Die Permeabilität von Substanzen über Biomembranen erfolgt auf Basis ihrer Größe und Lipophilie, wird jedoch auch zu einem großen Anteil vom aktiven Transport bestimmt. Speziell im menschlichen Verdauungstrakt ist dieser Transportmechanismus neben seinen essentiellen physiologischen Aufgaben, wie den Transport von Nährstoffen, an einer Resistenz gegen exogene Stoffe und Xenobiotika beteiligt, der die Aufnahme in den Organismus über einen Rücktransport in das Darmlumen limitiert. Dabei hat die membranständige Effluxpumpe p-Glykoprotein (p-GP) als ein Baustein dieses Schutzmechanismus auch einen großen Einfluss auf die Arzneimitteltherapie. Über eine Modulierung der Pharmakokinetik von Arzneistoffen beschränkt sie die Aufnahme von Medikamenten und senkt dadurch deren Bioverfügbarkeit. Es wird auch für pflanzliche Inhaltsstoffe aus der Gruppe der Polyphenole ein möglicher Einfluss auf dieses Transportprotein diskutiert. Diese Beeinflussung kann sich entweder in einer Induktion oder einer Inhibition des Proteins äußern, was positive wie negative Effekte haben kann. Eine Hemmung des Transportproteins führt zu einer erhöhten Aufnahme einiger Arzneistoffe, die mit einer erhöhten Bioverfügbarkeit und einer potentiellen Dosissenkung einhergeht. Induziert man p-GP dagegen, so wird es beispielsweise ermöglicht, potentiell schädliche Xenobiotika noch intensiver auszuscheiden und nachteilige Plasmaspiegel zu verhindern. Im Rahmen der vorliegenden Arbeit sollte daher der Einfluss ausgewählter Polyphenole auf die Funktionalität und die Genexpression im CaCo-II-Zellkulturmodell näher untersucht, sowie vorab charakteristische Eigenschaften der pflanzlichen Inhaltsstoffe - Taxifolin, Silibinin, M1, Urolithin A, Urolithin B, Urolithin C, Isourolithin A, racemisches Hydnocarpin D, (+)-Hydnocarpin D, (-)-Hydnocarpin D - vergleichend bestimmt werden. Diese stoffspezifischen Charakteristika umfassten die Zytotoxizität, die Stabilität und die antioxidative Kapazität. Vor allem die Zytotoxizität und die Stabilität sind essentielle Parameter für aussagekräftige Resultate. Die Substanzen waren in der eingesetzten Konzentration von 50 µM mehrheitlich, mit Ausnahme des Hydnocarpins D, nicht-toxisch innerhalb der relevanten Versuchszeiträume, 4 h und 24 h, und den verwendeten Kulturmedien, DMEM-Pest und HBSS. Vor allem im Hinblick auf die Genexpressionsversuche war es die Basis für valide Ergebnisse, den Zeitraum bis 24 h als nicht-toxisch sicherstellen zu können. Hinsichtlich der Stabilität waren nur Taxifolin (27 % Restkonzentration) und der M1 (0 % Restkonzentration) nach 24 h in Zellkulturmedium kritisch. Auf Basis ihrer antioxidativen Kapazität werden pflanzlichen Inhaltsstoffen eine Reihe von gesundheitsförderlichen Merkmalen nachgesagt, weswegen dieser Aspekt für die Testsubstanzen zusätzlich vergleichend evaluiert wurde. Der Eintritt von Pathogenen kann Zusammenfassung 377 zum Beispiel durch oxidative Schädigung des Darmepithels erleichtert werden, was zusätzlich zu einem Effekt auf p-GP durch die Polyphenole unter Umständen positiv beeinflusst werden kann. Taxifolin, der M1 sowie die Urolithine A und C konnten so als antioxidativ aktive Stoffe erstmals vergleichend analysiert und die Resultate sinnvoll zu bestehenden Daten in Relation gesetzt werden. Sie konnten nach antioxidativer Potenz in der Reihenfolge Urolithin C > M1 > Taxifolin > Urolithin A geordnet werden. Zur Analyse des Einflusses der ausgewählten Polyphenole auf die Funktionalität von p-GP sollten Transportversuche über einen CaCo-II-Monolayer mit Rhodamin 123 als Markersubstanz durchgeführt werden. Diese Untersuchungen benötigen typischerweise eine vorbereitende Kulturzeit der Zellen von insgesamt drei Wochen, sodass sich eine Verkürzung dieser Zeitspanne aus Zeitersparnis- und Kostengründen positiv auf den Durchsatz der Versuche auswirken würde. In einem umfassenden Ansatz mit kombinierter Bestimmung der Qualifizierung der Zellschichten im Hinblick auf Qualität des Monolayers (TEER-Messung, Lucifer-Yellow-Transportrate, Fluoreszenzfärbung der Tight-junctions) sowie der Funktionalität und Expression von p-GP gelang der Nachweis, dass 14 Tage hinreichend und sinnvoll waren. Zentraler Bestandteil war in der vorliegenden Arbeit die Identifizierung der Effekte der Urolithine auf sowohl p-GP direkt, als auch auf die Genexpression dieses Transportproteins. Diese Polyphenole werden im menschlichen Verdauungstrakt über einen bakteriellen Metabolismus aus Ellagtanninen und Ellagsäure hergestellt und sind aufgrund ihrer vielfältigen gesundheitsförderlichen Charakteristiken in der Forschung von steigendem Interesse. Hierfür konnten nach unserem Kenntnisstand mit den gewählten Versuchsansätzen neue Erkenntnisse gewonnen werden. In den Transportversuchen mit Rhodamin 123 als Modellsubstrat von p-GP konnten die Urolithine den p-GP-vermittelten Transport positiv beeinflussen. Die Urolithine B (Papp-Ratio 1,98), C (Papp-Ratio 2,15) und das Isourolithin A (Papp-Ratio 1,63) steigerten den Rhodamintransport signifikant und lediglich für Urolithin A (Papp-Ratio 1,45) konnte keine Signifikanz belegt werden. Der Einfluss der Urolithine lag jeweils im Bereich des Modellinduktors Dexamethason. Ebenso konnte eine positive Modulierung der Genexpression nach 24 h detektiert werden. Die Hochregulierungen durch die Urolithine A (zwei- bis dreifach), B (1,4-fach) und C (1,8-fach) waren konsistent und statistisch signifikant. Urolithin A konnte hierbei als potentester Induktor charakterisiert werden, wohingegen sein Isomer Isourolithin A keinerlei signifikante Beeinflussung der Expression zeigte. In diesen Inkubationsversuchen wurde die Eigenschaft zur Erhöhung der Genexpression über den Einfluss auf den p-GP-vermittelten Rhodamintransport bestätigt. Die Urolithine A, B, C und Isourolithin A konnten nach einer Vorinkubation über 24 h und 48 h auch den Transport von Rhodamin 123 nochmals signifikanter zu den klassischen E Zusammenfassung 378 Transportversuchen ohne Vorinkubation steigern. Relevanz hierfür hatte der erste Zeitraum über 24 h, da hier ein deutlicher Anstieg der Rhodamintransportrate zu erkennen war. Nach 48 h stieg der Rhodamintransport nur noch geringfügig an oder ging sogar leicht zurück (Urolithin B). Hinsichtlich der Genexpression konnte nach 48 h nur noch Urolithin C p-GP signifikant hochregulieren, allerdings sind diese Erkenntnisse auf Basis der Zytotoxizität der Substanzen über diesen Zeitraum kritisch zu betrachten. In der Analyse des Effektes der weiteren Polyphenole auf die Genexpression von p-GP konnten für die meisten Stoffe nur zufällige Zusammenhänge hinsichtlich Hoch- und Herunterregulierung bestimmt werden. In den Transportversuchen konnte jedoch (+)-Hydnocarpin (Papp-Ratio 0,48) den Transport in gleichem Ausmaß wie der Modellinhibitor Verapamil (Papp-Ratio 0,48) hemmen. Durch Modifizierung des Versuchsmediums zur Annäherung an physiologischeren Bedingungen (Gallensäuren, pH 6) konnte für manche Substanzen ein deutlich verändertes Verhalten beobachtet werden. Die Rhodamintransportrate nahm unter Einfluss von Urolithin B, Isourolithin A und dem M1 signifikant nun ab und bei Urolithin C signifikant zu. Dies legt nahe, dass mit dem klassischen Transportversuchsmodell lediglich Tendenzen für die Substanzen bestimmt werden können. Weitere Untersuchungen näher an der Physiologie des Verdauungstraktes sind nötig, um ein genaueres Bild des Stoffeinflusses zu gewinnen. Die Frage nach zeitlichem Einsetzen beziehungsweise der Kontinuität des Effektes auf p� GP konnte mit den Urolithinen A, B und C sowie Dexamethason geklärt werden. Eine Substanzexposition von lediglich fünf Minuten war nicht ausreichend, um in den nachfolgenden zwei Stunden einen Effekt zu beobachten. Dies legt eine Reversibilität der zugrundeliegenden Mechanismen und eine notwendige dauerhafte Anwesenheit der Substanzen über die Versuchszeit nahe. Neben Rhodamin 123 wurden noch Transportversuche mit dem Fluorchinolonantibiotikum Ciprofloxacin als Modellsubstanz durchgeführt, da es aufgrund dessen Substratcharakters für p-GP von therapeutischer Relevanz sein kann, wenn das Transportverhalten durch Polyphenole beeinflusst wird. Im Gegensatz zu Rhodamin 123 wurde der Transport von Ciprofloxacin durch die vier Urolithine verringert, was für diese Metabolismusprodukte eine zusätzliche Wirkung auf weitere Transportproteine nahelegt, weil Ciprofloxacin unter anderem auch über BRCP transportiert wird. Mittels des bakteriellen Endotoxins LPS konnte eine Schädigung des CaCo-II-Monolayers erzeugt werden, welche sich über erniedrigte TEER-Werte und einen erhöhten Rhodamintransport nachweisen ließ. Eine Vorinkubation der vier Urolithine war nicht in der Lage, diese Schädigung abzumildern, jedoch nicht komplett zu verhindern. Die TEER- Zusammenfassung 379 Werte konnten zwar wieder etwas gesteigert werden, jedoch maskierte die starke Stimulation dieser Pflanzenstoffe auf p-GP und den damit verbundenen Transport von Rhodamin 123 mögliche positive Effekte auf diese oxidative Stresssituation. Zusammenfassend war es mit der vorliegenden Arbeit erstmals durch systematische vergleichende Untersuchung und Kombination von Charakterisierungsansätzen möglich, eine deutliche Beeinflussung der Genexpression und Funktionalität des p-Glykoproteins durch vor allem die Urolithine aufzuzeigen, was eine Relevanz sowohl des Mikrobioms als auch der Ernährung in der Arzneimitteltherapie nahelegt. Zudem gelang es den klassischen Transportassay durch Verkürzung um eine Woche zu verbessern. / The permeability of substances across biomembranes is dependent on their size and lipophilicity, but is also determined to a large extent by active transport. This transport mechanism is involved in resistance to exogenous substances in the human digestive tract. It limits absorption into the organism via a reverse transport into the intestinal lumen. In addition, there are some essential physiological functions, such as the transport of nutrients. In this context, the membrane-bound efflux pump P-glycoprotein (P-gp), as a part of this protective mechanism, also has a major influence on drug therapy. The uptake of drugs is limited and their bioavailability is reduced by modulating the pharmacokinetics. Herbal compounds from the class of polyphenols are discussed to potentially influence this transport. This influence can present itself either in an induction or an inhibition of the protein. Inhibition of the transport protein leads to increased uptake of some drugs, which is associated with increased bioavailability and a potential dose reduction. Inducing P-gp support, excreting potentially harmful xenobiotics and to prevent toxic plasma levels, for example. In the context of the present study, the influence of selected polyphenols on the functionality and gene expression in the CaCo-II cell culture model was to be investigated. Characteristic properties of plant derived compounds - taxifolin, silibinin, M1, urolithin A, urolithin B, urolithin C, isourolithin A, racemic hydnocarpin D, (+)-hydnocarpin D, (-)-hydnocarpin D - were be determined comparatively. The substance-specific characteristics included cytotoxicity, stability and antioxidant capacity. Cytotoxicity and stability are essential prerequisites for meaningful results. With the exception of hydnocarpine D, the compounds were mostly non-toxic within the relevant experimental periods, 4 h and 24 h, and the culture media used, DMEM-Pest and HBSS. The basis for valid results in the gene expression experiments were non-toxic effects over 24 h. In the stability experiments, only taxifolin (27 % residual concentration) and M1 (0 % residual concentration) were critically unstable after 24 h in cell culture medium. Plant constituents are discussed to have a number of health-promoting characteristics based on their antioxidant capacity. An entry of pathogens is facilitated by oxidative damage at the intestinal epithelium, which can possibly be influenced by polyphenols in addition to an effect on P-gp. Taxifolin, M1 and urolithins A and C were analysed indirect comparison for the first time. They were active as antioxidants and the results were consistent with existing data. Their antioxidant potency ranked in the order urolithin C > M1 > taxifolin > urolithin A. Transport experiments via a CaCo-II monolayer with rhodamine 123 as marker substance were to be performed to analyse the influence of the selected polyphenols on the functionality of P-gp. These investigations typically require a preparatory culture time of the cells of three weeks. A reduction of this period would have a positive effect on the throughput and costs of the experiments. In a comprehensive approach with combined determination of the qualification of the cell layers with regard to the quality of the monolayer (TEER measurement, Lucifer yellow transport rate, fluorescence staining of the tight junctions) as well as the functionality and expression of P-gp, it was possible to prove that 14 days of culture time was sufficient and reasonable. A central component of the present work was the identification of the effects of urolithins on the functionality and on the gene expression of P-gp. Urolithins are produced in the human digestive tract via a bacterial metabolism from ellagtannins and ellagic acid and are of increasing interest in research due to their diverse health-promoting characteristics. To the best of our knowledge, new insights were gained with the selected experimental approaches. In transport experiments with rhodamine 123 as a model substrate of P-gp, the urolithins enhanced the P-gp-mediated transport. Urolithins B (Papp ratio 1.98), C (Papp ratio 2.15) and isourolithin A (Papp ratio 1.63) significantly increased rhodamine transport. Only urolithin A (Papp ratio 1.45) failed to show significant effects. The influence of the urolithins was in the range of the model inducer dexamethasone. A positive modulation of gene expression after 24 h was also detected. The upregulations by urolithins A (two- to threefold), B (1.4-fold) and C (1.8-fold) were consistent and statistically significant. Urolithin A was characterised as the most potent inducer, whereas its isomer isourolithin A showed no significant effect on expression. The increase of gene expression was confirmed via the influence on P-gp-mediated rhodamine transport. After a pre-incubation period of 24 h and 48 h, the urolithins A, B, C and isourolithin A also increased the transport of rhodamine 123 even more significantly than in the classical transport experiments without pre-incubation. The first period over 24 h was relevant, as a clear increase in the rhodamine transport rate was seen. After 48 h, the rhodamine transport further increased only slightly or even slightly decreased (urolithin B). Only urolithin C significantly upregulated the gene expression of p-GP after 48 h. However, these findings must be viewed critically on the basis of the cytotoxicity of the substances over this period. In the analysis of the effect of the other polyphenols on the gene expression of P-gp, only random correlations with regard to up- and down-regulation were determined for most compounds. (+)-Hydnocarpine (Papp ratio 0.48) was able to inhibit transport to the same extent as the model inhibitor verapamil (Papp ratio 0.48).

Flavonoide sind weitverbreitete sekundäre Pflanzeninhaltsstoffe. Ihr Beitrag zur Prävention von chronischen Erkrankungen wird zu großen Teilen auf immunmodulatorische und neuroprotektive Effekte zurückgeführt. Eine Voraussetzung für die Nutzung dieser Eigenschaften der Flavonoide stellt die Erfassung cytotoxischer Effekte dar. Mit Ausnahme von Xanthohumol und Quercetin ist für alle im Rahmen der vorliegenden Arbeit untersuchten Flavonoide, Hispidulin, Baicalein, Scutellarein, Hesperetin, Chrysin, Apigenin, Naringenin, Catechin, Pelargonidinchlorid und EMD 21388, sowohl in T-Zellen (Jurkat) als auch in neuronalen (SK-N-SH)-Zellen nach 24-stündiger Inkubation eine geringgradige Cytotoxizität festzuhalten. Für Xanthohumol bzw. Quercetin wird ein halbmaximaler Verlust der Zellvitalität je nach Modell in Konzentrationen von 33-45 µM bzw. 118-208 µM erreicht. Der weiterführenden Charakterisierung (zVAD, DNA-Laddering) ist zu entnehmen, dass die zellulären Veränderungen substanzabhängig differieren und sowohl nekrotische Mechanismen (Xanthohumol) als auch apoptotische Vorgänge (Quercetin) einschließen. Eine erhöhte Lipidperoxidation im oberen Dosisbereich lässt darüber hinaus auf eine Beteiligung von oxidativem Stress an den von Xanthohumol-induzierten nekrotischen Prozessen schließen. Eine positive Einflussnahme auf die Zellvitalität durch Antioxidantien wie GSH und NAC lässt des Weiteren vermuten, dass die erfassten Flavonoid-induzierten Prozesse jeweils sensitiv zum Redoxzustand der Zelle sind. Während die Effekte von Xanthohumol auch in anderen Zellmodellen (HL-60) nachweisbar bleiben, verhält sich Quercetin nicht durchgehend vitalitätsmindernd. Unterschiede zwischen den Testsubstanzen bestehen auch hinsichtlich antioxidativer Effekte. Das Eliminieren freier Radikale zählt zu den wichtigsten Mechanismen, die bei Flavonoid-vermittelter Neuroprotektion eine Rolle spielen. Insgesamt sind alle diesbezüglich untersuchten Substanzen als starke Superoxidanionen-Radikalfänger einzustufen. Im Co-Inkubationsversuch zeigt Scutellarein den stärksten Effekt, gefolgt von Quercetin, Hispidulin und Xanthohumol. Im Prä-Inkubations-Versuchsmodell liegen in der Reihenfolge ihrer Effektstärken Xanthohumol vor Quercetin, Hispidulin und schließlich Scutellarein. Die modellabhängigen Konstanten können, unter Beteiligung einer passiven Diffusion der hydrophoben Flavonoidaglykone, auf eine substanzgebundene Membranpermeabilität zurückzuführen sein. Das antioxidative Potential der Flavonoide resultiert u.a. aus einer komplexen Einflußnahme auf die Genexpression in der Zelle. In der vorliegenden Arbeit sind anhand von cDNA-Arrays für mehrere Vertreter übereinstimmend Wechselwirkungen mit Genen der zellulären Abwehr dargestellt. Demnach führen Scutellarein, Hispidulin, Quercetin und Xanthohumol zu einer deutlich reduzierten Expressionsstärke von STK4, CHD4, ARHGDIB, IL16, ISG20, PFN1 und SOD2. Unter den Flavonoid-induzierten Veränderungen ragen die Effekte auf ADAR1 heraus, dessen Genexpression von Scutellarein bis auf ein 0,1-faches der Referenzwerte reduziert wird. Gleichsinnige Auswirkungen von Scutellarein auf die Expression von ADAR1-Protein in Western Blots unterstreichen diese Interaktion und legen nahe, dass ADAR-vermittelte enzymatische Deaminierungen durch Flavonoide moduliert werden können. Diese Beobachtung wird ergänzt durch den nachgewiesenen Effekt von Flavonoiden auf die Expression einer Reihe weiterer Gene (ADAR2, APOBEC3B, APOBEC3C, APOBEC3F und APOBEC3G), die analoge posttranskriptionale Mechanismen steuern und gleichermaßen in Immunabwehr und Neuroprotektion eingebunden sind. Zu den wichtigsten Substraten von ADAR zählen Glutamatrezeptoren. Erwartungsgemäß ist nach der Einwirkung von Scutellarein auf humane Zellen, die Glutamatrezeptoren exprimieren, ein Rückgang der Deaminierung im Bereich der Glutamatrezeptoruntereinheit GluR 2 zu verzeichnen (Q/R-Position). Dem entspricht in elektrophysiologischen Modellen eine gesteigerte Ca2+-Permeabilität der jeweiligen Ionenkanäle und eine veränderte neuronale Exzitabilität. Hieraus ergibt sich ein breites Spektrum zusätzlicher Optionen für die Induktion von gesundheitsrelevanten Flavonoidfunktionen in der Zelle. So spielt die Modulation von Deaminierungen zugleich eine entscheidende Rolle im Vermehrungszyklus viraler Erreger. Die Annahme einer möglichen antiviralen Qualität von Scutellarein wird durch ein HBV-Infektionsmodell anhand drei Parameter der Virusreplikation (Virus-DNA-Konzentration, HBs- bzw. HBe-Antigenproduktion) bestätigt. Offen bleibt auch nach ausführlicher Prüfung, ob der deutliche antivirale Effekt als das Produkt von Flavonoid-induzierten Veränderungen der Deaminierungsraten oder als Folge eines Effekts auf die virale Polymerase zu interpretieren ist. Die hier dargestellten Wirkmechanismen leisten einen Beitrag zum Verständnis der Bedeutung von Flavonoiden für neue Anwendungen in Neuroprotektion und Immunabwehr. / Flavonoids are common secondary plant metabolites that confer numerous nutritional health effects. Their role in preventing chronic diseases is attributed to immunmodulatory and neuroprotective effects among others. In order to fully exploit these properties the limitations imposed by the compounds cytotoxic profiles must be addressed. For the majority of compounds investigated, hispidulin, baicalein, scutellarein, hesperetin, chrysin, apigenin, naringenin, catechin, pelargonidinchloride and EMD 21388, the present study confirms minimal cytotoxicity in T-cells (Jurkat) and in neuronal cells (SK-N-SH). As for xanthohumol and quercetin a 50% decline in cell-vitality is observed at concentrations of 33-45 µM and 118-208 µM, respectively. Further characterization using zVAD and DNA-laddering indicate that cell-vitality may be compromised both by necrotic mechanisms (xanthohumol) and by apoptotic effects (quercetin). An increase in lipidperoxidation in the upper dose range suggests that oxidative stress may be involved in xanthohumol toxicity. As this is counteracted by antioxidants such as GSH and NAC, these flavonoids impact on cell-vitality is likely codetermined by the cells redox state. While the effects of xanthohumol extend to other cell models, quercetin toxicity in HL-60 cells is less pronounced. Test compounds are also found to differ with regard to antioxidative profiles. The elimination of free radicals is a key mechanism in flavonoid-induced neuroprotection and is shown to vary with different incubation protocols. In short incubation experiments (5 min; co-incubation) scutellarein is identified as the most powerful scavenger, followed by quercetin, hispidulin and xanthohumol. In prolonged incubations (24 hrs; prä-incubation) xanthohumol and quercetin are followed by hispidulin and scutellarein. Model-specific constants suggest that passive diffusion of the hydrophobic flavonoid-aglyca may occur across cell membranes, alongside with other modes of permeation. Flavonoids antioxidative potential is mediated by complex effects on gene expression. The present work uses data from cDNA-arrays to highlight interactions with genes involved in cellular defense. Specifically, scutellarein, hispidulin, quercetin and xanthohumol downregulate expression for STK4, CHD4, ARHGDIB, IL16, ISG20, PFN1 and SOD2. In addition, flavonoids consistently downregulate ADAR1-expression, which drops to 0,1-fold of reference values and is paralleled by scutellarein-effects on ADAR1-protein-expression. Together, these findings indicate, that ADAR-mediated enzymatic deamination may be modulated by flavonoids. Similar effects are noted on related genes (ADAR2, APOBEC3B, APOBEC3C, APOBEC3F and APOBEC3G), relevant to posttranscriptional processing underlying immune defense and neuroprotection. Glutamate receptors count among the most important neuronal substrates of ADAR. Following exposure to scutellarein a decrease in deamination rates is confirmed with respect to the glutamate receptor subunit GluR 2 (Q/R-site). As a result, an enhanced Ca2+-permeability of the respective ion channels is anticipated, and modified neuronal excitability. Overall, the regulation of enzymatic deamination by flavonoids offers opportunities for multilevel balancing of cell homeostasis. Thus deaminations may interfere with the replication cycle of viral pathogens. Using an ex-vivo HBV-infection model and three parameters of viral replication (viral load, HBs and HBe indices), antiviral properties of scutellarein are illustrated. Despite extensive investigation, it remains to be seen whether these effects can be ascribed to deaminations of viral DNA or to an interaction with other substrates, e.g. the viral polymerase. In summary, the present observations serve to foster our understanding of flavonoids roles in neuroprotection and immune defense.

Aufgrund ihrer gut dokumentierten, umfangreichen gesundheitsfördernden biologischen Aktivitäten wird den Flavonoiden eine große Bedeutung zugeordnet. Die Ergebnisse von in vitro- und Tierstudien deuten zudem darauf hin, dass diese Verbindungen bei der Prävention und Therapie von Erkrankungen wie Krebs oder Alzheimer Krankheit (AD) positive Effekte zeigen. Zur besseren Charakterisierung der Interaktion von Flavonoiden mit Krebszellen wurden von uns die Cytotoxizität verschiedener Flavonoide auf T-Lymphoblastomzellen untersucht und Strukturelemente identifiziert, welche für einen Flavonoid-induzierten Zelltod relevant sind. Weitere Studien waren der potentiell neuroprotektiven Wirkung von Flavonoiden gewidmet. Die sowohl in neuronalen Zellkulturen als auch in transgenen Alzheimer-Mäusen (TgAPPsw) festgestellte Erhöhung der sAPPα Produktion und Reduktion von Aβ-Bildung wurden mit Aktivitäts- und Expressionssteigerung der α-Sekretase ADAM-10 assoziiert. Um herauszufinden, ob Flavonoide eine neuroprotektive Wirkung zeigen, wurden erste Vorbereitungen für ein Flavonoid-Screening mit einer sowohl hAPP alsauch ADAM-10 stabil transfizierten HT1080 Zellen getroffen. Dies beinhaltete die Suche nach einer potenten siRNA/shRNA und einem effektiven Flavonoid. Im zweiten Teil der Arbeit wurden Experimente durchgeführt, um die Rolle von Heparansulfat (HS) bei der foamyviralen Anbindung an die Wirtszelle zu untersuchen. Foamyviren (FV) sind Spumaviren und gehören zur Familie der Retroviren. Bei unseren Studien wurde die Bindung von FV an Heparin, die Korrelation der Suszeptibilität verschiedener Zellen mit zellulärem HS und die Reduktion der Infektion durch lösliches Heparin sowie durch enzymatischen HS-Abbau festgestellt. / Flavonoids have well-documented, beneficial biological effects. Furthermore different in vitro- and animal-experiments indicate that these compounds demonstrate positive effects in prevention and therapy of diseases, like cancer or neurodegenerative diseases. In order to characterize the interactionsbetween flavonoids and cancer cells, we examined the cytotoxicity of different flavonoids on a human leukemia cell line and identified structure elements that could be associated to flavonoid-induced cell death. Our further studies were dedicated to the potentially neuroprotective activity of flavonoids. It has been described that in neuronal cells as well as in transgenic AD mice EGCG increases levels of sAPPα- and reduces Aβ-production. This influence of EGCG was associated with enhanced activity and expression of the α-secretase, ADAM-10. To find out, if flavonoids showed neuroprotective activity, preliminary work for a later flavonoid screening on hAPP and ADAM-10 wit stably-transfected HT1080 cells was done. This includes the determination of flavonoid candidates and research of effective siRNAs towards ADAM-10. Furtherrmore we investigated the role of heparansulfat (HS) in attachment of FV to host cell. Foamyviruses (FV) are retroviruses and belong to the subfamily of spumaretroviruses. In our studies we discovered the binding of FV on heparin, the correlation between susceptibility of different cells and cellular HS and the reduction of infectivity through soluble heparin and through enzymatic HS-degradation.

Diversas espécies de Passifloras já foram estudadas visando as atividades ansiolítica, anti-inflamatória e cicatrizante com ênfase para P. alata Curtis e P. edulis Sims. Entretanto, não se encontraram estudos farmacológicos envolvendo P. setacea D.C e P. tenuifila Killip; os estudos referentes a estas duas espécies restringem-se ao campo agronômico e fitoquímico. No presente trabalho foram estudadas a eficácia e segurança destas duas espécies de Passiflora, provenientes do Cerrado. Avaliou-se a atividade antiúlcera aguda, utilizando o modelo de indução por etanol acidificado. P. setacea apresentou áreas relativas de lesão de 11,64%, 2,39%**, 0,43%**, respectivamente, para as doses de 100 mg/kg, 200 mg/kg, 400 mg/kg e P. tenuifila, de 16,44%, 12,42%, 10,84 , para as doses de 100 mg/kg, 200 mg/kg, 400 mg/kg (** p≤0,01 em relação ao controle negativo, ANOVA seguida de Tukey). Como P. setacea foi a espécie que apresentou melhores resultados no ensaio antiúlcera agudo, ela foi eleita para dar continuidade aos demais ensaios. O teor de flavonoides presentes no extrato bruto de P. setacea foi de 3,10 mg por g de extrato, através da metodologia do AlCl3. A quantificação de compostos polifenólicos totais presentes no extrato de P. setacea foi realizada utilizando o método do reagente de Folin-Dennis e obteve-se 27,05 mg de compostos polifenólicos por grama de extrato. Avaliou-se a atividade antioxidante do extrato bruto de P.setacea pelo método do DPPH radical, utilizando Trolox® como substância de referência, obteve-se 11,65 µmol de Trolox® por grama de extrato. Determinou-se o perfil cromatográfico do extrato de P. setacea, através de cromatografia líquida de alta eficiência. O cromatograma apontou a presença de compostos flavonoídicos. Avaliou-se potencial mutagênico do extrato bruto de P. setacea segundo o teste de Ames, e não se observaram-se sinais de mutagenicidade. A atividade anti Helicobacter pylori foi avaliada em modelo in vitro e obteve-se concentração inibitória mínima de 250 µg/mL. A toxicidade do extrato de P. setacea também foi avaliada, através de ensaios agudo e sub-agudo. No ensaio agudo utilizou-se o modelo de administração de dose única de 2000mg/kg por animal e observação por 14 dias de sinais e sintomas que indicassem uma possível intoxicação, durante e ao final do experimento. Ao se necropsiar os animais, não se notou nenhum sinal que apontasse para toxicidade nos animais. No ensaio de toxicidade subaguda, utilizou-se o modelo de administração reiterada do extrato de P. setacea aos animais por 28 dias em três doses (1200, 800 e 400mg/kg) e um grupo controle (água). Durante o experimento não se observou nenhum indício de intoxicação nos animais. Ao final do experimento houve coleta de sangue e posterior necropsia para coleta de órgãos. Nas análises hematológicas e bioquímicas, não se observou diferença significativa entre os grupos que receberam extrato e o grupo controle. Em relação aos órgãos coletados, macroscopicamente não se observou diferença entre os grupos tratados e controle. Conclui-se que P. setacea apresenta atividade antiúlcera bastante promissora em ratos, atividade antioxidante, que pode ser devida à presença de flavonoides e compostos polifenólicos. Apresenta atividade anti H. pylori in vitro e não apresenta toxicidade, nem potencial de mutagenicidade in vitro. / Several species of Passiflora have been tested to anxiolytic activity, anti-inflammatory and healing, mainly with emphasis on P. alata Curtis and P. edulis Sims. However, there are pharmacological studies involving P. setacea D.C and P. tenuifila Killip. Studies based on these two species are restricted to agronomic and phytochemical field. In the present work, we evaluate the efficacy and safety of these two species of Passiflora, that are native from Cerrado. We evaluated the acute anti-ulcer activity, using acidified ethanol induction model. P. setacea presented relative lesion areas of injury 11.64%, 2.39% ** and ** 0.43%, respectively, at doses of 100 mg/kg, 200 mg/kg, 400 mg/kg and for P .tenuifila, 16.44%, 12.42%, 10.84%, respectively, at doses of 100mg / kg, 200mg / kg, 400mg / kg (** p ≤0,01 compared to negative control, ANOVA followed by Tukey). As P. setacea was the species that showed better results in acute anti-ulcer test, it was elected to be used on the continue to the other tests. The flavonoid content in the crude extract was 3,10 mg per g of P. setacea extract using AlCl3 method. Total polyphenolic content was determined by Folin Dennis method yelding 27.05 mg of phenolics per g of dried extract. Antioxidant activity of the crude extract was evaluated by DPPH radical method, using Trolox as reference, each g of crude extract equivalated of 11.65 µg of Trolox. P. setacea chromatographic profile was determined using High Performance Liquid Chromatography. The chromatogram showed the presence of flavonoid compounds. Mutagenic potential of P. setacea crude extract was evaluated using Ames test. No signs of mutagenicity were observed. Anti Helicobacter pylori activity was evaluated in vitro model and yelding Minimum Inhibitory Concentration of 250 µg / ml. Toxicity of P. setacea extract was also evaluated through acute and sub-acute tests. Acute test was performed using a single dose of 2000 mg/kg orally given to each animal. Toxicity signs and symptoms were recorded for 14 days, during and after the experiment. After necropsy, no toxicity signs were shown. Subacute test was performed using the repeated doses model. P. setacea extract was orally given to animals for 28 days in three doses, control received water. No toxicity signs were shown during the experiment. At the endpoint, blood was collected and necropsy was performed to collect the organs. No significant difference was observed between test and control regarding hematological and biochemical analysis In conclusion, P. setacea shows promising antiulcer activity in rats. Also, its antioxidant activity may be related to the presence of flavonoids and phenolics. Additionally, it presented anti H. pylori activity in vitro and did not show toxicity, neither mutagenic potential in vitro.

München, Techn. Univ., Diss., 2002.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-09-26T18:39:07Z No. of bitstreams: 1 texto completo.pdf: 802816 bytes, checksum: 10866709d3f58803d87ed3c58d0fd097 (MD5) / Made available in DSpace on 2016-09-26T18:39:07Z (GMT). No. of bitstreams: 1 texto completo.pdf: 802816 bytes, checksum: 10866709d3f58803d87ed3c58d0fd097 (MD5) Previous issue date: 2001-02-20 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Foi avaliado em coelhos e ratos diabéticos, durante 28 dias, o efeito do flavonóide naringenina no peso e constituintes sangüíneos. Foi ainda avaliado o efeito da farinha da fruta- de-lobo (Solanum lycocarpum A.F.C.P. Saint-Hilaire) em coelhos. Diabetes foi induzida através de administração intravenosa de aloxano. A determinação do peso, consumo alimentar e da concentração plasmática de glicose, colesterol, triacilgliceróis, creatinina e albumina foi realizada semanalmente. Nos coelhos, as amostras de sangue foram retiradas do plexo capilar ocular e nos ratos, da extremidade final da cauda. Visando determinar a dose ideal de aloxano para indução de diabetes em coelhos adultos, machos, da raça Albino Nova Zelândia, realizou-se um experimento, onde os -1 tratamentos constituíram-se de 5 doses de aloxano (90, 105, 120, 135 e 150 mg.kg de peso vivo) e a testemunha (normal), com 8 repetições. Em todos os coelhos, injetou-se 10 mL de glicose, 4, 8 e 12 horas após a administração do aloxano, para evitar hipoglicemia. Aos 4 dias -1 após a aplicação do aloxano, observou-se que as doses de 90 e 105 mg.kg de aloxano causaram menor número de mortes nos animais tratados e propiciaram maior número de sobreviventes diabéticos, sendo então recomendadas para a indução de diabetes em coelhos. Coelhos foram considerados diabéticos quando o nível de glicose sangüínea foi igual ou -1 superior a 180 mg.dL . Não houve relação linear entre as doses de aloxano e os níveis sangüíneos de glicose, triacilgliceróis e colesterol, devido a grande variabilidade da resposta dos coelhos a cada dose de aloxano. Coelhos diabéticos receberam diariamente uma cápsula de 20 mg de naringenina ou de 40 mg de farinha de fruta-de-lobo. Não foi observada diferença significativa, entre os tratamentos aplicados aos coelhos diabéticos. Em ratos, o diabetes foi induzido com 60 mg.kg -1 de aloxano. Utilizaram-se ratos albinos, machos da raça Wistar, pesando entre 200 e 300 gramas. Estudou-se o efeito de dietas e de flavonóides no peso e constituintes sangüíneos. O delineamento experimental foi o inteiramente casualisado. Foram conduzidos 3 experimentos, nos quais ratos diabéticos receberam dieta básica (AIN-93G) ou modificada contendo 47,95% de sacarose, com ou sem 20 mg de naringenina diariamente. Não houve variação no peso, glicemia e nem em outros constituintes sangüíneos avaliados, nos ratos diabéticos, recebendo dieta básica ou modificada, acrescida ou não de naringenina. Os ratos normais que receberam apenas dieta modificada apresentaram maiores ganhos de peso e da glicemia em relação aos que receberam apenas dieta básica, evidenciado apenas em um experimento devido à alta variabilidade das características avaliadas. Cerca de quarenta dias após o início da administração das dietas, 12 ratos diabéticos que haviam recebido dieta básica sem naringenina, e outros 12 que receberam dieta rica em sacarose foram submetidos à inversão de dietas por 14 dias. Os ratos que passaram a receber dieta básica apresentaram redução do nível de triacilglicerol. Já os ratos que passaram a receber dieta rica em sacarose apresentaram aumento do peso final e do consumo alimentar. Concluindo, embora as diferenças não tenham sido estatisticamente significativas, nos ratos diabéticos recebendo dieta básica acrescida de naringenina, houve uma tendência a reduzir os níveis de glicose sangüínea e a aumentar o peso comparado a ratos diabéticos sem naringenina. / It was evaluated the effect of the flavonoid naringenin in body weight and blood constituents in diabetic rabbits and rats, for 28 days. In rabbits it was also evaluated the effect of the wolf-fruit (Solanum lycocarpum A.F.C.P. Saint-Hilaire). Diabetes were induced by intravenous administration of alloxan. The measurements of the body weight, food consumption and the concentration of plasma glucose, cholesterol, triacylglycerol, creatinin and albumin were measured at weekly intervals. In rabbits, blood was collected from the ocular capillary plexus and in rats, from the end of the tail. In order to determine the ideal dose of alloxan for diabetes induction in adult male -1 rabbits, Albino New Zealand, 6 alloxan doses of alloxan (0, 90, 105, 120, 135 and 150 mg.kg of live weight, with 8 animals per group) were administred. In all rabbits, 10 mL of glucose was injected, 4, 8 and 12 hours after the administration of the alloxan, to avoid hypoglycemia. Four -1 days later, it was observed that the doses of 90 and 105 mg. kg caused smaller number of deaths in the treated animals and resulted in larger number of diabetic survivors. Therefore, these doses were recommended for induction of diabetes in rabbits, in future experiments. Rabbits were considered diabetics when the levels of blood glucose were equal or greater than -1 180 mg.dL . There was no linear relationship among the alloxan doses and the blood levels of glucose, triacylglycerol and cholesterol, due to great variability of the response of the rabbits to each alloxan dose. Diabetic rabbits received a daily dose of 20 mg of naringenin or 40 mg of fruit-of-wolf flour. No significant difference was observed among the treatments applied to the diabetic rabbits. -1 Diabetes was induced using 60 mg.kg alloxan in albino Wistar males rats weighing between 200 and 300 g. It was studied the effect of diets and of flavonoids in body weight and blood constituents. The experimental design was entirely randomized. Three experiments were carried out, in which, diabetic rats, received basal (AIN-93G) or modified diet with 47.95% of sucrose, with or without 20 mg of naringenin daily. No difference was observed in body weight, glycemia and other blood constituents in diabetic rats, receiving basal or modified diet, with or without added naringenin. The normal rats that received modified diet showed body higher weight gain and glycemia in relation to those that received basal diet, however, it was evidenced in only one experiment due to the high variability of the characteristics evaluated. After 40 days in their diets, 12 diabetic rats that received basal diet without naringenin and 12 that received diet rich in sucrose were crossed over and maintained in their diets for another 14 days period. The rats that changed to basal diet presented reduction on triacylglycerol levels. The rats that changed to rich in sucrose diet presented an increase on final body weight and food consumption. In conclusion, althougt no significant difference has been observed among treatments, diabetic rats receiving basal diet with added naringenin showed a trend lowering blood levels of glucose and incresed body weight compared to diabetic rats without naringenin. / Não foi localizado o cpf do autor.

Submitted by LUIS FERNANDO MERCES CHAVES SILVA null (luisfernando.mchaves@gmail.com) on 2016-06-02T00:52:25Z No. of bitstreams: 1 TESE MESTRADO VERSÃO FINAL (LUIS FERNANDO M. C. SILVA).pdf: 2997186 bytes, checksum: 711036d97c3d4d1e8cce675ab6ad1477 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-06-06T14:47:13Z (GMT) No. of bitstreams: 1 silva_lfmc_me_bot.pdf: 2997186 bytes, checksum: 711036d97c3d4d1e8cce675ab6ad1477 (MD5) / Made available in DSpace on 2016-06-06T14:47:13Z (GMT). No. of bitstreams: 1 silva_lfmc_me_bot.pdf: 2997186 bytes, checksum: 711036d97c3d4d1e8cce675ab6ad1477 (MD5) Previous issue date: 2016-04-27 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O surgimento das biotecnologias aplicadas ao sêmen equino, como a refrigeração e congelação, representaram um grande avanço para equinocultura, principalmente por permitir um melhor aproveitamento do ejaculado, otimizando a utilização de animais considerados geneticamente superiores. Entretanto a aplicação dessas técnicas imprime estresse térmico às células espermáticas, resultando em injúrias físicas e químicas, dentre elas uma elevação significativa na produção de espécies reativas do oxigênio (EROs). Quando em excesso, estes agentes instáveis são capazes de reagir e promover lesão às enzimas, lipídeos de membrana e ácidos nucleicos, reduzindo a viabilidade dos espermatozoides criopreservados. Desta maneira surge a necessidade de se introduzir elementos que desempenhem função antioxidante nos meios de refrigeração e congelação. A quercertina, um polifenol do grupo dos flavonoides, é considerada um poderoso antioxidante por reduzir o surgimento das principais EROs, quelar íons metálicos formadores de radicais livres e estabilizar a peroxidação lipídica. Apesar disto, a eficiência da aplicação deste antioxidante sobre o ejaculado equino durante o processo de criopreservação ainda é controversa e pouco referenciada, sendo necessário mais estudos para que se determine a influência de sua utilização no ejaculado de garanhões durante a refrigeração e congelação. / The emergence of biotechnologies applied to the equine semen as cooling and freezing process represented a major breakthrough for Equine, especially for allowing a better use of the ejaculate, optimizing the use of genetically superior animals. However the application of these techniques induces thermal stress on sperm cells, resulting in physical and chemical injuries, among them a significant rise in the production of reactive oxygen species (ROS). These unstable agents when in excess, are able to react and promote damage to enzymes, lipid membrane and nucleic acids, thereby reducing the viability of cryopreserved sperm. Thus is required to introduce elements that perform antioxidant function in cooling and freezing extenders. The quercertina, a polyphenol of the flavonoids group, is considered a potent antioxidant by reducing the emergence of the main ROS, chelate metal ions producers of free radicals and stabilize the lipid peroxidation. Despite this, the efficiency of application of this antioxidant on the equine ejaculate during the process of cryopreservation is still controversial and poorly referenced, requiring further studies in order to determine if there is influence of their use in the ejaculate of stallions during cooling and freezing. / FAPESP: 2014/01681-4

Orientador: Frederico Ozanam Papa / Resumo: O surgimento das biotecnologias aplicadas ao sêmen equino, como a refrigeração e congelação, representaram um grande avanço para equinocultura, principalmente por permitir um melhor aproveitamento do ejaculado, otimizando a utilização de animais considerados geneticamente superiores. Entretanto a aplicação dessas técnicas imprime estresse térmico às células espermáticas, resultando em injúrias físicas e químicas, dentre elas uma elevação significativa na produção de espécies reativas do oxigênio (EROs). Quando em excesso, estes agentes instáveis são capazes de reagir e promover lesão às enzimas, lipídeos de membrana e ácidos nucleicos, reduzindo a viabilidade dos espermatozoides criopreservados. Desta maneira surge a necessidade de se introduzir elementos que desempenhem função antioxidante nos meios de refrigeração e congelação. A quercertina, um polifenol do grupo dos flavonoides, é considerada um poderoso antioxidante por reduzir o surgimento das principais EROs, quelar íons metálicos formadores de radicais livres e estabilizar a peroxidação lipídica. Apesar disto, a eficiência da aplicação deste antioxidante sobre o ejaculado equino durante o processo de criopreservação ainda é controversa e pouco referenciada, sendo necessário mais estudos para que se determine a influência de sua utilização no ejaculado de garanhões durante a refrigeração e congelação. / Abstract: The emergence of biotechnologies applied to the equine semen as cooling and freezing process represented a major breakthrough for Equine, especially for allowing a better use of the ejaculate, optimizing the use of genetically superior animals. However the application of these techniques induces thermal stress on sperm cells, resulting in physical and chemical injuries, among them a significant rise in the production of reactive oxygen species (ROS). These unstable agents when in excess, are able to react and promote damage to enzymes, lipid membrane and nucleic acids, thereby reducing the viability of cryopreserved sperm. Thus is required to introduce elements that perform antioxidant function in cooling and freezing extenders. The quercertina, a polyphenol of the flavonoids group, is considered a potent antioxidant by reducing the emergence of the main ROS, chelate metal ions producers of free radicals and stabilize the lipid peroxidation. Despite this, the efficiency of application of this antioxidant on the equine ejaculate during the process of cryopreservation is still controversial and poorly referenced, requiring further studies in order to determine if there is influence of their use in the ejaculate of stallions during cooling and freezing. / Mestre

Diversas espécies de Passifloras já foram estudadas visando as atividades ansiolítica, anti-inflamatória e cicatrizante com ênfase para P. alata Curtis e P. edulis Sims. Entretanto, não se encontraram estudos farmacológicos envolvendo P. setacea D.C e P. tenuifila Killip; os estudos referentes a estas duas espécies restringem-se ao campo agronômico e fitoquímico. No presente trabalho foram estudadas a eficácia e segurança destas duas espécies de Passiflora, provenientes do Cerrado. Avaliou-se a atividade antiúlcera aguda, utilizando o modelo de indução por etanol acidificado. P. setacea apresentou áreas relativas de lesão de 11,64%, 2,39%**, 0,43%**, respectivamente, para as doses de 100 mg/kg, 200 mg/kg, 400 mg/kg e P. tenuifila, de 16,44%, 12,42%, 10,84 , para as doses de 100 mg/kg, 200 mg/kg, 400 mg/kg (** p≤0,01 em relação ao controle negativo, ANOVA seguida de Tukey). Como P. setacea foi a espécie que apresentou melhores resultados no ensaio antiúlcera agudo, ela foi eleita para dar continuidade aos demais ensaios. O teor de flavonoides presentes no extrato bruto de P. setacea foi de 3,10 mg por g de extrato, através da metodologia do AlCl3. A quantificação de compostos polifenólicos totais presentes no extrato de P. setacea foi realizada utilizando o método do reagente de Folin-Dennis e obteve-se 27,05 mg de compostos polifenólicos por grama de extrato. Avaliou-se a atividade antioxidante do extrato bruto de P.setacea pelo método do DPPH radical, utilizando Trolox® como substância de referência, obteve-se 11,65 µmol de Trolox® por grama de extrato. Determinou-se o perfil cromatográfico do extrato de P. setacea, através de cromatografia líquida de alta eficiência. O cromatograma apontou a presença de compostos flavonoídicos. Avaliou-se potencial mutagênico do extrato bruto de P. setacea segundo o teste de Ames, e não se observaram-se sinais de mutagenicidade. A atividade anti Helicobacter pylori foi avaliada em modelo in vitro e obteve-se concentração inibitória mínima de 250 µg/mL. A toxicidade do extrato de P. setacea também foi avaliada, através de ensaios agudo e sub-agudo. No ensaio agudo utilizou-se o modelo de administração de dose única de 2000mg/kg por animal e observação por 14 dias de sinais e sintomas que indicassem uma possível intoxicação, durante e ao final do experimento. Ao se necropsiar os animais, não se notou nenhum sinal que apontasse para toxicidade nos animais. No ensaio de toxicidade subaguda, utilizou-se o modelo de administração reiterada do extrato de P. setacea aos animais por 28 dias em três doses (1200, 800 e 400mg/kg) e um grupo controle (água). Durante o experimento não se observou nenhum indício de intoxicação nos animais. Ao final do experimento houve coleta de sangue e posterior necropsia para coleta de órgãos. Nas análises hematológicas e bioquímicas, não se observou diferença significativa entre os grupos que receberam extrato e o grupo controle. Em relação aos órgãos coletados, macroscopicamente não se observou diferença entre os grupos tratados e controle. Conclui-se que P. setacea apresenta atividade antiúlcera bastante promissora em ratos, atividade antioxidante, que pode ser devida à presença de flavonoides e compostos polifenólicos. Apresenta atividade anti H. pylori in vitro e não apresenta toxicidade, nem potencial de mutagenicidade in vitro. / Several species of Passiflora have been tested to anxiolytic activity, anti-inflammatory and healing, mainly with emphasis on P. alata Curtis and P. edulis Sims. However, there are pharmacological studies involving P. setacea D.C and P. tenuifila Killip. Studies based on these two species are restricted to agronomic and phytochemical field. In the present work, we evaluate the efficacy and safety of these two species of Passiflora, that are native from Cerrado. We evaluated the acute anti-ulcer activity, using acidified ethanol induction model. P. setacea presented relative lesion areas of injury 11.64%, 2.39% ** and ** 0.43%, respectively, at doses of 100 mg/kg, 200 mg/kg, 400 mg/kg and for P .tenuifila, 16.44%, 12.42%, 10.84%, respectively, at doses of 100mg / kg, 200mg / kg, 400mg / kg (** p ≤0,01 compared to negative control, ANOVA followed by Tukey). As P. setacea was the species that showed better results in acute anti-ulcer test, it was elected to be used on the continue to the other tests. The flavonoid content in the crude extract was 3,10 mg per g of P. setacea extract using AlCl3 method. Total polyphenolic content was determined by Folin Dennis method yelding 27.05 mg of phenolics per g of dried extract. Antioxidant activity of the crude extract was evaluated by DPPH radical method, using Trolox as reference, each g of crude extract equivalated of 11.65 µg of Trolox. P. setacea chromatographic profile was determined using High Performance Liquid Chromatography. The chromatogram showed the presence of flavonoid compounds. Mutagenic potential of P. setacea crude extract was evaluated using Ames test. No signs of mutagenicity were observed. Anti Helicobacter pylori activity was evaluated in vitro model and yelding Minimum Inhibitory Concentration of 250 µg / ml. Toxicity of P. setacea extract was also evaluated through acute and sub-acute tests. Acute test was performed using a single dose of 2000 mg/kg orally given to each animal. Toxicity signs and symptoms were recorded for 14 days, during and after the experiment. After necropsy, no toxicity signs were shown. Subacute test was performed using the repeated doses model. P. setacea extract was orally given to animals for 28 days in three doses, control received water. No toxicity signs were shown during the experiment. At the endpoint, blood was collected and necropsy was performed to collect the organs. No significant difference was observed between test and control regarding hematological and biochemical analysis In conclusion, P. setacea shows promising antiulcer activity in rats. Also, its antioxidant activity may be related to the presence of flavonoids and phenolics. Additionally, it presented anti H. pylori activity in vitro and did not show toxicity, neither mutagenic potential in vitro.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A presente dissertação apresenta o estudo fitoquímico e avaliação biológica do extrato etanólico das folhas de Aegiphila integrifolia (Jacq.) pertencente à família Lamiaceae. O estudo fitoquímico realizado com eluato clorofómico do extrato etanólico das folhas levou ao isolamento e identificação de uma mistura de ácidos graxos na forma de seus ésteres metílicos, uma mistura de, β-sitosterol, estigmasterol e lupeol glicosilados, um flavonoide conhecido como Pectolinarigenina. As misturas de ésteres metílicos foram analisadas e determinadas por Cromatografia Gasosa. As determinações estruturais foram realizadas por meio de métodos espectrométricos mono e bidimensionais como IV, RMN de 1H (500 MHz), RMN de 13C (125 MHz), HSQC, HMBC, ESI-EM e comparação com dados da literatura. O estudo biológico do extrato etanólico apresentou um bom resultado quanto à atividade antioxidante pelo método DPPH comparado ao padrão quercetina. A investigação da toxicidade, de acordo com o teste realizado frente à Artemia salina indicou a baixa toxidade (DL 50% superior a 500 μg mL-1) e quando comparado com valores encontrados na Literatura confirma a baixa toxicidade do gênero Aegiphila frente à Artemia salina. Os ensaios da MIC para os fungos filamentosos, bactérias Gram-Positivas e Gram-Negativas e levedura, apresentaram baixa inibição comparadas com os antibióticos Ampicilina, exceto a levedura Cândida albicans. Esta apresentou inibição satisfatória, 96% aproximadamente para a amostra RR05 (do flavonoide) e 92% para amostra R06 (extrato etanólico) comparando aos antibióticos Nistatina e Miconazol (91%). / This dissertation presents the phytochemical study and biological evaluation of the ethanol extract of Aegiphila integrifolia (Jacq.) belonging to the family Lamiaceae. The phytochemical study of clorofómico eluate of ethanol leaf extract led to the isolation and identification of a mixture of fatty acids as their methyl esters, a mixture of, β - sitosterol, stigmasterol and lupeol glycosylated a flavonoid known as Pectolinarigenin. Mixtures of methyl esters were analyzed and determined by gas chromatography. Structural determinations were made by means of spectroscopic methods such as mono-and two-dimensional IR, 1H NMR (500 MHz), 13C NMR (125 MHz), HSQC, HMBC, ESI-MS and comparison with literature data. The biological study of the ethanol extract showed a good result for antioxidant activity by DPPH method compared to standard quercetin. The investigation of the toxicity of the test conducted according front Artemia salina indicated low toxicity (LD 50 % up to 500 mg mL-1) and compared to values found in literature confirming the low toxicity of the genus Artemia salina front Aegiphila. The MIC tests for filamentous fungi, Gram - Positive and Gram-Negative Bacteria and yeast showed low inhibition compared with Ampicillin antibiotics except the yeast Candida albicans. This inhibition was satisfactory, approximately 96% to RR05 sample (the flavonoid) and 92% for sample R06 (ethanol extract) comparing antibiotic Nystatin and Miconazole (91%).