JP4950571B2 - Composition having ability to promote collagen production - Google Patents

Description

  The present invention relates to a useful novel composition having the ability to promote collagen production 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 as described below in connective tissue.

  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 the dermal dermal extracellular matrix, collagen fibers form a mesh-like bundle to maintain the tissue morphology. Collagen fibers become thick and linear fiber bundles when they mature and proliferate and crosslink formation proceeds, giving moderate elasticity in young skin. However, in aging skin, collagen fibers in the dermal extracellular matrix are remarkably reduced, and the inherent elasticity is lost. As a result, 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 and the like are known. Ascorbic acids are known to have a cell activation effect by promoting the synthesis of biological components such as collagen.

Furthermore, it has also been reported that collagen synthesis promoting action is enhanced by using low-molecular-weight betaine in combination with ascorbic acids (Patent Document 3) or by using δ-tocopheryl retinoate together (Patent Document 4). Yes.
JP-A-7-194375 JP 2001-206835 A JP 2005-239645 A JP 2005-263794 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.

  As a result of intensive studies to solve the above problems, the present inventors have used ascorbic acids in combination with specific peptides having a specific amino acid sequence, so that collagen in a cell can be used rather than using ascorbic acids alone. The inventors have found that the ability to promote production is significantly promoted, and have completed the present invention.

Accordingly, the present invention provides the following.
(1) (A) at least one selected from the group consisting of ascorbic acid, derivatives thereof and salts thereof; and (B) a peptide represented by Leu-Glu-His-Ala (formula I), derivatives thereof and the like A composition comprising at least one selected from the group consisting of salts of:
(2) (A) at least one selected from the group consisting of ascorbic acid, derivatives thereof and salts thereof, and (B) one or more amino acids in the amino acid sequence of Leu-Glu-His-Ala (formula I) A composition comprising a peptide having a conservative substitution, deletion and / or addition and having the ability to promote collagen production in cells, at least one selected from the group consisting of derivatives thereof and salts thereof.
(3) The composition according to item (2), wherein the peptide has an amino acid length of 3 to 10 residues.
(4) The composition according to any one of items (1) to (3), which can be used to promote collagen production in cells.
(5) The composition according to any one of items (1) to (4), which is an external composition.

  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.

  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) at least one selected from the group consisting of ascorbic acid, derivatives thereof and salts thereof, (B) a peptide represented by Leu-Glu-His-Ala (formula I), derivatives thereof, and A composition containing at least one selected from the group consisting of salts thereof.

  The present invention also includes (A) at least one selected from the group consisting of ascorbic acid, derivatives thereof and salts thereof, and (B) one or more amino acid sequences in the amino acid sequence of Leu-Glu-His-Ala (formula I). It is a composition comprising at least one selected from the group consisting of peptides having amino acid conservative substitutions, deletions and / or additions and having the ability to promote collagen production in cells, derivatives thereof, and salts thereof.

  Ascorbic acid, its derivatives, and salts thereof used in the present invention (hereinafter sometimes collectively referred to as “ascorbic acids” in the present specification) are used in the fields of pharmaceuticals, quasi drugs, cosmetics, and foods. If it can be used in, it will not specifically limit. In the present invention, any of D-form, L-form and DL-form of ascorbic acids can be used, but preferably L-form is used.

  Specific examples of the ascorbic acid derivatives include L-ascorbic acid phosphates such as L-ascorbic acid monophosphate, L-ascorbic acid diphosphate and L-ascorbyl triphosphate; ascorbyl palmitate, Fat-soluble ascorbic acid derivatives such as ascorbyl stearate and ascorbyl dipalmitate; sugar derivatives such as ascorbic acid glucoside; L-ascorbic acid-2-sulfate, L-ascorbic acid-2-sulfonate, ascorbyl tetraisopalmitate (VCIP), ascorbic acid-2-phosphate-6-palmitate (APPS) and the like can be used.

  Specific examples of salts of ascorbic acid and ascorbic acid derivatives include various alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium, calcium and barium, and polyvalent metal salts such as aluminum. Metal salts: Ammonium salts such as ammonium and tricyclohexylammonium, various alkanolamine salts such as monoethanolamine, diethanolamine, triethanolamine, monoisopropanolamine, diisopropanolamine, and triisopropanolamine can be used.

  In the present invention, ascorbic acid, derivatives thereof and salts thereof are preferably L-ascorbic acid, L-ascorbic acid phosphate derivatives, ascorbyl palmitate, ascorbyl glucoside, L-ascorbic acid-2-sulfate. , Ascorbic acid-2-glucoside, ascorbyl tetraisopalmitate (VCIP), ascorbyl 2-phosphate-6-palmitate (APPS) and salts thereof (for example, L-ascorbic acid monophosphate sodium salt, L-ascorbyl magnesium phosphate, etc.) is used. When the composition of the present invention is used as a composition for external use, L-ascorbic acid, L-ascorbic acid monophosphate ester and salts thereof (particularly from the viewpoint of safety and high effect on the skin and mucous membranes) For example, L-ascorbic acid monophosphate sodium salt), ascorbyl palmitate, ascorbyl 2-glucoside, ascorbyl tetraisopalmitate (VCIP), ascorbyl 2-phosphate-6-palmitate (APPS) L-ascorbyl magnesium phosphate is used.

  In the present invention, the above ascorbic acid, derivatives thereof and salts thereof may be used singly or in any combination of two or more.

  The amount of ascorbic acid compounded in the composition of the present invention is not particularly limited as long as the effect of the present invention can be achieved, but is usually 0.00001% by weight or more, preferably 0.001% by weight or more, based on the whole composition. More preferably, it is 0.01% by weight or more, more preferably 0.1% by weight or more, particularly preferably 1% by weight or more, and the upper limit of blending is preferably 35% by weight or less, more preferably 25% by weight or less It is preferably 20% by weight or less, particularly preferably 15% by weight or less.

  The present invention further includes at least one selected from the group consisting of peptides represented by Leu-Glu-His-Ala (formula I), derivatives thereof and salts thereof, or Leu-Glu-His-Ala (formula I). At least one selected from the group consisting of a peptide having a conservative substitution, deletion and / or addition of one or more amino acids in the amino acid sequence and having the ability to promote collagen production in cells, derivatives thereof and salts thereof [Hereinafter, these may be collectively referred to as “specific peptides” in the present specification].

  In the present specification, the “peptide derivative” means, for example, acetylation, palmitoylation, myristylation, amidation, acrylation, dansylation, biotinylation, phosphorylation, succinylation, anilideation, benzyloxylation of peptides. Carbonylation, formylation, nitration, sulfonation, aldehyde formation, cyclization, glycosylation, monomethylation, dimethylation, trimethylation, guanidylation, amidineation, maleylation, trifluoroacetylation, carbamylation, trinitrophenyl Derivatized, nitrotroponylated or acetoacetylated derivatives. Among these, palmitoylation is preferable because it is expected to have high permeability to cells, and N-terminal acetylation, C-terminal amidation, and C-terminal methylation are exopeptidases that degrade peptides from the terminal. It is preferable because it is expected to be resistant to, and to be stable in the living body.

  In the present specification, a salt of a peptide or a derivative thereof refers to any pharmacologically acceptable salt (including an inorganic salt and an organic salt) of the peptide or a derivative thereof, for example, a sodium salt of the peptide or a derivative thereof. , Potassium salt, calcium salt, magnesium salt, ammonium salt, hydrochloride, sulfate, nitrate, organic acid salt (acetate, citrate, maleate, malate, oxalate, lactate, succinate , Fumarate, propionate, formate, benzoate, picrate, benzenesulfonate, etc.), preferably ammonium salts, hydrochlorides, sulfates and acetates, more preferably Are ammonium salts and acetates.

  As used herein, the term “amino acid conservative substitution” refers to a substitution between amino acids in each group of Table 1 below.

  Among these, preferred conservative substitutions of amino acids include substitution between aspartic acid (D) and glutamic acid (E), substitution between arginine (R), lysine (K), and histidine (H). Substitution between tryptophan (W) and phenylalanine (F), substitution between phenylalanine (F) and valine (V), between leucine (L), isoleucine (I) and alanine (A) And substitution between glycine (G) and alanine (A).

  A conservative substitution of one or more amino acids preferably refers to a conservative substitution of one or several amino acids, more preferably 1 to 3, more preferably 1 to 2, even more preferably 1. Conservative substitutions of amino acids.

  The deletion of one or more amino acids is preferably a deletion of one amino acid.

  The addition of one or more amino acids is preferably 1 to 6, more preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, and still more preferably 1 amino acid. Refers to addition.

  Conservative substitution or deletion of one or more amino acids in Leu-Glu-His-Ala (in this specification, the peptide sequence may be represented by a single letter abbreviation of the amino acid, for example, the peptide may be referred to as LEHA) Peptides having and / or additions include, for example, those containing one or more amino acid conservative substitutions in the LEHA sequence (eg, IEHA, LDHA, LDKA, LEKA, etc.), lacking one or more amino acids in the LEHA sequence. Including loss (eg, LEH, EHA, LHA, LEA, etc.), and one or more amino acids added to the LEHA sequence (eg, LEHAW, LEHAF, LEHAV, LEHAL, LEHAI, LEHAM, LEHAG, LEHAS, LEHAT, , ALEHA, GLEHA, SLEHA, MLEHA, TLE HA, FLEHA, GLEHAL, DLEHAL, QLEHAK, SLEHAD, QLEHAR, EFLEHA, LEHAVV, DPLELEHA, HLEHAAS, LEHASVD, etc.), but not limited thereto. Among these, preferred peptides include IEHA, LDHA, LDKA, LEKA, LEH, LEHAF, LEHA, GLEHAL, DLEHAL, QLEHAK, SLEHAD, QLEHAR, EFLEHA, LEHAVV, DPELEHA, HLEHAS, A, etc. , LDHA, LEH, LEHAF.

  Further, for example, peptides having conservative substitutions and additions of amino acids in the LEHA peptide are not particularly limited, and preferred examples include IEHAF, LDHAF, LDKAF, LEKAF and the like.

  As used herein, the term “having the ability to promote collagen production in cells” means that when a test substance is allowed to act on cells, the amount of collagen produced in the cells is lower than when the test substance is not allowed to act on cells. Means to increase. In certain embodiments, the cell in the term refers to fibroblasts, and in certain embodiments refers to dermal fibroblasts.

  The peptides used in the present invention can be prepared by methods known in the art. For example, the peptide of the present invention may be synthesized by a chemical synthesis method (eg, a solid phase method (eg, Fmoc method), a liquid phase method, etc.), or may be produced by a method such as gene recombinant expression. . In addition, although the amino acid which comprises the peptide of this invention may be a L body or a D body, Preferably it is a L body.

  Furthermore, the peptide of the present invention can also be prepared by cutting out a peptide comprising the target amino acid sequence from the amino acid sequence of the protein containing the target amino acid sequence by a known means such as protease treatment. For example, examples of the protein containing the LEHA sequence include proteins shown in Table 2 below.

  A person skilled in the art can appropriately select an appropriate protease in order to cut out a peptide comprising the target amino acid sequence from the amino acid sequence of the protein containing the target amino acid sequence in consideration of the sequence specificity of the protease. For example, in order to excise the LEHA sequence from the carrot-derived sequence, thermolysin (derived from Bacillus thermoproteolyticus) and chymotrypsin (derived from bovine pancreas) can be used in combination. Further, for example, in order to cut out the LEHA sequence from the potato-derived sequence, protease M “Amano” G (derived from Aspergillus oryzae: manufactured by Amano Enzyme Co., Ltd.) and the aforementioned thermolysin can be used. Further, for example, in order to cut out the LEHA sequence from the rice-derived sequence, the protease M “Amano” G and the thermolysin are used in combination. Further, for example, in order to cut out the LEHA sequence from the soybean-derived sequence, use of the thermolysin is exemplified. In addition, for example, in order to cut out the LEHA sequence from the kidney bean-derived sequence, the chymotrypsin and the thermolysin are used in combination. Moreover, for example, in order to cut out the LEHA sequence from the cassava-derived sequence, the chymotrypsin and the thermolysin are used in combination. Further, for example, in order to excise the LEHA sequence from the sequence derived from the river clover, it is possible to use trypsin (derived from porcine pancreas) and the thermolysin together. The combination of the protein containing the target amino acid sequence and the protease is not limited to the above combination, but a preferable combination includes a combination of soybean protein and thermolysin.

  The reaction conditions used when the protein is hydrolyzed with a protease 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 a commercially available protease, reaction conditions can be selected according to the instructions for use. 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, particularly preferably 12 to 18 hours may be used. The reaction pH is preferably near the optimum pH of the protease to be used. The means for stopping the reaction is not particularly limited, and known means can be used. Examples of such means include heat treatment such as heating at 85 ° C. for 15 minutes and heating at 100 ° C. for 5 minutes.

  After hydrolysis with protease, the target peptide can be obtained by purification by means known in the art. As such known means, for example, a strongly acidic ion exchange resin, octadecyl silica (ODS) resin, or the like can be used. For example, the peptide of interest can be purified by adsorbing an aqueous peptide solution after protease treatment to an ODS resin and eluting it with an organic solvent of any concentration (for example, acetonitrile). Alternatively, for example, an aqueous peptide solution after protease treatment is adsorbed onto a strongly acidic ion exchange resin, and an eluate having a salt concentration of about 0.18 M to 0.25 M (for example, sodium chloride, potassium chloride, etc.), more preferably about 0. The target peptide can be purified by elution with an eluent having a salt concentration of 20 M to 0.23 M.

  As described above, a peptide obtained by hydrolyzing a natural protein with a protease is more advantageous from the viewpoint of cost than a case where it is produced by a chemical synthesis method. Furthermore, it is considered that a peptide obtained by hydrolyzing a natural protein with a protease is safer for a living body. Therefore, the peptide thus obtained can be suitably used for internal preparations, foods, sensitive skin cosmetics, feeds, and the like that require higher safety for application to living bodies.

  The derivative of the peptide of the present invention is prepared by a method known in the art according to a solid phase synthesis method by Fmoc method (LACarpino, GYHan, J. Am. Chem. Soc., 92, 5748 (1970)). obtain.

  Salts of the peptides of the present invention can also be made by those skilled in the art by any method known in the art.

  In the present invention, the specific peptides as described above may be used alone or in any combination of two or more.

  Although the compounding quantity of the specific peptides mix | blended with the composition of this invention is not restrict | limited especially if the effect of this application can be show | played, Usually, it is good to set it as about 0.00001 to 70 weight% with respect to the whole composition. In particular, when the composition of the present application is an external composition, the blending amount may be small, for example, 0.00001 to 5% by weight, more preferably 0.0001 to 1% by weight, and still more preferably 0.0001. A sufficient effect of the present invention can be obtained even when the content is ˜0.1% by weight, particularly preferably 0.001 to 0.1% by weight.

  In one embodiment, in the composition of the present invention, the specific peptides are, for example, 0.05% by weight or more, preferably 0.08% by weight or more, more preferably 0.1% by weight or more, and further preferably 0.12. What was once refine | purified so that it may become weight% or more can be prepared by mix | blending so that it may become the said compounding quantity.

  In addition, the weight ratio of ascorbic acid and the specific peptide 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 specific peptide is 0.00001 to 1 part by weight of ascorbic acid. It is good to set it in the range of 1500 weight part, Preferably it is 0.0001-1000 weight part, More preferably, it is 0.001-700 weight part, More preferably, it is 0.01-600 weight part.

  In addition to the ascorbic acids and specific peptides as described above, the composition of the present invention is added for the purpose of enhancing or supplementing the action of ascorbic acids or specific peptides, or adding other useful effects to the present composition. Whitening ingredients, anti-inflammatory ingredients, antibacterial ingredients, cell activation ingredients, astringent ingredients, antioxidant ingredients, acne-improving ingredients, biogenic synthesis promoting ingredients such as hyaluronic acid, blood circulation promoting ingredients, moisturizing ingredients, anti-aging ingredients, etc. These various components can be blended alone or in 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, Japanese apricot, amethyst, asenyaku, aseibarabi, hinoki oyster, enoki, oyster (Diospyros kaki), cowpea, black bean, gentian, genzin, salsa, sweet bean, shokuma, jujuu, sage, senko, radish, azalea, tsuku Ingredients derived from plants such as shigigi, toshin, nigaki, parsley, holly, hops, malbahagi, clove and licorice can be mentioned. 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, Hops, Clove, Licorice and Toki, and more preferably Iris (Iris), Aloe, Ginkgo, Ages, Ogon, Oulen, Otogi Riso, gardenia, cucumber, rice, rice bran, saishin, peony, senkyu, sakuhakuhi, tea, touki, eucalyptus, hamamelis, safflower, buttonpi, amethyst, asenyaku, enoki, oyster (Diospyros kaki), sage, radish, azalea, azalea , Hops, licorice and Yokuinin plant-derived components. 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 5% by weight, more preferably 0.5 to 5% by weight. .

  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 oils and fats, the stability of ascorbic acids 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 and the like. 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, if it is a semi-solid agent, in addition to ascorbic acids and specific peptides as described above, the above-mentioned optional components are blended and mixed as necessary, and other solvents and commonly used compositions for external use as necessary. By blending the base of the product, various desired forms such as paste, mousse, gel, liquid, emulsion, cream, sheet (supporting substrate), aerosol, spray, etc. are prepared. be able to.

  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 may be used as an internal use composition or an external composition, but is preferably used as an external composition.

  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 is a composition for treating or preventing various disorders associated with collagen reduction (corneal disorders such as corneal ulcer, rheumatism, arthritis, osteoarthritis, joint disorders such as osteoarthritis, inflammatory disorders, etc.). A composition for preventing or treating cosmetic problems (for example, a composition for preventing and / or improving skin wrinkles or talmi due to UV exposure, aging, etc., against reduction in skin elasticity or elasticity It can be suitably used as a composition for prevention and / or improvement.

  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 LEHA peptide The peptide was synthesized by a solid phase synthesis method by the Fmoc method using an automatic peptide synthesizer (manufactured by Shimadzu Corporation: PSSM8). Subsequently, LEHA peptide was obtained by purifying by removing unreacted substances by preparative HPLC.
The purified product thus obtained was subjected to reverse phase high performance liquid chromatography for analysis [column: μBondasphere 5 μC18-100Å (inner diameter: 3.9 mm, length: 150 mm), manufactured by Waters; mobile phase: solvent A (0.1% Tri Fluoroacetic acid) and solvent B (0.1% trifluoroacetic acid, 90% acetonitrile) gradient (0 min (solvent B = 12%) to 20 min (solvent B = 17%)); flow rate: 1 ml / min; Detection method: Absorbance at a wavelength of 220 nm] showed a single sharp peak at 12.8 minutes and a purity of 99%.

Example 2
Collagen production assay in skin fibroblasts Human normal skin-derived fibroblasts (CRL-1836) were cultured in 48-well culture plates. 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 (DMEM) was used at 400 μl per well. Subsequently, the medium was replaced with the above-mentioned culture solution without adding FBS, that is, a serum-free medium, 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. On the other hand, 400 μl of a serum-free medium to which neither L-ascorbic acid nor LEHA peptide 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 3.

From this result, the amount of collagen produced in the cells cultured in a culture solution using both L-ascorbic acid and LEHA peptide was significantly increased as compared with the case where the cells were cultured in a culture solution containing L-ascorbic acid alone. -It has been demonstrated that the ability of ascorbic acid to promote collagen production can be dramatically enhanced.
It was also recognized that the collagen production promoting action obtained by combining L-ascorbic acid and LEHA peptide was synergistic.

  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 in which L-ascorbic acid and LEHA peptide were used in combination, and it was confirmed that the cells can be safely used for a living body. .

Example 3
Preparation of Other Specific Peptides In order to investigate whether other specific peptides other than LEHA can enhance the ability of L-ascorbic acid to promote collagen production, in addition to LEHA peptides, Derivatives and peptides having conservative substitutions, deletions and / or additions of one or more amino acids in the amino acid sequence of the LEHA peptide, respectively, were prepared.

  N-terminal acetylated LEHA and C-terminal amidated LEHA were prepared by conventional methods as LEHA peptide derivatives. Moreover, LEH, LEKA, LDHA, and LEHAF were prepared by a conventional method as peptides having conservative substitutions, deletions and / or additions of one or more amino acids in the amino acid sequence of the LEHA peptide. Specifically, both the above derivative and the above peptide are synthesized by a solid phase synthesis method (LA Carpino, GYHan, J. Am. Chem. Soc., 92, 5748 (1970)) well known in the art, Preparation was done by removing unreacted material and purifying by preparative HPLC.

Example 4
Collagen production promoting ability test of other specific peptides Using the other specific peptides prepared in Example 3, collagen production promoting ability was assayed.
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 each of D-MEM medium supplemented with test specific peptides shown in Table 4 below at each concentration was added. In addition, what added 500 microliters of culture media which do not add specific peptides was used as 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 4.

  As shown in the above results, other specific peptides other than the LEHA peptide, that is, peptides having one or more amino acid conservative substitutions, deletions and / or additions in the amino acid sequence of the derivative or LEHA peptide Even when used, it was recognized that the ability to promote collagen production of L-ascorbic acid could be enhanced in the same manner as the LEHA peptide demonstrated in Example 2.

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

Formulation Example 1: Emulsion [component] [ratio]
Ascorbic acid glucoside 2.0
LEHA peptide 0.01
Squalane 2.0
Liquid paraffin 5.0
Cetanol 0.5
Glyceryl monostearate 2.0
POE (25) cetyl ether 2.0
Triethanolamine 0.8
Glycerin 4.0
1,3-butylene glycol 6.0
Preservative appropriate amount Fragrance proper amount
Purified water
100.0% by weight

Formulation Example 2: Cream [ingredients] [Ratio]
Ascorbyl tetraisopalmitate (VCIP) 1.0
LEHA peptide 0.05
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
Triethanolamine 1.0
Glycerin 6.0
1,3-butylene glycol 8.0
Preservative appropriate amount Fragrance proper amount
Purified water
100.0% by weight

Formulation Example 3: Lotion [Ingredient] [Ratio]
L-ascorbic acid monophosphate sodium salt 0.2
LEHA peptide 0.2
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 4: Beverages [Ingredients] [Ratio]
L-ascorbic acid 0.5
LEHA peptide 0.1
Vitamin B2 0.005
Erythritol 10.0
Acidulant 1.0
Sweetener 1.0
Fragrance 0.01
Purified water remaining
100.0% by weight

SEQ ID NO: 1 is a peptide that can be used in the present invention.
SEQ ID NO: 2 is a peptide that can be used in the present invention.
SEQ ID NO: 3 is a peptide that can be used in the present invention.
SEQ ID NO: 4 is a peptide that can be used in the present invention.

Claims (5)

(A) Ascorbic acid, its phosphate ester derivative, fat-soluble ascorbic acid derivative, sugar derivative, L-ascorbic acid-2-sulfate, L-ascorbic acid-2-sulfonate, ascorbyl tetraisopalmitate (VCIP) And at least one selected from the group consisting of ascorbic acid-2-phosphate-6-palmitate (APPS) and salts thereof;
(B) a peptide represented by Leu-Glu-His-Ala (formula I), and having one or more amino acid conservative substitutions, deletions and / or additions in the amino acid sequence of formula I; Peptides having an amino acid length of 10 residues and capable of promoting collagen production in cells, and acetylation, palmitoylation, myristylation, amidation, acrylation, dansylation, biotinylation, phosphorylation, succinylation, anilide , Benzyloxycarbonylation, formylation, nitration, sulfonation, aldehyde formation, cyclization, glycosylation, monomethylation, dimethylation, trimethylation, guanidylation, amidination, maleylation, trifluoroacetylation, carbamylation trinitro phenylation, Nitorotoroponiru reduction, or were acetoacetylated induction And at least one collagen production accelerating composition containing a selected from the group consisting of salts thereof. The composition for promoting collagen production is a composition for preventing and / or improving skin wrinkles or tarmi, or for preventing and / or improving skin elasticity or reduction of elasticity. The composition as described . The composition according to claim 1 or 2 , which is a composition for external use. A peptide represented by Leu-Glu-His-Ala (formula I), and 3 to 10 residues having conservative substitutions, deletions and / or additions of one or more amino acids in the amino acid sequence of formula I Peptides having the ability to promote collagen production in cells, and their acetylation, palmitoylation, myristylation, amidation, acrylation, dansylation, biotinylation, phosphorylation, succinylation, anilide, benzyl Oxycarbonylation, formylation, nitration, sulfonation, aldehyde formation, cyclization, glycosylation, monomethylation, dimethylation, trimethylation, guanidylation, amidineation, maleylation, trifluoroacetylation, carbamylation, trinitro Phenylated, nitrotroponylated or acetoacetylated derivatives Ascorbic acid, its phosphate ester derivative, fat-soluble ascorbic acid derivative, sugar derivative, L-ascorbic acid-2-sulfate, L-ascorbic acid, comprising at least one selected from the group consisting of those salts as an active ingredient -2-sulfonic acid ester, tetraisopalmitic acid ascorbyl (VCIP), ascorbic acid-2-phosphate-6-palmitic acid ester (APPS), and at least one ascorbic acid selected from the group thereof Enhances the ability to promote collagen production. A peptide represented by Leu-Glu-His-Ala (formula I), and 3 to 10 residues having conservative substitutions, deletions and / or additions of one or more amino acids in the amino acid sequence of formula I Peptides having the ability to promote collagen production in cells, and their acetylation, palmitoylation, myristylation, amidation, acrylation, dansylation, biotinylation, phosphorylation, succinylation, anilide, benzyl Oxycarbonylation, formylation, nitration, sulfonation, aldehyde formation, cyclization, glycosylation, monomethylation, dimethylation, trimethylation, guanidylation, amidineation, maleylation, trifluoroacetylation, carbamylation, trinitro Phenylated, nitrotroponylated or acetoacetylated derivatives Ascorbic acid, its phosphate ester derivative, fat-soluble ascorbic acid derivative, sugar derivative, L-ascorbic acid-2-sulfate, L-ascorbic acid-2-sulfone is selected from the group consisting of these salts Adding to at least one ascorbic acid selected from the group consisting of acid esters, ascorbyl tetraisopalmitate (VCIP), ascorbyl 2-phosphate-6-palmitate (APPS), and salts thereof A method for enhancing the ability of ascorbic acids to promote collagen production by the method.