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Alkaloids from Selected Species of Amaryllidaceaefamily, their Toxicity and Biological Activity (in vitro study) I.
Krejčí, Andrea ; Cahlíková, Lucie (advisor) ; Grančai, Daniel (referee) ; Kokoška, Ladislav (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate Mgr. Andrea Krejčí, née Kulhánková Supervisor Ing. Lucie Cahlíková, Ph.D. Title of Doctoral Thesis Alkaloids from selected species of Amaryllidaceae family, their toxicity and biological activity (in vitro study) I. Zephyranthes robusta and Chlidanthus fragrans bulbs were chosen for phytochemical study as biologically active Amaryllidaceae alkaloids source, based on bio-guided study using spectrophotometric Ellman's method and GC-MS analysis of alkaloid extracts. Bulbs were extracted with ethanol and the summary extract was fractionated by column chromatography with the use of Al2O3 as a stationary phase and gradient elution chloroform - ethanol. Column chromatography, preparative TLC and crystallisation led to isolation of pure compounds the chemical structure of which was elucidated by MS and D1- and D2-NMR analyses. From fresh bulbs of Chlidanthus fragrans 11 alkaloids were isolated of which deoxypretazzetine, belladine, 3-epimakronine, ismine, undulatine, buphanisine and ambelline were reported for the first time from the genus Chlidanthus. Compounds were evaluated for their human AChE, BuChE and POP inhibitory activity. From Zephyranthes robusta bulbs 14 alkaloids of...
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The Role of Cytoskeleton and Phospholipids in signaling During Plant Defense Response
Angelini, Jindřiška ; Dušková, Jiřina (advisor) ; Jahodář, Luděk (referee) ; Grančai, Daniel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Department of pharmaceutical botany and ecology Candidate PharmDr. Jindřiška Matoušková Supervisor Ass. Prof. RNDr. Jiřina Dušková, Csc Title of Doctoral Thesis The role of cytoskeleton and phospholipids in signaling during plant defense response Upon abiotic and biotic cues, plant surveillance system simultaneously triggers a variety of signaling events to develop an effective defense response within a few hours. On the molecular level, signaling period is the chain of many consecutive and fast biochemical reactions. Tracking of one particular signaling event e.g. during plant-pathogen interactions is quite difficult task. Presented work is focused on several components of this intricate defense network; namely on the role of salicylic acid, phospholipid signaling and cytoskeleton dynamics and their mutual interaction coupled with downstream changes of plant metabolom. Live imaging can help to elucidate localization and dynamics of particular, fluorescently labeled proteins during defense response. Plenty of important events occurring in plant- -pathogen interactions take place in plasma membrane: where pathogen perception, exocytosis, endocytosis occure and ion channels and carriers are located. Also phosphatidic...
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The Role of Cytoskeleton and Phospholipids in signaling During Plant Defense Response
Angelini, Jindřiška ; Dušková, Jiřina (advisor) ; Jahodář, Luděk (referee) ; Grančai, Daniel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Department of pharmaceutical botany and ecology Candidate PharmDr. Jindřiška Matoušková Supervisor Ass. Prof. RNDr. Jiřina Dušková, Csc Title of Doctoral Thesis The role of cytoskeleton and phospholipids in signaling during plant defense response Upon abiotic and biotic cues, plant surveillance system simultaneously triggers a variety of signaling events to develop an effective defense response within a few hours. On the molecular level, signaling period is the chain of many consecutive and fast biochemical reactions. Tracking of one particular signaling event e.g. during plant-pathogen interactions is quite difficult task. Presented work is focused on several components of this intricate defense network; namely on the role of salicylic acid, phospholipid signaling and cytoskeleton dynamics and their mutual interaction coupled with downstream changes of plant metabolom. Live imaging can help to elucidate localization and dynamics of particular, fluorescently labeled proteins during defense response. Plenty of important events occurring in plant- -pathogen interactions take place in plasma membrane: where pathogen perception, exocytosis, endocytosis occure and ion channels and carriers are located. Also phosphatidic...
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Alkaloids from Selected Species of Amaryllidaceaefamily, their Toxicity and Biological Activity (in vitro study) I.
Krejčí, Andrea ; Cahlíková, Lucie (advisor) ; Grančai, Daniel (referee) ; Kokoška, Ladislav (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate Mgr. Andrea Krejčí, née Kulhánková Supervisor Ing. Lucie Cahlíková, Ph.D. Title of Doctoral Thesis Alkaloids from selected species of Amaryllidaceae family, their toxicity and biological activity (in vitro study) I. Zephyranthes robusta and Chlidanthus fragrans bulbs were chosen for phytochemical study as biologically active Amaryllidaceae alkaloids source, based on bio-guided study using spectrophotometric Ellman's method and GC-MS analysis of alkaloid extracts. Bulbs were extracted with ethanol and the summary extract was fractionated by column chromatography with the use of Al2O3 as a stationary phase and gradient elution chloroform - ethanol. Column chromatography, preparative TLC and crystallisation led to isolation of pure compounds the chemical structure of which was elucidated by MS and D1- and D2-NMR analyses. From fresh bulbs of Chlidanthus fragrans 11 alkaloids were isolated of which deoxypretazzetine, belladine, 3-epimakronine, ismine, undulatine, buphanisine and ambelline were reported for the first time from the genus Chlidanthus. Compounds were evaluated for their human AChE, BuChE and POP inhibitory activity. From Zephyranthes robusta bulbs 14 alkaloids of...
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The Influence of Compounds from Selected Higher Plant Taxons on Platelet Aggregation
Štejnarová, Eliška ; Opletal, Lubomír (advisor) ; Dršata, Jaroslav (referee) ; Grančai, Daniel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Pharmaceutical Botany and Ecology Candidate: Mgr. Eliška ŠTEJNAROVÁ Supervisor: Doc. RNDr. Lubomír OPLETAL, CSc. Title of doctoral thesis: The Influence of Compounds from Selected Higher Plant Taxons on Platelet Aggregation As a part of phytochemical study of secondary metabolites of plants from family Asteraceae, 32 species from this taxonomical unit were tested for platelet aggregation and antioxidant activity. Platelet aggregation was evaluated in vitro on human thrombocytes in platelet rich plasma. The primary used concentration of extracts was 500 µg·mL-1 , arachidonic acid (AA), adenosine diphosphate (ADP), collagen (COL), and thrombin (TR) were used as agonists. Out of the tested species, Leuzea carthamoides (leaves) was the only species which exhibited antiplatelet activity, the extract inhibited platelet aggregation induced by ADP and COL by 33 ± 6 % and 95 ± 5 %, respectively. None of the tested taxons showed proaggregatory effect. From 10.5 kg of dried leaves of L. carthamoide the extracts were prepared sequentially; five compounds: LC-1 (cynaropicrine), LC-2 (chlorojanerin), LC-3 (repdiolid), LC-4 (jaceosidin), LC-5 (hispidulin) were isolated from the most active extract (chloroform) by column and thin layer...
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Study of the Metabolism in in Vitro Cultures of Canabis Sativa L.
Peč, Jaroslav ; Dušek, Jaroslav (advisor) ; Grančai, Daniel (referee) ; Suchý, Václav (referee)
I. Summary The literature review concerning Cannabis sativa L. was prepared with focus on the biologically active compounds. The scope of cell cultures biotechnology and application of modern instrumental analytical methods in the field of metabolomics was discussed. Based on the NMR and MS results, no cannabinoids were detected in the control or elicited cell suspension cultures of Cannabis sativa L. Also other secondary metabolites were not detected by NMR. Other more sensitive analytical methods and their combinations should be used for absolute evaluation of other secondary metabolites, if they are present. It can be concluded, that biosynthesis of the cannabinoids was not induced by pectin or jasmonic acid as elicitors in the suspension, callus or embryogenic tissue cultures of Cannabis sativa L. Main metabolites of certain growt phases were evaluated based on the quantification of produced metabolites and prepared growth phase curves. The impacts of elicitations were interpreted by PCA statistical method, with subsequent metabolite identification or structure elucidation. Maxima of possible 1 H NMR signals were identified and 2D NMR experiments were applied for signals of unknown compounds. The results from NMR measurements combined with PCA analysis brought comprehensive picture of the cultures'...
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Heterologous Expression of ABC Transporter in Catharanthus Roseus (L.) G. Don
Pomahačová, Barbora ; Dušková, Jiřina (advisor) ; Tůmová, Lenka (referee) ; Grančai, Daniel (referee)
In this thesis, the regulation of transport is investigated as a limiting factor in biosynthesis of plant secondary metabolites. CjMDR1 in Catharanthus roseus (L.) G. Don. has been selected as a model system of active transporters. CjMDR1 is a member of ABC (ATP-Binding Cassette) protein superfamily. Especially, in previous reports it was found to transport preferably berberine in its producing plant, Coptis japonica. In order to look into the role of the ABC transporter berberine was exogenously fed to Catharanthus roseus cell line and evaluate the transport of the compound. Based on a series of experiments in this study it can be concluded as follows. 1. Transport of berberine in wild Catharanthus roseus was not clearly associated with ATP-dependency. Using diverse inhibitors it was proved that berberine was not an exclusive substrate of native ABC transporters in Catharanthus. Alternatively berberine might be transported via H+-antiport or efflux transporters from MATE (multidrug and toxic compound extrusion) group which was previously found in other heterologous plants (Yazaki, 2005). In reality in Catharanthus some effect of pH on plasmatic membrane on berberine transport has been observed. However, the exact mechanism of berberine transport on vacuolar and cell level was not fully uncovered...
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Investigation of Biologically Active Plant Secondary Metabolites by Antioxidant and Anti-Aggregative Tests
Řeháková, Zuzana ; Jahodář, Luděk (advisor) ; Semecký, Vladimír (referee) ; Grančai, Daniel (referee)
The effort to find compounds with significant antioxidant properties and with some other therapeutically useful biological activity (e.g. anti-platelet activity) from the wide group of sturcturally different naturally occuring compouds (or plant extracts) or their synthetic derivatives, was the purpose of this work. Radical Scavenging activity of tested compounds or plant extracts was measured by modified DPPH test use program SIA (sequential injection analysis). Singificant radical scavenging activity exhibited polyfenols, namely gallic acid (EC50= 0,0025 ± 0,002 mg/ml) and ethyl-gallate (EC50= 0,0038 ± 0,001 mg/ml). Also the pure latex of Croton lechleri exhibited significantly radical scavenging activity (EC50=0,0347 ± 0,018 mg/ml). From the group of tested methylcoumarins, the excelent antioxidant aktivity exhibited ortho-dihydroxy-4-methylcoumarins, espicially 7,8-dihydroxy-4- methylcoumarin and its derivatives (EC50= 24,9 ± 2,7 μM). Further was the antioxidant activity tested by FRAP methods modified to used micropalates P 400 μl. This spectrofothometric method based on ability of compounds to reduce Fe3+ to Fe2+, confirmed the antioxidant activity of ortho-dihydroxy-4-methylcoumarins. The antiplatelet activity of pure compounds and herbal extracts was assayed in vitro on the model of human...
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Bioassay Guided Separation of Metabolites of the Selected Plant Species - Evolvulus Alsinoides
Červenka, František ; Jahodář, Luděk (advisor) ; Grančai, Daniel (referee) ; Patočka, Jiří (referee)
9. SUMMARY 134 This experimental work deals with pharmaco-botanical evaluation of the aerial part of taxon Evolvulus alsinoides L. The plant is a component of formulations with nootropic and adaptogenic activity. A subject of the investigation was a dried herb of the plant, which is of Indian origin. The aim of this work was to extend the knowledge about compounds of the taxon, to evaluate biological activity of the fractions, isolated substances and to determine characteristic metabolites of the plant. In the first part of the project the crude extract of the plant E. alsinoides was prepared by percolation of 8.8 kg of herb with 95% ethanol. The extract was partitioned into three extracts by extracting by two solvents of different polarity (petroleum ether, ether). The first fraction of weight 92.1 g was obtained by extracting of methanolic solution of crude extract with petroleum ether. The second fraction of weight 69.7 g was obtained by extracting of water solution of the crude extract with ether. The remaining solvent was evaporated from the crude extract and this one represented the third part - polar residue, yield 231 g. The fractions were processed by the methods of column chromatography (CC). The bioassay-guided separation of selected parts of extract (test of acute toxicity, fototoxicity,...
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