Structural and physical-chemical analyses of sulfated polysaccharidesfrom the sea lettuce Ulva lactuca and their effects on thrombin generation / Análises estrutural e físico-química de polissacarídeos sulfatados da alface do mar Ulva lactuca e seus efeitos sobre geração de trombina

Ulva lactuca (Chlorophyceae) has biotechnologically-important sulfated-polysaccharides (Ul-SPs), but their potentials on thrombin generation (TG) are unknown. This study analyzed the structural and physicalchemical features of the Ul-SPs as modulators of TG. Proteolytic digestion yielded (13.13%) extract containing sulfate (20.43%) and total sugars (65.72%), besides ulvan consisting of rhamnose, xylose, glucose, glucuronic acid and α-/β-types glycosidic linkages as characterized by one-/two-dimensions nuclear magnetic resonance (NMR) experiments. Fractionation of the Ul-SPs by DEAE-cellulose chromatography yielded Ul-SP1 and Ul-SP2 (0.50 and 0.75 M NaCl, respectively) showing sulfation (15.72-18.04%) and total sugars (59.73-60.58%) consistent with the charge density pattern by combination of agarose/polyacrylamide gel eletrophoresis using sequential staining with toluidine blue and stains-all, although with slight differences in their sizes (40 and >100 kDa, respectively). By both activated partial thromboplastin time (APTT) and prothrombin time (PT) tests, anticoagulation of the fractions was virtually detected by APTT (0.39 and 0.43 IU, respectively) against heparin (193 IU). Fractions acted differently on both intrinsic/extrinsic pathways in TG using 60-fold diluted human plasma, with 50% efficacies up to 8.3 μg, whereas at high concentrations suggested intrinsic hypercoagulability since heparin abolished both systems at low amounts. Ul-SPs block TG, but predicting thrombosis in increasing doses.

Clorofícea Ulva lactuca possui polissacarídeos sulfatados (Ul-PSs) importantes biotecnologicamente, porém são desconhecidos seus potenciais sobre geração de trombina (GT). Analisaram-se as características estruturais e físico-químicas dos Ul-PSs como moduladores de GT. Digestão proteolítica rendeu (13,13%) extrato contendo sulfato (20,43%) e açúcares totais (65,72%), além de ulvana, como caracterizada por experimentos de ressonância magnética nuclear uni-/bi-dimensionais, consistindo de ramnose, xilose, glucose, ácido glucurônico e ligações glicosídicas tipos-α/-β. Fracionamento dos Ul-PSs por cromatografia de DEAE-celulose rendeu Ul-PS1 e Ul-PS2 (0,50 e 0,75 M de NaCl, respectivamente) mostrando sulfatação (15,72-18,04%) e açúcares totais (59,73- 60,58%) consistentes com o grau de densidade de carga por combinação de eletroforese em gel de agarose/poliacrilamida usando coramento sequencial com azul de toluidina e "stains-all", embora com diferenças quanto aos seus tamanhos (40 e >100 kDa, respectivamente). Por ambos os testes do tempo de tromboplastina parcial ativada (TTPA) e do tempo de protrombina, anticoagulação das frações foi detectada virtualmente pelo TTPA (0,39 e 0,43 UI, respectivamente) frente heparina (193 UI). Frações atuaram diferentemente sobre ambas as vias intrínsica/extrínsica na GT usando plasma humano diluído 60 vezes, com eficácias de 50% até 8,3 μg, enquanto em concentrações maiores sugeriram hipercoagulabilidade intrínsica visto que heparina aboliu ambos os sistemas em quantidades baixas. Ul-PSs bloqueiam GT, porém prevendo trombose em doses crescentes.

Role of central opioid on the antinociceptive effect of sulfated polysaccharide from the red seaweed Solieria filiformis in induced temporomandibular joint pain.

This study aimed to investigate the effect of sulfated polysaccharide from red seaweed Solieria filiformis (Fraction F II) in the inflammatory hypernociception in the temporomandibular joint (TMJ) of rats. Male Wistar rats were pretreated (30min) with a subcutaneous injection (s.c.) of vehicle or FII (0.03, 0.3 or 3.0mg/kg) followed by intra-TMJ injection of 1.5% Formalin or 5-hydroxytryptamine (5-HT, 225µg/TMJ). In other set of experiments rats were pretreated (15min) with an intrathecal injection of the non-selective opioid receptors Naloxone, or µ-opioid receptor antagonist CTOP, or δ-opioid receptor Naltridole hydrochloride, or κ-opioid receptor antagonist Nor-Binaltorphimine (Nor-BNI) followed by injection of FII (s.c.). After 30min, the animals were treated with an intra-TMJ injection of 1.5% formalin. After TMJ treatment, behavioral nociception response was evaluated for a 45-min observation period, animals were terminally anesthetized and periarticular tissue, trigeminal ganglion and subnucleus caudalis (SC) were collected plasma extravasation and ELISA analysis. Pretreatment with F II significantly reduced formalin- and serotonin-induced TMJ nociception, inhibit the plasma extravasation and inflammatory cytokines release induced by 1.5% formalin in the TMJ. Pretreatment with intrathecal injection of Naloxone, CTOP, Naltridole or Nor-BNI blocked the antinociceptive effect of F II in the 1.5% formalin-induced TMJ nociception. In addition, F II was able to significantly increase the ß-endorphin release in the subnucleus caudalis. The results suggest that F II has a potential antinociceptive and anti-inflammatory effect in the TMJ mediated by activation of opioid receptors in the subnucleus caudalis and inhibition of the release of inflammatory mediators in the periarticular tissue.

Analgesic and anti-inflammatory actions on bradykinin route of a polysulfated fraction from alga Ulva lactuca.

We investigated structural features of polysaccharides from Ulva lactuca and their effects on the classical models of nociception and inflammation. Crude extract was obtained by enzymatic digestion and isolated by ion exchange chromatography on DEAE-cellulose. The fraction with higher yield was used in the tests (SP-Ul). Swiss mice received SP-Ul (1, 3 or 9mg/kg; i.v.), 30min prior to injection of 0.8%-acetic acid or 1%-formalin or prior to a thermal stimulus. At same doses, SP-Ul was tested on Wistar rats on paw edema elicited by different irritants (carrageenan, dextran, bradykinin, histamine or serotonin). The results of infrared characterization indicated the presence of hydroxyl groups, sulfate, uronic acid and glycosidic linkages in all SP fractions spectrums. SP-Ul decreased significantly the antinociception in response to acetic acid or formalin (second phase), but not in the hot-plate test, suggesting that its analgesia occurs through a peripheral mechanism. SP-Ul did not reduce carrageenan-induced paw edema as supported by both histological and myeloperoxidase activity assessments. However, SP-Ul (1mg/kg; s.c.) reduced dextran-elicited edema, showing vascular anti-inflammatory effect, with bradykinin as major target because it did not reduce histamine- and serotonin-induced paw edemas. Therefore, SP-Ul acts on bradykinin pathway in its antinociceptive and anti-inflammatory responses.

Dual effects of a lectin from the green seaweed Caulerpa cupressoides var. lycopodium on inflammatory mediators in classical models of inflammation.

OBJECTIVE: Wide biotechnological investigations of only a limited number of seaweed lectins have been performed. We previously demonstrated the anti-nociceptive and anti-inflammatory effects of a lectin isolated from the green seaweed Caulerpa cupressoides var. lycopodium (CcL). Herein, we further studied the mechanisms of action of CcL. METHODS: Classical acute inflammation models induced by different flogistic agents were used to evaluate the anti-inflammatory action of CcL. CcL was injected locally into the rat paw to verify a possible pro-inflammatory outcome. RESULTS: CcL (0.1, 1 or 10 mg/kg; i.v.) reduced the carrageenan-induced rat paw edema and neutrophilic infiltration, which was not altered by either mucin (inhibitor of CcL carbohydrate-binding site) or ZnPP-IX (specific HO-1 inhibitor). Immunohistochemical analyses showed that CcL (1 mg/kg) reduced the expression of the cytokines IL-1ß, TNF-α, IL-6 and COX-2. CcL (0.1, 1 or 10 mg/kg) inhibited dextran, and CcL (1 mg/kg) inhibited histamine-induced rat paw edema. Both effects were reversed by mucin inhibition. CcL (1 mg/kg) was ineffective for the treatment of serotonin- and bradykinin-induced rat paw edema. When injected via the i.pl. route, CcL (10 mg/kg) elicited rat paw edema involving a wide range of mediators. CONCLUSIONS: The anti-inflammatory action of CcL involves the inhibition of IL-1ß, TNF-α, IL-6 and COX-2 expression and histamine H1 receptors. When locally administered, CcL exerts pro-inflammatory actions.

Mechanisms involved in the anti-inflammatory action of a polysulfated fraction from Gracilaria cornea in rats.

The anti-inflammatory mechanisms of the sulfated polysaccharidic fraction obtained from red marine alga Gracilaria cornea (Gc-FI) were investigated using a paw edema model induced in rats by different inflammatory agents (carrageenan, dextran, serotonin, bradykinin, compound 48/80 or L-arginine). Gc-FI at the doses of 3, 9 or 27 mg/kg, subcutaneously--s.c., significantly inhibited rat paw edema induced by carrageenan and dextran, as confirmed by myeloperoxidase and Evans' blue assessments, respectively. Gc-FI (9 mg/kg, s.c.) inhibited rat paw edema induced by histamine, compound 48/80 and L-arginine. Additionally, Gc-FI (9 mg/kg, s.c.) inhibited Cg-induced edema in animals with intact mast cells but did not inhibit that with degranulated mast cells by compound 48/80, revealing a protective role on mast cell membranes. Gc-FI down-regulated the IL-1ß, TNF-α and COX-2 mRNA and protein levels compared with those of the carrageenan group, based on qRT-PCR and immunohistochemistry analyses. After inhibition with ZnPP IX, a specific heme oxygenase-1 (HO-1) inhibitor, the anti-inflammatory effect of Gc-FI was not observed in Cg-induced paw edema, suggesting that the anti-inflammatory effect of Gc-FI is, in part, dependent on the integrity of the HO-1 pathway. Gc-FI can target a combination of multiple points involved in inflammatory phenomena.

A lectin from the green seaweed Caulerpa cupressoides reduces mechanical hyper-nociception and inflammation in the rat temporomandibular joint during zymosan-induced arthritis.

Seaweed lectins have been widely investigated as anti-nociceptive and anti-inflammatory agents. This study analyzed the anti-nociceptive and anti-inflammatory responses of a lectin from the green seaweed Caulerpa cupressoides (CcL) on zymosan-induced arthritis of the rat temporomandibular joint (TMJ). Rats received i.v. CcL 30 min prior to injection of zymosan (2mg/art.) or 0.9% saline into the left TMJ. Mechanical hyper-nociception was measured by the electronic von Frey method at baseline and 4h after zymosan injection. Animals were euthanized 6h after zymosan injection and the synovial fluid was collected for leukocyte counting and myeloperoxidase activity assessment. Other animals were treated with ZnPP-IX (3mg/kg; s.c.), a specific heme oxygenase-1 pathway inhibitor, and naloxone (10 µg/art.), a nonselective opioid receptor antagonist. TMJ tissues were excised to perform histopathological and immunohistochemistry analyses. CcL (0.1, 1 or 10mg/kg) significantly reduced zymosan-induced hyper-nociception (81, 83 and 89.5%, respectively) and inhibited the leukocyte influx (77.3, 80.7 and 98.5%, respectively) compared with the zymosan-only group, as confirmed by myeloperoxidase activity; however, treatment with naloxone or ZnPP-IX did not revert the effects of CcL (10mg/kg), suggesting that the naloxone-sensitive opioid and heme oxygenase-1 pathways are not involved. CcL also reduced the leukocyte influx and the expression of IL-1ß and TNF-α in the TMJ, based on histopathological and immunohistochemistry analyses, respectively. Therefore, CcL reduces TMJ hyper-nociception and inflammation with a mechanism that is partially dependent on TNF-α and IL-1ß inhibition. CcL reveals a potentially valuable alternative tool for future studies of TMJ disorders.

Peripheral antinociception and anti-inflammatory effects of sulphated polysaccharides from the alga Caulerpa mexicana.

Sulphated polysaccharides from marine algae are widely used in biotechnological and pharmaceutical areas. In this study, we evaluated the effects of sulphated polysaccharides from the green marine alga Caulerpa mexicana (Cm-SPs) in nociceptive and inflammatory models in rodents. Cm-SPs (10 or 20 mg/kg), administered i.v. in Swiss mice, significantly reduced nociceptive responses, as measured by the number of writhes in response to acetic acid. Cm-SPs (10 or 20 mg/kg) also reduced second-phase responses in the formalin test, but did not exhibit a significant antinociceptive effect in the hot plate test, suggesting that its antinociceptive action occurs through a peripheral mechanism. Cm-SPs (5, 10 or 20 mg/kg), administered s.c. in wistar rats 1 hr before carrageenan, dextran, histamine or serotonin, were tested in paw oedema models. Cm-SPs (10 or 20 mg/kg) reduced carrageenan-induced paw oedema and myeloperoxidase activity in the paw. In addition, Cm-SPs (20 mg/kg) inhibited dextran- or histamine-induced paw oedema, but not serotonin-induced oedema, suggesting that histamine is the major target of Cm-SPs anti-oedematogenic activity. Finally, Cm-SPs (20 mg/kg) administered in mice did not show significant signs of toxicity. In conclusion, Cm-SPs appear to be promising natural modulatory agents for pain and inflammatory conditions.

The involvement of the HO-1 pathway in the anti-inflammatory action of a sulfated polysaccharide isolated from the red seaweed Gracilaria birdiae.

OBJECTIVES: The aim of this study was to investigate the involvement of the hemoxigenase-1 (HO-1) pathway in the anti-inflammatory action of a sulfated polysaccharide from the red seaweed Gracilaria birdiae (SP-Gb). METHODS: SP-Gb (5, 10 and 20 mg/kg) was administered to Wistar rats in a peritonitis model using carrageenan or a paw edema model using carrageenan or dextran. To analyze the involvement of HO-1 in the anti-inflammatory activity of SP-Gb, the animals were pretreated subcutaneously with a specific HO-1 inhibitor (ZnPP IX). To evaluate the systemic effects, SP-Gb (10 mg/kg) was administered to mice intraperitoneally before waiting for 48 h or for 14 days. RESULTS: SP-Gb (10 mg/kg) caused an anti-inflammatory effect that was evidenced by a decrease in leukocytes in the peritoneal cavity. SP-Gb also reduced the paw edema induced by carrageenan and inhibited the paw edema induced by dextran in the first half-hour. After being inhibited by ZnPP IX, the anti-inflammatory effect of SP-Gb on carrageenan-induced rat paw edema was not observed. SP-Gb did not cause mortality or significant changes in the biochemical, hematological and histopathological parameters. CONCLUSION: SP-Gb may be used as a tool for further investigations into the inflammatory processes associated with the hemoxigenase-1 pathway.

Isolamento, fracionamento e avaliação toxicológica in vivo de polissacarídeos sulfatados de Hypnea musciformis / Isolation, fractionation and in vivo toxicological evaluation of sulfated polysaccharides from Hypnea musciformis

Objetivou-se isolar, fracionar e avaliar a toxicidade in vivo dos polissacarídeos sulfatados (PSs) da rodofícea Hypnea musciformis, quando obtidos por três métodos de extração (M I; M II e M III). Os PSs foram extraídos com papaína em tampão acetato de sódio 100mM (pH 5,0), contendo cisteína e EDTA (5mM) (M I) ou água (25-80°C (M II); 80°C (M III)) e, em seguida, determinados sua composição química de carboidratos totais, sulfato livre (SL) e proteínas contaminantes (PCs). Os PSs foram submetidos à cromatografia de troca iônica (DEAE-celulose) usando um gradiente de cloreto de sódio, sendo avaliado o grau de homogeneidade e densidade de carga por eletroforese em gel de agarose das frações obtidas e comparadas à heparina. O ensaio in vivo foi realizado em grupos (n=6) de camundongos Swiss machos e fêmeas (24-33g), os quais receberam: PSs (9mg kg-1; i.p.) isentos do PCs (M I) e salina 0,9 por cento (0,1mL 10g-1; i.p.), durante 14 dias consecutivos. No 15o dia, os animais foram anestesiados e sacrificados para coletas de sangue e órgãos, os quais foram utilizados para dosagens bioquímicas e correlações com suas massas corpóreas, respectivamente. O teor de SL (31,05±0,53 por cento) (P<0,05) e o fracionamento, em DEAE-celulose, indicaram o M I mais eficiente na obtenção de PSs, comparado ao M II e M III. Os animais mostraram-se tolerantes aos PSs do M I e não se observou alteração de ordem hepática ou renal (P>0,05).

This study aimed to isolate, fractionate and evaluate the in vivo toxicity of sulfated polysaccharides (SPs) from Hypnea musciformis (Rhodophyta), when obtained by three extraction methods (M I, M II and M III). SPs were extracted with papain in 100mM sodium acetate (pH 5.0) containing cysteine and EDTA (5mM) (M I) or water (25-80°C (M II), 80°C (M III)), and then their chemical composition of total carbohydrates, free sulfate (FS) and contaminant proteins (CPs) was determined. SPs were submitted to ion-exchange chromatography (DEAE-celulose) using a sodium chloride gradient, being the degree of homogeneity and charge density evaluated by agarose gel electrophoresis of the fractions obtained and compared to heparin. The in vivo assay was performed using groups (n=6) of male and female Swiss mice (24-33g), which received: SPs (9mg kg-1, i.p.) absence of CPs (M I) and 0.9 percent saline (0.1mL 10g-1, i.p.), for 14 consecutive days. On the 15th day, collect blood and organs for biochemical dosages and corporal mass correlation, respectively, from the animals anesthetized and sacrificed were performed. The sulfate content of FS (31.05±0.53 percent) (P<0.05) and the fractionation by DEAE-cellulose showed M I more effectiveness in obtaining SPs compared to M II and M III. The animals were tolerable to SPs from M I, and it wasn't observed hepatic or renal alteration (P>0.05).

Isolamento, fracionamento e atividade anticoagulante de iota-carragenanas da Solieria filiformis / Isolation, fractionation and anticoagulant activity of iota-carrageenans from Solieria filiformis

Este estudo teve como objetivo isolar, fracionar e avaliar o potencial anticoagulante de iota-carragenanas (i-CARs) da rodofícea Solieria filiformis, quando obtidas por dois métodos de extração (M I e M II). As i-CARs foram isoladas com papaína bruta em tampão acetato de sódio 0,1M (pH 5,0), contendo cisteína 5mM e EDTA 5mM (M I) ou água (80°C) (M II) e, em seguida, determinada sua composição química de carboidratos totais, sulfato livre (SL) e proteínas contaminantes. As i-CARs foram submetidas à cromatografia de troca iônica (DEAE-celulose) usando um gradiente de cloreto de sódio, sendo avaliado o tempo de tromboplastina parcial ativada (TTPA) e tempo de protrombina das frações obtidas e comparadas à heparina (193UI mg-1). Uma fração anticoagulante também foi submetida ao procedimento de eletroforese em gel de agarose a 0,5 por cento. A diferença no rendimento de i-CARs entre os métodos foi 10,14 por cento. A composição química de SL (29,40 por cento) e o fracionamento, por DEAE-celulose, indicaram o M I mais eficiente na obtenção de i-CARs, comparado ao M II. O TTPA também foi somente alterado para as i-CARs do M I. Contudo, a atividade anticoagulante in vitro de uma fração rica (8,52UI mg-1) foi inferior à da heparina.

This study aimed to isolate, fractionate and evaluate the anticoagulant potential of iota-carrageenans (i-CARs) from Solieria filiformis when two extraction methods (M I and M II) were used. i-CARs were isolated with papain in 0.1M sodium acetate (pH 5.0) containing 5mM cystein and 5mM EDTA (M I) or water (80°C) (M II), and then their chemical composition of total carbohydrates, free sulfate (FS) and contaminant proteins were determined. i-CARs were submitted to anion-exchange chromatography (DEAE-cellulose) using a sodium chloride gradient,being evaluated the activated partial thromboplastin time (APTT) and prothrombin time of obtained fractions and compared to heparin (193IU mg-1). A rich fraction of anticoagulant was also submitted to 0.5 percent agarose gel electrophoresis procedure. The difference of yield between methods was 10.14 percent. The chemical composition of FS (29.40 percent) and the fractionation by DEAE-cellulose showed M I more effectiveness in the obtaining of i-CARs compared to M II. The APTT was also modified for i-CARs from M I. However, the in vitro anticoagulant activity of a rich fraction (8.52IU mg-1) was inferior to heparin.