Composition for promoting EGF activity comprising compound k

Abstract

The present disclosure relates to a composition for promoting the activity of epidermal growth factor (EGF), comprising Compound K. Specifically, the composition for promoting the activity of EGF, according to the present disclosure, promotes the activity of EGF, which hardly permeates into the stratum corneum of the skin and has very low stability in cosmetic formulations, making it difficult to expect its effectiveness as a cosmetic ingredient, and thus when mixed with a low content of EGF, which alone does not exhibit an effect, excellent skin efficacy may be exhibited due to the epidermal cell proliferation activity of EGF.

Description

Composition for promoting the activity of EGF containing compound K

[0001] The present disclosure relates to a composition for promoting the activity of epidermal growth factor (EGF) containing compound K.

[0002] Cross-reference to related applications

[0003] This application claims priority to Republic of Korea Patent Application No. 10-2024-0052786, filed April 19, 2024, the entire contents of which are incorporated herein by reference.

[0004] EGF (epidermal growth factor) is an epidermal growth factor that acts on EGF receptors to promote their activity, directly promoting the proliferation and division of epidermal cells and demonstrating excellent effects in improving skin hydration, elasticity, and transparency. EGF receptors decrease in expression and activity with aging or due to external stimuli such as ultraviolet rays. This decrease in EGF receptor expression and activity is a major cause of skin aging due to decreased epidermal cell growth caused by aging or ultraviolet rays. In particular, the inhibition of epidermal cell growth causes an imbalance in the proliferation and differentiation of epidermal cells, leading to a loss of barrier function and severe skin dryness. Furthermore, by delaying the turnover cycle of epidermal cells, it thickens the stratum corneum and makes the shape and size of epidermal cells irregular, resulting in light being scattered instead of properly reflected, reducing skin radiance and transparency.

[0005] Using EGF, a cosmetic ingredient that directly promotes epidermal cell proliferation, could be a solution to these various skin aging symptoms. However, EGF barely penetrates the stratum corneum, and even when used with various skin absorption-enhancing carriers, only a very small percentage (approximately 1 / 20) is reported to be able to penetrate the skin and take effect. Furthermore, its stability in cosmetic formulations is very low. For these reasons, it is difficult to expect direct efficacy from EGF as a cosmetic ingredient.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] (Patent Document 1) Republic of Korea Patent Publication No. 10-2014-0003198

[0009] The present invention aims to provide a composition that exhibits excellent skin efficacy according to the epidermal cell proliferation activity of EGF when mixed with a low-content EGF that does not exhibit an effect alone by promoting the activity of EGF, which hardly penetrates the stratum corneum of the skin and has very low stability in cosmetic formulations, making it difficult to expect efficacy as a cosmetic ingredient.

[0010] In order to achieve the above-mentioned purpose, one embodiment of the present invention provides a composition for promoting the activity of EGF containing compound K.

[0011] The composition for promoting the activity of EGF according to the present disclosure promotes the activity of EGF, which hardly penetrates the stratum corneum of the skin and has very low stability in cosmetic formulations, making it difficult to expect effective efficacy as a cosmetic ingredient. Therefore, when mixed with a low-content EGF that does not exhibit an effect alone, it can exhibit excellent skin efficacy according to the epidermal cell proliferation activity of EGF.

[0012] Figures 1 and 2 show the results of confirming the activity promoting effect of EGF on wound repair of compound K according to Experimental Example 1.

[0013] Figures 3a and 3b show the results of confirming the promoting effect of EGF activity on DNA synthesis of compound K according to Experimental Example 2.

[0014] Figures 4a and 4b show the results of confirming the activity-promoting effect of EGF on collagen 17A1 expression of compound K according to Experimental Example 4.

[0015] Figure 5 shows the results of confirming the EGF receptor activity promotion effect of compound K according to Experimental Example 5.

[0016] Figure 6 shows the results of confirming the effect of compound K and EGF on improving proliferation and differentiation of aged epidermal cells according to Experimental Example 6.

[0017] Figure 7 shows the results of confirming the repair improvement effect of compound K and EGF on skin wound tissue according to Experimental Example 7.

[0018] Figure 8 shows the results of confirming the skin barrier improvement effect of compound K and EGF according to Experimental Example 8.

[0019] Hereinafter, the present invention will be described in detail.

[0020] In one aspect, the present invention may relate to a composition for promoting the activity of epidermal growth factor (EGF) containing compound K.

[0021] In one aspect, the present invention may relate to a method for promoting the activity of EGF, comprising applying an effective amount of compound K to a subject in need thereof.

[0022] In one aspect, the present invention may relate to the use of compound K for preparing a composition for promoting the activity of EGF.

[0023] In one aspect, the present invention may relate to a compound K for use in promoting the activity of EGF.

[0024] In one aspect, the present invention may relate to non-therapeutic uses of compound K for stimulating the activity of EGF.

[0025] In one aspect, the present invention may relate to a cosmetic composition containing compound K and epidermal growth factor (EGF) as effective ingredients.

[0026] In one aspect, the present invention may relate to a pharmaceutical composition containing compound K and epidermal growth factor (EGF) as active ingredients.

[0027] In one aspect, the present invention may relate to a method for promoting proliferation of epidermal cells, comprising applying an effective amount of compound K and epidermal growth factor (EGF) to a subject in need thereof.

[0028] In one aspect, the present invention may relate to a method for improving a skin barrier comprising applying an effective amount of compound K and epidermal growth factor (EGF) to a subject in need thereof.

[0029] In one aspect, the present invention may relate to a method for caring for skin damage, comprising applying an effective amount of compound K and epidermal growth factor (EGF) to a subject in need thereof.

[0030] In one aspect, the present invention may relate to the use of compound K and epidermal growth factor (EGF) for preparing a cosmetic composition for promoting epidermal cell proliferation.

[0031] In one aspect, the present invention may relate to the use of compound K and epidermal growth factor (EGF) for preparing a pharmaceutical composition for promoting epidermal cell proliferation.

[0032] In one aspect, the present invention may relate to the use of compound K and epidermal growth factor (EGF) for preparing a cosmetic composition for improving a skin barrier.

[0033] In one aspect, the present invention may relate to the use of compound K and epidermal growth factor (EGF) for preparing a cosmetic composition for skin repair.

[0034] In one aspect, the present invention may relate to the use of compound K and epidermal growth factor (EGF) for preparing a cosmetic composition for skin damage care.

[0035] In one aspect, the present invention may relate to compound K and epidermal growth factor (EGF) for use in promoting epidermal cell proliferation.

[0036] In one aspect, the present invention may relate to compound K and epidermal growth factor (EGF) for use in improving skin barrier.

[0037] In one aspect, the present invention may relate to the use of compound K and epidermal growth factor (EGF) for skin repair purposes.

[0038] In one aspect, the present invention may relate to compound K and epidermal growth factor (EGF) for use in skin damage care.

[0039] In one aspect, the present invention may relate to a non-therapeutic cosmetic use of compound K and epidermal growth factor (EGF) for promoting epidermal cell proliferation.

[0040] In one aspect, the present invention may relate to a non-therapeutic cosmetic use of compound K and epidermal growth factor (EGF) for improving the skin barrier.

[0041] In one aspect, the present invention may relate to non-therapeutic cosmetic uses of compound K and epidermal growth factor (EGF) for skin repair.

[0042] In one aspect, the present invention may relate to a non-therapeutic cosmetic use of compound K and epidermal growth factor (EGF) for skin damage care.

[0043]

[0044] In one embodiment, the compound K may be represented by the following chemical formula 1.

[0045]

[0046] In one embodiment, the composition for promoting the activity of EGF can be added to a cosmetic composition containing EGF.

[0047] In one embodiment, the composition for promoting the activity of EGF can be added to a pharmaceutical composition containing EGF.

[0048] In one embodiment, the compound K may be added to the cosmetic or pharmaceutical composition in an amount of 100 to 2,000 times the weight of the EGF. More specifically, the compound may be added to the cosmetic or pharmaceutical composition in an amount of 100 times or more, 150 times or more, 200 times or more, 250 times or more, 300 times or more, 350 times or more, 400 times or more, 450 times or more, 500 times or more, 550 times or more, 600 times or more, 650 times or more, 700 times or more, 750 times or more, 800 times or more, 850 times or more, 900 times or more, 950 times or more, 1,000 times or more, 1,100 times or more, 1,200 times or more, 1,300 times or more, 1,400 times or more, 1,500 times or more, 1,600 times or more, 1,700 times or more, 1,800 times or more, or 1,900 times or more relative to the weight of the EGF, Additionally, the compound may be added to the cosmetic or pharmaceutical composition in an amount of 2,000 times or less, 1,900 times or less, 1,800 times or less, 1,700 times or less, 1,600 times or less, 1,500 times or less, 1,400 times or less, 1,300 times or less, 1,200 times or less, 1,100 times or less, 1,000 times or less, 950 times or less, 900 times or less, 850 times or less, 800 times or less, 750 times or less, 700 times or less, 650 times or less, 600 times or less, 550 times or less, 500 times or less, 450 times or less, 400 times or less, 350 times or less, 300 times or less, 250 times or less, 200 times or less, or 150 times or less relative to the weight of the EGF.

[0049] In one embodiment, the EGF may be contained in an amount of 0.00001 wt% or less relative to the total weight of the cosmetic or pharmaceutical composition. More specifically, the EGF may be contained in an amount of 0.00001 wt% or less, 0.000001 wt% or less, 0.0000001 wt% or less, 0.00000001 wt% or less relative to the total weight of the cosmetic or pharmaceutical composition, and further, the EGF may be contained in an amount of 0.0000000001 wt% or more, or 0.000000001 wt% or more relative to the total weight of the cosmetic or pharmaceutical composition.

[0050] A composition for promoting the activity of EGF according to one embodiment of the present invention can be added to a cosmetic or pharmaceutical composition containing a very small amount of EGF to promote the activity of EGF, thereby realizing the efficacy of EGF on the skin.

[0051] In one embodiment, the compound K can activate the EGF receptor.

[0052] In one embodiment, the compound K can promote the activity of EGF by activating the EGF receptor.

[0053] In one embodiment, the daily dose of the compound K may be from 0.1 mg / kg body weight to 10 mg / kg body weight. Specifically, the daily application amount or usage of the compound K is 0.1 mg / kg (body weight) or more, 0.2 mg / kg (body weight) or more, 0.3 mg / kg (body weight) or more, 0.4 mg / kg (body weight) or more, 0.5 mg / kg (body weight) or more, 1 mg / kg (body weight) or more, 1.5 mg / kg (body weight) or more, 2 mg / kg (body weight) or more, 2.5 mg / kg (body weight) or more, 3 mg / kg (body weight) or more, 3.5 mg / kg (body weight) or more, 4 mg / kg (body weight) or more, 4.5 mg / kg (body weight) or more, 5 mg / kg (body weight) or more, 5.5 mg / kg (body weight) or more, 6 mg / kg (body weight) or more, 6.5 mg / kg (body weight) or more, 7 mg / kg (body weight) or more, 7.5 The daily application amount of the compound K may be 10 mg / kg (body weight) or less, 8 mg / kg (body weight) or more, 8.5 mg / kg (body weight) or more, 9 mg / kg (body weight) or more, or 9.5 mg / kg (body weight) or more, and further, the daily application amount of the compound K may be 10 mg / kg (body weight) or less, 9.5 mg / kg (body weight) or less, 9 mg / kg (body weight) or less, 8.5 mg / kg (body weight) or less, 8 mg / kg (body weight) or less, 7.5 mg / kg (body weight) or less, 7 mg / kg (body weight) or less, 6.5 mg / kg (body weight) or less, 6 mg / kg (body weight) or less, 5.5 mg / kg (body weight) or less, 5 mg / kg (body weight) or less, 4.5 mg / kg (body weight) or less, 4 mg / kg (body weight) or less, 3.5 mg / kg (body weight) or less, 3 It can be less than or equal to 1 mg / kg of body weight, less than or equal to 2.5 mg / kg of body weight, less than or equal to 2 mg / kg of body weight, less than or equal to 1.5 mg / kg of body weight, less than or equal to 1 mg / kg of body weight, less than or equal to 0.5 mg / kg of body weight, less than or equal to 0.4 mg / kg of body weight, less than or equal to 0.3 mg / kg of body weight, or less than or equal to 0.2 mg / kg of body weight.

[0054] In one embodiment, the compound K may be contained in an amount of 0.001 to 5 wt% based on the total weight of the cosmetic or pharmaceutical composition. Specifically, the compound K may be contained in an amount of 0.001 wt% or more, 0.002 wt% or more, 0.003 wt% or more, 0.004 wt% or more, 0.005 wt% or more, 0.006 wt% or more, 0.007 wt% or more, 0.008 wt% or more, 0.009 wt% or more, 0.01 wt% or more, 0.02 wt% or more, 0.03 wt% or more, 0.04 wt% or more, 0.05 wt% or more, 0.06 wt% or more, 0.07 wt% or more, 0.08 wt% or more, 0.09 wt% or more, 0.1 wt% or more, 0.5 wt% or more, 1 wt% or more, 2 wt% or more, 3 wt% or more, or 4 wt% or more, based on the total weight of the cosmetic or pharmaceutical composition, and further, the compound K may be contained in an amount of 0.001 wt% or more, 0.002 wt% or more, 0.003 wt% or more, 0.04 wt% or more, 0.05 wt% or more, 0.06 wt% or more, 0.07 wt% or more, 0.08 wt% or more, 0.09 ... It may be included in an amount of 5 wt% or less, 4 wt% or less, 3 wt% or less, 2 wt% or less, 1 wt% or less, 0.5 wt% or less, 0.1 wt% or less, 0.09 wt% or less, 0.08 wt% or less, 0.07 wt% or less, 0.06 wt% or less, 0.05 wt% or less, 0.04 wt% or less, 0.03 wt% or less, 0.02 wt% or less, 0.01 wt% or less, 0.009 wt% or less, 0.008 wt% or less, 0.007 wt% or less, 0.006 wt% or less, 0.005 wt% or less, 0.004 wt% or less, 0.003 wt% or less, or 0.002 wt% or less, relative to the total weight of the cosmetic or pharmaceutical composition.

[0055] In one embodiment, compound K and EGF as active ingredients of the cosmetic or pharmaceutical composition can promote proliferation of epidermal cells.

[0056] In one embodiment, the compound K and EGF can increase skin radiance and clarity by promoting proliferation of epidermal cells.

[0057] In one embodiment, the compound K and EGF can improve skin sagging by promoting proliferation of epidermal cells.

[0058] In one embodiment, the compound K and EGF can improve the skin turnover cycle by promoting proliferation of epidermal cells.

[0059] The cosmetic composition containing the EGF according to one embodiment of the present invention or the cosmetic composition containing the compound K and EGF as active ingredients may contain a cosmetically or dermatologically acceptable medium or base. This may be provided in any formulation suitable for topical application, for example, in the form of a solution, a gel, a solid, a pasty anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranule, or an ionic (liposome) and non-ionic vesicular dispersion, or in the form of a cream, a skin, a lotion, a powder, an ointment, a spray, or a concealer stick. These compositions may be prepared according to methods conventional in the art. The cosmetic composition may also be used in the form of an aerosol composition further containing a propellant compressed in the form of a foam.

[0060] The above cosmetic composition is not particularly limited in its formulation and may be appropriately selected depending on the intended purpose. For example, it may be manufactured in the form of a skin lotion, skin softener, skin toner, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, cleansing water, powder, body lotion, body cream, body oil, body cleanser, and body essence.

[0061] When the formulation of the above cosmetic composition is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0062] When the formulation of the above cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0063] When the formulation of the above cosmetic composition is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0064] When the formulation of the above cosmetic composition is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0065] When the formulation of the above cosmetic composition is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0066] The above cosmetic composition may further include functional additives and ingredients included in general cosmetic compositions in addition to the EGF or compound K. The functional additives may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts.

[0067] In addition to the functional additives, the composition may also contain ingredients typically found in cosmetic compositions, as needed. Other ingredients to be included include, but are not limited to, oil-based ingredients, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH regulators, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, and the like.

[0068] In one embodiment, the pharmaceutical composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

[0069] The formulation can be formulated and used in any form suitable for pharmaceutical preparations, including oral preparations such as tablets, capsules, powders or syrups, or external preparations such as ointments, gels, creams, patches or sprays, according to conventional methods.

[0070] It should be understood that the actual dosage of the active ingredient administered by the above pharmaceutical composition should generally be determined in light of various relevant factors, such as the severity of the symptoms, the selected route of administration, and the age, sex, weight, and health of the subject. In general, the dosage of the active ingredient may range from 0.0001 mg / kg / day to 3000 mg / kg / day.

[0071]

[0072] The present invention may provide, as an example, the following embodiments.

[0073] The first embodiment can provide a composition for promoting the activity of epidermal growth factor (EGF) containing compound K.

[0074] The second embodiment can provide a composition for promoting the activity of EGF, which is added to a cosmetic composition containing EGF in the first embodiment.

[0075] A third embodiment can provide a composition for promoting the activity of EGF, wherein in at least one of the first embodiment and the second embodiment, the compound K is added to the cosmetic composition in an amount of 100 to 2,000 times the weight of the EGF.

[0076] The fourth embodiment can provide a composition for promoting the activity of EGF, wherein in one or more of the first to third embodiments, the EGF is contained in an amount of 0.00001 wt% or less relative to the total weight of the cosmetic composition.

[0077] The fifth embodiment can provide a composition for promoting the activity of EGF, wherein the compound K activates the EGF receptor in at least one of the first to fourth embodiments.

[0078] The sixth embodiment can provide a composition for promoting the activity of EGF, wherein the daily application amount of the compound K is 0.1 mg / kg (body weight) to 10 mg / kg (body weight) in one or more of the first to fifth embodiments.

[0079] The seventh embodiment can provide a cosmetic composition containing compound K and epidermal growth factor (EGF) as active ingredients.

[0080] The eighth embodiment can provide a cosmetic composition according to the seventh embodiment, wherein the weight ratio of the compound K to the EGF is 100 to 2,000.

[0081] The ninth embodiment can provide a cosmetic composition according to at least one of the seventh to eighth embodiments, wherein the EGF is contained in an amount of 0.00001 wt% or less relative to the total weight of the cosmetic composition.

[0082] The tenth embodiment can provide a cosmetic composition according to at least one of the seventh to ninth embodiments, wherein the compound K is contained in an amount of 0.001 to 5 wt% based on the total weight of the cosmetic composition.

[0083] The eleventh embodiment can provide a cosmetic composition according to at least one of the seventh to tenth embodiments, wherein the effective ingredient promotes proliferation of epidermal cells.

[0084] The 12th embodiment can provide a cosmetic composition for skin barrier repair according to one or more of the 7th to 11th embodiments.

[0085] The 13th embodiment can provide a cosmetic composition for skin repair according to one or more of the 7th to 12th embodiments.

[0086] The 14th embodiment can provide a cosmetic composition for skin damage care according to one or more of the 7th to 13th embodiments.

[0087]

[0088] Hereinafter, the present invention will be described in more detail through experimental examples. However, these experimental examples are provided merely to facilitate understanding of the present invention, and the scope of the present invention is not limited to these experimental examples. Modifications, substitutions, and insertions commonly known in the art may be performed, and such modifications are also included within the scope of the present invention.

[0089]

[0090] [Experimental Example 1] Skin Damage Repair Effect Experiment

[0091] 1-1. Experiment on wound repair effect on normal skin keratinocytes

[0092] To confirm the activity promoting effect of EGF on wound repair of compound K, 3x10 per well of skin keratinocyte cell line (HaCaT) were seeded in 96-well plates. 4 After seeding, the cells were cultured for 24 hours and then cultured for 24 hours in DMEM medium containing 1% FBS. After scratching with a cell scratcher, EGF was treated alone at a concentration of 10 ng / ml or 1 ng / ml, or EGF 1 ng / ml and Compound K 0.1 μg / ml, 0.5 μg / ml, or 1 μg / ml together, and wound closure was measured for 48 hours using JuLI Stage (NanoEntek). The results are shown in Fig. 1.

[0093] From the results of Fig. 1, it was confirmed that there was no wound repair effect when EGF was treated alone at a concentration of 1 ng / ml. On the other hand, when EGF 1 ng / ml, which had no effect when treated alone, was treated together with Compound K 0.5 μg / ml or 1 μg / ml, it was confirmed that the wound was repaired to a significant level, and in particular, when EGF 1 ng / ml and Compound K 1 μg / ml were treated together, it was confirmed that a wound repair effect at a level similar to that when EGF was treated at 10 ng / ml was shown. From this, it was found that Compound K effectively promotes the activity of EGF for wound repair.

[0094] 1-2. Experimental study on the wound repair effect on aging skin keratinocytes.

[0095] To confirm the activity promoting effect of EGF on wound repair of aged skin keratinocytes in compound K, skin keratinocytes (normal human epidermal keratinocytes, NHEK) were seeded in a 96-well plate at 3x10 per well. 4After seeding, the cells were cultured for 24 hours, and then treated with a senescence factor (Senescence-associated secretory phenotype, SASP) to induce senescence, and then cultured in a serum-free medium for 24 hours. The senescence factor was prepared as follows. Normal human fibroblasts were continuously passaged until the population doubling level (PDL) reached 46 or higher, and serum-free media (Keratinocyte SFM (1X), gibco by lifetechnologies) was added. After 48 hours of culture, the media was collected and used as a senescence factor. After scratching with a cell scratcher, EGF 10 ng / ml and Compound K 1 μg / ml were treated separately or together, and wound closure for 48 hours was measured using JuLI Stage (NanoEntek). The results are shown in Fig. 2.

[0096] From the results in Fig. 2, it was confirmed that after inducing aging by treating keratinocytes with a senescence factor (SASP), no wound repair effect was observed even when EGF was treated, whereas a wound repair effect was observed even when compound K was co-treated. From this, it was found that compound K effectively promotes the activity of EGF for wound repair in aged keratinocytes.

[0097]

[0098] [Experimental Example 2] Experiment on the proliferation efficacy of labeled cells

[0099] We examined whether compound K enhances the proliferative effect of EGF in human epidermal keratinocytes by measuring the level of DNA synthesis. Specifically, 1x10 human epidermal keratinocytes were seeded on Lab-Tek Chamber slides. 6After seeding the cells, they were cultured for 24 hours. After that, EGF was treated alone at a concentration of 10 ng / ml and 1 ng / ml, or EGF 1 ng / ml and Compound K 1 μg / ml were treated together and cultured for 24 hours. After that, 10 μM EdU (5-ethynyl-2' -deoxyuridine) component was treated, and after 16 hours, the EdU component bound to replicating DNA was attached to a fluorescent probe (Click-iT Plus EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye, Invitrogen), and the amount of replicated DNA was measured using Zen software (Carl Zeiss, Jena, Germany) under a fluorescence microscope (Zeiss LSM700 confocal microscope). The results are shown in Fig. 3a and Fig. 3b.

[0100] From the results in Figures 3a and 3b, no significant increase in DNA synthesis was observed when EGF was treated alone at a concentration of 1 ng / ml. On the other hand, when EGF (1 ng / ml), which had no effect when treated alone, was treated together with Compound K (1 μg / ml), a significant increase in DNA synthesis was observed, and this was similar to the level observed when EGF was treated at 10 ng / ml. From this, it was found that Compound K effectively promotes the epidermal cell proliferation effect of EGF.

[0101]

[0102] [Experimental Example 3] Experiment to increase TIMP1 (TIMP metallopeptidase inhibitor 1) expression

[0103] Collagen 17A1 (Col17A1) is a protein involved in the dermal-epidermal junction (DEJ), which promotes adhesion between cells and acts as a pillar in epidermal differentiation, thereby enhancing the structural stability of the epidermis. The degradation of collagen 17A1 protein is mediated by matrix metalloproteinase 1 (MMP-1). When the expression of TIMP metallopeptidase inhibitor 1 (TIMP1), an inhibitory protein that blocks the degradation of MMP-1, increases, the degradation of collagen 17A1 is reduced, thereby strengthening the structural stability of the epidermis and enabling rapid recovery when the epidermis is damaged.

[0104] To determine whether compound K can promote the activity of EGF in increasing the expression of TIMP1, human epidermal keratinocytes were seeded in a 96-well plate at a density of 1x10 per well. 4 After seeding, the cells were cultured for 24 hours, and then treated with EGF alone at a concentration of 10 ng / ml and 1 ng / ml, or with 1 ng / ml of EGF and 1 μg / ml of compound K together, and cultured for 24 hours. Afterwards, RNA was extracted from the skin keratinocytes using RNeasy Mini KiT (Qiagen), and cDNA was synthesized from the extracted RNA using SuperScript VILO master mix (Invitrogen), and the change in the expression level of TIMP1 (Hs01092512_g1, Applied Biosystems, Foster City, CA, USA) was observed by real-time qPCR (a 7500 Fast Real-time PCR System, Thermo Fisher Scientific, Waltham, MA, USA). The results are shown in Table 1 below.

[0105] Sample treatment concentration TIMP1 expression level (Fold) Untreated group - 1 Compound K1 μg / ml 1.4 EGF 10 ng / ml 3.0 EGF 1 ng / ml 1.0 EGF + Compound K1 ng / ml + 1 μg / ml 2.7

[0106] From the results in Table 1, it was confirmed that when EGF was treated alone at a concentration of 1 ng / ml, there was no effect of increasing TIMP1 expression, whereas when the same concentration of EGF was treated together with 1 μg / ml of Compound K, the amount of TIMP1 expression increased significantly, and this was confirmed to be at a level similar to that when EGF was treated at 10 ng / ml. From this, it was found that Compound K effectively promotes the TIMP1 expression-increasing activity of EGF.

[0107]

[0108] [Experimental Example 4] Experiment to Increase Collagen 17A1 Expression

[0109] Immunofluorescence staining was used to determine whether compound K could promote the activity of EGF in increasing collagen 17A1 expression. Specifically, 1x10 skin keratinocytes were seeded on a Lab-Tek Chamber slide. 6 After seeding the cells, they were cultured for 24 hours. After that, EGF was treated alone at a concentration of 10 ng / ml and 1 ng / ml, or EGF 1 ng / ml and Compound K 1 μg / ml were treated together and cultured for 24 hours. Then, they were washed with PBS, fixed with 3.7% formaldehyde, reacted with collagen 17A1 antibody for 16 hours, and then reacted with fluorescent secondary antibody (Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Invitrogen) and analyzed using a fluorescence microscope (Zeiss LSM700 confocal microscope). The results are shown in Fig. 4a and Fig. 4b.

[0110] From the results of Figs. 4a and 4b, it was confirmed that when EGF was treated alone at a concentration of 1 ng / ml, the expression of collagen 17A1 did not increase, whereas when the same concentration of EGF was treated together with 1 μg / ml of compound K, the expression amount of collagen 17A1 increased significantly, and this was confirmed to be at a similar level to when EGF was treated at 10 ng / ml. From this, it was found that compound K effectively promotes the collagen 17A1 expression increasing activity of EGF.

[0111]

[0112] [Experimental Example 5] Experiment to promote EGF receptor activation and expression in aged epidermal cells.

[0113] NHEK cells were treated with senescence-associated secretory factor (SASP) for 48 hours to induce senescence. The cells were then treated with 10 ng / ml of EGF or 1 mg / ml of compound K for 10 minutes or 24 hours, respectively, and protein was extracted from the cells. 30 mg of protein was separated by size using 4-12% SDS-PAGE and blotted onto PDF (BioRad) membranes using iBlot2. The blot was blocked for 1 hour with superBlocking buffer (Thermofisher Scientific). The primary antibodies were phosphor-EGFR (Tyr1068), phosphoERK (Thr202 / Tyr204), and phosphoAKT (S473), and the secondary antibody was horse radish peroxidase-conjugated anti-mouse and rabbit IgG (Amersham). The reaction was performed using the enhanced chemiluminescence (ECL) kit from Amersham. The protein expression level of the reacted blot was confirmed using the LAS-3000 imaging system. The results are shown in Fig. 5.

[0114] From the results in Fig. 5, it was confirmed that aged skin keratinocytes did not activate the EGF receptor (EGFR) even when treated with EGF, and thus, cell proliferation signaling did not occur. On the other hand, when aged skin keratinocytes were treated with Compound K together with EGF, it was confirmed that the EGF receptor was activated and phosphorylated, and then cell proliferation signaling was activated by AKT, ERK, etc. From this, it was confirmed that Compound K has the effect of promoting the activation of the EGF receptor in aged epidermal cells. In addition, it was confirmed that Compound K also increased the expression of the EGF receptor.

[0115]

[0116] [Experimental Example 6] Experiment on the effect of improving proliferation and differentiation of aged epidermal cells.

[0117] NHEK cells were treated with senescence-associated secretory factor (SASP) for 48 hours to induce senescence. The cells were then treated with 10 ng / ml of EGF or 1 mg / ml of compound K for 10 minutes or 24 hours, respectively, and protein was extracted from the cells. 30 mg of protein was separated by size using 4-12% SDS-PAGE and blotted onto PDF (BioRad) membranes using iBlot2. The blot was blocked for 1 hour with superBlocking buffer (Thermofisher Scientific). Fillaggrin, Collagen17a1, Loricirin, p63, and TIMP1 were used as primary antibodies, and horse radish peroxidase-conjugated anti-mouse and rabbit IgG (Amersham) were used as secondary antibodies. The reaction was performed using the enhanced chemiluminescence (ECL) kit from Amersham. The protein expression level of the reacted blot was confirmed using the LAS-3000 imaging system. The results are shown in Fig. 6.

[0118] From the results in Fig. 6, it was confirmed that aged skin keratinocytes do not proliferate (p63, Collagen17a1) or differentiate (Fillaggrin, Loricrin), which damages the barrier and reduces skin radiance and transparency. In addition, it was confirmed that the skin sags, loses elasticity, and forms wrinkles because collagen degradation is not prevented (TIMP1). In comparison, when EGF and Compound K (CK) were treated together, it was confirmed that the proliferation (p63, Collagen17a1) and differentiation (Fillaggrin, Loricrin) of the aged epidermis were restored, improving skin radiance and transparency, and that by inhibiting the activity of collagen-decomposing enzymes (TIMP1), skin sagging, wrinkles, and elasticity could be improved.

[0119]

[0120] [Experimental Example 7] Experiment on the Improvement of Skin Damage Repair in Skin Biopsy Tissue

[0121] Skin biopsy tissues (36-year-old female, genoskin, 31100 Toulouse, France) were cultured in 24-well plates. EGF 200 ng / ml, EGF 10 ng / ml, or EGF 10 ng / ml + Compound K 10 mg / ml were treated, respectively, and cultured for 8 days. For the aging model, medium containing senescence factor (SASP) was used, and the aging model skin biopsy tissues were treated with EGF 200 ng / ml or Compound K 10 mg / ml and cultured in SASP medium for 8 days. The degree of wound repair was analyzed by taking tissue photographs for 8 days, and the wound changes in the photographs were analyzed using Image J. After 8 days, the tissues were fixed in formalin and stained with Heatoxylin & Eosin to analyze the wound changes, and the results are shown in Table 2 and Fig. 7 below.

[0122] Wound closure (%)P valuecontrol36.77±2.66EGF 10ng / ml39.230±4.13nsEGF 200ng / ml58.91±0.879***EGF 10ng / ml + Compound K(CK) 10mg / ml65.73±4.7***SASP25.88±3.148SASP + Compound K(CK) 10mg / ml36.1±1.32*SASP + EGF 200ng / ml26.88±3.9nsSASP + EGF 200ng / ml + Compound K(CK) 10mg / ml47.17±3.47**

[0123] From the results in Table 2 and Fig. 7, it was confirmed that the wounds in the skin tissue treated with SASP or EGF alone at 10 ng / ml did not show any wound repair compared to the untreated group, whereas when EGF 200 ng / ml was treated, wound repair progressed significantly by 60% compared to the untreated group. When EGF 10 ng / ml and Compound K 10 mg / ml were treated together, wound repair progressed significantly by about 70% compared to the untreated group, and this was confirmed to be a superior wound repair effect compared to when EGF was treated alone at a 20-fold concentration. In addition, when SASP was treated, EGF treatment at 200 ng / ml did not show a wound repair effect, whereas when EGF 200 ng / ml and Compound K 10 mg / ml were treated together to the SASP-treated wound tissue, a significant wound repair effect was confirmed.

[0124]

[0125] [Experimental Example 8] Experiment on Barrier Improvement Effects in Artificial Skin

[0126] Keratinocytes that were induced to age by treatment with senescence factors and those that were not were seeded in snap wells and cultured for two days in a CnT-Prime 3D barrier (CELLnTEC Advanced Cell Systems AG, Bern, Switzerland). After two days, the medium on the keratinocytes was removed, and the cells were exposed to air and cultured for 10 days to form an epidermal matrix. During the culture, the medium was continuously treated with 10 mg / ml of compound K. On the 8th day of air exposure, 200 ng / ml of EGF was treated. The tissues were fixed in formalin, and heatoxylin & eosin staining and immunostaining for filaggrin and keratin 10 were performed to analyze the proteins. The results are shown in Fig. 8.

[0127] From the results of Fig. 8, it was found that the expression of barrier markers Fillaggrin and keratin 10 decreased in keratinocytes treated with aging factors or when EGF was treated thereto. In comparison, when Compound K 10 mg / ml was treated or Compound K 10 mg / ml and EGF 200 ng / ml were treated together, it was confirmed that the expression of Fillaggrin and keratin 10, which had been decreased compared to the untreated group, was restored. From this, it was confirmed that the skin barrier improvement effect of Compound K was significantly increased when treated together with EGF compared to when treated alone.

[0128]

[0129] [Formulation Example 1] Lotion

[0130] Ingredients (weight) Compound K0.01 EGF0.000005 L-ascorbic acid-2-magnesium phosphate salt1.00 Soluble collagen (1% aqueous solution)1.00 Sodium citrate0.10 Citric acid0.05 Licorice extract0.20 1,3-butylene glycol3.00 Remaining tablet amount

[0131] [Formulation Example 2] Cream

[0132] Ingredients (weight) Compound K0.01 EGF0.000005 Polyethylene glycol monostearate 2.00 Glycerin 5.00 Cetyl alcohol 4.00 Squalene 4.00 Liquid paraffin 10.00 Tris-2-ethylhexane glyceryl 6.00 Sphingoglycolipid 1.00 1,3-butylene glycol 7.00 Remaining tablet amount

[0133] [Formulation Example 3] Pack

[0134] Ingredients (weight) Compound K0.01 EGF0.000005 Polyvinyl alcohol13.00 L-ascorbic acid-2-phosphate magnesium salt1.00 Lauroyl hydroxyproline1.00 Water-soluble collagen (1% aqueous solution)2.00 1,3-butylene glycol3.00 Ethanol5.00 Remaining tablet amount

[0135] [Formulation Example 4] Toner

[0136] Ingredients (weight) Compound K0.01 EGF0.000005 Glycerin3.0 Butylene Glycol2.0 Propylene Glycol2.0 Carboxyvinyl Polymer0.1 PEG-12 Nonylphenyl Ether0.2 Polysorbate-800.4 Ethanol10.0 Triethanolamine0.1 Preservative, Color, Fragrance Appropriate amount of purified water remaining

[0137]

[0138] While specific portions of this specification have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred implementation examples and do not limit the scope of this specification. Therefore, the substantial scope of this specification is defined by the appended claims and their equivalents.

Claims

1. A composition for promoting the activity of epidermal growth factor (EGF) containing compound K.

2. In paragraph 1, A composition for promoting the activity of EGF, which is added to a cosmetic composition containing EGF.

3. In paragraph 2, A composition for promoting the activity of EGF, wherein the compound K is added to the cosmetic composition in an amount of 100 to 2,000 times the weight of the EGF.

4. In paragraph 2, A composition for promoting the activity of EGF, wherein the EGF is contained in an amount of 0.00001 wt% or less relative to the total weight of the cosmetic composition.

5. In paragraph 1, The above compound K is a composition for promoting the activity of EGF, which activates the EGF receptor.

6. In paragraph 1, A composition for promoting the activity of EGF, wherein the daily application amount of the compound K is 0.1 mg / kg (body weight) to 10 mg / kg (body weight).

7. A cosmetic composition containing compound K and EGF (epidermal growth factor) as active ingredients.

8. In paragraph 7, A cosmetic composition wherein the weight ratio of the compound K to the EGF is 100 to 2,000.

9. In paragraph 7, A cosmetic composition, wherein the EGF is contained in an amount of 0.00001 wt% or less relative to the total weight of the cosmetic composition.

10. In paragraph 7, A cosmetic composition, wherein the compound K is contained in an amount of 0.001 to 5 wt% based on the total weight of the cosmetic composition.

11. In paragraph 7, A cosmetic composition wherein the above effective ingredient promotes proliferation of epidermal cells.

12. In paragraph 7, A cosmetic composition for improving the skin barrier.

13. In paragraph 7, A cosmetic composition for skin repair.

14. In paragraph 7, A cosmetic composition for skin damage care.

Images