BackgroundSnakebite 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).MethodsThe 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.ResultsTLC 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.ConclusionsThe 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.
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. (guaçatonga) 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.
Phenolic compounds from Dipteryx alata Vogel were assayed against the in vitro neurotoxic effect induced by Bothrops jararacussu (Bjssu) venom. Mutagenicity was assessed by the Ames test using Salmonella typhimurium strains TA98, TA97a, TA100, and TA102, in experiments with and without metabolic activation. Anti-bothropic activity was obtained by using mouse phrenic nerve-diaphragm (PND) preparation and myographic technique. Control experiments with physiological Tyrode solution were used for keeping the PND preparations alive (n = 4). Concentrations of phenolic compounds were as follow: protocatechuic and vanillic acids (200 µg/mL, n = 4), vanillin (50 µg/mL, n = 4). These compounds were used alone or pre-incubated with the venom (40 µg/mL), 30 min prior the addition to the organ bath (n = 4). Phenolic compounds significantly inhibited the neuromuscular blockade of Bjssu in the following order of potency: vanillic acid > protocatechuic = vanillin. Vanillic acid added 10 min after the Bjssu venom was also able to avoid the venomblockade evolution. The mutagenicity assay indicated that all phytochemicals were unable to increase the number of revertants, demonstrating the absence of mutagenic activity. This study * Corresponding author. E. H. Yoshida et al.2 demonstrated both the safety and therapeutical potential of the three phenolic compounds as novel complementary anti-bothropic agents.
In this study, we investigated the reproductive capacity of pregnant rats exposed to daily orally administered powder-dehydrated reconstituted of Agaricus brasiliensis (=Agaricus blazei sensu Murrill), the fetal organogenesis, and the development of the pups. Pregnant rats were exposed for the entire gestational period to water (control) and A. brasiliensis at 300 or 600 mg/kg/day. Fertility and body weight of dams were monitored. Pups were monitored for body weight, offspring vitality, morphology, and physical and neurobehavioral development. An increase in sternebrae agenesis was observed at the 600 mg/kg/day dose of A. brasiliensis, while incomplete ossification of sternebrae was seen even at a 300 mg/kg/day dose. In conclusion, this study is the first to demonstrate the impact of maternal exposure to A. brasiliensis on the fetal organogenesis and development of offspring in a rat model. The 600 mg/kg/day dose showed some negative effects, and low toxicity was observed at the 300 mg/kg/day dose.
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.
The ability of Terminalia fagifolia hydroalcoholic extract (Tf-HE) to neutralise the paralysis and myotoxicity induced by Bothrops jararacussu venom was assayed using mouse phrenic nerve-diaphragm (PND) preparation and two varieties of chick biventer cervicis (BC) preparations. Tf-HE 100 μg/mL and 500 μg/mL were tested against 40 and 200 μg of venom/mL in PND and BC preparations, respectively, using pre- and post-venom incubation treatments. The effects of Tf-HE against the myotoxicity caused by venom were evaluated via histological analysis (PND) and creatine kinase (CK) release (BC). Tf-HE was able to reverse the venom paralysis in both preparation types. The contractures to exogenous ACh in BC preparations showed that Tf-HE may act on extrinsic, preserving those intrinsic postsynaptic receptors. There was a positive correlation between CK and morphological changes. The high non-hemolytic saponin content can explain the Tf-HE efficacy against the toxic effects of B. jararacussu venom in vertebrate neuromuscular preparations.
Purpose: A silver nanoparticle obtained by reducing salts with solid dispersion of curcumin (130 nm, 0.081 mg mL −1 ) was used to counteract against the toxic -edematogenic, myotoxic, and neurotoxic -effects of Philodryas olfersii venom. Methods: The edematogenic effect was evaluated by plasma extravasation in rat dorsal skin after injection of 50 µg per site of venom alone or preincubated with 1, 10, and 100 µL of AgNPs; the myotoxicity was evaluated by measuring the creatine kinase released into the organ-bath before the treatment and at the end of each experiment; and neurotoxicity was evaluated in chick biventer cervicis using the conventional myographic technique, face to the exogenous acetylcholine (ACh) and potassium chloride (KCl) added into the bath before the treatment and after each experiment. Preliminarily, a concentration-response curve of AgNPs was carried out to select the concentration to be used for neutralizing assays, which consists of neutralizing the venom-induced neuromuscular paralysis and edema by preincubating AgNPs with venom for 30 min. Results: The P. olfersii venom-induced edema (n=6) and a complete neuromuscular blockade (n=4) that includes the total and unrecovered block of ACh and KCl contractures. AgNPs produced a concentration-dependent decrease the venom-induced edema (n=6) from 223.3% to 134.4% and to 100.5% after 10 and 100 µL AgNPs-preincubation, respectively. The preincubation of venom with AgNPs (1 µL; n=6) was able to maintain 46.5 ± 10.9% of neuromuscular response under indirect stimuli, 39.2 ± 9.7% of extrinsic nicotinic receptors functioning in absence of electrical stimulus and 28.3 ± 8.1% of responsiveness to potassium on the sarcolemmal membrane. The CK release was not affected by any experimental protocol which was like control. Conclusion: AgNPs interact with constituents of P. olfersii venom responsible for the edema-forming activity and neuromuscular blockade, but not on the sarcolemma membraneacting constituents. The protective effect of the studied AgNPs on avian preparation points out to molecular targets as intrinsic and extrinsic nicotinic receptors.
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