WO1997020539A1

WO1997020539A1 - Cosmetic preparation

Info

Publication number: WO1997020539A1
Application number: PCT/JP1996/003571
Authority: WO
Inventors: Keiichiro Okabe; Shouichiro Kondo; Aya Katsuyama
Original assignee: Kabushiki Kaisya Advance
Priority date: 1995-12-05
Filing date: 1996-12-05
Publication date: 1997-06-12
Also published as: JPH09157128A; JP3608059B2

Abstract

A cosmetic preparation which contains zinc ions and/or manganese ions that are an ingredient serving as a nourishment for useful indigenous dermal bacteria and accelerating the secretion of active substances functioning like SODs derived from indigenous bacteria, is harmless or less harmful to the skin, and functions to indirectly inhibit skin oxidation.

Description

Description Cosmetic composition Technical field

The present invention relates to a cosmetic composition, and more specifically, to maintain and enhance a healthy flora resident in the skin, and to provide a suboxyxide dismutase (SOD) -like active substance secreted by the bacteria resident in the skin. By increasing the amount, it decomposes and removes active oxygen on the surface of the skin, which is a great enemy of the skin, and is useful for skin conditioning and preventing skin aging, especially cosmetics expected to be used as external preparations for skin Composition. Background art

Although harmful active oxygen to living organisms is produced constitutively produced at the organism using 0 ₂ in the metabolic system, Super O wherein the door two on a kind of active oxygen - disproportionation of (0 ₂₎ Catalytic SOD is a typical antioxidant enzyme, and it is said that this enzyme activity protects cells from oxygen damage. In the skin health science, this active oxygen mediates the oxidation of skin lipids by solar ultraviolet rays, accelerates skin aging, and is also concerned with the involvement of skin troubles. Application to agents has been proposed (see, for example, JP-A-1-96107).

However, SOD derived from natural products purified from animals, plants, and microorganisms, or SOD derived from DNA recombinants are not suitable for quality control, such as storage stability in pharmaceuticals, from the viewpoint of production cost and storage stability in pharmaceuticals. Had problems.

Recently, bacteria that grow aerobically among the skin-resident bacteria that live on the skin surface It is strongly resistant to oxygen, and it is suggested that it is highly exposed to active oxygen because it is directly exposed to the sun's ultraviolet rays and has high antioxidant ability. In fact, Staphylococcus epidermides, the most predominant aerobic resident bacterium, is used in cosmetics containing the culture fluid or the crushed bacterial cells that exhibit antioxidant properties. External preparations have also been proposed (see, for example, JP-A-6-271851). However, problems remained with the instability of the formulation. Disclosure of the invention

As mentioned above, if SOD or cultured antioxidants are directly incorporated into cosmetics, the problem of quality control, such as the odor and color, and the storage stability of SOD activity during use remains to be solved. I was Although SOD and antioxidants have long paid attention to skin conditioning, there are still many issues to be solved in their application to cosmetics.

The purpose of the present invention is to grow most of the SOD or SOD-like active substance on the skin of healthy people instead of directly blending it into cosmetics so that the SOD or SOD-like active substance can always be stably expressed on the skin It. Provide an inexpensive and safe skin antioxidant cosmetic that exhibits an effective anti-aging effect on the skin and a skin-regulating effect by incorporating an active substance that efficiently secretes the SOD activity of useful bacteria resident in the skin. It is here.

According to the present invention, there is provided a cosmetic composition comprising at least one of a metal ion, a metal salt and a metal substance.

According to the present invention, the action of enhancing the growth of Staphylococcus epidermides, the most aerobic bacterium of the useful skin flora, and the function of producing and secreting Superoxide jism Cosmetic composition containing trace amounts of at least one of manganese and zinc, which are metal ions, and salts thereof, which have an effect of enhancing SOD-like active substances Is provided. BRIEF DESCRIPTION OF THE FIGURES

Hereinafter, the present invention will be described in detail with reference to the drawings.

Figure 1 shows the effect of metal ions (zinc and manganese) on the development of Staphylococcus epidermides.

Figure 2 shows the effect of metal ions (zinc and manganese) on the growth of Staphylococcus aureus. BEST MODE FOR CARRYING OUT THE INVENTION

In order to solve the above-mentioned problems, the present invention has conducted intensive studies to increase the extracellular SOD-like specific activity of Staphylococcus' epidermides, and to reduce the metal ions manganese and zinc even in poor nutrient media. The present inventors have found that by adding the same, the same bacterium can be grown in a short time and the extracellular SOD-like active substance can be maintained for a long period of time, and the present invention has been accomplished.

That is, the present invention is a cosmetic containing a substance capable of growing Staphylococcus epidermides resident on the skin in a short period of time and increasing the extracellular SOD-like specific activity. In addition, this cosmetic is a highly stable and safe cosmetic that achieves skin conditioning by normalizing the skin flora and enhancing the activity of its extracellular secretory SOD-like active substance. In particular, those in the aerobic bacteria on the skin of healthy human of the most dominant, vinegar evening off Lee Loco Kkasu. Cheap Joteki in 1 X 1 0 about ^four Z cm ² in Epiderumi Death resident skin Form a bacterial flora. The Staph I Loco Tsu dregs-E Biderumi death is established growth on the surface of the skin due to the high antioxidant capacity, and secrete the SOD-like active substances in the cells outside the body, the 0 ₂ on the skin caused Ri by such as on the results of ultraviolet rays It is thought to play a role in eliminating skin injuries by erasing, increasing skin health, and preventing other bacterial infections. In fact, skin with injuries such as atopic dermatitis (rough skin) or dry skin loses the balance of the bacterial bacterial flora and belongs to the genus Staphylococcus aureus. Harmful bacteria are found at a high rate. S. phylococcus as the dominant bacterium ■ Epidermides inhibits the growth of the above harmful bacteria, so that a slight skin injury can cause selective growth of Staphylococcus epidermides. Providing a cosmetic composition that can be used to reduce the number of pathogenic bacteria and improve skin damage can be expected.

In the cosmetic according to the first embodiment of the present invention, metal ions (for example, Mn, Zn2 ⁺ ) and metal salts (for example, manganese chloride, zinc chloride, zinc oxide, zinc stearate, paraffin) are used. Contains one or more of zinc norsulfonate, zinc laurate, manganese sorbet, and manganese sulfate.

The cosmetic according to the second aspect of the present invention has an effect of enhancing the growth of Staphylococcus epidermides, the most aerobic bacterium of the useful skin flora, and producing and secreting the cells. One of the metal ions manganese and zinc or their salts (eg, manganese chloride and zinc oxide) that have the effect of enhancing superoxide 'dismutase (SOD) -like active substances In trace amounts. The manganese ion preferably has a concentration in the range of 0.1 to 100 mM, more preferably 0.1 to 10 mM. On the other hand, the zinc ion preferably has a concentration in the range of 0.01 to 5 mM, and more preferably 0.1 to 1 mM.

Examples are described below to better explain the present invention.

(1) The microorganism used to prove the effectiveness of the present invention was Staphylococcus epider, a representative resident bacterium inhabiting the skin of healthy humans, as used in the following examples. A force that is a mide or a skin Staphylococcus ■ P. aureus is a representative of skin harmful bacteria. However, the bacterium used to carry out the present invention can be used irrespective of any bacterial species and strains, including those that can pass through the skin.

(2) As a medium used to prove the effectiveness of the present invention, not only a normal nutrient medium but also a minimum growth medium was used to examine the requirement for metal ions. That is, in the minimum growth medium, glucose and yeast extract were added as energy sources, and calcium carbonate was added to adjust the pH, which was used as the basal medium. Although the metal requirement of bacteria is known, several kinds of metal ions, such as manganese, which are the active centers of SOD, were added to the basal medium in advance, and the concentration at which growth was not inhibited was examined. Therefore, the minimum growth medium used in the present invention is defined as a medium to which a minimum concentration of metal capable of growing bacteria is added.

(3) The method of culturing bacteria used to prove the effectiveness of the present invention is as follows: using the above strains and medium, the bacteria are grown under aerobic conditions at 37 ° C under a rotary culture (120 rpm). ).

(4) The sample preparation method used to prove the effectiveness of the present invention is as follows. First, bacteria sufficiently grown in the above medium were separated into bacterial cells and culture supernatant at 4 ° C for 10 minutes using a cooling high-speed centrifuge. Subsequently, the SOD activity of the culture supernatant was measured and concentrated as follows. Add cold acetate to the culture supernatant to a final concentration of 75%, leave it in a freezer at 120 ° C, separate and collect the formed precipitate with a cooling centrifuge, and dissolve in a small amount of purified water Then, freeze drying was performed to obtain an acetone powder sample. The powder was dissolved in purified water and used as an extracellular secretory component sample.

(5) As a measurement of the SOD-like active substance used to prove the effectiveness of the present invention, the Nitrole tetrazolium (NBT) method was used. This measurement method is relatively simple among many measurement methods, has high sensitivity, and is on the order of ng. Detection is possible up to. Ri by the xanthine O Kishida one zero 〇 ₂ - the raised departure, 〇 2 - wavelength the color of the two-door Roburute tiger sledding ©-time by

It is measured at an absorbance of 560 nm. 0 The presence of SOD ₂ - is Ri Once disproportionation coloring inhibition of NBT is to put, SOD activity is determined from this inhibition rate. Using this method, SOD-like active substances secreted outside the cells can be measured with high sensitivity by acetonitrile concentration treatment of the culture supernatant sample.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples and Examples. However, it is needless to say that the scope of the present invention is not limited to these Examples.

Experimental Example 1 Influence of metal ions on the growth of S. huirococcus epidermides:

The basal medium used in the series of experiments was Davis's medium (sinium sodium hydrogen phosphate decahydrate, 8 g; potassium dihydrogen phosphate, 2 g; sodium citrate, 0.5 g). g; magnesium sulfate heptahydrate, 0.1 g; ammonium sulfate, 1 g; glucose, 0.5 g / liter of distilled water (pH 7)) plus 0.1% of yeast extract and 1% of carbonated calcium Was used. The inoculum was set at 1 × 10 ⁵ cells / ml, and the culture was performed in a thermostat at 37 ° C. The metals used in the experiments were copper sulfate, zinc sulfate, manganese chloride, zinc oxide, and ferrous sulfate. The concentrations were 100 / M, 1 mM, and 10 mM, respectively, and were added before the start of culture. After culturing for 24 hours, the culture solution was applied to a standard agar plate, and the number of viable cells was counted by a standard method. The results are shown in Table 1. Table 1: Development of Staphylococcal 'Epidermides'

Effect of metal ion on concentration of added metal ion (mM)

1 0 1 0.1 Copper sulfate

Zinc sulfate

Zinc oxide +

Manganese chloride + + tens + ferrous sulfate Note) Meaning of the expression of the growth status;

+ Z—: No increase / decrease, One: Decline, —: Death

As shown in Table 1, manganese chloride and zinc sulfate, zinc oxide, and ferrous sulfate had a growth promoting effect.

Experimental Example 2 Growth promotion and inhibition effects of zinc oxide and manganese chloride on bacteria isolated from skin: + +

In Example 1, the SOD-like active substance in the culture supernatant of Staphylococcus aureus + Ipidermides was measured.As a result, the experimental group to which zinc sulfate and manganese chloride were added showed higher values than the control group. The divalent metal

+ +

Attention. At this time, zinc oxide, which is widely used in cosmetics, was used for zinc sulfate, and ferrous sulfate was oxidized and discolored, and was excluded because it could adversely affect the skin. Therefore, the growth curve of Staphylococcus epidermides was examined using a medium supplemented with zinc oxide and manganese chloride. The concentration of both metals was 100 M, and the inoculum volume was 1 × 10 ^{s /} ml. The results are shown in Figure 1.

As shown in FIG. 1, a remarkable increase in the growth rate of Staphylococcus epidermides was observed in the mixture with zinc oxide and manganese chloride.

In addition, similar experiments were carried out on skin-resident bacteria other than Staphylococcus epidermides. A typical example is the staff The same experiment as described above was performed using Cassaureaus IDD671 strain, and the change in the number of bacteria was measured by the absorbance at a wavelength of 595 nm. The results are shown in FIG.

As shown in Figure 2, the harmful pathogen Staphylococcus aureus differs from the useful resident bacterium Staphylococcus epidermides by adding zinc oxide and chloride. No particular promotion of growth was observed with cancer, but rather a tendency toward inhibition.

Example 3 Influence of zinc oxide and manganese chloride on extracellular SOD-like activity of Staphy P coccus' Epidermides:

Next, the following studies were conducted to determine whether the development of extracellular SOD-like specific activity, zinc oxide and chlorinated manganese, which was found in Experimental Example 1, was related to the culture time. The culture was performed under the same conditions as in Experimental Example 1, and the SOD-like activity was measured over time. The number of samples in each experimental group was 3, and the average and standard deviation were calculated. Hereinafter, the experimental method of Experimental Example 3 will be described in more detail.

Sample preparation method: For sample preparation, culture was performed on a 100 ml flask at the scale of a 50 ml culture solution, and 5 ml was sampled each hour. The culture was immediately centrifuged (10,000 rpm, 4 ° C, 10 minutes) to separate cells and supernatant. Cool the collected supernatant with ice, add acetone at 20 ° C with stirring to a final concentration of 75%, and leave the mixture in ice for 2 hours. Thereafter, the precipitate was collected by centrifugation again (10, OOOrpnu 4 ° C, 10 minutes). Dissolve the precipitate in a small amount of purified water, freeze at 180 ° C and freeze-dry to remove acetate. Using the obtained dried powder dissolved in purified water as a sample, protein quantification and S0D-like activity were measured. Protein quantification method: Lowry method was used for quantification of protein. . A 0.4 ml sample containing 10 to 100 g of protein is placed in a test tube, and an alkaline copper solution (1 ml of 1% copper sulfate and 1 ml of 2% rossiel salt is mixed with 2% carbonic acid) Sodium, added to 100 ml of 0.1 N sodium hydroxide) Add 2 ml, stir, and leave at room temperature for 10 minutes. Immediately thereafter, add 0.2 ml of 1 N phenol reagent, stir, and leave at room temperature for 30 minutes. Thirty minutes later, colorimetric determination was performed at 750 nm. Bovine serum albumin (BSA, SIGMA) was used as the standard solution.

Measuring method of SOD-like active substance: Take 50 /] sample in a test tube, dissolve X0D (xanthine oxidase) solution in 0.5M ammonium sulfate, 1 mM EDTA-2Na solution at 20 ° C Dissolve immediately before use, and dilute so that the change in 0D560nm per minute is 0.0185), add 50/1, and keep in a 25 ° C water bath for 10 minutes without stirring. After heating to 25 ° C, solution A (0. ImM xanthine, 0.025 mM nitrobluete trazolyme, 0.1 lmM EDTA- 2 Na, 50 mM sodium carbonate buffer (ρΗΙΟ.2) After stirring for 2 minutes in a 25 ° C water bath, add 50 1 of copper chloride solution (0.2% CuCl ₂ , H ₂₀ ), stir and colorimetrically measure at 560 nm. For the control, purified water was added instead of the sample, and the blank was measured only with the sample and the 0.2 M ammonium sulfate solution excluding the X0D solution.The SOD activity was 1 when the change in absorbance was inhibited by 50%. Unit (U) was defined and calculated by the following formula.

S0D (U / ml) = [(control 0D-blank 0D) bar sample 0D blank 0D)-1] x 20 The results are shown in Table 2.

Table 2: Extracellular SOD-like specific activity (U / mg protein) Effect of metal ion on time course Culture (time)

Additive metal '19 26 No additive (control) 11.2 ± 4.16.2 Soil 37.9 ± 4.6 Zinc oxide 12.9 ± 2 * 13.2 ± 1.36.9 ± 4.6 Manganese chloride '29 .0 ± 2 * 37.8 ± 3 * 49.2 Sat 3.2 Compound * 23.5 ± 3.25.5 ± 7.34.5% 1.0 (zinc oxide + manganese chloride)

* ': Basal medium (ρΗ7.0) was prepared by adding yeast extract (0%) and calcium carbonate (1%) to Davis medium. Metal concentration: 0. ImM * ² : Average soil standard deviation (n-35

* P <0.05 ** P <0.01 U: Activity unit

As shown in Table 2, when zinc oxide and manganese chloride were added alone or as a mixture, a remarkable increase in the specific bacterial cell +/- outside SOD activity was observed in the initial stage of the culture.

Example 1 Skin irritation test (safety):

Based on the above results, an irritation test was conducted on cosmetics containing manganese and zinc. As an example of the composition of the cosmetic, the following (Table 3) solution was soaked in filter paper 20/1, and closed and taped on the inner skin of the forearm of an adult male (n21), and 24 hours later The skin irritation was observed, and the results shown in Table 4 were obtained.

Table 3: Example of cosmetic formulation

Formulation Example 3 (Control) Manganese chloride (0. ImM) +

Zinc oxide (0.1 mM) +

CaC0 ₃ (1%) one

Glycerin (5%) + +

Purified water (total 10 ml) Sample Nos. 1 and 2 are manganese / zinc combination and calcium carbonate Sample No. 3 was prepared as a control to examine the irritation properties of Pem. As a result of the judgment by the naked eye, there was no red spot or irritation, and no problem was found in safety.

Table 4: Skin irritation test results (24-hour patch)

Sake

Sample No.Mizuseo Maruno Removal 30 minutes after removal 24 hours after removal 48 hours after removal

Lead-free

1

2 Lugasin

3 (Control) Next, a formulation example of the cosmetic according to the present invention will be exemplified. The mixing ratio is

All are indicated by weight%. It goes without saying that the present invention is not limited to cosmetics according to the following formulation examples.

Example 2 Composition of lotion

Table 5 Composition Weight (%)

0. 002

0. 0008

5.0

8.0

13

Vitamin Mono Laulet

Proper

Example 3 Composition of Karamin Lotion

Oil and water

Phase composition

Ethanol

Glycerate HCl

Luchistacryli made by zinc oxide

Manga chloride subwater Ruchigimasesunobin

Lead calcium calcium carbonate

Benka La Gumi Lulte Gandundo

7J)) No

Deamimi camprate

Teziri

Purified water Rune

Acid arrow i ^ ίR

A Ί ^ A

^ Leri 1iE nX! One

o

Composition

Mangan chloride 1 OOO O Zinc oxide

Dipro Pilling Records

Purified water Example 5 Composition of skin cream

Table 8 Weight (%)

30.0

6.0

3.0

4.0

0. 0002 0. 00008

5.0 Experimental Example 4 Anti-peroxidative effects of UVA on squalene peroxidation of extracellular secretory S 0D of Staphylococcus' Epidermides:

In addition, peroxidation of extracellular SOD (0.16 U / mg) of S. phylococcus epidermides cultivated by adding manganese chloride (0. ImM) and zinc oxide (0.1 lmM) The lipid-suppressing effect was examined using a system that irradiates squalene with UVA.

As a control, a culture supernatant of the same strain, which had been cultured without the addition of the metal ion, to which was added the same weight of an acetone powder was used. SOD activity was hardly observed in these.

Lipid peroxide was quantified by the TBA (thiobarbituric acid) method shown below. Add 0.2 ml of the sample solution to 1.8 ml of an aqueous solution prepared by solubilizing 1.0% squalene with 10 mg / ml of Triton X-100, and apply ultraviolet light at 37 ° C (Toshiba FL20S-BL.352 nm 0.416 mW / cm ² ) was irradiated for 48 hours. 0.1 ml was taken out from the sample solution, 2.0 ml of TBA reagent (0.2% tiobarbituric acid, 5.0% acetic acid pH3.5) was added, heated at 100 ° C for 60 minutes, and then cooled with water. Furthermore, the final metabolite MDA (malonaldehyde) was extracted with n-butanol 2.Oiii and the absorbance at 530 nm was measured.

As a result, as shown in Table 9, the extracellular S0D (0.16 U / mg) induced by these metal ions showed a tendency to significantly (P and 0.01) inhibit lipid peroxide production as compared to the control group. Was. At this time, the effect was lower than the value obtained when the tocopherol (1 mgZml) as a positive control was added, but concentration dependence was observed. Table 9 Squalene Peroxidation Inhibition by UVA of Extracellular SOD of Star J Ilococca Spidermides Additive Sample (n = 3) Addition Amount Malonaldehyde Inhibition

(mg / ml) (nmol / mlZ48 hours) (%) Mean Soil standard deviation

Control (purified water) 1.0 43.5 soil 3.1 0.0 No metal ion 1.0 41.9 5.6 3.7 Subculture supernatant

Presence of metal ion 1.0 * 27.0 ± 3.3'37.9

Positive control

a Tocopherol 1.0 1.1 ± 0.2 ** 97.5

*: P <0.01 P <0.001 Industrial applicability

By using the cosmetics of the present invention containing appropriate amounts of the metal ions zinc and manganese, the healthy growth of useful normal flora of skin and the secretion of SOD-like active substances from the cells are promoted, and the growth of skin harmful bacteria It is expected to be effective in preventing skin cells, preventing active oxygen damage to skin cells, and preventing skin cell aging.

Claims

The scope of the claims

1. A cosmetic composition comprising at least one of a metal ion, a metal salt and a metal substance.

2. The effect of promoting the growth of Staphylococcus' epidermides, the most aerobic bacterium of the useful skin flora, and the superoxide. A cosmetic composition comprising at least a trace amount of manganese and zinc, which are metal ions, and a salt thereof, which has an action of enhancing the active substance.

3. The cosmetic composition according to claim 2, which contains manganese ions at a concentration of 3.0 to 100 mM.

3. The cosmetic according to claim 2, comprising zinc ion at a concentration of 4.0 to 5 mM.