Composition for improving skin conditions containing elder callus culture or its extract as an active ingredient

Abstract

The present invention relates to a composition for improving skin conditions, containing an elder callus culture or an extract thereof as an active ingredient. The elder callus culture or extract thereof according to the present invention inhibits collagen degradation or promotes cell proliferation, and has effects such as skin regeneration, skin firmness maintenance, skin moisturization, and wrinkle reduction. When used in combination with epidermal growth factor, it has a high synergistic effect, and can be used as a material for cosmetics, pharmaceuticals, etc.

Description

[Technical Field]

[0001] The present invention relates to a composition for improving skin conditions, which contains an elder callus culture or an extract thereof as an active ingredient. [Background technology]

[0002] The skin is the largest organ in the human body, accounting for approximately 16% of the total body volume. It is composed of the epidermis, dermis, and subcutaneous fat layer and is in direct contact with the external environment. It acts as the primary defense membrane, protecting internal organs from various external environmental stimuli such as temperature and humidity changes, ultraviolet rays, harmful microorganisms, and pollutants, and is also responsible for the biochemical functions necessary for the metabolism of the entire body.

[0003] In particular, the subcutaneous fat layer beneath the dermis is composed of lobules of fat cells, which act as a cushion to protect the organs within the skin by preventing thermal damage and absorbing shock from external environmental stimuli, and also functions as a nutrient reservoir by storing nutrients in the form of triacylglycerol and using them as an energy source when needed. However, as we age, aging progresses and the skin becomes damaged by natural factors and various external stimuli, resulting in the inability of skin cells to perform their original functions, leading to the formation of wrinkles, loss of firmness, and skin atrophy.

[0004] Collagen, a major component of the extracellular matrix, is the primary matrix protein produced by skin fibroblasts and present in the extracellular interstitium. It is an important protein, accounting for approximately 30% of the total body protein weight, and has a rigid triple helical structure. Collagen forms the majority of the organic material in skin, tendons, bone, and teeth, with particularly high concentrations in bone and skin (dermis). Collagen is also present as fibrous inclusion bodies in most other bodily structures. Collagen has relatively low immunogenicity, due in part to the shielding of potential epitopes by its helical structure, which also makes it resistant to proteolysis. Its main functions include providing mechanical toughness to skin, providing resistance to connective tissue, tissue cohesion, supporting cell adhesion, and guiding cell division and differentiation (during biological growth and wound healing) (Van der Rest et al., Ann NY Acad Sci, 1990). Collagen is known to decrease with age and photoaging due to UV exposure, which is closely related to the formation of wrinkles in the skin (Arthur K.Balin et al., Aging and the skin, 1989). Furthermore, collagen plays an important role in wound healing, promoting collagen synthesis in damaged epithelia and enabling wounds to heal quickly and without scarring.

[0005]

[0003] Conventionally, in order to utilize the effects of collagen on improving skin wrinkles and healing wounds, products containing collagen in external skin preparation compositions such as cosmetics or ointments have been commercially available. However, these products apply collagen itself to the skin surface, and since collagen, a polymeric substance, is difficult to absorb percutaneously, moisturizing effects or wound healing effects cannot be expected, and therefore, the essential functions of improving skin function and healing wounds cannot be achieved. This has led to increased interest in collagen synthesis promoters, and conventionally known collagen synthesis promoters include vitamin C, retinoic acid, and transforming growth factor (TGF, Cardinale G et al., Adv Enzymol, 41, p. 425, 1974). In addition, Japanese Patent Publication No. 8-231370 discloses a protein derived from animal placenta as a collagen synthesis substance, Japanese Patent Publication No. 8-208424 discloses betulinic acid, and Japanese Patent Publication Nos. 9-40523 and 10-36283 disclose chlorella extracts that promote fibroblast proliferation. However, these conventional substances have safety issues, such as irritation and redness when applied to the skin, and are limited in dosage or have only weak effects, so they cannot be expected to substantially improve skin function or wound healing. Therefore, there is a strong need for the development of new topical skin compositions that are safer to the body and more effective than existing topical skin compositions.

[0006] Elder (Sambucus nigra) is a general term for shrubs and their fruits in the genus Sambucus, a dicotyledonous plant in the family Lemnaceae, order Viburnum. It is a deciduous tree that grows naturally in humid areas of Europe, Asia, North Africa, and North America, producing purple-black elderberries and cream-white elderflowers. Elder plant components are known to have antioxidant, anti-inflammatory, antiviral, skin immune-boosting, skin trouble-relieving, and skin soothing effects, making it a highly valuable species for use in cosmeceuticals and biocosmetics.

[0007] On the other hand, "callus" is a wound-healing tissue that, when a plant is injured, cells regain their ability to divide, close the wound, and enlarge. It refers to a special cell mass that is produced by culturing tissue excised from a plant in a medium containing auxin, and interest in the use of such callus has been growing in recent years, and it is being researched in a variety of fields. Utilizing callus cultures, which are undifferentiated cell masses, is thought to be valuable as cosmetic ingredients with excellent physiologically active effects.

[0008] In this invention, we have developed a naturally derived elder callus extract from elder, which is highly useful as a biomaterial, as a safe and effective medicinal ingredient with skin moisturizing, regeneration, and collagen degradation inhibitory effects. We have also confirmed that when this elder callus extract is used in combination with the growth factor EGF (Epidermal Growth Factor), it has a synergistic effect on skin moisturizing, regeneration, and collagen degradation inhibitory effects compared to when used alone, and have completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention is directed to solving the above-mentioned problems and other related problems.

[0010] An object of the present invention is to provide a cosmetic composition for improving skin conditions, which contains an elder callus culture or an extract thereof as an active ingredient.

[0011] Another object of the present invention is to provide a quasi-drug composition for preventing or improving skin diseases, which contains an elder callus culture or an extract thereof as an active ingredient.

[0012] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating skin diseases, which contains an elder callus culture or an extract thereof as an active ingredient.

[0013] Another object of the present invention is to provide a method for preparing a composition for improving skin conditions containing an elder callus culture or an extract thereof.

[0014] Another object of the present invention is to provide a method for improving skin conditions, which comprises the step of applying a composition containing an elder callus culture or an extract thereof as an active ingredient to the skin or mucosa of an individual.

[0015] Another object of the present invention is to provide a method for preventing or treating skin diseases, which comprises the step of administering to an individual a composition containing an elder callus culture or an extract thereof as an active ingredient.

[0016] Another object of the present invention is to provide a use of a composition containing an elder callus culture or an extract thereof as an active ingredient for improving skin conditions.

[0017] Another object of the present invention is to provide a use of a composition containing an elder callus culture or an extract thereof as an active ingredient for the prevention or treatment of skin diseases.

[0018] Another object of the present invention is to provide use of a composition containing an elder callus culture or an extract thereof as an active ingredient for preparing a medicament for preventing or treating skin diseases.

[0019] The technical problems to be achieved by the technical ideas of the invention disclosed in this specification are not limited to the problems to be solved above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0020] This will be explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to each other description and embodiment. In other words, all combinations of various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of the present invention is not limited by the following specific description.

[0021] In one aspect to achieve the above object, the present invention provides a cosmetic composition for improving skin conditions, which contains an elder callus culture or an extract thereof as an active ingredient.

[0022] In the present invention, "elder (Sambucus nigra)" is a general term for shrubs and their fruits belonging to the dicotyledonous Viburnum order, family Lembriaceae, genus Sambucus. Elder is a deciduous tree that grows naturally in shady areas of Europe, Asia, North Africa, and North America, producing purple-black elderberries and cream-white elderflowers. It grows primarily in sunny locations under a variety of conditions, including moist and dry soil, and both the flowers and berries have long been used for culinary purposes, particularly in cordials and wines.

[0023] In this specification, "callus" refers to wound-healing tissue that occurs when a plant is wounded, and cells regain their ability to divide, close the wound, and enlarge. It refers to undifferentiated tissue or cell masses that are formed when excised tissue from a plant is cultured in an auxin-containing medium, or when a certain type of plant is wounded or the wound is treated with auxin. Callus can divide and proliferate indefinitely by subcultivating it in a new medium. Such callus is an amorphous tissue or cell mass that has lost the ability to undergo normal organogenesis or tissue differentiation. It is composed mostly of parenchyma cells. In a broader sense, it can also include plant tumor tissues caused by infection with bacteria such as Agrobacterium. In other words, once cells in a plant proliferate, they quickly determine their direction and regularly assemble into not only specific tissues but also organs. However, when external stimuli such as various plant growth hormones are applied, callus, an undifferentiated cell mass formed by tissue culture, is interpreted as releasing the previously maintained control and allowing cells to proliferate independently. When callus obtained by dedifferentiation is placed in a liquid medium of the same composition and cultured with shaking, the cells break apart and continue to grow in suspension. This callus or cultured cells can be subcultured on new medium, enabling them to divide and grow indefinitely, which is essentially different from differentiated cells, which age and die as a complete plant. When callus or cultured cells that have been subcultured for several generations are placed on solid medium free of various plant growth hormones, shoots and roots appear, and individual plants are regenerated. These regenerated plants are derived from the division and proliferation of a single cultured cell, and the origin of the cultured cell can be said to be derived from the hypocotyl or parenchyma.

[0024] In the present invention, the "callus" may be derived from any part of elder (Sambucus nigra), and specifically may be derived from stems, leaves, petals, calyx, fruit, ovary, part of the placenta, or cotyledons excised from germinated seedlings. Preferably, the callus may be derived from the leaves or stems of young elder.

[0025] In the present invention, the term "callus culture" refers to a callus cultured in a medium. The medium can be any medium commonly used for callus culture in the art, without limitation.

[0026] In the present invention, the term "extract" refers to a liquid component obtained by immersing a desired material, e.g., elder callus culture, in various solvents and then extracting it for a certain period of time at room temperature or under heated conditions, or a solid component obtained by removing the solvent from the liquid component, etc. It can also be interpreted as encompassing any diluted solution of the resultant product, concentrate thereof, crude product thereof, purified product thereof, etc.

[0027] The extract can be obtained by extraction with water or various organic solvents, and is not particularly limited, but is preferably extracted with water, an alcohol having 1 to 4 carbon atoms, and / or a mixed solvent thereof. The method for obtaining the extract is also not particularly limited, but preferably includes hot water extraction, in which a dried or processed product is immersed in the solvent and extracted at a temperature of 90 to 120°C, cold immersion extraction, in which extraction is performed at room temperature of 10 to 25°C, heat extraction, in which extraction is performed by heating at 40 to 100°C, ultrasonic extraction, in which extraction is performed by applying ultrasound, and reflux extraction using a reflux condenser. Hot water extraction is preferred.

[0028] The extraction solvent is preferably added at 1 to 20 times the weight of the extractant, and the extraction temperature is preferably, but not limited to, 90 to 120°C. The extraction time is preferably 1 to 4 hours, and most preferably, 2 hours, but is not limited to these. Drying is preferably performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, and more preferably, freeze drying, but is not limited to these.

[0029] Alternatively, it can be prepared by extraction using water, an organic solvent, or a mixture of these. The extracted liquid can be used immediately or after concentration and / or drying. When using an organic solvent, the organic solvent used can be methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixture of these solvents. Extraction can be performed at room temperature or with heating under conditions that minimize or prevent the destruction of the active ingredients of the herbal medicine. The degree of extraction and loss of the active ingredients of the medicinal herbs varies depending on the organic solvent used, so the appropriate organic solvent should be selected.

[0030] In the present invention, the extract may be used after being concentrated or diluted, and the distillate of the extract may be used, or the extract may be freeze-dried again and used in the form of powder.

[0031] In the present invention, the "improvement of skin condition" may be, but is not limited to, skin regeneration, wrinkle improvement, anti-aging of skin, maintaining skin firmness, moisturizing skin, or improvement of scars.

[0032] In the present invention, the term "improvement" refers to any action of improving or benefiting the condition of the skin using a composition containing the elder callus culture or an extract thereof according to the present invention.

[0033] The term "wrinkle reduction" in the present invention means maintaining or enhancing wrinkles and firmness of the skin. Collagen, which is a collagen fiber in the dermis layer of the skin, and elastin, which is an elastic fiber, are the main proteins that play this role and are responsible for skin firmness. Therefore, when a compound has an effect of inhibiting the activity of elastase and / or collagenase, which secrete these proteins, it can achieve a wrinkle reduction effect.

[0034] The term "skin regeneration" as used herein refers to the process of repairing skin tissue from damage caused by external and internal factors, such as ultraviolet rays, external pollutants, wounds, trauma, etc., and such damage caused by internal factors as stress, but is not limited to these.

[0035] The term "anti-skin aging" as used herein refers to the prevention or delay of both chronological aging (intrinsic aging), which is natural aging caused by physiological changes in the body over time, and photoaging (photoinduced aging), which occurs in areas exposed to sunlight. To prevent skin aging, it is necessary to protect the skin from harmful external environments, as well as to minimize wrinkle formation by activating skin cells and promoting the synthesis of biosynthetic proteins such as collagen and elastin. In particular, the dermis layer of the skin is composed of collagen, a collagenous fiber, and elastin, an elastic fiber, forming a meshwork structure. When this meshwork collapses, it is degraded by elastase, an elastin-degrading enzyme, causing skin sagging and wrinkles, resulting in intrinsic skin aging. Therefore, by inhibiting the degradation of elastin, one of the main causes of skin aging, skin aging can be inhibited.

[0036] The skin aging in the present invention may be, but is not limited to, photoaging caused by UV-B ultraviolet rays. Specifically, light with a wavelength of 280 nm to 40 nm contained in sunlight can accelerate photoaging of skin cells. In particular, exposure to ultraviolet rays such as UV-B with a wavelength in the 280 nm to 320 nm range can damage the skin and fibers, resulting in sunburn, which darkens the skin. UV-B exposure promotes the accumulation of reactive oxygen species (e.g., H2O2) and free radicals in skin cells, which can stimulate intracellular signaling pathways and induce oxidative stress in biomolecules such as DNA, proteins, and lipids, potentially resulting in damage to skin tissue. When oxidative stress in skin cells increases, irritation occurs in the keratinocytes of the epidermis and the fibroblasts of the dermis, which, through a series of intracellular signaling processes, can increase the expression of genes such as MMP (matrix metalloproteinase), a collagen-degrading enzyme. This can induce a decrease in collagen, a major component of the skin that accounts for 90% of the dermis and provides strength and tension to the skin, protecting it from external stimuli and forces, which can lead to skin aging and the formation of wrinkles.

[0037] The improvement of skin conditions of the present invention may be due to cell proliferation promoting activity, specifically, but not limited to, the promotion of human keratinocyte proliferation or the promotion of fibroblast proliferation.

[0038] The improvement of skin conditions in the present invention may be due to, but is not limited to, the inhibition of collagen degradation. Preferably, the improvement may be due to the inhibition of the activity of matrix metalloproteinases (MMPs), which are collagen-degrading enzymes. Specifically, the improvement may be due to the reduction of mRNA expression of matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), or matrix metalloproteinase 9 (MMP9).

[0039] Furthermore, the composition of the present invention may increase the expression of hyaluronan synthase or aquaporin in keratinocytes, and specifically, may increase the mRNA expression of hyaluronan synthase 1 (HAS-1), hyaluronan synthase 2 (HAS-2), hyaluronan synthase 3 (HAS-3), or aquaporin 3 (AQP-3).

[0040] The term "wound" as used herein refers to a disruption of the normal continuity of the skin structure due to physical bodily injury, and specifically refers to injuries to a part of an individual, such as contusion or bruise, laceration, avulsion, penetrating wound, non-healing traumatic wound, tissue destruction due to radiation, abrasion, osteonecrosis, gunshot wound, incision, burn, frostbite, skin ulcer, dry skin, skin keratosis, fissure, cleft, dermatitis, pain due to dermatophytosis, surgical wound, wound due to vascular disease, wounds such as corneal wound, pressure sore, depressed wound, conditions related to diabetes and poor circulation, chronic ulcer, suture site after cosmetic surgery, spinal injury wound, gynecological wound, chemical wound, and acne.

[0041] In the present invention, the term "moisturizing the skin" means increasing the moisture content of the skin and keeping it moist.

[0042] The composition containing the elder callus extract of the present invention may be non-cytotoxic to human keratinocytes or human fibroblasts.

[0043] The composition of the present invention may contain the elder callus culture or an extract thereof in various amounts, so long as it has the effects of wrinkle improvement, skin moisturizing, skin regeneration, or wound healing improvement, etc. Specifically, the elder callus culture or an extract thereof may be contained in an amount of 0.1 to 10 wt% based on the total weight of the composition. This ratio is merely an exemplary range, and it will be obvious to those skilled in the art that other ratios such as 20%, 30%, or more will also have excellent effects of improving skin condition, moisturizing the skin, and improving skin wrinkles.

[0044] In addition, the composition of the present invention may further contain epidermal growth factor (EGF), and when the elder callus culture or its extract is contained together with EGF, the effects of improving wrinkles, moisturizing the skin, regenerating the skin, or improving wound healing may be further enhanced.

[0045] Epidermal growth factor (EGF) is a polypeptide with a molecular weight of approximately 6 Ka, consisting of 53 amino acids and three disulfide bonds. EGF is a growth factor that specifically binds to the epidermal growth factor receptor (EGFR) present on the surface of cells to promote cell growth, proliferation, and differentiation. It plays a key role in skin regeneration by promoting fibroblast proliferation, promoting angiogenesis at the site of skin damage, inducing the secretion of other regeneration-promoting factors, and promoting the synthesis of fibronectin.

[0046] The cosmetic composition of the present invention can be formulated, in addition to the elder callus culture or an extract thereof, with various components commonly used in cosmetic compositions, such as water-soluble components, powder components, oils, surfactants, moisturizers, viscosity regulators, preservatives, antioxidants, fragrances, pigments, etc., as needed, within a range that does not reduce the effects of the present invention.

[0047] Non-limiting examples of usable surfactants include anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants. More specifically, anionic surfactants include alkylbenzene sulfonates, polyoxyalkylene alkyl sulfates, alkyl sulfates, olefin sulfonates, alkyl phosphates, polyoxyalkylene alkyl ether phosphates, dialkyl sulfosuccinates, and fatty acid salts. Nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, polyhydric alcohol fatty acid partial esters, polyoxyethylene polyhydric alcohol fatty acid partial esters, polyglycerin fatty acid esters, polyoxyethylene hydrogenated castor oil derivatives, and fatty acid diethanolamides. Cationic surfactants include tertiary aliphatic amine salts, alkyltrimethylammonium halides, and dialkyldimethylammonium halides. Amphoteric surfactants include amidobetaines, imidazolinium betaines, and sulfobetaines.

[0048] Examples of the moisturizing agent include glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, sorbitol, etc. Examples of the preservative include benzoic acid, dehydroacetic acid, parahydroxybenzoic acid esters (methyl parahydroxybenzoate, butyl parahydroxybenzoate, etc.), phenoxyethanol, etc. Examples of the antioxidant include ascorbic acid, BHA, etc. In addition, ultraviolet absorbers, anti-inflammatory agents, cooling agents, etc. may also be added.

[0049] The cosmetic composition of the present invention can be prepared into a dosage form selected from the group consisting of, but not limited to, a solution, an ointment for external use, a cream, a foam, a nourishing lotion, a softening lotion, a pack, a softening lotion, a milky lotion, a makeup base, an essence, a soap, a liquid cleanser, a bath additive, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleansing oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray.

[0050] The cosmetic composition of the present invention may further comprise one or more carriers acceptable for cosmetic formulations, including, but not limited to, conventional ingredients such as oils, water, surfactants, humectants, lower alcohols, thickeners, chelating agents, colorants, preservatives, and fragrances. Here, "carriers acceptable for cosmetic formulations" refers to compounds or compositions that are already known and used in cosmetic formulations, or compounds or compositions to be developed in the future, that are not toxic, unstable, or irritating to the extent that the human body can tolerate upon contact with the skin. The carrier may be present in the composition of the present invention in an amount of about 1% to about 99.99% by weight, based on the total weight of the composition, preferably about 90% to about 99.99% by weight of the composition. However, this ratio will vary depending on the formulation in which the composition of the present invention is prepared, the specific application site (e.g., face, neck, etc.), and the desired application amount, and therefore, this ratio should not be construed as limiting the scope of the present invention in any way.

[0051] The carriers acceptable for the cosmetic formulation contained in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.

[0052] When the dosage form of the present invention is an ointment, paste, cream, or gel, the carrier component may be, but is not limited to, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. These may be used alone or in combination of two or more.

[0053] When the dosage form of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. can be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether can be further included, but is not limited to these, and these can be used alone or in combination of two or more.

[0054] When the dosage form of the present invention is a solution or emulsion, a solvent, a solvent or an emulsifier can be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc., and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol aliphatic esters, polyethylene glycol or sorbitan fatty acid esters can be used, but are not limited to these, and these can be used alone or in combination of two or more.

[0055] When the dosage form of the present invention is a suspension, the carrier component may be, but is not limited to, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth, and these may be used alone or in combination of two or more.

[0056] When the dosage form of the present invention is a soap, the carrier component may be, but is not limited to, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. These may be used alone or in combination of two or more.

[0057] In one aspect to achieve the above object, the present invention provides a quasi-drug composition for preventing or ameliorating skin diseases, which contains an elder callus culture or an extract thereof as an active ingredient.

[0058] The "elder," "callus," "callus culture," "extract," and "composition" are the same as those described above.

[0059] The term "skin disease" as used herein is selected from the group consisting of, but not limited to, skin wounds, skin scars and skin pigmentation.

[0060] Non-limiting examples of the terms "skin wound" and "skin scar" of the present invention include abrasions and scars resulting from such abrasions.

[0061] The term "skin pigmentation" as used herein refers to all diseases caused by skin pigmentation due to increased melanin production. Non-limiting examples of skin pigmentation diseases include melasma, freckles, spots, solar pigmentation, post-drug hyperpigmentation, post-inflammatory hyperpigmentation, and pregnancy hyperpigmentation.

[0062] The term "prevention" as used herein means any action of suppressing or delaying skin diseases such as skin wounds, skin scars, and skin pigmentation by administering or applying the elder callus culture or extract thereof of the present invention to an individual.

[0063] The term "improvement" as used herein means any action of alleviating symptoms of skin diseases such as skin wounds, skin scars, and skin pigmentation by administering or applying the elder callus culture or extract thereof of the present invention to an individual.

[0064] In the present invention, the term "quasi-drug" refers to an article that is used for the purpose of diagnosing, curing, mitigating, treating, or preventing a disease in humans or animals, and is not a mechanism, machine, or device, and an article that is used for the purpose of exerting a pharmacological effect on the structure and function of humans or animals, excluding an article that is not a mechanism, machine, or device. One embodiment of the term may include an oral preparation, but is not limited to this. The formulation method, dosage, method of use, constituents, etc. of a quasi-drug may be appropriately selected from conventional techniques known in the art.

[0065] In addition to the above-mentioned components, the quasi-drug composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient, or diluent, as necessary. The pharmaceutically acceptable carrier, excipient, or diluent is not particularly limited as long as it does not impair the effects of the present invention, and may include, for example, a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, a lubricant, a sweetener, a flavoring agent, a preservative, etc.

[0066] In one aspect to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating skin diseases, which contains an elder callus culture or an extract thereof as an active ingredient.

[0067] The "elder," "callus," "callus culture," "extract," "skin disease," "prevention," and "composition" are the same as those described above.

[0068] The term "treatment" as used herein means any action of administering or applying the elder callus culture or extract thereof of the present invention to an individual to improve, alleviate or benefit the symptoms of skin diseases such as skin wounds, skin scars and skin pigmentation.

[0069] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. For example, the pharmaceutically acceptable carrier may further comprise a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc.

[0070] The parenteral carrier may also contain water, a suitable oil, saline, aqueous glucose, glycol, etc. Stabilizers and preservatives may also be included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl or propyl paraben, and chlorobutanol.

[0071] The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method, for example, orally or parenterally, and parenteral administration may be intravenous, intramuscular, intraarterial, central, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration, with central administration being preferred, but not limited thereto.

[0072] The pharmaceutical composition of the present invention can be formulated into a preparation for oral or parenteral administration by the administration route as described above. When formulated, one or more buffers (e.g., physiological saline or phosphate buffered saline (PBS)), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrants, surfactants, diluents, or excipients can be used.

[0073] Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions, and capsules, and these solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one or more excipients, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose, or gelatin, etc. For example, tablets or sugar-coated tablets can be obtained by blending the active ingredient with a solid excipient, pulverizing the mixture, adding suitable excipients, and then processing it into a granular mixture.

[0074] In addition to simple excipients, lubricants such as magnesium stearate talc (Talc) are also used. Oral liquid formulations include suspensions, oral solutions, emulsions, and syrups, and may contain various excipients such as wetting agents, sweeteners, flavoring agents, or preservatives in addition to commonly used simple diluents such as water and liquid paraffin. Furthermore, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives may also be added.

[0075] For parenteral administration, the pharmaceutical composition of the present invention can be formulated into injections, transdermal preparations, and nasal inhalants with a suitable parenteral carrier by methods known in the art. Injections must be sterilized and protected from microbial contamination, such as bacteria and fungi. Suitable carriers for injections include, but are not limited to, solvents or dispersion media such as water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferred carriers include Hank's solution, Ringer's solution, triethanolamine-containing PBS, or sterile water for injection, as well as isotonic solutions such as 10% ethanol, 40% propylene glycol, and 5% dextrose. To protect the injections from microbial contamination, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal, can be further included. In most cases, the injections can further contain an isotonic agent, such as sugar or sodium chloride.

[0076] Transdermal preparations include ointments, creams, lotions, gels, topical liquids, pastes, liniments, aerosols, etc. Here, "transdermal administration" means that a pharmaceutical composition is topically administered to the skin, and an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

[0077] For inhalation administration, the compositions used according to the present invention can be conveniently delivered in the form of an aerosol spray from a pressurized pack or a nebulizer using a suitable propellant, for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch. Dosage forms for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a formulary commonly known to all pharmaceutical scientists.

[0078] The amount of active compound in a unit dose of the pharmaceutical composition of the present invention may vary, but may be adjusted to about 0.01 mg to about 1 g per dose for an average 70 kg human. However, the dosage will vary depending on the requirements of the human or mammal, the severity of the disease being treated, and the final composition of the compound used. Determining the appropriate dosage for a particular situation is within the skill of one of ordinary skill in the art.

[0079] The pharmaceutical compositions of the present invention can be used alone or in conjunction with surgery, radiation therapy, hormone therapy, chemotherapy, or methods using biological response modifiers.

[0080] In one aspect for achieving the above-mentioned object, the present invention provides a method for preparing a composition for improving skin conditions containing an elder callus culture or an extract thereof, the method comprising the steps of: (a) inducing and culturing elder callus from elder plant tissue to prepare an elder callus culture; (b) preparing an elder callus extract from the elder callus culture; and (c) preparing a composition containing the elder callus culture or an extract thereof.

[0081] The "elder," "callus," "callus culture," "extract," "skin condition improvement," and "composition" are the same as those described above.

[0082] In the present invention, in step (a), the elder plant tissue may be, but is not limited to, a part of the stem, leaf, petal, calyx, fruit, ovary, or placenta tissue, and is preferably obtained by collecting young elder leaves or stems.

[0083] In the present invention, step (a) may include a step of collecting elder plant tissue and sterilizing it. The sterilization may be any sterilization method commonly used in the callus induction process in the same technical field, without any restrictions. Specifically, elder plant tissue may be collected, washed with running water, sterilized in 70% ethanol (EtOH) solution for 30 to 60 seconds, and then immersed in 2% sodium hypochlorite solution containing a small amount of Tween 20 for 3 to 7 minutes for sterilization.

[0084] Furthermore, step (a) may involve inducing elder callus from a medium containing picloram, 1-naphthaleneacetic acid, or 6-benzylaminopurine, preferably a medium containing 1 to 20 μM 1-naphthaleneacetic acid and 0.1 to 1 μM 6-benzylaminopurine, or a medium containing 1 to 20 μM picloram and 0.1 to 1 μM 6-benzylaminopurine, but is not limited to these.

[0085] In the present invention, step (a) may include a step of culturing elder callus on a solid medium followed by further culturing in a liquid medium. Specifically, the elder callus induced in step (a) may be cultured on a solid medium to select and stabilize good callus, followed by culturing in a medium containing 0.1-1 μM 6-benzylaminopurine and 1-20 μM picloram to grow the callus. Subsequently, the elder callus grown on the solid medium may be cultured in a liquid medium for mass cultivation, and the method may include a step of culturing a sufficient amount of callus by repeatedly inoculating the elder callus cultured on the solid culture medium in a 500 ml Erlenmeyer flask with the liquid medium and culturing it.

[0086] In step (a), a medium suitable for inducing and culturing elder callus can be selected, and any medium commonly used in the art for plant tissue culture can be used without limitation. For plants, MS medium and B5 medium are commonly used. For example, MS medium (per 1 L) contains 0.025 mg CoCl2·6H2O, 0.025 mg CuSO4·5H2O, 36.70 mg FeNaEDTA, 6.20 mg H3BO3, 0.83 mg KI, 16.90 mg MnSO4·H2O, 0.25 mg Na2MoO4·2H2O, 8.60 mg ZnSO4·7H2O, 332.02 mg CaCl2, 170.00 mg KH2PO4, 1900.00 mg KNO3, 180.54 mg MgSO4, and 1650.00 mg NH4NO3. Furthermore, the medium may contain zeatin, sucrose, agarose, etc.

[0087] In the present invention, the step (b) may include a step of drying the elder callus culture obtained in the step (a) to prepare a dried elder callus product, and mixing the dried product with purified water to prepare an extract.

[0088] In the present invention, in step (c), the elder callus culture obtained by culturing in step (a) can be directly prepared into a composition in an appropriate form, or it can be obtained in the form of an extract in step (b) and then prepared into a composition in an appropriate form.

[0089] In step (c), a composition in an appropriate form can be prepared according to the purpose using the known method described above.

[0090] In one aspect to achieve the above-mentioned object, the present invention provides a method for improving skin conditions, comprising the step of applying a composition containing an elder callus culture or an extract thereof as an active ingredient to the skin or mucosa of an individual.

[0091] In one aspect to achieve the above-mentioned object, the present invention provides a method for preventing or treating a skin disease, which comprises the step of administering to an individual a composition containing an elder callus culture or an extract thereof as an active ingredient.

[0092] The "elder," "callus," "callus culture," "extract," "composition," "improvement of skin condition," "skin disease," "prevention," and "treatment" are the same as those described above.

[0093] The individual may be a mammal, and specifically may be, but is not limited to, a human, cow, sheep, goat, horse, pig, dog, cat, rabbit, rat, mouse, fish, bird, etc.

[0094] In one embodiment for achieving the above object, there is provided use of a composition containing an elder callus culture or an extract thereof as an active ingredient for improving skin conditions.

[0095] In one embodiment for achieving the above object, there is provided use of a composition containing an elder callus culture or an extract thereof as an active ingredient for the prevention or treatment of skin diseases.

[0096] In one embodiment for achieving the above object, there is provided use of a composition containing an elder callus culture or an extract thereof as an active ingredient for the manufacture of a medicament for preventing or treating skin diseases.

[0097] The "elder," "callus," "callus culture," "extract," "composition," "improvement of skin condition," "skin disease," "prevention," and "treatment" are the same as those described above.

[0098] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention. [Effects of the Invention]

[0099] The elder callus culture or its extract according to the present invention inhibits collagen degradation or promotes cell proliferation, and has effects such as skin regeneration, maintaining skin firmness, moisturizing skin, and improving skin wrinkles. When used in combination with epidermal growth factor, it has a greater synergistic effect and can be used as a material for cosmetics, pharmaceuticals, etc. [Brief explanation of the drawings]

[0100] [Figure 1] FIG. 1 is a photograph showing the state of induced elder callus after it had been stabilized on solid medium after undergoing a selection process for good callus. [Figure 2] FIG. 2 shows the results of confirming whether or not elder callus extract has cytotoxicity to human keratinocytes and its cell proliferation effect. [Figure 3] FIG. 3 shows the results of a comparison of the cytotoxicity and cell proliferation effects of EGF on human keratinocytes. [Figure 4] FIG. 4 shows the results of confirming whether or not elder callus extract has cytotoxicity to human fibroblasts and its cell proliferation effect. [Figure 5] FIG. 5 shows the results of confirming whether or not a combination of elder callus extract and epidermal growth factor (EGF) causes cytotoxicity to human keratinocytes and the cell proliferation effect. [Figure 6] FIG. 6 shows the results of confirming whether or not the elder callus extract has cytotoxicity to human fibroblasts and the cell proliferation effect when treated with the combined use of elder callus extract and epidermal growth factor (EGF). [Figure 7] FIG. 7 shows the results of confirming the skin regeneration ability of human keratinocytes when treated with elder callus extract alone or with a combination of elder callus extract and epidermal growth factor (EGF). [Figure 8]Figure 8 shows the results of measuring the mRNA expression levels of matrix metalloproteinases (MMPs) in human fibroblasts when treated with elder callus extract alone or in combination with elder callus extract and epidermal growth factor (EGF). [Figure 9] Figure 9 shows the results of measuring the mRNA expression levels of hyaluronan synthase (HAS) and aquaporin (AQP), which are skin moisturizing factors, when human keratinocytes were treated with elder callus extract alone or with elder callus extract and epidermal growth factor (EGF). DETAILED DESCRIPTION OF THE INVENTION

[0101] The present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0102] Example 1: Elder callus induction and preparation of elder callus extract 1.1. Preparation of plant material Young elder plant tissues, such as leaves and stems, were harvested and washed with running water. They were then sterilized in 70% ethanol (Merck, cat. 603-002-00-5) for 60 seconds, followed by immersion in 2% sodium hypochlorite (Duksan, cat. 7681-52-9) for 7 minutes. The sterilized elder plant tissues were washed five times with sterile distilled water to remove any remaining sterilizing solution. Elder callus was induced using the sterilized tissues as follows.

[0103] 1.2. Elder callus induction and growth conditions The disinfected and washed tissue samples were cut into pieces, scratched, and then seeded on callus induction medium. The cancer cells were then cultured at a temperature ranging from 26 to 28°C. For callus induction, we used basal MS medium (Murashige and Skoog, Duchefa, Cat. M0222.0050) supplemented with picloram (Duchefas, Cat. P0914.0005), 6-benzylaminopurine (BA, Duchefa, Cat. B0904.0001), and 1-naphthaleneacetic acid (NAA, Duchefa, Cat. N0903.0025). The specific medium composition is shown in Table 1.

[0104] [Table 1]

[0105] 1.3. Stabilization and culture of elder callus The induced elder callus was stabilized on solid medium after a successful callus selection process (Figure 1). To grow the elder callus, it was cultured on basal MS medium supplemented with 1 μM and 10 μM of 6-benzylaminopurine (BA) and picloram, which are commonly used for plant callus growth.

[0106] 1.4. Elder callus liquid culture For mass cultivation of elder callus obtained by growth on solid medium, we switched to liquid medium cultivation. To do this, using the optimized cultivation conditions confirmed on solid medium, 100 ml of liquid medium was placed in a 500 ml Erlenmeyer flask, and elder callus equivalent to three well-cultured plates of solid medium was inoculated and cultivated at 25°C and 100 rpm. This cultivation was repeated to increase the callus volume and obtain a sufficient amount of callus.

[0107] 1.5. Preparation of Elder Callus Extract Elder callus extract was prepared from elder callus cultures obtained by liquid culture. The elder callus cultures were washed five times with purified water and then freeze-dried at -80°C to obtain dried elder callus. Five grams of freeze-dried elder callus was placed in 2 L of purified water and extracted with hot water at 100°C for two hours to produce elder callus extract. For experiments with precise concentrations, the extract was freeze-dried again to prepare callus extract powder.

[0108] (Example 2) Confirmation of safety and cell proliferation effect of elder callus extract on human keratinocytes 2.1. Culturing human keratinocytes Prior to the safety test, HaCaT, a cell line of keratinocytes, which are cells that make up human skin, was cultured in a 5% CO2 incubator at 37°C. The culture volume, which corresponds to 85-90% of the surface area of ​​the culture vessel, was measured by trypsin treatment to detach the cells and count them. 3 cells / cm 2 Subculture was carried out.

[0109] The cells were cultured in DMEM (Delbecco's Modified Eagle Medium, GIBCO) medium supplemented with 10% FBS (GIBCO), 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0110] 2.2. Safety and cell proliferation effects on human keratinocytes To determine whether the elder callus extract of the present invention can provide an ameliorative effect without skin irritation even after long-term use, its cytotoxicity to human keratinocytes was tested. Furthermore, to determine the concentration of epidermal growth factor (EGF) used during combined treatment with elderflower extract and elder extract, which serve as experimental groups for comparing the effects of elder callus extract, the cytotoxicity of elderflower extract and EGF to human keratinocytes was also tested.

[0111] 5 x 10 human keratinocytes in a 24-well plate 3 Cells were counted using a hemocytometer at the same time per well and then dispensed. After 48 hours of culture in DMEM containing 10% FBS, when the culture covered approximately 50% of the surface area of ​​the culture vessel, the culture was replaced with FBS-free DMEM containing elder callus extract or elderflower extract and further cultured for 24 hours. After incubation, 50 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma M5655) solution (2.5 mg / ml) was added and the culture was continued for another 3 hours. The cell culture medium was then discarded, and 200 μl of DMSO (dimethyl sulfoxide, Sigma D2650) was added to each well. After vortexing, 100 μl of each well was removed and the absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA). The degree of cytotoxicity was expressed as a percentage based on the absorbance intensity of the control group using pure water.

[0112] As a result, it was confirmed that the elder callus extract of the present invention was not cytotoxic at all concentrations tested on human keratinocytes. Furthermore, when callus was induced from elder and the elder callus extract was used, it was confirmed that a significantly superior cell proliferation effect was observed compared to elderflower extract (Figure 2). Furthermore, when EGF was used in combination treatment, it was confirmed that the EGF treatment concentration of 0.01 to 1 ppm did not show cytotoxicity to human keratinocytes, and that a cell proliferation effect was particularly observed at 0.5 ppm compared to the untreated group (Figure 3).

[0113] From the above results, it was confirmed that the elder callus extract of the present invention does not induce skin irritation due to cytotoxicity even when used for a long period of time, and at the same time, it has the effect of inducing human keratinocyte proliferation compared to elderflower extract.

[0114] (Example 3) Confirmation of safety and cell proliferation effect of elder callus extract on human fibroblasts 3.1. Cultivation of human fibroblasts Prior to the safety test, human fibroblast cells CCD-986sk were cultured at 37°C in a 5% CO2 incubator. The culture volume was 85-90% of the surface area of ​​the culture vessel. After detaching the cells with trypsin and counting, 5 x 10 3 cells / cm 2 The cells were cultured in Delbecco's Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% FBS (GIBCO), 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0115] 3.2. Safety and cell proliferation effects on human fibroblasts 5 x 10 CCD-986sk cells in a 24-well plate 3 Cells were counted at a cell / well ratio using a hemocytometer and then aliquoted for further culture. After culturing in DMEM containing 10% FBS for 48 hours, the cells were cultured until they covered approximately 50% of the surface area of ​​the culture vessel. The culture was then replaced with FBS-free DMEM containing elder callus extract or elderflower extract and cultured for an additional 24 hours. After further culturing, 50 μl of 2.5 mg / ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma M5655, USA) was added and the cells were cultured for an additional 3 hours. The cell culture medium was then discarded, and 200 μl of dimethyl sulfoxide (DMSO) (Sigma D2650, USA) was added to each well. After vortexing, 100 μl of each well was aliquoted and the absorbance at 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) at 570 nm. For relative evaluation, the degree of cytotoxicity was expressed as a percentage based on the absorbance intensity of the control group using FBS-free DMEM containing no extract.

[0116] As a result of the experiment, it was confirmed that the elder callus extract of the present invention has no cytotoxicity to human fibroblasts and exhibits a more potent proliferation effect on human fibroblasts than treatment with elderflower extract (Figure 4).

[0117] Based on the results of the cell safety tests in Examples 2 and 3, the concentrations of elderflower extract or elder callus extract used in combination with EGF were selected to allow comparison of synergistic effects without affecting the experiment, and were set at 500 ppm for elderflower extract or elder callus extract and 0.5 ppm for EGF.

[0118] Example 4: Confirmation of the cell safety and synergistic effect of a mixture of elder callus extract and EGF on cell proliferation The cytotoxicity and cell proliferation of a mixture of the elder callus extract of the present invention and epidermal growth factor (EGF) on human keratinocytes and human fibroblasts were evaluated. The test method for human keratinocytes was the same as in Example 2, and the test method for human fibroblasts was the same as in Example 3.

[0119] The results of the experiment showed that the combined treatment of elder callus extract and EGF was more effective than either extract alone or the combined treatment of elderflower extract and EGF. Specifically, when human keratinocytes were treated with elder callus extract and EGF, the cell viability rate was 132.71% compared to the untreated group, demonstrating significant benefits to cell proliferation. In contrast, the combined treatment of elderflower extract and EGF was 109.51%, indicating that the cell proliferation rate was at least 20% lower than when treated with callus extract and EGF (Figure 5).

[0120] Furthermore, when human fibroblasts were treated with elder callus extract and EGF in combination, the cell viability was confirmed to be 116.90% compared to the untreated group, and was also confirmed to be higher than when treated with elderflower extract and EGF in combination (104.19%) (Figure 6).

[0121] (Example 5) Synergistic effect of a mixture of elder callus extract and EGF on skin regeneration The extent of wound regeneration was investigated as one of the main physiological mechanisms of the skin regeneration and skin soothing cosmetic compositions. The skin regeneration activity of the elder callus extract of the present invention and the mixture of elder callus extract and EGF was evaluated as follows.

[0122] HaCaT, a keratinocyte cell line, was placed in a 6-well plate at 1 x 10 4 Keratinocytes were aliquoted at 100 cells / well and cultured in 10% FBS / DMEM medium for 48 hours to form a monolayer. The medium was then removed, and a cell-free zone (scratch wound) was created in each well using a wound marker. Each well was then washed three times with 1 ml of phosphate buffer (pH 7.4). 3 ml of elder callus extract, elderflower extract, EGF, and extract and EGF mixture diluted in FBS-free DMEM medium were added to each well and cultured in a CO2 incubator for 24 hours. The regenerative (healing) ability of the elder callus extract, elderflower extract, EGF, and extract and EGF mixture was measured, with a value of 100 for the control group treated with FBS-free DMEM medium alone. Regenerative ability was measured by comparing the wound area with that of the untreated group under a microscope after cell staining.

[0123] In skin regeneration tests, the elderflower callus extract of the present invention showed a skin regeneration effect in human keratinocytes compared to the untreated group.Furthermore, when the skin regeneration ability of human keratinocytes treated with a mixture of elderflower callus extract and EGF was examined, it was confirmed that the skin regeneration ability was significantly superior to that of treatment with each sample alone or treatment with elderflower extract and EGF in combination (Figure 7).

[0124] (Example 6) Synergistic effect of a mixture of elder callus extract and EGF on collagen degradation inhibition 6.1. Experimental Method 6.1.1. Human Fibroblast Culture and Sample Processing To measure the mRNA expression level of MMPs genes involved in collagen degradation in human fibroblasts, human dermal fibroblasts were first cultured. CCD-986sk human dermal fibroblast cells were cultured at 2 × 10 4 The cells were dispensed at 100 cells / well and cultured in 10% FBS-containing DMEM medium, covering approximately 70% or less of the surface area of ​​the culture vessel. Each well was then treated with elder callus extract, elderflower extract, EGF, and the extracts and EGF diluted in FBS-free DMEM.

[0125] 6.1.2. RNA extraction and cDNA synthesis After 24 hours of sample treatment, the samples were washed with phosphate buffered saline (PBS) and 1 mL of Trizol reagent was added to extract RNA. Prime Script RT Master Mix (×5) (Takara Bio, Japan) was added to the extracted RNA and incubated at 37°C for 15 minutes to synthesize cDNA, after which RT-PCR was performed.

[0126] RT-PCR For RT-PCR, 2 μl of each primer was added to 1 μl of synthesized cDNA, and 25 μl of EmeraldAmp GT PCR Master Mix (Takara Bio, Japan) and 20 μl of tertiary sterilized water were added and mixed well. Denaturation was performed at 95°C for 30 seconds, followed by extension at 72°C for 1 minute, for a total of 30 cycles. Annealing was performed at the optimal temperature for each primer.

[0127] The MMP-degrading enzymes identified in this experiment were MMP1, MMP2, and MMP9. The primer sequences for the MMPs used are shown in Table 2. The PCR products were electrophoresed on a 1.2% agarose gel to check for the presence of primer dimers and whether the PCR product sizes matched. The amount of product was quantitatively analyzed based on band density using the Image J program (National Institutes of Health, USA).

[0128] [Table 2]

[0129] 6.2. Confirmation of synergistic effect of mixture of elder callus extract and EGF on collagen degradation inhibition The stronger the inhibitory effect on the expression of collagen-degrading enzymes, MMPs, the better the anti-wrinkle effect. Based on this, when the mRNA expression levels of MMPs were measured after treatment with the elder callus extract of the present invention, it was confirmed that the mRNA expression level of MMP2 was particularly reduced in the elder callus extract-treated group compared to the untreated group. Furthermore, the mRNA expression level of MMP in human fibroblasts was reduced when treated with the mixture of elder callus extract and EGF compared to each of the individual substances. This confirmed that the synergistic effect on collagen degradation inhibition was superior to that of treatment with a mixture of elderflower extract and EGF (Figure 8).

[0130] From the above results, it was confirmed that the mixture of elder callus extract and EGF of the present invention inhibits collagenase in human fibroblasts, thereby having an anti-wrinkle effect.

[0131] (Example 7) Synergistic effect of a mixture of elder callus extract and EGF on skin moisturizing 7.1. Experimental Method 7.1.1. Human Keratinocyte Cultures and Sample Processing To measure the effect of the mixture of elder callus extract and EGF of the present invention on the expression of skin moisturizing factors, hyaluronic acid synthase (HAS) and aquaporin (AQP), in human keratinocytes at the mRNA level, a test was conducted to measure the expression level of moisturizing factors at the mRNA level. First, 1 × 10 human keratinocytes were placed in a 6-well plate. 4 The cells were dispensed at a rate of 100 cells / well, and when they were cultured in 10% FBS / DMEM medium covering approximately 70% or less of the surface area of ​​the culture vessel, each well was treated with the extract, EGF, or a mixture of the extract and EGF dissolved in FBS-free DMEM as a solvent.

[0132] 7.1.2. RNA extraction and cDNA synthesis After 24 hours of sample treatment, the samples were washed with phosphate buffered saline (PBS) and 1 mL of Trizol reagent was added to extract RNA. Prime Script RT Master Mix (5x) (Takara Bio, Japan) was added to the extracted RNA and incubated at 37°C for 15 minutes to synthesize cDNA, after which RT-PCR was performed.

[0133] RT-PCR For RT-PCR, 2 μl of each primer was added to 1 μl of synthesized cDNA, and the mixture was thoroughly mixed with 25 μl of EmeraldAmp GT PCR Master Mix (Takara Bio, Japan) and 20 μl of tertiary sterilized water. Denaturation was performed at 95°C for 30 seconds, followed by extension at 72°C for 1 minute, for a total of 30 cycles. Annealing was performed at the optimal temperature for each primer. The primer sequences for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HAS-1, HAS-2, HAS-3, and AQP-3 used in this experiment are listed in Table 3. PCR products were examined for nonspecific PCR products and primer dimers on a 1.2% agarose gel. Product amounts were quantified based on band density using the Image J program (National Institutes of Health, USA).

[0134] [Table 3]

[0135] 7.2. Confirmation of the skin moisturizing synergistic effect of a mixture of elder callus extract and EGF Measurement of the mRNA expression levels of skin moisturizing factors confirmed that treatment with elder callus extract increased the mRNA expression levels of HAS-1, HAS-2, HAS-3, and AQP-3 compared to the untreated group. Treatment with elder callus extract showed higher mRNA expression levels of moisturizing factors than treatment with elderflower extract, confirming the superior moisturizing effect of elder callus extract. Furthermore, compared to treatment with elderflower extract and EGF, combined treatment with the elder callus extract of the present invention increased the expression levels of moisturizing factors in human keratinocytes, confirming that the mixture of elder callus extract of the present invention and EGF has a synergistic effect on moisturizing factors (Figure 9).

[0136] Although specific portions of the present invention have been described in detail above, it is clear to those skilled in the art that such specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention should be defined by the appended claims and their equivalents.

Claims

1. A cosmetic composition for improving skin conditions, comprising an elder callus culture or an extract thereof as an active ingredient.

2. 2. The cosmetic composition for improving skin condition according to claim 1, wherein the improvement of the skin condition is improvement of wrinkles, maintenance of skin firmness, moisturizing of skin, regeneration of skin, prevention of skin aging, or improvement of scars.

3. The cosmetic composition for improving skin conditions according to claim 1, wherein the composition inhibits collagen degradation.

4. 2. The cosmetic composition for improving skin conditions according to claim 1, wherein the composition inhibits the activity of matrix metalloproteinases.

5. 2. The cosmetic composition for improving skin condition according to claim 1, wherein the composition increases the expression of hyaluronan synthase or aquaporin in keratinocytes.

6. The cosmetic composition for improving skin conditions according to claim 1, further comprising an epidermal growth factor.

7. A quasi-drug composition for preventing or improving skin diseases, comprising an elder callus culture or an extract thereof as an active ingredient.

8. The quasi-drug composition according to claim 7, wherein the skin disease is one or more selected from the group consisting of skin wounds, skin scars, and skin pigmentation.

9. A pharmaceutical composition for preventing or treating skin diseases, comprising an elder callus culture or an extract thereof as an active ingredient.

10. The pharmaceutical composition according to claim 9, wherein the skin disease is one or more selected from the group consisting of skin wounds, skin scars, and skin pigmentation.

11. A method for preparing a composition for improving skin conditions containing an elder callus culture or an extract thereof, comprising the following steps: (a) inducing and culturing elder callus from elder plant tissue to prepare an elder callus culture; (b) preparing an elder callus extract from the elder callus culture; and (c) preparing a composition containing the elder callus culture or an extract thereof.

12. 12. The method of claim 11, wherein step (a) comprises collecting and sterilizing young elder leaves, roots, flowers, fruits or stems.

13. 12. The method according to claim 11, wherein the step (a) involves inducing elder callus from a medium containing picloram, 1-naphthaleneacetic acid, or 6-benzylaminopurine.

14. The method according to claim 11, wherein the step (b) comprises drying the elder callus culture obtained in the step (a) to prepare a dried elder callus material, and mixing the dried material with purified water to prepare an extract.

15. The method according to claim 14, wherein the step of preparing an extract by mixing the dried material with purified water includes a step of preparing an extract by ultrasonic extraction or hot water extraction.

16. A method for improving skin conditions, comprising the step of applying a composition containing an elder callus culture or an extract thereof as an active ingredient to the skin or mucosa of an individual.

17. A method for preventing or treating a skin disease, comprising the step of administering to an individual a composition containing an elder callus culture or an extract thereof as an active ingredient.

18. 1. Use of a composition comprising an elder callus culture or an extract thereof as an active ingredient for improving skin conditions.

19. 1. Use of a composition comprising an elder callus culture or an extract thereof as an active ingredient for the prevention or treatment of skin diseases.

20. 1. Use of a composition comprising an elder callus culture or an extract thereof as an active ingredient for the preparation of a medicament for the prevention or treatment of skin diseases.

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