Ocotea daphnifolia: phytochemical investigation, in vitro dual cholinesterase inhibition, and molecular docking studies

This study aimed to evaluate the anticholinesterase activities of extracts and fractions of Ocotea daphnifolia in vitro and characterize its constituents. The effects of hexane, ethyl acetate, and ethanolic extracts on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity were determined with a spectrophotometry assay. All extracts inhibited cholinesterase activity, and the ethanolic extract (2 mg/mL) exhibited the highest inhibition of both enzymes (99.7% for BuChE and 82.4% for AChE). The ethanolic extract was fractionated by column chromatography resulting in 14 fractions that were also screened for their anticholinesterase effects. Fraction 9 (2 mg/mL) showed the highest activity, inhibiting AChE and BuChE by 71.8% and 90.2%, respectively. This fraction was analyzed by high-performance liquid chromatography high-resolution mass spectrometry which allowed the characterization of seven glycosylated flavonoids (containing kaempferol and quercetin nucleus) and one alkaloid (reticuline). In order to better understand the enzyme-inhibitor interaction of the reticuline toward cholinesterase, molecular modeling studies were performed. Reticuline targeted the catalytic activity site of the enzymes. Ocotea daphnifolia exhibits a dual cholinesterase inhibitory activity and displays the same pattern of intermolecular interactions as described in the literature. The alkaloid reticuline can be considered as an important bioactive constituent of this plant.

Determination of acute median lethal concentration and sublethal effects on AChE activity of Gymnotus carapo (Teleostei: Gymnotidae) exposed to trichlorfon / Determinação da concentração letal média e efeitos subletais da atividade AchE de Gymnotus carapo (Teleostei: Gymnotidae) exposto ao triclorfon

Trichlorfon (TRF) is a pesticide widely used in aquaculture to control fish ectoparasites. This pesticide is an inhibitor of acetylcholinesterase, an essential enzyme for termination of nerve impulses. High rates of TRF use generate risks to the environment and human health. In the environment, pesticides can affect the local fauna and generate an ecological breakdown. There are several studies performed with fish production; however, gaps are created for native fish with other commercial values. The tuvira (Gymnotus carapo) is a fish native to Brazilian fauna and has great commercial importance in sport fishing. The present study aimed to determine the lethal concentration of trichlorfon (Masoten) in Gymnotus carapo and its sublethal effects on the enzyme AChE. In this study, the acute toxicity (the concentrations to kill 50% of the fish LC50) of TRF in tuviras (Gymnotus carapo) and acetylcholinesterase inhibition in liver and muscle tissue of tuviras submitted to sublethal concentrations were evaluated. For the acute assay, concentrations of 0.0, 5.0, 7.5, 15, 22.5, 30, 37.5 and 45 mg L-1 were used for a period of 96 h. After the acute exposure period, a LC50 of 6.38 mg L-1 was determined. In the sublethal assay, concentrations of 0.0, 0.238, 0.438 and 0.638 mg L-1 were used, based on 10% of the LC50, over a period of 14 days. Two collections were performed: one at seven days and the other at the end (day 14). Inhibition of acetylcholinesterase in the liver was only shown (p < 0.05) for the treatment with 0.638 mg L-1 after 14 days of exposure. At seven days, muscle activity showed a significant difference only for the treatments 0.438 and 0.638 mg L-1, compared with the treatment 0.238 mg L-1 and control. At 14 days of exposure, only the treatment 0.638 mg L-1 showed significant differences in relation to the other groups, thus showing that enzyme recovery had occurred. The value found in the acute test allowed the conclusion that TRF presents moderately toxic characteristics to Gymnotus carapo. The toxicity parameter values calculated in the present study assisted in estimation of maximum allowable limits in bodies of water when combined with test data from other non-target organisms.(AU)

O triclorfon (TRC) é um pesticida muito utilizado na aquicultura para o controle de ectoparasitos de peixes. Este pesticida é um inibidor da acetilcolinesterase, uma enzima essencial para a finalização de impulsos nervosos. As altas concentrações utilizadas de TRC geram riscos ao meio ambiente e à saúde humana. No ambiente, os pesticidas podem afetar a fauna local e gerar um colapso ecológico. Existem vários estudos com peixes de produção, no entanto, há lacunas para peixes nativos com outros valores comerciais. A tuvira (Gymnotus carapo) é um peixe nativo da fauna brasileira e possui grande importância comercial na pesca esportiva. O presente trabalho, delineado para determinar a concentração letal de triclorfon (Masoten) em Gymnotus carapo e seus efeitos subletais na enzima AChE, avaliou a toxicidade aguda (concentrações para matar 50% dos peixes CL50) do TRC em tuviras (Gymnotus carapo) e a inibição da acetilcolinesterase no fígado e tecido muscular de tuviras. Para o ensaio agudo, foram utilizadas concentrações de 0,0, 5,0, 7,5, 15, 22,5, 30, 37,5 e 45 mg L-1por um período de 96 horas. Após o período de exposição aguda, foi determinado uma CL50 de 6,38 mg L-1. No ensaio subletal, foram utilizadas concentrações de 0,0, 0,238, 0,438 e 0,638 mg L-1, com base em 10% do CL50, durante um período de catorze dias. Foram realizadas duas colheitas: uma aos sete dias e a outra ao final (décimo quarto dia). A inibição da acetilcolinesterase no fígado foi demonstrada apenas (p <0,05) para o tratamento com 0,638 mg L-1 após catorze dias de exposição. Aos sete dias, a atividade muscular mostrou diferença significativa apenas para os tratamentos 0,438 e 0,638 mg L-1, em comparação com o tratamento 0,238 mg L-1 e controle. Aos catorze dias de exposição, apenas o tratamento 0,638 mg L-1 apresentou diferenças significativas em relação aos demais grupos, demonstrando a recuperação enzimática. O valor encontrado no teste agudo permitiu concluir que o TRC apresenta características moderadamente tóxicas para Gymnotus carapo. Os valores dos parâmetros de toxicidade calculados no presente estudo permitiram o estabelecimento da estimativa dos limites máximos permitidos em corpos d'água quando combinados com dados de testes de outros organismos não-alvo.(AU)

Niveles de colinesterasa sérica en caficultores del Departamento de Caldas, Colombia / Levels of serum cholinesterase in coffee growers from the Caldas Department, Colombia

RESUMEN Objetivo Determinar niveles de colinesterasa sérica en caficultores del departamento de Caldas y su asociación con factores demográficos y ocupacionales. Metodología Se realizó un estudio descriptivo, muestra de 1 098 agricultores del Alto Oriente y Centro Sur del Departamento de Caldas, por medio de una encuesta en la que se analizaron características del trabajador agrícola de tipo: sociodemográfico, ocupacional, clínicos y concentración de colinesterasa determinada con el método de Ellman. Resultados A nivel ocupacional, el 90,8 % de los agricultores refirió riesgo de exposición directa a plaguicidas. El 3,8 % de las determinaciones analíticas de colinesterasa fueron anormales, se relacionó que el 75,6 % de los agricultores preparan la mezcla del insecticida, el 22,2 % tienen una frecuencia de aplicación en el cultivo más de dos veces por semana, el 37,8 % no emplea ropa de protección durante la jornada de fumigación. El tiempo de la última aplicación fue dentro del rango de uno a diez días demostrando que a menor tiempo de aplicación del insecticida, se presenta mayor inhibición de la enzima. Los plaguicidas más reportados fueron los de tipo organofos-forado (58,6 %). Conclusiones El control de la exposición a plaguicidas se torna difícil porque la mayoría de trabajadores son de tipo informal. Se requiere fortalecer los programas de capacitación y campañas de sensibilización sobre los efectos de los plaguicidas en la salud, las medidas de higiene y seguridad en el trabajo. Los niveles bajos de colinesterasa sérica indican la absorción de una cantidad mínima de insecticidas inhibidores de la colinesterasa.(AU)

ABSTRACT Objective To determine the levels of serum cholinesterase in coffee growers from the Caldas department and its association with demographic and labor factors. Methodology A descriptive study was carried out in a sample of 1 098 farmers from the Upper East and South Center of the Caldas department, through a survey that analyzed characteristics such as sociodemographic, labor, and clinical conditions, as well as cholinesterase levels, determined by Ellman's method. Results Regarding the occupational aspect, 90.8 % of farmers reported a risk of direct exposure to pesticides. 3.8 % of the analytical determinations of cholinesterase were abnormal, which was related to the fact that 75.6 % of the farmers themselves prepare the mixture of the insecticide, 22.2 % spread the insecticide over their crops more than twice a week, and 37.8 % do not wear protective clothing during the fumigation. The last fumigation was within the range of one to ten days, revealing that the shorter the time of application of the insecticide, the greater the inhibition of the enzyme. The most frequent pesticides were organophosphates (58.6 %). Conclusions Controlling pesticide exposure is difficult because most workers are self-employed. It is necessary to strengthen training programs and awareness campaigns regarding the effect of pesticides on health, as well as health and safety measures at the workplace. Low levels of serum cholinesterase indicate the absorption of a minimal amount of cholinesterase inhibiting insecticides.(AU)

Investigations of anticholinestrase and antioxidant potentials of methanolic extract, subsequent fractions, crude saponins and flavonoids isolated from Isodon rugosus

BACKGROUND: Based on the ethnomedicinal uses and the effective outcomes of natural products in various diseases, this study was designed to evaluate Isodon rugosus as possible remedy in oxidative stress, alzheimer's and other neurodegenerative diseases. Acetylecholinestrase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of crude methanolic extract (Ir.Cr), resultant fractions (n-hexane (Ir.Hex), chloroform (Ir.Cf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq)), flavonoids (Ir.Flv) and crude saponins (Ir.Sp) of I. rugosus were investigated using Ellman's spectrophotometric method. Antioxidant potential of I. rugosus was determined using DPPH, H2O2 and ABTS free radicals scavenging assays. Total phenolic and flavonoids contents of plant extracts were determined and expressed in mg GAE/g dry weight and mg RTE/g of dry sample respectively. RESULTS: Among different fractions Ir.Flv and Ir.Cf exhibited highest inhibitory activity against AChE (87.44 ± 0.51, 83.73 ± 0.64%) and BChE (82.53 ± 0.71, 88.55 ± 0.77%) enzymes at 1 mg/ml with IC50 values of 45, 50 for AChE and 40, 70 µg/ml for BChE respectively. Activity of these fractions were comparable to galanthamine causing 96.00 ± 0.30 and 88.61 ± 0.43% inhibition of AChE and BChE at 1 mg/ml concentration with IC50 values of 20 and 47 µg/ml respectively. In antioxidant assays, Ir.Flv, Ir.Cf, and Ir.EtAc demonstrated highest radicals scavenging activities in DPPH and H2O2 assays which were comparable to ascorbic acid. Ir.Flv was found most potent with IC50 of 19 and 24 µg/ml against DPPH and H2O2 radicals respectively. Whereas antioxidant activates of plant samples against ABTS free radicals was moderate. Ir.Cf, Ir.EtAc and Ir.Cr showed high phenolic and flavonoid contents and concentrations of these compounds in different fractions correlated well to their antioxidant and anticholinestrase activities. CONCLUSION: It may be inferred from the current investigations that the Ir.Sp, Ir.Flv and various fractions of I. rugosus are good sources of anticholinesterase and antioxidant compounds. Different fractions can be subjected to activity guided isolation of bioactive compounds effective in neurological disorders.

Determinaçäo de resíduos de carbofuran em vegetais por cromatografia líquida de alta eficiência (HPLC) / Carbofuran residues determination in vegetables by high efficiency liquid chromatography (HPLC)

O pesticida carbofuran (2,3-dihydro-2,2-dimethyl-7-benzo-furanyl-N-methylcarbamate) é um inseticida-nematicida de amplo espectro de aplicaçao, sendo empregado em escala crescente em muitos países tropicais, devido a sua eficácia contra numerosos insetos e pestes de frutas, vegetais e graos. Apresenta natureza polar e aromaticidade que tornam a determinaçao por HPLC com detecçao no UV atrativa. A análise foi efetuada em cromatógrafo Shimadzu com detector espectrofotométrico ajustado em 275 nm. Na separaçao foi empregada uma coluna Nucleosil C-18 (250 x 4 mm, 5 mum) e metanol-água (50:50, v/v; l mL/min). O volume injetado foi de 20 muL. A eficiência do procedimento foi investigada com amostras de tomate e batata fortificadas com carbofuran em O,1 e 1,0 mg/kg. As amostras foram trituradas em metanol. Após procedeu-se extraçao líquido-líquido com diclorometano. Para remover coextrativos ácidos foi empregada lavagem com hidróxido de sódio diluído. As recuperaçoes variaram entre 94,7 e 98,3 por cento para tomate e entre 95,3 e 97,2 por cento para batata. O limite de detecçao de O,14 mg/L corresponde a concentraçao mínima detectável de O,01l mg/kg em vegetais após a extraçao de 25 g de amostra. Estes resultados indicam o grande potencial do método descrito na determinaçao de resíduos de carbofuran nos vegetais estudados.

Dibucaine number (DN) in duodenal ulcer.

Dibucaine Number (DN) was determined in 103 patients suffering from duodenal ulcer diagnosed radiologically and 214 normal subjects (control). Statistically significant (p less than 0.001) difference was observed in the distribution of DN in the two groups. Whereas 94% of the control subjects had normal DN (above 70), 6% had intermediate DN (between 40--70) and none had atypical DN, only 32% of patients suffering from duodenal ulcer had normal DN, 64% had intermediate DN and 4% had atypical DN. The results have been discussed.