KR100673051B1 - Endo-biopolymer isolated from submerged mycelial culture of pholiota nameko increasing immuno-stimulating activity - Google Patents

Abstract

Provided is an intracellular biopolymer isolated from a submerged-liquid culture of Pholiota nameko, which is effective in increasing anti-complement, macrophages, and spleen cells and is useful for immunostimulating pharmaceutical compositions. The Pholiota nameko-derived intracellular biopolymer having an increase effect of anti-complement, macrophages, and spleen cells is separated from a submerged-liquid culture of Pholiota nameko. The intracellular biopolymer consists of a fraction comprising 79.6% of saccharide containing mannose and glucose having a molecular weight of 1000kDa as main materials and 20.4% of protein containing glycine and lysine as main materials, and a fraction comprising 86.9% of saccharide containing mannose, galactose, and glucose having a molecular weight of 3.1kDa as main materials and 13.1% of protein containing aspartic acid, threonine, glutamic acid, and glycine as main materials.

Description

Endo-biopolymer isolated from submerged mycelial culture of Pholiota nameko increasing immuno-stimulating activity}

Figure 1 is a simplified diagram showing the process of separating the intracellular biopolymers from the mycelium liquid culture of the flavor mushrooms,

2 is a diagram showing the anti-complement activity of the intracellular biopolymers isolated from the mycelium liquid culture of taste mushrooms,

3 is a diagram showing anti-complement activity in the presence or absence of Ca ⁺⁺ and Mg ⁺⁺ of EN-PN-Fr. II, which is a fraction of intracellular biopolymers,

Figure 4 is a diagram showing the cross-electrophoresis (CIEP) form of C3 converted by EN-PN-Fr.II, a fraction of intracellular biopolymers under conditions of free Ca ⁺⁺ or divalent metal ions,

5 is a diagram showing the change of anti-complement activity by treatment of pronase or sodium periodate to EN-PN-Fr. II, a fraction of intracellular biopolymers,

Figure 6 is a diagram showing the lysosomal enzyme activity of macrophages at various concentrations of intracellular biopolymer fractions,

FIG. 7 is a diagram showing NO production by rat peritoneal macrophages of intracellular biopolymer fractions.

8 is a diagram showing the effect of EN-PN-Fr. II on the NO production of peritoneal macrophages in the presence or absence of IFN-γ,

9 is a diagram showing the effect of the intracellular biopolymer fraction on the NO production of peritoneal macrophages in mice in different cultures,

10 is a diagram showing the effect of the fraction of intracellular biopolymers on the proliferation of spleen lymphocytes by the lymphocytes reactant mixed at various concentrations,

11 is a diagram showing the effect of intracellular biopolymer fractions on the proliferation of spleen lymphocytes in different cultures,

12 is a diagram determining the molecular weight of the intracellular biopolymer fractions,

FIG. 13 shows FT-IR spectra of fractions of intracellular biopolymers. FIG.

The present invention relates to an intracellular biopolymer having a proliferative effect of anti-complement, macrophages and spleen lymphocytes isolated from mycelium liquid culture of Pholiota nameko .

Among various immune systems, the complement system consisting of enzymes in serum plays an important role in the first humoral regulation of antigen-antibody responses in host resistance. Complement systems are also involved in the induction and regulation of specific immune responses, such as the activation of macrophages and lymphocytes, the location and identity of antigens in the germinal center, the generation of memory B cells, Intercellular coordination and regulation of antibody production. Recently, anti-complement activity, cleavage-induced activity of lymphotoxin site (mitogenic activity), interferon-inducing activity, antitumor activity, as well as some pharmaceutical active are spicy peppers, ildanggwi (Angelica acutiloba), Bupleurum (Bupleurum falcatum ) Chinese Herbal Medicine, Herb ( Grifola umbelat ), Poria cocos fingering mushrooms, ( Coriolus versicolor ) It has been observed in polymers such as polysaccharides isolated from several foods, such as mushrooms.

Flavored Mushrooms ( Pholiota nameko ) is an economically important edible mushroom called "Nameko" in Japan and is a homo-basidiomycete that rots trees. Flavored mushrooms have a good gelatinous viscosity and aroma, and are usually cooked with miso soup, radish, or steamed in a pan. The mushrooms are generally known in Japan, China, Korea and Thailand.

However, neither of these documents teaches or discloses the immune activity of intracellular biopolymers isolated from mycelium liquid cultures of taste mushrooms.

Accordingly, the present inventors completed the present invention by confirming the anti-complementary activity of the intracellular biopolymers isolated from the mycelium liquid culture of taste mushrooms and the proliferative activity of macrophages and spleen lymphocytes.

An object of the present invention is to taste mushrooms ( Pholiota nameko ) provides intracellular biopolymers with proliferative effects of anti-complement, macrophages and splenic lymphocytes isolated from mycelium liquid cultures.

In order to achieve the above object, the present invention has a proliferation effect of anti-complement, macrophages and splenic lymphocytes ( Poliota) nameko ) provides intracellular biopolymers isolated from mycelial liquid cultures.

The intracellular biopolymer has a fraction consisting of 79.6% sugar and glycine and lysine 20.4% protein, mannose having a molecular weight of 1000 kDa, mannose, galactose and a molecular weight of 3.1 kDa. Glucose consists of 86.9% of sugar and aspartic acid, threonine, glutamic acid and glycine, and a fraction of 13.1% of protein.

Fraction having a molecular weight of the 1000kDa sugar methylation of 2,3,4,6-tetra-O-methyl-glucitol (2,3,4,6-tetra- O -methyl-glucitol ) and the second, 3,6-tree - O - methyl - consists of glucitol (2,3,6-tri- O -methyl-glucitol ), fractions having a molecular weight of 3.1kDa is a sugar methylation 2,3, 4, 6-tetra-O-methyl-glucitol (2,3,4,6-tetra- O -methyl-glucitol ), 2,3,6- tree-O-methyl-glucitol (2,3 , 6-tri- O -methyl-glucitol ), 2,4,6- tree-O-methyl-glucitol (2,4,6-tri- O -methyl-glucitol ), 2,4- di-O -methyl-glucitol (2,4-di- O -methyl-glucitol ), 2,4- di-O-methyl-mannitol (2,4-di- O -methyl-mannitol ), and 2,3, means that the methyl -D- go raksi Tall (2,3,4-tri- O -methyl-D -galacitol) consists in that the features of the intracellular biopolymer-4-tree-O.

Hereinafter, the present invention will be described in detail.

The method for separating the intracellular biopolymers of the present invention from the mycelium liquid culture of taste mushroom ( P. nameko ) is as follows.

The spawn culture of the taste mushrooms is carried out in a flask containing PDB (photato dextrose broth, pH 5.0) at 15-30 ° C., preferably 20-25 ° C. for 1-20 days, preferably 8-12 days. It can be cultured in a 200 rpm rotary shake incubator. Liquid culture was performed using mushroom complete medium (MCM) for the production of intracellular biopolymers. The composition of MCM consists of glucose, MgSO ₄ , KH ₂ PO ₄ , K ₂ HPO ₄ , yeast extract and peptone, and the pH before sterilization Can be adjusted to 4.0-6.0. For liquid culture, the flask containing the medium may be used with a rotary shaker in a condition of 20-30 ° C. for 10-30 days. Intracellular biopolymers can be recovered after centrifugation by centrifugation of the isolated mycelium by hot water extraction, and ethanol addition to the supernatant. The recovered intracellular biopolymers can be finally separated by dialysis and lyophilization.

The intracellular biopolymer may be separated into two fractions by Sepharose CL-6B gel chromatography, and the fractions each have a molecular weight of 1000 kDa and 3.1 kDa, and the fraction having a molecular weight of 1000 kDa is 79.6% of sugar. And 20.4% of protein, wherein the fraction having a molecular weight of 3.1kDa is composed of 86.9% of sugar and 13.1% of protein.

Fractions having a molecular weight of 3.1 kDa have anti-complement activity as a result of complement immobilization tests, which exhibit anti-complement activity in both the classical and alternative pathways in the complement system, and are characterized by inducing production of significantly superior NO. .

In addition, the fraction having a molecular weight of 1000 kDa has a useful effect on lysosomal enzyme activity of macrophages and proliferation of spleen lymphocytes.

Therefore, the intracellular biopolymer of the present invention may be usefully used as an immune enhancing pharmaceutical composition due to the activities of the two fractions.

In addition, the taste mushroom is a drug that has been used for a long time as a herbal medicine and edible, the intracellular biopolymer of the present invention extracted from them also have no problems such as toxicity and side effects.

The composition of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

The pharmaceutical composition comprising the biopolymer of the present invention may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The pharmaceutical compositions of the present invention may be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. have. Carriers, excipients, and diluents that may be included in compositions comprising biopolymers include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the composition. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 40 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food comprising the biopolymer and food acceptable food supplement additives exhibiting an immune enhancing effect. Examples of the food to which the biopolymer of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of immune boosting. At this time, the amount of the biopolymer in the food or beverage can be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml Can be.

Health functional food of the present invention includes the form of tablets, capsules, pills, liquids and the like.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compounds of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are only examples of the present invention, and the present invention is not limited thereto.

Example  1. Taste Mushrooms ( Pholiota nameko From mycelia liquid culture Intracellular  Isolation and Purification of Biopolymers

Taste mushroom ( P. nameko ) was sold by the Korean Agricultural Culture Collection.

Taste mushrooms were spawned in a 250 ml flask containing 100 ml PDB (photato dextrose broth, pH 5.0) at 25 ° C. for 10 days in a rotary shaker at 150 rpm.

Liquid culture was carried out in a mushroom complete medium (MCM) for the production of intracellular biopolymers. The composition of MCM was glucose 20g / l, MgSO ₄ 0.5g / l, KH ₂ PO ₄ 0.46g / l, K ₂ HPO ₄ 1.0g / l, yeast extract 2g / l, and peptone 2g / l and adjusted to pH 5.0 before sterilization. In water culture, a 500 mL flask containing 200 mL of medium was used with a rotary shaker (150 rpm, pH 5) at 25 ° C. for 20 days.

After that, the culture medium of the flavor mushrooms was centrifuged at a rate of 10,477 × g for 20 minutes, and the recovered mycelium was washed three times with distilled water, followed by repeated hot water extraction three times for 2 hours, and then centrifuged again (10,477 × g, 20 minutes). Then, four times the volume of ethanol was added to the separated supernatant, centrifuged (10,477 × g, 20 minutes), and the recovered precipitate was lyophilized and dissolved in 10 mg / ml deionized water (DIW). After dialysis for 3 days after performing centrifugation (10,477 × g, 20 minutes), the intracellular biopolymer (EN-PN) was isolated and purified by recovering the supernatant (see Figure 1).

Reference Example  1. Statistical Analysis

All experiments were repeated three times for statistical analysis. The results are expressed as ± SD (standard deviation).

Experimental Example  1. Complement of anti-complement activity

1-1. Intracellular  Biopolymer term Complement  Measurement of activity

Anti-complement activity was observed by complement fixation tests based on the level of erythrocyte lysis by complement consumption and residual complement. 50 μl of the intracellular biopolymer isolated in Example 1 was added GVB (gelatin veronal buffered saline, pH 7.4) containing normal human serum (NHS) and 500 mg of Mg ⁺⁺ and 150 mg of Ca ⁺⁺ . . The mixture was incubated at 37 ° C. for 30 minutes, and residual complement hemolysis (TCH ₅₀ ) was observed using EA cells (1 × 10 ⁸ cells / ml). NHS with DIW and GVB ⁺⁺ added as a positive control was incubated. Anti-complement activity of intracellular biopolymers was expressed as the inhibition rate of TCH ₅₀ of the control group. Calcium (Ca ⁺⁺⁾ ions is required for the activation of the classical complement in a path other than the alternate path. Alternative complement pathways are found in Platt-Mills and Ishizaka, Platt-Mills, TA, and K. Ishizaka, Activation of the alternative pathway of human complement by rabbit cell.Journal of Immunology , 113 , pp348-358, 1974. application of the method to GVB ^- was measured with a ₂ mM MgCl 2 10 mM EGTA containing the ^{- (Mg ++ -EGTA-GVB)} . Samples Mg ⁺⁺ -EGTA-GVB ^- and a NHS incubated at 37 ℃ for 30 min, and the residue was purified complement mixture Mg ⁺⁺ -EGTA-GVB ^- rabbit red blood cells incubated with the ^{(5 × 10 7 cells / ㎖} ) The hemolysis was observed.

As a result of the above experiment, as shown in FIG. 2, intracellular biopolymers were selected from those exhibiting the highest anti-complement activity, and Sepparos CL-6B chromatography was performed to fractions 26-58 to EN. -PN-Fr.I, fractions 59-86, were separated into two fractions of EN-PN-Fr.II.

The anti-complement activity of the two fractions increased in proportion to the concentration increase when compared at concentrations of 50, 100, 300 and 500 µg / ml. EN-PN-Fr. II showed higher activity than EN-PN-Fr. I at all concentrations. At the concentration of 500 μg / ml, EN-PN-Fr. I was 74.1%, and EN-PN-Fr. II was 99.1%, which showed higher activity than the positive control (LPS, 63.1%).

1-2. EN- PN - Fr Clause of .II Complement  Active measurement

In order to identify the activation pathway of the complement system, EN-PN-Fr.II was incubated with another buffer, and ITCH ₅₀ was measured by the same method as in Experimental Example 1-1.

Anti-complement activity is generally associated with hemolytic phenomena of both erythrocytes sensitized by human complement in the presence of Mg ⁺⁺ and Ca ⁺⁺ . It is known to be mediated through the classical and alternative complement pathways. Flat-Mills and Ishizaka used EGTA buffers to distinguish complement pathways requiring only Mg ⁺⁺ . Complement immobilization in the presence of EGTA is known to occur through alternative pathways. EGTA is strongly as EDTA in combination with Ca ⁺⁺ rather than Mg ^++, because weakly bound, EGTA buffer containing a sufficient amount of Mg ⁺⁺ in order to determine the activity of substantially the complement path while being the complement component C1 block It is used.

As a result of the above experiment, as shown in FIG. 3, under the condition of free Ca ⁺⁺ (Mg ⁺⁺ -EGTA ^- GVB-, only magnesium ion), 500 mg / mL of EN-PN-Fr.II Complement activity was 58.7%, which was significantly lower than 99.1% of GVB ⁺⁺ buffer (both calcium and magnesium ions). This result shows that complement system is activated by EN-PN-Fr.II through both engagement route (40.4%) and alternative route (58.7%).

Experimental Example  2. CIEP (crossed immunoelectrophoresis ) analysis

In order to confirm the results performed in Experimental Example 1-2, CIEP analysis was performed in the following manner.

Specific activation of C3 complement components by biopolymers in NHS was determined by comparative observation of C3 degradation. NHS is the Example 1 within the separation cell in the biopolymer solution and 10 mM EDTA equal volume (EDTA ^GVB-- also blocking the classical route and the alternate route as a buffer, Ca ⁺⁺ and Mg ⁺⁺ deficient) or (Sikkim activate an alternative path as a buffer containing Mg ^{++), -} GVB containing ^- Mg ⁺⁺ -EGTA-GVB The cells were incubated at 37 ° C. for 30 minutes in GVB ⁺⁺ (which activates the classical and alternative pathways as buffers containing Ca ⁺⁺ and Mg ⁺⁺ ). The mixture was subjected to crossed immuno-electrophoresis (CIEP) to confirm the location of the C3 digest. All samples (10 μl) were subjected to isoelectric focusing on a 1% agarose gel.

Two hours after the first run in a barbital buffer with an ionic strength of 0.025, pH 8.6 with 1% agarose, the second run was an anti-human complement C3 capable of recognizing both C3 and C3b. 15 hours was performed at a potential gradient of 15 mA / plate on a gel plate containing antiserum raised in goat, Sigma Co., USA. After electrophoresis, the plates were fixed and stained with 0.2% bromophenol blue to calculate the ratio between the heights of the C3 and C3b peaks.

In order to confirm the results performed in Experimental Example 1-2, GVB ⁺⁺ and to observe whether C3 activation occurs Mg ⁺⁺ -EGTA-GVB ^- the presence of a buffer after incubation with the NHS EN-PN-Fr.II was performed CIEP.

As a result of the experiment, the activated C3 complement component was decomposed into C3a and C3b while showing two sedimentation lines in the CIEP. The C3b fragment is located on the bacterial surface where the complement is activated. C3a and C3b were precipitated line of the buffer are the same shown in Figure 4 (see A, B), a sedimentation line GVB-EDTA ^{- -} GVB ⁺⁺ and Mg ⁺⁺ -EGTA-GVB not observed in the buffer system (See Figure 4C). From this result, the complement system of EN-PN-Fr.II was activated not only by the classical route but also through the alternative route, and the alternative route was observed as the main route. Therefore, the above results show that EN-PN-Fr.II has strong nonspecific immunopotentiating activity.

Experimental Example  3. Intracellular  Biopolymer Anticomplement  Determination of active ingredients

3-1. Intracellular  Biopolymer Pronase  By decomposition

40 mg of EN-PN-Fr.II isolated in Experimental Example 1-1 contained 10 mM CaCl ₂ . After dissolving in 50 mM Tris-HCl buffer (pH 7.9), 10 mg of pronase was added. The reaction mixture was incubated with a small amount of toluene for 48 hours. The reaction was terminated after 5 minutes of heating. The mixture was dialyzed with DIW for 2 days and lyophilized the undialyzed portion.

3-2. Intracellular  Biopolymer Periodate ( periodate ) Oxidation

40 mg of EN-PN-Fr.II isolated in Experimental Example 1-1 was dissolved in 40 ml of 50 mM acetate buffer (pH 4.5) and then 50 mM NaIO.₄ (10 mL) was added. The reaction mixture was incubated in the dark at 4 ° C. for 3 days. Ethylene glycol was added to destroy excess periodate and the mixture was dialyzed with DIW for 2 days. The non-dialysis solution was concentrated to 10 ml and 20 mg of sodium borohydride was added during continuous sterilization at room temperature for 12 hours. After the reaction mixture was neutralized with acetic acid, the boric acid contained in the sample was removed by repeated addition of methanol and evaporation to dryness. Finally the oxidized sample was obtained by lyophilization after dialysis.

As a result of the above experiments, as shown in FIG. 5, the pronase-treated EN-PN-Fr.II showed low anticomplement activity (94.7%) compared to the original fraction (99.1%).

However, the activity of the material obtained by periodate oxidation was markedly decreased, with an anti-complement activity of 30.6% at a concentration of 500 mg / ml. Therefore, it was confirmed that the carbohydrate component of EN-PN-Fr.II is essential for the activation of the complement system.

Experimental Example  4. Lysosomal Enzyme Activity Intracellular  Biopolymer Effect

Macrophages play an important role in the host defense system by digestion due to lysosomal enzymes when ingesting infectious microorganisms. The effect of the intracellular biopolymer fraction on the acidic ginseng activity of the peritoneal macrophages was investigated.

4-1. Preparation of Laboratory Animals

Six-week-old male C57BL / 6 and BALB / c mice weighing about 25 g were purchased from Daehan Biolink Co., LTD and divided into 5 groups. The mice were bred during the experiment with commercial breeding feed (Sam Yang Co., Korea) in a room with a humidity of 55 ± 5% and a temperature of 22 ± 2 ° C. under a 12 hour cycle.

4-2. Preparation of Mouse Macrophages

Macrophages were collected from mice three days after peritoneal administration of 3 ml of 10% thioglycolate medium. Cell concentration was about 1 × 10 ⁶ cells / ml in Dulbecco's modified engle medium (DMEM) buffer containing 10% fetal bovine serum. 200 μl of cell suspension (2 × 10 ⁵ cells / ml) was then incubated on 96 well plates. Attached macrophages were separated by incubation for 2 hours in the presence of carbon dioxide and subjected to stirring and washing to remove unattached macrophages. To determine NO (nitric oxide) production and activity of lysosomal enzymes, the cultures were cultured for 24 hours in a humid incubator with or without the addition of biopolymers, with or without IFN-γ (20 U / ml) or LPS. Incubated in the presence of 5% carbon dioxide.

4-3. Activity measurement of cellular lysosomal enzymes in macrophages

The activity of lysosomal enzymes was analyzed using 96 well flats with tissue culture plates. Macrophage monolayers (2 × 10 ⁶ cells / ml) of the microplates were liquefied by adding 25 μl of 0.1% Triton X-100. 150 mM of 10 mM p-nitrophenyl phosphate solution was added as substrate for acid phosphatase. The wells were then added with 50 μl citrate buffer. After incubation at 37 ° C. for 1 hour, 25 μl of 0.2 M borate buffer (pH 9.8) was added to the reaction mixture and the absorbance at 405 nm was measured.

As a result of the experiment, the activity of EN-PN-Fr. I and EN-PN-Fr. II showed relatively higher activity than LPS at various concentrations as shown in FIG. When macrophages were pretreated with 50 μg / ml EN-PN-Fr. I, the activity of lysosomal enzyme was found to be 54% higher than the positive control (LPS). Macrophages can be activated by regulators that kill tumor cells by producing lymphokines, endotoxins and various cell mediators and tumor necrosis factors. Therefore, EN-PN-Fr.I can be regarded as a strong substance for antitumor activity by activation of lysosomal enzymes in macrophages. In Figure 6, NC means a negative control as physiological saline, LPS means a positive control as lipopolysaccharide.

Experimental Example  5. In NO production Intracellular  Biopolymer Effect

NO is known as an effective factor for intracellular protozoa. Accumulation of nitrite is used as the induction of nitric oxide (NO) production. The procedure for measuring NO was applied to the Griess reaction. Peritoneal macrophages were isolated from male BALB / c mice and treated with intracellular biopolymers at concentrations of 10, 50 and 100 μg / ml for 48 hours.

100 μl of culture supernatant or sodium nitrite contained 0.1% (w / v) naphthyl ethylenediamine and 1% (w / v) sulfanilamide in 5% (w / v) phosphoric acid in the same volume ) Was mixed. After reacting at room temperature for 5 minutes, the absorbance at 550 nm was measured using an ELISA reader.

As a result of the above experiment, as shown in FIG. 7, when the concentration of the intracellular biopolymers was treated at 10, 50, and 100 µg / ml, the NO production by EN-PN-Fr. II of all concentrations was EN-PN. It was confirmed that it was higher than -Fr. I. During the screening step it was confirmed that the optimal concentration of IFN-γ for NO synthesis 20U / ㎖. Therefore, treatment of EN-PN-Fr. II was carried out both in the presence or absence of 20 U / ml, and the best NO production was observed at 200 µg / ml of IFN-µgγ (see FIG. 8, FIGS. 7 and 8). NC is negative control group as physiological saline, LPS is positive control group as lipopolysaccharide, IFN-γ is the activator of macrophages, + is present,-means absence.

In addition, as shown in Figure 9, macrophages that produced the highest NO (32μM) when incubated with EN-PN-Fr.II in different cultures with macrophages is EN-PN-Fr.II (200㎍ / ㎖) , LPS and IFN-γ was confirmed that the treatment. LPS and IFN-γ and EN-PN-Fr.II cooperated with each other to activate macrophages and secrete NO (in FIG. 9, DIW is non-ionized water and negative control, and LPS is lipopoly). As a saccharide, it is a positive control group, IFN-γ is an active factor of macrophages, A represents 200 µg / ml of EN-PN-Fr.I, and 600 µg / ml of EN-PN-Fr.II).

Experimental Example  6. Proliferative activity of splenic lymphocytes Intracellular  Biopolymer Effect

Splenocytes were prepared in the following manner.

After separating the spleen from mice, single cell suspensions were recovered using a 5 ml syringe.

The precipitate was then recovered by centrifugation (10 min, 300 x g). The precipitate was washed five times with red blood cell digestion buffer and incubated at room temperature for 5 minutes. Thereafter, RPMI-1640 culture was added, and the precipitate was separated by centrifugation and centrifuged three times in the same manner to wash single cell suspension. And spleen cells were prepared by measuring the cell concentration using 0.2% trypan blue.

Using the splenocytes prepared above, the immune response of splenocytes corresponding to allogenegens is determined by the mixed lymphocyte reaction (MLR).

Splenocytes (2 × 10 ⁷ cells / ml) isolated from BALB / c mice were incubated with 25 μg / ml of mitomycin-C for 30 minutes at 37 ° C., followed by 10% fetal bovine serum (FBS, fetal Washed twice with cold HBSS containing bovine serum) and washed 6-10 × 10 ⁷ cells / ml at 37 ° C. for 10 minutes. The cells treated with mitomycin-C as above were called stimulatory cells. Splenocytes were prepared at a concentration of 4 × 10 ⁷ cells / ml in C57BL / 6 mice, which were called response cells. In one-way mixed lymphocyte culture, a culture medium containing 2 × 10 ⁶ cells / ml stimulatory cells and 2 × 10 ⁶ cells / ml reactive cells in 200 μl of RPMI-1640 medium was prepared. With or without the addition of the polymer fraction, Con-A or LPS was added or not added together in 96-well plate and incubated for 92 hours at 5% CO2, 37 ℃. Then MTT (Methylthiazoletetrazolium) was added and incubated for 4 hours. At the end of the incubation, insoluble formazan was precipitated by centrifugation at 3,000 rpm for 20 minutes and the supernatant was removed. 0.1 ml of DMSO (dimethyl sulfoxide) was added to dissolve the formazon and absorbance was measured at 540 nm using an ELISA reader.

As a result of the experiment, as shown in Figure 10, the activity of splenic lymphocytes on the intracellular biopolymer fraction in lymphocytes was stimulated at various concentrations. Two fractions showed relatively high activity compared to the positive control (LPS). The highest activity of the spleen lymphocytes is due to 100 μg / ml EN-PN-Fr.I, which is a 14% increase compared to the positive control. The effect of EN-PN-Fr.I on different cultures is shown in Figure 11 (in Figure 11 DIW is non-ionized water, S is EN-PN-Fr.I and LPS is lipopolysaccharide As positive control for B lymphocytes, and Con-A as positive control for T-lymphocytes as Concanavalin-A). When lymphocytes were incubated with EN-PN-Fr.I and Con-A (Concanavalin-A), the proliferation of splenic lymphocytes was 10% inhibited compared to the group treated with EX-PN-Fr.I, whereas LPS In the case of simultaneous treatment with and EX-PN-Fr.I, the increase was 65%.

Con-A and LPS have generally been used as controls for limiting the mitogenic activity of T and B lymphocytes. The results show that EX-PN-Fr.I has a synergistic effect on the proliferation of lymphocytes with LPS. Therefore, EX-PN-Fr.I has strong cleavage activity of splenic lymphocytes.

Experimental Example  7. Determination of Molecular Weight of Biopolymer

The molecular weight of EN-PN-Fr.I and EN-PN-Fr.II isolated in Experimental Example 1-1 was measured by Sepharose CL-6B gel chromatography. Standard curves include glucose 180 and dextran series; T-500 (5 × 10 ⁵ ), T-70 (7 × 10 ⁴ ), T-40 (4 × 10 ⁴ ), and T-10 (1 × 10 ⁴ ) were used.

As a result, as shown in FIG. 12, molecular weights of EN-PN-Fr.I and Fr.II were observed at about 1,000 kDa and 3.1 kDa, respectively. In general, EN-PN-Fr.II had a relatively low molecular weight compared to water-soluble anticomplement polysaccharides in the range of 10 kDa-1000 kDa.

Experimental Example  8. Intracellular  Analysis of protein and sugar composition of biopolymer

8-1. Protein composition analysis

Total protein components of intracellular biopolymers were measured by applying the method Lowry using bovine serum albumin (BSA) (Lowry, OH, Rosebrough, NJ, Farr, L., and Randall, RJ, Protein measuremen with the Folin phenol reagent. J. Biol. Chem., 193, pp265-275, 1951). Proteins were hydrolyzed and the amino acid composition was analyzed using a Biochrom 20 (Aminochromium, Pharmacia Biotech. Ltd., USA) amino acid automated analyzer on a Na-form column.

As a result of the above experiment, total protein components of EN-PN-Fr.I and Fr.II are 20.40% and 13.10%, respectively. The amino acid composition of these fractions is shown in Table 1 below.

EN-PN-Fr.I is composed of 12.92% glycine, 12.14% lysine, 9.46% valine, 8.37% phenylalanine and 8.20% serine, and EN-PN-Fr.II is 15.40% glycine and aspartic acid. 13.45%, threonine 10.20%, glutamine 10.20%, alanine 9.31% was confirmed that.

Protein composition analysis result EN-PN-Fr.Ⅰ EN-PN-Fr.Ⅱ Total protein (%) 20.40 13.10 Amino acid (%) ^a Aspartic acid 6.31 13.45 Threonine 5.76 10.20 Sererin 8.20 8.90 Glutamic acid 6.12 10.20 Prorin - 0.67 Glycine 12.92 15.40 Alanine 8.01 9.31 Cysteine 5.38 1.10 Valine 9.46 5.65 Methionine 2.74 0.95 Isoleucine 3.05 3.16 Leucine 5.13 4.60 Phenylalanine 8.37 7.34 Histidine 3.45 3.78 Lysine 12.14 1.53 Arginine 2.96 3.79

8-2. Sugar composition analysis

Using the mixture of glucose and mannose 1: 1 as a standard, total phenolic components of biopolymers were analyzed using the phenol-sulfuric acid method (Dubois, M., Gilles et al., Colorimetric method for determination of sugar and related substance. Analyt. Chem ., 28, pp350-356, 1956). Sugar composition was determined using a Varian GC3600 gas chromatography with a flame-ionization detector (FID) on an SP ^TM -2380 capillary column (15m × 0.25mm id, 0.2-μm film: SUPELCO) based on hydrolysis and acetylation methods. Analyzed.

The total sugar components and composition of the intracellular biopolymer fractions are shown in Table 2 below. The total sugar components of EN-PN-Fr.I and Fr.II were 79.6% and 86.9%, respectively.

EN-PN-Fr.I mainly contained glucose and contained 0.01% of ribose, 0.01% of arabinose, 0.01% of xylose and 0.01% of mannose. In contrast, the main sugars in EN-PN-Fr.II are glucose, mannose and galactose. These intracellular biopolymer fractions contain glucose as the major saccharide that is maximally shared in the carbohydrate moiety and do not contain uronic acid.

Analysis result of sugar composition EN-PN-Fr.Ⅰ EN-PN-Fr.Ⅱ % Per total 79.60 86.90 Neutral sugars (molar ratio) ^a Rhamnos - 0.01 Ribose 0.01 0.01 Arabinos 0.01 0.01 Xylose 0.01 0.01 Mannos 0.01 0.44 Galactose a very small amount 0.31 Glucose 1.00 1.00

Experimental Example  9. Intracellular  Structural Analysis of Biopolymers

9-1. Infrared spectroscopy spectroscopy

1 mg of the intracellular biopolymer was mixed with 100 mg of pure potassium bromide and pressed with disc pellin. Total IR spectroscopy (4000-400 cm ^-1 ) was recorded on the Matson machine Fourier transform infrared spectrophotometer (FTIR) Genesis II, and the measured spectroscopic records were analyzed in comparison with published data.

The FT-IR spectra of intracellular biopolymers obtained from mycelia of taste mushrooms were measured in KBr pellets to determine the total IR spectra (4000-400 cm ^-1 ).

Results of the tests performed, as shown in FIG. 13 PN-EN-Fr.I and PN-EN-Fr.II of 914 and 880 cm ^- obvious absorption peaks at ¹ to indicate the presence of a cross-α and β configuration Could know. Both intracellular biopolymer fractions are 1250 and 1650 cm ^- represents a characteristic IR absorption at ^1, and their spectral patterns were found to be almost identical. In FT-IR spectroscopy of the two fractions, 1640 cm ^- in the carboxylic group in ¹ (C = O) band corresponding to the vibration it has shown that the carboxyl group (-CHO, -CO) are apparently bonded hydrogen .

In addition, the spectral characteristic is the natural poly wave number of ^{saccharide: OH stretching (3300-3500 cm -1)} , CH stretching (3000-2850 cm -1), carbonyl (-CHO, -CO) stretching (-1700 cm - ¹ ) showed absorbance. 1064 cm ^- prominent bands at ¹ is shown a ring vibration, and C-OH bending (bending). Absorbance around 1200 cm ⁻¹ reflects the presence of ester groups (COOR or COC).

9-2. Methylation

To measure glucoside binding, EN-PN-Fr.I and Fr.II were analyzed by GC-MS for converted alditol acetate after methylation and hydrolysis.

Intracellular biopolymers were methylated using Hakomori's method (Hakomori, SI A rapid permethylation of glycolipid, and polysaccharide catalyzed by methyl sulphinyl carbanion in dimethyl sulfoxide., Journal of Biochemicstry, 55, pp 205-204, 1964). 2 mg of intracellular biopolymers were dissolved in 0.1 ml DMSO (dimethyl sulfoxide) by ultrasound under nitrogen atmosphere. The solution was treated with 0.1 ml of methylsulfinyl carbanion for 4 hours at room temperature, followed by 0.1 ml of methyl iodide for 12 hours at room temperature.

Each methylated biopolymer was purified using Sep-pak C ₁₈ cartridge (Waters Assoc.). Permethylated biopolymers were hydrolyzed with 1.5 ml of trifluoroacetic acid (TFA) at 121 ° C. for 1 hour, and then reduced and acetylated with sodium borohydride. The resulting methylated alditol acetate was analyzed by gas liquid chromatography (GLC) and gas liquid chromatography-mass spectrometry (GLC-MS). GLC was performed on a Varian model STAR 3600CX gas chromatography with FID on an SP ^™ -2380 capillary column (30 m × 0.25 mm id, 0.2-μm film: SUPELCO). GLC-MS (70 eV) was performed with a Shimaju QP5050 instrument with the same capillary column as above. Peaks were identified based on relative retention time and cleavage pattern. The mol% for each sugar was adjusted using the peak site.

 The identification of each methylated alditol acetate was inferred based on the relative retention time and fraction pattern. The position of the glycosidic bond in the monosaccharide corresponds to the position of the methoxy group in the methylated sugar.

As a result of the above experiment, in Table 3, the remarkable peak of EN-PN-Fr.I is 2,3,4,6-tetra-O-methyl-glucitol (2,3,4,6-tetra-O -methyl-glucitol, 20.5%), 2,3,6-tri-O-methyl-glucitol (2,3,6-tri-O-methyl-glucitol, 79.5%). It was confirmed that EN-PN-Fr.I had a (1 → 4) bound glucopyranosyl unit as a skeleton and was linear. However, EN-PN-Fr.II is 2,3,4,6-tetra-O-methyl-glucitol (2,3,4,6-tetra-O-methyl-glucitol, 11.1%), 2,3 , 6-tri-O-methyl-glucitol (2,3,6-tri-O-methyl-glucitol, 17.5%), 2,4,6-tri-O-methyl-glucitol (2,4 , 6-tri-O-methyl-glucitol, 11.3%), 2,4-di-O-methyl-glucitol (2,4-di-O-methyl-glucitol, 12.6%), 2,4-di -O-methyl-mannitol (2,4-di-O-methyl-mannitol, 28.8%) and 2,3,4-tri-O-methyl-D-galaxitol (2,3,4-tri-O -methyl-D-galacitol (18.8%). It contains glucopyranosyl bound to (1 → 3) and (1 → 6) with EN-PN-Fr.II having a branching point, and galaccitopyranosyl residues bound to (1 → 3) and (1 → 6) there was.

Identification of Partially Methylated Alditol Acetate Methylated sugar Major volume spectral fragment (m / e) ^c Fr.Ⅰ (Mol% ^a ) Fr. II (Mol% ^a ) Combine ^b 2,3,4,6-tetra- O -Me-Glc 43,59,71,87,101,117,129,145,161,205 20.5 11.1 Glc ¹ → 2,3,6-tri- O- Me-Glc 43,71,87,101,117,129,161,189,233 79.5 17.5 → ⁴ Glc ¹ → 2,4,6-tri- O -Me-Glc 43,87,99,117,129,161,233 - 11.3 → ³ Glc ¹ → 2,4-di- O- Me-Glc 43,87,101,117,129,159,189 - 12.6 → ^3,6 Glc ¹ → 2,4-di- O -Me-Man 43,87,101,117,129,139,159,189 - 28.8 → ^3,6 Glc ¹ → 2,3,4-tri- O -Me-D-Gal 43,71,87,99,117,129,161,189 - 18.8 → ⁶ Glc ¹ →

As described above, the present invention relates to intracellular biopolymers (EN-PN) having an immunostimulating activity effect isolated from a liquid culture of Pholiota nameko mycelium. EN-PN-Fr.I in the intracellular biopolymer (EN-PN) fraction was excellent in the activity of lysosomal enzymes in macrophages and proliferation of splenocytes, and EN-PN-Fr.II was characterized by its anticomplement activity and nitric oxide. (NO) Has an excellent effect on production capacity. In addition, EN-PN-Fr.I having the activity of the present invention is a protein polysaccharide composed of major sugars of mannose and glucose, and major amino acids of glycine and lysine, and methylated sugar of 2,3,4,6-tetra. - O-methyl-glucitol (2,3,4,6-tetra- O -methyl-glucitol ) and 2,3,6-tree-O-methyl-glucitol (2,3,6-tri- O- methyl-glucitol), EN-PN-Fr.II is a protein polysaccharide consisting of mannose, galactose and glucose with major sugars, aspartic acid, threonine, glutamic acid and glycine the mainly 2,3,4,6-per-O-methyl-glucitol (2,3,4,6-tetra- O -methyl-glucitol ), 2,3,6- tree-O-methyl - glucitol (2,3,6-tri- O -methyl-glucitol ), 2,4,6- tree-O-methyl-glucitol (2,4,6-tri- O -methyl-glucitol ) , 2,4-di-O-methyl-glucitol (2,4-di- O -methyl-glucitol), 2,4-di-O-methyl-mannitol (2,4-di- O -methyl- mannitol), and 2,3,4-tri- O -methyl-D-galaxitol (2,3,4-tri O- methyl-D-galacitol). Therefore, the intracellular biopolymer of the present invention can be very useful in the pharmaceutical industry as a pharmaceutical composition for enhancing immune activity.

Claims (5)

Isolated from liquid mycelium of Pholiota name mycelium, a fraction consisting of 79.6% of sugar, glycine and lysine of 20.4% of protein, and a molecular weight of 3.1kDa of mannose and glucose with molecular weight of 1000kDa Mannose, galactose and glucose having anti-complement derived from flavored mushrooms, characterized in that 86.9% of the host is composed of sugars and fractions consisting of aspartic acid, threonine, glutamic acid and glycine of 13.1% of the host. complement), intracellular biopolymers with proliferative effects on macrophages and splenic lymphocytes. delete The method of claim 1, wherein the fraction having the molecular weight of the 1000kDa is a sugar methylation 2,3,4,6-tetra-O-methyl-glucitol (2,3,4,6-tetra- O -methyl -glucitol) and 2,3,6-tree - O - methyl - consists of glucitol (2,3,6-tri- O -methyl-glucitol ), fractions having a molecular weight of 3.1kDa was the methylation the 2,3,4,6-tetrahydro-per-O-methyl-glucitol (2,3,4,6-tetra- O -methyl-glucitol ), 2,3,6- tree-O-methyl-gluconic sitol (2,3,6-tri- O -methyl-glucitol ), 2,4,6- tree-O-methyl-glucitol (2,4,6-tri- O -methyl-glucitol ), 2 , 4-di-O-methyl-glucitol (2,4-di- O -methyl-glucitol ), 2,4- di-O-methyl-mannitol (2,4-di- O -methyl-mannitol ) , and 2,3,4-anticomplementary methyl -D- go raksi toll mushroom flavor derived, characterized in that consists of (2,3,4-tri- O -methyl-D -galacitol) (- O anti-complement), intracellular biopolymer with proliferative effect on macrophages and splenic lymphocytes. delete delete

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