Abstract Flavonoids are a class of secondary metabolites in plants that are involved in many important functions. Various flavonoid compounds have also been reported to be beneficial for human health. Bilberry (Vaccinium myrtillus L.) is the characteristic field layer species in boreal forests and the fruits of bilberry are rich in anthocyanin pigments, a subclass of flavonoids. In the present work, flavonoid biosynthesis was examined in different tissues of bilberry. The focus was on the developing fruits of wild type and natural color mutants of bilberry, and on effect of solar radiation on flavonoid biosynthesis in bilberry leaves. For the isolation of RNA for gene expression analysis, a method was optimized for different tissues of bilberry. The cDNA fragments of five genes from the flavonoid pathway, coding phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavanol 4-reductase and anthocyanidin synthase, were isolated from bilberry using polymerase chain reaction technique, sequenced, and labelled with dioxigenin-dUTP label. These homologous, bilberry-specific probes were used for determining the expression of the flavonoid pathway genes in bilberry fruits, flowers and leaves with a modified non-radioactive method developed in the course of the study. The anthocyanins, catechins, proanthocyanidins, flavonols and hydroxycinnamic acids in fruits, leaves and different fractions of bilberry were identified and quantified with high-performance liquid chromatography combined with a diode array detector and mass spectrometer. The results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. A correlation between flavonol and anthocyanin biosynthesis was detected in bilberry and also in previous literature collected from flavonol and anthocyanin analyses from other fruits. Accordingly, models for the connection between flavonol and anthocyanin synthesis in fruit species were suggested. Activation of the expression of flavonoid pathway genes and accumulation of flavonoids and hydroxycinnamic acids was detected in leaves growing under direct solar radiation, compared to the shadow leaves of the same plants. Based on the results, it is suggested that cyanidin of anthocyanins and flavonol quercetin play a predominant role in the defence against high solar radiation in Vaccinium leaves. The results give new information about the biosynthesis of flavonoids in bilberry at the gene level, in addition to the information of the composition and content of flavonoids during fruit development and in different parts of the bilberry plant. Also, new information was obtained of the roles of flavonoids in protecting plants from excess solar radiation.

Ftalocianina de alumínio-cloro (AlClPc) é um fotossensibilizador de segunda geração em terapia fotodinâmica (TFD) caracterizado por seu caráter anfifílico e tendência de auto-agregação em meio aquoso, o que prejudica seu potencial de aplicação. O aCHC é um substrato de transportadores de monocarboxilato (MCT) superexpresso em células de MCF-7. Objetivando a solubilização da AlClPc e aumento de internalização em tecidos neoplásicos nos propomos aqui o uso de DSPC e DOPC em diferentes proporções para formar vesículas lipidicas mistas (LV) na presença de aCHC como sistemas veiculadores de fármaco. Lv foi preparado pelo método de injeção etanólica e formou vesículas de dimensões nanométricas (aproximadamente 100 nm) com bom índice de polidispersão, valores negativos de potencial zeta e estáveis em meio aquoso por mais de 50 dias. AlClPc se complexou com o fosfato das LV o que conferiu uma localização interfacial às moléculas de AlClPc como demonstrado pelos resultados de supressão de fluorescência. Medidas de anisotropia, fluorescência estática e resolvida no tempo corroboram com estes resultados e demonstram que a auto-agregação da AlClPc ocorre mesmo em lipossomas. Entretanto, a veiculação da AlClPc por LV em carcinoma de células escamosas oral (OSCC) levou a um processo de desagregação demonstrado por (FLIM). Este incrível comportamento é novo e aumenta o conhecimento científico sobre o mecanismo intracelular de ação de fotossensibilizadores em TFD. Em TFD, ambos os sistemas LVIII+AlClPc e LVIII+AlClPc+aCHC não apresentaram toxicidade no escuro no período de incubação de 3 h com as concentrações de lipídios, AlClPc e aCHC iguais a 0,15 mmol/L, 0,5 umol/L e 10,0 umol/L, respectivamente. De maneira inesperada, o sistema LVIII+AlClPc foi mais eficiente em TFD que o sistema LVIII+AlClPc+aCHC, devido ao caráter antioxidante do aCHC. Estes resultados abrem uma nova perspectiva do potencial uso de LV-AlClPc para o tratamento fotodinâmico. / Aluminum phthalocyanine chloride (AlClPc) is a second-generation photodynamic therapy (PDT) photosensitizer characterized for its high hydrophobicity and self-aggregation tendency in aqueous media, which hampers its potential application. The aCHC is a monocarboxylate transporter substrate (MCT) overexpressed in MCF-7 line cell. Aiming at AlClPc solubilization and uptake increase we proposed here the use of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at different proportions to form mixed lipid vesicles (LV) in presence of aCHC as a drug delivery system. LV were prepared by ethanol injection method and formed nano-sized vesicles (about 100 nm) with suitable polydispersity index, negative zeta potential, and stable in aqueous medium for at least 50 days. AlClPc strongly interacts with LV (high binding constant values), especially due to aluminum-phosphate specific interactions, which gives a surface localization to AlClPc molecules as demonstrated by fluorescence quenching data. Anisotropy, static and time-resolved fluorescence measurements corroborated with these results and demonstrated that AlClPc self-aggregation occurred even in the liposomes. However, formulation uptake by oral squamous cell carcinoma (OSCC) the AlClPc suffered a disaggregation process demonstrated by fluorescence life-time imaging microscopy. This amazing behavior is new and increases the scientific knowledge about the intracellular mechanism of action of PDT photosensitizers. In PDT, both systems LVIII+AlClPc e LVIII+AlClPc+aCHC did not show toxicity in the dark at 3 h incubation period with lipid, AlClPc, and aCHC concentrations equal at 0,15 mmol/L, 0,5 umol/L, and 10,0 umol/L, respectively. Unexpectedly, the system LVIII+AlClPc was more effective in PDT that the LVIII+AlClPc+aCHC, devido ao caráter antioxidante do aCHC due to the antioxidant character of aCHC. These results open a new perspective to the potential use of ALClPc-LV formulations for photodynamic treatment.

O bagaço de cana-de-açúcar é um material rico em celulose, molécula que pode ser convertida enzimaticamente em glicose pela ação de celulases. No entanto, no bagaço, a celulose está associada à hemicelulose e à lignina, componentes que limitam a sua digestibilidade enzimática. A lignina é o principal limitante da hidrólise da celulose, reduzindo a acessibilidade das enzimas celulolíticas. A hemicelulose também limita a ação das celulases e a presença de ácidos hidroxicinâmicos, como ferúlico e cumárico contribuem para reduzir os níveis de hidrólise. Nesse trabalho, o bagaço de cana-de-açúcar foi tratado com clorito de sódio em meio ácido com o objetivo de remover seletivamente a lignina e produzir modelos com teores reduzidos desse componente. O bagaço de cana controle continha 22,8% de lignina e após a deslignificação por 4 horas, materiais com até 6,8% de lignina foram obtidos, mantendo quase inalteradas as frações celulósicas e hemicelulósicas. Os bagaços foram submetidos à hidrólise enzimática com celulases comerciais. Apenas 26% da celulose de bagaço controle foram convertidos em glicose e 11% da xilana foram convertidos em xilose, com 72 horas de hidrólise. Nos bagaços com menores teores de lignina, os valores de conversão de celulose e de xilana alcançaram 85% e 64% no mesmo tempo de hidrólise, respectivamente. Os resultados mostraram que existe uma correlação inversa entre o teor de lignina e a conversão de polissacarídeos e que a remoção de 60% da lignina confere níveis elevados de hidrólise. Para reduzir a inibição pelos produtos de hidrólise da celulose, foi adicionada β-glicosidase ao meio reacional. Com a adição dessa enzima, os bagaços mais deslignificados alcançaram 100% de conversão de celulose e 82% de xilana, com 48 horas de hidrólise. No presente trabalho, o efeito da lignina não ligada aos polissacarídeos também foi avaliado. Para isso, lignina de bagaço de cana foi adicionada ao cartão de celulose, e este foi submetido à hidrólise enzimática. Não houve diferença entre os níveis de hidrólise na presença e ausência de lignina, indicando que a lignina limita a hidrólise dos polissacarídeos quando ligada a eles. Foi utilizada a técnica de microespectrofotometria ultravioleta para detectar a lignina e os ácidos hidroxicinâmicos em fibras, vasos e células de parênquima das regiões de córtex e de medula, deslignificados e controle. Os espectros UV dos três tipos celulares mostraram bandas em 278 nm, associada à lignina, e em 315 nm, associada aos ácidos hidroxicinâmicos. Os vasos foram os tipos celulares mais lignificados, seguidos pelas fibras e células parenquimatosas. O tratamento com clorito causou uma rápida remoção dos ácidos hidroxicinâmicos das células de parênquima, enquanto que as fibras foram deslignificadas apenas com 4 horas de tratamento. As amostras de córtex e de medula também foram submetidas à hidrólise com celulases. A região de medula não tratada foi prontamente hidrolizada, alcançando 63% de conversão de celulose com 72 horas, enquanto que o córtex não tratado atingiu apenas 20%. O tratamento com clorito de sódio não aumentou os níveis de hidrólise da medula. No entanto, a remoção de lignina do córtex aumentou consideravelmente a conversão. / The sugarcane bagasse is a material rich in cellulose, wich can be enzymatically converted into glucose by the action of cellulases. However, in the bagasse, it is associated to hemicellulose and lignin, components that limit its enzymatic digestibility. Lignin is the main limitant of the enzymatic hydrolysis of cellulose, reducing the accessibility of cellulases. Hemicellulose also limits the cellulase action and the presence of the hydroxycinnamic acids, as ferulic and coumaric, contribute to reduce the hydrolysis levels. In this work, the sugarcane bagasse was treated with acid sodium chlorite, aiming to selective remove the lignin and produce models with lower contents of this component. The control bagasse had 22.8% of lignin and after a 4-hour delignification, materials with up to 6.8% of lignin were obtained, retaining the cellulosic and hemicellulosic fractions almost unchanged. The bagasses were submitted to the enzymatic hydrolysis with comercial cellulases. Only 26% of the cellulose of the control bagasse were converted into glucose and 11% of the xylan were converted into xylose, with 72 hours of hydrolysis. In the bagasses with lower lignin content, the cellulose and xylan conversion values reached 85% and 64% in the same time of hysylysis, respectively. The results showed that there is an inverse correlation between the lignin content and the enzymatic conversion of polysaccharides and that the removal of 60% of the lignin results in high levels of hydrolysis. To reduce the inhibition by the products of the cellulose hydrolysis, β- glucosidase was added to the reaction medium. With this enzyme, the most delignified bagasses reached 100% of cellulose and 82% of xylan convertion, with 48 hours of hydrolysis. In this work, the effect of non-linked lignin was also analysed. For this, sugarcane bagasse lignin was added to cellulose card, and it was submitted to the enzymatic hydrolysis. There were no differences between the hydrolysis levels in the presence or absence of lignin, suggesting that lignin only limits hydrolysis when linked to the polysaccharides. The ultraviolet microspectrophotometry technique was used to detect lignin and hydroxycinnamic acids in fiber, vessels and parenchyma cells in rind and pith regions, delignified and control. The UV spectra of the three cell types showed bands in 278 nm, related to lignin, and 315 nm, related to the hydroxycynamic acids. The vessels were the most lignified cell type, followed by fibers and parenchyma cells. The chlorite treatment caused a rapid removal of hydroxycynnamic acids from parenchyma cell walls, while fibers where delignified only with a 4-hour treatment. The rind and pith samples were also submitted to enzymatic hydrolysis. The untreated pith region was promptly hydrolysed, reaching 63% of cellulose convertion in 72 hours, while untreated rind reached only 20%. The sodium chlorite treatment did not enhance the pith hydrolysis levels. However, the lignin removal in rind samples substantially enhanced the conversion.

O bagaço de cana-de-açúcar é um material rico em celulose, molécula que pode ser convertida enzimaticamente em glicose pela ação de celulases. No entanto, no bagaço, a celulose está associada à hemicelulose e à lignina, componentes que limitam a sua digestibilidade enzimática. A lignina é o principal limitante da hidrólise da celulose, reduzindo a acessibilidade das enzimas celulolíticas. A hemicelulose também limita a ação das celulases e a presença de ácidos hidroxicinâmicos, como ferúlico e cumárico contribuem para reduzir os níveis de hidrólise. Nesse trabalho, o bagaço de cana-de-açúcar foi tratado com clorito de sódio em meio ácido com o objetivo de remover seletivamente a lignina e produzir modelos com teores reduzidos desse componente. O bagaço de cana controle continha 22,8% de lignina e após a deslignificação por 4 horas, materiais com até 6,8% de lignina foram obtidos, mantendo quase inalteradas as frações celulósicas e hemicelulósicas. Os bagaços foram submetidos à hidrólise enzimática com celulases comerciais. Apenas 26% da celulose de bagaço controle foram convertidos em glicose e 11% da xilana foram convertidos em xilose, com 72 horas de hidrólise. Nos bagaços com menores teores de lignina, os valores de conversão de celulose e de xilana alcançaram 85% e 64% no mesmo tempo de hidrólise, respectivamente. Os resultados mostraram que existe uma correlação inversa entre o teor de lignina e a conversão de polissacarídeos e que a remoção de 60% da lignina confere níveis elevados de hidrólise. Para reduzir a inibição pelos produtos de hidrólise da celulose, foi adicionada β-glicosidase ao meio reacional. Com a adição dessa enzima, os bagaços mais deslignificados alcançaram 100% de conversão de celulose e 82% de xilana, com 48 horas de hidrólise. No presente trabalho, o efeito da lignina não ligada aos polissacarídeos também foi avaliado. Para isso, lignina de bagaço de cana foi adicionada ao cartão de celulose, e este foi submetido à hidrólise enzimática. Não houve diferença entre os níveis de hidrólise na presença e ausência de lignina, indicando que a lignina limita a hidrólise dos polissacarídeos quando ligada a eles. Foi utilizada a técnica de microespectrofotometria ultravioleta para detectar a lignina e os ácidos hidroxicinâmicos em fibras, vasos e células de parênquima das regiões de córtex e de medula, deslignificados e controle. Os espectros UV dos três tipos celulares mostraram bandas em 278 nm, associada à lignina, e em 315 nm, associada aos ácidos hidroxicinâmicos. Os vasos foram os tipos celulares mais lignificados, seguidos pelas fibras e células parenquimatosas. O tratamento com clorito causou uma rápida remoção dos ácidos hidroxicinâmicos das células de parênquima, enquanto que as fibras foram deslignificadas apenas com 4 horas de tratamento. As amostras de córtex e de medula também foram submetidas à hidrólise com celulases. A região de medula não tratada foi prontamente hidrolizada, alcançando 63% de conversão de celulose com 72 horas, enquanto que o córtex não tratado atingiu apenas 20%. O tratamento com clorito de sódio não aumentou os níveis de hidrólise da medula. No entanto, a remoção de lignina do córtex aumentou consideravelmente a conversão. / The sugarcane bagasse is a material rich in cellulose, wich can be enzymatically converted into glucose by the action of cellulases. However, in the bagasse, it is associated to hemicellulose and lignin, components that limit its enzymatic digestibility. Lignin is the main limitant of the enzymatic hydrolysis of cellulose, reducing the accessibility of cellulases. Hemicellulose also limits the cellulase action and the presence of the hydroxycinnamic acids, as ferulic and coumaric, contribute to reduce the hydrolysis levels. In this work, the sugarcane bagasse was treated with acid sodium chlorite, aiming to selective remove the lignin and produce models with lower contents of this component. The control bagasse had 22.8% of lignin and after a 4-hour delignification, materials with up to 6.8% of lignin were obtained, retaining the cellulosic and hemicellulosic fractions almost unchanged. The bagasses were submitted to the enzymatic hydrolysis with comercial cellulases. Only 26% of the cellulose of the control bagasse were converted into glucose and 11% of the xylan were converted into xylose, with 72 hours of hydrolysis. In the bagasses with lower lignin content, the cellulose and xylan conversion values reached 85% and 64% in the same time of hysylysis, respectively. The results showed that there is an inverse correlation between the lignin content and the enzymatic conversion of polysaccharides and that the removal of 60% of the lignin results in high levels of hydrolysis. To reduce the inhibition by the products of the cellulose hydrolysis, β- glucosidase was added to the reaction medium. With this enzyme, the most delignified bagasses reached 100% of cellulose and 82% of xylan convertion, with 48 hours of hydrolysis. In this work, the effect of non-linked lignin was also analysed. For this, sugarcane bagasse lignin was added to cellulose card, and it was submitted to the enzymatic hydrolysis. There were no differences between the hydrolysis levels in the presence or absence of lignin, suggesting that lignin only limits hydrolysis when linked to the polysaccharides. The ultraviolet microspectrophotometry technique was used to detect lignin and hydroxycinnamic acids in fiber, vessels and parenchyma cells in rind and pith regions, delignified and control. The UV spectra of the three cell types showed bands in 278 nm, related to lignin, and 315 nm, related to the hydroxycynamic acids. The vessels were the most lignified cell type, followed by fibers and parenchyma cells. The chlorite treatment caused a rapid removal of hydroxycynnamic acids from parenchyma cell walls, while fibers where delignified only with a 4-hour treatment. The rind and pith samples were also submitted to enzymatic hydrolysis. The untreated pith region was promptly hydrolysed, reaching 63% of cellulose convertion in 72 hours, while untreated rind reached only 20%. The sodium chlorite treatment did not enhance the pith hydrolysis levels. However, the lignin removal in rind samples substantially enhanced the conversion.

Ftalocianina de alumínio-cloro (AlClPc) é um fotossensibilizador de segunda geração em terapia fotodinâmica (TFD) caracterizado por seu caráter anfifílico e tendência de auto-agregação em meio aquoso, o que prejudica seu potencial de aplicação. O aCHC é um substrato de transportadores de monocarboxilato (MCT) superexpresso em células de MCF-7. Objetivando a solubilização da AlClPc e aumento de internalização em tecidos neoplásicos nos propomos aqui o uso de DSPC e DOPC em diferentes proporções para formar vesículas lipidicas mistas (LV) na presença de aCHC como sistemas veiculadores de fármaco. Lv foi preparado pelo método de injeção etanólica e formou vesículas de dimensões nanométricas (aproximadamente 100 nm) com bom índice de polidispersão, valores negativos de potencial zeta e estáveis em meio aquoso por mais de 50 dias. AlClPc se complexou com o fosfato das LV o que conferiu uma localização interfacial às moléculas de AlClPc como demonstrado pelos resultados de supressão de fluorescência. Medidas de anisotropia, fluorescência estática e resolvida no tempo corroboram com estes resultados e demonstram que a auto-agregação da AlClPc ocorre mesmo em lipossomas. Entretanto, a veiculação da AlClPc por LV em carcinoma de células escamosas oral (OSCC) levou a um processo de desagregação demonstrado por (FLIM). Este incrível comportamento é novo e aumenta o conhecimento científico sobre o mecanismo intracelular de ação de fotossensibilizadores em TFD. Em TFD, ambos os sistemas LVIII+AlClPc e LVIII+AlClPc+aCHC não apresentaram toxicidade no escuro no período de incubação de 3 h com as concentrações de lipídios, AlClPc e aCHC iguais a 0,15 mmol/L, 0,5 umol/L e 10,0 umol/L, respectivamente. De maneira inesperada, o sistema LVIII+AlClPc foi mais eficiente em TFD que o sistema LVIII+AlClPc+aCHC, devido ao caráter antioxidante do aCHC. Estes resultados abrem uma nova perspectiva do potencial uso de LV-AlClPc para o tratamento fotodinâmico. / Aluminum phthalocyanine chloride (AlClPc) is a second-generation photodynamic therapy (PDT) photosensitizer characterized for its high hydrophobicity and self-aggregation tendency in aqueous media, which hampers its potential application. The aCHC is a monocarboxylate transporter substrate (MCT) overexpressed in MCF-7 line cell. Aiming at AlClPc solubilization and uptake increase we proposed here the use of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at different proportions to form mixed lipid vesicles (LV) in presence of aCHC as a drug delivery system. LV were prepared by ethanol injection method and formed nano-sized vesicles (about 100 nm) with suitable polydispersity index, negative zeta potential, and stable in aqueous medium for at least 50 days. AlClPc strongly interacts with LV (high binding constant values), especially due to aluminum-phosphate specific interactions, which gives a surface localization to AlClPc molecules as demonstrated by fluorescence quenching data. Anisotropy, static and time-resolved fluorescence measurements corroborated with these results and demonstrated that AlClPc self-aggregation occurred even in the liposomes. However, formulation uptake by oral squamous cell carcinoma (OSCC) the AlClPc suffered a disaggregation process demonstrated by fluorescence life-time imaging microscopy. This amazing behavior is new and increases the scientific knowledge about the intracellular mechanism of action of PDT photosensitizers. In PDT, both systems LVIII+AlClPc e LVIII+AlClPc+aCHC did not show toxicity in the dark at 3 h incubation period with lipid, AlClPc, and aCHC concentrations equal at 0,15 mmol/L, 0,5 umol/L, and 10,0 umol/L, respectively. Unexpectedly, the system LVIII+AlClPc was more effective in PDT that the LVIII+AlClPc+aCHC, devido ao caráter antioxidante do aCHC due to the antioxidant character of aCHC. These results open a new perspective to the potential use of ALClPc-LV formulations for photodynamic treatment.

No description available.

Plants produce a rich variety of natural products to face environmental constraints. Enzymes of the cytochrome P450 CYP98 family are key actors in the production of phenolic bioactive compounds. They hydroxylate phenolic esters for lignin biosynthesis in angiosperms, but also produce various other bioactive phenolics. We characterized CYP98s from a moss, a lycopod, a fern, a conifer, a basal angiosperm, a monocot and from two eudicots. We found that substrate preference of the enzymes has changed during evolution of land plants with typical lignin-related activities only appearing in angiosperms, suggesting that ferns, similar to lycopods, produce lignin through an alternative route. A moss CYP98 knock-out mutant revealed coumaroyl-threonate as CYP98 substrate in vivo and showed a severe phenotype. Multiple CYP98s per species exist only in the angiosperms, where we generally found one isoform presumably involved in the biosynthesis of monolignols, and additional isoforms, resulting from independent duplications, with a broad range of functions in vitro / Graduate / 2017-08-31

Made available in DSpace on 2016-08-10T10:29:40Z (GMT). No. of bitstreams: 1 Marlene Neves Antunes.pdf: 10408234 bytes, checksum: c1e51577817677c6ded3b53d4a4bbb59 (MD5) Previous issue date: 2009-11-05 / The medicinal plants have been a rich source for obtaining bioactive molecules, constituting one of the most successful strategies in the discovery of novel medicines. The specie Cochlospermum regium (Martius and Schrank) Pilger (Bixaceae), popularly known as algodãozinho-do-campo is a native plant, abundant in Brazilian Cerrado. After an ethnobotanical and ethnopharmacological research carried out at Tocantins State it was showed that this plant is used in folk medicine to treat several illnesses. Among the therapeutic indications the most frequents are: gynecological and renal inflammations, prostatitis, a few kinds of pains, fever, gastritis and skin affections, among others. This study aimed to contribute to the knowledge of the chemical constituents of C. regium. For this, extracts solutions were obtained by an exhausting way maceration of this plants roots in the solvents: hexane, dichloromethane, ethyl acetate and methanol, in this sequence increasing polarity, obtaining the following income rude mass macerated: 1,78%, 0,23%; 0,51% and 12,16%, respectively. The phytochemical investigation of the hexane extract (CrRH) allowed the isolation of substance initially identified as SA (7,0 mg), an unpublished chemical compound, still in characterization process and Substance SB (20,0 mg), identified as excelsina, a lignan reported for the first time in this specie. From the CrRH1 fraction originated by the fractionating chromatography column of the hexane extract was isolated the substance SC (6,0 mg) identified as a steroid, still in structural elucidation. From the dichloromethane extract (CrRD) the substance SD (9,0 mg) was isolated and identified as phidroxicinamic acid stereate, whose structures are not yet fully defined. Data obtained so far indicate the existence of a mixture of p-hidroxicinâmic acids, probably varying the size of the side chain ester. On the fractionation of ethyl acetate extract was obtained the substance SE (23mg) originated from the CrRAc1 fraction and identified as naringenin, a common flavanone in the genus Cochlospermum, have been also isolated in the stalk bark of this specie, not having, however, reports the presence of this flavanone in roots of C. regium. And finally, the SF substance (15,0 mg), originated of the CrRAc2 fraction, which was also identified as excelsina. / As plantas medicinais têm sido uma rica fonte para obtenção de moléculas bioativas, constituindo-se numa das mais bem sucedidas estratégias na descoberta de novos medicamentos. A espécie Cochlospermum regium (Martius e Schrank) Pilger (Bixaceae), popularmente conhecida como algodãozinho-do-campo, é uma planta nativa, abundante no cerrado brasileiro. No Tocantins, levantamento etnobotânico e etnofarmacológico realizado demonstrou que esta planta é utilizada na medicina popular no tratamento de várias enfermidades. Entre as indicações terapêuticas mais freqüentes estão: inflamações ginecológicas e renais, prostatites, dores diversas, febre, gastrite e afecções de pele, entre outras. Assim, este trabalho teve como objetivo contribuir para o conhecimento dos constituintes químicos de C. regium. Para tanto, foram obtidos extratos macerando-se diretamente, de forma exaustiva, as raízes da espécie nos solventes hexano, diclorometano, acetato de etila e metanol, nesta seqüência crescente de polaridade, obtendo-se o seguinte rendimento da massa bruta macerada: 1,78%, 0,23%; 0,51% e 12,16%, respectivamente. A análise fitoquímica do extrato hexano (CrRH) permitiu o isolamento das substâncias identificadas inicialmente como SA (7,0mg), composto inédito, ainda em processo de caracterização e Substância SB (20,0 mg), identificada como excelsina, uma lignana reportada pela primeira vez nesta espécie. Da fração CrRH1 proveniente do fracionamento cromatográfico do extrato hexânico, isolou-se a substância SC (6,0 mg) identificada como esteróide, ainda em fase de elucidação estrutural. Do extrato diclorometânico (CrRD) foi isolada a substância SD (9,0 mg), identificada como ácido p-hidroxicinâmico estereato, cuja estrutura ainda não está totalmente definida. Dados obtidos até o momento apontam a existência uma mistura de ácidos p-hidroxicinâmicos, provavelmente variando o tamanho da cadeia lateral do éster. Do fracionamento do extrato acetato de etila obteve-se a substância SE (23mg) originada da fração CrRAc1 e identificada como naringenina, flavanona comum no gênero Cochlospermum, tendo sido isolada, também, na casca do caule desta espécie, não havendo, entretanto, relatos da presença desta flavonona em raiz de C. regium. E finalmente, a substância SF (15,0 mg) originada da fração CrRAc2, que também foi identificada como excelsina.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-09-22T18:10:08Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_sousa_xavier.pdf: 3096603 bytes, checksum: dae91257943ac86316978d8deb31ff3e (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-10-09T13:42:33Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_sousa_xavier.pdf: 3096603 bytes, checksum: dae91257943ac86316978d8deb31ff3e (MD5) / Made available in DSpace on 2017-10-09T13:42:33Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_sousa_xavier.pdf: 3096603 bytes, checksum: dae91257943ac86316978d8deb31ff3e (MD5) Previous issue date: 2017 / A Leishmaniose Visceral Canina (LVC) ? considerada um problema de sa?de p?blica causada por um protozo?rio do g?nero Leishmania que acomete c?es, o qual no ciclo urbano de transmiss?o ? o principal reservat?rio. Neste trabalho, investigou-se o desenvolvimento de um imunossensor impedim?trico sobre eletrodos de grafite (EG) e eletrodos impressos de ?xido de grafeno (EIG) modificados com filmes polim?ricos para detec??o de Leishmaniose infantum em amostras de soros caninos. Os EG e o EIG foram modificados pela eletrodeposi??o do ?cido 2-hidroxicin?mico (2-HCA). A eletropolimeriza??o do 2-HCA foi realizada por voltametria c?clica (VC), utilizando-se solu??o monom?rica 2,50 mM preparada em H2SO4 0,50 M contendo 1,0 mL de etanol. Para o EG utilizou-se 100 ciclos de potencial na faixa de +0,20 a +1,20 V a 50 mV/s e para o EIG 15 ciclos consecutivos de potencial de 0,00 a +1,00V a 100 mV/s. Para ambos os eletrodos modificados o poli(2-HCA) apresentou caracter?sticas adsortivas e eletroativas. Estudos de Espectroscopia de Imped?ncia Eletroqu?mica (EIE) foram realizados para avaliar as propriedades el?tricas dos eletrodos modificados. Para o EG/2-HCA utilizou-se o circuito equivalente descrito como (Rs(RQp)(Qdl[RtcW]) onde obteve-se o valor de resist?ncia a transfer?ncia de carga (Rtc) de 13,2 k?, enquanto que para EIG/2-HCA o circuito equivalente proposto foi o de Randles, no qual o valor de Rtc foi de 4,2 k?. Visando a miniaturiza??o do sistema, optou-se pelo desenvolvimento do biossensor sobre os EIG. Desta forma, foram colocados sobre a superf?cie dos EIG/2-HCA, utilizando a t?cnica de adsor??o, 35 ?L de diferentes concentra??es do ant?geno L. infantum (Ag) (15, 30, 50, 70 e 100 ?g/mL), sendo realizados testes de intera??o com amostras de soros caninos contendo os anticorpos positivos (Ac+) de L. infantum em diferentes propor??es 1:80, 1:160, 1:320, 1:640 e 1:1280 dilu?dos em tamp?o HBS-EP pH 7,40 com 20 minutos de intera??o a 37 ?C. Os melhores resultados foram obtidos utilizando-se o ant?geno em concentra??o de 50 ?g/mL durante 20 min a 37 ?C e a dilui??o do soro de 1:320. Posteriormente, avaliou-se o tempo de imobiliza??o do Ag e de imunorrea??o Ag-Ac em 20, 40 e 60 minutos. Obteve-se 20 min de como tempo ideal de imobiliza??o de Ag e para imunorrea??o Ag-Ac+. Ap?s otimiza??o destes par?metros, avaliou-se a resposta do imunossensor na presen?a de amostras de soros caninos diagnosticados como positivos (Ac+) e negativos (Ac-) para Leishmaniose infantum, onde observou-se valores de Rtc para o sistema Ag-Ac+ 82% maior do que o obtido para o Ag-Ac-, demonstrando margem de seguran?a confi?vel no reconhecimento do analito de interesse. O imunossensor proposto para detec??o de Leishmaniose infantum apresentou boas perspectivas para aplica??o ao diagn?stico da doen?a, podendo-se tornar mais uma alternativa para realiza??o de testes r?pidos e de simples aplica??o para detec??o de LVC. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / Visceral Canine Leishmaniasis (LVC) is considered a public health issue caused by a protozoan of the Leishmania genre that attacks dogs, which is the main reservoir in the urban transmission cycle. In this work, we investigated the development of an impedimetric immunosensor on graphite electrodes (GE) and graphene oxide-modified screen-printed carbon electrode (GSP) both modified with polymeric films to detect Leishmaniose infantum in canine serum samples. The GE and GSP were modified by the electrodeposition of 2-hydroxycinnamic acid (2-HCA). Electropolymerization of 2-HCA was performed by cyclic voltammetry (CV) using a 2.50 mM monomeric solution prepared in 0.50 M H2SO4 containing 1.0 mL of ethanol. For the EG and EIG, 100 cycles of potential between +0.20 and +1.20 V at 50 mV/s and 15 cycles of potential between 0.00 and + 1.00 V at 100 mV/s were used, respectively. Poly(2-HCA) shown adsorptive and electroactive characteristics for both electrodes. Electrochemical Impedance Spectroscopy (EIS) studies were performed to evaluate the electrical properties of the modified electrodes. For the GE/2-HCA was used an equivalent circuit described as (Rs(RpQp)(Qdl[RtcW]), and a resistance of charge transfer (Rct) of 13.2 k? was obtained. For GSP/2-HCA the proposed equivalent circuit was that of Randles in which the value of Rct obtained was 4.2 k?. For system miniaturization it was developed the biosensor over GSP. Thus, 35 ?L of different concentrations of L. infantum antigen (Ag) (15, 30, 50, 70 e 100 ?g/mL) were placed on the GSP/2-HCA surface using the adsorption technique. Interaction tests were carried out with canine serum samples containing the positive antibodies (Ac+) of L. infantum in different proportions of 1:80, 1:160, 1:320, 1:640 e 1:1280, diluted in HBS-EP buffer pH 7.4 with 20 minutes of interaction at 37 ?C. The best results were obtained using the antigen at a concentration of 50 ?g/mL for 20 min at 37 ?C and a serum dilution of 1:320. Subsequently, the Ag immobilization and Ag-Ac immunoreaction time were evaluated in 20, 40 and 60 min. 20 min was obtained as the ideal time for Ag immobilization and Ag-Ac+ immunoreaction. After optimization of these parameters, the immunosensor response was evaluated in the presence of canine serum diagnosed as positive (Ac+) and negative (Ac-) for Leishmaniose infantum, where the Rct values for the Ag-Ac+ system were 82% higher than those obtained for Ag-Ac-, demonstrating a reliable safety margin in the recognition of the analyte of interest. The proposed immunoassay for the detection of Leishmaniasis infantum presented good prospects for application in the diagnosis of the disease, and it could become an alternative for performing rapid tests and simple application for the detection of LVC.

Ce travail de recherche a pour objectifs d’évaluer les activités antibactériennes, antiprolifératives et antioxydantes des extraits de feuilles de Solanum incanum L., S. schimperianum Hochst, S. nigrum L., Physalis lagascae Roem. & Schult. et Withania somnifera (L) Dunal. Plus précisément, les activités antibactériennes, anti-prolifératives et antioxydantes ont été déterminées à partir des extraits méthanoliques et des fractions de glycoalcaloïdes stéroïdiens (SGAF) de chaque plante. La sensibilité des bactéries, à Gram positif et à Gram négatif, était variable en présence de chacun des extraits (valeurs des IC50 dans la gamme de 15 > 1000 μg / mL). L'extrait méthanolique de la feuille de S. schimperianum a montré une activité anti-proliférative intéressante contre les lignées cellulaires humaines testées avec des valeurs de CI50 variant de 2,69 à 19,83 μg / mL tandis que l'activité la plus élevée des fractions de feuilles (SGAF) de W. somnifera a montré des valeurs IC50 variant de 1,29 à 5,00 μg / mL. Les fractions SGAF de toutes les espèces ont montré une activité antiradicalaire plus élevée que leurs extraits méthanoliques. La fraction SGAF de S. schimperianum a montré l'activité antioxydante la plus forte avec une valeur CI50 3,5 ± 0,2 μg / mL pour le test DPPH et 3,5 ± 0,3 μg / mL pour le test ABTS. L'analyse GC-MS des extraits méthanoliques et des fractions SGAF des espèces étudiées a révélé la présence d'alcaloïdes stéroïdiens, de saponines stéroïdiennes, de stéroïdes et d'autres composés comme des terpènes, des phénols et des alcanes. Leur répartition variait selon les espèces et, ce qui peut fournir des éléments pour évaluer les relations chimiotaxonomiques préliminaires. Douze dérivés de l'acide hydroxycinnamique ont été identifiés dans l'extrait méthanolique de la feuille de S. schimperianum et le composé N-caffeoyl agmatine était majoritaire. La présence d'alcaloïdes stéroïdiques comme la solanopubamine et la solanocapsine, ainsi que des dérivés des alcaloïdes 3-amino stéroïdes a été notée. De plus, trois composés, quercétine, kaempférol glycosylé et le β-sitostérol, ont été isolés et identifiés / This study aimed at the evaluation of in vitro antibacterial, antiproliferative and antioxidant activities of methanolic leaf extracts and steroidal glycoalkaloids fractions (SGAFs) of Solanum incanum L., S. schimperianum Hochst, S. nigrum L., Physalis lagascae Roem. & Schult. and Withania somnifera (L) Dunal. The sensitivity of Gram-positive and Gram-negative bacteria to each extract was variable (IC50 values in the range of 15->1000 µg/mL). The methanolic extract of S. schimperianum leaf demonstrated interesting anti-proliferative activity against the human cell lines tested with IC50 values in the range of 2.69 to 19.83 µg/mL while the highest activity from the SGAFs was obtained from W. somnifera leaf with IC50 values in the range of 1.29 to 5.00 µg/mL. The SGAFs of all species demonstrated higher scavenging activity than their respective methanolic extracts. The SGAF of S. schimperianum displayed the strongest antioxidant activity in both assays with IC50 value 3.5 ± 0.2DPPH and 3.5 ±0.3ABTS µg/mL. GC-MS analysis of methanolic and SGAFs extracts of the studied species revealed the presence of steroidal alkaloids, steroidal saponins, steroids and other compounds like terpenes, phenols and alkanes. Their distribution varied among the species and thus they could provide evidence to assess preliminary chemotaxonomic relationships. Twelve known hydroxycinnamic acid amides were tentatively identified from the methanolic extract of S. schimperianum leaf and N-caffeoyl agmatine appeared with the highest intensity. Moreover, the presence of steroid alkaloids solanopubamine and solanocapsine as well as dehydroderivatives of the 3-amino steroid alkaloids was suggested. Furthermore, three compounds quercetin, kaempferol glycoside and β-sitosterol were isolated and identified. In silico investigation of these three compounds for their potency against cancer revealed that β-sitosterol was found to be the most selective compound against human pregnane X receptor (PXR) and gave the highest binding energy (-11.2 kcal/mol). These results suggested that Solanaceae plants endogenous to Sudan could be a potential source of bioactive agents