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Chemoenzymatic separation of diastereoisomers of silybin
Purchartová, Kateřina ; Stiborová, Marie (advisor) ; Wimmer, Zdeněk (referee)
Silybin is major component of silymarin isolated from seeds of the milk thistle (Silybum marianum). This compound is widely used in human medicine against liver disorders and as a protectant against a number of hepatotoxins. It also exhibits other interesting activities as anticancer and chemoprotective, dermatoprotective and also hypocholesterolemic effects. Natural silybin is a nearly equimolar mixture of two diastereoisomers, silybin A and silybin B, whose analytical separation is quite feasible, but preparative separation is extremely complicated. The aim of this work was to find suitable method leading to separation of both silybin diastereoisomers. A library of hydrolases (lipases, esterases and proteases) was tested for their diastereoisomeric discrimination of the selective alcoholysis of 23-O-acetylsilybins. Novozym 435 (lipase B from Candida antarctica immobilized on acrylic resin) proved to be the most suitable enzyme for the preparative production of both optically pure silybin A and B by enzymatic hydrolysis. Under the optimized conditions, silybin A was obtained in 42 % yield and 97 % purity while silybin B was obtained in 67 % yield and 99 % purity. Covalent modifications of Novozym 435 (acetylation, succinylation, and hydroxyethylamidation), which should lead to improvement of...
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Enzymatic modifications of bioactive flavonoids
Rydlová, Lenka ; Tesařová, Eva (advisor) ; Wimmer, Zdeněk (referee)
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),...
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Chemoenzymatic separation of diastereoisomers of silybin
Purchartová, Kateřina ; Wimmer, Zdeněk (referee) ; Stiborová, Marie (advisor)
Silybin is major component of silymarin isolated from seeds of the milk thistle (Silybum marianum). This compound is widely used in human medicine against liver disorders and as a protectant against a number of hepatotoxins. It also exhibits other interesting activities as anticancer and chemoprotective, dermatoprotective and also hypocholesterolemic effects. Natural silybin is a nearly equimolar mixture of two diastereoisomers, silybin A and silybin B, whose analytical separation is quite feasible, but preparative separation is extremely complicated. The aim of this work was to find suitable method leading to separation of both silybin diastereoisomers. A library of hydrolases (lipases, esterases and proteases) was tested for their diastereoisomeric discrimination of the selective alcoholysis of 23-O-acetylsilybins. Novozym 435 (lipase B from Candida antarctica immobilized on acrylic resin) proved to be the most suitable enzyme for the preparative production of both optically pure silybin A and B by enzymatic hydrolysis. Under the optimized conditions, silybin A was obtained in 42 % yield and 97 % purity while silybin B was obtained in 67 % yield and 99 % purity. Covalent modifications of Novozym 435 (acetylation, succinylation, and hydroxyethylamidation), which should lead to improvement of...
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Nitrile hydrolysing enzymes: Fungal nitrilases
Vejvoda, Vojtěch ; Martínková, Ludmila (advisor) ; Wimmer, Zdeněk (referee) ; Vosáhlová, Simona (referee)
Charles University in Prague Faculty of Science Department of Biochemistry A thesis submitted for the degree of Doctor of Philosophy Nitrile-hydrolysing enzymes: Fungal nitrilases Mgr. Vojtěch Vejvoda Supervisor: Ing. Ludmila Martínková, CSc. Laboratory of Biotransformation, Institute of Microbiology, v.v.i. Academy of Sciences of the Czech Republic Consultant: Prof. RNDr. Karel Bezouška, DrSc. Faculty of Science, Department of Biochemistry, , Charles University in Prague Prague 2010 INTRODUCTION Nitriles are widespread in the environment - not only as the natural products of plants but also as the secondary metabolites of microorganisms or higher organisms. Synthetic nitriles and inorganic cyanides occur as pollutants from human activities (industrial wastes, herbicides, etc.). Both natural and man-made nitriles are poisonous to many organisms, the natural ones serving as a protection against enemies (e.g. herbivores). Their toxic activity is used in some herbicides. However, all of these compounds may exert negative effects on other beings living in the particular environment and create selection pressures, probably leading to the evolution of enzymes catalyzing the detoxification of nitriles, or, even better, utilizing them as the source of energy and nutrients. Nitrile-metabolizing enzymes are...
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Natural Sources of Chlorinated Volatile Hydrocarbons in Spruce Forest Undergrowth
Štangelová, Pavla ; Wimmer, Zdeněk ; Tesařová, E. ; Forczek, Sándor
Chlorine as one of the most frequently occurring elements in nature, is involved in biogeochemical\ncycles. Several anthropogenic and even natural sources of volatile chlorinated hydrocarbons of\nemission is known, yet natural sources have not been explored enough. The aim of this study is to\ngather information of potential sources of chlorinated organic hydrocarbons in spruce forest\nundergrowth. Emissions of chlorinated volatile hydrocarbons of selected species of bryophytes,\nferns and fungi were measured by SMPE sampling technique and analyzed with GC-MS analysis.\nSodium chloride solution was used as a stressor to simulate increased environmental chloride load.\nTrichloromethane (chloroform) was found in most natural samples, dichloromethane,\ntetrachloroethene and tetrachloromethane were found occasionally. Increased chloride\nconcentration caused elevated emissions of trichloromethane and tetrachloromethane in some plant\nspecies.
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Alternative Plant Utilization for Obtaining of Significant Nature Substances: Can Weed Find a Utilization?
Wimmer, Zdeněk ; Sajfrtová, Marie ; Sovová, Helena ; Pavlík, Milan ; Svobodová, Hana ; Jurček, Ondřej ; Wimmerová, Martina
Numbers of compounds and their mixtures, which are produced by plants, can be obtained by extracting plant materials by supercritical fluids. Phytosterols and phytoecdysterotds represent an important family of these plant products. They can be used in a synthesis of supramolecular structures with possible importance for targeted effect of drugs or other biological(y active compounds. Target organisms for the compounds investigated are: (a) insects - pests and human food competitors, also important as model system in investigation of convenient application forms of biologically active compounds; (b) cell cultures used during basic pharmacological research. Supramolecular structures may form convenient matrices for practical application of the biologically active compounds. It has already been proved that numbers of medicinal or cultural plants produce important natural compounds. The present question has appeared to be answered, if so far inutile weeds may be turned into important sources of pro-drugs for disease treatments.
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