Orientadores: Susanne Rath, Sonia Claudia do Nascimento de Queiroz / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-27T01:49:14Z (GMT). No. of bitstreams: 1 Porto_RafaelSilveira_M.pdf: 1983268 bytes, checksum: f89fcb853911e559df9889c577be5a8e (MD5) Previous issue date: 2015 / Resumo: O Brasil é um dos maiores produtores de frutas do mundo, no entanto, estima-se que doenças pós-colheita possam gerar perdas de até 50% em sua produção. A forma mais comum de tratamento para essas doenças envolve a aplicação de fungicidas sintéticos. Contudo, nos últimos anos, a demanda por tratamentos alternativos tem crescido, com destaque para o uso de biopesticidas, produtos desenvolvidos a partir de plantas, microrganismos e insetos. Este trabalho teve como objetivo investigar a presença dos compostos bioativos (4Z)-lachnophyllum lactona, (4Z,8Z)-matricaria lactona e (2Z,8Z)-matricaria ester nos espécimes brasileiros da planta Conyza canadensis, bem como avaliar a atividade antifúngica dessas substâncias isoladas contra diversos fungos associados a doenças pós-colheita de frutas. Por cromatografia flash preparativa foi possível isolar a (4Z)-lachnophyllum lactona e a (4Z,8Z)-matricaria lactona a partir de extratos da planta obtidos com diclorometano. Os compostos foram caracterizados por GC-MS/MS, NMR 1H e 13C, 1H-1H COSY e 1H-13C HSQC. Foram realizados ensaios de difusão em disco com 10 fungos filamentosos causadores de doenças pós-colheita em frutas. Os fungos Aspergillus niger, Cladosporium spp. e Penicillium digitatum se mostraram susceptíveis ao tratamento e, para eles, a concentração mínima inibitória dos compostos variou de 32 a 64 µg mL-1. Também foi desenvolvido um método de extração empregando água quente pressurizada, no qual foram otimizados os parâmetros de temperatura (100 °C), tempo de ciclo (1 min) e número de ciclos (quatro). Com essa técnica foi possível obter um rendimento de 1,46 mg g-1 e 0,24 mg g-1 para a (4Z)-lachnophyllum lactona e a (4Z,8Z)-matricaria lactona, respectivamente. O extrato aquoso da Conyza canadensis pode ser aplicado diretamente nos frutos com a vantagem de não conter resíduos de solventes orgânicos tóxicos / Abstract: razil is one of the largest fruit producers in the world. Nevertheless, it is estimated that postharvest diseases can lead to losses of up to 50% in its production. The most common treatment for these diseases involves the application of synthetic fungicides. Nonetheless, in recent years, the demand for alternative treatments has increased, especially for the use of biopesticides, products developed from plants, microorganisms and insects. This study aimed to investigate the presence of the bioactive compounds (4Z)-lachnophyllum lactone, (4Z,8Z)-matricaria lactone and (2Z,8Z)-matricaria ester in Brazilian specimens of the weed Conyza canadensis, as well as to evaluate the antifungal activity of these isolated substances against several fungi associated with postharvest diseases of fruits. With the use of preparative flash chromatography it was possible to isolate (4Z)-lachnophyllum lactone and (4Z,8Z)-matricaria lactone from plant extracts obtained with dichloromethane. The compounds were characterized by GC-MS/MS, NMR 1H e 13C, 1H-1H COSY and 1H-13C HSQC. Disk diffusion assays were performed in order to investigate the activity of the isolated compounds against 10 filamentous fungi regarded as common postharvest pathogens of fruits. Aspergillus niger, Cladosporium spp. and Penicillium digitatum proved susceptible to the treatment and, for them, the minimum inhibitory concentration of the compounds varied from 32 to 64 µg mL-1. An extraction method using pressurized hot water was also developed, in which the parameters of temperature (100 ° C), cycle time (1 min) and number of cycles (four) were optimized. By using this technique, it was possible to obtain a yield of 1.46 mg g-1 and 0.24 mg g-1 for the (4Z)-lachnophyllum lactone and (4Z,8Z)-matricaria lactone, respectively. The aqueous extract of Conyza canadensis can be applied directly on fruits with the advantage of not containing residues of toxic organic solvents / Mestrado / Quimica Analitica / Mestre em Química

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.

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.

Flavonoids are natural compounds widely distributed in plant kingdom. They are inseparable from human diet because they showed a protective effect against cancer, stroke and coronary heart diseases related to their antioxidant properties. Therefore, rapid and efficient extraction procedure prior to chromatographic analysis is required. The liquid extraction at elevated temperature and pressure – Pressurized fluid extraction (PFE) and Pressurized hot water extraction (PHWE), present fast, effective and environmentally friendly extraction methods for the determination of flavonoids in plant materials. PFE and PHWE followed by reversed phase high-performance liquid chromatography with UV-visible detection have been utilized for the determination of a group of flavonoids (rutin, myricetin, quercetin, luteolin, apigenin and kaempferol) in the leaves and berries of less common plants. The matrices were extracted by methanol, ethanol and water at higher temperature 40-120 oC and pressure 15 MPa during 15 minutes. The obtained results were compared with conventional Soxhlet extraction and ultrasound-assisted extraction, the same solvents were used. The most effective extraction of selected flavonoids was achieved by PHWE. PFE showed the extraction yields comparable to those of the Soxhlet extraction, and the lowest extraction power was displayed by ultrasound-assisted extraction.

The determination of anthocyanins in red grapes and wines has been of increasing interest in the last years, as they play an important role in colour quality of red wines revealing also many human health beneficial effects. They contribute e.g. to the reduction of coronary heart disease, but exhibit also antimutagenic, anti-inflammatory, anti-carcinogenic and antioxidant properties. In this doctoral thesis, the complex study of grape skin alcoholic and water extracts, prepared from Alibernet and St. Laurent wine grape varieties is presented. Extracts were prepared from three different amounts (0,5; 1,0 and 1,5 g) of lyophilized grape skin powder using the Pressurized fluid extraction (PFE) and the Pressurized Hot Water Extraction (PHWE) at different temperatures ranging from 40 up to 120°C and pressure of 15 MPa. Methanol, ethanol and water were used as a solvents. Total phenolic compound content (TPC) of individual extracts was determined using the Folin-Ciocalteau assay and their tristimulus color values (CIE Lab) were estimated, using the UV-VIS spectrophotometer. The identification and quantification of anthocyanins by high-performance liquid chromatography with diode array detection (HPLC-DAD) was performed. In addition, pH values of all extracts were also measured using the combinated glass electrode. Antioxidant activity of extracts was tested by EPR spectroscopy in Fenton system (H2O2/Fe2+) generating reactive radicals (•OH, O2-•, •CH3) followed by spin trapping technique, using 5,5-dimethylpyrroline-N-oxide (DMPO) as spin trap. In addition, radical scavenging activity of extracts was assessed applying 2,2-diphenyl-1-picrylhydrazyl (•DPPH) free radical and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) cation radical (ABTS•+) assays. All the experimental data were processed with principal component analysis (PCA) and canonical discriminant analysis (CDA) to specify the optimum extraction conditions for extract preparation from the perspective of the potential further application of the extracts as food supplements or food colour enhancers. The results indicated that grape skins of both varieties are a promising source of anthocyanins with prospective application in food industry.

Amount of rutin and quercetin in bark and leaves from different type of Sambucus nigra was determined in this diploma thesis. Records of amounts were compared with amounts in significant sources of these flavonoids. Matters from raw matrix were obtained with pressurized solvent extraction (PSE) and pressurized hot water extraction (PHWE). There was use temperature 120°C, pressure 150 bar, three times 5-min cycles for PSE. There were used 80°C, 150 bar, three 5-min cycles for rutin extracted with PHWE and the same condition were used for extraction of quercetin only the temperature was changed to 100 °C. Analysis of rutin and quercetin were carry out high performance liquid chromatography (HPLC) with diode array detection (DAD), where was used SUPELCOSILTM LC 8DB column (5µm; 250 x 4.6 mm). Methanol: water: formic acid (36: 61.5: 2.5, pH 2.17 2.28) was used like a mobile phase. The flow of the mobile phase was set up 0.7 ml/min. Size of sample was 10 µl. The most amount of rutin was determined in the leaves of wild kind of Sambucus nigra with methanol extraction and hot water too. Extraction with PHWE was more effectively. There were determined 5.58 mg of rutin in one gram of sample from wild Sambucus nigra. There were determined the smallest amount of rutin in Sambucus nigra Körsör. There was found only 0.13 mg/g with extraction PHWE. The quercetin wasn’t determined in any Sambucus nigra leaves. There were found both of the flavonoids (rutin and quercetin) in the branch of Sambucus nigra. The rutin was determined in the small amount in wild kind but it was found in the important amount in Sambucus nigra cultivar. Whereas the quercetin was found in higher concentration than rutin when was finding the ideal conditions for extraction of branch. The quercetin was chosen like more important in light of amount. In the end the quercetin wasn’t detected in any Sambucus nigra cultivars. The most amount of quercetin was determined in wild sambucus nigra, 0.24 mg/g. The most amount of rutin was found in Sambucus nigra Albida, 2.33 mg/g.

Aronia (Aronia melanocarpa) is a berry fruit with distinctive sensory characteristics and health-promoting properties. Polyphenols are the main bioactive compounds found in aronia including natural pigments – anthocyanins. Bioactive compounds are usually obtained from natural materials by extraction, in this work, two methods of extraction were compared – maceration and PHWE. Based on the total phenolic content, maceration was chosen as a more suitable method and then was optimized with statistical model – Design of Experiment. Optimal conditions were set to temperature of 30 °C, extraction agent 50 % ethanol, solid-solvent ratio 10 g per 50 ml and time of extraction for 30 minutes. Extract obtained under these conditions was characterised by total phenolic content – 1441 ± 90 mg/100 g DW, total anthocyanin content 943 ± 8 mg/100 g DW and antioxidation activity by ABTS 24,78 ± 0,09 molTE/g. In total of 25 volatile compounds were indentified in the optimal extract with content > 0,5 %. Major part created aldehydes and esters, with the main compounds being benzaldehyde (32,25 %) and methyl hexanoate (21,37 %).