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Analysis of milk thistle (\kur{Silybum marianum}) crop management in agricultural company
ŠINDELKOVÁ, Tereza
This diploma thesis deals with monitoring of milk thistle cultivation in selected farm in South Bohemia. The growth of the milk thistle was followed by fees for assessing growth and its developmental phase. Pre-harvest plant selections to estimate the number of flowers on the leaves and the number on the flowers. The feed yield, the tissue content for feeding, the water and dry matter content, the fat and N content, the total antioxidant activity were determined.
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HPLC determination of silybin diastereoisomers in plasma of laboratory animals
Kolářová, Petra ; Tesařová, Eva (advisor) ; Bednář, Petr (referee)
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...
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Preparative diastereomeric separation of silybin and its sulfates by RP-HPLC
Kolářová, Petra ; Tesařová, Eva (advisor) ; Kalíková, Květa (referee)
Silymarin is standardized extract isolated from fruits of Milk Thistle plant (Silybum marianum). The principal component of silymarin is silybin. This flavonolignan is mainly responsible for the medicinal effects of Milk Thistle fruits: antioxidant, hepatoprotective, anticancer and chemoprotective activities. Natural silybin exists as an equimolar mixture of diastereomers A and B whose preparative separation is very hard. It was shown that the biological activity of silybin A and B are different. Silybin in the blood conjugates mainly to sulfates. The structure or biological activity of the sulfates is not yet known. The aim of this work is to develop practically applicable method for preparative separation of diastereomers of silybin A and B, and sulfates, which are considered as one of the major metabolites of silybin. The preparative method for separation of silybin A and B in the mobile phase consisting of 50% MeOH on the chromatographic column Labio C18 25x250 mm, was developed. In addition, preparative method for separation of mixture of products accured from the sulphation of silybin-23-acetate in the mobile phase consisting of MeOH/H2O 60/40 (v/v) with addition of 10 ml/l HCOOH, was optimized.
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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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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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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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HPLC determination of silybin diastereoisomers in plasma of laboratory animals
Kolářová, Petra ; Tesařová, Eva (advisor) ; Bednář, Petr (referee)
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...
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Sulphated metabolites of silybin and their interactions with sulphatases
Ježková, Zuzana ; Weignerová, Lenka (advisor) ; Kavan, Daniel (referee)
This bachelor thesis solves the question of sulphated metabolites of silybin by their interactions with sulfatases. Silybin is flavonolignan prepared from the seeds of milk thistle (Silybum marianum). Sulfate of silybin (metabolite) has not been isolated from organism so far thus its exact structure has not been determined. In this work synthesis of silybin-7,23-disulfate from natural silybin is presented. Silybin disulfate was isolated and spectrally characterized (NMR, MS). In the next step the kinetics of the reaction pNPS with sulfatase from Helix pomatia was measured: Km = 0.0494 mmol/l a Vmax = 0.0325 mmol/dm3 /min. Conditions for sulfatase reaction from Helix pomatia were selected according to literature. One of the main goal was to determine, whether silybin-7,23-disulfate is a substrate and/or an inhibitor of sulfatase from Helix pomatia. Activity measurements of the sulfatase from Helix pomatia in the presence of various concentrations of silybin-7,23-disulfate enabled us to determine that silybin disulfate at concentration higher than 0.2 mM is a strong inhibitor of the sulfatase tested. Series of reactions with sulfatase from Helix pomatia were performed to determine whether silybin-7,23-disulfate is a substrate. Reactions were monitored by HPLC and it was demonstrated that the...
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Preparative diastereomeric separation of silybin and its sulfates by RP-HPLC
Kolářová, Petra ; Tesařová, Eva (advisor) ; Kalíková, Květa (referee)
Silymarin is standardized extract isolated from fruits of Milk Thistle plant (Silybum marianum). The principal component of silymarin is silybin. This flavonolignan is mainly responsible for the medicinal effects of Milk Thistle fruits: antioxidant, hepatoprotective, anticancer and chemoprotective activities. Natural silybin exists as an equimolar mixture of diastereomers A and B whose preparative separation is very hard. It was shown that the biological activity of silybin A and B are different. Silybin in the blood conjugates mainly to sulfates. The structure or biological activity of the sulfates is not yet known. The aim of this work is to develop practically applicable method for preparative separation of diastereomers of silybin A and B, and sulfates, which are considered as one of the major metabolites of silybin. The preparative method for separation of silybin A and B in the mobile phase consisting of 50% MeOH on the chromatographic column Labio C18 25x250 mm, was developed. In addition, preparative method for separation of mixture of products accured from the sulphation of silybin-23-acetate in the mobile phase consisting of MeOH/H2O 60/40 (v/v) with addition of 10 ml/l HCOOH, was optimized.
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