Molecular characteristics of polysulfated fractions from the green seaweed Caulerpa cupressoides and actions on thrombin generation in vitro

Caulerpa cupressoides produces sulfated polysaccharides (Cc-SPs) with serpin-dependent anticoagulant effect, but their actions on thrombin generation (TG) are unknown. This study aimed to partially characterize Cc-SPs and examine their potential as modulators of TG. Infrared analysis characterized extract containing three ulvan fractions (Cc-SP1, -SP2 and -SP3) separated by DEAEcellulose chromatography, with differences in the relative proportions of sulfate (10.99-18.38%) and total sugars (46.59-51.12%), without presenting proteins. Charge density patterns and nonSPs varying from 8 to > 100 kDa on agarose and polyacrylamide gel electrophoresis by sequential staining with toluidine blue and stains-all were also confirmed by gel permeation chromatography. The molecular weight of Cc-SP2 was not altered after treatment with 0.4 M HCl up to 5 h. Only Cc-SP2 altered the activated partial thromboplastin time (15 ± 0.3 IU) vs. heparin (193 IU) and abolished at high concentrations (> 4.1 μg) TG by intrinsic pathway in 60-fold diluted human plasma, while at 4.1 μg attenuated TG by 33.87% delaying the lag phase (32 min.) vs. control (28 min.). Cc-SP2 induced concentration-dependent TG in system without cephalin. Heparin abolished TG at 4.15-fold lower amount, but did not stimulate TG. Therefore, Cc-SPs express dual effects on thrombosis in vitro.

Extraction, physical-chemical characterization and in vitro inhibitorypotential of thrombin generation of crude sulfated polysaccharides from Brazilian tropical seaweeds / Extração, caracterização físico-química e potencial inibitório in vitro da geração detrombina de polissacarídeos sulfatados brutos de algas marinhas tropicais brasileiras

The biotechnological value of macroalgae for screening assays of thrombin generation-TG using sulfated polysaccharides-SPs as substitutes to heparin has been poorly explored. Five Brazilian species of macroalgae (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides and C. racemosa) wereanalyzed and compared for their abundance, physical-chemical characteristics and in vitro anticoagulant assays of activated partial thromboplastin time-APTT, prothrombin time-PT and TG. Papain extraction yielded (p 100 kDa. These procedures,combined with the use of Stains-All, also indicated nonSPs. APTTs ranged from 2.81 (A. muscoides) to 21.30 IU(Halymenia sp.) vs. heparin (193 IU), and were dependent on sulfation of the crude SPs. PT was not altered. Withrespect to TG assay, crude SPs modified concentration-dependent and independently from molecular mass TGby both intrinsic/extrinsic pathways in 60-fold diluted human plasma, with total intrinsic inactivation using crudeSPs from A. muscoides in parallel to heparin (p < 0.05). Thrombosis in vitro is differentially modulated by distinctcrude SPs from Brazilian seaweeds.

O valor biotecnológico das macroalgas para ensaios de varredura de geração de trombina-GT pouco tem sido explorado usando polissacarídeos sulfatados-PSs como substitutos à heparina. Foramanalisadas e comparadas cinco espécies brasileiras de macroalgas (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides e C. racemosa) quanto à abundância, às característicasfísico-químicas e os ensaios anticoagulantes in vitro de tempo de tromboplastina parcial ativada-TTPA, aotempo de protrombina-TP e a GT. A extração com papaína rendeu (p 100 kDa. Esses procedimentos,combinados ao uso de azul de toluidina/Stains-All, indicaram também polissacarídeos-não sulfatados. OsTTPAs foram dependentes da sulfatação dos PSs brutos e variaram de 2,81 (A. muscoides) a 21,30 UI (Halymenia sp.) vs. heparina (193 UI). O TP não foi alterado. Com respeito ao ensaio de GT, os PSs brutos modificaram, dependente de concentração e independentemente de massa molecular, GT pelas viasintrínseca/extrínseca no plasma humano diluído 60 vezes, com inativação intrínseca total usando PSs brutosde A. muscoides em paralelo à heparina (p < 0,05). A trombose in vitro é modulada diferencialmente porPSs brutos distintos de algas marinhas brasileiras.

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.

In vitro inactivation of thrombin generation by polysulfated fractions isolated from the tropical coenocytic green seaweed Caulerpa racemosa (Caulerpaceae, Bryopsidales) / Inativação in vitro da geração de trombina por frações polissulfatadas isoladas da macroalga verde cenocítica tropical Caulerpa racemosa (Caulerpaceae; Bryopsidales)

Pharmacological efficacy of Caulerpa racemosa (Chlorophyta) sulfated polysaccharidic (SPs) fractions (F I→III) on models of coagulation and inflammation has been demonstrated, but not their effects on thrombin generation (TG). This study examined fractions for composition and physical-chemical characteristics and in vitro inactivation of TG by F I and F II in 60-fold diluted human plasma using continuous method. Papain-extraction yield of 0.7% revealed F I→III by DEAE-cellulose chromatography, with differences among the relative proportions of sulfate (17.37-24.00%), total sugars (30.03-48.34%) and absence of proteins. Charge density patterns and molecular sizes > 100 kDa of the fractions were verified by both agarose/polyacrylamide analyses, respectively. These electrophoreses combined with toluidine blue/Stains-All also indicated nonSPs. Anticoagulant effects of 4.76 (F I), 12.00 (F II) and 2.32 (F II) IU mg-1 by activated partial thromboplastin time test were recorded against heparin (193 IU mg-1), without changes in prothrombin time. Diluted plasma treated with F I and F II reduced concentration-dependent and sulfation pattern TG by both intrinsic and extrinsic pathways, with 50% inactivation by intrinsic pathway of F II even at 4.1 µg. Heparin abolished TG at least 4-fold lower. Therefore, C. racemosa produces SPs with TG inhibition.

Eficácia farmacológica de frações (F I→III) polissacarídicas sulfatadas (PSs) da Chlorophyta Caulerpa racemosa sobre modelos de coagulação e inflamação tem sido demonstrada, exceto seus efeitos sobre geração de trombina (GT). Examinaram-se frações quanto à composição, características físico-químicas e inativação in vitro de GT por F I e F II, em plasma humano diluído 60 vezes usando método contínuo. Rendimento de extração-papaína (0,7%) revelou, por cromatografia de DEAE -celulose, F I→III com diferenças entre as proporções relativas de sulfato (17,37-24,00%), açúcares totais (30,03-48,34%) e ausência de proteínas. Foram verificados, por ambas as análises agarose/poliacrilamida, graus de densidade de carga e tamanhos moleculares > 100 kDa das frações, respectivamente. Também essas eletroforeses, combinadas com azul de toluidina/Stains-All, indicaram polissacarídeos não sulfatados. Foram registrados, pelo teste do tempo de tromboplastina parcial ativada, efeitos anticoagulantes de 4,76 (F I), 12,00 (F II) e 2,32 (F II) UI mg-1 contra heparina (193 UI mg- 1), porém não modificando tempo de protrombina. Plasma diluído tratado com F I e F II reduziu GT por ambas as vias intrinsíca/extrínsica, dependente de concentração e grau de sulfatação, com F II em 4,1 µg apresentando eficácia de 50% pela via intrínsica. Heparina, quatro vezes menos, aboliu GT. Portanto, C. racemosa produz PSs com inibição de GT.

An anti-dengue and anti-herpetic polysulfated fraction isolated from the coenocytic green seaweed Caulerpa cupressoides inhibits thrombin generation in vitro / Uma fração polissulfatada anti- dengue e anti- herpética isolada da alga marinha verde cenocítica Caulerpa cupressoides inibe geração de trombina in vitro

The antiviral potency of Caulerpa cupressoides (Chlorophyta) sulfated polysaccharidic fractions (Cc -SP1→3) associated with thrombin generation (TG) assay is unknown. This study analyzed the structure of Cc-SP1 and its effects against herpes viruses (HSV-1 and HSV-2), dengue-virus (DENV-1) , human-metapneumovirus (HMPV) and adenovirus (AdV) in Vero and mosquito C6/36 cell-lines and as inhibitor of TG in 60- fold diluted human plasma using continuous system. Infrared analysis indicated ulvan containing 11% sulfate, 40.16% total sugars and 6% uronic acid. Procedures of agarose/polyacrylamide gels electrophoresis revealed two major components presenting sulfate and non SPs, with molecular dispersion ranging from 8 to > 100 kDa, respectively, as confirmed by gel permeation chromatography. Cc-SP1 did not cause cytotoxicity up to 1000 µg mL-1 and was more effective inhibitor of DENV-1 (96%, 0.35 µg mL-1) compared with HSV-1 (90%, 27 µg mL- 1) and HSV-2 (99.9%, 0.9 µg mL-1) in Vero cell-line, whose selectivity indexes were > 714, > 9.2 and > 131, respectively, but was inactive against HMPV, AdV and C6/36 cell-line using plate reduction assay. Cc-SP1 required concentration 20.8-fold higher of SPs than heparin for abolishes intrinsically TG, but at concentrations so far the anti-HSV-1. Therefore, TG assay provides data to guide studies of Cc-SP1 on anticoagulant/antiviral effects.

A potência antiviral de frações polissacarídicas sulfatadas de Caulerpa cupressoides (Chlorophyta) (Cc-PS1→3) é desconhecida e associada com ensaio de geração de trombina (GT). Analisaram-se de Cc-PS1 estrutura e efeitos contra herpesviroses (HSV-1 e HSV-2), vírus da dengue (DENV-1), metapneumovírus-humano (MPVH) e adenovírus (AdV) em linhagens de células Vero e de mosquito C6/36 e como inibidora de GT em plasma humano diluído 60 vezes usando sistema contínuo. Análise de infravermelho indicou 'ulvana' contendo sulfato (11%), açúcares totais (40,16%) e ácido urônico (6%). Procedimentos de electroforese em géis de agarose/poliacrilamida revelaram dois componentes majoritários apresentando sulfato e polissacarídeos não sulfatados, com dispersão molecular variando de 8 a > 100kDa, respectivamente, confirmada por cromatografia de permeação em gel. Cc-PS1 não causou citotoxicidade até 1000 µg mL-1 e se mostrou, em linhagem de célula Vero usando ensaio de redução de placas, inibidora efetiva de DENV-1 (96%; 0,35 µg mL-1) comparada com HSV-1 (90%; 27 µg mL-1) e HSV-2 (99,9%; 0,9 µg mL-1), cujos índices de seletividade foram > 714; > 9,2 e > 278, respectivamente, enquanto, demonstrou-se ineficaz sobre HMPV, AdV e linhagem de célula C6/36. Foi requerida concentração 20,8 vezes maior de Cc-PS1 que heparina para abolir GT intrinsicamente, mas em concentrações diferentes da anti-HSV-1. Portanto, ensaio de GT fornece dados para estudos direcionados sobre efeitos anticoagulante/antiviral de Cc-PS1.

Anti-inflammatory and Anti-resorptive Effects of Atorvastatin on Alveolar Bone Loss in Wistar Rats

Abstract The aim of this study was to evaluate the anti-inflammatory and anti-resorptive effect of atorvastatin (ATV) in an experimental alveolar bone loss (ABL) model. Wistar rats were subjected to ligature placement around the maxillary second molar for 11 days. The animals received 0.9% saline (2 mL/kg) or ATV (0.3, 3 or 27 mg/kg) daily by gavage. ABL was evaluated by resorption area and histopathological analysis. Serum bone-specific alkaline phosphatase (BALP) activity was also evaluated. Leukogram was performed at 0 h, 6th h, 2nd, 7th and 11th days. Kidney and liver conditions and the body mass variation were analyzed. ATV (3 and 27 mg/kg) inhibited ABL by 39% and 56%, respectively. Histopathological analysis showed that ATV 27 mg/kg prevented ABL and cemental resorption, and inflammatory cell infiltration induced by ligature. ATV (27 mg/kg) prevented serum BALP levels reduction. ATV (27 mg/kg) prevented leukocytosis and did not affect either kidney or liver function nor body mass weight. ATV showed a protecting effect in the ligature-induced periodontitis, without affecting system parameters, by inhibition of inflammatory process and by its anabolic activity on the alveolar bone.

Resumo O objetivo do estudo foi avaliar o efeito anti-inflamatório e anti-reabsortivo da atorvastatina (ATV) no modelo de perda óssea alveolar experimental (POA). Para isto, ratos Wistar foram submetidos a inserção de ligadura ao redor do segundo molar maxilar durante 11 dias. Os animais receberam diariamente, por gavagem, soro fisiológico a 0,9% (2 mg/kg) ou ATV (0,3, 3 ou 27 mg/kg). A POA foi avaliada pela área de reabsorção e análise histológica. Dosagens séricas da atividade de fosfatase alcalina óssea foram avaliadas. Leucograma foi realizado a 0 h, na 6a h, 2o, 7o e 11o dias. Foram analisadas condições de rim, fígado e variação de massa corpórea. As ATV (3 e 27 mg/kg) inibiram POA em 39% e 56%, respectivamente. A análise histopatológica mostrou que a ATV 27 mg/kg preveniu POA e reabsorção de cemento, e o infiltrado celular inflamatório induzido por ligadura. ATV (27 mg/kg) preveniu a redução dos níveis séricos de fosfatase alcalina óssea. ATV (27 mg/kg) preveniu leucocitose e não afetou função renal e hepática ou o peso corporal. ATV mostrou um efeito protetor na periodontite induzida por ligadura, sem afetar os parâmetros sistêmicos, através da inibição do processo inflamatório e pela atividade anabólica no osso alveolar.

Mild-acid hydrolysis of a native polysulfated fraction from Acanthophora muscoides generates sulfated oligosaccharides displaying in vitro thrombin generation inhibition / Hidrólise ácida branda de uma fração polissulfatada de Acanthophora muscoides gera oligossacarídeos sulfatados mostrando inibição de geração de trombina in vitro

A. muscoides (Rhodophyta) has three polysulfated fractions (-1, -2 and Am-3). Am-2 displayed anti -inflammation and serpin-independent anticoagulation effects; however, no effect of oligomers on thrombin-generation (TG) has been demonstrated. This study employed mild-acid hydrolysis to obtain low-molecular-size derivatives from Am-2 and compared in vitro inhibitory effects between intact Am-2 and its hydrolysates on a TG assay. The polysaccharidic extract was fractionated by DEAE-cellulose that revealed Am-2 eluted with 0.75-M NaCl containing sulfate (23%), hexoses (51%) and absence of proteins, and indicating, by one-dimension nuclear magnetic resonance, structure of galactan similar to that of the extract. The depolymerization with HCl (0.02 or 0.04-M, 60°C) for different times progressively reduced the charge density and the molecular-size of Am-2 based on electrophoresis in agarose and polyacrylamide gels, respectively, where at higher acid concentration and critical time up to 5h yielded fragment of Ì´14-kDa similar to that of unfractionated heparin (UHEP). Regarding the TG assay, intact Am-2 inhibited concentration- dependent intrinsic pathway, whereas its hydrolysates abolished it like UHEP, except the analog fragment (92.87% inhibition), when in 60-fold diluted human plasma using chromogenic method in a continuous system. The results reveal an alternative approach for the production of oligosaccharides from A. muscoides with TG inhibition.

A rodofícea A. muscoides possui três frações polissulfatadas (-1, -2 e Am-3). Am-2 mostrou efeito anti-inflamação e anticoagulação independente de serpina. Entretanto, não se demonstrou efeito de oligômeros sobre ensaio de geração de trombina (GT) . Este estudo empregou hidrólise ácida branda para obter derivados de tamanho molecular baixo de Am -2 e os efeitos inibitórios in vitro entre Am-2 intacta e hidrolisados comparados sobre um ensaio de GT. O extrato polissacarídico, fracionado por DEAE-celulose, revelou Am-2 eluída com NaCl-0,75M contendo sulfato (23%), hexoses (51%) e destituída de proteínas. E, ainda, por ressonância magnética nuclear-unidimensional, indicando galactana semelhante a do extrato. A depolimerização com HCl (0,02 ou 0,04-M; 60°C) reduziu, progressivamente durante tempos diferentes, a densidade de carga e o tamanho molecular de Am-2 baseada nas eletroforeses em géis de agarose e de poliacrilamida, respectivamente, em que, concentração ácida elevada e tempo crítico de até 5h renderam fragmento de Ì´14-kDa semelhante ao da heparina não fracionada (HEPNF). Já no ensaio de GT, Am-2 intacta, quando em plasma humano diluído 60 vezes, usando método cromogênico por meio de sistema contínuo, quem inibiu a via intrínseca dependente de concentração, ao passo que seus hidrolisados aboliram como HEPNF, exceto fragmento análogo (inibição 92,87%). Os resultados revelam uma abordagem alternativa para produzir oligossacarídeos de A. muscoides com inibição de GT.

Analysis of two precipitation methods on the yield, structural features and activity of sulfated polysaccharides from Gracilaria cornea (Rhodophyta) / Análise de duas metodologias de precipitação sobre o rendimento, características estruturais e atividade de polissacarídeos sulfatados de Gracilaria cornea (Rhodophyta)

The global demand for natural products from seaweeds has increased worldwide; however, no description of the use of isoamly alcohol (IAA) for obtaining of sulfated polysaccharides (SPs) has been reported. We investigated the efficiency of two precipitation methods (M) in obtaining SPs from the red seaweed Gracilaria cornea. SPs enzymatically isolated were concentrated with cetylpyridinium chloride (M I) or IAA (M II) and extracts were examined with regard to their yield, structural features and in vitro effects on the activated partial thromboplastin time (APTT) using normal human plasma and standard heparin (193 IU mg-1). Yield difference reached 12.99%. Quantitative determination of sulfate was similar between the two methods (Ì´26%), but extracts revealed different pattern on charge density by agarose gel electrophoresis. Whereas both extracts revealed as agarocolloids, alternative M II was also efficient for lipids, proteins and nucleic acids according to the infrared analysis. Extracts had virtually no effect on APPT (1.95 and 2 IU mg-1 for M I and M II, respectively). The results revealed IAA as an alternative solvent for obtaining SPs from the red seaweed G. cornea, depending on the industry' usage criterion.

A demanda global de produtos naturais de algas marinhas tem aumentado mundialmente. Entretanto, a obtenção de polissacarídeos sulfatados (PSs) com álcool isoamílico (AIA) não é relatada. Investigou-se a eficiência de dois métodos (M) de precipitação de PSs da alga marinha vermelha Gracilaria cornea. Os PSs isolados enzimaticamente foram concentrados com cloreto cetilpiridimínio (M I) ou AIA (M II). Os extratos foram examinados, segundo seu rendimento, características estruturais e efeitos in vitro sobre o tempo de tromboplastina parcial ativada (TTPA) usando plasma humano normal e heparina padrão (193 UI mg-1). A diferença nos rendimentos foi 12,99% e semelhante determinação quantitativa de sulfato foi obtida entre os métodos (Ì´26%). A eletroforese em gel de agarose revelou diferenças em termos de densidade de cargas entre os extratos. Enquanto ambos os extratos revelaram agarocoloides, o método M II também se mostrou alternativo para lipídios, proteínas e ácidos nucleicos de acordo com a análise de infravermelho. Os extratos praticamente não modificaram o TTPA (1,95 e 2 UI mg-1 para M I e M II, respectivamente). Os resultados revelaram AIA como um solvente alternativo para obtenção de PSs da alga marinha vermelha G. cornea, dependendo do critério de utilização na indústria.

Inhibition of coagulation proteases and thrombosis and sub-chronic toxicological study of a sulfated polysaccharidic fraction from the red alga Gelidiella acerosa / Inibição de proteases da coagulação e trombose e estudo toxicológico subcrônico de uma fração polissacarídica sulfatada da alga vermelha Gelidiella acerosa

Metabolites isolated from Gelidiella species (Rhodophyta) have been few studied. We evaluated a sulfated polysaccharidic fraction from G. acerosa collected from two Brazilian beaches on the northwestern coast of Brazil (Flecheiras-F and Pedra Rachada-PR) on coagulation proteases and thrombosis. Their toxicity in vivo was also assessed. Enzymatic extractions yielded 1.40%, and similar chromatographic profiles (DEAE-cellulose) were obtained, with fractions (Ga-I→V) containing differences among the relative proportions of sulfate (5-42%), and revealing charge density patterns by electrophoresis. Ga-IV-PR had a discrete effect (3.01 IU mg-1) on normal human coagulation compared with heparin (193 IU mg-1) and was tested on coagulation proteases (thrombin and factor Xa) in the presence of antithrombin and in a model of venous thrombosis in rats using thromboplastin as the thrombogenic stimulus. The systems were inhibited; but at higher doses (>1.0 mg kg-1), this fraction reverted the antithrombotic effect. Regarding the toxicological study, consecutive Ga-IV (9 mg kg-1) for 14 days did not cause mortality in mice, but some biochemical and hematological parameters were discretely altered. Histopathological analysis revealed that increased liver and spleen sizes had no toxicological significance. Therefore, G. acerosa does not biochemically change its matrix polysaccharide composition and proved to be safe antithrombotic agent.

Poucos estudos mostram metabólitos isolados de rodofíceas de espécies Gelidiella. Avaliou-se uma fração polissacarídica sulfatada de G. acerosa coletada a partir de duas praias brasileiras do Nordeste do Brasil (Flecheiras-F e Pedra Rachada-PR) sobre proteases da coagulação e trombose, e em ensaio de toxicidade in vivo. Extrações enzimáticas renderam 1,40% e foram obtidos perfis cromatográficos semelhantes (DEAE-celulose), apresentando frações (Ga-I→V), contendo diferenças entre as proporções relativas de sulfato (5-42%), além de a eletroforese revelar diferenças na densidade de carga. A Ga-IV-PR apresentou discreto efeito (3,01 UI mg-1) sobre a coagulação humana normal comparada à heparina (193 UI mg-1) e foi testada sobre proteases da coagulação (trombina e fator Xa) na presença de antitrombina e em um modelo de trombose venosa em ratos usando tromboplastina com estímulo trombogênico, sendo inibidos esses sistemas. Entretanto, em elevadas doses (>1,0 mg kg-1) o efeito antitrombótico foi revertido. No estudo toxicológico, Ga-IV (9 mg kg-1) consecutiva durante 14 dias não causou mortalidade em camundongos, mas alterou discretamente alguns parâmetros bioquímicos e hematológicos. O aumento nos tamanhos do fígado e baço não apresentou significância toxicológica, segunda análise histopatológica. Portanto, G. acerosa não muda bioquimicamente a composição de polissacarídeo de sua matriz e detém agente antitrombótico seguro.

Analysis of some chemical nutrients in four Brazilian tropical seaweeds / Análise de alguns nutrientes químicos em quatro algas marinhas tropicais brasileiras

Seaweeds have various chemical components with beneficial effects for human use; however, their nutritional values in Brazilian species are not well known. This study aimed to evaluate the content of water, ash, protein, carbohydrate, and lipid in four seaweeds (Hypnea musciformis, Solieria filiformis, Caulerpa cupressoides and C. mexicana). Algal constituents were determined by difference, gravimetric or colorimetric method, being the values expressed as g 100 g-1 dehydrated weight (d.w.). Results revealed that the water (10.7 ± 0.18-15.06 ± 1.14 g 100 g-1 d.w.), ash (7.79 ± 0.87-15.12 ± 0.51 g 100 g-1 d.w), protein (17.12 ± 0.99-20.79 ± 0.58 g 100 g-1 d.w.), lipid (0.33 ± 0.01-3.77 ± 0.13 g 100 g-1 d.w.) and carbohydrate (38.07 ± 0.32-54.24 ± 0.157 g 100 g-1 d.w.) contents varied between the species (p < 0.05). H. musciformis and S. filiformis (Rhodophyta) had highest ash contents (14.14 ± 1.23-15.12 ± 0.51 g 100 g-1 d.w.), whereas lipids were higher for Caulerpa species (Chlorophyta) (1.52 ± 0.17-3.77 ± 0.13 g 100 g-1 d.w.) (p < 0.05). Protein and carbohydrate were the most sources in all the species. Therefore, the studied seaweeds could be a potential source of food ingredients for diets.

As algas marinhas possuem vários componentes químicos com efeitos benéficos para uso humano. Entretanto, não são conhecidos bem seus valores nutricionais em espécies brasileiras. Objetivou-se avaliar os conteúdos de água, cinza, proteína, carboidrato e lipídio em quatro algas marinhas (Hypnea musciformis, Solieria filiformis, Caulerpa cupressoides e C. mexicana). Os constituintes das algas foram determinados por método de diferença, gravimétrico ou colorimétrico, sendo os valores expressos em g 100 g-1 de peso desidratado (p.d.). Os resultados revelaram que os conteúdos de água (10,7 ± 0,18-15,06 ± 1,14 g 100 g-1 p.d.), cinza (7,79 ± 0,87-15,12 ± 0,51 g 100 g-1 p.d.), proteína (17,12 ± 0,99-20,79 ± 0,58 g 100 g-1 p.d.), lipídio (0,33 ± 0,01-3,77 ± 0,13 g 100 g-1 p.d.) e carboidrato (38,07 ± 0,32-54,24 ± 0,157 g 100 g-1 p.d.) variaram entre as espécies (p < 0,05). H. musciformis e S. filiformis (Rhodophyta) tiveram os maiores conteúdos de cinza (14,14 ± 1,23-15,12 ± 0,51 g 100 g-1 p.d.), enquanto as espécies Caulerpa (Chlorophyta) foram para lipídio (1,52 ± 0,17-3,77 ± 0,13 g 100 g-1 p.d.) (p < 0,05). Todas as espécies foram fontes maiores em proteína e carboidrato. Portanto, as algas marinhas estudadas poderiam ser uma fonte de ingrediente alimentar potencial para dietas.

Structural features and assessment of zymosan-induced arthritis in rat temporomandibular joint model using sulfated polysaccharide / Características estruturais e ensaio de artrite induzida por zimosam em modelo de articulação temporomandibular de ratos usando polissacarídeo sulfatado

The green seaweed Caulerpa cupressoides var. lycopodium contains three SPs fractions (Cc-SP1, Cc-SP2 and Cc-SP3). Cc-SP1 and Cc-SP2 had anticoagulant (in vitro), pro- and antithrombotic, antinociceptive and/or anti-inflammatory (in vivo ) effects. This study analyzed structural features and the antinociceptive and anti-inflammatory effects of Cc-SP1 on zymosan-induced acute arthritis of the rat temporomandibular joint (TMJ). Cc -SP1 was investigated by infrared technique. Male Wistar rats (200-240 g) received subcutaneously (s.c.) Cc-SP1 1h prior to intra-articular (i.art.) injection of zymosan (2 mg joint -1) or saline (0.9%) into the left TMJ. Mechanical hypernociception was measured by the electronic Von Frey method in the basal and 4h after zymosan injection. Animals were euthanized 6h after zymosan injection and the TMJ cavity was removed for total leukocyte counts from the synovial fluid and myeloperoxidase (MPO) activity assessment. Cc-SP1 (1, 3 or 9 mg kg-1) containing sulfate ester, galactose-6-sulfate, uronic acid and glycosidic linkages reduced zymosan-induced hypernociception (78.12, 81.13 and 87.43%, respectively, p < 0.01), and inhibited the total leukocyte influx (85, 88.14 and 89.95%, respectively, p < 0.01), being confirmed by MPO activity (p < 0.05). Therefore, Cc-SP1 reveals a pharmacological tool for treating inflammatory arthropathies.

A alga marinha verde Caulerpa cupressoides var. lycopodium contém três frações de PSs (Cc-PS1; Cc-PS2 e Cc-PS3). Cc-PS1 e Cc-PS2 apresentaram efeitos anticoagulante (in vitro), pró- e antitrombótico, antinociceptivo e/ou anti-inflammatório (in vivo). Analisaram-se as características estruturais e os efeitos antinociceptivo e anti-inflamatório de Cc-PS1 sobre artrite aguda induzida por zimosam na articulação temporomandibular (ATM) de ratos. Foi investigada a Cc-PS1 por técnica de infravermelho. Ratos machos Wistar (200-240 g) receberam subcutaneamente (s.c.) Cc-PS1 1h antes de injeção intra-articular (i.art.) de zimosam (2 mg articulação-1) ou salina (0,9%) na ATM esquerda. A hipernocicepção mecânica foi mensurada por método Von Frey elétrico em zero e 4h após injeção de zimosam. Os animais foram entanasiados após 6h de injeção de zimosam e a cavidade da ATM foi removida para contagem de leucócitos totais do fluído sinovial e ensaio da atividade de mieloperoxidase (MPO). A Cc-PS1 (1; 3 ou 9 mg kg-1) contendo éster sulfato, galactose-6-sulfato, ácido urônico e ligações glicosídicas reduziu a hipernocicepção induzida por zimosam (78,12; 81,13 e 87,4%, respectivamente; p < 0,01), além de inibir o influxo de leucócitos totais (85; 88,14 e 89,95%, respectivamente; p < 0,01), sendo, ainda, confirmado pela atividade de MPO (p < 0,05). Portanto, a Cc-PS1 revela como uma ferramenta farmacológica para tratar de artropatias inflamatórias.

Comparative study of sulfated polysaccharides from Caulerpa spp. (Chlorophyceae). Biotechnological tool for species identification? / Estudo comparativo dos polissacarídeos sulfatados de clorofíceas Caulerpa spp. (Chlorophyceae). Ferramenta biotecnológica na identificação de espécies?

Studies on macromolecules isolated from marine algae suggested sulfated polysaccharides (SPs) as possible molecular markers for species. We evaluated isolated and fractionated SPs from the green marine algae Caulerpa cupressoides, C. prolifera and C. racemosa collected at Pacheco Beach, as possible taxonomic molecular indicators. Total SPs were extracted with papain in 100 mM sodium acetate buffer (pH 5.0) containing cysteine and EDTA (both 5 mM), followed by ion-exchange chromatography on DEAE-cellulose using a NaCl gradient. The obtained fractions were analyzed by 0.5% agarose gel electrophoresis. Anticoagulant assays employing normal human plasma and standard heparin (193 IU mg-1) by the activated partial thromboplastin time (APTT) test were also performed as comparison parameters. Low yields, and similar chromatographic profiles were found among species' SPs, but electrophoresis revealed distinct SPs resolution patterns. The changes in APTT of SP fractions were dependent on charge density as showed by electrophoresis profiles. Activities were 17.37 (C. cupressoides), 22.17 (C. racemosa) and 25.64 (C. prolifera) IU mg-1, respectively, similar to a previous study using the first and second species. The results suggest that comparative studies of SPs isolated from seaweeds may be an important tool for the identification of Caulerpaceae.

A utilização de macromoléculas isoladas de organismos marinhos sugere correlacionar características em estudos taxonômicos e a investigação comparativa de polissacarídeos sulfatados (PSs) de algas despertam seu interesse como marcadores moleculares. Objetivou-se avaliar PSs isolados e fracionados das algas marinhas verdes Caulerpa cupressoides, C. prolifera e C. racemosa, coletadas na Praia do Pacheco, Estado do Ceará, como possíveis indicadores moleculares taxonômicos. Os PSs totais foram extraídos com papaína em tampão acetato de sódio 100 mM (pH 5,0) contendo cisteína e EDTA (ambos 5 mM), seguido por cromatografia de troca iônica em coluna de DEAE-celulose utilizando um gradiente de NaCl. As frações obtidas foram analisadas por eletroforese em gel de agarose a 0,5%. Ensaios anticoagulantes, utilizando o teste do tempo de tromboplastina parcial ativada (TTPA) com plasma humano normal e heparina padrão (193 UI mg-1), também foram realizados como parâmetros de comparação. Verificaram-se baixos rendimentos e semelhantes perfis cromatográficos entre os PSs das espécies, porém revelando, por eletroforese, diferenças moleculares marcantes. As alterações no TTPA das frações de PS foram dependentes da densidade de cargas negativas mostradas nos perfis eletroforéticos, cujas atividades foram 17,37 (C. cupressoides), 22,17 (C. racemosa) e 25,64 (C. prolifera) UI mg-1, respectivamente, e tal propriedade justificou um estudo já realizado utilizando a primeira e segunda espécies. Os resultados sugerem que estudos comparativos de PSs isolados de algas marinhas possam vir a ser uma ferramenta importante na identificação de Caulerpaceae.

Glicosaminoglicanos isolados da pele de palombeta (Chloroscombrus chrysurus) e guaiúba (Ocyurus chrysurus): características e implicações biológicas / Glycosaminoglycans isolated from the skin of 'palombeta' (Chloroscombrus chrysurus) and 'guaiúba' (Ocyurus chrysurus): features and biological implications

O Brasil abriga uma das maiores biodiversidades marinhas do mundo, favorecendo a descoberta de fontes alternativas de compostos farmacológicos. Desta forma, objetivou-se avaliar o potencial anticoagulante de glicosaminoglicanos (GAGs) isolados das peles da palombeta (Chloroscombrus chrysurus) e guaiúba (Ocyurus chrysurus). Os GAGs foram extraídos com papaína bruta em tampão acetato de sódio 0,1 M (pH 5,0) contendo cisteína 5 mM e EDTA 5 mM, seguido por cromatografia de troca iônica do extrato total em coluna de DEAE-celulose. As frações obtidas foram analisadas quanto à composição química (proteínas contaminantes e carboidratos totais) e os GAGs identificados por eletroforese em gel de agarose a 0,5%. Os ensaios de atividade anticoagulante foram realizados por meio do tempo de tromboplastina parcial ativada (TTPA) usando plasma humano normal e heparina-padrão (193,00 UI mg-1). O procedimento de obtenção e fracionamento dos GAGs mostrou-se eficiente, indicando semelhantes perfis cromatográficos entre as espécies avaliadas e, revelando para C. chrysurus, bandas com mobilidades semelhantes ao dermatam sulfato e com atividade de apenas 3,30 UI mg-1.

A great number of pharmacological compounds is found in the Brazilian marine diversity. This study evaluated the anticoagulant potential of glycosaminoglycans (GAGs) isolated from the skin of 'palombeta' Chloroscombrus chrysurus and 'guaiúba' Ocyurus chrysurus. GAGs were extracted with crude papain in 0.1 M sodium acetate buffer (pH 5.0) containing 5 mM cysteine and 5 mM EDTA, followed by ion exchange chromatography on DEAE-cellulose column. The chemical composition (contaminant proteins and total carbohydrates) and the analysis by 0.5% agarose gel electrophoresis of fractions were also determined. Anticoagulant assays were performed by activated partial thromboplastin time (APTT) using normal human plasma and standard heparin (193.00 IU mg-1). The obtaining and fractionation procedures of GAGs were effective and similar chromatographic profiles were verified between the species. A similar mobility to dermatan sulfate was revealed for C. chrysurus. This GAG also showed a low activity of 3.30 IU mg-1.

Antimicrobial effect of a crude sulfated polysaccharide from the red seaweed Gracilaria ornata

The aim of this study was to determine the yield, chemical composition, specific rotation (SR), infrared (IR) spectroscopy and the effect on bacterial growth of a crude sulfated polysaccharide (SP) from the red marine alga G. ornata (Go). Go-1 (25°C), Go-2 (80°C), and Go-3 (80°C) were sequentially extracted and yielded 9.2%. The contents of sulfate (5.88-10.3%) and proteins (0.1-3.7%) were small. The values of SR were [µ]D20°f -19.0, -51.0, and -56.5, respectively. IR spectrums showed the presence of galactose-4 sulfate and absence of 3,6-anydrogalactose-2 sulfate, galactose-6 sulfate and galactose-2 sulfate. SR and IR techniques confirmed SPs. Go-3 was tested on the growth of bacteria (Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogens, Escherichia coli, Pseudomonas aeruginosa, Salmonela choleraesuis and Salmonela typhi), but only E. coli was inhibited.

Analysis of two drying methods on the yield and activity of sulfated polysaccharides extracted from Halymenia sp (Rhodophyceae) / Análise de dois métodos de secagem sobre o rendimento e atividade de polissacarídeos sulfatados extraídos da rodofícea Halymenia sp

Sulfated polysaccharides (SPs) have attracted growing interest for various biotechnological applications. We evaluated the efficiency of two methods of drying SPs (M I and II) extracted from Halymenia sp Rhodophyceae in order to compare the yield, purification and anticoagulant activity. The total SPs (TSPs) were first extracted with papain in 100 mM sodium acetate (pH 5.0) containing cysteine and EDTA (5 mM). The TSPs obtained were dried in an oven (M I) or lyophilized (M II) and then examined by ion exchange chromatography (DEAE-cellulose) using the NaCl gradient technique. The fractions were analyzed by 0.5% agarose gel electrophoresis and the in vitro anticoagulant activity was evaluated by the activated partial thromboplastin time test using normal human plasma and compared to heparin (HEP) (193.00 IU mg-1). There was a difference in TSP yield of 19.05% and similar chromatographic SP profiles. Electrophoresis revealed fractions with distinct resolutions. The fractions eluted with 0.75 M of salt (M I and II) were the most active, measuring 27.40 and 72.66 IU mg-1, respectively, when compared to HEP. Therefore, obtaining SP with anticoagulant activity from Halymenia sp. is more efficient by freeze-drying.

Os polissacarídeos sulfatados (PS) têm despertado interesse crescente para diversas aplicações em biotecnologia. Avaliou-se a eficiência de duas metodologias de secagem de PSs (M I e II) extraídos da rodofícea Halymenia sp. a fim de se comparar o rendimento, a purificação e a atividade anticoagulante. Inicialmente, os PS totais (PSTs) foram extraídos com papaína em tampão acetato de sódio 100 mM (pH 5,0) contendo cisteína e EDTA (5 mM). Os PSTs foram secados em estufa (M I) ou liofilizados (M II), seguido por cromatografia de troca iônica (DEAE-celulose) utilizando um gradiente de NaCl. As frações foram analisadas por eletroforese em gel de agarose a 0,5% e a atividade anticoagulante "in vitro" avaliada por meio do teste do tempo de tromboplastina parcial ativada usando plasma humano normal e comparadas à heparina (HEP) (193.00 UI mg-1). Verificaram-se diferença no rendimento de PSTs (19,05%) e semelhantes perfis cromatográficos de PS, revelando, por eletroforese, frações de PS distintas em grau de resolução entre os métodos. As frações eluídas com 0,75 M de sal (M I e II) foram as mais ativas, com valores de atividade da ordem de 27,40 e 72,66 UI mg-1, respectivamente, em relação à HEP. Portanto, a secagem por liofilização seria uma forma mais eficaz na obtenção de PSs com atividade anticoagulante de Halymenia sp.

Effect of a crude sulfated polysaccharide from Halymenia floresia (Rhodophyta) on gastrointestinal smooth muscle contractility

The aim of this work was to study the effect of Halymenia floresia (Hf) on duodenum contractility, and on experimental protocols of gastric compliance (GC) in rats. Fraction Hf2s exhibited a concentration-dependent myocontractile effect (EC50 12.48 µg/ml), and an inhibitory effect after consecutive washing. The contractile response promoted by Hf2s in the duodenum strips was completely inhibited by verapamil, and the effects were prevented in the presence of Ca2+-free medium. The pretreatment with atropine prevented the Hf2s myocontractile effect. Hf2s was also capable to decrease the GC (from 3.8±0.06 to 3.4±0.13 ml, P<0.05), which did not return to basal levels after more 50 min of observation. These results indicated that the algal polysaccharide possessed in vitro and in vivo gastrointestinal effects.

Anticoagulant activity of a sulfated polysaccharide isolated from the green seaweed Caulerpa cupressoides

The aim of this study was to evaluate certain molecular characteristics of a sulfated polysaccharide (SPs) with anticoagulant properties, isolated from Caulerpa cupressoides (Chlorophyta). Crude SPs were extracted by proteolytic digestion (papain), followed by ion-exchange chromatography on a DEAE-cellulose column. The fractions obtained were analyzed for molecular mass, 0.5 percent agarose gel electrophoresis and chemical composition. The activated partial thromboplastin time (APTT) test was applied using normal human plasma and standard heparin (HEP) (193 IU mg-1). The yield was ~ 3 percent, and the chromatography procedure separated the material into three different SP fractions (F I, F II and F III), eluted at the concentrations of 0.50, 0.75 and 1.00 M of NaCl, respectively. Only fraction F II was active (24.62 IU mg-1), with high sulfate content (23.79 percent) and number of molecular mass peaks. Therefore, the APTT of a fraction isolated from C. cupressoides was less potent than HEP.

Anticoagulant properties of a crude sulfated polysaccharide from the red marine alga Halymenia floresia (Clemente) C Agardh / Propriedades anticoagulantes de um polissacarídeo sulfatado bruto da alga marinha vermelha Halymenia floresia (Clemente) C Agardh

Alternative sources of anticoagulants have arisen as a result of the increasing demand for safer anticoagulant clinical therapy, and the sulfated polysaccharides of seaweeds have gained attention in biomedicine. In this study, crude sulfated polysaccharide fractions (denominated Hf1, Hf2 and Hf3) were obtained from the red marine alga Halymenia floresia and the anticoagulant properties of a soluble crude polysaccharide fraction (Hf2s) were assayed. The three differential extractions yielded 38.6%. The polysaccharides are composed mainly of galactose, with small amounts of xylose and glucose. The anticoagulant properties of Hf2s containing 53.8% sulfate and 3% protein was also compared to those of heparin (193.0 IU mg-1) by assays of activated partial thromboplastin time (APTT) and thrombin time (TT) using normal human plasma. Hf2s showed a higher anticoagulant activity (68.4 IU mg-1) than those of Hf1s and Hf3s, whose activities were 37.6 and 36.6 IU mg-1, respectively. The compound was less active than heparin, but its anticoagulant mechanism suggested that it is dependent on cofactor heparin II to inhibit thrombin activity, but not on cofactors VIII and IX. Therefore, the polysaccharide from H. floresia interfered on coagulation cascade.

O aumento da demanda por anticoagulantes para a terapia clínica tem motivado a busca por fontes alternativas de anticoagulantes mais seguros e os polissacarídeos sulfatados de algas marinhas têm ganhado atenção na biomedicina. Objetivou-se obter frações de polissacarídeos sulfatados brutos (denominadas Hf1; Hf2 e Hf3) da alga marinha vermelha Halymenia floresia e para avaliar as propriedades anticoagulantes de uma fração polissacarídica bruta solúvel anticoagulante (Hf2s). As três extrações diferenciais renderam 38,60%. Os polissacarídeos são principalmente compostos de galactose com pequenas quantidades de xilose e glucose. As propriedades anticoagulantes da Hf2s, contendo 53,80% de sulfato e 3% de proteínas, foram também comparadas com a heparina (193,00 UI mg-1) pelo ensaio do tempo de tromboplastina parcial ativada (TTPA) e tempo de trombina (TT), usando plasma humano normal. A Hf2s apresentou maior atividade anticoagulante (68,40 UI mg-1) que Hf1s a Hf3s, cujas atividades foram 37,60 e 36,60 UI mg-1, respectivamente. O composto foi menos ativo que a heparina, mas sugere-se que o mecanismo anticoagulante seja dependente do cofator II da heparina para inibição da atividade da trombina, exceto pelos cofatores VIII a IX. Portanto, o polissacarídeo de H. floresia interferiu na cascata de coagulação.

Anticoagulant properties of a crude sulfated polysaccharide from the red marine alga Halymenia floresia (Clemente) C. Agardh - doi: 10.4025/actascibiolsci.v33i3.6402 / Anticoagulant properties of a crude sulfated polysaccharide from the red marine alga Halymenia floresia (Clemente) C. Agardh - doi: 10.4025/actascibiolsci.v33i3.6402

Alternative sources of anticoagulants have arisen as a result of the increasing demand for safer anticoagulant clinical therapy, and the sulfated polysaccharides of seaweeds have gained attention in biomedicine. In this study, crude sulfated polysaccharide fractions (denominated Hf1, Hf2 and Hf3) were obtained from the red marine alga Halymenia floresia and the anticoagulant properties of a soluble crude polysaccharide fraction (Hf2s) were assayed. The three differential extractions yielded 38.6%. The polysaccharides are composed mainly of galactose, with small amounts of xylose and glucose. The anticoagulant properties of Hf2s containing 53.8% sulfate and 3% protein was also compared to those of heparin (193.0 IU mg-1) by assays of activated partial thromboplastin time (APTT) and thrombin time (TT) using normal human plasma. Hf2s showed a higher anticoagulant activity (68.4 IU mg-1) than those of Hf1s and Hf3s, whose activities were 37.6 and 36.6 IU mg-1, respectively. The compound was less active than heparin, but its anticoagulant mechanism suggested that it is dependent on cofactor heparin II to inhibit thrombin activity, but not on cofactors VIII and IX. Therefore, the polysaccharide from H. floresia interfered on coagulation cascade.

Alternative sources of anticoagulants have arisen as a result of the increasing demand for safer anticoagulant clinical therapy, and the sulfated polysaccharides of seaweeds have gained attention in biomedicine. In this study, crude sulfated polysaccharide fractions (denominated Hf1, Hf2 and Hf3) were obtained from the red marine alga Halymenia floresia and the anticoagulant properties of a soluble crude polysaccharide fraction (Hf2s) were assayed. The three differential extractions yielded 38.6%. The polysaccharides are composed mainly of galactose, with small amounts of xylose and glucose. The anticoagulant properties of Hf2s containing 53.8% sulfate and 3% protein was also compared to those of heparin (193.0 IU mg-1) by assays of activated partial thromboplastin time (APTT) and thrombin time (TT) using normal human plasma. Hf2s showed a higher anticoagulant activity (68.4 IU mg-1) than those of Hf1s and Hf3s, whose activities were 37.6 and 36.6 IU mg-1, respectively. The compound was less active than heparin, but its anticoagulant mechanism suggested that it is dependent on cofactor heparin II to inhibit thrombin activity, but not on cofactors VIII and IX. Therefore, the polysaccharide from H. floresia interfered on coagulation cascade.

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).