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The effect of methylviologen on secondary metabolites production in in vitro culture of Fagopyrum esculentum, variety Spačinski
Vaicová, Nicole ; Tůmová, Lenka (advisor) ; Spilková, Jiřina (referee)
Plants are an important source of secondary metabolites, which are a valuable natural substance used in many fields. One way to increase their production is by the elicitation method. In this paper the effect of abiotic elicitor methylviologene in three different concentrations was studied on the rutin production in callus and suspension culture of Fagopyrum esculentum Moench variety Spačinki. The cultivation was carried out on Murashige and Skoog nutrient medium with the addition of a 1 mg/l 2,4- dichlorophenoxyacetic acid as growth regulator. Samples were taken at regular time intervals after 6, 12, 24, 48, 72 and 168 hours of elicitation. The rutin content was analyzed by HPLC. The maximum rutin production (0.3 mg/g DW) was recorded in the callus culture after 48 hours of methylviologene treatment at a concentration of 2.1929.10-4 mol/l. No increase in rutin content after methylviologene elicitation was observed in the suspension culture. The study also included monitoring of the rutin release into nutrient medium, but this was not demonstrated.
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The effect of methylviologen on secondary metabolites production in in vitro culture of Fagopyrum esculentum, variety Pyra
Zajačíková, Pavla ; Tůmová, Lenka (advisor) ; Kašparová, Marie (referee)
Elicitation is one of the methods used for increasing the production of secondary metabolites in vitro cultures. The aim of this work is to evaluate the effect of abiotic elicitor methylviologen (paraquat) on the production of flavonoids in callus and suspension cultures of Fagopyrum esculentum Moench., variety Pyra. The cultures were cultivated in Murashige and Skoog nutrient medium with addition of 2,4-D in concentration of 1 mg/l as a growth regulator. Elicitor was added as a solution in three different concentrations (c1 = 2.1929 · 10-4 mol/l, c2 = 2.1929 · 10-3 mol/l and c3 = 2.1929 · 10-2 mol/l). The effect of elicitation on rutin production was monitored in six time intervals: 6, 12, 24, 48, 72 and 168 hours. The rutin content was determined by HPLC analysis. No rutin was produced in callus and suspension cultures without the presence of elicitor. Even after the elicitation, there was no statistically significant increase in the production of rutin. The maximum rutin content was detected in the suspension culture after 12 hours of elicitor treatment in c2 concentration, the content was 0.1 mg/g DW. The release of rutin into the nutrient medium was also not observed.
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The effect of methylviologen on secondary metabolites production in in vitro culture of Fagopyrum esculentum, variety Bambi
Vlachová, Veronika ; Tůmová, Lenka (advisor) ; Karlíčková, Jana (referee)
10 ABSTRACT The subject of this study was to evaluate the efect of abiotc elicitor on rutn producton in callus and suspension cultures of buckwheat. The cultvar of buckwheat used for this research was Fagopyrum esculentum Moench var. Bambi, cultvated in Murashige and Skoog nutrient medium with the additon of growth regulator 2,4-dichlorfenoxyacetc acid (2,4-D) in concentraton of 1 mg/l. The elicitor used in this study was a soluton of methylviologen, 1 ml of it was added to the cultures in three diferent concentratons: c1 = 100.0 mg/100 ml, c2 = 10.0 mg/100 ml and c3 = 1.0 mg/100 ml. The elicitor was afectng the cultures for 6, 12, 24, 48, 72 or 168 hours. Afer the defned period of tme, cultures were collected, dried out and stored for further analysis of rutn content. To control samples (without elicitor treatment) 1 ml of ethanol 96% was added and they were collected afer 6, 24, 72 or 168 hours. Releasing of rutn into the nutrient medium was also investgated. Rutn content in each sample of cultures and in each sample of nutrient medium was later determined by HPLC. Any signifcant increase in the producton of rutn was not observed in this study. The maximum amount of rutn detected was 0.1 mg/g DW, thus the lowest quantty detectable, and was found in suspension cultures in three cases, afer the additon of...
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Biologically active phenolic compounds in small fruit.
LAXOVÁ, Lenka
This master´s thesis focuses at bioactive compounds in edible berries during its culinary processing. Elderberries (Sambucus nigra L.) were selected as a representative of edible berries variety. Elderberry is known for presence of significant bioactive compound content and usage in traditional medicine. Elderberries were processed acording to common culinary recipes. Amount od selected bioactive polyphenolic compounds (chlorogenic acid, quercetin, rutin, anthocyanes) was detected accordingly. Additionally content of ascorbic acid was determined. On the basis of the experimental determination, it was found that anthocyanes and rutin are the most common compounds found in elderberries. All analytes in juices and sirups gradually decreased during the treatments and long-term storage. On the contrary, the content of quercetin increased with gradual modifications and storage. The highest decrease was observed for vitamin C content. The lowest values in the products from elderberries were reached by quercetin and ascorbic acid.
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Enzymatic and Metabolic Transformation of Silybin and its Congeners
Purchartová, Kateřina ; Křen, Vladimír (advisor) ; Macek, Tomáš (referee) ; Vítek, Libor (referee)
Natural flavonoids and flavonolignans feature beneficial properties for living organisms such as antioxidant and hepatoprotective effects, anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activities. Their metabolism in mammals is complex, the exact structure of their metabolites still remains partly unclear and the standards are usually not commercially available. Hence, this project focused on the preparation of potential and defined biotransformation Phase II sulfated metabolites of silymarin flavonolignans: silybin, 2,3-dehydrosilybin, isosilybin, silychristin, silydianin and flavonoids quercetin, taxifolin, rutin and isoquercitrin. Pure sulfated derivatives were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and aryl sulfotransferase from rat liver. Using heterologously expressed PAPS (3'-phosphoadenosine-5'-phosophosulfate) - independent arylsulfotransferase from Desulfitobacterium hafniense and cheap p-nitrophenyl sulfate as sulfate donor, sulfated flavonolignans and flavonoids were obtained in high yields. Silymarin flavonolignans afforded exclusively monosulfates at the position C-20 (C-19 in the case of silychristin), except 2,3-dehydrosilybin that yielded also the 7,20-O-disulfated derivative. Isoquercitrin and rutin were selectively sulfated...
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Enzymatic and Metabolic Transformation of Silybin and its Congeners
Purchartová, Kateřina
Natural flavonoids and flavonolignans feature beneficial properties for living organisms such as antioxidant and hepatoprotective effects, anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activities. Their metabolism in mammals is complex, the exact structure of their metabolites still remains partly unclear and the standards are usually not commercially available. Hence, this project focused on the preparation of potential and defined biotransformation Phase II sulfated metabolites of silymarin flavonolignans: silybin, 2,3-dehydrosilybin, isosilybin, silychristin, silydianin and flavonoids quercetin, taxifolin, rutin and isoquercitrin. Pure sulfated derivatives were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and aryl sulfotransferase from rat liver. Using heterologously expressed PAPS (3'-phosphoadenosine-5'-phosophosulfate) - independent arylsulfotransferase from Desulfitobacterium hafniense and cheap p-nitrophenyl sulfate as sulfate donor, sulfated flavonolignans and flavonoids were obtained in high yields. Silymarin flavonolignans afforded exclusively monosulfates at the position C-20 (C-19 in the case of silychristin), except 2,3-dehydrosilybin that yielded also the 7,20-O-disulfated derivative. Isoquercitrin and rutin were selectively sulfated...
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Content of rutin in the biological waste material from plantations of elderberry
Boháčová, Hana ; Vránová, Dana (referee) ; Vespalcová, Milena (advisor)
This thesis is focused on determining the concentration of rutin in twigs elderberry (Sambucus nigra L.). The theoretical part provides an overview of the properties, occurrence and use of elderberry in folk medicine, medicine and food. Furthermore, there are the most important substances contained in elderberry, especially flavonoids, including rutin and quercetin as well. There are briefly described their properties, biological effects and possibilities of determination. The content of the experimental part of the thesis was developed cheap extraction methods to obtain the maximum yield of dried twigs rutin elderberry. Rutin content was observed in twigs cultivated elder varieties and wild elderberry. The identification and quantification of rutin in extracts was optimized and validated method of high performance liquid chromatography (HPLC). As a comparative method to develop extraction was used a pressurized hot water extraction (PHWE). There were tested three particle sizes of shredded twigs. The particles were extracted differently hot water varying duration. Most rutin was found in samples milled at 4 mm particles. The optimum time and temperature of extraction was 7 hours at 80°C. The highest amount of rutin in bred elderberry was determined in a sample Mladší 2014, digestion using 1,49 ± 0,004 mg.100g-1 and method PHWE 5,75 ± 0,015 mg.100g-1. There was determined the most rutin in a sample Září 2014 in the wild variant of elderberry. By digestion of ground twigs was obtained 0,90 ± 0,022 mg.100g-1 of rutin, using PHWE 2,98 ± 0,022 mg.100g-1.
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Assessment of rutin in elderberries
Kaňová, Kateřina ; Hrstka, Miroslav (referee) ; Vítová, Eva (advisor)
This work is focused on determination of the concentration of rutin in the fruits of elderberry (Sambucus nigra L.). The theoretical part provides an overview of the properties, occurrence and use of elderberry in natural healing, medicine and food industry. Furthermore, important substances contained in elderberry, especially flavonoids, including rutin, are mentioned. Properties, biological effects and the possibility of determination of rutin are briefly described. In the experimental part the optimization and validation of the method of high performance liquid chromatography (HPLC), which is used for identification and quantification of rutin in fruits of 18 elderberry species, was performed. The pressurized hot water extraction was used for the preparation and solid phase extraction (SPE) for purification of the sample. The largest amount of rutin was found in a variety of elderberry Albida, 6,70 mg per 1 g of dry matter. High concentrations of rutin included varieties Haschberg, Sambu, Pregarten, Sambo and Sampo. On the other hand, the lowest concentration was found in variety Aurea, 1,24 mg.g-1. Finally, rutin content in elderberries was compared with the content of rutin in elder leaves and branches andwith significant sources of rutin – buckwheat and rue.
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Content of rutin in selected elderberry cultivars
Píšťková, Magdalena ; Hrstka, Miroslav (referee) ; Vespalcová, Milena (advisor)
The aim of this diploma thesis was to determine the rutin content in springs and leaves of 19 elderberry species (Sambucus nigra L.) with use of liquid chromatography. Furthermore the rutin content was determined in its significant sources – rue and buckwheat. The observed values were compared in order to answer the question whether it is useful to use the elderberry spring and leaves as a new source of rutin. Theoretical part of the thesis describes elderberry in general, its chemical composition and its usage in medicine. Following part concentrates on important substances contained in elderberry – flavonoids. Also principles of extraction techniques and HPLC are discussed. In practice the rutin was gained from plant matrix with use of pressurized hot water extraction (PHWE) which was performed in three five minutes long cycles. Conditions during the PHWE were set up to 80 °C and 150 bars. Obtained extract was purified with use of SPE columns and analyzed by HPLC with DAD detector (wave length – 360 nm). HPLC was performed with SUPELCOSILTMLC-8DB column. The mobile phase was composed of MeOH, H2O, and HCOOH in ration of 36:61,5:2,5. The set up flow of mobile phase was 1 ml/min. In the leaves the highest rutin content was determined in Pregarten – 26,34 ± 0,50 mg/g. On the other hand the lowest rutin content in leaves was determined in Körsör – 3,74 ± 0,13 mg/g. In springs the obtained results were as follow. The highest content was determined in Samyl – 4,87 ± 0,01 mg/g and the lowest content was determined in Heidegg 13 springs – 0,56 ± 0,01 mg/g. Also the HPLC detection limit – 6,3 . 10-5 mg/ml and quantitation limit – 2,1 . 10-4 mg/ml were determined for rutin.
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