KR101421499B1 - An agent which promotes production of collagen, a cosmetic agent, a method for producing collagen - Google PatentsAn agent which promotes production of collagen, a cosmetic agent, a method for producing collagen Download PDF
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- KR101421499B1 KR101421499B1 KR1020070085020A KR20070085020A KR101421499B1 KR 101421499 B1 KR101421499 B1 KR 101421499B1 KR 1020070085020 A KR1020070085020 A KR 1020070085020A KR 20070085020 A KR20070085020 A KR 20070085020A KR 101421499 B1 KR101421499 B1 KR 101421499B1
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- Prior art keywords
- polyphosphoric acid
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- 102000008186 Collagen Human genes 0 title 2
- 108010035532 Collagen Proteins 0 title 2
- 229960005188 collagen Drugs 0 title 2
- 229920001436 collagens Polymers 0 title 2
- 239000003795 chemical substance by application Substances 0 title 1
- 239000002537 cosmetic Substances 0 title 1
- 238000005365 production Methods 0 title 1
The present invention relates to an agent for promoting the production of collagen containing polyphosphoric acid or the like as an active ingredient, a method for producing collagen, an application product thereof, and the like.
In aging skin, qualitative and quantitative changes of collagen fiber, elastin fiber and acidic mucopolysaccharide, which are epidermal cell epilepsy, occur due to decrease of fibroblast activity. Collagen fiber is originally rich in elasticity. However, as aging of human beings is promoted, crosslinking is formed in the collagen fibers, so elasticity disappears from the collagen fibers. As a result, the human skin loses its flexibility and wrinkles and sagging phenomena occur. Collagen production promoters have been developed for the purpose of preventing skin aging. However, all of them can not effectively promote the production of collagen, and there is also a problem that a drug as a raw material can not be easily obtained.
An object of the present invention is to provide an agent for facilitating the production of collagen using a raw material which is easy to obtain and which has excellent biocompatibility, and a method for producing collagen using such agent.
Basically, the present invention is based on the fact that a product containing an effective amount of polyphosphoric acid can effectively promote the production of collagen. The excellent biocompatibility of polyphosphoric acid has also been demonstrated in the fields of foods and the like. Therefore, when this agent is used, it is possible to promote the production of collagen by using an agent having excellent biocompatibility, and to prevent the aging phenomenon and the like, so that the agent of the present invention can be effectively used for cosmetics and the like.
A preferred form of the present invention relates to a cosmetic containing ultra phosphoric acid. Ultra-phosphoric acid, which is a net-shaped sodium polyphosphate, has a strong acidity. Because of this, ultra-phosphoric acid was never incorporated into cosmetics. However, ultraphosphoric acid has a strong acidity, so it exerts a peeling effect that reduces the dead skin of the skin. In addition, as demonstrated by the examples described below, ultra-phosphoric acid produces collagen. In other words, a preferred embodiment of the present invention is to provide a cosmetic composition containing an effective amount of ultrafinic acid, which can provide a peeling effect and accelerate the production of collagen.
A preferred form of the present invention relates to a method for producing collagen comprising a step of administering polyphosphoric acid, a salt thereof or a solvate thereof to a mammal other than human or human. In this method, ultraphosphoric acid is preferable as the polyphosphoric acid. Among mammals other than humans or humans, mammals other than humans are preferable, and examples thereof include pigs, monkeys, mice, rats and the like. That is, polyphosphoric acid or the like may be administered to a subject to produce collagen, and then the collagen produced may be appropriately recovered.
INDUSTRIAL APPLICABILITY According to the present invention, it is possible to effectively provide agents for promoting the production of collagen. Further, by containing ultra-phosphoric acid as an effective ingredient, it is possible to provide a cosmetic which can achieve a peeling effect and accelerate the production of collagen.
Since collagen is effective for whitening, prevention of aging and the like, cosmetic preparations and cosmetic packs containing the agent of the present invention are effective for whitening and prevention of aging.
The present invention relates to an agent for promoting the production of collagen containing polyphosphoric acid, a salt thereof, or a solvate thereof as an active ingredient. More specifically, it is an agent for promoting the production of collagen comprising an effective amount of polyphosphoric acid, a salt thereof, or a solvate thereof.
As described above, the agent of the present invention comprises polyphosphoric acid, a salt thereof, or a solvate thereof as an active ingredient. As the polyphosphoric acid of the present invention, a polyphosphoric acid having a structure in which two or more PO 4 tetrahedra share an oxygen atom at a vertex by dehydration condensation of orthophosphoric acid and have a structure in which they are in a chain form, a side chain Polyphosphoric acid, cyclic polyphosphoric acid, branched polyphosphoric acid (ultraphosphoric acid), or a mixture or derivative thereof.
The present invention relates to a polyphosphoric acid composition which comprises H 2 O and P 2 O 5 as constituent molecules and has a molar ratio (R) of H 2 O to P 2 O 5 of 2> R ≧ 1, Or polyphosphoric acid in the form of chains or rings as shown in (II).
H n +2 (PnO 3n +1) (I)
(HPO 3 ) n (II)
(In the formulas (I) and (II), each n independently represents an integer of 3 or more and 300 or less.) In addition, a form different from the above polyphosphoric acid is ultraphosphoric acid. Ultrathinic acid is a polyphosphoric acid having a high-order structure in the form of a mesh having a molar ratio (R) of H 2 O to P 2 O 5 constituting molecules of polyphosphoric acid of 1>R> 0. The number of phosphorus atoms in ultraphosphoric acid is not particularly limited, but may be from 3 to 300.
N in the formula (I) is preferably an integer of 3 to 130, more preferably 10 to 89. [
The average molecular weight of the polyphosphoric acid to be used is 240 to 25,000, preferably 810 to 7,300. On the other hand, among the total amount of polyphosphoric acid, it is preferable that 90% by weight or more of n in the formula (I) is in the range of 10 to 89 (preferably 20 to 80) because the ability to produce collagen increases.
Furthermore, polyphosphoric acid having a chain length of 1000 or more is not known to exist in the form of an aqueous solution, and is not preferable because it is considered to be poorly soluble in water. In addition, since the chain length of polyphosphoric acid in vivo is about 800, it is considered that polyphosphoric acid having a chain length of 800 or less has high effectiveness on various physiological functions in vivo (KD Kumble and A. Kornberg, Inorganic polyphosphate in mammalian cells and tissues, The Journal of Biological Chemistry, Vol.270, pp. 5818-5822, 1995).
The polyphosphoric acid of the present invention has an effect of promoting the production of collagen, so polyphosphoric acid having a chain length of 3 to 300 can be preferably used. In addition, as demonstrated in Examples described later, it may be said that polyphosphoric acid having a chain length of 20 or more is preferably used in order to accelerate the production of collagen.
The term "salt thereof " means a salt of polyphosphoric acid, particularly a salt of polyphosphoric acid, which is pharmaceutically acceptable. As used herein, " pharmaceutically acceptable " means not deleterious to the recipient. The polyphosphoric acid of the present invention can be converted into a salt by a conventional method. Examples of the salt include alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt and cobalt salt An inorganic salt such as an ammonium salt, a t-octylamine salt, a dibenzylamine salt, a morpholine salt, a glucosamine salt, a phenylglycine alkyl ester salt, an ethylenediamine salt, an N- methylglucamine salt, a guanidine salt, Triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloropropane salt, procaine salt, diethanolamine salt, N-benzyl-N-phenethylamine salt, piperazine salt , And organic salts such as tetramethylammonium salt and tris (hydroxymethyl) aminomethane salt. Of these, as the salt of polyphosphoric acid, an alkali metal salt is preferable, and a sodium salt is more preferable.
In the present specification, the " salt thereof " may include salicide as well as salicylate. Such salts are, for example, electrically dissociated in vivo and the like to function as polyphosphoric acid.
"Solvate" means a solvate of polyphosphoric acid. As the solvate, a hydrate can be mentioned. In addition, the agent of the present invention may be left in the air or recrystallized to absorb moisture, resulting in adsorption of water or hydration. The case where such a solvate is formed is also included in "the solvate ". Such a solvate is electrically dissociated in vivo and the like to function as polyphosphoric acid.
The polyphosphoric acid, the salt thereof, or the solvate thereof used in the present invention may be one kind, but may be a mixture of plural kinds. A plurality of polyphosphoric acids, salts thereof, or solvates thereof include polyphosphoric acid, salts or solvates thereof having different degrees of polymerization, polyphosphoric acids having different molecular structures, salts thereof or solvates thereof and polyphosphates having different metal ions.
Polyphosphoric acid, salts of polyphosphoric acid, and solvates of polyphosphoric acid can be produced by a commonly used production method such as a method of heating phosphoric acid, a method of dissolving phosphoric acid in phosphorus pentoxide, and the like.
Further, in particular, heavy chain polyphosphate having a chain length of 20 or more can be preferably prepared by the following method. First, hexametaphosphate is dissolved in water to a concentration of 0.1 to 20% by weight, preferably 9 to 11% by weight. 87 to 100% ethanol, preferably 96% ethanol, is added to the hexametaphosphoric acid aqueous solution in a volume of 1/10 to 1/3 of the total liquid amount after mixing the hexametaphosphoric acid solution and ethanol, that is, a hexametaphosphoric acid aqueous solution : Ethanol in an amount such that the volume ratio is 2: 1 to 9: 1. The mixed solution is sufficiently stirred, and the resulting precipitate is separated from the aqueous solution component by a separation method such as centrifugation or filter filtration. The precipitate thus separated is heavy chain polyphosphoric acid. The polyphosphoric acid is subsequently washed with 70% ethanol, and then dried. The average chain length of the polyphosphoric acid obtained in this separation operation is 60 to 70, and no single chain polyphosphoric acid of 10 or less is contained at all. Therefore, the molecular weight distribution is about 10 to 150 in terms of the number of phosphate residues.
Polyphosphoric acid is a process for producing polyphosphoric acid from phosphoric acid as described in Japanese Patent Specification No. 2004-537490, which comprises (a) providing a packed column, the packed column extending from the bottom to the top, (B) a first acid feed fluid comprising at least one phosphorus acid, wherein the first acid feed fluid comprises at least one first inlet opening in the vicinity of the top, and at least one second inlet opening disposed below the first inlet opening, (C) introducing a second acid feed fluid comprising phosphoric acid into the hot air fluid to form a fluid of thermal and acidic acids; (d) (E) polymerizing phosphoric acid in the first acid feed fluid and the second acid feed fluid to form polyphosphoric acid. The hexapolyphosphoric acid or octapolyphosphoric acid may be produced by, for example, a method disclosed in Japanese Patent Application Laid-Open No. 2004-035348.
The content of the polyphosphoric acid in the present invention is not particularly limited, but is preferably 0.001 to 0.5% by weight, and more preferably 0.002 to 0.1% by weight, for the cultured cells. When administered intra-tissue or percutaneously, a rather dark concentration is preferable to a concentration for treating cultured cells. Accordingly, the content of polyphosphoric acid in a tissue or percutaneous administration may be 0.1 to 10% by weight, more preferably 1 to 5% by weight, and most preferably 1 to 2% by weight. When polyphosphoric acid is used as a metal blocker or an antioxidant for cosmetics, it is generally used at a concentration of less than 1% by weight. For the purpose of increasing collagen production, it is effective to increase the production of collagen effectively by setting it to 1% by weight or more.
The polyphosphoric acid, the salt thereof, or the solvate thereof may be used individually or in combination with a pharmacologically acceptable carrier or diluent to promote the production of polyphosphoric acid. In addition, polyphosphoric acid, a salt thereof, or a solvate thereof may be mixed with a pharmaceutically acceptable additive and may be made into various materials in a form suitable for application to the affected part. Examples of the material form suitable for the present invention include those in the form of injections, external liquids (injecting agents, coating agents), solid agents (granules, fine granules, powders, ointments, tablets) It can be suitably prepared.
Pharmacologically acceptable additives include, for example, excipients, disintegrants or disintegration aids, binders, lubricants, coatings, pigments, diluents, bases, solubilizers or solubilizers, isotonic agents, pH adjusting agents, stabilizers, , Dispersants, emulsifiers, gelling agents, thickeners, pressure-sensitive adhesives, and compatibilizers.
Furthermore, the composition comprising the agent of the present invention and a pharmaceutically acceptable carrier or the like is a composition effective for prevention of skin aging and whitening. The composition can be administered orally or parenterally.
The pharmacologically acceptable carriers include those appropriately selected from excipients, diluents, lubricants, binders, disintegrators, stabilizers, and mating agents.
Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol, starch derivatives such as corn starch, potato starch, alpha starch and dextrin, cellulose derivatives such as crystalline cellulose, gum arabic, dextran, Organic excipients such as silicic anhydride, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and inorganic excipients such as calcium sulfate .
Examples of the lubricant include lubricants such as stearic acid metal salts such as stearic acid, calcium stearate, and magnesium stearate; talc; colloidal silica; waxes such as non-waxing and spermaceti; boric acid; adipic acid; sulfate such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; fatty acid sodium salts; lauryl sulfate such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and silicic acid hydrate; and the above starch derivatives.
Binders include, for example, compounds such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol and the above excipients.
Examples of the disintegrant include cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium and internal cross-linked carboxymethylcellulose sodium; carboxymethyl starch, carboxymethyl starch sodium, crosslinked polyvinylpyrrolidone And starch-celluloses such as starch and starch.
Examples of the stabilizer include paraoxine benzoic acid esters such as methylparaben and propylparaben, alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol, benzalkonium chloride, phenols such as phenol and cresol, thimerosal, dehydroacetic acid ; And sorbic acid. Examples of the mating agent include sweeteners, acidifiers, perfumes, and the like. Examples of the diluent include sterilized water, sterile organic solvent, and aqueous starch.
The amount of the agent of the present invention may be appropriately adjusted according to symptoms, age, sex, method of administration, and the like. For example, as demonstrated in the examples, it is preferable that polyphosphoric acid acts directly on the skin epidermis or near the dermis cells. Therefore, it is preferable to use a coating agent or a cosmetic as a formulation, (Preferably 5 mg / cm < 2 >) and 500 mg / cm < 2 > (preferably 50 mg /
A preferred embodiment of the present invention relates to a cosmetic composition containing the agent described above. Examples of cosmetics include lotion, milky lotion, cream, gel, essence and pack cosmetics. It is believed that the cosmetic comprising an effective amount of the agent of the present invention promotes the production of collagen at the site to which the cosmetic is applied, so that a young and healthy skin can be maintained.
"Lotion" is a transparent liquid cosmetic which is generally applied to the skin surface to keep the skin clean and healthy. The basic function of the lotion is to supply moisture and moisturizing ingredients to the stratum corneum of the skin, and lotion has the function of softening the skin. The cosmetic lotion is made by dissolving and stabilizing a substance that is difficult to dissolve in water to make the appearance transparent, a transparent or semitransparent one using microemulsion or lipid nanospheres, and an O / W oil (water-in-oil type) Emulsified opaque lotion, and water-soluble polymer blended therein. As the treatment method of the present invention, a known " lotion " can be suitably used depending on its use and the like.
The lotion contains, for example, the following components other than those of the present invention. In other words, cosmetics such as ion-exchange water, ion-exchanged water and the like are used to dissolve water-soluble components and to supply moisture to the stratum corneum; alcohols such as ethanol, propanol and the like for dissolving, sterilizing and sterilizing oil- Emollients (oil components that prevent water from evaporating), polyoxyethylene oleyl alcohol ethers, etc., for improving the moisture retention and feeling of use, such as ester oils and vegetable oils, etc., A buffer for adjusting the pH of a product such as citric acid, lactic acid, and amino acids; a flavoring agent such as vanillin, orange flavor, lemon flavor, milky flavor geraniol, Preservatives for inhibiting microorganisms such as methylparaben, phenoxyethanol, etc. to prevent decay, coloring agents for coloring, metal ion sequestrants, ultraviolet rays An antifogging agent for preventing discoloration or discoloration, and a medicament such as an astringent agent, a bactericide, a vitality agent, an anti-inflammatory agent or a whitening agent.
The amount of the polyphosphoric acid, the salt thereof or the hydrate thereof is preferably 0.00 to 20% by weight, more preferably 0.01 to 10% by weight, more preferably 0.1 to 5% by weight, most preferably 0.1 to 5% Should be about 0.2 to 2% by weight. The amount of each ingredient other than polyphosphoric acid constituting the lotion may be a known amount of the ingredient. For example, 10 to 90% by weight of purified water; 1 to 40% by weight of alcohol; 1 to 20% by weight of a moisturizer; 1 to 5% by weight of emollient; 0 to 1% by weight of solubilizing agent; 0 to 1% by weight buffer; And about 0 to 10% by weight of other drugs.
Basically, the lotion can be manufactured as follows. That is, the water-soluble component is dissolved at room temperature. The oil soluble component is heated to dissolve and purified water is added and mixed. This mixture is mixed with a water-soluble component. Then, a coloring agent may be added. Thereafter, purification is carried out by filtration. On the other hand, the components of the lotion may be adjusted by a known method depending on the use or the like.
"Milky lotion" is an emulsion that has an intermediate property between lotion and cream, and generally has fluidity. Latex is a cosmetic used for supplying moisture, moisturizing agent, oil and the like to the skin, in order to maintain the moisturizing balance, moisturizing property and flexibility of the skin. The components contained in the emulsion are similar to those contained in the cream described later, but the emulsion has a fluidity, so the amount of solid oil or lead is smaller than that of the cream. In the treatment method of the present invention, the known " milky lotion " can be suitably used depending on its use and the like.
The emulsion contains 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably 0.1 to 5% by weight, most preferably 0.2 to 2% by weight of polyphosphoric acid, its salt or its hydrate .
"Cream" is a kind of emulsion in which one of the liquids which do not mix with each other like water and oil is dispersed in a dispersed state in the other dispersion medium in a stable state. In the treatment method of the present invention, a known " cream " can be appropriately used depending on its use and the like. These creams include emolent creams to moisturize or soften the skin, massage creams to promote blood circulation, cleansing creams for skin cleansing, hair removers for hair removal, deodorant creams for deodorizing, Exfoliated cream, and the like.
The cream contains, for example, the following ingredients. Examples of the water component contained in the cream include purified water such as ion-exchanged water, alcohols for dissolving and sterilizing oil-soluble components such as ethanol and propanol, glycerin, PEG, and hyaluronic acid to moisturize the stratum corneum A moisturizer, and a mucus such as queen seed, pectin, and cellulose derivatives.
Examples of the oil component contained in the cream include hydrocarbons such as squalene, liquid paraffin, vaseline and solid paraffin, oils such as olive oil, almond oil, cacao fat, castor oil, etc., lead, lead, lanolin, jojoba oil, etc., stearic acid, oleic acid, palmitic acid And higher alcohols such as cetanol and stearyl alcohol; esters such as IPM, glycerin triester and pentaerythritol tetraester; and silicone oils such as polysiloxanes.
Examples of the surfactant (emulsifier) contained in the cream include nonionic emulsifiers such as glycerin monostearate, polyoxyethylene (POE) sorbitan fatty acid ester, sorbitan fatty acid ester, POE alkyl ether, POE polyoxypropylene (POP) block polymer; And anionic emulsifiers such as fatty acid soap or sodium aluminosulfate.
In addition, the cream may contain a buffer for adjusting the pH of the product, such as citric acid, lactic acid, and amino acids, a perfume such as geraniol or linalool, a microbial such as methylparaben or phenoxyethanol, A chelating agent such as EDTA, an alkali component such as potassium hydroxide or sodium hydroxide, a vitamin E, an antioxidant, an antioxidant, an antioxidant, a preservative for preventing decay, a coloring agent for coloring, Antioxidants such as vitamin C and dibutylhydroxytoluene, and agents such as astringents, fungicides, vitality agents, anti-inflammatory agents and whitening agents.
The amount of each component contained in the cream varies greatly depending on the type and application thereof, but known ones may be suitably used in the treatment method of the present invention.
Basically, the cream can be prepared as follows. That is, the water component is heated to about 70 캜. The oil phase component is dissolved by heating, and a flavor or the like is added and stirred. Then, the aqueous phase component and the aqueous phase component are stirred and emulsified at about 70 캜 using a homomixer or the like. Thereafter, degassing, filtration and cooling are carried out.
The gel is a cosmetic which is uniform in the appearance state such as a gel or a sol and is transparent to translucent. As a treatment method of the present invention, a known " gel " can be suitably used depending on its use and the like. A water-based gel for supplying moisture to the skin and moisturizing the skin, an oil-based gel for maintaining the moisturization of the skin and supplying oil, a water-based gel for massaging blood, and a cleansing gel. The gelo may be prepared by using a gelling agent containing a water-soluble polymer such as carboxyvinyl polymer or methylcellulose. In addition, a gel-like massage oil may be used.
The cream may contain 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably 0.1 to 5% by weight, and most preferably 0.2 to 2% by weight of polyphosphoric acid, its salt or its hydrate.
Essence (Essence) is basically a component such as the above-mentioned lotion, emulsion and cream, and includes those containing an emulsion, a solubilizing agent, a moisturizing agent, water, and other medicines. Known cosmetic liquids in the treatment method of the present invention can be used. Examples of the emulsion contained in the serum include natural vegetable oils such as olive oil, camellia oil, sesame oil, sunflower oil, sweet almond oil and jojoba oil; diglycerin esters or triglycerin esters of fatty acids such as capric acid, myristic acid, oleic acid, isostearic acid, And the like. These emulsions mainly function as emollients.
Examples of the solubilizing agent in the serum include propylene glycol fatty acid esters such as glycerin fatty acid esters, polyglycerin fatty acid esters, sorbitan fatty acid esters and propylene mono-stearic acid glycols, glycerin, diglycerin, ethylene glycol, propylene glycol, And polyhydric alcohols such as phenol and phenol. On the other hand, the solubilizing agent is preferable because it uses glycerin or diglycerin and improves the heat effect in massaging. Examples of the water contained in the serum include distilled water and deionized water. Other medicines include bactericides, preservatives, vitamins, natural extracts from plants, coloring matters, perfumes, and the like.
The amount of each component constituting the serum may be a known component amount. For example, when the total amount of the serum is 100 wt%, 0.001-20 wt%, preferably 0.01-10 wt%, more preferably 0.1-5 wt% of polyphosphoric acid, its salt or its hydrate, , And most preferably about 0.2 to 2 wt%. Also, water may be used in an amount of about 65 to 90% by weight of an oil, 1 to 10% by weight of a solubilizing agent, 1 to 30% by weight of a moisturizer, and 0 to 10% by weight of other medicines. The serum may be prepared according to a known production method.
The "pack cosmetic" is applied to the target site for the purpose of moisturizing the skin, promoting blood circulation, and cleansing the skin. The pack cosmetics generally include jelly, paste, and powder, and may be applied to the face to form a film, followed by peeling, wiping after application, and washing after application. When powder is used, it is used to dissolve uniformly in water or in suspension. Pack cosmetics in which a cosmetic material is impregnated into a nonwoven fabric or collagen sheet having a shape such as an eye mask or pack cosmetics in which the above nonwoven fabric is immersed in a cosmetic composition when used.
The "pack cosmetic preparation" contains 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably 0.1 to 5% by weight, and most preferably 0.2 to 2% by weight of polyphosphoric acid, its salt or its hydrate . The other composition may be a known composition used for pack cosmetics, and may be prepared according to a known production method.
Each of the above-mentioned cosmetic materials may be used in accordance with a commonly used method. In this case, the cosmetic contains an effective amount of polyphosphoric acid and the like, so that the production of collagen is promoted, and the effect of skin beauty and anti-aging effect is obtained.
Next, a method for producing collagen to which polyphosphoric acid, a salt thereof, or a solvate thereof is administered will be described. The method of extracting collagen from skin, etc. other than human is generally used, but using the production method of the present invention, collagen can be produced more effectively.
Specifically, the composition, etc. of the present invention is applied to skin such as livestock, and collagen is collected according to a conventional method. Thus, the skin coated with the agent of the present invention contains collagen more than ordinary skin, and thus collagen can be effectively produced.
The composition of the present invention or the composition of the present invention is appropriately applied to wounds of mammals other than humans or humans. Then, since the collagen production is promoted at the wound site, the wound is healed cleanly. The dose of Jenna may be as described above.
A typical method for measuring the absolute value of the average chain length of annular polyphosphoric acid is a titration method. This method is a kind of end-to-end measurement method and is mainly used for determining the molecular weight of the cyclic phosphate. In cyclic phosphate, there is one strongly acidic hydrogen and one slightly acidic hydrogen in the terminal group and one strongly acidic hydrogen in the middle group. That is, the average chain length of the chain-like phosphate can be obtained from the following formula (III) by obtaining the amount of strongly acidic hydrogen (SA) and the amount of weakly acidic hydrogen (WA).
That is, the average chain length = (2 SA / WA) ... (III).
As a method for obtaining the relative value of the average molecular weight and the molecular weight distribution, gel filtration chromatography using HPLC is generally used. The details of the gel filtration chromatography method and measurement results will be described later in Examples.
[Production Example 1]
Preparation of sodium polyphosphate
Sodium polyphosphate having various chain lengths was prepared as follows.
(i) 200 g of sodium hexametaphosphate (manufactured by Taihei Chemical Industry Co., Ltd.) as a food additive standard was dissolved in 1,000 ml of purified water, and 50 ml of 96% ethanol was gradually added thereto.
(ii) The obtained solution was well stirred and allowed to stand at room temperature for 1 hour or more, and then centrifuged (10,000 x g, 10 minutes, 25 캜) to separate the solution into two layers.
(iii) the upper layer was removed and transferred to another container.
(iv) A lower layer containing a high molecular weight (long chain) sodium polyphosphate was recovered and stored as a fraction (fraction 1) containing long-chain polyphosphate having the largest molecular weight.
(v) 50 ml of ethanol was added to the upper layer separated and collected by the operation of (iii) above. The liquids thus obtained were subjected to the operations described in (ii) to (iv) above. However, the lower layer thus obtained contained sodium polyphosphate having a molecular weight smaller than that of fraction 1, and this was stored as fraction 2.
(vii) The upper layer obtained by the above operation was subjected to the operations (v) and (vi), and the lower layer obtained was stored as fraction 3.
(viii) The above operation (vii) was repeated six more times, and the lower layer obtained at that time was stored. The obtained fractions were designated as fractions 4, 5, 6, 7, 8, and 9, respectively.
Determination of average molecular weight and molecular weight range of sodium polyphosphate
As a result of the separation operation, the respective fractions were divided into three groups and mixed, and each group was made of long-chain polyphosphate, sodium polyphosphate, and short-chain polyphosphate. The mixture of fractions 1 and 2 is long-chain polyphosphate. The mixture of fractions 3 to 5 is sodium polyphosphate, and the mixture of the remaining fractions 6 to 9 is monosodium polyphosphate. In addition, all of the fractions 1 to 9 were mixed to obtain a divided polyphosphate sodium containing all the molecular weights and having a large molecular weight distribution. The mixture of the respective fractions was lyophilized, and ethanol was completely removed to obtain sodium polyphosphate powder of various chain lengths. The average chain length and the molecular weight distribution were determined by the following titration method and gel filtration method.
The details and the results of the measurement of the average chain length of the above-mentioned long-chain, short-chain, short-chain sodium polyphosphate by the titration method are described below. 25 g each of a long chain, a long chain, and a short chain polyphosphate was dissolved in 500 ml of water, and 5 ml for decomposition and 40 ml for decomposition were collected. 0.04 ml of a 5N sodium hydroxide solution was added to the pre-decomposition solution to adjust the pH to about 12, and the pH was adjusted to about 2.5 with 0.5 mol / l hydrochloric acid. 48 ml of 5N hydrochloric acid and 280 ml of water were added to the solution for decomposition, and the mixture was hydrolyzed for more than 30 minutes, cooled, and then accurately milled to 400 ml. To 10 ml of this solution, 2.2 ml of 5N sodium hydroxide solution was added to adjust the pH to about 11, and the pH was adjusted to about 2.5 with 0.5 mol / l hydrochloric acid. An automatic titration device (Hiranuma Industry Co., Ltd., COM-1500) was used for titration.
When the amount of hydrochloric acid (W) of 0.5 mol / L from the first inflection point to the second inflection point of the titration curves of both solutions before and after decomposition is W and the amount of hydrochloric acid after decomposition (S) is S, The average chain length was as follows.
Long chain polyphosphate sodium = (2 x 5 x 1.56) /0.12 = 130
Sodium bisphosphate sodium = (2 x 5 x 1.32) /0.22 = 60.2
Mono-chain polyphosphate = (2 x 5 x 0.50) /0.35 = 14.3
The analytical results of the above-described long-chain, short-chain, short-chain sodium polyphosphate by gel filtration chromatography by HPLC are shown below. On the other hand, the analysis conditions in HPLC were as follows.
Analytical instrument (HPLC): LC2010C manufactured by Shimadzu Corporation
Column: Shodex OHpak SB-803 HQ
Column temperature: 30 ° C
Solvent: 0.1 M NaCl
Flow rate: 1 ml / min
Sample application amount: 0.01 ml
Sodium polyphosphate concentration in sample: 1%
As a result of the analysis by gel filtration under the above conditions, the elongation time of the long-chain polyphosphate was 7.95 minutes, the elution time of the sodium polyphosphate was 8.50 minutes, and the elution time of the monosodium polyphosphate was 9.17 minutes. From this value, a calibration curve of the relationship between the logarithm of average chain length (log) and solute time was prepared (Fig. 1). As a result, it was found that the relationship between the elution time and the logarithm of the chain length is represented by the following formula (VI). Chain length (log) = - 0.7987 x elution time +
Based on this calibration curve, the elution start time (high molecular weight side) and elution end time (low molecular weight side) in the gel filtration analysis of long-chain, long-chain and single-chain polyphosphate were calculated and the molecular weight ranges were calculated. The results are shown in Table 1. On the other hand, in Table 1, inability to calculate is outside the calibration curve and means inability to calculate.
Inability to calculate chain length 1.830955
Inability to calculate chain length 67.7571
Chain length 1.95076 1.47154
Chain length 89.281196 29.616927
Chain length 1.487514 Impaired
Chain length 30.726564 Output failure
From the analysis of the molecular weight range by the above titration method and gel filtration method, the long-chain polyphosphate mixed with the fractions 1 and 2 had an average chain length of 130, a chain length range of 67.8 or more, Sodium phosphate had an average chain length of 60 and a chain length range of 29.6 to 89.2. In addition, the single-chain sodium polyphosphate mixed with the remaining fractions 6 to 9 had an average chain length of 14 and a chain length range of 30.7 or less.
Identification of properties of sodium ultraphosphate
In the sodium ultrafine phosphate used in the present embodiment, the characteristic of its molecule is that when it is brought into an aqueous solution state, it exhibits strong acidity of pH 1 to 2, and when it is titrated with an alkali solution such as sodium hydroxide, It can be seen that it has a buffer region. From this, the aqueous sodium ultrafine phosphate solution was titrated in an aqueous solution of sodium hydroxide, and its pH change was measured and shown in Fig. As a result, it was confirmed that it exhibited a buffering effect at a pH of about 1 to 2 and had the property of sodium ultrafine phosphate.
Increase of collagen production by polyphosphoric acid
A collagen production evaluation test was performed on wound areas of rats using the divided sodium polyphosphate obtained according to the above Preparation Example.
3 is a conceptual diagram showing a wound healing model.
Six - week - old Wistar male rats were hindquartered under ether anesthesia and incised 20 mm to reach the fascia along the long axis of the body. Thereafter, a fusiform wound healing model was created by suturing one end of the fascia with the fascia so as to have a width of 5 mm (see Fig. 3). In the experimental group, 1% aqueous solution of sodium polyphosphate (sodium polyphosphate (average chain length: 60, chain length range: 3 to 300) containing all of the short chain, long chain long chain and short chain long chain polyphosphate prepared by the above- }, And 1 mg / ml of 1% phosphoric acid buffer was applied locally for 5 days per week. 3 days, and 7 days, skin samples were collected on the fascia as shown in Fig. 1 at a distance of 5 mm from the center of the wound. The collected skin specimens were fixed with formalin and then paraffin sections were prepared. Hematoxylin and eosin (HE) staining and AZAN staining were performed on the specimens on the 7th day after application. On the third day after the application, the amount of type I collagen mRNA expression was evaluated by in situ hybridization after deparaffinization.
Evaluation of I-type collagen mRNA expression by in situ hybridization
CDNA was synthesized from the RNA extracted from mouse tissue, and using the primer of FW: 5'-gagggggtttctgtgtcct-3 '(SEQ ID NO: 1) and RV: 5'-cgaggtagtctttcagcaacac-3' (SEQ ID NO: 2) PCR was carried out.
Thus, a DNA fragment containing a part of the mouse type I collagen gene was obtained.
The pCRII-R type 1-collagen plasmid was prepared by cloning the fragment with pCRII-TOPO vector (manufactured by Invitrogen). The plasmid was digested with restriction endonuclease Xho1 to form a chain, and Dig-tag type I collagen RNA probe was prepared by in vitro transcription. A sample (preparate) on the third day after the deparaffin application was treated with protease K (2 쨉 g / ml) for 10 minutes (room temperature) and hybridized at 60 캜 for 15 hours. The concentration of the probe at this time was 500 ng / ml and was performed in a solution of 5 x SSC (0.75 M NaCl, 60 mM Na 3 citrate, pH 7.0) containing 50% formamide (FA). Thereafter, cleaning was carried out in the order shown in Table 2 below, and the tissue portion in which type I collagen mRNA was expressed was specifically stained by treatment with BM-purple (manufactured by Lotshu).
[Table 2] Cleaning process
4 is a photograph showing the hematoxylin-eosin (HE) staining degree in place of the drawing.
4 (A) is an enlarged image of the HE staining solution of the control group. Fig. 4 (a-1) shows an HE-stained steel enlarged image of the portion surrounded by the square in Fig. 4A.
Fig. 4 (a-2) shows an enlarged image of the Azuki-colored streak of the portion surrounded by the square in Fig. 4 (A).
In the control group, the wound area tended to be tempered, but infiltration of inflammatory cells such as lymphocytes and macrophages was remarkable, and the epidermis had hardness of hardness. Also, as a result of Azan staining, it can be seen that I-type collagen production was not so much. Fig. 4 (B) is an enlarged image of the HE staining solution in the polyphosphoric acid coating group. Fig. 4 (b-1) shows the HE-stained steel enlarged image of the portion surrounded by the square in Fig. 4 (B). Fig. 4 (b-2) shows an enlarged image of the Azuki-colored steel enclosed by the square in Fig. 4 (B). In wound area, fibroblast proliferation, fiber growth and maturation were observed. From the results of Azan staining, it was confirmed that coarse collagen fibers were produced more clearly than the control group. Inflammatory cells were hardly recognized in the surface layer of the wound, and the wound was almost covered by the epidermis.
Figure 5 is a photograph that replaces the drawing showing the results of in situ hybridization. Fig. 5 (A) shows a control group, Fig. 5 (B) shows a group coated with polyphosphoric acid, and Fig. 5 (B) is a partially enlarged view of Fig.
In the control group (FIG. 5A), almost no staining was observed, and no remarkable type I collagen mRNA expression was observed. In the polyphosphoric acid group (FIG. 5B) , And FIG. 5B (enlarged view of the rectangular part of FIG. 5B), the expression of type I collagen mRNA was recognized in the cytoplasm of the fibroblast adjacent to the normal subcutaneous tissue at the wound end face end.
From the results of the in situ hybridization of the HE and the subarctic color and type I collagen mRNA, remarkable increase of collagen was confirmed by polyphosphoric acid, polyphosphoric acid has a function as a collagen production promoting material, It was found that the recovery of injured tissue was enhanced by the effect.
Polymerization Dependence of Collagen Production Promotion Effect of Human Dermal Fibroblast by Sodium Polyphosphate
The effect of promoting collagen production by sodium polyphosphate was examined using fibroblast (HDF) from human dermis. HDF was seeded on a 24-well plate at 25,000 per well and cultured (3 days) until the cells were fused with 5% CO 2 at 37 ° C using a D-MEM culture medium (Sigma). Subsequently, the culture medium was replaced with a D-MEM culture medium containing 1% bovine serum and cultured for 6 days or 9 days. Subsequently, a test piece of short chain, long span and long chain polyphosphate (average phosphoric acid degree of polymerization 14 (short chain), 60 (short chain), 130 (long chain)) prepared according to the above- Was added to the D-MEM culture so that the final concentration was 1%, and the treatment was carried out. In addition, with respect to the sodium polyphosphate (average chain length 60, chain length range 3 to 300) including both the single chain, the long chain, and the long chain polyphosphate prepared according to the above production example, the same treatment with the other sodium polyphosphate . On the other hand, a non-treated group was provided as a control and used as a control group. The treatment group treated with various polyphosphoric acids was shown as follows.
Treatment group A: Cells treated with sodium polyphosphate (average chain length 60, chain length range 3 to 300) containing both short chain, heavy chain and long chain polyphosphate.
Treatment group B: Cells treated with mono-chain polyphosphate.
Treated group C: Cells treated with medium sodium diphosphate.
Treatment group D: Cells treated with long chain polyphosphate.
Treated group E: cells treated with sodium ultrafine phosphate.
For the cells cultured for 6 days or 9 days by each treatment culture medium, I-type collagen was immunostained according to the following procedure. However, on the 4th day and 7th day of treatment, a fresh D-MEM culture medium containing the same test substance as the various treatment groups was used. The following operations are all for one hole of a 24-hole plate.
(i) The culture medium was removed, and cells were fixed with 0.5 ml of a 10% neutral buffered formalin solution (trade name: Tanaform, manufactured by Tanaka Co., Ltd.).
(ii) 1 ml of TBS-Ca (20 mM Tris-HCl pH 7.5, 0.15 M NaCl, 1 mM CaCl 2 ) was added to wash the cells.
(iii) 1 ml of methanol was added and left at -20 占 폚 for 30 minutes.
(iv) 1 ml of TBS-Ca was added and washed.
(v) 1 ml of TBS-Ca containing 5% skim milk was added and left for 50 minutes.
(vi) 1 ml of TBS-Ca was added and washed.
(vii) 0.15 ml of TBS-Ca containing 5% skim milk diluted 1/150 of anti-human type I collagen antibody (polyclonal antibody, product of Chemicon) was added and left for 50 minutes. (viii) Three times with 1 ml of TBS-Ca.
(ix) Subsequent operations were performed according to the protocol of the kit using the DAKO EnVision System HRP (DAB) kit (DacoCytomation, Inc.). The DAKO dextran polymer reagent bound with the secondary antibody was diluted 5-fold with TBS-Ca, added with 0.15 ml, and left for 30 minutes.
(x) 1 ml of TBS-Ca.
(xi) A mixture of DAKO substrate reaction mixture (0.15 ml) and DAKO color development substrate (3 μl) was added to perform color reaction for about 10 minutes.
(xii) The reaction was stopped by rinsing three times with distilled water, washed with ethanol, and the staining image was collected with a scanner. Each treatment group was subjected to analysis using Image-J (prewear), which is an image analysis software, and the degree of dyeing was quantified to be an index of evaluation.
Fig. 6 is a photograph in place of the drawing showing the immunofluorescence of HDF treated with sodium polyphosphate of each chain. Fig.
Fig. 7 is a graph that replaces the drawing showing the result of analyzing the dyed image of Fig. 6 with the image analysis software (Image-J) and quantifying the degree of dyeing.
For quantification, the intensity of the dye in the control group at each treatment day was set at 1, and the relative value of the dyeing intensity at the other treatments was taken as the collagen production rate.
7). On day 6 from FIG. 7, sodium polyphosphate (average chain length 60, chain length range 3 to 300) (treatment group A) containing all chain lengths (group A and group C) , And cells treated with sodium ultraphosphate (treatment group E) were found to have higher collagen production promoting ability than the long chain polyphosphate treated group (treated group D) or the untreated control group. On the 9th day of treatment, the single chain polyphosphate treated group (treatment group B) had the highest promoting ability (about 6.3 times of the control group), and the next, sodium hypophosphite (treated group C) About 4.9 times). In addition, sodium polyphosphate or sodium ultrafine phosphate (treatment group E) containing different molecular weights had about twice the collagen production promoting ability as compared with untreated cells (control group). From the above, it has been found that polyphosphoric acid has the ability to promote collagen production in the chain length range of 3 to 300, but the effect of promoting collagen production is particularly high in the short-chain and medium-sodium polyphosphate. In the untreated and phosphoric acid treated groups, the degree of staining was significantly lower than those of the other treated groups, and collagen production promotion was dependent on the sodium polyphosphate molecule itself.
1 is a graph showing the relationship between the elution time and the molecular weight in the gel filtration analysis for determining the molecular weight of sodium polyphosphate.
2 is a titration curve of sodium hypophosphite.
3 is a conceptual diagram showing a wound healing model.
Fig. 4 is a photograph showing the hematoxylin-eosin (HE) staining degree. 4 (A) is the enlarged image of the HE staining medium of the control group, 4 (a-1) represents the HE-stained enlargement of the area surrounded by the square of 4 A, 4 4 (B) shows an enlarged image of the HE staining in the polyphosphoric acid group, 4 (b-1) shows the HE stained enlargement image of the area surrounded by the square of 4 (B) , And 4 (b-2) represents an enlarged image of the Azan-colored streak of the portion surrounded by the square of 4 (B).
Figure 5 is a photograph that replaces the drawing showing the results of in situ hybridization. 5 (A) represents a control group, 5 (B) represents a group coated with polyphosphoric acid, and 5 (b) is a partial enlarged view of 5 (B).
Fig. 6 is a photograph in place of the drawing showing the immunofluorescence of HDF treated with sodium polyphosphate of each chain. Fig. Treated cells were treated with sodium polyphosphate (average chain length 60, chain length range 3 to 300) containing all of short chain, long chain long chain and short chain long chain polyphosphate as a control group, Cells treated with monosodium polyphosphate were treated with Group B, medium sodium hypophosphite, treated with Group C, and those treated with long chain polyphosphate were treated with Group D, sodium ultrafine phosphate Treated group E.
Fig. 7 is a graph that replaces the drawing showing the result of analyzing the dyed image of Fig. 6 with the image analysis software (Image-J) and quantifying the degree of dyeing. Cells treated with sodium polyphosphate (average chain length: 60, chain length range: 3 to 300) containing both single-stranded, double-stranded and long-chain sodium polyphosphate as control and cells treated with Group A , Cells treated with monosodium polyphosphate were treated with Group B, medium with sodium hypophosphite, treated with Group C, and those treated with long chain polyphosphate were treated with Group D, sodium ultrafine phosphate Treated group E was used.
<110> Regenetiss Inc. An agent which promotes the production of collagen, a cosmetic agent, a method for producing collagen <130> IN2007-PKR032 <150> JP2006-227237 <151> 2006-08-24 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 gagggggttt ctgtgtcct 19 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 cgaggtagtc tttcagcaac ac 22
- A cosmetic composition for promoting collagen production comprising polyphosphoric acid, a salt thereof or a solvate thereof as an active ingredient.
- The method according to claim 1,Wherein the polyphosphoric acid is a compound represented by the following general formula (I) or (II) wherein H 2 O and P 2 O 5 are constituent molecules and the molar ratio (R) of H 2 O to P 2 O 5 is 2> Wherein the cosmetic composition contains one or more kinds of chain-like or annular polyphosphoric acid.H n + 2 (P n O 3n + 1 ) (I)(HPO 3 ) n (II)(In the formulas (I) and (II), n independently represents an integer of 3 to 300.)
- The method of claim 2,Wherein n in the formulas (I) and (II) is an integer of 10 to 89, respectively.
- The method according to claim 1,Wherein said polyphosphoric acid is ultra-phosphoric acid.
- A method for producing collagen comprising the step of applying polyphosphoric acid, a salt thereof or a solvate thereof to the skin of a mammal other than a human.
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