CHARLES UNIVERSITY Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Diana Karaščáková Supervisor: prof. RNDr. Lubomír Opletal, CSc. Title of diploma thesis: Biological activity of plants secondary metabolites XXX.; Basic search of selected taxons on anticholinesterase activity As part of the study of the biological activity of secondary metabolites, 7 taxa of higher plants were selected. The study deals with basic research of summary and alkaloid extracts prepared from morphological parts of plants Annona muricata (leaves), Leonotis leonurus (perch), Turnera diffusa (perch), Hamelia patens (perch), Uncaria guianensis (bark), Allamanda cathartica (perch) and Morinda citrifolia (leaves). To elucidate the presence of the major types of secondary metabolites, extracts were prepared and subjected to detection reactions by TLC using ten detection reagents. After detection by Dragendorff's reagent of ethyl acetate extracts, alkaloids were significantly present only in Annona muricata. Alkaloids were not detected in the bark of Uncaria guianensis. Using the Ellman method, the extracts were tested for potential inhibitory activity against human brain cholinesterases, using both recombinant enzymes. No significant active substances were present in any of the measured...

Valová D.: Vinca minor L. alkaloids and their biological activity III. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2020, 75 p. Alzheimer's disease (AD) is a progressive, neurodegenerative disorder and it's the most common form of dementia. Because we're still not able to treat the cause of disease, searching for a new substance is relevant. This thesis is focused on isolation of alkaloids from a Vinca minor L. alkaloidal extract as a potential drug. The preparation and column chromatography of the summary extract were performed by Ing. Miroslav Ločárek as a part of his doctoral studies. Subsequent preparative TLC led to the isolation of three compounds. The chemical structures of the isolated alkaloids were elucidated by means of optical rotation, NMR and MS analyses and by comparison of the obtained data with those in the literature. One of the compounds was determined as(-)-vincine, other two compounds have not been isolated yet. Isolated compounds were also tested for their biological activity. Vincine, DV-1 a DV-3 were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Additionally, vincine and DV-3 were also tested for their inhibitory activity on prolyl...

Vašíčková A.: Alkaloids of Vinca minor L. and their biological activity (inhibition of human cholinesterases) V. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové 2020. From the selected fraction VM 215-258 were isolated 3 alkaloids by flash chromatography followed, preparative thin layer chromatography and crystallization. Their structure was determined by mass spectroscopy, NMR and optical rotation, and the obtained data were compared with those in literature. Strictamine and minovincinine belong to alkaloids previously isolated, the alkaloid AV-3 has not been isolated yet. These alkaloids were tested for their ability to inhibit human cholinesterases and their inhibitory activity was compared to standards galanthamine and physostigmine. Compound AV-3 showed mild inhibitory activity against BChE (IC50 μM > 86.3 ± 2.3), other alkaloids were considered inactive, their IC50 values against cholinesterases were > 100 μM. Key words: Vinca minor L. (Apocynaceae), vinca alkaloids, minovincinine, strictamine, Alzheimer's disease, acetylcholinesterase, butyrylcholinesterase.

Faschingbauer J.: Cytotoxic and cholinesterase inhibitory activity of extracts from selected species of the Centaurea L. genus. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2019. During the screening of biologically active secondary metabolites of plants carried out at the Department of Pharmaceutical Botany FAF UK, selected taxa of the genus Centaurea (Asteraceae) were investigated. This study is focused on a basic phytohemical research of extracts prepared from Centaurea cyanus, Centaurea jacea, Centaurea scabiosa, Centaurea pseudophrygia, Centuarea stoebe, Centaurea solstitialis a Centaurea benedicta. Extracts were prepared for evidence of the proof reactions of TLC and MS analysis (EI, ESI) to clarify a potential presence of alkaloids. EtOAc and ethanol extracts were evaluated for potential inhibitory activity against human erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE) and cytotoxicity against selected 9 tumor lines. C. cyanus alkaloid extract had interesting cholinesterase activity which selectively inhibited BChE (IC50 BChE = 22.62 ± 3.62 μg / ml, IC50 AChE = 221.50 ± 44.56 g / ml). Other EtOAc extracts of selected Centaurea species were considered inactive (IC50 > 100 μg/ml)....

The objectives of this two part prospective study were to evaluate the effects of statins on C-reactive protein (CRP), butyrylcholinesterase (BChE), lipids, and the relationships between these parameters. Subjects enrolled in this study were separated into two cohorts. The first group (study 1) consisted of 37 subjects converted from pravastatin to cerivastatin. The second group (study 2) consisted of 11 subjects with diabetes initiated on cerivastatin therapy. The subjects were followed for 12-weeks in the Lipid Clinic at David Grant Medical Center at Travis Air Force Base. CRP, BChE, total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides (TG) were measured at baseline, 6-weeks, and 12-weeks. In study 1, CRP (p = 0.704) and BChE (p = 0.746) remained relatively stable over 12-weeks. The lipid panel changed significantly with TC (p < 0.001) and LDL (p < 0.001) decreasing and HDL (p = 0.017) increasing. Although TG declined numerically, it was not statistically significant (p = 0.649). A significant negative correlation was detected at baseline (r = −0.353, p = 0.032), but lost at 6-weeks and 12-weeks. In study 2, CRP declined by 42.9%, but was not statistically significant (p = 0.178). BChE remained relatively stable over 12-weeks (p = 0.666). TC (p < 0.001) and LDL (p < 0.001) declined and TG (p = 0.035) fluctuated over the course of the study. HDL increased, but it was not statistically significant (p = 0.396). Significant positive correlations were seen between CRP and TG (r = 0.908, p < 0.001) and BChE and TC (r = 0.721, P = 0.012) at baseline and BChE and TG (r = 0.64, p = 0.034) at 12-weeks. These results suggest that statin effects on CRP are independent of the lipid-lowering effects and switching statins may not affect CRP disposition. Cerivastatin does not appear to have effects on BChE activity. Lastly, a possible competitive relationship may exist between CRP and BChE. This is suggested by the negative correlation seen in study 1 and with the gain in correlation between BChE and TG as the correlation was lost between CRP and TG in study 2.

Organophosphates are insecticides which result in acute adverse signs when exposed at toxic doses by animals and lead to death if left untreated. The current treatment for organophosphate toxicity includes atropine and the federally approved oxime 2-PAM. However, 2-PAM is not very effective at crossing the blood brain barrier which results in prolonged inactivation of acetylcholinesterase, which is the primary target of organophosphates, in the brain even after administration. The novel oximes, Oxime 15 and Oxime 20, are able to cross the blood brain barrier and reactivate the inhibited acetylcholinesterase. In this experiment with six animal species frequently used in toxicity studies, they were proven to be just as effective and sometimes better than 2-PAM at reactivating acetylcholinesterase or butyrylcholinesterase inhibited by paraoxon, chlorpyrifos-oxon, phorate-oxon, or dicrotophos. The detoxication enzymes butyrylcholinesterase, carboxylesterase, and paraoxonase were also studied as potential influences of the toxicity of the organophosphates in these different species.

Les pesticides et toxiques de guerre organophosphorés sont responsables d'intoxications qui se révèlent préoccupantes pour les autorités sanitaires. La recherche de solutions thérapeutiques pour pallier le manque de moyens efficaces pour contrer ces intoxications apparaît comme essentielle. L'acétylcholinestérase (AChE), enzyme de régulation de l'influx nerveux, en est la cible principale.Au cours de cette thèse, nous avons, d'une part, mis en place une stratégie de conception de molécules capables de réactiver les cholinestérases dites vieillies. Dans ce but, plusieurs dizaines de molécules ont été conçues et synthétisées. Leur évaluation in silico par docking moléculaire et in vitro par mesure d'affinité pour l'enzyme et par étude cristallographique n'a pas permis d'obtenir la réalkylation espérée, mais ce travail nous a offert quelques nouvelles perspectives.D'autre part, nos travaux de cristallographie de l'inhibition de la butyrylcholinestérase humaine par les agents V et le sarin montrent que cette cholinestérase a une énantiosélectivité altérée pour ces inhibiteurs chiraux par rapport à l'AChE humaine. Ceci implique la nécessité de quantités d'enzyme plus importantes pour atteindre la capacité protectrice désirée et donc un surcoût non négligeable. L'AChE humaine serait finalement un bioépurateur de neurotoxiques organophosphorés économiquement plus viable. / Organophosphate pesticides and chemical warfare are responsible for poisoning that pose to health security issues. The research of therapeutic solutions to overcome the lack of effective means to counter these poisonings is essential. Acetylcholinesterase (AChE), the enzyme involved in the regulation of nerve impulses, is the main target. ln this thesis, we have, first, set up a strategy for designing molecules that can reactivate aged cholinesterases. To this purpose, severa! dozen molecules have been designed and synthesized. Evaluation in silico by molecular docking and in vitro by rneasuring affinity for the enzyme and crystallographic study did not observe the desired realkylation. But this work opened new perspectives.Secondly, our crystallographic work on the inhibition of human butyrylcholinesterase by V-agents and sarin shows that cholinesterase has altered enantioselectivity for these chiral inhibitors of human AChE. This implies that larger amounts of enzyme are required for the desired protection and thus, a significant additional cost. Human AChE seems finally a more suitable neurotoxic bioscavenger.

Introduction: Cholinesterases and Paraoxonase 1 are mediators of poisoning process by organophosphate (OP). Monitor the activities of these enzymes and know the genetic variability of the agricultural population is of great importance in the evaluation of possible risk groups for poisoning OP. Objective: This case-control study aimed to investigate the association of genetic markers (tag SNPs) in BchE and PON1 genes with characteristic symptoms of exposure to OP pesticides in rural workers.Methodology: 427 patients, both sexes, mean age 40.96 years old, divided into 226 with and 201 without characteristic symptomatology of exposure to pesticides, were genotyped for three tag SNPs, rs1803274 (BChE gene), rs662 e rs854560 (PON1 gene). Also, socio-demographic and economic parameters were analyzed. BChE activity was demonstrated, as well as the profile of workers' health was evaluated. For the test of association of the categorical variables, the Chi-square test and Fisher's exact test. For the genetic models tests the Binary Logistic Regression was used in the Additive Model and Fisher's Exact Test and Chi-SquareTest in the Dominant and Recessive Models (p < .05). Results: Associationstatistically significant for place of residence with symptoms presentation; between the rs1803274 and symptoms, in the Recessive model. Muscular weakness was the most representative symptom, presenting a statistical association with rs1803274, in the Additive and Dominant model. BChE activity was associated with rs662 in theRecessive model, demonstrating that individuals with this SNP have a higher chance of presenting reduced activity for this enzyme. Conclusion: The results demonstrate that the intrinsic factors (SNPs) and extrinsic (residence) studied can modulate theprocess of intoxication by OR, increasing or reducing the susceptibility of the individuals involved. / Introdução: As colinesterases e a Paraoxonase 1 atuam como mediadores do processo de intoxicação por organofosforado (OF). Monitorar as atividades destas enzimas e conhecer a variabilidade genética da população agrícola é de grande importância na avaliação de possíveis grupos de risco para intoxicação por OF. Objetivo: Investigar associação dos polimorfismos genéticos rs1803274 (geneBChE) e rs662 e rs854560 (gene PON1) com sintomatologia característica de exposição a agrotóxicos OF em trabalhadores rurais. Metodologia: Estudo Casocontrole com abordagem transversal. Composto por 427 pacientes, ambos os gêneros, média de idade de 40,96 (±12,6) anos, divididos em: G1-226 indivíduos com presença de sintomas característicos de intoxicação por OF e G2-201 indivíduos sem presença de sintomas; foram genotipados para 3 SNPs: rs1803274 (gene BChE), rs662 e rs854560 (gene PON1). Além disso, os parâmetros socioeconômicos, sociodemográficos, perfil de saúde e a atividade de BChE foram analisados. Para teste de associação das variáveis categóricas, foi utilizado o teste Qui-Quadrado e Exato de Fischer. Para os testes dos modelos genéticos a Regressão Logística Binária foi utilizada no Modelo Aditivo e o Teste Exato de Fisher e teste Qui-Quadrado nos Modelos Dominante e Recessivo (p<0,05). Resultados: Associação estatisticamente significante para local de residência com apresentação de sintomas; entre o rs1803274 e sintomas, no modelo Recessivo. A fraqueza muscular se mostrou o sintoma mais representativo, apresentando uma associação estatística com o rs1803274, no modelo Aditivo e Dominante. A atividade de BChE apresentou associação com o rs662, no modelo Recessivo, demonstrando que indivíduos com este SNP tem maior chance de apresentar atividade reduzida para esta enzima. Conclusão: Os resultados demonstram que os fatores intrínsecos (SNPs) e extrínsecos (local de residência) estudados podem modular o processo de intoxicação por OF, aumentando ou reduzindo a susceptibilidade dos indivíduos envolvidos. / Lagarto, SE

Aquatic environments are ecosystems that most affected impacts caused by anthropogenic action, since it is the final compartments of various products produced by human activity. This study evaluated the fish specie Serrasalmus brandtii (pirambeba), collected at two sites in the Itaparica reservoir (Brazil) during March/12 to January/2013 through biochemical and genotoxic biomarkers. Around the reservoir resettlement areas exist with agricultural practices using agrochemicals. Thus, the study aimed to use S. brandtii as specie sentinel for this region of the Sao Francisco river. There was no significant difference in the physicochemical parameters measured such as temperature and dissolved oxygen over the sample period. Among the morphological parameters, the masses of the specimens varied between sampling period. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were quantify on the brain and muscle specimens collected from the two sites. Brain AChE from pirambeba collected at site 1 showed a significant decay in specific activity over the study period, however protein content did not show the same profile. Brain BChE specific activity had significant variation during the sampling period. On the contrary, pirambeba collected at site 2 showed no significant difference in the brain AChE specific activity and protein content. Brain BChE of these specimens showed significant variation on the specific activity values. On muscle tissue, pirambeba collected at site 1 showed significant variation in the values of AChE specific activity as well as on the protein content during the sample period. BChE specific activity on the muscle also had significant variation. Pirambeba collected at site 2 showed significant variation on muscle AChE specific activity and protein content values over the analyzed period. For muscle BChE activity,values, the results showed pattern similar to the one observed on site 1. Genotoxicity assays were performed on pirambeba erythrocyte cells collected at two sites. The results showed frequency of micronuclei (MN) and significant variation between the mean frequencies of MN over the study period was observed. Nuclear abnormalities (NA) were also analyzed and the results indicated that pirambeba the site 1 had higher nuclear abnormalities frequencies than the pirambeba from site 2. The Comet assay indicated that majority of erythrocyte nuclei analyzed from both sites shown class 1damage indicating slight DNA damage. In vitro studies were also performed for the characterization of brain AChE from pirambeba and kinetic assays showed values of 0.324 ± 0.04 mmol /L and 0.709 ± 0.014 mmol / min-1.mg protein for the apparent kinetic constants Kmapp and Vmáxapp respectively. Brain AChE inhibitory assays showed that muscle AChE (0.362±0.2 μM) was more sensitive to carbamate eserine that brain AChE (0.887 ± 0.1 μM) AChE and when compared brain AChE between eserine (0.887 ± 0,1μM) and glyphosate (2,276 ± 0,02μM), there was a greater sensitivity for brain AChE with eserine. We conclude that among the biomarkers genotoxic tested, the results showed that the MN test was considered the most sensitive; S. brandtii specie can be a specie sentinel for the submedium San Francisco region, since was present in all samples of the river, even during the most severe period of drought and low water level, but also by having a direct relationship with the AChE activity and forming MN. / Ambientes aquáticos são ecossistemas que mais sofrem impactos causados pela ação antropogênica, uma vez que constitui compartimentos finais de vários produtos gerados pela atividade humana. O presente trabalho avaliou a espécie de peixe Serrasalmus brandtii (pirambeba), coletada em dois sítios no reservatório Itaparica (Brasil) durante o período de março/12 a janeiro de 2013 através de biomarcadores bioquímicos e genotóxicos. No entorno do reservatório existem reassentamentos com práticas agrícolas empregando agroquímicos. Desta forma, o trabalho visou a utilização de S. brandtii como sentinela no rio Sao Francisco. Não houve diferença marcante nos parâmetros físico-químicos temperatura e oxigênio dissolvido mensurados ao longo do período amostral. Entre os parâmetros morfológicos, as massas dos espécimes variaram entre os meses de amostragem. Foi realizada quantificação de atividade acetilcolinesterase (AChE) e butirilcolinesterase (BChE) cerebral e muscular de espécimes coletados nos dois sítios. AChE cerebral de pirambeba coletada no sítio 1 apresentou decaimento significativo na atividade específica ao longo do período estudado, no entanto o conteúdo protéico não mostrou o mesmo perfil. A atividade específica BChE cerebral mostrou variação significativa durante o período de amostragem. Todavia, pirambeba coletada no sítio 2 não mostrou diferença significativa na atividade específica de AChE cerebral e conteúdo protéico. BChE cerebral nesses espécimes apresentou variação significativa nos valores de atividade específica. No tecido muscular, pirambeba coletada no sítio 1 apresentou variação significativa nos valores de atividade específica AChE, bem como conteúdo protéico durante período amostrado. A atividade específica de BChE muscular também teve variação significativa. Espécimes coletadas no sítio 2 apresentaram variação significativa na atividade específica da AChE muscular e no conteúdo protéico ao longo do período estudado. Para valores de atividade de BChE muscular, os resultados apresentaram um padrão semelhante ao observado no sítio 1. Os ensaios genotóxicos foram realizados em eritrócitos de pirambeba coletadas nos dois sítios. Os resultados mostraram frequência de micronúcleos (MN) e variação significativa entre as freqüências de MN foi observada ao longo do período estudado. Anormalidades nucleares (AN) também foram analisadas e resultados indicaram que espécimes do sítio 1 tiveram maiores freqüências de AN do que espécimes do sítio 2. O ensaio Cometa indicou que a maioria dos núcleos de eritrócitos analisados nos dois sítios apresentou dano na classe1, indicando danos leves no DNA das células. Estudos in vitro foram também realizados para caracterização de AChE cerebral de pirambeba e ensaios cinéticos mostraram valores de 0,324±0,04 mmol/L e 0,709±0,014 mmol/min-1.mg de proteína para constantes cinéticas aparentes Kmapp e Vmáxapp, respectivamente. Ensaios inibitórios de AChE mostraram que AChE muscular (0,362±0,2) foi mais sensível ao carbamato de eserina que a AChE cerebral (0,887±0,1) e quando comparados AChE cerebral entre a eserina (0.887±0,1μM) e o glifosato (2,276±0,02μM), verificou-se uma maior sensibilidade para a AChE cerebral com eserina. Concluímos que dentre os biomarcadores genotóxicos testados, os resultados mostraram que teste de MN foi considerado o mais sensível; a espécie S. brandtii pode ser a espécie sentinela para a região do submédio São Francisco, visto que esteve presente no rio em todas as coletas, mesmo durante o período mais rigoroso de estiagem e baixo nível da água, como também por apresentar relação direta com a atividade de AChE e a formação de MN.

Rýdlová Kateřina: Alkaloids Narcissus 'Dutch Master' (Amaryllidaceae) and their biological activity III. Diploma thesis 2017. Charles university in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology. The aim of this work was isolation of compounds from the selected fraction ND 3 - 5 obtained by column chromatography of a Narcissus 'Dutch Master' alkaloid extract. Preparation of the extract and its column chromatography was performed by Mgr. Daniela Hulcová as a part of her doctoral study. Two substances NDM-1 and NDM-2 were isolated from fraction ND 3 - 5 by column chromatography and preparative TLC. The structures were determined as (+)-masonine and (+)-homolycorine on the basis of NMR, GC-MS analysis, optical rotation and their comparison with literature data. Isolated alkaloids were tested on inhibitory activity against human erythrocyte acetylcholinesterase, plasma butyrylcholinesterase and prolyloligopeptidase. Activity of alkaloids was expressed as IC50 values: (+)-masonine (IC50 AChE = 305 ± 34 μM, IC50 BuChE = 229 ± 24 μM, IC50 POP = 314 ± 34 μM), (+)-homolycorine (IC50 AChE = 63.7 ± 4.3 μM, IC50 BuChE = 151 ± 20 μM, IC50 POP = 173 ± 41 μM). In comparison with standards of galanthamine (IC50 AChE = 1.710 ± 0.1 μM, IC50 BuChE = 42.3 ± 1.3 μM),...