Composition for enhancing immunity comprising gintonin which is red ginseng residue-derived glycolipoprotein complex

Abstract

The present invention relates to a composition for enhancing immunity, comprising gintonin, which is a red ginseng residue-derived glycolipoprotein complex comprising lysophosphatidic acid as a main ingredient, and, in particular, provides immune activity enhancement efficacy obtained by a red ginseng residue-derived glycolipoprotein complex composition, in which gintonin isolated from red ginseng residue induces the activity of macrophages, NK cells, and T cells, thereby increasing the secretion of cytokines, thus being able to improve immune activity. In addition, an immunity-enhancing product capable of enhancing immunity can be developed by using gintonin separated from red ginseng residue, and therefore, a low-cost, high-functional health functional product can be manufactured and effectively used for preventing or treating diseases.

Description

An immune-enhancing composition containing gintonin, a glycolipoprotein complex derived from red ginseng extract

[0001] The present invention relates to providing a red ginseng extract-derived glycolipoprotein complex composition capable of enhancing immune activity by increasing the secretion of cytokines by inducing the activity of macrophages, NK cells, and T cells using gintonin isolated from red ginseng extract.

[0002]

[0003] The immune system functions as the body's defense system against internal cancer cells and external invading bacteria and viruses. The recent global spread of coronavirus disease 2019 (COVID-19) has led to increased morbidity and mortality rates. The importance of immunity has been growing as it has been revealed that a weakened immune system contributes to the spread of COVID-19. Consequently, many researchers are developing various natural products that can modulate or strengthen the body's immune function.

[0004] Ginseng (Panax ginseng) contains numerous regulatory chemicals, such as ginsenosides, polysaccharides, peptides, and phytosterols, and possesses numerous biological activities, including anticancer, antioxidant, and immunomodulatory effects. Ginseng is commonly used as a tonic for longevity / life extension, or as an adaptogenic agent, known to enhance biological functions by combating various stresses, fatigue, cancer, and diabetes. Ginseng has been used for over a thousand years in Korea, China, and Japan, and has recently become one of the world's most popular medicinal herbs (Tyler, J. Pharm. Technol. 11, 214-220, 1995). Among the ginseng components, ginsenosides, also known as ginseng saponins, were isolated in the early 1960s and have been widely used in research since they became known as representative components in physiological / pharmacological studies (Shibata, et al. Tetraheadron Letters 1962, 1239-1245, 1963; Wagner-Jauregg and Roth, Pharm Acta Helv 37, 352-357, 1962). In addition, many studies have revealed that ginseng contains other unknown components such as polysaccharides, polyacetylenes, and water-soluble proteins (Nah, Kor. J. Ginseng Sci. 21, 1-12, 1997). Among these, gintonin, with a molecular weight of approximately 17 kDa, is actively being researched and developed as a G protein-coupled LPA receptor derived from ginseng with saponin removed. Gintonin is a new material newly isolated from red ginseng produced from ginseng and is a glycolipoprotein complex.The physiologically active component of gintonin is lysophosphatidic acid (LPA), and LPA acts as a G protein-coupled LPA receptor ligand to exert physiological activity. While ginseng gintonin has been reported to exhibit various efficacies, there have been no reports yet on its physiological role in improving immune function.

[0005] Korean red ginseng marc is the name for the by-product (residue) left after extracting red ginseng extract. Although the Ministry of Food and Drug Safety currently allows red ginseng marc to be used as a food additive, most of it is used as feed or compost, and the rest is disposed of as waste that requires disposal costs.

[0006] Accordingly, the inventors of the present invention discovered that red ginseng extract contains a large amount of LPA component, and studied the immune activity enhancing effect of a red ginseng extract-derived glycolipoprotein complex composition that can enhance immune activity by increasing the secretion of cytokines by inducing the activity of macrophages, natural killer cells (NK cells), and T cells by gintonin isolated from red ginseng extract.

[0007]

[0008] The problem to be solved by the present invention is to provide a pharmaceutical composition for enhancing immunity containing gintonin derived from red ginseng extract as an active ingredient.

[0009] In addition, the problem to be solved by the present invention is to provide a manufacturing method capable of manufacturing the composition for enhancing immunity more efficiently.

[0010] In addition, the problem to be solved by the present invention is to provide a health functional food for enhancing immunity, which contains gintonin derived from the red ginseng extract as an effective ingredient.

[0011] In addition, the problem to be solved by the present invention is to provide a method for preventing or treating an immune disease, which comprises a step of administering the compound to a subject in need thereof.

[0012] The present invention also provides a use of the above-described immune-enhancing pharmaceutical composition for preparing a medicament for use in the treatment or prevention of an immune disease.

[0013] The problems to be solved by the present invention are not limited to the problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0014]

[0015] In order to solve the above problem, according to one aspect of the present invention, an immune-enhancing pharmaceutical composition comprising red ginseng extract-derived gintonin as an active ingredient is provided.

[0016] According to another aspect of the present invention, a pharmaceutical composition for enhancing immunity is provided, characterized in that the gintonin comprises lysophosphatidic acid as an active ingredient.

[0017] According to one aspect of the present invention, an immune-enhancing pharmaceutical composition is provided, characterized in that the gintonin is isolated from red ginseng extract.

[0018] In order to solve the above problem, according to another aspect of the present invention, a method for producing an immune-enhancing pharmaceutical composition is provided, comprising the steps of: 1) adding ethanol to red ginseng powder and performing reflux cooling extraction to obtain an extract; 2) filtering the extract obtained in step 1); 3) repeating steps 1) to 2) 1 to 5 times to obtain a filtrate; and 4) vacuum concentrating the filtrate obtained in step 3, followed by dialysis and freeze-drying to obtain an ethanol extract containing crude ginseng.

[0019] According to another aspect of the present invention, a health functional food for enhancing immunity is provided, which comprises gintonin derived from red ginseng extract as an active ingredient.

[0020] According to one aspect of the present invention, a method for preventing or treating an immune disease is provided, comprising administering the compound to a subject in need thereof.

[0021] In addition, according to another aspect of the present invention, there is provided a use of the above-described immune-enhancing pharmaceutical composition for preparing a medicament for use in the treatment or prevention of an immune disease.

[0022]

[0023] The gintonin isolated from the red ginseng extract of the present invention can enhance immune activity by increasing the activity of NK cells and macrophages, increasing the toxicity of NK cells, increasing the activity of T cells, and increasing the expression of cytokines related to immune enhancement, and thus can be used as a composition for enhancing immunity.

[0024] According to the present invention, red ginseng marc, which is a by-product (residue) remaining after extracting red ginseng extract, can be utilized, and thus it is economical because waste resources can be utilized, and cost can be reduced, making it price competitive.

[0025] In addition, it is manufactured using only ethanol and water, is edible, can be put to practical use immediately without separate stability (toxicity) testing, and can be used as a health functional food composition.

[0026] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0027]

[0028] Figure 1 is a summary of the experimental process and a diagram showing the effects of gintonin on cell viability, body weight, and organ index.

[0029] Figure 2 is CD3 + T cells and CD4 + This is a diagram confirming the efficacy of gintonin on the distribution of T cells.

[0030] Figure 3 is CD8 + This is a diagram confirming the efficacy of gintonin in influencing the distribution of T cells.

[0031] Figure 4: CD3 in the spleen - CD161 + This is a diagram confirming the efficacy of gintonin on the differentiation of NK cells.

[0032] Figure 5 is a drawing confirming the effect of gintonin on the proliferation rate of T cells and B cells.

[0033] Figure 6 is a drawing confirming the efficacy of gintonin on the cytotoxicity of splenic NK cells against Yac-1 tumor cells.

[0034] Figure 7 is a drawing confirming the effect of gintonin on the expression of genes secreting IL-2, IL-6, and IFN-γ.

[0035] Figure 8 is a diagram showing the effect of gintonin on the regulation of iNOS, IL-1β, and GMCSF gene expression.

[0036] Figure 9 is a drawing confirming the effect of gintonin on changes in the concentrations of IL-2, IL-6, and IFN-γ.

[0037] Figure 10 is a diagram showing the experimental process using intraperitoneal macrophages and the effect of gintonin on macrophage viability.

[0038] Figure 11 is a drawing confirming the effect of gintonin on differentiation of M1-type macrophages expressing CD80.

[0039] Figure 12 is a diagram confirming the effect of gintonin on the expression of genes related to the immune activity of macrophages.

[0040]

[0041] Hereinafter, the present invention will be described in more detail. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0042] The terminology used in this specification is solely for the purpose of describing specific embodiments and is not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In the present specification, the term "comprising" or "including" a component does not exclude other components, but rather implies the inclusion of other components, unless otherwise specifically stated.

[0043] The term "extract" as used herein refers to an active ingredient isolated from a natural product, i.e., a substance exhibiting the desired activity. The extract may be obtained through an extraction process using water, an organic solvent, or a mixture thereof, and includes the dried powder form of the extract or any form formulated using the extract. Furthermore, the extract also includes fractions of the extract obtained through the extraction process.

[0044] The term "immunoenhancement" of the present invention is one of the important therapeutic strategies that reinforces the body's defense mechanisms against various diseases such as infectious diseases, inflammatory diseases, and cancer. It can achieve an immune-enhancing effect by increasing the activity of immune cells and stimulating the immune response. For example, phagocytes play a key role in the immune response. The main role of macrophages, phagocytosis, is to absorb microorganisms and other pyrogenic particles, and also stimulate the immune response by secreting cytokines such as TNF-α (tumor necrosis factor), IL-1β, and IL-12, and cytotoxic and inflammatory substances such as nitric oxide (NO). Therefore, increasing macrophage activity can be one means of immune enhancement. Since the occurrence of infections and diseases mainly occurs in a state of weakened immune function, various studies are being conducted to enhance these immune responses using immune-enhancing substances when the body's immune system function is weakened.

[0045] The above immunity may be innate immunity or acquired immunity, and preferably may mean both innate immunity and acquired immunity.

[0046] One aspect of the present invention provides a pharmaceutical composition for enhancing immunity, comprising gintonin derived from red ginseng extract as an active ingredient.

[0047] Before explaining, red ginseng is a type of ginseng that is made by carefully selecting fresh ginseng grown in suitable ginseng cultivation areas, steaming it for a long time without removing the epidermis, and then drying it (steaming and drying). Fresh ginseng dug up from the field is first classified into grades 1 to 5 based on size and shape, and then washed, steamed, and dried to become red ginseng. This red ginseng contains various saponins, and provides effects such as restoring energy, strengthening immunity, helping with nutrition and strengthening, and having antioxidant effects. Most red ginseng products have utilized saponin series (ginsenoside series) extracts manufactured by heating red ginseng with water or alcohol, and the ginsenoside content has been considered the quality standard for red ginseng products.

[0048] Meanwhile, the manufacturing method of red ginseng extract used in existing red ginseng products uses a method of extraction and concentration using hot water or alcohol (70% alcohol) depending on the solvent used. In the process of manufacturing this red ginseng extract, red ginseng by-product, red ginseng marc, is generated.

[0049] Despite the fact that multiple extractions were performed in the production of red ginseng extract, a significant amount of functional nutrients are not extracted from the red ginseng extract and are contained in the red ginseng extract, and the mass of the red ginseng extract is approximately 40 to 50% of that of red ginseng.

[0050] In other words, the red ginseng residue remaining after red ginseng processing contains a large amount of various functional substances comparable to saponins, and this red ginseng residue contains non-saponin functional substances such as acidic polysaccharides, polyacetylene components such as panaxytriol and panaxadiol, phenolic compounds, essential oil components, amino sugars, and vitamins.

[0051] However, despite the presence of functional ingredients contained in such red ginseng marc, most of the red ginseng marc is being discarded. However, the present invention utilizes red ginseng marc, and thus, by-products remaining after red ginseng production can be utilized, so that the remaining red ginseng marc can be used without being discarded.

[0052] The above gintonin is a new material newly isolated from red ginseng produced from ginseng and is a glycolipoprotein complex. The physiologically active component of gintonin is lysophosphatidic acid (LPA), and LPA acts as a G protein-linked LPA receptor ligand to exhibit physiological activity.

[0053] The gintonin according to the present invention provides a pharmaceutical composition for enhancing immunity, which includes lysophosphatidic acid as an active ingredient.

[0054] The above lysophosphatidic acid (LPA) is LPA C 18:2 , LPA C 18:1 , and LPA C 16:0 It may be one or more selected from, preferably LPA C 18:2 It may be. At this time, the LPA may be forming a complex through hydrophobic bonding with sugars and proteins.

[0055] The present invention provides an immune-enhancing pharmaceutical composition that enhances immune activity by inducing the activity of macrophages, NK cells, and T cells.

[0056] These macrophages, which play a role in innate immunity and are present in all tissues, perform essential functions in eliminating pathogens such as bacteria and viruses, as well as cancer cells. Based on their phenotypic markers, these macrophages are classified into immunosuppressive macrophages (M2 macrophages) and macrophages that eliminate pathogens such as bacteria and viruses (M1 macrophages).

[0057] In a specific example of the present invention, gintonin was administered to rats at various concentrations for macrophage activity analysis, and macrophage polarization (distribution) was analyzed through CD80 analysis. As a result, it was confirmed that the gintonin administration group increased, as shown in Fig. 11.

[0058] CD3 among peritoneal immune cells + CD4 T cells, CD3 + CD8 + T cells, CD3 + The distribution of T cells and granulocytes was analyzed phenotypically using flow cytometry.

[0059] CD3 above + T cells are CD8 + T cells and CD4 + T cells involved in activating T cells, CD4 + Helper T cells (T cells) are immune cells that promote humoral immunity by regulating the differentiation and activation of other immune cells. As shown in Figure 3, after administration of gintonin, splenic immune cells were isolated and T cell distribution was measured, confirming a significant increase in each distribution.

[0060] Additionally, gintonin inhibits CD8 in the spleen. + Increases differentiation of T cells. CD8 + T cells are cytotoxic T cells that recognize foreign antigens expressed by pathogens such as viruses and bacteria and tumors, and then eliminate abnormal cells expressing foreign antigens.

[0061] Gintonin is CD3 in the spleen - CD161 + Increases the differentiation of NK cells. Rat NK cells express CD161, a phenotypic NK receptor protein, on the cell membrane, but do not express CD3, so rat NK cells are CD3 - CD161 +It is an important immune cell responsible for primary immunity and is expressed as a cell. As shown in Figure 4, after gintonin administration, CD3 - CD161 + Analysis of the distribution of NK cells confirmed a significant increase.

[0062] Additionally, according to the present invention, the composition provides for inducing an increase in the expression of one or more cytokines selected from the group consisting of IL-2, IL-6, IL-10, IFN-γ, iNOS, GMCSF and IL-1β.

[0063] Cytokines are proteins secreted by immune cells and are important mediators that regulate the activity of immune cells and the immune system. In the present invention, we analyzed the levels of cytokine gene expression secreted by T cells, B cells, and macrophages in a group administered gintonin, a newly extracted extract from red ginseng extract.

[0064] IL-2 not only induces the differentiation, proliferation, and activity of T cells, but also increases the formation of NK cells and antibody production of B cells, which is effective in suppressing tumor metastasis. IL-6 is a cytokine that functions in both innate and adaptive immunity and promotes the proliferation and differentiation of T and B cells. IFN-γ is mainly secreted by T cells and NK cells, activates macrophages, and increases MHC expression.

[0065] As shown in Fig. 7, an increase in IL-2 mRNA expression, IL-6 mRNA expression, and IFN-γ mRNA expression were confirmed in the gintonin administration group. These results confirmed that gintonin administration was effective in producing IL-2, IL-6, and IFN-γ, which are related to immunity, especially the activity of NK cells and T cells.

[0066] Nitric oxide (NO) is produced in immune cells, and iNOS is expressed in various cells during bacterial infection. When iNOS is expressed, large amounts of NO are produced for a long period of time, and it is known as a substance that fights pathogens in the immune system. In the present invention, it was confirmed that the expression of iNOS-producing mRNA increased in a concentration-dependent manner as a result of administering gintonin at various concentrations. These results indicate that gintonin can exhibit immune activity by promoting NO production through iNOS expression.

[0067] GM-CSF is produced by osteoblasts and mesenchymal stem cells, participates in the formation of osteoclasts, stimulates monocyte production in the bone marrow, and activates the phagocytic and bactericidal action of cells against foreign substances. Therefore, to determine whether gintonin affects GM-CSF production, mRNA analysis was performed. As a result, as shown in Figure 8, GM-CSF mRNA expression was confirmed to be increased compared to the control group.

[0068] IL-1β is known as a representative pro-inflammatory cytokine that regulates inflammatory responses in vitro and in vivo. Therefore, mRNA analysis was performed to determine whether it affects the expression of IL-1β. As a result, as shown in Figure 8, it was confirmed that the amount of IL-1β mRNA expression increased compared to the control group. Therefore, the present invention revealed that gintonin stimulates immune cells and acts as a biological defense against infections such as invasion of foreign substances.

[0069] The pharmaceutical composition for enhancing immunity of the present invention can be used for the prevention or treatment of immune diseases through its immune enhancing effect, and can be used for the purpose of restoring a reduced immune state. The immune diseases may be, but are not limited to, colds, asthma, pneumonia, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergies, rheumatoid arthritis, Alzheimer's disease, autoimmune thyroid disease, inflammatory bowel disease, aplastic anemia, lupus erythematosus, or psoriasis.

[0070] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier or diluent, and may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. The pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, it includes diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Oral solid preparations include tablets, pills, powders, granules, capsules, etc., and these solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., and may include lubricants such as magnesium stearate, talc, etc. Oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc., and may include diluents such as water and liquid paraffin, wetting agents, sweeteners, fragrances, preservatives, etc. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0071] The pharmaceutical composition of the present invention can be administered to mammals such as livestock and humans by various routes, for example, oral, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, intrarectal, intrauterine, intradural or intracerebrovascular injection, and topical administration. Accordingly, the composition of the present invention can be formulated in various forms, such as tablets, capsules, aqueous solutions, or suspensions. In the case of tablets for oral administration, carriers such as lactose and corn starch and lubricants such as magnesium stearate can typically be added. In the case of capsules for oral administration, lactose and / or dried corn starch can be used as diluents. If an oral aqueous suspension is required, the active ingredient can be combined with an emulsifier and / or suspending agent. If desired, specific sweeteners and / or flavoring agents can be added. For intramuscular, intraperitoneal, subcutaneous, and intravenous administration, a sterile solution of the active ingredient is typically prepared, and the pH of the solution must be suitably adjusted and buffered. For intravenous administration, the total concentration of the solute should be adjusted to impart isotonicity to the formulation. The composition according to the present invention may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier, such as saline with a pH of 7.4. The solution may be introduced into the patient's intramuscular bloodstream by local injection.

[0072] The dosage of the active ingredient contained in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the form of the active ingredient, the route and period of administration, and can be appropriately adjusted depending on the patient. For example, the active ingredient may be administered at a dosage of 0.0001 to 300 mg / kg per day, preferably 50 to 300 mg / kg, and the administration may be administered once or twice a day. In addition, the pharmaceutical composition of the present invention may contain the active ingredient in a weight percentage of 0.001 to 90% based on the total weight of the composition.

[0073] The method for treating a disease caused by the above-mentioned immunodeficiency is to administer the composition to a human or an animal other than a human, particularly a mammal, for example, to an individual to be treated who has a disease caused by the above-mentioned immunodeficiency.

[0074] The subject of treatment may have a disease caused by the above-mentioned immune system deficiency.

[0075] The dosage, administration method, and number of administrations for the above treatment may refer to the dosage, administration method, and number of administrations of the pharmaceutical composition, medicine, veterinary pharmaceutical composition, or veterinary medicine.

[0076] In addition, the method for producing an immune-enhancing pharmaceutical composition containing ginseng extract derived from red ginseng marc of the present invention comprises the steps of 1) adding ethanol to red ginseng marc powder and extracting it by reflux cooling to obtain an extract; 2) filtering the extract obtained in step 1); 3) repeating steps 1) to 2) 1 to 5 times to obtain a filtrate; and 4) vacuum concentrating the filtrate obtained in step 3), followed by dialysis and freeze-drying to obtain an ethanol extract containing crude ginseng marc.

[0077] The above step 1) may be performed at 50°C to 100°C for 5 to 10 hours, preferably at 80°C for 8 hours, but is not limited thereto.

[0078] In addition, according to one aspect of the present invention, a health functional food for enhancing immunity is provided, which includes gintonin derived from red ginseng extract as an active ingredient.

[0079] The above “health functional food” refers to a food manufactured and processed using raw materials or ingredients with functionality useful to the human body according to the Health Functional Food Act, and “functionality” refers to consumption for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological action.

[0080] The health functional food of the present invention may contain conventional food additives, and its suitability as the above "food additive" is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0081] Items listed in the above "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to cotton, preservative preparations, and tar color preparations.

[0082] The health functional food of the present invention may contain the compound in a weight percentage of 0.01 to 95%, preferably 1 to 80%, based on the total weight of the composition, for the purpose of enhancing immunity to strengthen the immune system. In addition, the health functional food of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., for the purpose of enhancing immunity to strengthen the immune system.

[0083]

[0084] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0085]

[0086] Materials and Methods

[0087] Experimental design

[0088] The evaluation of the immune activation efficacy of gintonin was conducted using animals (rats), and the animal groups were randomly assigned to the following six groups.

[0089] 1) Normal control group (N / C): Animal group not treated with gintonin

[0090] 2) Positive control group: Levamisole, known as an immunostimulating drug, was injected intramuscularly at a dose of 10 mg / kg every 3 days for 14 days.

[0091] 3) 50 mg / kg gintonin group: 50 mg / kg of gintonin administered orally daily for 14 days.

[0092] 4) 100 mg / kg gintonin group: 100 mg / kg of gintonin administered orally daily for 14 days.

[0093] 5) 300 mg / kg gintonin group: 300 mg / kg of gintonin administered orally daily for 14 days.

[0094]

[0095] Red ginseng extract alcohol method

[0096] Water-extracted red ginseng powder was donated from Punggi Agricultural Cooperative. 1,000 g of red ginseng powder was added to 7 L of 70% (w / v) ethanol, and the extract was refluxed and cooled at approximately 80°C for 8 hours, filtered (the above process was repeated 3 times), concentrated in a vacuum concentrator, dialyzed (dialysis membrane pore size: 6,000–8,000), and freeze-dried to obtain 80 g of ethanol extract (EtOH ext.).

[0097]

[0098] Lysophosphatidic acid (LPA C) present in gintonin 18:2 ) Jeongrang method

[0099] Lysophosphatidic acids (LPAs) in crude gintonin were quantitatively analyzed using a liquid chromatography-mass spectrometry / mass spectrometry system (LC-MS / MS system, Agilent series 1100 (Palo Alto, CA, USA)). MS / MS was performed using an API 2000 (Applied Biosystems, Foster City, CA, USA) equipped with an electrospray ionization interface. Under liquid chromatography conditions, the column was separated using a C18 stainless column (ZORBAX Eclipse XDB, 2.1 mm ID × 100 mm, particle size: 3.5 μm, Agilent, USA), and the mobile phase was a mixture of 5 mM ammonium acetate and acetonitrile dissolved in double-distilled water at a volume ratio of 20:80 (pH 2.3). At this time, the flow rate was 0.2 ml / min, the injection amount was 10 μl, and the column temperature was maintained at 25°C.

[0100] The ionization method for mass spectrometry was electrospray ionization (ESI), and the detection method was analyzed in negative ion mode and multiple reaction monitoring (MRM) mode. The spray voltage was 5,000 V, the ion source temperature was maintained at 350°C, and nitrogen was used as the collision gas. The optimized parameter values ​​of MS / MS are as shown in [Table 1] below, and the obtained data were analyzed using Analyst 1.4.2 software (ABI, Inc.).

[0101]

[0102] AnalyteMRM ion (m / z)Precursor ion / Product ionDeclustering potential (V)Collision energyPotential (V)Collision exit potential (V)LPA C 18:2 433 → 79-26-78-4LPA C 18:0 (IS)437 → 152.9-91-36-4

[0103] *In the above, IS is used as an internal standard.

[0104] The quantification of LPAs was performed by injecting the test solution and standard solution into a liquid chromatography / mass spectrometer (LC / MS-MS) according to the above conditions, calculating the peak area ratio of each LPA to the peak area of ​​the internal standard from the obtained chromatogram, and calculating the concentration of LPAs contained in gintonin from the prepared calibration curve.

[0105]

[0106] Sample (Standard Spike amount, μg / ml)Calculated concentration (μg / mL) through LC-MS / MSAccuracy (%)LPA 18:2 (0.05)0.047194.3LPA 18:2 (0.1)0.099999.9LPA 18:2 (0.2)0.208104LPA 18:2 (0.5)0.51102LPA 18:2 (0.8)0.851106LPA 18:2 (1.0)0.93493.4

[0107] At this time, standard LPA C 18:2 Preparation of stock solution (1 mg / ml) and LPA C used as internal standard (IS) 18:0(1 mg / ml) was prepared using HPLC-grade methanol, and the prepared stock solution was diluted with HPLC-grade methanol and stored at 4°C until use. In addition, 0.1 to 2 μg / ml of calibration standard solutions was used. In addition, LPA C contained in crude gintonin obtained by 70% alcohol extraction from red ginseng extract 18:2 is as shown in [Table 3] below.

[0108]

[0109] SampleLPAC 18:2 (%) (average of n=4) Red ginseng extract 0.25

[0110]

[0111] Preparation of laboratory animals and approval of animal testing

[0112] For the experiment, male Sprague-Dayley (SD) rats (6 weeks, approximately 175 g) were purchased from Hanil Laboratory Animal Co., Ltd. (Korea) and fed standard rat diet. The rats were maintained in a rat animal room at 23+2℃, and after a one-week adaptation period for stabilization before administration of gintonin, the experiment was performed in accordance with the guidelines of the Chonbuk National University Experimental Animal Ethics Committee (IACUC Approval No.: NON2023-03-001-002).

[0113] The experimental animals were divided into five groups. The normal control (NTC) animals were fed standard feed and tap water daily, the positive control group received 10 mg / kg levamisole orally once daily for 14 days, and the gintonin administration groups at concentrations of 50, 100, and 300 mg / kg received gintonin at concentrations of 50, 100, and 300 mg / kg for 14 days. On the 15th day of the experiment, blood samples were collected, and the spleen and thymus indices were calculated as a percentage of body weight. In addition, T cell and NK cell analyses were performed, and serum samples were used for cytokine analysis.

[0114]

[0115] Isolation of immune cells from the spleen and peritoneal cavity

[0116] To isolate splenic immune cells from rats administered gintonin for 14 days, the animals were induced to undergo pre-anesthesia, and the left lower rib area was resected. The spleen was physically separated, filtered through a 200 μm filter (BD Biosciences, San Jose, CA, USA), and centrifuged at 3000 rpm for 10 min at 4 °C. After removing red blood cells, the obtained immune cells were washed with RPMI 1640 medium and centrifuged (3000 rpm, 10 min, 4 °C) to isolate splenic immune cells. The isolated spleen cells were cultured for each experiment in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotic-antimycotic in an incubator containing 5% CO2 at 37 °C. In addition, for the isolation of peritoneal macrophages, 1 ml of sterilized Difco fluid Thioglycollate medium was injected intraperitoneally into the rat, and 3 to 4 days later, 10 ml of saline was injected intraperitoneally to obtain peritoneal exudate, and then peritoneal immune cells were isolated. This process was repeated three times to isolate a large number of immune cells. Afterwards, red blood cells contained within the immune cells were removed using RBC lysis buffer, and only peritoneal immune cells were isolated and used.

[0117]

[0118] Splenic NK cell and T cell analysis

[0119] For NK cell and T cell measurement, spleen immune cell suspension was resuspended at 1x10 per mL in solution for FACS analysis. 6 Cells were prepared. After reacting with surface markers for 30 minutes, spleen immune cells were washed twice with 1X PBS, and spleen immune cells were analyzed using BD Accuri flow cytometry.

[0120]

[0121] T- / B-lymphocyte proliferation and NK cytotoxicity assays

[0122] To evaluate T- / B-lymphocyte proliferation, spleen immune cells (1X10 per mL) were first collected. 6 Cells) were dispensed into 96-well plates and cultured for 72 hours at 37°C in 5% CO2 conditions with 10 μg / mL concanavalin (ConA) as a T cell stimulator and 10 μg / mL lipopolysaccharides (LPS) as a B cell stimulator. These were used for T and B lymphocyte proliferation analysis, respectively. The cell group not treated with ConA or LPS was used as a negative control. After reacting with 10 μL WST-8 for 4 hours, the lymphocyte proliferation was analyzed by measuring the absorbance at 460 nm. The proliferation index of T- / B-lymphocytes was calculated as the difference in the final absorbance of the groups with or without ConA or LPS. For NK cell activity test, effector cells (1X10 per spleen immune cells / mL) were first cultured. 6 cells) 50 ml and target cells (1X10 per YAC-1 cell / ml) 4 NK cell activity test was performed by adding 50 μL of each of the effector:target cells to a 96-well plate to make the effector:target cell ratio 100:1, and then incubating at 37°C for 4 hours, adding 10 μL of WST-8, and then measuring the final absorbance at 460 nm (Epoch™ microplate spectrophotometer). NK cytotoxicity was calculated according to the formula below.

[0123] [formula]

[0124] NK cell activity (%) = (OD experimental - OD spontaneous) / (OD maximum - OD spontaneous) x 100

[0125]

[0126] Analysis of gene expression related to immune function activation

[0127] To evaluate the changes in immune activity gene expression in macrophages isolated from the spleen and peritoneum of rats administered with gintonin and the control group, genetic analysis was performed using the following method. That is, splenic immune cells (2X10) were isolated using TRIzol reagent and easy-spin™ total RNA extraction Kit. 6 After extracting RNA from peritoneal macrophages and peritoneal cells, cDNA was synthesized using a cDNA synthesis kit from 1 μg RNA. The synthesized cDNA was amplified by RT-PCR, and the final PCR reaction was performed on E-Gel® EX 2% Agarose, and the gel image was recorded using an E-Gel Power Snap electrophoresis device. The list of IL-2, IL-6, IFN-γ, iNOS, IL-1β, GMCSF, TNF-α, NFkB, IkBα, p65, Nos2, COX-2, JNK, p38, Ephb1, PERK, NLRP3, and Caspase1 primers used for analysis is shown in [Table 4] below.

[0128]

[0129] GeneSequenceAmpliconLenth(bp)IL-2F-AAGGAAACACAGCAGCCACCTR-CACAGTTGCTGGCTCATCAT350bpIL-6F-AGACTTCCAGCCAGTTGCCTTTR-ACGGAACTCCCAGAAGACCAGA343bpIFN-γF-AGGCCATCAGCAACAACAACATAAGTGR-GACAGCTTTGTGCTGGATCTGTG100bpiNOSF-TCCGGTTCACAGTCTGGGTGGR-ATTGCTGGGGCAGTCTCCAT216bpIL-1βF-AGAGCCCC GTCCTCTGTGACTR-TGGGGAACTGTGCAGACTCA407bpGMCSFF-ACTCTGCTCACGAAGGAACTCAGCR-CACAGCTCGGAAGAGCATCGCA300bpTNF-αF-AACTCGAGTGGACAAGCCCGTR-GCAAGGGCTCTTGATGGCAG282bpNFkBF-ACAACGGCCACACGTGTCTAR-TCCACGTTCCTTTGGCCACT430bpIkBαF-CAGGCTGCAGGAAGTACCTAR-GTTGGGTGGC. GTTCAGAC299bpp65F-CCAGCGGTTCGCAGTTTTCCR-AGCCAACTAGACCGCATCCC291bpNos2F-GTTCCTCAGGCTTTGGGTCTTR-ATATCCGAGGTGGCCTTGTGG402bpCOX-2 F-CTGGCTTCGGGAGCACAACAR-AACCCGGCCAGCAATCTGTC391bpJNKF-GAAGCTCAGCCGGCCATTTCR-TGCAGTCCTTGCCACTCCAA364bpp38F-AGATAATGCGTCTGACGG GGAR-GGCGGTGGCACAAAGCTAAT355bpEphb1F-CAGCATTACAGCACAGGCCGR-CGXTXAGAAATCCCGACGC265bpPERKF-CGTGAAGTCGAGAGGCGTCGR-CAGTGCGGCAATTCGTCCAT460bpNLRP3F-TGTGTGGTGACGCTCTGTF-CTAAAAGCGCCCTGGCTTGG267bpCaspase1F-AGGCATGCCGTGGGAGAAACR-CACCTTTCAGGGTTGGCA326bp

[0130]

[0131] Cytokine production analysis

[0132] To collect serum from animals administered gintonin, clotted blood was centrifuged at 2,500xg for 15 to 25 minutes, the supernatant was transferred to a tube and stored at -80°C, and cytokine quantification was performed via ELISA according to the protocol using a cytokine analysis kit.

[0133]

[0134] Analysis of M1 macrophage surface markers and intraperitoneal T cell activation

[0135] Macrophages, which play a role in innate immunity and are present in all tissues, perform essential functions in eliminating pathogens such as bacteria and viruses, as well as cancer cells. Based on their phenotypic markers, these macrophages are classified into immunosuppressive macrophages (M2 macrophages) and macrophages that eliminate pathogens such as bacteria and viruses (M1 macrophages).

[0136] M1 macrophage markers include CD11b / c, CD80, MHC-II, and CD45. + For analysis using monoclonal antibodies against RA, peritoneal immune cells were suspended in buffer with fluorochrome-conjugated antibodies, incubated at room temperature for 30 min, washed at least twice in buffer, and then analyzed for macrophage surface markers using BD Calibur (BD Bioscience).

[0137]

[0138] Analysis of gene expression of mediators that induce M1 macrophage immune activation

[0139] Macrophages produce nitric oxide (NO), COX to increase immune activity. -2, prostaglandin (PGE2), TNF-α, IL-1β, and IL-6, which are important mediators for maximizing the immune response. NLRP3 and NF-κB p65 (RelA) bind to the gene that produces IL-6, causing it to produce IL-6. NF-κB induces differentiation into M1 macrophages, and the activation of kb / MAPK signaling increases the expression of p38 and JNK, resulting in increased secretion of TNF-α and IL-6, which enhances immune function. In addition, caspase-1 activation induces differentiation from monocytes into macrophages. The Ephb1 receptor, which is related to Eph (erythropoietin-producing hepatocellular carcinoma) signaling, is involved in B cell and T cell activation and antibody production, and the PERK signal encoded by Eif2ak3 (endoplasmic reticulum stress kinase PERK) stimulates the activity of immune cells.

[0140]

[0141] Statistical analysis

[0142] The final data of the experiment for the present invention were statistically analyzed for the NTC group and the gintonin administration group and expressed as the mean + standard error. Statistical significance between groups was determined using the Bonferroni post-hoc test (nsp>0.05, * p<0.05; ** p<0.01; *** p<0.001) was evaluated using ANOVA analysis.

[0143] Experimental Example 1. Confirmation of animal survival rate and weight change and improvement in long-term indicators of gintonin.

[0144] After stabilizing the animals (rats), 50, 100, and 300 mg / kg of gintonin and 10 mg / kg of Levamisole, a positive control drug, were administered to each group for 14 days, and the immune activation efficacy of spleen immune cells and peritoneal macrophages was analyzed (see Figure 1A).

[0145] As a result, the groups administered 50, 100, and 300 mg / kg of gintonin showed no difference in survival rate compared to the normal control group (see Figure 1B), and it was confirmed that the body weight of the animals gradually increased with gintonin administration at different concentrations without significant difference from the control group (see Figure 1C). This means that oral intake of gintonin is safe without side effects in healthy SD rats.

[0146] In addition, the spleen and thymus indices significantly increased in the gintonin administration groups according to concentration (*p<0.001) (see Figure 1D). As a result, it was confirmed that gintonin administration functions as an immune enhancer in changing the activity of major organs of the immune system.

[0147]

[0148] Experimental Example 2. Confirmation of the efficacy of gintonin on the distribution of CD3+ T cells and CD4+ T cells.

[0149] After administering gintonin at various concentrations to experimental animals for 14 days, spleen immune cells were separated and the T cell distribution (populations) was analyzed using a flow cytometer (FACS). As a result, CD3 + T cells and CD4 + The proportion of T cells showed a significant difference compared to the normal control group that was not administered gintonin.

[0150] CD3 + T cells are CD8 + T cells and CD4 + As a T cell involved in activating T cells, the distribution was significantly increased in the 100 mg / kg and 300 mg / kg gintonin administration groups (*p<0.05). That is, compared to the normal control group, the CD3 + It was confirmed that the T cell distribution significantly increased (*P<0.05). (See Figure 2)

[0151] CD4 + T cells (helper T cells) are immune cells that promote humoral immunity by regulating the differentiation and activation of immune cells. In the 100 mg / kg and 300 mg / kg gintonin administration groups, CD4 + The distribution of T cells was significantly increased (*p<0.05). That is, compared to the normal control group, the distribution of CD3+ T cells was significantly increased in the 100 mg / kg gintonin and 300 mg / kg gintonin groups (*p<0.05, **p<0.01). (See Figure 2)

[0152]

[0153] Experimental Example 3. Confirmation of the effect of gintonin on the distribution of CD8+ T cells in the spleen.

[0154] CD8 after 14 days of gintonin administration to rats + T cells and CD4 + CD8 + Analysis of the distribution of T cells revealed that CD8 + It was confirmed that the T cell distribution ratio significantly increased in the group administered 300 mg / kg gintonin compared to the normal control group (*p<0.05) (see Figure 3).

[0155]

[0156] Experimental Example 4. Confirmation of the efficacy of gintonin on the differentiation of CD3-CD161+ NK cells in the spleen.

[0157] CD3 after 14 days of gintonin administration to rats - CD161 + Analysis of the distribution of NK cells revealed that CD3 - CD161 + It was confirmed that the NK cell distribution ratio significantly increased in all groups administered 50, 100, and 300 mg / kg gintonin (*p<0.01). That is, compared to the normal control group, the CD3 -CD161 + It was confirmed that the distribution of NK cells significantly increased (see Figure 4).

[0158]

[0159] Experimental Example 5. Confirmation of the effect of gintonin on the proliferation rate of T cells and B cells.

[0160] To determine whether administration of gintonin affects the cell proliferation effect of T cells and B cells, immune cells were isolated from the spleen of mice administered gintonin at various concentrations for 14 days, and the proliferation ability of T cells and B cells was measured using the T cell mitogen ConA (concanavalin A) and the B cell mitogen LPS, respectively.

[0161] As shown in Fig. 5, it was confirmed that the proliferation of T cells and B cells significantly increased in the groups administered 100 and 300 mg / kg gintonin (*p<0.01). That is, compared to the normal control group, T cell proliferation was significantly increased by 100.4% and 105.1% in the 100 mg / kg and 300 mg / kg gintonin administration groups, respectively. In addition, in the proliferation of B cells by gintonin treatment, compared to the normal control group, B cell proliferation was significantly increased by 50.1% and 67.5% in the 100 mg / kg and 300 mg / kg gintonin administration groups, respectively. However, it was confirmed that the proliferation of T cells and B cells in the 50 mg / kg gintonin administration group did not show a significant difference compared to the control group (ns>0.05).

[0162]

[0163] Experimental Example 6. Confirmation of the efficacy of gintonin on the cytotoxicity of splenic NK cells against Yac-1 tumor cells.

[0164] Splenic NK cells were used as effector cells (E) and Yac-1 cells were used as target cells (T) (E:T ratio 100:1) to confirm the cytotoxicity. As shown in Figure 6, compared to the normal control group, the activity (phagocytosis) of NK cells increased by 17.7% (*P<0.05), 17.8% (*P<0.05), and 20.9% (**P<0.01) in the 50, 100 mg / kg, and 300 mg / kg gintonin administration groups, respectively.

[0165]

[0166] Experimental Example 7. Confirmation of the effect of gintonin on the expression of genes secreting IL-2, IL-6, and IFN-γ.

[0167] We analyzed the expression levels of cytokine genes secreted by T cells, B cells, and macrophages in the group administered gintonin extracted from red ginseng extract. As a result, IL-2 mRNA expression increased by 27% and 107% in the 100 mg / kg and 300 mg / kg gintonin administration groups, respectively, compared to the normal control group (*p<0.01, see Fig. 7A). In addition, IL-6 mRNA expression increased by 31%, 59%, and 62% in the 50, 100 mg / kg, and 300 mg / kg gintonin administration groups, respectively (*p<0.01, see Fig. 7B). In addition, IFN-γ mRNA expression increased by 10%, 17%, and 59% in the 50, 100 mg / kg, and 300 mg / kg gintonin administration groups, respectively, compared to the normal control group (*p<0.01) (see Fig. 7C).

[0168] These results confirmed that administration of gintonin was effective in producing IL-2, IL-6, and IFN-γ, which are related to immunity, especially the activity of NK cells and T cells.

[0169]

[0170] Experimental Example 8. Confirmation of the effect of gintonin on the regulation of iNOS, IL-1β, and GMCSF gene expression.

[0171] When gintonin was administered to rats at various concentrations for 14 days, the expression of iNOS-producing mRNA increased in a concentration-dependent manner. That is, the iNOS mRNA expression level significantly increased from a concentration of 50 mg / kg gintonin compared to the control group. It was significantly increased in all 50, 100, and 300 mg / kg gintonin-administered groups (*p<0.01, see Figure 8A). In particular, the 300 mg / kg gintonin-administered group showed a significant increase compared to the normal control group. These results imply that gintonin can exhibit immune activity by promoting NO production through iNOS expression.

[0172] Additionally, mRNA analysis was performed to determine whether it affects the expression of IL-1β. As a result, IL-1β mRNA expression significantly increased starting from a concentration of 50 mg / kg of gintonin compared to the control group (*p<0.001, see Figure 8B).

[0173] To determine whether gintonin affects GM-CSF production, mRNA analysis was performed. Results showed that GM-CSF mRNA expression significantly increased at a gintonin concentration of 50 mg / kg compared to the control group (*p<0.001, see Figure 8C). These results confirmed that gintonin can stimulate immune cells and act as a defense mechanism against infections, including invasion by foreign substances.

[0174]

[0175] Experimental Example 9. Confirmation of the effect of gintonin on changes in the concentrations of IL-2, IL-6, and IFN-γ.

[0176] As a result of analyzing the concentration of cytokines in the blood according to the administration of gintonin, the concentration of IL-2 secreted by Th1 cells in the blood cytokine was increased by 55.1% and 160% in the 100 mg / kg and 300 mg / kg gintonin administration groups, respectively, compared to the normal control group (*p<0.05, **p<0.01, Fig. 9A). IFN-γ was increased by 51.6% and 125.7% in the 100 mg / kg and 300 mg / kg gintonin administration groups, respectively, compared to the normal control group (*p<0.05, **p<0.01, Fig. 9B).

[0177] In addition, the secretion of IL-6 secreted from Th2 cells was increased by 46.9% and 91.1% in the 100 mg / kg and 300 mg / kg gintonin administration groups, respectively (*p<0.05, **p<0.01, Fig. 9C). Based on the above results, it was confirmed that the concentrations of IL-2, IL-6, and IFN-γ related to T cell activity were increased by gintonin administration.

[0178]

[0179] Experimental Example 10. Effect of gintonin on intraperitoneal macrophage viability.

[0180] To analyze macrophage viability, RAW 264.7 cells, a macrophage cell line, were treated with 25 μg / ml to 500 μg / ml of gintonin for 24 hours, and peritoneal macrophages were isolated (see Figure 10A). The peritoneal macrophage viability was evaluated, and no difference in cell viability was observed compared to the normal group (see Figure 10B). The above results indicate that gintonin has no cytotoxic effect at the corresponding concentrations.

[0181]

[0182] Experimental Example 11. Confirmation of the efficacy of gintonin on differentiation of CD80-expressing M1-type macrophages.

[0183] The concentration of gintonin for macrophage activity analysis was determined as 50, 100, and 300 mg / kg by converting the safe concentration value in the cell experiment, and then administering 50, 100, and 300 mg / kg to rats for 14 days to analyze the activity of macrophages. M1 macrophages that exhibit immune activity phenotypically express CD80 on the cell membrane. Therefore, when the polarization (distribution) of M1 macrophages was analyzed through CD80 analysis, it increased by 262.5%, 238.1%, and 333.3% in the 50 mg / kg, 100 mg / kg, and 300 mg / kg gintonin administration groups, respectively (*p<0.01, see Figure 11).

[0184]

[0185] Experimental Example 12. Confirmation of the effect of gintonin on the expression of genes related to immune activity in macrophages.

[0186] The expression levels of TNF-α, IL-6, and IL-1β genes in peritoneal macrophages were significantly increased in the gintonin administration groups at different concentrations compared to the control group (*p<0.05, **p<0.01, Fig. 12A), and the expression levels of NF-κB, IkBα, and p65 in peritoneal macrophages were significantly increased in the gintonin administration groups at different concentrations compared to the normal control group (*p<0.05, **p<0.01, Fig. 12B), and the expression levels of Nos2 and Cox-2 in peritoneal macrophages were significantly increased in the gintonin administration groups at different concentrations compared to the normal control group (**p<0.01, Fig. 12C).

[0187] The expression levels of JNK and p38 in peritoneal macrophages were significantly increased in the gintonin administration groups at different concentrations compared to the normal control group (*p<0.05, **p<0.01, Fig. 12D). When gintonin was administered to rats, the expression levels of Ephb1 and PERK in peritoneal macrophages were significantly increased compared to the normal control group (*p<0.05, **p<0.01, Fig. 12E). It was confirmed that the expression levels of NLRP3 and caspase-1 in peritoneal macrophages were significantly increased compared to the normal control group when gintonin was administered to rats at different concentrations (*p<0.05, **p<0.01, Fig. 12F).

[0188]

[0189] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0190] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

[0191]

[0192] The present invention relates to an immune-enhancing composition comprising gintonin, a glycolipoprotein complex derived from red ginseng marc containing lysophosphatidic acid as its main component, and more specifically, to an immune-enhancing effect by a red ginseng marc-derived glycolipoprotein complex composition capable of enhancing immune activity by increasing the secretion of cytokines by inducing the activity of macrophages, NK cells, and T cells, as gintonin is isolated from red ginseng marc. According to the present invention, an immune-enhancing product capable of strengthening immunity can be developed by isolating gintonin from red ginseng marc, and through this, a low-cost, high-functionality health functional product can be manufactured, which can be effectively used for the prevention or treatment of diseases.

[0193] Therefore, it can be seen that the composition for enhancing immunity including gintonin, a glycolipoprotein complex derived from red ginseng extract containing lysophosphatidic acid as a main component according to one embodiment of the present invention, has industrial applicability.

Claims

1. Pharmaceutical composition for enhancing immunity containing gintonin derived from red ginseng extract as an active ingredient 2. In paragraph 1, The above gintonin is a pharmaceutical composition for enhancing immunity, characterized in that it contains lysophosphatidic acid as an active ingredient.

3. In paragraph 1, The above composition is a pharmaceutical composition for enhancing immunity, which enhances immune activity by inducing the activity of macrophages, NK cells, and T cells.

4. In paragraph 1, A pharmaceutical composition for enhancing immunity, wherein the composition induces an increase in the expression of at least one cytokine selected from the group consisting of IL-2, IL-6, IL-10, IFN-γ, iNOS, GMCSF, and IL-1β. 5.1) Step of obtaining an extract by adding ethanol to red ginseng powder and performing reflux cooling extraction; 2) A step of filtering the extract obtained in step 1); 3) A step of obtaining a filtrate by repeating steps 1) and 2) above 1 to 5 times; and 4) A step of vacuum concentrating the filtrate obtained in step 3 above, and then performing dialysis and freeze-drying to obtain an ethanol extract containing crude gintonin; Method for producing a pharmaceutical composition for enhancing immunity, comprising 6. In paragraph 5, A method for producing an immune-enhancing pharmaceutical composition, characterized in that the above step 1) is performed at 50°C to 100°C for 5 to 10 hours.

7. A health functional food for enhancing immunity, containing gintonin derived from red ginseng extract as an active ingredient.

8. In paragraph 7, The above gintonin is a health functional food for enhancing immunity, characterized in that it contains lysophosphatidic acid as an active ingredient.

9. In paragraph 7, The above composition is a health functional food for enhancing immunity, which enhances immune activity by inducing the activity of macrophages, NK cells, and T cells.

10. In paragraph 7, The composition is a health functional food for enhancing immunity, which induces an increase in the expression of one or more cytokines selected from the group consisting of IL-2, IL-6, IL-10, IFN-γ, iNOS, GMCSF, and IL-1β.

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