Introdução: A erva mate (Ilex paraguariensis St. Hilaire) é uma espécie nativa do Brasil, Paraguai e Argentina e tem importante apelo social, econômico e cultural. É utilizada na preparação de vários tipos de bebidas, como chimarrão, tererê e chá-mate, consumidas em toda a América Latina. O chá mate, assim como o chimarrão, apresenta potencial protetor à saúde devido à atividade antioxidante, ente outras, conforme demonstrado em diversos estudos. A alta concentração de compostos bioativos, como as saponinas, os compostos fenólicos e as metilxantinas respondem, em conjunto, pela sua atividade biológica. Objetivos: a) validar metodologia para isolar e quantificar as principais classes de compostos bioativos do chá mate (compostos fenólicos, xantinas, sapogeninas e melaninas); b) avaliar se há diferenças nos teores desses compostos nas diferentes marcas deste produto comercializado em São Paulo; c) avaliar a atividade biológica de cada classe por diferentes métodos in vitro. Metodologia: Foram analisados três lotes das três marcas de chá mate 100 por cento presentes em hipermercados no município de São Paulo. A validação da metodologia para quantificação, empregando CLAE, consistiu no cálculo da exatidão (recuperação), repetibilidade e sensibilidade para os compostos: ácido caféico, ácido-5-cafeoilquínico, cafeína, teobromina, acido ursólico e ácido oleanólico. Foram determinados os teores de fenólicos totais por método espectrofotométrico com o reagente Folin-Ciocalteu. As frações de fenólicos e xantinas, sapogeninas e melaninas foram isoladas e testadas quanto à atividade antioxidante pelo: sistema de oxidação -caroteno e ácido linoléico, teste da capacidade de absorbância de oxigênio radical ORAC (Oxygen Radical Absorbance Capacity), capacidade de seqüestrar o radical DPPH, e atividade ligante de ácidos biliares (ácidos glicocólico, cólico, deoxicólico e taurocólico). Resultados: Os coeficientes de determinação das curvas de calibração foram maiores que 0,99. A recuperação dos compostos variou de 84,2 a 115,88por cento e o coeficiente de variação médio foi 5 por cento. Uma xícara de chá mate (182 mL) apresentou, em média, 161,96 mg de fenólicos totais; 14,71 mg de ácido clorogênico (5CQA); 3,13 mg de ácido caféico ; 2,74 mg de teobromina e 15,16 mg de cafeína. Os teores de ácido ursólico e ácido oleanólico no chá mate (sachês), foram, em média 82,60 g/g e 54,08 g/g, respectivamente. As concentrações dos compostos variaram significativamente entre as marcas analisadas. A atividade antioxidante do chá mate (69,2 por cento de inibição da oxidação pelo sistema -caroteno e ácido linoléico; DPPH (IC50) = 0,014 mgSS/mL; ORAC = 9997molTrolox/xícara de chá), diferiu significativamente (p<0,05) entre as marcas apenas para o IC50. Para as frações houve diferença estatística apenas para a porcentagem de inibição da oxidação do ácido linoléico para o extrato melânico. Verificou-se correlação linear (p<0.05) positiva ou negativa entre a atividade antioxidante e a concentração de alguns compostos bioativos. A capacidade ligante de ácidos biliares foi semelhante ao controle positivo colestiramina para o chá mate, FS e FFX. O EM demonstrou baixa ou nenhuma capacidade de si ligar aos ácidos biliares. Conclusões: O chá mate é uma excelente fonte de compostos bioativos com atividade biológica. Ocorreu variação da concentração de compostos bioativos em função da marca analisada. Essa variação, no entanto, não influenciou na atividade antioxidante da bebida, conforme avaliada pelos métodos ora utilizados. / Introduction: Yerba maté (Ilex paraguariensis St. Hilaire) is a spontaneous species that grows in Brazil, Argentina and Paraguay and several beverages (chimarrão, terere, maté tea) are produced from its leaves. Maté tea, one of I. paraguariensis beverages present antioxidant activity and other biological effects. Compounds such as aponins, polyphenols and methylxanthines, which are present in considerable amounts, are responsible for the biological effects. Objectives: The aims of this work were: a) to validate analytical methodologies to evaluate the main bioactive compounds from maté tea (phenolic compounds, xanthines, sapogenins and melanins); b) to evaluate if there are differences on these compounds contents among maté tea brands commercialized in São Paulo; c) to evaluate biological activity of each class of compounds by different in vitro methodologies. Methods: Three lots from three commercial brands of maté tea present in 100 per cent of the main supermarkets in São Paulo city were analysed. HPLC methodology validation was assessed by determining accuracy (recovery), repeatability and sensibility (linearity, limits of detection and quantitation) for caffeic acid, 5- caffeoylquinic acid, caffeine, theobromine, ursolic and oleanolic acids. Total phenolic content was assessed with the Folin-Ciolcalteu´s reagent. Maté tea phenolic and methylxanthines (FFX), sapogenins (FS) and melanins (EM) fractions were isolated and tested for theirs in vitro antioxidantactivity by the following methods: -carotene and linoleic acid system, ORAC (Oxygen Radical Absorbance Capacity), radical scavenging capacity (DPPH). The ability of binding bile acids (glycocholic, cholic, taurocholic and deoxycholic acids) was also assayed. Results: Calibration curves determination coefficient (r 2) were higher than 0,99 for all compounds. Recovery ranged from 84,2 to 115,88per cent and repeatability average was 5per cent. One maté tea cup (182 mL) contains in average 161,96 mg5CQA of total phenolic compounds, 14,71 mg of chlorogenic acid (5CQA), 3,13 mg of caffeic acid, 2,74 mg of theobromine and 15,16 mg of caffeine. The roasted leaves contains 82,60 and 54,08 g/g of ursolic and oleanolic acids in average, respectively. Compounds contents varied significantly (p<0,05) among brands. The antioxidant activity assays showed no significant difference (p>0,05) among brands to maté tea, except for DPPH (IC50), and the average results for the maté tea are the following: 69,2 per cent in -carotene and linoleic acid system, DPPH (IC50) = 0,014mgSS/mL; ORAC = 9997,7molTrolox/tea cup. Antioxidant activity of the isolated fractions showed significant difference only in -carotene and linoleic acid system to melanic extract. A positive or negative correlations between the antioxidant activity and the bioactive compounds contents was observed (p<0.05). Maté tea, FS and FFX bile acid binding capacity were similar to the positive control cholestyramine. EM showed very little or no bile acid binding capacity. Conclusions: Maté tea is a source of bioactive compounds. Although concentration can vary significantly from brand to brand, this fact did not affect maté tea antioxidant activity evaluated by the methods used in this study.

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No. of bitstreams: 1 robertacristinanovaesreiscorrales.pdf: 3333393 bytes, checksum: a90614981046f5f6e0791845afd8c1fd (MD5) Previous issue date: 2011-06-17 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A tese de doutorado intitulada Síntese e Avaliação Biológica de Derivados de 6-Mercaptopurina, Carboidratos e Aminoálcoois está apresentada em três capítulos que descrevem a síntese e caracterização de compostos com potencial atividade antiparasitária (Leishmania, Plasmodium berghei), antibacteriana (bactérias Gram positiva e negativa, Mycobacterium tuberculosis) e em macrófagos peritoneais de mamíferos. Foram obtidos 53 compostos neste trabalho, sendo 30 inéditos, a saber: no capitulo 1 foi descrita a síntese de 27 compostos, sendo 14 derivados inéditos de 6mercaptopurina (6-MP); no capítulo 2 foi descrita a síntese de 14 compostos, sendo 6 derivados inéditos da D-glicose e 1 derivado inédito da D-ribonolactona; no capítulo 3 foi descrita a síntese de 14 compostos, sendo 9 aminoálcoois inéditos. O primeiro capítulo mostra a síntese de derivados de 6-MP contendo 1,2,3-triazol e derivados de esteróides. Os derivados triazólicos de 6-MP foram obtidos através de uma reação de cicloadição 1,3-dipolar tipo “click” usando um alcino terminal e um grupo azido. Os derivados de 6-MP contendo esteróides, sem o espaçador triazólico, foram obtidos através de uma reação de substituição nucleofílica entre o sal de 6-MP e mesilatos do ácido cólico e do ácido desoxicólico. Dentre os compostos submetidos à avaliação biológica, os derivados de 6-MP conjugados com esteróides apresentaram melhor atividade em Leishmania e a maioria apresentou importante atividade em P. berghei. Nenhum composto testado apresentou citotoxicidade in vitro para macrófagos peritoneais de camundongos até a máxima concentração de 48 µg/mL. O segundo capítulo mostra a síntese e caracterização de derivados da D-glicose contendo 1,2,3-triazol, obtidos através de reação tipo “click” e de derivados da D-gliconolactona e D-ribonolactona. Apesar dos compostos testados não terem apresentado atividade antiparasitária e antibacteriana efetiva, nenhum apresentou toxidez para os macrófagos de mamíferos. O terceiro capítulo descreve a síntese e caracterização de derivados aminoálcoois aromáticos com variada extensão de cadeia e de função química e apresentaram importante atividade biológica, principalmente em L. major. As estruturas dos produtos obtidos foram elucidadas pelos seus espectros na região do infravermelho, Ressonância Magnética Nuclear de 1H e 13C, Mapa de contornos homonuclear COSY, faixa de fusão e espectros de massas de alta resolução. / The doctoral thesis entitled Synthesis and Biological Evaluation of Derivatives of 6-Mercaptopurine, Carbohydrates and Aminoalcohol is presented in three chapters that describe the synthesis and characterization of compounds with potential antiparasitic activity (Leishmania, Plasmodium berghei), antibacterial (bacteria Gram positive and negative, Mycobacterium tuberculosis) and peritoneal macrophages of mammals. 53 compounds were obtained in this work, with 30 firsts, namely: Chapter 1 was described in the synthesis of compounds 27, 14 novel derivatives of 6-mercaptopurine (6MP), was described in Chapter 2 the synthesis of compounds 14, 6 being derived from unpublished 1 D-glucose and derived novel D-ribonolactona, was described in Chapter 3 the synthesis of compounds 14, 9 amino unpublished. The first chapter shows the synthesis of derivatives of 6-MP containing 1,2,3triazole derivatives and steroids. The triazole derivatives of 6-MP were obtained by a reaction of type 1,3-dipolar cycloaddition "click" using a terminal alkyne and an azide group. Derivatives of 6-MP containing steroids, without the spacer triazole, was obtained through a nucleophilic substitution reaction between the salt of 6-MP and mesylates cholic acid and deoxycholic acid. Among the compounds subjected to biological evaluation, derivatives of 6-MP in conjunction with steroids showed better activity in Leishmania and most showed a significant activity in P. berghei. No compound tested showed cytotoxicity in vitro for mouse peritoneal macrophages to g / mL.µthe maximum concentration of 48 µg/mL. The second chapter shows the synthesis and characterization of D-glucose derivatives containing 1,2,3-triazole, obtained by reaction type "click" derivatives of Dgliconolactona and D-ribonolactona. Although the compounds tested did not show effective antibacterial and antiparasitic activity, showed no toxicity to mammalian macrophages. The third chapter describes the synthesis and characterization of aromatic amino derivatives with varied chain length and chemical function and had significant biological activity, especially in L. Major. The structures of the products obtained were elucidated by their spectra in the infrared, 1H NMR and 13C, homonuclear COSY contour map, melting point and mass spectra with high resolution.

Abstract Liquid chromatography (LC) combined with mass spectrometry (MS) is one of the most widely used techniques in modern analytical laboratories. Remarkable developments during the two previous decades in both techniques has made LC-MS the method of choice in various environmental, pharmaceutical and biochemical laboratories due to selectivity, sensitivity and versatility. The main focuses in this study were to develop new LC-MS methods to identify and quantify phenolic secondary metabolites in bilberry, lingonberry and hybrid bilberry, to study the biosynthesis of the main secondary metabolites (hypericin and hyperforin and their derivatives) in St John’s wort (SJW) both in vitro and in vivo (in plant), to identify in vitro metabolites of hyperforin in human liver microsomes and to identify the cytochrome P450 (CYP) enzymes responsible for their formation. Both high-performance liquid chromatography (HPLC) and ultra high-performance liquid chromatography (U-HPLC) in combination with time-of-flight (TOF) and triple quadrupole (QqQ) mass spectrometry were used in this study. Identification of 52 phenolic compounds from the leaves of bilberry, lingonberry and hybrid bilberry was accomplished. In total, seven of the identified compounds were reported for the first time in Vaccinium plants and several other compounds were reported for the first time in the studied plants. Incorporation of valine and isoleucine into acyl side chains of phloroglucinols (hyperforin and adhyperforin) via biosynthesis in shoot cultures of SJW was confirmed by using isotopically labeled amino acids and HPLC-MS/MS. Also, 29 biosynthetic in vitro products for HpPKS2 enzyme originating from SJW were identified based on accurate mass data. The metabolism of hyperforin was studied in human liver microsomes (HLM) for the first time. 57 metabolites for hyperforin were identified in the incubations with HLMs, using a substrate concentration of 1 &#956;M. The phase I metabolism of hyperforin was suggested to rely mainly on CYP3A4 and on CYP2C family. / Tiivistelmä Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on yksi eniten käytetyistä analyysimenetelmistä nykyaikaisissa analytiikkalaboratorioissa. Viimeisten parin vuosikymmenen aikana LC-MS -laitteet ovat kehittyneet merkittävästi, ja nykyään LC-MS onkin paras menetelmä moniin ympäristö-, lääkeaine- ja biokemiallisiin laboratorioihin sen selektiivisyyden, herkkyyden ja monipuolisuuden vuoksi. Tässä väitöskirjassa kehitettiin uusia LC-MS –menetelmiä mustikan, puolukan ja mustikkapuolukan fenolisten sekundäärimetaboliittien tunnistamiseksi ja kvantitoimiseksi, mäkikuisman pääasiallisten sekundäärimetaboliittien (hyperisiini, hyperforiini ja niiden johdannaiset) tutkimiseksi in vitro- ja kasvinäytteistä sekä hyperforiinin aineenvaihduntatuotteiden tunnistamiseksi ja niitä muodostavien sytokromi P450 (CYP) entsyymien tunnistamiseksi ihmisen maksamikrosomeissa in vitro -menetelmin. Tässä työssä käytettiin sekä korkean erotuskyvyn nestekromatografia (HPLC) että ultra-korkean erotuskyvyn nestekromatografia (U-HPLC) yhdistettynä lentoaikamassaspektrometriin (TOF-MS) ja kolmoiskvadrupolimassaspektrometriin (QqQ-MS). Mustikan, puolukan ja mustikkapuolukan lehdistä tunnistettiin yhteensä 52 fenolista yhdistettä. Seitsemää näistä tunnistetuista yhdisteistä ei oltu aiemmin löydetty Vaccinium -suvun kasveista ja useat muut yhdisteistä löydettiin ensimmäistä kertaa nyt tutkituista kasveista. Valiinin ja isoleusiinin liittyminen floroglusinolien (hyperforiini ja adhyperforiini) asyylisivuketjuihin biosynteesin välityksellä varmistettiin isotoppileimattujen aminohappojen ja HPLC-MS/MS –mittausten avulla. Tässä työssä tunnistettiin myös 29 mäkikuismasta peräisin olevan HpPKS2 -entsyymin in vitro biosynteesituotetta tarkan massan mittausten avulla. Hyperforiinin metaboliaa tutkittiin ensimmäistä kertaa ihmisen maksamikrosomeissa (HLM). Hyperforiinille tunnistettiin yhteensä 57 aineenvaihduntatuotetta ihmisen maksamikrosomi-inkubaatioissa, kun hyperforiinin alkukonsentraatio oli 1 &#956;M. Tämän tutkimuksen tulosten perusteella hyperforiinin faasi I metabolia tapahtuu pääasiassa CYP3A4:n ja CYP2C-perheen välityksellä.

Terminalia glabrata et Rauvolfia nukuhivensis sont deux espèces végétales polynésiennes endémiques et menacées d’extinction. T. glabrata cohabite avec T. catappa, commune du Pacifique, ce qui contribuerait à sa vulnérabilité par interfertilité. R. nukuhivensis souffre de problèmes de régénération en raison de stress climatiques, de prédation et d’exploitation anthropique. Des approches de génétique et de chimie ont été developpées afin d’étudier ces deux espèces. Pour T. glabrata, les résultats de « barcoding » ont permis d’établir une très forte proximité génétique avec T. catappa. L’analyse métabolomique a révélé une forte variabilité intraspécifique. Les études génétiques de R. nukuhivensis ont permis de relier des espèces distinctes et d’origines géographiques différentes. Elles ont aussi démontré la présence d’un seul groupe de Rauvolfia dont les individus proviennent des îles de Nuku Hiva et de Ua Huka. Ces travaux ont permis d’établir des relations entre les diversités génétiques et chimiques. L’étude des métabolites secondaires de l’écorce de R. nukuhivensis et de la préparation médicinale a permis d’identifier 13 composés, appartenant à 4 familles chimiques (ajmalanes, sarpaganes, macrolines et  carbolines). 8 de ces composés sont nouvellement identifiés dans la nature et une hypothèse de biosynthèse inédite permettant d’expliquer leur co occurrence chez R. nukuhivensis a été établie. Enfin, ces produits ont été soumis à des tests d’activité pharmacologique. La préparation médicinale a stimulé la prolifération cellulaire et la cicatrisation (tissus FHN). Ces tests ont aussi montré la forte inhibition des canaux ioniques Kv11.1 par les nukuhivensiums. / Terminalia glabrata and Rauvolfia nukuhivensis are endangered Polynesian plant species and endemic. T. glabrata co-exists with a common species from the Pacific T. catappa, thus contributing to its vulnerability by interfertility. R nukuhivensis endures regeneration issues due to climate stress, predation and overexploitation. Hence, these species have been classified as protected species by the authorities and are subjected to conservation plans. Because of their heritage value and their traditional uses, they represent species of cultural importance for the country. Genetics and chemistry approaches were conducted for this study. Concerning T. glabrata, barcoding assays established great similarity with T. catappa. Metabolomics data showed infraspecific variability. Phylogenetic data of Rauvolfia species are consistent with their biogeography, and revealed the existence of an only group of individuals from Nuku Hiva and Ua Huka. Genetic diversity is linked to phytochemical occurence. Investigation of R. nukuhivensis bark metabolites and the traditional remedy led to identification of 13 isolated compounds within 8 new ones, belonging to the ajmalan, sarpagan, macroline and β-carboline skeleton. The co-occurrence of these alkaloid skeletons led to establish an unprecedented biosynthesis route. Finally, isolated compounds and the traditional remedy were submitted to bioassays. The traditional remedy induced cell proliferation and wound healing activities on FHN cells and ion channels Kv11.1 were strongly inhibited by

Fundação de Amparo a Pesquisa do Estado de São Paulo / Pithecoctenium echinatum belongs to the family Bignoniaceae, popularly known as comb monkey used in folk medicine for inflammations, rheumatism, skin infections, headaches, cleanses the blood and calming. The objective of this study was to characterize morphological and anatomical fruit of P. echinatum; to determine the physico-chemical quality of the plant drug, isolating chemical compounds of secondary metabolism, as well as to investigate antimicrobial, antioxidant and anti-inflammatory, and analyzing biochemical parameters of liver and kidney toxicity in the crude extract (CE) of seeds of P. echinatum. This species has bicarpelar ovary with the presence of trichomes, crystals and emergencies. It has parietal placentation. The rudiment seminal anatropous, unitegmic and tenuinucellate. The ring nectary has stomatal complexes and trichomes. The epicarp has cutin with the presence of lenticels, estomatíferos and complex emergencies. The outer mesocarp has parenchyma cells, stone cells, sclerified cells and crystals. The endocarp is represented by a single cell layer is not sclerified. The seed is winged and exotestal. The endothelium and the endosperm are the membranaceous embryo. The mesophyll has chemical compounds such as alkaloids, fixed oils, volatile oils, scarce presence of starch and consists of pectic walls. In physico-chemical characterization, using pharmacopoeia tests was used dried vegetable drug, noting that no significant variation of the samples collected in different locations. The hydroethanolic extract 70% seeds of P. echinatum presented secondary metabolic compounds such as steroids and/or triterpenes, flavonoids, alkaloids, anthraquinones, tannins, catechin, phenolic hydroxyl heterosides cardiotonic and volatile oils. Assay polyphenols, flavonoids and tannins were determined by spectroscopic in the CE and fractions. P. echinatum showed antioxidant potential (IC50 131.84 mg/ml) in acetate fraction by the DPPH method and antimicrobial activity to P. aeruginosa, P. zophii and C. albicans by the microdilution method (MIC). From the CE the seeds of P. echinatum we performed the isolation and identification of hesperetin and diometin. These flavonoids were isolated from P. echinatum first. The compounds were determined and analyzed by HPLC / MS and NMR and their spectra compared with the literature. The anti-inflammatory activity of the CE was assayed by the induction of inflammation for cotton rolls and serum taken from rats submitted to the bioassay, in which were administered orally by 300mg/kg/day of CE for 6 consecutive days and compared to nimesulide (5mg/kg/day) and negative control (propylene glycol 20% v/v). The results showed that the CE of P. echinatum showed significant inhibition of inflammation of 24.7%, similar to the inhibition obtained by the group of animals treated with nimesulide 21.4% compared to controls. The hematological parameters showed an increase in the number of leukocytes and decreased of corpuscular medium volume, while AST, ALT and creatinine showed no significant difference in the use of CE compared with the control group propylene glycol. Therefore, the physico-chemical parameters established for P. echinatum enable quality control of drugs of this kind, and this presented antimicrobial, antioxidant and anti-inflammatory activities. Furthermore, no evidence of a hepatotocidade and nephrotoxicity in the experimental conditions tested. / Pithecoctenium echinatum pertence à família Bignoniaceae, conhecida popularmente como pente-de-macaco e é utilizada na medicina popular para inflamações, reumatismo, infecções cutâneas, dor de cabeça, depurativo do sangue e calmante. O objetivo deste trabalho foi realizar a caracterização morfo-anatômica do fruto de P. echinatum; determinar parâmetros físico-químicos de qualidade da droga vegetal; isolar compostos químicos do metabolismo secundário, bem como investigar atividade antimicrobiana, antioxidante e anti-inflamatória, além de analisar parâmetros bioquímicos de toxicidade hepática e renal no extrato bruto das sementes de P. echinatum. Esta espécie possui ovário bicarpelar com presença de tricomas, emergências e cristais. Possui placentação parietal. O rudimento seminal é anátropo, unitegumentado e tenuinucelado. O anel nectarífero possui complexos estomáticos e tricomas. O epicarpo é cutinizado com presença de lenticelas, emergências e complexos estomatíferos. O mesocarpo externo possui células parenquimáticas, células pétreas, células esclerificadas e cristais. A semente é alada e exotestal. O endotélio e o endosperma constituem o envoltório membranáceo do embrião. O mesofilo cotiledonar possui compostos químicos como alcaloides, óleos fixos e voláteis, presença escassa de amido e é constituído de paredes pécticas. Na caracterização físico-química, por meio de testes farmacopeicos foi utilizado droga vegetal seca, constatando que não houve variação significativa das amostras coletadas em locais diferentes. O extrato hidroetanólico 70% (EB) das sementes de P. echinatum apresentou compostos do metabolismo secundário como esteróides e/ou triterpenos, flavonoides, alcaloides, antraquinonas, taninos catéquicos, oxidrila fenólica e heterosídeos cardiotônicos e óleos voláteis. Por doseamento espectroscópico foram determinados polifenóis, flavonoides e taninos no EB e frações. P. echinatum apresentou potencial antioxidante (CE50 131,84 μg/ml) na fração acetato pelo método do DPPH e atividade antimicrobiana à P. aeruginosa, P. zophii e C. albicans pelo método de microdiluição em caldo (CIM). A partir do EB das sementes de P. echinatum foi realizado o isolamento e a identificação de dois flavonoides diometina e hesperitina, agliconas flavona e flavanona, respectivamente. Estes flavonoides foram isolados de P. echinatum pela primeira vez. Os compostos foram determinados e analisados por CLAE/MS e RMN e seus espectros comparados com a literatura. A atividade anti-inflamatória do EB foi avaliada pelo método da indução da inflamação por cilindros de algodão, e realizadas dosagens séricas dos ratos submetidos ao bioensaio, nos quais foram administrados a três grupos de ratos, por via oral, 300mg/kg/dia do extrato bruto, 5mg/kg/dia de nimesulida e propilenoglicol 20% v/v, por 6 dias. Os resultados obtidos mostraram que o extrato bruto de P. echinatum apresentou inibição da inflamação significativa de 24,7%, semelhante à inibição obtida pelo grupo de animais tratados com nimesulida de 21,4%, em relação ao grupo controle. Os parâmetros hematológicos mostraram que houve aumento no número de leucócitos e diminuição no volume corpuscular médio (VCM), enquanto que AST, ALT e creatinina não apresentaram diferença significativa no uso do extrato de P. echinatum, quando comparados com o grupo controle propilenoglicol. Portanto, os parâmetros físico-químicos estabelecidos para P. echinatum possibilitam controle de qualidade da droga dessa espécie, e esta, apresentou atividades antimicrobiana, antioxidante e antiinflamatória. Além disso, não foram observados sinais de hepatotocidade e nefrotoxicidade nas condições experimentais testadas.

Sida rhombifolia L belongs to the family Malvaceae, it is native to the American continent and can be found in all regions of Brazil. It is popularly known as prickly sida , and used in traditional medicine as anti-inflammatory, antidiabetic and lipid lowering. The aim of this study was to determine and evaluate the physical and chemical parameters of quality in the four seasons and the aerial parts and roots of the plant drug; determining the content of total polyphenols, flavonoids and tannins in the extracts of the aerial parts and roots of S. rhombifolia in four seasons; evaluating the antimicrobial and antioxidant activity in these same extracts; investigating the hypoglycemic and lipid lowering activity of extracts that have a higher content of flavonoids, as well as to analyze biochemical parameters of liver, kidney and pancreatic toxicity and cytotoxicity in cells NCTC clone 929 by incorporation of neutral red dye. The drug was collected at the four seasons in Santa Maria, RS, separated into aerial parts and roots and made hydroethanolic extracts 70% from each of these collections. The physico-chemical parameters varied according to the plant part analyzed and the season. The values found for strange matter, determination of water and ash insoluble in hydrochloric acid are consistent with those established for herbs. The winter collection showed higher content of total ash, both to shoots (21.20%) and roots (17.63%). The swelling rates varied significantly among the aerial parts and roots, and the highest rates obtained for each of the samples were collected in summer (35.47%) and autumn (18.40%), respectively. There were significant differences between the content of total polyphenols and flavonoids throughout the year and these have focused differently from the aerial parts and roots. The content of tannins and did not suffer seasonal variation nor the distribution between the parties analyzed the plant. The extracts of the aerial parts (172.50 g / mL) and roots (374.08 g / mL) of winter presented the best antioxidant activity. Extracts of S. rhombifolia showed good antibacterial activity while showed no activity against fungi. The extracts of the aerial parts and roots of the summer of the winter showed no hypoglycemic and lipid lowering activity in healthy animals, the markers of liver, kidney and pancreatic damage did not vary in relation to the control group, and the IC50 obtained at the cytotoxicity assays were 8.88 mg/mL e 12.57 mg/mL, respectively. / Sida rhombifolia L. pertence à família Malvaceae, é nativa do continente americano e pode ser encontrada em todas as regiões do Brasil. É conhecida popularmente por guanxuma, e utilizada na medicina tradicional como antiinflamatória, hipoglicemiante e hipolipidemiante. O objetivo deste trabalho foi determinar e avaliar os parâmetros físico-químicos de qualidade nas quatro estações do ano e nas partes aéreas e raízes da droga vegetal; realizar o doseamento de polifenóis totais, flavonoides e taninos condensados nos extratos das partes aéreas e raízes de S. rhombifolia nas quatro estações; avaliar a atividade antioxidante e antimicrobiana nestes mesmos extratos; investigar a atividade hipoglicemiante e hipolipidemiante dos extratos que apresentarem maior conteúdo de flavonoides, assim como analisar parâmetros bioquímicos de toxicidade hepática, renal e pancreática e citotoxicidade em células NCTC clone 929 por incorporação do corante vermelho neutro. A droga vegetal foi coletada nas quatro estações do ano no município de Santa Maria, RS, separada em partes aéreas e raízes e feitos extratos hidroetanólicos 70% de cada uma destas coletas. Os parâmetros físico-químicos variaram de acordo com a parte da planta analisada e a estação. Os valores encontrados para matéria estranha, determinação de água e cinzas insolúveis em ácido clorídrico estão de acordo com os estabelecidos para drogas vegetais. A coleta de inverno apresentou maior conteúdo de cinzas totais, tanto para as partes aéreas (21,20%) quanto para as raízes (17,63%). Os índices de intumescência variaram significativamente entre as partes aéreas e raízes, e os maiores índices obtidos para cada uma das coletas foram nas amostras do verão (35,47%) e outono (18,40%), respectivamente. Houve diferença significativa entre os conteúdos de polifenóis totais e flavonoides ao longo do ano e estes concentraram se diferentemente entre as partes aéreas e raízes. O conteúdo de taninos não sofreu variação entre as partes analisadas da planta e de sazonalidade. Os extratos das partes aéreas (172,50 μg/mL) e das raízes (374,08 μg/mL) do inverno foram os que apresentaram melhor atividade antioxidante. Os extratos de S. rhombifolia apresentaram boa atividade antibacteriana e não apresentaram atividade contra fungos. Os extratos das partes aéreas do verão e das raízes do inverno não demonstraram atividade hipoglicemiante e hipolipidemiante em animais sadios. Os marcadores de danos hepáticos, renais e pancreáticos não variaram em relação ao grupo controle, e as IC50 obtidas nos testes de citotoxicidade das partes aéreas e raízes foram 8,88 mg/mL e 12,57 mg/mL, respectivamente.

L’ulcère de Buruli est une maladie nécrotique de la peau présente dans plus de trente pays dans le monde, et affectant principalement le continent africain et l’Océanie. L’infection est due à Mycobacterium ulcerans (M. ulcerans), un micro-organisme qui sécrète une exotoxine appelée mycolactone, représentant le premier polycétide isolé d’un pathogène humain. La maladie est caractérisée par la formation progressive de lésions nécrotiques combinée à une absence de réponse immunitaire et de sensation de douleur ; la mycolactone est connue pour être directement impliquée dans ce mécanisme biologique. A ce jour, aucun traitement totalement performant et spécifique contre l’ulcère de Buruli n’a été développé, ce qui révèle le manque crucial de connaissances sur les mécanismes chimique et biologique. Dans ce contexte, le projet développé s’intéresse à l’élucidation du mécanisme d’action des mycolactones en utilisant la synthèse totale comme outil principal. Pour cela, notre équipe a mis au point une voie de synthèse modulaire permettant la préparation de la toxine naturelle et de ses différents analogues en vue de les tester biologiquement et d’affiner ainsi notre compréhension mécanistique de cette infection. / Buruli ulcer is a necrotizing skin disease present in more than thirty countries in the world, located mainly in West and Central Africa but also in Australia and in Japan. This infection is caused by Mycobacterium ulcerans (M. ulcerans) that secretes a macrolide toxin called mycolactone, which is the first polyketide isolated from a human pathogen. The disease is characterized by the formation of painless progressive necrotic lesions combined with a lack of acute inflammatory response, and mycolactone is known to be directly involved in the biological mechanism. To date no specific and completely efficient treatment of Buruli ulcer has been developed which correlates with the dramatic lack of understanding of the associated chemical and biological mechanisms. In this context, this research project aims at a better understanding of mycolactone A/B molecular interactions by using total synthesis as main tool. To this end, our research team has developed an efficient synthetic pathway allowing the preparation of the natural toxin and its differents analogues for purposes of their biological evaluation and fine-tuning our mechanical understanding of this infection.

Le but principal de ce travail a été de développer de nouvelles méthodologies pour la synthèse de molécules d'intérêt biologique, telles que des azafluorénones, et des précurseurs d'analogues de la variolines B. Tout d'abord, nous avons synthétisé des diarylcétones, précurseurs pour nos cibles, par une séquence déprotocupration-aroylation. Ensuite, nous les avons impliquées dans une étape de couplage pallado-catalysé : les substrats ont subi une C-H activation pour aboutir aux azafluorénones. Nous nous sommes tournés par la suite vers la synthèse d'azafluorénones substituées. Ces dernières ont été synthétisées par un processus auto-tandem en combinant le couplage de Suzuki ou le couplage de Heck avec la cyclisation intramoléculaire catalysée par le palladium. Certaines de ces molécules ont été évaluées pour leurs propriétés biologiques et ont révélé des bonnes activités cytotoxique, antimalariale, antibactérienne et antifongique. Enfin, nous avons réussi à synthétiser des précurseurs d'analogues de variolines en seulement trois étapes à partir d'un produit commercial. / The main purpose of this work was to develop new methodologies for the synthesis of molecules of biological interest, such as azafluorenones and precursors of variolin B analogs. First, we synthesized diarylketones, precursors for our targets, via a deprotocupration-aroylation sequence. Then, we involved them in a pallado-catalyzed coupling step: some substrates underwent C-H activation to provide azafluorenones. Next, we turned our attention to the synthesis of substituted azafluorenones. The latter were synthesized by a tandem process combining Suzuki coupling or Heck coupling with intramolecular cyclization catalyzed by palladium. Some of these molecules were biologically evaluated and showed good biological activities: cytotoxic, antimalarial, antibacterial and antifungal. Finally, we succeeded in synthesizing precursors of variolin analogues in only three steps from a commercial product.

Des hormones gonadotropiques sont utilisées pour la maîtrise de la reproduction dans le domaine vétérinaire. Ces hormones sont actuellement extraites de tissus ou de fluides animaux. L’entreprise CEVA Santé Animale a récemment fait le choix stratégique de produire ces hormones par voie recombinante. L’objectif de cette étude était d’obtenir une gonadotropine chorionique équine recombinante, reCG, pure et biologiquement active, à partir d’une lignée de cellules mammifères CHO. Les étapes de production, de purification et de caractérisation de l’hormone recombinante ont été développées. Les cellules CHO ont été cultivées en fiole d’Erlenmeyer dans différents milieux de culture. Le suivi de la croissance cellulaire et de la quantité d’hormone produite a permis de sélectionner deux milieux. Le procédé de production, avec ces deux milieux, a été optimisé en bioréacteur en contrôlant les paramètres de culture (température, pH). Les protéines produites dans le surnageant, de ces deux cultures, ont été nommées reCG 1 et reCG 2. Un procédé de purification en 3 étapes a été mis au point pour la reCG 1. Plusieurs résines et conditions chromatographiques ont été criblées en microplaques. Les résines multimodales utilisées ont permis d’éliminer des contaminants majeurs grâce à leur sélectivité. Les agrégats de la reCG ont été éliminés grâce à une résine anionique. Le procédé de purification global a été validé pour la reCG 1 et la reCG 2. Il a permis d’obtenir une pureté de 98 % avec un rendement de 80 %. L’activité biologique de la reCG 1 et la reCG 2, in vitro et in vivo, est comparable à celle de la protéine naturelle. L’activité biologique in vivo des reCG est cohérente avec l’étude réalisée sur les glycosylations des hormones et notamment avec leur degré de sialylation. / The gonadotrophic hormones are used for reproduction control in farming animals. Up to now, these hormones were extracted from animal fluids or tissues. The company CEVA Santé Animal has recently decided to produce recombinant versions of these hormones. The objective of this study was to obtain a pure and biologically active recombinant equine chorionic gonadotropin (reCG) after expression in CHO mammalian cells. The production, purification and characterization steps have been developed. CHO cells were grown in Erlenmeyer flasks with different culture media. Two media were selected based on their cell growth potency and of the amount of reCG produced. By using a bioreactor to control key parameters (temperature, pH), the production process was then optimized. The recombinant proteins that accumulated in the supernatant of the two conditions were called reCG 1 and reCG 2. A 3-steps purification process was then developed using reCG 1. Several resins and chromatographic conditions were screened in microplates. Multimodal resins were used to eliminate the main contaminants thanks to their selectivity. reCG aggregates were efficiently eliminated by a chromatographic step with an anionic resin. The overall purification process was finally validated for reCG 1 and reCG 2. Purity and yield were respectively, 98 % and 80 % for the two reCG. We verified that the in vitro and in vivo activities of reCG 1 and reCG 2 were comparable to those of the CG extracted from natural sources. The in vivo assays also confirmed previous studies showing that the degree of glycosylation of an hormone, and most notably their level of sialytation, is important for their biological activity.

The occurrences of cyanotoxin and organic contaminants threaten drinking water sources and are a serious human health and environmental concern. The control of these problematic contaminants and the remediation of the associated contaminants are critical for ensuring safe drinking water to significant populations. Advanced oxidation processes (AOPs) have received considerable attention as a potential water treatment for various pollutants. In this dissertation, advanced oxidative degradation of four problematic water toxic contaminants (CYN, iopamidol, 4-methylcyclohexane methanol and propylene glycol phenyl ether) were studied to develop the fundamental understanding required to assess AOPs as a potential water treatment process. UV and visible light activated (VLA) TiO2 photocatalysis using nitrogen and fluorine-TiO2 (NF-TiO2), phosphorus and fluorine-TiO2 (PF-TiO2) and sulfur-TiO2 (S-TiO2) were employed for degradation of 6-hydroxymethyl uracil (6-HOMU), a model compound for the potent cyanotoxin cylindrospermopsin (CYN). NF-TiO2 exhibits the most photoactive, followed by marginally active PF-TiO2 and inactive S-TiO2 under visible light irradiation. Our results indicate that O2-• plays an important role in VLA TiO2 photocatalysis. Fe (VI), an environmentally friendly oxidant, was employed for the degradation of CYN and 6-HOMU over a range of pH (7 ~ 9.5). The second order rate constants for the reaction of Fe (VI) with CYN decrease from 38.83 ± 0.07 M-1s-1 at pH 7 to 5.02 ± 0.04 M-1s-1 at pH 9.5. Fe (VI) mediated reactions primarily occur via oxidation of the uracil ring in CYN. ELISA results demonstrate that Fe (VI) oxidation process leads to a significant decrease in the bioactivity of CYN as a function of treatment time. Fe (III)-oxalate/H2O2 process was employed for the remediation of iopamidol, a model for ICM, to determine the formation rates and steady concentrations of •OH and O2-• under UV and visible light irradiation. Reduction by CO2-• and oxidation by •OH contribute to the degradation pathways. Pulse and gamma radiolysis of 4-methylcyclohexane methanol (MCHM) and propylene glycol phenyl ether (PPh) were studied to determine the bimolecular rate constants and reaction pathways. •OH addition to ortho and para positions in PPh are the predominant reaction pathways; H-abstraction are the primary reaction mechanisms for ∙OH mediated oxidation of MCHM