JPH0426604A - Cosmetic and dermatic drug for external use - Google Patents

Abstract

PURPOSE:To provide a cosmetic containing hydrolyzed milk protein consisting of a mixture of peptide having a specific molecular weight and containing the major part of aromatic amino acids in the form of free amino acids, capable of imparting proliferation activity to dermatic cell of human and free from antigenicity of milk protein. CONSTITUTION:The objective cosmetic and dermatic drug for external use contains, as an active component, a hydrolyzed product and/or its salt consisting of a peptide mixture produced by the hydrolysis of milk protein, having a peptide molecular weight of <=1,000, containing >=90% of aromatic amino acids in the form of free amino acids, effective in activating the proliferation action of human dermatic cell and free from antigenicity of milk protein. The milk protein is dissolved in water at a concentration of 5-20% or a milk protein solution is concentrated and hydrolyzed with 3,000-5,000 active unit of an enzyme (e.g. pancreatine and exopeptidase) per 1g of milk protein at 40-55 deg.C. The cosmetic is effective in giving a smooth and fine skin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Definition of Terms] In the present invention, "milk protein" refers to casein, α,-casein, and β-casein obtained by fractionating casein.

γ-casein, ni-casein, whey protein, and σ-lactalbumin obtained by fractionating whey protein.

and β-lactoglobulin, one or a mixture of two or more thereof, and the "hydrolyzate" is a peptide mixture obtained by decomposing milk protein with a protease,
The molecular weight of the peptide is less than 1000, more than 90% (by weight, below) of the aromatic amino acids it contains are free amino acids, have a proliferation-stimulating effect on human skin cells, and have the ability to act as a milk protein. Hydrolysates and/or salts thereof that do not have antigenicity, and "cosmetics, etc." are products that are included in cosmetics, quasi-drugs, or pharmaceuticals as defined in the Pharmaceutical Affairs Law, and include lotions, creams, Cosmetics such as emulsions, packs, foundations, face washes, soaps, hair cosmetics, hair washing cosmetics, etc., and/or skin external preparations.

[Industrial Application Field] The present invention relates to cosmetics, etc., characterized by containing a milk protein hydrolyzate as an active ingredient. This invention relates to a sugar-sweetening agent that imparts activity and has the effect of recovering from rough skin, adjusting skin texture, and smoothing the skin.

[Technical Background and Description of Prior Art] Techniques for blending peptides or amino acids obtained by hydrolyzing milk proteins into cosmetics and the like have been known. For example, JP-A-57-209210, JP-A-58-50
0664, JP-A-59-152317, and JP-A-6.3-57515. These conventional techniques promote the solubilization of zinc pyrithione, suppress dental caries,
It is intended for use as an ingredient in oral products or bath additives. Also, JP-A-60-258102, JP-A-62-185100, JP-A-64-1
] and Japanese Unexamined Patent Publication No. 1-269499 disclose techniques for incorporating it into cosmetics and the like. The reason why these ingredients are blended into cosmetics and the like is primarily because of their beautifying effects due to their moisturizing, adsorption, film-forming, or protective effects.

These ingredients in the above-mentioned conventional technology are simply hydrolyzed milk proteins, and it is difficult to obtain decomposed products with specific physicochemical and biological properties. I didn't intend it.

Some of the present inventors have previously investigated the use of milk proteins for the purpose of solubilization of milk proteins, effectiveness of digestion and absorption, prevention and treatment of food allergies, treatment of aromatic amino acid metabolic disorders, and nutritional supplementation. Invented a non-antigenic hydrolyzate and its manufacturing method and filed a patent application (Japanese Patent Application No. 63-2910)
No. 90. (hereinafter referred to as "prior application"). The present inventors conducted various studies on the hydrolyzate obtained in the earlier application, and found that the hydrolyzate obtained by the method in the earlier application has the moisturizing effect, etc. of the previously known milk protein hydrolyzate. In addition to its skin beautifying effect, it was discovered that it has a proliferation-activating effect on human skin cells, and the present invention was completed.

[Object of the Invention and Summary of the Invention] An object of the present invention is to provide cosmetics and the like containing a hydrolyzate containing a peptide that imparts proliferation activity to human skin cells.

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

The present invention is a peptide mixture obtained by hydrolyzing milk protein, in which the molecular weight of the peptide is 1000 or less, and 90% or more of aromatic amino acids are free amino acids,
Cosmetics, etc., characterized by containing as an active ingredient a hydrolyzate of milk protein and/or a salt thereof that has a proliferation-stimulating effect on human skin cells and does not have antigenicity.

[Detailed Description of the Invention] The milk proteins used in the present invention are all commercially available products or easily available, and are usually prepared by dissolving them in water at a concentration of 5 to 20% or by concentrating them. .

There are no particular restrictions on the enzyme used to prepare the hydrolyzate.
Commercial products such as trypsin, chymotrypsin, subtilisin, elastase, proline-specific brotease, bu07-ase produced by a microorganism of the genus Streptoconcus, papain, pebun, and thermolysin can be used. In addition, as exopeptidase, carpoxypebutidase Y, protease produced by microorganisms of the genus Aspergillus, protease produced by microorganisms of the genus Streptomyces, protease produced by microorganisms of the genus Rhizopus, lactic acid bacteria extract, and lactic acid bacteria are produced. Protease etc. can be used. A lactic acid bacteria extract having 20,000 active units per gram can be obtained, for example, by the method described in Japanese Patent Publication No. 48-43878.

In addition, the amount of enzyme used for hydrolysis is 3 per gram of milk protein.
Pancreatin and exobeptidase or vancreatin and other protease and exopeptidase are mixed or added in portions at a ratio of 000 to 5000 activity units.

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

After completion of hydrolysis, the enzymes are inactivated 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 above-mentioned hydrolyzate are manufactured by conventional methods. The hydrolyzate is easily soluble in the water used for production at a concentration of 20% or less, and a predetermined amount is dissolved in purified water, for example.
Mixed, emulsified, and dispersed with other ingredients. When the concentration of the hydrolyzate exceeds 20%, the solubility gradually decreases as the concentration increases.

As the salt of the hydrolyzate, organic and inorganic salts such as soot, lium, potassium, etc., which are acceptable as pharmaceuticals, quasi-drugs, and cosmetics, are used.

If necessary, emulsifiers, fragrances, and other ingredients acceptable as pharmaceuticals, quasi-drugs, and cosmetics may also be used at the same time.

Next, the present invention will be explained in detail by showing test examples.

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

(1) Preparation of hydrolyzate 1) A 10% casein solution (pH 8,0) was mixed with an enzyme solution containing 1,000 active units each of lactic acid bacteria extract, Aspergillus-derived broteacetate, and vancreatin per 1 g of protein. It was added so that the sum of active units was 3000 units, decomposed at 50°C, and the degree of decomposition was measured over time, and the formol nitrogen/total nitrogen (%) was 21.32,4
] (%), heat inactivation at 90°C for 5 minutes.

After inactivation, filter until there is no precipitate and freeze-dry.

These freeze-dried products are hereinafter referred to as samples A and BC.

2) Add the enzyme of the above method to a 10% whey protein solution (pH 7.0) and adjust the degree of degradation to 30%,
A 41% decomposition product is prepared. These freeze-dried products are hereinafter referred to as samples D and E.

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

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

As is clear from Table 1, the release rate of aromatic amino acids increased as the degree of decomposition increased, and was found to be 90% or more in Samples C and E. However, the aromatic amino acid release rate of cosmetic casein peptides with an average molecular weight of 300 to 3,000 produced by the conventional method (the same method as Example 1 described in JP-A-1-269499) is less than 90%. .

(3) Measurement of molecular weight Samples C and E prepared in (1) above were measured at 0.5N using a 5ephadex G-25 column.
Molecular weight was determined by elution with acetic acid. As a result, the first fraction of samples C and E eluted by the gel filtration method eluted later than oxidonone (molecular weight 1000) used as a standard substance, so the molecular weight of samples C and E was less than 1000. It has been found.

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

The antigenicity of samples A to E prepared in Test Example 1 was determined by an ELISA inhibition test.

A 96-well plate (manufactured by Nunc) was coated with raw protein, and after washing, a mixture of rabbit antiserum prepared by sensitizing the raw protein and various hydrolysates was reacted, and after washing, alkaline phosphatase Labeled goat anti-Zagi TgG antibody (Zymed Label
After washing, the enzyme substrate p-nitrophenyl phosphate I.rium was added.
After 30 minutes, the reaction was stopped with 5N thorium hydroxide, and the reaction product was measured using a Microplay 1-reader (Journal of the Japanese Society of Pediatric Allergy, Vol. 1, p. 36, 1987). As a result, the antigenicity of the hydrolyzate decreased as the aromatic amino acid release rate increased. That is, the release rate is 31%
In sample A, the antigenicity was reduced to approximately 1/400 of casein, in sample B, 57%, to approximately 1/30,000 of casein, and in over 90% of sample C, it was confirmed that antigenicity had disappeared. Ta. Similarly, in whey protein, loss of antigenicity was observed in Sample E, which had an aromatic amino acid release rate of 90% or more. However, the casein antigenicity of casein clearly remained in cosmetic casein peptides with an aromatic amino acid release rate of less than 90% produced by the conventional method (the same method as in Example 1 described in JP-A-1269499).

, (Test Example 3) This test was conducted to examine the growth-stimulating activity of human skin cells.

Sample C1 commercially available bogus placenta extract prepared in Test Example 1 (manufactured by Potger, West Germany) and conventional method (same method as Example 1 described in JP-A-1269499)
The human skin cell proliferation activating activity of Sample F manufactured by the following method was tested.

Epithelial cells derived from normal human skin were cultured in modified KGM medium, and fibroblasts were cultured in modified M E containing 2% fetal bovine serum.
Each was cultured in M medium by a conventional method. Sample 01 commercially available bovine placenta extract and F were added in the medium at 1.10 and 1.
00 μg/mQ and continue culturing for 2 to 30 days,
Later, [3H]-labeled thymidine was added and the culture was continued for an additional 2 to 3 hours, after which [3H] was incorporated into the cells.
The amount of H1 was measured with a liquid chlorine counter. In addition, each culture 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 proliferation-stimulating effect on human skin cells, and is equivalent to or 1.6 times more effective than placenta extract, which is widely known to have a proliferation-stimulating effect on human skin cells. It had a revitalizing effect. On the other hand, sample F had the same effect as the control. still,
Similar to Sample C, Sample E prepared in Test Example 1 (1) was also found to have a significant proliferation-stimulating effect on human skin cells.

(Test Example 4) This test was conducted to examine the effects of cosmetics, etc., on the presence or absence of hydrolysates.

(1) Preparation of samples Seven samples (Samples 1 to 7) containing hydrolyzate were prepared using the same formulation and manufacturing method as in Examples 1 to 7, and seven samples not containing hydrolyzate were prepared as shown below. Samples (A to G: Control) were prepared by a conventional method. Incidentally, as is clear from the formulations of Examples and the following formulations, samples 1 and A1 and samples 2 and B correspond to each other.

l) Sample A A skin cream with the following formulation was manufactured.

Stearic acid 15.0 (%) Setanol 2.0 Squalane 3.0 Octyldodecyl myristate 5.0 Glycerin
l0101.3-Butylene glycol 4.0
Polyoxyethylene monostearate (20 mol) Sorbitan 3.0 Purified water
58.02) Sample B A lotion with the following formulation was manufactured.

Propylene glycol oleyl alcohol Hydrogenated human oil polyquine ethylene (60 mol) adduct ethanol purified water 10° 1.5 5゜ 83° (%) 3) Sample C A beauty serum with the following formulation was manufactured.

Sodium hyaluronate Blacenta extract glycerin purified water (%) 4) Sample sample A skin ointment with the following formulation was manufactured.

Vaseline 25 paraffin
5 (%) Cetostearyl alcohol 2.0 Propylene glycol 10.0 Polyoxyethylene
Polyquine Propylene Glycol Ether 3.0 Josei Ice
55.05) Sample E A hair treatment with the following formulation was produced.

Setanol 1.5 (%) 2
-Hexyldecanol 1.0+, 3-Pethylene glycol 3.0 Cationized cellulose 0,2 Polyoxyethylene stearyl ethyl 1.0 Purified water
93.36) Sample F A hair rinse with the following formulation was manufactured.

dialkyldimethylammonium Chloride 2, (%) in tanol polyoxyethylene stearyl Chill 1.3-butylene glycol cationized cellulose purified water 1.0 7) Sample G A hair shampoo with the following formulation was manufactured.

Lauryl sulfate triethanolamine 15.0 1.3-Butylene glyfur 2.0 Ethylene glycol monostearate 1-1.5 Purified water 81.5 (%) (2) Test method l) Test for skin cosmetics, etc. A panel of five experts in their 20s to 40s with rough skin were selected, and they thoroughly washed the inside of their left hand and arm with commercially available cosmetic soap, confirmed that there was no soap residue, and prepared samples 1 to 4.
and 0.5 g each of samples A-D (control), about 30 g each.
It was applied in a size of 15 mm x 15 mm and rubbed into the skin. This rubbing onto the skin was performed once a day for 4 consecutive weeks, and evaluation was made once a week by the following method.

Observe with the naked eye the area where each sample is rubbed, ■Prevent rough skin, ■Recover from rough skin, ■Smooth skin, ■Adjust skin texture, ■Tighten skin, ■Protect skin, ■Skin For each item of softening, +2 if it is significantly better than the control sample, +1 if it is slightly better than the control sample,
Evaluate on a five-point scale: 0 if it is equivalent to the control sample, -1 if it is slightly worse than the control sample, and -2 if it is considerably worse than the control sample.The average value is calculated and the effect is tested. l
child.

2) Testing of hair cosmetics, etc. 20-4 with obvious hair damage and dryness
A panel of 15 experts in the 0-year age group was selected and divided into three groups of five people each.The first group tested samples 5 and E, the second group tested samples 6 and F1, and the third group tested samples 7 and G. Wash the hair of the first and second group panels thoroughly with commercially available shampoo, make sure that no shampoo remains, and wash each sample twice.
An amount of 1 g was applied to approximately 50 mm x 80 mm of hair on both sides of the temporal region and rubbed in by hand. In the second group in which the hair rinse was tested, the hair was lightly washed with water immediately after applying the sample. Rub this into your hair once every 4 days, wash your hair on the 4th day, and apply once every 4 days.
0th time Judgment was made by the following method. In the third group in which the shampoo was tested, the hair was divided into left and right halves and washed with Samples 7 and G, without pre-washing with a commercially available shampoo.

Hair was washed once a day for 15 consecutive days, and the results were evaluated by the following method immediately before each day's hair washing.

Visually observe the application area of each sample to determine whether ■Recovery of damaged hair,■
Prevention of hair breakage/split ends, ■hair flexibility, ■smoothness after curling, ■disappearance of frizz, and ■hair gloss, if significantly better than the control sample, +2, control sample. It is evaluated on a five-point scale: +1 if it is slightly better than the control sample, 0 if it is similar to the control sample, -1 if it is slightly worse than the control sample, -2 if it is considerably worse than the control sample, and the average Values were calculated and effects tested.

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

Compared with Samples A and B, Samples I and 2 were found to be effective in recovering from rough skin by all five panelists, and were found to be effective in preventing skin tightening and rough skin by four panelists.

Compared to Sample C, Sample 3 was found to be effective in preventing rough skin, recovering from rough skin, adjusting skin texture, and smoothing the skin by 4 people.

Sample 4 was found to be effective in recovering from rough skin by all 5 panelists, and 4 participants were found to be effective in preventing rough skin and smoothing the skin, compared to sample 3.

Compared to Sample E, Sample 5 was found to be effective in recovering hair damage, preventing dry hair, and preventing hair breakage and split ends in 4 people.

Compared to Sample F, sample 6 was found to be effective in recovering damaged hair and softening hair by all 5 panelists, and 4 participants were found to have a smooth finish, prevent curly hair, and prevent hair breakage and split ends. It was done.

Compared to Sample G, Sample 7 was found to be effective in recovering damaged hair, giving a smooth finish, and preventing dry hair from 4 people.

As described above, by incorporating the hydrolyzate, excellent effects were observed in both skin products and hair products.

(Test Example 5) This test was conducted to determine the amount of hydrolyzate added.

(1) Preparation of Samples Skin creams and hair treatments were manufactured using the same formulations as in Examples 1 and 5.

However, the amount of hydrolyzate added is 0.0 as shown in Table 5.
The amount of purified water was adjusted to 1 to 50%, and the amount of purified water was increased or decreased accordingly. Note that the sample containing no hydrolyzate was Sample A of Test Example 4.
and E were prepared in the same manner as controls.

(2) Test method The same method as in Test Example 4 was used. However, for skin creams, the same applies to the following three items: ■Recovery from rough skin, ■Tightens the skin, and ■Prevents skin roughness.For hair treatments, the same applies to ■Recovery of damaged hair and ■Prevention of hair cut/split ends. Judgment is made.

(3) Test results The results of this test were as shown in Table 5.

For both skin cream and hair treatment, the above-mentioned effects are noticeable as the amount of hydrolyzate added increases, and the effect is maximum when the amount added is 1 to 20%, and from 30 to 20%.
It decreased by 50%. This decrease in effectiveness is presumed to be due to the inability to uniformly dissolve or disperse the hydrolyzate in the sample due to a decrease in the solubility of the hydrolyzate in water. Therefore, the desirable addition amount of the hydrolyzate is 20% or less, more preferably 0.01 to 20%.

(Test Example 6) This test was conducted to investigate the amount of hydrolyzate added to products other than those tested in Test Example 5.

(1) Preparation of Samples A lotion, a serum, an ointment for external use on the skin, a hair rinse, and a hair shampoo were manufactured using the same formulations as in Examples 2.3.4.6 and 7. However, the amount of hydrolyzate added was adjusted to 0.01%, 20%, and 50% as shown in Table 6, and the amount of purified water was increased or decreased accordingly. Note that samples containing no hydrolyzate were prepared in the same manner as Samples B, C, D, F, and G of Test Example 4 to serve 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 clear from Table 6, the desirable hydrolyzate additives in these products were 0.01 to 20%.

Next, an example of producing a hydrolyzate used in the present invention will be shown.

Reference Example 1 Suspend 200 g of commercially available casein in water to a concentration of 10%, and adjust the pH to 8.0 with a 10% hydroxide solution.
Adjusted to. After heat sterilizing the casein solution at 90°C for 10 minutes, it was cooled to 45°C.
g, and 4 g of the above lactic acid bacteria extract were added and hydrolyzed at 45° C. for 24 hours. The decomposition product was heated at 90°C for 5 minutes to inactivate the enzyme and filtered to remove the precipitate. This was freeze-dried to obtain 165 g of powdered hydrolyzed powder.

The resulting hydrolyzate was tested in the same manner as in Test Examples 1.2 and 3, and the release rate of aromatic amino acids was 90.5%.
It does not show the antigenicity of casein and has a molecular weight of 1000.
The effect of activating the proliferation of human skin cells was observed.

Reference Example 2 200 g of commercially available whey protein powder was dissolved in deionized water at a concentration of 8%. Filter to sterilize, adjust to 45°C, and add 5% thorium hydroxide aqueous solution to adjust pH.
Vancreatin F (Amano Pharmaceutical) while maintaining the
was added six times at 30-minute intervals, and after 15 hours, 2.0 g of atatinase AS (Kaken Pharmaceutical Co., Ltd.) was added, and the mixture was further decomposed for 5 hours.

The enzyme was inactivated by heating at 90°C for 5 minutes, and the precipitate was removed by filtration. This was freeze-dried to obtain 160 g of powdered hydrolyzed powder.

The resulting hydrolyzate was tested in the same manner as in Test Examples 1.2 and 3, and the release rate of aromatic amino acids was 90.4%, showing no antigenicity of whey protein, and the molecular weight was 1000.
The results were as follows, indicating an effect of stimulating the proliferation of human skin cells.

Next, the present invention will be explained in further detail by showing examples.

In addition, since each Example was manufactured by a conventional method, only the formulation is shown.

Example 1 A skin cream with the following formulation was manufactured.

Stearin #15゜cetanol 2゜squalane
3゜Octyldodenyl myristate
56 Glycerin 10゜13-
Butylene gelicol 4゜Polyoxyethylene monostearate (20 mol) Sorbitan 3゜Hydrolyzate of Reference Example 1 5゜Purified water
53° (%) Example 2 A lotion with the following formulation was produced.

Propylene glycol oleyl alcohol 10° 0° (%) Hardened castor 7 oil polyoxyedile (60 mol) adduct 1.5 ethane Hydrolyzate of Reference Example I 3゜ purified water 79° Example 3 A beauty serum with the following formulation was manufactured.

Sodium Hyaluronate O Placenta Extract l.

Glycerin l.

Hydrolyzate of Reference Example 2 4゜Purified water
93° (%) Example 4 A skin ointment having the following formulation was manufactured.

Vaseline 25 paraffin
5 Cetostearyl alcohol
2゜70 pyrene glycol 10゜Polyoxy, ethylene polyoxy7 fluorylene glycol ether 3゜Hydrolyzate of Reference Example 1 5 (%) Purified water 50.0 Example 5 Hair treatment mensetanol 2-hexyl/ Ludecanol 1.3-butyμ> glycol Cationized cellulose polyoxyethylene stearyl ether Hydrolyzate purified water of Reference Example 2 was produced.

1.5 (%) 1.0 3.0 0.2 1.0 4.0 Example 6 A hair rinse with the following formulation was manufactured.

Zoargyldimethylammoni Rum chloride Setanol Polyoxygen/ethylene stearyl leether 2゜ 1.0 (%) 1.3-7 ethylene glycol cationized cellulose Hydrolyzate of Reference Example 1 purified water Example 7 A hair shampoo with the following formulation was manufactured.

Lauryl sulfate triethanolamine 15.013-Butylene glycol 20 Ethylene glycol monostearach 15 Hydrolyzate of Reference Example 2 50 Purified water
76.5 (%) [Effects of the Invention 1 The effects achieved by the present invention are as follows.

(1) An excellent skin product that recovers from rough skin and smoothes the skin by adjusting the skin texture can be obtained, and (2) a hair product that is excellent in the effect of restoring damaged hair can be obtained.

Claims (1)

[Scope of Claims] [1] Cosmetics and external skin preparations characterized by containing a milk protein hydrolyzate and/or a salt thereof having the following characteristics (a) to (d): (a) It is a peptide mixture with a molecular weight of 1000 or less; (b) 90% (weight) or more of the aromatic amino acids are free amino acids; (c) It has a proliferation-stimulating effect on human skin cells. (d) It does not have the antigenicity of milk proteins.