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.

Shogaol-huprine hybrids: Dual antioxidant and anticholinesterase agents with beta-amyloid and tau anti-aggregating properties

Multitarget compounds are increasingly being pursued for the effective treatment of complex diseases. Herein, we describe the design and synthesis of a novel class of shogaolhuprine hybrids, purported to hit several key targets involved in Alzheimer"s disease. The hybrids have been tested in vitro for their inhibitory activity against human acetylcholinesterase and butyrylcholinesterase and antioxidant activity (ABTS.+, DPPH and Folin-Ciocalteu assays), and in intact Escherichia coli cells for their Aβ42 and tau anti-aggregating activity. Also, their brain penetration has been assessed (PAMPA-BBB assay). Even though the hybrids are not as potent AChE inhibitors or antioxidant agents as the parent huprine Y and [4]-shogaol, respectively, they still exhibit very potent anticholinesterase and antioxidant activities and are much more potent Aβ42 and tau anti-aggregating agents than the parent compounds. Overall, the shogaolhuprine hybrids emerge as interesting brain permeable multitarget anti-Alzheimer leads.

Shogaol-huprine hybrids: Dual antioxidant and anticholinesterase agents with beta-amyloid and tau anti-aggregating properties

Multitarget compounds are increasingly being pursued for the effective treatment of complex diseases. Herein, we describe the design and synthesis of a novel class of shogaolhuprine hybrids, purported to hit several key targets involved in Alzheimer"s disease. The hybrids have been tested in vitro for their inhibitory activity against human acetylcholinesterase and butyrylcholinesterase and antioxidant activity (ABTS.+, DPPH and Folin-Ciocalteu assays), and in intact Escherichia coli cells for their Aβ42 and tau anti-aggregating activity. Also, their brain penetration has been assessed (PAMPA-BBB assay). Even though the hybrids are not as potent AChE inhibitors or antioxidant agents as the parent huprine Y and [4]-shogaol, respectively, they still exhibit very potent anticholinesterase and antioxidant activities and are much more potent Aβ42 and tau anti-aggregating agents than the parent compounds. Overall, the shogaolhuprine hybrids emerge as interesting brain permeable multitarget anti-Alzheimer leads.

Shogaol-huprine hybrids: Dual antioxidant and anticholinesterase agents with beta-amyloid and tau anti-aggregating properties

Multitarget compounds are increasingly being pursued for the effective treatment of complex diseases. Herein, we describe the design and synthesis of a novel class of shogaolhuprine hybrids, purported to hit several key targets involved in Alzheimer"s disease. The hybrids have been tested in vitro for their inhibitory activity against human acetylcholinesterase and butyrylcholinesterase and antioxidant activity (ABTS.+, DPPH and Folin-Ciocalteu assays), and in intact Escherichia coli cells for their Aβ42 and tau anti-aggregating activity. Also, their brain penetration has been assessed (PAMPA-BBB assay). Even though the hybrids are not as potent AChE inhibitors or antioxidant agents as the parent huprine Y and [4]-shogaol, respectively, they still exhibit very potent anticholinesterase and antioxidant activities and are much more potent Aβ42 and tau anti-aggregating agents than the parent compounds. Overall, the shogaolhuprine hybrids emerge as interesting brain permeable multitarget anti-Alzheimer leads.

Shogaol-huprine hybrids: Dual antioxidant and anticholinesterase agents with beta-amyloid and tau anti-aggregating properties

Multitarget compounds are increasingly being pursued for the effective treatment of complex diseases. Herein, we describe the design and synthesis of a novel class of shogaolhuprine hybrids, purported to hit several key targets involved in Alzheimer"s disease. The hybrids have been tested in vitro for their inhibitory activity against human acetylcholinesterase and butyrylcholinesterase and antioxidant activity (ABTS.+, DPPH and Folin-Ciocalteu assays), and in intact Escherichia coli cells for their Aβ42 and tau anti-aggregating activity. Also, their brain penetration has been assessed (PAMPA-BBB assay). Even though the hybrids are not as potent AChE inhibitors or antioxidant agents as the parent huprine Y and [4]-shogaol, respectively, they still exhibit very potent anticholinesterase and antioxidant activities and are much more potent Aβ42 and tau anti-aggregating agents than the parent compounds. Overall, the shogaolhuprine hybrids emerge as interesting brain permeable multitarget anti-Alzheimer leads.

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.

Anticholinesterase-induced epileptiform activity in immature rat piriform cortex slices, in vitro

Purpose: Acute in vitro brain slice models are commonly used to study epileptiform seizure generation and to test anti-epileptic drug action. Seizure-like activity can be readily induced by manipulating external ionic concentrations or by adding convulsant agents to the bathing medium. We previously showed that epileptiform bursting was induced in slices of immature (P14–28) rat piriform cortex (PC) by applying oxotremorine-M, a potent muscarinic receptor agonist. Here, we examined whether raising levels of endogenous acetylcholine (ACh) by exposure to anticholinesterases, could also induce epileptiform events in immature (P12–14) or early postnatal (P7–9) rat PC brain slices. Methods: The effects of anticholinesterases were investigated in rat PC neurons using both extracellular MEA (P7–9 slices) and intracellular (P12–14 slices) recording methods. Results: In P7–9 slices, eserine (20 μM) or neostigmine (20 μM) induced low amplitude, low frequency bursting activity in all three PC cell layers (I–III), particularly layer III, where neuronal muscarinic responsiveness is known to predominate. In P12–14 neurons, neostigmine produced a slow depolarization together with an increase in input resistance and evoked cell firing. Depolarizing postsynaptic potentials evoked by intrinsic fibre stimulation were selectively depressed although spontaneous bursting was not observed. Neostigmine effects were blocked by atropine (1 μM), confirming their muscarinic nature. We conclude that elevation of endogenous ACh by anticholinesterases can induce bursting in early postnatal PC brain slices, further highlighting the epileptogenic capacity of this brain region. However, this tendency declines with further development, possibly as local inhibitory circuit mechanisms become more dominant.

A comparative study on the relationship between acetyl cholinesterase activity and acute toxicity in Daphnia magna exposed to anticholinesterase insecticides

Acetylcholinesterase (AChE) activity was measured in Daphnia magna that had been exposed to four organophosphates (OPs; parathion, chlorpyrifos, malathion, and acephate) and one carbamate (propoxur) for 48 h. These results were related to acute toxicity (median effective concentration [EC50] for immobility). For the four OPs, the EC50s were 7.03 pM, 3.17 pM, 10.56 pM, and 309.82 muM, respectively. The EC50 for propoxur was 449.90 pM. Reduction in AChE activity was directly related to an increase in immobility in all chemicals tested. However, the ratio between the EC50 and the AChE median inhibiting concentration ranged from 0.31 to 0.90. A 50% reduction in AChE activity generally was associated with detrimental effects on mobility. However, for acephate, high levels of AChE inhibition (70%) were observed in very low concentrations and were not associated with immobility. In addition, increasing the concentration of acephate further had a slight negative effect oil AChE activity but a Strong detrimental effect on mobility. Binding sites other than AChE possibly are involved in acephate toxicity to D. magna. Our findings demonstrate different associations between AChE inhibition and toxicity when different chemicals are compared. Therefore, the value of using AChE activity as a biomarker in D. magna will be dependent on the chemical tested.

A comparative study on the relationship between acetylcholinesterase activity and acute toxicity in Daphnia magna exposed to anticholinesterase insecticides

Acetylcholinesterase (AChE) activity was measured in Daphnia magna that had been exposed to four organophosphates (OPs; parathion, chlorpyrifos, malathion, and acephate) and one carbamate (propoxur) for 48 h. These results were related to acute toxicity (median effective concentration [EC50] for immobility). For the four OPs, the EC50s were 7.03 pM, 3.17 pM, 10.56 pM, and 309.82 microM, respectively. The EC50 for propoxur was 449.90 pM. Reduction in AChE activity was directly related to an increase in immobility in all chemicals tested. However, the ratio between the EC50 and the AChE median inhibiting concentration ranged from 0.31 to 0.90. A 50% reduction in AChE activity generally was associated with detrimental effects on mobility. However, for acephate, high levels of AChE inhibition (70%) were observed in very low concentrations and were not associated with immobility. In addition, increasing the concentration of acephate further had a slight negative effect on AChE activity but a strong detrimental effect on mobility. Binding sites other than AChE possibly are involved in acephate toxicity to D. magna. Our findings demonstrate different associations between AChE inhibition and toxicity when different chemicals are compared. Therefore, the value of using AChE activity as a biomarker in D. magna will be dependent on the chemical tested.

Anticholinesterase activity evaluation of alkaloids and coumarin from stems of Conchocarpus fontanesianus

Conchocarpus fontanesianus (A. St.-Hill.) Kallunki & Pirani, Rutaceae, popularly known as pitaguará, is a native and endemic tree from São Paulo and Rio de Janeiro States, Brazil. Based in the information that anticholinesterasic derivatives could act as new prototypes to treatment of Alzheimer disease, this work describes the fractionation guided by evaluation of the anticholinesterase activity of the ethanolic stems extract from C. fontanesianus. This procedure afforded the alkaloids dictamnine (1), γ-fagarine (2), skimianine (3), and 2-phenyl-1-methyl-4-quinolone (4), as well as the coumarin marmesin (5).

Mechanisms of anticholinesterase-induced myopathy and its prevention

In the introduction a brief outline of the possible mechanisms involved in the process of cellular necrosis with particular emphasis on skeletal muscle necrosis after antiChE is discussed. Ecothiopate (ECO), an antiChE, was shown to produce dose-dependent inhibition of both AChE and BuChE in diaphragm and blood of mice. Inhibition of AChE resulted in dose-dependent influx of calcium at the junctional region with the consequent development of morphological and biochemical alterations. Non-necrotising doses of ECO caused hypercontractions of varying severity, distorted end plate and slight elevation of serum creatine kinase (CK). Necrotising doses of ECO further caused contraction clumps, loss of striations and procion staining with high serum CK. The extent of ECO-induced myopathy depended on entry of extracellular calcium rather than the degree of AChE inhibition. The essential Ca2+ mediated process(es) in ECO-induced myopathy was thought to be the generation of superoxide and superoxide-derived free radicals and/or lipid peroxidation. Mitochondria and xanthine oxidase may be the major contributors to the generation of superoxide. No evidence was found for the depletion of high energy phosphates. ECO-induced myopathy could be successfully prevented by prior administration of pyridostigmine or various antioxidants, the most effective being Vit E or Vit E + N-acetylcysteine. Allopurinol or N-acetylcysteine alone were also effective. However, the use of a wide range of membrane end plate channel blockers or non-quantal release blockers were unsuccessful in the prevention of ECO-induced myopathy.

Methods for detection of anatoxin-a(s) by liquid chromatography coupled to electrospray ionization-tandem mass spectrometry

Anatoxin-a(s) is a potent irreversible inhibitor of the enzyme acetylcholinesterase with a unique N-hydroxyguanidine methylphosphate ester chemical structure. Determination of this toxin in environmental samples is hampered by the lack of specific methods for its detection. Using the toxic strain of Anabaena lemmermani PH-160 B as positive control, the fragmentation characteristics of anatoxin-a(s) under collision-induced dissociation conditions have been investigated and new LC-MS/MS methods proposed. Recommended ion transitions for correct detection of this toxin are 253 > 58, 253 > 159, 235 > 98 and 235 > 96. Chromatographic separation is better achieved under HILIC conditions employing a ZIC-HILIC column. This method was used to confirm for the first time the production of anatoxin-a(s) by strains of Anabaena oumiana ITEP-025 and ITEP-026. Considering no standard solutions are commercially available, our results will be of significant use for the correct identification of this toxin by LC-MS/MS. (C) 2009 Elsevier Ltd. All rights reserved.

Organophosphate and carbamate poisonings in the northwest of Parana state, Brazil from 1994 to 2005: clinical and epidemiological aspects

In the present study, clinical and epidemiological aspects of 529 intoxication cases of organophosphate or carbamate pesticides in the northwest of the state of Parana, Brazil, over a twelve-year period (1994-2005), are presented. One hundred-five of 257 patients (40.8%) who attempted suicide were admitted to Intensive Care Units (ICUs), with an average hospital stay of two days (range 1-40 days). Men corresponded to 56.4% of the cases of suicide attempts and sixteen individuals died. One hundred-forty patients intoxicated due to occupational exposure were all young adults and nine of them were admitted to ICU, with average hospital stays of eight days (range 1-16 days). Of these cases, two patients died. One hundred twenty-four patients intoxicated due to accidental exposure were mainly children and had a hospital average stay of four days. Twenty patients were admitted to the ICU, and one of them died. Overall complications included respiratory failure, convulsions, and aspiration pneumonia. Deliberate ingestion of organophosphates and carbamates Was much more toxic than occupational and accidental exposure. Men aged 15-39 years were the most likely to attempt suicide with these agents and had more prolonged ICU with significant complications and mortality

Supercritical carbon dioxide extraction of bioactive compounds from microalgae and volatile oils from aromatic plants

A discussion of the most interesting results obtained in our laboratories, during the supercritical CO(2) extraction of bioactive compounds from microalgae and volatile oils from aromatic plants, was carried out. Concerning the microalgae, the studies on Botryococcus braunii and Chlorella vulgaris were selected. Hydrocarbons from the first microalgae, which are mainly linear alkadienes (C(23)-C(31)) with an odd number of carbon atoms, were selectively extracted at 313 K increasing the pressure up to 30.0 MPa. These hydrocarbons are easily extracted at this pressure, since they are located outside the cellular walls. The extraction of carotenoids, mainly canthaxanthin and astaxanthin, from C. vulgaris is more difficult. The extraction yield of these components at 313 K and 35.0 MPa increased with the degree of crushing of the microalga, since they are not extracellular. On the other hand, for the extraction of volatile oils from aromatic plants, studies on Mentha pulegium and Satureja montana L were chosen. For the first aromatic plant, the composition of the volatile and essential oils was similar, the main components being the pulegone and menthone. However, this volatile oil contained small amounts of waxes, which content decreased with decreasing particle size of the plant matrix. For S. montana L it was also observed that both oils have a similar composition, the main components being carvacrol and thymol. The main difference is the relative amount of thymoquinone, which content can be 15 times higher in volatile oil. This oxygenated monoterpene has important biological activities. Moreover, experimental studies on anticholinesterase activity of supercritical extracts of S. montana were also carried out. The supercritical nonvolatile fraction, which presented the highest content of the protocatechuic, vanilic, chlorogenic and (+)-catechin acids, is the most promising inhibitor of the enzyme butyrylcholinesterase. In contrast, the Soxhlet acetone extract did not affect the activity of this enzyme at the concentrations tested. (C) 2011 Elsevier B.V. All rights reserved.