María C. Matulewicz

Sulfated Seaweed Polysaccharides as Antiviral Agents

Several sulfated seaweed polysaccharides show high antiviral activity against enveloped viruses, including important human pathogens such as human immunodeficiency virus, herpes simplex virus, human cytomegalovirus, dengue virus and respiratory syncytial virus. They can be obtained in major amounts and at low costs, have low toxicity and in some cases, lack anticoagulant effects. Even if the systemic applications have many drawbacks, their structure and mode of action indicate potential for topical uses to prevent virus infection. The herpes simplex viruses attach to cells by an interaction between the envelope glycoprotein C and the cell surface heparan sulfate (HS). The virus-cell complex is formed by ionic interactions between the anionic (mainly sulfate) groups in the polysaccharide and basic amino acids of the glycoprotein, and non-ionic ones depending on hydrophobic amino acids interspersed between the basic ones in the glycoprotein-binding zone. Hypothesis are advanced of the corresponding hydrophobic structures in the polysaccharides. The antiviral activity of the sulfated seaweed polysaccharides is based on the formation of formally similar complexes that block the interaction of the viruses with the cells. Correlations are established between different structural parameters and antiviral activity. The minimal, ionic and hydrophobic, structures in the seaweed polysaccharides were hypothesized by comparison of the polysaccharides with the known minimal binding structure in HS/heparin, together with a correlation between those structures of the polysaccharides and their antiviral activity.

Antiherpetic and anticoagulant properties of carrageenans from the red seaweed Gigartina skottsbergii and their cyclized derivatives: correlation between structure and biological activity

The antiviral activity against herpes simplex virus types 1 and 2 of kappa/l-, partially cyclized mu/v-, and lambda-carrageenans isolated from the red seaweed Gigartina skottsbergii and their cyclized derivatives was analyzed. lambda-Carrageenans and the partially cyclized mu/v-carrageenan were the most potent inhibitors of herpes viruses (including acyclovir-resistant variants and clinical isolates), with IC50 values lower than 1 microgram ml-1 against both serotypes and selectivity indices higher than 10(3). kappa/l-Carrageenans were slightly less effective than the other two types with IC50 values in the range 1.6-4.1 micrograms ml-1. Antiherpetic activity was directly correlated to the amount of alpha-D-galactose 2,6-disulfate residues in the natural carrageenans. The cyclization of the alpha-D-galactose 6-sulfate and 2,6-disulfate units into 3,6-anhydro-alpha-D-galactose and 3,6-anhydro-alpha-D-galactose 2-sulfate residues in these polysaccharides, in general, lowers the antiherpetic activity of the derivatives with respect to the natural carrageenans. Some carrageenans showed a very reduced anticoagulant activity only at concentrations that were considerably higher than the IC50, whereas others were totally devoid of anticoagulant properties. Among natural carrageenans, the mu/v-type IC3 shows the best relationship between antiviral efficacy and lack of anticoagulant action, resulting a very promising compound.

Antiherpetic activity and mode of action of natural carrageenans of diverse structural types

The lambda-carrageenan 1T1, the kappa/iota-carrageenan 1C1 and the mu/nu-type 1C3, isolated from the red seaweed Gigartina skottsbergii, proved to be potent and selective inhibitors of herpes simplex virus (HSV) types 1 and 2. The antiviral IC50 values determined by virus yield inhibition assay in different cell lines ranged from 0.4 to 3.3 microg/ml, and no cytotoxic effects, measured by trypan blue exclusion on stationary or proliferating cells, tetrazolium salt method or cell protein synthesis, were observed. Time of addition and attachment studies suggested that the main target for antiviral action of the three carrageenans was virus adsorption, whereas no effect on virus internalization, or early or late protein synthesis was detected. However, the lambda-carrageenan 1T1 was still significantly inhibitory when added any time after adsorption. The pretreatment of virions with the carrageenans showed that 1C1 and 1C3 lacked direct inactivating effect at concentrations near the antiviral IC50 but 1T1 exerted virucidal action. The cyclization of 1T1 to afford the derivative 1T1T1 maintained the antiviral activity but eliminated the virucidal properties. Thus, the structure of 1T1 seems to be responsible for its differential behavior from 1C1 and 1C3, probably allowing a more stable binding to HSV, leading to virion inactivation. In contrast, 1C1 and 1C3 fail to bind with high affinity to virus alone, but are able to interfere with the interaction between HSV particles and the cell.

Alkali-modification of carrageenans: mechanism and kinetics in the kappa/iota-, mu/nu- and lambda-series

Abstract The cyclization reaction (formation of 3,6-anhydro-α- d -galactose units from α- d -galactose 6-sulfate) of carrageenans follows a pseudo first-order kinetics, being 20–60 times faster for carrageenans of the kappa-family than for those of the lambda-family. In lambda-carrageenans the clustering of the sulfate groups around the hydroxyl on C-3 of the α-unit shields it from polarization or ionization, reducing the cyclization reaction rate. Furthermore, molecular models of lambda-carrageenans suggest an adequate geometry for the interaction between the hydroxyl group on C-3 of the α-unit and the sulfate group on C-2 of the β-unit. These observations also explain the lack of cyclized derivatives of lambda-carrageenans in nature. The ease with which the cyclization reaction occurs for carrageenans of the kappa-family indicates that the alkaline treatments used industrially could be carried out under milder conditions, giving products of high gel strength.

Carrageenan systems from tetrasporic and cystocarpic stages of Gigartina skottsbergii

Abstract Cystocarpic and tetrasporangial plants of Gigartina skottsbergii produced different systems of carrageenans. These carrageenans were fractionated with potassium chloride and analysed. The ranges of precipitation, the molar ratios Gal:3,6-AnGal: sulphate and the IR spectra of the fractions indicated that the cystocarpic system was composed of similar amounts of gelling and soluble carrageenans of the K/1 - and μ/ν-type, respectively. The tetrasporic system was composed of a major fraction of the λ- type precipitated at 0.60–1.00 M KCl and two minor fractions, one of the λ-type precipitated at 1.10–1.30 M KCl and the other soluble in 2.00 M KCl with a λ- like IR spectrum but μ/ν- like analytical data (ω-carrageenan). The major cystocarpic and tetrasporic carrageenan fractions were submitted to alkaline treatment and further fractionation with potassium chloride. The modified cystocarpic polysaccharide fractions gave products comprising mainly gelling carrageenans while the modified tetrasporic polysaccharide fractions yielded soluble carrageenans in agreement with previously assigned structures.

Sulfated polysaccharides from the red seaweed Georgiella confluens.

The water-soluble polysaccharides from Georgiella confluens, collected in Antarctica, were fractionated with cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration. The initially extracted polysaccharide and the major fraction isolated, soluble in 0.5 M NaCl, were studied. These are sulfated xylogalactans naturally rich in methylated sugar residues, comprising of 3,6-anhydro-2-O-methyl-L-galactose, 2-O-methyl-L-galactose and 6-O-methyl-D-galactose. Structural analysis was carried out by methylation, ethylation, desulfation-ethylation, desulfation-methylation, Smith degradation, 13C NMR spectroscopy and determination of the absolute configuration of monosaccharides by gas chromatography of diastereomeric derivatives produced by reductive amination. The results indicated the presence of an agaran backbone with an unusual substitution pattern: sulfation mainly at the 3-position of the alpha-L-galactose units and the presence of xylose side chains at the 4-position of the beta-D-galactose residues.

Determination of the structures of cystocarpic carrageenans from Gigartina skottsbergii by methylation analysis and NMR spectroscopy

The combined use of methylation analysis and high-field 1H and 13C NMR spectroscopy allows the determination of the fine structure of the carrageenans produced by the cystocarpic stage of Gigartina skottsbergii.

Agar from Gracilaria gracilis (Gracilariales, Rhodophyta) of the Patagonic coast of Argentina--content, structure and physical properties.

Milled summer thalli of Gracilaria gracilis from Argentina were sequentially extracted with water at room temperature (RTW1-3), 70 degrees C (W701-3) and 90 degrees C (W901-2). Both W701 and W901 consisted of high molecular weight polysaccharides (ca. 540,000Da), but polydispersity was higher for the major product W701 (yield, 72% of the recovered). Structural analyzes by methylation and (13)C NMR spectroscopy revealed that W701 was mainly agarose. Alkaline treatment, together with structural analyzes, indicated a negligible proportion of precursor l-galactose 6-sulfate residues in this product, while they were clearly detected in the (13)C NMR spectra of RTW2-3. The presence of floridean starch in W901 had an antagonistic effect on its gel strength, which resulted nearly three times lower than that of fraction W701. Ultrastructural observation by transmission electron microscopy showed that, after extraction with hot water, a partial loss of cell wall stratification and disorganization of the cuticle had occurred. Final cellular debris exhibited swelling in the microfibrillar component. After this first thorough study of the chemical composition and physical properties of the products of G. gracilis from Bahía Bustamante we conclude that a good quality agarose is obtained in high yield after extraction with water at 70 degrees C without the requirement of alkaline pretreatment, which usually produces degradation of the polysaccharide.

Structural analysis of antiviral sulfated α--(1 → 3)-linked mannans

Abstract The structures of two α-(1 → 3)-α- d -xylo- mannans were determined. The different antiviral activity of the xylo-mannans from Nothogenia fastigiata was explained on the basis of a flexible backbone, molecular size, content and distribution of sulfate groups and of the single stubs of β-(1 → 2)- linked d - xylose .

L-galactose containing galactans from the carrageenophyte Gigartina skottsbergii

Alkaline treatment of a partially cyclized mu/v-carrageenan and further fractionation with potassium chloride yielded a fraction soluble in 2 M KCl with negative optical rotation. We report the analysis and some structural features of this product, such as the presence of L-galactose and 3,6-anhydro-L-galactose.