Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objectives of this work were divided in two experiments phases. In the first phase, it was studied the presence of trans-resveratrol in temperate climate fruits and the variation of their concentration in these fruits during storage period, as well the amount of total phenols and total anthocyanins. It was evaluated total phenols, total anthocyanins and trans-resveratrol, before and after cold storage and under controlled atmosphere; and after five days shelf-life at 20°C in Bruno kiwifruit; Araza and Yvahe strawberries; Black mulberry; Galaxi , Gala and Fuji apples; persimmon Fuyu ; Bleugem blueberry, Isabel , Merlot and Niagara Pink grapes. It was detected trans-resveratrol in natura in flesh Bruno kiwifruit; Araza and Yvahe strawberries; Black mulberry. In other fruits, it was detected transresveratrol in natura in the skin. The cold storage stimulated trans-resveratrol synthesis in Bleugem blueberry, and Isabel and Merlot grapes. In the second phase, it was evaluated the induction of trans-resveratrol synthesis and its possible action as phytoalexin in Gala and Fuji apples stored in controlled atmosphere were evaluated. Experiments were with the application of abiotic elicitors: ultraviolet irradiation, phosphite and acibenzolar-S-methyl, applied before controlled atmosphere storage, and intermittent ozone applied during storage of to Gala and Fuji apples. Rot was controlled in another experiment with the same elicitors. In fruit bark it was analyzed trans-resveratrol; total phenols and total anthocyanins, as well as the diameter of injury after inoculation of Penicillium sp. The abiotic elicitors were induced in 'Fuji' apples trans-resveratrol synthesis in the following sequence: Acibenzolar-Smethyl > phosphite ³ UV-C irradiation ³ ozone, but it wasn t effective on 'Gala' apple. There was no correlation between the syntheses of trans-resveratrol and rotting control, with exception of phosphite was control of rot in 'Gala'. / Os objetivos deste trabalho foram alcançados em duas etapas. Na primeira etapa, o objetivo foi avaliar a presença de trans-resveratrol em frutos de clima temperado e a sua variação de concentração nesses frutos durante o período de armazenamento, assim como a evolução dos polifenóis totais e antocianinas totais. Avaliou-se o conteúdo de polifenóis totais, antocianinas totais e trans-resveratrol antes e após o armazenamento refrigerado e em atmosfera controlada, e após cinco dias de exposição a 20°C em kiwi Bruno , morangos Araza e Yvahe , amora (Morus nigra), maçãs Galaxi , Gala e Fuji , caqui Fuyu , mirtilo Bleugem , uvas Isabel , Merlot e Niágara Rosada . Detectou-se o trans-resveratrol in natura em polpa de kiwi Bruno , morangos Araza e Yvahe e amora Preta . Nos demais fruto, detectou-se in natura trans-resveratrol na casca. O armazenamento refrigerado estimulou a síntese de trans-resveratrol em mirtilo Bleugem , uvas Isabel e Merlot . Na segunda etapa, o objetivo foi avaliar a indução da síntese de trans-resveratrol e sua possível ação como fitoalexina em maçãs Gala e Fuji , armazenadas em atmosfera controlada. Realizaram-se experimentos com aplicação de elicitores abióticos: irradiação ultravioleta, fosfito e acibenzolar-S-metil, aplicados antes do armazenamento em atmosfera controlada, e ozônio aplicado de forma intermitente durante o armazenamento de maçãs Gala e Fuji . Realizou-se o controle de podridão em outro experimento com os mesmos elicitores. Na casca dos frutos analisaram-se trans-resveratrol, polifenóis totais e antocianinas totais, e o diâmetro de lesão após inoculação por Penicillium sp. Os elicitores abióticos induziram a síntese de trans-resveratrol em maçã Fuji na seguinte ordem: Acibenzolar-S-metil > Fosfito ³ irradiação UV-C ³ ozônio, mas não apresentaram efeito em maçãs Gala , durante o armazenamento em atmosfera controlada. Os elicitores abióticos não apresentaram efeito no controle de podridão, com exceção do fosfito que controlaram a podridão em maçã Gala . Não houve correlação entre a síntese de trans-resveratrol e o controle de podridão.

Stilbene phytoalexins such as resveratrol, 1, and the arachidins, including arachidin-1,2, are naturally synthesised by peanut (Arachis hypogaea) plants. The peanut phytoalexins are polyphenolic compounds consisting of a stilbene backbone, with a number of derivatives also possessing a prenyl moiety. These distinctive phytoalexins have gained attention, as they exhibit various biological activities, for instance arachidin-1, 2, has been reported to be more potent than resveratrol, 1, in the inhibition of lipopolysaccharide-induced expression of cyclooxygenase-2 (COX-2) and COX-2 mRNA, in vitro at doses that were low in cytotoxicity. Additionally the various arachidins have recently been shown to exhibit their anti-inflammatory properties, through the inhibition of a number of inflammatory mediator pathways. In this work, various routes into the synthesis of arachidin-1, 2, are described, via use of the Horner-Wadsworth-Emmons (HWE) reaction. Three different methodologies were explored, the first approach involving silyl ether (TIPS or TBDMS) protected benzaldehydes, proved unsuccessful due to cleavage of the silyl ether protecting groups, in basic and/or acidic conditions. This led to an alternative approach, whereby formation of the stilbene backbone proceeded via the regioselective demethylation of an acetal in the presence of sodium metal, subsequent electrophilic substitution using iodomethane and finally acetal hydrolysis of the acetal, gave the isolated aldehyde in moderate yield (52 %). Coupling of the aldehyde with the substituted benzylphosphonate, via the HWE reaction gave the desired trans-stilbene in good yield (86 %), however incorporation of the prenyl side chain proved to be challenging via the Wohl-Ziegler bromination. Further adaptation of the aforementioned route, whereby alkylation using diethyl iodomethylphosphonate, enabled the incorporation of the prenyl moiety and the subsequent construction of the trans-stilbene backbone, gave the 4-(3-methyl-but-1- enyl)-3,5,3',4'-tetramethoxystilbene, 3, albeit in poor yield (47 %). The final step involving demethylation using BBr3 gave arachidin-1, 2, also in poor yield (30 %), nevertheless this approach has been proved to be a successful route for the total synthesis of arachidin-1, 2, however optimised studies are required in order to obtain the desired compound in quantitative yields. Synthetic analogues of resveratrol, 1, are also known for their biological activities, including anti-inflammatory and chemopreventative properties. Recently, the anti-proliferative activity of a number of stilbenesulfonamides, against the National Cancer Institute's 60 (NCI-60) human tumour cell line has been reported. Furthermore, the anti-inflammatory effects of novel heterocyclic methylsulfone and sulfonamide analogues, via inhibition of the COX-2 protein have also been published, however both synthetic routes described require a total of six or seven steps, from the sulfanilamide and are limited to the synthesis of primary sulphonamides (SO2NH2). In this work, an efficient three step synthesis has been designed and successfully implemented, proceeding via chlorosulfonation of diethyl benzylphosphonate, to form the sulfonyl chloride intermediate. Aminolysis of the sulfonyl chloride intermediate was then performed, using a range of primary, secondary and cyclic alkyl amines, as well as aromatic amines; including ammonia, dimethylamine, morpholine and diphenylamine. Finally, formation of the stilbene backbone with various substituted aldehydes, via the HWE reaction offered a short, versatile and alternative route to the synthesis of novel primary, secondary and tertiary trans-stilbene benzenesulfonamides and heterocyclic analogues, in yields of 42 - 100 %. The activity of a selection of the synthesised stilbene benzenesulfonamides was evaluated against the human lung adenocarcinoma epithelial cell line (A549). Amongst the compounds tested, analysis of the data showed that the novel analogue, 4, was found to be the most potent compound, with a GI50 of 0.1 μM. Comparison with the previously published data found analogue, 4, to be approximately 500-fold more potent than the lead compound resveratrol, 1, (GI50 = 51.64 μM) and approximately twice as potent than 5-fluorouracil (GI50 = 0.189μM), a chemotherapy drug used to treat various forms of cancer 8. Overall, these results demonstrate that the total synthesis of trans-arachidin-1, 2, can be achieved via a five step methodology. A versatile route to the synthesis of novel stilbene benzenesulfonamides has also been successfully achieved, amongst the compounds synthesised one appears to show promising anticancer activity, and warrants further investigation (i.e. in vitro studies using other cancer cell lines, and the synthesis of additional compounds using analogue, 4, as a lead compound).

Magister Scientiae - MSc / This investigation focused on Rhoicissus tomentosa, belonging to the family, Vitaceae in an attempt to assess the phytochemistry of this plant which is widely used by traditional healers in South Africa to ensure the safe delivery during pregnancy and childbirth (Hutchings et al., 1996). / South Africa

Made available in DSpace on 2017-07-10T17:40:55Z (GMT). No. of bitstreams: 1 Edilaine_D_V_GoncalvesTrevisoli.pdf: 2707364 bytes, checksum: 7b7a664f9727d5909c470f377ee155e8 (MD5) Previous issue date: 2016-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The induction of resistance in plants to pathogens is an alternative method of disease control, wich involves activation of plant resistance mechanisms such as induction of phytoalexins. The elicitors molecules are able to induce and activate those responses, and therefore, techniques have sought to isolate and characterize fractions with elicitor character. The study aimed to purify, through ion exchange chromatography and gel filtration chromatography, eliciting molecules from pathogenic nematodes, and test them in phaseolin induction in beans hypocotyls beans and gliceolin in soybean cotyledons. The buffer solution Tris HCl 0.05 M (pH 6.8) was used as control and the acibenzolar-S-methyl (50 mg a.i. L-1) and Saccharomyces cerevisiae (20 mg mL-1) were used as induction standard treatments. Ion exchange chromatography (IEC) and gel filtration chromatography (GC) were performed to separate fractions with eliciting power from 500 female nematodes of Meloidogyne incognita and Meloidogyne javanica. For purification of elicitors from Meloidogyne javanica, through IEC, six glycidic fractions and six glycoproteins were obtained. These were purified on GC, obtaining sixty-three fractions. They have been classified according to their nature, as twenty-six glycidic and thirty-seven glycoprotein with molecular weights ranging from 29.19 to 2989.25 kDa. Regarding the elicitors purification of Meloidogyne incognita through IEC, nine glycidic and five glycoprotein fractions were obtained. From these fractions, a total of fifty-eight fractions was obtained through GC, twenty-five glycidic and thirty-three glycoprotein with molecular weights ranging from 37.42 to 200.32 kDa. From the fractions purified from Meloidogyne javanica eight had inducing potential of phaseolin. For gliceolin fifteen fractions showed inducing effect. Regarding the fractions purified from Meloidogyne incognita, no fraction has inductive potential of phaseolin superior to the standard treatment. However, twenty-two fractions suppressed phytoalexin inducing activity. For gliceolin ten fractions induced the same, whereas, twenty-three fractions suppressed the induction of gliceolin. Chromatography was efficient in the purification of elicitors compounds. Compounds with suppressing characteristics of gliceolin and phaseolin were checked in bioassays. For those fractions obtained through IEC, and then submitted to GC that did not induce phytoalexin, it is suggested that molecules need to act together to have elicitor effect and thus induce defense response in the plant / A indução de resistência em plantas contra patógenos é um método de controle alternativo de doenças, e que envolve a ativação dos mecanismos de resistência da planta, como a indução de fitoalexinas. As moléculas eliciadoras possuem a capacidade de induzir e ativar tais repostas, e assim sendo, técnicas têm buscado isolar e caracterizar frações com caráter eliciador. O trabalho teve por objetivo purificar, por cromatografia de troca iônica cromatografia de filtração em gel, moléculas eliciadoras a partir de nematoides fitopatogênicos, e testá-las na indução de faseolina em hipocótilos de feijoeiro e gliceolina em cotilédones de soja. O tampão Tris HCl 0,05 M (pH 6,8) foi utilizado como tratamento controle e o acibenzolar-S-metil (50 mg i.a. L-1) e o Saccharomyces cerevisiae (20 mg mL-1) foram utilizados como tratamento padrão de indução. Cromatografia de troca iônica (CTI) e cromatografia de filtração em gel (CFG) foram realizadas para separar frações com poder eliciador a partir de quinhentas fêmeas de nematoides de Meloidogyne incognita e Meloidogyne javanica. Para a purificação de eliciadores a patir de Meloidogyne javanica, por CTI, foram obtidos seis frações glicídicas e seis glicoproteicas. Estas, por sua vez, foram purificadas em CFG, sendo obtidos no total sessenta e três frações. As mesmas foram classificadas de acordo com sua natureza, sendo vinte e seis glicídicas e trinta e sete glicoproteicas, com massas moleculares variando de 29,19 a 2.989,25 kDa. Em relação a purificação de eliciadores de Meloidogyne incognita por CTI, foram obtidos nove frações glicídicas e cinco glicoproteicas. A partir destas, foram obtidos por CFG um total de cinquenta e oito frações, sendo vinte e cinco glicídicas e trinta e três glicoproteicas, com massas moleculares variando de 37,42 a 200,32 kDa. Das frações purificadas a partir de Meloidogyne javanica oito apresentaram potencial indutor de faseolina. Para gliceolina quinze frações mostraram efeito indutor. Em relação as frações purificadas a partir de Meloidogyne incognita, nenhuma fração apresentou potencial indutor de faseolina superior ao tratamento padrão. Entretanto, vinte e duas frações suprimiram a atividade de indução de fitoalexina. Para gliceolina dez frações induziram a mesma, enquanto que, vinte e três frações suprimiram a indução da gliceolina. A cromatografia foi eficiente na purificação de compostos eliciadores. Compostos com características supressoras de gliceolina e faseolina foram verificadas nos bioensaios. Para aquelas frações obtidas por CTI e posteriormente submetidas a CFG que não induziram fitoalexina, sugere-se que as moléculas necessitam atuar juntas para haver efeito eliciador e assim induzir a resposta de defesa no vegetal