Made available in DSpace on 2017-05-12T14:47:40Z (GMT). No. of bitstreams: 1 Tese Francielly_ Torres dos Santos.pdf: 2692597 bytes, checksum: db1b8713e9d8a55de139153f5a86a29e (MD5) Previous issue date: 2016-02-16 / Parsley has been widely traded as a flavoring in Brazil and worldwide. The quality to produce salsa depends on its cropping management. This, this study aimed to evaluate the effect of alternative organic substrates in productivity and nutritional quality of Petroselium crispum. The treatments consisted of five organic compounds obtained by agro-industrial waste composting process of broiler production chain in which they varied the main source of carbon, i.e, waste cotton carding, pruning of ground urban trees, sawdust, bagasse of milled cane and ground napier grass. In order to obtain the organic substrates, it was added 0, 15, 30, 45 and 60% biochar to each of the five organic compounds, a charcoal obtained from burning wood in boilers. The experimental design was completely randomized in a 5x5 factorial design, with four replications. The nutritional quality of salsa was determined by N, P, K evaluation, and antioxidant activity and productivity. Concerning substrates, functional groups and determination of humification degree were evaluated, correlated with parsley dry weight. It was concluded that the use of substrate from Napier grass ground does not favor Petroselium crispum production. Adding biochar associated with organic compost from urban tree pruning whose main source is carbon favors flavonoid content of Diosmetin-apiosilglucoside-isomer and apigenin-malonyl-glucoside in Petroselium crispum. The electrical conductivity of the organic substrate is the main factor that contains Petroselium crispum production while addition of biochar can minimize this effect / A salsa é amplamente comercializada como especiaria no Brasil e no mundo. A produção de salsa com qualidade depende da forma de cultivo. Assim, neste trabalho, objetivou-se avaliar o efeito de substratos orgânicos alternativos na produtividade e qualidade nutricional de salsa graúda portuguesa. Os tratamentos consistiram de cinco compostos orgânicos, obtidos pelo processo de compostagem de resíduos agroindustriais da cadeia produtiva do frango de corte em que se variou a principal fonte de carbono, a saber: resíduos de desfibrilação de algodão, resíduos de poda de árvores urbanas trituradas, serragem, bagaço de cana moído e capim-napier triturado. Para obtenção dos substratos orgânicos, a cada um dos cinco compostos orgânicos, acrescentaram-se 0, 15, 30, 45 e 60% de biochar, um carvão obtido da queima da madeira em caldeiras. O delineamento experimental adotado foi o inteiramente casualizado, em esquema fatorial 5x5, com quatro repetições. A qualidade nutricional da salsa foi determinada pela avaliação de N, P, K, atividade antioxidante e produtividade. Nos substratos, foram avaliados os grupos funcionais e a determinação do grau de humificação, correlacionados com a matéria seca da salsa. Concluiu-se que o uso de substrato originado de capim-napier triturado não favorece à produção de salsa graúda portuguesa. A adição de biochar associado ao composto orgânico, proveniente de poda de árvores urbanas como principal fonte de carbono, favorece o teor de flavonoides de diosmetin-apiosilglucosídeo isômero e apigenina-malonil-glucosídeo na salsa graúda portuguesa. A condutividade elétrica do substrato orgânico é o fator que mais limita a produção de matéria de salsa graúda portuguesa e a adição de biochar pode minimizar tal efeito.

Croton (Euphorbiaceae), é um gênero promissor para pesquisa de compostos bioativos, tanto pela variedade de compostos químicos encontrados, quanto pela diversidade de uso na medicina tradicional/popular. Estudos farmacológicos comprovaram atividades antimicrobianas, antivirais, anti-inflamatórias, antioxidantes e citotóxicas. Neste trabalho, foram obtidos extratos fracionados em Soxhlet (hexano, diclorometano, acetato de etila e metanol) de folhas e caules de C. pallidulus Baill var. pallidulus (Baill.) L.B. Smith & S.F. Smith. Esses extratos foram avaliados quanto às atividades 1) antibacteriana pelo método de microdiluição em caldo, utilizando Staphylococcus aureus - cepas sensível e resistente à vancomicina, Pseudomonas aeruginosa, Escherichia coli e Salmonella choleraesuis; 2) antioxidante (sequestro do radical livre DPPH); e quanto à toxicidade frente à Artemia salina. A fração mais ativa neste último ensaio foi selecionada para a avaliação da atividade citotóxica utilizando as células de sarcoma uterino humano. Os fenóis totais, e flavonóis e flavonas totais foram quantificados por métodos colorimétricos. O fracionamento dos extratos hexânico e diclorometânico foi feito por cromatografia em coluna de sílica, enquanto o dos extratos de acetato de etila e metanol, em coluna de PVPP. O isolamento das substâncias das frações ativas nos ensaios de atividade antibacteriana (S. aureus - sensível) e de toxicidade frente à A. salina foi realizado por CLAE semipreparativo. O teor de fenóis totais de C. pallidulus var. pallidulus foi de 1,95 g EAG/100g MS e de flavonóis e flavonas totais foi de 0,56 g EQ/100g MS. Todos os extratos apresentaram atividade contra S. aureus (sensível e resistente), com CBM de 2048 mg/L, exceto o extrato metanólico que apresentou atividade apenas contra S. aureus (sensível). Nenhum extrato apresentou atividade contra as demais cepas. As frações FH-5A - hexano (CBM de 256mg/L) e FA-7 - acetato de etila (CBM de 1024mg/L) foram as que apresentaram maior atividade antibacteriana. A fração FH-5A foi separada em coluna de sílica, e suas subfrações apresentaram atividades antibacterianas menores que a fração que lhe deu origem. Das sete substâncias majoritárias isoladas da fração FA-7, nenhuma apresentou atividade antibacteriana. Seis delas são flavonoides: três C-glicosídeos de apigenina, um O-glucosídeo de campferol, um O-galactosídeo de isoramnetina e um O-rutinosídeo de isoraminetina. Apenas o extrato diclorometânico apresentou atividade no ensaio de toxicidade frente à A. salina, com CL50 de 385mg/L. A fração com maior toxicidade foi a FD-4A, com CL50 de 101mg/L. As substâncias mais abundantes nesta fração foram isoladas e identificadas como 8,9-secocauranos, derivados da kongensina F. Quanto a atividade citotóxica, a atividade de FD-4A foi três vezes maior que a atividade do extrato diclorometânico. Os extratos de acetato de etila e de metanol apresentaram os maiores valores de atividade antioxidante: 12,11 e 11,77 gEQ/100g, respectivamente. Verificou-se que os flavonoides foram as substâncias fenólicas que mais influenciaram na atividade antioxidante das frações dos extratos de acetato de etila e de metanol. Com esses resultados verifica-se que C. pallidulus var. pallidulus possui ação antibacteriana, citotóxica e antioxidante, sendo uma potencial fonte para novos fármacos para tratamento de doenças infecciosas e câncer. Conseguiu-se também isolar três 8,9-secocauranos, derivados da kongensina F, que aparentemente não haviam ainda sido descritos / Croton (Euphorbiaceae), is a promising genre for investigation of bioactive compounds, both for the variety of compouds, as for the diversity of use in traditional medicine. Pharmacological studies have demonstrated antimicrobial, antiviral, anti-inflammatory, cytotoxic, and antioxidant activities. In this study, fractionated extracts of leaves and stems of C. pallidulus Baill var. pallidulus (Baill.) L.B. S.F. Smith & Smith were obtained throught Soxhlet (hexane, dichloromethane, ethyl acetate and methanol). The following activities of these extracts were tested: 1) antibacterial activity, through microbroth dilution assay using: Staphylococcus aureus - sensitive and vancomycin-resistant strains, Pseudomonas aeruginosa, Escherichia coli and Salmonella choleraesuis; 2) antioxidant activity (free radical DPPH scavenging) and Artemia salina toxicity evaluation. The most active fraction in the latter test was selected to evaluate the cytotoxic activity using human uterine sarcoma cells. The total phenols, and total flavonols and flavones were quantified by colorimetric methods. The fractionation of hexane and dichloromethane extracts was done through silica column chromatography, while PVPP column was used for the ethyl acetate and methanol extracts. The isolation of the active fractions major compounds in the antibacterial activity (S. aureus - sensitive) and A. saline toxicity assays were performed by semipreparative HPLC. The total phenolic content of C. pallidulus var. pallidulus was 1.95 g GAE/100g DM and total flavonols and flavones was 0.56 g QE/100g MS. All extracts showed activity against S. aureus (sensitive and resistant), with MBC of 2048 mg/L, except for the methanol extract, which showed activity only against S. aureus (sensitive). Extracts showed no activity against the other strains. Fractions FH-5A-hexane (MBC: 256mg/L) and FA-7-ethyl acetate (MBC: 1024mg/L) showed the highest antibacterial activity. The fraction FH-5A was separated on a silica column, and its subfractions showed smaller antibacterial activity than FH-5A. The seven major compounds isolated from the FA-7 fraction, showed no antibacterial activity. Six of them are flavonoids, three apigenin C-glycosides, one kaempferol O-glucoside, one isorhamnetin O-galactoside and one isorhamnetin O-rutinoside. Only the dichloromethane extract showed activity in the A. salina toxicity assay, LC50: 385mg/L. The fraction with the highest toxicity was the FD-4A, LC50: 101mg/L. The most abundant substances in this fraction were isolated and identified as 8,9 - secokauranes derived from kongensin F. As for the cytotoxic activity, the FD-4A activity was three times higher than the dichloromethane extract activity. The extracts of ethyl acetate and methanol showed the highest antioxidant activity: 12.11 and 11.77 gQE/100g, respectively. It was observed that flavonoids were the phenolic substances that most influenced the antioxidant activity of the fractions of the ethyl acetate and methanol extracts. With these results we suggest that C. pallidulus var. pallidulus has antibacterial activity, cytotoxic and antioxidant properties, being a potential source of new drugs for treatment of infectious diseases and cancer. We also isolated three 8,9-secokauranes derived from kongensin F, which apparently had not been described yet

Croton (Euphorbiaceae), é um gênero promissor para pesquisa de compostos bioativos, tanto pela variedade de compostos químicos encontrados, quanto pela diversidade de uso na medicina tradicional/popular. Estudos farmacológicos comprovaram atividades antimicrobianas, antivirais, anti-inflamatórias, antioxidantes e citotóxicas. Neste trabalho, foram obtidos extratos fracionados em Soxhlet (hexano, diclorometano, acetato de etila e metanol) de folhas e caules de C. pallidulus Baill var. pallidulus (Baill.) L.B. Smith & S.F. Smith. Esses extratos foram avaliados quanto às atividades 1) antibacteriana pelo método de microdiluição em caldo, utilizando Staphylococcus aureus - cepas sensível e resistente à vancomicina, Pseudomonas aeruginosa, Escherichia coli e Salmonella choleraesuis; 2) antioxidante (sequestro do radical livre DPPH); e quanto à toxicidade frente à Artemia salina. A fração mais ativa neste último ensaio foi selecionada para a avaliação da atividade citotóxica utilizando as células de sarcoma uterino humano. Os fenóis totais, e flavonóis e flavonas totais foram quantificados por métodos colorimétricos. O fracionamento dos extratos hexânico e diclorometânico foi feito por cromatografia em coluna de sílica, enquanto o dos extratos de acetato de etila e metanol, em coluna de PVPP. O isolamento das substâncias das frações ativas nos ensaios de atividade antibacteriana (S. aureus - sensível) e de toxicidade frente à A. salina foi realizado por CLAE semipreparativo. O teor de fenóis totais de C. pallidulus var. pallidulus foi de 1,95 g EAG/100g MS e de flavonóis e flavonas totais foi de 0,56 g EQ/100g MS. Todos os extratos apresentaram atividade contra S. aureus (sensível e resistente), com CBM de 2048 mg/L, exceto o extrato metanólico que apresentou atividade apenas contra S. aureus (sensível). Nenhum extrato apresentou atividade contra as demais cepas. As frações FH-5A - hexano (CBM de 256mg/L) e FA-7 - acetato de etila (CBM de 1024mg/L) foram as que apresentaram maior atividade antibacteriana. A fração FH-5A foi separada em coluna de sílica, e suas subfrações apresentaram atividades antibacterianas menores que a fração que lhe deu origem. Das sete substâncias majoritárias isoladas da fração FA-7, nenhuma apresentou atividade antibacteriana. Seis delas são flavonoides: três C-glicosídeos de apigenina, um O-glucosídeo de campferol, um O-galactosídeo de isoramnetina e um O-rutinosídeo de isoraminetina. Apenas o extrato diclorometânico apresentou atividade no ensaio de toxicidade frente à A. salina, com CL50 de 385mg/L. A fração com maior toxicidade foi a FD-4A, com CL50 de 101mg/L. As substâncias mais abundantes nesta fração foram isoladas e identificadas como 8,9-secocauranos, derivados da kongensina F. Quanto a atividade citotóxica, a atividade de FD-4A foi três vezes maior que a atividade do extrato diclorometânico. Os extratos de acetato de etila e de metanol apresentaram os maiores valores de atividade antioxidante: 12,11 e 11,77 gEQ/100g, respectivamente. Verificou-se que os flavonoides foram as substâncias fenólicas que mais influenciaram na atividade antioxidante das frações dos extratos de acetato de etila e de metanol. Com esses resultados verifica-se que C. pallidulus var. pallidulus possui ação antibacteriana, citotóxica e antioxidante, sendo uma potencial fonte para novos fármacos para tratamento de doenças infecciosas e câncer. Conseguiu-se também isolar três 8,9-secocauranos, derivados da kongensina F, que aparentemente não haviam ainda sido descritos / Croton (Euphorbiaceae), is a promising genre for investigation of bioactive compounds, both for the variety of compouds, as for the diversity of use in traditional medicine. Pharmacological studies have demonstrated antimicrobial, antiviral, anti-inflammatory, cytotoxic, and antioxidant activities. In this study, fractionated extracts of leaves and stems of C. pallidulus Baill var. pallidulus (Baill.) L.B. S.F. Smith & Smith were obtained throught Soxhlet (hexane, dichloromethane, ethyl acetate and methanol). The following activities of these extracts were tested: 1) antibacterial activity, through microbroth dilution assay using: Staphylococcus aureus - sensitive and vancomycin-resistant strains, Pseudomonas aeruginosa, Escherichia coli and Salmonella choleraesuis; 2) antioxidant activity (free radical DPPH scavenging) and Artemia salina toxicity evaluation. The most active fraction in the latter test was selected to evaluate the cytotoxic activity using human uterine sarcoma cells. The total phenols, and total flavonols and flavones were quantified by colorimetric methods. The fractionation of hexane and dichloromethane extracts was done through silica column chromatography, while PVPP column was used for the ethyl acetate and methanol extracts. The isolation of the active fractions major compounds in the antibacterial activity (S. aureus - sensitive) and A. saline toxicity assays were performed by semipreparative HPLC. The total phenolic content of C. pallidulus var. pallidulus was 1.95 g GAE/100g DM and total flavonols and flavones was 0.56 g QE/100g MS. All extracts showed activity against S. aureus (sensitive and resistant), with MBC of 2048 mg/L, except for the methanol extract, which showed activity only against S. aureus (sensitive). Extracts showed no activity against the other strains. Fractions FH-5A-hexane (MBC: 256mg/L) and FA-7-ethyl acetate (MBC: 1024mg/L) showed the highest antibacterial activity. The fraction FH-5A was separated on a silica column, and its subfractions showed smaller antibacterial activity than FH-5A. The seven major compounds isolated from the FA-7 fraction, showed no antibacterial activity. Six of them are flavonoids, three apigenin C-glycosides, one kaempferol O-glucoside, one isorhamnetin O-galactoside and one isorhamnetin O-rutinoside. Only the dichloromethane extract showed activity in the A. salina toxicity assay, LC50: 385mg/L. The fraction with the highest toxicity was the FD-4A, LC50: 101mg/L. The most abundant substances in this fraction were isolated and identified as 8,9 - secokauranes derived from kongensin F. As for the cytotoxic activity, the FD-4A activity was three times higher than the dichloromethane extract activity. The extracts of ethyl acetate and methanol showed the highest antioxidant activity: 12.11 and 11.77 gQE/100g, respectively. It was observed that flavonoids were the phenolic substances that most influenced the antioxidant activity of the fractions of the ethyl acetate and methanol extracts. With these results we suggest that C. pallidulus var. pallidulus has antibacterial activity, cytotoxic and antioxidant properties, being a potential source of new drugs for treatment of infectious diseases and cancer. We also isolated three 8,9-secokauranes derived from kongensin F, which apparently had not been described yet

La présente étude s’attache à étudier l’effet d’une source de carbone organique sélectionnée sur la croissance et la production de métabolites de la microalgue rouge Porphyridium marinum. Cette dernière s’est montrée incapable de se multiplier en hétérotrophie, en absence totale de lumière. Cependant, en condition mixotrophique la production de biomasse, lipides et de phycobiliprotéines par P. marinum a été améliorée en comparaison avec la condition autotrophique. Les teneurs en exopolysaccharides ont été presque similaires dans les deux conditions. Dans le but de valoriser ces métabolites, l’effet antioxydant, antibactérien, antibiofilm et anticancéreux ont été testés. L’exopolysaccharide de P. marinum ainsi que ses dérivés de faible poids moléculaire (EPS-2P et EPS-5P) ont tous présenté des activités antibactériennes et antibiofilm à différentes concentrations. Cependant l’EPS-2P et l’EPS-5P ont été jugés plus efficaces pour l’activité anticancéreuse contre les cellules de cancer de sein. D’autre part, la production du pigment majoritaire des microalgues rouges, la B-phycoerythrine (B-PE), a été optimisée moyennant des plans d’expériences adaptés. Une teneur de 40 mg/g MS a été obtenue en faisant varier la concentration en NaNO3, K2HPO4 et solution métallique ainsi que l’intensité lumineuse. Après purification, la molécule optimisée a montré une activité antioxydante en termes de piégeage des radicaux libres DPPH, chélation et réduction des ions de fer et inhibition de la décoloration du β-carotène. Nos données suggèrent alors que les métabolites produits par la microalgue rouge P. marinum peuvent être potentiellement utilisé dans plusieurs applications à savoir cosmétiques et pharmaceutiques. / The present study investigates the effect of a selected organic carbon source on the growth and production of metabolites of the red microalga Porphyridium marinum. The latter has been unable to multiply in heterotrophy, in total absence of light. However, in mixotrophic condition the productions of biomass, lipids and phycobiliproteins by P. marinum have been improved in comparison with the autotrophic condition. The contents of exopolysaccharide were almost similar under both conditions. In order to valorize these metabolites, the antioxidant, antibacterial, antibiofilm and anticancer effect were tested. The exopolysaccharide of P. marinum as well as its low molecular weight derivatives (EPS-2P and EPS-5P) have all exhibited antibacterial and antibiofilm activities at different concentrations. However, EPS-2P and EPS-5P were found to be more effective for anti-cancer activity against breast cancer cells. On the other hand, the production of the majority pigment of red microalgae, B-phycoerythrin (B-PE), has been optimized by means of adapted experimental plans. A content of 40 mg/g DW was obtained by varying the concentration of NaNO3, K2HPO4 and metal solution as well as the light intensity. After purification, the optimized molecule showed antioxidant activity in terms of free radical scavenging DPPH, chelation and reduction of iron ions and β-carotene bleachinginhibition. Our data suggest that the metabolites produced by the red microalga P. marinum may be potentially used in several applications namely cosmetic and pharmaceutical.

As peroxirredoxinas constituem uma família de tiol-proteínas, que reduzem peróxido de hidrogênio, peróxidos orgânicos e peroxinitrito a água, álcool e nitrito, respectivamente, utilizando equivalentes redutores fornecidos pela tiorredoxina, tiorredoxina redutase e NADPH. As peroxirredoxinas são enzimas abundantes (constituem aproximadamente 0,7 % do total de proteínas solúveis presentes em leveduras) e foram identificadas em diversas espécies de animais, plantas e bactérias, porém seu papel fisiológico ainda é discutido. Até recentemente, as peroxirredoxinas eram consideradas pouco eficientes para detoxificar peróxidos, em comparação às catalases e heme-peroxidases. De fato, as constantes de segunda ordem determinadas para as reações de peroxirredoxinas com peróxido de hidrogênio eram da ordem de 104-105 M-1 s-1, valores muito menores que os de hemeproteínas (~107 M-1 s-1). Neste trabalho, um método de cinética competitiva foi desenvolvido para re-determinar essas constantes de velocidade, utilizando a peroxidase de raiz forte como competidora das peroxirredoxinas de S. cerevisiae, Tsa1 e Tsa2. Este método foi validado e as constantes de velocidade determinadas para Tsa1 e Tsa2 foram da ordem de k~ 107 M-1 s-1 para a reação com peróxido de hidrogênio e da ordem de k~10105 M-1 s-1 para a reação com peroxinitrito. Utilizando a mesma metodologia, foi possível ainda determinar o pKa da cisteína peroxidásica da Tsa1 e Tsa2 (Cys47), como sendo 5,4 e 6,3, respectivamente. Paralelamente, o papel fisiológico das peroxirredoxinas foi examinado em linhagens de S. cerevisiae com deleção de Tsa1, Tsa2 ou de ambas isoformas. Os estudos foram realizados sob condições fermentativas e a linhagem tsa1Δtsa2Δ se mostrou mais resistente ao peróxido de hidrogênio (1 mM) e o consumiu mais rapidamente que a WT. Além disso, a linhagem tsa1Δtsa2Δ produziu quantidades mais altas do radical 1-hidroxietila, produto da oxidação do etanol, que é o principal metabólito da levedura em anaerobiose. O mecanismo de formação do radical 1-hidroxietila foi examinado e a quantificação da concentração de ferro quelatável, ferro total e cobre mostrou que a reação de Fenton não era sua principal fonte. Outro mecanismo investigado foi a formação do radical através da atividade peroxidásica da Sod1, cuja expressão e atividade se mostraram aumentadas cerca de 5 e 2 vezes, respectivamente, na linhagem tsa1Δtsa2Δ. Na linhagem mutante ainda foi observado que o tratamento com peróxido de hidrogênio aumentou a concentração de radicais derivados e adutos do DNA, detectados por imuno-spin trapping e incorporação de 14C derivado da glicose. Em conjunto, os resultados deste trabalho reforçam a importância das peroxirredoxinas na defesa antioxidante e mostram que as respostas compensatórias empregadas pela levedura para contornar as deleções de Tsa1 e Tsa2 podem ser deletérias longo prazo. / Peroxiredoxins constitute a family of cysteine-based peroxidases that are able to reduce hydrogen peroxide, organic peroxides and peroxinitrite to water, alcohol and nitrite, respectively, through the use of reducing equivalents provided by thioredoxin, thioredoxin reductase and NADPH. Peroxiredoxins are abundant enzymes (correspond to approximately 0.7% of total soluble protein in yeasts) and have been identified in several species ranging from animals, plants and bacteria, but their physiological role remains under scrutiny. Peoxiredoxins were regarded as less eficient enzymes in comparison with catalases and heme-peroxidases for detoxification of peroxides. Second-order rate constants determined for the reaction of peroxiredoxins with hydrogen peroxide were in the range of 104-105 M-1 s-1, which is quite low, as compared with those of heme-proteins (~107 M-1 s-1). In the present work, a competitive kinetic approach with horseradish peroxidase was developed in order to determine the second order rate constant of the reaction of peroxiredoxins with peroxynitrite and hydrogen peroxide. This method was validated and permitted for the determination of the second order rate constant value of the reaction of Tsa1 and Tsa2 with peroxynitrite (k~105 M-1 s-1) and hydrogen peroxide (k~ 107 M-1 s-1) at pH 7.4, 25 °C. It also permitted the determination of the pKa of the peroxidatic cysteine of Tsa1 and Tsa2 (Cys47) as 5.4 and 6.3, respectively. In parallel, the physiological role of peroxiredoxins was examined in S. cerevisiae strains with deletion of Tsa1, Tsa2 or of both isoforms. Under fermentative conditions, tsa1Δtsa2Δ cells were more resistant to 1 mM hydrogen peroxide than WT cells, and consumed it faster. In addition, tsa1tsa2 cells produced higher yields of the 1- hydroxyethyl radical from the oxidation of the glucose metabolite ethanol, as shown by spintrapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1Δtsa2Δ cells with respect to their levels of chelatable iron ions, total iron and copper ions, and of 1-hydroxyethyl radical produced in the presence of metal ion chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Sod1, whose expression and activity increased about five- and twofold, respectively, in tsa1Δtsa2Δ compared to WT cells. Relevantly, tsa1Δtsa2Δ cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of 14C from glucose into DNA, respectively. Taken together, our results reinforce the importance of peroxiredoxins in the antioxidant defense show that the compensatory responses employed by yeast to counterbalance the deletions of Tsa1 and Tsa2 may be deleterious in the long time range.

L'oligomérisation enzymatique de la rutine et esculine a donné lieu à cinq fractions d'oligomères de masse moléculaire moyenne entre 2127,42 et 8331,85 g/mol pour la rutine et 688,12 et 6973 g/mol pour l'esculine. L'analyse de ces fractions par FTIR montre que les fractions d'oligorutines sont obtenues à travers des liaisons C-C, C-O et C=O. Les fractions d'oligoesculines sont obtenues à travers des liaisons C-C. Une meilleure solubilité des oligorutines et des oligoesculines dans l'eau et une plus faible solubilité de ces oligomères dans l'éthanol comparé à leurs monomères a été mis en évidence. Une diminution de l'activité antiradicalaire vis-à-vis de DPPH., ABTS+. et OH. proportionnelle à la masse moléculaire moyenne des fractions d'oligorutines a été observé, contrairement aux fractions d'oligoesculines qui montrent un important pouvoir chélateur de ces mêmes radicaux comparé à leurs monomère. Une augmentation du pouvoir chélateur de fer, inhibiteur de la xanthine oxydase, réducteur du cuivre (CUPRAC), de l'activité antigénotoxique, ainsi que de l'activité stimulatrice de la prolifération des splénocytes, et des lymphocytes (B et T) proportionnelle au degré d'oligomérisation des oligomères étudiées a été noté. L'effet des fractions d'oligorutines et oligoesculines étudiées sur les macrophages en suivant la production de monoxyde d'azote (NO) montre un pouvoir anti-inflammatoire comparé à leurs monomères. L'étude de l'activité lysosomale induite par les fractions d'oligorutine révèle un pouvoir immunostimulateur proportionnelle à la masse moléculaire moyenne des oligorutines, et inversement proportionnelle à celle-ci pour les oligoesculines / Rutin and esculin have been polymerized by laccase. Five fractions with between 2127.42 and 8331.85 g/mol for oligorutins, and between 688.12 and 6973 g/mol for oligoesculins, were obtained. Fourier transformed infrared analysis showed that oligorutins were formed through C-C, C-O and C=O linkages, while oligoesculins were obtained through C-C linkages. Oligorutins and oligoesculins show a higher solubility in water and a lower solubility in ethanol compared to their monomers. The oligomerization of rutin decrease its antiradical capacity, while oligoesculin fractions demonstrated a high antiradical activity compared to monomeric esculin. Oligomer fractions showed a better iron chelating power, xanthine oxidase inhibition, copper reducing power (CUPRAC), antigenotoxic activity, and splenocytes stimulator activity compared to their monomers. Oligorutin and oligoesculin exhibited an important anti-inflammatory capacity through the nitric oxide inhibition. Moreover, oligorutin fractions demonstrated an immunostimulatory effect proportional to their degree of oligomerization, while oligoesculin fractions showed an immunostimulatory effect inversely proportional to their degree of oligomerization

As peroxirredoxinas constituem uma família de tiol-proteínas, que reduzem peróxido de hidrogênio, peróxidos orgânicos e peroxinitrito a água, álcool e nitrito, respectivamente, utilizando equivalentes redutores fornecidos pela tiorredoxina, tiorredoxina redutase e NADPH. As peroxirredoxinas são enzimas abundantes (constituem aproximadamente 0,7 % do total de proteínas solúveis presentes em leveduras) e foram identificadas em diversas espécies de animais, plantas e bactérias, porém seu papel fisiológico ainda é discutido. Até recentemente, as peroxirredoxinas eram consideradas pouco eficientes para detoxificar peróxidos, em comparação às catalases e heme-peroxidases. De fato, as constantes de segunda ordem determinadas para as reações de peroxirredoxinas com peróxido de hidrogênio eram da ordem de 104-105 M-1 s-1, valores muito menores que os de hemeproteínas (~107 M-1 s-1). Neste trabalho, um método de cinética competitiva foi desenvolvido para re-determinar essas constantes de velocidade, utilizando a peroxidase de raiz forte como competidora das peroxirredoxinas de S. cerevisiae, Tsa1 e Tsa2. Este método foi validado e as constantes de velocidade determinadas para Tsa1 e Tsa2 foram da ordem de k~ 107 M-1 s-1 para a reação com peróxido de hidrogênio e da ordem de k~10105 M-1 s-1 para a reação com peroxinitrito. Utilizando a mesma metodologia, foi possível ainda determinar o pKa da cisteína peroxidásica da Tsa1 e Tsa2 (Cys47), como sendo 5,4 e 6,3, respectivamente. Paralelamente, o papel fisiológico das peroxirredoxinas foi examinado em linhagens de S. cerevisiae com deleção de Tsa1, Tsa2 ou de ambas isoformas. Os estudos foram realizados sob condições fermentativas e a linhagem tsa1Δtsa2Δ se mostrou mais resistente ao peróxido de hidrogênio (1 mM) e o consumiu mais rapidamente que a WT. Além disso, a linhagem tsa1Δtsa2Δ produziu quantidades mais altas do radical 1-hidroxietila, produto da oxidação do etanol, que é o principal metabólito da levedura em anaerobiose. O mecanismo de formação do radical 1-hidroxietila foi examinado e a quantificação da concentração de ferro quelatável, ferro total e cobre mostrou que a reação de Fenton não era sua principal fonte. Outro mecanismo investigado foi a formação do radical através da atividade peroxidásica da Sod1, cuja expressão e atividade se mostraram aumentadas cerca de 5 e 2 vezes, respectivamente, na linhagem tsa1Δtsa2Δ. Na linhagem mutante ainda foi observado que o tratamento com peróxido de hidrogênio aumentou a concentração de radicais derivados e adutos do DNA, detectados por imuno-spin trapping e incorporação de 14C derivado da glicose. Em conjunto, os resultados deste trabalho reforçam a importância das peroxirredoxinas na defesa antioxidante e mostram que as respostas compensatórias empregadas pela levedura para contornar as deleções de Tsa1 e Tsa2 podem ser deletérias longo prazo. / Peroxiredoxins constitute a family of cysteine-based peroxidases that are able to reduce hydrogen peroxide, organic peroxides and peroxinitrite to water, alcohol and nitrite, respectively, through the use of reducing equivalents provided by thioredoxin, thioredoxin reductase and NADPH. Peroxiredoxins are abundant enzymes (correspond to approximately 0.7% of total soluble protein in yeasts) and have been identified in several species ranging from animals, plants and bacteria, but their physiological role remains under scrutiny. Peoxiredoxins were regarded as less eficient enzymes in comparison with catalases and heme-peroxidases for detoxification of peroxides. Second-order rate constants determined for the reaction of peroxiredoxins with hydrogen peroxide were in the range of 104-105 M-1 s-1, which is quite low, as compared with those of heme-proteins (~107 M-1 s-1). In the present work, a competitive kinetic approach with horseradish peroxidase was developed in order to determine the second order rate constant of the reaction of peroxiredoxins with peroxynitrite and hydrogen peroxide. This method was validated and permitted for the determination of the second order rate constant value of the reaction of Tsa1 and Tsa2 with peroxynitrite (k~105 M-1 s-1) and hydrogen peroxide (k~ 107 M-1 s-1) at pH 7.4, 25 °C. It also permitted the determination of the pKa of the peroxidatic cysteine of Tsa1 and Tsa2 (Cys47) as 5.4 and 6.3, respectively. In parallel, the physiological role of peroxiredoxins was examined in S. cerevisiae strains with deletion of Tsa1, Tsa2 or of both isoforms. Under fermentative conditions, tsa1Δtsa2Δ cells were more resistant to 1 mM hydrogen peroxide than WT cells, and consumed it faster. In addition, tsa1tsa2 cells produced higher yields of the 1- hydroxyethyl radical from the oxidation of the glucose metabolite ethanol, as shown by spintrapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1Δtsa2Δ cells with respect to their levels of chelatable iron ions, total iron and copper ions, and of 1-hydroxyethyl radical produced in the presence of metal ion chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Sod1, whose expression and activity increased about five- and twofold, respectively, in tsa1Δtsa2Δ compared to WT cells. Relevantly, tsa1Δtsa2Δ cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of 14C from glucose into DNA, respectively. Taken together, our results reinforce the importance of peroxiredoxins in the antioxidant defense show that the compensatory responses employed by yeast to counterbalance the deletions of Tsa1 and Tsa2 may be deleterious in the long time range.

PhD (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal Universtiy of Technology / The genus Tulbaghia has been used in traditional medicine to treat various ailments such as fever, earache, tuberculosis and esophageal cancer. However, there is limited scientific evidence to support its use. Therefore the objectives of this study were to perform phytochemical analysis, investigate the antioxidant, antimicrobial, anticancer, immunomodulatory activities and toxicity of crude acetone and water extracts from selected Tulbaghia species. Standard methods were used for preliminary phytochemical analysis. The total phenolic content of the plant extracts was determined using the folin ciocalteu method whereas the total flavonoids were determined by using the aluminium chloride colorimetric method. DPPH and ABTS assays were used to evaluate the antioxidant activity. The antimicrobial activity was assessed by agar well diffusion, microtiter dilution and time kill assays. For anticancer studies, the antiproliferative activity of the extracts was evaluated using the MTT assay on Hkesc-1 and KB cells. Morphological changes of the cancer cells treated with extracts were examined using light microscopy. Induction of apoptosis was assessed using fluorescence microscopy and acridine orange/ethidium bromide staining. Flow cytometry analysis was conducted to examine the multicaspase activity and cell cycle arrest. For immunomodulatory activity, the Greiss reagent and Luminex cytokine assays were used to determine the effect of the extracts on NO production and the concentration of the cytokines in the treated cells, respectively. Toxicity of selected Tulbaghia species was examined by investigating the effect of the extracts on the metabolic activity and cell membrane integrity on the treated RAW264.7 cells using the MTT and LDH assays, respectively. The zebrafish assay was used to evaluate the embryotoxicity and teratogenic effects of crude acetone and water extracts of T. violacea at 24 h intervals for 96 h post fertilisation (hpf). The percentage mortality, hatchability and heart rate were examined. Phytochemical screening of eight Tulbaghia species demonstrated the presence of flavonoids, glycosides, tannins, terpenoids, saponins and steroids. The amount of total phenol and flavonoid content varied in different plant extracts ranging from 4.50 to 11.10 milligrams gallic acid equivalent per gram (mg GAE/g) of fresh material and 3.04 to 9.65 milligrams quercetin equivalent per gram (mg QE/g) of fresh material respectively. The IC50 values based on DPPH and ABTS for T. alliacea (0.06 and 0.06 mg/mL) and T. violacea (0.08 and 0.03 mg/mL) were generally lower showing potential antioxidant activities. For antimicrobial activity, the acetone extracts of T. acutiloba, T. alliacea, T. leucantha, T. ludwigiana, T. natalensis and T. simmleri showed moderate antimicrobial activity against all test organisms while the water extracts showed moderate to no activity. One species, T. cernua, showed poor activity against all the tested microbes. The acetone and water extracts of T. violacea showed the greatest antibacterial and antifungal activity against all the tested microorganisms with minimum inhibitory concentration ranging from 0.1 mg/mL to 3.13 mg/mL. The acetone extracts of T. violacea also exhibited both bacteriostatic/fungistatic and bactericidal/fungicidal activity depending on the incubation time and concentration of the extract. The bactericidal/fungicidal activity was observed at x2 MIC. The results for anticancer activity showed that treatment of Hkesc-1 cells with acetone and water crude extracts had anti-proliferative activity with IC50 values of 0.4 mg/mL and 1.625 mg/mL, respectively while KB had 0.2 mg/mL and 1 mg/mL, respectively. Morphological changes such as blebbing, cell shrinkage and rounding were observed in the treated cells suggesting that apoptosis was taking place. AOEB staining showed that the level of apoptosis was dependent on the concentration of the extracts. The activation of multicaspase activity in both Hkesc-1 and KB treated cells was also concentration dependent leading to cell death by apoptosis and the induction of cell cycle arrest at the G2/M phase. Immunomodulatory activity results indicated that cell viability was above 80% when concentrations of 50 µg/mL or less of both acetone and water crude was used. Treatment with the acetone extract had no significant effect (p>0.05) on the LPS induced NO production in RAW264.7 cells except at 50 µg/mL where significant inhibition was observed. The water extract had no significant effect (p>0.05) on NO production at all the concentrations. Treatment of LPS–induced RAW264.7 cells with acetone extract stimulated the production of IL-1α, IL-6 and TNF-α, but had no significant effect (p > 0.05) on IL-1β. On the other hand, treatment with the water extracts stimulated the production of IL-1α, IL-6 but had no significant effect (p>0.05) on TNF-α and IL-1β. Treatment of LPS-induced RAW264.7 cells with the acetone extract had very little stimulatory effect on IL-4, IL-5 and IL-13 and no significant effect on IL-10 whereas for the water extract a significant stimulatory effect was only observed for IL-4 after 48 h of treatment. High concentrations (>10000 pg/mL) of MCP-1, MIP1-α, MIP1-β, MIP-2, GCSF, GM-CSF, RANTES and IP-10 were also observed in acetone and water extract treated RAW264.7 cells. For toxicity studies, acetone and aqueous crude leaf extracts from T. alliacea, T. simmleri, and T. violacea had a significant inhibitory (p<0.05) effect on the RAW264.7 cells after 48h treatment. Acetone extracts from T. alliacea, T. simmleri and T. violacea resulted in IC50 values of 0.48 mg/mL, 0.72 mg/mL and 0.1 mg/mL, respectively. Treatment with water extracts showed minimal toxic effect indicated by higher IC50 values of 0.95 mg/mL, 2.49 mg/mL and 0.3 mg/mL for T. alliacea, T. simmleri and T. violacea, respectively. The LDH release by macrophages after 24 h treatment with acetone extracts was observed to be concentration dependent while treatment with water extracts did not induce LDH release. The zebra fish assay showed a lethal dose (LD50) for the T. violacea acetone crude extract of 20 μg/mL whereas that for water extract was 85 μg/mL. The observed teratogenic effects included scoliosis, edema of the pericardial cavity, retarded yolk resorption, hook-like/bent tail and shorter body length. In conclusion, the results from this study indicate that the extracts from the eight Tulbaghia species examined contain phytochemicals that may have the antioxidant, antimicrobial, anticancer and immunomodulatory properties. Extracts from T. violacea were observed to be the most potent. This study thus supports the use of T. violacea in treating bacterial and fungal infections in traditional medicine. The results of this study also confirm the anticancer potential of T. violacea. The immunomodulatory activity of the acetone and water extracts from T. violacea indicated a dominantly pro-inflammatory activity. Traditional medicine prepared form T. violacea may be of benefit to individuals with weak immune systems. The toxicity of selected Tulbaghia species was observed to be concentration, extract and time dependent. Therefore, traditional medicine prepared from Tulbaghia extracts should be taken with caution preferably in small doses over a short period of time. Future studies will focus on the identification of the bioactive compound(s) responsible for the antimicrobial, anticancer and immunomodulatory activities.