Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Gabriela Dohnalová Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: Plant tissue cultures as a potential source of phenylpropanoids I Explant cultures are the source of plant secondary metabolites. However, the production of secondary metabolites is usually low in explant cultures. Production can be increased by a method called elicitation. The basic prerequisite for successful elicitation is, among other things, finding a suitable elicitor, its concentration and optimal time of elicitor action on plant culture in vitro. The aim of this study was to observe the influence of lead chloride and ferrous sulfate (in four concentrations) on the production of podophyllotoxin in the suspension cultures of Juniperus virginiana L. (variety Hetzii and Glauca). The culture was cultured on Schenk and Hildebrandt nutrient medium with addition of 3.0 mg.l-1 α-naphthylacetic acid, 0.2 mg.l-1 kinetin and 15 mg.l-1 ascorbic acid. Cultivation proceed in 25 řC temperature and 16 hours light/8 hours dark period. Subsequently, the determination of the content of podophyllotoxin by HPLC was performed. Juniperus virginiana L. variety Hetzii produces a higher amount of podophyllotoxin than the Glauca...

Podophyllotoxin is a natural medicine possessing an outstanding anti-tumour activity. It can be extracted from the rhizome of Podophillum peltatum (American Podophyllum). Volumetric heating of a packed bed of particles including solvent during the extraction can eliminate the solvent pre-heating time and provide uniform and quick heating of the bed. RF-assisted extraction has a potential to be a promising extraction alternative over conventional methods. The characterization and assessment of RF-assisted extraction of podophyllotoxin is crucial. Thermal properties including specific heat capacity, thermal conductivity, and thermal diffusivity of a packed bed of P. peltatum with and without ethanol solutions were determined and the associated multiples regression equations were obtained for the purpose of thermal analysis of RF-assisted packed bed extraction process and related modeling investigations.<p> The dielectric properties of the packed bed of rhizome particles were measured from 10 to 30 MHz using a precision LCR meter and a liquid test fixture. The effects of temperature, particle moisture content, volumetric concentration of ethanol and bed porosity on the dielectric constant, dielectric loss factor and power penetration depth were investigated. The dielectric loss factor significantly increased with the particle moisture content for the beds with 100% and 70% ethanol but not with 30% ethanol. The dielectric loss factor was proportional to temperature directly and to frequency inversely. With 30% ethanol (and therefore 70% water), the dielectric loss factor of the bed dramatically increased compared to 70% and 100% ethanol. Porosity had a significant effect on the dielectric constant but not on the dielectric loss factor. The power penetration depth of a packed bed with 100% ethanol was significantly larger than those of the packed bed with 30% and 70% ethanol. Empirical regression equations were developed for simulation and design of an RF-assisted packed bed extraction of podophyllotoxin.<p> A RF-transparent batch reactor was made of glass filled Teflon and the extraction kinetics of podophyllotoxin was characterized. The effects of temperature, ethanol volumetric concentration, solid/liquid ratio, RF heating and particle moisture content on the extraction rate and yield of podophyllotoxin were investigated at different extraction conditions. A generalized diffusion mathematical model taking into account three major particle geometries was developed and coupled with genetic algorithm for determination of effective diffusivity and partition coefficient through an inverse simulation approach. The approach was first verified by reported experimental data of andrographolide extraction followed by determining the effective diffusivity and partition coefficient of podophyllotoxin for different conditions. The optimum batch extraction condition was achieved with 30% ethanol-water solution at 53¢XC. A prototype was developed for RF-assisted extraction of podophyllotoxin using two optical and RF-transparent reactors with horizontal and vertical orientations. Applying the optimum conditions obtained from batch experiments, the potential of RF heating for providing a uniform temperature in the packed bed was evaluated. The effect of solvent dielectric loss factor on uniform RF heating was investigated and the chemical effect of NaCl used for increasing dielectric loss factor of the solvent on podophyllotoxin was assessed. The horizontal packed bed demonstrated a large temperature gradient across the thickness of the bed during RF heating; however, a uniform RF heating was achieved when the vertical packed bed reactor was used for RF-assisted extraction of podophyllotoxin. The concentration of 2.5 g NaCl/L of the solvent at the temperature controller set point of 40aC provided a relatively good uniform temperature of 50aC within the bed. Evaluating three flow rates of 130, 160 and 200 ml/min for the solvent of 30% ethanol with 2.5 g NaCl/L indicated that the flow rate of 160 ml/min could provide better temperature overlap of four positions of the bed height.

Thesis (M.D. with Distinction in Research) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 25-28.

Thesis (M.S.)--Mississippi State University. Department of Plant and Soil Sciences. / Title from title screen. Includes bibliographical references.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Nikola Urbanová Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: The study of secondary metabolites in plant tissue cultures II The aim of this work was to determine the effect of the potential elicitor chitosan on production of podophyllotoxin in callus and suspension cultures of Juniperus virginiana Glauca variety and suspension cultures of Juniperus virginiana Hetzii variety. Schenk and Hildebrandt medium supplemented with α-naphtylacetic acid (3.0 mg/l), kinetin (0.2 mg/l) and ascorbic acid (15.0 mg/l) was used for the cultivation. Chitosan solutions of various concentrations (0.001; 0.01; 0.1; 1 g/100 ml) were affecting the cultures for 6, 24, 48 and 168 hours. The content of chitosan was determined by HPLC. Higher values of podophyllotoxin content were measured in cultures derived from the Glauca variety. The best chitosan effect on podophyllotoxin production was manifested in callus cultures after 24 hours application of 0.001 g/100ml concentration. A podophyllotoxin content of 0.210 % was determined, which was about 320 % higher in comparison with the control. The maximum content (0.140 %) in suspension culture was induced by 24-hours application of a 0.1 g/100 ml concentration;...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Veronika Vargovčíková Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: Production of podophyllotoxin by plant tissue cultures of Juniperus virginiana Key words: Juniperus virginiana, callus cultures, suspension cultures, podophyllotoxin Elicitor and precursor adding method can be used to enhance the production of secondary metabolites in explantate cultures. This master's thesis is focused on the evaluation of the effects of biotic elicitor (salicylic acid) and phenylpropanoid metabolism precursor (cinnamic acid) on podophylotoxin production in Juniperus virginiana (var. 'Glauca' and 'Hetzii') suspension cell culture. Suspension cultures were cultivated on Schenk and Hildebrandt medium at 25řC and circadial period of 16 hours light and 8 hours dark. Medium contained 15 mg.l-1 ascorbic acid and growth regulators 3.0 mg.l-1 , α-naphthaleneacetic acid, 0.2 mg.l-1 kinetin. The effects of salicylic acid aqueous solution 0.01 mmol.l-1 , 0.10 mmol.l-1 , 1.00 mmol.l-1 , 10.0 mmol.l-1 and cinnamic acid 60% alcohol solution 0.10 mmol.l-1 , 1.00 mmol.l-1 , 10.0 mmol.l-1 , 100 mmol.l-1. were examined after 6, 24, 48 and 168 hours. Podophylotoxin content was evaluated using HPLC method. In 'Glauca'...

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<p> The objective of this study was to synthesize and biologically evaluate a series of novel nucleoside derivatives of the naturally occurring lignans, podophyllotoxin and 4'-demethylepipodophyllotoxin. Epipodophyllotoxin derivatives display two types of biological activity: while the naturally occurring compounds are potent inhibitors of microtubule polymerization, two semi-synthetic glycoside derivatives, viz VP16-213 (etoposide) and VM26 (teniposide), are specific inhibitors of mammalian DNA topoisomerase II. The latter two compounds have proven to be very useful in the treatment of many different types of cancer. In this study, the glycoside moiety of VP16-213/VM26 was replaced with either a thymidine or a 2'-deoxyadenosine group. The synthetic approach involved protective group chemistry, where functional groups on the nucleosides and DMEP were selectively blocked prior to their condensation in the presence of boron trifluoride etherate catalyst. These compounds are of interest because they involve substitution of the glucoside moiety with a nucleoside residue, whose effect on the biological activity of epipodophyllotoxin derivatives has not been examined. The biological activity of these compounds was assessed by examining their cross-resistance patterns towards a set of Chinese hamster ovary mutants resistant to podophyllotoxin VP16-213/VM26, mitotic index, and by a competition assay. From the cross resistance assay, all of the thymidine derivatives were found to be considerably less active than the parent podophyllotoxin and 4'-demethylepipodophyllotoxin molecules, while the 2'-deoxyadenosine derivatives were found to be completely inactive. The cross-resistance patterns of the thymidine derivatives suggests that these compounds display podophyllotoxin-like activity in vivo and show no VP16-213/VM26-like activity. Treatment of wild type cells with the active thymidine derivatives (compounds 1.2, 2.2, 2.3, and 2.4) increased the mitotic indices approximately ten-fold in a dose-dependent manner, which parallels the results of the dose-response curves in the initial cross-resistance assay. Furthermore, there was a marked increase in the levels of drug required to elevate the mitotic index in second-step mutants resistant to podophyllotoxin, again lending support to the initial results indicating that the cellular target of these compounds is tubulin. Only one compound (1.2), was found to out compete the binding of radiolabelled podophyllotoxin to purified calf brain tubulin. However, due to their low activity relative to podophyllotoxin, most of these compounds were insoluble at concentrations required to out compete the binding of radiolabelled podophyllotoxin. Molecular modelling studies have provided useful insights regarding the structure/activity relationships among the active and inactive nucleoside derivatives showing podophyllotoxin-like antimitotic activity. There appears to be steric limits for substituents attached at the C4 moiety that maintain the antimitotic activities of the parent molecules. The electrostatic potential and hydrophobic properties of these groups also seem to play a role in the degree of activity these compounds show, but remain unclear at this point. By simple comparison of the three-dimensional structures of these compounds there appears to be a very limited spacial and electrostatic requirement for the bulky glycosidic moiety attached to C4 which is essential for targeting VP16-213 and VM26 to DNA topoisomerase II. Thus, several important structural features which may distinguish the selectivity POD derivatives show for either tubulin or DNA topoisomerase II are described.</p> / Thesis / Master of Science (MSc)

Furo[3,4-c] pyranone is a unique bicyclic molecular structure found in bioactive sesquiterpene isobolivianine and in artificial cytotoxic stilbene derivatives. The structure of furo[3,4-c] pyranone is analogous to cyclopenta[c] pyran structure found in potent cytotoxic iridoids like catapol. Chiral substrates for intramolecular hetero-Diels-Alder (IHDA) reaction have been synthesized in yields ranging from 39% to 81%. These compounds undergo [4+2] cycloaddition via ambimodal/ bispericyclic process to give a mixture of furo[3,4-c] pyranone in yields ranging 40-70% and aryl tetralin lactone derivatives. Density functional theory (DFT) calculations have been performed to gain insight into the mechanism leading to the formation of these compounds. Podophyllotoxins are aryl tetralin lignans with great potential as lead compounds for anti-cancer and antibiotic agents. The bioactivity of these compounds is attributed to their unique stereochemistry that is not easy to synthesize. Chiral substrates for intramolecular dehydro-Diels-Alder (IDDA) reaction have been synthesized in moderate yields. These novel compounds have been used to synthesize aryl tetralin lactone cores by IDDA reaction. This work demonstrates the potential utility of asymmetric IHDA and IDDA reactions in the total synthesis of bioactive compounds featuring furo[3,4-c] pyranone core and aryl tetralin lactones found in anticancer podophyllotoxins.