JPH02121909A - Cosmetic for pimple - Google Patents

Abstract

PURPOSE:To obtain a cosmetic for pimple containing an egg yolk antibody prepared from an egg produced by a hen immunizing an acidic polysaccharide produced outside bacterium cell by Propionibacterium which is a bacterium always existing in a skin, free from stimulation and exhibiting action effective remedy for pimple. CONSTITUTION:A domestic fowl (preferably a hen of white leghorn, etc.) is subjected to immune operation using an acidic polysaccharide produced outside bacterium cell by Propionibacterium acness which is a bacterium always existing in skin to immunize the domestic fowl and an egg yolk antibody is obtained from an egg produced by the domestic fowl. The egg yolk antibody is blended at an amount of nearly 0.0001-0.5wt.% based on total amount of the composition to provide the cosmetic for pimple. The obtained cosmetic is free from stimulation to skin, has action effective to prevention and remedy for pimple and is stable even in the case of long-term storage and the commercial value is extremely high.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention is directed to Propionibacterium acnes (hereinafter referred to as P. acnes).
The present invention relates to a novel cosmetic for acne, characterized by containing an egg yolk antibody prepared from eggs of chickens immunized with acidic polysaccharides produced extracellularly by bacteria.

(The problem to be solved by the conventional technology and invention!! II)
Acne is an unsightly and often bothersome condition that affects areas of the skin that are rich in sebaceous glands, especially the face, neck, and skin.
Occurs on the back. The main causes of acne are ■ excessive secretion of sebum, ■ blockage of the hair follicle fatty drainage tube, and ■ sebum 1.
1.9 The presence of bacteria of the genus Propionibacterium (mainly P. acnes) in W. Depending on these factors, characteristic acne symptoms such as pimples, papules, and pimples appear.

Currently, the main methods of preventing, treating, or treating acne are: ■ Controlling excessive sebum secretion; ■
Remove blockages in hair follicles; ■ Inhibit bacterial growth and perform sterilization; ■ Prevent inflammatory effects;
, II, etc.

Examples of compositions using antibodies include antibodies and sprays containing the same as active ingredients (Japanese Patent Application Laid-Open No. 621754213).
・Method for passive immunization of lI+fiII using bovine antibodies and compositions therefor (Japanese Patent Application Laid-open No. 60-2.i'
4862 B), but Im bacteria alone can be isolated from birds such as chickens, cows, etc. tl+! Even if you immunize mammals, you cannot expect the resulting i to have a direct effect such as an antibacterial effect on bacteria in the body, and the current situation is that it is not necessarily effective against acne inflammation caused by P. acnes, etc. It is.

The object of the present invention is to provide a completely new cosmetic for acne that is non-irritating to the skin and has an effective treatment for acne.

(Means for Solving the Problems) The present invention uses egg yolk antibodies prepared from eggs produced by chickens immunized with acidic polysaccharides produced extracellularly by Propionibacterium acnes, which is a resident bacteria on the skin. A cosmetic for acne characterized by containing:

The egg yolk antibody according to the present invention is easily prepared from eggs produced by chickens immunized with the acidic polysaccharide produced extracellularly by P. acnes.

Here, P acnes used in the present invention is grown anaerobically at 37°C for 3 to 30 minutes using a liquid medium such as plain heart infusion medium (BHI medium), which is commonly used for culturing anaerobic bacteria. Culture is carried out by culturing for 10 days,
Easily obtained by centrifugation.

The acidic polysaccharide produced by P. acnes outside the bacterial cells is concentrated and recovered from the culture filtrate of P. acnes by operations such as ammonium sulfate precipitation, alcohol precipitation, and ultrafiltration. This acidic polysaccharide component was further subjected to column chromatography using Sephadex G-100, Sepharose CL-6 [3, etc., and was analyzed by immuno-diffusion method to obtain anti-P, acnes, etc.
Purification is possible by collecting fractions that show a positive reaction to eel serum antibodies. The obtained acidic polysaccharide can be stored by collecting, dialysis against distilled water, and freeze-drying. It is also possible to freeze and preserve it as it is.

There are no particular restrictions on the chicken used for the immunization procedure, but from the standpoint of mass production of antibodies, egg-producing chickens such as white leghorn are preferably used.

In addition, immunization methods include conventional methods such as subcutaneous injection, intraperitoneal injection, and intramuscular injection, as well as nasal drops, IJ injections, etc. This can be done by methods such as.

Furthermore, the amount of antigen to be administered may be appropriately selected so that the desired antibody value is obtained and does not have an adverse effect on the chickens. In addition, complete Freund's adjuvant, if necessary.
It may be used in combination with an adjuvant such as incomplete Freund's adjuvant.

After immunization, the antibody titer is measured at intervals of about two weeks by methods such as enzyme immunoassay and radioimmunoassay to track the progress. Usually, antibodies that specifically react with the administered antigen are obtained in eggs (egg yolks) within a few weeks after the first immunization, but the antibody titer is usually high enough to exert its effect as an antibody within about 3 months. Antibodies with this can be obtained. If a decrease in antibody titer is observed after immunization, the antibody titer can be increased by appropriately administering booster immunizations at appropriate intervals.

The egg yolk antibody according to the present invention is prepared by adding chloroform to the yolk of eggs produced by immunized chickens, shaking, stirring,
It is obtained from an aqueous supernatant solution after lipids are removed by centrifugation, and can be preserved by dialysis against distilled water, sterilization by filtration, freeze-drying, and other operations. The obtained egg yolk antibody exhibits a white to pale yellow color and is a protein component mainly composed of immunoglobulin (y-livetin).
It also contains other protein components such as β-1α-lihetin and ovalbumin.

Although the egg yolk antibody according to the present invention can be used in an unpurified state, an even higher antibody titer can be obtained by extracting and separating the immune protein "1 purine" contained in the egg yolk.

Extraction and separation methods include precipitation methods using salts such as ammonium sulfate and sodium sulfate, or polymeric compounds such as dextran sulfate and polyethylene glycol, and DB
Further purification is also possible by ion exchange chromatography using cellulose or the like, gel filtration chromatography using a gel filtration agent such as Sepharose or Sephadex. In addition, various commonly used methods for extracting and separating immunoglobulins can be used.

This fraction is water soluble and can dissolve up to 10% by weight. Other water containing alcohol? 8H can also be dissolved.

The mechanism of action of the egg yolk antibody according to the present invention against P acnes is thought to be as follows. The egg yolk antibody obtained by immunizing chickens with the acidic polysaccharide produced by P. acnes outside the bacterial body inhibits the production of the acidic polysaccharide of P. acnes, binds specifically to the surface of the bacterial cell, and inhibits bacterial activity. and inhibits proliferation.

Therefore, P.

As a result, the production of acidic polysaccharides, lipase, acid phosphatase, hyaluronidase, protease, neuraminidase, etc., which are produced outside the bacterial cells of P. acnes and are thought to enhance the inflammatory response within the skin, is suppressed, and the inflammation is suppressed. By inhibiting or reducing the ability to induce
It is presumed that it exhibits excellent acne treatment effects.

The blending amount of the egg yolk antibody according to the present invention is $4 for acne makeup (
Approximately 0.0001 to 0.000% based on the total amount of Mi products).
The range is 5% by weight (hereinafter abbreviated as wt%). Here, if the amount of the egg yolk antibody blended is less than 0.0001 wL%, the effect will not be sufficiently achieved, and even if it exceeds 0.5 wt%, an increase in the effect commensurate with the increased amount cannot be expected.

Furthermore, the cosmetic for acne of the present invention includes commonly used ingredients having a keratolytic effect such as redulucin, sulfur, salicylic acid, urea, isopropyl methylphenol, etc., and ingredients having a bactericidal effect such as gluconate lorcheni-1 sodine, Chlorhequine hydrochloride, Henzalkonium chloride,
Benzanium chloride and other ingredients such as glycyrrhizin, glycyrrhetinic acid, allantoin, zinc oxide, zinc sulfate, aluminum lactate, ethyl lactate, estradiol, camphor, biotin, etc. can be combined at the same time. In this case, further enhancement of the effect can be expected. Further, other additives such as antioxidants, fragrances, pigments, various vitamins, etc. can be appropriately prepared and blended within the range that achieves the first objective of the present invention.

The cosmetic for acne of the present invention can be prepared in the form of lawns, emulsions, creams, gels, stickies, pastes, etc. by a conventional method.

(Examples) Hereinafter, the present invention will be explained in detail based on the descriptions of Examples and Comparative Examples. The human skin patch (J test, acne treatment effect test described in Examples) is as follows.

ill Human Skin Patch Test 0.05 g of sample was applied to the skin of the forearm side of 25 subjects with a diameter of 1 mm. A 24-hour occlusive application was performed using a test plaster with a Qcm circular filter paper attached.

Next, bandage removal 1 according to the criteria in Table 1 below.
The 0 judgment results obtained after 24 hours and 24 hours were based on the evaluation of the stronger reaction, and were expressed as the number of people who gave an evaluation of (±) or higher among the 25 subjects.

Table 1 (2) Acne treatment efficacy test 111 The control product (
On the right side, a test product of an example or a comparative example was applied twice a day in the morning and evening for a period of one month. Next, the therapeutic effect of 7ft on the acne disease Φ area was determined by the single-head comparison method according to Table 2 below, El 241.
It has been decided. The judgment result is j・I! It is shown as the average value of the value.

Table 2 (Preparation of egg yolk antibodies) Examples of preparation of egg yolk antibodies, immunization against chickens, and preparation of P acnes and acidic polysaccharide produced extracellularly by P acnes according to Examples and Comparative Examples are shown.

1. Preparation of P. acnes bacterial cells B 111 liquid medium (manufactured by Difco, 51 ) using P.

P. acnes was cultured anaerobically at 37°C for 50 minutes, and the bacterial cells were removed from the culture by centrifugation. separated from R (wet weight of bacterial cells: 8.5
g).

2. Preparation of acidic polysaccharide produced by P. acnes extracellularly. - J. Iversen et al.'s method (Archives)
Of Dermatological Research, Volume 277, 225-
229, 1985), an acidic polysaccharide produced extracellularly by P. acnes was prepared. P. After culturing acne? Add ethanol to Ht oe to 70% at a final temperature of 4 degrees.
and left overnight at 4°C. The precipitate was collected by centrifugation and treated with DNase, RNase, ) ribsin, and pronase to remove protein and nucleic acid components and completely solubilize.

This fraction was further subjected to Sepharose CL-6B column chromatography (2.5X60cm, 0.15M N
Purification was performed using a Cl, p II 7.2).
It was.

By the ophthalony method, anti-P, acnes rabbit blood Yi
? As a result of examining antigen activity using antibodies, K a-v
Since an antigen peak was observed between = 0.23 and 0.53, these fractions were collected, concentrated, and lyophilized to obtain acidic polysaccharide (yield: 1679 mg). The analysis values for Motoichi are: Sugar content: 85.4% (phenol sulfuric acid method) Protein content: 8
．． 5% (Lori-Forin method) 3-1. Immunization of chickens with acidic polysaccharide The acidic polysaccharide produced by P. acnes extracellularly, obtained by the above method, was dissolved in physiological saline at a concentration of 5 mg/mβ. Next, the mixture was mixed with complete Freund's adjuvant to prepare a complete emulsion. These emulsions were injected intramuscularly into the left and right pectoral muscles of female chickens approximately 1.5 to 2 months old (10 birds per group).
One month later, the same amount of sample was injected intramuscularly again.

3-2. As a control for immunizing chickens with P. acnes whole cells, a separate experimental group was prepared using P. acnes whole cells LI-2 x IO9 co/mβ, which had been heat-treated in advance at 70°C for 3 hours, at 1 p as in the above immunization method. 110 chickens were immunized. After one month,
The same amount of sample was injected intramuscularly again.

4. Measurement of antibody titer Rabbits produced by immunized chickens during the period of 1 week before the start of immunization, 1 week from 2nd month 11, and 1 week from 3rd month were measured at 1 week before immunization, 2 months, and 3 months, respectively. It was used as a rabbit to measure antibody levels. Antibody fractions were prepared as follows. Egg yolks were collected, the same amount of physiological saline was added thereto, and the mixture was shaken at room temperature for 5 minutes. Chlorophorno, twice the amount of the original egg yolk, was then added, followed by shaking and stirring for 30 minutes. The resulting emulsion was left at room temperature for 30 minutes and heated at 3000 r.
After centrifugation at pm for 20 minutes, the upper 1# liquid was collected and sterilized by filtration to obtain an antibody fraction. The transition of the antibody titer was tracked by ELISA of the enzyme immunoassay according to the standard method, and the antibody titer before immunization, at the 2nd month, and at the 3rd month was measured at the 3rd month.
Shown in the table. As a result, the acidic polysaccharide produced outside the bacterial cells had a higher ability to increase the antibody titer, and after three months, the value of P. acnes was approximately four times higher than that of the whole bacterial cells.

Table 3. Preparation of egg yolk antibodies Next, collect the yolks of eggs produced by chickens during a period of one month from the third month after the start of immunization, and use the method described in section 4. Measurement of antibody titer J. Antibody fractions were obtained as described.

Furthermore, this antibody fraction was collected as a precipitate using ammonium sulfate at a 40% saturation concentration, dialyzed against physiological saline, recovered as a precipitate again using a 35% saturation concentration of ammonium sulfate, and dialyzed against distilled water for 48 hours. After dialysis, purified egg yolk antibodies were obtained by freeze-drying. The obtained dried antibody was redissolved in physiological saline to the same amount as the antibody fraction before precipitation with ammonium sulfate, and the antibody titer was measured by ELISA using this as a stock solution. The results are shown in Table 4.

Table 4 Examples 1 to 7, Comparative Examples 1 to 4 [Acne lotion] In the acne lotion base, P obtained in the above section (preparation of egg yolk antibody) and egg yolk against acidic poly1-9 of acnes acnes were added. antibody (Example, hereinafter abbreviated as acid-resistant polysaccharide antibody), egg yolk antibody against whole bacterial cells of P. acnes (comparative example,
Each of the formulations containing the anti-allergic tK compound (hereinafter abbreviated as anti-allergic tK compound) as shown in Table 5 was prepared and used in the test.

Table 5 (2) Preparation method After dissolving the ethanol, solubilizer, and propylene glycol shown in Table 5 in purified water by heating as necessary,
Cooled to 0°C. Next, an egg yolk antibody was added, and the remaining amount of purified water adjusted to have a total composition of 100% was added and mixed uniformly to prepare each acne lotion.

(3) Characteristics Table 6 shows the results of the acne treatment efficacy test for Examples 1 to 7 and Comparative Examples 1 to 4.

As shown in Table 6, the acne lotions containing the anti-acid polysaccharide antibodies of Examples 1 to 7 were clearly found to have high acne treatment effects. In addition, no skin irritation was observed in human skin patch tests. On the other hand, the acne lotions containing the anti-total bacterial antibodies of Comparative Examples 1 to 4 had low acne treatment effects and high skin irritation in human skin patch tests. Skin Cream for Acne] Each sample was prepared by blending the stamped antibody according to the present invention into a skin cream base for acne as shown in Table 7, and used in the test.

(1) Composition No. table Preparation method The B and C components in Table 7 are 8 each Heat to 0℃ After mixing the solutions, Cooled to 0°C.

Next, add the egg yolk antibody and pump it with cold IJ for 30 minutes while stirring.
Each acne skin cream was prepared by continuing to stir up to ℃.

(3) Properties Table 8 shows the results of the acne treatment efficacy test for Examples 8 to 14 and Comparative Examples 5 to 8.

As shown in Table 8, the skin creams for acne containing the anti-acidic polysaccharide antibodies of Examples 8 to 14 were clearly found to have high acne treatment effects. Also, skin in human skin patch test! No 11 severity was observed. On the other hand, the skin creams for acne containing anti-total bacterial antibodies of Comparative Examples 5 to 8 have low acne treatment effects, and skin stings in human skin patch tests (effect of the invention). It is non-irritating, has effective effects in the prevention and treatment of acne, and is stable even when stored for a long period of time, so its commercial value is extremely high.

Claims (1)

[Claims] A cosmetic for acne, characterized by containing an egg yolk antibody prepared from eggs produced by chickens immunized with acidic polysaccharides produced extracellularly by Propionibacterium acnes, which is a resident bacteria of the skin.