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.

Slight differences in sulfation of algal galactans account for differences in their anticoagulant and venous antithrombotic activities.

We compared sulfated galactans (SGs) from two species of red algae using specific coagulation assays and experimental models of thrombosis. These polysaccharides have an identical saccharide structure and the same size chain, but with slight differences in their sulfation patterns. As a consequence of these differences, the two SGs differ in their anticoagulant and venous antithrombotic activities. SG from G. crinale exhibits procoagulant and prothrombotic effects in low doses (up to 1.0 mg/kg body weight), but in high doses (>1.0 mg/kg) this polysaccharide inhibits both venous and arterial thrombosis in rats and prolongs ex-vivo recalcification time. In contrast, SG from B. occidentalis is a very potent anticoagulant and antithrombotic compound in low doses (up to 0.5 mg/kg body weight), inhibiting venous experimental thrombosis and prolonging ex-vivo recalcification time, but these effects are reverted in high doses. Only at high doses (>1.0 mg/kg) the SG from B. occidentalis inhibits arterial thrombosis. As with heparin, SG from G. crinale does not activate factor XII, while the polysaccharide from B. occidentalis activates factor XII in high concentrations, which could account for its procoagulant effect at high doses on rats. Both SGs do not modify bleeding time in rats. These results indicate that slight differences in the proportions and/or distribution of sulfated residues along the galactan chain may be critical for the interaction between proteases, inhibitors and activators of the coagulation system, resulting in a distinct pattern in anti- and procoagulant activities and in the antithrombotic action.

Αlpha-2 Adrenergic and Opioids Receptors Participation in Mice Gastroprotection of Abelmoschus esculentus Lectin.

Lectins are a heterogeneous group of proteins and glycoproteins with potential role as therapeutic and diagnostic tools to combat various diseases, besides some functions on human organism. Abelmoschus esculentus (Okra), a horticultural plant of African origin, is cultivated in northeastern Brazil, and used for different medicinal purposes. This work is aimed to elucidate the action mechanisms of Abelmoschus esculentus lectin (AEL) gastro protective effect on gastropathy induced by ethanol. Fasted mice treated with Ethanol 99.9% (0.2 ml/animal, p.o.) received previously AEL (0.01, 0.1, 1.0, 10 or 50 mg/kg, i.v.), saline (5 ml/kg; i.v.) or ranitidine (80 mg/kg, p.o.) in four experimental series, in which pharmacological tools (yohimbine, naloxone, L-NAME or indomethacin), were administered with the purpose of make clear possible molecular action mechanisms. Mice were euthanized 30 min after ethanol challenge to verify the stomach damages. Establishment of gastric oxidative stress, tissue hemoglobin (Hb) content and microscopic features (H&E) were taken in order to characterize the AEL gastro protective effect. AEL (1 mg/kg) was capable of protect mucosa against ethanol damages in presence of two (L-NAME and indomethacin) of four antagonists/inhibitors used. The AEL effect was reversed by naloxone and yohimbine, showing the involvement of opioids and Αlpha-2 adrenergic receptors on gastric protective effect of this lectin. Evaluation of microscopic features, oxidative stress, and Hb levels pointed the protective effects of AEL. This activity seems to be mediated by alpha-2 adrenergic and opioid receptors activation. Nitric oxide or prostaglandins were not involved. AEL simultaneously showed antioxidant effect that is probably implicated in its intricate defensive mechanism of action.

Structure and anticoagulant activity of a sulfated galactan from the red alga, Gelidium crinale. Is there a specific structural requirement for the anticoagulant action?

Marine red algae are an abundant source of sulfated galactans with potent anticoagulant activity. However, the specific structural motifs that confer biological activity remain to be elucidated. We have now isolated and purified a sulfated galactan from the marine red alga, Gellidium crinale. The structure of this polysaccharide was determined using NMR spectroscopy. It is composed of the repeating structure -4-alpha-Galp-(1-->3)-beta-Galp1--> but with a variable sulfation pattern. Clearly 15% of the total alpha-units are 2,3-di-sulfated and another 55% are 2-sulfated. No evidence for the occurrence of 3,6-anhydro alpha-galactose units was observed in the NMR spectra. We also compared the anticoagulant activity of this sulfated galactan with a polysaccharide from the species, Botryocladia occidentalis, with a similar saccharide chain but with higher amounts of 2,3-di-sulfated alpha-units. The sulfated galactan from G. crinale has a lower anticoagulant activity on a clotting assay when compared with the polysaccharide from B. occidentalis. When tested in assays using specific proteases and coagulation inhibitors, these two galactans showed significant differences in their activity. They do not differ in thrombin inhibition mediated by antithrombin, but in assays where heparin cofactor II replaces antithrombin, the sulfated galactan from G. crinale requires a significantly higher concentration to achieve the same inhibitory effect as the polysaccharide from B. occidentalis. In contrast, when factor Xa instead of thrombin is used as the target protease, the sulfated galactan from G. crinale is a more potent anticoagulant. These observations suggest that the proportion and/or the distribution of 2,3-di-sulfated alpha-units along the galactan chain may be a critical structural motif to promote the interaction of the protease with specific protease and coagulation inhibitors.

Purification of a lectin from the marine red alga Gracilaria cornea and its effects on the cattle tick Boophilus microplus (Acari: Ixodidae).

A lectin was purified from the seaweed Gracilaria cornea by hydrophobic interaction chromatography on phenyl-Sepharose CL-4B followed by affinity chromatography on immobilized mucin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of G. cornea lectin (GCL) revealed a single protein band of approximately 60 kDa, whereas by gel filtration on Sephadex G-100 its native molecular mass was 66 kDa. GCL exhibited a single isoeletric point of 4.3 and a 52.5% content of neutral sugars. Furthermore, the EDTA-treated lectin did not show any significant decrease in its ability to agglutinate trypsin-treated chicken erythrocytes. These data suggest that GCL is an acidic, monomeric glycoprotein that probably does not require divalent metal ions for its hemagglutinating activity. GCL hemagglutinating activity was not inhibited by any of the mono-, di-, and trisaccharides tested but was by the complex glycoproteins fetuin and porcine stomach mucin. Exposure of engorged females of the cattle tick (Boophilus microplus) to 0.1 mg mL(-1) GCL significantly (P < 0.05) reduced the female weight after the oviposition period, the egg mass weight, the hatching period, and the mean larvae survival time.

A report of a galactan from marine alga Gelidium crinale with in vivo anti-inflammatory and antinociceptive effects.

The sulfated galactan of the red marine alga Gelidium crinale (SG-Gc) was purified by ion exchange chromatography and tested by intravenous (i.v.) route in rodent experimental models of inflammation and nociception. The anti-inflammatory activity of SG-Gc (0.01, 0.1 and 1 mg/kg) was evaluated in the model of rat paw edema induced by different inflammatory stimuli, while SG-Gc (0.1, 1 and 10 mg/kg) antinociceptive effect was assessed in models of nociception/hyperalgesia elicited by chemical (formalin test), thermal (hot plate), and mechanical (von Frey) stimuli in mice. In addition, the toxicity was evaluated after rat treatment with SG-Gc (1 mg/kg; i.v.) during 10 days, followed by analysis of the wet weight of animal's body/organs and hematological/biochemical parameters. Sulfated galactan of G. crinale inhibited the time course of dextran-induced paw edema, at all doses, showing maximal effect at 1 mg/kg (42%) and that induced by carrageenan at 0.01 (18%) and 1 mg/kg (20%), but was ineffective on the edema elicited by zymosan. At the highest dose, SG-Gc also inhibited the paw edema induced by histamine (49%), compound 48/80 (32%), and phospholipase A(2) (44%). Sulfated galactan of G. crinale inhibited both neurogenic and inflammatory phases of the formalin test, at all doses, and at 10 mg/kg, the animals flinch reaction in the von Frey test in the 1st and 3rd h by 19 and 26%, respectively. Additionally, SG-Gc treatment was well tolerated by animals. In conclusion, SG-Gc presents anti-inflammatory effect involving the inhibition of histamine and arachidonic acid metabolites and also antinociceptive activity, especially the inflammatory pain with participation of the opioid system.

Peripheral antinociception and anti-edematogenic effect of a sulfated polysaccharide from Acanthophora muscoides.

BACKGROUND: Sulfated polysaccharides from red marine algae have presented a variety of potentially therapeutic biological effects, however, their antinocicpetive and anti-inflammatory properties are not well understood. METHODS: Male Swiss mice were pretreated with a sulfated polysaccharidic fraction obtained from the marine alga Acanthophora muscoides (AmII) (1, 3 or 9 mg/kg, iv) 30 min prior to either receiving an injection of 0.8% acetic acid or 1% formalin or prior to a thermal stimulus. AmII (1, 3 or 9 mg/kg, sc) was evaluated on carrageenan-, dextran- bradykinin-, histamine- and serotonin-induced rat paw edema models. AmII (500 µg, sc) was also injected into the paw. Additionally, mice were treated with the total sulfated polysaccharides from A. muscoides (Am-TSP) (20 mg/kg, ip) for 14 days. RESULTS: AmII reduced the number of acetic acid-induced writhes and licking time in the second phase of the formalin test, but it did not alter the response latency in the hot plate test, suggesting that its antinociceptive action occurs through a peripheral mechanism. AmII did not reduce carrageenan-induced paw edema and MPO activity. However, it reduced dextran-, histamine- and serotonin-induced paw edemas, but not bradykinin-induced edema, suggesting that histamine is the major target of AmII anti-edematogenic activity. AmII injected into the paw did not evoke local edema. Furthermore, Am-TSP induced no consistent signs of systemic damage, as revealed by body mass, organs wet weight and by biochemical, hematological and histopathological analyses. CONCLUSION: AmII has important antinociceptive and anti-inflammatory properties and represents an important therapeutic agent warranting future studies.

Antinociceptive and anti-inflammatory activities of a sulfated polysaccharide isolated from the green seaweed Caulerpa cupressoides.

BACKGROUND: Red and brown algae sulfated polysaccharides (SPs) have been widely investigated as antinociceptive and/or anti-inflammatory agents; however, no description of these biological properties concerning green algae SPs have been reported. Caulerpa curpressoides (Chlorophyta) presents three SPs fractions (Cc-SP1, Cc-SP2, and Cc-SP3). Anticoagulant (in vitro) and anti- and pro-thrombotic (in vivo) effects of Cc-SP2 had been recently reported. We evaluated the effects of Cc-SP2 using models of nociception and acute inflammation in vivo. METHODS: Male Swiss mice received Cc-SP2 (iv) 30 min prior to receiving 0.6% acetic acid (10 ml/kg, ip), 1% formalin (20 µl, sc) or were subjected to thermal stimuli (51 ± 1 °C). Cc-SP2 was injected sc to male Wistar rats in a peritonitis model or a paw edema model using carrageenan (ip or ipl, 500 µg). To analyze the systemic effects, Cc-SP2 (27 mg/kg, sc) was administrated to both genders mice before waiting for 14 days. RESULTS: Cc-SP2 (3, 9 or 27 mg/kg) reduced (p < 0.05) the number of writhes induced by acetic acid by 57, 89.9 and 90.6%, respectively, the licking time in the first (9 or 27 mg/kg with 42.47 and 52.1%, respectively) and the second (3, 9 or 27 mg/kg with 68.95, 82.34 and 84.61%, respectively) phases. In the hot-plate test, the antinociceptive effect of Cc-SP2 (9 mg/kg) was primarily observed at 60 min (26.7 ± 1.2 s), with its effect reversed by naloxone (8.6 ± 1.3 s), suggesting the involvement of the opioid system. Cc-SP2 (3, 9 or 27 mg/kg, sc, p < 0.05) showed anti-inflammatory effects by decreasing neutrophils migration by 64, 69 and 73%, respectively, and potently reduced the paw edema, especially at the second (0.16 ± 0.02, 0.16 ± 0.03 and 0.12 ± 0.05 ml) and third (0.16 ± 0.03, 0.18 ± 0.02 and 0.14 ± 0.04 ml) hours, respectively. Cc-SP2 did not cause hepatic or renal alterations or affect body mass or the macroscopy of the organs examined (p > 0.05). Histopathological analyses of the liver and kidney showed that both organs were affected by Cc-SP2 treatment, but these effects were considered reversible. CONCLUSION: The results indicate that the analgesic and anti-inflammatory effects of Cc-SP2 could be of biomedical applicability as a new, natural tool in pain and acute inflammatory conditions.

Antinociceptive and anti-inflammatory activities of sulphated polysaccharides from the red seaweed Gracilaria cornea.

Seaweeds have attracted special interest as good sources of sulphated polysaccharides (SP) for use in pharmaceutical industries and biotechnology. In this study, we evaluated the effects of SP from the red seaweed Gracilaria cornea (Gc-TSP) in nociceptive and inflammatory models. In mice, Gc-TSP (3, 9 or 27 mg/kg) significantly reduced nociceptive responses, as measured by the number of writhes, at all tested doses. In a formalin test, Gc-TSP significantly reduced licking time in both phases of the test at a dose of 27 mg/kg. In a hot-plate test, the antinociceptive effect was observed only in animals treated with 27 mg/kg of Gc-TSP, suggesting that the analgesic effect occurs through a central action mechanism at the highest dose. Gc-TSP (3, 9 or 27 mg/kg) caused only a slight reduction in neutrophil migration in the rat peritoneal cavity. However, lower doses of Gc-TSP (3 and 9 mg/kg) significantly inhibited paw oedema induced by carrageenan, especially at 3 hr after treatment. Reduction in oedema was confirmed by myeloperoxidase activity in the affected paw tissue. In addition, treatment (s.c.) of animals with different doses of Gc-TSP inhibited paw oedema induced by dextran within the first hour in all doses tested. After 14 consecutive days of intraperitoneal administration of Gc-TSP (9 mg/kg), we measured the wet weight of the liver, kidney, heart, spleen and thymus and performed biochemical, haematological and histopathological evaluations. No systemic damage was found. These results indicate that Gc-TSP possesses analgesic and anti-inflammatory effects and is a potentially important tool worthy of further study.

Heme oxygenase/carbon monoxide-biliverdin pathway may be involved in the antinociceptive activity of etoricoxib, a selective COX-2 inhibitor.

The aim of this study was to assess the interaction between the heme oxygenase-1/ biliverdin/carbon monoxide (HO-1/BVD/CO) and cyclooxygenase-2 (COX-2) pathways in the writhing test. Mice were pretreated with 0.1, 1 or 10 mg/kg, ip etoricoxib, a selective COX-2 inhibitor, or with one of the following HO-1/BVD/CO pathway modulators: 1, 3 or 9 mg/kg, sc ZnPP IX, a specific HO-1 inhibitor, 0.3, 1 or 3 mg/kg, sc hemin, a substrate of the HO-1/BVD/CO pathway; or 0.00025, 0.025 or 2.5 µmol/kg, sc DMDC, a CO donor. Mice pretreated with etoricoxib or one of the HO-1/BVD/CO pathway modulators received an injection of acetic acid (ip) after 30 and 60 min, respectively. Next, the number of writhes was quantified between 0 and 30 min after stimulus injection. In another series of experiments, ineffective doses of etoricoxib were co-administered with hemin or DMDC and an effective dose of etoricoxib with ZnPP IX, followed by an acetic acid injection. Four hours after the acetic acid injection, levels of bilirubin, which is a product of BVD conversion by the BVD reductase enzyme, in the peritoneal lavage were determined. Hemin or DMDC reduced (p<0.05) the number of writhes, but ZnPP IX potentiated (p<0.05) the effect of acetic acid by increasing (p < 0.05) the number of writhes. The co-administration of etoricoxib with hemin or DMDC reduced (p<0.05) the number of writhes. However, the analgesic effect of etoricoxib was not observed in the presence of ZnPP IX. Pretreatment with ZnPP IX reduced bilirubin levels, but etoricoxib pretreatment significantly increased the bilirubin concentration in peritoneal exudates. The data obtained from these experiments showed that the HO-1/BVD/CO pathway was activated in the acetic acid-induced abdominal writhing model. The analgesic effect of etoricoxib was at least partially dependent on the participation of the HO-1/BVD/CO pathway.

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.

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.