Immune-boosting deer antler enzyme-digested extract that significantly boosts immune cell activity, including NK cells

Abstract

The present invention relates to a composition for enhancing immunity, and more specifically, it contains an enzymatically hydrolyzed extract of velvet antler, which is rich in NK cell activity, as an active ingredient, thereby proliferating immune cells, and in particular, enhancing NK cell activity, thereby exhibiting an immunity enhancing effect, and therefore can be used as an immunity enhancing food composition, and ultimately as a functional health food or pharmaceutical composition.

Description

[Technical field]

[0001] The present invention relates to a composition for enhancing immunity, and more specifically, to a food composition for enhancing immunity containing an enzymatic hydrolysis extract of velvet, which is rich in NK cell activity, as an active ingredient, in particular, a health functional food for enhancing immunity and a pharmaceutical composition for enhancing immunity. [Background technology]

[0002] With the improvement of living standards and the westernization of eating habits, the incidence of lifestyle-related diseases (adult diseases) such as obesity, diabetes, high blood pressure, and heart disease caused by overnutrition and lack of exercise is increasing. In addition, interest in health promotion is growing as the average life expectancy increases and the country enters an aging society at an accelerated pace.

[0003] Health promotion refers to increasing resistance to disease, particularly chronic degenerative diseases and infectious diseases, and to ongoing stress, as well as increasing daily activity. Measures for this include managing one's entire lifestyle, such as improving nutrition, strengthening immune function, appropriate exercise and rest, and maintaining mental activity.

[0004] In particular, if you have a strong immune system, you are less likely to get sick, and even if you do get sick, you recover quickly. It is clear that if you continue to maintain this state of health as you age, your lifespan will increase. Therefore, immunity is directly linked to human health and lifespan, and it can be said that managing immunity is essential to maintaining health and extending lifespan.

[0005] Immunity refers to the mechanism by which the human or animal body physiologically recognizes and eliminates exogenous and endogenous foreign substances to maintain homeostasis. Here, the foreign substances are called antigens, and various types of white blood cells and proteins are involved in the process of eliminating them. Immunity is broadly divided into innate immunity, which we are born with, and acquired immunity, which we acquire by adapting to our lifestyles, etc.

[0006] Innate immunity is also called natural immunity or natural resistance. It responds non-specifically to antigens and has no special memory function. Innate immune systems include skin and mucus tissue that block the invasion of antigens, highly acidic stomach acid, and complements present in blood. Cells include macrophages and polymorphonuclear leukocytes that are responsible for phagocytosis, and NK cells that can kill infected cells. There are also cytokines produced by macrophages. In fact, most infections are defended against by the above-mentioned innate immunity.

[0007] The initial recognition of external infection is mainly performed by macrophages. During the process of phagocytosis of antigens, cytokines that mediate immune responses are produced.

[0008] Cytokines are glycoproteins that act as signaling substances to control and stimulate the body's defense system, and play important roles in immunity, infectious diseases, hematopoietic function, tissue recovery, and cell development and growth. Cytokines are also called inflammatory cytokines because they attract immune cells to the site of infection by inducing inflammation at the local site. The most important immune functions are performed by a group of cytokines known as interleukins, interferon gamma, and tumor necrosis factors. Among these, interleukins play an important role in the body's defense by acting at various stages of immune response and regulating the immune response, and there are various types such as IL-1, IL-6, and IL-12.

[0009] In addition, NK cells (natural killer cells) are cells that act on the front line of the body's immune system defense mechanism, eliminating host cells infected with tumor cells, bacteria, intracellular parasites, or viruses without prior sensitization with antigens, rejecting inappropriate bone marrow transplants, and regulating the immune response of T cells. It is known that when NK cells are highly active, their ability to attack and eliminate target cells also increases.

[0010] On the other hand, antler (Cervi Parvum Cornu, Antler) is made by cutting and drying the young antlers of the bucks of the Meihana deer, the Odocan, or the Baboon deer, which are densely covered with hair and have not yet ossified or have ossified to some extent. It refers to the antlers of bucks that are within two months of growing, have not yet keratinized, have soft tissue, and are evenly covered with hair. The antlers of deer that have not yet keratinized are somewhat soft, but by autumn they become hard and keratinized, reducing the efficacy of the antler. These are called antlers.

[0011] According to ancient documents such as the Donguibogam, velvet antler is known to have many pharmacological effects, such as tonicity, promotion of growth and development, blood replenishment, blood production, enhancement of the five internal organs, treatment of heart failure, enhancement of physical functions, enhancement of physical vitality, and strengthening of the diuretic function of the kidneys. Research on the effects of velvet antler using animals has reported that it has the following effects: growth promotion, blood production, lowering of serum cholesterol, promotion of protein synthesis, anti-aging, increase in spinal nerve enzyme activity in white rats, pain relief and anti-fatigue, increase in immune activity, and sedative effect.

[0012] The physiologically active components of velvet are known to include amino acids, calcium phosphate, calcium carbonate, collagen, phospholipids, chondroitin, glucosamine, pantocrin, gangliosides, and hyaluronic acid.

[0013] Conventionally, the main methods of taking velvet antler in traditional Chinese medicine are to mix dried velvet antler with various herbal medicines, crush it, extract it by heating with water as a solvent, and take the filtrate, or to crush dried velvet antler and herbal medicines into powder, then make it into pills and take it. The method of taking it by extraction makes it easy to absorb the active ingredients of velvet antler, but has the limitation that the active ingredients contained in the extraction residue cannot be utilized, and the pill form has the advantage of taking velvet antler itself, but has the problem that it is difficult to fully absorb the active ingredients in the body.

[0014] Korean Patent Publication No. 2005-0090041 describes a method for extracting antler by crushing the antler, extracting it, and treating it with proteolytic enzymes and hydrochloric acid. However, since the extraction, proteolytic enzymes, and hydrochloric acid treatments are performed in sequence, the process is complicated and there is a problem that physiologically active substances useful to the human body are decomposed.

[0015] In addition, Korean Patent Publication No. 2013-0078724 describes a method of treating antler with pepsin and lipolytic enzyme under acidic conditions, and then further treating it with proteolytic enzyme under neutral conditions. However, it only states that the free amino acid content and phospholipid content are increased, and does not describe the actual physiological activity or specific functionality. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] Korean Patent Publication No. 2005-0090041 [Patent Document 2] Korean Patent Publication No. 2013-0078724 Summary of the Invention [Problem to be solved by the invention]

[0017] The problem to be solved by the present invention is to provide an immune enhancing food composition containing as an active ingredient an enzyme-decomposed extract of velvet, which is rich in NK cell activity and is obtained by decomposing velvet with an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28.

[0018] Another problem that the present invention aims to solve is to provide a method for producing an enzymatically hydrolyzed extract of velvet antler with immune enhancing activity, the method comprising: preparing a freeze-dried velvet antler by freeze-drying raw or frozen velvet antler; and adding 50-500 parts by weight of water and 0.01-1 parts by weight of enzyme to 10 parts by weight of the freeze-dried velvet antler, and reacting at 30-60°C for 1-48 hours; the enzyme is an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28.

[0019] Another object of the present invention is to provide a pharmaceutical composition for enhancing immunity, comprising as an active ingredient an enzyme-decomposed extract of velvet, which is obtained by decomposing velvet with an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28 and is rich in NK cell activity. [Means for solving the problem]

[0020] In order to achieve the above-mentioned object, the present invention provides an immune enhancing food composition containing, as an active ingredient, an enzyme-decomposed extract of velvet obtained by decomposing velvet with an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28.

[0021] According to one embodiment of the present invention, the velvet enzymatic hydrolysis extract may be an velvet enzymatic hydrolysis extract obtained by hydrolyzing velvet with an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28.

[0022] According to one embodiment of the present invention, the enzyme of the IUBMB enzyme nomenclature class EC 3.4.11.1 may be a complex of fungal proteases and peptidases.

[0023] According to one embodiment of the present invention, the complex of fungal protease and peptidase may be a complex of endoprotease and exopeptidase derived from Aspergillus oryzae.

[0024] According to one embodiment of the present invention, the complex of endoprotease and exopeptidase may be Flavourzyme®.

[0025] According to one embodiment of the present invention, the enzyme of the IUBMB enzyme nomenclature class EC 3.4.24.28 may be a neutral protease derived from Bacillus.

[0026] According to one embodiment of the present invention, the Bacillus-derived neutral protease may be a neutral metallo(Zn)-endoprotease produced by submerged fermentation of Bacillus amyloliquefaciens.

[0027] According to one embodiment of the present invention, the neutral metallo(Zn) endoprotease may be Neutrase.

[0028] According to one embodiment of the present invention, the enzymatic decomposition extract of velvet may be an enzymatic decomposition extract of velvet antler obtained by hydrolyzing raw velvet antler or dried velvet antler obtained by freeze-drying frozen velvet antler through an enzymatic treatment.

[0029] The present invention also provides a method for producing an enzymatically hydrolyzed extract of velvet antler with immune enhancing activity, the method comprising: a preparation step of freeze-dried velvet antler, freeze-drying raw or frozen velvet antler; and an enzyme treatment step of adding 50-500 parts by weight of water and 0.01-1 parts by weight of enzyme to 10 parts by weight of the freeze-dried antler, and reacting at 30-60°C for 1-48 hours; the enzyme is an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28.

[0030] The present invention also provides a pharmaceutical composition for enhancing immunity, comprising as an active ingredient an enzymatically decomposed extract of velvet obtained by decomposing with an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28.

[0031] According to one embodiment of the present invention, the velvet enzymatic hydrolysis extract may be an velvet enzymatic hydrolysis extract obtained by hydrolyzing velvet with an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28. Effect of the Invention

[0032] The composition containing the enzymatic hydrolysis extract of velvet, which is rich in NK cell activity of the present invention, as an active ingredient proliferates immune cells and activates the functions of immune cells, and can therefore be used as an immune-enhancing food composition, and ultimately as a functional health food or pharmaceutical composition. [Brief description of the drawings]

[0033] [Figure 1] This is a graph showing the expression of immune-related cytokines analyzed using NanoString to confirm the effect of the enzymatic hydrolysis extract of velvet in enhancing the immune activity of antigen-stimulated leukocytes in Experimental Example 2. The more up-regulated the cytokines, the more blue they are. [Diagram 2]1 is a graph comparing the effect of nitric oxide production in macrophages according to the type and concentration of enzymatically hydrolyzed extract of velvet in Experimental Example 3. [Diagram 3] 1 is a graph comparing the expression level of iNOS gene in macrophages according to the type and concentration of enzymatic hydrolysis extract of velvet in Experimental Example 4. [Figure 4] 1 is a graph comparing the expression level of COX2 gene in macrophages according to the type and concentration of enzymatic hydrolysis extract of velvet in Experimental Example 4. [Diagram 5] 1 is a graph comparing the relative expression level of TNF-α depending on the type and concentration of enzymatic hydrolysis extract of velvet in Experimental Example 5. [Figure 6] 1 is a graph comparing the relative expression level of IFN-γ depending on the type and concentration of enzymatic hydrolysis extract of velvet in Experimental Example 5. [Figure 7] 1 is a graph comparing the relative expression level of IL-6 depending on the type and concentration of enzymatic hydrolysis extract of velvet in Experimental Example 5. [Figure 8] 1 is a graph comparing the relative expression level of IL-1β depending on the type and concentration of enzymatically hydrolyzed extract of velvet in Experimental Example 5. [Figure 9] 1 is a graph comparing macrophage proliferation ability according to the type and concentration of enzymatic hydrolysis extract of velvet and the change in culture time in Experimental Example 6. [Figure 10] 1 is a graph comparing NK cell activity according to the type and administration concentration of enzymatically hydrolyzed extract of velvet in an immunosuppressed animal model in Experimental Example 7. [Figure 11] 1 is a graph comparing TNF-α concentrations according to the type and administration concentration of enzymatically hydrolyzed extract of velvet in an immunosuppressed animal model in Experimental Example 7. [Figure 12] 1 is a graph comparing interferon gamma (IFN-γ) concentrations according to the type and administration concentration of velvet enzymatic hydrolysis extract in an immunosuppressed animal model in Experimental Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention will be described in detail below.

[0035] The inventors of the present invention have confirmed that an enzymatically hydrolyzed extract of velvet, particularly an enzymatically hydrolyzed extract of velvet obtained by hydrolyzing velvet with an enzyme of IUBMB enzyme naming class EC 3.4.11.1 or an enzyme of IUBMB enzyme naming class EC 3.4.24.28, preferably an enzymatically hydrolyzed extract of velvet obtained by hydrolyzing velvet with an enzyme of IUBMB enzyme naming class EC 3.4.11.1, has excellent NK cell activity and exhibits immune enhancing activity by promoting expression of immune-related cytokines.

[0036] The antlers mentioned above refer to unossified antlers of deer that are less than two months old.

[0037] The velvet antler may be dried naturally in a low humidity environment for several weeks, but preferably, the present invention uses dried velvet antler obtained by freeze-drying raw velvet antler or frozen velvet antler.

[0038] When raw or frozen antler is naturally dried or boiled before sterilization before freeze-drying, there is a problem that non-specific and non-uniform enzymatic decomposition occurs due to the action of endogenous enzymes before the temperature of the deep part of the antler rises to the enzyme inactivation temperature. Therefore, if raw or frozen antler is naturally dried or directly freeze-dried without boiling before freeze-drying, it is possible to produce an enzymatic decomposition extract of antler with uniform quality and rich immune enhancing activity. The dried antler that has been freeze-dried may be used after sterilization by a normal method, for example, after heat treatment at 120 to 130°C for 30 seconds to 20 minutes.

[0039] The enzymes in the IUBMB enzyme nomenclature class EC 3.4.23.1, such as pepsin and aspartic proteinase, do not show any immune enhancing effect when produced from an enzymatically decomposed extract of velvet. Pepsin is an aspartic endopeptidase that selectively cleaves hydrophobic, particularly aromatic, amino acid residues.

[0040] The enzymes of the IUBMB enzyme nomenclature class EC 3.4.11.1, such as A-LAP, acidic M17 leucine aminopeptidase, AcLAP, adipocyte-derived leucine aminopeptidase, Aminopeptidase, and "Flavourzyme" (Novozyme), show a much better immune enhancing effect than the conventional hot water extract or supercritical antler extract when preparing an enzymatically decomposed extract of antler by enzymatic treatment. The Flavozyme is a composite of endoprotease and exopeptidase produced by submerged fermentation of Aspergillus oryzae.

[0041] In addition, the enzymes of the IUBMB enzyme nomenclature class EC 3.4.24.28 are neutral proteases derived from Bacillus, such as bacillolysin, Bacillus metalloendopeptidase, anilozyme P10, Bacillus metalloproteinase, megateriopeptidase, and "Neutrase" (manufactured by Novozyme), when producing an enzymatically decomposed extract of velvet antler by enzyme treatment, show a much more excellent immune enhancing effect than conventional velvet antler hot water extract or supercritical velvet antler extract. The Neutrase is a neutral metallo(Zn)-endoprotease produced by submerged fermentation of Bacillus amyloliquefaciens. In addition to neutrase, enzymatically decomposed extracts of velvet antler treated with "Protease N 'Amano'" (manufactured by Amano Enzymes) and "Corolase N" (manufactured by AB Enzymes), which are neutral metalloproteinases of bacterial origin belonging to the above enzyme class, also show immune enhancing effects far superior to conventional velvet antler hot water extracts or supercritical velvet antler extracts.

[0042] However, enzymes of the UBMB enzyme nomenclature class EC 3.4.21.62, such as Alcalase, subtilisin, colorase N, ALK-enzymes; bacillopeptidase A; bacillopeptidase B; Bacillus subtilis alkaline proteinase bioprase; bioprase AL15; bioprase APL30; colistinase; subtilisin J; subtilisin S41; Subtilisin Sendai; subtilisin GX; subtilisin E; subtilisin BL; subtilisin A (Type VIII); genenase I; esperase; maxatase; thermoase PC10; protease XXVII; thermoase; superase; subtilisin DY; subtilopeptidase; SP266; savinase 8.0L; savinase 4.0T; kazusase; protease VIII; optimize; opticlean; Bacillus subtilis alkaline proteinase; protein A3L; savinase; savinase 16.0L; savinase 32.0L EX; orientase 10B; protease S and similar serine endopeptidases do not show immune-enhancing effects when an enzymatic hydrolysis extract of velvet is produced by enzymatic treatment.

[0043] In addition, in the case of "Protamex" (manufactured by Novozyme), a composite of an enzyme of IUBMB enzyme naming class EC 3.4.24.28 and an enzyme of IUBMB enzyme naming class EC 3.4.21.62, an enzymatically decomposed extract of velvet produced by enzyme treatment does not show any immune enhancing effect.

[0044] The enzymatic decomposition extract of velvet antler is prepared by adding an appropriate amount of velvet antler and enzyme to water for enzymatic treatment. For example, 50 to 500 parts by weight, preferably 100 to 400 parts by weight of water is added to 10 parts by weight of dried velvet antler such as freeze-dried velvet antler, and 0.01 to 1 part by weight, preferably 0.02 to 0.8 parts by weight, more preferably 0.05 to 0.5 parts by weight of enzyme can be added, but the amount of the enzyme can be changed as necessary.

[0045] The enzyme treatment temperature can be selected within an appropriate range depending on the optimum temperature of the selected enzyme, and the reaction can be carried out preferably at 30 to 60°C, more preferably at 40 to 55°C.

[0046] The enzyme treatment time can be appropriately selected depending on the amount of peptide produced by the enzyme treatment, the molecular weight of the peptide, and its profile, and is preferably 1 hour or more, more preferably 2 hours or more, and even more preferably 3 hours or more. Since enzyme treatment for a certain period of time or longer has little effect on the production yield and may only increase the process cost, the reaction is preferably carried out for 48 hours or less, more preferably 24 hours or less, and even more preferably 12 hours or less.

[0047] The enzyme treatment step may further include a step of inactivating the enzyme. Any method known to those skilled in the art may be applied for the enzyme inactivation, for example, by treating at a temperature of 85° C. or higher for 20 minutes or more.

[0048] The term "enzymatic hydrolysis extract of velvet antler" refers to a reaction product obtained by enzymatically treating velvet antler, fractions obtained by fractionating the reaction product, concentrates obtained by further concentrating the decomposition product or fraction, and purified products obtained by purifying or separating the reaction product, fraction, concentrate, or purified product, and also refers to a dried product obtained by drying the reaction product, fraction, concentrate, or purified product, or a powder obtained by pulverizing the reaction product, fraction, concentrate, or purified product.

[0049] Various further purification methods can be added, such as centrifugation to produce the concentrate or purified product, passage through ultrafiltration membranes with molecular weight cut-offs, or separation by various chromatographies (designed for separation by size, charge, hydrophobicity, or affinity).

[0050] The present invention relates to a food composition for enhancing immunity, which contains an enzymatically hydrolyzed extract of velvet as an active ingredient.

[0051] The "food composition" contains, in addition to the enzymatic hydrolysis extract of velvet antler as an active ingredient, food ingredients that are usable in food and are listed in the Food Standards and Regulations ("Food Code") commonly used in food manufacturing, and food additives that are listed in the Food Additives Code.

[0052] Although not particularly limited, examples include proteins, carbohydrates, fats, nutrients, seasonings and flavorings. The carbohydrates can be 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. The flavorings can be natural flavorings [thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavorings (saccharin, aspartame, etc.).

[0053] When a food composition is produced using the enzymatic hydrolysis extract of velvet antler as an active ingredient, the content of the enzymatic hydrolysis extract of velvet antler does not need to be particularly limited as long as it shows immune-enhancing efficacy, and may be, for example, 0.1 to 99 wt %, 0.5 to 95 wt %, 1 to 90 wt %, 2 to 80 wt %, 3 to 70 wt %, 4 to 60 wt %, or 5 to 50 wt %.

[0054] The amount of the velvet enzymatic decomposition extract, which is an active ingredient in the food composition, may vary depending on the condition, weight, and the presence or absence, severity, and duration of illness of the ingested person, and may be appropriately selected by a person skilled in the art. For example, the daily dosage may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, particularly preferably 20 to 800 mg, and most preferably 50 to 500 mg, and the number of administrations does not need to be particularly limited, but may be adjusted by a person skilled in the art within the range of 3 times a day to once a week. For long-term intake for the purpose of health and hygiene or health regulation, the amount may be less than the above range.

[0055] The food composition does not need to be particularly limited, and may be, for example, a powder, a granule, a tablet, a capsule, a pill, an extract, a jelly, a tea bag, or a beverage.

[0056] The enzymatic hydrolysis extract of velvet antler may also be added to general foods to impart immune enhancing functionality, and the foods to which it can be added are not particularly limited, and may be, for example, confectionery, bread or rice cakes, processed cocoa products or chocolates, processed meat or egg products, processed fish products, tofu or agar, noodles, tea, coffee, beverages, special-use foods, soy sauces, seasonings, dressings, kimchi, salted fish, salted foods, simmered foods, alcoholic beverages, dried foods, and other foods exemplified in the Food Standards and Specification for Livestock Products ("Livestock Products Standards") under Article 4 of the Livestock Products Sanitation Control Act. It may also be added to dairy products, processed meat products, packaged meat, and processed egg products exemplified in the Processing Standards and Specification for Livestock Products ("Livestock Products Standards") under Article 4 of the Livestock Products Sanitation Control Act.

[0057] On the other hand, since the enzymatic hydrolysis extract of velvet antler is rich in NK cell activity and enhances the expression of immune-related cytokines, a food composition containing the enzymatic hydrolysis extract of velvet antler as an active ingredient can be used alone as a "health functional food that helps enhance immunity."

[0058] The term "health functional food" refers to food that is manufactured (including processed) according to legal standards using raw materials or ingredients that have functional properties that are beneficial to the human body (Article 3, Paragraph 1 of the Health Functional Food Act). The term and scope of the term "health functional food" may differ depending on the country, but it may correspond to "dietary supplement" in the United States, "food supplement" in Europe, "health functional food" or "Food for Special Health Use (FoSHU)" in Japan, and "health food" in China.

[0059] The food composition or health functional food may further contain a food additive, and its suitability as a food additive shall be determined in accordance with the specifications and standards for the item in question, such as the general provisions and general test methods in the "Food Additives Code," unless otherwise specified.

[0060] In addition, the health functional food may contain a combination of the antler enzymatic hydrolysis extract and other health functional food ingredients related to immune enhancement, such as L-glutamine, germanium yeast, kumusameshimakobu, heat-treated dried powder of Enterococcus faecalis, mixed extract of Angelica sinensis, cordyceps sinensis spirit extract, spirulina, chlorella, culture product of Aspergillus oryzae (potassium poly-gamma glutamate), shiitake mushroom mycelium, yeast beta-glucan, and ginseng polysaccharide extract, which are notified or individually certified as functional ingredients used in health functional foods that help boost immunity.

[0061] The present invention relates to a pharmaceutical composition for enhancing immunity, which contains an enzymatically hydrolyzed extract of velvet as an active ingredient.

[0062] The "pharmaceutical composition" may contain, as an active ingredient, an enzymatic hydrolysis extract of velvet that is rich in NK cell activity and enhances immune-related cytokine expression, as well as suitable carriers, excipients and diluents that are commonly used in the manufacture of pharmaceutical compositions.

[0063] The "immune enhancing pharmaceutical composition" may be an immune anticancer agent or an anticancer adjuvant. The immune anticancer agent induces the expression of immune factors to increase the activity of immune cells and kill cancer cells, and the cancer cells may be solid cancer cells, and more specifically, the solid cancer may be selected from the group consisting of colon cancer, breast cancer, lung cancer, gastric cancer, epithelial ovarian cancer, brain cancer, skin cancer, and liver cancer, but is not limited thereto. The anticancer adjuvant may alleviate side effects caused by administration of an anticancer agent, and the side effects caused by administration of the anticancer agent may be selected from the group consisting of weight loss or immune factor loss, and cachexia.

[0064] The "pharmaceutical composition for enhancing immunity" may be an immunity enhancing agent that enhances the efficacy of an anti-viral vaccine. The immunity enhancing agent may be administered in combination with or sequentially to an anti-viral vaccine.

[0065] The anti-viral vaccine may be a human vaccine for the prevention of influenza, hepatitis A, hepatitis B, hepatitis C, herpes simplex virus (type 2), poliomyelitis, diphtheria, whooping cough, Haemophilus influenzae type B (Hib), measles, mumps, rubella, typhoid fever, chicken pox, dengue fever, Epstein-Paar virus infection, human papilloma virus infection, pneumococcal infection, meningococcal infection, Streptococcus pneumoniae infection, viral meningitis, rotavirus infection, tick-borne encephalitis, travel diarrhea, cholera, yellow fever or tuberculosis.

[0066] The "carriers" are compounds that facilitate the addition of compounds into cells or tissues. The "diluents" are compounds that are diluted in water that dissolve the compounds as well as stabilize the biologically active form of the compound of interest.

[0067] The carriers, excipients and diluents are not necessarily limited to, but may include, for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0068] The dosage of the pharmaceutical composition may vary depending on the age, sex, and weight of the patient, and will depend, inter alia, on the condition of the individual being treated, the particular category or type of disease being treated, the route of administration, and the identity of the therapeutic agent being used.

[0069] The pharmaceutical composition is appropriately selected depending on the absorbance and excretion rate of the active ingredient in the body, the age and weight, sex and condition of the patient or animal to be treated, the severity of the disease to be treated, etc., and may generally be administered at 0.1 to 1,000 mg / kg, preferably 1 to 500 mg / kg, more preferably 5 to 250 mg / kg, and most preferably 10 to 100 mg / kg per day. The unit dosage formulation thus formulated may be administered several times at regular time intervals as necessary.

[0070] The pharmaceutical compositions may be administered individually as a prophylactic or therapeutic agent, or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

[0071] Each of the pharmaceutical compositions may be formulated by a conventional method into oral dosage forms such as powders, granules, tablets, capsules, lozenges, suspensions, emulsions, syrups, and aerosols, and parenteral dosage forms such as sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories for use.

[0072] When the formulation is made, it may be prepared using a diluent or excipient that is commonly used, such as a filler, extender, binder, wetting agent, disintegrant, or surfactant.

[0073] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and such solid preparations can be prepared by mixing the enzymatic decomposition extract of velvet with at least one or more excipients, such as starch, calcium carbonate, sugar or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, tolerant solutions, emulsions, syrups, etc., and may contain various excipients, such as wetting agents, sweeteners, flavorings, preservatives, etc., in addition to water and liquid paraffin, which are often used as simple diluents.

[0074] For the preparation for parenteral administration, non-aqueous solvents may be used, and for suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. For the base of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. may be used.

[0075] The present invention will be described in more detail below with reference to preferred examples. However, it will be apparent to those skilled in the art that these examples are provided to more specifically explain the present invention and are not intended to limit the scope of the present invention.

[0076] Manufacturing Example 1: Manufacturing of antler powder Frozen antler (from New Zealand) containing blood was washed, crushed to an appropriate size, freeze-dried, and then crushed again and heat-treated at 123°C for 7 minutes to produce freeze-dried antler powder.

[0077] Production Example 2: Production of antler enzymatic decomposition extract 10 g of velvet powder from Preparation Example 1 was mixed with 199 mL of water. The mixture was stirred at 100 rpm in a water bath at the required temperature for each enzyme, for example, 60°C for Alcalase, and 50°C for Flavozyme, Neutrase and Protamex. 1 mL of each enzyme solution (100 mg / mL) was added at an enzyme:substrate ratio of 1:100, and the temperature was maintained for up to 23 hours while stirring for enzyme treatment. The enzyme was inactivated by holding the temperature at 85°C or higher for 20 minutes in a 95°C water bath, and then the cooled enzyme hydrolyzate was centrifuged at 25°C. The supernatant was made up to 200 mL with water, filtered under vacuum using Celite545, sterilized, dried and powdered. The powders were stored at -20°C for physiological activity analysis, and the samples were designated 1F, 2F, 3F and 5F, respectively.

[0078] In the case of pepsin, 10 g of velvet antler powder from Preparation Example 1 was mixed with 199 mL of dilute hydrochloric acid at pH 2. The mixture was stirred at 100 rpm in a 50°C water bath. 1 mL of pepsin enzyme solution (100 mg / mL) was added at an enzyme:substrate ratio of 1:100, and the mixture was enzymatically treated by stirring while maintaining the temperature for up to 23 hours. 8 mL of 6 M sodium hydroxide was added to adjust the pH to 5.5-6 to interrupt the decomposition, and the enzyme was inactivated by keeping the temperature at 85°C or higher for 20 minutes in a 95°C water bath, and then the cooled enzymatic decomposition product was centrifuged at 25°C. The supernatant was made to a constant volume of 200 mL with water, filtered using Celite545 under vacuum, sterilized, dried and powdered. The powder was stored at -20°C for physiological activity analysis, and this sample was designated as 4F.

[0079] Production Example 3: Production of control group As control groups for the enzymatic hydrolysis extract of velvet, the supercritical extract of velvet, the hot water extract of velvet and VARNZ RepaiRx were designated as C1, C2 and C3, respectively.

[0080] The velvet antler supercritical extract (C1) was produced by extracting the velvet antler powder of Production Example 1 under the conditions of carbon dioxide and ethanol as solvents, extraction pressure of 300 bar, extraction temperature of 40°C, separation pressure of 54 bar, separation temperature of 40°C, supply weight of 49.7 g, average carbon dioxide flow rate of 7.9 g / min, average ethanol flow rate of 2.0 g / min, carbon dioxide supply ratio of 22:1 and ethanol supply ratio of 3:1, and recovering the extract every 15 minutes and drying under vacuum reduced pressure.

[0081] To prepare the hot water extract of velvet antler (C2), 10 g of the powder of Preparation Example 1 was mixed with 200 mL of water and placed in a round-bottom flask, and extracted with hot water for 3 hours, then cooled on ice and centrifuged at 25,000 g for 30 minutes at 25° C. The supernatant was adjusted to 200 mL with water, filtered under vacuum using Celite 545, and the dried powder was stored at −20° C. for activity analysis.

[0082] VARNZ RepaiRx is manufactured according to US Pat. No. 07547761 and was diluted 1 / 25 and used as the C3 sample.

[0083] Experimental Example 1: Yield of velvet enzymatic decomposition extract The concentrations of the enzymatic decomposition extracts of velvet antler 1F to 5F produced in Production Example 2 and the control group C2 were measured, and the yields calculated from the results are shown in Table 1.

[0084] [Table 1] All of the enzymatically treated antler extracts from 1F to 5F showed higher yields than the hot water extract (C2). Among the enzymatically treated antler extracts, the extract using Flavozyme (2F), the extract using Neutrase (3F) and the extract using Protamex (5F) showed about 2-3 times higher yields than the hot water extract (C2) as well as the extract using other enzymes, Alcalase (1F) and Pepsin (4F).

[0085] Experimental Example 2: Effect of deer antler enzyme hydrolysis extract on enhancing immune activity of antigen-stimulated leukocytes White blood cells were isolated using blood samples provided by 12 donors (NZ Blood, Wellington, New Zealand). Red blood cell lysis reagent (TAC buffer: 85 mM Tris, 78 mM NH 4 Cl (Sigma-Aldrich, St. Louis, MO, USA) was preheated to 37°C before use. 45 mL of TAC buffer was added to 5 mL of whole blood, and the blood was placed in a 37°C water bath for 10 minutes. After centrifugation at 500xg for 10 minutes, the supernatant was removed and the blood was uniformly suspended in 25 mL of PBS (0.1M EDTA). After centrifugation in the same manner, the supernatant was removed and the blood was suspended in 5 mL of RPMI1640 (Sigma-Aldrich, 5% FCS added). The number of white blood cells used in this experiment was 1 x 10 7 cells / mL.

[0086] 50 μl of the separated leukocytes were dispensed into a 96-well plate, and then 25 μl of RPMI / FCS (control) or the velvet enzymatic decomposition extract of Production Example 2 and the control group, each diluted at 1 / 100, were added thereto, and the plates were incubated at 37° C. / 5% CO 2 The plate was placed in an incubator for 3 hours. 25 μl of stimulator was added, and the plate was incubated at 37° C. / 5% CO 2 The plates were placed in an incubator for 2 hours. The stimulators were SEB (200 ng / mL Staphylococcus enterotoxin B; Sigma-Aldrich), PWM (2 μg / mL pokeweed mitogen; Sigma-Aldrich), and lipopolysaccharide (LPS) (4 ng / mL LPS; Sigma-Aldrich). The cell lysate was prepared by centrifuging the plate at 350 x g, removing the supernatant, and resuspending the cells in 10 μl / well of Buffer RLT.

[0087] The effect of enhancing immune activity of antigen-stimulated white blood cells was analyzed by using NanoString to analyze the expression of immune-related cytokines, as shown in Figure 1. In Figure 1, up-regulation is indicated in red, and down-regulation is indicated in blue. In Figure 1, red and blue represent P<0.05, and orange and light blue represent P<0.1.

[0088] Regarding the immune-related gene enhancing effect of the velvet antler enzymatic hydrolysis extract depending on the type of enzyme, the velvet antler enzymatic hydrolysis extract (1F) using alcalase did not show any gene enhancing effect regardless of the manufacturing method of the velvet antler powder.

[0089] Among the velvet enzymatically hydrolyzed extracts using Flavozyme, the freeze-dried velvet enzymatically hydrolyzed extract (2F) increased the expression of a total of six genes, including 1FNB1, 1L12B, IL17A, IL2, IL4, and IL9.

[0090] Among the velvet antler enzymatic hydrolysis extracts using neutrase, the freeze-dried velvet antler enzymatic hydrolysis extract (3F) increased the expression of a total of six genes, including IFNB1, IL10, IL12B, IL17A, IL2, and IL9.

[0091] Among the extracts of velvet antler enzymatically decomposed using pepsin, the freeze-dried velvet antler enzymatically decomposed extract (4F) did not increase any gene expression.

[0092] Among the extracts of velvet enzymatically decomposed using Protamex, the freeze-dried velvet enzymatically decomposed extract (5F) did not increase any gene expression.

[0093] Control groups C1, C2 and C3 did not enhance any immune-related gene expression, and C3 enhanced only PTGS2 gene expression.

[0094] The activity of antler enzymatic hydrolysis extracts in promoting immune-related cytokine expression was high in the antler enzymatic hydrolysis extracts using flavozyme (2F) and neutrase (3F), while the activity of promoting immune-related cytokine expression was very low when treated with other enzymes or not treated with enzymes.

[0095] Subsequent experiments were carried out using velvet antler enzymatically hydrolyzed extract using flavozyme (2F), velvet antler enzymatically hydrolyzed extract using neutrase (3F), velvet antler enzymatically hydrolyzed extract using pepsin (4F), velvet antler enzymatically hydrolyzed extract using protamex (5F) and velvet antler hot water extract (C2).

[0096] Experimental Example 3: Effect of enzymatically decomposed extract of velvet on nitric oxide production in macrophages RAW264.7 cell line, a mouse-derived macrophage, was cultured in a medium containing 445 mL, 50 mL, and 5 mL of DMEM (Cat No. 11995-065, Gibco BRL, USA), FBS (Lot No. AE29155306, Hyclone, USA), and Antibiotic-antimycotic 100x (Batch No. ADD670076, Biopredic International, France), respectively, at 37°C and 5% CO. 2 Conditions of CO 2 The cells were cultured in an incubator (MCO-20AIC, Sanyo, Japan) and subcultured at 2- to 3-day intervals until use.

[0097] Before confirming the effect of the enzymatically decomposed extract of velvet on macrophage nitric oxide (NO) production, MTT assay was performed to confirm the cytotoxicity of the sample. Activated RAW 264.7 cell line was cultured at 3 × 10 4 Distribute cells / well into a 96-well plate and incubate at 37℃ for 24 hours in 5% CO 2 The samples were cultured in an incubator. The antler enzymatic decomposition extract samples were replaced with medium prepared according to the concentration at 0.001 to 100 mg / mL. After 24 hours of culture, 10 μl of cck-8 (Cat No. CK04-13, Dojindo, Japan) was added and the mixture was incubated at 37°C in CO 2The cells were further cultured in an incubator for 1 hour, and the absorbance was measured at a wavelength of 450 nm using a microplate reader to determine the cell viability of each sample at each concentration relative to the cell viability of the normal control group.

[0098] The enzymatically decomposed extract samples 2F, 3F, 5F and C2 of velvet antler did not show any cytotoxicity at concentrations below 0.5, 2.5, 6 and 50 mg / mL, respectively.

[0099] Nitric oxide production was evaluated by incubating RAW 264.7 cells at 5 × 10 5 The cells were dispensed at 1000 cells / well and cultured for 24 hours. The culture medium was removed and replaced with the positive control group and sample-containing medium prepared for each concentration, and then cultured for an additional 24 hours. The cell culture medium was collected and centrifuged at 1000 rpm for 5 minutes, and the supernatant was transferred to a new tube. The test was performed according to the protocol of the NO production kit (Cat No. ADI-917-020, Enzo, USA). The amount of NO produced was quantified using the absorbance value measured at a wavelength of 540 nm using 4-parameter logic regression of the standard curve, and is shown in Figure 2.

[0100] The normal control group (N) not treated with the test substance produced 14.89 μmole / L of nitric oxide, whereas the positive control group (LPS 1 μg / mL) produced 47.74 μmole / L of nitric oxide, a significant increase compared to the normal control group (p<0.01).

[0101] The amount of nitric oxide produced in the groups treated with flavozyme-treated enzymatic hydrolysis extract of velvet (2F) (100, 500 μg / mL) increased by values ​​and concentrations of 25.73 and 39.65 μmole / L, respectively, and was significantly increased compared to the normal control group (p<0.05 and p<0.01).

[0102] The amount of nitric oxide produced in the groups treated with antler enzymatic hydrolysis extract (3F) using Neutrase (500, 1000 μg / mL) was 30.42 and 29.39 μmole / L, respectively, which was significantly increased compared to the normal control group (p<0.01 and p<0.01). However, the increase was approximately 23.3% lower than that of the group treated with antler enzymatic hydrolysis extract (2F) using flavozyme at the same concentration of 500 μg / mL.

[0103] The groups treated with the Protamex enzymatic hydrolysis extract of velvet (5F) and the hot water extract of velvet (C2) showed no significant difference compared to the normal control group.

[0104] The amount of nitric oxide produced in macrophages is an indicator of whether or not macrophages, which act as a defense against foreign substances in the mammalian immune system, are activated. It was confirmed that the velvet antler enzymatic hydrolysis extract using flavozyme (2F) was the most effective in terms of the amount of nitric oxide produced, followed by the velvet antler enzymatic hydrolysis extract using neutrase (3F).

[0105] Experimental Example 4: Increasing effect of enzymatic hydrolysis extract of velvet on iNOS and COX2 gene expression To measure the expression levels of iNOS and COX-2 genes, RAW 264.7 cells were cultured in 6-well plates at 5 × 10 5 The cells were dispensed at 1000 cells / well and cultured for 24 hours. The culture medium was removed and replaced with medium containing the sample prepared for the positive control group and each concentration, and then cultured for an additional 24 hours. The cells were harvested and total RNA was extracted according to the protocol of the RNA extract kit (Cat No. 74106, Quigen, Germany). The extracted total RNA was quantified by measuring absorbance at wavelengths of 260 nm and 280 nm using Infinite 200Pro (TECAN, Switzerland), and then diluted according to the concentration. The gene expression levels of iNOS and COX2 were measured using a TOPreal One-step kit (Cat No. RT432M, enzymenomics, Korea). All reactions were corrected with β-actin and were calculated using a 2-fold increase from the normal control group. ΔΔ After normalization by the CT method, the relative expression levels of iNOS and COX2 are shown in FIG. 3 and FIG. 4, respectively.

[0106] In FIG. 3, the iNOS expression level in the positive control group (LPS, 1 μg / mL) increased 67.37-fold, which was a statistically significant increase compared to the normal control group (p<0.05).

[0107] The iNOS expression level in the groups treated with flavozyme-treated antler enzymatic hydrolysis extract (2F) (100, 500μg / mL) was 21.55 and 30.45 times higher than that in the normal control group, which was significantly increased depending on the concentration (p<0.05 and p<0.01).

[0108] The iNOS expression level in the group treated with antler enzymatic hydrolysis extract (3F) using Neutrase (500 μg / mL) was increased by 21.74 times compared to the normal control group (p<0.01), but the increase was approximately 28.4% lower than that in the group treated with antler enzymatic hydrolysis extract (2F) using flavozyme at the same concentration of 500 μg / mL.

[0109] The iNOS expression level in the group treated with velvet hot water extract (C2) (500 μg / mL) increased 1.93-fold compared to the normal control group, but was significantly lower than that of the velvet enzymatic hydrolysis extract using flavozyme (2F) and the velvet enzymatic hydrolysis extract using neutrase (3F).

[0110] On the other hand, in FIG. 4, the COX2 expression level in the positive control group (LPS, 1 μg / mL) increased 264.54-fold, which was a statistically significant increase compared to the normal control group (p<0.05).

[0111] The expression levels of COX2 in the groups treated with flavozyme-treated enzymatic hydrolysis extract of velvet (2F) (100, 500 μg / mL) were 7.82 and 17.49 times higher than in the normal control group, respectively, and were significantly increased depending on the concentration (p<0.01 and p<0.01).

[0112] The COX2 expression level in the group treated with antler enzymatic hydrolysis extract (3F) using Neutrase (500 μg / mL) was increased 8.86-fold compared to the normal control group (p<0.01), but the increase was approximately 49.3% lower than that in the group treated with antler enzymatic hydrolysis extract (2F) using flavozyme at the same concentration of 500 μg / mL.

[0113] The COX2 expression level in the group treated with hot water extract of velvet antler (C2) (500 μg / mL) was 0.98 times that of the normal control group, showing no significant difference.

[0114] iNOS is a gene involved in the production of NO by L-arginine in activated macrophages, and COX2 is a gene that is increased by NO production and other cytokines. It was confirmed that the expression levels of both genes were most significantly increased in the antler enzymatic hydrolysis extract using flavozyme (2F), followed by the second highest increase in the antler enzymatic hydrolysis extract using neutrase (3F).

[0115] Experimental Example 5: Increasing effect of immune-related cytokines on enzymatically decomposed extract of velvet The relative expression levels of immune-related cytokines TNF-α, IFN-γ, IL-6 and IL-1β were measured using the TOPreal One-step kit in the same manner as in Experimental Example 4, and are shown in Figures 5, 6, 7 and 8, respectively.

[0116] In FIG. 5, the expression level of TNF-α in the positive control group (LPS 1 μg / mL) was statistically significantly increased compared to the normal control group (N) (p<0.01).

[0117] The TNF-α expression levels in the groups treated with flavozyme-treated enzymatic hydrolysis extract of velvet (2F) (100, 500 μg / mL) were 3.17 and 3.58 times higher than in the normal control group (N), and were significantly increased depending on the concentration (p<0.01 and p<0.01).

[0118] The TNF-α expression levels in the groups treated with antler enzymatic hydrolysis extract (3F) using neutrase (500, 1000 μg / mL) were 3.2 and 3.38 times higher than the normal control group (N), and significantly increased depending on the concentration (p<0.01 and p<0.01).

[0119] The groups treated with pepsin-treated antler enzymatic hydrolysis extract (4F) (100, 500, 2000 μg / mL), protamex-treated antler enzymatic hydrolysis extract (5F) (100, 500, 2000 μg / mL), and antler hot water extract (C2) (100, 500 μg / mL) showed no significant difference in TNF-α expression levels compared to the normal control group at any treatment concentration.

[0120] Furthermore, in FIG. 6, the expression level of IFN-γ in the positive control group (LPS 1 μg / mL) was statistically significantly decreased compared to the normal control group (N) (p<0.05).

[0121] The IFN-γ expression levels in the groups treated with flavozyme-treated enzymatic hydrolysis extract of velvet (2F) (100, 500 μg / mL) were statistically significantly reduced by 0.44 and 0.59 times, respectively, compared to the normal control group (N) (p<0.05 and p<0.05).

[0122] The expression level of IFN-γ in the group treated with 3F (500 μg / mL) enzymatically hydrolyzed extract of velvet using neutrase was also significantly reduced by 0.54 times compared to the normal control group (N) (p<0.05).

[0123] However, the IFN-γ expression level in the group treated with the hot water extract of velvet antler (C2) (500 μg / mL) was not different from that in the normal control group (N).

[0124] Furthermore, in FIG. 7, the IL-6 expression level in the positive control group (LPS 1 μg / mL) was statistically significantly increased compared to the normal control group (N) (p<0.05).

[0125] The IL-6 expression levels in the groups treated with flavozyme-treated enzymatic hydrolysis extract of velvet (2F) (100, 500 μg / mL) increased by 2.0 and 2.97 times, respectively, depending on the concentration, compared to the normal control group (N), and the increase was statistically significant at the 500 μg / mL treatment concentration (p<0.01).

[0126] The IL-6 expression levels in the groups treated with 3F (500, 1000 μg / mL) containing an enzymatically hydrolyzed extract of velvet using neutrase were significantly increased by 1.85 and 1.83 times, respectively, depending on the concentration, compared to the normal control group (N) (p<0.05 and p<0.05).

[0127] The pepsin-treated velvet enzymatic hydrolysis extract (4F) group (100, 500, 2000 μg / mL), the protamex-treated velvet enzymatic hydrolysis extract (5F) group (100, 500, 2000 μg / mL), and the velvet hot water extract (C2) treated group (100, 500 μg / mL) showed no significant difference in IL-6 expression levels compared to the normal control group (N) at any treatment concentration.

[0128] Furthermore, in FIG. 8, the expression level of IL-1β in the positive control group (LPS 1 μg / mL) was statistically significantly increased compared to the normal control group (N) (p<0.05).

[0129] The expression levels of IL-1β in the groups treated with flavozyme-treated enzymatic hydrolysis extract of velvet (2F) (100, 500 μg / mL) increased by 3.18 and 6.9 times, respectively, depending on the concentration, compared to the normal control group (N), and the increase was statistically significant at the 500 μg / mL treatment concentration (p<0.01).

[0130] The IL-1β expression level in the group treated with antler enzymatic hydrolysis extract (3F) using Neutrase (500 μg / mL) was significantly increased by 2.82 times compared to the normal control group (N) (p<0.05), but the increase was approximately 59.1% lower than that in the group treated with antler enzymatic hydrolysis extract (2F) using flavozyme at the same concentration of 500 μg / mL.

[0131] In the group treated with the hot water extract of velvet antler (C2) (500 μg / mL), the expression level of IL-1β was not different from that of the normal control group (N) at any treatment concentration.

[0132] Experimental Example 6: Macrophage proliferation effect of enzymatically decomposed extract of velvet 5 × 10 activated RAW264.7 cell line 3 Distribute cells / well into a 96-well plate and incubate at 37℃ for 24 hours in 5% CO 2 The medium was replaced with a positive control group and a medium containing an enzymatically decomposed extract of velvet prepared at each concentration. After 24 hours of culture, 10 μl of cck-8 (Cat No. CK04-13, Dojindo, Japan) was added and incubated at 37°C in CO 2 After further culturing in an incubator for 1 hour, the absorbance was measured at a wavelength of 450 nm using Infinite 200Pro (TECAN, Switzerland) to determine the cell proliferation rate at each concentration relative to the cell proliferation rate of the normal control group, and the results are shown in FIG. 9.

[0133] The cell proliferation ability was calculated by calculating the cell proliferation rate for each time period after the treatment with the test substance based on the time before administration of the test substance (0 h), and statistical processing was performed based on the normal control group for each time point.

[0134] In the normal control group, cell proliferation increased by 2.35, 6.09, and 14.52 times at 24, 48, and 72 hours, respectively, while in the positive control group (LPS, 1 μg / mL), cell proliferation increased by 2.8, 11.3, and 17.7 times at 24, 48, and 72 hours, respectively, showing statistically significant differences compared to the normal control group at 24 and 48 hours (p<0.05 and p<0.01).

[0135] In the groups treated with the flavozyme-treated velvet enzymatic decomposition extract (2F) (100, 500μg / mL), the cell proliferation rate increased by 10.6 and 11.0 times, respectively, 48 hours after treatment with the test substance, compared to the normal control group after 48 hours, and the difference was statistically significant (p<0.01, p<0.01). At 72 hours, the cell proliferation rate increased by 21.68 and 20.71 times, respectively, compared to the normal control group after 72 hours.

[0136] The group treated with antler enzymatic hydrolysis extract (3F) using Neutrase (500 μg / mL) showed 11.2 and 22.4 times the normal values ​​at 48 and 72 hours, respectively, which was a statistically significant increase compared to the normal control group (p<0.01).

[0137] The group treated with the hot water extract of velvet antler (C2) (500 μg / mL) had values ​​2.18, 6.34, and 15.90 times higher at all measurement points, showing no difference from the normal control group.

[0138] Experimental Example 7: Immunity improvement effect of administration of enzymatically decomposed extract of velvet in immunosuppressed animal models In an animal model of compromised immunity, NK cell activity, interferon gamma (IFN-γ) concentration, and TNF-α concentration were measured to evaluate the effect of an enzymatic hydrolysis extract of velvet on improving immunity.

[0139] Balb / c mice were divided into 9 groups of 6 mice each, namely, a normal control group (G1), a negative control group (G2) and test substance administration groups (G3 to G9). The test substances were administered to the following groups: 50 mg / kg, 100 mg / kg and 200 mg / kg of velvet antler enzymatic hydrolysis extract (2F) using flavozyme (G3, G4 and G5, respectively); 100 mg / kg and 200 mg / kg of velvet antler enzymatic hydrolysis extract (3F) using neutrase (G6 and G7, respectively); and 100 mg / kg and 200 mg / kg of velvet antler hot water extract (C2) (G8 and G9, respectively). Cyclophosphamide (CP) was intraperitoneally administered at a dose of 70 mg / kg to the negative control group (G2) and the test substance administration groups (G3 to G9) except for the normal control group for 3 days before administration of the test substance and on the 13th day after administration of the test substance in order to induce immunosuppression. The test substance was orally administered once a day for 14 days, and the normal control group (G1) and the negative control group (G2) were orally administered with a vehicle instead of the test substance. All test groups were observed daily for general symptoms and weighed twice a week.

[0140] Body weight was decreased in the negative control group (G2) compared to the normal control group (G1), but the changes in body weight in the test substance administration groups (G3 to G9) were not statistically different from the negative control group (G2).

[0141] 1. Enhancement of NK cell activity Fourteen days after administration of the test substance, autopsy was performed to remove the spleen, and spleen cells were isolated to measure NK cell activity.

[0142] NK cell activity was measured using a commercial LDH assay kit (Cat. No.: 04744926001, Roche Diagnostics GmbH). Isolated spleen cells were used as effector cells, and YAC-1 cells, a mouse lymphoma cell line, were used as target cells. Effector cells (1 × 10 6 cells / well) and target cells (1 × 10 4 The effector cells (100:1) and effector cells (100:1) were mixed and cultured for 4 hours. The amount of LDH released by the effector cells in response to their attack on the target cells was measured and shown in Figure 10.

[0143] NK cells are known to play an important role in innate immunity, directly killing cells infected by microorganisms and cancer cells, and activating monocytes and dendritic cells by secreting cytokines.

[0144] As shown in FIG. 10, NK cell activity was 16.5% in the normal control group (G1) but 2.3% in the negative control group (G2), which was a statistically significant decrease compared to the normal control group (p<0.01).

[0145] In the G3, G4 and G5 groups administered 50, 100 and 200mg / kg of the velvet enzymatic decomposition extract (2F) using Flavozyme, the NK cell activity was 23.1, 26.4 and 23.7%, respectively, which was a statistically significant increase compared to the negative control group (G2) (p<0.01, p<0.01 and p<0.01), and was also statistically significantly increased compared to the same concentration of the velvet hot water extract (G8, G9) (p<0.01 and p<0.01).

[0146] In G6 and G7, which were administered 100 and 200mg / kg of antler enzyme decomposition extract (3F) using Neutrase, NK cell activity was 19.3 and 17.9%, respectively, which was a statistically significant increase compared to the negative control group (G1) (p<0.01 and p<0.01). However, the NK cell activity was lower than that of the antler enzyme decomposition extract (2F) using Flavozyme at the same concentration, and there was no statistically significant difference compared to the antler hot water extract (G8 and G9) at the same concentration.

[0147] G8 and G9, which were administered 100 and 200 mg / kg of hot water extract of velvet antler (C2), showed NK cell activity of 13.8 and 11.6%, respectively, which was a statistically significant increase compared to the negative control group, but was lower than the values ​​obtained with the same concentration of enzymatically hydrolyzed extract of velvet antler using flavozyme (2F) and enzymatically hydrolyzed extract of velvet antler using neutrase (3F).

[0148] 2. Analysis of immune cytokines in blood Representative cytokines secreted by Th1 cells are TNF-α and IFN-γ, which are known to increase the activity of macrophages and stimulate phagocytosis. The blood collected at the autopsy was left at room temperature for 15 to 30 minutes, centrifuged to separate the serum, and the IFN-γ concentrations were measured using TNF-α (Cat. No.: MTA00B, R&D systems) and IFN-γ (Cat. No.: MIF00, R&D systems) ELISA kits according to the manufacturer's manual, and the results are shown in Figures 11 and 12, respectively.

[0149] In FIG. 11, the blood TNF-α concentration in the normal control group (G1) was 9.7 pg / mL, while that in the negative control group (G2) was 12.2 pg / mL, showing a statistically significant increase in the negative control group (G2) in which immunosuppression was induced compared to the normal control group (G1) (p<0.01).

[0150] In G3, G4 and G5, which were administered 50, 100 and 200 mg / kg of antler enzymatic hydrolysis extract (2F) using Flavozyme, the TNF-α concentrations were 10.1, 10.3 and 10.0 pg / mL, respectively, and were significantly reduced in the low, medium and high concentration groups compared to the negative control group (G1) regardless of the administration concentration (p<0.05, p<0.05 and p<0.05, respectively).

[0151] In G6 and G7, which were administered 100 and 200 mg / kg of an enzymatically hydrolyzed extract of antler using Neutrase (3F), the TNF-α concentrations were 10.6 and 10.3 pg / mL, respectively. At the medium concentration, there was no significant difference from the negative control group (G2), and only at the high concentration, the TNF-α concentrations were significantly reduced compared to the negative control group (G2) (p<0.05).

[0152] Therefore, it was confirmed that the enzymatic hydrolysis extract of velvet using flavozyme (2F) significantly reduced the TNF-α concentration in the low concentration group of 50 mg / kg compared to the negative control group (G2), whereas the enzymatic hydrolysis extract of velvet using neutrase (3F) significantly reduced the TNF-α concentration in the high concentration group of 200 mg / kg compared to the negative control group (G2).

[0153] Furthermore, there was no significant difference in TNF-α concentration between G8 and G9, which were administered with 100 and 200 mg / kg of the hot water extract of velvet (C2), as compared with the negative control group (G1).

[0154] In FIG. 12, the blood IFN-γ concentration in the normal control group (G1) was 12.7 pg / mL, while that in the negative control group (G2) was 10.2 pg / mL, showing a statistically significant decrease in the negative control group (G2) in which immunosuppression was induced compared to the normal control group (G1) (p<0.01).

[0155] In G3, G4 and G5, which were administered 50, 100 and 200mg / kg of velvet enzymatic decomposition extract (2F) using Flavozyme, the IFN-γ concentration was 10.8, 11.4 and 11.7pg / mL, respectively, and increased depending on the concentration compared to the negative control group (G2). In particular, the increase was statistically significant in G4 and G5 administered at 100 and 200mg / kg (p<0.05 and p<0.01). The increase was also statistically significant compared to the same concentration of velvet hot water extract (G8 and G9) (p<0.05 and p<0.01).

[0156] G6 and G7, which were administered 100 and 200 mg / kg of antler enzymatic hydrolysis extract (3F) using Neutrase, and G8 and G9, which were administered 100 and 200 mg / kg of antler hot water extract (C2), showed no difference from the negative control group (G2).

[0157] The following describes examples of formulations of the composition containing the enzymatically decomposed extract of velvet of the present invention, but these are not intended to limit the present invention but are merely for illustrative purposes.

[0158] Formulation Example 1: Powder Production Production Example 2: Deer antler enzymatic decomposition extract powder 20mg Lactose 100mg Talc 10mg The above ingredients are mixed and packed into an airtight cloth to produce a powder.

[0159] Formulation Example 2: Tablet Production Production Example 2: Deer antler enzymatic decomposition extract powder 10mg Corn starch 100mg Lactose 100mg Magnesium stearate 2mg The above ingredients are mixed and then compressed into tablets by a conventional tablet manufacturing method.

[0160] Formulation Example 3: Production of capsules Production Example 2: Deer antler enzymatic decomposition extract powder 10mg Crystalline cellulose 3mg Lactose 14.8mg Magnesium stearate 0.2mg The above ingredients are mixed according to a conventional capsule manufacturing method, and the mixture is filled into a gelatin capsule to produce a capsule.

[0161] Formulation Example 4: Preparation of granules Production Example 2: Deer antler enzymatic decomposition extract powder 1,000mg Vitamin mixture (as needed) Vitamin A acetate 70μg Vitamin E 1.0mg Vitamin B1 0.13mg Vitamin B2 0.15mg Vitamin B6 0.5mg Vitamin B12 0.2 μg Vitamin C 10mg Biotin 10μg Nicotinamide 1.7mg Folic acid 50μg Calcium pantothenate 0.5mg Inorganic mixture appropriate amount Ferrous sulfate 1.75mg Zinc oxide 0.82mg Magnesium carbonate 25.3mg Dipotassium phosphate 15mg Dicalcium phosphate 55mg Potassium citrate 90mg Calcium carbonate 100mg Magnesium chloride 24.8mg The composition ratio of the above vitamin and mineral mixture is a preferred example of a mixture of ingredients that are suitable for functional health foods, but the mixing ratio may be modified as desired. After mixing the above ingredients using conventional functional health food manufacturing methods, granules can be produced and used to manufacture a functional health food composition using conventional methods.

[0162] Formulation Example 5: Production of beverage dosage form Production Example 2: Deer antler enzymatic decomposition extract powder 1,000mg Citric acid 1,000mg Oligosaccharides 100g Plum extract 2g Taurine 1g Add purified water to make a total of 900mL The above ingredients are mixed according to a conventional beverage manufacturing method, then stirred and heated at 85°C for about 1 hour, and the resulting solution is then filtered into a 2L sterilized container, sealed, sterilized, and stored in a refrigerator for use in the manufacture of the functional beverage composition of the present invention.

Claims

1. The present invention comprises an enzymatically decomposed extract of velvet obtained by decomposing velvet with an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28 as an active ingredient; The enzyme of the IUBMB enzyme nomenclature class EC 3.4.11.1 is a composite of endoprotease and exopeptidase produced by submerged fermentation of Aspergillus oryzae; and The food composition for enhancing immunity, wherein the enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28 is a neutral metallo(Zn) endoprotease produced by submerged fermentation of Bacillus amyloliquefaciens.

2. The food composition for enhancing immunity according to claim 1, wherein the enzyme is an enzyme of the IUBMB enzyme nomenclature class EC 3.4.11.

1.

3. The food composition for enhancing immunity according to claim 1, wherein the complex of endoprotease and exopeptidase is a flavourzyme.

4. The food composition for enhancing immunity according to claim 1, wherein the enzyme is an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.

28.

5. The food composition for enhancing immunity according to claim 1, wherein the neutral metallo(Zn)-endoprotease is Neutrase.

6. The immune-enhancing food composition according to any one of claims 1 to 5, characterized in that the velvet antler enzymatic decomposition extract is an velvet antler enzymatic decomposition extract obtained by hydrolyzing raw velvet antler or dried velvet antler obtained by freeze-drying frozen velvet antler through enzymatic treatment.

7. A freeze-dried antler preparation step in which raw or frozen antler is freeze-dried; and An enzyme treatment step of adding 50 to 500 parts by weight of water and 0.01 to 1 part by weight of enzyme to 10 parts by weight of the freeze-dried antler and reacting at 30 to 60° C. for 1 to 48 hours; The enzyme is an enzyme of the IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of the IUBMB enzyme nomenclature class EC 3.4.24.28, The enzyme of the IUBMB enzyme nomenclature class EC 3.4.11.1 is a composite of endoprotease and exopeptidase produced by submerged fermentation of Aspergillus oryzae; and The enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28 is a neutral metallo(Zn) endoprotease produced by submerged fermentation of Bacillus amyloliquefaciens, and is characterized in that the enzyme is an enzyme-decomposed extract of velvet having immune enhancing activity.

8. The method for producing an enzymatically decomposed extract of velvet having immune enhancing activity according to claim 7, wherein the enzyme is an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.

1.

9. The present invention comprises an enzymatically decomposed extract of velvet obtained by decomposing velvet with an enzyme of IUBMB enzyme nomenclature class EC 3.4.11.1 or an enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28 as an active ingredient; The enzyme of the IUBMB enzyme nomenclature class EC 3.4.11.1 is a composite of endoprotease and exopeptidase produced by submerged fermentation of Aspergillus oryzae; and The pharmaceutical composition for enhancing immunity, wherein the enzyme of IUBMB enzyme nomenclature class EC 3.4.24.28 is a neutral metallo(Zn) endoprotease produced by submerged fermentation of Bacillus amyloliquefaciens.

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