JPH02121910A - 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 having immunized ingredient of cell wall of Propionibacterium acnes which is a bacterium always existing in skin, free from stimulation to skin and effective on prevention and remedy for pimple. CONSTITUTION:The aimed cosmetic for pimple containing an egg yolk antibody prepared by subjecting bacterium cell of Propionibacterium acness (P-acnes) which is a bacterium always existing in skin to supersonic crushing and then centrifugation to remove uncrushed bacterium cell and treating the supernatant thereof to provide ingredient of cell wall and using an egg produced by a hen immunized by the resultant ingredient of cell wall. The egg yolk antibody which is an active ingredient is specifically bonded to the surface of P-acnes to suppress activity and proliferation of bacterium. P-acnes is produced outside the bacterium cell and inhibits or lowers excision ability of inflammation in skin and exhibits excellent effect for treating pimple.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to Propionibacterium acnes (hereinafter referred to as
3. The present invention relates to a novel cosmetic for acne, characterized in that it contains an egg yolk antibody prepared from eggs produced by chickens immunized with cell wall components of acnes (abbreviated as acne).

(Problem B that the Prior Art and Inventions Try to Solve) Acne, which disfigures the appearance of the skin and is often accompanied by troublesome symptoms, occurs in areas of skin rich in sebaceous glands, especially on the face, neck, and back. The causes of acne are mainly ■ Excessive secretion of sebum, ■ Blockage of the pilosebaceous drainage duct, and ■ Propionibacterium niobacterium (mainly 1″, acnes) bacteria (Y Due to these outbreaks, characteristic symptoms such as acne scars, papules, and pimples appear.

The methods of preventing, treating, or treating acne, which is currently the most prevalent type of acne, are: - Applying 1 lb of excess sebum;
■Remove blockages in hair follicles, ■Inhibit bacterial growth,
Further examples include applying sterilization treatment, and (2) eliminating inflammatory effects.

Examples of compositions using antibodies include antibodies and sprays containing the same as active ingredients (JP-A-62175-126), methods for passive immunization of mammals using avian or bovine antibodies, and compositions therefor (JP-A-62175=126); Firstly, even if birds such as chickens or mammals such as cows are immunized with bacteria alone, the resulting antibodies cannot be expected to have a direct effect such as bacteriostatic effect against the bacteria. Currently, it is not necessarily effective for acne inflammation caused by P. acnes and the like.

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 effect on the treatment of acne.

(Means for Solving the Problems) The present invention is characterized in that it contains an egg yolk antibody prepared from rabbits produced by chickens immunized with cell wall components of Propionibacterium acnes, which is a resident bacteria on the skin. Acne makeup r:1.

The Ruri 11 yellow antibody according to the present invention is easily prepared from eggs produced by chickens immunized with cell wall components of P. acnes.

Here, P used in the present invention, ゛? P. acnes, using a liquid medium such as plain heart infusion medium (BHI Iff medium), which is normally used for culturing a tolerogenic culture, by culturing anaerobically at 37°C for 3 to 100 g. can be easily obtained by centrifugation.

The cell wall components of P. acnes are 2.
It is obtained in the supernatant fraction after ultrasonic disruption for 0 minutes to 1 hour and removal of undisrupted bacterial cells by centrifugation. This fraction is
DNase, RNase.

It is completely solubilized by treatment with trypsin and Zorolize to remove protein and nucleic acid components. This fraction was further subjected to column chromatography using Sephadex G100, Sepharose CL-6B, etc., and fractions showing positive reactions to anti-P and acnes rabbit serum antibodies were collected by immuno-diffusion method. Purification is possible. The obtained cell wall components can be collected, dialyzed against distilled water, and stored by freeze-drying.

It is also possible to freeze and preserve as 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 can be carried out by conventional methods such as subcutaneous injection, intraperitoneal injection, and intramuscular injection, and methods such as nasal and eye drops.

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 determined at intervals of about 2 weeks by methods such as enzyme immunoassay and radioimmunoassay, and the progress is tracked.Usually, within a few weeks after the first immunization, the antibody titer becomes specific to the administered antigen. A reactive antibody is obtained in the egg (egg yolk), and it usually takes about 3 months to obtain an antibody with a high enough titer to be effective as an antibody.Please note that after immunization, the antibody titer decreases. If this is observed, the antibody titer can be increased by boosting at appropriate intervals.

The egg yolk antibody according to the present invention is prepared by adding chloroform to the egg yolk produced by the immunized chicken, 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 has a white to pale yellow color and is a protein component mainly composed of immunoglobulin (r-lihetin).
It also contains protein components such as β-2α-livetin and ovalbumin.

Although the egg yolk antibody according to the present invention can be used in an unpurified state, a higher antibody titer can be obtained by extracting and separating the immunoglobulins 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 datetran sulfate and polyethylene glycol;
Further purification is also possible by ion exchange chromatography using EAIE 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!1 fft%. It can also be dissolved in aqueous solutions containing other alcohols.

The mechanism of action of the egg yolk antibody according to the present invention against P acnes is thought to be as follows. Egg yolk antibodies obtained by immunizing chickens with the cell wall components of P. acnes bind specifically to the surface of P. acnes and suppress the activity and proliferation of the bacteria. Therefore, P.

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

The blending amount of the egg yolk antibody according to the present invention is approximately 0.00% based on the total amount of the acne cosmetic (composition). It is in the range of OOOl to 0.5 mold type% (hereinafter abbreviated as wt%). here,
If the amount of egg yolk antibody added is less than 0.0001 wt%, the effect will not be sufficiently achieved, and if it exceeds 0.5 wt%, no improvement in effect commensurate with the increased amount can be expected.

Furthermore, the cosmetic for acne of the present invention contains commonly used ingredients with a keratolytic effect such as resolunon, sulfur, nalicylic acid, urea, and isopropylmethylphenol, and ingredients with a bactericidal effect such as chlorhexidine gluconate and chloride hydrochloride.・＼Kinjin, henzalkonium chloride,
It is also possible to combine henzathonium chloride and other ingredients such as glycyrrhizin, glycyrrhizic acid, allantoin, zinc oxide, zinc sulfate, aluminum lactate, ethyl lactate, estradiol, camphor, biotin, etc. In that case, the The effect can be further enhanced. Further, other additives such as antioxidants, fragrances, pigments, various vitamins, etc. can be appropriately prepared and blended within a range that achieves the object of the present invention.

The cosmetic for acne of the present invention can be prepared in the form of lotions, emulsions, creams, gels, sticks, 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 test and acne treatment effect test described in the Examples are as follows.

il+ Human skin patch test vl, Test #40.05 was applied to the skin of the side of the forearm of 25 people.
g was occlusively applied for 24 hours using a test adhesive plaster with a circular filter paper of 1.9 cm in diameter.

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

Table 1 (2) Acne treatment effect test A control product (base only composition) was placed on the left side of a test subject with facial acne symptoms, and 10 test items of Examples or Comparative Examples were placed on the right side. I applied it continuously for 1 month, twice in the morning and once in the evening. Next, the therapeutic effect on acne diseased areas was evaluated using the criteria t in Table 2 below.
According to p, the 0 judgment result judged by the half-time comparison method is
It is shown as the average value of evaluation points.

Table 2 [Preparation of egg yolk antibody] P, acnes and P according to Examples and Comparative Examples.

Examples of preparation of cell wall components of Knes, immunization of chickens, and preparation of yellow antibodies are shown.

1. Preparation of P. acnes bacteria according to the method of G. Pabguchi et al. (Journal of Investigative Dermatology, 63S, pp. 231-238, 1974). 51) using P.

P. acnes was cultured anaerobically at 37°C for 5 days, and the bacterial cells were separated from the 18 culture supernatant by centrifugation (wet weight of bacterial cells: a,
5g).

2. Preparation of cell wall components of P. acnes Next, the method of G. Webster et al. (Journal of
Investigative Germatology Volume 84, 49
6-500, 1985), P. acnes cell wall components were prepared. 5 g of P. acnes was suspended in physiological saline and subjected to ultrasonic disruption (100 mW, 1 hour). Next, centrifugation was performed at 200 rpm for 10 minutes to remove unbroken bacterial cells in the precipitate fraction. The supernatant fraction was centrifuged at 15,000 rpm for 30 minutes to obtain a cell wall fraction among the precipitate fractions.

This fraction was treated with DNase, RNase, trypsin, and pronase to remove protein and nucleic acid components and completely solubilized, and then dialyzed against physiological saline. This fraction was further purified by Sepharose CLdB column chromatography (2.5 x 60 cm, 0.5 x 60 cm).
Purification was carried out by I 5 M Na CR, pH 7,2). As a result of examining the antigen activity using rabbit serum antibodies against whole P. acnes cells by the ophthalony method, an antigen peak was observed at K a V = 0.21-0.50, so this fraction was collected. It was concentrated and lyophilized to obtain a thin wall fraction (yield: 536 mg). Motoichi's analytical values showed that the sugar content was 281.2% (phenol-sulfuric acid method);
Protein content 11.4% (Lori-Forin method).

3-1. Immunization of cell wall components to chickens The cell wall components of P. acnes obtained by the above method were
m g / sn j! Dissolved in physiological saline at a concentration of The mixture was then mixed with complete Freund's adjuvant to prepare a complete emulsion. Apply these emulsions to the left and right pectoral muscles of female chickens approximately 1.5 to 2 months old.
After 1 month, 1.5 ml of muscular 1.j. The same amount of sample was again injected into the muscle.

3-2. As a control for immunizing chickens with P. acnes whole cells, 1) P. acnes whole cells, which had been heat-treated in advance at 70°C for 3 hours, were immunized at 2XIO'co/m1 in the same manner as the above immunization method, and in another experimental group. Ten chickens were immunized. One month later, the same site samples were injected intramuscularly again.

4. Dull immunized chickens with constant antibody titers were tested 1 week before the start of immunization and 1 week after the 2nd month.
Eggs produced during the period of one week from the third month and the third month were used to measure the antibody level as eggs before immunization, eggs from the second month, and eggs from the third month, respectively. Antibody fractions were prepared as follows.

The yolks of the eggs were collected, during which time one volume of physiological saline was added, and the mixture was shaken at room temperature for 5 minutes. Then, twice the amount of chloroform as the original yolk was added, and the mixture was shaken and stirred for 30 minutes. The resulting emulsion was left at room temperature for 30 minutes and then heated at 3000 rpm.
The supernatant was collected and filter-sterilized to obtain an antibody fraction. The changes in the antibody titer were tracked using ELIS 8 of enzyme immunoassay according to the conventional method, and the antibody titers before immunization, 2 months, and 3 months are shown in Table 3. As a result, the cell wall component showed a higher rise in antibody titer, and after 3 months, the value of P. acnes was about 4 times higher than that of the whole bacterial cells.

Table 3. Preparation of egg yolk antibodies Next, the yolks of eggs produced by chickens during a period of 1 month from the 3rd month after the start of immunization were collected, and the yolks were collected as described in ``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 the precipitation procedure with ammonium sulfate, and the antibody titer was measured by ELISA using this stock solution. . The results are shown in Table 4.

Table 4 Examples 1 to 7, Comparative Examples 1 to 4 [Acne lotion] Acne Kawaguchi-Noyon base and the above (preparation of eggshell tiL body)
1), egg yolk antibody encapsulated in acne cell wall components (example, hereinafter abbreviated as anti-cell wall antibody), as described in section 1);
Alternatively, 1), egg Cα antibodies (comparative examples, hereinafter abbreviated as anti-bacterial antibodies) against all bacterial cells of P. acnes were prepared and tested as shown in Table 5.

(1) Composition Table 5 (2) Preparation method After dissolving the ethanol, solubilizer, and propylene glycol shown in Table 5 in purified water by heating as necessary,
Next, the egg yolk antibody was added to the mixture, which was cooled to 0° C., and the remaining amount of purified water adjusted to have a total composition of 100 wt% 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-cell wall 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.

Examples 8 to 14, Comparative Examples 5 to 8 [Skin cream for acne] Each sample was prepared by blending the eggplant antibody according to the present invention into a skin cream base for acne as shown in Table 7 (11 Composition Table 7) (2) Preparation method Ingredients B and C in Table 7 were heated and dissolved at 80°C and combined in 1×1 volume, and then cooled to 50°C with stirring.

Next, component A was added and stirred to 30° C. (Ir was continued) to prepare each suton cream for acne.

(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-cell wall antibodies of Examples 8 to 14 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, Comparative Examples 5 to 8
A skin cream for acne containing anti-all-bacterial antibodies had low acne treatment efficacy and high skin irritation in human skin patch tests.

(Effects of the Invention) The cosmetic for acne of the present invention is non-irritating to the skin, has effective effects on preventing and treating acne, and is stable even when stored for a long period of time. The 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 cell wall components of Propionibacterium acnes, which is a resident bacteria of the skin.