Vellozia flavicans Mart. ex Schult. hydroalcoholic extract inhibits the neuromuscular blockade induced by Bothrops jararacussu venom

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOBackground: Snakebite is a significant public health issue in tropical countries. In Brazil, some of the most common snake envenomations are from Bothrops. Bothrops bites trigger local and systemic effects including edema, pain, erythema, cyanosis, infections, and necrosis. Vellozia flavicans is a plant from the Brazilian " cerrado" (savanna) that is popularly used as an anti-inflammatory medicine. Since inflammation develops quickly after Bothrops bites, which can lead to infection, the aim of the present study was to observe possible anti-snake venom and antimicrobial activities of V. flavicans (Vf). Methods: The chromatographic profile of the main constituents from the Vf leaf hydroalcoholic extract was obtained by thin-layer chromatography (TLC). The anti-snake venom activity was measured by Vf's ability to neutralize the in vitro neuromuscular blockade caused by Bothrops jararacussu venom (Bjssu) in a mouse phrenic nerve-diaphragm model (PND). After a 20 min incubation, preparations of PND were added to Tyrode's solution (control); Vf (0.2, 0.5, 1, and 2 mg/mL); 40 μg/mL Bjssu; pre-incubation for 30 min with Bjssu and 1 mg/mL Vf; and a Bjssu pretreated preparation (for 10 min) followed by 1 mg/mL Vf. Myographic recording was performed, and the contractile responses were recorded. The antimicrobial activity (minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC]) was obtained for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, using gentamicin and vancomycin as positive controls. Results: TLC analysis yielded several compounds from Vf, such as flavonoids (quercetin) and phenolic acids (chlorogenic acid). Bjssu completely blocked the contractile responses of PND preparations, while Vf preserved 97% (±10%) of the contractile responses when incubated with Bjssu. In the PND pretreated with Bjssu, Vf was able to inhibit the neuromuscular blockade progress. MIC and MBC of Vf ranged from 2.5 to 5.0 mg/mL for P. aeruginosa and S. aureus strains, while no antimicrobial activity was observed for E. coli and E. faecalis.Conclusions: The hydroalcoholic extract from Vf leaves was able to neutralize and decrease the in vitro neuromuscular blockade caused by Bjssu. However, it did not show significant antimicrobial activity against the tested bacteria. © 2014 Tribuiani et al.; licensee BioMed Central Ltd.Snakebite is a significant public health issue in tropical countries. In Brazil, some of the most common snake envenomations are from Bothrops. Bothrops bites trigger local and systemic effects including edema, pain, erythema, cyanosis, infections, and necrosis. Vellozia flavicans is a plant from the Brazilian "cerrado" (savanna) that is popularly used as an anti-inflammatory medicine. Since inflammation develops quickly after Bothrops bites, which can lead to infection, the aim of the present study was to observe possible anti-snake venom and antimicrobial activities of V. flavicans (Vf). The chromatographic profile of the main constituents from the Vf leaf hydroalcoholic extract was obtained by thin-layer chromatography (TLC). The anti-snake venom activity was measured by Vf's ability to neutralize the in vitro neuromuscular blockade caused by Bothrops jararacussu venom (Bjssu) in a mouse phrenic nerve-diaphragm model (PND). After a 20 min incubation, preparations of PND were added to Tyrode's solution (control); Vf (0.2, 0.5, 1, and 2 mg/mL); 40 μg/mL Bjssu; pre-incubation for 30 min with Bjssu and 1 mg/mL Vf; and a Bjssu pretreated preparation (for 10 min) followed by 1 mg/mL Vf. Myographic recording was performed, and the contractile responses were recorded. The antimicrobial activity (minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC]) was obtained for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, using gentamicin and vancomycin as positive controls. TLC analysis yielded several compounds from Vf, such as flavonoids (quercetin) and phenolic acids (chlorogenic acid). Bjssu completely blocked the contractile responses of PND preparations, while Vf preserved 97% (±10%) of the contractile responses when incubated with Bjssu. In the PND pretreated with Bjssu, Vf was able to inhibit the neuromuscular blockade progress. MIC and MBC of Vf ranged from 2.5 to 5.0 mg/mL for P. aeruginosa and S. aureus strains, while no antimicrobial activity was observed for E. coli and E. faecalis. The hydroalcoholic extract from Vf leaves was able to neutralize and decrease the in vitro neuromuscular blockade caused by Bjssu. However, it did not show significant antimicrobial activity against the tested bacteria14FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2004/09705-8; 2007/53883-6; 2008/52643-4; 2008/11005-5; 2012/08271-0Neglected tropical diseases: Snakebite, , http://www.who.int/bloodproducts/animal_sera/Rabies.pdf?ua=1, WHO - World Health Organization(2009) Textos Básicos de Saúde (Cadernos de Atenção Básican. 22), , Health surveillance:zoonoses, Brasil. Ministério da SaúdeTeixeira, R., Forma grave do acidente por ofídios da sub-família Crotalinae (1979) An Acad Med Bahia, 2, pp. 109-135Vellard, J.A., Serpentes venenosas (1945) Terapêutica Clínica, pp. 265-273. , Buenos Aires: Libreria y Editorial 'El Ateneo, Cardini C, Beretervide JJAlves, E., (1956) Medicina de Urgência, pp. 1052-1055. , Rio de Janeiro: Livraria AtheneuBrazil, V., (1901) Do envenenamento ophidico e seu tratamento, pp. 31-55. , Colletanea dos trabalhos Instituto ButantanRodrigues-Simioni, L., Borgese, N., Ceccarelli, B., The effects of Bothrops jararacussu venom and its components on frog nerve-muscle preparation (1983) Neuroscience, 10, pp. 475-489Souza, C.D., Felfili, J.M., The utilization of medicinal plants in the region of Alto Paraíso of Goiás, GO, Brazil (2006) Acta Bot Bras, 20, pp. 133-142Stannard, B.L., (1995) Flora of the Pico das Almas: Chapada da Diamantina - Bahia, Brazil, pp. 43-78. , Great Britain: Whitstable Litho LtdBranco, A., Pinto, A.C., Braz Filho, R., Chemical constituents from Vellozia graminifolia (Velloziaceae) (2004) An Acad Bras Cienc, 76, pp. 505-518Harborne, J.B., Williams, C.A., Greenham, J., Eagles, J., Variations in the lipophilic and vacuola flavonoids of the genus Vellozia (1994) Phytochemistry, 35, pp. 1475-1480Williams, C.A., Harborne, J.B., Greenham, J., Eagles, J., Differences in flavonoids patterns between genera within the Velloziaceae (1994) Phytochemistry, 36, pp. 931-940Pinto, A.C., Scofield, T.C.V., Braz-Filho, R., Two new diterpenes with a rosane skeleton from Velloziaceae (1983) Tetrahedron Lett, 24, pp. 5043-5046Pinto, A.C., Queiroz, P.P.S., Garcez, W., Diterpenes from Vellozia bicolor (1991) J Braz Chem Soc, 2, pp. 25-30Pinto, A.C., Rezende, C.M., Antunes, O.A.C., Correia, C.R.D., Three isomeric diterpenes from Vellozia flavicans (1996) Phytochemistry, 42, pp. 767-769Peixoto, E.M., Pinchin, R., Pinto, A.C., Constituintes químicos de Vellozia piresiana (1979) Ciênc Cult, 32, pp. 125-127Barnes, R.A., Pereira, A.L., Scofield, T.C.V., Braz-Filho, R., Pinto, A.C., A new triterpene from Vellozia compacta (1984) Chem Pharm Bull, 32, pp. 3674-3677Dharmappa, K.K., Kumar, R.V., Nataraju, A., Mohamed, R., Shivaprasad, H.V., Vishwanath, B.S., Anti-inflammatory activity of oleanolic acid by inhibition of secretory phospholipase A2 (2009) Planta Med, 75, pp. 211-215Magalhães, A., Santos, G.B., Verdam, M.C., Fraporti, L., Malheiro, A., Lima, E.S., Dos-Santos, M.C., Inhibition of the inflammatory and coagulant action of Bothrops atrox venom by the plant species Marsypianthes chamaedrys (2011) J Ethnopharmacol, 134, pp. 82-88Sampaio, S.C., Sousa-e-Silva, M.C.C., Borelli, P., Curi, R., Cury, Y., Crotalus durissus terrificus snake venom regulates macrophage metabolism and function (2001) J Leukoc Biol, 70, pp. 551-558(2002) Portuguese Pharmacopoeia, , Lisboa: Infarmed Editors, Portugal, Portuguese Pharmacopoeia CommitteeHarborne, J.B., (1998) Phytochemical Methods: A Guide to Modern Techniques of Plants Analysis, , London: Chapman & HallCintra Francischinelli, M., Silva, M.G., Andréo Filho, N., Cintra, A.C.O., Leite, G.B., Cruz Höfling, M.A.D.A., Rodrigues Simioni, L., Oshima Franco, Y., Effects of commonly used solubilizing agents on a model nerve-muscle synapse (2008) Lat Am J Pharm, 27, pp. 721-726Siles Villaroel, M., Rolim Rosa, R., Zelante, F., Furlanetto, R.S., Padronização da avaliação da potência de antivenenos botrópicos, em camundongos (1978) Mem Inst Butantan, 42-43, pp. 325-336Bülbring, E., Observation on the isolated phrenic nerve diaphragm preparation of the rat (1946) Br J Pharmacol, 1, pp. 38-61Ferraz, M.C., Parrilha, L.A.C., Moraes, M.S.D., Amaral Filho, J., Cogo, J.C., Dos Santos, M.G., Franco, L.M., Oshima Franco, Y., The effect of lupane triterpenoids (Dipteryx alata Vogel) in the in vitro neuromuscular blockade and myotoxicity of two snake venoms (2012) Curr Org Chem, 16, pp. 2717-2723Puebla, P., Oshima-Franco, Y., Franco, L.M., Santos, M.G., Silva, R.V., Rubem-Mauro, L., Feliciano, A.S., Chemical constituents of the bark of Dipteryx alata Vogel, an active species against Bothrops jararacussu venom (2010) Molecules, 15, pp. 8193-8204Gottlieb, O., Kaplan, M.A., Das plantas medicinais aos fármacos naturais (1993) Cienc Hoje, 15, pp. 51-54Souza Brito, A.R.M., How to study the pharmacology of medicinal plants in underdeveloped countries (1996) J Ethnopharmacol, 54, pp. 131-138Martini, N.D., Katerere, D.R., Eloff, J.N., Biological activity of five antibacterial flavonoids from Combretum erythrophyllum (Combretaceae) (2004) J Ethnopharmacol, 93, pp. 207-212Rates, S.M.K., Plants as source of drugs (2001) Toxicon, 39, pp. 603-613Soares, A.M., Ticli, F.K., Marcussi, S., Lourenço, M.V., Januário, A.H., Sampaio, S.V., Giglio, J.R., Pereira, O.S., Medicinal plants with inhibitory properties against snake venoms (2005) Curr Med Chem, 12, pp. 2625-2641Mors, W.B., Do Nascimento, M.C., Pereira, B.M.R., Pereira, N.A., Plant natural products active against snake bite - the molecular approach (2000) Phytochemistry, 55, pp. 627-642Barbosa, A.M., Villaverde, A.B., Guimarães Souza, L., Ribeiro, W., Cogo, J.C., Zamuner, S.R., Effect of low-level laser therapy in the inflammatory response induced by Bothrops jararacussu snake venom (2008) Toxicon, 1 (51), pp. 1236-1244Oshima-Franco, Y., Hyslop, S., Cintra, A.C., Giglio, J.R., da Cruz-Höfling, M.A., Rodrigues-Simioni, L., Neutralizing capacity of commercial bothropic antivenom against Bothrops jararacussu venom and bothropstoxin-I. (2000) Muscle Nerve, 23, pp. 1832-1839Oshima Franco, Y., Rosa, L.J.R., Silva, G.A.A., Amaral Filho, J., Silva, M.G., Lopes, P.S., Cogo, J.C., Da Cruz Höfling, M.A., Antibothropic action of Camellia sinensis extract against the neuromuscular blockade by Bothrops jararacussu snake venom and its mais toxin, bothropstoxin-I (2012) Pharmacology, pp. 469-489. , Croatia: Intech, Gallelli LMilani Júnior, R., Jorge, M.T., de Campos, F.P., Martins, F.P., Bousso, A., Cardoso, J.L., Ribeiro, L.A., Warrell, D.A., Snake bites by the jararacuçu (Bothrops jararacussu): clinic pathological studies of 29 proven cases in São Paulo State (1997) Brazil. QJM, 90, pp. 323-334Petricevich, V.L., Teixeira, C.F.P., Tambourghi, D.V., Gutiérrez, J.M., Increments in serum cytokine and nitric oxide levels in mice injected with Bothrops asper and Bothrops jararaca snake venom (2000) Toxicon, 38, pp. 1253-1266(2007) Rabies and envenomings: a neglected public health issue, , http://www.who.int/bloodproducts/animal_sera/Rabies.pdf, Available from: WHO - World Health OrganizationMalomouzh, A.I., Mukhtarov, M.R., Nikolsky, E.E., Vyskocil, F., Lieberman, E.M., Urazaev, A.K., Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction (2003) J Neurochem, 85, pp. 206-213Rubem Mauro, L., Rocha, D.S., Barcelos, C.C., Varca, G.H., Andréo Filho, N., Barberato Filho, S., Oshima Franco, Y., Phenobarbital pharmacological findings on the nerve-muscle basis (2009) Lat Am J Pharm, 28, pp. 211-218. , Vila MMDCCintra-Francischinelli, M., Silva, M.G., Andréo-Filho, N., Gerenutti, M., Cintra, A.C.O., Giglio, J.R., Leite, G.B., Oshima-Franco, Y., Antibothropic action of Casearia sylvestris Sw. (Flacourtiaceae) extracts (2008) Phytother Res, 22, pp. 784-790Camargo, T.M., Nazato, V.S., Silva, M.G., Cogo, J.C., Groppo, F.C., Oshima-Franco, Y., Bothrops jararacussu venom-induced neuromuscular blockade inhibited by Casearia gossypiosperma Briquet hydroalcoholic extract (2010) J Venom Anim Toxins incl Trop Dis, 16, pp. 432-441Nazato, V.S., Rubem Mauro, L., Vieira, N.A.G., Rocha Junior, D., Dos, S., Silva, M.G., Lopes, P.S., Oshima Franco, Y., vitro antiophidian properties of Dipteryx alata Vogel bark extracts (2010) Molecules, 15, pp. 5956-5970Melo, R.F., Farrapo, N.M., Rocha Junior, D.S., Silva, M.G., Cogo, J.C., Dal Belo, C.A., Rodrigues Simioni, L., Oshima Franco, Y., Antiophidian mechanisms of medicinal plants (2009) Flavonoids: Biosynthesis, Biological Effects and Dietary Sources, pp. 249-262. , New York: Nova Science Publishers, Keller RBFarrapo, N.M., Silva, G.A.A., Costa, K.N., Silva, M.G., Cogo, J.C., Dal Belo, C.A., Dos Santos, M.G., Oshima Franco, Y., Inhibition of Bothrops jararacussu venom activities by Plathymenia reticulata Benth extracts (2011) J Venom Res, 2, pp. 52-58Cos, P., Vlietinc, A.J., Berghe, D.V., Maes, L., Anti-infective potential of natural products: how to develop a stronger in vitro 'proof-of-concept' (2006) J Ethnopharmacol, 103, pp. 290-302Mishra, U.S., Mishra, A., Kumari, R., Murthy, P.N., Naik, B.S., Antibacterial activity of ethanol extract of Andrographis paniculata (2009) Indian J Pharm Sci, 71, pp. 436-438Premendran, S.J., Salwe, K.J., Pathak, S., Brahmane, R., Manimekalai, K., Anti-cobra venom activity of plant Andrographis paniculata and its comparison with polyvalent anti-snake venom (2011) J Nat Sci Biol Med, 2, pp. 198-204Mahadeswaraswamy, Y.H., Nagaraju, S., Girish, K.S., Kemparaju, K., Local tissue destruction and procoagulation properties of Echis carinatus venom: inhibition by Vitis vinifera seed methanol extract (2008) Phytother Res, 22, pp. 963-969Oliveira, D.A., Salvador, A.A., Smânia, A., Smânia, E.F., Maraschin, M., Ferreira, S.R., Antimicrobial activity and composition profile of grape (Vitis vinifera) pomace extracts obtained by supercritical fluids (2013) J Biotechnol, 164, pp. 423-43

Doppia stabilizzazione per il trattamento della lussazione coxo-femorale nel cane: contributo clinico

Viene presentato lo studio preliminare di una tecnica di riduzione e stabilizzazione della lussazione traumatica dell\u2019anca nel cane. L\u2019articolazione \ue8 stata esposta e ridotta mediante un approccio caudale. Utilizzando un nastro sintetico di B\ufchner \ue8 stato sostituito il legamento rotondo intrarticolare e contemporaneamente \ue8 stato ottenuto un rinforzo del tetto acetabolare per aumentare la stabilizzazione articolare anche in direzione ventro-dorsale. I risultati conseguiti, assenza di zoppia e di dolore, nessuna recidiva e un precoce recupero funzionale, incoraggiano l\u2019applicazione di questa tecnica presupponendo che su un numero maggiore di casi reclutati si riduca la possibilit\ue0 di recidiva descritta in bibliografia

A Highly Polar Phytocomplex Involving Rutin Is Responsible For The Neuromuscular Facilitation Caused By Casearia Sylvestris (guaçatonga)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Of the various biological activities ascribed to extracts from Casearia sylvestris (guaçatonga), its facilitatory activity, i.e., ability to increase skeletal muscle contractile amplitude, has promising therapeutic applications. In this work, we investigated the components responsible for the previously described neurofacilitation caused by C. sylvestris leaves. Methods: The methanolic fraction of C. sylvestris leaves was initially fractionated by column chromatography and partitioned in a MeOH:H2O gradient. The resulting fractions were analyzed by analytical HPLC and yielded fraction 5:5 (F55) that was subjected to solid phase extraction and preparative HPLC. Of the seven resulting subfractions, only F55-6 caused muscle facilitation. Subfractions F55-6 and F55-7 (similar in composition to F55-6 by TLC analysis, but inactive) were analyzed by 1H-NMR to identify their constituents. Results: This analysis identified a rutin-glycoside phytocomplex that caused neurofacilitation, a property that commercial rutin alone did not exhibit. Conclusion: F55-6 apparently caused neurofacilitation by the same mechanism (presynaptic action) as the methanolic fraction since its activity was also inhibited in tetrodotoxin-pretreated preparations. © 2016 Bentham Science Publishers.17151360136804/09705-8, FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo07/53883-6, FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo08/11005-5, FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo08/50669-6, FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo12/08271-0, FAPESP, Fundação de Amparo à Pesquisa do Estado de São PauloFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

The facilitatory effect of casearia sylvestris Sw. (guacatonga) fractions on the contractile activity of mammalian and avian neuromuscular apparatus

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOMany natural products influence neurotransmission and are used clinically. In particular, facilitatory agents can enhance neurotransmission and are potentially useful for treating neuromuscular diseases in which muscular weakness is the major symptom. In this work, we investigated the facilitatory effect of apolar to polar fractions of Casearia sylvestris Sw. (guacatonga) on contractility in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) neuromuscular preparations exposed to indirect (via the nerve; 3 V stimuli) and direct (30 V stimuli) muscle stimulation in the absence and presence of pharmacological antagonists. Methanolic and ethyl acetate fractions, but not hexane or dichloromethane fractions, exerted a facilitatory effect on PND (indirect stimulation). The methanolic fraction was chosen for further assays to assess the involvement of: 1) presynaptic sites (axons or nerve terminals), 2) postsynaptic sites (cholinergic receptors, sarcolemma or T-tubules), and 3) the synaptic cleft (acetylcholinesterase enzyme). In preparations treated with d-tubocurarine, the methanolic fraction did not cause facilitation in response to direct stimuli; this fraction was also unable to reverse dantrolene-induced blockade (indirect stimulation). In curarized preparations, the methanolic fraction either restored neuromuscular transmission (mimicking the effect of neostigmine) or failed to cause any recovery of neurotransmission. In the presence of 3,4-diaminopyridine (3,4-DAP), the methanolic fraction decreased twitch amplitude, whereas at a high frequency of stimulation (40 Hz) there was an increase in tetanic tension. In BC preparations, the methanolic fraction did not affect contractures to exogenous acetylcholine or potassium chloride. Incubation with atropine showed there was certain modulation by prejunctional nicotinic receptors, whereas treatment with nifedipine showed that the neurofacilitation required the entry of extracellular calcium. Tetrodotoxin did not prevent the facilitatory effect of 3,4-DAP or neostigmine, but antagonized the response to the methanolic fraction. These findings indicate that neuronal sodium channels have an important role in the facilitatory response to the methanolic fraction, with extracellular calcium entry via calcium channels modulating this neurofacilitation. Possible modulation of prejunctional cholinoceptors was not excluded, particularly in view of certain antagonism by the methanolic fraction at muscarinic receptors. Since facilitation by the methanolic fraction involved enhanced acetylcholine release, use of this fraction could be potentially beneficial in neuromuscular diseases and in the reversal of residual paralysis in the post-operative period or after local anaesthesia.Many natural products influence neurotransmission and are used clinically. In particular, facilitatory agents can enhance neurotransmission and are potentially useful for treating neuromuscular diseases in which muscular weakness is the major symptom. In this work, we investigated the facilitatory effect of apolar to polar fractions of Casearia sylvestris Sw. (guacatonga) on contractility in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) neuromuscular preparations exposed to indirect (via the nerve; 3 V stimuli) and direct (30 V stimuli) muscle stimulation in the absence and presence of pharmacological antagonists. Methanolic and ethyl acetate fractions, but not hexane or dichloromethane fractions, exerted a facilitatory effect on PND (indirect stimulation). The methanolic fraction was chosen for further assays to assess the involvement of: 1) presynaptic sites (axons or nerve terminals), 2) postsynaptic sites (cholinergic receptors, sarcolemma or T-tubules), and 3) the synaptic cleft (acetylcholinesterase enzyme). In preparations treated with d-tubocurarine, the methanolic fraction did not cause facilitation in response to direct stimuli; this fraction was also unable to reverse dantrolene-induced blockade (indirect stimulation). In curarized preparations, the methanolic fraction either restored neuromuscular transmission (mimicking the effect of neostigmine) or failed to cause any recovery of neurotransmission. In the presence of 3,4-diaminopyridine (3,4-DAP), the methanolic fraction decreased twitch amplitude, whereas at a high frequency of stimulation (40 Hz) there was an increase in tetanic tension. In BC preparations, the methanolic fraction did not affect contractures to exogenous acetylcholine or potassium chloride. Incubation with atropine showed there was certain modulation by prejunctional nicotinic receptors, whereas treatment with nifedipine showed that the neurofacilitation required the entry of extracellular calcium. Tetrodotoxin did not prevent the facilitatory effect of 3,4-DAP or neostigmine, but antagonized the response to the methanolic fraction. These findings indicate that neuronal sodium channels have an important role in the facilitatory response to the methanolic fraction, with extracellular calcium entry via calcium channels modulating this neurofacilitation. Possible modulation of prejunctional cholinoceptors was not excluded, particularly in view of certain antagonism by the methanolic fraction at muscarinic receptors. Since facilitation by the methanolic fraction involved enhanced acetylcholine release, use of this fraction could be potentially beneficial in neuromuscular diseases and in the reversal of residual paralysis in the post-operative period or after local anaesthesia165468481FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP [04/09705-8, 07/53883-6, 08/50669-6, 08/52643-4, 08/11005-5, 12/08271-0, 12/20591-0]04/09705-8, 07/53883-6; 08/50669-6; 08/52643-4; 08/11005-5; 12/08271-0; 12/20591-0sem informaçãosem informaçã