Camellia-derived flavonoids

Abstract

To provide a novel kaempferol compound obtained by enzymatically treating kaempferol saccharides extracted from Camellia oleifera seed cake, which is discarded after oil extraction from camellia seeds, and a skin protection composition containing the same that is safe for the human body.SOLUTION: An enzyme-treated kaempferol compound has an increased molecular weight as sugar is additionally linked to the precursor kaempferol saccharide, and can exhibit a skin protection effect.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] This specification relates to a novel camellia-derived flavonoid, and more specifically, to a kaempferol glycoside compound that is safe for the human body and has excellent skin protective effects, and a skin protective composition containing the same. [Background technology]

[0002] Plant-derived flavonoids have a C6-C3-C6 carbon skeleton with two phenyl groups attached via a C3 chain, and often exist in the form of glycosides bound to various sugars. Each plant produces specific glycoside structures through its own biosynthetic pathway.

[0003] These flavonoid glycosides exhibit various physiological activities, including antibacterial, anticancer, and anti-inflammatory activities, and the type and strength of the physiological activities vary depending on the sugar modification position in the flavonoid structure, the sugar chain structure, and the molecular weight.

[0004] However, many of the conventional plant-derived skin protection ingredients have issues such as the need to use large amounts of plant resources, or low efficacy and low value due to special molecular weights or structures. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent Publication No. 10-2022-0036148 [Patent Document 2] Korean Patent Publication No. 10-2021-0037643 [Non-patent literature]

[0006] [Non-Patent Document 1] B.-C.Liu et al. / Industrial Crops and Products 95(2017)296-304 Summary of the Invention [Problem to be solved by the invention]

[0007] In one aspect of the present invention, an object of the present invention is to provide an enzyme-treated kaempferol glycoside compound obtained from Camellia oleifera seed cake, which is a resource discarded after the extraction of camellia seed oil, and a skin protection composition containing the same.

[0008] In another aspect of the present invention, it is an object of the present invention to provide an enzyme-treated kaempferol glycoside compound with an adjusted molecular size, which is obtained by enzymatically treating an extract of Camellia oleifera seed cake, which contains a large amount of kaempferol glycoside compound, and a skin protection composition containing the same.

[0009] In yet another aspect of the present invention, an object is to provide a composition which contains an enzyme-treated kaempferol glycoside compound as an active ingredient, which is safe for the human body, has few side effects, and has excellent skin protective effects.

[0010] In yet another aspect of the present invention, an object is to provide a skin protection composition which contains an enzyme-modified kaempferol glycoside compound as an active ingredient and inhibits the activity of 3-phosphoinositide-dependent kinase 1 (PDK1). [Means for solving the problem]

[0011] The present specification provides an enzyme-treated kaempferol glycoside compound represented by any one of the following general formulas 1 to 6, a stereoisomer thereof, a pharma- ceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

[0012] [ka]

[0013] [ka]

[0014] [ka]

[0015] [ka]

[0016] [ka]

[0017] [ka]

[0018] The present specification provides a skin protection composition containing the above-mentioned enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharma- ceutical acceptable salt, its hydrate or its solvate as an active ingredient. Effect of the Invention

[0019] In one aspect, the enzymatically processed kaempferol glycoside compounds disclosed herein can inhibit the activity of 3-phosphoinositide-dependent kinase 1 (PDK1).

[0020] In one aspect, the enzymatically modified kaempferol glycoside compound disclosed herein can inhibit the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and exert a skin protective effect. [Brief description of the drawings]

[0021] [Figure 1]1 is a 365 nm ultraviolet absorption chromatogram for a camellia oil cake extract and its enzymatically converted product, an enzyme-treated kaempferol compound, according to one embodiment of the present invention. [Diagram 2] 2 is a 365 nm ultraviolet absorption chromatogram of a glycoside mixture obtained by enzymatic treatment of camelliaside A and its separated and purified products according to one embodiment of the present invention. [Diagram 3] 2 is a 365 nm ultraviolet absorption chromatogram of a glycoside mixture obtained by enzymatic treatment of camelliaside B and its separated and purified product according to one embodiment of the present invention. [Figure 4] 1 is a 365 nm ultraviolet absorption chromatogram of a glycoside mixture obtained by enzymatically treating nicotiflorin and its separated and purified products according to one embodiment of the present invention. [Diagram 5] 1 is a graph showing the inhibitory effect of DMSO (blank), BX-795 (control), COSCE (Camellia oil cake extract), COSCE-P (purified Camellia oil cake extract), and COSCE-PeP (purified Camellia oil cake extract after enzymatic treatment). The units above the bars are ug / mL. [Figure 6] FIG. 1 is a graph showing the inhibitory effect of 3-phosphoinositide-dependent kinase 1 (PDK1) activity against DMSO (blank), BX-795 (control), CamA (camelliaside A), CamB (camelliaside B), and EM (enzymatically modified) according to one embodiment of the present invention. The units of values ​​above the bars are ug / mL. EM-CamA includes compounds of general formulas 1 to 3, and EM-CamB includes compounds of general formulas 4 to 6. [Figure 7]FIG. 1 is a graph showing the inhibitory effect of 3-phosphoinositide-dependent kinase 1 (PDK1) activity against DMSO (blank), BX-795 (control), AG4 (1 molecule per camelliaside A+), AG5 (2 molecules per camelliaside A+), AG6 (3 molecules per camelliaside A+), AG7 (4 molecules per camelliaside A+), BG4 (1 molecule per camelliaside B+), BG5 (2 molecules per camelliaside B+), BG6 (3 molecules per camelliaside B+), BG7 (4 molecules per camelliaside B+), NG4 (2 molecules per nicotiflorin+), NG5 (3 molecules per nicotiflorin+), NG6 (4 molecules per nicotiflorin+), and NG7 (5 molecules per nicotiflorin+). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In this specification, when a part "comprises" a certain component, this does not mean that other components are excluded, but that other components may also be included, unless otherwise specified.

[0023] The present invention will be described in detail below.

[0024] In an exemplary embodiment of the present disclosure, there is provided an enzyme-treated kaempferol glycoside compound represented by any one of the following general formulas 1 to 6, its stereoisomer, its pharma- ceutically acceptable salt, its hydrate or its solvate.

[0025] [ka]

[0026] [ka]

[0027] [ka]

[0028] [ka]

[0029] [ka]

[0030] [ka]

[0031] In yet another exemplary embodiment of the present invention, there is provided a method for protecting the skin, comprising administering an effective amount of a composition containing, as an active ingredient, a kaempferol glycoside compound represented by any one of general formulas 1 to 6, its stereoisomer, its pharma- ceutical acceptable salt, its hydrate or its solvate to a subject in need thereof.

[0032] In yet another exemplary embodiment of the present invention, there is provided a use of a kaempferol glycoside compound represented by any one of general formulas 1 to 6, its stereoisomer, its pharma- ceutically acceptable salt, its hydrate, or its solvate for the manufacture of a skin protective composition.

[0033] In yet another exemplary embodiment of the present invention, there is provided a non-therapeutic use of a kaempferol glycoside compound represented by any one of general formulas 1 to 6, its stereoisomer, its pharma- ceutical acceptable salt, its hydrate or its solvate for protecting the skin.

[0034] As used herein, "isomer" includes in particular optical isomers (e.g., essentially pure enantiomers, essentially pure diastereomers or mixtures thereof), but also conformation isomers (i.e., isomers that differ only in the angle of at least one chemical bond), position isomers (especially tautomers) or geometric isomers (e.g., cis-trans isomers).

[0035] As used herein, "essentially pure," when used in connection with, for example, an enantiomer or diastereomer, means that a particular compound, exemplified by an enantiomer or diastereomer, is present in an amount of about 90% or more, preferably about 95% or more, more preferably about 97% or more or about 98% or more, even more preferably about 99% or more, and most preferably about 99.5% or more (w / w).

[0036] As used herein, "pharmaceutical acceptable" means capable of being approved, approved by a governmental or equivalent regulatory agency, listed in a pharmacopoeia, or other commonly recognized pharmacopoeia, for use in animals, and more particularly, in humans, by avoiding significant toxic effects when used in normal medicinal dosages.

[0037] As used herein, the term "pharmacologically acceptable salt" refers to a salt according to an embodiment of the present invention that is pharma- ceutically acceptable and has the preferred pharmacological activity of the parent compound. The salts are formed from (1) inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or from (2) acetic acid, propionic acid, hexanoic acid, cyclopentenepropionic 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-hydroxyethanes ... or (2) acid addition salts formed from organic acids such as benzoic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; or (3) salts formed when an acidic proton present in the parent compound is replaced.

[0038] As used herein, the term "hydrate" refers to a compound to which water is bound, and is a broad concept that includes an endocannabinoid compound in which there is no chemical bonding between the water and the compound.

[0039] As used herein, the term "solvate" refers to a higher-order compound formed between molecules or ions of a solute and molecules or ions of a solvent.

[0040] Camelliaside A, which is a precursor of the compound represented by any one of general formulas 1 to 3 according to an embodiment of the present invention, is represented by the following general formula A. The results of nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by general formula A are shown in Table 1 below.

[0041] [ka]

[0042] [Table 1]

[0043] The results of nuclear magnetic resonance analysis of the enzyme-treated kaempferol glycoside compound represented by general formula 1 are shown in Tables 2A and 2B.

[0044] [Table 2A]

[0045] [Table 2B]

[0046] The results of nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by general formula 2 are shown in Tables 3A and 3B.

[0047] [Table 3A]

[0048] [Table 3B]

[0049] The results of nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by general formula 3 are shown in Tables 4A and 4B.

[0050] [Table 4A]

[0051] [Table 4B]

[0052] The kaempferol heptaglycoside compound, which is obtained by adding four sugars to the above-mentioned camelliaside A as a precursor, is represented by the following general formula A-1. The results of the nuclear magnetic resonance analysis of the compound of general formula A-1 are shown in Tables 5A and 5B below.

[0053] [ka]

[0054] [Table 5A]

[0055] [Table 5B]

[0056] Camelliaside B, which is a precursor of the compound represented by any one of general formulas 4 to 6 according to an embodiment of the present invention, is represented by the following general formula B. The results of the nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by general formula B are shown in Table 6 below.

[0057] [ka]

[0058] [Table 6]

[0059] The results of nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by general formula 4 are shown in Tables 7A and 7B.

[0060] [Table 7A]

[0061] [Table 7B]

[0062] The results of nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by general formula 5 are shown in Tables 8A and 8B.

[0063] [Table 8A]

[0064] [Table 8B]

[0065] The results of nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by general formula 6 are shown in Tables 9A and 9B.

[0066] [Table 9A]

[0067] [Table 9B]

[0068] The kaempferol heptaglycoside compound, which is obtained by adding four sugars to the above-mentioned camelliaside B as a precursor, is represented by the following general formula B-1. The results of the nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by general formula B-1 are shown in Tables 10A and 10B.

[0069] [ka]

[0070] [Table 10A]

[0071] [Table 10B]

[0072] In one embodiment of the present invention, nicotiflorin may be used as a precursor, and nicotiflorin is represented by the following general formula N. The nuclear magnetic resonance analysis result of the kaempferol glycoside compound represented by general formula N is shown in Table 11.

[0073] [ka]

[0074] [Table 11]

[0075] The results of nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by the following general formula N-1, which was produced using the compound represented by the above general formula N as a precursor, are shown in Tables 12A and 12B.

[0076] [ka]

[0077] [Table 12A]

[0078] [Table 12B]

[0079] The results of nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by the following general formula N-2, which was produced using the compound represented by the above general formula N as a precursor, are shown in Tables 13A and 13B.

[0080] [ka]

[0081] [Table 13A]

[0082] [Table 13B]

[0083] The results of nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by the following general formula N-3, which was produced using the compound represented by the above general formula N as a precursor, are shown in Tables 14A and 14B.

[0084] [ka]

[0085] [Table 14A]

[0086] [Table 14B]

[0087] The results of nuclear magnetic resonance analysis of the kaempferol glycoside compound represented by the following general formula N-4, which was prepared using the compound represented by the above general formula N as a precursor, are shown in Tables 15A and 15B.

[0088] [ka]

[0089] [Table 15A]

[0090] [Table 15B]

[0091] The composition according to one embodiment of the present invention may realize reaction conditions for producing kaempferol trisaccharides extracted from Camellia oleifera seed cake, which is discarded after oil is extracted from camellia seeds, i.e., kaempferol 4-6 glycosides to which glucose has been added using a compound represented by general formula A or general formula B as a precursor.

[0092] Compared to the composition of kaempferol triglycoside compound extracted from camellia oil cake, it has a significantly superior skin protection effect.

[0093] Camellia oil cake extract consists of terpin glycosides, flavonoid glycosides, sugars and proteins. In particular, the flavonoid glycosides in camellia oil cake are composed of triglycosides (general formula A and general formula B). In the present invention, it was confirmed that eight types of tetraglycosides to hexaglycosides (general formulas 1 to 6) in which glucose is sequentially bound to kaempferol triglycoside derived from camellia oil cake as a precursor have excellent skin protection effects.

[0094] In one embodiment, the enzymatically treated kaempferol glycoside compound may be an enzymatically treated kaempferol triglycoside.

[0095] In one embodiment, the kaempferol triglycoside may be an extract derived from Camellia sinensis.

[0096] Specifically, the extract and the kaempferol triglycoside compound may be obtained by obtaining camellia oil cake obtained after squeezing oil from camellia seeds by a pressing method, and then extracting the kaempferol glycoside compound from the camellia oil cake using 70% ethanol.

[0097] In one embodiment, the enzyme may be cyclodextrin glucanotransferase.

[0098] In an exemplary embodiment of the present invention, a skin protecting composition is provided that contains an enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharma- ceutical acceptable salt, its hydrate or its solvate as an active ingredient.

[0099] In one embodiment, the daily dosage of the active ingredient may be 1 to 1000 mg / Kg. For example, the daily dosage of the active ingredient may be 1 mg / Kg or more, 5 mg / Kg or more, 10 mg / Kg or more, 50 mg / Kg or more, 100 mg / Kg or more, 300 mg / Kg or more, 500 mg / Kg or more, 700 mg / Kg or more, or 900 mg / Kg or more, or 1000 mg / Kg or less, 800 mg / Kg or less, 600 mg / Kg or less, 400 mg / Kg or less, 200 mg / Kg or less, 100 mg / Kg or less, 50 mg / Kg or less, 10 mg / Kg or less, or 5 mg / Kg or less.

[0100] If the daily application amount of the active ingredient is less than 1 mg / Kg, the skin protective effect may be insignificant, and if it exceeds 1000 mg / Kg, cytotoxicity may occur.

[0101] In one embodiment, the composition may inhibit the activity of 3-phosphoinositide-dependent kinase 1 (PDK1).

[0102] According to Patent Document 1, when PDK1 activity is inhibited, aged and growth-suppressed cells can grow again and damaged cells can be restored. Specifically, inhibition of PDK1 in aged human dermal fibroblasts can eliminate aging characteristics and inhibit NF-κB and mTOR signaling, thereby restoring the cell generation cycle and restoring or promoting skin regeneration ability. Furthermore, inhibition of PDK1 reverses dermal aging associated with replicative aging and chemically induced aging, as demonstrated by increased MMP1, decreased collagen fibers, and decreased procollagen secretion. In the case of epidermal homeostasis, the balance between keratinocyte proliferation and differentiation is controlled by dermal fibroblasts. Reversal of aged dermal fibroblasts by inhibition of PDK1 affects keratinocyte proliferation and differentiation. Inhibition of PDK1 restores the reduced abundance of these keratinocyte differentiation markers and components of the dermal-epidermal junction found in skin equivalents of aged NHDF.

[0103] According to Patent Document 2, PDK1 is a protein that simultaneously controls mTOR and NF-kB, among the major signaling proteins involved in cellular senescence, and is a core protein that determines the secretory phenotype associated with cell growth, division, and senescence. Transient inhibition of PDK1 effectively reverses a portion of senescent cells into normal young cells, and the senescent cell phenotype is no longer observed in these cells. PDK1 inhibitors inhibit the transcriptional activity of mTOR (mammalian Target Of Rapamycin) and NF-kB (Nuclear Factor Kappa B). Inhibiting PDK can induce reverse senescence.

[0104] In one embodiment, the composition may be for improving the appearance of wrinkles on the skin.

[0105] In one embodiment, the composition may be for promoting skin regeneration.

[0106] In one embodiment, the composition may be for improving atopic skin.

[0107] In one embodiment, the composition may be for regulating the skin's keratinogenesis cycle.

[0108] According to one aspect of the present invention, the composition can promote colony formation and differentiation of epidermal cells to shorten the keratinogenesis cycle or turnover cycle of epidermal cells.

[0109] In another embodiment, the composition may be for regulating skin keratin, for promoting skin keratin turnover, or for normalizing the skin keratin turnover cycle.

[0110] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and improving skin wrinkles.

[0111] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and promoting skin regeneration.

[0112] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) to improve atopic skin or for improving, preventing or treating atopic dermatitis.

[0113] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and regulating the keratinogenesis cycle of the skin.

[0114] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and regulating skin keratin.

[0115] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and promoting skin keratin turnover.

[0116] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and normalizing the skin keratin turnover cycle.

[0117] In one embodiment, the composition may be for improving sagging skin.

[0118] In one embodiment, the composition may be for increasing expression of Col7A1.

[0119] In one embodiment, the composition may be for increasing expression of Col17A1.

[0120] The Col7A1 and Col17A1 are proteins that constitute the DEJ (dermoepidermal junction), and a decrease in Col7A1 and Col17A1 reduces the function of the DEJ, which firmly holds epidermal cells and strengthens the binding force between the epidermis and dermis, causing skin sagging. Therefore, restoration of the expression of reduced Col7A1 and Col17A1 can prevent and improve structural deformation of the DEJ and rete ridges, thereby preventing and improving skin sagging.

[0121] According to Patent Document 1, when investigating whether reversal of senescent dermal fibroblasts by inhibition of PDK1 affects proliferation and differentiation of keratinocytes, it was confirmed that inhibition of PDK1 restores the decrease of components at the dermal-epidermal junction, such as COL17A1.

[0122] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and improving sagging skin.

[0123] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and increasing the expression of Col7A1.

[0124] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and increasing the expression of Col17A1.

[0125] In one embodiment, the composition may be for improving skin barrier function.

[0126] In one embodiment, the composition may be for improving skin moisturization.

[0127] In one embodiment, the composition may be for increasing keratin 10 expression.

[0128] In one embodiment, the composition may be for increasing the expression of filaggrin.

[0129] Keratin 10 and filaggrin are genes that regulate the keratinization of skin cells to normalize epidermal keratinization, and are known to be involved in skin barrier function, skin moisturization, skin wrinkles, aging, etc. Therefore, increased expression of keratin 10 and filaggrin can promote moisture retention in skin tissue and strengthen the skin barrier that prevents moisture loss in skin tissue.

[0130] According to Patent Document 1, when investigating whether reversal of senescent dermal fibroblasts by inhibition of PDK1 affects proliferation and differentiation of keratinocytes, it was confirmed that inhibition of PDK1 restores the expression of reduced keratinocyte differentiation markers such as keratin 10 and filaggrin.

[0131] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and improving skin barrier function.

[0132] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and improving skin moisturization. In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and increasing the expression of keratin 10.

[0133] In another embodiment, the composition may be for inhibiting the activity of 3-phosphoinositide-dependent kinase 1 (PDK1) and increasing the expression of filaggrin.

[0134] In one embodiment, the composition may be a cosmetic composition.

[0135] The appearance of the cosmetic composition according to an embodiment of the present invention may comprise a cosmetically or dermatologically acceptable vehicle or base. It may be in any formulation suitable for topical application, for example in the form of a solution, gel, solid, paste-free anhydrous product, emulsion obtained by dispersing an oily phase in an aqueous phase, suspension, microemulsion, microcapsule, microgranule or vesicular dispersion of ionic (liposome) and non-ionic type, or in the form of a cream, lotion, milk, powder, ointment, spray or stick concealer. These compositions may be prepared according to the usual methods in the art. The composition according to the present invention may also be used in the form of a foam or in the form of an aerosol composition further comprising a compressed propellant.

[0136] The cosmetic composition according to an embodiment of the present invention is not particularly limited in its formulation, and may be formulated into a cosmetic product such as, for example, a softening lotion, an astringent lotion, a nourishing lotion, a nourishing cream, a massage cream, a beauty serum, an eye cream, an eye essence, a cleansing cream, a cleansing foam, a cleansing water, a pack, a powder, a body lotion, a body cream, a body oil, or a body essence.

[0137] When the dosage form according to an embodiment of the present invention is a paste, cream or gel, the carrier component may be animal fiber, vegetable fiber, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide.

[0138] When the dosage form according to an embodiment of the present invention is a powder or spray, the carrier component may be lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder, and, particularly when it is a spray, may further include a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether.

[0139] When the dosage form according to one embodiment of the present invention is a solution or emulsion, the carrier component may be a solvent, solvating agent or emulsifier, such as water, ethanol, isopropanol, diethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, aliphatic glycerin esters, polyethylene glycol or fatty acid esters of sorbitan.

[0140] When the dosage form according to one embodiment of the present invention is a suspension, the carrier component may be 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.

[0141] When the dosage form according to an embodiment of the present invention is a surfactant-containing cleanser, the carrier component may be fatty alcohol sulfate, fatty 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, lanolin derivative, or ethoxylated glycerol fatty acid ester.

[0142] The cosmetic composition according to an embodiment of the present invention may further include functional additives and ingredients contained in general cosmetic compositions in addition to the active ingredient. The functional additives may include an ingredient selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, sphingolipids, and seaweed extracts.

[0143] The cosmetic composition according to an embodiment of the present invention may further contain components contained in general cosmetic compositions together with the functional additives, if necessary, such as oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, colorants, fragrances, blood circulation enhancers, cooling agents, antiperspirants, purified water, etc.

[0144] Furthermore, the composition according to one embodiment of the present invention may be a topical skin preparation, which is a general term that may include anything that is applied to the skin, and may include cosmetics and pharmaceuticals in various dosage forms.

[0145] In one embodiment, the composition may be a non-therapeutic transdermal composition.

[0146] In one embodiment, the composition may be a food composition for improving skin moisture status.

[0147] In one embodiment, the composition may be an oral composition.

[0148] In one embodiment, the composition may be a non-therapeutic oral composition.

[0149] The food composition or oral composition according to one embodiment of the present invention may be in a liquid or solid dosage form, and may be in the form of tablets, capsules, soft capsules, pills, granules, beverages (drinkables), diet bars, chocolates, caramel or confectionery, and the dosage form is not particularly limited.

[0150] The food composition or oral composition of the present invention may contain, in addition to the above-mentioned active ingredient, excipients, sugars, flavorings, colorants, fats and oils, proteins, and the like, as necessary.

[0151] In an exemplary embodiment of the present invention, a method for producing an enzyme-treated kaempferol glycoside compound is provided, comprising the steps of obtaining a kaempferol glycoside compound from camellia oil cake, purifying the kaempferol glycoside compound, enzyme-treating the purified kaempferol glycoside compound, and purifying the enzyme-treated enzyme-treated kaempferol glycoside compound.

[0152] In one embodiment, the purified kaempferol glycoside compound may be a kaempferol triglycoside compound.

[0153] In one embodiment, the enzyme may be cyclodextrin glucanotransferase.

[0154] The present invention will be described in more detail with reference to the following examples. Note that the following examples are merely illustrative for the purpose of aiding understanding of the present invention, and are not intended to limit the scope and confines of the present invention.

[0155] [Example] The camellia oil cake used in the experiment was obtained after oil was produced from camellia seeds (produced in China) by squeezing. An extract was produced from the camellia oil cake using 70% ethanol, and it was confirmed that the camellia oil cake extract was composed of terpine glycosides, flavonoid glycosides, sugars, and proteins. In particular, the flavonoid glycosides in camellia oil cake are composed of kaempferol triglycoside (general formulas A and B), and in this specification, it was confirmed that eight types of kaempferol 4-6 glycosides (general formulas 1-6) produced by enzyme reaction using two types of kaempferol triglycoside from camellia oil cake as precursors have excellent inhibitory effects on skin PDK1 activity.

[0156] 1) Purification of kaempferol glycoside fraction from camellia oil cake 1 kg of camellia seed oil cake, which had been extracted at low temperature to remove the oil, was added with 8 kg of 70% ethanol and extracted for 8 hours at 60°C using a heated extractor. The extract obtained by filtration using a filter press was concentrated under reduced pressure at 50-60°C using a rotary vacuum concentrator until the solid content was approximately 50%, yielding 200 g of concentrated liquid. The concentrated liquid was diluted with purified water to a solid content of 10%.

[0157] This solution was loaded onto a glass tube column packed with 2 L of ion exchange resin (HP-20), and then washed with 10 L of purified water to remove impurities and sugar components. Next, 10 L of 30% ethanol was poured in, and the eluate was concentrated under reduced pressure and freeze-dried to obtain about 20 g of Tsubakikaempferol glycoside fraction powder.

[0158] 2) Purification of kaempferol triglycoside from camellia oil cake The kaempferol triglycoside fraction derived from camellia oil cake was separated using preparative HPLC. The solvent conditions used were a mixture of water, acetonitrile, and methanol, and a concentration gradient or a fixed ratio mixture of these solvents was used, resulting in the separation of the kaempferol triglycoside compound components of general formulas A and B (Figure 1).

[0159] 3) Production and purification of kaempferol glycosides with added sugars derived from kaempferol triglycoside The camellia oil cake extract obtained in item 2) was dissolved in an acidic phosphate buffer (pH 5, 50 mM) at a concentration of 10 mg / mL, water-soluble starch at 100 mg / mL, and cyclodextrin glucanotransferase at 100 units / mL, and the enzyme reaction was started with stirring at a temperature condition of 50 to 70 °C. The enzyme reaction was terminated within 1 to 24 hours by adding a 30% aqueous ethanol solution, centrifuged (4000 g, 10 minutes) to remove the supernatant, and filtered and concentrated using a 0.45 micron filter. The concentrated filtrate was adsorbed with substances using a preparative chromatograph and a reverse phase column (C18), and then unnecessary substances were washed with pure water, and a wide range of peaks absorbing at UV280 nm were separated using a concentration gradient of acetonitrile. The separated solution was concentrated, freeze-dried, and prepared as a powder (Figure 1). For comparison, as starting materials, the pure substances camelliaside A (compound A), camelliaside B (compound B), and nicotiflorin (compound N) were subjected to an enzymatic reaction at 2 mg / mL and similarly separated.

[0160] 4) Purification of kaempferol 4-7 glycosides Pure substances camelliaside A and camelliaside B were prepared at a concentration of 2 mg / mL, and compositions with increased molecular weights were obtained by enzymatic reaction as in the example of 3) above. These were separated into single peaks using preparative chromatography and an adsorption column (C18), and four glycosides derived from camelliaside A (compound A) (compounds 1 to 3 and A-1) and four glycosides derived from camelliaside B (compound B) (compounds 4 to 6 and B-1) were obtained in pure form (Figures 2 and 3).

[0161] 5) Structural identification of purely isolated substances The structures of compounds A, B, and N used as precursors, and compounds 1 to 3 and A-1, and compounds 4 to 6 and B-1 obtained in 4) above, were identified by a nuclear magnetic resonance analyzer.

[0162] Comparative Example In order to confirm that the kaempferol triglycoside derived from camellia oil cake is useful as a precursor, kaempferol diglycoside (nicotiflorin; kaempferol rutinoside) (compound N) was used as a precursor to produce four glycosides (chemical substances N-1 to N-4) with increased molecular weights in the same manner as in Examples 3 and 4, which were purified and used in the same manner as in the test examples (Figure 4). The structures of the precursor compound N and the reactants compounds N-1 to N-4 were identified using a nuclear magnetic resonance analyzer as described above.

[0163] Test Example statistics In all the following experiments, statistical significance between samples was analyzed using student t-test, and p-values ​​of 0.05 or less were considered to be statistically significant (*p<0.05, **p<0.01, ***p<0.001).

[0164] Test Example 1: Analysis of PDK1 3-Phosphoinositide-dependent kinase 1 (PDK1) activity was measured using recombinant purified PDK1 enzyme and ADP-Glo+PDK1 Kinase Enzyme System (Promega, Madison, WI, USA). After dispensing a mixture of substrate, ATP, and buffer, test substances (1, 3, 10 μM) were dispensed into each well. Immediately after that, PDK1 was dispensed and reacted for 60 minutes. After the reaction, ADP-glo was added and incubated at room temperature for 40 minutes, and then kinase detection buffer was added and reacted for 30 minutes, after which the activity was measured using a luminescence reader (Wallac Victor2, Perkin Elmer).

[0165] Figure 5 is a graph showing the inhibitory effect of DMSO (blank), BX-795 (control), COSCE (Camellia oil cake extract), COSCE-P (purified Camellia oil cake extract), and COSCE-PeP (purified Camellia oil cake extract after enzymatic treatment). The units above the bars are ug / mL.

[0166] Specifically, the extract contains camelliasides A and B (both triglycosides) as the main components, and as a result of enzymatic treatment, it contains 4, 5, 6, 7 or more glycosides, including the triglycosides. Since it is not a separated and purified product, only triglycosides are present in the extract and its purified product, while the enzymatically treated product and its purified product contain a mixture of substances with 4-fold or more glycosides, including 3-fold glycosides.

[0167] Referring to FIG. 5, it is confirmed that the enzyme-treated product derived from the camellia oil cake extract of the present invention exhibits an excellent inhibitory effect on PDK activity.

[0168] Referring to Figure 6, it is confirmed that EM-CamA containing compounds of general formulas 1 to 3 and EM-CamB containing compounds of general formulas 4 to 6 have superior PDK inhibitory effects compared to the comparative compounds Compound A (CamA in Figure 6) and Compound B (CamB in Figure 6).

[0169] Referring to FIG. 7, it is confirmed that the compounds of general formulas 1 to 6 of the present invention (AG4 to AG6 and BG4 to BG6) have a more excellent PDK activity inhibitory effect than compounds A-1 (FIG. 7, AG7), B-1 (FIG. 7, BG7), and compounds N-1 to N-4 (FIG. 7, NG3 to NG6).

[0170] Specifically, it was confirmed in Fig. 7 that the compounds of the present invention represented by the general formulas 1 to 6 inhibited PDK1 activity more significantly than DMSO. The inhibition of PDK1 activity by the control compounds NG4 to NG7 was not as great as that of the compound (1b) represented by the general formulas 1 to 6.

[0171] Furthermore, among the kaempferol 4-7 glycoside compounds, the degree of inhibition of two kaempferol 7 glycosides (compounds A-1 AG7 and B-1 BG7) was similar to that of the control compounds, suggesting that there is a limit to the molecular size and the degree of activity inhibition.

[0172] Furthermore, the control compound kaempferol 4-6 glycoside (Figure 7, compounds NG4 to NG6) has a similar molecular size to the test compound kaempferol 4-6 glycoside, but there is a difference in activity inhibition, suggesting that the structural specificity of the molecule affects activity inhibition.

Claims

1. An enzyme-treated kaempferol glycoside compound represented by any one of the following general formulas 1 to 6, its stereoisomer, its pharma- ceutically acceptable salt, its hydrate or its solvate. 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】

2. The enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharmaceutical acceptable salt, its hydrate or its solvate as described in claim 1, wherein the enzyme-treated kaempferol glycoside compound is an enzyme-treated kaempferol triglycoside.

3. The enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharma- ceutical acceptable salt, its hydrate or its solvate as described in claim 2, wherein the kaempferol triglycoside is an extract derived from camellia.

4. The enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharma- ceutically acceptable salt, its hydrate or its solvate as described in claim 2, wherein the enzyme is cyclodextrin glucanotransferase.

5. A skin protection composition comprising an enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharma- ceutical acceptable salt, its hydrate or its solvate according to any one of claims 1 to 4 as an active ingredient.

6. The composition for protecting skin according to claim 5, wherein the daily application amount of the active ingredient is 1 to 1000 mg / kg.

7. The composition for protecting skin according to claim 5, wherein the composition is for improving wrinkles on the skin.

8. The composition for protecting skin according to claim 5, wherein the composition is for promoting skin regeneration.

9. The composition for protecting skin according to claim 5, wherein the composition is for improving atopic skin.

10. The composition for protecting the skin according to claim 5, wherein the composition is for regulating the skin keratinogenesis cycle.

11. 6. The composition for protecting skin according to claim 5, wherein the composition inhibits the activity of 3-phosphoinositide-dependent kinase 1 (PDK1).

12. The composition for protecting skin according to claim 5, wherein the composition is for improving sagging of the skin.

13. The composition for protecting skin according to claim 5 , wherein the composition is for increasing the expression of at least one of Col7A1 and Col17A1.

14. The composition for protecting skin according to claim 5 , wherein the composition is for improving skin barrier function.

15. 6. The skin protective composition of claim 5, wherein the composition is for improving skin moisturization.

16. The skin protective composition according to claim 5, wherein the composition is for increasing the expression of at least one of keratin 10 and filaggrin.

17. The composition for protecting skin according to claim 5 , wherein the composition is a cosmetic composition.

18. The skin protective composition according to claim 5, wherein the composition is a food composition.

19. A method for producing the enzyme-treated kaempferol glycoside compound according to any one of claims 1 to 4, comprising the steps of: obtaining kaempferol glycoside compound from camellia oil cake; purifying the kaempferol glycoside compound; A step of enzymatically treating the purified kaempferol glycoside compound; A method for producing an enzyme-treated kaempferol glycoside compound, comprising a step of purifying the enzyme-treated enzyme-treated kaempferol glycoside compound.

20. The method for producing an enzyme-treated kaempferol glycoside compound according to claim 19, wherein the purified kaempferol glycoside compound is a kaempferol triglycoside compound.

21. A method for producing an enzyme-treated kaempferol glycoside compound according to claim 19, wherein the enzyme is cyclodextrin glucanotransferase.

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