In vitro biological screening of the anticholinesterase and antiproliferative activities of medicinal plants belonging to Annonaceae

The aim of this research was to investigate the antiproliferative and anticholinesterase activities of 11 extracts from 5 Annonaceae species in vitro. Antiproliferative activity was assessed using 10 human cancer cell lines. Thin-layer chromatography and a microplate assay were used to screen the extracts for acetylcholinesterase (AchE) inhibitors using Ellman's reagent. The chemical compositions of the active extracts were investigated using high performance liquid chromatography. Eleven extracts obtained from five Annonaceae plant species were active and were particularly effective against the UA251, NCI-470 lung, HT-29, NCI/ADR, and K-562 cell lines with growth inhibition (GI50) values of 0.04-0.06, 0.02-0.50, 0.01-0.12, 0.10-0.27, and 0.02-0.04 µg/mL, respectively. In addition, the Annona crassiflora and A. coriacea seed extracts were the most active among the tested extracts and the most effective against the tumor cell lines, with GI50 values below 8.90 µg/mL. The A. cacans extract displayed the lowest activity. Based on the microplate assay, the percent AchE inhibition of the extracts ranged from 12 to 52%, and the A. coriacea seed extract resulted in the greatest inhibition (52%). Caffeic acid, sinapic acid, and rutin were present at higher concentrations in the A. crassiflora seed samples. The A. coriacea seeds contained ferulic and sinapic acid. Overall, the results indicated that A. crassiflora and A. coriacea extracts have antiproliferative and anticholinesterase properties, which opens up new possibilities for alternative pharmacotherapy drugs.

Neostigmine in the treatment of snake accidents caused by Micrurus frontalis: report of two cases

Antivenom in order to be effective in the treatment of coral snake accidents must be injected very soon after the bite owing to the rapid rate of absorption of the venom neurotoxins. As this is not always possible, other forms of treatment besides serotherapy must be employed to avoid asphyxia and death. Neostigmine and artificial respiration are used for this purpose. Neostigmine restores neuromuscular transmission if the venom-induced blockade results from a reversible interaction of its neurotoxins with the end-plate receptors. This is the mechanism of the neuromuscular blockade produced by the venom of M. frontalis snakes from centereastern and southern Brazil, and Argentine. Neostigmine is able, therefore, to antagonize the blockade, and has been shown to be very effective in the treatment of the experimental envenomation of dogs and monkeys. In the present communication, two cases of M. frontalis accidents treated with antivenom and neostigmine are reported. In both, neostigmine was successful in producing regression of the paralysis, confirming the effectiveness shown in the treatment of the poisoning induced in animals by M. frontalis venom.

Bites by coral snakes (Micrurus spp.) in Campinas, State of São Paulo, Southeastern Brazil

Coral snakes (Micrurus spp.) are the main representatives of the Elapidae in South America. However, bites by these snakes are uncommon. We retrospectively reviewed the data from 11 individuals bitten by coral snakes over a 20-year period; four were confirmed (snake brought for identification) and seven were highly suspected (neuromuscular manifestations) cases of elapid envenoming. The cases were classified as dry-bite (n = 1, caused by M. lemniscatus; did not receive antivenom), mild (n = 2, local manifestations with no acute myasthenic syndrome; M. frontalis and Micrurus spp.), moderate (n = 5, mild myasthenia) or severe (n = 3, important myasthenia; one of them caused by M. frontalis). The main clinical features upon admission were paresthesia (local, n = 9; generalized, n = 2), local pain (n = 8), palpebral ptosis (n = 8), weakness (n = 4) and inability to stand up (n = 3). No patient developed respiratory failure. Antivenom was used in ten cases, with mild early reactions occurring in three. An anticholinesterase drug was administered in the three severe cases, with a good response in two. No deaths were observed. Despite the high toxicity of coral snake venoms, the prognosis following envenoming is good. In serious bites by M. frontalis or M. lemniscatus, the venom of which acts postsynaptically, anticholinesterases may be useful as an ancillary measure if antivenom is unavailable, if there is a delay in obtaining a sufficient amount, or in those patients given the highest recommended doses of antivenom without improvement of the paralysis or with delayed recovery.

Reduction of QTc interval dispersion. Potential mechanism of cardiac protection of pyridostigmine bromide

OBJECTIVE: Parasympathetic dysfunction is an independent risk factor in individuals with coronary artery disease, and cholinergic stimulation is a potential therapeutical option. We determined the effects of pyridostigmine bromide, a reversible anticholinesterase agent, on electrocardiographic variables of healthy individuals. METHODS: We carried out a cross-sectional, double blind, randomized, placebo-controlled study. We obtained electrocardiographic tracings in 12 simultaneous leads of 10 healthy young individuals at rest before and after oral administration of 45 mg of pyridostigmine or placebo. RESULTS: Pyridostigmine increased RR intervals (before: 886±27 ms vs after: 1054±37 ms) and decreased QTc dispersion (before: 72±9ms vs after: 45±3ms), without changing other electrocardiographic variables (PR segment, QT interval, QTc, and QT dispersion). CONCLUSION: Bradycardia and the reduction in QTc dispersion induced by pyridostigmine may effectively represent a protective mechanism if these results can be reproduced in individuals with cardiovascular diseases.

Atividade anticolinesterásica e perfil químico de uma fração cromatográfica ativa do extrato etanólico das flores Bellis perennis L. (Asteraceae)

This work describes the isolation of an active flavonoid fraction and identification of isorhamnetin 3-O-β-D-(6''-acetyl)-galactopyranoside from flowers of B. perennis, and also the evaluation of anticholinesterase (AChE) activity of ethanolic extract from flowers (EEF) and the active fraction. The chemical structure of the flavonoid was defined on the basis of spectroscopic ¹H NMR, IR and UV data. EEF or flavonoid reduces AChE activity in vivo, while flavonoid also reduces AChE activity in vitro, showing a value of 1.49 mM for 50% inhibitory concentration (IC50), suggesting potential use as an insecticide or in the treatment of neurodegenerative diseases such as Alzheimer's disease.

Antinociceptive effect of intrathecal neostigmine evaluated in rats by two different pain models

The analgesic efficacy of cholinergic agonists and anticholinesterase agents has been widely recognized. The analgesic effect obtained by activating cholinergic mechanisms, however, seems to depend on the experimental pain model utilized for its evaluation. The antinociceptive effect of intraspinal neostigmine was examined in rats submitted concurrently to the tail flick and formalin tests. Neostigmine (8.25 and 16.5 nmol) produced a dose-dependent antinociceptive effect in the tail flick test (a model of phasic pain) and reduced the first phase (phasic pain) of the animal response to formalin also in a dose-dependent manner. The second phase (tonic pain) of the response to formalin, however, was slightly reduced after a longer period of time only by the higher dose of the anticholinesterase. The effect of neostigmine was not significantly different when the drug was injected into rats submitted exclusively to the tail flick test. The second phase of the animal response to formalin was slightly reduced by neostigmine (8.25 nmol) and strongly inhibited by the higher dose of the anticholinesterase when injection was made after the first phase. We conclude that phasic and tonic pain can both be controlled by high doses of neostigmine. In addition, we show that inhibition by a lower dose of neostigmine of the formalin-induced phasic pain did not prevent the subsequent occurrence of tonic pain produced by the irritant

Cholinergic stimulation with pyridostigmine reduces the QTc interval in coronary artery disease

Parasympathetic dysfunction is an independent risk factor in patients with coronary artery disease; thus, cholinergic stimulation is a potential therapeutic measure that may be protective by acting on ventricular repolarization. The purpose of the present study was to determine the effects of pyridostigmine bromide (PYR), a reversible anticholinesterase agent, on the electrocardiographic variables, particularly QTc interval, in patients with stable coronary artery disease. In a randomized double-blind crossover placebo-controlled study, simultaneous 12-lead electrocardiographic tracings were obtained at rest from 10 patients with exercise-induced myocardial ischemia before and 2 h after the oral administration of 45 mg PYR or placebo. PYR increased the RR intervals (pre: 921 ± 27 ms vs post: 1127 ± 37 ms; P<0.01) and, in contrast with placebo, decreased the QTc interval (pre: 401 ± 3 ms vs post: 382 ± 3 ms; P<0.01). No other electrocardiographic variables were modified (PR segment, QT interval, QT and QTc dispersions). Cholinergic stimulation with PYR caused bradycardia and reduced the QTc interval without important side effects in patients with coronary disease. These effects, if confirmed in studies over longer periods of administration, may suggest a cardioprotection by cholinergic stimulation with PYR.