JP2020132540A - Elastase inhibitors - Google Patents

Abstract

To provide elastase inhibitors containing sericin as the effective ingredient, and external preparations for skin containing the inhibitors.SOLUTION: Disclosed is an elastase inhibitor containing such sericin that when the sericin is subjected to SDS-PAGE, at least one band is detected in a range corresponding to the molecular weight of 150 kDa or more. The sericin is obtainable by extracting silkworm cocoons or raw silk with an inorganic salt-containing solvent.SELECTED DRAWING: None

Description

The present invention relates to elastase inhibitors. More specifically, the present invention relates to an elastase inhibitor and an external preparation for skin containing the inhibitor.

Silkworm thread is mainly composed of two kinds of proteins, sericin and fibroin. Of these, fibroin is an insoluble protein that constitutes silkworm cocoon fibers, and sericin is a water-soluble protein that covers the outside of the fibers formed by fibroin in layers and is involved in its adhesion.

For example, sericin is obtained from the waste liquid when fibroin is taken out from silk thread in the silk thread refining step, but the sericin obtained in the refining step is hydrolyzed to a low molecular weight. Various studies have been conducted on sericin that has undergone such treatment.

For example, Patent Document 1 discloses that a specific protein hydrolyzate has an elastase inhibitory action, and silk is mentioned as an example of a protein-containing raw material. Further, Patent Document 2 discloses that silk sericin extracted from cocoons with hot water and then precipitated in a specific solvent to deodorize is excellent in elastase inhibitory action.

Japanese Unexamined Patent Publication No. 2005-343887 Korean Publication No. 10-2000-0056836

An object of the present invention is to provide an elastase inhibitor having good anti-elastase activity and an external preparation for skin containing the inhibitor.

As mentioned above, sericin is known to be extracted as a hydrolyzate, but sericin is originally a high molecular weight protein, and there are still many unclear points about its physiological action. Under these circumstances, the present inventors have found that a fraction containing a high molecular weight sericin has a superior elastase inhibitory effect than a low molecular weight sericin, and have completed the present invention.

That is, the present invention relates to the following [1] to [5].
[1] An elastase inhibitor containing sericin in which at least one band is detected in a range corresponding to a molecular weight of 150 kDa or more by the SDS-PAGE method.
[2] An elastase inhibitor containing sericin obtained by a method including a step of extracting from silkworm cocoons or raw silk with an inorganic salt-containing solvent.
[3] The above-mentioned [1] or [2], wherein sericin is a sericin that forms a gel at 4 ° C. within 12 hours by adding 10% by mass of ethanol to an aqueous solution having a concentration of 0.5% by mass. Elastase inhibitor.
[4] An external preparation for skin containing the elastase inhibitor according to any one of [1] to [3] above.
[5] The agent according to the above [4], which is used for improving or preventing wrinkles and / or sagging.

According to the present invention, it is possible to provide an elastase inhibitor having an excellent elastase inhibitory action.

In addition, the elastase inhibitor of the present invention can exert an excellent effect of inhibiting elastase existing in the skin and increasing the elasticity of the skin, and can improve or prevent wrinkles and sagging of the skin. It is a thing.

FIG. 1 is a diagram showing the results of SDS-PAGE of an aqueous sericin solution. In the figure, M1 and M2 indicate molecular weight markers.

The elastase inhibitor of the present invention is characterized by containing a specific sericin as an active ingredient. Hereinafter, such sericin will be described as the sericin of the present invention.

The sericin of the present invention, for example, in one embodiment, in the SDS-PAGE method, a clear band is detected rather than a band cannot be confirmed or becomes a smear. Specifically, those in which at least one band is detected in the range corresponding to the molecular weight of 150 kDa or more can be mentioned. The band is preferably 150 kDa or more, but more preferably 160 kDa or more, still more preferably 180 kDa or more, and it is desirable to detect the band in a range corresponding to the molecular weight. Further, the upper limit of the number of bands detected in the above range is not particularly limited as long as it is at least one. Specifically, for example, a band is detected in the vicinity of 250 kDa. The SDS-PAGE method can be carried out according to the method described in Examples described later, and the molecular weight corresponding to the detected band can be determined by comparison with the band of the molecular weight fraction of the marker protein. Further, the composition ratio of the band can be calculated from the density of the band, but the composition is not particularly limited as long as the sericin of the present invention has elastase inhibitory activity.

Further, the sericin of the present invention preferably has a gelling ability. The term "having a gelling ability" as used herein means, for example, increasing the viscosity of the solution when it is in a solution state. For such gelling ability, for example, when a 0.5% by mass aqueous solution is prepared and 10% by mass of ethanol is added thereto, the gelling ability is obtained at 4 ° C. within 24 hours, preferably within 12 hours, more preferably within 6 hours, and further. Whether or not the gel is formed within 2 hours, more preferably within 1 hour can be evaluated as an index.

In addition, examples of the sericin of the present invention include those obtained by a method including a step of extracting from silkworm cocoons or raw silk with an inorganic salt-containing solvent.

The silkworm is not particularly limited as long as it produces the cocoon or raw silk. In addition to the above, insects such as silkworms such as Bombyx mandarin and Bombyx mandarin, silkworms such as silkworms, silkworms, silkworms, and tasarsan, honeybees, bees, spiders, and caddisflies are also used because they produce silk. You may. The production area is not particularly limited. In addition, the silkworm may be natural or mutated or genetically engineered. For example, since there is a tendency to efficiently extract high-molecular-weight sericin, silkworms with inferior fibroin-producing ability can be used. Specifically, silkworms whose fibroin-producing ability has decreased due to mutation (for example, sericin). Hope (sericin C), sericin N, Nd silkworm, Nd-s silkworm, etc.), silkworms lacking fibroin-producing ability by genetic manipulation (for example, transformed silkworms described in JP-A-2018-007569) should be used. Can be done.

The silkworm cocoon is a structure formed by the silkworm larva secreting silk protein in the silk gland during pupation, and is obtained by separating the silkworm from the cocoon. In addition, the raw silkworm thread is obtained by combining several threads (silk fibers) taken out from the cocoon into a thread shape.

The method for extracting the sericin of the present invention from the cocoon or raw silk is not particularly limited, but for example, the cocoon or raw silk is extracted as it is, or a processed product such as cutting, crushing, or drying is immersed in an extraction solvent for extraction. be able to.

The extraction solvent is not particularly limited, and water and / or an organic solvent or the like can be used. As the organic solvent, lower alcohols such as methanol, ethanol and isopropanol; glycols such as propylene glycol and diethylene glycol can be used, and aprotic such as dimethyl sulfoxide, dimethylformamide, dimethylacetamide and N-methylmorpholine-N-oxide. It may be an aprotic solvent. Further, in the present invention, it is preferable to use an inorganic salt from the viewpoint of extracting a high molecular weight sericin. The inorganic salt is not particularly limited, and for example, salts of alkali metals and alkaline earth metals can be used, and specifically, lithium bromide, potassium bromide, calcium bromide, lithium chloride, potassium chloride, etc. Examples thereof include calcium chloride, lithium thiocyanate and potassium thiocyanate. Specific examples of the extraction solvent include, for example, lithium bromide aqueous solution, potassium bromide aqueous solution, lithium chloride aqueous solution, potassium chloride aqueous solution, urea aqueous solution, lithium thiocyanate aqueous solution, potassium thiocyanate aqueous solution, calcium chloride aqueous solution, calcium chloride / ethanol solution. , Strongly electrolyzed water, calcium nitrate / methanol solution, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylmorpholin-N-oxide, etc., and these may be used alone or in combination of two or more. it can. Among these, an aqueous solution of lithium bromide is preferable.

The concentration of the inorganic salt in the extraction solvent is not particularly limited as long as sericin can be efficiently extracted and sericin is not or is not hydrolyzed. For example, 3M or more, 4M or more, 5M or more, 6M or more, 7M. As described above, 8M or more can be appropriately set.

The pH of the extraction solvent is preferably pH 5 to 8, more preferably pH 5.5 to 7.5. The amount of the extraction solvent used can be appropriately set to be 20 to 60 times the mass ratio and 30 to 50 times the mass ratio of the silkworm cocoon or raw silk.

The extraction temperature is preferably 50 ° C. or lower, more preferably 40 ° C. or lower from the viewpoint of suppressing hydrolysis, and 0 ° C. or higher from the viewpoint of extraction efficiency. The extraction time can be appropriately set to, for example, 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, etc., depending on the amount of the raw material used for extraction and the apparatus. The lower limit can be set to, for example, one hour or more.

The present invention has found that the extract thus obtained has elastase inhibitory activity. Conventionally, sericin has been obtained by hydrolyzing silkworm cocoons or raw silk by a hydrolysis method such as acid hydrolysis, alkaline hydrolysis, or enzymatic hydrolysis, and eluting it. However, the sericin of the present invention can be obtained, for example, by extracting from silkworm cocoons or raw silk without the above-mentioned hydrolysis method. Therefore, the sericin of the present invention may be referred to as "non-hydrolyzed sericin" or "non-hydrolyzed sericin". At the time of the extraction, the sericin of the present invention may be hydrolyzed as long as the effect of the present invention is not impaired. It may also contain fibroin.

As a method for extracting high molecular weight sericin, for example, a method for extracting from a cocoon or the like using an aqueous solution of lithium bromide (for example, Journal of the Silk Society of Japan, 20, 89-94 (2012)) is known. In addition to or modified from the extraction methods described above, these can be referred to as appropriate.

The obtained extract may be used as it is as long as it has elastase-inhibiting activity, but filtration, centrifugation, purification, concentration, ultrafiltration, dialysis, lyophilization, pulverization, and minutes It is preferable to carry out one or more treatments selected from the group consisting of images according to a known method. If necessary, the treatment may be carried out under normal pressure or reduced pressure. By the above treatment, for example, insoluble substances such as inorganic salts used at the time of extraction can be removed. For example, when extracted from an aqueous solution of lithium bromide, the extract may be obtained by performing a treatment such as dialysis to remove lithium bromide. Further, since sericin is acidic and easily aggregates, for example, Gly-NaOH (about pH 9) may be added to the obtained extract to adjust the liquid property.

The content of sericin of the present invention in the extract is not unconditionally set depending on the extraction conditions, and is exemplified by 0.0001 to 100% by weight, for example. In the present specification, the content means, for example, when the object is a liquid, a dry solid which indicates the weight of the target component as a percentage of the total weight of the dried product obtained when the liquid is dried by an evaporator or the like. It is a physical conversion value, and may be described as dry weight or simply content.

The shape of the elastase inhibitor of the present invention is not particularly limited, and may be any of powdery, solid, and liquid shapes. The extract may be used as it is, or it may be used as a granular solid obtained by granulating the extract by a known method. When the extract is liquid, the elastase inhibitor is a liquid by dissolving the extract itself, its concentrate or dilution, or the powdery extract in a liquid such as water or alcohol. May be. At that time, since sericin is acidic and easily aggregates, the pH of the solution may be adjusted to be weakly alkaline (about pH 9).

The elastase inhibitor of the present invention has an action of inhibiting elastase. Elastase is a proteolytic enzyme that hydrolyzes elastin, and the type of elastin to be targeted is not limited as long as it inhibits the hydrolyzing action. Since elastin has a function of supporting collagen fibers in skin tissue, for example, the elastase inhibitor of the present invention can be used, for example, to improve and / or prevent wrinkles and sagging of the skin.

In addition, the elastase inhibitor of the present invention not only has an action of inhibiting elastase, but also has an action of inhibiting collagenase. Thereby, for example, it can be used more effectively to improve and / or prevent wrinkles and sagging of the skin.

The present invention also provides a composition containing the sericin of the present invention, which is the active ingredient of the present invention. Since the composition of the present invention contains the active ingredient, it can act as an inhibitor of elastase. The composition of the present invention is not particularly limited as long as the active ingredient can be applied to the surface of a living body or ingested into the body, and is, for example, a pharmaceutical composition, a food or drink composition, or a cosmetic composition. It is preferably used as a composition.

Hereinafter, as one aspect of the composition of the present invention, a skin external preparation in the form of being applied to the surface of a living body will be described. The external preparation for skin in the present invention is not particularly limited, and includes those classified as quasi-drugs in the definition of medicated cosmetics and the like in the Pharmaceutical Affairs Law.

The shape of the external preparation for skin of the present invention is not particularly limited, and examples thereof include a liquid state, a fluid state, a semi-solid state, and a solid state. The form of the external preparation for skin of the present invention is not particularly limited, and for example, a liquid agent, a suspension agent, an emulsion, a cream agent, an ointment agent, a plaster agent, a gel agent, a liniment agent, a lotion agent, an aerosol agent, or a sheet agent. (Including film agents, cast film agents, sponge agents, gel sheet agents, sheet agents obtained by impregnating non-woven fabrics with chemicals), solid agents, powder agents and the like. Among these, since the sericin of the present invention has a gelling ability, it is preferably a liquid agent, a suspension agent, an emulsion, a cream agent or the like, and more preferably a liquid agent, an emulsion, a cream agent or the like.

The specific use of the external skin preparation of the present invention is not particularly limited, but for example, a lotion, a cosmetic emulsion, a cosmetic oil, a gel, a cream, a beauty liquid, a pack, a mask, a hand cream, a body lotion, and a body cream. Basic cosmetics such as; Makeup base, foundation, face powder, whitewash, eye shadow, lipstick, lip cream, cheek red, various colors, etc. Makeup cosmetics; shampoo, dry shampoo, conditioner, rinse, rinse-in shampoo, treatment, hair tonic , Hair styling products, hair oils, pomades, hair coloring agents and other hair care products.

As long as the external preparation for skin of the present invention contains the sericin of the present invention or the elastase inhibitor of the present invention as a raw material component, other components are not particularly limited. The blending amount of sericin of the present invention contained in the external preparation for skin of the present invention can be appropriately selected depending on other blending components and the like, and is not particularly limited, but for example, 3% by weight or less, 1.5% by weight or less, 1.0. It can be set to weight% or less. When it is 3% by weight or less, gelation is unlikely to occur at the time of preparing the external preparation, and the good condition of the external preparation can be maintained. Further, in the case of a sheet agent, a solid agent or a powder agent, for example, it may be set to 1% by weight or more, 10% by weight or more, preferably 50% by weight or more.

As the external preparation for skin of the present invention, for example, in addition to the sericin, water, and organic solvent of the present invention, ingredients commonly used in cosmetics and external preparations can be blended according to the purpose of use and dosage form. Examples of other components include, but are not limited to, water-soluble polymers, oils, moisturizers, thickeners, emulsifiers, powders, ultraviolet absorbers, ultraviolet scattering agents, and the like.

Examples of water-soluble polymers include quince seed, carrageenan, mannan, curdran, galactan, locust bean gum, xanthan gum, purulan, arabic rubber, benzoin rubber, dammar rubber, Irish moss, karaya rubber, carob rubber, guar gum, text run, and tragant gum. Mucopolymers such as starch, chitin, chitosan, pectin, gelatin, casein, collagen, alginic acid and salts thereof, hyaluronic acid, chondroitin sulfate, dermatane sulfate, heparin and salts thereof; natural succinyl chitosan, carboxymethyl chitin, chitosan and the like. Polymeric substances: Semi-synthesis of cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl hydroxypropyl cellulose, soluble starch, cationized cellulose, cationized guar gum, hydroxyethyl guar gum, carboxymethyl guar gum, etc. Polymer substances; carboxyvinyl polymers, polyvinyl alcohols, polyvinylpyrrolidones, polyacrylates, polyethyleneimines, highly polymerized polyethylene glycols, polyvinylmethyl ethers, polyvinyl methacrylates, polyalkylene oxides such as polyethylene oxides and polypropylene oxides, or crosslinked polymers thereof. , Polyoxyethylene / polyoxypropylene copolymer, vinyl alcohol / vinyl acetate copolymer, alkyl methacrylate copolymer, polyvinylpyrrolidone, maleic acid, vinyl acetate, styrene, methacrylate, acrylic acid, methacrylate and Examples thereof include synthetic polymer substances such as copolymers such as derivatives of acrylic acid, and one or more of these can be used.

As the oil agent, those usually used for skin external preparations such as cosmetics and non-medicinal products are preferably used, and the origins of animal oils, vegetable oils, synthetic oils, etc., and solid, semi-solid oils, liquid oils, volatilization Regardless of the properties of the oil, examples thereof include hydrocarbons, fats and oils, waxes, ester oils, hydrogenated oils, fatty acids, higher alcohols, silicone oils, lanolin derivatives, oily gelling agents and the like. .. Specifically, hydrocarbons such as paraffin wax, ceresin wax, microcrystallin wax, montan wax, fishertroph wax, liquid paraffin, squalane vaseline; mokuro, mink oil, olive oil, avocado oil, castor oil, macadamian nut oil. Oils and fats such as; waxes such as beeswax, carnauba wax, candelilla wax, gay wax; pentaerythritol rosinate, jojoba oil, tri (2-ethylhexanoic acid) glyceryl, isotridecyl isononanoate, 2-ethylhexanoic acid Cetyl, isopropyl myristate, isopropyl palmitate, octyldodecyl myristate, glyceryl trioctate, polyglyceryl diisostearate, glyceryl triisostearate, diglyceryl triisostearate, polyglyceryl triisostearate, diisostearyl malate, neopentyl diethylhexanate Esters such as glycols; fatty acids such as oleic acid, isostearic acid, stearic acid, lauric acid, myristic acid, and behenic acid; higher alcohols such as stearyl alcohol, cetyl alcohol, lauryl alcohol, behenyl alcohol, and oleyl alcohol. Kind: Methylpolysiloxane, Methylphenylpolysiloxane, trimethylsiloxysilicate, crosslinked polyether-modified methylpolysiloxane, methacryl-modified methylpolysiloxane, oleyl-modified methylpolysiloxane, polyvinylpyrrolidone-modified methylpolysiloxane, polyether-modified polysiloxane, etc. Silicone oils; lanolin derivatives such as lanolin, lanolin acetate, lanolin fatty acid isopropyl, lanolin alcohol; oily gelling agents such as aluminum isostearate, calcium stearate, 12-hydroxystearic acid, etc., and one of them. Alternatively, two or more types can be used.

As the moisturizing agent, those usually used for skin external preparations such as cosmetics and non-medicinal products are preferably used, for example, glycerin, diglycerin, polyglycerin, propylene glycol, dipropylene glycol, ethylene glycol, diethylene glycol. , Polyhydric alcohols such as polyethylene glycol, hexylene glycol, 1,3-butylene glycol, 1,2-pentanediol, isoprene glycol; sucrose, lactose, maltose, mannitol, erythritol, xylitol, martitol, sorbitol, trehalose. Sugars such as: NMF components such as sodium lactate and sodium pyrrolidone carboxylate; amino acids such as glycerin, serine, methionine, leucine, tyrosine, proline, isoleucine, tryptophan, hydroxyproline, aspartic acid, glutamine, glutamate, arginine, histidine, lysine, etc. And salts thereof, etc., and one or more of these can be used.

Examples of thickeners include arabic gum, carrageenan, carrageenan, tragacanth gum, carob gum, quince seed (malmero), casein, dextrin, gelatin, sodium pectinate, sodium aragite, methyl cellulose, ethyl cellulose, CMC, hydroxyethyl cellulose, hydroxypropyl. Cellulose, PVA, PVM, PVP, Sodium Polyacrylate, Carrageenan Polymer, Locust Bean Gum, Guar Gum, Tamarint Gum, Dialkyldimethylammonium Sulfate Cellulose, Xanthan Gum, Aluminum Magnesium Silate, Bentonite, Hectrite, AlMg Silate (Beagum), Examples thereof include raponite and silicic anhydride, and one or more of these can be used.

As the emulsifier, those usually used for skin external preparations such as cosmetics and non-pharmaceutical products are preferably used. For example, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, polyoxyethylene glycerin fatty acid ester. , Solbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbit fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene sterol, polyoxyethylene lanolin, polyoxyethylene honey wax , Hydrogenated sterol, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene copolymer, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkyl phenyl ether, sucrose fatty acid ester, maltitol hydroxy Nonionic surfactants such as fatty acid ether, alkylated polysaccharides, fatty acid diethanolamide, fatty acid isopropanolamide, alkyl glucoside; fatty acid soap, alkyl sulfate, alkyl sulfonate, polyoxyethylene alkyl sulfate, polyoxyethylene alkyl phenyl ether Sulfate, alkylbenzene sulfonate, α-olefin sulfonate, alkyl ether sulfonate, alkyl ether phosphate, alkyl ether carboxylate, polyoxyethylene alkyl sulfosuccinate, N-acylsarcosineate, N N-acyl fatty acid salts such as -acyl-N-methyl-β-alaninate, N-acyl glutamate, N-acyl-N-methyl-taurine, acylated hydrolyzed collagen salt, acylated caseine salt, acylated hydrolyzed silk Anionic surfactants such as salts, salts of acylated proteins such as acylated hydrolyzed casein salts; amide amino acid types such as alkylamide hydroxyethylaminoacetic acid, alkyldimethylaminoacetic acid betaine, alkyldihydroxyethylaminoacetic acid betaine and the like. Carbobetaine type, amide betaine type such as fatty acid amide propyl dimethylaminoacetic acid betaine, sulfobetaine type such as lauryl sulfobetaine, coconut oil fatty acid alkyl sulfobetaine, lauryl hydroxy sulfobetaine, hydroxy sulfo such as coconut oil fatty acid alkyl hydroxy sulfobetaine. Betain type, alkylamide dimethylhi Amidosulfobetaine type such as droxypropyl sulfobetaine, 2-alkyl-N-carboxyethyl-N-hydroxyethyl imidazolinium betaine, 2-alkyl-N-sodium carboxymethyl-N-carboxymethyloxyethyl imidazolinium Imidazolinium betaine type such as betaine, laurylaminopropionic acid or a salt thereof, laurylaminodipropionic acid or a salt thereof and an amino acid type such as N-alkyl-β-alanine, laurylphosphobetaine, coconut oil fatty acid alkylphosphobetaine and the like. Phosphobetaine type, 2- (N-dodecyl-N, N-dimethylammonio) ethyl phosphate ester, 2- (N-tetradecyl-N, N-dimethylammonio) ethyl phosphate ester, 2- (N-hexadecyl-N) , N-Dimethylammonioethyl) phosphate ester, 2- (N-octadecyl-N, N-dimethylammonioethyl) phosphate ester, and phosphate ester-type amphoteric surfactants such as salts thereof; lauryltrimethyl Monoalkyl quaternary ammonium salts such as ammonium salt, myristyltrimethylammonium salt, cetyltrimethylammonium salt, stearyltrimethylammonium salt, behenyltrimethylammonium salt, cocoyltrimethylammonium salt, dilauryldimethylammonium salt, dimyristyldimethylammonium salt, Dialkyl-type quaternary ammonium salts such as disetyldimethylammonium salt, distearyldimethylammonium salt and dicocoyldimethylammonium salt, tertiary fatty acid amine salts such as diethylaminoethylamide stearate and diethylaminopropylamide stearate, lauryldimethylbenzyl Cationic surfactants such as trialkylbenzylammonium salts such as ammonium salts, myristyldimethylbenzylammonium salts, cetyldimethylbenzylammonium salts, stearyldimethylbenzylammonium salts, betanyldimethylbenzylammonium salts, cocoyldimethylbenzylammonium salts; soybeans, Examples thereof include phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and the like obtained from egg yolk and the like, and one or more of these can be used.

The powder is not particularly limited by the shape such as spherical, plate-like, needle-like, particle size such as fumes, fine particles, pigment grade, and particle structure such as porous and non-porous, and inorganic powders, Glittering powders, organic powders, dye powders, metal powders, composite powders and the like can be mentioned. Specifically, white inorganic pigments such as titanium oxide, zinc oxide, cerium oxide, and barium sulfate; colored inorganic pigments such as iron oxide, carbon black, chromium oxide, chromium hydroxide, dark blue, and ultramarine; talc, white mica, and gold. Mica, red mica, black mica, synthetic mica, silk mica (serisite), synthetic sericite, kaolin, silicon carbide, bentonite, smectite, aluminum oxide, magnesium oxide, zirconium oxide, antimony oxide, silicate soil, aluminum silicate , White constitution powders such as aluminum magnesium metasilicate, calcium silicate, barium silicate, magnesium silicate, calcium carbonate, magnesium carbonate, hydroxyapatite, boron nitride, silica, titanium dioxide coated mica, titanium dioxide coated bismuth oxychloride, Glittering powders such as iron oxide-coated mica titanium, iron oxide mica, dark blue-treated mica titanium, carmine-treated mica titanium, bismuth oxychloride, fish scale foil; polyamide-based resin, polyethylene-based resin, polyacrylic resin, polyester-based resin, Fluorine-based resin, cellulose-based resin, polystyrene-based resin, copolymer resin such as styrene-acrylic copolymer, organic polymer resin powder such as polypropylene-based resin, silicone resin, urethane resin, zinc stearate, N-acyllysine, etc. Organic low molecular weight powder; Natural organic powder such as starch, silk powder, cellulose powder; Red 201, Red 202, Red 205, Red 226, Red 228, Orange 203, Orange 204, Blue Organic pigment powders such as No. 404 and No. 401, red No. 3, red No. 104, red No. 106, orange 205, yellow No. 4, yellow No. 5, green No. 3, blue No. 1 and the like zirconium, barium or Organic pigment powder such as aluminum lake; Furthermore, metal powder such as aluminum powder, gold powder, silver powder; fine particle titanium oxide coated mica titanium, fine particle zinc oxide coated mica titanium, barium sulfate coated mica titanium, titanium oxide silicon dioxide, zinc dioxide dioxide Composite powders such as silicon; polyethylene terephthalate / aluminum / industrial poxy laminated powder, polyethylene terephthalate / polyolefin laminated film powder, polyethylene terephthalate / polymethylmethacrylate laminated film powder powder; tar dye, natural dye, etc. One kind or two or more kinds can be used. These powder components may be one of fluorine-based compounds, silicone-based compounds, metal soaps, lecithin, hydrogenated lecithin, collagen, hydrocarbons, higher fatty acids, higher alcohols, esters, waxes, squalanes, waxes, surfactants, etc. Surface treatment may be performed using two or more types.

Examples of the ultraviolet absorber or ultraviolet scattering agent include paraaminobenzoic acid-based ultraviolet absorbers, anthranylic acid-based ultraviolet absorbers, salicylic acid-based ultraviolet absorbers, cinnamic acid-based ultraviolet absorbers, benzophenone-based ultraviolet absorbers, and sugar-based ultraviolet absorbers. , 3- (4'-Methylbenzylidene) -d-Phenyl, 3-Benzylidene-dl-Camfer, urocanic acid, urocanic acid ethyl ester, 2-phenyl-5-methylbenzoxazole, 2- (2'-hydroxy- 5'-t-octylphenyl) benzotriazole, 2- (2'-hydroxy-5'-methylphenyl) benzotriazole, dibenzalazine, dianisoilmethane, 4-methoxy-4'-t-butyldibenzoylmethane, 5- (3,3-Dimethyl-2-norbornylidene) -3-pentan-2-one, 2-hydroxy-4-methoxybenzophenone, octyldimethylparaaminobenzoate, ethylhexylmethoxycinnamate, titanium oxide, zinc oxide and the like can be mentioned. One or more of the above can be used.

In addition, the external preparation for skin of the present invention includes, for example, preservatives (methylparaben, ethylparabenbutylparaben, phenoxyethanol, etc.) and various extracts (for example, pearl oyster, auren, shikon, shakyaku, assembly, birch, sage, biwa, carrot). , Aloe, Zeniaoi, Iris, Grape, Yokuinin, Hechima, Yuri, Saffron, Senkyu, Shoukiyu, Otogirisou, Ononis, Garlic, Pepper, Chinpi, Touki, Seaweed, etc.), pH adjuster, other medicinal ingredients, fragrance, etc. You may.

Further, the base material that can be impregnated with the sericin of the present invention or the elastase inhibitor of the present invention is not particularly limited as long as it is a known non-woven fabric. For example, natural cellulose fibers such as cotton, pulp and hemp, regenerated cellulose fibers such as rayon, synthetic fibers such as polyester fibers, polypropylene fibers and nylon fibers, and composite fibers in which cellulose fibers and synthetic fibers are blended, etc. The material can be mentioned.

The external preparation for skin of the present invention can be produced by a known method as long as it contains the sericin of the present invention or the elastase inhibitor of the present invention. For example, the sericin of the present invention and the above-mentioned additives are mixed and stirred to obtain a uniform mixture, and then a liquid agent, a suspension agent, an emulsion, a cream agent, an ointment, a plaster, a gel agent, and a liniment agent. , Lotions, aerosols, sheet agents (including film agents, cast film agents, sponge agents, gel sheet agents, sheet agents impregnated with chemicals in non-woven fabrics, etc.), solid agents, powder agents, etc. It can be manufactured by converting.

Since the external preparation for skin of the present invention contains sericin of the present invention having excellent elastase inhibitory activity, it can be suitably used for improving and / or preventing wrinkles and sagging of the skin. In addition, the external preparation for skin of the present invention can be suitably used for improving and / or maintaining the elasticity of the skin. In addition, the external preparation for skin of the present invention can be suitably used for improving the elasticity of the skin.

The present invention also includes the following aspects.
A method for improving and / or preventing wrinkles and sagging of the skin, which comprises the step of applying the elastase inhibitor or external preparation for skin of the present invention to the skin.
-Use of the elastase inhibitor or external preparation for skin of the present invention for improving and / or preventing wrinkles and sagging of the skin.
-Use of sericin of the present invention to improve and / or prevent wrinkles and sagging of the skin.
-Use of the elastase inhibitor of the present invention to produce an agent that improves and / or prevents wrinkles and sagging of the skin.
-Use of sericin of the present invention to produce agents that improve and / or prevent skin wrinkles and sagging.

Although the preferred embodiment of the present invention has been described, a configuration in which each preferred embodiment is combined is also one of the preferred embodiments.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

Example 1
A flask was charged with 1 g of sericin cocoon obtained from a silkworm (Japanese Patent Laid-Open No. 2018-7569) lacking the fibroin-producing ability produced by gene recombination, and 20 mL of an 8M LiBr (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) aqueous solution. The mixture was stirred at ° C. for 5 hours (pH 6). The obtained solution was transferred to a centrifuge tube, and the inside of the flask was washed with 5 mL of Gly-NaOH buffer (pH 9) and combined with the centrifuge tube. Centrifugation was carried out at 4 ° C. and 9,500 × g for 5 minutes, and the mixture was poured into a dialysis membrane (Spectra / Por 4) to start dialysis. First, ion-exchanged water was used as the external liquid, and the external liquid was exchanged after 2.5 hours and 5.5 hours had elapsed. Next, the external solution was exchanged using 0.1 mM carbonate buffer in the morning of the next day, the evening of the next day, and the morning of the next day. After a total of 48 hours, dialysis was stopped, transferred to a centrifuge tube, and the solution was centrifuged at 4 ° C., 9,500 × g for 1 hour. The supernatant was recovered, the obtained aqueous solution was heated and dried, and the concentration was measured. As a result, it was 1.1 wt%.

Example 2
A sericin extract was obtained in the same manner as in Example 1 except that the cocoon was changed to 1 g of a sericin hope cocoon (purchased from Kogen Co., Ltd.) obtained from a silkworm produced by mutation. As a result of heating and drying the obtained aqueous solution and measuring the concentration, it was 1.0 wt%.

Comparative Example 1
3 g of normal cocoons (obtained from N & P Co., Ltd.) were finely crushed, washed with warm water (40 ° C. for 10 minutes), and dried at room temperature overnight. 50 mL of a 0.2% aqueous sodium carbonate solution was added, and the mixture was heat-treated in an autoclave at 120 ° C. for 1 hour to obtain Whatman No. 1 Sericin aqueous solution was obtained by suction filtration with filter paper. As a result of heating and drying the obtained aqueous solution and measuring the concentration, it was 1.75 wt%.

Test Example 1
The molecular weight distributions of Examples 1 and 2 and Comparative Example 1 were evaluated by the SDS-PAGE method.

Specifically, an aqueous solution obtained by diluting each of the solutions obtained in Examples 1 and 2 and Comparative Example 1 with ion-exchanged water 5 times was used. 15 μL of each aqueous solution was taken, mixed with 15 μL of 2 × loading buffer (manufactured by Wako Pure Chemical Industries, Ltd.), and heated at 100 ° C. for 10 minutes. Then, it was cooled and a 5-20% gradient gel manufactured by PAGEL (manufactured by Atto Co., Ltd.) was used, and 5 μL of each sample was applied and electrophoresed at 20 mA for 75 minutes. As molecular weight markers, Multicolor Protein Ladder (10 to 350 kPa) (manufactured by Nippon Gene) "M1" and HiMark ^TM United (manufactured by Invitrogen) "M2" were used. This was washed with water, stained with CBB Stain One (manufactured by Nacalai Tesque, Inc.) with shaking, and washed twice with shaking with water. The results of the implementation are shown in FIG.

From FIG. 1, a clear band was confirmed in the sericin obtained in Examples 1 and 2, and unhydrolyzed sericin was confirmed, but the band disappeared in the sericin obtained in Comparative Example 1 and smeared. It was found that the hydrolysis was in progress.

Test Example 2
The gelling ability of Examples 1 and 2 and Comparative Example 1 was evaluated.

Specifically, 0.5% by mass aqueous solutions of Examples 1 and 2 and Comparative Example 1 were prepared, 10% by mass of ethanol was added thereto, mixed, and then allowed to stand at 4 ° C. until gelation. The time and degree of gelation were evaluated using the following indicators. The results are shown in Table 1.
<Indicator of gelation>
◯: Good gelling ability (the shape hardly changes even if the container is tilted, the viscosity of the solution increases and a good gel is formed)
Δ: Gelable (the shape changes when the container is tilted, but the viscosity of the solution increases and gel is formed)
X: No gelling ability (state in which the viscosity of the solution does not increase and gel is not formed)

From Table 1, the sericin aqueous solution obtained in Examples 1 and 2 had a gelling ability, but the sericin aqueous solution obtained in Comparative Example 1 did not have a gelling ability.

Test Example 3
The elastase inhibitory activity of Examples 1 and 2 and Comparative Example 1 was evaluated.

Specifically, first, 24.02 g of trishydroxymethylaminomethane (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 58.4 g of NaCl are dissolved in 800 g of water, and the pH is adjusted to 8.5 with hydrochloric acid or an aqueous sodium hydroxide solution. The solution was increased to 1 L to obtain a 200 mM Tris-HCl buffer. 50 μg of neutrophil-derived elastase (manufactured by Kishida Chemical Co., Ltd.) was dissolved in 9.5 mL of the above buffer solution. 5 mg of 1 mM Suc (OMe) -Ala-Ala-Pro-Val-MCA (manufactured by Peptide Institute, Ltd.) was dissolved in 7.96 mL of the above buffer solution. The Suc (OMe) -Ala-Ala-Pro-Val-MCA solution was diluted 10-fold with 200 mM Tris-HCl buffer.

Next, 100 μL of 1 wt% aqueous solutions of Examples 1 and 2 and Comparative Example 1 and 50 μL of elastase solution were added to a 96-well plate, and the mixture was incubated at 37 ° C. for 20 minutes. Then, 50 μL of a 10-fold diluted solution of the above Suc (OMe) -Ala-Ala-Pro-Val-MCA solution was added, and the mixture was incubated at 37 ° C. for 30 minutes. After that, the fluorescence intensity was measured (measurement excitation wavelength 360 nm, measurement fluorescence wavelength 460 nm). The increase in fluorescence in the control (water) was defined as 100%, and the effect of suppressing the increase in fluorescence in each sample was defined as the elastase inhibitory activity (%). The results are shown in Table 2.

From Table 2, it was clarified that the sericin aqueous solutions of Examples 1 and 2 showed higher elastase inhibitory activity than the low molecular weight sericin aqueous solution of Comparative Example 1.

Test Example 4
An effect confirmation test was conducted in humans.

Specifically, as subjects, 10 men aged 20 to 60 years were allowed to use the external preparations for the skin of the test product and the comparative product twice a day (morning and night) for one consecutive month, and the condition of the application site. Was compared before and after the test to investigate the improvement effect. In this test, the external preparation for skin shown in Formulation Example 1 was used as a test product (Example 3), and for the comparative product, the skin was replaced with purified water by removing the sericin aqueous solution from the external preparation for skin shown in Formulation Example 1. An external preparation was prepared (Comparative Example 2), and the effect of its use was investigated. The evaluation was performed according to the criteria shown in Table 3. While using an external preparation for skin containing sericin every day, the condition of the skin was tabulated by questionnaire before the start of application and after application for 1 month, and the effect was confirmed. The results are shown in Table 4.

As shown in Table 4, the test product containing sericin for external use on the skin was found to have a tendency to increase the elasticity of the skin as compared with the comparative product in 7 out of 10 patients. Therefore, the sericin of the present invention was added. The high effect of doing so was demonstrated.

Next, a formulation example containing the sericin of the present invention will be shown, but the present invention is not limited thereto. Hereinafter, a prescription example of the external preparation for skin according to the present invention will be shown. The content is mass%. The manufacturing method is a conventional method.

<Prescription Example 1> Toner 1,3-butylene glycol ・ ・ ・ 6.00
Glycerin ・ ・ ・ 2.00
Polyoxyethylene oleyl ether (20E.0.) ... 0.15
Ethanol ・ ・ ・ 4.00
Example 1 Aqueous solution ... 10.0
Methylparaben ・ ・ ・ 0.2
Purified water ・ ・ ・ Total balance ・ ・ ・ 100

<Prescription Example 2> Emulsion Example 1 Aqueous solution ... 1.0
Squalene ... 6.0
Vaseline ・ ・ ・ 2.0
Polyoxyethylene oleyl ether (20E.0.) ... 2.0
Carboxyvinyl polymer ・ ・ ・ 0.2
1,3-butylene glycol ・ ・ ・ 0.5
Sodium hydroxide ・ ・ ・ 0.1
Ethanol ・ ・ ・ 4.0
Methylparaben ・ ・ ・ 0.2
Purified water ・ ・ ・ Total balance ・ ・ ・ 100

<Prescription Example 3> Cream Example 1 Aqueous solution ... 1.0
Vaseline ・ ・ ・ 2.0
Stearyl alcohol ・ ・ ・ 5.0
Stearic acid ・ ・ ・ 8.0
Squalene ・ ・ ・ 10.0
Self-emulsifying glyceryl monostearate ・ ・ ・ 3.0
Polyoxyethylene cetyl ether (20E.0.) ... 1.0
Propylene glycol ・ ・ ・ 5.0
Sodium hydroxide ・ ・ ・ 0.3
Methylparaben ・ ・ ・ 0.2
Purified water ・ ・ ・ Total balance ・ ・ ・ 100

Since the elastase inhibitor of the present invention can exhibit high elastase inhibitory activity, it can be suitably used, for example, for improving and / or preventing wrinkles and sagging of the skin.

Claims (5)

An elastase inhibitor containing sericin in which at least one band is detected in a range corresponding to a molecular weight of 150 kDa or more by the SDS-PAGE method. An elastase inhibitor containing sericin obtained by a method comprising the step of extracting from silkworm cocoons or raw silk with an inorganic salt-containing solvent. The elastase inhibitor according to claim 1 or 2, wherein sericin is a sericin that forms a gel at 4 ° C. within 12 hours by adding 10% by mass of ethanol to an aqueous solution having a concentration of 0.5% by mass. An external preparation for skin containing the elastase inhibitor according to any one of claims 1 to 3. The agent according to claim 4, which is for improving or preventing wrinkles and / or sagging.