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Background: Motor dysfunctions are some of the characteristic symptoms of organophosphate (OP) poisoning and they have been associated with decreased levels of cholinesterase inhibition within motor areas of the brain. Objectives: The current study aims to investigate the potential neuroprotective effects of Nigella sativa oil (NSO) in alleviating chlorpyrifos (CPF) induced toxicity in the cerebella and motor cortices of the rat brains using combined behavioural, biochemical and histochemical methods. Methods: Thirty-two rats were randomly divided into four groups (eight rats per group), exposed to 1ml/kg of normal saline, 14.9 mg/kg of CPF, 14.9 mg/kg of CPF plus 1ml/kg of NSO and 1ml/kg of NSO respectively for 14 consecutive days. The rats were each exposed to a single trial of the Open Field Test (OFT) on day 13 of the experiment. This experimental test measured locomotor activity levels (line crossing frequency (LCF)) and exploratory (rearing frequency (RF)) activities in the rats studied. The rats were euthanized on day 15 of the experiment and the brains were subsequently excised. The cerebella cortices of five brains were removed and homogenised to analyse for total reactive oxygen species (ROS), nitric oxide (NO) levels and acetylcholinesterase (AChE) activity. The motor and cerebella cortices from three other brains in each group were processed for histology (Nissl stain) and proliferative activity (Ki67 immunohistochemistry). Results: Rats exposed to CPF experienced a significant increase in cerebella NO and ROS levels, depletion in AChE activity, neurogenic cells loss and subsequent reduction in locomotor and exploratory behaviours respectively (LCF and RF). However, interventional treatment with NSO depleted markers of oxidative damage (NO and ROS), reduced AChE inhibition, preserved neurogenic (Ki67) cells distribution and motor functions. Conclusion: These results demonstrate the potential efficacy of NSO in OP poisoning and the roles of neurogenic and oxidative functions in the pathophysiology and treatment of motor dysfunction in OP neurotoxicity.

Prirodna niskotemperaturna eutektička otapala, nova zelena otapala, istražuju se kao alternativna otapala za otapanje djelatnih farmaceutskih tvari. Razvoj učinkovitog antidota za otrovanja organofosfornim (OP) spojevima, ireverzibilnim inhibitorima esencijalnog enzima acetilkolinesteraze (AChE), predmet je mnogih istraživanja. U središtu pozornosti su tzv. centralno-aktivni oksimski reaktivatori fosfilirane AChE, kao što je, u vodi slabo topljivi oksim RS-194B. U ovom radu je pomoću programa COSMOtherm predviđena, a zatim i eksperimentalno određena topljivost oksima RS-194B u prirodnim niskotemperaturnim eutektičkim otapalima tekućinskom kromatografijom visoke djelotvornosti. Najbolja topljivost postignuta je u otapalu limunska kiselina:glukoza=1:1 s 30 %-tnim udjelom vode. Praćena je stabilnost formulacije oksima RS-194B u odabranom i referentnom otapalu tijekom mjesec dana na temperaturama 4 °C i 25 °C pri čemu nije zapažena razlika u stabilnosti tih formulacija. Prije određivanja kinetičkih parametara reaktivacije AChE inhibirane OP spojevima sarinom i VX-om pomoću formulacije oksima RS-194B u odabranom otapalu, ispitan je utjecaj samog otapala na AChE. Pokazalo se da otapalo inhibira aktivnost AChE u potpunosti i njegov udio mora se održavati ispod 0,2 % kako bi enzim zadržao 90 % aktivnosti. Nadalje, afinitet AChE i fosfilirane AChE je veći za vezanje oksima otopljenog u odabranom otapalu nego u vodi. Oksim je, bez obzira na otapalo bolji reaktivator VX-om nego sarinom fosfilirane AChE. Međutim, brzina reaktivacije fosfilirane AChE je manja s RS-194B u odabranom otapalu nego s RS-194B otopljenim u vodi. Stoga, iako je primjenom odabranog otapala postignuta bolja topljivost oksima RS-194B u usporedbi s vodom kao referentnim otapalom i poboljšan je afinitet AChE za RS-194B, otapalo limunska kiselina:glukoza=1:1, 30 % vode, nije najbolji izbor za pripremu formulacije RS-194B oksima za in vivo upotrebu zbog inhibitornog učinka na enzim te viskoznosti i niske pH vrijednosti samog otapala. Natural deep eutectic solvents, new green solvents, are being investigated as alternative solvents for dissolving active pharmaceutical ingredients. The development of an effective antidote for poisoning by organophosphates (OPs), irreversible inhibitors of the essential enzyme acetylcholinesterase (AChE), has been a subject of many research. The focus is on the so-called centrally active oxime reactivators of phosphylated AChE like poorly water-soluble oxime RS-194B. In this study, the solubility of RS-194B oxime in natural deep eutectic solvents has been predicted by the COSMOtherm program and experimentally determined using high efficient liquid chromatography. Natural deep eutectic solvent citric acid: glucose=1:1 with 30 % water content has resulted with best solubility of oxime RS-194B. The stability of oxime RS-194B formulation in the selected natural deep eutectic solvent has been monitored during one month period at temperatures of 4 °C and 25 °C where no difference in the stability of these formulations was observed. Before determining the effect of RS-194B oxime formulation in the selected solvent on the reactivation kinetics of AChE inhibited by OPs sarin and VX, the effect of the solvent itself on the AChE was determined. It was found that the selected solvent inhibited AChE completely, and in order for AChE to maintain more than 90 % activity, its content has to be kept below 0,2 %. The affinity of AChE and phosphylated AChE is higher for the binding of oxime dissolved in the selected solvent than in water. The oxime, regardless of the solvent, is a better reactivator of VX- than sarin-inhibited AChE. However, the maximal first-order reactivation rate of phosphylated AChE is lower with RS-194B in the selected solvent than with RS-194B dissolved in water. Therefore, although the use of the selected solvent resulted in better solubility of RS-194B oxime compared to water as the reference solvent and improved the affinity of AChE for RS-194B, the solvent citric acid: glucose=1:1, 30 % water, is not suitable for preparing the RS-194B formulation for in vivo application due to its inhibitory effect on the enzyme, viscosity and low pH.

Pterodon pubescens (Benth.) Benth is native species from the Brazilian Cerrado, popularly known as “sucupira-branca,” and rich in bioactive metabolites. This study aimed to assess the inhibitory activity of the enzyme acetylcholinesterase by leaf extracts of P. pubescens Benth. Phenolic constituents and flavonoids were analyzed by LC-DAD and LCMS. The qualitative and quantitative enzymatic inhibitory profile was performed by 96-well microplate in ELISA reader. The statistical analysis was reached with a significance limit of p < 0.05 applied by the Tukey’s test. Bioactive components detected were terpenoids, phenolic acids (gallic acid, ferulic acid, and rosmarinic acid), and flavonoids (kaempferol, luteolin, apigenin, rutin, and quercetin). There were no significant differences of inhibition between the crude methanolic and ethyl acetate extracts and between the hydroalcoholic and ethyl acetate phases at the concentration of 80 µg mL-1. The phytochemical profile in LCMS are related to the acetylcholinesterase inhibitory effect. This species has an importance for future in vivo studies, purification, and isolation of molecules with a possible biopesticidal effect. Pterodon pubescens (Benth.) Benth. es una especie nativa del Cerrado brasileño, conocida popularmente como “sucupira-branca” y rica en metabolitos bioactivos. El estudio tuvo como objetivo evaluar la actividad inhibidora de la enzima acetilcolinesterasa en el extracto de hojas de P. pubescens Benth. Los constituyentes fenólicos y flavonoides se analizaron mediante LC-DAD y LCMS. La evaluación inhibidora enzimática cuantitativa se realizó en una microplaca de 96 pocillos en un lector de ELISA. El análisis estadístico se realizó con un límite de significancia de p < 0,05 y se aplicó la prueba de Tukey. Los compuestos bioactivos detectados fueron terpenoides, ácidos fenólicos (ácidos gálico, ferúlico y rosmarínico) y flavonoides (caempferol, luteolina, apigenina, rutina y quercetina). No hubo diferencia significativa en la inhibición entre el extracto metanólico crudo y el acetato de etilo, y entre sus respectivas fases hidroetanólicas y el acetato de etilo a una concentración de 80 µg mL-1. El perfil fitoquímico identificado por LCMS se informó a través del efecto inhibidor de la acetilcolinesterasa. La especie es de gran importancia para futuros estudios in vivo, de purificación y aislamiento de moléculas con posible efecto biopesticida. Pterodon pubescens (Benth.) Benth. é uma espécie nativa do Cerrado brasileiro, popularmente conhecida como “sucupira-branca,” e rica em metabólitos bioativos. O estudo teve como objetivo avaliar a atividade inibitória da enzima acetilcolinesterase do extrato das folhas de P. pubescens. Constituintes fenólicos e flavonoides foram analisados por LC- DAD e LCMS. A avaliação inibitória enzimática quantitativa foi realizada em microplaca de 96 poços em leitor ELISA. A análise estatística foi realizada com limite de significância p < 0,05 e aplicou-se o teste de Tukey. Os compostos bioativos detectados foram os terpenóides, ácidos fenólicos (ácido gálico, ferúlico e ácido rosmarínico) e flavonoides (caempferol, luteolina, apigenina, rutina e quercetina). Não houve diferença significativa de inibição entre o extrato bruto metanólico e acetato de etila, e entre suas respectivas fases hidroetanólica e acetato de etila na concentração de 80 µg mL-1. O perfil fitoquímico identificado pelo LCMS foi relatado através do efeito inibitório pela acetilcolinesterase. A espécie apresenta grande importância para futuros estudos in vivo, de purificação e isolamento de moléculas com possível efeito biopesticida.

Naresuan University Journal: Science and Technology, 28, 1, 65-81

Morávková, Martina. Comparison of original Ellman's method and its modification using microplate reader. Diploma thesis 2015. Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology. Supervisor: Ing. Kateřina Macáková, Ph.D. Key words: Ellman's method, acetylcholinesterase, butyrylcholinesterase, spectrophotometer, microplate reader. Ellman's method is often used procedure for measuring an inhibition of cholinesterases acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The principle is based on hydrolysis of acetylthiocholiniodide and butyrylthiocholiniodide to give an appropriate acid and thiocholine. Amount of thiocholine is determined spectrophotometrically by reaction with 5,5'-dithiobis-2-nitrobenzoic acid. Results of measurement from two different devices (spectrophotometer and microplate reader) and at two different temperatures (room temperature and 37 řC) were compared in this thesis. Substances with different amount of inhibition of both enzymes were chosen: allocryptopine, californidine iodide, eserine, galanthamine, huperzine A and tazettine. For californidine iodide (AChE, BuChE), eserine (BuChE) and huperzine A (AChE) were detected statistically significant differences during measurement on different devices and at...

Among a large number of different types of pesticides available, organophosphates (OP) are the most frequent causes of professional, suicidal or accidental intoxications. It is generally known that OPs act as irreversible inhibitors of cholinesterases (ChE/AChE), yet there are still some open questions about their non-specific effects. Another issue concerning OP poisoning is the inability of conventional therapies to provide adequate protection. The present study was undertaken to examine the impact of possible oxidative stress on OP toxicity and the efficacy of the applied therapy. Rats were injected subcutaneously with sublethal dose of paraoxon, and treated intraperitoneally 1 min later with a combination of oxime K048 (25 % of its LD50) and atropine (10 mg kg-1). Plasma and brain samples were analysed for ChE/AChE activity and concentration of thiobarbituric reactive substances (TBARS) at four different time points (0.5 h, 1 h, 6 h and 24 h) following the treatment. It is important to stress that cholinesterase activity in both plasma and brain did not return to normal even after 24 h following the exposure to paraoxon. A combination of K048 and atropine afforded high potency in restoring the activity of ChE in plasma (50 % to 60 % up to 6 h) while an increase in AChE activity in the brain did not surpass 20 %. Although applied therapy efficiently counteracted paraoxon poisoning, determined TBARS concentrations suggested that these acute treatments resulted with free radical-mediated lipid peroxidation.

Made available in DSpace on 2014-06-11T19:30:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-04-03Bitstream added on 2014-06-13T19:19:25Z : No. of bitstreams: 1 mataqueiro_mi_dr_jabo.pdf: 565768 bytes, checksum: 9c312d8045e79c1a6f25b2c153b020c0 (MD5) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Universidade Estadual Paulista (UNESP) O objetivo deste trabalho foi avaliar a toxicidade do triclorfom em curto prazo no pacu (Piaractus mesopotamicus), determinando a atividade da colinesterase no plasma e cérebro dos peixes, além de determinar a quantidade residual de triclorfom no plasma e na água através de cromatografia gasosa. O delineamento experimental foi inteiramente casualizado, com 4 concentrações (tratamentos) e 3 repetições (caixas) por tratamento, com 10 peixes por caixa. As concentrações de triclorfom usadas foram: 0,0; 0,025; 0,05 e 0,1 mg/L. Os efeitos subagudos do triclorfom nos peixes foram avaliados 1, 2, 7 e 15 dias após ter adicionado o triclorfom às caixas com o fluxo de água interrompido por 24 horas, mantendo a oxigenação. A atividade da colinesterase no plasma e no cérebro foi inibida pelo triclorfom, especialmente nas concentrações 0,05 e 0,1 mg/L, no 1º e 2º dias após exposição dos peixes ao praguicida, mas esse efeito desapareceu a partir do 7º dia. Para a análise cromatográfica, o éster correspondente da derivação do triclorfom apresentou limite de detecção de 3,0 g/L. O lindane foi usado como padrão interno na concentração de 50 pg/ L. O tempo de retenção do triclorfom foi de 2,12 minutos e do padrão interno 5,56. Ótima linearidade foi obtida na faixa de 2 a 100 pg/ L. Não foi detectado resíduo de triclorfom nas amostras de plasma durante todo o período experimental, mas somente traços em algumas amostras da maior concentração (0,1mg/l), sendo o valor encontrado menor que o limite de detecção. The aim of this research was to evaluate the toxicity in a short time of the trichlorfon to pacu (Piaractus mesopotamicus) determining cholinesterase activity in plasma and in fish brain, and the residual amount of trichlorfon in fish plasma and water by gas chromatography. Experimental design was all fortuitous with 4 concentrations (treatment) and 3 repetitions (boxes) per treatment, with 10 fish each box. Used concentrations were 0.0; 0.025; 0.05 and 0.1 mg/L. Subacute effects of trichlorfon in fish were estimated 1, 2, 7 and 15 days after added trichlorfon to boxes with interrupted water flow by 24 hours after applications, keeping the aeration rate. Cholinesterase activity in plasma and brain was inhibited by trichlorfon, specially in concentrations 0.05 and 0.1 mg/L in the first and second days after exposition of fish to the pesticides, but this effect desappeared in the 7th day. To the chromatography analysis, esther corresponds from trichlorfon derivation, that showed detection limit of 3 g/L. Lindane was used as intern standard in concentration of 50 pg/ L, Retention time of trichlorfon was 2.12 minutes and for the intern standard was 5.56. Great linearity was obtained between 2 to 100 pg/ L. It was not detected trichlorfon residues in plasma samples of higher concentration (0.1 mg/L) and the value obtained was lesser than detection limit.

The activity and properties of cholinesterase (ChE) of the motor endplate and its fractions were studied in isolated human skeletal muscle. This preparation was used since the ChE activity of the membrane preparation was localized only in the motor endplate. The endplate ChE was stable in the isolated membrane for 4 weeks at 4 degrees C. The specific activity of the extracted ChE of human muscle membrane was 29.6% higher than that of the original membrane. Studies with specific substrates and ChE inhibitors indicated that most of the ChE of human muscle membrane and its fractions was acetylcholinesterase, and that the minor component was pseudocholinesterase. A Michaelis-Menten constant of 3.82 mM was estimated in the endplate ChE, and 0.88 mM in the extracted ChE of the endplate. The extracted human endplate ChE was separated into three fractions by Sephadex G-200 chromatography, and into two fractions by acrylamide gel electrophoresis.

Cholinesterase activity was localized solely in the motor endplate of the membrane in rate intercostal muscle. The diameter of rat motor endplates in the gradient dimension was 31.9 micrometers. The cholinesterase activity per unit protein of the soluble fraction of rat muscle membrane was 35.6% higher than the original membrane. From studies with specific substrates and cholinesterase inhibitors, the cholinesterase activity of rat muscle membrane and its soluble fraction consists of more than 90% acetylcholinesterase and less than 10% pseudocholinesterase.