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Screening of New Iron - and Copper - Chelating Substances - in vivo and in vitro Studies
Říha, Michal ; Mladěnka, Přemysl (advisor) ; Patočka, Jiří (referee) ; Tůmová, Lenka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Michal Říha, MSc. First Supervisor: Assoc. Prof. Přemysl Mladěnka, Ph.D. Second Supervisor: Prof. Radomír Hrdina, M.D., Ph.D. Title of Doctoral Thesis: Screening of new iron- and copper-chelating substances - in vivo and in vitro studies Iron and copper are essential trace elements which play a role in a series of physiological processes in human organism. Homeostasis of these transition metals is meticulously regulated since free or loosely bound iron and copper can catalyse the production of free radicals. Hereditary hemochromatosis, transfusion hemosiderosis and Wilson's disease are associated with absolute iron and copper overload in the organism. Transition metal chelators have crucial significance for the treatment of these states. There are several other diseases with documented involvement of iron and/or copper in their pathophysiology. Examples are primarily neurodegenerative diseases, cardio- vascular diseases, tumours and diabetes mellitus. Various chelating compounds are examined in these possible indications. The aim of this doctoral thesis was to perform a screening of iron- and copper- chelating substances and to study their properties in detail using in vitro and...
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In vitro cultures of medicinal plants IXX
Seidlová, Markéta ; Tůmová, Lenka (advisor) ; Martin, Jan (referee)
In vitro cultures of medicinal plants - IXX The subject of this thesis is the evaluation of secondary metabolites production in in vitro cultures of Silybum marianum L. after elicitor treatment. In this study selenium dioxide as elicitor in concentrations of 9,012.10-3 mol/l; 9,012.10-4 mol/l; 9,012.10-5 mol/l was used. The samples were taken after 6, 12, 24, 48, 72 and 168 hours of elicitor treatment. The effect of elicitor was compared with control samples, which were cultured without elicitation. The content of taxifolin and flavonolignans was determined by the method of HPLC. The results showed, that almost all observed metabolites were released into a nutrient medium. Cells of callus and suspension cultures produced only small amounts of taxifolin (0.01 mg/g DW). Taxifolin and silymarin complex releasing into nutrient media was observed as in control and also in the elicitated samples of callus and suspension cultures. Selenium dioxide elicitation caused statistically significant increases in releasing taxifolin and silymarin complex into the nutrient medium. The statistically significant releasing of flavonolignans (2.2 mg/100 ml) to the medium of suspension culture was reached after 72 hours of treatment with selenium dioxide in concentration of c1 (9,012.10-3 mol/l). The statistically...
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Transport mechanisms of secondary metabolites across membranes of plant cells
Kubeš, Jan ; Tůmová, Lenka (advisor) ; Babula, Petr (referee) ; Obložinský, Marek (referee)
Faculty of Pharmacy in Hradec Králové, Charles University Department of pharmacognosy Candidate: Mgr. Jan Kubeš Supervisor: PharmDr. Lenka Tůmová, CSc. Title of Doctoral thesis: Transport mechanisms of secondary metabolites across membranes of plant cells An attention was focused on the metabolism of isoflavones in recent years, including their transport within plants and cultures in vitro. A research is focused on the transport mechanism identification across cell or vacuolar membrane. There are localized different transporters as or the transport of some metabolites can take place through membrane vesicules. The release of five isoflavones (genistin, genistein, biochanin A, daidzein and formononetin) into nutrient medium was observed in suspension culture of Genista tinctoria L and Trifolium pratense L. These cultures were treated with NH4VO3 solutions (1 a 10 M concentration) when isoflavon content in medium was higher after 24 hours. This increased volume of isoflavones was supressed by different transport mechanism inhibitors. The transport of isoflavones in T. pratense was influenced by ABC inhibitors from MRP subfamily, but genistein concentration in medium was lower after treatment with MDR subfamily inhibitors. Brefeldin A, which blocks vesicular transport, also decreased concentration of...
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Phytochemical study of individual plant species of Bergenia genus
Hendrychová, Helena ; Tůmová, Lenka (advisor) ; Nagy, Milan (referee) ; Šmejkal, Karel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Mgr. Helena Hendrychová Supervisor: doc. PharmDr. Lenka Tůmová, CSc. Title of Doctoral Thesis: Phytochemical study of individual plant species of Bergenia genus Bergenia, native to central Asia, is promising source of medicinal compounds mainly in the system of Chinese and Indian traditional medicine. These species have been used for centuries in therapy of various diseases. They are valued for their ability to dissolve kidney or bladder stones. Bergenia plants are used for therapy of pulmonary diseases and cold. They have demonstrated antioxidant, antibacterial, antiviral, immunostimulant, anti-inflammatory, anticancer, antidiabetic, antitussive, hepatoprotective and diuretic effects. The main aim of this thesis was to determine the content of secondary metabolites (arbutin, total polyphenols, bergenin and anthocyanins) in three taxons of Bergenia genus - Bergenia crassifolia (L.) Fritsch, Bergenia ciliata (Haw.) Sternb. a Bergenia x ornata Stein., to compare their biological activities and to identify the most appropriate kind of Bergenia plants with the greatest potential for use in therapy. Plant hybrid B. x ornata was evaluated on the content of phenolic compounds and biological activity...
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Production of secondary metabolites in the explant cultures of Trichocereus pachanoi (Cactaceae)
Urbanová, Lucie ; Martin, Jan (advisor) ; Tůmová, Lenka (referee)
Production of secondary metabolites in the explant cultures of Trichocereus pachanoi (Cactaceae) Lucie Urbanová Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Pharmacy Key words: Trichocereus pachanoi, the explant cultures, mescaline, secondary metabolites, prodrugs, elicitation, cinnamic acid, sodium cinnamate, tryptophan, ozone, methylene blue The aim of this thesis was the research of effect of selected precursors and elicitors for secondary metabolite production of mescaline in explant cultures Trichocereus pachanoi. Suspension cultures were cultivated on the Murashige and Skoog medium with addition of the growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. To observe changes in production these precursors: cinnamic acid, sodium cinnamate and tryptophan, and these elicitors: ozone and methylene blue, were selected. Precursors were prepared and used in concentrations of 50, 100 and 500 mg·l-1. Ozone was added in two time slots, 5 and 10 seconds. Methylene blue has been used in concentrations of 1, 10 and 100 mg·l-1. Cultivation with added compounds lasted for 24 and 168 hours. The content of mescaline was determined by HPLC analysis. A statistically significant effect on the production of mescaline in vitro culture was proved only with...
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Screening of New Iron - and Copper - Chelating Substances - in vivo and in vitro Studies
Říha, Michal ; Mladěnka, Přemysl (advisor) ; Patočka, Jiří (referee) ; Tůmová, Lenka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Michal Říha, MSc. First Supervisor: Assoc. Prof. Přemysl Mladěnka, Ph.D. Second Supervisor: Prof. Radomír Hrdina, M.D., Ph.D. Title of Doctoral Thesis: Screening of new iron- and copper-chelating substances - in vivo and in vitro studies Iron and copper are essential trace elements which play a role in a series of physiological processes in human organism. Homeostasis of these transition metals is meticulously regulated since free or loosely bound iron and copper can catalyse the production of free radicals. Hereditary hemochromatosis, transfusion hemosiderosis and Wilson's disease are associated with absolute iron and copper overload in the organism. Transition metal chelators have crucial significance for the treatment of these states. There are several other diseases with documented involvement of iron and/or copper in their pathophysiology. Examples are primarily neurodegenerative diseases, cardio- vascular diseases, tumours and diabetes mellitus. Various chelating compounds are examined in these possible indications. The aim of this doctoral thesis was to perform a screening of iron- and copper- chelating substances and to study their properties in detail using in vitro and...
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In vitro cultures of medicinal plants - XV
Slavík, Marek ; Tůmová, Lenka (advisor) ; Martin, Jan (referee)
The subject of this study is the evaluation of secondary metabolites production in Hypericum perforatum L. cultures in vitro after elicitor treatment. The aim was to find if orthosilicic acid as abiotic elicitor increases the flavonoid and hypericin production in Hypericum perforatum L. cultures in vitro. Experiment was carried out in callus and suspension cultures of H. perforatum using Murashige - Skoog nutrient medium78 supplemented with 10 mg. ml-1 α-naphtylacetic acid as growth regulator. The elicitor was added in the form of solution in 3 different concentrations (C1 = 10.4047∙10-3 mol l-1 , C2 = 10.4047∙10-4 mol l-1 , C3 = 10.4047∙10-5 mol l-1 ), it was affecting 6, 12, 24, 48, 72 and 168 hours. The content of flavonoids and hypericin was determined by HPLC. Secondary metabolites release into nutrient medium was also a part of this study. The increasing flavonoid and hypericin production in callus cultures after elicitor application at any concentrations was not observed. The maximum flavonoid content (0.04 mg g-1 DW) in suspension culture was detected after 72 h of elicitor treatment in concentration of C1 where the maximum hyperoside production was observed. The maximum hypericin production (0.21 mg g-1 DW) in suspension culture was detected after 12 h of elicitor application in...
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In vitro cultures of medicinal plants XX
Chreňová, Kateřina ; Tůmová, Lenka (advisor) ; Kašparová, Marie (referee)
The study is aimed to enhance in vitro production of secondary metabolites in Genista tinctoria L. via elicitor treatment. The different levels of elicitor concentration - selenium dioxide were utilized to affect the quantity of isoflavonoids occurred in cultures. Experiment was perfomed in callus and suspension cultures on MS nutrient media supplemented with 10 g l-1 of NAA (α-naphtylacetic acid) as growth regulator. The elicitor was added in the form of solution in concentrations of 9,012.10-3 mol l-1 ; 9,012.10-4 mol l-1 and 9,012.10-5 mol l-1 . It was exposed for 6, 12, 24, 48, 72 and 168 hours. The content of isoflavonoids was determined by HPLC in dry weight (DW) and medium. The most effective production of genistin (6,20 mg. g-1 DW, 8,30 mg. g-1 DW) in callus culture was measured. It was reached in concentrations of 9,012.10-4 mol l-1 and 9,012.10-5 mol l-1 after 168 h elicitor treatment. The second most satisfactory genistin level 5,20 mg g-1 DW was detected after elicitor application in concentration of 9,012.10-4 mol l-1 after 6 h. The content of genistein, daidzein and formononetin in callus culture was low and in the most cases equal zero compared to control samples. The content of biochanin A was equal zero compared to control samples. The most efficient daidzein production (37,10 mg...
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In vitro cultures of medicinal plants XVIII
Bremertová, Iva ; Tůmová, Lenka (advisor) ; Kašparová, Marie (referee)
Genista tinctoria, family Fabaceae, is a potent source of isoflavonoids (genistin, genistein, daidzein, formononetin, biochanin A) with a wide spectrum of potential medical impact. Genista also contains quinolizidin alkaloids (cytisin, anagyrin, lupanin, spartein, etc.), which are toxic. The reason why in vitro cultures are used is an absence of toxic alkaloids production and higher yield of isoflavonoids in comparison with intact plant. For an increase of isoflavonoid production method of elicitation is beeing used. Isoflavonoids are studied for their phytoestrogenic effects, for which they could be used in treatment of postmenopausal symptoms and even in treatment of hormon-dependent tumours. The elicitor ethephon in concentration of 7000 µM, 700 µM and 70 µM was used in this work. The effect of ethephon inhibitor (AgNO3) in concentration of 120 µM was investigated too. Samples were examined after 24, 48, 72, 96 and 168 hours and then analysed by HPLC method. An effect of ethephon and its combination with AgNO3 was observed in callus and suspension cultures. Release of isoflavonoides into culture media was studied too. Daidzein production was the highest of all isoflavonoids in the callus culture after the treatment of ethephon in concentration of 700 µM after 96 hours (45,10 mg/g DW). The most...
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Isolation of plant organelles and study of transport mechanisms
Kettnerová, Dana ; Martin, Jan (advisor) ; Tůmová, Lenka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Diploma thesis Author: Dana Kettnerová Supervisor: PharmDr. Jan Martin, Ph.D. Title of diploma thesis: Isolation of plant organelles and study of transport mechanisms Key words: isolation, chloroplast, protoplast, vacuole, cell wall Isolation of plant organelles and other cellular components is essential for the study of physiological and pathological processes within the plant cell. It is possible to analyze cell structures, detect accumulation of certain metabolites, ions, enzymes and other substances thanks to the isolation. The goal of this diploma thesis was to provide an overview of isolation methods used for the isolation of cell wall, protoplasts, chloroplasts and vacuoles of plant cells. Isolation processes used for individual types of cell structures, the pros and cons of the various isolation methods, components of used media and their functions, as well as the structure and function of individual plant structures were described.
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