Exploring the carbohydrate-binding ability of Canavalia bonariensis lectin in inflammation models.

Lectins are a group of proteins of non-immune origin recognized for their ability to bind reversibly to carbohydrates. Researchers have been intrigued by oligosaccharides and glycoconjugates for their involvement as mediators of complex cellular events and then many biotechnological applications of lectins are based on glycocode decoding and their activities. Here, we report a structural and biological study of a ConA-like mannose/glucose-specific lectin from Canavalia bonariensis seeds, CaBo. More specifically, we evaluate the binding of CaBo with α-methyl-D-mannoside (MMA) and mannose-1,3-α-D-mannose (M13) and the resultant in vivo effects on a rat model of acute inflammation. A virtual screening was also carried out to cover a larger number of possible bindings of CaBo. In silico analysis demonstrated the stability of CaBo interaction with mannose-type ligands, and the lectin was able to induce acute inflammation in rats with the participation of the carbohydrate recognition domain (CRD) and histamine release. These results confirm the ability of CaBo to interact with hybrid and high-mannose N-glycans, supporting the hypothesis that CaBo's biological activity occurs primarily through its interaction with cell surface glycosylated receptors.

Polysaccharide rich fractions from barks of Ximenia americana inhibit peripheral inflammatory nociception in mice Antinociceptive effect of Ximenia americana polysaccharide rich fractions

Abstract Ximenia americana L., Olacaceae, barks are utilized in folk medicine as analgesic and anti-inflammatory. The objective was to evaluate the toxicity and antinociceptive effect of polysaccharides rich fractions from X. americana barks. The fractions were obtained by extraction with NaOH, followed by precipitation with ethanol and fractionation by ion exchange chromatography. They were administered i.v. or p.o. before nociception tests (writhing, formalin, carragenan-induced hypernociception, hot plate), or during 14 days for toxicity assay. The total polysaccharides fraction (TPL-Xa: 8.1% yield) presented 43% carbohydrate (21% uronic acid) and resulted in two main fractions after chromatography (FI: 12%, FII: 22% yield). FII showed better homogeneity/purity, content of 44% carbohydrate, including 39% uronic acid, arabinose and galactose as major monosaccharides, and infrared spectra with peaks in carbohydrate range for COO- groups of uronic acid. TPL-Xa (10 mg/kg) and FII (0.1 and 1 mg/kg) presented inhibitory effect in behavior tests that evaluate nociception induced by chemical and mechanical, but not thermal stimuli. TPL-Xa did not alter parameters of systemic toxicity. In conclusion, polysaccharides rich fractions of X. americana barks inhibit peripheral inflammatory nociception, being well tolerated by animals.

Modulator effect of a polysaccharide-rich extract from Caesalpinia ferrea stem barks in rat cutaneous wound healing: Role of TNF-α, IL-1ß, NO, TGF-ß.

ETHNOPHARMACOLOGICAL RELEVANCE: In folk medicine stem barks of Caesalpinia ferrea (Caesalpinioideae) are used to treat enterocolitis, rheumatism and wounds and in experimental procedures, its aqueous extracts demonstrated antiulcer, anti-inflammatory, analgesic, and healing effects. AIM OF THE STUDY: The healing mechanism of the polyssacharide-rich extract of C. ferrea stem barks (TPL-Cf) was investigated in a model of excisional cutaneous wound in Wistar rats. MATERIALS AND METHODS: Excisional wounds received topical treatment with TPL-Cf (0.025-0.1%) during 21 days. Hypernociception, macroscopical, histological and immunohistochemical parameters were evaluated and analyzed by ANOVA, Bonferroni and Kruskal-Wallis tests, followed by Dunn and Chi-Square tests. RESULTS: TPL-Cf (0.1%) reduced wound area and hypernociception, and increased wound contraction. TPL-Cf reduced leukocyte infiltration and vascular permeability, and stimulated fibroblasia, angiogenesis, well formed granulation tissue, collagen deposition and epithelial layer formation. TPL-Cf reduced TNF-α expression and the levels of PGE2 (73%-day 5), IL-1 (42%-day 2), MDA (38%-day 5), total protein (53%-day 2; 73%-day 5) and MPO activity (53%-day 2), but increased the expression of i-NOS (days 5 and 7), TGF-ß (day 5) and the levels of NO (3.6 fold-day 5). CONCLUSION: The polysaccharide-rich extract of C. ferra stem barks accelerates wound healing by the control of the inflammatory phase and attenuates hypernociception via modulation of inflammatory mediators (TNF-α, IL-1ß, NO, TGF-ß).

Purified polysaccharides of Geoffroea spinosa barks have anticoagulant and antithrombotic activities devoid of hemorrhagic risks.

Polysaccharides were extracted from the barks of Geoffroea spinosa, purified using anion exchange chromatography and characterized by chemical and methylation analysis, complemented by infrared and NMR spectroscopies. These polysaccharides were tested for their anticoagulant, antithrombotic and antiplatelet activities and also for their effects on bleeding. Unfractionated polysaccharide contains low levels of protein and high levels of carbohydrate (including hexuronic acid). The purified polysaccharides (fractions FII and FIII) are composed of arabinose (Ara), rhamnose (Rha), hexuronic acid, small amounts of galactose, but no sulfate ester. They have highly complex structure, which was partially characterized. NMR and methylation analysis indicate that the polysaccharides have a core of α-Rhap and branches of 5-linked α-Araf. Residues of 4-linked α-GalpA are also found in the structure. The unfractionated (TPL) and fraction FIII, but not fractions FI and FII, prolonged the activated partial thromboplastin time (aPTT). TPL, FII and FIII inhibited the platelet aggregation induced by ADP. More significantly, both unfractionated and purified fractions exhibited potent antithrombotic effect (31-60%) and the fractions did not modify the bleeding tendency. These plant polysaccharides could be alternative source of new anticoagulant, antiplatelet and antithrombotic compounds devoid of the undesirable risk of hemorrhage.

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.

Venom's antinociceptive property in the primitive ant Dinoponera quadriceps.

ETHNOPHARMACOLOGICAL RELEVANCE: In northeastern Brazil, Dinoponera (Ponerinae) ants macerate are used to treat ear ache and its sting, rheumatism, and back pain. Such a popular use is a relevant fact that called for experimental evaluation of the antinociceptive activity of Dinoponera venom. MATERIALS AND METHODS: Dinoponera quadriceps venom (DqV; 5-500 µg/kg; i.v.) or morphine (3.4 mg/kg; s.c.) were evaluated in mice models of nociception (n=8 animals/group). Negative controls received sterile saline (0.9% NaCl; i.v.). RESULTS: DqV showed 64% protein content and exhibited antinociceptive activity, without affecting motor function, in the tests: formalin (72%), writhing (52%), von Frey (71%) and hot plate (45%). The antinociceptive activity was abolished under protein denaturant conditions. CONCLUSIONS: This study provided the first demonstration of the antinociceptive property of Dinoponera quadriceps venom in mice models of chemical, mechanical and thermal nociception, corroborating the popular use and suggesting its potential therapeutic utilization in painful conditions.

Anti-inflammatory polysaccharides of Azadirachta indica seed tegument

Azadirachta indica A. Juss., Meliaceae, or Indian neem is a plant used to treat inûammatory disorders. Total polysaccharide (TPL) and FI (fractioned by ion exchange chromatography) from the seed tegument of A. indica were evaluated in models of acute inflammation (paw edema/peritonitis) using Wistar rats. Paw edema (measured by hydroplethysmometry) was induced s.c. by Λ-carrageenan (300 µg), histamine (100 µg), serotonin (20 µg), compound 48/80 (10 µg), prostaglandin (PGE2 30 µg) or L-arginine (15 µg). Peritonitis (analyzed for leukocyte counts/protein dosage) was induced i.p. by carrageenan (500 mg) or N-formyl-methionyl-leucyl-phenylalanine (fMLP 50 ng). Animals were treated i.v. with TPL (1 mg/kg) or FI (0.01, 0.1, 1 mg/kg) 30 min before stimuli. FI toxicity (at 0.1 mg/kg, i.v. for seven days) was analyzed by the variation of body/organ mass and hematological/biochemical parameters. TPL extraction yielded 1.3%; FI, presenting high carbohydrate and low protein content, at 0.1 mg/kg inhibited paw edema induced by carrageenan (77%), serotonin (54%), PGE2 (69%) and nitric oxide (73%), and the peritonitis elicited by carrageenan (48%) or fMLP (67%), being well tolerated by animals. FI exhibited potent anti-inflammatory activity, revealing to be important active component in traditionally prepared remedies to treat inflammatory states.

A novel antinociceptive sulphated polysaccharide of the brown marine alga Spatoglossum schroederi.

Sulfated polysaccharides (SP) of brown algae (Phaeophyta) are composed mainly of alpha- L-fucose, being classified as fucans, with recognized role in inflammation but not in nociception, which was already described for SP obtained from red algae. Here the SP of the brown marine alga S. schroederi (named Ss-SP) was isolated and assayed for the antinociceptive effect. Ss-SP was isolated by DEAE-cellulose, analyzed by agarose gel electrophoresis and evaluated in nociception models (Formalin, Hot plate, Von Frey) using Swiss mice (20-25g). Anion exchange chromatography provided four major fractions being F1 (Ss-SP) that of highest metachromatic activity and sugar content. Ss-SP inhibited both phases of the formalin test. In the first phase the paw licking (55.2 +/- 8.07s) was reduced by 45% (30.5 +/- 6.51s) and 40% (32.85 +/- 8.66s) at 0.1 and 1 mg/kg, respectively. In the second phase, Ss-SP was also inhibitory about 39%, but only at 1 mg/kg (83.0 +/- 15.70s) compared to formalin (136.8 +/- 10.27s). This inhibitory effect suggests a mixed mechanism similar to morphine, which was not confirmed in the hot plate test, a model of pain associated with central neurotransmission. However, Ss-SP reduced the animal reaction in response to stimulation withVon Frey filament at the 2nd and 3rd h (20.8 +/- 6.86% versus carrageenan: 47.9 +/- 5.83%; 33.3 +/- 7.71% versus carrageenan: 62.5 +/- 9.83%). Accordingly, the paw edema induced by carrageenan (0.08 +/- 0.01g) was potently reduced in 45.35% by Ss-SP pre-treatment (0.02 +/- 0.003g), corroborating the anti-inflammatory activity demonstrated for brown seaweed polysaccharides. In conclusion our data revealed for the first time the antinociceptive effect of Ss-SP which could be used as a new source of analgesic substances.

Purification and partial characterization of a new pro-inflammatory lectin from Bauhinia bauhinioides Mart (Caesalpinoideae) seeds.

A new galactose-specific lectin, named BBL, was purified from seeds of Bauhinia bauhinioides by precipitation with ammonium sulfate, followed by two steps of ion exchange chromatography. BBL haemagglutinated rabbit erythrocytes (native and treated with proteolytic enzymes) showing stability even after exposure to 60 °C for an hour. The lectin haemagglutinating activity was optimum between pH 8.0 and 9.0 and inhibited after incubation with D-galactose and its derivatives, especially α-methyl-D-galactopyranoside. The pure protein possessed a molecular mass of 31 kDa by SDS-PAGE and 28.310 Da by mass spectrometry. The lectin pro-inflammatory activity was also evaluated. The s.c. injection of BBL into rats induced a dose-dependent paw edema, an effect that occurred via carbohydrate site interaction and was significantly reduced by L-NAME, suggesting an important participation of nitric oxide in the late phase of the edema. These findings indicate that BBL can be used as a tool to better understand the mechanisms involved in inflammatory responses.

In vivo anti-inflammatory effect of a sulfated polysaccharide isolated from the marine brown algae Lobophora variegata.

CONTEXT: Lobophora variegata J.V. Lamouroux (Dictyotaceae) is a brown marine alga widely encountered in the Brazilian sea coast that presents high content of fucans. Anti-inflammatory effects of fucans are reported mostly in models in vitro, but little is known about its effects in vivo. OBJECTIVE: To investigate vascular and cellular effects of a sulfated polysaccharide from the brown marine algae L. variegata (SP-Lv) in acute inflammatory models. MATERIALS AND METHODS: SP-Lv was isolated by DEAE-cellulose and analyzed by agarose gel electrophoresis and evaluated for its inhibitory effect on paw edema, vascular permeability, leukocyte migration and peritoneal nitrite content induced by zymosan in Wistar rats. Anticoagulant activities and possible systemic toxicity were also evaluated. RESULTS: SP-Lv inhibited the paw edema (120 min: 1.42 ± 0.11 vs. 0.95 ± 0.05 mL), plasma exudation (21.53 ± 0.62 vs. 11.96 ± 0.68 µg/g), nitrite content (4.42 ± 0.33 vs. 2.86 ± 0.003 µM) and leukocyte migration (5.15 ± 1.21 vs. 1.99 ± 0.16 cells/10(3) mL) induced by zymosan. SP-Lv and L-NAME reduced the paw edema (60-120 min) elicited by L-arginine. However, at 180 min SP-Lv effect was more accentuated and sustained until 240 min, while that of L-NAME was abolished. Similarly to indomethacin, SP-Lv inhibited the entire edema time-course induced by phospholipase A(2), except for the time of 60 min. DISCUSSION AND CONCLUSION: The anti-edematogenic effect of SP-Lv seems to occur via inhibition of nitric oxide synthase and cyclooxygenase activities. These results suggest a potential applicability of polysaccharides from alga origin in acute inflammatory conditions.

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.

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.