KR100757220B1 - Composition comprising the extract of salicis radicis cortex for immune activity - Google Patents

Abstract

A composition for enhancing immune activity comprising the extract of Salicis radicis cortex is provided to increase production of NO2 which inhibit growth of cancer cells, expression of cytokine related to T-cell and expression of CD14 without toxicity to human. A composition for enhancing immune activity comprises 0.1-50 wt.% of the extract of Salicis radicis cortex which is prepared by extracting with a polar solvent selected from water, C1-C4 lower alcohol or a mixed solvent thereof, wherein the daily dosage of Salicis radicis cortex extract is 0.0001-100 mg/kg, preferably 0.001-10 mg/kg and the composition is a pharmaceutical or health food composition having a formulation selected from tablet, capsule, pill and solution. The food or drink for enhancing immune activity comprises 0.01-50 wt.% of the health food composition.

Description

Composition comprising the extract of Salicis radicis cortex for immune activity}

1 is a diagram showing the amount of NO produced by the root extract

Figure 2 is a diagram showing the amount of TNF-α expression by the administration of the root extract.

Figure 3 is a diagram showing the expression of the cell surface molecules of macrophages by administration of the myopic extract

Figure 4 is a diagram showing the cytokine expression by the administration of the root extract.

The present invention relates to a composition for enhancing immune activity containing an extract of Salicis Radicis Cortex.

In bacterial, viral infections or inflammatory reactions, regulation of macrophage and lymphocyte activity plays a central role in the determination of the therapeutic effect of a medicinal product. The production of reactive oxygen species such as superoxide anion (O ₂ −), hydrogen peroxide (H ₂ O ₂ ) and nitric oxide (NO) by activated macrophages is nonspecific It is an important cytotoxic and cytostatic mechanism of immunity. Many studies have been conducted on which natural compounds affect the production of ROS and NO by macrophages. Macrophages are antigen-presenting, tumor-tumoring, and microbicidal cells that play a central role in cell-mediated or humoral immunity. As a regulatory cell, NO produced in activated macrophages exhibits macrophages, which are nonspecific host defense mechanisms, and proliferation inhibitory activity of bacteria and cancer cells.

In addition, hematopoietic stem cells are damaged during the hematopoietic process during chemotherapy, such as radiation therapy or chemotherapy, thereby reducing successively related hematopoietic cells and immune cells, which often causes disorders in the formation of hematopoietic and immune action. As a result, patients often experience anemia and lymphocytosis, thrombocytopenia or granulocytopenia, which can lead to serious and fatal infections and increase mortality in patients. Clinically, bone marrow growth factors such as G-CSF, GM-CSF, IL-1-12, M-CSF and EPO are used after chemotherapy or bone marrow transplantation. Promotes cell differentiation and production from BFU, increases cell migration, phagocytosis, superoxide production, and cytotoxicity of leukocytes following antibody stimulation.

The roots of Salicis Radicis Cortex, a deciduous tree of the Salicaceae, are about 20m high and about 80cm in diameter. The bark is black brown, shallowly split, and the small branches are yellowish green and beneath the hair, but hairs disappear. Leaves are alternate, basalate or long oval, 5-12cm long, 7-20cm butterfly. The tip is pointed and sawtooth is curved in at the edge. Petiole is 2-10mm long, without hairs or slightly fumbling. There are about 300 varieties of flowering, female and female, and fruiting in May. There are about 300 varieties, mainly distributed in the northern hemisphere, in one and the southern hemisphere.

The drug's temper is high, cold, and acts on the liver, kidney, and bladder. It has the effect of clearing, removing moisture, cutting wind and cutting swelling, so that cystitis, jaundice inflammatory eye disease, and customary arthritis It has been used to treat urine, eczema, toothache and diuretic, antipyretic, diuretic, lung abscess, stomatitis and gingivitis. Aspirin is also derived from willow roots.

Chemical components include salicylic acid, tutin, naringenin-7-glucoside, naringenin-5-glucoside, and quercitrin. It is known that it contains quercetrin, but pharmacological action has been reported to have central repression and coercive action (Balbaa SI et al., US Dispensatory , 25 Ed , p98, 1983; et al., Chinese Herbal Medicine , 15 (12) , p540, 1984), the effect on the immune enhancing effect of the root bark of the willow root bark has not been identified.

Therefore, the inventors of the present invention, while identifying a substance having an immune enhancing effect from natural drug resources, NO ₂ inhibiting the proliferation of cancer cells of the extract of the root of the present invention The present invention was completed by confirming the effect of increasing production, increasing the expression of cytokines associated with T-cells and increasing the expression of CD14.

It is an object of the present invention to provide a composition for enhancing immunity comprising a myodermal extract having no toxicity to the living body and exhibiting an immune enhancing effect.

In order to achieve the above object, the present invention is not toxic to the living body and exhibits immune increasing activity ( Salicis) Radicis cortex) provides an immune enhancing composition comprising the extract as an active ingredient.

The extract includes a polar solvent selected from water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably an extract available in water.

The root extract of the present invention may be prepared by the following manufacturing process.

Dried root skin may be prepared according to a conventional extraction method, for example, 1 to 30 times the dry weight of dried root skin, preferably 7 to 13 times the volume of C ₁ such as methanol, ethanol, butanol To C ₄ lower alcohols or mixed solvents having a mixing ratio of about 1: 0.1 to 1:10, preferably 1 to 10 times, preferably 2 to 5 hours, preferably with water added thereto, preferably 0.5 to 10 hours. Is repeated 2 to 5 times by cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction and the like, preferably hot water extraction, the extract is filtered under reduced pressure, and then the filtrate is concentrated and freeze-dried Root skin extract of the invention can be obtained.

The present invention provides an immune enhancing pharmaceutical composition containing the extract of Root root skin obtained by the above method as an active ingredient.

Immune enhancing pharmaceutical composition of the present invention, the extract comprises 0.1 to 50% by weight based on the total weight of the composition.

The pharmaceutical composition of the present invention is used for the prevention and treatment of diseases caused by a decrease in immune function by chemotherapy and radiation therapy, or a disease caused by immunosuppression after bone marrow transplantation, AIDS due to immune system damage and a decrease in immune function. Can be used.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents which may be formulated and used 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 rose, 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 in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts 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 preferred effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. Administration may be once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

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

The present invention provides a health functional food for immuno-enhancing comprising the above-mentioned roots extract as an active ingredient exhibiting an immune enhancing effect.

Examples of the food to which the extract of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing immunity lowering. The amount of the extract in the food or beverage is generally 0.01 to 50% by weight of the total food weight, preferably It may be added at 0.01 to 15% by weight, and the health beverage composition may be added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml.

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

The health beverage composition of the present invention is not particularly limited in the other components except the above-mentioned 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 are monosaccharides such as disaccharides such as glucose and fructose, for example polysaccharides such as maltose and sucrose, and conventional sugars such as dextrin, cyclodextrin and the like. And sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

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

Below, The invention is illustrated in detail by the following examples and experimental examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, the contents of the present invention is not limited by the following Examples, Reference Examples and Experimental Examples.

Example  One. Root skin  Preparation of Extract

The dried root of natural roots was finely chopped, finely dried and dried by 3 kg of dried root roots, 100% of water and 120 liters of heat extraction were performed three times, followed by filtration and concentration under reduced pressure, followed by freeze drying to obtain 635.5 g of roots of root extract.

Experimental Example  One. Rooted NO ₂  Secretion effect and cytotoxicity confirmation

RAW 264.7 cells were dispensed in 96-well microplates at 4 × 10 ⁵ per well, and then treated with 50 μg / ml or 100 μg / ml of the root extract, followed by incubation at 37 ° C. and 5% CO ₂ for 24 hours. Subsequently, the supernatant was taken, and the concentration of nitrite ions (NO ₂ −) was determined using a Gries's reagent by measuring the degree of color development based on NaNO ₂ .

In summary, the same amount of Griess reagent (0.1% (w / v) N- (1-naphthyl) ethylenediamine dihydrochloride + 1% (w / v) sulfanilamide in 5% (v) in 100 μl of cell culture supernatant / v) phosphoric acid) was added and mixed and left at room temperature for 20 minutes, after which the absorbance was measured at 540 nm using Ultrospec 4000 spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). Shown in

In addition, MTS assay was performed under the same conditions to examine cytotoxicity. In addition, the experiment was performed to isolate the primary peritoneal macrophage (primary peritoneal macrophage) in order to determine the effect of the extract from the roots of the roots of NO ₂ and the ability to secrete TNF-α as an inflammatory mediator, As shown, inflammatory mediators were increased in both RAW 264.7 cells and primary macrophages.

Experimental Example  2. Effects on Cell Surface Molecule Expression in Mouse Macrophages

Mouse primary macrophages were isolated, 1 × 10 ⁶ cells were treated with 100 μg / ml of myofibril extract for 24 hours, and then blocked with 1% BSA, followed by FACS analysis with CD86 and CD80 cell surface antibodies. The amount of each antibody was treated with 0.25 μg / 1 × 10 ⁶ and then compared with the positive control treated with LPS 10 μg / ml to analyze the results. As a result, as shown in FIG. 3, the expression of CD86, a B7 molecule expressed in antigen presentation cells, was increased in comparison with the negative control group (arch and clove), and LPS and LBP complex (complex). Expression of CD14, a co-receptor, is significantly increased.

Experimental Example  3. Interferon-gamma in mouse serum ( IFN -γ) production effect

After 7 days, mice were intraperitoneally injected with 4 mg / 500 μl of water extract, and after 72 hours, blood from the mice was collected from the heart to separate serum. And the cytokine secretion in the serum was confirmed using a CBA kit (cytokine assay kit, BD Science).

As shown in FIG. 4, the expression of cytokines such as TNF-α, IFN-γ, IL-2, IL-4, and IL-5 increased in serum when intraperitoneally infused with myofibril extract.

Experimental Example  4. Acute Toxicity  exam

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats. Two animals of each group were orally administered with the root extract of the present invention at a dose of 100 mg / kg. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. As a result, the extract of the present invention did not show a toxicity change even in rats up to 100 mg / kg, respectively, the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of more than 100 mg / kg.

One example of the formulation of the pharmaceutical composition comprising the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail.

Formulation example  One. Powder  Produce

20 mg of root extract

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

Radical Skin Extract 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation example  3. Manufacture of capsule

Radical Skin Extract 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2mg

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

Formulation example  4. Preparation of Injectables

Radical Skin Extract 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

_{Na 2 HPO 4 · 12H 2 O} 26 ㎎

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

20 mg of root extract

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of healthy food

Root Skin Extract 1000mg

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

50 μg folic acid

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  7. Manufacture of health drinks

Euphorbia Extract 100mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B ₁

0.3 g of vitamin B ₂

Water quantification

After mixing the above components in accordance with the conventional healthy beverage manufacturing 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 sterilization and refrigerated Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

Extract of Salicis Radicis Cortex of the present invention is NO ₂ to inhibit the proliferation of cancer cells As a result of increased production and increased cytokine expression associated with T-cells, it can be used as a medicine and health functional food for enhancing immunity to patients with reduced immune system and damaged immune system.

Claims (5)

Immunity enhancing pharmaceutical composition comprising Salicis Radicis Cortex water extract. delete delete Immuno-enhancing health functional foods comprising water extract of Yu-geun-huh, which shows an immune enhancing effect. delete

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