Made available in DSpace on 2014-06-11T19:26:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-24Bitstream added on 2014-06-13T19:54:31Z : No. of bitstreams: 1 souza_co_me_botfm.pdf: 403996 bytes, checksum: 1bf83541f86766588143610433c5d720 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Silibinina é um flavonóide quimicamente definido e o principal componente ativo da silimarina, um complexo polifenólico obtido de frutos e sementes de Silybum marianum, que possui propriedades anti-inflamatória, hepatoprotetora e anti-carcinogênica. Objetivo: O objetivo deste trabalho foi avaliar os efeitos da silibinina sobre a performance reprodutiva de ratas prenhes Wistar e as anomalias e/ou malformações de seus fetos. Métodos: Foram utilizadas ratas Wistar prenhes estratificadas em quatro grupos experimentais: controle (n = 6), ratas tratadas com 50mg/kg/dia de silibinina (Grupo I, n = 6), ratas tratadas com 100mg/kg/dia de silibinina (Grupo II, n = 6) e ratas tratadas com 200mg/kg/dia de silibinina (Grupo III, n = 6). Os animais receberam tratamento por via oral (gavage). Durante toda a prenhez, o consumo de ração e a ingestão de água, bem como o ganho de peso corporal foram avaliados. No dia 21 de prenhez as ratas foram anestesiadas A cesárea foi realizada no 21º dia, quando os recém-nascidos foram mortos e processados para análise das variações e/ou malformações. Resultados: os grupos controle e tratados não apresentaram diferenças significativas em relação aos parâmetros fetais, não se observando sinais de toxicidade materna com as diferentes concentrações de silibinina empregadas. Conclusão: O tratamento com silibinina não determinou efeitos deletérios na performance reprodutiva das ratas, mesmo quando utilizada em altas doses (200 mg/Kg/dia). / Silibinin is a chemically defined flavonoid and the main active component of silymarin, a polyphenolic complex from fruits and seeds of Silybum marianum, which has anti-inflammatory, hepatoprotective and anticarcinogenic properties. Objective: The aim of the present study was to evaluate the dose-dependent effect of silibinin treatment on maternal reproductive performance and on abnormalities and/or fetal malformations. Methods: Pregnant Wistar rats were distributed in four experimental groups: control, not treated with silibinin (n=6), rats treated with silibinin 50mg/kg/day (Group I, n = 6), rats treated with silibinin 100mg/kg/day (Group II, n = 6) and rats treated with silibinin 200mg/kg/day (Group III, n = 6). Silibinin was administrated by gavage from day 0 to 20 of pregnancy. During the period of pregnancy food and water intake, as well as weight gain were evaluated. On day 21 of pregnancy, the rats were anesthetized and submitted to cesarean section to analyse maternal reproductive performance and fetal malformations. Results: The control and silibinin-treated groups did not show significant differences in relation to the fetal parameters evaluated and no maternal toxicity effects of silibinin were detected in the concentrations employed. Conclusion: Silibinin treatment did not determined deleterious effect on the maternal reproductive performance and fetal outcome even when employed in high dose (200 mg/Kg).

Alzheimer’s disease is a complex network of several pathological hallmarks. These characteristics always occur concomitantly and cannot be taken as distinct features of the disease. While there are hypotheses trying to explain the origin and progression of the illness, none of them is able to pinpoint a definitive cause. This fact challenges researchers not to focus on one individual hallmark but, bearing in mind the big picture, target two or more indications at once. This work, therefore, addresses two of the major characteristics of AD: the cholinergic hypothesis and neurotoxic oxidative stress. The former was achieved by targeting the postsynaptic muscarinic M1 acetylcholine receptor to further investigate its pharmacology, and the latter with the synthesis of neuroprotective natural antioxidant hybrids. The first aim was the design and synthesis of dualsteric agonists of the muscarinic M1 acetylcholine receptor. Activation of this receptor was previously shown to improve AD pathologies like the formation of Aβ and NFTs and protect against oxidative stress and caspase activation. Selectively targeting the M1 receptor is difficult as subtypes M1 – M5 of the muscarinic AChRs largely share the same orthosteric binding pocket. Orthosteric ligands are thus unsuitable for selective activation of one specific subtype. Secondary, allosteric binding sites are more diverse between subtypes. Allosteric ligands are, however, in most cases dependent on an orthosteric ligand to cause downstream signals. Dualsteric ligands thus utilize the characteristics of both orthosteric and allosteric ligands in form of a message-address concept. Bridged by an alkylene-linker, the allosteric part ensures selectivity, whereas the orthosteric moiety initiates receptor activation. Two sets of compounds were synthesised in this sense. In both cases, the orthosteric ligand carbachol is connected to an allosteric ligand via linkers of different chain length. The first set utilizes the selective allosteric M1 agonist TBPB, the second set employs the selective M1 positive allosteric modulator BQCA. Six compounds were obtained in twelve-step syntheses each. For each one, a reference compound lacking the carbachol moiety was synthesised. The dualsteric ligands 1a-c and 2a c were tested in the IP1 assay. The assay revealed that the TBPB-dualsterics 1 are not able to activate the receptor, whereas the respective TBPB-alkyl reference compounds 27 gave signals depending on the length of the alkylene-linker, suggesting allosteric partial agonism of alkyl compounds 27 and no dualsteric binding of the putatively dualsteric compounds 1. The dualsteric BQCA molecules 2, however, activated the receptor as expected. Efficacy of the C5 linked compound 2b was the highest, yet C3 and C8 compounds (2a and 2c) also showed partial agonism. In this case, the reference compounds 31 showed no receptor activation, implying the intended dualsteric binding mode of the BQCA-carbachol compounds 2. Further investigations will be conducted by the working group of Dr. Christian Tränkle at the Department of Pharmacology at the University of Bonn to confirm binding modes and determine affinities as well as selectivity of the synthesised dualsteric compounds. The second project dealt with the design, synthesis and biological evaluation of neuroprotective esters of the flavonolignan silibinin. While silibinin is already a potent antioxidant, it has been observed that the 7-OH group has a pro-oxidative character, making this position attractive for functionalisation. In order to obtain more potent antioxidants, the pro-oxidative position was esterified with other antioxidant moieties like ferulic acid 35 and derivatives thereof. Seventeen esters of silibinin 32, including pure diastereomers of 7 O feruloylsilibinin (43a and 43b) and a cinnamic acid ester of 2,3-dehydrosilibinin 46, were synthesised by regioselective esterification using acyl chlorides under basic conditions. The physicochemical antioxidant properties were assessed in the FRAP assay. This assay revealed no improvement of the antioxidant properties except for 7-O-dihydrosinapinoylsilibinin 39b. These results, however, do not correlate with the neuroprotective properties determined in the HT-22 hippocampal neuronal cell model. The assay showed overadditive neuroprotective effects of the esters exceeding those of its components and equimolar mixtures with the most potent compounds being 7-O-cinnamoylsilibinin 37a, 7-O-feruloylsilibinin 38a and the acetonide-protected caffeic acid ester 40a. These potent Michael system bearing compounds may be considered as “PAINS”, but the assays used to assess antioxidant and neuroprotective activities were carefully chosen to avoid false positive readouts. The most potent compounds 37a and 38a, as well as the diastereomers 43a and 43b, were further studied in assays related to AD. In vitro ischemia, inhibition of microglial activation, PC12 cell differentiation and inhibition of Aβ42 and τ protein aggregation assays showed similar results in terms of overadditive effects of the synthesised esters. Moreover, the diastereomers 43a and 43b showed differences in their activities against oxytosis (glutamate-induced apoptosis), inhibition of Aβ42 and τ protein aggregation, and PC12 cell differentiation. The stereospecific effect or mode of action against Aβ42 and τ protein aggregation is more pronounced than that of silybin A (32a) and silybin B (32b) reported in literature and needs to be elucidated in future work. Stability measurements in cell culture medium revealed that the esters do not only get hydrolysed but are partially oxidised to their respective 2,3-dehydrosilibinin esters. Because dehydrosilibinin 45 itself is described as a more potent antioxidant than silibinin 32, 7 O cinnamoyl-2,3-dehydrosilibinin 46 was expected to be even more potent than its un-oxidised counterpart 37a in terms of neuroprotection. The oxytosis assay, however, showed that the neurotoxicity of 46 is much more pronounced, especially at higher concentrations, reducing its neuroprotective potential. Dehydrosilibinin esters are therefore inferior to the silibinin esters for application as neuroprotectants, because of the difficulty of their synthesis and their increased neurotoxicity. A synergistic effect of both species (silibinin and the oxidised form) might, however, be possible or even necessary for the pronounced neuroprotective effects of silibinin esters. As the dehydro-species show distinct neuroprotective properties at low concentrations, their continuous formation over time might make an essential contribution to the overall neuroprotection of the synthesised esters. Due to solubility issues for some of the ester compounds, 7-O-cinnamoylsilibinin 37a was converted into a highly soluble hemisuccinate. The vastly improved solubility of 7 O cinnamoyl-23-O-succinylsilibinin 48 was confirmed in shake-flask experiments. Contrary to expectation, stability examinations showed that the succinyl compound 48 is not cleaved to form 7-O-cinnamoylsilibinin 37a. Neuroprotection assays confirmed that 48 is not a prodrug of the corresponding ester. It was determined that the main site of hydrolysis is the 7-position, cleaving 37 to silibinin 32 and cinnamic acid thus reducing the compound’s neuroprotective effects. Nevertheless, the compound still showed neuroprotection at a concentration of 25 µM. The improved solubility might be more beneficial than the higher neuroprotection of the poorly soluble parent compound 37a in vivo. 7 O Cinnamoylsilibinin 37a was further investigated to reduce Aβ25 35 induced learning impairment in mice. While tendencies of improved short-term and long-term memory in the animals were observed, the effects are not yet statistically significant in both Y-maze and passive avoidance tests. A greater number of test subjects is necessary to ensure correctness of the preliminary results presented in this work. However, an effect of ester 37a is observable in vivo, showing blood-brain barrier penetration. The esters synthesised are a novel approach for the treatment of AD as they show strong neuroprotective effects and their hydrolysis products or metabolites are only non-toxic natural products. / Mehrere Hypothesen versuchen die Ursprünge und den Fortschritt der Alzheimer’schen Krankheit zu erklären. Eine definitive Ursache konnte allerdings bisher nicht festgestellt werden, da die Merkmale der Krankheit grundsätzlich nebeneinander auftreten. Wissenschaftler müssen also auf der Suche nach Therapien die verschiedenen pathologischen Vorgänge gleichzeitig behandeln. In dieser Arbeit wurden daher zwei der prominenteren Anzeichen der Alzheimer’schen Krankheit bearbeitet. Zum einen wurde die cholinerge Hypothese, mit Adressierung des postsynaptischen muskarinischen M1 Acetylcholinrezeptors, thematisiert. Und zum anderen wurde neurotoxischer oxidativer Stress, mit der Entwicklung von neuroprotektiven natürlichen Antioxidantien, behandelt. Das erste Projekt beschäftigte sich mit dem Design und der Synthese von dualsterischen Agonisten des muskarinischen M1 Acetylcholinrezeptors. In der Literatur wurde gezeigt, dass die Aktivierung dieses Rezeptors eine Verbesserung des Krankheitsverlaufs nach sich ziehen kann. Es wurde unter anderem demonstriert, dass Verminderungen der Aggregation von Aβ und τ Proteinen, wie auch von oxidativem Stress, eintreten. Die selektive Aktivierung des M1 Rezeptors gestaltet sich jedoch als sehr schwierig, da sich die fünf Subtypen M1 – M5 der muskarinischen Acetylcholinrezeptoren nur geringfügig in ihrer Substratbindestelle unterscheiden. Die Anwendung von rein orthosterischen Liganden ist daher ungenügend. Sekundäre bzw. allosterische Bindestellen, unterscheiden sich zwischen den Subtypen stärker. Allosterische Liganden sind allerdings oft von der Anwesenheit eines orthosterischen Agonisten abhängig, um den Rezeptor zu aktivieren. Die Einbeziehung beider Bindestellen gleichzeitig erlaubt jedoch die Anwendung eines Signal-Adresse-Konzepts, bei welchem ein orthosterischer Ligand (Signal) mit einem allosterischen Liganden (Adresse) über einen Linker verknüpft wird. Auf diese Weise wurden zwei Sätze dualsterischer Verbindungen hergestellt. Der orthosterische Ligand Carbachol wurde jeweils über unterschiedlich lange Alkyllinker mit einem allosterischen Liganden verbunden. Als allosterische Liganden, selektiv für den M1-Rezeptor, wurden zum einen TBPB, ein allosterischer Agonist, und zum anderen BQCA, ein positiver allosterischer Modulator, gewählt. Sechs Verbindungen wurden in Synthesen mit jeweils zwölf Schritten hergestellt. Außerdem wurden für jede der dualsterischen Verbindungen Referenzsubstanzen, ohne die Carbachol-Einheit, synthetisiert. Die dualsterischen Liganden 1a-c und 2a-c wurden im IP1 Assay getestet und es zeigte sich, dass die TBPB-Verbindungen 1 nicht in der Lage waren den Rezeptor zu aktivieren. Im Gegensatz dazu, konnten die entsprechenden Referenzverbindungen 27, abhängig von der Kettenlänge, Rezeptorantworten auslösen. Dieser Befund zeigt allosteren Agonismus der Referenzen 27 und lässt Grund zur Annahme, dass die Verbindungen 1 keinen dualsterischen Bindemodus eingehen. Die BQCA-Substanzen 2, auf der anderen Seite, aktivierten den Rezeptor wie erwartet. Die Verbindung mit dem C5-Linker 2b löste das stärkste Signal aus, aber auch die C3- und C8-Verbindungen (2a und 2c) zeigten Partialagonismus. In diesem Fall aktivierten die Referenzsubstanzen 31 den Rezeptor nicht, was auf einen dualsterischen Bindemodus der Zielstrukturen 2 schließen lässt. Weitere Untersuchungen dieser Verbindungen werden in der Arbeitsgruppe von Dr. Christian Tränkle am Institut für Pharmakologie und Toxikologie der Universität Bonn durchgeführt und sollen näheren Aufschluss über Bindemodi, Affinitäten und Selektivität geben. Das zweite Projekt beschäftigte sich mit dem Design, der Synthese und biologischen Evaluierung von neuroprotektiven Estern des Flavonolignans Silibinin 32. Obwohl Silibinin 32 selbst ein starkes Antioxidans ist, wurde festgestellt, dass seine 7-OH Gruppe pro-oxidativen Charakter besitzt. Diese Position ist somit ein attraktives Ziel für Funktionalisierungen zur Verstärkung der antioxidativen Eigenschaften. Um Substanzen mit verbesserten antioxidativen Effekten herzustellen, wurde diese Gruppe mit anderen Antioxidantien, wie etwa Ferulasäure 35 und strukturell ähnlichen Derivaten, verestert. Dabei konnten siebzehn Ester, inklusive den reinen Diastereomeren von 7-O-Feruloylsilibinin (43a und 43b) und einem Zimtsäureester von 2,3-Dehydrosilibinin 46, durch regioselektive Veresterung mit den entsprechenden Säurechloriden im basischen Milieu hergestellt werden. Die physikochemischen antioxidativen Eigenschaften wurden mit Hilfe des FRAP-Assays bestimmt. Hier trat im Allgemeinen keine Verbesserung der Eigenschaften auf. Nur 7 O Dihydrosinapinoylsilibinin 39b zeigte gesteigertes antioxidatives Verhalten. Diese Ergebnisse stimmten jedoch nicht mit den neuroprotektiven Eigenschaften, die in einem Zellmodel mit HT-22 hippocampalen neuronalen Zellen (Oxytose Assay) getestet wurden, überein. Dieser Assay konnte beweisen, dass die dargestellten Ester ihre einzelnen Komponenten, und eine Mischung dieser, hinsichtlich ihrer Eigenschaften übertreffen und überadditive neuroprotekive Effekte besitzen. Die wirksamsten Verbindungen waren 7 O Cinnamoylsilibinin 37a, 7-O-Feruloylsilibinin 38a und der Acetonid-geschützte Kaffesäureester 40a. Diese Substanzen besitzen alle ein Michael-System und könnten deshalb als sogenannte „PAINS“ angesehen werden. Allerdings wurden die verwendeten Assays sorgfältig ausgewählt, um falsch-positive Ergebnisse zu vermeiden. Die wirksamsten Substanzen, inklusive der beiden Diastereomeren 43a und 43b, wurden in weiteren, für die Alzheimer’sche Krankheit relevanten, Experimenten untersucht. Ein in vitro Ischämie Modell, die Inhibition von Mikroglia Aktivierung, die Aktivierung von PC12 Zell-Differenzierung und die Inhibition von Aβ und τ Protein Aggregation bestätigen die überadditiven Effekte der Ester. Die einzelnen Diastereomere 43a und 43b zeigten unterschiedlich stark ausgeprägte Effekte gegenüber Oxytose, Inhibition von Aβ und τ Protein Aggregation und PC12 Zell-Differenzierung. Dieser stereospezifische Effekt oder Wirkmechanismus war auffallender als die in der Literatur beschriebenen Effekte der Diastereomere Silybin A (32a) und Silybin B (32b) und muss in Zukunft näher untersucht werden. Stabilitätsuntersuchungen in Zellkulturmedium zeigten, dass die Ester nicht einfach nur hydrolysiert, sondern teilweise zu den entsprechenden Dehydrosilibininestern oxidiert werden. Da Dehydrosilibinin 45 selbst als stärkeres Antioxidans als Silibinin 32 beschrieben wird, wurde erwartet, dass der Zimtsäure-Dehydrosilibininester 46 höhere Wirksamkeit als der Silibininester 37a zeigt. Der Neuroprotektions-Assay bewies jedoch, dass 46 ausgeprägtere Neurotoxizität besitzt und seine potentielle Neuroprotektion dadurch verschlechtert wird. Dehydrosilibininester sind den Silibininestern daher bei dieser Anwendung unterlegen, auch weil ihre Synthese deutlich aufwendiger ist. Ein synergistischer Effekt beider Spezies (Silibinin und Dehydrosilibinin) könnte allerdings möglich und vielleicht sogar nötig sein. Die kontinuierliche Generierung der Dehydrosilibininester mit der Zeit könnte ein wichtiger Faktor für die neuroprotektiven Eigenschaften der Silibininester sein, da die neuroprotektiven Effekte der Dehydro-silibininester bei niedrigen Konzentrationen sehr ausgeprägt sind. Aufgrund der problematischen Löslichkeit der hergestellten Substanzen, wurde die Löslichkeit der Ester verbessert, indem 7 O-Cinnamoylsilibinin 37a, als Modell, mit gut löslichen Hemisuccinaten verbunden wurde. Die stark verbesserte Löslichkeit von 7 O Cinnamoyl-23-O-succinylsilibinin 48 wurde in shake-flask Experimenten bewiesen. Stabilitätsuntersuchungen und der Neuroprotektions-Assay zeigten allerdings, dass die Succinylverbindung 48 hauptsächlich an Position 7, und damit in Zimtsäure und 23 O Succinylsilibinin 50, gespalten wurde. Hemisuccinat 48 zeigte Neuroprotektion nur bei einer Konzentration von 25 µM und ist somit kein Pro-Drug von 7 O Cinnamoylsilibinin 37a. Die erhöhte Löslichkeit könnte jedoch in vivo nutzbringender sein als die gesteigerte Neuroprotektion des schlechter löslichen Derivats 37a. 7-O-Cinnamoylsilibinin 37a wurde außerdem darauf getestet Aβ25 35 induzierte Gedächtnisbeeinträchtigungen in Mäusen zu verbessern. Obwohl Tendenzen zu verbessertem Kurzzeit- und Langzeitgedächtnis erkenntlich wurden, konnten noch keine statistisch signifikanten Verbesserungen sowohl im Y-maze Test als auch im passive avoidance Test gezeigt werden. Mehr Testsubjekte sind nötig, um die Ergebnisse zu überprüfen. Dennoch sind Effekte in vivo zu beobachten, die Blut-Hirn-Schranken Penetration zeigen. Die dargestellten Ester sind somit ein neuer Ansatz für die Behandlung der Alzheimer’schen Krankheit, da sie ausgeprägte neuroprotektive Effekte zeigen und ihre Spaltprodukte und Metaboliten ausschließlich nicht-toxische Naturstoffe sind.

Orientador: Maria Terezinha Serrão Peraçoli / Banca: José Carlos Peraçoli / Banca: Joélcio Francisco Abbade / Banca: Nilton Hideto Takiuti / Resumo: Silibinina é um flavonóide quimicamente definido e o principal componente ativo da silimarina, um complexo polifenólico obtido de frutos e sementes de Silybum marianum, que possui propriedades anti-inflamatória, hepatoprotetora e anti-carcinogênica. Objetivo: O objetivo deste trabalho foi avaliar os efeitos da silibinina sobre a performance reprodutiva de ratas prenhes Wistar e as anomalias e/ou malformações de seus fetos. Métodos: Foram utilizadas ratas Wistar prenhes estratificadas em quatro grupos experimentais: controle (n = 6), ratas tratadas com 50mg/kg/dia de silibinina (Grupo I, n = 6), ratas tratadas com 100mg/kg/dia de silibinina (Grupo II, n = 6) e ratas tratadas com 200mg/kg/dia de silibinina (Grupo III, n = 6). Os animais receberam tratamento por via oral (gavage). Durante toda a prenhez, o consumo de ração e a ingestão de água, bem como o ganho de peso corporal foram avaliados. No dia 21 de prenhez as ratas foram anestesiadas A cesárea foi realizada no 21º dia, quando os recém-nascidos foram mortos e processados para análise das variações e/ou malformações. Resultados: os grupos controle e tratados não apresentaram diferenças significativas em relação aos parâmetros fetais, não se observando sinais de toxicidade materna com as diferentes concentrações de silibinina empregadas. Conclusão: O tratamento com silibinina não determinou efeitos deletérios na performance reprodutiva das ratas, mesmo quando utilizada em altas doses (200 mg/Kg/dia). / Abstract: Silibinin is a chemically defined flavonoid and the main active component of silymarin, a polyphenolic complex from fruits and seeds of Silybum marianum, which has anti-inflammatory, hepatoprotective and anticarcinogenic properties. Objective: The aim of the present study was to evaluate the dose-dependent effect of silibinin treatment on maternal reproductive performance and on abnormalities and/or fetal malformations. Methods: Pregnant Wistar rats were distributed in four experimental groups: control, not treated with silibinin (n=6), rats treated with silibinin 50mg/kg/day (Group I, n = 6), rats treated with silibinin 100mg/kg/day (Group II, n = 6) and rats treated with silibinin 200mg/kg/day (Group III, n = 6). Silibinin was administrated by gavage from day 0 to 20 of pregnancy. During the period of pregnancy food and water intake, as well as weight gain were evaluated. On day 21 of pregnancy, the rats were anesthetized and submitted to cesarean section to analyse maternal reproductive performance and fetal malformations. Results: The control and silibinin-treated groups did not show significant differences in relation to the fetal parameters evaluated and no maternal toxicity effects of silibinin were detected in the concentrations employed. Conclusion: Silibinin treatment did not determined deleterious effect on the maternal reproductive performance and fetal outcome even when employed in high dose (200 mg/Kg). / Mestre

A fenilcetonúria (PKU) é uma doença metabólica causada pela deficiência da enzima fenilalanina hidroxilase, levando ao acúmulo de fenilalanina. As principais características clínicas dos pacientes com PKU não tratados são o comprometimento neuropsicológico e o retardo no desenvolvimento. O estresse oxidativo tem sido detectado em muitos erros inatos do metabolismo, incluindo PKU. A silibinina é um flavonoide proveniente da planta cardo de leite (Silybum marianum) que apresenta propriedades antioxidantes e que, após administração, é amplamente distribuída pelos tecidos. Neste trabalho, nós investigamos os efeitos da silibinina in vivo e in vitro contra o estresse oxidativo causado por elevados níveis de fenilalanina. Ratos machos e fêmeas, com 12 dias de vida no início dos experimentos, receberam injeções subcutâneas de α- metilfenilalanina e fenilalanina para realizar o modelo agudo de hiperfenilalaninemia, e o tratamento com silibinina consistiu em injeções intraperitoniais da substância na dose de 20 mg/kg. Os animais foram mortos no 14° dia de vida. Para realizar os experimentos in vitro, homogeneizados de córtex cerebral de ratos de 14 dias de vida foram incubados com fenilalanina e silibinina. In vitro e in vivo, a silibinina foi capaz de prevenir a inibição provocada pela fenilalanina nas atividades das enzimas catalase, glutationa peroxidase e glicose-6-fosfato desidrogenase. Não foram verificadas diferenças entre os grupos nas atividades da superóxido dismutase e da glutationa redutase. Além disso, a silibinina preveniu as alterações provocadas pela fenilalanina no conteúdo de carbonilas proteicas, nas substâncias reativas ao ácido tiobarbitúrico e na produção de espécies reativas. A silibinina preveniu o dano oxidativo induzido pela fenilalanina e pode ser uma potencial terapia complementar para o tratamento da PKU. / Phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of phenylalanine hydroxylase, leading to accumulation of phenylalanine. The main clinical features of non-treated PKU patients are neuropsychological impairment and developmental retardation. Oxidative stress has been related to many inborn errors of metabolism including PKU. Silibinin is a flavonoid derived from the herb milk thistle (Silybum marianum) which presents antioxidant properties and is widely distributed into tissues after administration. In this study, we investigated the in vivo and in vitro effects of silibinin against oxidative stress caused by high levels of phenylalanine. Male and female rats, 12 days old at the beginning of experiments, received subcutaneous injections of α- methylphenylalanine and phenylalanine to produce hyperphenylalaninemia, and intraperitoneal injections of 20 mg/kg silibinin. The animals were killed on the 14th day of life. To perform in vitro experiments, cerebral cortex homogenates of 14 days old rats were incubated with phenylalanine and silibinin. In vivo and in vitro, silibinin was able to prevent the inhibition provoked by phenylalanine on the activities of catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase. No differences were found among the groups in the activities of superoxide dismutase and glutathione reductase. Moreover, silibinin prevented the alterations provoked by phenylalanine on protein carbonyl content, thiobarbituric acid-reactive substances and production of reactive species. Silibinin prevented oxidative damage induced by phenylalanine and may be a potential adjunctive therapy to PKU treatment.

A fenilcetonúria (PKU) é uma doença metabólica causada pela deficiência da enzima fenilalanina hidroxilase, levando ao acúmulo de fenilalanina. As principais características clínicas dos pacientes com PKU não tratados são o comprometimento neuropsicológico e o retardo no desenvolvimento. O estresse oxidativo tem sido detectado em muitos erros inatos do metabolismo, incluindo PKU. A silibinina é um flavonoide proveniente da planta cardo de leite (Silybum marianum) que apresenta propriedades antioxidantes e que, após administração, é amplamente distribuída pelos tecidos. Neste trabalho, nós investigamos os efeitos da silibinina in vivo e in vitro contra o estresse oxidativo causado por elevados níveis de fenilalanina. Ratos machos e fêmeas, com 12 dias de vida no início dos experimentos, receberam injeções subcutâneas de α- metilfenilalanina e fenilalanina para realizar o modelo agudo de hiperfenilalaninemia, e o tratamento com silibinina consistiu em injeções intraperitoniais da substância na dose de 20 mg/kg. Os animais foram mortos no 14° dia de vida. Para realizar os experimentos in vitro, homogeneizados de córtex cerebral de ratos de 14 dias de vida foram incubados com fenilalanina e silibinina. In vitro e in vivo, a silibinina foi capaz de prevenir a inibição provocada pela fenilalanina nas atividades das enzimas catalase, glutationa peroxidase e glicose-6-fosfato desidrogenase. Não foram verificadas diferenças entre os grupos nas atividades da superóxido dismutase e da glutationa redutase. Além disso, a silibinina preveniu as alterações provocadas pela fenilalanina no conteúdo de carbonilas proteicas, nas substâncias reativas ao ácido tiobarbitúrico e na produção de espécies reativas. A silibinina preveniu o dano oxidativo induzido pela fenilalanina e pode ser uma potencial terapia complementar para o tratamento da PKU. / Phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of phenylalanine hydroxylase, leading to accumulation of phenylalanine. The main clinical features of non-treated PKU patients are neuropsychological impairment and developmental retardation. Oxidative stress has been related to many inborn errors of metabolism including PKU. Silibinin is a flavonoid derived from the herb milk thistle (Silybum marianum) which presents antioxidant properties and is widely distributed into tissues after administration. In this study, we investigated the in vivo and in vitro effects of silibinin against oxidative stress caused by high levels of phenylalanine. Male and female rats, 12 days old at the beginning of experiments, received subcutaneous injections of α- methylphenylalanine and phenylalanine to produce hyperphenylalaninemia, and intraperitoneal injections of 20 mg/kg silibinin. The animals were killed on the 14th day of life. To perform in vitro experiments, cerebral cortex homogenates of 14 days old rats were incubated with phenylalanine and silibinin. In vivo and in vitro, silibinin was able to prevent the inhibition provoked by phenylalanine on the activities of catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase. No differences were found among the groups in the activities of superoxide dismutase and glutathione reductase. Moreover, silibinin prevented the alterations provoked by phenylalanine on protein carbonyl content, thiobarbituric acid-reactive substances and production of reactive species. Silibinin prevented oxidative damage induced by phenylalanine and may be a potential adjunctive therapy to PKU treatment.

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No. of bitstreams: 2 Tese - Flavio Fernandes Veloso Borges - 2015.pdf: 2110689 bytes, checksum: 595ebd21ecf4f13568b0e3179e801f99 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Silymarin (SM) is a standardized extract from the seeds and leaves of milk thistle Silybum marianum (L.) Gaertn. It is composed mainly of flavonolignans, with silibinin (SB) being its principal active constituent. Known mainly as antioxidant and hepatoprotector, SM and SB were found to be clinically effective in the treatment of a variety of liver disorders, including acute and chronic viral hepatitis, toxin and drug-induced hepatitis and cirrhosis. Due to the wide biological activities presented by SM and SB, the present study aimed to evaluate their antimutagenic activities using the Ames mutagenicity test in Salmonella typhimurium, their antigenotoxic activities using the mouse bone marrow micronucleous test and the alkaline comet assay, and to assess their effect on the gene expression pattern of some genes associated with the process of carcinogenesis and chemoprevention. To assess antimutagenicity, bacterial suspensions of Salmonella typhimurium (TA98 and TA100 strains) were treated with different concentrations of SM or SB simultaneously with the appropriate positive controls for each strain. To assess antigenotoxicity, Swiss mice were orally treated with different concentrations of SM or SB simultaneously with a single intraperitoneal dose of mitomycin C (MMC) for the micronucleus test, and human blood lymphocytes were cotreated with SM or SB and methyl methanesulfonate (MMS) for the alcaline comet assay. To investigate the role of SM and SB in modulating gene expression, we conducted microarray analysis. The results showed that SM was not significantly effective in reducing the number of frameshift mutations in strain TA98, while SB demonstrated significant protection at higher doses (p < 0.05). Regarding strain TA 100, SM and SB significantly decreased mutagenicity (point mutations) (p < 0.05). The results of the antigenotoxic evaluation demonstrated that SM and SB significantly reduced the frequency of micronucleated polychromatic erythrocytes (MNPCE) (p < 0.05). The results also indicated that SM and SB significantly attenuated MMC induced cytotoxicity (p < 0.05). In the comet assay, SM and SB significantly reduced the genotoxicity of MMS (p < 0.05), with a stronger antigenotoxic activity exerted by the extract complex (SM) than the one exerted by the isolated main active constituent (SB). The expression array analysis of five genes related to DNA damage, carcinogenesis and/or chemoprevention mechanisms demonstrated an up-regulation of PTEN and BCL2, down-regulation of BAX and ABL1 and no significant change in ETV6 expression levels.In conclusion, our results demonstrated that both SM and SB presented antimutagenic and antigenotoxic actions, as well as modulated the expression levels of genes analysed under the experimental conditions of this study. / A silimarina (SM) é um extrato padronizado obtido a partir das sementes e folhas de Silybum marianum (L.) Gaertn. SM é composta principalmente de flavonóides, sendo a silibinina (SB) seu principal componente ativo. Conhecidas principalmente como antioxidantes e hepatoprotetoras, SM e SB foram consideradas clinicamente eficazes no tratamento de uma variedade de doenças do fígado, incluindo hepatites virais agudas e crônicas, hepatites induzidas por toxinas e/ou drogas e cirrose. Assim, devido à ampla gama de atividades biológicas apresentadas pela SM e SB, o presente estudo teve como objetivo avaliar suas atividades antimutagênicas utilizando o teste de Ames em Salmonella typhimurium, suas atividades antigenotóxicas pelo teste do micronúcleo em medula óssea de camundongos e pelo teste do cometa em linfócitos humanos e avaliar seus efeitos nos perfis de expressão gênica de alguns genes associados ao processo de carcinogênese e quimioprevenção. Para a avaliação da antimutagenicidade, suspensões bacterianas de Salmonella typhimurium (cepas TA98 e TA100) foram co-tratadas com diferentes concentrações de SM ou SB e os controles positivos adequados para cada cepa. Para a avaliação de antigenotoxidade, camundongos Swiss foram tratados oralmente com diferentes concentrações de SM ou SB concomitantemente a uma única dose intraperitoneal de mitomicina C (MMC) para o teste do micronúcleo, e linfócitos humanos foram tratados simultaneamente com SM ou SB e metil-metanossulfonato (MMS) para o ensaio do Cometa. Os resultados mostraram que a SM não foi significativamente efetiva em reduzir o número de mutações com deslocamento de quadro de leitura na cepa TA 98, enquanto que a SB apresentou uma proteção significativa nas doses maiores (p < 0.05). Em relação à cepa TA100, SM e SB reduziram significativamente a mutagenicidade (mudanças de pares de bases) (p < 0.05). Na avaliação de antigenotoxidade, SM e SB reduziram significativamente a frequência de eritrócitos policromáticos micronucleados (EPCMN) (p<0,05). Os resultados também mostraram que a citotoxicidade causada pela MMC foi significativamente atenuada pela SM e SB (p<0,05). No ensaio do cometa, SM e SB reduziram significativamente a genotoxicidade provocada pelo MMS (p<0.05), com uma atividade antigenotóxica maior exercida pelo extrato complexo (SM) do que pelo principal componente ativo isolado (SB). A análise dos níveis de expressão de cinco genes relacionados ao dano no DNA, mecanismos de carcinogênese e/ou quimioprevenção demonstrou um aumento na expressão de PTEN e BCL2, diminuição na expressão de BAX e ABL1 e ausência de mudança significativa nos níveis de expressão do ETV6. Com base nesses resultados, conclui-se que a SM e a SB apresentaram ações antimutagênicas e antigenotóxicas, e também modularam os níveis de expressão dos genes analisados sob as condições experimentais deste estudo.

A fenilcetonúria (PKU) é uma doença metabólica causada pela deficiência da enzima fenilalanina hidroxilase, levando ao acúmulo de fenilalanina. As principais características clínicas dos pacientes com PKU não tratados são o comprometimento neuropsicológico e o retardo no desenvolvimento. O estresse oxidativo tem sido detectado em muitos erros inatos do metabolismo, incluindo PKU. A silibinina é um flavonoide proveniente da planta cardo de leite (Silybum marianum) que apresenta propriedades antioxidantes e que, após administração, é amplamente distribuída pelos tecidos. Neste trabalho, nós investigamos os efeitos da silibinina in vivo e in vitro contra o estresse oxidativo causado por elevados níveis de fenilalanina. Ratos machos e fêmeas, com 12 dias de vida no início dos experimentos, receberam injeções subcutâneas de α- metilfenilalanina e fenilalanina para realizar o modelo agudo de hiperfenilalaninemia, e o tratamento com silibinina consistiu em injeções intraperitoniais da substância na dose de 20 mg/kg. Os animais foram mortos no 14° dia de vida. Para realizar os experimentos in vitro, homogeneizados de córtex cerebral de ratos de 14 dias de vida foram incubados com fenilalanina e silibinina. In vitro e in vivo, a silibinina foi capaz de prevenir a inibição provocada pela fenilalanina nas atividades das enzimas catalase, glutationa peroxidase e glicose-6-fosfato desidrogenase. Não foram verificadas diferenças entre os grupos nas atividades da superóxido dismutase e da glutationa redutase. Além disso, a silibinina preveniu as alterações provocadas pela fenilalanina no conteúdo de carbonilas proteicas, nas substâncias reativas ao ácido tiobarbitúrico e na produção de espécies reativas. A silibinina preveniu o dano oxidativo induzido pela fenilalanina e pode ser uma potencial terapia complementar para o tratamento da PKU. / Phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of phenylalanine hydroxylase, leading to accumulation of phenylalanine. The main clinical features of non-treated PKU patients are neuropsychological impairment and developmental retardation. Oxidative stress has been related to many inborn errors of metabolism including PKU. Silibinin is a flavonoid derived from the herb milk thistle (Silybum marianum) which presents antioxidant properties and is widely distributed into tissues after administration. In this study, we investigated the in vivo and in vitro effects of silibinin against oxidative stress caused by high levels of phenylalanine. Male and female rats, 12 days old at the beginning of experiments, received subcutaneous injections of α- methylphenylalanine and phenylalanine to produce hyperphenylalaninemia, and intraperitoneal injections of 20 mg/kg silibinin. The animals were killed on the 14th day of life. To perform in vitro experiments, cerebral cortex homogenates of 14 days old rats were incubated with phenylalanine and silibinin. In vivo and in vitro, silibinin was able to prevent the inhibition provoked by phenylalanine on the activities of catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase. No differences were found among the groups in the activities of superoxide dismutase and glutathione reductase. Moreover, silibinin prevented the alterations provoked by phenylalanine on protein carbonyl content, thiobarbituric acid-reactive substances and production of reactive species. Silibinin prevented oxidative damage induced by phenylalanine and may be a potential adjunctive therapy to PKU treatment.

Le cancer colorectal est la deuxième cause de mortalité due au cancer en Europe et aux États-Unis. Etant donné l’efficacité limitée et la toxicité élevée des agents de chimiothérapie, de nouvelles approches sont nécessaires. Le flavanolignane silybine représente le principal constituant actif du chardon-marie (Silybum marianum). Les mécanismes moléculaires des propriétés anticancéreuses de la silybine ont été étudiés dans un modèle cellulaire de progression du cancer colorectal humain : les cellules SW480 issues d’un adénocarcinome, et leurs dérivées métastatiques les cellules SW620. Les effets chimiopréventifs de la silybine ont été étudiés dans un modèle de cancérogenèse colique induite par l’azoxyméthane chez le rat. La silybine induit une mort apoptotique avec activation de la caspase-3 dans les deux lignées. L’expression des récepteurs de mort TRAIL est augmentée, et la caspase-8 activée. Le potentiel mitochondrial est perturbé provoquant une libération du cytochrome c et une activation de la caspase-9. En plus de l’activation des voies apoptotiques extrinsèque et intrinsèque la silybine induit une réponse autophagique. La combinaison de la silybine et de TRAIL, un agent anti-cancéreux prometteur, provoque une mort cellulaire synergique dans les deux lignées. Un effet synergique est aussi observé avec la combinaison de la silybine et des inhibiteurs des histones déacétylases (HDAC) : TSA et SAHA. Dans le modèle chez le rat, la silybine réduit de 50% le nombre des lésions prénéoplasiques. En conclusion, la silybine est un agent naturel intéressant pour la prévention du cancer colorectal et dans le cadre d’une combinaison avec TRAIL/des inhibiteurs d’HDACs. / Colorectal cancer (CRC) is the second most common cause for cancer-related deaths in Europe and in the USA. Because of the limited efficacy and considerable toxicity of chemotherapeutic agents, new approaches are needed. The hepatoprotective flavonolignan silibinin is the major biologically active compound of the milk thistle (Silybum marianum).The molecular mechanisms of the anticancer properties of silibinin in CRC were studied in an in vitro model of cancer progression consisting of the adenocarcinoma cell line SW480 and its derived metastatic cell line SW620. Its chemopreventive effects were assessed in an in vivo model of azoxymethane-induced colon carcinogenesis in the rat. Silibinin induced apoptotic cell death with activation of caspase-3 in both cell lines. The expression of death receptors was upregulated, and caspase-8 was activated. The potential of the mitochondrial membrane was perturbed permitting the release of cytochrome c and the activation of caspase-9. Besides the activation of the extrinsic and the intrinsic apoptotic pathway, silibinin induced an autophagic response. Combination of silibinin and TRAIL, a promising anticancer agent selectively inducing apoptosis in cancer cells, induced synergistic cell death in both cell lines. Synergy in cell death induction was also observed by the combination of silibinin and the histone deacetylase (HDAC) inhibitors TSA and SAHA. In the preclinical model in the rat, silibinin administration was able to reduce by half the number of preneoplastic lesions present in the colon. In conclusion, silibinin is a promising natural agent for colon cancer chemoprevention and for combination therapy with TRAIL/HDAC inhibitors.

La stéatohépatite non alcoolique (NASH) est une pathologie du foie dont l’amplitude et les répercussions sont de plus en plus préoccupantes dans le monde médical ou biomédical. Elle est associée à l’obésité, au syndrome métabolique et au diabète sucré de type II. La recherche de la thérapie optimale pour le NASH est un domaine en plein essor puisqu’aucun traitement n’est suffisamment efficace à ce jour. La présente étude fait le point sur de nouvelles possibilités de traitements qui se sont avérés efficaces pour contrer les différentes lésions métaboliques et cellulaires rencontrées dans un modèle in vivo chez le rat où le NASH est induit par l’ingestion d’une diète riche en gras. Cette étude démontre, tout d’abord, que les traitements durant six semaines avec l’acide ursodéoxycholique (UDCA) et son dérivé le NCX 1000, possédant des propriétés donatrices de monoxyde d’azote, à doses équimolaires, protègent de manière équivalente le foie contre le stress oxydatif, l’hyperinsulinémie, l’inflammation et la fibrose causés par la stéatohépatite. De plus, la combinaison d’une plus faible dose de NCX 1000 avec un antioxydant lipophile tel que la vitamine E offre une protection similaire, particulièrement au niveau des paramètres du stress oxydatif. Par ailleurs, l’étude illustre aussi que la silibinine, composé polyphénolique actif du chardon marie (Silybum marianum) et utilisé en traitement pendant 5 semaines, possède un pouvoir hépatoprotecteur, des propriétés antioxydantes et un effet hypoinsulinémique dans ce modèle de stéatohépatite d’origine nutritionnelle. Le potentiel thérapeutique de ces composés en fait des candidats de choix pour le traitement du NASH qui méritent de faire l’objet d’études cliniques poussées. / Nonalcoholic steatohepatitis (NASH) is a serious liver condition related to the metabolic syndrome, obesity, and type II diabetes mellitus whose prevalence is drastically rising in developed countries and worldwide. Several remedies were investigated for the treatment of NASH but an efficient therapy has yet to be developed. In the present study, we explored novel therapeutic possibilities that were thought to be effective for the treatment of experimental high-fat diet-induced NASH the in rat. Our results show that a chronic six week treatment with a high dose of NCX 1000, a derivative of ursodeoxycholic acid (UDCA) with nitric oxide (NO) donating properties, is efficient at reversing steatosis, oxidative stress, inflammation, insulin resistance and fibrosis; major hallmarks of experimental NASH. We also demonstrated that the mother molecule, UDCA, is as efficacious in controlling the same parameters at equimolar doses. Moreover, our study demonstrates that NCX 1000 at lower doses can exert similar potent properties when combined with lipophilic antioxidants like vitamin E. On the other hand, we found that a 5-week treatment with silibinin, the major active component of milk thistle extract, improved liver steatosis and inflammation and decreased NASH-induced oxidative stress, insulin resistance, and fibrosis. These compounds have therefore the potential for being developed for the treatment of NASH. Clinical evidences are needed.

L'infection par le virus de l'hépatite C (VHC) est un problème de santé publique majeur car en absence de vaccin et de thérapie suffisamment efficace, l’infection peut dégénérer en carcinome hépatocellulaire. Il est alors important d’identifier de nouvelles cibles thérapeutiques et de développer de nouveaux antiviraux. Ainsi, nous avons étudié l'activité anti-VHC de différents composés : l'arbidol (Arb), la silymarine (SM) et les molécules la composant, notamment la silibinine (SbN). Ces composés ont l'avantage d'être déjà utilisés en médecine humaine depuis de nombreuses années et ont ainsi prouvé leur innocuité. Ils présentent un large spectre antiviral et inhibent plusieurs étapes du cycle viral, dont la fusion membranaire. Cette étape du cycle est intéressante à cibler car le virus serait bloqué précocement, avant de provoquer des dommages cellulaires.Nous avons approfondi notre connaissance du mécanisme d'inhibition de la fusion par Arb en montrant par différentes stratégies qu'il s'associe avec les phospholipides à l'interface membranaire et interagit avec des résidus aromatiques. Cela suggère que Arb pourrait former durant le processus de fusion un complexe entre glycoprotéine virale et membrane, permettant d'inhiber les changements conformationnels de la glycoprotéine, nécessaires à la fusion. De même SM et ses composés inhibent la fusion de pseudoparticules de HCV, probablement en stabilisant les membranes impliquées dans le processus. Enfin, nous avons observé une activité antivirale et anti-inflammatoire très différente entre deux formulations de SbN. Tous ces résultats sont discutés dans le contexte actuel d'un arsenal thérapeutique anti-HCV qui reste limité. / Infection by the hepatitis C virus (HCV) is a major public health problem since the infection can lead to hepatocellular carcinoma in the current absence of vaccine and effective treatment. It is therefore important to identify new therapeutic targets and to develop novel antiviral drugs. Here we studied the anti-HCV activity of two compounds : arbidol (Arb), the herbal extract silymarin (SM) and molecules therein, including silibinin (SbN). These compounds are already in use in human medicine for several years and have proven safety. They display a broad antiviral spectrum and inhibit several steps of the virus life cycle, including membrane fusion. This step is very interesting to target, since the virus could be blocked upstream the cellular damages it could induce. Using different biophysical strategies, we showed that Arb associates with phospholipids at the membrane interface and interacts with aromatic residues. This suggests that Arb could form during the fusion process a complex between viral glycoprotein(s) and membrane, leading to the inhibition of the conformational changes within the glycoprotein that are required during the fusion process. SM and its components inhibit fusion of HCV pseudoparticles, probably by stabilizing the membranes involved in this process. Finally, we observed different antiviral and anti-inflammatory activities between two different formulations of SbN. Knowledge of these antiviral mechanisms should lead to innovative therapeutic strategies against HCV.