JP2945054B2 - External preparation for skin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to cosmetics and quasi-drugs having a fair-skin / cell activating function using a solution obtained by hydrolyzing cuttlefish proteins and glycoproteins as an active ingredient. It relates to a skin external preparation.

[Conventional technology]

Conventionally, many cosmetics containing animal and plant extracts are known.

For example, human or mammalian placenta extracts and animal / bird liver / spleen extracts are used in cosmetics as animal extracts, and various crude drugs, herbal medicines, herbs, and seaweed extracts are used as plant extracts. Has been widely used to show functions such as fairness, moisturizing, and cell activation.

In addition, as a simple component that exhibits a color effect, ascorbic acid, colloidal sulfur, kojic acid, hydroquinone,
And unsaturated fatty acids are known, and have been used as external preparations such as creams, lotions, emulsions, packs, ointments, cataplasms and the like containing these fair-skin substances as active ingredients.

On the other hand, molecular weight 25,0 obtained from squid or octopus sumi
Japanese Patent Application Laid-Open Nos. 57-4998 and 64-63523 discloses that skin cosmetics containing natural melanin granules are disclosed in JP-A-57-4998 and JP-A-64-63523. Japanese Unexamined Patent Publication No. 49-71149 further discloses a makeup cosmetic containing sepia melanin as a black pigment.
No. 211, each of which utilizes the melanin granules contained in squid as an anti-inflammatory agent or cosmetic, and those which are expected to have the fair-skin effect aimed at by the present invention. It is different, and there is no technical idea that a protein or glycoprotein of cuttlefish obtained by removing the melanin granules from the components into low molecular weight by hydrolysis is used as an active ingredient.

[Problems to be solved by the invention]

The present applicant has developed a complexion for external use containing kojic acid as a complexion component early on and disclosed in, for example, JP-B-56-18569 and JP-A-53-3538.

The present inventor has been studying such external preparations that show such fairness, moisturizing, and cell activating functions.In this research, special treatment was carried out on squid stains that had been discarded until now. Have been found to exhibit excellent melanin production inhibitory activity and fibroblast activation and proliferation, and have completed the present invention based on this finding.

[Means for solving the problem]

According to the present invention, there is provided an external preparation for skin containing, as an active ingredient, a solution obtained by hydrolyzing cuttlefish proteins and glycoproteins.

As a raw material, squid / octopus sumi is used, and squid sumi is preferably used because it is easily available in large quantities. Although the type of squid is not particularly limited, a squid of Mongouika will be described as an example.

Fresh squid is collected, frozen and thawed when necessary. Thawed sumi is diluted by adding water, filtered or centrifuged to remove melanin and other insoluble substances, which are black pigment particles, to obtain a transparent viscous liquid, which is furthermore pH 3 ~.
An isoelectric precipitation is performed between 4 and the protein and glycoprotein precipitates are collected and redissolved in neutral water. Since this solution is unstable as it is because it has many high molecular weights,
After filtration as required, this is subjected to proteolysis.
The decomposition may be carried out by heat hydrolysis of an acid or alkali, but is preferably carried out with a mild protease.

The enzyme may be any of actinase (pronase), papain, pepsin, trypsin, etc.
It may be performed under pH and temperature conditions.

As the conditions, for example, in the case of actinase,
pH8-9, temperature 45-55 ° C for 5 hours, papain pH6.5-7.
5, 5 hours at 40 to 50 ° C, pH 2.0 to 3.0 for pepsin, 5 hours at 35 to 45 ° C, pH 7.0 to 8.0 for trypsin, temperature 3
Conditions such as 5 hours at 5 to 45 ° C can be exemplified, but it goes without saying that other proteases can be appropriately degraded.

After the decomposition is completed, the pH is returned to neutral, and the pH is 10
Heat for a minute to inactivate the proteolytic enzymes, filter after cooling,
The product is sterilized by a membrane filter to obtain a product (hereinafter referred to as sepia protein).

Production Example A specific production example is shown below.

Add 500 ml of purified water (3 times the amount of squid) to 500 g of frozen mongo squid and thaw.
Centrifuge at G to remove insolubles. The pH of the supernatant is adjusted to 3.5 by adding 10% lactic acid, and the resulting precipitate is left overnight at 5 ° C. to collect a precipitate. 500 ml of water was added to the precipitate,
The pH is adjusted to 8.0 by adding a 10% aqueous solution of sodium hydroxide. Add the proteolytic enzyme (actinase E, 1 × 1
( ⁶ PU / g, manufactured by Kaken Pharmaceutical Co., Ltd.) and stirred at 50 ° C. for 5 hours for decomposition. After decomposition, the enzyme is inactivated by heating at 95 ° C. for 10 hours, cooled to room temperature, adjusted to pH 7.0, and sterilized and filtered through a 0.45 μm membrane filter to obtain a product. Yield 450m
l around.

The sepia protein thus obtained has the following physical properties.

1. Specific gravity 1.002 2. pH 7.27 3. Total nitrogen 0.02% 4. Carbohydrate 0.08% 5. Evaporation residue 0.26% 6. Free amino acid composition Figures in parentheses indicate w / w% of free amino acids. Leucine (15.4%), phenylalanine (9.7%), tyrosine (9.1%), isoleucine (8.7%), arginine (6.9%)
%), Valine (6.7%), alanine (6.2%), serine (5.2%), methionine (4.9%), threonine (4.
7), asparagine (4.7%), lysine (4.3%), histidine (3.7%), glutamine (3.1%), aspartic acid (2.0%), glutamic acid (1.7%), glycine (1.4%)
%), Citrulline (0.7%), proline (0.4%), cystine (0.3%), taurine (0.2%) As the external preparation for skin in the present invention, ointments, emulsions,
Lotions, creams, emulsions, lotions, packs, cataplasms and other preparations generally applied to the skin can be mentioned. The mixing ratio of sepia protein is 0.01 to 50 with respect to the base of these external preparations for skin. % By weight, preferably 0.5-2
0.0% by weight.

The sepia protein used in the present invention is appropriately blended and used with a general base used for the above-mentioned external preparation together with other auxiliaries (for example, antioxidants, preservatives, UV absorbers, chelating agents, etc.). However, when the external preparation for skin is an emulsifier type, sepia protein is added to the aqueous layer.

Preparation example of skin external preparation ・ Ointment Sepia protein and a hydrophilic component consisting of a humectant such as glycerin or sorbite are added to purified water to form an aqueous phase.

On the other hand, solid oil components such as beeswax, paraffin, microcrystalline wax, ceresin, higher fatty acids, and hardened oils,
Oil components such as preservatives and surfactants are blended with semi-solid oils such as petrolatum, lanolin and glyceride, and liquid oils such as squalane, liquid paraffin and various ester oils to form an oil phase.

Next, the aqueous phase is heated, and when the temperature is raised to about 80 to 85 ° C., the oil phase heated to the same temperature is gradually added and emulsified to form an ointment.

・ Lotion A lotion is prepared by dissolving sepia protein, a humectant such as glycerin or propylene glycol, or a skin nutrient in alcohol, in purified water at room temperature.

・ Cream Sepia protein and hydrophilic components such as glycerin and sorbitol are added to purified water to prepare an aqueous phase. Separately, solid oils such as beeswax, paraffin, microcrystalline wax, ceresin, higher fatty acids, and hardened oils, liquid oils such as squalane, liquid paraffin, various ester oils, and oily components such as preservatives and surfactants To prepare an oil phase.

Next, similarly to the ointment, the aqueous phase was gently heated with stirring, and when the temperature reached about 80 to 85 ° C., the oil phase heated to the same temperature was gradually added. Emulsify to make a cream.

In other skin external preparations, if the formulation is a mixture of an aqueous phase and an oil phase, sepia protein is previously mixed in the aqueous phase, and then mixed with the oil phase under heating. In the case of an external preparation for skin composed only of the aqueous phase, the desired external preparation is prepared by blending sepia protein at an arbitrary time.

Confirmation of Sepia Protein Effect As shown on the next page, the molecular weight of sepia protein prepared by using actinase E, papain, pepsin, and trypsin as proteolytic enzymes was measured by HPLC. It is shown in comparison with the molecular weight.

Result Squid protein / glycoprotein before degradation MW less than 10,000 40% 10,000 to less than 30,000 56% 30,000 or more 4% Sepia protein (0.005% actinase E) (pH 9.0, stirred at 50 ° C for 5 hours) MW Less than 10,000 100% Sepia protein (0.05% papain) (pH 7.0, stirred at 45 ° C for 5 hours) MW less than 10,000 93% 10,000 to less than 30,000 7% sepia protein (0.1% pepsin) (pH2.0, stirred at 40 ° C for 5 hours) MW less than 10,000 93% 10,000 to less than 30,000 7% sepia protein (50 units / ml trypsin) (pH 8.0, stirring at 40 ° C for 5 hours) MW less than 10,000 93% 10,000 to less than 30,000 7% Next, the whitening effect of various sepia proteins is determined by the cultured cells. The following method evaluated.

I. B16 cell whitening action 1) Preparation of sample The sample was freeze-dried and used as a medium-added sample.

2) Preparation of medium Eagle's MeM containing 10% fetal calf serum was used. The freeze-dried sample was added to the above-mentioned basic medium in an amount converted to the liquid volume before freeze-drying so as to have each concentration.

 A basal medium was used as a control.

3) Cell culture Mouse melanoma B16 cells were used. 1.0 × 10 ⁵ cells were seeded on a plastic petri dish (Falcon 3003) containing a test medium, and cultured at 37 ° C. for 5 days under the condition of 5% CO ₂ . Medium exchange was performed once on the third day.

4) Judgment method The culture solution and the cells were separated for 5 days and then centrifuged to prepare a pellet, which was compared with a control to determine the whiteness.

II. Proliferation of fibroblasts 1) Preparation of sample The sample was freeze-dried to obtain a sample added with a medium.

2) Preparation of medium Eagle's MEM containing 2% fetal calf serum was used as a basic medium. A sample freeze-dried to the above-mentioned basic medium was added and adjusted to each concentration in an amount converted to a liquid amount before freeze-drying.

 A basal medium was used as a control.

3) Cell culture Mouse-derived Balb / 3T12-3 fibroblasts were used as cells. 2.0 × 10 ⁴ cells were seeded on a plastic petri dish (Falcon 3002) containing a test medium, and cultured for 5 days. Culture exchange was performed once during this time.

4) Measurement of cells After culturing for 5 days, the cells were detached with trypsin, and the cells were measured using an automatic blood cell counter (manufactured by Toa Medical Electronics Co., Ltd.).

The cell proliferation action was expressed as a multiple of the control cell number.

 The results are shown below.

Claims (2)

(57) [Claims] An external preparation for skin, characterized in that a liquid obtained by hydrolyzing cuttlefish protein or glycoprotein is used as an active ingredient. 2. The external preparation for skin according to claim 1, wherein the hydrolysis is caused by actinase, papain, pepsin or trypsin.