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.

Resumo A atividade carrapaticida in vitro do extrato metanólico (EM) e da fração de alcaloides (FA) de Prosopis juliflora foi investigada, frente ao Rhipicephalus microplus, e relacionada com a inibição da enzima acetilcolinesterase (AChE). A predição in silico das interações de alcaloides dessa fração com a AChE1 de R. microplus (RmAChE1) foi realizada por acoplamento molecular. A atividade carrapaticida foi avaliada, utilizando-se os ensaios de imersão de adultos e larvas. Maior efeito sobre parâmetros reprodutivos de teleóginas foi verificado para a FA, com valor de Concentração Efetiva 50% (CE50) (31.6 mg/mL), quatro vezes menor do que o valor do EM (121 mg/mL). A FA induziu mortalidade de larvas (Concentração Letal de 50% - CL50 = 13,8 mg/mL). A inibição da atividade da AChE de larvas do carrapato foi observada para EM e FA, sendo a FA mais ativa (Concentração Inibitória 50%- CI50 de 0,041mg/mL). As análises químicas da FA permitiram a identificação dos alcaloides prosopina (1), juliprosinina (2) e juliprosopina (3). No ensaio in silico, observou-se que esses alcaloides podem interagir com o sítio ativo da RmAChE1, principalmente por ligações de hidrogênio e interações cátion-pi. Os alcaloides de P. juliflora têm atividade carrapaticida contra R. microplus, atuando através do mecanismo anticolinesterásico.

Abstract We investigated the in vitro acaricide activity of the methanolic extract (ME) and alkaloid-rich fraction (AF) of Prosopis juliflora on Rhipicephalus microplus and correlated this effect with acetylcholinesterase (AChE) inhibition. The acaricide activity was evaluated using adult and larval immersion tests. Also, we studied the possible interaction mechanism of the major alkaloids present in this fraction via molecular docking at the active site of R. microplus AChE1 (RmAChE1). Higher reproductive inhibitory activity of the AF was recorded, with effective concentration (EC50) four times lower than that of the ME (31.6 versus 121 mg/mL). The AF caused mortality of tick larvae, with lethal concentration 50% (LC50) of 13.8 mg/mL. Both ME and AF were seen to have anticholinesterase activity on AChE of R. microplus larvae, while AF was more active with half-maximal inhibitory concentration (IC50) of 0.041 mg/mL. The LC-MS/MS analyses on the AF led to identification of three alkaloids: prosopine (1), juliprosinine (2) and juliprosopine (3). The molecular docking studies revealed that these alkaloids had interactions at the active site of the RmAChE1, mainly relating to hydrogen bonds and cation-pi interactions. We concluded that the alkaloids of P. juliflora showed acaricide activity on R. microplus and acted through an anticholinesterase mechanism.

RESUMO: O objetivo deste estudo foi avaliar a atividade anti-helmíntica in vitro do ácido succínico (AS) isolado do resíduo de sisal sobre nematódeos gastrointestinais de caprinos, utilizando os ensaios de inibição da eclosão de ovos e motilidade larval. Além disso, a citotoxicidade do AS em culturas de células Vero foi investigada empregando-se o teste de MTT (brometo de 3-4,5-dimetiltiazol-2-ilo, brometo de 2,5-difeniltetrazólio). O AS promoveu redução significativa no percentual de eclosão de ovos (P<0,05) em todas as concentrações testadas (60 a 250μg mL-1). As médias (± desvio padrão) da CE50 e CE90 (Concentração efetiva 50% e 90%) sobre ovos foram de 90.3±2.8 e 130±3.5μg mL-1, respectivamente. O AS não apresentou atividade larvicida. O AS foi menos tóxico para as células Vero, com média do percentual de viabilidade celular igual a 85±6.2% na concentração de 130μg mL-1. Os resultados sugerem que o AS tem potencial efeito anti-helmíntico, embora sejam necessários a realização de estudos in vivo para confirmar seu uso terapêutico.

ABSTRACT: This study was conducted to evaluate the in vitro anthelmintic activity of the succinic acid (SA) isolated from sisal waste against gastrointestinal nematodes of goats, using the egg hatching and larvae motility assays. In addition, potential cytotoxicity of SA on Vero cell cultures was investigated by means of MTT (3-4,5-dimethylthiazol-2-yl, 2,5diphenyltetrazolium bromide) test. The SA induced a significant inhibition of egg hatching (P<0.05) at all concentrations tested (60 to 250µg mL-1), and the concentrations to inhibit 50% (EC50) and 90% (EC90) values (mean ± standard deviation) were 90.3±2.8 and 130.6±3.5µg mL-1, respectively. The SA has not shown larvicidal activity. The SA was less toxic to the Vero cells, with the mean percentage of cell viability equal to 85±6.2% at the concentration of 130µg mL-1. The results suggested that SA has potential anthelmintic effect; although, more research is needed to confirm its activity in vivo.

Foi avaliada a atividade anti-helmíntica do suco de Agave sisalana (sisal) contra nematódeos gastrintestinais e possíveis efeitos tóxicos em caprinos. Nos testes in vitro, encontrou-se redução superior a 95% na contagem de larvas do gênero Haemonchus spp. nas concentrações entre 86,5 e 146,3 mg.mL-1. In vivo, o percentual de redução de larvas de quarto (L4) e quinto (L5) estágios de Haemonchus, Oesophagostomum e Trichostrongylus foi inferior a 95% para o GI e GII, e entre 80 e 90% para o GIII. O suco de A. sisalana nas concentrações testadas in vitro foi efetivo contra nematódeos gastrintestinais de caprinos, apresentando, no entanto, reduzida eficácia anti-helmíntica quando administrado nos animais.

It was evaluated the anthelmintic activity of Agave sisalana (sisal) juice against gastrointestinal nematodes and its potential toxic effects in goats. In vitro tests showed more than 95% reduction in larval counts of the genus Haemonchus spp. at concentrations between 86.5 and 146.3 mg.mL-1. In vivo the percent reduction of larvae of the fourth (L4) and fifth (L5) stages of Haemonchus, Oesophagostomum and Trichostrongylus was less than 95% in groups GI and GII, and between 80 and 90% in group GIII. A. sisalana juice at the concentrations tested in vitro was effective against gastrointestinal nematodes in goats; however, its anthelmintic efficacy was reduced when administered to animals.

O objetivo deste trabalho foi avaliar os efeitos do suco de alho sobre nematódeos gastrintestinais de caprinos. Foram utilizados 20 animais, distribuídos em dois grupos tratados com o suco de alho, um grupo tratado com ivermectina e um grupo controle sem tratamento. Os percentuais de redução do número de ovos e larvas de Strongyloidea foram inferiores a 95% para todos os grupos. O tratamento de caprinos com o suco de alho não foi eficiente no controle de nematódeos gastrintestinais.

The objective of this work was to evaluate the effects of the garlic juice on goat gastrointestinal nematodes. Twenty animals were alocated into two groups and treated with garlic juice. One group was treated with ivermectin and one control group was not treated. The percentage reductions in egg and larvae counts of the Strongyloidea were under 95% for all groups. The treatment of goats with garlic juice was not an effective control of gastrointestinal nematodes.