KR20050036117A - Composition comprising the extract of african ganoderma mushroom for the treatment and protection of immune disease - Google Patents

Abstract

The present invention relates to a composition containing an extract of Ganoderma genus mushroom having an immunological activity, the African Ganoderma genus extract of the present invention has an immuno-enhancing effect, so as to medicines for the treatment and prevention of immune diseases or It is useful for health functional food.

Description

 Composition comprising the extract of African Ganoderma mushroom for the treatment and protection of immune disease}

The present invention relates to a pharmaceutical composition for the treatment and prevention of immune diseases containing African Ganoderma lucidum extract having immunopotentiating efficacy.

Colds are the most frequently treated diseases in primary health care. About a third to a quarter of patients who visit general hospitals are acute upper respiratory tract infections called colds. Pediatric airway infections are particularly serious and can be life threatening. In children under five, airway infections are known to be the most important cause of death from lung disease. Infectious diseases such as these play an important role in immune function. Therefore, boosting immunity can be a good way to prevent and treat colds.

The immune system can be divided into natural resistance, nonspecific immune system and specific immune system. Natural resistance (primary defense) refers to anatomical physiological elements that block all invaders, including microorganisms, regardless of their type. Nonspecific immunity (secondary defense) is a phagocyte that breaks through natural resistance and removes invaders. The constructed defense system, and the specific immune system (tertiary line of defense) refer to the immune system composed of lymphocytes, which are highly developed immune systems with memory and the ability to distinguish between self and nonmagnetic.

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. Macrophages contain many lysosomes, which contain acidic hydrolase and peroxidase. In addition, it strongly adheres to the glass surface and plastic surface, and actively detects microorganisms and tumor cells. This cell has cytokine receptors such as IFN-γ. They produce cytokines such as complement, interferon, interleukin-1, and tumor necrosis factor and can be enhanced by several cytokines produced from T-cells. T-cells, also a type of white blood cell, make up about 70% of blood lymphocytes. T-cells differentiate in the thymus and have a T-cell antigen receptor (TCR). Peripheral blood T-cells are divided into CD4 positive TH cells and CD8 positive TC cells. CD4 + TH cells are activated by recognizing antigens bound to MHC class II molecules. They help B cells to enable antibody production or to help other T cells function. CD4 + TH cells can again be classified into TH1 and TH2 based on the cytokines they produce. TH1 cells of mice secrete interleukin-2 (IL-2), interferon-gamma (IFN-γ), etc., whereas TH2 cells produce IL-4, IL-5, IL-6, IL-9 Secrete IL-10, IL-13, and the like. However, in humans, the production of IL-2, IL-6, IL-10 and IL-13 is not strictly distinguished. TH1 cells are involved in cellular immune responses and activate cytotoxic and inflammatory responses. Cytokines produced in TH2 cells promote antibody formation, particularly the production of IgE and enhance the proliferation and function of eosinophils. Thus, TH2 cytokines are commonly found in antibody production and allergic reactions. It has been shown that TH1 and TH2 cytokines have mutually inhibitory functions and can change the course of the disease with an anti-IL-4 antibody and an anti-IFN-γ antibody in infectious diseases in animals, and IFN-γ in patients with rheumatoid arthritis In some cases, improvement of symptoms was observed by injection.

African Reishi mushroom (Ganoderma genus) of the present invention is a mushroom growing to parasitic bulrochogwa (Ganodermataceae), it belongs to the genus bulrocho (Ganoderma), such as broad-leaved trees growing wild in the wild state in alpine jungle of Kenya. Although the ecological feature of this area is tropical near the equator, it is an alpine area of 5000m above sea level and has the optimal environment for mushroom growth. It grows on dead wood and stump of hardwood hardwood, and grows at right angle to stump. The shape is round in shape of a horseshoe. The surface is smooth and has the shape of a kidney, semicircle. The front is yellowish white at first, but grows from reddish brown to reddish brown. The back side is yellowish white and has a lot of bureaucracy. The stem is slightly curved with the same color as the surface of the lampshade. It is called an artist conk because light spots show brown spots on the surface of white pores (see references).

It is known to be effective in preventing memory loss, inhibiting aging, physiologically active cell activity, anti-inflammatory, antibacterial action, blood pressure strengthening action, anti allergy, antihistamine, anti-male hormone action. In addition, it has anti-cancer effects, and the extract has also been proved by clinical trials to have excellent therapeutic and preventive effects on liver cancer, lung cancer and many other cancers.

In recent years, the market for alternative medicine for the prevention and treatment of colds is growing rapidly. In the United States, about one-third of the population relies on alternative medicines, not vaccines and chemicals. . In light of this trend, there is an urgent need for the development of a medicine or a food composition having an effect of increasing immune function as an alternative medicine such as herbal medicine.

Accordingly, the present inventors have completed the present invention by confirming the immune enhancing effect of the African Ganoderma lucidum extract, as a result of performing the immunological activity test of the African Ganoderma lucidum extract.

An object of the present invention to provide a pharmaceutical and dietary supplement as a composition for the prevention and treatment of immune diseases containing African Ganoderma lucidum extract.

In order to achieve the above object, the present invention provides a composition for the prevention and treatment of immune diseases containing African Ganoderma genus extract.

The ganoderma lucidum mushroom includes ganoderma lucidum native to Southeast Africa, preferably Kenya, such as Africa Kenya, Uganda and Tanzania.

The Ganoderma lucidum extract includes a crude extract and a non-polar solvent soluble extract, and the crude extract of the present invention comprises a lower alcohol such as water, ethanol, methanol, butanol, or a mixed solvent thereof, preferably an extract available in water or methanol. By nonpolar solvent soluble extract means an extract soluble in a nonpolar solvent such as hexane, dichloromethane or ethyl acetate, preferably hexane or ethyl acetate.

In addition, the African Ganoderma lucidum extract includes an African Ganoderma lucidum hydrothermal extract having a polymer polysaccharide (molecular weight 12,000 or more) fraction and an African Ganoderma lucidum mushroom extract having a macromolecular protein (molecular weight 12,000 or more) fraction.

Hereinafter, the present invention will be described in detail.

The ganoderma lucidum extract of the present invention, a nonpolar solvent soluble extract, a hydrothermal extract having a polymer polysaccharide fraction, and a cold extract having a polymer protein fraction can be obtained as follows.

After collecting the Ganoderma lucidum mushroom of the present invention, washed with water and dried, pulverized using a homogenizer and the like, and then powdered, Ganoderma lucidum mushroom has a volume of about 2 to 30 times the dry weight, preferably about 15 to 30 times Lower alcohols such as water, methanol, ethanol and butanol or mixed solvents having a mixing ratio of about 1: 0.1 to 1:10, preferably with an extraction temperature of 20 to 100 캜, preferably 50 to 70 캜 with methanol Extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for about 0.5 hours to 2 days, preferably 1 hour to 1 day, preferably 1 to 5 times, preferably 3 Continuous extraction and filtration under reduced pressure, and the filtrate was concentrated under reduced pressure at 20 to 100 ℃, preferably 20 to 70 ℃ with a vacuum rotary concentrator to water, lower alcohol or a mixed solvent thereof, preferably It can be obtained as a crude extract of Ganoderma lucidum extract available in coming.

The non-polar solvent soluble extract of the present invention, the suspension of the Ganoderma lucidum crude extract in water, and then extracted using a non-polar solvent such as hexane, ethyl acetate, dichloromethane, chloroform to obtain the Ganoderma lucidum non-polar solvent soluble extract of the present invention. Can be. More specifically, in the Ganoderma lucidum crude extract, preferably Ganoderma lucidum methanol extract, add about 2 to 30 times the dry weight of Ganoderma lucidum mushroom, preferably about 15 to 30 times the volume of water, and then stir well to dissolve it. After mixing a certain amount of hexane or ethyl acetate, and repeated three to four times, fractionation can be obtained to obtain a non-polar solvent soluble extract such as each hexane soluble extract and ethyl acetate soluble extract.

In addition, the African Ganoderma lucidum mushroom hydrothermal extract is washed with water and dried African Ganoderma lucidum with water, and then pulverized dried Ganoderma lucidum using a homogenizer, and then about 2 to 30 times the dry weight of Ganoderma lucidum mushroom, preferably Is about 3 to 10 times the volume of water and hot water extraction for about 1 to 24 hours, preferably 6 hours using a heating mantle, and then about 1 to 5 times, preferably in the hydrothermal extract obtained by filtration Preferably, three times lower alcohols, preferably cold ethanol, are added to precipitate for about 12 to 48 hours, preferably 24 hours at about 0 to 10 DEG C, preferably 4 DEG C, and then the supernatant is After removing and centrifuging to collect the precipitate to evaporate the solvent, the process of centrifugation by re-dissolving the pellet (pellet) with distilled water at about 50 to 100 ℃, preferably 60 ℃ The supernatant obtained by repeating five times was dialyzed with distilled water at about 0 to 10 ℃, preferably 4 ℃, for about 12 to 72 hours, preferably 48 hours, and then lyophilized to obtain a polymer polysaccharide (molecular weight: 12,000 Above) African Ganoderma lucidum hydrothermal extract of the present invention having a fraction can be obtained.

In addition, the African Ganoderma lucidum mushroom cold sediment extract of the present invention, after collecting the African Ganoderma lucidum mushroom washed with water and dried, and then crushed the dried Ganoderma lucidum finely using a homogenizer, adding a buffer solution about 0 to 10 After cold extraction with standing at 12 DEG C., preferably at 4 DEG C for about 12 to 24 hours, the extract was filtered and added with a protein precipitant to a salt such as ethanol, acetone or ammonium sulfate, preferably ammonium sulfate, and saturated. After centrifugation, the supernatant was removed, and the precipitate obtained was redissolved with a buffer solution, and the redissolved extract was then buffered at about 0 to 10 ° C., preferably at 4 ° C., at about 50 to 100 ° C. African ganoderma lucidum having a high molecular weight protein (molecular weight of 12,000 or more) fraction by dialysis for a period of time, preferably 74 hours, Pop tall can be obtained naengchim extract.

The present inventors isolated mouse spleen cells from the immune activity of African Ganoderma lucidum mushrooms, and cultured in a medium to which the African Ganoderma lucidum extract obtained by the above method was added, and confirmed the excellent immune enhancing effect of African Ganoderma lucidum mushrooms. .

The present invention provides an extract from Africa Ganoderma lucidum effective for the prevention and treatment of immune diseases obtained by the method.

In addition, the present invention provides an effective composition for the prevention and treatment of immune diseases containing the African Ganoderma lucidum extract of the present invention as an active ingredient.

The composition for the prevention and treatment of immune diseases of the present invention comprises 0.1 to 80% by weight of the extract, preferably 1.0 to 50% by weight, based on the total weight of the composition.

Pharmaceutical dosage forms of the extracts 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 fractions, as well as in any suitable collection.

The composition comprising the African Ganoderma lucidum extract of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Compositions comprising extracts according to the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories or sterile injectable solutions according to conventional methods. Can be used. Specifically, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used when formulated. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate and sucrose in the extract. ) Or lactose, gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use 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. have. 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.

The amount of African Ganoderma lucidum extract may vary depending on the age, sex, and weight of the patient, but in general, the amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 to 200 mg / kg Can be administered in several divided doses. In addition, the dosage of the extract may be increased or decreased depending on the route of administration, the type and severity of the disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present 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.

African Ganoderma lucidum extract itself of the present invention has little toxicity and side effects, so can be used with confidence even for prolonged administration for prophylactic purposes.

The present invention provides a health functional food comprising a food supplement acceptable food additives in addition to the African Ganoderma lucidum extract exhibiting the effects of preventing and improving the immune diseases described above.

Compositions comprising the extracts of the present invention can be used in a variety of food and beverages for the prevention and improvement of immune diseases. Foods to which the African Ganoderma lucidum extract of the present invention can be added include various foods, for example, candy, chocolate, beverages, gums, teas, vitamin complexes, health functional foods, and the like, powders, granules, tablets, It can be used in the form of a capsule or a beverage.

At this time, the amount of the extract in the food or beverage, in the case of the health functional food composition of the present invention can generally be added to 0.01 to 50% by weight, preferably 0.1 to 20% by weight of the total food weight, In the case of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional beverage composition of the present invention is not particularly limited in the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrin and the like; Conventional sugars such as and sugar alcohols such as xylitol, sorbitol, erythritol. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

Other flavors may be advantageously used natural flavors (tautin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.). The composition of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof, Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, etc. Other compositions of the present invention may be used in natural fruit juices and fruit juice beverages and vegetable beverages. The components may be used independently or in combination The ratio of such additives is not critical but is zero per 100 parts by weight of the composition of the present invention. It is generally selected from the range of about 20 parts by weight.

The present invention is described in more detail based on the following examples and experimental examples, but the present invention is not limited to the examples or experimental examples.

Example 1 Preparation of Hot Water Extract of African Ganoderma Lucidum Mushroom

10 kg of African Ganoderma lucidum mushrooms (from Kenya, Africa) were collected, washed with water, dried (dried in shaded shades), and 20 g of dried Ganoderma lucidum mushrooms were crushed finely using a homogenizer. Then, 1 liter of water per 20 g of Ganoderma lucidum was added to a heating mantle, followed by hot water extraction at 100 ° C. for 6 hours, followed by three times with 0.8 L of hot water extract obtained by filtering with filter paper. Cold 95% ethanol was added and precipitated at 4 ° C. for 24 hours, then the supernatant was removed and centrifuged at 2800 rpm, 4 ° C. for 20 minutes to collect only the precipitate. After evaporation of ethanol, the supernatant obtained by re-dissolving the pellet (pellet) with distilled water at a high temperature of 60 ° C. and repeating three times was dialyzed using distilled water at 4 ° C. for 48 hours (dialysis membrane Mw). More than 12,000) After lyophilization, 1 g of African Ganoderma lucidum hydrothermal extract having a polymer polysaccharide (molecular weight of 12,000 or more) fraction was obtained.

Example 2 Preparation of African Ganoderma Lucidum Mushroom Extract

Ten kilograms of African Ganoderma lucidum mushrooms (collected from Kenya, Africa) were collected, washed with water, dried (dried in shaded shades), and then 50 g of dried Ganoderma lucidum was ground finely using a homogenizer. After adding 0.2 L of 50 mM Tris-HCl (pH 8.0) buffer solution per 20 g of Ganoderma lucidum mushroom, it was allowed to stand overnight at 4 ° C, followed by cold extraction. The extract was filtered and added with ammonium sulfate (Ammonium Sulfate). After saturation, the supernatant was removed by centrifugation (8000 g, 20 minutes), and the obtained precipitate was redissolved with 50 mM tris-hydrochloric acid (pH 8.0) and 50 mM sodium chloride buffer, and then the redissolved extract. Dialysis (at dialysis membrane Mw 12,000 or more) for 72 hours using 50 mM tris-hydrochloric acid (pH 8.0) and 50 mM sodium chloride buffer solution, and after quantitative dialysis, the amount of protein was measured. minute The yield with the African Ganoderma lucidum extract naengchim 15 ㎖ (concentration 300 ㎍ / ㎖).

Example 3. Preparation of Crude Mushroom Extracts from Africa

Take an African Ganoderma lucidum mushroom (collected from Kenya, Africa), wash it with water, dry it (dried in shaded shade), and 200 g of dried Ganoderma lucidum using a homogenizer, and then heat it. Into a mantle (heating mantle) and methanol 4 ℃, the extract obtained by extracting three times at regular intervals (12 h) at 79 ℃ repeatedly filtered and then filtered using a rotary vacuum evaporator (rotary vacuum evaporator) Concentration under reduced pressure gave 7 g of ganoderma lucidum methanol extract.

Example 4. Preparation of African Ganoderma lucidum hexane soluble fraction

The Ganoderma lucidum crude extract obtained in Example 3, that is, 6.5 g of Ganoderma lucidum methanol extract was dissolved in 300 ml tertiary distilled water and stirred for about 1 hour, and then mixed with 1.5 L of hexane and repeatedly fractionated three times to obtain a hexane-soluble fraction. Then, this hexane soluble fraction was filtered and concentrated under reduced pressure to obtain 0.5 g of Ganoderma lucidum hexane soluble extract, which was used as a sample while being stored in a 4 ° C refrigerator.

Example 5 Preparation of Ethyl Acetate Soluble Fraction of African Ganoderma Lucidum Mushroom

The Ganoderma lucidum crude extract obtained in Example 3, that is, 6.5 g of Ganoderma lucidum methanol extract was added to 300 ml of tertiary distilled water and stirred for 1 hour, and then dissolved by adding 1.5 L of ethyl acetate, followed by mixing three times, fractionating and ethyl acetate. After the soluble fraction was obtained, the ethyl acetate soluble fraction was concentrated under reduced pressure after filtration to obtain 2.5 g of Ganoderma lucidum ethyl acetate soluble extract, which was used as a sample, and used as a sample while being stored in a 4 ° C. refrigerator.

Experimental Example 1. Ganoderma lucidum immune activity test

When immune cells are activated, DNA synthesis is primarily increased, cell volume is increased, and secondly, division and proliferation function as effector cells. The primary increased cells are called lymphoblastoma (lymphoblast), the schizophrenic effect is called the lymphoblastogenic effect (lymphoblastogenic effect).

In this experiment, mouse spleen cells were isolated from the immune activity of mushrooms, cultured in medium containing mushroom extracts, and the volume of immune cells was measured using a flow cytometer. The mice were analyzed by XTT proliferation assay. The proliferative effect of splenic white blood cells was examined.

1-1. Mouse Spleen Leukocyte Suspension Preparation

Place a 100-stainless steel mesh in a petri dish with a diameter of 45 mm, add 5 ml of RPMI 1640 medium, transfer the spleen of BALB / c mice killed by cervical dislocation to the mesh, and chop them with surgical scissors. The tissues were ground by rubbing lightly into the bottom of a mm glass vessel. After pipetting up and down the cells and tissues through the net several times, washed with RPMI 1640 medium, left for 5 minutes in an ice bath (ice bath) and only the upper cell suspension was transferred to a new container. After centrifugation, 1 ml of 0.83% ammonium chloride was added to the cell precipitate and suspended, and red blood cells were hemolyzed by standing in a water bath at 37 ° C. for 3 minutes. To this was added 2X PBS, filled with 1X PBS to the maximum dose and washed twice, which was counted with a hemocytometer and diluted to 4X106 cells / ml to prepare a splenic leukocyte suspension.

1-2. XTT Proliferation Assay

The spleen leukocyte suspension was washed once with RPMI 1640 medium without phenol red, and then 50 µl was added to a 96 well culture plate, and different fractions (hot water extract, cold extract, ethyl acetate extract, Hexane extract, crude extract) was added 5 mg / ㎖ each of the incubator for 48 hours at 37 ℃, 5% CO ₂ incubator. N-methyl dibenzopyrane methyl sulfate (PMS) solution in which XTT was dissolved at 1 mg / ml in RPMI 1640 medium without phenol red and dissolved at 1.25 mM in PBS after incubation. 50 ml of each well was added and mixed immediately before use, followed by incubation for 8 hours in a 37 ??, 5% CO ₂ incubator. After incubation, the absorbance was measured at 490 nm with an ELISA reader, and the results are shown in Tables 1 to 3.

XTT Proliferation First Experimental Results drugs OD ₄₉₀ Lymphoblastic lymphoma effect (pharmaceutical OD ₄₉₀ / control OD ₄₉₀ ) x100 Remarks One Control (PBS) 0.168 100% 2 Hydrothermal extraction 0.208 124% 3 Cold extraction 0.442 264% 4 Ethyl acetate extract 0.182 109% 5 Hexane extract 0.169 101% 6 Crude extract 0.183 109%

2nd experiment result of XTT proliferation method drugs OD ₄₉₀ Lymphoblastic lymphoma effect (pharmaceutical OD ₄₉₀ / control OD ₄₉₀ ) x100 Remarks One Control (PBS) 0.187 100% 2 Hydrothermal extraction 0.223 120% 3 Cold extraction 0.428 229% 4 Ethyl acetate extract 0.232 124% 5 Hexane extract 0.193 103% 6 Crude extract 0.194 104%

3rd experiment result of XTT proliferation method drugs OD ₄₉₀ Lymphoblastic lymphoma effect (pharmaceutical OD ₄₉₀ / control OD ₄₉₀ ) x100 Remarks One Control (PBS) 0.704 100% 2 Hydrothermal extraction 0.984 135% 3 Cold extraction 2.796 397% 4 Ethyl acetate extract 0.760 108% 5 Hexane extract 0.746 106% 6 Crude extract 0.751 107%

1-3. Flow Cytometry of Spleen Leukocyte Suspensions

100 μl of a spleen leukocyte suspension prepared at ⁴ × 10 ⁶ cells / ml was dispensed for each well, and 100 μl of RPMI 1640 medium containing the drug was added (the final concentration of the drug was 5 mg / ml), followed by 37 ° C., 5% CO _2. 48 hours incubation in the incubator. After incubation, the cell suspension was transferred to a fluorescence-activated cell sorter (FACS) tube, centrifuged to remove the supernatant, and the cell precipitate was dissolved in 200 μl of PBS. Mouse CD4 and CD8-FITC single cell antibodies were added thereto, reacted in an ice bath for 30 minutes, washed twice with PBS, suspended in 500 μl of PBS, and flow cytometry was performed using a flow cytometer. As a result, splenic leukocyte cells showed a marked increase in cell volume in cold extracts, and the proportion of CD4 cells in hot water extracts, hexane extracts and crude extracts was increased, and the increase in CD8 cell ratios was observed in hot water extracts.

Experimental Example 2. Determination of Toxicity of African Ganoderma lucidum Extract

2-1. Cytotoxicity of African Ganoderma Lucidum Extract

African to measure the cytotoxicity of the acid Ganoderma lucidum extract, a sensitive mammalian origin in enterovirus cell line ^{RD (Rabdomyosaarcoma, 6 × 10 4} / cell) using the in the laboratory African Ganoderma each extract, that is, hot water Cytotoxicity of the extract, crude extract, hexane extract, ethyl acetate extract was measured by MTT assay (MTT assay), wherein the concentration of each extract was 1 mg / ㎖.

First, the cell monolayer (6 × 10 ⁴ / cell) was incubated overnight at 37 ° C., and then the African Ganoderma lucidum sample was diluted 10-fold, 100 μl of the diluted sample was added, and then cultured at 37 ° C. for 3 days. Then, 50 μl of MTT staining (MTT staining) was added, incubated at 37 ° C. for 2 hours, and then the medium was discarded and 150 μl of DMSO was added, stirred for 1 hour, and the absorbance was measured at 540 nm. And the OD value compared to the average of the cell control (PBS) of each extract was compared, and the average of the cell control is represented by a constant line, based on which the higher than this line is not cytotoxic, the lower than this line is determined to be cytotoxic It was.

As a result, as shown in Table 4 showing the cytotoxicity of the hydrothermal extract of African Ganoderma lucidum mushroom and Table 5 below showing the cytotoxicity of the crude extract, hexane extract, ethyl acetate extract, African Ganoderma lucidum No significant cytotoxicity of each extract was found (see Figures 5 and 6).

number Titer process Average O.D One Cell control 0 mg / ml 0.409 2 Dilution 10 mg / ml 0.0905 3 Sample 10 ^(-1) 1 mg / ml 0.205 4 Sample 10 ^(-2) 100 μg / ml 0.187 5 Sample 10 ^(-3) 10 μg / ml 0.168 6 Sample 10 ^(-4) 1 μg / ml 0.326 7 Sample 10 ^(-5) 100 ng / ml 0.337 8 Sample 10 ^(-6) 10 ng / ml 0.374 9 Sample 10 ^(-7) 1 ng / ml 0.382 10 Sample 10 ^(-8) 100 pg / ml 0.346 11 Sample 10 ^(-9) 10 pg / ml 0.336 12 Sample 10 ^(-10) 1 pg / ml 0.530

number Titer process Average O.D Crude extract Hexane extract Ethyl acetate extract One Cell control 0 mg / ml 0.630 0.630 0.630 2 Sample 10 ^(-1) 1 mg / ml 0.100 0.249 0.059 3 Sample 10 ^(-2) 100 μg / ml 0.700 0.770 0.953 4 Sample 10 ^(-3) 10 μg / ml 0.640 0.733 0.785 5 Sample 10 ^(-4) 1 μg / ml 0.821 0.703 0.748 6 Sample 10 ^(-5) 100 ng / ml 0.759 0.652 0.810 7 Sample 10 ^(-6) 10 ng / ml 0.810 0.714 0.846 8 Sample 10 ^(-7) 1 ng / ml 0.748 0.640 0.816 9 Sample 10 ^(-8) 100 pg / ml 0.679 0.645 0.671 10 Sample 10 ^(-9) 10 pg / ml 0.755 0.830 0.642 11 Sample 10 ^(-10) 1 pg / ml 0.615 0.713 0.834

2-2. Histotoxicity of African Ganoderma Lucidum Extract

In order to measure the histotoxicity of African Ganoderma lucidum mushroom extract, crude Ganoderma lucidum extract of the Ganoderma lucidum extract using normal mouse (Nude mouse: normal colon), which was found to be normal tissue by staining of tissues extracted during surgery In order to measure the tissue toxicity experiments of the hexane extract and the ethyl acetate extract, MTT assay was performed, and it was determined that the extract was toxic only when the inhibition rate was 30% or more in consideration of various errors. Inhibition rate was calculated by the following equation (1).

       Inhibition rate (%) = 100-((O.D of the extract / O.D of the control) × 100]

As a result, the histotoxicity of the crude Ganoderma lucidum extract in Table 6a and 7a, the histotoxicity of the hexane extract in Table 6b and 7b, the histotoxicity of the ethyl acetate extract in Table 6c and 7c. In this case, the time when the inhibition rate (IR) is 30% is indicated by a constant line, and it was determined that the extract having the rod above this line was toxic. As can be seen in Tables 6a to 6c (see FIGS. 7a to 7c), the respective extracts of African Ganoderma lucidum mushrooms were not toxic in normal tissues because they showed no inhibition of more than 30%.

number process Crude extract IR Survival rate One PBS   0 % 100% 2   1 mg -16% 116% 3 100 μg -18% 118% 4  10 μg  20%  80% 5   1 μg  0 % 100%

number process Hexane extract IR Survival rate One PBS   0 % 100% 2   1 mg  29%  71% 3 100 μg  25%  75% 4  10 μg  -3%  103% 5   1 μg   0 % 100%

number process Ethyl acetate extract IR Survival rate One PBS   0 % 100% 2   1 mg  3%  97% 3 100 μg  6%  94% 4  10 μg  15%  85% 5   1 μg   2 %  98%

REFERENCE EXAMPLE 1 Identification of African Ganoderma lucidum Mushroom

In order to identify the type of African Ganoderma lucidum mushroom of the present invention, DNA sequencing was analyzed by Micro ID Inc., and ITS rDNA sequencing was identified. In this case, first, the% similarity value is obtained, and the evolution is obtained by Kimuras two-parameter model (Kimura, MA, J. Mol. Evol., 16 , pp111-120, 1980). After calculating the evolutionary distance, the tree tree (Neighbor-joining method (Saitou, N. and Nei, M., Mol. Biol. Evol., 4 , pp406-425, 1987) phylogenetic tree). On the other hand, the tree's scale bar represents a substitution per site of 0.1 or 0.01, and the number next to each branch in the tree represents the bootstrap percentage. it means. For reference In the case of ITS nucleotide sequences in the genus ITS1 and ITS2 are frequently registered in a database independently, two nucleotide sequences of the same strain were downloaded and analyzed into a single nucleotide sequence. The 5.8S rDNA sequences present between ITS1 and ITS2 were excluded from the analysis.

As a result, the determined number of nucleotide sequences was 567 bp and the nucleotide sequences are shown in FIG. 8 and SEQ ID NO: 1. This strain is Ganoderma It was found to be a fungus belonging to the genus Ganoderma genus .

African Ganoderma lucidum extract of the present invention can be administered in the following formulations, the following formulation examples are merely to illustrate the present invention, thereby not limiting the contents of the present invention.

Formulation Example 1 Preparation of Injection

100 mg of African Ganoderma lucidum extract of Example 3

Sodium Metabisulfite 3.0 mg

Methylparaben 0.8 mg

Propylparaben 0.1 mg

Appropriate sterile distilled water for injection

The above ingredients are mixed and prepared in a conventional manner to have a final volume of 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

African Ganoderma lucidum extract of Example 3 200 mg

Lactose 100 mg

Starch 100 mg

Magnesium stearate proper amount

A tablet is prepared by mixing and tableting the above components according to a conventional tablet manufacturing method.

Formulation Example 3 Preparation of Capsule

100 mg of African Ganoderma lucidum extract of Example 3

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate appropriate amount

According to a conventional capsule preparation method, the above ingredients were mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Liquid

African Ganoderma lucidum extract of Example 3 1000 mg

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Purified water was added to adjust the total volume to 1000 ml. According to the conventional method for preparing a liquid, the above components were mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation Example 5 Preparation of Healthy Food

African Ganoderma lucidum extract of Example 3 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 6 Preparation of Healthy Drink

African Ganoderma lucidum extract of Example 3 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilized and stored in the refrigerator and then Used to prepare the healthy beverage composition of the invention.

Ganoderma lucidum extract of the present invention has immunopotentiating efficacy. In addition, since it can be safely administered for a long time because there are no side effects in vivo and does not cause drug resistance, it can be usefully used as a pharmaceutical composition for the treatment and prevention of immune diseases.

1 is a diagram showing the results of the third experiment of the proliferation method using XTT.

Figure 2 is a diagram showing the distribution of cell volume distribution by the fraction extract in the flow cytometry of the splenic leukocyte suspension,

3 is a diagram showing the results of flow cytometry using a mouse CD4-FITC single cell antibody,

4 is a diagram showing the results of flow cytometry using a mouse CD8-FITC single cell antibody,

5 is a diagram measuring the cytotoxicity of the hot water extract of African Ganoderma lucidum extract,

6 is a diagram measuring the cytotoxicity of each solvent extract of African Ganoderma lucidum mushroom,

Figure 7a to 7c is a measure of the histotoxicity of each solvent extract of the African Ganoderma lucidum mushroom, Figure 7a is a measure of the histotoxicity of the crude extract, Figure 7b is a measure of the histotoxicity of the hexane extract, 7c is a diagram measuring the histotoxicity of the ethyl acetate extract,

8 is a diagram showing the nucleotide sequence of the African Ganoderma lucidum.

<110> KIM, JIN-DONG LEE, JONG-SUNG <120> Composition comprising the extract of African Ganoderma mushroom for the treatment and protection of immune disease <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 567 <212> DNA <213> Ganoderma genus <400> 1 gaaggatcat tatcgaattt ttgaccgggt tgtagctggc cttccgaggc atgtgcacgc 60 cctgctcaat ccactctaca cctgtgcact tactgtgggt gacggatcgc aaagcgggct 120 tcttgtccgt tataaagcgc atctgtggcc tgcgtttatc acaaactctt tgaaagtact 180 agaatgtaat attgggatat aatagatcta tatacaactt tcagcaacgg atctcttggc 240 tctcgcatcg atgaagaacg cagcgaaatg cgataagtaa tgtgaattgc agaattcagt 300 gaatcatcga atctttgaac gcaccttgcg ctccttggta ttccgaggag catgcctgtt 360 tgagtgtcat gaaatcttca acttgcaacc tctttgcgga gtttgtaggc ttggacttgg 420 agggcttgtc ggcctttaac ggtcggctcc tcttaaatgc attagcttga ttccttgcgg 480 atcggctgtc ggtgtgataa aatgtctacg ccgtgaccgt gaagcgtttg gatgagcttc 540 caaccgtctt gcttcaaaga caacttt 567

Claims (10)

Composition for the prevention and treatment of immune diseases containing African Ganoderma genus extract. The composition of claim 1, wherein the ganoderma lucidum mushroom is a ganoderma lucidum extract native to Kenya, Africa. The composition of claim 1, wherein the Ganoderma lucidum is Ganoderma lucidum having the nucleotide sequence of SEQ ID NO: 1. The composition of claim 1, wherein the Ganoderma lucidum extract is a crude extract or a nonpolar solvent soluble extract. The composition of claim 4, wherein the crude extract is extracted with water or methanol. The composition of claim 4, wherein the nonpolar solvent soluble extract is extracted with hexane or ethyl acetate. The composition of claim 1, wherein the extract is Ganoderma lucidum hydrothermal extract having a polymer polysaccharide (molecular weight of 12,000 or more) fraction. The composition of claim 1, wherein the extract is ganoderma lucidum cold extract having a high molecular protein fraction (molecular weight of 12,000 or more). Health functional foods for the prevention and improvement of immune diseases, including food supplements, which are food acceptable in addition to African Ganoderma lucidum extract. The health functional food according to claim 9, which is a health beverage.