Maria Eugênia R. Duarte

Inhibitory effect of sulfated galactans from the marine alga Bostrychia montagnei on herpes simplex virus replication in vitro.

Sulfated polysaccharides exhibit many biological properties such as antiviral and anticoagulant activities. Herein, we report the antiviral activity of sulfated galactans extracted from the red sea-weed Bostrychia montagnei against herpes simplex virus types 1 (strain F and the thymidine kinase-deficient strains Field and B2006) and 2 (strain G). Two crude extracts obtained with cold and hot water as well as some fractions obtained by anion exchange chromatography, inhibited significantly the replication of the different strains of herpesviruses as determined by plaque reduction assays. The inhibitory effect of the compounds studied here took place only when they were added during the adsorption period. They were found to be highly selective antiviral substances, causing no impairment of Vero cell viability. Furthermore, they had no direct inactivating effect on virions by incubation in a virucidal assay. The antiviral activity could be correlated with the molecular weight and sulfate content of the polysaccharides. Although sulfated polysaccharides are generally endowed with anticoagulant properties, the results of the activated partial thromboplastin time and the thrombine time assays indicated that the natural sulfated polysaccharides from Bostrychia montagnei have very low anticoagulant activity, confirming that there is no relation between the antiviral and anticoagulant properties.

Chemical structure and antiviral activity of the sulfated heterorhamnan isolated from the green seaweed Gayralia oxysperma

A homogeneous sulfated heterorhamnan was obtained by aqueous extraction, then by ultrafiltration from the green seaweed Gayralia oxysperma. Besides alpha-L-rhamnose it contains glucuronic and galacturonic acids, xylose and glucose. The structure was established by methylation analyses of the carboxyl-reduced, carboxyl-reduced/desulfated, carboxyl-reduced/Smith-degraded, and carboxyl-reduced/Smith-degraded/desulfated products and 1D, 2D NMR spectroscopy analyses. The heterorhamnan backbone is constituted by 3- and 2-linked rhamnosyl units (1.00:0.80), the latter being approximately 50% substituted at C-3 by side chains containing 2-sulfated glucuronic and galacturonic acids and xylosyl units. The 3- and 2-linked rhamnosyl units are unsulfated (20%), disulfated (16%), and mostly monosulfated at C-2 (27%) and C-4 (37%). The branched and sulfated heterorhamnan had high and specific activity against herpes simplex virus.

Effects of sulfated polysaccharide and alcoholic extracts from green seaweed Ulva fasciata on anthracnose severity and growth of common bean ( Phaseolus vulgaris L.)

Seaweed compounds can play important roles in either plant growth promoting or plant-pathogen interactions. The aim of this study was to evaluate the potential of crude extracts and sulfated polysaccharides isolated from the green macroalga Ulva fasciata in the control of bean anthracnose caused by Colletotrichum lindemuthianum as well the influence in the plant growth. Sulfated polysaccharides, called ulvan, were extracted with hot water, precipitated in ethanol and identified by chemical and spectroscopic methods (13C NMR). The contents of sulfate, uronic acid, protein and monosaccharides were determined. In order to obtain the crude extracts, the dried alga was extracted with methanol in Soxhlet apparatus or with ethanol at room temperature. The effect of ulvan (0.1; 1; and 10 mg ml−1) and crude extracts were tested in vitro on conidial germination and mycelial growth of C. lindemuthianum and also on the seed germination and seedling length of bean. Under greenhouse conditions, bean plants (Phaseolus vulgaris L.) were sprayed twice with ulvan or extracts and inoculated two days after the second application. The anthracnose severity was evaluated 7 days and aerial plant dry weight 14 days after inoculation. The soluble methanolic extract inhibited the mycelial growth of C. lindemuthianum in vitro, but did not reduce the disease severity under greenhouse conditions. Foliar spray of methanolic extracts enhanced the dry weight of bean plants by 20%. In contrast, ulvan increased in vitro the mycelium growth and the conidia germination of the fungus, but in greenhouse, the spray of 10 mg ml−1 ulvan reduced the anthracnose severity by 38% without affecting plant growth. The results indicate that ulvan is probably able to induce resistance to bean anthracnose.ZusammenfassungVerbindungen aus Algen können eine wichtige Rolle bei der Förderung des Pflanzenwachstums und bei Pflanze-Pathogen Interaktionen spielen. Ziel dieser Arbeit war es daher, das Potenzial von Rohextrakten und sulfonierten Polysacchariden aus der grünen Macroalge Ulva fasciata für die Bekämpfung der durch Colletotrichum lindemuthianum verursachten Brennfleckenkrankheit an Buschbohne (Phaseolus vulgaris L.) und für die Stimulation des Wachstums dieser Pflanze auszuwerten. Die sulfonierten Polysaccharide, nämlich Ulvane, wurden mit warmem Wasser extrahiert, mit Ethanol gefällt und mit Hilfe von spektroskopischen Methoden (13C-NMR) identifiziert. Der Gehalt an Sulfat, Uronsäure, Proteinen und Monosacchariden wurde bestimmt. Um die Rohextrakte zu gewinnen, wurden die getrockneten Algen mit Methanol im Soxhlet-Gerät oder mit Ethanol bei Raumtemperatur extra-hiert. Die in-vitro-Wirkung von Ulvan (0,1, 1 und 10 mg ml−1) und der Rohextrakte, sowohl auf die Konidienkeimung und das Myzelwachstum von C. lindemuthianum als auch auf die Keimungsfähigkeit und die Länge der Sämlingen von Bohnen, wurde ermittelt. Unter Gewächshausbedingungen wurden die Bohnenpflanzen zweimal mit Ulvan oder Rohextrakten besprüht und zwei Tage danach inokuliert. Der Krankheitsbefall und die pflanzliche Trockenmasse wurden 7 bzw. 14 Tagen nach der Inokulation ausgewertet. Der methanolische Extrakt reduzierte das pilzliche Myzelwachstum zwar in vitro, die Blattapplikation der methanolischen Extrakte wies aber keinen Effekt auf den Krankheitbefall im Gewächhaus auf. Die Trockenmasse der Bohnenpflanzen wurde um 20% erhöht. Im Gegensatz dazu verbesserte Ulvan das Myzelwachstum und die Konidienkeimung des Pilzes in vitro, während seine Blattapplikation im Gewäschshaus bei einer Konzentration von 10 mg ml−1 den Krankheitsbefall um 38% reduzierte, ohne Auswirkungen auf das Pflanzenwachstum zu haben. Diese Ergebnisse deuten darauf hin, dass Ulvan möglicherweise die Resistenz von Buschbohnenpflanzen gegen die Brennfleckenkrankheit induziert.

Brown algae overproduce cell wall polysaccharides as a protection mechanism against the heavy metal toxicity.

Brown algae are often used as heavy metal biomonitors and biosorbents because they can accumulate high concentrations of metals. Cation-exchange performed by cell wall polysaccharides is pointed out as the main chemical mechanism for the metal sequestration. Here, we biochemically investigated if the brown alga Padina gymnospora living in a heavy metal contaminated area would modify their polysaccharidic content. We exposed non-living biomass to Cd and Pb and studied the metals adsorption and localization. We found that raw dried polysaccharides, sulfate groups, uronic acids, fucose, mannose, and galactose were significantly higher in contaminated algae compared with the control ones. Metal concentrations adsorbed by non-living biomass were rising comparatively to the tested concentrations. Electron microscopy showed numerous granules in the cell walls and X-ray microanalysis revealed Cd as the main element. We concluded that P. gymnospora overproduces cell wall polysaccharides when exposed to high metal concentrations as a defense mechanism.

Selective sulfation of carrageenans and the influence of sulfate regiochemistry on anticoagulant properties

Sulfated polysaccharides are recognized for their broad range of biological activities, including anticoagulant properties. The positions occupied by the sulfate groups are often related to the level of the inherent biological activity. Herein the naturally sulfated galactans, kappa-, iota- and theta-carrageenan, were additionally sulfated by regioselective means. The anticoagulant activity of the resulting samples was then studied using the aPTT in vitro assay. The influence of sulfate regiochemistry on the anticoagulant activity was evaluated. From kappa-carrageenan three rare polysaccharides were synthesized, one of them involved a synthetic route with an amphiphilic polysaccharide intermediate containing pivaloyl groups. Iota- and theta-carrageenan were utilized in a selective C6 sulfation at β-D-Galp units to produce different structures comprising trisulfated diads. All the samples were characterized by NMR (1D and 2D). The resulting aPPT measurements suggested that sulfation at C2 of 3,6-anhydro-α-D-Galp and C6 of β-D-Galp increased the anticoagulant activity.

Chemical structure of the complex pyruvylated and sulfated agaran from the red seaweed Palisada flagellifera (Ceramiales, Rhodophyta).

A homogeneous agaran fraction from Palisada flagellifera (Laurencia complex, Rhodomelaceae, Ceramiales) was obtained by aqueous room-temperature extraction, followed by ion-exchange chromatography. This galactan presents a highly complex structure with at least 18 different types of derivatives. The A units were found mostly pyruvylated, 2-sulfated (∼34%), and 6-methylated (∼34%), with the latter partially 2- and 2,4-sulfated. Minor amounts of β-D-galactopyranosyl units 2-, 6- and 2,6-sulfated, 6-glycosylated, and non-substituted are also present. The B-units are L-sugars composed predominantly of their cyclized derivatives, 3,6-anhydrogalactose and 3,6-anhydro-2-O-methylgalactose (∼56%). The former are linked to β-D-galactosyl (6-methyl) (6-glycosylated) units, as well as to 4,6-O-(1-carboxyethylidene)-β-D-galactose 2-sulfate in the proportion of 3:1.8, respectively. A significant amount (∼18%) of the α-L-galactopyranosyl units are linked to pyruvylated β-D-galactose 2-sulfate residues. An important part of the B-units (20%) is represented by α-L-galactose 6-sulfate substituted on C-3 by xylosyl, galactosyl and/or 2,3-di-O-methylgalactose units or sulfate groups that preclude their cyclization to 3,6-anhydrogalactosyl derivative. The precursor units are present in relatively low percentages. Kinetic studies suggest that in P. flagellifera agaran the cyclizable units are linked to 6-O-methyl-β-D-galactosyl and/or β-D-galactosyl units (6-glycosylated). The structural complexity of this polysaccharide is increased by the presence of 2- and 3,6-sulfated α-L-galactoses, with the latter additionally 2-O-methylated. Therefore, the major subfraction obtained from the cold extract contains structurally complex sulfated, methylated, and pyruvylated agaran.

The structure of a galactan sulfate from the red seaweed Bostrychia montagnei

The sulfated, methylated galactan isolated from the red seaweed Bostrychia montagnei, showed an unusually narrow structural dispersion. This agaran has the defining linear backbone of alternating 3-linked beta-D-galactopyranosyl units and 4-linked alpha-L-galactopyranosyl and 3,6-anhydrogalactopyranosyl residues. The D-units have C-6 methylation, C-6 single stubs of xylopyranosyl and minor to trace amounts of (possible) C-6 linked single stubs of galactopyranosyl. These units are mainly sulfated on C-4 with lesser sulfation at C-6 and minor at C-2. The L-residues are mainly methylated on C-2 of the 3,6-anhydrogalactopyranosyl and sulfated on C-3 of the L-galactopyranosyl; minor amounts of 2,3- and 3,6-disulfated and 2-O-methyl or 2-O-glycosyl 3-sulfated L-galactopyranosyl were also found.

Differential inhibition of dengue virus infection in mammalian and mosquito cells by iota-carrageenan

The antiviral activity against dengue virus-2 (DENV-2) of carrageenans reported here has shown a differential susceptibility of C6/36 HT and Vero cells, taken as models of mosquito and mammalian cells, depending on the structural class of polysaccharides: all polysaccharides blocked DENV-2 infection in monkey Vero cells, but only iota-carrageenans were virus inhibitors in mosquito cells. However, iota-carrageenans were less effective in mosquito cells in comparison with mammalian cells with effective concentration 50 % (EC(50)) values in C6/36 HT cells 4.9-17.5-fold higher than in Vero cells, as determined by virus yield reduction assay. The mode of action of iota-carrageenan in both cell types was strikingly different: in Vero cells the inhibitory activity was exerted only at the initiation of the cycle, affecting virion binding, whereas in mosquito cells DENV-2 adsorption was not affected and comparable levels of inhibition were obtained if the compound was added to cells together with the virus, after 8 h of infection or by cell pre-treatment before infection. Furthermore, iota-carrageenans induced a subtle alteration in mosquito cells, detected by cell proliferation and protein synthesis analyses, suggesting that a probable cellular target may be responsible for the refractory state of mosquito cells to DENV-2 infection produced by this class of polysulfates. The failure of iota-carrageenan to block DENV-2 adsorption to mosquito cells appeared to be related to the low presence of adequate heparan sulfate (HS) in C6/36 HT cell surface and is indicative of a differential participation of HS residues for DENV-2 entry in both types of cells.

Dihydropyridine C-glycoconjugates by organocatalytic Hantzsch cyclocondensation. Stereoselective synthesis of α-threofuranose C-nucleoside enantiomers

The Hantzsch reaction of C-glycosyl aldehyde/enamino ester/beta-ketoester systems under l-proline catalysis to give dihydropyridine C-glycoconjugates is reported. Asymmetric cyclocondensations of differentially substituted enamine and beta-dicarbonyl components with formyl alpha-L-C-threofuranoside and with the alpha-D-isomer were also carried out. Each reaction occurred with high yet opposite stereoselectivity (de >95%) so that the pair of alpha-threofuranose C-nucleoside enantiomers was prepared.

Alkali modification of carrageenans. Part IV. Porphyrans as model compounds

Abstract The rate of alkaline cyclization of porphyran is in the same order as those of carrageenan model compounds containing non-sulfated β- d -galactose units, showing that the cyclization of the α-galactosyl units does not depend on the β- d -adjacent sugar residues when they carry no sulfate groups. It also suggests that there is no influence of the α- d -galactose 2-sulfate on the cyclization rate, in spite of its change from the equatorial to the axial position during the conversion of the 4 C 1 to 1 C 4 chair conformation.