JPH09278639A - Cosmetic containing collagen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a cosmetic good in extensibility and excellent in water retainability and homogeneity by using collagen extracted from a specific fish or its specific treated product or using the collagen together with hyaluronic acid. SOLUTION: This cosmetic is obtained by compounding cosmetic materials used for skin or hair with collagen extracted from a fish (especially preferably salmon, trout or codfish) living in a cold ocean current. The collagen can be obtained from the skin of the salmon, trout, codfish, etc., contained in marine wastes, and is denaturated into gelatin at a denaturation temperature of 16-19 deg.C. The collagen capable of being denaturated at a temperature near to room temperature is added to cosmetics and is used as milky lotions, skin lotions, concentrated jullies, hair liquids, lip sticks or creams for skin or hair. The collagen may be converted into atherocollagen with pepsin. The collagen may be used as succinylated collagen capable of being dissolved in a weakly acidic to neutral region. The simultaneous employment of both the collagen and hyaluronic acid exhibits an excellent moisture-retaining action.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to cosmetics containing collagen, which is a major protein of connective tissues of animals, that is, skin, blood vessels, tendons, bones, teeth and the like.

[0002]

2. Description of the Related Art Conventionally, collagen has been applied to artificial skin, artificial blood vessels, etc. because of its excellent applicability to the human body, and collagen has been incorporated into cosmetics.

[0003]

However, the origin of the collagen used in these is cow and pig, and the heat denaturation temperature to form a jelly-like substance was 35 to 40 ° C. Therefore, in cosmetics which are generally stored at room temperature of around 20 ° C and used for parts exposed to the outside air such as skin and hair, collagen particles are solidified and do not spread, so that they are sufficient for skin and hair. It was not possible to form a uniform film, allowed the moisture to escape, and could not obtain a uniform makeup surface. Therefore, an object of the present invention is to provide a cosmetic product which, when mixed with collagen, which has a heat denaturation temperature near room temperature, is well spread, and has excellent water retention and homogeneity.

[0004]

To achieve this object, the present invention has the following features.

According to the first aspect of the present invention, a collagen-containing cosmetic product is characterized in that collagen extracted from cold-dried fish is added to a cosmetic material used for skin and hair.

According to a second aspect of the present invention, in the first aspect of the invention, the cold-flowing fish is salmon, trout or cod, which is a cosmetic product containing collagen.

According to a third aspect of the present invention, in the first aspect of the present invention, a cosmetic product containing collagen is characterized in that the cosmetic material is mixed with the collagen extracted from one or more kinds of the cold-water fish. I am trying.

According to a fourth aspect of the present invention, in the first aspect of the present invention, the cosmetic material is mixed with the collagen to give an emulsion.
The cosmetic containing collagen is characterized in that it is one of a lotion, a jelly concentrate, a hair liquid, a lipstick, and a cream for skin and hair.

A fifth aspect of the present invention provides a cosmetic product containing collagen according to any one of the first to fourth aspects, wherein the collagen is atherolated by a pepsin treatment.

The invention of claim 6 is the invention of claim 1, 4 or 5.
In the invention, the collagen is a cosmetic product containing collagen, which is a succinylated collagen which is soluble in a weakly acidic to neutral region.

According to a seventh aspect of the present invention, in the first, fourth, fifth or sixth aspect of the invention, the collagen is used in combination with hyaluronic acid to provide a collagen-containing cosmetic product.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described based on Examples, but it goes without saying that the present invention is not limited thereto.

A large amount of fish skins, bones generated in the marine product processing process,
Most of the head and internal organs are treated as marine waste, and the amount is increasing year by year. Among these marine wastes, salmon,
We focused on collagen obtained from the skins of cold-dried fish such as trout and cod. It is known that the denaturation temperature of collagen varies depending on the living temperature of the organism from which it originates. Cold-water fish live in cold water, and the protein collagen that makes up the tissue is 16-19. Denature into gelatin at denaturation temperature of ° C. Therefore, it is a collage derived from cold-current fish that heat-denatures near room temperature
I thought about mixing Gen with cosmetics.

[0014] In the following, collagen was extracted from salmon rind, which was obtained in a large amount and was relatively easy to treat, from salmon of cold-current fish.
The physical properties of the extracted collagen will be clarified.

Experimental Example 1 Since collagen from salmon skin is heat-denatured at around room temperature, its temperature is 4 ° C. for extraction, and the temperature is kept at 0 ° C. or lower. I tried according to G.

Experimental method 1. It was washed thoroughly with distilled water to remove the remaining meat, scales, etc. from the salmon skin. 2. To improve the extraction efficiency, 300 g of salmon skin was minced using a mincing device. 3. Chloroform 450 ml (Metric), Me
The mixture was added to a mixture of 900 ml of OH (methanol) and 183 ml of water, stirred for 10 minutes and allowed to stand for 10 minutes to extract lipids. 4.3. Chloroform (450 ml) was added to the extraction residue of 1.
Stir for 1 minute, add 450 ml of water, stir for 6 minutes,
Lipid extraction was performed. 5. Lipids were filtered with a Buchner funnel, and the residue was washed with 450 ml of chloroform and then with MeOH to extract collagen. 6.5. After wrapping the collagen obtained in step 1 in a gauze and washing it thoroughly in running water, 0.2 M (mol) AcOH (acetic acid) at 4 ° C.
2 liters were added and the mixture was left at 4 ° C. for 72 hours. 7. Centrifuge for insoluble matter (HIMAC SCR20B manufactured by Hitachi, Ltd.)
/ 18) after centrifugation under a load of 3,000 g for 10 minutes, the supernatant is dialyzed against distilled water, and freeze-dried to obtain acid-soluble non-atelocollagen (the later-mentioned atelocollagen. (To distinguish it from).

Experimental Results The non-atherogenic collagen obtained was a white sponge-like solid, which was acid-soluble. The yield was 6 to 7% based on the original wet weight, but the yield can be improved by extracting the residue several times by the method described below.

Experimental Example 2 7. of Experimental Example 1. The residue obtained by the centrifugation performed in step 1) is further treated with pepsin to remove telopeptides, and acid-soluble atelocollagen as well as non-atelocollagen is obtained.

Experimental method 1. The residue extracted with acetic acid and centrifuged was 0.2 M (mol)
Dispersed in AcOH. 2. 1% pepsin was added to the weight of collagen, and the mixture was allowed to stand at a predetermined temperature for a predetermined time. 3. The residue was removed by centrifugation. 4. DEAE- Cellulose (Whatman DE52) 15W / V
0.5N (specified) HC so that (weight / volume)
1 (hydrochloric acid) aqueous solution, and stirred for 30 minutes. 5. Wash with distilled water until the pH reaches 4.0, then 15W / V
To 0.5N NaOH and stirred for 30 minutes. 6.4. And 5. The above procedure was repeated 4 times each, and finally washed with distilled water until neutral. 7. 15 g of DEAE-cellulose that has been equilibrated as above
(Gram) packed in an open column, 0.005M Ac
The atherolated collagen was dissolved in OH so that the concentration was 65 mg / ml, and the mixture was poured into a column and left overnight. 8. Dialysis and freeze-drying were carried out to obtain an atelolated acid-soluble atelocollagen collagen.

Experimental Results It was found that 74% of pepsin was removed by one purification with DEAE-cellulose, and further purification was carried out to almost 1%.
00% of pepsin was removed. Collagen has a helical structure with a structure called telopeptide at both ends. The telopeptide structure contains 12 to 27 amino acid residues, and the antigenicity of collagen is said to be expressed by this portion. The telopeptides are removed by this pepsin treatment to obtain atelocollagen without antigenicity.

As described above, the acid-soluble collagens obtained in Experimental Examples 1 and 2 are non-atelocollagen and the latter are atelocollagen.

<Molecular weight of salmon skin collagen and its distribution>
The molecular weight distribution was measured by SDS-PAGE electrophoresis. In this method, a protein is passed through a polyacrylamide gel, and a constant voltage is applied, whereby the protein is divided into the same molecular weights according to the balance between the gel pore size and the protein size. As described above, collagen has a different molecular weight distribution depending on its type, and can be identified by this method.

Experimental method 1. The following solutions were prepared. Solution A: 29.2 g of acrylamide and 0.8 g of N, N'-methylenebisacrylamide were dissolved in pure water to prepare 100
ml. Solution B: Trishydroxymethylaminotan (hereinafter referred to as Tris) 18.2 g and sodium dodecyl sulfate (SD
S) 0.4g, add 2ml of HCl to 100ml of pure water
And C solution: Tris 6.1 g, SDS 0.4 g, HCl 4. m
Pure water was added to 1 to make 100 ml. Solution D: 10% ammonium persulfate 2. A solution 3 ml, B solution 4.5 ml and pure water 10.5 m
After mixing 1, 0.01 ml of N, N, N ', N'-tetramethylethylenediamine (TEMED) and 0.08 of D solution
ml was added, mixed gently and poured between glass plates. 3.1 hours later, 0.9 ml of solution A, 1.5 ml of solution C,
D solution 0.02 ml, TEMED 0.01 ml, pure water 3.6
ml was mixed, poured between glass plates and left for 30 minutes. 4. A sample was prepared. SDS 0.1g, 2-mercaptoethanol 0.1ml, C solution 1ml, glycerin 2ml to 10ml with distilled water, dissolve the sample to 0.05%, add Pyroline Y, leave at 100 ° C for 3 minutes did. 5. Add the sample solution to the acrylamide gel and
An electric current was applied for 30 minutes at mA and 90 minutes at 20 mA. 6.0.25% Coomassie Brilliant Blue (KOD
AC), the gel was dipped in a mixed solution of 10% AcOH and 30% MeOH and allowed to soak overnight. 7. After decolorizing several times with a mixed solution of 10% AcOH and 30% MeOH, it was dried. 8. Specifically, this electrophoresis method is shown in FIG.

Experimental Results Salmon skin collagen, atherotized salmon skin collagen at 5 ° C,
According to the migration pattern (not shown) obtained in the experiment of the atherotized salmon skin collagen at room temperature, the salmon skin collagen atherotized at 5 ° C showed the same migration pattern as the original salmon skin collagen. Therefore, it can be said that atherification hardly progresses at low temperatures. The salmon skin collagen that was atherotized at room temperature showed bands not in the original pattern around the molecular weights of 210,000, 120,000, and 80,000. It is considered that this is because the telopeptide was detached due to the atelolation and the molecular weight was decreased.
Further, from the migration pattern of salmon skin collagen, it was found that the acid-soluble collagen obtained from salmon skin was type I among various types (Miyao Miyata, Journal of the Textile Society, 39, 11, 427 (1983)).

<Amino acid analysis and elemental analysis of salmon skin collagen> The amino acid composition is an important factor controlling the properties of the protein, and various properties such as the substituents and isoelectric point of the protein can be predicted. . Table 1 was obtained as a result of analysis by the Instrument Analysis Center of Hokkaido University.

[0026]

[Table 1] According to this, all of these collagens have a characteristic composition in which about 1/3 of all amino acid residues are glycine.

<Compatibility of succinylated collagen with various raw materials>

[Table 2]

[Table 3]

[Table 4] <Solubility> * Test method A diluted sodium hydroxide solution was added to the collagen solution adjusted to pH 2.5, and the results of measuring the absorbance at each pH are shown in FIG. It is shown that the higher the absorbance, the more the collagen fiber is deposited and the lower the solubility is.

* Results and Discussion Although general collagen is not soluble in the weakly acidic and neutral regions, it is observed that succinylated collagen is sufficiently soluble in these regions. That is, succinylated collagen is a special collagen that is made to dissolve in a weakly acidic to neutral region. When blending many commercially available collagens into cosmetics,
Although complicated operations such as pH adjustment and addition of salts were required, this succinylated collagen can save these troubles.

<Moisture retention> * Test method Using a skin layer stratum corneum water content measuring device (high frequency impedance meter Model IB-335: IBS Co., Ltd.), outer diameter 6 mm, center electrode diameter The electrical conductivity of the skin was measured with a 1 mm electrode. The measurement site is the inside of the forearm flexion side (diameter 1 cm), the electrical conductivity of the measurement site is measured beforehand, then the test solution is applied to the measurement site, and then the electrical conductivity is measured every one minute (25 ° C Humidity 55
%, Measured in a windless room). This is shown in FIG.

* Results and Discussion It can be seen from FIG. 4 that the succinylated collagen has excellent moisturizing properties. Further, it is recognized that a higher moisturizing effect is exhibited by using it together with hyaluronic acid. Therefore, succinylated collagen is a cosmetic material suitable for application as a moisturizing agent, and particularly when used in combination with hyaluronic acid, it exhibits an excellent moisturizing effect, and therefore can be incorporated into many cosmetic formulations.

<N-succinylation of salmon skin collagen> Generally, succinylated collagen having a high anticoagulant property and a wider dissolution pH region than unmodified collagen (acid-soluble collagen or atelocollagen). I tried to synthesize Gen.

* Experimental method 1. Salmon skin collagen 500mg 10% AcOH 17ml,
It was completely dissolved in 67 ml of MeOH. 6.4 g of succinic anhydride was added to the solution of 2.1, and the solution was allowed to stand at 4 ° C. for 24 hours after the succinic anhydride was completely dissolved. It was dialyzed at 3.4 ° C., the precipitate was collected by centrifugation and freeze-dried.

* Experimental Results The distilled water used for dialysis is slightly acidic (pH 4.5 to 5.0) because it contains carbon dioxide in the air. Since unreacted salmon skin collagen is soluble in this pH range, it is considered that what has precipitated is a product of some reaction. Since the reaction could not be confirmed from the IR spectrum of FIG. 5, the reaction rate was determined by quantifying the free amino group by the TNBS (trinitro bennzene sulfon-ate) method. The result is shown in Fig. 6.
Shown in According to this, it can be seen that the amount of free amino groups in the product is reduced to 32.1% of the unreacted product. This shows that the amino group in salmon skin collagen was amidated by the carboxyl group of succinic acid.

When the solubility of the product in each pH range was measured, a dissolution curve (FIG. 7) completely different from that of unreacted salmon skin collagen was obtained. According to FIG. 7, pH 4 ~
The solubility significantly decreases between 5 and increases rapidly over pH 5 to 6 and reaches 100% at pH 6 and above. Therefore, salmon-derived collagen is not soluble in weakly acidic and neutral as shown by white circles, but succinylated salmon-derived collagen as shown by black circles needs to be adapted to human skin, for example. It dissolves well in these weakly acidic and neutral regions, which are the conditions.

It can be seen that the isoelectric point was shifted between pH 4 and 5 because the solubility was greatly reduced as indicated by the curve indicated by the black circles. And in other words, that much salmon skin color-
It means that Gen has become anionic. From this result, it is considered that the cationic amino group decreased and the anionic carboxyl group increased. From the above, it can be said that the product is the reaction product shown in FIG. <Measurement of collagenase activity> * Preparation of reagent Collagen solution Atelo-collagen (salmon-derived), non-atelo-collagen (salmon-derived) and cowhide collagen were each lyophilized to 0. Dissolve in 0.01N HCl aqueous solution,
A 5 mg / ml solution was made. Buffer for collagen solution (Tris buffer) 0.1M Tris-HCl, pH 7.8, 0.4M N
aCl, 0.01M CaCl _2, 1M glucose, 0.04% NaN ₃ collagenase buffer solution (Tris-hydrochloric acid buffer solution) 0.05M Tris, pH 7.5 After adjusting pH to 7.5 when measuring activity. So that a small amount of 0.0
1N HCl, a buffer for collagen solution, and a collagenase buffer were taken and mixed at 1: 1: 2, 1N HCl was added in an appropriate amount to adjust the pH, and a corresponding amount of HCl was added to the collagenase buffer. Added to. Collagenase solution 0.01 mg / ml with collagenase buffer prepared above
Prepare the collagenase solution. In addition, collagenase is
Nitta Gelatin's COLLAGENASE N-2 from Streptomycespar
vulus subsp.citrinus was used. Reaction inhibitor 0.1M Disodium ethylenediaminetetraacetic acid (ET
A) Turbidity measuring solution 60% trichloroacetic acid (TCA) * Measuring method 1. For each collagen, a collagen solution and a buffer for collagen solution were mixed at a ratio of 1: 1, and the mixture was placed in a constant temperature bath at 37 ° C for 30 minutes.
I was soaked for more than a minute. 2. Add the collagenase solution to 1. The solution was mixed in the same amount with the solution, and the reaction time was set to 0. 3. An appropriate amount of the reaction solution was taken at each time, and ETA and the solution were mixed at a ratio of 1: 2 to stop the reaction. 4. After the measurement, the solution in which the reaction was stopped and TCA were mixed at a ratio of 3: 1, and after 30 minutes, the turbidity was measured at 340 nm. 5. Similarly, the collagenase activity was examined at 17 ° C. The composition of each solution after the completion of the activation reaction was collagen solution: collagen buffer: collagenase solution: ETA = 1: 1: 2: 2. The target for turbidity measurement was a separately prepared reaction-terminated mixed solution containing no collagen.

* Results Table 5 shows the measured results.

[0037]

[Table 5] From the data of Table 5, as shown in FIGS. 8 and 9, the turbidity was shown with respect to time. According to this, at 37 ° C. shown in FIG. 8, immediately after the start of the reaction, non-atelolated, atelolated, and bovine-derived collagens were gelatinized to become water-soluble and were sufficiently dissolved to have high turbidity. After about 20 minutes, the reaction proceeds and the molecule grows and precipitates and settles down.

On the other hand, at 17 ° C. shown in FIG. 9, both the non-atelolated and atelolated collagens began to dissolve in the solution after the start of the reaction and showed high turbidity, but the reaction did not proceed greatly. The amount of precipitation is also small over time, showing a slight downward line. On the other hand, cow-derived collagen is not water-soluble at such low temperature because it is not gelatinized, and its molecular weight is largely precipitated and its turbidity is low.

[0039]

As described above, it was found that salmon-derived collagen was heat-denatured in the vicinity of room temperature. Collagen obtained from cold-water fish has the same living temperature and the same physical properties. I understood. The present invention in which this collagen is applied to cosmetics has the following effects.

Human skin and hair, etc. that live in temperate zones
Generally, it is exposed at 16 to 19 ° C, and by mixing gelatin which becomes gelatinized and becomes water-soluble at around this temperature, absorption of collagen into skin and hair is good, spread is good, water retention, It is possible to provide a cosmetic product having excellent homogeneity.

By succinylation, a cosmetic product suitable for human skin, which is easier to use in the weakly acidic to neutral region, can be obtained.

Further, when used in combination with hyaluronic acid, an excellent moisturizing effect can be exhibited.

[Brief description of drawings]

FIG. 1 is an extraction flowchart of salmon-derived collagen.

FIG. 2 is an explanatory diagram of an electrophoresis method.

FIG. 3 is a graph showing the solubility of collagen with respect to pH.

FIG. 4 is a graph showing the moisturizing properties of collagen, which is a combination of collagen and hyaluronic acid.

FIG. 5 is a graph showing the IR spectrum of succinyl salmon collagen.

FIG. 6 is a graph showing the degree of blocking of aminoglucocollagens with compounds.

FIG. 7 is a graph showing the solubility of salmon collagen derivatives.

FIG. 8 is a graph showing collagenase activity at 37 ° C.

FIG. 9 is a graph showing collagenase activity at 17 ° C.

Claims (7)

[Claims] 1. A cosmetic product containing collagen, characterized in that collagen extracted from cold-dried fish is added to a cosmetic material used for skin and hair. 2. The cold drift fish according to claim 1,
Cosmetics with collagen characterized by being salmon, trout or cod. 3. The cosmetic product containing collagen according to claim 1, wherein the collagen material extracted from one or more cold-flowing fish is mixed with the cosmetic material. 4. The emulsion according to claim 1, wherein the cosmetic material is blended with the collagen, a lotion, a lotion, and a jelly concentrate.
A cosmetic containing collagen, which is one of a hair liquid, a lipstick, and a cream for skin and hair. 5. The cosmetic product containing collagen according to any one of claims 1 to 4, wherein the collagen is atherolated by a pepsin treatment. 6. The cosmetic product containing collagen according to claim 1, 4 or 5, wherein the collagen is a succinylated collagen which is soluble in a weakly acidic to neutral region. 7. The cosmetic product with collagen according to claim 1, 4, 5 or 6, wherein the collagen is used in combination with hyaluronic acid.