Au stade de carcinome in situ, la tumeur mammaire peut être retirée chirurgicalement. mais, à des stades plus avancés, les cellules tumorales peuvent subir une transition épithélio-mésenchymateuse, devenir invasive et chimioresistante. Les produits naturels représentent la majorité de nos médicaments et La biodiversité marine, dont les micro-organismes, est à l'origine de plusieurs médicaments sur le marché, en oncologie. Notre stratégie consiste en la caractérisation de molécules extraites de 70 souches fongiques associées aux algues brunes. Les extraits bruts et les molécules sont selectionnées sur leur capacité à inhiber la prolifération et la migration des cellules tumorales. Un test de viabilité a mis en évidence un extrait issu d'une souche de Paradendryphiella arenaria, très actif sur la lignée épithéliale MCF7 (IC50 de 0.37 µg / mL) et la lignée invasive MCF7-Sh-WISP2 (0.19 µg /mL). La purification de son extrait a permis l'isolement de la hyalodendrine, très cytotoxique (IC50 de 0.07 µg / mL sur les mcf7 et 0.046 µg / mL sur les MCF7-Sh-WISP2), ainsi qu'un nouveau dérivé de pentanorlanostane et du methoxycarbonyl methyl cholate. L'étude du mécanisme d'action de la hyalodendrine a montre des modifications des protéines p53, HSP60, HSP70 et PRAS40. Parallèlement, un test de migration des cellules MCF7-Sh-WISP2 simplifié en plaque 96 puits, a identifié un extrait brut très actif mais non cytotoxique, de Penicillium echinatum. Les molecules de l'extrait one été etudiee de manière déréplicative par élaboration d'un réseau moléculaire. L'ensemble de ces résultats ont montré le fort potentiel thérapeutique de champignons marins par leurs activités anti-metastatiques. / At the in situ carcinoma stage, the breast tumor can be surgically removed. But at later stages, tumor cells can undergo an epithalial-mesenchymal transition, become invasive and chemoresistant. Natural products represent the vast majority of our drugs on the market, especially in oncotherapy. Our experimental strategy consists in isolating and characterizing molecules extracted from 70 fungal strains associated to brown algae. Crude extracts and molecules are then selected for their capacity to inhibit cancer cell proliferation and migration. A first viability assay highlited a crude extract derived from the fungus Paradendryphiella arenaria, very active against the epithelial cancer cell line MCF7 (IC50 of 0.37 µg / mL) and its invasive counterpart MCF7-Sh-WISP2 (0.19 µg / mL). The purification of its extract allowed the isolation of the very cytotoxic hyalodendrin (IC50 of 0.07 µg / mL on MCF7 and 0.046 µg / mL on MCF7-Sh-WISP2), as well as a new pentanorlanostan derivative and the methoxycarbonyl methyl cholate. The mechansim of action of the hyalodendrin revealed p53, HSP60, HSP70 and PRAS40 protein modifications. In parallel, a simplified 96 well plate migration assay let identify a very active but non cytotoxic crude extract, from Penicillium echinatum. The molecules of this extract were studied by dereplication using a molecular network. Together, these results showed the strong therapeutic potential of marine fungi trhough their anti-metastatic activities.

The aplyronines are a family of 24-membered macrolides of polyketide origin, isolated from the Japanese sea hare Aplysia kurodai. They exhibit an exceptional biological activity profile, acting through an actin and tubulin dual-targeting mechanism, with subnanomolar growth inhibitory potency against a diverse range of cancer cell lines. These characteristics render the aplyronines ideal payloads for antibody-drug conjugates but their prohibitively low natural abundance calls for an efficient total synthesis to overcome the supply issue. This dissertation describes the efforts towards developing a second-generation Paterson synthesis of the macrocyclic core of the aplyronines, focused on improving the scalability and selectivity of key transformations. Chapter 1 details the isolation, biological background and previous synthetic efforts towards the aplyronines to illustrate their therapeutic potential and the challenges associated with material sourcing by chemical synthesis. Chapter 2 presents the existing body of work on the aplyronine project within the Paterson group, highlighting the lessons learned over the past two decades and shortcomings to be addressed. Chapter 3 discusses a revised protecting group strategy towards the C1-C27 macrocyclic alcohol 159 with fewer manipulation steps. A refined reaction sequence featuring titanium aldol methodology and an enzymatic desymmetrisation process delivered multigram stocks of the C15-C27 aldehyde 161 upon scale- up, testifying to the robustness of the devised route. Synthesis of the C1-C14 northern fragment 253 closely followed the existing boron aldol approach with optimisation of the C11-C12 alkylation step, geared towards enhancing the regioselectivity. Chapter 4 describes the coupling of the two major fragments using an Horner-Wadsworth-Emmons reaction to assemble the C1-C27 backbone of the cyclic aplyronine core and suitably adjusted endgame steps to enable a one-step oxidative unmasking of the macrolactonisation sites. The first-generation intermediate 159 was accessed via site-specific Yamaguchi esterification and orthogonal deprotection of the C27 allyl carbonate. Discussion in Chapter 5 includes the appendage of the C28-C34 side chain 118, prepared by the known sequence, and suggestions for the future direction of the second-generation route with the outlook of linker appendage for the purposes of antibody-drug conjugate development.

L. japonicus, o popular \"rubim\", pertence à família Lamiaceae. O presente trabalho tem como objetivo contribuir e complementar trabalho realizado anteriormente sobre a mesma espécie, em relação à caracterização farmacobotânica, aspectos químicos e farmacológicos, incluindo atividade antiúlcera, antioxidante e antimicrobiana. A droga, constituída de folhas e caule, foi caracterizada macro e microscopicamente. O extrato hidroetanólico 70% liofilizado (EHEL), preparado com as partes aéreas, obtido através de percolação, apresentou na triagem fitoquímica presença de flavonóides, taninos, saponinas e 0,06% de óleos essenciais. Os taninos presentes na droga vegetal e no extrato foram quantificados e os teores foram respectivamente de 0,06% e de 0,35%. O teor de flavonóides na droga vegetal foi de 0,76% e no EHEL, de 2,3%. O extrato foi fracionado por solventes de polaridades diferentes como clorofórmio, acetato de etila, etanol 50% e etanol. Foi determinado o perfil cromatográfico para o extrato e frações. O extrato apresentou atividade antiúlcera extremamente significativa, no modelo de indução por etanol acidificado, administrado na dose de 400 mg/kg, por via oral, reduzindo índice de Lesão, Área Total de Lesão e Área Relativa de Lesão, sendo mais evidente no próprio extrato e nas frações clorofórmica e acetato e etila, comparativamente ao controle. Não houve inibição de crescimento de Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Campylobacter coli em uma concentração de até 2.000 µg/mL, verificado através da determinação da concentração mínima inibitória pelo método de difusão em placa. O extrato não apresentou resultado significativo em relação a atividade antioxidante testada através do método que reduz o radical 2,2\'-difenil-1-picrilhidrazil (DPPH), e após o equilíbrio da reação, permite calcular a quantidade de antioxidante gasta para reduzir 50% do DPPH. / L. japonicus, popularly known as \"rubim\", belongs to the Lamiaceae family. The objective of the present study is to contribute and complement previous investigations on the same species, in relation to pharmacobotanical characterization, chemical and pharmacological aspects, including antiulcer, antioxidant e antibiotic activities. The drug, formed of leaves and stalk, was macro e microscopically qualified. Flavonoids, tannins, saponins and 0.06% of essential oils are present in the lyophilized 70% hydroalcoholic extract (HE), obtained through percolation. Tannins are present at 0,06% in the vegetable drug and at 0,35% in the extract. The content of flavonoids in the drug was at 0,76% and in the extract (HE), at 2,3%. The extract was fractioned by solvents of different polarities as chloroform, ethyl acetate, absolute ethanol and 50% ethanol. The chromatographic profile of the extract and fractions was determined. The extract, orally administered at the dose of 400 mg/kg, presented extremely significant antiulcer activity in the acidified ethanol induction model, by reduction of the Index of Lesion, Total Area of Lesion and Relative Area of Lesion. The values of the extract and of chloroformic and ethyl acetate fractions were most evident, in relation to control. There was no inhibition of growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Campylobacter coli at a concentration up to 2.000 µg/mL, verified through the the minimum inhibitory concentration by the plate diffusion method. The extract didn\'t present significant result in relation to antioxidant activity tested through the method that reduces the radical 2,2\'-Diphenyl-1-picrylhydrazyl (DPPH), and after the balance of the reaction, it allows to calculate the amount of antioxidant necessary to reduce 50% of DPPH.

Os organismos marinhos constituem uma fonte potencial de metabólitos secundários biologicamente ativos. Neste contexto, os micro-organismos isolados de algas marinhas, dentre eles fungos endofíticos, representam alvos para a pesquisa de novas substâncias com potencial farmacológico pronunciado. Substâncias naturais provenientes de espécies de fungos associados às algas marinhas vêm sendo bastante utilizadas em formulações fotoprotetoras devido à ação antioxidante e ao potencial contra a radiação solar. Deste modo, o presente trabalho teve como objetivo a investigação biológica e química dos fungos endofíticos marinhos pertencentes à família Xylariaceae, o Annulohypoxylon stygium, o Cladosporium sp. e o Acremonium implicatum (Hypocreaceae). A princípio, foi realizado um screening para avaliar a absorção de luz ultravioleta na faixa do UVA e UVB pelos extratos obtidos em escala piloto destes fungos endofíticos associados às algas marinhas. O extrato do fungo A. stygium apresentou intensa absorção na região do UV, mostrando-se promissor para a produção de metabólitos secundários com ação fotoprotetora. Além do ensaio proposto, foi realizada a avaliação do potencial antibacteriano e antifúngico da espécie A. stygium. O estudo químico em escala ampliada deste fungo proporcionou o isolamento e identificação de uma substância inédita da classe derivada da 2,5- dicetopiperazina, 3-benzilideno-2-metil-hexahidro-pirrolo [1,2-?] pirazina-1,4-diona (Sf3), e além desta, foram isolados mais quatro metabólitos como, os diasteroisômeros 1-fenil-1,2- propanediol (Sd2) e 1-fenil-1,2-propanediol (Sd3), 1,3-benzodioxole-5-metanol (Sc1), 1,2- propanodiol-1-(1,3-benzodioxol-5-il) (Se1). Ainda foi possível a desreplicação de substâncias via cromatografia gasosa acoplada à espectrometria de massas (CG-EM), entre elas o ácido palmítico, palmitato de metila, ácido metil linoléico, ácido oléico, álcool benzílico e o piperonal. Quanto ao estudo da atividade biológica, não foi observado potencial antibacteriano e antifúngico para os extratos e frações do fungo. Entretanto, notouse um potencial como fotoprotetor in vitro para as frações n-Hexano/AcOEt (2:3) e n- Hexano/AcOEt (1:4) obtidas a partir do extrato do cultivo de 28 dias do fungo A. stygium, extraído com solventes diclorometano/metanol (CH2Cl2/MeOH 2:1) e para a substância (Sf3) isolada do mesmo. Desta forma, o estudo químico e biológico do fungo Annulohypoxylon stygium demonstrou potencial para a produção de metabólitos secundários com atividade fotoprotetora, visto que uma estrutura inédita com esta atividade foi isolada e identificada como produto natural. / Marine organisms consist of a potential source of biologically active secondary metabolites. In this context, the microorganisms isolated from marine algae, including fungal endophytes represent targets for the search of new substances with pronounced pharmacological potential. Natural substances from fungal species associated with marine algae, have been widely used in sunscreens formulations due to the antioxidant action and the potential against solar radiation. Thus, this study aimed to research biological and chemical profile of marine endophytic fungi belonging to the Xylariaceae family, the Annulohypoxylon stygium, the Cladosporium sp. and Acremonium implicatum (Hypocreaceae). First of all, a screening was performed to evaluate the absorption of ultraviolet light in the range of UVA and UVB by extracts obtained in pilot scale of these endophytic fungi associated with seaweed. The fungus extract A. stygium showed intense absorption in the UV region, being promising for the production of secondary metabolites with sunscreen action. In addition to the proposed test was performed to evaluate the antibacterial and antifungal potential of the species A. stygium. The chemical studies on increased scale of this fungus provided the isolation and identification of a novel compound of the class derived from 2,5-diketopiperazine, 3- benzylidene-2-methylhexahydropyrrolo [1,2-?] pyrazine-1,4-dione (Sf3), and beyond, four metabolites were isolated like of the diasteroisomers 1-phenyl-1,2-propanediol (Sd2) and 1- phenyl-1,2-propanediol (Sd3), 1,3-benzodioxole-5-methanol (Sc1), 1,2-propanodiol-1-(1,3- benzodioxol-5-il) (Se1). Furthermore was possible to dereplicate substances by means of gas chromatography-mass spectrometry (CG-MS) analyses: palmitic acid, methyl palmitate, methyl linoleic acid, oleic acid, benzyl alcohol and piperonal. Regarding to biological activity evaluation, it was not observed antibacterial and antifungal potential for extracts and fractions of the fungus. However, it was observed as a potential sunscreen in vitro for fractions n-Hexane/AcOEt (2:3) and n-Hexane/AcOEt (1:4) obtained from extract after 28 days growth culture of the fungus A. stygium. The mycelia were extracted with solvents dichloromethane/methanol (CH2Cl2/MeOH 2:1) and the compound Sf3 was isolated from the same extract. Thus, the chemical and biological study of the fungus Annulohypoxylon stygium demonstrated the potential for production of secondary metabolites with sunscreen activity, since a novel structure displaying this potential was isolated and identified as a natural product.

Os trabalhos realizados e apresentados nesta tese de doutorado compreendem diversos estudos computacionais e experimentais aplicados ao planejamento de candidados a novos fármacos para o tratamento do câncer, de uma metodologia inovadora para investigar a formação de complexos proteicos e de uma base de compostos naturais reunindo parte da biodiversidade brasileira com a finalidade de incentivar e auxiliar a descoberta e o desenvolvimento de novos fármacos no país. No primeiro capítulo, são descritos estudos que permitiram a identificação e o desenvolvimento de novas moléculas com atividade anticâncer, através da integração de ensaios bioquímicos e métodos de modelagem molecular na área de química medicinal. Dessa forma, estudos de modelagem molecular e ensaios bioquímicos utilizando uma base de compostos disponibilizada pela colaboração com o Laboratório de Síntese de Produtos Naturais e Fármacos (LSPNF) da UNICAMP, permitiram identificar uma série de moléculas da classe ciclopenta-β-indóis como inibidores da polimerização de microtúbulos com considerável atividade anti-câncer. Estes compostos apresentaram-se capazes de modular a polimerização de microtúbulos em ensaios in vitro frente ao alvo molecular e a células cancerígenas, com valores de IC50 na faixa de 20 a 30 μM. Além disso, estudos experimentais permitiram identificar o sítio da colchicina na tubulina como a região de interação desta classe e ensaios de migração celular comprovaram sua atividade antitumoral. A partir dos resultados obtidos, estudos mais aprofundados de docagem e dinâmica molecular permitiram elucidar as interações moleculares envolvidas no processo de ligação à proteína tubulina, e a utilização destes modelos moleculares no planejamento, síntese e avaliação de uma nova série de compostos. Com base nos dados obtidos por estudos computacionais, modificações foram propostas e novos inibidores da polimerização de tubulina foram planejados, sintetizados e avaliados, resultando na identificação de um inibidor de elevada atividade e perfil farmacodinâmico superior dentre as moléculas planejadas, com IC50 de 5 μM. Concomitantemente, ensaios de citotoxicidade in vitro demostraram uma interessante seletividade destes compostos por células cancerígenas em comparação a células saudáveis. Os estudos desenvolvidos com inibidores de tubulina aqui apresentados permitiram identificar moduladores da polimerização de microtúbulos com excelente perfil anti-câncer, que servirão como modelo para o desenvolvimento de novos tratamentos eficazes contra o câncer. No segundo capítulo é apresentado um novo método para predizer modificações conformacionais e a formação de complexos multiméricos em sistemas proteicos. Este método foi elaborado durante os estudos desenvolvidos ao longo de um programa de intercâmbio no laboratório The Center for Theoretical and Biological Physics (CTBP, Rice University, Estados Unidos), sob orientação do professor Dr. José Nelson Onuchic. Durante este projeto, estudos de modelagem computacional foram realizados utilizando métodos computacionais modernos desenvolvidos no próprio CTBP, tal como o método de Análise de Acoplamento Direto (DCA, do inglês Direct-Coupling Analysis) e um método de simulação conhecido como Modelagem Baseada em Estrutura (SBM, do inglês Structure-Based Modeling). Nos estudos aqui apresentados, os métodos DCA e SBM desenvolvidos no CTBP foram combinados, modificados e ampliados no desenvolvimento de uma nova metodologia que permite identificar mudanças conformacionais e elucidar mecanismos de enovelamento e oligomerização em proteínas. Os resultados obtidos através da predição de diversos complexos proteicos multiméricos com uma alta precisão mostram que este sistema é extremamente eficaz e confiável para identificar regiões de interface de contato entre proteínas a a estrutura quaternária de complexos macromoleculares. Esta nova metodologia permite a elucidação e caracterização de sistemas proteicos incapazes de serem determinados atualmente por métodos puramente experimentais. No terceiro capítulo desta tese de doutorado, é descrito a construção de uma base virtual de dados em uma iniciativa pioneira que tem como principal objetivo reunir e disponibilizar o máximo possível de toda a informação já obtida através do estudo da biodiversidade brasileira. Esta base, intitulada NuBBE DataBase, reúne diversas informações como estrutura molecular 2D e 3D e informações de atividades biológicas de diversas moléculas já isoladas pelo Núcleo de Bioensaios Biossíntese e Ecofisiologia de Produtos Naturais (NuBBE), localizado na Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP). A NuBBEDB será de grande utilidade para a comunidade científica, fornecendo a centros de pesquisa e indústrias farmacêuticas informações para estudos de modelagem molecular, metabolômica, derreplicação e principalmente para o planejamento e a identificação de novos compostos bioativos. / The work developed during a doctorate program and shown here as a PhD thesis reports the accomplishment of a series of computational and experimental studies focused on the development of new anticancer agents, an innovative methodology for the investigation of protein complexes formation and of a new database for natural products based on the Brazilian biodiversity, in an effort to assist and encourage the discovery and development of new pharmaceutical drugs inside country. The first chapter describes studies that resulted in the identification and development of new molecules with anticancer activity through the integration of biochemical experiments and molecular modeling methods in the area of medicinal chemistry. Thus, molecular modeling studies and biochemical assays using a library of compounds provided by collaboration with the Laboratório de Síntese de Produtos Naturais e Fármacos (LSPNF) from the University of Campinas (Unicamp) have identified a number of molecules of the cyclopenta-b-indole class as inhibitors for microtubule polymerisation, with substantial anti-cancer activity. These compounds showed to be able to modulate microtubule polymerisation on in vitro assays against the molecular target and cancer cells with IC50 values in the range of 20 to 30 μM. Moreover, experimental studies have identified the colchicine site of tubulin as in the region of interaction of this class and cell migration assays have proven their antitumour activity. Based on these results, further studies using molecular docking and molecular dynamics allowed to elucidate the molecular interactions involved in the binding process to tubulin protein, and these molecular models were used to guide the design, synthesis and evaluation of a novel series of compounds. From the data obtained by computational studies, modifications were proposed to design, synthesise and evaluate new tubulin polymerisation inhibitors, resulting in identification of a high-activity inhibitor and superior pharmacodynamic profile and IC50 of 5 μM. Alongside, in vitro cytotoxicity assays demonstrated an interesting selectivity of these compounds for cancer cells when compared to healthy cells. The studies presented here with tubulin inhibitors allowed to identify modulators of microtubule polymerisation with excellent anti-cancer profile, that will provide a valuable scaffold for the development of new effective treatments against cancer. The second chapter presents a new method for predicting changes on the conformational and the formation of multimeric protein complexes. This method was developed during the studies carried out over an exchange program in the Center for Theoretical and Biological Physics (CTBT, Rice University, USA), under the supervision of professor Dr. José Nelson Onuchic. During this project, computer modeling studies were carried using modern methods developed in the CTBT itself, such as Direct-Coupling Analysis (DCA) and a simulation method known as Modeling Based Structure (SBM). In the studies presented here, the DCA and SBM methods developed in CTBP were combined, modified and expanded to develop a new methodology able to identify the conformational changes and to elucidate mechanisms folding and oligomerization of proteins. The results obtained through prediction of various multimeric protein complexes with high accuracy show that this system is extremely effective and reliable to identify interface contacts between proteins and to predict the quaternary structure of macromolecular complexes. This new method allows the characterization and elucidation of protein systems that are currently unable to be solely determined by experimental methods. The third chapter of this doctoral thesis describes the construction of a virtual database in a pioneering initiative that aims to gather and make available all the information already obtained through the study of Brazilian biodiversity. This database, entitled NuBBE DataBase, brings together various information such as 2D and 3D molecular structure and biological activity of several molecules already isolated by the Núcleo de Bioensaios Biossíntese e Ecofisiologia de Produtos Naturais(NuBBE), located at the Universidade Estadual Paulista Julio de Mesquita Filho (UNESP). The NuBBEDB will be useful to the scientific community, providing research and pharmaceutical centers information for molecular modeling studies, metabolomics, derreplication and principally for the planning and identification of new bioactive compounds.

O presente trabalho mostra a utilização de métodos computacionais e os resultados obtidos na determinação estrutural de produtos naturais, neste caso sesquiterpenos lactonizados. Nosso grupo de pesquisa tem desenvolvido nos últimos anos o projeto denominado SISTEMAT para a determinação estrutural de produtos naturais. Este sistema é composto de programas utilizados para a construção de bancos de dados capazes de armazenar qualquer tipo de informação referente a substâncias orgânicas, e programas que permitem a recuperação e análise das informações contidas nos bancos de dados (programas aplicativos). Foi também construído um programa para a previsão de espectros de RMN 13C utilizando modelos tridimensionais para a representação das moléculas. O programa considera cada carbono e sua vizinhança química em três dimensões como uma subestrutura. Para encontrar o deslocamento químico equivalente a uma nova subestrutura, ele procura no banco de dados um carbono com uma vizinhança equivalente. O banco de dados criado para o SISTEMAT contém 1300 espectros de RMN 13C de sesquiterpenos lactonizados, enquanto que o utilizado para o programa simulador de espectros de RMN 13C possui 1600 substâncias codificadas. / This work shows the use of computational methods and the results obtained in the structure determination of sesquiterpene lactones. Our research group have developed in the last years the expert system named SISTEMAT, to structure determination of natural products. This system have programs used to construct a database able to store any kind of information about the compounds and programs used to retrieve and analyse informations obtained in the database. It was also planned a program to make predictions of 13C NMR spectra of sesquiterpene lactones. It uses a codification working with tridimensional substructures. The program treats each carbon atom and its neighbouring as a substructure. It can scan all the database for other equal or similar chemical shift values that can be used to predict the 13C NMR spectrum for a new compound. The current database contains about 1300 13C NMR spectra of sesquiterpene lactones. The other database used in the program for 13C NMR prediction has 1600 compounds.

Hyperuricemia is the hallmark of gout. Pathogenic mechanisms of hyperuricemia include uric acid overproduction in the liver or underexcretion in the kidney. Current antihyperuricemic agents include xanthine oxidase inhibitors in which allopurinol is the most often prescribed. Inhibitors of renal urate reabsorption such as probenecid and benzbromarone are also employed. However, these existing antihyperuricemic agents possess some undesirable effects such as hypersensitivity towards allopurinol and hepatotoxicity associated with benzbromarone. Therefore, search for alternative antihyperuricemic agents with a more favorable toxicological profile or via mechanisms other than the above two mentioned is highly warranted. / The present project represents such an effort. Four in vitro experimental models were developed for the screening of new antihyperuricemic agents. The effects of the potential compounds from natural sources on the activities of phosphoribosyl pyrophosphate synthetase, hypoxanthine-guanine phosphoribosyl transferase and xanthine oxidase, as well as the uptake of urate through rat renal brush border membrane vesicles were investigated. Several compounds emerged with strong urate uptake inhibitory activities in which morin (3, 5, 7, 2', 4'-pentahydroxyflavone) was the most potent. Interestingly some of these compounds including morin were also demonstrated to be xanthine oxidase inhibitors. The subsequent in vivo experiment showed that morin indeed exhibited hypouricemic and uricosuric actions in an acute oxonate-induced hyperuricemic rat model. The uricosuric action of morin was hirther studied in transfected HEK293 cells expressing the human urate anion transporter 1 (hURATI) which is believed to regulate blood urate level by mediating urate reabsorption. In hURAT1-expressing HEK293 cells, urate uptake was significantly increased as compared to the non-transfected parental cells. Incorporation of morin into the uptake buffer could dose-dependently inhibit urate uptake in the transfected cells. Taken together our data indicated that morin is a potentially useful antihyperuricemic agent which acts by inhibiting xanthine oxidase and inhibiting urate reabsorption. In addition, the favorable safety profile of this natural compound makes it a potential candidate worthy of further investigations. / Yu Zhifeng. / "June 2006." / Advisers: Christopher H. K. Cheng; Wing Ping Fong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1584. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 155-169). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

alpha-Glucosidase from yeast was used to screen for alpha-glucosidase inhibitory activities in Chinese herbal medicines. Seventy crude extracts were studied. The extracts of Semen Fagopyri Esculenti, Herba Euphorbiae Humifusae, Radix Polygoni Multiflori, Cortex Cinnamomi, Radix Paeoniae Rubra, and Radix Paeoniae Alba exhibited alpha-glucosidase inhibitory activities. These herbs have high potential for finding active compounds to develop into new antidiabetic drugs. / In this study, an assay technique involving brush border membrane vesicles was developed to screen for glucose uptake inhibitory actions in sixteen compounds from natural sources. Two compounds, namely naringenin and desoxyrhaponticin, were demonstrated to exhibit moderate inhibitory action on glucose uptake in rabbit intestinal brush border membrane vesicles, and showed very strong inhibitory action in rat everted intestinal sleeves. The kinetics study indicated that they behave as competitive inhibitors on glucose uptake. Moreover, they could reduce the level of the glucose uptake in the diabetic rat intestinal and renal membrane vesicles. In vivo study further demonstrated that desoxyrhaponticin could significantly reduce the glucose levels after a single oral administration of glucose in neonatal streptozotocin-induced diabetic rats, but not naringenin. These results suggest that naringenin and desoxyrhaponticin may be useful in the control of hyperglycemia. They act by inhibiting glucose uptake in the intestine and glucose reabsorption in the renal proximal tubules. / On the other hand, several synthetic compounds based on the structure of valienamine were found to show strong inhibition on intestinal alpha-glucosidases such as sucrase, glucoamylase and maltase. The strongest inhibitor was further studied. It could reduce the postprandial plasma glucose level of neonatal streptozotocin-induced diabetic rats. These results demonstrated that it has the potential to develop inter an oral antihyperglycemic agent. / The objective of this study is to improve the postprandial hyperglycemic conditions of diabetes by two approaches: (1) inhibiting the digestive enzymes (alpha-glucosidases), and (2) inhibiting active glucose transport in the small intestine. We have screened for new inhibitors of alpha-glucosidase and monosaccharide cotransporters from natural products and their derivatives. These compounds may be useful in the management of type 2 diabetes and diabetic complications. / Type 2 diabetes mellitus accounts for 90-95% of all diabetic cases and has become a major health concern over the world. There is increasing evidence that postprandial hyperglycemia, a hallmark of diabetes, plays a critical role in the development of type 2 diabetes and cardiovascular complications. Therefore the early identification of postprandial hyperglycemia and its effective control can offer the potential for early intervention and prevention of diabetic complications. / Li Jianmei. / "August 2006." / Adviser: Christopher H. K. Cheng. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1592. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 161-180). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Uma das maneiras de obtenção de extratos comercialmente viáveis é a utilização dos procedimentos clássicos de maceração. Apesar de várias substâncias serem extraídas por esses métodos, moléculas mais sensíveis à degradação oxidativa podem não resistir. Neste trabalho foi avaliada a atividade antioxidante de diferentes extratos de espécies de macroalgas marinhas, obtidos por meio das marchas fitoquímicas clássicas. Os extratos não mostraram atividade significante nos modelos experimentais utilizados. Além disso, os ácidos graxos e esteróides das algas estudadas foram analisados por cromatografia gasosa. Os resultados indicaram uma maior quantidade de ácidos graxos insaturados e algumas das algas apresentaram o colesterol como o esteróide majoritário. Apesar de ser verificada a presença de carotenóides remanescentes, estes foram encontrados em concentrações baixas e nenhuma outra molécula resistente aos métodos de extração empregados foi detectada. Desta maneira, partiu-se para o desenvolvimento de metodologias de análise de carotenóides, que são substâncias com conhecida atividade antioxidante. Para isso, foi padronizado um método por HPLC-UV-EC, que separou um total de 16 pigmentos e os dados obtidos em ambos detectores foram comparados. Apesar do detector eletroquímico ser geralmente mais sensível, obteve-se melhores resultados utilizando-se o detector DAD. Com a metodologia padronizada, foi possível obter o perfil de pigmentos de cada espécie deste estudo. Dando continuidade ao desenvolvimento de metodologias para a análise de carotenóides, padrões dessa classe de substâncias, além de outras moléculas contendo polienos em sua estrutura, foram estudados por espectrometria de massas, elucidando-se seus mecanismos de ionização por diferentes fontes. Um balanço entre a formação de íons radicalares e protonados foi proposto para as xantofilas quando ionizadas por ESI, enquanto que nessa mesma fonte foram obtidos íons protonados para retinóides e moleculares para o ß-caroteno. Ao lado de cálculos teóricos de energia de ionização, sugere-se que a formação do íon molecular, exceção para as análises em ESI, é dependente da extensão da conjugação e está relacionada à presença de oxigênio na molécula. Estudos utilizando-se a fonte nanoSpray mostraram resultados opostos, obtendo-se maior intensidade do íon protonado para as xantofilas, mesmo na ausência de ácido. Visando suportar os resultados obtidos em ESI, moléculas sintéticas com cadeia poliênica de diferente extensão e uma porção flavonoídica foram analisadas. Aquela com maior número de conjugações apresentou baixa intensidade do íon molecular e reações de dissociação na fonte, de maneira análoga ao que é observado para o ß-caroteno, enquanto que a estrutura com a menor cadeia poliênica apresentou um íon molecular mais estável. Além disso, a energia de ionização calculada para a molécula maior foi menor, corroborando com os dados experimentais, o que sugere que o \"Caro-Flavo\" com maior cadeia poliênica deve apresentar atividade antioxidante mais próxima ao ß-caroteno que o de cadeia curta. Essas mesmas substâncias sintéticas, ao lado de astaxantina e epicatequina, foram testadas quanto à sua atividade em inibir a lipoperoxidação induzida por radiação UVA ou UVB, sendo que o \"Caro-Flavo\" de cadeia maior atuou como melhor antioxidante quando irradiado com UVB, apresentando maior atividade na menor dose de radiação. Porém, quando irradiado com UVA, apresentou atividade pró-oxidante mais pronunciada ainda que a da astaxantina. Nestes experimentos, foi verificada atividade atividade pró-oxidante em todas as condições de radiação para o \"Caro-Flavo\" menor. Outras moléculas sintéticas, derivadas do a-tocoferol também foram testadas e não apresentaram diferenças estatisticamente significantes desta vitamina. / Classic maceration is one of the most used processes to obtain commercially viable extracts. Despite several substances can be extracted by using these methods, molecules that are susceptible to oxidative degradation may not resist. In this work it was evaluated the antioxidant activity of extracts of different species of marine macroalgae, obtained through phytochemistry classic methods. The extracts showed no significant activity in the experimental models used. In addition, fatty acids and steroids from algae were analyzed by gas chromatography. Results indicated greater amount of unsaturated fatty acids and some of the algae showed the cholesterol as the major steroid. Although some carotenoids remained in the algae extract, they were found in low concentrations and no other molecule resistant to the methods of extraction employed was detected. Thus, it was turned to the development of methodologies for carotenoids analysis, which are known substances with antioxidant activity. A method was standardized by HPLC-UV-EC, which separated a total of 16 pigments and the data obtained in both detectors were compared. Even though the electrochemical detector is generally more sensitive, better results were obtained using the DAD detector. With a standardized methodology, it was possible to obtain the profile of pigments for each species studied. To continue the development of methodologies for carotenoids analysis, standards of this class of substances and other polyene-containing molecules were studied by mass spectrometry. Ionization mechanisms were elucidated by using different sources. A balance between the formation of radical and protonated ions was proposed for xanthophylls when ionized by ESI. When ESI was used to ionize retinoids and carotenes, only protonated and radical ions, respectively, were found. Besides theoretical calculations of ionization energy, it was suggested that the formation of the molecular ion, except for the analyses in ESI, is dependent on the conjugation extension and is related to the presence of oxygen in the molecule. Studies using the nanoSpray source showed opposite results. In this case, it was obtained higher intensity of the protonated ions for xanthophylls, even in the absence of acid. To support the results obtained in ESI, synthetic molecules containing different length of polyene chain and a flavonic portion were analyzed. Those with the greatest conjugation chain showed low intensity of the molecular ion and decoupling reactions at the source. This effect was similar to that observed for ß- carotene in the same conditions. The structure with the smaller polyene chain presented a more stable molecular ion. Furthermore, the ionization energy calculated for the larger molecule was lower, corroborating with the experimental data and suggesting that the larger \"Caro-Flavo\" should present a better antioxidant activity. These synthetic substances, along with astaxanthin and epicatechin, were tested according to their activity in inhibiting UVA or UVB induced lipoperoxidation. The largest \"Caro-Flavo\" showed better antioxidant activity when UVB-irradiated in the lowest irradiation dose. Nevertheless, when this molecule is UVA-irradiated, a pro-oxidant activity is even more pronounced than the astaxanthin pro-oxidant activity. In these experiments, a pro-oxidant activity for the smallest \"Caro-Flavo\" was verified for all irradiation conditions. Other synthetic molecules, derived from α-tocopherol were also tested and showed no statistically significant differences, when compared to this vitamin.

Fungos endofíticos estão presentes em plantas de diversos ambientes e produzem compostos com amplas propriedades químicas e aplicações, tanto na área médico-farmacológica quanto na agronômica. Entretanto, ainda há muito a ser investigado sobre seu potencial biotecnológico, principalmente em locais pouco explorados. As ilhas apresentam um ambiente particular e altamente vulnerável, tornando-as locais promissores na busca de organismos pouco comuns ou ainda endêmicos. Sendo assim, neste trabalho foi realizado o isolamento e estudo químico e biológico de fungos endofíticos isolados de 2 espécies plantas do gênero Anthurium da Ilha de Alcatrazes-SP. Para isto, fragmentos foliares das plantas A. loefgrenii (HRCB 46467) e A. alctrazense (HRCB 46465 - endêmica da ilha) foram inoculados em 10 meios de cultivo com diferentes composições, resultando no isolamento de 106 fungos endofíticos. Por meio de análises químicas por MALDI-TOF-MS e ensaio biológico contra fitopatógenos, foram selecionados 3 fungos para estudo. Estes foram identificados por técnicas morfológicas e moleculares como sendo Penicillium citrinum (P2MSF2F3), Penicillium simplicissimum (P210-4F2) e Aureobasidium melanogenum (P7AF2F3). No estudo dos metabólitos secundários de P. citrinum foi isolado o composto citrinina, o qual apresentou atividade inibitória do crescimento micelial dos fitopatógenos Colletotrichum gloeosporioides (MIC= 125 μg mL-1), Colletotrichum lindemuthianum (MIC= 0,48 μg mL-1), Phomopsis sojae (MIC= 250 μg mL-1) e Fusarium oxysporum f. sp. phaseoli (MIC= 125 μg mL-1). Outras frações obtidas do meio metabólico de P. citrinum (fração F3a3 e citrinina) apresentaram atividade inibitória (100% de inibição) à formas promastigotas de Leishmania infantum. No estudo dos metabólitos secundários de P. simplicissimum foi obtida a fração F2b, ativa contra L. infantum (100% de inibição), da qual foram isolados os compostos andrastina A e penicisoquinolina, sendo o primeiro relato de sua produção por esta espécie, além de outros 5 compostos ainda não identificados. No estudo dos metabólitos secundários de A. melanogenum foi isolado o composto metil-orselinato, relatado pela primeira vez para este fungo. Do mesmo fungo foi obtida a fração F1d2l ativa contra L. infantum (100% inibição), da qual foram isolados 2 compostos ainda não identificados. Este é o primeiro relato de fungos isolados de antúrios da ilha de Alcatrazes, bem como do estudo de seus metabólitos secundários. Este trabalho apresenta contribuição no conhecimento sobre fungos endofíticos e seu potencial metabólico, com aplicações nas áreas agronômica e médico-farmacológica. / Endophytic fungi are present in plants in various environments and produce compounds with wide chemical properties and applications, both in the medical and in agronomic field. However, much remains to be investigated about their biotechnological potential, especially in unexplored places. Islands have a particular and highly vulnerable environment, making them promising sites in search of unusual or endemic organisms. Thus, this work represents the isolation and chemical and biological study of endophytic fungi isolated from 2 species of plants of the genus Anthurium of Alcatrazes island-SP. For this, leaf fragments from plants A. loefgrenii (HRCB 46467) and A. alcatrazense (HRCB 46465 - endemic plant from the Island) were inoculated onto 10 culture media with different composition, resulting in the isolation of 106 endophytic fungi. Three strains were selected to be studied through chemical analysis by MALDI-TOF-MS and bioassay against phytopathogens. These were identified by morphological and molecular techniques as Penicillium citrinum (P2MSF2F3), Penicillium simplicissimum (P210-4F2) and Aureobasidium melanogenum (P7AF2F3). In the study of secondary metabolites of P. citrinum it was isolated the compound citrinin, which showed inibitory activity against the plant pathogens Colletotrichum gloeosporioides (MIC= 125 μg mL-1), Colletotrichum lindemuthianum (MIC= 0.48 μg mL-1), Phomopsis sojae (MIC= 250 μg mL-1) and Fusarium oxysporum f. sp. phaseoli (MIC= 125 μg mL-1). Other fraction obtained from the metabolic extract of P. citrinum (F3a3 fraction and citrinin), showed inibitory activity (100% inibition) to Leishmania infantum promastigotes. In the study of secondary metabolites of P. simplicissimum it was obtained F2b fraction, active against L. infantum (100% inhibition), which were isolated andrastin A and penicisoquinoline compounds, the first report of its production for this species, as well 5 unidentified compounds. In the study of secondary metabolites from A. melanogenum was isolated the methyl-orsellinate compound, first reported for this fungus. From the same strain was obtained F1d2l fraction, active against L. infantum (100% inhibition), from which were isolated 2 unidentified compounds. This is the first report of fungi isolated from Alcatrazes Island anthuriums and the study of their secondary metabolites. This study presents contribution to knowledge of endophytic fungi and their metabolic potential with applications in the medical and agronomic fields.