KR20130140338A - Complement-activating polysaccharides from fomes fomentarius - Google Patents

Abstract

The present invention relates to a water soluble polysaccharide derived from Fomes fomentarius having an effect of activating a channel of complement protein in plasma of the human body. Complement-activating polysaccharide is provided by separating and refining polysaccharide, and an anti-cancer composition and functional food are provided by using the same. The water soluble polysaccharide contains 60-99.9% of xylose.

Description

{Complement-Activating Polysaccharides from Fomes fomentarius}

The present invention relates to a water-soluble polysaccharide derived from horsetail mushroom which has an effect of activating pathway of complement protein in human plasma.

The complement system is likely to be involved not only in defense against infection, inflammation defenses, allergic reactions, and immune responses. Therefore, understanding of the complement system has a potential importance in diagnosing and treating infections, allergic diseases and autoimmune diseases, and it is noteworthy that it is directly involved in the immune activity of the human body. Among the natural products, the substances (or polysaccharides) that activate the complement system (or polysaccharide) in the polymer fractions (mainly polysaccharides or protein polysaccharides) rather than the low molecular fractions are developed as immunotherapies for improving or treating the immunodeficiency state of the living body. And the anti-complement activity stimulates immune cells such as macrophages in vivo to induce the secretion of cytokines necessary for lymphocyte proliferation and activation, thereby being useful for cancer treatment (Anderson J., 1972. Induction of immunoglobulin and antibody synthesis in vitro by lipopolysaccharide. Eur. J. Immunol. 2: 349) , (Mitsuhiko N., S. 1998. Activation of macrophages by crude polysaccharide fractions obtained from shoots of Glycyrrhiza glabra and hairy roots of Glyc yrrhiza uralensis in vitro. Bio. Pharm. Bull. 21 (109: 1110-1112).

Complement system is composed of about 20 kinds of circulating proteins including basic components of C1 to C9 as a factor that plays a part of the humoral immune system in higher organisms. The complement system is a biologic defense mechanism that is activated nonspecifically at the time of antigen invasion and decomposes the target cells such as bacteria and virus and removes the pathogenic bacteria through the activation of macrophages and lymphocytes, chemotaxis and opsonization. In particular, they are important defenses in that they respond immediately before the host is immunized (Hames, BD and Glover, DM, 1983, The complement system, In Molecular Immunology, IRL Press, Oxford. , 1986, Activations of the complement system, Allergy., 6: 61-66). Activation of the complement system is divided into a classical pathway, a lectin pathway and an alternative pathway depending on the pathway leading to C3 activation, which is known to play the most important role.

The anti-complement activity substances known so far are mainly pectic arabinogalactan (Angelica acutiloba) isolated from plants (Yamada, H., 1987, Structural characterization of an anti-complementary arabinogalactan from the roots of Angelica acutiloba KITAGAWA. Chrbohydr. 159: 275), arabinan, arabinogalactan, glucan of Bupleurum falcatum (Yamada, H., 1988, Chracterization of anti-complementary neutral polysaccharides from the roots of Bupleurum falcatum. Phytochem., 27: 3,163), Colocasia (Ginkgo biloba), Capsella bursapastoris (Shin, KS, 1993, Screening of complementsystem activating polysaccharide from edible plants and its action mode. Food Sci. Technol., 25: 197) Complement system activity has been reported.

Particularly, interferon inducing activity, antitumor activity, anticoagulant activity, antiinflammatory activity, pharmacological enhancing activity and activity of B lymphocyte have been revealed among natural polymeric substances, and its active ingredients have been confirmed as polysaccharides. Therefore, attempts have been made to select polysaccharides from mushrooms as biological response modifiers and use them as pharmaceuticals or functional foods. B-lymphocyte-stimulating polysaccharide from mushroom Phellinus linteus. Chem. Pharm. Bull 43 (12): 2105-2108), and mushroom mushroom (Agaricus bispours) (Yang HC, 1998. Structural characterization of the anti-complementary and macrophage activating polysaccharides isolated from Agaricus bisporus. Korean J. Food Sci. Technol. 30 (3): 709-716), Coriolus versicolor 28 (5): 601-607), which was prepared by the method described in Wang et al., J. Biol. Chem. macrophage activity, lentinan of Lentinusedodes (Song CH, 1998, Antimicrobial activity of endopolymers produced from submerged mycelial culture of higher fungi with particular reference to Lentinus edodes. Biotechnology Letters, 20 (8): 741 744) has been reported from the discovery that anti-complement activity and anti-cancer active substance. Most of these materials have been widely used for food or medicinal purposes since ancient times, and they can be said to be materials that are stable to the human body.

Fomes fomentarius , widely distributed in the temperate north of the Northern Hemisphere, is a mushroom belonging to the Polyporaceae Fomes, which grows for many years in hardwoods or raw trees of broad-leaved trees. The umbrella is semicircular, bell-shaped or horseshoe-shaped, the surface is gray, thick, covered with a hard shell, and has yellowish brown or blackish brown wavy patterns or transversely intense grooves. In the private sector, antitumor, anti-gram-positive bacteria, antioxidant, antipyretic, diuretic and anti-diabetic effects are known, but systematic studies on their pharmacological agents, especially pharmacological polysaccharides, are insufficient.

Although an invention relating to a therapeutic agent for an immune disease including a horseshoe mushroom (Korean Patent Application Publication No. 2006-0044888) has been disclosed, it has been disclosed that it is useful as a therapeutic agent for circulatory diseases including a horseshoe mushroom, a mushroom, a chaga mushroom, , Immune system diseases, and the like, and it is not known which mechanism of action is due to which ingredient, which makes it difficult to use.

Korean Patent Application Publication No. 2006-0044888 (May 19, 2006)

The present invention provides a complement-activating polysaccharide having xylose as a main constituent sugar by separating and purifying a water-soluble polysaccharide derived from horseradish to mushroom having an effect of activating pathway of complement protein in human plasma, We want to provide food.

The present invention provides a polysaccharide comprising a molecular weight of 10,000 to 20,000 daltons, 60.0 to 99.9% xylose.

The present invention provides a complement activator oligosaccharide obtained by hydrolyzing the polysaccharide.

The present invention provides an immunoconjugate composition comprising said polysaccharide.

The complement-activating polysaccharide according to the present invention stimulates the innate immune system, so that antitumor compositions and functional foods using the same can be provided.

Fig. 1 shows the separation process of a water-soluble polysaccharide from horseradish mushroom.
FIG. 2 shows the results of fractionation of the horseradish mushroom extract by DEAE-Sepharose FF column chromatography.
Fig. 3 shows the result of fractionation of Connavalin A Sepharose column fractions of Horseradish Mushroom Extract.
Figure 4 shows the antifouling activity of purified polysaccharide from the fruiting body of horseradish to mushroom.
Figure 5 shows the result of two-dimensional cross-immunoelectrophoresis of the purified polysaccharide fraction from horseradish to activate the complement pathway.
FIG. 6 shows the production ability of interleukin IL-6 by treating MFKF-AP1-? And MFKF-AP1-? Purified from the horseradish mushroom with macrophages.
FIG. 7 shows the production ability of interleukin IL-12 by treating macrophages with the purified polysaccharide fractions MFKF-AP1-? And MFKF-AP1-? From horseradish mushroom.
FIG. 8 is an analysis of the polysaccharide fractions MFKF-AP1-alpha and MFKF-AP1-beta purified from horseradish mushroom by gas chromatography. [(A); Per standard, (B); MFKF-AP1?, (C); MFKF-AP1?]
9 compares the complement activity of xylose derived from horseradish mushroom and virgin xylan.
Figure 10 shows the molecular weight of MFKF-AP1?, The main antiphlogistic active polysaccharide in the polysaccharide fraction purified from horseradish to mushroom, by HPLC
Fig. 11 is a graph of the molecular weights of two kinds of neutral polysaccharides and acidic polysaccharides derived from horseshoe mushroom, analyzed by Toyopearl HW65F column (2.5 cm in diameter and 100 cm in length).

The present invention provides a water-soluble polysaccharide composed of xylose having a molecular weight of 10,000 to 20,000 daltons, 60.0 to 99%.

The molecular weight of the polysaccharide may be from 10,000 to 20,000 daltons, from 10,000 to 18,000, from 10,000 to 15,000, or from 10,000 to 12,000 daltons.

The polysaccharide contains xylose as a main constituent sugar and includes at least one of nose, galactose, arabinose, fucose, rhamnose and glucose.

More specifically, the polysaccharide is composed of 97 to 98% of xylose, 1 to 2% of arabinose, 0.1 to 0.5% of mannose, 0.01 to 0.03% of rhamnose, 0.001 to 0.020% of galactose and 0.05 to 0.10% of glucose.

A polysaccharide composed of 99.01 to 99.99% xylose, 0.5 to 1.0% arabinose, 0.1 to 0.3% glucose, and a rhomboose amount;

Which comprises 69 to 70% of xylose, 9 to 10% of mannose, 7 to 8% of galactose, 4 to 5% of fucose, 3 to 4% of rhamnose and 3 to 4% of glucose and 1 to 2% of arabinose Polysaccharide; And

A polysaccharide composed of 89 to 90% of xylose, 5 to 6% of mannose, 2 to 3% of rhamnose, and 2 to 3% of fucose.

The polysaccharide is characterized by exhibiting anti-complement activity and interleukin production promoting activity.

The polysaccharide may be derived from horseradish mushroom.

The present invention relates to a method for preparing a crude polysaccharide, which comprises subjecting a horseshoe mushroom powder to hot water extraction, precipitating with alcohol, dialyzing and lyophilizing to obtain crude polysaccharide; (MFKF-NP, MFKF-AP1-α, and MFKF-AP1) were separated by ion exchange resin (DEAE Sepharose FF column) chromatography and then by affinity (Concanavalin A Sepharose column) -β, MFKF-AP2-α, MFKF-AP2-β).

DEAE-sephadex and DEAE-sepharose-based resins may be used as the ion exchange resin chromatography, and Concanavaline A-sepharose may be used for affinity chromatography.

The polysaccharide is characterized in that it exhibits anti-complement activity and interleukin production promoting activity.

The anticomplement activity is decomposed into C3a and C3b, which are the most important components in activation of the complement system by activation of the classical pathway and subpaths of the complement system, and thereafter, the activation of C5 and the binding reaction of C6 to C9 to the antigen are continuously performed, Is activated due to activation of the complement system.

The interleukin may more particularly be IL-12 or IL-6.

The present invention can provide a complement activator oligosaccharide obtained by hydrolyzing the polysaccharide.

The xylooligosaccharide can be obtained by partial hydrolysis of xylan derived from horseradish mushroom according to the present invention with an acid, xylocidase or xylanase.

The partial hydrolysis may be a commercial xylanase enzyme treatment.

The xylooligosaccharides obtained according to the above method can be oligomeric oligosaccharides with a lower molecular weight than the conventional xylooligosaccharides.

The present invention provides an immunoconjugate composition comprising a complement pathway activating polysaccharide.

The present invention activates the complement pathway and enhances the production of interleukin. The activity of interleukin-6 (IL-6) is related to the production of T cells and B cells. The activity of interleukin 6 (IL-6) and interleukin-12 (IL- Mediated immune responses such as activation of cytotoxic T lymphocytes (CTLs) through induction of responses. In particular, since IL - 12 is directly involved in NK cell activation, which is a cancer cell lethal effect in the presence of cancer cells, it is considered to be an essential cytokine for inducing anticancer activity. Therefore, this sample can effectively inhibit cancer cell growth.

The present invention also provides a functional food containing the composition for enhancing immunity comprising the complement pathway-activating polysaccharide as an active ingredient.

Functionality can be categorized into physical properties and physiological functions. When the composition for enhancing immunity comprising a polysaccharide derived from horsetail mushrooms according to the present invention is added to a general food, the physical properties and physiological functions of the general food will be improved. For example, an anticancer agent using the immunity enhancing effect of the immunoconjugate composition and a functional food for enhancing post-surgical immune function can be produced.

In addition, the dosage of the composition of the present invention to the human body may be varied depending on the age, weight, sex, dosage form, health condition and disease severity of the subject.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Horseshoe  Separation and purification

The process for separating the polysaccharide from the horseradish mushroom was as shown in Fig. First, the horseshoe mushrooms collected from Gangwon-do, Odae-san, were washed with water, cut into small pieces and then cut into small pieces. Five grams of 80% ethanol was added to the horseshoe mushroom. The pieces were dipped at room temperature for one day and then ethanol soluble materials were removed. 10 L of water was added and the mixture was heated at 100 ° C for more than 3 hours. The liquid was filtered through a Whatman No.4 filter paper (Rectman), and 2.5-fold (v / v) 100% ethanol was added to the solution. And allowed to stand overnight at 4 占 폚. The next day, the precipitate was recovered by centrifugation (6000 rpm, 20 min, 4 ° C), dissolved in distilled water and dialyzed for 3 days using a dialysis membrane (MW cut-off 12,000, Spectrum) To obtain MFKF, a water-soluble crude polysaccharide fraction.

1. DEAE-Sepharose column chromatography

500 mg of crude polysaccharide MFKF isolated from horseradish mushroom was dissolved in 10 mM Tris-HCl (pH 7.0), filtered through a syringe filter (0.45 μm), and then eluted with DEAE-Sepharose FF (Cl- The adsorbed fractions were separated by elution with 10 mM Tris-HCl (pH 7.0), and then adsorbed on 0.4 M and 1 M NaCl in 10 mM Tris-HCl (pH 7.0) And the adsorbed fractions were individually separated by elution in a stepwise manner. The fractions eluted from the anion exchange chromatography column were fractionated using a fraction collector (Model 2110, Bio-Rad, USA) and elution peaks were determined by measuring the total sugar, acid group and protein content, respectively . The total sugar content was determined by the phenol-sulfuric acid method using galactose as the standard substance, the acid sugar content by the m-hydroxy biphenyl method using the β-D-galacturonic acid as the standard substance and the protein content using the bovine serum albumin as the standard substance Bradford method (Blumenkrantz N, 1973. New method for quantitative determination of uronic acid. Anal Biochem 54: 484-489), (Bradford MM. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein using the principle of protein dyebinding. Anal Biochem 72).

One non-adsorptive fraction neutral polysaccharide (MFKF-NP) and two adsorptive fractionated acid polysaccharides (MFKF-AP1 and MFKF-AP2) were obtained from the crude polysaccharide fraction MFKF through this ion exchange resin (FIG. 2). Each of the fractions was dialyzed and lyophilized and used in the subsequent experiments (Fig. 1).

2. Concanavalin A-Sepharose 4B Chromatography

MFKF-AP1 and MFKF-AP2 fractions, which had excellent anti-complement activity, were subjected to affinity chromatography using Concanavalin A-Sepharose CL4B column (Sigma, 1.5 × 14 cm). 100 mg of the MFKF-AP1 and MFKF-AP2 fractions were dissolved in the first elution buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.4) and then filtered through a syringe filter (0.45 袖 m). Concanavalin A was equilibrated with the same buffer After elution with sufficient elution to completely dissociate the β-type polysaccharide that is not compatible with Concanavalin A from the first elution buffer, a second elution buffer solution containing 50 mM α-methyl-D-Glucoside (Sigma), 50 mM phosphate 7.0), and the α-type polysaccharide having strong affinity with Concanavalin A was isolated. The eluate was fractionated into 60 fractions of 5 ml each, and each fraction was subjected to phenol-sulfuric acid method (Dubois M et al., 1951, A colorimetric method for determination of sugars. Nature 168: 167), and two fractions with different affinities MFKF-AP1-α. β and MFKF-AP2-α. beta] (Fig. 3). Each of the fractions was dialyzed and lyophilized and used in the subsequent experiments.

< Example  2> Horseshoe Mushroom  Derived polysaccharide Anti-complement  Active measurement

 Antibody activity was measured by Meyer's method (Kabat, EE and Meyer, MM, 1964, Complement and complement fixation, Chales, C. In Experimental Immunolochemistry. Thomas Publisher, Illinois. ) Were measured by the complement fixation test method based on the degree of erythrocyte hemolysis due to the remaining complement. The samples dissolved in distilled water at various concentrations were mixed with 50 μL each of GVB ++ (containing gelatin veronal buffer, pH 7.4, 0.1% gelatin, 0.15 mM Ca ++, 0.5 mM Mg ++) and normal human serum. . 350 μL of GVB ++ was added to the reaction solution, which was serially diluted to 10-160 times. Then, 750 μL of GVB ++ and IgM-sensitized sheep erythrocyte (EA cell, 1 × 108 cells / mL, , Japan) was added and reacted at 37 ° C for 60 minutes. 2.5 mL of PBS (phosphate buffered saline, pH 7.4) was added to stop the reaction. The reaction solution was centrifuged at 2,000 rpm for 10 minutes, and the absorbance of the supernatant was measured at 412 nm to measure the residual hemolytic activity. Anti-complement activity was expressed as inhibition of 50% total complement hemolysis (ITCH 50,%) against the total complement hemolysis (TCH 50,%) of the negative control group reacted only with normal human serum and GVB ++ and distilled water . As a positive control, PSK (polysaccharideK), an immunity enhancer derived from Mushroom mushroom, was used.

As a result of assaying the complement activity of the four kinds of purified polysaccharides purified in Example 1, the inhibition of total complement hemolysis (ITCH) value was 70% or more even at a concentration of 20 μg / ml of MFKF-AP1β, And more than 50 times the activity of PSK (polysaccharide-K, ITCH value of 70% at 1 mg / ml), which is a polysaccharide. In addition, the activity of MFKF-AP1α was about 20-fold, and the activity of MFKF-AP2α and MFKF-AP2β was 10-fold (FIG. 4).

< Example  3> MFKF - AP1 of -β Complement path  analysis

The activation of C ₃ was confirmed by two - dimensional immunoelectrophoresis in order to confirm whether the complement activity of the sample confirmed by the Meyer method was due to complement system activation or complement inhibitor. The reaction of MFKF-AP1-β, which has the most excellent anti-complement activity, in the basic reaction system and the reaction system in which the specific metal ion was removed was reacted, and the decomposition of the factor C ₃ was observed. In the steady-state reaction system (Fig. 5 (A)) in which both Ca ⁺⁺ and Mg ⁺⁺ exist, the second settling line is relatively larger than the first settling line from the well. Considering that the first sedimentation line from the well is caused by C ₃ and the second sedimentation line by the degradation products C ₃ a and C ₃ b, it is considered that the complement activity of MFKF-AP1-β is not a complement inhibitor Activation. On the other hand, in the reaction system in which all of the metal ions have been removed (FIG. 5C), only the first sedimentation line appears clearly and no second sedimentation line is observed. On the other hand, a second sedimentation line was observed in the reaction system in which only Ca ⁺⁺ was selectively removed (Fig. 5 (B)), and the height thereof was slightly lower than that of the normal system. This suggests that activation of C ₃ was induced only in the subpath with the classical pathway inhibited. Thus, activation of the complement system by MFKF-AP1-β confirmed activation of both the classical pathway and the subpopulation.

< Example  4> Horseshoe Mushroom  Interleukin of derived polysaccharide Production capacity  analysis

In order to analyze the interleukin production ability of the polysaccharide derived from the horseshoe mushroom, the production ability of IL-6 (Interleukin-6) and IL-12 (Interleukin-12) was tested by treating the horseshoe mushroom-derived polysaccharide with macrophages.

In Example 2, the production of IL-6 and IL-12 was confirmed by treating macrophages with two kinds of main anticonvulsant polysaccharide (MFKF-AP1?, MFKF-AP1? In the case of IL-6, MFKF-AP1-alpha was found to increase in a concentration-dependent manner, but production stimulating activity was generally low (Fig. 6). In the case of MFKF-AP1-β, an increase in the concentration-dependent production was observed from 1.6 μg / mL to 200 μg / mL. These results suggest that MFKF-AP1-β, a polysaccharide derived from horseradish mushroom, has a relatively effective activity in inflammatory immunity rather than MFKF-AP1-α. The minimum concentration of this substance is several μg / mL .

On the other hand, in the case of IL-12, MFKF-AP1-? And MFKF-AP1-? Both showed a production ability equivalent to about 60% of the positive control LPS at 1.6 μg / mL (FIG. This indicates that cytotoxic T lymphocytes (CTLs) can be produced by stimulating macrophages to produce interleukins-6 and 12 at a relatively low concentration even when the two complement system-activated polysaccharides (MFKF-AP1-? And MFKF- In particular, IL-12 is directly involved in NK cell activation, which is a cancer cell lethal effect in the presence of cancer cells, and thus it is likely to act efficiently on the cancer cells of the present invention . Therefore, active polysaccharide derived from horseshoe mushroom stimulates immune cells to produce various cytokines, which is effective for application to an immune system of an organism constituting a complex network.

< Example  5> Horseshoe Mushroom  Compositional analysis of the resulting polysaccharide

(Albersheim, P, 1994, Structure and function studies of plant cell wall polysaccharide. Biochem. Soc. Trans., 22: 374 ). 2 mg of polysaccharide sample was hydrolyzed in 2M trifluoroacetic acid (TFA) at 121 ° C for 1.5 hours, dissolved in 1 ml of 1M NH 4 OH and reduced with 10 mg NaBH 4 for 4 hours. Acetic acid was added to remove remaining NaBH 4, Acetic acid was removed and converted to the corresponding alditols and aldonic acids. Each alditol was reacted with 1 ml of acetic anhydride at 121 ° C for 3 hours to convert it to alditol acetic acid. Per-0-acethlyated alditol was extracted with a hexane / H2O 2 phase solvent system. And used as a sample for GLC analysis. GC analysis of the alditol acetic acid derivative was carried out in Splitless mode and the following temperature conditions using a Hewlett-Packard 5890A GC equipped with a SP-2380 Capillary column (Supelco, 0.25 nm × 30 m, 0.2 μm film). 215 ° C (18.8 min), 215 ° C → 250 ° C (80 ° C / min), 250 ° C (5 min)] molar ratio and mole % Were calculated by calculating the peak area, molecular weight, molecular weight and molecular response factor for the corresponding derivative (Table 1).

(Mole%) Constitutive
monosaccharide kinds of polysaccharides MFKF-
NP MFKF-
AP1-a MFKF-
AP1-beta MFKF-AP2-alpha MFKF-AP2-? Rhamnose - a very small amount a very small amount 3.9 2.6 Fucose 5.5 - - 4.5 2.5 Arabinose 13.2 1.7 0.7 1.9 - Xylose 7.7 97.8 99.1 69.7 89.4 Mannose 8.7 0.4 - 9.5 5.5 Galactose 17.3 a very small amount - 7.5 - Glucose 47.6 0.1 0.2 3.0 -

MFKF-AP1-α and MFKF-AP1-β, which showed excellent anti-complement activity in the process of horseradish mushroom purification, did not contain protein. As shown in Table 1, MFKF-AP1-alpha and MFKF-AP1-beta were hydrolyzed to an alditol acetic acid derivative and the compositions of the constituent sugars were examined by GC. As a result, Rose contains 97% or more of the highest content, and arabinose contains 0.66% and 1.66% of each, respectively. In addition, only a small amount of nose and glucose were detected. Based on the results per constitution, their structure is presumed to be xylan linked with xylose (Fig. 8).

The anti-complement activity of xylan polysaccharide (xylose content 90%) isolated from the birch sold as a reagent was 42% and 24% at 1,000 μg / mL and 500 μg / mL, respectively, The mushroom-derived MFKF-AP1-β polysaccharide can be expected to have a structure different from that of the existing xylan (Fig. 9).

< Example  6> Horseshoe Mushroom  Molecular weight measurement of derived polysaccharide

The molecular weights of the polysaccharides MFKF-NP, MFKF-AP1 and MFKF-AP2 isolated by DEAE-Sepharose were determined by Toyopearl HW-65F column chromatography (diameter 2.5 cm, length 100 cm). Each retention time was determined using a pullulan series (Showa, Japan, P-800, 400, 200, 100, 50, 20, 10 and 5) The molecular weight was determined from the standard curve obtained by calculating the Kav value (Fig. 11). Further, for the measurement of the molecular weight of MFKF-AP1-beta exhibiting high purification and excellent immunoactivity, HPLC (Varian, Model 356-LC, manufactured by Varian, Inc.) equipped with Superdex ^TM 75 10 / 300GL packed column (GE Healthcare, USA, 0.76 x 30 cm) ) Was eluted with 50 mM ammonium formate buffer (pH 5.5), and the average molecular weight was calculated based on a standard curve (FIG. 10).

The molecular weights of the polysaccharides MFKF-NP, MFKF-AP1 and MFKF-AP2 isolated by DEAE-Sepharose were determined by Toyopearl HW-65 column chromatography. In the case of MFKF-NP, the molecular weight was about 20,000, and the molecular weight of MFKF-AP1 and MFKF-AP2 was about 10,000. In addition, MFKF-AP1-β, which exhibited high anti-complement activity during purification of affinity chromatography, was confirmed to have relatively pure refinement as a result of performing HPLC to confirm purity and molecular weight, Molecular weight measurement using a pullulan series showed a polysaccharide having a molecular weight of 10,000 or more. These results indicate that polysaccharides that activate physiological activities are mostly poly- meric substances with a molecular weight of several hundred thousand or more, whereas MFKF-AP1-β derived from horseradish mushroom is a relatively low-molecular-weight soluble polysaccharide and activates complement system proteins It is expected as a substance.

These results suggest that this polysaccharide is a xylan with a specific binding that has not been reported so far.

Claims (8)

A water-soluble polysaccharide comprising xylose having a molecular weight of 10,000 to 20,000 Daltons and 60.0 to 99.9%. The method of claim 1,
The polysaccharide comprises xylose as the main constituent; Polysaccharide comprising at least one of mannose, galactose, arabinose, fucose, rhamnose and glucose. 3. The method of claim 2,
The polysaccharide comprises a polysaccharide consisting of 97 to 98% of xylose, 1 to 2% of arabinose, 0.1 to 0.5% of mannose, 0.01 to 0.03% of rhamnose, 0.001 to 0.020% of galactose, and 0.05 to 0.10% of glucose;
Polysaccharide consisting of 99.01 to 99.99% of xylose, 0.5 to 1.0% of arabinose, 0.1 to 0.3% of glucose, trace amount of rhamnose;
Xylose 69 to 70%, mannose 9 to 10%, galactose 7 to 8%, fucose 4 to 5%, rhamnose 3 to 4%, glucose 3 to 4% consisting of arabinose 1 to 2% Polysaccharides; And
Polysaccharide, characterized in that selected from polysaccharides consisting of 89 to 90% of xylose, 5 to 6% of mannose, 2 to 3% of rhamnose, 2 to 3% of fucose. The method of claim 1,
The polysaccharide is characterized in that it exhibits anti-complementary activity and interleukin production promoting activity. 5. The method according to any one of claims 1 to 4,
The polysaccharide is a polysaccharide derived from a horseshoe mushroom. Complement activated xyloligosaccharide obtained by hydrolyzing the polysaccharide of any one of claims 1 to 4. An immunopotentiating composition comprising the complement pathway-activated polysaccharide of any one of claims 1 to 4. Functional food comprising the immuno-enhancing composition of claim 6.

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