Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Alena Novýsedláková Supervisor: PharmDr. Jana Pourová, PhD. Title of Thesis: In vitro effects od selected isoflavonoids on isolated rat aorta Abstract: The aim of this study was to determine whether the selected isoflavonoids are able to vasodilate aorta, what structural features might be responsible for a relaxing activity and estimate the mechanism of action. The experiment we carried out the classical in vitro method on the isolated rat aorta, and the results evaluated by the computer program GraphPad. Only glycitin did not show statistically significant relaxation of blood vessels, which can be attributed to ineffective glycosidic form. Other izoflavonoids- glycitein, daidzein and tectorigenin seem to be promising in terms of potential vasodilatation. Further studies would be needed to verify also the in vivo effects. Likely structural features include the hydroxy group at 7-position and a methoxy group at position 8 of the core od isoflavonoids. The mechanism of action is not yet known, but most likely appears endothelium-dependent mechanisms, alpha 1 receptor antagonism and agonism of muscarinic M3 receptor. It may be also involved in the effect of the antioxidant and...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Bc. Lenka Jančíková Supervisor: PharmDr. Jana Pourová, Ph.D. Title of diploma thesis: Cardiovascular effects of isoflavonoids Background: The aim of this thesis is to summarize existing findings about natural vegetable substances known as phytoestrogens-isoflavones and to map the results of latest studies focused on cardiovascular effects of isoflavones. Main findings: Available data suggest that isoflavones and their metabolites have positive effect on human organism. These include antiaterosclerotic, antimenopausal and anticarcinogenic effects and a positive effect on osteoporosis. Positive effect on cardiovascular system includes antihypertensive, anti-inflammatory, anti-angiogenic, antiproliferative and antiaggregating effects. On the other hand, there are studies that point out possible risks related to mainly long-term use of isoflavones. Nevertheless, even though the findings of cardiovascular effects are not always consistent, most of them confirm positive effects of isoflavones on cardiovascular system. Conclusion: Isoflavones and their metabolites are a very interesting group of substances with natural origin and carry a potential for possible development of new drugs. Therefore, they...

Elicitation is one of possible methods for increasing the secondary metabolites production in plant cell cultures. This paper explores the potential effect of methylviologene on isoflavonoids production in Genista tinctoria L. suspension and callus cultures. Schenk and Hildebrandt nutrient medium was used with the addition of growth regulators 2,4- D in concentration of 5 mg/L and kinetin of 1 mg /L of medium. Elicitor was added in the form of ethanol solution at concentration of 2.19·10-2 mol l-1 , 2.19·10-3 mol l-1 , 2.19·10-4 mol l-1 . Samples were taken after 6, 12, 24, 48, 72 and 168 hours of elicitor exposure. Control samples were collected at 6, 24, 72 and 168 hours. The isoflavonoid content in suspension and callus cultures and in nutrient medium was evaluated by high performance liquid chromatography. The biggest increase of isoflavonoid content in callus culture was reached in daidzein content after 48 hours elicitor treatment in concentration of 2.19·10-3 mol l-1 (8.5 mg/g DW) and 2.19·10-4 mol l-1 (1.6 mg/g DW). The production of genistin, genistein and formononetin was slightly increased or zero compared to controls. Biochanin A was almost absent in the samples. The highest level of isoflavonoids was measured in the suspension culture after 48 hours elicitor application in the...

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Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Bc. Pavlína Šramarová Diploma thesis supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of thesis 2015/2016: Metabolism of isoflavonoids in human organism, pp. 54 Isoflavonoids are subgroup of flavonoids, belonging to the phytoestrogenic compounds. These compounds occur especially in leguminous, mainly in soya (Glycine max (L.) Merr). They have a positive impact on human health, especially for its estrogenic effects, due to which are applied in the fight against osteoporosis, cardiovascular diseases, atherosclerosis and for moderation of menopausal symptoms. Furthermore they exhibit antibacterial activity and protect cells against damage of DNA. This thesis deals with their metabolism and action in human and animal organism, also with the influence of intestinal microflora to transform these isoflavonoids into metabolites, as well as the effects of these metabolites. There are also mentioned these isoflavonoids: daidzein, genistein, biochanin A, formononetin, calycosin, prunetin, puerarin, cladrin, tectorigenin and kakkalid. Key words: isoflavonoids, metabolism, human organism

1 Abstract Veronika Machová Effect of 2-benzylthiopyridine-4-carbothioamide derivatives on isoflavonoids and flavonoids accumulation in suspension culture of Trifolium pratense L. The paper examinate the effect of three concentration of 2-(2-fluoro-6- nitrobenzylsulfanyl) pyridine-4-carbothioamide on the production of flavonoids and isoflavonoids by the Trifolium patense L. (variety DO-8, variety Tempus). The culture were cultivated in the Gamborg nutrient media with addition of 2 mg·l-1 2,4- dichlorophenoxyacetic acid a 2 mg·l-1 6-benzylaminopurine, at the temperature of 25řC, 16-hr light/8-hr dark period. The elicited and the inspection samples underwent the photometric determination of flavonoids in accordance with the Czech Pharmacopoeia 2009 ant the determination of isoflavonoids via the HPLC method. The results show that the maximum content of flavonoids in suspension cultures of Trifolium pratense L. (variety DO-8) was caused by 48-hour elicitation by 100 µmol·l-1 solution of 2-(2-fluoro-6-nitrobenzylsulfanyl) pyridine-4-carbothioamide, when the a statistically significant increase in production was 93% compared to control cultures. The maximum content of flavonoids in variety Tempus was caused by 48-hour elicitation by 10 µmol·l-1 solution of 2-(2-fluoro-6-nitrobenzylsulfanyl) pyridine-4-...

Isoflavonoids are a class of plant secondary metabolites which have multiple biological roles in plants as pest feeding deterrents, phytoalexins and signals to rhizobial microbes. Some isoflavonoids, or their breakdown products, are estrogenic when ingested by animals, and pastures with high levels of the isoflavonoid formononetin can cause sterility in ewes. White clover has low levels of isoflavonoids and is susceptible to pests like the clover root weevil. The overall aim of this project was to test whether isoflavonoids could be manipulated in white clover through metabolic engineering. The genes of the key isoflavonoid biosynthesis enzymes have been cloned from a range of legumes and three major genes, chalone reductase (CHR), isoflavone synthase (IFS) and isoflavonoid O-methyltransferase (IOMT), were cloned from white clover in this study. The white clover IFS2_12 gene was expressed in transgenic tobacco. Genistein, an isoflavonoid that is not naturally present in tobacco, was detected in the IFS-expressing tobacco, thus confirming the functionality of the IFS2_12 gene. Tobacco plants were transformed with ANT1, a transcription factor that induces the production of anthocyanins that share precursors with the isoflavonoid biosynthesis pathway. When IFS was expressed in red tobacco leaves, where anthocyanin biosynthesis was occurring, the levels of genistein were greater than in anthocyanin-free green leaves. White clover was transformed to overexpress the cloned IFS2_12 gene and some transformants had greater levels of IFS gene expression, up to 12.9 times the average wild type level. However, these transformants did not produce formononetin levels greater than the wild-type. A gene fusion of alfalfa chalcone isomerase (CHI), which produces the precursors naringenin and liquiritigenin, and soybean IFS, which converts the precursors to genistein and daidzein, respectively, was received from the Noble Foundation. Transgenic white clover plants expressing IFS/CHI were produced using a novel method that also regenerated wild-type clones of the transgenic plants. When compared with their wild-type clones, two IFS/CHI transformants produced higher levels of formononetin, thus supporting the suggestion that isoflavonoid levels can be increased in white clover through overexpression of isoflavonoid biosynthesis genes.

Charles University in Prague Faculty of Pharmacy in Hradci Králové Department of Pharmaceutical Botany and Ecology Candidate: Kamila Ambrožová Supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of Thesis: Copper reducing activity of isoflavonoids Copper in our body appears only in trace amounts. Despite this fact is an important component of many enzymes, which are responsible for right function of the whole organism. The most important are antioxidant activity, collagen and conective tissue synthesis, formation of pigmentation and cell respiration. Main target of the organism is control the homeostasis of copper, because excess and deficiency of the element could be pathological. Increase level of copper can cause many of civilization diseases. Isoflavones are the plant metabolites of polyfenolic structure which can positively affect different processes in our body. Research is dedicated to anti-inflammatory and estrogenic effects, antioxidant and anticancer aktivity. The elimination and inactivation of radical elements is performed becase of ability of isoflavones to reduce transition elements or chelate them. The aim of this thesis was to determine the reducing activity of seven isoflavonoids - daidzein, genistein, biochanin A, genistin, glycitein, formononetin and prunetin. The highest reduction...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Hana Novotná Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: Secondary metabolites of plant tissue culture of Trifolium pratense L. Explant cultures are perspective sources of secondary metabolites. Nevertheless production of flavonoids and isoflavonoids by the suspension culture of Trifolium pratense L. is not high. Elicitation is one of the methods used to enhance the biosynthesis of secondary metabolites. Elicitation induces physiological changes, stimulates defensive or stress-induced reactions in plants and subsequently triggers the synthesis of secondary metabolites. The objective of this study was to observe the influence of two elicitors - abscisic acid and ascorbic acid - on the production of flavonoids and isoflavonoids by the Trifolium pratense L. suspension culture (Sprint variety). The culture was cultivated in Gamborg medium to which 2 mg.l-1 of 2,4- dichlorophenoxyacetic acid and 2 mg.l-1 of 6-benzylaminopurine were added, at the temperature of 25 řC and 16 hours light / 8 hours dark period. The best elicitation effect of abscisic acid on the production of flavonoids and isoflavonoids was observed after a 6-hour application of the highest 500 µmol.l-1...

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.