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Vliv ostropestřece mariánského (Silybum marianum) na stravitelnost živin u koní
Tomíšková, Lucie
The experiment was performed on horses in Našiměřice. The whole experiment took place in the period 2020-2021, and was divided into 7 periods, each lasting 28 days. A predetermined feed ration was fed throughout one period. Feed rations differed in the compositin in every period. Milk thistle (Silybum marianum) was fed to the horses in various forms. It was served in the form of milk thistle seedcake (as component of the granulated mixture), whole seeds and with/without the addition of a mineral premix and/or oil. The aim of the diploma thesis was to find diferences between varitions of milk thistle administration to horses affect the digestibility of crude fat, crude fiber, crude ash, crude protein, BNLV and digestible energy. Monitoring of the digestibility of flavonolignans was another aim of the thesis. The results were obtained from analyze determinig the nutrients in the individual components of feed ration and faeces. Statistically significant differences were found both in the digestibility of nutrients and in some flavonoligans. The highest average digestibility of crude ash was 58,92 ± 1,43%, crude protein 72,26 ± 0,62%, crude fat 65,40 ± 0,78%, crude fiber 73,73 ± 1,27% and nitrogen-free substances yield 84,06 ± 1,15%. Daily balance digestibility of silichristin was 2,39 mg/g, silybin A 1,09 mg/g and isosilybin A + B was digestibility 1,69 mg/g. From the results it could be concluded that the fedding of milk thistle positively affected the digestibility of nutrients.
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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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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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Cultivation Technology of Milk Thistle (Silybum Marianum)in Relation to the Product Quality and its Processing
GRAMANOVÁ, Hana
Silybum marianum has been a plant known for millenniums thanks to its pharmacological effects especially in fields of liver, gallbladder or even colon cancer treatment. A complex of effective components is called silymarin. Its amount and structure in milk thistle seeds are very important. That{\crq}s why there{\crq}s a tendency to develop methods which could increase silymarin quantity together with silymarin quality in this herb. This was also the aim of this thesis. One of the possibilities to level up the content of effective components in drugs is to bring this plant in stress. There were realized two ground-plot experiments. The stress agens was acetatonsalicylic acid (ASA) of different concentrations applicated on leaves of these plants, concretely in the concentrations of 10-5 mol.l-1, 10-4 mol.l-1, 10-3 mol.l-1. In the case of the application ASA concentration 10-3 mol.l-1 there was proved an effective action. The increase of effective components in seeds reached approximately 116,5 % ratio compared with control application of distilled water on the Silybum marianum leaves. The silymarin complex was determined by High Performance Liquid Chromatography. The preparation of the extract was practised in different ways {--} using ethyl alcohol extract and distilled water in combination with various temperatures of extracts storage. These methods were compared one another. As the best one has been turned out to be using 60% ethanol concentration for the duration of 96 hours in the storage temperature 20°C.
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