JPH07242687A - Compound having melanin-formation inhibiting activity - Google Patents

Abstract

PURPOSE:To obtain the subject compound for a cosmetic, an agrochemical, a pharmaceutical, etc., having melanin-formation-inhibiting activity against a melanin-producing actinomyces and a melanoma cell and mushroom tyrosinase- inhibiting activity by culturing a specific strain of Trichoderma. CONSTITUTION:Trichoderma 74N7 strain (FERM-P-14050) is inoculated on a medium and this is cultured at 28 deg.C for 7 days under stirring at 120-150rpm to obtain a seed strain. The obtained seed strain is transplanted in a jar- fermenter and this is cultured at 28 deg.C for T day under stirring at 120rpm. The obtained fermentation broth is filtered and the filtrate is extracted with n-butyl acetate. The solvent is removed from the organic phase by a reduced pressure distillation. The dried product is dissolved in a solvent and purified through a silica gel column to finally obtain the objective product. The product comprises an isonitrile compound having a maximum absorption at 208nm in UV spectroscopy (30% methanol solution). This compound has melanin- formation-inhibiting activity against a melanin-producing actinomyces and mushroom tyrosinase-inhibiting activity and useful for a beautifying agent, an agrochemical, an insecticide, a melanoma-curing agent, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a melanin production inhibitory compound and its use as a whitening agent, a pesticide, an insecticide and a therapeutic agent for melanoma. More specifically, this compound is Trichoderma 7
It can be obtained by culturing the 4N7 strain in a nutrient medium.

[0002]

BACKGROUND OF THE INVENTION Japanese Patent Application Laid-Open No. 2-145189 discloses a Trichoderma h.
arzianum ) 3422 strain produces a substance exhibiting inhibitory activity against mushroom tyrosinase. It has been shown that this substance has substantially no antibacterial activity against several kinds of bacteria and is particularly effective as a whitening agent for cosmetics.

[0003] However, as described in that publication, in order for a specific substance to exert the action effect as a whitening agent, the production inhibiting or decolorizing action of melanin pigment which causes coloring of human skin is in vivo. It needs to be effective. Considering various biochemical reaction mechanisms involved in the production process of the melanin pigment, it is obvious that a compound having a strong tyrosinase inhibitory activity should be provided, and a compound which may exhibit various biochemical actions. Would also be desired.

Therefore, the object of the present invention is to show a tyrosinase inhibitory activity of a different type from that described in the above publication,
It is to provide a compound that inhibits the production of melanin.

[0005]

DETAILED DESCRIPTION OF THE INVENTION The present inventors have found that Streptomyces bikiniensis ( Streptmyces) , which is a melanin-producing actinomycete.
omyces bikiniensis ) NRRL B
Method for screening melanin production inhibitor using -1049 (K. Tomita et al., J. Antibiotic)
s, 43 , 12, 1601-1605, 1990).
According to the above, various soil fungi have been screened from the viewpoint that a compound different from that disclosed in JP-A-2-145189 may be available.

As a result, they have found that a strain belonging to the genus Trichoderma produces a compound having both a strong melanin production inhibitory activity and a strong tyrosinase inhibitory activity, and arrived at the present invention.

Therefore, the above object is according to the present invention:
A melanogenesis-inhibiting active compound that can be produced by Trichoderma strain 74N7,
(A) The main absorption bands (cm ⁻¹ ) by IR spectroscopy are about 3427, 2926, 211.4, 1736.7, 16
31.1, 1452.8, 1380.6, 1314.1, 1
122.4, 1066.7, 1021.4, 968.9, 8
67.2, 620.4, 493.2, 472.1,
(B) The main absorption bands (δ, ppm, external standard TSP) by the ¹ H-NMR method (in solvent D ₂ O, 400 MHz) are
About 1.38 (3H, d), 1.52 (3H, d), 2.9
8 (1H, d-t) to 3.43 (1H, d to t), 2.9
8 (1H, d to t) to 4.17 (1H, d to t), 6.6
6 (1H, t), (C) UV absorption (30% methanol) has a maximum absorption at 208 nm, and (D) FAB-MS peak (m / z) is (M +).
H) ⁺ for 439, 630.7; (M−H) ⁻ for 375, 437, 567, 721, and (E)
Thin layer chromatography (Kieselgel 60F)
₂₅₄ , manufactured by Merck & Co .; developing solvent: chloroform / methanol (10/1)) to provide a compound having an Rf value of 0.37.

According to the present invention, there is also provided a whitening agent containing the above compound as an active ingredient.

From the above-mentioned various spectral data, it is considered that the compound is an isonitrile (RN-C) compound, and a known isonitrile antibiotic (for example,
It is presumed to be an analog of JP-A-53-15345).

The above compound belongs to the genus Trichoderma and is isolated from soil in the semi-arid region of Pucarpa, Peru, South America,
The strain designated by the present inventors as strain 74N7 can be cultured in a nutrient medium and isolated from the culture by an appropriate separation / purification means.

This 74N7 strain is characterized by its mycological properties, which will be described later, and A REVISION OF TH from Rifai.
Since it is judged to be a strain belonging to the genus Trichoderma when collated with the items described in E GENUS TRICHORDERMA, 1969, etc., it was named Trichoderma 74N4 as described above. In addition, this strain was deposited at the Institute of Biotechnology, Institute of Industrial Science on January 11, 1994, and was designated as FERM P-
Commissioned at 14050.

The bacteriological properties of this deposited strain are shown below.

1. Morphological properties (1) Potato dextrose agar medium Growth on potato dextrose agar medium (Kyokuto Pharmaceutical Co., Ltd.) is fast. The colonies grow rapidly, initially appearing white in color and forming sparse mats of mycelium. Immediately, the mycelium of wool extends and covers the surface of the colony, and rises as it approaches the center. A conidial group is formed from the center of the colony, and the color is initially light green. Then it becomes bright green,
It turns dark green at the end. Conidia stalk branches from aerial mycelium,
Furthermore, it produces a large number of side branches and has a dendritic appearance. Therefore, the colony looks like a wheel-like, somewhat sparse lawn-like colony. Conidia are spherical or spheroidal, light green and have a diameter of 4.
It is 0 to 5.0 μm × 4.5 to 5.0 μm, and the conidium wall surface is smooth.

The surface of the colony is white with no pigment production.

(2) Malt extract agar medium Growth on malt extract agar medium is fast.

The aerial mycelium extends about 8 mm long, covers the front surface of the medium loosely, and presents a white fluffy colony. Conidia stalks are produced from aerial hyphae, and spore stalks are branched like wheels. A short infarct occurs at the tip of the infarct, and conidia are produced in a lump form from the tip of each infarct, giving a tree-like appearance. The conidia are spherical or spheroidal, green and have a diameter of 3.5-4.5 μm × 4.5-5.5 μm,
Conidia wall surface is smooth.

Therefore, when conidia are settled, a ring-shaped green conidiospore group is formed in a ring shape at the center of the colony.

The back of the colony is white with no pigment production.

2. Growth conditions pH: Grows well in a wide range of pH 2 to pH 9. In particular, it grows very well at pH 3 to pH 6 and prefers the acidic side.

Temperature: Grow well at 20 ° C to 30 ° C. 1
The growth at 5 ° C is somewhat good, and the growth at 10 ° C is weak.

On the other hand, at 37 ° C, the growth is somewhat good, but 45
No growth occurs at ℃.

The above strain can accumulate the compound of the present invention in the culture by culturing in a nutrient medium in which the strain belonging to the genus Trichoderma can be usually cultured. As the nutrient medium, xylose, arabinose,
About 2 to 10 carbon sources such as glucose, mannose, fructose, galactose, sucrose, cellobiose, trehalose, maltose, lactose or starch.
% (Wt%, the same below), ammonium sulfate, polypeptone, sodium nitrate, baker's yeast extract, brewer's yeast extract or potato extract, such as a nitrogen source of about 0.1 to 1%,
Magnesium source such as magnesium sulfate about 0.01 to 0.0.
05%, phosphorus and potassium sources such as potassium monohydrogen phosphate and potassium dihydrogen phosphate 0.01 to 0.1%, other inorganic salts such as ferric sulfate, ferric chloride and sodium chloride about 0.001 Media containing ~ 0.005% can be used.

In the present invention, the strain is inoculated into a medium,
It is preferable to carry out under aerobic conditions in which an oxygen-containing gas such as air is introduced by means such as aeration. Also, culture is 1
It is suitable to carry out at a temperature of 0 to 40 ° C., preferably 20 to 35 ° C. for 2 to 7 days.

To collect the desired compound from the culture thus obtained, the culture is centrifuged or filtered to remove the cells, and the culture supernatant or filtrate is then mixed with an organic solvent immiscible with water, such as acetic acid. Extraction treatment with n-butyl etc., the solvent was distilled off from the organic phase, the residue was subjected to adsorption chromatography on silica gel activated carbon, etc., and gel filtration treatment was carried out if necessary.
High-performance liquid chromatography treatment for fractionation may be performed.

The compound of the present invention thus isolated is
It has the activity of strongly inhibiting the melanin production of Streptomyces bikiniensis, and also has the activity of strongly inhibiting the tyrosinase produced by mushrooms. Further, a composition comprising this compound as an active ingredient,
It can be used as a whitening agent, other pesticides, insecticides and melanoma therapeutic agents. Hereinafter, the formulation will be specifically described by taking a whitening agent as an example, but those skilled in the art can easily infer the composition of other agents by referring to the following description.

The whitening agent of the present invention may contain various components usually used in cosmetics, pharmaceuticals and the like, in addition to the above-mentioned compound in a predetermined amount depending on its dosage form. As these components, for example, oils, ultraviolet absorbers, antioxidants, surfactants, moisturizers, fragrances, water, alcohols, thickeners, coloring materials, skin nutrients (tocopherol acetate, pantotenyl ethyl ether, Glycyrrhizinate) and the like.

The dosage form of the present invention is arbitrary and may be, for example, a solubilizing system such as lotion, an emulsifying system such as emulsion or cream, or an ointment or dispersion liquid. The whitening agent of the present invention, the above compound, based on the total composition weight,
It can be included in an amount of about 0.01 to 1.0% by weight, preferably 0.05 to 0.1% by weight. Thus, there is provided a whitening agent which can prevent a desired whitening effect, for example, the generation of stains on the face and the like, and can be used for decolorizing already generated stains. When the compound of the present invention is used as a pesticide, an insecticide or a therapeutic agent for melanoma, the dose may be adjusted appropriately.

[0028]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited by these.

Example 1 (1) Culture Trichoderma ( Trichod ) stored in PD agar medium
erma ) 74N7 strain (FERM P-14050)
While maintaining the agar of 1 cm x 1 cm, add 500 mL of CD medium (Zapeck Dox medium, pH 6.0) to 1000 m.
The seed culture was obtained by inoculating the medium that had been sterilized and cooled in an L-baffled Erlenmeyer flask and cultivated at 28 ° C. and 120 to 150 rpm for 7 days.

Then, after sterilizing 20 L of the CD medium using a 50 L jar fermenter, 74N7 which was seed-cultured
(500 mL PD agar medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) / 10
00mL Erlenmeyer flask, 74N7 1cm × 1cm agar inoculation, 28 °, 120rpm, 3 days culture) 500m
L, inoculated, 28 °, 120 rpm, 0.5 vvm, 7
Cultured for a day.

The composition of the above medium is as follows.

[0032] (2) Purification The fermentation broth obtained in (1) is a filter aid (perlite).
And filtered to obtain 10 L (pH 5.03) of filtrate.
The filtrate was extracted with 10 L of n-butyl acetate. The solvent was distilled off under reduced pressure from the organic phase, and then the dried solid was dissolved or dispersed in chloroform. Silica gel (Wako gel C-2
Sample (about 200 mg) was loaded onto a column (10 mm × 300 mm) filled with 00) and chloroform / methanol was 100/0 (fraction I), 200/1 (fraction II), 50/1 (fraction III) 10/1.
(Fraction IV), 0/100 (fraction V)
After elution with 100 ml of each of the eluents, the weight of each fraction and the melanin production inhibitory activity were measured. The activity was found in fraction III.

Using the active fraction after silica gel chromatography as a sample, Capcell Pak C ₁₈
(Shiseido, 4.6 x 250 mm) 40% using column
Elution with methanol was carried out to collect the active fraction.
Furthermore, the same column was used to elute with 30% methanol and the activity peak was collected. Detection was carried out at UV 220 nm.

In this way, a white powdery compound of the present invention was obtained. This compound is FTS- manufactured by Bio-Rad.
IR absorption spectrum with a 40-type IR spectrophotometer and ¹ with a JNM-EX400-type NMR measuring instrument manufactured by JEOL Ltd.
When the 1 H-NMR absorption spectrum was measured, it showed the above main absorption band.

This compound showed excellent activity in each test of Example 2 below.

Example 2 (1) Melanin production inhibitory activity test The above purification steps were monitored by the following melanin production inhibitory activity test using actinomycetes.

As an indicator bacterium, Streptomyces bikiniensis ( Strepto) , which is a melanin-producing actinomycete, is used.
myces bikiniensis ) JCM 401
1 (hereinafter abbreviated as S. bikiniensis) was used to carry out a melanin production inhibitory activity test.

By the method of Tomita et al. (Supra), S.
Bikiniensis storage slant (Papav
iza's YDYA agar slant), and cultured at 25 ° C. for 1 week. Fully grown S. bi
The cells were suspended in sterilized water using kiniensis (1 platinum loop / mL). 0.2 mL of the bacterial solution was added to the following tyrosine agar medium containing 0.1 mg / l of copper sulfate (CuSO ₄ .5H ₂ O), spread with a Conradi stick and dried. Place a paper disc (diameter 8 mm) on a petri dish, soak the sample in an amount of 50 μL, and incubate at 25 ° C. for 2 days. Then, determine the diameter of the white band around the paper disc (diameter 8 mm) resulting from the inhibition of melanin production. It was measured. A sample having a white band with a diameter of 10 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.

Composition of Tyrosine Agar Medium Glycerin 15.0 g L-Tyrosine 0.5 g L-Asparagine 1.0 g K ₂ HPO ₄ 0.5 g MgSO ₄ 0.5 g NaCl 0.5 g FeSO ₄ .7H ₂ O 0.01 g Yeast extract 2.0g trace salt solution _{1mL FeSO 4 · 7H 2 O 0.1g} MnCl 2 · 4H 2 O 0.1g ZnSO 4 · 7H 2 O 0.1g distilled water 100mL agar _{15~20g CuSO 4 · 5H 2 O 0} . 1 mg Distilled water 1000 mL pH 7.2 to 7.4 (2) Preparation of tyrosinase inhibitory activity test reagent Tyrosinase solution (500 Unit / mL) Mushroom tyrosinase (2000 Unit / m
g: SIGMA) 10 mg 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 Na ₂
Mix 49.1 mL of HPO ₄ to make 100 mL.

Equipment used Titer Tech Twin Reader Plus (Type38
1: Flow Laboratories, Inc.) Test method 0.1M phosphate buffer (pH 6.) in each well of a 96-well plate.
8) Dispense 100 μL each and add 20 μL of sample solution
Added. Next, 30 μL of each tyrosinase solution is added, the absorbance at 492 nm is measured with a twin reader, and the value of the absorbance at 0 hour of reaction is defined as T ₀ . Then, 50 μL of L-tyrosine solution is added to each well and reacted 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, 0.1M phosphate buffer (pH 6.8) instead of the sample solution
20 μL is used, and the absorbance at 0 hour of the reaction is defined as C ₀ . Similarly, incubate at 37 ° C for 60 minutes, and after 60 minutes, 4
The absorbance at 92 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 (3) Melanin production inhibition test of B16 melanoma cultured cells (i) cells and Culturing method Mouse-derived B16 melanoma cultured cells were used. B
16 melanoma cultured cells are cultured petri dishes (diameter 35 m
10% FBS (fetal bovine serum: Gener)
al Scientific Laboratorie
s Ltd.) and, CO ₂ in theophylline (0.09 mg / mL) Eagle MEM culture containing (manufactured by Nissui Pharmaceutical)
In an incubator (95% air, 5% carbon dioxide),
It was cultured under the condition of 37 ° C.

(Ii) Addition of sample 100,000 cells were implanted in a 35 mm petri dish and cultured 24
After a lapse of time, the medium was replaced with a medium containing 20 μL of the sample, and the culture was further continued for 3 days. Four days after cell implantation (three days after drug addition), the cells were peeled off by the following method to measure the number of cells, and then the amount of melanin was measured.

(Iii) Cell number measurement method The cell number was measured by adding 0.6 mL of 0.25% trypsin / PBS (Takara Shuzo) solution and 0.02% EDTA / PBS.
Remove cells from petri dish with 0.6 ml of solution, 10%
Stop the action of trypsin by adding 0.8 ml of FBS-added Eagle MEM medium to make a cell suspension of 500,000 to 1,000,000 cells / mL, measure the number of cells with a hemocytometer, and calculate the number of cells per dish. did.

(Iv) Method for measuring the amount of melanin in cells The amount of melanin in cells was measured according to the method of Cilchrest et al. Using a microplate reader (Bio-RA).
D: MODEL 3550). (Ii
After the cell number was measured by the method of i), the cell number was adjusted to 1,000,000, centrifuged at 1000 rpm for 10 minutes, and stored in a freezer. When measuring the amount of melanin, the cells that had been frozen and stored were thawed, air-dried, 0.4 mL of a 1N NaOH solution was added, and the mixture was boiled at 100 ° C. for 30 minutes to dissolve it, and the absorbance at 475 nm was measured. From the above results, the cell growth rate in the sample addition system, the melanin production rate per 1 million cells, and the melanin production rate per dish (the number of cells per dish and Calculated from the amount of melanin per unit).

Example 3 (Preparation) Creamy whitening cosmetic composition weight% (1) Stearyl alcohol 4.0 (2) Stearic acid 5.0 (3) Isopropyl myrstate 18.0 (4) Glycerin monostearate 3 0.0 (5) Propylene glycol 10.0 (6) Compound obtained in Example 1 0.1 (7) Sodium hydroxide 0.2 (8) Sodium hydrogen sulfite 0.01 (9) 2,4-Dihydroxybenzophenone 0.1 (10) perfume Appropriate amount (11) the compound obtained was added with purified water balance and sodium hydroxide propylene glycol preparation purified water and example 1, and heated after stirring kept at 70 ° C. (aqueous phase). Other components are mixed by stirring and heated to 70 ℃
Keep (oil phase). The oil phase is gradually added to the water phase, and after the addition is completed, the temperature is kept for a while to cause the reaction. Then, it was uniformly emulsified with a homogenizer and cooled to 30 ° C. with thorough stirring to obtain a cream whitening cosmetic composition.

The creamy whitening cosmetic composition had an excellent whitening effect.

[0049]

INDUSTRIAL APPLICABILITY According to the present invention, a compound having a strong melanin production inhibitory activity of actinomycetes and a mushroom tyrosinase inhibitory activity can be provided. In view of such inhibitory activity, the compound of the present invention can be used as an active ingredient of a whitening agent, a pesticide, an insecticide and a therapeutic agent for melanoma.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // (C12P 1/02 C12R 1: 885) (72) Inventor Shinya Yamamoto Kohoku Ward, Yokohama City, Kanagawa Prefecture 1050 Shinba-cho Shiseido Daiichi Research Center, a stock company

Claims (2)

[Claims] 1. Trichoderm
a ) a melanin production inhibitory compound that can be produced by strain 74N7, which has a major absorption band (c) by (A) IR spectroscopy
m ^-1 ) is about 3427, 2926, 2114.4, 17
36.7, 1631.1, 1452.8, 1380.6, 1
314.1, 1122.4, 1066.7, 1021.4,
968.9, 867.2, 620.4, 493.2, 47
2.1, (B) ¹ H-NMR method (in solvent D ₂ O, 40
The main absorption band (δ, ppm, external reference TSP) by 0 MHz) is about 1.38, 1.52, 2.98 to 3.43,
2.98-4.17, 3.59, 3.75, 6.47, 6.6
6, the absorption maximum by (C) UV spectroscopy (30% methanol) is at 208 nm, and (D) FAB-MS.
The peak (m / z) by the method is (M + H) ⁺ , 4
39, 630.7; for (M-H) ^- , 375, 43.
7, 567, 721, and (E) thin layer chromatography (Kieselgel 60F ₂₅₄ , manufactured by Merck Ltd.); developing solvent: chloroform / methanol (10 /
The compound having an Rf value of 0.37 in 1)). 2. A whitening agent comprising the compound according to claim 1 as an active ingredient.