Production, purification and characterisation of polysaccharides from pleurotus ostreatus with antitumor activity

Background: Mushroom polysaccharides play an important role in functional foods because they exhibit biological modulator properties such as antitumour, antiviral and antibacterial activities. The present study involved the production, purification and characterisation of intracellular and extracellular free and protein-bound polysaccharides from Pleurotus ostreatus and the investigation of their growth-inhibitory effect on human carcinoma cell lines. Results: Several fermentation parameters were obtained: batch polysaccharide productivities of 0.013 +/- 8.12 x 10-5 and 0.037 +/- 0.0005 g L-1 day-1 for intracellular and extracellular polysaccharides respectively, a maximum biomass concentration of 9.35 +/- 0.18 g L-1, Pmax = 0.935 +/- 0.018 g L-1 day-1, µmax = 0.218 +/- 0.02 day-1, YEP/X = 0.040 +/- 0.0015 g g-1 and YIP/X = 0.014 +/- 0.0003 g g-1. Some polysaccharides exhibited superoxide dismutase (SOD)-like activity of 50-200 units. Fourier transform infrared analysis of the polysaccharides revealed absorption bands characteristic of such biological macromolecules. Cytotoxicity assays showed that both intracellular and extracellular polysaccharides exhibited antitumour activity towards several tested human carcinoma cell lines in a dose-dependent manner. Conclusion - The polysaccharides of P. ostreatus exhibited high SOD-like activity, which strongly supports their biological effect on tumour cell lines. The extracellular polysaccharides presented the highest antitumour activity towards the RL95 carcinoma cell line and should be further investigated as an antitumour agent.

Protein-Polysaccharides of trametes versocolor: production and biological activities

Extracellular-(E-PPS) and intracellular-protein-polysaccharides (I-PPS) complexes were produced by Trametes versicolor in submerged cultures with different carbon sources. The highest extracellular-(EPS) and intracellular-polysaccharide (IPS) concentration in the complexes was obtained with tomato pomace culture. DPPH radical scavenging for E-PPS and I-PPS produced by liter of culture was equivallent to 2.115 +/- A 0.227 and 1.374 +/- A 0.364 g of ascorbic acid, respectively. These complexes showed a protector effect in the oxidation of erythrocyte membranes and had ability to inhibit the hemolysis and methemoglobin synthesis in stressed erythrocytes. These results suggest that extracellular- and intracellular- polysaccharides produced are important bioactive compounds with medicinal potential.

Supercritical carbon dioxide extraction of bioactive compounds from microalgae and volatile oils from aromatic plants

A discussion of the most interesting results obtained in our laboratories, during the supercritical CO(2) extraction of bioactive compounds from microalgae and volatile oils from aromatic plants, was carried out. Concerning the microalgae, the studies on Botryococcus braunii and Chlorella vulgaris were selected. Hydrocarbons from the first microalgae, which are mainly linear alkadienes (C(23)-C(31)) with an odd number of carbon atoms, were selectively extracted at 313 K increasing the pressure up to 30.0 MPa. These hydrocarbons are easily extracted at this pressure, since they are located outside the cellular walls. The extraction of carotenoids, mainly canthaxanthin and astaxanthin, from C. vulgaris is more difficult. The extraction yield of these components at 313 K and 35.0 MPa increased with the degree of crushing of the microalga, since they are not extracellular. On the other hand, for the extraction of volatile oils from aromatic plants, studies on Mentha pulegium and Satureja montana L were chosen. For the first aromatic plant, the composition of the volatile and essential oils was similar, the main components being the pulegone and menthone. However, this volatile oil contained small amounts of waxes, which content decreased with decreasing particle size of the plant matrix. For S. montana L it was also observed that both oils have a similar composition, the main components being carvacrol and thymol. The main difference is the relative amount of thymoquinone, which content can be 15 times higher in volatile oil. This oxygenated monoterpene has important biological activities. Moreover, experimental studies on anticholinesterase activity of supercritical extracts of S. montana were also carried out. The supercritical nonvolatile fraction, which presented the highest content of the protocatechuic, vanilic, chlorogenic and (+)-catechin acids, is the most promising inhibitor of the enzyme butyrylcholinesterase. In contrast, the Soxhlet acetone extract did not affect the activity of this enzyme at the concentrations tested. (C) 2011 Elsevier B.V. All rights reserved.

Synthesis of optimization of [11C] Pittsburgh compound B by the captive solvent method

Mestrado em Medicina Nuclear.

Nutritional status and overhydration: can bioimpedance spectroscopy be useful in haemodialysis patients?

Background: Protein-energy wasting (PEW), associated with inflammation and overhydration, is common in haemodialysis (HD) patients and is associated with high morbidity and mortality. Objective: Assess the relationship between nutritional status, markers of inflammation and body composition through bioimpedance spectroscopy (BIS) in HD patients. Methods: This observational, cross-sectional, single centre study, carried out in an HD centre in Forte da Casa (Portugal), involved 75 patients on an HD programme. In all participating patients, the following laboratory tests were conducted: haemoglobin, albumin, C-reactive protein (CRP) and 25-hydroxyvitamin D3 [25(OH)D3]. The body mass index of all patients was calculated and a modified version of subjective global assessment (SGA) was produced for patients on dialysis. Intracellular water (ICW) and extracellular water (ECW) were measured by BIS (Body Composition Monitor®, Fresenius Medical Care®) after the HD session. In statistical analysis, Spearman’s correlation was used for the univariate analysis and linear regression for the multivariate analysis (SPSS 14.0). A P value of <.05 was considered statistically significant. Results: PEW, inversely assessed through the ICW/body weight (BW) ratio, was positively related to age (P<.001), presence of diabetes (P=.004), BMI (P=.01) and CRP (P=.008) and negatively related to albumin (p=.006) and 25(OH)D3 (P=.007). Overhydration, assessed directly through the ECW/BW ratio, was positively related with CRP (P=.009) and SGA (P=.03), and negatively with 25(OH)D3 (P=.006) and BMI (P=.01). In multivariate analysis, PEW was associated with older age (P<.001), the presence of diabetes (P=.003), lower 25(OH)D3 (P=.008), higher CRP (P=.001) and lower albumin levels (P=.004). Over-hydration was associated with higher CRP (P=.001) and lower levels of 25(OH)D3 (P=.003). Conclusions: Taking these results into account, the ICW/BW and ECW/BW ratios, assessed with BIS, have proven to be good markers of the nutritional and inflammatory status of HD patients. BIS may be a useful tool for regularly assessing the nutritional and hydration status in these patients and may allow nutritional advice to be improved and adjusted.

Molecular mobility, composition and structure analysis in glycerol plasticised chitosan films

This study was developed with the purpose to investigate the effect of polysaccharide/plasticiser concentration on the microstructure and molecular dynamics of polymeric film systems, using transmission electron microscope imaging (TEM) and nuclear magnetic resonance (NMR) techniques. Experiments were carried out in chitosan/glycerol films prepared with solutions of different composition. The films obtained after drying and equilibration were characterised in terms of composition, thickness and water activity. Results show that glycerol quantities used in film forming solutions were responsible for films composition; while polymer/total plasticiser ratio in the solution determined the thickness (and thus structure) of the films. These results were confirmed by TEM. NMR allowed understanding the films molecular rearrangement. Two different behaviours for the two components analysed, water and glycerol were observed: the first is predominantly moving free in the matrix, while glycerol is mainly bounded to the chitosan chain. (C) 2013 Elsevier Ltd. All rights reserved.

Generation of high-affinity monoclonal antibodies of IgG class against native β-d-glucans from basidiomycete mushrooms

β-d-glucans from basidiomycete strains are powerful immunomodulatory agents in several clinical conditions. Therefore, their assay, purification and characterization are of great interest to understand their structure-function relationship. Hybridoma cell fusion was used to raise monoclonal antibodies (Mabs) against extracellular β-d-glucans (EBGs) from Pleurotus ostreatus. Two of the hybridoma clones (1E6-1E8-B5 and 3E8-3B4) secreting Mabs against EBGs were selected. This hybridoma cell line secreted Mabs of the IgG class which were then purified by hydroxyapatite chromatography to apparent homogeneity on native and SDS-PAGE. Mabs secreted by 1E6-1E8-B5 clone were found to recognize a common epitope on several β-d-glucans from different basidiomycete strains. This Mab exhibited high affinity constant (KA) for β-d-glucans from several mushroom strains in the range of 3.20 × 109 ± 3.32 × 103-1.51 × 1013 ± 3.58 × 107 L/mol. Moreover, they reacted to some heat-treated β-d-glucans in a different mode when compared with the native forms; these data suggest that this Mab binds to a conformational epitope on the β-d-glucan molecule. The epitope-binding studies of Mabs obtained from 1E6-1E8-B5 and 3E8-3B4 revealed that the Mabs bind to the same epitope on some β-d-glucans and to different epitopes in other antigen molecules. Therefore, these Mabs can be used to assay for β-d-glucan from basidiomycete mushrooms. © 2015 Elsevier Ltd. All rights reserved.