Skin topical agent composition

Abstract

To provide a skin topical agent composition containing a component highly effective in alleviating xerosis.SOLUTION: A skin topical agent composition comprises a heparin-like substance having a molecular weight distribution as determined by size-exclusion high-performance liquid chromatography at a measurement wavelength of 210 nm, the molecular weight distribution being as follows. (A) The proportion of components with a molecular weight of 20,000 or less is 35 to 70 mol%. (B) The proportion of components with a molecular weight exceeding 20,000 is 30 to 65 mol%.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition for external use on the skin containing a heparin-like substance.

Background Art

[0002] Heparin-like substances are known to have effects such as promoting blood flow, anti-inflammatory effects, moisturizing effects, and improving the skin structure, and are widely used in cosmetics, quasi-drugs for external use, or pharmaceuticals for external use. Among the above-mentioned pharmacological effects, skin care compositions having even higher moisturizing effects and effects of improving xerosis have been developed. There are formulations that combine heparin-like substances with other active ingredients (tocopherol or its derivatives) (Patent Document 1), and plant extract components that enhance the skin's moisturizing ability and barrier function by enhancing ceramide production have been proposed (Patent Document 2). However, development focusing on the molecular weight of heparin-like substances has not been carried out so far.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a composition for external use on the skin containing a component having high moisturizing and xerosis-improving effects.

Means for Solving the Problems

[0005] As a result of intensive studies to achieve the above object, the present inventors have found that in size exclusion high performance liquid chromatography with a measurement wavelength of 210 nm, a heparin-like substance having a specific molecular weight distribution enhances the expression level of the gene Claudin-1 related to the moisturizing function, and have found that it is excellent in moisturizing and improving dry skin, thus leading to the completion of the present invention.

[0006] Accordingly, the present invention provides the following external preparation composition for skin. 1. An external preparation composition for skin containing a heparin-like substance having the following molecular weight distribution in the molecular weight distribution by size exclusion high performance liquid chromatography with a measurement wavelength of 210 nm. (A) The proportion of a molecular weight of 20,000 or less is 35 to 70 mol% (B) The proportion of a molecular weight exceeding 20,000 is 30 to 65 mol% 2. The external preparation composition for skin according to 1, wherein the (B) has the following molecular weight distribution. (B1) Molecular weight exceeding 20,000 and 40,000 or less: 10 to 63 mol% (B2) Molecular weight exceeding 40,000: 2 to 20 mol% 3. The external preparation composition for skin according to 2, having the following molecular weight distribution in the molecular weight distribution. (A) Molecular weight 20,000 or less: 35 to 60 mol% (B1) Molecular weight exceeding 20,000 and 40,000 or less: 20 to 60 mol% (B2) Molecular weight exceeding 40,000: 5 to 20 mol% 4. The external preparation composition for skin according to any one of 1 to 3, wherein the weight average molecular weight of the heparin-like substance by size exclusion high performance liquid chromatography with a measurement wavelength of 210 nm is 7,000 to 40,000.

Effects of the Invention

[0007] According to the present invention, it is possible to provide an external preparation composition for skin containing components having high moisturizing and dry skin improving properties.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

Embodiments for Carrying Out the Invention

[0009] Hereinafter, the present invention will be described in detail. Heparinoid is a polysulfated mucopolysaccharide such as chondroitin polysulfate, and is known to have a moisturizing effect, an effect of improving dry skin, an effect of increasing blood flow, and the like.

[0010] The origin of the heparinoid used in the present invention is not particularly limited. For example, those obtained by polysulfating mucopolysaccharides, those extracted from the tissues of edible animals (for example, lungs including tracheal cartilage of cows, pigs, etc.), and the like can be mentioned.

[0011] The heparinoid of the present invention is a heparinoid having the following molecular weight distribution in the molecular weight distribution by size exclusion high performance liquid chromatography at a measurement wavelength of 210 nm. When the total amount of the heparinoid is 100 mol%, (A) The proportion of molecules with a molecular weight of 20,000 or less is 35 to 70 mol% (B) The proportion of molecules with a molecular weight exceeding 20,000 is 30 to 65 mol% That is. A heparinoid having such a molecular weight distribution enhances the expression level of the gene Claudin-1 related to the moisturizing function and is excellent in moisturizing and improving dry skin (A) The proportion of molecules with a molecular weight of 20,000 or less is preferably 35 to 60 mol%, more preferably 40 to 55 mol%. (B) The proportion of molecules with a molecular weight exceeding 20,000 is preferably 40 to 65 mol%, more preferably 45 to 60 mol%.

[0012] In particular, the range of (B) is (B1) Over 20,000 and 40,000 or less in molecular weight: 10 to 63 mol% is preferred, 20 to 60 mol% is more preferred, and 30 to 55 mol% is even more preferred. (B2) Over 40,000 in molecular weight: 2 to 20 mol% is preferred, 5 to 20 mol% is more preferred, and 5 to 15 mol% is even more preferred.

[0013] The weight-average molecular weight of the heparin-like substance is preferably 7,000 to 40,000, more preferably 15,000 to 30,000, and even more preferably 20,000 to 28,000. In the present invention, it is not simply a matter of having a low weight-average molecular weight, but the molecular weight distribution is important. Hereinafter, the measurement of the weight-average molecular weight, the measurement and calculation of the molecular weight distribution will be described.

[0014] Details of the conditions for size exclusion high performance liquid chromatography at a measurement wavelength of 210 nm are based on the examples described later, and are as follows. Detector: PDA (210 nm) Column: OHpak SB-806M HQ manufactured by Shodex, particle size 13 μm, inner diameter 8 mm × length 300 mm, or equivalent thereto Column temperature: Constant temperature around 40 °C Mobile phase: 0.1 mass% aqueous NaCl solution Standard substance: Pullulan (Standard P-82 for Shodex Standard GFC (aqueous GPC) column), or equivalent thereto

[0015] [Calculation of weight-average molecular weight] Let the molecular weight of the component eluting at time \(t\) be \(M(t)\), and the Intensity at that time be \(I(t)\). Since the heparin - like substance has a certain repeating structure, its molar extinction coefficient is proportional to the molecular weight. Also, from Lambert - Beer's law, since the absorbance is proportional to the molar extinction coefficient and the compound concentration, the number of compound molecules eluting at time \(t\) can be expressed as \(N(t)=I(t) / kM(t)\) (\(k\) is the proportionality constant). By integrating \(N(t)\) over the time period when the peak occurs, the total number of substances can be obtained. Since the total area value of the peak (the integral value of \(I(t)\)) is proportional to the total weight of the substance, \(\sum I(t) / \sum kN(t)\) gives the average molecular weight. The molecular weight of the component eluting at each Retention Time is determined from the calibration curve. The sample used is desalted and freeze - dried.

[0016] [Calculation of molecular weight distribution abundance ratio (mol%)] Using \(\sum N(t)\) in the time period when the peak occurs as the denominator, divide \(M(t)\) within the range into molecular weights of 20,000 or less, greater than 20,000 and 40,000 or less, and greater than 40,000, and calculate the total value of \(N(t)\) in each molecular weight range.

[0017] The heparin - like substance of the present invention enhances the expression level of the gene Claudin - 1 related to the moisturizing function. This evaluation method will be described in detail in the examples. The present invention improves the mRNA transcription level of the gene Claudin - 1 related to moisturizing in an experimental system of monolayer two - dimensional cell culture where percutaneous absorption can be ignored. That is, in this evaluation system, the effect due to percutaneous absorption is excluded, and the effect of the present invention is not due to the improvement of percutaneous absorption. Claudin - 1 is a tight junction factor present in epidermal keratinocytes. Since an increase in the mRNA amount of the tight junction factor involved in the skin barrier function can be obtained, not only the moisturizing and xeroderma improvement effects, but also the improvement effect on sensitive skin and chronic itching improvement can be expected.

[0018] The production method of the heparin-like substance of the present invention is not particularly limited, as long as the desired molecular weight distribution can be obtained. For example, it can be obtained by subjecting a known heparin-like substance to strong alkali treatment. As the treatment water used for the strong alkali treatment, for example, a 2 (mol / L) sodium hydroxide aqueous solution or the like may be used. The treatment temperature and time are appropriately selected from, for example, 60°C, 5 to 8 hours, etc., depending on the heparin-like substance as the raw material, the desired molecular weight distribution, etc.

[0019] In addition, the heparin-like substance of the present invention can also be obtained by mixing the heparin-like substance before treatment, the heparin-like substance subjected to strong alkali treatment, and the fractionated heparin-like substance.

[0020] From the viewpoint of the moisturizing effect, the content of the heparin-like substance of the present invention is preferably 0.1% by mass or more, more preferably 0.1 to 0.4% by mass, and even more preferably 0.1 to 0.3% by mass in the skin external preparation composition.

[0021] [Other components] In addition to the above-mentioned heparin-like substance of the present invention, the skin external preparation composition of the present invention may be blended with various active ingredients (sometimes referred to as other active ingredients) used in pharmaceuticals, quasi-drugs, cosmetics, etc., as long as the effects of the invention are not impaired as necessary. Examples of other active ingredients include anti-inflammatory agents, antihistamines, antipruritics, wound healing agents, local anesthetics, vitamins, moisturizers, cooling agents, bactericides, vasoconstrictors, amino acids, etc. These can be used in appropriate amounts alone or in combination of two or more.

[0022] Examples of anti-inflammatory agents include, for example, glycyrrhetinic acid and pharmaceutically acceptable salts thereof (e.g., glycyrrhetinic acid, stearyl glycyrrhetinate, etc.), glycyrrhizic acid and pharmaceutically acceptable derivatives thereof, tranexamic acid and pharmaceutically acceptable derivatives thereof, placenta, licorice extract, steroid compounds (hydrocortisone, prednisolone, methylprednisolone, clobetasone, betamethasone, dexamethasone, cortisone, fluocinolone acetonide, beclomethasone, fluticasone and pharmaceutically acceptable derivatives thereof), indomethacin, ibuprofen, ibuprofen piconol, bufexamac, ufenamate, piroxicam, ketoprofen, salicylic acid and pharmaceutically acceptable derivatives thereof, dimethylisopropylazulene, toki extract, shikonin extract, etc. When formulating an anti-inflammatory agent, the content is preferably 0.001 to 15% by mass in the topical skin composition.

[0023] Examples of antihistamines include, for example, diphenhydramine, chlorpheniramine, mequitazine, azelastine, emedastine, ketotifen and pharmaceutically acceptable salts or pharmaceutically acceptable derivatives thereof, etc. Preferably, diphenhydramine hydrochloride, chlorpheniramine maleate, etc. are mentioned. When formulating an antihistamine, the content is preferably 0.01 to 15% by mass in the topical skin composition, and more preferably 0.01 to 5% by mass.

[0024] Examples of antipruritics include, for example, crothiamide, salicylic acid and pharmaceutically acceptable derivatives thereof, nonivamide, capsaicin, benzyl nicotinate, capsicum tincture, etc. When formulating an antipruritic, the content is preferably 0.001 to 10% by mass in the topical skin composition.

[0025] Examples of wound healing agents include, for example, allantoin, zinc oxide, zinc chloride, etc. When formulating a wound healing agent, the content is preferably 0.1 to 30% by mass in the topical skin composition.

[0026] Examples of local anesthetics include lidocaine and its pharmaceutically acceptable salts, dibucaine and its pharmaceutically acceptable salts, procaine, methyl aminobenzoate, ethyl aminobenzoate, and the like. When formulating a local anesthetic, the content is preferably 0.001 to 10% by mass in the topical composition for skin.

[0027] Examples of vitamin agents include, for example, vitamin A compounds [retinol and its pharmaceutically acceptable derivatives (e.g., retinal, retinoic acid, retinol palmitate, vitamin A oil, etc.)], vitamin B1 compounds [thiamine and its pharmaceutically acceptable derivatives (e.g., thiamine hydrochloride, thiamine nitrate, etc.)], vitamin B2 compounds [riboflavin and its pharmaceutically acceptable derivatives (e.g., riboflavin phosphate, sodium riboflavin phosphate, riboflavin butyrate, and flavin adenine dinucleotide sodium, etc.)], vitamin B3 compounds [nicotinic acid and its pharmaceutically acceptable derivatives (nicotinamide, nicotinic acid tocopherol, benzyl nicotinate, etc.)], vitamin B5 compounds [pantothenic acid and its pharmaceutically acceptable derivatives (e.g., calcium pantothenate, panthenol, pantothenyl ethyl alcohol)], vitamin B6 compounds [pyridoxine, pyridoxal and pharmaceutically acceptable salts], vitamin B 12Classes [cobalamins and pharmaceutically acceptable derivatives thereof (e.g., cyanocobalamin, mecobalamin, and hydroxocobalamin hydrochloride, etc.)], biotin, folic acid and pharmaceutically acceptable salts thereof, vitamin C [ascorbic acid and pharmaceutically acceptable derivatives thereof (e.g., erythorbic acid, sodium ascorbate, palmitic acid ascorbic acid, L-ascorbic acid-2-glucoside, etc.)], vitamin D [calciferol and pharmaceutically acceptable derivatives thereof (e.g., ergocalciferol, cholecalciferol, etc.)], vitamin E [tocopherol, ubiquinone and pharmaceutically acceptable derivatives thereof (e.g., tocopherol acetate, succinic acid tocopherol calcium, etc.)], other vitamins (e.g., hesperidin, carnitine, ferulic acid, γ-oryzanol, orotic acid, rutin, eriocitrin, inositol, and pharmaceutically acceptable salts thereof), etc. may be mentioned. When formulating a vitamin agent, the content is preferably 0.01 to 20% by mass, more preferably 0.01 to 10% by mass, and even more preferably 0.02 to 3% by mass in the topical skin composition.

[0028] Examples of the humectant include petrolatum, polyhydric alcohols (glycerin, 1,3-butylene glycol, etc.), hyaluronic acid and pharmaceutically acceptable derivatives thereof, high molecular compounds (collagen, chitosan, etc.), amino acids (glycine, alanine, aspartic acid, etc.), natural moisturizing factors (sodium lactate, urea, etc.), ceramide, plant extracts (chamomile extract, aloe extract, etc.). When formulating a humectant, the amount is preferably 0.001 to 15% by mass in the topical skin composition.

[0029] Examples of the cooling agent include l-menthol, camphor, borneol and related substances thereof, perilla oil, eucalyptus oil, peppermint oil, etc.

[0030] Examples of the bactericide include isopropylmethylphenol, chlorhexidine hydrochloride, benzethonium chloride, benzalkonium chloride, cetrimide, chlorhexidine gluconate, resorcinol, hinokitiol, etc.

[0031] Examples of the vasoconstrictor include naphazoline, tetrahydrozoline, methylephedrine, and pharmaceutically acceptable salts thereof.

[0032] Examples of the amino acids include glutamic acid, aspartic acid, glycine, alanine, serine, aminoethylsulfonic acid (taurine), and pharmaceutically acceptable salts thereof. When amino acids are formulated, the amount is preferably 0.001 to 10% by mass in the topical skin preparation composition. By containing 0.001% by mass in the topical skin preparation composition, a moisturizing effect can be obtained, and thus the usability (moist feeling and residual feeling on the skin) is further improved.

[0033] The skin preparation composition of the present invention can be prepared by formulating the heparin-like substance of the present invention and, if necessary, other active ingredients together with various additives described below according to a conventional method. Any components used as pharmaceutical additives and cosmetic raw materials that can be formulated in external preparations can be used for preparing the skin preparation composition of the present invention.

[0034] Examples of the other optional components (various additives) include oily agents, surfactants, polyhydric alcohols (including those overlapping with the above), high molecular compounds (including those overlapping with the above), stabilizers, preservatives, pH adjusters, fragrances, water, and the like.

[0035] Examples of the oily agent include hydrocarbons such as petrolatum, paraffin, liquid paraffin, squalane, ceresin, gelled hydrocarbon, and microcrystalline wax; higher fatty acids such as stearic acid, isostearic acid, myristic acid, oleic acid, and behenic acid; higher fatty alcohols such as cetanol, stearyl alcohol, cetostearyl alcohol, octyldodecanol, and behenyl alcohol; fatty acid esters such as isopropyl myristate, octyldodecyl myristate, isopropyl palmitate, and diisopropyl adipate; and triglyceride fatty acid glycerides such as glyceryl triisooctanoate and glyceryl tri(caprylyl / caprate). The amount when formulating the oily agent is preferably 0.05 to 30% by mass in the topical composition for skin.

[0036] Examples of the surfactant include nonionic surfactants, ionic surfactants, and the like. Examples of nonionic surfactants include glycerin fatty acid esters such as glyceryl monostearate and glyceryl monooleate; polyglycerin fatty acid esters such as polyglyceryl-10 monolaurate, polyglyceryl-10 monostearate, polyglyceryl-10 monooleate, and polyglyceryl-10 pentaoleate; polyoxyethylene glycerin fatty acid esters such as polyoxyethylene(15) glyceryl monostearate; sorbitan fatty acid esters such as sorbitan monostearate, sorbitan monooleate, and sorbitan sesquioleate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene(20) sorbitan monolaurate (polysorbate 20), polyoxyethylene(20) sorbitan monostearate (polysorbate 60), and polyoxyethylene(20) sorbitan monooleate (polysorbate 80); polyoxyethylene sorbit fatty acid esters such as polyoxyethylene(6) sorbit monolaurate, polyoxyethylene(60) sorbit tetrastearate, and polyoxyethylene(60) sorbit tetraoleate; polyoxyethylene castor oil such as polyoxyethylene(60) castor oil; polyoxyethylene hydrogenated castor oil such as polyoxyethylene(10) hydrogenated castor oil and polyoxyethylene(60) hydrogenated castor oil; polyethylene glycol fatty acid esters such as polyethylene glycol(10EO) monolaurate, polyethylene glycol(10EO) monooleate, polyethylene glycol(40EO) monostearate, polyethylene glycol(45EO) monostearate, and polyethylene glycol(55EO) monostearate; polyoxyethylene alkyl ethers such as polyoxyethylene(9) lauryl ether (lauromacrogol), polyoxyethylene(10) cetyl ether, and polyoxyethylene(15) oleyl ether; and polyoxyethylene alkyl ethers such as polyoxyethylene(20) polyoxypropylene(4) cetyl ether and polyoxyethylene(20) polyoxypropylene(8) cetyl ether.In addition, the numerical value at the end in the notation of the polyglycerol fatty acid ester exemplified above represents the average degree of polymerization of glycerin, and the numerical value within parentheses in the notation of the nonionic surfactant exemplified above represents the average degree of polymerization of ethylene oxide or propylene oxide, that is, the average number of repetitions of oxyethylene groups or oxypropylene groups.

[0037] Examples of the anionic surfactant include sodium lauryl sulfate, sodium cetyl sulfate, etc. The amount when formulating the surfactant is preferably 0.1 to 10% by mass in the topical skin composition.

[0038] Examples of the polyhydric alcohol include glycerin, propylene glycol, 1,3 - butylene glycol, and polyethylene glycol (macrogol 300, macrogol 400, macrogol 1500, macrogol 4000, macrogol 6000, macrogol 20000), etc. Among them, from the viewpoint of the usability (ease of spreading) of the topical skin composition, glycerin, propylene glycol, 1,3 - butylene glycol, and macrogol 400 are preferable. The amount when formulating the polyhydric alcohol is preferably 0.05 to 40% by mass in the topical skin composition, and more preferably 1 to 30% by mass.

[0039] Examples of the high - molecular compound include carboxyvinyl polymer, hydroxypropyl cellulose, hypromellose, hydrophobized hydroxypropyl methylcellulose, acrylated starch 300, polyvinyl pyrrolidone, gellan gum, gum arabic, karaya gum, xanthan gum, carob gum, guar gum, guaiac resin, quince seed, damar gum, tragacanth, benzoin gum, locust bean gum, casein, agar, alginic acid, dextrin, dextran, carrageenan, gelatin, collagen, pectin, starch, polygalacturonic acid, chitin and its derivatives, chitosan and its derivatives, elastin, heparan sulfate, hyaluronic acid, chondroitin sulfate, etc. The amount when formulating the high - molecular compound is preferably 0.01 to 5% by mass in the topical skin composition.

[0040] Examples of stabilizers include sodium chloride, sodium thiosulfate, sodium sulfite, sodium edetate, boric acid, borax, etc. Among them, boric acid, borax, and sodium edetate can be preferably used. The amount when formulating a stabilizer is preferably 0.01 to 5% by mass, more preferably 0.02 to 1% by mass in the skin external preparation composition.

[0041] Examples of preservatives include methyl paraben, propyl paraben, butyl paraben, sodium benzoate, chlorobutanol, phenoxyethanol, dibutylhydroxytoluene, benzalkonium chloride, and benzethonium chloride, etc. The amount when formulating a preservative is preferably 0.01 to 5% by mass, more preferably 0.05 to 1% by mass in the skin external preparation composition.

[0042] Examples of pH adjusters include diisopropanolamine, triethanolamine, sodium hydroxide, hydrochloric acid, citric acid, sodium citrate, lactic acid, sodium lactate, malic acid, sodium malate, potassium dihydrogen phosphate, and sodium hydrogen phosphate, etc. The amount when formulating a pH adjuster may be an amount to achieve the target pH. For example, it is preferably 0.01 to 5% by mass, more preferably 0.05 to 1% by mass in the skin external preparation composition.

[0043] Examples of the fragrance include compound fragrances such as citrus fragrance, floral fragrance, and rose fragrance, and essential oils such as eucalyptus oil, bergamot oil, star anise oil, rose oil, mint oil, peppermint oil, spearmint oil, rosemary oil, lavender oil, and lemon oil. Representative components contained in these fragrances include terpenoid compounds. Terpenoid compounds include terpene hydrocarbons, terpene alcohols, terpene aldehydes, and terpene ketones. Also, depending on the number of carbon atoms, there are monoterpenes, sesquiterpenes, diterpenes, triterpenes, and tetraterpenes, but the most representative component is monoterpene. Preferred terpenoid compounds include l-menthol, dl-menthol, d-camphor, dl-camphor, d-borneol, dl-borneol, citral, limonene, citronellol, geraniol, cineol, linalool, nonanal, eugenol, iso-eugenol, indole, benzaldehyde, vanillin, limonene, galaxolide, γ-nonalactone, γ-methyl ionone, iso-E-super, Z-3-hexenyl salicylate, γ-undecalactone, 5-cyclohexadecene-1-one, 2-methyl-4-phenyl-2-butanol, methyl dihydrojasmonate, methyl-β-naphthyl ketone, cinnamyl acetate, benzyl acetate, α-terpineol, hexyl cinnamic aldehyde, γ-undecalactone, etc. The amount in the case of blending the fragrance is preferably 0.001 to 1% by mass, more preferably 0.01 to 0.1% by mass in the skin external preparation composition.

[0044] The ratio in the case of blending water is appropriately selected from the range of 50 to 98% by mass in the skin external preparation composition according to the dosage form.

[0045] [Manufacturing method] The skin external preparation composition of the present invention can be obtained, for example, by mixing the heparin-like substance of the present invention and, if necessary, any components (other active ingredients, various additives).

[0046] [Skin external preparation composition] The property of the topical skin preparation composition of the present invention may be any of liquid, solid, semi-solid (gel, ointment, paste, foam, etc.). Further, it may be in any dosage form such as topical pharmaceutical products, quasi-drugs for external use on the skin, cosmetics, skin cleansers, etc.

[0047] The dosage form of the topical skin preparation composition of the present invention is not particularly limited. For example, ointments, creams, lotions, gels, emulsions, liquids, patches, sprays that spray in mist form, pump foams that spray in foam form, aerosol agents that spray in mist, powder, foam or paste form, pack agents, etc. may be mentioned. Among them, liquids, gels, emulsions, creams, pump foams or aerosol agents are preferred. In addition, the emulsified state of creams, emulsions, etc. is not particularly limited, and it may be any of W / O, O / W, W / O / W, O / W / O.

[0048] The upper limit value of the pH of the topical skin preparation composition of the present invention at 20°C is preferably 8 or less, more preferably 7.5 or less, and even more preferably 7 or less. The lower limit value of the pH is preferably 3 or more, preferably 4 or more, and even more preferably 5 or more. Further, the lower limit value and the upper limit value of the pH are preferably 3 to 7.5, more preferably 4 to 7, and even more preferably 5 to 7. In the present invention, the pH is measured according to the General Test Methods of the Japanese Pharmacopoeia, 18th Revision (measurement temperature: 20°C).

[0049] The topical skin preparation composition of the present invention is not particularly limited as long as it is for external use, and the skin includes the scalp.

[0050] The skin external preparation composition of the present invention is a skin external medicine having effects and efficacy such as xeroderma, dry skin of children, rough fingers, cracks and red streaks on hands and feet, keratosis of elbows, knees, heels and ankles, heat rash (excluding sagging), scarring and tightening of the skin after wounds and burns (excluding the face), swelling, muscle pain and joint pain after bruises and sprains, etc., and prevents skin roughness, roughness, sweating, heat rash, cracks, red streaks, acne, prevents oily skin and razor burn, prevents freckles and chloasma caused by sunburn, prevents flushing after sunburn and snowburn, tightens the skin, cleanses the skin, improves the skin, keeps the skin healthy, moisturizes the skin, protects the skin, prevents skin dryness, improves the texture of the skin, prevents skin roughness, replenishes and maintains the moisture and oil content of the skin, maintains the flexibility of the skin, softens the skin, gives firmness to the skin, makes the skin smooth, improves the skin after shaving, prevents sunburn, prevents freckles and chloasma caused by sunburn, and makes fine wrinkles caused by dryness less noticeable, etc. It can be used for quasi-drugs and cosmetics having such effects and efficacy. In addition to these, effects such as "improvement of wrinkles, dullness, firmness, and enlarged pores", "maintenance of skin moisture and lasting moisture", "improvement of skin structure", "improvement of barrier function", and "restoration of water retention capacity of aging skin" can also be obtained. Since the skin external preparation composition of the present invention contains the heparin-like substance of the present invention, in particular, it has high effects on xeroderma, dry skin of children, moisturizing the skin, preventing skin dryness, improving the texture of the skin, preventing skin roughness, making fine wrinkles caused by dryness less noticeable, maintaining skin moisture and lasting moisture, improving skin structure, and improving barrier function.

Example

[0051] Hereinafter, examples and comparative examples will be shown to specifically explain the present invention, but the present invention is not limited to the following examples. In the following examples, unless otherwise specified, "%" of the composition indicates mass%, and the amounts of each component in the table are amounts in terms of pure components.

[0052] [Preparation method of Sample A] 50 mg of a heparin analogue (manufactured by Tokyo Riki Co., Ltd., an unauthorized “heparin analogue”) was dissolved in 2.5 mL of purified water and desalted using PD-10 (a gravity / centrifugation column for desalting and buffer exchange, manufactured by Cytiva). Thereafter, it was frozen at -80°C and then a dried sample was obtained using a freeze dryer.

[0053] [Method for preparing Samples B and C] 100 mg of a heparin analogue (manufactured by Tokyo Riki Co., Ltd., an unauthorized “heparin analogue”) was dissolved in 25 mL of a 2M aqueous sodium hydroxide solution. It was heated in an incubator at 60°C for 5 hours (Sample B) and 7.5 hours (Sample C) with different reaction times respectively. After cooling to room temperature, it was adjusted to pH 5 - 8 with 10% hydrochloric acid while stirring. Ethanol was added so that the volume percentage of ethanol became 60% by volume, and the treated heparin analogue was precipitated. Thereafter, centrifugation was performed at 15,000 rpm for 10 minutes. After removing the supernatant, it was dried at room temperature. The obtained dried product was dissolved in 2.5 mL of purified water and desalted using PD-10 (a gravity / centrifugation column for desalting and buffer exchange, manufactured by Cytiva). Thereafter, it was frozen at -80°C and then a dried sample was obtained using a freeze dryer.

[0054] Regarding Samples A, B, and C obtained above, water was added to make it 0.5 w / v%, and the following measurements and evaluations were performed. [Size exclusion high performance liquid chromatography at a measurement wavelength of 210 nm] Detector: PDA (210 nm) Column: Shodex OHpak SB-806M HQ, particle size 13 μm, inner diameter 8 mm × length 300 mm Column temperature: A constant temperature near 40°C Mobile phase: 0.1 mass% aqueous NaCl solution Standard substance: Pullulan (Standard P-82 for Shodex Standard GFC (aqueous GPC) column)

[0055] [Calculation of weight average molecular weight] Data on each Retention Time (minutes) and I(t) which is Intensity were obtained from the chromatogram by the above measurement. Based on the calibration curve obtained using pullulan as a standard substance, the molecular weight M(t) was calculated from the Retention time (minutes) of each sample (M(t) = 8.703×10 8 ×e (-1.184t) , where e is the base of the natural logarithm.). Next, kN(t) = I(t) / M(t) was calculated (k is a proportionality constant), and the weight-average molecular weight was calculated by ΣI(t) / ΣkN(t).

[0056] [Calculation of Molecular Weight Distribution] Using ΣN(t) in the time zone where peaks occur as the denominator, and dividing the M(t) within the range into molecular weights of 20,000 or less, more than 20,000 and 40,000 or less, and more than 40,000 respectively, the total value of N(t) in each molecular weight range was calculated.

[0057] The weight-average molecular weight and molecular weight distribution are shown in Table 1 below. The size exclusion high performance liquid chromatography charts of Samples A, B, and C are shown in Figure 1, and the graphs showing the molecular weight distributions of Samples A, B, and C are shown in Figure 2.

[0058]

Table 1

[0059] [mRNA Transcription Level Evaluation Test] An increase in the mRNA amount of Claudin-1, a tight junction factor present in epidermal keratinocytes, was confirmed. Normal human epidermal keratinocytes (NHEK) were seeded in a 96-well plate at 2×10 4 cells / well. Two days after seeding the cells, the medium was replaced with a sample adjusted to a predetermined concentration in the medium, and the cells were cultured at 37°C and 5% CO2 in a humidified state for 24 hours. The concentrations of the test substances (Samples A to C) were adjusted to a concentration of 0.1% by mass. Subsequently, RNA was extracted from the cells for each sample using the Superprep Cell Lysis & RT Kit for qPCR (TOYOBO SCQ-101). After performing a reverse transcription reaction using a known method to obtain cDNA, it was quantified using a real-time PCR apparatus (reverse transcription; ReverTra Ace2, TOYOBO, Polymerase; KOD SYBR qPCR mix, TOYOBO). The sequences of the primers used are shown below. The amount of Claudin-1 mRNA was calculated as the target gene and corrected with the amount of GAPDH mRNA as the reference gene.

[0060] [Table 2]

[0061] The results are shown in the following table together with the growth rate (%) = [(mRNA amount of sample B or C) / (mRNA amount of sample A) - 1] × 100 with respect to the results of sample A as the reference.

[0062] [Table 3]

[0063] From the above results, the heparin-like substance of the present invention having a specific molecular weight distribution enhanced the expression level of the gene Claudin-1 related to the moisturizing function.

[0064] As a skin external preparation composition containing the heparin-like substance of the present invention, an aqueous cream (O / W preparation), an oil-in-water emulsion (W / O preparation), and a lotion of the following formulation were prepared according to a conventional method. The obtained products were excellent in moisturizing and improving dry skin.

[0065] [Table 4]

[0066] [Table 5]

[0067]

Table 6

[0068] The raw materials used in preparing the examples, comparative examples, and formulation examples are shown below. Note that the descriptions "Japanese Pharmacopoeia", "Non-Pharmacopoeial Drugs", "Pharmaceutical Additives", and "External Ingredients" below mean that the raw materials conform to the 18th Revised Japanese Pharmacopoeia Standards, Japanese Pharmacopoeia Non-Pharmacopoeial Drugs Standards, Pharmaceutical Additives Standards (2018), and External Ingredients for Quasi-Drugs Standards (2021), respectively. · Heparin-like substance: Non-Pharmacopoeial Drugs, Non-Pharmacopoeial Drugs Heparin-like substance, manufactured by Toiri Co., Ltd. · Pyridoxine hydrochloride: Japanese Pharmacopoeia, Pyridoxine hydrochloride, manufactured by DSM Co., Ltd. · Dipotassium glycyrrhizinate: Non-Pharmacopoeial Drugs, Dipotassium glycyrrhizinate, manufactured by Maruzen Pharmaceutical Co., Ltd. · Allantoin: Non-Pharmacopoeial Drugs, Allantoin, manufactured by Pharmachem Asia Co., Ltd. · Sodium ascorbate: Pharmaceutical Additives Standards, Sodium ascorbate, manufactured by DSM Co., Ltd. · Sodium hyaluronate: Japanese Pharmacopoeia, Purified Sodium Hyaluronate JP-E, manufactured by Bloomage Biotechnology Japan Co., Ltd. · Tocopherol acetate: Japanese Pharmacopoeia, Japanese Pharmacopoeia Tocopherol acetate, manufactured by Mitsubishi Chemical Foods Co., Ltd. · Squalane: External Ingredients for Quasi-Drugs Standards, Squalane, manufactured by Nikko Chemicals Co., Ltd. · Glycerin: Japanese Pharmacopoeia, Pharmacopoeia Glycerin, manufactured by Sakamoto Yakuhin Kogyo Co., Ltd. · Propylene glycol: Japanese Pharmacopoeia, Propylene glycol, manufactured by ADEKA Co., Ltd. · 1,3-Butylene glycol (1,3-BG): Pharmaceutical Additives Standards, 1,3-Butylene glycol, manufactured by Daicel Chemical Industries, Ltd. · Macrogol 20000: Japanese Pharmacopoeia, Macrogol 20000, manufactured by Sanyo Chemical Industries, Ltd. · Ethanol: Standards of the Alcohol Association, General Alcohol 95% Synthetic Non-Denatured, manufactured by Nippon Synthetic Alcohol Co., Ltd. · Polyoxyethylene sorbitan monostearate (Polysorbate 60): Pharmaceutical additive grade, NIKKOL TS-10MV, manufactured by Nippon Surfactant Industry Co., Ltd. · Polyoxyethylene hydrogenated castor oil 10: Pharmaceutical additive grade, HCO-10, manufactured by Nippon Surfactant Industry Co., Ltd. · Polyoxyethylene (9) lauryl ether (Lauromacrogol): Japanese Pharmacopoeia grade, NIKKOL BL-21, manufactured by Nippon Surfactant Industry Co., Ltd. · Glyceryl fatty acid ester: Food additive grade, NIKKOL Decaglyn 5-OV, manufactured by Nippon Surfactant Industry Co., Ltd. · Stearyl alcohol: Pharmaceutical additive grade, Calcocol 8098, manufactured by Kao Corporation. · Octyldodecanol: Pharmaceutical additive grade, NDJCOL 200A, manufactured by Shin Nippon Rika Co., Ltd. · Isopropyl myristate: Pharmaceutical additive grade, NIKKOL IPM-100, manufactured by Nippon Surfactant Industry Co., Ltd. · Xanthan gum: Pharmaceutical additive grade, Echo Gum T, manufactured by DSP Gohsei Food & Fine Chemical Co., Ltd. · Carbomer: Pharmaceutical additive grade, CRBOPOL980, manufactured by Noveon Co. · Sodium edetate hydrate: Japanese Pharmacopoeia grade, Sodium edetate, manufactured by Chubu Kirest Co., Ltd. · Methyl paraben: Japanese Pharmacopoeia grade, Methyl paraben, manufactured by Ueno Pharmaceutical Co., Ltd. · Propyl paraben: Japanese Pharmacopoeia grade, Propyl paraben, manufactured by Ueno Pharmaceutical Co., Ltd. · Sodium citrate: Japanese Pharmacopoeia grade, Sodium citrate hydrate, manufactured by Fuso Chemical Industry Co., Ltd. · Citric acid: Japanese Pharmacopoeia grade, Citric acid hydrate, manufactured by Three F Co., Ltd. · Diisopropanolamine: Pharmaceutical additive grade, Diisopropanolamine, manufactured by Mitsui Chemicals Fine Co., Ltd. · Borax: Japanese Pharmacopoeia grade, Borax, manufactured by Kosakai Pharmaceutical Co., Ltd. · Fragrance: Fragrances a to d described in paragraphs

[0065] to

[0071] of JP-A-2002-128658, A to E described in JP-A-2003-73249, or those appropriately selected from 1 to 4 described in

[0016] to

[0023] of Japanese Patent Application No. 2019-023940.

Sequence free text

[0069] Array number 1 in the sequence listing: Forward primer used for PCR (hClaudin-1) Array number 2 in the sequence listing: Reverse primer used for PCR (hClaudin-1) Array number 3 in the sequence listing: Forward primer used for PCR (hGAPDH) Array number 4 in the sequence listing: Reverse primer used for PCR (hGAPDH)

Claims

1. A topical skin composition comprising a heparin analogue having the following molecular weight distribution in the molecular weight distribution by size exclusion high performance liquid chromatography at a measurement wavelength of 210 nm. (A) The proportion of a molecular weight of 20,000 or less is 35 to 70 mol% (B) The proportion of a molecular weight exceeding 20,000 is 30 to 65 mol%

2. The topical skin composition according to claim 1, wherein the (B) has the following molecular weight distribution. (B1) Molecular weight exceeding 20,000 and 40,000 or less: 10 to 63 mol% (B2) Molecular weight exceeding 40,000: 2 to 20 mol%

3. The topical skin composition according to claim 2, having the following molecular weight distribution in the molecular weight distribution. (A) Molecular weight 20,000 or less: 35 to 60 mol% (B1) Molecular weight exceeding 20,000 and 40,000 or less: 20 to 60 mol% (B2) Molecular weight exceeding 40,000: 5 to 20 mol%

4. The topical skin composition according to any one of claims 1 to 3, wherein the weight average molecular weight of the heparin analogue by size exclusion high performance liquid chromatography at a measurement wavelength of 210 nm is 7,000 to 40,000.

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