Bothrops jararacussu venom-induced neuromuscular blockade inhibited by Casearia gossypiosperma Briquet hydroalcoholic extract

The hydroalcoholic extract of Casearia gossypiosperma Briquet (Flacourtiaceae) was standardized for the first time through quality control procedures including pharmacognostic methods, fingerprint chromatograms, defined amounts of marker substances and physicochemical characteristics. The pharmacological activity of C. gossypiosperma (Cg) hydroalcoholic extract was assayed by a traditional in vitro test, which involved irreversible neuromuscular blockade induced by Bothrops jararacussu (Bjssu) venom (60 μg/mL) in mouse phrenic nerve-diaphragm preparations. Bjssu venom blocked muscle activity for 26 (± 2.0) minutes (n = 6). Cg extract (0.1 mg/mL) induced changes on the baseline muscle activity without impairing the muscle function and inhibited 87.6% (± 1.8) (n = 6) of the Bjssu venom-induced blockade. Both flavonoids (0.624 g%) and polyphenols (4.63 g%) from the extract were spectrophotometrically quantified. Therefore, the present study confirms the antibothropic activity of Cg extract, supporting the ethnomedical use of Casearia sp. in the treatment of snakebite victims.

Geographical isolation helps reinforce the need of traditional and local medical practices, selecting natural resources for the treatment of many diseases, including snakebites (28).In addition, recognizing the potential of a medicinal plant could also contribute to preserve it.
In this study, Casearia gossypiosperma Briquet, a native tree from Brazil and popularly known as "pau-de-espeto", was pharmacognostically and pharmacologically assessed for the first time.
In vivo myotoxic effects and in vitro irreversible neuromuscular blockade effects of crude venom from the snake Bothrops jararacussu are well known pharmacological methods used to study drugs showing antivenom properties (29,30).Thus, the aim of the present study was to verify the capability of Casearia gossypiosperma hydroalcoholic extract to neutralize the neuromuscular blockade induced by Bothrops jararacussu venom.

Plant Material and Extraction
Leaves from an adult C. gossypiosperma Briquet (Cg) tree were collected from the herbarium at the Higher School of Agriculture Luiz de Queiroz (ESALQ) of the University of São Paulo, Piracicaba, São Paulo, Brazil, in January 2006; the species was previously identified in 1988 (protocol IAC 38115).Cg leaves were dried at 35°C to 40°C for 24 hours.The dried leaves were then powdered, ground in a mill, macerated (200 g, during five days) in 2 L of 70% ethanol and the suspension was percolated (under protection against light) at 20 drops/minute, resulting in a 10% (m/v) hydroalcoholic extract.

Quality Control Assays of the Medicinal Plants Ash and humidity tests
To observe elementary physical and chemical characteristics, Cg powder was subjected to ash and humidity tests (31).Briefly, 100 g of the specimen powder were placed in six calibrated melting pots, which were warmed until total carbonization of the powders.The melting pots were kept at 650°C and the ashes were then weighed.Results are expressed as grams of ashes/100 g of sample.The humidity test was performed by placing 1 g of specimen powder in six calibrated porcelain capsules, which were heated at 105ºC during four hours and then weighed.

Flavonoid content
The flavonoid content was determined in Cg hydroalcoholic extract as described elsewhere (33).The method is based on the UV absorption of AlCl 3 -flavonoid complexes, expressed as total quercetin content.Briefly, 80% methanol (50 mL) was added to 10 mL extract, and 5 mL solution were transferred to volumetric flasks and diluted again with 80% methanol (50 mL).Four aliquots (2 mL) of solution were mixed with 2 mL of 5% anhydrous aluminum chloride solution (AlCl 3 ; complexing agent) and adjusted to 10 mL with 80% methanol.After 15 minutes, the absorbance of samples was read at 420 nm, considering a blank sample containing 80% methanol (8 mL) and 5% AlCl 3 (2 mL).The percentage of flavonoids (%) was calculated from a standard curve of quercetin (0, 4, 8, 12, and 16 µg/mL) prepared with methanol.

Polyphenol content
The polyphenol content in Cg hydroalcoholic extract was determined as previously described (34).Briefly, 5 mL extract was poured into a volumetric flask and distilled water was added to 250 mL; then, a 1 mL aliquot was transferred to another volumetric flask and distilled water added to 25 mL (final solution).Aliquots (1 mL) of the final solution received 1 mL of phosphomolybdotungstic reagent and the final volume (10 mL) was adjusted with 15% sodium carbonate solution.After 30 minutes, the absorbance of samples was read at 720 nm, considering a blank sample containing 15% sodium carbonate solution.The percentage of polyphenols (%) was determined from a standard curve (5,10,15,20,25,30,35, and 40 µg/mL) of pyrogallol (Sigma Chemical Co., USA).

Animals
Male Swiss white mice (26-32 g) were supplied by Anilab Animais de Laboratório (Brazil).Animals were housed at 25 ± 3°C under a 12-hour light-dark cycle with access to food and water ad libitum.This study was approved (protocol number A009/2006 CEP) by the Research Ethics Committee of the Vale do Paraiba University (UNIVAP) and all experiments were performed according to the guidelines of the Brazilian Society of Laboratory Animal Science (SBCAL/ CEUA).

Statistical analysis
Each pharmacological protocol was repeated at least five times.Results were expressed as the mean ± standard deviation (SD).ANOVA and Tukey's (post hoc) tests were used for data comparison.The significance level was set at 5%.

RESULTS AND DISCUSSION
The chemical composition of plant extracts is very complex and variable as it depends on the climate and/or the soil composition (36).Thus, physicochemical control is indispensable for phytotherapics.Drug discovery techniques applied to the standardization of herbal medicines have revealed numerous analytical marker compounds.In the present study, quality control tests such as pharmacognostic methods, characteristic fingerprint chromatograms, defined amounts of marker substances and physicochemical characteristics were used for the extract standardization (37).
According to Brazilian Pharmacopeia (31), the usual values for extract ashes are up to 2% and for humidity, 14%.The results (mean ± SD, in grams/100 g) obtained in the ash and humidity tests for Cg were 3.67 ± 0.48 and 11.47 ± 1.02, respectively.The high ash content found in Cg powder indicates the presence of inorganic compounds due to the soil composition, although no correlation between elemental composition of medicinal plants and their curative properties have been established (38).The humidity level found for Cg powder confirms that no enzymatic reaction occurred during sample collection.
The Rf values obtained using solvent system B were 0.307, 0.387, 0.493, 0.547 and 0.880 for Cs; 0.181, 0.403 and 0.458 for Cg; and 0.663 and 0.400 for apigenin and quercetin, respectively, whereas rutin was not displaced by the solvent.In this solvent system, Cg spots are suggestive of rutin, quercetin and another flavonoid not identified (orange coloration).
Orange and blue compounds in the plants, visualized by TLC, are suggestive of flavonoids and polyphenol compounds and here were quantitatively confirmed as 0.624 g% (quercetin calibration curve Y = 0.0632X + 0.0035, r = 0.998) and 4.63 g% (pyrogallol calibration curve Y = 0.1693X -0.0004, r = 0.999) for Cg (32).The latter has very similar flavonoid content but twice more polyphenol concentration compared to C. sylvestris (0.640 g% and 2.22 g% for flavonoids and polyphenols, respectively); these parameters were measured by the methodology described elsewhere (39).This variation in plant compounds can change medicinal properties (40).Parameters such as ash production, humidity, flavonoids and phenols are important for phytochemical characterization and maintenance of a physicochemical standard of plant extracts.In the present study, the plant assays showed reproducibility, which could indicate that the extracts had good standardization.
The major constituents (flavonoids and polyphenols) of Cg hydroalcoholic extracts were identified by TLC and spectrophotometrically quantified, and their capability to inhibit neuromuscular activities of bothropic venom was demonstrated in vitro.Crude Bjssu venom induces neurotoxicity in vitro and causes muscle necrosis in vivo, resulting in irreversible tissue loss and limb amputation in severe accidents (29,30).Muscle necrosis is usually attributed to bothropstoxin-I, which is a myotoxic Lys49 PLA 2 homologue and the main toxic component of Bjssu venom (41).The myotoxic components of snake venoms can reduce the initiation of muscle contractures, muscle response after direct stimulation or elevated potassium exposure (42).In this work, the addition of Bjssu venom (60 µg/ mL or 0.3 mg, n = 6) alone to the bath produced total muscle paralysis starting at 26 ± 2 minutes until the end of the experiment.
Figure 2 shows the bioassays on the muscle activity of mouse phrenic nerve-diaphragm preparations using 0.05, 0.1, 1 and 2 mg/mL (or 0.25, 0.5, 5 and 10 mg, respectively) Cg hydroalcoholic extract over 120 minutes.Except 0.05 mg/mL, all other Cg extract concentrations significantly decreased the muscle activity.However, in all assays, a total recovery of the muscle activity was verified after total removal of extracts by washing the preparations with fresh Tyrode solution.The concentrations of 0.05, 0.1 and 1 mg/mL Cg extract were assayed with crude Bjssu venom.
Figure 3 shows the neutralization of Bjssu venom by Cg extract mixed with venom in the bath kept at 37 o C for 120 min.Curiously, after decreasing muscle activity, 0.1 mg/mL Cg extract significantly (p < 0.05) reduced muscle paralysis by 87.6±1.8%(n = 6).The protective action     against crude venom was confirmed by a total recovery after washing (W).
Using the same experimental model, two organ preparations were observed, pretreatment: preincubation with Cg extract followed by addition of crude Bjssu venom (Figure 4, n = 6); and post-treatment: addition of Cg extract after 15 minutes of Bjssu venom exposure (Figure 5, n = 6).Preincubation with Cg extract protected the muscle against the blockade induced by Bjssu venom, similarly to the results shown in Figure 3. Post-treatment with 0.1 mg/mL Cg extract did not change the blockade profile (not shown), but 1 mg/mL Cg extract partially and significantly (p < 0.05) protected against the total blockade induced by Bjssu venom.This result could indicate that 1 mg/mL Cg extract could prevent the venom activity propagation.Since only higher Cg concentrations showed pharmacological effect, the bioactive compounds could be concentrated in the extract, which is interesting for a future C. gossypiosperma bio-prospection.
Our experience with C. sylvestris (27, 39) using the same experimental model and all attempts to identify the bioactive compounds (mainly rutin) showed that this plant protected the muscle against the toxic effects of Bjssu venom due to phytocomplex formation.Different molecules in the plant constitution inhibit Bjssu venom activity in different ways, for example, coumarin from Mikania laevigata and tannin from Plathymenia reticulata.In commercial phytochemical preparations, these molecules (coumarin and tannic acid) confirmed the capability to revert Bjssu venom-induced muscle blockade by pharmacological (M.laevigata) or protein precipitation (P.reticulata) (48).We also observed that the capability to protect against the neuromuscular blockade induced by Bjssu venom does mean protection against the paralysis caused by Crotalus durissus terrificus venom (data not published), which indicates that the mechanism of action of the venoms must be considered and the plant and venom mixing is not enough to neutralize the toxic effects.
In the present study, the neuromuscular blockade induced by Bjssu venom was inhibited by Cg hydroalcoholic extract, suggesting the presence of compounds with the ability to reduce neurotoxicity and effectively inhibit the myotoxic action of the venom.Based on the similarity of the constituents found in C. gossypiosperma to those seen in C. sylvestris, we suggest the same mechanism of action for both, i.e., phytocomplex formation between venom and antioxidants (flavonoids and polyphenols).This is the first attempt to show the antivenom activity of C. gossypiosperma and prove its ethnobotanical use.Further studies are needed to identify the bioactive compound(s) responsible for the antineurotoxic and antimyotoxic effects of Cg.Raposo Tavares, km 92,5, Sorocaba, SP, 18.023-000, Brasil.Phone: +55 15 2101 7041.Fax: +55 15 2101 7112.Email: yoko.franco@prof.uniso.br.

Figure 4 .
Figure 4. Twitch tension of mouse isolated phrenic nerve-diaphragm preparations under indirect stimuli, in percentage (each point represents the mean ± SEM, n = 6).Pretreatment of preparations with 0.1 mg/ mL (or 0.5 mg) Cg hydroalcoholic extract for 30 minutes, followed by 60 μg/mL (or 300 mg or 0.3 mg) Bjssu venom addition during 120 minutes.* p < 0.05 compared to Tyrode control for all periods (except 0).# p < 0.05 compared to Cg extract alone or to pretreatment with Cg extract.

Figure 5 .
Figure 5. Twitch tension of mouse isolated phrenic nerve-diaphragm preparations under indirect stimuli, in percentage (each point represents the mean ± SEM, n = 6).Post-treatment of preparations with 1 mg/ mL (or 5 mg) Cg hydroalcoholic extract after 15 minutes, followed by 60 μg/mL (or 300 mg or 0.3 mg) Bjssu venom addition.* p < 0.05 compared to Tyrode control for all periods (except 0).# p < 0.05 compared to treatment with Bjssu during 15 minutes, followed by Cg extract addition.