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Analysis of sulfated flavonoids by HPLC-MS/MS
Petránková, Barbora ; Bojarová, Pavla (advisor) ; Kozlík, Petr (referee)
The aim of this work is to find the optimal separation conditions in high performance liquid chromatography for the analysis of sulfated polyphenolic substances, especially flavonoids, phenolic acids and catechols. Three chromatographic columns were used in the development of the method. ZIC-HILIC column with polar organic phase and C18 and Kinetex PFP columns with reverse phase. We focused in particular on a short time of separation, sharp tailless peaks and separation of all components of the mixture. Using a Kinetex PFP column, the optimal method for separating the test mixtures was found. The use of a mobile phase of 10 mM ammonium acetate/0,1% fromic acid and methanol was shown to be the most appropriate, with a mobile phase flow rate of 0,6 ml/min. By modifying the gradient and temperature, all components of the tested mixtures were separated, the peaks were sharp, and the separation time in most cases ranged do not exceed 10 min. The method is also directly applicable in conjunction with a mass detector. Keywords: High Performance Liquid Chromatography (HPLC), flavonoids, polyphenolic substances, sulfation, separation
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Effects of selected natural substances on the antioxidant system of an organism
Hodková, Anna ; Eybl, Vladislav (advisor) ; Mayer, Otto (referee) ; Anzenbacherová, Eva (referee)
of study named: Effects of selected natural substances on the antioxidant system of an organism Developed: Mgr. Anna Hodková Department of Pharmacology and Toxikology, Faculty of Medicine in Pilsen, Charles University in Prague Pilsen 2016 The aim of this study was to compare the effects of selected natural substances on the antioxidant defense system under comparable conditions, focusing on influencing the activity of selenoenzymes thioredoxin reductase (TrxR-1) and glutathione peroxidase (GPx-1). Experiments were performed in rats (Wistar, male). Livers, and in some cases kidneys were collected in all experiments. Homogenates were created from the collected organs and subsequently the activity of TrxR-1 and GPx-1, glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD), and reduced glutathione (GSH) and lipid peroxidation (LP) levels were determined. We demonstrated significant effects of selected natural substances on the redox system, including influences of selenoenzymes thioredoxin reductase and glutathione peroxidase. The biggest influence on the activity of selenoenzymes thioredoxin reductase and glutathione peroxidase had hydroxytyrosol (HT) and oleuropein (OLEU). In rat liver tissue there was a significant decrease of the activity of both above mentioned enzymes after...
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Use of capillary electrophoresis to determine a selected group of flavonoids
Tran Xuan, Tiep ; Mravcová, Ludmila (referee) ; Řezáčová, Veronika (advisor)
In recent years, the use of natural medicinal properties of plants has again come to the forefront of interest of the public. Because of their therapeutic properties, these plants are commonly used in alternative treatments for minor ailments. This thesis deals with the determination of polyphenolic acids in extracts from aronia, echinacea, tetradenia and centella. One of the aims of the thesis was to compare the amount of polyphenolic acids in individual plants with their antioxidant properties. The theoretical part includes a general introduction to flavonoids, introduction to polyphenolic acids, introduction to the principles of capillary zone electrophoresis and a summary of selected foreign studies for the determination of flavonoids. An important chapter of the theoretical part is a description of individual plants with an explanation of their possible therapeutic use. The experimental part is devoted to the process of optimization of electrophoretic determination of selected polyphenolic acids and evaluation of their content. It was found that in the extracts six representatives of polyphenolic acids occur in minimal amounts, often below the limit of quantification or detection of the method.
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Characterization of blackthorn products
Červinková, Zuzana ; Veselá, Mária (referee) ; Kovalčík, Adriána (advisor)
This bachelor thesis deals with the preparation and the chemical characterization of ethanolic extracts and products from the blackthorn (Prunus spinosa L.) fruit. The theoretical part of the work contains the characterization of the blackthorn and describes the chemical composition of the fruit. Furthermore, the theoretical part states possible effects and possible uses of the Prunus spinosa L. fruit in the food industry, folk medicine and pharmacy. The experimental part describes the preparation of the extracts (40% ethanol) and the products (i.e., homemade sloe wine and homemade blackthorn liqueur). The content of reducing sugars, polyphenols and flavonoids was quantified in samples of wine, liqueur, juice and ethanol extracts. Furthermore, the antioxidant activity of individual samples was determined. Two spectrophotometric methods for the determination of reducing sugars were used and compared, namely the Somogyi-Nelson (SN) assay and the 3,5-dinitrosalicylic acid (DNS) assay. The DNS assay provided slightly overestimated results, while the results from the SN assay were closer to the actual content of reducing sugars. Polyphenols and flavonoids are substances that have antioxidant properties. A strong correlation was observed between antioxidant activity and the concentration of flavonoids.
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Effect of dihydromyricetin on ethanol metabolism
Skotnicová, Aneta
Dihydromyricetin (DHM), also ampelopsin, is a flavonoid compound which exhibits a broad spectrum of positive effects on the human body. Herbal extracts containing this compound have been widely used in traditional Chinese medicine mainly for their hepatoprotective properties. DHM also helps with alcohol intoxication and reduces the signs of hangover or abstinence. Given the fact that the mechanism of DHM effects on the ethanol metabolism has not been clarified yet, the effect of dihydromyricetin on the expression and activity of alcohol dehydrogenase (ADH), one of the most important enzymes involved in ethanol metabolism, was therefore studied in this thesis. The cultivation conditions of primary hepatocytes which were isolated from unpretreated and ethanol-pretreated rats and subsequently exposed to EtOH and DHM were optimized. While determining the degree of cell damage caused by EtOH in the presence of DHM, no significant trend in the protective effect of DHM was found. On the other hand, the protective effect of ethanol in hepatocytes cultivated in EtOH and DHM was detected by technique of ELISA (the determination of alanine transaminase). The Western blot technique followed by immunodetection did not detect the induction of ADH expression in hepatocytes. Furthemore, the modulation effect of...
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Enzymatic and Metabolic Transformation of Silybin and its Congeners
Purchartová, Kateřina
Natural flavonoids and flavonolignans feature beneficial properties for living organisms such as antioxidant and hepatoprotective effects, anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activities. Their metabolism in mammals is complex, the exact structure of their metabolites still remains partly unclear and the standards are usually not commercially available. Hence, this project focused on the preparation of potential and defined biotransformation Phase II sulfated metabolites of silymarin flavonolignans: silybin, 2,3-dehydrosilybin, isosilybin, silychristin, silydianin and flavonoids quercetin, taxifolin, rutin and isoquercitrin. Pure sulfated derivatives were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and aryl sulfotransferase from rat liver. Using heterologously expressed PAPS (3'-phosphoadenosine-5'-phosophosulfate) - independent arylsulfotransferase from Desulfitobacterium hafniense and cheap p-nitrophenyl sulfate as sulfate donor, sulfated flavonolignans and flavonoids were obtained in high yields. Silymarin flavonolignans afforded exclusively monosulfates at the position C-20 (C-19 in the case of silychristin), except 2,3-dehydrosilybin that yielded also the 7,20-O-disulfated derivative. Isoquercitrin and rutin were selectively sulfated...
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The study of secondary metabolites in plant tissue cultures I
Blahušová, Adriana ; Kašparová, Marie (advisor) ; Siatka, Tomáš (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Adriana Blahušová Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: The study of secondary metabolites in plant tissue cultures I A principal precondition for successful elicitation used to increase the production of secondary metabolites is, among other, finding a suitable elicitor, its concentration and the optimal period of time of the action of the elicitor on the plant culture in vitro, which was the aim of the present diploma thesis. The effect was examined of a 6, 24, 48 and 168 hour action of the solution of chitosan (in four concentrations) on the production of flavonoids in the suspension culture Trifolium pratense L. (variety Sprint, Tempus and DO-8). The culture was cultivated in Gamborg medium to which 2 mg/l of 2,4- dichlorophenoxyacetic acid and 2 mg/l of 6-benzylaminopurine were added, at the temperature of 25řC and 16 hours light/8 hours dark period. The best elicitation effect of chitosan on the production of flavonoids was the lowest concentration of 1 mg/100ml in all three studied varieties after 48 hours of application.
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The study of secondary metabolites in plant tissue cultures I
Blahušová, Adriana ; Kašparová, Marie (advisor) ; Siatka, Tomáš (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Adriana Blahušová Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: The study of secondary metabolites in plant tissue cultures I A principal precondition for successful elicitation used to increase the production of secondary metabolites is, among other, finding a suitable elicitor, its concentration and the optimal period of time of the action of the elicitor on the plant culture in vitro, which was the aim of the present diploma thesis. The effect was examined of a 6, 24, 48 and 168 hour action of the solution of chitosan (in four concentrations) on the production of flavonoids in the suspension culture Trifolium pratense L. (variety Sprint, Tempus and DO-8). The culture was cultivated in Gamborg medium to which 2 mg/l of 2,4- dichlorophenoxyacetic acid and 2 mg/l of 6-benzylaminopurine were added, at the temperature of 25řC and 16 hours light/8 hours dark period. The best elicitation effect of chitosan on the production of flavonoids was the lowest concentration of 1 mg/100ml in all three studied varieties after 48 hours of application.
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Secondary metabolites and their utilisation in Urtica dioica agg.
Pekařová, Michaela ; Urfus, Tomáš (advisor) ; Hojka, Jakub (referee)
Stinging nettle (Urtica dioica) is a very well-known taxon of genus Urtica, but still its taxonomy is not satisfactory resolved. An obvious reason of taxonomic obscurity of entire aggregate is enormous morphological variation. Polyploidization significantly contributes to this variability. Polyploidy probably is a reason of successful expansion of tetraploid cytotype in synanthropic habitats. Urtica dioica is rich in secondary metabolites, especially fatty acids, phenolic acids and flavonoids. Secondary metabolites are isolated for various purposes. They are widely applied especially in pharmacy and subsequently used for various medications. Despite the undoubtful economic importance a linkage between secondary metabolites composition and polyploidy has not been studied yet.
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The effect of pyrazine derivatives on secondary metabolites content in plant cultures in vitro II.
Hanzlíková, Tereza ; Tůmová, Lenka (advisor) ; Karlíčková, Jana (referee)
The main purpose of the theses was to find if the abiotic elicitor 2,4,6-trimethyl-N-(pyrazine- 2-yl)benzenesulfonamide has any influence on the secondary metabolites production in callus and suspension cultures of Hypericum perforatum L. The cultivation was taking place in the Murashige a Skoog (MS) nutrient medium enriched by the growth regulator - alpha-naphthyl acetic acid at the concentration of 1 mg.L-1 . The elicitor was added to the cultures at the three levels of concentration: c1= 100.0 mg/100 ml; c2= 10.0 mg/100 ml; c3= 1.00 mg/100 ml. The individual samples were taken after 6, 24, 48, 72 and 168 hours of the elicitor application. The control samples without the elicitor were taken after 6, 48 and 168 hours. The collected samples were dried and subsequently transformed into methanol extracts in order to determine secondary metabolites content (rutin, hyperoside and quercetin) by HPLC method. Release of these metabolites into nutrient media was also subject of this observation. The elicitation has influenced production of the secondary metabolites, particularly in callus cultures, wherein several statistically significant values, characterizing increase in their production, were measured. The highest content of rutin (0.169 mg.g-1 DW) was determined in callus culture after 168 hours when...
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