In Vitro Antiophidian Properties of Dipteryx alata Vogel Bark Extracts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Extracts from Dipteryx alata bark obtained with different solvents (hexane, dichloromethane, ethyl acetate and methanol) were mixed in vitro with Bothrops jararacussu (Bjssu, 40 mu g/mL) and Crotalus durissus terrificus (Cdt, 15 mu g/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 similar to 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.15959565970Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)PROBIC/UNISOConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [Proc. FAPESP 04/09705-8, 07/53883-6]FAPESP [07/51414-9, 08/05459-3]CNPq [Proc. 302206/2008-6

The neuromuscular activity of paradoxin: A presynaptic neurotoxin from the venom of the inland taipan (Oxyuranus microlepidotus)

The inland taipan is the world's most venomous snake. However, little is known about the neuromuscular activity of the venom or paradoxin (PDX), a presynaptic neurotoxin from the venom. Venom (10 mu g/ml) and PDX (65 nM) abolished indirect twitches of the chick biventer cervicis and mouse phrenic nerve diaphragm preparations. The time to 90% inhibition by PDX was significantly increased by replacing Ca2+ (2.5 mM) in the physiological solution with Sr2+ (10 mM). In the biventer cervicis muscle, venom (10 mu g/ml), but not PDX (65 nM), significantly inhibited responses to ACh (1 mM) and carbachol (20 mu M), but not KCl (40 mM). In the mouse diaphragm (low Ca2+; room temperature), the inhibitory effect of PDX (6.5 nM) was delayed and a transient increase (746 +/- 64%; n = 5) of contractions observed. In intracellular recording experiments using the mouse hemidiaphragm, PDX (6.5-65 nM) significantly increased quantat content and immature endplate potential frequency prior to blocking evoked release of acetylcholine. In extracellular recording experiments using the mouse triangularis sterni, PDX (2.2-65 nM) significantly inhibited the voltage-dependent K+, but not Na+, waveform. In patch clamp experiments using B82 mouse fibroblasts stably transfected with rKv 1.2, PDX (22 nM; n = 3) had no significant effect on currents evoked by 10 mV step depolarisations from -60 to +20 mV. PDX exhibits all the pharmacology associated with beta-neurotoxins, and appears to be one of the most potent, if not the most potent beta-neurotoxin yet discovered. (c) 2007 Elsevier Ltd. All rights reserved.5251229123

In Vitro Antiophidian Mechanisms of Hypericum brasiliense Choisy Standardized Extract: Quercetin-Dependent Neuroprotection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The neuroprotection induced by Hypericum brasiliense Choisy extract (HBE) and its main active polyphenol compound quercetin, against Crotalus durissus terrificus (Cdt) venom and crotoxin and crotamine, was enquired at both central and peripheral mammal nervous system. Cdt venom (10 mu g/mL) or crotoxin (1 mu g/mL) incubated at mouse phrenic nerve-diaphragm preparation (PND) induced an irreversible and complete neuromuscular blockade, respectively. Crotamine (1 mu g/mL) only induced an increase of muscle strength at PND preparations. At mouse brain slices, Cdt venom (1, 5, and 10 mu g/mL) decreased cell viability. HBE (100 mu g/mL) inhibited significantly the facilitatory action of crotamine (1 mu g/mL) and was partially active against the neuromuscular blockade of crotoxin (1 mu g/mL) (data not shown). Quercetin (10 mu g/mL) mimicked the neuromuscular protection of HBE (100 mu g/mL), by inhibiting almost completely the neurotoxic effect induced by crotoxin (1 mu g/mL) and crotamine (1 mu g/mL). HBE (100 mu g/mL) and quercetin (10 mu g/mL) also increased cell viability in mice brain slices. Quercetin (10 mu g/mL) was more effective than HBE (100 mu g/mL) in counteracting the cell lysis induced by Cdt venom (1 and 10 mu g/mL, resp.). These results and a further phytochemical and toxicological investigations could open new perspectives towards therapeutic use of Hypericum brasiliense standardized extract and quercetin, especially to counteract the neurotoxic effect induced by snake neurotoxic venoms.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundo de Apoio ao Ensino, a Pesquisa e Extensao (FAEPEX)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPES [063/2010

New evidence for a presynaptic action of prednisolone at neuromuscular junctions

The action of prednisolone at the neuromuscular junction was studied in mouse isolated phrenic nerve-diaphragm and rat external popliteal/sciatic nerve-tibialis anterior muscle preparations. Prednisolone (0.03 mM and 0.3 mM) did not alter the twitch-tension in phrenic nerve-diaphragm preparations after 120 min, but increased the frequency (170 +/- 4%) and amplitude (200 +/- 13%) of miniature end-plate potentials. Quantal content was not influenced by the glucocorticoid treatment. Prednisolone (400 mug/kg) did not change the twitch-tension in rat external popliteal/sciatic nerve-tibialls anterior muscle preparations. However, this steroid (0.3 mM) prevented the neuromuscular blockade by d-tubocurarine (1.45 muM) in mouse preparations by 70 +/- 10% (P < 0.05). A similar effect (82 +/- 6% protection, P < 0.05) occurred in rats treated with prednisolone (400 mug/kg) before d-tubocurarine (225 mug/kg). In phrenic nerve-diaphragm preparations, prednisolone (0.3 mM) increased (13 4%, p < 0.05) the twitch-tension in the presence of &beta;-bungarotoxin (1 &mu;M), and prevented the blockade produced by this toxin (0.15 &mu;M) in its third phase of action. This presynaptic facilitatory effect may contribute to the usefulness of prednisolone in myasthenia gravis. (C) 2002 Wiley Periodicals, Inc.261374

Determination of the amino acid sequence of a new phospholipase A(2) (MIDCA1) isolated from Micrurus dumerilii carinicauda venom

A new Phospholipase A(2) (PLA(2)) from Micrurus dumerilii carinicauda venom was isolated and its primary structure determined. This new PLA(2) showed a low enzymatic activity when compared with other PLA(2)s and it is moderately basic with an isoelectric point of 8.0. Its amino acid sequence showed the presence of 120 amino acid residues and its sequence was: NLIQFLNMIQCTTPGREPLVAFANYGCYCGRGGSGTPVDELDRCCQVHDNCYDTAKKVFGCSPYFTMYSYDCSEGKLTCKDNNTKCKAAVCNCDRTAALCFAKAPYNDKNYKIDLTKRCQ. The structural model of MIDCA1, when compared with other strong neurotoxic PLA(2)s, such as Naja naja, showed significant differences in the beta-wing and neurotoxic sites, despite the high level of amino acid sequence similarity. These observations indicate a dissociation between the biological and catalytic activity of this new PLA(2), supporting the view that other regions of the protein are involved in the biological effects.24314715

The presynaptic activity of bothropstoxin-I, a myotoxin from Bothrops jararacussu snake venom

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 (Mn2+), a presynaptic blocker, to investigate a possible presynaptic action of bothropstoxin-I. At concentrations of 0.9 mM and 1.8 mM, Mn2+ produced 50% neuromuscular blockade in less than 4 min., which was spontaneously reversible at the lower concentration. Bothropstoxin-I (1.4 muM) irreversibly inhibited neuromuscular blockade by 50% in 31+/-4 min. (mean+/-S.E.M., n=9). Pretreating preparations with 0.9 mM Mn2+ prevented the blockade by bothropstoxin-I. When added after bothropstoxin-I, Mn2+ produced its characteristic blockade and, after washing, the twitch tension returned to pre-Mn2+ levels, indicating that bothropstoxin-I caused irreversible damage before the addition of Mn2+. Electrophysiological measurements showed that a concentration of bothropstoxin-I (0.35 muM), 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 Mn2+ (0.9 mM, 30 min.) were protected against the depolarizing action of bothropstoxin-I (0.7 muM). These results show that, in addition to its well-known myotoxic effect, bothropstoxin-I also has a presynaptic action.95417518

Mutagenicity induced by the hydroalcoholic extract of the medicinal plant Plathymenia reticulata Benth

Plathymenia reticulata Benth has an anti-inflammatory effect and is capable of neutralizing the neuromuscular blockade induced by Bothrops jararacussu or Crotalus durissus terrificus venoms, probably by precipitating venom proteins (an effect caused by plant tannins). The present study aimed to evaluate the mutagenic activity of P. reticulata by using the Salmonella mutagenicity assay (Ames test) and the micronucleus test in CHO-K1 cells. P. reticulata extract concentrations of 2.84, 5.68, 11.37, and 19.90 mg/plate were assayed by the Ames test using TA97a, TA98, TA100 and TA102 bacterial strains, with (+S9) and without (-S9) metabolic activation. Concentrations of 5, 1.6 and 0.5 &#956;g/mL of P. reticulata extract were used for the micronucleus test. P. reticulata extract was mutagenic to TA98 (-S9) and showed signs of mutagenic activity in TA97a and TA102 (both -S9) strains. Micronucleus test CBPI values showed that the endogenous metabolic system increased the number of viable cells when compared to the non-activated samples and the micronucleus frequency increased when the cells were treated in the absence of S9. We concluded that P. reticulata extract may present direct mutagenic properties