoholic extract of C. sylvestris protected against the paralysis induced by BthTX-I remains unclear.Further purification and identification of the active fractions of the extract will contribute to our understanding of the pathways involved.

Figure 3
Figure 3 shows the effects of aqueous (A) and hydroalcoholic (B) extracts of C. sylvestris leaves on mouse PND preparations at concentrations of 2, 4, 8, and 12 mg/ml (n=3-6 and 3-12 for the aqueous and hydroalcoholic extracts, respectively).

Figure
Figure4Ashows the neuromuscular blockade caused by BthTX-I (20 µg/ml, n=4, p<0.05) compared to the Tyrode control (n=5) and the response to a mixture of