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Study of Activity of Enzyma and Low Molecular Weight Antioxidant Systems
Macuchová, Simona ; Kráčmar, Stanislav (referee) ; Čertík, Milan (referee) ; Márová, Ivana (advisor)
Oxidative processes play important role in cell physiology and pathology as well. Balance of these processes is supplied by cooperating antioxidative systems; function of antioxidant defense systems depens on high levels of antioxidants in organism. Presented work is focused on developement and optimization of methods for analysis of important enzyme and non-enzyme antioxidants as well as total antioxidant capacity of selected types of biological material. Extractions and analyses of vitamin E, carotenoids, superoxide dismutase, catalase, peroxidase and lipoxygenase in barley and malt were optimized. RP-HPLC and HPLC/ESI-MS were used for analysis of vitamin E, phenolic and carotenoid content, spectrophotometry was used for enzymes activity analysis. A new methods for catalase and lipoxygenase activities were developed and compared with direct UV methods. Superoxide dismutase activity was determined by commercial diagnostic kit. A colorimetric method was used for peroxidase activity determination. Some kinetic parameters of enzymes were provided too. Optimized methods were used in the analyses of antioxidants in plant material - in barley and malt - in sets of samples of 6 varieties cultivated in four different locations for two years. Content of individual antioxidants differed depending on the variety, but usually were not found significant differences in the levels, depending on growing location. Perhaps climatic conditions have the greatest influence on levels of low molecular weight and enzymatic antioxidants at the specific location; oxidation processes are influenced both the quantity of moisture, both by sunlight, which induces oxidative processes in cultivated plants. The activity of antioxidants in barley caryopses is rapidly increasing during the malting process; an elevated temperature and moistness first induces activation the enzyme systems including antioxidant. In caryopsis is metabolic activity increased during which we can expect an increased production of radicals; for this purpose can antioxidant systems be activated that protect cells from damage by oxidative stress. In the second part of work optimized methods were applied in two clinical trials focused on study of the influence of exogenous antioxidants intake on metabolic and antioxidant status in human organism. In the first clinical study influence of food supplement containing polyunsaturated fatty acids and vitamin E on metabolism of hyperlipidaemics was evaluated. After 3-month supplemenation a lipid profile was improved and serum antioxidant levels increased. The second experiment was focused on enzyme and non-enzyme antioxidant levels in healthy subjects after temporarily intake of specific foods rich in antioxidants. After two-month intake plasma phenolic substances were slightly increased. Total antioxidant capacity and activities of enzyme antioxidants were not affected. Results of both clinical exeriments showed that supplying of antioxidants in natural form or in the form of food supplements does not markedly affect metabolism of healthy subjects, while in patients with chronic diseases antioxidant supplementation can positively influence metabolic status. Results of this work showed that optimized methods are suitable for analyses of antioxidant status parameters and also for monitoring of exogenous antioxidant intake.
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Changes of enzyme activities in fruits during long-term storage
Ferdová, Jitka ; Melounová,, Jitka (referee) ; Márová, Ivana (advisor)
This study is focused on study of changes of enzyme and low-molecular weight antioxidants in different fruits during long-term storage. In theoretical part individual low-molecular weight antioxidants and enzymes are described. The main causes of fruit decay and some possibilities of fruit preservation and storage are summarized. As biological material some common fruits were chosen - green and red apples, peaches, plums and white grapes. The fruits were stored in laboratory, cellar, in refrigerator and in freezer. In freezing experiments some ways of fruit preparation and processing were tested and their influence on fruit antioxidant status was compared. Shortened storage experiment was applied on blueberries, cranberries, raspberries and strawberries too. In fruits some group parameters – total antioxidant status, dry mass content, ascorbate level, total flavonoids and total phenolics were analyzed spectrophotometrically. Individual flavonoids and phenolics were determined by RP-HPLC/UV-VIS and on-line LC/PDA/ESI-MS. Antioxidant enzyme activities (superoxide dismutase SOD, catalase CAT, polyphenol oxidase PPO and lipoxygenase LOX) were measured by spectrophotometry. The surface microscopy and cultivation of moulds from fruit surface were performed too. Influence of storage conditions on biological activities is dependent on fruit sort. Freezing is the most suitable procedure for long-term storage without significant changes of active substance content. Long-term storage in controlled temperature conditions and/or atmosphere is usable for fruits with longer storage period. In these fruits stabile levels of antioxidant enzymes are stored for relatively long time. Some of enzymes act synergistically. Enzyme activities differed according to storage phase; at the beginning mainly high SOD and LOX activities were observed. CAT and PPO are probably activated as defence systems in rippened and/or damaged fruits. Levels of total as well as individual low molecular weight antioxidants varied during storage in all sorts, generally, increased course with longer storage period can be observed.
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Influence of storage conditions on content of biologically active substances in apple fruits.
Ferdová, Jitka ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
This study deals with antioxidants in diet and their effects on human organism. Further, it summarizes the agents affect the quality of apples in the course of long-term storage and it outlines the possibility of defence against them. In the experimental part methods of determination of antioxidant enzymes superoxid dismutase (SOD), catalase and polyphenol oxidace (PPO) in apples were introduced. The enzymes were measured in apples tissues in liquid nitrogen after 158 days in normal or modified atmosphere. In Apple the quantitative and qualitative analysis of proteins was realized. Further, some low molecular antioxidants (total phenolics, total flavonoids and vitamin C) as well as total antioxidant status were measured in frozen raw juice. This values were compared with values from apples analyzed immediately after the harvesting. Artificial inoculation with fungi was made in the last part and the fruitfulness was observed.
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The effect of metribuzine on oxidative stress and antioxidant enzymes of signal crayfish
LIDOVÁ, Jaroslava
The aim of this study was to investigate effects of the triazine herbicide metribuzine on oxidative stress level and antioxidant enzymes activity in gills, muscle and hepatopancreas of signal crayfish (Pacifastacus leniusculus Dana) and also extension of knowledge about effect of metribuzine on the environment. The experiment took up 60 days. Crayfish were exposured to metribuzine concentrations of 0.52 micrograms.l-1 (real environmental concentration) and 3.06 mg.l-1 (10% 96hLC50) for the first 30 days. Then a second phase followed depuration without metribuzine (30 days). Changes in the oxidative stress level (TBARS), superoxiddismutase (SOD) activity and catalase (CAT) activity were observed in all examined tissues. Changes in glutathionreductase (GR) activity were observed only in hepatopancreas. Chronic exposure of metribuzine demonstrated an oxidative damage of cell lipids, proteins and also changes in antioxidant activity in examined crayfish tissues.The results of this study suggest that crayfish are a very suitable organisms for toxicological tests and simultaneously extend knowledge about effect of metribuzine on the environment.
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Effect of oxidative stress on antioxidant enzymes in \kur{Drosophila melanogaster}.
SZAKOSOVÁ, Klára
Oxidative stress reflects an imbalance between reactive oxygen species and antioxidant defense, which can result in cellular damage. The reactive radicals can interact with cell components, including proteins, lipids and nucleic acid. Antioxidant enzymes are the main defense against oxidative stress, representing by two the most important enzymes, catalase and superoxide dismutase. In this thesis I studied the effect of oxidative stress on activity of antioxidant enzymes in Drosophila melanogaster and Locusta migratoria, after exposure to the herbicide paraquat.
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