JPH06271595A - New melanogenesis-inhibiting substance, microbial strain producing the substance and its production - Google Patents

Abstract

PURPOSE:To obtain the subject new substance separable from a cultured product of a microbial strain belonging to the genus Streptomyces and capable of producing a melanogenesis-inhibiting substance, having excellent melanogenesis-inhibiting action, free from side effect and useful for pharmaceuticals, cosmetics, etc. CONSTITUTION:A microbial strain belonging to the genus Streptomyces and capable of producing OH-3984K1 having melanogenesis-inhibiting action [e.g. Streptomyces sp. OH-3984 (FERM P-13068)] is inoculated on a medium and cultured at 27 deg.C for 72hr under shaking to obtain a seed strain. The seed strain is transplanted to a medium in a jar fermenter and cultured under aeration and agitation. The cultured liquid is mixed with ethyl acetate, stirred and subjected to centrifugal separation to separate an extract liquid. The ethyl acetate layer is concentrated under reduced pressure, hexane is added to the residue and the hexane-insoluble fraction is subjected to chromatography to obtain the objective melanogenesis-inhibiting substance having an elemental composition of C18H26O4, a molecular weight of 306 and a specific rotation [alpha]p of -154.9 deg. (C=0.366, C2H5OH), scarcely soluble in water and soluble in acetone, ethyl ether, methanol, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel melanin pigmentation-inhibiting substance, a bacterium producing the same, and a method for producing the same. More specifically, it belongs to the genus Streptomyces, an antibiotic which suppresses melanin production in B16 mouse melanoma cells. The present invention relates to a producing bacterium and a manufacturing method thereof.

[0002]

2. Description of the Related Art Stain,
Freckles, pigmentation on the skin after sunburn, etc. are less likely to occur, increase or disappear with age, and are a cause of concern for middle-aged and older people. Although the pathogenic mechanism of these pigmentation diseases has not been clarified yet, it is considered that the melanin-synthesizing function of epidermal melanocytes is generally enhanced by the action of sunlight, especially ultraviolet rays and melanocyte-stimulating hormone.

Further, it is considered that the delay of keratinization of epidermal keratinocytes (keratinocytes) with aging causes an increase in the density of melanin granules in the epidermis, that is, a clinically symptomatic increase in pigmentation. These pigmented areas are localized and may cause an apparent difference from the surrounding normal skin color.

There is a demand for the development of a drug capable of restoring the pigment (ie, melanin) deposits thus acquired to the normal skin color, and many drugs have been commercialized so far. For example, in recent years, cosmetics containing a vitamin C (L-ascorbic acid) derivative having excellent reducing ability have been used. However, this has a difficulty in stability, and is hardly effective when applied externally. On the other hand, in Europe and America, hydroquinone is used as a medicine for treating spots and whitening black skin, but this also has a problem in the safety of the substance itself (irritation, allergenicity),
Further, there is a problem in compounding it as a drug in that it may cause vitiligo. In addition, various melanin inhibitors, for example, isoflavone derivatives (Japanese Patent Laid-Open Publication No. 5 (1999) -58242)
8-225004), p-hydroxycinnamic acid as a cinnamic acid derivative (JP-A-59-196813), p-
Hydroxycinnamic acid amide derivative (JP-A-62-5645)
No. 9) and the like are known.

However, a substance capable of effectively suppressing the production of melanin pigment and thus sufficiently improving pigmentation has not yet been known.

Therefore, it has been desired to develop a drug capable of effectively suppressing melanin pigment formation to prevent pigmentation and reducing side effects.

[0007]

[Means for Solving the Problems] As a result of intensive studies in view of such circumstances, the present inventors have found that the melanin production inhibitory action of B16 mouse melanoma cells in a culture of strain OH-3984 newly isolated from soil is suppressed. The inventors have found that the substances possessed by them are produced and have completed the present invention.

That is, first, the present invention provides OH-3984 K ₁ which is a melanin pigment formation inhibitor having the following physicochemical properties (1) to (8). (1) The elemental composition is C ₁₈ H ₂₆ O ₄ . (2) The molecular weight is 306. (3) Specific rotation [α] _D is −154.9 ° (C = 0.3
66, in ethanol). (4) The ultraviolet absorption spectrum (in methanol) shown in FIG. 1 is given. (5) The infrared absorption spectrum (in chloroform) shown in FIG. 2 is given. (6) It is sparingly soluble in water and soluble in acetone, ethyl acetate, ethyl ether, hexane, methanol, ethanol, chloroform and benzene. (7) The nuclear magnetic resonance spectra (in deuterated chloroform) shown in FIGS. 3 and 4 are given. (8) The color reaction to sulfuric acid and iodine is positive.

Secondly, the present invention provides a melanin pigment production inhibitor OH having the following physicochemical properties (1) to (8).
And it provides a -3 984 K _2. (1) The elemental composition is C ₁₈ H ₂₈ O ₄ . (2) The molecular weight is 308. (3) Specific optical rotation [α] _D is −167.9 ° (C = 0.9
27, in ethanol). (4) The ultraviolet absorption spectrum (in methanol) shown in FIG. 5 is given. (5) The infrared absorption spectrum (in chloroform) shown in FIG. 6 is given. (6) It is sparingly soluble in water and soluble in acetone, ethyl acetate, ethyl ether, hexane, methanol, ethanol, chloroform and benzene. (7) The nuclear magnetic resonance spectra (in deuterated chloroform) shown in FIGS. 7 and 8 are given. (8) The color reaction to sulfuric acid and iodine is positive.

Thirdly, the present invention belongs to the genus Streptomyces and has the above-mentioned OH-3984 K ₁ or OH-3984.
The present invention provides a strain capable of producing K ₂ .

Fourthly, the present invention comprises culturing the above-mentioned producing strain in a medium and adding OH-3984K ₁ or OH-398 to the culture.
4 K ₂ was accumulated, and the above-mentioned OH-398 was collected from the culture.
There is provided a method for producing OH-3984 K ₁ or OH-3984 K ₂ and collecting the 4K ₁ or OH-3984 K _2.

First, as long as it belongs to the genus Streptomyces as a bacterium producing the melanin pigmentation-inhibiting substances OH-3984 K ₁ and OH-3984 K ₂ and has the ability to produce the melanin pigmentation-inhibiting substance. Although not particularly limited, examples thereof include strain OH-3984 newly isolated by the present inventors.

The mycological properties of this strain OH-3984 are shown below. (A) Morphological properties Vegetative hyphae develop well on various agar media and fragmentation is not observed. Aerial mycelium abundantly grows on glucose / asparagine agar, starch / inorganic salt agar, etc., and exhibits a white color. When observed under a microscope, the aerial mycelium exhibited a spiral shape, and 2
A chain of 0 or more spores is recognized. The spore size is 0.8 × 1.1 μm and the shape is oval. The surface of spores is spiny. No sclerotia, sporangia and zoospores are found.

(B) Properties on various media EB Shirrin
g) and the method of D. Gottlieb (International Journal of Sististic Bacteriology, Vol. 16, 313,
Table 1 shows the culturing properties of this production strain measured according to (1966).
~ Shown in Table 3. The color tone is determined using the standard color as described in the Color Harmony Manual 4th edition (Container Corporation of America Chicago, 1958), and the code is also shown in parentheses along with the name of the color chart. did. The following are the results of observation in each medium at 27 ° C. for 2 weeks unless otherwise specified.

[0015]

[Table 1]

[0016]

[Table 2]

[0017]

[Table 3]

(C) Physiological properties (A) Formation of melanin pigment (a) Tyrosine agar positive (b) Peptone-yeast iron agar positive (c) Glucose-peptone-gelatin medium positive (21-23 ° C) (d) ) Tryptone yeast solution positive (B) tyrosinase reaction positive (C) hydrogen sulfide production positive (D) nitrate reduction negative (E) gelatin liquefaction (21 to 23 ° C) negative (glucose peptone gelatin medium) (F ) Starch hydrolysis positive (G) skim milk coagulation (37 ° C) negative (H) skim milk peptone (37 ° C) positive (I) growth temperature range 12 to 46 ° C (J) availability of carbon source ( Pre-Dam Gothrieve Agar) Use: glucose, arabinose, xylose, raffinose, melibiose, mannitol, rhamnose, boar Lumpur, sucrose slightly Use: fructose (K) degradation negative cellulose

(D) Cell wall composition Diaminopimelic acid in the cell wall is of the LL type.

From the above, the bacteriological characteristics of this bacterium are summarized as follows. Diaminopimelic acid in the cell wall is LL
It is a type. The aerial hyphae are spiral-shaped and have long spore chains. The surface of spores is spiny. As a property of culture, vegetative hyphae have an ivory color tone, and aerial hyphae have a white color tone. Soluble pigments produce melanin pigments in tyrosine agar, peptone / yeast extract agar, etc.

From these results, this strain is a strain belonging to the genus Streptomyces, and is classified into Pridham and Tresner (Vergis, Manual of Determinant Bacteriology, 8th Edition, 748 to 829,
1974) and is considered to belong to the white series. This strain is Streptomyces
SP OH-3984 ( Streptomyces
sp. OH-3984), which has been deposited with the Institute of Microbiology, Institute of Industrial Science and Technology [Ministry of Microbiological Research, No. 13068 (F
ERM P-13068)]].

As mentioned above, the antibiotics OH-3984 Ks (K
₁ and K ₂ , which may be referred to below) The production bacteria have been explained. However, the general characteristics of actinomycetes are such that the mycological characteristics are extremely variable, and they are naturally or commonly used for UV irradiation, It is a well-known fact that mutation is carried out by X-ray irradiation or an artificial mutagenesis method using a mutagenesis agent such as N-methyl-N-nitro-N-nitrosoguanidine and ethyl methanesulfonate. Therefore, all strains belonging to the genus Streptomyces and having the ability to produce the antibiotics OH-3984 K ₁ and K ₂ can be used in the present invention regardless of the above artificial mutants and natural mutants. .

The method for producing the antibiotics OH-3984 K ₁ and OH-3984 K ₂ using the above-mentioned producing bacterium of the present invention will be described below.

In the production method of the present invention, first, an antibiotic OH-3984 K type bacteria belonging to the genus Streptomyces is cultured in an appropriate medium. In culturing the bacterium, the usual culturing method for actinomycetes may be followed. As the medium, it is preferable to use a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and, if necessary, an inorganic salt. Assimilable carbon sources, glucose, molasses, starch, dextrin, cellulose, corn
Steep liquor, glycerin, organic acid and the like are used alone or in combination. As a digestible nitrogen source,
Peptone, meat extract, yeast extract, dry yeast, soybean powder,
Corn steep liquor, cottonseed meal, casein, soybean protein hydrolyzate, amino acids, organic nitrogen sources such as urea, nitrates,
Inorganic nitrogen compounds such as ammonium salts are used alone or in combination. In addition, if necessary, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, and phosphate may be added. In addition, the medium may contain growth of the bacterium or the antibiotic OH-3984 as necessary.
Micronutrients, growth promoting substances, precursors, etc. that promote the production of Ks may be appropriately added.

Culturing is usually carried out under aerobic conditions such as shaking or aeration culture medium. From the industrial point of view, deep aeration stirring culture is preferred. It is preferable to culture the medium at a pH of around neutral. The culturing temperature may be 20 to 37 ° C., but it is usually 24 to 30 ° C., preferably around 27 ° C.
As for the culture time, in the case of liquid culture, if the culture is usually carried out for 3 to 6 days, this antibiotic is produced and accumulated. Preferably, the culture may be terminated when the accumulated amount in the culture reaches the maximum. These culture composition, the liquidity of the medium, the culture temperature, the stirring speed, the culture conditions such as the aeration amount are appropriately adjusted to obtain preferable results depending on the type of the strain to be used and external conditions,
It goes without saying that it will be selected. When foaming occurs in liquid culture, a defoaming agent such as silicone oil, vegetable oil, or surfactant may be used as appropriate.

Since the antibiotics accumulated in the culture thus obtained are contained in the culture filtrate and the cultured cells, the culture may be treated with a filter aid such as Celite, if necessary. A high-flow super cell or the like is added to the mixture, which is then filtered or centrifuged to separate the culture filtrate and the microbial cells, and the mixture of the culture filtrate and the organic solvent extract of the microbial cells is concentrated to give the antibiotic OH-3984 K ₁ And it is advantageous to collect K ₂ .

Further, the culture filtrate and the bacterial cells can be directly extracted with the non-hydrophilic organic solvent without separation. Further, when the content of the antibiotics OH-3984 K is extremely high in either the culture filtrate or the bacterial cells, it is preferable to extract from the larger one. In order to isolate and purify the antibiotics OH-3984 K from the cultured cells, the culture filtrate and a concentrated product of the organic solvent extract of the cells or a mixture or a non-immersive solvent such as ethyl acetate or chloroform is usually used. The OH-3984 K antibiotics may be redissolved in an organic solvent by extraction with the like. The organic solvent layer thus obtained is dehydrated by adding various dehydrating agents such as anhydrous sodium sulfate and bead gel, if necessary, and then the organic solvent is distilled off under reduced pressure. In this concentration operation, the antibiotic OH-3984 was used.
Although Class K is a stable substance, the heating temperature is usually 50
It is preferable that the temperature is not higher than C. An organic solvent such as hexane or petroleum ether is added to the residue to prepare the antibiotic OH-39 of the present invention.
84 Ks can be precipitated.

The precipitate obtained is washed several times with hexane or the like, then suction filtered or centrifuged to give the antibiotic OH-3984.
A crude product of class K can be collected. In order to purify the above crude product, many differences in solubility between antibiotics OH-3984K and contaminants, difference in partition between two immiscible liquid phases, and difference in adsorption force for various adsorption carriers are used. Means can be used. In particular, chromatography is an effective method for purifying antibiotics OH-3984K. Chromatography effective for the purification of the antibiotics OH-3984 K, such as adsorption chromatography using silica gel, alumina, activated carbon cellulose, hydroxyapatite HP-20 and other adsorption resins, silanized silica gel, octadecyl silanized silica gel, etc. is used. Examples thereof include reverse phase partition chromatography, Sephadex LH-20, gel filtration chromatography based on molecular sieve using Toyopearl, ion exchange chromatography and the like.

The antibiotics OH-3984 K of the present invention can be obtained by using these chromatography, electrophoresis, countercurrent distribution, ultrafiltration, etc., individually or in combination or repeatedly in any order. , Can be separated and purified. For example, the above crude product is dissolved in a small amount of chloroform and adsorbed on silica gel, column chromatography is performed using a chloroform-acetone mixed solution, and the active fraction is concentrated under reduced pressure and then dissolved in a small amount of methanol, which is dissolved in methanol. Antibiotic OH-39 was obtained by performing gel filtration chromatography based on molecular sieves.
84 K ₁ and K ₂ can be separated and purified.

The antibiotic OH-of the present invention thus obtained
The physicochemical properties of 3984 K ₁ and OH-3984 K ₂ are as described above. Among the various physicochemical properties,
(1) Elemental composition is measured by high-resolution mass spectrum, and (2) molecular weight is fast atom bombardment (F
AB) Measured by mass spectrum.

[0031]

INDUSTRIAL APPLICABILITY The antibiotic of the present invention OH-3984 K ₁
And OH-3984 K ₂ exhibit remarkable melanin pigmentation-inhibiting activity, and are expected to be applied to a drug and / or cosmetics for preventing melanin pigmentation. Further, by using the above substance-producing bacterium of the present invention, high-purity OH-3984Ks can be easily produced on an industrial scale.

[0032]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

Example 1 2.0 wt% glucose (hereinafter simply referred to as%), peptone 0.5%, yeast extract 0.3%, meat extract 0.5%, sodium chloride 0.5 in a 500 ml Erlenmeyer flask.
%, Calcium carbonate 0.3% (pH 7.
0) 100 ml sterilized agar slope containing starch 1%, NZ amine 0.3%, yeast extract 0.1%, meat extract 0.1%, calcium carbonate 0.3%, agar 1.0%. One platinum loop was inoculated from the slant culture medium of Streptomyces OH-3984 (FERM P-13068) cultured on the culture medium at 27 ° C for 6 days, and shaken at 27 ° C for 72 hours on a rotary shaker rotating 220 rpm. After culturing, the seed mother was obtained. Next, in a 50 l jar fermenter, glucose 2.0%, peptone 0.5%, yeast extract 0.3%,
After sterilizing by adding 30 liters of a medium containing 0.5% of meat extract, 0.5% of sodium chloride and 0.3% of calcium carbonate,
600 ml of the seed seed obtained by the above method was aseptically transplanted, and agitated and cultured to obtain about 30 l of the seed seed. Ethyl acetate was added to this culture solution, and the mixture was sufficiently stirred, and then the ethyl acetate extract was separated by centrifugation. Hexane was added to the oily residue obtained by concentrating the ethyl acetate layer under reduced pressure to obtain a hexane-soluble fraction and a hexane-insoluble fraction. This hexane-insoluble fraction was filled with chloroform in advance and had an inner diameter of 30 m.
Adsorbed on a silica gel (70-230 mesh, manufactured by Merck) column having a length of m and a length of 390 mm, chloroform-
Chromatography was performed by eluting with an acetone mixed solvent. The obtained active fraction was concentrated under reduced pressure to obtain OH-3984.
87 mg of crude material I containing K ₁ was obtained.

The crude material I containing the antibiotic OH-3984 K ₁ was then applied to silica gel (230-400 mesh,
Merck) was subjected to column chromatography using a column previously packed with chloroform. A column having an inner diameter of 15 mm and a length of 130 mm was used, and the crude product I dissolved in a small amount of chloroform was passed through the column, which was developed and eluted with a mixed solvent of chloroform-acetone, and the antibiotic OH-3 was used.
60 mg of crude material II containing 984 K ₁ was obtained. This was dissolved in a small amount of methanol and subjected to high performance liquid chromatography (YMC-Pack D-ODS-5, φ20 × 250).
mm), and the active fraction was collected using an 80% methanol solution as a mobile phase to obtain the antibiotic OH-3984 K.
44 mg of a pure product of ₁ was obtained.

Further, a 100 l jar fermenter was newly charged with 70 l of the medium having the same composition as described above, and the mixture was cultured under aeration and stirring under the same conditions to obtain about 70 l of a culture seed. Ethyl acetate was added to this culture solution, and the mixture was sufficiently stirred, and then the ethyl acetate extract was separated by centrifugation. The oily residue obtained by concentrating the ethyl acetate layer under reduced pressure was preliminarily filled with chloroform, and the silica gel (70 to 2
It was adsorbed on a column of 30 mesh, manufactured by Merck & Co., Inc., and subjected to chromatography eluting with a chloroform-acetone mixed solvent. The obtained active fraction was concentrated under reduced pressure and OH-
320 mg of crude material III containing 3984 K ₂ were obtained.

Crude material III containing the antibiotic OH-3984 K ₂ was then subjected to column chromatography using a column pre-packed with Sephadex LH-20. A column with an inner diameter of 20 mm and a length of 780 mm was used as the column, and the crude purified product III dissolved in a small amount of methanol was passed through the column and developed and eluted with methanol.
268 mg of crude substance IV containing the antibiotic OH-3984 K _2.
Got This was dissolved in a small amount of methanol and subjected to high performance liquid chromatography (YMC-Pack D-ODS-
5, Φ20 × 250 mm) and the active fraction was fractionated using a 70% methanol solution as a mobile phase to obtain 175 mg of a pure product of the antibiotic OH-3984 K ₂ .

Test Example 1 Antibiotics of the present invention OH-3984 K ₁ and OH-39
To examine the inhibitory activity of 84 K ₂ for melanin pigment formation,
Each substance was allowed to act on B16 melanoma (malignant melanoma), and the minimum inhibitory concentration (MIC) was measured. As a result, the MIC of OH-3984K ₁ was 7.5 μg / ml, while the MIC of OH-3984 K ₂ was 3.8 μg / ml. From these results, it can be seen that the antibiotics of the present invention have extremely good melanin pigment formation inhibitory activity.

[Brief description of drawings]

FIG. 1 is a drawing showing an ultraviolet absorption spectrum (in methanol) of OH-3984 K ₁ of the present invention.

FIG. 2 is a drawing showing an infrared absorption spectrum (in chloroform) of OH-3984 K ₁ of the present invention.

FIG. 3 is a drawing showing a ¹ H ⁺ nuclear magnetic resonance spectrum of OH-3984 K ₁ of the present invention (in deuterated chloroform, 300 MHz).

FIG. 4 is a drawing showing the ¹³ C nuclear magnetic resonance spectrum of OH-3984 K ₁ of the present invention (in deuterated chloroform, 75 MHz).

FIG. 5 is a drawing showing an ultraviolet absorption spectrum (in methanol) of OH-3984 K ₂ of the present invention.

FIG. 6 is a drawing showing an infrared absorption spectrum (in chloroform) of OH-3984 K ₂ of the present invention.

FIG. 7 is a drawing showing a ¹ H nuclear magnetic resonance spectrum of OH-3984 K ₂ of the present invention (270 MHz in deuterated chloroform).

FIG. 8 is a drawing showing a ¹³ C nuclear magnetic resonance spectrum of OH-3984 K ₂ of the present invention (67.8 MHz in deuterated chloroform).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical display location (C12P 7/00 C12R 1: 465) (72) Inventor Masahiko Hayashi 5-9 Shirokane, Minato-ku, Tokyo No. 1 Inside Kitasato Research Center (72) Inventor Satoshi Takamatsu 5-9-1 Shirokane, Minato-ku, Tokyo Inside Kitasato Research Center (72) Inventor Yukihiro Yada 1-1-1 Hisudada Nishi, Ninomiyacho, Haga-gun, Tochigi Prefecture (72) Inventor Genji Imokawa 1022-89 Himurocho, Utsunomiya City, Tochigi Prefecture

Claims (4)

[Claims] 1. A melanin pigment production inhibitor OH-3984 K ₁ having the following physicochemical properties (1) to (8). (1) The elemental composition is C ₁₈ H ₂₆ O ₄ . (2) The molecular weight is 306. (3) Specific rotation [α] _D is −154.9 ° (C = 0.3
66, in ethanol). (4) The ultraviolet absorption spectrum (in methanol) shown in FIG. 1 is given. (5) The infrared absorption spectrum (in chloroform) shown in FIG. 2 is given. (6) It is sparingly soluble in water and soluble in acetone, ethyl acetate, ethyl ether, hexane, methanol, ethanol, chloroform and benzene. (7) The nuclear magnetic resonance spectra (in deuterated chloroform) shown in FIGS. 3 and 4 are given. (8) The color reaction to sulfuric acid and iodine is positive. 2. A melanin pigment formation inhibitor OH-3984 having the following physicochemical properties (1) to (8):
K ₂ . (1) The elemental composition is C ₁₈ H ₂₈ O ₄ . (2) The molecular weight is 308. (3) Specific optical rotation [α] _D is −167.9 ° (C = 0.9
27, in ethanol). (4) The ultraviolet absorption spectrum (in methanol) shown in FIG. 5 is given. (5) The infrared absorption spectrum (in chloroform) shown in FIG. 6 is given. (6) It is sparingly soluble in water and soluble in acetone, ethyl acetate, ethyl ether, hexane, methanol, ethanol, chloroform and benzene. (7) The nuclear magnetic resonance spectra (in deuterated chloroform) shown in FIGS. 7 and 8 are given. (8) The color reaction to sulfuric acid and iodine is positive. 3. The method according to claim 1, which belongs to the genus Streptomyces.
OH-3984 K ₁ or OH- according to claim 2.
A strain capable of producing 3984 K ₂ . 4. The strain according to claim 3 is cultivated, and the melanin pigment production inhibitor accumulated in the culture is collected, and OH-3984K _{1 according to} claim ₁ or claim 2. process for the preparation of OH-3984 K _2.