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The Role of Uncoupling in Down-regulation of Reactive Oxygen Species
Ježek, Jan ; Žáčková, Markéta (advisor) ; Modrianský, Martin (referee) ; Drahota, Zdeněk (referee)
Uncoupling attenuates Complex I-derived superoxide production by accelerating electron flux and proton pumping within Complex I. However, under circumstances leading to hampered proton pumping pathway within Complex I, e.g. due to aberrant mutations of mtDNA encoding either ND2, ND4 or ND5 Complex I H+-pumping subunit, therapeutic strategy based simply on uncoupling would fail. Experimentally, hydrophobic amiloride EIPA mimicks the model of disabled H+-pumping. We show for the first time that EIPA is a real inhibitor of H+-pumping of mitochondrial Complex I. We searched for an agent that, unlike uncoupling, would be able to counteract oxidative stress associated with obstructed proton pumping of Complex I. Mitochondria-targeted ubiquinone MitoQ10 proved to be an effective antioxidant for this purpose when the rate of superoxide formation was high due to the electron flow retardation within Complex I. Because of its pro-oxidant properties, targeted delivery of MitoQ10 as a cure to the pathological tissue is necessary. Activation of mitochondrial phospholipase iPLA2 by mild oxidative stress can provide free fatty acid hydroperoxides as the cycling substrates for UCP2 that initiates mild uncoupling leading to the attenuation of reactive oxygen species (ROS) production, as a part of feedback regulatory loop of...
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Investigation of Biologically Active Plant Secondary Metabolites by Antioxidant and Anti-Aggregative Tests
Řeháková, Zuzana ; Jahodář, Luděk (advisor) ; Semecký, Vladimír (referee) ; Grančai, Daniel (referee)
The effort to find compounds with significant antioxidant properties and with some other therapeutically useful biological activity (e.g. anti-platelet activity) from the wide group of sturcturally different naturally occuring compouds (or plant extracts) or their synthetic derivatives, was the purpose of this work. Radical Scavenging activity of tested compounds or plant extracts was measured by modified DPPH test use program SIA (sequential injection analysis). Singificant radical scavenging activity exhibited polyfenols, namely gallic acid (EC50= 0,0025 ± 0,002 mg/ml) and ethyl-gallate (EC50= 0,0038 ± 0,001 mg/ml). Also the pure latex of Croton lechleri exhibited significantly radical scavenging activity (EC50=0,0347 ± 0,018 mg/ml). From the group of tested methylcoumarins, the excelent antioxidant aktivity exhibited ortho-dihydroxy-4-methylcoumarins, espicially 7,8-dihydroxy-4- methylcoumarin and its derivatives (EC50= 24,9 ± 2,7 μM). Further was the antioxidant activity tested by FRAP methods modified to used micropalates P 400 μl. This spectrofothometric method based on ability of compounds to reduce Fe3+ to Fe2+, confirmed the antioxidant activity of ortho-dihydroxy-4-methylcoumarins. The antiplatelet activity of pure compounds and herbal extracts was assayed in vitro on the model of human...
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Mitochondrial subpopulations in rat myocardium - effect of chronic hypoxia
Kovalčíková, Jana ; Nováková, Olga (advisor) ; Žurmanová, Jitka (referee)
Adaptation to chronic hypoxia induces endogenous cardioprotection and increases the heart resistance to ischemia/reperfusion injury. The heart mitochondria, which produce reactive oxygen species (ROS) in addition to ATP, play an important role in these processes. During ischemia/reperfusion, ROS are produced in excessive amounts and damage the cells. However, in lower concentrations, ROS are involved in the signalling pathway of cardioprotection induced by adaptation to chronic hypoxia. In the heart, two mitochondrial subpopulations have been observed, subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IMFM), which differ in cell localization as well as in morphological and biochemical properties. The aim of this work was to introduce the method of SSM and IMFM isolation in our laboratory and to analyse their antioxidative capacity after adaptation to chronic hypoxia. Adult male Wistar rats were kept either under normoxic conditions or exposed to intermittent high-altitude hypoxia (IHA; 7000 m, 5 days a week/8 hours a day, totally 25 exposures). Mitochondrial subpopulations were isolated from heart left ventricle and their functionality was verified by measuring oxygen consumption and enzyme activities. The IMFM had higher oxygen consumption in comparison with SSM and activities...
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Positive and negative substances in guarana
Hrdinová, Lucie ; Vitoulová, Eva (referee) ; Hrstka, Miroslav (advisor)
This bachelor work theoretically describes Southern American plant named guarana (Paullinia cupana), its history, cultivation and traditional preparation. The work deals with effects of significant alkaloids (caffeine, theophylline, theobromine), tannins and saponins included in guarana, as well as general activity of guarana seeds. Contemporary studies are in agreement with strong antibacterial and antioxidant effects of guarana seed extracts, genotoxic and mutagenic activity of guarana in prokaryotic organisms and toxic affects on dogs, which are extremely sensitive on methylxanthines. In studies on human volunteers there was not shown any adverse effects, but there were recorded some cases of new-onset seizures in adults following heavy consumption of energy drinks, rich in caffeine, taurine, and guarana seed extract. The studies generally proved helpful activity against obesity and positive influence on lipids metabolism. There were confirmed positive psychoactive and mild antidepressant effects of guarana as well as adstringent, lipolytic and gastroprotective properties.
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Study of extraction of the biologically active substances from elderberry marc
Lošková, Tereza ; Diviš, Pavel (referee) ; Vespalcová, Milena (advisor)
This bachelor thesis deals with the determining of anthocyanin pigments in the extracts of elderberry marc (Sambucus nigra L.). The theoretical part is divided into four main chapters. In the first chapter there is botanical characteristics of elderberry, its origin and occurrence, chemical composition and the possibilities of usage in a food industry. The second part is devoted to characterization of phenolic compounds in berry fruits. Bigger attention is given to anthocyanin dyes, its occurrence, properties, health and antioxidant effects and the possibilities of usage in food industry. The third chapter is devoted to methods of determination of discussed dyes and phenolic compounds. The fourth chapter deals with the possibilities of disruption of plant cell walls. In the experimental part a series of elderberry marc's extraction was made. The purpose was to optimize the extraction course and to get the highest yield of dyes in an economically and operationally acceptable way. Time dependence of extractions was studied and a kind of extraction solvent and a ratio of its components were optimized. Distilled water or its mixture with ethanol was used as a solvent. During the water extraction the temperatures 25 °C and 50 °C were maintained and with the higher temperature the higher yields were obtained. Apart from this another supporting components as cellulolytic enzyme, ultrasound or shaker were used. The most effective method of extraction proved to be the extraction by the mixture water-ethanol (50:50) in the volume 80 mL. The total anthocyanins yield of this extraction was, expressed as cyanidin-3-glucoside equivalent, 914,4 ± 3,8 mg100 g-1 dry weight after 10 hours and the total phenolics yield was, expressed as gallic acid equivalent, 1957,0 ± 9,9 mg100 g-1 dry weight at the same time. The usage of ultrasound increased the efficiency of the extraction by water at 50 °C by 12 %. On the contrary, the usage of cellulolytic enzyme didn't increase the anthocyanin yield, because the enzyme was inactive.
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Development of an analytical method for evaluation of prodrug- boronyl salicylaldehyde isonicotinoyl hydrazone in a biological material
Bureš, Jan ; Kovaříková, Petra (advisor) ; Sochor, Jaroslav (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Candidate: Jan Bureš Supervisor: PharmDr. Petra Kovaříková, Ph.D. Title of diploma thesis: Development of an analytical method for evaluation of pro-drug - boronyl salicylaldehyde isonicotinoyl hydrazone in biological material High performance liquid chromatography is nowadays one of the most frequently used and progressive analytical method in pharmaceutical analysis. It can be used for both qualitative and quantitative analysis of complicated samples also in a biological matrix. Iron is a vital element for all organisms. It's essential for a proper function of variety of crucial enzymes. On the other hand it's excess in the body is harmful and it is connected with progress of various pathological conditions. Hence iron chelation therapy is an effective tool to treat and protect living cells against injurious effect of iron. Iron chelators were originally used to treat iron overload disease. However, thanks to their interesting antioxidative and antiproliferative properties, their therapeutic potential has been significantly widened. These compounds are currently investigated as promising drugs for the treatment of Parkinson's and Alzheimer's diseases, cancer, malaria,...
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Design of New Antibacterial Active Molecules
Vavříková, Eva ; Vinšová, Jarmila (advisor) ; Opletalová, Veronika (referee) ; Čižmáriková, Růžena (referee)
The main task of the thesis was a design and synthesis of new potential antimycobacterial active molecules. Presently, the appearance of MDR-TB strains is alarming and the development of new therapeutical agents is necessary. The work is divided into two parts; first one is related to aminopolysaccharide chitosan and its connection with appropriate antimycobacterial drugs or dyes, second part is concerned with modifications of current antituberculotics. Due to the structure and physico-chemical properties, chitosan has been found as an interesting drug carrier in biomedicinal chemistry. It is used in drug delivery system with control release of the drug in the target cells or tissues. The component of the first part of the thesis was to review molecular modelling of chitosan, especially its usage as a prodrug or carrier in a field of antibacterial, antitumor and antioxidant activity. Derivatives of chitosan linked with the first or second line antituberculotics were prepared for the purpose of decreasing hepatotoxicity of used drugs. Their antimycobacterial activity against M. tbc., M. avium and M. kansasii was 125 µg/mL for all strains. Unexpectedly, O-carboxymethyl chitosan as an intermediate showed better MIC against M. tbc. and M. avium. It means that biological activity of chitosan derivatives...
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Application of analytical techniques and chemometry for authentification of wine
Pořízka, Jaromír ; Karovičová, Jolana (referee) ; Jarošová, Alžběta (referee) ; Diviš, Pavel (advisor)
This PhD. thesis is focused on application of different analytical techniques for determination of elemental composition of Moravian wine combined with analysis of selected polyphenol compounds. Two spectrometric techniques were used for elemental analysis of wine – Mass spectrometry and Optical emission spectrometry with inductively coupled plasma (ICP-MS and ICP-OES). Analysis of organic constituents of wine was evaluated by High performance liquid chromatograph with diode array detector (HPLC-DAD). Data from analysis of wine were used for construction of mathematical models for determining geographical origin of wine and for determining type of varietal wine. Data handling was performed by usage of multivariate statistical methods (cluster analysis, principal component analysis and canonical discriminant analysis). For the discrimination of wine by the geographical origin, two separate models for white and red wine were constructed. Effectiveness of discrimination of Moravian wine (with knowledge about origin) into 4 wine regions was 100 % successful for white wine and 96,43 % for red wine samples. Those models were used for cross validation test of unknown samples with result 95 % successfully classified samples of white wine and 82,15% of red wine. Besides that, mathematical model for discrimination and classification of varietal wine was made. Müller Thurgau, Riesling and Gruner Veltliner were discriminated by the usage of 8 elemental parameters. Discrimination success rate of known samples was evaluated by canonical discriminant analysis with 95,83% of correctly classified known samples and 70,83% of correctly classified unknown varietal wine (cross validation test). In addition to these part of research, impact of different grape production systems on elemental composition of wine and Vitis Vinifera was studied. The core of the study was to find the connections between the type of agriculture and chemical properties of wine and Vitis Vinifera. Two agriculture systems were assessed – Organic agriculture and Integrated pest management. . Results of this study showed that quantity and manner of pesticide application is very important factor affecting the final elemental composition of the grapevine and wine, especially the content of copper, zinc and manganese. That knowledge can be used in the future for creation of classification model of wine according to grape production system. For comparison of radical scavenging activity, electron paramagnetic resonance spectroscopy (EPR) with DPPH method was used. In the case of this study, the influence of vine growing under different farming systems on the total antioxidant activity of wine was found to be insignificant.
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The electrochemical study of the degradation of the natural dyes from historical objects
Ramešová, Šárka ; Schwarzová, Karolina (advisor) ; Navrátil, Tomáš (referee)
The oxidation of quercetin (3,3',4',5,7-pentahydroxyflavone) and luteolin (3',4',5,7-tetrahydroxyflavone) has been studied. These intensively coloured polyphenolic compounds belong to a large group of naturally occuring flavonoid compounds. They were used with advantage as colourants in old tapestries. The study concerns the analysis of degradation processes of the original dyes. Degradation products formed during the ageing process can be used as fingerprints for the determination of the original colours. The degradation processes of flavonoid compounds were studied by electrochemical methods, absorption spectrometry and separation methods in aqueous media. The oxidation mechanism involves electron transfer coupled with chemical reaction and is influenced by the acidobasic properties of quercetin. The dissociation constants pK1 and pK2 of quercetin and pK1 and pK2 of luteolin were determined by combining of independent methods: acid-base titration and UV-Vis spectroscopy. The stability of both compounds significantly depends on the presence of the oxygen. The distribution of the oxidation products of quercetin in aqueous media within the exposure to oxygen was monitored by LC-MS/MS and LC- DAD analysis.
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Purification of patatin proteins from tubers of selected potato (\kur{Solanum tuberosum} L.) cultivars with subsequent characterization of isolated patatin proteins
ŠVEHLOVÁ, Eva
The complex of patatin proteins were extracted from potato tubers (Solanum tuberosum L.) and its purification process is described in this work. The initial material for patatin protein isolation were potato fruit juices extracted from cultivars Laura, Sibu and Westamyl. Based on the known patitin´s properties, the ion-exchange chromatography (DEAE medium) coupled with affinity chromatography was applied. We used the column containing biological ligant of Concanavalin A, which was able to bind the carbohydrates in the molecule of patatin proteins. The used purification process were successful and according to the SDS-PAGE analysis we obtained patatin proteins of a high purity from each the potato cultivars. The antioxidant activity was observed for all samples and range from 0,007 to 0,086 mg ascorbic acid concentration / g protein.
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