Orientador: Luis Octavio Regasini / Resumo: As micoses superficiais e cutâneas são infecções fúngicas que acometem a camada mais externa do estrato córneo da pele e seus anexos. São as micoses mais frequentes dentre todas as infecções fúngicas e estão distribuídas por todo o mundo, afetando todas as faixas etárias. O impacto primário destas micoses é seu aspecto visual com estética negativa. Contudo, não se restringem apenas ao aspecto estético, vindo a ser um grave problema de saúde na população neonatal, senescente e de imunocomprometidos. Além disso, muitos fármacos exibem toxicidade alta, interações medicamentosas e alto custo econômico. As recidivas por meio do tratamento convencional costumam ser frequentes, as quais podem ser ocasionadas por cepas resistentes. Esse cenário torna premente a busca por antifúngicos inovadores. No presente estudo avaliou-se atividade antifúngica de 19 curcuminoides, provenientes da composteca do Laboratório de Antibióticos e Quimioterápicos (LAQ), contra fungos filamentosos dermatofíticos dos gêneros Trichophyton, Epidermophyton e Microsporum e leveduriformes (Candida spp.), de cepas de referência ATCC e de isolados clínicos, provenientes da coleção do Laboratório de Microbiologia da Faculdade de Medicina de São José do Rio Preto (FAMERP), bem como avaliou-se toxicidade dos curcuminoides mais potentes contra células de queratinócitos humanos (HaCat) e larva G. mellonella. Afim de verificar a atividade antifúngica dos compostos, foram determinados os valores: Concentração Inibitória ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Superficial and cutaneous mycoses are fungal infections that affect the outermost layer of the stratum corneum of the skin and its attachments. They are the most frequent mycoses among all fungal infections and are distributed all over the world. The first impact of these mycoses is their visual appearance with a negative aesthetic. However, they are not restricted to the aesthetic aspect, becoming a serious health problem in the neonatal, senescent and immunocompromised population. In addition, many drugs exhibit high toxicity, drug interactions and high economic cost. Relapses through conventional treatment are often frequent, which can be caused by resistant strains. This scenario demands the development of potent but safer compounds. Herein, we described the antifungal activity of a novel curcuminoid against dermatophyte and Candida species. A series of nineteen curcuminoid analogs was submitted to antifungal assay to assess MIC (minimum inhibitory concentration) and MFC (minimum fungicidal concentration) determined by the broth microdilution method. First, all compound were tested at 31.2 µg mL-1 in order to select the most active compound. The compound 3, 3'-Dimethoxycurcumin (DMC) showed the highest growth-inhibitory activity the largest spectrum of action against dermatophyte clinical isolates and reference strains of Candida tropicalis ATCC and Trichophyton rubrum CBS, with MIC values range of 1.9±31.2 µg mL-1. DMC exhibited fungistatic activity. Inverted microscope ... (Complete abstract click electronic access below) / Mestre

<p>Zemlji&scaron;te predstavlja bogat izvor različitih mikroorganizama čiji<br />produkti metabolizma mogu biti od izuzetnog značaja za čoveka.<br />Dosada&scaron;nja ispitivanja mikrobnog diverziteta u zemlji&scaron;tu su<br />otkrila bogati biosintetski potencijal za proizvodnju novih<br />prirodnih proizvoda kod velikog broja mikroorganizama, naročito<br />kada je u pitanju klasa Actinobacteria. Među zemlji&scaron;nim izolatima,<br />rod Streptomyces prednjači po broju identifikovanih bioaktivnih<br />molekula u odnosu na sve ostale bakterije. Stoga je jedan od<br />ciljeva u okviru ove doktorske disertacije izolacija streptomiceta iz<br />rizosfera medicinski značajnih biljaka sakupljenih na teritoriji<br />Republike Srbije (Papaver rhoeas, Matricaria chamomilla, i Urtica<br />dioica) i ispitivanje njihovog antifungalnog potencijala na različite<br />vrste kandida. Morfolo&scaron;ki različiti izolati (ukupno 103) su izolovani<br />iz uzoraka rizosfera i okarakterisani kao streptomicete. Dve<br />različite podloge i dve procedure za ekstrakciju su kori&scaron;ćene da bi<br />se pospe&scaron;ila detekcija antifungalnih jedinjenja. Ispitan je uticaj<br />ukupno 412 ekstrakata na rast Candida albicans disk difuzionim<br />esejem pri čemu je utvrđeno da 42% (43/103) izolata imaju<br />sposobnost proizvodnje antifungalnih jedinjenja pri ispitivanim<br />uslovima. Pojedini ekstrakti su inhibirali rast važnih humanih<br />patogena poput Candida krusei, Candida parapsilosis, i Candida<br />glabrata. Na osnovu stepena i spektra antifungalne aktivnosti<br />devet izolata je odabrano za dalja istraživanja. Ispitana je<br />sposobnost njihovih ekstrakata da inhibiraju rast kandida u tečnoj<br />kulturi i u formi biofilma, a takođe je ispitan i njihov uticaj na već<br />formirane biofilmove kandide u koncentracijama od 8 do 250<br />pg/ml. Hromatografski profili ovih ekstrakata i uvid u njihovu<br />metaboličku raznolikost dobijeni su kori&scaron;ćenjem tečne<br />hromatografije visokih performansi. Tri ekstrakta sa specifičnom<br />antifungalnom aktivno&scaron;ću podvrgnuta su hemijskim analizama s<br />ciljem da se detektuju i strukturno okarakteri&scaron;u molekuli koji su<br />nosioci antifungalne aktivnosti. Na osnovu rezultata nuklearnomagnetno-<br />rezonantne spektroskopije otkriveno je da su aktivni<br />molekuli genistein, daidzein i staurosporin. Genistein i daidzein<br />koji su poznati fitoestrogeni poreklom iz sojinog bra&scaron;na za koje je<br />poznato da inhibiraju ključne enzime u biosintetskom putu<br />steroida. Njihovo prisustvo je u ovom istraživanju detektovano<br />usled kori&scaron;ćenja sojinog bra&scaron;na u hranljivoj podlozi. Kako<br />streptomicete u čijim ekstraktima su detektovani ovi molekuli<br />pokazuju sposobnost oslobađanja ovih važnih jedinjenja iz<br />kompleksne hranljive podloge, mogu se uzeti u razmatranje za<br />biotehnolo&scaron;ku proizvodnju fitoestrogena. Staurosporin je<br />detektovan kao nosilac antifungalne aktivnosti kod ekstrakta soja<br />Streptomyces sp. BV410. Staurosporin je inhibitor protein kinaza i<br />njegovi derivati i analozi se koriste u kao antitumorski agensi.<br />Biosinteza ovog molekula je optimizovana do prinosa od 36,94<br />mg/l nakon 14 dana gajenja u hranljivoj podlozi koja sadrži<br />glukozu, skrob, manitol i sojino bra&scaron;no (JS). Dalja optimizacija<br />hranljive podloge za biosintezu staurosporina ukazala je na<br />sledeći sastav hranljive podloge: 20 g/l glukoze, 0,36 g/l skroba,<br />21,46 g/l manitola, 17,32 g/l sojinog bra&scaron;na. Primenom<br />definisanih optimalnih vrednosti i kori&scaron;ćenjem odgovarajućih<br />matematičkih modela, predviđeno je da će se na ovaj način<br />postići prinosi od 46,88 mg/l staurosporina i 12,05 mg/ml<br />biomase. Validnost predviđenih rezultata potvrđena je<br />izvođenjem bioprocesa u optimizovanoj hranljivoj podlozi (JSSta).<br />Ispitana je kinetika biosinteze staurosporina i produkcije biomase,<br />kao i potro&scaron;nje izvora ugljenika i razvijeni su odgovarajući<br />procesni modeli. Dodatna optimizacija je podrazumevala dodatak<br />suplemenata koji prema literature stimuli&scaron;u sekundarni<br />metabolizam streptomiceta (joni cinka, gvožđa, fosfati, metil<br />oleat, ulje semenki grožđa). Ovi eksperimenti su izvođeni na tri pH<br />vrednosti (6,5, 7,5 i 8,5) a uspe&scaron;nost bioprocesa je procenjivana 7.,<br />10. i 14. dana gajenja. Dodatna optimizacija je dovela do podatka<br />da dodatak soli gvožđa značajno pospe&scaron;uje biosintezu<br />staurosporina sa povećanjem prinosa od 25%. Dobijeni rezultati<br />potvrđuju da su rizosfere medicinski značajnih biljaka značajan<br />izvor streptomiceta koje proizvode komponente sa<br />antifungalnom aktivno&scaron;ću. Izolacija novog proizvođača<br />staurosporina i optimizacija procesa njegove biosinteze<br />omogućiće dalja istraživanja ovog jedinjenja koje može biti<br />osnova za razvoj novih antifungalnih i jedinjenja koja inhibiraju<br />angiogenezu. Rezultati dobijeni u okviru ovih istraživanja<br />predstavljaju početni korak ka potencijalnoj industrijalizaciji<br />proizvodnje staurosporina.</p> / <p>Different soils are still a source of remarkable microbial diversity<br />which also reflects in the unexplored chemical diversity. Recent<br />advances in assessment of microbial diversity from soil have<br />revealed the extraordinarily rich biosynthetic potential for the<br />production of new natural products among different microbial<br />strains, especially within the group of Actinobacteria. Among<br />bacterial soil isolates, representatives of Streptomyces genus are<br />the most prolific producers of bioactive compounds. One of the<br />objectives of the present study was to isolate Streptomyces spp.<br />from the rhizosphere soils of three ethno-medicinal plants<br />collected in Serbia (Papaver rhoeas, Matricaria chamomilla, and<br />Urtica dioica) and to screen their antifungal activity against<br />Candida spp. Morphologically different sporulating isolates (103<br />in total) were collected from rhizosphere soil samples and<br />determined as Streptomyces spp. Two different media and two<br />extraction procedures were used to induce the production and<br />facilitate identification of antifungals. Overall, 412 crude cell<br />extracts were tested against Candida albicans using disk<br />diffusion assays, with 42% (43/103) of the strains showing the<br />ability to produce antifungal agents. Also, extracts inhibited<br />growth of other important human pathogens: Candida krusei,<br />Candida parapsilosis, and Candida glabrata. Based on the<br />established degree and range of antifungal activity, nine isolates<br />were selected for further testing. Their ability to inhibit Candida<br />growth in liquid culture, to inhibit biofilm formation, and to<br />disperse pre-formed biofilms was assessed with active<br />concentrations from 8 to 250 pg/ml. High-performance liquid<br />chromatographic profiles of extracts derived from selected<br />strains were recorded, revealing moderate metabolic diversity.<br />The most potent extracts were subjected to comprehensive<br />identification and structural characterization of antifungal<br />compounds. Applying a bioactivity-guided isolation approach,<br />active compounds of three extracts were separated, and based<br />on NMR structure elucidation it was shown that active<br />compounds were genistein, daidzein and staurosporine.<br />Genistein and daidzein, soy phytoestrogens, are known to inhibit<br />key enzymes in the steroid metabolism pathway and were<br />coming from the fermentation medium containing soy flower.<br />Since isolated Streptomyces spp. showed good ability to extract<br />these molecules from complex medium, they can be further<br />considered for biotechnological production of these<br />phytoestrogens. One of the isolates, Streptomyces sp. BV410,<br />was characterized as an efficient staurosporine producer.<br />Staurosporine is a potent inhibitor of protein kinases and is<br />considered in anticancer therapy. The biotechnological<br />production of staurosporine by strain BV410 was optimized to<br />yield 36.94 mg/l after 14 days of incubation in soy flowerglucose-<br />starch-mannitol based fermentation medium (JS).<br />Further optimization of medium for biosynthesis of<br />staurosporine indicated the following optimal values of the<br />examined factors: the content of glucose of 20 g/l, starch 0.36<br />g/l, mannitol 21.46 g/l, soy flower 17.32 g/l. By applying the<br />defined optimal values and using the appropriate mathematical<br />models, the following responses were predicted: concentration<br />of staurosporine 46.88 mg/l and biomass yield 12.05 mg/ml. The<br />validity of the results was confirmed by performing the<br />biosynthesis of the staurosporine in the medium with optimal<br />composition (JSSta). Kinetics of staurosporine and biomass<br />production and carbon source consumption were examined and<br />process models were developed. Additionally, optimization of<br />staurosporine production was performed with different<br />supplements which, according to literature data, had stimulative<br />effect on secondary metabolism (Zn, Fe and P salts, methyl<br />oleate, grape seed oil). In order to improve the production of<br />staurosporine, effects of pH (6.5, 7.5 and 8.5) and incubation time<br />(7, 10 and 14 days) were also examined. It was found that<br />addition of FeS04 significantly improved the staurosporine yield<br />in comparison to the starting conditions (increase of 25%). Our<br />results proved that rhizosphere soils of ethno-medicinal plants<br />are a prolific source of streptomycetes, producers of compounds<br />with good antifungal activity. Isolation of the new staurosporine<br />producing strain, allowed for its detailed bioactivity assessment.<br />Staurosporine scaffold might serve as a lead structure for the<br />development of new antifungal and antiangiogenic agents. Also,<br />results obtained within this research represent the basis for the<br />further scale-up and potential industrialization of the proposed<br />production process.</p>

>Magister Scientiae - MSc / Nanotechnology is spreading rapidly across the world as an extremely powerful technology. Nanoscience and nanotechnology are innovative scientific advancements that have been introduced only in this century. Nanotechnology has developed as the scientific advancement to grow and transform the entire agri-food area, with the potential to elevate global food production, in addition to the nutritional value, quality, and safety of food and food products. It has gained recognition due to its variability in shape, size, and dimension and how it correlates to its possibilities. One of those functions is nanoparticles’ (NPs) ability to have antimicrobial activity, more specifically its antifungal activity. One particular pathway of synthesising NPs is through phytonanotechnology which is the use of biomaterial to synthesis the NPs. / 2024

The main aim of this study was to isolate and characterise antifungal and antibacterial compounds from leaf extracts of Combretum molle which belonging to the Combretaceae family. C. molle is one of the commonly used medicinal plants in southern Africa for numerous ailments. Three animal fungal pathogens, namely, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus and five plant fungal pathogens, namely, Aspergillus niger, Aspergillus parasiticus, Fusarium oxysporum, Penicillium janthinellum, Rhizoctonia solani and four nosocomial bacteria Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa were used as test microorganisms for bioactive compounds in leaf extracts of C.molle. Experiments for phytochemical analysis were done using different C. molle leaf extracts which were made using acetone, methanol, ethanol, ethyl acetate, chloroform, butanol and hexane as extractants. Thin Layer Chromatography (TLC) fingerprints of different leaf extracts were developed in three mobile phase systems, EMW, CEF and BEA and detected with vanillin-sulphuric acid spraying agent. The different extracts of C. molle showed the presence of many different compounds with distinct retardation factors (Rf), separated according to their polarities. Bioautography was carried out to determine the number of active compounds and their Rf values. The TLC plates were developed in three mobile systems, each sprayed with either fungal or bacterial strains. In BEA bioautograms of A. fumigatus, clear zones of inhibition were observed at Rf values of 0.12, 0.23, and 0.40. In EMW bioautogram of C. albicans, clear zones of inhibition were observed at Rf value of 0.73, 0.81, 0.87. C. neoformans had weak growth inhibition. Most of the fungal and bacterial strains tested in the bioautography displayed susceptibility to the active compounds, with P. janthinellum and P. aeruginosa showing exceptional sensitivity. The minimum inhibitory concentrations (MIC) values ranged from 0.02 to 2.5 mg/ml against the tested pathogens. The acetone and ethyl acetate extracts had the best inhibitory activity against P. janthinellum with an MIC value of 0.02 mg/ml. The acetone extract of C. molle gave the highest total activity (775 ml/g) against P. janthinellum. C. albicans was the most resistant pathogen with an average MIC value of 0.56 mg/ml compared with the other tested strains. Extracts were active against both Gram-positive and Gram-negative strains. P. aeruginosa extracts had the highest average MIC value (0.24 mg/ml) among the tested bacterial strains. In general, there was good overall inhibitory activity by different extracts of C. molle. Bioassay-guided fractionation of DCM extract of the leaves of C. molle yielded 32 fractions. Further fractionation led to the isolation of five compounds (C1, C2, C3, C4 and C5). Compound C1 was selected for structure elucidation due a larger quantity isolated and higher antimicrobial activity compared with the other isolated compounds. Nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (MS) was used to show that compound C1 was taraxerol, belonging to the taraxerane group. Antimicrobial activity of the isolated compound against P. janthinellum had an MIC value of 0.08 ug/ml. Although the compound taraxerol have been discovered in other plant species, it is reported for the first time from C. molle in the study. The results illustrate that crude extracts and compound taraxerol from C. molle can be used as either an antibacterial or antifungal, and warrants further investigation. / Dissertation (MSc)--University of Pretoria, 2017. / Paraclinical Sciences / MSc / Unrestricted

Strains MS455 and MS389, endophytic bacteria, were isolated from healthy soybean plant growing adjacent to a patch of plants affected by charcoal rot disease, caused by the fungal pathogen Macrophomina phaseolina. The complete genomes of both strains were sequenced and identified as Burkholderia species Strain MS455 exhibits broad-spectrum antifungal activities against economically important pathogens, including Aspergillus flavus. Random and site-specific mutations were employed in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA-seq analysis demonstrated ORF1, a homolog to the ambR1 LuxR-type regulatory gene, not only regulates occidiofungin biosynthesis in MS455, but also involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate and corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Strain MS389 shows significant antifungal and antibacterial activities as well. Mutagenesis study identified that the TatC gene, an important unit of twin-arginine translocase (Tat) secretion system, and the LysR-type transcriptional regulatory gene were essential for the antifungal activity of strain MS389. RNA-seq analysis implied that the pyrrolnitrin biosynthesis gene cluster and an uncharacterized NRPS / PKS gene cluster were involved in antifungal activity. By comparing several endophytic bacteria of Burkholderia, including MS455 and MS389, to pathogenic Burkholderia species, endophytic bacteria were observed to harbor multiple antimicrobial biosynthesis genes but lack certain pathogenic or virulence genes. The potential endophytic behavior related genes and characteristics related to antibiotic resistance, secretion system, and CRISPR-Cas profiles were well established. The research findings on strains MS455 and MS389 have provided important genetic clues for understanding their molecular mechanism of antimicrobial activities and exhibited their potential possibility as biocontrol agents.

Orientadora: Lourdes Campaner dos Santos / Banca: Isabele Rodrigues Nascimento / Banca: Marcelo Aparecido da Silva / Resumo: Paepalanthus é um dos gêneros mais representativos da família Eriocaulaceae e consta na literatura que já foram isoladas naftopiranonas e flavonoides, substâncias com comprovadas atividades como antirradicalar, antiúlcera e mutagênica. Apesar dos estudos realizados até o momento com espécies de Eriocaulaceae, é importante destacar que ainda existe um grande número de espécies que não foram estudados química e biologicamente. Portanto, neste trabalho descrevemos o estudo químico e biológico do extrato metanólico dos capítulos de Paepalanthus acanthophyllus. A estratégia de fracionamento por HPLC-PDA em escala semipreparativa do extrato metanólico permitiu isolar as substâncias 6-metoxicanferol-3-O-(6"-p-cumaroil)-β-D-glucopiranosil-7-O-β-D-glucopiranosídeo, Pa2, 6-metoxicanferol-3-7-di-O-β-D-glucopiranosídeo,Pa1, 6-metoxicanferol-3-O-β-D-glucopiranosídeo, Pa3, Paepalantina-9-O-β-D-glucopiranosideo, Pa4 e a Paepalantina, Pa5. A padronização do extrato metanólico foi realizada por meio da quantificação dos derivados do canferol glicosilados (Pa1, Pa2 e Pa3) e do derivado da paepalantina glicosilada (Pa4) por HPLC-PDA. A metodologia permitiu avaliar os parâmetros de linearidade, limites de detecção e quantificação.Os limites de detecção (12,22 μg.mL-1) e quantificação (30,03 μg.mL-1) foram satisfatórios para as condições analisadas para os derivados do canferol. Para o derivado da paepalantina glicosilada observou-se os valores de limite de detecção 25,54μg.mL-1 e de quantifica... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Paepalanthus is one of the most representative genus of the Eriocaulaceae family and it is known in literature that naphthopyrano nes and flavonoids were isolated. These substances are proven to have antiradical, antiulcer and mutagenic biological activities. Despite available studies of the Eriocaulaceae species, it is important to emphasize that there is still a large number of sp ecies that have not been studied chemically and biologically. Therefore, in this work we describe the chemical and biological study of the m ethanol extract of the capitula from Paepalanthus acanthophyllus . The fractionation strategy by HPLC - PDA semiprepa rative scale of the methanolic extract allowed us to isolate substances 6 - methoxykaempferol - 3 - O - (6" - p - coumaroyll) - β - D - glucopyranosyl - 7 - O - β - D - glucopyranoside, Pa2, 6 - methoxyKaempferol - 3 - 7 - di - O - β - D - glucopyranoside, Pa1, 6 - methoxyKaempferol - 3 - O - β - D - glucopyr anoside, Pa3, p aepalantina - 9 - O - β - D - glucopyranoside, Pa4 and p aepalantin e Pa5 . The standardization of the methanol extract was performed by quantification of glycosides kaempferol derivatives and paepalantina glycosylated by HPLC - PDA. The methodology allow ed us to evaluate the parameters: linearity, limits of detection and quantification. The detection limits (12.22 μ g.mL - 1 ) and quantitation (30.03 μ g.mL - 1 ) were satisfactory for the conditions tested for the kaempferol derivatives. For the derivative of the glycosylated paepalantina, the detection limit (25.54 μ g.mL - 1 ) and quantization limit (77.39 μ g.mL - 1 ) were observed. The content of substances found in the met hanolic extract of the capitula were 2.39 % ( Pa1 ), 1.22 % ( Pa2 ), 2.52 % ( Pa3 ) and 8.38 % ( Pa4 ) . The total phenols content obtained was 126.41 m g. g - 1 in the extract of the capitulae and the content of the total flavonoid founded were 122.15 mg.g - 1. At the same time we evaluated the... / Mestre

Os compostos lipofílicos da própolis vermelha brasileira (PVB) têm recebido atenção especial devido a interessantes relatos referentes às suas atividades biológicas. Neste contexto, este estudo teve três etapas: a primeira objetivou investigar a atividade do extrato bruto etanólico da PVB e respectivas frações (obtidas com solventes de polaridade crescente, por meio de maceração em temperatura ambiente) contra cepas de Candida não-albicans (CNA) - C. krusei, C. glabrata, C. tropicalis e C. parapsilosis. A concentração inibidora mínima (CIM) foi determinada pelo método de microdiluição em caldo, com concentrações variando entre 1,9 a 500 μg/mL. Dentre as frações testadas, a fração hexânica (HEX) apresentou maior potencial antifúngico, alcançando valor de CIM de 1,95 μg/mL contra espécies de C. parapsilosis. Para cepas de C. glabrata, C. krusei e C. tropicalis, os valores de CIM obtidos foram variáveis (1,95-250 μg/mL). Ensaio colorimétrico de MTT foi utilizado para confirmar o dano celular e os resultados variaram de 80,66 a 94,44%, com extensa morte celular causada pela HEX, incluindo efeitos contra cepas resistentes de CNA, principalmente C. glabrata e C. parapsilosis. Esta potencial capacidade antifúngica está relacionada com compostos lipofílicos, provavalmente benzofenonas polipreniladas (BPPs), anteriormente descritas para PVB. A segunda etapa teve como objetivo avaliar a composição química do extrato hexânico da PVB (HEXred) por CLUE-DAD-EM. A investigação química resultou, principalmente, na identificação de BPPs (oblongifolina A, gutiferona E e/ou xantochimol). Após, um método de CLAE-UV isocrático foi validado para a determinação do teor total de BPPs (a 260 nm) expresso como garcinol, um diasterorisômero da gutiferona E disponível comercialmente. O método mostrou ser específico, exato, preciso e linear (0,1 a 10 μg/mL) para a determinação de BPPS na HEXred e na nanoemulsão (NE) contendo HEXred, bem como em amostras de pele e mucosa suínas, após estudos de permeação/retenção. O efeito matriz foi determinado para todas as matrizes complexas, demonstrando baixo efeito durante a análise. A estabilidade do método foi verificada por meio da exposição da HEXred às condições de estresse ácida, alcalina, oxidativa e térmica. Não houve interferência dos produtos de degradação durante a análise, indicando que o método analítico/bioanalítico proposto provou ser simples e confiável para a determinação de BPPs na presença de diferentes matrizes. Como terceiro passo nós buscamos otimizar a incorporação de BPPs em NE destinada ao tratamento de infecções fúngicas mucocutâneas. A otimização da NE foi realizada por meio de experimento de Box-Behnken, que permitiu avaliar simultaneamente a influência das concentrações do fosfolipídeo da lecitina de gema de ovo, do lípido catiónico DOTAP e das BPPs nas propriedades físico-químicas da NE, bem como na eficiência de associação (EA) das BPPs. Utilizando o software Mini-Tab®, a formulação ótima foi selecionada com base no menor tamanho de gotícula e maiores potencial zeta e eficiência de associação, exibindo um tamanho médio de 140,56 ± 5,22 nm, potencial zeta de + 60,72 ± 3,07 e 99,55 ± 1,09% de EA. Células de difusão do tipo Franz foram usadas para avaliar a distribuição das BPPs através da pele e mucosa suínas, sendo encontradas BPPs nas camadas de ambos os tecidos (principalmente na derme). Uma maior quantidade de BPPs (até 3 vezes) foi detectada na pele e mucosa lesadas o que demonstra o efeito da integridade do tecido na distribuição das BPPs, como sugerido por imagens de microscopia confocal de fluorescência. As BPPs foram detectadas no fluido receptor apenas quando a mucosa esofágica foi lesada. A atividade antifúngica das formulações foi investigada contra espécies de CNA - C. krusei, C. glabrata, C. tropicalis e C. parapsilosis. Os valores de CIM variaram de 0,654 a 2,617 μg/mL, com dano celular superior a 78% como verificado por ensaio de MTT. Tais resultados sugerem que a NE otimizada possui potencial promissor para ser utilizada topicamente para o tratamento de infecções fúngicas mucocutâneas causadas por espécies de CNA. / Lipophilic compounds of Brazilian red propolis (BRP) have received increasing attention due to some interesting findings regarding their biological activities. This study had three steps: first we aimed to investigate the activity of BRP crude ethanolic extract and their fractions (obtained with increasing polarity solvents, through maceration at room temperature) against non-albicans Candida (NAC) strains. Minimal inhibitory concentration (MIC) was determined by the broth microdilution method, with concentrations ranging from 1.9 to 500 μg/mL. Among the tested fractions, n-hexane fraction (HEX) showed higher antifungal potential, achieving MIC values of 1.95 μg/mL against C. parapsilosis strains. On C. glabrata, C. krusei and C. tropicalis strains, variable MIC values were obtained (1.95 to 250 μg/mL). MTT colorimetric assay was employed to confirm the cell damage and the results ranged from 80.66 to 94.44%, with extensive cell death caused by HEX, including the effects against NAC resistant strains, mainly C. glabrata and C. parapsilosis. This potential antifungal capacity showed by HEX is related to the lipophilic compounds, probably polyprenylated benzophenones (PPBs), previous described for BRP. In the second step we aimed to evaluate the chemical composition of BRP n-hexane extract (HEXred) by UPLC-PDA-MS. Chemical investigation mainly resulted in the identification of PPBs in this extract, named oblongifolin A, guttiferone E, and/or xanthochymol. After that, an isocratic HPLC-UV method was validated for the determination of total content of PPBs (at 260 nm) expressed as garcinol, a commercially available guttiferone E diastereoisomer. The method showed to be specific, precise, accurate, and linear (0.1 to 10 μg/mL) for the determination of PPBs in HEXred, BRP-loaded nanoemulsions (NE), as well as, in porcine skin and mucosa samples after permeation/retention studies. The matrix effect was determined for all complex matrices, demonstrating low effect during the analysis. The stability-indicating method was verified by submitting HEXred to acidic, alkaline, oxidative, and thermal stress conditions. No interference of degradation products was detected during analysis, indicating that the proposed analytical/bioanalytical method proved to be simple and reliable for the determination of PPBs in the presence of different matrices. As third step we aimed to optimize the incorporation of PPBs into NE intended for the treatment of mucocutaneous fungal infections. The optimization of NE was performed by means of a Box-Behnken Design, which allowed evaluating simultaneously the influence of the phospholipid egg-lecithin, the cationic lipid DOTAP and PPBs concentrations on the physicochemical properties of NE, as well as on the association efficiency (AE) of PPBs. By using the Mini Tab® software, the optimal formulation was selected based on the smallest droplet size and highest zeta potential and AE, exhibited a mean average size of 140.56±5.22 nm, zeta potential of +60.72±3.07 and AE of 99.55±1.09%. Franz-type diffusion cells were used to evaluate PPBs distribution through porcine skin and mucosa being PPBs found in both mucosa and skin layers (mainly in the dermis). A higher amount of PPBs (up to 3-fold) was detected in impaired skin and mucosa demonstrating the effect of the integrity of the tissue on PPBs distribution, as suggested by confocal fluorescence microscopy images. PPBs were detected in the receptor fluid only when esophageal mucosa was impaired. The antifungal activity of the formulations was investigated against NAC species – C. krusei, C. glabrata, C. tropicalis and C. parapsilosis. MIC values varied from 0.654 to 2.617 μg/mL, with cell damage higher than 78 % as verified by MTT assay. Such results suggest that the optimized NE have promising potential to be used topically for the treatment of mucocutaneous fungal infections caused by NAC strains.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O Cajueiro (Anacardium occidentale L.) à uma planta com uma grande importÃncia social e econÃmica no Nordeste do Brasil. O bagaÃo do pedÃnculo do caju à uma das maiores fontes de resÃduos (90-94%) produzidos pela indÃstria cajueira. Neste estudo, foram preparadas cinzas do bagaÃo e submetidas à anÃlise da composiÃÃo e a testes de atividade antifÃngica in vitro contra espÃcies de Fusarium. Esta anÃlise indicou uma cristalinidade em torno de 73%, correspondendo Ãs seguintes fases solÃveis: carbonato Ãcido de potÃssio - KHCO3 (39,54%), sulfato de potÃssio - K2SO4 (24,87%), e estruvita-K - MgKPO4 â 6H2O (8,59%). As fases amorfas (cerca de 27%) foram identificadas como a fraÃÃo insolÃvel de cinzas. A soluÃÃo apresentou alta atividade antifÃngica contra F. oxysporum, F. moniliforme e F. lateritium. Sua aÃÃo foi maior do que o Cercobin (tiofanato metÃlico), indicando uma possÃvel utilizaÃÃo como um agente antifÃngico nÃo tÃxico. / Cashew (Anacardium occidentale L.) is a plant with a highly social and economic importance in Northeast Brazil. Cashew peduncle bagasse is one of the greatest sources of residues (90â94%) produced by the cashew agronomic industry. In this study, we prepared cashew peduncle bagasse ashes and submitted them to compositional analysis and tests for antifungal activity in vitro against Fusarium species. This analysis indicated a crystallinity around 73%, corresponding to the following soluble phases: acid potassium carbonate- KHCO3 (39.54%), potassium sulfate - K2SO4 (24.87%), and struvite-K - MgKPO4Â6H2O (8.59%). The amorphous phases (around 27%) were identified as the insoluble fraction of ashes. The solution showed high antifungal activity against F. oxysporum, F. moniliforme and F. lateritium. This activity of this product was greater than that of Cercobin (thiophanate-methyl), indicating a possible use of this material as a non-toxic antifungal agent.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-05-08T02:43:19Z No. of bitstreams: 1 127110045.pdf: 1527824 bytes, checksum: 960729a0a23069d5d40ec997cc3034b8 (MD5) / Made available in DSpace on 2015-05-08T02:43:19Z (GMT). No. of bitstreams: 1 127110045.pdf: 1527824 bytes, checksum: 960729a0a23069d5d40ec997cc3034b8 (MD5) Previous issue date: 2014-07-17 / O gênero Cuphea, popularmente conhecido no Brasil por “sete-sangrias”, tem seu uso medicinal reconhecido devido aos efeitos diurético, hipotensor e cardioprotetor. No sul do Brasil, em região característica do bioma Pampa, foi encontrada a espécie Cuphea glutinosa Cham. & Schltdl. Embora o uso popular, esta espécie é pouco descrita na literatura. O presente trabalho tem como objetivos o estudo da composição química dos extratos de C. glutinosa e a avaliação das atividades antifúngica e anti-hipertensiva. O material vegetal foi coletado na cidade de Uruguaiana (RS, Brasil), identificado e depositado em herbário. Após secagem e trituração do material vegetal, foram obtidos os extratos hidroetanólicos através de maceração exaustiva com etanol 40% (v/v) para folhas e etanol 70% (v/v) para raízes. Para a infusão, utilizou-se água a 80oC. As análises cromatográficas foram realizadas em equipamento cromatógrafo a líquido Prominence Shimadzu, em técnica por CLAE e CLUE. Utilizou-se sistema de fase reversa, eluição por gradiente com fase móvel composta por acetonitrila:metanol (4:1) e ácido fórmico 0,1% pH 3,0, coluna C18 analítica e fast, e detecção por UV-DAD e ESI-MS. Os teores de polifenóis totais e de flavonóides foram determinados por método colorimétrico, seguindo metodologia padronizada. A atividade antifúngica in vitro foi realizada utilizando o método de microdiluição em caldo, determinando-se a CIM, in-vitro, contra diferentes isolados clínicos. Para avaliação do potencial anti-hipertensivo in vivo, foram realizadas medições da pressão sanguínea pelo método de monitoramento hemodinâmico invasivo, através da inserção de cateter na artéria carótida. Os resultados de teor de fenólicos totais indicaram predominância destes componentes em extratos obtidos de folhas e por maceração, conforme os valores obtidos: 1,8501 mg EAG/mL (folhas) e 0,8467 mg EAG/mL (raízes) para infusão, e 3,7284 mgEAG/mL (folhas) e 2,6266 mg EAG/mL (raízes) para maceração. Quanto ao teor de flavonóides, os resultados quantitativos foram: 7,0959 mg/g (folhas) e 0,5664 mg/g (raízes) para a infusão, e 7,9511 mg/g (folhas) e 0,5994 mg/g (raízes) para maceração. Na análise cromatográfica, os extratos obtidos das folhas de C. glutinosa apresentaram picos cromatográficos bem separados, em perfil reprodutível. Na determinação por CLUE-MS, os dados de íon molecular e fragmentos de massa indicaram a composição predominante em flavonóides, sugerindo-se os componentes quercetina-3-O-glicosídeo, quercetina-3- arabinosídeo, quercetina-3-glicuronídeo, isoramnetina e quercetina-5-O-β-glicopiranosídeo. Para o potencial antifúngico, os extratos das folhas e raízes apresentaram atividade in vitro contra Candida parapsilosis, Candida tropicalis e Trichosporon asahii, com CIM variando na faixa de 1,9-62,5 μg/mL. Nos testes hemodinâmicos realizados, os extratos das folhas não apresentaram efeito significativo sobre a pressão arterial. A identificação dos componentes em C. glutinosa, derivados de quercetina, torna promissora novas investigações a fim de aprofundar o conhecimento a respeito desta espécie, em especial na busca de respostas para a relatada ação anti-hipertensiva. / The Cuphea genus, popularly known in Brazil as "sete-sangrias", is used traditionally due the diuretic, hypotensive and cardioprotective effects. In southern Brazil, in characteristic region of Pampa biome, it was found the species Cuphea glutinosa Cham. & Schltdl. Although used popularly, this species is few reported in the literature. The present work aimed to study the chemical composition of extracts from C. glutinosa and to evaluate the antifungal and anti -hypertensive activities. The plant material was collected in the city of Uruguaiana (RS, Brazil), identified and deposited in a herbarium. After dryness and milling, the hydroethanolic extracts were obtained through exhaustive maceration using ethanol 40% (v/v) for leaves and ethanol 70% (v/v) for roots. The infusions were prepared using water at 80 °C. The chromatographic analyses were performed in liquid chromatography Prominence Shimadzu, for HPLC and UPLC assays. The method was conducted using reverse phase system, gradient elution with mobile phase composed by acetonitrile:methanol (4:1) and formic acid 0.1% pH 3.0, C18 analytical and fast column, and detection by UV-DAD and MS. The polyphenols and flavonoids contents were determined by colorimetric method. The in vitro antifungal activity was conducted by using the broth microdilution method, determining the MIC against different clinical isolates. For evaluation of in vivo anti-hypertensive potential, the blood pressure was measured by the method of invasive hemodynamic monitoring, through of insertion the catheter into the carotid artery. The results of phenolic content indicated the high concentration of these compounds in leaves extracts obtained by maceration: 1.8501 mgEAG/mL (leaves) and 0.8467 mgEAG/mL (roots) for infusion, and 3.7284 mgEAG/mL (leaves) and 2.6266 mgEAG/mL (roots) for maceration. For flavonoids, the contents were: 7.0959 mg/g (leaves) and 0.5664 mg/g (roots) for infusion, and 7.9511 mg/g (leaves) and 0.5994mg/g (roots) for maceration. In the chromatographic analyses, the leaf extracts from C. glutinosa presented chromatographic peaks well separated and reproducible. In the determination by UPLC-MS, the molecular ion and mass fragments indicated the predominant composition in flavonoids, suggesting the compounds quercetin-3- O-glucoside, quercetin-3-arabinoside, quercetin-3-glucuronide, isorhamnetin and quercetin-5- O-β-glucopiranoside. For the antifungal potential, the leaf and roots extracts presented activity against Candida parapsilosis, Candida tropicalis e Trichosporon asahii, with MIC values ranging 1,9-62,5 μg/mL. In the hemodynamic tests performed, the leaves extracts did not present significant effect in the arterial pressure, although a tendency in pressure reduction could be observed. The identification of quercetin derivatives in C. glutinosa becomes promisor further investigations about this species, mainly in respect to the anti-hypertensive action.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-07T12:58:36Z No. of bitstreams: 1 HEMERSON SILVA DA ROSA.pdf: 1232470 bytes, checksum: b64c04e3acdc1a67c9bd8559b68fb261 (MD5) / Made available in DSpace on 2016-04-07T12:58:36Z (GMT). No. of bitstreams: 1 HEMERSON SILVA DA ROSA.pdf: 1232470 bytes, checksum: b64c04e3acdc1a67c9bd8559b68fb261 (MD5) Previous issue date: 2013-01-24 / Sida tuberculata (Malvaceae), conhecida popularmente como “guanxuma”, é uma espécie vegetal de porte herbáceo, bem representada na região sul do Brasil. Na cultura popular é utilizada para tratamento de diversas enfermidades, em especial àquelas relacionadas ao diabetes e ao colesterol elevado. Para algumas espécies, existem relatos de eventual potencial antimicrobiano. Considerando a ausência de estudos sobre esta planta, o presente trabalho investigou a composição química dos extratos brutos de S. tuberculata e avaliou seu potencial antifúngico in vitro. Após a coleta e identificação, o material vegetal foi submetido aos processos de secagem e trituração. Submeteu-se a extração a frio por percolação, utilizando-se como solvente solução hidroetanólica a 40% para folhas e 70% para raízes. Para fins de comparação foram feitas extrações aquosas por infusão. Na sequência, foram determinados os teores de fenólicos totais e flavonóides totais. Posteriormente, as amostras foram analisadas através de método por CLAE-UV, com seleção das condições cromatográficas de melhor eficiência de separação. Sendo estas estabelecidas, efetuou-se análise por LC-MS em modo ESI positivo. Para os ensaios da atividade antifúngica foram utilizados os protocolos de microdiluição em ágar para determinação da concentração inibitória mínima (CIM) e concentração fungicida mínima (CFM). Também foi aplicada a metodologia para avaliação do potencial de remoção de biofilme em Cateter Venoso Central (CVC). Os resultados obtidos demonstraram um maior teor de fenólicos e flavonóides totais nos extratos das folhas. As análises por LC-UV-MS permitiram a identificação e proposição de cinco compostos, entre ecdisteróides, flavonóides e alcalóides. Nos ensaios de atividade antifúngica os extratos aquosos apresentaram atividade contra linhagens de Candida krusei, com valores de CIM variando entre 3.9 - 62.5 μg/ml para folhas e 1.95 - 31.25 μg/ml para raízes. No teste de remoção de biofilme, os extratos aquosos das folhas demonstraram um maior potencial de remoção. Os dados de composição química obtidos, nas variantes de diferentes partes da planta, bem como a atividade antimicrobiana detectada, geram expectativas quanto a novos estudos de exploração do potencial biológico de S. tuberculata. / Sida tuberculata (Malvaceae), popularly known as "guanxuma", is an herbaceous plant species present in southern Brazil. In popular culture, it is used for the treatment of several diseases, such as those related to diabetes and high cholesterol level. For some species of Sida, there are also reports about the antimicrobial potential. Considering the lack of studies at this species, this study proposed an investigation about the chemical composition of Sida tuberculata extracts and their in vitro antifungal activity. After collection and identification, the plant material was submitted to dryness and powdered. Then, it was submitted to extraction by percolation using hydroethanolic solution at 40% and 70% as solvent, to leaves and roots respectively. For comparison, aqueous extracts were obtained by infusion. The total phenolic and flavonoid contents of extracts were determined. The samples were also evaluated by HPLC-UV, testing the chromatographic conditions that promote the better separation efficiency. After, for the identification procedure, the analysis by LC-ESI-MS in positive mode was conducted using previously established conditions. For the antifungal activity assay, the agar microdilution protocols were used for determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). In addition, the extracts were tested for potential biofilm removal in Central Venous Catheter (CVC). The results demonstrated a higher concentration of total phenolic and flavonoids compounds in the leaves extracts. LC-MS analysis allowed the identification of five components, between ecdysteroids, flavonoids and alkaloids. In the assay of antifungal activity, the aqueous extract had activity against Candida krusei strains, with the MIC values varying between 3.9 - 62.5 μg/ml for leaves and 1.95 - 31.25 μg/ml for roots. In the CVC biofilm removal testing, the aqueous leaves extracts presented a greater potential. The chemical composition data obtained in this work, considering the different parts of plant, as well as the antimicrobial activity detected, bring perspectives of exploring this species concerning its biological potential.