JPH08143445A - Cosmetic - Google Patents

Abstract

PURPOSE: To obtain a cosmetic consisting essentially of cow placenta extract which is subjected to fractionation of molecular weight and an acidic polysaccharide of shellfishes and excellent in high safety, beautifying and whitening action, cell-activating action and moisture retention. CONSTITUTION: Impurities such as blood are removed from healthy cow placenta in third to fourth month of pregnancy to afford pulpy placenta and a water-soluble ingredient is extracted under low temperature from the pulpy placenta and subjected to centrifugation and germ-free filtration to provide a water-soluble placenta extract, which is then subjected to fractionation of molecular weight within the range of a limit molecular weight of 900-1100 by ultrafiltration or gel filtration and ingredients exceeding the limit molecular weight are removed. The liquid and acidic polysaccharide obtained by decomposing meat of shellfishes such as pearl oyster with proteolytic enzyme, deproteinizing the decomposed product, removing low molecular substance therefrom, adding q quaternary ammonium salt thereto to precipitate and separate the acidic polysaccharides are blended with cosmetic to provide the objective cosmetic. The cosmetic obtained by blending both of placenta extract and acidic polysaccharides of shellfish meats with cosmetic hardly has sensitization and exhibits excellent moisture retention than a cosmetic obtained by singly blending.

Description

Detailed Description of the Invention

[0001]

[Industrial application] Cosmetics that are highly safe and have excellent whitening, cell activating, and moisturizing effects.

[0002]

2. Description of the Related Art The use of water-soluble substances obtained from the placenta of cattle for cosmetics has been performed for a long time. Its components include water-soluble vitamins: thymine, riboflavin, pyridoxine, cyanocobalamin, nicotinic acid, pantothenic acid, paraaminobenzoic acid, biotin, focic acid, inositol, and amino acid groups: arginine, cystine, leucine,
Histidine, glutamic acid, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, serine, alanine, hydroxyproline, asparagine, and mineral groups: calcium, nallium, potassium, chlorine, silicic acid, magnesium, copper, cobalt, iron ,, Other components: Alkaline phosphatase, cholesterol, cholesterol ester, deoxyribonucleic acid, etc. are known to be contained. And, as the effect, the action of tissue respiration of the skin; the inhibition of melanogenesis; the action of softening the skin; the improvement of fine wrinkles, rough skin, etc. have been reported. To date, various studies have been conducted with a focus on polymer components as in Japanese Patent Application No. 62-16519 to improve the effectiveness of the placenta extract.

On the other hand, the acid polysaccharide of shellfish meat is disclosed in JP-A-2-1.
It is known as a cosmetic raw material in No. 31417.

[0004]

As a result of the allergy test of a water-soluble substance obtained from the placenta of a conventional cow, the present inventor showed a positive reaction in any of the samples, and sensitization It turns out that it contains a sex substance. However, it was feared that the removal of this allergenic source would also reduce the effectiveness of the placenta extract. An object of the present invention is to provide a cosmetic having less sensitizing substances than these biologically-derived raw materials and having an excellent whitening effect, cell activating effect, moisturizing effect and the like.

[0005]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventor has found that the sensitizing substance can be removed while maintaining the effectiveness by the molecular weight fractionation of the water-soluble placenta extract. I found it. Furthermore, we have screened and examined substances that have already been used for food for many years and have been confirmed to be safe for the human body, and examined those that are useful as moisturizers. As a result, it was found that a very strong moisturizing effect is exhibited by mixing the extract with the acidic polysaccharide of shellfish meat.

That is, according to the present invention, after washing and removing impurities such as blood from the placenta of a healthy cow, it becomes a porcelain placenta, and only water-soluble components are extracted at low temperature, and further centrifugal separation and sterile filtration are performed. A water-soluble placenta extract is obtained by subjecting the extract to ultrafiltration or gel filtration to perform molecular weight fractionation. The molecular weight fraction is limited to a range of 900 to 1100 to remove components above the molecular weight limit. A cosmetic containing an extract and an acidic polysaccharide of shellfish meat. The placenta is preferably a healthy cow placenta from 3 to 4 months of gestation. Since animal extract components are mainly composed of proteolytic products, it is expected that components exceeding a certain molecular weight will have sensitizing properties. However, on the other hand, removing components with a certain molecular weight may result in removing part of the effectiveness of the water-soluble placenta extract. The present inventor tried to remove components having a molecular weight of more than about 1000 from the water-soluble placenta extract, and was able to eliminate sensitization, and also cell activation, melanin formation inhibition, skin softening action, fine lines and rough skin. It was proved by various experiments that the effectiveness of the improvement was maintained.

Next, the method for producing each raw material will be described in detail. [Cow placenta extract] Blood and other impurities are washed away from the placenta of healthy cows 3 to 4 months pregnant. Since the placenta is usually obtained in a frozen form, it is thawed and then minced by a method capable of mincing the tissue, for example, a mincing machine, a mixer, a homodisper or the like. All these operations must be carried out at a low temperature of 5 ° C or lower. Therefore, there is no method for generating heat even when crushing, and since there is a step of removing impurities such as blood in the tissue after this, it is not preferable to grind too much to cause outflow of the active ingredient. The step of removing impurities such as blood is preferably performed with a press. In the final step,
Since there is an ultrafiltration step, it is not necessary to strictly perform it. Thereafter, an aqueous butanol solution is added as an extraction solvent, but the concentration of butanol is preferably 15 to 50% by weight. The amount of the solvent to be added is appropriately 2 to 5 times the wet weight of the placenta. After adding the butanol aqueous solution, thoroughly stir and homogenize, and extract the water-soluble components at low temperature. Then, the undissolved portion of the solid phase or the like is removed by filtration or centrifugation, left to stand to remove the butanol layer, adjusted to weak acidity with acetic acid, citric acid or the like, preferably pH 4.5 to 5.0, and stirred. To do. Precipitates, suspended matters and the like generated by this operation are removed by centrifugation. Furthermore, in order to completely remove butanol, concentration under reduced pressure is performed. This concentration is performed so that the concentration becomes one third to one tenth of the original amount. The preservative and ethanol are added to the obtained liquid, but this is not necessary when the final step is a freeze-drying step to obtain a powder. Ethanol may be added during storage of this cosmetic raw material because it may precipitate due to the effects of fatty acids and the like, but its concentration is 2-1.
0 wt% is suitable. From this solution, molecular weight fractionation is performed by ultrafiltration or gel filtration in order to remove sensitizing components. In this molecular weight fractionation, the limiting molecular weight is set to about 1000, but considering the accuracy of ultrafiltration and the like, the limiting molecular weight may be between 900 and 1100. If the limiting molecular weight is less than 900, the active ingredient as a cosmetic raw material may be lost, and if it exceeds 1100, the sensitizing component may remain, which is not preferable. After that, bacterial filtration is performed using a membrane filter or the like to obtain the cosmetic raw material of the present invention. It is also possible to finally carry out a method such as freeze-drying to make the powder non-powder. All these steps are carried out at low temperatures, preferably below 5 ° C.

[Acid Polysaccharide of Shellfish Meat] The acid polysaccharide of shellfish meat may be used in any type of shellfish meat, but the pearl pearl oyster used for pearl cultivation is not edible except for scallops and is edible after pearl cultivation. Because of problems such as contamination, the present inventors consider that it is better to use pearl oysters. That is, the present invention, after decomposing shellfish with a protease, deproteinization,
A method for producing a cosmetic raw material containing an acidic polysaccharide as a main component, which comprises removing a low-molecular substance and then adding a quaternary ammonium salt to precipitate and decompose the acidic polysaccharide. Dialysis, gel filtration, ultrafiltration, or an organic solvent that precipitates acidic polysaccharides is used to remove low-molecular substances. Further, in order to purify the acidic polysaccharide separated by precipitation with a quaternary ammonium salt, an inorganic salt aqueous solution is added to the precipitate and dissolved, and then reprecipitated with an alcohol aqueous solution, or by repeating this method. You can It will be described in more detail. In the first step, shellfish meat from which shells have been removed is decomposed by a protease. In order to facilitate this treatment, it is preferable to heat and denature and then pulverize with a mixer or the like. When pearl oysters are used, the mucus associated with the shell meat is also used as a raw material, and only mucus may be used. At this time, it can be used regardless of taking out pearls as in the case of using shell meat, the raw material can be easily collected, and the crushing step is not necessary. The proteolytic enzyme is not particularly limited, but one that has no substrate specificity and strong decomposing power is preferable. For example, one or a mixture of two or more of papain, actinase, samoase, denazyme and the like is used for decomposition. In the second step, deproteinization is performed to remove undegraded proteins, nucleic acids and enzymes. The method for deproteinization is not particularly limited, but generally, trichloroacetic acid or perchloric acid is used at a concentration of 10% or Se is used.
The vag method is used. In the third step, low molecular weight substances such as deproteinizing agents, amino acids, peptides and salts are removed. As the removal method, there are a dialysis method, gel filtration, ultrafiltration, and a method of adding an organic solvent in which an acidic polysaccharide is precipitated, and the dialysis method is preferable. In the fourth step, neutral polysaccharides and glycoproteins that are present together with acidic polysaccharides are separated. As a removing means, an aqueous solution of a quaternary ammonium salt is added. Since the acidic polysaccharide binds to the quaternary ammonium salt and precipitates, it is separated by decantation or centrifugation. Fourth
The primary ammonium salt may be one having one or more alkyl groups having 12 or more carbon atoms. Also in the pyridinium type, it is sufficient that the alkyl group bonded to nitrogen has 12 or more carbon atoms. For example, lauryl trimethyl ammonium salt, stearyl trimethyl ammonium salt, cetyl pyridinium chloride, etc. can be illustrated. By the fourth stage, the acidic polysaccharide can be obtained, but since this precipitate contains a quaternary ammonium salt and other salts, further purification is preferable. For purification, an inorganic salt aqueous solution is added to this precipitate to dissolve it, and the complex is dissociated and dissolved, and then alcohol is added to reprecipitate the acidic polysaccharide and the quaternary ammonium salt is separated and removed in the liquid. To do. This precipitate is dissolved in purified water, water is added so that the alcohol concentration becomes 80%, and the mixture is stirred and left to stand to obtain a precipitate by centrifugation. It is preferable to repeat this step 2-3 times. Any inorganic salt can be used as long as it dissociates and dissolves the complex of the quaternary ammonium salt of acidic polysaccharide. Examples thereof include sodium chloride and potassium chloride. The salt form used here determines the salt form of the final product. For example, if sodium chloride is used, the final product will be the sodium salt. The concentration of the inorganic salt is a concentration required to dissociate the complex, and sodium chloride needs to be 1.5 M or more.
There is no particular upper limit, but if the concentration is too high, the desalting operation will take time, waste of reagents will occur, and the limit is about 4M. If you want to remove the remaining proteins and dyes, add the Lloyd's reagent and cations to an aqueous solution prepared by dissolving the precipitate in water,
Adsorbs and removes dyes and proteins. After removing the adsorbent by centrifugation and filtration, the acidic polysaccharide is freeze-dried. When the acidic polysaccharide obtained in this manner is incorporated into cosmetics,
It has high water retention and moisturizing properties like hyaluronic acid, so it moisturizes the skin and hair, prevents dryness, and exhibits a unique lubricity effect due to its excellent permeability and tissue affinity. . It also increases the viscosity of the cosmetics themselves,
Improving stability and usability are also important effects.

[Manufacturing Example] The present invention will be described below in more detail with reference to a manufacturing example, but the present invention is not limited to the manufacturing example. % Are based on dry weight. (Production Example 1) [Cow placenta extract] A frozen product of 100 kg of fresh placenta of a healthy cow from 3 to 4 months of gestation was thawed and then minced with a mincing machine. Remove the blood etc. with a squeeze machine and add purified water 50
Add 4 liters of butanol and 100 liters of butanol, 4 ℃
Then, the mixture was homogenized for 10 hours. After centrifugation, the sedimented portion was removed and the supernatant was filtered. This was left to stand and the butanol layer was discarded. The pH was adjusted to 4.7 with acetic acid, and the mixture was stirred for 3 hours, centrifuged, and allowed to stand. After centrifugation, the sedimented portion was removed and the supernatant was filtered. This was concentrated under reduced pressure to 200 liters. The liquid having a molecular weight of more than 1000 was removed by ultrafiltration. This component having a molecular weight of 1000 or less was used as a placenta extract obtained in the examples of cosmetic raw materials described below. The whole water-soluble placenta extract before this ultrafiltration was designated as Comparative Production Example 1,
Ingredients having a molecular weight of 10,000 or more are compared to Comparative Production Example 2, molecular weight 1
000 to 10,000 components in Comparative Production Example 3, molecular weight 10
The components of 00 or less are referred to as Production Example 1. (Production Example 2) [Acid polysaccharide of shellfish meat] a. 100kg of pearl oysters with shells removed are heated,
After denaturing, it was pulverized with a mixer. b. Add 100g of actinase E to this, 45 ℃
The mixture was left to stand for 24 hours while being stirred. c. 10 kg of trichloroacetic acid was added, and the mixture was stirred, allowed to stand for 1 hour and then centrifuged to remove the precipitate, and deproteinize. Further, dialysis with cellophane tube was carried out for 48 hours to remove low molecular weight substances. d. Then, 1 kg of cetylpyridinium chloride was added, and the mixture was stirred, allowed to stand for 1 hour, and then centrifuged to obtain a precipitate of cetylpyridinium salt of acidic polysaccharide. e. 2M sodium chloride was added to this precipitate to dissolve the precipitate. Ethyl alcohol (3 times) is added thereto, and the mixture is stirred, left standing for 1 hour and then centrifuged to obtain a precipitate. f. A small amount of purified water is added to this precipitate to dissolve the precipitate,
Ethyl alcohol is added to have a concentration of 80%, the mixture is stirred, left for 1 hour and then centrifuged to obtain a precipitate. g. f was repeated 3 times. The yield was 512 g. (Production Example 3) a. 100kg of pearl oysters with shells removed are heated,
After denaturing, it was pulverized with a mixer. b. After adjusting this to pH 6-6.5, 100 g of Samoases and 500 g of Denazyme were added, and the mixture was left standing for 100 minutes with stirring at 60 ° C. c. Centrifuge to remove insoluble matter, add 10 kg of trichloroacetic acid, stir, leave for 1 hour, centrifuge,
After removing the precipitate, low molecular weight substances were removed by ultrafiltration with a molecular weight of 10,000. d. To this, 10 kg of purified water was added and dissolved, then 1 kg of cetylpyridinium chloride was added and stirred, and the mixture was left standing for 1 hour and then centrifuged to obtain a precipitate of acidic polysaccharides. e. 2M potassium chloride was added to this precipitate to dissolve the precipitate. Add 3 times ethyl alcohol to this and stir,
After standing for 1 hour, centrifugation was performed to obtain a precipitate of acidic polysaccharide. f. A small amount of purified water is added to this precipitate to dissolve the precipitate,
Ethyl alcohol was added so as to have a concentration of 80%, the mixture was stirred, allowed to stand for 1 hour and then centrifuged to obtain a precipitate. g. f was repeated 3 times. h. To this, 10 kg of purified water was added and dissolved, then 500 g of kaolin was added and stirred, centrifuged to remove precipitates, filtered through a 5 micron filter, and freeze-dried. The yield was 485 g. (1) Sensitization test (Maximization Tes)
t) 15 Hatley white guinea pigs were used, 10 for sensitization treatment, and 5 as control at the time of induction. (The control group is a group for comparing with the sensitized group to see if there is a reaction (primary stimulus) other than sensitization when the sample is applied to the skin.) (Sensitized 1) Above the scapula The skin is shaved and an adjuvant (immunity enhancer) specimen and specimen + adjuvant are intradermally injected in 0.05 ml each in two places on each side. (1 week has passed) (Sensitization 2) 10% lauryl sodium sulfate (SL)
S) is applied, and after 24 hours, 0.2 ml of sample is added to 4
The occlusion patch was applied at 8:00. (2 weeks passed) (Induction) The ventral side was shaved, and 0.2 ml of the sample was spread and applied. (24 hours have elapsed) (Induction refers to observing whether or not an allergic reaction occurs by applying antigen again after antibody is produced in the body by antigen-antibody reaction in sensitization 1 and 2.) ) Judge 1, 24, 48, 72 hours after removal of the sticking. (Judgment Criteria) Score 0: No macroscopic change 1: Mild or sparse erythema 2: Moderate erythema 3: Severe erythema and edema 10 Scores are totaled and the average value is displayed. (2) Cytotoxicity and cell activation test Eagle was placed on a 6 cm chalet with a cover glass.
Dispense 5 ml each of MEM (fetal bovine serum 20%) medium JTC-17 cells (toxicity test = 300,000 cells, activation note test =
(200,000 cells) added suspension 5% CO ₂ , culture at 36 ° C. for 48 hours Buffer PBS (−) (NaCl 8.0 g, KCl)
0.2 g, Na ₂ PO _4, 1.15 g, KH2PO4
Wash twice with buffer containing 0.2 g) Toxicity test Add 3 ml of sample diluted to each concentration with PBS (-) and add 3
After culturing at 6 ° C for 60 minutes, 7 ml of Eagle MEM (fetal bovine serum 20%) medium was dispensed, and 5% CO _{2 was} incubated at 36 ° C for 8 hours, fixing, staining, and determination Activation test Eagle MEM (serum-free) medium Add 5 ml of sample diluted to each concentration and 0.05 ml of fetal bovine serum, and add 5% CO
₂ 36 ° C., 8 h culture, fixed, stained, determination ◇ cytotoxic-cell stimulatory test criteria (1) determined by the number of cells (including abnormal cell number) [2] Judgment based on cell morphology In Tables 3 and 4, the first number indicates the determination result based on the number of cells, and the numbers in parentheses indicate the determination result based on the cell morphology. (3) Melanin production inhibition test 10% fetal bovine serum and sample in Eagle MEM medium And add as the sample. Send a sample to a 6 cm Petri dish, B
Add 16 cells 1 × 10 ⁵ cells / 0.1 ml and add 5%
Cultivate at 37 ° C. in CO ₂ and exchange the sample on the 3rd day. (Since the cultured cells are adherent, it means that on the third day of culture, only the culture solution is discarded using the culture bottle, and the culture solution containing the sample is put into the culture bottle and cultured again. ) On the 6th day of culture, the cells are detached (physically detached because they are adherent) and centrifuged to observe the degree of whitening. (Judgment Criteria) Judgment by Whiteness Score −: No self-coloring +: Slightly whitening ++: Clear whitening ++: Strong whitening ++ means that melanin production is suppressed, and the more +, the more melanin It shows that the production suppression ability is strong. The analyte concentration in the following tests is% solids.

Next, examples of cosmetics containing the placenta extract and acidic polysaccharides produced by the production examples are shown, and the results of the test for confirming the effectiveness of the cosmetics are also shown. (Example 1) Emollient cream 1 (% by weight) A liquid paraffin 5.0 octyldodecyl myristate 3.0 jojoba oil 2.0 lanolin 2.0 cetanol 5.0 stearic acid 4.0 palmitic acid 4.0 glycerin mono Stearate 3.0 Polyoxyethylene (20 mol) cetyl ether 1.0 Antioxidant 0.2 B Purified water 51.1 Triethanolamine 1.0 1,3 Ptyrene glycol 7.0 Propylene glycol 5.0 Antiseptic Agent 0.2 C Placenta extract obtained in Production Example 1 5.0 Acidic polysaccharide obtained in Production Example 2 1.0 D Perfume 0.5 A and B were weighed separately and heated to about 70 ° C. Then, stir A and gradually add B, and when the temperature reaches 60 ° C., add C and D and cool to room temperature. (Example 2) Lotion A Fragrance 0.1 Squalane 0.2 Polyoxyethylene (25 mol) oleyl ether 3.0 Polyoxyethylene (20 mol) sorbitan monooleate 2.0 B Purified water 68.5 Glycerin 8. 0 Dipropylene glycol 2.0 Polyethylene glycol 1000 5.0 Carboxyvinyl polymer (1.0% aqueous solution) 5.0 Placental extract obtained in Production Example 5.0 Acidic polysaccharide obtained in Production Example 2 1.0 Preservative 0.2 A is weighed, heated to about 70 ° C., and cooled with stirring. A
Add B while stirring. (Example 3) Lotion A lotion prepared in the same manner as in Example 2 by replacing Production Example 2 of Example 2 with Production Example 3.

Comparative Example 1 Production Example 1 of Example 1
The placenta extract obtained in step 1 was replaced with the placenta extract produced by Company A, and the other procedures were performed in the same manner as above to prepare an emollient cream. (Effectiveness Confirmation Method) Example 1 and Comparative Example 1 1
The test was conducted on 02 women (18-54 years). The test method was randomly divided into 51 people, one group for each of Example 1
On the other hand, Comparative Example 1 was used for one month. As a result, the number of people who had irritation was 10 in the Example and 13 in the Comparative Example.

Usage test Seven women were divided into left and right faces, one of which was used as an example and the other was used as a comparative example, which was used once or more daily.
One week later, I did a questionnaire. Comparative Example 1-Excluding the acidic polysaccharide from Example 1 Comparative Example 2-Excluding the placenta extract from Example 1 Comparative Example 3-Excluding the acidic polysaccharide and placenta extract from Example 1 Comparison Example 4-Example 2 from which acid polysaccharides were removed Comparative Example 5-Example 2 from which placenta extract was removed Comparative Example 6-Example 2 from Example 2 from which acid polysaccharides and placenta extract were removed Comparative Example 7. The placenta extract was removed from Example 3. The criteria are as follows, and the results of the questionnaire are summarized in the table below. Example is much better 3 Example is much better 2 Example is slightly better 1 No difference 0 Comparative example is slightly better -1 Comparative example is much better -2 Comparative example Is much better -3

[0013]

[0014]

EFFECTS OF THE INVENTION According to the test results of each component obtained by fractionating the placenta extract of the production example in terms of molecular weight, the cell activating activity is the same as that of the whole extract and other components having a molecular weight of more than 1000. Is much weaker than the whole extract and other components. Regarding the melanin production inhibitory effect, the inhibitory ability is stronger than that of the whole extract and other components. With respect to the sensitizing property which is the object of the present invention, the components having a molecular weight of 1000 or less produced by the method of the present invention have no sensitizing properties while other components have intermediate sensitizing properties. Absent. Furthermore, a significant difference in moisturization was observed as compared with the cosmetics using the placenta extract of bovine and the acidic polysaccharide of shellfish meat alone. That is, according to the present invention, by combining the molecular weight fractionated bovine placenta extract and shellfish meat acidic polysaccharide together in cosmetics, there is less sensitization and the effect of superior moisturizing effect compared to the case of mixing them alone. Demonstrate. In addition, naturally, each of them also exerts a whitening effect, a cell activating effect and the like.

Claims (2)

[Claims] 1. Washing and removing impurities such as blood from the placenta of a healthy cow to form a porcelain placenta, extracting only water-soluble components at low temperature, further centrifuging and sterile filtering. A water-soluble placenta extract was obtained, and the extract was subjected to ultrafiltration or gel filtration to perform molecular weight fractionation. The molecular weight fraction was limited to 900 to 1100, and components above this molecular weight limit were removed. An acidic polysaccharide characterized by decomposing the liquid and shellfish meat with a proteolytic enzyme, deproteinizing and removing low-molecular substances, and then adding a quaternary ammonium salt to precipitate and decompose the acidic polysaccharide. Cosmetics containing as a main component. 2. After washing and removing impurities such as blood from the placenta of a healthy cow, it becomes a porcelain placenta, only water-soluble components are extracted at low temperature, and further centrifugation and aseptic filtration are performed. A water-soluble placenta extract was obtained, and the extract was subjected to ultrafiltration or gel filtration to perform molecular weight fractionation. The molecular weight fraction was limited to 900 to 1100, and components above this molecular weight limit were removed. After decomposing the liquid and shellfish meat with proteolytic enzymes, deproteinizing and further dialysis, a quaternary ammonium salt is added to precipitate and separate the acidic polysaccharide, and an inorganic salt aqueous solution is added to this precipitate. A cosmetic product whose main component is a purified acidic polysaccharide, which is prepared by dissolving it in water and then re-precipitating it by adding an aqueous alcohol solution.