Oral composition for inhibiting elastase and its use, elastase inhibitor, and method for inhibiting elastase activity by orally taking elastase inhibitor

Abstract

Provided are an oral composition for inhibiting elastase and its use, an elastase inhibitor, and a method for inhibiting elastase activity by orally ingesting the elastase inhibitor, wherein the oral composition is used to restore the reduction of elastin in biological tissues caused by external factors such as ultraviolet rays and active oxygen, reduced metabolic function of the organism, etc., improve aging symptoms of the skin, etc. A composition for inhibiting elastase, characterized in that it contains a processed product of seeds of Abelmoschuse sculentus or a specific compound derived from seeds of Abelmoschuse sculentus as an active ingredient.

Description

Technical Field

[0001] The present invention relates to an oral composition for inhibiting elastase and use thereof, an elastase inhibitor, and a method for inhibiting elastase activity by orally ingesting the elastase inhibitor. The oral composition for inhibiting elastase is characterized by containing a processed product of Abelmoschus esculentus seeds.

[0002] In particular, the present invention relates to an oral composition for inhibiting elastase containing the cyclic peptide "OF4949-II" as an active ingredient and its use, an elastase inhibitor, and a method for inhibiting elastase activity by orally ingesting the elastase inhibitor. Background Art

[0003] The biological tissues of animals and fish are generally divided into epithelial tissues such as the epidermis of the skin and the surface of the organs, muscle tissue that mainly constitutes muscles, neural tissue that is distributed throughout the body, and connective tissue that maintains and supports the body structure. Connective tissue is also called supporting tissue, and in a broad sense, bones, cartilage, blood, fat, etc. are sometimes also classified as connective tissue.

[0004] Among them, research and development efforts have been made on epithelial tissues and supporting tissues in the areas of skin, bones, and fat since ancient times. In recent years, for example, as part of the aging symptoms of the skin, the mechanism of the decrease in skin tension and elasticity (wrinkles, sagging, etc.) has been gradually clarified, and it is said that elastin (elastic fiber) is closely related to wrinkles and sagging of the skin.

[0005] Elastin is a type of fibrous high molecular weight protein present in connective tissue. It binds collagen, a high molecular weight protein, to each other in a lattice shape, and forms an extracellular matrix structure in the gaps between them. The extracellular matrix structure contains various extracellular components represented by mucopolysaccharides such as hyaluronic acid and chondroitin sulfate. The extracellular matrix structure plays the role of supporting cells and skin tissues, retaining water in the intercellular spaces, maintaining the lubricity and softness of the skin, and protecting the skin tissues from external factors such as ultraviolet rays, dry environments, mechanical stimulation, damage, and microbial infections. It is known that these proteins and extracellular components are produced by fibroblasts in vivo (Non-Patent Document 1).

[0006] It is generally believed that the content of elastin in biological tissues is about 78-80% in the nuchal ligament, about 50% in the artery, about 20% in the lung, and about 2-5% in the dermis. In addition, one of the characteristics of elastin is that, unlike collagen and hyaluronic acid, the amount of elastin is basically 0% when a person is born, increases as they grow, and reaches a peak at around 25 years old. After that, it turns to decrease and decreases to less than 50% after the age of 40. This indicates that with the increase of age, metabolic changes, ultraviolet rays, and other factors may cause the decomposition or denaturation of elastin in biological tissues. It is speculated that one of the reasons is the hyperactivity and excessive induction of elastase, an enzyme that decomposes elastin.

[0007] From this point of view, it is disclosed that an aqueous component obtained from the seeds of C. niger is used as an effective component for promoting the proliferation of fibroblasts (Patent Document 1). In addition, in order to prevent the reduction of the content of elastin, collagen, etc. in the dermis, various studies have been conducted, and there are various reports and proposals. As an example, retinol glycoside is disclosed as an effective component of cosmetics for improving skin aging (wrinkles, elasticity) (Patent Document 2). However, retinol derivatives are also known to have side effects caused by excessive administration, and are not sufficient in terms of safety.

[0008] In addition, there have been many attempts to explore substances that inhibit the activity of elastase. To date, as plant-derived ingredients and extracts, extracts of Angelica dahurica (Patent Document 3), extracts of Orobanchaceae plants containing phenylethanol glycosides (echinacoside and acteoside) (Patent Document 4), resveratrol extracted from grape buds and vines (Patent Document 5), powder and / or extracts of pomegranate flowers (Patent Document 6), extracts of Ganoderma lucidum (Patent Document 7), and soy protein hydrolyzates (Patent Document 8) have been proposed.

[0009] In the above-mentioned prior art documents, it is recorded that these ingredients and extracts are combined into cosmetics and skin external preparations and applied to the skin for application. However, when these ingredients and extracts are used in cosmetics and skin external preparations, there are doubts and difficulties in terms of the transdermal absorbability and easy cleanability of the ingredients. As a countermeasure for the aging symptoms of the skin, the desired effect cannot be sustained, and the physiological function of the skin tissue cannot be improved in essence. In addition, when oral ingestion of peptides, there is a risk of deterioration and decomposition in the gastrointestinal tract, and there are few materials that can show effectiveness in practical terms. Therefore, there is a need for effective materials that can improve the above-mentioned skin aging symptoms.

[0010] It should be noted that Abelmoschus esculentus is a plant belonging to the genus Abelmoschus of the Malvaceae family, and grows in tropical and temperate regions of the world, and has a long history of being cultivated and eaten as a vegetable. Generally, the fruit (pod) containing white to yellowish-white immature seeds is eaten as a fresh vegetable.

[0011] As attempts to industrially utilize extracts and ingredients obtained by processing Ampelopsis affine, the above-mentioned Patent Document 1, a hyaluronic acid synthesis promoter containing an Ampelopsis affine extract and cosmetics, foods and beverages blended therewith (Patent Document 9), and an anti-aging skin external preparation containing an oligopeptide derived from Ampelopsis affine seeds and a specific plant extract (Patent Document 10) have been proposed. However, there is no mention of the relationship between Ampelopsis affine seeds and processed products and elastin and elastase.

[0012] Prior art literature

[0013] Non-patent literature

[0014] Non-patent document 1: Hattori Michihiro, "Skincare Chemistry" (Skin Care Science), pages 6 to 14 and 15 to 83, Soukabo Co., Ltd., published on February 25, 1997

[0015] Patent Literature

[0016] Patent Document 1: Japanese Patent Application Publication No. 2018-115143

[0017] Patent Document 2: Japanese Patent Application Laid-Open No. 10-158290 (Claim 1, etc.)

[0018] Patent Document 3: Japanese Patent Application Publication No. 2012-201615

[0019] Patent Document 4: Japanese Patent Application Publication No. 2009-263279

[0020] Patent Document 5: Japanese Patent Application Publication No. 2008-239576

[0021] Patent Document 6: Japanese Patent Application Publication No. 2005-53873

[0022] Patent Document 7: Japanese Patent Application Publication No. 2005-23021

[0023] Patent Document 8: Japanese Patent Application Publication No. 2004-182687

[0024] Patent Document 9: Japanese Patent Application Publication No. 2004-51533

[0025] Patent Document 10: Japanese Patent Application Publication No. 2008-74757

[0026] Patent Document 11: Japanese Patent Application Laid-Open No. 02-288890 Summary of the invention

[0027] Technical issues

[0028] In view of the above-mentioned situation, the object of the present invention is to provide an oral composition for inhibiting elastase and its use, an elastase inhibitor, and a method for inhibiting elastase activity by orally ingesting the elastase inhibitor, wherein the composition is used to restore the decrease of elastin in biological tissues caused by external factors such as ultraviolet rays and active oxygen, and the decrease of metabolic function of the organism, and to improve the aging symptoms of the skin.

[0029] Technical Solution

[0030] To solve the above problems, the present inventors have conducted intensive studies on materials that inhibit the activity of fibroblast-derived elastase and methods for processing the materials, and found that processed materials of Ampelopsis foetida seeds are effective.

[0031] Furthermore, the present inventors continued to research and develop components that inhibit the activity of fibroblast-derived elastase and methods for purifying the components, and as a result, found that a cyclic peptide "OF4949-II" obtained from mature seeds of Ampelopsis pilosa L. is an active ingredient, thereby completing the present invention.

[0032] That is to say, the main features of the present invention are roughly the following aspects.

[0033] (1) A composition for inhibiting elastase derived from skin fibroblasts, comprising a processed product of Abelmoschus esculentus seeds.

[0034] Here, the seeds are preferably mature seeds, the processed product is preferably any one of an extract, an extraction residue, an enzymatic decomposition product or a seedling of the coffee sunflower seed, the extraction treatment is preferably hot water extraction, and the enzymatic hydrolysis treatment is preferably hydrolysis using alkaline protease and / or neutral protease. In addition, the composition is preferably taken orally or administered orally.

[0035] (2) A composition for inhibiting elastase derived from skin fibroblasts, characterized by containing a specific compound derived from seeds of Ampelopsis pilosa L. as an active ingredient.

[0036] Here, the seeds are preferably mature seeds. In addition, the specific compound is preferably a cyclic peptide "OF4949-II", and the peptide is preferably the following formula I.

[0037]

[0038] (3) A use of a composition, characterized in that the activity of elastase is inhibited by ingesting or administering the composition.

[0039] Here, the composition is preferably taken orally or administered orally and applied, and the oral composition is preferably in the form of food and drink. In addition, the application of the above composition is preferably used for increasing elastin based on the elastase activity inhibition of skin tissue, and is further preferably used for improving the aging symptoms of skin tissue.

[0040] (4) The use of the cyclic peptide "OF4949-II", characterized in that it inhibits the activity of elastase.

[0041] Here, preferably the composition containing the peptide is taken orally or administered orally and applied, and the oral composition containing the peptide is preferably in the form of food and drink. In addition, the application of the above composition is preferably used for increasing elastin based on the elastase activity inhibition of skin tissue, and is further preferably used for improving the aging symptoms of skin tissue.

[0042] (5) An elastase inhibitor, characterized in that it is formulated with the above-mentioned composition for inhibiting elastase.

[0043] (6) A method for inhibiting elastase activity, characterized in that the elastase inhibitor is orally administered.

[0044] Effects of the Invention

[0045] The processed products obtained from the mature seeds of the coffee ambrette of the present invention, namely the hot water extract, the residue after hot water extraction, the protein hydrolyzate and the seedling, contain the cyclic peptide "OF4949-II", a specific compound derived from the coffee ambrette seeds, as an active ingredient, and have a strong inhibitory effect on the elastase derived from the skin fibroblasts even in a trace amount.

[0046] Therefore, the composition for inhibiting elastase and the elastase inhibitor of the present invention containing the above peptide can promote skin renewal, thereby improving skin problems and aging symptoms (wrinkles, dullness, sagging, etc.), and contribute to skin beauty. In addition, it can be expected that the elastin in the skin tissue can be enhanced, which can help maintain the health of the skin.

[0047] This effect is significantly manifested by oral ingestion or oral administration of the composition for inhibiting elastase of the present invention. Therefore, the composition for inhibiting elastase of the present invention and the inhibitor formed by the composition can be effectively used for skin improvement in the fields of food, medicine, animal feed, etc., directly in the form of the above-mentioned preparation or in the form of various conventional products in the above-mentioned fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : is a flow chart showing the process of obtaining an elastase inhibitory active component from a solution of an extract of mature seeds of Ampelopsis pilosula.

[0049] Figure 2 This is the chromatogram when component F is separated at a concentration of 4 mg / mL.

[0050] Figure 3 This is the chromatogram when component F is separated at a concentration of 4 mg / mL.

[0051] Figure 4 This is a chromatogram of the purified product when fraction (5)-1 was isolated at a concentration of about 10 mg / mL. DETAILED DESCRIPTION

[0052] The present invention is described in detail below. First, the composition for inhibiting elastase of the present invention is characterized in that it contains a processed product of Ampelopsis pilosa seeds, and the composition has an effect of inhibiting elastase, an enzyme that decomposes elastin present in biological tissues, particularly dermal tissues of the skin.

[0053] In addition, the composition for inhibiting elastase of the present invention contains the cyclic peptide "OF4949-II", which is a specific compound derived from Ampelopsis cerevisiae seeds, as an active ingredient.

[0054] The coffee sunflower generally has varieties with pods in the shape of pentagons, octagons, circles, etc., and varieties with colors of green, purple, white, etc. The coffee sunflower of the present invention is not limited to these varieties, and any coffee sunflower can be used.

[0055] In the present invention, the mature seeds of the coffee sunflower are preferably used as raw materials. The mature seeds refer to seeds harvested from fruits (pods) grown from the plant body or naturally generated scattered seeds. The mature seeds are dark green to dark brown, with a diameter of about 5 mm, and have the ability to germinate as long as they are sown.

[0056] On the other hand, immature seeds are white to yellowish white seeds with a diameter of about 2 to 4 mm contained in the pods in the process of growth, and are usually edible together with the pods. The mature seeds of coffee sunflower are commercially available from seedling companies for the cultivation of coffee sunflower, and are easy to use.

[0057] The Ampelopsis cerasifera seeds are preferably collected as processed products, namely, extracts, extract residues, enzymatic hydrolyzates or buds of Ampelopsis cerasifera seeds.

[0058] The extract of the seeds of the coffee sunflower can be prepared, for example, by the following processing. The peeled or unpeeled seeds are coarsely or finely ground, and after adding water to form a dispersion, the dispersion is allowed to stand at room temperature or under heating for extraction or is subjected to extraction while appropriately stirring. Next, the extract is treated by conventional methods such as centrifugation and filtration to remove insoluble matter, and more preferably, it is concentrated and dried or freeze-dried at room temperature or below, thereby producing the extract of the present invention.

[0059] In the above-mentioned extraction step, when preparing a hot water extract, the aqueous dispersion is set to about 30 to about 90° C., more preferably about 40 to about 60° C., and the extraction treatment is performed for about 10 minutes to several days, more preferably about 30 minutes to several hours. When preparing a low-temperature extract, the extraction treatment is performed by immersing at about 10 to about 30° C., more preferably about 15 to about 25° C., for about 30 minutes to several days, more preferably several hours to 1 day.

[0060] The extraction residue of the sunflower seeds can be prepared by concentrating and drying the insoluble matter removed in the above extraction step or freeze-drying it by a conventional method. In this case, in order to prevent contamination caused by the generation of molds, etc., it is preferred that the degree of drying is such that the moisture content is 5% by mass or less. It should be noted that in terms of the relationship with the desired effect of the present invention, the above residue of the present invention is preferably a residue separated when preparing the above low-temperature extract (low-temperature extraction residue).

[0061] The enzymatic decomposition product of the coffee yellow sunflower seeds can be processed as follows to make it. That is, the coffee yellow sunflower seeds are crushed to form an aqueous dispersion, and a protein hydrolase equivalent to about 0.1 to about 5% by mass, more preferably about 0.5 to about 2% by mass of the seed mass is added thereto, and the mixture is allowed to stand or appropriately stirred at room temperature or heated (about 30 to about 90°C, more preferably about 40 to about 60°C), and a hydrolysis treatment is performed for about 10 minutes to several days, more preferably 30 minutes to several hours. After the treatment, the enzyme is heated by a conventional method to inactivate it, and then centrifuged or filtered to separate the insoluble matter, preferably concentrated at room temperature or a low temperature below room temperature, dried or freeze-dried, thereby making the enzymatic decomposition product of the coffee yellow sunflower seeds. It should be noted that in the present invention, a liquid obtained by dissolving or dispersing the extract or extraction residue of the coffee yellow sunflower seeds obtained by the above-mentioned treatment in water can also be used instead of the above-mentioned aqueous dispersion, and the enzyme treatment is performed in the same manner.

[0062] The above-mentioned proteolytic enzyme can use any kind or type of enzyme. In the present invention, alkaline protease and / or neutral protease are preferred, and alkaline protease is further preferred. Here, alkaline protease can be used as long as it is a protease that shows an effect when the pH of the substrate is about 6.5 to 12, and neutral protease has no problem as long as it is a protease that shows an effect when the pH of the substrate is about 5 to 8. In addition, each protease can be used not only one kind, but also two or more kinds can be used in combination, and it is simple to use a commercially available product described later.

[0063] The following specific examples are given as alkaline proteases, but the present invention is not limited to these specific examples.

[0064] "Protin SD-AY10", "Protease P "AMANO" 3SD", "Prolease (registered trademark) FG-F" (all manufactured by Amano Enzyme Co., Ltd.), "Alkaline (registered trademark) 2.4LFG" (manufactured by Novozymes), "Orienter (registered trademark) 22BF" (manufactured by HBI (Industry)), "Aloaze (registered trademark) XA-10" (manufactured by Yakult Pharmaceutical Co., Ltd.), "Sumitium (registered trademark) MP" (manufactured by Shin Nippon Chemical Co., Ltd.), "Bioprase OP, SP-20FG, AL-15FG, 30G, APL-30 and 30L" (manufactured by Nagase ChemteX (manufactured by Co., Ltd.), "OPTIMASE (registered trademark) PR89L", "MALTIFE CT (registered trademark) PR6L" (all manufactured by DaniscoUS), "Protease K", "Chymotrypsin" (all manufactured by Roche), etc.

[0065] Examples of the neutral protease include the following, but the present invention is not limited to these.

[0066] "Flavourzyme (registered trademark)", "PROTAMEX (registered trademark) MG", "Neutrase" (all manufactured by Novozymes), "Pantidalze (registered trademark) P and MP", "Aloareze (registered trademark) AP-10, NP-10 and NS" (all manufactured by Yakult Pharmaceutical Industries, Ltd.), "Nukreishin (registered trademark)", "Olyenteraze (registered trademark) 10NL and 90N" (all manufactured by HBI Corporation), "Protin P" (manufactured by Yamato Chemical Industry Co., Ltd.), "Protease A "AMANO" G", "Papain W40", "Promeline F" (both manufactured by Amano Enzyme Co., Ltd.), "Sumitome (registered trademark) LP and PL" (manufactured by Shin Nippon Chemical Co., Ltd.), "Purified Papain for Foods", "DENATYME AP" (both manufactured by Nagase ChemteX Co., Ltd.), "Trypsin" (manufactured by Roche), etc.

[0067] The sprouts of the coffee sunflower seeds can be prepared by the following method: the seeds are germinated by a known method, grown to a radish seedling type (kaiwa type), and cultivated until green leaves grow and the stem length reaches about 5 to 15 cm, harvested, washed with water, and then dried or freeze-dried.

[0068] The processed product obtained by the above treatment is an extremely complex composition containing various components such as proteins, polysaccharide-protein complexes, polysaccharides, etc. contained in coffee sunflower seeds and / or peptides, amino acids, oligosaccharides, monosaccharides or combinations of oligosaccharides and peptides or amino acids obtained by hydrolyzing them.

[0069] The inventors conducted an in-depth study on the complex composition constituting the processed coffee ambrette seed product, and found that it contained a cyclic peptide "OF4949-II" as an active ingredient. The cyclic peptide "OF4949-II" is known to strongly inhibit the activity of aminopeptidase B and is produced by a microorganism belonging to the genus Penicillium (Patent Document 11), but there is no report that the cyclic peptide "OF4949-II" is contained in mature coffee ambrette seeds.

[0070] The cyclic peptide "OF4949-II" can be produced, for example, by the purification treatment described below. The mature seeds of the coffee sunflower are allowed to stand under heating for extraction or are subjected to extraction while being appropriately stirred. Next, the extract is treated by conventional methods such as centrifugation and filtration to remove insoluble matter, and more preferably, the extract is concentrated and dried or freeze-dried at room temperature or below, thereby obtaining an extract. The obtained extract is purified by repeatedly using flash chromatography or high performance liquid chromatography to concentrate the target component.

[0071] In the above purification step, the peptide was finally identified by mass spectrometry and nuclear magnetic resonance equipment, and was confirmed to be the cyclic peptide "OF4949-II".

[0072] In the present invention, not only the cyclic peptide "OF4949-II" derived from the seeds of C. coffee ambrette, but also the cyclic peptide "OF4949-II" obtained from the microorganisms as described above can be used as the active ingredient with elastase inhibitory effect of the present invention, and the same effect can be expected.

[0073] In the present invention, the processed product of the sunflower seeds can be used directly or in combination with known excipients such as dextrin, cellulose, gelatin, purified water, etc. to prepare a liquid, powder, granular, or capsule inhibitor containing the composition for inhibiting elastase of the present invention. The content of the processed product of the sunflower seeds in the preparation is approximately 20% by mass or more. If it is lower than this content, it is difficult to show the desired effect of the present invention. Therefore, the processed product of the sunflower seeds / excipient (mass ratio) is preferably 100 to about 20 / 0 to about 80, and more preferably 100 to about 50 / 0 to about 50.

[0074] The composition for inhibiting elastase of the present invention and the elastase inhibitor containing the composition inhibit the action of elastase in skin tissue and prevent the decrease of elastin by oral ingestion or oral administration. Therefore, it can be used as a cosmetic means for improving the above-mentioned skin problems, restoring aging symptoms, and promoting skin beauty.

[0075] As a preferred embodiment, the elastase inhibitor of the present invention can be formed into solid (powder, granules), gel (colloid), paste or liquid (beverage, wine) food, medicine, animal feed and other products. In addition, known additives (surfactants, thickeners, antioxidants, colorants, spices, etc.) used to make these products and known materials (fibroblast proliferation promoting effect, collagen, hyaluronic acid and other extracellular component production promoting effect, anti-skin aging effect, skin beauty promoting effect, etc.) can be appropriately combined to make the above-mentioned various products by conventional methods.

[0076] Example

[0077] Next, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. It should be noted that, below, parts and % are by mass unless otherwise specified.

[0078] Example 1

[0079] (Example of producing processed products from coffee sunflower seeds)

[0080] Production example 1

[0081] Mature seeds of Amaranthus serratus produced in Ibusuki, Kagoshima Prefecture, were coarsely ground with a grinder, and 300 mL of distilled water was added to 100 g of the coarsely ground seeds. The mixture was heated to 58°C while stirring to prepare a dispersion (pH 7.5). One portion of a protease (manufactured by HBI (Inc.), trade name: "Olyenter 22BF", alkaline protease) was added to the dispersion, and the mixture was stirred slowly for 5 hours. Then, the enzyme was inactivated (at 80°C for 30 minutes), the insoluble matter was removed by centrifugation, and freeze-dried to obtain an enzymatic decomposition product (Sample 1).

[0082] Comparative sample 1

[0083] As Comparative Sample 1, a commercial product derived from litchi seeds (manufactured by ORIZA YUKA CO., LTD., trade name: “Lychee Seed Extract-WSP”) was used, which is a known material having an elastase inhibitory effect.

[0084] Comparative sample 2

[0085] As Comparative Sample 2, a commercially available composition containing resveratrol (manufactured by BHN Co., Ltd., trade name: “Resveratrol ε”) which is a known material having an elastase inhibitory effect was used.

[0086] Comparative sample 3

[0087] As Comparative Sample 3, a commercially available product of Peucedanum japonicum Leaf Powder (manufactured by BHN Co., Ltd., trade name: “Peucedanum japonicum Leaf Powder”) which is a known material derived from a plant was used.

[0088] Comparative sample 4

[0089] As Comparative Sample 4, a commercially available sesame leaf powder (manufactured by BHN Co., Ltd., trade name: “sesame leaf powder”) which is a known material derived from plants was used.

[0090] Comparative sample 5

[0091] As Comparative Sample 5, a commercially available product of Ambretteia odorata seed powder (sold by Matsumoto Kosho Co., Ltd., trade name: “Myoxonol LS 9736”) which is a known material derived from Ambretteia odorata was used.

[0092] Production example 2

[0093] The mature seeds used in Preparation Example 1 were coarsely ground, and 800 mL of water was added to 100 g of the coarsely ground seeds. After heating at 80 to 90°C for 60 minutes, the mixture was cooled to room temperature, and then finely filtered (filter paper) and the filtrate was collected. 600 mL of water was added to the filtration residue again, and the filtrate was collected by the same treatment. The two filtrates were combined, concentrated under reduced pressure, freeze-dried, and ground to prepare an extract (Sample 2).

[0094] Production example 3

[0095] The mature seeds used in Preparation Example 1 were coarsely ground, and 800 mL of water was added to 100 g of the coarsely ground seeds. After soaking at 15 to 25°C overnight, the mixture was finely filtered (filter paper) and the filtrate was collected, concentrated under reduced pressure, freeze-dried and ground to prepare an extract (Sample 3).

[0096] Production example 4

[0097] The residue after filtration described in Preparation Example 3 was freeze-dried and pulverized to prepare an extraction residue (Sample 4).

[0098] Production example 5

[0099] The same mature seeds as those in Preparation Example 1 were immersed in water at 0°C to room temperature for 8 to 20 hours and then the water was removed. The seeds were sown in a cultivation container and stored in a dark room at room temperature to 30°C and a humidity of 75 to 90%. The seeds were sprayed with water appropriately to allow them to germinate. After germination, the seeds were transferred indoors and sprayed with water appropriately under sunlight at room temperature of 25 to 30°C. After 5 to 7 days, radish seedling-type (kaiware-type) seedlings were obtained. The seeds were washed with water, freeze-dried, and pulverized to produce a seedling pulverized product (Sample 5).

[0100] Comparative production example 1

[0101] In Production Example 1, the same treatment was carried out except that the mature seeds were replaced with immature seeds to obtain an enzymatic decomposition product (Comparative Sample 6).

[0102] Comparative Example 2

[0103] In Preparation Example 1, the same treatment was performed except that the mature seeds were replaced with a commercial product derived from the edible part of Ampelopsis affine (pods containing immature seeds, the same below) (manufactured by El Co., Ltd., trade name: "Ampelopsis affine powder") to obtain an enzymatic decomposition product (Comparative Sample 7).

[0104] Comparative Example 3

[0105] An extract (Comparative Sample 8) was obtained by performing the same treatment as in Preparation Example 2 except that the mature seeds were replaced with a commercial product derived from the edible part of Ampelopsis pilosa L. (manufactured by El Co., Ltd., trade name: Ampelopsis pilosa L. powder) .

[0106] Test example

[0107] The effects of the above samples on the activity of elastase were examined by the following method.

[0108] Test Example 1: Elastase Inhibition (Part 1)

[0109] As test samples for measuring elastase activity, the above-mentioned sample 1 and comparative samples 1 to 4 were used. Each test sample was dissolved in 100 mM Tris hydrochloric acid buffer (pH 8.0) to prepare a test solution with a final concentration of 40.0 to 1000.0 μg / ml. At this time, if it was difficult to dissolve, DMSO within 10% was added.

[0110] 25 μl of the test solution and 25 μl of an elastase solution derived from human skin fibroblasts (here, the elastase solution is prepared by pre-culturing human skin fibroblasts (manufactured by Kurabo Co., Ltd.) in a confluent manner, culturing with a DMEM medium containing 2% FBS for 24 hours, washing twice with PBS (-), adding 0.1% triton X-100, and dissolving by ultrasonic treatment. The dissolved solution was centrifuged at 12,000 rpm for 5 minutes, the supernatant was recovered, and the freeze-dried powder was used as a crude enzyme powder, which was dissolved with a buffer solution each time in each test.) were mixed and added to a 96-well microplate, and pre-heated at 37°C for 5 minutes.

[0111] As a control, a mixed solution obtained by adding only 100 mM Tris-HCl buffer and DMSO to the elastase solution was subjected to the same treatment.

[0112] Then, 50 μl of a solution containing N-succinyl-tripalaninyl-P-nitroaniline as a substrate (the substrate was dissolved in a minimum amount of DMSO and 100 mM Tris-HCl buffer (pH 8.0) was added to 5 mM) to start the reaction. After 2 hours, the nitroaniline released by the action of elastase was measured under 405 nm wavelength light using a spectrophotometer (Microplate Reader SH9000 Lab, manufactured by CORONA Electric Co., Ltd.), and the elastase inhibition rate was calculated based on the absorbance of the test sample relative to the absorbance of the control to which the test sample was not added. The elastase inhibition rate was calculated by the following calculation formula.

[0113] Elastase inhibition rate (%) = {1-(control-sample) / (control-blank)}×100

[0114] The results are shown in Table 1. In the table, the degree of inhibition rate of fibroblast-derived elastase is expressed as a relative value when the value of a control test performed simultaneously is set to 0.

[0115] From the data in this table, it can be seen that when the mature seeds were treated with enzymes (Sample 1), extremely high elastase inhibition was shown at any concentration of the test sample. In contrast, the lychee seed extract (Comparative Sample 1) and the composition containing resveratrol (Comparative Sample 2), which are known to inhibit elastase, showed increased inhibition at high concentrations, but had a low inhibition at low concentrations. In addition, the plant-derived Peucedanum leaf powder (Comparative Sample 3) and Sesame leaf powder (Comparative Sample 4) showed weak inhibition at any concentration.

[0116]

[0117] Test Example 2: Elastase Inhibition (Part 2)

[0118] In this test example, the effects of processed products other than enzymatic decomposition products of Ambretteia odorifera seeds and processed products other than mature seeds on the inhibitory effect of elastase were examined. Here, the determination of the elastase inhibitory activity and the evaluation of the degree of the inhibitory effect were carried out by the same method as in Test Example 1, but the final concentration of the test solution was set to 0.4 to 10.0 μg / ml. In addition, the test samples used were the above-mentioned sample 1, samples 2 to 5, and comparative samples 5 to 8.

[0119] The results are shown in Table 2. In the table, the degree of elastase inhibition (inhibition rate) is expressed as a relative value when the value of a control test performed simultaneously is set to 0.

[0120] According to the data in this table, it was confirmed that the extracts (Samples 2 and 3), extraction residues (Sample 4) and seedlings (Sample 5) which are processed products of mature seeds of Ampelopsis affine showed strong elastase inhibitory effects to the same extent as the enzymatic decomposition product (Sample 1). On the other hand, in the case of the powder derived from the seeds of Ampelopsis affine (Comparative Sample 5), the elastase inhibitory effect was poor, and almost no elastase inhibitory effect was confirmed in the case of enzymatic decomposition of immature seeds (Comparative Sample 6), the case of enzymatic treatment of the edible part (pods containing immature seeds) (Comparative Sample 7) and the case of hot water extraction (Comparative Sample 8).

[0121]

[0122] Test Example 3: Human Monitoring Test

[0123] 40 adult female volunteers (30-55 years old, average age: 44.5 years old) who agreed to the following test were divided into two groups, each with 20 people. One group took sample 1 (100 mg) orally twice a day, and the other group took a placebo (100 mg dextrin) orally twice a day for 4 weeks. A questionnaire survey was conducted to evaluate whether there was any improvement in skin changes before and after taking the product, including skin dryness, spots, wrinkles, firmness, dullness, sagging, nasolabial folds, makeup adhesion, and enlarged pores.

[0124] As a result, in the sample 1 group, 17 volunteers' skin improved, 2 volunteers' skin remained unchanged, and 1 volunteer's skin deteriorated compared to before taking it. In the placebo group, 2 volunteers' skin improved, 14 volunteers' skin remained unchanged, and 4 volunteers' skin deteriorated. The 17 volunteers whose skin changed selected two or more items of improvement, especially wrinkles, firmness, sagging, and nasolabial folds. In addition, they also selected improvements in dry skin and enlarged pores, which were not seen in the placebo group.

[0125] The results suggest that the processed product of mature seeds of Ampelopsis foetida of the present invention is useful for improving skin by inhibiting the activity of elastase.

[0126] Example 2

[0127] (Example of preparation of cyclic peptide "OF4949-II")

[0128] 10 mL of distilled water was added to 5 g of the extract obtained from the mature seeds of Amaranthus cerevisiae and stirred thoroughly, ultrasonicated, centrifuged or allowed to stand, and the supernatant was recovered as a separation sample. 1 mL of the separation sample was loaded into a chromatographic column tube equipped with preparative separation filler TOYOPEARL HW-40F (Tosoh), and a certain amount of 20% methanol was passed through.

[0129] like Figure 1As shown in the flowchart, the eluted solutions were separated into glass test tubes at 3 mL each.

[0130] Test example

[0131] The effects of the above components on the activity of elastase were examined by the method described below.

[0132] Test example: Elastase inhibition

[0133] As test samples for measuring the elastase activity, the above-mentioned components were used.

[0134] The test method was carried out in the same manner as in the above-mentioned Test Example 2, except that the final concentration of the test solution was set to 0.4 to 10.0 μg / ml.

[0135] The results are shown in Table 3. In the table, the degree of elastase inhibition rate of each fraction is expressed as a relative value when the value of a control test performed simultaneously is set to 0.

[0136] It can be seen from the data in this table that component F shows extremely high elastase inhibition effect (%) at any concentration of other components.

[0137]

[0138] Then, the above-mentioned fraction F was treated and purified as follows. The above-mentioned fraction F was concentrated, dried, and dissolved in distilled water to a concentration of about 1 to 5 mg / mL. After dissolution, high performance liquid chromatography (HPLC) was used at a flow rate of 0.7 mL / min using a C18 column 5C 18 -MS-II (Nacalai Tesque) gradient analysis was performed to obtain 5 fractions ((1) to (5) in elution order). The elastase inhibitory activity of each fraction was measured in the same manner, and the results confirmed that fraction (5) had the highest inhibitory activity.

[0139] In the gradient analysis, the mobile phase of HPLC used water (mobile phase A) containing 0.1% formic acid and acetonitrile (mobile phase B) as the gradient, and the ratio of mobile phase B was 0-5 (min): 0%, 15 (min): 50%, and 15-25 (min): 80%.

[0140] Furthermore, when fraction (5) was separated into three fractions ((5)-1 to 3) under the same conditions, high inhibitory activity was substantially confirmed only in (5)-1. The results are shown in Table 4.

[0141] The chromatogram when fraction F was separated at a concentration of 4 mg / mL is shown in FIG. Figure 2 , Figure 3 .exist Figure 2 , 3 In the figure, the vertical axis is the UV absorption intensity (mV) and the horizontal axis is the retention time (min).

[0142]

[0143] The same operation was repeated for fraction (5)-1 having high inhibitory activity to obtain a sufficient amount, and then gradient analysis was performed by HPLC at a flow rate of 0.6 mL / min using Intrada Amino Acid (Imtakt) as an amino column, and the peak at around 10.5 minutes was recovered for purification.

[0144] In the gradient analysis, the mobile phase of HPLC used water (mobile phase A) containing 0.1% formic acid and acetonitrile (mobile phase B) as the gradient, and the ratio of mobile phase B was 0-3 (min): 90%, 10 (min): 0%, and 10-15 (min): 0%.

[0145] For example, the chromatogram of the purified product when fraction (5)-1 was fractionated at a concentration of about 10 mg / mL is shown in FIG. Figure 4 .exist Figure 4 In the figure, the vertical axis is the UV absorption intensity (mV) and the horizontal axis is the retention time (min).

[0146] The purified product obtained in the above treatment was injected into direct injection / mass spectrometry (DI / MS) and nuclear magnetic resonance analysis (NMR), and the molecular weight was estimated to be 472 and the molecular formula was C 22 H 24 N4O8 was confirmed to be "OF4949-II" (CAS No.: 93375-50-9) of the cyclic peptide represented by the above formula I.

[0147] Trial Example 1: Hard Capsule Preparation

[0148] The sample 1 was put into a capsule filling machine, and a gelatin-coated hard capsule preparation with a content of 100 mg per capsule was prepared by a conventional method. When the hard capsule preparation (preparation containing mature sunflower seeds of Preparation Example 1) was orally ingested as a monitoring test, it was found that it was useful for improving the skin. Therefore, the hard capsule preparation can be used as a nutritional supplement food, medicine or animal feed that can be orally ingested for improving the skin.

[0149] Trial Example 2: Tablets

[0150] The raw materials shown below were tableted by conventional methods to prepare tablets. Here, any one of the above samples 1 to 5 was used as mature ambrette seeds. These tablets were stable and easy to take, and can be used as nutritional supplements and pharmaceuticals.

[0151]

[0152] Trial Example 3: Nutritional Supplementary Food

[0153] 100 mg of the sample 1, 400 mg of leucine, 250 mg of isoleucine, 200 mg of valine and 300 mg of arginine were mixed and sieved, and the powder thus obtained was packaged and filled to prepare a nutritional supplementary food. The nutritional supplementary food can be used not only for humans but also for pets.

[0154] Trial Example 4: Food (Noodles)

[0155] 10 g of the sample 1, 350 g of buckwheat flour, and 150 g of high-gluten flour were mixed and sieved, and 150 ml of water were added to make noodles. The noodles had a flavor and a texture that were not unpleasant compared to commercial products.

[0156] Trial Example 5: Refreshing Drinks and Drinks

[0157] 500 mg or 100 mg of the above sample 1 or sample 2 was added to 100 mL of commercially available refreshing water or beverage and mixed thoroughly to prepare a beverage. The beverage had no color in flavor and storage stability compared to the original refreshing water or beverage. It can be used as a refreshing beverage or beverage.

[0158] Industrial Applicability

[0159] Processed products obtained from the mature seeds of Ambretteia odorata, especially the cyclic peptide "OF4949-II" obtained from the mature seeds of Ambretteia odorata, have the effect of inhibiting the activity of elastase derived from fibroblasts. Therefore, by oral ingestion or administration, they can be effectively used as food and beverages, medicines, animal feed, etc. that help restore the original physiological functions of the skin, improve skin problems, promote skin beauty, etc.

Claims

1. A composition for inhibiting elastase derived from skin fibroblasts, characterized in that: A processed product derived from the seeds of Ampelopsis affine or a specific compound of the seeds of Ampelopsis affine as an active ingredient, wherein the specific compound is a cyclic peptide "OF4949-II": 。 2. The composition according to claim 1, characterized in that The seeds are mature seeds.

3. The composition according to claim 1 or 2, characterized in that The processed product is an extract, an extraction residue, an enzymatic decomposition product or a seedling of Ampelopsis nigra seeds.

4. The composition according to claim 3, characterized in that The extract is a hot water extract.

5. The composition according to claim 3, characterized in that The enzymatic degradation product is a degradation product obtained by hydrolysis with alkaline protease and / or neutral protease.

6. The composition according to claim 4, characterized in that The enzymatic degradation product is a degradation product obtained by hydrolysis with alkaline protease and / or neutral protease.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a composition for inhibiting the activity of elastase.

8. An elastase inhibitor, characterized in that A composition comprising the composition according to any one of claims 1 to 6.

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