Neuromuscular Blocking Action Research Articles

Extraction and phytochemical investigation of Calotropis procera: Effect of plant extracts on the activity of diverse muscles

Context: Calotropis procera (Ait.) R.Br. (Asclepiadaceae) is a shrub or small tree that grows wild in Egypt. Calotropis acts as a purgative, anthelmintic, anticoagulant, palliative (in problems with respiration, blood pressure), antipyretic, and analgesic, and induces neuromuscular blocking activity. Little research has been done to study the electrophysiological effects of this plant’s extracts on cardiac, smooth, and skeletal muscle activities.Objective: The present study was conducted to determine the phytochemical composition and the effect of the total alcohol extract of the shoot of the plant, which contains almost all of C. procera’s cardiac glycosides, flavonoids, and saponins. Also, this study attempted to throw more light on the electrophysiological effects of the plant extracts on cardiac, smooth, and skeletal muscle activities and to clarify the mechanism(s) of their observed action(s).Materials and methods: The aerial parts of the plant were air dried and their ethanol extracts partitioned with successive solvents. Cardiac, smooth, and skeletal muscles were used in this study to investigate the physiological and pharmacological effects of the plant extracts from different solvents. The data were analyzed by paired t-test.Results: The phytochemical investigation of Calotropis procera revealed the presence of cardenolides, flavonoids, and saponins. The effects of ethanol, n-butanol, and ethyl acetate (EtOAc) extracts were each evaluated on isolated toad heart and their mechanisms of action determined. Perfusion with 2 μg/mL ethanol, 0.2 μg/mL butanol, and 0.2 μg/mL EtOAc extracts caused a significant decrease in heart rate (bradycardia), significant increase in the force of ventricular contraction, and increase in T-wave amplitude. In addition, the effects of different extracts of the studied plant on smooth muscle and skeletal muscle were investigated in this study. The different extracts and latex of C. procera induced a negative chronotropic effect and decreased the heart rate (HR) of isolated toad heart. The different extracts increased the power of contraction of the duodenum (trace a). Pretreatment with atropine sulfate as a muscarinic receptor blocker abolished the stimulatory effect of the different plant extracts and latex of C. procera (trace b).Discussion: The present data suggest that ethanol, butanol, and EtOAc extracts of Calotropis procera have negative chronotropism and positive inotropism. Verapamil could abolish the inotropic effect of ethanol as well as that of butanol and EtOAc extracts. Meanwhile, atropine did not abolish the observed negative chronotropic effect. The ethanol extract increased the power of contraction of rabbit duodenum, but atropine abolished this effect. It also decreased the skeletal muscle contraction; this effect could be through blocking of the nicotinic receptors. Butanol and EtOAc extract data for smooth and skeletal muscles are very close to those for the corresponding ethanol extract of the studied plant. The present data for C. procera indicate its direct action on the myocardium, its increase of smooth muscle motility, and its relaxation of skeletal muscle contraction. The chemical constituents could directly affect the cell membrane probably through receptors coupling to G proteins. They regulate the ion channel physiology as in the myocardium.Conclusion: The present data on the extracts of C. procera indicate a direct action on the myocardium, stimulatory effect on smooth muscle motility, and relaxant action on skeletal muscle contraction. Chemical constituents could directly affect the cell membrane probably through receptors coupling to G proteins. They regulate the ion channel physiology as in the myocardium.

Synthesis and neuromuscular blocking activity of 16-(2- and 3-pyridylmethylene) dehydroepiandrosterone derivatives

The interonium distance plays a major role in neuromuscular blocking activity of bis-quaternary ammonium compounds. In this study we tried to alter the distance between two quaternary nitrogens in some of the steroidal derivatives synthesized and evaluated them for neuromuscular blocking activity using in vivo (in chicks) and in vitro models (rectus abdominus and chick biventer cervis muscle) for their mechanism of action. All the synthesized compounds have shown to possess good depolarizing, competitive neuromuscular blocking activity, particularly the 17-acetoxy derivative and the increase in the distance between two quaternary nitrogens decreased the activity.

Effect of pantoprazole and its interactions with vecuronium on the neuromuscular junction

Objective:To study the effect of pantoprazole on neuromuscular transmission and its interactions with vecuronium at the neuromuscular junction (NMJ).Materials and Methods:Effect of pantoprazole on neuromuscular transmission (2 μM – 16 mM) and reversal of neuromuscular blockade by pantoprazole and vecuronium with neostigmine (3.3 μM), 3,4-diaminopyridine (0.25 mM), KCl (6 mM), and CaCl2 (10 mM) were studied by the indirect and direct stimulated preparation of rat phrenic nerve hemidiaphragm. Cumulative reponse curves (CRC) of vecuronium (1 μM to 32 μM) were studied in the absence and presence of 32 μM, 64 μM, and 128 μM pantoprazole. Time for head drop by vecuronium infusion was recorded in the absence and presence of acute and chronic administration of pantoprazole (1.9 mg/kg) in rabbits.Results:Pantoprazole potentiated the basal contractile twitch responses at a lower concentration followed by neuromuscular blockade at a higher concentration. The neuromuscular blockade was not reversed by neostigmine (3.3 μM), 3,4-diaminopyridine (0.25 mM), KCl (6 mM), and CaCl2 (10 mM). Pantoprazole potentiated the vecuronium-induced neuromuscular blockade. It decreased the total time for complete blockade in rat phrenic nerve hemidiaphragm preparation (P < 0.05) and decreased the time for the head drop in rabbits with vecuronium infusion (P < 0.0001).Conclusion:Pantoprazole has a direct neuromuscular blocking action. It has the potential to interact with vecuronium.

Effects of sepsis on the neuromuscular blocking actions of d-tubocurarine on rat adductor and abductor laryngeal muscles

We evaluated the effects of sepsis on the neuromuscular blocking actions of d-tubocurarine (dTc) in the lateral cricoarytenoid (LCA) and posterior cricoarytenoid (PCA) muscles, an adductor muscle and an abductor muscle of the vocal cords, respectively, in vitro. Sepsis was induced in rats by cecal ligation and puncture (CLP) to elicit panperitonitis. Electromyograms (EMGs) and endplate potentials (EPPs) were recorded from the LCA and PCA muscles of CLP-operated septic rats and sham-operated nonseptic rats, using extracellular and intracellular microelectrodes, respectively. EMG and EPP (amplitude and quantum content) were depressed by dTc, but the dTc-induced neuromuscular blocking effects were attenuated by sepsis. The suppressive effects of dTc on EMG and EPP (amplitude and quantum content) were less intense in the LCA muscle than in the PCA muscle under both sepsis and nonsepsis conditions. Our study shows that sepsis has a depressive effect on dTc-induced neuromuscular blocking actions at both the adductor and abductor muscles of vocal cords in the larynx.

Adenosine and adenosine uptake inhibitors potentiate the neuromuscular blocking action of rocuronium mediated by adenosine A1 receptors in isolated rat diaphragms

Adenosine and adenosine uptake inhibitors potentiate the neuromuscular blocking action of rocuronium mediated by adenosine A1 receptors in isolated rat diaphragms

Adenosine and adenosine uptake inhibitors potentiate the neuromuscular blocking action of rocuronium mediated by adenosine A1 receptors in isolated rat diaphragms

Adenosine, which pre-junctionally modulates neuromuscular transmission, and adenosine uptake inhibitors, which increase extracellular adenosine, have been used clinically. We investigated the effects of adenosine, dipyridamole and midazolam on the neuromuscular blocking action of rocuronium. Isometric twitch tensions of rat nerve-hemidiaphragm preparations elicited by indirect (phrenic nerve) supra-maximal stimulation at 0.1 Hz were evaluated (n=6 in all data). Pre-treatments with adenosine (0.1 and 1 microM) and CCPA (1 microM, adenosine A(1) receptor agonist), but not that with CGS21680 (0.5 microM, A(2) receptor agonist), shifted the rocuronium concentration-twitch tension curves to the left and decreased the rocuronium concentration for 50% twitch depression (IC(50)) compared with the control (P<0.01). The leftward shift induced by 1 microM adenosine was inhibited by pre-treatments with theophylline (50 microM, non-selective adenosine receptor antagonist) and DPCPX (0.2 microM, A(1) receptor antagonist) but not by that with DPMA (5 microM, A(2) receptor antagonist). Pre-treatments with dipyridamole and midazolam, adenosine uptake inhibitors, shifted the curve to the left and decreased IC(50) at supra-therapeutic concentrations (10 and 2.5 microM, respectively) but not at clinical concentrations (2 and 0.5 microM, respectively), and the leftward shifts were inhibited by pre-treatment with DPCPX (0.2 microM). The results indicate that adenosine potentiates the neuromuscular blocking action of rocuronium mediated by adenosine A(1) receptors and that supra-therapeutic concentrations of dipyridamole and midazolam also potentiate the action of rocuronium by increasing endogenous adenosine concentration.

Botulinum type A2 neurotoxin (150 kDa) is stronger than type A1 progenitor toxin (900 kDa) in the inhibition activity of neuromuscular transmission of isolated mouse phrenic nerve-hemidiaphragm

Introduction : The botulinum neurotoxin type A complex is the drug of choice for neurological disorders in the world. Botulinum toxin has the ability to inhibit muscle activity by blocking the release of acetylcholine at neuromuscular junctions. In this study, we evaluated type A2 150 kDa neurotoxin (A2NTX) and type A1 progenitor toxin (A1LL) by the twitch tension assay with isolated mouse phrenic nerve–hemidiaphragm. Methods : The twitch tension of isolated phrenic nerve–hemidiaphragm preparations induced by electrical stimulations of the phrenic nerve at pH 7.4 was recorded before and after the preparations were exposed to A2NTX (Kaketsuken) and A1LL, respectively. In addition, assuming the possible muscle pH conditions of patients on muscle contraction, these twitch tension responses were compared under an acidic condition of pH 6.8 using lactic acid. Results and discussion : In the electrical stimulation of the phrenic nerve, each botulinum toxin preparation caused attenuation of the twitch tension of the diaphragm ex vivo. In comparing the linear range of the dose–response relationship between NTX and A1LL, it was revealed that the neuromuscular blocking activity of A2NTX was approximately 2 times more potent at pH 7.4, and was 4 times more potent at pH 6.8. These results suggested that NTX shows marked potency as a pharmacological agent compared with the A1 type progenitor toxin.

Lack in Effects of Therapeutic Concentrations of Dexmedetomidine and Clonidine on the Neuromuscular Blocking Action of Rocuronium in Isolated Rat Diaphragms

We investigated the effects of the alpha-2 adrenoceptor agonists clonidine and dexmedetomidine on the neuromuscular blocking effect of rocuronium in vitro. Isometric twitch tensions of rat nerve-hemidiaphragm preparations elicited by indirect (phrenic nerve) supramaximal stimulation at 0.1 Hz were evaluated. Clonidine and dexmedetomidine 50 microM (n = 6 each), but not 0.05 microM, shifted the rocuronium concentration-twitch tension curves to the left and decreased the rocuronium concentration for 50% twitch depression (IC50) compared with control (n = 9, P < 0.01). The leftward shift induced by clonidine 50 microM or dexmedetomidine 50 microM was not antagonized by yohimbine 50 microM, an alpha-2 adrenoceptor antagonist. Twitch tensions partially depressed by 7 microM rocuronium, to about 65% of the control, were further suppressed in a concentration-dependent manner by clonidine (n = 6) and dexmedetomidine (n = 9) at concentrations of 30 microM or more (P < 0.01). These results indicate that very high, but not therapeutic, concentrations of clonidine and dexmedetomidine enhance the neuromuscular blocking action of rocuronium, but this is not mediated by an agonist action on alpha-2 adrenoceptors.

Safety evaluation of a standardized phytochemical composition extracted from Bacopa monnieri in Sprague–Dawley rats

BacoMind™ is an enriched phytochemical composition derived from Bacopa monnieri, a common medicinal plant having multiple uses in the traditional system of medicine and particularly used as a memory enhancing agent for centuries. The plant and its extracts have been evaluated for anti-inflammatory, cardio tonic, sedative and neuro-muscular blocking activities. In view of the extensive use of this plant, BacoMind™, standardized to bioactive compounds was evaluated in a series of toxicity studies, to confirm the safety of its usage. BacoMind™, on single oral administration had a median lethal dose of 2400 mg/kg in Sprague–Dawley rats. In a 14 day repeated dose oral toxicity study in rats, except for mild lowering in body weight gain in male rats, it was found to be tolerated well up to the dose of 500 mg/kg. A subchronic oral toxicity study for 90 days in rats at the dose levels of 85, 210 and 500 mg/kg did not reveal any evidence of toxicity with respect to clinical signs, neurological examination, food consumption, body weight gain, haematological and blood biochemistry parameters. The absolute and relative organ weight of vital organs did not differ significantly from that of the control. Necropsy and histopathological examination, did not reveal any remarkable and treatment related changes. A no-observed adverse effect level of 500 mg/kg body weight was established in rats.

Neutralization of snake venom phospholipase A 2 toxins by aqueous extract of Casearia sylvestris (Flacourtiaceae) in mouse neuromuscular preparation

Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A 2 (PLA 2) toxins. In this work we evaluated the influence of C. sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA 2s (crotoxin from C. durissus terrificus, bothropstoxin-I from B. jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 μM) and all other PLA 2 toxins (1.0 μM) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA 2 toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 °C) significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80–95%). Higher concentration of the C. sylvestris extract (1:10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA 2 inhibitors.

Biological and Structural Characterization of Crotoxin and New Isoform of Crotoxin B PLA2 (F6a) from Crotalus durissus collilineatus Snake Venom

A new crotoxin B isoform PLA2 (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using μ-Bondapack C-18 column analytic. The new crotoxin B isoform PLA2 (F6a), complex crotoxin, the catalytic subunit crotoxin B isoform PLA2 (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus. The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit of isoforms of PLA2 (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: crotoxin B: 14,943.14 Da, crotapotin F3: 8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new crotoxin B isoform PLA2 (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other PLA2s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region with other myotoxic PLA2s. The new crotoxin B isoform PLA2 (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced by crotoxin and new crotoxin B isoform PLA2 (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole crotoxin was devoid of cytolytic activity upon myoblasts and myotubes in vitro, whereas new crotoxin B isoform PLA2 (F6a) was clearly cytotoxic to these cells.

Bacopa monniera

The plant is used in India as well as several countries since several centuries for treating different types of ailments. The plant is an important constituent of the Ayurvedic Materia Medica and finds mention in several ancient texts including Caraka Sanhita (<TEX>$6^{th}$</TEX> century A.D.) and the Bhavprakasa (<TEX>$16^{th}$</TEX> century A.D.). The scientific studies on this plant have reported several activities of this plant. Though the plant has cardiotonic, vasoconstrictor, sedative, neuro-muscular blocking, and anticancer activities, it is more popular as memory enhancer. Traditionally, a poultice made of the boiled plant is placed on the chest in acute bronchitis and coughs of children. The plant contains saponins: bacosides A and B, hersaponin, sapogenins: bacogenin <TEX>$A_{1}$</TEX>, <TEX>$A_{2}$</TEX>, and <TEX>$A_{3}$</TEX> stigmasterol, and flavonoids: luteolin and luteolin-7 glucoside, nicotine, brahmine, and herpestine. This review focuses on the scientific data published since 1931.

An Initial Assessment of the Systemic Pharmacokinetics of Botulinum Toxin

Botulinum toxin is an extraordinarily potent molecule that has an unusually long duration of action. Despite this, there is little information available on natural mechanisms for metabolism or elimination and virtually no information on pharmacologically induced mechanisms for metabolism and elimination. Therefore, a number of experiments were performed on laboratory animals that addressed two major issues: 1) the effect of blood on the structure, function, and biologic half-life of the toxin, and 2) the effect of neutralizing antibodies on half-life and elimination of circulating toxin. In the first series of studies, the metabolic transformation of toxin was assessed by incubating it in blood for varying lengths of time. At each time point, aliquots were examined to determine the amount of toxin, the structure of toxin, the catalytic activity of toxin, and the neuromuscular blocking activity of toxin. This work demonstrated that blood did not alter any characteristic of the toxin molecule. Experiments were also done in which toxin was administered to mice and rats at doses that produced clinical poisoning. The results demonstrated that the elimination half-life for native (nonmetabolized) toxin in blood and serum was 230 to 260 min. During the second series of studies, the rate of elimination of circulating toxin was studied in the presence of antibodies directed against the carboxyl-terminal half of the toxin molecule. This work demonstrated that neutralizing antibodies 1) enhanced clearance of toxin from the circulation and 2) enhanced tissue accumulation of toxin, particularly in liver and spleen.

Influência da nifedipina no bloqueio neuromuscular produzido pelo atracúrio e pelo cisatracúrio: estudo em preparações nervo frênico-diafragma de rato

Calcium channel blockers may interact with neuromuscular blockers, increasing its effects. Research studies about this interaction display controversial results. In some studies these drugs produced neuromuscular blockage, or contracture, or no effect at all was proved over skeletal neuromuscular response. This study assessed the nifedipine effects over muscular responses and its possible interaction with neuromuscular blockers in rat diaphragm. A number of 25 rats were used, weighing between 250 and 300 g and sacrificed under anesthesia with intraperitoneal pentobarbital (40 mg.kg-1). Preparation was mounted according to the technique described by Bulbring. Diaphragm was kept under tension, connected to an isometric transducer and subjected to an indirect stimulation of 0.1 Hz frequency. Diaphragm contractions were registered on a physiograph. In order to evaluate the effect of these drugs on neuromuscular transmission, they were added separately or associated to the preparation, on the following concentrations: nifedipine (4 microg.mL-1); atracurium (20 microg.mL-1); cistracurium (3 microg.mL-1). On phrenic-nerve preparation, the assessed items were: 1) the extent of diaphragm muscle response to indirect stimulation, before and 45 minutes after adding nifedipine and neuromuscular blockers separately and after the association of both drugs; 2) nifedipine effects on membrane potentials (MP) and miniature end-plate potentials (MEPP). Employed separately, nifedipine did not alter the extent of muscular responses, but it did significantly increase the neuromuscular blocking activity of atracurium and cistracurium. Nifedipine did not alter the membrane potential and caused an initial increase on MEPP frequencies, followed by a blockage. Nifedipine, on the employed concentration, increased the neuromuscular blockage produced by atracurium and cistracurium. Electrophysiological studies demonstrate the existence of presynaptic action and absence of depolarizing action over the muscle fiber.

6H,13H-Pyrazino[1,2-a;4,5-a′]diindole analogs: Probing the pharmacophore for allosteric ligands of muscarinic M2 receptors

A series of 6H,13H-pyrazino[1,2-a;4,5-a′]diindole analogs was synthesized in order to probe the pharmacophore hypothesis for allosteric ligands of muscarinic M2 receptors. The 3D structure of the novel ring system was determined by means of NMR spectroscopy and X-ray diffraction revealing a totally flat geometry. Low binding affinities for the [3H]N-methylscopolamine-occupied M2 receptors (reflected by EC50,diss) indicated that the spatial arrangement of the pharmacophore elements (two aromatic rings flanked by two cationic centers) incorporated in the bisquaternary analogs 5 and 6 is unfavorable for strong ligand–receptor interactions. Due to the structural similarity of the novel compounds to neuromuscular-blocking agents, their affinities (reflected by Ki) to the muscle type of nicotinic acetylcholine receptors were also determined. The dimethyl and diallyl analogs 5 and 6 exhibited rather high affinities to the muscle type of nicotinic acetylcholine receptors, suggesting a pronounced neuromuscular-blocking activity. Compound 5 showed a 34-fold higher affinity for the muscle type nAChR than for the allosteric site of M2 receptors.

Arthur A. Demarest, The Petexbatun Regional Archaeological Project. A Multidisciplinary Study of the Maya Collapse. (Nashville: Vanderbilt University Press (Vanderbilt Institute of Mesoamerican Archaeology Series, Volume 1), 2006, 211 pp., 56 figs, 3 colour plates, hbk, ISBN 0 8265 1443 X)

Seventy-five ASA Grades I-III patients (18-85 years, 45-90 kg) were randomized into five groups. All patients received N2O/O2 (2/1) and alfentanil: loading dose (LD) 0.015 mg kg-1 and maintenance dose (MD) 0.045 mg kg-1 h-1 (groups 1-4). Group 1 received propofol (LD 2 mg kg-1 and MD 6 mg kg-1 h-1); Group 2 etomidate (LD 0.3 mg kg-1 and MD 0.6 mg kg-1 h-1); Group 3 midazolam (LD 0.2 mg kg-1 and MD 0.120 mg kg-1 h-1); Group 4 methohexitone (LD 1.5 mg kg-1 and MD 4 mg kg-1 h-1); Group 5 dehydrobenzperidol 0.05-0.23 mg kg-1 and alfentanil (LD 0.100 mg kg-1 and MD 0.060 mg kg-1 h-1). The neuromuscular block induced by pipecuronium (50 micrograms kg-1) was evaluated. No statistically significant differences were found between the five groups as concerned degree of block (expressed as % twitch amplitude in response to the first of the TOF stimuli (Ta1) at intubation, T1 minimum and recovery to Ta1 = 20%, 25% and 75%. Slightly faster intubation was possible when midazolam was used in comparison with propofol, methohexitone or NLA and when etomidate was used in comparison with propofol. A wide range of individual values of maximal neuromuscular blocking activity was found.

Synthesis and Neuromuscular Blocking Activity of 16β-Piperidino Epiandrosterone Derivatives

This study reports the synthesis of steroidal quaternary ammonium compounds 11 and 12, with quaternary nitrogen at position 3 and 16 of the steroid nucleus in 5α-epiandrosterone series; along with their neuromuscular blocking activity using chick biventer cervicis muscle preparation. The compound 12 was found to be five times more potent than 11 in reducing twitch response to nerve stimulations, indicating the importance of extended interonium distances and 17-acetoxy function for potent antagonist activity.

Ethyl acetate extract from black tea prevents neuromuscular blockade by botulinum neurotoxin type A in vitro

Botulinum neurotoxin produced by Clostridium botulinum is the strongest neurotoxin and causes botulism in mammals. The current study aimed to find an inactivator for botulinum neurotoxin in black, oolong, roasted, and green teas. The ability of the four teas to inactivate the neuromuscular blocking action of botulinum neurotoxin was determined. Water extracts from black, oolong, and roasted teas protected against the toxicity of botulinum neurotoxin type A in mouse phrenic nerve–diaphragm preparations. The order of potency of the water extracts was black tea > oolong tea > roasted tea > green tea (no effect). The effects of several organic solvent extracts of black tea water extract were examined, and the order of potency was ethyl acetate extract > butanol extract = remaining extract > chloroform extract (no effect). Ethyl acetate extracts from oolong, roasted, and green tea water extracts also exhibited a stronger protecting effect than chloroform, butanol, and remaining extracts from these teas, but they had weaker protective effect than ethyl acetate extract from black tea water extract. These protective effects occurred only when each extract was pre-mixed with the toxin before the assay, and they were not modified by mixing each extract with bovine serum albumin (BSA) before adding the toxin. These results indicate that ethyl acetate extract from black tea is the best source for searching for tea-derived inactivating substance(s) of botulinum neurotoxin.

Central nervous system effects of the crude extract of Erythrina velutina on rodents

The aqueous extract (AE) of Erythrina velutina prolonged the sleep duration induced by sodium pentobarbital (control: 6.4 ± 1.2 min; extract 10 mg/kg, 47.1 ± 3.9 min; extract 100 mg/kg, 109.4 ± 7.2 min; F = 243, P < 0.001). In the open field, the extract at the doses of 10 and 50 mg/kg did not changed the number of crossings, rearings nor groomings. On the other hand, at the dose of 200 mg/kg it reduced the number of crossings ( q = 6.25, P < 0.05) and groomings ( q = 3.91, P < 0.05). When exposed during three consecutive days to the open field, the control animals showed habituation for crossings ( F = 17.56, P < 0.001) and rearings ( F = 14.01, P < 0.001). The same was not true for animals treated with 10 mg/kg of the extract (crossings: F = 3.59, P > 0.05; rearings: F = 3.62, P > 0.05). At the same dose, the extract blocked the acquisition of foot shock memory ( P = 0.9219) when compared to the control values ( P = 0.0078). Our data showed that the crude extract of Erythrina velutina at lower doses interferes with mnemonic process for different tasks, while at higher doses, the sedative and neuromuscular blocking actions are the main effects.

Molecular evolution and structure-function relationships of crotoxin-like and asparagine-6-containing phospholipases A2 in pit viper venoms.

Some myotoxic or neurotoxic PLA2s (phospholipases A2) from pit viper venoms contain characteristic N6 substitutions. Our survey of the venoms of more than ten pit viper genera revealed that N6-PLA2s exist only in limited Asian pit vipers of two genera, Protobothrops and Gloydius, and exist as either monomers or the basic subunits of heterodimers in some New World pit vipers. For the newly identified N6-PLA2s, the neuromuscular blocking activities were assayed with the chick biventer cervicis neuromuscular tissue, whereas the increased serum creatine kinase level assessed their myotoxicities. The purified N6-PLA2s from Protobothrops mangshanensis and Gloydius intermedius saxatilis were found to be presynaptic neurotoxins. In contrast, all N6-PLA2s from the venoms of Sistrurus miliarius strackeri, S. m. barbouri, Crotalus viridis viridis, C. lepidus lepidus, Cerrophidion godmani and Bothreichis schlegelii were myotoxins without neurotoxicity even in the presence of crotoxin A. Crotoxin-like complexes were for the first time purified from the venoms of Sitrurus catenatus tergeminus, C. mitchelli mitchelli, C. horridus atricaudatus, C. basiliscus and C. durissus cumanensis. The cDNAs encoding six novel N6-PLA2s and subunits of the crotoxin-like complex from S. c. tergeminus were cloned and fully sequenced. Phylogeny analysis showed that two structural subtypes of N6-PLA2s with either F24 or S24 substitution have been evolved in parallel, possibly descended respectively from species related to present-day Protobothrops and Gloydius. Calmodulin binds all the N6-PLA2s but crotoxin A may inhibit its binding to crotoxin B and to other neurotoxic N6-PLA2s. Structure-activity relationships at various regions of the PLA2 molecules were extensively discussed.