JP3236970B2 - Cosmetics - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cosmetic containing a stable lysozyme modified with a high molecular weight copolymer, and more particularly, it can maintain its activity in an aqueous system for a long time,
The present invention relates to cosmetics containing modified lysozyme having a wide range of application.

[0002]

2. Description of the Related Art Lysozyme is composed of N-acetylmuramic acid (MurNAc) and N
-A lytic enzyme that hydrolyzes the β1 → 4 bond between acetylglucosamine (GlcNAc). This enzyme is widely found in the animal and plant kingdoms, of which hen egg white lysozyme is the most studied. Its molecular weight is 14,300, 12
It is a relatively small protein consisting of 9 amino acids. It has been subjected to X-ray crystallography for the first time as an enzyme, and its three-dimensional structure has been clarified.

The major pharmacological actions of lysozyme include (1) antiviral action against viral diseases, (2) antiinfective action against bacterial and protozoan infections, (3) hemostatic action, (4) antitumor and antiinflammatory actions (5) Tissue repair action is known. Lysozyme applications include anti-virus, anti-infection,
Attempts have been made to use tissue-repairing effects as cosmetics in facial cleansers, basic cosmetics, etc.In addition, it is used in toothpaste and gargles to control bacteria that cause dental caries, prevent viral stomatitis, and infection from the oral cavity There are attempts to prevent it.

[0004]

However, when lysozyme is directly incorporated into an aqueous system, the enzyme activity decreases,
There are problems such as easy irritation and allergy to the skin. In the application of lysozyme, various attempts have been made such as a method of leaving it in a dry state immediately before use and mixing it with an aqueous solution at the time of use, liposome formation, microencapsulation, and immobilization with a polymer compound. The method of microencapsulation is a stabilization by cutting off the contact between lysozyme and a base such as an aqueous solution until immediately before use, and does not solve an essential problem. Also,
The method of adding saccharides such as polyhydric alcohol, maltose, and sorbitol is effective for stabilizing other enzymes such as protease, but has almost no effect on lysozyme. However, other methods have not been able to obtain a sufficiently satisfactory stabilizing effect.

[0005] Therefore, an object of the present invention is to provide a cosmetic containing modified lysozyme which can maintain its activity in an aqueous system for a long time and has a wide application range.

[0006]

Means for Solving the Problems The present inventors have found that lysozyme, in which a polymer having both an acid anhydride group and a polyoxyalkylene group in the same molecule, is chemically bonded, has a long activity even in cosmetics. The present invention was found to last, and the present invention was reached. That is, in the present invention, the molar ratio of the alkenyl ether represented by the formula (1), maleic anhydride and another monomer is 5%.
A cosmetic comprising 0.0001 to 10% by weight of modified lysozyme chemically bonded to a copolymer having a molecular weight of from 60:20 to 90: 0 to 30.

[0007]

Embedded image

(Z is a residue of a compound having 2 to 8 hydroxyl groups, AO is one or a mixture of two or more oxyalkylene groups having 2 to 18 carbon atoms. May be added at random or may be added at random.
¹ represents an alkenyl group having 2 to 5 carbon atoms, and R ² represents 1 to 24 carbon atoms.
A, b and c are each an average number of added moles of an oxyalkylene group of 0 to 600, and m is 1
An integer of 0 to 7, n is an integer of 0 to 6, m + n = 1 to 7, n /
(1 + m + n) ≦ １ ／ and a + bm + cn = 1 to 10
00).

In the alkenyl ether of the formula (1) used in the present invention, compounds having 2 to 8 hydroxyl groups having Z as a residue include ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, styrene glycol. C8 to C18 alkylene glycols, glycols such as neopentyl glycol, glycerin, diglycerin, polyglycerin; trimethylolethane, trimethylolpropane, 1,3
5-pentanetriol, erythritol, pentaerythritol, dipentaerythritol, sorbitol,
Sorbitan, sorbide, condensate of sorbitol and glycerin, adonitol, arabitol, xylitol,
Polyhydric alcohols such as mannitol, or partially etherified or esterified products thereof; xylose, arabinose, ribose, rhamnose, glucose, fructose, galactose, mannose, sorbose, cellobiose, maltose, isomaltose, trehalose, sucrose, raffinose, gentianose ,
Sugars such as melezitose, or partially etherified or esterified products thereof; catechol, resorcinol,
Examples include phenols such as hydroquinone and phloroglucin.

The oxyalkylene group represented by AO includes oxyethylene, oxypropylene, oxybutylene, oxytetramethylene, oxystyrene, oxydecylene, oxytetradecylene, oxyhexadecylene. And an oxyoctadecylene group. These may be added alone, or two or more may be added simultaneously. When two or more types are added at the same time, block-shaped addition or random addition may be performed.

The oxyalkylene group is necessary to increase the affinity between the copolymer and lysozyme and to improve the stability of lysozyme. If the amount is too large, the weight of the maleic anhydride unit in the copolymer is too large. As the ratio becomes lower and the reaction with the amino group in lysozyme hardly occurs,
a + bm + cn must not exceed 1000. R
^As the alkenyl group having 2 to 5 carbon atoms represented by ¹ , a vinyl group, an allyl group, a methallyl group, 1,1-dimethyl-2-
There are a propenyl group and a 3-methyl-3-butenyl group.

Examples of the alkyl group having 1 to 24 carbon atoms represented by R ² include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group and a hexyl group. , Isoheptyl, 2-ethylhexyl, octyl, isononyl, decyl, dodecyl, isotridecyl, tetradecyl, hexadecyl, isocetyl, octadecyl, isostearyl, oleyl, octyldodecyl, docosyl, There are decyltetradecyl group, benzyl group, cresyl group, butylphenyl group, dibutylphenyl group, octylphenyl group, nonylphenyl group, dodecylphenyl group, dioctylphenyl group, dinonylphenyl group, styrenated phenyl group, etc. Acetic acid, propionic acid, butyric Acid, isobutyric acid, caprylic acid, 2-ethylhexanoic acid, isononanoic acid, capric acid, lauric acid, myristic acid, palmitic acid, isopalmitic acid, stearic acid, isostearic acid, arachidic acid, behenic acid, palmitoleic acid, oleic acid And acyl groups derived from linoleic acid, linolenic acid, erucic acid, benzoic acid, hydroxybenzoic acid, cinnamic acid, gallic acid and the like.

In order for the copolymer of the alkenyl ether represented by the formula (1) and maleic anhydride to sufficiently bind to lysozyme, an acid anhydride structure is required, and an alkenyl ether having a large amount of free hydroxyl groups is used. And an ester bond with an acid anhydride unit at the time of polymerization, so that the reactivity with lysozyme decreases or the solubility of the copolymer in a solvent decreases, so that n / (1 + m + n) ≦ 1 / 2.

The copolymer used in the present invention can be easily obtained by copolymerizing the alkenyl ether represented by the formula (1) with maleic anhydride using a radical polymerization catalyst. At that time, another monomer may be added for copolymerization. Other monomers include unsaturated carboxylic acids such as acrylic acid, methacrylic acid, itaconic acid, crotonic acid, aromatic vinyl compounds such as styrene and methylstyrene, vinyl chloride compounds such as vinyl chloride and vinylidene chloride, and isobutylene. And olefins such as diisobutylene, and vinyl acetate, acrylonitrile, and acrylamide. These can be added at the time of improving physical properties such as imparting tackiness to the copolymer, but if the ratio becomes too large, the content of oxyalkylene groups or acid anhydride groups required for lysozyme modification is increased. And the modification cannot be performed successfully, so that the proportion of other monomers must be 30 mol% or less of all monomers.

The copolymer used in the present invention can obtain copolymers of various degrees of polymerization by changing the type of polymerization initiator or the structure of the alkenyl ether of the formula (1). The average molecular weight is from 10 to 2,000,000, preferably from 3000 to 100,000. In addition, a copolymer having high hydrophilicity can be obtained by using an oxyalkylene group to which a large number of oxyethylene groups are added.

The method for obtaining the modified lysozyme used in the present invention is a method utilizing an amino group in lysozyme. The target lysozyme includes lysozyme derived from chicken egg white, egg white such as turkey and duck. , Papaya, figs and other plants, and Bacillus subtilis (B
A variety of microorganisms such as those derived from microorganisms such as acillus subtilis) can be used, but are not particularly limited thereto.

The modified lysozyme used in the present invention can be obtained by reacting a copolymer with lysozyme. The ratio when reacting the copolymer with lysozyme cannot be specified unconditionally because it varies depending on the content of amino groups in lysozyme or acid anhydride groups in the copolymer, but it cannot be specified unconditionally. If the amount of the copolymer is too small, the modification time decreases, and the intended temporal stability becomes insufficient.If the amount of the copolymer is too large, the initial activity of the modified lysozyme is significantly reduced. Is 50 to 40 copolymers per 100 parts by weight of lysozyme.
0 parts by weight.

The reaction between the two is preferably carried out by directly adding the copolymer to an aqueous solution of lysozyme when the copolymer is water-soluble, and when the copolymer is hardly soluble in water or insoluble in water. Is an easy and preferred method of dissolving the copolymer in advance in a solvent such as acetone which is compatible with water and then adding it to the aqueous lysozyme solution. Also,
The pH during the reaction is from 6 to 10, preferably from 8 to 9. This is because when the pH is on the acidic side, the amount of free amino groups in lysozyme decreases, and the modification rate decreases.

On the other hand, if the reaction temperature is too high, the activity of lysozyme decreases in the modification step, and the hydrolysis reaction of the acid anhydride group is more likely to occur than the reaction between the amino group and the acid anhydride group of lysozyme. Because the rate decreases, 0-10
C is a preferred range. Examples of the cosmetics of the present invention include facial cosmetics such as facial cleansers, creams and emulsions, and basic cosmetics for limbs; hair cosmetics such as hair styling and hair treatment; makeups such as foundation, white powder, blush, eyeshadow and lipstick. Cosmetics may be mentioned, but any may be used as long as the modified lysozyme used in the present invention can be blended.

The content of the modified lysozyme in the cosmetic of the present invention is 0.0001 to 10% by weight, particularly preferably 0.001 to 10% by weight.
1.01.0% by weight, and in addition to the modified lysozyme, commonly used cosmetic ingredients can be blended according to the product type or cosmetic purpose. Examples of such cosmetic ingredients include oils and raw materials such as oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, lanolin, and silicone oils.
Powder materials such as white pigments, coloring pigments, extender pigments, metallic soaps, surfactants, polyhydric alcohols, water-soluble polymers, water,
Examples include antioxidants, ultraviolet absorbers, preservatives, tar pigments, cosmetic ingredients, and fragrances.

[0021]

The present invention relates to a cosmetic containing a novel modified lysozyme which is a reaction product of a copolymer of an alkenyl ether having a polyoxyalkylene group with maleic anhydride and lysozyme. It has a long lasting activity, has low skin irritation, and has excellent cosmetic effects such as moist feeling after use and prevention of rough skin.

[0022]

EXAMPLES The present invention will be described with reference to production examples and examples of modified lysozyme. Production Example 1 The following compound was dissolved in 1 liter of toluene, and a polymerization reaction was carried out at 80 ± 2 ° C. for 7 hours under a nitrogen atmosphere.

CH ₂ = CHCH ₂₀ (C ₂ H ₄ O) ₃₃ CH ₃ 1524 g (1 mol) Maleic anhydride 103 g (1.05 mol) Benzoyl peroxide 4.8 g (0.02 mol) Then toluene and unreacted maleic anhydride 10 to 30
Distillation at 100 ± 10 ° C under reduced pressure of mmHg, 1450g of copolymer
No.1 was obtained. The obtained copolymer No. 1 was a pale yellow waxy solid, having a melting point of 45 ° C. and a saponification value of 68.5.

Lysozyme (derived from chicken egg white, titer 1.64)
(g / g) was dissolved in 20 g of a borate buffer (Note 1) at pH 8.6, and the temperature of the system was maintained at 3 ° C. No.1 copolymer
Was added to the system in the form of a fine powder, and the mixture was stirred at 3 ± 1 ° C. for 30 minutes. Then, 1 g was further added and stirring was continued at the same temperature for 30 minutes. After the reaction is completed, pH is adjusted with 0.1N-sodium hydroxide aqueous solution.
Was adjusted to 7.0, inorganic salts were removed using a reverse osmosis membrane, and dried at 35 ° C. under reduced pressure to obtain 2.0 g of modified lysozyme.

The activity of the obtained modified lysozyme was measured using Micrococcus lysodeikt.
icus) The result of measurement by a method using dried cells (Note 2) was 0.41 mg / g, and the residual activity rate by modification of lysozyme was 75% in terms of pure content. (Note 1) Boric acid buffer solution (pH 8.6) 0.2M-sodium hydroxide aqueous solution 12.00ml 0.2M-boric acid / potassium chloride aqueous solution 50.00ml The above aqueous solutions were mixed, and purified water was added to make a total volume of 200ml.

A 0.2 M boric acid / potassium chloride aqueous solution is composed of 12.4 g of boric acid and 14.9 g of potassium chloride in 1 liter of the solution.
It is an aqueous solution containing g. (Note 2) Activity measuring method Substrate solution (Note 3) 3.0m pre-incubated at 37 ° C
Then, 0.1 ml of a sample solution (Note 4) was added to the mixture, and the mixture was stirred. The mixture was transferred to an optical cell (1 ml), and the decrease in absorbance at a wavelength of 640 nm due to lysis was measured over time using a Hitachi spectrophotometer U-3210. The same procedure was applied to the standard lysozyme product (Note 5), and the amount of lysozyme in the sample was calculated according to the following equation.

[0027] (Note 3) Micrococcus lysodicticus (M.lyso
deikticus) to 75 mM sodium phosphate (pH
6.2) (Note 6), shake, suspend, and add dry cells or the same buffer to the same buffer as a control so that the absorbance at a wavelength of 640 nm becomes 1.00.

(Note 4) Dissolve in purified water so that the amount of lysozyme is about 5 to 15 μg / g. (Note 5) National health laboratory standard control No.831 (Note 6) 75mM Na ₂ HP0 ₄ and 75mM NaH ₂ P0 ₄ were mixed, pH6.2
Adjust to FIGS. 1 and 2 show the infrared absorption spectra of the modified lysozyme and the raw material lysozyme, respectively.

FIG. 3 shows these gel permeation chromatograms together with the copolymer No. 1. It can be seen that the molecular weight of the modified lysozyme is increased. The conditions for gel permeation chromatography are as follows. The above two are connected.

Sample concentration: lysozyme 0.5 mg / ml Modified lysozyme 1.0 mg / ml Sample injection amount: 100 μl Solvent: 0.1 M phosphate buffer (pH 7.0) Flow rate: 0.5 ml / min Detector: Showa Denko KK RI differential calorimeter SE-61.

Production Example 2 6.5 g of t-butylperoxy-2-ethylhexanoate
(0.03 mol) was dissolved in 1 liter of benzene, the temperature was raised to 70 ° C. while stirring under a nitrogen atmosphere, and then a mixture having the following composition was added dropwise to carry out a polymerization reaction at 70 ± 2 ° C. CH ₂ = CHCH ₂ 0 (C ₃ H ₆ O) ₅ (C ₂ H ₄ O) ₁₅ CH ₃ 1022 g (1 mol) 98 g of maleic anhydride (1 mol) Benzene 3 liter After dropping the whole amount, keep at the same temperature for 3 hours After that, benzene and unreacted maleic anhydride were removed under reduced pressure of 10 to 30 mmHg.
Distillation was performed at 120 ± 10 ° C. to obtain 1028 g of copolymer No. 3. Copolymer No. 3 was a viscous liquid and had a saponification value of 101.5.

Lysozyme (derived from chicken egg white, titer 1.64)
mg / g) was dissolved in 20 g of a pH 8.0 phosphate buffer solution (Note 7), and the temperature was maintained at 3 ° C. To this, the copolymer No. 3 of 0.
Add 5g (20% acetone solution) and stir at 3 ± 1 ° C for 30 minutes, then add 0.5g (20% acetone solution) and at the same temperature
Stir for 30 minutes. Next, water is distilled off at 35 ° C. under reduced pressure,
2.1 g of modified lysozyme was obtained. The activity of the resulting modified lysozyme was 0.435 mg / g, and the residual activity of the modified lysozyme by modification was 63% in terms of a pure component. (Note 7) Phosphate buffer solution (pH 8.0) 0.2M-aqueous sodium hydroxide solution 46.85ml 0.2M-potassium dihydrogen phosphate aqueous solution 50.00ml The above aqueous solutions were mixed, and purified water was added to make a total volume of 200ml.

Production Examples 3 to 7 Hereinafter, copolymers No. 2 and
Nos. 4 to 8 were produced, and using these copolymers,
The modified lysozymes shown in Table 4 were obtained.
It was also described in.

[0034]

[Table 1]

[0035]

[Table 2]

In the table, Note 1) BPO: benzoyl peroxide, LPO: lauroyl peroxide, tBEH: t-butylperoxy-2-ethylhexanoate, AIBN: azobisisobutyronitrile,

[0037]

[Table 3]

[0038]

[Table 4]

Note 1) Unit: mg / g 2) Modified lysozyme activity rate (%) relative to lysozyme activity (titer 1.64 mg / g) 3) Activity rate (%) relative to initial activity of modified lysozyme 4) Phosphoric acid Dissolved in a system buffer (containing methyl 4-hydroxybenzoate) 5) A solution obtained by adding 30% of sorbitol to an aqueous lysozyme solution.

Stability Stability Test With respect to the modified lysozyme obtained in each of the production examples, the stability with time was examined by the following method. 0.5 g of modified lysozyme at pH 7.
Dissolved in 100 ml of a phosphate buffer (Note 8) (containing 0.1% methyl 4-hydroxybenzoate as a preservative), kept at 40 ° C for a predetermined time, measured the activity of the modified lysozyme, and examined the stability over time. Was.

As a comparative solution, a solution obtained by dissolving 0.5 g of lysozyme in 100 ml of a phosphate buffer at pH 7.0 (containing 0.1% of methyl 4-hydroxybenzoate as a preservative) (Reference Example 1)
The stability with time was also examined for a sample to which 30% sorbitol was added (Reference Example 2). Table 4 shows that the modified lysozyme used in the present invention has excellent temporal stability in an aqueous system. (Note 8) Phosphate-based buffer (pH 7.0) pH adjusted to 7.0 by mixing 10 mM disodium hydrogen phosphate aqueous solution and 10 mM monosodium hydrogen phosphate aqueous solution.

Examples 1 to 8 and Comparative Examples 1 to 3 Using the modified lysozyme obtained in Production Example 1, lotions were prepared at the compounding ratios shown in Table 5. In Comparative Examples, lysozyme as a raw material was used instead of modified lysozyme. The lotion is
First, components other than lysozyme were mixed and dissolved, and then modified lysozyme or lysozyme as a raw material was added and dissolved.

[0043]

[Table 5]

Note 1) Phosphate buffer (pH 7.0) A mixture of 10 mM disodium phosphate aqueous solution and 10 mM monosodium hydrogen phosphate aqueous solution adjusted to pH 7.0. Stability test with time Each of the lotions shown in Table 5 was stored at 40 ° C., and the activity of the modified lysozyme was determined every month by the above-mentioned measurement method. Show.

[0045]

[Table 6]

From Table 6, it can be seen that the modified lysozyme of the lotion of the present invention is stable over time. Sensory test A sensory test was performed by 10 specialized panels on the three months after preparation of each lotion shown in Table 5. The results were evaluated according to the following evaluation criteria and determined according to the following criteria based on the average score of 10 persons. Table 7 shows that the lotion of the present invention is less irritating and has an excellent skin roughness improving effect. Understand.

[Evaluation Criteria] 5 points: Very no irritation (very good effect of improving rough skin) 4 points: No irritation (a slight effect of improving rough skin) 3 points: Both 2 points: Slight irritation (no significant effect on skin roughening) 1 point: Extremely irritating (no effect on skin roughening) [Judgment criteria] 4.0 points or more ○ 2.1 to 3.9 Points △ 2.0 points or less ×

[0048]

[Table 7]

Example 9 A cream having the following composition was prepared. That is, component 2
8 and components 9 to 12 were mixed and heated to 70 ° C. and dissolved, and then both were mixed.
Ingredient 1 was added at 0 ° C and mixed well. This cream was stable even after 4 months from the preparation, and retained the effect of moisturizing the skin.

Composition (% by weight) Component 1: Modified lysozyme of Preparation Example 1 0.5 $ 2: Stearic acid 1.0 $ 3: Cetyl alcohol 3.5 $ 4: Glyceryl monostearate 3.0 $ 5: Macadamia Nut oil 6.0 $ 6: Squalane 10.0 $ 7: Sorbitan sesquioleate 1.0 $ 8: Perfume appropriate amount $ 9: Sodium hydroxide 0.05 $ 10: Carboxyvinyl polymer 0.1 $ 11: 4 -Methyl hydroxybenzoate 0.1 $ 12: Remaining purified water Example 10 An emulsion was prepared with the following composition. That is, components 1 to 5 and components 7 to 9 were uniformly mixed and heated and dissolved at 70 ° C. On the other hand, components 10 to 13 were mixed and heated to 70 ° C, then both were mixed and stirred, then cooled, and component 6 was added at 25 ° C and mixed well. This emulsion was stable after 4 months from the preparation and was an emulsion having excellent hypoallergenicity and an effect of smoothing the skin.

Composition (% by weight) Component 1: N-stearoyl-L-glutamic acid 0.2 〃2: cetyl alcohol 0.5 〃3: glyceryl monostearate 0.5 〃4: liquid paraffin 9.0 〃5: glyceryl trioctanoate 2.5 〃6: production Modified lysozyme of Example 2 1.0 〃7: Methyl 4-hydroxybenzoate 0.1 〃8: Sorbitan monostearate 0.2 〃9: Fragrance 0.1 〃10: Sodium hydroxide 0.05 〃11: Carboxyvinyl polymer 0.1 〃12: Glucerin 3.0 〃13 : Remaining purified water

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of lysozyme modified with copolymer No. 1.

FIG. 2 is an infrared absorption spectrum diagram of lysozyme.

FIG. 3 is a gel permeation chromatogram of lysozyme, copolymer No. 1 and modified lysozyme.

Continuation of front page (56) References JP-A-63-174935 (JP, A) JP-A-1-283206 (JP, A) JP-A-1-96107 (JP, A) JP-A-1-128910 (JP) JP-A-2-96510 (JP, A) JP-A-1-153,088 (JP, A) JP-A-5-328971 (JP, A) JP-A-5-51313 (JP, A) 5-284967 (JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) A61K ^7/ 00-7/50 C12N 11/02-11/12

Claims (1)

(57) [Claims] (1) The molar ratio of the alkenyl ether represented by the formula (1), maleic anhydride and another monomer is from 5 to 60:
A cosmetic comprising 0.0001 to 10% by weight of lysozyme modified with a copolymer having a ratio of 20 to 90: 0 to 30. Embedded image (Z is a residue of a compound having 2 to 8 hydroxyl groups, AO is one or a mixture of two or more oxyalkylene groups having 2 to 18 carbon atoms, and if two or more, it is added in a block form. R ¹ may have 2 carbon atoms.
An alkenyl group of 5 to 5, R ² is a hydrocarbon group or an acyl group having 1 to 24 carbon atoms, a, b and c are each an average number of added moles of an oxyalkylene group of 0 to 600, m is an integer of 1 to 7,
n is an integer of 0 to 6, m + n = 1 to 7, n / (1 + m +
n) ≦ １ ／ and a + bm + cn = 1 to 1000. )