Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-09-22T14:51:20Z No. of bitstreams: 1 DISS_2014_Ruberlei Godinho de Oliveira.pdf: 1976037 bytes, checksum: 1bc4af52537ae6c49276a4a8287308cc (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-09-26T14:23:02Z (GMT) No. of bitstreams: 1 DISS_2014_Ruberlei Godinho de Oliveira.pdf: 1976037 bytes, checksum: 1bc4af52537ae6c49276a4a8287308cc (MD5) / Made available in DSpace on 2017-09-26T14:23:02Z (GMT). No. of bitstreams: 1 DISS_2014_Ruberlei Godinho de Oliveira.pdf: 1976037 bytes, checksum: 1bc4af52537ae6c49276a4a8287308cc (MD5) Previous issue date: 2014-02-27 / CAPES / Dilodendron bipinnatum Radlk. (Sapindaceae), conhecida popularmente como “mulher-pobre”, é uma árvore nativa do Pantanal de Mato Grosso, Brasil. A entrecasca do caule de Dilodendron bipinnatum é utilizada pela população, nas formas de decocto e macerado, para o tratamento de inflamações. Não há estudos na literatura que comprovem a atividade anti-inflamatória desta planta e o seu respectivo mecanismo de ação. Esse trabalho tem como objetivo avaliar a atividade e o mecanismo de ação anti-inflamatório do extrato hidroetanólico da entrecasca de Dilodendron bipinnatum (EHDb) em modelos experimentais in vivo e in vitro. A entrecasca foi macerada em solução hidroetanólica 70% (1:3, p/v) durante 7 dias, filtrada, concentrada em evaporador rotativo e o solvente residual eliminado em estufa a 40ºC, obtendo-se assim o EHDb. Os taninos condensados e hidrolisáveis foram identificados e quantificados por cromatografia líquida de alta eficiência (CLAE). A citotoxicidade do EHDb em macrófagos murinos (células RAW 264.7) foi avaliada pelo método de azul de Alamar. A atividade anti-inflamatória in vivo do EHDb (20, 100 e 500 mg/kg v.o.) foi avaliada no modelo de peritonite induzida por lipopolissacarídeo (LPS), em camundongos Swiss cujo lavado peritoneal foi recolhido para determinações de TNF-α, IL-1β e IL-10, usando-se kits comerciais para ELISA. A atividade anti-inflamatória in vitro (1, 5 e 20 μg/mL) foi avaliada em células RAW 264.7 estimuladas com LPS e/ou INF-γ, coletando-se o sobrenadante para determinações das mesmas citocinas observadas in vivo, assim como de óxido nítrico pelo método de Griess. O efeito do EHDb sobre a expressão gênica de MAPK p38, ERK 1/2 e COX-2 foi avaliado em ensaio de western blotting em células RAW 264.7 estimuladas com LPS. O EHDb não alterou a viabilidade em células RAW 264.7, apresentando uma concentração inibitória mínima (CI50%) > 200 ± 0,38 μg/mL. No modelo de peritonite induzida por LPS, o EHDb reduziu a migração de leucócitos totais e neutrófilos, em todas as doses, atingindo o maior efeito (45% e 64%, p<0,001, respectivamente) com 20 mg/kg, em ambos os casos. A ação anti-inflamatória foi devida, pelo menos em parte, pela inibição da migração, citocinas do tipo Th1, expressão de MAPK p38 e COX-2, e estimulação de citocinas do tipo Th2, sem afetar a via do NO. Pode-se sugerir que os taninos respondem, pelo menos em parte, pela atividade anti-inflamatória do EHDb. / Dilodendron bipinnatum Radlk. (Sapindaceae), popularly known as “mulher-pobre”, is a native tree of the Pantanal of Mato Grosso, Brazil. The inner stem bark of Dilodendron bipinnatum is used by the population, in the forms of decoction and maceration in the treatment of inflammatory conditions. There is no information in the literature demonstrating the anti-inflammatory activity of Dilodendron bipinnatum and its respective mechanism of action. This study aimed to evaluate the anti-inflammatory activity and mechanism of action of the hydroethanolic extract of the stem bark of Dilodendron bipinnatum (HEDb) using in vivo and in vitro experimental models. The stem bark of Dilodendron bipinnatum was macerated in 70% hydroethanolic solution (1:3, w/v) for 7 days, filtered, concentrated on a rotary evaporator and the residual solvent removed in oven at 40 ° C, thus obtaining HEDb. The effect of HEDb on cell viability in murine macrophages (RAW 264.7 cells) was assessed by the Alamar blue assay. The in vivo anti-inflammatory activity and mechanism of action of HEDb (20, 100 and 500 mg/kg) was evaluated in lipopolysaccharide (LPS) -induced peritonitis model in Swiss mice. The peritoneal lavage was collected for the determination of TNF-α, IL-1β and IL-10, using commercial ELISA kits. The in vitro anti-inflammatory studies activity of HEDb (1, 5 and 20 μg/ml) was determined in RAW 264.7 cells stimulated with LPS and/or INF-γ. The supernatant of the stimulated cells was used for the determination of the aforementioned cytokines as well as oxide nitric (NO) by the Griess method. The effect of HEDb on the gene expression of p38 and ERK 1/2 MAPKs and COX-2 was assessed using Western blot assay in RAW 264.7 cells stimulated with LPS. Preliminary phytochemical analysis of HEDb was analyzed by high performance liquid chromatography (HPLC). HEDb showed very low cytotoxicity with IC50>200 ± 0.38 μg/mL. In LPS-induced peritonitis model, HEDb reduced total leukocytes and neutrophils migration, at all doses, reaching maximum effect (45% and 64%, p<0.001 respectively) at 20 mg/kg in both cases. HEDb also attenuated increase in the concentrations of the pro-inflammatory cytokines (IL-1β and TNF-α) and increased level of anti-inflammatory cytokine IL-10 in peritonitis. HEDb had no demonstrable effect on the increased concentrations of TNF-α, IL-10 and NO in activated RAW 264.7 cells. HEDb caused in vitro inhibition of gene expression of MAPK p38 and COX-2. HPLC analyses identified some condensed and hydrolysable tannins, with epigallocatechin gallate being the major compound. HEDb presented a novel multitargeted anti-inflammatory action and mechanism, and low in vitro cytotoxicity. Its anti-inflammatory action was due, at least in part, to the inhibition of cell migration, Th1 type cytokines, expressions of p38 MAPK and COX-2, and to the stimulation of Th2 type cytokine, without affecting NO pathway. It can be suggested that tannins account at least in part to the anti-inflammatory activity of HEDb.

<div>Grand Sable Dunes, as a perched dune field on the shore of Lake Superior, is a sensitive</div><div>ecosystem subject to continual disturbance. Repeated natural disturbances necessitate specialized</div><div>plant communities to develop. There were two objectives of my research in this system that are</div><div>treated in separate chapters. They include: 1) the quantification of successional changes in the</div><div>plant community over time, and the identification of population demography changes for rare</div><div>species within the dunes and 2) the evaluation of evaluate in pollinator species for two plants</div><div>Hieracium caespitosum (Yellow Hawkweed) and Lithospermum caroliniense (Carolina</div><div>Puccoon).</div><div>For the first objective, target plant community composition and structures (i.e. richness,</div><div>diversity) were quantified in 2011 and 2018 across Grand Sable Dunes in 1 m² quadrats.</div><div>Additionally, two relatively rare plant species (Cirsium pitcheri and Tanacetum bipinnatum)</div><div>were selected to quantify demographic (i.e. flowering, non-flowering) patterns and changes over</div><div>time. Samples for C. pitcheri and T. bipinnatum were acquired via circle-plots 2.5 m in diameter.</div><div>Population comparisons between 2011 and 2018 illustrate minimal change in community</div><div>structure (richness and diversity). Composition increased slightly with eight species occurring in</div><div>2018, but not 2011. Additionally, community similarity was high (~78%) between the two years.</div><div>C. pitcheri occurrence was inversely related to presence of other species. Plant community</div><div>composition in eastern and western survey zones within the dunes appear to be diverging. Minor</div><div>changes in the plant community composition and structures indicate successional changes have</div><div>occurred, but without major disturbance. This divergence in community composition may be</div><div>related to weather related incidents associated with Lake Superior disturbance potential.</div><div>The secondary objective concerns pollinator species on two similar plant species found in</div><div>the Grand Sable Dunes. Individuals of H. caespitosum and L. caroliniense were observed and all</div><div>floral visitors were identified to family. The majority of arthropod families were observed</div><div>10</div><div>visiting both H. caespitosum and L. caroliniense, with an absence of typically important families</div><div>(e.g. Apidae, Bombiliidae). Halictidae, Muscidae and Syrphidae were the most common visitors,</div><div>with L. caroliniense attracting far more Muscidae than their H. caespitosum competitors.</div><div>Overlap in visitors for both species was observed, which may lead to decreased reproduction in</div><div>L. caroliniense.</div>