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In vitro assessment of copper-chelating properties of isolated pure compounds from silymarine
Vítková, Martina ; Karlíčková, Jana (advisor) ; Polášek, Miroslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Mgr. Martina Vítková Supervisor: PharmDr. Jana Karlíčková, Ph.D. Consultant: doc. Ing. Kateřina Valentová, Ph.D. Title of Thesis: In vitro assessment of copper-chelating properties of isolated pure compounds from silymarin Copper is a trace element. Copper is important in many enzymatic reactions, for haemopoiesis, sugar metabolism, bone formation and for the nervous system. Disorder of copper homeostasis in organism will lead to Wilson disease, Menkes disease, Alzheimer's disease or Prion disease. Chelation agent binds redundant copper from tissues during chelation. The subject of this thesis was to in vitro designate effectivity of chelation cupric and cuprous ions by selected pure compounds isolated from silymarin and sulphates which are arised in organism as a metabolite. I used two spectrophotometric methods - hematoxylin method (to detect if the compound chelate copper) and bathocuproin method (more competitive assay) at various pH levels. All tested compounds chelated cupric and cuprous ions in hematoxylin method. 2,3-dehydrosilybin A+B and 2,3-dehydrosilychristin had the highest chelation effect in bathocuproin method. Keywords: silymarin, flavonolignans, copper, chelation, hematoxylin,...
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The effect of abiotic elicitors on secondary metabolites content in plant cultures in vitro - II.
Tomaidesová, Barbora ; Tůmová, Lenka (advisor) ; Karlíčková, Jana (referee)
The Effect of Abiotic Elicitors on the Content of Secondary Metabolites in In vitro Plant Cultures - II. The method of elicitation is used for the increased production of secondary metabolites in in vitro plant cultures. In this study, there the effect of pyridine derivate N-(5-chlorpyridin-2-yl)-4-ethylbenzamide in three concentration - 3.845.10-3 mol/l; 3.845.10-4 mol/l and 3.845.10-5 mol/l for the production of flavonolignans in callus and suspension cultures of Silybum marianum was tested. The evaluation of elicitation effect proceeded after 6, 24, 48, 72 a 168 hours against control samples without the elicitor treatment after 24 and 168 hours. In the same intervals the samples of culture medium were taken and assessed. The cells were cultivated on Murashige and Skoog medium with 10 mg/ml of α-naphtylacetic acid as growth regulator. The obtained and adjusted samples were analysed by the HPLC method. From the parts of silymarin complex were detected silychristin, silybin A, isosylibin A, isosilybin B. Flavonoid taxifolin wasn̕ t detected in any case. In callus culture, there was achieved a maximal production of all detected components of silymarin complex (30.508 μg/g DW) after 168 hours of elicitor treatment in concentration of 3.845.10-4 mol/l, it was about only substance - silychristin. The...
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Interaction of chalcones with transition metals I.
Faberová, Jana ; Macáková, Kateřina (advisor) ; Karlíčková, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidat: Jana Faberová Supervisor: doc. Ing. Kateřina Macáková, Ph.D. Title of Thesis: Interaction of chalcones with transition metals I. Iron is a biogenic metal element with unpaired d-electrons that plays a key role in the survival of unicellular organisms, plants and mammals. The electron shell of the iron atom is able to easily bind and release electrons and thus contribute to the proper function of the organism. Iron is necessary to ensure the transport of respiratory gases, cell differentiation and the functioning of some specific enzymes. However, despite the clearly positive effect of iron on human health, inappropriately low or high level of iron in the body can cause the development many diseases. Chalcones are plant polyphenols belonging to the family of flavonoids, secondary metabolites of plants. The term chalcone generally refers to a chemical structure with an ,-unsaturated ketone system based on 1,3-diphenylprop-2-en-1-on. These substances have a wide spectrum of biological activities and in the right amount can be good for human health. The aim of this diploma thesis was to monitor the iron-chelating and iron-reducting activity of selected chalcones: isoliquiritigenin, licochalcon A,...
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Can essential amino acids be considered as copper chelators?
Holotíková, Nikola ; Karlíčková, Jana (advisor) ; Tůmová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Nikola Holotíková Supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of Thesis: Can essential amino acids be considered as copper chelators? Copper is a trace element playing an important role in the human organism. Copper levels in the body have to be carefully controlled because disruption of copper homeostasis can cause oxidative damage which may lead to various pathologies. Chelation of copper can potentially be used as a therapeutic tool. Medical chelators should ideally mimic chelators, especially peptides and proteins which are important for regulation of copper homeostasis. Their structure is formed by amino acids linked by peptide bonds. For this reason, the study is focused on selected amino acids. Proteinogenic amino acids are classified into essential and nonessential. Essential amino acids must be obtained from the diet. The aim of this study was to compare the copper chelation and reduction activity of essential amino acids L-histidine and L-methionine, and nonessential amino acids L-cysteine, its dimer L-cystine, L-aspartic acid and L-glutamic acid. Also a L-cysteine-derived compound, N-acetylcysteine, was tested. All of them were tested using spectrophotometric methods at pHs which...
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Biotransformation of flubendazole and albendazole in plant cells
Seidlová, Helena ; Skálová, Lenka (advisor) ; Karlíčková, Jana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Helena Seidlová Supervisor: Prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Biotransformation of flubendazole and albendazole in plant cells Benzimidazole anthelmintics, the drugs against parasitic worms, are widely used in human as well as in veterinary medicine. Following excretion, these substances may persist in the environment and impact non-target organisms. Phytoremediation might be a suitable method for elimination of anthelmintics from the environment. In order to test detoxification abilities of plants, biotransformation pathways of albendazole (ABZ) and flubendazole (FLU) were studied in reed (Phragmites australis) in vitro. It was found that reed cells were able to uptake and biotransform both anthelmintics. These drugs did not significantly affect viability of the reed cells. Using HPLC/MS 10 ABZ metabolites and 5 FLU metabolites were found. Some atypical biotransformation metabolites (glucosylglucosides, acetylglucosides and xylosylglucosides), which have not been previously reported were identified. Based on the obtained results, the schemes of metabolic pathways of ABZ and FLU in reed were proposed. The amount of main metabolites (reduced FLU, ABZ-sulfoxide,...
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Interactions of tamarixetin and isorhamnetin with iron
Lavičková, Barbora ; Karlíčková, Jana (advisor) ; Carazo Fernández, Alejandro (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Mgr. Barbora Lavičková Supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of Rigorous Thesis: Interaction of tamarixetin and isorhamnetin with iron Iron is an important element in the human body that is essential for many biological functions. For example, it is a part of enzymes. This element is able to receive or deliver electrons and occur in the body in two oxidation states Fe2+ and Fe3+ ).. Under certain circumstances it can lead to the free radicals. Tamarixetin and Isorhamnetin are quercetin metabolites classified as flavonoids. Their structure is similar, containing 4 hydroxyl groups, differing only in the position of methoxy group of pfenyl, once in the 4' position of tamarixetin, and the second in the 3' position of isorhamnetin. Both substances have significant antioxidant activity. In this rigorous thesis, we investigate the interaction of two flavonols - tamarixetin and isorhamnetin - with iron ions at different (pato) physiologically significant pHs. Namely we tested chelating and reducing activity of the substances. The chelation efficiency was spectrophotometrically measured using ferrozine, which is a selective indicator of ferrous ions with which it forms a violet-colored complex...
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The effect of pyrazine derivatives on secondary metabolites content in plant cultures in vitro II.
Hanzlíková, Tereza ; Tůmová, Lenka (advisor) ; Karlíčková, Jana (referee)
The main purpose of the theses was to find if the abiotic elicitor 2,4,6-trimethyl-N-(pyrazine- 2-yl)benzenesulfonamide has any influence on the secondary metabolites production in callus and suspension cultures of Hypericum perforatum L. The cultivation was taking place in the Murashige a Skoog (MS) nutrient medium enriched by the growth regulator - alpha-naphthyl acetic acid at the concentration of 1 mg.L-1 . The elicitor was added to the cultures at the three levels of concentration: c1= 100.0 mg/100 ml; c2= 10.0 mg/100 ml; c3= 1.00 mg/100 ml. The individual samples were taken after 6, 24, 48, 72 and 168 hours of the elicitor application. The control samples without the elicitor were taken after 6, 48 and 168 hours. The collected samples were dried and subsequently transformed into methanol extracts in order to determine secondary metabolites content (rutin, hyperoside and quercetin) by HPLC method. Release of these metabolites into nutrient media was also subject of this observation. The elicitation has influenced production of the secondary metabolites, particularly in callus cultures, wherein several statistically significant values, characterizing increase in their production, were measured. The highest content of rutin (0.169 mg.g-1 DW) was determined in callus culture after 168 hours when...
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Interactions of alkaloids with transition metals IV.
Loskotová, Lenka ; Macáková, Kateřina (advisor) ; Karlíčková, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Bc. Lenka Loskotová Supervisor: doc. Ing. Kateřina Macáková, Ph.D. Title of Thesis: Interactions of alkaloids with transition metals IV. Copper is a biogenic trace element that participates in the proper function of an organism. It is part of a number of enzymes and participates in metabolic processes in the human body. The level of copper in the organism should be regulated to avoid its excess or deficit, as pathologies could occur. Alkaloids are natural nitrogenous substances of alkaline nature. We find many biological activities in them. The aim of this thesis was to measure copper-chelating and copper- reducing activity of isoquinoline alkaloids: corypalmine, thalictricavine, 8-oxoberberine, fumarilin, norisocorydine and laurotetanine. Alkaloid activity was measured at different pH environments (4.5; 5.5; 6.8 and 7.5) and in the DMSO environment by a spectrophotometric method using hematoxyline and acid disodium salt bathocuproindisulfonic. Based on the results, structure-effect relationships were derived. In experimental measurement, it was found that none of us tested alkaloids showed chelating activity. Reduction activity has been demonstrated in all test substances. The lowest activity...
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Interactions of reduced and oxidized glutathione with copper, iron and zinc
Salanciová, Ingrid ; Karlíčková, Jana (advisor) ; Jenčo, Jaroslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Ingrid Salanciová Supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of Thesis: Interactions of reduced and oxidized glutathione with copper, iron and zinc Copper (Cu), iron (Fe) and zinc (Zn) are important trace elements that are necessary for proper function of the body. Excess but also the lack of these metals may lead to pathological conditions. Glutathione is the main antioxidant in the human body so it is expected to protect the organism against concequences of metal excess. Glutathione occurs in the reduced (GSH) and oxidized state as glutathione disulfide (GSSG) in the organism, both states forming an important redox system. On the other hand, glutathione can reduce these metals so that could participate in the formation of free radicals (metal based Fenton reaction). In this diploma thesis, was tested the ability of reduced and oxidized glutathione to interact with Fe, Cu and Zn in various (patho) physiological pH conditions was tested by using in vitro spectrophotometric competitive methods. Interactions include not only the chelation of Cu, Fe and Zn ions, but also their reductive activity toward Cu2+ and Fe3+ cations. Hematoxylin, bathocuproinedisulfonic acid disodium salt, ferrozine...
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Preparation of a novel method for screeing of cobalt chelators
Moravcová, Monika ; Mladěnka, Přemysl (advisor) ; Karlíčková, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Monika Moravcová Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of thesis: Preparation of a Novel Method for Screening of Cobalt Chelators Cobalt as a structural part of the vitamin B12 is an essential microelement for living organisms including humans. However, its excess is associated with pathological conditions. Cobalt poisoning can be caused for example by exposure to cobalt metal dust during the production of hard metals or follow the corrosion of metal hip prosthesis. Patients intoxicated by cobalt can develop different manifestations including neurological impairment, hypothyroidism or cardiomyopathy. The aim of this work is to prepare a standardized, rapid, cheap but precise method for the screening of cobalt chelators. For this purpose, spectrophotometric detection using 1-nitroso-2-nafphhol-3,6-disulfonic acid disodium salt as the indicator was used. Firstly, it was found that the addition of cobalt ions led to a clear bathochromic shift of the maximum absorbance of the indicator. The relationship between the absorbance and cobalt concentration was highly linear from 470 to 560 nm at all 4 tested pH conditions (4.5, 5.5, 6.8 and 7.5). The sensitivity of the method...
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