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Isolation of alkaloids of the species Geissospermum vellosii Allemão and study of their biological activity I.
Buláková, Anna ; Šafratová, Marcela (advisor) ; Siatka, Tomáš (referee)
Buláková, A: Isolation of alkaloids of the species Geissospermum vellosii Allemão and study of their biological activity I. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmacognosy, Hradec Králové, 2020. Key words: Geissospermum vellosii, bark, alkaloidal extracts, isolation of alkaloids, GC/MS analysis, biological activity, acetylcholinesterase, butyrylcholinesterase. Geissospermum vellosii as a plant belongs to the Apocynaceae family, whose bark is characterized by the content of indole alkaloids, which can also act as acetylcholinesterase inhibitors. The aim of this thesis was to process the pool of ethereal and chloroformic alcaloids from the plant Geissospermum vellosii. By means of column chromatography, the ethereal pool was processed into subfractions GV-1 to GV-16. Taking advantage of preparational method TLC, the subfraction GV-11 was processed, of which one alcaloid-based substance was isolated. Its structure was determined based on GC-MS and NMR analysis and after comparing obtained data with the literature, the substance was identified as Normacusin B. The substance, we extracted, was of unstable nature and because of that, we could not research its biological activity further in relation to AChE and BuChE. Due to the instable nature of...
The effect of abiotic elicitors on secondary metabolites content in plant cultures in vitro - I.
Teplá, Klára ; Tůmová, Lenka (advisor) ; Siatka, Tomáš (referee)
Plants are a source of a wide range of secondary substances, which due to their effects find use in many areas of focus. By a method called elicitation, we can achieve their higher and thus more efficient production. This diploma thesis aimed to determine whether the abiotic elicitor 2-(4-chlorophenyl)-N-(5-chloropyridin-2-yl)acetamide can positively affect the production of the flavonoid hyperoside in callus and suspension culture of Hypericum perforatum L. The elicitor was added to the in vitro cultures in three concentrations: C1 = 3,571.10-3 mol/l; C2 = 3,571.10-4 mol/l and C3 = 3,571.10-5 mol/l. A sample was taken at regular intervals after 6, 24, 48, 72 and 168 hours of elicitor treatment. Control samples were taken after 24 and 168 hours. The content of hyperoside produced was subsequently determined using High Performance Liquid Chromatography. Simultaneously, the amount of hyperoside released into the nutrient media of both plant cultures was also monitored. Maximum hyperoside production was recorded in suspension culture after 6 (17,7 µg/g DW) and 48 hours (3,69 µg/g DW) of elicitor treatment with the lowest concentration of C3 (3,571.10-5 mol/l). The content of hyperoside in the first case was 1770 % higher compared to the control sample. There was a significant release of hyperoside...
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.
Sulfates as phase II metabolites of natural phenolic compounds
Hetman, Anastasiia ; Mladěnka, Přemysl (advisor) ; Siatka, Tomáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anastasiia Hetman Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Co-supervisor: Assoc. Prof. Kateřina Valentová, Ph.D. Title of diploma thesis: Sulfates as phase II metabolites of natural phenolic compounds Natural monophenolic compounds, which both occur in plants and are formed during gut microbiota metabolism of polyphenols, are absorbed and pass through phase II metabolism. This results in the formation of various conjugated metabolites, such as sulfates, whose biological activity was not studied as yet. This work focused on the preparation and isolation of pure sulfated metabolites of phenolic compounds, namely 4- methylcatechol, protocatechuic, homoprotocatechuic, and 2,3,4-trihydroxybenzoic acid, and phloroglucinol, using the aryl sulphotransferase from Desulfitobacterium hafniense. As a result, two sulfated products of sufficient purity were prepared, namely 4- methylcatechol-O-sulfate (an inseparable mixture of 4-methylcatechol-2-O-sulfate and 4- methylcatechol-1-O-sulfate) and phloroglucinol-O-sulfate. Their antioxidantactivity was evaluated and compared with the activity of the parent compounds using the following six in vitro tests: DPPH (1-diphenyl-2-picrylhydrazyl) and...
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.
Alkaloids of Narcissus species cultivars and their biological activity I.
Tkáčová, Beáta ; Breiterová, Kateřina (advisor) ; Siatka, Tomáš (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of Pharmaceutical Botany (16-16130) Author: Tkáčová Beáta Supervisor: PharmDr. Kateřina Breiterová, Ph.D. Title of thesis: Alkaloids of Narcissus species cultivars and their biological activity I. Key words: Narcissus, alkaloids, biological activity, Alzheimer's disease. The main aim of this diploma thesis was to obtain summary alkaloid extracts from bulbs of five species cultivars (Narcissus cv. Acropolis, Delta, White Marvel, Kedron, Scarlet Gem) and one variety (Narcissus albus var. plenus odoratus), labeled as AL-450, AL-457, AL-460, AL-463, AL-467 and AL-508, from which samples were subsequently prepared for GC-MS analysis and screening of biological activities (cholinesterase inhibitory activity and cytotoxicity). Totally, 18 alkaloids were identified by GC-MS analysis, comparing their mass spectra. Identified alkaloids include 3-O-demethylmaritidine, 11,12-dehydroanhydrolycorine, assoanine, dehydroassoanine, galanthamine, galanthine, haemanthamine, hippeastrine, incartine, lycoramine, lycorine, narwedine, norpluvine, pancracine, pluvine, pseudolycorine and tazettine. Samples of cultivars AL-457, AL-460, AL-463 and AL-508, showed relatively promising hAChE inhibitory activity in screening assay (c = 50 µg/ml),...
Isolation of alkaloids of the species Geissospermum vellosii Allemão and study of their biological activity I.
Buláková, Anna ; Šafratová, Marcela (advisor) ; Siatka, Tomáš (referee)
Buláková, A: Isolation of alkaloids of the species Geissospermum vellosii Allemão and study of their biological activity I. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmacognosy, Hradec Králové, 2020. Key words: Geissospermum vellosii, bark, alkaloidal extracts, isolation of alkaloids, GC/MS analysis, biological activity, acetylcholinesterase, butyrylcholinesterase. Geissospermum vellosii as a plant belongs to the Apocynaceae family, whose bark is characterized by the content of indole alkaloids, which can also act as acetylcholinesterase inhibitors. The aim of this thesis was to process the pool of ethereal and chloroformic alcaloids from the plant Geissospermum vellosii. By means of column chromatography, the ethereal pool was processed into subfractions GV-1 to GV-16. Taking advantage of preparational method TLC, the subfraction GV-11 was processed, of which one alcaloid-based substance was isolated. Its structure was determined based on GC-MS and NMR analysis and after comparing obtained data with the literature, the substance was identified as Normacusin B. The substance, we extracted, was of unstable nature and because of that, we could not research its biological activity further in relation to AChE and BuChE. Due to the instable nature of...
The effect of pyrazine derivatives on secondary metabolites content in plant cultures in vitro I.
Graclíková, Bohumila ; Tůmová, Lenka (advisor) ; Siatka, Tomáš (referee)
1 Abstract Elicitation method is one way to increase the production of secondary metabolites in in vitro of plant cultures and this work deals with this problem. Its aim was to find the positive effect of 2-hydroxy-4-(imidazo[1,2-a]pyrazine-2-carboxamide)benzoic acid from the group of pyrazine derivates on the production of isoflavonoids in in vitro cultures of Genista tinctoria L. Isoflavonoids play an important role in therapy of climacteric problems or they are promising substances of therapy various cancer types due to their affinity to estrogens β-receptors. This plant or rather its in vitro cultures are a potencial sources of isoflavonoids. Suspension and callus cultures of Genista tinctoria L. were used for elicitation. They were cultivated on Schenk & Hildebrant medium with the addition of 2,4-dichlorophenoxyacetic acid (2,4-D) as growth regulator at a concentration of 1 ml/l. The elicitor solution was added to the cultures at three concentrations: 3.3642 × 10-3 mol/l; 3.3642 × 10-4 mol/l; 3.3642 × 10-5 mol/l. The samples were taken at intervals of 6, 24, 48, 72 and 168 hours and then they were analyzed by HPLC method. At the same time intervals the samples of medium, where isoflavonoids were released, were analyzed. The biggest production of isoflavonoid was reached in suspension culture. The...
Podophyllotoxin in eastern red cedar (Juniperus virginiana)
Olšar, Jiří ; Siatka, Tomáš (advisor) ; Kastner, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Jiří Olšar Supervisor: doc. PharmDr. Tomáš Siatka, CSc. Title of diploma thesis: Podophyllotoxin in eastern red cedar (Juniperus virginiana) This thesis describes the optimisation of the extraction process for quantity determination of podophyllotoxin in the leaves of Juniperus virginiana L. This determination was conducted through three different methods: the extraction in ultrasound bath, the extraction in the orbital shaker and the maceration in cold environment of 4 řC. Extractions were affected by various factors which were monitored, for instance time of extraction, temperature, and extraction solvents. The most efficient method appears to be the extraction in ultrasound bath with the use of methanol or a combination of phosphate buffer with ethyl-acetate.
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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