Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Martina Vítková Supervisor: RNDr. Jitka Vytlačilová, Ph.D. Title of diploma thesis: The biological activity of the selected drugs of traditional Chinese medicine The subject of this diploma thesis was to prepare aqueous extracts of five selected drugs from traditional Chinese medicine and deal with their biological effects. Namely it was Magnoliae officinalis cortex, Houttuyniae herba, Paeoniae radix alba, Psoraleae fructus and Typhae pollen. These drugs have a high potential for use in our medicine due to their beneficial effects on the human organism. The theoretical part contains a description of individual drugs and their therapeutic effects. Also there are described methods of toxicity testing. The experimental part describes the course of the acute toxicity test of used drugs on the model organism Artemia salina. LC50 was used as the endpoint. Testing with Artemia salina showed low toxicity of the tested drugs. For Magnoliae officinalis cortex the LC50 was found to be 0,6613 mg/ml, Psoraleae fructus 1,777mg/ml, Houttuyniae herba 4,079 mg/ml and Typhae pollen 4,34 mg/ml. No toxicity was detected for Paeoniae radix alba even at the highest measured concentration which was 15...

Bibliographical identification: Author's first name and surname: RNDr. Lucie Brulíková (nee Spáčilová) Title: Synthesis, reactivity and biological activity of C-5 substituted uracil analogues Type of thesis: Ph.D. thesis Department: Department of Organic Chemistry, Faculty of Science, Palacký University Olomouc Advisor: prof. RNDr. Antonín Holý, Dr.Sc., Dr.hc. mult. Advisor-consultant: doc. RNDr. Jan Hlaváč, Ph.D. The year of presentation: 2011 Abstract: The presented thesis is focused on the synthesis of various C-5 modified uracil analogues, the study of their reactivity and biological activity, especially cytotoxic activity. In the first part, the brief survey of described results for selected 5-alkoxymethyluracil analogues is performed. The second part of the presented thesis deals with the synthesis of novel uracil analogues modified at the C-5 position, the development and optimizing of procedure leading to the desired compounds, the study of biological activity and the evaluation of structure- activity relationship (SAR). This part presents the synthesis of a series of 5-[alkoxy(4- nitrophenyl)methyl)]uracil and 5-alkoxymethyluracil analogues and extended SAR studies depending on a substitution of metylene bridge directly attached at the C-5 position as well as alkoxy chain length. The last part of...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Adéla Potůčková Supervisor: doc. Ing. Lucie Cahlíková, Ph.D. Title of diploma thesis: Derivatives of Amaryllidaceae alkaloids and their biological activity The plants of Amaryllidaceae family are source of a large amount of biologically active substances called Amaryllidaceae alkaloids. Their effects include cytotoxic, antifungal, antiviral, antibacterial or antimalaric activity and, last but not least, the inhibition of cholinesterases. The Amaryllidaceae alkaloids, galanthamine type also includes alkaloid chlidanthine, which is a positional isomer of the clinically practice used alkaloid galanthamine. The sources of chidanthine are Chlidanthus fragrans, Haemanthus multilflorus and Hippeastrum aulicum. The object of this thesis was the preparation of chlidanthine derivatives, and the study of their biological activity connected to the therapy of Alzheimer's disease and cancer. Ten aromatic ester derivatives of chlidanthine were prepared. The chemical structures were elucidated by NMR, MS experiments and optical rotation. Most derivatives were screened for their AChE and BuChE inhibitory potential and their cytotoxic potential against a panel of tumor and non-tumor cell lines. The most...

Mišáková K: Alkaloids of the Amaryllidaceae family and their biological activity 1. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové, 2014, 63 p. The aim of the diploma thesis was a preparation of alkaloidal extract from Nerine Bowdenii to izolation of 2 pure alkaloids by column and thin layer chromatography. Subsequently these two alkaloids were subjected to structural MS and NMR analysis and tested for biological activity against human cholinesterases (HuAChE and HuBuChE) and for antioxidant and cytotoxic activity. Isolated substances were identified as ambelline and undulatine alkaloids, which have been already described previously in this plant. In the test for determining cholinesterase inhibitory activity of ambelline and undulatine the following values were measured; for ambelline: IC50, HuAChE = 169 ± 7 μM a IC50, HuBuChE = 985 ± 25 μM, for undulatine: IC50, HuAChE = 23,52 ± 1,19 μM a IC50, HuBuChE > 1000 μM. Galanthamin (IC50, HuAChE = 1,71 ± 0,007 μM, IC50, HuBuChE = 42,30± 1,30 μM), huperzin A (IC50, HuAChE = 0,033 ± 0,001 μM, IC50, HuBuChE > 1000 μM) and eserin (IC50, HuAChE = 0,063 ± 0,001 μM, IC50, HuBuChE = 0,130 ± 0,004 μM) were used like a positive controls. In the test for...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Daniela Hulcová Consultant: Prof. RNDr. Lubomír Opletal, CSc. Title of Diploma Thesis: Biological aktivity of plants metabolites. XXVII. Alkaloids of Fumaria officinalis L. and their effect on acetylcholinesterase and butyrylcholinesterase The summary ethanolic and diethylether extract were prepared from the herbs of a plant Fumaria officinalis L. We have obtained 201 fractions from this extract by column chromatography on the neutral Al2O3 (aluminium oxide). Joined fraction 68-76 were processed by thin layer chromatography, and 3 substances were obtained in pure state: DH-1, DH-2, DH-3. These 3 compounds were identified as protopine, (+)-fumariline and N- methylcorydaldine by the comparison with the literature and results of MS and NMR. These alkaloids were tested for the inhibitory activity against human erythrocytic acetylcholinesterase and plasmatic butyrylcholinesterase by Ellman`s method. The isolated alkaloids did not show any significant inhibitory activity (IC50, µM) compared with the standard galanthamine (IC50, µM; AChE 1,710 ± 0,065, BuChE 42,30 ± 1,30): protopin: AChE: 345,42 ± 31,12, BuChE: 239,66 ± 20,89, (+)-fumarilin: AChE: 2939,2 ± 309,41, BuChE: 330,62 ±...

Vítovcová, A.: Biological activity of secondary plants metabolites IV. Alkaloids of Vinca minor L. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2016. Extract of Vinca minor L. was separated to fractions in column chromatography. Flash chromatography, preparative TLC and crystalisation led to isolation of two alkaloids from fraction number two and number five. Alkaloids were identified by GC/MS as (+)-vincaminorine and vincorine. These alkaloids were tested for their inhibition activity towards cholinesterase (AChE and BuChe). Obtained activities IC50 were compared with standards (galanthamine, huperzine A). The most interesting activity against galanthamine (AChE 1,710 ± 0,065 µM, BuChE 42,30 ± 1,30 µM) and huperzine A (AChE 0,033 ± 0,001 µM, BuChE > 1000 µM) showed vincorine (AChE > 1000 µM, BuChE 9,75 ± 0,45 µM). His activity toward BuChE is higher than the activity of both standards. Inhibition activities of (+)-vincaminorine (AChE 746,5 ± 84,13 µM, BuChE 684,32 ± 70,66 µM) are negligible. Key words: Alzheimerʼs disease, cholinesterase, Apocynaceae, Vinca minor L., indole alkaloids

1 ABSTRACT Kučerová J.: Biological activity of secondary plants metabolites VI. Alkaloids of Vinca minor L. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of pharmaceutical botany and ecology, Hradec Králové 2016, 67 pages. Isolation of alkaloids was done from extract of Vinca minor, family Apocynaceae. Individual substances obtained were examined for their ability to inhibit human acetylcholinesterase and butyrylcholinesterase. The basic extract was after an initial extraction with 95% ethanol pre-purified by extraction with chloroform. This chloroform extract was divided further by column chromatography on a total of 531 fractions. The fractions obtained were, based on the similarities identified by thin layer chromatography, interconnected in a final number of 17 fractions. The mobile phase for column chromatography were mixtures of chloroform and medical gasoline, chloroform and ethanol, and ethanol itself. For further testing were used fractions number 2 and 5. From the fraction 2 was by preparative thin layer chromatography and subsequent crystallization isolated substance number 1. After analysis of this substance by GC/MS was found that it is a (+) - vincaminorein. Further was examined it's biological activity towards cholinesterases, which found a...

Este trabalho apresenta uma análise comparativa da atividade biológica das células imobilizadas em um reator anaeróbio, tratando esgoto sanitário. Os materiais utilizados como suporte para o desenvolvimento do biofilme foram: um polimérico poroso (espuma de poliuretano), um polímero menos poroso (PVC), uma cerâmica porosa (cerâmica especial, desenvolvida pelo DEMA-UFSCar) e uma cerâmica menos porosa (tijolo refratário). A concepção do reator anaeróbio possibilitou o desenvolvimento do biofilme de forma igual, assim como a retirada desse suporte mantendo íntegra a biomassa aderida. Alguns suportes e o lodo retirados do reator anaeróbio foram colocados em reatores diferenciais para determinação dos parâmetros cinéticos. Esses parâmetros foram utilizados para avaliar a atividade biológica, tanto do biofilme aderido aos suportes quanto do lodo. As células imobilizadas nos materiais poliméricos apresentaram maior atividade biológica. Não foi verificada nenhuma diferença significativa na remoção dos constituintes da matéria orgânica (proteínas, carboidratos e lipídeos) entre as células imobilizadas. As análises microscópicas não constataram diferenças na predominância de algum gênero nos suportes ou lodo. O reator foi operado durante 183 dias, com remoção média de DQO de 50,5%, a taxa de carregamento orgânico média foi de 0,193 kg DQO.m3.dia-1. / This work presents a comparative analysis of the biological activity of immobilized biomass in a fixed-bed anaerobic reactor treating domestic sewage. The matrices used for biofilm growth were a porous polymer (polyurethane foam), a polymer with low porosity (PVC), a porous ceramic (special ceramic) and a ceramic with low porosity (refractory brick). The conception of the fixed-bed anaerobic reactor warranty the structure and integrity of the biofilm when samples of support material must be taken. Moreover, the conditions for biofilm growth were similar for all the materials used. After start-up period, some supports were taken from the fixed-bed reactor and transferred to differential reactors, where kinetic studies were carried out. Kinetic parameters were estimated for the different supports and for granulated sludge and used to evaluate the biological activity in a comparative way. The cells adhered to the polymeric supports presented higher activities and the highest activity was obtained when polyurethane foam was used. No differences were verified for organic matter consumption (as proteins, carbohydrates and lipids) when different matrices were used for cell immobilization. Microscopic analysis indicated that the morphological types in the biofilms were similar in ali support materiais. The fixed-bed reactor was operated for 183 days with mean COD removal efficiency of 50.5%, subjected to a organic loading rate of 0.193 Kg COD.m3.dia-1.

O estudo fitoquímico das folhas de Ocotea catharinensis (Lauraceae) resultou no isolamento da neolignana tetraidrofurânica veraguensina (1) e do flavonóide glicosilado afzelina (10), ainda não descritas para a espécie, além de quatro neolignanas hexaidrobenzofurânicas (2-5) anteriormente descritas para mesma. Nos embriões somáticos in vitro constatou-se o acúmulo de duas neolignanas hexaidrobenzofurânicas (2, 3) e duas biciclo[3.2.1]octânicas (6, 7), dois sesquiterpenos (8, 9) e um fenilpropanóide (11). A neolignana biciclo[3.2.1]octânica (7S,8R,1\'R,2\'R,3\'R)-2\'-acetoxi-3,4-metilenodioxi-3\',5\'-dimetoxi-4\'-oxo-Δ1,3,5,5\',8\'-8.1\',7.3\'-neolignana (7), o sesquiterpeno (-)-eudesm-11-em-4α-ol (9) e o fenilpropanóide 6-metóxieugenol (11) não haviam sido isolados anteriormente desta espécie. O perfil dos metabólitos secundários incluiram análises do óleo essencial das folhas cuja análise indicou a predominância de mono- e sesquiterpenos e ausência de fenilpropanóides. Além disso, foram avaliadas atividades antifúngica e antioxidante nos extratos e substâncias isoladas. A fração de CHCl3 do extrato etanólico mostrou atividade antifúngica contra Cladosporium cladosporioide ou C. sphaerospermum. As frações de CHCl3 e de AcOEt do extrato etanólico, as neolignanas hexaidrobenzofurânica (5), biciclooctânica (6), o flavonóide glicosilado (10) e o fenilpropanóide (11) apresentaram atividade antioxidante através do método de DPPH (1,1-difenil-2-picril-hidrazila). A avaliação de hipóteses biossintéticas envolvidas na formação de neolignanas em O. catharinensis fizeram uso de culturas embriogênicas como modelo experimental. Foram realizados diversos estudos de bioconversões de precursores como o isoeugenol e 5-metoxieugenol utilizando-se suspensões celulares e frações enzimáticas porém as conversões in vivo utilizando-se precursores marcados e os embriões foram melhor sucedidas. Entre os precursores radioativos, a L-[U-14C]-fenilalanina foi incorporada às neolignanas hexaidrobenzofurânicas (2, 0,30% e 3, 0,19%) e biciclooctânica (6, 0,12%). No caso do ácido [8-14C]-ferúlico, esse foi incorporado à neolignana hexaidrobenzofurânica (2, 0,17%). Por outro lado, o uso de L-[1-13C]-fenilalanina e análise por espectrometria de massas-massas confirmou o enriquecimento de 13C nas neolignanas hexaidrobenzofurânicas (2 e 3). A análise por RMN de 13C indicou o enriquecimento de 4,3 e 5,0% nos carbonos C9 e C9\', respectivamente. Desta maneira, através dos dados de incorporação desses precursores, desvenda-se parte da via biossintética de neolignanas em O. catharinensis. / The phytochemical study of Ocotea catharinensis (Lauraceae) leaves resulted in the isolation of tetrahydrofuran neolignan veraguensin (1) and glycosylated flavonoid afzelin (10), not yet described for this species, besides four hexahydrobenzofuran neolignans (2-5), which had been previously described. The in vitro somatic embryos showed the accumulation of two hexahydrobenzofuran (2, 3) and two bicyclo[3,2,1]octane (6, 7) neolignans, two sesquiterpenes (8, 9) and one phenylpropanoid (11). Among these compounds (7S,8R,1\'R,2\'R,3\'R)-2\'-acetoxy-3,4-methylenedioxy-3\',5\'-dimethoxy-4\'-oxo-Δ1,3,5,5\',8\'-8.1\',7.3\'-neolignan (7), (-)-eudesm-11-en-4α-ol (9) and 6-methoxy-eugenol (11) have not been previously described for this species. The profile of secondary metabolite included the analysis of essential oil of leaves which indicated the predominance of mono- and sesquiterpene and no phenylpropanoids. In addition, antifungal and antioxidative activities were performed with extracts and isolated compounds. The CHCl3 fraction of ethanol extract exhibited antifungal activities against Cladosporium cladosporioide or C. sphaerospermum. The CHCl3 and EtOAc fractions of ethanol extract, the hexahydrobenzofuran (5) and a bicyclo[3.2.1]octane (6) neolignans, afzelin (10) and 6-methoxy-eugenol (11) displayed antioxidant activities using DPPH (1,1-diphenyl-2-picryl-hydrazyl). The evaluations of biosynthetic hypothesis for neolignan formation in O. catharinensis were based on the use of embriogenic culture as an experimental model. Several assessment for conversion of putative precursors such as eugenol and 5-methoxyeugenol were evaluated using suspension cells and cell free preparations but none of them were as effective as in vivo conversion using labeled precursors directly in embryos. Among the radioactive precursors L-[U-14C]-phenylalanine was incorporated to hexahydrobenzofuran (2, 0.30%; 3, 0.19%) and bicyclo[3.2.1]octane (6, 0.17%) neolignans while [8-14C]-ferulic acid was incorporated only to the hexahydrobenzofuran neolignan (2, 0,17%). The tandem mass spectrometry analysis confirmed the incorporation of 13C atoms in the neolignans (2, 3) indicating the incorporation of one or two molecules of L-[1-13C]-phenylalanine. The analysis of 13C NMR data revealed the enrichment of 4.3 and 5.0% at the positions C9 and C9\', respectively. In summary, the labeling experiments have contributed to unravel the biosynthesis of neolignans in O. catharinensis.

Além das funções mecânicas, sabe-se que o colágeno do tipo I é um dos componentes mais ativos na modulação das atividades biológicas que ocorrem na matriz extracelular (MEC) tais como a adesão celular, a coagulação e a mineralização do osso. Estes eventos ocorrem nos limites do período D, que é provavelmente o domínio funcional que mais prevalece em relação à transferência da informação extracelular para o interior da célula. O objetivo deste trabalho foi estudar as correlações existentes entre as atividades biológicas da MEC e as características estruturais do colágeno do tipo I, principalmente em relação à distribuição topográfica dos aminoácidos (aa) ácidos, básicos e hidrofóbicos. A metodologia empregada foi baseada em informações já existentes sobre a estrutura colágeno do tipo I e suas atividades relacionadas com a MEC. Os resultados mostraram que aa ácidos, básicos e hidrofóbicos formam duas regiões distintas. Em uma delas, aa ácidos e básicos se alojam em áreas delimitadas por aa hidrofóbicos; na outra, aa básicos, ácidos e hidrofóbicos formam aglomerados localizados principalmente nas proximidades dos limites Overlap:Gap. Exceto para os eventos de natureza estrutural são as regiões do colágeno do tipo I mais envolvidas na modulação das atividades da MEC, com a interface Overlap:Gap funcionando como um divisor do controle destas atividades. Enquanto 83,2% das atividades de adesão estão localizadas na zona do Overlap, 93,3% das atividades de natureza estruturais ocorrem a partir da zona limite Overlap:Gap e em direção à zona Gap. Embora nenhuma correlação possa ter sido estabelecida para a resultante de carga, sugere-se que a participação do colágeno do tipo I nas atividades da MEC, possa estar associada a diferenças de interações dos ligantes (carga, massa molecular e hidrofobicidade) com gradientes eletrostático e hidrofóbico existentes ao longo do período D. / Besides mechanical function, it is known today that type I collagen plays a major role in the modulation of many of the activities occurring in the extra-cellular matrix (ECM) such as cell adhesion, coagulation cascade and mineral deposition during bone formation. All these events occur by interaction of cells and ligands within the D-period of collagen microfibrill assembly, which is perhaps the most prevalent module of ECM information in the body. The purpose of this work was the study of correlations between the biological activities of ECM and the structural features of collagen associated with the distribution of acidic, basic and hydrophobic amino acids (aa) within the D period. The methodology used was based on existing information with respect to the structure of type I collagen and its function with respect to ECM activities. The results showed that acidic, basic and hydrophobic aa are found in two major distinct regions: one in which acidic and basic aa are located in areas defined by hydrophobic aa barriers; the other in which acidic, basic and hydrophobic aa forming clusters located preferentially in the boundaries of the Overlap:Gap zones. Except for the structural event, these clusters are the major sites for the interaction involving cell adhesion. The results also suggest that the Overlap:Gap interface function as a borderline for collagen interactions with ECM since 83,2% of the activities related to cell adhesion are located in the Overlap region while 93,3% of the activities of structural nature are enclosed within the limiting zone of Overlap:Gap to the direction of the Gap zone. Although no correlation could be established for net charge along the D period it is suggested that type I collagen participation in the activities of the ECM may also be associated to differences in ligand interactions (charge, molecular mass and hydrophobicity) with the electrostatic and the hydrophobic gradients that exists within the D period.