Polysaccharide suitable to modulate immune response

Abstract

Objective of the present invention is to provide polysaccharides which modulate immune response, and which can be used as ingredients in edible products or pharmaceutical compositions. The present invention provides such polysaccharides obtained from the species Camellia sinensis, which comprise a rhamnogalacturonan-I core, and wherein the molar ratio of galacturonyl acid residues to rhamnosyl residues in the backbone of the polysaccharide is close to 1:1. The present invention also provides edible products or pharmaceutical compositions containing such polysaccharides, in order to modulate immune response.

Claims

1. A method of stimulating immune response in a mammal, comprising administering to a mammal in need of stimulation of its immune response a product selected from a food product, a beverage, a dietary food product, a clinical food product and a dietary supplement, the product comprising an aqueous sugar beet extract that is enriched in polysaccharide having a molecular weight of at least 70 kDa and a backbone comprising alternating rhamnogalacturonan-I cores and alpha(1,4)-linked polygalacturonic acid or alpha(1,4)-linked oligogalacturonic acid cores, wherein the molar ratio of galacturonyl acid residues to rhamnosyl residues in the backbone of the polysaccharide ranges from 2.5:1 to 1:1, wherein the rhamnogalacturonan-I core comprises one or more side chains substituted at the 4-OH position of the rhamnosyl residues and having a backbone of at least one or more alpha(1,5)-linked arabinosyl residues, wherein the rhamnogalacturonan-I core comprises one or more side chains substituted at the 4-OH position of the rhamnosyl residues and having a backbone of at least one or more beta(1,4)-linked galactosyl residues, wherein the molar ratio of arabinosyl residues to rhamnosyl residues is between 50:1 and 1.1:2, and wherein the composition comprises 0.0001 to 25% by weight of the polysaccharide, which is administered at a daily dose of between 10 and 10,000 mg.

2. The method according to claim 1, wherein the polysaccharide has a molecular weight between 70 and 2,000 kDa.

3. The method according to claim 1, wherein the molar ratio of galacturonyl acid residues to rhamnosyl residues in the backbone of the polysaccharide ranges from 2:1 to 1:1.

4. The method according to claim 1, wherein the molar ratio of arabinosyl residues to rhamnosyl residues is between 30:1 and 1:2.

5. The method according to claim 1, wherein the molar ratio of galactosyl residues to rhamnosyl residues is between 80:1 and 1:1.

6. The method according to claim 1 for treating or increasing natural resistance of a subject against common cold.

7. The method according to claim 1, wherein the polysaccharide is administered as an adjuvant for a vaccine.

8. The method according to claim 1, wherein the composition is an edible product or pharmaceutical composition.

9. The method according to claim 1, wherein the composition comprises 0.0002% to 25% by weight of the polysaccharide.

10. The method according to claim 9, wherein the composition comprises 0.5% to 25% by weight of the polysaccharide.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Schematic representation of a preferred polysaccharide according to the invention:

(2) 1: galacturonyl acid residue in RG-I core

(3) 2: rhamnosyl residue in RG-I core

(4) 3: galactosyl residue in side chain, connected to 4-OH position of the rhamnosyl residue

(5) 4: arabinosyl residue in side chain, connected to 4-OH position of the rhamnosyl residue

(6) FIG. 2: Immune modulating effect of polysaccharides, obtained from the fruit of the species Malus domestica (apple); whole blood cell assay.

(7) Concentration of extract in microgram per milliliter (x-axis) versus percentage phagocytosis (y-axis).

(8) FIG. 3: Immune modulating effect of polysaccharides, obtained from the bean of the species Glycine max (soyabean); HL60 cell assay.

(9) Concentration of extract in microgram per milliliter (x-axis) versus percentage phagocytosis (y-axis).

EXAMPLES

(10) The following non-limiting examples illustrate the present invention.

(11) Methods

(12) Separation of Polysaccharides by Molecular Weight

(13) Polysaccharides obtained by boiling water extraction are separated by gel filtration chromatography on a column of Superdex 200 (130 cm; GE Healthcare). The polysaccharide samples are dissolved in 0.1 M ammonium bicarbonate and aliquots of 5-15 mg were run on the column in the same buffer. Eluting components are detected by absorption at 214 nm.

(14) The polysaccharides are pooled into three fractions: M.sub.W>110 kDa, 70-110 kDa and 40-70 kDa. The pooling limits are determined by comparing to elution positions of Dextran standards 40 kDa, 70 kDa and 110 kDa (ex GE Healthcare).

(15) Substitution and Composition Analysis of Polysaccharides

(16) The composition of polysaccharide fractions as obtained from the extraction process are determined using NMR analysis. Prior to NMR analysis, dry polysaccharide samples (1-10 mg) are dissolved in deuterium oxide (D.sub.2O) and dried in a vacuum centrifuge. Samples are then dissolved in 600 microliter of D.sub.2O. Spectra are collected on a Varian Unity 500 NMR spectrometer at 296 K and referenced to internal acetone standard (2.225 ppm).

(17) Referring to table 1, the polysaccharide substitution level and the molar composition of the building units are analysed by using the NMR data as follows:

(18) Rhamnogalacturonan Substitution Level

(19) The ratio of 4-substituted rhamnosyl units and nonsubstituted units is estimated by integrating the splitted rhamnose CH.sub.3 signals. The CH.sub.3 protons in 4-substituted rhamnosyl units resonate around 1.32 ppm, while those of the nonsubstituted rhamnosyl units at 1.25 ppm.

(20) Molar Ratio of Galactans and Arabinans

(21) The molar amount of galactans and arabinans is analysed by integrating the observed 1,4-galactan H-1 signal (4.64 ppm) and the arabinan H-1 signals (5Ara H-1, 5.09 ppm; 3,5Ara H-1 5.12 ppm; terminal Ara1-3 5.15 ppm; terminal Ara1-2 5.18 ppm; 2,3,5Ara H-1 5.26 ppm. These integration values are compared to the integrated rhamnose CH.sub.3 signals.

(22) Polygalacturonic Acid/RG-I Ratio

(23) The ratio of polygalacturonic acid to RG-I is analysed as follows: The total amount of galacturonic acid H-4 signals is integrated between 4.42 and 4.47 ppm, and the amount of rhamnose is obtained by integration of the CH.sub.3 signals (1.25-1.32 ppm). The H-4 signal of the RG-I specific GalA1-2 unit is located in the same 4.42-4.47 signal, and its portion has to be deducted from the total H-4 signal. This value is the same as rhamnose amount, as RG-I is a 1:1 polymer of Rha and GalA. The remaining H-4 signal represents the share of polygalacturonic acid type GalA1-4 H-4 signal.

(24) In vitro Assays

(25) Two assays are used to determine the immunomodulating response of the polysaccharides in vitro, both based on phagocytosis activity. These assays are:

(26) Whole Blood Cell Assay

(27) Phagocytosis activity in whole blood is evaluated using the Phagotest kit of Orpegen Pharma (Heidelberg, Germany) using an adjusted protocol. Fresh blood is obtained from healthy human volunteers in sodium heparin vacutainers (BD Biosciences). 30 microliter of whole blood and 5 microliter of the ingredient are incubated in duplicates for 30 minutes in a polypropylene 96-well plate at 37 C. in a water bath. Control incubations consisted of PBS (=basal phagocytosis activity) or 100 ng/mL E. coli-lipopolysaccharide (LPS) (=positive control sample) in triplicate measurements. After the incubation step, 10 microliter of FITC-labeled E. coli (white blood cell to E. coli ratio of 25:1) is added. This incubation at 37 C. is stopped after 6.5 minutes by adding 50 microliter of ice-cold quencher solution. The cells are washed three times by adding 230 microliter of ice-cold wash-buffer and centrifugation for 3 min at 300g (4 C.). The erythrocytes are lysed using 290 microliter of lysis buffer. After incubation in the dark for 20 minutes at room temperature, the cells are centrifuged for 5 min at 300g (4 C.). Cells are resuspended in 150 microliter of wash-buffer and stained with propidium iodide. Analysis is performed by flow cytometry (Coulter FC500 MPL flow cytometer, Beckman Coulter Nederland BV, Mijdrecht). Within the leucocytes, granulocytes are gated according to the FSC/SSC profile. The percentage of phagocytosing cells in the granulocyte population is determined. The results are normalized to the dynamic range between basal and LPS-stimulated phagocytosis and expressed as a relative percentage phagocytosis activity. A normalized percentage of more than 40% is considered to be positive.

(28) HL60 Cell Assay

(29) The human promyelocytic leukaemia cell line HL60 is used to determine the phagocytosis-enhancing capacity of the ingredients. This cell line can be differentiated towards the monocyte lineage with vitamin D3 and the cells subsequently obtain phagocytic capacity. 48 hours before the start of the assay, the HL60 cells are differentiated along the monocytic lineage by the addition of 1,25-dihydroxyvitamin D3 (VitD3) to the medium. Upon differentiation, 200 microliter of HL60 cells (810.sup.5 cells/ml) are transferred to 96-wells flat-bottom plates in triplicates. Fluorescent labeled microspheres (ratio beads: cells, 19:1) are added and incubated for 24 hrs at 37 C. Control incubations consist of differentiated HL60 cells in PBS (=basal phagocytosis level) or 100 ng/mL E. coli-lipopolysaccharide (LPS) (=positive control sample) in triplicate measurements. After the incubation period, the cells are transferred to a 96-wells V-bottom plate, washed three times and fixed with formaldehyde. For analysis, the cells are transferred to a 96-wells clear-bottom plate and fluorescence intensity is analyzed using a Flex Station II fluorometer. The data are normalized using the positive control and expressed as a relative percentage phagocytosis activity. A normalized percentage of more than 40% is considered to be positive.

Example 1

Isolation and Characterisation of Polysaccharides

(30) Various plant materials were used to extract polysaccharides. Polysaccharides were obtained from the following raw materials: leaves of the species Camellia sinensis (tea); the composition of polysaccharides from several samples was determined; roots of the species Daucus carota subsp. sativus (carrot); fruit of the species Malus domestica (apple); root of the species Beta vulgaris L. (sugar beet); bean of the species Glycine max (soyabean), source of the polysaccharides is water-soluble soya polysaccharides ex Fuji Oil (Japan).

(31) The procedure to obtain the materials was as follows. 25 g of methanol insoluble plant material was washed 2 times with 200 ml of 85% ethanol (VWR Prolabo) in water for 2.5 hours at 80 C. and 1 time with 200 ml of 85% ethanol in water for 1.5 hours at 80 C. After decanting the ethanol, the pellet was dried overnight in a fuming cabinet. The polysaccharides were extracted by adding 200 ml of demineralised (MilliQ) water and boiling for 3 hours at atmospheric pressure. After centrifugation at 2,000g for 20 min at room temperature (RT), the pellet was re-suspended in 200 ml of demineralised (MilliQ) water and boiled again for 3 hours. The supernatants of the first and second extraction were collected, freeze dried and stored at room temperature.

(32) From the polysaccharide enriched freeze dried extracts a 2% (w/w) suspension in demineralised water was made and autoclaved at a temperature of about 121 C. during about 15 minutes. The suspension was centrifuged at 2,000g for 30 minutes at room temperature. The supernatants were further purified by filtering through a 0.2 micrometer filter, divided in small portions and stored at 20 C.

(33) Subsequently, about 650 mg of extract was dissolved in 30 ml of 20 mM Tris-HCl, pH 7.5, and allowed to dissolve with frequent mixing for 1 hour. Insoluble matter was removed by centrifugation and the clear supernatant was subjected to anion-exchange chromatography on a column of DEAE-sepharose (50150 mm, ca. 290 ml) equilibrated with 20 mM Tris-HCl, pH 7.5, at a flow rate of 10 ml/min. After injection, the column was run isocratically with the equilibration buffer for 30 min, followed by a gradient of 0-1 M NaCl over 30 min, and 1 M NaCl for additional 40 min. Absorbance at 214 nm was recorded and fractions of 25 ml collected. The acid fraction was concentrated and desalted by ultrafiltration over a 10 kDa membrane prior to GPC. The yield was not measured at this point.

(34) The acid fractions obtained above were combined (from multiple runs) and then subjected to gel-filtration chromatography on a column of Superdex 200 (5 cm diameter, 95 cm length). Superloop was used in the injection due to large sample volumes. The column was eluted at a flow rate of 5 ml/min with 100 mM ammonium bicarbonate and the absorbance at 214 nm was recorded. Fractions of 25 ml were collected.

(35) Polysaccharides from apple were obtained in the following way. Fresh apples were grated and treated with an experimental pectolytic enzyme preparation, Rapidase C600 (0.02% w/w, 4 h at 45 C.; enzyme obtained from Gist Brocades, Delft, the Netherlands). The soluble part was recovered by centrifugation, and subjected to ultrafiltration on a 60 kDa molecular weight cut-off membrane, and then lyophilized. This fraction was further saponified to remove labile methyl and acetyl esters: a sample of 50.4 mg of apple RG was dissolved in 5 ml of 0.2 M sodium carbonate, pH 10, and allowed to react for 18 h at room temperature. The reaction mixture was then dialyzed for 24 h with three solution changes against 50 mM ammonium bicarbonate in MWCO 6000-8000 dialysis tubing. Finally, the sample was subjected to SPE in C-18 silica.

(36) The following compositions of polysaccharides obtained from various sources were determined.

(37) TABLE-US-00001 TABLE 1 Substitution and composition analysis of polysaccharides obtained from various sources; polysaccharide fractions separated on Superdex 200. Material and Rha 4-OH M.sub.W fraction subst..sup.(1) 1-4Gal.sup.(2) Ara.sup.(3) 1-4GalA.sup.(4) Tea #7 >110 kDa 45% 3 5 .sup.(5) 70-110 kDa 45% 2 3 .sup.(5) 40-70 kDa (comparative as 65% 3 3 .sup.(5) M.sub.W <70) Tea #15 >110 kDa 50% 6.5 0.5 .sup.(5) 70-110 kDa 70% 8 1.5 .sup.(5) 40-70 kDa (comparative as 60% 9 0.8 .sup.(5) M.sub.W <70 kDa) Tea #218 >110 kDa 35% 17 19 .sup.(5) 70-110 kDa (comparative 70% 8 20 46 as GalA:Rha >2.5) Tea #244 >110 kDa 37.50% 7.5 11 .sup.(5) 70-110 kDa (comparative 50% 3 55 as GalA:Rha >2.5) Apple >110 kDa 80% 3 3 .sup.(5) 70-110 kDa 60% 1.5 1 1.2 40-70 kDa (comparative as 80% 2 0.7 1.5 M.sub.W <70 kDa) Sugar beet >110 kDa 60% 1.7 29 .sup.(5) 70-110 kDa 60% 1.9 20 .sup.(5) 40-70 kDa (comparative as 45% 2.1 11 .sup.(5) M.sub.W <70 kDa) Carrot >110 kDa 55% 15 17 .sup.(5) 70-110 kDa (comparative 70% 5 10 16 as GalA:Rha >2.5) 40-70 kDa (comparative as 40% 1.5 6 16 M.sub.W <70 kDa) Soya >110 kDa 55% 44 23 70-110 kDa 50% 26 13 40-70 kDa (comparative as 40% 6 4 3 M.sub.W <70 kDa) Legend: .sup.(1)Rha 4-OH subst.: molar fraction of the Rha moieties in the RG-I core which is substituted at the C-4 position with a side chain; as measured from the Rha CH.sub.3 signal shift; .sup.(2)1-4Gal: molar ratio of beta(1,4)-linked Gal as compared to Rha (mol/mol); relates to the length of side chains containing beta(1,4)-linked galactan residues; .sup.(3)Ara: molar ratio of alpha(1,5)-linked arabinan as compared to Rha (mol/mol); relates to the length of side chains containing alpha(1,5)-linked arabinosyl residues; .sup.(4)1-4GalA: alpha-1,4-galacturonyl acid residues vs. rhamnosyl residues (mol/mol); i.e. this number indicates the molar ratio between GalA residues in the alpha(1,4)-linked polygalacturonic acid or alpha(1,4)-linked oligogalacturonic acid cores in the polysaccharide and Rha residues in the RG-I core of the polysaccharide; .sup.(5)Below measurable level; i.e. the amount of alpha(1,4)-linked GalA residues present in the polysaccharide originating from the alpha(1,4)-linked polygalacturonic acid or alpha(1,4)-linked oligogalacturonic acid cores is so low that it is not detectable. If this is the case, then the ratio GalA:Rha is 1.

Example 2

Immunomodulating Effect of Polysaccharides from Various Sources

(38) The immuno-modulating effect of various samples have been determined at various concentrations, using the two assays as described above. The results are given in the following table.

(39) Polysaccharides were obtained from the following raw materials: leaves of the species Camellia sinensis (tea); the immuno modulating effect of a pooled mix of samples of tea #7, tea #15, tea #218, tea #244 (see table 1) is indicated in the table below; roots of the species Daucus carota subsp. sativus (carrot); fruit of the species Malus domestica (apple); root of the species Beta vulgaris L. (sugar beet); bean of the species Glycine max (soyabean).

(40) TABLE-US-00002 TABLE 2 In vitro immune modulating activity of various samples, tested using whole blood cell assay, and/or HL60 cell assay. Concentration of extract (microgram per milliliter) obtained according to method above, versus phagocytosis activity of assay. whole blood cell assay HL60 cell assay 3 30 0.3 3 Material and microgram/ microgram/ microgram/ microgram/ M.sub.W fraction ml ml ml ml Tea >110 kDa + ++ + ++ 70-110 kDa ++ + Apple >110 kDa ++ ++ ++ ++ 70-110 kDa ++ + 40-70 kDa (comparative as M.sub.W <70 kDa) Sugar beet >110 kDa + ++ Carrot >110 kDa ++ ++ ++ Legend: ++ very positive effect (% phagocytosis >80%) + positive effect (% phagocytosis 40%-80%) no effect blank: not tested

(41) FIGS. 2 and 3 indicate the measured phagocytosis percentage, for some of the polysaccharide samples:

(42) FIG. 2: Apple (as in table 1 and 2), whole blood cell assay.

(43) FIG. 3: Soyabean; HL60 cell assay; this sample not in table 2, soyabean polysaccharides: Rhamnogalacturonan (Soy Bean) ex Megazyme International Ireland Ltd. (Wicklow, Ireland).

(44) Two of the samples obtained from the tea plant (Camellia sinensis) were also tested in the two tests, at lower concentrations this time. These samples were tea #7 and tea #15. Also the samples from carrot and apple were tested. The structural features of these materials have already been given in table 1. The results of these tests are the following:

(45) TABLE-US-00003 TABLE 3 In vitro immune modulating activity of two samples obtained from the plant Camellia sinensis, and from carrot and apple, tested using whole blood cell assay, and/or HL60 cell assay. Concentration of extract (microgram per milliliter) obtained according to method above, versus phagocytosis activity of assay. whole blood cell assay HL60 cell assay 0.03 0.3 0.003 0.03 Material and microgram/ microgram/ microgram/ microgram/ M.sub.W fraction ml ml ml ml Tea #7 >110 kDa ++ ++ 70-110 kDa ++ ++ 40-70 kDa ++ Tea #15 >110 kDa ++ ++ ++ ++ 70-110 kDa ++ ++ 40-70 kDa ++ Carrot >110 kDa 70-110 kDa 40-70 kDa Apple >110 kDa 70-110 kDa 40-70 kDa Legend: ++ very positive effect (% phagocytosis >80%) + positive effect (% phagocytosis 40%-80%) no effect blank: not tested

(46) This example shows that the extracts obtained from the plant Camellia sinensis showed very high immunostimulating activity in vitro. Already at concentrations as low as 0.003 microgram per millileter, already a very high immunostimulating activity was measured in the whole blood cell assay, as compared to various other polysaccharides obtained from other vegetable materials. Polysaccharides obtained from apple or from carrot did not show immunostimulating effect in the whole blood cell assay at concentrations of 0.03 and 0.3 microgram per millilter.

Example 3

Properties of Polysaccharide

(47) During the isolation of polysaccharides from carrot, it was observed that these polysaccharides did not lead to thickening of fluids in which it was dissolved. This is in contrast to normal pectins, wherein similar concentrations of polymer lead to thickening of the fluids.