JP7542865B2 - Oligopeptide having collagen gel contraction promoting effect and its use - Google Patents

Description

The present invention relates to an oligopeptide and its derivatives that have a collagen gel contraction promoting effect or a fibroblast activating effect. The present invention also relates to an agent for preventing or improving wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin, an agent for promoting collagen gel contraction, and an agent for activating fibroblasts.

In recent years, more and more people, regardless of age or gender, are becoming interested in beauty. In particular, for anti-aging purposes, surgical treatments, topical medications, and cosmetics containing functional ingredients that improve wrinkles, sagging, and lack of firmness in the skin (hereinafter also referred to as "skin") and smooth the skin texture or texture are gaining popularity.

Surgical treatment involves inserting extremely fine absorbable sutures into the subcutaneous fascial layer to lift the skin and muscles, but although it is a symptomatic treatment, it is an invasive procedure and is performed under the supervision of a doctor, placing a heavy burden on the patient.

In addition, there are cosmetics on the market that use moisturizing agents to temporarily reduce the appearance of fine lines around the eyes, but once use is stopped, the fine lines become noticeable again over time. For this reason, there is a strong demand for topical medications, functional cosmetics, or food compositions for beauty that can improve wrinkles, sagging, and loss of firmness in the skin over the long term with daily use.

Hyaluronic acid exists in the skin as one of the extracellular matrices involved in water retention and viscosity. Hyaluronic acid has a water-retaining effect, and when applied to the skin, it increases the moisture content of the stratum corneum for a long time, improving skin firmness, so topical medicines and cosmetics containing hyaluronic acid have been used. However, hyaluronic acid is metabolized very quickly, and it is said that about half of the hyaluronic acid is decomposed in the skin every day. Therefore, even if hyaluronic acid is applied to the skin every day, it is constantly metabolized, and the improvement in skin firmness cannot be fully felt.
In addition, it is known that topical agents are formulated to improve skin firmness by incorporating ingredients that promote the production of extracellular matrix such as hyaluronic acid and collagen when applied to the skin, and ingredients that inhibit the breakdown of elastin, an extracellular matrix. However, these ingredients have the drawback of causing inflammation and irritating the skin.

On the other hand, peptides are desirable as active ingredients in topical agents because they are metabolized relatively slowly and do not irritate the skin. Bovine lactoferrin and its partial peptides are known to be peptides that are expected to improve skin firmness.

For example, Non-Patent Document 1 reports that bovine lactoferrin enhances phosphorylation of myosin light chains in fibroblasts and increases their collagen gel contractile activity.
Lactoferrin is an iron-binding glycoprotein found in exocrine fluids such as breast milk, tears, sweat, and saliva, and was first reported in 1939 as a "red protein" found in cow's milk. It was subsequently purified from human and cow's milk in 1960, and its amino acid sequence was determined. It was found to consist of 689 amino acids in cow's milk and 692 amino acids in humans, with two globular domains called the N-lobe and C-lobe linked by a single polypeptide, and each lobe was found to strongly bind to one iron ion. Lactoferrin has been shown to have various functions, such as anti-inflammatory, antioxidant, antibacterial, and cell proliferation regulation, in addition to promoting collagen gel contraction.

Non-Patent Document 2 reports that the C-terminal fragment obtained by decomposing bovine lactoferrin with trypsin at a molar ratio of 1/200 for 45 minutes at 37°C has a significantly stronger collagen gel contraction promoting activity than full-length lactoferrin, and an activity promoting phosphorylation of myosin light chain in fibroblasts. Among these bovine lactoferrin C-terminal fragments, the p44 fragment corresponds to the portion consisting of amino acids 341-689 of bovine lactoferrin, the p36 fragment corresponds to the portion consisting of amino acids 1-284 of bovine lactoferrin, and the p51 fragment corresponds to the portion consisting of amino acids 285-689 of bovine lactoferrin, and the molecular weights of these fragments are 44,000, 36,000, and 51,000, respectively.

The skin is divided into the epidermis, dermis, and subcutaneous tissue, of which the dermis contains more than 70% collagen fibers, which are extremely important for maintaining the skin structure, forming flexible and elastic dermal tissue. Proteins that make up collagen and other fibers are produced by fibroblasts, and the interaction of fibroblasts with collagen fibers forms flexible and elastic dermal tissue.
Collagen gel contraction is a phenomenon in which fibroblasts pull on collagen fibers and cause them to contract. When collagen extracted from tissue is placed at 37°C under neutral and physiological ionic strength, the collagen fibers are reconstituted. This is called a collagen gel, and when fibroblasts are cultured in the collagen gel, the fibroblasts pull on the collagen fibers and contract, orienting the collagen fibers, and the reconstituted collagen gel becomes more flexible, elastic, and strong, and takes on a structure similar to that of the dermis. Therefore, the contracted gel obtained by collagen gel contraction by fibroblasts is considered to be a model for dermal tissue (Non-Patent Document 3).

It has been reported that fibroblasts derived from elderly individuals have a lower collagen gel contraction activity than fibroblasts derived from young individuals, and it is believed that aging reduces the gel contraction activity of fibroblasts (Non-Patent Document 4). This suggests that if the collagen gel contraction activity is reduced by aging, it will no longer be possible to contract the dermal tissue and maintain it in a firm state as in younger individuals (Patent Document 1).
That is, wrinkles, sagging, and loss of firmness in the skin that accompanies aging are caused by a decrease in the tissue contractile force of dermal fibroblasts, resulting in a loss of flexibility, elasticity, and strength. It has been reported that it is expected that wrinkles, sagging, and loss of firmness in the skin can be improved by increasing the collagen fiber contractile activity of dermal fibroblasts (Patent Document 2).

However, the bovine lactoferrin taught in Non-Patent Document 1 and the partial peptide of bovine lactoferrin taught in Non-Patent Document 2 both have large molecular weights, making them difficult to absorb through the skin, and are highly immunogenic, which may cause allergies. Therefore, it is desirable for the components of topical agents to be oligopeptides with small molecular weights that have a collagen gel contracting effect or a fibroblast activating effect.

Patent No. 5923410 Patent No. 4225588

Takayama Y, Mizumachi K. Effects of lactoferrin on collagen gel contractile activity and myosin light chain phosphorylation in human fibroblasts. FEBS lett. 2001, 508 (1) : 111-116. Takayama Y, Mizumachi K, Takezawa T. The bovine lactoferrin region responsible for promoting the collagen gel contractile activity of human fibroblasts. Biochem Biophys Res Comn. 2002, 299 (5) : 813-817. Bell E, Sher S, Hull B et al. The reconstitution of living skin. J Invest Dermatol. 1983, 81 : 2s-10s. Yamato M, Yamamoto K, Hayashi T. Age-related changes in collagen gel contraction by cultured human lung fibroblasts resulting in cross-over of contraction curves between young and aged cells. Mech Ageing Dev. 1993, 67(1-2): 149 -158.

The present invention aims to provide an oligopeptide and its derivatives that have the effect of promoting collagen gel contraction, as well as an agent for preventing or improving wrinkles, sagging, sagging, loss of firmness, or loss of elasticity of the skin, which contains the oligopeptide and its derivatives as active ingredients.

In order to solve the above problems, the present inventors focused on lactoferrin, subjected bovine lactoferrin to limited hydrolysis with trypsin, fractionated the limited hydrolysis product using a preparative ODS column, and further fractionated the fraction having collagen gel contraction-promoting activity by gel filtration, discovering two oligopeptides consisting of eight amino acids and one oligopeptide consisting of five amino acids that have collagen gel contraction-promoting activity.
In addition, peptides were synthesized by removing one amino acid residue each from the N-terminus or C-terminus of these oligopeptides, and the collagen gel contraction promoting effect of these synthetic peptides was confirmed, and it was found that each of the dipeptides AV and DG is a core amino acid sequence that exhibits the collagen gel contraction promoting effect. This also suggested that any oligopeptide containing up to 8 amino acids as long as it contains the amino acid sequence of AV or DG has a collagen gel contraction promoting effect, and it was confirmed that among them, each of the oligopeptides No. 2 to 10 containing AV and each of the peptides No. 12 to 24 containing DG have a collagen gel contraction promoting effect. The oligopeptides with each number are as shown in the "Oligopeptide" section of "Mode for Carrying Out the Invention".
Furthermore, it was found that the collagen gel contraction promoting effect of these oligopeptides was not lost even when they were modified, such as in the form of esters.

The present invention has been completed based on the above findings, and provides the following [1] to [29].
[1] Any one of the following oligopeptides (a), (b), (c), and (d) or a derivative thereof:
(a) an oligopeptide consisting of the amino acid sequence of AV, SCHTAVDR, CHTAVDR, CHTAVD, HTAVD, CHTAV, TAVD, HTAV, TAV, or AVD
(b) An oligopeptide consisting of an amino acid sequence in which one amino acid is substituted in AV, SCHTAVDR, CHTAVDR, CHTAVD, HTAVD, CHTAV, TAVD, HTAV, TAV, or AVD (including AV), and having the effect of promoting collagen contraction.
(c) an oligopeptide consisting of the amino acid sequence of DG, LLCLDGTR, LCLDGTR, CLDGTR, LDGTR, LDGT, DGTR, LDG, DGT, SVDGK, SVDG, VDGK, DGK, or VDG
(d) an oligopeptide consisting of an amino acid sequence in which one amino acid is substituted in DG, LLCLDGTR, LCLDGTR, CLDGTR, LDGTR, LDGT, DGTR, LDG, DGT, SVDGK, SVDG, VDGK, DGK, or VDG (including DG), and having a collagen contraction-promoting effect; [2] The oligopeptide or derivative thereof according to [1], wherein the derivative is an N-terminal modified form of the oligopeptide.
[3] An agent for preventing or improving wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin, comprising at least one of the oligopeptide and its derivatives according to [1] or [2].
[4] The agent for preventing or improving wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin according to [3], which is an external composition or a food composition.
[5] A collagen gel contraction promoter comprising at least one of the oligopeptide and its derivatives according to [1] or [2].
[6] The collagen gel contraction promoter according to [5], which is a composition for external use or a food composition.
[7] A fibroblast activator comprising at least one of the oligopeptide and its derivative according to [1] or [2].
[8] The fibroblast activator according to [7], which is a composition for external use or a food composition.

[9] Use of at least one of the oligopeptide and its derivatives according to [1] or [2] for the manufacture of an agent for preventing or improving wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin.
[10] The use according to [9], wherein the agent for preventing or improving wrinkles, sagging, sagging skin, loss of firmness, or loss of elasticity is a composition for external use or a food composition.
[11] Use of at least one of the oligopeptide and its derivatives according to [1] or [2] for the production of a collagen gel contraction promoter.
[12] The use according to [11], wherein the collagen gel contraction promoter is a composition for external use or a food composition.
[13] Use of at least one of the oligopeptide and its derivatives according to [1] or [2] for the production of a fibroblast activator.
[14] The use according to [13], wherein the fibroblast activator is a composition for external use or a food composition.

[15] Non-therapeutic use of a composition containing at least one of the oligopeptides and derivatives thereof according to [1] or [2] as an agent for preventing or improving wrinkles, sagging, sagging, loss of firmness, or loss of elasticity of the skin.
[16] The use according to [15], wherein the composition is a composition for external use or a food composition.
[17] Non-therapeutic use of a composition containing at least one of the oligopeptide and its derivatives according to [1] or [2] as a collagen gel contraction promoter.
[18] The use according to [17], wherein the collagen gel contraction promoter is a composition for external use or a food composition.
[19] Non-therapeutic use of a composition comprising at least one of the oligopeptide and its derivatives according to [1] or [2] as a fibroblast activator.
[20] The use according to [19], wherein the composition is a composition for external use or a food composition.

[21] The oligopeptide or derivative thereof according to [1] or [2] for use in preventing or improving wrinkles, sagging, sagging, loss of firmness, or loss of elasticity of the skin.
[22] The oligopeptide or derivative thereof according to [1] or [2] for use in promoting collagen gel contraction.
[23] The oligopeptide or a derivative thereof according to [1] or [2] for use in fibroblast activation.

[24] A method for preventing or improving wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin, comprising the step of administering to a human an effective amount of at least one of the oligopeptides and derivatives thereof according to [1] or [2].
[25] The method according to [24], wherein the application is by application, rubbing, spraying, or patching to the skin, or oral administration.
[26] A method for promoting collagen contraction in the skin, comprising the step of applying an effective amount of at least one of the oligopeptide and its derivatives according to [1] or [2] to a human.
[27] The method according to [26], wherein the application is by application, rubbing, spraying, or patching to the skin, or oral administration.
[28] A method for activating skin fibroblasts, comprising the step of applying an effective amount of at least one of the oligopeptide and its derivatives according to [1] or [2] to a human.
[29] The method according to [28], wherein the application is by application, rubbing, spraying, or patching to the skin, or oral administration.

The oligopeptide and its derivatives of the present invention have a collagen gel contraction promoting effect or a fibroblast activation effect. In the dermis, fibroblasts contract collagen fibers to maintain a firm state, so that the oligopeptide and its derivatives of the present invention, when applied to human skin, are thought to be able to prevent or improve wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin by promoting collagen contraction by fibroblasts in the dermis.
In addition, unlike proteins, the oligopeptide and its derivatives of the present invention have a small molecular weight, and therefore are easily absorbed through the skin, and are easily absorbed from the digestive tract when administered orally. The molecular weights of human lactoferrin and bovine lactoferrin are about 83,000, and when applied to the skin, they remain on the skin surface, and do not penetrate through the stratum corneum to the epidermis and dermis, and when administered orally, they have low absorption efficiency, but the oligopeptide and its derivatives of the present invention have a significantly higher penetration into the epidermis and dermis and absorption efficiency from the digestive tract than human lactoferrin and bovine lactoferrin.
Furthermore, unlike proteins, the oligopeptides and derivatives thereof of the present invention have low immunogenicity and are therefore excellent in that they are less likely to cause allergies.
Furthermore, unlike the case where an extracellular matrix such as hyaluronic acid, collagen, elastin, or glycosaminoglycan is applied to the skin, when the oligopeptide and its derivative of the present invention are applied, rubbed, sprayed, or attached to the skin, they are unlikely to be metabolically lost after being absorbed into the skin. Also, unlike conventional ingredients, they are not or only slightly irritating to the skin. In these respects, the oligopeptide of the present invention is superior to conventional ingredients used to improve wrinkles or firmness of the skin.

The present invention will be described in detail below.
Oligopeptides
The oligopeptide of the present invention is any one of the following oligopeptides (a), (b), (c), and (d):
(a) No. Oligopeptide consisting of the amino acid sequence 1 to 10
(b) An oligopeptide having an amino acid sequence in which one amino acid has been substituted in the amino acid sequence of No. 2 to 10 (including AV), and having a collagen contraction promoting effect.
(c) Oligopeptide consisting of the amino acid sequence of No. 11 to 24
(d) An oligopeptide having an amino acid sequence in which one amino acid is substituted in the amino acid sequence of No. 12 to 24 (including DG) and having a collagen contraction promoting effect.

The fact that the oligopeptides (b) and (d) have the collagen contraction promoting effect is confirmed by the method described in Example 1, and it is determined that they have the collagen contraction promoting effect when the collagen gel area is smaller than that of the control.

The oligopeptide numbers of the present invention are shown below.
No. 1: AV
No. 2: SCHTAVDR (SEQ ID NO: 1)
No. 3: CHTAVDR (SEQ ID NO: 2)
No. 4: CHTAVD (SEQ ID NO: 3)
No. 5: HTAVD (SEQ ID NO: 4)
No. 6: CHTAV (SEQ ID NO: 5)
No. 7: TAVD (SEQ ID NO: 6)
No. 8: HTAV (SEQ ID NO: 7)
No. 9: TAV
No. 10: A.V.D.
No. 11: DG
No. 12: LLCLDGTR (SEQ ID NO: 8)
No. 13: LCLDGTR (SEQ ID NO: 9)
No. 14: CLDGTR (SEQ ID NO: 10)
No. 15: LDGTR (SEQ ID NO: 11)
No. 16: LDGT (SEQ ID NO: 12)
No. 17: DGTR (SEQ ID NO: 13)
No. 18: LDG
No. 19: DGT
No. 20: SVDGK (SEQ ID NO: 14)
No. 21: SVDG (SEQ ID NO: 15)
No. 22: VDGK (SEQ ID NO: 16)
No. 23: DGK
No. 24: VDG

The amino acid substitutions in the oligopeptides Nos. 2 to 10 and 12 to 24 can be made between amino acids having similar structures or properties, and it is reasonably predicted that the substitutions will still have the effect of promoting collagen gel contraction.
Examples of amino acids that can be substituted for each other include acidic amino acids E (Glu) and D (Asp), basic amino acids R (Arg), K (Lys), and H (His), and neutral amino acids such as G (Gly), A (Ala), V (Val), L (Leu), and I (Ile) having alkyl chains (particularly, G (Gly) and A (Ala) having linear alkyl chains, and V (Val), L (Leu), and I (Ile) having branched alkyl chains), S (Ser) and T (Thr) having hydroxy groups, C (Cys) and M (Met) having sulfur atoms, N (Asn) and Q (Gln) having amide groups, and F (Phe), Y (Tyr), and W (Trp) having aromatic rings.
For example, since it has been demonstrated that HTAV has the effect of promoting collagen gel contraction, it is reasonably predicted that HTAL also has a similar effect of promoting collagen gel contraction.

As shown in the Examples section, the oligopeptide of the present invention was discovered based on the partial amino acid sequence of bovine lactoferrin, and it was confirmed that the amino acid sequence SCHTAVDR (No. 2) containing AV is present not only at positions 475 to 482 in bovine lactoferrin but also at positions 458 to 465 in human lactoferrin. In other words, Nos. 1 to 10 are amino acid sequences present in bovine lactoferrin and human lactoferrin.
It was also confirmed that the amino acid sequence of LLCLDGTR containing DG (No. 12) is present at positions 590 to 597 in bovine lactoferrin but is not present in human lactoferrin. In other words, Nos. 11 to 19 are amino acid sequences present in bovine lactoferrin.
It was also confirmed that the amino acid sequence of SVDGK containing DG (No. 20) is present in bovine lactoferrin but not in human lactoferrin. That is, Nos. 20 to 24 are amino acid sequences present in bovine lactoferrin.
Therefore, the oligopeptide of the present invention consisting of an amino acid sequence present in human or bovine lactoferrin can be prepared by trypsin digestion of human or bovine lactoferrin. Since lactoferrin is abundant in milk serum (whey), the oligopeptide consisting of an amino acid sequence present in bovine lactoferrin may be obtained by trypsin digestion of milk, cheese, or yogurt, or by trypsin digestion of whey such as milk whey, cheese whey, or yogurt whey.
Alternatively, each of the oligopeptides of the present invention can be prepared synthetically.

Oligopeptide Derivatives The oligopeptides of the present invention may be derivatives (modified forms) such as salts.
Examples of the salt include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, salts with metals such as zinc and aluminum, ammonium salts, basic amino acid salts, and salts with amines such as triethanolamine.
In addition, it may be a salt with an inorganic acid or an organic acid.
Examples of inorganic acid salts include salts of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like.
Examples of the organic acid salts include salts of aliphatic monocarboxylic acids (fatty acid salts) such as acetic acid, propionic acid, butanoic acid (butyric acid), isobutanoic acid, 6-heptanoic acid, hexanoic acid (caproic acid), palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, octanoic acid (caprylic acid), and decanoic acid (capric acid); salts of aliphatic polycarboxylic acids such as fumaric acid, maleic acid, succinic acid, and malonic acid; salts of aliphatic oxycarboxylic acids such as lactic acid, tartaric acid, and citric acid; salts of aromatic carboxylic acids such as nicotinic acid; and salts of organic sulfonic acids such as methanesulfonic acid, toluenesulfonic acid, tosylic acid, and napadisilic acid.
The derivative may be one in which any of the N-terminus, C-terminus, or side chain of the oligopeptide of the present invention has been modified, or two or more of these may have been modified. Although the derivative has collagen gel contraction promoting activity regardless of which part has been modified, an N-terminus modification is preferred in terms of good transdermal absorbability.

Composition The oligopeptide and its derivative of the present invention can be used as an active ingredient in an agent for preventing or improving wrinkles, sagging, sagging, loss of firmness, or loss of elasticity of the skin, an agent for promoting collagen gel contraction, or an agent for activating fibroblasts (hereinafter sometimes abbreviated as "the agent of the present invention").
These agents can be compositions for external use (cosmetics, quasi-drugs) or food compositions. The compositions can be prepared by mixing at least one of the oligopeptides of the present invention and their derivatives with additives or other physiologically or pharmacologically active ingredients, which are added as necessary.

The concentration of the oligopeptide and/or its derivative of the present invention in the composition can be 0.00001% by weight or more, 0.0001% by weight or more, 0.001% by weight or more, 0.01% by weight or more, or 0.1% by weight or more, relative to the total amount of the composition, and can be 10% by weight or less, 1% by weight or less, 0.1% by weight or less, or 0.01% by weight or less. Within this range, the collagen gel contraction promoting effect can be sufficiently obtained.

The topical composition may in particular be a topical composition for skin application. The skin includes the scalp. In the present invention, the skin also includes mucous membranes.
The external composition may be in either a solid or liquid form. The solid form includes a form having plasticity that can be deformed by applying force, and a freeze-dried form. The liquid form includes a form having viscosity, such as a flowable form.
Examples of dosage forms include lotions, creams, emulsions, gels, ointments, liquids, solutions, suspensions, sprays or aerosols, foams, patches in which these are impregnated or applied to a base material, powders, dosage forms in which powders are solidified, etc. Emulsion compositions such as creams and emulsions may be of the O/W type, W/O type, W/O/W type, O/W/O type, etc.
When the composition for external use is a cosmetic product, it can be basic cosmetics such as lotion, milky lotion, cream, gel, beauty essence, sunscreen cosmetic, pack, mask, hand cream, body lotion, or body cream, as well as makeup cosmetics such as foundation, lipstick, or blusher.

Additives for topical compositions include bases, surfactants, thickeners, preservatives or preservatives, pH adjusters, stabilizers or chelating agents, UV absorbers or UV scattering agents, antioxidants, irritation mitigators, colorants, fragrances, etc. One or more types of additives can be blended.

As other physiologically or pharmacologically active ingredients, ingredients that improve skin firmness other than the oligopeptide or derivative thereof of the present invention can be preferably blended.
Ingredients that improve skin firmness include ingredients that have the effect of promoting collagen gel contraction, as well as ingredients that have the effect of promoting the production of extracellular matrix (hyaluronic acid, collagen, elastin, glycosaminoglycan, etc.), ingredients that have the effect of inhibiting the decomposition of extracellular matrix, ingredients that have the effect of promoting the proliferation of fibroblasts, and ingredients that have the effect of promoting the turnover of keratinocytes.
Ingredients that have such effects include lactoferrin, artichoke, Argania spinosa, aloe vera, nettle, turmeric, prickly pear, okra, ginseng, kakkon, obliquus, raspberry, goldfinch, kudzu, gardenia, mulberry, chlorella, alpinia speciosa, carambola, cherry blossom, pomegranate, Japanese pepper, lithospermum, peony, birch, star fruit, sage, soybean, Chinese laurel, clove, Centella asiatica, Angelica acutiloba, corn, Eucommia jujube, carrot, passion fruit, Job's tears, Belamcanda chinensis, bilberry, loquat, grape, beech, peony, Peony stalk, hops, mugwort, and purple , extracts of plants such as evening primrose, peach, cornflower, eucalyptus, yuzu, European beech, astragalus, rosemary, Roman chamomile, and burnet, pearl protein hydrolysate, hydrolyzed conchiolin protein, γ-oryzanol, rice bran oil, Haematococcus pluvialis extract, glycylglycine, astaxanthin, dipotassium glycyrrhizinate, β-sitosterol, stem cell culture supernatant, hydrolyzed eggshell membrane, umbilical cord extract, ceramide, hydroxyproline, acetylhydroxyproline, mevalonolactone, γ-aminobutyric acid, ectoine, acetylglucosamine, and copper tripeptide-1.
In addition, hyaluronic acid, collagen, elastin, glycosaminoglycan, chondroitin sulfate, heparinoids, proteoglycan, or decomposition products, derivatives, and salts thereof can also be blended, which can also improve the firmness of the skin.

Further, examples of physiologically active or pharmacologically active ingredients include whitening ingredients, immunostimulants, astringents, antioxidants, blood circulation promoters, antibacterial agents, warming agents, vitamins, amino acids, wound healing promoters, cell activators, and enzymes.
Such ingredients include retinol, vitamin A and its derivatives, vitamin C and its derivatives, salicylic acid and its derivatives, resorcinol and its derivatives, phospholipids such as phosphatidylserine, phosphatidic acid, cyclic phosphatidic acid, tranexamic acid and its derivatives, niacinamide, hydroquinone and its derivatives, trehalose, trehalose sulfate, and the like.
One or more physiologically active or pharmacologically active ingredients other than the oligopeptide or derivative thereof of the present invention may be blended.

Food Composition The food composition may be an oral administration preparation known as a supplement.
Examples of solid oral preparations include tablets, powders, granules, fine granules, pills, capsules (soft capsules, hard capsules), etc. The solid preparations may contain one or more additives such as excipients, binders, disintegrants, lubricants, flow agents, colorants, flavorings, sweeteners, flavorings, preservatives, or antiseptics.
Examples of liquid preparations for oral administration include solutions, elixirs, suspensions, emulsions, lemonades, syrups, etc. Liquid preparations may contain one or more additives such as pH adjusters, buffers, thickeners, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, colorants, and flavors.
In addition, the oral preparation may contain one or more physiologically or pharmacologically active ingredients other than the oligopeptide or its derivative of the present invention. Examples of such physiologically or pharmacologically active ingredients include those exemplified for the topical composition.

Furthermore, the food composition may be a composition in which the oligopeptide of the present invention and/or its derivatives are blended with general food. For example, the oligopeptide of the present invention and/or its derivatives may be blended with solid foods such as jelly, agar confectionery, gum, candy, baked goods (cookies, biscuits, etc.), or liquid foods such as energy drinks, tea drinks, coffee drinks, milk drinks, fruit and vegetable juices, and soft drinks, and these allow the oligopeptide of the present invention and/or its derivatives to be easily ingested.

The food composition may be used as a nutritional supplement, a health supplement, a nutritionally adjusted food, a food for specified health uses, a food with nutritional functions, a food with functional claims, etc.

Methods of Use The present invention provides (i) a method for preventing or improving wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin, (ii) a method for promoting collagen contraction in the skin, and (iii) a method for activating fibroblasts in the skin, each of which comprises the step of applying an effective amount of the oligopeptide and/or derivative thereof of the present invention described above to a human.
An effective amount is an amount effective for each of (i) preventing or improving wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin, (ii) promoting collagen contraction in the skin, and (iii) activating fibroblasts in the skin.

The oligopeptide and/or its derivative of the present invention may be applied to humans as the agent of the present invention (topical composition or food composition) described above. In the case of a topical composition, "application" can be coating, rubbing, spraying, or pasting on the skin, depending on the dosage form. The target skin can be the face, neck, arms, fingers, feet, toes, trunk, etc., and among these, it can be parts where wrinkles, sagging, sagging, loss of firmness, and/or loss of elasticity have occurred or parts that are in the pre-stage of such. In the case of a food composition, "application" can be oral administration or ingestion.

The target human to which the agent of the present invention is applied may be any of those who have wrinkles, sagging, loose skin, loss of firmness, and/or loss of elasticity, those who are in the pre-stage of such, and those who have healthy skin. The skin in this case includes the face, neck, arms, fingers, feet, toes, and torso, but is particularly suitable for those who have wrinkles, sagging, loose skin, loss of firmness, and/or loss of elasticity in the skin of the face and/or neck, and those who are in the pre-stage of such.

When the agent of the present invention is a composition for external use, the composition for external use can be applied to the skin so that the amount of the oligopeptide and/or derivative thereof of the present invention applied per ^cm2 of skin per day is 0.0001 mg or more, 0.001 mg or more, 0.01 mg or more, 0.1 mg or more, or 1 mg or more, and is 100 mg or less, 10 mg or less, 1 mg or less, or 0.1 mg or less.
When the agent of the present invention is a food composition, the food composition can be administered or ingested so that the daily administration or intake amount of the oligopeptide and/or its derivative of the present invention is 0.05 mg or more, 0.5 mg or more, 5 mg or more, 50 mg or more, or 500 mg or more, and 50,000 mg or less, 5,000 mg or less, 500 mg or less, or 50 mg or less.
The above-mentioned daily application amount to the skin, and daily administration or intake amount are effective amounts for (i) preventing or improving wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin, (ii) promoting collagen contraction in the skin, and (iii) activating fibroblasts in the skin.
Additionally, the number of applications per day can be 1-5 times, 1-4 times, 1-3 times, 1-2 times, or 1 time.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.
Example 1 (Preparation of oligopeptides and evaluation of collagen gel contraction promoting activity)
(Preparation of trypsin-limited hydrolysate of bovine lactoferrin)
1 g of bovine lactoferrin and 0.01 g of trypsin were dissolved in 20 mL of a pH 8.0 buffer solution, and the solution was left to stand at 50°C for 16 hours for limited hydrolysis. The hydrolysis product was heated at 100°C for 20 minutes to inactivate the enzyme, and a bovine lactoferrin hydrolysis solution was obtained. The bovine lactoferrin hydrolysis solution was fractionated into 40 portions using an ODS column and freeze-dried. A phosphate buffer solution (pH 7.4) solution with a concentration of 5 mg/mL was prepared from each freeze-dried fraction, and the collagen gel contraction promoting activity was evaluated. As a result, fractions No. 36 and No. 37 were found to have collagen gel contraction promoting activity, and these were fractionated using a gel filtration column to estimate the approximate molecular weight. As a result, four candidate sequences (SCHTAVDR (No. 2), LLCLDGTR (No. 12), SVDGK (No. 20), and DLLFK (No. 25) (SEQ ID NO: 17)) were considered, and these peptides were synthesized and solutions with a concentration of 5 mg/mL were prepared using a solvent (DMSO:PBS = 1:1). As a result of evaluating the collagen gel contraction promoting activity of these solutions, each of the peptides SCHTAVDR (No. 2), LLCLDGTR (No. 12), and SVDGK (No. 20) was found to have collagen gel contraction promoting activity, so peptides were synthesized by removing one amino acid each from the N-terminus or C-terminus of each of these peptides, and their collagen gel contraction promoting activity was evaluated.

(Evaluation of collagen gel contraction promoting activity)
To each well of a 24-well plate, 100 μL of a medium (0.94 g of Minimum Essential Medium (MEM), 500 μL of 1 mol/L NaHCO ₃ , 29.2 mg of L-glutamine, and 1 mL of bovine serum were added to pure water to prepare a 20 mL volume) at a concentration 5 times the manufacturer's specified concentration, 2.5 μL of a test sample prepared to 5 mg/mL, 365 μL of type I collagen (IAC-30), 12.5 μL of 1 mol/L NaHCO ₃ , and 20 μL of human fibroblasts (2.5 million cells/mL) were added in this order. The well plate was then cultured at 37° C. for 1 hour to prepare a collagen gel. The gel attached to the wall of the well plate was peeled off and placed in the well, and 500 μL of Dulbecco's Modified Eagle Medium (DMEM) containing 1% FBS and a test sample adjusted to 5 mg/mL was added to the top of the gel. This was further cultured at 37° C., and the area of the gel after 3 days from the start of culture was measured by measuring the diameter of the gel with a digital caliper.

As a control sample, cultivation and observation were carried out in the same manner as above, except that the solvent used in preparing the test sample was used instead of the test sample.
Bovine lactoferrin was used as a positive control sample.
Three gels were prepared for each sample and incubated (n=3), and the gel areas were measured and the average and standard deviation were calculated.
If the gel area of the test sample is smaller than that of the control, it is found that the test sample has the activity of promoting collagen gel contraction by cultured human fibroblasts. Therefore, by measuring the gel area using the above method, it is possible to evaluate whether the test sample has an effect of improving wrinkles, sagging, and loss of firmness in the skin.

The results are shown in Table 1. The collagen gel area ratio in Table 1 is a relative value when the collagen gel area of the control is set to 1.

By adding the oligopeptide SCHTAVDR (No. 2) or LLCLDGTR (No. 12), the area of the collagen gel was significantly reduced, i.e., collagen gel contraction was promoted, compared to the control, and it was confirmed that these oligopeptides have the effect of improving wrinkles, sagging, and lack of firmness in the skin. The positive control bovine lactoferrin also significantly promoted collagen gel contraction compared to the control. In addition, no significant difference was observed between the collagen gel contraction promotion activity of each of the oligopeptides No. 2 and No. 12 and the collagen gel contraction promotion activity of bovine lactoferrin.
Many of the oligopeptides having amino acid sequences obtained by removing one amino acid each from the N-terminus or C-terminus of each of the oligopeptides No. 2 and No. 12 significantly promoted collagen gel contraction compared to the control, but some of them did not have significant collagen gel contraction-promoting activity.

The oligopeptide SVDGK (No. 20) promoted the contraction of collagen gel. Moreover, oligopeptides having amino acid sequences obtained by deleting one amino acid each from the N-terminus or C-terminus of the oligopeptide No. 20 promoted the contraction of collagen gel significantly more than the control.
DLLFK (No. 25), which consists of the amino acid sequence of 316 to 320 of bovine lactoferrin, was not found to have any collagen gel contraction promoting activity.

Example 2 (Effective Concentration of Oligopeptide for Promoting Collagen Gel Contraction)
To each well of a 24-well plate, 100 μL of a medium with a concentration 5 times the manufacturer's specified concentration {0.94 g of MEM, 500 μL of 1 mol/L NaHCO ₃ , 29.2 mg of L-glutamine, and 1 mL of bovine serum were added to pure water to prepare a volume of 20 mL}, 2.5 μL of oligopeptide of TAV (No. 9) prepared at each concentration, 365 μL of type I collagen (IAC-30), 12.5 μL of 1 mol/L NaHCO ₃ , and 20 μL of human fibroblasts (2.5 million cells/mL) were added in this order. The well plate was then cultured at 37° C. for 1 hour to prepare a collagen gel. The gel attached to the wall of the well plate was peeled off and placed in the well, and 500 μL of DMEM containing 1% FBS containing oligopeptide of TAV prepared at each concentration was added on top of the gel. This was further cultured at 37° C., and the area of the gel 3 days after the start of culture was measured by measuring the diameter of the gel with a digital caliper.

As a control sample, cultivation and observation were carried out in the same manner as above, except that the solvent used in preparing the test sample was used instead of the test sample.
Bovine lactoferrin was used as a positive control sample.
Three gels were prepared for each sample and incubated (n=3), and the gel areas were measured and the average and standard deviation were calculated.

０．１ μｇ／ｍＬ、１ μｇ／ｍＬ、１０ μｇ／ｍＬ、２５ μｇ／ｍＬ、５０ μｇ／ｍＬ、及び１００μｇ／ｍＬのＴＡＶのオリゴペプチドをそれぞれ添加することにより、コントロールに比べて、有意なコラーゲンゲルの面積の減少、すなわちコラーゲンゲルの収縮促進効果が認められた。このオリゴペプチドを０．１～１００μｇ／ｍＬの濃度範囲で含む組成物は、肌のシワ、たるみ、ハリのなさの改善効果があることが確認された。 The results are shown in Table 2. The oligopeptide concentrations in Table 2 are the final concentrations in the culture medium in the wells.

The addition of 0.1 μg/mL, 1 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL, and 100 μg/mL of the TAV oligopeptide resulted in a significant reduction in the collagen gel area, i.e., an effect of promoting collagen gel contraction, compared to the control. It was confirmed that a composition containing this oligopeptide in a concentration range of 0.1 to 100 μg/mL has an effect of improving wrinkles, sagging, and lack of firmness in the skin.

Example 3 (Evaluation of collagen gel contraction promoting activity of modified oligopeptides)
The collagen gel contraction promoting activity of N-terminal acetyl-modified, N-terminal succinyl-modified, and N-terminal palmitoyl-modified oligopeptides of CHTAVDR (No. 3) (each manufactured by Biologica Co., Ltd.) was evaluated.
To each well of a 24-well plate, 100 μL of medium (0.94 g of MEM, 500 μL of 1 mol/L NaHCO ₃ , 29.2 mg of L-glutamine, and 1 mL of bovine serum were added to pure water to prepare a 20 mL volume) at a concentration 5 times the manufacturer's specified concentration, 2.5 μL of a test sample prepared to 20 mg/mL, 365 μL of type I collagen (IAC-30), 12.5 μL of 1 mol/L NaHCO ₃ , and 20 μL of human fibroblasts (2.5 million cells/mL) were added in this order. The well plate was then cultured at 37° C. for 1 hour to prepare a collagen gel. The gel attached to the wall of the well plate was peeled off and placed in the well, and 500 μL of 1% FBS-containing DMEM containing a test sample prepared to 20 mg/mL was added to the top of the gel. This was further cultured at 37° C., and the area of the gel 3 days after the start of culture was measured by measuring the diameter of the gel with a digital caliper.

As a control sample, cultivation and observation were carried out in the same manner as above, except that the solvent used in preparing the test sample was used instead of the test sample.
Three gels were prepared for each sample and incubated (n=3), and the gel areas were measured and the average and standard deviation were calculated.

ＣＨＴＡＶＤＲのオリゴペプチドのＮ末端アセチル修飾体、Ｎ末端スクシニル修飾体、及びＮ末端パルミトイル修飾体を、最終濃度１００ μｇ／ｍＬとなるようにそれぞれ添加することにより、コントロールに比べて有意なコラーゲンゲルの面積の減少、すなわちコラーゲンゲルの収縮促進が確認されたため、これらの修飾体に肌のシワ、たるみ、ハリのなさの改善効果があることが確認された。 The results are shown in Table 3.

By adding N-terminal acetyl-modified, N-terminal succinyl-modified, and N-terminal palmitoyl-modified CHTAVDR oligopeptides to a final concentration of 100 μg/mL, a significant reduction in the collagen gel area was observed compared to the control, i.e., promotion of collagen gel contraction, demonstrating that these modifications have the effect of improving wrinkles, sagging, and loss of firmness in the skin.

Example 4 (Comparison of collagen gel contraction promoting activity with other wrinkle, sagging and firmness improving agents)
The collagen gel contraction promoting activity of niacinamide (Sigma-Aldrich Co.) and palmitoyl tripeptide-5 "(Pal)-Lys-Val-Lys-[OH]" (PH Japan Co., Ltd.), both of which have already been confirmed to have anti-wrinkle effects, was compared with that of the oligopeptide AVD (No. 10).
To each well of a 24-well plate, 100 μL of medium (0.94 g of MEM, 500 μL of 1 mol/L NaHCO ₃ , 29.2 mg of L-glutamine, and 1 mL of bovine serum were added to pure water to prepare a 20 mL volume) at a concentration 5 times the manufacturer's specified concentration, 2.5 μL of a test sample prepared to 20 mg/mL, 365 μL of type I collagen (IAC-30), 12.5 μL of 1 mol/L NaHCO ₃ , and 20 μL of human fibroblasts (2.5 million cells/mL) were added in this order. The well plate was then cultured at 37° C. for 1 hour to prepare a collagen gel. The gel attached to the wall of the well plate was peeled off and placed in the well, and 500 μL of 1% FBS-containing DMEM containing a test sample prepared to 20 mg/mL was added to the top of the gel. This was further cultured at 37° C., and the area of the gel 3 days after the start of culture was measured by measuring the diameter of the gel with a digital caliper.

As a control sample, cultivation and observation were carried out in the same manner as above, except that the solvent used in preparing the test sample was used instead of the test sample.
Bovine lactoferrin was used as a positive control sample.
Three gels were prepared for each sample and incubated (n=3), and the gel areas were measured and the average and standard deviation were calculated.

最終濃度１００ μｇ／ｍＬのＡＶＤのオリゴペプチド、最終濃度１００ μｇ／ｍＬのナイアシンアミドの添加により、コントロールに比べて有意なコラーゲンゲルの面積の減少、すなわちコラーゲンゲルの収縮促進が確認されたため、これらには肌のシワ、たるみ、ハリのなさの改善効果があることが確認された。また、ＡＶＤのオリゴペプチドによるコラーゲンゲルの面積の減少変化とナイアシンアミドによるコラーゲンゲルの面積の減少変化との間には有意差があり、ＡＶＤのオリゴペプチドによるコラーゲンゲルの収縮促進効果はナイアシンアミドによるコラーゲンゲルの収縮促進効果よりも有意に大きいことが確認された。一方、最終濃度１００ μｇ／ｍＬのパルミトイルトリペプチド－５にコラーゲンゲルの収縮促進効果は認められなかった。 The results are shown in Table 4.

The addition of AVD oligopeptide at a final concentration of 100 μg/mL and niacinamide at a final concentration of 100 μg/mL confirmed a significant reduction in collagen gel area, i.e., collagen gel contraction promotion, compared to the control, and confirmed that these have the effect of improving wrinkles, sagging, and lack of firmness of the skin.In addition, there is a significant difference between the reduction in collagen gel area caused by AVD oligopeptide and the reduction in collagen gel area caused by niacinamide, and it was confirmed that the collagen gel contraction promotion effect of AVD oligopeptide is significantly greater than that of niacinamide.On the other hand, palmitoyl tripeptide-5 at a final concentration of 100 μg/mL did not show any collagen gel contraction promotion effect.

These results suggest that administering the oligopeptide and its derivatives of the present invention to humans can improve wrinkles, sagging, loose skin, loss of firmness, and loss of elasticity.

The embodiments and examples disclosed above should be considered to be illustrative in all respects and not restrictive.

The oligopeptide and its derivatives of the present invention are highly useful in that they have excellent percutaneous absorption and are low-irritant to the skin, yet can effectively improve wrinkles, sagging, loose skin, loss of firmness, and loss of elasticity.

Claims (4)

A collagen contraction promoter in the skin, containing a tripeptide consisting of the amino acid sequence of AVD. The collagen contraction promoter for skin according to claim 1, which is a topical composition or a food composition. The agent for preventing or improving wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin contains a tripeptide consisting of the amino acid sequence of AVD, and prevents or improves wrinkles, looseness, sagging, loss of firmness, or loss of elasticity of the skin by promoting collagen contraction by fibroblasts. The agent for preventing or improving wrinkles, sagging, looseness, loss of firmness, or loss of elasticity of the skin according to claim 3, which is an external composition or a food composition.