29 resultados para agarose


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The sulfated polysaccharides (SP) from the edible red seaweed Gracilaria birdiae were obtained using five different condition extraction (GB1: Water; GB1p: Water/proteolysis; GB1s: Water/sonication; GB1sp: Water/sonication/proteolysis; GB2s: NaOH/sonication; GB2sp: NaOH/sonication/proteolysis. The yield (g) increased in the following order GB2sp>GB1sp>GB1p>GB2s>GB1s>GB1. However, the amount of SP extracted increased in different way GB2sp>GB1p>GB1>GB1sp>GB1s>GB2s. Infrared and electrophoresis analysis showed that all conditions extracted the same SP. In addition, monosaccharide composition showed that ultrasound promotes the extraction of other polysaccharides than SP. In the prothrombin time (PT) test, which evaluates the extrinsic coagulation pathway, none of the samples showed anticoagulant activity. While in the activated partial thromboplastin time (aPTT) test, which evaluates the intrinsic coagulation pathway, all samples showed anticoagulant activity, except GB2s. The aPTT activity decreased in the order of GB1sp>GB2sp>GB1p>GB1>GB1s>GB2s. Total capacity antioxidant (TCA) of the SP was also affected by condition extraction, since GB2s and GB1 showed lower activity in comparison to the other conditions. In conclusion, the conditions of SP extraction influence their biological activities and chemical composition. The data showed NaOH/sonication/proteolysis was the best condition to extract anticoagulant and antioxidant SPs from Gracilaria birdiae.

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Chondroitin sulfate (CS) is a naturally glycosaminoglycan found in the extracellular matrix of connective tissues and it may be extracted and purified those tissues. CS is involved in various biological functions, which may be related to the having structural variability, despite the simplicity of the linear chain structure from this molecule. Researches in biotechnology and pharmaceutical field with wastes from aquaculture has been developed in Brazil. In recent decades, tilapia (Oreochromis niloticus), native fish from Africa, has been one of the most cultivated species in various regions of the world, including Brazil. The tilapia farming is a cost-effective activity, however, it generates large amount of wastes that are discarded by producers. It is understood that waste from tilapia can be used in research as a source of molecules with important biotechnological applications, which also helps in reducing environmental impacts and promote the development of an ecofriendly activity. Thus, nile tilapia viscera were subjected to proteolysis, then the glycosaminoglycans were complexed with ion exchange resin (Lewatit), it was fractionated with increasing volumes of acetone and purified by ion exchange chromatography DEAE-Sephacel. Further, the fraction was analyzed by agarose gel electrophoresis and nuclear magnetic resonance (NMR). The electrophoretic profile of the compound together the analysis of 1H NMR spectra and the HSQC correlation allow to affirm that the compound corresponds to a molecule like chondroitin sulfate. MTT assay was used to assess cell viability in the presence of CS tilapia isolated and showed that the compound is not cytotoxic to normal cells such as cells from the mouse embryo fibroblast (3T3). Then, this compound was tested for the ability to reduce the influx of leukocytes in model of acute peritonitis (in vivo) induced by sodium thioglycolate. In this context, it was done total and differential leukocytes counting in the blood and peritoneal fluid collected respectively from vena cava and the peritoneal cavity of the animals subjected to the experiment. The chondroitin sulfate for the first time isolated from tilapia (CST ) was able to reduce the migration of leukocytes to the peritoneal cavity of inflamed mice until 80.4 per cent at a dose 10µg/kg. The results also show that there was a significant reduction (p<0.001) of the population of polymorphonuclear leukocytes from peritoneal cavity in the three tested doses (0.1µg/kg; 1µg/kg and 10µg/kg) when it was compared to the positive control (just thioglycolate). Therefore, since the CST structure and mechanism of action has been completely elucidated, this compound may have potential for therapeutic use in inflammatory diseases

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Since the first description of sulfated polysaccharides from seaweeds, the biological activities of these compounds have been evaluated under different aspects and experimental procedures. Among the broad biological activities presented by seaweed polysaccharides, anticoagulant action appears as a promising function. In this present study we have obtained sulfated polysaccharides from the green seaweed Codium isthmocladium by proteolytic digestion, followed by separation into five fractions (0.3, 0.5, 0.7, 0.9 and 1.2) by sequential acetone precipitation. The chemical analyses have demonstrated that all fractions are composed mainly by sulfated polysaccharides. The anticoagulant activity of these fractions was determined by activated partial thromboplastin time (aPTT) and prothrombin time test (PT) using citrate normal human plasma. None fraction has shown anticoagulant activity by PT test. Furthermore, all of them have shown anticoagulant activity by aPTT test. These results indicated that the molecular targets of these sulfated polysaccharides are mainly in the intrinsic via of the coagulation cascade. Agarose gel electrophoresis in 1,3-diaminopropane acetate buffer, pH 9.0, stained with 0.1% toluidine blue showed the presence of two or three bands in several fractions while the fraction 0.9 showed a single spot. By anion exchange chromatography, the acid polysaccharides from the 0.9 acetone fraction were separated into two new fractions eluted respectively with 2.0 and 3.0 M NaCl. These compounds showed a molecular weight of 6.4 and 7.4 kDa respectively. Chemical analyses and infrared spectroscopy showed that Gal 1 and Gal 2 are sulfated homogalactans and differ one from the other in degree and localization of sulfate groups. aPPT test demonstrated that fractions 2,0 and 3,0M (Gal1 and Gal 2, respectively) have anticoagulant activity. This is the first time that anticoagulant sulfated homogalatans have been isolated from green algae. To prolong the coagulation time to double the baseline value in the aPTT, the required amount of sulfated galactan 1 (6,3mg) was similar to low molecular heparin Clexane®, whereas only 0,7mg of sulfated galactan 2 was needed to obtain the same effect. Sulfated galactan 2 in high doses (250mg) induces platelet aggregation. These results suggest that these galactans from C. isthmocladum have a potential application as an anticoagulant drug

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Four different sponge species were screened using Ouchterlony agarose gel and immunodiffusion tests to identify cross-reactivity with the polyclonal antibody IgG anti-deglicosilated CvL, a lectin from Cliona varians. Crude extract from the sponge Cinachyrella apion showed cross-reactivity and also a strong haemmaglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglicosilated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA) gel filtration on a Superose 6 10 300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A and O erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide D-lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass determined by FPLC-AKTA gel filtration on a Superose 12 10 300 column and SDS gel electrophoresis was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was relatively heat- and pH-stable. Leishmania chagasi romastigotes were agglutinated by CaL, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the physiological defense roles of CaL and its possible use in the antibiosis of pathogenic protozoa

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Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

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In recent years, sulfated polysaccharides (SP) from marine algae have emerged as an important class of natural biopolymers with potential pharmacology applications. Among these, SP isolated from the cell walls of red algae have been study due to their anticoagulant,antithrombotic and anti-inflammatory activities. In the present study, three sulfated polysaccharides fractions denominated F1.5v, F2.0v and F3.0v were obtained from seaweed G. caudate by proteolysis followed to acetone fractionation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9,0, stained with 0.1% toluidine blue, showed the presence of SP in all fractions. The chemical analysis demonstrated that all the fractions are composed mainly of galactose. These compounds were evaluated in anticoagulant, antioxidant and antiproliferative activities. In anticoagulant activity evaluated through aPTT and PT tests, no one fractions presented anticoagulant activity at tested concentrations (0.1 mg/mL; 1.0 mg/mL; 2.0 mg/mL).The antioxidant activities of the three fractions were evaluated by the following in vitro systems: Total antioxidant capacity, superoxide and hydroxyl radical scavenging, ferrous chelating activity and reducing power. The fractions were found to have different levels of antioxidant activity in the systems tested. F1.5v shows the highest activity, especially in the ferrous chelating system, with 70% of ferrous inhibiting at 1.0 mg.mL-1. Finally, all the fractions showed dose-dependent antiproliferative activity against HeLa cells. The fractions F1.5v and F2.0v presented the highest antiproliferative activity at 2.0 mg/mL with 42.7% and 37.0% of inhibition, respectively. Ours results suggests that the sulfated polysaccharides from seaweed G. caudata are promising compounds in antioxidant and/or antitumor therapy

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Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

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In recent years, sulfated polysaccharides from marine algae have emerged as an important class of natural biopolymers with potential application in human and veterinary health care, while taking advantage of the absence of potential risk of contamination by animal viruses. Among these, fucans isolated from the cell walls of marine brown alga have been study due to their anticoagulant, antithrombotic, anti-inflammatory and antiviral activities. These biological effects of fucans have been found to depend on the degree of sulfation and molecular size of the polysaccharide chains. In the present study, we examined structural features of a fucan extracted from brown alga Dictyota menstrualis and its effect on the leukocyte migration to the peritoneum. The sulfated polysaccharides were extracted from the brown seaweed by proteolytic digestion, followed by sequential acetone precipitation producing 5 fractions. Gel lectrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9.0, stained with 0.1% toluidine blue, showed the presence of sulfated polysaccharides in all fractions. The chemical analyses demonstrated that all fractions are composed mainly of fucose, xylose, galactose, uronic acid, and sulfate. Electrophoresis in agarose gel in three different buffers demonstrated that the fraction 2.0v have only one population of fucan. This compound was purify by exclusion molecular. It has shown composition of fucose, xilose, sulfate and uronic acid in molar ration of 1.0: 1.7: 1.1: 0.5 respectively. The effect of this heterofucan on the leukocyte migration was observed 6h after zymozan (mg/g) administration into the peritoneum. The heterofucan showed higher antimigratory activity, it decrease the migration of leukocyte in 83.77% to peritoneum. The results suggest that this fucan is a new antimigratory compound with potential pharmacological appications

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The exopolysaccharides are extracellular compounds produced by some species of fungi and bacteria. It is suggested that these molecules, even when in the form of complex polysaccharide-peptide, are the main bioactive molecules of many fungus. Some of the biological activities displayed by these compounds can be accentuated and others may arise when you add chemically polar or nonpolar groups to polysaccharides. The fruiting body of Pleurotus sajor-caju produces a heteropolysaccharide with antineoplastic and antimicrobial activity, but other biological activities of this polymer have not been evaluated. In this work the exopolysaccharide of Pleurotus sajor-caju was sulfated chemically and structurally characterized. We also evaluated the antiproliferative, antioxidant and anticoagulant activities from native exopolysaccharide (PN) and its sulfated derivated (PS). Polyacrylamide gel electrophoresis, infrared spectroscopy and nuclear magnetic resonance (¹³C) proved successful in sulfation of PN to obtain PS. Analysis by gas chromatography-mass spectroscopy showed that PN and PS are composed of mannose, galactose, 3-O-methyl-galactose and glucose in proportion percentage of 44,9:16,3:19,8:19 and 49, 7:14,4:17,7:18,2, respectively. The percentage of sulfate found in PS was 22.5%. Antioxidants assays revealed that the sulfation procedure affects differently the activities of exopolysaccharides, while the total antioxidant capacity, the scavenging activity of superoxide radical and ferric chelating were not affected by sulfation, on the other hand the chemical modification of PN enhanced the scavenging activity of hydroxyl radical and reducing power. PS also showed anticoagulant activity in a dose-dependent manner and clotting time was 3.0 times higher than the baseline value in APTT at 2 mg/mL. The exopolysaccharide not presented antiproliferative activity against HeLa tumor cells, but PS affects the cellular proliferation in a time-dependent manner. After 72 h, the inhibition rate of PS (2.0 mg/mL) on HeLa cells was about 60%. The results showed that PN sulfation increase some of their activities.

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The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (¹H and ¹³C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 μg of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods, pharmaceuticals and chemical industries.

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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

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Fucan is a term used to denominate L-fucose rich sulfated polysaccharides. The fucans have been studied due their pharmacological activities like antithrombotic, antiproliferative and antioxidant. We have extracted three fucan fractions from the brown seaweed Spatoglossum schröederi. These fucans were denominated Fuc B 1, Fuc B 1.5 and Fuc B 2. The chemical analyzes show that the fucans have very similar composition as demonstrated by agarose electrophoresis gel, sugar and sulfate content. The antiproliferative effect was determined by MTT and BrdU methodologies in CHO cells. The inhibition of proliferation effect of the three fractions was about 40%. Therefore this we proceed just with the Fuc B 2 due the higher yield. There is no apoptosis indication using the anexin V/propidium iodide test. We found a cell cycle phase G1 arrest. The western blotting show that the PKC; pFAK; pERK 1/2 are activated when the cells were treated with fucans. The treatement with inhibitor of MAPK PD98059 extinguished the fucan effect. These results indicates that fucan act by the ERK pathway inducing the cell death.

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Heparin is a pharmaceutical animal widely used in medicine due to its potent anticoagulant effect. Furthermore, it has the ability to inhibit the proliferation, invasion and adhesion of cancer cells to vascular endothelium. However, its clinical applicability can be compromised by side effects such as bleeding. Thus, the search for natural compounds with low bleeding risk and possible therapeutic applicability has been targeted by several research groups. From this perspective, this study aims to evaluate the hemorrhagic and anticoagulant activities and citotoxic effect for different tumor cell lines (HeLa, B16-F10, HepG2, HS-5,) and fibroblast cells (3T3) of the Heparin-like from the crab Chaceon fenneri (HEP-like). The HEP-like was purified after proteolysis, ion-exchange chromatography, fractionation with acetone and characterized by electrophoresis (agarose gel) and enzymatic degradation. Hep-like showed eletroforetic behavior similar to mammalian heparin, and high trisulfated /Nacetylated disaccharides ratio. In addition, HEP-like presented low in vitro anticoagulant activity using aPTT and a minor hemorrhagic effect when compared to mammalian heparin. Furthermore, the HEP-like showed significant cytotoxic effect (p<0.001) on HeLa, HepG2 and B16-F10 tumor cells with IC50 values of 1000 ug/mL, after incubation for 72 hours. To assess the influence of heparin-like on the cell cycle in HeLa cells, analysis was performed by flow cytometry. The results of this analysis showed that HEP-like influence on the cell cycle increasing S phase and decreasing phase G2. Thus, these properties of HEP-like make these compounds potential therapeutic agents

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Rheumatoid arthritis (RA) is systemic auto imune disorder. It is caracterized by chronic inflammation of joints leading to progressive erosion of cartilage and bone. We investigated the effect of the administration of fucoidan, sulfated polysaccharides, from algae Fucus vesiculosus in the acute (6h) in zymosan-induced arthritis (AZy). Wistar rats (180-230 g) were used for all groups experimental. Non-treated animals received just intraarticular injection of 1 mg the zymosan, control group received intraarticular injection of 50 µL the saline, groups received either fucoidan of Fucus vesiculosus (15, 30, 50 or 70 mg/Kg) or parecoxib (1 mg/Kg) 1 hour after injection of zymosan. After 6 h, the articular exudates were collected for evaluation of the cell influx and nitrite (Griess reaction) release. The sinovial membranes and articular cartilages were excised for histopathological analysis and by determination of the glycosaminoglycan (GAG), respectively. ZyA led to increased NO and cell influx into the joints. Therapeutic administration of the fucoidan or parecoxib did significantly inhibited the cell influx and the synovitis, as compared to non-treated rats (p<0,05), though being able to reduced NO release. Representative agarose gel electrophoresis of the GAGs, the content of condroitin-sulphate was observed during the process. These findings suggest that the fucoidan from Fucus vesiculosus has potential anti-inflammatory activity