JPH07242525A - Whitening agent containing isonitrile compound - Google Patents

Abstract

(57) [Summary] [Composition] A whitening agent containing an isonitrile antibiotic produced by filamentous fungi as an active ingredient. [Effect] The isonitrile antibiotic has a strong mushroom tyrosinase inhibitory activity and a melanin production inhibitory activity of actinomycetes, and suppresses melanin production of B16 melanoma cultured cells.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a whitening agent containing an isonitrile antibiotic produced by filamentous fungi.

[0002]

2. Description of the Related Art Melanin pigments that cause skin coloration are produced in melanin-producing granules (melanosomes) in melanocytes (melanocytes) between the epidermis and the dermis, and the produced melanin is adjoined by osmotic action. Spread to cells. The biochemical reaction in this melanocyte is currently estimated to be as follows. The process in which tyrosine, which is an essential amino acid, becomes dopaquinone by the action of the enzyme tyrosinase, and this is converted to black melanin through the red and colorless pigments by the enzymatic or non-enzymatic oxidation action is the process of melanin pigment formation.

Therefore, it is considered that the production of melanin can be suppressed by suppressing the action of tyrosinase, which is the first step of the reaction, or by reducing the intermediate quinones. Already, as a means for suppressing this, a substance that binds to copper, which is the active center of tyrosinase (eg thiourea, cysteine, kojic acid), a substance that can be a competitive substrate with tyrosine that is a substrate of tyrosinase (eg N-acetyltyrosine, γ- Pyrone, hinokitiol), a substance that prolongs the induction period of the enzymatic reaction between tyrosinase and a substrate (such as Tween 20), a substance that selectively binds to an o-dihydroxy group such as dopa (such as molybdenum ion), o
-Substances that bind to quinones (for example, aniline), reducing agents for o-quinones (for example, ascorbic acid, hydroquinone and its derivatives), etc. have been proposed.

Some of these compounds are effective as a whitening agent, but in view of the above-mentioned melanin pigment formation process, there is a possibility that they may have a unique mechanism of action. It may be desirable to provide certain compounds (or compositions).

From the above viewpoints, the present inventors have screened tyrosinase-inhibiting active substances having a whitening effect from microbial sources. And first, Trichoderma ( Tri
It was found that a strain belonging to the genus choderma produces a tyrosinase inhibitory active substance, and proposed a method for producing the substance (see Japanese Patent Laid-Open No. 2-145189). Since this substance has no antibacterial activity but has a considerable tyrosinase inhibitory activity, its use as a whitening agent is also of interest.

[0006]

However, there remains a need for more effective whitening agents. Therefore, an object of the present invention is to provide a whitening agent containing a compound screened from a new point of view (which may have a mechanism of action different from the conventional one).

[0007]

Means for Solving the Problems The present inventors have tyrosinase (mushroom-derived) inhibitory activity and
Talaromyces ( Talaromyces ) has been extensively screened for compounds having melanogenesis-inhibiting activity of Streptomyces biliniensis, which is a melanin-producing bacterium, and melanogenesis-inhibiting activity of mouse-derived B16 melanoma culture cells. It has been found that a compound having an isonitrile group produced by one strain FO-3182 belonging to the genus exhibits each of the above inhibitory activities and can be advantageously used as a whitening agent.

The compounds produced by the above FO-3182 strain were identified.

[0009]

[Chemical 9]

It was found to be a so-called isonitrile antibiotic represented by The present inventors
It was named O-3182. FO-3182 is a known compound which is also produced by filamentous fungi belonging to the genus Penicillium, as described in JP-A-60-155116. According to this patent publication, the above compounds are known to have anti-cancer effects.

Based on the above findings, the tyrosinase inhibitory activity and the melanin production inhibitory activity of other known isonitrile antibiotics widely produced by filamentous fungi were examined. Three
It has been found to exhibit a similar effect to 182.

Therefore, according to the present invention, there is provided a whitening agent containing as an active ingredient an isonitrile antibiotic produced by a filamentous fungus or a physiologically acceptable salt or derivative when they have a carboxyl group.

The filamentous fungus referred to in the present invention is Trichoderma,
Strains belonging to the genus Penicillium and Talaromyces are included, and their isonitrile antibiotics include isonitrines A, B,
C and D, and isonitrinic acid (Isonitrinic
acid) E and F (eg A. Fujiwara et al., Agri
c. Biol. Chem., 46 (7), 1803-1809,
1982; JP-A Nos. 53-26395 and 53-153.
45), FO-3182 or antibiotic No.
2188 (see Japanese Patent Laid-Open No. 59-17989), Dermadin (US Pat. No. 3,627,882)
No.), and trichobilidein (Japanese Patent Publication No. 4)
5-15435). Further, typical examples of these derivatives include esters of the above antibiotics having a carboxyl group (see, for example, JP-A-60-152488). The salt is a salt formed by a salt-forming reaction with a carboxyl group in the molecule, and examples thereof include alkali metal salts such as sodium and potassium, and physiologically acceptable salts with various organic amines. Is mentioned.

Among these compounds, specific compounds suitable for use as a whitening agent include those represented by the following formula (I).

[0015]

[Chemical 10]

In the formula (I), R ¹ and R ² together represent a single bond, R ³ represents a hydrogen atom, and R ⁴ and R ^{5 respectively.}
Are expressions together

[0017]

[Chemical 11]

And R ⁶ and R ⁸ together represent --O.
A group (and corresponding to isonitrine A) in which R ⁷ and R ⁹ each represent a hydrogen atom; R ¹ and R ² together represent a single bond, R ³ represents a hydrogen atom, R ⁴ Is an expression

[0019]

[Chemical 12]

R ⁵ represents a hydroxyl group, R ⁵ represents a hydroxyl group, and R ⁶ and R
⁸ represents together -O- group, and R ⁷ and R ⁹
Each represents a hydrogen atom (corresponding to isonitrine B); R ¹ and R ² together represent a —O— group, R ³ represents a hydrogen atom, and R ⁴ represents a formula.

[0021]

[Chemical 13]

R ⁵ represents a hydroxyl group, R ⁵ represents a hydroxyl group, and R ⁶ and R
⁸ represents together -O- group, and R ⁷ and R ⁹
Represents a hydrogen atom (corresponding to isonitrine C); R ¹ and R ⁹ together represent a single bond, R ² and R ³ each represent a hydrogen atom, and R ⁴ represents a formula.

[0023]

[Chemical 14]

R ⁵ and R ⁶ together represent a single bond, and R ⁷ and R ⁸ each represent a hydrogen atom (corresponding to isonitrine D); R ¹ and R ⁹ together Represents a single bond, R ² and R ³ each represent a hydrogen atom, and R ⁴ represents a formula.

[0025]

[Chemical 15]

R ⁵ and R ⁶ together represent --O.
A group each representing a hydrogen atom, and R ⁷ and R ⁸ each represent a hydrogen atom (corresponding to isonitrinic acid E); R ¹ and R ² together represent a single bond, R ³ represents a hydrogen atom, R ^{3 4} and R ⁵ together form an expression

[0027]

[Chemical 16]

A compound (isonitrinic acid F) in which R ⁶ , R ⁷ , R ⁸ and R ⁹ each represent a hydrogen atom; and R ¹ and R ⁹ together represent a --O-- group. , R ² and R ³ each represent a hydrogen atom, and R ⁴ is a formula.

[0029]

[Chemical 17]

R ⁵ and R ⁶ together represent --O.
Represents a group, and R ⁷ and R ⁸ each represent a hydrogen atom (equivalent to FO-3182).

The above-mentioned antibiotics are compounds known per se, and can be isolated from the culture by culturing the filamentous fungus described in the above publication in a nutrient medium. In addition, it can be produced using a strain screened from soil fungi with the melanin production inhibitory activity of melanin-producing Streptomyces bikini as described below as an index.

For example, the FO-3182 strain of the filamentous fungus isolated from the soil by the present inventors accumulates several kinds of compounds having the above melanin production inhibitory activity in the culture when it is cultured in a nutrient medium. Specifically, the ethyl acetate extract of the culture supernatant was purified by silica gel thin layer chromatography [plate: Kieselgel 60F ₂₅₄ (manufactured by Merck),
A developing solvent: chloroform / methanol (6/1) 1% acetic acid] shows a chromatogram as shown in FIG. Of these components, the separation zone 12 corresponds to FO-3182 which is the active ingredient of the present invention.

The mycological properties of this FO-3182 strain are shown below.

1) Morphological Properties This strain grows rapidly on corn meal agar medium, oatmeal agar medium, YpSs agar medium and the like. A large number of ascomycetes are formed, and conidia are well established. When the colonies grown on the YpSs agar medium are observed under a microscope, the hyphae are transparent and have septa, and they form soft, ascomycetes wrapped in yellow hyphae. Ascomycetes spherical with no mouth, diameter 1
It is from 00 to 400 μm. Ascos are almost spherical and colorless, 8 spores, 8-10 μm in diameter. Ascospores are yellow, oval, 3-4 × 4-5 μm, and the surface is spiny.

The conidia peduncle is directly grown from the basal hyphae. Penicillus is a compound unicycle and rarely includes a monocycle. 2 to 2
Three rings are formed and the size is 7.5 to 10.0 × 2.5 to 3 μm. Fluoride is a pen-tip type, and has 2 to 5 wrinkles, 7.5 to 12.
It is 5 μm. Initially, one phialioconidia settles on the apical end of the fluoride and becomes chain-like with the lapse of culture time, and finally this chain reaches around 200 μm. Conidia are subspherical, 2-2.5 × 2.5-3 μm, and the surface is smooth.

2) Culture properties The following table shows the results of macroscopic observation when the cells were cultured on various agar media at 25 ° C for 14 days.

[0037]

[Table 1]

In these media, formation of secretory fluid and sclerotia associated with the growth of the fungus was not observed.

3) Physiological properties (1) Optimal growth conditions The optimum growth conditions for this strain are pH 5 to 5 in YpSs medium.
7. The temperature is 14 to 31 ° C.

(2) Growth conditions The growth range of this strain is pH 2-8 and temperature 10-34 ° C. in YpSs medium.

(3) Distinction between aerobic and anaerobic aerobic strains Based on the morphological characteristics, culture characteristics and physiological characteristics of the FO-3182 strain, an attempt was made to compare it with known bacterial strains. As a result, this strain was found to be Talaromyces ) Could be identified as a single bacterial species belonging to the genus. Therefore, this strain was designated as Taraomyces sp. It was named FO-3182. This bacterium is Talaromyces sp. FO-3182 (Talaro
myces sp. FO-3182), February 16, 1994
It has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science as of the date of accession number 14150 (FERM P-14150).

The FO-3182 strain is a general nutrient medium for filamentous fungi, for example, glucose 2% by weight, yeast extract 0.1%.
Aeration using 2% by weight, MgSO ₄ .7H ₂ O 0.05% by weight, polypeptone 0.5% by weight, KH ₂ PO ₄ 0.1% by weight and agar 0.1% by weight (adjusted to pH 6.0). It can be cultured under a suitable jar fermenter. From the culture cultivated at 28 ° C. for about 3 days, the target compound can be isolated by removing the microbial cells and then subjecting it to various chromatographies such as solvent extraction, adsorption, ion exchange and gel filtration. The isolated material was confirmed to have the above structural formula by ¹ H and ¹³ C NMR, IR, UV and MS spectra.

It can be judged by the following activity test that the FO-3182 thus obtained and the above-mentioned known compounds can be used as active ingredients of a whitening agent.

(A) Melanin production inhibitory activity test using radiogenic bacteria As an indicator bacterium, Streptomyces bikiniensi, which is a melanogenic radiobacterium, is used.
s) This test was performed using JCM 4011 (hereinafter abbreviated as S. bikiniensis).

Timita et al. Antibiotics, 43 , 1
S. 601-1605 (1990).
Slant for storage of bikiniensis (Papavizas'YDYA
agarslant) and cultured at 25 ° C. for 1 week. Fully grown S. The cells were suspended in sterile water using bikiniensis (1 platinum loop / ml). Copper sulfate (CuSO _4.5 / 5H
₂ O) 0.1 mg / l added agar medium (ISP medium 7+
0.2 ml of the yeast solution was added to 0.2% yeast extract), spread with a large stick and dried. Place a paper disk (diameter 8 mm) on a Petri dish, soak 50 μl of the sample, incubate at 25 ° C for 2 days, and then measure the diameter of the white band around the paper disk (diameter 8 mm) resulting from the inhibition of melanin production. It was measured. The diameter of the white belt is 10
A sample having a size of mm or more was determined to be positive. In addition, the diameter of the growth inhibition circle resulting from the inhibition of the growth of the bacterial cells was measured to determine the antibacterial activity.

(B) Preparation of mushroom tyrosinase inhibitory activity test reagent Tyrosinase solution (500 Unit / ml) Mushroom tyrosinase (2000 Unit / ml: S
10 mg (manufactured by IGMA) is precisely weighed and dissolved in 40 ml of 0.1 M phosphate buffer (pH 6.8).

L-Tyrosine solution L-tyrosine (manufactured by Wako Pure Chemical Industries, Ltd.) 0.0453 g was precisely weighed,
Dissolve in 100 ml of 0.1 M phosphate buffer (pH 6.8).

0.1M phosphate buffer (pH 6.8) 0.19M NaH ₂ PO _{4 (} 50.9 ml) and 0.1M NaH ₂ P
Mix 49.1 ml of O ₄ to make 100 ml.

Equipment used Titer Tech Twin Leader Brass (Type 381:
Flow Laboratories, Inc.) Test method 96-well plate 0.1M phosphate buffer (pH 6.
8) is dispensed by 100 μl and 20 μl of the sample solution is added. Next, add 30 μl of tyrosinase solution,
The absorbance at 492 nm is measured with a twin reader, and the value of the absorbance at 0 hour of reaction is designated as T ₀ . After that, L- in each hole
Add 50 μl of tyrosine solution and react at 37 ° C. for 60 minutes. After 60 minutes, the absorbance at 492 nm is measured, and the value is designated as T. As a control, 20 μl of 0.1 M phosphate buffer (pH 6.8) is used instead of the sample solution, and the absorbance at 0 hour of reaction is defined as C ₀ . Similarly,
The reaction is carried out at 37 ° C. for 60 minutes, and after 60 minutes, the absorbance at 492 nm is measured, and the value is designated as C. Tyrosinase inhibitory activity was calculated by the equation shown below. A sample having an inhibitory activity of 70% or more was determined to be positive.

Tyrosinase inhibition (anti-tyrosinase) activity (%) = [1- (T-T ₀ ) / (C-C ₀ )] × 100 (C) Melanin production inhibitory effect test of B16 melanoma cultured cells. The cells were seeded and cultured in growth culture for 24 hours, and then replaced with a new medium containing a sample solution.
A sample solution prepared by dissolving in DMSO was used, and 20 μl was added to 5 ml of the medium. After further culturing for 3 days, 4 days after the start of culturing, cells are collected by trypsin treatment, and the number of cells and the amount of melanin are measured, and the cell growth rate in the sample addition system is higher than that in the case where no sample is added. Was calculated.

The number of cells was measured using a hemocytometer, and the number of cells per dish was calculated. To measure the amount of melanin in cells, a cell suspension was prepared so as to contain 1 million cells, washed twice with PBS solution, and then washed with 1N.
NaOH was added, the mixture was incubated at 100 ° C. for 30 minutes and dissolved, and the absorbance at 475 nm was measured.

The above-mentioned test method is as described in Nisshinkai, 101
(6), 609-613, 1991, "Membrane production inhibitory effect of arbutin B16 melanoma cell biochemical study", which is a partial modification of the method.

The isonitrile antibiotic used in the present invention is
All of them show activity in each of the above tests with some differences, and can be used as an active ingredient of a whitening agent. In addition to these active ingredients, those that can be contained in the whitening agent of the present invention are those that do not adversely affect the action of the coexisting isonitrile antibiotics, and are components commonly used in cosmetics, medicines, etc. Not limited. As these components, oil, an ultraviolet absorber, an antioxidant, a surfactant,
Activators, moisturizers, fragrances, water, alcohols, thickeners, coloring materials, skin nutrients (tocopherol acetate, pantotenyl ethyl ether, glycyrrhizinate) and the like can be mentioned.

The dosage form of the present invention is arbitrary, and for example, a solubilizing system such as a lotion, an emulsifying system such as an emulsion or a cream, or an ointment or a dispersion can be adopted. The whitening agent of the present invention, the above compound, based on the total composition weight,
About 0.01 to 30% by weight, preferably 0.1 to 20% by weight
Can be included in. Such a whitening agent may be applied to the skin in need of treatment based on its tyrosinase inhibitory activity. In this way, it is possible to prevent the desired whitening effect, for example, the generation of stains on the face, etc., and to use it for decolorizing the stains that have already formed.

[0055]

EXAMPLES The present invention will be described in more detail with reference to FO-3182, but the present invention is not limited thereto.

Example 1 Various Inhibitory Activities of FO-3182 FO-3182 powder was diluted with ethanol water as shown in the following table to prepare diluted samples.

With respect to these samples, the melanin production inhibitory activity and the mushroom tyrosinase inhibitory activity of the radiobacterium measured according to the above method are shown in the following table.

[0058] Concentration (mM) Melanin production inhibitory activity Tyrosinase inhibitory activity 25 52 (nm) 100 (%) 2.5 35 96 0.25 10 94 0.025 <8 79 In addition, regarding the melanin production inhibitory effect of B16 melanoma cultured cells, FO -3182 has a final concentration of 0.01 mM
Then, the cells (1.
The amount of melanin per 0 × 10 ⁶ pieces is 25%, and the dish (d
The amount of melanin per ish) was suppressed by 30%.

Example 2 Whitening effect test Preparation of emulsion Polyoxyethylene (20 moles) 1.0 Polyoxypropylene (2 moles) Cetyl alcohol Silicone KF96 (20cs) 2.0 (Shin-Etsu Chemical) Liquid paraffin (medium) 3. 0 N, N-dimethyl PABA 5.0 Octyl ester (UV absorber) Propylene glycol 5.0 Glycerin 2.0 Ethyl alcohol 15.0 Carboxy vinyl polymer 0.3 Hydroxypropyl 0.1 Cellulose 2-Aminomethylpropanol 0.1 1 Preservative A suitable amount FO-3182 Ion-exchanged water Residual (production method) FO-3182 is dissolved in ion-exchanged water and ethanol under heating, and water-soluble components below propylene glycol are dissolved and kept at 70 ° C (water phase) . Other oily ingredients are mixed, heated and melted and kept at 70 ° C. (oil phase). The oil phase was added to the aqueous phase, preliminarily emulsified, homogeneously emulsified with a homomixer, and then after emulsification, cooled to 30 ° C. with good stirring.

Judgment of Whitening Effect With respect to the above-mentioned emulsion, 40 patients (females) with melasma were divided into 2 groups of 20 and the whitening effect when FO-3182 was blended was evaluated. As a control, the above emulsion containing no FO-3182 was used.

In the judgment test, the subject milk was applied to the affected area of the face twice a day for 12 weeks (0.2 to 0.3 g / time), and 12 times
The degree of improvement in the affected area after a week was visually evaluated.

The results are shown below.

Emulsion containing FO-3182 FO-3182 Emulsion not containing FO-3182 Pigmentation extinction 0 0 Significantly improved pigmentation 3 0 Significantly improved pigmentation 8 0 Slightly improved pigmentation 7 3 Pigmentation unchanged 2 17 Pigmentation deterioration 0 0 Total 20 20. No skin irritation was observed in the emulsion of the present invention.

[0064]

According to the present invention, there is provided a whitening agent containing as an active ingredient an isonitrile antibiotic known to have antibacterial activity and the like.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a thin layer chromatogram showing the components of the ethyl acetate extract of the culture supernatant derived from the FO-3182B strain.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] May 11, 1994

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction content]

[0009]

[Chemical 9]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yuki Shibata 1050 Shinba-cho, Kohoku-ku, Yokohama-shi, Kanagawa Inside Shiseido Daiichi Research Center (72) Inventor Kazuo Komatsu 1050 Shinba-cho, Kohoku-ku, Yokohama-shi, Kanagawa Incorporated company Shiseido Daiichi Research Center (72) Inventor Satoshi Omura 5-9-1 Shirokane, Minato-ku, Tokyo Kitasato Institute (72) Inventor Isuro Masuma 5-9-1 Shirokane, Minato-ku, Tokyo Kitasato Research (72) Inventor Hiroki Komiyama Kitasato Institute 5-9-1 Shirokane, Minato-ku, Tokyo

Claims (2)

[Claims] 1. A whitening agent comprising as an active ingredient an isonitrile antibiotic produced by filamentous fungi or a physiologically acceptable salt or derivative thereof when they have a carboxyl group. 2. The whitening agent according to claim 1, wherein the isonitrile antibiotic is a compound represented by the following formula (I): Formula (I) In the above formula, R ¹ and R ² together represent a single bond, R ³ represents a hydrogen atom, and R ⁴ and R ⁵ together represent the formula: And R ⁶ and R ⁸ together represent a —O— group,
And does R ⁷ and R ⁹ each represent a hydrogen atom; R ¹
And R ² together represent a single bond, R ³ represents a hydrogen atom, and R ⁴ is of the formula: Represents a group, R ⁵ represents a hydroxyl group, R ⁶ and R ⁸ represent together -O- group, and either R ⁷ and R ⁹ each represents a hydrogen atom; R ¹ and R ² together Represents a —O— group, R ³ represents a hydrogen atom, and R ⁴ represents a group of the formula Represents a group, R ⁵ represents a hydroxyl group, R ⁶ and R ⁸ represent together -O- group, and either R ⁷ and R ⁹ each represents a hydrogen atom; R ¹ and R ⁹ together Represents a single bond, R ² and R ³ each represent a hydrogen atom, and R ⁴ represents the formula: R ⁵ and R ⁶ together represent a single bond, and R ⁷ and R ⁸ each represent a hydrogen atom; R ¹ and R ⁹ together represent a single bond; ² and R ³ each represent a hydrogen atom, and R ⁴ is of the formula: And R ⁵ and R ⁶ together represent a —O— group,
And does R ⁷ and R ⁸ each represent a hydrogen atom; R ¹
And R ² together represent a single bond, R ³ represents a hydrogen atom, R ⁴ and R ⁵ together represent the formula: Or R ⁶ , R ⁷ , R ⁸ and R ⁹ each represent a hydrogen atom; or R ¹ and R ⁹ together are
Represents an O-group, R ² and R ³ each represent a hydrogen atom, and R ⁴ is of the formula: And R ⁵ and R ⁶ together represent a —O— group,
R ⁷ and R ⁸ each represent a hydrogen atom.