Inhibitor of decreased decomposition of denatured elastin, agent for maintaining normal elastin fibers, inhibitor of formation of elastin-elafin complex, and method for screening substance having inhibitory effect on formation of elastin-elafin complex

Abstract

The present invention aims to provide an elastin-elafin complex formation inhibitor capable of inhibiting the formation of elastin-elafin complexes, a composition for inhibiting the formation of elastin-elafin complexes, a method for screening substances having the effect of inhibiting the formation of elastin-elafin complexes, a method for inhibiting the formation of elastin-elafin complexes, a method for inhibiting the decreased degradation of denatured elastin, and a method for maintaining normal elastin fibers, etc. The present invention relates to an inhibitor of the decreased degradation of denatured elastin, which comprises as an active ingredient one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althaea extract, white willow extract, calamus extract, peach kernel extract, and soybean extract.

Description

[Technical field]

[0001] The present invention relates to an agent for suppressing the decreased decomposition of denatured elastin, an agent for maintaining normal elastin fibers, and an agent for suppressing the formation of an elastin-elafin complex. The present invention also relates to a composition for suppressing the decreased decomposition of denatured elastin, a composition for maintaining normal elastin fibers, and a composition for suppressing the formation of an elastin-elafin complex. The present invention further relates to a method for screening a substance having an effect of suppressing the formation of an elastin-elafin complex. The present invention also relates to a method for suppressing the formation of an elastin-elafin complex, a method for suppressing the decreased decomposition of denatured elastin, and a method for maintaining normal elastin fibers. The present invention also relates to the use of a plant extract for suppressing the formation of an elastin-elafin complex, the use of a plant extract for suppressing the decreased decomposition of denatured elastin, and the use of a plant extract for maintaining normal elastin fibers. [Background technology]

[0002] Elastin is the main component of elastin fibers (elastic fibers), and is widely expressed in tissues such as the skin, blood vessels, and lungs, where elasticity is essential for the expression of their functions. For example, in the dermis of the skin, elastin fibers provide elasticity to the skin and maintain its firmness, but they are denatured and lose their elasticity due to factors such as aging and ultraviolet rays. In particular, when the skin is exposed to ultraviolet rays for many years, denatured elastin accumulates and deposits in the dermis, which is believed to cause solar elastosis, which is poor in elasticity and has many wrinkles.

[0003] One of the causes of the local accumulation of denatured elastin, as seen in solar elastosis, is thought to be a decrease in the degradation of denatured elastin. Colocalization of elastin fibers with elafin has been observed in the dermis exposed to UV light, suggesting that elastin and elafin form a protein complex. Elafin is an elastase inhibitor, and when elastin binds to elafin to form a complex (elastin-elafin complex), it becomes resistant to degradation by elastase.

[0004] It has been reported that some plant extracts have various useful effects. For example, Patent Document 1 describes white mustard extract as a melanin production inhibitor. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2005-154375 A Summary of the Invention [Problem to be solved by the invention]

[0006] When elastin and elafin are combined to form an elastin-elafin complex as described above, the complex is resistant to elastase. Therefore, the formation of an elastin-elafin complex reduces the decomposition of elastin and slows down the turnover of elastin fibers. If the formation of an elastin-elafin complex can be inhibited, for example, the decrease in the decomposition of denatured elastin can be inhibited, and the turnover of elastin fibers can be promoted. In addition, it is expected that the accumulation of denatured elastin can be inhibited or improved, making it possible to maintain normal elastin fibers. Patent Document 1 describes white mustard extract as a melanin production inhibitor, but has not investigated any substance that has an effect of inhibiting the formation of an elastin-elafin complex.

[0007] The present invention aims to provide an elastin-elafin complex formation inhibitor and a composition for inhibiting the formation of elastin-elafin complexes, which are capable of inhibiting the formation of elastin-elafin complexes. Another object of the present invention is to provide a method for screening a substance that has an effect of inhibiting the formation of elastin-elafin complexes. Another object of the present invention is to provide a method for inhibiting the formation of elastin-elafin complexes, a method for inhibiting the decrease in decomposition of denatured elastin, a method for maintaining normal elastin fibers, etc. [Means for solving the problem]

[0008] Means for Solving the Problems The present inventors have conducted intensive research to solve the above problems and have found that extracts from specific plants such as passion fruit have an inhibitory effect on the formation of elastin-elafin complexes, thereby completing the present invention.

[0009] The present invention includes, but is not limited to, the following elastin-elafin complex formation inhibitors, etc. [1] An agent for inhibiting the decreased decomposition of denatured elastin, comprising as an active ingredient one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract and soybean extract. [2] An agent for maintaining normal elastin fibers, comprising as an active ingredient one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract, and soybean extract. [3] The agent according to [1] or [2] above, which inhibits the formation of an elastin-elafin complex. [4] An agent for inhibiting the formation of elastin-elafin complexes, comprising as an active ingredient one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract, and soybean extract. [5] The elastin-elafin complex formation inhibitor according to [4] above, which is used to promote the turnover of elastin fibers by inhibiting the formation of an elastin-elafin complex. [6] The agent according to any one of [1] to [5] above, wherein the plant extract is one or more selected from the group consisting of passion fruit extract, calendula officinalis extract, and white mustard extract. [7] A composition for suppressing the decrease in the decomposition of denatured elastin, comprising an inhibitor of the decrease in the decomposition of denatured elastin described in [1], [3] or [6] above. [8] A composition for maintaining normal elastin fibers, comprising the agent for maintaining normal elastin fibers described in [2], [3] or [6] above. [9] A composition for inhibiting the formation of an elastin-elafin complex, comprising the elastin-elafin complex formation inhibitor described in any one of [4] to [6] above.

[10] The composition according to any one of [7] to [9] above, which is an external preparation for skin.

[11] The composition according to any one of the above [7] to

[10] , which is a cosmetic.

[12] The composition according to any one of the above [7] to [9], which is a food or drink.

[13] A method for screening for a substance having an inhibitory effect on the formation of an elastin-elafin complex, comprising: (a) contacting elafin with immobilized elastin in the presence and absence of a test substance; (b) detecting inhibition of binding between the elafin and immobilized elastin due to the presence of the test substance; and (c) selecting the test substance as a substance having an inhibitory effect on the formation of an elastin-elafin complex when binding between elafin and immobilized elastin is inhibited by the presence of the test substance.

[14] A method for suppressing the formation of elastin-elafin complexes, comprising administering one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract, and soybean extract.

[15] A method for suppressing the decreased decomposition of denatured elastin, comprising administering one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract, and soybean extract.

[16] A method for maintaining normal elastin fibers, comprising administering one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract, and soybean extract.

[17] Use of one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract and soybean extract to inhibit the formation of elastin-elafin complex.

[18] Use of one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract, and soybean extract to inhibit the degradation of denatured elastin.

[19] Use of one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract, and soybean extract for maintaining normal elastin fibers. Effect of the Invention

[0010] According to the present invention, it is possible to provide an elastin-elafin complex formation inhibitor capable of inhibiting the formation of an elastin-elafin complex, a composition for inhibiting the formation of an elastin-elafin complex, etc. Furthermore, according to the present invention, it is possible to provide a method for screening a substance having an effect of inhibiting the formation of an elastin-elafin complex.

[0011] By inhibiting the formation of elastin-elafin complexes, it is possible to inhibit the degradation of denatured elastin. In addition, inhibiting the formation of elastin-elafin complexes can also contribute to maintaining normal elastin fibers. According to the present invention, it is possible to provide a method for inhibiting the formation of elastin-elafin complexes, a method for inhibiting the degradation of denatured elastin, and a method for maintaining normal elastin fibers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described below. However, the present invention is not limited to the following embodiments, and can be appropriately modified and applied within the scope of the present invention.

[0013] The elastin-elafin complex formation inhibitor of the present invention contains as an active ingredient one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract and soybean extract.

[0014] As shown in the examples below, the above-mentioned plant extract has the effect of inhibiting the binding of elastin and elafin to form a complex (elastin-elafin complex).The above-mentioned plant extract is used for inhibiting the formation of an elastin-elafin complex.The inhibition of the formation of an elastin-elafin complex can also be referred to as the inhibition of the binding of elastin and elafin. As described above, the formation of elastin-elafin complex reduces the degradation of elastin. By inhibiting the formation of elastin-elafin complex, the reduction in the degradation of denatured elastin can be inhibited. By inhibiting the reduction in the degradation of denatured elastin, it is expected that the turnover of elastin fibers will be promoted, and the accumulation of denatured elastin in tissues such as the skin will be inhibited or improved. It is also expected that normal elastin fibers will be able to be maintained.

[0015] In the present invention, normal elastin fibers refer to elastin fibers that have the inherent functions and physical properties (strength, viscoelasticity, etc.) of elastin fibers, while denatured elastin refers to elastin fibers and their constituent factors (tropoelastin, elastin degradation products, etc.) that have lost their inherent functions and physical properties. In the present invention, when simply referring to elastin, and in the elastin-elafin complex, it includes tropoelastin which is the source of elastin fibers, elastin contained in elastin fibers, and tropoelastin and elastin fibers which have undergone partial structural changes, decomposition or fragmentation due to aging or stimulation. "Maintaining normal elastin fibers" means suppressing the decrease in the amount of normal elastin fibers and their original functions and physical properties. "Suppressing the decrease in the amount of normal elastin fibers and their original functions and physical properties" means suppressing the decrease in the amount of normal elastin fibers and their original functions and physical properties compared to when the plant extract used as an active ingredient in the present invention is not used.

[0016] In one aspect, the elastin-elafin complex formation inhibitor of the present invention is preferably used to promote the turnover of elastin fibers by inhibiting the formation of elastin-elafin complexes.In addition, since the above-mentioned plant extract inhibits the formation of elastin-elafin complexes, it can be used to inhibit the degradation of denatured elastin and maintain normal elastin fibers. The present invention also encompasses an agent for inhibiting the degradation of denatured elastin and for maintaining normal elastin fibers, which comprises as an active ingredient one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract and soybean extract.

[0017] The elastin-elafin complex formation inhibitor, the inhibitor of reduced decomposition of denatured elastin, and the agent for maintaining normal elastin fibers of the present invention are hereinafter collectively referred to as complex formation inhibitors, etc.

[0018] In the complex formation inhibitor of the present invention, two or more of the above plant extracts may be used in combination as active ingredients. Among them, one or more plant extracts selected from the group consisting of passion fruit extract, calendula officinalis extract, and white mustard extract are preferred because they have a high inhibitory effect on the formation of elastin-elafin complexes.

[0019] In the present invention, passion fruit refers to the fruit of Passiflora edulis (scientific name) of Passifloraceae Passiflora family. Calendula officinalis (scientific name) of Asteraceae Calendula family. Calendula officinalis is also known as Calendula. Sinapis alba (scientific name) of Brassicaceae Sinapis family. Althaea refers to Althaea officinalis (scientific name) of Malvaceae Althaea family. Althaea is also known as Marshmallow. White willow refers to Salix alba (scientific name) of the Salicaceae Salix genus. Calamus refers to Acorus calamus (scientific name) of the Acoraceae Martinov Acorus genus. Peach kernel refers to the seeds (peach kernels) of Prunus persica (or Prunus persica var. davidiana) (scientific name) of the Rosaceae Amygdalus genus. Soybean refers to Glycine max (scientific name) of the Fabaceae Glycine genus.

[0020] Passion fruit extract is an extract of the fruit of Passiflora edulis. Peach kernel extract is an extract of peach seeds. Extracts of Calendula officinalis, White Mustard, Althaea, White Willow, Calamus and Soybean can be produced by extracting any part of these plants (e.g., roots, rhizomes, stems, leaves, bark, flowers, fruits, seeds) with a solvent. A combination of multiple parts may be used for extraction. The calendula officinalis extract is preferably an extract of calendula officinalis flowers. The white mustard extract is preferably an extract of white mustard seeds. The Althaea extract is preferably an extract of Althaea roots. The white willow extract is preferably an extract of white willow bark. The calamus extract is preferably an extract of calamus rhizomes. The soybean extract is preferably an extract of soybean sprouts.

[0021] The method of extracting a plant to obtain a plant extract is not particularly limited, and can be obtained by a normal extraction method used for extracting plant components. The extraction method can be set appropriately, and the extraction conditions are not particularly limited. For example, it is preferable to obtain a plant extract by extracting the above-mentioned plant with a solvent (extraction solvent) at room temperature or under heating. In preparing a plant extract, the above-mentioned plant as a raw material may be subjected to the extraction process as it is, or may be subjected to the extraction process after being crushed, cut or dried. In the present invention, the plant extract may be an extract obtained by extracting the above-mentioned plant as it is, or may be an extract obtained by extracting the plant after crushing, cutting or drying. Preferably, it is an extract obtained by crushing, cutting or drying the above-mentioned plant.

[0022] The extraction solvent used in the preparation of the plant extract can be appropriately selected, and those usually used for the extraction of plant components can be used. Examples of the extraction solvent include water; monohydric alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propanol, and butanol; polyhydric alcohols having 2 to 5 carbon atoms, such as ethylene glycol, propylene glycol, 1,2-butylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, and 2,3-butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; and squalane. These may be used alone, or a mixed solvent (mixture) of two or more types may be used. As the monohydric alcohols having 1 to 5 carbon atoms, those having 1 to 4 carbon atoms are preferable, those having 2 to 4 carbon atoms are more preferable, and ethanol is even more preferable. As the polyhydric alcohol having 2 to 5 carbon atoms, a dihydric or trihydric alcohol having 2 to 4 carbon atoms is preferred, a dihydric alcohol is more preferred, and 1,3-butylene glycol is even more preferred. Among them, as the extraction solvent, water, a monohydric alcohol having 1 to 5 carbon atoms, a polyhydric alcohol having 2 to 5 carbon atoms, or a mixed solvent of two or more of these is preferred, water, a monohydric alcohol having 2 to 4 carbon atoms or an aqueous solution thereof, a dihydric alcohol having 2 to 4 carbon atoms or an aqueous solution thereof is more preferred, water, ethanol, an aqueous ethanol solution, 1,3-butylene glycol, or an aqueous 1,3-butylene glycol solution is even more preferred, and water, an aqueous ethanol solution, or an aqueous 1,3-butylene glycol solution is particularly preferred. In one aspect, the aqueous ethanol solution and the aqueous 1,3-butylene glycol solution have an ethanol or 1,3-butylene glycol concentration of 10 to 98% by volume, more preferably 30 to 90% by volume, and even more preferably 30 to 70% by volume. The above-mentioned solvent extract can be suitably used as the plant extract in the present invention.

[0023] In one embodiment, the passion fruit extract, calendula extract, and Althaea extract are preferably extracts obtained by extracting the above plants with an aqueous 1,3-butylene glycol solution.The white willow extract, calamus extract, and peach kernel extract are preferably extracts obtained by extracting the above plants with an aqueous ethanol solution.The white mustard extract and soybean extract are preferably extracts obtained by extracting the above plants with water (preferably hot water).

[0024] An acid or alkali may be added during extraction to adjust the pH of the extraction solvent. After extraction, it is preferable to remove the plant residue (the plant body or a part thereof after extraction) from the extract. The method for removing the plant residue from the extract is not particularly limited, and includes known separation means such as filtration and centrifugation.

[0025] As an example of a plant extraction method, for example, the following method can be used. The plant is crushed as is or dried, and 0.1 to 30 times by weight of an extraction solvent is added, and extraction is performed at room temperature under normal pressure for preferably 10 minutes to 15 days, more preferably 30 minutes to 10 days, and even more preferably 1 hour to 7 days, or extraction is performed near the boiling point of the extraction solvent for preferably 10 minutes to 1 day (more preferably 10 minutes to 2 hours), and then filtration is performed to obtain a filtrate. During extraction, the mixture may be left to stand or may be appropriately stirred. The obtained filtrate (plant extract) may be used as a plant extract as is, or may be diluted, concentrated, dried, or the like as necessary.

[0026] In the present invention, the plant extract obtained by extraction can be used as it is as a plant extract. In addition, as long as the effect of the present invention is not impaired, it may be diluted, concentrated or dried by a known method to form a diluted solution, concentrate or powder, or prepared in a paste form for use. Examples of drying methods include freeze-drying and spray-drying. Furthermore, the above-mentioned plant extract, its concentrate or dried powder, etc. may be further purified, such as by deodorization and decolorization, as necessary, within a range that does not impair the effect of the present invention. Such a purification method may be performed by any ordinary means selected at will. The plant extract of the present invention includes various solvent extracts obtained by the above-mentioned extraction methods, their dilutions, their concentrates, their dried powders, and their purified products. The extract can also be used after diluting or dissolving in a solvent different from the extraction solvent.

[0027] In addition, the above-mentioned plant extracts are commercially available, and commercially available products can also be used.

[0028] The complex formation inhibitor of the present invention may consist of the above-mentioned plant extract, and may further contain other ingredients, additives, etc. and be used in the form of a composition. The elastin-elafin complex formation inhibitor, inhibitor of reduced decomposition of denatured elastin, and agent for maintaining normal elastin fibers of the present invention can be used as an elastin-elafin complex formation inhibitor, inhibitor of reduced decomposition of denatured elastin, and agent for maintaining normal elastin fibers by themselves. They can also be used by being incorporated into a composition for inhibiting elastin-elafin complex formation, etc., described below. The complex formation inhibitor, etc. of the present invention are suitably used as an active ingredient of a composition for inhibiting elastin-elafin complex formation, etc.

[0029] The present invention also includes a composition for inhibiting the formation of an elastin-elafin complex, comprising the inhibitor of elastin-elafin complex formation of the present invention.The present invention also includes a composition for inhibiting the decrease in the degradation of denatured elastin, comprising the inhibitor of the decrease in the degradation of denatured elastin of the present invention.The present invention also includes a composition for maintaining normal elastin fibers, comprising the agent for maintaining normal elastin fibers of the present invention. The composition for inhibiting the formation of elastin-elafin complexes, the composition for inhibiting the degradation of denatured elastin, and the composition for maintaining normal elastin fibers of the present invention are collectively referred to as compositions for inhibiting complex formation, etc. The compositions for inhibiting complex formation, etc. of the present invention contain the above-mentioned plant extract as an active ingredient. The plant extract and preferred embodiments thereof are as described above. The complex formation inhibitors and compositions for inhibiting complex formation of the present invention can be suitably used, for example, to inhibit the formation of elastin-elafin complexes in the skin, to inhibit the reduced decomposition of denatured elastin in the skin, or to maintain normal elastin fibers in the skin.

[0030] The complex formation inhibitor and the composition for inhibiting complex formation of the present invention can be used for either therapeutic applications (medical applications) or non-therapeutic applications (non-medical applications). The complex formation inhibitor etc. and the composition for inhibiting complex formation etc. of the present invention, which contain the above-mentioned plant extract as an active ingredient, can be used, for example, for preventing or improving a condition or symptom in which inhibition of elastin-elafin complex formation is desired. In one aspect, the complex formation inhibitor etc. and the composition for inhibiting complex formation etc. of the present invention can be used, for example, for preventing or improving photoelastosis etc. by inhibiting elastin-elafin complex formation. In addition, the complex formation inhibitor etc. and the composition for inhibiting complex formation etc. of the present invention are also useful for cosmetic purposes such as maintaining skin elasticity and preventing or improving wrinkles and / or sagging by inhibiting elastin-elafin complex formation. Prevention includes prevention of onset, delay of onset, reduction of onset rate, etc. Improvement includes alleviation of symptoms, improvement of symptoms, inhibition of progression of symptoms, cure of symptoms, etc.

[0031] The composition for inhibiting complex formation of the present invention can be used for various applications such as cosmetics, food and beverages, medicines, quasi-drugs, etc. The composition for inhibiting complex formation of the present invention may itself be a cosmetic, food and beverage, medicine, or quasi-drug for inhibiting the formation of elastin-elafin complexes, inhibiting the degradation of denatured elastin, or maintaining normal elastin fibers, or it may be a material or preparation to be blended with the cosmetic, food and beverage, medicine, quasi-drug, etc.

[0032] The complex formation-inhibiting composition of the present invention is preferably used, for example, as an external preparation for the skin. External preparations for the skin include cosmetics, pharmaceuticals, and quasi-drugs, and are preferably cosmetics. In one embodiment, when used to inhibit the formation of elastin-elafin complexes in the skin, to inhibit the degradation and decrease of denatured elastin in the skin, or to maintain normal elastin fibers in the skin, the complex formation-inhibiting composition is preferably used as an external preparation for the skin, and more preferably as a cosmetic. The complex formation-inhibiting composition of the present invention can also be used as a medicine or quasi-drug other than an external preparation for the skin. In another embodiment, the complex formation-inhibiting composition etc. may be prepared as a food or drink. The case where the complex formation-inhibiting composition etc. of the present invention is prepared as a cosmetic, a medicine, an external preparation for skin such as a quasi-drug, a food or drink, etc. will be described below.

[0033] When the complex formation inhibiting composition of the present invention is used as a skin external preparation, the dosage form is not particularly limited, and may be any form such as a solution, emulsion, cream, gel, powder, aerosol, mist, capsule, and sheet. The skin external preparation is preferably a cosmetic. The product form of the cosmetic is also not particularly limited, and examples thereof include skin care cosmetics such as face wash, makeup remover, lotion, serum, pack, emulsion, cream, and sunscreen; makeup cosmetics such as foundation, makeup base, lipstick, eye shadow, eyeliner, mascara, eyebrow, blusher, and nail enamel; hair cosmetics such as hair shampoo, hair rinse, hair styling agent, hair dye, and hair growth agent; cleansing agents such as soap and body soap; and bath additives.

[0034] The above-mentioned cosmetic preparation may appropriately contain one or more of the following components, such as carriers and additives acceptable for cosmetic preparations, within the scope of the present invention, for example, water, alcohols, oils, surfactants, thickeners, metal soaps, gelling agents, powders, chelating agents, water-soluble polymers, film-forming agents, resins, inclusion compounds, antibacterial agents, deodorants, salts, pH adjusters, UV absorbers, extracts derived from animals, plants, and microorganisms other than the above, keratolytic agents, enzymes, hormones, other vitamins, moisturizers, bactericides, anti-inflammatory agents, and fragrances. The cosmetic preparation may be produced by a general production method. For example, the cosmetic preparation may be obtained by mixing the above-mentioned plant extract with one or more of the above-mentioned components that can be used in the cosmetic preparation, and then processing the mixture into a desired form.

[0035] When the skin topical preparation is a drug or quasi-drug, components such as carriers and additives acceptable for drugs or quasi-drugs may be used within the scope that does not impair the effects of the present invention. Examples of such components include one or more of excipients, binders, disintegrants, lubricants, antioxidants, colorants, etc., which can be used as needed.

[0036] The composition for inhibiting complex formation of the present invention can be a medicine or quasi-drug other than the above-mentioned skin topical preparation. Such medicine or quasi-drug may be administered orally or parenterally. The medicine or quasi-drug can be in the form of an oral preparation (internal preparation) or a parenteral preparation. Examples of the dosage form of an oral preparation include liquids, tablets, powders, fine granules, granules, sugar-coated tablets, capsules, suspensions, emulsions, chewables, etc. Examples of the dosage form of a parenteral preparation include injections and infusions. Such medicines or quasi-drugs may contain components such as carriers and additives that are acceptable for medicines or quasi-drugs. Examples of such components include one or more of excipients, binders, disintegrants, lubricants, antioxidants, colorants, flavorings, etc., which can be used as needed. Medicines and quasi-drugs can be manufactured by a general manufacturing method. For example, the above-mentioned plant extract can be mixed with one or more ingredients that can be used in pharmaceuticals or quasi-drugs, and then processed into a desired form to obtain the composition.

[0037] When the composition for inhibiting complex formation of the present invention is used as a food or drink, the food or drink is not particularly limited, and examples thereof include general food or drink, health food, functional food, and food for specified health uses. The form of the food or drink is also not particularly limited. The above-mentioned health food, functional food, and food for specified health uses can be in various formulation forms, such as liquids, tablets, powders, fine granules, granules, sugar-coated tablets, capsules, suspensions, emulsions, chewable agents, and liquid diets.

[0038] The above-mentioned food and drink may contain components acceptable for food and drink, such as other food and drink ingredients, additives to be added to food and drink, etc., within the scope of not impairing the effects of the present invention. Such food and drink can be produced by a general production method. For example, the above-mentioned plant extract may be added to food and drink ingredients, etc., in the production of the food and drink.

[0039] When the composition for inhibiting complex formation of the present invention is in the form of a skin topical preparation, a drug or quasi-drug other than a skin topical preparation, or a food or drink, the content of the plant extract in the composition is, for example, preferably 0.0000001 to 100% by weight, more preferably 0.000001 to 100% by weight, and even more preferably 0.00001 to 10% by weight, calculated as the weight of the dry matter of the extract. When two or more kinds of plant extracts are contained, the above content is the total content thereof.

[0040] The amount of the composition for inhibiting complex formation of the present invention is not particularly limited, and may be an amount that can obtain the effect of inhibiting the formation of elastin-elafin complex, and may be appropriately set according to the condition, body weight, sex, age, or other factors of the subject. In the case of a skin external preparation such as a cosmetic, the amount is preferably, for example, 0.01 to 500 mg of the above plant extract (converted into dry matter) per adult (60 kg) per day. In the case of oral administration of a pharmaceutical or quasi-drug, the amount is preferably, for example, 0.01 to 500 mg of the above plant extract (converted into dry matter) per adult (60 kg) per day. In the case of a food or drink, the intake amount is preferably, for example, 0.01 to 500 mg of the above plant extract (converted into dry matter) per adult (60 kg) per day. The above amount of plant extract may be ingested or administered in a single dose or in multiple doses. There are no particular limitations on the timing of applying (administering) the skin topical agent containing the above-mentioned plant extract to the skin, or the timing of ingesting or administering the food, drink, medicine or quasi-drug.

[0041] The subject of application (which may also be referred to as the subject of administration) of the composition for inhibiting complex formation of the present invention is not particularly limited, and may be applied to humans or non-human mammals, etc., with humans being preferred. Examples of subjects of application include subjects who desire or require inhibition of elastin-elafin complex formation, subjects who desire or require inhibition of decreased degradation of denatured elastin, subjects who desire or require maintenance of normal elastin, etc. Examples of such subjects include men and women who suffer from ultraviolet exposure (particularly excessive exposure to ultraviolet light) and unwanted skin changes associated with aging, particularly wrinkles and / or sagging.

[0042] The present invention also encompasses the following uses and methods. Use of one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract and soybean extract for inhibiting the formation of elastin-elafin complexes, inhibiting the degradation of denatured elastin, or maintaining normal elastin fibers. A method for inhibiting the formation of elastin-elafin complexes by administering one or more plant extracts selected from the group consisting of passion fruit extract, calendula extract, white mustard extract, althea extract, white willow extract, calamus extract, peach kernel extract and soybean extract; a method for inhibiting the decreased decomposition of denatured elastin by administering the above-mentioned plant extract; a method for maintaining normal elastin fibers by administering the above-mentioned plant. The above-mentioned plant extract is preferably applied to the skin and is suitably used for inhibiting the formation of elastin-elafin complexes in the skin, inhibiting the degradation of denatured elastin in the skin, or for maintaining normal elastin fibers in the skin.

[0043] In the above uses and methods, the plant extract, the subject to which it is administered, and their preferred aspects are the same as those in the above-mentioned complex formation inhibitor and composition for inhibiting complex formation of the present invention. The plant extract may be used as is or in combination with other ingredients. The complex formation inhibitor or composition for inhibiting complex formation of the present invention may be used. The plant extract may be used in the form of the above-mentioned skin topical agent, food, drink, etc. The use may be a therapeutic use or a non-therapeutic use. The method may be a therapeutic method or a non-therapeutic method, the latter being a concept that does not include medical procedures, i.e., surgery, treatment or diagnosis of humans.

[0044] Other aspects of the present invention include the use of the above-mentioned plant extract for producing an elastin-elafin complex formation inhibitor or a composition for inhibiting the formation of elastin-elafin complexes; the use of the above-mentioned plant extract for producing an inhibitor of the reduction in the degradation of denatured elastin or a composition for inhibiting the reduction in the degradation of denatured elastin; and the use of the above-mentioned plant extract for producing an agent for maintaining normal elastin fibers or a composition for maintaining normal elastin fibers. The plant extract and preferred aspects thereof are the same as those in the above-mentioned complex formation inhibitor and composition for inhibiting complex formation of the present invention.

[0045] The present invention also encompasses a method for screening for a substance that has an inhibitory effect on the formation of an elastin-elafin complex. Next, the screening method of the present invention will be described. The screening method of the present invention for a substance having an inhibitory effect on the formation of an elastin-elafin complex comprises the steps of: (a) contacting elafin with immobilized elastin in the presence and absence of a test substance; (b) detecting inhibition of the binding between the elafin and immobilized elastin due to the presence of the test substance; and (c) selecting the test substance as a substance having an inhibitory effect on the formation of an elastin-elafin complex when the binding between elafin and immobilized elastin is inhibited due to the presence of the test substance. The screening method of the present invention is an in vitro screening method.

[0046] In step (a), elafin is contacted with immobilized elastin in the presence and absence of a test substance. Elastin is used after being immobilized on a solid support in advance, which makes washing and other operations easier. The elastin to be immobilized is preferably tropoelastin, α-elastin, or the like. In particular, it is preferable to use the above-mentioned human elastin. Human elastin is commercially available, and commercially available products can be used. Elastin to which a modification such as a tag peptide has been added may also be used. The solid phase carrier is not particularly limited, and may be in the form of, for example, a microplate, a microchip, a slide glass, etc. The material of the solid phase carrier is not particularly limited, and may be made of, for example, plastic, glass, ceramics, metal oxide, etc. For example, when a 96-well microplate or the like is used as the solid-phase carrier, elastin can be solidified by dispensing an elastin solution prepared by dissolving or suspending elastin in a buffer solution into each well of the microplate and leaving the solution at 4 to 37° C. for 0.5 to 30 hours. The buffer solution for preparing the elastin solution is not particularly limited, and buffer solutions such as carbonate buffer and phosphate buffered saline (PBS) can be used. The concentration of elastin in the elastin solution is preferably 1 to 100 μg / mL. The immobilized elastin and solid support are preferably washed with a buffer such as PBS to remove unbound elastin before carrying out step (a). After removing unbound elastin, it is preferable to add a blocking solution (PBS containing 1 to 10% skim milk, albumin, etc.) to perform blocking.

[0047] It is preferable to use human elafin as the elafin. Human elafin and the like are commercially available, and commercially available products can be used. The elafin may be any that binds to immobilized elastin, and may be modified with a tag peptide or the like. Elafin is not limited to a full-length protein, and a protein that binds to elastin and has an amino acid sequence in which one or more amino acids (e.g., 1 to 9, 1 to 5, 1 to 3, 1 to 2, or 1) have been deleted, substituted, inserted, and / or added in the amino acid sequence of the full-length elafin protein may be used. The test substance is not particularly limited, and examples thereof include plant extracts, cell extracts, cell culture supernatants, fermentation products, proteins, peptides, vitamins, synthetic compounds, etc. These may be known substances or novel substances.

[0048] The method of contacting elafin with immobilized elastin in the presence of a test substance is not particularly limited, and can be performed, for example, by incubating elafin and immobilized elastin in a solution containing the test substance. When step (a) is performed in the absence of a test substance, elafin and immobilized elastin may be incubated in a solution not containing the test substance. A buffer solution such as PBS can be used for the solution. The concentration of elafin in the solution is not particularly limited, but is preferably 0.1 to 10 μg / mL. The concentration of the test substance may be appropriately set, for example, 1 to 100 μg / mL. The pH (25° C.) of the solution is preferably 3 to 12. Serum or the like may be added to the solution used in step (a). The temperature when contacting elafin with immobilized elastin is preferably 4 to 40° C. The time for contacting elafin with immobilized elastin is usually 15 minutes to 24 hours, and preferably 30 minutes to 2 hours. After step (a) and before step (b), it is preferable to wash with a buffer solution such as PBS to remove unreacted test substance and elafin.

[0049] In step (b), inhibition of binding between the elafin and immobilized elastin due to the presence of the test substance is detected. The inhibition of binding can be detected by quantifying the amount of elafin bound to immobilized elastin in the presence and absence of the test substance in step (a), and comparing the amount of elafin bound to immobilized elastin in the presence of the test substance with the amount of elafin bound to immobilized elastin in the absence of the test substance.

[0050] The method for quantifying elafin bound to immobilized elastin is not particularly limited, and can be performed by enzyme linked immunosorbent assay (ELISA) or the like. For example, the immobilized elastin that has been subjected to step (a) is reacted with anti-elafin antibody, washed to remove unreacted antibody, and then reacted with a secondary antibody that reacts with the anti-elafin antibody, washed to remove unreacted secondary antibody. For the secondary antibody, for example, an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), or an antibody that reacts with anti-elafin antibody labeled with a fluorescent dye can be used. A substrate corresponding to the enzyme such as HRP or AP bound to the secondary antibody is reacted to produce color or light emission, or fluorescence is produced by excitation, and the color or light emission or fluorescence is quantified using a plate reader or the like, thereby quantifying the amount of elafin bound to immobilized elastin. In another embodiment, the immobilized elastin that has been subjected to step (a) is reacted with an enzyme such as HRP or AP, or an anti-elafin antibody labeled with a fluorescent dye, and after washing off the unreacted antibody, a substrate corresponding to the enzyme such as HRP or AP that was bound to the antibody is reacted to produce color or luminescence, or fluorescence is produced by excitation, and the color or luminescence or fluorescence is quantified using a plate reader or the like, thereby quantifying the amount of elafin bound to the immobilized elastin. As the fluorescent dye, Alexa Fluor (registered trademark) 488, Alexa Fluor (registered trademark) 596 (Thermo Fisher Scientific Inc.), etc. can be used. The amount of elafin bound to immobilized elastin in the presence and absence of the test substance is compared, and if the amount of elafin bound to immobilized elastin is less in the presence of the test substance compared to its absence, the test substance is evaluated as having inhibited the binding of elafin to immobilized elastin.

[0051] In the above step (b), when it is detected that the amount of elafin bound to immobilized elastin is smaller in the presence of the test substance than in the absence of the test substance, it is determined that the binding between elafin and immobilized elastin is inhibited by the presence of the test substance.In step (c), when the binding between elafin and immobilized elastin is inhibited by the presence of the test substance, the test substance is selected as a substance having an effect of inhibiting the formation of elastin-elafin complex. According to the screening method of the present invention, a substance having an inhibitory effect on the formation of an elastin-elafin complex can be screened simply and efficiently. EXAMPLES

[0052] The present invention will be described below based on examples, but the present invention is not limited to these examples.

[0053] <Example 1> The following plant extracts were used as samples: (1) Passion fruit extract: Extract of passion fruit (fruit of Passiflora edulis) (2) Calendula officinalis extract: Calendula officinalis flower extract (3) White mustard extract: Extract of white mustard seeds (4) Althea extract: extract of Althea root (5) White willow extract: Extract of the bark of white willow (6) Calamus extract: Extract of Calamus rhizome (7) Peach seed extract: Peach seed extract (8) Soybean extract: extract of soybean sprouts

[0054] The above plant extracts were obtained by removing plant residues from the extract obtained by extracting the above plants with a solvent through filtration, concentrating the filtrate with an evaporator, and drying it. In the above (1), (2), and (4), the crushed plant material was immersed in 1,3-butylene glycol aqueous solution in an amount ten times the weight thereof, and extracted for 10 minutes to 7 days at room temperature with stirring once a day. In the above (5), (6), and (7), a 30 to 90 vol% ethanol aqueous solution was used instead of the 1,3-butylene glycol aqueous solution, and extraction was performed by the above method. In the above (3) and (8), hot water was used instead of the 1,3-butylene glycol aqueous solution, and extraction was performed by the above method. The obtained powdered plant extracts were used for the following evaluations.

[0055] The inhibitory effect of the samples on the formation of elastin-elafin complexes was evaluated by a solid phase binding assay. The solid phase binding assay was performed as follows. Phosphate-buffered saline (PBS) containing 0.5% serum albumin was used as the dilution buffer. The plant extract (powder) was dissolved in dimethyl sulfoxide (DMSO) before use. Tropoelastin and serum albumin were diluted to 20 μg / mL with bicarbonate buffer (14 mM sodium bicarbonate, 6 mM sodium carbonate), and 100 μL of each was added to a 96-well plate. After sealing the lid with Parafilm, the reaction was allowed to proceed at 4°C for 24 hours. The reaction solution was removed, and 150 μL of wash buffer (0.5% Tween20 in PBS) was added to each well, and the wash buffer was then removed. This was repeated three times. This step is hereinafter referred to as "washing". Next, 100 μL of blocking buffer (5% skim milk in PBS) was added to each well, and the reaction was allowed to proceed at room temperature for 1 hour. After washing the blocking buffer, a complex formation reaction was carried out. In the complex formation reaction of the sample, 50 μL of complex reaction solution (elafin (final concentration 1 μg / mL), the above plant extract (final concentration 10 μg / mL), and DMSO (final concentration 0.1%) in dilution buffer) was added to each well, and the reaction was allowed to proceed at 37°C for 1 hour.

[0056] After washing the complex reaction solution, an elafin-recognizing primary antibody reaction solution was added and reacted at 37°C for 1 hour. After washing the primary antibody reaction solution, a horseradish peroxidase-labeled specific secondary antibody reaction solution was added and reacted at room temperature for 1 hour. After washing the secondary antibody reaction solution, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) solution was added per well and reacted at room temperature for 30 minutes. After adding 100 μL of 1 M phosphoric acid per well to stop the reaction, the absorbance was measured using a microplate reader (wavelength 450 nm).

[0057] As a negative control, in the complex formation reaction, instead of the complex reaction solution containing elafin and plant extract, 50 μL of dilution buffer containing elafin (final concentration 1 μg / mL) and DMSO (final concentration 0.1%) was added to each well and the reaction was performed. As a background, in the complex formation reaction, instead of the complex reaction solution containing elafin and plant extract, 50 μL of dilution buffer (not containing elafin and plant extract) containing DMSO (final concentration 0.1%) was added to each well and the reaction was performed. Other than the above, the reaction with the antibody reaction solution was performed in the same manner as the sample to which the plant extract was added, and the absorbance was measured using a microplate reader (wavelength 450 nm).

[0058] All reagents used in the above assays were commercially available. For tropoelastin, we used unmodified full-length human tropoelastin (Sigma-Aldrich, #T0706), and for elafin, we used full-length human elafin with a polyhistidine tag (His tag) added to the C-terminus (Sino Biological Inc., #12187-H08H).

[0059] The inhibition rate of elastin-elafin complex formation (%) was calculated from the absorbance at 450 nm of the plant extract (sample), negative control, and background using the following formula: The above test was performed with n=4 to 6 for each of the plant extract, negative control, and background, and the inhibition rate of complex formation was calculated from the average absorbance at 450 nm. Complex formation inhibition rate (%)=100-100×(Ab(S)-Ab(NC)) / (Ab(PC)-Ab(NC))

[0060] In the above formula, Ab(S) is the absorbance at 450 nm of the plant extract (sample), Ab(NC) is the absorbance at 450 nm of the background, and Ab(PC) is the absorbance at 450 nm of the negative control. The significance test was performed by Dunnett's test (vs. negative control) (significance level: p<0.05).

[0061] The inhibition rates of elastin-elafin complex formation by the plant extracts are shown in Table 1. Significant differences (p<0.05) were observed in the inhibition rates of complex formation by the passion fruit extract, calendula extract, and white mustard extract compared to the negative control.

[0062] [Table 1]

[0063] In the method described in Example 1, any test substance can be used instead of the above-mentioned plant extract to screen for a substance having an effect of inhibiting the formation of an elastin-elafin complex. A test substance selected as a substance having an effect of inhibiting the formation of an elastin-elafin complex can be used as an active ingredient for inhibiting the degradation of denatured elastin, maintaining normal elastin fibers, or inhibiting the formation of an elastin-elafin complex.

Claims

1. An agent for preventing or improving solar elastosis, comprising passion fruit extract as an active ingredient.

2. The agent described in claim 1, which inhibits the decrease in decomposition of denatured elastin.

3. The agent according to claim 1 or 2, which inhibits the formation of an elastin-elafin complex.

4. A composition for preventing or ameliorating solar elastosis, comprising the agent for preventing or ameliorating solar elastosis according to any one of claims 1 to 3.

5. The composition according to claim 4, which is an external skin preparation.

6. The composition according to claim 4 or 5, which is a cosmetic preparation.

7. The composition according to claim 4, which is a food or drink.

Images