KR101039110B1 - A water composition comprising Broussonetia kazinoki Sieb. for immunopotentiating - Google Patents

Abstract

The present invention relates to a composition for enhancing immune function containing the water extract of the mulberry, specifically, the mulberry water extract of the present invention promotes the growth of splenocytes and macrophages of mice and promotes the production of NO, the mulberry water of the present invention It was confirmed that the extract has an immune activation and augmentation effect, the water extract of the mulberry of the present invention can be usefully used as a composition for enhancing immune function, health food for enhancing immune function, and a therapeutic agent for diseases caused by reduced immune function.

Paper mulberry, enhance immune function.

Description

A water composition comprising Broussonetia kazinoki Sieb. for immunopotentiating}

The present invention relates to a composition for enhancing immune function containing the water extract of mulberry, a health food for enhancing the immune function containing the water extract of mulberry and a therapeutic agent for a disease due to a decrease in immune function containing the water extract of mulberry.

Paper mulberry ( Broussonetia) kazinoki ) is called kanji in Chinese. Scientific name is Broussonetia kazinoki SIEB., a deciduous broad-leaved shrub of the Moraceae family, found on both sides of the mountain, with small hairs growing on the branches, but disappearing as they grow, the bark is grayish brown, about 3 m high. The leaves are ovate or oblong in shape, with sharp teeth on the edges, and are torn in the shape of the insect as eaten. Flowers are reddish purple and discharged in the form of male to female in May-June. Fruits are nucleus spherical and mature in September. The mulberry is collected from the oriental medicine in the summer and autumn together with young branches and leaves. Leaves are regenerated, egg-shaped or egg-shaped oval, long apex, round or subcardiac, 5-20 cm long, with sharp ends, sometimes deeply split, with sharp teeth on the edges. Young trees have 2 to 3 missing lobes. The surface is rough and the back is initially hairy. The petiole is 1-2cm long, with curly hairs but gradually disappears (National Species Information System, http://www.nature.go.kr/plant/plantGuide/). The old mulberry tree was once used to weave a kind of skin called a follicle and used as a support fee. To process the paper mulberry as a support material, first cut the harvested stems into 1 to 2 m lengths and steam them in a closed pot. After two hours of steaming, it smells like earth and gives off a sweet scent. Dried as it is called black skin or black skin (粗 皮), and can be processed again to get white skin (白皮). It is commonly used as a refining fee, but the black skin scraped from the manufacture of white skin is also used as a raw material for the lower grade (造林 技術 (金 容 寬, 英 崙, 1962), 特 用 林 産物 栽培 and 利用 (尹國炳, 英 崙). 1963), Specialty Water Cultivation (Lim Kyung-bin, Hyangmun-sa, 1976), Agricultural Products Handbook (National Association of Agricultural Cooperatives, 1983), 韓國 樹木 圖鑑 (林業 試驗場, 1966) Federation, 1973). Its pharmacological action is known to be effective against roughness, diuresis, blood loss, rheumatism, pain, edema, bruises and dermatitis.The chemical composition of Kazinol A, Kazinol B, Kazinol D, Kazinol E, Kazinol F, Kazinol K, Kazinol H, Kazinol Q, Kazinol R, broussonetine W, broussonetine X, broussonetine M1, broussonetine U1, broussonetine J3, broussonetine J2, broussonetine R, broussonetine S, broussonetine T, broussoneone O, broussonetine O, brousus It is known to contain broussonol B, broussonol C, broussonol D, and broussonol E, broussonetinine A, broussonetinine B, 7,4'-dihydroxyflavan, and oleanolic acid.

Representative methods of verifying immune activation are macrophage proliferation effects (Friedl R, Moeslinger T, Kopp B, and Spiecke-rmann PG.Stimulation of nitric oxide synthesis by the aqueous extract of Panax ginsena root in Raw 264.7 cells.British Journal of pharma -cology, 2001; 134: 1663-1670.) and the proliferative effects of splenocytes were selected. NO is a marker of macrophage activation. NO produced in macrophages acts to eliminate invading pathogens and activate basic host defenses (Farrell AJ, Blake DR. Nitric oxide. Annals of the Rheumatic Dieases. 1996; 55: 7-20.). Particularly, when Thl and Th2 are divided in the ThO state during the differentiation of Th cells, it is known to have a function of helping to differentiate into Thl. In addition, cytotoxic activity of killing cancer cells, parasites or bacteria among leukocyte activity has been reported to be released by the release of NO (Nathan C. Nitric oxide as a secretory product of mammalian cells.FASEB Journal. 1992; 6: 3051-3064) .). In addition, NO induced immunomodulating between T-cell and endothelial cells (Bingisser RM, Holt PG Immunomodulating mechanism in the lower respiratory tract; nitric oxide mediated intraction between alveolar macrophage, epithelial cell, and T-cell, SWISS Medical Weekly. 2001; 131; 171-179.) It is known that there is a potent platelet aggregation inhibitory effect.

Accordingly, the present inventors completed the present invention by confirming that the water extract of mulberry promotes the proliferation of splenocytes and macrophages, and induces the production of NO.

An object of the present invention is a composition for enhancing immune function containing the water extract of the mulberry as an active ingredient, health foods for enhancing the immune function containing the water extract of mulberry as an active ingredient and a therapeutic agent for diseases caused by a decrease in immune function using the same as an active ingredient To provide.

The present invention provides a composition for enhancing immune function, which contains the extract of the mulberry extract as water as an active ingredient.

In addition, the present invention provides a health food for enhancing immune function containing the composition for enhancing immune function as an active ingredient.

In addition, the present invention provides a method for producing the composition for enhancing immune function.

In addition, the present invention provides a therapeutic agent for diseases caused by a decrease in immune function containing the composition for enhancing immune function as an active ingredient.

In addition, the present invention provides a method for the treatment of diseases caused by impaired immune function.

Since the water extract of the mulberry of the present invention increases the growth of splenocytes and macrophages in mice, and increases the production of NO, it is effective for strengthening immune function, and thus, composition for enhancing immune function, health food for enhancing immune function, and lowering immune function. It is useful to use as a therapeutic agent for diseases caused by.

Hereinafter, the present invention will be described.

The present invention provides a composition for enhancing immune function, which contains the extract of the mulberry extract as water as an active ingredient.

In addition, the present invention provides a health food for enhancing immune function containing the composition for enhancing immune function as an active ingredient.

In addition, the present invention provides a therapeutic agent for diseases caused by a decrease in immune function containing the composition for enhancing immune function as an active ingredient.

In addition, the present invention provides a method for the treatment of diseases caused by impaired immune function.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for enhancing immune function, which contains the extract of the mulberry extract as water as an active ingredient.

It is preferable to obtain the above-mentioned stems and use about 1/5 to 1 times, preferably about 1/3 times, water as the extraction solvent. The extraction is also performed at room temperature for about 1 to 24 hours, preferably 4 to 15 hours, most preferably 2 to 3 hours. The extraction method is preferably hot water extraction, cold extraction, reflux cooling extraction, ultrasonic extraction and steam extraction, more preferably by performing the lyophilization after steam extraction can be obtained the water extract of the mulberry tree of the present invention.

The present inventors investigated the effect of the water extract of the mulberry extract as described above on the growth of spleen cells, macrophages of mice. As a result, it induces the proliferation of splenocytes (see Fig. 1 and Table 1), showed an excellent proliferation effect also in macrophages, it was confirmed to be superior to the positive control (see Fig. 2 and Table 2).

The inventors then investigated the effect of the water extract of the mulberry on NO production. As a result, it was confirmed that the production of NO also effectively effective in boosting immunity (see FIG. 3 and Table 3).

Therefore, the water extract of the mulberry of the present invention can be efficiently used in the composition for enhancing immune function.

The composition for enhancing immune function of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions. Carriers, excipients, and diluents that may be included in compositions containing wood vinegar 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. 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 the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose and gelatin. 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, solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, injections. 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.

In addition, the present invention provides a health food for enhancing immune function containing the composition for enhancing immune function as an active ingredient.

Examples of foods to which the water extract may be added include, for example, various foods, dairy products, beverages, gums, teas, vitamin monosaccharides, dietary supplements, etc. Can be.

It may also be added to foods or beverages for the purpose of enhancing immune function. At this time, the amount of the extract in the food or beverage can be added in 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 1 g based on 100 ml Can be.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the above-mentioned mulberry water extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. 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 said 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 mulberry water extract of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, such as flavoring agents, coloring and neutralizing agents (cheese, chocolate, etc.), 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, and the like. In addition, the mulberry water extract of the present invention may contain a flesh for preparing natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the mulberry of the present invention.

In addition, the present invention provides a therapeutic agent for diseases caused by a decrease in immune function containing the composition for enhancing immune function as an active ingredient.

The disease caused by the reduced immune function is preferably any one selected from the group consisting of infectious diseases such as cold and inflammatory diseases, allergic diseases such as atopic dermatitis, chronic fatigue, cancer and the like, but is not limited thereto. All diseases resulting from a known decrease in immune function are included in the present invention. Preferred dosages of the water extract of Methanol 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 water extract of the mulberry of the present invention is preferably administered at 0.0001 to 500 mg / kg, preferably at 0.001 to 500 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.

In addition, the present invention provides a method for the treatment of diseases caused by impaired immune function.

The method of treatment comprises the following steps: administering the water extract of the mulberry of the present invention to the subject as an active ingredient.

In the above method, the mulberry water extract of the present invention may be administered by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Formulation Examples.

However, the following Examples, Experimental Examples, and Formulation Examples specifically illustrate the present invention, and the content of the present invention is not limited to Examples, Experimental Examples, and Formulation Examples.

Example 1 Broussonetia kazinoki  Sieb.) Water Extract Manufacturer

The mulberry steam extract was collected from Cheonyang Paper Co., Ltd. during the manufacturing process of Hanji and used by lyophilization at Jeonju Biomaterials Research Institute. Cut 1 ~ 1.5 m long stems, trim them, and tie them together in small steps of about 15-20 kg. Fill a 2 m diameter, 1 m deep steel cylinder with 1/3 of water, and then cut them up. About 450-600 kg was put up and fixed firmly to move the circumference. The outside was covered with vinyl to prevent steam from escaping and heated for 2-3 hours by steam supply. Sufficiently steamed mulberry was removed, and the extract of the mulberry under the cylinder was recovered, concentrated under reduced pressure, and lyophilized to be used for the experiment. The yield of dried mulberry water extract was 1.3%.

Experimental Example 1 Investigation of Cell Proliferation Effect of Mouse Spleen Cells

Seven-week-old male C57BL / 6 mice (Korean Biolink, Chungbuk Negative) were extracted after the cervical spine bone. The extracted spleens were washed with HBSS (Hank's Balanced Salts Modified.Gbico BRL.), Centrifuged and separated to remove red blood cells with RBC lysing buffer (Sigma), followed by 10% FBS (fetal bovine serum, Gibco BRL. ) And penicillin (penicillin, Gibco BRL.) And streptomycin (streptomycin, Gibco BRL.) And suspended in RPMI 1640 (Hyclone) medium containing and cultured at 5% CO ₂ , 37 ℃. In order to detect the effect of the water extract of the mulberry prepared in Example 1 on the spleen cell proliferation, the splenocytes separated and cultured in a 96-well plate were divided into 5 × 10 ⁵ cells / well and the mulberry water extract was It was added by dissolving in PBS at a concentration of 1-100 μg / ml. As a comparison material for the cell proliferation effect, ConA (Concanavalin A, Sigma Co., USA) and LPS (lipopolysaccharide, Sigma Co., USA) were used as 2 μg / ml, respectively. After 48 hours of incubation, the solution was added with a solution of tetrazolium salt CCK-8 kit (Cell Counting Kit-8, Dojindo Laboratories, Tokyo, Japan) for 4 hours, followed by ELISA reader (Ceres UV 900C, Bio-tech instrument, USA). Absorbance was measured at 450 nm using. The ratio of the absorbance of drug addition wells to the absorbance of control wells was calculated in% and the average value of several wells was used. The calculation is as follows.

As a result, the proliferation effect of the splenocytes was increased concentration-dependently when treated with 1 ~ 100 μg / ml to the spleen cells isolated from the rat water extract (March) (Fig. 1). This resulted in an increase of 203.4 ± 5.1 and 224.9 ± 17.7% of the control group when 2 μg / ml of LPS and ConA were used as the positive control group. .

Mice of Methanol Water Extract Splenocyte  Investigation of cell proliferation effect Cell growth rate (%) Positive control group LPS 2 µg / ml 203.47 ± 5.06 2 μg / ml ConA 224.94 ± 17.69 Paper mulberry water extract of Example 1-1 1 μg / ml 133.18 ± 5.35 5 μg / ml 177.68 ± 6.17 10 μg / ml 185.66 ± 2.56 50 μg / ml 210.01 ± 2.66 100 μg / ml 202.41 ± 3.66

< Experimental Example  2> cell proliferation of mouse macrophage lines and NO ( nitric oxide Investigate the creation effect

<2-1> Investigation of Cell Proliferation in Mouse Macrophage Cell Line

Raw 264.7 cell line, a Murin macrophage cell line, was dispensed in 96-well plates at 5 × 10 ³ cells / well and treated with water extract at a concentration of 1-100 μg / ml. As a comparison material, ConA (Sigma Co., USA) and LPS (Sigma Co., USA) were used at 2 μg / ml, respectively. After 48 hours of incubation, add tetrazolium salt CCK-8 kit (Cell Counting Kit-8, Dojindo Laboratories, Tokyo, Japan) solution and incubate for 4 hours more, then use ELISA reader (Ceres UV 900C, Bio-tech instrument, USA). Absorbance at 450 nm was measured. The ratio of the absorbance of drug addition wells to the absorbance of control wells was calculated in% and the average value of several wells was used. The calculation is as follows.

As a result, when the mulberry water extract was treated with 1 ~ 100 μg / ml in the RAW 264.7 cell line, which is a mouse macrophage, the cell proliferation effect was higher than that of the LPS used as the positive control group. It was confirmed that it is effective for boosting immunity (FIG. 2).

Investigation of the Cell Proliferation Effect of Macrophage in Mice Cell growth rate (%) Positive control group LPS 2 µg / ml 123.14 ± 2.09 ConA 2µg / ml 63.80 ± 0.88 Paper mulberry water extract of Example 1-1 1 μg / ml 152.06 ± 5.24 5 μg / ml 163.88 ± 2.23 10 μg / ml 163.56 ± 1.49 50 μg / ml 155.80 ± 1.32 100 μg / ml 147.61 ± 1.51

<2-2> NO Production Effect of Mouse Macrophage Cell Line

Raw 264.7 cells were aliquoted into 2.5 × 10 ⁵ cells / well in 48-well plates, incubated in CO ₂ incubator for 24 hours, and treated with LPS by concentration as a positive control. In the drug treatment group, water extract was treated with 1-100 μg / ml, the supernatant was separated after 18 hours of incubation, and NO production was measured. NO production was reacted using Griess reagent (Promega, USA) and the absorbance was measured at 535 nm.

As a result, when the water extract of the mulberry tree was treated with 1 ~ 100 μg / ml in the RAW 264.7 cell line, which is a mouse macrophage, the NO production promoting effect was excellent. It was confirmed that the water extract of mulberry not only induces the proliferation of macrophages but also promotes the production of NO in the macrophages and is effective in enhancing immunity (FIG. 3).

Mice of Methanol Water Extract Splenocytes  undergarment NO  Generating Effect Survey NO production amount (μM) Control group (No sample group) - 0.80 ± 0.10 Positive control group LPS 0.4 μg / ml 38.93 ± 0.53 LPS 4 μg / ml 42.92 ± 1.71 Paper mulberry water extract of Example 1-1 1 μg / ml 0.60 ± 0.05 10 μg / ml 0.95 ± 0.05 100 μg / ml 24.43 ± 0.85

According to the above results, the water extract of the present invention showed the excellent effect on the cell proliferation of the mouse splenocytes, the proliferation of macrophages, the promotion of NO production of macrophages, the water extract of the present invention is the immune activation and immune function enhancement It can be used for.

Examples of formulations for the composition of the present invention are illustrated below.

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Preparation of powders

2 g of young water extract of the present invention

1 g lactose

After mixing the above components, the airtight cloth was filled to prepare a powder.

<1-2> Preparation of Tablet

100 mg of water extract according to the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<1-3> Preparation of Capsule

100mg of Methanol Water Extract of the Invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

<1-4> Preparation of Injection Solution

10 ㎍ / ml of the water extract of the mulberry according to the present invention

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

Dissolve the wood mushroom mycelium powder according to the present invention in an appropriate volume of sodium chloride BP for injection, adjust the pH of the resulting solution to pH 3.5 with dilute hydrochloric acid BP, and adjust the volume with sodium chloride BP for injection and sufficiently Mixed. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

< Formulation example  2> Manufacture of food

Foods comprising the water extract of the mulberry of the present invention according to the present invention were prepared as follows.

<2-1> Preparation of Flour Food

0.1 ~ 10.0 parts by weight of the water extract of the mulberry of the present invention was added to the flour, and using the mixture, bread, cake, cookies, crackers and noodles were prepared in a conventional manner to prepare foods for health promotion.

<2-2> Preparation of Soups and Gravys

0.1-1.0 parts by weight of the water extract of Methanol of the present invention was added to soups and gravy to prepare meat products for health promotion, soup of noodles and gravy in a conventional manner.

<2-3> Preparation of Ground Beef

10 parts by weight of the water extract of the mulberry of the present invention was added to the ground beef to prepare a ground beef for health promotion in a conventional manner.

<2-4> dairy products (dairy products )

0.1-1.0 parts by weight of the water extract of Methanol of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared in a conventional manner using the milk.

<2-5> Preparation of Wire

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The water extract of the present invention was concentrated under reduced pressure and concentrated in a vacuum concentrator, and dried by spraying and drying with a hot air dryer to grind the dried product to a particle size of 60 mesh using a grinder to obtain a dry powder.

The grains, seeds and the mycelium mycelium mycelium powder according to the present invention were prepared in the following ratio to prepare a conventional method.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Methanol water extract of the present invention (1 part by weight),

(0.5 part by weight),

Foxglove (0.5 part by weight)

< Formulation example  3> Manufacturing of beverage

Drinks containing the water extract of the mulberry of the present invention was prepared as follows.

<3-1> Preparation of Health Beverage

Instant sterilization was carried out by homogeneously mixing the ingredients such as liquid fructose (0.5%), oligosaccharides (2%), sugar (2%), salt (0.5%), water (75%) and the water extract of the mulberry according to the present invention. Then, it was packaged in a small packaging container such as glass bottles, plastic bottles to prepare a healthy beverage.

<3-2> Preparation of Vegetable Juice

0.5 g of the water extract of the present invention was added to 1,000 ml of vegetable juices such as tomatoes or carrots to prepare vegetable juice for health promotion in a conventional manner.

<3-3> Preparation of Fruit Juice

0.1 g of the water extract of the mulberry of the present invention was added to 1,000 ml of fruit juices such as apples or grapes to prepare fruit juice for health promotion in a conventional manner.

1 is a graph showing the proliferative effect of splenocytes according to the treatment of the water extract of the present invention in the mouse splenocytes.

Figure 2 is a graph showing the proliferation effect of the macrophage cell line according to the treatment of the water extract of the present invention in the mouse macrophage line.

Figure 3 is a graph showing the NO production inducing effect according to the treatment of the water extract of the present invention in the mouse macrophage line.

Claims (5)

delete delete Health food for strengthening immune function, containing medicinal extracts of water extracts from water. delete delete

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