JPH02270808A - Cosmetic - Google Patents

Abstract

PURPOSE:To obtain a cosmetic imparting smoothness and gloss to skin and hair by mixing modified protease obtained by bonding polysaccharide introduced off reaction-active group using cyanogen bromide with protease and having specific modified fraction. CONSTITUTION:Protease is bonded with polysaccharide activated by cyanogen bromide to obtain modified protease containing >=15% modified fraction of surface amino group of the same protease measured with TNBS method and the resultant modified protease is contained in the subject cosmetic. As the protease, protease derived from genus Bacillus, etc., is preferable and pullulan, etc., is preferable as the polysaccharide. The subject cosmetic is readily used without giving stimulation or allergy to skin. Furthermore, bad odor or discoloration is not generated in preserving of the cosmetic. Senile cuticle of skin is removed and the skin is made smooth.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to cosmetics containing a modified protease, and more specifically, cosmetics that are storage stable and can impart smoothness and luster to the skin or hair through the action of the protease. The present invention relates to a novel cosmetic with excellent properties, skin stability, and practical properties.

[Conventional technology]

Conventional skin stains caused by 9 hydrolytic enzymes such as proteases
Various enzyme-containing cosmetics have been proposed that utilize ingredients and the decomposition action of aged keratin.

For example, in JP-A-58-77808 ``Cream Composition,'' a cosmetic composition in which a hydrolytic enzyme is blended into an emulsion is proposed, but if the enzyme is simply blended into an emulsion containing water, the enzyme may This is undesirable because cosmetics may cause discoloration and odor when taken orally, and may cause skin irritation or allergies.

Also, JP-A-63-130415 ``Powder cosmetics''
Powdered cosmetics containing hydrolytic enzymes have been proposed, but they are not easy to use because they must be mixed with water to develop the enzyme activity, and the enzymes may cause irritation to the skin. It is undesirable because it causes allergies.

In addition, in order to improve such drawbacks, the present inventors proposed an emulsified cosmetic containing powdered immobilized hydrolytic enzyme in Japanese Patent Application No. 82022/1988 entitled "Emulsified Skin Cosmetic". However, these cosmetics have the disadvantage that they give a rough feeling when used, making them unpleasant to use.

[Problem to be solved by the invention]

As a result of intensive research aimed at improving the drawbacks of the above-mentioned conventional techniques, the present inventors discovered that the above-mentioned drawbacks could be solved by cosmetics obtained by blending specific modified enzymes described below, and completed the present invention. However, the purpose of the present invention is to provide a product that is easy to use, highly safe so as not to cause irritation or allergies to the skin, and which does not cause odor or discoloration even when administered orally, and which is suitable for the skin. Removes aging keratin and gives smoothness, and gives shine and finish to hair.]
Our aim is to provide cosmetics that provide the desired benefits.

[Means to solve the problem]

The present invention provides multi-t
i[ and the modification rate of the surface amino groups of the protease is T
This cosmetic is characterized by containing 15% or more of a modified protease as measured by the NBS method.

Next, the configuration of the present invention will be explained in detail.

The modified protease used in the present invention includes protease,
It is obtained by combining it with a polysaccharide activated by cyanogen bromide.

Examples of the protease used here include animal-derived proteases such as trypsin and chymotrypsin, microbial-derived proteases, and the like. All of the modified proteases of the present invention have suppressed antigenicity and skin sensitization, and also have greatly improved stability. However, the relative stability varies depending on the protease. In terms of stability, many proteases derived from microorganisms are superior to proteases derived from animals. Therefore, preferably a protease derived from a microorganism, particularly preferably a protease derived from the genus Bacillus, can be used with good results.

In addition, polysaccharides such as agarose, guar gum, inulin, starch, dextran, pullulan, xanthan gum, carrageenan, pectin, alginic acid and other natural polysaccharides and their derivatives, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose,
Examples include carboxymethylcellulose. Among them, dextran and pullulan have a low solution viscosity even when using a substance with a fairly high molecular weight, and the reaction operation is easy, and
The performance of the resulting modified protease is also stable and gives uniform favorable results.

More than $7! As long as the molecular weight of TFI is not extremely small, the stability of the modified protease will give good results, but antigenicity etc. are significantly affected by the molecular weight.
Its average molecular weight is 10. Good results can be obtained by using a value of OO0 or more, particularly preferably 40,000 or more.

In order to obtain the modified protease used in the present invention, a polysaccharide may first be activated with cyanogen bromide, and the activated form and protease may be combined according to a commonly used method.

The degree of modification of the surface amino groups of the protease in the modified protease obtained in this way varies depending on the type of multi-IJN used, the mixing ratio, and the reaction conditions, but the degree of modification depends on the safety and stability of the obtained modified protease. In order to ensure performance, it is necessary that this modification rate be 15% or more as measured by the 'I'' N 13 S method.

Furthermore, in the binding reaction between poly$Ms and protease activated by cyanogen bromide, the activated polysaccharide is
It is necessary to use at least three times the weight of protease. If the amount is less than 3 times, it will not be possible to obtain a modified prothiase with sufficient suppression of antigenicity and skin sensitization, and good results will not be obtained.

Furthermore, even if an excessive amount of polysaccharide is added, the performance of the resulting modified protease will be saturated, so the amount used is preferably 20 times or less.

In addition, in the above production method, the activation reaction of the polysaccharide is p
Hl0~11.25℃ or less, and the binding reaction is performed at pH
It is preferable to carry out the reaction at a temperature of 8.5 to 9.5.5°C or lower.

Furthermore, the enzyme concentration in the binding reaction between polysaccharide and protease also influences the properties of the obtained modified protease. If the enzyme concentration is too high, antigenicity and skin sensitization may not be completely suppressed, so the enzyme concentration is preferably 1% by weight or less.

After the binding reaction between the 6-protease and the activated form, stable quality can be obtained by adding lysine, glycine, aminoethanol, etc. to the excess active groups of the polysaccharide and performing post-treatment. . Further, the obtained modified protease can be purified by ultrafiltration, gel filtration liquid chromatography, or the like.

In the present invention, the amount of modified protease blended is preferably 0,0001 to 5 parts by weight, based on 100 parts by weight of the total amount of the cosmetic. If the amount is less than 0.0001 part by weight, the protease in the cosmetic may not function sufficiently. Furthermore, even if the amount exceeds 5 parts by weight, it is not expected to produce much effect commensurate with the amount incorporated.

The cosmetics of the present invention include humectants, water-soluble polymers, surfactants, water, oils, waxes, fragrances, colorants, preservatives, antioxidants, bactericides, amino acids, vitamins, hormones, and ultraviolet absorbers. Ingredients commonly used in cosmetics can be appropriately blended.

The cosmetics of the present invention include skin cream, skin milk, cleansing cream, cleansing milk, cold cream, cream sorb, makeup base, skin lotion, milky lotion, punk, calamine lotion, T zone essence, hand cream,・
Nosesis powder, whitening powder, powder soap, bar soap, transparent soap, lip balm, lipstick, nutritional essence, creamy foundation, face powder, powder eye shadow, powder foundation, nail remover, hair tonic, hair riki nodo, Applicable to hair cream, hair treatment, scalp treatment, shampoo, conditioner, hair spray, sun oil, sunscreen, shaving foam, shaving cream, heavy oil, etc.

〔Example〕

The present invention will be specifically described below with reference to Examples.

In addition, in the present invention, the surface amino group modification rate, thermal stability, antigenicity, and storage stability of protease.

Skin sensitization and practical properties were evaluated as follows.

(Surface amino group modification rate of Ij protease by the method of Itaynes et al.
es, R, eLal.

Biochemistry, 6. 541 (1
967) ) to trinitrohenzenesulfonic acid (
The amount of unreacted amino groups on the surface of the modified protease was measured as the reaction amount of TNBS), and the modification rate of the surface amino groups was calculated from the ratio to the amount of surface amino groups of the unmodified product.

(2) After incubating the thermally stable sample at 60°C for 6 hours,
Enzyme activity was measured and calculated using the following formula.

Thermal stability (%) - (3) 0.4 ml of antiserum prepared separately in advance was added to 0.4 ml of the antigenic sample, and incubation was performed at 30°C for 2 hours. The generated precipitate was collected by centrifugation, washed three times with 1 mA of 75 mM phosphate buffer (pH 7, 8), and then washed with 0.0 mA of 75 mM phosphate buffer (pH 7, 8). lN NaOH3mj! was added to dissolve it, and the absorbance at 285 nm was measured.

Antigenicity was determined according to the following criteria.

(4) Storage stability The samples were sealed and left in a constant temperature bath at 45"C for 3 months, protected from light, and then observed for changes in color and odor.

(5) Skin sensitization maximization method (
Bertil.

M and Albert, M, K, + J, I
nvest, Derm, 52(3), 268
(1969)), a skin sensitization test was conducted.

The samples were used as they were for both induction and challenge. The degree of skin sensitization was determined from the average score shown below.

(6) 20 practical property specialist inspectors conducted practical tests 101 times over three consecutive days and answered the questionnaire regarding the following test items.

■Easy to use: Number of people who answered that it was easy to use.

■Roughness during use: Number of people who said they felt rough when using it.

■Irritation after use: Number of people who said they felt irritation on their skin (scalp) after using the survey.

■Smoothness after use: Number of people who said their skin (hair) became smoother after use.

■Gloss after use: Number of people who said their skin (hair) became shiny after using it.

Example 1 Skin lotion A skin lotion with the following formulation was prepared by a conventional method.

The properties of the obtained skin lotion are shown in Table 1. 1st
As is clear from the table, the skin lotion of the present invention had excellent thermal stability, practical properties, and storage stability, and had low antigenicity and skin sensitization.

(Formulation of skin lotion) (Preparation of modified protease) 1.0g of dextran (average molecular weight 6-9X10') in 20m7! It was dissolved in water. 1NN aOH to this
was added to adjust the pH to 10.4. Dissolve 259 mg of cyanogen bromide in 3 ml of water and add this solution. The solution was gradually added to the dextran aqueous solution at room temperature, and at the same time, 1N NaOH was added to maintain the pH at around 10. Do this operation for about 15
After a minute, stir for about 10 minutes and add 4% NaHCO.
Aqueous solution was added to give p I+9. Next, this activated dextran solution was added to Bacillus licheniformis (B.
100 mg of protease derived from A. acilus licheniformis (manufactured by Novo, trade name: Esperase) (hereinafter referred to as Esperase) was added, and the mixture was allowed to react at 4°C for 24 hours. Add 4.0 mg of glycine to this reaction solution.
After treatment for 2 hours, the solution was ultrafiltered, purified and concentrated, and then frozen and transferred.

The surface amino group modification rate of the obtained modified protease was 17
%, and the enzyme activity retention rate was 27%.

Example 2 Skin Lotion A skin lotion of the present invention was obtained in the same manner as in Example except that a modified protease prepared as described below was used instead of the modified protease of Example 1.

Its characteristics are shown in Table 1. As is clear from Table 1, the skin lotion of the present invention had excellent thermal stability, practical properties, and storage stability, and had low antigenicity and skin sensitization.

(Preparation of modified protease) Pullulan (average molecular weight 50.000) was used instead of dextran (average molecular weight 6 to 9×10') used in Example 1 (preparation of modified protease), and esperase was used as protease. A modified protease was prepared in the same manner as in Example 1 (preparation of modified protease), except that Bioplase (manufactured by Nagase Biochemical Co., Ltd.) was used.

The surface amino group modification rate of the obtained modified protease was 22
%, and the activity retention rate was 33%.

Example 3 Skin Lotion A skin lotion of the present invention was obtained in the same manner as in Example 1, except that a modified protease prepared as described below was used instead of the modified protease in Example 1.

Its characteristics are shown in Table 1. As is clear from Table 1, the skin lotion of the present invention had excellent thermal stability and storage stability, and had low antigenicity and sensitization.

(Preparation of modified protease) The procedure of Example 1 (modified Modified protease was prepared in the same manner as in (Preparation of protease).

The surface amino group modification rate of the obtained modified protease was 20
%, 'activity retention rate was 30%.

Example 4 Skin lotion A skin lotion of the present invention was obtained in the same manner as in Example 1, except that a modified protease prepared as described below was used instead of the modified protease of Example I.

Its characteristics are shown in Table 1. As is clear from Table 1, the skin lotion of the present invention had excellent thermal stability, practical properties, and storage stability, and had low antigenicity and sensitization.

(Preparation of modified protease) In the same manner as in Example 1 (preparation of modified protease) except that inulin (average molecular weight 50,000) was used instead and chymotrypsin (manufactured by Sigma) was used instead of esperase as protease. A modified protease was prepared.

The surface amino group modification rate of the obtained modified protease was 18
%, and the activity retention rate was 27%.

Comparative Example 1 Skin Lotion A comparative skin lotion was prepared in the same manner as in Example 1, except that unmodified protease was used instead of the modified protease in Skin Lotion Example 1.

Its properties are shown in Table 1. As is clear from Table 1,
A comparative skin lotion containing unmodified protease had extremely poor thermal stability, antigenicity and skin sensitization, caused irritation during use, and had poor storage stability.

Comparative Example 2 Skin Lotion A comparative skin lotion was prepared in the same manner as in Example 1, except that an immobilized protease prepared as described below was used instead of the modified protease in Skin Lotion Example 1.

Its characteristics are shown in Table 1.

As is clear from Table 1, the comparative skin lotion using immobilized protease felt rough upon use and was not desirable.

(Preparation of immobilized protease) 20 parts by weight of calcium chloride was dissolved in 20 parts by weight of water, 80 parts by weight of methanol was mixed therewith, and 5 parts by weight of nylon powder (average particle size 6 to 10 μm) was added and dispersed. , and stirred at 50°C for 30 minutes. After collecting this and washing with water,
．． It was immersed in 100 parts by weight of 5M hydrochloric acid and stirred at 45°C for 50 minutes. After washing with water, 0.00ml containing 10% glutaraldehyde.
It was immersed in 50 parts by weight of 1 M sodium borate buffer (pH 8.5) and then washed with the same buffer. This treated powder was mixed with 0.05 M containing 1 part by weight of esperase.
Soluium phosphate buffer (p)-17,' 5)
The mixture was added to 50 parts by weight, reacted at 10°C for 5 hours, and washed with water to obtain carrier-bound immobilized protease powder.

Example 5 Skin Cream Using the modified protease prepared in Example 1, a skin cream with the following formulation was prepared.

(Preparation method for skin cream) The oil phase components were uniformly heated and dissolved at 80°C, and
Add the aqueous phase components uniformly heated and dissolved at 0°C, cool while stirring, add modified protease at 40°C, and cool to 30°C to obtain the skin of the present invention (skin cream formulation). Practical characteristics and storage stability are the second priority.
Shown in the table. As is clear from Table 2, the skin cream of the present invention had excellent practical properties and storage stability.

Example 6 Hair Cream Using the modified protease prepared in Example 2, a hair cream having the following formulation was prepared.

(Preparation method for hair cream) The oil phase component was uniformly heated and melted at 80°C, the aqueous phase component that was also uniformly heated and dissolved at 80°C was added, cooled with stirring, and the modified protease was added at 40°C. In addition, 30℃
The hair cream of the present invention (formulation of hair cream) was cooled to
Shown in the table. As is clear from Table 2, the hair cream of the present invention had excellent practical properties and storage stability.

Example 7 Cleansing Milk Using the modified protease prepared in Example 3, a cleansing milk with the following formulation was prepared.

(Preparation method for cleansing milk) Heat and dissolve the oil phase components uniformly at 80°C, and then
Add the aqueous phase components uniformly heated and dissolved at 0°C, cool while stirring, add modified protease at 40°C, and cool to 30°C to prepare the cleansing milk of the present invention (prescription of cleansing milk). Practical properties and storage stability are shown in Table 2. As is clear from Table 2, the cleansing milk of the present invention had excellent practical properties and storage stability.

Example 8 Cream Soap Using the modified protease prepared in Example 4, a cream soap having the following formulation was prepared.

(Preparation method for cream soap) After uniformly heating and dissolving the aqueous phase components at 80°C for 1 hour, cooling while stirring, adding modified protease at 40°C.
Cool to 0℃ and add modified protease at 40℃ for 30 minutes.
Cool to ℃ and cream soap (cream soap formulation) The practical properties and storage stability of the obtained cream soap
Shown in the table. As is clear from Table 2, the cream soap of the present invention had excellent practical properties and storage stability.

Example 9 Powder Foundation Using the modified protease prepared in Example 1, raw materials of the following formulation were uniformly stirred and mixed to obtain a powder foundation.

(Powder foundation prescription) Practical properties of the obtained powder foundation.

Storage stability is shown in Table 2. As is clear from Table 2,
Practical use of the powder foundation of the present invention Table 1 [Effects of the invention] As described above, the cosmetic of the present invention is easy to use, does not cause irritation or allergies to the skin, and does not cause odor or discoloration even when stored. It removed aged keratin and imparted smoothness to the skin, and its action and effects were remarkable.

Claims (1)

[Claims] A modified protease obtained by combining a polysaccharide into which a reactive group has been introduced with cyanogen bromide and a protease, and in which the modification rate of surface amino groups of the protease is 15% or more as measured by the TNBS method. Cosmetics characterized by: