The effect of four sub-extracts prepared from the lyophilized hydroalcoholic bark of Dipteryx alata (Leguminosae-Papilionoideae) dissolved in a methanol-water (80:20) mixture through a liquid-liquid partition procedure has been investigated against the neuromuscular blockade of the venom of the snake Bothrops jararacussu. The active CH2Cl2 sub-extract has been extensively analyzed for its chemical constituents, resulting in the isolation of four lupane-type triterpenoids: lupeol (1), lupenone (2), 28-hydroxylup-20(29)-en-3-one (3), betulin (4), nine isoflavonoids: 8-O-methylretusin (5), 7-hydroxy-5,6,4’-trimethoxyisoflavone (6), afrormosin (8), 7-hydroxy-8,3’,4’-trimethoxyisoflavone (9), 7,3’-dihydroxy-8,4’-dimethoxyisoflavone (10), odoratin (11), 7,8,3’-trihydroxy-4’-methoxyisoflavone (13), 7,8,3’-trihydroxy-6,4’-dimethoxyisoflavone (15), dipteryxin (17), one chalcone: isoliquiritigenin (7), one aurone: sulfuretin (14) and three phenolic compounds: vanillic acid (12), vanillin (16), and protocatechuic acid (18). Their chemical structures were elucidated on the basis of spectroscopic analysis, including HRMS, 1D- and 2D-NMR techniques.

Bothropstoxin-I from Bothrops jararacussu snake venom is a lysine-49 phospholipase A 2 with myotoxic and neurotoxic activities. In this study, we used mouse phrenic nerve-diaphragm preparations in the absence and presence of manganese (Mn 2π ), a presynaptic blocker, to investigate a possible presynaptic action of bothropstoxin-I. At concentrations of 0.9 mM and 1.8 mM, Mn 2π produced 50% neuromuscular blockade in less than 4 min., which was spontaneously reversible at the lower concentration. Bothropstoxin-I (1.4 mM) irreversibly inhibited neuromuscular blockade by 50% in 31∫4 min. (mean∫S.E.M., nΩ9). Pretreating preparations with 0.9 mM Mn 2π prevented the blockade by bothropstoxin-I. When added after bothropstoxin-I, Mn 2π produced its characteristic blockade and, after washing, the twitch tension returned to pre-Mn 2π levels, indicating that bothropstoxin-I caused irreversible damage before the addition of Mn 2π . Electrophysiological measurements showed that a concentration of bothropstoxin-I (0.35 mM), which did not produce neuromuscular blockade, caused the appearance of giant miniature end-plate potentials with no change in the membrane resting potential but increased the quantal content. Preparations preincubated with Mn 2π (0.9 mM, 30 min.) were protected against the depolarizing action of bothropstoxin-I (0.7 mM). These results show that, in addition to its well-known myotoxic effect, bothropstoxin-I also has a presynaptic action.

Extracts from Dipteryx alata bark obtained with different solvents (hexane, dichloromethane, ethyl acetate and methanol) were mixed in vitro with Bothrops jararacussu (Bjssu, 40 μg/mL) and Crotalus durissus terrificus (Cdt, 15 μg/mL) snake venoms, and applied to a mouse phrenic nerve-diaphragm preparation to evaluate the possible neutralization of venom effects. Cdt venom neurotoxic effect was not inhibited by any of the extracts, while the neurotoxic and myotoxic actions of Bjssu venom were decreased by the methanolic extract. This inhibition appears to be augmented by tannins. Dichloromethane bark extract inhibited ~40% of Bjssu venom effects and delayed blockade induced by Cdt. The methodology used to determine which extract was active allows inferring that: (i) phenolic acids and flavonoids contained in the methanolic extract plus tannins were responsible mostly for neutralization of Bjssu effects; (ii) terpenoids from the dichloromethane extract may participate in the anti-Cdt and anti-Bjssu venom effects; (iii) a given extract could not inhibit venoms from different species even if those belong to the same family, so it is improper to generalize a certain plant as antiophidian; (iv) different polarity extracts do not present the same inhibitory capability, thus demonstrating the need for characterizing both venom pharmacology and the phytochemistry of medicinal plant compounds.

Casearia sylvestris Sw., popularly known in Brazil as 'guaçatonga', has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C. sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects.

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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.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA 2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (−S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.