JP2008056568A - External preparation for skin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an external preparation for skin having moisturizing action and cell-activating action and having high permeability. <P>SOLUTION: The external preparation for skin comprises at least arginine, aspartic acid, isoleucine, leucine, lysine and threonine or salts thereof among aminoacids and comprises a hydrolyzed silk. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an external preparation for skin that can be applied to various kinds of pharmaceuticals, quasi drugs, cosmetics (preparations used for humans and other animals) and the like.

The skin is covered with a stratum corneum, which is a thin biological protective film, and it is possible to live in a dry atmosphere without losing moisture, because the stratum corneum exists on the skin in contact with the outside world, that is, the epidermis. It is. The stratum corneum keeps the moisture from the body from being lost, and at the same time, makes various adjustments so that the skin is kept in a normal state.
However, the skin is damaged and hardened due to trauma (burn, inflammation caused by various stimulating factors such as ultraviolet rays, etc.), the stratum corneum is cracked and peeled off, and a large amount of water is lost compared to normal skin. Will happen.

On the other hand, a decrease in skin water content is often caused or involved in one of the causes for losing the flexibility, extensibility, and strength necessary for the stratum corneum. A functionally defective stratum corneum does not have enough water binding (retention) force to keep the stratum corneum flexible, and conversely allows a large amount of moisture to pass through and lose skin.
In general, when the moisture content decreases to 10% or less of the dry weight of the stratum corneum, it loses the softness and softness of the skin that has been maintained until now, and it becomes hard and brittle, so-called crisp, moist feeling It can be said that the moisture content of the skin stratum corneum is very important because it results in no dry skin (skin).

  Therefore, conventionally, in order to make up for this point, cosmetics have been ideally reproduced on the skin with substances similar to those on the skin surface. Many of them are used. In recent years, a stratum corneum component called NMF (Natural Moisturizing Factor) has attracted attention as a third cosmetic component, and research and development of amino acids and their derivatives as NMF components have been actively conducted. (Moisturizing / cleaning / ultraviolet absorption / antibacterial action, etc.).

  Oligopeptides obtained by hydrolyzing natural proteins have a chemical structure similar to that of natural peptides in the epidermis, and thus have been widely used as humectants. For example, hydrolyzed collagen and hydrolyzed silk can be mentioned. However, it has not been sufficiently studied as a material that is combined with an amino acid to improve the skin permeability of the amino acid and carry it into the epidermis.

On the other hand, various cell activators have been proposed in order to keep the skin healthy. For example, it is known that the aforementioned amino acids have a cell activation effect.
However, with regard to cell activators, etc., only the activation effect of the compound alone is taken into consideration, and skin permeability is not taken into consideration.In addition, when the additive is an amino acid, for example, there are many types of amino acids. There was a problem with the combination.

JP-A 61-289016 JP-A-6-279227

As described above, amino acids have been known for various uses in the cosmetics field, but there is still room for improvement in terms of formulation. In addition, problems remain in the development of useful ingredients that take into consideration the permeability to the skin.
Therefore, the present inventors have intensively researched the additive materials and blending that are useful for pharmaceuticals, quasi-drugs, and cosmetics that have a cell activation effect and have excellent moisture retention.
The present invention provides a skin external preparation having a moisturizing action and a cell activating action and having high permeability.

The above object was achieved by the following items 1 to 7.
1. A skin external preparation containing at least arginine, aspartic acid, isoleucine, leucine, lysine, threonine, or salts thereof among amino acids and hydrolyzed silk.
2. Regarding the addition amount of amino acids or salts thereof, the addition amount of arginine is 20 to 400 microweight percent, the addition amount of aspartic acid is 3 to 60 microweight percent, the addition amount of isoleucine is 30 to 600 microweight percent, and the addition of leucine 1. The amount is 30-600 micro weight percent, the addition amount of lysine is 30-600 micro weight percent, and the addition amount of threonine is 25-500 micro weight percent. Topical skin preparation.
3. 1. Addition amount of hydrolyzed silk is 5 to 3000 micro weight percent. Topical skin preparation.
4). Furthermore, it includes at least one of glycine, histidine, serine, valine, tyrosine, cysteine, phenylalanine and salts thereof among amino acids. ~ 3. Topical skin preparation.
5. Contains vitamin Bs ~ 4. Topical skin preparation.
6). 4. B vitamins are vitamin B1 or vitamin B6 Topical skin preparation.
7). 1. Containing hyaluronic acid or a salt thereof ~ 6. Topical skin preparation.
8). 1. The topical skin preparation is a cosmetic or quasi-drug. ~ 7. Topical skin preparation.
9. The topical skin preparation is lotion, emulsion, cream, hair nourishing agent, pack. Topical skin preparation.

  ADVANTAGE OF THE INVENTION According to this invention, it has a moisturizing effect and a cell activation effect, and can provide the skin external preparation with high permeability.

The amino acids used as essential in the present invention are arginine, aspartic acid, isoleucine, leucine, lysine and threonine, which may be optically active or racemic, but all of them are particularly preferred.
The amount of amino acids added as essential in the present invention will be described in detail.
The amount of arginine added is preferably 20 to 400 microweight percent, more preferably 40 to 200 microweight percent, and most preferably 60 to 120 microweight percent.

The amount of aspartic acid added is preferably 3 to 60 micro weight percent, more preferably 8 to 30 micro weight percent, and most preferably 12 to 20 micro weight percent.
The amount of isoleucine added is preferably 30 to 600 microweight percent, more preferably 50 to 300 microweight percent, and most preferably 80 to 200 microweight percent.
The amount of leucine added is preferably 30 to 600 micro weight percent, more preferably 50 to 300 micro weight percent, and most preferably 80 to 200 micro weight percent.

The amount of lysine added is preferably 30 to 600 microweight percent, more preferably 50 to 300 microweight percent, and most preferably 80 to 200 microweight percent.
The amount of threonine added is preferably 25 to 250 micro weight percent, more preferably 40 to 150 micro weight percent, and most preferably 60 to 120 micro weight percent.

  The hydrolyzed silk used in the present invention is obtained by hydrolyzing silk protein with an acid, enzyme or other method, but there is no particular limitation on the hydrolysis method. The amount of hydrolyzed silk added is preferably 5 to 3000 microweight percent, more preferably 10 to 1000 microweight percent, and most preferably 20 to 200 microweight percent.

  Next, amino acids used in addition to the essential amino acids in the present invention will be described. Further, the amino acids used by addition are glycine, histidine, serine, valine, tyrosine, cysteine, and phenylalanine, which may be optically active or racemic, but all L-forms are particularly preferred. At least one of these amino acids is preferably added, more preferably at least three types are added, and most preferably all eight types are added.

Next, the amount of amino acid added and used in the present invention will be described.
The amount of glycine added is preferably 6 to 120 micro weight percent, more preferably 16 to 60 micro weight percent, and most preferably 20 to 40 micro weight percent.
The amount of histidine added is preferably 8 to 160 microweight percent, more preferably 20 to 90 microweight percent, and most preferably 30 to 60 microweight percent.
The amount of serine added is preferably 8 to 160 micro weight percent, more preferably 20 to 90 micro weight percent, and most preferably 30 to 60 micro weight percent.
The amount of threonine added is preferably 30 to 500 microweight percent, more preferably 50 to 200 microweight percent, and most preferably 70 to 150 microweight percent.
The amount of valine added is preferably 30 to 500 micro weight percent, more preferably 50 to 200 micro weight percent, and most preferably 70 to 150 micro weight percent.

The amount of tyrosine added is preferably 0.08 to 1.6 microweight percent, more preferably 0.2 to 0.9 microweight percent, and most preferably 0.3 to 0.6 microweight percent.
The amount of cysteine added is preferably 15 to 300 microweight percent, more preferably 30 to 150 microweight percent, and most preferably 40 to 80 microweight percent.
The amount of phenylalanine added is preferably 0.12 to 2.4 microweight percent, more preferably 0.2 to 1.5 microweight percent, and most preferably 0.3 to 1.0 microweight percent.

Vitamins have been used in various skin external preparations due to their coenzyme activity, but vitamins belonging to the vitamin B group are particularly preferred when combined with the skin external preparation of the present invention. The vitamin B used in the present invention is specifically vitamin B1, vitamin B2, vitamin B3, vitamin B4, vitamin B5, vitamin B6, vitamin B12, vitamin B13, vitamin B15, vitamin B17, vitamin BT, vitamin Bx, vitamin Bc, vitamin B1 related to metabolism of sugar, vitamin B2 related to metabolism of lipid and vitamin B6 related to metabolism of protein, and vitamin B1 and vitamin B6 are more preferable.
The amount of vitamin B to be used in the present invention is preferably 0.8 to 16 microweight percent, more preferably 2 to 9 microweight percent, and most preferably 3 to 6 microweight percent.

  In the present invention, it is particularly preferable to add hyaluronic acid in order to maintain skin elasticity. The hyaluronic acid may be a sodium salt or a potassium salt, or may be a free acid. When the skin external preparation of the present invention is a cosmetic, addition of hyaluronic acid is particularly preferable. The amount of hyaluronic acid used in the external preparation for skin is preferably 150 to 3000 microweight percent, more preferably 300 to 1500 microweight percent, and most preferably 400 to 800 microweight percent.

The skin external preparation of the present invention can be used in combination with various medicinal agents. Such medicinal agents are, for example, active oxygen scavengers, antioxidants, cell activators, anti-inflammatory agents, tyrosinase activity inhibitors, UV absorbers and humectants. Specific examples of the medicinal agent include those shown below, but of course not limited thereto.
The active oxygen scavenger or antioxidant that can be used in the present invention is not particularly limited, but is preferably extracted or purified from natural products rather than those obtained by chemical synthesis.

  Examples of the active oxygen scavenger include β-carotene, α-carotene, lycopene, lutein, astaxanthin, capsanthin, fucoxanthin, heteroxanthine, loroxanthin, luteoxanthine, lycophy, lycoxanthin, neochrome, neoxanthine, rhodopine, rhodopinal. , Rhodopinol, rhodovibrin, trolixanthine, xanthophyll, zeaxanthin and other carotenoids, superoxide dismutase, mannitol, rutin and derivatives thereof, bilirubin, cholesterol, tryptophan, histidine, quercetin, quercitrin, catechin, catechin derivatives, gallic acid and derivatives thereof , Glutathione and its derivatives, and their salts, hornon extract, ginkgo biloba extract, keiketto extract, hawthorn extract Plant extracts containing flavonoids, such as extracts, mica squid extract, cypress extract, melissa extract, genno shoko extract, button pi extract, parsley extract, tormentilla extract, rakan fruit extract, yashajitsu extract and dicoppi extract Etc.

  Antioxidants include, for example, retinol and its derivatives such as retinol palmitate and retinol acetate, retinal and its derivatives, vitamin A such as dehydroretinal; thiamines such as thiamine hydrochloride and thiamine sulfate, riboflavin, pyridoxine hydrochloride , Pyridoxines such as pyridoxine dioctanoate, flavin adenine nucleotide, cyanocobalamin, folic acid, nicotinic acid amide, nicotinic acid such as benzyl nicotinate, calcium pantothenate, D-pantothenyl alcohol, pantothenyl ethyl ether, acetyl pantothenyl Pantothenic acids such as ethyl ether, vitamin B such as choline and inositol; L-ascorbic acid, L-ascorbyl palmitate, L-ascorbyl dipalmitate, isopalmitic acid L Ascorbyl, L-ascorbyl diisopalmitate, L-ascorbyl tetraisopalmitate, L-ascorbyl stearate, L-ascorbyl distearate, L-ascorbyl isostearate, L-ascorbyl diisostearate, L-ascorbyl myristate, dimyristin L-ascorbyl acid, L-ascorbyl isomyristate, L-ascorbyl diisomyristate, L-ascorbyl oleate, L-ascorbyl dioleate, L-ascorbyl 2-ethylhexanoate, sodium L-ascorbate phosphate, L-ascorbic acid potassium phosphate, L-ascorbic acid phosphate magnesium, L-ascorbic acid phosphate calcium, L-ascorbic acid phosphate aluminum , Sodium L-ascorbate sulfate, potassium L-ascorbate sulfate, magnesium L-ascorbate sulfate, calcium L-ascorbate sulfate, aluminum L-ascorbate sulfate, sodium L-ascorbate, L- Vitamin C such as ascorbic acid and its derivatives such as potassium ascorbate, magnesium L-ascorbate, calcium L-ascorbate, aluminum L-ascorbate, and their salts; ergocalciferol, cholecalciferol, dihydroxystanal, etc. Vitamin D of dl-α (β, γ) -tocopherol, dl-α-tocopherol acetate, nicotinic acid-dl-α-tocopherol, linoleic acid-dl-α-tocopherol, succinic acid Examples include tocopherols such as dl-α-tocopherol and derivatives thereof, salts thereof, vitamin E such as ubiquinones, dibutylhydroxytoluene, butylhydroxyanisole, and the like.

Among the above active oxygen scavengers and antioxidants, mannitol, beta carotene, astaxanthin, rutin and its derivatives, ginkgo biloba extract, urgonum extract, quette extract, yukinoshita extract, melissa extract, gennoshoco Extracts, Ages extract, vitamins C and their derivatives and salts thereof, vitamin E and its derivatives and salts thereof, and more preferable are beta carotene, astaxanthin, ginkgo extract, vitamins C and Their derivatives and their salts, vitamin E and its derivatives and their salts.
The concentration of the active oxygen removing agent or antioxidant in the external preparation for skin of the present invention is preferably 0.00001 to 50% by weight, more preferably 0.0001 to 30% by weight. However, when using a plant extract, there is no problem as long as the dry solid content is within the above range. The active oxygen scavenger or antioxidant can be used alone or in combination of two or more.

(Cell activator)
Examples of the cell activator include adenylate derivatives such as deoxyribonucleic acid and salts thereof, adenosine triphosphate and adenosine monophosphate and salts thereof, ribonucleic acid and salts thereof, guanine, xanthine and derivatives thereof and salts thereof. Nucleic acid-related substances such as serum deproteinized extract, spleen extract, placenta extract, chicken crown extract, royal jelly extract, etc .; yeast extract, lactic acid fermentation extract, bifidobacteria extract, ganoderma extract Extracts derived from microorganisms such as foods; extracts derived from plants such as carrot extract, assembly extract, rosemary extract, agaric extract, garlic extract, hinokitiol, cephalanthin; α- or γ-linolenic acid, Eicosapentaenoic acid and their derivatives, succinic acid and its derivatives and their salts, estradiol and its derivatives And α-hydroxy acids such as lactic acid, glycolic acid, citric acid, malic acid, salicylic acid, and derivatives thereof, and salts thereof.

Among these cell activators, particularly preferred are deoxyribonucleic acid and its salt, adenosine triphosphate and its salt, serum deproteinized extract, placental extract, yeast extract, lactic acid fermentation extract, carrot extract, Hinokitiol, succinic acid and its derivatives and their salts.
The concentration of the cell activator in the external preparation for skin of the present invention is preferably 0.0001 to 5% by weight, more preferably 0.001 to 3% by weight. However, when using a plant extract, there is no problem as long as the dry solid content is within the above range. Moreover, a cell activator can be used 1 type or in combination of 2 or more types.

(Anti-inflammatory agent)
Anti-inflammatory agents include glycyrrhizic acid, glycyrrhetinic acid, mefenamic acid, phenylbutazone, indomethacin, ibuprofen, ketoprofen, allantoin, guaiazulene and their derivatives and their salts, ε-aminocaproic acid, zinc oxide, diclofenac sodium, aloe extraction Products, salvia extract, arnica extract, chamomile extract, birch extract, hypericum extract, eucalyptus extract, mukuroji extract and the like.
Among these anti-inflammatory agents, particularly preferred are glycyrrhizic acid, glycyrrhetinic acid, guaiazulene and derivatives thereof, and salts thereof, ε-aminocaproic acid, aloe extract, chamomile extract.
The concentration of the anti-inflammatory agent is generally 0.0001 to 1%, preferably 0.01 to 0.5% in the composition. However, when using a plant extract, there is no problem as long as the dry solid content is within the above range. Moreover, an anti-inflammatory agent can be used individually or in combination of two or more.

(Tyrosinase activity inhibitor)
Examples of tyrosinase activity inhibitors include cysteine and derivatives thereof (for example, N, N′-diacetylcystine dimethyl, etc.) and salts thereof, Sempukuka extract, quette extract, sunpens extract, Sohakuhi extract, Toki extract, Ibukitorano extract Products, Clara extract, hawthorn extract, white lily extract, hop extract, Neubara extract, Yokuinin extract and the like.
The concentration of the tyrosinase activity inhibitor is preferably 0.0001 to 2%, particularly preferably 0.001 to 0.5%. However, when using a plant extract, there is no problem as long as the dry solid content is within the above range. In addition, these can be used 1 type or in combination of 2 or more types.

(Humectant)
As the humectant, not only synthetic compounds such as urea, but also low molecular weight compounds such as amino acids known as natural moisturizing factors, pyrrolidone carboxylic acid and lactate can be used. In addition, mucopolysaccharides and / or proteins that are constituents of the skin and are conventionally blended in cosmetics can be used.
Among these, amino acids described in “Development of Newly Useful Cosmetics”, Masato Suzuki / Supervision, CMC Publishing, September 2004, pages 16 to 19, can be mentioned.
Examples of the mucopolysaccharide include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate, and salts thereof. Hyaluronic acid, chondroitin sulfate, and salts thereof can be preferably used. .
Examples of the protein include collagen, elastin, keratin and derivatives thereof, and salts thereof, and collagen is particularly preferable. There is no restriction | limiting in particular about the origin of each of these components, Any of animal origin, microorganism origin, and a synthetic product may be sufficient. There are no particular restrictions on the extraction method and the purification treatment method in the case of natural origin.
These moisturizing components may be of one type, or two or more types can be added and used as appropriate.
Furthermore, the amount of the moisturizing agent varies depending on the combination of the components, but generally 0.0001 to 5% is preferable, and 0.001 to 3% is more preferable.

  Compositions of amino acids, vitamin B and hydrolyzed silk comprising the amino acids of the present invention or their salts can be prepared by blending with various forms of bases known as ordinary external compositions according to conventional methods. . That is, oil agents (natural animal and vegetable oils, semi-synthetic oils and fats, hydrocarbon oils, higher fatty acids, ester oils, silicone oils, fluorine-based oil agents, etc.), gelling agents, metal soaps, surfactants (anionic, cationic, amphoteric, Powders (inorganic powders, organic powders, pigments, etc.), alcohols (higher alcohols, polyhydric alcohols, sterols, etc.), water-soluble polymers (animal and vegetable systems, microbial systems, synthetic systems), film forming agents, resins, Preservatives, antibacterial agents, fragrances, essential oils, salts, water (purified water, hot spring water and deep water), pH adjusters, fresheners, rough skin improvers, blood circulation promoters, skin astringents, antiseborrheic agents, amino acids Nucleic acid-related substances, enzymes, hormones, inclusion compounds, plant extracts, animal and microbial extracts, UV scattering agents and the like can be added.

  For amino acids used as essential in the present invention, hydrolyzed silk, amino acids used additionally added, vitamin Bs, and hyaluronic acid, the amount used exceeding the upper limit of the amount added of the present invention has a particularly negative physiological effect. However, the positive effect is saturated near the upper limit amount presented in the present invention, and the benefit of increased addition amount is not produced. Further, addition exceeding the upper limit of the present invention may lead to deterioration of the use ring such as stickiness.

  Hereinafter, the present invention will be described in detail based on examples, but it goes without saying that the present invention is not limited thereto.

Example 1 (Skin permeability test)
Skin for Permeation Test Abdominal skin of a 10-20 week old male male hairless mouse (Kyudo Co., Ltd.) was removed and used after removing subcutaneous fat.

Skin Permeation Test Skin samples were mounted in a Kesany-Chien type diffusion cell. 1 ml of the test solution (described later) was added to the donor phase, and the upper part was sealed with parafilm. 0.01 mol / l phosphate buffered saline (Wako Pure Chemical Industries, Ltd.) is added to the receptor phase, stirred, incubated at 32 ° C., and after 24 hours, the amount of amino acid is quantified by HPLC, and the transmittance is calculated. did.

Amino acid quantification method The HPLC amino acid analysis system (Prominence) of Shimadzu Corporation was used and quantified by the post-column fluorescence detection method using OPA (orthophthalaldehyde) as a reaction reagent.

Test solution 20 kinds of amino acids (Wako Pure Chemicals: valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, Serine) in an amount of 0.1% by weight, 1,3-butylene glycol (Wako Pure Chemicals) in an amount of 1% by weight, and 1 weight of hydrolyzed silk (Seiwa Kasei: Promois Silk) as an additive % And 0.01 mol / l phosphate buffered saline (Wako Pure Chemical Industries, Ltd.) were mixed to prepare Test Solution 1.

  Instead of hydrolyzed silk, glucose (Wako Pure Chemical), trehalose (Wako Pure Chemical), marine collagen (Nitta Gelatin, Maringen S-06), marine collagen (Nitta Gelatin, Maringen SP-03), Pig dermis-derived collagen (Nitta Gelatin, Collagen P) was mixed in the same manner to prepare test solutions 2, 3, 4, 5, and 6.

  The test solution was tested by the above-mentioned skin permeation test method, and the results of measuring the transmittance of all permeated amino acids are shown in Table 1.

The hydrolyzed silk of the present invention showed a permeation promoting effect superior to that of glucose and trehalose saccharides, and a permeation promoting effect superior to other hydrolyzed peptides.
The transmittance of each amino acid when the additive of the present invention is hydrolyzed silk is shown in Table 2.

  Leucine, isoleucine, arginine, lysine, aspartic acid, and threonine show excellent skin permeability in the presence of hydrolyzed silk under the test conditions. This is due to the synergistic effect of these amino acids and hydrolyzed silk. It suggests the possibility of being infiltrated.

Example 2 (Human normal keratinocyte proliferation test)
Preparation of Purified Type IV Collagen-Acetic Acid Solution According to the following protocols (1) to (16), 8 mg of purified polymer type IV collagen was obtained from the porcine eyeball as a raw material. The following protocol was performed at 4 ° C.

(1) Remove the cornea from the eyeball using scissors and remove the lens from the inside of the eyeball.
(2) Remove insoluble parts such as vitreous adhering to the lens as much as possible with scissors.
(3) Add 1 tablet of complete protease inhibitor cocktail (Roche) to 50 ml of cold PBS (phosphate buffered saline), add the lens capsule, and stir for 2 hours.
(4) Centrifugation (2000 g, 10 minutes, 4 ° C.) to remove unnecessary sites present in the supernatant.
(5) The precipitate is suspended in 25 ml of 0.5 M acetic acid in which a complete protease inhibitor cocktail half tablet (Roche) is dissolved.
(6) Crush finely using a homogenizer (IKA).
(7) The finely crushed lens capsule is stirred for 3 days to extract type IV collagen.
(8) Centrifugation (2000 g, 10 minutes, 4 ° C.) to separate the supernatant (acetate-soluble collagen) and the precipitate.
(9) Repeat this agitation extraction and centrifugation once more.
(10) To the supernatant obtained by centrifugation, add NaCl in which crystals are ground as much as possible in a mortar to a final concentration of 1.7M.
(11) Stir overnight to precipitate collagen.
(12) Centrifugation (5000 g, 30 minutes, 4 ° C.) to collect the precipitate.
(13) Add 0.5 M acetic acid to the precipitate and dissolve it sufficiently.
(14) An acidic collagen aqueous solution is placed in a dialysis tube (Sanko Junyaku) and dialyzed using 0.5 M acetic acid.
(15) Further, dialysis is performed with 2 mM hydrochloric acid.
(16) Collect the collagen solution after dialysis to obtain a purified type IV collagen solution.

Procedure for culture on collagen film The aforementioned type IV collagen-acetic acid solution (1 mg / ml) is diluted 10-fold with sterilized ultrapure water, and 50-100 μl per square centimeter is poured into a 48-well culture dish or plate.
2. Extend thinly to spread over the entire culture surface.
3. Open the lid of the culture dish or plate in the clean bench and dry at 25 ° C or lower.
4). After drying, in order to remove the acid from the type IV collagen film, it is washed with a medium three times.
5. 3500 normal keratinocytes (Sanko Junyaku CC-2503-NZ) are seeded per square centimeter.
6). Place IV collagen plate in CO ₂ incubator and start culture.
7. Change medium every 2 days and incubate for 8 days.

Skin cell proliferation test The proliferation test was performed using the MTT Cell Growth Assay Kit.
Reagent A: MTT, (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrasodium bromide), 50 mg / vial.
Reagent B: PBS pH 7.4, 15 mL
Reagent C: Color development solution (isopropanol with 0.04N HCl), 100 mL
Add 10 ml of reagent B to reagent A and mix well.
Leave in a cool dark place overnight.
The AB reagent is sterilized by filtration through a 0.22 μm filter.
Add 30 μl of AB reagent per cm ^{2 and} mix gently.
Incubate for 4 hours in a CO ₂ incubator.
After culture, the culture supernatant is transferred to a tube.
Add 300 μl of Reagent C per cm ² .
After mixing, transfer to a tube containing the culture supernatant.
Absorbance (570 nm) is measured within 1 hour.

Test medium Each additive (Wako Pure Chemical Industries) was added to Brett Kit KGM-2 Medium (Proliferation Medium for Epidermal Keratinocytes) Sanko Pure Chemical CC-1307-NZ so as to have the concentration shown in Table 3-15. The test medium was used. The test medium shown in Table 13 has a composition similar to that shown in the known examples of JP-A-61-289016.

Results of skin cell proliferation test The absorbance at the time of no addition was defined as 100, and the relative concentrations relative thereto were shown in Tables 16 and 17.

The composition 2-1 of the present invention promotes the growth of human epidermal keratinocytes on the type IV collagen plate, and its effect is enhanced by vitamin B6 and vitamin B1 (the composition 2-2 of the present invention). (The composition 2-3 of the present invention).
The influence of the concentration of each amino acid is shown in Compositions 2-4 to 2-13 of the present invention. The cell growth effect was substantially saturated at the upper limit indicated for each amino acid. When the amount of hydrolyzed silk is 5 μg / ml, there is a growth effect (Invention composition 2-14), but when it is reduced to 3 μg / ml, the effect is lost (Comparative composition 2-1). There is no growth effect even if the kind is added (Comparative composition 2-1).
When the amino acid of the present invention was not added, no cell proliferation effect was obtained (Comparative Examples 2-3 to 2-8).
Human epidermal cell proliferation effects were not observed only with known amino acid combinations (Comparative Examples 2-9 to 2-11).

(Cosmetics test)
Reference example 1
A skin care lotion was made with the following composition.
Each component was mixed at 70 ° C. so as to have the concentrations shown in Table 18 below to obtain an aqueous solution, and then cooled to room temperature.

Reference example 2
A skin care emulsion was prepared with the following composition.
A liquid Each component was mixed at 70 degreeC so that it might become a composition of Table 19, and it was set as the aqueous solution.

B liquid Each component was mixed at 70 degreeC so that it might become a composition ratio of Table 20.

Production Method 65 ml of Liquid A and 15 g of Liquid B were mixed at 70 ° C., 20 ml of xanthan gum (2% aqueous solution) was added, and mixed at 70 ° C. until uniform. Then, it cooled to room temperature.

Reference example 3
A skin care cream was prototyped with the following composition.
A liquid Each component was mixed at 70 degreeC so that it might become a composition of Table 21, and it was set as the aqueous solution.

B liquid Each component was mixed at 70 degreeC so that it might become the composition ratio of Table 22. FIG.

Manufacturing method 51 ml of liquid A and 40 g of liquid B were mixed at 70 ° C., 1.0 g of triethanolamine was added, and the mixture was mixed at 70 ° C. until the emulsion became uniform. Then, it cooled to room temperature.

Example 3
Skin Care Lotion Evaluation Method Test Panel 10 healthy women aged 27-35 years, average age 34.2 years Location Room with a temperature of about 24 ° C and humidity of about 55 percent.
Evaluation method It was applied to a random position inside the forearm after washing, and the feeling of use (sensory feeling) was tested. The sensory test results were scored according to the following criteria.

4 Feels very well 3 Well feels well 2 Doesn't feel much 1 Cannot feel

  And the average of the evaluation score of 10 panelists was calculated and ranked as follows.

A 3.2 or more B 2.7 or more and less than 3.2 C 2.2 or more and less than 2.7 D 1.7 or more and less than 2.2 E less than 1.7

Skin Care Lotion Comparative Example 3-1 with the composition of Table 18 as the present invention except for the hydrolyzed silk, Comparative Example 3 with hydrolyzed silk replaced with marine collagen (Nitta Gelatin, Maringen SP-03) -2, The substitute for porcine dermis-derived collagen (Nitta Gelatin, Collagen P) was used as Comparative Example 3-3.

  The result of inquiring about the impression after about 5 minutes after applying the film so as to be sufficiently applied is shown below.

  As is apparent from the table, the amino acid lotion combined with the hydrolyzed silk of the present invention has a strong penetrating feeling, is excellent in moistness and stretch, and is an excellent cosmetic product overall.

Example 4 (moisturizing effect)
The evaluation panel is the same as before, with an average age of 32.7 years.
The moisturizing effect was evaluated by measuring the amount of horny moisture by the high frequency impedance method. (Asahi BioMed's high-sensitivity angular layer thickness moisture meter ASA-MX, double frequency phase difference amplitude detection method)
Emulsion for skin care Comparative example 4-1 excluding hydrolyzed silk in Table 19; comparative example 4-2 replacing hydrolyzed silk with marine collagen (Nitta Gelatin, Maringen SP-03), porcine dermis The substitute for the derived collagen (Nitta Gelatin, Collagen P) was designated as Comparative Example 4-3.

  After application so as to be sufficiently applied, the skin conductivity was measured about 18 hours later, and the increase rate after application was determined.

  As apparent from the table, the composition of the present invention showed excellent moisture retention.

Example 5
Evaluation method The evaluation panel is the same as before, with an average age of 36.4 years.
Each day, twice in the morning and noon, after washing both hands, it was applied to the back of the hand and used for 2 weeks.
The test results were scored according to the following criteria.
Skin activation effect

4 I feel a lot of improvement in skin tension and gloss 3 Well, I feel 2 I don't feel much 1 I can't feel

  And the average of the evaluation score of 10 panelists was calculated and ranked as follows.

A 3.2 or more B 2.7 or more and less than 3.2 C 2.2 or more and less than 2.7 D 1.7 or more and less than 2.2 E less than 1.7

  Rough skin suppression effect

4 I feel the improvement of roughness and rough skin 3 Well, I feel 2 I don't feel much 1 I can't feel

  And the average of the evaluation score of 10 panelists was calculated and ranked as follows.

A 3.2 or more B 2.7 or more and less than 3.2 C 2.2 or more and less than 2.7 D 1.7 or more and less than 2.2 E less than 1.7

  The composition of Table 21 is the present invention, except that the hydrolyzed silk is removed as Comparative Example 5-1, and the hydrolyzed silk is replaced with marine collagen (Nitta Gelatin, Maringen SP-03) as Comparative Example 5-2. A substitute for porcine dermis-derived collagen (Nitta Gelatin, Collagen P) was used as Comparative Example 5-3.

  As is apparent from the table, the composition of the present invention showed excellent skin activation and rough skin suppression effect.

Claims (9)

  A skin external preparation containing at least arginine, aspartic acid, isoleucine, leucine, lysine, threonine or salts thereof among amino acids and hydrolyzed silk.   Regarding the addition amount of amino acids or salts thereof, the addition amount of arginine is 20 to 400 microweight percent, the addition amount of aspartic acid is 3 to 60 microweight percent, the addition amount of isoleucine is 30 to 600 microweight percent, and the addition of leucine The external preparation for skin according to claim 1, wherein the amount is 30 to 600 microweight percent, the addition amount of lysine is 30 to 600 microweight percent, and the addition amount of threonine is 25 to 500 microweight percent.   2. The skin external preparation according to claim 1, wherein the amount of hydrolyzed silk added is 5 to 3000 microweight percent.   Furthermore, the skin external preparation in any one of Claims 1-3 containing at least 1 sort (s) among glycine, histidine, serine, valine, tyrosine, cysteine, phenylalanine, and those salts among amino acids.   The skin external preparation in any one of Claims 1-4 containing vitamin B.   The topical skin preparation according to claim 5, wherein the vitamin B is vitamin B1 or vitamin B6.   The skin external preparation in any one of Claims 1-6 containing a hyaluronic acid or its salt.   The skin external preparation according to any one of claims 1 to 7, wherein the skin external preparation is a cosmetic or a quasi-drug.   The skin external preparation according to claim 8, wherein the skin external preparation is a lotion, an emulsion, a cream, a hair nourishing agent, or a pack.