ABSTRACT
Four pyrazines were isolated from the n-butanol fraction of Hypecoum erectum L. by using various chromatographic methods, including MCI gel, ODS, silica gel and semi-preparative HPLC. The structures of the isolated compounds were identified as hyperectpyrazin A (1), 1′S-(6-methylpyrazin-2-yl)-ethane-1′,2′-diol (2), 2-hydroxymethyl-6-methylpyrazin (3) and pyrazine-2-carboxylic acid (4) by spectroscopy methods (1D NMR, 2D NMR, UV, IR, MS, etc.). The absolute configuration of compound 2 was determined by using the Mo2(OAc)4 induced CD analysis for the first time. Compound 1 was a new compound, compounds 2-4 were isolated from H. erectum for the first time. Compounds 1-4 were evaluated for their inhibition against acetylcholinesterase and nitric oxide generation induced by lipopolysaccharide-RAW264.7 macrophage cells. At a concentration of 50 μmol·L-1, compounds 2 and 4 displayed inhibitory effects on acetylcholinesterase with the inhibition rates of 44.40% and 43.99%, respectively.
ABSTRACT
A novel pair of Z/E isomeric compounds with unprecedented carbon skeleton were isolated from an aqueous extract of Aspongopus chinensis Dallas by macroporous resin, silica gel, and semi-preparative high performance liquid chromatography (HPLC). Their structures were identified by nuclear magnetic resonance (NMR), Infrared spectroscopy (IR), Mass spectroscopy (MS) and other spectroscopic methods as (Z)-3-(but-1″-en-1″-yl)-1-(2ʹ-hydroxyethyl)-4-propylpyridin-1-ium, namely aspongopyridine A, and (E)-3-(but-1″-en-1″-yl)-1-(2ʹ-hydroxyethyl)-4-propylpyridin-1-ium, namely aspongopyridine B, respectively. Besides, the anti-inflammatory, anti-tumor, acetylcholinesterase inhibition and butyrylcholinesterase inhibition activities of the compounds 1 and 2 were evaluated. The results showed that compounds 1 and 2 have no anti-inflammatory, anti-tumor, and butyrylcholinesterase inhibition activities instead of weak acetylcholinesterase inhibition activity.
ABSTRACT
Introducción: Las colinesterasas son enzimas que se encargan de hidrolizar la acetilcolina en ácido acético y colina, poniéndole fin a la transmisión nerviosa a lo largo de la sinapsis de las uniones neuromusculares. La medición de la actividad de la colinesterasa sérica constituye un indicador del efecto causado por la exposición prolongada a los organofosforados. Objetivo: Determinar los niveles de colinesterasa sérica y factores relacionados con la exposición a organofosforados en agricultores de la vereda de Páramo Lagunas de San Pablo de Borbur, Boyacá, Colombia. Metodología: Estudio prospectivo, de corte transversal, con una muestra de 57 trabajadores. A cada individuo se le aplicó una encuesta de datos sociodemográficos y factores laborales, posteriormente se les tomó una muestra de sangre venosa en ayuno de 8-12 horas; con el fin de determinar las concentraciones de colinesterasa sérica (kit Colinesterasa Butiriltiocolina Biosystems®) con el equipo automatizado de química clínica A-15 de Biosystems®. Resultados: El rango de edad de los participantes estuvo entre los 22 y 64 aflos, el 72 % de los individuos pertenecía al sexo masculino. El 3,5 % (2 varones) presentó valores inferiores al intervalo biológico de referencia (IBR), el 88 % de las personas afirmó realizar tareas con plaguicidas y el 54 % afirmó no utilizar los elementos de protección personal (EPP) al trabajar con estas sustancias. Conclusiones: Se evidenció la falta de escolaridad y la ausencia del acompaflamiento técnico en esta zona, lo que induce a que estos agricultores realicen procesos agrícolas relacionados con la aplicación de plaguicidas sin el adecuado conocimiento y sin la utilización adecuada de EPP.
Introduction: Cholinesterases are enzymes responsible for hydrolyzing acetylcholine in acetic acid and choline, which ends nerve transmission along the synapse of neuromuscular junctions. Measurement of serum cholinesterase activity acts as an indicator of the effect caused by prolonged exposure to organophosphates and carbamates Objective: To determine serum cholinesterase levels and factors related to exposure to organophosphates in farmers from the county of Páramo Lagunas in San Pablo de Borbur, Boyacá, Colombia. Methodology: Prospective, cross-sectional study, with a sample of 57 agricultural workers, a survey of sociodemographic data and labor factors was applied to each individual and a venous blood sample was taken in an 8-12 hour fasting, Serum cholinesterase concentrations were determined (Biosystems® Butyrylthiocholine Cholinesterase kit), by means of the Biosystems® A-15 automated clinical chemistry kit. Results: The age range of the participants was between 22 and 64 years old, 72% of the individuals belonged to the male sex. 3.5% (2 male individuals) presented values lower than the biological reference interval (BRI); 88% of the people affirmed to carry out tasks with pesticides and 54% of them affirmed not to use personal protective equipment (PPE) when working with these substances. Conclusions: The lack of schooling was evidenced in most of the farmers, as well as the absence of technical support to this area, which induces these farmers to carry out agricultural processes such as pesticide application, without adequate knowledge and without the proper use of PPE.
ABSTRACT
Alzheimer's disease, which is a progressive neurologic disorder, is the most common form of dementia. Although there are various treatment options for Alzheimer’s disease, there is no definite treatment for this disease yet. In this study it was aimed to investigate the treatment potentials of three bis(3-(4-nitrophenyl)acrylamide) derivatives, two of which are known and one is new, for Alzheimer's disease. The study consists of three parts; in the first part of the study, synthesis and characterization studies of the investigated compounds were carried out. In the characterization of the compounds, IR, 1H-NMR, 13C-NMR, LC-MS and elemental analysis techniques were used. In the second part of the study, the compounds were investigated computationally with the assistance of various computational techniques including density functional theory (DFT) calculations, molecular docking and molecular dynamics simulations. In this part, binding free energy calculations were also performed on the investigated compounds. Results of computational studies showed that synthesized compounds interacted with AChE effectively and can be promising structures as AChE inhibitors. In the last part of the study, antioxidant and antimicrobial properties of the compounds were investigated. Antioxidant activities were determined by DPPH? and ABTS?? radical scavenging methods. According to the DPPH? test, the most active compound was found to be 2, while the most active compound was found to be 3 according to the ABTS? test, showing that these methods for antioxidant assay were not significantly correlated with each other. On the other hand, the results of the antimicrobial activity tests showed that compound 3 was the most active compound, which exhibited both antioxidant and antimicrobial activity.
ABSTRACT
Alzheimer's disease, which is a progressive neurologic disorder, is the most common form of dementia. Although there are various treatment options for Alzheimer’s disease, there is no definite treatment for this disease yet. In this study it was aimed to investigate the treatment potentials of three bis(3-(4-nitrophenyl)acrylamide) derivatives, two of which are known and one is new, for Alzheimer's disease. The study consists of three parts; in the first part of the study, synthesis and characterization studies of the investigated compounds were carried out. In the characterization of the compounds, IR, 1H-NMR, 13C-NMR, LC-MS and elemental analysis techniques were used. In the second part of the study, the compounds were investigated computationally with the assistance of various computational techniques including density functional theory (DFT) calculations, molecular docking and molecular dynamics simulations. In this part, binding free energy calculations were also performed on the investigated compounds. Results of computational studies showed that synthesized compounds interacted with AChE effectively and can be promising structures as AChE inhibitors. In the last part of the study, antioxidant and antimicrobial properties of the compounds were investigated. Antioxidant activities were determined by DPPH? and ABTS?? radical scavenging methods. According to the DPPH? test, the most active compound was found to be 2, while the most active compound was found to be 3 according to the ABTS? test, showing that these methods for antioxidant assay were not significantly correlated with each other. On the other hand, the results of the antimicrobial activity tests showed that compound 3 was the most active compound, which exhibited both antioxidant and antimicrobial activity.
ABSTRACT
Aims@#This study was aimed to investigate the anti-inflammatory and anti-rheumatoid effects of the Bacillus amyloliquefaciens derived surfactin.@*Methodology and results@#Crude and biosurfactant extracts were analyzed using thin-layer chromatography to determine the presence of biosurfactant. Both extracts were evaluated for their inhibitory effects against the acetylcholinesterase and 5-lipoxygenase enzymes. Human synovial cells were induced with TNF-α and IL-1β. The percentages of the cell viability for both normal and induced cells were determined with an MTT assay. Results showed that surfactin was detected in the biosurfactant extract and demonstrated higher inhibitory effects compared to the crude extract against both inhibitory enzymes acetylcholinesterse (IC50=30.60 μg/mL) and lipoxygenase (IC50=110.10 μg/mL). Both crudes showed no cytotoxic effects at the highest concentration used (50 μg/mL) against normal human synovial cells but showed active reactions against the induced cells. The anti-proliferative effects of biosurfactant and crude extracts were in dose-dependent manner.@*Conclusion, significance and impact of study@#Notably, surfactin obtained from B. amyloliquefaciens has shown an inhibitory effect against pro-inflammatory enzymes and cell viability of the induced rheumatoid arthritis cell line. These results highlighted the therapeutic potential of surfactin application as an anti-inflammatory agent for arthritis treatment. Further study is needed to elucidate the mechanisms underlying the anti-inflammatory effect of surfactin.
Subject(s)
Bacillus amyloliquefaciens , Surface-Active Agents , Anti-Inflammatory Agents , Rheumatoid FactorABSTRACT
Tetradenia riparia (Hochst.) Codd (Lamiaceae) is a shrub, commonly known as ginger bush or false myrrh, and several studies have shown that T. riparia exhibits a variety of biological properties. This study aimed to determine the chemical composition of T. riparia essential oil and its fractions, investigate their anticholinesterase activity, and assess their larvicidal activity against the cattle tick Rhipicephalus microplus and the mosquito Aedes aegypti. Eleven essential oil fractions were obtained by fractionation and analyzed by gas chromatography/mass spectrometry. Larvicidal activity against R. microplus and third-instar A. aegypti was assessed using a larval packet test and a larval immersion test, respectively. Anticholinesterase activity was determined by a bioautographic method. Forty-nine compounds were identified in the essential oil, of which the major classes were oxygenated sesquiterpenes (45.95%) and sesquiterpene hydrocarbons (35.20%) and the major components were isospathulenol (17.40%), ß-caryophyllene (15.61%), 14-hydroxy-9-epi-caryophyllene (10.07%), 14-hydroxy-α-muurolene (8.32%), and 9ß,13ß-epoxy-7-abietene (5.53%). Bioassays showed that T. riparia essential oil (LC50 = 1.56 µg/mL) and FR3 (LC50 = 0.30 µg/mL) were the most active against R. microplus and A. aegypti larvae, respectively. The essential oil and FR1, FR2, and FR3 exhibited acetylcholinesterase inhibitory activity. These results indicate that T. riparia essential oil and its fractions hold promise in the development of novel, environmentally safe agents for the control of R. microplus and A. aegypti larvae.
Subject(s)
Ticks , Aedes , Lamiaceae/toxicity , Lamiaceae/chemistry , LarvicidesABSTRACT
Background: Pternandra galeata belongs to the family Melastomataceae. It is a native flowering plant in Borneo Island that serve as food for monkey habitat. There has been limited study on the medicinal and chemical properties of this plant. Objectives: We investigated the acetylcholinesterase inhibitory activity and evaluated the antioxidant activity of the ethanolic extract of Pternandra galeata stem. The total phenolic content in the sample was also determined. Methods: The acetylcholinesterase inhibitory assays were performed using Ellman's method. Two different methods were used to evaluate the antioxidant activity of the extract by 2,2-diphenyl-1-picryl hydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The total phenolic content was determined by the Folin-Ciocalteu method by employing gallic acid as a reference. Results: The ethanolic extract of the P. galeata stems inhibited the AChE enzyme with an IC50 value of 74.62 ± 0.89 µg/mL.The sample exhibited antioxidant activity in the DPPH assay with an IC50 value of 20.21 ± 0.08 µg/mL and 7.68 ± 0.09 µg/mL in the ABTS scavenging assay. The total phenolic content was 164.71 ± 3.33 mg GAE/g extract. Conclusion: The ethanolic extract of the P. galeata stem can be a promising cholinesterase inhibitor and antioxidant for treating Alzheimer's disease
Antecedentes: Pternandra galeata pertenece a la familia Melastomataceae. Se trata de una planta con flores nativa de la isla de Borneo que sirve de alimento para el hábitat de los monos. Se han realizado pocos estudios sobre las propiedades medicinales y químicas de esta planta. Objetivos: Se investigó la actividad inhibidora de la acetilcolinesterasa y se evaluó la actividad antioxidante del extracto etanólico del tallo de Pternandra galeata. También se determinó el contenido fenólico total de la muestra. Métodos: Los ensayos de inhibición de la acetilcolinesterasa (AChE) se realizaron mediante el método de Ellman. Se utilizaron dos métodos diferentes para evaluar la actividad antioxidante de los ensayos de 2,2-difenil-1-picril hidrazilo (DPPH) y 2,2'-azinobis-(ácido 3-etilbenzotiazolina-6-sulfónico) (ABTS). El contenido fenólico total se determinó por el método de Folin-Ciocalteu empleando el ácido gálico como referencia. Resultados: El extracto etanólico de los tallos de P. galeata inhibió la enzima AChE con un valor IC50 de 74.62 ± 0.89 µg/mL. La muestra mostró actividad antioxidante en el ensayo DPPH con un valor IC50 de 20.21 ± 0.08 µg/mL y 7.68 ± 0.09 µg/mL en el ensayo de barrido ABTS. El contenido fenólico total fue de 164.71 ± 3.33 mg GAE/g de extracto. Conclusión: El extracto etanólico del tallo de P. galeata puede ser un prometedor inhibidor de la colinesterasa y antioxidante para el tratamiento de la enfermedad de Alzheimer.
Subject(s)
Humans , Alzheimer Disease , Cholinesterase Inhibitors , Phenolic Compounds , AntioxidantsABSTRACT
Background: Diabetes mellitus is associated with cognitive, neurophysiological, and structural changes in the central nervous system. Aim and Objective: The aim of the study was to evaluate the effect of Ficus benghalensis on cognitive behavior and acetylcholinesterase levels in brain of diabetic rats, and to compare with Piracetam and Glimepiride. Material and Methods: Wistar rats of either sex weighing 150–200 g were randomized into ten groups of ten each (five groups of diabetic rats and five groups of non-diabetic rats) where one group of diabetic and one group of non-diabetic rats each received F. benghalensis dose I (50 mg/kg), F. benghalensis dose II (100 mg/kg), Piracetam (200 mg/kg) and Glimepiride (0.5 mg/kg), and one group of diabetic rats and one group of non-diabetic rats served as the control group. The blood glucose levels were assessed at 0 and 30th days. The assessment of acquisition phase of each cognitive behavior test was done on 0, 14th, and 29th days, whereas retention phase was assessed on 1st, 15th, and 30th days. Results: In comparison with diabetic control group, F. benghalensis at both doses showed significant decrease in blood glucose levels as well as acquisition and retention of Transfer Latency in elevated plus maze on 29th and 30th days, respectively. Further, both doses exhibited significant increase in retention of step-down latency (SDL) on 30th in continuous avoidance apparatus, but only dose II showed significant increase in acquisition of SDL on 29th day. Similarly, significant increase in retention of Quadrant-time in Morris Water Maze was also observed with both doses of F. benghalensis and other groups compared to controls on 30th day. However, significant decrease in brain AChE level, was observed with only F. benghalensis dose II. Conclusion: Overall, the positive effects of F. benghalensis on cognition were comparable to other two groups, namely, Piracetam and Glimepiride. Hence, it can be concluded that F. benghalensis might be effective in alleviating the behavioral and biochemical changes in diabetes mellitus.
ABSTRACT
Phenytoin, a drug of choice for Epilepsy is also known for its adverse effects. The most common adverse effect due to phenytoin is cognition impairment. Cognition impairment is a serious problem in society as it debars the person's social life. Thus to overcome such a problem demand for a solution arises. Huperzine, sesquiterpene alkaloids having immense neuroprotective properties. Thus in this study, it was aimed to evaluate the effectiveness of huperzine on Phenytoin- induced Cognition Impairment. The protective effect of huperzine on phenytoin-induced cognition impairment was evaluated in rats. The effect of Huperzine on phenytoin-induced cognitive impairment was evaluated by behavioral, biochemical, and histopathological studies. The co-administration of huperzine with phenytoin showed significant results. The treatment of Huperzine with phenytoin resulted in significant improvement in learning and memory. The oxidative stress induced by Phenytoin was reversed by huperzine. A significant decrease in cholinesterase activity was also observed. The histopathology showed damaged neuronal cells in periventricular regions and cortex due to phenytoin which was altered by Huperzine. Thus, the present study demonstrates the protective effect of huperzine on phenytoin-induced cognition impairment.
ABSTRACT
Abstract The ß-carboline-1,3,5-triazine hydrochlorides 8-13 were evaluated in vitro against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The analysed compounds were selective to BuChE, with IC50 values in the range from 1.0-18.8 µM being obtained. The N-{2-[(4,6-dihydrazinyl-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-ß-carboline-3-carboxamide (12) was the most potent compound and kinetic studies indicate that it acts as a competitive inhibitor of BuChE. Molecular docking studies show that 12 strongly interacts with the residues of His438 (residue of the catalytic triad) and Trp82 (residue of catalytic anionic site), confirming that this compound competes with the same binding site of the butyrylthiocholine
Subject(s)
Triazines/adverse effects , In Vitro Techniques/methods , Pain , Acetylcholinesterase/pharmacology , Butyrylcholinesterase/pharmacology , Butyrylthiocholine/adverse effects , Carbolines/agonists , Cholinesterase Inhibitors/administration & dosage , Molecular Docking Simulation/instrumentationABSTRACT
OBJECTIV E To investigate the effect s and mechanism of the ethanol extract of Tiarella polyphylla （“TPE”）on learning and memory impairment in mice. METHODS Male Kunming mice were randomly divided into normal group ，model group，positive group （donepezil hydrochloride 4 mg/kg）and TPE low-dose ，medium-dose and high-dose groups （150，300，600 mg/kg），with 10 mice in each group. Drug administration groups were given relevant medicine intragastrically once a day ，and normal group and model group were given water intragastrically once a day ，for consecutive 22 d. On the 17th day ，administration groups and model group were intraperitoneally injected with scopolamine hydrobromide （3 mg/kg）to establish a model of learning and memory impairment. The learning and memory ability of the mice were evaluated by the Morris water maze. Hematoxylin-eosin （HE） staining was used for morphological observation of hippocampus cells of the mice. The levels of acetylcholinesterase （AChE），choline acetyltransferase （ChAT），superoxide dismutase （SOD），malondialdehyde（MDA），interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）in cerebral tissue as well as the relative expression of phosphorylated Tau protein （p-Tau），β-site amyloid precursor protein cleaving enzyme 1（BACE1）and amyloid precursor protein （APP）in hippocampus tissue were all detected. RESULTS The escape latency of mice in positive group ，TPE medium-dose and high-dose groups were all significantly shortened than the model group on the 4th to 5th day of training ，while the times of crossing platform and the percentage of movement distance in target quadrant were significantly increased （P＜0.05）. Compared with model group ，the neurons in the hippocampal CA 1 region of mice were increased to var ying degrees in administration groups ，the ne urons in solidified and atrophic state decreased ，and the arrangement of neurons tended to be close；the levels of ChAT and SOD in cerebral tissue were significantly increased in positive group and TPE medium-dose and high-dose groups ；the levels of AChE ，MDA，IL-6，the levels of TNF-α and relative expression of p-Tau ，BACE1 and APP in hippocampus tissue were decreased significantly （P＜ 0.05）. CONCLUSIONS TPE can improve the learning and memory impairment induced by scopolamine in mice ，and the mechanism may be related to balancing the brain cholinergic system ，alleviating oxidative stress injury ，improving inflammatory response，and inhibiting the overexpression of p-Tau ，BACE1 and APP .
ABSTRACT
Objective: To examine the protective effect of Vitamin D3 against Type 3 diabetes-induced cognitive dysfunction in rats. Materials and Methods: Type 3 diabetes was induced by a high-fat diet plus streptozotocin in rats. Rats were divided into seven groups: negative control, positive control, Vitamin D3 groups (100, 500 and 1000 IU/kg/day), Vitamin D3 plus rivastigmine, and rivastigmine monotherapy. A radial arm maze test was used to assess cognitive function. Levels of acetylcholinesterase (AChE), dopamine (DA), nerve growth factor, neurotrophin-3 (NT-3), and glial cell line-derived neurotrophic factor (GDNF) in the hippocampus were estimated by the enzyme-linked immunosorbent assay kits. Results: Chronic treatment with Vitamin D3 significantly (P < 0.05) and dose dependently alleviated cognitive deficits, with enhancing cholinergic transmission pathway activity through attenuated hippocampal AChE and increased DA level (P < 0.001). Moreover, Vitamin D3 significantly increased (P < 0.001) neurotrophin levels as an underlying mechanism for the resulted improvement. Conclusion: Vitamin D3 plus rivastigmine (combined group) is better than Vitamin D (100 and 500 mg/kg/day) for improvement of AChE, DA, NT-3, and GDNF levels. Vitamin D (500 and 1000 IU/kg/day) was effective as a combined group in terms of the behavioral test.
ABSTRACT
The venom of the krait (Bungarus sindanus), an Elapidae snake, is highly toxic to humans and contains a great amount of acetylcholinesterase (AChE). The enzyme AChE provokes the hydrolysis of substrate acetylcholine (ACh) in the nervous system and terminates nerve impulse. Different inhibitors inactivate AChE and lead to ACh accumulation and disrupted neurotransmission. Methods: The present study was designed to evaluate the effect of palladium(II) complex as antivenom against krait venom AChE using kinetics methods. Results: Statistical analysis showed that krait venom AChE inhibition decreases with the increase of Pd(II) complex (0.025-0.05 µM) and exerted 61% inhibition against the AChE at a fixed concentration (0.5 mM) of ACh. Kinetic analysis using the Lineweaver Burk plot showed that Pd(II) caused a competitive inhibition. The compound Pd(II) complex binds at the active site of the enzyme. It was observed that K m (Michaelis-Menten constant of AChE-ACh into AChE and product) increased from 0.108 to 0.310 mM (45.74 to 318.35%) and V max remained constant with an increase of Pd(II) complex concentrations. In AChE K Iapp was found to increase from 0.0912 to 0.025 µM (29.82-72.58%) and did not affect the V maxapp with an increase of ACh from (0.05-1 mM). K i (inhibitory constant) was estimated to be 0.029µM for snake venom; while the K m was estimated to be 0.4 mM. The calculated IC50 for Pd(II) complex was found to be 0.043 µM at constant ACh concentration (0.5 mM). Conclusions: The results show that the Pd(II) complex can be deliberated as an inhibitor of AChE.(AU)
Subject(s)
Animals , Bungarus , Elapid Venoms/toxicity , Synthetic Biology , Palladium , AcetylcholinesteraseABSTRACT
A guanitoxina (GNT) é uma neurotoxina produzida por algumas cepas de cianobactérias dos gêneros Dolichospermum e Sphaerospermopsis>. A GNT é o único organofosforado natural, capaz de causar a morte de animais selvagens e domésticos devido à inibição irreversível da acetilcolinesterase. Apesar de sua alta toxicidade, o diagnóstico da GNT em amostras biológicas ainda é um grande desafio. A dificuldade para sua detecção está diretamente ligada à sua instabilidade em altas temperaturas e pH alcalino, tornando difícil seu monitoramento em corpos d'água. Por isso, esta pesquisa objetivou estudar a estabilidade e biodisponibilidade da GNT em amostras aquosas, com intuito de obter mais informações sobre a natureza química e biológica dessa potente neurotoxina. Para realizar este estudo, a cepa ITEP-24 (S. torques-reginae) produtora de GNT foi cultivada em laboratório sob condições controladas, para obter biomassa para os experimentos de extração, semi-isolamento, estabilidade, ensaio in vitro e identificação por LC-MS/MS. Primeiramente foram realizados testes de extração da GNT partir de células liofilizadas da cepa ITEP-24 utilizando água, metanol e etanol em pH ácido. Depois utilizou-se dois métodos de extração em fase sólida (SPE) com cartuchos preenchidos com fases estacionarias C18 em fase reversa e sílica gel em fase normal, com objetivo de avaliar qual método de SPE seria melhor para extrair e concentrar a GNT. Nós também testamos métodos para lisar as células com sondas de ultrassom, misturador e centrifugação. Além dos métodos de extração, nós avaliamos a estabilidade da toxina em diferentes temperaturas, para isso a biomassa seca contendo a GNT ficou condicionada a 4 °C, 23 °C, -20 °C, -80 °C durantes seis meses, e análises de identificação foram realizadas dentro período de 150 dias em uma sequência de 30 dias. A estabilidade da toxina foi analisada também a partir de extrações em soluções com diferentes valores de pH (1,5; 3,0; 5,0; 7,0; 8,5; 10,5) e temperatura (23 ºC e 37 ºC). Depois, analisou-se a biodisponibilidade da GNT em células frescas da linhagem ITEP-24 através de teste de dissolução in vitro. O objetivo deste teste foi avaliar a liberação da toxina intracelular em meio simulado do conteúdo gástrica e intestinal com e sem enzimas digestivas para compreender e estimar a disponibilidade da GNT in vivo. Os resultados de todos experimentos descritos neste estudo, foram obtidos a partir de análises por cromatografia líquida de interação hidrofílica (HILIC) acoplado ao espectrômetro de massas do tipo triplo quadrupolo LC-QqQ-MS/MS utilizando as transições 253>58, 253>159 e 159>58 [M+H]+ utilizando coluna com fase estacionária zwitteriônica (ZIC). A identificação da GNT foi realizada também por cromatografia líquida acoplada ao espectrômetro de massas de alta resolução (LC-HR-QTOF-MS) com coluna Luna C18, Hydro-RP C18 e ZIC-HILIC. Dos protocolos de extração testados, a combinação de metanol/água (70:30 v/v) com ácido acético (0.3%) extraiu maior quantidade relativa da GNT a partir de células frescas e liofilizadas da cepa ITEP-24 e a concentração da toxina foi maior em amostras de células frescas. Em relação aos métodos de lise celular, as extrações realizadas em sonda de ultrassom com banho-maria e centrifugação por 1h foram estatisticamente significantes para liberar a toxina intracelular. Não houve diferença significativa entre os testes de SPE, no entanto, a semipurificação da toxina foi melhor com cartucho preenchido com sílica gel em fase normal e adaptação desse método em coluna aberta permitiu obter uma fração enriquecida com GNT. A GNT mostrou ser mais estável em pH ácido, sendo o pH 3,0 o melhor para manter e extrair a toxina em amostras aquosas e a toxina intracelular presente em células secas podem degradar em temperatura de 23 °C por um período de 150 dias mesmo em solução com pH 3,0. Durante os testes de extração e purificação foi observado também a degradação da toxina em processos de secagem e ressuspensão. As análises realizadas no LC-HR-QTOF-MS com diferentes métodos cromatográficos possibilitou a identificação da GNT, porém o método realizado com coluna ZIC-HILIC mostrou melhor resolução cromatográfica dos picos relativos m/z e tempo de retenção de toxina. Os resultados obtidos nos testes de dissolução in vitro mostraram que a GNT fica mais disponível no simulado gástrico com e sem a enzima pepsina, mas também pode ser absorvida no intestino. Portanto, o teste de dissolução in vitro pode ser uma ferramenta útil para a avaliação de risco de cianotoxinas in vivo, devido ao seu potencial de monitorar qualitativa e quantitativamente substâncias dissolvidas em fluidos gastrointestinais. Os resultados apresentados neste estudo fornecem informações valiosas para uma melhor compreensão da estabilidade e biodisponibilidade do GNT. Além disso, os métodos apresentados neste estudo podem ser úteis para diversas aplicações projetadas para identificar a toxina em amostras ambientais, bem como orientações para procedimentos de purificação da GNT
Guanitoxin (GNT) is a neurotoxin produced by some strains of cyanobacteria of the genus Dolichospermum and Sphaerospermopsis. GNT is the only natural organophosphate, capable of causing the death of animals from wild and domestic animals due to irreversible inhibition of acetylcholinesterase. Despite its high toxicity, the diagnosis of GNT in biological samples is still a significant challenge. The difficulty in its detection is directly linked to its instability at high temperatures and alkaline pH, making it difficult to monitor in bodies of water. Therefore, this research aimed to study the stability and bioavailability of GNT in aqueous samples to provide more information about the chemical and biological nature of this molecule. The strain ITEP-24 (S. torques-reginae) producing GNT was grown in the laboratory under controlled conditions to obtain biomass for the extraction, semi-isolation, stability, in vitro tests, and toxin identification by LC-MS/MS. Firstly, tests were carried out to extract GNT from lyophilized cells strain ITEP-24 using water, methanol, and ethanol at acidic pH and, two SPE methods in cartridges with stationary phases of C18 reverse phase and normal phase gel silica, to evaluate which would be better to extract and concentrate the GNT. We also tested different methods of cell lysis, such as ultrasound probes, mixers, and centrifugation. In addition to the extraction methods, the stability of the toxin was evaluated at different temperatures, for this, the dry biomass containing the toxin was conditioned at 4 °C, 23 °C, -20 °C, -80 °C for 150 days and analysis of the identification of the GNT was carried out within that period in a sequence of 30 days. The toxin stability was also analyzed from extractions in solutions with different pH values (1.5; 3.0; 5.0; 7.0; 8.5; 10.5) and temperature (23 ºC and 37 ºC). In addition, we performed dissolution tests with fresh cells of the ITEP-24 strain to evaluate the bioavailability of GNT in simulated gastric and intestinal fluids with and without digestive enzymes to understand and estimate the availability of GNT in vivo. The results of all experiments described in this study were obtained from analyzes by hydrophilic interaction liquid chromatography (HILIC) coupled to the LC-QqQ-MS/MS triple quadrupole mass spectrometer using the transitions m/z 253> 58, m/z 253> 159 and m/z 159> 58 [M + H]+ using a column with the zwitterionic stationary phase (ZIC). Liquid chromatography coupled to the high-resolution mass spectrometer (LC-HR-QTOF-MS) with Luna column C18, Hydro-RP C18, and ZIC-HILIC carried out the identification of the GNT. From the extraction protocols tested, the combination of methanol/water (70:30 v/v) with acetic acid (0.3%) extracted a greater relative amount of GNT from fresh and lyophilized ITEP-24 cells, and the concentration of the toxin is higher previously fresh. Concerning cellular methods, the ultrasound probe with a water bath and centrifugation for 1h ware statistically significant to release the intracellular toxin. There was no significant difference between the SPE tests. However, the semi-purification of the toxin was better with a cartridge filled with gel silica in the normal phase and adaptation of this method in an open column allowed to obtain a fraction enriched with GNT. GNT was more stable at acid pH, with pH 3.0 being the best to maintain and the intracellular toxin present in dry cells can degrade at a temperature at 23 °C for 150 days even in pH 3.0 solution. The toxin can also hydrolyze in the drying and resuspension processes. The analyzes carried out in LC-HR-QTOF-MS with different chromatographic methods made it possible to identify the GNT itself, however, the ZIC-HILIC column method showed excellent chromatographic resolution of the relative m/z peaks and toxin retention time. The results obtained in the in vitro dissolution tests showed that GNT is more available in the gastric simulation with and without the enzyme pepsin, but it can also be absorbed in the intestine. Thus, in vitro dissolution tests can be used as a useful tool for the risk assessment of cyanotoxins in vivo due to their potential to qualitatively and quantitatively monitor substances dissolved in gastrointestinal fluids. The results presented in this study provide valuable information for a better understanding of the stability and bioavailability of GNT. Besides, the methods presented in this study can be useful for various applications designed to identify the toxin in environmental samples, as well as guidance on procedures for purifying GNT
Subject(s)
Acetylcholinesterase/adverse effects , Mass Spectrometry/methods , Diagnosis , Methods , Organophosphorus Compounds/antagonists & inhibitors , In Vitro Techniques/methods , Chromatography, Liquid/methods , Cyanobacteria/metabolism , Solid Phase Extraction/instrumentation , Hydrophobic and Hydrophilic Interactions , Hydrogen-Ion ConcentrationABSTRACT
Abstract This study reports the action of essential oils (EO) from five plants on the activity of native and recombinant acetylcholinesterases (AChE) from Rhipicephalus microplus. Enzyme activity of native susceptible AChE extract (S.AChE), native resistant AChE extract (R.AChE), and recombinant enzyme (rBmAChE1) was determined. An acetylcholinesterase inhibition test was used to verify the effect of the EO on enzyme activity. EO from Eucalyptus globulus, Citrus aurantifolia, Citrus aurantium var.dulcis inhibited the activity of S.AChE and R.AChE. Oils from the two Citrus species inhibited S.AChE and R.AChE in a similar way while showing greater inhibition on R.AChE. The oil from E. globulus inhibited native AChE, but no difference was observed between the S.AChE and R.AChE; however, 71% inhibition for the rBmAChE1 was recorded. Mentha piperita oil also inhibited S.AChE and R.AChE, but there was significant inhibition at the highest concentration tested. Cymbopogon winterianus oil did not inhibit AChE. Further studies are warranted with the oils from the two Citrus species that inhibited R.AChE because of the problem with R. microplus resistant to organophosphates, which target AChE. C. winterianus oil can be used against R. microplus populations that are resistant to organophosphates because its acaricidal properties act by mechanism(s) other than AChE inhibition.
Resumo Este estudo relata a ação de óleos essenciais de cinco plantas na atividade de acetilcolinesterases (AChE) nativas e recombinantes de Rhipicephalus microplus. A atividade enzimática do extrato de acetilcolinesterase nativa suscetível (S.AChE) e resistente (R.AChE) e da enzima recombinante (rBmAChE1) foi determinada. Um teste de inibição da AChE foi utilizado, para verificar o efeito dos óleos essenciais sobre a atividade enzimática. Óleos essenciais de Eucalyptus globulus, Citrus aurantifolia, Citrus aurantium var. dulcis inibiram a atividade de S.AChE e R.AChE. Os óleos das duas espécies de Citrus inibiram S.AChE e R.AChE de maneira semelhante, mas mostraram maior inibição sobre R.AChE. O óleo de E. globulus inibiu a AChE nativa, mas sem diferença entre a S.AChE e a R.AChE; no entanto, 71% de inibição para rBmAChE1 foi observada. O óleo de Mentha piperita também inibiu S.AChE e R.AChE, mas houve inibição significativa apenas nas concentrações mais altas testadas. O óleo de Cymbopogon winterianus não inibiu a AChE. Estudos adicionais são necessários com os óleos das duas espécies de Citrus que inibiram a R.AchE, devido ao problema de R. microplus resistente aos organofosforados ter como alvo AChE. O óleo de C. winterianus pode ser usado contra populações de R. microplus, que são resistentes a organofosforados, porque suas propriedades acaricidas agem por mecanismos diferentes.
Subject(s)
Animals , Oils, Volatile/pharmacology , Cholinesterase Inhibitors/pharmacology , Cymbopogon , Rhipicephalus/enzymology , Acaricides/pharmacology , Acetylcholinesterase , LarvaABSTRACT
O Brasil é o maior consumidor global de agrotóxico tendo como consequência intoxicações de um grande número de trabalhadores, com altos gastos com a saúde e prejuízos ambientais. Objetivo geral: avaliar os efeitos do uso de agrotóxicos sobre a saúde de trabalhadores atuantes na agricultura familiar. Método: estudo transversal, analítico e comparativo entre o Grupo (1) composto por 80 trabalhadores rurais atuantes na agricultura familiar, expostos diretamente a agrotóxicos e o Grupo (2) composto por 60 usuários dos Programas de Saúde da Família (PSF) dos municípios de Capitólio e Pimenta - Minas Gerais - não diretamente expostos a agrotóxicos sendo o grupo controle por meio da análise de biomarcadores colinesterase eritrocitária e plasmática. Resultados: dos 140 participantes deste estudo, a maioria (51,2%) não referiu problemas de saúde e tratamento medicamentoso, 6,3% deles, expostos a agrotóxicos, referiram intoxicações e 42,5% relataram terem algum sinal e/ou sintoma de intoxicação com predominância (33,8%) para a dor de cabeça. O biomarcador colinesterase eritrocitária apresentou normalidade em 63,8% dos trabalhadores rurais e 36,2% com alteração na butirilcolinesterase. Conclusão: a butirilcolinesterase encontrada em 1/3 dos participantes expostos a agrotóxicos correlacionam-se à presença dos sintomas dor de cabeça e irritabilidade/nervosismo. Existe, portanto, a necessidade da educação e capacitação dos agricultores no uso de agrotóxicos para limitar níveis de exposição e efeitos nocivos à saúde para prevenir, diagnosticar e tratar possíveis disfunções causadas por esses produtos químicos
Brazil is the largest global consumer of pesticides, resulting in the intoxication of a large number of workers, with high expenditures on health and environmental damage. General objective: to evaluate the effects of the use of pesticides on the health of rural workers working in family farming in terms of intoxication through the evaluation of the erythrocyte and plasma cholinesterase biomarkers. Method: cross-sectional, analytical and comparative study between Group (1) of rural workers working in family farming, directly exposed to pesticides and Group (2) of users of Family Health Programs (PSF), in the municipalities of Capitólio and Pimenta - Minas Gerais State (Brazil) - not directly exposed to pesticides, being a control group through the analysis of biomarkers. Results: in this study of 140 participants, the most of them did not report health problems and drug treatment, 6.3% of participants, exposed to pesticides, reported poisoning and 42.5% reported having any signs and/or symptoms of intoxication with predominance for headache. The erythrocyte cholinesterase biomarker showed normality in 63.8% rural workers and 36.2% with alteration in butyrylcholinesterase. Conclusion: butyrylcholinesterase alteration was proven in 36.2% of participants exposed to pesticides and these results correlate with the presence of headache and irritability/nervousness symptoms. There is, therefore, a need for education and training of farmers to limit levels of exposure and harmful health effects to prevent, diagnose and treat possible dysfunctions caused by these chemicals applied to agriculture
Subject(s)
Humans , Female , Adult , Middle Aged , Occupational Risks , Biomarkers , Rural Health , Occupational Exposure , Pesticide Exposure , AgricultureABSTRACT
portuguêsO Brasil é o maior consumidor global de agrotóxico tendo como consequência intoxicações de um grande número de trabalhadores, com altos gastos com a saúde e prejuízos ambientais. Objetivo geral: avaliar os efeitos do uso de agrotóxicos sobre a saúde de trabalhadores atuantes na agricultura familiar. Método: estudo transversal, analítico e comparativo entre o Grupo (1) composto por 80 trabalhadores rurais atuantes na agricultura familiar, expostos diretamente a agrotóxicos e o Grupo (2) composto por 60 usuários dos Programas de Saúde da Família (PSF) dos municípios de Capitólio e Pimenta - Minas Gerais - não diretamente expostos a agrotóxicos sendo o grupo controle por meio da análise de biomarcadores colinesterase eritrocitária e plasmática. Resultados: dos 140 participantes deste estudo, a maioria (51,2%) não referiu problemas de saúde e tratamento medicamentoso, 6,3% deles, expostos a agrotóxicos, referiram intoxicações e 42,5% relataram terem algum sinal e/ou sintoma de intoxicação com predominância (33,8%) para a dor de cabeça. O biomarcador colinesterase eritrocitária apresentou normalidade em 63,8% dos trabalhadores rurais e 36,2% com alteração na butirilcolinesterase. Conclusão: a butirilcolinesterase encontrada em 1/3 dos participantes expostos a agrotóxicos correlacionam-se à presença dos sintomas dor de cabeça e irritabilidade/nervosismo. Existe, portanto, a necessidade da educação e capacitação dos agricultores no uso de agrotóxicos para limitar níveis de exposição e efeitos nocivos à saúde para prevenir, diagnosticar e tratar possíveis disfunções causadas por esses produtos químicos
Brazil is the largest global consumer of pesticides, resulting in the intoxication of a large number of workers, with high expenditures on health and environmental damage. General objective: to evaluate the effects of the use of pesticides on the health of rural workers working in family farming in terms of intoxication through the evaluation of the erythrocyte and plasma cholinesterase biomarkers. Method: cross-sectional, analytical and comparative study between Group (1) of rural workers working in family farming, directly exposed to pesticides and Group (2) of users of Family Health Programs (PSF), in the municipalities of Capitólio and Pimenta - Minas Gerais State (Brazil) - not directly exposed to pesticides, being a control group through the analysis of biomarkers. Results: in this study of 140 participants, the most of them did not report health problems and drug treatment, 6.3% of participants, exposed to pesticides, reported poisoning and 42.5% reported having any signs and/or symptoms of intoxication with predominance for headache. The erythrocyte cholinesterase biomarker showed normality in 63.8% rural workers and 36.2% with alteration in butyrylcholinesterase. Conclusion: butyrylcholinesterase alteration was proven in 36.2% of participants exposed to pesticides and these results correlate with the presence of headache and irritability/nervousness symptoms. There is, therefore, a need for education and training of farmers to limit levels of exposure and harmful health effects to prevent, diagnose and treat possible dysfunctions caused by these chemicals applied to agriculture
Subject(s)
Humans , Male , Female , Adult , Occupational Risks , Biomarkers , Cholinesterases/toxicity , Occupational Exposure/adverse effects , Pesticide Exposure , FarmersABSTRACT
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.
Subject(s)
In Vitro Techniques/instrumentation , Cholinesterase Inhibitors/analysis , Lauraceae/classification , Ocotea/adverse effects , Molecular Docking Simulation/instrumentation , Plants, Medicinal/anatomy & histology , Acetylcholinesterase/adverse effects , Spectrophotometry/instrumentation , Flavonoids , Butyrylcholinesterase/adverse effects , AlkaloidsABSTRACT
Hippeastrum puniceum is a species that belongs to the Amaryllidaceae family. A particular characteristic of this family is the consistent and very specific presence of isoquinoline alkaloids, which have demonstrated a wide range of biological activities such as antioxidant, antiviral, antifungal, antiparasitic, and acetylcholinesterase inhibitory activity, among others. In the present work, fifteen alkaloids were identified from the bulbs of Hippeastrum puniceum (Lam.) Kuntz using a GC-MS approach. The alkaloids 9-O-demethyllycoramine, 9-demethyl-2α-hydroxyhomolycorine, lycorine and tazettine were isolated through chromatographic techniques. The typical Amaryllidaceae alkaloids lycorine and tazettine, along with the crude and ethyl acetate extract from bulbs of the species were evaluated for their inhibitory potential on α-amylase, α-glucosidase, tyrosinase and acetylcholinesterase activity. Although no significant inhibition activity was observed against α-amylase, α-glucosidase and tyrosinase from the tested samples, the crude and ethyl acetate extracts showed remarkable acetylcholinesterase inhibitory activity. The biological activity results that correlated to the alkaloid chemical profile by GC-MS are discussed herein. Therefore, this study contributed to the knowledge of the chemical and biological properties of Hippeastrum puniceum (Lam.) and can subsidize future studies of this species