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Previous issue date: 2016-07-29 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Macroalgas marinhas s?o importantes fontes de polissacar?deos neutros e negativos com importante potencial industrial e farmacol?gico. A macroalga marrom Lobophora variegata ? conhecida por apresentar polissacar?deos sulfatados (heterofucanas) e neutros (laminarinas). Neste trabalho, mostra-se a obten??o de 6 fra??es polissacar?dicas (F0.3; F0.5; F0.8; F1; F1.5 e F2). A partir dos dados de caracteriza??o f?sico-qu?mica das fra??es, escolheu-se a F1 para purifica??o de seus componentes polissacar?dicos. An?lises de cromatografia l?quida de exclus?o molecular levaram a observa??o que F1 apresenta polissacar?dicos de baixa (~ 5 kDa) at? alta (> 100 kDa) massa molecular. Estes componentes foram separados de acordo com sua massa molecular, utilizando-se dispositivos de separa??o por tamanho molecular. Com isso, obteve-se subfra??es polissacar?dicas com tamanhos m?dios de 5, 20, 40, 75 e acima 100 kDa, denominadas de F1-5, F1-20, F1-40. F1-75 e F1>100 kDa, respectivamente. Estas subfra??es foram analisadas f?sico-qu?micamente por eletroforese em gel de agarose, cromatografia l?quida de alta efici?ncia, assim como, por an?lises de suas propriedades antioxidantes, imunoestimulantes e citot?xicas/antiproliferativas. Como resultados, detectou-se um teor de a??cares totais acima de 55% em todas as subfra??es, j? prote?nas e compostos fen?licos n?o foram observados. As fra??es F1-5, F1-20 e F1-40 apresentaram somente glucose (glucanas), j? F1-75 e F1>100 apresentam glucose, galactose e fucose (heterofucanas). Todas as subfra??es apresentaram atividade quelante de metais, mas somente F1-75 e F1>100 foram mais efetivas em sequestrar radicais. A produ??o/libera??o de ?xido n?trico (NO) por c?lulas RAW 264.7 ficou aumentada apenas na presen?a F1-5 (10 ?M, p<0,05), F1-75 (10 ?M, p<0,001) e, com destaque, F1>100 (10 ?M, p<0,001) cuja presen?a aumentou em cerca de 12 vezes a quantidade de NO. Entretanto, quando estas c?lulas foram ativadas com LPS, somente F1-20 e F1>100 (10 ?M) (p<0,001) proporcionaram produ??o/libera??o de ON. As demais fra??es n?o interferiram na a??o do LPS. Somente F1-5 e F1>100 estimularam a produ??o/libera??o de citocinas. Somente F1>100 (10 ?M) foi citot?xica para a linhagem 3T3 (fibroblastos) (p<0,01). Nenhuma das subfra??es apresentaram efeito citot?xico para linhagem de c?lulas RAW 264.7 (macr?fagos) ou para linhagem de c?lulas tumorais Hep-G2 (carcinoma hep?tico). J? com a linhagen de c?lulas 786-0 (adenocarcinoma renal) somente a glucana F1-40 n?o apresentou atividade citot?xica (p>0,05). Os maiores valores de citotoxidade foram obtidos com a subfra??o F1-20, no caso contra c?lulas B16F10 (melanoma). Dados de citometria de fluxo indicaram que esta subfra??o promove uma parada das c?lulas na fase G1 do ciclo celular (10 ?M, p<0,01). An?lises de resson?ncia magn?tica nuclear levaram a proposta de que F1-20 ? uma ?-glucana formada por ?-(1?3) glucoses com ramifica??es, compostas por um res?duo de glicose, na posi??o 6. A propor??o ? de nove res?duos de glucose 1?3 ligados para cada ponto de ramifica??o. Os dados apontam F1-20, por ser antioxidante, imunomoduladora e citot?xico, como um composto pluripotente cujo potencial deva ser melhor investigado. / Seaweeds are important sources of neutral and negative polysaccharides with important industrial and pharmacological potential. The brown macroalgae Lobophora variegata is known for presenting sulfated polysaccharides (heterofucans) and neutral (laminarins). In this work, it is shown getting 6 polysaccharide fractions (F0.3, F0.5, F0.8, F1, F1.5 and F2). From the data of physicochemical characterization of fractions, F1 was chosen for next purification steps. Liquid chromatography of molecular exclusion analysis showed that F1 contain polysaccharide from low (~ 5 kDa) to high (> 100 kDa) molecular weight. These components were separated according to their molecular weight using separation by molecular size devices. Thus, it was obtained subfractions with an average polysaccharide size of 5, 20, 40, 75 and above 100 kDa, named F1-5, F1-20, F1-40. F1-75 and F1>100 kDa, respectively. These subfractions were analyzed physico-chemically by agarose gel electrophoresis, high-performance liquid chromatography, as by analysis of their antioxidant, immunomodulatory and cytotoxic/antiproliferative properties. As result, total sugar content in all subfractions was above 55%, since proteins and phenolic compounds were not observed. The fractions F1-5, F1-20 and F1-40 showed only glucose (glucans), as F1-75 and F1> 100 exhibit glucose, galactose and fucose (heterofucans). All subfractions showed chelating activity of metals, but only F1-75 and F1>100 were more effective as radical scavenger. The production/release of nitric oxide (NO) by RAW cells was increased only in the presence F1-5 (10 ?M, p <0.05), F1-75 (10 ?M, p<0.001) and, especially, F1>100 (10 ?M, p <0.001) whose its presence increased about 12 times the amount of NO. However, when these cells were activated with LPS only F1-20 and F1>100 (10 ?M) (p<0.001) yielded production/release of NO. The other fractions did not interfere in the LPS action. Only F1-5 and F1>100 stimulated the production/release of cytokines. Only F1>100 (10 ?M) was cytotoxic to line 3T3 (fibroblast) (p<0.01). None of subfractions showed cytotoxicity effect to cell line RAW 264.7 (macrophages) or tumor cell line Hep-G2 (hepatocellular carcinoma). On the other hand, only F1-40 did not showed cytotoxic activity (p>0.05) against 786-0 renal carcinoma cells. The highest cytotoxicity values were obtained with the subfraction F1-20, in the case against B16F10 cells (melanoma). Flow cytometry data indicate that this subfraction stopped of cells in the G1 phase of the cell cycle (10 ?M, p<0.01). Nuclear magnetic resonance analysis led to the proposal that F1-20 is a ?-glucan formed by ?-(1?3) glucosides with branches, comprising a glucose residue at position 6. The ratio is nine glucose residues 1,3 connected to each branching point. The data point F1-20, being an antioxidant, immunomodulating and cytotoxic as a compound pluripotent whose potential should be further investigated.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/22054 |
| Date | 29 July 2016 |
| Creators | Paiva, Almino Afonso de Oliveira |
| Contributors | 76111830449, Dore, Celina Maria Pinto Guerra, 03383860418, Albuquerque, Ivan Rui Lopes de, 91237386420, Costa, Leandro Silva, 04647720446, Nobre, Leonardo Thiago Duarte Barreto, 06494949460, Silveira, Raniere Fagundes de Melo, 05241130411, Rocha, Hugo Alexandre de Oliveira |
| Publisher | PROGRAMA DE P?S-GRADUA??O EM BIOQU?MICA, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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