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This thesis entitled "Effect of gloeosporium rot as gaseous constituents of apple varieties" deals with microorganisms in terms of storage and processing. Characterized mainly epiphyte microflora occurring on apples, mainly sponge Gloeosporium spp. Specifies how does this affect rot on the gaseous components in the fruit, how it affects breathing and the formation of ethylene and what risks may cause the processing. Features mold and products of their metabolism, concentrations and health risks. The conclusion of this paper describes the experimental determination of the invading microorganisms fruit during storage.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of biophysics and physical chemistry Candidate: Šárka Slezáková Supervisor: Ing. Vladimír Kubíček, CSc. Title of diploma thesis: HPLC Determination of Benzimidazoles In this thesis we have investigated the possibility of establishing benzimidazole with HILIC chromatography using a chromatographic column Ascentis Express HILIC 10.0 cm x 3.0 mm; 2.7 microns. Two groups of benzimidazoles were tested. The first one was focused on albendazole and its metabolites. Experiments with these substances did not produce satisfactory results, because the mobile phase composition which enables separation of the studied analytes was not found. The second group was formed by flubendazole and its reduced and hydrolyzed form. In this case, several mobile phase compositions were tested. Finally, distribution of individual analytes in a mixture, using a mobile phase ACN:HCOOH 0.03 mol/l (90:10), was successfully achieved. Ricobendazol was chosen as an internal standard. When separation conditions were found, calibration curve for the determination of reduced flubendazole in biological samples was subsequently constructed using ricobendazol as the internal standard with the use of fluorescence detection. Keywords: HILIC, HPLC, albendazole,...

Silybin is the main component of silymarin, a standardized extract obtained from the seeds of milk thistle (Silybum marianum). Flavonolignan silybin has antioxidant, hepatoprotective, chemoprotective and antitumor activities. Natural silybin occurs as an approximately equimolar mixture of two diastereoisomers - silybin A and silybin B. Analytical separation of these diastereoisomers is possible but preparative separation is complicated. The biological activity of the silybin A is different from the silybin B. Silybin diastereoisomers are mainly conjugated to glucuronides and sulfates in organism. A mixture of both silybin diasteroisomers is used in the majority of reported biological, chemical and pharmacokinetic studies. For the first time, optically pure silybin A and silybin B were used for pharmacokinetic study in this thesis. The object of this work was determination of the concentration of free and total silybin in rats plasma in relation to time. Theoretical introduction describes the current state of the problem of chemistry, pharmacology and pharmacokinetics of silybin diastereoisomers. Second part is focused on the selection of appropriate analytical column, chromatographic method and suitable procedure for preparation of biological samples for the determination of the silybin...

Extract from milk thistle (Silybum marianum (L.) Gaertn., synonym Carduus marianus L., Asteraceae) silymarin contains among others primarily bioactive flavonolignans. They have hepatoprotective and antioxidative effects and also anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activity. This thesis focuses on the preparation of metabolites of the second phase of biotransformation unexplored flavonolignans 2,3-dehydrosilybin (DHSB), silychristin (SCH), 2,3-dehydrosilychristin (DHSCH). Pure sulfated derivatives were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and p-nitrophenyl sulfate (p- NPS) as a donor. Flavonolignans yield exclusively monosulfates at the position C- 20 (C-19 in the case of silychristin and 2,3-dehydrosilychristin), except for 2,3- dehydrosilybin that gives also the 7,20-disulfated derivatives. For all samples were made antioxidant tests - DPPH assay (the highest activity had 2,3-dehydrosilychristin sulfate: IC50= 7,87 µM), Folin-Ciocalteau reduction assay (the highest activity had 2,3-dehydrosilychristin: 1,58 ekvivalents of gallic acid), ABTS+ scavenging (the highest activity had silychristin: 1,50 ekvivalents of vitamin C), inhibition of microsomal lipid peroxidation (the highest activity had 2,3-dehydrosilybin: IC50 = 10,6 µM),...

This thesis studies the effects of various concentrations of sodium chloride on growth and the content of secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii. The total content of phenolic compounds, flavonoids and total antioxidant capacity was analyzed by spectrophotometry. In the context of growth, also the content of photosynthetic pigments was determined. Further, the amount of selected metabolites was determined by HPLC/MS-MS. Cultivation of microalgae with increased NaCl inhibited cell growth and production of photosynthetic pigments. Conversely, higher levels of NaCl have proven to stimulate the synthesis of complete phenolic compounds and flavonoids. Similarly, the amount of phenolic acids was significantly influenced by the effect of increasing NaCl concentration, while the total antioxidant capacity of the microalgae also increased. These results confirm the involvement of phenolic compounds in the defense mechanism of unicellular algae Ch. reinhardtii against the observed stress factor.

Microalgae are increasingly involved in the diet due to their beneficial effects on human health and therefore they are more often studied. Nowadays, there is a trend of using nanotechnology. The undesirable effects of copper oxide nanoparticles (CuO) attract more and more attention today. Since, there is an increasing demand for fossil fuels and simultaneously the amount of CO2 is increasing in the air, microalgae could serve in future as renewable fuels thanks to its rapid grow properties. In the diploma thesis, I dealt with the amount of carotenoid and polyphenolic substances in the algae Chlorella vulgaris after the influence of CuO nanoparticles. Experimental part of this work summarizes the results, revealing that CuO nanoparticles caused decrease of chlorophyle a by up to 60 % with CuO nanoparticles concetration of 25 mg/l, together with significant decrease of chlorophyle b and carotenoids. The amount of polyphenols and flavonoids in biomass has been changed only minimaly. In contrast, with usage of increasing concentration of CuO nanoparticles I found out by the SPOT test the increase of biomass in algae. Significant changes in selected secondary metabolites has been observed. The greatest statistically significant (p<0,01) growth with concetration 5 mg/l (1,08 ± 0,05 µg/g dry weight) has been observed for pOHbenzaldehyde, with increase by 42 % compared to control. In contrast rapid decrease has been noted with concentration 25 mg/l (0,06 ± 0,04 µg/g dry weight)

This thesis studies effect of cultivation conditions on the production of secondary me-tabolites in the green microalgae Scenedesmus quadricauda. During the experiment was studied influence of pH, UVC radiation, light intensity and various temperature conditions on the amount of biologically active compounds (polyphenols, flavonoids, antioxidants). Metabolites were determined using biochemical analytical methods. The total content of polyphenols was determined using the Folin-Ciocalteu method, which is based on chemical reaction of phenols with Folin-Ciocalteu reagent. The total con-tent of flavonoids was determined spectrophotometrically using AlCl3.6H2O with the addition of NaNO2 and 1 M NaOH. The total antioxidant capacity (TAC) was deter-mined using phosphomolybdenun antioxidant assay. Antioxidant activity was stimu-lated by UVC radiation. The highest value being recorded when samples were exposed to UVC for 5 minutes. Light intensity and the temperature changes did not have significant effect on antioxidant activity of studied samples. There was an increase in TAC in samples cultured in acid pH medium (4 and 5). The content of polyphenolic and flavonoid compounds with high light intensity (3500, 4000 lx) and high tempera-tures (35 °C, 40 °C) decreased. Conversely, changes in pH and UV-C stimulate biosyn-thesis of both polyphenols and flavonoids. The greatest increase of these compounds was observed in the medium with pH = 4 and the time of exposure to UV-C for 5 minutes a day.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical botanic and ekology Candidate: Nikol Vavřichová Supervisor: Ing. Kateřina Macáková Ph.D. Title of diploma thesis: Iron - chelating effects of flavonoid metabolites - small polyphenolic substances (2015/2016) Iron is a trace element which is necessary for function of an organism. There is no specific excretion mechanism for iron, therefore any deflection causes deficiency or abundance. Iron abundance leads to hemochromatosis. An application of iron chelators is a treatment of choice for hemochromatosis. There are three clinically used chelators for treatment of iron overload: deferoxamine, deferasirox and deferiprone. These drugs are inappropriate for therapy (side effects, dosage form). Therefore there is a place for serching for new types of medicine for treatment of hemochromatosis. Flavonoids are natural substances with positive influence on a human organism. The most investigated effects are antioxidant and chelating activity. As flavonoids are metabolised in a human body, there is an arising question if flavonoid metabolites are also able to chelate iron ions. For this work we chose derivates of benzoic acid: benzoic acid, 3 - hydroxybenzoic acid, 4 - hydroxybenzoic acid, 2,4 - dihydroxybenzoic...

Increasing of human life comfort and safety requires development and application of new chemical substances, which, unfortunately, cannot be completely removed by WWTPs. Thus, these compounds, their metabolites and TPs formed during human/animals/bacteria metabolism, wastewater treatment and natural environmental processes are tend to contaminate all the environmental compartments. Appearance of advanced analytical instrumentation and techniques enable to perform research on emerging environment contaminants fate. This thesis was focused on application of high resolution mass spectrometer and new generation of triple quadrupole for analysis of the environmental pollutants. All studies were mainly devoted to such a group of contaminants as pharmaceuticals because of their high consumption, frequent occurrence in all environmental matrices and potential to cause adverse effects for non-targeted organism due to their biological activity. The first part of the thesis is related to method development for identification and quantification of metabolites in the different tissues of rainbow trout exposed to diltiazem. As a result, 17 diltiazem metabolites were tentatively identified by HRMS and spectral interpretation software. The complex metabolic pathway in metabolic phase I for diltiazem and tissue specific bioconcentration in fish were proposed in the study. The second part of the work is mainly focused on investigation of pharmaceuticals fate in the soil environment. The transformation of several pharmaceuticals, such as atenolol, metoprolol, carbamazepine, sulfamethoxazole, trimethoprim, and clindamycin in 13 different soils was studied under laboratory conditions. The parent compounds were analyzed in all studied soils and dissipation curves were plotted. Finally, 15 known and new metabolites were detected and identified. Moreover, identity of 6 of them was confirmed by available analytical standards. It was shown that almost the same metabolites profile was observed in all soils, but formed amount and degradation rate of all detected metabolites were soil type dependent. Several parent compounds and metabolites were shown to be persistent in the soils. Consequently, pharmaceuticals can be transported via ground water to plant. Further fate of pharmaceuticals must be studied as it can have adverse effect on crop consumers. The third part of the dissertation dealt with an important issue of pharmaceuticals transport between different aquatic environmental compartments. Water, sediments and fish from a pond affected with TWW from a local WWTP were analyzed within this project. 18 pharmaceuticals and 7 their metabolites were found in all studied matrices. Solid water distribution coefficients for sediments and bioaccumulation factors for fish liver were obtained in this study under real conditions. Seasonal variations in all studied parameters were investigated for one year period. This study help to extend a limited available information on fate of wide range of compound, especially metabolites in the aquatic environment affected by treated wastewater.