Alberto S. Cerezo

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.

Antiviral properties of fucoidan fractions from Leathesia difformis.

Three fractions of fucoidans isolated from the brown seaweed Leathesia difformis (Ee, Ec and Ea) were found to be selective antiviral agents against herpes simplex virus (HSV) types 1 and 2 and human cytomegalovirus. Fraction Ea was the most active, with IC50 values in the range 0.5-1.9 microg/ml without affecting cell viability at concentrations up to 400 microg/ml. The antiherpetic activity of Ea was assessed by three different methods, plaque reduction, inhibition of virus yield and prevention of HSV-2 induced shut-off of cell protein synthesis, demonstrating that the inhibitory effect was independent of the antiviral assay and the multiplicity of infection. The mode of action of Ea could be ascribed to an inhibitory action on virus adsorption. The fucoidans did not inhibit the blood coagulation process even at concentrations exceeding more than 100 times the IC50 value.

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.

Novel DL-Galactan Hybrids from the Red Seaweed Gymnogongrus Torulosus are Potent Inhibitors of Herpes Simplex Virus and Dengue Virus:

A novel series of DL-galactan hybrids extracted from the red seaweed Gymnogongrus torulosus, was evaluated for its in vitro antiviral properties against herpes simplex virus type 2 (HSV-2) and dengue virus 2 (DEN-2). These compounds were very active against both viruses with inhibitory concentration 50% (IC50) values in the range 0.6–16 μg/ml for HSV-2 and 0.19–1.7 μg/ml for DEN-2, respectively, as determined in a virus plaque reduction assay in Vero cells. The DL-galactans lacked of cytotoxic effects, on stationary as well as on actively dividing cells, and anticoagulant properties. Some of the compounds showed a variable level of direct inactivating effect on both virions, with virucidal concentration 50% values exceeding the IC50s obtained by plaque reduction assay. Full inhibitory activity was achieved when the galactans were present during virus adsorption period, suggesting that the mode of action of these compounds is an interference in the binding of the surface envelope glycoprotein with the cell receptor.

High-field NMR spectroscopy of cystocarpic and tetrasporic carrageenans from Iridaea undulosa

Abstract High-field 1 H and 13 C NMR spectroscopy has been used to characterize the fine structure of the cystocarpic and tetrasporic carrageenans from Iridaea undulosa . The proportion of different carrageenans from the κ-family in cystocarpic samples were calculated. In the 13 C NMR spectra of the tetrasporic samples, twelve signals of approximately equal intensity were observed, indicating a nearly “perfect” λ-structure.

The systems of carrageenans from cystocarpic and tetrasporic stages from Iridaea undulosa: fractionation with potassium chloride and methylation analysis of the fractions

Abstract The carrageenans produced by the cystocarpic and tetrasporic stages from Iridaea undulosa were fractionated by potassium chloride precipitation and analyzed. The cystocarpic system is composed of similar amounts of a gelling carrageenan with κ/ι-characteristics, and a soluble, partially cyclized, μ/gn-carrageenan. The tetrasporic system is composed mainly of a λ-carrageenan but also with a small proportion of a galactan sulfate containing l -galactose. The structures of the components of both were examined by methylation analysis. Correlation with previous data from carrageenans of the same and different sources allows us to investigate some of the problems in the chemistry of these polysaccharides, namely: (a) the formal pattern of the systems biosynthesized by each stage of the seaweed and the relationships between the components, (b) general relationships between the primary structure and solubility in potassium chloride solutions, and (c) the solubility of carrageenan mixtures versus that of “pure” compounds in these solutions.

The system of low-molecular-weight carrageenans and agaroids from the room-temperature-extracted fraction of Kappaphycus alvarezii.

The room-temperature-extracted fraction from the red seaweed Kappaphycus alvarezii consists mainly of low-molecular-weight carrageenans, with structural dispersion around a basic kappa-pattern. This dispersion results from: (a) low percentages of 3,6-anhydrogalactose and the presence of precursor units; (b) important quantities of 6-O-methyl beta-D-galactose (4-sulfate) residues; (c) significant amounts of iota-repeating structure, and (d) small amounts of non-sulfated and disulfated beta-D-galactose residues. Significant quantities of alpha-L-galactose units suggest the presence of agaroids, as it has been reported in several other carrageenophytes.

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.