Camellia-derived flavonoids

Abstract

The present application provides: a novel kaempferol compound obtained by enzyme-treating a kaempferol glycoside extracted from a Camellia seed meal which is discarded after extraction of oil from Camellia seeds; and a composition for protecting the skin, which contains the novel kaempferol compound and is safe to the human body. The enzyme-treated kaempferol compound has an increased molecular weight by using a kaempferol glycoside as a precursor and further binding a sugar, and thus can exhibit a skin-protecting effect.

Description

Technical Field

[0001] The present application relates to a novel flavonoid derived from camellia, and specifically discloses a kaempferol glycoside compound which is safe for human body and has excellent skin protection effect, and a skin protection composition containing the same. Background Art

[0002] Flavonoids derived from plants have a C6-C3-C6 carbon skeleton structure consisting of two phenyl groups connected by a C3 chain, and usually exist in the form of glycosides bound to various sugars. Different individuals produce specific glycoside structures through the inherent biosynthetic pathway of plants.

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

[0004] In addition, many existing plant-derived skin protection ingredients have the problem that a large amount of plant resources are required or that their efficacy is low due to the specificity of molecular weight and molecular structure, and their utilization value is low.

[0005] [Prior art literature]

[0006] [Patent Literature]

[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 application aims to provide an enzyme-treated kaempferol glycoside compound obtained from camellia oleifera seed cake, and a skin protection composition containing the same. Camellia oleifera seed cake is a resource discarded after camellia seed oil is extracted.

[0010] In another aspect, the present application aims to provide an enzyme-treated kaempferol glycoside compound whose molecular size is adjusted by enzymatically treating a camellia oil cake extract containing a large amount of kaempferol glycoside compounds, and a skin protection composition containing the same.

[0011] In another aspect, the present application aims to provide a composition comprising an enzyme-treated kaempferol glycoside compound as an effective ingredient, which is safe to the human body due to its small side effects and has excellent skin protection effect.

[0012] In another aspect, the present application provides a skin protection composition comprising an enzyme-treated kaempferol glycoside compound as an active ingredient to inhibit the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1, 3-Phosphoinositide-dependent kinase 1).

[0013] Technical Solution

[0014] The present application provides an enzyme-treated-kaempferol glycoside compound represented by any one of the following Chemical Formulas 1 to 6, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

[0015]

Chemical formula 1

[0016]

[0017]

Chemical formula 2

[0018]

[0019]

Chemical formula 3

[0020]

[0021]

Chemical formula 4

[0022]

[0023]

Chemical formula 5

[0024]

[0025]

Chemical formula 6

[0026]

[0027] The present application provides a skin protection composition, which comprises the above-mentioned enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate as an active ingredient.

[0028] Beneficial Effects

[0029] In one aspect, the enzyme-treated kaempferol glycoside compound provided herein can inhibit the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0030] In another aspect, the enzyme-treated kaempferol glycoside compound provided herein can exhibit a skin protection effect by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The 365 nm ultraviolet absorption chromatogram of the camellia oil cake extract and its enzyme conversion product (enzyme-treated-kaempferol compound) according to one embodiment of the present application is shown.

[0032] Figure 2 The 365nm ultraviolet absorption chromatogram of a glycoside mixture obtained by enzymatic treatment of camellia glycoside A and its separated and purified product according to one embodiment of the present application is shown.

[0033] Figure 3 The 365 nm ultraviolet absorption chromatogram of a glycoside mixture obtained by enzymatically treating camelliaside B according to one embodiment of the present application and its separated and purified product is shown.

[0034] Figure 4 Shown is a 365nm ultraviolet absorption chromatogram of a glycoside mixture obtained by enzymatically treating nicotinic acid according to an embodiment of the present application and its separated and purified product.

[0035] Figure 5 A graph showing the inhibitory effects of DMSO (blank), BX-795 (control), COSCE (camellia oil meal extract), COSCE-P (purified camellia oil meal extract), and COSCE-PeP (purified camellia oil meal extract after enzyme treatment) on 3-phosphoinositide-dependent protein kinase 1 (PDK1) activity according to one embodiment of the present application. The unit of the numbers above the bars is ug / mL.

[0036] Figure 6 A chart showing the inhibitory effects of DMSO (blank), BX-795 (control), CamA (Camellia glycoside A), CamB (Camellia glycoside B), and EM (enzymatically modified) on the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1) according to one embodiment of the present application. The unit of the numbers above the bars is ug / mL. EM-CamA contains compounds of chemical formulas 1 to 3, and EM-CamB contains compounds of chemical formulas 4 to 6.

[0037] Figure 7A graph showing the inhibitory effects of DMSO (blank), BX-795 (control), AG4 (camelliaside A + 1 sugar), AG5 (camelliaside A + 2 sugars), AG6 (camelliaside A + 3 sugars), AG7 (camelliaside A + 4 sugars), BG4 (camelliaside B + 1 sugar), BG5 (camelliaside B + 2 sugars), BG6 (camelliaside B + 3 sugars), BG7 (camelliaside B + 4 sugars), NG4 (nicotinosidin + 2 sugars), NG5 (nicotinosidin + 3 sugars), NG6 (nicotinosidin + 4 sugars), and NG7 (nicotinosidin + 5 sugars) on the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1) according to one embodiment of the present application. DETAILED DESCRIPTION

[0038] In the present application, when a part “includes” a certain component, unless otherwise specifically stated, it means that the part may also include other components, rather than excluding other components.

[0039] Hereinafter, the present invention will be described in detail.

[0040] In an exemplary embodiment, the present application provides an enzyme-treated-kaempferol glycoside compound represented by any one of the following Chemical Formulas 1 to 6, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

[0041]

Chemical formula 1

[0042]

[0043]

Chemical formula 2

[0044]

[0045]

Chemical formula 3

[0046]

[0047]

Chemical formula 4

[0048]

[0049]

Chemical formula 5

[0050]

[0051]

Chemical formula 6

[0052]

[0053] In other exemplary embodiments, the present application provides a method for protecting the skin, the method comprising applying an effective dose of a composition to a subject in need thereof, the composition comprising as an effective ingredient a kaempferol glycoside compound represented by any one of the above Chemical Formulas 1 to 6, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

[0054] In other exemplary embodiments, the present application provides use of a kaempferol glycoside compound represented by any one of the above Chemical Formulas 1 to 6, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof in preparing a skin protection composition.

[0055] In other exemplary embodiments, the present application provides a non-therapeutic use of a kaempferol glycoside compound represented by any one of the above Chemical Formulas 1 to 6, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof in protecting the skin.

[0056] In the present application, the term "isomer" includes not only optical isomers, for example, essentially pure enantiomers, essentially pure diastereomers or mixtures thereof, but also conformational isomers (i.e., isomers that differ only in the angle of one or more chemical bonds), positional isomers (especially tautomers) or geometrical isomers (e.g., cis-trans isomers).

[0057] In the present application, "essentially pure" means, for example, when the relevant enantiomers or diastereomers are used, the specific compound exemplified by the enantiomers or diastereomers accounts for 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 further preferably about 99.5% (w / w) or more.

[0058] In the present application, "pharmaceutically acceptable" means that when used in conventional pharmaceutical dosages, it avoids significant toxic effects and can be used in animals (more specifically humans), and this is approved by or obtained from a government or equivalent regulatory agency, or is recorded in the pharmacopoeia or known through other general pharmacopoeias.

[0059] In the present application, "medically acceptable salt" refers to a salt according to one aspect of the present application that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. The salt may include: (1) an acid addition salt formed by an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or an acid addition salt formed by an inorganic acid 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-ethanesulfonic acid, (2) an acid addition salt formed by an organic acid such as 1,2-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-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; or, (2) a salt formed by replacing an acidic proton present in the parent compound.

[0060] In the application, the term "hydrate" refers to a compound combined with water, and is used in a broad sense to include inclusion compounds in which there is no chemical bond between water and the compound.

[0061] In the present application, the term "solvate" refers to a higher order compound formed between molecules or ions of a solute and molecules or ions of a solvent.

[0062] As a precursor of the compound represented by any one of Chemical Formulas 1 to 3 according to an embodiment of the present application, camelliaside A can be represented by the following Chemical Formula A, and the nuclear magnetic resonance analysis results of the kaempferol glycoside compound represented by Chemical Formula A are shown in Table 1 below.

[0063]

Chemical formula A

[0064]

[0065]

Table 1

[0066] D2O Camellia glycoside A 1 H- and 13 C-NMR data

[0067]

[0068] The results of nuclear magnetic resonance analysis of the enzyme-treated kaempferol glycoside compound represented by Chemical Formula 1 are shown in Table 2.

[0069]

Table 2

[0070] D2O Camellia glycoside A-G4 1 H- and 13 C-NMR data

[0071]

[0072] The nuclear magnetic resonance analysis results of the kaempferol glycoside compound represented by the chemical formula 2 are shown in Table 3.

[0073]

Table 3

[0074] D2O Camellia glycoside A-G5 1 H- and 13 C-NMR data

[0075]

[0076] The nuclear magnetic resonance analysis results of the kaempferol glycoside compound represented by the chemical formula 3 are shown in Table 4.

[0077]

Table 4

[0078] D2O Camellia glycoside A-G6 1 H- and 13 C-NMR data

[0079]

[0080]

[0081] The kaempferol heptaglycoside compound obtained by using the above-mentioned camelliaside A as a precursor and combining four sugars can be represented by the following chemical formula A-1. The nuclear magnetic resonance analysis results of the compound represented by chemical formula A-1 are shown in Table 5.

[0082]

Chemical formula A-1

[0083]

[0084]

Table 5

[0085] D2O Camellia glycoside A-G7 1 H- and 13 C-NMR data

[0086]

[0087]

[0088] As a precursor of the compound represented by any one of Chemical Formulas 4 to 6 according to one embodiment of the present application, camelliaside B can be represented by the following Chemical Formula B, and the nuclear magnetic resonance analysis results of the kaempferol glycoside compound represented by Chemical Formula B are shown in Table 6.

[0089]

Chemical formula B

[0090]

[0091]

Table 6

[0092] D2O Camellia glycoside B 1 H- and 13 C-NMR data

[0093]

[0094] The nuclear magnetic resonance analysis results of the kaempferol glycoside compound represented by the chemical formula 4 are shown in Table 7.

[0095]

Table 7

[0096] D2O Camellia glycoside B-G4 1 H- and 13 C-NMR data

[0097]

[0098]

[0099] The nuclear magnetic resonance analysis results of the kaempferol glycoside compound represented by the chemical formula 5 are shown in Table 8.

[0100]

Table 8

[0101] D2O Camellia glycoside B-G5 1 H- and 13 C-NMR data

[0102]

[0103]

[0104] The nuclear magnetic resonance analysis results of the kaempferol glycoside compound represented by the chemical formula 6 are shown in Table 9.

[0105]

Table 9

[0106] D2O Camellia glycoside B-G6 1 H- and 13 C-NMR data

[0107]

[0108]

[0109] The kaempferol heptaglycoside compound obtained by using the above-mentioned camelliaside B as a precursor and combining four sugars can be represented by the following chemical formula B-1. The nuclear magnetic resonance analysis results of the kaempferol glycoside compound represented by chemical formula B-1 are shown in Table 10.

[0110]

Chemical formula B-1

[0111]

[0112]

Table 10

[0113] D2O Camellia glycoside B-G7 1 H- and 13 C-NMR data

[0114]

[0115]

[0116] In one embodiment of the present application, nicotinic acid glycoside (Nicotiflorin) can be used as a precursor. Nicotiflorin can be represented by the following chemical formula N. The nuclear magnetic resonance analysis results of the kaempferol glycoside compound represented by chemical formula N are shown in Table 11.

[0117]

Chemical formula N

[0118]

[0119]

Table 11

[0120] Nicotinic acid in DMSO-d6 1 H- and 13 C-NMR data

[0121]

[0122] The kaempferol glycoside compound represented by the following chemical formula N-1 was prepared by using the compound represented by the above chemical formula N as a precursor. The nuclear magnetic resonance analysis results are shown in Table 12.

[0123]

Chemical formula N-1

[0124]

[0125]

Table 12

[0126] Nicotinamide-G4 in D2O 1 H- and 13 C-NMR data

[0127]

[0128]

[0129] The kaempferol glycoside compound represented by the following chemical formula N-2 was prepared by using the compound represented by the above chemical formula N as a precursor. The nuclear magnetic resonance analysis results are shown in Table 13.

[0130]

Chemical formula N-2

[0131]

[0132]

Table 13

[0133] Nicotinamide-G5 in D2O 1 H- and 13 C-NMR data

[0134]

[0135]

[0136] The kaempferol glycoside compound represented by the following chemical formula N-3 was prepared by using the compound represented by the above chemical formula N as a precursor. The nuclear magnetic resonance analysis results are shown in Table 14.

[0137]

Chemical formula N-3

[0138]

[0139]

Table 14

[0140] Nicotinamide-G6 in D2O 1 H- and 13 C-NMR data

[0141]

[0142]

[0143] The kaempferol glycoside compound represented by the following chemical formula N-4 was prepared by using the compound represented by the above chemical formula N as a precursor. The nuclear magnetic resonance analysis results are shown in Table 15.

[0144]

Chemical formula N-4

[0145]

[0146]

Table 15

[0147] Nicotinamide-G7 in D2O 1 H- and 13C-NMR data

[0148]

[0149]

[0150] The composition according to one aspect of the present application can achieve the following reaction conditions, using kaempferol trisaccharides extracted from camellia oleifera seed cake discarded after oil extraction from camellia seeds, that is, a compound represented by chemical formula A or chemical formula B as a precursor, and combining it with glucose to generate kaempferol tetra-hexaglycosides.

[0151] Compared with the composition of kaempferol triglycoside compounds extracted from camellia oil cake, it can show significantly excellent skin protection effect.

[0152] The camellia oil meal extract contains terpene glycosides, flavonoid glycosides, sugars and proteins. In particular, the flavonoid glycosides of camellia oil meal are composed of triglycosides (chemical formula A, chemical formula B), and according to the present application, 8 tetra- to hexaglycosides (chemical formulas 1 to 6) formed by using kaempferol triglycoside derived from camellia oil meal as a precursor and sequentially combining glucose have excellent skin protection effects.

[0153] In one embodiment, the enzyme-treated kaempferol glycoside compound can be obtained by enzymatically treating kaempferol triglycoside.

[0154] In one embodiment, the kaempferol triglycoside may be an extract derived from Camellia sinensis.

[0155] Specifically, the extract and kaempferol triglycoside compound can be obtained by producing oil from camellia seeds by pressing to obtain camellia oil cake, and then extracting kaempferol triglycoside compound from the camellia oil cake using 70% ethanol.

[0156] In one embodiment, the enzyme may be cyclodextrin lucanotransferase.

[0157] In an exemplary embodiment, the present application provides a skin protection composition comprising, as an effective ingredient, an enzyme-treated-kaempferol glycoside compound, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

[0158] 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, and 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.

[0159] If the daily application amount of the active ingredient is less than 1 mg / Kg, the skin protection effect may be minimal, and if it exceeds 1000 mg / Kg, cytotoxicity may be exhibited.

[0160] In one embodiment, the composition can inhibit the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0161] Referring to Korean Patent Publication No. 10-2022-0036148, when the activity of PDK1 is inhibited, cells whose growth is inhibited due to aging can grow again, and damaged cells can also be restored. Specifically, by inhibiting PDK1 in aging human dermal fibroblasts, aging characteristics can be eliminated and NF-κB and mTOR signaling can be inhibited, thereby restoring the cell generation cycle, thereby restoring or promoting skin regeneration ability. In addition, by inhibiting PDK1, replicative aging characterized by increased MMP1, decreased collagen fibers, and decreased procollagen secretion, as well as dermal aging associated with chemically induced aging, can be reversed. For epidermal homeostasis, the balance between keratinocyte proliferation and differentiation is regulated by dermal fibroblasts. Aged dermal fibroblasts are reversed by inhibiting PDK1, thereby affecting the proliferation and differentiation of keratinocytes. The inhibition of PDK1 restores the reduced abundance of such keratinocyte differentiation markers and components connecting the dermis and epidermis that appear in the skin equivalents of aged NHDF.

[0162] Referring to Korean Patent Publication No. 10-2021-0037643, PDK1 is a protein that simultaneously regulates mTOR and NF-кB, which are the main signaling proteins involved in cell aging. It is a key protein that determines cell growth, division, and aging-related secretory phenotypes. When PDK1 is temporarily inhibited, some senescent cells are effectively reversed to 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 Rapamy cin) and NF-кB (Nuclear Factor Kappa B). Reversal of aging can be induced by inhibiting PDK.

[0163] In one embodiment, the composition can be used to improve skin wrinkles.

[0164] In one embodiment, the composition can be used to promote skin regeneration.

[0165] In one embodiment, the composition can be used to improve atopic skin.

[0166] In one embodiment, the composition can be used to regulate the skin keratinogenesis cycle.

[0167] According to one aspect of the present application, the composition can promote the colony formation of epidermal cells and promote differentiation, thereby shortening the skin keratin production cycle or the epidermal cell renewal cycle.

[0168] In another embodiment, the composition can be used to regulate skin keratin, or to promote skin keratin renewal, or to normalize the skin keratin renewal cycle.

[0169] In another embodiment, the composition can be used to improve skin wrinkles by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0170] In another embodiment, the composition can be used to promote skin regeneration by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0171] In another embodiment, the composition can be used to improve atopic skin, or improve, prevent or treat atopic dermatitis by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0172] In another embodiment, the composition can be used to regulate the skin keratinogenesis cycle by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0173] In another embodiment, the composition can be used to regulate skin keratinization by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0174] In another embodiment, the composition can be used to promote skin keratin renewal by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0175] In another embodiment, the composition can be used to normalize the skin keratin renewal cycle by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0176] In one embodiment, the composition can be used to improve skin sagging.

[0177] In one embodiment, the composition can be used to increase the expression of Col7A1.

[0178] In one embodiment, the composition can be used to increase the expression of Col17A1.

[0179] The Col7A1 and Col17A1 are proteins that constitute DEJ (dermoepidermal junction). The functions of DEJ include firmly fixing epidermal cells and strengthening the bonding between the epidermis and the dermis. The reduction of Col7A1 and Col17A1 leads to the impairment of the above functions of DEJ, thereby causing skin sagging. Therefore, restoring the reduced expression of Col7A1 and Col17A1 can prevent and improve the deformation of DEJ and rete ridges structures, thereby preventing and improving skin sagging.

[0180] Referring to Korean Patent Publication No. 10-2022-0036148, it was investigated whether reversing senescence of dermal fibroblasts by inhibiting PDK1 affects the proliferation and differentiation of keratinocytes. The results confirmed that inhibition of PDK1 restored the reduction of dermal-epidermal junction components such as COL17A1.

[0181] In another embodiment, the composition can be used to improve skin sagging by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0182] In another embodiment, the composition can be used to increase the expression of Col7A1 by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0183] In another embodiment, the composition can be used to increase the expression of Col17A1 by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0184] In one embodiment, the composition can be used to improve skin barrier function.

[0185] In one embodiment, the composition can be used to improve skin moisturization.

[0186] In one embodiment, the composition can be used to increase keratin 10 expression.

[0187] In one embodiment, the composition can be used to increase the expression of filaggrin.

[0188] Keratin 10 and filaggrin are genes that normalize epidermal keratinization by regulating keratin formation in skin cells, and are known to be involved in skin barrier function, skin moisturizing, skin wrinkles, aging, etc. Therefore, by increasing the expression of keratin 10 and filaggrin, it is possible to promote moisture retention in skin tissue and strengthen the skin barrier that prevents moisture loss in skin tissue.

[0189] Referring to Korean Patent Publication No. 10-2022-0036148, it was investigated whether reversing senescent dermal fibroblasts by inhibiting PDK1 affects the proliferation and differentiation of keratinocytes. The results confirmed that inhibiting PDK1 restored the reduced expression of keratinocyte differentiation markers such as keratin 10 and filaggrin.

[0190] In another embodiment, the composition can be used to improve skin barrier function by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0191] In another embodiment, the composition can be used to improve skin moisturization by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0192] In another embodiment, the composition can be used to increase the expression of keratin 10 by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0193] In another embodiment, the composition can be used to increase the expression of filaggrin by inhibiting the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0194] In one embodiment, the composition may be a cosmetic composition.

[0195] The profile of the cosmetic composition according to one embodiment of the present invention can include cosmetics or dermatologically acceptable medium or matrix.It can be prepared into all dosage forms suitable for topical application, for example, can be prepared into solution, gel, solid, pasty anhydrous product, by the emulsion obtained by dispersing the oil phase in the aqueous phase, suspension, microemulsion, microcapsule, microgranule ball or ionic (liposome) and nonionic vesicle dispersant form, or frost, toner, emulsion, powder, ointment, spray or concealer stick form.These compositions can be prepared by the conventional methods of this area.Composition according to the present invention can also be used in the form of foam (foam) or in the form of aerosol composition further comprising a compressed propellant.

[0196] The dosage form of the cosmetic composition according to one embodiment of the present invention is not particularly limited. For example, the cosmetic composition can be prepared as a skin softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, facial mask, powder, body lotion, body cream, body oil and body essence.

[0197] When the dosage form according to one embodiment of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, polysiloxane, bentonite, silicon dioxide, talc or zinc oxide, etc. can be used as the carrier component.

[0198] When the dosage form according to one embodiment of the present invention is a powder or a spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component. In particular, when the dosage form is a spray, it can further contain a propellant such as chlorofluorocarbons, propane / butane or dimethyl ether.

[0199] When the dosage form according to one embodiment of the present invention is a solution or an emulsion, a solvent, a solvating agent or an emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol fatty acid ester, polyethylene glycol or fatty acid ester of sorbitan.

[0200] When the dosage form according to one embodiment of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth gum, etc. can be used as a carrier component.

[0201] When the dosage form according to one embodiment of the present invention is a facial cleanser containing a surfactant, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazoline derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glyceryl fatty acid esters can be used as carrier components.

[0202] In the cosmetic composition according to one embodiment of the present invention, in addition to the active ingredients, functional additives and ingredients contained in a common cosmetic composition may also be included. The functional additives may include ingredients selected from water-soluble vitamins, oil-soluble vitamins, high molecular peptides, high molecular polysaccharides, sphingolipids and seaweed extracts.

[0203] In addition, in the cosmetic composition according to one embodiment of the present invention, in addition to the functional additives, the ingredients contained in the general cosmetic composition can also be mixed as needed. In addition, the mixed ingredients can be oil components, moisturizers, emollients, surfactants, organic pigments and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH regulators, alcohols, pigments, fragrances, blood circulation promoters, coolants, antiperspirants, purified water, etc.

[0204] In addition, the composition according to one embodiment of the present invention may be a skin external preparation, which is a general term for any preparation form applied to the outside of the skin, including cosmetics and pharmaceuticals of various preparation forms.

[0205] In one embodiment, the composition may be a non-therapeutic transdermal composition.

[0206] In one embodiment, the composition may be a food composition for improving the moisture status of the skin.

[0207] In one embodiment, the composition may be an oral composition.

[0208] In one embodiment, the composition may be a non-therapeutic oral composition.

[0209] The food composition or oral composition according to one embodiment of the present invention may be in liquid or solid dosage form, and may be in the form of tablets, capsules, soft capsules, pills, granules, beverages (drinks), slimming bars, chocolate, caramel dosage forms or biscuit dosage forms, and the dosage form is not particularly limited.

[0210] The food composition or oral composition of the present invention may contain appropriate amounts of excipients, sugars, flavors, pigments, fats and oils, proteins, etc., in addition to the active ingredients.

[0211] In an exemplary embodiment, the present application provides a method for preparing an enzyme-treated kaempferol glycoside compound, which comprises the following steps: obtaining a kaempferol glycoside compound from camellia oil cake; purifying the kaempferol glycoside compound; enzymatically treating the purified kaempferol glycoside compound; and purifying the enzyme-treated kaempferol glycoside compound.

[0212] In one embodiment, the purified kaempferol glycoside compound may be a kaempferol triglycoside compound.

[0213] In one embodiment, the enzyme may be cyclodextrin lucanotransferase.

[0214] Hereinafter, the present invention will be described in more detail by the following examples. However, the purpose of providing the following examples is only to help understand the present invention, and the category and scope of the present invention are not limited thereto.

[0215] Example

[0216] The camellia oil cake used in the experiment was obtained by producing oil from camellia seeds (produced in China) by pressing. An extract was prepared from the camellia oil cake using 70% ethanol, and it was confirmed that the camellia oil cake extract is composed of terpene glycosides, flavonoid glycosides, sugars and proteins. In particular, the flavonoid glycosides of the camellia oil cake are composed of trisaccharides of kaempferol (chemical formula A, chemical formula B). According to the present application, two types of kaempferol trisaccharides of the camellia oil cake are used as precursors and eight types of kaempferol tetra-hexasaccharides (chemical formulas 1 to 6) are generated by enzyme reaction, which have excellent inhibitory effects on skin PDK1 activity.

[0217] 1) Purification of kaempferol glycoside fraction from camellia oil cake

[0218] 8 kg of 70% ethanol was added to 1 kg of camellia oil cake deoiled by low temperature oil pressing, and extracted at 60°C for 8 hours using a heating extractor. The extract obtained by filtering through a filter press was concentrated under reduced pressure at 50°C to 60°C using a rotary vacuum concentrator until the solid content was about 50%, thereby obtaining 200 g of a concentrate. The concentrate was diluted with purified water to a solid content of 10%.

[0219] The solution was loaded into a glass column containing 2 L of ion exchange resin (HP-20), and then washed with 10 L of purified water to remove impurities and sugar components. Then, 10 L of 30% ethanol was passed through, and the eluate was concentrated under reduced pressure and freeze-dried to obtain about 20 g of camellia kaempferol glycoside fraction powder.

[0220] 2) Purification of kaempferol trisaccharide from camellia oil cake

[0221] Preparative HPLC was used to separate the kaempferol triglycoside fraction from camellia oil cake. The solvent conditions were a mixed solvent of water, acetonitrile, and methanol, and a concentration gradient or a mixture of these solvents in a certain ratio was used. As a result, the kaempferol triglycoside compound components of chemical formulas A and B were separated ( Figure 1 ).

[0222] 3) Production and purification of sugar-bound kaempferol glycosides derived from kaempferol triglycosides

[0223] The camellia oil cake extract obtained in 2) above was dissolved to a concentration of 10 mg / mL, the water-soluble starch was dissolved to a concentration of 100 mg / mL, and the cyclodextrin glucosyltransferase was dissolved to a concentration of 100 unit / mL using citrate-phosphate buffer (pH 5, 50 mM), and the enzyme reaction was started while stirring at a temperature of 50 to 70°C. A 30% ethanol aqueous solution was added to terminate the enzyme reaction within 1 to 24 hours, and the supernatant was collected by centrifugation (4000g, 10 minutes), and filtered and concentrated using a 0.45 micron filter. The concentrated filtrate was adsorbed using a preparative chromatography and a reverse phase column (C18), and then the unnecessary substances were washed away with pure water, and then the wide range absorption peak at UV280nm was separated using an acetonitrile concentration gradient. The aliquot was freeze-dried after concentration to prepare a powder. ( Figure 1 ) For comparison, pure substances camelliaside A (Compound A), camelliaside B (Compound B) and nicotinic acid glycoside (Compound N) were used as starting materials, and the enzyme reaction was carried out at 2 mg / mL and the samples were fractionated in the same manner.

[0224] 4) Purification of kaempferol tetra-heptaglycosides

[0225] The pure substances camellia glycoside A and camellia glycoside B were subjected to the same enzyme reaction as in 3) at a concentration of 2 mg / mL to obtain a composition with increased molecular weight, which was separated peak by peak using preparative chromatography and an adsorption column (C18), thereby obtaining four glycosides (compounds 1 to 3 and A-1) derived from camellia glycoside A (compound A) and four glycosides (compounds 4 to 6 and B-1) derived from camellia glycoside B (compound B). Figure 2 , Figure 3 )

[0226] 5) Structural identification of purified and isolated substances

[0227] The structures of Compound A, Compound B, Compound N used as a precursor; and Compounds 1 to 3 and A-1, Compounds 4 to 6 and B-1 obtained in the above 4) were identified using a nuclear magnetic resonance analyzer.

[0228] Comparative Example

[0229] In order to confirm the significance of kaempferol triglycosides derived from camellia oil cake as precursors, kaempferol diglycosides (nicotin; kaempferol rutin) (compound N) were used as precursors, and four glycosides (chemical substances N-1 to N-4) with increasing molecular weight were generated and purified in the same manner as in the above 3) and 4) to be used in the same experimental example ( Figure 4 ). In the same manner as above, the structures of the compound N as a precursor and the compounds N-1 to N-4 as reactants were identified using a nuclear magnetic resonance analyzer.

[0230] Experimental example

[0231] statistics

[0232] In the analysis of all the following experiments, the statistical significance between samples was tested using the student t-test, and p values ​​below 0.05 were considered statistically significant. (*p<0.05, **p<0.01, ***p<0.001)

[0233] Experimental Example 1: Analysis of PDK1

[0234] The activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1) was measured by using recombinant purified PDK1 enzyme and ADP-Glo ​​+ PDK1 kinase enzyme system (PDK1 Kinase Enzyme System; Promega, Madison, Wisconsin, USA). After the separation of substrate, ATP, and buffer mixture, the experimental substances (1μM, 3μM, 10μM) were separated into each well. Then, PDK1 was separated immediately and reacted for 60 minutes. After the reaction, ADP-glo was added and incubated at room temperature (incubation) for 40 minutes, and then the kinase detection buffer was added and reacted for 30 minutes, and then measured with a luminescence reader (Luminescence reader; Wallac Victor2, Perkin Elmer).

[0235] Figure 5 The graph shows the inhibitory effects of DMSO (blank), BX-795 (control), COSCE (camellia oil meal extract), COSCE-P (purified camellia oil meal extract), and COSCE-PeP (purified camellia oil meal extract after enzyme treatment) on 3-phosphoinositide-dependent protein kinase 1 (PDK1) activity. The unit of the numbers above the bars is ug / mL.

[0236] Specifically, the extract contains camellia glycosides A and B (both are triglycosides) as main components, and after enzyme treatment, contains tetra-, penta-, hexa-, hepta- and higher glycosides including the triglycoside. Since no separation and purification is performed, only triglycosides exist in the extract and its purified product, and tetra- and higher glycoside substances including triglycosides are mixed in the enzyme-treated product and its purified product.

[0237] Reference Figure 5 It is clear that the enzyme-treated product derived from the camellia oil cake extract of the present application has a more excellent inhibitory effect on PDK activity.

[0238] Reference Figure 6 It can be seen that compared with the comparative example compound A ( Figure 6 CamA) and compound B ( Figure 6 EM-CamA containing the compounds represented by Chemical Formulas 1 to 3 and EM-CamB containing the compounds represented by Chemical Formulas 4 to 6 have better inhibitory effects on PDK activity.

[0239] Reference Figure 7 It can be seen that compared with compound A-1 ( Figure 7 AG7), compound B-1( Figure 7 BG7) and compounds N-1 to N-4 ( Figure 7 NG4 to NG7), the compounds represented by Chemical Formulas 1 to 6 of the present application (AG4 to AG6 and BG4 to BG6) have a more excellent inhibitory effect on PDK activity.

[0240] Specifically, if Figure 7 As shown, compared with DMSO, the compounds represented by Chemical Formulas 1 to 6 of the present application can significantly inhibit PDK1 activity. The inhibition degree of NG4 to NG7 as the control group on PDK1 activity failed to reach the inhibition degree of the compounds represented by Chemical Formulas 1 to 6.

[0241] In addition, among the tetra- to hepta-glycoside compounds of kaempferol, the inhibition levels of two kaempferol hepta-glycosides (compound A-1, i.e., AG7, and compound B-1, i.e., BG7) were similar to those of the control group compounds, indicating that they were limited in terms of molecular size and activity inhibition.

[0242] In addition, the kaempferol tetra-hexaglycosides ( Figure 7 Although the molecular sizes of the compounds NG4 to NG6 are similar to those of the experimental group compounds, the inhibitory activities are different, indicating that the structural specificity of the molecules has an impact on the inhibitory activity.

[0243] Implementation

[0244] Embodiment 1: An enzyme-treated kaempferol glycoside compound represented by any one of the following chemical formulas 1 to 6, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof,

[0245]

Chemical formula 1

[0246]

[0247]

Chemical formula 2

[0248]

[0249]

Chemical formula 3

[0250]

[0251]

Chemical formula 4

[0252]

[0253]

Chemical formula 5

[0254]

[0255]

Chemical formula 6

[0256]

[0257] Embodiment 2: According to the enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate as described in embodiment 1, wherein the enzyme-treated kaempferol glycoside compound is obtained by enzymatically treating kaempferol trisaccharide.

[0258] Embodiment 3: The enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate according to embodiment 1 or 2, wherein the kaempferol triglycoside is an extract derived from Camellia sinensis.

[0259] Embodiment 4: The enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate according to any one of Embodiments 1 to 3, wherein the enzyme is cyclodextrin glucosyltransferase.

[0260] Embodiment 5: Use of the enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate according to any one of Embodiments 1 to 4 in preparing a skin protection composition.

[0261] Embodiment 6: The use according to embodiment 5, wherein the daily administration amount of the active ingredient is 1 to 1000 mg / Kg.

[0262] Embodiment 7: The use according to embodiment 5 or 6, wherein the composition is used to improve skin wrinkles.

[0263] Embodiment 8: The use according to any one of embodiments 5 to 7, wherein the composition is used to promote skin regeneration.

[0264] Embodiment 9: The use according to any one of embodiments 5 to 8, wherein the composition is used to improve atopic skin.

[0265] Embodiment 10: The use according to any one of embodiments 5 to 9, wherein the composition is used to regulate the keratin production cycle of the skin.

[0266] Embodiment 11: The use according to any one of embodiments 5 to 10, wherein the composition inhibits the activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

[0267] Embodiment 12: The use according to any one of embodiments 5 to 11, wherein the composition is used to improve skin sagging.

[0268] Embodiment 13: The use according to any one of embodiments 5 to 12, wherein the composition is used to increase the expression of at least one of Col7A1 and Col17A1.

[0269] Embodiment 14: The use according to any one of embodiments 5 to 13, wherein the composition is used to improve skin barrier function.

[0270] Embodiment 15: The use according to any one of embodiments 5 to 14, wherein the composition is used to improve skin moisturization.

[0271] Embodiment 16: The use according to any one of embodiments 5 to 15, wherein the composition is used to increase the expression of at least one of keratin 10 and filaggrin.

[0272] Embodiment 17: The use according to any one of embodiments 5 to 16, wherein the composition is a cosmetic composition.

[0273] Embodiment 18: The use according to any one of Embodiments 5 to 17, wherein the composition is a food composition for improving the moisture status of the skin.

[0274] Embodiment 19: The method for preparing an enzyme-treated kaempferol glycoside compound according to any one of embodiments 1 to 4, comprising the following steps:

[0275] Obtaining kaempferol glycoside compounds from camellia oil cake;

[0276] Purifying the kaempferol glycoside compound;

[0277] enzymatically treating the purified kaempferol glycoside compound; and

[0278] The enzyme-treated kaempferol glycoside compound was purified.

[0279] Embodiment 20: The method for preparing an enzyme-treated kaempferol glycoside compound according to Embodiment 19, wherein the purified kaempferol glycoside compound is a kaempferol trisaccharide compound.

[0280] Embodiment 21: The method for preparing an enzyme-treated kaempferol glycoside compound according to embodiment 19 or 20, wherein the enzyme is cyclodextrin glucosyltransferase.

Claims

1. An enzyme-treated kaempferol glycoside compound represented by any one of the following chemical formulas 1 to 6, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, 【Chemical formula 1】 【Chemical formula 2】 【Chemical formula 3】 【Chemical formula 4】 【Chemical formula 5】 【Chemical formula 6】 2. The enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate according to claim 1, wherein: The enzyme-treated kaempferol glycoside compound is obtained by subjecting kaempferol triglycoside to an enzyme treatment.

3. The enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate according to claim 2, wherein: The kaempferol triglycoside is an extract derived from camellia sinensis.

4. The enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate according to claim 2, wherein: The enzyme is cyclodextrin glucosyltransferase.

5. Use of the enzyme-treated kaempferol glycoside compound, its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate according to any one of claims 1 to 4 in the preparation of a skin protection composition.

6. The use according to claim 5, wherein The daily administration amount of the enzyme-treated-kaempferol glycoside compound, its stereoisomer, its pharmaceutically acceptable salt, its hydrate or its solvate is 1 to 1000 mg / Kg.

7. The use according to claim 5, wherein The composition is used for improving skin wrinkles.

8. The use according to claim 5, wherein The composition is used to promote skin regeneration.

9. The use according to claim 5, wherein The composition is used for improving atopic skin.

10. The use according to claim 5, wherein: The composition is used for regulating the keratin production cycle of the skin.

11. The use according to claim 5, wherein: The composition inhibits the activity of 3-phosphoinositide-dependent protein kinase 1.

12. The use according to claim 5, wherein: The composition is used for improving skin sagging.

13. The use according to claim 5, wherein: The composition is used to increase the expression of at least one of Col7A1 and Col17A1.

14. The use according to claim 5, wherein The composition is used to improve the skin barrier function.

15. The use according to claim 5, wherein: The composition is used to improve skin moisturization.

16. The use according to claim 5, wherein: The composition is used to increase the expression of at least one of keratin 10 and filaggrin.

17. The use according to claim 5, wherein: The composition is a cosmetic composition.

18. The use according to claim 5, wherein The composition is a food composition for improving the moisture state of skin.

19. The method for preparing an enzyme-treated kaempferol glycoside compound according to any one of claims 1 to 4, comprising the following steps: Obtaining kaempferol glycoside compounds from camellia oil cake; Purifying the kaempferol glycoside compound; enzymatically treating the purified kaempferol glycoside compound; and The enzyme-treated kaempferol glycoside compound was purified.

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

21. The method for preparing an enzyme-treated kaempferol glycoside compound according to claim 19, wherein: The enzyme is cyclodextrin glucosyltransferase.

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