Miklová V.: Cytotoxic and cholinesterase inhibitory activity of extracts from selected species of the Centaurea L. genus II. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2020. This Diploma Thesis was carried out at the Department of Pharmaceutical Botany FAF UK and it is a part of a screening of biologically active secondary metabolites of selected on taxa of the genera Centaurea L. and Psephellus L. in the family Asteraceae. Secondary metabolites are responsible for various effects on the human body. The study is focused on the phytochemical research of extracts prepared from aerial parts (cauloms with leaves) of Centaurea cyanus L., Centaurea stoebe L., Cyanus montanus (L.) Hill, Centaurea benedicta L., Centaurea jacea L., Centaurea macrocephala Muss. Puschk. ex. Willd, Centaurea solstitialis L., Centaurea nigra L., Centaurea scabiosa L., Psephellus bellus (Trautv.) Wagenitz, Centaurea pannonica (Heuff.) Hayek and Psephellus dealbatus (Willd) K. Koch. Both ethyl acetate and summary ethanolic extracts were prepared for detection of individual groups of secondary metabolites by thin-layer chromatohraphy. Consistently with previously published results, sesquiterpenes, flavonoids, coumarins, phenolic compounds,...

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)....

La pléiocarpamine est un alcaloïde indolo-monoterpénique de la famille des mavacurans. L’intérêt de cette substance naturelle réside dans sa capacité d’assemblage avec divers alcaloïdes indoliques donnant lieu à des bisindoles de structures complexes possédant des activités biologiques significatives. Plusieurs stratégies ont été étudiées pour la synthèse totale de ce composé pentacyclique. Notamment, la contraction d’un cétolactame a été envisagée pour la formation du cycle E. De manière inattendue, cette stratégie a conduit à la première synthèse totale de la (–)-17-nor-excelsinisine, un alcaloïde zwitterionique avec des activités antivirales. En parallèle, cette substance naturelle a été obtenue par cyclisation oxydante bioinspirée entre le formylester et l’atome d’azote aliphatique de la geissoschizine. La quaternarisation de ce dernier a ensuite permis de guider la chimiosélectivité de cette réaction vers le noyau indolique. Ainsi le couplage oxydant avec l’atome d’azote indolique a généré la liaison signature des mavacurans. Grâce à cette stratégie, plusieurs alcaloïdes de cette famille : (+)-taberdivarine H, (+)-16-hydroxyméthyl-pléiocarpamine et (+)-16-épi-pléiocarpamine, ont été synthétisés. Par la suite, une synthèse totale racémique de cette dernière a également été réalisée en seulement neuf étapes grâce à l’addition 1,4 intermoléculaire diastéréosélective d’un iodure de vinyle sur un indolylacrylate tétracyclique et à la fermeture finale du cycle D par substitution nucléophile. Le développement d’une version énantiosélective a été initié. Finalement, une étude a été réalisée pour la synthèse de la pycnanthinine, un bisindole dérivé de la pléiocarpamine. / Pleiocarpamine is a monoterpene indole alkaloid of the mavacuran family. The main interest of this natural product is its ability to assemble with various indole alkaloids to give structurally complex bisindoles with significant biological activities. Several strategies have been studied for the total synthesis of this pentacyclic compound. Especially, the ring contraction of a ketolactam was contemplated for E-ring formation. Unexpectedly, this strategy led to the first total synthesis of (–)-17-nor-excelsinisine, a zwitterionic alkaloid with antiviral activities. Simultaneously, this natural product was obtained via a bioinspired oxidative cyclization between the formylester and the aliphatic nitrogen atom of geissoschizine. Quaternarization of the latter allowed to guide the reaction chemoselectivity towards the indole nucleus. Therefore the oxidative coupling with the indolic nitrogen atom etablished the signature bond of the mavacurans. Thanks to this strategy, several alkaloids of this family: (+)-taberdivarine H, (+)-16-hydroxymethyl-pleiocarpamine and (+)-16-epi-pleiocarpamine, were synthetized. Subsequently, a racemic total synthesis of the latter was completed in only nine steps via a diastereoselective intermolecular 1,4 addition of a vinyl iodide into a tetracyclic indolylacrylate and a final D-ring closure by nucleophilic substitution. The development of an enantioselective version has been initiated. Finally, a study was performed for the assembly of pycnanthinine, a pleiocarpamine-derived bisindole.

The first semi-synthesis of (-)-melodinine K was completed from the monoterpene indole alkaloid (-)-tabersonine through regioselective enzymatic hydroxylation and Polonovski-Potier coupling. Regioselective borylation strategies were used to achieve meta-oxygenated derivatives of (-)-tabersonine for analysis in a Drosophila fruit fly Alzheimer’s phenotype model. A library of oxygenated and epoxidized bis-indole alkaloid analogs in the taberyunine class were prepared to conduct a structure activity relationship (SAR) study of the bis-indole framework against colon cancer and melanoma cell lines. Alkaloid aryl trifluoroborates were synthesized to increase nucleophilicity of arenes for unfavored alpha-arylations of tertiary amines on route to melosuavine bis-indole alkaloids. Overall, nine new bis-indole alkaloids were synthesized in the melodinine, taberyunine, and melosuavine class that showed strong cytotoxicity against a variety of melanoma, colon cancer, and renal cancer cell lines. / Chemistry

Bacterial membranes act as protective barriers and help to regulate molecular interactions between a cell and its surrounding environment. External chemical and physical influences have the potential to alter the properties of bacterial membranes and therefore impact the viability of the cell. This can stem from natural or seasonal changes to the local environment (e.g., temperature, pH, and salinity), or even deliberate application of an antimicrobial agent. Regardless, understanding exactly how such external stimuli influence bacterial membrane properties is of fundamental importance, both in terms of basic microbiology as well as for designing pharmaceutical interventions. Experimentally, this is a non-trivial task as this requires selective isolation of a signal arising from the membrane, which is typically buried in the overwhelming background response of the surrounding bulk environment. In particular, our lab has previously developed the surface-sensitive nonlinear optical method, second harmonic light scattering (SHS), as a means of interrogating molecular interactions at the membrane surfaces of living cells, even for multimembrane systems (e.g., Gram-negative bacteria). In this dissertation, time-resolved SHS was employed to study a variety of membrane properties across two separate projects, including 1) chemical and physical induced changes in membrane permeability and 2) temperature-induced membrane permeability changes. Specifically, in the first project (Chapter 4), the influence of the signaling molecule, indole, on the permeability of the bacterial cytoplasmic membrane was quantified. It was revealed that the interaction of indole with the tryptophan specific transporting protein, Mtr permease, resulted in enhanced passive diffusion across the membrane. For the second project (Chapter 5), we examined the influence of temperature on the rate of passive diffusion across a membrane, both in model systems (liposomes) and in living cells (E. coli). For both bacterial and liposome systems, increasing temperatures resulted in a modest increase in passive diffusion rates across the membrane. However, when the temperature range included a phase transition, passive diffusion increased by an order of magnitude. Therefore, by monitoring transport rate in relation to temperature, membrane phase transitions can be quantitatively determined based on the characteristic discontinuities in the measured trend. / Chemistry

Two series of stable palladium and silver complexes ligated to hemilabile ligands were prepared. The stability and applicability of these well-defined complexes in promoting various reactions (cycloaddition, hydroamination, cross-coupling reactions, etc.) was investigated. Structure-activity studies with a series of related ligands led us to find a pronounced ligand effect on these reactions. The dichotomous reactivity of triethyl phosphonium hydrobromide salt with dialkyl acetals was used for the synthesis of alkoxy phosphonium salts. Reactivity and applications of these phosphonium salts were described for the synthesis of the biologically active cinnamyl triazoles and ethyl indole-2-carboxylates. Total synthesis of the natural product nostodione A, was developed in eight chemical steps and with 21.6% overall yield from ethyl-2-indolecarboxylates. The synthesis of a mini-panel of structural analogs allowed for the discovery of anti-parasitic biological activity of nostodione A and its analogues for the first time. / Thesis / Doctor of Science (PhD)

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La croissance et la capacité d'absorption d'azote de la microalgue d'eau douce Chlorella vulgaris co-immobilisée dans des billes d'alginate, en présence de la bactérie promotrice de croissance des microalgues Azospirillum brasilense Cd, ont été étudiés. La co-immobilisation des microalgues avec A. brasilense conduit à deux phénomènes indépendants directement affectés par des facteurs de culture, tels que la forme azotée, le pH et la présence d'une source de carbone. Premièrement, la croissance de la population de microalgues augmentait sans une augmentation de la capacité d'absorption d'azote par les cellules seules, ou deuxièmement, la capacité des cellules à absorber l'azote augmentait sans une augmentation de la population entière de microalgues. Ces phénomènes étaient dépendants de la densité de la population des microalgues, qui était à son tour affectée par les facteurs de culture. Cela renforce la conclusion selon laquelle la taille de la population de microalgues contrôle l'absorption d'azote chez les cellules de C. vulgaris -plus la population est élevée (peu importe les paramètres expérimentaux), moins les cellules absorbent d'azote. Les résultats ont montré que YAzospirillum affectait la croissance de la Chlorella vulgaris. L'un des processus proposés par lequel XAzospirillum promeut la croissance des plantes supérieures, est la production de phytohormones, principalement 1' acide 3-indole acétique (auxine ou AI A). Par conséquent, l'effet de cette phytohormone, produite par plusieurs souches de VAzospirillum et favorisant la croissance de microalgue, a été étudié. Cinq souches de type sauvage â!Azospirillum {A. brasilense souches Cd, Sp6, 8-1 et Sp245 et A. lipoferum JA4) et leurs mutants déficients en production d'AIA : A. brasilense SpM 7918 (modifié de A. brasilense Sp6), A. brasilense FAJ0009 (modifié de A. brasilense Sp245), A. brasilense S-l-gfp (modifié de A. brasilense 8-1) et A. lipoferum 3A4::ngfp\5 (modifié de A. lipoferum JA4) ont été co-immobilisés avec C. vulgaris en billes d'alginate. Aucune production d'AIA n'est possible sans l'apport de tryptophane, nos résultats corroborant des observations antérieures selon lesquelles la production d'AIA chez Azospirillum est induite par la présence de tryptophane. Les taux élevés de tryptophane étaient délétères sur la croissance des pousses de C. vulgaris utilisées. Cette recherche montre que que l'AIA produit par 1' Azospirillum spp. est impliqué dans la croissance du C vulgaris immobilisé dans des billes d'alginate, et que la co-immobilisation avec différentes souches d'Azospirillum spp. atténue les effets délétères du tryptophane sur le C. vulgaris. Co-immobilisation de Chlorella vulgaris avec Azospirillum brasilense affecte l'absorption d'ammonium par les microalgues par le biais de métabolismes indéfinis. Les résultats présentés dans ce travail montrent clairement qu'il y a un effet & Azospirillum sur les activités spécifiques de la Glutamine synthétase (GS) et de la Glutamate déshydrogénase (GDH) de Chlorella. Lorsque Chlorella coimmobilisée absorbe plus d'ammonium que Chlorella seule, les deux enzymes fonctionnent en même temps, et les activités spécifiques par cellule de la GS et de la GDH sont plus élevées. Toutefois, dans les cas où seule l'activité GS augmente, mais pas celle de la GDH, l'activité GS plus élevée n'est pas suffisante pour atteindre une absorption d'ammonium plus élevée au niveau de la culture. En résumé, il y a un effet positif d'A. brasilense sur l'activité de la glutamine synthétase et de la glutamate déshydrogénase chez Chlorella. La régulation et la relation de l'activité enzymatique avec la concentration initiale en ammonium demeurent floues .Les données présentées dans ce chapitre dans leur ensemble montrent que les activités plus élevées de GS et de GDH augmentent l'absorption d'ammonium par Chlorella. / Growth and capacity to absorb nitrogen in the freshwater microalgae Chlorella vulgaris when co-immobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense Cd and the mechanisms involved were studied in this work. Co-immobilization of the microalgae with A. brasilense leads to two independent phenomena directly affected by culturing factors, such as nitrogen species, pH, and presence of a carbon source. First, growth of the microalgae population increased without an increase in capacity of the single cells to absorb nitrogen; second, the capacity of cells to absorb nitrogen increased without increasing the microalgae population. These phenomena were dependent on population density of the microalgae, which was, in turn, affected by culturing factors. This supports the conclusion that the size of the microalgae population controls absorption of nitrogen in C. vulgaris cells—the higher the population (whatever the experimental parameters), the less nitrogen each cell takes up. The results show that Azospirillum affects the growth of Chlorella vulgaris. One of the mechanisms proposed by which Azospirillum promotes growth of higher plants is the production of phytohormones, mainly the auxin indole-3-acetic acid (IAA); therefore the effect of this phytohormone produced by several strains of Azospirillum in promoting growth of the microalgae was studied. Five wild type strains of Azospirillum {A. brasilense strains Cd, Sp6, 8-1, and Sp245 and A. lipoferum JA4) and their indole-3-acetic-acid déficient mutants, A. brasilense SpM 7918 (modifïed from A. brasilense Sp6), A. brasilense FAJ0009 (modified from A. brasilense Sp245), A. brasilense %-\-gfp (modified from A, brasilense 8-1), and A. lipoferum lA4::ngfp\5 (modified from A. lipoferum JA4) were coimmobilized with C. vulgaris in alginate beads. No IAA production occurred without tryptophan. Our results corroborate previous observations that IAA production in Azospirillum is induced by the presence of tryptophan. It was found that high levels of tryptophan are deleterious to the growth of the C. vulgaris strain we used. This study demonstrated that IAA produced by Azospirillum spp. is involved in growth promotion of C. vulgaris immobilized in alginate beads, and that co-immobilization with several strains of Azospirillum spp. alleviated the deleterious effects of tryptophan in C. vulgaris. Co-immobilization of Chlorella vulgaris with Azospirillum brasilense affects ammonium absorption by the microalgae via undetermined metabolism. The results presented in this work show clearly that there is an effect of Azospirillum on the specific activity of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella. When coimmobilized Chlorella absorbs more ammonium than Chlorella alone, both enzymes are operating at the same time, and the specific activity per cell of GS and GDH are higher. However, in cases when only the activity of GS increases, but not of GDH, this GS higher activity was not sufficient to yield a higher absorption of ammonium at the culture level. There is a positive effect of A. brasilense on the activity of glutamine synthetase and glutamate dehydrogenase in Chlorella. However, the regulation and the relation of the enzymatic activity with initial ammonium concentration are still unclear.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24557号 / 薬科博第174号 / 新制||薬科||19(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 高須 清誠, 教授 大宮 寛久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM