Anti-respiratory syncytial virus and anti-herpes simplex virus activity of chemically engineered sulfated fucans from Cystoseira indica.

Seaweed polysaccharides, particularly sulfated ones, exhibited potent antiviral activity against a wide variety of enveloped viruses, such as herpes simplex virus and respiratory viruses. Different mechanisms of action were suggested, which may range from preventing infection to intracellular antiviral activity, at different stages of the viral cycle. Herein, we generated two chemically engineered sulfated fucans (C303 and C304) from Cystoseira indica by an amalgamated extraction-sulfation procedure using chlorosulfonic acid-pyridine/N,N-dimethylformamide and sulfur trioxide-pyridine/N,N-dimethylformamide reagents, respectively. These compounds exhibited activity against HSV-1 and RSV with 50 % inhibitory concentration values in the range of 0.75-2.5 µg/mL and low cytotoxicity at concentrations up to 500 µg/mL. The antiviral activities of chemically sulfated fucans (C303 and C304) were higher than the water (C301) and CaCl2 extracted (C302) polysaccharides. Compound C303 had a (1,3)-linked fucan backbone and was branched. Sulfates were present at positions C-2, C-4, and C-2,4 of Fucp, and C-6 of Galp residues of this polymer. Compound C304 had a comparable structure but with more sulfates at C-4 of Fucp residue. Both C303 and C304 were potent antiviral candidates, acting in a dose-dependent manner on the adsorption and other intracellular stages of HSV-1 and RSV replication, in vitro.

Chemically modified galactans of Grateloupia indica: From production to in vitro antiviral activity.

Herpes simplex viruses (HSVs) have an affinity for heparan sulfate proteoglycans on cell surfaces, which is a determinant for virus entry. Herein, several sulfated galactans that mimic the active domain of the entry receptor were employed to prevent HSV infection. They were produced from Grateloupia indica using chlorosulfonic acid-pyridine (ClSO3H.Py)/N,N-dimethylformamide reagent (fraction G-402), SO3.Py/DMF reagent (G-403), or by aqueous extraction (G-401). These galactans contained varied molecular masses (33-55 kDa), and sulfate contents (12-20 %), and have different antiviral activities. Especially, the galactan (G-402) generated by using ClSO3H.Py/DMF, a novel reagent, exhibited the highest level of antiviral activity (EC50 = 0.36 µg/mL) compared to G-403 (EC50 = 15.6 µg/mL) and G-401 (EC50 = 17.9 µg/mL). This most active sulfated galactan possessed a linear chain containing ß-(1 â†’ 3)- and α-(1 â†’ 4)-linked Galp units with sulfate group at the O-2/4/6 and O-2/3/6 positions, respectively. The HSV-1 and HSV-2 strains were specifically inhibited by this novel 33 ± 15 kDa galactan, which also blocked the virus from entering the host cell. These results highlight the significant potential of this sulfated galactan for antiviral research and drug development. Additionally, the reagent used for the effective conversion of galactan hydroxy groups to sulfate during extraction may also be useful for the chemical transformation of other natural products.

Synthesis, molecular features and biological activities of modified plant polysaccharides.

Plant polysaccharides represent a natural source material characterized by high ubiquity, abundance and biological activities that can be utilised as organic matter in numerous manufacturing processes including pharmaceutical. The goal-oriented modification of polysaccharides by chemical reactions brings about a substantial refinement in their molecular features and biological activity. An improved understanding of the intrinsic molecular features of plant polysaccharides, as well as the structure-activity relationship eventually caused by chemical alterations, may be fundamental for the development of technologies supporting their biomedical oriented exploitation. Herein, we attempt to provide a broader view on these topics and to illustrate methodologies of chemical modification. These are intended to enhance the functionality of plant polysaccharides, such as their modification-induced biological activities, finally making the products available for translational purposes.

The heparin-mimicking arabinogalactan sulfates from Anogeissus latifolia gum: Production, structures, and anti-herpes simplex virus activity.

Herpes simplex virus type 1(HSV-1) attaches to cell surface heparan sulfate aiming to enter into susceptible cells. In this work, we utilized a sulfur trioxide-pyridine in N,N-dimethylformamide (SO3·Pyr/DMF) based amalgamated extraction-sulfation procedure for producing arabinogalactan sulfates from Anogeissus latifolia gum. Chemical, chromatographic, spectroscopic and chemical data revealed that the derived polymers contained varying molecular masses (31-69 kDa) and degrees of sulfation (0.1-0.5), but similar saccharide compositions. The highly active polymer (HSV-1: IC50 and SI, respectively, of 127 µg/mL and 15.7) was a 69 kDa arabinogalactan holding sulfates at O-5 of arabinofuranosyl residues and showed no cytotoxicity as far as 2 mg/mL concentration. This chemically sulfated macromolecule acted by obstructing viral attachment and entry. Thus, SO3·Pyr/DMF is suitable for producing new molecules with varied structures and altered pharmacological activities from plant sources.

Chemically sulfated arabinoxylans from Plantago ovata seed husk: Synthesis, characterization and antiviral activity.

Limited options for the treatments of diseases triggered through viral infections revealed the quest for novel antiviral drugs. Polysaccharide sulfates owing to their unique mode of action are prominent antiviral drug candidates. Herein, the arabinoxylan of Plantago ovata seed husk was simultaneously extracted and chemically sulfated using sulphur trioxide-pyridine reagent in N,N-dimethylformamide solvent (SO3⋅Py/DMF). Thus, three arabinoxylan sulfates (IS1201-IS1203) holding variable degrees of sulfation (DS: 0.1-0.9), molar masses (18.4-31.3 kDa) and glycosyl makeup (Ara: Xyl::10-19:81-90; molar ratio) were produced and then characterized. According to the results, these polymers displayed anti-herpes simplex virus type 1 activity and their potency depends upon DS. The utmost effective compound (IS1203, IC50: 2.9 µg mL-1) was a 18.4 kDa arabinoxylan possessing sulfate groups at O-3 and O-2,3 positions of xylopyranosyl (Xylp), and O-5 of arabinofuranosyl (Araf) residues. Besides, this polymer showed no cytotoxicity at concentration up to 1000 µg mL-1. Given that polysaccharide sulfates have antiviral activities, synthesis of new molecules possessing diverse structures will be a useful addition to the arsenal of antivirals.

Antiviral Strategies Using Natural Source-Derived Sulfated Polysaccharides in the Light of the COVID-19 Pandemic and Major Human Pathogenic Viruses.

Only a mere fraction of the huge variety of human pathogenic viruses can be targeted by the currently available spectrum of antiviral drugs. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has highlighted the urgent need for molecules that can be deployed quickly to treat novel, developing or re-emerging viral infections. Sulfated polysaccharides are found on the surfaces of both the susceptible host cells and the majority of human viruses, and thus can play an important role during viral infection. Such polysaccharides widely occurring in natural sources, specifically those converted into sulfated varieties, have already proved to possess a high level and sometimes also broad-spectrum antiviral activity. This antiviral potency can be determined through multifold molecular pathways, which in many cases have low profiles of cytotoxicity. Consequently, several new polysaccharide-derived drugs are currently being investigated in clinical settings. We reviewed the present status of research on sulfated polysaccharide-based antiviral agents, their structural characteristics, structure-activity relationships, and the potential of clinical application. Furthermore, the molecular mechanisms of sulfated polysaccharides involved in viral infection or in antiviral activity, respectively, are discussed, together with a focus on the emerging methodology contributing to polysaccharide-based drug development.

Conjugation reaction with ferulic acid boosts the antioxidant property of arabinogalactan-protein and enhances its ability to form complex with ß-lactoglobulin.

Ferulic acid was chemically grafted onto the arabinogalactan protein of Aegle marmelos fruit gum using 1,1'-carbonyldiimidazole as coupling reagent. Thus, grafted polysaccharides with different degrees of substitution were prepared and then characterized by gas chromatography/mass spectrometry, size exclusion chromatography, and ultraviolet-visible, infra-red, and nuclear magnetic resonance spectroscopic investigations. Fluorescence spectroscopic investigation showed hydrophobic microdomain formation in grafted polymers. The antioxidant activities of the derivatives, as determined by the 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl radical assay, were strong and increases with increasing the degree of feruloylation. Compared to parental arabinogalactan protein (K = 2.38 × 106 M-1), these grafted polymers bind more strongly with ß-lactoglobulin (K = 11.4 × 106 M-1 and 8.19 × 106 M-1). Given that gum polysaccharides are valuable component in functional foods, synthesis of antioxidative graft polymer possessing good compatibility with ß-lactoglobulin may have important implication.

Isolation, structural features, in vitro antioxidant activity and assessment of complexation ability with ß-lactoglobulin of a polysaccharide from Borassus flabellifer fruit.

This research was intended to investigate the structural feature, antioxidative activity and interaction with ß-lactoglobulin (ß-lg) of a polysaccharide (P) isolated from Borassus flabellifer fruit thru aqueous extraction, protein elimination and chromatographic techniques. Polysaccharide P (molecular weight: 21,000 g mol-1) was constituted of arabinose, galactose, glucose, and rhamnose in a 50:24:20:6 M ratio alongside 9% (w/w) galacturonic acid. It encompassed a petite backbone entailing galacturonopyranosyl and rhamnopyranosyl units substituted with sizable side chains comprising of arabinofuranosyl, galactopyranosyl and esterified coumaric acid (CA) residues. Various series of oligosaccharides including (i) Gal1,2,4-9Ac5-29, (ii) Ara2-3Ac6-8, (iii) Gal3Ara1-3Ac13-17, (iv) Gal4-6Ara2Ac18-24, (v) Gal6Ara1Ac22 and (vi) Gal1Ara2CA1Ac7 and Gal1Ara3CA1Ac9 epitomizing polysaccharide structure were generated and characterised. Fraction P exhibited dose-dependent antioxidant activity and possessed a strong ß-lactoglobulin binding capability. Accordingly, B. flabellifer fruit offers an antioxidative polysaccharide having novel structure that can associate with ß-lg and, hence, useful in formulating novel food possessing adjustable composition.

The heteropolysaccharide of Mangifera indica fruit: Isolation, chemical profile, complexation with ß-lactoglobulin and antioxidant activity.

A 91 kDa heteropolysaccharide (F2) was isolated from Mangifera indica fruit via extraction with H2O, purification by C2H5OH, starch removal and ion exchange chromatography. This polymer was made up mostly of Ara, Gal, Glc, Rha, Xyl, and GalA in a 37: 29: 9:3:2:19 molar proportion. It inherited a small backbone containing GalpA and Rhap units substituted with very large side chains containing differently linked Ara and Gal units plus esterified gallic acid (GA) residue. Several enzymes generated oligosaccharides including (i) Ara2-10Ac6-22, (ii) Gal1-8Ac5-26 and (iii) GA1Gal1Ac7 were characterized. This polysaccharide, which showed dose dependent antioxidant activity, exhibited synergism with gallic acid, and formed a complex (K = 1.2 × 106 M-1) with ß-lactoglobulin. Accordingly, H2O treatment produces a polysaccharide with desired biochemical properties; this could be effective in designing innovative functional food with flexible makeup.

Exploiting the Amazing Diversity of Natural Source-Derived Polysaccharides: Modern Procedures of Isolation, Engineering, and Optimization of Antiviral Activities.

Naturally occurring polysaccharide sulfates are highly diverse, owning variations in the backbone structure, linkage pattern and stereochemistry, branching diversity, sulfate content and positions of sulfate group(s). These structural characteristics bring about diverse sulfated polymers with dissimilar negative charge densities and structure-activity relationships. Herein, we start with a short discussion of techniques needed for extraction, purification, chemical sulfation, and structural characterization of polysaccharides. Processes of isolation and sulfation of plant-derived polysaccharides are challenging and usually involve two steps. In this context, we describe an integrated extraction-sulfation procedure that produces polysaccharide sulfates from natural products in one step, thereby generating additional pharmacological activities. Finally, we provide examples of the spectrum of natural source-derived polysaccharides possessing specific features of bioactivity, in particular focusing on current aspects of antiviral drug development and drug-target interaction. Thus, the review presents a detailed view on chemically engineered polysaccharides, especially sulfated derivatives, and underlines their promising biomedical perspectives.

Chemically sulfated polysaccharides from natural sources: Assessment of extraction-sulfation efficiencies, structural features and antiviral activities.

The provisioning of compound libraries with a high degree of diversity and attractive pharmacological properties is a limiting step in drug development. This study reports the production of highly bioactive sulfated polysaccharides, originally present in a nonsulfated, dormant state in natural sources, and demonstrates their antiviral activity (human cytomegalovirus EC50 values of 2.34-7.77 µg/mL) at a low degree of cytotoxicity. Furthermore, data strongly suggested the inhibition of virus entry as the main mode of antiviral action. Remarkably, the utilized oleum-DMF reagent was able to generate a range of sulfated polysaccharides from various natural sources, possessing varying saccharide compositions, degrees of sulfation (0.4-1.7) and molecular masses (38-94,000 g/mol). Typically, in a matter of minutes, this reagent not only solubilized polysaccharides but also chemically converted their hydroxyl functionality into sulfates. The most active sulfated polysaccharide (EC50 of 2.62 µg/mL) proved to be a 94,000 g/mol branched glucan with sulfates at C-6/C-3,6/C-2,3,6 positions. In conclusion, the important determinants of such compounds' antiviral activity are: (i) degree of sulfation, (ii) molecular mass and (iii) structural features. Thus, our approach offers a huge prospect for the improvement of natural source-derived libraries based on biologically active polysaccharides with diversified chemical profiles.

Assessment of antiherpetic activity of nonsulfated and sulfated polysaccharides from Azadirachta indica.

Azadirachta indica leaf is used by Indian population for the healing of various diseases including viral infection. Herein, we analyzed the antiherpetic (HSV-1) activity of two polysaccharides (P1 and P2) isolated from the leaf of A. indica and their chemically sulfated derivatives (P1S and P2S). The molecular weights of P1S and P2S are 41 and 11 kDa, respectively. Sulfate groups are located at positions C3 of the Araf and C6 of both Galp and Glcp residues of the most active polysaccharide (P1S). These compounds were not cytotoxic in HEp-2 cells, up to 1000 µg/mL. Both P1S and P2S exhibited antiviral activity when used simultaneously to HSV-1, with 50% inhibitory concentration/selectivity index, respectively, of 31.1 µg/mL/>51.4 and 80.5 µg/mL/>19.8. P1S showed better inhibitory effect (91.8%) compared to P1 (50%), P2 (71.1%) and P2S (70%) at 200 µg/mL. Synthesis of viral protein showed a dose-dependent response and the nucleic acid synthesis was inhibited up to 25 µg/mL, by P1 and P1S and up to 50 µg/mL, by P2 and P2S. The antiviral effect is probably due to the interference of polysaccharides at the early stages of HSV-1 replication, including adsorption. Further studies are under way to get insight into the mechanism of action of the substances.

Functional exploration of Pseudoalteromonas atlantica as a source of hemicellulose-active enzymes: Evidence for a GH8 xylanase with unusual mode of action.

To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a ß-xylosidase, a α-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end.

Polysaccharides from Thymus vulgaris leaf: Structural features, antioxidant activity and interaction with bovine serum albumin.

Thymus vulgaris is used for various health benefits and culinary, nevertheless, report on its macromolecules is absent. Here, we report chemical compositions of the polysaccharides isolated from its leaf by sequential extraction with inorganic solvents. In particular, chemical profile of a unique rhamnogalacturonan I type polysaccharide containing ester linked phenolic acids has been described. Sugar compositional, TLC, UV-spectrometric and ESI-MS analyses of oligosaccharides generated from this polysaccharide by enzyme digestion, controlled acid hydrolysis and Smith degradation revealed atypical fine structural details. Biochemical analysis demonstrated dose-dependent antioxidant activity. A combination of large neutral side chains of the ramified region and ester linked phenolic acids are regarded as the functional sites. Ultraviolet spectrometric and fluorimetric analyses showed that this polysaccharide forms a homogeneous water-soluble complex with bovine serum albumin (binding constant, K = 2.91 × 106/M). Consequently, water extraction affords a polysaccharide which induces pharmacological effect; this underlines the impact of thyme as natural dietetic antioxidant.

Chemical profile of a polysaccharide from Psidium guajava leaves and it's in vivo antitussive activity.

Decoction of Psidium guajava leaves has been used as medication for chronic coughs and breathlessness for ages. Despite demonstration of antitussive activity, the specific molecule responsible for this remains unidentified. Herein, we report chemical profile and antitussive activity of its water extract (WE) and a polysaccharide (F1) present therein. This polysaccharide (F1), purified from WE by precipitation with ethanol and then through Cu(II)acetate, contains Ara, Gal, Rha, Glc and GalA residues, and has a molecular mass of 156 kDa. It comprises of terminal-, (1,5)- and (1,3,5)-linked Araf; (1,3)-, (1,6)- and (1,3,6)-linked Galp alongside (1,2)- and (1,2,4)-linked Rhap residues. Oligosaccharides indicating polysaccharide structure have been generated by Smith degradation and characterized. The WE fraction suppressed citric acid induced cough efforts in guinea pigs in the dose of 50 mg kg-1. Assessment of antitussive activity of fractions prepared from WE namely F1 (polysaccharide) and F2 (ethanol soluble fraction) revealed that polysaccharide is the active component. Remarkably, tested samples do not alter the specific airway smooth muscle reactivity in animals significantly. The simple extraction method, prominent activity and favorable reactions profile suggest that this macromolecule could be an antitussive drug candidate.

Green seaweed Enteromorpha compressa (Chlorophyta, Ulvaceae) derived sulphated polysaccharides inhibit herpes simplex virus.

The herpes simplex virus (HSV) diseases represent a relevant medical and social problem due to their communicability and recurrence following latency. The green algae are rich source of polysaccharides referred to as ulvans, reported as being biologically and pharmacologically active. In this work, we analyzed the activity of seven chemically modified polysaccharides from Enteromorpha compressa (Chlorophyta, Ulvaceae), against HSV. Only the derivative named SU1F1 showed satisfactory viral inhibition activity, with a high selectivity index, and, therefore, it was submitted to analysis of the probable mechanism of action and structure. SU1F1 is a sulphated (22% w/w) heteroglycuronan with an apparent molecular mass of 34kDa. The antiviral activity was assayed by plaque reduction assay under the protocols of the time-of-addition (from 3h before infection to 16h after infection), the inhibition of virus adsorption and penetration, and the virucidal effects. SU1F1 showed a high viral activity at the time 0h. We demonstrated that its inhibitory effect was maintained until 4h post-treatment with 100% of viral inhibition at 100µg/ml. No effect was observed in additional protocols (the pre-treatment, the inhibition of adsorption and penetration and virucidal assays). Reverse Transcriptase associated PCR (RT-PCR) results were in accordance with plaque reduction assay and demonstrated the activity of SU1F1 at the initial stages of HSV replication.

Structural insight of an antioxidative arabinogalactan protein of Aegle marmelos fruit gum and it's interaction with ß-lactoglobulin.

The complication of arabinogalactan protein (AGP) structure, a significant ingredient of gum polysaccharides, not merely hinders the allocation of its role, but restricts its utilization as well. Here, we describe structural details of an AGP purified from Aegle marmelos fruit gum. This AGP (310×103g/mol), which is water-soluble, contains ß-1,3-linked galactopyranosyl main chain substituted at O-6 position with side chains containing galactose and arabinose residues. Also data on sugar composition, ring size, glycosidic linkage pattern, anomeric configuration and sequence of monosaccharide units of a number of oligosaccharides produced from this AGP by chemical and enzymatic methods were acquired. Biochemical analysis reveals resemblance in antioxidative potential between this arabinogalactan protein and standard antioxidants. Moreover, mixture of AGP and ß-lactoglobulin form stable water soluble complex having binding constant K=2.38×106/M. As gum polysaccharides are important raw materials of food industry discovering an antioxidative gum with the ability of creating stable water soluble complex with ß-lactoglobulin may have important implication.

Additionally sulfated xylomannan sulfates from Scinaia hatei and their antiviral activities.

Herpes simplex viruses (HSVs) display affinity for cell-surface heparan sulfate proteoglycans with biological relevance in virus entry. This study demonstrates the potential of chemically engineered sulfated xylomannans from Scinaia hatei as antiHSV drug candidate. Particularly, a dimethylformamide -SO3/pyridine based procedure has been employed for the generation of anionic polysaccharides. This one-step procedure has the power of providing a spectrum of xylomannans with varying molecular masses (<12-74kDa), sulfate content (1-50%) and glycosyl composition. Especially, the sulfated xylomannans S1F1 and S2F1 possessed altered activity against HSV-1 and HSV-2 compared to the parental compound (F1) and that too in the absence of drug-induced cytotoxicity. Regarding methodological facet, the directive decoration of hydroxyl functionality with sulfate group plus changes in the molecular mass and sugar composition during isolation by the used reagent opens a door for the production of new molecular entity with altered biological activity from other natural sources.

Isolation and structural elements of a water-soluble free radical scavenger from Nyctanthes arbor-tristis leaves.

The leaves of Nyctanthes arbor-tristis L. (Oleaceae) are used in Ayurvedic medicine for the management of a range of diseases, but reports on its phytochemicals and pharmacological properties are inadequate. Herein, we report purification of an antioxidative polysaccharide (F2) extracted from its leaves by water. The presence of a highly branched polysaccharide (75 kDa) containing esterified phenolic acids was revealed by chemical, chromatographic and spectroscopic analyses. Particularly, ESMS analysis of per acetylated oligomeric fragments derived by Smith degradation provides important structural information on a spectrum of glycerol tagged oligosaccharides. This polysaccharide showed dose dependent free radical scavenging capacity as evidenced by DPPH and Ferric reducing power assay. This pharmacologically active compound (F2) formed a water soluble complex with bovine serum albumin over pH 4.0-7.4. Accordingly, traditional aqueous extraction method provides a molecular entity that induces a pharmacological effect: this could epitomize a smart approach in phytotherapeutic management.

Isolation and structural features of an antiradical polysaccharide of Capsicum annuum that interacts with BSA.

Red peppers, Capsicum annuum, are used worldwide as spices, foods and medicines. Herein, we have analyzed an antiradical polysaccharide isolated from red peppers through successive acetate buffer extraction. This macromolecule was purified using graded precipitation with ethanol, α-amylase treatment, deproteination and anion-exchange chromatography. This highly-branched polysaccharide (360 kDa) was esterified with phenolic acids and contained a (1,3)-linked-ß-Galp chain substituted at O-6 by (1,6)-linked-ß-Galp residues. The latter was substituted at O-3 by (1,5)- and (1,3,5)-linked-α-Araf residues, and non-reducing end-units of α-Araf and ß-Galp. The antiradical potential of this polysaccharide was comparable to standard antioxidants. The phenolic acid residues were the functional sites. This polysaccharide could form complex with bovine serum albumin having binding constant K = 5.24 × 10(6)/M and change its microenvironment. Thus, aqueous extraction method provides a macromolecule that stimulates biological responses; this emphasizes the significance of red pepper as dietary antioxidant.