Bulvová, L.: Alkaloids of Papaver rhoeas L. (Papaveraceae) and their biological activity related to Alzheimerʼs disease I. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2017. The aim of this study was to process the summary alkaloidal extract of aerial parts of Papaver rhoeas L.; to isolate contained alkaloids using chromatographical methods; to identify them and to determine their inhibitory activity towards human enzymes acetylcholinesterase, butyrylcholinesterase and prolyloligopeptidase. Two alkaloids (+)-rhoeagenine and LB-2 were isolated, and the structure of LB-2 (its absolute configuration) is being determined nowadays. In vitro biological assays of these alkaloids found the following results: (+)-rhoeagenine (IC50 AChE ˃ 1000 μM, IC50 BuChE = 230 ± 10 μM, IC50 POP = 878 ± 45 μM) and LB-2 (IC50 AChE ˃ 1000 μM, IC50 BuChE = 314 ± 13 μM, IC50 POP = 706 ± 2 μM). The determined IC50 values of isolated alkaloids were compared with inhibitory standards of cholinesterases galanthamine (IC50 AChE = 1,71 ± 0,065 μM, IC50 BuChE = 42,30 ± 1,30 μM), huperzine A (IC50 AChE = 0,033 ± 0,001 μM, IC50 BuChE > 1000 μM, IC50 POP > 1000 μM) and rivastigmine (IC50 AChE = 0,037 ± 0,001 μM, IC50 BuChE = 0,0033 ± 0,0003...

Čakurdová, M.: Alkaloids of Papaver rhoeas L. (Papaveraceae) and their biological activity related to Alzheimer's disease II. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové, 2017. The aim of this work was focused on isolation of alkaloids from the fraction 1 (joined fractions 20-27), which was obtained from the summary alkaloid extract of Papaver rhoeas L. (Papaveraceae). The alkaloids were identified by structural analysis NMR, GC-MS, optical activity and melting point. Two alkaloids were identified as (-)-stylopine and (+)-rhoeadine. Isolated substances were tested on ability to inhibit the enzymes acetylcholinesterase, butyrylcholinesterase and prolyloligopeptidase. Obtained data were expressed as IC50 values: (-)-stylopine (IC50 AChE = 522 ± 67 µM, IC50 BuChE = >1000 µM, IC50 POP = >790 µM); (+)-rhoeadine (IC50 AChE = 915 ± 64 µM, IC50 BuChE = >1000 µM, IC50 POP = >790 µM). None of the isolated substances showed so potent cholinesterase inhibitory activity as the alkaloids galanthamine (IC50 AChE = 1.71± 0.065 µM, IC50 BuChE =42.30 ± 1.30 µM) and huperzine A (IC50 AChE = 0.033 ± 0.001 µM, IC50 BuChE = >1000 µM) or rivastigmine (IC50 AChE = 0.037 ± 0.001 µM, IC50 BuChE = 3.3 ± 0.3 nM)....

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...

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In recent years there has been an increase in researches for new enzyme inhibitors on medicinal plants used in the treatment of disorders such as schizophrenia, anxiety, amnesia, different stages of depression and bipolar affective disorder. The study of enzymatic inhibitors of prolyl oligopepetidase (POP) and acetylcholinesterase (AChE) are directly related to the treatment of central nervous sistem diseases. In a research with the species Hypericum brasiliense, native from Brazil, it was isolated as main secondary metabolite the chlorogenic acid, a compound able to inhibit POP and AChE. From this observation, three series of chlorogenic, caffeic and cinnamic acids derivatives were obtained through simple derivatization reactions and coupling with the amino acid proline. The derivatives were obtained by usual acetylation, methylation and esterification reactions with satisfactory yields of 50-90%. The couplings with the proline methyl ester were performed using either O-(7- azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU)/ diisopropylethylamine (DIEA) in dimethylformamide or isobutyl chloroformate/ Nmethylmorpholine in tetrahydrofuran. The derivatives were tested against the enzymes POP, AChE and dipeptidyl oligopeptidase (DPP IV). Also, a study was conducted to determine the antibacterial and antifungal activities of all derivatives. The capacity of the tested compounds to inhibit DPP IV and AChE was not exceptional. In contrast, the derivatives methyl ester and 1,7-acetonide obtained from chlorogenic acid, and caffeic acid and its methyl ester derivative showed selectivity and satisfactory performance as POP inhibitors, with IC50 values of 3 to 14 μM. All compounds showed moderate antimicrobial activity. / Nos últimos anos observa-se uma intensificação nas pesquisas por novos inibidores enzimáticos produzidos por plantas medicinais usadas no tratamento de transtornos mentais, tais como esquizofrenia, ansiedade, amnésia, diferentes estágios de depressão e o transtorno afetivo bipolar. O estudo de inibidores das enzimas prolil oligopepetidase (POP) e acetilcolinesterase (AChE), estão diretamente relacionados ao tratamento de tais enfermidades do sistema nervoso central (SNC). Em um trabalho com a planta medicinal Hypericum brasiliense, nativa do Brasil, foi isolado como um dos principais metabólitos secundários o ácido clorogênico, que demonstrou ter capacidade de inibir a POP e a AChE. A partir desta observação, foram obtidas três séries de derivados do ácido clorogênico, do ácido cafeico e do ácido cinâmico através de técnicas simples de derivatização e acoplamento com a prolina metil éster. Os derivados foram obtidos através de técnicas usuais de acetilação, metilação e esterificação, com rendimentos satisfatórios de 50-90 %. Já os acoplamentos com a prolina metil éster foram realizados com hexafluorofosfato de O-(7-azabenzotriazol-1-il)-N,N,N',N'- tetrametiluronio (HATU)/ diisopropiletilamina (DIEA) em dimetilformamida ou cloroformato de isobutila/ N-metilmorfolina em tetraidrofurano. Estes compostos foram avaliados quanto à capacidade inibitória das enzimas POP e AChE, bem como foram testados frente a enzima dipeptidil peptidade IV (DPP IV). Também foi realizado um estudo da atividade antimicrobiana dos derivados obtidos. Os resultados obtidos indicaram que, frente à DPP IV e AChE, os compostos avaliados não demonstraram significativa capacidade inibitória. Entretanto, o derivado metil éster e 1,7-acetonídeo obtidos a partir do ácido clorogênico, o cafeoato de metila e o próprio ácido cafeico mostraram capacidade inibitória seletiva para a POP, com valores de IC50 entre 3 e 14 μM. Com relação à atividade antimicrobiana, os derivados apresentaram moderada ação bactericida, destacando-se os compostos derivados do ácido cinâmico por serem fungicidas.

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),...

Drabbová, A.: Title of Diploma Thesis: Centaurea cyanus L. (Asteraceae) alkaloids and their biological activity related to Alzheimer's disease. Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové 2020. Ethyl acetate and chloroform alkaloids extracts were prepared from Centaurea cyanus L. seeds. A novel alkaloid AD-1 (Adrabbin) was isolated from the ethyl acetate extract by common chromatografic methods (column chromatography, preparative TLC). Its structure was elucidated by mass spektrometry, NMR analysis and determination of optical rotation. The alkaloid AD-1 (Adrabbin) possesses a unique structure which consists of a cyclic indole moiety. Also LC-MS analysis of fractions obtained by flash chromatography was performed. In those fractions were detected molecular ions related to compounds previously isolated from other Centaurea species. Alkaloid AD-1 (Adrabbin) was tested on ability to inhibit human cholinesterases, prolyl oligopeptidase and gylcogen synthase kinase 3β. The compound was considered against human cholinesterases inactive (IC50 values > 100 µM). Interestibgly, the alkaloid inhibited prolyl oligopeptidase the same intensity as a standard berberine (AD-1: IC50 143,0 ± 6,0 µM; berberin: IC50 142,0 ± 21,0 µM). The novel compound...

V. Hojgrová: Alkaloids of Vinca minor L. and their biological activity VIII. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany. Number of pages 69. This diploma thesis deals with the isolation of alkaloids from Vinca minor L. from the family Apocynaceae. Separation of alkaloids from the selected fraction (VM 215-258) or from their subfractions (VM 34-41, VM 86, VM 87-113) was performed by preparative TLC. Two pure alkaloids were isolated from the subfraction (VH 34-41). The first VH-1 alkaloid not yet isolated and the second VH 2 alkaloid: (-)-raucubainin. Alkaloids were identified by EI-MS, LC-MS, NMR and optical rotation and were compared with data in the literature. Isolated alkaloids were tested for acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and prolyloligopeptidase (POP) inhibitory activity and for cytotoxicity. Both substances did not show significant cholinesterase inhibitory activity IC50 against AChE after measurement, only (-)-raucubainin showed a slight activity against BuChE (IC50 = 94 ± 7 μM), VH-1 was found to be inactive (IC50 > 100 μM). POP inhibitory activity has so far only been tested for (-)-raucubainin; was found to be inactive (IC50 > 1000 µM). The results of the cytotoxic activity of the alkaloids...

La flexibilité est une caractéristique intrinsèque des protéines qui doivent, dès le mo- ment de leur synthèse, passer d’un état de chaîne linéaire à un état de structure tridimen- sionnelle repliée et enzymatiquement active. Certaines protéines restent flexibles une fois repliées et subissent des changements de conformation de grande amplitude lors de leur cycle enzymatique. D’autres contiennent des segments si flexibles que leur structure ne peut être résolue par des méthodes expérimentales. Dans cette thèse, nous présentons notre application de méthodes in silico d’analyse de la flexibilité des protéines : • À l’aide des méthodes de dynamique moléculaire dirigée et d’échantillonnage pa- rapluie, nous avons caractérisé les trajectoires de liaison de l’inhibiteur Z-pro- prolinal à la protéine Prolyl oligopeptidase et identifié la trajectoire la plus pro- bable. Nos simulations ont aussi identifié un mode probable de recrutement des ligands utilisant une boucle flexible de 19 acides aminés à l’interface des deux domaines de la protéine. • En utilisant les méthodes de dynamique moléculaire traditionnelle et dirigée, nous avons examiné la stabilité de la protéine SAV1866 dans sa forme fermée insérée dans une membrane lipidique et étudié un des modes d’ouverture possibles par la séparation de ses domaines liant le nucléotide. • Nous avons adapté auproblème de la prédiction de la structure des longues boucles flexibles la méthode d’activation et de relaxation ART-nouveau précédemment uti- lisée dans l’étude du repliement et de l’agrégation de protéines. Appliqué au replie- ment de boucles de 8 à 20 acides aminés, la méthode démontre une dépendance quadratique du temps d’exécution sur la longueur des boucles, rendant possible l’étude de boucles encore plus longues. / Flexibility is an intrinsic characteristic of proteins who from the moment of synthesis into a linear chain of amino acids, have to adopt an enzymatically active tridimensionnel structure. Some proteins stay flexible once folded and display large amplitude confor- mational changes during their enzymatic cycles. Others contain parts that are so flexible that their structure can’t be resolved using experimental methods. In this thesis, we present our application of in silico methods to the study of protein flexibility. • Using steered molecular dynamics and umbrella sampling, we characterized the binding trajectories of the Z-pro-prolinal inhibiter to the Prolyl oligopeptidase pro- tein and we identified the most probable trajectory. Our simulations also found a possible ligand recrutement mechanism that involves a 19 amino acids flexible loop at the interface of the two domains of the protein. • Using traditional and steered molecular dynamics, we examined the stability of the SAV1866 protein in its closed conformation in a lipid membrane and we studied one of its proposed opening modes by separating its nucleotide binding domains. • We also adapted the activation-relaxation technique ART-nouveau which was pre- viously used to study protein folding and aggregation to the problem of structure prediction of large flexible loops. When tested on loops of 8 to 20 amino acids, the method demonstrate a quadratic execution time dependance on the loop length, which makes it possible to use the method on even larger loops.

Adamcová, M.: Study of biological activity of alkaloids isolated from Argemone grandiflora (Papaveraceae) I. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2015. The aim of this study was isolation of substances from total diethyl ether alkaloid extract of Argemone grandiflora Sweet, their identification and assessment of their inhibition activity towards acetylcholinesterase, butyrylcholinesterase and prolyl oligopeptidase. Using common chromatografic methods, four alkaloids were isolated, that was identified as (+)-laudanosine, protopine, (-)-argemonine a (-)-platycerine. These substances was tested for their inhibition activity IC50: (+)-laudanosine (IC50 AChE = 617,00 ± 46,55 μM, IC50 BuChE = 644,77 ± 55,52 μM, IC50 POP = not mesured yet); protopine (IC50 AChE = 229,98 ± 21,02 μM, IC50 BuChE = 208,87 ± 17,67 μM, IC50 POP ˃ 1000 μM); (-)-argemonine (IC50 AChE = 4677,75 ± 1241,08 μM, IC50 BuChE = 885,45 ± 119,50 μM, IC50 POP = 337 ± 83,1 μM); (-)-platycerine (IC50 AChE = 223,65 ± 19,61 μM, IC50 BuChE = 1651,25 ± 327,7 μM, IC50 POP = 687 ± 74 μM). In comparison with the standards galanthamine (IC50 AChE = 1,710 ± 0,065 μM, IC50 BuChE = 42,30 ± 1,30 μM) and huperzine A (IC50 AChE = 0,033 ± 0,001...

Dvořáková Zdeňka: Alkaloids of Narcissus 'Dutch Master' (Amaryllidaceae) and their biological activity II. Diploma thesis 2016, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology. The content of this work was isolation of compounds from the selected fraction ND-6 obtained by column chromatography of Narcissus 'Dutch Master' alkaloid extract. Preparation of extract and its column chromatography was performed by Mrg. Daniela Hulcová as part of her doctoral studies. By the means preparative TLC was from fraction ND- 6 homolycorine type alkaloid (+)-O-methyllycorenine gained. Its structure was determined on the basis NMR, GC-MS analysis and optical rotation. The obtained data were compared with facts in known literature. By the isolated alkaloid was determined its cholinesterase inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Its inhibitory activity was expressed as IC50 (M) and compared with known standards galanthamine, physostigmine, and Huperzine A. This alkaloid is inactive against cholinesterase (IC50 AChE > 1000 M, IC50 BChE > 1000 M). On the basis of gained results, we can evaluate this alkaloid from the point of view of cholinesterase inhibition as potentially unusable in the treatment of AD. Key...