Composition for enhancing innate immune function comprising lipid extract of arctoscopus japonicus eggs as active ingredient

Abstract

The present invention relates to a composition for enhancing an innate immune function, comprising, as an active ingredient, a lipid extract of Arctoscopus japonicus eggs. By analyzing the fatty acid composition of the lipid extract of Arctoscopus japonicus eggs and the effects thereof on spleen index, spleen lymphocyte proliferation, NK cell activity promotion, and an increase in NO production, it was confirmed that the lipid extract of Arctoscopus japonicus eggs, of the present invention, can be used for enhancing immunity.

Description

Composition for enhancing innate immune function containing extract of sea squirt egg lipid as an active ingredient

[0001] The present invention relates to a composition for enhancing innate immune function comprising a lipid extract of sea squirt eggs as an effective ingredient.

[0002] The immune system consists of cells and chemicals that play a specific role in defending against infection. The immune system is divided into two categories: innate immunity and adaptive immunity. The immune system protects the body from microorganisms and foreign antigens. Many diseases, such as autoimmune diseases, inflammatory diseases, and even cancer, are caused by problems with the immune system. Cyclophosphamide (CY) is a chemotherapy drug commonly used to treat malignant tumors and autoimmune diseases. However, cyclophosphamide has several serious side effects, including nausea, fatigue, and immunosuppression. CY-induced immunosuppressed mice have been used in numerous studies to evaluate the immunomodulatory effects of functional substances. These studies assessed immune function by measuring immune organ indices, such as the spleen and thymus, and the activation of immune-related cells, such as macrophages, lymphocytes, and natural killer (NK) cells. Macrophages are phagocytes derived from monocytes and play a crucial role in both adaptive and innate immunity. In inflammation, the functional response of macrophages is involved in protecting the host through phagocytosis, antigen presentation, and immune regulation through the release of various cytokines and growth factors.

[0003] Lipids and fatty acids are key structural components of cell membranes. They are essential for various metabolic processes and some cellular functions. Fish eggs are rich in polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, C20:5n3) and docosapentaenoic acid (DPA, C22:5n3), which provide the energy fish need to survive and reproduce.

[0004] Fatty acids are used as building blocks for cell membrane biosynthesis, serve as intracellular signaling molecules, and act as precursors for the production of other cellular components. Fatty acids may indirectly be effective in many inflammatory conditions and diseases, and have been shown to reduce inflammatory responses in conditions such as Alzheimer's disease, cancer, cardiovascular disease, and anti-inflammatory conditions. In immune cells, fatty acids are responsible for energy production, gene expression regulation, and the production of bioactive lipid mediators. Recent studies have shown that DHA can improve immunomodulatory activity and molecular mechanisms in RAW264.7 cells by stimulating GPR120 and MAPKs via the NF-κB pathway. Fish oil containing EPA and DHA has a positive effect on the immune system in breast cancer patients. Fish oil supplementation in early infancy influences the development of the infant immune response. An appropriate dietary DHA / EPA ratio helps improve the growth performance of marine fish and enhances their immune response. Additionally, monounsaturated fatty acids (MUFAs), particularly oleic acid (C18:1n9), can modulate inflammation and promote wound repair. Saturated fatty acids (SFAs), such as palmitic acid (C16:0) and stearic acid (C18:0), also play a role in immune responses.

[0005] The sandfish (Arctoscopus japonicus) belongs to the Trichodontidae family and has a wide distribution, including the northwestern Pacific, the east coast of Korea, north-central Japan, Sakhalin, and Alaska. It is a popular commercial fish in South Korea due to its texture, flavor, and health benefits. It has been reported that sandfish and its roe contain functional peptides with antioxidant and anti-inflammatory properties. Peptides derived from enzymatic hydrolysates have been shown to have anti-inflammatory effects in RAW264.7 cells. The major fatty acids found in sandfish roe are palmitic acid, oleic acid, docosahexaenoic acid (DHA), and EPA. Previous studies have shown that lipids isolated from sandfish roe, particularly those rich in EPA and DHA, possess immunobiological activity in mouse RAW264.7 macrophages. However, lipids from sandfish roe have not been shown to exhibit in vivo immune-enhancing activity in immunosuppressed mouse models.

[0006] Accordingly, the inventor of the present invention completed the present invention by confirming that the acorn jellyfish egg lipid extract can be used for the purpose of enhancing immunity through analysis of the fatty acid composition of the acorn jellyfish egg lipid extract, its effect on spleen index, its effect on spleen lymphocyte proliferation, its effect on promoting NK cell activity, and its effect on increasing NO production.

[0007] The purpose of the present invention is to provide an immune-enhancing food composition containing a lipid extract of acorn jelly as an effective ingredient.

[0008] Another object of the present invention is to provide a health functional food composition for enhancing immunity, which contains a lipid extract of sea squirt as an active ingredient.

[0009] Another object of the present invention is to provide a pharmaceutical composition for enhancing immunity, which comprises a lipid extract of the egg of the sea buckthorn as an active ingredient.

[0010] Another object of the present invention is to provide a cosmetic composition for enhancing immunity, which contains a lipid extract of acorn jelly as an active ingredient.

[0011] Another object of the present invention is to provide a method for enhancing immunity comprising a step of administering the pharmaceutical composition to a subject.

[0012] In order to achieve the above object of the present invention, the present invention provides an immune-enhancing food composition comprising a lipid extract of acorn jellyfish eggs as an effective ingredient.

[0013] In addition, the present invention provides a health functional food composition for enhancing immunity, which comprises a lipid extract of sea squirt as an effective ingredient.

[0014] In addition, the present invention provides a pharmaceutical composition for enhancing immunity, which comprises a lipid extract of the egg of the sea buckthorn as an active ingredient.

[0015] In addition, the present invention provides a cosmetic composition for enhancing immunity, which comprises a lipid extract of the egg of the sea squirt as an effective ingredient.

[0016] The present invention also provides a method for enhancing immunity comprising administering the pharmaceutical composition to a subject.

[0017] The present invention relates to a composition for enhancing innate immune function, which comprises a sea squirt egg lipid extract as an active ingredient. Through analysis of the fatty acid composition of the sea squirt egg lipid extract, the effect on spleen index, the effect on spleen lymphocyte proliferation, the effect on NK cell activity promotion, and the effect on increasing NO production, it was confirmed that the sea squirt egg lipid extract can be used for the purpose of enhancing immunity, and thus it can be usefully used in related businesses.

[0018] Figure 1 is a diagram showing the fatty acid composition of the lipid extract of the sea squirt egg.

[0019] Figure 2 is a diagram showing changes in spleen size according to treatment with various concentrations of acorn jelly lipid extract in spleen cells.

[0020] Figure 3 is a diagram showing changes in spleen indices according to treatment with various concentrations of acorn extract lipid in spleen cells.

[0021] Figure 4 is a diagram showing the proliferation rate of spleen lymphocytes according to treatment with various concentrations of acorn jelly egg lipid extract in spleen cells.

[0022] Figure 5 is a diagram showing the activity of splenic NK cells according to treatment with various concentrations of acorn jellyfish egg lipid extract in spleen cells.

[0023] Figure 6 is a diagram showing the effect of the lipid extract of the sea squirt on IL-1β mRNA expression in spleen lymphocytes.

[0024] Figure 7 is a diagram showing the effect of the lipid extract of the sea squirt on IL-2 mRNA expression in spleen lymphocytes.

[0025] Figure 8 is a diagram showing the effect of the lipid extract of the sea squirt on IL-4 mRNA expression in spleen lymphocytes.

[0026] Figure 9 is a diagram showing the effect of the lipid extract of the sea squirt on IL-6 mRNA expression in spleen lymphocytes.

[0027] Figure 10 is a diagram showing the effect of the lipid extract of the sea squirt on TNF-α mRNA expression in spleen lymphocytes.

[0028] Figure 11 is a diagram showing the effect of the lipid extract of the sea squirt on IFN-γ mRNA expression in spleen lymphocytes.

[0029] Figure 12 is a diagram showing the effect of the lipid extract of the sea squirt on TLR4 mRNA expression in spleen lymphocytes.

[0030] Figure 13 is a diagram showing the results of analysis of the T lymphocyte subtype ratio of splenic lymphocytes in the normal group.

[0031] Figure 14 is a diagram showing the results of analysis of the T lymphocyte subtype ratio of splenic lymphocytes in the CY treatment group.

[0032] Figure 15 is a diagram showing the results of analysis of the T lymphocyte subtype ratio of spleen lymphocytes in the ginseng treatment group.

[0033] Figure 16 is a diagram showing the results of analysis of the T lymphocyte subtype ratio of splenic lymphocytes in the levamisole treatment group.

[0034] Figure 17 is a diagram showing the results of analysis of the T lymphocyte subtype ratio of splenic lymphocytes in the PEG 6000 treatment group.

[0035] Figure 18 is a diagram showing the results of analysis of the T lymphocyte subtype ratio of spleen lymphocytes in the AJ-PEG 25 mg / kg BW treatment group.

[0036] Figure 19 is a diagram showing the results of analysis of the T lymphocyte subtype ratio of spleen lymphocytes in the AJ-PEG 50 mg / kg BW treatment group.

[0037] Figure 20 is a diagram showing the results of analysis of the T lymphocyte subtype ratio of spleen lymphocytes in the AJ-PEG 75 mg / kg BW treatment group.

[0038] Figure 21 is a diagram showing the results of analysis of the T lymphocyte subtype ratio of spleen lymphocytes in the AJ-PEG 100 mg / kg BW treatment group.

[0039] Figure 22 is a diagram showing the change in the ratio of CD 4+ / CD 8+ T cells according to treatment with the lipid extract of the sea squirt egg.

[0040] Figure 23 is a diagram showing changes in the macrophage proliferation rate in peritoneal macrophages according to treatment with the lipid extract of the sea squirt egg.

[0041] Figure 24 is a diagram showing changes in NO production in peritoneal macrophages according to treatment with a lipid extract of sea squirt eggs.

[0042] Figure 25 is a diagram showing changes in phagocytosis in peritoneal macrophages according to treatment with a lipid extract of sea squirt eggs.

[0043] Figure 26 is a diagram showing the effect of the lipid extract of the sea squirt on iNOS mRNA expression in peritoneal macrophages stimulated by LPS.

[0044] Figure 27 is a diagram showing the effect of the lipid extract of the sea squirt on COX-2 mRNA expression in peritoneal macrophages stimulated by LPS.

[0045] Figure 28 is a diagram showing the effect of the lipid extract of the sea squirt egg on IL-1β mRNA expression in peritoneal macrophages stimulated by LPS.

[0046] Figure 29 is a diagram showing the effect of the lipid extract of the sea squirt egg on IL-6 mRNA expression in peritoneal macrophages stimulated by LPS.

[0047] Figure 30 is a diagram showing the effect of the lipid extract of the sea squirt egg on TNF-α mRNA expression in peritoneal macrophages stimulated by LPS.

[0048] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments of the present invention. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0049] In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and this may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0050] The present invention provides an immune-enhancing food composition comprising a lipid extract of sea squirt eggs as an effective ingredient.

[0051] The extract according to the present invention can be obtained by extracting and separating from nature using extraction and separation methods known in the art, and the "extract" defined in the present invention is extracted from the eggs of the sea squirt using an appropriate solvent, and includes, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract. Any pharmaceutically acceptable organic solvent may be used as a suitable solvent for extracting the extract from the eggs of the acorn, and water or an organic solvent may be used, and is not limited thereto, for example, purified water, alcohols having 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane, and various solvents may be used alone or in combination. As an extraction method, any one of hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, dissolution, and pressing may be selected and used. Additionally, the desired extract may be subjected to additional conventional fractionation processes and purified using conventional purification methods.

[0052] There is no limitation on the method for preparing the extract of the present invention, and any known method can be used. For example, the extract included in the composition of the present invention can be prepared in a powder form by additional processes such as reduced pressure distillation and freeze drying or spray drying of the primary extract extracted by the above-mentioned hot water extraction or solvent extraction method. In addition, the primary extract can be further purified to obtain a fraction using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. Therefore, in the present invention, the extract is a concept that includes all extracts, fractions, and purified products obtained at each stage of extraction, fractionation, or purification, as well as their dilutions, concentrates, or dried products.

[0053] The food composition of the present invention may contain, in addition to containing the effective ingredient of the present invention, various flavoring agents or natural carbohydrates as additional ingredients, like conventional food compositions.

[0054] Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-mentioned flavoring agent can advantageously use natural flavoring agent (thaumatin), stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0055] In addition, the food composition may contain, in addition to the active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks.

[0056] In one embodiment of the present invention, the acorn seed lipid extract may be characterized by being extracted using a solvent selected from the group consisting of distilled water, C1 to C4 lower alcohol, lower alcohol aqueous solution, hexane, chloroform or acetic acid, but is not limited thereto.

[0057] In one embodiment of the present invention, the acorn seed lipid extract may include, but is not limited to, saturated fatty acids, monounsaturated fatty acids, or polyunsaturated fatty acids.

[0058] In one embodiment of the present invention, the saturated fatty acid may be, but is not limited to, palmitic acid (C16:0) or stearic acid (C18:0).

[0059] In one embodiment of the present invention, the monounsaturated fatty acid may be at least one selected from the group consisting of palmitoleic acid (C16:1n7), oleic acid (C18:1n9), vaccenic acid (C18:1n7), and eicosenoic acid (C20:1), but is not limited thereto.

[0060] In one embodiment of the present invention, the polyunsaturated fatty acid may be at least one selected from the group consisting of linoleic acid (C18:2n6), linolenic acid (C18:3n3), eicosatrienonic acid (C20:3n3), eicosapentaenoic acid (EPA, eicosapentaenoic acid C20:5n3), docosapentaenoic acid (DPA, docosapentaenoic acid C22:5n3), and docosahexaenoic acid (DHA, docosahexaenoic acid C22:6n3), but is not limited thereto.

[0061] In one embodiment of the present invention, the extract of the lipid of the sea squirt may have an activity of increasing spleen size and spleen index, but is not limited thereto.

[0062] In one embodiment of the present invention, the extract of the lipid of the sea buckthorn may have an activity of promoting the proliferation of lymphocytes in the spleen, but is not limited thereto.

[0063] In one embodiment of the present invention, the acorn seed lipid extract may enhance NK cell activity in the spleen, but is not limited thereto.

[0064] In one embodiment of the present invention, the acorn seed lipid extract may have an activity that promotes the expression of immune-related factors in spleen lymphocytes, but is not limited thereto.

[0065] In one embodiment of the present invention, the immune-related factor may be at least one selected from the group consisting of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interferon γ (IFN-γ), and Toll-like receptor 4 (TLR4), but is not limited thereto.

[0066] In one embodiment of the present invention, the acorn seed lipid extract may exhibit immune-enhancing activity by activating Th1 and Th2 cytokines, but is not limited thereto.

[0067] In one embodiment of the present invention, the acorn seed lipid extract may increase the proportion of CD4+ T lymphocytes in the spleen, but is not limited thereto.

[0068] In one embodiment of the present invention, the acorn seed lipid extract may promote the proliferation of macrophages, but is not limited thereto.

[0069] In one embodiment of the present invention, the acorn seed lipid extract may promote phagocytosis of macrophages, but is not limited thereto.

[0070] In one embodiment of the present invention, the acorn seed lipid extract may have an activity that promotes the expression of immune-related factors in macrophages, but is not limited thereto.

[0071] In one embodiment of the present invention, the immune-related factor may be at least one selected from the group consisting of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), but is not limited thereto.

[0072]

[0073] In addition, the present invention provides a health functional food composition for enhancing immunity, which comprises a lipid extract of sea squirt as an effective ingredient.

[0074] The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. In the present invention, the term "health functional food composition" refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means to be consumed 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 effects. The health functional food of the present invention may include conventional food additives, and whether it is suitable as a food additive is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the above "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sulphate pigment, and guar gum; Examples thereof include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline agents, preservative preparations, and tar color preparations. For example, health functional foods in tablet form can be prepared by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives, granulating the mixture using a conventional method, and then adding a lubricant, etc. to compress and molding the mixture, or directly compress and molding the mixture. In addition, the health functional foods in tablet form can contain a maturing agent, etc., if necessary. Among health functional foods in capsule form, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The soft capsules can contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., if necessary.The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can be coated with white sugar or other coating agents, or the surface can be coated with a substance such as starch or talc. The granular health functional food can be manufactured into a granular form using a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can contain a flavoring agent, a flavoring agent, etc.

[0075]

[0076] In addition, the present invention provides a pharmaceutical composition for enhancing immunity, which comprises a lipid extract of the egg of the sea buckthorn as an active ingredient.

[0077] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete or incomplete adjuvant may be further included to increase immunogenicity.

[0078] The pharmaceutical composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0079] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0080] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0081] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0082] The above pharmaceutical composition can be formulated into various oral or parenteral dosage forms.

[0083] Oral dosage forms include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrups, and granules. These dosage forms may further contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). In addition, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidine, and, if desired, disintegrants or effervescent mixtures such as starch, agar, alginic acid or its sodium salt, and / or absorbents, coloring agents, flavoring agents, and sweetening agents. The above formulation can be prepared by conventional mixing, granulating or coating methods.

[0084] In addition, representative parenteral administration formulations include injectable preparations, and solvents for injectable preparations include water, Ringer's solution, isotonic saline solution, or suspensions. Sterile fixed oils for the injectable preparations can be used as solvents or suspension media, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose.

[0085] Additionally, the above injectable formulation may use a fatty acid such as oleic acid.

[0086]

[0087] In addition, the present invention provides a cosmetic composition for enhancing immunity, which comprises a lipid extract of the egg of the sea squirt as an effective ingredient.

[0088] The composition comprising the lipid extract of the sea buckthorn of the present invention can be used in various ways, such as for the prevention or improvement of skin diseases. Products to which the composition can be added include, for example, cosmetics such as various creams, lotions, skin toners, essences, shampoos, rinses, cleansers, facial cleansers, soaps, treatments, packs, and beauty solutions.

[0089] The cosmetic of the present invention comprises a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts.

[0090] Any water-soluble vitamin that can be mixed into cosmetics may be used, but preferably, vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinamide, folic acid, vitamin C, vitamin H, etc., and their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbate-2-phosphate, magnesium ascorbate-2-phosphate, etc.) are also included in the water-soluble vitamins that can be used in the present invention. Water-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microbial culture, enzymatic method, or chemical synthesis method.

[0091] Any vitamin that can be incorporated into cosmetics may be used as the useful vitamin, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol), and their derivatives (ascorbic acid palmitate, ascorbic acid stearate, ascorbic acid dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherol nicotinate, vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl ether, etc.) are also included in the useful vitamins used in the present invention. The useful vitamins can be obtained by conventional methods such as microbial transformation, purification from a microbial culture, enzymatic or chemical synthesis, etc.

[0092] Any polymer peptide that can be incorporated into cosmetics may be used, but preferred examples include collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. Polymer peptides can be purified and obtained by conventional methods such as purification from microbial cultures, enzymatic methods, or chemical synthesis methods, or can be purified and used from natural products such as the dermis of pigs or cows, or silkworm fibers.

[0093] Any polymer polysaccharide that can be incorporated into cosmetics may be used, but preferred examples include hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate, or a salt thereof (e.g., sodium salt). For example, chondroitin sulfate or a salt thereof can usually be purified from mammals or fish and used.

[0094] Any sphingolipid that can be incorporated into cosmetics may be used, but preferred examples include ceramide, phytosphingosine, and sphingoglycolipids. Sphingolipids can be purified by conventional methods from mammals, fish, shellfish, yeast, or plants, or obtained by chemical synthesis.

[0095] Any seaweed extract that can be mixed into cosmetics may be used, but preferably, brown seaweed extract, red seaweed extract, green seaweed extract, etc. are used. In addition, calagenan, arginic acid, sodium arginate, potassium arginate, etc. purified from these seaweed extracts are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained by purifying seaweed using a conventional method.

[0096] In addition to the above essential ingredients, the cosmetic of the present invention may contain other ingredients commonly contained in cosmetics, if necessary. In addition, examples of ingredients that may be added include fat components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, and the like. Examples of fat components include ester-based fats, hydrocarbon-based fats, silicone-based fats, fluorine-based fats, animal fats, and plant fats.

[0097] As ester-based fats and oils, tri-2-ethylhexanoate glyceryl, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, butyl stearate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, diisopropyl adipate, isoalkyl neopentanoate, tri(caprylic, capric) glyceryl, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, tetra-2-ethylhexanoate pentaellistolate, cetyl caprylate, decyl laurate, Hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinoleate, isostearyl laurate, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, cetostearyl 2-ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprate, di(caprylic, capric) propylene glycol, propylene glycol dicaprylate, neopentyl glycol dicaprate, neopentyl glycol dioctanoate, glyceryl tricaprylate, Glyceryl triundecyl acid, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerol oleate ester, polyglycerol isostearate ester, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malate, hydroxystearic acid 2-Ethylhexyl, di-2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate,Examples include esters such as dioctyl sebacate, cholesterol stearate, cholesteryl isostearate, cholesterol hydroxystearate, cholesterol oleate, dihydrocholesteryl oleate, phytosteryl isostearate, phytosteryl oleate, isocetyl 12-stearoylhydroxystearate, stearyl 12-stearoylhydroxystearate, and isostearyl 12-stearoylhydroxystearate.

[0098] Examples of hydrocarbon-based fats include squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybutene, microcrystalline wax, and vaseline.

[0099] Examples of silicone-based oils include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane and methylcetyloxysiloxane copolymers, dimethylsiloxane and methyl stearoxysiloxane copolymers, alkyl-modified silicone oils, and amino-modified silicone oils.

[0100] Examples of fluorine-based oils include perfluoropolyether.

[0101] Examples of animal or plant fats include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, sunflower oil, soybean oil, corn oil, rapeseed oil, almond oil, palm kernel oil, palm oil, castor oil, sunflower oil, grape seed oil, cottonseed oil, coconut oil, kukui nut oil, wheat germ oil, rice germ oil, shea butter, colostrum oil, marc deimia nut oil, meadowsweet oil, egg yolk oil, beef tallow, horse oil, mink oil, orange rapeseed oil, jojoba oil, candelilla wax, carnauba wax, liquid lanolin, and hydrogenated castor oil.

[0102] Examples of moisturizers include water-soluble low-molecular-weight moisturizers, fat-soluble molecular-weight moisturizers, water-soluble polymers, and fat-soluble polymers.

[0103] Examples of water-soluble low-molecular-weight moisturizers include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone-carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n = 2 or higher), polypropylene glycol (polymerization degree n = 2 or higher), polyglycerin B (polymerization degree n = 2 or higher), lactic acid, and lactate salts.

[0104] Examples of fat-soluble low-molecular-weight moisturizers include cholesterol and cholesterol esters.

[0105] Examples of water-soluble polymers include carboxyvinyl polymer, polyaspartate, tragacanth, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, and dextrin.

[0106] Examples of lipid-soluble polymers include polyvinylpyrrolidone and eicosene copolymers, polyvinylpyrrolidone and hexadecene copolymers, nitrocellulose, dextrin fatty acid esters, and high molecular silicones. Examples of emollients include long-chain acylglutamic acid cholesteryl ester, cholesteryl hydroxystearate, 12-hydroxystearic acid, stearic acid, rosin acid, and lanolin fatty acid cholesteryl ester.

[0107] Examples of surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

[0108] Nonionic surfactants include self-emulsifying monostearate glycerin, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbitan fatty acid ester, POE glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hydrogenated castor oil, POE castor oil, POE and POP (polyoxyethylene and polyoxypropylene) copolymer, POE and POP alkyl ether, polyether-modified silicone, lauric acid alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, etc.

[0109] Examples of anionic surfactants include fatty acid soaps, alpha-acyl sulfonates, alkyl sulfonates, alkyl allyl sulfonates, alkyl naphthalene sulfonates, alkyl sulfates, POE alkyl ether sulfates, alkyl amide sulfates, alkyl phosphates, POE alkyl phosphorus acids, alkyl amide phosphates, alkyloyl alkyl taurine salts, N-acyl amino acids, POE alkyl ether carboxylates, alkyl sulfosuccinates, sodium alkyl sulfoacetate, acylated hydrolyzed collagen peptide salts, and perfluoroalkyl phosphate esters.

[0110] Examples of cationic surfactants include alkyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium bromide, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzylammonium chloride, behenyl trimethyl ammonium bromide, benzalkonium chloride, diethylaminoethyl amide stearate, dimethylaminopropyl amide stearate, and quaternary ammonium salts of lanolin derivatives. Examples of amphoteric surfactants include carboxy betaine type, amide betaine type, sulfo betaine type, hydroxysulfo betaine type, amide sulfo betaine type, phospho betaine type, aminocarboxylate type, imidazoline derivative type, and amide amine type.

[0111] Organic and inorganic pigments include inorganic pigments such as silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium-coated mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine, chromium oxide, chromium hydroxide, calamine, and complexes thereof; Examples thereof include polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluororesin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene and styrene copolymers, silk powder, cellulose, CI pigment yellow, CI pigment orange, and composite pigments of inorganic pigments and organic pigments.

[0112] Examples of organic powders include metal soaps such as calcium stearate; alkyl phosphate metal salts such as sodium zinc cetylphosphate, zinc laurylate, and calcium laurylate; acylamino acid polyvalent metal salts such as calcium N-lauroyl-beta-alanine, zinc N-lauroyl-beta-alanine, and calcium N-lauroylglycine; amide sulfonic acid polyvalent metal salts such as calcium N-lauroyl-taurine and calcium N-palmitoyl-taurine; N-acyl basic amino acids such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoyl lysine, N-alpha-paritoyl olnitine, N-alpha-lauroyl arginine, and N-alpha-hydrogenated beef tallow fatty acid acylarginine; N-acyl polypeptides such as N-lauroyl glycyl glycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene and styrene copolymers, and ethylene tetrafluoride.

[0113] UV absorbers include para-aminobenzoic acid, para-aminobenzoic acid ethyl, para-aminobenzoic acid amyl, para-aminobenzoic acid octyl, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, benzyl cinnamic acid, 2-ethoxyethyl para-methoxycinnamic acid, octyl para-methoxycinnamic acid, mono-2-ethylhexaneglyceryl dipara-methoxycinnamic acid, isopropyl para-methoxycinnamic acid, diisopropyl and diisopropyl cinnamic acid ester mixtures, urocanic acid, ethyl urocanic acid, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and its salts, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenone disulfonate, dihydroxybenzophenone, Examples include tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p-(carbo-2'-ethylhexyl-1'-oxy)-1,3,5-triazine, and 2-(2-hydroxy-5-methylphenyl)benzotriazole.

[0114] Examples of disinfectants include hinokitiol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zincpyrithione, benzalkonium chloride, photosensitive agent No. 301, sodium mononitroguaiacol, and undecylenic acid.

[0115] Antioxidants include butylated hydroxyanisole, propyl gallic acid, and ellisorbic acid.

[0116] Examples of pH adjusters include citric acid, sodium citrate, malic acid, sodium malate, formaldehyde, sodium formaldehyde, succinic acid, sodium succinate, sodium hydroxide, and sodium hydrogen phosphate.

[0117] As for alcohol, we can mention high-grade alcohols such as cetyl alcohol.

[0118] In addition, the compounding ingredients that may be added are not limited to these, and any of the above ingredients may be compounded within a range that does not impair the purpose and effect of the present invention.

[0119] The cosmetic of the present invention can take the form of a solution, an emulsion, a viscous mixture, etc.

[0120] The ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions as active ingredients, and include, for example, conventional auxiliary agents and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0121] The cosmetic composition of the present invention can be manufactured in any formulation commonly manufactured in the art, and examples thereof include emulsion, cream, toner, pack, foundation, lotion, cosmetic solution, hair cosmetic, etc.

[0122] Specifically, the cosmetic composition of the present invention includes formulations of skin lotion, skin softener, skin toner, milk lotion, astringent, lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, hair lotion, hair tonic, hair essence, hair shampoo, hair rinse, hair treatment, body lotion, and body cleanser.

[0123] When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0124] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0125] In the case where the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0126] When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as carrier components.

[0127] When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0128]

[0129] The present invention also provides a method for enhancing immunity comprising administering the pharmaceutical composition to a subject.

[0130] The term "subject" as used in the present invention refers to a subject requiring a method for preventing, controlling, or treating a disease, and may be used without limitation as a human, dog, monkey, cat, rodent, such as a mouse, genetically modified mouse, etc. More specifically, it refers to a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc.

[0131] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0132]

[0133] <Example 1> Analysis method

[0134] 1-1. Preparing the mouse

[0135] Six-week-old BALB / c mice weighing 21–23 g were purchased from Central Lab Animal Inc., Seocho-gu, Seoul, South Korea. All mice were housed at 22 ± 2°C, with a relative humidity of 55–60% and a 12-h light / dark cycle, with free access to food and water during the experiment. Analyses were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (Approval Number: GWNU-2021-12).

[0136]

[0137] 1-2. Lipid separation and fatty acid composition analysis

[0138] Arctoscopus japonicus was obtained from the East Sea of ​​Gangwon Province, South Korea. The eggs were separated, freeze-dried, and pulverized for lipid extraction. The egg lipids (AJ, A. japonicus lipids) were extracted using a modified method of Bligh and Dyer. Gas chromatography-amine ionization detection (GC-FID) analysis was then used to determine the fatty acid profile. The yield of the extracted lipid was 2.18 g, corresponding to 6.92% of the dry matter, and it was dissolved in ethanol.

[0139]

[0140] 1-3. Preparation of AJ-PEG (Aster Jelly Egg Lipid Extract) combined with PEG6000

[0141] AJ-PEG was prepared from A. japonicus lipids (AJ) and PEG6000. Saline is generally used for oral administration, but AJ does not dissolve in saline. Therefore, hydrophobic AJ was converted into AJ-PEG using PEG6000 and dissolved in saline. A mixture of AJ and PEG6000 (1:1, w / w) was dissolved in a constant temperature water bath at 60°C, and the solvent was removed using a rotary evaporator at 40°C for 2 hours at 45 rpm. The dried sample was stored at -20°C for 24 hours. AJ-PEG was prepared by crushing the sample, sieving it through a 100 mesh sieve, and then storing it in a desiccator.

[0142]

[0143] 1-4. Establishment of an immunosuppressed mouse model

[0144] Mice that had completed environmental adaptation were randomly divided into nine groups, with five mice per group, as shown in Table 1 below.

[0145]

[0146]

[0147] Levamisole and ginseng were used as positive controls, and the PEG6000 group was used as a model control group. From day 1 to day 10, daily gavage was performed on the mice, and cyclophosphamide (CY) 80 mg / kg BW was administered intraperitoneally to all mice except the normal group. The mice were euthanized within 24 hours after the last administration.

[0148]

[0149] 1-5. Preparation of peritoneal macrophages

[0150] The Ray method was used to collect peritoneal macrophages. Cells were harvested by peritoneal lavage using 5 mL of ice-cold phosphate-buffered saline (PBS) and 3% fetal bovine serum (FBS). The cell suspension was centrifuged and washed twice with 1X PBS buffer. Peritoneal macrophages were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin (PS) at 1 X 10 6 Dispersed at a density of 10 cells / mL.

[0151]

[0152] 1-6. Isolation of splenic lymphocytes

[0153] To extract splenocytes, the spleens of BALB / c mice were gently crushed. The spleen index was calculated based on the spleen weight (mg) and body weight (g). After weighing the spleen on a scale, the spleen was isolated using 1X RBC Lysis Buffer (eBioscience, San Diego, CA, USA). The isolated spleen was centrifuged, washed twice with 1X PBS buffer, and the cells were cultured in RPMI-1640 medium supplemented with FBS and 1% PS at 2 X 10 6 Dispersed at a density of cells / mL.

[0154]

[0155] 1-7. Analysis of peritoneal macrophage proliferation and nitric oxide (NO) production

[0156] Peritoneal macrophages were cultured with or without 1 μg / mL lipopolysaccharides (LPS). After 24 h of culture, 100 μL of the culture supernatant was mixed with Griess reagent (Sigma-Aldrich, Saint Louis, MO, USA) and reacted at room temperature for 10 min. The absorbance at 540 nm was measured to determine whether the culture supernatant contained nitrate.

[0157] Cell proliferation was measured using the EZ-Cytox Cell Viability Assay Kit (Daeil Lab Service, Seoul, Republic of Korea). 100 μL of water-soluble tetrazolium bromide (WST) solution was added to each well, and after incubation at 37°C for 1 h, the absorbance was measured at a wavelength of 450 nm using a microplate reader from BioTek Instruments (Santa Clara, CA, USA).

[0158]

[0159] 1-8. Analysis of peritoneal macrophage phagocytosis

[0160] The degree of phagocytosis was measured using a neutral red uptake assay. Neutral red solution (0.09% mass ratio of the solute) was added to each well. After incubation at 37°C for 30 min, the cells were washed with 1X PBS buffer to remove excess dye. Subsequently, 100 μL of 50% EtOH and 1% glacial acetic acid were added. The absorbance was then measured at a wavelength of 540 nm using a microplate reader (BioTek Instruments, USA).

[0161]

[0162] 1-9. Proliferation analysis of splenic lymphocytes

[0163] Splenocytes (2 Х 10 6cells / mL) were stimulated with concanavalin A (Con A, 5 μg / mL) as a T cell mitogen and LPS (10 μg / mL) as a B cell mitogen, and cultured at 37°C for 48 h in a humidified incubator containing 5% CO2. The degree of cell proliferation was determined using the EZ-Cytox Cell Viability Assay Kit (DaeilLab Service, Seoul, Republic of Korea).

[0164]

[0165] 1-10. Analysis of NK cell activity in spleen cells

[0166] Splenocytes were treated with YAC-1 cells at a ratio of 50:1. After incubation at 37°C for 24 h, cell activity was analyzed using the CytoTox 96® Non-Radioactive cytotoxicity assay (Promega, Madison, WI, USA) according to the manufacturer's instructions.

[0167]

[0168] 1-11. Analysis of immune-related gene expression

[0169] Total RNA was isolated from cells using the Universal RNA Extraction Kit (Takara Bio Inc., Tokyo, Japan) according to the manufacturer's instructions. cDNA was prepared using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Saint Louis, MO, USA). Immune-related gene expression was measured using the QuantStudio® 3 Flex Real-Time PCR System (Applied Biosystems, USA). PCR reactions were performed by mixing 5 ng / μL of cDNA, TB Green® Premix Ex Taq™ II (Takara Bio Inc., Tokyo, Japan), and the specific primers listed in Table 2 below.

[0170]

[0171]

[0172] 1-12. Analysis of T lymphocyte subsets in spleen cells

[0173] Splenocyte suspensions were incubated with 0.5 μL of anti-CD3e-PE, 1.5 μL of anti-CD4-APC, and 1 μL of anti-CD8-FITC antibodies at 4°C for 20 min. Cells were then centrifuged, washed twice with 1 x PBS buffer, and resuspended in FACs buffer (2% FBS and 0.1% sodium azide in 1 x PBS buffer). The number of CD4+ and CD8+ T cells was measured using a CytoFLEX Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA).

[0174]

[0175] 1-13. Statistical Analysis

[0176] All data analyses were performed using SPSS 23.0 software (SPSS, Armonk, NY, USA). Data were compared using one-way ANOVA and Duncan's multiple range test, with significance set at p < 0.05. Values ​​are expressed as mean ± standard deviation (SD).

[0177]

[0178] <Example 2> Analysis results

[0179] 2-1. Fatty acid composition of A. japonicus egg lipid extract (AJ-PEG) combined with PEG6000

[0180] The fatty acid composition of AJ-PEG manufactured by Example 1-3 was analyzed using the GC-FID analysis method of Example 1-2.

[0181] Figure 1 shows the results of GC-FID analysis. AJ-PEG was found to be composed of 28.3 ± 0.5% saturated fatty acid (SFA), 29.6 ± 0.3% monounsaturated fatty acid (MUFA), and 42.1 ± 0.9% polyunsaturated fatty acid (PUFA), with 16:0 (24.4%), DHA (20.6%), 18:1n9 (19.9%), EPA (15.9%), 18:1n7 (6.7%), and 18:0 (4.0%) accounting for the largest proportions in that order.

[0182]

[0183] 2-2. Analysis of spleen size and spleen index

[0184] When AJ-PEG was treated in mice according to the above Examples 1-4, changes in spleen cells were confirmed. Figures 2 to 5 show changes in spleen cells when AJ-PEG was treated in mice. Figure 2 shows changes in the size of spleen cells, and Figure 3 shows changes in spleen index. Looking at Figures 2 and 3, it can be confirmed that the spleen size and spleen index of the CY (cyclophosphamide) group were significantly reduced compared to the normal group. However, the spleen index of the AJ-PEG group was significantly higher than that of the CY treatment group, confirming that AJ-PEG can increase the spleen size and spleen index of mice immunosuppressed by CY. However, the PEG6000 group, 25 mg / kg BW AJ-PEG group, and CY group did not show significant differences in spleen size or spleen index, and the spleen index of the levamisole group and ginseng group was significantly higher than that of the CY group.

[0185]

[0186] 2-3. Analysis of spleen lymphocyte proliferation rate

[0187] According to the above Example 1-9, the change in the proliferation rate of spleen lymphocytes was confirmed. Figure 4 shows the change in the proliferation rate of spleen lymphocytes when AJ-PEG was treated in mice. Looking at Figure 4, the proliferation rate of spleen lymphocytes in the CY group was significantly lower than that in the normal control group, and it can be confirmed that the proliferation rate of spleen lymphocytes in the CY group significantly decreased in response to T cell and B cell mitogens. In addition, when Con A or LPS was used, the AJ-PEG group showed a significantly higher spleen lymphocyte proliferation rate than the CY group, and the AJ-PEG 25, 50, 75, or 100 mg / kg BW treatment groups showed a dose-dependent increase in the spleen lymphocyte proliferation rate compared to the CY group. In addition, in the AJ-PEG 75 mg / kg BW treatment group, the Con A and LPS stimulation groups showed similar results to the normal group.

[0188] The above results confirmed that AJ-PEG has the activity of promoting spleen lymphocyte proliferation.

[0189]

[0190] 2-4. Analysis of NK cell activity in spleen cells

[0191] According to the above Examples 1-10, when AJ-PEG was treated in mice, the cytotoxic activity of spleen cells against NK-sensitive YAC-1 cells was confirmed. Figure 5 shows the change in cytotoxicity of spleen NK cells when mice were treated with AJ-PEG. As shown in Figure 5, spleen NK cell activity was significantly suppressed in the CY group compared to the normal group. NK cell activity was found to increase in a dose-dependent manner in the AJ-PEG group. AJ-PEG 75 mg / kg BW showed cytotoxic activity in the CY treatment group at a level similar to that of the normal group. The above results confirmed that AJ-PEG has the activity of promoting NK cell activity in spleen cells.

[0192]

[0193] 2-5. Gene expression analysis of splenic lymphocytes

[0194] When AJ-PEG was treated in mice according to the above Examples 1-11, changes in mRNA expression in spleen lymphocytes were confirmed. Figures 6 to 12 show changes in mRNA expression levels in spleen lymphocytes according to AJ-PEG treatment. The CY group showed lower expression levels of immune-related genes in spleen lymphocytes compared to the normal group. The above results confirm that CY treatment significantly suppressed the expression of immune-related genes in response to T-cell and B-cell mitogens.

[0195] In the spleen lymphocytes of the groups treated with AJ-PEG at various concentrations (25-100 mg / kg BW), the expression levels of immune-related genes such as interleukin-1 beta (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), and Toll-like receptor 4 (TLR4) were significantly higher than those in the CY group. In particular, cells stimulated with Con A showed significantly higher expression levels of these genes than cells stimulated with LPS.

[0196] The above results confirm that AJ-PEG can restore immunosuppressed mice by activating Th 1 and Th 2 cytokines.

[0197]

[0198] 2-6. Analysis of T lymphocyte subtype ratios in splenic lymphocytes

[0199] CD3+ was selected through flow cytometry according to the above Examples 1-12, and the expression of CD4+ and CD8+ T lymphocytes was analyzed. The analysis results are shown in Figures 13 to 21, and the change in CD4+ / CD8+ ratio is shown in Figure 22. The ratio of CD4+ and CD8+ T lymphocytes and the CD4+ / CD8+ ratio were significantly lower in the CY group than in the control group. However, the CD4+ / CD8+ ratio was significantly higher in the AJ-PEG (75 and 100 mg / kg BW) group than in the CY treatment group.

[0200]

[0201] 2-7. Analysis of peritoneal macrophage proliferation and NO production

[0202] When AJ-PEG was treated according to the above Example 1-7, the proliferation of peritoneal macrophages and changes in NO production were confirmed. Figures 23 and 24 show the changes in peritoneal macrophage proliferation and NO production when AJ-PEG was treated. The macrophage proliferation rate and NO production were higher in the AJ-PEG group than in the CY group, and the macrophage proliferation rate and NO production showed an AJ-PEG dose-dependent pattern of increase. In addition, AJ-PEG 75 and 100 mg / kg BW were found to significantly increase peritoneal macrophage proliferation and NO production to a level similar to that of the normal group.

[0203] The above results confirmed that AJ-PEG has the activity of promoting peritoneal macrophage proliferation and NO production.

[0204]

[0205] 2-8. Analysis of phagocytosis of peritoneal macrophages

[0206] According to the above Examples 1-8, when AJ-PEG was treated, changes in phagocytosis of peritoneal macrophages were confirmed. Figure 25 shows changes in phagocytosis of peritoneal macrophages when treated with AJ-PEG. The phagocytosis rate of normal mouse peritoneal macrophages was considered 100% by absorbing neutral red. The peritoneal macrophage phagocytosis of the CY group was significantly lower than that of the normal control group. Compared to the CY group, the peritoneal macrophage phagocytosis rates of the ginseng group, levamisole group, PEG6000 group, and the groups treated with AJ-PEG (25-100 mg / kg BW) were significantly higher. In particular, AJ-PEG 75 mg / kg BW was shown to restore the macrophage phagocytosis rate to a higher level than that of the normal control group. The above results confirmed that AJ-PEG has the activity of promoting phagocytosis of peritoneal macrophages.

[0207]

[0208] 2-9. Gene expression analysis of peritoneal macrophages

[0209] According to the above Examples 1-11, in order to confirm the effect of AJ-PEG on peritoneal macrophage activation in CY-treated mice, the expression levels of immune-related genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were measured using real-time qPCR. The results of gene expression level measurement are shown in Figs. 26 to 30. As can be seen from Figs. 26 to 30, it can be confirmed that the mRNA expression levels of these genes in the CY group are significantly reduced compared to the normal control group. In the AJ-PEG (25, 50, 75, 100 mg / kg BW) groups, the expression of several immune-related genes was significantly higher than in the CY group. In addition, peritoneal macrophages treated with 75 mg / kg BW of AJ-PEG showed gene expression levels similar to or higher than those of the positive control group. These results confirmed that AJ-PEG has the activity of promoting immune-related gene expression in peritoneal macrophages.

[0210]

[0211] Through the above examples, the spleen index enhancing activity, spleen lymphocyte proliferation promoting activity, spleen NK cell activity promoting activity, immune-related gene enhancing activity, peritoneal macrophage proliferation promoting activity, and NO production enhancing activity of the sea squirt egg lipid extract were confirmed, and it was confirmed that the sea squirt egg lipid extract can be used for immune enhancement purposes.

Claims

1. A food composition for enhancing immunity, comprising a lipid extract from the egg of the sea squirt as an effective ingredient.

2. In paragraph 1, A composition characterized in that the above-mentioned dorumuk egg lipid extract is extracted using a solvent selected from the group consisting of distilled water, lower alcohols having C1 to C4, lower alcohol aqueous solutions, hexane, chloroform or acetic acid.

3. In paragraph 1, A composition wherein the above-mentioned acorn seed lipid extract comprises a saturated fatty acid, a monounsaturated fatty acid or a polyunsaturated fatty acid.

4. In paragraph 3, A composition wherein the saturated fatty acid is palmitic acid (C16:0) or stearic acid (C18:0).

5. In paragraph 3, A composition wherein the monounsaturated fatty acid is at least one selected from the group consisting of palmitoleic acid (C16:1n7), oleic acid (C18:1n9), vaccenic acid (C18:1n7), and eicosenoic acid (C20:1).

6. In paragraph 3, A composition wherein the polyunsaturated fatty acid is at least one selected from the group consisting of linoleic acid (C18:2n6), linolenic acid (C18:3n3), eicosatrienonic acid (C20:3n3), eicosapentaenoic acid (EPA, eicosapentaenoic acid C20:5n3), docosapentaenoic acid (DPA, docosapentaenoic acid C22:5n3), and docosahexaenoic acid (DHA, docosahexaenoic acid C22:6n3).

7. In paragraph 1, A composition wherein the above-mentioned extract of the acorn jelly has the activity of increasing spleen size and spleen index.

8. In paragraph 1, A composition wherein the above-mentioned extract of the acorn jelly has the activity of promoting the proliferation of lymphocytes in the spleen.

9. In paragraph 1, A composition wherein the above-mentioned acorn seed lipid extract enhances NK cell activity in the spleen.

10. In paragraph 1, A composition wherein the above-mentioned extract of the acorn jelly has the activity of promoting the expression of immune-related factors in spleen lymphocytes.

11. In paragraph 10, A composition wherein the above immune-related factor is at least one selected from the group consisting of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon γ (IFN-γ), and toll-like receptor 4 (TLR4).

12. In paragraph 1, A composition wherein the above-mentioned extract of the acorn jelly exhibits immune-enhancing activity by activating Th1 and Th2 cytokines.

13. In paragraph 1, A composition wherein the above-mentioned extract of the acorn jelly increases the proportion of CD4+ T lymphocytes in the spleen.

14. In paragraph 1, A composition wherein the above-mentioned acorn extract promotes the proliferation of macrophages.

15. In paragraph 1, A composition wherein the above-mentioned extract of the acorn jelly promotes the phagocytosis of macrophages.

16. In paragraph 1, A composition wherein the above-mentioned extract of the acorn jelly has the activity of promoting the expression of immune-related factors in macrophages.

17. In paragraph 16, A composition wherein the immune-related factor is at least one selected from the group consisting of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α).

18. A health functional food composition for enhancing immunity, containing a lipid extract from the egg of the sea squirt as an effective ingredient.

19. A pharmaceutical composition for enhancing immunity, comprising a lipid extract from the egg of the sea buckthorn as an effective ingredient.

20. A cosmetic composition for enhancing immunity, comprising a lipid extract from the egg of the sea buckthorn as an effective ingredient.

21. A method for enhancing immunity, comprising the step of administering to a subject the pharmaceutical composition of claim 19.

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