JP5084393B2 - Composition having collagen production promoting ability and / or fibroblast proliferation promoting ability - Google Patents

Description

  The present invention relates to a useful novel composition having an ability to promote collagen production, a useful novel composition having an ability to promote 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. Among these, collagen plays an important role in the living body as described later.

  Collagen is a major protein that constitutes the connective tissue of animals, and collagen occupies nearly 30% of the total protein of the human body. Since the main function of collagen is in the formation of a skeletal structure of living tissue, it is widely distributed in skin, cartilage tissue, cornea, heart, liver, etc. as the main component constituting the skeletal structure of animal tissue form. Collagen acts specifically on cell adhesion, cell differentiation and proliferation, and also has a role as a regulator of cell function. Collagen reduction is caused by corneal disorders such as corneal ulcer, rheumatism, It may cause various diseases such as arthritis, osteoarthritis, joint disorders such as osteoarthritis, and inflammatory diseases. In recent years, it has been found that collagen also has a function of suppressing the progression of ulcers in the digestive organs and suppressing an increase in blood pressure.

  In the cutaneous dermal extracellular matrix, collagen fibers form a mesh-like bundle to maintain the tissue morphology. Collagen fibers form thick and elastic fiber bundles as they mature and moderate cross-linking proceeds, giving moderate tension in young skin. However, in aging skin, collagen fibers in the dermal extracellular matrix are remarkably reduced, and the inherent elasticity is lost. According to one theory, although collagen in humans occupies 70% of the dermis of the skin peaking at around 20 years old, it is said that it decreases to about 20% of the peak in old age. And when the amount of collagen decreases in this way, wrinkles and tarmi are formed on the skin. Changes in the structure of collagen fiber bundles in hairless mice due to photoaging have been examined in detail (see Non-Patent Document 1). Collagen fiber bundles are formed so that wrinkles are formed in hairless mice irradiated with UVB and coincide with the formation of wrinkles. It has been shown that the structure collapses and skin elasticity decreases. It is also known that collagen is excellent in water retention function.

  In order to improve the state caused by the decrease in collagen, various collagen synthesis promoting substances have been found. For example, plant extracts such as retinoic acid (Non-patent document 2), three amino acids composed of glycine, proline and alanine (Patent document 1), licorice, Sakuhakuhi, aloe, sugina, goldfish, grasshopper, oyster or gentian (patent document) 2), TGF-β, ascorbic acids, collagen degradation products (Patent Document 3) and the like are known.

  The fibroblasts, which are the main cells of the dermis, include collagen as described above, elastin that maintains the elasticity of the skin, and mucopolysaccharides that significantly affect the skin's moisturizing power (eg, hyaluronic acid, chondroitin) Sulfuric acid etc.). 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-A-7-194375 JP 2001-206835 A JP 2000-309521 A Fragrance Journal, 4, p36-37, 1998 R. Marks et al., British Journal of Dermatology, 122, 91-98, 1990

  In view of the importance of the role of collagen in the living body as described above, development of a further useful new composition having a remarkable ability to promote collagen production is desired. In view of such conventional problems, an object of the present invention is to provide a useful novel composition having a remarkable ability to promote collagen 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 decomposed soybean protein with a specific protease called thermolysin, and at least one selected from the group consisting of collagen, gelatin, and degradation products thereof. By using in combination with the resulting product, the ability to promote collagen production in cells is significantly promoted compared to using at least one selected from the group consisting of collagen, gelatin, and their degradation products alone. As a result of further finding and further investigation, it has been found that the combined use of both the above components synergistically promotes the proliferation of fibroblasts, and the present invention has been completed.

Accordingly, the present invention provides the following.
(1) (A) at least one selected from the group consisting of collagen, gelatin, and degradation products thereof;
(B) A composition containing a thermolysin degradation product of soybean protein.
(2) The composition according to item (1), which can be used to promote collagen 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 collagen production. The composition of the present invention can be used to promote collagen production in cells, such as 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 prophylactic or therapeutic composition. The present invention also provides a useful novel composition having the ability to promote fibroblast proliferation. In addition, as shown in the examples described later, it is suggested that the dramatically high collagen production promoting action obtained by the composition of the present application is not due only to the growth promoting effect of fibroblasts producing collagen. ing.

  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 relates to a composition containing (A) at least one selected from the group consisting of collagen, gelatin, and a degradation product thereof, and (B) a thermolysin degradation product of soybean protein.

  At least one selected from the group consisting of collagen, gelatin (denatured collagen), and degradation products thereof (collagen degradation product or gelatin degradation product) used in the present invention [hereinafter, these are collectively referred to as “ "Collagens" may be used as long as it can be used in the fields of pharmaceuticals, quasi drugs, cosmetics, and foods.

  As collagen or gelatin, collagen obtained by extracting from connective tissues such as skin, bones, tendons, etc. of animals such as cattle, pigs, fish, chickens, etc., gelatin obtained by heat denaturing those collagens, etc. can be used. . The type of collagen is not particularly limited, and collagens such as type I to type XIII, or some of these collagen mixtures can be used. Commercially available collagen or gelatin can also be used.

  Collagen degradation products or gelatin degradation products can be obtained by degrading collagen or gelatin by any method. For example, collagen or gelatin can be degraded by proteolytic enzymes, decomposed with acid, alkaline It can be obtained by decomposing using a method or a method using a combination of these. Preferably, a degradation product obtained by degrading collagen or gelatin with a proteolytic enzyme is used.

  Such an enzyme is not particularly limited. For example, collagenase (for example, collagenase derived from bacteria such as Clostridium histolyticum, Streptomyces parvulus, actinomycetes or fungi, etc. can be used, and gene recombination technology can be used. May be a collagenase obtained by producing these collagenases by other fungi), trypsin, chymotrypsin, subtilisin, elastase, proline-specific protease, protease produced by Streptococcus microorganisms, papain, pepsin, Thermolysin, carboxypeptidase Y, protease produced by Aspergillus, protease produced by Streptomyces microorganisms, protease produced by Rhizopus microorganisms, protease produced by lactic acid bacteria It can be used singly or in combination of two or more. Preferably, a degradation product hydrolyzed by collagenase is used in the present invention. Particularly preferably, the amino acid sequence (Gly-XY) n specific to collagen (wherein Gly represents a glycine residue, X and Y represent any amino acid residue other than a glycine residue, and A hydrolyzate hydrolyzed by collagenase that specifically cleaves the amino terminal side of the glycine residue (which may be the same or different), and most preferably a bacterium such as Clostridium histolyticum or Streptomyces parvulus A hydrolyzate hydrolyzed with collagenase having cleavage specificity as described above derived from actinomycetes or fungi is used.

  The degradation product of collagen or gelatin used in the present invention includes (Gly-XY) n (wherein Gly represents a glycine residue, and X and Y represent any amino acid residue other than a glycine residue. It may be the same as or different from each other, and n is preferably an integer of 1 to 3). A tripeptide having a (Gly-XY) sequence is preferably 20 to 100% by weight, and more preferably 25 to 100% by weight, based on the entire collagen or gelatin degradation product. Such a degradation product of collagen or gelatin causes collagenase that specifically cleaves the amino terminal side of the glycine residue of collagen-specific amino acid sequence (Gly-XY) n to act on collagen or gelatin. For example, it can be produced by a method known in the art according to the method described in JP-A-7-82299.

  The degradation product of collagen or gelatin used in the present invention is not particularly limited as long as the effect of the present invention can be obtained, but preferably has a molecular weight of 100 or more, more preferably has a molecular weight of 100 to 20000, more preferably 280 to 15000. More preferably, it is preferable to use those within the range of 280-9800. The average molecular weight is not particularly limited as long as the effect of the present invention can be achieved, but is usually a degradation product of collagen or gelatin that is in the range of 280-20000, preferably 280-15000, more preferably 280-9800. Should be used. The molecular weight and the average molecular weight can be measured by any method known to those skilled in the art, and can be easily measured by, for example, a gel filtration chromatography (GFC) method or a gel permeation chromatography (GPC) method. Moreover, the method of fractionating the decomposed product based on the molecular weight or the average molecular weight can also be performed by any fractionation method known to those skilled in the art.

  Commercially available products can also be suitably used for the collagen or gelatin degradation product used in the present invention. Examples of commercially available degradation products of collagen or gelatin include, for example, collagen tripeptide F, collagen tripeptide M30, M60, M90, HACP-01, HACP-02, HACP-GR, HACP manufactured by Zerais Co., Ltd. -FL1, HACP-U2, HACP-S3, Collagen Peptide 700F from Nitta Gelatin Co., Collagen Peptide SCP-5000, Fish Collagen Peptide WP from Maruha Co., Ltd., Marine from Nippon Kayaku Food Techno Co., Ltd. Examples include, but are not limited to, collagen-rich 500 and C-LAP manufactured by Nippon Ham.

  In this invention, at least 1 type selected from the group which consists of said collagen, gelatin, and those decomposition products may be used individually by 1 type, and 2 or more types may be used in arbitrary combinations.

  The amount of collagen to be 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 0.00001 to 20% by weight, more preferably 0.0001 to 10% with respect to the entire composition. % By weight, more preferably 0.0001 to 5% by weight, particularly preferably 0.001 to 3% by weight.

  In the present invention, a thermolysin degradation product of soybean protein is further used (hereinafter, this product 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 the ability to promote collagen production in cells and / or the ability to promote proliferation of fibroblasts 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 / absence of the ability to promote collagen production and / or the presence / absence of the ability to promote fibroblast proliferation can be confirmed, for example, as described in Examples below.

In this specification, the term “having the ability to promote collagen production in cells” means that when a test substance is allowed to act on cells, the production of collagen in the cells is less than when the test substance is not allowed to act on cells. Means that the amount increases. In certain embodiments, the cell in the term refers to fibroblasts, and in certain embodiments refers to dermal fibroblasts.
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. For example, it can be easily measured by gel permeation chromatography (GPC) as described in the Examples below. it can.

  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%, even more preferably 0.01 to 20 wt%.

  In addition, the weight ratio of the collagens to the soybean thermolysin degradation product in the composition of the present invention is not particularly limited as long as the effect of the present invention can be achieved, but usually the soybean thermolysin degradation product is 0. It is good to set it within the range of 001 to 500 parts by weight, preferably 0.01 to 250 parts by weight, more preferably 0.1 to 100 parts by weight.

  In addition to the collagens and soybean thermolysin degradation product as described above, the composition of the present invention has the purpose of enhancing or supplementing the action of collagens or soybean thermolysin degradation product, or other useful effects on the composition of the present application. Whitening ingredients, anti-inflammatory ingredients, antibacterial ingredients, cell activation ingredients, astringent ingredients, antioxidant ingredients, acne improving ingredients, hyaluronic acid and other bio-component synthesis promoting ingredients, blood circulation promoting ingredients, moisturizing ingredients, anti-aging Various components such as components can be blended in one or a combination of two or more. 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 the whitening component include placenta; arbutin; kojic acid; ellagic acid; phytic acid; lucinol; chamomile ET; vitamins such as vitamin A or a derivative thereof, pantothenic acid or a derivative thereof, and the like. Among these, as a preferable thing, pantothenic acid or its derivative (s), ellagic acid, phytic acid, vitamin A or its derivative (s) 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, and Licorice, and more preferably Iris (Iris), Aloe, Ginkgo, Ages, Ogon, Ouren, Hypericum , Gardenia, kujin, rice, rice bran, saishin, peony, senkyu, sakuhaku, tea, touki, kansenka, hamameris, safflower, buttonpi, amethyst, asenyaku, enoki, oyster (Diospyros kaki), sage, radish, azalea, azalea, azalea The plant origin component of a hop, a licorice, and a Yokuinin is mentioned. 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: vitamins such as retinol, thiamine, riboflavin, pyridoxine hydrochloride and pantothenic acids: α-hydroxy acids such as glycolic acid and lactic acid: tannin, Examples include flavonoids, saponins, allantoin, and photosensitizer 301. Preferred are amino acids such as γ-aminobutyric acid and ε-aminocaproic acid: vitamins such as retinol, 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.

  Antiaging components include retinoids (retinol, retinoic acid, retinal, etc.), pangamic acid, kinetin, ursolic acid, turmeric extract, sphingosine derivatives, silicon, silicic acid, N-methyl-L-serine, mevalonolactone, and the like. Preferred are retinoid (retinol, retinoic acid, retinal, etc.) and 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, when the composition of the present invention is a composition for external use, the usability of the preparation can be further improved by blending a surfactant, a solubilizing component or fats and oils. Moreover, when it is an external composition, it is preferable to mix | blend a transdermal absorption promotion component further.

  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 above-described collagens and soybean thermolysin degradation product, the above-mentioned optional components are blended and mixed as necessary, and other solvents and externally used externally 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.)), In addition, makeup cosmetics such as foundation, lipstick, mascara, eye shadow, eyeliner, eyebrow, and nail polish; lotion (lotion and milky lotion) Etc.), basic cosmetics such as creams, oils, packs, gels (gel-like serums, gel creams, etc.); washings such as facial cleansers, cleansings, body washings, bathing agents, and the like.

  The composition of the present invention can be suitably used as a composition for internal use or as an external composition. However, in order to exert a higher direct effect on skin aging symptoms, it is preferably used as an external composition. Used.

  Since the composition of the present invention has a remarkable ability to promote collagen production, it can be suitably used to promote collagen production in cells. For example, the composition of the present invention can be used to treat various disorders associated with reduction or degeneration of collagen (corneal disorders such as corneal ulcer, rheumatism, arthritis, osteoarthritis, joint disorders such as osteoarthritis, inflammatory disorders, etc.) Composition for prevention, composition for treatment or prevention of digestive organ ulcer, composition for treatment or prevention for suppressing an increase in blood pressure, for prevention or treatment of cosmetic problems caused by decrease or degeneration of collagen, etc. Composition (for example, a composition for preventing and / or improving skin wrinkles or tarmi caused by reduction or degeneration of collagen due to UV exposure, aging, etc., or skin caused by reduction or degeneration of collagen, etc. The composition can be suitably used as a composition for preventing and / or improving the decrease in elasticity or elasticity.

  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
Collagen production assay in skin fibroblasts (1)
Human normal skin-derived fibroblasts (CRL-1836; ATCC) were cultured in a 48-well culture plate until subconfluent. More specifically, the cells were seeded at a density of 1.0 × 10 ⁴ cells / well and cultured at 37 ° C. in an environment of 5% carbon dioxide gas and 95% air for 2 days. As a culture solution, a medium containing 10% by weight of fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (D-MEM) at a concentration of 10% by weight was used in 400 μl per well. Subsequently, the culture medium was replaced with a serum-free medium from which FBS was removed, and further cultured for 1 day. Thereafter, the culture medium was removed, and the test drugs shown in Table 1 below were replaced with 400 μl of serum-free medium in which each concentration was dissolved and cultured. On the other hand, 400 μl of a serum-free medium to which neither collagens nor soybean thermolysin degradation product was added was used as a control. After culturing for 3 days, the culture solution is collected, and the concentration of type I collagen secreted in the culture solution is quantified by an enzyme-linked immunoassay (Anti-Human Procollagen type C-peptide EIA Kit; manufactured by Takara Bio Inc.). did. Based on the quantitative results, the amount of collagen in each test culture was calculated with the amount of type I collagen in the control culture as 100%. The results are summarized in Table 1.

  The collagen tripeptide F (also referred to as CTP-F; manufactured by Zerais Co., Ltd.) used in this example is a collagen degradation product obtained by degrading fish-derived (fish skin-derived) collagen with collagenase. Yes, (Gly-XY) (wherein Gly represents a glycine residue, and X and Y represent any amino acid residue other than a glycine residue, and may be the same or different from each other) ) Is known to contain about 30% tripeptides (see Fragrance Journal, 3, p54-60, 2006; Fragrance Journal, 11, p61-67, 2003).

Moreover, the molecular weight of CTP-F was measured. The measurement was performed by the GFC method under the following measurement conditions. The molecular weight distribution was determined using a calibration curve prepared based on the elution time and molecular weight of a molecular weight standard product. Molecular weight standards include (1) Gly-Gly-Gly (molecular weight 189, SIGMA), (2) Gly-Gly-Tyr-Arg (molecular weight 451, SIGMA), (3) Angiotensin II, Human (molecular weight 1050, Calbiochem ], (4) Cytochrome C from equine heart [molecular weight 12500, SIGMA] was used.
[Measurement condition]
Detector: UV spectrophotometer SPD-10A (manufactured by Shimadzu Corporation)
Column: TSKgel G2500PW ^XL (manufactured by Tosoh Corporation, φ7.8mm300mm)
-Column temperature: rt
・ Mobile phase: A mixture of water, acetonitrile and trifluoroacetic acid (55: 45: 0.1 (v / v / v))
・ Flow rate: 0.5 ml / min ・ Measurement wavelength: 220 nm
・ Measurement range: 0.2AUFS
Sample concentration: 0.2 mg / ml
・ Injection volume: 20 μl
As a result, the molecular weight of CTP-F was estimated to be in the range of about 100 to about 9800.

  From the results shown in Table 1 above, in the cells cultured in the culture solution using both the collagen degradation product and the soybean thermolysin degradation product, the collagen produced in the cells is better than when cultured in the culture solution of the collagen degradation product alone. It was demonstrated that the degradation product obtained by treating soybean protein with a specific protease called thermolysin can remarkably enhance the collagen production promoting ability of the collagen degradation product.

Further, the number of viable cells was counted by WST-8 method for the cells after collecting the culture solution. More specifically, the medium was changed to that added with WST-8 reagent at a ratio of 40 μl in 400 μl medium, and then cultured for 35 minutes, and then 200 μl of the supernatant was taken and the absorbance was measured. As a result, it was confirmed that there was no significant decrease in the number of viable cells by culturing in a culture solution in which a collagen degradation product and a soybean thermolysin degradation product were used in combination, and that it was safe to use for living bodies It was.
Example 3
Preparation of molecular weight fraction of soybean thermolysin degradation product In order to investigate whether the ability to promote collagen production by collagens can be enhanced using the molecular weight fraction of soybean thermolysin degradation product, as a test material in Example 1 In addition to the prepared soybean thermolysin degradation product, a molecular weight fraction of the soybean protein thermolysin degradation product was prepared.

  Specifically, 1 g of defatted soybean powder (trade name Profam 974, manufactured by ADM Far East Co., Ltd.) was dispersed in 40 ml of distilled water, and adjusted to pH 8.5 with 0.1 N NaOH. 50 mg of thermolysin (product name “Samoase PC10F”, manufactured by Daiwa Kasei Co., Ltd.) was added thereto, and decomposition was performed at 60 ° C. for 15 hours. After the reaction, thermolysin was inactivated by boiling at 100 ° C. for 10 minutes. After allowing to cool, 1 g of a filter aid (Radiolite 500, Showa Chemical Industry Co., Ltd.) was added and stirred, followed by filtration.

  After passing 40 ml of the filtrate obtained as described above through a 150 ml column packed with a strongly acidic ion exchange resin (trade name “Dowex 50W × 2, H + form, 50-100 mesh”, manufactured by Dow Chemical Company), Was washed with 5 volumes of deionized water to remove non-peptide components. A 2M ammonia solution was passed through to elute the column adsorbing component, and the peptide fraction was collected. Ammonia was removed using an evaporator and further concentrated to dryness. 5 ml of water was added to dissolve the dried solid, and then centrifugation (10,000 rpm, 30 minutes) was performed to remove insoluble matters. As a result of freeze-drying the supernatant, 125 mg of crude peptide was obtained as a thermolysin hydrolyzate of soy protein.

  Subsequently, molecular weight fractionation was performed by GPC method. A column (φ2.6 × 100 cm) packed with Sephadex G-25 (Medium type, manufactured by Amersham Biosciences) was equilibrated with 0.1 M sodium phosphate buffer (pH 7.0). Next, the entire amount of the crude peptide solution obtained by dissolving 125 mg of the crude peptide after lyophilization in 2.0 ml of 0.1 M sodium phosphate buffer (pH 7.0) was loaded onto this column at a flow rate of 1.0 ml / min. Eluted. As a peptide sample having a known molecular weight, the same sample as that used in Example 1 was used, and the peptide was detected at 214 nm. Molecular weight distribution and average molecular weight were estimated from the elution time. Fraction (F1) with molecular weight distribution 3000-3400 (average molecular weight about 3200), fraction (F2) with molecular weight distribution 2000-3000 (average molecular weight about 2500), fraction (F3) with molecular weight distribution 1000-2000 (average molecular weight about 1500) And a fraction (F4) having a molecular weight distribution of 100 to 1000 (average molecular weight of about 550) was obtained. These were desiccated and lyophilized to obtain 26.0 mg (F1), 32.8 mg (F2), 16.4 mg (F3) and 4.2 mg (F4) powder.

Example 4
Collagen production promoting ability assay of molecular weight fraction of soybean thermolysin degradation product The collagen production promoting ability was assayed using the molecular weight fraction prepared as described above.
First, human normal skin-derived fibroblasts (CRL-1836; ATCC) were cultured in 48-well culture plates. More specifically, the cells were seeded at a density of 12500 cells / 1 cm ² and cultured at 37 ° C. in an environment of 5% carbon dioxide gas and 95% air for about 72 hours. As the culture solution, 500 μl of each medium containing Dulbecco's Modified Eagle Medium (D-MEM) containing fetal bovine serum (FBS) at a concentration of 10% by weight was used. When the cells became confluent, the culture solution was removed, and 500 μl of D-MEM medium supplemented with 100 μg / ml of the test substance shown in Table 2 below was added. In addition, 500 μl of a medium to which neither the soybean thermolysin degradation product prepared in Example 1 nor the molecular weight fraction prepared in Example 3 was added was used as a control. After culturing for 72 hours, the culture solution was collected, and the concentration of type I collagen secreted in the culture solution was quantified by an enzyme-linked immunoassay (Anti-Human Procollagen type C-peptide EIA Kit; manufactured by Takara Bio Inc.). did. Based on the quantification results, the amount of type I collagen in each test medium was calculated with the amount of type I collagen in the control medium as 100%. The results are summarized in Table 2.

  As shown in the above results, even when the molecular weight fraction of the soybean thermolysin degradation product is used, the ability of collagens to promote collagen production is enhanced in the same manner as the soybean thermolysin degradation product demonstrated in Example 2. It was recognized that it was possible.

Example 5
Collagen production assay in dermal fibroblasts (2)
Using collagens different from those in Example 2, the effect of combined use with soybean thermolysin degradation product was further confirmed.
First, human normal skin-derived fibroblasts (NHDF: CRL-2089) were cultured in a 48-well culture plate. More specifically, the cells were seeded at a density of 1.0 × 10 ⁴ cells / well and cultured at 37 ° C. in an environment of 5% carbon dioxide gas and 95% air for 2 days. As a culture solution, a medium containing 10% by weight of fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (D-MEM) at a concentration of 10% by weight was used in 400 μl per well. Subsequently, the culture medium was replaced with a serum-free medium from which FBS was removed, and further cultured for 1 day. Thereafter, the culture solution was removed, and the test drug shown in Table 3 below was replaced with 400 μl of serum-free medium in which each concentration was dissolved and cultured. Here, in this example, a collagen degradation product (average molecular weight 1500) obtained by degrading fish-derived (fish scale-derived) collagen was used as the collagen. On the other hand, 400 μl of a serum-free medium to which neither collagens nor soybean thermolysin degradation product was added was used as a control. After culturing for 3 days, the supernatant was removed and washed 3 times with serum-free medium, and then 400 μl of serum-free medium was added to each well and further cultured for 2 days. Thereafter, the culture solution was collected, and the concentration of type I collagen secreted in the culture solution was quantified by an enzyme-linked immunoassay (Anti-Human Procollagen type I-C-peptide EIA Kit; manufactured by Takara Bio Inc.). Based on the quantitative results, the amount of collagen in each test culture was calculated with the amount of type I collagen in the control culture as 100%. The results are summarized in Table 3.

  As shown in the above results, even when collagens different from those in Example 2 were used, the combined use of collagens and soybean thermolysin degradation product compared to the case where each was used alone. It was confirmed that the amount of collagen produced in the cell increased dramatically.

Example 6
Skin Fibroblast Growth Promotion Test To investigate in detail the action mechanism of collagen production promotion effect by the combined use of collagens and soybean thermolysin degradation product, the influence of both components on the proliferation of fibroblasts was examined.
First, human normal skin-derived fibroblasts (NHDF; CRL-2089) were cultured in a 96-well culture plate. More specifically, the cells were seeded at a density of 1600 cells / well, and cultured at 37 ° C. in an environment of 5% carbon dioxide gas and 95% air for 24 hours. As the culture solution, 100 μl of Dulbecco's Modified Eagle Medium (DMEM) was used. Thereafter, the culture solution was removed, and the test drug shown in Table 4 below was replaced with a 200 μl serum-free medium in which each concentration was dissolved and cultured. In this example, the same collagen as that used in Example 5 (that is, a collagen decomposition product (average molecular weight 1500) obtained by degrading fish-derived (fish scale-derived) collagen was used as the collagen. It was. On the other hand, 200 μl of a serum-free medium to which neither collagens 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 counted by the WST-8 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 4.

  From this result, when cultured in a culture solution in which collagens and soybean thermolysin degradation product are used in combination, the number of fibroblasts can be increased synergistically compared to the case where each is cultured alone. Admitted. However, the synergistic fibroblast proliferation-promoting effect confirmed here was considered to be somewhat milder than the dramatically enhanced collagen production-promoting effect of the combined use of both. Therefore, although the fibroblast proliferation promoting action may play a part in the dramatically high collagen production promoting mechanism achieved by the combined use of both components, there is only the fibroblast proliferation promoting action. It is unlikely that the collagen production promoting effect was so high, and it was suggested that other action mechanisms are also involved in the collagen production promoting action by the combined use of both components.

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

Formulation Example 1: Emulsion [component] [ratio]
Collagen degradation product 0.1
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.1
Squalane 2.0
Liquid paraffin 5.0
Cetanol 0.5
Glyceryl monostearate 2.0
POE (25) cetyl ether 2.0
Glycerin 4.0
1,3-butylene glycol 6.0
pH adjuster appropriate amount preservative appropriate amount perfume appropriate amount
Purified water
100.0% by weight

Formulation Example 2: Emulsion [component] [ratio]
Collagen degradation product 1.0
Soybean protein thermolysin degradation product (average molecular weight 1500) 2.0
Gluco-oligosaccharide 0.5
Acid xylo-oligosaccharide 0.5
1,3-butylene glycol 12.0
Jojoba oil 6.0
Glycerin 4.0
Vaseline 3.5
Polyoxyethylene (40) hydrogenated castor oil 1.5
Glyceryl stearate 1.2
Allantoin 0.1
Carboxyvinyl polymer 0.1
Xanthan gum 0.1
Cetanol 0.6
Panthenol 1.0
Triethanolamine appropriate amount
Purified water
100.0% by weight

Formulation Example 3: Cream [ingredients] [Ratio]
Collagen degradation product 0.01
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.5
Vaseline 1.0
Squalane 5.0
Liquid paraffin 10.0
Stearic acid 1.5
Stearyl alcohol 2.0
Glyceryl monostearate 2.0
POE (20) cetyl ether 3.0
Glycerin 6.0
1,3-butylene glycol 8.0
pH adjuster appropriate amount preservative appropriate amount perfume appropriate amount
Purified water
100.0% by weight

Formulation Example 4: Lotion [Ingredient] [Ratio]
Collagen degradation product 1.0
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.5
1,3-butylene glycol 12.0
Glycerin 3.0
Gluco-oligosaccharide 2.0
Allantoin 0.1
Polyquaternium-51 0.5
Sodium hyaluronate 0.01
PEG-1500 0.5
pH adjuster appropriate amount preservative appropriate amount perfume appropriate amount
Purified water
100.0% by weight

Formulation Example 5: Lotion [Ingredient] [Ratio]
Collagen degradation product 0.005
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.1
POE (20) sorbitan monoisostearate 0.3
Succinic acid 0.2
Sodium succinate 0.5
Edetate trisodium 0.05
1,3-butylene glycol 6.0
Preservative appropriate amount Fragrance proper amount
Purified water
100.0% by weight

Formulation Example 6: Gel agent [component] [ratio]
Collagen degradation product 0.05
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.2
Gluco-oligosaccharide 0.5
Alkyl-modified carboxyvinyl polymer 0.5
1,3-butylene glycol 5.0
Phenylmethylpolysiloxane 1.0
Lavender oil 0.1
Sodium hyaluronate 0.1
Menthol 0.1
Arginine Suitable amount Preservative Suitable amount Fragrance Suitable amount
Purified water
100.0% by weight

Formulation Example 7: Beverage [ingredients] [Ratio]
Thermolysin degradation product of soybean protein (average molecular weight 1500) 1.0
Fish-derived collagen degradation product 0.5
Vitamin B2 0.005
Erythritol 10.0
Acidulant 1.0
Sweetener 1.0
Fragrance 0.01
Purified water remaining
100.0% by weight

Formulation Example 8: Tablets for internal use (in 1 tablet)
[Ingredient] [Ratio]
Soybean protein thermolysin degradation product (average molecular weight 1500) 50.0mg
Pig-derived collagen degradation product 20.0mg
Vitamin B1 1.0mg
Vitamin B2 0.5mg
Fragrance 0.625mg
Sweetener 0.625mg
Sucrose fatty acid ester 3.75mg
Maltitol 48.5mg
125.0mg

Claims (4)

(A) at least one selected from the group consisting of collagen, gelatin, and degradation products thereof;
(B) Thermolysin degradation product of soybean protein (wherein the thermolysin is a hydrophobic amino acid residue having at least one large side chain selected from the group consisting of isoleucine, leucine, valine, phenylalanine, methionine and alanine) It is a protease that cleaves the peptide bond on the amino group side of
A composition for promoting collagen production . (A) at least one selected from the group consisting of collagen, gelatin, and degradation products thereof;
(B) Thermolysin degradation product of soybean protein (wherein the thermolysin is a hydrophobic amino acid residue having at least one large side chain selected from the group consisting of isoleucine, leucine, valine, phenylalanine, methionine and alanine) It is a protease that cleaves the peptide bond on the amino group side of
A composition for promoting fibroblast proliferation, comprising: (A) at least one selected from the group consisting of collagen, gelatin, and degradation products thereof;
(B) Thermolysin degradation product of soybean protein (wherein the thermolysin is a hydrophobic amino acid residue having at least one large side chain selected from the group consisting of isoleucine, leucine, valine, phenylalanine, methionine and alanine) It is a protease that cleaves the peptide bond on the amino group side of
A composition for preventing and / or improving skin wrinkles or talmi. (A) at least one selected from the group consisting of collagen, gelatin, and degradation products thereof;
(B) Thermolysin degradation product of soybean protein (wherein the thermolysin is a hydrophobic amino acid residue having at least one large side chain selected from the group consisting of isoleucine, leucine, valine, phenylalanine, methionine and alanine) It is a protease that cleaves the peptide bond on the amino group side of
The composition for prevention and / or improvement with respect to the reduction | decrease of the elasticity or elasticity of skin containing these.