|
Cytotoxic effect of some cyanobacterial crude extracts and metabolites against selected human cancer lines, especially focusing on pancreatic cancer cell line PaTu
VICKOVÁ, Kateřina
Cancer is disease that affects human population for many thousands of years. Fortunately fighting cancer is more and more effective. This is mainly thanks to combining different strategies of therapy and progress in novel drug development and research. Cyanobacteria are emerging as an important source of novel bioactive secondary metabolites which provides an opportunity for new drug discovery. The aim of this study was evaluation of three cyanobacterial strains for in vitro cytotoxicity against human cancer cell lines, with special focus on pancreatic cancer cells. Crude extracts and chromatographic fractions were tested for their possible use (effectiveness) as selective cytotoxic and/or pro-apoptotic agents. Probably novel compound with anti-proliferative activity was found and isolated by HPLC-MS techniques.
|
|
A study of the possibility of waste pastries using for the bioproduction of selected metabolites
Hudečková, Helena ; Vránová, Dana (referee) ; Babák, Libor (advisor)
The aim of this diploma thesis was to study the possibility of using waste bread to bioproduction of selected metabolites. As bakery waste was used waste bread that came from coffee-house “Zastávka”. Bread was pre-treated by grinding into small particles and then it was made to form 15% w/v suspension, which was subjected to enzymatic hydrolysis. For the hydrolysis has been used the -amylase for liquefaction of the substrate and that was followed by a glucoamylase which sacharificated the substrate. There have been several methods of hydrolysis from which was chosen the optimal method for pre-treatment of the substrate prior to fermentation. The effectivity and a process of hydrolysis were determined spectrophotometrically by Somogyi-Nelson method. Final yields of glucose from hydrolysis were determined by HPLC method. Enzymatic hydrolysis was followed by fermentation, which was carried out in two ways, namely by adjusting the pH of the hydrolyzate to pH 5, and without pH adjustment. During the fermentation was carried out sampling in which was determined the content of glucose, glycerol and ethanol by HPLC method. The yeasts Saccharomyces cerevisiae were used for the fermentation which was performed at 30 °C. High yield of glucose was achieved by hydrolysis in two steps (70,28 gl-1), but for the fermentation was used mixed hydrolysis (second method of mixed hydrolysis) with yield 67,94 gl-1. High ethanol yield was achieved during fermentation without treatment pH, namely 31,5 gl-1.
|
| |
|
HPLC Method for Simultaneous Determination of Liposolubile Vitamines A, D and E
Nová, Alena ; Matysová, Ludmila (referee) ; Solich, Petr (advisor)
In this work the new HLPC method for simultaneous determination of vitamins D2, D3 and metabolite 25(OH)D3, vitamin A (retinol) and vitamin E (_-tocopherol) using the internal standard was developed. During the suggested assessment the monolith column Chromolith Performance RP-18e, 100 x 4,6 mm and Chromolith Performance RP-18e, 50 x 4,6 mm were used. The detection was carried out with the help of a diode array detector at wavelenght 264 nm for vitamins D2, D3 and metabolite 25(OH)D3, 295 nm for _-tocopherol and the internal standard tocol and 325 nm for retinol. The mixture of methanol : water : 2- propanol (75 : 15 : 10, v/v) was used as the mobile phase A in time 0,0-3,0 minutes. As the mobil phase B the mixture of methanol : water (95 : 5, v/v) was used in flow rate 3,5 ml/min in time 3,0-6,5 minutes. The injection volume of the sample was 20 μl. The total time of the analysis was 6,5 minutes including the equilibration of the column. This method was developed and partially validate. With the method will be continue on biological material.
|
|
HPLC Analysis of Benzimidazoles Using Core-shell Columns I
Oslejová, Kateřina ; Kubíček, Vladimír (advisor) ; Lázníčková, Alice (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of biophysics and physical chemistry Candidate: Kateřina Oslejová Supervisor: ing. Vladimír Kubíček, CSc. Title of diploma thesis: HPLC Analysis of Benzimidazoles Using Core-shell Columns I The aim of this work was to transfer the HPLC method of determination of flubendazole and its metabolite, i.e. reduced flubendazole, from a classical C18 column (250×3,0 mm; 5 µm) to core-shell column Ascentis® Express C18 (10×3,0; 2,7 µm) and to find a suitable internal standard. Experiments were performed using liquid chromatograph Agilent 1200 Series® with both DAD and fluorescence detection. Flubendazole was detected with the DAD detector. Low concentrations of reduced flubendazole were detected with the fluorimetric detector. Several mobile phase compositions were tested. The mobile phase consisting of phosphate buffer (pH = 3,0; 0,025 mol/l) and acetonitrile (70/30, v/v) appeared to be a good choice for the separation under study. Two internal standards were exploited for the quantification, depending on the detection principle. Mebendazole served as the internal standard in the case of DAD detection, while oxibendazole was utilized for fluorimetric detection. The proposed method was successfully applied for analyses of real samples.
|
|
HPLC analysis of drug candidates from the group of aroylhydrazones II.
Stariat, Ján ; Kovaříková, Petra (advisor) ; Kučera, Radim (referee)
1. ABSTRACT High performance liquid chromatography (HPLC) is one of the most frequently used analytical techniques for the analysis of drugs. Although iron is a vital element, excessive amounts in the body are highly toxic. The search for highly selective and effective iron chelating agents has been mainly inspired by the need to mobilize iron from tissues that are chronically overloaded with iron. However, recent investigations focused on the possibility to use iron chelators for the treatment of many other pathologies. Salicylaldehyde isonicotinoyl hydrazone (SIH), a biocompatible iron chelator derived from aroylhydrazone, is under extensive investigation as a promising drug candidate. Besides ability to bind iron, it shows interesting pharmacological effects: antioxidative, antiproliferative, cardioprotective, antimalarical and antimicrobic. The aim of this study was to develop optimal HPLC conditions for the separation of SIH and its potential metabolites (isoniazide, acetylisoniazide, salicylaldehyde) and to apply the method to the study focused on the isolation of analytes from rabbit urine using SPE. The best chromatographic analysis was achieved on a HPLC column (Phenomenex 250 4.6 mm I. D.) packed with Prodigy 5u ODS3 100A (5 μm) as a stationary phase. The mobile phase was composed of methanol :...
|
|
Bioanalytical methodical approaches for the disposition monitoring of a new potential drug in organism using HPLC-PDA-MS-MS
Císař, Přemysl ; Nobilis, Milan (advisor) ; Chládek, Jaroslav (referee) ; Wsól, Vladimír (referee)
1. Summary The aim of this study consisted in the development and validation of a bioanalytical method for the identification and determination of dimefluron and its metabolites in biomatrices. For the purposes of this study, six expected potential dimefluron metabolites were prepared and identified by NMR and MS. Higher homologue of dimefluron (homodimefluron) was selected and synthesized as an internal standard. The chromatographic columns with various types of the stationary phases were tested. The best results were achieved using a pentafluorophenylpropylsilyl silica gel column rinsed with the mobile phase of acetonitrile- phosphate buffer pH = 3 in a gradient mode. After the validation of the bioanalytical method, this chromatographic system was applied to the disposition study of dimefluron in rats. After an intragastric administration of dimefluron to rats, samples of urine and faeces were collected each 24 hours. The elimination of dimefluron and its phase I and phase II metabolites in urine and faeces was studied. Maximum concentrations of dimefluron derivatives in the excrements were found in the time interval of 24 - 48 hours after the administration. 9-O-Desmethyldimefluron and 3-O-desmethyldimefluron were the principal metabolites found in the rat faeces, while the metabolic products of...
|
|
Determination of methotrexate and 7-hydroxymethotrexate by HPLC and correlation with imunochemical determination of total methotrexate
Suchánska, Iveta ; Dršata, Jaroslav (advisor) ; Kukačka, Jiří (referee)
The aim of this work was to verify the correlation at determination of methotrexate by high performance liquid chromatography and imunochemically determination of whole methotrexate. Methotrexate belongs to the chemotherapeutic agent commonly used in the treatment of acute lymphoblastic leukemia. Methotrexate was determined chromatographicly with UV detection at 303 nm after deproteinization with trichloracetic acid. Fluorescence polarization immunoassays of methotrexate was measured on TDx FLx analyzer. The data obtained were analyzed utilizing the PrismGraph Pad 5.0 software. The methotrexate measurements were evaluted employing nonparametric paired t-test (p-value <0,05). Our data indicate good correlation between methotrexate levels > 1 μmol/l determined by high performance liquid chromatography and fluorescence polarization immunoassays. While the concentration of methotrexate < 1 μmol/l measured by fluorescence polarization immunoassays were overestimated. This could be done because of cross reactivity with metabolites 7-hydroxymethotrexate and 2,4-diamino-N10 -methylpteroic acid. These metabolites could influence the determination of methotrexate, because of the close stuctural similarities.
|
|
Analytical evaluation of drugs and novel drug candidates using HPLC
Kovaříková, Petra ; Klimeš, Jiří (advisor) ; Nobilis, Milan (referee) ; Blešová, Marie (referee)
High performance liquid chromatography (HPLC) is one of the most frequently used analytical techniques for the analysis of drugs. HPLC methods are widely employed in all fields of the modern pharmaceutical analysis - new drugs development, quality control and assurance, the analysis of drugs and metabolites in a biological material (e.g. therapeutic drug monitoring, bioequivalence). The theoretical part of this thesis deals with the main specifics and aspects associated with HPLC analysis of drugs. The experimental part is consisted of six original research papers with appropriate comments divided in to two sections (The analysis of the drug candidates from the group of aroylhydrazone iron chelators and The stability study on selected drugs). The first section concerns with development, validation and application of new HPLC methods in the analysis of drug candidates from the group of iron chelators - pyridoxal isonicotinoyl hydrazone (PIH), salicylaldehyde isonicotinoyl hydrazone (SIH) and pyridoxal 2-chlorobenzoyl hydrazone (o-108). Aroylhydrazone iron chelators are under the investigation as promising drug candidates. Despite these chelators have demonstrated number of interesting pharmacological effects, in fact there were no modern analytical techniques suitable for the analysis of these drug...
|
|
Srovnání citlivosti 17 ekotoxikologických bioestů pro detekci cyanotoxinů
Maršálek, Blahoslav ; Bláha, L.
Detection potential of some bioassays used for cyanotoxin detection seems to be promising, but the real comparison of results and the sensitivity to cyanotoxins is imposible, because different authors use different cyanobacterial sample with uncomparable toxin composition, different methods for sample preparation, different design of bioassay, media, etc. A critical comparison of alternative bioassays for cyanotoxins detection, which is important for water management is up to date still missing. The aim of the present study was to compare the sensitivity of 17 bioassays for cyanotoxins detection (with respect to the content of hepatotoxic microcystins )using the same cyanobacterial biomass, and the same sample preparation . Additional aims of this study was as follow: i) can the fractionation of cyanobacterial biomass improve the sensitivity and decrease the variability of bioassays? , ii) which bioassay can more precisely dicrimine the presence of microcystins - is there any dose-response relationships? Cyanobacterial biomass was fractionated by SPE-C18 columns. Crude aqueous extracts, "non-toxic" pigment fraction without microcystins and "toxic" fractions (concentrated microcystins) were compared. This fractionation allowed to determine if the toxicity is due to microcystins, or some other not monitored metabolites present in the complex crude extract.
|
guest ::
