Sulphated polysaccharide composition

Abstract

The invention concerns a composition made from at least one sulphated polysaccharide, in particular from an algae, and combined with at least one food ingredient. This composition can be used, in particular, in the field of food or in the treatment or prevention of an infection caused by microsporidia in humans or animals. It has useful properties as an antiparasitic agent in humans or animals; for the treatment or prevention of an infection caused by at least one microsporidia in humans or animals; in particular for the treatment or prevention of an infection caused in bees by the microsporidia Nosema, preferably Nosema ceranae or Nosema apis, or indeed for stimulating the immune defences of bees.

Claims

1. A method for treating or preventing an infection caused in honey bees by the microsporidium Nosema, comprising administering to honey bees in need thereof an effective amount of a sulphated polysaccharide selected from the group consisting of: a sulphated polysaccharide derived from a red alga; a sulphated polysaccharide derived from a green macroalga of the Chlorophyceae species that produces polysaccharides; a sulphated polysaccharide derived from a brown macroalga of the Pheophyceae group that produces sulphated fucans; and a mixture thereof.

2. A method for stimulating immune defence systems of honey bees, comprising administering to honey bees in need thereof an effective amount of a sulphated polysaccharide selected from the group consisting of: a sulphated polysaccharide derived from a red alga; a sulphated polysaccharide derived from a green macroalga of the Chlorophyceae species that produces polysaccharides; a sulphated polysaccharide derived from a brown macroalga of the Pheophyceae group that produces sulphated fucans; and a mixture thereof.

3. The method according to claim 1, wherein the sulphated polysaccharide is included in a composition further comprising at least one food ingredient.

4. The method according to claim 1, wherein the sulphated polysaccharide is included in a composition further comprising at least one food ingredient selected from a dietary protein agent, a sugar including saccharose, a sugar including sucrose, honey or mixtures thereof.

5. The method according to claim 1, wherein the sulphated polysaccharide is derived from a macroalga and obtained by a method comprising the following steps: preparing a solution of the sulphated polysaccharide from the macroalgae, by heating under reflux in water at a temperature ranging from 50° C. to 100° C.; precipitating the sulphated polysaccharide using a polar solvent or by dialysis; and drying the sulphated polysaccharide.

6. The method according to claim 1, wherein the sulphated polysaccharide is derived from a microalga and obtained by a method comprising: culturing the microalga; concentrating the culture medium, after extraction of the biomass, in vacuo and at a temperature ranging from 30° C. to 50° C.; extracting the sulphated polysaccharide by diafiltration, precipitation or dialysis; and drying the sulphated polysaccharide.

7. The method according to claim 1, wherein: the sulphated polysaccharide is obtained by chemical modification of a polysaccharide; or the sulphated polysaccharide is prepared by a method including a step of sulphation; or the sulphated polysaccharide has a rate of sulphation on a weight basis that is greater than or equal to 5%; or the sulphated polysaccharide is comprised in a composition wherein the mass concentration of sulphated polysaccharide ranges from 50 to 1,000 mg/mL; or the sulphated polysaccharide is comprised in a composition further comprising at least one food ingredient, and wherein the sulphated polysaccharide/food ingredient ratio by weight ranges from 0.0001 to 0.002; or the sulphated polysaccharide is comprised in a composition further comprising at least one food ingredient, and wherein the food ingredient is selected from the group consisting of saccharose, sucrose, fructose, glucose, maltose, honey, and mixtures thereof; or the sulphated polysaccharide is comprised in a composition at a concentration by weight ranging from 0.01% to 2%; or the sulphated polysaccharide is comprised in a composition further comprising at least one food ingredient at a concentration by weight ranging from 98% to 99.99%.

8. The method according to claim 1, wherein the sulphated polysaccharide is derived from: a red macroalga selected from the Rhodophyceae species that produce agar, carrageenans, porphyrans, furonans, or complex sulphated galactans; or a red microalga selected from Porphyridium marinum, Porphyridium purpureum, and the Rhodella species.

9. The method according to claim 1, wherein the microsporidium Nosema is at least one of Nosema ceranae or Nosema apis.

Description

EXAMPLE 1

Evaluation of Cytotoxicity of Sulphated Polysaccharides According to the Invention

(1) Culturing of HFF (Human Foreskin Fibroblast Cells in 96-Well Plate From a 75 cm.sup.2 flask of HFF cells at confluence, the culture medium was removed, and then 2 mL of trypsin-EDTA (0.025% trypsin and 0.01% EDTA—Ethylenediaminetetraacetic acid) was added (at 37° C.). The flask was then briefly rinsed and the trypsin was removed. 2 mL of trypsin-EDTA was subsequently added and the flask was incubated for 5 minutes in an oven at 37° C.

(2) 5 ml of culture medium (minimal essential medium—MEM (PARA Laboratories GmbH)+Glutamine (2 mM)+Fungizone (2.5 μg/mL)+Penicillin (100 units/mL)+Streptomycin (100 μg/mL)+decomplemented Foetal Calf Serum 15%) was added and then the flask was rinsed with a pipette. The medium and cells were recovered in a tube. Counting on the Malassez counting chamber was then performed.

(3) The cells were subsequently centrifuged at 200 g, for 7 minutes, and then the supernatant was removed. The pellet was taken up in the culture medium in a manner so as to obtain 10.sup.5 cells/mL.

(4) 200 μL of this suspension per well was distributed, while leaving empty the wells of columns 1 and 12 and rows A and H. The wells that were left empty were filled with 200 μL of culture medium in order to prevent evaporation.

(5) Addition of Sulphated Polysaccharides

(6) When the cells had reached confluence in the wells (about 48 hours), the medium was aspirated by making use of a vacuum extractor pump and then 200 μL of culture medium containing the various polysaccharides to be tested (the latter are tested at 50, 100, and 200 μg/mL) was added.

(7) Each concentration was tested in triplicate. Each plate had to contain a negative control (culture medium without extract), a positive control (culture medium containing 20% of DMSO), and a fumagillin control (1 μg/mL). The results are summarised in Table 1 here below.

(8) TABLE-US-00001 TABLE 1 Viability (%) Conclusion Healthy Cells Control 100 +/− 2.3  Control validated DMSO Positive Control 6.8 +/− 1.4 Cytotoxic: control validated Fumagillin Control 99.1 +/− 12.2 Non-cytotoxic: control validated
Test of Cytotoxicity

(9) After 96 hours of incubation, the medium contained in the wells was aspirated with a vacuum extractor pump and then 200 μL of fresh culture medium and 50 μL of TCA (Trichloroacetic acid) at 50% were added and the mixture was left for 5 minutes at ambient temperature and then for a period of 2 hours at 4° C.

(10) The supernatant was removed with a vacuum extractor pump and washed 5 times (by overturning the 96-well plate) with 100 μL of distilled water and then the plate was dried (1 hour in the vacuum extractor pump, or 2 hours overturned on the bench). Once dried the plate was able to be stored at ambient temperature until subsequent use thereof.

(11) 100 μL of sulforhodamine B (0.4% in 1% acetic acid) was added and the mixture was incubated for 20 minutes at ambient temperature. The sulforhodamine B was removed by making use of a vacuum extractor pump. A wash cycle repeated 5 times (by overturning the 96-well plate) with 100 μL of 1% acetic acid was carried out and then the plate was dried for 5 minutes at ambient temperature by overturning it.

(12) The proteins were solubilised by using 200 μL of Tris-Base (10 mM) for 5 minutes on a plate with stirring at ambient temperature. The OD (Optical Density) at 550 nm was measured by making use of a microplate reader (Multiskan FC357, Thermo Scientific).

(13) The results are presented in Table 2 here below.

(14) The mean of the OD and the standard deviation have been calculated for each sample tested in order to arrive at a conclusion with respect to the toxicity of the products.

(15) TABLE-US-00002 TABLE 2 Viability Viability Viability (%) at 50 (%) at 100 (%) at 200 μg/mL μg/mL μg/mL Conclusion SP1 119.1 +/− 1.8  156 +/− 8  110 +/− 5  Non-cytotoxic SP2  95.6 +/− 13.7 89.2 +/− 6.sup.  69.2 +/− 7.8 Non-cytotoxic up to 100 μg/ mL SP3 101.3 +/− 8.5  108.1 +/− 20.8 .sup. 110 +/− 13.5 Non-cytotoxic SP4 94.2 +/− 5.2  98.7 +/− 20.8 97.3 +/− 8.8 Non-cytotoxic SP5 81.1 +/− 2.9 74.3 +/− 10   87.5 +/− 15.6 Considered to be non- cytotoxic SP6 84.4 +/− 13  .sup. 89 +/− 6.4 63.7 +/− 5.sup.  Non-cytotoxic up to 100 μg/ mL SP7 144.7 +/− 30.6 136.7 +/− 21.8 139.6 +/− 18.2 Non-cytotoxic SP8 143.7 +/− 35.7 142.2 +/− 31.sup.   96.7 +/− 22.3 Non-cytotoxic SP9 149.4 +/− 16.1 141.4 +/− 18.3 124.5 +/− 20.2 Non-cytotoxic

(16) The results show that the sulphated polysaccharides according to the invention present no cytotoxicity, in particular up to 100 μg/mL.

EXAMPLE 2

Evaluation of the Anti-Parasitic Activity In Vitro on the Microsporidium Encephalitozoon Cuniculi, of Sulphated Polysaccharides According to the Invention

(17) Culturing of HFF (Human Foreskin Fibroblast) Cells in 48-Well Plates

(18) From a 75 cm.sup.2 flask of HFF cells at confluence, the culture medium was removed, and then 2 mL of trypsin-EDTA (0.025% trypsin and 0.01% EDTA—Ethylenediaminetetraacetic acid) (at 37° C.) was added.

(19) The flask was then briefly rinsed, and then the trypsin was removed. 2 mL of trypsin-EDTA (0.025% trypsin and 0.01% EDTA) was subsequently added and the plate was incubated for 5 minutes in an oven at 37° C.

(20) 5 ml of culture medium (MEM (PARA Laboratories GmbH)+Glutamine (2 mM)+Fungizone (2.5 μg/ml)+Penicillin (100 units/mL)+Streptomycin (100 μg/mL)+decomplemented Foetal Calf Serum 15%) were added and the dish was rinsed using the pipette. The medium and cells were recovered in a tube. Counting on the Malassez counting chamber was then performed. The cells were subsequently centrifuged at 200 g, for 7 minutes. The supernatant was then removed and the pellet was taken up in the culture medium in a manner so as to obtain 10.sup.5 cells/mL.

(21) 400 μL of this suspension per well was distributed, while leaving empty the wells of columns 1 and 8 and rows A and F. The wells that were left empty were filled with 400 μL of culture medium in order to prevent evaporation.

(22) ELISA Test on Microsporidia

(23) When the cells had reached confluence in the wells (about 48 hours), the medium was aspirated by making use of a vacuum extractor pump. Then 400 μL of culture medium containing the various polysaccharides to be tested (the latter were tested at the highest non-cytotoxic concentration, that is to say 100 or 200 μg/mL) was added, and then incubated for 2 hours in an oven at 37° C.

(24) In parallel, a solution of spores of Encephalitozoon cuniculi (obtained from culture supernatant maintained in the laboratory) at a concentration of 5.10.sup.5 spores/mL was pre-incubated for 2 hours at 37° C. in the culture medium containing the polysaccharides to be tested. The spores were also pre-incubated under the same conditions but without polysaccharide, the said spores having served to infect the cells used to produce the negative control cited farther below. The latter was subsequently brought into contact for 1 hour at 37° C. with the cells, the polysaccharide being still present.

(25) After a wash with PBS (Phosphate Buffered Saline) in order to remove the non-adherent spores, the various polysaccharides were added and thus left for the time period of the test operation (5 days). Each polysaccharide was tested in triplicate. Each plate contained an uninfected “healthy cells” control, a negative control (infected cells with no treatment) and a positive control (infected cells incubated in the presence of fumagillin at 1 μg/mL).

(26) At the end of the test operation, the culture medium of each well was removed and a wash cycle with 200 μL of PBS was carried out. The wells were fixed individually with 100 μL of methanol for 20 minutes at −80° C. and then the cells were saturated with 100 μL of a blocking solution (100 mM Tris, 2% Bovine Serum Albumin BSA) for an entire night at 4° C.

(27) The blocking solution was subsequently removed by making use of a vacuum extractor pump, then 100 μL of naturally infected rabbit serum (primary antibody) diluted to 1/1,000.sup.th in the dilution buffer for antibodies (10 mM Tris pH=7.4, 150 mM NaCl, 0.05% Tween 20, 0.2% BSA) was added. The mixture was incubated for a period of 2 hours at 37° C. Then a wash cycle repeated 5 times with 200 μL of the washing solution (0.05% Tween 20, 10 mM Tris pH=9.8) was carried out. 100 μL of the anti-rabbit IgG secondary antibody (IgG Alkaline Phosphatase AP-conjugate Promega 1 mg/mL) coupled with alkaline phosphatase diluted to 1/10,000.sup.th in the dilution buffer for antibodies was added. The mixture was incubated for a period of 1 hour at 37° C., and then a wash cycle repeated 5 times with 200 μL of the washing solution was carried out.

(28) 200 μL of 10 mM MUP (4-methylumbelliferyl phosphate) diluted to 1/100.sup.th in the revelation solution (1 mM MgCl.sub.2, 50 mM Na.sub.2CO.sub.3 pH=9.8) was added, and then the mixture was incubated for 30 minutes in the dark, under gentle agitation conditions.

(29) The reading was carried out with fluoroscan with an excitation wavelength and an emission wavelength of 355 nm and 460 nm, respectively.

(30) The results are presented in Table 3 here below.

(31) The parasite growth inhibition data were calculated by taking into consideration the following information: the “healthy cells” control corresponds to the blank sample, the negative control (infected cells with no treatment) corresponds to 100% of parasite growth.

(32) The inhibition capacity of the polysaccharides tested was evaluated on the basis of the following scale: <30%: low inhibition; between 30% and 40%: average inhibition; between 40% and 50%, satisfactory inhibition; between 50% and 70%: strong inhibition; between 70% and 90%: very strong inhibition; >90%: excellent inhibition.

(33) TABLE-US-00003 TABLE 3 Inhibition of parasite Product growth/development (%) Conclusion SP1 (200 μg/mL) 34.5 +/− 5.sup.  average inhibition SP2 (100 μg/mL)  34.1 +/− 11.9 average inhibition SP3 (200 μg/mL) 46.8 +/− 8.7 satisfactory inhibition SP4 (200 μg/mL) 29.9 +/− 4.sup.  average inhibition SP5 (200 μg/mL) 99.4 +/− 1.1 excellent inhibition SP6 (100 μg/mL)  90.3 +/− 16.7 excellent inhibition SP7 (200 μg/mL)  48.3 +/− 11.3 satisfactory inhibition SP8 (200 μg/mL) 35.7 +/− 7.4 average inhibition SP9 (200 μg/mL) 35.9 +/− 0.7 average inhibition

(34) The results show that the sulphated polysaccharides according to the invention exhibit satisfactory antiparasitic activity on microsporidia. The sulphated polysaccharides derived from a microalga of the genus Porphyridium show excellent growth inhibitory activity.

EXAMPLE 3

Evaluation of the Inhibitory Activity In Vivo of Sulphated Polysaccharides According to the Invention on Nosema in Honey Bees

(35) The results presented here have been obtained from the summer honey bees.

(36) Retrieval/Recovery of Emerging (Newborn) Honey Bees and Maintenance in Beekeeping Box (Super)

(37) After recovering one brood chamber frame (or more as per the needs in honey bees) from the apiary by means of a glass frame holder, the latter was placed in an oven at 34° C. for a period of 24 to 48 hours in a manner so as to allow the honey bees to emerge from their cells. Then, the young honey bees were retrieved from the frame by making use of a pair of entomologist's forceps and placed in the beekeeping boxes (supers) on the basis of 50 honey bees/box. ⅙.sup.th of a tube stick of PseudoQueen (Contech Enterprises, Canada) was added in each of the beekeeping boxes.

(38) Feeding of the Honey Bees

(39) Each polysaccharide treatment was carried out in triplicate (three boxes/treatment). Each test operation contained a “healthy honey bees” control (honey bees not infected and not treated), an “infected honey bees” control (honey bees infected experimentally and not treated) and a fumagillin control; with these controls also being in triplicate.

(40) The honey bees were fed for a period of 3 days prior to infection with the syrup alone (“healthy honey bees” control and “infected honey bees” control), with syrup supplemented with 1 mg/ml fumagillin (fumagillin control), or with syrup containing the various sulphated polysaccharides to be tested (at the highest non-cytotoxic concentration, that is to say 100 or 200 μg/mL). The syrup was constituted of 50% of saccharose, supplemented with 1% of proteins (Provita′Bee, Laboratoires Corylis, France).

(41) The feeding of the honey bees was accomplished by means of 5 mL transparent plastic tubes drilled at the ends thereof in order to enable the honey bees to feed themselves.

(42) Individual Infection of the Honey Bees by Nosema ceranae

(43) The honey bees had been kept in fasting condition for 30 to 60 minutes prior to infection. During this time, a solution of Nosema ceranae spores was prepared by diluting the latter in the feeding syrup on the basis of 125,000 spores for 3 μL. Each bee was thus infected with 125,000 spores.

(44) The honey bees were subsequently gassed with CO.sub.2 (about 1 minute/box). When honey bees began to wake up, they were withdrawn by making use of a pair of entomologist's forceps. The honey bees were subsequently individually infected via the oral route, by making use of a pipette containing 3 μL of the prepared solution of spores (by ensuring that the honey bees drank the 3 μL solution in its entirety). The same test operation was carried out with the boxes of the “healthy honey bees” control while ensuring that only the feeding syrup alone was given. 45 honey bees per box were placed in the supers.

(45) After the infection step, to each of the boxes the feeding tubes were added so as to correspond to the different conditions (only syrup for the “healthy honey bees” control and “infected honey bees” control, syrup supplemented with 1 μg/mL fumagillin for the fumagillin control, syrup containing the various sulphated polysaccharides). Honey bees were placed in the oven at 33° C. (humidity 60-70%), and fed continuously for a period of 20 days.

(46) Monitoring of the Test Operation During the Post-Infection Phase

(47) Over the course of the post-Infection phase (a time period of 20 days), the dead honey bees were counted and removed on a daily basis from each of the boxes in order to track and monitor mortality. The feeding tubes were replaced every two days until the end of the test operation. It was possible to weigh the feeding tubes with a view to monitoring the consumption of the various different feed treatments by the honey bees.

(48) At the end of 20 days, 30 live honey bees (10 honey bees/box) were sacrificed for each of the conditions tested, in order to carry out a count of the Nosema ceranae spores with the objective of assessing the parasite load in the honey bees. For this, the intestine and rectal ampulla of each of the honey bees were dissected and then ground in a Potter mill in 100 μL of PBS. After 3 washes, the counting was performed on Kovacs chambers.

(49) The results are presented in Table 4 here below.

(50) TABLE-US-00004 TABLE 4 Mortality at 20 days Change in post-infection (%) parasite load “Infected Honey Bees” 53.3 +/− 13.6 None Control Fumagillin Control 25.9 +/− 16.7 Decrease of 63.4% SP1 60 +/− 8  Decrease of 26.4% (200 μg/mL) SP3 41.5 +/− 19.sup.  Decrease of 0.6% (200 μg/mL) SP5 43.7 +/− 6.8  Decrease of 20.4% (200 μg/mL) SP6 35.6 +/− 10.2 Decrease of 33.8% (100 μg/mL) SP1-SP5 Mixture 54.8 +/− 26.5 Decrease of 6.25% (100 μg/mL each)

(51) The results showed a significant decrease in mortality of the honey bees when they were fed with a composition comprising a sulphated polysaccharide according to the invention.

(52) The sulphated polysaccharide SP6 derived from Porphyridium marinum in particular shows a significant decrease in mortality among the honey bees.

(53) Thus, the sulphated polysaccharides according to the invention provide the ability to limit the development and growth of the parasite (illustrated by the decline in the spore count) and thereby to significantly reduce the mortality rate among honey bees infected with nosema disease.