JP2014208607A - Novel peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel material having a promoting effect of expression of integrin, collagen, and the like in vivo.SOLUTION: Provided is a peptide consisting of an amino acid sequence expressed by the following formula (I): Gly-Arg-Ile-Arg-Val-Leu (SEQ ID NO: 1), or a peptide consisting of an amino acid sequence having an addition, a deletion, and/or a conservative substitution of one or some amino acids in the amino acid sequence expressed by the formula (I), or derivatives of those peptides, or salts thereof.

Description

  An object of this invention is to provide the novel substance which has the effect of promoting the expression of integrin, collagen, etc. in the living body. The present invention further relates to a technique for enhancing the activity of functional peptides such as growth factors and hormones by utilizing cell adhesion via cell adhesion factors such as integrins.

  In living tissue, cells adhere to and interact with extracellular matrix, and signals are transmitted to the inside of cells to induce or suppress various factors such as genes and proteins, thereby differentiating and proliferating cells. Control of cell traits and secretion of metabolites are performed. In addition, the extracellular matrix itself such as collagen also adheres to the cells, thereby strengthening and stabilizing the lattice-like three-dimensional structure of the collagen fiber bundle and maintaining the firmness and elasticity of the skin. Therefore, if the adhesion between the cells and the extracellular matrix is weakened due to external factors such as ultraviolet rays or aging, signal transmission to the cells is not successful, and the cell functions (production of various factors, etc.) Not only weakens, but also changes in the matrix structure cause skin firmness and elasticity, leading to skin aging symptoms such as wrinkles and tarmi. Furthermore, laminin, a major protein that forms the basement membrane, an extracellular matrix that exists between the epidermis and dermis in skin tissue, is known to interact with integrins on the cell surface and participate in cell adhesion. . And, through the bond between laminin and integrin, the basement membrane and the cell are firmly adhered to each other, so that trophic factors and growth factors move from the dermis to the epidermis, which is an important role of the basement membrane. Becomes smooth and may lead to maintaining a healthy skin condition. Therefore, if the adhesion ability between the basement membrane and the cells is weakened, the overall health condition of the skin may be deteriorated, leading to thinning of the epidermis, and the movement of various factors through the basement membrane. It is hindered to cause a turnover failure, and it is likely to cause stains (melanin accumulation) and dullness. Therefore, it is very important to enhance the function of the factor that adheres the cells to the extracellular matrix.

  As an adhesion factor between a cell and an extracellular matrix, an integrin existing on a cell membrane is well known. In addition, factors such as fibronectin and laminin involved in adhesion to integrins in the extracellular matrix are also called cell adhesion factors in a broad sense. Therefore, substances that can increase the expression level of these factors are useful.

  By the way, it is considered that the use of a cell adhesion promoting factor is beneficial also when a functional factor having physiological activity on cells such as growth factors, hormones, antagonists and agonists is allowed to act on a living body. That is, by using a cell adhesion promoting factor together with a functional peptide intended to act on a cell, the probability that the functional factor interacts with the cell can be increased, and a high effect can be expected.

  In view of the above, there is a need for a search for a substance that can promote cell adhesion factor expression and promote adhesion to cells.

  Hitherto, peptides having a specific sequence (Patent Documents 1 and 2) are known as substances that promote adhesion between cells and collagen. A TGFP-CAP peptide containing a specific amino acid sequence derived from TGF-β, which is a functional peptide, and a cell-attached amino acid sequence has also been proposed (Patent Document 3). However, there is a need for the development of further useful new substances that can promote the adhesion to cells by increasing the expression level of the cell adhesion factor.

US Patent Application Publication No. 2004/0141939 International Publication No. 2012/144546 Pamphlet International Publication No. 2008/130082 Pamphlet

  The present invention has been made in view of the prior art, and an object of the present invention is to provide a novel substance that can increase the expression level of cell adhesion factors such as integrins and is also useful for promoting the production of collagen and the like. And

  As a result of intensive studies to solve the above problems, the present inventors have found that short-chain peptides having a specific amino acid sequence are cell adhesion factors such as integrin, fibronectin or laminin, collagen, emilin (EMILIN), and hyaluronic acid synthesis. It is possible to increase the expression level of components present in the extracellular matrix such as enzymes (hyaluronan synthase), elastin, TIMP metallopeptidase inhibitors, or components involved in the formation thereof. It has been found that integrin and / or fibronectin production promoting activity can also be exhibited. Furthermore, the present inventors have repeatedly studied, and by making the peptide a fusion peptide fused with a functional peptide that acts on cells, it is easy to exert the action of the functional peptide on cells. As a result, the present invention has been completed.

That is, the present invention provides the following.
[1] The following formula (I):
Gly-Arg-Ile-Arg-Val-Leu (SEQ ID NO: 1)
Or a peptide comprising an amino acid sequence having addition, deletion and / or conservative substitution of one or several amino acids in the amino acid sequence represented by the formula (I), or a peptide thereof Derivatives thereof, or salts thereof.
[2] Gly-Arg-Ile-Arg-Val-Leu (SEQ ID NO: 1), Gly-Arg-Ile-Arg-Val (SEQ ID NO: 4), Arg-Ile-Arg-Val-Leu (SEQ ID NO :) 5), Gly-Arg-Ile-Arg (SEQ ID NO: 6), Gln-Tyr-Gly-Arg-Ile-Arg (SEQ ID NO: 8), Tyr-Gly-Arg-Ile-Arg-Val (SEQ ID NO: 9), Arg-Ile-Arg-Val-Leu-Gln (SEQ ID NO: 10), Ile-Arg-Val-Leu-Gln-Arg (SEQ ID NO: 7), Arg-Val-Leu-Gln-Arg-Phe (SEQ ID NO: 11), Tyr-Gly-Arg-Ile-Arg-Val-Leu (SEQ ID NO: 3), or Gly-Arg-Ile-Arg-Val Leu-Gln (SEQ ID NO: 2), peptide [1], or a derivative of the peptide, or a salt thereof.
[3] The peptide according to [1] or [2], which has an amino acid length of 4 to 8 residues, or a derivative of the peptide, or a salt thereof.
[4] A composition comprising the peptide of any one of [1] to [3], a derivative of the peptide, or a salt thereof.
[5] The composition according to [4], which is used for beauty.
[6] The composition according to [4] or [5], which is used for improving or preventing skin wrinkles, tarmi, reduced elasticity, reduced elasticity, poor turnover, spots, dullness, or thinning of the skin.
[7] The composition according to any one of [4] to [6], which is used for tightening the skin.
[8] The composition according to any one of [4] to [7], which is used for promoting production of at least one selected from the group consisting of collagen, integrin, and fibronectin.
[9] The composition according to any one of [4] to [8], wherein expression of a gene encoding a cell adhesion factor, a protein in an extracellular matrix or a protein involved in the formation thereof is increased.
[10] A fusion peptide obtained by fusing a functional peptide that functions to cells and any peptide of [1] to [3], or a derivative of the fusion peptide, or a salt thereof.
[11] The fusion peptide of [10], or a derivative of the fusion peptide, wherein the functional peptide is one or more selected from the group consisting of growth factors, hormones, antagonists, agonists, and parts thereof, or Their salt.
[12] The fusion peptide of [10] or [11], or a derivative of the fusion peptide, wherein the functional peptide is a fragment peptide derived from transforming growth factor (TGF) or a fragment peptide derived from melanocyte stimulating hormone (MSH) Or their salts.
[13] The fusion peptide of any one of [10] to [12], wherein the functional peptide and any peptide of [1] to [3] are fused via a linker, or the fusion peptide Derivatives or their salts.
[14] The fusion peptide of any of [10] to [13], or a derivative of the fusion peptide, or a salt thereof, wherein the peptide of any of [1] to [3] adheres to a cell.
[15] A composition comprising the fusion peptide according to any one of [10] to [14], or a derivative of the fusion peptide, or a salt thereof.

  The peptide of the present invention or a derivative thereof, or a salt thereof has an activity of increasing the expression level of a cell adhesion factor such as integrin, a component in an extracellular matrix such as collagen or a component involved in the formation thereof. Therefore, the present invention provides a novel substance that can promote adhesion between cells and extracellular matrix and promote production of collagen, integrin and / or fibronectin. By using this new substance, adhesion between the cells and the extracellular matrix is promoted. As a result, skin wrinkles and tarmi, reduced elasticity, reduced elasticity, poor turnover and accompanying stains, dullness, thin skin The composition can be effectively improved, helps to tighten the skin, and the like, and a composition useful for beauty can be prepared.

  Furthermore, by using the above-mentioned novel substance that enhances the cell adhesion effect, the activity of functional peptides (eg, growth factors, hormones, antagonists, agonists) that act on cells can be improved.

It is a graph which shows the evaluation result of the collagen production promotion effect of Example 3. 6 is a graph showing the evaluation results of the cell adhesion test of Example 4. It is a figure which shows the expression promotion of integrin alpha3 by the peptide of this invention of Example 6. FIG. It is a figure which shows the expression promotion of the fibronectin by the peptide of this invention of Example 6. FIG.

  Hereinafter, the present invention will be described in detail. Throughout this specification, it should be understood that expression in the singular also includes the concept of the plural unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. In the present specification, amino acids may be represented by well-known three-letter abbreviations or one-letter abbreviations.

<Peptides that can promote the expression of integrins and collagen>
The present invention provides the following formula (I):
Gly-Arg-Ile-Arg-Val-Leu (single letter abbreviation: GRIRVL, SEQ ID NO: 1) peptide consisting of an amino acid sequence, or one or several amino acids in the amino acid sequence represented by the above formula (I) A peptide comprising an amino acid sequence having addition, deletion and / or conservative substitution of the above, or a derivative of the peptide, or a salt thereof is provided.

  The peptide used in the present invention may be a GRIRVL peptide represented by the above formula (I), or an amino acid having one or several amino acid additions, deletions, and / or conservative substitutions in the amino acid sequence It may be a peptide consisting of a sequence (hereinafter also referred to as a peptide variant). Preferred is a GRIRVL peptide, GRIRV peptide or RIRVL peptide, and more preferred is a GRIRVL peptide.

  Here, the conservative substitution of amino acids refers to substitution between amino acids having the same properties as the amino acid substituted with the original amino acid. More specifically, the hydrophobic amino acids include tryptophan (W), phenylalanine (F), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), and alanine (A ): Between amino acids in arginine (R), lysine (K), and histidine (H) as basic amino acids; glycine (G), alanine ( A), serine (S), methionine (M), substitution between amino acids in threonine (T);

  The peptide variant used in the present invention may be a variant having any one of addition, deletion, or conservative substitution in the amino acid sequence represented by the formula (I) as long as the effects of the present invention can be exhibited. Alternatively, the amino acid sequence may have two or more modifications such as addition and deletion, addition and conservative substitution, or deletion and conservative substitution. In order to obtain the effect of the present invention more reliably, a peptide variant having an addition and / or deletion in the amino acid sequence represented by the formula (I) is preferably used, and more preferably the formula (I) Peptide variants having additions and / or deletions at the N-terminus and / or C-terminus of the amino acid sequence represented by

  Furthermore, the peptide variant of the present invention is a peptide variant comprising any amino acid sequence having one or several amino acid additions, deletions, and / or conservative substitutions in the amino acid sequence represented by formula (I). Preferably, it is a peptide variant consisting of an amino acid sequence having an addition, deletion and / or conservative substitution of preferably 1 to 3, more preferably 1 to 2, particularly preferably 1 amino acid.

  Moreover, the length of the amino acid residue of the modified peptide used in the present invention is not particularly limited as long as the effect of the present invention can be obtained, but is preferably 4 to 8 residues, more preferably 5 to 7 residues.

  More specifically, examples of the peptide variants used in the present invention include those having an addition at the N-terminus or C-terminus of the amino acid sequence represented by formula (I) (for example, GRIRVLQ (SEQ ID NO: 2), YGRIRVL ( SEQ ID NO: 3), those having a deletion at the N-terminal or C-terminal of the amino acid sequence represented by formula (I) (for example, GRIRV (SEQ ID NO: 4), RIRVL (SEQ ID NO: 5), GRIR (SEQ ID NO: 6 )), Having deletions and additions at the N-terminus and C-terminus of the amino acid sequence represented by formula (I) (for example, IRVLQR (SEQ ID NO: 7), QYGRIR (SEQ ID NO: 8), YGRIRV (SEQ ID NO: 9)) RIRVLQ (SEQ ID NO: 10), RVLQRF (SEQ ID NO: 11)) and the like. From the viewpoint that it can be expected that a high effect can be exhibited more reliably, preferably GRIRVLQ (SEQ ID NO: 2), YGRIRVL (SEQ ID NO: 3), GRIRV (SEQ ID NO: 4), RIRVL (SEQ ID NO: 5), IRVLQR (SEQ ID NO: 5) 7), more preferably GRIRVLQ (SEQ ID NO: 2), YGRIRVL (SEQ ID NO: 3), GRIRV (SEQ ID NO: 4), RIRVL (SEQ ID NO: 5).

  In the present specification, the “peptide derivative” means, for example, acetylation, palmitoylation, myristylation, amidation, acrylated, dansylated, biotin of peptides (including peptide variants and fusion peptides described later). , Phosphorylation, Succinylation, Anilidation, Benzyloxycarbonylation, Formylation, Nitration, Sulfonation, Aldelation, Cyclization, Glycosylation, Monomethylation, Dimethylation, Trimethylation, Guanidylation, Amidineation, Maleyl Derivatized, trifluoroacetylated, carbamylated, trinitrophenylated, nitrotroponylated or acetoacetylated derivatives.

  In the present specification, the term “salt” refers to any pharmacologically acceptable salt (including inorganic salts and organic salts) of peptides (including peptide variants and fusion peptides described below) or derivatives thereof. For example, sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, hydrochloride salt, sulfate salt, nitrate salt, organic acid salt (acetate salt, trifluoroacetate salt, citrate salt, maleic acid) Salt, malate, oxalate, lactate, succinate, fumarate, propionate, formate, benzoate, picrate, benzenesulfonate, etc.), An ammonium salt, hydrochloride, sulfate, acetate, or trifluoroacetate is preferable, and an ammonium salt, acetate, or trifluoroacetate is more preferable.

  Hydrate or anhydrate may be sufficient as the peptide of this invention (a peptide variant and a fusion peptide mentioned later are included) or its derivative (s), or those salts. Moreover, the solvate or non-solvate may be sufficient as the peptide of this invention, its derivative (s), or those salts.

  The peptides of the present invention can be produced 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. . The amino acid constituting the peptide of the present invention may be L-form or D-form, but is preferably L-form.

  Furthermore, the peptide of the present invention may 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.

  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. 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 technical knowledge. After hydrolysis with protease, the target peptide can be purified by purification by means known in the art, if necessary.

  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. Here, food includes all foods and drinks including beverages.

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

  The salts of the peptides of the present invention can also be easily prepared by those skilled in the art by any method known in the art.

  In the present invention, any of the peptides having the specific sequence described above or derivatives thereof, or salts thereof can be used. However, in order to obtain the effect of the present application more reliably, a peptide or a salt thereof is preferably used. Particularly preferred are peptides.

  The peptide of the present invention or a variant thereof, or a derivative of the peptide, or a salt thereof is a protein encoding a cell adhesion factor such as integrin or an extracellular matrix such as collagen or a protein involved in the formation thereof. Expression can be increased. Hereinafter, the peptides of the present invention or variants thereof, or derivatives of these peptides, or salts thereof are referred to as peptides of the present invention. As a protein in the cell adhesion factor or extracellular matrix whose expression is increased by the peptides of the present invention or in the formation thereof, specifically, integrin, fibronectin, laminin, Examples include collagen, elastin, TIMP metallopeptidase inhibitor, elastin microfibril interfacer (also referred to as Emilin), hyaluronan synthase, and the like.

  Integrins are one of cell surface proteins and are involved in adhesion to extracellular matrix and signal transduction from extracellular matrix. Fibronectin is one of the extracellular matrices and is involved in adhesion with integrins. Fibronectin also adheres to extracellular matrix components such as collagen and plays a role in adhering cells to the extracellular matrix. Laminin is a major protein of the basement membrane (a kind of extracellular matrix), and is known to interact with integrins and participate in cell adhesion and the like. Collagen is a major protein of the extracellular matrix, specifically acts on adhesion of various cells, differentiation and proliferation of cells, and also has a role as a regulator of cell function. Collagen reduction may cause various diseases such as corneal disorders such as corneal ulcers, rheumatism, arthritis, osteoarthritis, joint disorders such as osteoarthritis, and inflammatory diseases. Further, in the extracellular matrix of the skin dermis, the collagen fibers form a reticulated bundle to give an appropriate elasticity. There are various types of collagen such as types I to XIX, and the peptides of the present invention are particularly type I collagen (collagen type I), type IV collagen (collagen type IV), type XVIII collagen (collagen type XVIII). ) Expression can be increased. Elastin is a component of elastic fibers that constitute the extracellular matrix. TIMP metallopeptidase inhibitors have the effect of inhibiting the activity of peptidases involved in extracellular matrix degradation, so that TIMP suppresses excessive degradation of extracellular matrix and plays an important role in tissue homeostasis. I'm in charge. Emilin is one of the multimeric glycoproteins that make up the extracellular matrix, and is thought to exist between elastin and microfibers and participate in elastic fiber formation and structural stabilization. . Hyaluronic acid synthase is an enzyme localized in the cell membrane and synthesizes hyaluronic acid, which is one of the main components of the extracellular matrix. Hyaluronic acid is known to affect the elasticity and firmness of the skin because it supports the structure of the extracellular space, is involved in the transport of nutrient factors and waste materials, and has a strong water retention effect.

  Since the above-mentioned various proteins are directly or indirectly involved in the adhesion between the extracellular matrix and the cells, when the production amount of these proteins increases, for example, the cell adhesion factor increases, and the extracellular matrix further increases. Adhesion between skin cells such as fibroblasts and keratinocytes and the extracellular matrix is promoted.

  Furthermore, the peptides of the present invention can increase or decrease the expression of genes encoding proteins that are not directly involved in cell-extracellular matrix adhesion but can act on the skin. Here, acting on the skin can effectively improve, for example, skin wrinkles and tarmi, reduction in elasticity, decrease in elasticity, poor turnover and accompanying stains, dullness, thinning of the skin, etc. In addition to having a good effect on the skin such as helping to tighten the skin, it also means having an unfavorable effect on the skin such as inducing skin inflammation and melanin production. The peptides of the present invention may increase the expression of genes that have a positive effect on the skin while decreasing the expression of genes that have a negative effect on the skin.

  Examples of proteins that can have a good effect on the skin and whose gene expression is increased by the peptides of the present invention include catalase, amphiregulin, sirtuin, telomerase, etc. It is done.

  Catalase is a hydrogen peroxide-degrading enzyme, which decomposes hydrogen peroxide that is harmful to the skin caused by ultraviolet irradiation or the like. Therefore, an increase in the expression level can be expected to have a good effect on the skin. Amphiregulin is one of the genes of the EGF family and regulates cell proliferation and differentiation, and can be expected to promote epidermal turnover and anti-wrinkle effect. Sirtuins are also called anti-aging genes. When this sirtuin gene is activated, an effect of extending cell life can be expected. Telomerase is a telomere elongating enzyme that can also be expected to have an effect of extending cell life.

  For example, when normal human dermal fibroblasts are cultured in the presence of the peptides of the present invention, for example, in a medium containing the peptides of the present invention at a concentration of 0.5 mg / ml or 1 mg / ml, The amount of mRNA produced by a gene encoding a cell adhesion factor such as integrin or a protein in an extracellular matrix such as collagen or a protein involved in the formation thereof and a protein capable of exerting a good effect on the skin is preferably about 10% or more, preferably More than about 15%, more preferably about 20% or more, more preferably about 25% or more, particularly preferably about 30% or more.

  In addition, as a protein that can have an adverse effect on the skin and whose gene expression is reduced by the peptides of the present invention, prostaglandin endoperoxide synthase, prostaglandin-endoperoxide synthase, And leukin-6 (IL-6: interleukin 6).

  Prostaglandin endoperoxide synthase (prostaglandin synthase) and interleukin-6 (IL-6) are enzymes and cytokines that act in the inflammatory system, respectively. By reducing the expression level of these genes, it is expected to help suppress inflammation. Moreover, since these gene expression products directly or indirectly promote melanin production, a whitening effect can be expected by reducing the expression level and suppressing melanin production.

  For example, when normal human dermal fibroblasts are cultured in the presence of the peptides of the present invention, for example, in a medium containing the peptides of the present invention at a concentration of 0.5 mg / ml or 1 mg / ml, The amount of mRNA production of a gene encoding a protein that may have an adverse effect on the skin is about 20% or more, preferably about 30% or more, more preferably about 40% or more, more preferably about 70% or more, particularly preferably Decrease by about 80% or more.

  Whether or not the expression of these genes increases and the amount thereof increases or decreases and the amount thereof decreases, for example, in normal human dermal fibroblasts in the presence and absence of the peptides of the present invention. Cultured cells, extracted RNA from the cells, measured the amount of RNA using a DNA chip on which a gene or fragment thereof that is likely to act on the skin is immobilized, and cultured in the presence of the peptides of the present invention What is necessary is just to compare the RNA amount in the cell cultured in absence and the amount of RNA in the inside. For example, a human skin chip (SKNH-LX) Genopal (registered trademark) (Mitsubishi Rayon Co., Ltd.) can be used as a DNA chip on which a gene or fragment thereof that may act on the skin is immobilized. . Whether or not gene expression increases and the amount thereof increases or decreases and whether or not the amount decreases, for example, quantitative PCR (qPCR) is performed on RNA extracted by processing in the same manner as described above. Can also be evaluated.

  With the peptides of the present invention, when the amount of protein involved in adhesion between cells and extracellular matrix increases, cells such as fibroblasts and keratinocytes adhere to the extracellular matrix in the dermis or epidermis of the living body, Strengthens the bond between cells and extracellular matrix. In particular, the integrin on the surface of the fibroblast adheres to the extracellular matrix in the dermis of the living body, thereby strengthening the bond between the fibroblast and the extracellular matrix. In addition, fibroblasts adhere to collagen in the extracellular matrix in the dermis of a living body, and collagen firmly forms a fiber bundle structure, thereby strengthening the binding between fibroblasts and extracellular matrix. As a result, the dermis is tightened by increasing the tensile strength of the cells and the extracellular matrix, and the firmness and elasticity of the skin are maintained. Therefore, the peptides of the present invention can be used to improve skin wrinkles and talmi caused by a decrease in the adhesive ability between cells and extracellular matrix, or to restore skin elasticity and elasticity. Furthermore, when the epidermal keratinocytes adhere to the extracellular matrix of the basement membrane and firmly adhere the epidermis to the basement membrane, sagging (for example, sagging pores) and wrinkles are suppressed, and keratinocyte proliferation occurs. Can be used to promote turnover and improve spots and dullness. Here, the turnover means that keratinocytes of the epidermis are produced in the basal layer of the skin, form a horny layer, and finally fall off in the skin. The cells of the epidermis are replaced with new cells by turnover. The turnover time is shortened by promoting the turnover. In addition, by promoting adhesion between the dermis or epidermis and the basement membrane, the nutrient supply to the epidermis and the transfer of transcription factors, etc., become smoother, and a healthier skin condition (for example, no thinning of the epidermis has occurred) State). Therefore, the peptides of the present invention can be used for sagging, wrinkles, reduced turnover, spots, dullness, and thinning of the epidermis due to decreased adhesion ability between fibroblasts or keratinocytes and the extracellular matrix (particularly the basement membrane). It can also be used to improve conversion.

  Furthermore, the peptides of the present invention increase the expression of genes that can have a positive effect on the skin, while decreasing the expression of genes that can have a negative effect on the skin. As a result, the skin condition can be made healthier and better.

  The present invention also includes a composition comprising the above peptides as an active ingredient. The composition is, for example, a pharmaceutical composition, a cosmetic composition, a food composition or a feed composition, and further for elucidation of physiological conditions related to adhesion between cells and extracellular matrix, or skin condition. It can be suitably used as a research reagent for elucidation of Preferably, it is a pharmaceutical composition, cosmetic composition, food composition or feed composition used for cosmetic purposes. In the present invention, a composition used for beauty (ie, a beauty composition) refers to a composition used for the purpose of making the face and body beautiful (for example, beauty such as a skin-beautifying effect on the skin). .

  Examples of the pharmaceutical composition include a prophylactic and / or therapeutic agent for diseases caused by a decrease in adhesion ability between cells and extracellular matrix in mammals including humans. Specifically, the pharmaceutical composition of the present invention includes, for example, a preventive agent for skin wrinkles or talmi caused by a decrease in the adhesion ability between fibroblasts or keratinocytes caused by aging or the like and an extracellular matrix, and As a therapeutic agent, as a prophylactic and / or therapeutic agent for skin elasticity or a decrease in elasticity due to the decrease in adhesion ability, or as a skin tightening agent, poor skin turnover due to the decrease in adhesion ability It can be used as a prophylactic and / or therapeutic agent for blemishes or dullness associated therewith, or as a prophylactic and / or therapeutic agent for thinning of the skin caused by the reduced adhesive ability. The pharmaceutical composition containing the peptides of the present invention is useful for the prevention and / or treatment of wrinkles, tarmi, elasticity and reduced elasticity as described above, skin tightening, etc. It is also clear from the results of the collagen, integrin and / or fibronectin production promoting effect shown in 3 or 6.

  The cosmetic composition can be used, for example, as a cosmetic for preventing and / or improving a condition caused by a decrease in the adhesion ability between cells and extracellular matrix in mammals including humans. Specifically, the cosmetic composition of the present invention can prevent skin wrinkles or tarmi caused by, for example, a decrease in the adhesion ability between fibroblasts or keratinocytes and extracellular matrix caused by aging and the like. As a cosmetic for improvement, as a cosmetic for prevention and / or improvement of skin firmness or a decrease in elasticity due to a decrease in the adhesive ability, or as a cosmetic for tightening the skin, As a cosmetic for the prevention and / or improvement of skin turnover failure and stains or dullness accompanying it due to a decrease in adhesion ability, or for the prevention and / or improvement of epidermis thinning due to the decrease in adhesion ability Can be used as cosmetics. The cosmetic composition containing the peptides of the present invention is useful for the prevention and / or improvement of wrinkles, tarmi, elasticity and reduction in elasticity, tightening of the skin, etc. described later. It is also clear from the results of the collagen, integrin and / or fibronectin production promoting effect shown in Example 3 or 6.

  As a food composition, for example, it can be used as a food for preventing and / or ameliorating a condition caused by a decrease in adhesion ability between cells and extracellular matrix in mammals including humans. Specifically, the food composition of the present invention is capable of preventing and / or preventing skin wrinkles or talmi caused by, for example, a decrease in adhesion ability between fibroblasts or keratinocytes and extracellular matrix caused by aging or the like. Alternatively, as a food for improvement, as a food for prevention and / or improvement of skin elasticity or a decrease in elasticity caused by the decrease in the adhesive ability, or as a food for tightening the skin, Used as a food for the prevention and / or improvement of skin turnover failure and stains or dullness associated therewith, or as a food for prevention and / or improvement of thinning of the skin caused by the above-mentioned decrease in adhesion ability Can be done. The food composition containing the peptides of the present invention is useful for the prevention and / or improvement of wrinkles, tarmi, elasticity and elasticity reduction, skin tightening, etc. as described below. It is also clear from the results of the collagen, integrin and / or fibronectin production promoting effect shown in 3 or 6.

  The food composition of the present invention includes health food, food for specified health use, nutritional functional food, health supplement food and the like. These compositions include cosmetic health foods. The food for specified health is a food that is ingested for the purpose of specific health in the diet, and indicates that the purpose of the health can be expected by the intake. These food products can be provided as food products with a label indicating that they are used for the above-mentioned purposes in a specific embodiment. In other words, it is used to display the indication that it is used for beauty, the indication that it has a beautifying effect, the indication that it is used for improving the skin quality, and to restore the elasticity and elasticity of the skin. An indication that it is used, an indication that it is used for prevention or improvement of skin wrinkles and talmi, an indication that it is used for tightening the skin, poor skin turnover, and It can be provided as a food with a label indicating that it is used for the prevention or improvement of accompanying stains or dullness and a label indicating that it is used for the prevention or improvement of skin thinning.

  As feed, for example, for prevention of a state caused by a decrease in adhesion ability between cells and extracellular matrix in domestic animals such as cattle, pigs, chickens, sheep, horses, and pet animals such as dogs and cats, and / or Examples include feed for improvement, feed for prevention and / or improvement of a state caused by a decrease in the amount of collagen, integrin and / or fibronectin in vivo.

  The content of the peptides of the present invention in the present composition varies depending on the dosage form of the composition, etc., but generally, the effect of promoting adhesion between high cells and extracellular matrix, or collagen, integrin and / or From the viewpoint of obtaining the effect of promoting fibronectin production, it is preferably 0.0001 to 100% by weight, more preferably 0.001 to 95% by weight, still more preferably 0.01 to 90% by weight, and particularly preferably 0.1 to 80% by weight.

  The composition of the present invention includes, in addition to the peptides of the present invention, carriers, bases, and / or additives that are usually used in the fields of pharmaceutical preparation, food and the like within the scope of achieving the object of the present invention. It can mix and prepare suitably.

  As the carrier, for example, saccharides, celluloses, poorly water-soluble gums, crosslinked vinyl polymers, lipids and the like can be used alone or in combination of two or more. As the base, for example, water, fats and oils, mineral oils, waxes, fatty acids, silicone oils, sterols, esters, metal soaps, alcohols and the like may be used alone or in combination of two or more. . Examples of additives include surfactants, solubilizing components, emulsifiers, oils, stabilizers, thickeners, preservatives, binders, lubricants, dispersants, pH adjusters, humectants, UV absorbers. , Chelating agents, percutaneous absorption enhancers, antioxidants, disintegrating agents, plasticizers, buffering agents, vitamins, amino acids, coloring agents, fragrances and the like may be used alone or in combination.

  Furthermore, in order to add other useful actions as necessary, the composition of the present invention includes a whitening component, an anti-inflammatory component, an antibacterial component, a cell activation component, an astringent component, an antioxidant component, an acne improving component, Various components such as a biocomponent synthesis promoting component such as collagen, a blood circulation promoting component, a moisturizing component, and an anti-aging component may be used alone or in combination.

  The composition of the present invention may be in any dosage form such as an internal preparation (including food and feed) or an external preparation (including cosmetics). For internal use (including food and feed), for example, tablets, pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (drinks, suspensions, syrups) ), Gel agent, liposome agent, extract agent, tincture agent, lemonade agent, jelly agent and the like.

  In the case of food, bread, noodles, prepared foods, processed meat foods (eg, ham, sausage, etc.), processed fishery products, seasonings (eg, dressing, etc.), dairy products, confectionery (eg, biscuits, candy, Jelly, ice cream, etc.), soup, juice, etc., and can be provided as a food form contained in any common food. In such a form, the hydrolyzate can be appropriately blended by a method known to those skilled in the art depending on the properties of the target food.

  As an external preparation, for example, it can be used in any form such as liquid, emulsion, cream, lotion, paste, mousse, gel, sheet (substrate-supported), aerosol or spray.

Cosmetics include, for example, basic cosmetics such as lotions, emulsions, creams, oils, packs, makeup cosmetics such as foundations, blushers and lipsticks, as well as cleansers such as facial cleansers, cleansings, body cleansers, and bath preparations. Etc. may be used in any form.
The feed is not particularly limited because it can be used in any form.

The present invention further includes a method for promoting adhesion between a cell and an extracellular matrix, and a method for promoting production of collagen, integrin and / or fibronectin, wherein the peptide is used. In the method of the present invention, the peptides are used in an effective amount that can achieve an effect of promoting adhesion between cells and extracellular matrix, or an effective amount that can produce an effect of promoting production of collagen, integrin and / or fibronectin. What is necessary is just to use by quantity. That is, the amount of the peptides used in the method of the present invention is usually about 0.001 to 10,000 mg / day, more preferably about 1 to 1000 mg / day, more preferably about 1 to 1000 mg / day per adult body weight in the case of internal use. Preferably it is about 1-100 mg / day. In the case of an external preparation, the amount used is generally about 0.1 μg to 2 g / day, preferably about 50 kg per adult body weight. Further, when used as an external preparation, the amount of the peptides applied to the skin is preferably about 1 ng to 500 μg / cm ² , more preferably about 0.01 to 50 μg / cm ² , and still more preferably about 0.1 to 10 μg / cm ² . is there.

The present invention further provides the use of said peptides for the manufacture of a composition for promoting adhesion between cells and extracellular matrix. Furthermore, the present invention also provides use of the peptides for producing a composition for promoting production of collagen, integrin and / or fibronectin.
What is necessary is just to use the usage-amount of the said peptides so that it may become content in the said composition.

<Fusion peptide with functional peptide>
As shown in the results of Examples described later, it has been clarified that the peptide consisting of the amino acid sequence represented by formula (I) or a peptide variant thereof can increase the expression level of integrin in cells. Furthermore, it has been confirmed that the peptide consisting of the amino acid sequence represented by the formula (I) or a modified peptide thereof can actually adhere to cells. Therefore, it is expected that the activity of a functional peptide intended to act on cells can be enhanced by using the peptide of the present invention or a peptide variant thereof.

  Therefore, from another viewpoint, the present invention comprises (A) a functional peptide that functions on cells (hereinafter also referred to as (A) element) and (B) an amino acid sequence represented by formula (I). Also provided is a fusion peptide comprising a peptide or a peptide consisting of an amino acid sequence having an addition, deletion, and / or conservative substitution of one or several amino acids in the amino acid sequence (hereinafter also referred to as (B) element) To do. In such a fusion peptide, since the (B) element promotes cell adhesion via integrin and the like and can exert a cell adhesion promoting effect, the (A) element can increase the probability of contacting the cell, It becomes easier to exert the action of the element (A) on cells more effectively. That is, the peptide consisting of the amino acid sequence represented by the formula (I) of the present invention or a fusion peptide of the peptide variant and a functional peptide acts on the cell, and the cell or peptide variant of the present invention acts on the cell. In, the expression of integrin increases when the fusion protein is present in the vicinity of the cell. Furthermore, the peptide consisting of the amino acid sequence represented by the formula (I) of the present invention or a peptide variant thereof directly adheres to cells. As a result, the probability that the fusion protein will come into contact with the cell due to the cell adhesion effect is increased. Can demonstrate.

  The functional peptide (A) serving as a component of the fusion peptide of the present invention may be any peptide intended to exert a function on cells, specifically, a growth factor, a hormone, It may be an antagonist, an agonist, or a part thereof.

  A growth factor is also called a growth factor or a cell growth factor, and is a general term for proteins that affect the proliferation and differentiation of specific cells. Specifically, transforming growth factor (TGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), brain-derived neurotrophic factor ( BDNF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) and the like. Preferred are transforming growth factor, epidermal growth factor, fibroblast growth factor, insulin-like growth factor, and hepatocyte growth factor. For the fusion peptide of the present invention, a peptide that is a part of the growth factor may be used. Examples of such peptides include fragment peptides derived from TGF-β as described in WO 2008/130082 (for example, the Ile-Trp-Ser-Leu-Asp-Thr-Gln-Tyr sequence ( Peptide) having SEQ ID NO: 12) and the like can be used.

  Hormones are physiologically active substances that act on cells and regulate cell activity. The hormone used as the functional factor in the fusion protein of the present invention is preferably a peptide hormone. Peptide hormones specifically include melanocyte stimulating hormone (MSH), atrial natriuretic peptide (ANP), atrial natriuretic factor (ANF), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicular stimulation Examples include hormone (FSH), human chorionic gonadotropin (hCG), oxytocin, vasopressin and the like. Preferably, it is a melanocyte stimulating hormone. For the fusion peptide of the present invention, a peptide which is a part of the above peptide hormone may be used. Examples of such a peptide include α as described in US Pat. No. 6,254,342 and US Pat. No. 7,737,119. A fragment peptide of -MSH (for example, a peptide having a Glu-His-Phe-Arg-Trp-Gly sequence (SEQ ID NO: 13)) or the like can be used.

  An antagonist is a general term for substances that act on a receptor of a cell and have an action of inhibiting the binding of a ligand to the receptor. Specific examples include RAGE antagonists. RAGE antagonists act as antagonists to receptors (terminal receptors for AGE) of terminal glycation products (also called AGEs) generated by glycation of proteins, which is considered to be one of the aging phenomena, and inhibit ligand binding It is a substance. In the fusion peptide of the present invention, a part of the RAGE antagonist can also be used, and as such a part of the peptide, a part of the RAGE antagonist as described in US Patent Application Publication No. 2010/0249038, etc. Can be used.

  An agonist is a general term for substances that act on a receptor of a cell and exert the same action as a ligand that acts on the receptor. A peptide that is a part of such an agonist can also be used for the fusion peptide of the present invention.

  In the fusion peptide of the present invention, the order of fusion between the functional peptide as the element (A) and the peptide represented by the formula (I) as the element (B) or a peptide variant thereof is not particularly limited. , (A) element may be on the N-terminal side and (B) element may be on the C-terminal side, or vice versa. In the fusion peptide, the N-terminal peptide may be referred to as a head peptide and the C-terminal peptide as a tail peptide.

  Further, the fusion peptide of the present invention may be a fusion peptide composed only of the (A) element and the (B) element, and includes a further amino acid sequence other than the (A) element and the (B) element. It may be. Furthermore, the fusion peptide of the present invention may be a fusion peptide in which the (A) element and the (B) element are directly linked, or an amino acid sequence serving as a linker between the (A) element and the (B) element. It may be a fusion peptide containing When the (A) element and the (B) element are fused via a linker, the structural stability of each of the (A) element and the (B) element increases, and it can be expected that higher effects can be easily exhibited. . Accordingly, the fusion peptide of the present invention preferably contains a linker between the (A) element and the (B) element. In addition, when the fusion peptide of the present invention contains a plurality of functional peptides as the element (A), it is preferable that the functional peptides are also configured to include a linker.

The linker contained in the fusion peptide of the present invention can be any linker sequence as long as the function of each of the (A) element and (B) element is not affected. As an example, examples of the linker include a linker composed of amino acids. Here, the amino acid that can be used for the linker is an organic compound having both an amino group (—NH ₂ group) and a carboxyl group (—COOH group) in the same molecule, and the amino group and the carboxyl group have the same carbon. It may be an α-amino acid (for example, glycine, serine, alanine (α-alanine), threonine, methionine), β-alanine, aminobutyric acid (for example, 4-aminobutyric acid), aminovaleric acid (Eg 5-aminovaleric acid), aminohexanoic acid (eg 6-aminohexanoic acid), aminoheptanoic acid (eg 7-aminoheptanoic acid), aminooctanoic acid (eg 8-aminooctanoic acid), aminononane Acids (9-aminononanoic acid), aminodecanoic acid (eg 10-aminodecanoic acid), aminoundecanoic acid (eg 11-aminoundecanoic acid), aminolauric acid ( For example, 12-aminolauric acid), aminotridecanoic acid (eg 13-aminotridecanoic acid), aminomyristic acid (eg 14-aminomyristic acid), aminopentadecanoic acid (eg 15-aminopentadecanoic acid), An amino palmitic acid (for example, 16-amino palmitic acid) or the like having about 2 to 50 carbon atoms, preferably about 2 to 30 carbon atoms, more preferably about 2 to 20 carbon atoms, and further preferably about 2 to 15 carbon atoms. An aminocarboxylic acid may be used. Preferably, a linker composed of glycine, serine and / or an aminocarboxylic acid having 2 to 20 carbon atoms can be mentioned, and more preferably composed of glycine and / or an aminocarboxylic acid having 2 to 15 carbon atoms. A linker can be mentioned, More preferably, the linker comprised only with glycine can be mentioned.

  The amino acid length of the linker is not particularly limited, but is generally 1 to 20 residues, preferably 1 to 15 residues, more preferably 1 to 10 residues, and still more preferably 1 to 5 residues. Group, more preferably 1 to 3 residues.

  The fusion peptide of the present invention can be more easily prepared by a chemical synthesis method known in the art (for example, a solid phase method (for example, Fmoc method), a liquid phase method, etc.). When chemically synthesizing a fusion peptide, (A) element, (B) element, and if necessary, other peptides such as a linker may be separately prepared and then fused, or (A) element and (B) A series of amino acid sequences of a fusion peptide including elements and, if necessary, a linker are designed in advance, and the amino acid sequence of the fusion peptide so designed is sequentially synthesized from the N-terminus or C-terminus. It may be prepared in the form.

  The fusion peptide of the present invention comprises (A) a DNA encoding a functional peptide as an element and (B) an element represented by formula (I) or a DNA encoding a peptide variant thereof in frame. Can be prepared as a recombinant peptide by genetic engineering by introducing the fusion DNA into an expression vector and introducing the vector into an appropriate host cell for expression. At this time, elements such as a promoter and a terminator may be linked in a functionable manner. When a linker composed only of genetically encoded amino acids is arranged, it is represented by (A) DNA encoding a functional peptide that is an element and (B) element (I). The DNA encoding the linker peptide may be interposed between the DNA encoding the peptide or its peptide variant.

  In addition, a derivative of the fusion peptide of the present invention or a salt of the fusion peptide of the present invention or a derivative thereof can be used for the same purpose as described above. The derivative of the fusion peptide, or the salt of the fusion peptide or derivative thereof may be in the form of a derivative or salt similar to the aforementioned peptides of the present invention.

  The present invention also includes a composition comprising the above fusion peptide. The composition can be used as a composition for allowing a functional peptide to act on cells. The blending amount of the fusion peptide in the composition, the type and blending amount of other components (carrier, base, additive, etc.) that can be blended, the form of the composition, etc. are the same as those of the peptides of the present invention described above. It is the same as the composition containing. Further, the use of the composition may be the same as the composition containing the peptides of the present invention described above, or may be used for another use depending on the effect of the functional peptide. Good. For example, good effects on the skin (eg prevention and / or improvement of skin wrinkles, tarmi, reduced elasticity, reduced elasticity, poor turnover, spots, dullness, or thinning of the skin; skin tightening and / or whitening Prevention and / or improvement of inflammation in the skin; prevention and / or improvement of skin aging symptoms such as protein glycation (AGEs generation) in the skin) In addition to the action of the peptides of the present invention itself, it can also be used advantageously to obtain an effect based on the action of the functional peptide.

  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 GRIRVL (Gly-Arg-Ile-Arg-Val-Leu) peptide (1) Peptide synthesis:
The GRIRVL peptide was synthesized by a solid phase synthesis method by Fmoc method using an automatic peptide synthesizer (manufactured by Shimadzu Corporation: PSSM8). The specific procedure is as follows: First, the C-terminus of Fmoc-Leu-OH is bound to a solid phase synthesis resin, and then the protecting group (Fmoc) is removed by piperidine treatment, and then the resin is neutralized. -After washing, Fmoc-Val-OH was introduced into the N-terminus of Leu. Next, the protecting group (Fmoc) was removed by piperidine treatment, the resin was neutralized and washed again, and Fmoc-Arg (Pmc) -OH was introduced into the N-terminus of Val. Next, the protecting group (Fmoc) was removed by piperidine treatment, and this resin was neutralized and washed again, and Fmoc-Ile-OH was introduced into the N-terminus of Arg. Next, the protecting group (Fmoc) was removed by piperidine treatment, the resin was neutralized and washed again, and Fmoc-Arg (Pmc) -OH was introduced into the N-terminus of Ile. Next, the protecting group (Fmoc) was removed by piperidine treatment, the resin was neutralized and washed again, and Fmoc-Gly-OH was introduced into the N-terminus of Arg. Next, the protecting group (Fmoc) was removed by piperidine treatment, and the peptide chain was excised from this resin. Deprotection was performed by cleaving the Pmc group of Arg with TFA (trifluoroacetic acid). Finally, GRIRVL was obtained by purification by removing unreacted substances by preparative HPLC.

(2) Peptide purity test:
The purified product thus obtained was analyzed by reversed-phase high-performance liquid chromatography [column: Inertsil ODS-3 (inner diameter: 4.6 mm, length: 250 mm), manufactured by GL Sciences; mobile phase: solvent A (0.05% trifluoroacetic acid) ) And solvent B (0.05% trifluoroacetic acid, 100% acetonitrile) gradient (0 min (solvent B = 0%) to 30 min (solvent B = 11%)); flow rate: 1 ml / min; detection method: wavelength Absorbance at 220 nm] showed a single sharp peak at 5.5 minutes with a purity of 99.9%.

Example 2: Measurement of gene expression by microarray Normal human dermal fibroblasts (KF-4009; Kurabo Industries) were used in a 12-well microplate using Dulbecco's modified MEM (DMEM) (GIBCO) containing 10% calf serum (FBS). The cells were seeded at a cell density of 3.0 × 10 ⁴ cells / cm ² . 24 hours after seeding, the GRIRVL peptide prepared in Example 1 was replaced with DMEM containing a predetermined concentration (0.5 mg / ml or 1 mg / ml) and cultured for 48 hours. Separately, as a control (untreated group), a well exchanged with DMEM not containing the test peptide was prepared and cultured in the same manner for 48 hours. Subsequently, RNA was extracted from the fibroblasts of the GRIRVL peptide-treated group and the untreated group using RNeasy Mini Kit (manufactured by QIAGEN) and QIA shredder (manufactured by QIAGEN). For each extracted RNA, compare the gene expression pattern of GRIRVL peptide-treated group and untreated group using human skin chip (SKNH-LX) Genopal (registered trademark) (Mitsubishi Rayon Co., Ltd.) did. In the human skin chip, genes associated with skin formation and metabolism and 139 genes that may act on inflammation and toxicity are immobilized. Of the 139 genes whose expression was increased or decreased by treatment with GRIRVL peptide, the expression level in the GRIRVL peptide treatment group, when the expression level in the untreatment group was set to 100, was calculated as follows: It shows to Tables 1-4. The genes shown in Table 1 and Table 2 are genes that can be directly or indirectly involved in the adhesion between cells and the extracellular matrix, and the genes shown in Table 3 and Table 4 are the adhesion between cells and the extracellular matrix. It is a gene that is not directly involved in but can have a good or bad effect on the skin.

  As shown in the results of Tables 1 and 2 above, in fibroblasts treated with GRIRVL peptide, in addition to cell adhesion factors such as integrin, fibronectin, laminin, TIMP metallopeptidase inhibitor, elastin that suppresses collagen and its degrading enzyme In addition, the expression level of components present in the extracellular matrix such as emilin and hyaluronic acid synthase or components involved in the formation thereof has been increased. Among them, a high expression level was observed for type I collagen.

  From the above results, the peptide of the present invention increases the expression level of the cell adhesion factor such as integrin and the component in the extracellular matrix or the component involved in the formation thereof, and the interaction between the cell and the extracellular matrix is increased. It has been found that it can be beneficial and used to promote cell adhesion.

  In addition, as shown in the results of Tables 3 and 4 above, in fibroblasts treated with GRIRVL peptide, the expression level of genes that can have a good effect on the skin, such as catalase, amphiregulin, sirtuin, and telomerase, increased. It was. On the other hand, the expression levels of genes such as prostaglandin endoperoxide synthase (prostaglandin synthase), interleukin-6 (IL-6) and the like that can have an adverse effect on the skin were decreased.

  From the above results, the peptide of the present invention increases the expression level of genes that can have a good effect on the skin and decreases the expression level of genes that can have a bad effect on the skin, resulting in a better action on the skin. It has been clarified that it can be useful for improving skin aging symptoms.

Example 3 Evaluation of Collagen Production Promotion Effect Normal human dermal fibroblasts (KF-4009; Kurabo Industries) were added to a 96-well microplate using Dulbecco's modified MEM (DMEM) (GIBCO) containing 10% calf serum (FBS). The cells were seeded at a cell density of 6.25 × 10 ⁴ cells / cm ² . 24 hours after seeding, the GRIRVL peptide prepared in Example 1 was replaced with DMEM containing a predetermined concentration. Separately, a well exchanged with DMEM not containing the test peptide was also prepared as a control (untreated group). After the GRIRVL peptide-containing group and the untreated group were cultured for about 48 hours, the culture supernatant was collected and subjected to Collagen Type I ELISA. The primary antibody reaction uses Affinity Purified Anti-Collagen Type I (Rabbit) (ROCKLAND), and the secondary antibody reaction uses Histofine Simple Stain MAX-PO (R) (Rabbit) (Nichirei). Type I collagen concentration was measured. Cells were lysed with 0.5% Triton X-100 solution, total protein amount was quantified using BCA Protein Assay Reagent Kit (Thermo Scientific), and type I collagen production per cell protein amount was compared with untreated group .

  The result is shown in FIG. As is clear from the results of FIG. 1, type I collagen production increased in the GRIRVL peptide-treated group, and in particular, a significant increase about 1.5 times that in the untreated group was observed in the 200 μg / ml treated group. From the above results, it is clear that the peptide of the present invention can exert a high collagen production promoting effect and can be beneficially used for prevention and / or improvement against skin wrinkles, tarmi, firmness and reduced elasticity. It became.

  Moreover, it can be understood from this test result that the increase or decrease in the gene expression level confirmed in Example 2 is actually associated with the increase or decrease in the protein amount.

Example 4: Evaluation of cell adhesion using peptide chip In order to evaluate the cell adhesion effect of the peptide of the present invention, the following test was conducted.
(1) Preparation of peptide chip:
Using an automatic peptide synthesizer (ASP222, Intavis AG, Koeln, Germaniy), a peptide chip was prepared by an ordinary method using Fmoc solid phase synthesis. First, esterify the C-terminus of Fmoc-β-Alanin-OH to the cellulose membrane, then remove the protecting group (Fmoc) by piperidine treatment, and then neutralize and wash this resin, then Fmoc-11-Aminoundecanoic acid It was combined with the C terminus. Subsequently, the protecting group (Fmoc) was removed by piperidine treatment, the resin was neutralized and washed, and then the C-terminus of Fmoc-Leu-OH was bound. Subsequently, the protecting group (Fmoc) was removed by piperidine treatment, and after this resin was neutralized and washed, Fmoc-Val-OH was introduced into the N-terminus of Leu. Next, the protecting group (Fmoc) was removed by piperidine treatment, the resin was neutralized and washed again, and Fmoc-Arg (Pmc) -OH was introduced into the N-terminus of Val. Next, the protecting group (Fmoc) was removed by piperidine treatment, and this resin was neutralized and washed again, and Fmoc-Ile-OH was introduced into the N-terminus of Arg. Next, the protecting group (Fmoc) was removed by piperidine treatment, the resin was neutralized and washed again, and Fmoc-Arg (Pmc) -OH was introduced into the N-terminus of Ile. Next, the protecting group (Fmoc) was removed by piperidine treatment, the resin was neutralized and washed again, and Fmoc-Gly-OH was introduced into the N-terminus of Arg. The protecting group (Fmoc) was then removed by piperidine treatment. Furthermore, deprotection was performed by cleaving the Pmc group of Arg with TFA (trifluoroacetic acid). Peptide variants having one or several amino acid additions and / or deletions in the amino acid sequence of GRIRVL peptide include GRIRVLQ (SEQ ID NO: 2), YGRIRVL (SEQ ID NO: 3), GRIRV (SEQ ID NO: 4), RIRVL (sequence) No. 5), GRIR (SEQ ID NO: 6), IRVLQR (SEQ ID NO: 7), QYGRIR (SEQ ID NO: 8), YGRIRV (SEQ ID NO: 9), RIRVLQ (SEQ ID NO: 10), RVLQRF (SEQ ID NO: 11) A peptide chip was prepared by the procedure described above. Finally, simple sterilization treatment with methanol was performed, and then used for cell assay.

(2) Cell adhesion evaluation:
The peptide chip produced as described above was perforated using BIOPSY PUNCH (KAI medical, Gifu, Japan) and transferred to a 96-well microplate. Normal human dermal fibroblasts (KF-4009; Kurabo) on a peptide chip, cell density of 2.0 × 10 ⁴ cells / well using Dulbecco's modified MEM (DMEM) (SIGMA) containing 10% calf serum (FBS) And cultured for 3 hours at 37 ° C. and 5% CO ₂ . Thereafter, non-adherent cells were removed by washing with physiological saline. The number of adherent cells was evaluated using a fluorescent dye calcein AM (Molecular Probes, Leiden, Netherland) (Ex: 485 nm, Em: 538 nm). Specifically, after cell seeding, the fluorescence intensity of the cellulose membrane was measured, and the number of adherent cells was calculated from a calibration curve. The fluorescence intensity of the membrane seeded with cells at a density of 2.0 × 10 ⁴ cells / well was taken as 100, and the ratio of the number of adherent cells (the number of remaining cells) to that was determined.

  The result is shown in FIG. As is clear from the results of FIG. 2, the GRIRVL peptide (SEQ ID NO: 1) and the peptides of SEQ ID NO: 2 to 11 with one or several amino acids added and / or deleted based on the amino acid sequence of SEQ ID NO: 1 When the modified product was used, a high cell adhesion effect was observed. Therefore, it can also be seen that by using the peptides of the present invention, the activity of a functional peptide desired to exert its effect on cells can be enhanced.

Example 5: Measurement of gene expression by quantitative PCR GRIRVL peptide (SEQ ID NO: 1) prepared in Example 1 above and GRIRV (SEQ ID NO: SEQ ID NO: 1) prepared by Fmoc method solid phase synthesis in the same procedure as Example 1 4) Using the peptide and RIRVL peptide (SEQ ID NO: 5), the quantitative PCR (qPCR) method was used to evaluate changes in gene expression when these peptides were allowed to act on normal human dermal fibroblasts.

First, normal human dermal fibroblasts (KF-4009; Kurabo Industries) were used in Dulbecco's modified MEM (DMEM) (GIBCO) containing 10% Fatal bovine serum (FBS) in a 96-well microplate at 1 x 10 ⁴ cells. The cells were seeded at a cell density of / well, cultured at 37 ° C. under 5% CO ₂ for about 24 hours, and replaced with DMEM (GIBCO) containing 0.5% FBS. Four hours later, the above three test peptides were exchanged with those dissolved at a predetermined concentration (0.5 mg / ml) using DMEM (GIBCO) containing 0.5% FBS. Separately, as a control (untreated group), a well exchanged with 0.5% FBS-containing DMEM not containing the test peptide was also prepared. After 24 hours, RNA samples were collected and used for qRT-PCR. Cells Direct One Step qRT-PCR Kit (Life Technologies) was used for sample recovery and qRT-PCR preparation. For detection of mRNA expressed from each gene, the following Taqman (registered trademark) Gene Expression Assays (Life Technologies) primers and probe sets were used [Fn1 gene: Hs00365058_m1, Timp2 gene: Hs00234278_m1, Cat gene: Hs00156308_m1, 18s rRNA (Reference gene): Hs99999901_s1]. qRT-PCR instrument is Applied Biosystems ABI PRISM 7000 Sequence Detection System, 50 ℃ 15 minutes (1 cycle), 95 ℃ 2 minutes (1 cycle), 95 ℃ 15 seconds, 60 ℃ 40 seconds (50 cycles) I went there. Relative quantification was performed by the ΔΔCT method using CT values obtained by the crossing point method, and the control group and the test peptide-treated group were compared. Table 5 below shows the results of calculating the ratio of each gene expression level in the cells of each test peptide treatment group when the control group (non-treatment group) is 100.

  Each target gene shown in Table 5 above is a gene whose expression level was increased in fibroblasts when treated with the GRIRVL peptide of the present invention in the evaluation using the microarray of Example 2 above. As is clear from these results, in this evaluation using the quantitative PCR method, not only the GRIRVL peptide but also its peptide variants GRIRV peptide and RIRVL peptide increase the expression levels of these genes. Was confirmed.

Example 6: Evaluation of integrin and fibronectin production promoting effect Normal human dermal fibroblasts (Kurabo) were cultured in 8-well culture slides (Thermo) using Dulbecco's modified MEM (DMEM) (GIBCO) containing 10% calf serum (DS Pharma). ) At a cell density of 3.0 × 10 ⁴ cells / cm ² . 24 hours after sowing, the medium was replaced with a serum-free DMEM medium. After another 24 hours, the GRIRVL peptide prepared in Example 1 was replaced with DMEM containing 1 mg / ml and cultured for 48 hours. Separately, as a control (untreated group), a well exchanged with DMEM not containing the test peptide was prepared and cultured in the same manner for 48 hours. Methanol cooled to -20 ° C was fixed for 20 minutes, and blocking treatment was performed at room temperature for 90 minutes with PBS containing 2% BSA.Anti-integrin α3 antibody (AB1920: Millipore) or anti-fibronectin antibody (ab23750 : abcam) was used for the primary antibody reaction. Each antibody was diluted 200-fold with PBS containing 2% BSA, and the reaction was allowed to stand overnight at 4 ° C. Thereafter, a secondary antibody reaction was performed using a goat-producing anti-rabbit antibody (Molecular Probe) to which alexa488 label was added. The secondary antibody was diluted 200-fold with PBS containing 2% BSA, and the reaction was allowed to proceed for 90 minutes at room temperature. Thereafter, it was treated with 1 ug / ml nuclear staining dye (Hoechst33258) for 5 minutes at room temperature, sealed with a commercially available mounting agent (Aqua Poly / Mount (Polysciences)), and observed using a fluorescence microscope. In the liquid exchange, the next reaction solution was added after washing twice with PBS.

  The resulting immunostained images (fluorescence microscope: 100 magnification) are shown in FIG. 3 and FIG. FIG. 3 is an immunostained image showing the expression of integrin α3. As is clear from FIG. 3, in the control (FIG. 3A), blue cell nuclei stained with Hoechst (Hoechst33258) are observed, but the presence of integrin α3 that develops a green color is hardly observed. On the other hand, when treated with the GRIRVL peptide of the present invention (FIG. 3B), an image of integrin α3 that develops green color can be clearly confirmed along with cell nuclei that develop blue color. That is, it can be seen that the expression of integrin α3 protein is promoted by treatment with the GRIRVL peptide. Similarly, in the immunostained image in which the expression of fibronectin was confirmed, an image of fibronectin colored green was recognized, and it was confirmed that the expression of fibronectin protein was actually promoted by using the peptide of the present invention ( FIG. 4).

Example 7: Evaluation of fusion peptide with αMSH-derived fragment peptide
ΑMSH-derived fragment peptide (Glu-His-Phe-Arg-Trp, which is known to have a melanin production-promoting effect as a functional peptide, in order to evaluate a fusion peptide obtained by fusing a GRIRVL peptide and a functional peptide -Gly: EHFRWG, SEQ ID NO: 13). As test peptides, there is a peptide (EHFRWG) in which nothing is fused to a fragment peptide derived from αMSH, a peptide in which an RGD sequence known as a cell adhesion motif sequence is fused at the N-terminus (RGD-EHFRWG), and the GRIRVL of the present invention Three types of peptides (GRIRVL-EHFRWG) were prepared by fusing peptides to the N-terminus.

First, B16 melanoma cells derived from mice were cultured in a 6-well culture plate. More specifically, the cells were seeded at a density of 7,000 cells / 1 cm ² and cultured at 37 ° C. in an environment of 5% carbon dioxide and 95% air for 24 hours. As the culture solution, 500 μl of each medium containing Dulbecco's modified MEM (DMEM) 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 3 ml of a medium containing the above-mentioned three kinds of test peptides added to DMEM at a concentration of 1 μg / ml was added. In addition, a medium supplemented with 3 ml of a medium to which no peptide was added was used as a control. After 48 hours of incubation, wash twice with PBS, then add 3 ml of WST-8 reagent diluted 100-fold with DMEM, and incubate at 37 ° C for 2 hours in an environment of 5% carbon dioxide and 95% air Went. Each supernatant was transferred to a 96-well culture plate and the number of cells was measured by measuring the absorbance at 450 nm. After the cells remaining in the 6-well culture plate were washed twice with PBS, lysed with 280 μl of a 10% DMSO solution containing 20% NaOH, transferred to a 1.5 ml tube, and reacted at 60 ° C. for 2 hours with a plate heater. Extraction was performed. This was transferred to a 96-well culture plate and the absorbance was measured at 475 nm to measure the amount of melanin. Based on this measured value, the ratio of the amount of melanin in each test peptide treatment group when the amount of melanin in the control was 100% was calculated. The results are shown in Table 6 below.

  As shown in Table 6 above, the αMSH-derived fragment peptide itself (EHFRWG) also increases the amount of melanin production compared to the control. And it turns out that the amount of melanin production increases further by using the peptide (RGD-EHFRWG) which fused this with the motif arrangement | sequence (RGD) known to have a cell adhesion effect | action to this. Furthermore, in the fusion peptide (GRIRVL-EHFRWG) in which GRIRVL, which is the peptide of the present invention, was fused to the N-terminus, a larger amount of melanin production was observed than when the RGD motif sequence was fused. That is, it can be seen from this result that the fusion peptide obtained by fusing the peptide of the present invention is excellent in the effect of improving the action of the functional peptide on cells.

Example 8: Evaluation of fusion peptide with fragment peptide derived from TGF-β
In order to further evaluate a fusion peptide obtained by fusing a GRIRVL peptide and a functional peptide, a TGF-β-derived fragment peptide (Ile-Trp-Ser-) known to have a collagen production promoting effect as a functional peptide The test was performed using Leu-Asp-Thr-Gln-Tyr: IWSLDTQY, SEQ ID NO: 12). As a test peptide, the GRIRVL peptide of the present invention is placed at the C-terminal across a peptide (IWSLDTQY) that is not fused to a fragment peptide derived from TGF-β and a linker (a three-residue linker composed of glycine). Two types of peptides, fused peptides (IWSLDTQY-GGG-GRIRVL), were used.

First, normal human dermal fibroblasts (KF-4009; Kurabo) were added to a 96-well microplate using Dulbecco's modified MEM (DMEM) (GIBCO) containing 10% calf serum (FBS), 6.25 × 10 ⁴ cells / Seeding at a cell density of cm ² . After 24 hours of seeding, the medium was replaced with DMEM. After further incubation for 24 hours, the above two kinds of test peptides were exchanged with DMEM containing 4 μM concentration. Separately, a well exchanged with DMEM not containing the test peptide was also prepared as a control (untreated group). After culturing the test peptide-containing group and the untreated group for about 48 hours, the culture supernatant was collected and used in the Procollagen type I C-peptide (PIP) EIA Kit (TakaRa), and human procollagen I in the culture supernatant was collected. The type C-terminal peptide (PIP) concentration was measured. The cells were lysed with 0.5% Triton X-100 solution, and the total protein amount was quantified using BCA Protein Assay Reagent Kit (Thermo Scientific). Subsequently, the amount of PIP production per cell protein amount was calculated from these quantitative results. Based on the calculated value, the ratio of the production amount of PIP per cell protein amount in each test peptide treatment group when the PIP amount per cell protein amount in the control was taken as 100% was determined. The results are shown in Table 7 below.

  As shown in Table 7, the TGF-β-derived peptide itself (IWSLDTQY) also increases the amount of PIP production per cell compared to the control. In addition, the fusion peptide (IWSLDTQY-GGG-GRIRVL) in which GRIRVL, which is the peptide of the present invention, is fused to the C-terminus via a three-residue glycine linker was found to further increase the amount of PIP production per cell. It was. From this result, it can be seen that even if a fusion peptide obtained by fusing a functional peptide and a GRIRVL peptide via a linker is used, the action of the functional peptide on cells can be improved.

  The peptide of the present invention or a derivative thereof, or a salt thereof can be used to promote cell adhesion and to exert a cosmetic effect based on the production promoting effect of collagen, integrin and / or fibronectin. it can. Furthermore, using the peptides of the present invention, the activity of a functional peptide intended to act on cells can be enhanced.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

SEQ ID NOs: 1-13

Claims (15)

Formula (I) below:
Gly-Arg-Ile-Arg-Val-Leu (SEQ ID NO: 1)
Or a peptide comprising an amino acid sequence having addition, deletion and / or conservative substitution of one or several amino acids in the amino acid sequence represented by the formula (I), or a peptide thereof Derivatives thereof, or salts thereof.   Gly-Arg-Ile-Arg-Val-Leu (SEQ ID NO: 1), Gly-Arg-Ile-Arg-Val (SEQ ID NO: 4), Arg-Ile-Arg-Val-Leu (SEQ ID NO: 5), Gly-Arg-Ile-Arg (SEQ ID NO: 6), Gln-Tyr-Gly-Arg-Ile-Arg (SEQ ID NO: 8), Tyr-Gly-Arg-Ile-Arg-Val (SEQ ID NO: 9), Arg-Ile-Arg-Val-Leu-Gln (SEQ ID NO: 10), Ile-Arg-Val-Leu-Gln-Arg (SEQ ID NO: 7), Arg-Val-Leu-Gln-Arg-Phe (SEQ ID NO: 10) 11), Tyr-Gly-Arg-Ile-Arg-Val-Leu (SEQ ID NO: 3), or Gly-Arg-Ile-Arg-Val-L u-Gln (SEQ ID NO: 2) is a peptide of claim 1, or a derivative of the peptide, or a salt thereof.   The peptide according to claim 1 or 2, which has an amino acid length of 4 to 8 residues, or a derivative of the peptide, or a salt thereof.   A composition comprising the peptide according to any one of claims 1 to 3, or a derivative of the peptide, or a salt thereof.   The composition according to claim 4, which is used for cosmetic purposes.   The composition according to claim 4 or 5, which is used for improving or preventing skin wrinkles, tarmi, reduced elasticity, reduced elasticity, poor turnover, spots, dullness, or thinning of the skin.   The composition according to any one of claims 4 to 6, which is used for tightening skin.   The composition according to any one of claims 4 to 7, which is used for promoting production of at least one selected from the group consisting of collagen, integrin, and fibronectin.   The composition according to any one of claims 4 to 8, wherein expression of a gene encoding a cell adhesion factor, a protein in an extracellular matrix or a protein involved in the formation thereof is increased.   The fusion peptide which fuse | melted the functional peptide which functions with respect to a cell, and the peptide in any one of Claims 1-3, the derivative of the fusion peptide, or those salts.   The functional peptide is at least one selected from the group consisting of growth factors, hormones, antagonists, agonists, and parts thereof, or a fusion peptide according to claim 10, or a derivative of the fusion peptide, or Salt.   The fusion peptide according to claim 10 or 11, wherein the functional peptide is a fragment peptide derived from transforming growth factor (TGF) or a fragment peptide derived from melanocyte-stimulating hormone (MSH), or a derivative of the fusion peptide, or Salt.   The fusion peptide according to any one of claims 10 to 12, or a derivative of the fusion peptide, wherein the functional peptide and the peptide according to any one of claims 1 to 3 are fused via a linker, or Their salt.   The fusion peptide according to any one of claims 10 to 13, or a derivative of the fusion peptide, or a salt thereof, wherein the peptide according to any one of claims 1 to 3 adheres to a cell.   A composition comprising the fusion peptide according to any one of claims 10 to 14, or a derivative of the fusion peptide, or a salt thereof.

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