JP5171752B2 - Topical skin preparation - Google Patents

Description

    The present invention comprises a moisturizer, a whitening agent, an anti-inflammatory agent, an anti-aging agent, characterized by containing an antifreeze protein obtained from a fish of the order Salmoniformes, Salmonidae, Salvelinus, It relates to an external preparation for skin.

    Conventionally, in order to provide a moisturizing ingredient that prevents the skin from drying and moisturizes the skin, various active ingredients have been studied. Moreover, as factors of skin symptoms such as aging, diseases, stress, wrinkles due to ultraviolet rays, blemishes, reduced skin elasticity, dryness, cellular function decline, melanin production and pigmentation due to ultraviolet rays, decrease and degeneration of dermal matrix components, ultraviolet rays Oxidative damage of cells due to the above. In order to prevent and improve such skin symptoms, various active ingredients have been screened and formulated. In particular, naturally-occurring components are known to have various pharmacological and cosmetic effects, and so far, applications of many extracts of animals, plants and fungi to skin preparations have been studied.

    For example, in order to obtain an external preparation for skin having anti-aging and improving effects on skin, moisturizing effect and safety of skin such as essence of Ponkan (see Patent Document 1) as a component having an activity of dermal fibroblast activation or proliferation. As an excellent moisturizing agent, an extract of the genus Halibut (see Patent Document 2) and the like, as a whitening agent, an extract of the white crane reishi (see Patent Document 3) and the like, as an antioxidant, the plant of the genus Sarogase As a plant extract having an anti-inflammatory action, such as an extract of sucrose (see Patent Document 4), a dried product of soy leaf, cacao and musk or hot water extract thereof (see Patent Document 5), a solvent extract of a cannaceae plant ( In the topical skin preparation containing fish components, the cosmetics containing collagen extracted from cold-flowing fish (see Patent Literature 7), the brain and / or nerves of fish belonging to the mackerel family A cosmetic composition characterized by blending an extract (see Patent Document 8), a rough skin improving agent characterized by containing a component extracted by treating the ovarian membrane of salmon with a proteolytic enzyme (Patent Document) 9) and the like.

    Antifreeze protein is a protein that has been found in many organisms such as fish, insects, plants, fungi, and bacteria that are generally adapted to low-temperature environments, and has the effect of inhibiting the growth of ice crystals by binding to ice crystals. Say. It is thought that the antifreeze protein has such an effect because it has a hydroxyl group in the amino acid side chain and entraps water through this hydroxyl group. Therefore, it is easily dissolved in water and has a very high stability, so that it can be applied to high-temperature heat treatment.

    Examples of fish-based antifreeze proteins include fish-derived antifreeze proteins (see Patent Document 10), methods for producing frozen compositions (see Patent Document 11), freeze-drying methods (see Patent Document 12), and starch aging. An inhibitor, a starch product (see Patent Document 13), and the like are disclosed, and applied to food preservation and storage.

JP 2001-131045 A JP 2007-77072 A JP 2003-89630 A Japanese Patent Laid-Open No. 10-182413 JP 2003-201246 A JP-A-4-270224 Japanese Patent No. 3628100 Japanese Patent No. 4143509 Japanese Patent No. 3899116 Japanese Patent No. 4228068 Japanese National Patent Publication No. 10-508759 JP 2003-250506 A JP 2003-253263 A

    Thus, various naturally derived components have been applied so far. However, among naturally derived components, the effect is not sufficient, and the development of better components has been demanded. Therefore, the present invention is to provide an excellent skin external preparation having a moisturizing effect, a whitening effect, an anti-inflammatory effect, and an anti-aging effect.

    We found that antifreeze proteins obtained from salmonid salmonid fishes have excellent moisturizing, whitening, anti-inflammatory and anti-aging effects, and moisturizing, whitening, anti-inflammatory and anti-aging agents It came to provide the skin external preparation.

    According to the present invention, a moisturizer, a whitening agent, an anti-inflammatory agent, an anti-aging agent, and a skin external preparation having an excellent effect are provided by incorporating an antifreeze protein obtained from salmonid salmonid fish. can do.

FIG. 1 is a diagram of electrophoresis obtained by purifying antifreeze protein. FIG. 2 is a diagram of an antifreeze protein confirmation test utilizing the freeze concentration phenomenon.

    The salmonid salmonids used in the present invention live in the coldest waters of the uppermost stream of Japanese rivers and are carnivorous, falling from zooplankton, aquatic insects, other fish, and riverside trees. Eat insects and other small animals on the bottom. In addition, in the genus Iwana, it includes char (Salvelinus leucomaenis), American trout (Ezoiwana) (Salvelinus leucomaenis leucomaenis), Japanese char (Salvelinus leucomaenis pluvius), Yamatowana (Salvelinus leucomaenis jabronicus) The seed is known.

    The harvest time of the salmonid salmonids used in the present invention is preferably from the end of October to the beginning of May, and particularly preferably from the winter to the beginning of April.

    In the present invention, when using a salmonid salmonid fish, it is preferable to remove the head and abdomen and bones and use the body part excluding the skin.

    Extraction is to remove the head, abdomen and bones of salmonid salmonids, add water to the body part excluding the skin, 1) leave at 4 ° C or less overnight, 2) centrifuge And the step of collecting the supernatant, 3) the step of heat treatment, 4) the steps of 2 and 3) are repeated, and the step of obtaining the protein by centrifugation is included. The heat treatment is preferably 70 to 80 ° C. and 10 to 15 minutes, and the centrifugation is preferably 5000 to 13000 rpm and 20 to 50 minutes.

    The extract of the salmonid salmonid fish used in the present invention with the above solvent can be used as it is, but it may be left to stand for a certain period of time and matured, or concentrated and dried. The product can be used again by dissolving it in water or a polar solvent. Alternatively, it may be used after performing purification treatment such as decolorization, deodorization, and desalting, and fractionation treatment by column chromatography or the like within a range not impairing these physiological functions. The said extract of plant and fish, its processed material, and the fractionation thing can also be used after freeze-drying after each processing and fractionation, and melt | dissolving in the solvent at the time of use. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

    Antifreeze protein obtained from salmonid salmonid fishes has excellent moisturizing effect, whitening effect, anti-inflammatory effect, anti-aging effect, moisturizing agent, whitening agent, anti-inflammatory agent, anti-aging agent, skin It can be used as an external preparation.

    A moisturizing agent containing an antifreeze protein obtained from salmonid salmonid fish as an active ingredient has a filaggrin synthesis promoting action and exhibits an excellent moisturizing effect.

    Whitening agent with antifreeze protein obtained from salmonid salmonid fishes as an active ingredient has an inhibitory effect on epidermal melanocyte tyrosinase activity, and is excellent in preventing and improving pigmentation, spots, freckles, etc. Demonstrate whitening effect.

    A whitening agent containing antifreeze protein obtained from salmonid salmonid fishes as an active ingredient has a melanin production inhibitory action using B16 melanoma cells, preventing and improving pigmentation, spots, freckles, etc. Exhibits excellent whitening effect.

    Whitening agent with antifreeze protein obtained from salmonid salmonid fishes as an active ingredient has an inhibitory effect on epidermal melanocyte tyrosinase activity, and is excellent in preventing and improving pigmentation, spots, freckles, etc. Demonstrate whitening effect.

    An anti-inflammatory agent containing an antifreeze protein obtained from salmonid salmonid fish as an active ingredient has an IL-1α production inhibitory action and exhibits an excellent anti-inflammatory effect.

    An anti-aging agent comprising an antifreeze protein obtained from salmonid salmonid fish as an active ingredient has an activating effect on human dermal fibroblasts, and exhibits an excellent effect in preventing and improving aging symptoms.

    An external preparation for skin containing antifreeze protein obtained from salmonid salmonid fishes exhibits excellent moisturizing effect, whitening effect, anti-inflammatory effect, anti-aging effect and the like.

    Each of these agents is not limited at all in terms of its form and the presence or absence of other components as long as it contains an antifreeze protein obtained from salmonid salmonid fish as an active ingredient. As for the form, any form such as liquid, paste, gel, solid or powder can be selected according to its use and the like, vehicle (excipient), solvent, Or other general additives (an antioxidant, a coloring agent, a dispersing agent etc.) can be included arbitrarily.

    Here, the external preparation for skin means all external compositions applied externally to the skin or hair such as cosmetics, quasi-drugs or external pharmaceuticals.

    The dosage form of the external preparation for skin is arbitrary, and can be provided, for example, as a solubilization system such as lotion, a dispersion system such as calamine lotion, or an emulsification system such as cream or emulsion. Furthermore, it can also be provided in various dosage forms such as aerosol form, ointment or poultice filled with propellant.

    Specifically, various cosmetics such as emulsions, creams, lotions, lotions, packs, cosmetic liquids, cleaning agents or makeup cosmetics; liquids, ointments, powders, granules, aerosols, patches or poultices Various forms of cosmetics, quasi-drugs, or external medicines can be exemplified.

    In addition to the antifreeze protein obtained from salmonid salmonid fish, the topical skin preparations include pharmaceuticals, quasi-drugs, skin cosmetics, hair cosmetics, and cleansing agents, as needed. Any component that is usually blended in, for example, water, oily component, moisturizer, powder, pigment, emulsifier, solubilizer, gelling agent, cleaning agent, UV absorber, anti-inflammatory agent, thickener, pH A regulator, a chelating agent, a drug (medicinal component), a fragrance, a resin, a fungicide, a fungicide, an antioxidant, an alcohol, or the like can be appropriately blended. Furthermore, other moisturizers, whitening agents, anti-inflammatory agents, anti-aging agents, fish other than the salmonid salmonaceae, or extracts thereof, or other animal and plant extracts, as long as the effects of the present invention are not impaired. Can also be used together.

    The amount of antifreeze protein obtained from salmonid salmonid fish can be adjusted depending on the type and purpose, but the solid content of the total amount can be adjusted from the standpoint of efficacy and stability. In terms of conversion, it is preferably 0.001 to 5.0% by mass, more preferably 0.01 to 5.0% by mass, and still more preferably 0.1 to 5.0% by mass.

    Below, preparation examples of antifreeze protein obtained from salmonid salmonid fish, test methods for evaluating moisturizing effect, whitening effect, anti-inflammatory effect, and anti-aging effect, prescription example as a skin external preparation However, the technical scope of the present invention is not limited by these.

[Example 1]
<Extract 1>
The head, abdomen, and bones of salmonid salmonids were removed, the body part excluding the skin was crushed with a mixer, an equal amount of water was added, and the mixture was allowed to stand at 4 ° C. or less overnight. Thereafter, the mixture was centrifuged with a swing rotor at 5000 rpm for 30 minutes, and the supernatant was collected. The supernatant was treated at 70 ° C. for 10 minutes and allowed to stand on ice. Then, after dispensing into a 1.5 mL tube, centrifugation was performed at 13000 rpm for 20 minutes, and the supernatant was collected. The supernatant was treated at 80 ° C. for 15 minutes, transferred to a 1.5 mL tube, centrifuged at 13000 rpm for 50 minutes, and the supernatant was collected.

[Example 2]
<Purification of protein>
The supernatant was fractionated by SDS-PAGE. A VIVASPIN6 column was used to purify a protein around 7 kDa containing antifreeze protein (AFPIII). The result is shown in FIG.
Extract 1 (FIG. 1, lane 2)
After purification on VIVASPIN6 column (Figure 1, lanes 5 and 7)

[Example 3]
<Purified protein>
The supernatant obtained in Example 1 was freeze-dried for 24 hours and used as a crude protein product.

[Example 4]
<Antifreeze protein confirmation test>
A confirmation test of antifreeze protein using the freeze concentration phenomenon was conducted. Antifreeze protein has a function to hold water, thereby suppressing the crystallization of ice, and as shown below, when antifreeze protein is added together with inks and other contaminants and frozen, the solute is uniform even when frozen. A distributed state can be maintained. The results are shown in FIG.
* Ink + water Crystal grows using only water molecules, and other solutes are physically excluded, so the solute and ice separate (freeze concentration phenomenon). After thawing, the solute floated on the water surface or accumulated at the bottom.
* Ink + water + 7 kDa protein solution Freezing concentration phenomenon did not occur, and the solute was kept in a clean dispersed state as before freezing.
From the above, it was confirmed that the purified protein contained antifreeze protein.

    Each effect was evaluated using the said extract. Note that * and ** described in each evaluation result indicate that the significance probability P value in the t-test is less than 5% (P <0.05), and less than 1% significance (P <0.01). ) Is represented by **.

[Example 4]
<Moisturizing effect (evaluation of filaggrin synthesis promoting action)>
1. Human epidermal keratinocyte HaCaT cells were cultured in Dulbecco's MEM medium containing 10% FCS under conditions of 37 ° C. and 5% CO ₂ .
2. When it reached a confluent state, it was further cultured for 24 hours in Dulbecco's MEM medium supplemented with Extract 1, and then total RNA was extracted. RNA protect Cell Reagent and RNeasy Protect Cell Mini Kit (QIAGEN) were used for extraction of total RNA.
3. The expression level of filaggrin mRNA was measured by RT-PCR based on the total RNA extracted from HaCaT cells.
4). GAPDH was used as an internal standard. Using the expression level of GAPDH mRNA as a correction value, the expression level of filaggrin mRNA was determined as a ratio to the mRNA expression level in blank (only medium-treated) cells.
The evaluation results are shown in Table 1.

    As is clear from the results in Table 1, Extract 1 showed a significant moisturizing effect.

[Example 5]
<Whitening effect (evaluation of human epidermal melanocyte tyrosinase activity inhibitory action)>
Normal human epidermal melanocytes manufactured by Kurabo Industries Co., Ltd. were seeded in a 96-well microplate so that the number of cells was 3.0 × 10 ⁴ per well. As a seeding medium, Medium154S manufactured by Kurabo Industries Co., Ltd. was used. After 24 hours, the medium was replaced with a crude protein product culture medium adjusted to each concentration with Medium 154S, and further cultured for 48 hours. Next, it was replaced with 75 μL of a phosphate buffer containing 1% by weight Triton-X to completely lyse the cells, and 50 μL was used as a crude enzyme solution. To the crude enzyme solution, 50 μL of a 0.05% by mass L-dopa-containing phosphate buffer as a substrate was added and allowed to stand at 37 ° C. for 2 hours. The absorbance at 405 nm was measured immediately after the addition of the substrate and at the end of the reaction, and the amount of dopamelanin produced was determined by introducing the difference between the two measured values into the following equation.
Amount of dopamelanin produced = {(405 nm value after reaction−405 nm value before reaction) −2.166} /5.238
In addition, the amount of protein in each well was measured with a BCA protein assay kit manufactured by PIERCE, and the amount of dopamelanin produced per unit protein was determined. Tyrosinase activity inhibitory action was evaluated from the relative value when the control value was 100. The results are shown in Table 2.

    As is clear from the results in Table 2, the protein crude product had a significant whitening effect.

[Example 6]
<Whitening effect (evaluation of B16 mouse melanoma cell melanin production inhibitory effect)>
B16 mouse melanoma cells (B16F0 cells) were seeded in a 90 mm dish so that there were 18000 cells per dish. Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS) was used as the seeding medium. After 24 hours, the medium was replaced with a culture solution to which a crude protein purified product had been added to a concentration shown in Table 3 in a DMEM medium supplemented with 5% mass FBS, and further cultured for 5 days. After completion of the culture, cells were peeled off by trypsin treatment, transferred to a 1.5 mL microtube, and centrifuged to obtain a cell precipitate. The obtained precipitate was visually determined based on the following determination table. In the evaluation, a 5% FBS-added DMEM medium containing 5% by mass FBS was used as a negative control, and a 5% by mass FBS-added DMEM medium containing 50 mM sodium lactate was used as a positive control. These naked eye determination results were determined to be determination 5 and determination 1, and used as an index for sample determination. The naked eye evaluation was evaluated in five stages as shown below.
Judgment and Standard Judgment Criterion 1 Same as positive control (almost white)
2 Slightly darker than the positive control (light brown)
3 Between positive control and negative control (brown)
4 Blackening is slightly suppressed compared to the negative control (blackish brown)
5 Same level as negative control (almost black)
At the same time, a tissue solubilizer (trade name Solvable) was added to the precipitate, boiled, returned to room temperature, 150 μL was added to a 96-well microplate, and 500 nm was measured with a spectrophotometer (HITACHI spectrophotometer U-3010). Was measured to determine the total amount of melanin. The evaluation results are shown in Table 3.

    As apparent from the results in Table 3, the protein crude product had a significant whitening effect.

[Example 7]
<Anti-inflammatory effect (evaluation of IL-1α production inhibitory effect)>
1. Human epidermal keratinocyte HaCaT cells were cultured in Dulbecco's MEM medium containing 10% FCS under conditions of 37 ° C. and 5% CO ₂ .
2. When it reached a confluent state, it was further cultured for 24 hours in Dulbecco's MEM medium supplemented with Extract 1, and then total RNA was extracted. RNA protect Cell Reagent and RNeasy Protect Cell Mini Kit (QIAGEN) were used for extraction of total RNA.
3. The expression level of filaggrin mRNA was measured by RT-PCR based on the total RNA extracted from HaCaT cells.
4). GAPDH was used as an internal standard. Using the GAPDH mRNA expression level as a correction value, the expression level of IL-1α mRNA was determined as a ratio to the mRNA expression level in blank (only the medium treated) cells.
The evaluation results are shown in Table 4.

    As is clear from the results in Table 4, Extract 1 showed a significant anti-inflammatory effect.

[Example 8]
<Anti-aging effect (Evaluation of human dermal fibroblast type I collagen production promoting effect)>
Normal human dermal fibroblasts were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells per well. Dulbecco's modified Eagle medium (DMEM) supplemented with 5 wt% fetal bovine serum (FBS) was used as the seeding medium. After 24 hours, the medium was replaced with a culture solution to which a crude protein purified product had been added to a concentration shown in Table 5 in a 0.5% by mass FBS-added DMEM medium, and further cultured for 24 hours. The ELISA method was used for the quantification of type I collagen secreted into the culture supernatant, and finally 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt against labeled peroxidase. (ABTS) and hydrogen peroxide were added and reacted, and then the absorbance at 405 nm was measured with a microplate reader. In the evaluation, 0.5% by mass containing 0.5% by mass FBS-added DMEM medium as a negative control in addition to the sample culture solution and 50 μM L-ascorbic acid phosphate magnesium salt (VCPMg) as a positive control. FBS-added DMEM medium was used. The evaluation was performed by obtaining a relative value when the cell activation effect in the control was set to 100. Further, the number of cells in each well or the amount of collagen produced per unit protein was determined using a Coulter Counter manufactured by BECKMAN or a BCA protein assay kit manufactured by PIERCE. The evaluation was performed by obtaining a relative value when the collagen production amount per unit of the negative control was 100.

    As is clear from the results in Table 5, the protein crude product had a significant anti-aging effect.

    Then, the formulation example of the skin external preparation which mix | blended the extract 1 obtained by the said preparation method or the protein rough refined material is shown.

[Example 9]
Emulsion (1) Squalane 10.0 (mass%)
(2) Methylphenylpolysiloxane 4.0
(3) Hydrogenated palm kernel oil 0.5
(4) Hydrogenated soybean phospholipid 0.1
(5) Polyoxyethylene monostearate
Sorbitan (20E.O.) 1.3
(6) Sorbitan monostearate 1.0
(7) Glycerin 4.0
(8) Methyl paraoxybenzoate 0.1
(9) Carboxyvinyl polymer 0.15
(10) Purified water 100 (11) Crude protein purified product 1.0
(12) Arginine (1% by weight aqueous solution) 20.0
Production method: The oil phase components (1) to (6) are heated and dissolved at 80 ° C. On the other hand, the aqueous phase components (7) to (11) are dissolved by heating at 80 ° C. The oil phase component is added to this while stirring and uniformly emulsified with a homogenizer. After cooling, add (12) sequentially at 40 ° C. and mix uniformly.

[Example 10]
Lotion (1) Ethanol 15.0 (mass%)
(2) Polyoxyethylene (40E.O.) hydrogenated castor oil 0.3
(3) Fragrance 0.1
(4) Purified water 100 (5) Citric acid 0.02
(6) Sodium citrate 0.1
(7) Glycerin 1.0
(8) Hydroxyethyl cellulose 0.1
(9) Protein crude product 0.5
Production method: (2) and (3) are dissolved in (1). Further, after sequentially adding (4) to (8), the mixture is sufficiently stirred, and (9) is added and mixed uniformly.

[Example 11]
Cream (1) Squalane 10.0 (mass%)
(2) Stearic acid 2.0
(3) Hydrogenated palm kernel oil 0.5
(4) Hydrogenated soybean phospholipid 0.1
(5) Cetanol 3.6
(6) Lipophilic glyceryl monostearate 2.0
(7) Glycerin 10.0
(8) Methyl paraoxybenzoate 0.1
(9) Arginine (20 mass% aqueous solution) 15.0
(10) Remainder 100 (11) Carboxyvinyl polymer (1% by weight aqueous solution) 15.0
(12) Protein crude product 1.0
Production method: The oil phase components (1) to (6) are heated and dissolved at 80 ° C. On the other hand, the aqueous phase components (7) to (10) are dissolved by heating at 80 ° C. The oil phase component is added to this while stirring and uniformly emulsified with a homogenizer. Add (11), stir, cool and add (12) at 40 ° C. and mix uniformly.

[Example 12]
Essence (1) Purified water 100 balance (mass%)
(2) Glycerin 10.0
(3) Sucrose fatty acid ester 1.3
(4) Carboxyvinyl polymer (1% by weight aqueous solution) 17.5
(5) Sodium alginate (1% by weight aqueous solution) 15.0
(6) Polyglyceryl monolaurate 1.0
(7) Macadamia nut oil fatty acid phytosteryl 3.0
(8) N-lauroyl-L-glutamic acid di (phytosteryl-2-octyldodecyl) 2.0
(9) Hardened palm oil 2.0
(10) Squalane (from olive) 1.0
(11) Behenyl alcohol 0.75
(12) Beeswax 1.0
(13) Jojoba oil 1.0
(14) 1,3-butylene glycol 10.0
(15) L-arginine (10% by mass aqueous solution) 2.0
(16) Protein crude product 1.0
Production method: The aqueous phase components (1) to (6) are mixed and dissolved by heating at 75 ° C. On the other hand, the oil phase components (7) to (14) are mixed and dissolved by heating at 75 ° C. Next, the oil phase component is added to the aqueous phase component and preliminary emulsification is performed, followed by uniform emulsification with a homomixer. After cooling, add (15) at 50 ° C and (16) at 40 ° C and mix uniformly.

[Example 13]
Aqueous gel (1) Carboxyvinyl polymer 0.5 (mass%)
(2) The balance made into purified water 100 (3) Sodium hydroxide (10 mass% aqueous solution) 0.5
(4) Ethanol 10.0
(5) Methyl paraoxybenzoate 0.1
(6) Fragrance 0.1
(7) Polyoxyethylene (60E.O.) hydrogenated castor oil 1.0
(8) Protein crude product 0.5
Manufacturing method: (1) is added to (2), and after stirring uniformly, (3) is added. After stirring uniformly, (5) previously dissolved in (4) is added. After stirring uniformly, (6) to (7) previously mixed are added, finally (8) is added, and the mixture is stirred and mixed uniformly.

[Example 14]
Cleansing fee (1) Squalane 81.0 (mass%)
(2) Polyoxyethylene glyceryl isostearate 15.0
(3) Purified water 100 (4) Crude protein product 1.0
Manufacturing method: (1) and (2) are uniformly dissolved. (3) and (4) are sequentially added to this and mixed uniformly.

[Example 15]
Facial cleansing foam (1) Stearic acid 16.0 (mass%)
(2) Myristic acid 16.0
(3) Lipophilic glyceryl monostearate 2.0
(4) Glycerin 25.0
(5) Sodium hydroxide 7.5
(6) Palm oil fatty acid amidopropyl betaine 1.0
(7) Purified water, the rest (8) Protein crude product 0.1
Production method: The oil phase components (1) to (4) are heated and dissolved at 80 ° C. On the other hand, the aqueous phase components (5) to (7) are heated and dissolved at 80 ° C., and mixed and stirred uniformly with the oil phase components. After cooling, add (8) at 40 ° C. and mix uniformly.

[Example 16]
Makeup base cream (1) Squalane 10.2 (mass%)
(2) Cetanol 2.0
(3) Glycerin tri-2-ethylhexanoate 2.5
(4) Lipophilic glyceryl monostearate 1.0
(5) Propylene glycol 11.0
(6) Sucrose fatty acid ester 1.3
(7) The balance which makes 100 purified water (8) Titanium oxide 1.0
(9) Bengala 0.1
(10) Yellow iron oxide 0.4
(11) Fragrance 0.1
(12) Protein crude product 1.0
Production method: The oil phase components (1) to (4) are mixed and dissolved by heating at 75 ° C. On the other hand, the aqueous phase components (5) to (7) are mixed and dissolved by heating at 75 ° C., and the pigments (8) to (10) are added thereto and dispersed uniformly with a homomixer. The oil phase component is added to the water phase component and emulsified with a homomixer. After cooling, add components (11) and (12) at 40 ° C. and mix uniformly.

[Example 17]
Emulsion foundation (1) Methylpolysiloxane 2.0 (mass%)
(2) Squalane 5.0
(3) Octyldodecyl myristate 5.0
(4) Cetanol 1.0
(5) Polyoxyethylene (20E.O.)
Sorbitan monostearate 1.3
(6) Sorbitan monostearate 0.7
(7) 1,3-butylene glycol 8.0
(8) Xanthan gum 0.1
(9) Methyl paraoxybenzoate 0.1
(10) The balance of purified water 100 (11) Titanium oxide 9.0
(12) Talc 7.4
(13) Bengala 0.5
(14) Yellow iron oxide 1.1
(15) Black iron oxide 0.1
(16) Fragrance 0.1
(17) Protein crude product 0.1
Production method: The oil phase components (1) to (6) are mixed and dissolved by heating at 75 ° C. On the other hand, the aqueous phase components (7) to (10) are mixed and dissolved by heating at 75 ° C., and the pigments (11) to (15) are added thereto and uniformly dispersed with a homomixer. Add oil phase ingredients and emulsify. After cooling, components (16) and (17) are added sequentially at 40 ° C. and mixed uniformly.

[Example 18]
Water-in-oil emollient cream (1) Liquid paraffin 34.0 (mass%)
(2) Microcrystalline wax 2.0
(3) Vaseline 5.0
(4) Diglycerin oleate 5.0
(5) Sodium chloride 1.3
(6) Potassium chloride 0.1
(7) Propylene glycol 3.0
(8) 1,3-butylene glycol 5.0
(9) Methyl paraoxybenzoate 0.1
(10) The balance of 100 purified water (11) Fragrance 0.1
(12) Protein crude product 1.0
Production method: Dissolve (5) and (6) in a part of (11) to 50 ° C., and gradually add to (4) heated to 50 ° C. with stirring. After mixing this, it disperse | distributes uniformly to (1)-(3) heated and melt | dissolved at 70 degreeC. (7) to (9) are added to the remainder of (10) heated and dissolved at 70 ° C. with stirring, and emulsified with a homomixer. After cooling, add (11) and (12) at 40 ° C and mix uniformly.

[Example 19]
Pack (1) Remainder water (100%)
(2) Polyvinyl alcohol 12.0
(3) Ethanol 17.0
(4) Glycerin 9.0
(5) Polyethylene glycol (average molecular weight 1000) 2.0
(6) Protein crude product 1.0
(7) Fragrance 0.1
Production method: (2) and (3) are mixed, heated to 80 ° C, and then dissolved in (1) heated to 80 ° C. After evenly dissolving, add (4) and (5) and cool with stirring. Add (6) and (7) at 40 ° C. and mix uniformly.

[Example 20]
Bath agent (1) Fragrance 0.3 (mass%)
(2) Protein crude product 3.0
(3) Sodium bicarbonate 50.0
(4) Sodium sulfate 46.7
Production method: (1) to (4) are mixed uniformly.

[Example 21]
Hair wax (1) Stearic acid 3.0 (mass%)
(2) Microcrystalline wax 2.0
(3) Cetyl alcohol 3.0
(4) Highly polymerized methylpolysiloxane 2.0
(5) Methylpolysiloxane 5.0
(6) Poly (oxyethylene / oxypropylene)
Methylpolysiloxane copolymer 1.0
(7) Methyl paraoxybenzoate 0.1
(8) 1,3-butylene glycol 7.5
(9) Arginine 0.7
(10) Purified water 100 (11) Crude protein purified product 1.0
(12) Fragrance 0.1
Production method: The oil phase components (1) to (6) are mixed and heated and dissolved at 75 ° C. On the other hand, the aqueous phase components (7) to (10) are dissolved by heating at 75 ° C., the oil phase component is added, and the mixture is emulsified with a homomixer. After cooling, add components (11) and (12) at 40 ° C. and mix uniformly.

[Example 22]
Hair artic (1) Ethanol 50.0 (mass%)
(2) The balance of 100 purified water (3) Extract 1 1.0
(4) Fragrance 0.1
Production method: Components (1) to (4) are mixed and homogenized.

    The skin external preparations shown in Examples 8 to 22 were compositions having a moisturizing effect, a whitening effect, an anti-inflammatory effect, and an anti-aging effect.

    The antifreeze protein obtained from the salmonid salmonid fish of the present invention is useful for use in skin external preparations such as skin cosmetics, hair cosmetics or cleaning agents, pharmaceuticals, and quasi drugs. It is. In addition, since the antifreeze protein obtained from the fish of the genus Ivana according to the present invention is a naturally derived component, it is easily considered to be highly safe and has great significance as an external preparation for skin. It is useful as a new external preparation for skin.

Claims (5)

    A moisturizer characterized by containing antifreeze proteins obtained from Salmoniformes, Salmonidae and Salvelinus fishes.     Whitening agent characterized by containing antifreeze protein obtained from Salmoniformes, Salmonidae and Salvelinus fishes.     An anti-inflammatory agent characterized by containing an antifreeze protein obtained from a fish of the Salmoniformes Salmonidae and Salvelinus species.     An anti-aging agent characterized by containing antifreeze protein obtained from Salmoniformes, Salmonidae and Salvelinus fishes.     A skin external preparation characterized by containing antifreeze protein obtained from Salmoniformes, Salmonidae, and Salvelinus fish.

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