Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-11T18:07:24Z No. of bitstreams: 1 Tietbohl, Luis Armando Candido [Dissertação, 2014].pdf: 8958883 bytes, checksum: 41b0a4341bcfc24a0012440a901a42f7 (MD5) / Made available in DSpace on 2017-04-11T18:07:24Z (GMT). No. of bitstreams: 1 Tietbohl, Luis Armando Candido [Dissertação, 2014].pdf: 8958883 bytes, checksum: 41b0a4341bcfc24a0012440a901a42f7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A espécie Myrciaria floribunda (H.West ex Willd.) O. Berg., Myrtaceae, popularmente conhecida como camboim-amarelo, foi coletada no Parque Nacional da Restinga de Jurubatiba (RJ, Brasil). Esta espécie é rica em estruturas secretoras de óleo essencial. Neste trabalho, é descrita a análise da composição química dos óleos essenciais de folhas, caules e flores. O monoterpeno 1,8 cineol apresenta-se como componente majoritário nos óleos de folhas (38,4%) e flores (22,8%). No óleo de caules a substância majoritária foi o acetato de farnesila (19,9%). Além das substâncias já descritas para o óleo essencial de folhas desta espécie, foram identificadas pela primeira vez: α-pineno (0,4%), mirceno (0,7%), (Z)-β-ocimeno (0,8%), γ-terpineno (0,8%), acetato de geranilo (1,5%), γ-himachaleno (7,0%), valenceno (1,8%), δ-amorpheno (4,0%), zonareno (4,6%), selina-3.7(11)-dieno (3,8%), neo-intermedeol (0,8 %) e (2E, 6Z)-farnesol (1,4%). As análises dos óleos essenciais de caules e flores ainda não haviam sido descritas para esta espécie. Também foram isolados e identificados pela primeira vez na espécie o triterpeno ácido betulínico e o flavonoide miricetina 3-O-β-galactosídeo da fração acetato de etila de folhas. O óleo essencial de folhas foi avaliado quanto as atividades: anticolinesterásica, antimicrobiana e inseticida. Os óleos de folhas, flores e caules foram avaliados quanto a atividade anticolinesterásica, mostrando um IC50 de 1583 e 681 μg/mL para flores e folhas, respectivamente. O óleo de caules não apresentou atividade. O teste de susceptibilidade antimicrobiana do óleo essencial das folhas utilizando cepas de Staphylococcus aureus ATCC 29213, apresentou um CMI de 250 μg/mL. A atividade inseticida do óleo essencial das folhas foi avaliada frente aos fitófagos Oncopeltus fasciatus e Dysdercus peruvianus e os óleos essenciais das folhas e flores foram avaliados frente ao percevejo hematófago da espécie Rhodnius prolixus. Os tratamentos dos insetos com os óleos essenciais de M. floribunda induziram mortalidade, atraso no desenvolvimento e inibição de muda. Para avaliar a atividade hipoglicemiante e integridade do fêmur em ratos machos, foi utilizado o extrato aquoso das folhas de M. floribunda a uma concentração de 5 % (p/v), onde foi possível verificar ao final dos 42 dias de tratamento uma diferença significativa na glicemia e resultados similares para o fêmur. O grupo tratado apresentou resultados significativamente aumentados de colesterol, proteína total, fósforo, magnésio e aspartato aminotransferase, massa de fígado menor e massa dos rins maiores quando comparados com o grupo controle. / The species Myrciaria floribunda (H.West ex Willd.) O. Berg., Myrtaceae, popularly known as “camboim-amarelo”, was collected in the “Parque Nacional da Restinga de Jurubatiba” (RJ, Brazil). This species is rich in secretory structures essential oil. This work describes the analysis of the chemical composition of the essential oil of leaves, stems and flowers in that the monoterpene 1.8-cineole present as the major component in the oil leaves and flowers corresponding 38.4% 22.8%, respectively. Already at oil from stems the substance majority was farnesyl acetate (19.9%). In addition to the substances already described for the essential oil from leaves of the species were first identified the following substances : α-pinene (0.4%), myrcene (0.7%), (Z)-β-ocimene (0.8%), γ-terpinene (0.8%), geranyl acetate (1.5%), γ-himachalene (7.0%), valencene (1.8%), δ-amorphene (4.0%), zonarene (4.6%), 3.7-selina (11) -diene (3.8%), neo-intermedeol (0.8%) and (2E, 6Z)-farnesol (1.4%). The analysis of essential oils from stems and flowers had not yet been described. Were also isolated and identified for the first time in the kind triterpene betulinic acid and flavonoid myricetin 3-O-β-galactoside of ethyl acetate fraction of leaves. The essential oil from leaves was evaluated for activities: acetylcholinesterase, antimicrobial and insecticidal. The oils from leaves, flowers and stems were evaluated for anticholinesterase activity, showing an IC50 of 1583 and 681 mg/mL for flowers and leaves, respectively, and stems showed no activity. The antimicrobial susceptibility testing essential oil from leaves using Staphylococcus aureus ATCC 29213, filed an IMC 250 μg/mL. Insecticidal activity of the essential oil from the leaves was evaluated against phytophagous Oncopeltus fasciatus and Dysdercus peruvianus, and the essential oil from the leaves and flowers were evaluated against the Rhodnius prolixus. All treatments of insects with essential oils of M. floribunda induced mortality, developmental delay and inhibition of molting. To evaluate the hypoglycemic activity and femur health in male rats, were evaluated with the aqueous extract of leaves of M. floribunda at a concentration of 5%, which was verified at the end of the 42 days of treatment the infusion decreased the glycemia e maintenance results similar to the femur. Treated group apresented results significantly increased to cholesterol, total protein, phosphorus, magnesium and aspartate aminotransferase, mass minor liver and kidney mass was increased compared with the control group

Cancer is one of the leading causes of morbidity and mortality in developed and developing nations. It is estimated that 86% of new cases and 64% of death due to cancer are from Africa and 13.1 million deaths are estimated to occur worldwide by the year 2030. Cancer death rates have not subsided despite recent advances in cancer drug development and treatment. Present cancer drug regimens are limited due to unpredictable efficiency, severe side effects ,resistance and high cost. Plants provide a vast array of natural compounds such as terpenoids, phenolics and alkaloids with antiproliferative pro-apoptotic and antioxidant effects. Plants are principal sources of compounds for drug discovery and development of several clinically proven useful anticancer drugs. The present study focused on the isolation of compounds from the Holarrhena floribunda (H. Floribunda) leaves for their potential anticancer activities. Standard methods were employed to assess the antiproliferative potential, apoptosis, cell cycle analysis and reactive oxygen species of the methanolic leaf extract (MLE) of H. floribunda. The standard methods of isolation such as column chromatography, thin layer Chromatography, high performance liquid chromatography (hplc) and nuclear magnetic Resonance (nmr) were used to isolate and purify bioactive compounds from the leaves. To Elucidate the mechanism of cytotoxicity of the isolated compounds, apoptosis effect was studied by flow cytometry analysis using the Apopercentagetm dye, Annexin-V/PI stain, induction of cspase-3 using the Caspase-3/7 glo assay kit and Parp-1 deactivation using Western blot analysis. The mode of action was further assessed by evaluating reactive oxygen species (ros), mitochondrial toxicity, light and fluorescent microscopic morphological evaluations of F-actin and topoisomerase-I relaxation assay. In addition, potential cancer prevention of the plant was also evaluated by assessing the antioxidant activity of the flavonoids compounds isolated from the mle. The results of the present study show that the MLE of H. floribunda inhibited the proliferation of the cancer cell lines (HeLa, HT-29 and MCF-7) in a dose- and timedependent manner. The anti-proliferative activity of the methanolic extract is selective towards cancer cells more than normal KMST-6 fibroblast cells used in the study. The extract showed cytotoxicity, cell cycle arrest, induced generation of ROS and apoptosis. The methanolic extract of the leaves led to the isolation of two pure steroidal alkaloid and four flavonoid compounds. The two steroidal alkaloids - holamine and funtumine were found to be selectively cytotoxic to human cancer cells more than the normal fibroblasts. The cytotoxicity of the two steroidal alkaloids was mediated through the induction of apoptosis. The apoptosis induction was found to be triggered by the activation of caspase-3, deactivation of PARP-1, increased ROS, cell cycle arrest at G0/G1 and G2/GM phases, mitochondria toxicity, F-actin disorganization and topoisomerase-I inhibition. However, four flavonoids (kaempferol-3-O-rutinoside, quercetin-3-galactoside/glucoside, quercetin-3-O-glucoside and kaempferol-3-O-glucoside) isolated were subjected to antioxidant activity assay using oxygen radical absorbance capacity (ORAC), ferric reducing/antioxidant power (FRAP), trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation inhibition. Two flavonoids with a quercetin nucleus were found to be active in the entire antioxidant spectrum, except pro-oxidant activity while the remaining two compounds with a kaempferol nucleus were selective in their activity. Structure-activity relationship analysis shows that the activity of the flavonoids depends on the ortho dihydroxyl group on the B-ring of the flavonoids. The present study on H. floribunda has shown that the leaves contain bioactive compounds with potential as anticancer agents and cancer protective and preventive activity. More work is still needed to elucidate other possible anticancer mechanisms that might be relevant to the cytotoxic effects of the two isolated alkaloids. / Philosophiae Doctor - PhD

Philosophiae Doctor - PhD / Cancer is one of the leading causes of morbidity and mortality in developed and developing nations. It is estimated that 86% of new cases and 64% of death due to cancer are from Africa and 13.1 million deaths are estimated to occur worldwide by the year 2030. Cancer death rates have not subsided despite recent advances in cancer drug development and treatment. Present cancer drug regimens are limited due to unpredictable efficiency, severe side effects, resistance and high cost. Plants provide a vast array of natural compounds such as terpenoids, phenolics and alkaloids with antiproliferative pro-apoptotic and antioxidant effects. Plants are principal sources of compounds for drug discovery and development of several clinically proven useful anticancer drugs. The present study focused on the isolation of compounds from the Holarrhena floribunda (H. floribunda) leaves for their potential anticancer activities. Standard methods were employed to assess the antiproliferative potential, apoptosis, cell cycle analysis and reactive oxygen species of the methanolic leaf extract (MLE) of H. floribunda. The standard methods of isolation such as column chromatography, thin layer chromatography, high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) were used to isolate and purify bioactive compounds from the leaves. To elucidate the mechanism of cytotoxicity of the isolated compounds, apoptosis effect was studied by flow cytometry analysis using the ApopercentageTM dye, Annexin-V/PI stain, induction of caspase-3 using the Caspase-3/7 Glo assay kit and PARP-1 deactivation using Western blot analysis. The mode of action was further assessed by evaluating reactive oxygen species (ROS), mitochondrial toxicity, light and fluorescent microscopic morphological evaluations of F-actin and topoisomerase-I relaxation assay. In addition, potential cancer prevention of the plant was also evaluated by assessing the antioxidant activity of the flavonoids compounds isolated from the MLE.

Allanblackia floribunda est un arbre des forêts denses humides tropicales valorisé pour la teneur élevée en acides gras de ses graines, essentiellement constitués d’acides stéarique et oléique dont l’efficacité dans la réduction du mauvais cholestérol de l’organisme humain a été prouvée. Pour cette raison, les graines de A. floribunda collectées en milieu naturel sont commercialisées. Toutefois, les travaux sur la culture de cette espèce sont encore à leur phase initiale. Nous avons déterminé la possibilité d’amélioration génétique de cette espèce en échantillonnant 17 à 40 fruits par arbre de 70 arbres distribués sur quatre sites en milieu naturel. La matière grasse a été extraite des graines, et les teneurs en acides stéarique et oléique estimées à l’aide de méthodes développées au cours de cette étude. La variation phénotypique des traits des fruits et des graines a été caractérisée dans et entre les arbres, et entre les sites. Les estimations de répétabilité des caractères mesurés ont été effectuées. Des corrélations phénotypiques entre les traits étudiés ont aussi été estimées, et quatre traits ont été retenus pour effectuer la sélection multi-caractère de 20 arbres-plus qui constitueront la population d’amélioration de cette espèce pour la production des graines. Nous avons par la suite isolé 10 marqueurs moléculaires de type microsatellite polymorphes à partir de A. floribunda, et sept de ces marqueurs étaient polymorphes à la fois chez Allanblackia gabonensis et Allanblackia stanerana. La variation de huit loci microsatellites a permis de caractériser la structure génétique neutre de 10 populations de A. floribunda de zone de forêt naturelle du Cameroun, puis d’inférer l’histoire récente des forêts humides d’Afrique Centrale. Aucune différence significative n’a été observée entre les paramètres génétiques de la population d’amélioration et celle existant en milieu naturel indiquant qu’une amélioration de cette espèce à partir des 20 arbres sélectionnés ne réduirait pas sa diversité génétique neutre. Toutefois, une légère augmentation du taux de consanguinité a été observée dans la population d’amélioration, et des recommandations sont formulées pour la conservation des ressources génétiques durant l’amélioration de A. floribunda. / Allanblackia floribunda or tallow tree is a tropical forest-tree species that is valued for its seeds, which are rich in hard fat consisting mostly of stearic and oleic acids, reported to lower plasma cholesterol levels, thus reducing the risks of heart attack. Owing to this fat profile, Allanblackia oil is used for margarine production and in soap and ointments manufacture, and seeds extracted from Allanblackia fruits by local communities are traded. We determined whether the species could be genetically improved for fruit/seed production by sampling 17 to 40 fruits from each of 70 trees that were distributed among four sites in wild stands. Fat was extracted from the seeds, and stearic and oleic acid content of the fat was estimated using methods developed in this study. Phenotypic variation in fruit/seed traits was assessed within- and among-trees, and among sites. Repeatabilities were estimated for measured characters, and relationships between these characters investigated. Twenty “plus trees” were selected for breeding, and implications for improvement discussed. Then we isolated and characterized ten microsatellite primer pairs for A. floribunda. Seven of these microsatellite loci were polymorph for both Allanblackia gabonensis and Allanblackia stanerana species as well. Using eight informative microsatellite loci, we have characterized the genetic structure of A. floribunda natural populations from Cameroon, and inferred the recent history of rainforests from Central Africa. No significant difference was identified in genetic parameters between wild stands and the breeding population, indicating that breeding A. floribunda from 20 trees would not reduce nuclear genetic diversity. However, a slight increase in inbreeding was observed in the breeding population, and recommendations for genetic diversity conservation during tree improvement in the species are made.

Chondroitin sulfate proteoglycans (CSPGs) are found in specialized brain extracellular matrix structures termed perineuronal nets (PNNs). The chondroitin sulfate chains of these CSPGs are thought to have a strong effect on neuroplasticity, along with development, injury, and diseased states of the brain. Wisteria floribunda agglutinin (WFA) is a plant lectin used to identify PNN via staining; the pattern of this staining is changed upon schizophrenia. As such, one powerful method of probing the identity of the CS chains of PNNs and addressing what changes in CS identity occur during schizophrenia is to characterize the features of the CS which bind to the lectin. Methods for characterization of WFA-CS binding and their biological relevance were developed and evaluated. Commercially available CS was used to probe the binding affinity of the agglutinin to various regions of CS via hemagglutination inhibition assays and affinity gradient elution of CS bound to WFA. The size, sulfation extent, and fragment location in the CS chain from these eluates were determined using HILIC-LC-MS. As commercial sources can be used to elucidate the binding specificity of WFA, but not the actual relevant binding partner of WFA within the brain, PNN CS extractions were performed with a modified method aimed at reducing the timescale at which PNN CS can be obtained so as to allow similar experimentation on CS directly from PNN. The results pave the way for further determination of WFA-CS binding.

Made available in DSpace on 2015-04-20T12:31:53Z (GMT). No. of bitstreams: 1 Raquel da Silva Correa.pdf: 2133386 bytes, checksum: 50c647396bc7dfa86c0445d6a28b0b4e (MD5) Previous issue date: 2011-05-12 / Fundação de Amparo à Pesquisa do Estado do Amazonas / The pepper is a vegetable of great importance socioeconomic in Brazil. Is among the ten most vegetables grown in country. Like most crops, many arthropods pests are associated with that culture, including some species of mites such as Tetranychus desertorum, reported infesting crops in Manaus. In most cases the mites are controlled by chemicals, which although effective can lead to problems both human health and the environment. In that sense, this work aimed to study the toxicity of extracts of Derris rariflora Benth. and Derris floribunda Benth. on T. desertorum leaf Pepper, aiming to offer products safer, cheaper and with less environmental impact. Plants were collected in natural areas, washed, dried and ground. Were then diluted in water distilled ethanol and acetone at concentrations of 0.5, 1, 10, 20 and 30%, resulting in aqueous extract, ethanol and acetone, these extracts were analyzed by High Performance Liquid Chromatography Efficiency. For bioassays, discs of pepper leaves were treated with extracts by dipping each disk and then received eight female mites. The commercial rotenone (98%) was also tested on the mites in concentrations of 0.1, 0.5 and 1%. It was found that rotenone is not present in aqueous extracts of the species. The extracts acetone and ethanol was found a rotenone content of 4.5 and 4% for D. floribunda and 4,3 e 5% for D. rariflora, not among them. The species D. rariflora was more toxic to the mite when compared to D. floribunda. Among the extracts, the ethanol was the most toxic followed by and aqueous acetone. The concentrations of 20 and 30% caused more mortality rates. The lowest LC50 was observed for the extract ethanolic D. rariflora. Rotenone commercial was not toxic to T. desertorum. However, except the aqueous extract of acetone and D. floribunda, which failed to achieve 50% mortality, the others were promising for control of T. desertorum on pepper leaves / O pimentão é uma hortaliça de grande importância socioeconômica no Brasil. Está entre as dez hortaliças mais cultivadas no país. Assim como a maioria das plantas cultivadas, muitos artrópodes pragas estão associados a essa cultura, entre eles algumas espécies de ácaros, como Tetranychus desertorum, relatado infestando plantios no município de Manaus. Na maioria das vezes os ácaros são controlados por produtos químicos, que apesar de eficientes podem acarretar problemas tanto a saúde humana quanto ao meio ambiente. Nesse sentido, o presente trabalho teve como objetivo estudar a toxicidade dos extratos de Derris rariflora Benth. e Derris floribunda Benth. sobre T. desertorum em folha de pimentão, visando à oferta de produtos mais seguros, menos onerosos e de menor impacto ambiental. As plantas foram coletadas em áreas naturais, lavadas, desidratadas e moídas. Em seguida, foram diluídas em água destilada, álcool etílico e acetona, nas concentrações de 0,5, 1, 10, 20 e 30%, resultando no extrato aquoso, etanólico e acetônico, esses extratos foram analisados por Cromatografia Líquida de Alta Eficiência. Para os bioensaios, discos de folhas de pimentão foram tratados com os extratos por meio de imersão e em seguida cada disco recebeu 8 ácaros fêmeas. A rotenona comercial (98%) também foi testadas sobre os ácaros, nas concentrações de 0,1, 0,5 e 1%. Foi verificado que a rotenona não está presente nos extratos aquosos das espécies estudadas. Nos extratos acetônico e etanólico foi verificado um teor de rotenona de 4,5 e 4% para D. floribunda e 4,3 e 5% para D. rariflora, não diferindo entre si. A espécie D. rariflora foi mais tóxica para o ácaro quando comparada a D. floribunda. Entre os extratos, o etanólico foi o mais tóxico seguido do aquoso e acetônico. As concentrações de 20 e 30% causaram maior percentual de mortalidade. A menor CL50 foi observada para o extrato etanólico de D. rariflora. A rotenona comercial não foi tóxica para T. desertorum. Contudo, com exceção ao extrato aquoso e acetônico de D. floribunda, que não atingiram 50% de mortalidade, os demais foram promissores para o controle de T. desertorum em folhas de pimentão