Dissertations / Theses on the topic 'Acetylcholinesterase – analyse'

La galanthamine est un alcaloïde isoquinoléique, utilisé dans le monde entier pour le traitement palliatif de la maladie d’Alzheimer en raison de son pouvoir inhibiteur de l’acétylcholinestérase. Cet alcaloïde est extrait à partir de bulbes d’Amaryllidaceae, Leucojum aestivum, Galanthus nivalis, et Narcissus sp. ou obtenu par synthèse chimique. La difficulté principale de cette dernière réside dans le respect de la configuration des centres d'asymétrie. La culture de tissus in vitro pourrait constituer une alternative intéressante pour obtenir ce composé à haute valeur ajoutée. Le premier objectif de ce projet, vise à améliorer l’accumulation de cet alcaloïde par les biais des biotechnologies. Le second objectif est de rechercher par criblage phytochimique (HPLC, LCMS, GCMS, et HPTLC-MS) de bulbes in vitro et in vivo d’Amaryllidaceae, de nouveaux alcaloïdes biologiquement actifs. Le troisième objectif porte sur l’étude de la voie de biosynthèse en vue de réaliser une synthèse biomimétique de la galanthamine. Nous avons établi des cultures in vitro de 3 espèces d’Amaryllidaceae. La variation des paramètres exogènes a conduit à une accumulation accrue d’alcaloïdes (0,02 à 0,2 % MS). Le criblage phytochimique a conduit à l’identification d’alcaloïdes nouveaux issus des cultures in vitro, n’existant pas in vivo, et présentant un puissant pouvoir inhibiteur de l’acétylcholinestérase (40 à 80 % Inh). L’ajout, de la 4’-Ométhyl-d3-norbelladine aux cultures in vitro a conduit à sa métabolisation en trois types d’alcaloïdes deutérés. Une stimulation de la synthèse et du relargage de la galanthamine native (0,15 % MS et 0,16 % Milieu) a été observée en présence du précurseur deutéré
Galanthamine is an Amaryllidaceae alkaloid used worldwide for the symptomatic treatment of Alzheimer’s disease because of his capacity to inhibit the acetylcholinesterase enzyme. There are two galanthamine sources for medical applications. One is the total synthesis, a complicated process because galanthamine has three asymmetric carbons, requiring stereochemically controlled synthesis. Galanthamine is also extracted from bulbs of some Amaryllidaceae such as Leucojum aestivum, Galanthus nivalis, and Narcissus sp.. The first aim of this work is to improve the accumulation of this alkaloid using biotechnologies. The second aim consists on the phytochemical screening (HPLC, LCMS, GCMS, et HPTLCMS) of in vivo and in vitro Amaryllidaceae bulbs, in order to identify new alkaloids with important pharmacological activities. Finally, the third aim concerns the study of the biosynthesis pathway in order to establish a biomimetic synthesis of galanthamine. Therefore, we established in vitro cultures of three Amaryllidaceae species. The variation of exogenousparameters led to the obtainment of high galanthamine accumulation (0.02 to 0.2 % DW). The phytochemical screening showed new alkaloids in extracts of in vitro cultures, which did not exist in in vivo extracts, and possessing high acetylcholinesterase activity (40 to 80 % Inh). The 4’-O-methyl-d3-norbelladine is incorporated into three different groups ofAmaryllidaceae alkaloids. The addition of the labelled precursor to shoot cultures stimulated the synthesis of native galanthamine (0.15 % DW and 0.16 % Culture medium)

Les travaux présentés dans ce manuscrit concernent le développement d‘une approche originale permettant la détermination de plusieurs composés (principalement aflatoxines et pesticides organophosphorés), à l‘aide de biocapteurs électrochimiques basés sur l‘inhibition de l‘acétylcholinestérase. Dans un premier temps, un nouveau biocapteur potentiométrique utilisant des transistors à effet de champ sensibles au pH (pH-FETs) comme transducteurs a été développé pour la détermination de l‘aflatoxine B1 (AFB1) et différent paramètres d‘élaboration et de fonctionnement du biocapteur ont été optimisés. Le biocapteur proposé est caractérisé par une stabilité opérationnelle élevée and bonne reproductibilité du signal en cours d‘utilisation et de stockage. Le biocapteur a ensuite été évalué pour l‘analyse d‘échantillons réels (blé, sésame, noix et pois) et une simulation mathématique de la réponse du biocapteur potentiométrique à l‘AFB1 a été proposée pour la première fois et validée. Dans un deuxième temps, un biocapteur conductimétrique utilisant des microélectrodes interdigitées en or a été développé. La sensibilité de ce biocapteur aux aflatoxines ainsi qu‘à d‘autres classes de substances toxiques, tels que les pesticides organophosphorés, les métaux lourds, les glycoalkaloïdes, et les surfactants, a été déterminée. Une nouvelle procédure originale, permettant la détermination sélective de toxines multiclasses par application successive de solutions de réactivation visant spécifiquement des inhibiteurs irréversibles ou réversibles, a été finalement proposée. En utilisant cette méthode, il a été montré que les biocapteurs enzymatiques pouvaient être appliqués à l‘analyse des aflatoxines et des pesticides organophosphorés, ainsi qu‘à la détermination de la toxicité globale des échantillons
Investigations reported in this manuscript are focused on the development of an original approach for the detection of several toxic compounds, mainly aflatoxins and organophosphorus pesticides, using acetylcholinesterase (AChE)-based inhibitory electrochemical biosensors. In a first step, a new potentiometric biosensor using pH Sensitive Field-Effect Transistors (pH-FETs) as transducers was investigated for aflatoxin B1 (AFB1) determination and different elaboration and working parameters were optimized. The proposed biosensor was characterized by high operational stability and reproducibility of the signal during the work as well as during the storage. The biosensor was further evaluated for real samples analysis (wheat, sesame, walnuts and peas) and a mathematical simulation of the potentiometric biosensor response to aflatoxin B1 was proposed for the first time and validated. In a second step, a conductometric biosensor using interdigitated gold microelectrodes was developed. The sensitivity of the biosensor to aflatoxins and other classes of toxic substances, such as organophosphorus pesticides, heavy metals ions, glycoalkaloids, and surfactants, was determined. A new and original procedure, enabling the selective determination of multiclass toxins by applying successive reactivation solutions targeting either irreversible or reversible inhibitors, was finally proposed. Using this method, the electrochemical enzyme inhibitory biosensors could be applied to the analysis of aflatoxins and organophosphorus pesticides, as well as for the determination of total toxicity of the samples

Introduction: Malgré l'absence d'innervation cholinergique, les vaisseaux sont très réactifs à la présence d'acétylcholine (ACh). De plus, ce neurotransmetteur est couramment utilisé pour évaluer la fonction endothéliale des vaisseaux. Cependant, les informations sur les cholinestérases vasculaires (ChE), les enzymes qui mettent fin à l'action de l'ACh, sont rares. Le principal objectif de cette thèse de doctorat était de caractériser les ChE vasculaires et l'ensemble du système cholinergique de l'aorte dans des conditions normales et pathologiques. Méthodes: Des rats Wistar mâles adultes et des rats spontanément hypertendus (SHR) nourris avec une alimentation régulière ou riche en graisses ont été utilisés dans le cadre du projet. L'expression relative des enzymes et des transporteurs étudiés a été déterminée par la méthode RT-qPCR. Les activités de ChE dans les extraits de tissus ont été mesurées par la méthode d'Ellman, la coloration de l'activité a été réalisée par la méthode de Tsuji et la localisation des protéines a été faite par double immunohistochimie. Les formes moléculaires de ChE ont été séparées par des gradients de saccharose. Résultats et conclusion: Toutes les enzymes et tous les transporteurs impliqués dans la synthèse, le stockage, la libération et la dégradation de l’ACh ont été détectés dans l'aorte du rat au niveau de l'ARNm et au niveau des protéines. Cela confirme que l'aorte est un tissu cholinergique non neuronal, capable de soutenir pleinement le cycle de vie des ACh. Les ChE sont présents principalement sous forme de formes ancrées dans la PRiMA dans chaque partie de l'aorte, tandis que la butyrylcholinestérase (BChE) est l’enzyme dominante, localisée principalement sur le muscle lisse. Dans les rat SHR, des niveaux plus faibles de BChE ont été détectés, accompagnés d'une diminution des expressions relatives de la carnitine acétyltransférase et des transporteurs de cations organiques. Cela suggère une signalisation cholinergique plus faible dans l'aorte de la SHR par rapport aux rats normotendus. Dans l'expérience pharmacologique, l'inhibition du BChE et l'alimentation riche en graisses ont toutes deux entraîné une prise de poids significative et une augmentation des taux sériques de TAG. De plus, un régime riche en graisses a induit une augmentation des niveaux d'ARNm du BChE, indiquant son implication dans le métabolisme des lipides
Introduction: Despite the lack of cholinergic innervation, vessels are highly reactive to the presence of acetylcholine (ACh). Moreover, this neurotransmitter is commonly used to assess the endothelial function of vessels. However, information about vascular cholinesterases (ChE), the enzymes that terminate ACh action, is spares. The main aim of this dissertation thesis was to characterize vascular ChE and overall non-neuronal cholinergic system in the aorta under physiological and pathological conditions. Methods: Adult male Wistar rats and spontaneously hypertensive rats (SHR) were used in the project, fed either with regular or high-fat diet. Relative expression of studied enzymes and transporters were determined by RT-qPCR method. ChE activities in tissue extracts were measured by Ellman's assay, activity staining was performed by Tsuji’s method and proteins localizations were done by dual immunohistochemistry. Molecular forms of ChE were studied by sucrose gradients. Results and conclusion: The enzymes and transporters necessary for ACh synthesis, storage, release, and degradation were detected in the rat aorta at mRNA and at protein levels. This confirms that aorta is a non-neuronal cholinergic tissue, capable to fully support the ACh life cycle. ChE are present mainly as PRiMA-anchored forms in each part of the aorta, while butyrylcholinesterase (BChE) is the dominant ChE, localized primarily in the smooth muscle. In SHR, lower levels of BChE were detected, accompanied by decreased relative expressions of carnitine acetyltransferase and organic cation transporters. This suggests lower cholinergic signaling in SHR aorta as compared to normotensive rats. In the pharmacological experiment, both inhibition of BChE and high-fat diet resulted in significant weight gain and increased serum TAG levels. Moreover, a high-fat diet induced mRNA expression of BChE. Our data suggest BChE involvement in lipid metabolism
Úvod: Napriek absencii cholínergickej inervácie, cievy sú vysoko reaktívne na prítomnosť acetylcholínu (ACh). Okrem toho, práve tento neurotransmiter sa využíva vo fyziologických experimentoch na sledovanie funkčnosti endotelu. Napriek tomu však chýbajú informácie o prítomnosti cholínesteráz (ChE), enzýmov, ktoré ukončujú jeho účinok v cievach. Cieľom tejto dizertačnej práce bola detailne charakterizovať tieto enzýmy a kompletne preštudovať prítomnosť komponentov neneuronálneho cholínergického systému v aorte normotenzných a spontánne hypertenzných potkanov (SHR). Metódy: V experimentoch boli použité 12-týždňové potkany rodu Wistar a SHR, ktoré boli kŕmené buď štandardnou alebo vysoko-tukovou stravou. Relatívna expresia študovaných enzýmov a transportérov bola stanovená metódou RT-qPCR. Aktivity ChE boli stanovené v tkanivových extraktoch pomocou Ellmanovej metódy, aktivitné farbenie bolo prevedené podľa Tsujiho metódy. Na vizualizáciu a lokalizáciu bola využitá metóda dvojfarebnej immunohistochémie. Molekulové formy ChE boli charakterizované pomocou metódy sacharózového gradientu. Výsledky a diskusia: Všetky enzýmy a transportéry, ktoré sú potrebné na syntézu, uchovávanie, vylučovanie a degradáciu ACh boli potvrdené nielen na úrovni mRNA, ale aj na úrovni proteínov. Tieto zistenia potvrdzujú, že aorta patrí medzi neneuronálne cholínergické tkanivá. ChE sú prítomné primárne v PRiMA-kotvenej forme v každej časti aorty, pričom butyrylcholínesteráza (BChE) je dominantná a je prítomná hlavne v hladkom svalstve. V spontánne hypertenznom modeli bola detegovaná nižšia BChE aktivita a tiež nižšia relatívna expresia karnitínacetyltransferázy a organických katiónových transportérov. Vo farmakologickom experimente, aj inhibícia BChE, aj vysoko-tuková strava spôsobila signifikantný prírastok hmotnosti a zvýšenie sérových hladín triacylglycerolov. Okrem toho, vysoko-tuková strava indukovala zvýšenie hladín mRNA pre BChE, čo naznačuje dôležitú úlohu tohto enzýmu nielen vo fyziológii ciev, ale aj v metabolizme lipidov

The current economic and social conditions in many rural areas of the world are threatening the precious heritage of ethnobotanical knowledge and traditional farming practices.This can cause loss of precious cultural heritage and reduction in plant biodiversity, as ancient crops tend to disappear. The main aim of this thesis is to document traditional uses of plants by different ethnic groups (Tibeto-Burman and Indo-Aryan) living in certain rural areas of the Kavrepalanchok District in Central Nepal. In the study area, due to distance from urban centres and difficulty in accessing the government healthcare system, people still rely heavily on the use of local plants for various purposes, above all for primary healthcare. Through interviews with 32 informants, most of whom were key informants, we explored uses of 116 plant species, of which 101 were plants with medicinal value employed in the treatment of human and veterinary diseases. Some unusual uses of medicinal plants and original recipes were also reported. The data document the richness of the local flora and traditional knowledge of medicinal plant species used by ethnic communities in these rural areas. Therefore, future projects will have to involve local people in the improvement and conservation of the biological and cultural heritage.There is also a need for an ecological strategy for integrated management of land, water and living resources. Another aim of the research presented in this thesis is to better characterise some plants found to be of particular interest among those surveyed in the study area. With this in mind, we have focused our attention on those plants used by informants in the treatment of nervous system disorders, such as two species belonging to Caprifoliaceae (formerly Valerianaceae): Valeriana jatamansi Jones ex Rob. and Nardostachys jatamansi (D. Don) DC. These plants are widely used in traditional medicine for their sedative and anxiolytic properties in Nepal and in many other Asian countries. The pharmacognostic and phytochemical profile and the biological effects of essential oils (EOs) of these species were compared with those of Valeriana officinalis L., a species whose phytotherapeutic use is widespread in Western medicine. The multidisciplinary approach used represents a way to avoid adulteration of herbal drugs and allows evaluation of the effectiveness of EOs that could be used for a wide range of therapeutic applications. Overall, the results of this research could be useful for enhancing knowledge of the potential of still little-known medicinal plants for the possible formulation of new pharmaceutical products, eventually contributing to the economic development of local communities.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
The phytochemical investigation of methanol crude extract of the stem bark of the species Helietta apiculata Benth and Zanthoxylum fagara (L.) Sarg., Rutaceae, provided the isolation of thirty-one compounds. The species H. apiculata were isolated eight furoquinolínicos alkaloids (8, 9, 10, 11, 12, 13, 15 and 24) and one quinolinone (22), six coumarins (51, 52, 53, 58, 115 and 116), 116 first identified in the literature, three cinnamates (121, 122 and 123), two lignans (98 and 123), a limonoid (92), a long-chain acid (126), a steroid (80) and a mixture terpenes (T39). Two benzophenantridine alkaloids (33 and 34), five amides (69, 70, 117, 118 and 119), and a lignan (124) have been identified in species Z. fagara, along with the steroid 80. The cinnamate 120, 123 and the lignan 119 were first identified as a natural product. The cinnamate 122 and coumarin 115 are first described in H. apiculata species, while the amide 115 is first described in the genus Zanthoxylum, and 119 for the first time in the species Z. fagara. Lignans 98, 123 and 124 are identified in the Rutaceae family first time. The limonoid 92 was subjected to structural modifications through aminolysis reactions with various amines, providing fifteen derivatives, which when subjected to evaluation of the antimicrobial potential demonstrated greater power to inhibition of microorganisms that limonoid. Other derivatives obtained during this study was the acetylation and oxidation products of tembamida (69) amide and methylating the flindersiamina (15) alkaloid. Extracts, fractions and products were subjected to analysis of antimicrobial potential and inhibition of AChE enzyme. The alkaloids showed inhibition potential only for gram-positive bacteria, whereas coumarins were compounds which had the largest range of antimicrobial activities front fungi, Gram-negative and Gram-positive. Inhibition of acetylcholinesterase enzyme was significant only for fractions: ether basic H. apiculata, basic butanol, final aqueous and aqueous extract from the leaves Z. fagara with inhibition values ranging from 58,24% to 74,34% while for isolates the results were not significant.
A investigação fitoquímica do extrato bruto metanólico das cascas do caule das espécies Helietta apiculata Benth e Zanthoxylum fagara (L.) Sarg., Rutaceae, levaram ao isolamento de trinta e um compostos. Da espécie H. apiculata foram identificados oito alcaloides furoquinolínicos (8, 9, 10, 11, 12, 13, 15 e 24), um quinolinona (22), seis cumarinas (51, 52, 53, 58, 115 e 116) sendo a 116 identificada pela primeira vez na literatura, três cinamatos (121, 122 e 123), duas lignanas (98 e 123), um limonoide (92), um ácido de cadeia longa (126), um esteroide (80) e uma mistura de terpenos (T39). Dois alcaloides benzofenantridínicos (33 e 34), cinco amidas (69, 70, 117, 118 e 119) e uma lignana (124) foram identificados na espécie Z. fagara, juntamente com o esteroide 80. O cinamato 120, a lignana 123 e a amida 119 foram identificados pela primeira vez como produto natural. O cinamato 122 e a cumarina 115 são descritos pela primeira vez na espécie H. apiculata, enquanto a amida 115 é descrita pela primeira vez no gênero Zanthoxylum, e 119 pela primeira vez na espécie Z. fagara. As lignanas 98, 123 e 124 são identificadas na família Rutaceae pela primeira vez. O limonoide 92 foi submetido a modificações estruturais através de reações de aminólise com diferentes aminas, proporcionando quinze derivados, que quando submetidos à avaliação do potencial antimicrobiano demonstraram maior poder de inibição dos microrganismos que o limonoide. Outros derivados obtidos durante este trabalho foram os produtos da acetilação e a oxidação da amida tembamida (69), e a metilação do alcaloide flindersiamina (15). Os extratos, frações e produtos obtidos foram submetidos a analise do potencial antimicrobiano e de inibição da enzima AChE. Os alcaloides apresentaram potencial de inibição apenas para bactérias gram-positivas, enquanto as cumarinas foram os compostos que apresentaram o maior leque de atividades antimicrobianas frente a fungos, bactérias gram-negativas e gram-positivas. A inibição da enzima Acetilcolinesterase foi relevante apenas para as frações: etérea básica de H. apiculata, butanólica básica, aquoso final e extrato aquoso a partir das folhas de Z. fagara com valores de inibição entre 58,24% a 74,34% enquanto para os produtos isolados os resultados não foram significativos.

A esclerose múltipla é a principal doença desmielinizante do sistema nervoso central (SNC). É considerada a principal causa de incapacidade neurológica em adultos jovens. O comprometimento cognitivo é muito comum nessa doença, envolvendo o aprendizado, a memória e a organização cortical do movimento, funções vitais que são reguladas pelo sistema colinérgico. O modelo de desmielinização tóxica induzida pelo brometo de etídio (BE) foi utilizado neste estudo, para avaliar a atividade da enzima acetilcolinesterase (AChE) no estriado (ST), hipocampo (HP), córtex cerebral (CC), hipotálamo (HY) e ponte (PN) associado ao tratamento com interferon beta (IFN-b), ciclosporina A (CsA), vitamina E (vit E) e ebselen (Ebs). Além disso, também foi investigado o efeito in vitro do BE na atividade da AChE, juntamente com os parâmetros cinéticos dessa enzima no ST, HP, CC e CB de ratos adultos. Os resultados demonstraram que o BE inibiu significativamente a atividade da AChE no ST, HP, CC e CB nas concentrações de 0,00625, 0,0125, 0,025, 0,05 e 0,1mM e a análise dos dados cinéticos mostraram uma inibição do tipo incompetitiva no ST, HP e CC, enquanto no CB a inibição foi do tipo mista. No estudo in vivo, foi observado uma inibição na atividade da AChE no CC, ST, HP, HY, PN e CB nos diferentes períodos avaliados pós-injeção do BE (3, 7, 15, 21 e 30 dias). Quando ratos desmielinizados com BE foram tratados com IFN-b e CsA, não houve alteração na atividade dessa enzima. Por outro lado, o tratamento com Vit E e Ebs foram capazes de aumentar a atividade da AChE no ST, CC e HP. Estudos imuno-histoquímicos demonstraram que nos ratos tratados com Vit E e Ebs as lesões induzidas pelo BE foram menores, sugerindo que estes compostos interferem no desenvolvimento de lesões desmielinizantes. Foi também avaliado o efeito per se do IFN-b, da CsA, da Vit E e do Ebs na atividade da AChE, os quais demonstraram um efeito inibitório sobre a atividade dessa enzima. Em plaquetas de ratos desmielinizados, foi observado uma diminuição na atividade da NTPDase e o tratamento com a Vit E e o Ebs modularam a hidrólise dos nucleotídeos de adenina. O presente trabalho demonstrou que o BE é um potente inibidor da atividade da AChE in vitro e os resultados in vivo demonstraram que a atividade dessa enzima está alterada após um evento de desmielinização tóxica no SNC. Os resultados deste estudo ajudaram a confirmar que drogas utilizadas no tratamento de pacientes com esclerose múltipla tais como o IFN-b e a CsA causam efeitos na atividade da AChE. A Vit E e o Ebs, além de demonstrarem uma interação com a neurotransmissão colinérgica, também modularam a hidrólise dos nucleotídeos de adenina em plaquetas de ratos, contribuindo no controle da coagulação plaquetária em processos desmielinizantes. Neste contexto, sugere-se que o IFN-b, a CsA, a vitamina E e o ebselen podem ser investigados em estudos futuros com a intenção de encontrar uma melhor terapia para beneficiar pacientes com patologias desmielinizantes.
Multiple sclerosis is the main demyelinating disease of the central nervous system (CNS). It is the most common cause of neurological disability among young adults. The cognitive impairment is very commum in this illness, involving learning, memory and cortical organization of the movement, vital functions regulated by the cholinergic system. The model of toxic demyelination induced by ethidium bromide (EB) was used to evaluate brain acetylcholinesterase (AChE) activity in the striatum (ST), hippocampus (HP), cerebral cortex (CC), cerebellum (CB), hypothalamus (HY) and pons (PN), associated with treatment with interferon beta (IFN-b), ciclosporine A (CsA), vitamin E (Vit E) and ebselen (Ebs). In addition, the per se effect of EB on AChE activity was studied in vitro together with the kinetic parameters of this enzyme in the ST, HP, CC and CB of adult rats. The results showed that EB in vitro significantly inhibited AChE activity in the ST, HP, CC and CB at concentrations of 0.00625, 0.0125, 0.025, 0.05 and 0.1mM. The kinetic analysis demonstrated an uncompetitive inhibition in the ST, HP and CC, whereas in the CB the inhibition type was mixed. In relation to the in vivo results, AChE activity was inhibited after demyelination by EB in the CC, ST, HP, HY, PN and CB at the post-injection points of time evaluated (3-7-15-21 and 30 days). When demyelinated rats were submitted to the treatment with IFN-b and CsA, the results demonstrated that these compounds did not alter AChE activity. However, Vit E and Ebs were able to increase AChE activity in the ST, CC and HP. In addition, immunohistochemistry studies demonstrated that in Vit E and Ebs treated rats, the lesions induced by EB were smaller suggesting that these compounds somehow interfered in the development of the lesions. The per se effect of IFN-b, CsA, Vit E and Ebs were also evaluated, demonstrating that these compounds have an inhibitory effect on AChE activity. Platelets of the demyelinated rats demonstrated a reduction in NTPDase activity and the treatments with Ebs and Vit E modulated adenine nucleotide hydrolysis. The present investigation demonstrated that EB is a strong inhibitor of AChE activity in vitro and the results in vivo showed that the activity of this enzyme is altered after an event of toxic demyelination in the CNS. The results this study help the confirm that drugs used in the treatment of the patients with multiple sclerosis such as IFN-b and CsA cause effects in the AChE activity. The Vit E and Ebs besides of interaction with the cholinergic neurotransmission also modulated adenine nucleotide hydrolysis in platelets of the rats, contributing to the control of the platelet coagulant status in the demyelinating process. In this context, we can suggest that IFN-b, CsA, Vit E and Ebs may be investigated in future studies with the intention of finding a better therapy for to improve patients with demyelinating pathologies.

Ethanol extracts of “jenipapo” (Genipa americana Linnaeus), “umbu" (Spondia tuberosa Arruda), “siriguela” (Spondia purpurea Linnaeus) and “mangaba” (Hancornia speciosa Gomes) were prepared from separate pulp, seeds and peel; except mangaba which are used pulp and peel. The investigation of antioxidant capacity of the ethanol extracts were carried out by the methods of determining the content of ascorbic acid and total phenolics; scavenging 2, 2 -diphenyl-1-picrilhydrazyl and 2, 2’- azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical, antioxidant capacity of reduction of iron and copper and lipid peroxidation using a biomimetic membrane system, and measurement enzymatic of catalase and superoxide dismutase. It was also analyzed the acetylcholinesterase inhibitory activity, cytotoxic effect on corneal epithelial cells of sheep, as well as phytochemicals assays and identification of phenolic compounds and organic acids present in the extracts by UPLC - MS. The highest values of total phenolic content were obtained with peel and seed extracts and to ascorbic acid in the seed of siriguela, showing better antioxidant activities of seeds and peel extracts of siriguela and umbu. Lipid peroxidation assays indicated that genipap pulp is a promising antioxidant. Genipap pulp and siriguela seed ethanol extracts presented an acetylcholinesterase inhibition zone similar to that of the positive control, carbachol. The investigation of phenols and organic acid contents revealed the presence of quercetin (48,38 to 3,88 μg.g-1), quinic acid (43,28 to 41,88 μg.g-1) and citric acid (3,78 to 0,43 μg.g-1) in several extracts and chlorogenic acid with the highest amount found in siriguela seeds (356,93 μg.g-1). These data suggest new uses for these foods as potential antioxidant supplements for the food, pharmaceutical and cosmetic industries.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A partir das cascas, polpas e sementes do jenipapo (Genipa americana Linnaeus), umbu (Spondia tuberosa Arruda), siriguela (Spondia purpurea Linnaeus) e mangaba (Hancornia speciosa Gomes) foram preparados extratos etanólicos, com exceção da mangaba onde o extrato foi preparado utilizando casca e polpa. Os extratos foram submetidos à investigação da capacidade antioxidante através dos métodos de determinação do conteúdo de ácido ascórbico e fenóis totais, sequestro do radical 2,2-difenil-1-picril-hidrazil e 2,2´- azinobis (3-etilbenzotiazolina-6-ácido sulfônico), capacidade antioxidante de redução do ferro e cobre, lipoperoxidação utilizando um sistema de membranas biomimético e mensuração enzimática da catalase e superóxido dismutase. Também foi avaliada a atividade inibitória da enzima acetilcolinesterase, o efeito citotóxico em células epiteliais da córnea de ovelhas, além da realização de ensaios fitoquímicos e a identificação de compostos fenólicos e ácidos orgânicos presentes nos extratos. Os maiores teores de fenóis totais foram obtidos nos extratos das cascas e sementes e no referente ao ácido ascórbico na semente da siriguela, apresentando melhor atividade antioxidante os extratos das sementes e cascas da siriguela e do umbu. Porém, no ensaio de peroxidação lipídica, o extrato etanólico da polpa de jenipapo demonstrou ser um antioxidante promissor. Os extratos etanólicos da polpa do jenipapo e da semente da siriguela apresentaram uma zona de inibição da enzima acetilcolinesterase semelhantes ao controle positivo, carbacol. Na investigação dos compostos fenólicos e ácidos orgânicos por UPLC-MS verificou-se a presença nos extratos de quercetina (48,38 a 3,88 μg.g-1), ácido quínico (43,28 a 41,88 μg.g-1), ácido cítrico (3,78 a 0,43 μg.g-1) em vários extratos. E ácido clorogênico, na semente da siriguela (356,93 μg.g-1). Os resultados obtidos desses extratos sugerem novas formas de utilização desses alimentos como potenciais suplementos antioxidantes na indústria alimentícia, farmacêutica e cosmética.

Biological activity of plant metabolites XXVIII. Alkaloids from selected cultivars of Narcissus cyclamineus REDOUTÉ and their influence on acetylcholinesterase and butyrylcholinesterase. JANURA M.: Diploma work, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2015, Czech Republic. Abstract: The summary extracts of alkaloids from bulbs of N. cyclamineus esp. N. cyclamineus cv. Jenny, N. cyclamineus cv. Little Witch, N. cyclamineus cv. Rapture, N. cyclamineus cv. Surfside, N. cyclamineus cv. Peeping Tom, N. cyclamineus cv. Warbeld and N. cyclamineus cv. Skater's Waltz were obtained by the procedures common for this type of compounds. The alkaloid extracts were analyzed by GC/MS and the probable occurrence of individual alkaloid substances was observed. These extracts were also assayed for HuAChE and HuBuChE inhibitory activity. N. cyclamineus cv. Surfside with IC50 = 61,26 ± 6,42 µg/ml and N. cyclamineus cv. Warbeld with IC50 = 85,43 ± 11,13 µg/ml have the best results in inhibition of HuBuChE. The best inhibitory activity on HuAChE has N. cyclamineus cv. Warbeld with IC50 (HuAChE) = 36,82 ± 4,50 µg/ml. Key words: Narcissus, Acetylcholinesterase, Butyrylcholinesterase, GC/MS, Alzheimer's disease.

Farkašovský M.: Biological activity of plant metabolites XXIX. Alkaloids of selected cultivars of Narcissus triandrus L. and their influence on acetylcholinesterase and butyrylcholinesterase. Diploma thesis. Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of pharmaceutical botany and ecology, Hradec Králové 2015, 70 pages Screening of seven bulb samples of Narcissus genus was performed. It included cultivars Narcissus triandrus cv. Hawera, Narcissus triandrus cv. Ice Wings, Narcissus triandrus cv. Stint, Narcissus triandrus cv. Tresamble, Narcissus cyclamineus cv. February Gold, Narcissus cyclamineus cv. Greenlet and Narcissus cyclamineus cv. Itzim. Primary extract was prepared by boiling crushed bulbs in ethanol 95% and then it was condensated. After extraction in diethyl ether and ethyl acetate was pure alkaloid extract dried with air flow in water bath. Isolated alkaloid extracts were tested for their inhibition activity on acetylcholinesterase and butyrylcholinesterase. Also GC-MS analysis was provided to identify alkaloids. These alkaloids were identified with GC-MS analysis - epinorgalanthamine, galanthamine, galanthine, haemanthamine, hippeastrine, homolycorine, cherylline, incartine, lycoramine, lycoramine-acetate, lycorine, narwedine, neruscine, sanguinine...

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Sabina Tanková Supervisor: PharmDr. Daniela Hulcová, PhD. Title of diploma thesis: Alkaloids of the genus Narcissus and their biological activity. Key worlds: Narcissus pseudonarcissus L. cv. Dutch Master, Amaryllidaceae, alkaloids, AChE, BuChE, POP, GSK-3β, biological activity. Alkaloid extract obtained from bulbs of Narcissus pseudonarcissus L. cv. Dutch Master was extracted by ethanol and was purified by liquid-liquid extraction and fractionated by column chromatography to individual fractions. At the end, were obtained 11 pooled fractions, which were used to isolate pure alkaloids. The ND 3-5 / 7 fraction was processed by preparative thin layer chromatography followed by crystallization of pure substances. In total, 5 alkaloid substances of ST1D2, ST1D3, ST2A, ST2B1 and ST3C were obtained from this fraction in various amounts. These substances were determined by GC-MS analysis, NMR analysis and optical rotation. Subsequently, the obtained data were compared with the NIST library spectra and the literature. Isolated substances have been identified as caranine, O-ethyllycorenine, narwedine, pluviine and N-demethylhomolycorine. The alkaloids obtained in sufficient amounts were subsequently subjected to...

Puskásová Dominika: Alkaloids of the Amaryllidaceae family and their biological activity I. Diploma thesis 2019. Charles university in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmacognosy. The aim of this thesis was to isolate alkaloids from herbal extract, which was obtained from Narcissus pseudonarcissus 'Dutch Master' plant. The preparation and column chromatography of the extract were performed by PharmDr. Daniela Hulcová, Ph.D. as a part of her doctoral study. Using the preparative TLC method, 2 alkaloids marked as No.2.1 and 2.2.2 were isolated from the fraction No.4. Their structure was determined by using the NMR, GC-MS analysis and optical rotation. After comparing the data obtained with literature, the compounds were identified as (+)-homolycorine and (+)-masonine. Both homolycorine and masonine were subsequently subject to testing of inhibitory activity against acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), prolyloligopeptidase (POP) and glycogensynthase kinase 3β (GSK-3β). The activity was expressed as IC50 and was compared to IC50 of the reference substances. Galanthamine (IC50 AChE = 1,7 ± 0,1 μM, IC50 BuChE = 42,3 ± 1,3 μM) and huperzine A (IC50 AChE = 0,033 ± 0,001 μM, IC50 BuChE > 500 μM) were used as standards to compare the inhibitory activity...

Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of Pharmacognosy (16-16180) Author: Štefan Kosturko Supervisor: PharmDr. Marcela Šafratová, Ph.D. Advisor: PharmDr. Daniela Hulcová, Ph.D. Thesis title: Alkaloids of the family Amaryllidaceae and their biological activity II. Key words: Narcissus pseudonarcissus dutch master; bulbs; alkaloidal extracts; GC/MS analysis; biological activity; acetylcholinesterase; butyrylcholinesterase. The main aim of the thesis ‚,Alkaloids of the family Amaryllidaceae and their biological activity II.'' was the isolation of alkaloids, as a pure substances, in sufficient quantities to identify their structure and to test their biological aktivity in vitro. Alkaloids were separated from subfraction number 82 - 97 of weigh 2,3268 g. This subfraction was a part of the total plant extract, which was prepared by PharmDr. Daniela Hulcová Ph.D., as a part of her dissertation thesis and who also performed primary extraction and separation work. A total and concentrated alkaloid extract weighing 58 g, which included the aforementioned subfraction, was obtained. The already mentioned alkaloid subfraction, was divided by preparative thin-layer chromatography into five separates, which were subjected to further phytochemical work, and five pure...

Růžička, L. Isolation of alkaloids from Geissospermum vellosii Allemão and study of their biological activity. Diploma thesis, Department of pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic, 2020. The aim of this diploma thesis are alkaloids from Geissospermum vellosii. This tree native in South America is commonly used for treatment of various diseases, including cognitive deficits in elder people1 . The study is based on previous research that was focused on inhibition of human cholinesterases and glycogensynthase 3 β(GSK-3 β), which can be used in treatment of Alzheimer disease. Incidence of this disease is rising up in the last decades and it represents a big burden for both health service and economy of developed countries2 . Isolation was carried out from crude crushed stembark. After extraction and agitation, 50.4 g of thick yellowish ether extract was obtained. This extract showed activity against cholinesterases (IC50 AChE = 15.19 ± 0.96 μg/ml and IC50 BuChE = 0.37 ± 0.049 μg/ml). Later, this extract was separated to 16 fractions by column chromatography. Fraction GV9 was chosen for additional research. Thin layer chromatography was carried out for purification and extraction of white crystalline alkaloid. Structure was determined by...

Eliška Emrichová: Isolation of alkaloids of the species Geissospermum vellosii Allemão and study of their biological activity IV. Diploma thesis 2020. Charles University, faculty of Pharmacy in Hradec Králové, Department of Pharmacognosy. Key words: Geissospermum vellosii, bark, alkaloidal extracts, isolation of alkaloids, GC/MS analysis, biological aktivity, acetylcholinesterase, butyrylcholinesterase. The aim of this study was to isolate at least one pure alkaloid from the extract of Geissospermum vellosii Alemão bark. The whole process involved bark processing, to obtain summary and alkaloid extract and subsequent column chromatography. GV-4, one of the 16 obtained fractions, was separated into 5 subfractions. The GV-4bsubfraction was used to isolate pure alkaloids, processed by preparative thin layer chromatography and crystallization of pure compound. The structure of pure compound was determined by using NMR, GC-MS analysis and optical rotation. This compound was identified as anhydropereirine and was tested its inhibitory activity against human cholinesterases, AChE and BuChE. The alkaloids GV-1-a, GV-8-3-B, GV-9-c were isolated in the course of further work on the extract. Their inhibitory activity against GSK-3β was tested as well as their possibility to cross the blood-brain-barieer with...

Indiana University-Purdue University Indianapolis (IUPUI)
Approximately 2.8% of the world population is currently infected with hepatitis C virus (HCV). Neutralizing antibodies (nAbs) are often generated in chronic hepatitis C patients yet fail to control the infection. In the first two chapters of this study, we focused on two alternative routes of HCV transmission, which may contribute to HCV’s immune evasion and establishment of chronic infection. HCV was transmitted via a cell-cell contact-mediated (CCCM) route and in the form of exosomes. Formation of HCV infection foci resulted from CCCM HCV transfer and was cell density-dependent. Moreover, CCCM HCV transfer occurred rapidly, involved all four known HCV receptors and intact actin cytoskeleton, and led to productive HCV infection. Furthermore, live cell imaging revealed the temporal and spatial details of the transfer process. Lastly, HCV from HCV-infected hepatocytes and patient plasma occurred in both exosome-free and exosome-associated forms and the exosome-associated HCV remained infectious, even though HCV infection did not significantly alter exosome secretion. In the third chapter, we characterized HCV interaction with astrocytes, one of the putative HCV target cells in the brain. HCV infection causes the central nervous system (CNS) abnormalities in more than 50% of chronically infected subjects but the underlying mechanisms are largely unknown. We showed that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the free virus form or through cell-cell contact. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. In summary, we showed that CCCM HCV transfer and exosome-mediated HCV infection constituted important routes for HCV infection and dissemination and that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction. These findings provide new insights into HCV infection of hepatocytes and astrocytes and shall aid in the development of new and effective strategies for preventing and treating HCV infection.