JPH04217627A - Tyrosinase activity inhibitor and melanism depressant - Google Patents

Abstract

PURPOSE:To obtain a tyrosinase activity inhibitor and melanism depressant comprising dehydrodieugenol, mixable with medicines, quasi-drugs, cosmetic, etc., in order to beautify skin. CONSTITUTION:A clove oil is subjected to steam distillation, a residual fraction thereof is eluted by silica gel chromatography to give an ethyl acetate fraction, which is further purified by silica gel chromatography to give dehydrodieugenol shown by the formula. Since the compound shows high effects with a low concentration and high safety, the compound is mixable with cosmetics of preventing formation of stain and freckles and is usable for drugs of treating and preventing chromatosis.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a tyrosinase activity inhibitor and melanin production inhibitor that can be incorporated into pharmaceuticals, quasi-drugs, cosmetics, etc. for the purpose of whitening the skin.

0002

[Prior Art and Problems to be Solved by the Invention] Melanin is thought to be produced by the action of tyrosinase in pigment cells, where tyrosine is changed into dopa and dopaquinone, and then through dopachrome, etc. It exists in the skin and plays an important role in protecting the body from ultraviolet rays. However, overproduction of melanin causes the formation of age spots and freckles and accelerates skin aging, so recently, drugs aimed at preventing overproduction of melanin due to ultraviolet rays have been developed.

Hydroquinone and NBEH (monobenzyl ether of hydroquinone) have traditionally been used to achieve fair and beautiful skin, but they cause degeneration and death of pigment cells and impair the skin's natural physiological functions. It has defects that cause side effects such as irreversible white spots, pigment abnormalities, and rashes.

[0004] Focusing on tyrosinase, which is an enzyme involved in the production of melanin mentioned above, vitamin C and its derivatives have been used for the purpose of reducing the amount of melanin in the skin. However, these tyrosinase activity inhibitors have low activity and poor stability because they are drugs that utilize reducing power. In addition, there were many problems with safety itself.

[0005] Accordingly, an object of the present invention is to provide a highly safe drug that effectively inhibits tyrosinase activity in small amounts and suppresses melanin production to a small amount.

[0006]

[Means for Solving the Problems] In order to achieve the above-mentioned purpose, the present inventors conducted a tyrosinase activity inhibition test and searched for natural products that have an inhibitory effect on melanin production, and found that clove oil was steam distilled, Regarding the residual fraction, the inventors further purified the ethyl acetate fraction eluted by silica gel chromatography, and found that dehydrodieigenol had strong physiological activity, and completed the present invention.

[0007] The present invention is based on the structural formula

This is a tyrosinase activity inhibitor and melanin production inhibitor consisting of dehydrodiogenol represented by the following formula.

[0008] Dehydrodieigenol is a known compound (Japanese Chemical Journal, Vol. 87, No. 9, p. 110-11)
2, 1966), and its high safety has been confirmed (Japanese Patent Application No. 01-207581).

The tyrosinase activity inhibitor and melanin production inhibitor of the present invention can be applied to, for example, pharmaceuticals, quasi-drugs, cosmetics, and the like.

[0010] The amount applied varies depending on the system used.
Although it cannot be generalized, as is clear from the following examples,
The concentration may be equivalent to or considerably lower than existing substances of this kind.

[0011] Next, an example will be shown to demonstrate the effects of dehydrodiogenol on inhibiting tyrosinase activity and inhibiting melanin production.

0012

Examples Example 1 Tyrosinase Activity Inhibition Test The measurement principle is to measure dopachrome produced by the action of tyrosinase on tyrosine.

[0013] The reaction composition liquid contains L-tyrosine (0.3 mg/
ml) 1.0ml, McCleben buffer (pH 6.8)
Prepare a total volume of 2.9 ml (1.0 ml, 0.9 ml of dehydrodieugenol), incubate at 37°C for 10 minutes, add 0.1 ml of tyrosinase (1 mg/ml, mushroom, manufactured by Sigma), ℃，
Incubation was performed for 15 minutes, and absorbance at 475 nm was measured. Dehydrodieigenol is poorly soluble in water, so it was dissolved in 1% ethanol to a final concentration of 0.6.
, 1.5, and 3.0 mM samples were prepared and used. In addition, since it is possible that the enzyme was inactivated by ethanol, we also conducted a study using the same concentration to confirm that there was no enzyme deactivation.

[0014] The inactivation of tyrosinase enzyme inhibitory activity was calculated using the following formula and is shown in Table 1.

[0015]

[Math 1]

(A): Absorbance when buffer is added instead of sample (B): Absorbance when sample is added (C):
Absorbance when adding buffer instead of tyrosinase (
D): Absorbance when buffer solution is added instead of tyrosinase and sample

Table 1

From the results shown in Table 1, it was found that dehydrodiogenol inhibits tyrosinase activity in a concentration-dependent manner and is effective at low concentrations.

Example 2 Melanin Suppressing Effect Test Melanin suppressing effect test on pigment cells was carried out by injecting B16 melanoma cells (1×10 6 cells) into Eage 1 minimum essential medium 5 containing 10% fetal bovine serum supplemented with dehydrodieigenol.
The cells were cultured in a Falcon petri dish (φ60 mm) for 4 days under conditions of 5% CO2 in ml and 37°C to react.

After culturing, the cells were detached with 0.05% trypsin. Furthermore, the cells were washed three times with phosphate buffered saline, and the number of cells (cell increase rate) was calculated using a Neuhauer hemocytometer.

On the other hand, the cells were fixed in a Petri dish with 10% neutral formalin for 20 minutes, washed three times with 50 mM phosphate buffer (pH 6.8), and washed with the same buffer containing 5 mM L-dopa for 30 minutes. After incubation for 5 hours at
After thorough washing, cells whose entire cells were stained black were counted as DOPA-positive cells.

For sample preparation, with 1% ethanol,
After confirming that there was no effect on cells, samples were prepared with the same concentration of ethanol to a final concentration of 0.1, 0.01, and 0.001mM, and the suppression rate (%) for the increase in B16 melanoma cells without the addition of the sample. ).

As a comparative example, ascorbic acid and hydroquinone were used. The results are shown in Table 2.

Table 2 Suppression rate (%
)

From the results shown in Table 2, dehydrodieigenol had a higher melanin suppressing effect than ascorbic acid, and its suppressing effect was comparable to that of hydroquinone. On the other hand, the cell increase rate of hydroquinone at 0.1mM is 1/1 compared to that of dehydrodieigenol.
It was about 5. That is, this indicates that the melanin production inhibiting effect of hydroquinone is largely due to cytotoxicity, which poses a major safety problem. On the other hand, dehydrodieigenol is superior in safety in terms of cytotoxicity.

[0026]

[Effects of the Invention] As described above, the present invention has a high tyrosinase activity inhibiting effect and a melanin production suppressing effect at low concentrations, and is also highly safe. It is clear that a useful tyrosinase activity inhibitor and melanin production inhibitor can be provided, which can be incorporated into cosmetics to prevent pigmentation, and can be used in medicines as a treatment and preventive agent for pigmentation disorders.

Claims (1)

[Claims] [Claim 1] A tyrosinase activity inhibitor consisting of dehydrodieigenol represented by the following structural formula: [Claim 1]