Composition for improving skin condition containing camellia callus-derived exosomes as an active ingredient

Abstract

This invention relates to a cosmetic composition containing camellia callus-derived exosomes as an active ingredient, which has anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement, and skin soothing effects. The camellia callus-derived exosomes provided by this invention, as a cosmetic or pharmaceutical composition for anti-inflammatory, atopic dermatitis improvement, wound healing, and wound healing promotion, exhibit excellent effects.

Description

Technical Field

[0001] This invention relates to a product containing camellia ( Camellia japonica A composition for improving skin condition, with exosomes derived from callus tissue as the active ingredient. Background Technology

[0002] The aging process occurs in all organs of the body, and the skin also gradually ages with age. Wrinkles are the most important and common symptom of aging skin, serving as a standard skin appearance for assessing the degree of aging. In recent years, with improved living standards and increased attention to skin aesthetics, people have become increasingly concerned about skin aging. While it's impossible to stop skin aging, various attempts are being made to inhibit and delay skin aging and its representative symptom, wrinkles.

[0003] Based on the factors influencing aging, skin aging is divided into endogenous and exogenous factors. Among the endogenous factors of skin aging, the loss of the extracellular matrix (ECM) in the dermis, composed of collagen, elastin, fibronectin, proteoglycans, etc., is considered the biggest cause. Dermal aging leads to a decline in ECM function, resulting in thinning, flattening of the skin, wrinkles, reduced elasticity, and skin atrophy and dryness. Furthermore, during skin aging, the function of skin appendages such as hair follicles, sweat glands, and sebaceous glands also declines, thus delaying wound healing caused by external stimuli.

[0004] Regarding ultraviolet (UV) radiation as an exogenous factor, it has been reported that when skin is exposed to UV rays, it promotes melanin production, leading to skin aging and damage. Furthermore, reactive oxygen species (ROS) generated through UV radiation and respiration dominate damage to skin cells and tissues. Excessive ROS disrupt the body's antioxidant defense system, becoming a major factor in causing inflammation, erythema, burns, immunosuppression, and DNA damage, ultimately accelerating skin aging. In addition, exogenous and endogenous stimuli, due to decreased moisture content in the stratum corneum and reduced natural moisturizing factors, lead to decreased skin hydration, exacerbating dryness and even damaging the skin barrier. Such unbalanced skin undergoes changes in its physicochemical properties, resulting not only in wrinkles, pigmentation, decreased hydration, and skin barrier damage, but also inflammation and reduced cell regeneration capacity.

[0005] Therefore, to delay and inhibit skin aging, it is necessary not only to establish an antioxidant defense system within the organism but also within the skin to inhibit and effectively remove excess reactive oxygen species. Consequently, there is a growing interest in materials for skin improvement, particularly in natural materials for protecting the skin barrier and preventing skin aging. While the demand for natural materials for commercial use is increasing, plants are unsustainable resources, requiring stable harvests. Plant tissue culture, unaffected by external environmental factors such as location and temperature, allows for stable and continuous production, making it a high-value technology for industrial-scale utilization. Leveraging this advantage, research is actively underway in cosmetics, health functional foods, and pharmaceuticals for large-scale propagation and the development of transgenic plants.

[0006] Camellia ( Camellia japonica Camellia is an evergreen tree belonging to the genus Camellia in the family Theaceae. Approximately 100 species of Camellia are known to be distributed in Southeast Asia, with one species found in southern Korea. Furthermore, in Korea, horticultural varieties with altered flower morphology, color, and leaves are widely used as ornamental trees. They grow to a height of 3–7 meters and mainly grow in the mountains and fields of Jeju Island and the southern region of Korea. Camellia seeds contain 9.1–11.5% saturated fatty acids, 85.6–89.4% oleic acid (an unsaturated fatty acid), and 1.3–2.9% linoleic acid. It is a non-drying oil with a high oleic acid content and has been used since ancient times as hair oil, precision machinery oil, and edible oil. According to the *Compendium of Materia Medica*, the flowers were ground into powder and used to treat burns. Research on the components of camellia has primarily been conducted in Japan. Reports indicate that the leaves, seeds, and flowers of camellia contain a variety of compounds that contribute to its medicinal properties, including camellin, pipecolic acid, eugenol, camelliasaponin A and B, triterpenoids, tannins, benzenoids, steroids, flavonoids, and phenylpropanoids. In Japan, dried camellia buds are traditionally used as a hemostatic agent for hemoptysis and intestinal bleeding. Safflower powder is taken with children's urine, ginger juice, and alcohol, and has been reported to possess antiparasitic, antispasmodic, tartar-inhibiting, alcohol-absorbing, and skin-whitening effects.

[0007] In recent years, exosomes have been applied in various fields such as biotechnology, pharmaceuticals, and cosmetics manufacturing. Exosomes are small, membrane-bound vesicles secreted by various cells, including mammals, bacteria, and plants. As intercellular communication substances, exosomes are nanoscale materials with a size of 50–200 nm within extracellular vesicles. These nanoscale exosomes can transport water-insoluble membrane proteins. Membrane proteins are proteins located in the phospholipid bilayer, which, due to their hydrophobicity, are difficult to transport between cells via the bloodstream. In contrast, exosomes are composed of a phospholipid bilayer, thus enabling them to transport membrane proteins. By encapsulating their contents within the phospholipid bilayer, exosomes can protect their contents from external degradation. Furthermore, they readily penetrate cells and perform various physiological and pathological functions, such as immune responses and signal transduction. Due to these characteristics, exosomes have recently been widely used in cosmetic compositions. Therefore, research on the extraction of exosomes is actively underway.

[0008] In recent years, research has been ongoing on the various functions of plant-derived exosomes. Unlike animal cells, plants possess not only cell membranes but also cell walls, forming a space between the cell wall and the cell membrane called an apoplast. Within the apoplast, not only do nutrients and water move, but numerous extracellular vesicles (EVs) or exosomes (nanovesicles) accumulate. Like animal-derived exosomes, plant-derived exosomes contain nucleic acids (DNAs / RNAs), proteins (cytokines, growth factors, etc.), and, most importantly, a wide variety of plant-specific secondary metabolites. Furthermore, plants contain various lipids, and plant-derived exosomes exhibit a more diverse composition and higher content of lipids compared to animal-derived exosomes, thus promising a range of physiological activities. Plant-derived exosomes, as natural products or vegan derivatives, can be actively absorbed by animal cells as heterogeneous organisms, exhibiting higher biocompatibility than synthetic carriers. Therefore, research utilizing them as core materials is expanding.

[0009] Human cell-derived extracellular vesicles have limited functionality, and large-scale production requires significant costs and equipment. Furthermore, the use of animal-derived substances such as fetal bovine serum (FBS) raises safety concerns. In contrast, plant-derived exosomes are not affected by safety issues, and if their physiological activity is proven, they hold great value as functional foods, pharmaceuticals, and cosmetics.

[0010] Research on extracting various plant-derived substances from callus tissue and producing useful substances such as exosomes is expanding. In South Korea, patents published include: Korean Patent Publication No. 10-2023-0144973, "High-Yield Production Method of Plant-Derived Exosomes and Functional High-Purity Exosomes Produced Therefrom"; Korean Patent Publication No. 10-2461314, "Composition for Skin Condition Improvement Containing Apple Callus-Derived Exosomes"; and Korean Patent Publication No. 10-2023-0063541, "Preparation Method of Aloe Vera Peel Callus-Derived Nano-Exosomes and Composition for Skin Improvement Containing Aloe Vera Peel Callus-Derived Nano-Exosomes Prepared Therefrom."

[0011] However, there is currently no method of utilizing camellia ( Camellia japonica Research on cosmetic or pharmaceutical compositions in which exosomes obtained from callus tissue can simultaneously impart anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, wound healing, skin barrier improvement, atopic dermatitis improvement and skin soothing effects.

[0012] Therefore, the inventors discovered camellia ( Camellia japonica The exosomes derived from this invention possess the following activities: inhibiting nitric oxide production, reducing reactive oxygen species generated by UV radiation, inhibiting extracellular melanin production, inhibiting MMP-1 (matrix metalloproteinase-1) gene expression, increasing aquaporin 3 (AQP3) gene expression, reducing intercellular gaps, increasing filaggrin gene expression, inhibiting TARC (thymus and activation-regulated chemokine) production, and reducing vascular endothelial growth factor (VEGF) gene expression. Therefore, these exosomes can simultaneously impart anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, wound healing, skin barrier improvement, atopic dermatitis improvement, and skin soothing effects, thus completing this invention. Summary of the Invention

[0013] Technical problems to be solved The purpose of this invention is to provide a skin condition improvement composition containing exosomes derived from camellia callus tissue as an active ingredient.

[0014] Another object of the present invention is to provide a cosmetic or pharmaceutical composition for anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement and skin soothing, or for anti-inflammatory, atopic dermatitis improvement, wound healing and wound healing promotion, comprising exosomes derived from camellia callus tissue as an active ingredient.

[0015] Solution to the problem To achieve the above objectives, the present invention provides a cosmetic composition containing exosomes derived from camellia callus tissue as an active ingredient, which has anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement and skin soothing effects.

[0016] In this invention, the camellia callus-derived exosomes are characterized in that they are isolated and purified from camellia-induced callus or the culture medium of callus.

[0017] In this invention, the callus tissue is characterized in that it is induced from any part selected from the group consisting of camellia flowers, leaves, stems, roots, seeds and fruits.

[0018] In this invention, the exosomes are characterized in that they are isolated from one or more of the group consisting of exosomes derived from callus tissue, fragments of callus tissue, and extracts of callus tissue.

[0019] In this invention, the exosomes are characterized as extracellular vesicles with an average diameter of 50 to 200 nm.

[0020] In this invention, the composition is characterized by being selected from any one of the following groups: shampoo, soap, conditioner, cleansing products containing surfactants, skin, toner, cream, lotion, emulsion, serum, essence, suspension, emulsion, ointment, tonic, treatment, conditioner, paste, gel, oil, wax, spray, aerosol, mist, and powder.

[0021] Furthermore, the present invention provides a pharmaceutical composition for anti-inflammatory, atopic dermatitis improvement, wound healing and wound healing promotion, comprising exosomes derived from camellia callus tissue as an active ingredient.

[0022] In this invention, the composition is characterized in that it is administered or treated by injection, microneedling, iontophoresis, topical application, or a combination thereof.

[0023] In this invention, the composition is characterized in that it is selected from any one of the group consisting of liquid formulations, patch formulations, injectable formulations, infusion formulations and spray formulations.

[0024] The effects of the invention The exosomes derived from camellia callus provided by this invention exhibit the following activities: inhibiting nitric oxide production; reducing reactive oxygen species generated by UV radiation; inhibiting extracellular melanin production; inhibiting MMP-1 (matrix metalloproteinase-1) gene expression; increasing aquaporin 3 (AQP3) gene expression; reducing intercellular gaps; increasing filaggrin gene expression; inhibiting TARC (thymus and activation-regulated chemokine) production; and reducing vascular endothelial growth factor (VEGF) production. It enhances the expression activity of the VEGF gene, thus exhibiting excellent anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, wound healing, skin barrier improvement, atopic dermatitis improvement, and skin soothing effects. Therefore, it is highly effective as a cosmetic or pharmaceutical composition for anti-inflammatory, atopic dermatitis improvement, wound healing, and wound healing promotion. Attached Figure Description

[0025] Figure 1 This is a diagram showing the process of inducing camellia callus and mass-producing the callus through liquid culture.

[0026] Figure 2 This is a graph showing the results of measuring the size and concentration of exosomes in Examples 1-1 to 1-3, Example 2, Example 3 and Comparative Example 1 using a Nanoparticle Tracking Analyzer (NTA, Particle Metrix).

[0027] Figure 3The exosomes of Examples 1-1 to 1-3 and Comparative Example 1 were photographed using a transmission electron microscope (TEM).

[0028] Figure 4 The figure shows the results of confirming the cell activity of Examples 1-1 to 1-3 and Comparative Example 1 in mouse macrophages (RAW264.7), mouse melanoma cells (B16F10), human keratinocytes (HaCaT), and human fibroblasts (CCD-986sk) according to the present invention.

[0029] Figure 5 The figure shows the results of confirming the cell activity of the cells in mouse macrophages (RAW264.7), mouse melanoma cells (B16F10), human keratinocytes (HaCaT), and human fibroblasts (CCD-986sk) according to Examples 2, 3, and 1-3 of the present invention.

[0030] Figure 6 The graph shows the level of nitric oxide production in mouse macrophages (RAW 264.7) to confirm the anti-inflammatory effects of Examples 1-1 to 1-3 and Comparative Example 1 according to the present invention.

[0031] Figure 7 This is a graph showing the level of nitric oxide production in mouse macrophages (RAW 264.7) to confirm the anti-inflammatory effects of Examples 2, 3 and 1-3 of the present invention.

[0032] Figure 8 The figure shows the results of confirming the intracellular antioxidant effect of Examples 1-1 to 1-3 and Comparative Example 1 according to the present invention in human keratinocytes (HaCaT).

[0033] Figure 9 The graph shows the degree of melanin production in mouse melanoma cells (B16F10) to confirm the whitening effect of Examples 1-1 to 1-3 and Comparative Example 1 according to the present invention.

[0034] Figure 10 This is a graph showing the degree of melanin production in mouse melanoma cells (B16F10) to confirm the whitening effect of Examples 2, 3 and 1-3 of the present invention.

[0035] Figure 11This is a graph showing the mRNA expression level of matrix metalloproteinase-1 (MMP-1) in human fibroblasts (CCD-986sk) to confirm the anti-wrinkle effect of Examples 1-1 to 1-3 and Comparative Example 1 according to the present invention.

[0036] Figure 12 This is a graph showing the mRNA expression level of matrix metalloproteinase-1 (MMP-1) in human fibroblasts (CCD-986sk) to confirm the anti-wrinkle effect of Examples 2, 3 and 1-3 of the present invention.

[0037] Figure 13 This is a graph showing the results of confirming the moisturizing effect of Examples 1-1 to 1-3 and Comparative Example 1 according to the present invention, and the results of confirming the expression level of aquaporin 3 (AQP3) mRNA in human keratinocytes (HaCaT).

[0038] Figure 14 The figure shows the results of a wound healing experiment performed in human fibroblasts (CCD-986sk) to confirm the wound healing effect of Examples 1-1 to 1-3 and Comparative Example 1 according to the present invention.

[0039] Figure 15 This is a graph showing the results of confirming the skin barrier improvement effect of Examples 1-1 to 1-3 and Comparative Example 1 according to the present invention, and displaying the results of confirming the mRNA expression level of filaggrin (FLG) in human keratinocytes (HaCaT).

[0040] Figure 16 This is a graph showing the results of confirming the atopic improvement effect of Examples 1-1 to 1-3 and Comparative Example 1 according to the present invention, showing the results of confirming the amount of TARC (Thymus and Activation-regulated Chemokine) production in human keratinocytes (HaCaT).

[0041] Figure 17 This is a graph showing the results of VEGF (Vascular Endothelial Growth Factor) gene expression levels in human keratinocytes (HaCaT) to confirm the skin irritation improvement effect of Examples 1-1 to 1-3 and Comparative Example 1 according to the present invention. Detailed Implementation

[0042] The best way to implement an invention Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used in this specification is well-known and commonly used in the art.

[0043] Throughout this application specification, when a part is referred to as "containing" a certain constituent element, unless otherwise stated, it means that other components are not excluded, but other constituent elements may also be included.

[0044] As used in this specification, the term "callus" refers to various unorganized clusters of thin-walled cells in a plant, which typically contain cells with typical functions. Furthermore, callus can form in various parts of a plant, including flowers, leaves, stems, roots, seeds, and fruits. Callus formation and induction are generally carried out according to formally recognized methods in the field. More specifically, this callus formation and induction can be achieved by culturing plant tissues or explants in tissue culture media.

[0045] As used in this specification, the term "exosomes" refers to membrane-structured, nano-sized vesicles secreted or released from plant cells or plant stem cells into the extracellular space. Preferably, these are extracellular vesicles with an average particle size of 50–200 nm.

[0046] The term "extracellular vesicle" as used in this manual is a general term for membrane-bound vesicles secreted by cells for intercellular signal transduction, including exosomes, microvesicles, apoptotic bodies, etc.

[0047] Therefore, according to one embodiment of the present invention, the present invention relates to a cosmetic composition for anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement and skin soothing purposes, comprising exosomes derived from camellia callus tissue as an active ingredient.

[0048] According to one embodiment of the present invention, the exosomes derived from camellia callus can be isolated and purified from camellia-induced callus or the culture medium of callus.

[0049] The callus can be induced from any part of the group consisting of camellia flowers, leaves, stems, roots, seeds and fruits.

[0050] According to one embodiment of the present invention, the exosomes may be isolated from one or more selected from the group consisting of exosomes derived from callus tissue, fragments of callus tissue, and extracts of callus tissue.

[0051] Methods for obtaining exosomes from plants include exosomes derived from plant callus, exosomes derived from plant juice, and exosomes derived from ground plant material. However, in this invention, exosomes derived from plant callus are used as the active ingredient. The activity of the exosomes was confirmed using various methods, including exosomes derived from plant juice and ground plant material. The results showed that when exosomes were isolated and induced extracellularly through plant callus culture, they exhibited excellent activity because they preserved the main core components of plant cells.

[0052] According to one embodiment of the present invention, the exosomes may be extracellular vesicles with an average diameter of 50 to 200 nm.

[0053] According to one embodiment of the present invention, the composition may be any one selected from the group consisting of shampoo, soap, conditioner, cleansing products containing surfactants, skin, toner, cream, lotion, emulsion, serum, essence, suspension, emulsion, ointment, tonic, treatment, conditioner, paste, gel, oil, wax, spray, aerosol, mist, and powder, more preferably, it may be an emulsion or cream.

[0054] In cosmetic compositions containing camellia callus-derived exosomes of the present invention as active ingredients, which are used for anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement, and skin soothing purposes, the camellia callus-derived exosomes may be present in a concentration of 1.0 x 10⁻⁶. 8 Up to 1.0X10 12 The concentration of particles / mL contained in the composition is preferably 2.0 x 10⁻⁶. 9 Up to 2.0X10 10 The particle / mL concentration includes, but is not limited to, any amount sufficient to provide an effective quantity of exosomes derived from the preferred camellia callus tissue.

[0055] The cosmetic composition containing camellia callus-derived exosomes as an active ingredient, which has anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement, and skin soothing effects, can be appropriately combined with ingredients commonly used in cosmetic compositions, such as moisturizers, antioxidants, oily ingredients, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powder ingredients, colorants, aqueous ingredients, water, and various skin nutrients, etc., as needed, without compromising the effects of the present invention.

[0056] Cosmetic compositions containing camellia callus-derived exosomes as active ingredients for anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement, and skin soothing purposes, in addition to camellia callus-derived exosomes, can be used in combination with skin improvers and / or moisturizers used in the prior art, to the extent that their effects (e.g., anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement, skin soothing, etc.) are not impaired.

[0057] The cosmetic composition containing camellia callus-derived exosomes as an active ingredient, which has anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement, and skin soothing effects, can be applied in various forms such as patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, oils, face packs, sprays, aerosols, mists, foundations, powders, and oil paper.

[0058] Cosmetic compositions containing camellia callus-derived exosomes as active ingredients for anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement, and skin soothing purposes can be used for anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement, and skin soothing purposes. The cosmetic dosage form can be formulated into any dosage form commonly prepared in the art. For example, it can be made into patches, mask packs, mask sheets, skin softeners, nutrition lotions, astringent lotions, nutrition creams, massage creams, eye creams, cleansing creams, essences, eye serums, cleansing lotions, cleansing foams, cleansing waters, sunscreens, lipsticks, soaps, shampoos, surfactant-containing cleansers, bath agents, body lotions, body creams, body oils, body essences, body cleansers, and hair dyes. Hair dye, hair tonic, etc., but not limited to these.

[0059] This invention relates to a cosmetic composition containing camellia callus-derived exosomes as an active ingredient, which has anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, skin barrier improvement, atopic dermatitis improvement, and skin soothing effects. The composition includes ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, as well as carriers. Furthermore, for various dosage forms of the cosmetic composition, other ingredients can be easily and appropriately selected and combined by those skilled in the art, depending on the type of cosmetic composition or its intended use.

[0060] As previously mentioned, exosomes derived from camellia callus exhibit activities that inhibit nitric oxide production, reduce reactive oxygen species generated by UV radiation, inhibit extracellular melanin production, inhibit MMP-1 (matrix metalloproteinase-1) gene expression, increase aquaporin 3 (AQP3) gene expression, reduce intercellular gaps, increase filaggrin gene expression, inhibit TARC (thymus and activation-regulated chemokine) production, and reduce vascular endothelial growth factor (VEGF) production. This product enhances the expression activity of the VEGF (Vitamin B12) gene, thus exhibiting excellent anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, wound healing, skin barrier improvement, atopic dermatitis improvement, and skin soothing effects. Therefore, it is highly effective as a cosmetic or pharmaceutical composition for anti-inflammatory, atopic dermatitis improvement, wound healing, and wound healing promotion purposes. Furthermore, compared to simple extracts or culture media, it has a lower likelihood of containing impurities.

[0061] Furthermore, the present invention relates to a pharmaceutical composition for anti-inflammatory, atopic dermatitis improvement, wound healing and wound healing promotion, comprising exosomes derived from camellia callus as an active ingredient.

[0062] According to one embodiment of the present invention, the composition is characterized by being administered or treated by injection, microneedling, iontophoresis, topical application, or a combination thereof.

[0063] According to one embodiment of the present invention, the composition may be any one selected from the group consisting of liquid formulations, patch formulations, injection formulations, infusion formulations and spray formulations.

[0064] When used in the pharmaceutical compositions of the present invention for anti-inflammatory, atopic dermatitis improvement, wound healing, and wound healing promotion, the compositions may contain pharmaceutically acceptable carriers, excipients, or diluents. Examples of such carriers, excipients, and diluents include lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, but are not limited thereto. Furthermore, the effective amount of the pharmaceutical composition in the embodiments of the present invention refers to the amount required to administer the drug in order to achieve the desired anti-inflammatory, atopic dermatitis improvement, wound healing, and wound healing promotion effects.

[0065] Therefore, the exosomes derived from camellia callus provided by this invention have the following activities: inhibiting nitric oxide production, reducing reactive oxygen species generated by UV radiation, inhibiting extracellular melanin production, inhibiting MMP-1 (matrix metalloproteinase-1) gene expression, increasing aquaporin 3 (AQP3) gene expression, reducing intercellular gaps, increasing filaggrin gene expression, inhibiting TARC (thymus and activation-regulated chemokine) production, and reducing vascular endothelial growth factor (VEGF) production. It enhances the expression activity of the VEGF gene, thus exhibiting excellent anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, skin regeneration, wound healing, skin barrier improvement, atopic dermatitis improvement, and skin soothing effects. Therefore, it is highly effective as a cosmetic or pharmaceutical composition for anti-inflammatory, atopic dermatitis improvement, wound healing, and wound healing promotion.

[0066] Methods of implementing the invention The present invention will now be described in more detail through embodiments. These embodiments are merely for illustrating the invention more specifically, and it will be apparent to those skilled in the art that the scope of the invention is not limited to these embodiments, as they embody the spirit of the invention.

[0067] <Example 1> In vitro introduction and callus induction of camellia Camellia used in this invention ( Camellia japonicaLeaves were collected in Yeosu, Jeollanam-do, South Korea in October. After soaking in 95% ethanol for 60 seconds, the leaves were disinfected with a disinfectant solution (50% sodium hypochlorite + 0.1% Tween-20) for 20 minutes, and then washed with sterile water at least three times. Explants measuring 2*2 mm in length and width were prepared from the washed leaf tissue using a sharp knife. A combination of 2,4-D (2,4-dichlorophenoxyacetic acid, Sigma-Aldrich) and 6-BA (6-benzylaminopurine solution, Sigma-Aldrich) was used as a plant growth regulator to test callus induction rate. When comparing the effects of different combinations of growth regulators on callus formation rate and proliferation rate, the culture medium containing 0.3 mg / L 2,4-dichlorophenoxyacetic acid and 1 mg / L 6-benzylaminopurine as growth regulators showed significantly higher callus formation rate and proliferation rate compared to the comparative study which used 2 mg / L 2,4-dichlorophenoxyacetic acid (an auxin-based hormone) and 0.2 mg / L kinetin (a cytokinin-based hormone). This indicates that the types and amounts of growth regulators used in this study were optimally applied to promote rapid callus formation and proliferation. Table 1 below shows the callus formation rate and proliferation rate of Camellia japonica based on the growth regulators. Callus was induced by dark culture in a growth chamber at 25±2℃ and 60% humidity in basal MS medium containing 3% sucrose as a carbon source and 0.4% gelrite as a medium solidifying agent. The callus induction status was observed using a microscope (zoom stereo microscope head & dual light, KS-306). Superior cell lines were selected through 2–3 subcultures every 4 weeks in solid medium.Liquid culture was conducted for 4 weeks in a 10L bioreactor at 25±2℃ in MS liquid medium containing 0.3 mg / L of 2,4-D (2,4-dichlorophenoxyacetic acid, Sigma-Aldrich) and 1 mg / L of 6-BA (6-benzylaminopurine solution, Sigma-Aldrich) hormone, with an aeration rate of 0.2 vvm. Figure 1 ).

[0068] Table 1

[0069] a The callus induction rate is defined as: callus induction rate (%) = (number of explants that produce callus / total number of explants cultured) × 100%.

[0070] b The callus proliferation rate was recorded based on the daily growth of callus after induction and was divided into three grades by comparison: very good (+++), moderate (++), and poor (+).

[0071] <Example 1-1> Isolation of exosomes from callus tissue derived from camellia leaves The camellia leaf-derived callus culture harvested after large-scale cultivation in Example 1 was filtered to separate callus tissue and culture medium, yielding only callus tissue. 20% by weight of the obtained callus tissue was mixed with 80% by weight of sterile distilled water and then ground to obtain a grinding liquid. The grinding liquid was centrifuged to remove impurities. The centrifuge was performed sequentially at 1,000–2,000 x g for 10 minutes and then at 3,000–4,000 x g for 10–20 minutes to remove the precipitate, collecting only the supernatant. The supernatant obtained in this process was centrifuged at 10,000–15,000 x g for 30–60 minutes using an ultracentrifuge. To further improve the purity of the supernatant, it was filtered to separate and remove residues, thereby further improving purity. Exosomes present in the high-purity supernatant were centrifuged at 100,000–150,000 x g for 2–4 hours using an ultracentrifuge to precipitate the exosomes in the sediment layer. The exosomes in the final sediment layer were resuspended and dispersed in sterile distilled water to prepare exosomes. Finally, the exosomes were sterilized by passing them through a 0.2 μm syringe-type membrane (Syringe filter) to prepare the final camellia callus-derived exosomes.

[0072] <Examples 1-2> Isolation of exosomes from callus culture medium derived from camellia leaves The camellia callus culture collected in Example 1 after extensive culturing was filtered to separate the callus tissue and culture medium, yielding only the culture medium. Exosomes were prepared from the obtained culture medium using the same method as in Examples 1-1 above.

[0073] <Examples 1-3> Isolation of exosomes from callus tissue and culture medium mixture derived from camellia leaves The camellia callus culture obtained after large-scale culture in Example 1 was filtered to separate the callus tissue and culture medium. 20% by weight of the callus tissue and 80% by weight of the culture medium were then mixed and ground to obtain a ground liquid. The obtained ground liquid was used to prepare exosomes using the same method as in Examples 1-1 above.

[0074] <Example 2> Isolation of exosomes from callus tissue derived from camellia Using the same method as in Example 1 above, camellia flowers were introduced in vitro and callus tissue was induced, which was then cultured in a 10L bioreactor. After harvesting a large number of camellia callus cultures, the cultures were filtered to separate the callus tissue and culture medium. 20% by weight of the callus tissue and 80% by weight of the culture medium were mixed and then ground to obtain a grinding solution. The obtained grinding solution was used to prepare exosomes using the same method as in Example 1-1 above.

[0075] <Example 3> Isolation of exosomes from callus tissue derived from camellia seeds Using the same method as in Example 1 above, camellia seeds were introduced in vitro and callus tissue was induced, then cultured in a 10L bioreactor. The camellia seed callus cultures harvested after a large-scale culture were filtered to separate the callus tissue and culture medium. 20% by weight of the callus tissue and 80% by weight of the culture medium were mixed and then ground to obtain a grinding solution. The obtained grinding solution was used to prepare exosomes using the same method as in Example 1-1 above.

[0076] <Comparative Example 1> Isolation of exosomes from camellia leaves 20% by weight of the disinfected and washed tea leaves from Example 1 were mixed with 80% by weight of sterile distilled water and then ground to obtain a grinding liquid. The resulting grinding liquid was then used to obtain exosomes using the same method as in Examples 1-1 above.

[0077] <Experimental Example 1> Confirmation of Exosome Granules To perform quantitative and physical evaluation of the exosomes isolated from Examples 1-1 to 1-3, Example 2, Example 3, and Comparative Example 1, analysis was conducted using a Nanoparticle Tracking Analysis (NTA, manufactured by Particle Metrix). The NTA is a device used to determine the number and size of exosome particles; a Zetaview Zeta potential model from Particle Metrix was used. For the determination, the exosome solution was diluted appropriately in sterile distilled water, and the laser wavelength was set to 488 nm with a conductivity of 1.00 μS / cm.

[0078] The result, such as Figure 2 As shown, the particle number and particle size were confirmed, and the high purity of the particles was confirmed by the SPAN value. Based on this confirmed particle number, a sample diluted to a concentration of 2.0E+9 particles / ml was used as 100% for the experiment.

[0079] On the other hand, to analyze the morphology of Examples 1-1 to 1-3 and Comparative Example 1, analysis was performed using a transmission electron microscope (JEOL, JEM 1010). Specifically, 10 μL of the isolated samples from Examples 1-1 to 1-3 and Comparative Example 1 were dropped onto a carbon-plated copper grid and deposited at room temperature for 1 to 5 minutes. For negative staining of exosomes, 2% uranyl acetate was used as a control for 1 to 2 minutes. The stained exosomes were then photographed using a transmission electron microscope.

[0080] The result, such as Figure 3 As shown, it can be confirmed that the morphology of exosomes is spherical with a phospholipid bilayer.

[0081] <Experimental Example 2> Cytotoxicity Assessment: WST-1 Assay In order to conduct in vitro (Examples 1-1 to 1-3, Example 2, Example 3 and Comparative Example 1) in-vitro In the efficacy experiment, different concentrations were first applied to confirm whether it was toxic to each cell line.

[0082] Cytotoxicity experiments were performed on mouse macrophages (RAW 264.7), mouse melanoma cells (B16F10), human keratinocytes (HaCaT), and human fibroblasts (CCD-986sk). RAW 264.7, B16F10, and HaCaT cells were cultured in DMEM (Dulbecco's Modified Essential Medium) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S), respectively. CCD-986sk cells were cultured in IMDM (Iscove's Modified Dulbecco's Medium) containing 10% FBS and 1% P / S in an incubator at 37°C and 5% CO2. The cultured cells were aliquoted into 96-well plates at appropriate concentrations and cultured in an incubator at 37°C and 5% CO2 to allow them to adhere to the plates. After 24 hours, the medium was replaced with a 0-10% dilution of the medium used for Examples 1-1 to 1-3, Examples 2, Examples 3, and Comparative Example 1. After another 24 hours, the medium was removed, and each well was treated with 10% WST-1 reagent. After reacting in the incubator for 2 hours, the absorbance was measured at 450 nm using an ELISA reader (BioTeck, USA), and cell viability was confirmed according to the following mathematical formula 1.

[0083] [Mathematical Expression 1] Cell viability (%) = [Absorbance of the sample treatment group / Absorbance of the blank sample treatment group] × 100 The result, such as Figure 4 As shown, Figure 4 a represents the cytotoxicity assessment results of mouse macrophages (RAW 264.7). Figure 4 b represents the cytotoxicity assessment results of mouse melanoma cells (B16F10). Figure 4 c represents the cytotoxicity assessment results of human keratinocytes (HaCaT). Figure 4 d represents the cytotoxicity assessment results of human fibroblasts (CCD-986sk). When Examples 1-1 to 1-3 and Comparative Example 1 were treated with RAW 264.7, B16F10, HaCaT, and CCD-986sk, no cytotoxicity was observed at any of the treated concentrations.

[0084] In addition, such as Figure 5As shown, the results of cytotoxicity assessment of exosomes isolated from callus induced from different parts of Camellia in Examples 1-3, 2, and 3, on mouse macrophages (RAW 264.7), mouse melanoma cells (B16F10), human keratinocytes (HaCaT), and human fibroblasts (CCD-986sk) confirmed that no cytotoxicity was observed at any of the treated concentrations.

[0085] <Experimental Example 3> Anti-inflammatory efficacy test: Nitric oxide production inhibition activity To understand the anti-inflammatory effects of Examples 1-1 to 1-3, Example 2, Example 3 and Comparative Example 1, the production of nitric oxide (NO) as an indicator of inflammatory response was measured using RAW 264.7 cells, which are mouse macrophages.

[0086] RAW 264.7 cells were planted at a density of 2 × 10⁶ cells per well. 5 After aliquoting the samples into 24-well plates, the cells were incubated at 37°C and 5% CO2 for 24 hours to allow cell adhesion. Samples were diluted to different concentrations in DMEM medium containing 1 μg / ml of lipopolysaccharide (LPS), an inflammatory inducing factor that promotes NO production. As a positive control, 50 μM N-monomethyl-L-arginine acetate (L-NMMA) was diluted for use. After removing the culture medium from the cultured cells, the prepared samples (Examples 1-1 to 1-3, Example 2, Example 3, Comparative Example 1, and the positive control) were treated and incubated at 37°C and 5% CO2 for 24 hours. The culture medium was recovered and dispensed into 96-well plates at 100 μL per well. Equal volumes of Griess reagent A (Naphthylethylene-diamine buffer) and Griess reagent B (Sulfanilamide buffer) were then added to bring the total volume to 200 μL. After reacting for 10 minutes, the absorbance was measured at 540 nm. The amount of nitric oxide (NO) produced was confirmed using a standard curve prepared with nitrite diluted at different concentrations.

[0087] The result, such as Figure 6As shown, it was confirmed that NO generation was reduced through Examples 1-1 to 1-3 and Comparative Example 1, and the NO generation inhibition activity of Examples 1-1 to 1-3 was superior to that of Comparative Example 1. In particular, when treated with a 10% concentration, the NO generation reduction effect was confirmed to be 54.19% for Example 1-1, 49.85% for Example 1-2, 56.73% for Example 1-3, and 20.27% for Comparative Example 1.

[0088] In addition, such as Figure 7 The results show the effectiveness of exosomes isolated from callus induced from different parts of Camellia in inhibiting NO production in Examples 1-3, 2, and 3. A reduction in NO production was confirmed. When each example was treated with a 10% concentration, Examples 1-3 showed a 55.90% reduction in NO production, Example 2 showed 31.83%, and Example 3 showed 29.52%. The NO production inhibition effect of Examples 1-3 was particularly significant.

[0089] <Experiment Example 4> Antioxidant Efficacy Test: Confirmation Test of Intracellular Reactive Oxygen Species (ROS) Reduction To confirm the antioxidant effects of Examples 1-1 to 1-3 and Comparative Example 1, the intracellular antioxidant effects were confirmed using HaCaT cells of human keratinocytes via the H2DCFDA assay.

[0090] HaCaT cells were used at a rate of 1×10 4 Cells were dispensed into 96-well plates and cultured for 24 hours at 37°C with 5% CO2 to allow cell adhesion. The culture medium was then replaced with serum-free medium and cultured for another 24 hours. Next, the cells were reacted with 5 mM CM-H2DCFDA at 37°C for 30 minutes. Then, cells diluted appropriately (Examples 1-1 to 1-3, Comparative Example 1, and 2 μM Trolox as a positive control) were treated and cultured for 1 hour at 37°C with 5% CO2. Following this, the cells were treated with 500 mJ / cm² UVB to induce reactive oxygen species (ROS) generation and cultured for 1 hour at 37°C with 5% CO2. After the reaction, fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 528 nm, and antioxidant capacity was assessed according to the following mathematical formula 2.

[0091] [Mathematical Expression 2] Intracellular reactive oxygen species production (%) = fluorescence intensity of the sample treatment group / fluorescence intensity of the blank sample treatment group × 100 The result, such as Figure 8As shown, it can be confirmed that reactive oxygen species generated by UV radiation are reduced through Examples 1-1 to 1-3 and Comparative Example 1, and the reactive oxygen species reduction effect of Examples 1-1 to 1-3 is superior to that of Comparative Example 1. In particular, when treated with a 10% concentration, Example 1-1 showed a reactive oxygen species reduction effect of 34.87%, Example 1-2 33.03%, Example 1-3 37.33%, and Comparative Example 1 28.80%.

[0092] <Experimental Example 5> Whitening Efficacy Test: Analysis of Extracellular Melanin Content To confirm the whitening effect of Examples 1-1 to 1-3, Example 2, Example 3 and Comparative Example 1, an extracellular melanin content assay was performed using mouse melanoma B16F10 cells.

[0093] B16F10 cells were spaced at 3 × 10⁶ cells per well. 4 After aliquoting the samples into 24-well plates, the cells were cultured at 37°C and 5% CO2 for 24 hours to allow cell adhesion. Examples 1-1 to 1-3, Examples 2, Examples 3, and Comparative Example 1 were diluted to different concentrations in DMEM medium (phenol red-free) containing α-Melanocyte Stimulation Hormone (α-MSH) to induce melanin production. As a positive control, β-arbutin was diluted to 200 μg / ml. After removing the culture medium from the cultured cells, the prepared samples diluted to different concentrations and the positive control were processed and incubated at 37°C and 5% CO2 for 48–72 hours. After culture, the culture medium was recovered, centrifuged, and aliquoted into 96-well plates. The absorbance was measured at 490 nm, and the extracellular melanin production rate was calculated using the following mathematical formula 3.

[0094] [Mathematical Expression 3] Melanin production rate (%) = Melanin amount in the sample treatment group / Melanin amount in the blank sample treatment group × 100 The result, such as Figure 9 As shown, it was confirmed that the amount of melanin secreted into the extracellular space was reduced through Examples 1-1 to 1-3 and Comparative Example 1. Compared with Comparative Example 1, the extracellular melanin reduction effect of Examples 1-1 to 1-3 was superior. In particular, when treated with a 10% concentration, the extracellular melanin reduction effect was confirmed to be 32.81% for Example 1-1, 33.60% for Example 1-2, 34.60% for Example 1-3, and 22.80% for Comparative Example 1.

[0095] In addition, such as Figure 10 As shown, after processing exosomes isolated from callus tissue induced from different parts of Camellia japonica in Examples 1-3, 2, and 3, the amount of melanin secreted extracellularly was confirmed. All examples showed a reduction in melanin secretion. When treated with a 10% concentration, Examples 1-3 showed a 35.87% reduction in extracellular melanin secretion, Examples 2 showed 25.92%, and Examples 3 showed 23.82%. Among these, Examples 1-3 demonstrated the most significant reduction in extracellular melanin secretion.

[0096] <Experimental Example 6> Wrinkle Improvement Efficacy Test: Determination of MMP-1 mRNA Expression Level To understand the wrinkle-improving effects of Examples 1-1 to 1-3, Example 2, Example 3 and Comparative Example 1, the mRNA expression level of matrix metalloproteinase-1 (MMP-1), a collagen-degrading enzyme, was measured using human fibroblasts.

[0097] Human fibroblast CCD-986sk cells were cultured at 2 × 10⁶ cells per well. 4 Cells were dispensed into 24-well plates and cultured at 37°C with 5% CO2 for 24 hours to allow cell adhesion. The culture medium was then removed, and DPBS was added. The cells were then irradiated with UVA at 1 J / cm². Treatments were performed using cells diluted appropriately in serum-free medium in Examples 1-1 to 1-3, Examples 2, Examples 3, and Comparative Example 1. As a positive control, hTGF-β (human Tumor Growth Factor-β) was treated at a concentration of 5 ng / mL. RNA was then extracted from the cells of each treated sample according to the procedures outlined in the NucleoSpin RNA Kit from Macherey-Nagel. The extracted RNA was quantified as total RNA at 260 nm using a Nanodrop spectrophotometer, and cDNA was synthesized using an appropriate amount of RNA in an amplification instrument. Real-time polymerase chain reaction (PCR) was performed on a real-time PCR machine using a mixture of target gene-specific primers and SYBR Green supermix (as an anthocyanin dye) added to the synthesized cDNA to ultimately assess the expression level of the target gene. The primer sequences and reaction conditions for the target gene are shown in Table 2 below. Gene expression levels were finally analyzed after correction for the β-actin gene.

[0098] Table 2

[0099] The result, such as Figure 11 As shown, Examples 1-1 to 1-3 and Comparative Example 1 confirmed that they reduced MMP-1 expression in a concentration-dependent manner, and the MMP-1 reduction activity of Examples 1-1 to 1-3 was superior to that of Comparative Example 1. In particular, when treated with a 10% concentration, compared with the untreated group, the MMP-1 reduction effect of Example 1-1 was confirmed to be 35.39%, Example 1-2 33.62%, Example 1-3 38.61%, and Comparative Example 1 18.36%.

[0100] In addition, such as Figure 12 As shown, the expression levels of MMP-1 mRNA by exosomes isolated from callus induced from different parts of Camellia were confirmed in Examples 1-3, 2, and 3. When each example was treated with a 10% concentration, Examples 1-3 showed a 38.61% reduction in MMP-1 mRNA expression, Examples 2 showed 27.00%, and Examples 3 showed 24.00%. Among them, Examples 1-3 showed the most excellent reduction in MMP-1 mRNA expression.

[0101] <Experimental Example 7> Skin Moisturizing Activity Assay: Determination of Aquaporin-3 (AQP-3) mRNA Expression To confirm the moisturizing improvement effects of Examples 1-1 to 1-3 and Comparative Example 1, the mRNA expression level of aquaporin 3 (AQP3) was confirmed using human keratinocyte HaCaT cells.

[0102] Human keratinocyte HaCaT cells were used at a density of 1 × 10⁻⁶ cells per well. 5 Cells were dispensed into 24-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion. Then, cells were treated with serum-free medium diluted to appropriate concentrations as in Examples 1-1 to 1-3 and Comparative Example 1, and reacted for 24 hours. RNA was then extracted following the procedures outlined in the Macherey-Nagel NucleoSpin RNA Kit. The extracted RNA was quantified as total RNA at 260 nm using a Nanodrop spectrophotometer, and cDNA was synthesized using an appropriate amount of RNA in an amplification instrument. Real-time polymerase chain reaction (PCR) was performed on the synthesized cDNA using a mixture of target gene-specific primers and SYBR Green supermix as an anthocyanin dye to ultimately assess the expression level of the target gene. The primer sequences and reaction conditions for the target gene are shown in Table 3 below. Gene expression levels were finally analyzed after correction for the β-actin gene.

[0103] Table 3

[0104] The result, such as Figure 13 As shown, Examples 1-1 to 1-3 and Comparative Example 1 confirmed that AQP-3 expression was increased in a concentration-dependent manner, and the expression-increasing activity of Examples 1-1 to 1-3 was superior to that of Comparative Example 1. In particular, when treated with a 10% concentration, compared with the untreated group, it was confirmed that Example 1-1 increased by 45.18%, Example 1-2 by 41.63%, Example 1-3 by 46.28%, and Comparative Example 1 by 14.87%.

[0105] <Experimental Example 8> Skin Regeneration and Wound Healing Improvement Efficacy Test: Wound Healing Assay To understand the skin regeneration and wound healing effects of Examples 1-1 to 1-3 and Comparative Example 1, a wound healing experiment was conducted using human fibroblasts.

[0106] Human fibroblasts CCD-986sk were used at 5 × 10⁶ cells per well. 4 Cells were dispensed into 24-well plates containing wound healing inserts and cultured at 37°C with 5% CO2 for 24 hours. Once cell confluence reached 80%, the wound healing inserts were removed, and the cells were washed with DPBS. Examples 1-1 to 1-3 and Comparative Example 1, diluted appropriately in serum-free medium, were treated. After 48 hours of reaction, the degree of filling of artificially created gaps between cells was assessed under a microscope to confirm cell migration, and the expanded area was calculated using the following mathematical formula 4.

[0107] [Mathematical Expression 4] Invaded Area (%) = 100 - (Area of ​​treated sample group / Area of ​​blank sample group) × 100 The result, such as Figure 14 As shown, it can be confirmed that the intercellular spaces narrowed through Examples 1-1 to 1-3 and Comparative Example 1, and the degree of intercellular space recovery in Examples 1-1 to 1-3 was superior compared to Comparative Example 1, thus demonstrating the efficacy of improving skin regeneration and wound healing. In particular, when each sample was treated with a 10% concentration, it was confirmed that the intercellular space was reduced by 57.67% in Example 1-1, 71.81% in Example 1-2, 72.22% in Example 1-3, and 24.03% in Comparative Example 1.

[0108] <Experiment Example 9> Skin Barrier Improvement Effect Test: Determination of Filagrin mRNA Expression To confirm the skin barrier improvement effect of Examples 1-1 to 1-3 and Comparative Example 1, the mRNA expression level of filaggrin (FLG) was measured using human keratinocytes.

[0109] Human keratinocyte HaCaT cells were used at a density of 1 × 10⁻⁶ cells per well. 5 Cells were dispensed into 24-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion. Then, cells were treated with serum-free medium diluted to appropriate concentrations as in Examples 1-1 to 1-3 and Comparative Example 1, and reacted for 24 hours. RNA was then extracted following the procedures outlined in the Macherey-Nagel NucleoSpin RNA Kit. The extracted RNA was quantified as total RNA at 260 nm using a Nanodrop spectrophotometer, and cDNA was synthesized using an appropriate amount of RNA in an amplification instrument. Real-time polymerase chain reaction (PCR) was performed on the synthesized cDNA using a mixture of target gene-specific primers and SYBR Green supermix as an anthocyanin dye to ultimately assess the expression level of the target gene. The primer sequences and reaction conditions for the target gene are shown in Table 4 below. Gene expression levels were finally analyzed after correction for the β-actin gene.

[0110] Table 4

[0111] The result, such as Figure 15 As shown, Examples 1-1 to 1-3 and Comparative Example 1 confirmed that they increased FLG expression in a concentration-dependent manner, and compared with Comparative Example 1, Examples 1-1 to 1-3 showed superior FLG expression-increasing activity. In particular, when treated with a 10% concentration, compared with the untreated group, Examples 1-1 showed an increase of 31.59%, Examples 1-2 an increase of 32.89%, Examples 1-3 an increase of 35.35%, and Comparative Example 1 an increase of 15.29%.

[0112] <Experimental Example 10> Improvement of Atopic Dermatitis: TARC Production Inhibition Test To confirm the atopic improvement effect of Examples 1-1 to 1-3 and Comparative Example 1, a TARC (Thymus and Activation-regulated Chemokine) production confirmation test was performed using human keratinocyte HaCaT cells.

[0113] HaCaT was applied at a rate of 1×10 per well. 5 Cells were dispensed into 24-well plates and cultured at 37°C with 5% CO2 for 24 hours to allow cell adhesion. Examples 1-1 to 1-3 and Comparative Example 1 were diluted to different concentrations in serum-free DMEM medium containing 10 ng / ml of inflammatory inducing factors TNF-α and INF-γ. As a positive control, TGF-β (Transforming Growth Factor-β) was diluted to 10 ng / ml. The culture medium was then removed, and the prepared samples of Examples 1-1 to 1-3, Comparative Example 1, and the positive control were treated and cultured at 37°C with 5% CO2 for 24 hours. The culture medium was then recovered, and TARC production was measured. To determine TARC production, ELISA analysis was performed according to the procedure of the ELISA kit (Biolegend, 441104). The TARC production was converted to a percentage using the TARC standard curve obtained from the ELISA analysis.

[0114] The result, such as Figure 16 As shown, it was confirmed that the amount of TARC generated was reduced through Examples 1-1 to 1-3 and Comparative Example 1, and the TARC generation reduction activity of Examples 1-1 to 1-3 was superior to that of Comparative Example 1. In particular, when treated with a 10% concentration, the TARC generation reduction effect was confirmed to be 21.21% for Example 1-1, 21.68% for Example 1-2, 23.62% for Example 1-3, and 11.82% for Comparative Example 1.

[0115] <Experimental Example 11> Skin Soothing and Improvement Effect Test: Determination of VEGF mRNA Expression Level To confirm the skin-soothing and improving effects of Examples 1-1 to 1-3 and Comparative Example 1, the mRNA expression level of vascular endothelial growth factor (VEGF) was measured using human keratinocyte HaCaT cells.

[0116] Human keratinocyte HaCaT cells were used at a density of 1 × 10⁻⁶ cells per well. 5Cells were dispensed into 24-well plates and cultured at 37°C with 5% CO2 for 24 hours to allow cell adhesion. Subsequently, for skin stimulation, cells were irradiated with UVA at a concentration of 0.5 J / cm². Then, cells from Examples 1-1 to 1-3 and Comparative Example 1, diluted appropriately in serum-free medium, were treated and reacted for 24 hours. RNA was then extracted according to the Macherey-Nagel NucleoSpin RNA Kit. The extracted RNA was quantified as total RNA at 260 nm using a Nanodrop spectrophotometer, and cDNA was synthesized using an appropriate amount of RNA in an amplification instrument. Real-time polymerase chain reaction (PCR) was performed on the synthesized cDNA using a mixture of target gene-specific primers and SYBR Green supermix as an anthocyanin dye to ultimately assess the expression level of the target gene. The primer sequences and reaction conditions for the target genes are shown in Table 5 below. The gene expression levels were finally analyzed after correction for the β-actin gene.

[0117] Table 5

[0118] The result, such as Figure 17 As shown, Examples 1-1 to 1-3 and Comparative Example 1 confirmed that they reduced VEGF mRNA expression in a concentration-dependent manner, and compared with Comparative Example 1, Examples 1-1 to 1-3 showed superior VEGF mRNA expression reduction activity. In particular, when treated with a 10% concentration, compared with the untreated group, Examples 1-1 showed a reduction of 34.62%, Examples 1-2 a reduction of 31.16%, Examples 1-3 a reduction of 37.44%, and Comparative Example 1 a reduction of 12.36%.

[0119] The foregoing has described specific parts of the present invention in detail. It will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the invention. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents.

[0120] Industrial availability Because the camellia callus-derived exosomes provided by this invention have excellent anti-inflammatory, antioxidant, whitening, anti-wrinkle, moisturizing, wound healing, skin barrier improvement, atopic dermatitis improvement, and skin soothing effects, they can be effectively applied to cosmetics or pharmaceutical compositions for anti-inflammatory, atopic dermatitis improvement, wound healing, and wound healing promotion.

Claims

1. An anti-inflammatory, anti-oxidative, whitening, anti-wrinkle, moisturizing, skin regenerating, skin barrier improving, atopic dermatitis improving, and skin soothing cosmetic composition comprising camellia callus-derived exosomes as an effective ingredient.

2. The anti-inflammatory, anti-oxidative, whitening, anti-wrinkle, moisturizing, skin regenerating, skin barrier improving, atopic dermatitis improving, and skin soothing cosmetic composition according to claim 1, wherein the camellia callus-derived exosomes are isolated and purified from a callus induced from camellia or a culture solution of the callus.

3. The anti-inflammatory, anti-oxidative, whitening, anti-wrinkle, moisturizing, skin regenerating, skin barrier improving, atopic dermatitis improving, and skin soothing cosmetic composition according to claim 2, wherein the callus is induced from any one part selected from the group consisting of flowers, leaves, stems, roots, seeds, and fruits of camellia.

4. The anti-inflammatory, anti-oxidative, whitening, anti-wrinkle, moisturizing, skin regenerating, skin barrier improving, atopic dermatitis improving, and skin soothing cosmetic composition according to claim 1, wherein the exosomes are isolated from one or more selected from the group consisting of callus-derived exosomes, a callus disruptate, and a callus extract.

5. The anti-inflammatory, anti-oxidative, whitening, anti-wrinkle, moisturizing, skin regenerating, skin barrier improving, atopic dermatitis improving, and skin soothing cosmetic composition according to claim 1, wherein the exosomes are extracellular vesicles having an average diameter of 50 to 200 nm.

6. The anti-inflammatory, anti-oxidative, whitening, anti-wrinkle, moisturizing, skin regenerating, skin barrier improving, atopic dermatitis improving, and skin soothing cosmetic composition according to claim 1, wherein the composition is any one selected from the group consisting of a shampoo, a soap, a hair conditioner, a surfactant-containing cleansing product, a cosmetic water, a toner, a cream, an emulsion, an emulsin, an essence, a serum, a suspension, a suspoemulsion, an ointment, a skin tightening water, a hair care serum, a hair conditioner, a paste, a gel, an oil, a wax, a spray, an aerosol, a spray, and a powder.

7. An anti-inflammatory, atopic dermatitis improving, wound healing, and wound healing promoting pharmaceutical composition comprising camellia callus-derived exosomes as an effective ingredient.

8. The anti-inflammatory, atopic dermatitis improving, wound healing, and wound healing promoting pharmaceutical composition according to claim 7, wherein the composition is administered or treated by injection, microneedle, iontophoresis, application, or a combination thereof.

9. The anti-inflammatory, atopic dermatitis improving, wound healing, and wound healing promoting pharmaceutical composition according to claim 7, wherein the composition is any one selected from the group consisting of a liquid preparation, a patch preparation, an injection preparation, an infusion preparation, and a spray preparation. ​ ​ ​ ​ ​ ​ ​

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