Composition for treating, improving and / or preventing skin aging

Abstract

To provide a new composition capable of effectively improving the state of the skin caused by aging and treating, improving and / or preventing various symptoms accompanied by skin aging.SOLUTION: The present inventors have found that skin aging is remarkably improved by administering a coicis semen extract of adlay to an aged mouse. The present invention provides a composition for treating, improving and / or preventing skin aging, which comprises adlay as an active ingredient. The skin aging is at least one selected from the group consisting of progression of skin dryness due to aging, a decrease in skin moisture content, an increase in wrinkles, an increase in sagging, a decrease in skin elasticity, skin thinning, and a decrease in skin regeneration ability.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition for treating, ameliorating and / or preventing skin aging. [Background technology]

[0002] It is known that the amount of moisture in the skin decreases with age. On the other hand, the amount of sebum in the skin increases with age when we are young, peaks in our 30s, and then decreases with age. It is known that both moisture and sebum in the skin decrease rapidly when we reach our 40s. This makes skin more susceptible to dryness as we age.

[0003] Dry skin associated with aging is clearly different from normal dry skin in terms of its causes, symptoms, and histological findings. Dry skin associated with aging is caused by age-related changes in the skin, which result in a decrease in sebum secretion and a decline in the skin's moisturizing ability. Dry skin associated with aging is characterized by the prominent appearance of fine wrinkles and sagging, a decrease in skin elasticity, and a tendency to wrinkle.

[0004] Skin is also composed of the epidermis, the outermost layer, the dermis located below the epidermis, and subcutaneous tissue located below the dermis. The subcutaneous tissue is composed of fat cells and other components, and is important for skin firmness, moisture retention, and elasticity. The epidermis provides the skin's barrier function and prevents moisture loss. The dermis stores moisture and is responsible for skin moisture retention and elasticity. The subcutaneous tissue contains a fat layer, which is important for skin firmness, moisture retention, and elasticity. It is known that the epidermis, dermis, and subcutaneous tissue (fat layer) become thinner (thinning) as skin ages.

[0005] It has been reported that the main cause of skin thinning with aging is a decline in regenerative ability due to a decrease in skin stem cells with age (Non-Patent Documents 1 and 2). The decrease in stem cells reduces the number of cells present in the epidermis, dermis, and subcutaneous fat, resulting in thinning of the skin. Furthermore, as epidermal and dermal cells age and decrease, the amount of moisturizing components such as hyaluronic acid, as well as components that maintain skin elasticity and structural stability, such as collagen and elastin, produced by these cells, decreases. As a result, the amount of keratin and sebum that prevent moisture evaporation decreases, causing the skin to dry out and leading to the appearance of wrinkles and sagging.

[0006] On the other hand, normal dry skin can occur regardless of age due to environmental and genetic factors. It can also be associated with exposure to ultraviolet light, excessively dry air, high temperatures, hand and face washing techniques, atopic skin, allergies, exposure to irritants, and skin care methods. Normal dry skin can exhibit itching and redness, as well as skin sensitivity. Furthermore, the decrease in sebum secretion in normal dry skin is less pronounced than in dry skin associated with aging. Furthermore, normal dry skin can exhibit flaking and peeling. One of the most notable features of normal dry skin is the frequent involvement of inflammation. As a result, the epidermis rapidly proliferates and thickens, and the dermis also thickens due to the production of tissue fluid and extracellular matrix. The thickened epidermis is composed of immature cells and therefore cannot adequately prevent moisture loss from the skin. In contrast, dry skin associated with aging undergoes epidermal thinning, which contributes to skin dryness. Therefore, the causes and mechanisms of normal dry skin and age-related dry skin are completely different.

[0007] As described above, there is a clear difference between dry skin due to aging and normal dry skin, and the methods for improving normal dry skin may not be effective or sufficient for dry skin due to aging. Therefore, in order to improve dry skin due to aging, it is necessary to use a composition and a method that can effectively improve the skin condition due to aging in addition to or instead of the methods used for normal dry skin. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Aesthetic Dermatology, 2013, 23 No.1 [Non-patent document 2] Akamatsu H, Hasegawa S, Yamada T, Mizutani H, Nakata S, Yagami A, Matsunaga K. Age-related decrease in CD271(+) cells in human skin. J Dermatol 2016; 43: 311-313 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, the present invention aims to provide a novel composition that can effectively improve the deterioration of skin condition due to aging and can treat, improve and / or prevent various symptoms associated with skin aging. [Means for solving the problem]

[0010] To solve the above problems, the present inventors compared young and old mice and found that the skin structure of mice changes with age, and that old mice exhibited characteristics of skin aging, such as increased dryness, increased wrinkles, and decreased skin elasticity. Administration of Job's tears coix seed extract to treat such skin aging significantly inhibited the age-related decrease in skin moisture content and also inhibited the decrease in skin elasticity. Furthermore, the inventors found that the coix seed extract significantly inhibited age-related skin thinning.

[0011] These results suggest that Job's tears coix seed extract may have the effect of inhibiting skin aging or promoting skin regeneration. Therefore, immunostaining of skin tissue was performed using the cell proliferation marker Ki67. The results showed that coix seed extract significantly inhibits the age-related decline in the proliferation ability of basal cells, which play a role in epidermal turnover. Furthermore, it was shown that coix seed extract inhibits the age-related decline in epidermal stem cells, which play a major role in maintaining the proliferation ability of basal cells. Furthermore, it was shown that coix seed extract inhibits the age-related decline in expression of HAS2, a hyaluronic acid synthase that contributes to maintaining skin moisture retention and elasticity. Furthermore, it was shown that coix seed extract inhibits the age-related decline in collagen in skin tissue and the accumulation of senescent cells due to age. The present invention was based on these findings.

[0012] The present invention provides a composition for treating, improving and / or preventing skin aging, which contains Job's tears as an active ingredient.

[0013] The present invention also provides a composition for treating, improving, and / or preventing skin aging, wherein the skin aging is at least one selected from the group consisting of age-related progression of dry skin, decreased skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, skin thinning, and decreased skin regeneration ability.

[0014] The present invention also provides a composition for treating, improving and / or preventing skin aging, wherein the Job's tears are Job's tears seeds from which the husk and astringent skin have been removed.

[0015] The present invention also provides a composition for treating, improving and / or preventing skin aging, which contains Job's tears extract as an active ingredient.

[0016] The present invention also provides a composition for treating, improving, and / or preventing skin aging, wherein the skin aging is at least one selected from the group consisting of age-related progression of dry skin, decreased skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, skin thinning, and decreased skin regeneration ability.

[0017] The present invention also provides a composition for treating, improving and / or preventing skin aging, wherein the Job's tears are Job's tears seeds from which the husk and astringent skin have been removed.

[0018] The present invention also provides a food or feed composition for improving and / or preventing skin aging, which contains Job's tears as an active ingredient.

[0019] The present invention also provides a food or feed composition for improving and / or preventing skin aging, wherein the skin aging is at least one selected from the group consisting of age-related progression of dry skin, decreased skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, skin thinning, and decreased skin regeneration ability.

[0020] The present invention also provides a food or feed composition for improving and / or preventing skin aging, wherein the Job's tears are seeds from which the husk and astringent skin have been removed. [Effects of the Invention]

[0021] The present invention can effectively improve skin conditions caused by aging, and can treat, improve, and / or prevent various symptoms associated with skin aging, such as the progression of dry skin due to aging, decreased skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, thinning of the skin, and decreased skin regeneration ability. [Brief explanation of the drawings]

[0022] [Figure 1] Graph showing typical skin conditions 4 months after the start of the test in the Young group, Aged (normal diet) group, and Coix seed-administered group. [Figure 2] Graph showing changes in skin moisture content in the Young group, Aged (normal diet) group, and Coix seed-administered group. [Figure 3] Graph showing R7 values ​​of the young group, aged (normal diet) group, and coix seed-administered group. [Figure 4] A graph showing the R2 values ​​for the young group, aged (normal diet) group, and coix seed-administered group. [Figure 5] Graph showing cross sections of the skin of the Young group, the Aged (normal diet) group, and the Coix seed-administered group. [Figure 6] This is a graph comparing the thickness of the epidermis, dermis, and fat layer of the skin of the young group, aged (normal diet) group, and coix seed administration group. [Figure 7] Graph showing Ki67-positive cells in the epidermis of the young group, the aged (normal diet) group, and the coix seed-administered group. [Figure 8] Graph showing the number of Ki67-positive cells / mm in the Young group, the Aged (normal feed) group, and the Coix seed-administered group. [Figure 9] Graph showing nuclear staining of skin tissue from the young group, aged (normal diet) group, and coix seed-administered group. [Figure 10] Graph showing the number of basal cells in the Young group, Aged (normal diet) group, and Coix seed-administered group. [Figure 11] FIG. 1 shows the results of fluorescent staining of skin tissue using CD271 antibody. [Figure 12]A graph showing the relative fluorescence intensity of CD271 in the epidermis of the young group, the aged (normal diet) group, and the coix seed-administered group. [Figure 13] Graph showing the mRNA expression level of HAS2 ( / GAPDH expression level) in the skin 4 months after the start of the test for the Young (normal diet) group, the Aged (normal diet) group, and the Coix seed administration group. [Figure 14] Graph showing the results of Masson trichrome staining of skin tissues from the Young (normal diet) group, the Aged (normal diet) group, and the 3% coix seed administration group. [Figure 15] Graph showing collagen area ratio in skin tissue of the Young (normal diet) group, the Aged (normal diet) group, and the Yokuinin administration group (1%, 3%). [Figure 16] FIG. 1 shows the results of immunohistochemical staining of the cellular senescence marker p21 in skin tissue from the Young (normal diet) group, the Aged (normal diet; NT) group, and the 3% coix seed administration group. [Figure 17] Graph showing the number of p21-positive cells in the epidermis, papillary dermis, and full dermis thickness. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides a composition for treating, improving and / or preventing skin aging, which contains Job's tears as an active ingredient.

[0024] As used herein, "skin aging" refers to changes that occur in skin due to aging, and refers to a change from healthy, youthful skin caused by a decline in skin cell function due to age. Skin aging can refer to changes that occur in skin due to, for example, a decline in epidermal cell proliferation, a decline in basal cell count, or a decrease in epidermal stem cells due to aging. Skin aging can also refer to changes that occur in skin due to, for example, a decrease in hyaluronic acid or collagen in the skin due to aging, or the accumulation of senescent cells due to aging. Skin aging includes, for example, the progression of dry skin, a decrease in skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, skin thinning, and a decrease in skin regeneration ability due to aging. Skin experiencing skin aging, such as the progression of dry skin, a decrease in skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, skin thinning, and a decrease in skin regeneration ability due to aging, is also referred to as "aging skin" in this specification. In other words, the composition of the present invention can suppress the progression of age-related dry skin, decreased skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, thinning of the skin, and decreased skin regeneration ability, and can maintain or improve the regeneration ability of the skin (aging skin).

[0025] As used herein, the term "treatment" includes inhibiting, alleviating or alleviating a disease or symptom, inhibiting, suppressing, slowing or eliminating the progression of a disease or symptom, achieving remission, and achieving an improved prognosis.

[0026] As used herein, the term "amelioration" includes alleviating or alleviating a disease or symptom, as well as inhibiting, slowing or eliminating the progression of a disease or symptom.

[0027] As used herein, the term "prevention" includes inhibiting, suppressing or delaying the occurrence (development) of a disease or condition, and inhibiting, suppressing or delaying the recurrence of a disease or condition.

[0028] As used herein, "inhibiting" includes inhibiting, alleviating, or alleviating a particular disease, symptom, or condition; inhibiting, slowing, or eliminating the progression of a particular disease, symptom, or condition; and inhibiting or slowing the onset of a particular disease, symptom, or condition.

[0029] Job's tears (Coix) is an annual plant of the genus Coix (Poaceae). The Job's tears used in the present invention are not particularly limited and may be Job's tears leaves, branches, bark, trunks, stalks, fruits, seeds, seed husks, seed astringent skin, seeds with the husk or astringent skin or both removed, flowers, roots, or a mixture thereof. The Job's tears used in the present invention may be, for example, Job's tears seeds with the husk and astringent skin removed. Job's tears seeds with the husk and astringent skin removed may be, for example, a herbal medicine called Job's tears. The composition of the present invention may contain Job's tears or Job's tears extract as an active ingredient. The Job's tears used in the present invention are not particularly limited and may be Job's tears seeds with the husk and astringent skin, Job's tears seeds with the husk or astringent skin or both removed, a herbal powder obtained by simply grinding Job's tears, or an extract from Job's tears.

[0030] The "extract" in the present invention includes an extract obtained by adding a solvent to a raw material and extracting it, a squeezed liquid obtained after subjecting a raw material to a compression treatment, an extract obtained by adding a solvent to the residue after compressing the raw material and extracting it, and a dried product obtained by drying these extracts or squeezed liquids.

[0031] The parts used as the extraction raw material are not particularly limited, and may be Job's tears leaves, branches, bark, trunks, stalks, fruits, seeds, seed husks, seed astringent skins, seeds with the husks or astringent skins or both removed, flowers, roots, and mixtures thereof. In this specification, "extract" can be replaced with "extracted".

[0032] The Job's tears and Job's tears extract used in the present invention can be produced by known methods. The Job's tears extract used in the present invention can be produced, for example, by extraction at room temperature or by extraction with heat using an extraction solvent. If necessary, extraction may be carried out under reduced pressure or pressure. The obtained extract may be used as is, or may be used after being concentrated or lyophilized to dryness.

[0033] The extract may be subjected to hydrolysis after extraction with an extraction solvent. Hydrolysis may be carried out using any conventionally known method. Alternatively, enzymatic hydrolysis may be carried out before extraction with an extraction solvent. Enzymatic hydrolysis may be carried out using any conventionally known method.

[0034] The extract can be obtained by extracting with an extraction solvent and then removing solid matter by filtration, centrifugation, and / or decantation, etc. Filtration, centrifugation, and decantation can be carried out using any conventionally known method.

[0035] Examples of extracting solvents that can be used include water, physiological saline, alcohols (e.g., lower alcohols such as methanol, absolute ethanol, and ethanol, and polyhydric alcohols such as propylene glycol and 1,3-butylene glycol), ketones such as acetone, esters such as ethyl ether, dioxane, acetonitrile, and ethyl acetate, xylene, benzene, and chloroform. Among these, it is preferable to use water, alcohols such as methanol and ethanol, or mixed solvents of these as the extracting solvent. The above-mentioned extracting solvents may be used alone or in combination of two or more.

[0036] The extract can be pH adjusted as necessary and subjected to gel filtration and / or ultrafiltration to remove macromolecules without losing its activity. The extract or its fractions may be concentrated by methods such as vacuum concentration, ultrafiltration and / or freeze concentration. The extract may also be dried by methods such as freeze drying, spray drying and / or plate drying, resulting in a dried product, such as a powder.

[0037] The composition of the present invention can be in any form of preparation. For oral administration, the composition of the present invention can be in the form of tablets such as sugar-coated tablets, buccal tablets, coated tablets and chewable tablets; lozenges; pills; powders; capsules including hard capsules and soft capsules; granules; and liquids such as suspensions, emulsions, syrups and elixirs.

[0038] The composition of the present invention can be administered parenterally, for example, by intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, nasal administration, pulmonary administration, enteral administration, buccal administration, or transmucosal administration. The main protease inhibitor of the present invention can be administered, for example, in the form of an injection, a transdermal absorption tape, an aerosol, or a suppository.

[0039] The composition of the present invention may also be in a form suitable for consumption, such as a solid, liquid, granular, particulate, powder, capsule, cream, or paste.

[0040] The composition of the present invention may further contain any ingredient commonly used in pharmaceuticals, quasi-drugs, cosmetics, and foods, such as a pharmaceutically acceptable base, carrier, excipient, binder, disintegrant, lubricant, and colorant.

[0041] Examples of carriers and excipients used in the compositions of the present invention include lactose, glucose, sucrose, mannitol, dextrin, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, and crystalline cellulose.

[0042] Examples of binders include starch, gelatin, syrup, tragacanth gum, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, and the like.

[0043] Examples of disintegrants include starch, agar, powdered gelatin, crystalline cellulose, calcium carbonate, sodium bicarbonate, sodium alginate, sodium carboxymethylcellulose and calcium carboxymethylcellulose.

[0044] Examples of lubricants include magnesium stearate, hydrogenated vegetable oil, talc, macrogol, etc. As the coloring agent, any coloring agent permitted to be added to pharmaceuticals, quasi-drugs, and foods can be used.

[0045] Furthermore, the composition of the present invention may be coated with one or more layers of sucrose, gelatin, purified shellac, gelatin, glycerin, sorbitol, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, methyl methacrylate, methacrylic acid polymers, or the like, if necessary.

[0046] Furthermore, the composition of the present invention may contain, as necessary, pH adjusters, buffers, stabilizers, preservatives, antiseptics, diluents, coating agents, sweeteners, flavoring agents, solubilizers, and the like.

[0047] The content of Job's tears in the composition of the present invention may be any amount that can fully exert the effects of the present invention, and can be appropriately set depending on the subject, purpose, and administration method (intake method) of the composition. For example, when the composition is orally ingested by humans, the content of Job's tears is preferably an amount that results in a daily intake of 0.001 to 10 mass% on a solid content basis, and more preferably an amount that results in a daily intake of 0.1 to 2 mass%.

[0048] The composition of the present invention can be, for example, a pharmaceutical product, a cosmetic product, an external skin preparation, etc. The composition of the present invention can be, for example, a pharmaceutical product, a cosmetic product, or an external skin preparation for treating, improving, and / or preventing skin aging.

[0049] Cosmetics include, for example, skin cosmetics such as lotions, emulsions, creams, ointments, gels, lotions, oils, packs, mists, and face and body cosmetic sheets; makeup cosmetics such as foundations, lipsticks, blushers, eye shadows, eyeliners, and mascaras; cleansing compositions such as body soaps, hand soaps, and soaps; and hair cosmetics such as shampoos, rinses, treatments, and setting agents.

[0050] The external preparation for skin is not particularly limited, and may be in the form of, for example, skin cosmetics, skin patch sheets, cosmetic sheets, etc. The external preparation for skin may be a quasi-drug, a designated quasi-drug, an external medicine, etc. The dosage form of the external preparation for skin can be selected arbitrarily depending on the purpose.

[0051] In addition to Job's tears, pharmaceuticals, cosmetics, and topical skin preparations can be blended with substances appropriately selected from the following depending on the required efficacy and effect: For example, various cosmetic ingredients commonly used can be blended, such as oils and fats, surfactants, moisturizers, whitening agents, pH adjusters, binders, thickeners, polyhydric alcohols, essential oils, fragrances, preservatives, antioxidants, ultraviolet absorbers, pigments, crushed plants, herbal medicines, inorganic salts, inorganic acids, detergents, and emulsifiers.

[0052] The oils and fats may be ingredients commonly used in cosmetics, such as vegetable oils and fats such as soybean oil, almond oil, paraffin, cetanol, avocado oil, olive oil, jojoba oil, coconut oil, palm oil, rice bran oil, egg yolk oil, castor oil, squalane, lanolin, liquid paraffin, and white petrolatum; animal oils and fats such as beef tallow, lard, horse fat, turtle oil, mink oil, Purcellin oil, and squalane; and synthetic oils and fats such as methylpolysiloxane, behenyl alcohol, glyceryl tricaprate, glyceryl trioctanoate, liquid paraffin, DHA, and EPA.

[0053] Examples of surfactants that can be used include anionic surfactants such as sodium lauryl sulfate, triethanolamine lauryl sulfate, and lauric acid diethanolamide; cationic surfactants such as stearyl trimethylammonium chloride, cetyl trimethylammonium chloride, and benzalkonium chloride; nonionic surfactants such as glyceryl monostearate, sorbitan monostearate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene hydrogenated castor oil, sucrose esters, and fatty acid amides; polymeric surfactants, fluorine-based surfactants, silicone-based surfactants, polypeptide derivatives, and natural surfactants.

[0054] Examples of moisturizing agents that can be used include synthetic moisturizing agents such as sodium pyrrolidone carboxylate and pantetheine-S sulfonate; and natural moisturizing agents such as hyaluronic acid, elastin, keratin, dermatan sulfate, collagen, placenta extract, royal jelly and their hydrolysates, microbial fermentation liquids such as chitin, chitosan, pectin, glucosamine and N-acetylglucosamine, other plant and animal extracts, vegetable oils and their esters, and derivatives.

[0055] Examples of whitening agents that can be used include ascorbic acid and its salts or derivatives, arbutin and its derivatives, placenta extracts, and other plant- or animal-derived extracts.

[0056] Examples of pH adjusters that can be used include organic acids such as citric acid, lactic acid, salicylic acid, tartaric acid, malic acid, benzoic acid, sodium citrate, fumaric acid, sodium monohydrogen phosphate, and potassium dihydrogen phosphate, as well as inorganic acids and their salts.

[0057] As the binder and thickener, those generally used in cosmetics, such as sodium carboxymethylcellulose, methylcellulose, casein, pectin, starch, sodium alginate, polyvinyl alcohol, polyvinylpyrrolidone, locust bean gum, agar, carbopol, xanthan gum, and bentonite, can be used.

[0058] Examples of polyhydric alcohols that can be used include glycerin, propylene glycol, sorbitol, polyethylene glycol, 1,3-butylene glycol, dipropylene glycol, and polyoxyethylene glycerin (26E.O).

[0059] Examples of essential oils and fragrances that can be used include natural and synthetic fragrances such as lavender oil, jasmine oil, rose oil, lemon oil, orange oil, peppermint oil, thyme oil, calamus oil, fennel oil, cedar oil, hiba oil, raspberry oil, cypress oil, rose oil, eucalyptus oil, camphor, peppermint oil, spearmint oil, geraniol, mandarin oil, spruce oil, citronellol, terpenes, coffee oil, tea oil, camellia oil, and raspberry ketone.

[0060] Examples of preservatives that can be used include paraoxybenzoic acid esters such as methylparaben, ethylparaben, propylparaben, and butylparaben, phenoxyethanol, ethanol, and dehydroacetic acid.

[0061] Examples of antioxidants that can be used include EDTA4Na, EDTA2Na, butyloxytoluene, hydroxybutyltoluene, butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT), and derivatives thereof.

[0062] The ultraviolet absorber is not particularly limited as long as it has the property of selectively absorbing ultraviolet light, and examples that can be used include oxybenzone, oxybenzonesulfonic acid, tetrahydroxybenzophenone, dihydroxymethoxybenzophenone sodium sulfonate, dihydroxymethoxybenzophenone, dihydroxybenzophenone, cinoxate, methyl diisopropylcinnamate, octyl methoxycinnamate, glyceryl para-aminobenzoate, and octyl para-aminobenzoate.

[0063] Examples of pigments that can be used include red iron oxide, yellow iron oxide, black iron oxide, titanium oxide, nylon powder, zinc oxide, sericite, mica, talc, carbon, and medicinal charcoal.

[0064] Examples of crushed plants and herbal medicines that can be used include crushed, dried, and extracts of lemon peel, seaweed, cypress, Japanese cypress, fucus, rice bran, calamus, ginger, licorice, tangerine peel, yuzu, angelica tree, carrot, mint, cinnamon bark, Ubai, mugwort, Houttuynia cordata, peach blossom, chamomile, aloe, jasmine, rose hip, lavender, guava, Scutellaria root, wolfberry, lychee, elderberry, Angelica tree, Aralia aralia, burdock, black sesame, black rice, Fujisanchi ginseng, Korean ginseng, Panax notoginseng, Sekkotsu grass, and Cnidium rhizome.

[0065] Examples of inorganic salts and inorganic acids that can be used include sodium chloride, sodium hydrogencarbonate, sodium carbonate, boric acid, borax, sodium sulfate, sodium sulfide, potassium sulfide, sodium nitrate, calcium nitrate, ammonium sulfate, sodium thiosulfate, calcium hydrogenphosphate, potassium chloride, ammonium chloride, sodium phosphate, sodium hyposulfite, calcium thiosulfate, sulfur, sodium sesquicarbonate, magnesium sulfate, magnesium chloride, silicic anhydride, metasilicic acid, boric acid, magnesium carbonate, magnesium hydroxide, magnesium dihydride, and dried seawater.

[0066] The detergent may be, for example, a fatty acid soap, an anionic surfactant such as sodium lauryl sulfate or alkyl ether carboxylate, or an aliphatic detergent.

[0067] The emulsifier may be a substance known as a cosmetic raw material, and examples thereof include sucrose fatty acid esters such as sucrose laurate, sucrose myristate, sucrose palmitate, sucrose stearate, sucrose oleate, and sucrose erucate; tetraglycerin monostearate, tetraglycerin monooleate, tetraglycerin tristearate, hexaglycerin monolaurate, hexaglycerin monomyristate, hexaglycerin monostearate, hexaglycerin tristearate, decaglycerin monolaurate, decaglycerin monomyristate, decaglycerin monostearate, decaglycerin distearate, decaglycerin tristearate, decaglycerin pentastearate, decaglycerin monolaurate, decaglycerin monomyristate, decaglycerin monostearate, decaglycerin distearate, decaglycerin tristearate, decaglycerin pentastearate, decaglycerin monos ... Polyglycerol fatty acid esters such as sorbitan monoisostearate, decaglycerol diisostearate, decaglycerol pentaisostearate, decaglycerol monooleate, decaglycerol trioleate, and decaglycerol pentaoleate; sorbitan branched fatty acid esters such as sorbitan monoisostearate, sorbitan sesquiisostearate, and sorbitan triisostearate; lecithins such as soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, lysolecithin and / or hydrogenated lysolecithin obtained by enzymatically treating these lecithins to form monoacylated forms, and hydroxylated hydroxylecithin; and glycerol branched fatty acid esters such as glyceryl monoisostearate, glyceryl sesquiisostearate, and glyceryl diisostearate.

[0068] The above-exemplified components may be used singly or in combination of two or more.

[0069] In addition to the ingredients mentioned above, pharmaceuticals, cosmetics, and topical skin preparations may contain, as needed, milk-derived ingredients such as lactose, milk, and condensed milk; inorganic pigments such as titanium and talc; disinfectants such as isopropylmethylphenol, chlorhexidine hydrochloride, and chlorhexidine gluconate; vitamins and coenzymes such as vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin P, CoQ10, CoQ9, CoQ8, and thioctic acid, fluidizing agents such as silicic anhydride and synthetic aluminum silicate; and tar-based pigments for pharmaceuticals, quasi-drugs, and cosmetics.

[0070] In addition to the above-mentioned ingredients, various cosmetic ingredients such as medicinal ingredients for the purposes of beauty, facial beauty, and skin treatment can be appropriately blended into the pharmaceuticals, cosmetics, and topical skin preparations.

[0071] Medicinal ingredients include, for example, avocado extract, hydrangea extract, althea extract, arnica extract, apricot extract, apricot kernel extract, ginkgo extract, fennel extract, turmeric extract, oolong tea extract, echinacea leaf extract, phellodendron bark extract, barley extract, Dutch mustard extract, orange extract, hydrolyzed wheat powder, hydrolyzed silk, chamomilla extract, carrot extract, artemisia capillaris extract, kalkade extract, kiwi extract, cinchona extract, cucumber extract, guanosine, kumazasa extract, walnut extract, grapefruit extract, clematis extract, yeast extract, comfrey extract, cowberry extract, bupleurum extract, umbilical cord extract, salvia extract, soapwort extract, bamboo grass extract, hawthorn extract, shiitake mushroom extract, rehmannia root extract, lithospermum root extract, linden extract, meadowsweet extract, calamus root extract, white birch extract, horsetail extract, honeysuckle Examples of useful extracts that can be used include Japanese whiting, common weed extract, hawthorn extract, elderberry extract, yarrow extract, peppermint extract, mallow extract, Swertia japonica extract, Chinese laurel extract, thyme extract, clove extract, Imperata cylindrica extract, tomato extract, natto extract, wild rose extract, hibiscus extract, Bakumondo extract, parsley extract, Parietaria extract, Japanese knotweed extract, bisabolol, coltsfoot extract, butterbur honey, Japanese gourd extract, Poria columbine extract, butcher's broom extract, grape extract, propolis, loofah extract, peppermint extract, linden extract, hop extract, pine extract, horse chestnut extract, Asian skunk cabbage extract, Soapberry extract, peach leaf extract, cornflower extract, eucalyptus extract, apple extract, lettuce extract, astragalus extract, rose extract, rosemary extract, and Roman chamomile extract.

[0072] In addition, pharmaceuticals, cosmetics, and topical skin preparations contain anti-inflammatory agents such as ε-aminocaproic acid, glycyrrhizinic acid, dipotassium glycyrrhizinate, β-glycyrrhetinic acid, lysozyme chloride, guaiazulene, and hydrocortisone; blood circulation promoters such as vitamin C, vitamin D, calcium pantothenate, biotin, nicotinamide, vitamin E, and vitamin E derivatives; vitamins such as β-carotene and carotenoid derivatives; active ingredients such as allantoin, diisopropylamine dichloroacetate, and 4-aminomethylcyclohexanecarboxylic acid; antioxidants such as flavonoids, tannins, lignans, and saponins; wound healing agents such as γ-oryzanol and protease; cepharanthine, capsicum tincture, and hinokitiol. The following may be optionally blended: ethanol, iodized garlic extract, pyridoxine hydrochloride, nicotinic acid, nicotinic acid derivatives, acetyl pantothenyl ethyl ether, estradiol, ethinyl estradiol, capronium chloride, diphenhydramine hydrochloride, camphor, nonylic acid vanillylamide, nonanoic acid vanillylamide, piroctone olamine, glyceryl pentadecanoate, l-menthol, menthol pyrrolidone carboxylate, mononitroguaiacol, urea, resorcinol, γ-aminobutyric acid, benzethonium chloride, mexiletine hydrochloride, auxin, female hormones and female hormone-like substances, cantharides tincture, cyclosporine, analgesics, tranquilizers, antihypertensives, antibiotics, antihistamines, and antibacterial substances.

[0073] The method for producing the pharmaceuticals, cosmetics, and topical skin preparations is not particularly limited, and any conventionally known production method can be used. Job's tears can be added as appropriate in any step for preparing the pharmaceuticals, cosmetics, and topical skin preparations.

[0074] The present invention also provides a food composition containing Job's tears as an active ingredient for improving and / or preventing skin aging. The present invention also provides a feed composition containing Job's tears as an active ingredient for improving and / or preventing skin aging. The food and feed compositions of the present invention can be constructed in the same manner as the above-mentioned compositions.

[0075] As used herein, "food compositions" include not only general foods and beverages, but also foods for the sick, health foods, functional foods, foods for specified health uses, nutritional supplements, and supplements. General foods and beverages include, for example, various beverages, various foods, confectioneries, seasonings, liquid foods (such as soups), nutritional drinks, and processed foods. Supplements include, for example, tablet-type supplements.

[0076] Examples of beverages include water, tea, coffee, fruit juice drinks, carbonated drinks, cocoa, dairy drinks, alcoholic beverages, jelly drinks, sports drinks, and energy drinks. The beverage may be in the form of powder, granules, or tablets that are dissolved before consumption. The beverage may also be a teabag-type beverage that is extracted and consumed. The beverage may be a packaged beverage. The containers used for the packaged beverage may be those typically used for beverages, such as plastic bottles, metal cans, paper cartons, and bottles.

[0077] The various foods include, for example, bread, noodles, rice, tofu, oily foods, and dairy products.

[0078] Examples of confectioneries include candy, chewing gum, candy, gummy candies, tablets, chocolates, tablet sweets, cookies, biscuits, snacks, baked goods, cakes, rice crackers, yokan, puddings, jellies, jams, creams, ice creams, popsicles, sherbets, crackers, and chips.

[0079] The seasonings include, for example, miso, soy sauce, dressing, mayonnaise, sweeteners, and food additives.

[0080] As used herein, the term "processed food" refers to natural ingredients (such as animals and plants) that have been processed and / or cooked, and includes, for example, processed meat products, processed vegetables, processed fruit products, frozen foods, retort foods, canned foods, bottled foods, and instant foods.

[0081] The food composition of the present invention may be a food labeled with the claim that it improves and / or prevents skin aging, or that it improves and / or prevents the progression of dry skin, decreased skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, and thinning of the skin due to aging. The food composition of the present invention may also be provided in a sealed form, such as a bag or container. The bag or container used in the present invention may be any bag or container commonly used for food.

[0082] The food composition may contain any of the following ingredients, if necessary, as long as the object of the present invention is not impaired: for example, carbohydrate sweeteners (monosaccharides such as fructose, glucose, tagatose, and arabinose, oligosaccharides such as lactose, oligosaccharides, maltose, and trehalose, powdered starch syrup, dextrin, and sugar alcohols), high-intensity sweeteners (sucralose, acesulfame K, and stevia), polysaccharides such as starch, oils and fats, dairy products, stabilizers, emulsifiers, flavors (vanillin, linalool, and natural flavors), pigments, coloring agents, acidulants, flavoring ingredients (eggs, coffee, tea, cocoa, fruit juice and pulp, yogurt, and alcoholic beverages), flavorings (raspberry flavor, apple flavor, and the like), and the like. flavors and coffee flavors, etc.), anti-humectants, electrolytes, preservatives, humectants, proteins, amino acids, peptides, dietary fiber, organic acids (citric acid, malic acid, fumaric acid, malonic acid, succinic acid, tartaric acid, lactic acid, etc.), vitamins (L-ascorbic acid, dl-α-tocopherol, vitamin B1, vitamin B2, niacin, pantothenic acid, vitamin B6, vitamin B12, folic acid, biotin, inositol, etc.), gold, silver, platinum, minerals (zinc, iron, calcium, magnesium, chromium, selenium, potassium, sodium, etc.). ), marine collagen, hydrolyzed collagen, hyaluronic acid, fermented yeast extract, glucosamine, yeast, eggshell membrane, lycopene, astaxanthin, other carotenoids, silk, chondroitin, ceramide, placenta extract, shark fin extract, deep sea shark extract, squalene, gamma-aminobutyric acid, casein dodecapeptide, raw chestnut skin extract, chestnut leaf extract, chestnut bur extract, chestnut pulp extract, chestnut bark extract, fermented cabbage extract, rose petal extract, grape leaf extract, grape seed extract, apple polyphenols, chamomile extract, lychee seed Extract, gotu kola extract, shell ginger leaf extract, lotus germ extract, star fruit leaf extract, mulberry leaf extract, guava tea extract, red wine, green tea, black tea, oolong tea, coffee, cocoa, chocolate, black sesame, beans, soy milk, nuts, mushrooms, green and yellow vegetables, egg-derived ingredients such as iodine eggs, catechins, other polyphenols, raspberry ketone, low molecular weight alginic acid, psyllium seed coat, ginkgo leaf extract, pine bark extract, nattokinase, plant sterols, diacylglycerol, chitosan, hyaluronic acid, methylsulfonylmethane,These include kojic acid, ellagic acid, arbutin, rucinol, magnolignan, magnesium L-ascorbate phosphate, CoQ10, and α-lipoic acid.

[0083] As used herein, the term "feed composition" refers to a substance that is ingested as food by animals other than humans, such as livestock, poultry, companion animals, and laboratory animals. Livestock includes, but is not limited to, mammals such as cows, horses, donkeys, pigs, sheep, and goats. Poultry includes, but is not limited to, birds such as ducks, chickens, geese, turkeys, ducks, and quails. Pets include, but are not limited to, dogs, cats, hamsters, and rabbits. Laboratory animals include, but are not limited to, monkeys, mice, rats, and guinea pigs.

[0084] The feed composition of the present invention may contain any ingredients typically incorporated into feed, as needed, provided that the purpose of the present invention is not impaired. The feed composition may contain, for example, a main feed such as a plant feed or an animal feed, moisture, oil, a pH adjuster, an antioxidant, a preservative, a coloring material, a flavoring, an excipient, vitamins, hormones, amino acids, antibiotics, and antibacterial agents.

[0085] The feed composition of the present invention can be used to improve or prevent skin aging in non-human animals, and can improve or prevent the progression of age-related dry skin, decreased skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, thinning of the skin, or decreased skin regeneration ability in non-human animals.

[0086] The present invention also provides a method for treating, improving, and / or preventing skin aging in a subject, comprising administering Job's tears to the subject. In the method of the present invention, Job's tears can be administered in the form of the above-mentioned composition.

[0087] In the method of the present invention, the amount of Job's tears administered may be any amount that can exert its effect, and can be appropriately set depending on the subject, purpose, and administration method (intake method). For example, when administered orally to humans, Job's tears can be administered so that the daily intake is preferably 0.001 to 10% by mass, and more preferably 0.1 to 2% by mass, based on the solid content.

[0088] Subjects to which the method of the present invention can be applied include mammals such as humans, cows, horses, donkeys, pigs, sheep, and goats; pet animals such as dogs, cats, hamsters, and rabbits; laboratory animals such as monkeys, mice, rats, and guinea pigs; and birds such as ducks, chickens, geese, turkeys, wild ducks, and quails.

[0089] The subject to which the present invention is applied is not particularly limited, and may be a subject in need of treatment, improvement, and / or prevention of skin aging. In the case of humans, the subject to which the present invention is applied is not particularly limited, and may be, for example, a subject in his / her 30s or older, preferably a subject in his / her 40s or older.

[0090] The present invention also provides Job's tears for use in the treatment, improvement and / or prevention of skin aging.The present invention also provides use of Job's tears for the treatment, improvement and / or prevention of skin aging.

[0091] The present invention also provides use of Job's tears for producing a composition for treating, improving and / or preventing skin aging, a food composition for improving and / or preventing skin aging, or a cosmetic for improving and / or preventing skin aging. [Example]

[0092] (Yokuinin extract) This shows the manufacturing method for dried powder of Job's Tears extract. The outer and inner skins of dried Job's Tears are removed, and 10 times the amount of purified water is added. Heat extraction is carried out at 95°C or above for 2 hours, and then filtered. The filtrate is centrifuged to obtain an extract (primary extract). Eight times the amount of purified water is added to the residue, and heat extraction is carried out at 95°C or above for 1 hour, and then filtered in the same way. The filtrate is centrifuged to obtain an extract (secondary extract). The extracts are combined and concentrated under reduced pressure at 65°C or below. The concentrated solution is heat sterilized and then spray-dried to obtain dry powder of Job's Tears extract. 1g of extract powder is equivalent to approximately 20g of the original herbal medicine.

[0093] (animal) C57BL / 6J (male), 10-week-old and 60-week-old, were obtained from Jackson Laboratory Japan.

[0094] The mice were divided into the following four groups (n=10). (1) Young group (10 weeks old) (2) Aged (normal diet) group (60 weeks old) (3) Aged + 1% coix seed mixed diet group (1% group) (4) Aged + 3% coix seed mixed diet group (3% group)

[0095] The (1) Young group and (2) Aged (normal diet) group were fed a normal diet of CE-2 solid feed (manufactured by CLEA Japan, Inc.), while the (3) 1% group and (4) 3% group were fed a diet prepared by mixing CE-2 powder feed (manufactured by CLEA Japan, Inc.) with 1% or 3% coix seed extract (manufactured by Oriental Yeast Co., Ltd.).

[0096] (Test Method) Each group was fed either a normal diet or a diet containing coix seed for five months. Skin moisture content was measured every month. After five months, the study was terminated and skin samples were collected.

[0097] (Skin moisture measurement) One week before the measurements, the mice were depilated. One week after the depilation, the skin moisture content was measured five times at each of three locations on the back of the mice: the neck, center, and buttocks, using a Corneometer CM825 (Courage+Khazaka, Germany). The average value of the three locations was used as the measurement value.

[0098] (Measurement of the thickness of the epidermis, dermis and fat layer) The collected skin was fixed in 4% PFA (Wako). The PEA-fixed skin tissue was prepared into paraffin blocks, sliced ​​at 4 μm thickness, stained with HE staining, and observed under a Zeiss microscope. The thickness of the epidermis, dermis, and fat layer was calculated by dividing the area of ​​each layer by the length of the basal layer.

[0099] (Ki67 immunostaining) The collected skin was fixed in 4% PFA (Wako). The PFA-fixed skin tissue was then cut into paraffin blocks at 4 μm thickness and immunostained for Ki67 using the following procedure. First, the paraffin-embedded sections were deparaffinized and washed with distilled water. They were then immersed in 0.01 M citrate buffer, pH 6.0, and autoclaved at 121°C for 5 minutes for activation. After 1 hour, they were washed with TBST. They were then incubated in 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature to treat endogenous peroxidase, followed by washing with TBST.

[0100] The sections were then incubated in 5% goat serum (Life Technologies) / TBST for 1 hour at room temperature for blocking. After overnight incubation at 4°C in primary antibody Ki67 (Abcam) diluted 1:1000 in antibody dilution buffer (Dako), the sections were washed with TBST. The sections were then incubated in goat anti-rabbit IgG (Dako) for 1 hour at room temperature for secondary antibody reaction, followed by washing with TBST.

[0101] After developing the color with the color-developing substrate solution DAB (Dako) (150 seconds), the reaction was stopped by immersion in distilled water, and the specimen slide was immersed in hematoxylin, a counterstaining reagent (nuclear stain), (1 to 1.5 minutes), and then the color was developed in PBS (1 minute).

[0102] The sections were dehydrated with ethanol, cleared with xylene, and mounted in a non-aqueous mounting medium (Quick-Mount). After drying, the skin tissue was photographed using a Zeiss microscope. The number of Ki67-positive cells in the epidermis was counted, and the number of Ki67-positive cells / mm was calculated using the following formula: Number of Ki67-positive cells / mm = Number of Ki67-positive cells / length of basal layer

[0103] (CD271 fluorescent staining) The collected skin was fixed in 4% PFA (Wako). The PFA-fixed skin tissue was then cut into paraffin blocks at a thickness of 4 μm and fluorescently stained for CD271 using the following procedure. First, the paraffin-embedded sections were deparaffinized, and washed with distilled water. Next, the sections were immersed in Immunosaber (Nissin EM Co., Ltd.) solution diluted 200-fold with distilled water at 98°C for 45 minutes, followed by washing with TBST.

[0104] Afterwards, the sections were incubated in 5% goat serum (Life Technologies) / TBST for 1 hour at room temperature for blocking. Then, the sections were incubated overnight at 4°C in the primary antibody CD271 (Abcam) diluted 1:500 in antibody dilution buffer (Dako), and washed with TBST. Then, the sections were incubated in the secondary antibody (1 / 1000, CF TM The sections were incubated in a solution of 488A Goat Anti-Rat IgG (H+L) (Biotium) and Hoechst 33342 (1 / 2000, Donji) at room temperature for 1 hour in the dark to carry out the secondary antibody reaction, and then washed with TBST.

[0105] The sections were mounted on a slide using ProLong Glass (Invitrogen) anti-fade mounting medium and dried overnight at 4°C. After drying, the skin tissue was photographed under a microscope (10x objective). The number of cells along the basal layer in the photographed skin images was counted, and the number of basal cells / mm was calculated using the following formula: Basal cell count / mm = number of cells along the basal layer / length of the basal layer

[0106] (Total RNA extraction and cDNA synthesis) Total RNA was extracted from mouse skin tissue using the GenElute Mammalian Total RNA Miniprep Kit (Cat# RTN70, Sigma) according to the attached protocol. The extracted RNA solution was quantified using a Biospec nano and adjusted to 500 ng / 10 μL of RNA before cDNA synthesis. cDNA synthesis was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remove (Cat# FSQ-301, Toyobo) according to the attached protocol.

[0107] (HAS2 gene expression analysis by real-time PCR) The expression level of the HAS2 gene was measured using the StepOnePlus Real-Time PCR System (Applied Biosystems Japan). GAPDH was used as an endogenous control, and relative gene expression levels were quantified using the ΔΔCt method. The composition of the reaction solution, reaction conditions, and primer sequences used are as follows:

[0108] (Reaction solution composition) TB Green Premix Ex Taq II(2X): 10 μL Primer (Forward, 50 μM): 0.2 μL Primer (Reverse, 50 μM): 0.2 μL ROX Reference Dye (50X): 0.4 μL milliQ: 8 μL cDNA (10-fold dilution): 2 μL (final concentration: 10 ng)

[0109] (PCR reaction conditions) Initial denaturation: 95°C, 30 seconds (1 cycle) Cycle conditions: 95°C, 5 seconds → 60°C, 30 seconds (40 cycles)

[0110] (Primer sequences used) [Table 1]

[0111] (Masson trichrome staining and collagen area measurement) The collected mouse skin tissue was fixed using 4% PFA (Wako). The PFA-fixed skin tissue was prepared into paraffin blocks, sliced ​​at 4 μm thickness, stained with Masson trichrome, and photographed using a Zeiss optical microscope. Five fields of view were randomly selected from the obtained Masson trichrome-stained images, and image analysis was performed using Fiji (ImageJ) image analysis software. The blue-stained collagen area in the image was extracted by thresholding, and the collagen area ratio (area ratio) in the skin tissue was evaluated by calculating the ratio of the collagen area to the total tissue area in each field of view.

[0112] (p21 immunohistological staining) PFA-fixed mouse skin tissue was prepared into paraffin blocks and sliced ​​at 4 μm thickness. Then, immunohistochemistry (IHC) for the cellular senescence marker p21 was performed as follows:

[0113] (1) Paraffin-embedded sections were treated in xylene for 10 minutes three times, then in 100% ethanol for 5 minutes three times and in 70% ethanol for 5 minutes to deparaffinize them, and then washed in distilled water for 5 minutes twice.

[0114] (2) For antigen retrieval, the sections were immersed in Immunosaber (diluted 1:200) and heated at 98°C for 45 minutes. After cooling to room temperature, the sections were washed three times with TBST for 5 minutes each.

[0115] (3) To suppress endogenous peroxidase activity, the sections were immersed in 3% hydrogen peroxide (in methanol) at room temperature for 10 minutes, and then washed twice with TBST for 5 minutes.

[0116] (4) To prevent nonspecific antibody binding, the sections were incubated for 1 hour at room temperature in a blocking solution prepared by diluting 5% goat serum (Life Technologies) in TBST.

[0117] (5) For the primary antibody reaction, anti-p21 antibody (rabbit monoclonal, ab188224, Abcam) was diluted 1:500 in antibody dilution buffer (Dako), added dropwise to the sections, and incubated overnight at 4°C. The next day, the sections were washed three times for 5 minutes with TBST.

[0118] (6) For the secondary antibody reaction, HRP-labeled goat anti-rabbit IgG (Dako, K4003) was used, and the sections were incubated at room temperature for 1 hour, followed by washing with TBST for 5 minutes three times.

[0119] (7) DAB chromogenic substrate (Dako) was used for color development. After 4 minutes of incubation, the reaction was stopped with distilled water. The sections were then immersed in hematoxylin for 1 minute as a nuclear stain, and washed with PBS for 5 minutes to develop the color.

[0120] (8) After staining, the sections were dehydrated with ethanol, cleared with xylene, and then mounted using a non-aqueous mounting medium (Quick-Mount).

[0121] The dried slides were observed and photographed using a Zeiss optical microscope. In the images, p21-positive cells present in the epidermis, dermis (papillary dermis and full dermis), and subcutaneous fat layer were counted, and the number of positive cells per unit length was calculated using the following formula: Number of p21-positive cells / mm = Number of p21-positive cells / length of basal layer.

[0122] (statistical analysis) Test results were expressed as mean ± standard deviation. Statistical comparisons were performed using Tukey's or Dunnett's test. Significant differences were considered when the risk level was less than 5%.

[0123] [Test 1: Effect on aging skin conditions] Figure 1 shows typical skin conditions four months after the start of the study in the Young group, the Aged (normal diet) group, and the Coix Seed-treated group. The skin of the Young group was observed to have a regularly spaced texture and be moisturized. On the other hand, the skin of the Aged (normal diet) group was observed to have deep wrinkles and irregular texture, lacking moisture and being dry. Sagging due to a decrease in elasticity was also observed. The skin of the 1% and 3% Coix Seed-treated groups was moisturized, had a regular texture, and showed almost no deep wrinkles. The dryness seen in the Aged (normal diet) group was improved, and almost no sagging was observed.

[0124] Therefore, it was shown that coix seed has the effect of improving age-related skin conditions, such as wrinkles, irregular skin texture, sagging, and dryness.

[0125] [Test 2: Effect on skin moisture content due to aging] It is known that aging causes a decrease in moisturizing ingredients and a weakening of the skin barrier, leading to increased dryness of the skin. A similar phenomenon has been reported in animals. In this study, we investigated the effect of coix seed oil on age-related dry skin.

[0126] Figure 2 shows the changes in skin moisture content over time in the Young group, the Aged (normal diet) group, and the Coix Seed-treated group (Means ± SD, n = 7-8, *p < 0.05, ***p < 0.001 vs. Aged group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Young group, Tukey's test). (1) Skin moisture content in the Young group was 51.2 μS before the start of the study (month 0), 48.5 μS after one month, 49.1 μS after two months, and 47.9 μS after four months, showing a slight, though not significant, tendency to decrease. On the other hand, (2) in the Aged (normal diet) group, skin moisture content clearly decreased from 43.1 μS before the start of the study (month 0) to 35.8 μS after four months. (3) The skin moisture content of the 1% and (4) 3% coix seed administered groups showed a slight decrease in the 1% group at the third month, but in the 3% group, the condition at the start of the study was consistently maintained throughout the study period.

[0127] Therefore, it was shown that administering coix seed significantly inhibits the decrease in skin moisture content due to aging. These results suggest that coix seed can improve dry skin conditions caused by aging and / or prevent the progression of dry skin conditions caused by aging.

[0128] [Test 3: Effect on skin elasticity] Skin elasticity was measured using a Cutometer CT580MP (Courage+Khazaka, Germany) by applying a negative pressure of 300 hPa to the skin surface at the center of the back of each 4-month-old mouse for 2 seconds and then releasing it. The skin deformation during 2 seconds of negative pressure (Uf: maximum suction height; maximum extension), the amount of skin return immediately (2 seconds after release of negative pressure) (Ur: instantaneous return), and the amount of skin return within 2 seconds after release of negative pressure (Ua: final return) were measured. The instantaneous return rate of the skin relative to maximum extension (R7) and the final return rate of the skin relative to maximum extension (R2) were calculated as follows: R7=Ur / Uf R2=Ua / Uf

[0129] Figure 3 shows the R7 values ​​for the Young group, the Aged (normal diet) group, and the Coix Seed-treated group, and Figure 4 shows the R2 values ​​for each group (Means ± SD, n = 6-8, $<0.1, #p<0.05, ###p<0.001 vs. Aged group, Dunnett's test). The Aged (normal diet) group had lower R7 and R2 values ​​than the Young group, indicating a decrease in skin elasticity. On the other hand, the 1% and 3% groups had higher R7 and R2 values ​​than the Aged (normal diet) group, indicating a suppression of the decrease in skin elasticity. These results suggest that Coix Seed-treated group has the effect of suppressing the decrease in skin elasticity due to aging.

[0130] [Test 4: Effect on age-related skin thinning] For each group, HE-stained skin tissue was observed and the thickness of the epidermis, dermis, and fat layer was measured. Figure 5 shows cross-sections of the skin from the Young, Aged (normal diet), 1% and 3% diet groups. Figure 6 shows a graph comparing the thickness of the epidermis, dermis, and fat layer of each group (Means ± SD, n = 7-8, vs. Aged group, ##P < 0.01, ###P < 0.001, Tukey's test). Compared to the Young group, the Aged (normal diet) group showed approximately 1.5-fold thinner epidermis, dermis, and fat layer (skin thinning). On the other hand, the 1% and 3% groups showed thicker epidermis than the Aged (normal diet) group. In particular, the 3% group showed thicker dermis and fat layer than the Aged (normal diet) group, indicating that thinning of each layer was improved to a similar level as the Young group. These results suggest that coix seed extract has the effect of significantly suppressing skin thinning due to aging.

[0131] [Test 5: Effect on age-related changes in cell proliferation] These results suggest that Coix seed oil may have the effect of suppressing skin aging or promoting skin regeneration. Therefore, we performed immunohistochemistry of skin tissue using Ki67, a cell proliferation marker.

[0132] Figure 7 shows the number of Ki67-positive cells in the epidermis of each group. Figure 8 is a graph showing the number of Ki67-positive cells / mm in each group (Means ± SD, n = 7-8, vs. Young group, ***P < 0.001; vs. Aged group, #P < 0.05, ###P < 0.001, Tukey's test). In the epidermis of mice in the aged (normal diet) group, the number of Ki67-positive cells was approximately two-fold lower than that in the Young group. On the other hand, the number of Ki67-positive cells in the coix seed-treated group was significantly increased, and in particular, the number of positive cells in the 3% group was almost the same as that in the Young group. These results suggest that coix seed has the effect of improving the age-related decline in epidermal cell proliferation (epidermal cell regeneration), in other words, the age-related decline in epidermal turnover.

[0133] [Test 6: Effect on age-related decline in basal cell count] It is known that as we age, the regenerative ability of skin cells declines, the epidermis and dermis become thinner, and the fat layer also shrinks. It has generally been reported that basal cells in the basal layer of the epidermis decrease in aging skin. Basal cells continually divide to produce new skin cells and play a role in skin regeneration.

[0134] Therefore, to count the number of basal cells in each group of mice, we counted the number of nucleated cells in the basal layer. Nucleated cells include keratinocytes and their precursor cells, stem cells, stromal cells, pigment cells, and cells of the immune system. However, unless the tissue is inflamed, more than 90% are keratinocyte-type cells. The reason for using nucleated cells as an index is that we can measure basal cells excluding cells in the upper layer of the epidermis, where nuclei have disappeared, and because many cells have unclear boundaries between cells, counting nuclei provides the most accurate value analytically.

[0135] Figure 9 shows the nuclear staining of skin tissue in each group. Figure 10 shows the number of basal cells in each group (means ± SD, n = 7-8, vs. Young group, ***P < 0.001; vs. aged (normal diet) group, ##P < 0.05, ###P < 0.001, Tukey test). In the aged (normal diet) group, the number of nucleated basal cells was reduced to approximately 68% compared to the Young group. On the other hand, the cell number was significantly improved in the 1% and 3% coix seed administration groups. In particular, the cell number in the 3% coix seed administration group was shown to be almost the same as that in the Young group.

[0136] Therefore, it was shown that coix seed extract significantly suppresses the age-related decline in basal cell number, i.e., significantly improves the age-related decline in basal cell proliferation. Considering this together with the result that coix seed extract effectively improves the age-related decline in the number of proliferating cells in the epidermis, it was shown that coix seed extract has the effect of promoting the regeneration and / or maintenance of the keratinocyte layer that forms the epidermis by activating and promoting the proliferation of keratinocyte precursor cells or epidermal stem cells in the basal layer of the epidermis.

[0137] [Test 7: Effect on age-related decrease in stem cell marker CD271] Generally, new cells in each layer of the skin are produced by stem cells. It is known that stem cells produce keratinocyte precursor cells, which differentiate into mature keratinocytes to form the epidermal skin layers. It has been reported that the number of stem cells decreases with age. The decrease in stem cells is known to be the main cause of the decline in skin cell proliferation ability with aging. Therefore, we performed immunostaining and quantification of skin tissue using an antibody against CD271, which is specifically localized on the membrane of skin stem cells.

[0138] Figure 11 shows the results of fluorescent staining of skin tissue using CD271 antibody. Figure 12 shows the relative fluorescence intensity of CD271 in the epidermis of each group (means ± SD, n = 7–8; vs. Young group, ***P < 0.001; vs. aged (normal diet) group, ##P < 0.01, ###P < 0.001, Tukey's test). Expression of CD271 protein (green) around basal cells in the aged (normal diet) group was significantly reduced compared to the Young group. On the other hand, the 3% coix seed oil group showed a higher number of expressing cells and higher expression levels than the aged (normal diet) group. Furthermore, the aged group showed a significantly reduced relative fluorescence intensity of CD271 compared to the Young group. On the other hand, the 1% and 3% groups showed an improvement in the reduced relative fluorescence intensity of CD271.

[0139] Therefore, it was shown that coix seed has the effect of suppressing the age-related decrease in the stem cell marker CD271, suggesting that coix seed has the effect of promoting the regeneration of the epidermal layer through the activation of stem cells.

[0140] [Test 8: Effect on age-related decrease in expression of hyaluronic acid synthase HAS2] Hyaluronan plays an important role in maintaining skin moisture retention and elasticity due to its high hydrophilicity and interaction with the extracellular matrix structure. Hyaluronan synthase HAS2, which plays an important role in the synthesis of hyaluronan in the skin, is a transmembrane enzyme present across the cell membrane and has the function of synthesizing and secreting hyaluronan extracellularly using a substrate.

[0141] The expression levels of HAS2 mRNA (GAPDH expression levels) in the skin of the Young (normal diet) group, Aged (normal diet) group, and Coix seed administration group (1% group and 3% group) four months after the start of the study were measured using real-time PCR.

[0142] Figure 13 is a graph showing the expression levels of HAS2 mRNA in the skin 4 months after the start of the study for the Young (normal diet) group, the Aged (normal diet) group, and the Coix Seed Insect-treated group (Means ± SD, n = 8 to 12, vs. the Aged (normal diet) group, #P < 0.05, ##P < 0.01, ###P < 0.001, Tukey test).

[0143] The expression level of HAS2 was shown to be significantly decreased in the Aged (normal diet) group compared to the Young (normal diet) group.On the other hand, the 3% group administered 3% coix seed oil showed a significant increase in expression level compared to the Aged (normal diet) group not administered coix seed oil.

[0144] Therefore, it was suggested that by suppressing the age-related decrease in expression of hyaluronan synthase, coix seed oil can maintain the amount of hyaluronan in the skin, thereby contributing to the maintenance of skin moisture retention, elasticity, and structural integrity.

[0145] [Test 9: Effect on decrease in collagen area due to aging] Collagen is important for maintaining the structural stability and elasticity of the skin, preventing the progression of sagging and wrinkles associated with aging, and also plays an important role in maintaining skin firmness and elasticity.

[0146] Skin tissue from each group was stained using Masson trichrome staining four months after the start of the study, and the collagen area ratio in the skin tissue was calculated from microscopic images. Figure 14 shows the results of Masson trichrome staining of skin tissue from the Young (normal diet) group, the Aged (normal diet) group, and the 3% coix seed treatment group. Figure 15 is a graph showing the collagen area ratio in skin tissue from the Young (normal diet) group, the Aged (normal diet) group, and the coix seed treatment groups (1% and 3%) (Means ± SD, n = 8-12, vs. the Aged (normal diet) group, #P < 0.05, ##P < 0.01, ###P < 0.001, Tukey's test).

[0147] It was shown that the collagen area ratio in skin tissue significantly decreased with age in the Aged (normal diet) group compared to the Young (normal diet) group.It was also shown that the collagen area ratio significantly increased in the Coix Seeds Group compared to the Aged (normal diet) group.

[0148] Therefore, it was suggested that by inhibiting the decrease in collagen in skin tissue due to aging, coix seed oil can maintain the structural stability and elasticity of the skin, contributing to the prevention of skin aging and the maintenance of healthy skin function.

[0149] [Test 10: Effect on accumulation of senescent cells due to aging] It has been reported that with aging, cells exhibiting cellular senescence (senescent cells) accumulate in skin tissue. While irreversibly arresting the cell cycle, senescent cells continuously secrete senescence-associated secretory phenotype (SASP) factors such as inflammatory cytokines, causing chronic inflammatory responses and adversely affecting surrounding cells. It has also been reported that the accumulation of senescent cells accelerates skin aging.

[0150] Senescent cells are characterized by increased expression of cell cycle inhibitors such as p16INK4A and p21WAF1, increased senescence-associated β-galactosidase activity, and decreased expression of nuclear HMGB1 and lamin B1. These factors are used as markers of cellular senescence.

[0151] In this study, skin tissue from each group was immunostained 4 months after the start of the study using p21, a marker of cellular senescence. In the stained images for each group, p21-positive cells were counted in the epidermis, papillary dermis, and full-thickness dermis, and the number of positive cells per unit length was calculated. Figure 16 shows the results of immunohistochemical staining for the cellular senescence marker p21 in skin tissue from the Young (normal diet) group, the Aged (normal diet; NT) group, and the 3% coix seed treatment group. Figure 17 is a graph showing the number of p21-positive cells in the epidermis, papillary dermis, and full-thickness dermis (Means ± SD, n = 8–12, vs. the Aged (normal diet) group, #P < 0.05, ##P < 0.01, ###P < 0.001, Tukey's test).

[0152] Compared with the Young (normal diet) group, the Aged (normal diet) group showed a significant increase in the number of p21-positive cells in the epidermis and dermis due to aging. On the other hand, the Yokuinin-administered group showed a significant decrease in the number of p21-positive cells compared with the Aged (normal diet) group.

[0153] Therefore, it was suggested that by suppressing the accumulation of senescent cells due to aging, coix seed oil may contribute to improving age-related skin thinning, loss of skin elasticity, and dry skin. [Industrial Applicability]

[0154] The present invention can be suitably used in pharmaceuticals, cosmetics, foods, and the like for treating, improving, and / or preventing skin aging.

Claims

1. A composition for treating, improving and / or preventing skin aging, comprising Job's tears as an active ingredient.

2. 2. The composition according to claim 1, wherein the skin aging is at least one selected from the group consisting of age-related progression of dry skin, decreased skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, thinning of the skin, and decreased skin regeneration ability.

3. 3. The composition according to claim 1, wherein the Job's tears are seeds from which the husk and astringent skin have been removed.

4. A composition for treating, improving and / or preventing skin aging, comprising an extract of Job's tears as an active ingredient.

5. 5. The composition according to claim 4, wherein the skin aging is at least one selected from the group consisting of age-related progression of dry skin, decreased skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, thinning of the skin, and decreased skin regeneration ability.

6. 6. The composition according to claim 4, wherein the Job's tears are seeds from which the husk and astringent skin have been removed.

7. A food or feed composition for improving and / or preventing skin aging, which contains Job's tears as an active ingredient.

8. 8. The food or feed composition according to claim 7, wherein the skin aging is at least one selected from the group consisting of age-related progression of dry skin, decreased skin moisture content, increased wrinkles, increased sagging, decreased skin elasticity, thinning of the skin, and decreased skin regeneration ability.

9. 9. The food or feed composition according to claim 7, wherein the Job's tears are seeds from which the husk and astringent skin have been removed.

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