A piplartina é um alcaloide natural conhecido por apresentar diversas atividades biológicas, onde se destaca a ação anticancerígena. Esse produto natural apresentou atividade seletiva frente a vários tipos de células cancerígenas, sendo assim considerado promissor para o desenvolvimento de fármacos. O conhecimento do metabolismo de produtos naturais bioativos é uma importante e necessária etapa para avaliar a eficácia e segurança dessas substâncias. Os micro-organismos são amplamente utilizados em estudos de metabolismo, uma vez que catalisam reações quimio-, régio-, e estereoespecíficas, que muitas vezes são semelhantes às catalisadas pelos seres humanos. Nesse contexto, esse trabalho teve o objetivo de estudar o metabolismo microbiano da piplartina pelos fungos endofíticos Papulaspora immersa SS13 e Penicillium crustosum VR4, de solo Mucor rouxii NRRL 1894, e de coleção comercial Cunninghamella echinulata ATCC 8688a e Beauveria bassiana ATCC 7159. Os experimentos de biotransformação foram monitorados por UPLC-DAD-MS e UPLC-DAD-MS/MS. Todos os fungos utilizados biotransformaram a piplartina, sendo que 14 substâncias majoritárias foram identificadas como produtos de biotransformação nos experimentos em pequena escala. A piplartina e seus derivados apresentaram fragmentações características em IES-EM/EM que foram explicadas utilizando cálculos computacionais. O estudo dessas fragmentações permitiu a identificação e proposição das alterações estruturais que ocorreram nos metabólitos formados. Os fungos P. crustosum VR4 e B. bassiana ATCC 7159 foram selecionados para realizar os experimentos de biotransformação em escala ampliada, pois foram capazes de formar a maior diversidade de derivados da piplartina. Cinco substâncias foram isoladas e identificadas por RMN de 1H, RMN de 13C, HMQC, HMBC, COSY e HRESIMS. Essas substâncias não tinham sido obtidas por biotransformação microbiana anteriormente, sendo que uma ainda não foi descrita na literatura. Foram identificados principalmente produtos formados a partir de reações semelhantes às do metabolismo humano de fase I, como reduções, hidroxilações e hidrólises. Dessa forma, podemos concluir que as culturas microbianas são uma ferramenta útil para estudos preliminares de metabolismo, e para obter padrões de metabólitos que podem ser formados pelo metabolismo humano. / Piplartine is a natural alkaloid recognized by its biological properties, especially the anticancer activity. This natural product showed selective activity against several cancer cells lines, thus being considered a promising hit for drug development. Studies of bioactive natural products metabolism are an important and necessary step for the evaluation of their efficacy and safety. Microorganisms have been widely employed in metabolism studies, since they may catalyze chemo-, regio- and stereospecific reactions that are similar to human metabolism. This work aimed to study the microbial metabolism of piplartine by different fungal strains: the endophytes Penicillium crustosum VR4 and Papulaspora immersa SS13, the soil strain Mucor rouxii NRRL 1894, and the commercial collection strains Cunninghamella echinulata ATCC 8688a and Beauveria bassiana ATCC 7159. Biotransformation experiments were monitored by UPLC-DAD-MS and UPLC-DADMS/ MS. All the screened fungi were able to biotransform piplartine, and 14 compounds were identified as major biotransformation products in the small scale experiments. Piplartine and its derivatives showed characteristics fragmentations on ESI-MS/MS, which were explained using computer calculations. These fragmentation studies allowed the identification and structural proposition of piplartine metabolites. The fungi P. crustosum VR4 and B. bassiana ATCC 7159 were selected to perform the large scale biotransformation experiments, since they were capable to produce a large diversity of piplartine derivatives. Five compounds were isolated and identified by 1H NMR, 13C NMR, HMQC, HMBC, COSY and HRESIMS data. The isolated products had never been previously identified by microbial biotransformation, and one of them was found to be novel in the literature. All the identified and isolated compounds have been produced by reactions similar to those that occur in phase I of human metabolism, such as reduction, hydroxylation and hydrolysis reactions. Thus, we can conclude that the microbial cultures are useful tools for preliminary metabolism studies, and to obtain chemical standards similar to those produced by human metabolism

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-10-11T14:08:37Z No. of bitstreams: 1 jessicaleirasmotaconegundes.pdf: 1763849 bytes, checksum: 374bcafcbd3cb3795cf0a0c6af6419ee (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-10-16T14:06:30Z (GMT) No. of bitstreams: 1 jessicaleirasmotaconegundes.pdf: 1763849 bytes, checksum: 374bcafcbd3cb3795cf0a0c6af6419ee (MD5) / Made available in DSpace on 2018-10-16T14:06:30Z (GMT). No. of bitstreams: 1 jessicaleirasmotaconegundes.pdf: 1763849 bytes, checksum: 374bcafcbd3cb3795cf0a0c6af6419ee (MD5) Previous issue date: 2017-06-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A espécie Siparuna guianensis Aublet. (Siparunaceae), conhecida como negramina, capitu, folha santa e limão-bravo, é amplamente distribuída no território nacional. A espécie tem sido tradicionalmente utilizada no combate de sintomas de algumas patologias como febre, inflamação, dores de cabeça e no corpo. O presente estudo teve como objetivos avaliar a atividade antioxidante, antinociceptiva, anti-inflamatória e toxidez oral aguda da partição em diclorometano (PDCM) das folhas de S. guianensis, bem como determinar o perfil químico de PDCM e suas frações. Para tal, as folhas secas foram submetidas à extração em metanol por maceração estática. O extrato metanólico foi então particionado, primeiramente com hexano, seguido de diclorometano, obtendose a PDCM que, por sua vez, foi fracionada e forneceu 14 frações (FD1 a FD14). O perfil químico da PDCM e suas frações foi determinado por cromatografia em camada delgada (CCD) e cromatografia líquida de alta eficiência (CLAE), possibilitando a identificação de constituintes terpênicos e flavonoides. Com relação à atividade antioxidante in vitro, a PDCM foi eficaz em reduzir o radical DPPH (CI50 25 μg/mL); no método do fosfomolibidênio foi encontrado o valor de 0,34 mg/mg de PDCM em equivalentes de ácido ascórbico; no método do TBA a amostra na concentração de 7,5 mg foi capaz de inibir a peroxidação lipídica de forma estatisticamente igual à substância de referência (BHT), na mesma concentração. In vivo, a atividade anti-inflamatória foi avaliada em camundongos pelo método do edema de orelha induzido pelo óleo de cróton e pelo modelo de peritonite induzida por LPS. No primeiro modelo PDCM administrada por via oral (v.o.) foi capaz de reduzir significativamente o edema nas doses de 100 e 300 mg/kg em 54 e 52%, respectivamente. Enquanto que no segundo ensaio, PDCM na dose de 100 mg/kg foi capaz de inibir a migração leucocitária em 53%. O estudo toxicológico agudo, durante 14 dias, mostrou que esta amostra é segura para administração por via oral. PDCM v.o. apresentou ação antinociceptiva em dois dos três modelos utilizados. No modelo de contorções abdominais induzidas por ácido acético, as doses de 100, 200 e 300mg/kg foram capazes de inibir o número de contorções em 67, 71 e 83%, respectivamente. No teste da formalina, foi observada atividade tanto na nocicepção neurogênica, quanto na nocicepção inflamatória. Na primeira, todas as doses (100, 200 e 300 mg/kg) foram eficazes reduzindo em 56, 54 e 48%, respectivamente, o tempo de lambida. Do mesmo modo, na segunda fase todas as doses foram eficazes, porém de forma menos significativa que na primeira fase, reduzindo o tempo de lambida em 29, 38 e 44%, respectivamente. Devido à natureza comportamental dos ensaios antinociceptivos, o teste do campo aberto mostrou que a amostra não tem atividade sobre a locomoção dos animais. Os resultados indicam que a PDCM de S. guianensis possui potencial antioxidante, antinociceptiva e anti-inflamatória, provavelmente relacionados aos constituintes terpênicos e flavonoides presentes na espécie. / The species Siparuna guianensis Aublet. (Siparunaceae), known as negramina, capitú, folha-santa e limão-bravo, is widely distributed around the national territory. Species have traditionally been used to combat the symptoms of some pathologies fever, inflammation, headaches and body aches, and other disorders. The present study aimed to evaluate the antioxidant, antinociceptive and anti-inflammatory activities and acute oral toxicity profile of the dichloromethane partition (PDCM), as well as to determine the chemical profile of PDCM and its fractions. For this, the dry leaves were subjected to methanol extraction by static maceration. The methanolic extract was subject to liquidliquid partition, firstly with hexane, followed by dichloromethane, to obtain the dichloromethane partition (PDCM). Then, PDCM was fractionated, and 14 fractions (FD1 – FD14) were collected. The PDCM and fractions chemical profiles was performed using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), which allowed the identification of terpenes and flavonoids. According to the in vitro tests, PDCM was effective in reducing the DPPH radical (IC50 25 μg/mL); the value of 0.34 mg/mg of PDCM in ascorbic acid equivalents was determined using the fosfomolybdenium assay; the sample at 7.5 mg was able to inhibit lipid peroxidation with no statistical difference compared to the reference substance (BHT) at the same concentration in the TBA assay. Besides, the in vivo anti-inflammatory activity was investigated in mice by the cróton oil-induced ear edema and by the model of peritonitis induced by LPS. PDCM was capable to reduce the ear edema at 100 and 300 mg/kg by 54% and 52%, respectively. Also, PDCM inhibited the leucocyte migration by 53% at 100 mg/kg. The acute toxicological evaluation, during 14 days, suggested that the oral administration of this sample is safe. PDCM also showed antinociceptive action in two of the three models used. The number of writhing was reduced by 67%, 71% and 83% at 100, 200 and 300 mg/kg, respectively, in acetic acid-induced writhing test. The neurogenic and inflammatory antinociceptive activity was also observed by the formalin test. All doses (100, 200 and 300 mg/kg) were effective, reducing by 56%, 54% e 48% the licking time during the neurogenic inflammation, respectively. All doses (100, 200 and 300 mg/kg) were also effective in the second phase, however in a less significant manner, reducing the licking time by 29%, 38%, and 44%, respectively. Due to the behavioral nature of the antinociceptive assays, the open field test was carried out to ensure that the sample has no interference on animal locomotion. The results indicated that PDCM from S. guianensis is endowed with antioxidant, antinociceptive and antiinflammatory potential, probably related to the terpenes and flavonoids presented in this species.

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-23T22:59:17Z (GMT). No. of bitstreams: 1 Rocha_DanieleFernandadeOliveira_D.pdf: 6414836 bytes, checksum: 154b421c3fabef860e5439b2a47d4972 (MD5) Previous issue date: 2013 / Resumo: Opiliões são aracnídeos encontrados em todos os continentes, com mais de 6000 espécies descritas, e secretam uma mistura de compostos voláteis que atuam principalmente como defesa contra predadores naturais. A família Gonyleptidae, pertence à subordem Laniatores, é a mais diversa química e morfologicamente e está presente em todo o território brasileiro. Assim, o estudo da identidade e da origem biossintética dos compostos presentes no exudato de espécimes desta família fornece informações filogenéticas, além de ser uma fonte de novos produtos naturais. Neste trabalho foram caracterizados os exudatos de cinco espécies de opiliões de diferentes subfamílias. As espécies Cobania picea, Roeweria virescens e Serracutisoma proximum secretam uma mistura benzoquinonas. Por outro lado, Iporangaia pustulosa e Neosadocus maximus produzem 1-hepten-3-ona e 1-(6-butil-3,4-diidro-2H-piran-2-il)-pentan-1-ona. Este último foi descrito pela primeira vez na literatura, revelando uma nova classe de compostos com esqueleto piranil em opiliões, originados da reação de hetero-Diels-Alder in vivo de duas moléculas de vinil cetona. Também foi determinada a configuração absoluta da (R)-4-metil-1-hexen-3-ona produzida por Acanthogonyleptes pulcher e Gonyleptes saprophilus. Outras 4-metil-3-cetonas de insetos possuem configuração (S). Adicionalmente foi realizado o primeiro estudo biossintético com opiliões através da incorporação de precursores marcados com C e espectroscopia de RMN de C. Foram estudados I. pustulosa, que produz vinil cetona, e Magnispina neptunus, que produz benzoquinona, revelando que ambas as classes químicas são formadas através de unidades acetato e propionato pela via policetídica / Abstract: Harvestmen are arachnids widespread in all continents, with more than 6,000 described species. They secrete a mixture of volatiles compounds with the main function of defense against natural predators. The family Gonyleptidae belongs to the suborder Laniatores, is the most diverse in chemistry and morphology and is predominant in Brazil. Therefore, studying the identity and biosynthetic origin of this family exudate components gives phylogenetic information and is a source of new natural products. In this work five species exudate from different subfamilies were characterized. The species Cobania picea, Roeweria virescens and Serracutisoma proximum secrete a mixture of benzoquinones, while Iporangaia pustulosa and Neosadocus maximus produce 1-hepten-3-one and 1-(6-butil-3,4-dihydro-2H-piran-2-yl)-pentan-1-one. The latter was described for the first time, and belongs to a new class of harvestman metabolites with piranyl moiety in harvestmen, which were rationalized as the hetero-Diels-Alder adduct of two vinyl ketone molecules. Additionally the absolute configuration of (R)-4-methyl-1-hexen-3-one from Acanthogonyleptes pulcher and Gonyleptes saprophilus was determined. Analogous 4-methyl-3-cetones from insects have S configuration. It was also performed the first biosynthetic investigation with harvestmen by C labeled precursors incorporation and C NMR. The studied species were /.pustulosa and Magnispina neptunus, which produce vinyl ketones and benzoquinones, respectively. The results revealed that these chemical classes are biosynthesized with acetate and propionate units via polyketide pathway / Doutorado / Quimica Organica / Doutora em Ciências

Actuellement, la tuberculose (TB) est la deuxième maladie infectieuse au monde avec une forte prévalence et un taux de mortalité annuel élevé. L'Organisation Mondiale de la Santé (OMS) a récemment dévoilé sa nouvelle stratégie, appelée «Mettre fin à la tuberculose», pour la prévention, le contrôle et l'éradication de cette maladie. Les résultats doivent mener à une éradication totale d'ici au 2035. Dans le cadre des objectifs spécifiques de cette stratégie, l'OMS a intégré la recherche de nouvelles connaissances et innovations scientifiques qui améliorent et augmentent l'efficacité des traitements qui sont utilisés contre cette maladie.Deux des stratégies pharmaceutiques qui ont été les plus étudiées pendant ces dernières années ayant comme objectif principal d'augmenter l'efficacité globale des régimes thérapeutiques utilisés dans la tuberculose comprennent: i) l'incorporation de médicaments antituberculeux de première et de seconde ligne dans des systèmes de libération contrôlé, en particulier les nanoparticules polymères (NP); et, ii) l'utilisation de produits naturels (par exemple, des extraits de plantes ou des composés isolés) comme adjuvants, c'est-à-dire des substances qui, lorsqu'elles sont administrées conjointement avec des médicaments existants, augmentent leur activité.Dans ce contexte, ce travail de recherche a conduit au développement de deux stratégies pour rendre le traitement de la tuberculose plus efficace. D'une part, on a proposé le développement d´une nanomédecine à base de NP biodégradables contenant un principe actif antituberculeux de deuxième ligne, la clofazimine (CFM). D´autre part, on a conçu le développement des systèmes adjuvants provenant d'extraits végétaux obtenus à partir de l´arbre Schinus molle, lesquels ont été aussi incorporés dans des NP (biodégradables et non biodégradables). Dans cette deuxième stratégie, l'objectif principal a été l'obtention de potentielles nanoformulations qui permettent une application sûre de ces extraits par diverses voies d'administration (p. ex. par voie intraveineuse, pulmonaire ou orale).Dans ce travail, le Chapitre I est une revue de la littérature sur l'étude des produits naturels (spécifiquement des extraits de plantes) qui ont été proposés comme des agents potentiellement antibactériens contre Mycobacterium tuberculosis [etc...] / Tuberculosis is a chronic infection located in the lungs during the early stages of this disease. The World Health Organization, annually, registers about 9 million new cases and 1.5 million deaths. In addition, the development of multi-drug resistant strains of Mycobacterium tuberculosis has complicated the global control of tuberculosis. An effective control for this epidemic can be based on two main pharmaceutical strategies. First, the development of novel formulations based on controlled release systems for antitubercular drugs which could be used for establishing more effective therapeutic schemes. A second approach can be focused on development of natural products nanoformulations (e.g. natural extracts) for their application as adjuvants for tuberculosis treatment. In this context, the present research work was focused on the design and development of a nanomedicine based on biodegradable nanoparticles and an antitubercular drug of second line (clofazimine). In addition, organic extracts obtained from Schinus molle were formulated into nanoparticles in order to use them as adjuvants in tuberculosis treatment. The characterization of the nanoformulations established a direct relationship between the physicochemical properties (i.e. particle size, surface charge and release profile) of the active-loaded polymeric nanoparticles (with drug or extract) and the increase of the antitubercular activity in vitro. Particularly, additional in vitro tests showed that nanoencapsulation of S. mole extract decreased their toxicity as compared to a non-encapsulated extract. In conclusion, nanoformulations loaded with clofazimine or extract of S. molle showed to have a high potential to be applied in efficient therapeutic schemes for tuberculosis treatment

Les peptides Cα-thioester jouent un rôle central dans la synthèse de protéines par voie chimique : ils sont des partenaires clés dans la ligation chimique native (NCL), réaction qui a révolutionné ce domaine. L’accès à ces peptides par Fmoc-SPPS, méthode largement utilisée dans les laboratoires, est encore restreint à des experts. Cette limitation représente à l’heure actuelle un frein à la démocratisation de la synthèse de protéines par NCL. Le premier volet de cette thèse décrit la conception et le développement d’une nouvelle méthode bioinspirée de synthèse de précurseurs de peptides thioester, fondée sur un dispositif de thioestérification intramoléculaire de type N-(2-hydroxy-5-nitrobenzyl)-cystéine (N-Hnb-Cys). La synthèse des peptides portant ce dispositif a été totalement automatisée à partir de réactifs peu couteux et ne requière pas d’étapes post-SPPS avant la NCL. La conception biomimétique du dispositif –ce dernier opérant via un réarrangement par transfert d’acyle N→S–, résulte en des cinétiques de NCL rapides à pH neutre. Un large éventail de précurseurs de thioester a été synthétisé, ceci nous a permis d’explorer le potentiel et les limites de la méthodologie, tout en soulignant son efficacité même pour des « séquences difficiles » et de longs peptides. Cette approche a été appliquée à la synthèse par ligation d’une gamme représentative de peptides naturels riches en cystéines, issus du venin d’un serpent, de mollusques, de primates ou encore de plantes. En particulier, nous avons décrit la première synthèse d’une Big-défensine (93 acides aminés), issue de l’huitre et dont l’activité biologique est en cours d’évaluation. Par ailleurs, une voie synthétique originale pour accéder à des peptides comprenant une cystéine C-terminale a été proposée. Elle repose sur l’introduction de cet acide aminé par NCL, évitant les réactions secondaires inhérentes aux approches existantes. Nous avons également appliqué notre approche à la synthèse par NCL intramoléculaire du squelette peptidique cyclique de plusieurs produits naturels, avec des rendements remarquables. L’ensemble de nos résultats est extrêmement encourageant pour la généralisation de notre approche. / Peptide Cα-thioesters play a prominent role in the chemical synthesis of proteins: they are key partners in native chemical ligation (NCL), a reaction that has revolutionized the field. Nonetheless, access to such peptides via the widely used Fmoc-SPPS is still limited to experts. This limitation is currently an obstacle to further popularization of NCL-based protein synthesis. The first part of this thesis describes the design and optimization of a new bio-inspired methodology for the synthesis of peptide thioester precursors, based on an N-(2-hydroxy-5-nitrobenzyl)-cysteine intramolecular thioesterification device (N-Hnb-Cys). Synthesis of peptides bearing this device was fully automated, starting from inexpensive materials. Importantly, no post-SPPS steps are required prior to NCL. The biomimetic design of the device –that operates through an N→S acyl shift–, results in fast NCL kinetics at neutral pH. A broad range of thioester precursors has been synthesized; this allowed us to explore the scope and limitation of the methodology, while stressing its efficiency even for demanding sequences and long peptides. This approach has been applied to the ligation-based synthesis of a representative variety of naturally occurring disulfide-rich peptides (DRP) sequences, from snake venom, molluscs, primates and plants. In particular, we have described the first synthesis of a Big-defensin (93 amino acids), discovered in oyster and whose biological activity is currently under evaluation. Furthermore, an original method for the synthesis of C-terminal cysteine-containing DRP has been proposed. It is based on the introduction of this amino acid through NCL, avoiding side reactions inherent to existing approaches. We have also applied our approach to the intramolecular NCL-based synthesis of the cyclic backbone of several DRP, with remarkable yields. Altogether, our results are extremely encouraging for the generalization of this methodology.

Malaria is one of the three most deadly diseases in Africa. Although there are available treatments, their efficacy has been greatly reduced over the past two decades due to the development of resistance to currently available drugs. This has necessitated the search for new and effective antimalarial agents. This project approached the search for new antimalarial compounds in two ways: (i) by screening natural products isolated from marine algae against the Plasmodium parasite and (ii) by modification of selected isolated active compounds to target 1-deoxY-đ-xylulose 5-phosphate reductoisomerase (DXR), an enzyme found in the nonmevalonate isoprenoid biosynthetic pathway of Plasmodium Jalciparum. It was envisaged that such a compound would exhibit dual action on the Plasmodium parasite. Extracts obtained from 22 marine algae were prefractionated by solvent partitioning and were screened for anti plasmodial activity against the chloroquine sensitive (CQS) P. Jalciparum D 10 strain. Overall, 50% of the algae screened produced at least one crude fraction with activity against P. Jalciparum. Extracts of the algae Sargassum heterophyllum, Plocamium cornutum, Amphiroa ephedrea and Pterosiphonia cloiophylla gave the most promising results. Fractionation of S. heterophyllum afforded three tetraprenyltoluquinols (3.1, 3.2 and 3.5) and an all-trans-fucoxanthin (3.6). Three new compounds (4.5, 4.6 and 4.7) and two known halogenated monoterpenes (4.1 and 4.4) were isolated from P. cornutum. Each of the isolated compounds from both S. heterophyllum and P. cornutum showed antiplasmodial activity with IC₅₀ values ranging from 2.0 - 15.3 μM for S. heterophyllum and 13 - 230 μM for P. cornutum. Attempts to synthetically modify halogenated monoterpene 4.4 by dihydroxylation and phosphorylation in order to inhibit the DXR enzyme was unsuccessful. However, the hemiterpene analogue (5.42) of the halogenated monoterpenes was successfully phosphorylated and dihydroxylated to give compound 5.45 which showed promising activity against DXR. The result obtained indicated that the proposed phosphorylation and dihydroxylation of the halogenated monoterpene 4.4 would result in the synthesis of a potent DXR inhibitor and therefore a potential antimalarial agent with dual mode of action on the Plasmodium parasite.

The role of antioxidants in the prevention of neurodegenerative diseases has been well documented. The use of synthetic antioxidants has decreased due to the ssociation of these compounds with certain cancers. Thus, the search for novel natural antioxidants has gained much focus in research. Most common methods of determining antioxidant capacity are the radical generated assays and biological assays such as lipid peroxidation and the nitroblue tetrazolium assay. Electrochemical methods have been proposed for the determination of bio-active compounds such as antioxidants. The electrochemical methods of cyclic voltammetry and square wave voltammetry were evaluated for the determination of antioxidant capacity initially examining known antioxidants and then using plant extracts of Sutherlandia frutescens as a case study. The antioxidant properties determined by electrochemical methods were validated utilising the non-biological methods of the DPPH, TEAC, ferrozine and FC assay and biological pharmacological methods. The results indicated that Sutherlandia frutescens contains potent antioxidant compounds that are able to reduce lipid peroxidation. The electrochemical techniques of square wave voltammetry and cyclic voltammetry were applied for the screening of a large number of extracts of various algae for the detection of antioxidant compounds. The results indicated that electrochemistry can be used as a preliminary method for the rapid screening of a large number of crude samples for antioxidant compounds. Electrochemical methods were also evaluated as a method for guiding the isolation and purification of antioxidant metabolites in Sargassum elegans. Solvent partitioning and fractionation of the marine alga allowed for the purification of antioxidant compounds. At each step of purification electrochemical methods were utilized to determine which fractions contained the more potent antioxidant compounds and thus guide further purification. The purified antioxidant compounds were elucidated using NMR to determine the structure of the antioxidant compounds.

The chemicals produced by biological systems, whether proteins, peptides, or terpenes, will always provide an intriguing topic for researchers. Invisibly controlling every aspect of nature, these molecules are responsible for life, evolution, and death. Specifically, here is described the secondary metabolites produced by Antarctic marine organisms as well as others, and how they are used to defend or attract other animals while potentially providing health benefits to mankind. This is done through collection, extraction, and separation of individual specimens. The respective mixtures of compounds after isolation are then analyzed via spectroscopic methods such as nuclear magnetic resonance spectroscopy, mass spectrometry, and X-ray crystallography. Once identified, these compounds are tested in biological assays to provide a hypothesis for their use in nature or evidence that there may be a use for them in medicine. For this thesis, the Antarctic organisms described are an alga, Pocamium cartilagineum, an amphipod, Paradexamine fissicauda, a sponge, Dendrilla membranosa, and one undescribed and two known deep sea coral species, Briareopsis aegeon and Plumarella delicatissima. Beyond these specific specimens, their chemistry as well as natural products from other origins were combined to create a diverse compound library for biological screening against human pathogens. This was done using computational modeling and statistical analysis of the compound library and its comparison to other known chemical libraries. The diversity and impact of these molecules are assessed.

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No. of bitstreams: 1 jordanadeabreulazzarini.pdf: 11340904 bytes, checksum: 748a672389e8977fcf91c77687db2f7b (MD5) Previous issue date: 2015-07-30 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente trabalho buscou caracterizar fitoquimicamente, avaliar o potencial antifúngico frente às principais linhagens causadoras de esporotricose no Brasil; a atividade antioxidante e a citotoxicidade preliminar dos óleos essenciais adquirido comercialmente (OCM) e obtido por extração no laboratório de Farmacognosia da Faculdade de Farmácia da Universidade Federal de Juiz de Fora (OEX) e do extrato hidroetanólico 95% (v/v), obtidos a partir das folhas de Pimenta pseudocaryophyllus (Gomes) Landrum (Myrtaceae). O screening fitoquímico realizado com o extrato hidroetanólico resultou na identificação de flavonoides, taninos e proantocianidinas. A quantificação destes metabólitos secundários na droga vegetal, por espectrofotometria UV-VIS, resultou em 18,77% de constituintes fenólicos totais, 10,69% de taninos e 0,52% de flavonoides. O perfil cromatográfico obtido por CGEM do OEX indicou elevados teores de monoterpenos (48,83%) e fenilpropanoides (39,08%), tendo como constituinte majoritário o eugenol (34,38%). O OCM apresentou elevados teores de sesquiterpenos (25,64%) e fenilpropanoides (70,05%), e também apresentou o eugenol como constituinte majoritário (66,93%). Extrato e frações apresentaram notável atividade antioxidante pelo método de redução do DPPH, sugere-se que a mesma seja atribuída aos altos teores de compostos fenólicos, flavonoides e taninos encontrados. Promissores resultados de atividade antioxidante foram encontrados para os OEX e OCM, os quais possuem como constituinte majoritário o eugenol. Para a avaliação da atividade antifúngica foram utilizadas as seguintes linhagens fúngicas: Sporothrix schenckii ATCC 1099- 18, Sporothrix schenckii IPEC 15383, Sporothrix brasiliensis ATCC 5110 e Sporothrix brasiliensis IPEC 17943 e duas linhagens clínicas de Sporothrix schenckii, denominadas genericamente como A e B. O OEX apresentou atividade antifúngica frente a todas as linhagens em concentrações que variaram de 260,46 a 520,90 μg/mL. Foi observada atividade fungicida frente a todas as linhagens em concentrações acima de 520,90 μg/mL. O OCM foi capaz de inibir todas as linhagens em concentrações que variaram de 65,52 a 260,10 μg/mL. Foi observada atividade fungicida frente a todas as linhagens em concentrações acima de 262,10 μg/mL. O eugenol apresentou atividade antifúngica frente a todas as linhagens em concentrações que variaram de 34,36 a 137,48 μg/mL. Foi observada atividade fungicida frente a todas as linhagens em concentrações acima de 137,48 μg/mL. Ao ajustar os resultados de atividade antifúngica a 100% do constituinte majoritário eugenol, verificou-se a existência de sinergismo entre os constituintes do OEX, que apresentou maior atividade antifúngica (CIM=89,54 μg/mL) do que o eugenol (CIM=136,11 μg/mL) para quatro das seis linhagens estudadas. Foi observado que somente duas das linhagens estudadas apresentaram susceptibilidade à anfotericina b e três linhagens foram consideradas resistentes ao cetononazol com valores de CIM de 4,0 μg/mL. Em relação ao itraconazol, todas as linhagens estudadas foram consideradas resistentes, com valores de CIM>128 μg/mL. Estes resultados sugerem indícios de resistência fúngica das linhagens estudadas em relação aos fármacos de referência empregados no tratamento da esporotricose. As eletromicrografias das linhagens fúngicas revelaram que, tanto nos fungos expostos aos tratamentos experimentais, quanto naqueles expostos aos fármacos de referência, pode-se observar deformidades na estrutura fúngica quando comparadas ao grupo não tratado. Em relação aos ensaios de citotoxicidade realizados com fibroblasto murino (L929) pelo método de redução do MTT, o extrato não apresentou citotoxicidade nas concentrações avaliadas (10 a 100 μg/mL), o eugenol não apresentou citotoxicidade em concentrações menores ou igual a 125 μg/mL, o OCM não foi tóxico em concentrações menores ou igual a 250 μg/mL. Já em relação ao OEX concentrações inferiores ou igual a 62,5 μg/mL não apresentaram citotoxicidade. / The purpose of this work is to carry out the phytochemical study, evaluate the potential antifungal against the major strains causing sporotrichosis in Brazil, the antioxidant activity and the preliminary cytotoxicity of essential oils obtained commercially (CMO) and obtained by extraction in Pharmacognosy Laboratory of Pharmacy Faculty of the Federal University of Juiz de Fora (OEX) and hydroethanolic extract 95% (v/v), obtained from the leaves of Pimenta pseudocaryophyllus (Gomes) Landrum (Myrtaceae). The phytochemical screening performed with the hydroethanolic extract resulted in the identification of flavonoids, tannins and proanthocyanidins. The quantification of these secondary metabolites in plant drug, by UV-VIS revealed the presence of phenolic constituents (18.77%), tannins (10.69%) and flavonoids (0.52%). The chromatographic profile obtained by GC-MS indicated the OEX high levels of monoterpenes (48.83%) and phenylpropanoids (39.08%) having as a major constituent eugenol (34.38%). The OCM had high levels of sesquiterpenes (25.64%) and phenylpropanoids (70.05%), with as major constituent eugenol (66.93%). Extract and fractions showed notable antioxidant activity by reduction of DPPH method, it is suggested that it be attributed to the high concentration of phenolic compounds, flavonoids and tannins found. Promising results of antioxidant activity were found for the OEX and OCM, which have as major constituent eugenol. For evaluating the antifungal activity the following fungal strains were used: Sporothrix schenckii ATCC 1099-18, Sporothrix schenckii IPEC 15383, Sporothrix brasiliensis ATCC 5110 and Sporothrix brasiliensis IPEC 17943 and two clinical strains, known generically as A and B. The OEX showed activity antifungal against all strains at concentrations ranging from 260.46 to 520.90 μg/mL. It was observed fungicidal activity against all strains at concentrations above 520.90 μg/mL. The OCM was able to inhibit all strains at concentrations ranging from 65.52 to 260.10 μg/mL. It was observed fungicidal activity against all strains at concentrations above 262.10 μg/mL. The eugenol showed antifungal activity against all strains at concentrations ranging from 34.36 to 137.48 μg/mL. It was observed fungicidal activity against all strains at concentrations above 137.48 μg/mL. By adjusting the results of antifungal activity to 100% of major constituent eugenol, it was found that there is synergy between the constituents of the OEX, with the highest antifungal activity (MIC = 89.54 μg/mL) than eugenol (MIC = 136.11 μg/mL) for four of the six strains studied. It was noted that only two of the studied strains were susceptible to amphotericin B and three strains were considered resistant cetononazol with MIC values of 4.0 μg/mL. Compared to itraconazole, all the strains studied were considered resistant with MIC values> 128 μg/mL. The micrographs revealed that the fungal strains in both fungi exposed to the experimental treatments, as those exposed to the reference drug, one can observe fungal deformities in structure when compared to the untreated group. In relation to the cytotoxicity assays performed with murine fibroblasts (L929) by the MTT reduction method, the extract showed no cytotoxicity at concentrations tested (10 to 100 μg/mL), eugenol showed no cytotoxicity at concentrations less than or equal to 125 μg/mL, OCM was not toxic at concentrations less than or equal to 250 μg/mL. In relation to the OEX concentrations below or equal to 62.5 μg/mL showed no toxicity.

Com o surgimento da metagenômica, a descoberta de compostos bioativos aumentou. A Arrabidaea chica é uma planta trepadeira, usada em tatuagens pelos índios. O enriquecimento da extração dos antocianosídeos através da fermentação das folhas com xilanase de Bacillus pumilus foi estudado anteriormente. A análise qualitativa da produção de xilanase por clones de bibliotecas metagenômicas e B. pumilus SG-32 e B. firmus P1-1 foi feita com a finalidade de elaborar um método miniaturizado para encontrar novas fontes dessa enzima. Bem como avaliar o seu potencial enzimático sobre os antocianosídeos. Os clones e o B. firmus não expressaram xilanases em meio sólido de xilana de bétula. Porém, o B. pumilus SG-32 expressou, como confirmado pelo atividade xilanolítica. Por isso, o caldo enzimático desta espécie foi utilizado como inóculo para o tratamento enzimático das folhas de A. chica que liberou suas antocianidianas, como confirmado pelo método de Bial e CLAE-DAD. Uma nova fonte de xilanase foi descoberta com atividade hidrolítica sobre os antocianosídeos de A. chica. / With the advent of metagenomics, the discovery of bioactive compounds increased. The Arrabidaea chica is a climbing plant, used in tattoos by the Indians. The extraction of anthocyanosides enrichment through fermentation of the leaves with xylanase from Bacillus pumilus has been studied previously. Qualitative analysis of xylanase production by clones of metagenomics libraries and B. pumilus SG-32 and B. firmus P1-1 was made in order to develop a miniaturized method to find new sources of this enzyme. And to evaluate the potential enzymatic on the anthocyanosides. The clones and the B. firmus xylanases did not express in solid birch xylan. However, B. pumilus SG-32 expressed as confirmed by the xylanolytic activity. Therefore, the broth enzymatic of this specie was used as inoculum for the enzymatic treatment of the leaves of A. chica that liberated their anthocyanidines, as confirmed by Bial method and HPLC-DAD. A new source of xylanase was discovered with hydrolytic activity on anthocyanosides from A. chica.