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Secondary metabolites of plant cultures in vitro I
Zlochová, Pavlína ; Kašparová, Marie (advisor) ; Siatka, Tomáš (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Pavlína Zlochová Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: Secondary metabolites of plant cultures in vitro I. Key words: Suspension cultures, Trifolium pratense, phytoestrogens, flavonoids, isoflavonoids Explant cultures are source of plant secondary metabolites. Nevertheless the production of secondary metabolites is usually low in explant cultures. This production can be increased by elicitation metods. Suitable elicitor is added into the cultivate medium and leads to gene expression and production of secondary metabolites. The aim of this study was to observe the influence of sodium chloride on the production of flavonoids and isoflavonoids by the Trifolium pratense L. suspension culture (Tempus variety). The culture was cultivated on Gamborg nutriet medium with addition of 2 mg.l-1 2,4- dichlorophenoxyacetic acid and 2 mg.l-1 6-benzylaminopurine. Cultivation proceed in 25řC temperature and 16 hours light/8 hours dark period. Then determination of flavonoids in according to Czech Pharmacopoeia 2009 and the determination of isoflavonoids by HPLC method was performed. The best elicitation effect on the production of flavonoids was 175 mmol.l-1 of sodium chloride after a 3...
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Enzymatic and Metabolic Transformation of Silybin and its Congeners
Purchartová, Kateřina ; Křen, Vladimír (advisor) ; Macek, Tomáš (referee) ; Vítek, Libor (referee)
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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Flavonoids in some cultivars of Sambucus nigra L. flowers.
Krulišová, Markéta ; Spilková, Jiřina (advisor) ; Martin, Jan (referee)
The aim of this work was to find out the content of flavonoids in the flowers of eleven cultivars of black elder (Sambucus nigra L.) and determine if their contents differ according to the statistical significance. Preparing the list of effects and substances that are contained in the flowers was the goal as well. The drug Sambuci nigrae flos is mostly used for its content of flavonoids and hydroxycinnamic acids in the therapy of colds and diseases of the urinary and respiratory tract. Its benefits were confirmed also by studies testing antibacterial and antiviral activity, diabetes and obesity, effects on the immune system and also protection against UV radiance. The content of flavonoids was established by the spectrophotometric method that is listed in the Czech Pharmacopoeia 2009 in the article Sambuci nigrae flos as the method for the determination of content. The statistic evaluation of the differences in the flavonoids content between the cultivars was made by ANOVA and the Bonferroni test. The demand of Czech Pharmacopoeia 2009 on the minimal content of flavonoids is 0,80%. This requirement was fulfilled in the flowers of these cultivars: Albida, Heidegg 13, Riese auß Voßloch, Sambu, Samdal, Sampo and Samyl. On the opposite side, flowers of the cultivars Allesö, Aurea, Dana and Juicy did...
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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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Study of effect of flavonoids on biotransformation enzymes expression
Koucká, Kamila ; Moserová, Michaela (advisor) ; Mrízová, Iveta (referee)
Flavonoids belong to the group of phenolic compounds included in the secondary metabolites of plants, which are represented mainly in fruits and vegetables. These compounds have antioxidant and anti-carcinogenic effects. Some of them can affect biotransformation enzymes, which include the cytochrome P450 and can interfere with the metabolism of xenobiotics. In the present work we studied effect of dihydromyricetine on the gene expression of cytochrome P450 1A1, 1A2 and cytochrome b5 in livers, lungs, kidneys, colon and small intestine of laboratory rats. The small intestine was divided into three parts namely a proximal, middle and distal. At first, isolation of total RNA was made using commercial reagents and isolated RNA was converted to cDNA by reverse transcription. Finally, using polymerase chain reaction in real time (RT-PCR), the relatively gene expression of genes observed in the organs of laboratory rats pretreated dihydromyricetine and control laboratory rats (untreated) to a reference gene β-actin was determined. It was found that dihydromyricetine did not significantly affect the gene expression of studied genes in most organs. However, a significant decrease of gene expression of CYP1A1 and CYP1A2 was observed in the lungs. On the contrary, an increase of gene expression of CYP1A2 was...
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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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Properties of plant extracts
Bělonožníková, Kateřina ; Ryšlavá, Helena (advisor) ; Liberda, Jiří (referee)
Plants synthesize a wide variety of compounds called secondary metabolites. These compounds comprise products of the phenylpropanoid pathway (flavonoids, stilbens, anthocyanines and tannins), isoprenoids, and alkaloids. Since plant secondary metabolites seem to affect human health in a positive way, decrease the risk of some diseases such as cancer, diabetes type 2 and disorders of the cardiovascular system, their studies attract a lot of attention. The mechanism of the effect of individual compounds on human health has not been clarified yet, the positive effect is probably caused by their antioxidant capacity. In this work 22 extracts from different kinds of fruit and vegetables were prepared. The total content of phenolic compounds and flavonoids, the antioxidant capacity by methods FRAP (ferric ion reducing antioxidant power) and ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6- sulphonic acid)] were determined. High content of phenolic compounds was established in the extract from strawberry, cherry and kiwi, the highest antioxidant capacity was found in the extract from strawberry. Another studied attribute of extracts from fruit and vegetables was their influence on the activity of digestive enzymes pancreatic lipase and α-amylase. The extracts from lemon, red currant, jostaberry, lime and...
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Analytical approaches of identification and quantification of flavonoids
Dušková, Miroslava ; Hudeček, Jiří (advisor) ; Hodek, Petr (referee)
The first part of this thesis summarizes the available information about flavonoids and gives deeper insight into their classification, biosynthesis, occurrence in human diet and beneficial effects on human health. In the next part, the most widely used analytical approaches for identification and quantification of these compounds are described in detail. The aim of experimental part of this thesis was to determine the purity of two samples of flavonoid compounds - myricetin and dihydromyricetin. As the first step, ultraviolet-visible spectroscopy was used. The determined wavelengths of absorbance maxima are in accordance with literary data for both compounds. Next, the reversed-phase high-performance liquid chromatography was used for determination of eventual impurities. The chromatographs obtained with optimized elution protocols are demonstrating the purity of both samples, corresponding to the manufacturers' certificates.
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Antioxidant activity of some cultivars of Sambucus nigra L. fruits.
Nehybová, Monika ; Spilková, Jiřina (advisor) ; Tůmová, Lenka (referee)
8. Abstract The fruit of the European elder, Sambucus nigra L., is used in therapy as a drug Sambuci fructus for its diaphoretic, diuretic and laxative effects. Cultivars of European elder are being studied for the use in the food industry and pharmacy. The fruit contains especially large amount of anthocyanins and flavonoids that are known for their antioxidant activity. The aim of this work is to measure the antioxidant activity of the drug Sambuci fructus and of the fruit of the European elder cultivars Samdal, Mammut, Sambo, Sambu, Sampo, Bohatka, Haschberg, Weihenstephan, Allesö, and Samyl. An extract containing anthocyanins was obtained from fruit by a process based on extraction of frozen material with acidified water at high temperature, followed by separation with polymer non-ionogenic polystyrene-divinylbenzene sorbent. The content of anthocyanins in the extracts was determined spectrophotometrically using a modified pH-differential method. The absorbance was measured along the range of 350 - 600 nm. The content of the anthocyanins was quantified in units of mg of cyanidin 3-glucoside in 1 ml of extract. The content ranged from 0.33 - 2.76 mg cyanidin 3-glucoside/ml of extract. The highest content was in the extract from the fruit of the cultivar Samdal and the lowest was of the cultivar Mammut....
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Studium interakcí doplňků stravy s enzymy biotransformace xenobiotik
Bebová, Michaela ; Hodek, Petr (advisor) ; Hansíková, Hana (referee)
Currently, an increasing attention is being paid to phytochemicals as one of the most widely used chemopreventive compounds, generally considered as health-promoting and safe. Flavonoids representing a large group of phytochemicals are present in many dietary supplements formulated from natural sources. The consumption of these concentrated phytochemicals has dramatically increased in the recent decade. It appears, however, that the ingestion of flavonoids might be associated with some adverse effects. Some flavonoids are known modulators of enzymes involved in phase I and phase II metabolism of xenobiotics biotransformation, thus their induction may result in an increase of carcinogen activation. In this study, the effects of selected flavonoid compounds -naphthoflavone, - naphthoflavone, myricetin, and dihydromyricetin, and carcinogens (BaP, PhIP) on phase II metabolism enzymes, sulfotransferases (SULT), have been investigated. To determine the induction of SULT, antibodies for their immunodetection have been developed. Peptide antigens derived from sequences of selected rat sulfotransferases rSULT1A1, 1B1, 1C1, 1C2, 1C1/2, 1E1, and 2A1, were used as KLH conjugates for hen immunization to obtain yolk anti-peptide antibody (IgY). Fractions of IgY were isolated from eggs yolks by simple...
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