Skin condition improvement composition comprising glutathione-containing exosome-like vesicles

Abstract

The present invention relates to a skin condition improvement composition comprising glutathione-containing exosome-like vesicles. The glutathione-containing exosome-like vesicles in which glutathione, which is a skin useful substance, is loaded in exosome-like vesicles isolated from plant callus, have an anti-inflammatory effect superior to that of the application of glutathione alone, and thus the composition according to one aspect can be effectively used as a skin condition improvement composition.

Description

Composition for improving skin condition comprising glutathione-containing exosome-like vesicles

[0001] The present invention relates to a composition for improving skin condition comprising glutathione-containing exosome-like vesicles.

[0002] Glutathione, a tripeptide containing glutamate, cysteine, and glycine, is known as a valuable bionutrient with powerful antioxidant properties. Consequently, glutathione is used as a raw material in health functional foods, pharmaceuticals, and cosmetics. However, despite its usefulness, glutathione has a very low ability to penetrate cell membranes, making it difficult to absorb.

[0003] Exosomes are extracellular vesicles (EVs) measuring tens to hundreds of nanometers in size and composed of a phospholipid bilayer membrane identical in structure to the cell membrane. Exosomes are intercellular signaling mediators secreted by cells and are known to regulate various cellular behaviors, and active research is being conducted on their components and functions. Meanwhile, plant callus is a mass of cells formed to regenerate wounded plant tissues. This callus has the potential to differentiate into entire plants through treatment with plant growth regulators, and is therefore also called plant stem cells.

[0004] Recently, research is being conducted to use exosomes derived from these plant calli as cosmetic materials (Korean Patent Publication No. 10-2023-0052827), but attempts to use them as a carrier that can stably retain glutathione are minimal.

[0005] One aspect is to provide a cosmetic composition for improving skin condition, comprising exosome-like vesicles containing glutathione.

[0006] Another aspect is to provide a composition for external application of skin for improving skin condition, comprising exosome-like vesicles containing glutathione.

[0007] Another aspect is to provide a health functional food composition for improving skin condition, which comprises an exosome-like vesicle containing glutathione.

[0008] Another aspect is to provide a pharmaceutical composition for preventing or treating an inflammatory disease comprising an exosome-like vesicle containing glutathione.

[0009] Another aspect provides the use of exosome-like vesicles containing glutathione for the preparation of a cosmetic composition for improving skin condition.

[0010] Another aspect provides the use of exosome-like vesicles containing glutathione for the preparation of a topical skin composition for improving skin condition.

[0011] Another aspect provides the use of exosome-like vesicles containing glutathione for the preparation of a health functional food composition for improving skin condition.

[0012] Another aspect provides the use of exosome-like vesicles containing glutathione for the manufacture of a medicament for the prevention or treatment of inflammatory diseases.

[0013] Another aspect provides a method of improving, preventing, or treating a skin condition comprising administering to a subject in need thereof an effective amount of exosome-like vesicles containing glutathione.

[0014] One aspect provides a cosmetic composition for improving skin condition, comprising exosome-like vesicles containing glutathione.

[0015] The term “Glutathione” refers to a tripeptide containing glutamate, cysteine, and glycine, and is generally reported to be a physiologically active substance with strong antioxidant activity.

[0016] The term “extracellular vesicle (EV)” refers to vesicles secreted from cells and released into the extracellular space, and may have a membrane structure in which the inside and the outside are separated by a phospholipid bilayer membrane identical to the structure of the cell membrane, and may contain genetic materials such as cytokines, growth factors, miRNAs, DNA, proteins, and bioactive factors inside. The extracellular vesicles may act as extracellular messengers that bind to other cells to deliver membrane components, mRNAs, miRNAs, etc., and deliver these messengers to recipient cells, thereby mediating cell-to-cell communication. In an exemplary embodiment, the extracellular vesicles may include vesicles formed when cells burst due to turgor pressure to capture active ingredients inside, or vesicles in which the membrane or the composition of active ingredients inside is reconstituted when multivesicles burst due to turgor pressure.

[0017] The term “exosome-like vesicle (ELV)” may be used interchangeably with the term “exosome-like nanovesicle”, which refers to an extracellular vesicle (EV) of tens to hundreds of nanometers in size, and is the broadest concept that includes not only exosomes but also vesicles whose nano-sized vesicle structure and composition are similar to exosomes. In an exemplary embodiment, the extracellular vesicle may be an exosome-like vesicle.

[0018] In one specific example, the exosome-like vesicles may be plant-derived exosome-like vesicles, for example, plant callus-derived exosome-like vesicles.

[0019] The term "plant" may refer to a whole plant or to a part of a plant, specifically including plant tissue, plant cells, and plant seeds.

[0020] In one specific example, the plant may be any one part selected from the group consisting of a flower, leaf, stem, branch, fruit, fruit peel, and root of the plant.

[0021] In one specific example, the plant may be one or more selected from the group consisting of apple, grape, desert rose, purslane, and edelweiss.

[0022] The term “callus” refers to an unorganized mass of plant cells, which means cells with totipotent potential, and may include somatic embryos and plant stem cells, and may also be induced by transferring a part of a plant tissue to a tissue culture medium and culturing it, and may be induced from any one part selected from the group consisting of a flower, leaf, stem, branch, fruit, fruit peel, and root of a plant. The induction of the callus may be performed through a conventional method known in the art. More specifically, the induction of the callus may be performed by culturing a plant tissue or a fragment thereof in a tissue culture medium. As the above tissue culture medium, various basic plant media can be used, such as MS (Murashige and Skoog, 1962) medium, SH Medium (Duchefa, Haarlem, Netherlands), N6 (Chu et al., 1975), B5 (Gamborg et al., 1968), NN (Nitsch andNitsch, 1967) medium, and WHITE medium. In addition, the tissue culture medium may be used with a plant growth regulator added.The plant growth regulator may use a substance known in the art, and for example, auxins such as 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), Dicamba (2-methoxy-3,6-dichlorobenzoic acid), IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), MCPA (2-methyl-4-chlorophenoxyacetic acid), NAA (1-naphthylacetic acid), NOA (2-naphthyloxyacetic acid), and Picloram (4-amino-2,5,6-trichloropicolinic acid); Cytokinins such as BA (6-benzylaminopurine), 2iP (N6-(2-isopentyl)adenine), Kinetin (6-furfurylaminopurine), Thidiazuron (1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea), Zeatin (4-hydroxy-3-methyl-trans-2-butenylaminopurine), and derivatives thereof may be included. In addition, alkaloids, terpenoids, carotenoids, carbohydrates, phenol compounds, and natural extracts that act on cell differentiation and proliferation may be additionally added to tissue culture media.

[0023] In one specific example, the plant callus may be cultured in an MS medium containing NAA (1-naphthylacetic acid) and BA (6-Benzylaminopurine).

[0024] In one specific example, the plant callus may be derived from any one part selected from the group consisting of a flower, leaf, stem, branch, fruit, fruit peel, and root of the plant.

[0025] In one specific example, the exosome-like vesicles may be isolated from a lysate of the plant. Specifically, the exosome-like vesicles may be isolated from a lysate of the plant callus. In one specific example, the plant callus lysate may be a lysate of a mixed plant callus culture. In another specific example, the plant callus lysate may be a lysate of a callus recovered by removing a culture supernatant from a mixed plant callus culture. In yet another specific example, the plant callus lysate may be a lysate of a culture supernatant recovered by removing callus from a mixed callus culture. A standard sieve and mesh net can be used to separate callus or culture supernatant from the above plant callus mixed culture, and the pore size may be appropriately 100 ㎛ to 200 ㎛, but is not limited thereto, and a conventional method used in the art to separate callus and culture supernatant, such as a centrifuge, a filter device, or a qualitative filter paper, can be used.

[0026] The term “callus mixed culture” may refer to the entire medium including callus, its metabolites, and extra nutrients, etc., obtained by culturing callus for a certain period of time in a medium that can supply nutrients so that callus can grow and survive in vitro. For example, it may be obtained by culturing callus in a medium at a temperature of more than 10°C or less than 40°C for a certain period of time, for example, 1 to 40 days. Those skilled in the art may appropriately select or modify the culture medium and culture conditions for culturing the callus.

[0027] The term “culture supernatant” can mean only the liquid in the upper layer after leaving the culture solution still for a certain period of time, excluding the portion that has settled to the lower layer, or can mean only the liquid in the upper layer after centrifuging the culture solution, excluding the sediment at the bottom.

[0028] The term “crusted material” may refer to a product obtained by crushing with physical force. In one specific example, the crushed material may be crushed using pressure (turgor pressure), and may be, for example, a crushed material obtained using a high-pressure homogenizer. When glutathione is added during the process of physically obtaining a crushed material of callus by applying pressure to the plant callus using a high-pressure homogenizer, glutathione can be induced to be encapsulated within exosome-like vesicles during the crushing process, and thus, the exosome-like vesicles isolated from the crushed material of plant callus can be usefully used as a carrier encapsulating glutathione therein.

[0029] The term “glutathione-containing” may be used interchangeably with the terms “glutathione-encapsulated,” “glutathione-loaded,” or “glutathione-encapsulated.”

[0030] In one specific example, the skin condition improvement may be anti-inflammatory.

[0031] The term "anti-inflammatory" may be used interchangeably with "inhibiting or improving inflammation" and may refer to any action that alleviates an inflammatory condition.

[0032] The term "improvement" may mean any action that reduces at least the severity of a symptom, for example, a parameter related to the alleviation or treatment of a condition.

[0033] In one specific example, the exosome-like vesicles may be separated by ultrafiltration with a MWCO (molecular weight cutoff) filter of 20 kDa to 500 kDa. Specifically, the exosome-like vesicles may be separated by ultrafiltration from a high-pressure homogenized product of plant callus, and more specifically, may be separated by ultrafiltration from a sample obtained by centrifuging a high-pressure homogenized product of a mixed culture of plant callus to remove cell-related debris and filtering the obtained supernatant through a 0.45 μm filter.

[0034] In one specific example, the exosome-like vesicles may have a diameter of 20 to 400 nm. For example, 20 nm to 350 nm, 20 nm to 300 nm, 20 nm to 250 nm, 20 nm to 200 nm, 50 nm to 400 nm, 50 nm to 350 nm, 50 nm to 300 nm, 50 nm to 250 nm, 50 nm to 200 nm, 80 nm to 400 nm, 80 nm to 350 nm, 80 nm to 300 nm, 80 nm to 250 nm, 80 nm to 200 nm, 80 nm to 150 nm, 80 nm to 130 nm, 80 nm to 120 nm, 80 nm to 100 nm, 100 nm to 400 nm, 100 nm to 350 nm, 100 nm to 300 nm, 100 nm to 250 nm, 100 nm to 200 nm, 100 nm to 150 nm, 100 nm to 130 nm, 100 nm to 120 nm, 150 nm to 400 nm, 150 nm to 350 nm, 150 nm to 300 nm, 150 nm to 250 nm, 150 nm to 200 nm, 200 nm to 400 nm, 200 nm to 350 nm, 200 nm to 300 nm, 200 nm to 250 nm, 250 nm to 400 nm, 250 nm to 350 nm, 250 nm to 300 nm, 300 nm to 400 nm, or 300 nm to 350 nm.

[0035] In one specific example, the glutathione may be contained in an amount of 0.00001-50 wt%, for example, 0.00001-40 wt%, 0.00001-30 wt%, or 0.00001-20 wt%, based on the total weight of the exosome-like vesicles. If the glutathione content is less than the above content, it is difficult to exhibit a significant level of effect, and if the content exceeds the above content, problems may arise with exosome stability.

[0036] The composition is present in an amount of 0.000000001 wt% to 80 wt%, for example, 0.000000001 wt% to 60 wt%, 0.000000001 wt% to 40 wt%, 0.000000001 wt% to 30 wt%, 0.000000001 wt% to 20 wt%, 0.000000001 wt% to 10 wt%, 0.000000001 wt% to 5 wt%, 0.000000001 wt% to 0.001 wt%, 0.000000001 wt% to 0.0001 wt%, 0.000000001 wt% to 0.0001 wt%, 0.0001 wt% to 80 wt%, 0.0001 wt% to 60 wt%, 0.0001 wt% to 40 wt%, 0.0001 wt% to 30 wt%, 0.0001 wt% to 20 wt%, 0.0001 wt% to 10 wt%, 0.0001 wt% to 5 wt%, 0.001 wt% to 80 wt%, 0.001 wt% to 60 wt%, 0.001 wt% to 40 wt%, 0.001 wt% to 30 wt%, 0.001 wt% to 20 wt%, 0.001 wt% to 10 wt%, 0.001 wt% to 5 wt%, 0.01 wt% to 80 wt%, 0.01 wt% to 60 wt%, 0.01 wt% to 40 The exosome-like vesicles may comprise glutathione-containing exosomes in an amount of 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.1 wt% to 80 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt%.

[0037] In one specific example, the composition comprises 10 glutathione-containing exosome-like vesicles. 310 to 100 pcs / ml 12 dog / ml, for example, 10 3 10 to 100 pcs / ml 11 dog / ml, 10 3 10 to 100 pcs / ml 10 dog / ml, 10 3 10 to 100 pcs / ml 9 dog / ml, 10 3 10 to 100 pcs / ml 8 dog / ml, 10 3 10 to 100 pcs / ml 7 dog / ml, 10 3 10 to 100 pcs / ml 6 dog / ml, 10 3 10 to 100 pcs / ml 5 dog / ml, 10 3 10 to 100 pcs / ml 4 dog / ml, 10 4 10 to 100 pcs / ml 11 dog / ml, 10 4 10 to 100 pcs / ml 10 dog / ml, 10 4 10 to 100 pcs / ml 9 dog / ml, 10 4 10 to 100 pcs / ml 8 dog / ml, 10 4 10 to 100 pcs / ml 7 dog / ml, 10 4 10 to 100 pcs / ml 6 dog / ml, 10 4 10 to 100 pcs / ml 5 dog / ml, 10 5 10 to 100 pcs / ml 11 dog / ml, 10 5 10 to 100 pcs / ml 10 dog / ml, 10 5 10 to 100 pcs / ml 9 dog / ml, 10 5 10 to 100 pcs / ml 8 dog / ml, 10 5 10 to 100 pcs / ml 7 dog / ml, 10 5 10 to 100 pcs / ml 6 dog / ml, 10 6 10 to 100 pcs / ml 12dog / ml, 10 6 10 to 100 pcs / ml 11 dog / ml, 10 6 10 to 100 pcs / ml 10 dog / ml, 10 6 10 to 100 pcs / ml 9 dog / ml, 10 6 10 to 100 pcs / ml 8 dog / ml, 10 6 10 to 100 pcs / ml 7 dog / ml, 10 7 10 to 100 pcs / ml 12 dog / ml, 10 7 10 to 100 pcs / ml 11 dog / ml, 10 7 10 to 100 pcs / ml 10 dog / ml, 10 7 10 to 100 pcs / ml 9 dog / ml, 10 7 10 to 100 pcs / ml 8 dog / ml, 10 8 10 to 100 pcs / ml 12 dog / ml, 10 8 10 to 100 pcs / ml 11 dog / ml, 10 8 10 to 100 pcs / ml 10 dog / ml, 10 8 10 to 100 pcs / ml 9 dog / ml, 10 9 10 to 100 pcs / ml 12 dog / ml, 10 9 10 to 100 pcs / ml 11 dog / ml, 10 9 10 to 100 pcs / ml 10 dog / ml, 10 10 10 to 100 pcs / ml 12 dog / ml, 10 10 10 to 100 pcs / ml 11 dog / ml, 10 11 10 to 100 pcs / ml 12It may be included at a concentration of 100 mg / ml. If the glutathione-containing exosome-like vesicles are included in the composition in an amount less than the above range, a significant skin condition improvement effect may not be observed, and if they are included in an amount exceeding the above range, the formulation stability may be reduced.

[0038] The above cosmetic composition can be prepared in a formulation including a toner (skin lotion), skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, eye cream, hand cream, foundation, essence, nourishing essence, eye essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cream, body cleanser, suspension, gel, powder, paste, mask pack, or sheet or aerosol composition. A composition in such a formulation can be prepared according to a method conventional in the art.

[0039] The ingredients included in the above cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the composition as an active ingredient, and may include, for example, conventional auxiliary agents and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0040]

[0041] Another aspect provides a composition for external application for skin improvement comprising exosome-like vesicles containing glutathione. In the composition for external application for skin, glutathione, exosome-like vesicles, skin condition improvement, and the composition are as described above.

[0042] In one specific example, the exosome-like vesicles may be plant-derived exosome-like vesicles, for example, plant callus-derived exosome-like vesicles. Plants, callus, etc. are as described above.

[0043] The above-mentioned external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The above-mentioned external preparation for skin may be appropriately mixed with ingredients commonly used in external preparations for skin such as cosmetics or medicines, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or a combination thereof, as needed. The above skin external preparation may also appropriately contain metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glabridin, hot water extract of the fruit of Calin; various herbal medicines; tocopheryl acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof; and sugars such as vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0044]

[0045] Another aspect provides a health functional food composition for improving skin condition, comprising exosome-like vesicles containing glutathione. In the health functional food composition, glutathione, exosome-like vesicles, skin condition improvement, and the composition are as described above.

[0046] In one specific example, the exosome-like vesicles may be plant-derived exosome-like vesicles, for example, plant callus-derived exosome-like vesicles. Plants, callus, etc. are as described above.

[0047] The above health functional food composition can be used alone or in combination with other foods or food ingredients, including the exosome-like vesicles containing the glutathione, and can be used appropriately according to a conventional method. The amount of the active ingredient mixed can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present specification can be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the type of the health functional food. Among the types of health functional foods, the beverage composition can contain various flavorings or natural carbohydrates as additional ingredients, like a conventional beverage. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used. The health food composition may also contain nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, or a combination thereof. The health functional food composition may also contain fruit pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, or a combination thereof.

[0048]

[0049] Another aspect provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising exosome-like vesicles containing glutathione. In the pharmaceutical composition, glutathione, exosome-like vesicles, wounds, and the composition are as described above.

[0050] In one specific example, the exosome-like vesicles may be plant-derived exosome-like vesicles, for example, plant callus-derived exosome-like vesicles. Plants, callus, etc. are as described above.

[0051] The above "inflammatory disease" may be an inflammatory skin disease, an allergic inflammatory disease, an inflammatory eye disease, an inflammatory osteoarthritis, an inflammatory muscle disease, or an inflammatory bowel disease, and the above "inflammatory skin disease" may be any one selected from the group consisting of atopic dermatitis, psoriasis, contact dermatitis, eczematous dermatitis, actinic dermatitis, seborrheic dermatitis, dermatitis herpetiformis, lichen planus, lichen sclerosus, pyoderma gangrenosum, pemphigus, epidermolysis bullosa, allergy, and hypersensitivity.

[0052] The term "prevention" can comprehensively refer to preventing a disease in advance or reducing the likelihood or frequency of occurrence by administering the composition in a pharmaceutically effective amount. For example, it can reduce the probability of developing a disease or the probability of recurrence in a patient who is likely to develop the disease or has previously developed the disease. The "pharmaceutically effective amount" can be readily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the route of administration, the method of administration, the number of administrations, the treatment period, the combination, or concurrently used drugs.

[0053] The term "treatment" may comprehensively refer to improving a disease by administering the composition in a pharmaceutically effective amount, may provide relief or cure of the symptoms of the disease in a shorter period of time compared to natural healing, and may improve one or most symptoms caused by the disease. The pharmaceutically effective amount is as described above.

[0054] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a glidant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium dihydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0055]

[0056] Another aspect provides the use of exosome-like vesicles containing glutathione for the preparation of cosmetic compositions for improving skin condition. In the above use, overlapping details, such as exosome-like vesicles containing glutathione, skin condition improvement, and cosmetic compositions, are as described above.

[0057] Another aspect provides the use of exosome-like vesicles containing glutathione for the preparation of topical skin compositions for improving skin condition. In the above use, overlapping details, such as exosome-like vesicles containing glutathione, skin condition improvement, and topical skin compositions, are as described above.

[0058] Another aspect provides the use of exosome-like vesicles containing glutathione for the manufacture of a health functional food composition for improving skin condition. In this use, overlapping details, such as exosome-like vesicles containing glutathione, skin condition improvement, and skin topical compositions, are as described above.

[0059] Another aspect provides the use of exosome-like vesicles containing glutathione for the manufacture of a medicament for the prevention or treatment of inflammatory diseases. In this use, overlapping content regarding exosome-like vesicles containing glutathione, inflammatory diseases, prevention, treatment, and medicament is as described above.

[0060] Another aspect provides a method for improving, preventing, or treating a skin condition, comprising administering to a subject in need thereof an effective amount of exosome-like vesicles containing glutathione. In the method, overlapping aspects of the exosome-like vesicles containing glutathione, and the improvement, prevention, and treatment of skin conditions are as described above.

[0061] The terms "administering," "introducing," and "implanting" are used interchangeably and may refer to placement of a composition according to one embodiment into a subject by a method or route that results in at least partial localization of the composition to a desired site according to one embodiment.

[0062] Administration may be by any method known in the art. Administration may be administered directly to the subject by any means, including intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. Administration may be systemic or local.

[0063] The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an object in need of improving skin condition, for example, an effect of suppressing skin inflammation.

[0064] The term "effective amount" may mean an amount effective enough to produce the effect mentioned above.

[0065] The above administration may be 0.1 mg to 1,000 mg of the composition according to one specific example per subject per day, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or twice or more within the range of clinically acceptable side effects, and the administration site may be one or more than two sites, and the total number of administration days may be from 1 to 30 days per treatment, daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after an appropriate period. For animals other than humans, the same dosage as for humans per kg may be administered, or the above dosage may be converted into an amount based on the volume ratio (e.g., average value) of the organs (heart, etc.) of the target animal and humans.

[0066] Exosome-like vesicles containing glutathione according to the daily aspect can effectively deliver glutathione into cells, have excellent anti-inflammatory effects, and can be usefully used as a composition for improving skin condition.

[0067] Figure 1 shows the results of confirming the expression level of IL-1β mRNA after treating keratinocytes with glutathione-containing plant callus-derived exosome-like vesicles (Example) or glutathione (Comparative Example 1) at a concentration of 1.0%, respectively.

[0068] Figure 2 shows the results of confirming the expression level of TSLP mRNA after treating keratinocytes with glutathione-containing plant callus-derived exosome-like vesicles (Example) or glutathione (Comparative Example 1) at a concentration of 1.0%, respectively.

[0069] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0070]

[0071] Example. Preparation of exosome-like vesicles derived from plant callus containing glutathione.

[0072] (1) Induction of apple callus

[0073] Apples (Malus domestica) used in the experiment were provided by the Neunggeum Agricultural Cooperative. The apples were washed in running water and sterilized in 70% ethanol for 1 min. After sterilization in 1% sodium hypochlorite for 10 min, they were washed three times with sterilized distilled water. The sterilized fruit tissue was cut into 0.5 cm pieces. 2After cutting to size, they were cultured on MS medium containing 4.5 mg / L NAA (1-naphthylacetic acid) and 2.5 mg / L BA (6-Benzylaminopurine) as growth regulators. The pH of the medium was adjusted to 5.8, and the culture was performed in the dark at an external temperature of 27 ± 3 °C. After that, 4 g of the induced apple callus was inoculated into an Erlenmeyer flask containing 50 mL of a liquid medium with the same medium composition, maintained in the dark, and cultured in a shaker (J-MBB2, JISICO, Republic of Korea) at 90 rpm. The culture was performed for a total of 3 to 4 weeks.

[0074] (2) Preparation of glutathione-containing exosome-like vesicles derived from apple callus

[0075] An isotonic solution was added to the apple callus mixed culture medium cultured in the above Example (1) and pulverized using a homogenizer (Homogenizer, T50D, IKA, Aachen, Germany). Glutathione was added to the obtained pulverized material, and the process of applying a pressure of 1000 bar using a high-pressure homogenizer (Microfludizer, MN600P-300, Picomax, Korea) was repeated three times, so that the cell wall, cell membrane, etc. were crushed by the turgor pressure, and exosome-like vesicles inside the plant cells were separated. At the same time, the separated exosome-like vesicles were ruptured by the turgor pressure, capturing glutathione to form vesicles encapsulating glutathione. The obtained mixed culture lysate was centrifuged at 10,000Хg at 4℃ for 30 minutes to remove cell wall debris and obtain a supernatant. The obtained supernatant was filtered through a 0.45 μm sterilizing filter to further remove residual cell debris. The filtered supernatant was then ultrafiltered (Pellicon® 2 and 3 Mini Holder, Merck, Germany) with a MWCO (molecular weight cutoff) of 100 kDa to isolate and concentrate glutathione-containing exosome-like vesicles with a molecular weight cutoff of 100 kDa or greater. In addition, sterilization filtration was performed through a 0.2 μm sterilizing filter to remove impurities. The finally isolated glutathione-containing exosome-like vesicles were diluted 200-fold and used in the following tests.

[0076]

[0077] Comparative Example 1. Preparation of glutathione aqueous solution

[0078] Glutathione in the same amount as in the above example was dissolved in water to prepare an aqueous solution.

[0079]

[0080] Comparative Example 2. Preparation of glutathione-containing human stem cell-derived exosome-like vesicles.

[0081] Glutathione-encapsulated human stem cell-derived exosome-like vesicles were prepared in the same manner as in the above example, except that human stem cell-derived exosome-like vesicles were used instead of plant callus-derived exosome-like vesicles in the above example.

[0082] Specifically, human adipose-derived stem cells were cultured under 5% CO2, 37°C conditions according to a cell culture method known in the art to which the present invention pertains. Then, after washing with phosphate-buffered saline (purchased from ThermoFisher Scientific), the cells were cultured by replacing the medium with a serum-free, phenol red-free medium. After culturing the human adipose-derived stem cells for 72 hours, the same amount of glutathione as in the example was added to the culture medium, and the process of applying a pressure of 1000 bar using a high-pressure homogenizer (Microfludizer, MN600P-300, Picomax, Korea) was repeated three times to induce the formation of vesicles encapsulating glutathione. This was separated and concentrated into glutathione-containing exosome-like vesicles with a molecular weight cutoff (MWCO) of 100 kDa or more by ultrafiltration (Pellicon® 2 and 3 Mini Holder, Merck, Germany). In addition, sterilization filtration was performed through a 0.2 μm sterilizing filter to remove impurities. The finally separated glutathione-containing exosome-like vesicles were diluted 200-fold and used in the following tests.

[0083]

[0084] Experimental Example 1. Evaluation of anti-inflammatory effect

[0085] In order to confirm the anti-inflammatory effect of the glutathione-containing plant callus-derived exosome-like vesicles prepared in the above example, keratinocytes were treated with the glutathione-containing plant callus-derived exosome-like vesicles of the example, and then the expression levels of the inflammatory cytokines IL-1β (Interleukin-1β) and TSLP (Thymic stromal lymphopoietin) were measured at the mRNA level.

[0086] 1-1. Human keratinocyte culture

[0087] Human keratinocyte HaCaT cells were cultured in 100 mm dishes containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin. Samples were prepared by filtering through a 0.2 μm syringe filter, and each sample was added to the cell culture medium to a final concentration of 1% (v / v). 1 μM dexamethasone, which has an anti-inflammatory effect, was used as a positive control. The culture medium containing the samples was cultured for 24 hours in an incubator maintained at 37°C, 5% CO2 concentration, and 95% humidity, and then treated with immunostimulants, Poly I:C (10 μg / ml) and IL-4 (10 ng / ml), and cultured for 4 hours.

[0088] 1-2. Analysis of expression of inflammatory cytokines

[0089] The cells cultured in Experimental Example 1-1 above were harvested, washed with chilled phosphate buffer solution (PBS), and total RNA was extracted using PureLink® RNA Mini Kit (Thermo Fisher Scientific). The specific RNA extraction was performed according to the manual provided with the product. Gene expression changes were measured using qRT-PCR (quantitative real-time PCR), which binds a fluorescent material to the DNA product amplified in polymerase chain reaction (PCR) and continuously detects the fluorescent material. To quantify the expression of the inflammatory response factor IL-1β and TSLP genes, the fluorescence value emitted by the PCR product was measured using SYBR Green (Thermo Fisher Scientific). A reaction solution was prepared by mixing 0.2 μM primers, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl2, and 1× SYBR Green in a PCR tube. After primary denaturation at 94°C for 3 minutes using a PCR machine, denaturation, annealing, and polymerization were performed for a total of 40 cycles: 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, and the fluorescence intensity was measured after each cycle. The fluorescence intensity of the IL-1β and TSLP genes was standardized to the actin value, converted to 100% of the control group, and the difference was compared to measure the expression level of each gene, and the results are shown in Figures 1 and 2. The primer sequences used in the experiment are as follows in Table 1.

[0090] Gene Forward primer Reverse primer IL-1β 5'-GTCATTCGCTCCCACATTCT-3' (SEQ ID NO: 1) 5'-ACTTCTTGCCCCCTTTGAAT-3' (SEQ ID NO: 2) TSLP 5'-ATGTTCGCCATGAAAACTAAGGC-3' (SEQ ID NO: 3) 5'-GCGACGCCACAATCCTTGTA -3' (SEQ ID NO: 4) Actin 5'-GGCCATCTCTTGCTCGAAGT-3' (SEQ ID NO: 5) 5'-GACACCTTCAACACCCCAGC-3' (SEQ ID NO: 6)

[0091] Fig. 1 shows the results of confirming the expression level of IL-1β mRNA after treating keratinocytes with glutathione-containing plant callus-derived exosome-like vesicles (Example) or glutathione (Comparative Example 1) at a concentration of 1.0%, respectively. Fig. 2 shows the results of confirming the expression level of TSLP mRNA after treating keratinocytes with glutathione-containing plant callus-derived exosome-like vesicles (Example) or glutathione (Comparative Example 1) at a concentration of 1.0%, respectively. As a result, as shown in Figs. 1 and 2, it was confirmed that the expression of inflammatory factors was significantly reduced when glutathione was loaded into plant-derived exosome-like vesicles (Example) and treated compared to when glutathione was treated alone at the same concentration (Comparative Example 1). This means that when glutathione is encapsulated in plant callus-derived exosome-like vesicles and treated, the active ingredient glutathione can be delivered more effectively into cells than when glutathione is treated alone, thereby effectively suppressing inflammation.

[0092]

[0093] Experimental Example 2. Stability Evaluation by Transmitter

[0094] In order to evaluate the stability of glutathione according to the delivery vehicle, the glutathione-containing plant callus-derived exosome-like vesicles of the example, the glutathione-containing human stem cell-derived exosome-like vesicles of Comparative Example 2, and the glutathione aqueous solution of Comparative Example 1 were stored for 1 year under light-protected storage conditions at 25°C, and the concentration of the encapsulated glutathione was measured using high-performance liquid chromatography (HPLC) analysis. Reduced glutathione was used as a standard. Glutathione is a powerful antioxidant that rapidly oxidizes and removes reactive oxygen species when it comes into contact with them, which causes the instability of glutathione itself. Encapsulating glutathione within a structure such as an exosome-like vesicle can improve stability by reducing the opportunity for exposure to reactive oxygen species. Therefore, the stability of glutathione loaded within exosome-like vesicles was confirmed through the degree of oxidation of reduced glutathione.

[0095] Specifically, reduced glutathione (GSH) standard (500 ppm) was dissolved in 3-distilled water in a 50 ml conical tube and then diluted to 500 ppm, 250 ppm, 125 ppm, 62.5 ppm, and 31.25 ppm to prepare. The diluted standard, Comparative Example 1 diluted in 3-distilled water, Example diluted in 3-distilled water, and Comparative Example 2 diluted in 3-distilled water were each filtered with a 0.2 ㎛ syringe filter and placed in an HPLC vial. 10 ul was injected into a C18 column and the values ​​were measured using a DAD detector while flowing at a flow rate of 1 ml per minute. The results are shown in Table 2 below.

[0096] Reduced glutathione (GSH) relative concentration Immediately after loading After 6 months of loading After 1 year of loading Comparative example 11.000.210.06 Comparative example 21.000.810.73 Example 1.000.920.85

[0097] As a result, as shown in Table 2, in the case of glutathione not loaded in exosome-like vesicles (Comparative Example 1), the concentration of glutathione significantly decreased to about 20% of the initial concentration after 6 months, and decreased to about 6% after 1 year. However, in the case of glutathione loaded in exosome-like vesicles (Example and Comparative Example 2), the concentration was maintained at about 70% or more. In particular, when plant callus-derived exosome-like vesicles were used as a glutathione carrier (Example), a very high concentration of about 85% or more was secured, confirming that glutathione was stably maintained for a long period of time. This means that plant callus-derived exosome-like vesicles according to one aspect are effective carriers that can stably retain glutathione inside them for a long period of time.

Claims

1. A cosmetic composition for improving skin condition comprising exosome-like vesicles containing glutathione.

2. A cosmetic composition for improving skin condition according to claim 1, wherein the exosome-like vesicles are plant-derived exosome-like vesicles.

3. A cosmetic composition for improving skin condition according to claim 2, wherein the plant is any one part selected from the group consisting of a flower, a leaf, a stem, a branch, a fruit, a fruit peel, and a root of the plant.

4. A cosmetic composition for improving skin condition according to claim 1, wherein the improvement in skin condition is anti-inflammatory.

5. A cosmetic composition for improving skin condition according to claim 1, wherein the plant-derived exosome-like vesicles are separated from a fragment of the plant.

6. A cosmetic composition for improving skin condition according to claim 1, wherein the exosome-like vesicles are separated by ultrafiltration with a MWCO (molecular weight cutoff) filter of 20 kDa to 500 kDa.

7. A composition for external application of skin for improving skin condition, comprising exosome-like vesicles containing glutathione.

8. A health functional food composition for improving skin condition, comprising an exosome-like vesicle containing glutathione.

9. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising an exosome-like vesicle containing glutathione.

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