KR101832357B1

KR101832357B1 - A composition for the enhancement of immune system comprising extracts of soybean sprouts as an active ingredient and the method of preparation thereof

Info

Publication number: KR101832357B1
Application number: KR1020150157785A
Authority: KR
Inventors: 김소영; 이상명; 김가람
Original assignee: 대한민국(농촌진흥청장); 전북대학교산학협력단
Priority date: 2015-11-10
Filing date: 2015-11-10
Publication date: 2018-02-27
Also published as: KR20170055100A

Abstract

The present invention relates to an extract for enhancing an immune response derived from soybean sprouts and a method for producing the extract. The immune activity was confirmed in the bean sprouts extract, and the immunological activity was confirmed by mixing the bean sprouts extract and the lactic acid bacterium. As a result, Lactobacillus lambosus It was confirmed that the mixture of Leah and bean sprouts extracts had better immunity than that of the single treatment. When the bean sprouts and extraction conditions were examined, It was confirmed that the immunoactivity was the most excellent when the extract was performed 2 to 3 times and the mixture of the bean sprouts extract and the bean sprouts extract and the lactic acid bacteria showed immunological activity in mouse spleen cells. The composition containing the active ingredient is useful as an immunostimulating composition Can be used.

Description

TECHNICAL FIELD The present invention relates to an extract for the enhancement of immunity derived from soybean sprouts and to a method for preparing the same for extracting soybean sprouts,

The present invention relates to an extract for enhancing immunity derived from soybean sprouts and a method for producing the same, and aims to find a material excellent in immunological activity among the domestic agricultural food sources frequently consumed by the people and utilize it as a health functional food material .

Currently, the global health functional food market is estimated to be US $ 89 billion in 2011 and will exceed US $ 120 billion in 2016 (Lee et al., 2012) KHIDI Brief, 36, 1-8).

Immunization refers to a reaction that maintains the homeostasis of the body by distinguishing between self and non-self in the body, recognizing naturally occurring substances in the body or recognizing harmful substances from the outside (Cannon., 2000. News Physiol Sci, 15, 298-303). This reaction plays a very important role in resisting harmful bacteria such as viruses, bacteria and parasites that invade the human body from the outside, and in resisting or removing cancer cells inside.

Recently, as diseases related to immune system such as cancer, immunodeficiency, atopy and autoimmune are increasing due to various factors such as dietary change, aging, stress, smoking and drinking, and exposure to environmental pollution, Immunization studies using food materials are on the rise. In addition, not only natural materials but also lactic acid bacteria, which are present as intestinal microorganisms in healthy persons, also protect the host such as human body through the immune system. However, studies on the development of functional products and related products by the combination of plant material with high immunity activity and lactic acid bacteria are in short supply.

Soybean sprouts are germinated soybeans, which can be easily grown in a short time regardless of season, making them economical and nutritional supplements. Its color is white or light yellow, and it contains a lot of vitamin B ₁ , vitamin B ₂ , and vitamin C. Soybean, which is a raw material of bean sprouts, is rich in dietary fiber, isoflavone, and phenolic compounds and is known to be effective for diabetes, arteriosclerosis, heart disease, and anti-cancer effect (Madar Z., Am. J. Clin. Nutr., 43, pp388-396, 1984; Kim, Sook Hee et al., Physiological Activity of Chungkukjang, Food Industry and Nutrition, 4 (2), pp40-46, 1999).

Accordingly, the present inventors have found that a mixture of soybean sprout extract, lactic acid bacterium and bean sprouts exhibits an immunostimulating effect while trying to find an immunologically active material showing immunity enhancing effect and a lactic acid bacteria complex using the immunostimulating effect among foods frequently consumed by Korean people, Thus completing the present invention.

It is an object of the present invention to provide an extract for immune enhancement derived from soybean sprouts and a method for producing the same.

In order to achieve the above object, the present invention provides a food composition for enhancing immunity, which comprises soybean sprouts extract as an active ingredient.

The present invention also provides a food composition for enhancing immunity, comprising a bean sprouts extract, and a mixture of lactic acid bacteria or a culture solution of said lactic acid bacteria as an active ingredient.

The present invention also provides a feed additive for bee immune enhancement containing as an active ingredient a soybean sprout extract alone or a mixture of the soybean sprout extract and a lactic acid bacterium or a culture solution of the lactic acid bacterium.

The present invention also provides a pharmaceutical composition for immunoenhancing comprising a soybean sprout extract alone or a mixture of the soybean sprout extract and a lactic acid bacterium or a culture solution of the lactic acid bacterium as an active ingredient.

The present invention also provides a food composition for preventing and / or ameliorating hypomagnesa, which comprises a soybean sprout extract alone or a mixture of the soybean sprout extract and a lactic acid bacterium or a culture solution of the lactic acid bacterium as an active ingredient.

The present invention also provides a feed additive for preventing and treating hypo-immunodeficiency comprising as an active ingredient a soybean sprout extract alone or a mixture of the soybean sprout extract and a lactic acid bacterium or a culture solution of the lactic acid bacterium.

The present invention also provides a pharmaceutical composition for preventing and treating hypo-immunodeficiency comprising as an active ingredient a soybean sprout extract alone, or a mixture of the soybean sprout extract and a lactic acid bacterium or a culture solution of the lactic acid bacterium.

The present invention relates to an immunoconjugate composition containing an extract of soybean sprouts as an active ingredient, wherein when the soybean sprout ethanol extract is treated with RAW-Blue ™ cells, the NF-κB activity is increased and the NO production ability and the cytokine (TNF-α and IL-1β), and the mixing conditions of soybean sprout extract and lactic acid bacteria were examined. Weissella cibaria It was confirmed that the combination of JW15 and bean sprouts extract samples at 1: 0.25 ratio showed synergistic effects as well as increased production of immune-enhancing cytokines as well as NO production ability by proliferating macrophages more than when they were treated alone, The extraction method of extracting 70% ethanol with a solvent 2-3 times at 25 ° C temperature is superior to the cotyledon part of the cotyledon of the bean sprouts, and the immunological active component of the soybean sprout extract is extracted the highest, Confirming that the mouse spleen cells exhibit excellent immunosuppressive activity, the bean sprouts extract of the present invention can be effectively used as a composition for improving immunity.

1 is a graph comparing NF-κB activity by mixing soybean sprouts extract with lactic acid bacteria.
FIG. 1A shows NF-.kappa.B activity according to the soybean sprout extract, Lactobacillus plantarum 425 strain, and bean sprouts extract and the lactic acid bacteria mixing ratio.
1B is a graph showing NF-κB activity according to the mixing ratio of the bean sprouts extract, Lactobacillus rhamnosus GG strain, and bean sprouts extract and the lactic acid bacteria.
Figure 1c shows the results of a bean sprout extract, Weissella cibaria JW15 strain, and bean sprouts extract, and NF-κB activity according to the mixing ratio of the lactic acid bacteria.
Fig. 2 is a graph comparing NO production amounts by mixing soybean sprouts extract and lactic acid bacteria.
FIG. 2 (a) is a graph showing NO production amount according to the mixing ratio of the bean sprouts extract, Lactobacillus plantarum 425 strain, and soybean sprout extract and the lactic acid bacteria.
FIG. 2B is a graph showing the NO production amount according to the mixing ratio of the soybean sprout extract, Lactobacillus rhamnosus GG strain, and bean sprouts extract and the lactic acid bacteria.
FIG. 2c is a graph showing the results of extracting bean sprouts, Weissella cibaria JW15 strain, and bean sprouts extract, and the amount of NO produced according to the mixing ratio of the lactic acid bacteria.
FIG. 3 is a graph comparing TNF-.alpha. Production by mixing soybean sprout extract and lactic acid bacteria.
FIG. 3A shows the amount of TNF-α produced by the bean sprouts extract, Lactobacillus plantarum 425 strain, and soybean sprout extract and the lactic acid bacteria mixing ratio.
FIG. 3B is a graph showing the amount of TNF-α produced by the bean sprouts extract, Lactobacillus rhamnosus GG strain, and bean sprouts extract and the lactic acid bacteria mixing ratio.
Fig. 3c is a graph showing the results of extracting bean sprouts, Weissella cibaria JW15 strain, and bean sprouts extract, and the amount of TNF-α according to the mixing ratio of the lactic acid bacteria.
FIG. 4 is a graph comparing the amounts of IL-1? Produced by mixing soybean sprout extract and lactic acid bacteria.
FIG. 4A is a graph showing IL-1β production according to soybean sprout extract, Lactobacillus plantarum 425 strain, and soybean sprout extract and the lactic acid bacteria mixing ratio.
FIG. 4B is a graph showing the amount of IL-1? Produced according to the mixing ratio of soybean sprout extract, Lactobacillus rhamnosus GG strain, and soybean sprout extract and the lactic acid bacteria.
FIG. 4c shows the results of the production of the bean sprouts extract, Weissella cibaria JW15 strain, and bean sprouts extract, and the amount of IL-1? Produced according to the mixing ratio of the lactic acid bacteria.
Fig. 5 is a view for confirming the immunological activity depending on the bean sprouts extraction site.
5A is a graph comparing NF-κB activity according to bean sprouts.
FIG. 5B is a graph comparing NO production amounts according to bean sprouts. FIG.
FIG. 5C is a graph comparing TNF-.alpha. Production according to bean sprouts. FIG.
Fig. 6 is a graph showing immunoactivity according to the bean sprout extraction temperature. Fig.
6A is a graph comparing NF-κB activity according to bean sprouts extraction temperature.
FIG. 6B is a graph comparing NO production amounts according to bean sprouts extraction temperature. FIG.
6C is a graph comparing TNF-a production amounts according to bean sprouts extraction temperature.
Fig. 7 is a chart for confirming the immunoactivity according to the bean sprout extracting solvent.
7A is a graph comparing NF-κB activity according to a bean sprout extraction solvent.
FIG. 7B is a graph comparing NO production amounts according to bean sprouts extraction solvents. FIG.
FIG. 7C is a graph comparing TNF-.alpha. Production according to a bean sprout extraction solvent. FIG.
FIG. 8 is a graph showing immunoactivity according to the number of bean sprouts extracted. FIG.
8A is a graph comparing NF-κB activity according to the number of bean sprouts extracted.
FIG. 8B is a graph comparing NO production amounts according to the number of bean sprouts extracted. FIG.
8C is a graph comparing TNF-a production amounts according to the number of bean sprouts extracted.
Fig. 9 is a graph showing the change in the index component according to the bean sprouts extraction conditions. Fig.
9A is a diagram showing a standard curve of a method for measuring total polyphenol content using tannic acid as a standard substance.
FIG. 9B is a graph showing changes in the index component according to the extraction temperature. FIG.
FIG. 9c is a graph showing the change in the index component according to the extraction solvent.
FIG. 9D is a diagram showing a change in the index component according to the number of extraction times. FIG.
FIG. 10 shows the results of measurement of T cell proliferative activity of bean sprouts, lactic acid bacteria and bean sprouts complex using flow cytometry.
FIG. 11 shows the results of measuring the B cell proliferative activity of bean sprouts, lactic acid bacteria and bean sprouts complexes using flow cytometry.
12 is a graph comparing the T cell activity of soybean sprout extract, lactic acid bacterium alone, and lactic acid bacterium and bean sprouts complex in mouse spleen cells.
Figure 12a shows CD25 expression of CD4 + T cells.
12B is a graph showing CD69 expression of CD4 + T cells.
Fig. 13 is a graph comparing the B cell activity of soybean sprout extract, lactic acid bacterium alone, and lactic acid bacterium and bean sprouts complex in mouse spleen cells.
Figure 13a shows B220 + IgM + expression of B cells.
FIG. 13B shows MHC II + expression of B cells. FIG.
14 is a graph comparing Macrophage bacterium performance of soybean sprout extract, lactic acid bacterium alone, and lactic acid bacterium and bean sprout complex in mouse spleen cells.
Figure 14a shows CD86 expression of CD11b + cells.
14B is a diagram showing MHC expression of CD11b + cells.
Fig. 15 is a graph showing the amounts of bean sprouts extract, lactic acid bacterium alone, and the combination of lactic acid bacteria and bean sprouts in mouse spleen cells.
FIG. 15A is a graph showing the amount of IL-6 produced. FIG.
Fig. 15B is a graph showing the amount of TNF-a produced.
15C is a graph showing the amount of IFN gamma production measured.
FIG. 16 is a graph showing the amounts of bean sprouts extract, lactic acid bacteria alone, and immune cell active substances produced in a mixture of lactic acid bacteria and bean sprouts in mouse spleen cells. FIG.
16A is a graph showing the amount of IL-10 produced.
16B is a graph showing the amount of IL-17 produced.
16C is a graph showing the amount of IL-12 produced.

Hereinafter, the present invention will be described in detail.

The present invention provides a food composition for enhancing immunity which contains soybean sprouts extract as an active ingredient.

The bean sprouts may be cultivated or commercially available. The bean sprouts may be used without limitation. Any part of the hair or roots may be used, but according to the specific embodiment of the present invention, the immunity component of the hair (cotyledon) is superior.

The bean sprouts extract can be extracted with water, 100% ethanol, 70% ethanol, etc. According to a preferred embodiment of the present invention, the extract extracted with 70% ethanol is excellent in immunological activity.

The extraction temperature of the bean sprouts extract may be 25 to 70 캜. According to a specific embodiment of the present invention, extraction at 25 캜 is excellent in immunological activity.

The number of times of extraction of the bean sprouts extract may be 1 to 3 times, and according to a specific embodiment of the present invention, extraction is most preferably 2 to 3 times.

The bean sprouts extract may be mixed with lactic acid bacteria to increase the immunological activity.

Preferably, the lactic acid bacterium is Lactobacillus plantarum , Lactobacillus rhamnosus and Weissella cibaria . According to a specific embodiment of the present invention, lactobacillus rhamnosus and wi It is most preferred that it is Selashibarians.

The mixing ratio of the mixture of the lactic acid bacteria and the bean sprouts extract is preferably 1: 0.25 to 1: 1.

In a specific embodiment of the invention, the present inventors has confirmed that the immune activity in the bean sprouts extract (see Table 1 to Table 4), bean sprouts extract, lactic acid bacteria main Lactobacillus Planta volume (Lactobacillus plantarum), Lactobacillus ramno suspension (Lactobacillus rhamnosus and Weissella cibaria were mixed with each other to confirm the immunological activity. When Lactobacillus lambus and Wysselacivia were mixed with the bean sprouts extract, the immunological activity was confirmed to be better than that when treated alone (See FIGS. 1 and 4), immunological activity was checked according to the bean sprouts and the extraction conditions. As a result, the immunoreactivity was better in the cotyledon than in the tailed part (see FIGS. 5 and 5) (See FIG. 6 and Table 6), 70% ethanol, 100 ethanol and tertiary As a result, it was confirmed that the extract of 70% ethanol had the best immunological activity (see FIG. 7 and Table 7). Extraction with 2 to 3 times of extraction showed that the immune activity was the best (See FIG. 8 and Table 8), confirming that the mixture of the bean sprouts extract and the bean sprouts extract and the lactic acid bacterium mixture exhibited immunological activity in mouse spleen cells, the composition containing the bean sprouts extract of the present invention as an active ingredient, And the like.

The food composition according to the present invention includes all forms such as a general food, a functional food, a nutritional supplement, a health food, and a food additive. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.

For example, as the health food, the bean sprouts extract of the present invention and the mixture of the lactic acid bacteria or the culture liquid of the lactic acid bacteria itself may be prepared in the form of tea, juice and drink to be consumed, or the mixture may be paste, granulated, encapsulated, And can be ingested.

In addition, the bean sprouts extract and the mixture of the lactic acid bacteria or the culture liquid of the lactic acid bacteria may be prepared in the form of a powder or a concentrated liquid and added to food for the purpose of promoting growth of infants and adolescents. For example, beverages, fruits and their processed foods (eg canned fruits, bottles, jams, maalmalade), fish, meat and their processed foods (eg ham, sausage and confection), breads and noodles (Such as udon, buckwheat noodle, ramen noodle, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, dairy products such as butter, cheese, edible vegetable oil, margarine, vegetable protein, retort food, : Miso, soy sauce, sauce, etc.).

The content of the soybean sprout extract and the mixture of the lactic acid bacteria or the culture broth of the lactic acid bacteria in the food composition of the present invention is not particularly limited and may be 0.01 to 90%, preferably 0.1 to 50% . Furthermore, the food composition of the present invention may further contain a minor amount of minerals, vitamins, lipids, saccharides, and components having known immunological activity in addition to a soybean sprout extract or a mixture of lactic acid bacteria and bean sprouts.

The minerals may contain nutrients necessary for growing period, such as calcium and iron. Vitamins may include vitamin C, vitamin E, vitamin B ₁ , vitamin B ₂ and vitamin B ₆ . The lipid may include alkoxyglycerol or lecithin, and the saccharides may include fructo-oligosaccharides and the like.

The bean sprouts extract according to the present invention and the mixture of the lactic acid bacteria or the culture broth of the lactic acid bacteria may be added to the health supplement or health functional food such as food, In this case, the bean sprouts extract and the mixture of the lactic acid bacteria or the culture broth of the lactic acid bacteria may be added in an amount of 0.01 to 20% by weight, preferably 0.1 to 5% by weight based on the raw material when used as a food additive. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount exceeding the above range. The bean sprouts extract, and the mixture of the lactic acid bacteria or the culture broth of the lactic acid bacteria may be used together with other food or food ingredients and suitably used according to a conventional method.

There is no particular limitation on the kind of the food. Examples of the food to which the above extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, , An alcoholic beverage and a vitamin complex, and includes all the health foods in a conventional sense.

Various flavors or natural carbohydrates may be used in the food composition of the present invention. The natural carbohydrates include sugar monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As sweeteners, natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 part by weight, preferably 0.02 to 0.03 part by weight per 100 parts by weight of the composition according to the present invention.

In addition to the above, the composition according to the present invention may further comprise various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid concentrating agents, pH adjusting agents, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the composition according to the present invention may contain flesh for the production of natural fruit juices and vegetable drinks. These components can be used independently or in combination, and the proportion of the additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition according to the present invention.

Preferably, the lactic acid bacterium is Lactobacillus plantarum , Lactobacillus rhamnosus and Weissella cibaria . According to a specific embodiment of the present invention, lactobacillus rhamnosus and wi It is most preferred that it is Selashibarians.

The feed additive composition of the present invention may contain a phosphate or polyphenol such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, or organic acids such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate), catechin, Alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and the like.

Various adjuvants such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes, living microbial agents and the like are used as auxiliary components of the above feed additive, for example, cereals such as crushed or crushed wheat, oats, barley, corn And rice; Vegetable protein feedstuffs, for example, based on rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; A sugar ingredient and a milk product, for example, a dry ingredient consisting of various powdered milk and whey powder, and a dry additive are all mixed and then mixed with a liquid ingredient and a component which becomes a liquid after heating, that is, an animal fat And vegetable fats and the like can be used together with materials such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

The feed additive may be administered to the animal alone or in combination with other feed additives in an edible carrier. In addition, the composition for animal feed additives can be administered as top dressing or they can be mixed directly with the livestock feed or separately from the feed, in separate oral formulations, by injection or transdermal or in combination with other ingredients. Typically, a single daily dose or a divided daily dose can be used as is well known in the art.

When the feed additive is administered separately from an animal feed, the dosage form of the composition, as is well known in the art, can be prepared in an immediate release or sustained release formulation in combination with their non-toxic pharmaceutically acceptable edible carrier . Such edible carriers may 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 a tablet, capsule, powder, troche or emulsion or top-dressing in finely divided form. When a liquid carrier is used, it may be in the form of a soft gelatin capsule, or a syrup or suspension, emulsion or solution. In addition, the dosage form may contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoting agents and the like.

In addition, the feed comprising the feed additive may be any protein-containing organoleptic fraction commonly used to meet animal dietary needs. These protein-containing flours usually consist mainly of corn, soy flour or corn / soy flour mix.

The feed additive may be added to the animal feed by immersion, spraying or mixing. The present invention is applicable to a number of animal diets including mammals, poultry, and fish. More specifically, the diets may be used in commercial mammals, such as pigs, cows, sheep, goats, laboratory animals (e.g., rats, mice, hamsters and gervilus rats), fur- , And zoo animals (e.g., monkeys and tailless monkeys), as well as livestock (eg, cats and dogs). Common commercially important poultry include chickens, turkeys, ducks, geese, pheasants and quails. Commercial breeding fish such as trout can also be included.

In the method for mixing animal feed containing the feed additive according to the present invention, the animal feed additive is incorporated into animal feed in an amount of about 1 g to 100 g per 1 kg of feed on a dry weight basis. Also, after the feed mixture is thoroughly mixed, lightly viscous granulation or granulation material is obtained depending on the degree of crushing of the components. This is supplied as a mesh, or is formed into a desired discrete shape for further processing and packaging. At this time, in order to prevent separation during storage, it is preferable to add water to the livestock feed, followed by a usual pelleting, expansion, or extrusion process. Excess water can be dried off.

The composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term " pharmaceutically acceptable "as used herein means that the composition is free of toxicity to cells or humans exposed to the composition. Compositions comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, But may be at least one selected from the group consisting of polyvinylpyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, But are not limited to, ordinary carriers, excipients or diluents. The components can be added to the fermented rumba extract as an active ingredient independently or in combination.

Solid formulations for oral administration may include tablet pills, powders, granules, capsules and the like, which may contain one or more excipients, such as starch, calcium carbonate, sucrose or lactose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may also be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, Or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. There is no particular restriction on the dosage, and it may vary depending on the body's absorption, body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease and the like. The pharmaceutical composition of the present invention is prepared in consideration of an effective dose range, and the unit dosage formulations thus formulated are classified according to the judgment of the expert who monitors or observes the administration of the drug, if necessary, Or may be administered several times at a predetermined time interval. Preferably, the composition of the present invention may be administered at a dose of 0.5 to 5000 mg / kg, preferably 50 to 500 mg / kg, more preferably 50 mg / kg, per day based on the amount of the bean sprouts extract, The above administration may be carried out once a day or several times.

Hereinafter, the present invention will be described in detail with reference to 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 70% ethanol extract of bean sprouts

In order to evaluate the immunological activity of soybean sprouts extract, 70% ethanol extracts were prepared from soybean sprouts purchased from large grocery stores in Suwon area.

Specifically, bean sprouts were subjected to lyophilization (5-7 days, 20 torr, rack temp. -45 ° C, trap temp. -70 ° C) using a freeze dryer (Ilshin Lab Co., To form a powder. The extract was prepared by weighing a predetermined amount of powdered leather extract, adding 70% ethanol in an amount corresponding to 10 times the weight of the extract, and stirring at room temperature for 24 hours at 170 rpm using a stirrer (Jeiotech, Shaker, SK-71). The supernatant was collected by filtration (Advantec No. 6). The supernatant was collected by filtration twice using a rotary vacuum evaporator (5 to 7 days, 20 torr, rack temp. -45 ° C, trap temp. -70 ° C). The solvent was removed by centrifugation (EYELA, CCA-110, Tokyo, Japan). The dried bean sprout ethanol extracts thus obtained were stored in a deep freezer at -70 ° C. until the start of the experiment and were prepared according to the concentration conditions described below.

As a result, a bean sprout extract was obtained with a yield of 22.3%.

&Lt; Example 2 > Cell culture

To evaluate the immunological activity of soybean sprouts extract, RAW-Blue ™ cells were cultured by the following method.

Specifically, RAW-Blue ™ cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco-Invitrogen) medium containing 10% FBS (fetal bovine serum, Gibco-Invitrogen) and 1% penicillin-streptomycin (Gibco) And cultured at 5% CO ₂ . Transfection was performed once every two days and RAW-Blue ™ cells were cultured once a week using DMEM supplemented with Zeocin ™ (InvivoGen, USA).

<Experimental Example 1> Confirmation of NF-κB transcription factor activity

The expression of NF-κB / AP1 expressed by treating the RAW-Blue ™ cells cultured by the method of Example 2 with soybean sprouts extract prepared in Example 1 and a control (LPS treated and untreated) The activity of the transcription factor was measured. The RAW-Blue ™ cells were transfected with the NF-κB / AP-1 reporter cell line (RAW-Blue ™ cells, InvivoGen, USA) (Pink → blue / violet) caused by an indicator (Quanti blue), and it can be judged whether or not it is activated.

Specifically, RAW-Blue ™ cells were seeded in 96-well plates at a concentration of 1 × 10 ⁵ cells / ml for 24 hours, then treated with 100 μg / ml of soybean sprout extract and cultured for another 24 hours. Then, 20 μl of the supernatant and 200 μl of Quanti blue (InvivoGen, USA) reagent were mixed and reacted in a dark room for 10 minutes, and the absorbance was measured at 650 nm using a microplate reader (SpectraMax M2). The positive control group was LPS (lipopolysaccharide, Escherichia coli O11: B4, Sigma) at a concentration of 100 ng / ml was used as a negative control, and LPS-untreated was used as a negative control.

As a result, as shown in Table 1, the bean sprouts extract was highly active when treated with LPS (Table 1).

group

NF-kB activity (OD _{650 nm} ), Mean ± SD
LPS stimulation
(LPS stimulated) LPS non-stimulation
(LPS non-stimulated) The bean sprouts extract (100 占 퐂 / ml) 1.11 + 0.02 0.08 ± 0.00 Control group
LPS (+) 0.41 ± 0.00 LPS (-) 0.07 ± 0.00

Experimental Example 2 Determination of Nitric Oxide (NO) Production

To evaluate the immunological activity of soybean sprouts extract, the amount of nitric oxide (NO) production was determined.

Specifically, RAW-Blue ™ cells cultured by the method of Example 2 were divided into 96-well plates at a concentration of 1 × 10 ⁵ cells / ml, cultured for 24 hours, and treated with 100 μg / ml of sample extract And cultured for another 24 hours to obtain a supernatant. 50 μl of the supernatant was mixed with 100 μl of a reagent prepared by mixing the same amount of Griess reagent (Promega) I (Sulfanilamide solution) and II (NED solution) and reacted at room temperature for 10 minutes. Using a microplate reader (SpectraMax M2) Absorbance was measured at 540 nm. The concentration of nitric oxide was calculated using the standard curve of sodium nitrite.

As shown in Table 2, NO production was higher at 5.59 ± 0.20 μM / ㎖ than soybean sprout extract alone, and higher than that of LPS-treated control (3.94 ± 0.08 μM / ㎖) Respectively.

group

Nitric oxide (μM / ml) LPS stimulation (LPS stimulated) LPS Non-stimulated (LPS non-stimulated) Bean sprouts extract
(100 [mu] g / ml) 4.44 0.32 5.59 ± 0.20 Control group
LPS (+) 3.94 ± 0.08 LPS (-) 1.19 + 0.04

<Experimental Example 3> Cytokine measurement

In order to evaluate the immunological activity of the soybean sprouts extract, the amount of TNF-α and IL-1β produced in the macrophages treated with LPS was measured.

Specifically, RAW-Blue ™ cells cultured by the method of Example 2 were divided into 96-well plates at a concentration of 1 × 10 ⁵ cells / ml and cultured for 24 hours. Then, the cells were cultured in the same manner as in Example 1 One bean sprout extract was treated at 100 ㎍ / ㎖ and cultured again for 24 hours. Then, only the supernatant was taken and the cytokine content was measured by an ELISA kit (ebioscience, San Diego, California, USA) using Enzyme-linked immunosorbent assay (ELISA). To each well plate coated with an antibody of cytokine (TNF-α and IL-1β) (Mouse TNF-a ELISA Ready-Set Go and Mouse IL-1B ELISA Ready-Set Go, eBioscience), 100 μl of supernatant After incubation at room temperature for 2 hours, the supernatant was removed and washed 5 times with washing buffer consisting of PBS and Tween 20 (Sigma). Detection antibody solution was added to react with antibody. Then, Horseradish peroxidase (HRP) enzyme conjugated with Avidin was added and reacted at room temperature for 15 minutes. After that, TMB solution was added as a substrate for the HRP enzyme, and the change in color was confirmed. The cytokine (TNF-α and IL-1β) production was measured by this change because cytokine was produced and present in the sample. stop solution (H ₂ SO ₄ ) was added to terminate the reaction between the HRP enzyme and the TMB substrate, and the absorbance was measured at 450 nm using a microplate reader (SpectraMax M2).

As shown in Table 3, when the bean sprout ethanol extract was treated with LPS, the amount of TNF-α produced was 1509.55 ± 1.38 pg / ㎖, and the amount of TNF-α produced was 292.02 ± 3.27 pg / .

sample

TNF-α production (pg / ml) LPS stimulation
(LPS stimulated) LPS non-stimulation
(LPS non-stimulated) Bean sprouts extract
(100 [mu] g / ml) 1,509.55 ± 1.38 292.02 + - 3.27 Control group
LPS (+) 879.80 ± 3.27 LPS (-) 12.77 ± 2.28

As shown in the following Table 4, when the amount of IL-1β produced was measured, the amount of pg / ml was 54.56 ± 1.08.

sample

IL-1? Production (pg / ml) LPS stimulation
(LPS stimulated) LPS non-stimulation
(LPS non-stimulated) Bean sprouts extract
(100 [mu] g / ml) 54.56 ± 1.08 Non-detection Control group
LPS (+) 1.264.07 ± 0.96 LPS (-) 19.83 + - 5.51

Data are expressed as means ± SD (n = 3).

<Experimental Example 4> Evaluation of immune activity by mixing soybean sprout extract and lactic acid bacteria

In the above Experimental Examples 1 to 3, the effect of the ethanol extract of soybean sprouts was confirmed, and the immunological activity was evaluated by mixing with the lactic acid bacteria.

<4-1> Preparation of lactic acid bacteria sample

The Weissella < RTI ID = 0.0 > The isolated strains of cibaria JW15 (KACC 91811P) and Lactobacillus plantarum 4-25 (KACC 91936P) were purchased from the College of Veterinary Medicine of Chungbuk National University and were selected as lactic acid bacteria with probiotic function and immunological activity in the previous study. And used as a comparative strain. Lactobacillus rhamnosus GG is a commercial strain and well known as a strain with immune function, and was used as a control strain in this experiment. The strains were subcultured every 2 weeks in lactobacilli MRS broth (Difco, USA) for culture of lactic acid bacteria in a refrigerator at 4 ° C. The cells were suspended in a medium containing 50% glycerol sterilized before use and stored at -70 ° C. Of deep freezers.

Lactobacillus rhamnosus GG, Lactobacillus plantarum 4-25, and Weissella cibaria JW15 were cultured in MRS liquid medium at 37 ° C for 24 hours. Then, each strain was divided into live cells and dead cells. The culture broth was centrifuged at 12,000 rpm for 5 minutes (Gyrogen, Labogene, 1580R), and the supernatant was removed. The supernatant was removed and the absorbance (OD660nm) was measured by diluting with PBS. One viable cell was heat-treated at 100 ° C for 15 minutes to remove the supernatant, diluted with PBS, and adjusted to a concentration of 0.5 in absorbance.

<4-2> Measurement of NF-κB transcription factor activity

The activity of the expressed NF-κB / AP1 transcription factor was measured by mixing RAW-Blue ™ cells cultured by the method of Example 2 with the soybean sprout extract prepared in Example 1 above with lactic acid bacteria.

Specifically, RAW-Blue ™ cells were seeded in a 96-well plate at a concentration of 1 × 10 ⁵ cells / ml and cultured for 24 hours. Then, 100 μg / ml of soybean sprout extract prepared in Example 1 and OD Value of 0.5 were individually treated or mixed with lactic acid bacteria and sample extracts at a ratio of 1: 1, 1: 0.5, 1: 0.25, and then re-cultured for 24 hours. 20 μl of the cultured supernatant and 200 μl of Quanti blue (InvivoGen, USA) reagent were mixed and reacted for 10 minutes in a dark room. Absorbance was measured at 650 nm using a microplate reader (SpectraMax M2).

As a result, as shown in FIG. 1, when soybean sprout extracts were mixed with L. plantarum 4-25 at a mixing ratio of 1: 0.25, synergistic effect was shown to be highest at all ratios with dead bacteria, and Lactobacillus rhamnosus GG strain at 1: 0.25 ratio showed significant synergy effect. Weissella In the case of cibaria JW15 strain, there was no effect of NF-κB activation by mixing both live cells and dead cells (FIG. 1).

<4-3> Measurement of nitric oxide (NO) production

The bean sprouts extract prepared in Example 1 was mixed with lactic acid bacteria and treated with RAW-Blue ™ cells cultured by the method of Example 2 to confirm the change in NO production amount.

Specifically, RAW-Blue ™ cells cultured by the method of Example 2 were divided into 96-well plates at a concentration of 1 × 10 ⁵ cells / ml and cultured for 24 hours. Then, LPS was added to 100 ng / ml as a positive control And treated with PBS as a negative control. As a test group, bean sprouts extract at a concentration of 100 ㎍ / ㎖ and lactic acid bacteria with an OD value of 0.5 at 660 nm were individually treated, or the sample extract and lactic acid bacteria were mixed at a ratio of 1: 1, 1: 0.5, 1: 0.25 And cultured again for 24 hours. 100 μl of the reagent prepared by mixing the same amount of Griess reagent (Promega) Ⅰ (Sulfanilamide solution) and Ⅱ (NED solution) was added to 50 μl of the cultured supernatant, and the mixture was reacted at room temperature for 10 minutes, followed by using a microplate reader (SpectraMax M2) And the absorbance at 540 nm was measured. The concentration of nitric oxide was calculated using the standard curve of sodium nitrite.

As a result, as shown in Fig. 2, no increase in NO production was observed due to the similarity between soybean sprouts and Lactobacillus plantarum 425 strain alone and in the mixed group, and only when the ratio of soybean sprout extract and Lactobacillus rhamnosus GG mixture was 1: 0.25 , And it was confirmed that synergistic effect was significantly higher when bean sprouts extract and JW15 dead cells were mixed at a ratio of 1: 0.25 (FIG. 2).

<4-4> Cytokine measurement

The bean sprouts extract prepared in Example 1 was mixed with lactic acid bacteria and treated with RAW-Blue ™ cells cultured by the method of Example 2 to confirm changes in cytokine production amount.

Specifically, RAW-Blue ™ cells cultured by the method of Example 2 were divided into 96-well plates at a concentration of 1 × 10 ⁵ cells / ml and cultured for 24 hours. Then, 100 μg / ml of sample extracts Lactic acid bacteria with an OD value of 0.5 at 660 nm were treated with a combination treatment group consisting of a single treatment group, a sample extract and a lactic acid bacterium at a ratio of 1: 1, 1: 0.5, 1: 0.25, and cultured for another 24 hours. The cytokine content was measured with an ELISA kit (ebioscience, San Diego, California, USA) using an enzyme-linked immunosorbent assay (ELISA) in the same manner as in Experiment 3 with the supernatant cultured for 24 hours Respectively.

As a result, as shown in FIG. 3 and FIG. 4, the TNF-α production was slightly higher at a mixing ratio of 1: 0.25 with the soybean sprout extract and Lactobacillus plantarum 425, but there was no statistical significance and no mixing effect was observed in IL-1β production (Figs. 3 and 4).

In addition, bean sprouts extract, Lactobacillus rhamnosus when mixed GG strain live cells and dead cells, TNF-α production and IL-1β production is eopeotjiman mixing effect with the bacteria, Lactobacillus rhamnosus GG dead cells and 1: 0.25 high synergy significantly from the mixing ratio (Figs. 3 and 4).

Sprouts Extract and Weissella The amount of IL-1β produced when mixed with cibaria JW15 strain was significantly higher than that of JW15 bacteria at a mixing ratio of 1: 0.25 Showing synergy (Figs. 3 and 4).

Based on the results of in vitro phase immunoactivity functional evaluation described above, as compared with the positive control strain Lactobacillus rhamnosus GG strain, Weissella cibaria It was confirmed that the complex mixture of JW15 deadspring and bean sprouts extract mixture at 1: 0.25 ratio showed synergistic effects as well as increased production of immune enhancing cytokines and NO production ability by proliferating macrophages.

< Experimental Example 5> Comparison of immunological activity according to bean sprouts

The immunological activities of the bean sprouts were compared.

Specifically, soybean sprout samples were divided into cotyledon (head) and hypocotyl (hypocotyl), which were used for the experiment. (5-7 days, 20 torr, rack temp. -45 ° C, trap temp. -70 ° C) using a freeze dryer (Ilshin Lab Co., Ltd., Korea) 70% ethanol was added to the sample, and the mixture was extracted twice with 170 rpm for 24 hours at 25 ° C using an agitator, filtered through a filter paper (Advantec No. 6), and the solvent was removed using a rotary vacuum concentrator And then lyophilized. The extracted dry powder thus obtained was stored in a deep freezer at -70 ° C. In order to examine the immunological activity of each extract, NF-κB / AP-1 pathway transcription factor activation assay was performed in the cells of Example 2 in the same manner as Experimental Examples 1 to 3, (NO), IL-1β, and TNF-α cytokines.

As a result, as shown in Fig. 5, the comparison of NF-κB / AP1 transcription factor activity was about 3.5 times higher than that of untreated control group of cotyledon, and about twice as high as that of the control group. Cotyledon was significantly higher (Fig. 5A). In addition, the highest NO production was observed in the control group treated with LPS. The NO production capacity of the sample was higher than that of the cotyledon (2.23 ± 0.02 uM / ㎖) Significantly higher NO was produced, and cotyledon produced higher NO than control without LPS (FIG. 5b). As shown in Fig. 5C, both of the bean sprouts showed higher production of TNF-α than the control without LPS treatment, and the cotyledon (3110.03 ± The production of TNF-α was significantly higher than that of the pellet (2985.43 ± 24.70 pg / ml) (FIG. 5c) IL-1β production was 12.46 ± 3.95 pg / ㎖ higher than cotyledon (1.06 ± 1.00 pg / ㎖) and control (1.78 ± 1.32 pg / ㎖), respectively (Table 5).

sample 1L-1 [beta] production (pg / ml) The control (control) ND ^c) LPS 1.78 ± 1.32 ^c ⁾ all 15.65 + 2.01 ^a ⁾ Cotyledon 12.46 + 3.95 ^b ⁾ Hypocotyl 1.06 ± 1.00 ^c ⁾

Data are expressed as means ± SD (n = 3).

** Other characters at the top of the bar indicate significant differences (P <0.05).

When the immunological activity evaluation test of each sample part was integrated, the best part of the immunity activity was the cotyledon part, and the functional component of the cotyledon part was considered to affect the immune activation function. The results of NF-κB / AP1 transcription factor activity were 0.29 and 0.25 (OD _650nm ), respectively, and NO production was 1.40 ± 0.04 uM / ㎖ and 2.23 ± 0.02 uM / Respectively. On the other hand, the amount of TNF-α in the cytokine was higher than that of the cotyledon (3,110.03 ± 5.50 pg / ml) and the total fraction (16.36 ± 0.37 pg / ml) ) Was slightly higher than cotyledon (12.46 ± 3.95 pg / ml). Therefore, bean sprouts are thought to contain some components related to immunological activity in the cotyledon of the head region.

< Experimental Example 6> Activation of macrophages by extraction conditions

<6-1> Activation of macrophages by extraction temperature

Extracts of soybean sprout samples were prepared at 25 ℃ and boiling point of 70 ℃ with different extraction temperature and treated with macrophages.

Specifically, the product was lyophilized (5-7 days, 20 torr, rack temp. -45 ° C, trap temp. -70 ° C) using a freeze dryer (Ilshin Lab Co., Ltd., Korea) The extract was filtered twice with filter paper (Advantec No. 6) at 70 rpm, 70% ethanol, and extracted twice at 170 rpm at room temperature (25 ° C) or 70 ° C for 24 hours with a stirring extractor. The solvent was removed using a rotary vacuum concentrator and then lyophilized. The extracted dry powder thus obtained was used in the experiment while being stored in a deep freezer at -70 ° C, and the activity of NF-κB transcription factor, nitric oxide (NO) production, and cell activity were measured in the same manner as in Experimental Examples 1 to 3 The material was measured.

As a result, as shown in FIG. 6A, the activity of 25 ° C and 70 ° C was higher than that of LPS-treated control. However, the activity of 25 ° C and 70 ° C was higher than that of control without LPS. Activity was higher (Fig. 6A).

As shown in FIG. 6B, the amount of NO produced by treating each extract was 3.03 ± 0.02 uM / ml in the control group treated with LPS, and the highest NO production was observed in the LPS treated control group and the respective extracts (Fig. 6B).

The results of the experiments comparing the amount of cytokine produced to determine the macrophage activity according to the extract temperature are shown in FIG. 6C and Table 6. Compared with the control (3296.74 ± 3.42 pg / ㎖) treated with LPS, all extracts showed higher production of TNF-α, especially 3800.88 ± 558.33 pg / ㎖ at 25 ℃ But no statistical significance (FIG. 6c). However, IL-1β produced 16.36 ± 0.37 pg / ㎖ in extracts at 25 ℃, which was much higher than control (1.78 ± 1.32 pg / ㎖) (Table 6).

sample 1L-1 [beta] production (pg / ml) ^* The control (control) N.D. LPS 1.78 ± 1.32 ^b ⁾ 25 ℃ 16.36 ± 0.37 ^a ⁾ 70 ℃ N.D.

Data are expressed as means ± SD (n = 3).

When the immunoperoxidase activity was evaluated by the extraction temperature, the best extraction temperature was 25 ℃.

<6-2> Activation of macrophages by extraction solvent

The soybean sprout samples were extracted with various extraction solvents such as 70% ethanol, 100% ethanol and third distilled water, and the extracts were prepared by freeze-drying and treated with RAW-Blue cells to evaluate the immunoreactivity.

Specifically, the powdery sample was lyophilized by adding 10 times 70% ethanol, 100% ethanol and third distilled water. The mixture was stirred at room temperature (25 ° C) for 24 hours at 170 rpm twice The extract was filtered with filter paper (Advantec No. 6), and then the solvent was removed using a rotary vacuum concentrator and freeze-dried. The extracted dry powder thus obtained was used in the experiment while being stored in a deep freezer at -70 ° C, and the activity of NF-κB transcription factor, nitric oxide (NO) production, and cell activity were measured in the same manner as in Experimental Examples 1 to 3 The material was measured.

As shown in FIG. 7, the NF-κB / AP1 transcription factor activity was higher in the 70% ethanol extract and the third distilled water extract than in the 100% ethanol extract, but there was no significant difference between the two solvents 7a). The results of NO production of the extracts compared with the control group showed that the 70% ethanol extract showed the highest production of 1.40 ± 0.04 uM / ㎖, and the 100% ethanol extract and the third distilled water extract showed the highest production of 1.17 ± 0.03 uM / Ml and 1.19 + 0.04 uM / ml, respectively (Fig. 7b). Comparing the amount of TNF-α produced in cytokines, all extracts showed higher production than the LPS-treated control, and the 100% ethanol extract and the third distilled water were higher than the 70% ethanol extract, (Fig. 7c). As shown in Table 7, the amount of IL-1β produced in the 70% ethanol extract was 16.36 ± 0.37 pg / ㎖ in the control group (1.78 ± 1.32 pg / ml) . However, IL-1β was not produced in the 100% ethanol extract and the third distilled water extract (Table 7).

sample 1L-1 [beta] production (pg / ml) ^* The control (control) N.D. LPS 1.78 ± 1.32 ^b ⁾ Distilled water N.D. 70% ethanol 16.36 ± 0.37 ^a ⁾ 100% ethanol N.D.

Data are expressed as means ± SD (n = 3).

The most excellent extract for the immune activity was 70% ethanol extract.

<6-3> Comparison of activation of macrophages with extraction frequency

The extracts of bean sprouts were extracted once, twice, and three times to compare the immunoreactivity between the extracts.

Specifically, the powdery sample was lyophilized in 70% ethanol (10 times the weight), and extracted with a stirring extractor at room temperature (25 ° C) for 24 hours at 170 rpm for 1, 2, and 3 times After filtration with a filter paper (Advantec No. 6), the solvent was removed using a rotary vacuum concentrator, followed by lyophilization. The extracted dry powder thus obtained was used in the experiment while being stored in a deep freezer at -70 ° C, and the activity of NF-κB transcription factor, nitric oxide (NO) production, and cell activity were measured in the same manner as in Experimental Examples 1 to 3 The material was measured.

As a result, as shown in FIG. 8, the NF-κB / AP1 transcription factor activity was the highest in the control group treated with LPS, and the extracts showed higher activity than the extracts of 3 times in the 1st and 2nd extracts There was no significant difference between the 1st and 2nd extracts (Fig. 8a). The NO production among the extracts was the highest at 3.03 ± 0.02 uM / ㎖ in the control group treated with LPS, and the extracts were 1.40 ± 0.11 uM / ㎖, 1.40 ± 0.04 uM / Ml, and 1.55 ± 0.15 uM / ml, respectively, but there was no significant difference between the samples (FIG. 8b). As a result of comparing the amount of cytokine produced, the amount of TNF-α produced was slightly higher than that of the control group treated with LPS but not statistically significant (FIG. 8C ) And IL-1β cytokine production was found to be as high as 15.65 ± 2.01 pg / ㎖ and 20.03 ± 4.79 pg / ㎖ in the 2nd and 3rd extracts, respectively, as shown in Table 8 below, (Table 8).

sample 1L-1 [beta] production (pg / ml) ^* The control (control) N.D. LPS 1.78 ± 1.32 ^c ⁾ One-time extraction N.D. 2 times extraction 15.65 + 2.01 ^b ⁾ 3 times extraction 20.03 + - 4.79 ^a ⁾

Data are expressed as means ± SD (n = 3).

The highest number of immunological activity was obtained two or three times when the immunoperoxidase activity was evaluated by the number of times of extraction. The amount of the component influencing the immune activity seems to increase as the number of times of extraction increases.

Based on the results of the standardization studies on the soybean sprout samples, it can be expected that the extraction conditions having high immunological activity can be highly active when the cotyledon of bean sprouts is used and extracted three times at room temperature with 70% ethanol.

<6-4> Change of surface composition according to extraction condition

The total polyphenol content was measured as an indicator component in order to evaluate the immune activity and the change of the useful substance in the process of producing the bean sprouts extract.

Specifically, the total polyphenol content measurement method is a modification of the Folin-Denis method. A 100-ml round flask containing 100 mg of the sample is filled with distilled water and extracted with ultrasonic waves. By taking the 1 ㎖ extract and the absorbance is measured at 765 nm was introduced into distilled water for 7 ㎖ 0.5 ㎖ Folin-Ciocalteu's phenol reagent with saturated Na ₂ CO ₃ 60 minutes at room temperature was added sequentially 1 ㎖ reaction. Tannic acid was used as a reference material.

The results of comparing the total polyphenol contents according to the production conditions of the bean sprouts extract are shown in FIG. (6.72 ± 0.77 μg / mL and 11.37 ± 0.83 μg / mL at 70 ° C. and 25 ° C., respectively) at room temperature (FIG. 9). The total polyphenol contents of the extracts were 70.3%, 9.40 ± 0.92, and 8.27 ± 0.60 μg / mL, respectively, in the order of 70% ethanol, hot water and 94% ethanol. Finally, the total polyphenol content of the extracts was 9.24 ± 0.55 μg / mL at 1 time of extraction and 11.37 ± 0.83 and 10.52 ± 0.50 μg / mL at 2 to 3 times of extraction, respectively, but there was no statistical significance.

Based on the above results, the total content of polyphenols in soybean sprout extracts was determined according to the production conditions (extraction temperature, solvent, number of times). As a result, when extracting 2 to 3 times with 25% And the results were in good agreement with those of the previous immunoperoxidase activities.

< Example 7> Activation of spleen extract in mouse spleen immune cells

<7-1> Sample preparation of 70% ethanol extract of soybean sprouts

In order to evaluate the immunological activity of soybean sprouts extract, 70% ethanol extracts were prepared from soybean sprouts purchased from large grocery stores in Suwon area.

<7-2> Lactic acid bacteria sample preparation

Weissella The isolated strain of cibaria JW15 (KACC 91811P) was purchased from the College of Veterinary Medicine of Chungbuk National University and used for the experiment. The strains were subcultured every 2 weeks in lactobacilli MRS broth (Difco, USA) for 4 days. The cells were suspended in 50% glycerol sterilized before use and stored at -70 ° C deep freezer.

<7- 3> Mouse Splenocyte Separation, culture and sample treatment

The spleen was aseptically extracted from Balb / c mice (samtako), washed with RPMI 1640 solution, and then disrupted to obtain cells. The separated cell suspension was passed through a 200 mesh stainless steel sieve, centrifuged at 1,200 rpm for 3 minutes at 4 ° C, and the cell pellet was suspended in ACK buffer for 5 minutes to remove erythrocytes. The spleen cells were suspended in RPMI 1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin at a concentration of 1 × 10 ⁶ cells / ml, and 500 μl of each was dispensed into 48 well plates. 1: 0.25, 1: 0.5, 1: 1, 1: 0.25, 1: 0.5 and 1: 1, respectively, of the lactic acid bacteria prepared in Examples <7-1> and <7-2> and the soybean sprout extracts of 100, 200 and 400 ㎍ / : 1 mixed treatment group, and lipopolysaccharide (LPS) treated group. Cells treated as above were incubated at 37 ° C in a 5% CO ₂ incubator for 72 hours and the supernatant of the culture was stored at -20 ° C for cytokine production.

&Lt; 7-4 > Mouse spleen immune cells Proliferative ability Check measurement

The mouse spleen cells were treated with ethanol extract of soybean sprouts to confirm CD4 + T cell proliferation induction ability after 72 hours.

Specifically, the mouse spleen cells were washed twice with PBS, counted the number of cells, and injected into a 15 ml tube in an amount of 0.2 μl per 1.0 × 10 ⁷ cells / ml of spleen cells CFSE (ebioscience, cat # 65-0850-84) was added to a final concentration of 1 μM. Then, the tube was wrapped with a foil, allowed to react at room temperature for 10 minutes, and then a volume of RPMI-1640 medium having a volume of 4 times was added thereto, followed by reaction on ice for 5 minutes. After the reaction, the supernatant was centrifuged at 4 ° C and 1200 rpm for 8 minutes, and the supernatant was removed. The RPMI-1640 medium was added to 5 ml of the tube, and the cells were released. Washed three times. Then, the cells were transferred to a 96-well plate at 1.0 × 10 ⁶ cells / well and cultured for 72 hours in accordance with each treatment condition. The plate was then centrifuged to allow the cells to settle, washed once with FACS medium, and stained with CD4 + T cells using anti-mouse CD4 PerCP, and the degree of proliferation of the cells was analyzed.

As a result, as shown in Fig. 10, Weissella cibaria The JW15 dead cells had a higher CD4 + T cell proliferation inducing ability than the live cells, and the efficacy was better than that of LGG dead cells, but no synergistic effect was observed by addition of the bean sprouts extract (FIG. 10).

In addition, as shown in Fig. 11, the strain of Weissella cibaria JW15 was larger than that of live cells and had better efficacy than that of LGG, but no synergistic effect by the addition of the bean sprouts extract (Fig. 11) .

<7-5> Activation of mouse spleen immunocytes

The activities of T cells, B cells and macrophages were evaluated according to the combination of soybean sprouts extract and lactobacilli and soybean sprouts.

Specifically, the mouse spleen cells were separated by the method of Experimental Example < 7-3 >, followed by treatment of the combined treatment group of the bean sprouts extract and the mixture of the lactic acid bacteria and the bean sprouts. After 72 hours, spleen cells were analyzed for the activity of T cells, B cells and macrophages using fluorescence-activated cell sort (FACS). FACS analysis was performed as follows. The cultured spleen cells were collected, stained with trypan blue, and then washed with FACS medium. Monoclonal antibodies to the markers to be labeled were added to the cells, and the cells were treated with an ice bath for 60 minutes. T cells were analyzed for the expression patterns of activated biomarker molecules CD25 and CD69. B cells were analyzed for IgD and IgM expression and B220 (CD45R) and MHC class II (antigen delivery molecule) Expression of CD80, CD86 (co-stimulatory molecule) and MHC class II was measured using FACS.

As a result, as shown in Fig. 12, the control group treated with LPS had the highest expression level of CD25, and the next highest expression was Weissella cibaria JW15 live bacteria and soybean sprout extract (400 ㎍ / ml) at a ratio of 1: 1, but there was no statistical significance (Fig. 12). In the case of the live cells, expression was higher in the 1: 1 ratio than in the case of the isolate alone, but the expression was decreased in the ratio of 1: 0.5 (200 μg / ml) and 1: 0.25 (100 μg / ml) It looked. On the other hand, in the case of dead cells, the expression level of CD25 was similarly high in the combination of Weissella cibaria JW15 alone and the bean sprout mixture at a ratio of 1: 0.5, and the expression level of the remaining complexes was decreased. In the case of bean sprouts extract, there was no significant difference according to concentration. Another CD4 + T cell activation marker, CD69, is initially expressed in activated cells, and the result of the amount of CD69 expression is shown in FIG. 12b. The highest CD69 expression was observed when the bean sprouts extract was treated at a concentration of 400 μg / ml, and the soybean sprout samples also showed a dose-dependent increase in the expression level of CD69 (FIG. 12b). In addition, the JW15 strain also showed a synergistic effect on the activation of T cells by mixing with the bean sprouts extract sample, since the expression of CD69 was increased when the strain and the sample were treated together.

B cells were evaluated by measuring the proportion of mature B cells and measuring the expression level of MHC II. First, the results of measuring the proportion of mature B cells are shown in FIG. The above experiment measured the immature B cells (immature B cells) present in the spleen differentiated into mature B cells (mature B cells) when they were activated by the cell active substances. The highest proportion of mature B cells was found in Weissella cibaria JW15 live bacteria alone, followed by a high proportion of Weissella cibaria JW15 bacterium was significantly higher than other treatment groups. However, the ratio of mature B cells was decreased in the case of the mixture of the strain and the bean sprout extract sample, and the proportion of the B cell was gradually decreased as the concentration of the bean sprout extract alone increased (Fig. 13a) . This suggests that JW15 isolate alone activates immature B cells rather than mixed with bean sprouts extracts, and no synergistic effect on the complex was observed.

The results of measurement of MHC Ⅱ expression level, another B cell activation marker, are shown in FIG. 13B. The highest expression was the control group treated with LPS, followed by a single treatment of the bean sprouts extract, but the expression level gradually increased with increasing concentration, but there was no statistical significance (FIG. 13b). In the case of treatment with JW15 alone, the expression of MHC Ⅱ was increased in the combination of strain and bean sprouts extract samples compared to that of the strain alone, but it was significantly increased only in the mixture with dead cells. However, I did.

The activity of CD11b + macrophages was assessed by measuring the expression levels of CD86 and MHC Ⅱ. First, CD86 expression, a macrophage activation marker, is shown in FIG. 14A. The expression of LPS was significantly higher in the control group than in the LPS treated group, and the expression level of the mixture of Weissella cibaria JW15 and soybean sprout extract was slightly increased. However, the bacterial or bacterial isolate and the strain and the sample were mixed There was no statistical significance between one composite. In the case of bean sprouts extract samples, the expression level of CD86 was not changed even when the concentration was increased (FIG. 14A).

When macrophages are activated, the expression level of MHC class Ⅱ protein increases. This result is shown in Fig. 14B. All groups showed high expression similar to the control group, but among them, Weissella The amount of MHC class II expression was increased in the case of treatment with both live bacteria and dead cells of cibaria JW15 alone and with 1: 1 mixture of dead bacteria and bean sprouts extract samples, but the statistical significance was low, so there was no synergy (Fig. 14B). As a result of treatment with soybean sprout extract alone, the expression of MHC class II was gradually lowered as the concentration increased.

Based on these results of the above, in the case of the surface molecules related to T cell activation marker of CD25 and CD69 expression levels, Weissella Synergistic effect was obtained by increasing the expression level of the mixture of the strain and the bean sprouts extract 1: 1, rather than the sole treated with the cibaria JW15 strain alone. On the other hand, the results of the evaluation of the action performance of B220 + B cell showed that Weissella cibaria JW15 isolate alone was the highest proportion, and Weissella cibaria JW15 It was found that the dead cells activated B cells and differentiated into mature B cells. The expression of MHC Ⅱ, another B cell activation marker, was higher than that of the extract of soybean sprout extract alone. As a result of comparing macrophage activity, CD86 expression level was not significantly different from that treated with the strains alone and with the mixture of strains and samples. The expression level of MHC class Ⅱ protein was higher than that of the complex alone Higher. Therefore, when we examined the effect of bean sprouts extract and JW15 strain on the activation of splenocytes, the JW15 strain showed immune enhancement effect by activating immune cells in both live bacteria and dead bacteria, and when mixed with bean sprouts extract samples at high concentration, A slight increase in immunity enhancement could be expected.

&Lt; 7-5 > Active substance of mouse spleen immunocytes ( Cytokine ) Production amount measurement

Mouse spleen immunocytes were treated with soybean sprout extract and lactic acid bacterium to determine the amount of active substance produced.

Specifically, mouse spleen cells were isolated by the method of Experimental Example < 7-3 > to remove red blood cells, and then added to the cell culture medium at a concentration of 1 × 10 ⁶ cells / ml per 100 ml of the culture medium. (1: 0.25, 1: 0.5, 1: 1) and lipopolysaccharide (LPS) alone were used as the control. Each plate was incubated with cytokines (TNF-α, IFN-γ, and IFN-γ) in an ELISA kit (ebioscience, San Diego, California, USA) using an enzyme-linked immunosorbent assay (ELISA) IL6, IL10, IL17, IL12) were measured. 100 μl of the supernatant was added to a well plate coated with antibodies to cytokines (TNF-α, IFN-γ, IL6, IL10, IL17 and IL12) and reacted at room temperature for 2 hours. Tween 20 (Sigma), and the antibody information used in the experiment is shown in Table 9 below. and incubate for 15 minutes at room temperature. Add Avidin-conjugated Horseradish Peroxidase (HRP) to the reaction mixture. Then, TMB solution is added as a substrate for the HRP enzyme and reacted to confirm the color change. IL-10, IL-17, IL-16, IL-16, IL-16, IL-16, IL12 < / RTI > Stop solution (H ₂ SO ₄ ) was added to terminate the reaction between the HRP enzyme and the TMB substrate and the absorbance was measured at 450 nm using a microplate reader (SpectraMax M2).

Antibody Product Name manufacturer Mouse TNF alpha ELISA Ready-SET-Go! ®,
Cat # 88-7324-88 ebioscience Mouse IFN gamma ELISA Ready-SET-Go! ®,
Cat # 88-7314-88 ebioscience Mouse IL-6 ELISA Ready-SET-Go! ®,
Cat # 88-7604-88 ebioscience Mouse IL-17A (homodimer) ELISA Ready-SET-Go! ®, Cat # 88-7371-88 ebioscience Mouse IL-12 p70 ELISA Ready-SET-Go! ®,
Cat # 88-7121-88 ebioscience Mouse IL-10 ELISA Ready-SET-Go !,
Cat # 88-7104 ebioscience

As a result, as shown in Fig. 15A, Weissella IL-6 production was measured by treating cibaria JW15 strain and bean sprout sample alone or in combination. IL-6 production was the highest in control group treated with LPS alone, . However, when soybean sprout samples were treated alone, almost no IL-6 was produced even when the concentration of the sample was increased. In the case of treatment of a mixture of bacteria and a sample, there was no synergistic effect of the complexes, as IL-6 production was reduced rather than treated alone (Fig. 15a). IL-6 is a type of cytokine secreted by T cells and macrophages during immune stimulation.

TNF-α is produced by activation of macrophages and is also produced by other cells such as CD4 + T cells or NK cells, and its main role is to regulate immune cells. The result of measuring the amount of TNF-α produced is shown in FIG. 15B. The highest production was observed when JW15 dead cells and live cells were treated alone, and the production was significantly higher than that of the control. TNF-α production was decreased in the case of the combination of the bacteria and the sample, and when the sample was treated alone, almost no TNF-α was produced even when the concentration of the sample increased (FIG. 15B ). As a result, treatment of the strain alone increased the amount of TNF-α produced, and there was no synergistic effect of the mixture of the strain and the sample.

IFNγ is a cytokine that is secreted in T cells and NK cells and leads to a cell mediated immune response. It enhances macrophage activation mainly involved in innate immunity and induces MHC Ⅱ expression. The results of measuring the amount of IFN gamma production are shown in FIG. When JW15 dead cells were treated alone, the production was highest, and JW15 live bacteria also showed high production. IFN gamma production was decreased in the case of the combination of bacteria and sample. On the other hand, when the sample was treated alone, IFN gamma production was hardly generated even when the concentration of the sample increased (FIG. 15C). As a result, the production of IFNγ could be enhanced by treating the strain alone.

IL-10 is a cytokine involved in immunomodulation and inflammation that increases antibody production, regulates NF-κB activity, and regulates the JAT-STAT signaling system. The amount of IL-10 produced is shown in Figure 16a. IL-10 production was the highest in the control group treated with LPS, and the amount of IL-10 production among the complexes treated with the isolate alone, the strain and the sample mixed at a ratio of 1: 1 and 1: 0.5 (Fig. 16A). When the bean sprouts extract was treated alone, the amount of IL-10 produced was almost insignificant regardless of the concentration of the sample, so that the synergy between the strain and the sample did not appear.

IL-17 is an inflammatory cytokine that acts as an immune system response when exogenous pathogens enter the body and is known to synergize with TNF and IL-1. The amount of IL-17 produced is shown in FIG. 16B. The production of IL-17 was much higher in the case of the mixture of 1: 1 and 1: 0.5 ratio of soybean sprout extracts than when JW15 was treated alone. However, in the case of dead cells, Respectively. In the case of soybean sprout extract alone, no IL-17 was produced at all concentrations (Fig. 16B). Therefore, IL-17, which is not present in the single component in the mixture of JW15 and bean sprouts at a concentration of 100 ㎍ / ㎖, is a result of JW15 broth mixed with bean sprouts promoting T cell activation and producing more IL-17 .

IL-12 is a T-cell stimulating factor that stimulates T cell function and growth and activates IFNγ and TNF-α production. The results of measuring the amount of IL-12 produced are shown in Fig. 16c. The highest IL-12 production was treated with JW15 broth, and the JW15 broth and bean sprout extracts were slightly higher than those of single broth at 1: 1 ratio, but there was no statistical significance compared with single broth. On the other hand, the JW15 dead cells showed a much lower production than the live cells, but the production amounts of the dead cells and the sample were similar, and the production of IL-12 was not observed in the soybean sprout samples regardless of the concentration (FIG. As a result, when JW15 strain alone was treated with live bacteria, IL-12 production was high.

Claims

1. A food composition for immunomodulation comprising, as an active ingredient, a mixture of an extract obtained by extracting soybean sprouts with 60 to 80% ethanol and a culture solution of a lactic acid bacterium,
The lactic acid bacterium is any one selected from Lactobacillus rhamnosus or Weissella cibaria ,
Wherein the ratio of the bean sprouts extract and the lactic acid bacteria culture solution is 1: 0.15-0.35.

The food composition according to claim 1, wherein the bean sprouts are cotton cotyledons.

The food composition according to claim 1, wherein the bean sprouts extract is extracted 2 to 3 times at a temperature of 23 to 27 占 폚.

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The food composition according to claim 1, wherein the culture medium for lactic acid bacteria is heat-treated at 95 to 100 ° C for 10 to 20 minutes.

An animal feed additive comprising an extract of soybean sprouts with 60 to 80% ethanol and a mixture of lactic acid bacterium culture liquid as an active ingredient,
Wherein the lactic acid bacterium is any one selected from Lactobacillus lambatus or Wyselasia bivalves,
Wherein the ratio of the bean sprouts extract and the lactic acid bacteria culture solution is 1: 0.15-0.35.

A pharmaceutical composition for enhancing immunity, comprising as an active ingredient, a mixture of a fermented soybean sprout extract and an extract obtained by extracting soybean sprout with 60 to 80% ethanol,
Wherein the lactic acid bacterium is any one selected from Lactobacillus lambatus or Wyselasia bivalves,
Wherein the ratio of the bean sprouts extract and the lactic acid bacteria culture solution is 1: 0.15-0.35.

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