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HPLC-high resolution mass spectrometry analysis of in vitro and in vivo metabolism of scoparone
Novák, Filip ; Matoušková, Petra (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Eastern Finland in Kuopio, School of Pharmacy Department of Pharmaceutical Chemistry Candidate: Filip Novák Supervisor: Ing. Petra Matoušková, Ph.D. Consultant: prof. Seppo Auriola Title of Diploma thesis: HPLC-high resolution mass spectrometry analysis of in vitro and in vivo metabolism of scoparone Scoparone is an active ingredient of Artemisia scoparia, a medicinal plant used in traditional Chinese medicine. It has been studied for various pharmacological effects such as upregulation of conjugation enzymes included in excretion of bilirubin, reduction of proinflammatory cytokines, lowering of plasma lipids levels and inhibition of platelet aggregation. In this thesis, metabolism of scoparone was studied by LC-MS method using Q-ToF device. Scoparone was incubated with liver microsomes obtained from 6 different mammal species to study in vitro oxidation. In total, six metabolites were detected in the incubation samples. Scopoletin and isoscopoletin were identified as major metabolites in every species, however, the rates of scoparone oxidation as well as a ratio of formed isoscopoletin and scopoletin varied. Furthermore, in vivo metabolites in human were studied in urine samples obtained from...
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Modulatory effect of humulene, caryophyllene and caryophyllene oxide on selected biotransformation enzymes in human liver cells
Trnčáková, Veronika ; Boušová, Iva (advisor) ; Matoušková, Petra (referee)
1 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Bc. Veronika Trnčáková Supervisor: doc. PharmDr. Boušová Iva, Ph.D. Title of diploma thesis: Modulatory effect of humulene, caryophyllene and caryophyllene oxide on selected biotransformation enzymes in human liver cells Sesquiterpenes are substances of natural origin, produced mainly by higher plants. They are mainly found in food supplements and natural medicines. Large structural diversity is a feature of these substances. The anti-inflammatory, antiparasitic and anti-cancer effects were also reported. The aim of this diploma thesis was to investigate the effect of selected sesquiterpenes, α-humulene (HUM), β-caryophyllene (KAR) and β-caryophyllene oxide (CAO), on gene and protein expression of selected phase I drug-metabolizing enzymes, which were carbonyl reductase 1 (CBR1), aldo/keto reductase 1C (AKR1) and several cytochrome P450 isoforms, namely CYP3A4, CYP2B6 and CYP2C. The effect of sesquiterpenes on the gene and protein expression of enzymes was studied using precision-cut human liver slices (8 mm diameter, 150-180 µm thickness). Liver samples were received from hepatic surgery of five subjects of both sexes and at 58-69 years of age. Liver slices were incubated with studied compounds (HUM, KAR,...
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Effect of anthelmintics on expression of selected plant enzymes
Navrátilová, Martina ; Matoušková, Petra (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Martina Navrátilová Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of anthelmintics on expression of selected plants enzymes Fenbendazole (FEN) is a broad-spectrum benzimidazole anthelmintic that is widely used to prevent and treat helminthoses in farm animals. The use of anthelmintics is associated with the risk of their transfer to the environment, where they can potentially affect non-target organisms, including plants. They come into contact with anthelmintics and their metabolites mainly due to the fertilization of farmland with manure and/or come directly into contact with the excrements of treated animals on pastures. There have not been many studies focused on the cell physiology and cell metabolism of plants affected by anthelmintics, which lead to our participation in a study which dealt, in part, with the influence of FEN on the transcriptome and gene expression in model plant Arabidopsis thaliana. A. thaliana is a monocotyledonous plant, which is a very common model organism of higher plants. Its genome was also published in 2000, allowing extensive research into gene expression. The aim of this work was to monitor expression of selected A....
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The effect of alpha-humulene and carophyllene oxide in ovarian cancer cells
Žáková, Kateřina ; Matoušková, Petra (advisor) ; Svobodová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Žáková Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: The effect of alpha-humulene and caryophyllene oxide in ovarian cancer cells. Sesquiterpenes α-humulene and β-caryophyllene oxide are substances with antiproliferative activity against number of tumor cell lines. In this study, the antiproliferative efect of α-humulene and β-caryophyllene oxide was tested alone and in combination with doxorubicin, using neutral red uptake assay. Doxorubicin- sensitive cell line A2780 was more sensitive to the action of doxorubicin and sesquiterpenes than SKOV3 cell line, which is naturally doxorubicin-resistant. To determine the interactions between sesquiterpenes and doxorubicin, software program CalcuSyn which is based on Chou-Talay method was used. The combination indexes ofsesquiterpenes with doxorubicin were determined. Both sesquiterpenes had antagonistic effect on doxorubicin in A2780 cell line and synergistic effect on SKOV3 cell line. The mechanism of the antiproliferative effect of all three tested substances is explained by the production of ROS. For the determination of pro-oxidant effects of the tested substances the DCF assay was used. Our results showed good...
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Effect of monepantel on the expression of selected enzymes in Haemonchus contortus
Jelenková, Klára ; Matoušková, Petra (advisor) ; Raisová Stuchlíková, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Klára Jelenková Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of monepantel on the expression of selected enzymes in Haemonchus Contortus Barber's pole worm (Haemonchus contortus) is parasitic nematode which is the cause of haemonchosis, worldwide distributed disease of small ruminants leading to decrease in productivity and in many cases even to animals death. For prevention and treatment of this disease are used drugs for a long time - anthelmintics. Due to their frequent use and lack of innovation in this group of drugs parasites have gradually built an extensive resistance to these drugs. New hope in therapy of haemonchoses is monepantel, drug belonging to aminoacetonitrile derivatives which have unique mechanism of action. This drug was launched more than 25 years after registration of the last drug from the group of anthelmintics. Despite the initial great success information about occurrence of resistance often appear in recent years. Current knowledge about the mechanisms of resistance development and the factors that influence them is as well as information about drug action at the molecular level still insufficient. To limit the development of...
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Identification of new potent inhibitors of selected mycobacterial enzyme
Komrsková, Jitka ; Novotná, Eva (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Jitka Komrsková Supervisor: Mgr. Eva Novotná, Ph.D. Title of Diploma Thesis: Identification of new potent inhibitors of selected mycobacterial enzyme Mycobacterium tuberculosis remains one of the deadly pathogens, which are threat for people around the world. The combination of antituberculotic drugs is effective but the treatment is prolonged and sometimes it is not successful because of multiresistant forms of tuberculosis and also co-infection with HIV, that are the problems in last a few years. Therefore, it is necessity to find a new drug against tuberculosis, which will be effective against resistant forms of mycobacterium and can be used for HIV therapy. Enzyme isocitratelyase performs a potentially drug target against Mycobacterium tuberculosis. This enzyme plays a key role of glyoxylate pathway, where catalyzes the conversion of isocitrate to succinate and glyoxylate. This enzyme is necessary for mycobacterial survive during acute phase of the infection. The aim of this work is to determine that the activity of isocitratelyase is inhibited by S-benzylisothiosemicarbazones and flavonoids and so they could be new potentially antituberculotic drugs. Activity of this enzyme was...
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Influence of TNF-α on hENaC subunits expression
Martan, David ; Svobodová, Hana (advisor) ; Matoušková, Petra (referee)
University of Vienna Faculty of Life Sciences Department of Pharmacology and Toxicology Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: David Martan Supervisors: Prof. Dr. Rosa Lemmens-Gruber, PharmDr. Hana Bártíková, Ph.D. Title of the diploma thesis: Influence of TNF-α on hENaC subunits expression The human amiloride-sensitive epithelial sodium channel (hENaC) is a type of ion channel which has the ability to control salt and water homeostasis. Therefore it is one of the main driving forces for the reabsorption of water through the alveolar epithelium. A dysfunction of this channel, respectively of this control mechanism, leads to a very severe disease - pulmonary edema and several other pathological conditions. Previous studies tested a drug called AP301, recently named solnatide. AP301 is a cyclic protein comprising the human tumour necrosis factor lectin-like domain sequence. This drug was recently developed as a potential treatment of pulmonary edema. The principle is that it activates hENaC by increasing the open probability. It was also shown that AP301 transiently increases the expression of hENaC subunits in mammalian cells. In this study we used the Western blot method to test the influence of tumour necrosis factor α...
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Role of disulphide bonds in hA2A subtype adenosine receptor
Zappe, Lukáš ; Matoušková, Petra (advisor) ; Bárta, Pavel (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lukáš Zappe Supervisor: Ing. Petra Matoušková Ph.D., PD Dr. Anke C. Schiedel Title of diploma thesis: Role of disulphide bonds in hA2A subtype adenosine receptor The adenosine A2A receptor belongs to the G protein coupled receptor family (GPCR). GPCRs are the targets of almost 40% of the drugs on the market. GPCRs are characterized by seven transmembrane helices, which are linked by three extracellular and three intracellular loops (ECL and ICL). The structure of the receptor has been revealed by crystallography, hence we know that ECL1 and ECL2 are connected by several disulphide bonds. The ECL2 is believed to be involved in ligand binding and recognition. In order to understand the relevance of those disulphide bonds involved in this process, four adenosine A2A receptor mutants were generated by one-site direct mutagenesis, in which the cysteine residues were replaced with serine residues (C146S, C159S, C166S and C146S- C159S). These receptor mutants were expressed in the mammalian cell line, CHO K1(Chinese Hamster Ovary) and the receptor expression was tested with ELISA (Enzyme- linked immunosorbent assay). The determination of ligand binding has been carried out by radioligand...
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