doctoralThesis
Prospecção de polissacarídeos bioativos da alga Lobophora variegata e elucidação estrutural de uma de suas β-glucanas
Fecha
2016-07-29Registro en:
PAIVA, Almino Afonso de Oliveira. Prospecção de polissacarídeos bioativos da alga Lobophora variegata e elucidação estrutural de uma de suas β-glucanas. 2016. 122f. Tese (Doutorado em Bioquímica) - Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, 2016.
Autor
Paiva, Almino Afonso de Oliveira
Resumen
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 μM, p <0.05), F1-75 (10 μM, p<0.001) and, especially, F1>100 (10 μM, 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 μM) (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 μM) 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 μM, p<0.01). Nuclear magnetic resonance analysis led to the proposal that F1-20 is a β-glucan formed by β-(13) 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.