KR20120071779A - Composition for enhancing imunological activity - Google Patents

Abstract

PURPOSE: An immunoenhancer composition containing a Canavalia gladiate fermentation is provided to promote macrophage proliferation and to enhance immunity. CONSTITUTION: An immunoenhancer composition contains: a fermentation which is prepared by sealing a mixture of Canavalia gladiata and ethanol solution and fermenting at room temperature(20-30°C) for 50-70 days; or a fermentation prepared by maturing the fermented material at low temperature(3-10°C) for 40-50 days. The composition additionally contains Sapindus mukorossi Gaertn. Leaf extract. The ethanol solution contains 20-40%(v/v) of ethanol. The composition is a pharmaceutical or food composition.

Description

Immunity Enhancer Composition {Composition for Enhancing Imunological Activity}

The present invention relates to an immune enhancer composition.

Immunity is roughly divided into innate immunity and acquired immunity.

Innate immunity, also called spontaneous immunity, refers to immunity that nonspecifically responds to an antigen without special memory. Innate immune systems include skin, mucous tissue, stomach acid, blood complement, macrophages, polymorphonuclear leukocytes, natural killer cells (NK cells), and the like.

Acquired immunity refers to an immune response that removes antigens by reacting specifically to the invading antigens, when they invade again. Humoral immunity and cell-mediated immunity Separated by.

Humoral immunity during acquired immunity refers to an immune response that removes antigens by antibodies secreted by B lymphocytes that recognize antigens, which are present in body fluids as glycoproteins, and include IgG, IgM, IgA, IgD, and IgE. Known.

Cellular immunity refers to an immune response in which T lymphocytes derived from the thymus or macrophages activated by lymphokine secreted by T lymphocytes remove cells infected by a virus or the like.

When the immune function is lowered, various immune diseases such as asthma, rhinitis, conjunctivitis, atopic dermatitis, urticaria and hemolysis of red blood cells may occur.

Recently, natural products having various immune enhancing activities have been developed, for example, polysaccharides obtained from Baekkukchae (Domestic Patent Registration No. 310059), Hyunsam Extract (Domestic Patent Registration No. 553267), and Octagonal Extract (Domestic Patent Registration No. 535268). , Root extract of Korea (Korean patent registration No. 757220) and the like.

The present invention is completed by confirming the proliferation promoting activity of T lymphocyte cells or macrophages associated with cellular immunity.

It is an object of the present invention to provide an immune enhancer composition.

The specific object of the present invention will be presented below.

As confirmed in the following Examples and Experimental Examples, the present invention is the immune treatment when the soybean fermented to EL-4 and macrophage RAW264.7 T-lymphoma cell line immune-related cells In addition to promoting the proliferation of the cells involved, it was confirmed that the cytokine secretion of TNF-α, IL-1β, IL-6 and the like of the macrophages. The fermented soybeans used in the experiment was obtained by mixing the soybeans with 30% ethanol, sealing them, and fermenting them at room temperature (20 ~ 30 ℃) for 60 days at room temperature.The fermentation broth was sealed at low temperature (3 ~ 10 ℃) for 45 days. It is obtained by aging for days.

The immune enhancer composition of the present invention is provided based on the results of these experiments, and (I) a fermentation product obtained by fermenting at room temperature (20 to 30 ° C.) for 50 to 70 days by sealing a mixture of soybean and ethanol aqueous solution, or ( Ⅱ) Fermentation obtained by sealing a mixture of aqueous soybean ethanol solution and fermenting at room temperature (20 ~ 30 ℃) for 50 ~ 70 days, and then aging for 40 ~ 50 days at low temperature (3 ~ 10 ℃) while maintaining the sealed state. It is characterized by including water as an active ingredient.

As used herein, the term "immune enhancement" means enhancement of acquired immunity, and preferably means enhancement of cellular immunity, in particular, promotion of proliferation of T lymphocytes and macrophages.

In addition, in the present specification, the "active ingredient" refers to a component that can exhibit the activity alone or in combination with a carrier having no activity in itself.

In addition, in this specification, a "fermented substance" is meant to include the fermentation stock solution after completion | finish of fermentation or fermentation and maturation, the filtrate of this fermentation stock solution, and the liquid or solid concentrate obtained by concentrating this filtrate.

Fermented soybeans, which are included as an active ingredient in the immune enhancer composition of the present invention, soybeans ( Canavalia) gladiata ) is obtained by mixing, sealing and fermenting an aqueous solution of ethanol, where the aqueous solution of ethanol is in the range of 20-40% (v / v) where the preferred ethanol content is a mixture of ethanol and water, where% means volume percentage. Equal to). It is because if less than 20% unintended fermentation may proceed by the various bacteria, and if more than 40% fermentation may not proceed or the fermentation may proceed very slowly. Although not shown in the following Examples and Experimental Examples, fermentation with 50% aqueous ethanol solution was very insignificant, and the fermentation with 13% aqueous ethanol solution caused undesirable odor in the fermentation broth. .

In addition, in the preparation of fermented soybeans, which are included as an active ingredient in the immune enhancer composition of the present invention, the fermentation-maturation period is determined in relation to the fermentation-maturation temperature, but when the fermentation temperature is 20 ° C, the fermentation period is about 70 days. In the case where the fermentation temperature is 30 ° C., the fermentation period is preferably about 50 days. In addition, the ripening period is preferably 50 days when the ripening temperature is 3 ℃, the ripening period is preferably 40 days when the ripening temperature is 10 ℃. If the fermentation temperature and the fermentation period is out of the above range, unintended fermentation and ripening proceeds, or the fermentation and maturation degree is low, so that the desired effect of fermentation and ripening cannot be obtained.

The immune enhancing composition of the present invention may include any effective amount (effective amount) according to the use, formulation, formulation purpose, etc. as long as the active ingredient can exhibit the immune enhancing activity, and the conventional effective amount may be based on the total weight of the composition. When determined to be in the range of 0.001% to 99.990% by weight. Here, the "effective amount" refers to the amount of the active ingredient that can induce an immune enhancing effect. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

Subjects to which the compositions of the invention can be applied (prescribed) are mammals and humans, in particular humans.

In a preferred embodiment, the immune enhancing composition of the present invention is characterized in that it further comprises a leafless leaf extract in addition to the bean extract as an active ingredient.

The following experimental example shows that the immune enhancing activity is increased when the leafless tree leaf extract is included.

The leafless tree leaf extract is obtained by immersing the leaves of Sapindus mukorossi Gaertn. As an extraction solvent in water, ethanol or a mixture thereof, and concentrates the extract stock, the filtrate of the extract stock, and the filtrate. It is the meaning containing the liquid or solid concentrate obtained by making it.

The composition of the present invention may be regarded as a pharmaceutical composition in specific embodiments.

Pharmaceutical compositions of the present invention, in addition to its active ingredients, include pharmaceutically acceptable carriers, excipients, and the like, oral formulations (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterile injectable aqueous solutions). Or oily suspensions), topical formulations (creams, lotions, ointments (semi-solid external preparations), microroemulsions, gels, pastes, transdermal agents (TTS) (such as patches, bandages, etc.), and the like.

As used herein, "pharmaceutically acceptable" means that the subject of application (prescription) is not as toxic as applicable, without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, derivatives thereof (such as sodium carboxymethyl cellulose, ethylcellulose, etc.), malt, gelatin, talc, Solid lubricants (such as stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, etc.), alginic acid, emulsifiers (such as TWEENS), wetting agents (Such as sodium lauryl sulfate), colorants, flavors, tableting agents, stabilizers, antioxidants, preservatives, water, saline, phosphate buffer solutions, and the like. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

Excipients may be selected and used according to the formulation of the pharmaceutical composition of the present invention, for example, when the pharmaceutical composition of the present invention is prepared with an aqueous suspending agent, suitable excipients are sodium carboxymethyl cellulose, methyl cellulose, hydropropylmethylcellulose And suspending agents and dispersing agents such as sodium alginate and polyvinylpyrrolidone. Suitable excipients when prepared from injection solutions include Ringer's solution, isotonic sodium chloride, and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably topically.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, and the severity of the patient, the dosage may limit the scope of the present invention in any aspect. It should not be understood as.

The composition of this invention can be grasped | ascertained by food compositions, such as a functional drink, in another specific aspect.

The food composition of the present invention may include sweeteners, flavoring agents, bioactive ingredients, minerals, etc. in addition to the active ingredients.

Sweeteners may be used in amounts that give the food a suitable sweet taste, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavoring agents can be used to enhance the taste or aroma, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

As the physiologically active substance, catechins such as catechin, epicatechin, gallocatechin, epigallocatechin, vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, riboflavin, and the like can be used.

As minerals, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc and the like can be used.

In addition, the food composition of the present invention may contain a preservative, an emulsifier, an acidulant, a thickener, and the like, in addition to the sweetener.

Such preservatives, emulsifiers and the like are preferably added and used in very small amounts as long as the use to which they are added can be achieved. By trace amount is meant numerically expressed in the range of 0.0005% to about 0.5% by weight based on the total weight of the food composition.

Examples of preservatives that can be used include sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), and the like.

Emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like.

Examples of acidulants that may be used include lead acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Such acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing taste.

Thickeners that can be used include suspending implements, sedimenters, gel formers, swelling agents and the like.

As described above, it is possible to provide an immunoadjuvant composition according to the present invention. The immune enhancing composition of the present invention may be commercialized as a drug or food.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Small beans Fermented  Produce

Example 1 Fermented Soybean Preparation Example 1

500 g of soybeans were mixed with 1.5 L of 30% ethanol (30% ethanol aqueous solution; the same as below), put into a container, sealed, and fermented at room temperature (20-30 ° C.) for 60 days to obtain a soybean fermentation broth. The fermentation broth was filtered through a 2 μm filter paper and then lyophilized to obtain a solid fermentation product.

Example 2 Fermented Soybean Leaf Preparation Example 2

The fermented soybean fermentation broth obtained in <Example 1> was kept at a low temperature (3-10 ° C.) for 45 days to prepare a ripe soybean fermentation broth. The fermentation broth was filtered through a 2 μm filter paper and then lyophilized to obtain a solid fermentation product.

< Comparative example > Preparation of Pea Extract

500 g of bean beans were immersed in 1.5 L of 70% ethanol (70% ethanol aqueous solution) and extracted with stirring for 24 hours. After extraction, the extract was filtered with 2 탆 filter paper and then lyophilized to obtain a solid extract.

< Reference Example > Tree  Preparation of Leaf Extract

500 g of leafless leaf cuttings were immersed in 1.5 L of 70% ethanol (70% ethanol aqueous solution) and extracted with stirring for 24 hours. After extraction, the extract was filtered with 2 탆 filter paper and then lyophilized to obtain a solid extract.

< Experimental Example > Immune enhancing activity experiment

Experimental Example 1 Evaluation of Proliferation Promoting Activity of Immune-Related Cells

The immune enhancing activity of the soybean fermented product and the extracts of the examples and comparative examples was evaluated as proliferation promoting activity against immune-related cell lines using the MTT evaluation method.

Immune-related cell lines used in the experiment were purchased from Korea Cell Line Bank (KCLB) as EL-4 murine T lymphoma cell line and macrophage line RAW264.7.

Each cell line was treated at 37 ° C., 5% CO ₂ using DMEM medium (Invitrogen, USA) containing 10% fetal calf serum (FBS) and 1% penicillin-streptomycin (100 units / mL). The incubator was adapted and passaged.

Each cell line was dispensed with 1.0 × 10 ⁴ cells in a 96 well plate filled with 90 μl per well, and the same amount of each fermentation product of the Example, the extract of Comparative Example, or the same mixture of the extract of Example 2 and Reference Example was 0.1. Prepared to a concentration of mg / ㎖ and added to 10 μl, incubated for 48 hours, and then MTT solution (3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide; Sigma prepared at a concentration of 5 mg / ㎖; , USA) 10 μl were added and further incubated for 4 hours. Afterwards, the medium was removed by centrifugation, and 150 µl of DMSO per well was added for reaction for 30 minutes at room temperature, and the absorbance was measured at 540 nm using a microplate reader (Bio-TEK instrumenis. Inc., USA). The degree of cell proliferation promotion was compared as a percentage of the control group compared to the absorbance value for the no control group.

The results are shown in Table 1 below as MEAN ± SD of three replicate experiments. Statistical treatment was performed by t-test, and statistically determined that the p value was 0.05 or less.

Promoting Proliferation of Immune-Related Cell Lines division EL-4 RAW264.7 Example 1 29.3 ± 3.4 * 35.4 ± 3.6 * Example 2 45.6 ± 2.7 * 59.6 ± 2.4 * Comparative example 18.2 ± 2.8 * 15.5 ± 2.8 * Example 2+ Reference Example 68.4 ± 3.1 * 75.7 ± 4.2 * Example 2 + Reference Example is a mixture of the same amount of the fermentation product of Example 2 and the extract of Reference Example
-* P <0.05

The results of Table 1 show that both the soybean fermented product and the soybean extract showed the proliferation promoting activity of the immune-related cell lines EL-4 and RAW264.7, among them, the proliferation promoting activity of the soybean fermented product of <Example 2>, In particular, the mixture of the soybean fermented product of <Example 2> and the leafless tree leaf extract of the reference example shows the highest growth promoting activity.

Experimental Example 2 Evaluation of Secretion Promoting Activity of Cytokines

The macrophage line RAW264.7 was dispensed at 1.0 × 10 ⁴ cells in a 96 well plate filled with 90 μl per well, and the same amount of each fermentation product, the extract of Comparative Example, or the same amount of the extract of Example 2 and Reference Example. Was prepared to a concentration of 0.1mg / ㎖ and added to 10ul, incubated for 24 hours, the culture medium was centrifuged at 12,000 rpm for 3 minutes to measure the cytokine secretion of macrophages for 3 minutes Got it. The cytokines TNF-α, IL-1β and IL-6 in the supernatants were measured using an ELISA assay kit (R & D Systems Inc., USA). Table 1]. Statistical treatment was performed by t-test, and statistically determined that the p value was 0.05 or less.

Evaluation of secretion promoting activity of cytokines division TNF-α (ng / ml) IL-1β (ng / ml) IL-6 (ng / ml) Control (no treatment) 0.67 ± 0.07 0.42 ± 0.03 0.31 ± 0.02 Example 1 2.96 ± 0.17 * 2.14 ± 0.25 * 1.97 ± 0.21 * Example 2 4.36 ± 0.28 * 3.94 ± 0.24 * 3.53 ± 0.24 * Comparative example 1.23 ± 0.28 * 0.72 ± 0.14 * 0.61 ± 0.05 * Example 2+ Reference Example 5.93 ± 0.19 * 5.15 ± 0.24 * 5.08 ± 0.18 * Example 2 + Reference Example is a mixture of the same amount of the fermentation product of Example 2 and the extract of Reference Example
-* P <0.05

The results of [Table 2] were generally similar to the results of [Table 1], and Saito in the order of a mixture of a soybean extract, a soybean fermented product, a soybean fermented product of <Example 2> and a non-berry extract of a reference example Caine secretion promoting activity was high.

Claims (6)

(I) a fermentation product obtained by sealing a mixture of soybeans and an ethanol aqueous solution and fermenting at room temperature (20 to 30 ° C.) for 50 to 70 days, or
(II) Sealing the mixture of soybean and ethanol aqueous solution to ferment at room temperature (20 ~ 30 ℃) for 50 ~ 70 days, and then aged for 40 ~ 50 days at low temperature (3 ~ 10 ℃) while maintaining the sealed state Immune enhancer composition comprising the fermented product as an active ingredient.
The method of claim 1,
The composition is an immune enhancer composition, characterized in that it further comprises a leafless tree leaf extract.
The method of claim 1,
The fermented product (I) is a fermented product obtained by fermenting a soybean for 30 days at room temperature (20 ~ 30 ℃) by sealing a mixture of 30% ethanol aqueous solution,
The fermentation product of (II) is fermented for 60 days at room temperature (20 ~ 30 ℃) by sealing the mixture of 30% ethanol aqueous solution of the soybeans, and then maintain the sealed state for 45 days at low temperature (3 ~ 10 ℃) Characterized in that it is a fermented soybean fermented by aging for a while
Immune enhancer compositions.
The method of claim 1,
The ethanol aqueous solution is an immune enhancer composition, characterized in that the ethanol content 20% (v / v) to 40 (v / v)%.
The method of claim 1,
The composition is an immune enhancer composition, characterized in that the pharmaceutical composition.
The method of claim 1,
The composition is an immune enhancer composition, characterized in that the food composition.