KR20080026924A - Compositions containing low molecular weight hyaluronic acid or salts thereof - Google Patents

Abstract

The present invention relates to a composition containing a low molecular weight hyaluronic acid or a salt thereof having a weight average molecular weight of 500 to 10,000 daltons having a moisturizing, wrinkle improvement, wound healing and anti-inflammatory effect, and the composition of the present invention is compared with a conventional polymer hyaluronic acid. It is easy to absorb the skin and can be usefully used as a skin external preparation composition and a cosmetic composition.

Description

A composition comprising low molecular weight hyaluronic acid or the salt thereof

1 is a diagram showing the skin barrier recovery effect according to the treatment of low molecular weight hyaluronic acid or low molecular weight sodium hyaluronate salt in animal experiments,

2 is a diagram showing the cell survival rate by concentration of low molecular weight hyaluronic acid or low molecular weight sodium hyaluronate salt,

3 is a diagram showing the iNOS production inhibitory effect of low molecular weight hyaluronic acid or low molecular weight sodium hyaluronate salt.

The present invention relates to a composition containing a low molecular weight hyaluronic acid or a salt thereof having a weight average molecular weight of 500 to 10,000 Daltons.

Hyaluronic acid (hyaluronic acid) is a biopolymer of water-soluble polysaccharides with high viscosity, moisture retention, biocompatibility, etc. It is one of the three major elements of skin along with collagen and elastin. Hyaluronic acid is a linear long polymer substance composed of β-D-glucuronic acid and β-DN-acetylglucosamine formed by mutual beta linkage, and its molecular weight is 0.1 × 10 ⁶ depending on the origin, preparation and purification method. ~ 10 × 10 ⁶ Daltons, very diverse. It is viscous, elastic and moisturizing in aqueous solution, and this property depends on the molecular weight and concentration of hyaluronic acid. Among mucopolysaccharides, they are relatively easy to obtain, have unique physicochemical and physiological properties, and their own or derivatives thereof are widely used in various fields. Commonly used polymer hyaluronic acid is mainly used for ophthalmic surgery, the treatment of knee joints of race horses, as a moisturizer for cosmetics (US Patent Application No. 844,833 and US Patent Application No. 467,925) and joint diseases It is used for therapeutic agents (international patent application WO99 / 59603).

On the other hand, the molecular weight of hundreds of millions of polymer hyaluronic acid is difficult to use at high concentrations due to the high viscosity is limited in the concentration in use in pharmaceuticals such as cosmetics or injections, and has a disadvantage in that the bioavailability is lowered when used as food.

On the other hand, low molecular weight hyaluronic acid, which has a molecular weight of several thousand to tens of thousands, can be used in high concentration due to the increased solubility compared with that of the conventional high molecular weight, and has no characteristics such as non-stickiness, and the polymerization degree of sugar is 10-14 sugars. It has been reported that low molecular weight hyaluronic acid and its salts promote angiogenesis (slevin M. et al., J Biol Chem ., 25; 277 (43) , pp41046-59, 2002).

Various methods are known as low molecular weight methods of high molecular weight hyaluronic acid, using high temperature heating and ultrasonic waves (Motohashi et al., Journal of Chromatography , 435 , pp335-342, 1988; Bothner et al., Int . J. Biol . Macrmol . , P10 , 1998), a method using an acid or a base catalyst (Japanese Patent Laid-Open No. 63-57602, Japanese Patent Application Laid-Open No. 1-266102), a method using a chlorine-based oxidizing agent such as hypochlorous acid (Japan Japanese Patent Application Laid-Open No. 2-245193), a method using a decomposition catalyst such as ammonium persulfate (Korean Patent No. 10-0369517) and a method of obtaining a low molecular weight hyaluronic acid using a hyaluronidase enzyme (A. Sattor et al. , J. Invest. Dermatol . 103 , pp576-579, 1994, Japanese Patent Application Laid-Open No. 62-79790, Japanese Patent Application Laid-Open No. 11-124401) and the like.

However, none of the above documents teaches or discloses that the composition of the present invention is excellent in moisturizing, wrinkle improvement, wound healing and inflammation, and excellent skin permeability compared to high molecular weight hyaluronic acid.

Therefore, the present inventors have a low-molecular weight hyaluronic acid or its salt or a formulation containing the same, moisturizing, wrinkle improvement, wound healing and inflammation experiments, skin permeability experiments using the skin of hairless mice and Caco-2 cells Through the cell permeability experiment, the present invention was completed by confirming that the low molecular weight hyaluronic acid or salt thereof of the present invention is far superior to other compositions known to have an excellent skin permeability compared to high molecular weight hyaluronic acid.

An object of the present invention is easier to penetrate the skin than conventional high molecular weight hyaluronic acid containing a low molecular weight hyaluronic acid or a salt thereof having a weight average molecular weight of 500 ~ 10,000 Daltons or its salt as an active ingredient having wrinkle improvement, moisturizing, wound healing and anti-inflammatory effect It is to provide an external skin composition and a cosmetic composition.

The present invention is easier to penetrate the skin than polymer hyaluronic acid, moisturizing, wrinkle improvement, moisturizing, wrinkle improvement and low molecular weight hyaluronic acid having a weight average molecular weight of 500 to 10,000 Daltons or its salt as an active ingredient Provided is an external skin composition for preventing and treating wound healing and inflammation.

Low molecular weight hyaluronic acid or a salt thereof as defined herein specifically includes a compound represented by the following general formula (I) or a salt thereof.

(Ⅰ)

(Ⅰ)

N is an integer from 1 to 25;

R is a hydrogen atom or a pharmaceutically acceptable salt.

Preferred compound groups of the present invention include compound groups where n is an integer of 1 to 20, more preferably n is an integer of 1 to 10, and the R substituent includes a pharmaceutically acceptable salt.

Pharmaceutically acceptable salts of the above formula (I) include salts of acidic groups which may be present in compounds of formula (I) unless otherwise indicated, and salts of acidic groups include lithium, sodium, potassium Alkali metal salts such as alkali metal salts, magnesium, calcium, ammonia or ethylenediamine, cyclohexylamine, aniline, ethanolamine, diethanolamine, triethanolamine, ethylamine, propylamine, arylamine, diethylamine, butyl Salts with organic amines such as amine, isobutylamine, triethylamine, pentylamine, t-butylamine, and isopropylamine, and preferably salts with sodium, potassium, calcium or ammonia, and physiologically acceptable Salts with possible amines, and these salts can be prepared by methods or processes for preparing salts known in the art.

The low molecular weight hyaluronic acid or salt thereof has a weight average molecular weight of 500 to 10,000 Daltons, preferably 500 to 5,000 Daltons, more preferably 500 to 1,000 Daltons.

As a result of conducting cell experiments and animal experiments for the composition of the present invention, it was confirmed that the effect of improving wrinkles, moisturizing and inflammation, the composition of the present invention can be usefully used as a topical skin composition and cosmetic composition.

Inflammation as defined herein includes common inflammatory diseases, including various dermatitis, systemic lupus erythematosus, retinitis, gastritis, hepatitis, enteritis, pancreatitis, nephritis, preferably dermatitis, the allergy being anaphylaxis, allergy Allergic rhinitis, asthma, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, insect allergies, food allergies, drug allergies, preferably allergies Sexual dermatitis, atopic dermatitis, contact dermatitis.

The external skin pharmaceutical composition containing the composition of the present invention is a skin external preparation having wrinkle improvement, moisturizing and inflammatory effects, such as a cream, gel, patch, spray, ointment, warning agent, lotion agent, linen agent, pasta agent or cataplasmase agent. It can be prepared and used as a pharmaceutical composition in the form of external preparations for the skin, but is not limited thereto.

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

In addition, the present invention is easier to penetrate the skin than the polymer hyaluronic acid, moisturizing, wrinkles containing a low molecular weight hyaluronic acid or a salt having a weight average molecular weight of 500 to 10,000 Daltons as an active ingredient having moisturizing, wrinkle improvement, wound healing and anti-inflammatory effect It provides a cosmetic composition for the improvement, prevention of wound healing and inflammation.

It is easier to penetrate the skin than polymer hyaluronic acid, and has a low molecular weight hyaluronic acid or its salt having a weight average molecular weight of 500 to 10,000 Daltons represented by the general formula (I), which has moisturizing, wrinkle improvement, wound healing and anti-inflammatory effects as an active ingredient. Cosmetic composition for preventing and improving moisturizing, wrinkle improvement, wound healing and inflammation

The composition of the present invention can be used in a variety of cosmetics and cleansing agents and the like having moisturizing, wrinkle improvement, wound healing and improvement activities for inflammation.

Products to which the composition can be added include, for example, cosmetics such as lotion, skin, lotion, nourishing lotion, nourishing cream, massage cream, essence and pack, and cleansing, face wash, soap, treatment, essence, etc. have.

Cosmetics of the present invention comprises a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract.

The water-soluble vitamins may be any compound that can be incorporated into cosmetics, but preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H, and the like. Their salts (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-sodium phosphate salt, ascorbic acid-2-magnesium phosphate salt, etc.) may also be used in the water-soluble vitamins that can be used in the present invention. Included. The water-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microorganism culture, enzyme or chemical synthesis.

The oil-soluble vitamin may be any compound that can be formulated into cosmetics, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol), and the like. And derivatives thereof (ascorbic palmitate, ascorbic stearate, ascorbic acid dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherolvitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl) Ethers, etc.) are also included in the oil-soluble vitamins used in the present invention. Oil-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification of microorganism culture, enzyme or chemical synthesis.

The polymer peptide may be any compound as long as it can be incorporated into cosmetics. Preferably, collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin, and the like can be given. Polymeric peptides can be purified and obtained by conventional methods such as purification from microbial cultures, enzymatic methods or chemical synthesis methods, or can be purified and used from natural products such as dermis and pig silk such as pigs and cattle.

The polymer polysaccharide may be any compound as long as it can be incorporated into cosmetics. Preferably, hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt, etc.) may be mentioned. For example, chondroitin sulfate or its salt can be normally purified from a mammal or fish.

The sphingolipid may be any compound as long as it can be blended into cosmetics. Preferably, ceramide, phytosphingosine, sphingosaccharide lipid, etc. may be mentioned. Sphingolipids can usually be purified from mammals, fish, shellfish, yeasts or plants by conventional methods or obtained by chemical synthesis.

The seaweed extract may be any compound as long as it can be blended into cosmetics. Preferably, the seaweed extract may include brown algae extract, red algae extract, green algae extract, and the like. Also, calginine, arginic acid, sodium arginate, Potassium arginate and the like are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained by purification from seaweed by conventional methods.

In addition to the said essential component, you may mix | blend the cosmetics of this invention with the other components normally mix | blended with cosmetics as needed.

Other components that may be added include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavorings, Blood circulation accelerators, cooling agents, restriction agents, purified water and the like.

Examples of the fat or oil component include ester fats, hydrocarbon fats, silicone fats, fluorine fats, animal fats, and vegetable fats and oils.

As ester fats and oils, glyceryl tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl mystinate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, and stearic acid Butyl, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl acid isocetyl, isostyl acid isostearyl, isostaryl palmitate, octylate acid octyldodecyl, isostearic acid isetyl, diethyl sebacate, adipine Acid isopropyl, isoalkyl neopentane, tri (capryl, capric acid) glyceryl, trimethyl ethyl trimethylol propane, triisostearic acid trimethylol propane, tetra 2-ethylhexanoic penta erythritol Cetyl caprylate, lauric acid decyl, hexyl laurate, decyl myristin, myristin acid myristyl, myritic acid cetyl, stearyl stearate, decyl oleate, rininooleic acid Teyl, isostearyl laurate, isotridecyl myristin, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyl dodecyl oleate, octyl dodecyl linoleate, isopropyl isopropyl acid, 2-ethylhexanoate cetostearyl, 2-ethylhexanoate stearyl, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicapric acid, propylene glycol di (capryl, capric acid), Dicaprylic acid propylene glycol, dicapric acid neopentyl glycol, dioctanoate neopentyl glycol, tricaprylate glyceryl, triundecyl glyceryl, triisopalmitinate glyceryl, triisostearate glyceryl, octylate octadodode Sil, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isstearyl isostearate, Sothete octylate, polyglycerol oleate, polyglycerine isostearate, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myritic lactate, octyl lactate, octyl lactate Ethyl, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malic acid, 2-ethylhexyl hydroxystearate, di2-ethylhexyl succinate, diisobutyl adipicate, diisopropyl sebacinate, Dioctyl sebacate, cholesteryl stearate, cholesteryl isostearic acid, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, physteryl isostearate, phytic oleate, 12-Steloylhydroxystearate isocetyl, 12-Steloylhydroxystearate stearyl, 12-steal One hydroxy stearic acid and the like esters such as cetearyl source.

Hydrocarbon-based fats and oils, such as squalene, a liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, a liquid isoparaffin, polybutene, microcrystal wax, and a vaseline, etc. are mentioned as a hydrocarbon-type fats and oils.

Examples of the silicone-based oils and fats include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane, methylcetyloxysiloxane copolymer, dimethylsiloxane and methylsteoxysiloxane copolymer, and alkyl. Modified silicone oil, amino modified silicone oil and the like.

Perfluoro polyether etc. are mentioned as fluorine-based fats and oils.

Animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, soybean oil, soybean oil, corn oil, rapeseed oil, almond oil, palm kernel oil, palm oil, castor oil, sunflower oil, grape seed oil. , Cottonseed oil, Palm oil, Cucumber nut oil, Wheat germ oil, Rice germ oil, Shea butter, Walnut colostrum oil, Marker demia nut oil, Meadow home oil, Egg yolk oil, Uji, Horse oil, Mink oil, Orange rape oil, Jojoba oil And animal or plant fats and oils such as candeler wax, carnava wax, liquid lanolin and hardened castor oil.

Examples of the moisturizing agent include a water-soluble low molecular moisturizer, a fat-soluble molecular moisturizer, a water-soluble polymer, and a fat-soluble polymer.

Water-soluble low molecular humectants include serine, glutamine, sorbitol, mannitol, pyrrolidone-sodium carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n = 2 or more), polypropylene Glycol (polymerization degree n = 2 or more), polyglycerol B (polymerization degree n = 2 or more), lactic acid, lactic acid salt, etc. are mentioned.

Examples of the fat-soluble low molecular humectants include cholesterol and cholesterol esters.

Examples of the water-soluble polymer include carboxyvinyl polymer, polyasparaginate, tragacanth, xanthan gum, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, water soluble chitin, chitosan, and dextrin. Can be.

Examples of the fat-soluble polymers include polyvinylpyrrolidone-eicosene copolymers, polyvinylpyrrolidone-hexadecene copolymers, nitrocellulose, dextrin fatty acid esters, polymer silicones, and the like.

Examples of the emollient include long-chain acyl glutamic acid cholesteryl esters, hydroxy stearic acid cholesterol, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesteryl esters, and the like.

As surfactant, nonionic surfactant, anionic surfactant, cationic surfactant, amphoteric surfactant, etc. are mentioned.

Examples of nonionic surfactants are self-emulsifying glycerin monostearate, propylene glycol fatty acid esters, glycerin fatty acid esters, polyglycerol fatty acid esters, sol nonfatty acid esters, POE (polyoxyethylene) sorbitan fatty acid esters, POE sorbitan fatty acid esters, and POE. Glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hardened castor oil, POE castor oil, POE / POP (polyoxyethylene / polyoxypropylene) copolymer, POE / POP alkyl ether, polyether modified silicone, lauric acid Alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, etc. are mentioned.

Anionic surfactants include fatty acid soaps, alpha-acyl sulfonates, alkyl sulfonates, alkyl allyl sulfonates, alkyl naphthalene sulfonates, alkyl sulfates, POE alkyl ether sulfates, alkylamide sulfates, alkyl phosphates, POE alkylphosphates, and alkylamides. Phosphates, alkyloylalkyltaurine salts, N-acylamino acid salts, POE alkyl ether carboxylate salts, alkyl sulfosuccinate salts, sodium alkyl sulfo acetates, acylated hydrolyzed collagen peptide salts, perfluoroalkyl phosphate esters, and the like. have.

As cationic surfactant, alkyl trimethylammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium bromide, behenyl trimethyl ammonium chloride, chloride Benzalkonium, diethylaminoethyl stearate, dimethylaminopropyl stearate, lanolin derivatives, quaternary ammonium salts, and the like.

Examples of amphoteric surfactants include the carboxybetaine type, the amide betain type, the sulfobetain type, the hydroxysulfobetain type, the amide sulfobetain type, the phosphobetaine type, the aminocarboxylate type, the imidazoline derivative type, and the amideamine type. An amphoteric surfactant etc. are mentioned.

Organic and inorganic pigments include silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium film mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, oxide Inorganic pigments such as aluminum, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine blue, chromium oxide, chromium hydroxide, coloramine and composites thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene, styrene copolymer, Organic pigments such as silk powder, cellulose, CI pigment yellow, CI pigment orange, and composite pigments of these inorganic pigments and organic pigments;

As organic powder, Metal soaps, such as a calcium stearate; Alkyl phosphate metal salts such as sodium cetylinate, zinc lauryl acid and calcium laurate; Acylamino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroylglycine calcium; Amide sulfonic acid polyvalent metal salts, such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N-epsilon-lauroyl-L-lysine, N-epsilon-palmitolyzine, N-alpha-paratoylol nitin, N-alpha-lauroyl arginine, N-alpha-cured fatty acid acyl arginine -Acyl basic amino acid; N-acyl polypeptides, such as N-lauroyl glycyl glycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; Polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride and the like.

Examples of the ultraviolet absorber include paraaminobenzoic acid, ethyl paraaminobenzoate, amyl paraaminobenzoic acid, octyl paraaminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomentyl salicylic acid and benzyl cinnamic acid. , Paramethoxy cinnamic acid-2-ethoxyethyl, paramethoxy cinnamic acid octyl, diparamethoxy cinnamic acid mono-2-ethylhexaneglyceryl, paramethoxy cinnamic acid isopropyl, diisopropyl diisopropyl cinnamic acid ester mixture, wuro Canonic acid, ethyl urokanoate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and salts thereof, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone sodium sulfonate, dihydroxybenzophenone , Tetrahydroxybenzophenone, 4- tert -butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino- p- (carbo-2'-ethylhexyl-1'-oxy) -1 , 3,5-triazine, 2- (2-hi And the like can be mentioned hydroxy-5-methylphenyl) benzotriazole.

As fungicides, hinokithiol, trichloric acid, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, ginxitlionone, benzalkonium chloride, photosensitive Sodium No. 301, sodium mononitroguicol, undecylenic acid, and the like.

Examples of the antioxidant include butylhydroxyanisole, propyl gallic acid, and erythorbic acid.

Examples of the pH adjuster include citric acid, sodium citrate, malic acid, sodium malate, fmaric acid, sodium pmarate, succinic acid, sodium succinate, sodium hydroxide, sodium dihydrogen phosphate, and the like.

Examples of the alcohol include higher alcohols such as cetyl alcohol.

Moreover, the compounding component which may be added other than this is not limited to this, In addition, although all the said components can be mix | blended within the range which does not impair the objective and effect of this invention, Preferably it is 0.01-5% weight with respect to gross weight. Percentage, more preferably 0.01-3% by weight.

The cosmetic of the present invention may take the form of a solution, an emulsion, a viscous mixture, or the like.

Ingredients included in the cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the compound as an active ingredient, for example, conventional auxiliary agents such as stabilizers, solubilizers, vitamins, pigments and flavorings. And carriers.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, and includes, for example, milky lotion, cream, lotion, pack, foundation, lotion, essence, hair cosmetic, and the like.

Specifically, the cosmetic composition of the present invention skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, Formulations of packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers.

When the formulation of the present invention is a paste, cream or gel, animal carriers, vegetable fibers, waxes, paraffins, starches, tracantes, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide, etc. may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the dosage form of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide ether. Sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The low molecular weight hyaluronic acid or salt thereof of the present invention may be used in an amount of 0.001 to 90% by weight, preferably 0.01 to 10% by weight, based on the total weight of the external skin pharmaceutical composition or cosmetic composition.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example  1. Preparation of composition for efficacy evaluation test

A cosmetic composition (composition examples 1 and 2 and comparative composition example 1) was prepared in the composition example of Table 1 below.

number Raw material Composition example 1 Composition example 2 Composition example 3 Comparative composition example 1 (weight %) One   Low molecular weight hyaluronic acid (weight average molecular weight 5,000 daltons) 0.5 - - - 2   Low molecular weight sodium hyaluronate salt (weight average molecular weight 5,000 daltons) - 0.5 - - 3 High molecular weight hyaluronic acid (weight average molecular weight 1.1 × 10 ⁶ daltons) - - 0.5 - 4 Stearic acid 0.5 0.5 0.5 0.5 5 Cetyl alcohol 0.8 0.8 0.8 0.8 6 Glyceryl Monostearate 2.0 2.0 2.0 2.0 7 Polyoxyethylene sorbitan monostearate 0.5 0.5 0.5 0.5 8 Sorbitan sesquioleate 0.2 0.2 0.2 0.2 9 Squalane 8.0 8.0 8.0 8.0 10 Wax 0.6 0.6 0.6 0.6 11 Carboxy Vinyl Polymer 0.1 0.1 0.1 0.1 12 glycerin 2.0 2.0 2.0 2.0 13 Triethanolamine 0.1 0.1 0.1 0.1 14 Purified water Remaining amount Remaining amount Remaining amount Remaining amount system 100.0 100.0 100 100

Experimental Example  1. Skin Barrier Recovery Test

1-1. Preparation of Laboratory Animals

Six-week-old male hairless mice (Skh / hrl) were supplied from Samtaco (Osan, Gyeonggi-do, Korea) and adapted to a clean cage for about 5 days. Water and feed (Samtaco, Osan, Gyeonggi-do) Korea) was freely ingested, temperature (23 ± 2 ℃), humidity (55 ± 10%) and contrast period was automatically adjusted to 12 hours.

1-2. Skin Barrier Recovery Test

In order to evaluate the effect on the recovery of the skin barrier on the damaged skin of Composition Example 1, Composition Example 2, Composition Example 3 and Comparative Composition Example 1 prepared by the composition examples of Table 1, the hair loss of Experimental Example 1-1 Acetone was applied to the back of the mouse (2 × 2 cm, 4 compartments) twice a day for 5 days to impair the barrier function of the back skin of the hairless mice, and then percutaneously with a vaometer (Vapometer, delfin). The recovery of skin barrier was evaluated by measuring the amount of water loss (TEWL, transepidermal water loss). The TEWL of the first (0 hour) of damaged skin was measured, and then 2.5 ul / cm ^{2 of} Composition 1, Composition 2, Composition 3, and Comparative Composition 1 were obtained. After each application, it was dried and the TEWL of each coating site was measured with a vapor meter at 1, 7, 12, 24, 36 and 48 hours. The unit of measurement was g / m ² hr. All TEWL experiments were conducted at 23-25 ℃ and 38-42% relative humidity.

As a result of the experiment, it was confirmed that the treatment groups coated with Composition Examples 1 and 2 recovered barrier damage faster and returned to normal skin than the group coated with Comparative Composition 1. On the other hand, the treatment group coated with Composition Example 3 was only slightly better than the treatment group coated with Comparative Composition Example 1, and the damage recovery effect was inferior to the treatment groups coated with Composition Examples 1 and 2. This may be due to the fact that the skin absorption of high molecular weight hyaluronic acid is lower than that of low molecular weight hyaluronic acid. (See Figure 1)

Experimental Example  2. Skin moisture  exam

The skin moisturizing power of the cosmetic composition prepared in the composition example 1, composition example 2, composition example 3 and comparative composition example 1 prepared in the composition example of Table 1 of Example 1 was measured as follows. The skin moisturizing power was evaluated by measuring the electrostatic load capacity of the skin according to moisture content using a corneometer (Corneometer CM820: Courage, Khazaka) as a measuring device.

Each subject was rinsed with water before the experiment and allowed to adjust to external environment for 30 minutes. Experimental environmental conditions were kept the same at room temperature 23-25 ℃ and relative humidity 38-42%. 10 ul each sample was applied to the inside of the upper arm (2 × 2 cm), dried for 1 minute, and then 20 ul of purified water was applied to each application site, and at 0 minutes at 1 minute intervals immediately after wiping off the moisture with a tissue. The capacitance of the skin surface of each application site up to 10 minutes was measured with a coneometer and the conductivity (%) was calculated according to the following Equation 1.

Conductivity (%) = average Cv value measured 5 times per minute-Cv value before sample application / immediately after sample application (0 minutes)-Cv value before sample application × 100

As a result of the experiment, as shown in Table 2, Composition Example 3 containing high molecular weight hyaluronic acid generally known to have excellent moisturizing power showed the best skin moisturizing power, and then Composition Example 1 and Composition 2 Although slightly lower than that of Composition Example 3, it showed excellent skin moisturizing ability than Comparative Composition Example 1, and it was found that low molecular weight hyaluronic acid also has excellent skin moisturizing ability.

         Sample time (minutes) Composition example 1 Composition example 2 Composition example 3 Comparative Composition 1 0 100.00 100.0 100.00 100.0 One 75.21 73.43  80.27 67.09 2 64.29 57.70  72.14 57.61 3 62.33 57.26 68.97 48.80 4  51.29 48.21 59.65 37.14 5 44.90 42.92 51.28 32.02 6 41.74 39.87 46.24 25.42 7 39.54 36.89 44.12 20.13 8 35.31 32.94 40.86 20.60 9 33.89 31.45 39.27 19.20 10 31.67 30.88 39.42 19.71

Experimental Example  3. Cytotoxicity Test

MTT assay [3- (4,5-dimethylthylthiazol-2-yl) -2,5 by culturing fibroblasts (Human Skin fibroblast, ATCC, CRL-2310) to investigate the cytotoxicity of low molecular weight hyaluronic acid and its salts -diphenyltetrazolium bromide reduction test] was performed using the method described in the literature (T. Mosmann et al, J. Immunol. Methods , 65 , p55, 1983).

3-1. Cell culture

Human fibroblasts were seeded in 24-well culture plates at a concentration of 5 × 10 ⁴ / ml. Medium was used DMEM (Dubelcco'S Modified Eagle Medium; BRL, USA) containing 10% fetal bovine serum (FBS).

24 hours after the inoculation, the cells were replaced with DMEM containing 2% fetal bovine serum, and samples of low molecular weight hyaluronic acid and salts thereof were added at different concentrations , and then cultured in a 37 ° C., 5% CO ₂ incubator for 3 days.

3-2. Cell viability measurement

The supernatant of the cultured cells of Experimental Example 3-1 was removed and washed again by adding 200ul of 5% phosphate buffered saline (PBS).

MTT (Sigma, USA) solution was added 1.0 ml per well, MTT was removed after 4 hours and DMSO (dimethyl sulfoxide) was added 1.0 ml per well, followed by incubation at 37 ° C. for 12 hours and absorbance at 570 nm. Was measured.

Cell viability was calculated according to the following equation.

Cell viability (%) = sample OD _{570 nm} value / control OD _{570 nm} value × 100

As a result, it was found that low molecular weight hyaluronic acid and its salts were not toxic to fibroblasts in the concentration range of 1,000 ug / ml. Therefore, the experiment was then performed with the highest concentration of 1,000 ug / ml in the cell experiment. (See Figure 2)

Experimental Example  4. Elastase  Inhibitory effect

The low-molecular-weight hyaluronic acid and its salts were measured as follows according to the method of elastase inhibitory activity (Lee et al. J. Cosmetic Science , 49 , pp 285-297, 1998).

The buffer solution was 0.267M Tris solution adjusted to pH 8.0 with 0.267M hydrochloric acid (HCl) solution, and the substrate solution was the elastase substrate Succ-Ala-Ala-Ala-p-nitroanilide. A standard product (Sigma, USA) was prepared at 8.8 mM, and the enzyme solution was prepared such that porcine pancreatic elastase standard product was 10 μg / ml.

In order to measure the elastase activity, 60 μl of the buffer solution was mixed with 20 μl of substrate solution and 100 μl of sample, and 20 μl of enzyme solution was mixed and shaken to react for 15 minutes at 25 ° C., and the amount of p -nitroaniline was produced. Absorbance (B) was measured and measured at a wavelength of 410 nm.

In addition, absorbance (C) was measured using 60 µl of the buffer solution, 20 µl of the substrate solution, 20 µl of purified water and 100 µl of the sample.

Separately, the absorbance (A) was measured using 100 μl of purified water instead of the sample, which was obtained by the same method as the test solution.

 120 μl of purified water was added instead of the enzyme solution and the sample solution, and the absorbance (D) was measured using the solution obtained by the same method as the sample solution as the color correction solution.

Elastase inhibition rate (%) was calculated according to the following Equation 3, and the experimental results are shown in Table 3 below.

% Elastase Inhibition = [1-(B-C) / (A-D)] × 100

sample Weight average molecular weight IC ₅₀ (ug / ml) Low molecular weight hyaluronic acid 2,050 453 5,000 510 8,300 572 Low Molecular Weight Sodium Hyaluronate 2,100 461 5,000 497 8,100 583

Experimental Example  5. Collagenase  Inhibitory effect

In order to determine the inhibitory effect on collagenase enzyme in vitro, the experiment was performed by applying a method known in Korea Patent No. 10-0515418.

Experimental concentrations of low molecular weight hyaluronic acid and its salts were set to 500 ug / ml and the highest experimental concentration of 1,000 ug / ml. 500 mg of the sample was added to 5 mg of azo dye-impregnated collagen (Sigma) and 1 ml of 0.1 M potassium phosphate buffer (pH 7.8), and the total volume was made to be 2 ml with purified water.

Thereafter, pretreatment was performed at 37 ° C. for 1 minute, and collagenase (type IA, Sigma Co., Ltd.) was added to a final 0.01 unit, followed by reaction for 20 minutes.

After cooling in ice water, the absorbance of the supernatant was measured at 520 nm by centrifugation at 3,500 rpm for 10 minutes. As a control, 500 ul of DMSO (sample dissolved solvent) was added instead of the sample, and the total volume was 2 ml with purified water.

Using the measured absorbance, the collagenase inhibition rate (%) was calculated according to Equation 4 below. (See Table 4)

The collagenase inhibitory effect of each sample of low molecular weight hyaluronic acid and its sodium salt was not significantly different, and all showed an inhibitory effect of more than 50% at the concentration of 500 ug / ml. It was.

Collagenase inhibition rate (%)

= [1-(enzyme activity of each concentration sample / enzyme activity of control)] × 100

sample Weight average molecular weight Collagenase Inhibition Rate (%) 500 ug / ml 1,000 ug / m l Low molecular weight hyaluronic acid 2,050 72.3 75.4 5,000 51.3 66.8 8,300 50.2 57.9 Low Molecular Weight Sodium Hyaluronate 2,100 65.2 72.7 5,000 50.1 60.9 8,100 51.3 58.6

Experimental Example  6. MMP -One mRNA  Expression inhibitory effect

To investigate the gene expression inhibitory effect on Matrix Metallo-Preoteinase-1 that promotes collagen breakdown in the skin, the method described in the literature was applied to reverse transcriptase polymerase chain reaction (RT). -PCR, Reverse transcription-polymerase chain reaction (Choe T. et al., J. Cosmet . Sci ., 54 , pp 229-238, 2003).

Sodium Nitroprusside (SNP) is used as a substance that generates nitric oxide in cells (Brune B and Lapetina EG., J. Biol. Chem ., 264 , pp8455-8458, 1989). , Increases the expression of MMP-1 and 2 in human fibroblasts. In addition, nitric oxide acts as a multifunctional messenger, and when MMP-1 and SNP are activated by inducible nitric oxide synthase (iNOS), the amount of intracellular nitric oxide increases and thus the expression of MMP is increased. (Choe T et al., J. Cosmet . Sci ., 54 , pp . 229-238 , 2003).

6-1. Cell culture

Fibroblasts were dispensed at a density of 1 × 10 ⁶ cells / ml in 100 mm dishes and then incubated at 37 ° C. for 24 hours. Sodium nitroprusside 50 uM was added to the medium using a source of nitric oxide and low molecular weight hyaluronic acid and its salt were added to a concentration of 1,000 ug / ml, and EGCG 25 uM was added as a control for 24 hours each. Incubated at 37 ° C.

6-2. RNA analysis

Cells were removed from the medium and 1 ml of Trizol (Invitrogen) was added to separate RNA according to the RNA separation method of Invitrogen. After quantification at 260nm using an ultraviolet detector, reverse transcription polymerase chain reaction was performed. Reverse transcription polymerase chain reaction was used an all-in-one reverse transcription polymerase chain reaction kit (All-in-one RT-PCR kit; Superbio), the primers and the reaction conditions are shown in Table 5 and Table 6 below, all-in-one reverse transcription polymerization The experiment was carried out according to the manual of the enzyme chain reaction kit.

MMP-1 Primer sense Seq. ID 1: 5'-TGGGAGCAAACACATCTGA-3 ' Antisense Seq. ID 2: 5'-ATCACTTCTCCCCGAATCGT-3 ' Reaction conditions After reverse transcription at 50 ° C. for 30 minutes, the reverse transcriptase was inactivated at 96 ° C. for 3 minutes, followed by polymerization at 29 cycles of 94 ° C. for 1 minute, 48 ° C. for 1 minute, and 72 ° C. for 1 minute.

Actin primer sense Seq. ID 3: 5'-GAGACCTTCAACACCCCAGCC-3 ' Antisense Seq. ID 4: 5'-GGCCATCTCTTGCTCGAAGTC-3 ' Reaction conditions After reverse transcription at 50 ° C for 30 minutes, inactivation of reverse transcriptase at 96 ° C for 3 minutes, polymerization reaction at 22 cycles of 94 ° C for 1 minute, 62 ° C for 1 minute, and 72 ° C for 1 minute

Experimental results of the MMP-1 expression inhibitory effect was shown by correcting for actin, as shown in Table 7 below, low-molecular-weight hyaluronic acid and its salts to show the activity of MMP-1 induced by sodium nitroprusside This excellent inhibition was confirmed.

sample Weight average molecular weight MMP-1 expression level (%, corrected for actin) Control 100 Sodium Nitroprusside (SNP) 133 SNP + Low Molecular Hyaluronic Acid 1,000 ug / ml 2,050 57 5,000 63 8,300 65 SNP + Low Molecular Hyaluronic Acid Salt 1,000 ug / ml 2,100 61 5,000 68 8,100 68 SNP + EGCG 25uM 60

Experimental Example  7. Inflammation inhibitory effect

Raw264.7 cells, which are macrophages of mice, were used to measure the inhibitory effect of inflammation as described below (Green L. et al., Anal. Biochem ., 126 , pp131-138, 1982).

     Inflammation was induced with lipopolysaccharide, and the activity of inducible nitric oxide producing enzyme and COX-2 (Cyclooxygenase-2) gene expression were confirmed.

7-1. Cell culture

8 x 10 ⁵ cells / ml of the cells were suspended in DMEM containing 10% fetal bovine serum without phenol-red and inoculated in a 24-well culture plate. One day later, the samples were treated at 100 ug / ml, 500 ug / ml and 1,000 ug / ml concentrations for 18 hours, and 1 ug / ml lipopolysaccharide (LPS, lipopolysaccharide; Sigma Co.) was treated for cellular inflammation. After induction, the cells were incubated for 24 hours.

7-2. Nitric Oxide Activity Measurement

     The supernatant of the cells cultured by the method of Experimental Example 7-1 was recovered, and 100 ul each was added to a 96-well plate, and the same amount of Greases reagent (Sigma) was added thereto, and gently shaken at room temperature for 10 minutes, followed by 570 nm. Absorbance was measured. A calibration curve was prepared using sodium nitrate (Aldrich, USA) as a standard, and the amount of nitric oxide produced in the culture solution of each sample at 100 ug / ml, 500 ug / ml and 1,000 ug / ml was determined. It was. The amount of nitrogen oxide produced in the lipopolysaccharide-treated group was 100% to determine the amount of each sample produced.

      As a result, as shown in Figure 3, it was confirmed that low molecular weight hyaluronic acid and its salts inhibit the production of inducible nitric oxide synthase (iNOS) in a concentration-dependent manner.

7-3. COX-2 gene expression measurement

     Remove the medium of the cells cultured by the method of Experimental Example 7-1 and add 1 ml of Trizol (Trizol; Invitrogen) to separate RNA according to the RNA separation method of Invitrogen, 260nm using an ultraviolet detector After quantification at, reverse transcription polymerase chain reaction was performed. Reverse transcription polymerase chain reaction was used for the All-in-one reverse transcription polymerase chain reaction kit (All-in-one RT-PCR kit; Superbio), the reaction conditions with the primers are shown in Table 8 below All-in-one reverse transcription polymerase chain reaction kit Experiment according to the manual.

COX-2 primer sense Seq. ID 5: 5'-CTGAAGCCCACCCCAAAC-3 ' Antisense Seq. ID 6: 5'-AACCCAGGTCCTCGCTTATG-3 ' Reaction conditions After reverse transcription at 50 ° C. for 30 minutes, the reverse transcriptase was inactivated at 96 ° C. for 3 minutes, followed by polymerization at 32 cycles of 94 ° C. 30 seconds, 55 ° C. 30 seconds, and 72 ° C. for 1 minute.

As a result of measuring the inhibitory effect of COX-2 expression was shown by correcting for actin, as shown in Table 9, low-molecular-weight hyaluronic acid and its salts inhibit the activity of lipopolysaccharide-induced COX-2. Sikkim could be confirmed.

sample Weight average molecular weight Inhibitory effect of COX-2 expression (%, corrected for actin) Control 100 Lipopolysaccharide 148 Lipopolysaccharide + low molecular weight hyaluronic acid 1,000 ug / ml 2,050 108 5,000 126 8,300 129 Lipopolysaccharide + Low Molecular Hyaluronic Acid Sodium Salt 1,000 ug / ml 2,100 119 5,000 128 8,100 131

Experimental Example  8. Wound Healing Experiment

Formulation example  8-1. Placebo

(The first)

Propylene Glycol 100.0mg

Hydroxypropylmethylcellulose 15.5mg

Water 884.5mg

The components are mixed by vigorous stirring.

Formulation example  8-2. Low molecular weight  Hyaluronic acid (weight average molecular weight 2,000) 0.25%

(Formulation 2) 2.5 mg of low molecular weight hyaluronic acid (weight average molecular weight 2,050)

Propylene Glycol 99.0 mg

Hydroxypropylmethylcellulose 15.5 mg

883 mg of water

The low molecular weight hyaluronic acid as an active ingredient is added to water with vigorous stirring, and then other ingredients are added thereto.

Formulation example  8-3. Low molecular weight  Hyaluronic acid (weight average molecular weight 5,000) 0.25%

Preparation 3 low molecular weight hyaluronic acid (weight average molecular weight 5,000) 2.5 mg

Propylene Glycol 99.0 mg

Hydroxypropylmethylcellulose 15.5 mg

883 mg of water

The low molecular weight hyaluronic acid as an active ingredient is added to water with vigorous stirring, and then other ingredients are added thereto.

Wound healing experiments were carried out by applying the method described in the literature using the formulation examples of Formulation Examples 8-1, 8-2 and 8-3 containing the low molecular weight hyaluronic acid of the present invention (Takami , M. et al., Science, 53 (3) , pp 185, 1993).

Forty six-week-old male Wistar rats (Samtako, Korea) with an average body weight of 150 g were divided into four groups of ten animals each for comparison, group 1 (placebo), group 2, and Experiment was carried out as the formulation 3 group. After anesthesia with ether, the flanks were pulled and sterilized, and then wounds were made by using a punch to remove two circular skin flakes (1 cm in diameter) from the skin of the chest. Healing was started once a day 1 day after the above surgical procedure.

The comparative group was a dry gauze pad, and the formulation 1 group (placebo group), the formulation 2 group and the formulation 3 group were wound with the gauze pad moistened with the components of Formulation Examples 8-1, 8-2 and 8-3, respectively, of the test formulation. Covering the site and fixed with adhesive tape 10 days after the experiment, the wound was checked for complete healing.

Treatment group Number of animals used Healing Animals Comparison 10 3 Formulation group 1 10 5 Formulation group 2 10 8 Formulation group 3 10 7

Compared to the wounds of the comparative group and the two wounds treated with placebo (Group 1), the wounds treated with Formulation Group 2 and Group 3 were faster. (See Table 10)

Experimental Example  9. Caco With -2 cells Low molecular weight  Cell Permeability Experiment of Hyaluronic Acid and Its Salts

9-1. Cell culture

Caco-2 cells were suspended in DMEM containing 10% fetal bovine serum and inoculated in a 24 well culture plate by suspending 3.6 x 10 ⁴ cells / well into HMEM-Transwell with an area of 0.33 cm ² . The cells were cultured in an insert (Costar, Cambridge, USA). The medium contains non-essential amino acids (JHR Biosciences, USA), 1% L-glutamine (Aldrich, USA), 100 U / ml penicillin G (Aldrich, USA) and 100 ug / ml of streptomycin (JHR). Biosciences, USA). The medium was changed every two days and incubated under 37 ° C., 95% relative humidity and 5% CO ₂ conditions. Monolayer culture was carried out for about 21 days with cells having passages of 20 to 40, and the experiment was performed by selecting only cells having a TEER value of 400Ωcm ² or more. TEER values were measured by Millicell-ERS (Millipore, USA).

10-2. Low molecular weight  Of hyaluronic acid and its salts Caco -2 cell permeation experiment

Before performing the experiment, 10 mM HEPES (N-2-hydroxyethylpiperazine-

N'-2-ethanesulfonic acid; Sigma, USA) was replaced with HBSS buffer medium containing, and the upper and lower compartments were confirmed to be pH 7.4. Monolayer cultured cells were washed twice with buffer solution and used after confirming the integrity of the TEER value.

The low molecular weight hyaluronic acid and its salt were made at a concentration of 500 ug / ml, and the weight average molecular weight of 1.1 million high molecular weight hyaluronic acid sodium salt (Bioland, Korea) was used as a control. The solution volume of the upper compartment was 0.2 ml and the solution volume of the lower compartment was 0.6 ml and incubated at 37 ° C. for 2 hours. After 2 hours, the concentrations of low molecular weight hyaluronic acid, its salts, and high molecular weight hyaluronic acid in the lower compartments were analyzed by GPC (gell permeation chromatography).

9-3. Gel Penetration  Chromatography ( GPC ) analysis

Gel permeation chromatography (GPC) was performed using a VP-10AD, RID-10A (shimadzu, Japan) instrument, and the column was ultrahydrogel ^TM 120. 250 (7.8 × 300 mm, WATERS, USA). As the eluate, 0.1 M sodium nitrate solution was used, and the flow rate was 0.5 ml / min. Ethylene glycol polymer was used as a standard for molecular weight analysis.

9-4. Low molecular weight  Of hyaluronic acid and its salts Caco -2 cell permeability calculation

Cell permeability was calculated according to Equation 5 below with the concentrations of low molecular weight hyaluronic acid and salts thereof penetrating Caco-2 cells obtained by GPC analysis, and the high molecular weight sodium hyaluronate salt as a control.

Cell Permeability = Lower Section Final Sample Concentration / Upper Section Initial Sample Concentration × 100

As a result, as shown in Table 11, the high molecular weight hyaluronic acid sodium salt has a cell permeability close to zero, while the low molecular weight hyaluronic acid and the low molecular weight hyaluronic acid sodium salt have a significantly higher cell permeability than the high molecular weight hyaluronic acid. It can be confirmed that excellent.

sample Weight average molecular weight Cell permeability  Low molecular weight hyaluronic acid 2,050 30.24 ± 0.45 5,000 28.31 ± 0.38 8,300 18.78 ± 0.56  Low Molecular Weight Sodium Hyaluronate 2,100 33.95 ± 0.32 5,000 29.43 ± 0.18 8,100 20.85 ± 0.35  Polymeric hyaluronic acid sodium salt 1.1 × 10 ⁶ 0.32 ± 0.08

Experimental Example  10. Using mouse skin Low molecular weight  Skin Permeability Experiment of Hyaluronic Acid and Its Salts

10-1. Preparation of the Skin of Rats

Eight weeks-old female ICR mice (ICR mouse; Samtako, Korea), who had no pregnancy experience, were anesthetized with ether, and then the cervical bones were separated and their hair was shaved to 1 mm or less. Thereafter, the skin of the back and abdomen was removed. The skin is up to the dermis, where the dermis is removed.

Removal of the dermis was performed as follows by trypsinizing and dissolving the protein of the dermis with a trypsin solution. Filter paper was placed on Petri dish, 1% trypsin (Aldrich, USA) solution using pH 8 phosphate buffer solution was saturated and absorbed by filter paper, and the dermis of skin was adhered to filter paper and maintained at 37 ℃ incubator for 4 hours. I was.

After removing the skin from the incubator, the deionized water was gently stirred and the trypsin solution was washed away. After washing several times, the above method was repeated once more. The skin prepared in the same manner as above was dried under vacuum and stored frozen (-20 ° C.).

10-2. Low molecular weight  Skin Permeation Experiment of Hyaluronic Acid and Its Salts

In the skin permeation experiment of low molecular weight hyaluronic acid and its salt, Franz diffusion cell (Iwaki, Japan) was used. The volume of the collection part was 4.6 ml, and the permeation area was 0.64 cm ² . The skin was fixed at 37 ° C. with the epidermis fixed in the lower compartment and stirred with a stirrer. The lower compartment was filled with a phosphate buffer solution of pH 7.4, and the upper compartment was mixed with 1 mg of low molecular weight hyaluronic acid in 1 ml of deionized water.

The skin permeability of low molecular weight hyaluronic acid and salts thereof was determined after 48 hours in the lower compartment by analyzing the concentration by GPC. As a control, a weight average molecular weight of 1.1 million polymer hyaluronic acid sodium salt (Bioland, Korea) was used, and the amount of use and the experimental method were the same. GPC analysis was performed in the same manner as in Experiment 9-3.

10-3. Low molecular weight  Calculation of skin permeability of hyaluronic acid and its salts

Permeability was calculated according to the following equation (6) as the concentration of the sample obtained by GPC analysis.

Skin Permeability = Lower Section Final Sample Concentration / Upper Section Initial Sample Concentration × 100

As a result of the experiment, as shown in Table 12, the high molecular weight hyaluronic acid sodium salt has a skin permeability close to zero, while the low molecular weight hyaluronic acid and the low molecular weight hyaluronic acid sodium salt are high as shown in the results of the cell permeability experiment. Very good results were obtained compared to the molecular weight sodium salt of hyaluronic acid.

sample Weight average molecular weight Skin permeability  Low molecular weight hyaluronic acid 2,050 39.87 ± 0.26 5,000 33.41 ± 0.29 8,300 29.02 ± 0.35  Low Molecular Weight Sodium Hyaluronate 2,100 41.47 ± 0.38 5,000 31.89 ± 0.49 8,100 30.16 ± 0.32  Polymeric hyaluronic acid sodium salt 1.1 × 10 ⁶ 0.53 ± 0.13

Experimental Example  11. The human body Patch experiment (Patch test)

In order to determine the degree of skin irritation with respect to the composition according to the present invention, skin patch experiments were carried out on the composition examples 1 and 2 and the comparative composition example 1 of Example 1 as follows.

The subjects were selected to have no skin disease at the test site of 20 ~ 50 years old. After wiping the upper arm of the patch area with 70% ethanol and drying, petroleum jelly was used as a blank test using a fin chamber. It was patched for 24 hours, the skin reaction was examined within 1 hour after the patch was removed, and then examined again the next day (after 48 hours).

The skin response was determined by the test criteria of Table 13 below, and the evaluation of the skin irritation probability was performed using the average response rate calculated according to Equation 7 below. Skin patch test results for the cosmetic according to the following are shown in Table 14.

Average response rate (%) = [Σ (Number of response subjects after 24 hours × score) + Σ (Number of response subjects after 48 hours × score)] × 100 / (Total number of tests × maximum score × total number of subjects)

Skin reaction score Reaction - 0 no response ± One Weak positive reaction (erythema) + 2 Positive reaction (erythema) ++ 3 Strong positive reaction (erythema, edema) +++ 4 Severe positive reactions (erythema, edema, blisters)

Test substance Number of subjects who responded after 24 hours (persons) Number of subjects who responded after 48 hours (persons) Average response rate (%) 0 One 2 3 4 0 One 2 3 4 Composition 1 14 One 0 0 0 14 One 0 0 0 0.06 Composition 2 15 0 0 0 0 14 One 0 0 0 0.06 Comparative Composition 1 15 0 0 0 0 15 0 0 0 0 0.00

As a result of the human patch test, it was confirmed that the composition containing the low molecular weight hyaluronic acid or a salt thereof is a substance that is safe for the human body, belonging to the non-irritating range.

Hereinafter, the preparation examples of the external preparation for skin and the cosmetic composition containing the composition of the present invention will be described, but the present invention is not intended to be limited thereto but merely to be described in detail.

Formulation example  1. Hydrophilic Ointment

number Raw material Formulation Example 1 (weight%) One   Low molecular weight hyaluronic acid 0.5 2   White vaseline 25.0 3   Stearyl alcohol 22.0 4   Ethyl (or methyl) p-oxybenzoate a very small amount 5   Propylene glycol 12.0 6   Sodium lauryl sulfate 15.0 7   Propyl p-oxybenzoate a very small amount system 100

Formulation example  2. flexible lotion

number Raw material Formulation Example 2 (weight%) One   Low molecular weight hyaluronic acid 0.5 2 Butylene Glycol 4.0 3 glycerin 8.0 4 Carboxy Vinyl Polymer 0.06 5 Disodium ethylenediamineteracetic acid 0.01 6 ethanol 7.0 7 Ethanolamine 0.2 8 Sorbitan monostearate 0.8 9 Hydrogenated Castor Oil 0.4 10 Phenylmethicone 3.5 11 Paraoxybenzoic acid propyl 0.2 12 Methyl paraoxybenzoate 0.2 13 Purified water Remaining amount system 100.0

Formulation example  3. Nutritional Cosmetics

number Raw material Formulation Example 3 (weight%) One   Low molecular weight hyaluronic acid 0.5 2 Stearic acid 1.0 3 Cetyl alcohol 1.0 4 Glyceryl Monostearate 1.0 5 Polyoxyethylene sorbitan monostearate 1.0 6 Sorbitan sesquioleate 1.0 7 Liquid paraffin 4.0 8 Carlylic / Capricglycerides 4.0 9 Squalane 3.0 10 Carboxy Vinyl Polymer 0.1 11 Butylene glycol 5.0 12 Ethanolamine 0.2 13 Paraoxybenzoic acid propyl 0.2 14 Methyl paraoxybenzoate 0.2 15 Purified water Remaining amount system 100.0

Formulation example  4. Nutrition Cream

number Raw material Formulation Example 4 (weight%) One   Low molecular weight hyaluronic acid 0.5 2 Stearic acid 2.0 3 Cetyl alcohol 2.0 4 Glyceryl Monostearate 2.0 5 Polyoxyethylene sorbitan monostearate 0.5 6 Sorbitan sesquioleate 0.5 7 Glyceryl Monostearate / Glyceryl Stearate / Polyoxyethylene Stearate 1.0 8 Wax 1.0 9 Liquid paraffin 4.0 10 Carlylic / Capricglycerides 4.0 11 Squalane 4.0 12 Carboxy Vinyl Polymer 0.3 13 Butylene glycol 5.0 14 glycerin 3.0 15 Ethanolamine 0.5 16 Paraoxybenzoic acid propyl 0.2 17 Methyl paraoxybenzoate 0.2 18 Purified water Remaining amount system 100.0

Formulation example  5. Essence

number Raw material Formulation Example 5 (weight%) One   Low molecular weight hyaluronic acid 0.5 2 Cytostolol 1.70 3 Polyglyceryl 2-oleate 1.50 4 Ceramide 0.1 5 Ceteares-4 1.2 6 cholesterol 1.5 7 Dicetylphosphate 0.4 8 Concentrated glycerin 5.0 9 Sunflower oil 15.0 10 Carboxy Vinyl Polymer 0.2 11 Xanthan Gum 0.2 12 antiseptic a very small amount 13 Spices a very small amount 14 Purified water Remaining amount system 100.0

Formulation example  6. Oil painting Foundation

number Raw material Formulation Example 6 (weight%) One   Low molecular weight hyaluronic acid 2.0 2 Beeswax 2.0 3 Pyromethicone 2.0 4 Liquid paraffin 5.0 5 Squalane 5.0 6 Stearic acid 2.0 7 Lipophilic monostearic acid glycerin 3.0 8 Caprylic / Capricglycerides 4.0 9 glycerin 4.0 10 Propylene glycol 3.0 11 Butylene glycol 3.0 12 Ethanolamine 1.0 13 Aluminum Magnesium Silicate 0.5 14 Pigment 12 15 antiseptic a very small amount 16 Spices a very small amount 17 Purified water Remaining amount system 100.0

The composition containing the low molecular weight hyaluronic acid or a salt thereof having a weight average molecular weight of 500 ~ 10,000 Daltons of the present invention has a moisturizing, wrinkle improvement, wound healing and anti-inflammatory effect and is easier to penetrate the skin than the polymeric hyaluronic acid composition, and It can be usefully used as a cosmetic composition.

<110> BIOLAND LTD. <120> A composition comprising low molecular weight hyaluronic acid or          the salt young <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> sense for MMP-1 primer <400> 1 tgggagcaaa cacatctga 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense for MMP-1 primer <400> 2 atcacttctc cccgaatcgt 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sense for ACTIN primer <400> 3 gagaccttca acaccccagc c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> antisense for ACTIN primer <400> 4 ggccatctct tgctcgaagt c 21 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> sense for COX-2 primer <400> 5 ctgaagccca ccccaaac 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense for COX-2 primer <400> 6 aacccaggtc ctcgcttatg 20

Claims (10)

It is easier to penetrate skin than polymer hyaluronic acid, and has low molecular weight hyaluronic acid or its salt having a weight average molecular weight of 500 to 10,000 Daltons represented by the following general formula (I), which has moisturizing, wrinkle improvement, wound healing and anti-inflammatory effects as an active ingredient. External skin composition for moisturizing, wrinkle improvement, wound healing and prophylaxis and treatment containing:

(Ⅰ) N is an integer from 1 to 25; R is a hydrogen atom or an alkali metal salt of lithium, sodium or potassium, an alkali metal salt of magnesium or calcium, ammonia or ethylenediamine, cyclohexylamine, aniline, ethanolamine, diethanolamine, triethanolamine, ethylamine, propylamine And salts with organic amines such as arylamine, diethylamine, butylamine, isobutylamine, triethylamine, pentylamine, t-butylamine and isopropylamine. The composition for external application for skin according to claim 1, wherein n is a compound having an integer of 1 to 25. According to claim 1, wherein the low molecular weight hyaluronic acid and the external composition of the skin, characterized in that the molecular weight of 500 to 10,000 Daltons. The salt according to any one of claims 1 to 3, wherein the salt is an alkali metal salt of lithium, sodium, potassium, alkaline earth metal salt such as magnesium, calcium, ammonia or ethylenediamine, cyclohexylamine, aniline, ethanolamine Salts with organic amines such as diethanolamine, triethanolamine, ethylamine, propylamine, arylamine, diethylamine, butylamine, isobutylamine, triethylamine, pentylamine, t-butylamine and isopropylamine. A skin external composition for use. The composition for external application for skin according to claim 1, wherein the composition is a cream, gel, patch, spray, ointment, warning, lotion, linen, pasta or cataplasma.    It is easier to penetrate skin than polymer hyaluronic acid, and has low molecular weight hyaluronic acid or its salt having a weight average molecular weight of 500 to 10,000 Daltons represented by the following general formula (I), which has moisturizing, wrinkle improvement, wound healing and anti-inflammatory effects as an active ingredient. Cosmetic composition for the prevention and improvement of moisturizing, wrinkle improvement, wound healing and inflammation containing.

(Ⅰ) The cosmetic composition according to claim 6, wherein n is an integer of 1 to 25 and a salt thereof. 7. The cosmetic composition according to claim 6, wherein the low molecular weight hyaluronic acid and its salt have a molecular weight of 500 to 10,000 Daltons. The salt of R of the general formula (I) is an alkali metal salt of lithium, sodium, potassium, alkaline earth metal salt such as magnesium, calcium, ammonia or ethylenediamine, cyclohexylamine, aniline, ethanolamine Salts with organic amines such as diethanolamine, triethanolamine, ethylamine, propylamine, arylamine, diethylamine, butylamine, isobutylamine, triethylamine, pentylamine, t-butylamine and isopropylamine. Cosmetic composition characterized in that.    According to claim 6, wherein the composition is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, A cosmetic composition, characterized in that it has at least one formulation selected from the group consisting of packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers.

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