F-fucoidan, desulfated F-fucoidan, and its processed derivatives in terms of desulfated oligo-fucose as inhibitors of gastrointestinal infection

Abstract

The subject of this invention are native forms of fucoidan polysaccharide, desulfated fucoidan and derivatives obtained by processing to the level of fucose-oligosaccarides containing preferentially, but not exclusively less than 20 monosaccaride fucose units alpha-linked by a glycosidic bond, with an average molecular weight of preferentially less than 3 kDa. The poly- and oligosaccarides that are described according to the invention are natural and safe, and are used as a dietary supplement for the use in prevention and treatment of pathologies associated with Noroviruses or Rotaviruses, Salmonella sp. or Pseudomonas aeruginosa or Campylobacter jejuni and other enteroviruses and enteric pathogens that cause bacterial gastroenteritis, as well as in the food production process. The invention also relates to a method for the desulfation and fragmentation of fucoidans to generate desulfated poly- or oligo-fucoses.

Claims

1. F-fucoidan comprising desulfated oligosaccharides comprising: terminal fucose alpha-linked by a glycosidic bond as constituents, wherein the oligosaccharides with structural elements Fuc1-2Fuc, Fuc1-3Fuc, Fuc1-4Fuc, Fuc1-2Fuc1-3Fuc, Fuc1-2(Fuc1-4)Fuc, Fuc1-2(Fuc1-3)Fuc, Fuc1-3(Fuc1-2)Fuc and Fuc1-2Fuc1-3Fuc(Fuc1-4)Fuc, and the terminal fucose being exposed at a valency due to branching of oligo- or polysaccharides, wherein the F-fucoidan have anti-viral activity due to competitive inhibitory effect on the norovirus/pathogen binding to blood-group-related glycans on host epithelial mucin, inhibiting pathogen binding to blood-group H related oligosaccharides on epithelia of the gastrointestinal tract, for pathogens causing infections, exhibit competitive activity and accordingly inhibitory effects on binding of enteropathogenic Noroviruses or Rotaviruses, Salmonella sp., Pseudomonas aeruginosa and Campylobacter jejuni, to human H- and Lewis-b blood-group positive epithelial mucins, wherein the desulfation is achieved by inorganic or organic acids, at temperatures and for reaction times that allow effective desulfation and simultaneous fragmentation to the level of oligosaccharides in the size range between 2 and 20 monosaccharide units, or combinations of chemical desulfation/fragmentation with enzymatic treatments with fucoidanases and sulfatases carried out by dialysis from the heated reaction mixture using a soluble, polystyrenesulfonic acid, thereby separating continuously the oligosaccharide products of low masses below a defined membrane cutoff.

2. The F-fucoidan according to claim 1, wherein the F-fucoidan being isolated from Fucus vesiculosus, brown algae, seaweed and other marine animals or plants, desulfated and/or processed by partial hydrolysis of the fucoidan polysaccharide that exhibits activity either in native state of sulfation or with a complete or partial loss of sulfate after solvolysis or processing by partial acid hydrolysis.

3. The F-fucoidan according to claim 1, wherein the desulfation is carried out by solvolysis of neutralized, dry F-fucoidan with dimethyl sulfoxide/pyridine, 5:12 (v:v) for 9h at 100° C., followed by dialysis of organic chemicals and drying.

4. The F-fucoidan according to claim 1, wherein F-fucoidan have anti-viral activity due to competitive inhibitory effect on the norovirus/pathogen binding to blood-group-related glycans on host epithelial mucin for the treatment of organisms in industrial farming for the food industry.

5. The F-fucoidan according to claim 1, wherein F-fucoidan is a processed Fucoidan.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a line chart showing results from a duplicate assay at varying fucoidan concentrations;

(2) FIG. 2 is a bar chart depicting B, C, Binding of GII.4 (Sydney 2012) VLPs to human gastric mucin (HGM) at pH 7.4 (B) and at pH 1.5 to 5.5 (C);

(3) FIG. 3 is a bar chart depicting D, Variation of blocking conditions for binding assays of GII.4 to HGM (5% skim milk/PBS vs. 5% BSA/PBS;

(4) FIG. 4 is a bar chart depicting Triplicate assay with fixed inhibitor concentration (10 mg/ml);

(5) FIG. 5 is a bar chart depicting Triplicate assay with fixed inhibitor concentration (5 mg/mL);

(6) FIG. 6 is line chart depicting neutralization and solubilization in capsid binding buffer (0.01% Tween-20/PBS) at varying concentrations;

(7) FIG. 7 is bar chart depicting Inhibition of GII.4 (2012) (A), and GIL 17 (2015) (B) VLP binding to HGM by processed fucoidan;

(8) FIG. 8 is a bar chart depicting Time kinetics of fucoidan processing in dilute hydrochloric acid based on binding inhibition assay with GII.4 (Sydney, 2012) VLPs;

(9) FIG. 9 is a bar chart depicting Comparative evaluation of different work-up procedures including solid-phase extraction on graphitized carbon (150 mg—Carbograph cartridges) and ultrafiltration on cellulose membranes (cutoff 10 kDa);

(10) FIG. 10 is bar chart depicting various Fucoidan tested for inhibitory activity in VLP binding assay with GII.4 capsids;

(11) FIG. 11 is bar chart depicting Inhibition of GII.4 VLP binding to HGM by polystyrenesulfonic acidprocessedfucoidan (PSSA);

(12) FIG. 12 is a bar chart depicting Fractionation of fucoidan processing products on BioGel-P2;

(13) FIG. 13 is a line chart depicting MALDI mass spectrometry of fucoidan processing products;

(14) FIG. 14 is a line chart depicting B, MALDI-MS/MS spectrum (Post-Source Decay spectrum in the LIFT modus) of a nonameric oligofucose at m z 1551.

(15) FIG. 15 is a line chart depicting Linkage analysis by GCMS of partially methylated alditol acetates (PMAA) from fucoidan processing products.

BEST MODE OF CARRYING OUT THE INVENTION

(16) Thus, with Fucoidan, desulfated Fucoidan and the Fucoidan-derived desulfated oligosaccharides that are the subject of this invention, infections by Noroviruses, Rotaviruses, Salmonella sp., Pseudomonas aeruginosa or Campylobacter jejuni can efficiently be prevented and/or treated.

(17) This invention also relates to the use of native Fucoidan, desulfated Fucoidan or the derived desulfated oligosaccharides as prophylactic and therapeutic agents (food additives) that can be applied during epidemic phases in hospitals, or outside, in order to prevent infections caused by Noroviruses, as they block viral binding to gastrointestinal epithelia. Fucoidan, desulfated Fucoidan or the derived desulfated oligosaccharides can also be used as prophylactic and therapeutic agents (food additives) during other infections caused by other pathogens, such as Rotaviruses, Salmonella sp., Pseudomonas aeruginosa and Campylobacter jejuni, which generally have the same pathogen entry route mediated by blood group H-like carbohydrate structures. The Norovirus contamination of oysters can also be abolished by washing the oysters with solutions of native, desulfated or processed Fucoidans, which should make them highly valuable in industrial (oyster) application.

(18) The following examples illustrate the inventive concept, in accordance with the description of the invention revealed in this patent, regarding the competitive affinity of desulfated oligosaccharides and providing the inhibition of pathogen binding whenever the infection is mediated by blood-group H-like carbohydrate structures, in order to reduce intestinal retention of the pathogens such as Noroviruses, Rotaviruses, Salmonela sp., Pseudomonas aeruginosa or Campylobacter jejuni. It is to be understood that these examples are provided by way of illustration only, and nothing therein should be taken as a limitation upon the overall scope of the invention.

(19) The invention will be illustrated further on by the embodiments without intending to limit on them.

Example 1—Binding of GII.4 VLPs on Immobilized Native F-Fucoidan

(20) A, Binding of GII.4 (Sydney 2012) VLPs on immobilized native F-fucoidan.

(21) Fucoidan was immobilized by drying from an ammonium hydrogencarbonate solution onto polystyrene microtitration plates. Blocking was performed with 5% BSA/PBS. VLPs were dispersed in 0.05% Tween-20/PBS (10 μg/ml according to protein content). Incubation conditions of binding assay were throughout 1 h at 37° C. for VLPs, primary antibody (anti-GII.4, rabbit polyclonal, 1:3000), and secondary antibody (anti-rabbit-Ig-alkaline phosphatase, 1:5000). Substrate p-nitrophenylphosphate (5 mg/ml) was incubated generally for 30 min at RT and color formation was measured at 405 nm. The figure (FIG. 1.) shows results from a duplicate assay at varying fucoidan concentrations.

(22) B, C, Binding of GII.4 (Sydney 2012) VLPs to human gastric mucin (HGM) at pH 7.4 (B) and at pH 1.5 to 5.5 (C); (FIG. 2.)

(23) D, Variation of blocking conditions for binding assays of GII.4 to HGM (5% skim milk/PBS vs. 5% BSA/PBS. (FIG. 3.)

Example 2—Inhibition of GII.4 (Sydney, 2012) VLP Binding to HGM (10 μg/Ml) by Native F-Fucoidan, and Desulfated F-Fucoidan from Fucus Vesiculosus

(24) Triplicate assay with fixed inhibitor concentration (10 mg/ml). (FIG. 4.)

Example 3—Inhibition of GII.4 (Sydney, 2012) VLP Binding to HGM (10 μg/Ml) by Processed F-Fucoidan (Processed by Partial Acid Hydrolysis in 0.01 M HCl, 4 h, 60° C., Followed by Neutralization)

(25) Triplicate assay with fixed inhibitor concentration (5 mg/ml). (FIG. 5.)

Example 4—Inhibition of GII.4 (Sydney, 2012) VLP Binding to HGM (10 μg/Ml) by Different Charges/Preparations of Native and Processed Fucoidan

(26) F-fucoidan stored for over 10 years at 5° C. and a fresh sample (both from Sigma) were tested in duplicate assays (see “old” and “new” charges) after partial acid hydrolysis (0.01 N HCl, 60° C., 4 h), neutralization and solubilization in capsid binding buffer (0.01% Tween-20/PBS) at varying concentrations. (FIG. 6.)

Example 5—Inhibition of GII.4 (2012) (A), and GII.17 (2015) (B) VLP Binding to HGM by Processed Fucoidan

(27) Fucoidan from Fucus vesiculosus was processed by partial acid hydrolysis with 0.01 N HCl for 4 h at 60° C., neutralized and solubilized after drying in the capsid binding buffer (0.01% Tween-20 in PBS). (FIG. 7.)

Example 6—Time Kinetics of Fucoidan Processing in Dilute Hydrochloric Acid Based on Binding Inhibition Assay with GII.4 (Sydney, 2012) VLPs

(28) Fucoidan from Fucus vesiculosus was incubated with 0.01 N HCl at 60° C. for increasing reaction times (0-16 h), neutralized with NaOH and tested for inhibitory activity in VLP binding assay with GII.4 capsids as described above. (FIG. 8.)

Example 7—Comparative Evaluation of Different Work-Up Procedures

(29) Workup procedures included solid-phase extraction on graphitized carbon (150 mg-Carbograph cartridges) and ultrafiltration on cellulose membranes (cutoff 10 kDa). Processed fucoidan (concentrations indicated in the graph, FIG. 9.) was tested for inhibitory activity in VLP binding assay with GII.4 capsids as described above.

Example 8—Variation of Acids, their Concentrations and Incubation Times

(30) Parameters were 0.01 M HCl (4 h, 60° C.), 0.1 M formic acid (FA) (16 h, 60° C.), 0.1 M acetic acid (AA) (16 h, 60° C.). Processed fucoidan (5 mg/ml) was tested for inhibitory activity in VLP binding assay with GII.4 capsids as described above. (FIG. 10.)

Example 9—Inhibition of GII.4 VLP Binding to HGM by Polystyrenesulfonic Acid Processed Fucoidan (PSSA)

(31) 30 mg fucoidan was solubilized in 3 ml water containing 46.7 μl PSSA, filled into a dialysis bag (1 cm diameter, 12 cm length, 6-8 kDa cutoff) and heated to 60° C. over a time period of 18 h. The filtrate was continuously cycled over 580 mg graphitized carbon (flow rate 24 ml/h).

(32) Elution from graphitized carbon was performed with 80% acetonitrile in water. Most of the filtered oligofucose remained unbound in the dialysis bag (65%), whereas 35% was eluted from the GC column. Each fraction was tested separately (filtrate: 7 mg/ml; GC eluate: 2.9 mg/ml). (FIG. 11.)

Example 10—Fractionation of Fucoidan Processing Products on BioGel-P2

(33) The fractionation on BioGel-P2 column (9.2 ml, 1.4×6 cm) is shown together with results from binding inhibition exerted by glycans in fractions eluted at 5-9 ml in GII.4 VLP binding assay on HGM. The insert (upper panel) shows results from a colorimetric assay of sugars (phenol-sulphuric acid assay). (FIG. 12.)

Example 11—MALDI Mass Spectrometry of Fucoidan Processing Products

(34) A, MALDI mass spectrometry on an Ultraflextreme TOF/TOF instrument of permethylated fucoidan processing products after ultrafiltration. Positive ion survey spectrum (MS1). The mass increment of 174 corresponds to a methylated deoxyhexose (indicated by “F”). Oligosaccharides with up to about 20 monosaccharide units were detectable. (FIG. 13.)

(35) B, MALDI-MS/MS spectrum (Post-Source Decay spectrum in the LIFT modus) of a nonameric oligofucose at m/z 1551. The mass increment of 174 corresponds to a methylated deoxyhexose (indicated by “F”). (FIG. 14.)

Example 12—Linkage Analysis by GCMS of Partially Methylated Alditol Acetates (PMAA) from Fucoidan Processing Products

(36) Six different PMAAs were identified in the GC chromatogram (TIC, total ion current) using full-scan spectra and single ion monitoring at m/z 118, 175 (terminal fucose, Fuc), m/z 190 and 247 (2-Fuc, 3-Fuc), m/z 203 (4-Fuc), m/z 262 (2,3-Fuc), and m/z 275 (3,4-Fuc). (FIG. 15.)