JP2844088B2 - Tyrosinase activity inhibitor - Google Patents

Description

The present invention relates to a novel tyrosinase activity inhibitor derived from a natural product and having high safety.

<Background of the Related Art and the Invention> It is considered that pigment (melanin) is deeply involved in the darkening of skin (skin). In other words, it is considered that melanin is produced in the skin tissue of the skin under external stimuli such as ultraviolet rays, which promotes darkening of the skin and causes various symptoms such as spots, freckles, and black and white.

There are several mechanisms of action for preventing skin whitening or darkening, and one of them is proposed to suppress the activity of tyrosinase (enzyme) involved in the production of melanin.

Conventionally, glutathione, for example, is known to be effective as a known substance capable of suppressing the activity of tyrosinase. However, when glutathione is exposed to wet conditions, glutathione is easily oxidized and easily induces discoloration and odor. there were. In addition, tyrosinase activity inhibitors derived from chemicals (chemically synthesized products) have problems in safety and the like, and various restrictions are imposed on their uses such as pharmaceuticals and cosmetics. From such a viewpoint, development of a tyrosinase activity inhibitor derived from a natural product has been demanded. However, tyrosinase activity inhibitors present in natural products are:
Each of them has various advantages in safety such as little irritation, but has a problem that the tyrosinase activity inhibitor is low. Therefore, it is a substance derived from natural products,
The discovery and development of substances with high tyrosinase activity inhibitors and excellent stability, safety, storage stability, etc. are desired.

By the way, as a tyrosinase activity inhibitor derived from a natural product, its presence has been reported in extracts of crude drugs, wind and beach windbreaks, and agaricus (Agaricus bisporous). (JP-B-59-48808) Further, the inventors have previously reported that water or / and organic solvents from various fruiting bodies (mushrooms) of Ningyo-Tame, Shogenji, Hon-Shimeji, Usumurasakihatsu, Usumurasakihokutaketake and Maitake mushroom were used. "Cosmetic" based on the discovery that the extract exerts various other effects such as tyrosinase activity inhibitory effect
Filed a patent application for (Japanese Patent Application No. 1-125216) However, as long as other natural indigenous fruiting bodies such as the above-mentioned gingko mushrooms are used, there are various restrictions in the sampling area, sampling time, and the like, and they can be extracted from natural indigenous fruiting bodies. There is a problem that the quality of the tyrosinase activity-inhibiting substance itself is not uniform, such as the potency and content, and there are various problems that the production amount and the yield are limited, and thus they are not suitable for mass production. Further, a substance (such as an extract) containing such a tyrosinase activity inhibitor effect derived from a natural product
No tyrosinase activity inhibitor itself has been purified or isolated, and its chemical structure has not been elucidated. Therefore, there is a high possibility that the conventional tyrosinase activity inhibitor derived from natural products contains impurities, and the efficiency of inhibiting tyrosinase activity is reduced.

<Problems to be Solved by the Invention> The present invention aims to solve the above-mentioned problems and meet the above-mentioned needs.

That is, in order to search and develop a useful tyrosinase activity inhibitor which is a natural product or a substance derived from a natural product, which is safe with little irritation and which is safe,
The search for the presence or absence of a tyrosinase activity inhibitor has been performed for various extracts of various natural products, especially various mushrooms (bearing bodies) (Japanese Patent Application No. 1-125216). Obtaining a tyrosinase activity inhibitor,
Further, in order to obtain a tyrosinase activity inhibitor that can be stably supplied, the present invention has been completed by succeeding in purifying and isolating the tyrosinase activity inhibitor of the fruit body of Amanita mushroom, and determining the chemical structure. As a result, the tyrosinase activity inhibitor (hereinafter, referred to as “compound I”) of the Amanita mushroom was 5-farnesyl-6-methyl-resorcinol (5-farnesyl-6-methyl-resocinol).
rcinol) [alias: neogrifolin /
Or compound name by international nomenclature: E, E-5-methyl-4
-(3,7,11-trimethyl-2,6,10-dodecatrienyl)
-1,3-benzenediol {E, E-5-methyl-4- (3,
7,11-trimethy1-2,6,10-dodecatrienyl) -1,3-ben
zen-diol｝]. The structure of neoglycolin was confirmed by various physicochemical properties, but all of them have been reported in the literature [(1) Phytochemistry 16 , 1409 (1977) / (2) Che.
m. Pharm. Bull., 36 , 2918 (1988)].

In addition, the literature [(1) Phytochemistry 16 , 1409 (19)
77), / (2) Chem. Pharm. Bull., 36 , 2918 (1988)]
All mention only the property that the neoglypholine and its isomer, glypholine, are antibiotics, and a substance having an inhibitory effect on tyrosinase activity derived from an extract from the fruit body of Amanita mushroom for the first time by the present inventors. Is neoglypholine.

<Means for Solving the Problems> In order to achieve the above object, in the present invention, "5-
Farnesyl-6-methyl-resorcinol (5-farn
A tyrosinase activity inhibitor comprising, as an active ingredient, esyl-6-methyl-resorcinol) (alias: neogrifolin). ].

The separation, purification, and isolation of the compound neogrifolin (hereinafter, referred to as “compound I”) will be described.

Preparation of crude extract from raw materials: As a natural product for extraction, gingko mushroom [polyporus
Fruiting body (mushroom) of confluence (Polyporus confluens Fr.) (hereinafter simply referred to as "ginger mushroom")
Use As a raw material for the extraction of the Aging mushroom, a living body (mushroom) of a fruit body (mushroom) or a dried product can be used, and the fruit body (mushroom) is dried at room temperature or in the sun for about 2 to 10 days. It is preferable to use one in which rot is prevented.

In the extraction of the active ingredient, the raw material of the fruit material of Aingyotake is crushed or pulverized and extracted using water / organic solvent as a solvent for extraction. The extraction solvent can be used singly among the water and the organic solvent, or a mixed solution of two or more people can be used.

Examples of the organic solvent that can be used include C 1-22 such as methanol, ethanol and isopropanol.
Linear monohydric alcohols and / or side chain monohydric alcohols, ketones such as acetone, various esters such as ethyl acetate and ethyl esters of fatty acids, and halogenated alkanes such as dichloromethane (mainly chlorides); Various polar organic solvents and / or various non-polar organic solvents, such as various ethers such as diethyl ether, and other aromatic hydrocarbons such as benzene and toluene, can be used. Above all, the monohydric alcohols have 1 carbon atoms such as methanol, ethanol, isopropanol and the like.
Lower alcohols, such as diethyl ether for the ethers, acetone, ethyl methyl ketone, isopropylmethyl keto for the ketones, ethyl acetate for the esters, and dichloromethane, chloroform, dichloroethane for the halogenated alkanes. Among the aromatic hydrocarbons, organic solvents having a large polarity or organic solvents having a medium polarity such as benzene are particularly preferable. The solvent used for extracting the active ingredient is not particularly limited, but a volatile solvent is preferred from the viewpoint of the operation of purification and isolation after extraction of compound I according to the present invention.

As the extraction conditions, the extraction solvent was added to the crushed material / fine powder of fruiting bodies (mushrooms) (living cells versus <live cells> or / and dried cells), and the mixture was heated on a boiling water bath under reflux cooling. The extraction for about 1 hour each time is repeated 3 to 5 times, all the extracts are combined, the extract is dehydrated, and then the solvent is distilled off (preferably under reduced pressure) to obtain a crude extract.

Separation / purification / isolation method of a substance having a tyrosinase activity inhibitory effect (compound I: neogrifolin) from the crude extract: Separation / fractionation of a tyrosinase activity inhibitory substance of the crude extract obtained from the fruiting body of Amanita bamboo Purification can be performed by various known chromatography, for example, various column chromatography (hereinafter referred to as “CC”), high-performance liquid chromatography (High-Performance Liquid C).
hromatography (hereinafter referred to as “HPLC”)) and the like. At this time, in the separation and fractionation by various chromatography, the presence or absence of the tyrosinase activity inhibitory effect and the elution category of the active ingredient are confirmed by monitoring and measuring the tyrosinase activity inhibitor ratio in each fraction solution in parallel.

The tyrosinase activity inhibition rate is measured by the following method. That is, if necessary, each sample for the various separation / fractionation liquids of the crude extract of the fruit body of the Aingyotake mushroom is, for example, 30%
1,3-butylene glycol aqueous solution (hereinafter simply referred to as “30% 1,3-
BG solution. An appropriate solvent (e.g., the same applies hereinafter) or the like at an appropriate concentration (for example, a concentration of about 0.1 to 10%, etc.
Is adjusted to a concentration within a range where the absorbance of the sample shows an appropriate value).

On the other hand, L-tyrosine solution (concentration: 0.3
mg / ml) and McIlvain Buffer
er Solution) (pH 6.8), 0.9 ml each of the “test solution for measurement” and the 30% 1,3-BG solution for blank test was added to each of these test tubes, and this was added to each test tube. ° C
Incubate for 10 minutes in a water bath.

0.1 ml of a tyrosinase solution (concentration: 1 mg / ml in McKilvain buffer) was added to the incubated cells, and the mixture was stirred well. Immediately after that, each reaction solution was set on a spectrophotometer.
Is measured over time. (As the absorbance value at each measurement point, the suffix ₀ is added to the absorbance value immediately after the addition of the tyrosinase solution, and the suffix x is added to the absorbance value after X minutes of incubation after the addition.) The value is substituted into the following equation to calculate the tyrosinase activity inhibition rate (%). In the calculation of the tyrosinase activity inhibition rate of the present invention, the absorbance value 10 minutes after the introduction of the reaction solution (x = 10) is used.

B ₀ : Absorbance value at 0 minutes after blank solution Bx: Absorbance value at X minutes after blank solution A ₀ : Absorbance value at 0 minutes after test solution Ax: Absorbance value at X minutes after test solution The value is measured by the amount of dopachrome (a precursor of melanin) produced.

Then, the purity of the target fraction (i.e., the fraction containing Compound I) obtained by eluting and fractionating the crude extract by various column chromatography or / and high performance liquid chromatography is obtained. In order to determine whether or not the substance is a substance, various types of chromatography, for example, thin-layer chromatography (hereinafter referred to as “TLC”), gas chromatography (Gas-Liquid Chromatogr
aphy (hereinafter referred to as "GLC")) and the like.

The structure of the tyrosinase activity inhibitor (compound I) derived from the fruit body of Amanita bamboo, which has been confirmed as a single substance as a result of the separation and purification, is determined by the following known method.

First, the molecular weight of the tyrosinase activity inhibitor derived from the fruit body of Amanita bamboo was determined by the high molecular mass spectrum (Mass
Spectrum (hereinafter referred to as “MS”)). Various known analytical techniques can be used to measure the physicochemical properties required for determining the chemical structure of the tyrosinase activity inhibitor derived from the fruit body of the Amanita mushroom. That is, the ultraviolet absorption spectrum (Ultraviolet Spectrum <hereinafter “U
V ”)), Infrared Spec
trum (hereinafter referred to as “IR”), Proton Nuclear Magnetic Resonance Spec
tra (hereinafter referred to as “ ¹ H-NMR”), carbon-13 nuclear magnetic resonance spectrum (Carbon-13 Nuclear Magnetic Resonan)
ce Spectra (hereinafter referred to as " ^13C -NMR")) and the like can be used.

The inventors have extracted, separated, purified, and isolated a substance having an inhibitory effect on tyrosinase activity from the fruiting body of Amanita bamboo and determined its chemical structural formula.

As will be described and analyzed in detail in the examples described below, data relating to extraction, separation, purification, and isolation of a substance having an inhibitory effect on tyrosinase activity from the fruiting body of Amanita bamboo and the determination of its chemical structure, and the tyrosinase activity inhibitor (compound I) An overview of the chemical substances, physicochemical properties, and biochemical properties of is described.

As a raw material for extraction, the fruit body of Amanita bamboo is collected from those naturally grown in the natural world, and the dried or viable cells (live cells) are crushed or pulverized. Ethyl acetate is added to the crushed and pulverized fruit body of Amanita mushroom, and extraction is repeated three times for 1 hour each time under a reflux condition on a boiling water bath, and all the extracts are combined to obtain an extract. After dehydration of this extract, the solvent for extraction (ethyl acetate) is distilled off under reduced pressure to obtain a crude extract of the fruit body of Aingyotake mushroom.

The crude extract of the body of the fruiting body of Amanita bamboo is subjected to alumina column chromatography (eluting solvent system: benzene-ethyl acetate-methanol system) and silica gel chromatography (eluting solvent system: benzene-ethyl acetate system) to elute the tyrosinase activity. Separate the fractionation parts. Thereafter, the eluted fraction is separated and fractionated by high performance liquid chromatography (HPLC), and a fraction having an inhibitory effect on tyrosinase activity is fractionated. The substance contained in the fraction thus obtained is an isolated tyrosinase activity inhibitor (compound I) showing one spot on thin layer chromatography (TLC) and one peak on gas chromatography (GLC). . This compound I is an odorless syrup-like substance which is colorless or faint yellow in appearance.

 The physicochemical properties of the compound I will be described.

The high-resolution mass spectrum (MS) of Compound I is as shown in FIG. 1, and the experimental value of the molecular weight is 328.2422, from which the molecular formula C ₂₂ H ₃₂ O ₂ (calculated molecular weight = 328.24
01).

The ultraviolet absorption spectrum of Compound I is as shown in FIG. 2, and the maximum absorption wavelength λ _max when the dissolving solvent is methanol is 206 nm and 282 nm. The molecular extinction coefficient E (log ε) at 282 nm is 3.59.

The infrared absorption spectrum (IR) of the compound I is as shown in FIG. 3, and the wave number of the hydroxy group (OH group) is 34.
00cm ^-1 and fractions 1610 cm ^-1 indicating the benzene ring, 1600 cm ^-1,
Absorption is observed at 1510 cm ^-1 . On the other hand, the infrared absorption spectrum of the acetylated compound of Compound I is as shown in FIG. 4 and shows a fraction of 1775 cm ⁻¹ , 1200 c
m ^-1 and 1620 cm ^-1 indicating the benzene ring, 1590cm ^-1, 1490cm
^-1 is observed, whereas the absorption at a wave number of 3400 cm ^-1 indicating the hydroxy group (OH group) observed in the compound I disappears.

Proton nuclear magnetic resonance spectrum of compound I ( ¹ H-NM
R) and carbon-13 nuclear magnetic resonance spectrum ( ^13C -NM)
The results of (R) are as shown in Table 1 below. The ¹ H-NMR spectrum of Compound I is as shown in FIG.

The following can be analyzed based on Table 1 and FIG. That is, from the results of ¹ H-NMR, compound I has (a) a methyl group (-CH) attached to a total of four double bonds at 1.57 ppm (6H), 1.67 ppm (3H), and 1.78 ppm (3H). ₃ ), (b) 1.95 to 2.10 ppm (8H), methylene group (-CH
_{2 -), (c) 2.21ppm} (3H) methyl groups attached to the benzene ring _{(-CH 3), (d)} 3.27ppm (2H) methylene group sandwiched double bond (-CH ₂ -), (E) 5.04 to 5.11 ppm (4H) with 3 protons of olefin [methine group (-CH =)] and 1 proton of hydroxy group (OH group); (f) 6.20 ppm (1H), 6.25 ppm In (1H), protons of the benzene ring are observed.

According to the results of ¹³ C-NMR, compound I contained (g) 16.0 ppm, 16.2 ppm, 17.6 ppm, 25.7 ppm, and 20.0 ppm in total of 5 carbons of methyl group (—CH ₃ ), (h) 25.0 ppm , 26.4ppm, 26.7ppm, 39.6ppm a total of four methylene group (-CH ₂ -) carbon atoms of, (i) 101.0ppm, 109.7ppm, 122.0ppm, 123.7ppm, 124.4ppm
(J) 116.0ppm, 138.5ppm, 155.2ppm, 154.2ppm, 131.3pp
A total of 7 (> C <) carbons are found at m, 135.4 ppm, and 137.3 ppm, respectively.

When the chemical structure of Compound I is analyzed and analyzed in detail based on such physicochemical properties of Compound I, the molecular formula is C ₂₂ H ₃₂ O ₂ from the data of high-resolution mass spectrum (MS). From the nuclear spectra of IR and NMR, the hydroxyl group (OH
Group) and a benzene ring, and the IR spectrum (1775 cm ⁻¹ , 1
200 cm ^-1 ) and ¹ H-NMR spectrum (about 2.3 ppm
According to the present invention, the compound has two phenol acetates. From the ¹ H-NMR and ¹³ C-NMR nuclear spectra of Compound I, it is confirmed that one methyl group (about 2.2 ppm) and a farnesyl group bonded to the benzene ring are present. Is suggested.

Therefore, compound I has a farnesyl group (C
It is presumed that the compound has one substituted ₁₅ H ₂₅ -group, one methyl group (-CH ₃ group) and two hydroxy groups (-OH group).

On the other hand, from the ¹ H-NMR spectrum, the proton of the benzene ring of the compound I according to the present invention showed 6.20 ppm and 6.25 ppm
It appears at pm and is meta-coupled to each other at J = 2.5 Hz. This signal is down-shifted to 6.7 ppm and 6.8 ppm due to the acetylation of Compound I.

Comprehensively judging from such data, the chemical structural formula of compound I according to the present invention is as follows: 5-Farnesyl-6-methyl-resorcinol (5-
farnesyl-6-methyl-resorcinol) [alias: neogrifolin / or compound name by international nomenclature: E, E-5-methyl-4- (3,7,
11-trimethyl-2,6,10-dodecatrienyl) -1,3-
Benzenediol ｛E, E-5-methyl-4- (3,7,11-t
rimethy1-2,6,10-dodecatrienyl) -1,3-benzen-di
ol｝].

The chemical structure of compound I and the physicochemical properties of compound I are described in the above-mentioned literature [(1) Phytochemistry 16 , 1409 (1977) / (2) Che.
m. Pharm. Bull., 36 , 2918 (1988)].

<Action> The biochemical properties of compound I (neoglyfoline) according to the present invention will be described.

The compound I (neoglyphorin) according to the present invention is
As is clear from the process of separation and purification from the raw material of the fruit body of Amanita bamboo, it has a significant tyrosinase activity inhibitory action as a biochemical property. In addition, Compound I showed a negative result in a sensitization test and the like, indicating that the compound I was excellent in safety.

As the biochemical properties of the compound I, the degree of the tyrosinase activity inhibitory effect can be determined by measuring the amount of dopachrome (a precursor of melanin) as described in detail in (a) above and (b) It is measured by measuring the melanocyte production rate by the method of culturing melanocytes using “clone M-3 cells (derived from mouse melanoma)” that produces melanin. Table 2 below shows an example of the measurement results of the amount of dopachrome (precursor of melanin) produced in the above (a) among the measurement results of the tyrosinase activity inhibitory action / effect of the compound I (neoglypholine) according to the present invention. Show. According to the result example of Table 2, the concentration of Compound I was 10 pp.
m solution has a tyrosinase activity inhibitory effect of 56.7%,
A 50 ppm solution of Compound I suggests a remarkable biochemical effect of having a 100% tyrosinase activity inhibitory effect.

Further, the test method and the measurement result by the above-mentioned (b) “clone M-3 cell (derived from mouse melanoma)” culture method will be described in detail in the Examples section below.

In addition, the melanin production inhibitory rate of the compound I according to the present invention is as shown in Table 3 below based on the measurement results obtained by the above-mentioned (b) “clone M-3 cell (derived from mouse melanoma)” culture method. In comparison with the “negative control group” to which no compound I was added (the amount of compound I added: 0.00 mg), the amount of compound I added: 0.005 mg, 0.025 mg, and 0.05 mg in each test group 52% to 66 melanin production
%.

A sensitization test was performed on the safety of compound I (neoglyphorin) according to the present invention. As a result, the concentration of Compound I was negative at 1000 ppm, demonstrating that Compound I according to the present invention is extremely safe.

<Example> An example of the present invention will be described below, but it is needless to say that the present invention is not limited to this example.

I) The raw material of the material Aingyotake.

As the raw material of the fruiting body of the Amanita bamboo, those naturally occurring in nature were collected. The fruiting body of the Amanita bamboo shoot used in the present invention was collected in October 1988 in the mountains of Toyohira-cho, Yamagata-gun, and Chiyoda-cho, Hiroshima-prefecture. ) Or air-dried cells for 3 to 10 days at room temperature.

II) Extraction / purification / isolation of compound I from raw materials.

(1) Preparation of a crude extract from fruiting bodies of A. mushrooms: A live cell (100 g) of a fruiting body of A. mushroom or a dried cell (20 g) of a fruiting body of A. mushroom is finely crushed or powdered. Extraction was performed three times for 1 hour with 100 ml of an extraction solvent (ethyl acetate) under boiling water and reflux cooling. The extracts were combined, dehydrated with anhydrous sodium sulfate, and then, if possible, the solvent for extraction (ethyl acetate) was distilled off under reduced pressure to obtain a crude extract. The crude extract (1.3 g) was obtained from the viable cells (100 g).
g, 2.1 g of the crude extract was obtained from the dried cells (20 g).

(2) Fractionation / Purification / Isolation of Compound I from Crude Extract: Fractionation by Alumina Column Chromatography: Column Conditions [(20 mmφ × 155 mm-L), I
Use CN Adsorbentien 4580 / 50g]. As an elution solvent, a system of benzene: ethyl acetate: methanol: acetic acid (benzene 100% → stepwise mixture of benzene + ethyl acetate → ethyl acetate 100% → stepwise mixture of ethyl acetate + methanol → methanol 100% → methanol + Acetic acid). Then, a part of the eluate is always eluted and fractionated carefully by measuring the amount of dopachrome produced for each elution section and confirming and monitoring the tyrosinase activity inhibition rate of the elution section to obtain the target tyrosinase activity. Separate the elution category that has the inhibitory effect. As a category having a tyrosinase activity inhibitory action according to the present invention, a category of an elution solvent of ethyl acetate / methanol = 50/50 was fractionated.

Fractionation by silica gel column chromatography: The tyrosinase activity inhibitor (applied amount = 0.7 g) of the alumina column chromatography fraction collected above was
The reaction was performed under column conditions (20 mmφ × 250 mm-L), using silica gel / 20 g as a column carrier. As an elution solvent,
Benzene: dichloromethane: ethyl acetate system (benzene
100% → stepwise mixture of benzene + dichloromethane → stepwise mixture of benzene + ethyl acetate). Then, as in the column chromatography described above, for a part of the eluate, the amount of dopachrome produced is measured for each elution section at all times, and the tyrosinase activity inhibition rate of the elution section is checked and monitored, and the elution and fractionation are performed carefully. Then, an elution fraction having an intended tyrosinase activity inhibitory action is collected. As the category having the tyrosinase activity inhibitory action according to the present invention, the category of the elution solvent of benzene / ethyl acetate = 99.0 / 1.0 was fractionated.

Purification by high performance liquid chromatography: The sample collected above is further purified by high performance liquid chromatography. The column conditions used were: column: Shim-pack PREP-ODS (2.0 cmφ × 250 mm), eluent: methanol / water = 95/5, and the target tyrosinase activity inhibitor elution category (elution including compound I) Classification)
In the preparative experiment, the tyrosinase activity inhibition rate was measured for each of the elution sections in the same manner as in the above and above, and data on the section (peak) from which the tyrosinase activity inhibitor was eluted was collected in advance. Then, the eluate fraction of the target tyrosinase activity inhibitor (that is, containing compound I) was fractionated. The solvent in this eluate was concentrated under reduced pressure, transferred to n-hexane, and the insolubles were collected to obtain a colorless to slightly yellow syrup-like odorless compound I.

Confirmation of isolation of compound I: (a) Confirmation by thin-layer chromatography (TLC): Isolation of a tyrosinase activity inhibitor (compound I) fractionated and purified by the above-mentioned (2) or each of the following operations
Confirmed by silica gel TLC.

That is, thin-layer carrier: silica gel 70-plate Wako (commercially available / prepared product / manufactured by Wako Pure Chemical Industries, Ltd.) Developing solvent: benzene / ethyl acetate = 3/1 Color developing solution: 10% sulfuric acid (ethanol solution) As a result of TLC, a single spot was detected near Rf ≒ 0.6.

(B) Confirmation by Gas Chromatography (GLC): Isolation of the tyrosinase activity inhibitory substance (compound I) fractionated and purified by the above-mentioned (2) or each of the following operations was carried out.
It was also confirmed by GLC.

Column: 3.0mmφ × 2.6m Filler carrier: Chromosolve (pickled, treated with dimethylchlorosilane, 60-80 mesh) Column filler: 1.5% silicon OV-1 Carrier gas: Nitrogen, 50ml / min Column temperature: 150 ℃ → 250 ℃ (8 ℃ / min) → 250 ℃ Constant at 250 ℃ Inlet and detector temperature: 280 ℃ Detector: FID (hydrogen flame ionization detector) The GLC result under the above conditions is Rt Detected a single peak at about 14-16 minutes.

Thus, a single compound (Compound I) could be isolated on TLC and GLC.

III) Measurement of physicochemical properties of Compound I and Compound I
Determination of the chemical structural formula.

(1) High-resolution mass spectrum (MS) of compound I: FIG. 1 shows a high-resolution mass spectrum (MS) of compound I.
FIG. From the results of the MS, it is found that the molecular weight (experimental value) of the compound I according to the present invention is 328.242, the molecular formula is C ₂₂ H ₃₂ O ₂ , and the calculated molecular weight is 328.2401.

(2) Example of ultraviolet absorption spectrum (UV) of compound I: FIG. 2 shows an ultraviolet absorption spectrum (UV) of compound I.
FIG. As is clear from FIG.
Is the maximum absorption λmax = 206 nm in a methanol solvent.
And λmax = 282 nm, and the molecular extinction coefficient E (logε) at 282 nm is found to be 3.59.

(3) Example of infrared absorption spectrum (IR) of compound I: FIG. 3 shows an infrared absorption spectrum (IR) of compound I.
FIG. 4 is an example, and FIG. 4 is an example of an infrared absorption spectrum (IR diagram) of compound I of the acetylated compound of compound I.

The presence of a benzene ring and a hydroxy group (OH group) was confirmed from the IR diagram of compound I in FIG.
From the IR diagram of the acetylated product, the presence of two phenol acetates was confirmed.

(4) Examples of proton nuclear magnetic resonance spectrum ( ¹ H-NMR) and carbon-13 nuclear magnetic resonance spectrum ( ¹³ C-NMR) of compound I and an acetylated product of compound I: Table 1 shows compound I and compound I results examples of the proton nuclear magnetic resonance spectrum of the acetylated compound ⁽¹ H-NRM) Yobi carbon-13-nuclear magnetic resonance spectrum ⁽¹³ C-NMR) is. FIG. 5 is an example of a ¹ H-NMR diagram of Compound I.

Based on the various physicochemical properties described above, Compound I
-Farnesyl-6-methyl-resorcinol (5-fa
rnesyl-6-methyl-resorcinol) [alias: neogrifolin / or compound name according to international nomenclature: E, E-5-methyl-4- (3,7,11-trimethyl-2,
6,10-dodecatrienyl) -1,3-benzenediol {E, E-5-methyl-4- (3,7,11-trimethy1-2,6,10
-Dodecatrienyl) -1,3-benzen-diol {], suggesting that the chemical structural formula is as follows.

Chemical structure of compound I: IV) Biochemical properties of compound I: (1) Example of measurement result of compound I tyrosinase activity inhibition rate by measuring the amount of dopachrome produced.

The tyrosinase activity inhibitory effect of compound I is measured by the method described above. Compound I was prepared in a 30% 1,3-BG solution at a predetermined concentration (47% when measuring the tyrosinase activity inhibitory effect).
(The concentration suitable for showing an appropriate absorbance value at 5 nm).

To measure the tyrosinase activity inhibition rate of Compound I, each absorbance was measured after 10 minutes, and the tyrosinase activity inhibition rate (%) at each concentration of Compound I was calculated by the following formula. The results of the tyrosinase activity inhibition rate of each extract were
It is shown in the table.

B ₀ : Absorbance value at 0 minutes after blank solution B ₁₀ : Absorbance value at 10 minutes after blank solution A ₀ : Absorbance value at 0 minute after test solution A ₁₀ : Absorbance value at 10 minutes after test solution From the above results, Compound I exhibited a tyrosinase activity inhibition rate of 56.7% at a concentration of 10 ppm and a tyrosinase activity inhibition rate of 100% at a concentration of 50 ppm. Compound I has a very remarkable tyrosinase activity inhibitory effect. Suggest that.

(2) Test example of measuring the melanin production inhibition rate of compound I by measuring the amount of melanocytes produced by clone M-3 cells derived from mouse black tumor.

Preparation of sample and culture medium: (a) Preparation of sample of compound I: 100 g of water was added to 1 g of dried fruit body of Amanita bamboo, heated for 1 hour on a boiling water bath under reflux conditions, cooled, and filtered. Then, an aqueous extract of fruiting bodies of A. niger was prepared. 90 ml of ethanol was added to 10 ml of this water extract and mixed (90% ethanol solution), and the resulting precipitate (mainly polysaccharide) was collected. About 8 ml of distilled water was added to the precipitate to dissolve it. To this was added 0.5 mg of Compound I dissolved in a small amount of ethanol, and the ethanol was evaporated under reduced pressure (rotary evaporator).
Was used as a sample.

As a negative control (blank), a compound to which the compound I in the sample preparation step was not added was used.

(B) Test cell culture: "Clone M-3 cells" derived from melanin-producing mouse melanoma were used as test culture cells.

(C) Medium used (medium composition): • Ham's F-10: 87.5 wt% (Flow Laboratories) • Fetal bovine serum (FBS): 2.5 wt% • Horse serum (Horse Serum) 1) 15.0wt% medium was used.

Culturing experiment method: (a) Clone M-3 cells were uniformly dispersed in a medium to a concentration of 1 × 10 ⁶ cells / ml, and this was placed in a 12-well petri dish (culture area: about 4.52 cm ² / well). After dispensing ml, the medium was added to each well in an amount of 1.9 ml, and clone M-3 cells were cultured at 37 ° C for 1 day.

(B) After culturing, discard all medium, and then add 1m of medium
l and the test amounts (0.1 ml, 0.5 ml, 1.0 ml) of the sample (compound I sample and blank sample) were added, and the total volume was 2 ml each in phosphate buffered saline (PBS).
(PH = 7.2) was added and cultured at 37 ° C. for 2 days.

(C) After the culture, the same operation as in the above (b) was performed, and the cells were further cultured at 37 ° C. for 2 days.

Method for measuring the amount of melanin and the amount of protein: (a) 1 ml of a 0.25% trypsin solution is added to each well of the above-mentioned petri dish, and after leaving it for about 30 seconds, the trypsin solution is discarded.

(B) About 5 minutes after the treatment of (a), a phosphate buffer (PB
S) The cells were peeled off with 1 ml (pH = 7.2), dispersed, and further treated with ultrasonic waves (20 KHz, 20 seconds) to uniformly pulverize the cultured cells.

(C) In order to quantify the amount of melanin produced, a wavelength of 40
The absorbance at 0 nm was measured.

(D) In order to quantify the amount of the produced protein, color was developed with a color developing agent “Protein Assay” (manufactured by Bio-Rad) and the absorbance at a wavelength of 595 nm was measured.

Since the number of cells and the amount of protein are correlated,
By measuring the amount of protein instead of the number of cells, the rate of production of melanin per unit of protein was measured. Thus, in this experimental method, the rate of inhibition of melanin production of Compound I was measured.

Table 3 shows an example of measurement test results of the melanin production inhibitory rate of compound I by measuring the melanocyte production rate produced by clone M-3 cells derived from mouse black tumor. The numerical values indicate the absorbance at each wavelength, and the values indicate the average of three values.

V) Safety test of compound I: (1) Sensitization test: Test animals: Hartley white guinea pig males weighing 300 g were subjected to the test.

Number of animals: 10 animals per group.

Rearing conditions: Three cages are housed in stainless steel cages (350 × 420 × 200 mm) in a rearing room set at a temperature of 22 ± 2 ° C. and a humidity of 60 ± 10%, and a sample (CLEA Japan CG-3) and tap water Was freely fed and bred.

Test sample: A solution of Compound I at a concentration of 100 ppm in a 1,3-BG solution was used as a test substance stock solution, and the test solution stock solution was used as a sample.

Test method: Performed as follows according to the Maximization method.

A) The scapula of the animal was shaved to 4 × 6 cm, and the next intradermal injection was performed at the shaved site.

(A) Adjuvant ^{* 1} 0.1 ml (0.05 ml left and right 2 places) (b) Sample 0.1 ml (0.05 ml left and right 2 places) (c) Sample adjuvant ^{* 1} 0.1 ml (0.05 ml
B) Six days after the intradermal injection, the hair at the same site was shaved, and 10% sodium lauryl sulfate ^{* 2} was applied.

C) On the 7th day, the sample was applied to a filter paper of 2 × 4 cm, the filter paper was applied to the same site, and a closed application was performed for 48 hours.

D) Two weeks after application of the closure, the sides of the body are shaved to 5 x 5 cm,
The sample was applied to a circular filter paper having a radius of 2 cm, the filter paper was applied to the same site, and the sample was closed and adhered for 24 hours.

E) The judgment was made 24 hours after the removal of the adhesive.

Judgment criteria: No response ... 0 Mild or sporadic erythema ... 1 Moderate erythema ... 2 Severe erythema and edema ... 3 Note) ^{* 1} = Freund's complete adjuvant (manufactured by Wako Pure Chemical Industries) ^{* 2} = 10% sodium lauryl sulfate in white petrolatum
What was dispersed so that it might become weight%.

<References: “New skin physiology and safety” Seiji Shoin
Issue> Results: Compound I sensitization test results are 1000 ppm
At no concentration was "no response" (negative) referred to in the above criteria.

This result suggested that Compound I was an extremely safe compound.

<Effect of the Invention> Compound I (neoglyphorin), which has been extracted, separated, purified, and isolated from the fruiting body of the Aingyotake mushroom according to the present invention and whose chemical structure has been clarified, has a remarkable tyrosinase activity inhibitory effect, In addition, the compound I has high safety. In addition, since the chemical structure of the tyrosinase activity inhibitor derived from the fruiting body of A. mushroom is determined by the present invention, the way of chemically synthesizing the same substance as the tyrosinase activity inhibitor derived from a natural product (derived from the fruiting body of A. mushroom) is opened up. A tyrosinase activity inhibitor with uniform and uniform quality, high tyrosinase activity, and excellent safe properties can be stably mass-produced. It has remarkable effects to achieve the object of the invention, such as being applicable to whitening cosmetics that are safe and have a high tyrosinase activity inhibition rate.

[Brief description of the drawings]

FIG. 1 shows a compound I according to the present invention, which was extracted, separated, purified, isolated and determined for its chemical structure from the fruiting body of Amanita bamboo.
FIG. 2 is an ultraviolet absorption spectrum of the compound I according to the present invention, FIG. 3 is an infrared absorption spectrum of the compound I according to the present invention, and FIG. FIG. 5 shows an infrared absorption spectrum of the acetylated compound of Compound I according to the present invention, and FIG. 5 shows a proton nuclear magnetic resonance spectrum of Compound I according to the present invention.

Continued on the front page (72) Inventor Hisao Uehara 1-17-117 Ebie, Fukushima-ku, Osaka-shi, Osaka (72) Inventor Hiroshi Tanaka 43-1, Midoricho, Neyagawa-shi, Osaka (58) Field surveyed (Int.Cl . ^6, DB name) A61K 31/05 A61K 7/00 CA (STN ) REGISTRY (STN) WPIDS (STN)

Claims (1)

(57) [Claims] A tyrosinase activity inhibitor comprising 5-farnesyl-6-methyl-resorcinol (alias: neogrifolin) as an active ingredient.