JP5118399B2 - Composition having hyaluronic acid production promoting ability and / or fibroblast proliferation promoting ability - Google Patents

Description

  The present invention relates to a useful novel composition having the ability to promote hyaluronic acid production, a useful novel composition having the ability to promote the proliferation of fibroblasts, and an invention related thereto.

  Conventionally, it has been known that connective tissues of animals contain collagen, hyaluronic acid, elastin, chondroitin sulfate, heparan sulfate, dermatan sulfate, laminin and the like as main components. Of these, hyaluronic acid plays an important role in the connective tissue as described below.

  Hyaluronic acid is a kind of acidic mucopolysaccharide present in skin, cartilage, joint fluid, umbilical cord, ophthalmic vitreous, and other connective tissues. Hyaluronic acid is known to have a variety of roles in the body. For example, hyaluronic acid is a main component of joint fluid, and serves as a cushion in joints and has a function of repairing worn cartilage. Are known. Hyaluronic acid is also known to have an anti-inflammatory effect in vivo.

  Furthermore, in the skin epidermis, hyaluronic acid is widely distributed from the basal layer to the granular layer, supporting the structure of the extracellular space of the epidermis, involved in the transport of substances such as nutrients and waste products from the epidermal basal layer to the horny layer, It is known to act as a trigger to promote epidermal cell turnover. It is also known that hyaluronic acid has a strong water-retaining effect that can hold as much as about 6 L of water with only 1 g, and has a role of holding water in the cell gap by that action. Hyaluronic acid is known to gradually decrease with age, and this decrease may lead to the formation of skin wrinkles and tarmi, skin elasticity and elasticity, or skin aging such as skin dryness and rough skin. It is a cause.

  For these reasons, it is desirable to supplement and increase the amount of hyaluronic acid that has decreased due to aging, etc., but since hyaluronic acid is a polymer compound, it is supplied to the living body from outside the skin. It is not easy to do. Therefore, in order to supply hyaluronic acid in vivo (for example, between epidermal cells), it is said that promotion of biosynthesis of hyaluronic acid in vivo is important.

  Various hyaluronic acid synthesis promoting substances have been found to improve the state caused by the reduction of hyaluronic acid. For example, aloe extract, okra extract, water-soluble β-1,3-glucan derivative, yeast extract (patent document 1), seaweed extract belonging to the genus Tokamodori (patent document 2), lavender extract ( Known are Patent Document 3), extracts of seaweeds belonging to the genus Davilia (Patent Document 4), retinoic acid, and the like.

  Fibroblasts, which are the main cells of the dermis, produce mucopolysaccharides containing hyaluronic acid as described above, collagen that keeps the skin firm, and elastin that keeps the skin elastic. However, due to the effects of external factors such as ultraviolet light irradiation, drastic air drying, excessive skin washing, and aging, the proliferation ability of fibroblasts itself is reduced, and the moisture retention function and elasticity of the skin are reduced. It is known that aging of the skin (for example, reduction in elasticity or elasticity of the skin, wrinkles or sagging of the skin, etc.) is accelerated.

Therefore, it is considered that promoting the proliferation of fibroblasts also contributes to the prevention and / or improvement against a decrease in skin elasticity or elasticity, and the prevention and / or improvement of skin wrinkles or talmi.
JP 2004-051533 A JP 2000-136147 A JP-A-10-182402 Japanese Patent Laid-Open No. 09-176036

  In view of the importance of the role of hyaluronic acid in the living body as described above, development of a further useful new composition having a remarkable ability to promote hyaluronic acid production is desired. In view of the conventional problems, an object of the present invention is to provide a useful novel composition having a remarkable ability to promote hyaluronic acid production. Another object of the present invention is to provide a useful novel composition having the ability to promote fibroblast proliferation.

  As a result of intensive studies to solve the above problems, the present inventors have used retinols alone by using retinols in combination with a product obtained by decomposing soybean protein with a specific protease called thermolysin. It has been found that the ability to promote hyaluronic acid production in cells is significantly promoted compared to that used in the above, and further studies have shown that the combined use of both components synergistically promotes the proliferation of fibroblasts. And the present invention has been completed.

Accordingly, the present invention provides the following.
(1) A composition comprising (A) at least one selected from the group consisting of retinol and derivatives thereof, and (B) a thermolysin degradation product of soybean protein.
(2) The composition according to item (1), which can be used to promote hyaluronic acid production in cells.
(3) The composition according to item (1) or (2), which can be used to promote fibroblast proliferation.

  The present invention provides a useful novel composition having a remarkable ability to promote hyaluronic acid production. The composition of the present invention can be used to promote hyaluronic acid production in cells. For example, an anti-wrinkle composition, an anti-tarmi composition, a composition for preventing or treating joint disorders, an inflammatory disease It can be beneficially used as a preventive or therapeutic composition. The present invention also provides a useful novel composition having the ability to promote fibroblast proliferation.

  Hereinafter, the present invention will be described in detail. It should be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

  The present invention is a composition comprising (A) at least one selected from the group consisting of retinol and derivatives thereof, and (B) a thermolysin degradation product of soybean protein.

  The retinol and derivatives thereof used in the present invention (in the present specification, these are sometimes collectively referred to as “retinols”) are those that can be used in the field of pharmaceuticals, quasi drugs, cosmetics or foods. If there is no particular limitation.

  Retinol derivatives include: retinoic acid; retinal; retinol, retinoic acid, or an isomer of retinal; retinol or an ester derivative of the isomer; retinol or an ether derivative of the isomer; an ester derivative of retinoic acid or the isomer; , Retinoic acid, retinal or their isomer epoxy derivatives; retinol, retinoic acid, retinal or their isomers with hydrogen atoms eliminated or hydrogen atoms added; and the like.

Examples of the isomers of retinol, retinoic acid or retinal include geometric isomers (cis-trans isomers or E, Z isomers), and the bonding position and number of double bonds and the steric structure thereof are not particularly limited.
Specific examples of the isomers of retinol, retinoic acid, or retinal include all-trans form, 13-cis form, 11-cis form, 9-cis form, 7-cis form, 7, retinol, retinoic acid, or retinal form. Examples thereof include 9-di-cis isomer, 7,13-di-cis isomer, 11,13-di-cis isomer, 9,13-di-cis isomer, 9,11,13-tri-cis isomer, and the like. Among these isomers, all-trans isomer or 13-cis isomer is preferable, and all-trans isomer is particularly preferable.

  Examples of the ester derivative of retinol or its isomer include esters of retinol or its isomer with acids such as aliphatic carboxylic acids and aromatic carboxylic acids.

Examples of the aliphatic carboxylic acid include saturated or unsaturated aliphatic carboxylic acids having 1 to 22 carbon atoms, preferably saturated or unsaturated aliphatic carboxylic acids having 2 to 18 carbon atoms. Specific examples include formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, pivalic acid, hexanoic acid, heptanoic acid, octanoic acid, octylic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid , Myristic acid, pentadecanoic acid, palmitic acid, isopalmitic acid, heptadecanoic acid, stearic acid, isostearic acid, behenic acid, acrylic acid, propiolic acid, methacrylic acid, crotonic acid, isocrotonic acid, undecylenic acid, myristoleic acid, palmitoleic acid Aliphatic carboxylic acids such as oleic acid, ricinolenic acid, linoleic acid, linolenic acid, arachidonic acid, icosapentaenoic acid, docosahexaenoic acid, cyclopentanecarboxylic acid, cyclohexanecarboxylic acid; oxalic acid, malonic acid, succinic acid, glutaric acid, Adipic acid, pimelin , Suberic acid, azelaic acid, sebacic acid, fumaric acid, maleic acid and other aliphatic dicarboxylic acids or monoesters thereof (eg, methyl, ethyl, propyl, isopropyl, butyl, s-butyl, t-butyl, isobutyl, pentyl, C1-C6 alkyl esters such as hexyl); and retinoic acid. In addition, the aliphatic carboxylic acid may be substituted, and the substituent may be an alkoxy group having 1 to 6 carbon atoms (eg, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, s-butoxy group). Group, t-butoxy group, isobutyloxy group, pentyloxy group, hexyloxy group, etc.), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom), amino group, hydroxy group and the like.
As aromatic carboxylic acid, C7-12 aromatic carboxylic acid is mentioned, Preferably it is C7-10 aromatic carboxylic acid. Specific examples include aromatic monocarboxylic acids such as benzoic acid, naphthoic acid and cinnamic acid; aromatic dicarboxylic acids such as phthalic acid, isophthalic acid and terephthalic acid. In addition, the aromatic carboxylic acid may be substituted, and the substituent is an alkyl group having 1 to 6 carbon atoms (eg, methyl group, ethyl group, propyl group, isopropyl group, butyl group, s-butyl group). , T-butyl group, isobutyl group, pentyl group, hexyl group, etc.), C1-C6 alkoxy group (eg, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, s-butoxy group, t -Butoxy group, isobutyloxy group, pentyloxy group, hexyloxy group, etc.), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom), amino group, hydroxy group and the like.

  Specific examples of ester derivatives of retinol or its isomers include retinol acetate, retinol propionate, retinol butyrate, retinol pivalate, retinol heptanoate, retinol octylate, retinol nonanoate, retinol laurate, retinol myristate, palmitic acid Examples thereof include retinol, retinol stearate, retinol heptadecanoate, retinol oleate, retinol linolenate, retinol linoleate, retinol cyclopentanecarboxylate, retinol succinate, L-lactic acid retinol and the isomers thereof.

Examples of the ether derivative of retinol or its isomer include those in which the hydrogen atom of the hydroxy group of retinol or its isomer is substituted with an alkyl group, alkenyl group, aryl group, sugar residue or the like.
As an alkyl group, a C1-C22 alkyl group is mentioned, Preferably it is a C1-C6 alkyl group. Specific examples include methyl group, ethyl group, propyl group, isopropyl group, butyl group, s-butyl group, t-butyl group, isobutyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group. , Undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group and the like.
Examples of the alkenyl group include alkenyl groups having 2 to 12 carbon atoms, preferably alkenyl groups having 2 to 6 carbon atoms. Specific examples include vinyl group, allyl group, butenyl group (eg, 1-butenyl, 2-butenyl) and the like.
These alkyl group and alkenyl group may be substituted, and as the substituent, an alkoxy group having 1 to 6 carbon atoms (eg, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, s-butoxy group) Group, t-butoxy group, isobutyloxy group, pentyloxy group, hexyloxy group, etc.), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom), amino group, hydroxy group, oxo group, phenyl group Etc.

Examples of the aryl group include aryl groups having 6 to 12 carbon atoms. Specific examples include a phenyl group and a naphthyl group (1-naphthyl group, 2-naphthyl group). In addition, the aryl group may be substituted, and examples of the substituent include alkyl groups having 1 to 6 carbon atoms (eg, methyl group, ethyl group, propyl group, isopropyl group, butyl group, s-butyl group, t -Butyl group, isobutyl group, pentyl group, hexyl group, etc.), C1-C6 alkoxy group (eg, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, s-butoxy group, t-butoxy group) Group, isobutyloxy group, pentyloxy group, hexyloxy group, etc.), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom), amino group, hydroxy group and the like.
Examples of sugar residues include monosaccharide residues and oligosaccharide residues. Specific examples of monosaccharide residues include glucose residues, fructose residues, galactose residues, mannose residues, talose residues, idose residues, altrose residues, allose residues, growth residues, xylose residues. , Ribose residues, arabinose residues, rhamnose residues, fucose residues, glucuronic acid residues and the like. Examples of oligosaccharide residues include oligosaccharide residues composed of 2 to 10 monosaccharides. Constituent monosaccharides of oligosaccharide residues include glucose, fructose, galactose, mannose, talose, idose, altrose, allose, gulose, xylose, ribose, arabinose, rhamnose, fucose, glucuronic acid, etc. Or they may be different. The hydrogen atom of the hydroxy group of the sugar residue may be substituted, and as the substituent, an acyl group having 1 to 7 carbon atoms (eg, formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, valeryl group) Group, isovaleryl group, pivaloyl group, etc.), alkyl group having 1 to 6 carbon atoms (eg, methyl group, ethyl group, propyl group, isopropyl group, butyl group, s-butyl group, t-butyl group, isobutyl group, pentyl) Group, hexyl group, etc.).

  Specific examples of the ether derivative of retinol or its isomer include 15-deoxy-15-methoxyretinol, 15-deoxy-15-ethoxyretinol, O- (β-D-glucopyranosyl) retinol, O- (β-D- Glucuronopyranosyl) retinol, O- (β-D-maltosyl) retinol and the isomers thereof.

Examples of ester derivatives of retinoic acid or its isomers include esters of retinoic acid with aliphatic or aromatic alcohols, tocopherols and the like.
Examples of the aliphatic alcohol include aliphatic alcohols having 1 to 22 carbon atoms, preferably aliphatic alcohols having 1 to 18 carbon atoms. Specific examples include methanol, ethanol, propanol, isopropanol, butanol, s-butanol, t-butanol, isobutanol, pentanol, hexanol and the like. In addition, the aliphatic alcohol may be substituted, and as the substituent, an alkoxy group having 1 to 6 carbon atoms (eg, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, s-butoxy group, t-butoxy group, isobutyloxy group, n-pentyloxy group, n-hexyloxy group, etc.), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom), amino group, phenyl Groups and the like.
Examples of the aromatic alcohol include aromatic alcohols having 6 to 12 carbon atoms, and specific examples include benzyl alcohol, 1-phenylethyl alcohol, 2-phenylethyl alcohol and the like. In addition, the aromatic alcohol may be substituted, and the substituent includes an alkyl group having 1 to 6 carbon atoms (eg, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, s -Butyl group, t-butyl group, isobutyl group, n-pentyl group, n-hexyl group, etc.), an alkoxy group having 1 to 6 carbon atoms (eg, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, s-butoxy group, t-butoxy group, isobutyloxy group, n-pentyloxy group, n-hexyloxy group, etc.), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom) , Amino group, hydroxy group and the like.
Examples of tocopherols include α-tocopherol, β-tocopherol, and δ-tocopherol.

  Specific examples of ester derivatives of retinoic acid or its isomer include methyl retinoic acid, ethyl retinoic acid, α-tocopheryl retinoate, β-tocopheryl retinoate, δ-tocopheryl retinoate and the isomers thereof. Can be mentioned.

Examples of epoxy derivatives of retinol, retinoic acid, retinal or their isomers include those in which the double bond of retinol, retinoic acid, retinal or their isomers is epoxidized. There is no particular limitation. Moreover, the epoxy derivatives of retinol, retinoic acid, retinal or their isomers may be further esterified and etherified, respectively.
Specific examples include 5,6-dihydro-5,6-epoxyretinol, 5,6-epoxy-5,6-dihydroretinol acetate, 5,8-dihydro-5,8-epoxyretinol, 11,14-epoxy. -11,14-dihydroretinol, 5,6; 11,14-diepoxy-5,6,11,14-tetrahydroretinol, 5,6-dihydro-5,6-epoxyretinoic acid and the isomers thereof. Be

In the derivative from which hydrogen atoms of retinol, retinoic acid, retinal or their isomers are eliminated, the position and number of the hydrogen atoms to be eliminated are not particularly limited. Moreover, in the derivative | guide_body which added the hydrogen atom to retinol, retinoic acid, retinal, or those isomers, the position and number of the hydrogen atom to add are not specifically limited. The derivatives of retinol, retinoic acid, retinal or their isomers from which hydrogen atoms are eliminated or hydrogen atoms are added may be further esterified or etherified, respectively.
Specific examples include 3,4-didehydroretinol, 7,8-didehydroretinol, 7,8-didehydroretinol acetate, 5,6-dihydroretinol acetate, 5,6-dihydroretinol, 3,4-di- Examples include dehydroretinal, 5,6-dihydroretinal, 7,8-dihydroretinal, 9,10-dihydroretinal, 3,4-didehydroretinoic acid, and the isomers thereof.

  Moreover, the retinol derivative of the present invention may further have a substituent, and the substitution position and the number of substituents are not particularly limited. Examples of the substituent include alkyl groups having 1 to 6 carbon atoms (eg, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, s-butyl group, t-butyl group, isobutyl group, n -Pentyl group, n-hexyl group, etc.), C1-C6 alkoxy group (eg, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, s-butoxy group, t-butoxy group) Group, isobutyloxy group, n-pentyloxy group, n-hexyloxy group, etc.), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom), amino group, hydroxy group, carboxy group, oxo group, etc. Is mentioned.

  The retinol derivative may be a salt, and as such a salt, a pharmacologically and physiologically acceptable salt is preferable. Specific examples include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as magnesium salts and calcium salts; ammonium salts; alkanolamine salts such as monoethanolamine salts;

  In the present invention, all-trans retinol, retinol acetate, retinol palmitate, and δ-tocopheryl retinoate are preferably used as retinol and derivatives thereof.

  In this invention, said retinol and its derivative (s) may be used individually by 1 type, or may be used in the state of a mixture combining 2 or more types arbitrarily. In addition, for example, in the present invention, as retinol and derivatives thereof, fatty oils or extracts (such as vitamin A oil) obtained from tissues of marine animals containing retinol and derivatives thereof can be used.

  The amount of retinol compounded in the composition of the present invention is not particularly limited as long as the effect of the present invention can be obtained, but is usually 0.1 IU to 5 million IU, more preferably 50 IU to 1.5 million IU per 100 g of the composition. More preferably, it is blended so as to contain 300 IU to 1 million IU, 1000 IU to 500,000 IU of retinol.

  Here, IU is an international unit relating to the amount of fat-soluble vitamins such as vitamin A, as can be generally understood by those skilled in the art. For example, 1 IU for retinol corresponds to about 0.30 μg of retinol, 1 IU for retinol acetate corresponds to about 0.34 μg of retinol acetate, and 1 IU for retinol palmitate to about 0.55 μg of retinol palmitate. Corresponding to 1 IU = about 0.72 μg of δ-tocopheryl retinoate in the case of δ-tocopheryl retinoate.

  Furthermore, the thermolysin degradation product of soybean protein is used in the present invention [in this specification, this may be referred to as “soybean thermolysin degradation product”].

  Soybean (Glycin max) is a leguminous annual plant with a height of about 60-70 cm. It is known that the seeds are often used as edible beans or processed into tofu, miso, soy sauce, etc.

  The soy protein used in the present invention can be any protein derived from such a soybean plant, but preferably can be any protein derived from the seed of a soybean plant.

  In the present invention, as a source of soybean protein, soybean plant itself, soybean plant seed itself, or a crushed or crushed product of the plant or the seed may be used. Preferably, all components in the soybean plant are used. From which the protein component is separated and purified, more preferably, the protein component is separated and purified from all the components in the seeds of soybean plants. Soy protein obtained by separating and purifying in this way can be used for substantially all kinds of soybean plant or soybean plant seeds as long as the product obtained by decomposing it with thermolysin can achieve the effect of the present application. Proteins may be included, or some types of proteins may be included.

  As the soybean protein, commercially available products can also be suitably used. For example, Nisshin Cosmo Foods Co., Ltd., ADM Far East Co., Ltd., Showa Sangyo Co., Ltd., Fuji Oil Co., Ltd., Koyo Shokai Co., Ltd. Readily available from manufacturers or suppliers.

  In the present specification, the seeds of soybean plants not only refer to the whole structure commonly referred to as soybean seeds, but are obtained from, for example, dehulled soybean seeds, defatted soybean seeds (powdered ones, etc.) and whole soybean seeds. It can be snow flora (okara), soy milk, and the like.

  Thermolysin (EC 3.4.24.4) used in the present invention is well known as a heat-resistant protease produced by a heat-resistant bacterium called Bacillus thermoproteolyticus. Thermolysin is generally known to cleave peptide bonds on the amino group side of hydrophobic amino acid residues having large side chains (for example, isoleucine, leucine, valine, phenylalanine, methionine, alanine, etc.).

  Commercially available products can be suitably used for thermolysin, and for example, they can be easily obtained from manufacturers such as Daiwa Kasei Co., Ltd.

  In the present invention, a protease known in the art as a protease having peptide cleavage characteristics (such as cleavage sequence specificity) equivalent to thermolysin can also be used as thermolysin.

  The reaction conditions used when degrading soybean protein with thermolysin are not particularly limited, and can be appropriately selected by those skilled in the art according to common general technical knowledge. For example, when using commercially available thermolysin, it can be used according to the instruction manual. As a specific example, soy protein concentration is generally 0.1 to 30% (w / v), preferably 1 to 10% (w / v) in a solvent such as water. Soy protein or a raw material containing soy protein is suspended, and this suspension is generally about 0.001 to 3% (w / v), preferably about 0.01 to 0.125% (w / v). The aspect which adds thermolysin so that it may become and performs a decomposition reaction is mentioned. In general, reaction temperatures of 30-80 ° C, preferably 40-70 ° C, more preferably 50-60 ° C may be used. In general, a reaction time of 2 to 30 hours, preferably 3 to 24 hours, more preferably 10 to 20 hours, and even more preferably 12 to 18 hours may be used. The pH of the reaction solution is preferably near the optimum pH of thermolysin, and is preferably around 7.0 to 8.5, for example.

  The means for stopping the reaction is not particularly limited, and known means can be used. Examples of such means include heat treatment. For example, the thermolysin contained in the reaction product can be deactivated by heat-treating the reaction product at a temperature of about 80 to 100 ° C. for 3 to 20 minutes, preferably 5 to 15 minutes. For example, examples of such heat treatment include heat treatment at 85 ° C. for 15 minutes and heat treatment at 100 ° C. for 5 minutes.

  The soybean thermolysin degradation product obtained by the above degradation reaction can be further processed by any method known to those skilled in the art, if necessary. For example, it is preferable to remove large solid particles in the decomposition product by a treatment such as filtration. Filtration conditions and the like are not particularly limited and may be appropriately selected by those skilled in the art according to common general technical knowledge. For example, when the filter paper is likely to be clogged, a filter aid or the like can be suitably used.

  Further, the decomposition product can be pulverized by concentrating under reduced pressure and then freeze-drying. The conditions and equipment used in the concentration under reduced pressure and lyophilization are not particularly limited and can be appropriately selected by those skilled in the art according to common general technical knowledge. The decomposition product pulverized in this way can be used as it is or after being dissolved in a solvent such as water.

  The degradation product used in the present invention may be in a state containing substantially all of a wide variety of peptides generated by degrading soybean protein with thermolysin, or such a wide variety of peptides, It may be a portion obtained by further fractionation and purification by a known method using the presence or absence of hyaluronic acid production promoting ability in cells and / or fibroblast proliferation promoting ability as an index. However, for convenience, the soy protein is used as it is in a state of containing substantially all of a wide variety of peptides obtained by decomposing the protein with thermolysin. The presence or absence of hyaluronic acid production promoting ability and / or the presence or absence of fibroblast proliferation promoting ability can be confirmed, for example, as described in Examples and the like described later.

In this specification, the term “having the ability to promote hyaluronic acid production in a cell” means that when a test substance is allowed to act on a cell, hyaluronic acid in the cell is compared with a case where the test substance is not allowed to act on the cell. This means that the production amount of increases. In a particular embodiment, the cell in the term means a keratinocyte, and in a particular embodiment means an epidermal keratinocyte.
Further, in the present invention, the term “having the ability to promote fibroblast proliferation” means that when a test substance is allowed to act on fibroblasts, the proliferation of fibroblasts is greater than when the test substance is not allowed to act. It means being promoted. In certain embodiments, fibroblasts in the term refer to dermal fibroblasts.

  The average molecular weight of the thermolysin decomposition product of soybean protein used in the present invention is preferably 300 to 10,000. The average molecular weight is more preferably 400 to 5000, still more preferably 500 to 3500, and even more preferably 550 to 3200, from the viewpoint of increasing the permeability to cells and obtaining a higher effect. Preferably it is 1000-2000. The average molecular weight of the degradation product can be measured by any method known to those skilled in the art, and for example, it can be easily measured by gel permeation chromatography (GPC) as described in the Examples below. .

  The blending amount of the soybean thermolysin degradation product blended in the composition of the present invention is not particularly limited as long as the effect of the present invention can be obtained, but is usually 0.0001 to 70% by weight, more preferably 0.001 based on the entire composition. ˜50 wt%, more preferably 0.001 to 30 wt%, and even more preferably 0.01 to 20 wt%.

Moreover, the blending ratio of the soybean thermolysin degradation product and the retinols in the composition of the present invention is not particularly limited as long as the effect of the present application can be achieved, but usually 1 × 10 ⁻⁸ IU to 1 × per 100 μg of soybean thermolysin degradation product. 10 ⁸ IU, preferably 1 × 10 ⁻⁷ IU to 1 × 10 ⁶ IU, more preferably 1 × 10 ⁻⁶ IU to 1 × 10 ⁴ IU, and even more preferably 1 × 10 ⁻⁵ IU to 1 × 10 ³ IU Particularly preferably, it should be in the range of 1 × 10 ⁻⁵ IU to 1 × 10 ⁻⁴ IU.

  In addition to the retinols and soybean thermolysin degradation product as described above, the composition of the present invention has the purpose of enhancing or supplementing the action of retinols or soybean thermolysin degradation product, or other useful effects on the composition of the present application. Whitening component, anti-inflammatory component, antibacterial component, cell activation component, astringent component, antioxidant component, acne improving component, biosynthesis synthesis component such as collagen, blood circulation promoting component, moisturizing component, anti-aging component Such various components can be blended in one kind or in combination of two or more kinds. Preferred are one or more of whitening components, anti-inflammatory components, antibacterial components, cell activation components, astringent components, antioxidant components, anti-aging components or moisturizing components. These components are not particularly limited as long as they can be used in the fields of pharmaceuticals, quasi drugs, foods, and cosmetics, and arbitrary components can be appropriately selected and used.

  Examples of whitening ingredients include placenta; arbutin; kojic acid; ellagic acid; phytic acid; lucinol; vitamins such as chamomile ET, pantothenic acid or derivatives thereof. Among these, as a preferable thing, pantothenic acid or its derivative (s), ellagic acid, and phytic acid can be mentioned. These whitening components may be used alone or in combination of two or more.

  A plant component having a whitening effect may be used as a whitening component, such as Iris (iris), almond, aloe, ginkgo, oolong tea, ages, ogon, lauren, hypericum, licorice, seaweed, cuckoo, gardenia, Kujin, chlorella, gobaishi, wheat, rice, rice haiga, oryzanol, rice bran, saishin, salamander, perilla, peony, senkyu, sakuhaku, soybeans, natto, tea, toki, pearl millet, garlic, hamamelis, safflower, buttonpi, yokuinin, Amethyst, asenyaku, acebi bracken, oyster, enoki, oyster (Diospyros kaki), cattle, black bean, gentian, genzin, salsa, string beans, shokuma, jujuro, sage, senko, radish, azalea, azalea, Ingredients derived from plants such as Toshin, Nigaki, Parsley, Holly, Hop, Marubahagi, Clove, and Licorice. Preferably, Iris (Iris), Aloe, Ginkgo, Oolong tea, Ages, Ogon, Oulen, Hypericum, Olysam, Seaweed, Cuckoo, Gardenia, Kujin, Gobaishi, Wheat, Rice, Rice bran, Saishin, Salamander, Perilla, Peonies, Senkyu , Sakuhakuhi, Tea, Toki, Toki-Senka, Hamelis, Safflower, Buttonpi, Yokuinin, Amethyst, Acalysia, Enoki, Oyster (Diospyros kaki), Cattle, Black Bean, Gentian, Salsa, Soyagen, Juju, Sage, Zenko, Daikon, Tsuji It is a plant-derived component of Tsukuhashigi, Toshin, Nigaki, Parsley, Holly, Hop, Clove, Licorice, and more preferably Iris (Iris), Aloe, Ginkgo, Ages, Ogon, Oulen, Hypericum, Hypericum Chinese pear, kujin, rice, rice bran, saishin, peony, senkyu, sakuhakuhi, tea, touki, eucalyptus, hamamelis, safflower, buttonpi, amethyst, asenyaku, enoki, oyster (Diospyros kaki), sage, radish, azalea, parsley, parsley , Plant-derived components of licorice and yakuinin. When these plant components are used in the composition of the present invention, the form of the plant component is not particularly limited, but can usually be used in the form of a plant extract (plant extract) or an essential oil. In addition, the inside of () described in the said plant component is the scientific name of the plant, an alias, or a crude drug name.

  When the whitening component is used, the proportion to be blended in the composition of the present application is preferably 0.0003 to 10% by weight, more preferably 0.01 to 5% by weight.

  When a plant component having a whitening effect is used as the whitening component, one or two or more kinds can be used in any combination depending on the purpose. When the plant component is used as a whitening component, the blending ratio in the composition of the present application is usually 0.00001 to 20% by weight, preferably 0.0001 to 15% by weight, more preferably in terms of extracts such as extracts and essential oils. Is 0.001 to 10% by weight.

  Anti-inflammatory components include allantoin, calamine, glycyrrhizic acid or derivatives thereof, glycyrrhetinic acid or derivatives thereof, zinc oxide, guaiazulene, tocopherol acetate, pyridoxine hydrochloride, menthol, camphor, turpentine oil, indomethacin, salicylic acid or derivatives thereof. . Preferred are allantoin, glycyrrhizic acid or a derivative thereof, glycyrrhetinic acid or a derivative thereof, guaiazulene, or menthol.

  When using the said anti-inflammatory component, the ratio mix | blended with this-application composition becomes like this. Preferably it is 0.0003 to 10 weight%, More preferably, it is 0.01 to 5 weight%.

  Antibacterial components include chlorhexidine, salicylic acid, benzalkonium chloride, acrinol, ethanol, benzethonium chloride, cresol, gluconic acid and derivatives thereof, popidone iodine, potassium iodide, iodine, isopropylmethylphenol, triclocarban, triclosan, photosensitizer 101 Photosensitive element 201, paraben, phenoxyethanol, 1,2-pentanediol, alkyldiaminoglycine hydrochloride and the like. Preferably, benzalkonium chloride, benzethonium chloride, gluconic acid and its derivatives, isopropylmethylphenol, triclocarban, triclosan, photosensitizer 101, photosensitizer 201, paraben, phenoxyethanol, 1,2-pentanediol, alkyldiamino hydrochloride Examples include glycine. More preferred are benzalkonium chloride, gluconic acid and its derivatives, benzethonium chloride, and isopropylmethylphenol.

  When using the said antibacterial component, the ratio mix | blended with this-application composition becomes like this. Preferably it is 0.0003 to 10 weight%, More preferably, it is 0.01 to 5 weight%.

  Cell activation components include amino acids such as γ-aminobutyric acid and ε-aminocaproic acid: thiamine, riboflavin, pyridoxine hydrochloride, pantothenic acids and other vitamins: glycolic acid, α-hydroxy acids such as lactic acid: tannin, flavonoids, Examples include saponin, allantoin, and photosensitive element 301. Preferred are amino acids such as γ-aminobutyric acid and ε-aminocaproic acid: vitamins such as thiamine, riboflavin, pyridoxine hydrochloride and pantothenic acids.

  When using the said cell activation component, the ratio mix | blended with this-application composition becomes like this. Preferably it is 0.0003 to 10 weight%, More preferably, it is 0.01 to 5 weight%.

  Examples of the astringent component include metal salts such as alum, chlorohydroxyaluminum, aluminum chloride, allantoin aluminum salt, zinc sulfate, and aluminum potassium sulfate; organic acids such as tannic acid, citric acid, lactic acid, and succinic acid. Alum, chlorohydroxyaluminum, aluminum chloride, allantoin aluminum salt, aluminum potassium sulfate, and tannic acid are preferred.

  When the astringent component is used, the ratio of the composition of the present application is usually 0.0003 to 10% by weight, preferably 0.01 to 5% by weight, more preferably 0.01 to 5% by weight.

  Antioxidant components include butylhydroxyanisole, dibutylhydroxytoluene, sodium bisulfite, erythorbic acid and its salts, flavonoids, glutathione, glutathione peroxidase, glutathione-S-transferase, catalase, superoxide dismutase, thioredoxin, taurine, thiotaurine, hypotaurine Etc. Preferred are thiotaurine, hypotaurine, thioredoxin, and flavonoid.

  When using an antioxidant component, the proportion of the composition of the present application is usually 0.0001 to 10% by weight, preferably 0.0001 to 5% by weight, and more preferably 0.001 to 5% by weight.

  Examples of the anti-aging component include pangamic acid, kinetin, ursolic acid, turmeric extract, sphingosine derivative, silicon, silicic acid, N-methyl-L-serine, and mevalonolactone. Preferably, it is kinetin.

  When using the said anti-aging component, the ratio mix | blended with this-application composition becomes like this. Preferably it is 0.0003 to 10 weight%, More preferably, it is 0.01 to 5 weight%.

  As moisturizing ingredients, amino acids such as alanine, serine, leucine, isoleucine, threonine, glycine, proline, hydroxyproline, glucosamine, theanine and derivatives thereof; polyvalent such as glycerin, 1,3-butylene glycol, propylene glycol, polyethylene glycol, etc. Examples include alcohols; sugar alcohols such as sorbitol; phospholipids such as lecithin and hydrogenated lecithin; mucopolysaccharides such as propylene glycol hyaluronate, heparin and chondroitin; and NMF-derived components such as lactic acid, sodium pyrrolidonecarboxylate, and urea. Preferred are alanine, serine, glycine, proline, hydroxyproline, glucosamine, theanine, collagen, collagen peptide, glycerin, 1,3-butylene glycol, hydrogenated lecithin, propylene glycol hyaluronate, heparin, chondroitin, lactic acid, pyrrolidone carboxylic acid Sodium acid.

  When using a moisturizing component, the proportion to be blended in the composition of the present application is usually 0.1 to 10% by weight, preferably 0.5 to 7.5% by weight, more preferably 0.5 to 5% by weight. Can do.

  In addition to the above components, the composition of the present invention may be appropriately mixed with components usually used in the field of pharmaceuticals, quasi drugs, cosmetics or foods, depending on the use or dosage form of the composition. The component that can be blended is not particularly limited, but for example, amino acids, alcohols, polyhydric alcohols, saccharides, gums, polysaccharides and other high molecular compounds, surfactants, solubilizing components, fats and oils, transdermal Absorption-promoting ingredients, antiseptic / antibacterial / bactericidal agents, pH adjusters, chelating agents, antioxidants, enzyme components, binders, disintegrating agents, lubricants, fluidizing agents, cooling agents, minerals, cell activation Agents, nourishing tonics, excipients, thickeners, stabilizers, preservatives, isotonic agents, dispersants, adsorbents, disintegration aids, wetting agents or wetting regulators, dampening agents, coloring agents, wearing Perfume or fragrance, fragrance, reducing agent, solubilizer, solubilizer, foaming agent, thickener or thickener, solvent, base, emulsifier, plasticizer, buffer, brightener, etc. be able to. In particular, by adding a surfactant, a solubilizing component or fats and oils, the stability of retinols in the preparation can be increased, and when the composition is for external use, the usability of the preparation should be further improved. Can do. When the composition of the invention is an external composition, it is preferable to further blend a transdermal absorption promoting component.

  Examples of the surfactant used herein include POE-branched alkyl ethers such as polyoxyethylene (hereinafter referred to as POE) -octyldodecyl alcohol and POE-2-decyltetradecyl alcohol; POE-oleyl alcohol ether and POE-cetyl alcohol. POE-alkyl ethers such as ethers; sorbitan esters such as sorbitan monooleate, sorbitan monoisostearate and sorbitan monolaurate; POE-sorbitan monooleate, POE-sorbitan monoisostearate, and POE-sorbitan monolaurate POE-sorbitan esters such as glycerol; glycerol fatty acid esters such as glycerol monooleate, glycerol monostearate, and glycerol monomyristate; POE-glycerol monoo POE-cured fatty acid esters such as reate, POE-glycerin monostearate, and POE-glycerin monomyristate; POE-cured such as POE-dihydrocholesterol ester, POE-cured castor oil, and POE-cured castor oil isostearate Castor oil fatty acid ester; POE-alkyl aryl ether such as POE-octylphenyl ether; Glycerin alkyl ether such as monoisostearyl glyceryl ether and monomyristyl glyceryl ether; POE such as POE-monostearyl glyceryl ether and POE-monomyristyl glyceryl ether -Glycerin alkyl ethers; diglyceryl monostearate, decaglyceryl decastearate, decaglyceryl decaisostearate, and diglyceride Various nonionic surfactants such as polyglycerin fatty acid esters such as rildiisostearate: or naturally derived surfactants such as lecithin, hydrogenated lecithin, saponin, surfactin sodium, cholesterol, bile acids, etc. be able to. These surfactants may be used alone or in any combination of two or more.

  When a surfactant is used, the blending ratio in the composition of the present application is not particularly limited as long as it does not interfere with the effects of the present invention, and usually 0.01 to 30% by weight in the composition of the present application. Can be appropriately selected and used within the range included in the above. From the viewpoint of the stability of the active ingredient in the composition of the present application, it is preferably 0.1 to 20% by weight, more preferably 0.5 to 10% by weight.

  Examples of solubilizing components include lower alcohols such as ethanol, polyhydric alcohols such as glycerin and ethylene glycol, hydrogenated soybean phospholipid, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene lanolin alcohol, polyoxyethylene castor oil, poly Examples thereof include oxyethylene hydrogenated castor oil, polyoxyethylene sterol, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, and the like. Preferably, ethanol, glycerin, ethylene glycol, diethylene glycol, dipropylene glycol, hydrogenated soybean phospholipid, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene lanolin alcohol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxy Ethylene alkyl ether, more preferably ethanol, glycerin, ethylene glycol, diethylene glycol, dipropylene glycol, and hydrogenated soybean phospholipid. These solubilizing components may be used alone or in any combination of two or more.

  When using these solubilizing components, the blending ratio in the composition of the present application is not particularly limited as long as it does not interfere with the effects of the present invention, and usually 0.01 to 70% by weight in the composition of the present application. Can be appropriately selected and used within such a range that is included in the ratio. From the viewpoint of the stability of the active ingredient in the present application composition, it is preferably 0.1 to 50% by weight, more preferably 0.1 to 30% by weight.

  Examples of fats and oils include synthetic fats and oils such as medium-chain fatty acid triglycerides; soybean oil, rice oil, rapeseed oil, cottonseed oil, sesame oil, safflower oil, castor oil, olive oil, cacao oil, coconut oil, sunflower oil, palm oil, flax oil Vegetable oil such as perilla oil, perilla oil, shea oil, monkey oil, palm oil, tree wax, jojoba oil, grape seed oil, and avocado oil; animal fat such as mink oil, egg yolk oil, beef tallow, milk fat, and pork fat; beeswax Waxes such as whale wax, lanolin, carnauba wax, candelilla wax; hydrocarbons such as liquid paraffin, squalene, squalane, microcrystalline wax, ceresin wax, paraffin wax, petrolatum; lauric acid, myristic acid, stearic acid, olein Natural and synthetic fatty acids such as acids, isostearic acid and behenic acid; cetanol, stearyl alcohol Natural and synthetic higher alcohols such as ru, hexyl decanol, octyl decanol, lauryl alcohol; esters and ethers such as isopropyl myristate, isopropyl palmitate, octyldodecyl myristate, octyldodecyl oleate, cholesterol oleate; silicone oil, etc. It is done. These fats and oils may be used alone or in any combination of two or more.

  When these fats and oils are used, the blending ratio in the composition of the present application is not particularly limited as long as the effect of the present invention is not hindered, and usually 0.01 to 70% by weight in the composition of the present application. It can be appropriately selected and used within a range that is included in a proportion. From the viewpoint of the stability of the active ingredient in the composition of the present application, the range is preferably 0.1 to 60% by weight, more preferably 0.1 to 50% by weight.

  The composition of the present invention can take a dosage form usually used for pharmaceuticals, quasi drugs, cosmetics or foods, and is usually a solid, semi-solid or liquid. Specifically, tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules , Dry syrups, liquids (including drinks, suspensions, syrups), gels, liposomes, extracts, tinctures, lemonades, ointments, jellys, etc. . In addition, pastes, mousses, gels, liquids, emulsions, creams, sheets (substrate support), aerosols can be added by blending other solvents and commonly used bases as necessary. It can be prepared in various desired forms such as spray.

  These dosage forms can be produced by conventional methods in the art. For example, in the case of a semi-solid agent, in addition to the retinols and soybean thermolysin decomposition product as described above, the above-mentioned optional components are blended and mixed as necessary, and other solvents and commonly used externally used as necessary. Prepared in various desired forms such as paste, mousse, gel, liquid, emulsion, cream, sheet (substrate support), aerosol, spray, etc. by blending composition base can do.

  The composition of the present invention is not particularly limited as long as the effects of the present invention are achieved. For example, pharmaceuticals, quasi-drugs, foods (including confectionery, health foods, nutritional supplements (balance nutritional foods, supplements, etc.)), Including nutritional functional foods and foods for specified health use, etc., and in cosmetics, makeup cosmetics such as foundations, lipsticks, mascaras, eye shadows, eyeliners, eyebrows and beauty nails; emulsions, creams, lotions, Basic cosmetics such as oils and packs; cleansing agents such as facial cleansers, cleansing and body cleaning agents, bathing agents, and the like.

  The composition of the present invention can be suitably used as an internal composition or an external composition.

  Since the composition of the present invention has a remarkable ability to promote hyaluronic acid production, it can be suitably used to promote hyaluronic acid production in cells. For example, the composition of the present invention is a composition for treating or preventing various disorders (rheumatoid, arthritis, osteoarthritis, joint disorders such as osteoarthritis, inflammatory disorders, etc.) associated with reduction or degeneration of hyaluronic acid, hyaluronic acid A composition for preventing or treating cosmetic problems caused by acid reduction or denaturation (for example, prevention of skin wrinkles or tarmi caused by reduction or modification of hyaluronic acid due to UV exposure, aging, etc.) And / or a composition for improvement, a composition for preventing and / or improving the skin elasticity or elasticity due to reduction or modification of hyaluronic acid, and the like.

  In addition, since the composition of the present invention has the ability to promote proliferation of fibroblasts, for example, a composition for preventing or treating cosmetic problems associated with, for example, reduction or inactivation of fibroblasts (for example, exposure to ultraviolet rays) Composition for prevention and / or improvement of skin wrinkles or tarmi caused by decrease or inactivation of fibroblasts due to aging or aging, etc., or skin caused by reduction or inactivation of fibroblasts The composition can be suitably used as a composition for preventing and / or improving the decrease in elasticity or elasticity.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1
Preparation of Thermolysin Degradation Product of Soy Protein 50 g of powdered separated soy protein (product name “PR-800”, manufactured by Fuji Oil Co., Ltd.) was dispersed in 2 L of distilled water and adjusted to pH 8.0 with 0.1 N NaOH. 500 mg of thermolysin (EC3.4.24.4, derived from Bacillus thermoproteolyticus, product name “Samorose PC10F”, manufactured by Daiwa Kasei Co., Ltd., 100 units / mg) was added, and degradation was performed at 60 ° C. for 15 hours. After the reaction, the thermolysin was inactivated by boiling at 100 ° C. for 10 minutes. After allowing to cool, 25 g of filter aid (Radiolite 500, Showa Chemical Industry Co., Ltd.) was added and stirred, followed by filtration. The obtained filtrate was concentrated under reduced pressure to 500 ml, and then freeze-dried to finally obtain about 26 g of a soybean protein thermolysin degradation product.

  The average molecular weight of the decomposition product thus obtained was measured by the GPC method. 100 mg of soybean thermolysin degradation product after lyophilization was dissolved in 2.0 ml of 0.1 M sodium phosphate buffer (pH 7.0) to prepare a test solution. A column (φ2.6 × 100 cm) packed with Sephadex G25 (Medium type, manufactured by Amersham Biosciences) was equilibrated with the same 0.1 M sodium phosphate buffer (pH 7.0). The column was loaded with 2.0 ml of the test solution and eluted at a flow rate of 1.0 ml / min. Insulin (bovine pancreas origin, Sigma, molecular weight 5733), Insulin A chain (bovine pancreas origin, Sigma, molecular weight 2532), and Bradykinin (Sigma, molecular weight 1050) are used as peptide preparations with known molecular weights. It was. Peptides were detected at 214 nm, and the molecular weight distribution and average molecular weight were estimated from the elution time. As a result, the average molecular weight of the soybean thermolysin degradation product obtained by the above method was estimated to be about 1500.

Example 2
Hyaluronic acid production assay in epidermal keratinocytes Human normal epidermal keratinocytes (NHEK / B-3, Kurashiki Boseki Co., Ltd.) were cultured in 48-well culture plates. More specifically, the cells were seeded at a density of 0.8 × 10 ⁴ cells / well and cultured at 37 ° C. in an environment of 5% carbon dioxide gas and 95% air for 4 days. As a culture solution, HuMedia KG-2 (manufactured by Kurashiki Boseki Co., Ltd.) was used in an amount of 400 μL per well. Thereafter, the culture solution was removed, and the medium was replaced with a 400 μL medium in which the test drug shown in Table 1 below was dissolved, and cultured. On the other hand, a medium supplemented with 400 μL of neither retinol nor soybean thermolysin degradation product was used as a control. After culturing for 2 days, the culture solution was collected, and the hyaluronic acid concentration secreted in the culture solution was quantified by an enzyme-linked immunoassay (hyaluronic acid measurement kit; manufactured by Seikagaku Corporation). Based on the quantification results, the amount of hyaluronic acid in each test culture was calculated with the amount of hyaluronic acid in the control culture as 100%. The results are summarized in Table 1.

This result shows that hyaluronic acid production is significantly increased in cells cultured in a culture solution containing both retinol and soybean thermolysin degradation product, compared to when cultured in a retinol alone culture solution. It was demonstrated that the degradation product obtained by treating with protease can remarkably enhance the ability of retinol to promote hyaluronic acid production.
It was also recognized that the hyaluronic acid production promoting action obtained by combining retinol and soybean thermolysin degradation product was synergistically remarkably high.

  Furthermore, the number of viable cells was counted by WST-1 method for the cells after collecting the culture solution. More specifically, the medium was changed to that added with WST-1 reagent at a ratio of 10 μl in 400 μl medium, and after culturing for 45 minutes, 200 μl of the supernatant was taken and the absorbance was measured. As a result, no significant decrease in the number of viable cells was observed by culturing in a culture solution using a combination of retinol and soybean thermolysin degradation product, and it was confirmed that the cells can be safely used for a living body.

Example 3
Skin fibroblast proliferation promotion test Human normal skin-derived fibroblasts (NHDF; CRL-2089) were cultured in 96-well culture plates. More specifically, the cells were seeded at a density of 1280 cells / well and cultured at 37 ° C. in an environment of 5% carbon dioxide and 95% air for 24 hours. As the culture solution, 200 μl of a medium containing 10% by weight of fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) was used. Subsequently, the culture medium was replaced with a serum-free medium from which FBS was removed, and further cultured for 24 hours. Thereafter, the culture broth was removed, and the test drugs shown in Table 2 below were replaced with 200 μl of serum-free medium dissolved at the respective concentrations, and cultured (retinols were first dissolved in absolute ethanol with 200 because they are fat-soluble. After adjusting to a double concentration, the ethanol solution was adjusted by adding 1 μl of 200 μl). On the other hand, 200 μl of serum-free medium to which neither retinol nor soybean thermolysin degradation product was added was used as a control. After further culturing for 48 hours, the number of viable cells in each well was measured by the WST-8 method having higher sensitivity than the WST-1 method (Cell Counting Kit-8; manufactured by Dojindo Laboratories). Based on the measurement results, the number of viable cells (%) in each test drug addition group was calculated with the number of viable cells in the control as 100%. The results are summarized in Table 2.

  From these results, it was confirmed that the number of fibroblasts increased synergistically when cultured in a culture solution containing both retinol and soybean thermolysin degradation product, compared to the case where each was cultured alone. It was.

  Formulation examples are given below, but the present invention is not limited to these examples.

Formulation Example 1: Emulsion [component] [ratio]
All-trans retinol 0.07g
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.5g
Squalane 2.0g
Soybean oil 0.6g
Liquid paraffin 5.0g
Cetanol 0.5g
Glyceryl monostearate 2.0g
POE (25) cetyl ether 2.0g
Glycerin 4.0g
1,3-butylene glycol 6.0 g
pH adjuster appropriate amount preservative appropriate amount perfume appropriate amount
Purified water
100.0g

Formulation Example 2: Cream [ingredients] [Ratio]
Retinol palmitate 0.14g
Thermolysin degradation product of soybean protein (average molecular weight 1500) 1.0g
Vaseline 1.0g
Squalane 5.0g
Sunflower oil 0.1g
Liquid paraffin 10.0g
Stearic acid 1.5g
Stearyl alcohol 2.0g
Glyceryl monostearate 2.0g
POE (20) cetyl ether 3.0g
Glycerin 6.0g
1,3-butylene glycol 8.0g
Dibutylhydroxytoluene 0.1g
pH adjuster appropriate amount preservative appropriate amount perfume appropriate amount
Purified water
100.0g

Formulation Example 3: Cream [ingredients] [Ratio]
δ-Tocopheryl retinoate 0.1 g
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.1g
Glycerin 6.0g
Tri (caprylic acid / capric acid) glyceryl 0.9g
Carboxy vinyl polymer 0.8g
Polyoxyethylene cetyl ether 3.5g
3.5 g of glyceryl monostearate
Cetanol 3.0g
Dibutylhydroxytoluene 0.03g
pH adjuster appropriate amount preservative appropriate amount perfume appropriate amount
Purified water
100.0g

Formulation Example 4: Cream [ingredients] [Ratio]
δ-Tocopheryl retinoate 0.25g
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.1g
Glycerin 6.0g
Tri (caprylic / capric) glyceryl 2.25g
Carboxy vinyl polymer 0.8g
Polyoxyethylene cetyl ether 3.5g
3.5 g of glyceryl monostearate
Cetanol 3.0g
Dibutylhydroxytoluene 0.03g
pH adjuster appropriate amount preservative appropriate amount perfume appropriate amount
Purified water
100.0g

Formulation Example 5: Tablets for internal use (in 1 tablet)
[Ingredient] [Ratio]
Total trans-retinol 0.05mg
Soybean protein thermolysin degradation product (average molecular weight 1500) 50.0mg
Vitamin B1 1.0mg
Vitamin B2 0.5mg
Fragrance 0.625mg
Sweetener 0.625mg
Sucrose fatty acid ester 3.75mg
Maltitol 68.45mg
125.0mg

Claims (2)

(A) retinol and retinoic acid; retinal; ester derivatives of retinoic acid or its isomers; retinol, retinoic acid, or isomers of retinal; retinol or ester derivative of the isomer thereof; ether derivatives of retinol or its isomers; Retinol, retinoic acid, retinal or their epoxide derivatives; Retinol, retinoic acid, retinal or their isomers with hydrogen atoms removed or hydrogen atoms added; or C 1-6 alkyl groups Any one of the above derivatives further substituted with an alkoxy group having 1 to 6 carbon atoms, a halogen atom, an amino group, a hydroxy group, a carboxy group, or an oxo group, and a salt thereof. When,
(B) A composition containing a thermolysin degradation product having an average molecular weight of 300 to 10,000 of soy protein. The composition of claim 1, which can be used to promote hyaluronic acid production in cells.