KR101151484B1 - Composition comprising the extract of Puerariae Caulis for immunostimulatory activity - Google Patents

Abstract

The present invention relates to a composition for enhancing immune activity containing Puerariae Caulis extract, and more specifically, the present invention provides an effect of increasing the secretion capacity of NO, TNF-α, which is an immune enhancer in macrophage Raw 264.7 cells, In addition to showing an effect of increasing the production of IL-12 in the spleen cells, food, medicines and drugs having excellent immunopotentiating efficacy in the prevention, inhibition and treatment of immunosuppression due to the excellent immunity enhancing effect of the animal model that induced Salmonella gallinarum infection. It is useful as a feed additive.

Description

Composition comprising the extract of Puerariae Caulis for immunostimulatory activity

The present invention relates to a composition for enhancing immune activity containing galman extract or crude polysaccharide fraction.

[Reference 1] Kindt TJ et al ., KUBY Immunology 6 th , 2006.

[Reference 2] John MM et al . , Annu . Rev. Immunol ., 15 , pp. 323-350, 1997.

[Reference 3] Paul WE, Fundamental Immunology 6 th , pp. 547-569, 2008.

Document 4 Wagner H., Immunomodulatory Agent from Plants , pp. 1-39, 1999.

[Reference 5] Information Interpretation, Hyangjeomsa Dictionary, Younglimsa Temple , pp.704-706, 1998.

[Reference 6] Kinjo JE et al ., Chem . Pharm . Bull ., 35 , pp. 4846-4850, 1987.

[7] peanut, immunization and oriental medicine, open books , 1998.

[Reference 8] Yang XY et al ., Phytother. Res., 23 , pp. 1713-1720, 2009.

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

Document 10 Vilcek J., et al ., J. Biol . Chem . , 266 , pp.7313-7316, 1991.

[Reference 11] Wang M., et al . , Int . Immunopharmacol ., 4 , pp. 311-315. 2004.

[Reference 12] Waihenya RK et . al . , Journal of Ethnopharmacology , 79 , pp. 317-323, 2002.

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 phagocytic cell that breaks through natural resistance and removes invaders. The constructed defense system, and the specific immune system (tertiary line of defense) refers to the immune system composed of lymphocytes, which are highly developed immune systems with memory and the ability to distinguish between self and nonmagnetic (Kindt TJ et. al ., KUBY Immunology 6th, 2006).

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 nitric oxides (NO) in oxidative stress situations. In particular, iNOS expressed by stimulation such as lipopolysaccharide (LPS), cytokine, TNF-α in macrophages is known to produce a large amount of NO for a long time to promote NFκB 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 antigens, remove tumors, or kill microbial cells, and play a central role in cell-mediated or humoral immunity. As a regulatory cell, NO produced by activated macrophages shows macrophages, which are nonspecific host defense mechanisms, and proliferation inhibitory activity of bacteria and cancer cells (John MM et. al . , Annu. Rev. Immunol., 15 , pp. 323-350, 1997).

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 (Paul WE, Fundamental Immunology 6th, pp.547-569, 2008).

Traditional medicines, which have been used for the purpose of treating and preventing diseases since the beginning of human history, have accumulated long clinical experience, and still take a large part in the treatment and prevention of diseases. Traditional Chinese medicine is called Chinese medicine, and plants, animals, microorganisms, and minerals are made of natural products such as plants, and usually two or more medicines are mixed and administered as a prescription. Oriental medicine books, such as Dongbobogam and Chemopyeonpyeon, also mention the efficacy of herbal medicines listed as a collection of prescriptions to treat various diseases. Although the efficacy of Chinese medicine is still useful in the treatment of diseases today, the disease pattern of human beings was deficient diseases such as infectious diseases and malnutrition in the past, but as the standard of living improves due to economic development, diabetes, malignant tumors, arteriosclerosis, Patients with chronic degenerative diseases such as cardiovascular diseases are on the rise, and the emergence of endogenous strains due to the abuse of antibiotics and the emergence of new diseases such as AIDS are urgently required to develop new treatments. Chinese medicine is a useful resource for the development of therapeutic agents for such diseases, and the efficacy of Chinese medicine with immunopotentiating efficacy can be used for the prevention and treatment of infectious diseases and cancer caused by viruses. Among the components contained in Chinese medicine, low-molecular substances such as saponins, alkaloids, and quinones, and high-molecular substances such as lectins, peptides, and polysaccharides exhibit immuno-enhancing effects. Known (Wagner H., Immunomodulatory Agent from Plants, pp.1-39, 1999). In Chinese medicine, herbal medicines such as bodhisattva medicine, blood donation medicine, nourishing medicine and voicing medicine are used for the purpose of immunity enhancement, but their efficacy has not been scientifically proven. There is a need for scientific verification of the immune-enhancing effects of herbal medicines used for immuno-enhancing purposes and the discovery of new immune-enhancing substances (Pigranula, Immunology and Oriental Medicine, Open Books, 1998).

Galman (葛蔓; Puerariae Caulis) is kudzu (Pueraria belonging to leguminous (Leguminosae) A vine stem of lobata (Willd.) Ohwi.), distributed in mountainous fields nationwide (Information Disclosure, Korean Medicine Dictionary, pp.704-706, 1998). Dried roots of 갈 dried root, flowers are browned, fruit is grained or browned, leaves are brown leaves are used in oriental medicine. Galman is known to contain isoflavonoid compounds such as puerarin, daizein, daidzin, genistein and genistin. Kinjo JE et al ., Chem. Pharm. Bull., 35 , pp. 4846-4850, 1987).

Galman is an unused site that is not widely used in clinical practice and is not commercially available at all. The research on the components contained in galman is also extremely inferior to the roots, and the composition cannot be compared with each other. Brown root is known as a sulfur plant, but the stem, galman, has never been used for food. Therefore, if only the unused galman can be developed as an animal immunopotentiator, considering the ecological characteristics of 칡, in terms of industrial and environmental recycling in consideration of reducing forest destruction, increasing farm household income, and promoting public health, etc. It is believed to have a huge effect.

To date, however, no records related to the immunopotentiating effect of galman extract have been reported anywhere in this document.

Therefore, the inventors of the present invention, while searching for a substance having an immune enhancing effect by using an objective immunostimulating activity indicator, surprisingly, the galman extract and the extract-derived copolysaccharide fraction of the present invention is an immune enhancer in macrophage Raw 264.7 cells. In addition to increasing the secretion capacity of NO and TNF-α and increasing the expression of IL-12 cytokines in splenocytes, galman powder administration is effective in boosting immunity in animal models inducing Salmonella gallinarum infection. The present invention by confirming that the galman extract of the present invention and the extract-derived crude polysaccharide fraction can be usefully used as a component of food, medicine and feed having excellent immunopotentiating efficacy in the prevention, inhibition and treatment of immunosuppression. Was completed.

In order to achieve the above object, the present invention provides a pharmaceutical composition for immune enhancement containing a Galman (Puerariae Caulis) extract or crude polysaccharide fraction as an active ingredient.

The present invention provides a dietary supplement for immune enhancing containing Galmane (Puerariae Caulis) extract or crude polysaccharide fraction as an active ingredient.

In addition, the present invention provides an animal feed additive for enhancing immunity, which comprises a Puerariae Caulis extract or a crude polysaccharide fraction as an active ingredient.

"Extract" as defined herein includes extracts soluble in water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably water, wherein the extract-derived "copolysaccharide fraction" of the present invention is The precipitate obtained by cooling the mixture with water and ethanol or acetone and centrifuged the precipitate obtained by the dialysis step comprises a fraction containing a large amount of the polymer material obtained.

Galman extract and crude polysaccharide fraction of the present invention can be prepared by the following manufacturing process.

Dried galman can be prepared according to a conventional extraction method, C ₁ to C ₄ lower alcohols such as water, methanol and the like 1 to 20 times, preferably 5 to 15 times the volume of dried galman dry weight 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 galman extract of the present invention. .

In addition, the crude polysaccharide fraction of the present invention is a mixture of water and ethanol, preferably 70 to 100% ethanol mixed solvent, more preferably 95% ethanol or acetone to the brown extract obtained in the step 1 to 10 times the volume of the extract (v / v), preferably 1 to 4 times (v / v) volume, and the precipitate obtained by standing at 4 ° C. to room temperature for about 6 to 48 hours, preferably about 12 to 48 hours is filtered through a filter paper. The precipitate obtained after filtration or centrifugation can be obtained through the dialysis membrane using distilled water or the purification process using an ultrafilter to obtain the crude polysaccharide fraction of the present invention in which the low molecular material is substantially removed.

The present invention provides an immune enhancing pharmaceutical composition containing the root extract or crude polysaccharide fraction obtained as the active ingredient.

The pharmaceutical composition for enhancing immune composition of the present invention comprises 0.1 to 50% by weight of the extract or crude polysaccharide fraction based on the total weight of the composition.

The pharmaceutical composition of the present invention may be used for immunocompromise such as lowering of immune function by chemotherapy and radiation therapy or disease caused by immunosuppression after bone marrow transplantation, AIDS due to immune system damage and cancer disease due to lowering of immune function. It can be used for prevention and treatment.

Pharmaceutical compositions containing extracts or crude polysaccharide fractions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions containing extracts or crude polysaccharide fractions according to the invention, respectively, oral formulations, external preparations, suppositories, and sterile injections of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols etc. according to conventional methods Carriers, excipients and diluents which may be used in the form of solutions and may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia Rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Can be. 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 stearate 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 or crude polysaccharide fractions 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. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

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 health functional food for immuno-enhancement containing the galman extract or crude polysaccharide fraction.

Examples of the food to which the extract or crude polysaccharide fraction 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 to 0.01 to 50% by weight, preferably 0.01 to 15% by weight of the total food weight, the health beverage composition is 100 ml It can be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on the amount.

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 extract or crude polysaccharide fraction of the present invention, a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors 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 drinks 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.

The present invention also provides an animal feed additive for immuno-enhancement containing the galman extract or crude polysaccharide fraction or powder thereof 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 these composition-toxic pharmaceutically acceptable edible carriers. have. 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 extract 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, wherein the extract of the present invention is included as an animal feed additive, may be any protein-containing organic cereal meal commonly 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.

Extract of Galuer (Puerariae Caulis) of the present invention induces the production of immune enhancing factors NO, TNF-α secretion in macrophage Raw 264.7 cells, the effect of increasing IL-12 cytokine expression in splenocytes and Salmonella gallinarum infection As one animal model exhibits an immunostimulating effect, it can be used as a food, pharmaceutical and feed additive having excellent immunopotentiating efficacy in preventing, suppressing and treating immunocompromised and compromised immunocompromise.

1 is a diagram showing the effect of increasing the production of NO and TNF-α secretion in RAW264.7 cells and cell viability when treated with galman extract and crude polysaccharide fraction,
2 is a diagram showing the effect of increasing the production of IL-12 and cell viability in splenocytes by the crude polysaccharide fraction of Galman extract,
3 is a diagram showing spleen proliferation by immunization of the spleen against the challenge of pathogenic Salmonella gallinarum of galman powder;
Figure 4 is a diagram showing the effect of inhibiting the growth of bacteria and spleen in the liver against the challenge of pathogenic Salmonella gallinarum of galman powder,
5 is a diagram showing the protective and therapeutic effects of the challenge of the pathogenic Salmonella gallinarum of galman powder.

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

However, the following examples and experimental examples are 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 Galman Extract

After extracting and drying in Yasan, Iksan-si, Jeonbuk, 10 ℓ of distilled water was added to 1 kg of finely cut galman, repeated three times of heating extraction at 100 ° C for 3 hours, filtered and concentrated under reduced pressure and freeze-dried to obtain 320 g of galman extract. Obtained.

Example 2. Preparation of Crude Copolysaccharide Fraction

After dissolving the extract obtained in Example 1 in distilled water, and added 3 times the volume of 95% ethanol and left at 4 ℃ for 12 hours, centrifugal separator at 3000 rpm for 30 minutes (Combi-514R, Hanil Science Industry Co., Ltd., After centrifugation to obtain a supernatant and a precipitate, the precipitate was lyophilized and dialyzed with distilled water using a cellulose dialysis membrane (Viskase, MEMBRA-CEL MD25 14 × 100 CLR, USA) to remove low molecular weight material. Dialysis was carried out for 3 days by exchanging distilled water every day at 4 ℃, lyophilized by collecting the inside of the dialysis membrane to obtain 30 g of crude polysaccharide fraction.

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

In order to examine the effect on the increase effect of NO secretion and TNF-α secretion of the extract and crude polysaccharide fraction obtained in the above example, the experiment was performed as follows by applying the method disclosed in the literature (Yang XY et al ., Phytother. Res., 23 , pp. 1713-1720, 2009).

1-1. NO  Secretory effect

In order to measure the NO secretion effect, the concentration of nitrite ions (NO ₂ −) was measured using a Grease reagent (iNtRON Biotechnology, Nitric Oxide Detection Kit, Korea) based on NaNO ₂ as follows. It was.

RAW 264.7 cells (ATCC The Global Bioresource Center ^™ ) were dispensed in 96-well microplates at 1 × 10 ⁵ per well and coarsely extracted with the galman water extracts obtained in Examples 1 and 2 at 25, 50 and 100 μg / ml concentrations. The upper and crude polysaccharide fractions obtained during the production of the polysaccharide fraction and LPS (0.1 ㎍ / mL) (Sigma, L3024, USA) used as the positive control were treated with 37 ° C. and 5% CO ₂ , respectively. After culturing for 24 hours under the conditions, the supernatant was taken, and the same amount of Greries 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 allowed to stand at room temperature for 10 minutes, then at 550 nm using Inferit 200 microplate reader (Tecan, Gr, Austria). Absorbance was measured (Nakai M. et al ., Dig. Dis. Sci., 50 (9 ), pp. 1169-1676, 2005).

1-2. TNF -α secretion effect

Under the same conditions, 50 μl of cell culture supernatant was taken and subjected to ELISA test of inflammatory mediator TNF-α (tumor necrosis factor-α) (R & D Systems Quantikine Mouse TNF-α / TNFSF1A kit, USA; Vilcek J. et al ., J. Biol. Chem., 266 , pp.7313-7316, 1991).

1-3. Effect on Cell Viability

In addition, cell proliferation was measured by MTS method (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega Corp.) to determine whether the extracts and fractions affect cell proliferation (Wang M. et. al . , Int. Immunopharmacol., 4 , pp. 311-315. 2004).

As a result, as shown in FIG. 1, the concentration of nitrite ion (NO ₂ ⁻ ) in the RAW 264.7 cells increased 3.6 times compared to the control, and the crude polysaccharide fraction increased by 4.6 times, and treated with LPS alone as a positive control. By showing a similar increase with the group, it was confirmed that the galman extract increases the NO secretion, the increase in NO secretion effect of the galman extract was confirmed that the polysaccharide. In addition, TNF-α, an inflammatory mediator, was increased by 5.1 times compared to the control group in the RAW 264.7 cells, and the crude polysaccharide fraction was 6.5 times higher than that in the LPS-only treatment group. Accordingly, the Galman extract increased both the measured TNF-α and NO secretion, and found that the polysaccharide contained in the extract was the active substance.

When the Galman extract and fractions were treated with 25, 50, 100 μg / ml, no significant difference in cell viability was observed at all concentrations. That is, the concentration of the galman extract and fractions used in the present invention (25-100 ㎍ / ㎖) was confirmed that there is no cytotoxicity. (See Figure 1).

Experimental Example  2. In splenocytes IL  -12 expression increase effect

In order to examine the effect of the extract and the copolysaccharide fraction obtained in the above example on the IL-12 expression increase effect was carried out as follows by applying the method disclosed in the literature (Yang XY et al ., Phytother. Res., 23 , pp. 1713-1720, 2009).

To determine whether the galman crude polysaccharide fraction influences the production of IL-12, a T _H 1 cytokine secreted from splenocytes, 5 week old C57BL / 6N mouse females were adapted to the animal laboratory environment for 1 week. After sacrificing the cervical vertebra and taking the spleen, it was carefully ground using RPMI 1640 (Gibco Co., 22400, USA) medium and filtered through a nylon sieve (nylon mesh; BD Falcon, 352340, USA). The filtrate was suspended in RBS lysis solution (Sigma, R7757, USA) and allowed to stand for 1 minute to dissolve red blood cells. After repeated washing with RPMI 1640 medium, splenocytes were dispensed at 1 x 10 ⁶ / well in a 96 well microplate using a medium containing 10% FBS (Fetal bovine serum, Gibco. Co.) in Example 2 The obtained Galman crude polysaccharide fraction (25, 50 μg / ml) and LPS (5 μg / ml) (Sigma, L3024, USA) used as a positive control were treated for 24 hours and the IL-12 contained in the culture supernatant. The content was measured using ELISA kits (R & D systems).

In addition, under the same conditions, it was measured using the MTS method to determine the effect on the survival rate of splenocytes.

As a result, as shown in Figure 2, 50 ㎍ / ml of the galman crude polysaccharide fraction increased the IL-12 1.8 times than the control group, showing a similar enhancement effect to the LPS alone treated group used as a positive control group. In addition, the galman crude polysaccharide fraction did not significantly affect the splenocyte proliferation at the concentration used in the experiment (see FIG. 2).

Experimental Example  3. Salmonella Galina Room  Immune Enhancing Effects of Infection-Induced Animal Models

To investigate the effects of galman's immune-enhancing effects, the antimicrobial and mortality effects of Salmonella gallinarum on oral administration of galman powder obtained from flour mills were assessed against chicken pathogens. Performed together (Waihenya RK et . al . , Journal of Ethnopharmacology, 79 , pp. 317-323, 2002).

Salmonella gallinarum used ATCC # 9184 strain (prepared from the National Veterinary Research Institute), and experimental animals were purchased from Harim (Iksan, Korea) with 72 heads of 5 days old broiler broth. The test was divided into three groups, Salmonella gallinarum inoculation positive control group (non-treated group), two concentrations (0.5%, 0.1%) of galman powder, and three groups, Salmonella gallinarum inoculation group, and mortality was measured from each group to the lower group. The meat was divided into 14 heads in the group and 10 heads in the spleen and soy extract group.

Galman powder was divided into two concentrations of 0.5% and 1% by weight and added to the feed for pre-treatment for 5 days, and the untreated positive control group supplied only the basic feed (modern feed, Korea) without antibiotic and anticoccidial agent. Each bronchus was challenged with oral administration of 0.5 ml (10 ⁸ CFU / ml) of Salmonella gallinarum culture. Five days after challenge, spleen weight, spleen, hepatic bacteria and 20 days of mortality were measured to investigate the antimicrobial and protective effects against pathogenic Salmonella gallinarum bacteria in galman powder chickens. Feed and water were freely consumed throughout the test period.

3 and 4, the Galman powder-administered group showed increased spleen weight compared to the positive control group after Salmonella gallinalum inoculation, while the spleen and hepatic bacterial counts decreased, resulting in immunization against the pathogenic Salmonella gallinarum. It is believed to have an enhancing and antibacterial effect.

In addition, as shown in FIG. 5, the positive control group showed mortality from 85 days of inoculation with Salmonella gallinarum, but the mortality rate was 85%. The mortality rate was 64% and 71%, respectively, and 36% and 29% survival rate, respectively, suggesting a protective effect against the pathogenic Salmonella gallinarum challenge.

Claims (8)

delete delete delete delete delete Puerariae Caulis Water Extract; Or immunity enhancing animal feed additives containing crude polysaccharide fraction containing a large amount of polymer material obtained by dialysis of the precipitate obtained after cooling and centrifuging the galman water extract with water and ethanol mixed solvent or acetone. 7. The animal feed additive according to claim 6, wherein the composition for animal feed additives is 20 to 90% high concentrate or powder or granule form. Feed comprising the animal feed additive of claim 6.

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