WO2017026697A1 - Composition containing extracellular polysaccharide produced by ceriporia lacerata as active ingredient for immunoregulation - Google Patents

Abstract

The present invention relates to an extracellular polysaccharide produced by Ceriporia lacerate, a culture medium of Ceriporia lacerata hyphae including the extracellular polysaccharide, a composition for immunoregulation containing, as an active ingredient, dried powder of the hyphae culture medium or an extract of the hyphae culture medium, a pharmaceutical composition for preventing or treating an immune disease, and a health food for immunoregulation. The composition of the present invention has an excellent effect on immune enhancement and overactive immune response inhibition.

Description

Immune modulating composition containing an extracellular polysaccharide produced by Ceriporia laccerata as an active ingredient

The present invention is an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ), mycelium culture medium of the seriporia laccerata comprising the extracellular polysaccharide, the dry powder of the mycelium culture medium, or the extract of the mycelium culture solution It relates to a composition for immunomodulation containing as an active ingredient.

The immune system can be divided into natural resistance, nonspecific immune system and specific immune system. Natural resistance (primary line of defense) refers to the anatomical physiology that prevents all invaders, including microorganisms of any kind, and the nonspecific immune system (secondary line of defense) removes invaders entering the body through natural resistance. The defense system consists of phagocytes, and the specific immune system (tertiary line of defense) refers to the immune system composed of lymphocytes, of which a highly developed immune system with memory and the ability to distinguish between self and nonmagnetic. (Richard A. Goldsby, et al., KUBY Immunology , 2000).

Leukocytes constitute a secondary or tertiary line of defense, breaking through the primary line of defense, and are responsible for foreign bodies entering the body.In the case of bacterial, viral infections, or inflammatory reactions, the regulation of macrophage and lymphocyte activity is necessary to determine the therapeutic effect of medicines. Play a pivotal role. Macrophage is a major cell involved in the immune response. It releases cytokines that regulate the function of immune cells to regulate immune responses, destroying microorganisms, antigens, and dead tissues by phagocytosis, body fluids in natural immunity. Activity as an effector cell in sexual immune responses, antigen removal in delayed type hypersensivity, destruction of tumor cells through the release of Tumor Necrosis Factor-α (TNF-α), and acquired immunity Antigen processing and presentation in the reaction, control the inflammatory response by secreting substances or cytokines involved in the inflammatory response, producing growth factors of fibroblasts and vascular endothelial cells to heal damaged tissues It is known to do. In addition, macrophages play a protective role against diseases such as infection and cancer, and secrete inflammatory cytokines and chemical mediators by inflammatory stimuli. Lipopolysaccharide (lipopolysaccharide), a membrane constituent of Gram-negative bacteria, activates macrophages among immune cells, such as TNF-α, interleukin-1 (IL-1), IL-6, IL-10, It is known to secrete prostanoids and nitric oxide.

Research is actively conducted to treat various diseases by enhancing the immunity to regulate the intracellular levels of the factors related to immunity. Initially, chemical substances were used to control the intracellular levels of the factors, but side effects are severe. Recently, there is an active research to control the intracellular levels of these factors using natural-derived materials that have no side effects or appear at very low levels.

In this regard, Korean Patent Laid-Open Publication No. 2014-0019113 discloses a therapeutic or prophylactic activity of a disease due to an immunomodulatory abnormality of alpha-galactosyl ceramide analogues, and Korean Patent No. 10-1417341 discloses a dandelion moss extract. And it discloses a composition for the prevention and treatment of inflammatory diseases or immune diseases comprising apo-9'-fucoxanthinone (apo-9'-fucoxanthinone) compound.

On the other hand, Ceriporia laccerata is a type of white fungus, and is known to perform co-metabolism called lignin decomposition in order to use carbon sources such as cellulose, hemicellulose, other polysaccharides, and glycerol in an ecosystem.

Regarding the medical treatment using Ceriporia laccerata, only the use of Ceriporia laccerata culture extract in the treatment of diabetes in Korean Patent No. 10-1031605 is known so far, and Ceriporia laccerata The immunomodulatory effect has not been reported until now.

Therefore, the present inventors have an immunomodulatory effect of the extracellular polysaccharide isolated from the seriporia laccerata, the mycelium culture medium of the seriporia laccerata containing the extracellular polysaccharide, the dry powder of the mycelium culture medium, or the extract of the mycelium culture medium. This invention was completed by confirming that it represents.

It is an object of the present invention to provide an immunomodulatory composition containing an active ingredient produced by Ceriporia laccerata, a pharmaceutical composition for the prevention or treatment of immune diseases, and a dietary supplement for immunomodulation.

Another object of the present invention is to provide an immunomodulatory method for administering an active ingredient produced by Ceriporia laccerata, and the use of the active ingredient for use in the manufacture of a medicament for immunomodulation.

In order to achieve the above object, the present invention is an extracellular polysaccharide produced by Seriphoria laccerata, mycelium culture medium of the Seriphoria laccerata comprising the extracellular polysaccharide, dry powder of the mycelium culture medium or the mycelium culture medium It provides an immunomodulatory composition containing the extract of as an active ingredient, a pharmaceutical composition for the prevention or treatment of immune diseases and health functional foods for immunomodulation.

The present invention also provides an immunomodulatory method for administering an active ingredient produced by Ceriphoria laccerata, and the use of the active ingredient for use in the manufacture of a medicament for immunomodulation.

The immunomodulatory composition of the present invention, the pharmaceutical composition for the prevention or treatment of immune diseases, and the dietary supplement for immunomodulation inhibit the excessive lymphocyte proliferation, and thus have an excellent effect of suppressing the immune excess due to excessive lymphocyte growth. In addition, the composition and health functional food of the present invention is excellent in immune enhancing activity such as increasing the proliferative capacity of splenocytes and increasing the expression of TNF-α to activate macrophages.

1 is a graph showing lymphocyte proliferation (%) after treatment of mouse-derived splenocytes at various concentrations with extracellular polysaccharide (EPS) produced by Seriphoria laccerata (** p <0.01, * p <). 0.05).

Figure 2 shows the results of agarose gel electrophoresis confirming the expression of TNF-α mRNA after treatment of the extracellular polysaccharide produced by Seriphoria lacserata to mouse-derived peritoneal macrophages at various concentrations.

Figure 3 is a Western blot result confirming the expression of TNF-α protein after treating the extracellular polysaccharide produced by Seriphoria lacserata to mouse-derived peritoneal macrophages at various concentrations.

The present invention is an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ), mycelium culture medium of the seriporia laccerata containing the extracellular polysaccharide, dry powder of the mycelium culture medium or extract of the mycelium culture solution It provides a composition for immunomodulation containing as an active ingredient.

As used herein, the term "extracellular polysaccharide (EPS)" is a part of the cell wall of a microorganism such as a fungus, and the polysaccharide is secreted to the outside of the cell to form a capillary around it or secreted into the periphery or medium as a slime. Means. The extracellular polysaccharide is secreted by the microorganism to protect itself from the external environment such as antibodies, toxic substances, protozoa and bacteriophage.

The extracellular polysaccharide is 40 to 60% by weight of sugar and 30 to 40% by weight of protein, 40 to 50% by weight of sugar and 32 to 38% by weight of protein, or 43 to 47% by weight of sugar and 33 to 36% by weight. % Protein, and more specifically about 45% sugar and about 34% protein.

The sugar may contain mannose, galactose and glucose.

The extracellular polysaccharide may have a molecular weight of 100 ~ 150 kDa, 110 ~ 140 kDa or 115 ~ 125 kDa, more specifically may have a molecular weight of about 120 kDa.

In one embodiment of the present invention, the extracellular polysaccharide comprises the steps of: (a) liquid culture of the seriporia laccerata mycelium to prepare a culture medium of the seriporia lacserata mycelium; (b) drying and powdering the Ceriporia laccerata mycelium culture solution; And (c) extracting the Ceriporia laccerata mycelium culture powder with a solvent, filtering the same, and concentrating under reduced pressure.

The medium for the liquid culture of the seriporia laccerata mycelium in the step (a) is sugar, glucose, starch, hydrate, soybean meal, soybean meal, magnesium sulfate (MgSO ₄ ), potassium phosphate (KH ₂ PO ₄ ), potassium diphosphate (K ₂ HPO ₄ ) and may include water, the hydrogen ion concentration may be pH 4.5 ~ 6.0.

Specifically, the medium, 0.2 to 3% by weight of sugar, 0.2 to 3% by weight of glucose, 0.2 to 4% by weight, 0.1 to 0.5% by weight of water, 0.1 to 0.5% by weight of wheat flour, 0.2 to 3 weight of soy flour %, 0.05% to 0.1% by weight of magnesium sulfate (MgSO ₄ ), 0.05% to 0.25% by weight of potassium monophosphate (KH ₂ PO ₄ ), 0.05% to 0.25% by weight of potassium diphosphate (K ₂ HPO ₄ ) and the balance of water Can be.

The liquid culture in step (a) may be performed under a blue LED light source, and specifically, the liquid culture may be performed by maintaining a concentration of carbon dioxide at 1,000 to 2,000 ppm under a blue LED light source.

The liquid culture, for example, at 20 ~ 28 ℃, hydrogen ion concentration (pH) 4.5 ~ 6.0, the light source maintains a blue LED, the illuminance is 0.1 ~ 0.8 LUX and air is injected at 0.5 ~ 2.0 kgf / ㎠ and carbon dioxide The concentration of may be performed for 8 to 13 days while maintaining at 1,000 to 2,000 ppm. Specifically, it may be performed for 5 to 15 days at the conditions of 20 ~ 25 ℃, pH 4.5 ~ 6.0, 0.5 ~ 2.0 kgf / ㎠, carbon dioxide concentration of 1,000 ~ 2,000 ppm. When the liquid culture under the conditions described above, the content of the extracellular polysaccharide produced is high.

As the parent strain in step (a), one excellent strain stored at 1 to 5 ° C. in PDA (Potato dextrose agar) medium was used in a shaker incubator using PDB (Potato dextrose broth) medium in an Erlenmeyer flask. Maintain a constant temperature of 25 ℃ can be used after 7 to 9 days incubation process. In addition, after culturing the parent strains, the culture solution or the obtained mycelium can be used as the inoculum. Specifically, the amount of mycelium to be added to the inoculum may be about 0.5% (w / v) based on the amount of the solution to be cultured. The amount of extracellular polysaccharides does not increase as the amount of mycelia (% / 100 ml, w / v) increases, so the medium composition has the highest content of extracellular polysaccharides, not the best nutritional ratio and environmental conditions for the growth of mycelia. It is preferable to apply the selective culture conditions to form.

The culture solution can be separated and purified into a mycelium and an aqueous solution. For the separation and purification, the solution from which the mycelium is removed by centrifugation may be repeatedly purified using a multi-sheet filter press and a vibration centrifugal separator (PALLSEP), and then irradiated with ultraviolet (UV) light for 1 minute. In addition, the culture solution may be stored after sealing to remove oxygen, which may bring a change in the content of the active ingredient due to the growth of the mycelia when the mycelia exist in the culture solution.

In step (b), the mycelia culture solution prepared in step (a) may be dried and powdered. The drying may be performed for 48 to 96 hours at a temperature of 40 ℃ or less, more specifically 30 ℃ or less to prevent the disappearance of the active material. In addition, the drying of step (b) is preferable to use a vacuum freeze dryer rather than a vacuum dryer that sets the evaporation temperature relatively high because the change in the effective substance content is minimized.

In the step (c), after extracting the dry powder of the mycelia culture solution obtained in step (b) with a solvent, the extracellular polysaccharide which is the active ingredient of the composition according to the present invention is separated.

Specifically, 100 ml of distilled water is added to the dry powder of 3 to 10 g of the mycelium culture medium, and then suspended well, followed by centrifugation at 5,000 to 10,000 rpm for 10 to 30 minutes to obtain a supernatant. An extraction solvent corresponding to a pear may be added and placed in a refrigerator at 1 to 5 ° C. for 10 to 15 hours. After only centrifuging the supernatant again at 5,000 to 10,000 rpm for 10 to 30 minutes in the stationary material, the precipitate may be recovered to prepare a crude extracellular polysaccharide. The crude extracellular polysaccharide can be vacuum-dried at 30 ° C. or lower to obtain extracellular polysaccharide.

The extraction solvent may be a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, acetone, ether, chloroform and ethyl acetate or a mixed solvent thereof, and more specifically, water, methanol, ethanol, butanol It may be a solvent selected from the group consisting of acetone and ethyl acetate or a mixed solvent thereof, and more specifically, may be water or 50 to 80% (v / v) ethanol aqueous solution.

The immunomodulatory composition may include 0.1 to 80% by weight of the total weight of the composition, specifically, 0.1 to 50% by weight of the extracellular polysaccharide, and the mycelium culture medium, the dry powder thereof or the mycelium culture medium of Seriphoria laccerata The extract may be appropriately included in an amount corresponding to the content of the extracellular polysaccharide. However, the effective amount of the extracellular polysaccharide, the culture solution containing the same, the dry powder of the culture solution or the extract of the culture solution may be appropriately adjusted according to the method of use and purpose of the composition.

In addition, the immunomodulatory composition may be used as a pharmaceutical composition or health functional food for the purpose of regulating immune activity and preventing, improving or treating immune diseases, and the amount of use and use form may be appropriately adjusted according to the purpose. .

The immunomodulatory composition may exhibit an immunopromoting effect and an immunosuppressive effect. The immunostimulating effect is an effect of increasing the expression of TNF-α in macrophages or enhancing the proliferation of splenocytes to enhance immunity. The immunosuppressive effect is an effect of inhibiting lymphocyte overexpression caused by a nonspecific stimulator in splenocytes to suppress immune excess. According to one embodiment of the present invention, the immunomodulatory composition of the present invention showed an excellent immunopotentiating activity to increase the expression of TNF-α in the macrophages of the administration group, and improve the proliferation of splenocytes depending on the EPS concentration. In addition, in the splenocytes of the administration group, it showed an immunosuppressive effect of suppressing lymphocyte overexpression by PMA, which is a nonspecific stimulus source, depending on EPS concentration.

The present invention comprises an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ), mycelium culture solution of Ceriporia laccerata , dried powder of the mycelia culture medium or extract of the mycelium culture solution as an active ingredient. To provide a pharmaceutical composition for the prevention or treatment of immune diseases.

The pharmaceutical composition of the present invention exhibits the prevention or treatment of various immune diseases through the above-described immune enhancing effect or effective immune suppression effect of the active ingredient.

The immune diseases include dermatitis, allergy, rhinitis, gout, spastic spondylitis, rheumatic fever, lupus, fibromyalgia, peritonitis, type 1 diabetes, scleroderma, degenerative neuropathy, type 2 diabetes, silicosis , Atherosclerosis, vitiligo, conjunctivitis and autoimmune diseases.

The autoimmune diseases include rheumatoid arthritis, systemic scleroderma, atopic dermatitis, psoriasis, asthma, Guilian-Barre syndrome, dermatomyositis, polymyositis, and multiple sclerosis. one or more diseases selected from the group consisting of multiple sclerosis, autoimmune encephalomyelitis, nodular polyarteritis nodosa, temporal arteritis, childhood diabetes, alopecia areata, bleb, aphthous stomatitis, Crohn's disease and Behcet's disease In particular, it may be one or more diseases selected from the group consisting of rheumatoid arthritis, atopic dermatitis, psoriasis, asthma, dermatitis, alopecia areata, blue swelling, aphthosis and Crohn's disease.

As described above, it is already known that increased expression of TNF-α in macrophages is effective for the treatment or prevention of rheumatoid arthritis and Crohn's disease among the diseases (Kim RB, and so on. J. Rheumatic Diseases). reference 324-34): 2013;:; 20 (3) 21 177-180 and Braun J, Baraliakos X. Curr Opin Rheumatol 2009... In addition, enhanced proliferation of spleen cells are known to be effective in the prevention and treatment of lupus and eczema (literature: Chae BS Nat Prod Sci 2011; 17:...... 354 and Kim KBWR, and so on J. see 1121-1127):... 44 ( 8); Korean Soc Food Sci Nutr 2015. Furthermore, suppression of lymphocyte overexpression of splenocytes is effective in the treatment or prevention of rheumatoid arthritis, systemic scleroderma, atopic dermatitis, diabetes, alopecia areata, psoriasis, bleb, asthma, aphthous stomatitis, multiple myositis, multiple sclerosis or autoimmune encephalomyelitis It is also well known (see related documents: Korean Patent Registration No. 10-1227819).

The pharmaceutical composition is an extracellular polysaccharide produced by Seriphoria laccerata, mycelium culture medium of the Seriphoria laccerata, containing the dry powder of the mycelium culture medium or extract of the mycelium culture medium as an active ingredient In addition it may further comprise suitable carriers, excipients and diluents commonly used in pharmaceutical compositions.

The pharmaceutical composition according to the present invention can be formulated and used in various forms according to conventional methods. Suitable formulations include, but are not limited to, tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, suppositories, and the like.

The pharmaceutical compositions according to the invention can be prepared in suitable formulations using pharmaceutically inert organic or inorganic carriers. That is, when the formulation is a tablet, coated tablet, dragee and hard capsule, it may include lactose, sucrose, starch or derivatives thereof, talc, calcium carbonate, gelatin, stearic acid or salts thereof. In addition, when the formulation is a soft capsule, it may include polyols of vegetable oils, waxes, fats, semisolids and liquids. In addition, when the formulation is in the form of a solution or syrup, it may include water, polyols, glycerol, vegetable oils and the like.

The pharmaceutical composition according to the present invention may further include a preservative, a stabilizer, a humectant, an emulsifier, a solubilizer, a sweetener, a colorant, an osmotic agent, an antioxidant, and the like in addition to the carrier.

The method of administering the pharmaceutical composition according to the present invention can be easily selected according to the dosage form and can be administered orally or parenterally. The dosage may vary depending on the age, sex, weight, severity, and route of administration of the patient, but is generally 5 to 1,000 mg / kg body weight based on the extracellular polysaccharide, which is an active ingredient, specifically 10 to 600 The amount of mg / kg body weight may be administered once to three times a day. However, the dosage does not limit the scope of the invention in any aspect.

The pharmaceutical composition according to the present invention not only provides excellent immune enhancing and immune suppression effect but also has little toxicity and side effects due to the drug, and thus can be used safely for long-term administration for the purpose of treating or preventing immune diseases.

In addition, the present invention is an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ), mycelium culture medium of the seriporia laccerata comprising the same, the dry powder of the mycelium culture solution or the extract of the mycelium culture solution active ingredient Provides a dietary supplement for immune regulation comprising.

The health functional food for immune regulation has an activity of maintaining homeostasis of immune function by enhancing immune activity or suppressing or improving excessive immunity.

The dietary supplement according to the present invention may be in the form of a powder, granule, tablet, capsule or beverage. In addition, the health functional food according to the present invention may be candy, chocolate, beverage, gum, tea, vitamin complex or health supplement.

In this case, the content of the extracellular polysaccharide according to the present invention contained in the health functional food, mycelium culture medium containing the same, the dry powder of the mycelium culture medium or the extract of the mycelium culture medium, usually 0.01 to 50% by weight of the total food weight Specifically, it may be included in 0.1 to 20% by weight. In addition, in the case of a health beverage composition, it may be included in an amount of 0.02 to 10 g, specifically 0.3 to 1 g based on 100 ml of the health beverage composition.

The food may further comprise a food supplement acceptable with the extracellular polysaccharide of the present invention, a mycelium culture medium of the seriporia laccerata containing the same, a dry powder of the mycelium culture medium or an extract of the mycelium culture medium. .

The present invention is an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ), mycelium culture medium of the seriporia laccerata comprising the extracellular polysaccharide, the dry powder of the mycelium culture medium, or the extract of the mycelium culture solution It provides a method of immunomodulation, comprising administering to a subject in need of immunostimulation or immune suppression.

The subject may be a mammalian animal, more specifically a human.

In addition, the immunomodulatory method is a disease that requires immunosuppression or hyperimmunity suppression, such as dermatitis, allergy, rhinitis, gout, spondylitis spondylitis, rheumatic fever, lupus, fibromyalgia, peritonitis, first It can be used for the prevention and treatment of type diabetes mellitus, scleroderma, degenerative neuropathy, type 2 diabetes, silicosis, atherosclerosis, vitiligo, conjunctivitis, autoimmune diseases and the like.

In addition, the present invention is an extracellular polysaccharide produced by Ceriporia lacerata for use in the manufacture of a medicament for immunomodulation, mycelium culture medium of the seriporia laccerata comprising the extracellular polysaccharide It provides a dry powder of the mycelium broth, or the use of the extract of the mycelium broth.

The extracellular polysaccharide produced by the Seriphoria laccerata, the mycelium culture medium of the Seriphoria laccerata containing the extracellular polysaccharide, the dry powder of the mycelium culture solution, or the extract of the mycelium culture medium is as described above.

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

[ Example ]

Production Example  One. Serifolia Lakserata  Mycelium broth, its dry powder, extract and Extracellular polysaccharide (exopolysaccharide; hereinafter referred to as "EPS")

1-1: Serifolia Lakserata  Preparation of Mycelia Culture

The seedlings grown in subcultured oak reimbursement in Sangju, Gyeongbuk were grown by subculture and stored frozen at -80 ℃. The cryopreserved strain was passaged 2-3 times in PDA (Potato dextrose agar) medium (87 plastic cultures; Difco, Becton Dickinson and Company) and stored the strain (hereinafter referred to as “PDA culture strain”) in a 4 ° C. refrigerator. Was used. Then, after preparing 600 ml of PDB (Potato dextrose broth) medium (Difco, Becton Dickinson and Company) in an Erlenmeyer flask, one PDA culture strain was added thereto, followed by shaking culture at 25 ° C. for 8 days to obtain a PDB culture strain.

On the other hand, 1.5% by weight of sugar, 0.5% by weight of glucose, 0.5% by weight of potato starch, 0.25% by weight of water, 0.25% by weight of soybean meal, 0.75% by weight of soy flour, 0.05% by weight of magnesium sulfate (MgSO ₄ ) , Liquid culture medium containing 0.05% by weight of potassium monophosphate (KH ₂ PO ₄ ), 0.05% by weight of potassium diphosphate (K ₂ HPO ₄ ) and the balance of water at 121 ° C. in an 800 L fermenter and air at 1.5 kgf / Injected in cm 2 sterilized for 20 minutes. This was inoculated with 600 ml of the PDB culture strain with a starter in a state of cooling to 23 ° C., while ventilating air at 0.5 to 1.5 kgf / cm 2, the light source maintained a blue LED, the illuminance was 0.5 LUX, and the concentration of carbon dioxide was Ceriporia lacserata mycelium culture was prepared by liquid culture of the seriporia lacserata mycelium at 2,000 ppm for 10 days at a constant temperature of 23 ° C.

1-2: Serifolia Lakserata  Preparation of Culture Powder Dry Powder

The dry powder of the Ceriporia laccerata mycelium culture medium was prepared by lyophilizing the powder of the Ceriporia laccerata mycelium culture medium prepared in Preparation Example 1-1 at 25 ° C. for 72 hours.

1-3: Serifolia Lakserata  Preparation of culture extract

100 ml of distilled water was added to 5 g of the dry powder of the Ceriporia laccerata mycelium culture medium prepared in Preparation Example 1-2, and then suspended well, and centrifuged at 8,000 rpm for 20 minutes. Ethanol corresponding to 2-3 times the volume of the supernatant was added to the supernatant, and the mixture was allowed to stand at 4 ° C. for 12 hours. Subsequently, the supernatant was taken from the stationary material to prepare an extract of the culture medium of the seriporia laccerata.

1-4 Serifolia Lakserata  Preparation of EPS from Culture

After centrifugation of the extract of the seriporia laccerata mycelium culture medium prepared in Preparation Example 1-3 again at 8,000 rpm for 20 minutes, the precipitate was recovered to obtain a crude EPS. The crude EPS was vacuum-dried at 25 ° C. for 72 hours using a vacuum freeze dryer to obtain EPS produced by Ceriporia laccerata.

Example  1. Characterization of EPS

1-1: Gel permeation  Chromatography (Gel Permeation Chromatography, GPC Molecular weight of EPS using

The EPS prepared in Preparation Example 1 was dissolved in 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ [adjusting pH to 4 with glacial acetic acid] to 1% (w / v) in a solution, and then at 4,000 rpm. After centrifugation for 0.5 hour, only the supernatant was filtered with a 0.45 μm syringe filter and analyzed by GPC.

For the GPC analysis, the refractive index was used as the detector, and the GPC column was adjusted to pH 4 with 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ [glacial acetic acid using OHpak SB 805 HQ (Shodex, Japan). ], The flow rate of the mobile phase was flowed at a rate of 1.0 mL / min. Standard curves were prepared using Dextran (American Polymer Corporation, USA) with different molecular weights (130 kDa, 400 kDa, 770 kDa, or 1200 kDa), and Knauer K-2310 (refractive index (RI) measuring instrument). Germany) to determine the molecular weight of EPS. The measurement conditions are summarized in Table 1 below.

	Measurement of molecular weight
GPC system	Knauer K-501 System
column	OHpak SB 805 HQ (Shodex, Japan)
Mobile phase	0.1 M Na 2 SO 4 / 0.05 M NaN 3 / pH 4
Flow rate	1.0 ml / min
Measuring instrument	RI (Knauer K-2310)

As a result, the molecular weight of EPS of the present invention was found to be about 120 kDa.

1-2: Determination of sugar and protein content of EPS

The EPS prepared in Preparation Example 1 was second-purified and treated with proteolytic enzymes to determine sugar and protein content.

Specifically, the super purified EPS (EPS prepared in Preparation Example 1) was dissolved in distilled water and centrifuged at 8,000 rpm for 20 minutes to separate the supernatant. Ethanol corresponding to 2-3 times the volume of the supernatant was added to the separated supernatant and placed in a 4 ° C. refrigerator for 12 hours. Thereafter, only the supernatant was again centrifuged at 8,000 rpm for 20 minutes, and the precipitate was recovered to obtain a second purified EPS. The second purified EPS was dissolved in distilled water, and the proteolytic enzyme alcalase was treated at 50 ° C. for 30 minutes at a concentration of 0.5% (w / v).

Sugar content was measured by the phenol-sulfuric acid method.

Specifically, 25 µl of 80% (w / v) phenol was added to 1 ml of the diluted sample, and 2.5 ml of sulfuric acid was added thereto, followed by cooling to room temperature. The absorbance was measured at 465 nm to calculate the sugar content.

In addition, the protein content was measured by the BCA method (Smith PK et al., Analytical Biochemistry , 150 (1): 76-85, 1985), and bovine serum albumin was used as a standard.

The sugar and protein content measured as described above are shown in Table 2 below, wherein the EPS content was found to be 45-51 wt% and the protein content was 33-34 wt%.

	yield(%)	Total sugar content (%)	Total protein content (%)
EPS	1.22 ± 0.03	45.32 ± 1.41	34.17 ± 0.73
Secondary Refined EPS	0.78 ± 0.01	50.49 ± 0.52	33.50 ± 2.79
Enzyme-treated EPS *	0.24 ± 0.06	51.39 ± 1.32	34.61 ± 1.51

^* Enzyme treatment: 0.5% Al Calle Izu, 50 ℃, 30 minutes.

Each figure is mean ± SE (n≥3).

In addition, the results of the sugar composition analysis of EPS, EPS mainly contained mannose, galactose and glucose.

Example  2. Confirm the effect of inhibiting excessive proliferation of lymphocytes

In order to evaluate the immunosuppressive effect of EPS of Preparation Example 1, mouse-derived splenocytes were treated with phorbol 12-myristate 13-acetate (PMA), a nonspecific stimulator, and at various concentrations of EPS. After the treatment was confirmed whether to inhibit excessive lymphocyte proliferation.

Specifically, female Balb / C mice, which were obtained at about 8 weeks of age and about 25 g of body weight, were purchased from Korean Hyochang Science and used in the experiment. After sacrificing the mouse by cervical dislocation, the spleen was aseptically removed and crushed on a 100 mesh cell strainer on RPMI 1640 solution to release the cells. The suspension containing free cells was carefully placed in a vessel containing Lymphoprep ™ and centrifuged at 15 ° C., 2,000 rpm for 15 minutes to remove precipitated erythrocytes. 200 μl of red blood cells depleted splenocytes (2 × 10 ⁶ cells / ml) were dispensed into 96-well microplates and treated with 250 ng of PMA per 1 ml of culture to induce lymphocyte proliferation of splenocytes. 0.05 μg, 0.15 μg, 0.5 μg, 1.5 μg or 5 μg was added dropwise to 1 ml of the culture solution. For comparison, no control was treated with EPS. The microplates were then incubated for 48 hours in an incubator maintained at 5% CO ₂ , 37 ° C. and sufficient humidity. To determine the proliferation capacity of cultured cells after incubation, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (Sigma, USA) assay was assayed in each well of the microplate. Was performed.

Specifically, the cultured cells were treated with MTT solution at a final concentration of 0.1 mg / ml and incubated for 4 hours while blocking light with aluminum foil to induce formazan crystal formation. The supernatant was removed, and each well was treated with 150 μl of dimethyl sulfoxide (DMSO) for 10 minutes, and then absorbance was measured at 540 nm using an ELISA reader. Lymphocyte proliferation of splenocytes was calculated by the following equation.

% Cell viability = (test group absorbance / control absorbance) × 100

The MTT assay was repeated three times, and the results are shown in Table 3 below.

	Treatment concentration	Absorbance (540 nm)			Absorbance Average and Deviation	Survival rate (%)
Voice control group (no processing)	0	0.1842	0.1526	0.1390	0.159 ± 0.0008	100
PMA	250 ng / ml	0.7287	0.6074	0.6034	0.647 ± 0.071	406.9
EPS (μg / ml) of Preparation Example 1	0.05	0.7075	0.5181	0.6831	0.636 ± 0.103	400.0
	0.15	0.5581	0.6609	0.5147	0.578 ± 0.075	363.5
	0.5	0.4818	0.5196	0.5127	0.505 ± 0.020	317.6
	1.5	0.3680	0.4890	0.4321	0.430 ± 0.061	270.4
	5	0.3013	0.4054	0.4779	0.395 ± 0.089	248.4

As shown in Table 3, EPS of the present invention inhibited lymphocyte proliferation increased concentration-dependently and significantly by treatment of PMA, a nonspecific stimulator in splenocytes. This is an effect of suppressing the immune excess due to excessive lymphocyte increase, the results show that the EPS can be effectively used for the prevention and treatment of autoimmune diseases.

Example  3. Splenocytes Proliferative capacity  Measure

Except not treated with PMA, which is a nonspecific stimulator, splenocyte proliferation was measured by EPS treatment in the same manner as in Example 2, and the results are shown in FIGS. 1 and 4.

	Treatment concentration	Absorbance (540 nm)			Absorbance Average and Deviation	Survival rate (%)
Voice control group (no processing)	0	0.1842	0.1526	0.1390	0.159 ± 0.0008	100
EPS (μg / ml) of Preparation Example 1	0.05	0.1726	0.2	0.2352	0.203 ± 0.010	127.7
	0.15	0.2016	0.2357	0.2238	0.220 ± 0.006	138.4
	0.5	0.2312	0.2665	0.2498	0.249 ± 0.006	156.6
	1.5	0.3128	0.2539	0.3182	0.295 ± 0.012	185.5
	5	0.2914	0.295	0.2598	0.282 ± 0.006	177.4

As shown in FIG. 1 and Table 4, the EPS of the present invention in splenocytes increased the proliferative capacity of the splenocytes in a concentration dependent and significantly. The proliferation of splenocytes as described above is an effect of activating the immune system, and the results show that the EPS can be effectively used for the prevention and treatment of immune diseases.

Example  4. TNF -α expression measurement

4-1: EPS Treatment Cell Manufacturing

In order to evaluate the immuno-enhancing effect of EPS of Preparation Example 1, TNF-α mRNA and protein expression were measured after treatment with various concentrations of EPS in mouse-derived peritoneal macrophages.

Specifically, as a Balb / C mouse, about 8 weeks of age and about 25 g of body weight were purchased from Korea Hyochang Science and used for the experiment. After the mice were intraperitoneally injected with 1 ml of 4% thioglycollate and sacrificed by cervical dislocation, the peritoneum was exposed, 10 ml of cold RPMI 1640 was injected into the abdominal cavity, and the abdominal cavity was lightly massaged. Thereafter, the washing solution was extracted from the abdominal cavity using a 10 ml syringe and centrifuged at 1,000 rpm for 10 minutes at 4 ° C. After removing the supernatant, 1 mL of fresh RPMI 1640 medium was added to the precipitate, and the cells were incubated for 3 hours by dispensing 3 × 10 ⁶ cells / well in a 96 well plate. After incubation, cells not adsorbed on the plate were removed, and the adsorbed cells were further cultured for 3 days to obtain mouse abdominal macrophages.

Thereafter, the cultured macrophages were put into 100 μl in 96 well plates and incubated for 24 hours in an incubator at 37 ° C. and 5% CO ₂ . Discard the medium and wash the surface of the culture cells with 1X PBS solution (Sigma, USA), each well treated with 0.5 μg / ml, 1.5 μg / ml or 5 μg / ml of EPS of Preparation Example 1 and incubated for 6 hours. To obtain EPS treated cells. For comparison, no control was treated with EPS.

4-2: TNF -α mRNA  Expression analysis

RNA was isolated from the EPS treated cells of Example 4-1 and polymerase chain reaction (PCR) was performed to analyze TNF-α mRNA expression.

Specifically, total RNA separation was performed according to the method of easy-BLUETM total RNA Extraction kit. Then, for cDNA synthesis, 1 μg of the total RNA, oligodT 18 and DEPC-DW (Bionia) were added to RT-PreMix (Bionia), followed by 60 minutes at 42 ° C and 5 minutes at 94 ° C to synthesize cDNA. . Then, 5 μl of the RT mixture containing cDNA was placed in PCR PreMix (Bionia), followed by PCR with TNF-α forward primer and reverse primer. MRNA was amplified by repeating 30 times at 95 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C under PCR conditions. The difference in expression of TNF-α mRNA was confirmed by electrophoresis on 1.5% agarose gel, and the results are shown in FIG. 2.

Forward primer (5'-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3 ', SEQ ID NO: 1) and reverse primer (5'-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3', SEQ ID NO: 2) of the TNF-α to perform the RT-PCR, A forward primer (5'-AGATCCACAACGGATACATT-3 ', SEQ ID NO: 3) and a reverse primer (5'-TCCCTCAAGATTGTCAGCAA-3', SEQ ID NO: 4) of GAPDH were used (Bionia, Korea).

As shown in Figure 2, compared to the macrophages not treated with EPS, EPS-treated macrophages of the present invention increased TNF-α mRNA expression depending on the EPS treatment concentration. This is a result of confirming the immunopotentiation activity of the EPS of the present invention, as the expression of TNF-α which activates macrophages to prevent the growth and replication of the virus is increased.

4-3: TNF -α protein expression analysis

The EPS treated cells of Example 4-1 were treated with protein extract solution (Protein Extraction Solution, Intron Biotechnology Co., Ltd.) and lysed. After centrifugation of this solution at 13,000 rpm for 15 minutes at 4 ° C, the upper protein was collected and the concentration of the protein was measured at 595 nm using a Bradford reagent, and the amount of protein in all samples was determined at 30 µg. It was. The total volume was made equal using NuPAGE LDS Sample Buffer (Novex, USA), then heated at 100 ° C. for 5 minutes and allowed to stand on ice for 15 minutes. Electrophoresis was performed on 10% SDS-polyacrylamide (SDS-PAGE) gel using this. The band on this SDS-PAGE gel was transferred to a nitrocellulose membrane at 4 ° C. and 300 mM for 1 hour 30 minutes. The membrane was then reacted with Tris-Buffer saline (TBS) containing 0.1% Tween 20 (Sigma, USA) and 5% skim milk for 1 hour to block nonspecific binding of the antibody, followed by TBS-T (Tris-Buffered Saline Tween 20) was washed three times at 15 minute intervals.

Then, for the first antibody, TNF-α (manufactured by abcam) antibody was added 1,000 times diluted with TBS-T containing 5% skim milk, and reacted at 4 ° C. for 1 hour, followed by TBS-T. 5 washes 15 minutes each. For secondary antibody reactions, anti-rabbit polyclonal antibody (Hell Signaling, Danvers, MA, USA) labeled horseradish peroxidase (HRP) on the membrane with 5% skim milk After adding a solution diluted 5,000-fold with the included TBS-T and reacted at room temperature for 1 hour, the chemiluminescent HRP (chemiluminescent HRP) substrate was evenly treated on the membrane to confirm the protein expression. The result is shown in FIG.

As shown in Figure 3, compared to the macrophages not treated with EPS, the EPS-treated macrophages of the present invention increased TNF-α protein expression depending on the EPS treatment concentration. This is a result of confirming the immunopotentiation activity of the EPS of the present invention, as the expression of TNF-α which activates macrophages to prevent the proliferation and replication of the virus is increased.

Claims (23)

1. Extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ), mycelium culture medium of the seriporia laccerata containing the extracellular polysaccharide, dry powder of the mycelium culture medium, or extract of the mycelium culture medium is an active ingredient It contains, the composition for immunomodulation.
2. The method of claim 1,

   The extracellular polysaccharide comprises 40 to 60% by weight of sugar and 30 to 40% by weight of protein and has a molecular weight of 100 to 150 kDa, the composition for immunomodulation.
3. The method of claim 2,

   The extracellular polysaccharide comprises 43 to 47% by weight of sugar and 33 to 36% by weight of protein and has a molecular weight of 115 to 125 kDa, the composition for immunomodulation.
4. The method of claim 2,

   The sugar contains mannose, galactose and glucose, the composition for immunomodulation.
5. The method of claim 1,

   The extracellular polysaccharide,

   (a) liquid culturing the Ceriporia laccerata mycelium to prepare a Ceriporia laccerata mycelium culture solution;

   (b) drying and powdering the Ceriporia laccerata mycelium culture solution; And

   (c) extracting the seriporia laccerata mycelium culture powder with a solvent, and then filtration and concentration under reduced pressure prepared by the manufacturing method, the composition for immunomodulation.
6. The method of claim 5,

   The medium for culturing the liquid is sugar, glucose, starch, hydrated water, soybean meal, soybean meal, magnesium sulfate (MgSO ₄ ), potassium monophosphate (KH ₂ PO ₄ ), potassium diphosphate (K ₂ HPO ₄ ) and water To include, the hydrogen ion concentration (pH) is 4.5 to 6.0, the composition for immunomodulation.
7. The method of claim 5,

   The liquid culture is carried out under a blue LED light source, and is carried out by maintaining the concentration of carbon dioxide at 1,000 ~ 2,000 ppm, the composition for immunomodulation.
8. The method of claim 1,

   The extracellular polysaccharide is included in an amount of 0.1 to 80% by weight relative to the total weight of the composition, the composition for immunomodulation.
9. Extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ), mycelium culture medium of the seriporia laccerata containing the extracellular polysaccharide, dry powder of the mycelium culture medium, or extract of the mycelium culture medium is an active ingredient It contains, as a pharmaceutical composition for the prevention or treatment of immune diseases.
10. The method of claim 9,

    The immune diseases are dermatitis, allergy, rhinitis, gout, spastic spondylitis, rheumatic fever, lupus, fibromyalgia, peritonitis, type 1 diabetes, scleroderma, degenerative neuropathy, type 2 diabetes, silicosis , Atherosclerosis, vitiligo, conjunctivitis and autoimmune disease is one or more diseases selected from the group consisting of, pharmaceutical composition for the prevention or treatment of immune diseases.
11. The method of claim 10,

    The autoimmune diseases include rheumatoid arthritis, systemic scleroderma, atopic dermatitis, psoriasis, asthma, Guilian-Barre syndrome, dermatomyositis, polymyositis, and multiple sclerosis. one or more diseases selected from the group consisting of multiple sclerosis, autoimmune encephalomyelitis, nodular polyarteritis nodosa, temporal arteritis, childhood diabetes, alopecia areata, bleb, aphthous stomatitis, Crohn's disease and Behcet's disease , Pharmaceutical composition for the prevention or treatment of immune diseases.
12. The method of claim 11,

    Wherein the autoimmune disease is at least one disease selected from the group consisting of rheumatoid arthritis, atopic dermatitis, psoriasis, asthma, dermatitis, alopecia areata, blue swelling, aphthous stomatitis and Crohn's disease, a pharmaceutical composition for preventing or treating immune diseases .
13. The method of claim 9,

    The extracellular polysaccharide comprises 40 to 60% by weight of sugar and 30 to 40% by weight of protein and has a molecular weight of 100 to 150 kDa, a pharmaceutical composition for preventing or treating immune diseases.
14. The method of claim 13,

    The extracellular polysaccharide comprises 43 to 47% by weight of sugar and 33 to 36% by weight of protein and has a molecular weight of 115 to 125 kDa, the pharmaceutical composition for preventing or treating immune diseases.
15. The method of claim 13,

    Pharmaceutical composition for the prevention or treatment of immune diseases, wherein the sugar contains mannose, galactose and glucose.
16. The method of claim 9,

    The extracellular polysaccharide,

    (a) liquid culturing the Ceriporia laccerata mycelium to prepare a Ceriporia laccerata mycelium culture solution;

    (b) drying and powdering the Ceriporia laccerata mycelium culture solution; And

    (C) a pharmaceutical composition for the prophylaxis or treatment of immune diseases, prepared by the manufacturing method comprising the step of extracting the seriporia laccerata mycelium culture powder with a solvent and then filtration and concentration under reduced pressure.
17. The method of claim 16,

    The medium for culturing the liquid is sugar, glucose, starch, hydrated water, soybean meal, soybean meal, magnesium sulfate (MgSO ₄ ), potassium monophosphate (KH ₂ PO ₄ ), potassium diphosphate (K ₂ HPO ₄ ) and water To include, the hydrogen ion concentration (pH) is 4.5 to 6.0, the pharmaceutical composition for preventing or treating immune diseases.
18. The method of claim 16,

    The liquid culture is carried out under a blue LED light source, and is carried out by maintaining the concentration of carbon dioxide at 1,000 to 2,000 ppm, a pharmaceutical composition for preventing or treating immune diseases.
19. Extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ), mycelium culture medium of the seriporia laccerata containing the extracellular polysaccharide, dry powder of the mycelium culture medium, or extract of the mycelium culture medium is an active ingredient Contains as a dietary supplement for immune regulation.
20. The method of claim 19,

    The health functional food is in the form of powder, granules, tablets, capsules or beverages, health functional foods for immune regulation.
21. The method of claim 19,

    The health functional food is candy, chocolate, beverages, gum, tea, vitamin complex or health supplement food, health functional food for immune regulation.
22. Immuno-enhancing the extracellular polysaccharide produced by Ceriporia lacerata , the mycelium culture solution of Ceriporia laccerata containing the extracellular polysaccharide, the dry powder of the mycelium culture solution, or the extract of the mycelium culture solution Or administering to a subject in need thereof.
23. Extracellular polysaccharide produced by Ceriporia lacerata for use in the manufacture of a medicament for immunomodulation, mycelium culture medium of Ceriporia laccerata comprising the extracellular polysaccharide, the mycelium culture medium Use of dry powder or extract of the mycelium culture.

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