JPH06199679A - Tyrosinase activity inhibitor - Google Patents

Abstract

PURPOSE:To provide the subject inhibitor containing an organic solvent-soluble material of fermented rice as the active component, useful for prevention of a spot or a freckle due to sunburn, capable of production from inexpensive recedily available raw materials and excellent in safety. CONSTITUTION:The objective inhibitor contains an organic solvent-soluble material of fermented rice as the active component preferably in an amount of >=0.0001wt.%. In addition, rice wine lees obtained as a by-product in production of refined rice wine is used as the raw material fermented rice in general. The organic solvent-soluble material of fermented rice is obtained, e.g. by dispersing rice wine lees in ethanol, homogenizing it using a homomixer, carrying out centrifugal separation, then evaporating ethanol from the resultant supernatant under a reduced pressure and subsequently further adding ethanol thereto.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a preparation for inhibiting the activity of tyrosinase.

[0002]

Tyrosinase is a metalloenzyme which catalyzes the oxidation of tyrosine or dopa and is widely distributed in microorganisms, plants, animals and the like having the ability to synthesize melanin. In humans, the melanin pigment is a melanocyte existing in the substratum of the epidermis and is synthesized by the action of tyrosinase induced by ultraviolet rays using tyrosine as a substrate (G.Prota, J. Inv.
est. Dermatol., 75, 122-127, 1980).

Melanin is divided into yellow to reddish-brown pheomelanin and black eumelanin. Melanin, which is usually called, is a mixture type of these. All of these are produced by the action of tyrosinase using tyrosine as a substrate (H. Rorsma
n, et al., Phenotypic Expression in Pigment Cells,
p.67, ed. by M. Seiji, Univ. of Tokyo Press).

Therefore, it is considered that inhibition of tyrosinase activity makes it possible to prevent pigmentation, spots and freckles due to sunburn. As suitable for these mechanisms of action, kojic acid is currently used as a quasi drug,
Arbutin, ascorbic acid derivatives and the like are used. However, since these tyrosinase activity inhibitors lack stability or are difficult to obtain raw materials,
It was very expensive and neither was satisfactory.

On the other hand, the organic solvent-soluble product of fermented rice requires its raw material for sake lees, which is a by-product of sake production. Sake lees have been used for food since ancient times, and are contained in non-sake liquor such as Dubroc, and their safety has been empirically verified for many years. In addition, the raw material sake lees themselves are very inexpensive and abundant. It has never been known that the organic solvent-soluble matter of this sake lees has a very strong tyrosinase activity inhibitory effect.

[0006]

Based on the above-mentioned conventional knowledge, the present invention is intended to provide an inhibitor of tyrosinase activity by using a raw material which is easily available and relatively inexpensive.

[0007]

That is, the present invention is characterized by containing an organic solvent-soluble product of a fermented rice product as an active ingredient, and the active ingredient is at least 0.000.
The present invention proposes a tyrosinase activity inhibitor that is contained at a concentration of 1% (W / V).

[0008]

In the present invention, the fermented rice product used as a starting material is sake lees which are mainly produced as a by-product during the production of sake. As this sake lees, sake of any fermentation process can be used, and all of them are commercially available or can be obtained at a sake manufacturing plant.

[0009]

[Examples] First, tyrosin-a soluble organic solvent of fermented rice
An experiment was conducted to examine the inhibitory effect on the zease activity. (1) Preparation of tyrosinase activity inhibitor Commercially available sake lees 1 kg (wet weight) was suspended in 3 times the amount of 99.5% ethanol and homogenized at room temperature for 10 minutes using a homomixer. This is centrifuged (3,000 rpm, 1
Then, the supernatant (ethanol-soluble fraction) was obtained. Ethanol was evaporated under reduced pressure. Then 300 ml of 9
Additional 9.5% ethanol was added and the lysate (about 40 g,
(Dry weight) was obtained and used as a tyrosinase activity inhibitor in the subsequent experiments.

(2) Experimental method and result 1) Measurement of tyrosinase activity inhibitory effect L-tyrosine (special grade, manufactured by Wako Pure Chemical Industries, Ltd.) was added to 0.1M phosphate buffer (pH 6.8) at 1.66 μmol / A substrate solution was prepared by dissolving so as to have a volume of ml. Tyrosinase is derived from Matsumura (3,000 units / manufactured by Sigma).
mg. solid) was dissolved in the above buffer solution to a concentration of 800 units / ml. The reaction was carried out as follows. MacIlvine buffer (pH 6.
8) 0.1 ml of sample and 0.1 ml of tyrosinase per 1 ml
Was added and mixed, and then pre-incubated at 37 ° C for 10 minutes. After that, 1 ml of substrate solution was added, and the temperature was 37 ° C.
And reacted for 90 minutes. The absorption value at 475 nm was measured with a spectrophotometer (this absorption value is referred to as B).

As a control, 0.1 ml of 99.5% ethanol was added in place of the sample, and the same operation was performed (this absorbance value is designated as A). As a blank, 0.1 ml of a 0.1 M phosphate buffer solution was added instead of the enzyme solution, and the same operation was performed (this absorption value is referred to as C). From these values, the tyrosinase activity inhibition rate (%) was calculated by the following formula. Inhibition rate (%) = 100 [1-((B-C) / A)]

2) Discoloration effect on black goldfish Studies on decolorizing agents such as hydroquinone revealed that black goldfish have high sensitivity to melanin production inhibitory effect, easy appearance observation, and pigment cells can be easily searched by removing scales. It is often used for things (Y. Mislima, et a
l., AnatomicalRecord, 137, 382, 1960., W. Chavin,
J. Pharmacol. Exp. Therap., 142, 275-290, 1963). Sample prepared by the method described in (1) above 0.02% (W
/ V), and black goldfish were bred for 30 days in breeding water containing the same, and the fading effect was examined. The bleaching effect during the experiment was macroscopically, and the scales were collected on the 30th day, and melanophore was measured under an optical microscope. Scales were collected over time during the breeding period, and 0.1 M phosphate buffer (pH 7.
After extracting tyrosinase in 2), the tyrosinase activity was measured according to the method described in 1) of (2).

3) Inhibitory effect on melanin synthesis of B-16 mouse melanoma cells B-16 mouse melanoma cells (1 * 10 ⁶ cells) were 5%.
Using RPMI1640 medium supplemented with fetal bovine serum (manufactured by Gibco), preculture was carried out for 48 hours under the conditions of 37 ° C. and 5% CO ₂ . The sample prepared by the method described in 1) of (2) above was added to the medium in an amount of 0 to 100 μg / ml so as to be a medium, and after irradiation with ultraviolet rays (A, B, C waves) of 160 μW / cm ² for 10 minutes. The cells were further cultured under the same conditions for 48 hours. The cells were taken out, thoroughly washed with a salinized phosphate buffer solution, and then 1 ml of 1N NaOH and 1 ml of 1% dimethyl sulfoxide were added to lyse the cells. Further, after filtering with a filter having a pore size of 0.45 nm, the absorption value at 475 nm was measured using a spectrophotometer.

(3) Experimental Results 1) Tyrosinase Activity Inhibitory Effect of Rice Fermented Soluble Organic Solvents The tyrosinase activity inhibitory rate of rice fermented organic solvent solubles shows a correlation with concentration. It was About 60 at a sample concentration of 3.9 μg
% Inhibition, with 19.5 μg completely inhibiting tyrosinase activity (see Table 1).

[Table 1]

2) Effect of fading on black goldfish Photo 1 is a photograph showing the conditions of the first day of black goldfish (A) and black goldfish (B) which were not treated with the organic solvent-soluble matter of the fermented rice. In this case, the discoloration was visually observed from the second day, and the discoloration progressed with the treatment period. The fading was systemic rather than local. Photo 2 shows the fading situation of B compared to A after 30 days.

[Photo 1]

[Photo 2] In addition, as can be seen in Photo 3 (no treatment) and Photo 4 (treatment) showing optical microscope images (× 30) of black goldfish scales in the biopsy after 30 days, melanophore (me) was observed in the treated ones.
lanophore) miniaturization, irregular distribution, and a marked decrease in dendrites.

[Photo 3]

[Photo 4]

At this time, the tyrosinase activity was about 60% of that in the untreated group, which was significantly decreased (see Table 2).

[Table 2]

3) Inhibitory effect on melanin synthesis of B16 mouse melanoma cells When melanoma cells were cultured in a medium containing rice fermented product soluble in organic solvent, melanin synthesis was inhibited depending on the concentration. The inhibition rate is 0.5 μg / ml. About 28% in total of the medium, 2 μg / ml. About 38% in medium, 10 μg /
ml. About 40% in the medium, 2 μg / ml. A plateau was reached at a concentration near the medium. The tyrosinase activity of the cells was also inhibited with the same tendency as the inhibition of melanin synthesis (Table 3).

[Table 3]

(4) The effect of the tyrosinase activity inhibitor of the present invention is strong not only when it is dissolved in an organic solvent but also when it is turbid in water (Table 1).
For example, it can be used in the form of liquid or powder for cosmetic base materials, bath agents and the like. Further, due to its safety and stability, it can be applied to agricultural products (mushrooms, etc.) that turn black due to the action of tyrosinase, and marine products (shrimp, crab, etc.), and can be used for maintaining freshness and ensuring quality. Even in this case, various forms and concentrations can be used depending on the purpose of use.

[0019]

EFFECTS OF THE INVENTION The tyrosinase activity inhibitor of the present invention is considered to be very effective in preventing pigmentation, spots and freckles caused by sunburn.

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[Procedure amendment]

[Submission date] December 8, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0015

[Correction method] Change

[Correction content]

2) Discoloration effect on black goldfish Among the black goldfish not treated with the organic solvent-soluble matter of the fermented rice and the treated black goldfish, the treated black goldfish was visually discolored from the second day. And the fading progressed with the treatment period. The fading was systemic rather than local. Comparing untreated and treated black goldfish scales in biopsies after 30 days also showed a reduction in melanophore size, an irregular distribution and a marked reduction of dendrites in the treated ones. Admitted.

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Claims (2)

[Claims] 1. A tyrosinase activity inhibitor comprising a fermented rice product soluble in an organic solvent as an active ingredient. 2. The active ingredient is at least 0.0001%.
The tyrosinase activity inhibitor according to claim 1, which is contained at a concentration of (W / V).