Anti-photoaging agent for the skin and agent for preventing and improving skin aging using the same

Abstract

To provide an anti-photo aging agent with a novel component as an effective component.SOLUTION: Features of the present invention for solving the above problems are as follows. 1. A cell atrophy inhibitor due to UVA irradiation on fibroblast cells with a Psoralea extraction as an effective component. 2. A cell atrophy inhibitor due to UVA irradiation in a fibroblast cells with bakuchiol as an effective component. 3. A collagen gel shrinkability increasing agent with the Psoralea extraction as an effective component. 4. A collagen gel shrinkability increasing agent with bakuchiol as an effective component. 5. An integrin α1 increasing agent with the Psoralea extraction as an effective component. 6. An integrin α1 increasing agent with bakuchiol as an effective component. 7. an anti-photo aging agent with a preparation described in any one of 1. to 6.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an anti-photoaging agent and an agent for preventing and improving skin aging using the same. The present invention is widely used in foods (including health foods such as functional foods and foods for specified health uses), medicines, quasi-drugs, cosmetics, skin care products, etc. [Background technology]

[0002] Aging is biologically the change that occurs in an individual organism over time, and in particular the functional decline and process that occurs until the organism dies. In particular, since the skin is exposed to ultraviolet light, which is an external environment, photoaging, which is an environmental factor, inevitably occurs simultaneously in addition to physiological aging. Physiological aging is skin aging that occurs with age, and is characterized by fine wrinkles and dryness to the naked eye. The skin becomes thinner, metabolism slows, and the skin turnover rate slows. This is due to a decrease in the amount of collagen in the dermis and a decrease in the elasticity of elastin, an elastic fiber. Proteins such as collagen and elastin combine with active sugars to cause the Maillard reaction (glycation reaction), which reduces the moisture retention capacity and skin elasticity. In addition, the barrier function of the epidermis decreases due to a decrease in sweat and natural moisturizing factors. This reduces the moisture content of the stratum corneum, leading to dryness. Photoaging is a change in the skin caused mainly by ultraviolet rays, and is characterized by deeper wrinkles compared to physiological aging. This is due to changes in the epidermal cells themselves, degeneration of elastic fibers, and peeling due to reduced adhesion between the epidermis and dermis. The dermis layer is composed of extracellular matrix (collagen, elastin, hyaluronic acid, etc.) and its producers, fibroblasts. It has been confirmed that these substances are reduced or disappeared by ultraviolet rays (UVA) that reach the dermis layer. Pigmentation, commonly known as blemishes, is also mainly caused by melanin pigments settling in the epidermis due to ultraviolet rays.

[0003] Furthermore, there are two types of wrinkles: deep wrinkles that originate in the dermis and shallow wrinkles that originate in the epidermis. Of these, deep wrinkles and sagging skin originating from the dermis are mostly caused by the influence of the extracellular matrix and fibroblasts in the dermis. Furthermore, it has been reported that UVA irradiation of fibroblasts causes the cytoskeleton to shrink, which in turn causes cell inactivation (reduced collagen production and activation of MMP1 (collagen-degrading enzyme)) (Non-Patent Document 1). This shrinkage of the cytoskeleton is said to be one of the causes of deep wrinkles originating from the dermis.

[0004] The dermis is mainly composed of collagen. This collagen is produced by fibroblasts, which are strongly bound to each other within the dermis. In this case, a protein present on the surface of the cell membrane called integrin functions as an adhesive between cells and collagen. It has been reported that integrin adheres not only to collagen but also to laminin 5 and elastin, which will be described later. It has been reported that this integrin decreases when irradiated with UVA, weakening the binding between cells and extracellular matrices such as collagen (Non-Patent Document 2). As a result, this causes sagging skin and deep wrinkles originating from the dermis. In addition, when fibroblasts are cultured in collagen gel, contraction of the collagen gel is observed. However, it has been reported that UVA irradiation reduces the contractile ability, which is a phenomenon caused by the aforementioned reduction in adhesive ability. Therefore, in order to suppress the aforementioned reduction in adhesive ability, it is necessary to suppress the reduction in collagen gel contractile ability or to increase the contractile ability.

[0005] Moreover, under normal fibroblast culture conditions, when fibroblasts are embedded and cultured in collagen gel, the collagen gel shrinks (Non-Patent Document 3). Collagen gel shrinkage varies depending on the number of cells and the amount of serum in the medium, and becomes more pronounced the greater the number of fibroblasts in the gel and the amount of serum in the medium. However, such shrinkage does not occur at all in gels made of collagen alone without fibroblasts or in gels embedded with floating cells such as lymphocytes.

[0006] Furthermore, with regard to skin aging, it is known that fibroblasts derived from older people have a lower contraction rate of collagen gel compared to cells derived from younger people, and it has been revealed that aging reduces the contractile ability of the gel (Non-Patent Document 4). In other words, a decrease in the contractile ability of collagen gel indicates that the dermal connective tissue of the skin loses its contractile force, strength, and elasticity, resulting in deep wrinkles and sagging derived from the dermis. Therefore, if the contractile ability of the collagen gel in which fibroblasts are embedded can be increased and the strength of the gel formed can be increased, the dermal connective tissue can be contracted, tightened, and its strength can be increased, which in turn can suppress the formation of sagging skin and deep wrinkles derived from the dermis due to aging, improve the elasticity and firmness of the skin that have decreased, and tighten the skin (Patent Document 1).

[0007] In addition, wrinkles and sagging skin originating from the dermis are caused by a decrease or degeneration of the extracellular matrix in the dermis. Elastin is formed by the polymerization of fibrillin 1, tropoelastin, fibrin 4, 5, etc., which are produced by fibroblasts. In addition, fibulin 4 is known to act like a stake, anchoring fibrillin and tropoelastin, which act as scaffolds in the formation of elastic fibers. By promoting the expression of fibulin 4, deep wrinkles originating from the dermis can be improved.

[0008] Furthermore, the epidermis and dermis are in close contact with each other through the epidermal basement membrane structure. The reason why the epidermis and dermis do not separate even when the skin is rubbed is because the epidermal basement membrane functions normally (Non-Patent Document 5). When this basement membrane structure is weakened, the adhesion between the epidermis and dermis weakens, which causes shallow wrinkles caused by the epidermis. The protein that acts as this adhesive is laminin 5. By promoting the expression of laminin 5, the epidermal basement membrane can function normally, and shallow wrinkles caused by the epidermis can be improved.

[0009] [Non-Patent Document 1] Yamada H., et al, Experimental dermatology, 25 (Suppl. 3), P45-51 (2016). [Non-Patent Document 2] Wongnapa N., et al, Photochemistry and Photobiology, 94(4) (2018). [Non-Patent Document 3] E. Bell et al. J. Invest. Dermatol.,81,2s(1983) [Non-Patent Document 4] M. Yamato, et al. Mech. Aging Dev.67,149(1993) [Non-Patent Document 5] Hiroshi Shimizu, New Dermatology, 2nd Edition, Nakayama Shoten [Patent Document 1] JP 2014-34567 A DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0010] Against this background, the present inventors evaluated the inhibitory effect of Hollandia extract and its main component, bakuchiol, on cell atrophy caused by UVA irradiation in fibroblasts, and found that they have the effect of inhibiting cell atrophy of fibroblasts, thereby completing the present invention. In addition, a 3D model of the dermis was created by culturing fibroblasts in collagen gel and evaluated in vitro. As a result, it was found that Dutch ragwort and bakuchiol have the effect of enhancing the contractile ability of collagen gel, thereby completing the present invention. Furthermore, as a result of evaluating the amount of integrin protein in fibroblasts, it was found that the compound has the effect of increasing integrin α1, thereby completing the invention. In addition, the expression level of fibulin 4 gene was evaluated using fibroblasts (TIG-103) derived from elderly people, and the expression level of laminin 5 was evaluated using normal human epidermal keratinocytes (NHEK) cells. As a result, it was found that the compound has an effect of promoting the expression of these genes, thereby completing the present invention. In other words, the present invention aims to provide an agent for inhibiting cell atrophy caused by UVA irradiation in fibroblasts, an agent for increasing collagen gel contractility, and an agent for increasing integrin α1, each of which contains a new active ingredient, as well as an anti-photoaging agent using these. Additionally, it is possible to provide a fibulin 4 gene expression promoter and a laminin 5 expression promoter that contain novel active ingredients, thereby aiming to provide a skin aging prevention / improvement agent that suppresses the formation of both deep wrinkles derived from the dermis and shallow wrinkles derived from the epidermis. [Means for solving the problem]

[0011] The features of the present invention for solving the above problems are as follows. 1. An agent that inhibits cell shrinkage caused by UVA irradiation in fibroblasts, containing extract of Hollandia as the active ingredient. 2. An agent containing bactiol as the active ingredient that inhibits cell shrinkage caused by UVA irradiation in fibroblasts. 3. A collagen gel contraction ability enhancer containing Hollandia extract as an active ingredient. 4. A collagen gel contraction enhancer containing bactiol as an active ingredient. 5. An integrin α1 increaser containing Hollandia extract as the active ingredient. 6. An integrin α1 increaser whose active ingredient is bactiol. 7. An anti-photoaging agent comprising any one of the agents described in 1. to 6. above as an active ingredient. 8. A fibrin 4 gene expression promoter containing Hollandia extract as an active ingredient. 9. A laminin 5 expression promoter containing Hollandia extract as the active ingredient. 10. A laminin 5 expression promoter containing bactiol as the active ingredient. 11. Hyaluronidase inhibitor containing Hollandia extract as the active ingredient. 12. A wrinkle improvement agent containing at least one of Hollandia extract and bakuchiol as an active ingredient. 13. The wrinkle improving agent according to claim 12, characterized in that the wrinkle improving agent improves both deep wrinkles originating from the dermis and shallow wrinkles originating from the epidermis. 14. The wrinkle improving agent according to claim 12, which is for oral administration or topical application. 15. An oral wrinkle improving agent comprising the agent according to 12. or 13. above. 16. A skin aging prevention / improvement agent containing as an active ingredient any one of the agents described in 1. to 14. above. 17. A non-polar solvent extract of Dutch holly, characterized in that the non-polar solvent extract of Dutch holly has been removed of psoralens. Effect of the Invention

[0012] According to the present invention, the extract of Hollandia japonica and bakuchiol have an inhibitory effect on cell shrinkage caused by UVA irradiation in fibroblasts. This suggests that Hollandia extract and bakuchiol are expected to suppress cell inactivation (e.g., decreased collagen production and activation of MMP1 (collagen-degrading enzyme)) associated with cytoskeleton atrophy. As a result, Hollandia extract and bakuchiol can be used as anti-photoaging agents, and in particular suppress the formation of deep wrinkles derived from the dermis caused by ultraviolet rays. Therefore, they are useful as agents for preventing and improving skin aging. In addition, according to the present invention, the extract of Hollandia japonica and bakuchiol have an effect of increasing integrin α1, which functions as an adhesive between cells and collagen. This can prevent the binding between cells and extracellular matrix such as collagen from weakening, and can increase collagen gel contraction. Therefore, it suppresses the decrease in collagen gel contraction caused by UVA irradiation. As a result, it can prevent the formation of sagging skin and deep wrinkles derived from the dermis, so it can be used as an anti-photoaging agent and is useful as an agent for preventing and improving skin aging. Furthermore, according to the present invention, the Hollandia extract has an effect of promoting the expression of fibulin 4, which is a protein important in the production of elastin and plays an important role in the formation of the extracellular matrix in the dermis layer, and which acts like a stake by connecting fibrillin and tropoelastin, which serve as scaffolds in the formation of elastic fibers, and can effectively prevent the reduction and degeneration of the extracellular matrix in the dermis layer. As a result, the Hollandia extract can improve deep wrinkles originating from the dermis, and is therefore useful as an agent for preventing and improving skin aging. In addition, Hollandia extract and bakuchiol promote the expression of laminin 5, which keeps the epidermal basement membrane structure that adheres the dermis to the epidermis functioning normally. This improves the adhesive function between the epidermis and dermis, improving sagging skin and shallow wrinkles caused by the epidermis. Therefore, they are useful as skin aging prevention and improvement agents. Furthermore, according to the present invention, the Hollandia extract has a hyaluronidase inhibitory effect and is therefore useful as an agent for preventing and improving skin aging. [Brief description of the drawings]

[0013] [Figure 1] This is a micrograph (fluorescent immunostaining of β-actin) showing the state of cellular shrinkage of normal diploid fibroblasts (NB1RGB cells) due to UVA irradiation. [Diagram 2] 1 shows micrographs (fluorescent immunostaining of β-actin, 100x) and graphs illustrating the evaluation results of the inhibitory effect of UVA irradiation on cell shrinkage. [Diagram 3] 1 shows micrographs (fluorescent immunostaining of β-actin, 400x) and graphs illustrating the evaluation results of the inhibitory effect of UVA irradiation on cell shrinkage. [Figure 4] 1 is a graph showing the effect of Hollandia extract and bakuchiol on collagen gel area value in a 3D dermis model. [Diagram 5] 1 is a graph showing the effect of Hollandia extract and bakuchiol on integrin α1 (fluorescence intensity of fluorescent immunostaining) in a 3D dermis model. [Figure 6]1 is a graph showing the results of evaluating the fibulin 4 gene expression promoting effect of an extract from Hollandia japonica. [Figure 7] 1 is a graph showing the results of evaluating the laminin 5 expression promoting effect of Hollandia extract and bakuchiol. [Figure 8] 1 is a graph showing the evaluation results of the hyaluronidase inhibitory effect of Hollandia japonica extract. [Figure 9] FIG. 1 shows a comparison of the HPLC charts of the secondary extract and the Hollandia extract of this example. [Figure 10] Photographs to explain wrinkle grades. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in detail below. The present invention is characterized in that it contains an extract of Dutch pipiens (Psoralea corylifolia L.) as an active ingredient. Psoralea corylifolia L. is an annual herb of the legume family, covered entirely with yellowish-white hairs and black-brown glandular dots. It grows in warm, humid climates, and is primarily grown in Sichuan and Henan (China). Its fruits (seeds) are wrapped in a pericarp and have a unique fragrance. The main component of the seeds has been reported to be bakuchiol, a monoterpene phenol derivative. The part of the hollyhock used in the present invention is not particularly limited, and for example, the fruit, seeds, leaves, etc. can be used, but it is preferable to use the fruit.

[0015] The extraction solvent used in producing the extract may be a non-polar solvent such as hexane, acetone, ethyl acetate, etc. Two or more of these solvents may be mixed. Preferably, hexane is used as the extraction solvent to efficiently extract the active ingredients.

[0016] The extraction temperature is preferably 20 to 80° C., and more preferably about 20 to 50° C. If the extraction temperature is too low, the active ingredients are difficult to extract, whereas if the extraction temperature is too high, the active ingredients decompose, resulting in a decrease in physiological activity (health functionality).

[0017] As the extraction method, any method such as stirring extraction, continuous extraction, immersion extraction, countercurrent extraction, and supercritical extraction can be adopted, and any apparatus can be used at room temperature or under reflux heating.

[0018] The specific extraction method is to put the raw material (such as the fruit of the Dutch pipiens) into a treatment tank filled with an extraction solvent, and dissolve the active ingredients while stirring. For example, when using hexane as the extraction solvent, about 2 to 100 times (by weight) the amount of extraction solvent as the extraction raw material is used, and extraction is carried out for about 30 minutes to 2 hours. After the active ingredients are dissolved in the solvent, the extract is obtained by filtering to remove the extraction residue.

[0019] Thereafter, the extract is subjected to treatments such as dilution, concentration, purification, drying, etc. according to conventional methods to obtain the skin aging prevention / improvement agent according to the present invention.

[0020] It is preferable to use the above-mentioned Hollandia extract from which coumarins such as psoralen and isopsoralen have been removed. This is because they have cytotoxicity against fibroblasts, and therefore a more excellent anti-photoaging agent can be obtained by not including these. The method for removing coumarins from the Hollandia extract is not particularly limited, and can be carried out by known methods such as a method using an adsorbent (e.g., JP-A-10-316991, JP-A-57-005794, JP-A-56-070096, JP-A-63-089594, etc.).

[0021] The present invention is also characterized in that it contains bakuchiol as an active ingredient. Bactiol is a compound represented by the following chemical formula (1). [ka]

[0022] The method for obtaining bakuchiol is not particularly limited, but it can be obtained from Psoralea corylifolia L. That is, it can be obtained by subjecting Psoralea corylifolia L. extract to treatments such as concentration, purification, and drying. Specifically, it is preferable to obtain it by the method described in the examples of the present specification.

[0023] The skin aging prevention and improvement agent of the present invention can be used as an ingredient in various foods and beverages. Examples of foods and beverages include general foods such as confectionery (gum, candy, caramel, chocolate, cookies, snacks, jellies, gummies, tablet candies, etc.), noodles (soba, udon, ramen, etc.), dairy products (milk, ice cream, yogurt, etc.), seasonings (miso, soy sauce, etc.), soups, beverages (juice, coffee, black tea, tea, carbonated drinks, sports drinks, etc.), health foods (tablets, capsules, etc.), and nutritional supplements (nutritional drinks, etc.). The skin aging prevention and improvement agent of the present invention may be appropriately blended into these foods and beverages.

[0024] These foods and beverages can contain various ingredients depending on their type, and the following food ingredients can be used: glucose, fructose, sucrose, maltose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid esters, polyglycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, gum arabic, carrageenan, casein, gelatin, pectin, agar, B vitamins, nicotinamide, calcium pantothenate, amino acids, calcium salts, colorants, flavorings, and preservatives.

[0025] As a specific manufacturing method, the skin aging prevention / improvement agent is spray-dried or freeze-dried together with powdered cellulose, and then made into a powder, granules, tablet, or solution, which can be easily added to food and beverages (instant foods, etc.). The skin redness improvement agent can also be dissolved in, for example, oils and fats, ethanol, glycerin, or a mixture thereof to make a liquid, which can be added to beverages or solid foods. If necessary, it can also be mixed with a binder such as gum arabic or dextrin to make a powder or granule, which can be added to beverages or solid foods.

[0026] When the skin aging prevention / improvement agent of the present invention is applied to food and beverages, the amount of the active ingredient added is preferably 1 to 20 wt% in total relative to the food and beverages, since the main purpose is disease prevention and health maintenance.

[0027] The skin aging prevention / improvement agent of the present invention may be used as a material for medicines (including pharmaceuticals and quasi-drugs). The skin aging prevention / improvement agent of the present invention can be appropriately mixed with raw materials for pharmaceutical preparations to produce the agent. Examples of raw materials for preparations that can be mixed with the skin aging prevention / improvement agent of the present invention include excipients (glucose, lactose, sucrose, sodium chloride, starch, calcium carbonate, kaolin, crystalline cellulose, cacao butter, hardened vegetable oil, kaolin, talc, etc.), binders (distilled water, physiological saline, ethanol water, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.), disintegrants (sodium alginate, agar, charcoal, etc.), and the like. Examples of suitable additives include disintegration inhibitors (sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearate monoglyceride, starch, lactose, powdered acacia, gelatin, ethanol, etc.), disintegration inhibitors (white sugar, stearin, cacao butter, hydrogenated oils, etc.), absorption enhancers (quaternary ammonium bases, sodium lauryl sulfate, etc.), adsorbents (glycerin, starch, lactose, kaolin, bentonite, silicic acid, etc.), lubricants (purified talc, stearates, polyethylene glycol, etc.).

[0028] The method of administration of the skin aging prevention and improvement agent of the present invention can be generally orally administered in the form of tablets, pills, soft or hard capsules, fine granules, powders, granules, liquids, etc., but may also be parenterally administered. When administered as a parenteral agent, it can be administered in the form of a solution or a dispersant, suspending agent, stabilizer, etc., by local tissue administration, intradermal, subcutaneous, intramuscular, or intravenous injection. It may also be in the form of a suppository, etc. Furthermore, it can be administered as an eye drop.

[0029] The dosage may vary depending on the administration method, the condition, the age of the patient, etc., but typically, for adults, 0.5 to 5000 mg of the active ingredient can be administered per day, and for children, 0.5 to 3000 mg. The blending ratio of the skin aging prevention / improvement agent can be changed appropriately depending on the dosage form, but it is usually about 0.3 to 15.0 wt% when administered orally or via mucosal absorption, and about 0.01 to 10 wt% when administered parenterally. Note that the dosage varies depending on various conditions, so in some cases a smaller dosage than the above is sufficient, and in other cases it is necessary to administer more than the range.

[0030] The skin aging prevention / improvement agent of the present invention can be expected to have an anti-aging / improvement effect even when used as an external skin preparation (including cosmetics, medicines and quasi-drugs). Examples of the forms of external skin preparations that can be blended with the skin aging prevention / improvement agent of the present invention include milky lotion, soap, facial cleanser, bath additive, cream, milky lotion, skin lotion, cologne, shaving cream, shaving lotion, cosmetic oil, suntan / sunscreen lotion, face powder, foundation, perfume, pack, nail cream, enamel, enamel remover, eyebrow ink, blusher, eye cream, eye shadow, mascara, eyeliner, lipstick, lip balm, shampoo, rinse, hair dye, dispersion, cleanser, etc. Examples of the forms of medicines or quasi-drugs that can be blended with the skin aging prevention / improvement agent of the present invention include ointments, creams, external liquids, etc.

[0031] In addition to the skin aging prevention / improvement agent of the present invention, the skin topical preparation in the above form may contain ingredients that are incorporated into skin topical preparations such as cosmetics and quasi-drugs, as long as the skin aging prevention / improvement effect is not impaired, such as oils, higher alcohols, fatty acids, ultraviolet absorbers, powders, pigments, surfactants, polyhydric alcohols / sugars, polymers, physiologically active ingredients, solvents, antioxidants, fragrances, preservatives, etc. Examples are listed below, but the present invention is not limited to these examples.

[0032] (1) Example of oil Ester-based oil phase components: glyceryl tri-2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, butyl stearate, butyl myristate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, diisopropyl adipate isopropyl, isoarachyl neopentanoate, tri(caprylic / capric)glyceryl, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, pentaerythritol tetra-2-ethylhexanoate, cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinoleate, isostearyl laurate, isotridecyl myristate, isocetyl myristate, isostearate Tearyl, isocetyl palmitate, isostearyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, cetostearyl 2-ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprate, propylene glycol dicaprylate, propylene glycol dicaprylate, dicaprin Neopentyl glycol acid, neopentyl glycol dioctanoate, glyceryl tricaprylate, glyceryl triundecylate, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerin oleate, polyglycerin isostearate, dipropyl carbonate,Dialkyl carbonate (C12-18), triisocetyl citrate, triisoarachyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malate, 2-ethylhexyl hydroxystearate, di-2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate propyl, dioctyl sebacate, cholesteryl stearate, cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, phytosteryl isostearate, phytosteryl oleate, isocetyl 12-stearoylhydroxystearate, stearyl 12-stearoylhydroxystearate, isostearyl 12-stearoylhydroxystearate, and the like. Hydrocarbon-based oil phase components: squalane, liquid paraffin, α-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybutene, microcrystalline wax, petrolatum, etc. Animal and vegetable oils and their hydrogenated oils, and naturally derived waxes: beef tallow, hydrogenated beef tallow, lard, hydrogenated lard, horse oil, hydrogenated horse oil, mink oil, orange roughy oil, fish oil, hydrogenated fish oil, egg yolk oil, and other animal oils and their hydrogenated oils, avocado oil, almond oil, olive oil, cocoa butter, kiwi seed oil, apricot kernel oil, kukui nut oil, sesame oil, wheat germ oil, rice germ oil, rice bran oil, safflower oil, shea butter, soybean oil, evening primrose oil, perilla oil, tea seed oil, camellia oil, corn oil, rapeseed oil, Examples of such oils include vegetable oils and their hydrogenated oils such as hydrogenated rapeseed oil, palm kernel oil, hydrogenated palm kernel oil, palm oil, hydrogenated palm oil, peanut oil, hydrogenated peanut oil, castor oil, hydrogenated castor oil, sunflower oil, grape seed oil, jojoba oil, hydrogenated jojoba oil, macadamia nut oil, medicamentoil, cottonseed oil, hydrogenated cottonseed oil, coconut oil, and hydrogenated coconut oil; and waxes such as beeswax, high acid value beeswax, lanolin, reduced lanolin, hydrogenated lanolin, liquid lanolin, carnauba wax, and montan wax. Silicone-based oil phase components: dimethylpolysiloxane, methylphenylpolysiloxane, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, methylhydrogenpolysiloxane, polyether-modified organopolysiloxane, dimethylsiloxane-methylcetyloxysiloxane copolymer, dimethylsiloxane-methylstearoxysiloxane copolymer, alkyl-modified organopolysiloxane, terminal-modified organopolysiloxane, amino-modified silicone oil, amino-modified organopolysiloxane, dimethiconol, silicone gel, acrylic silicone, trimethylsiloxysilicate, silicone RTV rubber, etc. Fluorine-based oil phase components: Perfluoropolyether, fluorine-modified organopolysiloxane, fluorinated pitch, fluorocarbon, fluoroalcohol, fluoroalkyl-polyoxyalkylene-co-modified organopolysiloxane, etc.

[0033] (2) Examples of higher alcohols Examples of the alcohol include lauryl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, isostearyl alcohol, oleyl alcohol, behenyl alcohol, 2-ethylhexanol, hexadecyl alcohol, and octyldodecanol.

[0034] (3) Examples of fatty acids Examples of the acid include caprylic acid, capric acid, undecylenic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, erucic acid, and 2-ethylhexanoic acid.

[0035] (4) Examples of UV absorbers Para-aminobenzoic acid, amyl para-aminobenzoate, dihydroxypropyl ethyl para-aminobenzoate, glyceryl para-aminobenzoate, ethyl para-aminobenzoate, octyl para-aminobenzoate, octyldimethyl para-aminobenzoate, ethylene glycol salicylate, octyl salicylate, triethanolamine salicylate, phenyl salicylate, butylphenyl salicylate, benzyl salicylate, homomenthyl salicylate, benzyl cinnamate, octyl para-methoxycinnamate, 2-ethylhexyl para-methoxycinnamate, glyceryl di-para-methoxycinnamate mono-2-ethylhexanoate, isopropyl para-methoxycinnamate, diethanolamine salt of para-methoxyhydrocinnamate, mixture of diisopropyl and diisopropyl cinnamate, urocanic acid, ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and its salts, di Hydroxymethoxybenzophenone, dihydroxymethoxybenzophenone sodium disulfonate, dihydroxybenzophenone, dihydroxydimethoxybenzophenone, hydroxyoctoxybenzophenone, tetrahydroxybenzophenone, butylmethoxydibenzoylmethane, 2,4,6-trianilino-p-(carbo-2-ethylhexyl-1-oxy)-1,3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzotriazole, methyl-O-aminobenzoate, 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, phenylbenzimidazole sulfate, 3-(4-methylbenzylidene)camphor, isopropyldibenzoylmethane, 4-(3,4-dimethoxyphenylmethylene)-2,5-dioxo-1-imidazolidinepropionate 2-ethylhexyl, and the like, as well as polymer derivatives and silane derivatives thereof.

[0036] (5) Examples of powders and pigments Dyes such as Red No. 104, Red No. 201, Yellow No. 4, Blue No. 1, Black No. 401, lake dyes such as Yellow No. 4 AL Lake, Yellow No. 203 BA Lake, nylon powder, silk powder, urethane powder, Teflon (registered trademark) powder, silicone powder, polymethyl methacrylate powder, cellulose powder, starch, silicone elastomer spherical powder, polyethylene powder and other polymers, yellow iron oxide, red iron oxide, black iron oxide, chromium oxide, carbon black, ultramarine, Prussian blue and other colored pigments, zinc oxide, titanium oxide, acid Examples of such pigments include white pigments such as cerium chloride, extender pigments such as talc, mica, sericite, kaolin, and plate-like barium sulfate, pearl pigments such as titanium mica, metal salts such as barium sulfate, calcium carbonate, magnesium carbonate, aluminum silicate, and magnesium silicate, inorganic powders such as silica and alumina, metal soaps such as aluminum stearate, magnesium stearate, zinc palmitate, zinc myristate, magnesium myristate, zinc laurate, and zinc undecylenate, bentonite, smectite, and boron nitride. There are no particular limitations on the shape (spherical, rod-like, needle-like, plate-like, irregular, flaky, spindle-like, etc.) and particle size of these powders. These powders may or may not have been previously surface-treated by a conventionally known surface treatment, such as a fluorine compound treatment, a silicone treatment, a silicone resin treatment, a pendant treatment, a silane coupling agent treatment, a titanium coupling agent treatment, an oil treatment, an N-acylated lysine treatment, a polyacrylic acid treatment, a metal soap treatment, an amino acid treatment, a lecithin treatment, an inorganic compound treatment, a plasma treatment, or a mechanochemical treatment.

[0037] (6) Examples of surfactants Anionic surfactants: fatty acid soaps, α-acylsulfonates, alkylsulfonates, alkylarylsulfonates, alkylnaphthalenesulfonates, alkyl sulfates, POE alkyl ether sulfates, alkylamide sulfates, alkyl phosphates, POE alkyl phosphates, alkylamide phosphates, alkyloylalkyl taurine salts, N-acylamino acid salts, POE alkyl ether carboxylates, alkylsulfosuccinates, sodium alkylsulfoacetates, acylated hydrolyzed collagen peptide salts, perfluoroalkyl phosphate esters, and the like. Cationic surfactants: alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide, cetostearyltrimethylammonium chloride, distearyldimethylammonium chloride, stearyldimethylbenzylammonium chloride, behenyltrimethylammonium bromide, benzalkonium chloride, behenic acid amidopropyldimethylhydroxypropylammonium chloride, stearate diethylaminoethylamide, stearate dimethylaminopropylamide, lanolin derivative quaternary ammonium salts, etc. Amphoteric surfactants: Examples of amphoteric surfactants include carboxybetaine type, amidobetaine type, sulfobetaine type, hydroxysulfobetaine type, amidosulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type, and amidoamine type. Nonionic surfactants: Propylene glycol fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, sorbitan fatty acid esters, POE sorbitan fatty acid esters, POE sorbit fatty acid esters, POE glycerin fatty acid esters, POE alkyl ethers, POE fatty acid esters, POE hydrogenated castor oil, POE castor oil, POE·POP copolymers, POE·POP alkyl ethers, polyether modified silicone lauric acid alkanolamide, alkylamine oxide, hydrogenated soybean phospholipids, etc. Natural surfactants: Examples include lecithin, saponin, sugar-based surfactants, etc.

[0038] (7) Examples of polyhydric alcohols and sugars Examples of suitable sugars include ethylene glycol, diethylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol, polypropylene glycol, glycerin, diglycerin, polyglycerin, 3-methyl-1,3-butanediol, 1,3-butylene glycol, sorbitol, mannitol, raffinose, erythritol, glucose, sucrose, fructose, xylitol, lactose, maltose, maltitol, trehalose, alkylated trehalose, mixed isomerized sugar, sulfated trehalose, pullulan, etc. Chemically modified versions of these sugars may also be used.

[0039] (8) Examples of polymers Acrylic acid ester / methacrylic acid ester copolymer (Plussize, manufactured by GOO Chemical Co., Ltd.), vinyl acetate / crotonic acid copolymer (Resin 28-1310, manufactured by NSC Co., Ltd.), vinyl acetate / crotonic acid / vinyl neodecanoate copolymer (28-2930, manufactured by NSC Co., Ltd.), methyl vinyl ether maleic acid half ester (Gantrez ES, manufactured by ISP Co., Ltd.), t-butyl acrylate / ethyl acrylate / methacrylic acid copolymer (Rubymer, manufactured by BASF Co., Ltd.), vinyl pyrrolidone / vinyl acetate / vinyl propionate copolymer (Ruviscol VAP, manufactured by BASF Co., Ltd.), vinyl acetate Vinyl acetate / crotonic acid copolymer (Rubyset CA, BASF), vinyl acetate / crotonic acid / vinyl pyrrolidone copolymer (Rubyset CAP, BASF), vinyl pyrrolidone / acrylate copolymer (Rubyflex, BASF), acrylate / acrylamide copolymer (Ultrahold, BASF), vinyl acetate / butyl maleate / isobornyl acrylate copolymer (Advantage, ISP), carboxyvinyl polymer (Carbopol, BFGoodrich), acrylic acid / alkyl methacrylate copolymer (Pemulen, BF Examples of such polymers include anionic polymers such as acetic acid amphoteric product of dialkylaminoethyl methacrylate polymer (Yukaformer, manufactured by Mitsubishi Chemical Corporation) and amphoteric polymers such as octylacrylamide acrylate / hydroxypropyl acrylate / butylaminoethyl methacrylate copolymer (AMPHOMER, manufactured by NSC Corporation); cationic polymers such as quaternary product of vinylpyrrolidone / dimethylaminoethyl methacrylate (GAFQUAT, manufactured by ISP Corporation) and methylvinylimidazolium chloride / vinylpyrrolidone copolymer (Rubycoat, manufactured by BASF Corporation); and nonionic polymers such as polyvinylpyrrolidone (Ruviscol K, manufactured by BASF Corporation), vinylpyrrolidone / vinyl acetate copolymer (Ruviscol VA, manufactured by BASF Corporation), vinylpyrrolidone / dimethylaminoethyl methacrylate copolymer (copolymer 937, manufactured by ISP Corporation), and vinylcaprolactam / vinylpyrrolidone / dimethylaminoethyl methacrylate copolymer (copolymer VC713, manufactured by ISP Corporation).Also suitable for use are naturally occurring polymeric compounds such as cellulose or derivatives thereof, keratin and collagen or derivatives thereof, calcium alginate, pullulan, agar, gelatin, tamarind seed polysaccharides, xanthan gum, carrageenan, high methoxyl pectin, low methoxyl pectin, guar gum, gum arabic, crystalline cellulose, arabinogalactan, karaya gum, tragacanth gum, alginic acid, albumin, casein, curdlan, gellan gum, and dextran.

[0040] (9) Examples of physiologically active ingredients Examples of the physiologically active ingredient include substances that impart some physiological activity to the skin when applied to the skin. For example, they include whitening ingredients, immune activators, anti-aging agents, UV protection agents, slimming agents, tightening agents, antioxidants, hair growth agents, hair restorers, moisturizers, blood circulation promoters, antibacterial agents, bactericides, drying agents, cooling agents, warming agents, vitamins, amino acids, wound healing promoters, irritation relievers, analgesics, cell activators, enzyme ingredients, etc. Examples of suitable ingredients include angelica extract, avocado extract, amacha extract, althea extract, arnica extract, aloe extract, apricot extract, apricot kernel extract, ginkgo extract, fennel extract, turmeric extract, oolong tea extract, eijitsu extract, echinacea leaf extract, scutellaria extract, phellodendron bark extract, coptis japonica extract, barley extract, hypericum extract, white nettle extract, watercress extract, orange extract, Dried seawater, seaweed extract, hydrolyzed elastin, hydrolyzed wheat powder, hydrolyzed silk, chamomile extract, carrot extract, artemisia capillaris extract, licorice extract, kalkade extract, kakyoku extract, cinchona extract, cucumber extract, guanosine, gardenia extract, kumazasa extract, sophora flavescens extract, walnut extract, grapefruit extract, clematis extract, chlorella extract, mulberry extract, gentiana extract, black tea extract, yeast extract, burdock extract, coix seed extract, Fermented rice bran extract, rice germ oil, comfrey extract, collagen, lingonberry extract, Chinese ivy extract, Chinese rhizome extract, umbilical cord extract, salvia extract, soapwort extract, bamboo extract, hawthorn extract, Japanese pepper extract, shiitake mushroom extract, rehmannia extract, lithospermum extract, perilla extract, linden extract, meadowsweet extract, peony extract, calamus root extract, white birch extract, horsetail extract, ivy extract, hawthorn extract, se Elderberry extract, yarrow extract, peppermint extract, sage extract, mallow extract, cnidium extract, swertia britannica extract, soybean extract, tsinga extract, thyme extract, tea extract, clove extract, imperata cylindrica extract, tangerine peel extract, angelica extract, calendula extract, peach kernel extract, spruce extract, houttuynia cordata extract, tomato extract, natto extract, carrot extract, garlic extract, wild rose extract, hibiscus extract,Examples of such extracts include burdock root extract, parsley extract, honey, witch hazel extract, parietaria extract, burdock root extract, bisabolol, loquat extract, coltsfoot extract, butterbur bud extract, poria extract, butcher's broom extract, grape extract, propolis, loofah extract, safflower extract, peppermint extract, linden extract, peony extract, hop extract, pine extract, horse chestnut extract, skunk cabbage extract, soapberry extract, melissa extract, peach extract, cornflower extract, eucalyptus extract, saxifrage extract, coix seed extract, mugwort extract, lavender extract, apple extract, lettuce extract, lemon extract, astragalus extract, rose extract, rosemary extract, Roman chamomile extract, and royal jelly extract. In addition, it contains biopolymers such as deoxyribonucleic acid, mucopolysaccharides, sodium hyaluronate, sodium chondroitin sulfate, collagen, elastin, chitin, chitosan, and hydrolyzed eggshell membrane, moisturizing ingredients such as amino acids, hydrolyzed peptides, sodium lactate, urea, sodium pyrrolidone carboxylate, betaine, whey, and trimethylglycine, oily ingredients such as sphingolipids, ceramides, phytosphingosine, cholesterol, cholesterol derivatives, and phospholipids, ε-aminocaproic acid, glycyrrhizic acid, β-glycyrrhetinic acid, lysozyme chloride, and guaiazure. immune stimulants such as glycerin, hydrocortisone, etc.; vitamins such as vitamin A, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, calcium pantothenate, biotin, nicotinamide, vitamin C ester, etc.; active ingredients such as allantoin, diisopropylamine dichloroacetate, 4-aminomethylcyclohexanecarboxylic acid, etc.; antioxidants such as tocopherol, carotenoids, flavonoids, tannins, lignans, saponins, etc.; cell stimulants such as alpha-hydroxy acids, beta-hydroxy acids, etc.; blood circulation promoters such as gamma-oryzanol, vitamin E derivatives, etc. Wound healing agents such as retinol and retinol derivatives, skin whitening agents such as arbutin, kojic acid, placenta extract, sulfur, ellagic acid, linoleic acid, tranexamic acid, and glutathione, cepharanthine, licorice extract, capsicum tincture, hinokitiol, iodized garlic extract, pyridoxine hydrochloride, DL-α-tocopherol, DL-α-tocopherol acetate, nicotinic acid, nicotinic acid derivatives, calcium pantothenate, D-pantothenyl alcohol, acetyl pantothenyl ethyl ether, biotin, allantoin, isopropyl methylphenol, estradiol, All, ethinyl estradiol, capronium chloride, benzalkonium chloride, diphenhydramine hydrochloride, tacanal, camphor, salicylic acid, vanillylamide nonylate, vanillylamide nonanoate, piroctone olamine, glyceryl pentadecanoate, L-menthol, mononitroguaiacol, resorcinol, gamma-aminobutyric acid, benzethonium chloride, mexiletine hydrochloride, auxin, female hormones, cantharides tincture, cyclosporine, zinc pyrithione, hydrocortisone, minoxidil, polyoxyethylene sorbitan monostearate, peppermint oil,Examples include hair growth agents such as Sasanishiki extract.

[0041] (10) Examples of antioxidants Examples of antioxidants include sodium bisulfite, sodium sulfite, erythorbic acid, sodium erythorbate, dilauryl thiodipropionate, tocopherol, tolylbiguanide, nordihydroguaiaretic acid, parahydroxyanisole, butylhydroxyanisole, dibutylhydroxytoluene, ascorbyl stearate, ascorbyl palmitate, octyl gallate, propyl gallate, carotenoids, flavonoids, tannins, lignans, saponin, and plant extracts such as apple extract and clove extract that have antioxidant effects.

[0042] (11) Examples of solvents Examples of suitable solvents include purified water, ethanol, lower alcohols, ethers, LPG, fluorocarbons, N-methylpyrrolidone, fluoroalcohols, volatile linear silicones, and next-generation fluorocarbons.

[0043] The skin aging prevention / improvement agent of the present invention can be used as a raw material for food and drink compositions, pharmaceutical compositions, and external skin preparations. The pharmaceutical raw materials that can be blended with these can be the same as those used in the above-mentioned skin aging prevention and improvement agent, and the manufacturing methods and administration methods can also be the same as those for the skin aging prevention and improvement agent. EXAMPLES

[0044] Examples of the present invention will be described below. Note that the following examples are described to confirm the various actions and effects of the skin aging prevention and improvement agent obtained by the present invention, and the scope of the present invention is not limited to these products and manufacturing methods.

[0045] Example: Preparation of Hollandia extract and bakuchiol The crushed dried fruit was used in n-hexane in an amount 5 times its weight, and the oil was extracted at 20-40°C and stirred for 1 hour. This operation was repeated twice to obtain primary and secondary extracts. Then, solid-liquid separation was performed using filter paper, and coumarins such as psoralen and isopsoralen were removed by adsorbent treatment. Hexane was then distilled, and deodorization and dehexane were performed under reduced pressure at 160°C to obtain the Hollandia extract of this example. The Hollandia extract of this example was analyzed by HPLC, and it was confirmed that psoralen had been removed from the Hollandia extract (Figure 9). In addition, bakuchiol was isolated from the Hollandia extract of this example by the following method. That is, the entire amount of the secondary extract was subjected to silica gel flash chromatography (2 L, Yamazen Co., Ltd.) and eluted with hexane:ethyl acetate [1:9 (5 min) → (5 min) → 3:7 (5 min) → (5 min) → 5:5 (5 min) → (5 min) → 0:10 (5 min)]. Detection was performed by UV: 260 nm. Fractions 10-14 [hexane:ethyl acetate (3:7) elution portion] were concentrated to obtain a crude bactiol fraction. The crude bactiol fraction (1.5 g) was purified by HPLC (Cosmosil C18 ARII, 90% methanol, 260 nm) to obtain bactiol (85 mg).

[0046] Test Example 1: Evaluation of the inhibitory effect of UVA irradiation on cell shrinkage (1) Cultivation method of normal diploid fibroblasts (NB1RGB cells) derived from human neonatal skin Human neonatal fibroblast cell line NB1RGB (RCB 0222) cells purchased from RIKEN were cultured and used for the experiment. The medium used was Dulbecco's Modified Eagles Medium (DMEM containing 1000 mg / L glucose) supplemented with 10 (v / v) % FBS (fetal bovine serum), 100 units / mL penicillin G, and 100 mg / mL streptomycin. The cells were cultured in a 75 cm 2The subculture was carried out in a culture flask at 37°C in the presence of 5% CO2. The cultured cells were washed twice with PBS (-) and then detached from the flask with a phenol red-containing 0.25 w / v% Trypsin-1 mM EDTA·4Na solution and used in the experiment. The results are shown in Figure 1. (2) Method for evaluating the inhibitory effect of UVA irradiation on cell shrinkage NB1RGB cells were seeded (9000 cells / well) in an ibidi 8-well plate and cultured for one day, after which the medium was replaced with serum-free DMEM containing the test substance and cultured for another day. After culture, the medium was replaced with PBS (-), the plate lid was removed, and the cells were irradiated with UVA (10 J / cm) using a CL-1000 UV Crosslinker (Analytik Jena AG, Germany). 2 ). After irradiation, the medium was replaced with serum-free DMEM and the cells were cultured for 3 hours. Then, fluorescent immunostaining of β-actin and nuclei was performed according to standard methods. Analysis of the fluorescence intensity and cell number (number of nuclei) after fluorescent immunostaining was performed using Image J. Retinol acetate, a vitamin A derivative, was used as a positive control. Analysis of the luminescence intensity and cell number (number of nuclei) after fluorescent immunostaining was performed using Image J. The results are shown in Figure 2 (100x) and Figure 3 (400x).

[0047] Measurement results and effects of the embodiment in Test Example 1 Normally, when fibroblasts are cultured in a monolayer, they expand their cytoskeleton (β-actin) as shown on the left side of Figure 1. However, it was confirmed that when UVA was irradiated, the skeleton contracted as shown on the right side of Figure 1. We then investigated the effects of Hollandia extract and bactiol, and found that Hollandia and bactiol inhibited fibroblast contraction caused by UVA irradiation (Figure 2, magnification x100; Figure 3, magnification x400). When fibroblasts were irradiated with UVA, not only was the cytoskeleton of the fibroblasts contracted, but there was also a decrease in cell death (absolute number). The graph reflects the results of dividing the fluorescence intensity of the cytoskeleton (actin) in the photograph by the number of cell nuclei and correcting it, so the actin emission intensity per cell is calculated. These results confirmed that Dutch ibis extract and bakuchiol have the effect of inhibiting fibroblast atrophy, suggesting that they inhibit the inactivation of fibroblasts (e.g., a decrease in collagen production and activation of MMP1 (collagen-degrading enzyme)). From the above, it was confirmed that the extract of Hollandia and bakuchiol are useful as agents for inhibiting cell atrophy caused by UVA irradiation and are useful as anti-photoaging agents.

[0048] Test Example 2: Evaluation of collagen gel contraction degree and integrin α1 increase effect using a dermis 3D model (collagen gel) Test Method A 3D model of the dermis was created by culturing NB1RGB cells in type I collagen gel (PureCol®) and evaluated in vitro. A suspension of NB1RGB cells was mixed with a collagen solution (pH 7.4) containing Hanks buffer, and the mixture was seeded (1.2 × 10 5 The cells were then cultured for 1 day at 37 °C (100 µL / well). Then, 10% FBS-containing DMEM was added onto the gel (500 µL / well) and cultured for 1 day. The medium was then replaced with 10% FBS-containing DMEM containing the test substance. After 2 days of culture, the medium was replaced with 10% FBS-containing DMEM, and the cells were cultured for an additional 5 days (medium replacement every 2 days). The area of ​​the collagen gel and fluorescent immunostaining of integrin α1 were then performed, and the fluorescence intensity was measured (calculated using Image J).

[0049] Results and Effects of the Examples in Test Example 2 As shown in Figure 4, it was confirmed that Hollandia extract and bakuchiol have the effect of increasing the collagen gel contraction ability (Figure 4). Furthermore, fluorescent immunostaining of integrin α1 was performed using the collagen gel, and as a result, it was confirmed that bakuchiol has the effect of increasing integrin α1 at concentrations of 1 and 3 μM, as shown in Figure 5 (Figure 5). These results confirmed that Hollandia extract and bakuchiol have the ability to contract collagen gel, and at the same time, an increase in integrin α1 was also confirmed. Therefore, it was confirmed that Hollandia extract and bakuchiol have the effect of increasing skin elasticity and anti-wrinkle action by increasing the adhesive ability between cells and collagen gel.

[0050] Test Example 3: Evaluation test of the effect of promoting the expression of elastin-related genes (fibulin 4) using fibroblasts (TIG 103) derived from elderly people TIG-103 (JCRB0528) cells purchased from the JCRB Cell Bank were seeded onto a 12-well plate (1.0 × 10 5 The cells were cultured at 1 mL (cells / well) for one day. The medium was then replaced with DMEM containing 10% FBS and the test substance. After two days of culture, RNA was extracted and RT-PCR was performed. *The culture method was the same as for NB1RGB. The results are shown in Figure 6.

[0051] Results and Effects of the Examples in Test Example 3 As shown in Figure 6, the extract of Hollandia japonica was found to increase the expression level of fibulin 4 gene in a concentration-dependent manner in TIG103 cells (Figure 6). This confirmed that the extract of Hollandia japonica is useful as an agent for promoting the expression of fibulin 4 gene.

[0052] Test Example 4 Evaluation of laminin 5 expression promoting effect using normal human epidermal keratinocytes (NHEK) cells (1) Method for culturing normal human epidermal keratinocytes (NHEK) NHEK cells were cultured and used for the experiment. The medium used was normal human epidermal keratinocyte growth medium (HuMedia-KG2). The cells were cultured on collagen-coated 75 cm2 The culture was carried out in a culture flask at 37°C under 5% CO2. For subculture, the cultured cells were washed twice with PBS (-) and then detached with 0.05 (w / v) % trypsin-0.53 mmol / l EDTA 4Na solution containing phenol red for use in the experiment. (2) Method for evaluating the effect of promoting laminin 5 expression Normal human epidermal keratinocytes (NHEK) cells were seeded onto a 6-well plate (coated with collagen) (2.0 × 10 5 The cells were cultured at 1 mL / well for 1 day. The medium was then replaced with a medium specifically for human epidermal cells (HuMedia-KG2) containing the test substance. After culturing for 2 days, protein extraction was performed and Western blotting was performed according to standard methods.

[0053] Results and Effects of the Examples in Test Example 4 As shown in Figure 7, the positive control retinoic acid and the test substances Hollandia extract and bakuchiol showed a tendency to increase the expression of laminin 5. It was confirmed that bakuchiol had the same effect as the positive control. From these results, it was confirmed that Hollandia extract and bakuchiol are useful as agents for promoting laminin 5 expression, and that Hollandia extract and bakuchiol increase the adhesion between the epidermis and dermis and have anti-sagging and anti-wrinkle effects, and therefore are useful as agents for preventing and improving skin aging.

[0054] Test Example 5: Evaluation of hyaluronidase inhibitory activity The buffer used was 0.1 M acetate buffer (pH 4.0). Compound 48 / 80 solution (20 μL / well, 0.5 mg / mL), hyaluronidase solution (10 μL / well, 400 units / mL), buffer solution containing the test substance (20 μL / well), and hyaluronic acid solution (50 μL / well, 400 μg / mL) were added to a 96-well plate, and the reaction was allowed to proceed at 37°C for 40 minutes. After that, NaOH solution (20 μL / well, 0.4 N) was added to stop the enzyme reaction, and the plate was immediately placed on ice. Next, boric acid solution (pH 9.1, 20 μL / well, 0.8 M) was added, and the plate was boiled (90°C) for 3 minutes. After boiling, the plate was left on ice, and the reaction solution was transferred to a new 96-well plate at 50 μL / well. P-DMBA solution (10 N hydrochloric acid: acetic acid = 1:8 mixed solution in which 4-Dimethyl aminobenzaldehyde was dissolved x 10 100 mg / mL P-DMAB solution was prepared, and this solution was diluted 10 times with acetic acid for use in the experiment) was added (200 μL / well) and the color reaction was carried out at 37 °C for 20 minutes. The OD value was then measured using a microplate reader (585 nm). The results are shown in Figure 8.

[0055] Results and Effects of the Examples in Test Example 5 As shown in Figure 8, the Hollandia extract had a hyaluronidase inhibitory effect (Figure 8). It was confirmed that the Hollandia extract is useful as a hyaluronidase inhibitor.

[0056] Test Example 6: Clinical trial evaluation of the anti-wrinkle effect of Hollandia extract Test Method (1) Study design This was an open-label study in which 20 healthy male and female subjects were asked to take the test product for four weeks. Before and after the four-week period, the subjects were evaluated by photographing wrinkles around the eyes, analyzing wrinkles using replicas of the wrinkles around the eyes, and completing a questionnaire. (2) Test specimen The test product was a hard capsule filled with 35 mg of the Hollandia extract containing 3% bakuchiol and 150 mg of porous oil-absorbing dextrin (Oil Q-50, manufactured by Nippon Starch Chemical Co., Ltd.). The subjects were asked to orally take one capsule per day freely during the four-week intake period. (3) Subjects After explaining the details of the study, the subjects were asked to participate in the study with their consent. The subjects were required to be healthy adult men and women, with the following exceptions: (1) those undergoing hormone therapy, (2) those who were pregnant or breastfeeding, (3) those who had an allergic reaction to the test product, and (4) those who had undergone cosmetic medical treatment that could affect the test area. (4)Measurement items Photographs of the corners of the eyes before and after the test, and replicas (squares) of the corners of the eyes were taken and 3D analysis of the replicas was performed. Measurements were taken twice, before intake (0W) and four weeks later (4W). Subjects were asked to remove their facial makeup 15 minutes before entering the measurement room, and were allowed to acclimate for 15 minutes in a room with a temperature of 25±1°C and humidity of 65%±5%, before measurements were taken. (5) Statistical analysis The measured values ​​for each item are shown as the mean ± standard deviation. Significant differences were tested by analysis of variance followed by a paired t-test comparing the values ​​before and after ingestion.

[0057] Results and Effects of the Examples in Test Example 6 (1) Whether or not there were dropouts No subjects dropped out throughout the study period, so the analysis consisted of 20 subjects. However, one subject could not be analyzed due to the inability to analyze the wrinkle replica. (2) Measurement results (2-1) Results of 3D wrinkle analysis of the entire subject The overall results (n=19) are shown in Table 1 below. [Table 1]

[0058] As shown in Table 1, after taking Hollandia extract, a tendency for improvement was observed in the following items: total volume ratio, wrinkle area ratio, wrinkle volume ratio, maximum wrinkle width, average wrinkle depth, and number of wrinkles.

[0059] (2-2) Stratified analysis results for wrinkle grades 2 to 3 The tester determined the wrinkle grade of the subjects, and the results of the stratified analysis of subjects with wrinkle grades 2 to 3 are shown in Table 2 below. The wrinkle grade is the wrinkle level evaluation standard set out in the "Guidelines for Evaluation of Anti-Wrinkle Products" (see Figure 10). Wrinkle grades 2 to 3 are conditions in which shallow wrinkles originating from the epidermis have formed.

[0060] [Table 2]

[0061] Stratified analysis of subjects with wrinkle grades 2 to 3 showed that the number of wrinkles was significantly reduced by taking Hollandia extract. This confirmed that oral intake of Hollandia extract can improve shallow wrinkles originating from the epidermis.

[0062] (2-3) Stratified analysis results for wrinkle grade 4 or higher The results of stratified analysis of subjects with wrinkle grade 4 or higher are shown in Table 3. Wrinkle grade 4 or higher refers to a state in which deep wrinkles originating from the dermis have been formed.

[0063] [Table 3]

[0064] As shown in Table 3 above, improvements were confirmed in the following items for wrinkle grade 4 or higher: total volume ratio, wrinkle area ratio, wrinkle volume ratio, maximum wrinkle width, and average wrinkle depth. This confirmed that orally ingested Dutch ibis extract has the effect of improving even deep wrinkles originating from the dermis.

[0065] The following are examples of formulations of the skin aging prevention and improvement agent (hollandia extract) according to the present invention. Note that the following formulation examples do not limit the present invention. Formulation example 1: Chewing gum Sugar 53.0wt% Gum base 20.0 Glucose 10.0 Syrup 16.0 Fragrance 0.5 Skin aging prevention and improvement agent 0.5 100.0wt%

[0066] Mixture Example 2: Gummies Reduced starch syrup 40.0wt% Granulated sugar 20.0 Glucose 20.0 Gelatin 4.7 Wednesday 9.68 Yuzu juice 4.0 Yuzu flavor 0.6 Dye 0.02 Skin aging prevention and improvement agent 1.0 100.0wt%

[0067] Mixture example 3: Candy Sugar 50.0wt% Syrup 33.0 Wednesday 14.4 organic acid 2.0 Fragrance 0.2 Skin aging prevention and improvement agent 0.4 100.0wt%

[0068] Mixture example 4: Yogurt (hard / soft) Milk 41.5wt% Skim milk powder 5.8 Sugar 8.0 Agar 0.15 Gelatin 0.1 Lactic acid bacteria 0.005 Skin aging prevention and improvement agent 0.4 fragrance trace amount water residue 100.0wt%

[0069] Formulation example 5: Soft drink Fructose glucose liquid sugar 30.0wt% Emulsifier 0.5 Skin aging prevention and improvement agent 0.3 Fragrance (appropriate amount) Purified water remainder 100.0wt%

[0070] Formulation example 6: Tablet confectionery Sugar 76.4wt% Glucose 19.0 Sucrose fatty acid ester 0.2 Skin aging prevention and improvement agent 0.5 Purified water 3.9 100.0wt%

[0071] Formulation example 7: Soft capsule Brown rice germ oil 47.0wt% Yuzu seed oil 40.0 Emulsifier 12.0 Skin aging prevention and improvement agent 1.0 100.0wt%

[0072] Formulation Example 8: Tablets Lactose 54.0wt% Crystalline cellulose 30.0 Starch decomposition product 10.0 Glycerin fatty acid ester 5.0 Skin aging prevention and improvement agent 1.0 100.0wt%

[0073] Formulation example 9: Cosmetic cream Squalane 20.0wt% Beeswax 5.0 Refined Jojoba Oil 5.0 Glycerin 5.0 Glycerin Monostearate 2.0 Polyoxyethylene (20) Sorbitan- Monostearate 2.0 Skin aging prevention and improvement agent 2.0 Preservatives (appropriate amount) Fragrance (appropriate amount) Purified water remainder 100.0wt%

[0074] Formulation example 10: Lotion Ethanol 5.0wt% Glycerin 2.0 1,3-Butylene glycol 2.0 Polyethylene oleyl ether 0.5 Sodium citrate 0.1 Citric acid 0.1 Skin aging prevention and improvement agent 0.1 Purified water remainder 100.0wt%

[0075] Formulation example 11: Body gel Macadamia nut oil 2.0wt% Octyldodecyl myristate 10.0 Methylphenylpolysiloxane 5.0 Behenyl Alcohol 3.0 Stearic Acid 3.0 Batyl alcohol 1.0 Glyceryl Monostearate 1.0 Polyoxyethylene sorbitol tetraoleate 2.0 Hydrogenated soy phospholipids 1.0 Ceramide 0.1 Retinol Palmitate 0.1 Preservatives (appropriate amount) Centella asiatica extract 1.0 Skin aging prevention and improvement agent 1.0 1,3-Butylene glycol 5.0 Purified water remainder 100.0wt%

[0076] Formulation example 12: Emulsion Squalane 4.0wt% Vaseline 2.5 Cetanol 2.0 Glycerin 2.0 Lipophilic glyceryl monostearate 1.0 Stearic acid 1.0 L-Arginine 1.0 Skin aging prevention and improvement agent 0.5 Potassium hydroxide 0.1 fragrance trace amount Purified water remainder 100.0wt%

[0077] Formulation example 13: Bath additive (liquid) Propylene glycol 50.0wt% Ethanol 20.0 Sodium sulfate 5.0 Skin aging prevention and improvement agent 0.5 Lanolin 0.5 Avocado oil 0.5 Dye 1.5 Fragrance 22.0 100.0wt% [Industrial Applicability]

[0078] As described above, the present invention can provide a fibulin 4 gene expression promoter and a laminin 5 expression promoter each containing a novel ingredient as an active ingredient, as well as an agent for preventing and improving skin aging using these.

Claims

1. An oral wrinkle improvement agent containing at least one of Hollandia extract and bakuchiol as active ingredients.

2. An oral agent for preventing and improving skin aging, comprising the agent described in claim 1 as an active ingredient.

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