Antitussive activity of Withania somnifera and opioid receptors.

Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways.

Sulfated xylogalactofucans from Spatoglossum asperum: Production, structural features and antiviral activity.

In cultured cells, herpes simplex virus (HSV) infectivity is successfully inhibited by sulfated polysaccharides. Herein, we utilized an amalgamated extraction-sulfation procedure to produce two xylogalactofucan sulfates (S203 and S204) from Spatoglossum asperum using ClSO3H.Pyr/DMF and SO3.Pyr/DMF reagents, respectively. Among these xylogalactofucans, the 17 ± 12 kDa polymer (S203) with 14 % sulfate exhibited activity on several HSV variants, including an acyclovir-resistant HSV-1 strain. This is the first report of the anti-HSV activity of a sulfated xylogalactofucan of S. asperum. The effective concentration 50 % (EC50) value of S203 against HSV-1 strain F was 0.6 µg/mL with a selectivity index of 833. The backbone of this polymer (S203) is made up mostly of (1 â†’ 4)-linked-α-l-Fucp units having sulfate groups typically at O-3 and sometimes at O-2 positions. Oligosaccharides containing Xyl, Gal and Fuc units confirms that they are an integral part of a single polymer, another novelty of this study. The EC50 values of the native xylogalactofucan (S202) and the SO3.Pyr/DMF modified polymer (S204), containing 2 % and 6 % sulfates, were >100 and 3.3 µg/mL, respectively. Introduction of sulfate groups enhanced their capability to inhibit the infection of cells by HSV-1. These findings suggest feasibility of inhibiting HSV attachment to cells by blocking viral entry with polysaccharide having specific structure.

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.

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.

Antioxidative carbohydrate polymer from Enhydra fluctuans and its interaction with bovine serum albumin.

Enhydra fluctuans leaves are traditionally sold on Indian markets for various health benefits. However, no phytochemical study on its high molecular weight compound has so far been performed. Chemical, chromatographic, ESI-TOF-MS, and NMR analyses of the water extracted carbohydrate polymer (CP) of E. fluctuans leaves showed the presence of a 24 kDa arabinogalactan having a (1,3)-linked ß-d-Galp main chain, substituted at O-6 by (1,6)-linked ß-d-Galp side chains. The latter residues were substituted at O-3 by (1,3)-, (1,5)-, and (1,3,5)-linked α-l-Araf chains, and nonreducing end-units of α-l-Araf and ß-d-Galp. This polymer contained esterified phenolic acids. Biochemical analysis revealed similarity in antioxidative potential between the identified carbohydrate polymer and known standard antioxidants. The highly branched side chains and the phenolic acid residues of the arabinogalactan might be the functional sites. Fluorimetric and ultraviolet spectrometric analyses showed that the studied carbohydrate polymer can form complex with bovine serum albumin having binding constant K = 2.42 × 10(6)/M and changes its microenvironment. Thus, traditional aqueous extraction method provides a carbohydrate polymer, which stimulates a fair biological response: this could represent an interesting approach in phytotherapeutic treatments.

Chemically engineered sulfated glucans from rice bran exert strong antiviral activity at the stage of viral entry.

Attachment and entry of many viruses are mediated by their affinity for polysaccharides present on the surface of target cells. In this paper, we demonstrate that sulfated glucans isolated from rice (Oryza sativa) can be utilized as experimental drugs exerting strong antiviral activity. In particular, oleum-DMF-based extraction is described as a procedure for the generation of chemically engineered glucans from commercially available rice bran. The one-step procedure has the potential to provide a spectrum of related glucans with varying molecular masses and modifications, including sulfation. The sulfated glucans P444, P445, and P446 possess increased antiviral activity compared to a previously described glucan (S1G). P444, P445, and P446 were highly active against human cytomegalovirus (HCMV), moderately active against other members of the family Herpesviridae, while not active against unrelated viruses. Specific experimentation with HCMV-infected cells provided evidence that antiviral activity was based on inhibition of viral entry and that inhibition occurred in the absence of drug-induced cytotoxicity. These findings underline the high potential of sulfated glucans for antiviral research and drug development. In addition, the procedure described for the efficient transformation of glucan hydroxy groups to sulfate groups may be similarly beneficial for the chemical alteration 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.

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.

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.

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.

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.

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.

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.

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.

Focus on antivirally active sulfated polysaccharides: from structure-activity analysis to clinical evaluation.

In recent years, many compounds having potent antiviral activity in cell culture have been detected and some of these compounds are currently undergoing either preclinical or clinical evaluation. Among these antiviral substances, naturally occurring sulfated polysaccharides and those from synthetic origin are noteworthy. Recently, several controversies over the molecular structures of sulfated polysaccharides, viral glycoproteins, and cell-surface receptors have been resolved, and many aspects of their antiviral activity have been elucidated. It has become clear that the antiviral properties of sulfated polysaccharides are not only a simple function of their charge density and chain length but also their detailed structural features. The in vivo efficacy of these compounds mostly corresponds to their ability to inhibit the attachment of the virion to the host cell surface although in some cases virucidal activity plays an additional role. This review summarizes experimental evidence indicating that sulfated polysaccharides might become increasingly important in drug development for the prevention of sexually transmitted diseases in the near future.

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.

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.

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.

Anti-herpetic activity of a sulfated xylomannan from Scinaia hatei.

Many viruses display affinity for cell surface heparan sulfate proteoglycans with biological relevance in virus entry. This raises the possibility of the application of sulfated polysaccharides in antiviral therapy. In this study we have analyzed polysaccharide fractions isolated from Scinaia hatei. The crude water extract (ShWE) as well as one fraction (F1) obtained by size exclusion chromatography had potent anti-HSV activity. Their inhibitory concentration 50% (IC50) values ranging from 0.5 to 4.6 microg/ml were much lower than the cytotoxic concentration 50% (CC50) values (1000 microg/ml). These fractions had very low anticoagulant activity. Furthermore, they had a weak inactivating effect on virions in a virucidal assay at concentrations in the range of 60-100 microg/ml. Chemical, chromatographic and spectroscopic methods showed that the major polysaccharide, which had 0.4 sulfate group per monomer unit and an apparent molecular mass of 160 kDa, contained a backbone of alpha-(1-->3)-linked D-mannopyranosyl residues substituted at C-6, C-4 and C-2 with single stub of beta-d-xylopyranosyl residues. Sulfate groups, when present, are located at C-4 of alpha-(1-->3)-linked D-mannopyranosyl units, and appeared to be very important for the anti-herpetic activity of this polymer.

Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides.

Isolated cell walls of Argania spinosa fruit pulp were fractionated into their polysaccharide constituents and the resulting fractions were analysed for monosaccharide composition and chemical structure. The data reveal the presence of homogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) in the pectic fraction. RG-I is abundant and contains high amounts of Ara and Gal, indicative of an important branching in this polysaccharide. RG-II is less abundant than RG-I and exists as a dimer. Structural characterisation of xyloglucan using enzymatic hydrolysis, gas chromatography, MALDI-TOF-MS and methylation analysis shows that XXGG, XXXG, XXLG and XLLG are the major subunit oligosaccharides in the ratio of 0.6:1:1.2:1.6. This finding demonstrates that the major neutral hemicellulosic polysaccharide is a galacto-xyloglucan. In addition, Argania fruit xyloglucan has no XUFG, a novel xyloglucan motif recently discovered in Argania leaf cell walls. Finally, the isolation and analysis of arabinogalactan-proteins showed that Argania fruit pulp is rich in these proteoglycans.