Effect of glucans from Caripia montagnei mushroom on TNBS-induced colitis.

In this study, we evaluated the effect of different doses of polysaccharides extracted from Caripia montagnei mushroom at different intervals of treatment on colonic injury in the model of colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The FT-IR analysis and NMR showed that the polysaccharides from this species of mushroom are composed of α- and ß-glucans. The colonic damage was evaluated by macroscopic, histological, biochemical and immunologic analyses. The results showed the reduction of colonic lesions in all groups treated with the glucans. Such glucans significantly reduced the levels of IL-6 (50 and 75 mg/kg, p < 0.05), a major inflammatory cytokine. Biochemical analyses showed that the glucans from C. montagnei acted on reducing levels of alkaline phosphatase (75 mg/kg, p < 0.01) and myeloperoxidase (p < 0.001), a result confirmed by the reduction of cellular infiltration observed microscopically. The increase of catalase activity possibly indicates a protective effect of these glucans on colonic tissue, confirming their anti-inflammatory potential.

Antioxidant and Anti-Inflammatory Properties of an Extract Rich in Polysaccharides of the Mushroom Polyporus dermoporus.

Polyporus dermoporus mushroom, native to Brazil, is produced under natural conditions in the unexplored reserve of Mata da Estrela-Rio Grande do Norte-RN. These mushrooms were delipidated with chloroform:methanol (2:1 v/v), extracted with water at 100 °C, and fractionated with ethanol (one and three volumes) and then centrifuged. The ethanol precipitation showed a high total sugar level of 64.8% and 1% of protein. This precipitate contained a high glucan level, characterized by chemical methods and by NMR of (13)C and ¹H and spectroscopy. The (13)C NMR spectrum of these mushroom extracts showed the presence of ß-glucose by a signal at 103.25 ppm. Studies with these glucans were made to elucidate antioxidant and anti-inflammatory activities. This extract of glucans inhibited the lipid peroxidation (42.9%) and superoxide radicals (83.3%) at 67 µg/mL. However, the inhibition of hydroxyl radical by the extract of this mushroom was 96% at 267 µg/mL. The action of this extract on induced pleurisy showed a 92.5% and 68.7% reduction in polymorphonuclears cells and nitric oxide, respectively, at 30 mg/kg. The glucans reduced the croton oil-induced ear edema by 65.6% at 30 mg/kg.

Evaluation of anti-nociceptive and anti-inflammatory activities of a heterofucan from Dictyota menstrualis.

Fucan is a term that defines a family of homo- and hetero-polysaccharides containing sulfated l-fucose in its structure. In this work, a heterofucan (F2.0v) from the seaweed, Dictyota menstrualis, was evaluated as an antinociceptive and anti-inflammatory agent. F2.0v (20.0 mg/kg) inhibits 100% of leukocyte migration into the peritoneal cavity after chemical stimulation. However, F2.0v does not alter the expression of interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6), as well as tumor necrosis factor alpha (TNF-α). F2.0v (20.0 mg/kg) has peripheral antinociceptive activity with potency similar to dipyrone. On the other hand, it had no effect on pain response on the hot plate test. Confocal microscopy analysis and flow cytometry showed that F2.0v binds to the surface of leucocytes, which leads us to suggest that the mechanism of action of anti-inflammatory and antinociceptive F2.0v is related to its ability to inhibit the migration of leukocytes to the site of tissue injury. In summary, the data show that F2.0v compound has great potential as an antinociceptive and anti-inflammatory, and future studies will be performed to further characterize the mechanism of action of F2.0v.

Antiangiogenic activity and direct antitumor effect from a sulfated polysaccharide isolated from seaweed.

Angiogenesis is a dynamic proliferation and differentiation process. It requires endothelial proliferation, migration and tube formation. In this context, endothelial cells are a preferred target for several studies and therapies. Anionic polysaccharides (SV1 and PSV1) from brown seaweed Sargassum vulgare were fractionated (SV1), purified (PSV1) and displayed with high total sugars, sulfate content and very low level of protein. The antiangiogenic efficacy of polysaccharides was examined in vivo in the chick chorioallantoic membrane (CAM) model by using fertilized eggs. Decreases in the density of the capillaries were assessed and scored. The results showed that SV1 and PSV1 have an inhibitory effect on angiogenesis. These results were also confirmed by the inhibition of tubulogenesis in rabbit aorta endothelial cell (RAEC) in matrigel. These compounds were assessed in an apoptosis assay (Annexin V-FITC/PI) and cell viability by MTT assay of RAEC. These polysaccharides did not affect the viability and did not have apoptotic or necrotic action. RAEC cell when incubated with SV1and PSV1 showed inhibition of VEGF secretion, observed when compounds were incubated at 25, 50 and 100 µg/µL. The VEGF secretion with the RAEC cell line for 24 h was more effective for PSV1 at 50 µg/µL (71.4%) than for SV1 at 100 µg/µL (75.9%). SV1 and PSV1 had an antiproliferative action (47%) against tumor cell line HeLa. Our results indicate that these sulfated polysaccharides have antiangiogenic and antitumor actions.

A sulfated polysaccharide, fucans, isolated from brown algae Sargassum vulgare with anticoagulant, antithrombotic, antioxidant and anti-inflammatory effects.

Fucan (SV1) sulfated polysaccharides from the brown algae Sargassum vulgare were extracted, fractionated in acetone and examined with respect to chemical composition, anticoagulant, anti-inflammatory, antithrombotic effects and cellular proliferation. These polysaccharides contain low levels of protein, high level of carbohydrate and sulfate. Monosaccharides analysis revealed that SV1 was composed of fucose, galactose, xylose, glucuronic acid and mannose. SV1 polysaccharide prolonged activated partial thromboplastin time (aPTT) and exhibited high antithrombotic action in vivo, with a concentration ten times higher than heparin activity. PSV1, a purified form in gel filtration showed very low biological activities. SV1 stimulated the enzymatic activity of FXa. Its action on DPPH radical scavenging activity was 22%. This polymer has no cytotoxic action (hemolytic) on ABO and Rh blood types in different erythrocyte groups. It displays strong anti-inflammatory action at all concentrations tested in the carrageenan-induced paw edema model, demonstrated by reduced edema and cellular infiltration.

Freshwater plants synthesize sulfated polysaccharides: heterogalactans from Water Hyacinth (Eicchornia crassipes).

Sulfated polysaccharides (SP) are found mainly in seaweeds and animals. To date, they have only been found in six plants and all inhabit saline environments. Furthermore, there are no reports of SP in freshwater or terrestrial plants. As such, this study investigated the presence of SP in freshwaters Eichhornia crassipes, Egeria densa, Egeria naja, Cabomba caroliniana, Hydrocotyle bonariensis and Nymphaea ampla. Chemical analysis identified sulfate in N. ampla, H. bonariensis and, more specifically, E. crassipes. In addition, chemical analysis, FT-IR spectroscopy, histological analysis, scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDXA), as well as agarose gel electrophoresis detected SP in all parts of E. crassipes, primarily in the root (epidermis and vascular bundle). Galactose, glucose and arabinose are the main monosaccharides found in the sulfated polysaccharides from E. crassipes. In activated partial thromboplastin time (APTT) test, to evaluate the intrinsic coagulation pathway, SP from the root and rhizome prolonged the coagulation time to double the baseline value, with 0.1 mg/mL and 0.15 mg/mL, respectively. However, SP from the leaf and petiole showed no anticoagulant activity. Eichornia SP demonstrated promising anticoagulant potential and have been selected for further studies on bioguided fractionation; isolation and characterization of pure polysaccharides from this species. Additionally in vivo experiments are needed and are already underway.

Sulfated fucans extracted from algae Padina gymnospora have anti-inflammatory effect

Sulfated polysaccharides were extracted with acetone from brown algae Padina gymnospora. The fraction precipitated with 1.5 volumes of acetone (F1.5) purified in Sephadex G-75 was characterized by infrared and nuclear magnetic resonance of 13C and ¹H, through which the presence of sulfate groups on the C4 of α-L-fucose could be observed. This polysaccharide showed that an MW of 25,000 Da was effective in reducing leukocyte influx into the peritoneal cavity in mice at 10 mg/kg and 25 mg/kg body weight, causing a decrease of 60 and 39 percent, respectively. In the present study, it was observed that this fucan has anti-inflammatory properties but no cytotoxic action, indicating its potential use in the pharmaceutical industry.

Polysaccharides from the fungus Scleroderma nitidum with anti-inflammatory potential modulate cytokine levels and the expression of Nuclear Factor kB

Several pharmacological properties are attributed to polysaccharides and glucans derived from fungi such as tumor, anti-inflammatory, and immunomodulatory activity. In this work, the anti-inflammatory potential of polysaccharides from the fungus Scleroderma nitidum and their possible action mechanism were studied. The effect of these polymers on the inflammatory process was tested using the carrageenan and histamine-induced paw edema model and the sodium thioglycolate and zymosan-induced model. The polysaccharides from S. nitidum were effective in reducing edema (73 percent at 50 mg/kg) and cell infiltrate (37 percent at 10 mg/kg) in both inflammation models tested. Nitric oxide, a mediator in the inflammatory process, showed a reduction of around 26 percent 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 percent) and IL-2 (22 percent). We observed the influence of polysaccharides on the modulation of the expression of nuclear factor κB. This compound reduced the expression of NF-κB by up to 64 percent. The results obtained suggest that NF-κB modulation an mechanisms that explain the anti-inflammatory effect of polysaccharides from the fungus S. nitidum.

Heterofucan from Sargassum filipendula induces apoptosis in HeLa cells.

Fucan is a term used to denominate a family of sulfated polysaccharides rich in sulfated l-fucose. Heterofucan SF-1.5v was extracted from the brown seaweed Sargassum filipendula by proteolytic digestion followed by sequential acetone precipitation. This fucan showed antiproliferative activity on Hela cells and induced apoptosis. However, SF-1.5v was not able to activate caspases. Moreover, SF-1.5v induced glycogen synthase kinase (GSK) activation, but this protein is not involved in the heterofucan SF-1.5v induced apoptosis mechanism. In addition, ERK, p38, p53, pAKT and NFκB were not affected by the presence of SF-1.5v. We determined that SF-1.5v induces apoptosis in HeLa mainly by mitochondrial release of apoptosis-inducing factor (AIF) into cytosol. In addition, SF-1.5v decreases the expression of anti-apoptotic protein Bcl-2 and increased expression of apoptogenic protein Bax. These results are significant in that they provide a mechanistic framework for further exploring the use of SF-1.5v as a novel chemotherapeutics against human cervical cancer.

Antioxidant and anti-inflammatory effect of polysaccharides from Lobophora variegata on zymosan-induced arthritis in rats.

This study analyzes the action of sulfated polysaccharides, fucans, from algae Lobophora variegata on zymosan-induced arthritis in rats. Groups of fucans, obtained after acetone fractionation (0.3-2.0 volumes), were denominated F0.3, F0.5, F0.8, F1, F1.5, and F2. The results that F1 contained a high yield in relation to other fractionated fucans. Chemical and structure analysis of F1 was performed by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopies. The in vitro antioxidant activities of the fraction F1 were also observed. Thus, 2 mg/mL of F1 inhibited the phosphomolybdate in the total antioxidant activity assay. The EC(50) values were 0.3 mg/mL and 0.12 mg/mL for superoxide and hydroxyl radicals, respectively. Fucan F1 (25, 50, and 75 mg/kg by body weight), diclofenac sodium (10 mg/kg), and L-NAME (25 mg/kg) were administered intraperitoneally (i.p.) in rats, according to body weight of different groups of animals (n=6). After 6 h, analyses of cell influx and nitrite levels were conducted. Then after 96 h, analysis of edema and concentration of serum TNF-α was carried out along with histopathological analysis. F1 at 25, 50, and 75 mg/kg i.p. by body weight reduced cell influx in 52.1-96.7% and nitric oxide level in 27.2-39% compared with the control group. The reduction of edema and serum TNF-α was observed at 50 mg/kg i.p. (p<0.001). These results suggest that this heterofucan from the brown algae L. variegata has potential anti-inflammatory activity in acute zymosan-induced arthritis in rats and that antioxidant activity promotes modulation in the cellular redox state.

Evaluating the possible genotoxic, mutagenic and tumor cell proliferation-inhibition effects of a non-anticoagulant, but antithrombotic algal heterofucan.

Fucan is a term used to denominate a family of sulfated polysaccharides rich in L-fucose. They are extracted mainly from brown seaweeds and echinoderms. The brown seaweed Spatoglossum schröederi (Dictyotaceae) synthesizes three heterofucans named A, B and C. Our research group purified a non-anticoagulant heterofucan (fucan A) which displays antithrombotic activity in vivo. However, its in vitro toxicity has yet to be determined. This work presents the evaluation of the potential cytotoxicity, mutagenicity and genotoxicity of this fucan. After 48 h incubation fucan A cytotoxicity was determinate using MTT assay. Tumor-cell (HeLa, PC3, PANC, HL60) proliferation was inhibited 2.0-43.7%; at 0.05-1 mg ml⁻¹ of the heterofucan, the 3T3 non-tumor cell line proliferation was also inhibited (3.3-22.0%). On the other hand, the CHO tumorigenic and RAW non-tumor cell lines proliferation were not affected by this molecule (0.05-1 mg ml⁻¹). We observed no mutagenic activity in Salmonella reversion assay when bacterial strains TA97a, TA98, TA100 and TA102 (with and without S9) were used.Comet assay showed that fucan A had no genotoxic effect (from 20 to 1000 mg ml⁻¹) on CHO cells. In conclusion, this study indicates that the S. schröederi fucan A was not found to be genotoxic or mutagenic compound; thus it could be used in new antithrombotic drug development.

Assessment of zymosan-induced leukocyte influx in a rat model using sulfated polysaccharides.

Fucoidan, a sulfated polysaccharide from the brown algae Fucus vesiculosus, has diverse biological properties, including anti-inflammatory, anticoagulant and antithrombotic activity. This study analyzed the therapeutic activity of total fucoidan (TF) from F. vesiculosus and that of purified fractions (F1 and F2) on zymosan-induced arthritis. Arthritis was induced by injecting zymosan into the knee joint. Thus, three fucoidan fractions were obtained by acetone fractionation. Due to the yield obtained from F3, we used only fucoidans F1 and F2 in the induced inflammation tests. Chemical analyses and electrophoretic characterization of these fractions demonstrated that they contain polysaccharides, sulfate ester and very low protein levels. The fucoidans obtained from TF showed only an electrophoretic band in agarose gel with much lower polydispersion. The F2 fraction showed a migration between fucoidans F1 and F3. We administered TF (15, 30, 50 mg/kg I. P.), F1 or F2 (10, 25 and 50 mg/kg I. P.), diclofenac sodium (10 mg/kg I. P.), lumiracoxib (5 mg/kg O. A.) or L-NAME (30 mg/kg I. P.), 1 hour after induction of articular inflammation. We analyzed cell influx and nitrite levels in addition to performing histopathological analysis. TF (total fucoidan) at 15, 30, 50 mg/kg I. P. and its fractions (F1 and F2 at concentrations of 25 and 50 mg/kg I. P.) significantly reduced cellular influx and nitric oxide concentration. Moreover, the articular inflammation in zymosan-induced arthritis caused a progressive loss in glycosaminoglycan content. This loss decreased when TF (30 mg/kg) was administered. These data suggest that fucoidan exerts anti-inflammatory action in a zymosan-induced model of acute inflammation in rats. Taken together with the fact that these natural compounds have minimal toxicity, this may have important therapeutic implications.

Glucans from the Caripia montagnei mushroom present anti-inflammatory activity.

Caripia montagnei is a basidiomycete species which contains polysaccharides with immunomodulatory properties. An extract of this mushroom underwent removal of the fat content by organic solvent and subsequently proteolysis. The aqueous phase obtained after proteolysis was precipitated with methanol yielding a fraction containing carbohydrates (98.7+/-3.3%) and protein (1.3+/-0.25%). Chemical analysis, infrared spectroscopy and nuclear magnetic resonance (NMR) showed that the carbohydrate fraction contained (63.3+/-4.1) of beta-glucans and proteins (2.2+/-0.3%). These glucans (50mg/kg of body weight) significantly reduced the inflammatory infiltrate produced by thioglycolate-induced peritonitis by 75.5+/-5.2%, when compared to Wy-14643 (60.3+/-6.1%), PFOA (37.8+/-2.8%) and clofibrate (52.2+/-3.2%), p<0.001, which are of the peroxisome proliferator-activated receptor (PPAR-alpha). L-NAME, a nitric oxide synthase inhibitor, reduced the plantar edema in Wistar rats by 91.4+/-1.3% (p<0.001). A significant reduction in nitric oxide (NO) levels was observed in the exudates when the glucans was used in comparison to carrageenan. The C. montagnei glucans did not present signs of inducing cytotoxicity. A decrease in IL-1ra, IL-10 and IFN-gamma in the peritonitis model was observed. Thus, the results suggest that glucans from the C. montagnei mushroom is an effective immunomodulator and may have potential for anti-inflammatory properties.

Heparinoids algal and their anticoagulant, hemorrhagic activities and platelet aggregation.

Polysaccharides extracted from brown marine algae represent a source of marine compounds with potential applications in medicine. Heparin-like compounds, fucoidans, have been proposed as alternatives to the anticoagulant heparin, which is prepared from mucous membrane of mammals. In this study, the activity of anticoagulant in activated partial thromboplastin time (APTT) and prothrombin time (PT) tests was assessed in the fucoidan (TF), from seaweed Fucus vesiculosus, partially desulfated fucoidans (PDF), desulfated fucoidans (DF) and purified fractions F1, F2 and F3 in acetone. Studies were also conducted to assess these polysaccharides for platelet aggregation and hemorrhagic activity. The APTT test showed high activity at 5 microg (> or = 240s) for TF, F1 and F2 (P<0.001). PT test showed high anticoagulant activity at 50 microg (> or = 120s) for F1 (P<0.001). Fraction F3, with low MW (15.2 kDa) and sulfate content (26.1%), had little effect in these two in vitro tests (P<0.001). These compounds demonstrated a two-phase response to platelet aggregation at 50 microg/mL. However, at a concentration of 0.1 mg/mL, a hypoaggregate profile was observed for all fractions tested (P<0.001). The analysis showed that fucoidans irreversibly induced platelet aggregation in high concentration. These polymers have low hemorrhagic effect when compared to heparin.

A xylogalactofucan from the brown seaweed Spatoglossum schröederi stimulates the synthesis of an antithrombotic heparan sulfate from endothelial cells.

The brown seaweed Spatoglossum schröederi (Dictyotaceae) contains three main fucans (fucans A, B and C) with different mobility in electrophoresis. The fucan with highest mobility (fucan C) was precipitated with 2.0 volumes of acetone, purified using a combination of ion exchange chromatography and electrophoresis. It showed an MW of 24 kDa determined by HPLC and Sephadex G-75 chromatography and migrates as a single band in three distinct electrophoretic systems. This fucan contains fucose, xylose, galactose and sulfate in a molar ratio 1 : 0.6 : 2:2.3. The fucan has neither anticoagulant (from 10 to 100 microg) nor hemorrhagic activities (100 microg/mL). In addition, fucan C is neither cytotoxic nor cytostatic. However, fucan C (100 microg/mL) stimulated the synthesis of an antithrombotic heparan sulfate from endothelial cells of rabbit aorta. The results suggest that fucan C might be used as an antithrombotic therapeutic compound.