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No. of bitstreams: 1 Tese-Jaqueline de A Bezerra.pdf: 7201878 bytes, checksum: c93d23f2fadb7f5d76a37178a4e187c6 (MD5) Previous issue date: 2014-05-08 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Piper peltatum L. and Commelina erecta L. are tropical species widely distributed in the Amazon region and heavily used by provincials Amazon, for its hardiness, ease of collection and cultivation, to curing several diseases, especially inflammatory. In this work has been performed phytochemical studies and evaluations of antioxidant activity of these species, aiming to contribute to it chemical knowledge, adding value to traditional knowledges. Essential oils from the leaves and hexane, ethyl acetate and ethanol extracts of the leaves, stems and inflorescences of P. peltatum were investigated. Similarly, were studied extracts of leaves and stems of C. erecta and also analyzed its hydro-alcoholic extract (30%) of its flowers. The essential oils of P. peltatum presented as main constituents β-caryophyllene (19.9 to 36.9%), germacrene D (5.8 to 18.8%), α-humulene (4.9 to 6.3%), bicyclogermacrene (3.3 to 10.8), caryophyllene oxide - (3.3-8.3%), E-nerolidol (2.9 to 6.1%) and phytol (1.7 to 8.4%). From the leaves extracts of P. peltatum were isolated, diterpene phytol, a mixture of steroid sitosterol, stigmasterol and campesterol, the flavonoid 5-hydroxy-4 ',7-dimethoxyflavone and identified the 4-nerolidylcatechol, the A, B, and C peltatols; from inflorescence were isolated 4-nerolidylcatechol and peltatol A, as well as identified the B and C peltatols and p-hydroxybenzoic acid; In the stems have been identified cinnamic acid, the uridine and adenosine nucleosides,the uracil base, 4-nerolidylcatechol and A, B and C peltatols. From the ethyl acetate extract of C. erecta stems were isolated the flavonoid luteolin and a mixture of steroids glycosides. In ethyl acetate and ethanol extracts from stems of C. erecta were identified saccharinic acid lactone, shikimic acid, flavonoids luteolin, quercitrin, isoquercitrin, the uridine nucleoside. In the ethyl acetate and ethanol extracts from stems, the saccharinic acid lactone and shikimic acid are major constituents, respectively. In the ethanol leaf extract were identified shikimic acid, 5-cafeoilshikimic acid, the flavonoids orientin, isoschaftoside, 8-C-pentosyl-luteolin, the base uracil and uridine and adenosine nucleosides, and in the hydroalcoholic extract of flowers were identified the isovitexin and vitexin flavonoids C-glycosides. The compounds structures were determined by extensive studies by nuclear magnetic resonance spectroscopy, one-dimensional (1D) and two dimensional (2D) and/or mass spectrometry. The most effective extracts as the scavenging activity of DPPH and ABTS radicals were the ethanol 96% of the inflorescence of P. peltatum (IC50 37.8 and 34.1 μg/mL) and leaves of C. erecta (IC50 50.0 and 65.0 μg/mL). The constitution of these extracts presents phenolic compounds that contribute to antioxidant activity, as 4-nerolidylcatechol for P. peltatum, orientin and luteolin for C. erecta, compatible with the popular use of the species, stimulating the continuation of their investigations. / Piper peltatum L. e Commelina erecta L. são espécies tropicais largamente distribuídas na região Amazônica e muito utilizadas pelos interioranos do Amazonas, devido a sua rusticidade, facilidade de coleta e cultivo, para cura de diversas enfermidades, em especial, processos inflamatórios. Neste trabalho, foram realizados estudos fitoquímicos e avaliação da atividade antioxidante, visando contribuir para o conhecimento químico destas espécies, agregando valor ao conhecimento tradicional. Foram investigados os óleos essenciais das folhas e os extratos hexânicos, em acetato de etila e etanólicos das folhas, caules e inflorescências de P. peltatum. Similarmente, foram estudados os extratos de folhas e caules de C. erecta e analisado o extrato hidroalcoólico (30%) de suas flores. Análises dos óleos essenciais de P. peltatum reveleram como principais constituintes: β-cariofileno (19,9 - 36,9%), germacreno D (5,8 - 18,8%), α-humuleno (4,9 - 6,3%), biciclogermacreno (3,3 - 10,8), óxido de cariofileno (3,3 - 8,3%), E-nerolidol (2,9 - 6,1%) e fitol (1,7 - 8,4%). A partir dos extratos das folhas de P. peltatum foram isolados o diterpeno fitol, uma mistura dos esteroides (sitosterol, estigmasterol e campesterol), o flavonoide 5-hidroxi-4’,7-dimetoxiflavona e identificados, o 4-nerolidilcatecol e os peltatóis A, B e C; da inflorescência foram isolados o 4-nerolidilcatecol e o peltatol A, bem como identificados os peltatóis B, C e o ácido p-hidroxibenzóico; nos caules foram identificados o ácido cinâmico, os nucleosídeos uridina e adenosina, e a base uracila, 4-nerolidilcatecol e os peltatóis A, B e C. A partir do extrato em acetato de etila dos caules de C. erecta foram isolados o flavonoide luteolina e uma mistura de esteroides glicosilados. A partir dos extratos em acetato de etila e etanólico dos caules de C. erecta foram identificados a lactona do ácido sacarínico, o ácido chiquímico, os flavonoides quercitrina e isoquercitrina, bem como o nucleosídeo uridina. Nos extratos em acetato de etila e etanólico dos caules, a lactona do ácido sacarínico e o ácido chiquímico são os constituintes majoritários, respectivamente. No extrato etanólico das folhas foram identificados os ácidos chiquímico, 5-cafeoilchiquímico, os flavonoides orientina, isoschaftoside, 8-C-pentosil-luteolina, a base uracila e os nucleosídeos uridina e adenosina, e no extrato hidroalcoólico das flores, os flavonoides C-glicosídeos vitexina e isovitexina. Os compostos tiveram suas estruturas determinadas por extensivos estudos de espectroscopia de Ressonância Magnética Nuclear unidimensional (1D) e bidimensional (2D) e/ou Espectrometria de Massas. Os extratos mais eficientes quanto à atividade sequestrante dos radicais DPPH. e ABTS+. foram os etanólicos a 96% da inflorescência de P. peltatum (CI50 37,8 e 34,1 μg/mL) e das folhas de C. erecta (CI50 50,0 e 65,0 μg/mL). Estes extratos apresentam em sua constituição compostos fenólicos, como o 4-nerolidilcatecol para P. peltatum, luteolina e orientina para C. erecta, aos quais podem ser atribuídas atividades antioxidantes. Esses resultados são compatíveis com o uso popular das espécies, o que estimula a continuação dos estudos.

O oceano apresenta uma vasta diversidade de espécies, entre elas as algas marinhas, as quais são usadas principalmente como fonte de alimentos, de produtos industriais e para uso medicinal. Considerando a biodiversidade encontrada, são poucos os estudos que verificam a composição química e atividade biológica de algas. Desta forma, o presente trabalho descreve especialmente compostos do metabolismo primário (lipídios, proteínas e aminoácidos), composição química volátil, e potencial antioxidante e antimicrobiano de quatro espécies de algas vermelhas da costa brasileira (Laurencia filiformis, Laurencia intricata, Plocamium brasiliense e Ochtodes secundiramea). As análises de lipídeos revelaram que estas algas são ricas em ácidos graxos poliinsaturados ω3 e ω6, mas que apresentam o ácido palmítico como majoritário. O teor de proteínas encontrado é considerável e aproximadamente 50% da composição de aminoácidos é de aminoácidos essenciais. Para extrair os compostos voláteis das algas selecionadas para o estudo, três métodos foram utilizados: arraste a vapor, extração por solvente e HS-SPME. A caracterização química dos compostos voláteis deu-se principalmente pela utilização de cromatografia gasosa acoplada à espectrometria de massas (CG-EM). Ainda foram isolados e identificados dois compostos majoritários do óleo essencial de L. filiformis, o (-)-7-epi-silfiperfolan-6β-ol e o (-)-silfiperfolan-7β-ol, e quatro compostos do extrato acetona/água de P. brasiliense, o 3,4-eritro-7-diclorometil-3-metil-3,4,8-tricloro-1,5(E),7(E)-octatrieno; o 3,4-eritro-7-diclorometil-3-metil-3,4,8-tricloro-1,5(E),7(Z)-octatrieno; o 3,4-eritro-1-bromo-7-diclorometil-3-metil-3,4,8-tricloro-1(E),5(E),7(E)-octatrieno e o 3,4-eritro-1-bromo-7-diclorometil-3-metil-3,4,8-tricloro-1(E),5(E),7(Z)-octatrieno, utilizando diferentes técnicas cromatográficas, como CCDP e CLAE, para isolamento, e técnicas espectroscópicas (RMN uni e bidimensionais) e espectrométricas (HRMS e EIMS) para análise. A atividade antioxidante dos óleos essenciais, dos extratos e das substâncias isoladas foi verificada utilizando-se dois métodos (DPPH e quimioluminescência). Os extratos diclorometano de L. filiformis (IC50 de 48,5 µg/mL) e L. intricata (IC50 de 58,0 µg/mL) mostraram-se como os mais potentes. As mesmas amostras não apresentaram potencial antimicrobiano em concentrações de até 500 µg/mL frente aos nove microrganismos testados. / The ocean provides large diversity of species, among them the seaweeds, which are mainly used as food, industrial products and as medicine. Considering the biodiversity, there are only few studies which analize the algae volatile compounds and their biological activity. So that, this work describes specially compounds from the primary metabolism (lipids, proteins and amino acids), chemical volatile composition, and antioxidant and antimicrobial potencies of four red algae of the Brazilian coast (Laurencia filiformis, Laurencia intricata, Plicamium brasiliense and Ochtodes secundiramea). The lipid analysis showed that these algae have ω3 and ω6 polyunsaturated fatty acids, but the palmitic acid is the most abundant. The protein content observed is considerable and approximately 50% of the amino acid composition is of essential amino acids. To extract the volatile organic compounds from the algae selected for this study, three methods were used: hydrodestilation, solvent extraction and HS-SPME. For chemical characterization of the volatile compounds, the technique used was gas chromatography coupled with mass spectrometry (GC-MS). In addition, the two most abundant compounds from the essential oil of L. filiformis, the (-)-7-epi-silphiperfolan-6β-ol and the (-)-silphiperfolan-7β-ol, and four compounds of the aceton/water extract of P. brasiliense the 3,4-erythro-7-dichloromethyl-3-methyl-3,4,8-trichloro-1,5(E),7(E)-octatriene; the 3,4-erythro-7-dichloromethyl-3-methyl-3,4,8-trichloro-1,5(E),7(Z)-octatriene; the 3,4-erythro-1-bromo-7-dichloromethyl-3-methyl-3,4,8-trichloro-1(E),5(E),7(E)-octatriene and the 3,4-erythro-1-bromo-7-dichloromethyl-3-methyl-3,4,8-trichloro-1(E),5(E),7(Z)-octatriene, were isolated and identified using different chromatographic techniques like preparative TLC and HPLC for isolation and spectroscopic (NMR uni and bidimensional) and spectrometric techniques (HRMS and EIMS) for analysis. The antioxidant activity of the essential oils, of the extracts and of the isolated compounds was verified by two methods (DPPH and chemiluminescence). The dichloromethane extracts of L. filiformis (IC50 of 48.5 µg/mL) and L. intricata (IC50 de 58.0 µg/mL) showed higher potency. The same samples do not have antimicrobial activity in concentrations until 500 µg/mL up against the nine microorganisms tested.

Os frutos de Iryanthera paraensis Huber foram separados em suas partes constituintes e de seu pericarpo foi obtido o extrato clorofórmico que, por cromatografia e purificação em CLAE, forneceu cinco substâncias inéditas (uma neolignana e quatro γ-butanolidos): Eu-IP: reI (7S,8R)-4-hidroxi-3,5\'-dimetoxi-Δ1,3,5,1\',3\',5\',7- 7.O.3\',8.O.4\'-neolignana. A-1: (2S,3S,4S)-2-(9\'-fenil-n-nonil)-3-hidroxi-4-metilbutanolido. A-2: (2S,3R,4S)-2-(9\'-fenil-n-nonil)-3-hidroxi-4-metilbutanolido. B-1: (2S,3S,4S)-2-(11\'-fenil-n-undecil)-3-hidroxi-4-metilbutanolido. B-2: (2S,3R,4S)-2-(11\'-fenil-n-undecil)-3-hidroxi-4-metilbutanolido. A determinação das estruturas destas substâncias foi baseada principalmente em técnicas espectrométricas (EM, IV, RMN de 1H e de 13C). / The pericarps were removed from fruits of Iryanthera paraensis Huber, dried, ground and percolated with chloroform. The chloroform extract submitted to chromatographic techniques followed by HPLC purification, yielded a new neolignan and four new butanolides: Eu-IP: reI (7S,SR)-4-hydroxy-3,5\'-dimethoxy-Δ1,3,5,1\',3\',5\',7-7.O.3\',S.O.4\'-neolignan. A-1: (2S,3S,4S)-2-(9\'-phenyl-n-nonyl)-3-hidroxy-4-methylbutanolide. A-2: (2S,3R,4S)-2-(9\'-phenyl-n-nonyl)-3-hidroxy-4-methylbutanolide. B-1: (2S,3S,4S)-2-(11\'-phenyl-n-undecyl)-3-hidroxy-4-methyl-butanolide. B-2: (2S,3R,4S)-2-(11\'-phenyl-n-undecyl)-3-hidroxy-4- methyl-butanolide. The structural determination of these substances were based mainly on spectrometric methods (MS, IR, 1H and 13C NMR)

As espécies Aspidosperma polyneuron Muell. Arg. e Aspidosperma cylindrocarpon Muell. Arg. pertencentes à família Apocynaceae, de ocorrência natural no estado de São Paulo, foram objeto de investigação química e estabelecimento de culturas in vitro. Como não havia relatos da composição dos alcalóides presentes nas folhas de ambas as espécies, nossos estudos foram concentrados nessa parte do vegetal. Adicionalmente, foi analisado a composição dos óleos voláteis destas espécies. Como são descritas diversas atividades biológicas para os alcaloides indólicos, foram analisados as atividades antitumoral, antimicrobiana e antimalárica dos extratos de alcalóides totais. Os alcalóides das folhas de A. polyneuron foram obtidos através de partição ácido-base com solventes orgânicos e foram isolados por técnicas cromatográficas de adsorção, sendo analisadas por CG/MS. Na espécie A. polyneuron foram identificados 5 alcalóides indólicos do tipo aspidospermatano: aspidospermina, desmetil-aspidospermina, desmetóxi-aspidospermina, cilindrocarina e pirifolidina. O estudo químico das folhas de A. cylindrocarpon, através de técnicas cromatográficas e CG/MS permitiu a identificação de alcalóides indólico do tipo aspidospermatano (aspidospermina, N-benzoíl-cilindrocarina, N-benzoíl-20-hidróxi-cilindrocarina, N-cinamoíl-20-hidróxi-cilindrocarina) do tipo erbunano (16-epi-vincamina) e do tipo hetero-ioimbano (tetra-hidro-alstonina). Os óleos voláteis foram obtidos das folhas por destilação por arraste a vapor. O óleo de A. polyneuron apresentou um constituinte majoritário, o diterpeno caureno (73,7%), e aldeídos de cadeia longa. No óleo volátil das folhas de A cylindrocarpon foram identificados 25 constituintes que correspondem a 96,1 % do óleo bruto. Principalmente sesquiterpenos hidrocarbonados (germacreno-D, β-cariofileno) e sesquiterpenos oxigenados (espatulenol, epi-globulol e globulol), além de monoterpenos hidrocarbonados (α-pineno) e oxigenado (linalol) e aldeídos de cadeia longa. Na cultura de células in vitro de A. polyneuron, foi possível apenas a indução de calos friávies. A cultura de células da A. cylindrocarpon foi estabelecida desde as células não diferenciadas. Dos calos até as suspensões celulares. As culturas apresentram fase lag com aproximadamente 4 dias, seguida de uma fase de crescimento exponencial e de uma fase com crescimento linear, com duração de 15 dias. Após essa fase as células entram numa fase estacionária de crescimento. Nos ensaios de autobiografia para determinação da atividade antifúngica frente aos fungos Clasdosporium cladosporiodes e Clasdosporium sphaerospermum, ambos extratos de alcalóides totais apresentaram respostas positivas. A atividade antimalárica foi avaliada em cepas mutantes de Saccharomyces cerevisiae deficientes em sistema de reparo do DNA (RS321, Rad 52Y). Neste ensaio, apenas os alcalóides das folhas de A. cylindrocarpon apresentaram atividade. A atividade antitumoral foi determinada in vitro com isolados de Plasmodium falciparum sensíveis a cloroquina (K1) e resistentes (Palo Alto). Como havia relato dessa atividade para A. polyneuron foram feitos testes apenas para A. cylindrocarpon (folhas e ramos). Os extratos alcaloídicos apresentaram uma forte inibição do desenvolvimento dos parasitas. A Cl50 para o isolado K1 foi de 9,0µg/ml para os ramos e folhas 8,0µg/ml e para o isolado Palo Alto, ramos foi de 13,3µg/ml e folhas 10,5µg/ml. / Aspidosperma polyneuron Muell. Arg. and Aspidosperma cylindrocarpon Muell. Arg. belong to Apocynaceae and naturally occur in São Paulo State (Brazil). Both species were chemically investigated and in vitro cultures established. As there were no previous studies concerning the alkaloid composition from the leaves, our studies were focused on this plant part. Moreover, the volatile oil composition was determined for both species. As several biological activities have been described for indole alkaloids, the antimicrobial, antitumor and antimalarial activities were assayed for the total alkaloids. The leaf alkaloids were extracted by acid/base partitioning with organic solvents and isolated by adsorption chromatography techniques. The isolated alkaloids were analysed by GC-MS. A. polyneuron leaves afforded 5 aspidospermatane alkaloids: aspidospermine, demethoxy-aspidospermine, demethylaspidospermine cylindrocarine and pyrifolidine. In A. cylindrocarpon leaves different indole alkaloid skeletons were identified, 4 aspidospermatanes (aspidospermine, N-benzoyl-cylindrocarine, N-benzoyl-20-hydroxy-cylindrocarine, N-cynamoyl-20-hydroxy-cylindrocarine), 1 eburnane (16-epi-vincamine) and 1 heteroyohimbane (tetrahydroalstonine). The volatile oils were extracted from the leaves by steam distillation. A. polyneuron oil consisted of one single major compound, the diterpene kaurene (73,7%), and several long chain aldehydes. On the other hand, from the A. cylindrocarpon oil 25 constituents could be identified corresponding to 96.1 % of the crude oil. The major components were sesquiterpene hydrocarbons (germacreno-D, β-caryophyllene), oxygenated sesquiterpenes (spathulenol, epi-globulol, globulol), a monoterpene hydrocarbon (α-pinene), an oxygenated monoterpene (linalool) and also several long chain aldehydes. For A. polyneuron, callus cultures were induced, while for A. cylindrocarpon was also possible the establishment of cell suspension cultures. A. cylindrocarpon cell suspension cultures showed a lag phase of growth for approximately 4 days. The lag phase was followed by a growth phase for 15 days. After 20 days of culture the cells showed a stationary growth. Alkaloid extract from both species showed a antifungal activity in the bioautography assay with Clasdosporium cladosporiodes and C. sphaerospermum. The antitumor activity was evaluated with mutant Saccharomyces cerevisiae strains deficient in the DNA repair system (RS321 and Rad 52Y). In this assay, only the leaf alkaloids of A. cylindrocarpon presented activity. In vitro antimalarial assay were performed with Plasmodium falciparum chloroquine sensitive (K1) and resistant (Palo Alto). As such an activity was previously reported for A. polyneuron, only A. cylindrocarpon alkaloid extracts from leaves and stems were tested. Both alkaloid extracts showed strong inhibition of the parasite development. The IC50 for the K1 isolate were 9.0 µg/ml for the leaves and 8,0 µg/ml for the stems. The Palo Alto isolated presented a higher IC50, 13,3 µg/ml stems and 10,5 µg/ml.

Les cyanoglucosides non-cyanogènes se trouvent dans de nombreuses plantes en particulier médicinales, mais n'ont été qu'assez peu étudiés. En particulier la Ménisdaurine, dont la synthèse n'a jamais été réalisée, nous a paru un objectif intéressant. Le produit de départ, choisi en fonction de travaux antérieurs, est un dérivé de la 7-oxanorbornanone. La première voie étudiée avait comme étape-clé l'addition électrophile sur une double liaison mais n'a pas abouti. Nous avons ensuite envisagé l'ouverture d'un époxyde suivie de réduction. Ayant observé que le cycle époxy peut être ouvert par migration intramoléculaire d'un groupement méthoxy en présence d'iode, nous avons réalisé la même réaction avec un groupement phenylthio. L'ouverture de l'époxyde a ainsi été effectuée de façon totalement régio- et stéréo-sélective dans des conditions pratiquement neutres, ce qui n'avait jusque là pas été rapporté dans la littérature. Une étude mécanistique a confirmé que cette réaction se déroulait bien en deux étapes : formation d'un hémimercaptal sur la fonction cétone suivie d'une ouverture de l'époxyde par attaque exclusivement intramoléculaire. Le remplacement de l'iode par l'iodure de zinc conduit à des rendements encore supérieurs. Le groupement phenylthio étant facilement enlevé par réduction, cette voie est très efficace pour réduire sélectivement un époxyde en alcool. La suite de la synthèse conduit à l'aglycone protégée souhaitée puis la glycosidation a été réalisée avec un rendement de 80%. La(-)-Ménisdaurine naturelle a été obtenue (et caractérisée sous forme de son pentaacétate) en 10 étapes et 3% de rendement global. / Non-cyanogenic cyanoglucosides are found in many plants, specially in medicinal species but have been the subject of only few studies. Particularly, Menisdaurin seemed us an interesting target and we decided to work on its first total synthesis. The starting material, chosen by reference to previous work in the laboratory, is a derivative of 7- oxanorbornanone. The first synthetic route had as key-step an electrophilic addition on a double bond but, unfortunately, was unsuccessful. Then we chose as an alternative the nucleophilic opening of an epoxide ring followed by reduction. We noticed that this epoxide ring could be opened by the intramolecular migration of a methoxy group in the presence of iodine, and therefore we tried, successfully, to perform the same reaction with a phenylthio group. So the opening of the epoxide ring was carried out with total regio- and stereo-selectivity in almost neutral conditions, which had not been hitherto reported in literature. Mechanistic studies confirmed that this reaction consisted of two steps : the formation of an hemimercaptal on the ketone followed by the opening of the epoxide by an intramolecular attack. Replacement of iodine by zinc iodide afforded even better yields. The phenylthio group being easily removed by reduction, this procedure is very efficient to obtain selectively an alcohol from an epoxide. Then, the following synthetic steps afforded the desired protected aglycone which was glycosidated with a 80% yield. Natural (-)­-Menisdaurin was obtained (and characterized as its pentaacetate) in 10 steps with a global 3% yield.

The relationship between the endocannabinoid system in the renal medulla and the long-term regulation of blood pressure is not well understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase (FAAH), PF-3845. Treatment of MMICs with PF-3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. HPLC analyses of lipid extracts of MMIC culture medium revealed a 205nm-absorbing peak that showed responsiveness to PF-3845 treatment. The biologic activities of the PF-3845-induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically-instrumented mice. Intramedullary and intravenous infusion of PIP at low dose rates (0.5-1 AU/10 min) stimulated diuresis and natriuresis, whereas at higher doses, these parameters returned toward baseline but mean arterial pressure (MAP) was lowered. Whereas intravenous bolus doses of PIP stimulated diuresis, GFR and medullary blood flow (MBF) and reduced or had no effect on MAP, an intraperitoneal bolus injection of PIP reduced MAP, increased MBF, and had no effect on urinary parameters. Genetic or pharmacological ablation of the cannabinoid type 1 receptors in mice completely abolished the diuretic and vasodepressor properties of intramedullary infused PIP, suggesting that the PF-3845-induced product requires the presence of CB1 receptors in order to elicit its renal effects. In a radioactive competition binding assay, using Chinese hamster ovary cells expressing CB1 receptors, PIP successfully displaced the CB1 selective inverse agonist [3H] SR141716A, revealing that the lipid extract was able to compete for binding to CB1 receptors. Finally, we investigated the tubular location of diuretic activity that the PF-3845-induced lipid fraction exhibits. In a renal function in vivo experiment, we pre-treated anesthetized mice with an intramedullary infusion of one of four well-known diuretics. This procedure was followed by an intramedullary infusion of PIP (1AU). Only inhibition of the proximal tubule sodium reabsorption diminished the diuretic activity of the PF-3845-induced product, suggesting that the lipid fraction requires a physiologically intact proximal tubular reabsorption mechanism for it to produce diuresis. These data support a model whereby PF-3845 treatment of MMICs results in increased secretion of a neutral lipid which acts directly to promote diuresis and natriuresis and indirectly through metabolites to produce vasodepression. Efforts to identify the structure of the PF-3845-induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.

Background This thesis investigated the efficacy and safety of Bacopa monnieri in improving memory in healthy Australians over the age of 55-years. A review of the literature showed that memory impairment and dementia are increasingly prevalent in the current demographic climate of an ageing population. As well as the pathological cognitive loss of neurodegenerative disease, many older persons are experiencing memory loss as part of the physiological process of ageing. Bacopa monnieri is a herbal medicine used since antiquity in the traditional Ayurvedic medical system of India for its cognitive enhancing effects. A number of pre-clinical and clinical studies support this traditional usage. Laboratory studies have demonstrated antioxidant and cholinergic actions in the brain as well as improved memory and cognitive performance in animal models. Human trials of Bacopa have also demonstrated improved memory performance. Some of these trials are limited by methodological flaws such as lack of blinding, small sample sizes, or use of outcome measurements which are not well validated. However, a small number of well designed human trials provide evidence for efficacy in cognitive and memory performance improvement. The current study was employed to extend on previous findings by assessing the efficacy and safety of Bacopa in the aged population specifically, as it is in this population that memory impairment becomes apparent. Aims 1. To assess the efficacy of Bacopa monnieri in improving memory in healthy Australians over the age of 55-years. 2. To assess whether the use of Bacopa is associated with side-effects Design A 12-week, randomised, double-blind, placebo-controlled, parallel group clinical trial. Participants Participants were self selected from the general population. They were aged 55-years or over at the commencement of the trial. Participants were without dementia, depression or other serious health conditions and did not use psychotropic medications. Intervention Participants were randomised to one of two treatment conditions, either a tableted extract of Bacopa monnieri called Bacomind™ (300mg/day, standardised to contain at least 40% bacosides), or an identical placebo. Participants attended three clinical evaluations: the first an initial screening session, the second a baseline evaluation of neuropsychological function and subjective memory performance at the commencement of the trial and the third, an end-of-trial outcome evaluation at 12-weeks, during which neuropsychological function and subjective memory performance were again assessed along with side-effects and study compliance. Primary Outcome Measures Rey Auditory Verbal Learning Test (AVLT), Rey-Osterrieth Complex Figure Test (CFT), Memory Complaint Questionnaire (MAC-Q), and Trail Making Test (TMT) Results From 136 people who elected to participate, 103 people met study entry criteria and 98 of these commenced the trial. Of these, 81 participants completed the trial and provided evaluable data for the end point analysis. Bacopa monnieri versus placebo significantly improved verbal learning as well as delayed recall as measured by the AVLT (p<.05). Though improvements were noted in the CFT, MAC-Q and TMT, there were no significant differences between placebo and active groups found for these tests. The Bacopa group reported a higher incidence of gastro-intestinal (GIT) side-effects than the placebo group, these predominantly being increased stool frequency, abdominal cramps and nausea. No other significant adverse effects were found. Conclusions A clinical trial was carried out to assess the effects of 12-weeks administration of Bacopa monnieri (300mg/day) on memory performance in people over the age of 55-years. Primary outcome measures were well validated neuropsychological tests that objectively measured verbal and visual memory and a memory complaint questionnaire that measured subjective memory complaints. The results demonstrated that Bacopa significantly improved memory acquisition and retention in older Australians. This concurs with findings from previous human and animal studies, as well as supports traditional Ayurvedic claims and uses. The beneficial effects on memory observed may be due to previously demonstrated antioxidant and cholinergic effects of the herb on the central nervous system. The use of Bacopa was associated with GIT side-effects, particularly increased bowel movements, nausea and abdominal cramping, findings infrequently reported previously. Possible explanations for these side-effects include GIT irritation by the saponin constituents of the herb, or cholinergic stimulation of autonomic and motor responses in the GIT, or a combination of both of these factors. The side-effects observed in the current study provide supportive evidence that Bacopa may increase cholinergic activity in humans. A worthwhile future extension of the current study would be to assess whether the finding of Bacopa’s efficacy for improving memory performance is replicable in populations with either mild cognitive impairment or early dementia.

<p>Pancreatic cancer is the fourth most lethal cancer and ranks as the eighth most commonly diagnosed cancer worldwide. This is due to its rapid proliferation, strong metastatic potential and its delayed detection. One major risk factor for developing pancreatic cancer is the aggressive inflammatory disease chronic pancreatitis. Chronic inflammation frequently precedes the development of certain pancreatic cancers.</p><p>Inflammation is a protective and necessary process by which the body can alert the immune system of the existence of a wound or infection and mount an immune response to remove the harmful stimuli and start wound healing. The cross-talking of cells of the immune system and infected cells happens through cytokines, soluble proteins that activate and recruit other immune cells to increase the system’s response to the pathogen. Failure to resolve the injury can result in persistent cytokine production that in turn allows a cell that is damaged or altered to survive when in normal conditions it would be killed. Inflammation is thought to create a microenvironment that facilitates the initiation and/or growth of pancreatic cancer cells.</p><p>Cytokines use two important kinases for their signaling: Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs). The JAKs are activated upon the binding of cytokines to their corresponding receptors. When activated, the JAKs activate STATs through tyrosine phosphorylation. The STATs transduce signals to the nucleus of the cells to induce expression of critical genes essential in normal physiological cellular events such as differentiation, proliferation, cell survival, apoptosis and angiogenesis. STAT3 (a member of the STAT family) is constitutively activated in some pancreatic cancers, promoting cell cycle progression, cellular transformations and preventing apoptosis. Therefore, STAT3 is a promising target for cancer treatment. Novel therapies that inhibit STAT3 activity in cancers are urgently needed. Natural products are a very good resource for the discovery of new drugs against pancreatic cancer.</p><p>Covering more than 70% of the Earths surface, The Ocean is an excellent source of bioactive natural products. Harbor Branch Oceanographic Institute’s Center for Marine Biomedical and Biotechnology Research (HBOI-CMBBR) situated in Florida, aims to find new marine natural products useful in disease prevention and drug therapy. Their current focus is to look for novel treatments for preventing both the formation of new pancreatic tumors and the metastasis of existing tumors.</p><p>The hypothesis of this degree project was that novel inhibitors of STAT3 useful in the treatment of pancreatitis and/or pancreatic cancer could be found from marine-natural products. The first specific aim of this degree project was to set up an assay to identify bioactive marine natural products as inhibitors of inflammation. Furthermore the assay was validated using a commercially available inhibitor of inflammation (Cucurbitacin I). The last aim was to further validate the assay by screening pure compounds and peak library material from the HBOI marine specimen collection.</p><p>At the end of the experimentation time, the assay still was not set-up as there were difficulties in proper cell culture techniques and the cell line did not respond as advertised. While the results were not as expected, the work performed resulted in familiarization with research laboratory practices and increased laboratory skills. Moreover, the results from the assays point to future directions to accomplish this project.</p> / Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer

According to WHO, leishmaniasis is endemic in 98 countries, and has been placed ninth in a global analysis of infectious diseases. Treatment of leishmaniasis is based on pentavalent antimonials but toxicity and developing resistance have been reported. Traditional medicine and scientific studies have shown that the extract of Galipea longiflora Krause (Evanta) exhibits antileishmanial activity. We hypothesized that the healing observed when using this plant might not only be due to the direct action on the parasite, but possibly to a parallel effect on the host immune response. We found that an alkaloid extract of Evanta (AEE) inhibited the growth of Leishmania braziliensis promastigotes while viability of eukaryotic cells was practically not affected. We also found that AEE interfered with polyclonal activation or Leishmania-specific re-stimulation of lymphocytes, as revealed by a reduction of in vitro cellular proliferation and IFN-g production. More important, AEE treatment of mice hosting L. braziliensis showed that AEE is able to control both inflammation and parasite load. Additionally, the healing process was improved when AEE and meglumine antimoniate were administered simultaneously. Dendritic cells (DCs) play a pivotal role in T-cell stimulation and polarization of naïve T cells. Therefore, we investigated if AEE could alter the activation of DCs and if allostimulatory DCs properties were altered if activated in the presence of AEE. DCs activated in the presence of AEE reduced the production of IL-12p40 and IL-23. When we analyzed the allostimulatory capacity of AEE-treated DCs, we found that allogeneic CD4+ T-cells secreted lower levels of IFN-γ. In conclusion, this thesis provides valuable insight into the effects of Evanta derived extract. The dual effect found for AEE, on Leishmania parasite and on the immune response, suggests that AEE may be useful in controlling the parasite burden and preventing over-production of inflammatory mediators and subsequently avoiding tissue damage. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Accepted. Paper 3: Submitted.</p>

Soy contains many bioactive molecules known to elicit anti-cancer effects. One such peptide, Lunasin, has been shown to selectively act on newly transformed cells while having no cytotoxic effect on non-tumorigenic or established cancer cell lines. In this study we attempt to understand the developmental effects of Lunasin overexpression in vivo and create reagents that will help us understand Lunasin’s anti tumorigenic effects in an intact organism. cDNA encoding lunasin and EGFP-lunasin were cloned into pUAST and microinjected into Drosophila embryos. Tissue-specific overexpression of EGFP-Lun in the resulting transgenic lines was accomplished by crossing transgenics to various GAL4 driver lines. Progeny were assessed for phenotypic alterations and no phenotypic abnormalities were observed in tissues expressing EGFP-Lunasin, supporting current studies that show Lunasin does not affect normal cells. Previous studies have localized Lunasin to the nuclear compartment. To test if this was the case for EGFP-Lun, subcellular localization of EGFP-Lun was determined via fluorescence microscopy. Salivary glands from EGFP-Lun expressing individuals were dissected, fixed, and mounted in Vectashield® with the nuclear stain, DAPI. Our results demonstrate that EGFP-Lun, like native Lunasin, is localized to the nucleus. Eight transgenic lines were mapped to specific chromosomes and EGFP-Lun transgenic line GEJ1-L2 was balanced in preparation for use in tumor suppression studies. In summary, we have created and characterized transgenic flies capable of overexpressing Lunasin under the control of the GAL4/UAS system. Localization of EGFP-Lunasin to the nucleus and data on the phenotypic consequence of its overexpression in flies is presented. Finally, reagents created as part of this thesis will aid experiments aimed at understanding the effects of Lunasin on benign and invasive tumors.