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...

A solid phase extraction (SPE) method was developed for simultaneous extraction of dicarboxylic acids and diols formed during hydrolysis of poly(butylene succinate), PBS, and poly(butylene adipate), PBA. The developed SPE method and subsequent GC-MS analysis were used to extract, identify and quantify low molecular weight products migrating from linear and branched poly(butylene adipate) (PBA) and poly(butylene succinate) (PBS) during aging in aqueous media. The combination of SPE and GC-MS proved to be a sensitive tool, able to detect small differences in the degradation rate during early stages of hydrolysis before any significant differences were observed by weight loss and molecular weight measurements. The detected low molecular weight products included monomers i.e. adipic acid and 1,4-butanediol for the PBA polymers and succinic acid and 1,4-butanediol for PBS. Several dimers and trimers i.e. hydroxybutyl adipate, hydroxybutyl succinate, di(hydroxybutyl) adipate, di(hydroxybutyl) succinate and hydroxybutyl disuccinate were also detected. Best extraction efficiency for 1,4-butanediol and succinic acid was achieved with a hydroxylated polystyrene-divinylbenzene resin as solid phase. Linear range for the extracted analytes was 1-500 ng/ml for adipic acid and 2-500 ng/ml for 1,4-butanediol and succinic acid. Detection and quantification limits for all analytes were between 1-2 ng/ml (S/N=3) and 2-7 ng/ml (S/N=10) respectively. Relative standard deviations were between 3 % and 7 %. Comparison of measured weight loss and the amount of monomeric products showed that weight loss during early stages of hydrolysis was mainly caused by the release of water-soluble oligomers that on prolonged ageing were further hydrolyzed to monomeric species. Significant differences in degradation rate could be assigned to degree of branching, molecular weight, aging temperature and degradation medium. Linear and branched PBA was mixed with PVC in solution cast films to study the effects of molecular weight and branching on plasticizer efficiency. Used as polymeric plasticizer, PBA formed a semi-miscible two-phase system with PVC where the amorphous part exhibited one single glass transition temperature and the degree of polyester crystallinity was dependent on molecular weight, degree of branching and blend composition. Plasticizing efficiency was favored by higher degree of branching and a 40 weight-percent polyester composition. / QC 20101209

Los insectos saproxílicos, que dependen de la presencia de madera muerta y/o en descomposición, de hongos de la madera, o bien de la presencia de otros organismos saproxílicos, en algún momento de su ciclo vital, desempeñan una función clave para el correcto funcionamiento de los ecosistemas de bosque, actuando como recicladores de los nutrientes contenidos en la madera en descomposición. La diversidad de insectos saproxílicos, y más concretamente de coleópteros, así como la estructura de los ensambles que conforman, se encuentran condicionadas por una serie de variables de carácter físico, químico y biótico. Comprender cómo este amplio conjunto de variables afecta a los patrones espacio-temporales de las comunidades, resulta esencial a la hora de elaborar estrategias de manejo de los ecosistemas, que permitan preservar este tipo de fauna, en muchas ocasiones, amenazada. Con este marco de fondo, el objetivo general de esta tesis doctoral reside en analizar el conjunto de factores ambientales que afectan a la diversidad y a la distribución espacio-temporal de los ensambles de coleópteros saproxílicos que habitan los bosques mediterráneos de roble melojo ubicados en el oeste de la península ibérica para, de este modo, poder proponer una serie de directrices a seguir, a la hora de manejar este tipo de ecosistemas. Este objetivo general se divide en una serie de objetivos específicos, cada uno de los cuales ha derivado en la redacción de un artículo científico y que pueden ser consultados de manera individualizada en los capítulos II, III y IV. En el capítulo II se evaluó la influencia de un total de 24 variables ambientales a nivel de paisaje y a nivel de árbol, muchas de ellas consecuencia del manejo tradicional del bosque en el oeste ibérico por parte del hombre, analizando la riqueza observada de especies y empleando los estimadores de diversidad verdadera. El estudio permitió demostrar que, son muchas las variables que deben ser tenidas en cuenta a la hora de aplicar estrategias de manejo de los ecosistemas forestales y que no todos los métricos empleados para evaluar la diversidad de los ensambles saproxílicos responden de la misma manera frente a esas variables derivadas del manejo. De acuerdo con los resultados obtenidos, es posible establecer una serie de recomendaciones para la gestión forestal de áreas sometidas a técnicas tradicionales de manejo: favorecer en cierta medida la apertura del medio, reducir la carga ganadera y la explotación de madera a nivel vecinal, mantener en el paisaje un elevado volumen de madera en avanzado estado de descomposición, realizar un trasmoche selectivo de cierta cantidad de árboles para acelerar la aparición de oquedades y conservar los denominados “árboles hábitat”, que presenten microhábitats potenciales para este tipo de fauna, ya sean oquedades o tumores. El capítulo III se centró en analizar la posible influencia de los factores abióticos de temperatura y humedad relativa, en los patrones intranuales mostrados por los ensambles de coleópteros saproxílicos que habitan los bosques de roble melojo en la península ibérica, tanto en términos de riqueza como de composición de especies. Además, se abordó el estudio del tamaño corporal, como un rasgo que puede afectar tanto a la fisiología como a la eficacia biológica (fitness) de las especies y que podría explicar, al menos en parte, la dinámica temporal de estos ensambles. Este estudio permitió demostrar que la dinámica temporal de los ensambles de coleópteros saproxílicos que habitan los bosques de roble en el oeste ibérico está condicionada, al menos en gran medida, por las fluctuaciones anuales de las variables abióticas de temperatura y humedad relativa. Considerando el marco actual de calentamiento global, el hecho de conocer cómo ambas variables abióticas afectan a la estructura y diversidad de estos ensambles, permitirá predecir algunas de las consecuencias que el cambio climático puede tener en la distribución temporal de estas comunidades faunísticas. Además, se observó que no todas las especies de coleópteros saproxílicos muestran la misma plasticidad a la hora de adaptarse a esas fluctuaciones de temperatura y que muchas de ellas pueden permanecer activas únicamente bajo un cierto rango térmico. En este sentido, se recomienda profundizar en el estudio de los rangos térmicos de las especies, ya que proporcionará información muy valiosa a la hora de tratar de entender los patrones temporales mostrados por estos ensambles. Finalmente, en el capítulo IV, se estudió la influencia que pueden tener las emisiones de compuestos orgánicos volátiles (COVs) por parte del roble melojo, en los ensambles de coleópteros saproxílicos que emplean esta especie arbórea como planta hospedadora. Para ello, se partió de la premisa de que los árboles añosos (generalmente de mayor tamaño y en un estado de descomposición avanzado) son más visitados por los insectos saproxílicos, que en el caso de los árboles más jóvenes (generalmente de menor porte y en un estado de descomposición nulo o poco avanzado); las emisiones de COVs podrían explicar, al menos en parte, ciertos aspectos de la etología de este grupo de insectos. A pesar de la ausencia de significación estadística, los resultados mostraron una tendencia clara, que indicó que los árboles de mayor porte (árboles añosos, en un estado de descomposición avanzado) emiten este tipo de compuestos con una mayor intensidad que los árboles de menor tamaño. Si tenemos en cuenta, además, que este estudio demostró que los árboles añosos, de mayor perímetro (generalmente en un estado de senescencia más avanzado) albergaron una mayor riqueza de especies de coleópteros saproxílicos, se llega a la conclusión de que la emisión de COVs debe ser tenida en cuenta como una variable más (dentro del amplio conjunto de las ya comentadas en apartados previos), que puede estar condicionando la dinámica espacio-temporal de los ensambles de coleópteros saproxílicos que habitan los bosques de roble melojo en la península ibérica. Este estudio abre una novedosa vía de investigación que permitirá profundizar en el estudio de la relación directa que puede existir entre los compuestos químicos emitidos por los árboles y la fauna saproxílica circundante.

Historical pharmaceutical preparations analyzed in this thesis were a senna extract more than 200 years old, an ointment "Naso-Merfen" 75 years old, and an ointment "Sulfathiazol" 42 years old. The active substances in the analyzed samples were sennosides A and B (senna extract), ephedrine and menthol (the ointment "Naso-Merfen"), and sulfathiazole (the ointment "Sulfathiazol"). The senna extract was analyzed by RP-HPLC and HPLC-MS. Separation conditions were optimized, especially for separation of the sennoside A and B enantiomers. The active substances were not detected in the sample. One degradation product and substances characteristic for senna were identified. Their presence in the historical and contemporary sample was compared. Detailed ESI− -MSn fragmentation mechanisms of sennoside A and B have been proposed. The sample of the ointment "Naso-Merfen" was analyzed by HILIC-UV, HPLC-MS, GC-MS, and AAS. Separation conditions were optimized. The active substances were quantified. Degradation products of the active substances were not detected in the sample. The sample of the ointment "Sulfathiazol" was analyzed by RP-HPLC and HPLC-MS. Separation conditions were optimized. The active substance was quantified. Degradation products of the active substance were not detected. The authenticity of...

Les nanotubes de carbone (NTCs) sont des charges particulièrement intéressantes car ils présentent des facteurs de forme (longueur/diamètre) très élevés. Cependant, le développement de ces applications à haute valeur ajoutée a été freiné par les problèmes de mise en oeuvre des NTCs (résultant de la difficulté de les disperser dans un milieu polymère) et par la formation d’agrégats de nanotubes en « fagots », ne permettant pas l’obtention de mélanges homogènes. La solution réside dans la fonctionnalisation des nanotubes avec des chaînes polymères afin de réduire l’effet des interactions entre NTCs et d’assurer une meilleure comptabilisation avec le polymère hôte au cours du mélange. Ici, nous nous sommes intéressés à la fonctionnalisation des nanotubes de carbone par des polyoléfines en utilisant une procédure de greffage radicalaire de type « grafting onto ». / Carbon nanotubes (CNTs) as filler are particularly interesting because they possess very high aspect ratio (length/diameter), typically up to 10,000. Hence, they can form conductive path in polymer matrix at much lower concentrations (below 5%), whereas in case of carbon black filler more than 20wt% loading is needed. However, the development of applications based on nanotubes with high value addition has been hampered by processing limitations resulting from the difficulty of dispersing in a polymeric medium. The formation of aggregates or bundles of nanotubes into host polymer do not allow obtaining homogeneous mixtures. The solution lies in the functionalisation of nanotubes with polymer chains to reduce the effect of interactions between CNTs and better compatibility with the host polymer in the mixture. Here, in this study, we aim to functionalise carbon nanotubes by using a polyolefin grafting procedure involving radical ‘grafting onto’.

One of the main problems encountered in chemical analysis operations in the field is collecting sufficient sample from the source and transferring that sample to the measurement instrument for fast separation and identification. I have been involved in developing a field-portable gas chromatography-mass spectrometry (GC-MS) system with solid phase microextraction (SPME) sampling for point detection of chemical agents. The objective is to minimize the analysis time between sampling and detection of a potential chemical threat. SPME offers a convenient means for sampling gaseous and liquid samples, concentrating the analytes, and transferring the analytes to the injection port of a GC system for separation and identification. GC-MS has advantages of high efficiency, speed, and applicability for field analysis. Work was done to optimize the SPME fiber coating, capillary column dimensions, and GC operating conditions to provide complete analysis within 3 minutes. Since isothermal operation of the GC was a prior requirement, many components in the chromatograms were unresolved. Therefore, a peak de-convolution algorithm was applied to allow for identification and quantitation of poorly resolved and often completely obscured trace components. Details of the instrumentation and optimization of operating conditions are described in this thesis.

Inborn errors of metabolism (IEM) cause significant morbidity and mortality when left untreated. Urine organic acid (UOA) analysis is often a first-line investigation when an IEM is suspected. UOAs are usually qualitatively analyzed via the current gold standard, GC-EI-MS (Gas Chromatography-Electron Impact-Mass Spectroscopy). The Agilent 7890 GC in tandem with the Waters’ Xevo TQ-S MS contains an easily interchangeable LC-ESI (liquid chromatography-electrospray Ionization) and GC-APCI (Atmospheric Pressure Chemical Ionization) instrument set-up, while maintaining accuracy and sensitivity in both LC and GC applications. Utilizing this novel GC-APCI instrument, this project aims to develop and validate a new UOA method for clinical use. Furthermore, utilizing the machine’s MRM mode would increase sensitivities thus allowing for hopefully quantitative analysis. Chemical standards and patient urine samples were extracted via a liquid-liquid ether extraction and derivatized with BSTFA for proper GC elution. Results were compared on the current gold standard GC-EI-MS instrument and the new GC-APCI-MS instrument. Initial instrument suitability and method setup was then optimized. Source moisture levels were modified to explore the wet proton transfer and the dry charge transfer mechanism using [M+H]+ and [M+*]+ ion peak ratios, respectively. Elution times and APCI ion mass spectra profiles of UOA metabolites of interest were identified from full scan mode in preparation for MRM mode analysis. Exploration into the wet and dry mode settings of the APCI source determined that the former induced via methanol had greater peak areas and signal-to-noise ratios. Suitable MRMs were determined for clinically relevant organic acids from which a quantitative assay was developed for methyl malonic acid and several other compounds. The Waters’ Xevo TQ-S micro with Agilent 7890 GC demonstrated promising GC-APCI-MS detection of urine organic acids. With clear avenues for future work, the APCI technique hints at great benefits for biochemical genetic laboratories. / Thesis / Master of Science (MSc) / Inborn errors of metabolism (IEM) are a class of genetic diseases that when left untreated, cause reduced quality of life and sometimes death in newborns. Urine organic acid (UOA) analysis is used for detection using an instrument called GC-EI-MS (Gas Chromatography Electron Impact Mass Spectroscopy). This project explores how a new instrument, the Agilent 7890 GC and the Waters’ Xevo TQ-S MS, can detect these genetic diseases using a technique called APCI (Atmospheric Pressure Chemical Ionization) while still being accurate and sensitive. UOAs are isolated from urine and run through the new machine. When compared to the currently used technique, results were promising but further optimization is needed. Using the new machine, various UOA compounds that were elevated and/or decreased in newborns with genetics diseases were identified and quantified. With clear avenues for future work, the APCI technique can greatly improve newborn diagnosis of IEMs.