KR20110015040A - Animal feed additive and animal feed comprising the extract of puerariae radix for immune activity - Google Patents

Abstract

PURPOSE: A livestock feed additive containing a puerariae radix extract, and a feed using thereof are provided to secure the secretion function effect of TNF-a inside a macrophage Raw 264.7. CONSTITUTION: A livestock feed additive for improving the immunity contains a puerariae radix water extract as an active ingredient. A producing method of the puerariae radix water extract comprises the following steps: adding 10L of distilled water to 1kg of dried puerariae radix; heat-extracting the mixture for 3hours at100deg C, and repeating the process for 3times; and vacuum filtering, before compress-condensing and freeze-drying.

Description

Animal feed additive and animal feed comprising the extract of Puerariae Radix for immune activity

The present invention relates to a composition for enhancing immune activity containing a root extract.

[Reference 1] Richard A. Goldsby, et al., KUBY Immunology , 2000

2 Dawson ™, et al., Annu . Rev. Med , 47 , pp.219-227, 1996

Abul K. Abbas et al., Cellular and Molecular Immunology 6 th , pp. 298-301, 2007

4 Eisenberg et al., N. Eng. J. Med., 328 , pp.246-252

[Document 5] baegihwan, South Korea medicinal plants, gyohaksa, p.369, 2000

[Reference 6] Herbal Medicine School Reorganization Committee, Herbal Medicine, Dongmyeongsa , pp.100-102, 2006

[Reference 7] Korea Institute of Herbal Medicine, Herbology, Korean Pharmacy Society, pp.107-110, 1995

8 Nakai M., et al., Dig Dis Sci . , Sep ; 50 (9) , pp. 1669-1676, 2005

9 Vilcek J., et al., J. Biol . Chem . , 266 , p.7313, 1991

[Reference 10] Wang M., et al., Int Immunopharmacol , 4 , pp. 311-315. 2004

[11] Youn et al., Biochem Pharmacol , Jan 15; 75 (2) , pp. 494-502, 2008

12 Akira S. et al., Nat . Rev. Immunol , 4 (7) , pp. 499-511, 2004

[Document 13] Jong Man Kim et al., Journal of Agricultural Science Papers, 39 , pp.1-7, 1997

[Document 14] Toxicological Standards of the Korea Food and Drug Administration and Drugs, Korea Food and Drug Administration Notice 2005-60, 2005

The immune system can be divided into natural resistance, nonspecific immune system and specific immune system. Natural resistance (primary line of defense) refers to anatomical physiological elements that block all invaders, including microorganisms, regardless of their type. Nonspecific immunity (secondary line of defense) is a phagocyte that breaks through natural resistance and removes invaders. The constructed defense system, and the specific immune system (tertiary line) refers to an immune system composed of lymphocytes, which is 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 multifunctional cell that plays an important role in the immune system by producing various cytokines and oxygen monoxide (NO) in oxidative stress situations. In particular, iNOS expressed by stimulation such as lipopolysaccharide (LPS), cytokine, TNF-a in macrophages is known to produce a large amount of NO for a long time to promote NFB activity. Of reactive oxygen species (ROS) and nitric oxide (NO) such as superoxide anion (O ₂ −), hydrogen peroxide (H ₂ O ₂ ) by activated macrophages Production is an important cytotoxic and cytostatic mechanism in nonspecific immunity. Many studies have been conducted on which natural compounds affect the production of ROS and NO by macrophages. Macrophages are microbicidal cells that present antigen, present tumors, or kill microbial cells and play a central role in cell-mediated or humoral immunity. NO cells produced by activated macrophages show non-specific host defense mechanisms such as macrophages and proliferation inhibitory activity of bacteria and cancer cells (Dawson TM, et al., Annu . Rev. Med , 47 , pp.219 -227, 1996).

Macrophages contain many lysosomes, which contain acid hydrolases and peroxidases. In addition, it strongly adheres to the glass surface and plastic surface, and actively detects microorganisms and tumor cells. The cell has a cytokine receptor 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, 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 were divided into CD4-positive T _H cells (helper T-cell) and CD8-positive T _C cells (cytotoxic T-cell), CD4 + T _H cells recognize the antigen binds to MHC II class (class) molecule It is activated by helping B cells to produce antibodies or to help other T cells function. CD4 + T _H cells can again be classified into T _H 1 and T _H 2 based on the cytokines they produce. T _H 1 cells in mice secrete interleukin-2 (IL-2), interferon-gamma (IFN-γ), etc., whereas T _H 2 cells produce IL-4, IL-5, IL-6 Secrete IL-9, 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. T _H 1 cells are involved in cellular immune responses and activate cytotoxic and inflammatory responses. Cytokines produced in T _H 2 cells promote antibody formation, in particular T _H 2 cytokines are commonly found in antibody production and allergic reactions as they help produce IgE and enhance eosinophil proliferation and function. It has been shown that T _H 1 and T _H 2 cytokines have mutually inhibitory functions and that the disease course can be altered with an anti-IL-4 antibody and an anti-IFN-γ antibody in infectious diseases in animals, and patients with rheumatoid arthritis In some cases, symptomatic improvement was observed by injecting IFN-γ into (Aichard A. Goldsby, et al., KUBY Immunology . 2000).

Hematopoietic stem cells are damaged during chemotherapy, such as radiotherapy or chemotherapy, resulting in a decrease in hematopoietic and immune cells, which often leads to the formation of hematopoietic and immune function formation. As a result, patients experience anemia and lymphocytosis, thrombocytopenia or granulocytopenia, which can lead to serious and fatal infections and increase patient mortality. Clinically, bone marrow growth factors such as G-CSF, GM-CSF, IL-1 to 12, M-CSF, and EPO are used after chemotherapy or bone marrow transplantation. It promotes cell differentiation and production from BFU, increases cell migration, phagocytosis, superoxide production, and cytotoxicity of leukocytes following antibody stimulation (Abul K. Abbas et al., Cellular and Molecular Immunology 6th, pp. 298-301, 2007).

Currently, many herbal medicines are being used around the world to improve health through strengthening immunity. In the United States, herbal medicines are recognized not only as drugs but also as functional foods. It is known that not only in Asia, but a third of the American population take herbal medicine at least once in the form of alternative medicine (Eisenberg et al., N. Eng. J. Med., 328 , pp.246-252). . Drugs derived from herbal medicines have been focused on the identification of active ingredients for the prevention and treatment of diseases. Taxol, an anticancer agent obtained from the bark of the oleander, is a representative example. Most modern Western drug treatments are purified compounds that target specific biochemical pathways, but Korean herbal medicines used in the past by our country are complexes of various compounds and have therapeutic effects with little side effects.

Although the efficacy of herbal medicines is mentioned in past oriental medicine books, such as consent, the information from the literature is not associated with the current specific disease or symptom. In addition, it is not known which of the components of the herbal medicine has a direct effect. Among the herbal medicines listed in the consent agreement and Chinese medicine metabolism, the Chinese herbal medicines that are known to have an immune enhancing effect need to be verified by modern scientific research.

Puerariae Radix is a vine of Pueraria (Leguminosae). It is the root of lobata (Willd.) Ohwi.), grows in the foothills of Korea, and is distributed in Japan and China (Bae Gi-hwan, Korean medicinal plant, Kyohaksa, p.260, 2000). The components of the root roots contain saponin and starch such as flavonoid compounds such as puerarin, daizein, and daidzin, and soyasaponin. It is known to be reorganized by the Pharmacognomy School Reorganization Committee, Pharmacognosy, Dongmyeongsa, p. 100-102, 2006.

In the oriental medicine, the temper of the drug is persimmon, kidney, and quantity, and it is returned to the stomach and stomach, and the regression of anatomy and the shot margin Efficacy in the appearance of vulgaris, Saengjingegal, and Ascendant Branches. External signs, marginal imperfections, fever-galling, and heat stroke Iii), but it is used for the treatment of non-herbular diarrhea (Korea Herbal Medicine School Association, Herbology, Korean Pharmacy Society, pp.107-110, 1995). There is no disclosure that it is effective for augmentation effects.

Therefore, we tried to elucidate the immune enhancing effect and its mechanism of action in natural products used as herbal medicines by using modern objective immune activity indicators.

Therefore, the inventors of the present invention, while identifying a substance having an immune enhancing effect from more than 300 kinds of extracts derived from natural extracts of the present invention, the effect of increasing the secretion capacity of the immune enhancing factors NO, TNF-a, spleen in macrophage Raw 264.7 cells In addition to enhancing cell proliferation, cytokine expression, and innate immune receptors, TLR-4 (Toll-like receptor 4) enhances the activity in immune cells, as well as rotavirus-induced intracellular immune activity. And it has been found that the immune enhancing efficacy of the animal model that induces E. coli infections, the root extract of the present invention can be useful as a component of food, medicine and feed having excellent immunopotentiating efficacy in the prevention, inhibition and treatment of immunosuppression The present invention was completed by confirming the presence of the present invention.

In order to achieve the above object, the present invention provides a pharmaceutical composition for enhancing immunity containing the extract of Puerariae Radix as an active ingredient.

The present invention provides a dietary supplement for immune enhancement containing Puerariae Radix extract as an active ingredient.

The present invention also provides an animal feed additive for enhancing immunity containing the extract of Puerariae Radix as an active ingredient.

* The extract includes water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably extracts soluble in water.

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

Dried roots may be prepared according to a conventional extraction method, C ₁ to C ₄ lower alcohols such as water, methanol, etc., 1 to 20 times, preferably 5 to 15 times the volume of dried dry roots, or these Mixed solvent, preferably water, 0.5 to 10 hours, preferably 2 to 5 hours, 70 ℃ to 100 ℃, 1 to 10 times, preferably 2 to 5 times repeated cold extraction, hot water extraction In the extraction method, such as ultrasonic extraction and reflux cooling extraction, preferably hot water extraction, the extract is filtered under reduced pressure, and then the filtrate is concentrated and lyophilized to obtain the root extract of the present invention.

The present invention provides a pharmaceutical composition for immune enhancement containing the root extract as an active ingredient obtained by the above production method.

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 immunosuppressed, such as a disease 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 cancer disease due to a decrease in immune function. It can be used for the prevention and treatment of.

The pharmaceutical composition containing the extract of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical compositions containing extracts according to the present invention may be prepared 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 a conventional method. 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, 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. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . 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 administered 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 rats, mice, livestock, humans and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a dietary supplement for immune augmentation containing the root extract.

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 enhancing immunity. At this time, the amount of the extract in the food or beverage is generally added to the dietary supplement composition of the present invention 0.01 to 50% by weight, preferably 0.01 to 15% by weight of the total food weight, the health beverage composition is 100ml Can be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g.

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 include monosaccharides such as disaccharides such as glucose and fructose, for example polysaccharides such as maltose and sucrose, and conventional sugars such as dextrin and 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 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 in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides an animal feed additive for immuno-enhancement containing the root extract and a feed comprising the same.

The animal feed additive composition may be 20 to 90% high concentrate or powder or granule form.

The composition for animal feed additives of the present invention includes organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polymeric phosphate), polyphenols, and catechins (catechin) ), Alpha-tocopherol, rosemary extract (rosemary extract), vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and the like may further include any one or more than one.

Animal feed additives containing the composition of the present invention and feed comprising the same as a secondary component, various supplements such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, live microbial preparations, grains, for example Milled or crushed wheat, oats, barley, corn and rice; Vegetable protein feed, such as rape, soybeans and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Sugars and dairy products, for example, dry powders consisting of various powdered milks and whey powders, and drying additives, and then liquid components and components which become liquids after heating, that is, lipids, for example, animals liquefied by heating, for example. In addition to the main components such as fats and vegetable fats can be used with substances such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

The animal feed additive composition may be administered to an animal in combination with other feed additives in an edible carrier alone.

In addition, the composition for animal feed additives can be easily administered as a top dressing or directly mixed with the animal feed or separately from the feed, in a separate oral formulation, by injection or transdermal or in combination with other ingredients. Typically, a single daily dose or divided daily doses can be used, as is well known in the art.

When the composition for animal feed additives is administered separately from the animal feed, the dosage form of the composition, as is well known in the art, may be prepared in an immediate release or sustained release formulation in combination with their non-toxic pharmaceutically acceptable edible carrier. Can be. Such edible carriers can be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the compound may be tablets, capsules, powders, torokies or sugar-containing tablets or top dressings in microdisperse form. If a liquid carrier is used, it may be in the form of soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions. In addition, the dosage form may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like.

In addition, the animal feed in which the compound of the present invention is included as an animal feed additive may be any protein-containing organic cereal meal conventionally used to meet the dietary needs of animals. Such protein-containing flours typically consist mainly of corn, soy flour, or corn / bean flour mixtures.

The animal feed additive may be used by adding to the animal feed by dipping, spraying or mixing.

The invention is applicable to a number of animal diets, including mammals, poultry and fish. More specifically, the diet is commercially important mammals such as pigs, cows, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), furry animals (eg mink and fox), and Zoo animals (eg monkeys and tailless monkeys), as well as livestock (eg cats and dogs). Commercially important poultry typically include chickens, turkeys, ducks, geese, pheasants and quails. Commercially raised fish, such as trout, may also be included.

In the animal feed blending method comprising the composition according to the present invention, the composition is incorporated into the animal feed in an amount of about 1 g to 100 g per kg of feed on a dry weight basis.

In addition, the feed mixture is thoroughly mixed and then a viscous granulated or granular material is obtained depending on the degree of grinding of the components. It is fed as a mash or formed into the desired discrete shape for further processing and packaging. At this time, it is preferable to add water to the animal feed and then go through the usual pelletization, expansion, or extrusion process to prevent separation during storage. Excess water may be removed to dryness.

The extract of Puerariae Radix of the present invention is TLR, a receptor involved in the production of immune-enhancing factors NO, TNF-α, splenocyte proliferation, cytokine expression, and innate immunity in macrophage Raw 264.7 cells. -4 (Toll-like receptor 4) not only shows the enhanced activity in immune cells, but also rotavirus-induced intracellular immune activity And it can be used as a food, pharmaceutical and feed additive having excellent immunopotentiating efficacy in the prevention, inhibition and treatment of immunodeficiency and immunodeficiency impaired immune system shows the immune enhancing effect of the animal model inducing E. coli infection.

1 is a diagram showing the effect of increasing the production of NO and TNF-α secretion in RAW264.7 cells when treated with the root extract,
2 is a diagram showing the splenocyte proliferation effect by the extract of the root,
Figure 3 is a diagram showing the effect of increasing the production of IFN-γ and TNF-a of splenocytes by the root extract,
4 is a diagram showing the effect of increasing the expression of COX-2, IFNbeta and the activation of NFkB, IRF3 when the extract of the roots treated in RAW264.7 cells in a concentration-dependent manner,
FIG. 5 is a diagram showing a reduction effect of dominant-negative mutants of TRIF or MyD88, which are subsignal molecules of TLR4 and TLR4, of respective COX-2 expressions by the root extract;
6 is a diagram showing the effect of COX-2 expression by the root extract of NFkB inhibitor TPCK, p38 pathway inhibitor (SB203580) inhibitory effect and the reduction effect by the IkBa mutant of the super-repressor of NFkB,
7 is a diagram showing the effect of the root extract extract of enteritis caused by rotavirus and viral genes,
8 is a diagram showing the effect of increasing the production of IFN-γ induced by the specific immune response of rotavirus (RV) in splenocytes of splenocytes,
9 is a diagram showing the effect of increasing the IFN-γ production and lymphocyte proliferation in the lymph organs by the root extract,
10 is a diagram showing the effect of increasing the expression of the innate immune receptors TLR2 and TLR4 by the root extract in an animal model induced rotavirus infection,
11 is a diagram showing the effect of reducing mortality by the root extract in the animal model induced E. coli infection.

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

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1 Preparation of Root Extract

After drying the roots naturally, finely chopped, 10 liters of distilled water was added to 1 kg of dried roots, and the extraction was repeated three times at 100 ° C. for 3 hours, followed by vacuum filtration to obtain a filtrate, and the filtrate was concentrated under reduced pressure (EYELA, N-1000, Japan) and lyophilized to give 228 g of the root extract.

Experimental Example 1. Increase effect of NO secretion and TNF-α secretion

In order to measure the NO secretion effect, the concentration of nitrite ion (NO ₂ −) was measured as follows using a grease reagent based on NaNO ₂ .

After dispensing RAW 264.7 cells (ATCC The Global Bioresource Center ^TM ) into 96-well microplates at 1 × 10 ⁵ per well, and treating the root extract obtained in Example 1 at a concentration of 50, 100, 500 μg / ml, 37 ° C., 5% CO ₂ After culturing for 24 hours under the conditions, the supernatant was taken, and the same amount of grease reagent (Griess reagent, 0.1% (w / v) N- (1-naphthyl) ethylenediamine dihydrochloride + 1% (w) was added to 100 µl of the cell culture supernatant. / v) sulfanilamide in 5% (v / v) phosphoric acid) was mixed and left for 10 minutes at room temperature, then 550nm using Ultrospec 4000 spectrophotometer (Pharmacia Biotech, Uppsala, Sweden) Absorbance was measured at (Nakai M., et al., Dig Dis Sci. , Sep; 50 (9) , pp. 1669-1676, 2005).

In addition, 50 μl of cell culture supernatant was taken under the same conditions, and ELISA of tumor necrosis factor-α (inflammatory mediator) was performed (R & D Systems Quantikine Mouse TNF-α / TNFSF1A kit; Vilcek J., et al., J. Biol. Chem., 266 , p.7313, 1991).

As a result, as shown in FIG. 1, the concentration of nitrite ion (NO ₂ ⁻ ) in RAW 264.7 cells was increased by 4.2 times compared to that of the control group and showed a similar increase as the LPS-only treatment group used as a positive control group. In addition, it was confirmed that the secretion was increased. In addition, TNF-α, an inflammatory mediator, was increased by 5.1 times in the RAW 264.7 cells, showing a similar effect as the LPS-only treatment group used as a positive control group. , Root extract was found to increase both the measured TNF-α and NO secretion (see Figure 1).

Experimental Example 2. Splenocyte proliferation effect

To determine if the root extract affects cell proliferation, spleens are taken from C57BL / 6N mice to separate unsplenified splenocytes, and then 1 × 10 ⁶ splenocytes are dispensed into 96 well microplates. 100 μg / ml of the root extract obtained in Example 1 was treated for 24 hours and cell proliferation was measured by MTS method (CellTiter96 ™ AQueous Assay) (Wang M., et al., Int Immunopharmacol, 4 , pp.311 -315. 2004).

As a result, as shown in FIG. 2, 149% cell proliferation was observed in the group treated with the root extract, which showed an excellent proliferation effect than the control group (the group without the root extract), and thus the root extract increased the growth of splenocytes. It can be seen that the induced (see Figure 2).

Experimental Example 3. Increased cytokine expression in splenocytes

To determine whether the root extracts affect the production of T _H 1 cytokines secreted from splenocytes, spleens were taken from mice to separate unsplenified splenocytes and then placed into 1 × 10 ⁶ plates in 96-well microplates. Dispense splenocytes. The root extract obtained in Example 1 was treated for 24 hours in a concentration-dependent manner (50 and 100 μg / ml), and IFN-γ ELISA kit (mouseIFN-γ ELISA kit, BD) and TNF-αELISA kit (Quantikine Mouse TNF- The level of cytokine secretion was measured using α / TNFSF1A kit, R & D Systems).

As a result, as shown in FIG. 3, the group treated with the extract of the root extract compared to the control without the extract of the root extract showed that the IFN-γ and TNF-a secretion ability increased (see FIG. 3).

Experimental Example 4. Activity enhancing effect in congenital immune cells induced by the root extract

4-1. Gene Expression of COX-2, IFNbeta and Activation Effects of NFkB, IRF3

In order to study whether the extract of Brown root has an activity enhancing effect in innate immune cells, the changes of the immune cell activity indices were investigated as follows.

COX-2 and IFNβ are representative immunoproteins that increase when TLR4 is activated by bacteria or viruses in macrophages, which are innate immune cells, and NF-kB and IRF3 are representative transcription factors activated by TLR4 (Youn et al., Biochem Pharmacol, Jan 15; 75 (2) , pp. 494-502, 2008).

In Example 1 after injection of a luciferase reporter plasmid having COX-2-promoter, IFNβ-promoter, NF-kB binding site, and IRF3 binding site into mouse macrophage 1x10 ⁵ cells, respectively. The resulting root extract was treated for 8 hours concentration dependent (100 and 500 μg / ml). Obtain the cell extract using a luminase gene analysis kit (luciferase reporter assay system ^TM , Promega Co., Madison, Wis.) Expression at the gene level of COX-2 and IFNβ and activation of NF-kB and IRF3 were measured, respectively.

As a result, as shown in Figure 4, the root extract extract increases COX-2, IFNβ expression and NF-kB, IRF3 activation in a concentration-dependent manner at 100μg / ㎖, 500 μg / ㎖, respectively, these results It can be seen that the root extract increases the activation of innate immune cells by increasing the activity of transcription factors related to innate immunity and increasing the expression of immune proteins (see FIG. 4).

4-2. Increase in immune cell activity TLR -4 Activation Indicator Measurement

Based on the above results, it was investigated whether the effect of increasing the immune cell activity by the root extract is via TLR-4 activation.

Activation of TLR-4 upon ligand binding causes intracellular clustering of adapter molecules. This activates downstream signaling molecules to promote the production of immune mediators. The major adapter molecules of TLR-4 are MyD88 and TRIF, which specifically activates MyD88-dependent and MyD88-independent subsignal pathways. Sub signaling molecules are also specific to each pathway, but are specific to each pathway. kinase, and the transcription factor may also present to induce the expression of specific target gene (Akira S. et al., Nat . Rev. Immunol, 4 (7), pp.499-511, 2004).

Dominant-negative mutants for COX-2-promoter luciferase reporter plasmid and toll-like receptor 4 (TRL4), TRIF, and MyD88 in mouse macrophage 1x10 ⁵ cells, respectively. ) Was injected and the root extract obtained in Example 1 was treated with 500μg / ml for 8 hours. Obtain the cell extract using a luminescent enzyme gene analysis kit (luciferase reporter assay system ^TM , Promega Co., Madison, Wis.) By measuring the degree of COX-2 expression, it was determined whether each dominant-negative mutant inhibited the enhancement of immune cell activity by the root extract.

As shown in FIG. 5, COX-2 expression by the root extract was greatly reduced by the dominant-negative mutant of TLR-4, and TRIF or MyD88 was also dominant-negative. mutant), and these results indicate that TRIF, MyD88, the adapter molecules of TLR-4 and TLR-4, are involved in immune cell activation by the root extract. It can be seen that the immune cell activation effect mediated the activation of TLR-4 (see FIG. 5).

4-3. TLR Immune Cell Activation by Representative Signal Transduction System

In order to identify the intracellular TLR-4 signaling system activated by the root extract, the efficacy change of the root extract was measured after treatment with inhibitors for each signaling system.

After injection of COX-2-promoter luciferase reporter plasmid into mouse macrophage 1 × 10 ⁵ cells, the root extract obtained in Example 1 was concentration-dependent (100 and 500 μg / ml) for 8 hours. Treated. According to the experiment, NF-kB and p38 pathway inhibitors were treated before the root extract. Obtain the cell extract using a luminescent enzyme gene analysis kit (luciferase reporter assay system ^TM , Promega Co., Madison, Wis.) By measuring the expression level of COX-2, it was determined whether the inhibition of immune cell activity by the root extract is inhibited. We determined whether NF-kB and p38 pathways, TLR-4's representative signaling systems, contribute to immune cell activation by the root extract.

As a result, as shown in FIG. 6, the immune cell activation by the root extract is inhibited by TPCK treatment, which is an inhibitor of NF-kB, and by an IkBa mutant, which is a super-repressor of NF-kB. Results were obtained (see FIG. 6). These results suggest that TLR-4 activation by the root extract extracts induces activation of the NF-kB pathway, and that the p38 pathway, along with the NF-kB pathway, will be activated when immune cells are activated by the root extract. see.

Accordingly, it can be seen that the root extract has an effect of activating immune cells, and in particular, activating TLR-4 may induce activation of immune cells and enhancement of immune responses.

Experimental Example  5. Root Extract  Immune Activity Enhancement Effect by Rotavirus

5-1. Rooting  Effect of Extracts on Immune Responses in Rotavirus-induced Enteritis

To investigate the effect of the root extract on the immune response in rotavirus enteritis, the root extract was orally administered (10 mg / mouse) for 3 days to C57BL / 6 newborn mice (5-6 days old, n = 4). The virus (wild-type murine rotavirus, EC strain, distributed by Professor Harry B. Greenberg of Stanford Medical University, National Veterinary Research and Quarantine Service, Import Permit No. 2007-56) was infected with intestinalitis the day after the last dose of root extract. The amount of diarrhea caused by the virus (enteritis) and the level of the virus in the small intestine infected with the virus (enteritis) was confirmed.

As a result, as shown in FIG. 7, mice in the group ingesting the root extract were reduced in diarrhea compared to the control group, and in particular, the amount of the viral NSP3 gene in the small intestine was significantly reduced compared to the control group after 4 days of viral infection. Could be (see Figure 7).

5-2. Rooting  Effect of Extracts on Rotavirus Specific Immune Responses

To investigate the effects of the root extract on rotavirus specific immune responses, the root extract was orally administered (10 mg / mouse) for 3 days in C57BL / 6 newborn mice (5-6 days old, n = 4) and rotavirus (wild-). type murine rotavirus, EC strain, obtained from Professor Harry B. Greenberg of Stanford Medical University) 2 days after infection with the root extract, the spleens of mice were stimulated to stimulate the rotavirus (RRV strain) antigen. After 5 days mixed culture with splenocytes, cell culture medium was collected and subjected to IFN-γ ELISA (mouse IFN-γ ELISA kit, BD) method.

As a result, as shown in Figure 8 it was confirmed that the IFN-γ production was significantly increased in the mouse group of the group taking the root extract (see Figure 8).

5-3. Rooting  Extracts of Rotavirus Infected Mice Polyclonal  Antibodies ( polyclonal Impact on stimuli

To investigate the effect of the root extract on polyclonal stimulation of rotavirus-infected mice, the root extract was orally administered to C57BL / 6 newborn mice (5-6 days old, n = 4) for 3 days (10 mg / day). 2 weeks after infection with rotavirus (EC strain) the day after administration of the last root extract, the spleens of the mice were harvested and cultured in plate well 1x10 ⁶ coated with Anti-CD3 antibody, followed by IFN-γ. FACS staining (daily culture) and BrdU (4-day culture) were measured by an incorporation assay.

As a result, as shown in Figure 9 it was confirmed that the increased IFN-γ acid increase effect and lymphocyte proliferation (proliferation) effect in the lymph organs of the mouse group ingesting the root extract extract (see Figure 9).

5-4. Rooting  Effect of Extracts on the Expression of Innate Immunoreceptors After Rotavirus Infection

The root extract was orally administered to C57BL / 6 newborn mice (5-6 days old, n = 4) for 3 days (10 mg / mouse) and 4, 8 days after infection with rotavirus (EC strain) the day after the last root extract was administered. Mesenteric lymph nodes of mice were harvested to confirm gene expression of TLR2 and TLR4.

As a result, as shown in Figure 10, the expression of TLR2 and TLR4 in the group receiving oral root extract was increased on the fourth day after the virus infection, the difference in TLRs expression between the two groups on the eighth day of virus clearance (virus clearance) There was no. Accordingly, it can be seen that the root extract extract increases the immune immunity against rotavirus by enhancing the expression of TLR2 and TLR4 (see FIG. 10).

Experimental Example  6. Immune Enhancing Effect of Animal Model Inducing E. Coli Infection

To investigate the immune enhancing effect of extract E. coli was administered intraperitoneally in mice to evaluate the protective effect against pathogenic bacteria through the prevention of mortality caused by E. coli bacterial infection by the root extract (Kim Jong-man et al., 39 , pp.1-7). , 1997).

E. coli (E. coli; Escherichia coli) was used as a K88ab strain (University College of Veterinary Medicine, Seoul National University), and 30 animals of 6-week-old male ICR mice were purchased from Samtaco (Osan, Korea). For the test, 10 heads of each group were used in three groups, the control group, the E. coli inoculation positive control group, and the E. coli inoculation group after administration of the root extract. Dissolve the brown root extract with water and give oral administration twice a day at 500mg / kg dose to the mice in groups 2 and 3, and attack the mice with intraperitoneal administration of 0.5ml (10 MLD) of E. coli culture solution to each mouse after 24 hours. Inoculation. The mortality was recorded for 4 days after challenge and the rate of pathogenic bacterial protection of the root extract was examined.

As shown in FIG. 11, the animals of the E. coli inoculation positive control group (I) showed a mortality rate of 100% at 10 days after inoculation of E. coli (D1), but after the root extract was administered with the root extract E. coli inoculated group (III) had 6 deaths on day 1, 1 day on day 2, and 1 day on day 3 after E. coli inoculated. There were two surviving mice at the end of the final test. As shown by 20% survival rate in the protective effect experiment, it is determined that there is a bacterial defense effect (see FIG. 11).

Experimental Example  7. Oral Toxicity Test

In this experiment, 7-week-old ICR mice (Oriental Bio, Japan) were used to fast the mice for 4 hours before administering the root extract, weigh them, and measure the toxicity standards of the Korea Food and Drug Administration (KFDA, Drugs, etc.). According to Toxicity Test Criteria, Korea Food and Drug Administration 2005-60, 2005), the highest dose was set to 2,000 mg / kg, diluted with purified distilled water to prepare 5 mg / kg, 50 mg / kg and 500 mg / kg of test substance. Administration was oral administration, which is the main application route in the clinic, and the administration method was fixed directly by transcutaneous skin fixation and then injected directly into the stomach using a metal oral administration zone. Once daily, 10 ml / kg body weight was administered as a dose, and the feed was restrained for 2 hours after administration of the test substance to the test animals, and the feed and water were continuously supplied during the test period.

After administration in accordance with the Toxicological Test Standards of the Agency and the Acute Toxicity Testing Guidelines of the International Economic Cooperation Organization (OECD Guideline for the Testing Chemicals revised draft guideline 420, Organization for Economic Cooperation and Development (OECD), 2001). During the first six hours, with special attention, the skin, hair, eyes, mucous membranes, respiratory tract, circulatory system, autonomic nervous system, central nervous system, physical activity, behavioral patterns and tremors, convulsions, Salivation, diarrhoea, lethargy, sleep, coma and the like were observed. General clinical symptoms were observed once a day for 14 days after administration, and the individual body weights of the test animals were measured before and after administration of the test substance and once a day during the test period.

At 14 days after administration, ICR mice were anesthetized with ethyl ether, bleeded to death, and all organs were removed, and gross lesions of the individual test animals were observed and the weights of the organs were measured.

As a result of acute toxicity test of single root oral administration of the root extract which has an effect on immunity, no deaths were observed in the test animals used in this study and at the highest dose of 2,000 mg / kg administered under the conditions. The dose did not cause toxicity by the administration of the root extract in all the indicators observed in accordance with the Toxicological Standards of the Korea Food and Drug Administration. In conclusion, it was confirmed that the root extract is safe without toxicity even at the recommended maximum dose of 2,000 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

Brown root extract 20 mg

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

10 mg extract

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

10 mg extract

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 injections

10 mg extract

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

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

Brown root extract 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of health food

Brown root extract 1000mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

0.13 mg of vitamin B ₁

0.15 mg of vitamin B ₂

0.5 mg of vitamin B ₆

Vitamin B ₁₂ 0.2 g

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  7. Manufacture of health drinks

Brown root extract 100 mg

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

Vitamin B ₁ 0.25 g

Vitamin B ₂ 0.3 g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is a composition suitable for a preferred 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.

[National R & D project supporting this invention]

[Task unique number]

RTI05-03-02

[Name of Buddha]

Ministry of Knowledge Economy

[Name of research project]

Local Technology Innovation Project

[Name of Research Project]

Research and Development Project for Acquired Common Infectious Diseases

[Host]

Wonkwang University

[Research period]

May 01, 2005 ~ April 30, 2010

Claims (2)

Animal feed additive for immune enhancement containing Puerariae Radix water extract as an active ingredient.
A feed comprising the animal feed additive of claim 1.

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