JPS63188607A - Cosmetic - Google Patents

Abstract

PURPOSE:To obtain a cosmetic preventing aging of skin, showing reproduction promoting effects on damages, containing water-soluble protein extracted from bovine serum or various bovine organs such as udder, spleen, thymus, etc., without using an organic solvent, strong acid, strong alkali and enzyme. CONSTITUTION:A cosmetic containing water-soluble protein having specific molecular weight and isoelectric point, extracted from bovine serum or bovine udder, spleen, thymus, etc., without using an organic solvent, strong acid, strong alkali and enzyme. The cosmetic is not only effective for preventing aging of skin such as drying of epidermal layer part, increase in wrinkles, reduction in luster, occurrence of strain, etc., but also useful as a medical external preparation showing rapidly restoring action on damages ranging from epidermal layer to dermal layer in cuts, burns, etc. The water-soluble protein is obtained by combining conventional extracting methods of a organism components such as salting-out fractionation method, gel filtration method, centrifugal separation method, etc.

Description

[Detailed Description of the Invention] [A] Purpose of the Invention The present invention is directed to the use of bovine serum (blood) or various bovine organs (
The present invention relates to a new cosmetic containing water-soluble protein extracted from the breast, lung, thymus) without using organic solvents, strong acids, strong alkalis, or enzymes (proteolytic enzymes).

"Industrial Application Field: The present invention relates to a cosmetic containing a water-soluble protein extracted from bovine serum or various bovine organs and having a specified molecular weight and isoelectric point. Water-soluble proteins act directly on fibroblasts, activate them, prevent skin aging, and in the case of trauma or burns, work to regenerate granulation tissue and stimulate the epidermis. It reduces crater-like scars and makes the epidermis (skin) smooth, so it can also be used as a therapeutic agent applied externally to the skin.

In addition, in the present invention, although the raw materials to be used are shown based on bovine serum (blood), breast, lung, and thymus gland, if the extraction means shown in the present invention is adopted, other livestock M (animals),
For example, those obtained using various organs such as pigs and serum (blood) can also be used.

1. Prior art Since ancient times, extracts made from the blood and organs of animals such as livestock have been used in cosmetics and medicines. Regarding the efficacy or effect (action) of these extracts, it is said that they contain factors that have cell activators or tissue regeneration and repair effects, such as placenta, liver, kidney, and tablets. Extracts obtained by various methods from organs such as the heart, stomach, and intestines, as well as blood, have been successfully applied.

However, until now, it has not been possible to identify the main component that has cell activation or tissue regeneration and repair effects, and much remains unclear.

Among them, water-soluble extracts containing water-soluble proteins, water-soluble peptides, nucleic acids, or nuclear #related substances are frequently used in cosmetics, etc., and the water-soluble substances contained in these extracts are effective for cell activation. It has been presumed that it exhibits a tissue regeneration-promoting effect.

In other words, these estimates have been made by analyzing components distributed in cells and tissues, and have been considered to be one of the leading factors.

However, from these conventionally known extracts,
Regarding extracts containing water-soluble components, they are truly effective for cells.
At present, there are very few extracts that have been proven to have a direct activating effect.

Therefore, the present inventors sought and reevaluated the cell and tissue activation and regeneration and repair abilities of extracts derived from animal organs such as cows that have been used since ancient times, and developed even better extracts. In order to establish a test method for direct evaluation from the cellular level, we developed the method described below. The present inventors have already employed this new evaluation method, which is based on cultured fibroblasts, to quantitatively evaluate the growth (M increase) effect on the fibroblasts in cattle or humans. For example, from the placenta of cows, it is a water-soluble protein with a molecular weight of 80,000 (power cutoff point 6.
5-7.0), and the molecular weight is 25.000 (isoelectric point 5.000).
0 to 5.5') succeeded in isolating a certain substance, and in 1932
The application was filed with Patent Application No. 159713.

1. Problems to be Solved by the Invention The present inventors have continued to re-evaluate extracts derived from various animal organs. In other words, as mentioned above, although animal-derived extracts have long been said to have cell-activating and tissue-activating abilities, when it comes to extracts that actually have a direct activating effect on m-cells, It has been found that those obtained using conventional extraction methods have zero or very weak ability to proliferate cells.

In other words, although there are substances in each organ that have a direct activating effect on cells and exhibit proliferation ability, the extraction method is incomplete, and as a result, only components that have no effect are extracted. It is also assumed that extracts that contain these substances have been used. Here, we focused our development efforts on obtaining various organs and directly searching for substances that have physiological activity in cells.

“Evaluation method.

The main cells of the skin include epidermal cells (keratinocytes) and fibroblasts. For example, fibroblasts collected from guinea pigs (Hartley strain) flourish in Eagle's MEMum supplemented with 5% FBS. The cells undergo division and proliferation, and the time for multiple proliferation is approximately 48 hours, as shown in FIG.

To determine the cell proliferation rate, first, 4 to 5
X 10' cells are seeded and Eagle's MEM medium supplemented with low concentration of FBS is changed every 2-3 days. Cell counts are determined every 2-3 days using a Bürkertürk hemocytometer.

[Mouth] Structure of the Invention The present invention resides in a cosmetic containing an extract consisting of water-soluble protein under the following conditions, which has been confirmed to have a strong effect based on the above-mentioned evaluation method.

(1) The molecular weight obtained from cow blood is 75,000,
(Isoelectric point: around 6.0 to 6.5) (2) The molecular weights obtained from the cow's udder are 55.000 and 9.900 (blackout point: the former is around 6.0 to 6.5, the latter is around 7.0 to 7.5) (3) The molecular weight obtained from cow lung is 41,000,
(Power failure point: around 6.0 to 6.5) (4) The molecular weight obtained from the cow thymus was 46,000, (conductivity point: 6.0 to 6.
．． 5) However, the water-soluble extracts shown in (1) to (4) above are limited to those extracted by methods that do not use organic solvents, strong acids, strong alkalis, or enzymes, and do not undergo heat treatment. However, when used in cosmetics, (1) ~
The protein shown in (4) is not limited to the use of isolated proteins, but can be used in solutions containing any of (1) to (4), or in solutions containing any of (1) to (4). In any of the crude extracts or in any of the extracts (1) to (4),
A solution containing other stabilizers and various other agents can be incorporated into cosmetics and used.

Below, in order to show more concretely, the method for producing each extract specified in the present invention and the method for blending (formulating) it into cosmetics will be shown.

r Key points in the production method (extraction method)" The key points in the method for producing the extract used in the present invention are as follows (a) -
(b).

(a) The starting materials are those shown in (1) to (4) above,
In both cases, it is best to use fresh organs after slaughter and dissection, and it is usually best to freeze or store them at low temperatures and use them at appropriate times.As for blood, it is better to leave it still as a fresh barometer. It is possible to use both a state in which the serum and blood are separated, and a state in which the serum is in a hemolyzed state in which no separation is obtained.

(b) The key points in extraction are not to apply heat, to use muffled solvents,
It is essential to extract without using strong acids, strong alkalis, or enzymes.

That is, these processing operations are the object of the present invention.
This is because the activity will be deactivated. In other words, extracts containing water-soluble proteins extracted from animal-derived organs or blood that have been conventionally blended into cosmetics have been subjected to at least one of the above-mentioned operations before being finally obtained. However, extracts obtained by employing such treatments during the process no longer exhibit the ability to directly proliferate cells.

Therefore, according to this, by combining conventional methods for extracting biological components such as salting-out fractionation method, gel filtration method, centrifugation method, etc., the production (extraction) can be read. investigated the relationship between extracts obtained using various extraction methods and their active effects, and as a result found that the active substances that play a leading role differ depending on the organ. That is, although the main substance of the obtained extract is a water-soluble protein, it was confirmed by the evaluation test shown above that the molecular weight is different.

In other words, the extract (crude liquid) obtained under extraction conditions without heat treatment, organic solvent treatment, strong acid/alkaline treatment, or enzymatic decomposition treatment has no effect on any organ or blood. However, it was found that all of them contain water-soluble proteins that have the ability to proliferate cells. Then, based on the extracted extract obtained, the crude liquid (extract)
As a result of isolation (separation) and purification of the main substance of (
It was confirmed that the substance had the molecular weight shown in 1) to (4).

Therefore, with the discovery of the main active substance, extraction methods can be used to achieve the objectives described below by devising combinations of them based on various known means, without being limited to the method of the present inventors. It became possible to extract effective substances.

"Extraction example-1 = Extraction method from blood and serum" Ammonium sulfate is added to bovine blood (hemolyzed blood is also acceptable) or serum obtained by separating it from blood to a 20% saturation concentration. Thereafter, the supernatant is separated by centrifugation, ammonium sulfate is added again to the supernatant to a concentration of 60% saturation, and the precipitate is recovered by centrifugation.

Next, the precipitate is dissolved in a small amount of water or physiological saline, and then desalted. The desalting operation may be performed by any method such as gel filtration, ultrafiltration, or dialysis. For example, if a method is adopted in which the molecular weight is 75.
A liquid (crude liquid) containing a water-soluble protein having a molecular weight of 0.000 was obtained.

1 Extraction Example-2: Extraction method from udder Cut the cow's udder into small pieces without washing, homogenize with a homogenizer or the like at low temperature, and then add water or sodium chloride solution, or a buffer solution as shown in Table 1. Add and stir. Here, after adding water or a sodium chloride solution and stirring, it is left to stand for 6 hours - day and night, and then centrifuged to obtain a supernatant, which has a concentration of 40% saturation with respect to this supernatant. Add ammonium sulfate, centrifuge, take the supernatant, add ammonium sulfate again to the stage solution to a concentration of 80% saturation,
The following operation of collecting the precipitate by centrifugation is similar to that shown in Extraction Example 1 above, by dissolving it in a small amount of water or physiological saline, etc., and then desalting it to reduce the molecular weight. A liquid (crude liquid) containing water-soluble proteins at 55.000 and 9.900 can be obtained.

“Extraction Example-3: Extraction method from lungs j After cutting cow lungs into small pieces without performing any pretreatment such as washing,
Homogenize at low temperature. After homogenization, water or sodium chloride solution, or a buffer as shown in Table 1 is added, and the subsequent operations are preferably carried out in accordance with Extraction Example 2 above.However, in this case, the second When adding ammonium sulfate, the concentration is adjusted to 60% saturation. This results in a molecular weight of 41°0
A liquid containing water-soluble protein (crude liquid) having a pH of 0.00 is obtained.

1 Extraction Example-4: Extraction method from thymus j Cow thymus is immediately cut into pieces without pretreatment such as washing, and homogenized at low temperature. The subsequent operations are the same as those in Extraction Example 3. As a result, the molecular weight is 46.
．． A liquid containing a water-soluble protein (crude liquid) having a molecular weight of 0.000 is obtained.

"Table 1" Relationship between buffer solution in crude extraction and ion exchanger in purification The extract (crude solution) obtained by the method shown in Extraction Examples 1 to 4 above can be used as it is in the cosmetics targeted by the present invention. It can also be used in combination with the skin, and it shows direct proliferation ability for cells.In other words, if it is used in combination with the skin, it can smooth the metabolism of skin tissue cells. none,
It has anti-aging effects, prevents fine wrinkles, and gives you shiny, youthful skin. In addition, if there are small cuts on the skin, or if spots on the face of pregnant women (before and after childbirth) become noticeable, or if you have melasma, please check the pigmentation of these spots. It can be prevented to a minimum.

In addition, it is also good to purify and use each substance (protein with a specified molecular weight) that plays a leading role, but the purification method is, for example, the one obtained in Extraction Examples 1 to 4 above. The crude liquid can be purified by fractionation using a combination of known gel filtration, ion chromatography, and the like. or,
Carriers used for gel filtration include Sephadex,
Examples include agarose and sephacryl.

1 Purification Example - 1 Each of the crude liquids obtained in Extraction Examples 1 to 4 was treated under the conditions of [1] shown below, and the desired active fraction was further obtained as shown in [2]. Fractionation is performed using an ion exchanger (m fat). Examples of carriers used for ion exchange include cationic and anionic exchangers such as Sephadex, Sepharose, and Sephacryl. Here, for example, DEAB-Sepharose CL-6B was used.

[1] Resin: Sephacryl S-200 Super Fine, column size: 26X956+1111% solvent 20.1M
-0,05N Tris-HCl buffer CpH 8,0), flow rate: 3,3 mfi/Il″m″/h [2] Resin: DEAE-Sepharose CL-6B, solvent: 0.
IM-0,05N Tris-HCl buffer (pH 8,0)
, Elution conditions: NaC 1m degree 0-0.3M The active fraction obtained in the above [2] was further gel-filtered with Sephacryl S-200, thereby obtaining water-soluble active fractions having the respective specified molecular weights. Separate.

The activities (cell proliferation ability) of the extracts separated after the above purification and indicated by specific molecular weights are as shown in FIGS. 2 to 1, respectively.

In other words, Fig. 1 shows cell growth curves depending on the concentration of 5% FBS (fetal bovine serum), and when compared with this, all extracts show strong activity. be.

This effect is achieved by extracts obtained using conventional extraction methods.
Even if the extract contained components with similar molecular weights, it would have been an extract that had been deactivated.

'Therefore, we have thoroughly investigated the cause of this problem, although we do not show the results of the extracts obtained using the conventional method, with tables and figures for comparison.

As a result, active substances capable of cell proliferation are inactivated when exposed to the following conditions.

(1) When exposed to heat above body temperature for a long time under special conditions, (2) When exposed to organic solvents, (3) In the presence of strong acids, strong alkalis, or proteolytic enzymes. However, it was found that most of the extracts extracted through these treatments no longer show proliferative ability against am and become inactive extracts.

In other words, extracts extracted from organs derived from various animals, which have been used in conventional medicines and cosmetics, are processed by applying at least one of the above treatments during the process. uses antibacterial agents (preservatives, anti-inflammatory agents, etc.),
Alternatively, when using the raw material, it has been extracted after undergoing treatments such as blood removal and blood removal. However, even with these operations, the desired active substance is rapidly deactivated. In particular, washing with water during pretreatment causes the main active substance to flow out, and the result is an extract that has no effect at all, no matter how high the protein content of the water-soluble extract. Furthermore, it goes without saying that antibacterial agents themselves are toxic to cells, and cannot be expected to have any effect on cells. The same applies to the treatment of organic solvents. Moreover, using enzymes,
It was discovered that strong alkalis immediately denature and decompose active substances, deactivating them.

Therefore, although extracts obtained using these conventional methods have a remarkable moisturizing effect on the skin, they are known to have the effect of promoting cell and tissue metabolism, which has been reported since ancient times. It has come to be believed that the effect was small, or that it was obtained from a secondary effect as a nutritional supplement.

"Key Points of Formulation Method" Next, we will discuss the use of the present invention in cosmetics.
When blended into cosmetics, it can be used in the same manner as conventional extracts derived from various animals, but as mentioned above, due to the extraction method, the substances listed as causes of deactivation may be used. It is desirable to avoid formulating creams and emulsions as much as possible, and it is also desirable to avoid heat treatment as much as possible when formulating creams and emulsions.

When incorporated into liquid cosmetics such as lotions that do not require emulsification or suspension, heat treatment is often not required, and the product will not be deactivated. Therefore, the action is stably sustained, but the active substance (extract) shown in the present invention can be used for a longer period of time.
When formulating cosmetics containing , for example, extracts containing water-soluble pallor, peptides, amino acids, etc. obtained by hydrolysis of various animal-derived materials or treatment with organic solvents, etc., using conventional methods. The concomitant use with substances (extracts), certain Iha, mucopolysaccharides (hyaluronic acid, fundroitin sulfate, etc.), extracts containing them, aqueous solutions of chitosan or chitin, etc. will improve the sustainability of stability. .

In addition, these known substances (extracts) can be used not only in liquid products such as lotions that do not require emulsification or suspension, but also when used in the formulation of creams and emulsions with similar results. is obtained.

Therefore, in the present invention, for example, extraction examples 1 to
Using the residue after extracting the crude liquid shown in 4, this residual pickle is subjected to known hydrolysis (decomposition with strong acid, strong alkali, enzyme, etc.) or an extract extracted using an organic solvent. This may also be used as a stabilizer. Of course, the extract obtained from this residue exhibits excellent moisturizing effects on the skin. However, the ability to proliferate cells is zero.

1 Prescription Example - 1 = Lotion type agent" ■ The crude liquid obtained in extraction side 1 to 4, or ■ A liquid obtained by concentrating the crude liquid under reduced pressure to an appropriate concentration, or ■ Obtained from the crude liquid. The purified freeze-dried powder is added to the next formulation to produce a lotion using a conventional method.

1-20% for ■, 0.3-0.5% for ■, 0 for ■
．． 001-0.1% is added in the following formulation.

Aqueous solution containing carboxymethyl chitin...20% propylene glyfur...
・3% Brasenando V (place center extract)...5%
Make total volume 100 with purified water.

1. Prescription Example-2: Milk Lotion Type Agent" In advance, prepare 20. Sodium hyaluronate 0.3%
or R5C Liquid (cock's comb extracted mufu polysaccharide-containing aqueous solution) 20, the residue produced when extracting the crude liquid of any of Extraction Examples 1 to 4 was obtained by known hydrolysis. , an aqueous solution containing a water-soluble protein-derived extract (containing a total nitrogen content of 300 w/v%) is prepared in advance, and added to the following formulation by 5 to 20% (containing a total nitrogen content of 300 w/v%). When the temperature drops below 45℃, add it little by little).

Liquid paraffin・・・・・・・・・・・・・・・・・・
・・・13% squalane・・・・・・・・・・・・・・・
・・・・・・・・・・・・10% stearic acid...
・・・・・・・・・・・・・・・・・・15% Lanolin・・・・・・・・・・・・・・・・・・・
・・・・・・・・・4% white beeswax・・・・・・・・・
・・・・・・・・・・・・・・・2% sorbitan trioleate ・2% egg yolk lecithin (99% as total phospholipids) 2% paraben (3
Seed complex)・・・・・・・・・・・・・・・・・・Appropriate amount Fragrance・・・・・・・・・・・・・・・・・・・・・・・・・
......Appropriately, make the total amount 100 with the water at the end.

[C] Effects of the Invention It has been disclosed by the test using the above evaluation method that each extract obtained by the present invention has the ability to directly proliferate cells. Cosmetics containing extracts with such active effects can be used, for example, to treat cuts,
It works to rapidly repair damage from the epidermal layer to the dermal layer due to burns, etc., and has the effect of promoting regeneration in the damaged area. therefore,
It can be used not only in the cosmetics field but also as a medical external preparation. In either case, the extract requires complete sterilization in the extraction process.

The effect of its use (compounding) in cosmetics is to counteract skin aging. In other words, skin aging is caused by dryness of the epidermal layer,
Increased wrinkles, decreased luster, and appearance of stains can be seen in appearance. If this is observed from the dermal layer, it can be seen that the metabolic function of fibroblasts has decreased. In other words, fibroblasts are cells that work to regenerate surrounding tissue fibers (such as collagen) and maintain aqueous substances between the tissue fibers, and are deeply involved in maintaining the balance of mucopolysaccharides, etc. However, it can be thought that one of the major causes is that this activity is reduced.

Regarding this matter, for example, the present inventors have investigated 1.
By administering crude liquid), a state in which mucopolysaccharide production and water-soluble collagen production ability are increased.
This could be confirmed under a microscope.

Therefore, if the bacteria are used in cosmetics, they will gradually penetrate into the skin, making it possible to keep the skin youthful. In particular, given the historical background in which extracts extracted from the extremely thick organs of various animals have been incorporated into cosmetics and have been used with high expectations for their ability to activate cells and tissues, the present invention The use of extracts has been able to confirm its effectiveness by adopting a more specific evaluation method at the cellular level for its M production mechanism, which will be a major advance for the future development of cosmetics. I think that it will bring.

[Brief explanation of the drawing]

The first rXJ shows the cell proliferation ability of a solution containing 5% FBS (fetal bovine serum). Figures 2 to 6 show the cell proliferation ability of the water-soluble protein obtained by the present invention. Figure 2 shows water-soluble protein derived from cow serum (blood) (molecular weight: 75.000), Figure 3 shows water-soluble protein derived from cow udder (molecular weight, 55.0OO), and Figure 4 shows: Water-soluble protein derived from cow udder (molecular weight: 9.900), Figure 5 shows water-soluble protein derived from cow spleen (molecular weight: 41.0OO)
, Figure 6 shows water-soluble protein derived from bovine thymus (molecular weight: 46
．． 0OO). In Figures 2 to 6, A shows the growth curve when the added amount is 1 μg/ml), H shows the growth curve when the added amount is 0.1 μg/ml, and C shows the growth curve of the control group. t21i1ii Lo. j8I! Number of days for cultivation! s3 diagram culture days! 0 181118 legs

Claims (1)

[Claims] Cosmetics containing water-soluble proteins derived from cow blood or cow organs extracted without using organic solvents, strong acids, strong alkalis, or enzymes and without heating, the molecular weight of the water-soluble proteins contained therein. , and isoelectric point are as follows (1): 75,000 for extract derived from bovine serum, and its isoelectric point is 6.0.
- Around 6.5, (2): For extracts derived from cow udder, 5
5,000 and 9,900, their isoelectric points are 6,
Around 0 to 6.5, the latter around 7.0 to 7.5, (3)
: Extract derived from bovine spleen has a concentration of 41,000 and its isoelectric point is around 6.0 to 6.5. 6.0 to about 6.5, and contains one or more of (1) to (4).