A new composition containing exosomes derived from iris as an active ingredient

Abstract

The present invention provides a composition for improving skin elasticity, reducing skin wrinkles, regenerating skin, improving skin tone and brightness, skin whitening, anti-inflammation, wound healing and / or promoting wound healing, which comprises iris-derived exosomes as an active ingredient. The composition of the present invention is less likely to contain impurities such as residual solvents and growth regulators compared to conventional iris hot water extracts and solvent extracts, filtrates of such extracts and iris callus culture solutions, and has excellent effects of improving skin elasticity, reducing skin wrinkles, regenerating skin, improving skin tone and brightness, skin whitening, anti-inflammation, wound healing and / or promoting wound healing.

Description

Technical Field

[0001] The present invention relates to a new composition containing iris-derived exosomes as an active ingredient, and more particularly, to a composition for improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness, skin whitening, anti-inflammation, wound healing and / or promoting wound healing, which contains iris-derived exosomes as an active ingredient.

[0002] In addition, the present invention relates to a cosmetic composition for improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness and / or whitening, which contains the above composition, and a pharmaceutical composition for anti-inflammation, wound healing and / or promoting wound healing.

Background Art

[0003] When skin aging occurs, skin elasticity decreases and skin wrinkles increase. The decrease in skin elasticity and the formation of skin wrinkles are known to occur when the synthesis of collagen decreases and the expression of MMP (matrix metalloproteinase), an enzyme that decomposes collagen, is promoted.

[0004] In addition, in skin cells due to the progression of aging and ultraviolet rays, etc., it is known that the synthesis of prostaglandin E2 increases due to an increase in COX-2, an enzyme that generates inflammatory cytokines, and the generation of inflammatory factors increases. Due to the inflammatory reaction, the biosynthesis of MMP increases, collagen degradation occurs, and a decrease in skin elasticity and the formation of skin wrinkles appear. In particular, when sunlight or ultraviolet rays are directly irradiated on the skin, a large amount of free radicals are generated, and such free radicals damage the antioxidant defense system of the skin, increase wrinkles, relax the skin, etc., accelerating skin aging. Substances known to be effective in improving skin wrinkles include adenosine, retinoic acid, etc. However, adenosine has little clinical efficacy, and retinoic acid cannot be used by pregnant women and has side effects such as erythema.

[0005] Human skin color is determined by the concentration and distribution of melanin within the skin. Melanin is synthesized through a non-enzymatic oxidation reaction after being converted from tyrosine to DOPA and then to dopaquinone by tyrosinase. When melanin is overproduced, pigmentation occurs, resulting in freckles, spots, and chloasma on the face, neck, arms, etc., which is not aesthetically pleasing. Although skin whitening agents have been developed to improve freckles, chloasma, and dark skin tones, it is a reality that it is difficult to develop a skin whitening agent that is excellent in whitening effect and has no side effects. For example, a skin whitening agent that selectively attacks melanocytes that produce melanin has excellent whitening effects but has side effects of skin toxicity.

[0006] Inflammation is a defensive reaction of the living body that appears in response to a pathological state caused by physical or chemical trauma, infection by bacteria, fungi, or viruses, various allergy-inducing substances, etc. The inflammatory reaction is manifested as part of the innate immune response. Although various substances and physiological and chemical phenomena are involved in the inflammatory reaction, recent research has revealed that various inflammatory cytokines play important roles in the inflammatory reaction. Major cytokines involved in the inflammatory reaction include IL-1β, TNF-α, IL-6, IL-8, IL-12, IFN-β, etc. An increase and activation in the expression level and secretion amount of these are related to a series of complex physiological reactions such as the secretion of inflammatory mediators, infiltration of immune cells, cell migration, and tissue destruction, as well as symptoms such as erythema, edema, fever, and pain.

[0007] Generally, if the source of infection is removed and the damaged tissue is regenerated, the inflammatory response will not cause major problems and the diseased site will recover normally. However, if the source of infection is not removed or the inflammatory response occurs excessively or persistently due to internal substances, acute and chronic inflammatory diseases will be induced. Non-steroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs, neuropeptide antagonists, COX inhibitors, antihistamines, and immunosuppressants such as cyclosporine A are used to alleviate or treat the inflammatory response and the resulting inflammatory diseases, but there are problems such as side effects like skin atrophy, vasodilation, depigmentation, allergic reactions, resistance, and neutropenia. In addition, these drugs have limitations in that they are not a fundamental treatment but only help to adjust the symptoms to an appropriate level.

[0008] On the other hand, recently, studies have been reported that cell secretions (secretome) contain various bioactive factors that regulate cell behavior. In particular, cell secretions contain "exosomes" that have an intercellular signaling function, and research on their components and functions is actively underway.

[0009] Cells release various membrane-type vesicles into the extracellular environment, and usually, such released vesicles are called extracellular vesicles (EVs). Extracellular vesicles are also called vesicles derived from the cell membrane, ectosomes, shedding vesicles, microparticles, exosomes, etc., and in some cases, they may be used separately from exosomes.

[0010] Exosomes are vesicles with a size of several tens to several hundreds of nanometers, having a bilayer lipid membrane identical to the cell membrane structure, and containing proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo inside. The exosome cargo contains a wide range of signaling factors, and these signaling factors are known to be regulated to be specific to cell types and vary according to the environment of the secreting cells. Exosomes are an intercellular signaling medium secreted by cells, and various cell signals transmitted thereby are known to regulate cell behaviors including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Exosomes contain specific genetic materials and bioactive factors according to the properties and states of the originating cells. In the case of exosomes derived from proliferating stem cells, cell behaviors such as cell migration, proliferation, and differentiation are regulated, and the characteristics of stem cells related to tissue regeneration are reflected (Non-Patent Document 1).

[0011] That is, exosomes, called cell avatars, contain bioactive factors such as growth factors like cells, but play the role of a transporter that carries bioactive factors between cells, that is, the role of communication between cells. Exosomes are released not only from animal cells such as stem cells, immune cells, fibroblasts, and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae. For example, exosomes can be isolated from the culture fluids of cancer cells, immune cells, mesenchymal stem cells, etc., as well as from plant cell or plant stem cell culture fluids.

[0012] However, research on the isolation, purification, and characterization of exosomes derived from plant cells or plant stem cells is still in its initial stage, and most of these are at the level of marketing by claiming that extracellular vesicles simply mixed as a mixture in the filtrate of plant juice are exosomes. Therefore, more detailed research on the characteristics and functions of exosomes derived from plant cells is needed.

[0013] Iris (Iris sp.) is a perennial herbaceous plant of the genus Iris in the family Iridaceae of monocotyledonous plants. Iris is distributed in various regions depending on the species. For example, Iris nertschinskia is distributed in Korea, Japan, northeastern China, and eastern Siberia, and its natural habitat is dry places at the foot of mountains. Among Iris extracts, substances in the isoflavone series have been reported to exhibit anti-cancer or anti-inflammatory effects, but the components and mechanism of action of Iris extracts have not been clearly elucidated. Therefore, accurate component analysis of Iris and scientific elucidation of its pharmacological mechanism of action are necessary. The current technology is at the level of using extracts obtained by hot water extraction or solvent extraction of Iris flowers, leaves, stems, roots, etc. as cosmetic raw materials. However, solvent extracts have the problem of human harmfulness due to the residual extraction solvent.

[0014] In recent years, research on cosmetic materials using callus, which is called plant stem cells, has been carried out. In this regard, a technique using callus cultures obtained by culturing callus induced by injuring Iris flowers, leaves, etc. has been introduced, but this is also only at the level of using the callus culture itself or the extract obtained by extraction such as hot water extraction or solvent extraction after drying it as a cosmetic ingredient. In addition, callus cultures contain growth regulators and callus inducers, and it is difficult to say that they are natural cosmetic raw materials, and there is a risk that growth regulators and the like contained in the cultures may cause side effects such as skin problems.

[0015] The inventor has confirmed that exosomes derived from Iris are effective in improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness, skin whitening, anti-inflammation, wound healing and / or promoting wound healing, etc., and developed a cosmetic composition for improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness and / or whitening, containing exosomes derived from Iris as an active ingredient, and a pharmaceutical composition for anti-inflammation, wound healing and / or promoting wound healing.

[0016] On the one hand, it must be understood that the matters described as the above-mentioned background art are for promoting the understanding of the background of the present invention and are not cited as an approval that they can be used as the "prior art" of the present invention.

Prior Art Documents

Non-Patent Documents

[0017]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0018] An object of the present invention is to provide a composition for improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness, skin whitening, anti-inflammation, wound healing and / or promoting wound healing, which contains exosomes derived from iris as an active ingredient.

[0019] Another object of the present invention is to provide a cosmetic composition for improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness and / or whitening, which contains the above composition, and a pharmaceutical composition for anti-inflammation, wound healing and / or promoting wound healing.

[0020] However, the problems of the present invention as described above are exemplary, and the scope of the present invention is not limited thereby. Further, other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

Means for Solving the Problems

[0021] The present invention provides a composition for improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness, skin whitening, anti-inflammation, wound healing and / or promoting wound healing, which contains exosomes derived from iris as an active ingredient.

[0022] As used herein, the term "iris" means a plant of the genus Iris sp. For example, the plant of the genus Iris sp. may be Iris ensata, Iris pallida, Iris versicolor, Iris florentina, Iris nertschinskia, Iris pseudacorus, Iris setosa, Iris lactea, Iris germanica, or Iris odaesanensis.

[0023] As used herein, the term "exosomes" means nano-sized vesicles having a membrane structure secreted or released from plant cells into the extracellular space, and is also defined as exosome-like vesicles or exosome-like particles.

[0024] As used herein, the term "skin elasticity" means the property that the skin deformed by an external force easily returns to its original shape when the external force is removed. "Skin wrinkles" means fine wrinkles caused by skin aging, but can be induced by genetic causes, a decrease in collagen and elastin present in the skin dermis, the external environment, etc. Therefore, as used herein, the term "skin wrinkle improvement" means suppressing or inhibiting the formation of wrinkles on the skin, or relaxing already formed wrinkles.

[0025] On the other hand, as used herein, the term "skin tone" means the state of the degree of intensity of the skin color. "Skin whitening" includes increasing the lightness of the skin with a reduced lightness due to an excess of pigments such as melanin, or maintaining the lightness of the skin at a certain level.

[0026] As used herein, the term "anti-inflammatory" means preventing, suppressing, alleviating, improving, or treating inflammation. Examples of inflammatory diseases, without limiting the present invention, include dermatitis, atopic dermatitis, eczema, inflammation due to bacterial infection, viral infection or fungal infection, burns, inflammation caused by burns, wounds, inflammation caused by wounds, and the like.

[0027] As used herein, the term "wound" means a state in which a living body is damaged, and includes a pathological state in which tissues constituting the internal or external surface of the living body, such as skin, muscle, nerve tissue, bone, soft tissue, internal organs or vascular tissue, are severed or destroyed. Without limiting the present invention, examples of wounds or injuries include abrasions, lacerations, punctures, cuts, avulsions, pressure sores, bedsores, tissue destruction by radiation, penetrated wounds, gunshot wounds, burns, frostbite, surgical wounds, suture sites after plastic surgery, chemical injuries, etc., and may include damage to any part of an individual.

[0028] As used herein, the term "iontophoresis" means a method of passing an ionized active ingredient through the skin by an electro-repulsive force by applying a minute current to the skin to which the active substance is applied to create a potential difference and changing the electrical environment of the skin. The iontophoresis used in a specific example of the present invention may include a method in which a current from an external power source flows into an electrode patch on the skin to introduce a minute current into the skin, a method in which a battery is attached to the electrode patch itself to introduce a minute current into the skin, a method in which a minute current is introduced into the skin through a patch equipped with a reversed electrodialysis means that generates a current due to the ion concentration difference between a high-concentration electrolyte solution and a low-concentration electrolyte solution, and the like. However, the present invention is not limited thereto, and it goes without saying that various methods of iontophoresis can be used.

[0029] As used in the specification of the present invention, the term "exosomes derived from iris" means any exosomes that are separated from, for example, iris plant cell culture fluid, iris callus culture fluid, iris plant stem cell culture fluid, iris juice, or biological solutions of iris equivalent thereto, or are derived from iris plant cells or plant stem cells, such as those secreted and / or released.

[0030] In the composition of a specific example of the present invention, the exosomes derived from iris may be those separated and purified from a callus culture fluid derived from the roots of iris.

[0031] A composition containing exosomes derived from iris as an active ingredient in a specific example of the present invention may exhibit at least one efficacy among improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness, skin whitening, anti-inflammation, wound healing, and / or promoting wound healing.

[0032] The composition containing exosomes derived from iris as an active ingredient of the present invention may be a cosmetic composition or a pharmaceutical composition.

[0033] The present invention provides a cosmetic composition for improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness, and / or whitening, which contains exosomes derived from iris as an active ingredient. For example, the cosmetic composition may be shampoo, soap, rinse, surfactant-containing cleansing, cream, lotion, ointment, tonic, treatment, conditioner, suspension, emulsion, paste, gel, oil, wax, spray, aerosol, mist, or powder, and preferably may be lotion or cream.

[0034] In addition, the present invention provides a pharmaceutical composition for anti-inflammation, wound healing, and / or promoting wound healing, which contains exosomes derived from iris as an active ingredient.

[0035] As an exemplification of the present invention, the pharmaceutical composition of a specific example of the present invention can be administered or treated by injection, micro-needling, iontophoresis, application, or a combination thereof, but is not limited thereto. For example, the pharmaceutical composition may be in an injection dosage form, an infusion dosage form, a spray dosage form, a liquid dosage form, or a patch dosage form.

[0036] When the composition of a specific example of the present invention is used as a pharmaceutical composition, it can contain pharmaceutically acceptable carriers, excipients, diluents, and the like. Examples of the carriers, excipients, and diluents include, but are not limited to, lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Also, the effective amount of the pharmaceutical composition of a specific example of the present invention means the amount required for administration in order to expect an anti-inflammatory, wound healing, and / or wound healing promoting effect.

[0037] The mixing ratio of the pharmaceutical composition of a specific example of the present invention can be appropriately selected according to the types, amounts, forms, and the like of the additional components as described above. For example, based on the total amount of the injection, the pharmaceutical composition of the present invention may be contained in an amount of about 0.1 to 99% by weight, preferably about 10 to 90% by weight. Also, the appropriate dosage of the pharmaceutical composition of a specific example of the present invention can be adjusted according to the severity of the disease, the type of dosage form, the formulation method, the age, sex, weight, health status, diet, excretion rate, administration time, and administration method of the patient. For example, when administering the pharmaceutical composition of a specific example of the present invention to an adult, it can be administered in doses of 0.001 mg / kg to 100 mg / kg per day, divided into one to several times.

[0038] On the one hand, when the composition of a specific example of the present invention is manufactured as a cosmetic composition, within the range not impairing the effects of the present invention, components usually used in cosmetic compositions, such as humectants, antioxidants, oily components, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, etc., can be appropriately blended as needed.

[0039] In addition, the cosmetic composition of a specific example of the present invention, in addition to exosomes derived from iris, can be mixed and used together with conventionally used skin improvers, antioxidants, anti-aging agents, whitening agents, and / or humectants to the extent that their effects (such as improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness, skin whitening, skin beautification, etc.) are not impaired.

[0040] The cosmetic composition of a specific example of the present invention can be applied to various forms such as patches, mask packs, sheet masks, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, oils, packs, sprays, aerosols, mists, foundations, powders, oil papers, etc.

[0041] The cosmetic composition of a specific example of the present invention can be used for purposes such as improving skin elasticity, improving skin wrinkles, skin regeneration, improving skin tone and skin brightness, and / or skin whitening, and the cosmetic dosage form can be manufactured into any dosage form usually manufactured in the industry. For example, it can be formulated into patches, mask packs, sheet masks, softening lotions, nutritive lotions, astringent lotions, nutritive creams, massage creams, eye creams, cleansing creams, essences, eye essences, cleansing lotions, cleansing foams, cleansing waters, sunscreens, lipsticks, soaps, shampoos, surfactant-containing cleansers, bath salts, body lotions, body creams, body oils, body essences, body cleansers, hair dyes, hair tonics, etc., but is not limited thereto.

[0042] The cosmetic composition of a specific example of the present invention contains components commonly used in cosmetic compositions, and can include, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, and ordinary auxiliaries such as fragrances and carriers. Also, in each dosage form for the cosmetic composition, other components can be appropriately selected and formulated by those skilled in the art without difficulty according to the type of the cosmetic composition or the purpose of use, etc.

[0043] Another specific example of the present invention provides a beauty method for regulating the skin condition of mammals excluding for therapeutic use using the above cosmetic composition. In the beauty method of the present invention, the regulation of the skin condition means improving and / or prophylactically regulating the skin condition, and the improvement of the skin condition means a positive change that can be visually and / or tactilely perceived in the appearance and feel of the skin. For example, the improvement of the skin condition may be improvement of skin elasticity, improvement of skin wrinkles, skin regeneration, improvement of skin tone and skin brightness, and / or skin whitening.

[0044] A beauty method of a specific example of the present invention includes (a) directly applying the above cosmetic composition to the skin of a mammal, (b) contacting or attaching to the skin of a mammal a patch, mask pack or sheet mask coated or impregnated with the above cosmetic composition, or sequentially performing the above (a) and (b). In the step (a), lotions or creams can be used as the cosmetic composition.

[0045] As an alternative, a beauty method of a specific example of the present invention can further include (c) after the step (b), removing the patch, mask pack or sheet mask from the skin of the mammal and applying the above cosmetic composition to the skin of the mammal. In the step (c), lotions or creams can be used as the cosmetic composition.

[0046] In a beauty method of a specific example of the present invention, the mammal may be a human, dog, cat, rodent, horse, cow, monkey, or pig.

[0047] The present invention also provides a method for treating inflammation, treating wounds, or accelerating wound healing, which includes administering a therapeutically effective amount of the pharmaceutical composition to a mammal or applying the pharmaceutical composition to the skin, an inflamed site, or a wound site. The mammal may be a human, dog, cat, rodent, horse, cow, monkey, or pig.

Advantages of the Invention

[0048] Compared with conventional Iris hot water extracts, solvent extracts, filtrates of such extracts, and Iris calamus culture solutions, the composition of the present invention has a lower possibility of containing impurities such as residual solvents and growth regulators, and is excellent in improving skin elasticity, reducing skin wrinkles, promoting skin regeneration, improving skin tone and skin brightness, whitening the skin, anti-inflammation, wound healing, or accelerating wound healing effects.

[0049] On the other hand, the scope of the present invention is not limited by the above-described effects.

Brief Description of the Drawings

[0050]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0051] Hereinafter, the present invention will be described in more detail with the following examples. However, the following examples are merely illustrative of the content of the present invention and do not limit or restrict the scope of rights of the present invention. It is to be understood that what can be easily inferred by an ordinary technician in the technical field to which the present invention pertains from the detailed description and examples of the present invention belongs to the scope of rights of the present invention. The references cited in the present invention are incorporated herein by reference.

[0052] Throughout the specification, when a certain part states that a certain component "comprises", this means that other components can be further included, rather than excluding other components, unless otherwise stated to the contrary.

Examples

[0053] <Production of Iris Callus Culture Solution> By the method for producing and culturing plant stem cells known in the art, callus derived from the roots of iris is induced, and the induced iris callus is cell-cultured. Then, callus in a good growth state is selected and cultured in large quantities to prepare an iris callus culture solution.

Example

[0054] <Production of Iris-derived exosomes> An Iris callus culture solution [Iris germanica callus culture solution] produced by the same method as in Example 1 was obtained from Biospectrum Co., Ltd. (a supplier of Iris callus culture solution located in Gyeonggi-do, South Korea). The Iris callus culture solution was filtered through a 0.22 μm filter to remove impurities such as cell debris, waste products, and large particles. From the filtered culture solution, Iris-derived exosomes were separated using a tangential flow filtration (TFF) device.

[0055] The size and concentration of the separated Iris-derived exosomes were confirmed by nanoparticle tracking analysis (NTA) using NS300 (purchased from Malvern Panalytical) (Figure 1).

Example

[0056] <Confirmation of the ability of Iris-derived exosomes to be transferred to skin fibroblasts> To confirm whether exosomes derived from iris are transmitted into human dermal fibroblasts (purchased from ATCC), the following analysis was performed. To fluorescently stain the membrane of exosomes derived from iris prepared in Example 2, it was reacted with the PKH67 (purchased from Sigma-Aldrich) fluorescent dye. After the reaction, the reaction solution was fractionated with a MiniTrap-25 (purchased from Cytiva) column to remove the free PKH67 fluorescent dye that was not stained on the exosome membrane. For the negative control group, after reacting the PKH67 fluorescent dye with a buffer solution, the fractionated product using the MiniTrap-25 column was used. After culturing the exosomes stained with PKH67 together with human dermal fibroblasts prepared by pre-culturing, it was observed using a fluorescence microscope whether the exosomes were transmitted into the cells over time. Hoechst (purchased from Thermo Fisher) fluorescent dye was used to stain the nuclei of the cells, and CellMask Orange fluorescent dye (purchased from Thermo Fisher) was used to stain the cytoplasm. As a result of confirming whether the exosomes were transmitted into the cells, it was confirmed that the fluorescently stained exosomes were transmitted into the cells and green fluorescence accumulated inside the cells over time (Figure 2).

Example

[0057] <Confirmation of the effect of promoting collagen production> Human dermal fibroblasts (Human Dermal Fibroblast; purchased from ATCC) dispersed in DMEM medium containing fetal bovine serum were subcultured in a multi-well plate and then cultured for 24 hours. Then, the iris callus culture solution prepared in Example 1 and the iris-derived exosomes prepared in Example 2 were each diluted in serum-free medium and then treated with human dermal fibroblasts, and the human dermal fibroblasts were cultured for 24 hours. To confirm the collagen production efficacy using human dermal fibroblasts, the experimental groups were classified as follows. (1) Negative control group (indicated as "N.C." in Figure 3): The experimental group treated with only serum-free medium; (2) Iris calvus culture solution treatment group (indicated as "Iris calvus CM" in Fig. 3): An experimental group treated by diluting the Iris calvus culture solution prepared in Example 1 in a serum-free medium (treatment concentration - low concentration: 60 μg / mL, high concentration: 240 μg / mL); (3) Exosome treatment group derived from Iris (indicated as "Iris exosome" in Fig. 3): An experimental group treated by diluting the exosomes derived from Iris prepared in Example 2 in a serum-free medium (treatment concentration - low concentration: 60 μg / mL, high concentration: 240 μg / mL).

[0058] After treating each of the above experimental groups with human dermal fibroblasts and culturing for 24 hours, the culture solution was collected and centrifuged, and then the centrifuged culture solution was prepared. The amount of collagen synthesized from human dermal fibroblasts and accumulated in the culture solution was measured using an EIA kit for procollagen type I C-peptide (PIP) (purchased from Takara). The relative amount of collagen was determined by normalizing it by dividing by the total number of cells measured by an MTT assay kit (purchased from Sigma-Aldrich).

[0059] As a result, the Iris calvus culture solution treatment group had no effect of increasing collagen synthesis in human dermal fibroblasts compared to the negative control group. On the other hand, the exosomes derived from Iris of the present invention increased collagen synthesis in human dermal fibroblasts. In particular, when the exosomes derived from Iris were treated at a high concentration, it was confirmed that the collagen synthesis in human dermal fibroblasts was significantly increased (Fig. 3).

[0060] From the above experimental results, it can be seen that the exosomes derived from Iris of the present invention are superior to the Iris calvus culture solution in terms of the efficacy of increasing collagen synthesis, that is, improving skin elasticity, reducing wrinkles and / or skin regeneration efficacy.

[0061] According to the above results, it can be seen that the iris-derived exosomes of the present invention have functions useful as cosmetics for improving skin elasticity, reducing wrinkles, and / or regenerating skin, that is, an activity of increasing collagen synthesis. Therefore, the iris-derived exosomes of the present invention can be usefully utilized as an active ingredient of a cosmetic composition for improving skin elasticity, reducing wrinkles, and / or regenerating skin.

Example

[0062] <Confirmation of Skin Regeneration Efficacy Using Skin Fibroblasts> To evaluate whether the exosomes prepared as in Example 2 promote wound healing ability in human dermal fibroblasts (purchased from ATCC), a scratch-wound assay was performed. Human dermal fibroblasts dispersed in DMEM medium containing fetal bovine serum were subcultured at a density of 5,000 cells / well in a wound-inducing culture plate (ImageLock Plate; purchased from EssenBio), and cultured at 5% CO2 and 37 °C for 24 hours. After confirming a confluence of 90% or more, a scratch was induced using a wound maker (purchased from EssenBio). To confirm the skin regeneration efficacy using human dermal fibroblasts, the experimental groups were classified as follows. (1) Negative control group (indicated as "N.C." in Figure 4): The experimental group treated with only serum-free medium; (2) Positive control group (indicated as "P.C." in Figure 4): The experimental group treated with a culture medium containing 10% fetal bovine serum; (3) Iris callus culture solution treatment group (indicated as "Iris callus CM" in Figure 4): The experimental group treated by diluting the iris callus culture solution prepared in Example 1 in serum-free medium (treatment concentration - low concentration: 50 μg / mL, high concentration: 200 μg / mL); (4) Iris-derived exosome treatment group (indicated as "Iris exosome" in Figure 4): The experimental group treated by diluting the iris-derived exosomes prepared in Example 2 in serum-free medium (treatment concentration - low concentration: 50 μg / mL, high concentration: 200 μg / mL).

[0063] Thereafter, each of the experimental groups was treated with Scratch-Wound, and after culturing human dermal fibroblasts for 24 hours under the conditions of 5% CO2 and 37°C, the wound healing ability was measured using Incucyte (purchased from Sartorius).

[0064] As a result of measuring the wound healing ability, the iris callus culture solution did not increase the migration of human dermal fibroblasts compared to the negative control group, while the iris-derived exosomes of the present invention were confirmed to increase the migration of human dermal fibroblasts compared to the negative control group (Figure 4).

[0065] From the above experimental results, it can be seen that the iris-derived exosomes of the present invention are superior to the iris callus culture solution in terms of the ability to promote the migration of human dermal fibroblasts, that is, the wound healing ability or skin regeneration ability.

[0066] Therefore, the iris-derived exosomes of the present invention can be usefully utilized as an active ingredient in a cosmetic composition for improving skin elasticity, improving skin wrinkles and / or skin regeneration, and a pharmaceutical composition for wound treatment or promoting wound treatment.

Example

[0067] <Confirmation of the ability of iris-derived exosomes to transfer to melanoma cells> To confirm whether exosomes derived from iris are transmitted into mouse melanoma (B16F10; purchased from ATCC), the following analysis was performed. To fluorescently stain the membrane of exosomes derived from iris prepared in Example 2, it was reacted with a PKH67 (purchased from Sigma-Aldrich) fluorescent dye. After the reaction, the reaction solution was fractionated using a MiniTrap-25 (purchased from Cytiva) column to remove the free PKH67 fluorescent dye that was not stained on the exosome membrane. As a negative control group, a solution obtained by reacting the PKH67 fluorescent dye with a buffer solution and then fractionating it using a MiniTrap-25 column was used. After culturing the exosomes stained with PKH67 together with mouse melanoma cells prepared by pre-culturing, it was observed over time using a fluorescence microscope whether the exosomes were transmitted into the cells. A Hoechst (purchased from Thermo Fisher) fluorescent dye was used to stain the nuclei of the cells, and a CellMask Orange fluorescent dye (purchased from Thermo Fisher) was used to stain the cytoplasm. As a result of confirming whether the exosomes were transmitted into the cells, it was confirmed that the fluorescently stained exosomes were transmitted into the cells and the green fluorescence accumulated inside the cells over time (Figure 5).

Example

[0068] <Melanin production inhibitory effect of exosomes derived from iris> The whitening effect of exosomes derived from iris was confirmed by inhibiting melanin production in melanoma cells. The melanoma cells are cells derived from mouse melanoma (B16F10; purchased from ATCC) and are cells that secrete a black pigment called melanin. After subculturing the melanoma cells at 8,000 cells per unit area in a 48-well plate, they were cultured for 24 hours under conditions of 5% CO2 and 37°C.

[0069] Subsequently, the iris callus culture solution prepared in Example 1 and the iris-derived exosomes prepared in Example 2 were each diluted in a culture medium mixed with α-MSH, a melanin synthesis stimulant, and then treated with melanoma cells, and the melanoma cells were cultured for 48 hours. For the confirmation of the melanogenesis inhibitory efficacy using melanoma cells, the experimental groups were classified as follows. (1) Negative control group (labeled "N.C." in Fig. 6): The experimental group treated with a culture medium mixed with α-MSH, a melanin synthesis stimulant; (2) Positive control group (labeled "P.C." in Fig. 6): The experimental group treated with a culture medium mixed with α-MSH, a melanin synthesis stimulant, and arbutin (final concentration: 1 mM); (3) Iris callus culture solution treatment group (labeled "Iris callus CM" in Fig. 6): The experimental group treated with a culture medium mixed with the iris callus culture solution prepared in Example 1 and α-MSH, a melanin synthesis stimulant (treatment concentration - low concentration: 100 μg / mL, high concentration: 200 μg / mL). (4) Iris-derived exosome treatment group (labeled "Iris exosome" in Fig. 6): The experimental group treated with a culture medium mixed with the iris-derived exosomes prepared in Example 2 and α-MSH, a melanin synthesis stimulant (treatment concentration - low concentration: 100 μg / mL, high concentration: 200 μg / mL).

[0070] After collecting the melanoma culture media for each of the above experimental groups, the collected melanoma culture media were mixed with a CCK-8 assay reagent (purchased from Dojindo) and incubated for 2 hours under the conditions of 5% CO2 and 37 °C. Then, the supernatant was transferred to a 96-well plate, and the absorbance at 450 nm was measured to measure the total cell number. The melanoma cells in the 48-well plate were washed with a washing solution, and then 1N NaOH (purchased from Merck-Milipore) mixed with 10% DMSO was treated with the washed melanoma cells. After that, the plate was sealed and heated at 85 °C for 20 minutes to extract the melanin in the melanoma cells. The extracted melanin was measured for the absorbance at 405 nm to calculate the amount of melanin, and it was normalized with the absorbance value measured by the CCK-8 assay.

[0071] As a result, it was confirmed that the iris-derived exosomes of the present invention have the ability to inhibit melanin synthesis in melanoma cells in a concentration-dependent manner compared to the negative control group, and are superior to the iris callus culture solution in terms of the inhibitory effect on melanin synthesis (Figure 6).

[0072] Therefore, the cosmetic composition containing the iris-derived exosomes of the present invention as an active ingredient has a whitening effect, and the iris-derived exosomes of the present invention can be usefully utilized as an active ingredient of a cosmetic composition for improving skin tone, improving skin brightness and / or skin whitening.

Example

[0073] <Confirmation of the ability of iris-derived exosomes to be transferred to macrophages> In order to confirm whether iris-derived exosomes are transferred into mouse macrophages (RAW264.7; purchased from ATCC), the following analysis was performed. The membrane of the iris-derived exosomes prepared in Example 2 was reacted with a PKH67 (purchased from Sigma-Aldrich) fluorescent dye for fluorescent staining of the membrane. After the reaction, the reaction solution was fractionated using a MiniTrap-25 column (purchased from Cytiva) to remove the free PHK67 fluorescent dye that was not stained on the exosome membrane. As a negative control group, a solution obtained by reacting the PKH67 fluorescent dye with a buffer solution and then fractionating it using a MiniTrap-25 column was used. The exosomes stained with PKH67 were cultured with mouse macrophages prepared in advance, and then whether the exosomes were transferred into the cells over time was observed using a fluorescence microscope. A Hoechst fluorescent dye (purchased from Thermo Fisher) was used to stain the cell nuclei, and a CellMask Orange fluorescent dye (purchased from Thermo Fisher) was used to stain the cytoplasm. As a result of confirming whether the exosomes were transferred into the cells, it was confirmed that the fluorescently stained exosomes were transferred to the cells and the green fluorescence accumulated in the cells over time (Figure 7).

Example

[0074] <Evaluation of the anti-inflammatory efficacy of exosomes derived from Iris To confirm whether the exosomes derived from Iris prepared as in Example 2 exhibit anti-inflammatory efficacy, the effect of the exosomes derived from Iris on the production amount of IL-6 in RAW264.7 cells, which are mouse macrophages, was confirmed. RAW264.7 cells suspended in DMEM medium containing 10% FBS were subcultured into each well of a 48-well plate so as to have a confluency of 80 to 90%. Then, each of the Iris callus culture solution prepared in Example 1 and the exosomes derived from Iris prepared in Example 2 was mixed with a new medium containing LPS [DMEM medium containing 1% FBS and 200 nM lipopolysaccharide (LPS)] and used to treat RAW264.7 cells, and then the RAW264.7 cells were cultured for 24 hours. The experimental groups for the anti-inflammatory efficacy evaluation were classified as follows. (1) Negative control group (indicated as "N.C." in Fig. 8): The experimental group in which only the LPS medium was used to treat RAW264.7 cells; (2) Positive control group (indicated as "P.C." in Fig. 8): The experimental group in which dexamethasone (final concentration: 200 μM) was mixed with the LPS medium and used to treat RAW264.7 cells; (3) Iris callus culture solution treatment group (indicated as "Iris callus CM" in Fig. 8): The experimental group in which the Iris callus culture solution prepared in Example 1 was diluted with the LPS medium and used to treat RAW264.7 cells (treatment concentration - low concentration: 50 μg / mL, high concentration: 200 μg / mL); (4) Exosomes derived from Iris treatment group: The experimental group in which the exosomes derived from Iris prepared in Example 2 were diluted with the LPS medium and used to treat RAW264.7 cells (treatment concentration - low concentration: 50 μg / mL, high concentration: 200 μg / mL).

[0075] After the completion of culture, the culture supernatant was collected, and the production amount of IL-6, an inflammatory cytokine present in the culture supernatant, was measured using an IL-6 ELISA kit. The production amount of IL-6 (inflammatory cytokine) in the group treated with only LPS according to the manufacturer's manual of the ELSIA kit (purchased from R&D systems) and the production amount of IL-6 in the experimental groups treated by mixing dexamethasone, Iris californica culture solution, and exosomes derived from Iris with the LPS medium were confirmed (Figure 8).

[0076] As shown in Figure 8, in the experimental group in which RAW264.7 cells, which are mouse macrophages, were treated with the exosomes derived from Iris of the present invention together with LPS, the production of IL-6 was significantly suppressed compared to the negative control group in which only LPS was treated on RAW264.7 cells. On the other hand, in the experimental group in which the Iris californica culture solution was treated with LPS on RAW 264.7 cells, the effect of reducing the production of IL-6 did not appear. In addition, as the treatment concentration of the exosomes derived from Iris of the present invention increased, the production of IL-6 tended to be further suppressed, and it was confirmed that the exosomes derived from Iris of the present invention have a concentration-dependent anti-inflammatory effect.

[0077] From the above experimental results, it was found that the exosomes derived from Iris of the present invention are excellent in anti-inflammatory efficacy. Therefore, the exosomes derived from Iris of the present invention can be usefully utilized as an active ingredient of a pharmaceutical composition for anti-inflammation, wound treatment, and / or promotion of wound treatment.

[0078] As described above, the present invention has been described with reference to the above examples, but the present invention is not limited thereto. Those skilled in the art will be able to make modifications and changes without departing from the spirit and scope of the present invention, and it will be understood that such modifications and changes also belong to the present invention.

Claims

1. A cosmetic composition for improving skin elasticity, reducing skin wrinkles, promoting skin regeneration, improving skin tone or brightness, or whitening the skin, comprising exosomes derived from iris as an active ingredient.

2. The cosmetic composition according to claim 1 for improving skin elasticity, reducing skin wrinkles, promoting skin regeneration, improving skin tone or brightness, or whitening the skin, which is shampoo, soap, rinse, surfactant-containing cleansing, cream, lotion, ointment, tonic, treatment, conditioner, suspension, paste, gel, oil, wax, spray, mist, or powder.

3. The cosmetic composition according to claim 2 for improving skin elasticity, reducing skin wrinkles, promoting skin regeneration, improving skin tone or brightness, or whitening the skin, which is cream or lotion.

4. A pharmaceutical composition for anti-inflammation, wound healing, or promoting wound healing, comprising exosomes derived from iris as an active ingredient.

5. The pharmaceutical composition according to claim 4 for anti-inflammation, wound healing, or promoting wound healing, which is administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof.

6. The pharmaceutical composition according to claim 4 for anti-inflammation, wound healing, or promoting wound healing, which is in the form of injection, infusion, aerosol, liquid, or patch.

7. A beauty method for improving skin elasticity, reducing skin wrinkles, promoting skin regeneration, improving skin tone or brightness, or whitening the skin of mammals excluding for therapeutic use, using a cosmetic composition comprising exosomes derived from iris as an active ingredient.

8. The beauty method according to claim 7, comprising (a) directly applying the cosmetic composition to the skin of a mammal; (b) contacting or attaching to the skin of a mammal a patch, mask pack, or sheet mask coated or impregnated with the cosmetic composition; or sequentially performing (a) and (b).

9. The beauty method according to claim 8, wherein in step (a), lotion or cream is used as the cosmetic composition.

10. The beauty method according to claim 8 or 9, further comprising (c) after step (b), removing the patch, mask pack, or sheet mask from the skin of the mammal and applying the cosmetic composition to the skin of the mammal.

11. The beauty method according to claim 10, wherein in the step (c), a lotion or a cream is used as the cosmetic composition.

12. The mammal is a human, a dog, a cat, a rodent, a horse, a cow, a monkey, or a pig. The beauty method according to any one of claims 7 to 9.

13. A step of administering a therapeutically effective amount of a pharmaceutical composition containing exosomes derived from the iris as an active ingredient to a mammal excluding a human, or a step of applying a pharmaceutical composition containing exosomes derived from the iris as an active ingredient to the skin, an inflamed site, or a wound site of a mammal excluding a human. A method for treating inflammation, treating a wound, or promoting wound healing.

14. The mammal is a dog, a cat, a rodent, a horse, a cow, a monkey, or a pig. The method for treating inflammation, treating a wound, or promoting wound healing according to claim 13.

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