Avaliação das propriedades farmacológicas de polissacarídeos do fungo Scleroderma nitidum

Several pharmacological properties have been attributed to isolated compounds from mushroom. Recently, have these compounds, especially the polysaccharides derived from mushrooms, modulate the immune system, and its antitumor, antiviral, antibiotic and antiinflammatory activities. This study assesses the possible pharmacological properties of the polysaccharides from Scleroderma nitidum mushroom. The centesimal composition of the tissue showed that this fungus is composed mainly of fibers (35.61%), ash (33.69%) and carbohydrates (25.31%). The chemical analysis of the polysaccharide fraction showed high levels of carbohydrates (94.71%) and low content of protein (5.29%). These polysaccharides are composed of glucose, galactose, mannose and fucose in the following molar ratios 0.156, 0.044, 0.025, 0.066 and the infrared analysis showed a possible polysaccharide-protein complex. The polysaccharides from Scleroderma nitidum showed antioxidant potential with concentration-dependent antioxidant activity compared to ascorbic acid. The analysis scavenging of superoxide radical and inhibition of lipid peroxidation showed that the polysaccharides from S. nitidum have an IC50 of 12.70 mg/ml and EC50 10.4 μg/ml, respectively. The antioxidant activity was confirmed by the presence of reducing potential of these polysaccharides. The effect of these polymers on the inflammatory process was tested using the carrageenan or histamine-induced paw edema model and the sodium thioglycolate or zymosan-induced model. The polysaccharides were effective in reducing edema (73% at 50 mg/kg) and cell infiltrate (37% at 10 mg/kg) in both inflammation models tested. Nitric oxide, a mediator in the inflammatory process, showed a reduction of around 26% at 10 mg/kg of body weight. Analysis of pro- and anti-inflammatory cytokines showed that in the groups treated with polysaccharides from S. nitidum there was an increase in cytokines such as IL-1ra, IL-10, and MIP-1β concomitant with the decrease in INF-γ (75%) and IL-2 (22%). We observed the influence of polysaccharides on the modulation of the expression of nuclear factor κB. Thus, polysaccharides from S. nitidum reduced the expression of NF-κB by up to 64%. The results obtained suggest that NF-κB modulation is one of the possible mechanisms that explain the anti-inflammatory effect of polysaccharides from the fungus S. nitidum.

Novas propriedades do SKTI (Inibidor de tripsina de soja): inibição para elastase neutrofílica humana e efeitos no processo de injúria pulmonar aguda

Seeds from legumes including the Glycine max are known to be a rich source of protease inhibitors. The soybean Kunitz trypsin inhibitor (SKTI) has been well characterised and has been found to exhibit many biological activities. However its effects on inflammatory diseases have not been studied to date. In this study, SKTI was purified from a commercial soy fraction, enriched with this inhibitor, using anion exchange chromatography Resource Q column. The purified protein was able to inhibit human neutrophil elastase (HNE) and bovine trypsin. . Purified SKTI inhibited HNE with an IC50 value of 8 µg (0.3 nM). At this concentration SKTI showed neither cytotoxic nor haemolytic effects on human blood cell populations. SKTI showed no deleterious effects on organs, blood cells or the hepatic enzymes alanine amine transferase (ALT) and aspartate amino transferase (AST) in mice model of acute systemic toxicity. Human neutrophils incubated with SKTI released less HNE than control neutrophils when stimulated with PAF or fMLP (83.1% and 70% respectively). These results showed that SKTI affected both pathways of elastase release by PAF and fMLP stimuli, suggesting that SKTI is an antagonist of PAF/fMLP receptors. In an in vivo mouse model of acute lung injury, induced by LPS from E. coli, SKTI significantly suppressed the inflammatory effects caused by elastase in a dose dependent manner. Histological sections stained by hematoxylin/eosin confirmed this reduction in inflammation process. These results showed that SKTI could be used as a potential pharmacological agent for the therapy of many inflammatory diseases

Caracterização físico-química e efeitos de frações polissacarídicas da alga amansia multifida na coagulação, inflamação, radicais livres e viabilidade celular

Galactans are polysaccharides sulfated present in the cell wall of red algae. Carrageenans are galactans well known in the food industry as gelling polysaccharides and for induce inflammatory process in rodents as animal model. The extraction of polysaccharides from A. multifida has been carried out by proteolysis and precipitation in different volumes of acetone, which produced three fractions (F1, F2, and FT). Chemical and physical analyses revealed that these fractions are sulfated galactan predominantly. Results of the antioxidant activity assays showed that all of these fractions have antioxidant activity and that was associated with sulfate content of the analysis of reducing power and total antioxidant capacity. However, these fractions were not effective against lipid peroxidation. The fraction FT presented higher activity on the APTT test at 200 μg (> 240 s). The assessment of the hemolytic activity showed that the FT fraction has the best activity, increasing lyses by the complement system to 42.3% (50 μg) (p< 0,001). The fraction FT showed the best yield, anticoagulant and hemolytic activity between the three fractions and therefore it was choose for the in vivo studies. The Inflammation assessment using the FT fraction (50 mg / kg MB) showed that the cellular migration and the IL-6 production increased 670.1% (p< 0,001) and 531.8% (p< 0,001), respectively. These results confirmed its use as an inflammation inducer in animal model. Cytotoxicity assay results showed that all fractions have toxic effects on 3T3 and HeLa cells after exposition of 48 hours, except when 100 μg for both F1 and FT were used. These results arise the discussion whether these polysaccharides it should be used as additive in foods, cosmetics and medicines.

Atividades anticoagulante e antioxidante de extratos brutos ricos em polissacarídeos sulfatados das macroalgas marinhas marrons Canistrocarpus cervicornis, Dictyota mertensii e Dictyopteris delicatula e de Heterofucanas de Canistrocarpus cervicornis

In the present study, extracts rich-sulfated polysaccharides were obtained from three different species of Dictyotales (a class of brown macroalgae): Canistrocarpus cervicornis, Dictyota mertensii and Dictyopteris delicatula and their anticoagulant and antioxidant activities were evaluated. All extracts showed anticoagulant activity on aPTT assay, but not on PT assay. Extracts also exhibited total antioxidant activity, superoxide radical scavenging capacity and ferric chelating property. The extract from C. cervicornis showed the best results and was choose to have their sulfated polysaccharides fractioned and subsequently analysed. Thus, six fractions (CC-0.3, CC-0.5, CC-0.7, CC-1.0, CC-1.2 and CC-2.0) were obtained by proteolysis followed by sequential acetone precipitation. Agarose gel eletrophoresis stained with blue toluidine, confirmed the presence of sulfated polysaccharides in all fractions. Chemical analyses showed that all fractions presented heterofucans mainly constitued by fucose, galactose, glucuronic acid and sulfate. Any fraction changed the PT. However, all fractions were able to double the aPTT on a dose-dependent manner. CC- 0.3, CC-0.5, CC-0.7 and CC-1.0 needed only 0.100 mg/mL to double the aPTT, result only 1.25 times higher than the Clexane® (0.080 mg/mL), a commercial low molecular heparin. The heterofucans presented appreciable total antioxidant capacity, low capacity on scavenging hydroxyl radical and good efficiency on scavenging superoxide radicals (except CC-1.0). CC-1.2 showed 43.1 % on superoxide radical scavenging. This result was higher than that showed by the same concentration of gallic acid (41.8 %), a known antioxidant. Furthermore, the heterofucans showed excelent activity on ferrous chelating activity (except CC-0.3). CC-0.5, CC-0.7 and CC-1.0 showed the highest activities with 47.0 % of ferrous chelating activity, a result 2.0 times lesser than that exhibited by the same concentration of EDTA. These results clearly indicated the beneficial effects of heterofucans extracted from C. cervicornis as potential anticoagulant and antioxidant agents. However additional steps of purification, structural studies, besides in vivo experiments are needed for these fucans may be used as therapeutic agents

Propriedades antioxidante, anti-hemostástica e antiproliferativa de galactanas sulfatadas da alga vermelha hypnea musciformis (wulfen) j. V. Lamouroux

Marine algae are one of the major sources of biologic compounds. In extracellular matrix of these organisms there are sulfated polysaccharides that functions as structural components and provides protection against dehydration. The fraction 1.0 (F1.0) rich in sulfated galactans obtained from red seaweed Hypnea musciformis was physicochemical characterized and evaluated for pharmacologic activity through antioxidant activity, cytotoxic action on erythrocytes, anticoagulant, stimulatory action under antithrombotic heparan sulfate synthesis and their effects on cell proliferation and cycle cell progression. The main components of F1.0 were carbohydrates (49.70 ± 0.10%) and sulfate (44.59 ± 0.015%), presenting phenolic compounds (4.79 ± 0.016%) and low protein contamination (0.92 ± 0.001%). Fraction 1.0 showed polidisperse profile and signs in infrared analysis in 1262, 1074 and 930, 900 and 850 attributed to sulfate esters S=O bond, presence of a 3,6- anidrogalactose C-O bond, non-sulfated β-D-galactose and a C-O-SO4 bond in galactose C4, respectively. The fraction rich in sulfated galactans exhibited strong antioxidant action under lipid peroxidation assay with IC50 of 0.003 mg/mL. Besides the inhibition of hemolysis induced by H2O2 in erythrocytes treated with F1.0, this fraction did not promote significant cytotoxity under erythrocytes membranes. F1.0 exhibited low anticoagulant activity causing moderate direct inhibition of enzimatic activity of thrombin. This fraction promoted stimulation around of 4.6 times on this synthesis of heparan sulfate (HS) by rabbit aortic endothelial cells (RAEC) in culture when was compared with non treated cells. The fraction of this algae displayed antiproliferative action under RAEC cells causing incresing on cell number on S fase, blocking the cycle cell progression. Thus F1.0 presented cytostatic and no cytotoxic action under this cell lineage. These results suggest that F1.0 from H. musciformis have antioxidant potential which is a great effect for a compound used as food and in food industry which could be an alternative to food industry to prevent quality decay of lipid containing food due to lipid peroxidation. These polysaccharides prevent the lipid peroxidation once the fraction in study exhibited strong inhibitory action of this process. Furthermore that F1.0 present strong antithrombotic action promoting the stimulation of antithrombotic HS synthesis by endothelial cells, being important for thrombosis preventing, by its inhibitory action under reactive oxygen species (ROS) in some in vitro methods, being involved in promotion of hypercoagulability state.

Lectina da esponja marinha Tedania ignis: purificação, caracterização e interação com leishmanias

Lectin obtained from the marine sponge Tedania ignis was purified and characterized by extraction of soluble proteins (crude extract) in 50mM Borax, pH 7.5. The purification procedure was carried out by crude extract precipitation with ammonium sulfate 30% (FI). The precipitated was resuspended in the same buffer and fractionated with acetone 1.0 volume (F1.0). A lectin was purified from this specific fraction by using an affinity chromatography Sepharose 6B. This lectin preferentially agglutinated human erythrocytes from B type previously treated with papain enzyme. The hemagglutinating activity lectin was dependent of divalent Mn2+ cation and was inhibited by the carbohydrates galactose, xylose and fructose. SDS-PAGE analysis indicated a molecular mass of the lectin around 45 kDa. This protein showed stability until 40°C for 1 h. Further, it showed activity between pH 2.5 and 11.5, with an enhanced activity at pH 7.5. Leishmania chagasi promastigotes stained with Coomassie brilliant blue R-250 were agglutinated by F1,0 and in the presence of galactose this interaction was abolished. These results show that this lectin could be implicated in defense procedures and it will can be used as biological tools in studies with this protozoon

Ação de polissacarídeos sulfatados de Fucus Vesiculosus na Hemostasia e no sistema complemento

Fucans are a family of sulfated homo and teropolysaccharides respectively, composed mainly of a- (1®2) and a- (1®3) linked by L-fucose residues. Properties such as the ability to act as an anti-contraceptive, to reduce cholesterol levels, and to act as an anti-tumor agent are much related. We have focused our attention on the anticoagulant properties, platelet aggregation, hemorrhagic activity and complement system in vitro of commercial fucoidan (F) and their purified fractions (F1, F2 and F3) from Fucus vesiculosus obtained from fractionation of the fucoidan with different concentrations of acetone 1, 2 and 3v. These compounds were chemically characterized and the fucoidan (F) was modified by desulfation. The anticoagulant activity of the compounds was assessment by activated partial thromboplastin time (APTT) and prothrombine time assay (PT) using citrated normal human plasma. The results of APPT test showed that F, F1 and F2 have high anticoagulants activities 240.0 s (5 µg). The F3 showed 73.7 s in the same concentrations. The results obtained with PT test to F, F1, F2 and F3 were 81.5 s, 120.0 s, 57.1 and 32.5 s respectively with 50 µg. The dessulfated polymer showed a decrease in the anticoagulant activity in these two tests. Platelet aggregation assay was measured turbidimetrically with platelet aggregometer by method of Born. The aggregation platelet with F and fractions F1, F2 and F3 exhibited a two-phase answer in the concentration of 5 mg/mL with maximum aggregation of 76.36 ± 10.3% ; 69.54 ± 9.40%; 75.94 ± 9.01%; 51.13 ± 9.59% respectively. However, was observed a hipoaggregate profile F (15.17 ± 5.2%), F1 (7.40 ± 3.04 %), F2 (19.1 ± 5.41%) and F3 (5.09 ± 3.02%) at 0.1 mg/mL. The hemorrhagic activity assay was carried in Wistar rats and showed that these compounds have low hemorrhagic effect when compared to heparin. The complement system ( alternative pathway was made using non-sensibilized rabbit red blood cells The results of complement system essay showed that F , F2 and F3 have action inhibitory in relation to the group control 0.544, 0.697, 0.622 and 0.958 respectively The results showed that these compounds have action on this system. Interaction of the polisaccharides with proteins C3 and C4 showed that the fraction F1 stimulated the activity assay hemolytic using red blood cells

Purificação e caracterização parcial de duas n-acetil-B-hexosaminidases do equinoderma marinho echinometra lucunter

Two b-N-acetylhexosaminidases (F11 e F15) were purified from Echinometra lucunter gonads extracts. The purified enzymes were obtained using ammonium sulfate fractionation, followed by gel filtration chromatographies (Sephacryl S-200, Sephadex G-75 and Sephacryl S-200). The F11 fraction was purified 192.47 -fold with a 28.5% yield, and F15 fraction 85.41 -fold with a 32.3% yield. The molecular weights of the fractions were 116 kDa for F11 and 42 kDa for F15 using SDS-PAGE. In Sephacryl S-200, F15 was 84 kDa, indicating that it is a dimeric protein. When p-nitrophenyl-β-D-glycosaminide was used as substrate, we determined an apparent Km of 0.257 mM and Vmax of 0.704 for F11 and for F15 the Km was 0.235 mM and Vmax of 0.9 mM of product liberated by hour. Both enzymes have optimum pH and temperature respectively at 5.0 and 45 °C. The enzymes showed inhibition by silver nitrate, while the glucuronic acid was a potent activator. The high inhibition of F15 by N-etylmaleimide indicates that sulphydril groups are involved in the catalysis of synthetic substrate

Polissacarídeos sulfatados da Alga Marrom Dictyopteris justii como agentes antioxidantes e inibidores da formação de cristais de oxalato de cálcio

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