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Orientador: Prof. Dr. Eliezer Rodrigues de Souto Coorientador: Prof. Dr. Dauri José Tessmann Dissertação (mestrado em agronomia) - Universidade Estadual de Maringá, 2019 RESUMO: A Stevia (Stevia rebaudiana (Bert.) Bertoni) pertence a família Asteraceae e é uma planta originária da América do Sul. A Stevia vem ganhando cada vez mais espaço no mercado devido ao seu poder adoçante, que além de ser maior do que a própria sacarose é isento de calorias, carboidratos e com índice glicêmico zero. Sabe se que a germinação da Stevia através de sementes é baixa e pouco se sabe a respeito de fungos associados à mesma. Tendo em vista essa escassez de trabalhos associados à essas variáveis o presente trabalho objetivou buscar condições eficazes para o teste de sanidade da semente, definindo temperatura(20, 25 e 28°C) e período de incubação (5, 7 e 10 dias pós semeadura) ideais para avaliação, bem como, avaliar a incidência dos fungos encontrados e verificar a transmissão para plântulas e possíveis interferências no vigor e germinação das mesmas, com e sem assepsia, a partir da inoculação dos fungos identificados como mais importante no lote trabalhado.Foi observado a presença dos fungos Alternariaalternata,Bipolaris sp., Fusarium sp., Cladosporium sp., Phytomices sp. e Phoma sp., entretanto apenas Alternaria alternata, Bipolaris sp. e Fusarium sp. foram discutidos e submetidos a análise estatística. De maneira geral, a melhor condição observada foi em incubação feita a 20ºC, entre 7 e 10 dias de incubação. A assepsia realizada no teste de sanidade com as sementes inoculadas apontaram redução significativa na incidência dos fungos avaliados, apontando que o fungo havia apenas contaminado a semente. Em relação ao vigor das sementes foi observado diferença significativa apenas em sementes inoculadas com Fusarium sp. quando houve assepsia. No teste de germinação, concluiu-se que a inoculação dos fungos afetou negativamente a germinação apenas de sementes inoculadas com Fusarium sp.. As maiores taxas de transmissão aconteceram com os fungos A. alternatae Bipolaris sp. com 24 horas de contato da semente com a colônia, sem assepsia ABESTRACT: Stevia (Stevia rebaudiana (Bert.) Bertoni) belongs to the family Asteraceae and is a plant native to South America. Stevia has been gaining more and more space in the market due to its sweetening power, which in addition to being greater than itself sucrose is calorie free, carbohydrate and zero glycemic index. It is known that the germination of Stevia through seeds is low and little is known about fungi associated with it. The objective of this work was to search for effective conditions for the seed health test, defining temperature (20, 25 and 28 ° C) and incubation period (5, 7 and 10 days after sowing) ), as well as to evaluate the incidence of the fungi found and to verify the transmission to seedlings and possible interferences in their vigor and germination, with and without asepsis, from the inoculation of the fungi identified as most important in the lot worked. The presence of fungi Alternaria alternata, Bipolaris sp., Fusarium sp., Cladosporium sp., Phytomices sp. and Phoma sp., however only Alternaria alternata, Bipolaris sp. and Fusarium sp. were discussed and submitted to statistical analysis. In general, the best observed condition was incubation at 20ºC, between 7 and 10 days of incubation. The asepsis performed in the sanity test with the inoculated seeds indicated a significant reduction in the incidence of the evaluated fungi, indicating that the fungus had only contaminated the seed. Regarding seed vigor, a significant difference was observed only in seeds inoculated with Fusarium sp. when there was asepsis. In the germination test, it was concluded that the inoculation of the fungi negatively affected the germination only of seeds inoculated with Fusarium sp.. The highest transmission rates occurred with the fungi A. alternata and Bipolaris sp. with 24 hours contact of the seed with the colony, without asepsis

Orientador: Prof.ª Dr.ª Maria Ida Bonini Ravanelli Speziali Dissertação (mestrado em Ciências Fisiológicas)--Universidade Estadual de Maringá, 2017 Resumo: O esteviol é um diterpeno extraído das folhas de Stevia rebaudiana (Bertoni) Bertoni, uma planta nativa da América do Sul. Nas últimas décadas, os glicosídeos de esteviol, principalmente o esteviosídeo e o rebaudiosídeo A, têm sido utilizados como adoçantes em alimentos e bebidas e, após sofrerem metabolização no trato gastrointestinal, o esteviol é o principal metabólito encontrado no plasma. Além disso, diversos estudos têm atribuído propriedades terapêuticas aos produtos derivados de S. rebaudiana. No entanto, é escasso o número de trabalhos descrevendo a interação desses compostos com as membranas biológicas. O objetivo deste estudo foi avaliar o transporte do esteviol, isoesteviol e esteviolbiosídeo através da membrana lasmática de células 3T3-L1. Para isso, empregamos uma metodologia baseada em espectroscopia de fluorescência, utilizando as sondas FPE e BCECF. A ligação destes produtos, que são moléculas com grupamentos ionizáveis, à membrana plasmática e sua translocação (flip-flop) foram avaliadas por meio de medidas de fluorescência do FPE (sonda de cargas de superfície) e BCECF (sonda de pH intracelular), respectivamente. Nossos dados utilizando a sonda FPE demonstraram que há uma rápida ligação do esteviol e isoesteviol à monocamada externa da membrana plasmática (t1/2 < 2s), seguida de uma recuperação do sinal de volta aos valores iniciais. Por outro lado, o esteviolbiosídeo não apresentou o mesmo efeito. Os experimentos com a sonda BCECF mostraram que o esteviol, isoesteviol e esteviolbiosídeo sofreram translocação através da membrana plasmática (t1/2 < 2s). Em ambas as sondas, o isoesteviol causou quedas mais intensas na fluorescência. O efeito do esteviolbiosídeo foi consideravelmente menor do que o causado pelas agluconas, possivelmente em função da presença das moléculas de glicose associadas. Baseado em nossos resultados, concluímos que o esteviol e seu isômero isoesteviol são capazes de ligarem-se rapidamente à monocamada externa de células 3T3-L1 e serem translocados através da membrana plasmática alcançando a monocamada interna, sendo a resposta ao esteviolbiosídeo comparativamente menor Abstract: Steviol is a diterpene extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni, a plant native from South America. In the last decades, steviol glycosides, mainly stevioside and rebaudioside A, have been used as sweeteners in foods and beverages. However, these glycosides are metabolized by colonic bacteria into steviol, the main metabolite found in the plasma. In the last years, several studies have been attributed therapeutic properties to the glycosides from S. rebaudiana. However, information about the interaction of these compounds with biological membranes is scarce. Therefore, the aim of this study is to evaluate the transport of steviol, isosteviol and steviolbioside through the plasma membrane of 3T3-L1 cells. For this, we employed a methodology based on fluorescence spectroscopy. The binding and diffusion across the plasma membrane of the compounds, molecules with ionizable groups, were measured by the charge surface probe FPE and the intracellular pH probe BCECF, respectively. Our data with FPE demonstrated a rapid binding of steviol and isosteviol to the outer monolayer of the plasma membrane (t1/2 < 2s), measured as a reduction in FPE fluorescence emission, followed by a recovery of the signal back to the baseline. Experiments with BCECF also indicated a rapid translocation of steviol and isosteviol across the plasma membrane (t1/2 < 2s), measured as a reduction in BCECF fluorescence. Steviol caused more intense reductions in fluorescence in both probes. On the other hand, the effect of steviolbioside, a glycoside from steviol with an ionizable group, on BCECF and FPE was considerably lower than that caused by aglucones. These data are explained by the presence of glucose residues in the steviolbioside molecule, which probably reduce its binding to the plasma membrane. Based on our initial results, we conclude that steviol and its isomer isosteviol are able to rapidly bind and translocate across the plasma membrane of 3T3-L1 cells, besides, the response of steviolbioside was comparatively lower

Orientador: Prof.ª Dr.ª Kellen Brunaldi Dissertação (mestrado em Ciências Fisiológicas)--Universidade Estadual de Maringá, 2017 Resumo: Os glicosídeos da estévia apresentam potencial terapêutico anti-diabetogênico e anti-hipertensivo. Porém, não há um consenso na literatura quanto ao mecanismo de transporte dos glicosídeos da estévia e suas agluconas através de membranas celulares. Portanto, o objetivo deste trabalho foi avaliar o transporte das agluconas esteviol, isoesteviol e do glicosídeo esteviolbiosídeo através da bicamada lipídica por meio de espectroscopia de fluorescência. Lipossomas (large unilamellar vesicles - LUVs) de egg-PC foram formados por extrusão. As sondas fluorescentes piranina (sensível a pH) e calceína foram encapsuladas nas LUVs, enquanto que a sonda FPE (sensível a cargas de superfície) foi inserida seletivamente na monocamada externa da bicamada lipídica. As soluções estoque dos produtos da estévia foram preparadas em DMSO ou em BSA (2 mol de produto:1mol de BSA). A ligação dos produtos da estévia a BSA foi caracterizada pela fluorescência intrínseca da BSA (resíduos de triptofano). O raio hidrodinâmico das LUVs foi medido pela técnica de DLS (Dynamic light scattering). A ligação dos produtos da estévia à BSA foi evidenciada pela supressão da fluorescência intrínseca da BSA. A constante de ligação à 37 °C, calculada a partir dos dados experimentais, indicaram uma maior afinidade do esteviolbiosídeo pela BSA. Entretanto, o esteviol e isoesteviol, promoveram um deslocamento hipsocrômico (blue shift) no espectro de emissão da BSA, indicativo do aumento na hidrofobicidade ao redor dos resíduos de triptofano. O esteviol e o isoesteviol promoveram uma rápida (em segundos) redução e recuperação da fluorescência do FPE, condizentes com a ligação da forma ionizada e carregada negativamente à monocamada externa seguida de sua difusão (flip-flop), respectivamente. Nos ensaios com piranina, o esteviol, isoesteviol e em menor extensão o esteviolbiosídeo, causaram uma rápida (em segundos) redução da fluorescência da piranina, um indicativo de acidificação do meio intravesicular. Esses dados refletem a difusão da forma protonada dos produtos da estévia através da bicamada lipídica, com o posicionamento dos grupamentos ionizáveis na interface água/membrana e entrega de prótons para interior das LUVs. Os produtos da estévia não afetaram o raio hidrodinâmico das LUVs e não induziram o vazamento de calceína encapsulada, ou seja, não alteraram a organização estrutural da bicamada lipídica que justificassem a difusão transmembrana dessas moléculas assim como o vazamento das sondas FPE e piranina. Portanto, os resultados apresentados, inéditos na literatura, indicam que as agluconas esteviol e isoesteviol são capazes de se ligarem e atravessarem membranas lipídicas por difusão simples e que a permeabilidade da bicamada lipídica ao esteviolbiosídeo é bastante reduzida Abstract: The stevia glycosides present anti-diabetogenic and antihypertensive actions. However, there is no consensus in the literature regarding the transport mechanism of glycosides and their aglucones across cell membranes. Thus, the objective of this work was to evaluate the transmembrane transport of the steviol, isoesteviol and steviolbioside by means of fluorescence spectroscopy. Liposomes (large unilamellar vesicles - LUVs) of egg-PC were prepared by extrusion. Pyranin (pH probe) and calcein were encapsulated in LUVs. FPE (charge surface probe) was selectively inserted into the outer monolayer of the lipid bilayer. Stock solutions of stevia products were prepared in DMSO or BSA (2 mol of product: 1 mol of BSA). The binding of stevia products to BSA was characterized by the BSA intrinsic fluorescence (tryptophan residues). The hydrodynamic radius was measured by DLS (Dynamic light scattering). The binding of the stevia products to BSA caused fluorescence suppression. The steviolbioside binding constant at 37 °C, calculated from the experimental data, was higher than those calculated for steviol and isoesteviol. Furthermore, the aglucones induced a blue shift in the BSA emission spectrum probably due to an increase in hydrophobicity around the tryptophan residues. In the FPE assay, steviol and isosteviol promoted a rapid (in seconds) reduction in FPE fluorescence followed by a recovery towards to the base line. These results are explained by the binding and flip-flop of the ionized molecules. In the piranine assay, steviol, isosteviol and, to a lesser extent, steviolbioside, caused a rapid (in seconds) reduction of the pyranin fluorescence without recovery when BSA was used as vehicle. These results reflect the binding and flip-flop of the protonated molecules, with delivery of protons to the LUVs interior. The stevia products did not affect the LUV hydrodynamic radius and did not induce calcein leakage. These results indicate that the structural organization of the lipid bilayer was not affected by the binding of stevia products. Therefore, this study indicate that steviol and isosteviol are able to bind and cross lipid membranes by simple diffusion and the permeability of lipid bilayer to steviolbioside is quite reduced

Orientador: Prof.ª Dr.ª Cecília Edna Mareze da Costa Dissertação (mestrado em Ciências Fisiológicas)--Universidade Estadual de Maringá, 2017 Resumo: A Stevia rebaudiana além de ser uma fonte de adoçantes não calóricos é, também, importante fonte de moléculas bioativas. Este trabalho investigou, primeiramente, o efeito do extrato etanólico (EET) e das frações clorofórmica (FCL) e de acetato de etila (FAE), obtidas das folhas da stevia, na secreção de insulina de ilhotas isoladas de ratos. Os resultados obtidos nestes estudos iniciais e o fato da FAE ser rica em compostos fenólicos, apresentar alto conteúdo protéico e ser praticamente isenta de glicosídeos edulcorantes, determinou que esta fosse a fração selecionada para os testes subsequentes que foram realizados neste trabalho. Este é o primeiro trabalho que avalia os efeitos de uma fração de stevia com tais características na secreção de insulina e possíveis mecanismos, no metabolismo da glicose em hepatócitos isolados e na tolerância à glicose in vivo. Os resultados obtidos com as ilhotas isoladas de ratos normais mostraram que a FAE estimula a secreção de insulina apenas na presença de concentrações altas de glicose e de forma cálcio dependente, por mecanismos não totalmente esclarecidos, mas que parecem envolver canais de potássio sensíveis a ATP e receptores colinérgicos M3 e adrenérgicos a2. No estudo com hepatócitos, isolados de ratos em estado alimentado, foi observado que FAE estimula a glicólise, não interfere na glicogenólise e causa aumento na razão NADH/NAD+. O teste oral de tolerância à glicose mostrou que a FAE melhora a tolerância à glicose Abstract: Stevia rebaudiana, besides being a source of non-caloric sweeteners, is also an important source of bioactive molecules. This work first investigated the effect of ethanolic extract (ETE), chloroform (CLF) and ethyl acetate (EAF) fractions, obtained from stevia leaves, on the insulin secretion of islets isolated from rats. The results obtained from this study and the fact that FAE is rich in phenolic compounds, present high protein content and practically free of sweetening glycosides, determined that this was the fraction selected for the subsequent tests performed in this work. This is the first work to evaluate the effects of a stevia fraction with such characteristics on insulin secretion mechanisms, glucose metabolism in isolated hepatocytes, and in vivo glucose tolerance. The results obtained with islets isolated from normal rats showed that FAE stimulates insulin secretion only in the presence of high glucose and calcium dependent concentrations, by mechanisms not fully understood, but which seem to involve ATP-sensitive potassium channels and M3 cholinergic receptors and a2 adrenergic receptors. In the study with hepatocytes isolated from fed rats, it was observed that FAE stimulates glycolysis, does not interfere with glycogenolysis, and causes an increase in the NADH/NAD+ ratio. The oral glucose tolerance test has shown that FAE improves glucose tolerance

Στην παρούσα μεταπτυχιακή εργασία πραγματοποιήθηκε μελέτη του πολυμορφισμού της ινσουλίνης μέσω της κρυσταλλογραφίας ακτίνων-Χ. Για το σκοπό αυτό πραγματοποιήθηκαν πολυάριθμα πειράματα κρυστάλλωσης της ινσουλίνης υπό διαφορετικές συνθήκες (pH, οργανικοί προσδέτες). Οι κρύσταλλοι που προέκυψαν χρησιμοποιήθηκαν σε πειράματα περίθλασης ακτίνων-Χ τα οποία πραγματοποιήθηκαν στο Ευρωπαϊκό σύγχροτρον, ESRF, της Γαλλίας και στο Ελβετικό σύγχροτρον, SLS. Η τεχνική που ακολουθήθηκε για τα πειράματα περίθλασης ήταν αυτή του powder diffraction, η οποία τα τελευταία χρόνια έχει αναδειχθεί σε ένα σημαντικό εργαλείο χαρακτηρισμού των πρωτεϊνικών πολυκρυσταλλικών ιζημάτων. Από τη μελέτη αυτή, προέκυψαν διάφορα πολύμορφα της ινσουλίνης που χαρακτηρίζονται από διαφορετικές συμμετρίες, ενώ πραγματοποιήθηκε ο προσδιορισμός κάθε κρυσταλλικής φάσης στο επίπεδο της μοναδιαίας κυψελίδας. Ενδιαφέρον αποτελεί ο εντοπισμός δύο νέων κρυσταλλικών φάσεων της ινσουλίνης, μονοκλινούς συμμετρίας, οι οποίες ενδέχεται να είναι υψηλής φαρμακευτικής σημασίας. In this thesis the polymorphism of insulin via X-ray powder crystallography has been carried out. For this purpose numerous experiments of insulin crystallization have been performed under different conditions (pH, organic ligands). The resulting crystals were employed in X-ray diffraction experiments, which took place at the European synchrotron, ESRF, in France and at the Swiss synchrotron, SLS. The technique employed for the X- Ray diffraction experiments, was the powder diffraction method, which has been proven to be an important tool for the determination of polycrystalline protein precipitants during the last decade. From this study, a number of insulin polymorphs were identified in different crystal systems and furthermore, each crystalline phase was determined at the level of unit cell symmetry and dimensions. An interesting point occurs from the characterization of two novel insulin phases, belonging to monoclinic symmetry, which are potentially associated with high pharmaceutical importance.

Οι κυκλοδεξτρίνες είναι κυκλικοί ολιγοσακχαρίτες αποτελούμενοι από μόρια α-D-γλυκοπυρανόζης που συνδέονται με α-1-4 γλυκοζιτικούς δεσμούς και παράγονται από το άμυλο. Διαφέρουν μεταξύ τους από τον αριθμό των μονάδων γλυκοπυρανόζης της δομής τους. Οι κυκλοδεξτρίνες διακρίνονται σε δύο ομάδες: τις φυσικές και τις τροποποιημένες. Οι πιο κοινές φυσικές κυκλοδεξτρίνες είναι οι α-κυκλοδεξτρίνη, β-κυκλοδεξτρίνη και γ-κυκλοδεξτρίνη, που αποτελούνται από 6, 7 και 8 μονάδες γλυκοπυρανόζης, αντίστοιχα. Επίσης, οι τροποποιημένες κυκλοδεξτρίνες εμφανίζουν σημαντικά πλεονεκτήματα σε σχέση με τις φυσικές. Η δομή των κυκλοδεξτρινών προσομοιάζει το σχήμα κόλουρου κώνου με μια υδρόφιλη εξωτερική επιφάνεια και μια λιπόφιλη εσωτερική κοιλότητα. Η υδρόφιλη εξωτερική επιφάνεια εξασφαλίζει καλή υδατοδιαλυτότητα για το μόριο της κυκλοδεξτρίνης, ενώ η υδρόφοβη εσωτερική κοιλότητα δημιουργεί το κατάλληλο περιβάλλον για τον εγκλωβισμό ολόκληρου του μορίου μιας βιοδραστικής ένωσης ή τμήματος αυτού. Οι κυκλοδεξτρίνες μπορούν να αλληλεπιδράσουν με μόρια κατάλληλου μεγέθους για το σχηματισμό συμπλόκων έγκλεισης. Ο σχηματισμός των συμπλόκων έγκλεισης των κυκλοδεξτρινών με τις βιοδραστικές ενώσεις συχνά έχει ως αποτέλεσμα τη τροποποίηση των φυσικών και χημικών ιδιοτήτων του εγκλωβιζόμενου μορίου. Η συμπλοκοποίηση των κυκλοδεξτρινών έχει χρησιμοποιηθεί επιτυχώς για την αύξηση της διαλυτότητας, του ρυθμού διαλυτοποίησης, της χημικής σταθερότητας και βιοδιαθεσιμότητας ελάχιστα διαλυτών και αδιάλυτων βιοδραστικών ενώσεων. Επίσης, οι κυκλοδεξτρίνες μπορούν να χρησιμοποιηθούν για τη μείωση ή κάλυψη ανεπιθύμητων οσμών και δυσάρεστων γεύσεων, την αντιμετώπιση των προβλημάτων ασυμβατότητας μεταξύ των βιοδραστικών ουσιών και μεταξύ των βιοδραστικών ουσιών και των εκδόχων και για τη κάλυψη των παρενεργειών των φαρμάκων. Η ιτρακοναζόλη είναι μία τριαζόλη με αντιμυκητιασική δράση ευρέως φάσματος, η οποία είναι πρακτικά αδιάλυτη στο νερό σε φυσιολογικές συνθήκες και περισσότερο διαλυτή σε πολύ όξινες συνθήκες, με αποτέλεσμα να παρουσιάζει πολύ μικρή βιοδιαθεσιμότητα μετά από του στόματος χορήγηση. Η ιτρακοναζόλη είναι μία ασθενής βάση με πολύ μικρή υδατοδιαλυτότητα. Η υδατοδιαλυτότητα της ιτρακοναζόλης σε ουδέτερο pH είναι ~ 1 ng/ml και σε pH 1 είναι ~ 4μg/ml. Οι κυκλοδεξτρίνες που μελετήθηκαν στη παρούσα εργασία είναι οι : υδροξυπρόπυλο-β-CD (ΗP-β-CD), μέθυλο-β-CD (Με-β-CD), υδροξυαίθυλο-β-CD (HE-β-CD) και υδροξυπρόπυλο-γ-CD (ΗP-γ-CD). Στόχοι της παρούσας εργαστηριακής άσκησης είναι:  ο προσδιορισμός της επίδρασης των παραπάνων κυκλοδεξτρινών στην διαλυτότητα της ιτρακοναζόλης σε όξινα υδατικά διαλύματα  ο χαρακτηρισμός των στερεών συμπλόκων της ιτρακοναζόλης με τις προς εξέταση κυκλοδεξτρίνες που παρασκευάσθηκαν με διαφορετικές τεχνικές. Cyclodextrins are cyclic (α-1,4)-linked oligosaccharides of α-D-glucopyranose derived from starch. They differ from one another by the number of glucopyranose units in the structure. Cyclodextrins can be classified in two groups: natural and modified. The most common natural cyclodextrins are α-cyclodextrin, β–cyclodextrin andι γ-cyclodextrin which consist of six, seven and eight glucopyranose units respectively. Also, modified cyclodextrins offer significant advantages in comparison with natural cyclodextrins. The cyclodextrin structure provides a molecule shaped like a segment of a hollow cone with an exterior hydrophilic surface and interior hydrophobic cavity. The hydrophilic surface generates good water solubility for the cyclodextrin and the hydrophobic cavity provides a favorable environment in which the entire or part of the drug molecule is fitted. Cyclodextrins can interact with appropriately sized molecules to result in the formation of inclusion compounds. Inclusion complex formation between cyclodextrins and drugs often modifies the physical and chemical properties of the guest molecules. Cyclodextrin complexation has been successfully used to improve solubility, dissolution rate, chemical stability and bioavailability of sparingly soluble or insoluble drugs. Also, cyclodextrins can be used to reduce or eliminate unpleasant smell or tastes of drugs, prevent drug-drug or drug-additive interactions and reduce or prevent drug side effects. Itraconazole is an orally broad-spectrum triazole antifungal agent, which is practically insoluble in water at physiological conditions and more soluble only in extremely acidic media, leading to poor oral biovailability. ITZ is a very poorly water soluble, weak base. The aqueous solubility is estimated at ~ 1ng/ml at neutral pH and ~ 4μg/ml at pH = 1. [2] The chemical structure of ITZ is shown in Figure 1. The cyclodextrins tested in this work were : Hydroxypropyl-β-Cyclodextrin (HP-β-CD), Methyl-β-Cyclodextrin (Me-β-CD), Hydroxyethyl-β-Cyclodextrin (He-β-CD) and Hydroxypropyl-γ-Cyclodextrin (HP-γ-CD). The aims of this work are:  to evaluate the influence of above cyclodextrins on the solubility of ITZ at acified aqueous solutions  to characterize the solid complexes formed between cyclodextrins and ITZ prepared by different techniques.