KR20230168761A - Composition for enhancing immune response comprising alfalfa sprout hydrolysate or polysaccharide fraction thereof - Google Patents

Abstract

The present invention relates to a composition for enhancing immune function, and can enhance immune function by containing alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient. Additionally, since it is non-toxic, it can be consumed in food form.

Description

A composition for enhancing immune function comprising alfalfa sprout hydrolysate or polysaccharide fraction thereof as an active ingredient {Composition for enhancing immune response comprising alfalfa sprout hydrolysate or polysaccharide fraction thereof}

The present invention relates to a composition that can enhance immune function by containing alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

Immune function regulation is a term that comprehensively encompasses the action of restoring normal immune function or alleviating changes in the body's normal immune function caused by environmental pollutants, side effects of medicines, diseases, and aging, and is generally undesirable. It is classified into immunosuppressive action, which is a regulatory action that suppresses an increased immune response, and immune enhancing action, a regulatory action that increases a decreased immune response.

Among these, immunostimulation is one of the important therapeutic strategies that strengthens the body's defense mechanism against various diseases such as cancer and inflammatory diseases. It stimulates the immune response by increasing the activity of immune cells to achieve an immune enhancement effect. You can. For example, macrophages play a major role in the immune response. Phagocytosis, a major role of macrophages, absorbs microorganisms and other pyrogenic particles, and also produces tumor necrosis factor-α (TNF-α) and interleukin. -Immune response by secreting a number of cytokines such as interleukin (IL-1β) and interleukin-12 (IL-12) and cytotoxic and inflammatory substances such as nitric oxide (NO) (Wolf et al., 1994; Lee and Hong, 2011; Murray and Wynn, 2011). Therefore, increasing macrophage activity can be a means to enhance immunity.

Since infections and diseases mainly occur when immune function is reduced, various studies are being conducted on how to enhance immune responses through immune-enhancing substances when the body's immune system function is reduced.

Meanwhile, macrophages are known to be involved in host defense and homeostasis by participating in various host responses such as innate immunity as well as adaptive immunity, and during immune responses, IL-1β (interleukin-1β) and IL-6 ( It is known as a cell that plays an important role in biological defense in the early stages of infection by producing cytokines such as interleukin-6) and tumor necrosis factor-α (TNF-α) (Journal of Pharmaceutical Sciences 2013; 57(6): 394 ~399). In addition, Jeon Chang-bae et al. (Journal of Pharmaceutical Sciences 2013; 57(6): 394~399) describe the macrophage immune-boosting effect of Polygonum aviculare L. extract, and Heo Seon-mi et al. (Kor. J. Pharmacogn. 2011; 42(3): 246-252) describes the immune-boosting effect of lectin (KML-C) isolated from Korean mistletoe.

Therefore, there is a continuous need for agents that can safely and effectively enhance immunity.

Republic of Korea Patent Publication No. 2021-0087406 Republic of Korea Patent No. 0687468 Republic of Korea Patent No. 2073175

The purpose of the present invention is to provide a food composition for enhancing immune function containing alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

In addition, another object of the present invention is to provide a food composition for enhancing immune function containing alfalfa water extract as an active ingredient.

In addition, another object of the present invention is to provide a pharmaceutical composition for enhancing immune function containing alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

In addition, another object of the present invention is to provide a feed composition for enhancing immune function containing alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

The food composition for enhancing immune function of the present invention to achieve the above object may contain alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

The alfalfa sprout hydrolyzate may be obtained by treating alfalfa sprouts with a hydrolytic enzyme.

The hydrolytic enzymes include alcalase, amyloglucosidase (AMG), celluclast, flavozyme, nutrase, protamex, and termamyl ( It may be one or more types selected from the group consisting of termamayl), ultraflo, and viscozyme.

The hydrolytic enzyme may be alcalase, amyloglucosidase (AMG), or celluclast.

The hydrolytic enzyme may be used in an amount of 0.1 to 3 parts by weight based on 100 parts by weight of alfalfa sprouts.

The alfalfa sprout hydrolyzate may be obtained by hydrolyzing alfalfa sprouts and then extracting them with hot water.

The alfalfa sprout polysaccharide fraction is a group consisting of alfalfa sprout hydrolyzate water, alcohol with 1 to 4 carbon atoms, hexane, ethyl acetate, chloroform, dichloromethane, and a mixed solvent thereof. It may be obtained by fractionation with a solvent selected from .

In addition, the food composition for enhancing immune function of the present invention to achieve the other purposes described above may contain alfalfa water extract as an active ingredient.

In addition, the pharmaceutical composition for enhancing immune function of the present invention to achieve the above-described other object may contain alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

In addition, the feed composition for enhancing immune function of the present invention to achieve the above-described other object may contain alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

The composition for enhancing immune function containing alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient of the present invention has the ability to promote the production of NO, phagocytosis, IL-1β or TNF-α in macrophages; Because it can exhibit effects such as high expression levels of iNOS and MAPKs, it can be widely used in the development of agents for improving immunity.

Figure 1 shows the effects of alfalfa sprout hydrolyzed hot water extract (alfalfa sprout hydrolyzate) and alfalfa sprout polysaccharide fraction prepared according to Examples 12 to 14 on iNOS protein expression in macrophages at different concentrations. This is a Western blot showing the results.
Figure 2 shows the effect of alfalfa sprout hydrolyzed hot water extract (alfalfa sprout hydrolyzate) and alfalfa sprout polysaccharide fraction prepared according to Examples 12 to 14 on MAPKs protein expression in macrophages at different concentrations. This is a Western blot showing the results.

The present invention relates to a composition that can enhance immune function by containing alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The composition for enhancing immune function of the present invention may contain alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient. Additionally, alfalfa water extract may also be contained as an active ingredient.

The alfalfa sprout has been used as a pasture and green manure crop in Europe and the United States since ancient times and is a perennial herb of the legume family. It has a mild taste, grows well even at low temperatures, contains dietary fiber and various vitamins, and is not only effective in preventing constipation, but also has the effect of lowering blood cholesterol.

The alfalfa sprout hydrolyzate of the present invention may be an extract obtained by hydrolyzing alfalfa sprouts with a hydrolytic enzyme or by hot water extraction after hydrolysis.

For example, alfalfa sprout hydrolyzate is prepared by mixing alfalfa sprouts and solvent at 1:10 to 40 parts by weight, preferably 1:20 to 35 parts by weight, adding hydrolytic enzyme, and heating at 40 to 70° C., preferably 50 to 60° C. It is obtained by treatment at ℃ for 15 to 30 hours, preferably 20 to 25 hours. The solvent is water, lower alcohol having 1 to 4 carbon atoms, ethylene glycol, ethyl ether, or a mixed solvent thereof; The lower alcohol may include 20 to 80% methanol, ethanol, butanol, or propanol. The solvent is not particularly limited, but extracts extracted with water are preferred for enhancing immune function.

If the weight ratio of the alfalfa sprouts and the solvent is outside the above range, a small amount of the active ingredient of the alfalfa sprouts may be obtained in the hydrolyzate. Additionally, if the hydrolysis temperature is outside the above range, hydrolysis may not be performed, and if the hydrolysis time is outside the above range, the immune function enhancing effect may be reduced.

In another example, the alfalfa sprout hydrolyzate is prepared by mixing the hydrolyzate treated with the hydrolytic enzyme and water at a weight ratio of 1:5 to 25, preferably 1:10 to 20, at 30 to 120° C., preferably 30 to 30° C. It is obtained by extraction at 110°C for 1 to 5 hours, preferably 2 to 24 hours.

If the weight ratio of the alfalfa sprout decomposition product and water is outside the above range, the active ingredient of the alfalfa sprout decomposition product may be extracted in a small amount in the extract. Additionally, if the extraction temperature and extraction time are outside the above range, the immune function enhancing effect may be reduced.

Hydrolytic enzymes used in the present invention include alcalase, amyloglucosidase (AMG), celluclast, flavozyme, nutrase, and protamex. ), termamayl, ultraflo, and viscozyme may include one or more selected from the group consisting of; Preferably, it may be amyloglucosidase (AMG) or ultraflo.

The hydrolytic enzyme is used in an amount of 0.1 to 3 parts by weight based on 100 parts by weight of alfalfa sprouts. If the hydrolytic enzyme content is less than the lower limit, hydrolysis may not be performed, and if it exceeds the upper limit, the yield decreases. and no further improvement can be achieved.

The alfalfa sprout hydrolyzate obtained by hydrolyzing the alfalfa sprouts with an enzyme and then extracting them with hot water contains an acidic sugar consisting of 5 to 10 mole% galacturonic acid and 1 to 6 mole% glucuronic acid; and mannose 10 to 30 mole%, rhamnose 1 to 8 mole%, glucose 10 to 30 mole%, galactose 20 to 40 mole%, xylose 1 to 5. It contains neutral sugars consisting of 10 to 20 mole% mole% and arabinose.

In addition, the alfalfa sprout polysaccharide fraction of the present invention is prepared by hydrolyzing the alfalfa sprouts with an enzyme and then adding hot water-extracted alfalfa sprout hydrolyzate to the fractionation solvent and precipitating it for 15 to 25 hours, preferably 20 to 24 hours, at 3 to 8 ° C. It can be obtained by doing it.

The alfalfa sprout hydrolyzate and the fractionation solvent are used at a weight ratio of 1:2 to 10, preferably 1:2 to 4.

If the weight ratio of the alfalfa sprout hydrolyzate and the fractionating solvent is outside the above range, the active ingredient of the alfalfa sprout hydrolyzate may be extracted in a small amount in the extract. Additionally, if the precipitation temperature and precipitation time are outside the above range, the immune function enhancing effect may be reduced.

The fractionating solvent may include a solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, hexane, ethyl acetate, chloroform, dichloromethane, and mixed solvents thereof. Preferably, it can be fractionated by immersion in 20 to 80% ethanol in terms of enhancing immune function.

The alfalfa sprout polysaccharide fraction contains 5 to 10 mole% galacturonic acid and 0.5 to 5 mole% glucuronic acid; and mannose 20 to 40 Mole%, rhamnose 0.5 to 5 Mole%, glucose 0.5 to 5 Mole%, galactose 30 to 60 Mole%, xylose 1 to 5. It may contain a neutral sugar consisting of 1 to 8 mole% arabinose and 0.1 to 1 mole% fucose.

The term ‘fraction’ used herein when referring to alfalfa sprouts includes not only fractions obtained by treatment with an extraction solvent, but also processed products of alfalfa sprout polysaccharide fractions. For example, alfalfa sprout polysaccharide fraction can be prepared in powder form by distillation of alfalfa sprouts and further processes such as freeze-drying or spray-drying.

In addition, the alfalfa sprout hydrolyzate or polysaccharide fraction thereof of the present invention, in a broad sense, also includes alfalfa sprout processed products formulated so that alfalfa sprouts can be administered to animals, such as alfalfa sprout powder. Although the present invention conducted experiments with alfalfa sprout hydrolyzate or its polysaccharide fraction, those skilled in the art would be able to predict that the desired effect can be achieved even in the form of alfalfa sprout processed product.

Meanwhile, in this specification, the term ‘containing as an active ingredient’ means containing a sufficient amount to achieve the efficacy or activity of the alfalfa sprout hydrolyzate or its polysaccharide fraction. For example, the alfalfa sprout hydrolyzate or its polysaccharide fraction is used at a concentration of 1 to 20 mg/Kg, preferably 3 to 10 mg/kg. Since the alfalfa sprout hydrolyzate or its polysaccharide fraction is a natural product and has no side effects on the human body even when administered in excessive amounts, the upper quantitative limit of the alfalfa sprout hydrolyzate or its polysaccharide fraction contained in the composition of the present invention can be selected and implemented within an appropriate range by a person skilled in the art. there is.

The present invention relates to a food composition for enhancing immune function containing alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

The 'food composition' includes alfalfa sprout hydrolyzate or polysaccharide fractions thereof as active ingredients, as well as food raw materials and food additives that are listed in the Food Standards and Specifications ('Food Code') commonly used in food production. Contains the listed food additives.

There is no need to specifically limit it, but examples include proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include monosaccharides, such as glucose, fructose, etc.; Disaccharides such as maltose, sugar, lactose, etc.; Oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin, etc.; Sugar alcohols such as xylitol, sorbitol, erythritol, etc. can be used. The flavoring agent may be a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) or a synthetic flavoring agent (saccharin, aspartame, etc.).

When preparing a food composition using the alfalfa sprout hydrolyzate or its polysaccharide fraction as an active ingredient, there is no need to specifically limit the content of the alfalfa sprout hydrolyzate or its polysaccharide fraction as long as it can enhance immune function, for example, 0.1 to 99%. It may be included in weight%, 0.5 to 95% by weight, 1 to 90% by weight, 2 to 80% by weight, 3 to 70% by weight, 4 to 60% by weight, and 5 to 50% by weight.

The alfalfa sprout hydrolyzate or polysaccharide fraction thereof, which is an active ingredient in the above food composition, varies depending on the condition, body weight, presence, degree, and period of the disease of the consumer, but may be appropriately selected by a person skilled in the art. For example, based on the daily dosage, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, even more preferably 20 to 800 mg, and most preferably 50 to 500 mg. There is no need to specifically limit the number of administrations, but a person skilled in the art can adjust it within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.

The food composition does not need to be particularly limited, but may be, for example, powder, granule, tablet, capsule, pill, extract, jelly formulation, tea bag formulation, or beverage formulation.

In addition, the alfalfa sprout hydrolyzate or its polysaccharide fraction can be added to general foods to provide immune function enhancement functionality. Foods that can be added do not need to be specifically limited, but for example, foods according to Article 7 of the Food Sanitation Act. Confectionery, bread or rice cake, processed cocoa products or chocolate, processed meat or egg products, processed fish meat products, tofu or jelly, noodles, tea, coffee, beverages, special purpose foods, as exemplified in the standards and specifications ('Food Code of Conduct'). It can be added to sauces, seasoned foods, dressings, kimchi, salted seafood, pickled foods, stewed foods, alcoholic beverages, raisins, and other foods. In addition, it can be added to dairy products, processed meat products, packaged meat, and egg products as exemplified in the livestock product processing standards and ingredient specifications ('Livestock Product Code') pursuant to Article 4 of the Livestock Products Sanitation Management Act.

Meanwhile, the food composition containing the alfalfa sprout hydrolyzate or its polysaccharide fraction as an active ingredient can be used alone as a “health functional food for enhancing immune function.”

The above ‘health functional food’ refers to food manufactured (including processing) in accordance with legal standards using raw materials or ingredients with functional properties useful to the human body (Article 3, Paragraph 1 of the Health Functional Food Act). The terminology or scope of the above 'health functional food' may vary depending on the country, but it is also called 'Dietary Supplement' in the United States, 'Food Supplement' in Europe, 'Health Functional Food' or 'Health Functional Food' in Japan. It may be classified as ‘Food for Special Health Use (FoSHU)’ or ‘Health Food’ in China.

The above food composition or health functional food may additionally contain food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions of the ‘Food Additive Code’ and general test methods, etc., unless otherwise specified. Follow.

In addition, the above-mentioned health functional food contains the alfalfa sprout hydrolyzate or its polysaccharide fraction along with raw materials notified as ‘functional raw materials’ used in “health functional food for enhancing immune function” or individually recognized raw materials to enhance immune function. Functional foods can be manufactured.

The present invention relates to a feed composition for enhancing immune function comprising alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

In addition to alfalfa sprout hydrolyzate or polysaccharide fractions thereof, the 'feed composition' can use food raw materials usable as foods as described in the Food Standards and Specifications ('Food Code') and food additives described in the Food Additive Code as active ingredients. , Even if they are not food raw materials or food additives that can be used as food, raw materials that fall within the scope of single feed in Annex 1 of the ‘Standards and Specifications for Feed, etc.’ and raw materials that fall within the scope of supplementary feed in Annex Table 2 can be used.

The ‘feed composition’ may be an extractant among supplementary feeds in accordance with ‘Standards and specifications for feed, etc.’, or may be a compound feed containing the above supplementary feed.

When preparing a feed composition using the alfalfa sprout hydrolyzate or its polysaccharide fraction as an active ingredient, there is no need to specifically limit the content of the alfalfa sprout hydrolyzate or its polysaccharide fraction as long as it can enhance immune function, for example, 0.1 to 99%. It may be included in weight%, 0.5 to 95% by weight, 1 to 90% by weight, 2 to 80% by weight, 3 to 70% by weight, 4 to 60% by weight, and 5 to 50% by weight.

The alfalfa sprout hydrolyzate or polysaccharide fraction thereof, which is an active ingredient in the feed composition, varies depending on the condition, body weight, presence, degree, and period of disease of the ingesting animal, but may be appropriately selected by a person skilled in the art. For example, based on the daily dosage, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, even more preferably 20 to 800 mg, and most preferably 50 to 500 mg. There is no need to specifically limit the number of administrations, but a person skilled in the art can adjust it within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.

The present invention relates to a pharmaceutical composition for enhancing immune function containing alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

Additionally, the present invention relates to a pharmaceutical composition for animals for enhancing immune function comprising alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.

In addition, the present invention provides a new use of alfalfa sprout hydrolyzate or its polysaccharide fraction for the manufacture of medicines for enhancing immune function or medicines for animals.

The 'pharmaceutical composition', 'medicine', 'pharmaceutical composition for animals' or 'veterinary medicine' includes alfalfa sprout hydrolyzate or polysaccharide fractions thereof as active ingredients, as well as appropriate carriers and excipients commonly used in the production of pharmaceutical compositions, etc. And it may further include a diluent.

The ‘carrier’ is a compound that facilitates the addition of the compound into cells or tissues. The ‘diluent’ is a compound diluted in water that not only stabilizes the biologically active form of the target compound, but also dissolves the compound.

There is no need to specifically limit the carrier, excipient, and diluent, but for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The amount of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may vary depending on the age, gender, and weight of the patient or animal to be treated, and, above all, the condition of the subject to be treated, the specific category of the disease to be treated, or It will depend on the type, route of administration, and properties of the therapeutic agent used.

The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals is appropriately selected depending on the absorption rate of the active ingredient in the body, the excretion rate, the age, weight, sex, and condition of the patient or animal to be treated, and the severity of the disease to be treated. , it is generally preferred to administer 0.1 to 1,000 mg/kg per day, preferably 1 to 500 mg/kg, more preferably 5 to 250 mg/kg, and most preferably 10 to 100 mg/kg. The unit dosage form formulated in this way can be administered several times at regular time intervals as needed.

The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may be administered individually as a preventive or therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The pharmaceutical compositions, medicines, pharmaceutical compositions for animals, or medicines for animals can be used by formulating them into oral dosage forms such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods. there is. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations include the compound with at least one excipient, such as starch, calcium carbonate, sugar or lactose, and gelatin. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .

The dosage, administration method, and frequency of administration for the treatment may refer to the dosage, administration method, and frequency of administration of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and it is clear to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the attached patent claims.

[Alfalfa sprout hydrolyzate_by enzyme]

Control group 1.

Alfalfa sprouts and water were mixed at a weight ratio of 1:33 and stirred in a shaking water bath for 24 hours to obtain an alfalfa sprout extract.

Examples 1 to 9.

After mixing alfalfa sprouts and water at a weight ratio of 1:33, hydrolytic enzyme (1 part by weight per 100 parts by weight of alfalfa sprouts) was added and stirred in a shaking water bath for 24 hours under the conditions shown in Table 1 below. Then, the enzyme was inactivated by heating to 100°C and filtered to obtain alfalfa sprout hydrolyzate.

division enzyme pH Temperature (℃) Control group 1 - - 25 Example 1 alcalase 8.0 50 Example 2 Amyloglucosidase (AMG) 4.5 60 Example 3 celluclast 4.5 50 Example 4 Flavozyme 7.0 50 Example 5 nutrase 6.0 40 Example 6 protamex 6.0 40 Example 7 termamayl 6.0 60 Example 8 ultraflo 7.0 60 Example 9 viscozyme 4.5 50

<Test Example Ⅰ>

Macrophages (Raw 264.7 cells) were grown in DMEM (Gibco, USA) medium prepared by adding 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) in 5% CO ₂ at 37°C. Cultured under conditions. Cells were checked for confluence every two days and subcultured at a concentration of 1X10 ⁵ cells/dish when 70-80% confluence.

^### p<0.001 compared with Normal, ^* p<0.05, ^*** p<0.001 compared with LPS

Test Example 1. Toxicity measurement in Raw 264.7 cells

To confirm the cytotoxicity of the alfalfa sprout hydrolysates prepared in Control Group 1 and Examples 1 to 9, Raw 264.7 cells were distributed and cultured in a 96-well plate at a concentration of 2X10 ⁴ cells/100 μL/well. After culturing for 18 hours, the medium was replaced with serum-free DMEM without FBS, samples of each concentration were treated with lipopolysaccharide (LPS) at a concentration of 1 μg/mL, and cultured for 20 hours. After culturing, the medium was removed, treated with 100 μg/mL MTT solution diluted in the medium, and reacted in an incubator for 3 hours. After the reaction, the upper medium was removed, 100 μL of DMSO was added to each well to dissolve formazan, and the absorbance of the dissolved formazan was measured at 540 nm using a microplate reader.

division 0.5 μg/mL 1 μg/mL 2.5 μg/mL 5 μg/mL Normal 100.0 ± 1.1 100.0 ± 1.1 100.0 ± 0.8 100.0 ± 0.7 LPS 72.8 ± 2.4 ^### 72.8 ± 2.4 ^### 72.3 ± 0.2 ^### 73.9±0.2 ^### Control group 1 97.7 ± 1.9 ^*** 92.3 ± 1.0 ^*** 91.7 ± 1.9 ^*** 85.2 ± 0.5 ^*** Example 1 97.5 ± 0.7 ^*** 97.8 ± 2.8 ^*** 92.1±0.0 ^*** 86.5 ± 0.3 ^*** Example 2 96.8 ± 1.9 ^*** 97.7 ± 1.9 ^*** 96.9 ± 0.1 ^*** 88.8 ± 0.1 ^*** Example 3 95.0 ± 1.0 ^*** 95.7 ± 0.2 ^*** 94.0 ± 0.1 ^*** 91.4 ± 0.0 ^*** Example 4 94.7 ± 0.4 ^*** 95.6 ± 0.4 ^*** 95.3 ± 0.5 ^*** 90.5 ± 0.1 ^*** Example 5 97.6 ± 1.1 ^*** 97.4 ± 0.2 ^*** 97.7 ± 0.4 ^*** 83.1 ± 0.6 ^*** Example 6 98.5 ± 4.2 ^*** 98.9 ± 0.2 ^*** 97.5 ± 0.2 ^*** 91.0 ± 0.0 ^*** Example 7 97.1 ± 5.9 ^*** 96.6 ± 3.0 ^*** 94.3 ± 0.4 ^*** 91.9 ± 0.1 ^*** Example 8 96.8 ± 1.3 ^*** 96.9 ± 0.8 ^*** 92.9 ± 1.1 ^*** 87.3 ± 0.7 ^*** Example 9 99.4 ± 2.4 ^*** 99.4 ± 1.6 ^*** 88.9 ± 0.9 ^*** 83.7 ± 1.6 ^***

As shown in Table 2 above, the alfalfa sprout hydrolysates prepared according to Control Group 1 and Examples 1 to 9 were confirmed to have excellent cell viability at a concentration of 0.5 to 5 ug/ml.

Test Example 2. Measurement of the effect of promoting nitric oxide production

To confirm the effectiveness of the alfalfa sprout hydrolysates prepared in Control Group 1 and Examples 1 to 9 in enhancing nitric oxide (NO) production, Raw 264.7 cells were distributed and cultured in a 96-well plate at a concentration of 2X10 ⁴ cells/100μL/well. . After culturing for 18 hours, the medium was replaced with serum-free DMEM without FBS, treated with samples of each concentration and LPS at a concentration of 1 μg/mL, and cultured for 20 hours. After incubation, the upper layer of medium was taken and transferred to an extra 96-well plate, and the same amount of griess reagent was added and reacted for 20 minutes. The absorbance of the reaction product was measured at 570 nm using a microplate reader.

[Table 3] below shows the NO expression level by concentration of the alfalfa sprout hydrolyzate prepared according to Control Group 1 and Examples 1 to 9 of the present invention.

division 0.5 μg/mL 1 μg/mL 2.5 μg/mL 5 μg/mL Normal 100.0 ± 3.6 100.0 ± 3.6 100.0 ± 0.3 100.0 ± 0.3 LPS 305.5 ± 0.5 ^### 305.5 ± 0.5 ^### 294.5 ± 0.3 ^### 294.5 ± 0.3 ^### Control group 1 126.3 ± 1.3 ^*** 148.6 ± 2.3 ^*** 172.5 ± 1.9 ^*** 197.0 ± 7.5 ^*** Example 1 100.2 ± 2.8 ^*** 119.1 ± 2.4 ^*** 164.7 ± 1.2 ^*** 197.8 ± 7.0 ^*** Example 2 107.0 ± 3.2 ^*** 125.8 ± 2.9 ^*** 165.6 ± 0.3 ^*** 223.8 ± 3.8 ^*** Example 3 102.5 ± 3.9 ^*** 114.7 ± 1.4 ^*** 139.0 ± 1.6 ^*** 173.8 ± 1.5 ^*** Example 4 98.7 ± 3.9 ^*** 106.5 ± 2.5 ^*** 132.4 ± 3.8 ^*** 168.0 ± 5.1 ^*** Example 5 98.6 ± 3.6 ^*** 111.1 ± 3.1 ^*** 148.2 ± 2.1 ^*** 177.3 ± 0.2 ^*** Example 6 103.1 ± 1.2 ^*** 125.0 ± 1.7 ^*** 156.6 ± 0.6 ^*** 193.7 ± 1.0 ^*** Example 7 104.8 ± 2.3 ^*** 125.1 ± 4.6 ^*** 153.0 ± 0.3 ^*** 189.6 ± 0.7 ^*** Example 8 122.2 ± 1.6 ^*** 139.8 ± 2.6 ^*** 161.8 ± 3.9 ^*** 204.4 ± 6.9 ^*** Example 9 101.0 ± 0.9 ^*** 122.1 ± 4.3 ^*** 182.5 ± 4.3 ^*** 192.3 ± 0.1 ^***

As shown in Table 3 above, among the alfalfa sprout hydrolysates prepared according to Examples 1 to 9, it was confirmed that Examples 2 and 8 had higher NO expression levels than the other groups at all concentrations.

In particular, it was confirmed that Example 2 had a higher NO expression level compared to Control Group 1 at a concentration of 5 ug/ml, and that Example 1 had a lower NO expression level at all concentrations and even at a concentration of 5 ug/ml.

Generally, in the immune system, it is known that when immune effector cells are induced to produce a large amount of NO under the influence of cell activators, which are immune transmitters, a large amount of NO is released to the site of the immune inflammatory reaction, resulting in an immune response.

From the above results, it was analyzed that alfalfa sprout hydrolyzate stimulates immune cells to provide primary defense against infections such as invading foreign substances, and has an immune-enhancing effect on biological defense in the early stage of the immune response.

Test Example 3. Measurement of phagocytosis activity

To confirm the effectiveness of the alfalfa sprout hydrolysates prepared in Control Group 1 and Examples 1 to 9 in enhancing phagocytosis activity, Raw 264.7 cells were distributed and cultured in a black wall 96-well plate at a concentration of 2X10 ⁴ cells/100μL/well. After culturing for 18 hours, the medium was replaced with DMEM without FBS, and samples of each concentration were treated with LPS at a concentration of 1 μg/mL and incubated for 1 hour. After culture, phagocytosis assay was performed using EZCell ^TM Phagocytosis Assay Kit (Cat NO. K963-100, Biovision). Dispense 5 μL of E.coli slurry into each well of cells cultured with the sample and culture in an incubator for 2-3 hours. After incubation, remove the medium, dispense 100 μL of quenching solution diluted in assay buffer, and react for 2 minutes. Afterwards, the quenching solution was removed, the cells were washed with assay buffer, and the absorbance was measured at 490/520 nm (Ex/Em) using a microplate reader.

division 2.5 μg/mL 5 μg/mL Normal 100.0 ± 3.0 100.0 ± 1.2 LPS 164.2 ± 0.4 ^### 165.0 ± 2.1 ^### Control group 1 123.8 ± 2.4 ^*** 149.1 ± 2.4 ^*** Example 1 119.7 ± 7.1 ^*** 153.7 ± 1.1 ^** Example 2 143.2 ± 5.1 167.7 ± 0.6 Example 3 124.8 ± 2.7 ^*** 157.6 ± 1.2 ^* Example 4 118.0 ± 8.0 ^*** 148.2 ± 0.6 ^*** Example 5 130.9 ± 2.0 ^*** 149.3 ± 0.9 ^*** Example 6 126.4 ± 1.4 ^*** 157.6 ± 0.0 ^* Example 7 123.4 ± 0.5 ^*** 148.3 ± 0.3 ^*** Example 8 166.3 ± 3.1 178.0 ± 0.7 ^*** Example 9 148.8 ± 0.2 149.2 ± 0.3 ^***

As shown in Table 4 above, it was confirmed that the alfalfa sprout hydrolysates prepared according to Examples 1 to 9 had excellent phagocytosis activity compared to Control 1, and in particular, Examples 2 and Example 8 had phagocytosis activity at all concentrations. This was confirmed to be excellent.

Summarizing the results of Test Examples 1 to 3, the effect of using amyloglucosidase (AMG, Example 2) and Ultraflo (ultraflo L, Example 8) as hydrolytic enzymes was superior to that of the other groups. confirmed.

The amyloglucosidase is an enzyme derived from Aspergillus niger, and the Ultraflo is an enzyme derived from Humicola insolens.

[Alfalfa sprout hydrolyzate_hot water extraction status and enzyme content]

Example 10. Omission of hot water extraction_Enzyme 0.1 part by weight

An alfalfa sprout hydrolyzate was obtained in the same manner as in Example 2, except that amyloglucosidase (AMG) was used in an amount of 0.1 parts by weight based on 100 parts by weight of alfalfa sprouts.

Example 11. Hot water extraction of decomposition product_enzyme 0.1 part by weight

After mixing alfalfa sprouts and water at a weight ratio of 1:33, amyloglucosidase (AMG, 0.1 parts by weight per 100 parts by weight of alfalfa sprouts) was added and incubated in a shaking water bath at pH 4.5 at 60°C. After stirring for 24 hours, the enzyme was inactivated, extracted at 100°C for 2 hours, and filtered to obtain a hydrolyzed hot water extract of alfalfa sprouts.

Example 12. Hot water extraction

Alfalfa sprouts and water were mixed at a weight ratio of 1:33 and extracted at 100°C for 2 hours to obtain alfalfa sprout hot water extract.

Comparative Example 1. Omission of enzyme treatment

Alfalfa sprouts and water were mixed at a weight ratio of 1:33, stirred in a shaking water bath for 24 hours, and then extracted at 100°C for 2 hours to obtain an alfalfa sprout extract.

<Test Example Ⅱ>

^### p<0.001 compared with Normal, ^* p<0.05, ^*** p<0.001 compared with LPS

Test Example 4. Measurement of nitric oxide production promotion effect

The NO expression level was measured in the same manner as in Test Example 2.

Classification (unit: %) 2.5ug/ml 5ug/ml 10ug/ml 50ug/ml Normal 100 LPS 308.7 ^### Example 10 227.4 ± 2.1 ^* 303.1 ± 0.9 ^*** 314.4 ± 1.5 ^*** 328.6 ± 1.3 ^*** Example 11 248.8 ± 1.6 ^* 306.2 ± 1.5 ^*** 327.4 ± 0.9 ^*** 329.6 ± 1.1 ^*** Example 12 218.6 ± 0.7 ^* 301.1 ± 0.3 ^*** 309.8 ± 0.5 ^*** 326.7 ± 0.9 ^*** Comparative Example 1 210.8 ± 1.1 ^* 229.9 ± 0.5 ^* 266.7 ± 0.8 ^** 277.7 ± 0.3 ^**

As shown in Table 5 above, it was confirmed that the alfalfa sprout hydrolyzate prepared according to Examples 10 and 11 of the present invention had a higher NO expression level compared to Example 12 and Comparative Example 1. In particular, hot water extraction after hydrolysis It was confirmed that Example 11, which was performed, had a higher NO expression level than Example 10, where only hydrolysis was performed.

Test Example 5. Measurement of phagocytosis activity

The efficacy of enhancing phagocytosis activity was measured in the same manner as in Test Example 3.

Classification (unit: %△RFU 490/520 nm) 2.5ug/ml 5ug/ml 10ug/ml 50ug/ml Normal 100 LPS 221.8 ^### Example 10 142.7 ± 1.4 ^** 156.9 ± 0.8 ^*** 161.1 ± 0.6 ^*** 173.2 ± 1.1 ^*** Example 11 151.8 ± 0.9 ^** 163.5 ± 0.4 ^*** 176.8 ± 1.1 ^*** 184.5 ± 1.5 ^*** Example 12 135.4 ± 2.1 ^* 140.6 ± 1.8 ^* 153.4 ± 0.4 ^*** 152.8 ± 0.6 ^*** Comparative Example 1 136.8 ± 1.8 ^* 141.8 ± 1.1 ^* 150.6 ± 1.6 ^** 151.7 ± 0.2 ^**

As shown in Table 6 above, it was confirmed that the alfalfa sprout hydrolysates prepared according to Examples 10 and 11 of the present invention had superior phagocytosis activity compared to Example 12 and Comparative Example 1.

In particular, it was confirmed that Example 11, in which hot water extraction was performed after hydrolysis, had better phagocytosis activity than Example 10, in which only hydrolysis was performed.

[Alfalfa sprout polysaccharide fraction]

Example 12. Hot water extraction

As in Control Group 2, alfalfa sprouts and water were mixed at a weight ratio of 1:33 and extracted at 100°C for 2 hours to obtain a hot water extract of alfalfa sprouts.

Example 13. Hot water extraction of decomposed product_Enzyme 1 part by weight

After mixing alfalfa sprouts and water at a weight ratio of 1:33, amyloglucosidase (AMG, 1 part by weight per 100 parts by weight of alfalfa sprouts) was added and incubated in a shaking water bath at pH 4.5 at 60°C. After stirring for 24 hours, the enzyme was inactivated, extracted at 100°C for 2 hours, and filtered to obtain a hydrolyzed hot water extract of alfalfa sprouts.

Example 14. Alfalfa sprout polysaccharide fraction

The alfalfa sprout hydrolyzed hot water extract prepared in Example 12 and the 80% ethanol aqueous solution were mixed at a weight ratio of 1:0.5, soaked at 4°C for 24 hours, and then centrifuged at 14,000 rpm for 10 minutes to separate the precipitate and supernatant. Then, the precipitate was dissolved in bicarbonate water and freeze-dried to obtain an alfalfa sprout polysaccharide fraction.

<Test Example Ⅲ>

^### p<0.001 compared with Normal, ^* p<0.05, ^*** p<0.001 compared with LPS

Test Example 6. Toxicity measurement in Raw 264.7 cells

Cytotoxicity was measured in the same manner as Test Example 1 above.

division 5 μg/mL 10 μg/mL 50 μg/mL Normal 100.0 ± 1.6 100.0 ± 1.6 100.0 ± 1.6 LPS 71.3 ± 1.6 ^### Example 12 92.7 ± 0.5 ^*** 87.9 ± 0.8 ^*** 85.1 ± 1.5 ^*** Example 13 93.4 ± 1.1 ^*** 89.6 ± 0.8 ^*** 86.0 ± 0.4 ^*** Example 14 91.7 ± 3.5 ^*** 89.7 ± 1.7 ^*** 82.2 ± 1.4 ^***

As shown in Table 7 above, the alfalfa sprout hydrolysates prepared according to Examples 12 to 14 of the present invention were confirmed to have excellent cell viability at a concentration of 5 to 50 ug/ml.

Test Example 7. Measurement of nitric oxide production promotion effect

The NO expression level was measured in the same manner as in Test Example 2.

division 5 μg/mL 10 μg/mL 50 μg/mL Normal 100.0 ± 3.0 100.0 ± 3.0 100.0 ± 3.0 LPS 245.3 ± 1.0 ^### Example 12 104.2 ± 0.2 ^*** 127.1 ± 3.9 ^*** 156.8 ± 4.6 ^*** Example 13 148.0 ± 2.1 ^*** 167.8 ± 2.2 ^*** 199.8 ± 0.3 ^*** Example 14 172.2 ± 5.9 ^*** 194.0 ± 4.0 ^*** 231.4 ± 5.5 ^ns

As shown in Table 8 above, the alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14 of the present invention were confirmed to be superior because the NO expression level was concentration-dependently higher than that of Example 12.

In addition, it was confirmed that the alfalfa sprout polysaccharide fraction of Example 14 had a higher NO expression level than the alfalfa sprout hydrolyzed hot water extract of Example 13 at a concentration of 5 to 50 ug/ml.

Test Example 8. Measurement of phagocytosis activity

The efficacy of enhancing phagocytosis activity was measured in the same manner as in Test Example 3.

division 5 μg/mL 10 μg/mL 50 μg/mL Normal 100.0 ± 2.9 100.0 ± 2.9 100.0 ± 2.9 LPS 156.4 ± 5.1 ^### Example 12 125.9 ± 3.4 ^*** 132.9 ± 1.5 ^** 136.7 ± 1.9 ^* Example 13 140.7 ± 1.2 ^* 145.2 ± 4.5 ^ns 150.4 ± 2.6 ^ns Example 14 143.9 ± 7.1 ^ns 149.5 ± 4.1 ^ns 155.7 ± 0.7 ^ns

As shown in Table 9 above, the alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14 of the present invention were confirmed to have superior phagocytosis activity in a concentration-dependent manner compared to Example 12.

In addition, it was confirmed that the alfalfa sprout polysaccharide fraction of Example 14 had higher phagocytosis activity than the alfalfa sprout hydrolyzed hot water extract of Example 13 in a concentration-dependent manner.

Test Example 9. Measurement of the effect of promoting TNF-α production

The cultured cells were dispensed into a 6-well plate at a concentration of 1X10 ⁶ /well and cultured in a humidified incubator (5% carbon dioxide, 95% air, 37°C) for 48 hours. Thereafter, the alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 12 to 14 were treated at various concentrations (5, 10, or 50 μg/ml) and then cultured at 37°C for 48 hours. . Afterwards, the amount of TNF-α produced from each cultured cell line was measured using an ELISA kit (Quantikine/Mouse; R&D Systems, eBioscience, USA). At this time, the absorbance was determined by subtracting the value at 540 nm from the value at 450 nm using an ELISA reader (Bio-Tek Instruments, USA), and the concentration of TNF-α was measured using each standard curve in each well. did. Additionally, as a comparison group, the Raw264.7 cell line treated with LPS was used.

division 5 μg/mL 10 μg/mL 50 μg/mL Normal 100.0 ± 6.2 100.0 ± 6.2 100.0 ± 6.2 LPS 1178.0 ± 1.3 ^### Example 12 143.0 ± 0.1 ^*** 367.0 ± 12.9 ^*** 867.0 ± 6.2 ^*** Example 13 740.0 ± 10.2 ^*** 924.0 ± 1.6 ^*** 1098.0 ± 3.4 ^ns Example 14 911.0 ± 2.5 ^*** 929.0 ± 1.7 ^*** 1179.0 ± 4.8 ^ns

As shown in Table 10 above, the alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14 of the present invention were confirmed to increase the amount of TNF-α produced in a concentration-dependent manner compared to Example 12. did.

In general, TNF-α interacts with lymphocytes to regulate the activity and growth of lymphocytes. In particular, it is a cytokine that induces Th-1 cell responses and is known to enhance immunity in diseases such as cerebral infarction and cancer.

Therefore, from the results in Table 10, it was analyzed that the alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14 were effective in enhancing immunity by promoting TNF-α production.

Test Example 10. Measurement of the effect of promoting IL-1β (interleukin-1β) production

After culturing the Raw264.7 cell line treated with the alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 12 to 14, each cultured cell line was cultured using an ELISA kit (BioLegend, CA, USA). The amount of IL-1β produced was measured. At this time, the concentration of IL-1β was measured using each standard curve in each well. Additionally, as a comparison group, the Raw264.7 cell line treated with LPS was used.

division 5 μg/mL 10 μg/mL 50 μg/mL Normal 100.0 ± 6.2 100.0 ± 6.2 100.0 ± 6.2 LPS 112.9 ± 0.7 ^### Example 12 104.8 ± 0.5 ^** 105.8 ± 0.6 ^** 111.4 ± 0.4 ^** Example 13 109.9 ± 0.4 ^ns 112.3 ± 0.9 ^ns 116.7 ± 0.1 ^*** Example 14 113.9 ± 0.4 ^ns 116.7 ± 0.8 ^** 120.6 ± 1.1 ^***

As shown in Table 11 above, it was confirmed that the alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14 increased the amount of IL-1β produced in a concentration-dependent manner compared to Example 12.

In general, IL-1β is a cytokine produced by mononuclear leukocytes and macrophage cells and plays an important role in the cytokine network, such as activating T cells and enhancing the activity of cytokines such as IL-2 secreted by T cells. In addition, it is known to have the function of mediating local inflammatory responses and acting on vascular endothelial cells to increase the expression of surface molecules that mediate the adhesion of white blood cells.

Therefore, from the results in Table 11, it can be seen that the alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14 activate macrophages and promote IL-1β production to activate T cells and induce a local inflammatory response. It was analyzed to be effective in enhancing immunity by acting on vascular endothelial cells and mediating the adhesion of white blood cells.

Test Example 11. Measurement of intracellular iNOS and MAPKs transcription factors

Raw 264.7 cells were cultured in a 60 mm dish at a concentration ^{of 5} . After culturing for 18 hours, the medium was replaced with serum-free DMEM without FBS, treated with samples of each concentration and LPS at a concentration of 1 μg/mL, and cultured for 20 hours. After culturing, the medium was removed, washed twice with ice-cold DPBS, and 1 mL of ice-cold DPBS was added and the cells were recovered using a scraper. The recovered cells were centrifuged at 5,000 rpm for 5 minutes to cell-down, and then DPBS was removed. RIPA buffer containing protease inhibitor was added to the cell pellet, vortexed, and lysed on ice for 30 minutes. Afterwards, centrifuge at 12,000 rpm for 20 minutes to collect the supernatant, quantify protein through bradford assay, and prepare a loading sample. Samples were separated by SDS-PAGE, transferred to nitrocellulose membrane (NC), and blocked with 5% skim milk. After reacting with the primary antibody of the protein of interest overnight at 4°C, react with the secondary antibody specific to the primary antibody for 1 hour at room temperature. After reaction, it was washed with 1X TBST solution, reacted with ECL solution, and expression of the desired protein was observed in gel-doc.

Figure 1 shows the effects of alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 12 to 14 on iNOS protein expression in macrophages at different concentrations. This is a Western blot showing the results.

As shown in Figure 1, iNOS expression was confirmed to increase in macrophages treated with alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14 compared to Example 12.

In particular, when treated with alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14, iNOS expression increases in a concentration-dependent manner, so it is believed to exhibit an immune-enhancing effect through NF-κB signaling activity.

Figure 2 shows the effect of alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 12 to 14 on MAPKs protein expression in macrophages at different concentrations. This is a Western blot showing the results.

As shown in Figure 2, it was confirmed that the expression of MAPKs transcription factors increased in macrophages treated with alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14 compared to Example 12. .

In particular, it was confirmed that the expression of pERK, pJNK, pp38, and p38 increased in a concentration-dependent manner when treated with the alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14, which was confirmed in Examples 13 and 14. It is believed that alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to the method exhibit immune-enhancing effects through MAPKs signaling activity.

Test Example 12. Measurement of sugar components

The components of monosaccharides contained in alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 12 to 14 were measured.

Classification (unit: Mole%) Example 12 Example 13 Example 14 acid sugar galacturonic acid 2.3±0.1 6.2±0.1 ^*** 8.9±0.2 ^*** Glucuronic acid 0.9±0.5 4.3±2.0 ^*** 1.3±0.1 ^* Neutral Cathedral Mannose 46.8±0.4 15.5±0.5 ^** 29.3±0.1 ^*** Rhamnose - 4.6±0.0 ^*** 2.9±0.1 ^*** glucose - 23.9±0.3 ^*** 2.3±0.1 ^*** galactose 16.7±0.9 27.6±0.8 ^** 47.1±0.1 ^*** xylose - 2.8±0.2 ^*** 2.9±0.2 ^*** Arabinose 33.3±0.1 15.1±0.4 ^*** 4.9±0.2 ^** fucose - - 0.4±0.0 ^*** Sum 100 100 100

As shown in Table 12 above, the alfalfa sprout hydrolyzed hot water extract and alfalfa sprout polysaccharide fraction prepared according to Examples 13 and 14 of the present invention are different substances with different monosaccharide composition and content from Example 12. Confirmed.

Below, a formulation example of a composition containing the powder of the present invention is described, but the present invention is not intended to be limited, but merely explained in detail.

Formulation Example 1. Preparation of powder

500 mg of alfalfa sprout hydrolyzate obtained in Example 1

100 mg lactose

Talc 10 mg

The above ingredients are mixed and filled into an airtight bubble to prepare a powder.

Formulation Example 2. Preparation of tablets

300 mg of alfalfa sprout hydrolyzate obtained in Example 1

Corn starch 100 mg

100 mg lactose

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.

Formulation Example 3. Preparation of capsules

200 mg of alfalfa sprout hydrolyzate obtained in Example 1

3 mg crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a typical capsule manufacturing method.

Formulation Example 4. Preparation of injections

600 mg of alfalfa sprout hydrolyzate obtained in Example 1

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

It is prepared with the above ingredients per ampoule according to the usual manufacturing method for injections.

Formulation Example 5. Preparation of liquid formulation

4 g of alfalfa sprout hydrolyzate obtained in Example 1

10 g isomerized sugar

5 g mannitol

Proper amount of purified water

According to the usual liquid preparation method, add and dissolve each ingredient in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, adjust the total to 100g by adding purified water, and then fill it in a brown bottle and sterilize it. to produce a liquid.

Formulation Example 6. Preparation of granules

1,000 mg of alfalfa sprout hydrolyzate obtained in Example 1

Vitamin mixture dosage

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 ㎍

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Monobasic Potassium Phosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a mixture of components relatively suitable for granules in a preferred embodiment, but the mixing ratio may be modified arbitrarily. The above components are mixed according to a typical granule manufacturing method, and then the granules are mixed. It can be prepared and used to manufacture a health functional food composition according to a conventional method.

Formulation Example 7. Preparation of functional beverage

1,000 mg of alfalfa sprout hydrolyzate obtained in Example 1

1,000 mg citric acid

100 g oligosaccharides

2 g plum concentrate

1 g taurine

Add purified water to make a total of 900 mL.

After mixing the above ingredients according to a typical health drink manufacturing method, stirring and heating at 85°C for about 1 hour, the resulting solution was filtered, placed in a sterilized 2 L container, sealed, sterilized, stored in the refrigerator, and then refrigerated. Used for manufacturing the functional beverage composition of the invention.

The composition ratio is a preferred embodiment of mixing ingredients relatively suitable for beverages of preference, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, country of demand, and intended use.

Formulation Example 8: Preparation of feed composition

Example 1: 0.1 kg of alfalfa sprout hydrolyzate powder, 25.5 kg of corn, 15.04 kg of wheat, 8.15 kg of wheat flour, 7.4 kg of rice bran, 18 kg of soybean meal, 1 kg of jaggery, 14 kg of chicken by-products, 9 kg of animal fat, 0.3 kg of processed salt. , 0.3 kg of tricalcium phosphate, 1 kg of limestone, 0.01 kg of choline chloride, 0.05 kg of vitamins, 0.05 kg of minerals, and 0.1 kg of digestive enzymes were mixed to prepare an animal (dog, pet) feed composition.

Claims (14)

A food composition for enhancing immune function, characterized in that it contains alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient. The food composition for enhancing immune function according to claim 1, wherein the alfalfa sprout hydrolyzate is obtained by treating alfalfa sprouts with a hydrolytic enzyme. The method of claim 2, wherein the hydrolytic enzymes include alcalase, amyloglucosidase (AMG), celluclast, flavozyme, nutrase, and Protamex ( A food composition for enhancing immune function, characterized in that it is one or more selected from the group consisting of protamex, termamayl, ultraflo, and viscozyme. The food composition for enhancing immune function according to claim 2, wherein the hydrolytic enzyme is alcalase, amyloglucosidase (AMG), or celluclast. The food composition for enhancing immune function according to claim 2, wherein the hydrolytic enzyme is used in an amount of 0.1 to 3 parts by weight based on 100 parts by weight of alfalfa sprouts. The food composition for enhancing immune function according to claim 1, wherein the alfalfa sprout hydrolyzate is obtained by hydrolyzing alfalfa sprouts and then extracting them with hot water. The method of claim 1, wherein the alfalfa sprout hydrolyzate contains an acidic sugar consisting of 5 to 10 mole% of galacturonic acid and 1 to 6 mole% of glucuronic acid; and mannose 10 to 30 mole%, rhamnose 1 to 8 mole%, glucose 10 to 30 mole%, galactose 20 to 40 mole%, xylose 1 to 5. A food composition for enhancing immune function, characterized in that it contains a neutral sugar consisting of 10 to 20 mole% mole% and arabinose. The method of claim 1, wherein the alfalfa sprout polysaccharide fraction contains 5 to 10 Mole% of galacturonic acid and 0.5 to 5 Mole% of glucuronic acid; and mannose 20 to 40 Mole%, rhamnose 0.5 to 5 Mole%, glucose 0.5 to 5 Mole%, galactose 30 to 60 Mole%, xylose 1 to 5. A food composition for enhancing immune function, characterized in that it contains a neutral sugar consisting of 1 to 8 mole% of arabinose and 0.1 to 1 mole% of fucose. The method of claim 1, wherein the alfalfa sprout polysaccharide fraction is obtained by mixing alfalfa sprout hydrolyzate with water, alcohol having 1 to 4 carbon atoms, hexane, ethyl acetate, chloroform, dichloromethane and these. A food composition for enhancing immune function, characterized in that it is obtained by fractionation with a solvent selected from the group consisting of mixed solvents. A food composition for enhancing immune function, characterized in that it contains alfalfa water extract as an active ingredient. A pharmaceutical composition for enhancing immune function, characterized in that it contains alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient. The pharmaceutical composition for enhancing immune function according to claim 11, wherein the alfalfa sprout hydrolyzate is obtained by treating alfalfa sprouts with a hydrolytic enzyme. The method of claim 11, wherein the hydrolytic enzymes include alcalase, amyloglucosidase (AMG), celluclast, flavozyme, nutrase, and Protamex ( A pharmaceutical composition for enhancing immune function, characterized in that it is one or more selected from the group consisting of protamex, termamayl, ultraflo, and viscozyme. A feed composition for enhancing immune function, characterized in that it contains alfalfa sprout hydrolyzate or a polysaccharide fraction thereof as an active ingredient.