JP2895572B2 - Cosmetic and external preparation for skin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Definition of terms] In the present invention, "milk protein" is defined as casein, casein-fractionated _αs -casein, β-casein, γ-casein, and _K -casein, whey protein, whey. Α-lactalbumin and β-lactoglobulin obtained by fractionating proteins, and one or a mixture of two or more thereof. The “hydrolysate” is a peptide mixture obtained by degrading milk protein with a protease. Wherein the molecular weight of the peptide is 10
Not more than 00, and 90% (by weight) of the aromatic amino acids contained.
The same applies hereinafter) is a free amino acid, a hydrolyzate having a growth activating effect on human skin cells, and having no antigenicity as a milk protein and / or a salt thereof. , Cosmetics referred to in the Pharmaceutical Affairs Law, quasi-drugs, products included in any of the pharmaceuticals, lotion, cream, milky lotion, pack, foundation, face wash, soap,
Cosmetics such as hair cosmetics and hair wash cosmetics and / or external preparations for skin.

[Field of Industrial Application] The present invention relates to cosmetics and the like characterized by containing a hydrolyzate of milk protein as an active ingredient, and more specifically, does not show antigenicity of milk protein and proliferates on human skin cells. The present invention relates to cosmetics and the like which have an effect of imparting activity, recovering from rough skin, smoothing the skin by adjusting texture, and the like.

[Background of the Technology and Description of the Prior Art] Conventionally, a technique of blending a peptide or amino acid obtained by hydrolyzing milk protein into a cosmetic or the like is known. For example, there are JP-A-57-209210, JP-A-58-500664, JP-A-59-152317, and JP-A-63-57515. These prior arts are intended to promote the solubilization of zinc pyrithione, suppress dental caries, and be used as a component of oral products or solvents. In addition,
JP-A-60-258102, JP-A-62-185100, JP-A-64
No. -11 and Japanese Patent Application Laid-Open No. 1-269499 disclose a technique for blending in cosmetics and the like. The reason that these components are blended into cosmetics and the like is mainly due to the skin beautiful effect by the moisturizing, adsorbing, film-forming or protecting effects of these components.

These components in the prior art are merely hydrolyzed milk proteins, and it is intended to obtain a degraded product having specific physicochemical and biological properties and use it in cosmetics and the like. Not.

Some of the present inventors, previously, for the purpose of solubilization of milk protein, effectiveness of digestion and absorption, prevention and treatment of food allergy, treatment and nutritional supplementation of aromatic amino acid metabolism disorders,
A patent application was filed for inventing a hydrolyzate of protein of milk without antigenicity and a method for producing the same (Japanese Patent Application No. 63-291090).
issue. Hereinafter referred to as the earlier application). The present inventors conducted various studies on the hydrolyzate obtained by the prior application, and as a result, the hydrolyzate obtained by the method of the prior application was found to have a moisturizing effect, etc. possessed by a conventionally known milk protein hydrolyzate. In addition to the beautiful skin effect of, has been found to have a growth activating effect on human skin cells,
The present invention has been completed.

[Object of the Invention and Summary of the Invention] It is an object of the present invention to provide a cosmetic or the like containing a hydrolyzate containing a peptide that imparts proliferative activity to human skin cells.

Another object of the present invention is to provide a cosmetic or the like containing a hydrolyzate containing a peptide that does not exhibit the antigenicity of milk protein.

The present invention is a peptide mixture obtained by hydrolyzing milk protein, the molecular weight of the peptide is 1000 or less,
90% or more of the aromatic amino acids are free amino acids, have a growth stimulating effect on human skin cells, and contain a hydrolyzate and / or a salt thereof having no milk protein antigenicity as an active ingredient. And the like.

[Specific description of the invention] The milk proteins used in the present invention are all commercially available or easily available, and are usually prepared by dissolving or concentrating in water at a concentration of 5 to 20%. I do.

The enzyme used for preparing the hydrolyzate is not particularly limited, and can be trypsin, chymotrypsin, subtilisin, elastase, proline-specific protease, protease produced by a microorganism of the genus Streptococcus, papain,
Commercial products such as pepsin and thermolysin can be used. or,
Carboxypeptidase Y, as an exopeptidase
Proteases produced by microorganisms of the genus Aspergillus, proteases produced by microorganisms of the genus Streptomyces, proteases produced by microorganisms of the genus Rhizopus, lactic acid bacteria extracts, proteases produced by lactic acid bacteria, and the like can be used.
As the lactic acid bacteria extract, for example, a lactic acid bacteria extract having 20,000 activity units per gram can be obtained by the method described in JP-B-48-43878.

In addition, the amount of enzyme used for hydrolysis is 30 g / g of milk protein.
Pancreatin and exopeptidase or pancreatin and other proteases and exopeptidase are mixed or added in a ratio of 00 to 5000 activity units.

The temperature condition is 40 to 55 ° C., and the pH condition is adjusted to an optimum pH within a range that does not denature the raw material protein before addition of the enzyme, and preferably the pH is maintained for at least 1 hour.

After the completion of the hydrolysis, the enzyme is deactivated by heating, and after cooling, filtration, desalting, concentration and drying are performed as necessary to obtain a hydrolyzate.

The hydrolyzate used in the present invention is prepared as described above.

Cosmetics and the like using the hydrolyzate are produced by a conventional method. The hydrolyzate is easily soluble in water used for production at a concentration of 20% or less, and a predetermined amount is dissolved in, for example, purified water,
It is mixed, emulsified and dispersed with other components. When the concentration of the hydrolyzate exceeds 20%, the solubility gradually decreases as the concentration increases.

As salts of the hydrolyzate, organic and inorganic salts acceptable as pharmaceuticals, quasi-drugs and cosmetics such as sodium and potassium are used.

If necessary, emulsifiers, fragrances, and other components that are acceptable as pharmaceuticals, quasi-drugs, and cosmetics can also be used at the same time.

 Next, the present invention will be described in detail with reference to test examples.

(Test Example 1) This test was performed to examine the physicochemical properties of the hydrolyzate.

(1) Preparation of hydrolyzate 1) 1 g of an enzyme solution obtained by mixing a lactic acid bacterium extract, a protease derived from the genus Aspergillus, and pancreatin in a 10% casein solution (pH 8.0) in 1000 activity units each
Per unit so that the sum of the three active units per unit is 3000 units.
Decompose at 50 ° C and measure the degree of decomposition over time. When formal nitrogen / total nitrogen (%) becomes 21,32,41 (%), 90%
Deactivate by heating at ℃ for 5 minutes After inactivation, filter until no precipitate is present and freeze-dry. This freeze-dried product is hereinafter referred to as samples A, B, and C.

2) The enzyme of the above method is added to a 10% whey protein solution (pH 7.0), and a degradation product having a degree of degradation of 30% or 41% is prepared in the same manner as the above method. This lyophilized product is hereinafter referred to as Samples D and E.
And

(2) Measurement of aromatic amino acid release rate Total amino acids and free amino acids were analyzed by an automatic amino acid analyzer.

Table 1 shows the results of amino acid analysis of Samples A to E prepared in the above (1).

As is clear from Table 1, the release rate of aromatic amino acids increased with the increase in the degree of decomposition, and it was found that samples C and E were 90% or more. However, the aromatic amino acid release rate of the cosmetic casein peptide having an average molecular weight of 300 to 3000 produced by the conventional method (the same method as Example 1 described in JP-A-1-269499) was less than 90%.

(3) Measurement of molecular weight Samples C and E prepared in the above (1) were eluted with 0.5N acetic acid using a Sephadex G-25 column to measure the molecular weight. As a result, the first fractions of samples C and E eluted by the gel filtration method eluted later than the oxytocin (molecular weight 1000) used as the standard substance, so that the molecular weights of samples C and E are 1000 or less. There was found.

(Test Example 2) This test was performed to examine the antigenicity of the hydrolyzate.

The antigenicity of Samples A to E prepared in Test Example 1 was measured by an ELISA inhibition test. 96-well plate (N
unc Co.), coated with the starting protein, washed, reacted with a mixture of rabbit antiserum prepared by sensitizing the starting protein and various hydrolysates, washed, and washed with alkaline phosphatase-labeled goat anti-rabbit. IgG antibody (Zymed Labor
atories), and after washing, the enzyme substrate p
-Add sodium nitrophenyl phosphate and after 30 minutes 5N
The reaction was stopped with sodium hydroxide, and the reaction product was measured with a microplate reader (Toho Medical Association, No. 35
Vol., 509 pages, 1989). As a result, the antigenicity of the hydrolyzate decreased as the aromatic amino acid release rate increased. That is, in sample A having a release rate of 31%, about 1 /
In 400 and 57% of sample B, it was confirmed that the antigenicity was reduced to about 1/30000 of casein, and in sample C of 90% or more, antigenicity was lost. Similarly, in the whey protein, antigenic elimination was observed in sample E having a release rate of aromatic amino acids of 90% or more. However, in the casein peptide for cosmetics produced by the conventional method (the same method as Example 1 described in JP-A-1-269499) having an aromatic amino acid release rate of less than 90%, the antigenicity of casein was clearly left. .

(Test Example 3) This test was performed to examine the activity of stimulating the proliferation of human skin cells.

Sample C prepared in Test Example 1 above, a commercially available bovine placenta extract (manufactured by Botoger Co., West Germany), and sample E prepared by a conventional method (the same method as Example 1 described in JP-A-1-269499) are described below. Was used to test the growth stimulating activity of human skin cells.

Epithelial cells derived from human normal skin were cultured in a modified KGM medium and fibroblasts were cultured in a modified MEM medium containing 2% fetal bovine serum, respectively, by a conventional method. Sample C, commercially available bovine placenta extract and F contained 1, 10, and 100 μg / ml in the medium.
, The culture was continued for 2 to 3 days, then thymidine labeled with [ ³ H] was added, and the culture was continued for another 2 to 3 hours. Then, the amount of [ ³ H] incorporated into the cells was measured by liquid scintillation. Measured with a counter. Each medium to which nothing was added was used as a control.

 The results of this test were as shown in Table 2.

As is clear from Table 2, sample C has a remarkable growth activating effect on human skin cells, and has a stimulating effect that is equal to or 1.6 times or more that of bovine placenta extract, which has been widely known to have a growth activating effect on human skin cells. Had. In contrast, sample F was as effective as the control. still,
Sample E prepared in Test Example 1 (1) also showed a remarkable growth activating effect on human skin cells, similar to Sample C.

(Test Example 4) This test was performed to examine the effects of cosmetics and the like depending on the presence or absence of a hydrolyzate.

(1) Preparation of samples Seven samples (samples 1 to 7) containing a hydrolyzate were prepared by the same formulation and production method as in Examples 1 to 7, and seven types containing the hydrolyzate having the following formulation were used. (A to G: control) were prepared by a conventional method. In addition, as is clear from the composition of the example and the following composition, sample 1 corresponds to A and sample 2 corresponds to B.

1) Sample A A skin cream having the following composition was produced.

Stearic acid 15.0 (%) Cetanol 2.0 Squalane 3.0 Octyldodecyl myristate 5.0 Glycerin 10.0 1,3-butylene glycol 4.0 Polyoxyethylene monostearate (20 mol) Sorbitan 3.0 Purified water 58.0 2) Sample B Make up lotion with the following formulation did.

Propylene glycol 10.0 (%) Oleyl alcohol 0.1 Hydrogenated castor oil polyoxyethylene (60 mol) adduct 1.5 Ethanol 5.0 Purified water 83.4 3) Sample C A beauty serum having the following composition was prepared.

Sodium hyaluronate 0.1 (%) Placenta extract 1.5 Glycerin 1.0 Purified water 97.4 4) Sample D A skin external ointment having the following formulation was produced.

Vaseline 25.0 (%) Paraffin 5.0 Cetostearyl alcohol 2.0 Propylene glycol 10.0 Polyoxyethylene / polyoxypropylene glycol ether 3.0 Purified water 55.0 5) Sample E A hair treatment having the following composition was produced.

Cetanol 1.5 (%) 2-hexyldecanol 1.0 1,3-butylene glycol 3.0 cationized cellulose 0.2 polyoxyethylene stearyl ether 1.0 purified water 93.3 6) Sample F A hair rinse having the following composition was produced.

Dialkyldimethylammonium chloride 2.0 (%) Cetanol 1.0 Polyoxyethylene stearyl ether 1.0 1,3-butylene glycol 3.0 cationized cellulose 0.2 purified water 92.8 7) Sample G A hair shampoo having the following composition was produced.

Triethanolamine lauryl sulfate 15.0 (%) 1,3-butylene glycol 2.0 Ethylene glycol monostearate 1.5 Purified water 81.5 (2) Test method 1) Tests for skin cosmetics, etc. 20 to 40-year-olds with apparent skin roughness 5 expert panels, the inside of the left hand arm was thoroughly washed with a commercially available toilet soap, and it was confirmed that there was no residual soap. Samples 1-4 and Samples A-D (control) 0.5 g each , Each about 30mm x 15mm
And rubbed into the skin. This rubbing on the skin was performed once a day for four consecutive weeks, and the determination was performed once a week by the following method.

Observe the rubbing area of each sample with the naked eye to prevent rough skin,
For recovery from rough skin, smoothing skin, adjusting skin texture, applying skin tension, protecting skin, and softening skin, +2 if control sample is significantly better than control sample. + If slightly better than sample
1, 0 if equal to control sample, -1 if slightly worse than control sample,-if significantly worse than control sample
Evaluation was made in five steps of 2, the average value was calculated, and the effect was tested.

2) Testing of hair cosmetics, etc. 20 to 40 with obvious hair damage and pastsuki
We selected 15 panelists from the age group and divided them into 3 groups of 5
Samples 5 and E were tested in the first group, samples 6 and F in the second group, and samples 7 and G in the third group. The hairs of the panels of the first and second groups were thoroughly washed with a commercially available shampoo, and after confirming that no shampoo remained, 2 g of each sample was applied to hair of about 50 mm × 80 mm on both sides of the temporal region. Rubbed by hand. In the second group for testing the hair rinse, the hair was lightly washed with water immediately after the sample was rubbed. The rubbing of the hair was performed once every four days, the hair was washed on the fourth day, and the evaluation was performed 10 times every four days by the following method. In the third group in which the shampoo was tested, the hair was not pre-washed with a commercially available shampoo, and the sample 7 was used.
And G, the left and right hairs were washed separately. The hair was washed once a day for 15 consecutive days, and was evaluated by the following method immediately before daily hair washing.

Visually observe the rubbing area of each sample to recover damaged hair,
For each item of prevention of cut and split ends, flexibility of hair, smoothness after finishing, disappearance of pastsuki, and gloss of hair, +2 when considerably better than control sample, and slightly better than control sample Is evaluated in five steps: +1, 0 when equivalent to the control sample, -1 when slightly poorer than the control sample, and -2 when significantly worse than the control sample, calculate the average value, and evaluate the effect. Tested.

(3) Test results The results of this test are as shown in Tables 3 and 4.

In Samples 1 and 2, as compared with Samples A and B, all five panelists had an effect of recovering from rough skin, and four panelists had an effect of preventing skin tension and rough skin.

Compared with Sample C, Sample 3 showed an effect of preventing skin roughness, recovering from skin roughness, improving skin texture, and smoothing skin.

In Sample 4, as compared with Sample D, all five panelists had an effect of recovering from rough skin, and four had an effect of preventing rough skin and smoothing the skin.

In Sample 5, as compared with Sample E, the effects of recovery of hair damage, prevention of pastsuki, and prevention of cut and split ends were observed in 4 subjects.

In Sample 6, compared to Sample F, all five panelists were found to have the effect of recovering from damaged hair and softening the hair, and four panelists were found to have a smooth finish, prevent pastsuki, and prevent cut hair and split ends. Was.

In Sample 7, as compared with Sample G, four persons were found to be able to recover hair damage, have a smooth finish, and have an effect of preventing pastsuki.

As described above, the combination of the hydrolyzate showed an excellent effect for both skin products and hair products.

(Test Example 5) This test was performed to examine the amount of the hydrolyzate added.

(1) Preparation of Sample A skin cream and a hair treatment were manufactured according to the same formulation as in Examples 1 and 5. However, the amount of the hydrolyzate was adjusted to 0.01 to 50% as shown in Table 5, and the amount of purified water was increased or decreased accordingly. A sample containing no hydrolyzate was prepared in the same manner as Samples A and E of Test Example 4 and used as a control.

(2) Test method The same method as in Test Example 4 was used. However, skin cream was evaluated in the same way based on three items of recovery from rough skin and imparting tension to the skin and prevention of rough skin, and hair treatment was similarly evaluated based on two items of recovery of damaged hair and prevention of cut hair and split ends.

(3) Test results The results of this test were as shown in Table 5. With skin cream and hair treatment, the above effect was remarkably observed with an increase in the amount of the hydrolyzate added, and the effect was maximized in the range of 1 to 20%, and decreased at 30 to 50%. It is presumed that the decrease in this effect is caused by the fact that the solubility of the hydrolyzate in water cannot be uniformly dissolved or dispersed in the sample. Therefore, the desirable addition amount of the hydrolyzate is 20% or less, more preferably 0.01 to 20%.

(Test Example 6) This test was performed to examine the amount of the hydrolyzate added to products other than the product tested in Test Example 5.

(1) Preparation of Sample A lotion, a serum, a skin external ointment, a hair rinse, and a hair shampoo were produced by the same formulation as in Examples 2, 3, 4, 6, and 7. However, the amount of the hydrolyzate was adjusted to 0.01%, 20% and 50% as shown in Table 6, and the amount of purified water was increased or decreased accordingly. A sample containing no hydrolyzate was prepared in the same manner as samples B, C, D, F and G of Test Example 4 and used as a control.

(2) Test method The same method as in Test Example 5 was used.

(3) Test results The results of this test were as shown in Table 6. As is evident from Table 6, the desired hydrolyzate additive in these products was also 0.01-20%.

 Next, a production example of the hydrolyzate used in the present invention will be described.

Reference Example 1 200 g of commercially available casein was suspended in water to a concentration of 10%.
The pH was adjusted to 8.0 with a 10% aqueous sodium hydroxide solution.
After heat sterilizing the casein solution at 90 ° C. for 10 minutes, the solution was cooled to 45 ° C., and 10 g of pancreatin F (Amano Pharmaceutical), 2 g of protease N “Amano” (Amano Pharmaceutical), and 4 g of the lactic acid bacteria extract were added. Hydrolysis for hours. The digest was heated at 90 ° C. for 5 minutes to inactivate the enzyme and filtered to remove the precipitate.
This was freeze-dried to obtain about 165 g of a powder hydrolyzate.

The obtained hydrolyzate was tested in the same manner as in Test Examples 1, 2, and 3, and as a result, the release rate of aromatic amino acids was 90.5%, the casein did not show antigenicity, and the molecular weight was 1,000 or less. The effect of stimulating the proliferation of human skin cells was observed.

Reference Example 2 200 g of a commercially available whey protein powder was dissolved in deionized water at a concentration of 8%. The bacteria were removed by filtration through a filter, the temperature was adjusted to 45 ° C., and 2 g of pancreatin F (Amano Pharmaceutical) was added while maintaining the pH at 7.5 by adding a 5% aqueous sodium hydroxide solution.
The solution was added 6 times every 30 minutes, and after 15 hours, 2.0 g of actinase AS (Kaken Pharmaceutical) was added, followed by further decomposition for 5 hours. The enzyme was inactivated by heating at 90 ° C. for 5 minutes and filtered to remove the precipitate. This was freeze-dried to obtain about 160 g of a powdered hydrolyzate.

The obtained hydrolyzate was tested in the same manner as in Test Examples 1, 2 and 3, and as a result, the release rate of aromatic amino acids was 90.4%, it did not show the antigenicity of whey protein, and the molecular weight was 1000 or less. The effect of stimulating the proliferation of human skin cells was observed.

 Next, the present invention will be described in more detail with reference to examples.

In addition, since each Example was manufactured by the usual method, only the combination was shown.

Example 1 A skin cream having the following composition was produced.

Stearic acid 15.0 (%) Cetanol 2.0 Squalane 3.0 Octyldodecyl myristate 5.0 Glycerin 10.0 1,3-butylene glycol 4.0 Polyoxyethylene monostearate (20 mol) Sorbitan 3.0 Hydrolyzate of Reference example 1 5.0 Purified water 53.0 Example 2 Was prepared.

Propylene glycol 10.0 (%) Oleyl alcohol 0.1 Hydrogenated castor oil polyoxyethylene (60 mol) adduct 1.5 Ethanol 5.0 Hydrolyzate of Reference example 1 3.5 Purified water 79.9 Example 3 A beauty serum having the following composition was prepared.

Sodium hyaluronate 0.1 (%) Placenta extract 1.5 Glycerin 1.0 Hydrolyzate of Reference Example 2 4.0 Purified water 93.4 Example 4 A skin external ointment having the following formulation was produced.

Vaseline 25.0 (%) Paraffin 5.0 Cetostearyl alcohol 2.0 Propylene glycol 10.0 Polyoxyethylene / polyoxypropylene glycol ether 3.0 Hydrolyzate of Reference example 1 5.0 Purified water 50.0 Example 5 A hair treatment having the following composition was produced.

Cetanol 1.5 (%) 2-hexyldecanol 1.0 1,3-butylene glycol 3.0 cationized cellulose 0.2 polyoxyethylene stearyl ether 1.0 Hydrolyzate of Reference Example 2 4.0 Purified water 89.3 Example 6 A hair rinse having the following composition was produced.

Dialkyldimethylammonium chloride 2.0 (%) Cetanol 1.0 Polyoxyethylene stearyl ether 1.0 1,3-butylene glycol 3.0 Cationic cellulose 0.2 Hydrolyzate of Reference example 1 4.5 Purified water 88.3 Example 7 Production of hair shampoo having the following composition did.

Triethanolamine lauryl sulfate 15.0 (%) 1,3-butylene glycol 2.0 Ethylene glycol monostearate 1.5 Hydrolyzate of Reference Example 2 5.0 Purified water 76.5 [Effect of the Invention] The effects of the present invention are as follows. is there.

(1) An excellent skin product which recovers from rough skin and smoothes the skin by adjusting the texture of the skin is obtained.

(2) A hair product having an excellent effect of recovering damaged hair can be obtained.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/00 ADA A61K 37/18 ADA 38/17 37/16 (72) Inventor Seiji Kawaura 1 Doshomachi, Chuo-ku, Osaka-shi, Osaka 7--11, Iwase Cosfa Co., Ltd. (72) Inventor Yasuo Fukuwata 3-1-4-103 Nijigaoka, Aso-ku, Kawasaki City, Kanagawa Prefecture (72) Inventor Masanobu Nojiri 2-14-19 Tsukushino, Machida-shi, Tokyo (56 References JP-A-1-269499 (JP, A) JP-B-7-73507 (JP, B2) (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 7/00-7/50 A61K 37 / 18,37 / 16

Claims (2)

(57) [Claims] (1) The following characteristics (a) to (d): (a) a peptide mixture having a molecular weight of 1,000 or less as measured by elution from 0.5N acetic acid using Sephadex G-25, b) 90% (by weight) or more of the aromatic amino acids are free amino acids; (c) they have a growth activating effect on human skin cells; and (d) they do not have milk protein antigenicity. A cosmetic comprising a hydrolyzate of a milk protein and / or a salt thereof having the following. 2. The following characteristics (a) to (d): (a) a peptide mixture having a molecular weight of 1000 or less as measured by elution from 0.5N acetic acid using Sephadex G-25, b) 90% (by weight) or more of the aromatic amino acids are free amino acids; (c) they have a growth activating effect on human skin cells; and (d) they do not have milk protein antigenicity. An external preparation for skin, characterized by containing a hydrolyzate of milk protein and / or a salt thereof having the formula: