JPH03109319A - Tyrosinase activity inhibitor - Google Patents

Abstract

PURPOSE:To obtain a tyrosinase activity inhibitor, containing 5-farnesyl-6-methyl- resorcinol as an active ingredient, having a high tyrosinase activity inhibition rate, applicable to beautifying and whitening cosmetics, etc., and having high safety. CONSTITUTION:An inhibitor containing 5-farnesyl-6-methyl-resorcinol (another name; neogrifolin) as an active ingredient. The aforementioned compound is obtained by using a fruit body (mushroom) of 'NINGYO-TAKE' [Polyporus confluens Fr. (hereinafter simply referred to as 'NINGYO-TAKE')] as a natural product for extraction. Although a living body (in an intact raw state) or dried substance of the fruit body can be utilized as raw materials for extracting the 'NINGYO-TAKE', a fruit body dried at ambient temperature or in the sun about 2-10 days and preventing putrefaction is preferably used.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a novel rosinase activity inhibitor derived from natural products and highly safe.

<Prior Art and Background of the Invention> It is believed that the pigment melanin is deeply involved in the darkening of the skin (skin 1jl). That is, it is thought that melanin is produced in the skin tissues of the skin in response to external stimuli such as ultraviolet rays, which promotes the darkening of the skin and causes various symptoms such as age spots, freckles, and dark skin.

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

Conventionally, glutathione, for example, is considered to be effective as a known substance capable of suppressing tyrosinase activity. However, when glutathione is exposed to humid conditions, it is easily oxidized and easily causes discoloration and odor, which can be a fatal problem, especially when added to cosmetics. there were. Furthermore, tyrosinase activity inhibitors derived from chemicals (chemically synthesized products) etc. have safety issues, and various restrictions arise regarding their use in pharmaceuticals, cosmetics, etc. From this point of view, there is a demand for the development of tyrosinase activity inhibitors derived from natural products. However, although all of the tyrosinase activity inhibitors present in natural products have various advantages in terms of safety, such as low irritation, they have the problem of a low tyrosinase activity inhibition rate. Therefore, it is desired to discover and develop a substance derived from natural products that has a high tyrosinase activity inhibition rate and has excellent stability, safety, and preservability.

By the way, tyrosinase activity inhibitors derived from natural products have been conventionally used in herbal medicines, Kafu, Hamafu, and Tsukuritake mushrooms.
Its presence has been reported in extracts of Agaricus, bisporous). (Tokuko Showa 59-4880
No. 4) In addition, the inventors have previously discovered that extracts from the seed bodies (mushrooms) of Ningyotake, Shogenji, Honshimeji, Usmurasakihatsu, Usmurasakibōkitake, and Maitake using water and/or organic solvents suppress tyrosinase activity. Based on the discovery that it has various other effects, we filed a patent application for a ``cosmetic''. (
(Japanese Patent Application No. 1-125216) However, as long as fruiting bodies such as the above-mentioned Ningyotake and other naturally occurring fruiting bodies are used, there are various restrictions in terms of the area and timing of collection, and it is difficult to extract fruiting bodies from naturally occurring fruiting bodies. There is a problem that the quality of the tyrosinase activity inhibitor itself such as titer and content is not uniform, and there are also various problems that it is not suitable for mass production, such as there are limits to its production amount and yield. Furthermore, with respect to such substances (extracts, etc.) that have a tyrosinase activity inhibitory effect derived from natural products, the tyrosinase activity inhibitor itself has not been purified or isolated, much less its chemical structure has been clarified. Not yet. Therefore, there is a high possibility that conventional tyrosinase activity inhibitors derived from natural products contain impurities.

There is a problem that the efficiency of suppressing tyrosinase activity is also reduced.

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

That is, in order to search for and develop a beneficial tyrosinase activity inhibitor that is a natural product or a substance derived from a natural product and is less irritating, safe, and stable, the inventors selected a variety of natural products. Although various extracts of various mushrooms (fruiting bodies) have been searched for the presence or absence of tyrosinase activity inhibitors (Patent Application No. 1-125216), we have taken this research one step further to obtain even more efficient tyrosinase activity inhibitors. In addition, in order to obtain a tyrosinase activity inhibitor that can be stably supplied, the present invention was completed by successfully purifying and isolating a tyrosinase activity inhibitor from the fruiting body of N. chinensis and determining its chemical structure. As a result, the substance that inhibits the tyrosinase activity of P. chinensis (hereinafter referred to as "compound ■") was found to be 5-farnesyl-6-methyl-resorcinol
et'hyl-resorcinol) CAlternative name:
Neogrifalin /
Or compound name according to international nomenclature: B, B-5-methyl-4-(3,7,11-trimethyl-2,6,10-dodecatrienyl)-1,3-benzenediol (B,
B-5-methyl-4-(3,7,1l-tri
Methyl-2,6,1O-dodecatr 1e
ny 1) -1,3-1161ze1-dial)
] was confirmed. The structure of neogrifolin was confirmed by various physicochemical properties, but
All of the documents have already been reported [Q) Phytoc
hemistry 16.1409 (1977)
/(2) Chem, Pharm, Bull, 3
6.2918 (1988), etc.).

In addition, the above-mentioned document [(1) Phytochemistry
16.1409 (1977), /(2) Chem
, Pharm, Bull, 36.2918
(1988)], all of which only touch on the properties of neogrifolin and its isomer griffolin as antibiotics, and the inventors were the first to describe tyrosinase derived from an extract from the fruiting body of N. It has been confirmed that the substance that has an activity-suppressing effect is neogrifolin.

Means for Solving the Problems> In order to achieve the above objects, the present invention provides 5-farnesyl-6-methyl-resorcinol (5-farnesyl-6-methyl-resorcinol).
nesyl-6-methyl-resorcinol
) CAlso known as: neogrifolin
A tyrosinase activity inhibitor containing lin) ] as an active ingredient. ”.

Preparation of crude extract from raw materials to explain the separation, purification, and isolation of the compound neogrifolin (hereinafter referred to as "Compound I")! ! I: As a natural product for extraction, we use Polyporus confluence (Polyporus confluence).
nsPr, ) (hereinafter simply referred to as "Ningyoutake")]
Use the fruiting bodies (mushrooms) of The raw material for extracting this mushroom can be the living (raw) or dried fruiting body (mushroom), but the fruiting body (mushroom) is dried at room temperature or in the sun for about 2 to 10 days. It is also preferable to use it in a state where it has been protected from spoilage.

When extracting the active ingredient, the fruiting body of the Physcomitrium nigra as the raw material is crushed or finely powdered, and extracted using water and/or an organic solvent as an extraction solvent. The extraction solvent can be used alone among the water and/or organic solvents,
A mixture of two or more can also be used.

Examples of the organic solvents that can be used include methanol,
Straight chain monohydric alcohols and/or side chain monohydric alcohols with 1 to 22 carbon atoms such as ethanol and isopropanol, ketones such as acetone, various esters such as ethyl acetate and fatty acid ethyl ester, dichloromethane, etc. halogenated alkanes (mainly chlorides etc.),
Various polar organic solvents and/or non-polar organic solvents such as various ethers such as diethyl ether, aromatic hydrocarbons such as benzene and toluene, etc. can be used. Among these monohydric alcohols, methanol,
Lower alcohols having 1 to 3 carbon atoms such as ethanol and isopropanol; ethers such as diethyl ether; ketones such as acetone, ethyl methyl ketone, and isopropyl methyl ketone; esters such as ethyl acetate; Particularly suitable are organic solvents having high polarity or medium polarity, such as dichloromethane, chloroform, dichloroethane, etc. for alkanes, and benzene, etc. for the aromatic hydrocarbons. Although there are no particular restrictions on the solvent used to extract the active ingredient, volatile solvents are preferred as extraction conditions from the viewpoint of purification and isolation processing operations after extraction of Compound I according to this invention. The extraction solvent is added to the crushed/fine powder of fruiting bodies (mushrooms) (live fungal cells and/or dried fungal cells), and the mixture is heated on a boiling water bath and cooled under reflux for about 1 hour each time. Repeat the extraction for 3 to 5 times, combine all the extracts, dehydrate the extract, and then evaporate the solvent (preferably under reduced pressure) to obtain the crude extract.

Separation, purification, and isolation method of a substance having a tyrosinase activity inhibitory effect (compound I: neogrifolin) from the crude extract: Separation, fractionation, and separation of a tyrosinase activity inhibitory substance from a crude extract obtained from the fruiting body of Ningyotake Purification can be carried out using various known chromatographies, such as various column chromatographies (
Column Chromatography (hereinafter referred to as rccJ), high performance liquid chromatography (High
h-Performance Liquid Ch
romatography (hereinafter referred to as rHPLcJ)
) etc. can be used. At this time, in the separation/fractionation by various chromatography, the tyrosinase activity inhibition rate of each fraction is monitored and measured in parallel to confirm the presence or absence of the tyrosinase activity inhibition effect and the elution category of the active ingredient.

The tyrosinase activity inhibition rate is measured by the following method. That is, for example, 30% of each sample of the various separation/fractionation solutions of the crude extract of the fruiting body of N.
1,3-butylene glycol aqueous solution (hereinafter simply referred to as “30%
1.3-BG solution (hereinafter referred to as "BG solution"), etc., to an appropriate concentration (e.g., about 0.1 to 10%, etc., in a range where the absorbance at 475 nm shows an appropriate value). ) is diluted and adjusted as the “test solution for measurement”.
shall be.

Meanwhile, add L-tyrosine solution (concentration:
0.3 mg/mf) 1 mj! and 7-) Killvain Buffer 5
solution) (pH 6,8) lrnl, and add 0.9 - 0.9 - of the above-mentioned "test solution for measurement" and 30% 1,3-Bo solution for blank test to each of these test tubes. 1 in a constant temperature water bath at 37℃
Incubate for 0 minutes.

Add 0.1 m of tyrosinase solution (concentration: 1 mg/-・pine kilvain buffer) to the incubated material.
I! In addition, after stirring thoroughly, each reaction solution was immediately set in a spectrophotometer and the absorbance at 475 nm was measured over time. (As the absorbance value at each measurement point, the absorbance value immediately after the addition of the tyrosinase solution is indicated by the subscript 0, and the absorbance value after incubation for X minutes after the addition is indicated by the subscript X.) Each absorbance The tyrosinase activity inhibition rate (%) is calculated by substituting the value into the following 0 formula. In addition, the calculation of the tyrosinase activity inhibition rate of this invention requires 1 hour after adding the reaction solution.
The absorbance value after 0 minutes (x=10) is used.

Tyrosinase activity inhibition rate Bo: Absorbance value of blank solution after 0 minutes Bx Absorbance value of blank solution at X minute diameter Ao: Absorbance value of test solution after 0 minutes Ax: Absorbance value of test solution at X minute diameter The value is measured by the amount of dopachrome (a precursor to melanin) produced.

Then, the purity (singleness) of the target fractionated solution (that is, the fractionated solution containing compound Various types of chromatography, such as thin-layer chromatography, can be used to determine whether a substance is a substance or not.
phy <hereinafter referred to as rTLCJ>), Gas-Liquid Chromatogray (Gas-Liquid Chromatogray)
phy (hereinafter referred to as "GLC")), etc. can be used.

The structure of the tyrosinase activity-inhibiting substance (compound I) derived from the fruiting body of N. chinensis, which was thus isolated and purified and confirmed as a single substance, is determined by the following known method.

First, the molecular weight of the tyrosinase activity inhibitor derived from the fruiting body of N. chinensis was determined by high-resolution mass spectrometry (Mass
Spectrum (hereinafter referred to as rMsJ)). In addition, various known analytical techniques can be used to measure the physicochemical properties necessary for determining the chemical structure of the tyrosinase activity inhibitor derived from the fruiting body of N. chinensis. In other words, the ultraviolet absorption spectrum (1lltrav
iolet spectrum <hereinafter referred to as "UV">), infrared absorption spectrum (Infrared S
pectrum (hereinafter referred to as r I RJ).

Proton nuclear magnetic resonance spectrum (protonNucl)
ear Magnetic Re5onance
5pectra (hereinafter referred to as r'H-NMRJ)
, Carbon-13 nuclear magnetic resonance spectrum (Carbon-1
3Nuclear Magnetic Re5onan
ce 5pectra (hereinafter referred to as 113C-NMRJ)), etc., the inventors extracted, separated, purified, and isolated a substance that has the effect of suppressing tyrosinase activity from the fruiting body of N. gyotake, and determined its chemical structural formula. .

As will be explained and analyzed in detail in the Examples below, materials related to the extraction, separation, purification, and isolation of a substance that has a tyrosinase activity inhibitory effect from the fruiting body of N. chinensis and the determination of its chemical structure, as well as the tyrosinase activity inhibitory substance (
An overview of the chemical, physicochemical, and biochemical properties of compound ■).

As the raw material for extraction, the fruiting bodies of Ningyotake mushrooms are collected from the wild in nature, and dried or viable cells (raw cells) are crushed or pulverized. Add ethyl acetate to the crushed and crushed Ningyotake fruiting body,
Repeat the extraction three times for 1 hour each time under reflux conditions on a boiling water bath and combine all the extracts to obtain the extract. After this extract is dehydrated, the extraction solvent (ethyl acetate) is distilled off under reduced pressure to obtain a crude extract of the fruiting body of N. japonica.

The crude extract of the fruiting body of N. chinensis was subjected to alumina column chromatography (elution solvent system: benzene-ethyl acetate-methanol system) and silica gel chromatography (elution solvent system: benzene-ethyl acetate system) to determine the eluate that has the effect of inhibiting tyrosinase activity. Separate each section. [Then, the eluate fraction is separated and fractionated by high performance liquid chromatography (HPLC), and a fraction having the effect of inhibiting tyrosinase activity is collected.

The substance contained in the fraction thus obtained yields an isolated tyrosinase activity inhibitor (Compound I) which shows 1 spot on thin layer chromatography (TLC) and 1 peak on gas chromatography (GLC). .

The various physicochemical properties of Compound I, which is a colorless to slightly yellow and odorless sillage-like substance in appearance, will be explained below.

The high-resolution mass spectrum (MS) of Compound I is shown in Figure 1, with experimental molecular weights of 328 and 242.
2, and from this the molecular formula C22H3202 (calculated molecular weight = 328.2401) is determined.

The ultraviolet absorption spectrum of Compound I is as shown in FIG. 2, and the maximum absorption wavelengths λ and □ are 206 nm and 282 nm when the dissolution solvent is methanol. and,
The molecular extinction coefficient E (log ε) at 282 nm is 3
．． It is 59.

The infrared absorption spectrum (IR) of Compound I is as shown in FIG.
Absorption is observed at cl', 1600 cm-', and 1510 cm-'. On the other hand, the infrared absorption spectrum of the acetylated compound (1) is as shown in Figure 4, with wave numbers of 1775 cm-' and 12, indicating phenylacetate.
00 cm −' and 1620 cm − indicating the benzene ring.
', 1590cm-', 1490c+n-' were observed, whereas the hydroxy group (
The absorption at a wave number of 3400 cm −' indicating OH group disappears.

Proton nuclear magnetic resonance spectrum ('H-NM
R) and carbon-13-nuclear magnetic resonance spectra (“C-
NMR) results are shown in Table 1 below. The 'H-NMR spectrum of Compound I can be analyzed as follows based on Table 1 and FIG. 4, which are shown in FIG. That is, from the results of 'H-NMR, Compound I has (a) 1
．． 5'/ppm (6)1), 1.61pprn
(3H), 1. '18ppm (3H) with a total of 4 methyl groups (-CH3) attached to double bonds, et al. H, 95
~2.10ppm (8H), methylene group (-CH
2-), (c) Hensen ff at 2.21 ppm (3H)
Mini)Itame-f-)ItlE, (-(')f,), (d) Methylene group (-CH2-) sandwiched between double bonds at 3.27ppm (2H), (e) 5.04~ 5.1 lppm (4N) has 3 protons of olefin [methine group (-Ctl=)] and 1 proton of hydroxyl group (OH group), (f) 6.20ppm (LH), 6.25pp
A benzene ring proton is observed in m (LH), respectively.

From the results of 13C-NMR, Compound I was found to have (鵠16.
Oppm, 16.2ppm, 17.6ppm,
25.7ppm.

20.0 ppm with a total of 5 methyl group (-[:H3) carbons, (h) 25.0 ppm, 26.4 ppm,
26. lppm, 39.6 ppm with a total of 4 methylene group (-CH2-) carbons, (i) 101.0 ppm
, 109.7ppm, 122.0ppm, 123
．． 7ppm.

124.4ppm with a total of 5 (-CH) carbons, 3
;; (jH16, Oppm, 138.5ppm.

131.3ppm, 135.4ppm.

〉C) carbon, are recognized respectively.

155.2ppm, 154.2ppm.

Based on these physicochemical properties of Compound I, a detailed analysis of the chemical structure of the compound reveals that ■ Based on high-resolution mass spectrum (MS) data, ,
The molecular formula is C22H-202, ■IR and N
From each MR spectrum, it was found that it had a hydroxyl group (OH group) and a benzene ring. ■IR spectrum of acetylated compound I (1775
cm-', 1200 cm-') and 'H-NMR
From the spectrum (two around 2-3 ppm), it is found that there are two phenol acetates. From the 'H-NMR and 13C-NMR spectra of compound I, there is one methyl group (approximately 2 .2 ppm) and the presence of a farnesyl group.

Therefore, compound I has a farnesyl group (C,
,82,- group), one methyl group (-CH3 group), and two hydroxy groups (-OH group).

On the other hand, according to the 'H-NMR spectrum, the proton of the benzene ring of compound I according to the present invention is 6.20 ppm.
and 6.25 ppm, and are meta-coupled with each other at J=2.5H. Also, this signal is due to acetylation of compound (F) 6.7ppm and 6.8p
Shift downfield to pm.

Judging comprehensively from such data, the chemical structural formula of Compound I according to the present invention is 5-farnesyl-6-methyl-resorcinol (5-
farnesyl-6-methyl-resorci
nol ) [Also known as: neogrif
olin )/or compound window according to the International Nomenclature:
B, B-5-methyl-4-(3,7,11-)limethyl-2,6,10-dodecatrienyl)-1,3-benzenediol (B, B-5-methyl-4-(3
, 7,11-trimethyl-2,6,1O-do
decatr 1enyl) -1,3-benzan
-diol) ).

On the other hand, the chemical structure of compound ■ and the physicochemical properties of compound ■ are both based on the previously reported document 1.
:(1) Phytochemistry 18.14
09 (1977), / (2) Chem,
Pharm, Bull, 36.2918 (19
88) etc.].

Effects> The biochemical properties of Compound I (neogrifolin) according to the present invention will be described.

The compound ■ (neogrifolin) according to this invention is
As is clear from the separation and purification process from the raw material, the fruiting body of N. japonica, it has a remarkable biochemical property of inhibiting tyrosinase activity. Furthermore, Compound I showed negative results in sensitization tests, etc., suggesting that it is also excellent in safety.

As for the biochemical properties of the compound I, the degree of tyrosinase activity inhibitory effect can be determined by (a) measuring the amount of dopachrome (melanin precursor) produced as detailed above, and (b) as described below. It is determined by measuring the melanin cell production rate using a melanin cell culture method using "clone M-3 cells (derived from mouse melanoma tumor)" which produce melanin. Table 2 below shows an example of the measurement results of the amount of dopachrome (melanin precursor) produced in (a) above, among the measurement results of the tyrosinase activity inhibiting action and effect of the compound (neogrifolin) according to the present invention. show. According to the example results in Table 2, the concentration of compound ■ is 10
A ppm solution has a tyrosinase activity inhibitory effect of 56.7%, and a 50 ppm solution of Compound I has a tyrosinase activity inhibitory effect of 100%, suggesting extremely significant biochemical effects.

Further, the test method and measurement results using the culture method of (c) "clone M-3 cells (derived from mouse melanoma tumor)" will be described in detail in the Examples section below.

The melanin production inhibition rate of Compound I according to the present invention is as shown in Table 3 below based on the measurement results using the above-mentioned (b) "clone M-3 cells (derived from mouse melanoma tumor)" culture method. Compared to the "negative control group" in which Compound I was not added (amount of Compound ■ added: 0.00 mg), the amount of Compound I added was 0.005 mg, 0.02
In each test group containing 5+ng and 0.05 mg, melanin production was suppressed to 52% to 66% compared to the negative control group.

A sensitization test was conducted to determine the safety of the compound (1) (neogrifolin) according to the present invention. As a result, the test result was negative at a concentration of Compound (1) of 1000 ppH1, proving that Compound I according to the present invention is extremely safe.

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

■) Raw material: Ningyotake fruiting body.

The raw material, the fruiting bodies of Ningyotake mushrooms, were collected from those that grow naturally. The fruiting bodies of Ningyotake used in this invention were collected around October 1988 from growing wild in the mountains of Toyohira Town, Yamagata District, Hiroshima Prefecture, and Chiyoda Town, Hiroshima Prefecture. ) or dried bacterial cells air-dried at room temperature for 3 to 10 days.

■) Extraction, purification, isolation, etc. of compound I from raw materials.

(1) Preparation of a crude extract from the fruiting body of Ningyotake: Prepare the live cells (100 g) of the fruiting body of Ningyotake or the dry cells (20 g) of the fruiting body of Ningyotake, finely crushed or finely powdered. Extraction was carried out three times for 1 hour using 100 mff of extraction solvent (ethyl acetate) on a boiling water bath and under reflux cooling. The extracts were combined and dehydrated with anhydrous sodium sulfate, and then the extraction solvent (ethyl acetate) was distilled off under reduced pressure if possible to obtain a crude extract. Note that IJg of the crude extract was obtained from the live bacterial cells (100 g), and 2.1 g of the crude extract was obtained from the dried bacterial cells (20 g).

(2) Fractionation, purification, and isolation of compound I from crude extract: ■ Fractionation by alumina column chromatography: Column condition C (20 mmφ x 155 mm-L), ICN Adsorbentien 4580 /
using 50 g]. As an elution solvent, benzene: ethyl acetate: methanol: acetic acid system (benzene 1
00% → stepwise mixture of benzene + ethyl acetate → ethyl acetate 100% → stepwise mixture of ethyl acetate + methanol → stepwise mixture of methanol 100% - methanol + acetic acid) was used. And always for a portion of the eluate,
Measure the amount of dopachrome produced in each elution section, carefully elute and fractionate while checking and monitoring the tyrosinase activity inhibition rate of the elution section, and separate the elution section that has the desired tyrosinase activity inhibition effect. . As a category having a tyrosinase activity inhibiting effect according to this invention,
An elution solvent fraction of 50,150 ethyl acetate/methanol was collected.

■ Fractionation by silica gel column chromatography: The tyrosinase activity-inhibited sample (applied amount = 0.7 g) of the alumina column chromatography fraction collected in step (■) above was
The analysis was carried out under column conditions [(20 mm φ x 250 mm), using 20 g of silica gel as a column carrier].

As the elution solvent, a system of benzene: dichloromethane: ethyl acetate (100% benzene → graded mixture of benzene + dichloromethane → graded mixture of benzene + ethyl acetate) was used.

As with the column chromatography described in (2) above, the amount of dopachrome produced in each elution section is always measured for a portion of the eluate, and the tyrosinase activity inhibition rate of the elution section is carefully monitored while being carefully eluted and fractionated. death,
Collect the elution fraction that has the desired tyrosinase activity inhibiting effect. The category having the tyrosinase activity inhibiting effect according to the present invention is benzene/ethyl acetate = 99.
A fraction of the elution solvent of 0/1.0 was collected.

(2) Purification by high-performance liquid chromatography: The sample collected in (1) above is further purified by high-performance liquid chromatography. The column conditions used were: Column: Shim-
pack PROP-ODS (2,0cmφX250
mm), eluate: methanol/water = 9515, and the target tyrosinase activity inhibitor elution section (compound I
According to preliminary experiments,
As in the case of ■ and ■ above, measure the tyrosinase activity inhibition rate for each elution section, collect data on the section (peak) in which the tyrosinase activity inhibitor is eluted in advance, and then The eluate fraction containing the tyrosinase activity inhibitor (that is, Compound I) was collected. The solvent in this eluate fraction was reduced to a
The mixture was condensed, transferred to n-hexane, and insoluble matter was collected to obtain a colorless to slightly yellow, slag-like, odorless Compound I.

■ Confirmation of isolation of Compound I: (a) Confirmation by thin layer chromatography (TLC); Isolation of the tyrosinase activity inhibitor (Compound I) fractionated and purified by each operation of (2) (2) or (2) above. was confirmed by silica gel TLC.

Namely, thin layer carrier Nizirika gel 70-plate Wako (commercial product/prepared product/manufactured by Wako Pure Chemical Industries, Ltd.) Developing solvent: benzene/ethyl acetate = 3/1 coloring liquid = 10
% sulfuric acid (ethanol solution) The TLC results under the above conditions detected a single spot around Rf'=0.6.

) By gas chromatography (GLC)! Acknowledgment: The isolation of the tyrosinase activity inhibitor (compound I) that has been fractionated and purified by each of the operations in (2)
It was also confirmed by C.

That is, column: 3. Omm φ 25
0°C (8°C/m1n) → 250°C Constant at 250°C Inlet and detector temperature: 280°C Detector: FID (flame ionization detector) GLC results under the above conditions show that Rt is approximately 14 A single peak was detected at ~16 minutes.

In this way, a single compound (compound I) could be isolated on TLC and GLC.

■) Measurement of the physicochemical properties of Compound I and determination of the chemical structural formula of Compound ■.

(1) High-resolution mass spectrum (MS) of Compound I: FIG. 1 is an example of a high-resolution mass spectrum (MS diagram) of Compound I. From the results of this MS, the molecular weight (experimental value) of Compound I according to the present invention is 328, 2422,
From this, the molecular formula is C22H! It can be seen that □0□, and the calculated value of molecular weight = 328.2401.

(2) Example of ultraviolet absorption spectrum (UV) of compound ■: Figure 2 is an ultraviolet absorption spectrum (UV diagram) of compound I
This is an example. As is clear from FIG. 2, compound I has maximum absorption λmaX = 206 in methanol solvent.
nm and λmax = 282 nm, the 282
Molecular extinction coefficient E (logε) in nm = 3.59
It can be seen that it is.

(3) Example of infrared absorption spectrum (IR) of Compound I: Figure 3 is an infrared absorption spectrum (IR diagram) of Compound I.
As an example, Figure 4 shows the acetylated compound of compound I.
This is an example of an infrared film absorption spectrum diagram (IR diagram).

The presence of a benzene ring and a hydroxyl group (OH group) is recognized from the IR diagram of Compound I in Figure 3, and from the IR diagram of the acetylated compound I in Figure 4.

Table 1 where presence of two phenol acetates is observed: IH-NM of acetylated compounds of Compound I and Compound ■
R and "C-NMR (4) Proton nuclear magnetic resonance spectra ('H-N
MR) ・Carbon-13-nuclear magnetic resonance spectrum ("C
-NMR) Examples: Table 1 shows example results of proton nuclear magnetic resonance spectra ('H-NMR) and carbon-13-nuclear magnetic resonance spectra ("C-NMR) of Compound I and acetylated compounds of Compound I. Figure 5 is an example of 'H-NMR diagram of compound (1).

[Margins below this page]

Mountain *=One H of hydroquino group (OH group) is included in 4H.

Based on the various physicochemical properties mentioned above, Compound I has 5-
Farnesyl-6-methyl-resorcinol (5-fa
rnesyl-6-methyl-resorcino
l ) [Also known as: neogrifolin
olin) / or compound name according to international nomenclature:
B, B-5-methyl-4-(3,7,11-trimethyl-2,6,10-dodecatrienyl)-1,3-benzenediol (B, B-5-methyl-4-(
3,7゜11-trimethyl-2,6,1O-d
odecatrienyl) -1,3-benzene
-dial) ], suggesting that its chemical structural formula is as follows.

Chemical structural formula of compound f: ■) Biochemical properties of compound I: (1) Example of the measurement results of the tyrosinase activity inhibition rate of compound I by measuring the amount of dopachrome produced.

The tyrosinase activity inhibitory effect of Compound I is measured by the method described above. Compound I is 30% 1,3-EI
It was diluted with G solution to a predetermined concentration (concentration suitable for showing an appropriate absorbance value at 475 nm when measuring the tyrosinase activity inhibitory effect) and used for measurement.

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 using the formula 0 below. The results of the tyrosinase activity inhibition rate of each extract were
Shown in the table.

Tyrosinase activity inhibition rate [margin below this page] ...■Formula Bo Absorbance value Boo after 00 minutes of blank solution
Absorbance value Ao of the blank solution after 10 minutes: Absorbance value Ago of the test solution after 0 minutes: 1 of the test solution
This suggests that it has the effect of suppressing the absorbance value after 0 minutes.

(2) Example of a test for measuring the melanin production inhibition rate of compound (1) by measuring the amount of melanin cells produced by mouse melanoma tumor-derived clone M-3 cells.

From the above results, Compound I showed a tyrosinase activity inhibition rate of 56.7% at a concentration of 10 ppm, 501]1]
The tyrosinase activity inhibition rate was 100% at a concentration of m, and Compound I had an extremely significant tyrosinase activity ■Preparation of samples, culture media, etc.: (a) Preparation of sample of Compound I: Add 1 g of dried Ningyotake fruiting body to After adding 100 ml of water, the mixture was heated under reflux conditions on a boiling water bath for 1 hour, and after cooling, the mixture was filtered to prepare an aqueous extract of the fruiting body of N. chinensis. Add 90 ml of ethanol to 10 ml of this aqueous extract and mix (90% ethanol solution), and the resulting precipitate (
Mainly polysaccharides) were collected. Approximately 8 liters of distilled water is added to this sediment.
nl was added and dissolved. To this, compound 1005■ was dissolved in a small amount of ethanol and added.

After evaporating ethanol under reduced pressure (rotary evaporator), the volume was adjusted to 10 ml and used as a sample of Compound I.

In addition, as a negative control (blank), one in which Compound I was not added during the sample preparation step was used.

(b) Test cultured cells: "Clone M-3 cells" derived from mouse melanin tumors that produce melanin were used as test cultured cells.

(C) Medium used (medium composition): Hams P-10 (Ham's F-10)-87,
5wt% (manufactured by Plow Laboratories), fetal bovine serum (FBS)...2.5
wt%・Horse Serum ---
-15.0 wt% medium was used.

■Culture experiment method: (a) Clone M-3 cells were uniformly dispersed in the medium at a concentration of 1 x 106 cells/ml', and placed in a 12-well shear dish (culture area: approximately 4.52 cl/well). After dispensing 0.1 cells of the medium, 1.9 cells of the medium was added to each formula, and clone M-3 cells were cultured at 37° C. for 1 day.

(c) After culturing, discard all the medium, and then add 1 medium
mj! and the test amount (0.1 rnl) of the sample (compound I sample and blank sample).

0.5m! , 1.0 ml) and phosphate buffer solution (PBS) so that the total volume is 2 ml each.
(pH=7.2) and cultured at 37°C for 2 days.

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

■ Measuring method for melanin placement and protein amount: (a) 0.2
Add 1 mj2 of 5% trypsin solution to each of the petri dishes, leave for about 30 seconds, and then discard the trypsin solution.

) Approximately 5 minutes after the treatment in (a) above, a phosphate buffer solution (PBS
) (pH=7.2) 1 ml! The cells were detached with
0 seconds) Each cultured cell was homogeneously ground.

(C) Wavelength 4 to quantify the amount of melanin produced.
The absorbance at 00 nm was measured.

(d) To quantify the amount of protein produced, a color was developed using a coloring agent "Protein Assay" (manufactured by Bio-Rad), and the absorbance at a wavelength of 595 nm was measured.

In addition, since there is a correlation between the number of cells and the amount of protein, the production rate of melanin per unit amount of protein is measured by measuring the amount of protein instead of the number of cells. The melanin production inhibition rate was measured.

Table 3 shows the results of compound I by measuring the production rate of melanocytes produced by mouse melanoma tumor-derived clone M-3 cells.
Examples of test results for measuring melanin production inhibition rate are shown below. The numbers indicate the absorbance at each wavelength, and the values indicate the average of three values.

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■) Regarding the safety test of Compound I: (1) Sensitization test: ■ Test animal Two male Hartley white guinea pigs weighing 300 g were subjected to the test.

■Number of animals = 10 animals per group.

■ Breeding conditions: Three animals per cage are housed in stainless steel cages (350 x 420 x 200 mm) in a breeding room set at a temperature of 22 ± 2°C and a humidity of 60 ± 10%, and feed (Nippon Talea CG-3) and water supply are provided. The animals were kept with free access to water.

■Test sample: 1.3-Compound I in BG solution at 11,000
The test substance stock solution was dissolved to a concentration of pp, and the test solution stock solution was used as a sample as it was.

■Test method: Maximization
ion) method as follows.

^) The hair on the scapula of the animal was shaved to a size of 4 x 6 cm, and the following intradermal injections were performed at the shaved site.

(a) Adjuvant” 0.1m1 (0.05d left and right 2
(b) Samples 0 and 1rnf! (0.05 ml in 2 locations on the left and right) (C) 0.1 ml of adjuvant 1 emulsified with the sample, (0.05 rnI! 2 locations on the left and right) B) On the 6th day after intradermal injection, the hair at the same location was shaved and 10% Sodium lauryl sulfate 02 was applied.

C) Apply the sample to a 2 x 4 cm filter paper at 78th,
A filter paper was applied to the same site and closed for 48 hours.

D) Two weeks after the closed application, the side of the body was shaved to a size of 5 x 5 cm, the sample was applied to a circular filter paper with a radius of 2 cm, the filter paper was applied to the same area, and the closed application was performed for 24 hours B) Judgment was made 24 hours after the patch was removed.

■Judgment criteria: No reaction・・・・・・・・・ −・・・・
・ 0 Mild or scattered erythema 1 Moderate erythema 2 Severe erythema and edema 3 Note) I
I=Freund's complete adjuvant (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 92=Sodium lauryl sulfate dispersed in white petrolatum at a concentration of 10% by weight.

References: “Physiology and Safety of New Skin” Seishi Shoin
Issue> ■Results: The results of the sensitization test for Compound I were 1100.
"No reaction" according to the above criteria at a concentration of 0pp
(negative).

From this result, compound ■ is extremely cheap. Suggested to be a highly tonic compound It was done.

<Effects of the Invention> Compound I (neogrifolin), which has been extracted, separated, purified, and isolated from the fruiting body of Ningyota spp. and whose chemical structure has been clarified, has a remarkable effect of inhibiting tyrosinase activity. Moreover, the compound (1) is highly safe. In addition, this invention has determined the chemical structure of the tyrosinase activity inhibitor derived from the fruiting body of N. chinensis, paving the way for the chemical synthesis of the same substance as the tyrosinase activity inhibitor derived from natural products (derived from the fruiting body of N. chinensis).
It has become possible to stably mass-produce tyrosinase activity inhibitors with homogeneous and constant quality, high tyrosinase activity, and safe and excellent properties, and furthermore, when incorporated into cosmetics, it has never been possible before. It has a remarkable effect of achieving the purpose of the invention, such as being applicable to whitening cosmetics that are safe and have a high inhibition rate of tyrosinase activity.

[Brief explanation of drawings]

Figure 1 shows the compound according to the invention whose chemical structure has been extracted, separated, purified, isolated, and determined from the fruiting body of Ningyotake.
Figure 2 is a high-resolution mass spectrum diagram of (neogrifolin). Figure 2 is an ultraviolet absorption spectrum diagram of compound (2) according to the present invention. Figure 3 is an infrared absorption spectrum diagram of compound (1) according to the present invention, Figure 4 is an infrared absorption spectrum diagram of the acetylated compound Nuclear magnetic resonance spectrograms are shown respectively.

Claims (1)

[Claims] (1) 5-farnesyl-6-methyl-resorcinol (5-farnesyl-6-methyl-resorcinol)
rcinol) [Alternative name: neogrifolin (neogr
A tyrosinase activity inhibitor containing ``ifolin'' as an active ingredient.