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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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Secondary metabolites and their utilisation in Urtica dioica agg.
Pekařová, Michaela ; Urfus, Tomáš (advisor) ; Hojka, Jakub (referee)
Stinging nettle (Urtica dioica) is a very well-known taxon of genus Urtica, but still its taxonomy is not satisfactory resolved. An obvious reason of taxonomic obscurity of entire aggregate is enormous morphological variation. Polyploidization significantly contributes to this variability. Polyploidy probably is a reason of successful expansion of tetraploid cytotype in synanthropic habitats. Urtica dioica is rich in secondary metabolites, especially fatty acids, phenolic acids and flavonoids. Secondary metabolites are isolated for various purposes. They are widely applied especially in pharmacy and subsequently used for various medications. Despite the undoubtful economic importance a linkage between secondary metabolites composition and polyploidy has not been studied yet.
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The effect of pyrazine derivatives on secondary metabolites content in plant cultures in vitro II.
Hanzlíková, Tereza ; Tůmová, Lenka (advisor) ; Karlíčková, Jana (referee)
The main purpose of the theses was to find if the abiotic elicitor 2,4,6-trimethyl-N-(pyrazine- 2-yl)benzenesulfonamide has any influence on the secondary metabolites production in callus and suspension cultures of Hypericum perforatum L. The cultivation was taking place in the Murashige a Skoog (MS) nutrient medium enriched by the growth regulator - alpha-naphthyl acetic acid at the concentration of 1 mg.L-1 . The elicitor was added to the cultures at the three levels of concentration: c1= 100.0 mg/100 ml; c2= 10.0 mg/100 ml; c3= 1.00 mg/100 ml. The individual samples were taken after 6, 24, 48, 72 and 168 hours of the elicitor application. The control samples without the elicitor were taken after 6, 48 and 168 hours. The collected samples were dried and subsequently transformed into methanol extracts in order to determine secondary metabolites content (rutin, hyperoside and quercetin) by HPLC method. Release of these metabolites into nutrient media was also subject of this observation. The elicitation has influenced production of the secondary metabolites, particularly in callus cultures, wherein several statistically significant values, characterizing increase in their production, were measured. The highest content of rutin (0.169 mg.g-1 DW) was determined in callus culture after 168 hours when...
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Isolation of antimicrobial compounds from spent coffee grounds
Kurzová, Pavlína ; Veselá, Mária (referee) ; Kovalčík, Adriána (advisor)
Coffee grounds are one of the very valuable lignocellulosic wastes that have been able to be processed and used for isolated phenolic substances. Many phenolic substances isolated from lignocellulosic wastes have antimicrobial properties. Aim of this thesis is isolation phenolic substances from spent coffee grounds extract and their available antimicrobial properties. Two isolation ways were applied to receive phenolic substances from spent coffee grounds: 1) solvent extraction (hexane, 75% ethanol, 70% acetone, diethyl ether, and ethyl acetate) and 2) release of active substances by alcoholic fermentation. All isolated materials were characterized by the viewpoint of concentration of reducing sugars, polyphenols and flavonoids. Subsequently, their antimicrobial activity was determined by using agar diffusion and broth dilution methods. Two gram-positive bacteria (Bacillus subtilis and Micrococcus luteus), one gram-negative bacteria (Serratia marcescens) and two yeasts (Candida glabrata and Saccharomyces cerevisiae) were used for antimicrobial testing. High-performance liquid chromatography (HPLC) was used to identify phenolic substances in the extracts. First, the results showed that the isolated sample with the highest antimicrobial activity was 70% acetone extract. This extract contained chlorogenic acid, gallic acid, caffeic acid and coumaric acid according to HPLC. The ethyl acetate extract showed the lowest antimicrobial activity. Second, after lyophilization, the isolated materials also revealed high antimicrobial activity. The highest antimicrobial activity displayed the materials obtained by the extraction with 70% ethanol. This sample contained chlorogenic acid, gallic acid and caffeic acids. Next, samples with phenolic compounds were obtained by the alcoholic fermentation of spent coffee grounds. These samples showed similarly to the previous solution extracts significant antimicrobial activity. Interestingly, the unfiltered samples received directly after alcoholic fermentation also showed antifungal properties. The characterization of phenolic compounds by HPLC showed similarly as in previous examples that chlorogenic, caffeic and gallic acids were present in these samples.
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Isolation and characterizaation of oil from some types of coffee
Vašků, Barbora ; Hoová, Julie (referee) ; Skoumalová, Petra (advisor)
Coffee, extract, oil, phenolic compounds, flavonoids, antioxidant capacity, SPF, critical wavelenght.
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Study of natural compounds of elderberry flowers
Pončíková, Alžbeta ; Skoumalová, Petra (referee) ; Veselá, Mária (advisor)
The aim of this bachelor thesis was to determine the concentration of bioactive substances such as polyphenols and flavonoids and also the total antioxidant capacity in extracts from flowers of elder. The second part was the subject of an experiment to determine the antimicrobial activity. The theoretical part deals with the description of individual bioactive substances, the description of the elder and the bacteria used. In the experimental part, aqueous extracts were prepared, in which total polyphenols, flavonoids and oxidizing activity were determined. The results of the determination of polyphenols and flavonoids show that in order to achieve a higher content of flavonoids and polyphenols, it is more appropriate to use the highest possible initial temperature for leaching the base flower. No significant differences were found when comparing the antioxidant activity of solutions from the average values. The extracts did not show significant antimicrobial activity.
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Determination of athenticity of plant foods by molecular techniques
Plášková, Anna ; Mikulíková, Renata (referee) ; Němcová, Andrea (advisor)
The aim of presented diploma thesis was to determination of authenticity of fruit baby foods for early infant feeding using molecular methods. In the experimental part, isolation kit was used for isolation of plant DNA from fruits (strawberry, apricot, raspberry, apple) and from six commercial fruit products for children. Isolated DNA was characterized and verified using PCR methods with primers specific for plant rDNA (ITS2). Specific primer pairs were designed to amplify DNA for the detection of one fruit species. Primer specificity was assessed with four fruit species. A mixture of fruit puree from the two fruits was used to determine the sensitivity of the multiplex PCR assay. Six commercial fruit products were evaluated to verify the applicability of the multiplex PCR assay. The methodology of molecular detection of fruit DNA by qPCR and multiplex qPCR (duplex) includes approaches, which enable to detect two fruits (strawberry-raspberry, apricot-apple) in one reaction and thus reduces time and money requirements.
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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...
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Effect of dihydromyricetin on ethanol metabolism
Skotnicová, Aneta ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
Dihydromyricetin (DHM), also ampelopsin, is a flavonoid compound which exhibits a broad spectrum of positive effects on the human body. Herbal extracts containing this compound have been widely used in traditional Chinese medicine mainly for their hepatoprotective properties. DHM also helps with alcohol intoxication and reduces the signs of hangover or abstinence. Given the fact that the mechanism of DHM effects on the ethanol metabolism has not been clarified yet, the effect of dihydromyricetin on the expression and activity of alcohol dehydrogenase (ADH), one of the most important enzymes involved in ethanol metabolism, was therefore studied in this thesis. The cultivation conditions of primary hepatocytes which were isolated from unpretreated and ethanol-pretreated rats and subsequently exposed to EtOH and DHM were optimized. While determining the degree of cell damage caused by EtOH in the presence of DHM, no significant trend in the protective effect of DHM was found. On the other hand, the protective effect of ethanol in hepatocytes cultivated in EtOH and DHM was detected by technique of ELISA (the determination of alanine transaminase). The Western blot technique followed by immunodetection did not detect the induction of ADH expression in hepatocytes. Furthemore, the modulation effect of...
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Characterization of intermediates and waste products of some fermentation productions
Kilian, David ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
This bachcelor thesis was aimed on characterisation of intermediate and waste products of some fermentation productions. The theoretical part of this thesis is aiming on characterization of raw material and its fermentation, process of distillation, waste product and introduction of main active components of all these intermediates. In the experimental part were characterized intermediates and products regarding the contents of sugar, ethanol and polyphenol. All these extracts contain particular levels of polyphenol and sugar corresponding to individual phases of fruit distillates production. The chemical characteristics of extracts from different phases of fruit distillates production were compared. Parameters as total phenolics, flavonoids, glucose, fructose and ethanol were evaluated. Subsequently the changes between individual phases were described. In conclusion, a solution of further use of waste products formed during fruit fermentation process was suggested.
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