Compositions comprising camellia-derived flavonoids for inducing growth of skin cells whose growth is inhibited due to aging

Abstract

The present specification provides a skin-protecting composition containing a kaempferol glycoside compound as an effective ingredient, the kaempferol glycoside compound being extracted from Camellia japonica seed cake discarded after extracting oil from Camellia japonica seeds, thus being environmentally friendly while being safe to the human body due to few side effects, and excellent in skin regeneration and the like.

Description

Technical Field

[0001] This invention discloses a skin protection composition comprising kaempferol glycoside compounds that are safe for human use and have excellent skin protection effects. Background Technology

[0002] The skin is the body's first line of defense, protecting internal organs from external environmental stimuli such as temperature and humidity changes, ultraviolet rays, and pollutants. It also plays an important role in maintaining body homeostasis, such as regulating body temperature.

[0003] However, due to external factors such as excessive physical and chemical stimulation and stress, nutritional deficiencies, and aging, the normal function of the skin declines, promoting loss of elasticity, keratinization, and wrinkle formation. To prevent this and maintain healthier, more elastic skin, efforts have been made to use cosmetics enhanced with bioactive substances to maintain the skin's inherent functions and activate skin cells to effectively promote skin regeneration.

[0004] However, most existing cosmetic ingredients either have poor efficacy or, when the effect is too strong, can cause various skin side effects. Therefore, ingredients that are safe for the skin and can effectively promote skin regeneration and regulate the skin's keratinocyte production cycle are receiving increasing attention.

[0005] [Existing Technical Documents]

[0006] [Patent Documents]

[0007] (Patent Document 1) B.-C. Liau et al., Industrial Crops and Products 95 (2017) 296-304. Summary of the Invention

[0008] Technical issues

[0009] In one aspect, the present invention aims to provide a composition comprising kaempferol glycosides as an active ingredient, which is safe for the human body due to its few side effects and has excellent skin protection effects.

[0010] On the other hand, the present invention aims to provide a skin protection composition comprising kaempferol glycosides extracted from Camellia japonica seed cake as an active ingredient, utilizing waste resources discarded after the extraction of Camellia japonica seed cake.

[0011] On the other hand, the present invention aims to provide a composition that improves skin wrinkles or promotes skin regeneration.

[0012] In another respect, the present invention aims to provide a composition for inhibiting the expression of matrix metalloproteinase-1 (MMP-1).

[0013] In another respect, the present invention aims to provide a composition for improving atopic skin.

[0014] On the other hand, the present invention aims to provide a composition for regulating the skin keratinization cycle, regulating skin keratin, promoting skin keratin renewal, or normalizing the skin keratin renewal cycle.

[0015] In another respect, the present invention aims to provide a composition for improving atopic skin or improving, preventing or treating atopic dermatitis by inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1).

[0016] In another respect, the present invention aims to provide a composition that regulates the skin keratinization cycle, modulates skin keratinization, promotes skin keratin renewal, or normalizes the skin keratin renewal cycle by inhibiting the activity of inositol 3-phosphokinase 1 (PDK1).

[0017] Technical solution

[0018] This specification provides a skin protection composition comprising, as an active ingredient, a kaempferol glycoside compound, its isomer, its pharmaceutically acceptable salt, its hydrate, or its solvate, wherein the kaempferol glycoside compound is represented by any one of the following chemical formulas 1 to 4.

[0019] [Chemical Formula 1]

[0020]

[0021] [Chemical Formula 2]

[0022]

[0023] [Chemical Formula 3]

[0024]

[0025] [Chemical Formula 4]

[0026]

[0027] Beneficial effects

[0028] On one hand, the skin protection composition disclosed in this specification contains kaempferol glycosides as active ingredients, particularly kaempferol 4-glycosides, which inhibit the expression of matrix metalloproteinase-1 (MMP-1), thereby improving skin wrinkles and promoting skin regeneration.

[0029] On the other hand, the skin protection composition disclosed in this specification contains kaempferol 4-glycoside compound as an active ingredient, thereby inhibiting the activity of inositol 3-phosphokinase 1 (PDK1), thereby improving atopic skin or improving, preventing or treating atopic dermatitis, and regulating the skin keratinization cycle. Attached Figure Description

[0030] Figure 1 The observation results of the expression of the aging biomarker SA-β-gal according to an embodiment of the present invention are shown.

[0031] Figure 2a and Figure 2b A graph is shown illustrating the inhibitory effect of a composition according to an embodiment of the present invention on the activity of 3-phosphoinositol-dependent protein kinase 1 (PDK1).

[0032] Figure 3 A graph is shown illustrating the inhibitory effect of a composition according to an embodiment of the present invention on matrix metalloproteinase-1 (MMP-1) expression.

[0033] Figure 4 A graph is shown illustrating the effects of a composition according to an embodiment of the present invention on H&E, Ki-67, KRT10, FLG, and INV.

[0034] Figures 5a to 5b The results of the effect of the composition according to an embodiment of the present invention on epidermal thickness and Ki-67 are shown.

[0035] Figures 6a to 6c The results of the effects of the composition according to an embodiment of the present invention on KRT10, FLG and INVd are shown.

[0036] Figure 7 Information on kaempferol glycoside compounds represented by chemical formulas 1 to 4 is shown.

[0037] Figures 8a to 8b The diagram shows an experiment illustrating whether regeneration capacity is restored when PDK1 is suppressed.

[0038] Figures 9a to 9b The results show that skin cell function was restored by inhibiting PDK1.

[0039] Figures 10a to 10bResults showing that PDK1 affects skin regeneration are presented.

[0040] Figures 11a to 11b A graph showing the inhibition of SASP by PDK1 inhibition in the skin equivalent is shown. Detailed Implementation

[0041] In this specification, when a part "contains" a component, unless otherwise specified, it means that the part may also contain other components, rather than excluding other components.

[0042] The present invention will be described in detail below.

[0043] An exemplary embodiment of the present invention provides a skin protection composition comprising, as an active ingredient, a kaempferol glycoside compound, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, wherein the kaempferol glycoside compound is represented by any one of the following chemical formulas 1 to 4.

[0044] [Chemical Formula 1]

[0045]

[0046] [Chemical Formula 2]

[0047]

[0048] [Chemical Formula 3]

[0049]

[0050] [Chemical Formula 4]

[0051]

[0052] In this specification, the term "isomer" specifically includes not only optical isomers (e.g., essentially pure enantiomers, essentially pure diastereomers, or mixtures thereof), but also conformation isomers (i.e., isomers that differ only at the angle of one or more chemical bonds), position isomers (especially tautomers), or geometric isomers (e.g., cis-trans isomers).

[0053] In this specification, "essentially pure" means, for example, when using the relevant enantiomers or diastereomers, that the amount of a specific compound, such as an enantiomer or diastereomer, is about 90% (w / w) or more, preferably about 95% (w / w) or more, more preferably about 97% (w / w) or more, or 98% (w / w) or more, more preferably about 99% (w / w) or more, and even more preferably about 99.5% (w / w) or more.

[0054] In this instruction manual, "pharmaceutical acceptable" means that, when used at the usual medicinal dosage, significant toxic effects can be avoided, thereby obtaining or receiving approval from a government or equivalent regulatory body for use in animals, more specifically for use in humans, or being recognized as being disclosed in a pharmacopoeia or other general pharmacopoeia.

[0055] In this specification, "pharmaceuticalally acceptable salt" means a salt according to one aspect of the invention that is pharmaceutically acceptable and has the pharmacological activity required by the parent compound. The salt may include, (1) an acid addition salt formed from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or an acid addition salt formed from organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2,2,2]-oct-2-en-1-carboxylic acid, glucoheponic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecyl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, mucoconic acid, etc. (2) Salt formed by replacing acidic protons present in the parent compound.

[0056] In this specification, "hydrate" refers to a compound that is bound to water, and is used in a broad sense to include inclusion compounds in which there is no chemical bond between water and the compound.

[0057] In this specification, "solvent" refers to a higher-order compound formed between molecules or ions of the solute and molecules or ions of the solvent.

[0058] The IUPAC name of the kaempferol glycoside compound represented by chemical formula 1 is kaempferol-3-O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-xylopyranosyl-(1→2)-O-β-D-galactopyranoside, which exists as a pale yellow powder at room temperature.

[0059] Information regarding the kaempferol glycoside compound represented by chemical formula 1 is as follows.

[0060] Pale yellow powder; UV (HPLC-PDA) λ max 266, 347 nm; [α] 22 D -57.4 (c 0.10); ESI-Q-TOF-MS: m / z 859.2503 [M+H] + (C 37 H 47 O 23 + The calculated values ​​are 859.2508 and 881.2329 [M+Na]. + (C 37 H 46 O 23 Na + The calculated value is 859.2327). 1 H- (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125MHz) (see...) Figure 7 ).

[0061] The IUPAC name of the kaempferol glycoside compound represented by chemical formula 2 is kaempferol-3-O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-xylopyranosyl-(1→2)-O-β-D-glucopyranoside, which exists as a pale yellow powder at room temperature.

[0062] Information regarding the kaempferol glycoside compound represented by chemical formula 2 is as follows.

[0063] Pale yellow powder; UV (HPLC-PDA) λ max 266, 347 nm; [α] 22 D -63.7 (c 0.10); ESI-Q-TOF-MS: m / z 859.2505 [M+H] + (C 37 H 47 O 23 + The calculated values ​​are 859.2508 and 881.2325 [M+Na]. + (C 37 H 46 O 23 Na + The calculated value is 859.2327). 1 H- (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125MHz) (see...) Figure 7 ).

[0064] The IUPAC name of the kaempferol glycoside compound represented by chemical formula 3 is kaempferol-3-O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→2)-O-β-D-galactopyranoside, which exists as a pale yellow powder at room temperature.

[0065] Information regarding the kaempferol glycoside compound represented by chemical formula 3 is as follows.

[0066] Pale yellow powder; UV (HPLC-PDA) λ max 266, 347 nm; [α] 22 D -72.5 (c 0.2); ESI-Q-TOF-MS: m / z 889.2616 [M+H] + (C 38 H 49 O 23 +The calculated values ​​are 889.2613 and 911.2432 [M+Na]. + (C 38 H 48 O 24 Na + The calculated value is 859.2327). 1 H- (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125MHz) (see...) Figure 7 ).

[0067] The IUPAC name of the kaempferol glycoside compound represented by chemical formula 4 is kaempferol-3-O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→2)-O-β-D-gluctopyranoside (Kaempferol-3-O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→2)-O-β-D-gluctopyranoside), which exists as a pale yellow powder at room temperature.

[0068] Information regarding the kaempferol glycoside compound represented by chemical formula 4 is as follows.

[0069] Pale yellow powder; UV (HPLC-PDA) λ max 266, 347 nm; [α] 22 D -45.1 (c 0.10); ESI-Q-TOF-MS: m / z 889.2613 [M+H] + (C 38 H 49 O 23 + The calculated values ​​are 889.2613 and 911.2423 [M+Na]. + (C 38 H 48 O 24 Na + The calculated value is 859.2327). 1 H- (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125MHz) (see...) Figure 7 ).

[0070] The composition according to one aspect of the present invention comprises kaempferol tetraglycoside compound extracted from camellia seed oil meal discarded after oil extraction from camellia seeds as an active ingredient, and compared with existing compositions containing kaempferol glycoside compound as an active ingredient, it can show significantly superior effects in improving skin wrinkles, skin regeneration, improving, preventing or treating atopic dermatitis, and regulating the skin keratinization cycle.

[0071] Camellia seed oil meal extract is composed of terpene glycosides, flavonoid glycosides, sugars, and proteins. In particular, the flavonoid glycosides of camellia seed oil meal consist of tetrasaccharides (chemical formulas 1, 2, 3, 4) and trisaccharides (chemical formulas 5, 6, 7, 8) of kaempferol. The inventors have confirmed that four tetrasaccharides (chemical formulas 1, 2, 3, 4) of the kaempferol glycosides derived from camellia seed oil meal have excellent skin protective effects.

[0072] [Chemical Formula 5]

[0073]

[0074] [Chemical Formula 6]

[0075]

[0076] [Chemical Formula 7]

[0077]

[0078] [Chemical Formula 8]

[0079]

[0080] In one embodiment, the kaempferol glycoside compound can be extracted from camellia seed oil meal.

[0081] Specifically, the kaempferol glycoside compound can be obtained by: producing oil from camellia seeds by pressing to obtain camellia seed oil meal, and then extracting the kaempferol glycoside compound from the camellia seed oil meal using 70% ethanol.

[0082] In one embodiment, based on the total weight of the composition, the concentration of the active ingredient can be from 0.01 μM to 100 μM, for example, it can be 0.01 μM or more, 0.1 μM or more, 0.5 μM or more, 1 μM or more, 1.5 μM or more, 2 μM or more, or 2.5 μM or more, and 100 μM or less, 50 μM or less, 10 μM or less, 8 μM or less, 6 μM or less, or 4 μM or less.

[0083] When the concentration is less than 0.01 μM, the skin protection effects, such as promoting skin regeneration, improving atopic dermatitis, or regulating the skin keratinization cycle, may be minimal. However, when the concentration is greater than 100 μM, it may exhibit cytotoxicity.

[0084] In one embodiment, the composition can be used to improve skin wrinkles.

[0085] In one embodiment, the composition can be used to promote skin regeneration.

[0086] In one embodiment, the composition can inhibit the expression of matrix metalloproteinase-1 (MMP-1).

[0087] In another embodiment, the composition can be used to improve skin wrinkles by inhibiting the expression of matrix metalloproteinase-1 (MMP-1).

[0088] In another embodiment, the composition can be used to promote skin regeneration by inhibiting the expression of matrix metalloproteinase-1 (MMP-1).

[0089] In one embodiment, the composition can be used to improve atopic skin.

[0090] In one embodiment, the composition can be used to regulate the skin's keratinization cycle.

[0091] The skin keratinization cycle, also known as the epidermal cell turnover cycle, refers to the cycle in which keratinocytes degenerate from the bottom of the epidermis to the skin surface and become corneocytes.

[0092] The skin cell renewal cycle can be determined by two factors: one is by maintaining the growth potential of epidermal cells to measure colony formation efficiency, and the other is by promoting epidermal cell differentiation to maintain barrier enhancement to measure the degree of differentiation. According to one aspect of the invention, the composition comprises a kaempferol tetrasaccharide compound as an active ingredient, thereby promoting epidermal cell colony formation and differentiation, and thus shortening the skin keratinization cycle or epidermal cell renewal cycle.

[0093] In another embodiment, the composition can be used to regulate skin keratinization, promote skin keratinization renewal, or normalize the skin keratinization renewal cycle.

[0094] In one embodiment, the composition can inhibit the activity of 3-phosphoinositol-dependent protein kinase 1 (PDK1).

[0095] When PDK1 activity is inhibited by using a composition comprising a kaempferol glycoside compound represented by any one of the chemical formulas 1 to 4, cells whose growth is inhibited due to aging can regrow and damaged cells can be restored.

[0096] In other words, inhibiting PDK1 in aging human dermal fibroblasts can eliminate signs of aging and inhibit NF-κB and mTOR signaling, thereby restoring the cell cycle and thus restoring or promoting skin regeneration.

[0097] In another embodiment, the composition can be used to improve skin wrinkles by inhibiting the activity of inositol 3-phosphokinase 1 (PDK1).

[0098] In another embodiment, the composition can be used to promote skin regeneration by inhibiting the activity of inositol 3-phosphokinase 1 (PDK1).

[0099] In another embodiment, the composition can be used to improve atopic skin, or to improve, prevent or treat atopic dermatitis by inhibiting the activity of inositol 3-phosphokinase 1 (PDK1).

[0100] In another embodiment, the composition can be used to regulate the skin keratinization cycle by inhibiting the activity of inositol 3-phosphokinase 1 (PDK1).

[0101] In another embodiment, the composition can be used to regulate skin keratinization by inhibiting the activity of inositol 3-phosphate-dependent protein kinase 1 (PDK1).

[0102] In another embodiment, the composition can be used to promote skin cell turnover by inhibiting the activity of inositol 3-phosphokinase 1 (PDK1).

[0103] In another embodiment, the composition can be used to normalize the skin's keratinocyte turnover cycle by inhibiting the activity of inositol 3-phosphokinase 1 (PDK1).

[0104] In another embodiment, the composition can be used to improve the skin barrier by inhibiting the activity of inositol 3-phosphokinase 1 (PDK1).

[0105] In one embodiment, the composition may be a cosmetic composition.

[0106] The cosmetic composition according to one embodiment of the invention may comprise a cosmetically or dermatologically acceptable medium or matrix. It may be in any dosage form suitable for topical application, for example, it may be prepared as a solution, gel, solid, anhydrous paste, emulsion, suspension, microemulsion, microcapsule, microsphere, or ionic (liposome) and nonionic vesicle dispersion obtained by dispersing an oil phase in an aqueous phase, or in the form of cream, toner, lotion, powder, ointment, spray, or concealer stick. These compositions can be prepared by conventional methods in the art. The compositions according to the invention may also be used in the form of a foam or as an aerosol composition further comprising a compressible propellant.

[0107] The dosage form of the cosmetic composition according to one embodiment of the present invention is not particularly limited. For example, it can be prepared as a softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, serum, eye cream, eye serum, cleansing cream, cleansing foam, cleansing water, mask, powder, body lotion, body cream, body oil, and body essence, etc.

[0108] When the dosage form according to an embodiment of the present invention is a paste, cream or gel, the carrier component may include animal fiber, plant fiber, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicon, bentonite, silica, talc or zinc oxide, etc.

[0109] When the dosage form according to an embodiment of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component. In particular, when the dosage form is a spray, it may further contain a propellant such as chlorofluorocarbon, propane / butane or dimethyl ether.

[0110] When the dosage form according to an embodiment of the present invention is a solution or emulsion, a solvent, solvator or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol fatty acid ester, polyethylene glycol or fatty acid ester of sorbitol.

[0111] When the dosage form according to one embodiment of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene dehydrated sorbitol ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar or tragacanth gum, etc. can be used as carrier components.

[0112] When the dosage form according to an embodiment of the present invention is a facial cleanser containing a surfactant, the carrier component may include fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinate monoester, hydroxyethyl sulfonate, imidazoline derivative, methyl taurine, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, fatty alcohol, fatty acid glycerides, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester, etc.

[0113] In a cosmetic composition according to an embodiment of the present invention, in addition to the aforementioned active ingredient, functional additives and ingredients typically found in cosmetic compositions may also be included. The functional additives may include components selected from water-soluble vitamins, oil-soluble vitamins, high-molecular-weight peptides, high-molecular-weight polysaccharides, sphingolipids, and seaweed extracts.

[0114] Furthermore, in a cosmetic composition according to an embodiment of the present invention, in addition to the aforementioned functional additives, ingredients typically found in cosmetic compositions may be added as needed. These additional ingredients may include oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, coolants, antiperspirants, purified water, etc.

[0115] Furthermore, the composition according to one embodiment of the present invention may be a topical skin agent, and the term "topical skin agent" is a general term encompassing any dosage form applied externally to the skin, including various dosage forms of cosmetics and pharmaceuticals.

[0116] In one embodiment, the composition may be a pharmaceutical composition for improving, preventing, or treating atopic dermatitis.

[0117] The pharmaceutical composition according to one embodiment of the present invention may further comprise preservatives, stabilizers, wettable powders or emulsifiers, pharmaceutical adjuvants for controlling osmotic pressure such as salts and / or buffers, and other substances useful for treatment, and may be formulated into various oral or parenteral dosage forms according to conventional methods.

[0118] The oral dosage forms may include, for example, tablets, pills, hard capsules and soft capsules, liquids, suspensions, emulsions, syrups, powders, granules, pellets, and microcapsules. In addition to the active ingredient, these dosage forms may also contain surfactants, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), and lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and polyethylene glycol). Furthermore, tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethyl cellulose, and polyvinylpyrrolidone, and, depending on the specific circumstances, may also contain pharmaceutical additives such as disintegrants, absorbents, colorants, flavoring agents, and sweeteners, such as starch, agar, alginic acid, or their sodium salts. The tablets can be prepared using conventional mixing, granulation, or coating methods. In addition, the parenteral dosage form may be a transdermal dosage form, such as injection, drops, ointment, lotion, gel, cream, spray, suspension, emulsion, suppository, patch, etc., but is not limited to these.

[0119] The pharmaceutical composition according to one embodiment of the present invention can be administered via parenteral, rectal, topical, transdermal, or subcutaneous routes.

[0120] Those skilled in the art can determine the dosage of the active ingredient based on their level of knowledge. Although the daily dosage of the drug varies depending on various factors such as the degree of disease progression, time of onset, age, health status, and complications of the drug recipient, generally, on an adult basis, the dosage of the composition can range from 0.01 mg / kg / day to 100 mg / kg / day, for example, 0.01 mg / kg / day or more, 0.1 mg / kg / day or more, 0.5 mg / kg / day or more, 1 mg / kg / day or more, 1.3 mg / kg / day or more, or 1.5 mg / kg / day. / day or more, 1.7 mg / kg / day or more, 2 mg / kg / day or more, or 2.2 mg / kg / day or more, and 100 mg / kg / day or less, 50 mg / kg / day or less, 10 mg / kg / day or less, 7 mg / kg / day or less, 5 mg / kg / day or less, 3 mg / kg / day or less, or 2.5 mg / kg / day or less, preferably, 1 mg / kg to 5 mg / kg can be divided into 1 to 3 daily doses, and this dosage does not limit the scope of the invention in any way.

[0121] In one embodiment, the composition may be a food composition.

[0122] The food composition according to one embodiment of the present invention may be in liquid or solid form, and may be in the form of tablets, capsules, soft capsules, pills, granules, beverages (drinks), slimming bars, chocolate, caramel, or biscuits, with no particular limitation on the dosage form. In addition to the aforementioned active ingredients, the food composition of the present invention may also contain, as needed, appropriate amounts of excipients, sugars, flavorings, colorings, oils, proteins, etc.

[0123] In another exemplary embodiment of the present invention, a method for protecting the skin is provided, the method comprising administering an effective dose of a composition to a desired subject, the composition comprising, as an active ingredient, a kaempferol glycoside compound, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, the kaempferol glycoside compound being represented by any one of the following chemical formulas 1 to 4.

[0124] [Chemical Formula 1]

[0125]

[0126] [Chemical Formula 2]

[0127]

[0128] [Chemical Formula 3]

[0129]

[0130] [Chemical Formula 4]

[0131]

[0132] In another exemplary embodiment of the invention, the use of kaempferol glycoside compounds, their isomers, pharmaceutically acceptable salts, their hydrates or solvates thereof in the preparation of skin protective compositions is provided, said kaempferol glycoside compounds being represented by any one of the following chemical formulas 1 to 4.

[0133] [Chemical Formula 1]

[0134]

[0135] [Chemical Formula 2]

[0136]

[0137] [Chemical Formula 3]

[0138]

[0139] [Chemical Formula 4]

[0140]

[0141] In another exemplary embodiment of the invention, kaempferol glycoside compounds, their isomers, pharmaceutically acceptable salts, their hydrates or solvates are provided for non-therapeutic use in protecting the skin, said kaempferol glycoside compounds being represented by any one of the following chemical formulas 1 to 4.

[0142] [Chemical Formula 1]

[0143]

[0144] [Chemical Formula 2]

[0145]

[0146] [Chemical Formula 3]

[0147]

[0148] [Chemical Formula 4]

[0149]

[0150] In another exemplary embodiment of the invention, kaempferol glycoside compounds, their isomers, their pharmaceutically acceptable salts, their hydrates or solvates thereof are provided for improving, preventing or treating atopic dermatitis, said kaempferol glycoside compounds being represented by any one of the following chemical formulas 1 to 4.

[0151] [Chemical Formula 1]

[0152]

[0153] [Chemical Formula 2]

[0154]

[0155] [Chemical Formula 3]

[0156]

[0157] [Chemical Formula 4]

[0158]

[0159] The present invention will now be described in more detail through the following embodiments. However, the following embodiments are provided only to aid in understanding the present invention, and the scope and range of the present invention are not limited thereto.

[0160] Example

[0161] The camellia seed oil meal used in the experiment was obtained from camellia seeds from Camellia Village, Jeju Island, through pressing to produce oil. An extract was prepared from the camellia seed oil meal using 70% ethanol, and it was confirmed that the extract consisted of terpene glycosides, flavonoid glycosides, sugars, and proteins. Specifically, the flavonoid glycosides in the camellia seed oil meal consisted of tetrasaccharides (chemical formulas 1, 2, 3, and 4) and trisaccharides (chemical formulas 5, 6, 7, and 8) of kaempferol. This specification confirms that the four tetrasaccharides (chemical formulas 1, 2, 3, and 4) in the kaempferol glycosides of the camellia seed oil meal possess excellent skin anti-aging and regeneration effects.

[0162] 1) Purification of kaempferol glycoside fraction from camellia seed oil meal

[0163] Eight kilograms of 70% ethanol were added to 1 kilogram of camellia seed oil meal that had been deoiled after low-temperature pressing, and the mixture was extracted at 60°C for 8 hours using a heated extractor. The extract obtained after filtration was concentrated under reduced pressure using a rotary vacuum concentrator at 50°C to 60°C until the solids content was approximately 50%, yielding 200g of concentrate. The concentrate was then diluted with purified water to a solids content of 10%.

[0164] The solution was loaded into a glass column containing 2L of ion exchange resin (HP-20) and washed with 10L of purified water to remove impurities and sugar components. Then, 10L of 30% ethanol was passed through the column sequentially, and the eluent was concentrated under reduced pressure and lyophilized to obtain approximately 20g of camellia kaempferol glycoside fraction powder.

[0165] 2) Purification of kaempferol tetrasaccharides and trisaccharides from camellia seed oil meal

[0166] The elution time differences of the various components in the camellia kaempferol glycoside fraction were very small in the HPLC results, therefore preparative HPLC was used for separation. The solvent conditions were a mixture of water, acetonitrile, and methanol, using concentration gradients or constant ratios of these solvents. This resulted in the separation of kaempferol glycoside compounds of formulas 1 to 4 (tetrasaccharides) and formulas 5 to 8 (trisaccharides).

[0167] Experimental Examples

[0168] statistics

[0169] In all the following analyses, statistical significance between samples was determined using the Student's t-test. A p-value of 0.05 or lower was considered statistically significant. (*p<0.05, **p<0.01, ***p<0.001)

[0170] Experimental Example 1: Confirmation of the expression level of the aging biomarker SA-β-gal

[0171] Replace with DMEM medium diluted with the experimental material (2 μM or 3 μM) and incubate for 48 hours. Then, collect the supernatant and determine the amount of MMP-1 using a human total MMP-1 ELISA (R&D systems). The results are as follows: Figure 3 As shown.

[0172] Reference Figure 3 It can be confirmed that, compared with control groups such as kaempferol trisaccharide compounds (chemical formulas 5 and 7) and retinoic acid (RA), the kaempferol tetrasaccharide compounds (chemical formulas 1, 3 and 4) of the present invention have superior inhibition of MMP-1 expression.

[0173] Experimental Example 4: Confirmation of Skin Barrier Improvement

[0174] 1) 3D organ-type skin epidermis

[0175] Human epidermal models (Epiderm™ EPI-201; MatTek, Ashland, MA, USA) corresponding to young adults (21 years old, denoted as 21Y) and elderly adults (58 years old, denoted as 58Y) were purchased separately. The epidermal models corresponding to the elderly adults were treated with 3 μM of kaempferol tetrasaccharide (represented by Formula 1) for 4 days. After fixation in 10% neutral buffered formalin, paraffin blocks were prepared.

[0176] 2) Confirmation of skin barrier markers

[0177] After reacting with antibodies against KRT10 (Biolegend, San Diego, California), FLG (Novus Biologicals, Centnell, Colorado), epidermal protein, and Ki67 (Abcam), the expression of each antibody was analyzed under a microscope. Specifically, epidermal tissue from young donors (21 years old) and older donors (58 years old) was treated with the drugs, then stained with the aforementioned antibodies and quantified. The results are as follows: Figure 4 , Figures 5a to 5b and Figures 6a to 6c As shown, Figures 5a to 5b and Figures 6a to 6c Yes Figure 4 The results were quantified.

[0178] This confirms that, compared to younger epidermal tissue, older epidermal tissue has a reduced epidermal thickness. Figure 5a ), barrier function (KRT10- Figure 6a FLG- Figure 6b INV- Figure 6c The regeneration capacity marker (KI67-) weakens, and the regeneration capacity marker (KI67-) Figure 5bThe expression of ) was reduced. However, when aged epidermal tissue was treated with compounds represented by chemical formula 1, the expression of epidermal thickness, barrier function, and regenerative capacity markers in all items was restored to a level similar to that in young epidermal tissue.

[0179] Experimental Example 5: Recovery of regeneration capacity when PDK1 is inhibited

[0180] By analyzing the regulatory signaling network, PDK1 was predicted to be an inhibitory target that can transform senescent fibroblasts into quiescent fibroblasts. Figure 8a To verify this prediction, NHDF (normal human dermal fibroblasts) exposed to a PDK1 inhibitor were used. Figure 8b Experiments were conducted and it was confirmed that cellular senescence characteristics were removed and cell proliferation was restored in response to growth factors. Skin regeneration capacity was also confirmed to be restored in two-dimensional culture and three-dimensional skin equivalent models.

[0181] In summary, it was confirmed that inhibition of PDK1 reduced both cell growth and SASP, key processes for maintaining the senescence phenotype, and inactivated the major positive feedback loop related to cell growth, consisting of PDK1, AKT, IKBKB, and PTEN. These consequential changes in signal transduction led to the restoration of the quiescence phenotype.

[0182] In the following experiments, normal human dermal fibroblasts (NHDF; Lonza, Switzerland) were cultured in DMEM medium (Gibco 1210-0038) containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2. Cells at passages 6-9 were used as young cells and passaged at a ratio of 1:5 for 37 times to obtain senescent dermal fibroblasts.

[0183] 1) Analyze the effect of PDK1 inhibition on the functional recovery of skin cells through wound healing analysis.

[0184] First, to confirm that PDK1 inhibition or dual inhibition of mTOR and NF-κB not only reduces aging characteristics but also achieves true regenerative capacity, the ability of reversed cells to heal two-dimensional wounds and regenerate three-dimensional skin was investigated.

[0185] The specific experimental method is as follows.

[0186] Young NHDF cells were not subjected to senescence conditions or drug treatment. Young NHDF cells were analyzed when the culture reached approximately 80% confluence at PDL 12. The remaining NHDF cells were exposed to doxorubicin (100 ng / ml) and IGF-1 (100 ng / ml) for 7 days to induce senescence. For drug treatment conditions, senescent cells were further treated for 7 days with DMSO, rapamycin (100 nM), rapamycin and JSH (2.5 μM), or BX795 (100 nM). For conditioned medium treatment (BX795 + CM), cells were grown for 7 days in the presence of doxorubicin and IGF-1, then passaged for 7 days in the presence of doxorubicin, IGF-1, and BX795, and then cultured for another 3 days with conditioning medium extracted from senescent cells.

[0187] Reference Figures 9a to 9b The following are examples: (9a) Representative images are shown at 0 and 96 hours post-traumatic injury, with dashed lines indicating the wound site. Scale bar = 200 μm. (9b) Quantitative analysis of migration regions in the scratch wound test. Mean ± SD (n = 3), according to one-way ANOVA, ***P < 0.001 compared with senescent cells in the DMSO control group.

[0188] The findings indicate that PDK1 inhibition or dual inhibition of mTOR and NF-κB (not mTOR single inhibition) promotes the healing of NHDF monolayer scratch wounds. Figures 9a to 9b ).

[0189] 2) The effect of PDK1 on skin regeneration

[0190] 3D skin models (skin equivalents) were prepared using young NHDF (PDL 12) or senescent NHDF induced by doxorubicin and IGF-1 or repeated coating treatments (replicating senescence, PDL 57), and exposed to DMSO or BX795. The concentrations for each treatment were as follows: doxorubicin (100 mg / mL), IGF-1 (100 ng / mL), and BX-795 (100 nM).

[0191] Reference Figures 10a to 10b The results are as follows: (10a) Skin equivalent model sections were stained with hematoxylin and eosin (H&E) or Masson's Trichrome to detect collagen fibers. Labeled proteins were detected by immunohistochemistry. Results were obtained from three representative experiments. Scale bar = 50 μm.

[0192] Specifically, the experimental method is as follows.

[0193] Paraffin samples were dewaxed in xylene and rehydrated by decreasing ethanol (3 minutes for each solution). Epitope retrieval was performed for 10 minutes at 121°C using an antigen retrieval pot (64142; Electron Microscopy Sciences, Hatfield, Pennsylvania) with citrate buffer pH 6.0, followed by cooling of the slides before washing three times in PBS / 0.05% Tween 20. The slides were then incubated in water / 1% H2O2 for 30 minutes to block endogenous peroxidase, rinsed with water and PBS / 0.05% Tween 20 for 5 minutes, and incubated for 20 minutes in PBS / 10% goat serum (Dako, Gloucestershire, Denmark) before exposure to primary antibodies overnight at 4°C. The primary antibodies used were: anti-KRT10 (Biolegend, San Diego, CAN), anti-FLG (Abcam), anti-MMP-1 (Abcam), and anti-Ki67 (Abcam).

[0194] The samples were thoroughly washed with water for 10 minutes, then thoroughly washed with PBS / 0.05% Tween 20 for 5 minutes, and then incubated for 30 minutes with EnVision+System-horseradish peroxidase-labeled polymeric anti-mouse antibody (K4001; Dako, Carpintriia, CA). The samples were then washed with water and PBS for 3 to 5 minutes before incubation with a liquid DAB+ substrate chromogen system (K3468; Dako). The above reactions were terminated by rinsing with tap water, and the sections were counterstained in Mayer's hematoxylin for 3 to 5 minutes and rinsed in tap water for 1 minute, then rinsed in Scott's blue solution for 2 minutes.

[0195] Finally, the sections were dehydrated in a specified grade of ethanol (xylene removed) and mounted in Cytoseal 60 (ThermoScientific, Waltham, MA). Masson's trichrome staining was performed using a commercially available kit (BBCBiochemical, Seattle, WA). For immunofluorescence staining, antigen retrieval was performed by boiling the slides in Dako target retrieval solution (Dako) for 20 minutes. Nonspecific staining was blocked by incubation in PBS containing 10% donkey serum and 0.05% Triton X-100 for 30 minutes. Tissue sections were incubated overnight at 4°C in Dako fluorescent mounting medium (Dako) with COL17A1 (Abcam) and laminin 5 (Laminin 5; Santa Cruz Biotechnology), then incubated at room temperature for 2 hours with an appropriate secondary antibody conjugated to Alexa Fluor488 (Invitrogen-Molecular Probes). After washing with PBS, 4',6-diamidin-2'-phenylindole dihydrochloride (DAPI, Invitrogen-Molecular Probes) was added for nuclear counterstaining. The coverslip was then mounted onto the slide using fluorescent mounting medium (Thermo Electron).

[0196] (10b) In Figure 10b From top left to right, the data in section B are: quantification of skin thickness using ImageJ software; quantification of MMP1; quantification of pan-collagen fibers detected by Masson's trichrome staining; quantification of Ki-67 positive cells; quantification of epidermal thickness using ImageJ software; quantification of keratin 10; quantification of filaggrin; quantification of COL17A1; quantification of laminin 5; and quantification of type I procollagen secreted into the culture medium by ELISA. Data in section B are expressed as mean ± SD of three independent fields obtained from three independent samples. According to one-way ANOVA, compared with senescent cells in the DMSO control group, ***P < 0.001.

[0197] PDK1 inhibition reversed dermal aging associated with replication-induced aging and chemically induced aging, as evidenced by increased MMP1, decreased collagen fibers, and reduced procollagen secretion. Figures 10a to 10b Under epidermal homeostasis, the balance between keratinocyte proliferation and differentiation is regulated by dermal fibroblasts.

[0198] Therefore, this study investigated whether reversing senescent dermal fibroblasts through PDK1 inhibition affects keratinocyte proliferation and differentiation. BX-795 is a potent PDK1 inhibitor that blocks PDK1 / Akt signaling in tumor cells and inhibits adherence-dependent growth of various tumor cell lines in culture or induced apoptosis. The results showed that, in skin equivalents treated with the well-known PDK1 inhibitor BX795 (… Figures 10a to 10b The Ki-67 proliferation marker was significantly elevated in the epidermis of NDHF, indicating an increased proliferative capacity of keratinocytes that maintain epidermal thickness in the induced senescence NDHF skin equivalent. Figures 10a to 10b ).

[0199] Furthermore, a comparison of staining intensities of keratinocyte differentiation markers (keratin 10 and filaggrin) and basement membrane markers at the dermal-epidermal junction (COL17A1 and laminin 5) showed that PDK1 inhibition restored the reduced abundance of these keratinocyte differentiation markers and dermal-epidermal junction components found in the skin equivalents of senescent NHDF. Figure 10a ).

[0200] Observations on two-dimensional aging NHDF ( Figures 9a to 9b Similarly, in the conditioned medium of skin equivalents prepared from two types of aging NHDF treated with BX795, the contents of GRO, GROα, IL-6, IL-8, MCP-1 to 3, and RANTES were lower than those in the conditioned medium of skin equivalents extracted from vehicle-treated samples. Figures 11a to 11b ).

[0201] Specifically, the experimental method is as follows.

[0202] PDK1 inhibition in the skin equivalent inhibits SASP. The 3D skin model (skin equivalent) was prepared using young NHDF (PDL 12) or senescent NHDF induced by doxorubicin and IGF-1 or repeated coating treatments (replicating senescence, PDL 57), and exposed to DMSO or BX795. The concentrations for each treatment were as follows: doxorubicin (100 mg / mL), IGF-1 (100 ng / mL), and BX-795 (100 nM).

[0203] Conditioned culture medium (CM) from skin equivalents of chemically induced or replicated senescent NHDF cells treated with or without BX795 was measured using a cytokine antibody array and normalized with an internal positive control for each membrane. (11a) Secreted proteins of chemically induced NHDF cells treated with or without BX795 were measured in the cytokine array of the skin equivalents. (11b) Secreted proteins of replicated senescent NHDF cells treated with or without BX795 were measured in the cytokine array of the skin equivalents. According to one-way ANOVA, the mean ± SD (n=3) was significantly different from that of the DMSO control group of senescent cells; ***P<0.001.

[0204] In summary, these findings suggest that PDK1 inhibition transforms senescent dermal fibroblasts into quiescent fibroblasts, thereby enhancing epidermal regeneration and maintaining skin thickness, thus limiting age-related skin deterioration.

[0205] Specific examples

[0206] Specific Example 1: Use of kaempferol glycosides, their isomers, pharmaceutically acceptable salts, their hydrates or solvates in the preparation of skin-protecting compositions, wherein the kaempferol glycosides are represented by any one of the following chemical formulas 1 to 4:

[0207] [Chemical Formula 1]

[0208] ,

[0209] [Chemical Formula 2]

[0210] ,

[0211] [Chemical Formula 3]

[0212] ,

[0213] [Chemical Formula 4]

[0214] .

[0215] Specific Example 2: According to the use described in Specific Example 1, wherein the kaempferol glycoside compound is extracted from camellia seed oil meal.

[0216] Specific Example 3: According to the use described in Specific Example 1 or 2, wherein, based on the total weight of the composition, the concentration of the kaempferol glycoside compound, its isomer, its pharmaceutically acceptable salt, its hydrate or its solvate is from 0.01 μM to 100 μM.

[0217] Specific Example 4: The use according to any one of Specific Examples 1 to 3, wherein the skin protection is a composition that improves skin wrinkles; promotes skin regeneration; inhibits the expression of matrix metalloproteinase-1 (MMP-1); improves, prevents or treats atopic skin; regulates the skin keratinization cycle; or inhibits the activity of inositol 3-phosphate-dependent protein kinase 1.

[0218] Specific Example 5: The use according to any one of Specific Examples 1 to 4, wherein the composition is a cosmetic composition.

[0219] Specific Example 6: The use according to any one of Specific Examples 1 to 4, wherein the composition is a pharmaceutical composition for improving, preventing or treating atopic dermatitis.

[0220] Specific Example 7: According to the use described in Specific Example 6, the dosage of the kaempferol glycoside compound, its isomer, its pharmaceutically acceptable salt, its hydrate or its solvate is from 0.01 mg / kg / day to 100 mg / kg / day.

[0221] Specific Example 8: The use according to any one of Specific Examples 1 to 4, wherein the composition is a food composition.

Claims

1. Use of kaempferol glycoside compounds or pharmaceutically acceptable salts thereof in the preparation of compositions for inducing the growth of aging-inhibited skin cells, said kaempferol glycoside compounds being represented by any one of the following chemical formulas 1 to 4, wherein, The induction of aging-induced growth inhibition of skin cells is achieved by inhibiting the activity of inositol 3-phosphate-dependent protein kinase 1, and this induction includes restoring epidermal thickness or barrier function in aged epidermal tissue. [Chemical Formula 1] ， [Chemical Formula 2] ， [Chemical Formula 3] ， [Chemical Formula 4] 。 2. The use according to claim 1, characterized in that, The kaempferol glycoside compound was extracted from camellia seed oil meal.

3. The use according to claim 1, characterized in that, Based on the total weight of the composition, the concentration of the kaempferol glycoside compound or its pharmaceutically acceptable salt is from 0.01 μM to 100 μM.

4. The use according to claim 1, characterized in that, The induction of growth in skin cells whose growth is inhibited due to aging is achieved by inhibiting the expression of matrix metalloproteinase-1 or regulating the skin keratinization cycle.

5. The use according to claim 1, characterized in that, The composition is a cosmetic composition.

6. The use according to claim 1, characterized in that, The composition is a pharmaceutical composition.

7. The use according to claim 6, characterized in that, The dosage of the kaempferol glycoside compound or its pharmaceutically acceptable salt is from 0.01 mg / kg / day to 100 mg / kg / day.

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