WO2011068147A1

WO2011068147A1 - Enhancement of melanin synthesis and activation of tyrosinase

Info

Publication number: WO2011068147A1
Application number: PCT/JP2010/071548
Authority: WO
Inventors: 堅次杉林; 嘉寛徳留; 辰彦金
Original assignee: 株式会社シャローム
Priority date: 2009-12-03
Filing date: 2010-12-02
Publication date: 2011-06-09
Also published as: JP2011136976A; JP5017386B2

Abstract

Disclosed is a novel technique by which melanin synthesis can be enhanced. Specifically disclosed is a method for producing a melanin synthesis enhancer. One embodiment of the method comprises: a drying/pulverization step (S100) in which plant tissues of stevia serving as a starting material are dried and pulverized; a fermentation step (S102) in which the dried stevia powder obtained in the drying/pulverization step (S100) is fermented; an extraction step (S104) in which extraction from the fermented stevia powder (hereinafter referred to as "fermented stevia") is carried out using an aqueous ethanol solution, thereby obtaining a liquid extract; a first fractionation step (S106) in which the liquid extract of the fermented stevia is fractionated, thereby obtaining an ethanol-soluble fraction; and a second fractionation step (S108) in which the ethanol-soluble fraction is further fractionated, thereby obtaining a lipid-soluble fraction.

Description

Promotion of melanin synthesis and activation of tyrosinase

The present invention relates to a technique capable of promoting melanin synthesis, for example, a method for producing a melanin synthesis promoting substance for promoting melanin synthesis, a melanin synthesis promoting substance, an external preparation for skin, and a method for producing a tyrosinase active substance for activating tyrosinase. , Tyrosinase active substance, and pharmaceuticals.

Background of the Invention

Hair color is the color of melanin synthesized by melanocytes (pigment cells) in the hair matrix that produces hair. When the melanin synthesis ability of melanocytes decreases due to aging, stress, illness, inheritance, etc., the supply of melanin to the hair decreases, and the hair becomes whitened, so-called white hair.

Conventionally, various hair coloring agents (hair dyes) have been developed to make gray hair inconspicuous. For example, oxidation hair dyes are widely used as hair dyes. Oxidative hair dyes color hair by opening the epidermis (cuticle) of the hair and injecting a pigment into the hair. With an oxidative hair dye, the cuticle is opened and a pigment is injected, so that hair dyed with an oxidative hair dye is easily damaged, loses moisture and becomes uncomfortable.

Thus, hair dyes that improve the flexibility, moist feeling or smoothness of hair by containing a cationic polymer, an amphoteric polymer and an anionic polymer in an oxidative hair dye are disclosed (for example, Patent Document 1).

JP 2002-193772 A

However, even if the technique described in Patent Document 1 is used, it does not change that hair cannot be dyed unless a cuticle on the surface of the hair is opened and a pigment is injected, so it is difficult to completely eliminate damage to the hair. is there. Also, since the gray hair itself is not lost, when the hair grows, the color difference between the dyed part on the tip and the unstained part on the scalp side (white hair part) becomes conspicuous, creating a sense of incongruity. End up.

The object of the present invention is to provide a new technique that can promote the synthesis of melanin.

As an example of an embodiment of the present invention, a method for producing a melanin synthesis promoting substance that promotes melanin synthesis, the first fractionation step of fractionating an extract of fermented stevia to obtain an ethanol-soluble fraction; And a second fractionation step of further fractionating the ethanol-soluble fraction to obtain a fat-soluble fraction.

The inventor of the present application, as a result of earnest research, has the effect of promoting the synthesis of melanin in the fat-soluble fraction obtained by further fractionating the ethanol-soluble fraction obtained by fractionating the extract of fermented stevia. I found it. Therefore, for example, by applying the above fat-soluble fraction to the melanocytes of hair matrix cells that produce hair, melanin can be included in the hair, not from the surface of the hair, Whitening can be suppressed.

Before the first fractionation step, a fermentation step is performed in which yeast and water are added to the dried stevia plant tissue and fermentation is performed, and an extract is obtained from the fermentation stevia obtained by the fermentation step using an aqueous ethanol solution. An extraction step.

The fermented stevia extract obtained through the above fermentation step and extraction step is suitable as a fermented stevia extract for obtaining a melanin synthesis promoting substance by the above production method.

The ethanol-soluble fraction obtained in the first fractionation step may be a 30-100% ethanol-soluble fraction.

The fat-soluble fraction fractionated from the 30-100% ethanol-soluble fraction is particularly excellent as a melanin synthesis promoting substance.

According to another example of the embodiment of the present invention, a melanin synthesis promoting substance that promotes melanin synthesis was obtained by further fractionating an ethanol-soluble fraction obtained by fractionating an extract of fermented stevia. Contains fat-soluble fraction.

The external preparation for skin according to another example of the embodiment of the present invention includes a melanin synthesis promoting substance produced by the method for producing a melanin synthesis promoting substance. The skin external preparation may be a cosmetic, a quasi-drug, a pharmaceutical, or a gray hair preventing pharmaceutical.

As another example of the embodiment of the present invention, a method for producing a tyrosinase active substance that activates tyrosinase is disclosed. This production method includes a first fractionation step for fractionating an extract of fermented stevia to obtain an ethanol-soluble fraction, and a second fraction for further fractionating the ethanol-soluble fraction to obtain a fat-soluble fraction. And a process.

As a result of extensive research, the present inventor has found that the fat-soluble fraction obtained by further fractionating the ethanol-soluble fraction obtained by fractionating the extract of fermented stevia has a function of activating tyrosinase. It was. Since tyrosinase is an essential enzyme in the melanin synthesis process, it is possible to improve the ability of melanocytes to synthesize melanocytes by applying the above fat-soluble fraction that activates tyrosinase to melanocytes.

Also, the synthesis of melanin from tyrosine to L-DOPA (3,4-dihydroxy-L-phenylalanine) is performed by tyrosinase. L-DOPA is a precursor of dopamine, which is a neurotransmitter, and can pass through the blood-brain barrier, and thus is used as a useful drug for Parkinson's disease and the like. Therefore, the above fat-soluble fraction that activates tyrosinase can promote the synthesis of tyrosine to L-DOPA, and may be applicable as a drug suitable for Parkinson's disease in the future.

The tyrosinase active substance for activating tyrosinase according to an embodiment of the present invention is a fat-soluble fraction obtained by further fractionating an ethanol-soluble fraction obtained by fractionating an extract of fermented stevia. including.

A pharmaceutical product according to an embodiment of the present invention includes a tyrosinase active substance produced by the above-described method for producing a tyrosinase active substance.

The constituent elements based on the technical idea of the method for producing the melanin synthesis promoting substance and the explanation thereof can be applied to the melanin synthesis promoting substance, the external preparation for skin, the method for producing the tyrosinase active substance, the tyrosinase active substance, and the pharmaceutical. is there.

Depending on the embodiment, the synthesis of melanin can be promoted by activating tyrosinase, and melanin can be contained in the hair that grows in the future to suppress whitening of the hair. Embodiments include, for example, a method for producing a melanin synthesis promoting substance, a melanin synthesis promoting substance, an external preparation for skin, a method for producing a tyrosinase active substance, a tyrosinase active substance, and a pharmaceutical product.

It is explanatory drawing for demonstrating the manufacturing method of the melanin synthesis acceleration | stimulation substance which promotes the synthesis | combination of melanin according to one Example. It is explanatory drawing for demonstrating the biosynthesis pathway of melanin. It is explanatory drawing for demonstrating the experimental procedure of the comparative experiment of the amount of melanin synthesis using B16 melanoma. It is explanatory drawing for demonstrating the number of living cells of B16 melanoma which added the fat-soluble fraction, and control B16 melanoma. It is explanatory drawing for demonstrating the amount of melanins of B16 melanoma which added the fat-soluble fraction, and control B16 melanoma. It is explanatory drawing for demonstrating the experimental procedure for measuring the tyrosinase activity in a cell-free system. It is a figure which shows the formula for evaluating a tyrosinase activation rate. It is explanatory drawing for demonstrating the synthesis amount of the dopaquinone of the mushroom tyrosinase which added the fat-soluble fraction, and the control mushroom tyrosinase. It is explanatory drawing for demonstrating the experimental procedure for measuring the tyrosinase activity in a cell. It is a figure which shows the formula for evaluating a tyrosinase activation rate. It is explanatory drawing for demonstrating the synthesis amount of dopaquinone by the supernatant of a fat-soluble fraction, and the supernatant of a control.

DESCRIPTION OF PREFERRED EMBODIMENTS

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The dimensions, materials, and other specific numerical values shown in the examples are merely examples for facilitating the understanding of the invention, and do not limit the present invention unless otherwise specified. In the present specification and drawings, elements having substantially the same function and configuration are denoted by the same reference numerals, and redundant description is omitted, and elements not directly related to the present invention are not illustrated. To do.

<Example: Method for producing melanin synthesis promoting substance>
FIG. 1 is an explanatory diagram for explaining a method of manufacturing a melanin synthesis promoting substance that promotes melanin synthesis according to one embodiment. As shown in FIG. 1, the method for producing a melanin synthesis promoting substance according to this example includes a step of drying and crushing stevia plant tissue (dry crushing step: S100), and a dried stevia obtained by the dry crushing step S100. A step of fermenting yeast and water by adding yeast and water to the plant tissue (fermentation step: S102) and a step of extracting from the fermentation stevia obtained by the fermentation step S102 using an aqueous ethanol solution to obtain an extract (extraction step: S104) ), A step of fractionating the extract obtained in the extraction step (S104) to obtain an ethanol-soluble fraction (first fractionation step: S106), and a first fractionation step (S108). A step of further fractionating the ethanol-soluble fraction to obtain a fat-soluble fraction (second fractionation step: S108), and the reduced pressure of the fat-soluble fraction obtained by the second fractionation step (S108) , Step concentrated to dryness (concentrated to dryness step: S110) and a. Below, the structure of each process is demonstrated in detail.

(Drying and grinding step S100)
The drying and pulverizing step S100 is a step of drying and pulverizing the plant tissue of the stevia plant as a raw material. Stevia plant which is one of the raw materials of this example is a perennial plant belonging to the family Asteraceae originating from Paraguay in South America. The scientific name for Stevia plant is Stevia Rebaudiana Bertoni (scientific name). When stevia plants are cultivated in Japan, seeds, stock seedlings or cutting seedlings are often planted from February to May and cultivation is often started. Harvesting is often performed from October to early November when Stevia plants are fully mature.

In this example, the melanin synthesis promoting substance is a dry stevia plant tissue powder (hereinafter simply referred to as dry stevia powder) prepared by thoroughly drying the plant tissue of this stevia plant and finely grinding it. Used as Even if it says powder, it is sufficient if it grind | pulverizes so that the magnitude | size of each piece may be about 1 cm or less. Moreover, it is preferable to select and use the leaf part and stem part of a Stevia plant.

It is desirable to dry the leaves and stems of Stevia plants in a room with as much ventilation as possible at room temperature, and it is not preferable to apply heat suddenly.

(Fermentation process S102)
Fermentation process S102 is a process of fermenting the dry stevia powder obtained by dry crushing process S100. Fermentation of the dry stevia powder can be performed by adding water and yeast to the dry stevia powder, stirring and leaving it to stand. The amount of water to be added in the fermentation step S102 only needs to be sufficient for fermentation, and an amount sufficient to wet the whole is sufficient. As yeast, it is preferable to use Saccharomyces.

It is preferable to perform “completely” fermentation of the dry stevia powder. When you lick the unfermented stevia powder, it tastes sweet, but the fully fermented stevia powder has no sweetness. Therefore, it is desirable to proceed the fermentation until the sweetness is no longer felt. For complete fermentation, a standing period of about 1 to 4 weeks is required at room temperature.

Specifically, in the fermentation step S102, moisture is first added to the dried stevia powder, and yeast (for example, Saccharomyces) is further added, and fermentation is continued for 1 to 4 weeks at room temperature while supplying water. Then, when stevia powder is included in the mouth and the sweetness is no longer felt, it is considered to be “completely” fermented.

(Extraction step S104)
In this step, extraction is performed using an aqueous ethanol solution from the fermented stevia powder (hereinafter simply referred to as “fermentation stevia”) obtained in the fermentation step S102. The extract extracted from the fermented stevia obtained in the fermentation step S102 has a function of promoting melanin synthesis or a function of activating tyrosinase. In this example, it is important to use ethanol as the solvent used when extracting from fermentation stevia. This is to fractionate the ethanol-soluble fraction in the subsequent first fractionation step S108.

Extraction is performed by preparing an aqueous solution of about 30% ethanol, adding fermented stevia, and infiltrating at room temperature for several hours to several days with gentle stirring. A filtrate obtained by filtering fermented stevia into about 30% ethanol aqueous solution through a filter paper of about 140 mesh is used as an extract. In this embodiment, the extract is further concentrated under reduced pressure at about 60 ° C. to concentrate the extract.

(First fractionation step S106)
The first fractionation step S106 is a step of fractionating the concentrated extract obtained in the extraction step S104 (hereinafter simply referred to as a concentrated extract) to obtain an ethanol-soluble fraction. In the first fractionation step S106, an ion exchange resin (synthetic adsorbent) is used to fractionate the ethanol soluble fraction. In the first fractionation step S106 in this example, the ethanol-soluble fraction is fractionated from the concentrated extract by passing the concentrated extract through a column packed with DIAION (registered trademark) HP20.

The ethanol soluble fraction obtained in the first fractionation step S106 is preferably a 30-100% ethanol soluble fraction, more preferably a 70-100% ethanol soluble fraction, and even more preferably 85%. It is an ethanol soluble fraction.

(Second fractionation step S108)
The second fractionation step S108 is a step of further fractionating the ethanol-soluble fraction obtained in the first fractionation step S106 to obtain a fat-soluble fraction. In the second fractionation step S108, a weak anion exchange resin is used to fractionate the fat-soluble fraction. In the second fractionation step S108 in the present example, the fat-soluble fraction is fractionated from the ethanol-soluble fraction by passing the ethanol-soluble fraction through a column packed with DIAION (registered trademark) WA30.

In the second fractionation step S108 of this example, first, the ethanol-soluble fraction obtained in the first fractionation step S106 is passed through a column packed with DIAION (registered trademark) WA30, and then DIAION (registered trademark) WA30. Those that were not adsorbed on the surface were taken as the fat-soluble fraction. Such a fat-soluble fraction functions as a melanin synthesis promoting substance.

(Concentration drying step S110)
The concentration / drying step S110 is a step of concentrating and drying the fat-soluble fraction obtained in the second fractionation step S108. In the concentration and drying step S110, the fat-soluble fraction fractionated in the second fractionation step S108 is concentrated to dryness under reduced pressure so that it can be easily stored and transported.

The melanin synthesis promoting substance can be produced through the dry pulverization step S100, the fermentation step S102, the extraction step S104, the first fractionation step S106, the second fractionation step S108, and the concentration and drying step S110 described above. .

Also, the fat-soluble fraction according to the present example promotes melanin synthesis by activating tyrosinase, which is an enzyme essential in the melanin synthesis process. Therefore, the above fat-soluble fraction also functions as a tyrosinase active substance.

<Evaluation>
FIG. 2 is an explanatory diagram for explaining a biosynthesis pathway of melanin. As shown in FIG. 2, melanin is synthesized using tyrosine as a starting material. For example, in melanocytes, tyrosine supplied from the blood is oxidized by tyrosinase, which is a copper-containing enzyme, and synthesized into L-DOPA. Furthermore, L-DOPA is also oxidized by tyrosinase and synthesized into dopaquinone. Dopaquinone is automatically oxidized and synthesized into dopachrome and melanin.

Therefore, the following experiment was conducted to evaluate the promotion of melanin synthesis by the melanin synthesis promoting substance or tyrosinase active substance produced by the above production method.

(Measurement of melanin synthesis in B16 melanoma (mouse melanoma cell line))
FIG. 3 is an explanatory diagram for explaining an experimental procedure of a comparative experiment of the amount of melanin synthesis using B16 melanoma. As shown in FIG. 3, first, in a medium in which the fat-soluble fraction produced by the above production method was added to B16 melanoma, culture was performed at 0.3 × 10 ⁵ cells / well at 37 ° C. under 5% CO ₂ for 96 hours. Went. In addition, as a control, the culture was carried out under the same conditions as the above culture conditions in a medium in which no fat-soluble fraction was added to B16 melanoma.

The B16 melanoma to which the fat-soluble fraction was added and the control B16 melanoma were collected from the culture dish, and the number of viable cells was counted using the Trypan Blue staining method.

FIG. 4 is an explanatory diagram for explaining the number of viable cells of the B16 melanoma to which the fat-soluble fraction was added and the control B16 melanoma. In FIG. 4, error bars added to the bar graph indicate standard deviations (mean ± SD). As shown in FIG. 4, the number of viable cells of the control B16 melanoma (1.37 × 10 ⁶ cells / well (standard deviation 0.12)) and the number of viable cells of the B16 melanoma to which the fat-soluble fraction was added (1.17). Since there is almost no difference in × 10 ⁶ / well (standard deviation 0.15)), it can be seen that the addition of the fat-soluble fraction does not promote the death of B16 melanoma.

Further, 1N NaOH was added to the cultured B16 melanoma collected from the culture dish, and incubated at 75 ° C. for 90 minutes to dissolve the cultured B16 melanoma to obtain a lysate. Then, the absorbance of the solution was measured at a wavelength of 405 nm to calculate the concentration of melanin. In addition, 405 nm is an absorption wavelength of melanin. In addition, a calibration curve was created here using certified synthetic melanin.

FIG. 5 is an explanatory diagram for explaining the amount of melanin between a B16 melanoma to which a fat-soluble fraction is added and a control B16 melanoma. FIG. 5 (a) shows the amount of melanin synthesis per cell. (B) shows the amount of melanin synthesis per well. In FIG. 5, error bars added to the bar graph indicate standard deviation (mean ± SD). As shown in FIG. 5 (a), the control B16 melanoma contains 30.34pg (standard deviation 4.83) melanin per cell, and the B16 melanoma to which the fat-soluble fraction was added was 1 Contains 55.00 pg (standard deviation 6.93) of melanin per cell. Therefore, it can be seen that the B16 melanoma to which the fat-soluble fraction was added as compared with the control B16 melanoma synthesized about 1.6 times the amount of melanin per cell. Further, as shown in FIG. 5 (b), the control B16 melanoma contains 41.27 μg (standard deviation 3.47) of melanin per well, and the B16 melanoma added with the fat-soluble fraction is 1 well. It contains 63.48 μg (standard deviation 5.28) of melanin. Therefore, it can be seen that the B16 melanoma to which the fat-soluble fraction was added as compared with the control B16 melanoma synthesized about 1.5 times as much melanin per well.

Thus, it was found that the fat-soluble fraction produced by the above production method promotes the synthesis of melanin. Therefore, it becomes possible to improve the melanin synthetic ability of a melanocyte by applying the fat-soluble fraction (melanin synthesis acceleration | stimulation substance) manufactured with the said manufacturing method to a melanocyte. That is, whitening of hair can be suppressed by applying the fat-soluble fraction to melanocytes of hair matrix cells that produce hair.

(Measurement of tyrosinase activity in cell-free system)
FIG. 6 is an explanatory diagram for explaining an experimental procedure for measuring tyrosinase activity in a cell-free system. As shown in FIG. 6, first, the fat-soluble fraction was added to 0.1 M PBS (Phosphate Buffered Saline) (pH 6.) while irradiating ultrasonic waves so as to be 100 pg / mL, 100 ng / mL, and 100 μg / mL. Dissolved in 8). Then, 200 unit / mL mushroom tyrosinase was added to each of the 100 pg / mL, 100 ng / mL, and 100 μg / mL fat-soluble fraction solutions, and incubated at 37 ° C. for 10 minutes. As a control, 200 unit / mL mushroom tyrosinase was added to 0.1 M PBS (pH 6.8) and incubated at 37 ° C. for 10 minutes.

Then, 2mM L-DOPA was added to each of the fat-soluble fraction to which mushroom tyrosinase was added and the control to which mushroom tyrosinase was added, and kept at 37 ° C. After 20 minutes, the absorbance was measured at a wavelength of 475 nm every minute. It was measured. In addition, 475 nm is an absorption wavelength of dopaquinone.

FIG. 7 is a diagram showing an equation for evaluating the tyrosinase activation rate, and FIG. 8 is an explanatory diagram for explaining the tyrosinase activity rate of the fat-soluble fraction.

In the equation shown in FIG. 7, the absorbance of a mixed solution of mushroom tyrosinase and L-DOPA to which a fat-soluble fraction at 0 minutes was added (hereinafter simply referred to as a mixture of fat-soluble fractions), fat at 20 minutes Absorbance of a mixture of soluble fractions, absorbance of a mixture of mushroom tyrosinase and L-DOPA (hereinafter simply referred to as a control mixture) added with control at 0 minutes, and control mixture at 20 minutes By substituting the absorbance, the tyrosinase activation rate (%) of the fat-soluble fraction can be calculated.

As shown in FIG. 8, the fat-soluble fraction had a higher tyrosinase activity rate than the control (100% (standard deviation 9.90)). In addition, the tyrosinase activity rate when the fat-soluble fraction was added at 100 pg / mL was 105.99% (standard deviation 10.46), and the tyrosinase activity rate at 100 ng / mL was 114.30% (standard deviation 14.4). 08), the tyrosinase activity rate when added at 100 μg / mL was 118.06% (standard deviation 8.02). Therefore, it was found that the fat-soluble fraction activates tyrosinase in a concentration-dependent manner.

Thus, it was found that the fat-soluble fraction produced by the above production method activates tyrosinase in a cell-free system.

(Measurement of intracellular tyrosinase activity)
FIG. 9 is an explanatory diagram for explaining an experimental procedure for measuring tyrosinase activity in cells. As shown in FIG. 9, first, in a medium in which the fat-soluble fraction produced by the above production method was added to B16 melanoma to a concentration of 100 μg / mL, 0.3 × 10 ⁵ cells / well, 37 ° C., Culture was performed for 96 hours under 5% CO ₂ . In addition, as a control, the culture was carried out under the same conditions as the above culture conditions in a medium in which no fat-soluble fraction was added to B16 melanoma.

Then, the medium was removed, and each of the B16 melanoma to which the fat-soluble fraction was added and the control B16 melanoma were washed with PBS. Each of the B16 melanoma washed with PBS and added with the fat-soluble fraction and the control B16 melanoma were dissolved using a cell lysis protein extraction reagent (for example, Cell-LyEX1 manufactured by Toyo B-Net Co., Ltd.).

The cell lysate obtained using the cell lysis protein extraction reagent was collected and centrifuged at 15000 g at 4 ° C. for 15 minutes to obtain a supernatant.

The protein of the obtained supernatant was quantified (Lowly method), and it was confirmed that the serum contained in the medium was completely removed.

The supernatant of the cell lysate of B16 melanoma to which the obtained fat-soluble fraction was added (hereinafter simply referred to as the supernatant of the fat-soluble fraction), the supernatant of the cell lysate of B16 melanoma as a control, 2 mM, L-DOPA was added to each, and kept at 37 ° C., and absorbance was measured at a wavelength of 475 nm every 10 minutes until 60 minutes had elapsed.

FIG. 10 is a diagram showing an equation for evaluating the tyrosinase activation rate, and FIG. 11 is an explanatory diagram for explaining the tyrosinase activity rate of the supernatant of the fat-soluble fraction. The formula shown in FIG. 10 shows the absorbance of the mixture of the fat-soluble fraction supernatant and L-DOPA at 0 minutes, and the absorbance of the mixture of the fat-soluble fraction supernatant and L-DOPA at 60 minutes. By substituting the absorbance of the mixture of the control supernatant and L-DOPA at 0 minutes and the absorbance of the mixture of the control supernatant and L-DOPA at 60 minutes, the fat-soluble fraction was substituted. The tyrosinase activation rate (%) of the supernatant can be calculated.

As shown in FIG. 11, compared with the control (100% (standard deviation 2.66)), the tyrosinase activity rate of the fat-soluble fraction was 131.55% (standard deviation 19.67). Min supernatant was found to activate 131.55% of tyrosinase compared to control supernatant.

As described above, it was found that the fat-soluble fraction produced by the above production method activates tyrosinase even in cells. Since tyrosinase is an essential enzyme in the melanin synthesis process, it is possible to improve the ability of melanocytes to synthesize melanocytes by applying the above fat-soluble fraction that activates tyrosinase to melanocytes. Therefore, by applying the above fat-soluble fraction to the melanocytes of hair matrix cells that produce hair, melanin can be included in the hair, not from the surface of the hair. It becomes possible to suppress.

Also, the synthesis of melanin from tyrosine to L-DOPA is performed by tyrosinase. L-DOPA is a precursor of dopamine, which is a neurotransmitter, and can pass through the blood-brain barrier, and thus is used as a useful drug for Parkinson's disease and the like. Therefore, the above fat-soluble fraction that activates tyrosinase can promote the synthesis of tyrosine to L-DOPA, and may be applied as a drug (medicine) suitable for Parkinson's disease in the future. There is.

As described above, the details of the method for producing the melanin synthesis promoting substance and the method for producing the tyrosinase active substance according to this example and the experimental results demonstrating the effects of the melanin synthesis promoting substance and the tyrosinase active substance produced by the production method are shown. From the experimental results described above, it was shown that the obtained melanin synthesis promoting substance has a surprising melanin synthesis promoting effect, and that the obtained tyrosinase active substance has a surprising tyrosinase active effect. Therefore, either one or both of the above melanin synthesis promoting substance and tyrosinase active substance can be used in a wide range of skin external preparations such as cosmetics, quasi-drugs, pharmaceuticals, and gray hair prevention medicines. In particular, it is applicable to skin external preparations, cosmetics, quasi-drugs, pharmaceuticals, and gray hair preventives suitable for those with white hair or similar symptoms or those with neurological diseases such as Parkinson's disease or similar symptoms. I can expect.

Here, the term “white hair preventive drug” described in the present specification is a coined word, but means an agent that can promote the expression of the background color of the body hair such as hair (original hair color). Depending on the embodiment, the effect of preventing or delaying whitening of the hair or increasing the color can be expected due to the tyrosinase activity action or melanin synthesis promoting action of the agent. Usually, the color of body hair fades with age, and often turns white at the end, but depending on the example, the effect of preventing, delaying, or reversing the progress of such aging changes may be achieved. It can be expected. For this reason, in some examples, the agent utilizing the present invention may be referred to as an anti-aging agent for preventing aging.

Either one or both of the melanin synthesis promoting substance and the tyrosinase active substance according to the present invention is a pharmaceutical product for preventing white hair (an anti-aging hair agent for preventing hair aging) that returns the hair color to the background color in cosmetics. , Hair cosmetics, hair styling, hair nourishing, scalp, hair coloring, hair rinsing, hair rinse, skin cosmetics / skin lotion, cream, milky lotion, tanning, sunscreen, cleaning, shaving, shaving , Facial rinse, pack, cosmetic oil, body rinse, massage, finishing cosmetics / foundation, makeup base, funny, lipstick, eye makeup, cheek cosmetic, body makeup, cologne / perfume, bath cosmetic, nail makeup For powders, body powders, and pharmaceuticals, powders, fine granules, granules, tablets, capsules, pills, glazes, pencils, contents Agents, liquids for external use, extract, plasters, suppositories, aerosols, gas chemistry, chemical adsorbents, ophthalmic agents, injections, bandages and the like are widely available.

The preferred embodiments of the present invention have been described above with reference to the accompanying drawings, but it goes without saying that the present invention is not limited to these embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the scope of the claims and these are naturally within the scope of the present invention.

In addition, each process in the manufacturing method of the melanin synthesis promotion substance which accelerates | stimulates the synthesis | combination of melanin of this specification, and the tyrosinase active substance which activates tyrosinase does not necessarily need to process in time series along the order described as a flowchart. It is also possible to proceed in parallel.

The present invention is used for, for example, a method for producing a melanin synthesis promoting substance that promotes melanin synthesis, a melanin synthesis promoting substance, an external preparation for skin, a method for producing a tyrosinase active substance that activates tyrosinase, a tyrosinase active substance, and a pharmaceutical. be able to.

S100 ... Drying and grinding step S102 ... Fermentation step S104 ... Extraction step S106 ... First fractionation step S108 ... Second fractionation step S110 ... Concentration drying step

Claims

A method for producing a melanin synthesis promoting substance that promotes melanin synthesis,
A first fractionation step of fractionating the extract of fermented stevia to obtain an ethanol soluble fraction;
A second fractionation step of further fractionating the ethanol-soluble fraction to obtain a fat-soluble fraction;
A method for producing a melanin synthesis promoting substance comprising:
Before the first fractionation step,
A fermentation process in which yeast and water are added to the dried stevia plant tissue and fermented;
Extraction using an aqueous ethanol solution from the fermented stevia obtained by the fermentation step, and obtaining the extract,
The manufacturing method of the melanin synthesis acceleration | stimulation substance of Claim 1 containing this.
The method for producing a melanin synthesis promoting substance according to claim 1 or 2, wherein the ethanol-soluble fraction obtained in the first fractionation step is a 30 to 100% ethanol-soluble fraction.
A melanin synthesis promoting substance that promotes the synthesis of melanin, including a fat-soluble fraction obtained by further fractionating an ethanol-soluble fraction obtained by fractionating an extract of fermented stevia.
A skin external preparation containing a melanin synthesis promoting substance produced by the production method according to any one of claims 1 to 3.
The external preparation for skin according to claim 4, which is a cosmetic.
The external preparation for skin according to claim 4, which is a quasi-drug.
The external preparation for skin according to claim 4, which is a pharmaceutical product.
The external preparation for skin according to claim 4, which is a pharmaceutical product for preventing white hair.
A method for producing a tyrosinase active substance that activates tyrosinase, comprising:
A first fractionation step of fractionating the extract of fermented stevia to obtain an ethanol soluble fraction;
A second fractionation step of further fractionating the ethanol-soluble fraction to obtain a fat-soluble fraction;
A method for producing a tyrosinase active substance.
Before the first fractionation step,
A fermentation process in which yeast and water are added to the dried stevia plant tissue and fermented;
Extraction using an aqueous ethanol solution from the fermented stevia obtained by the fermentation step, and obtaining the extract,
The manufacturing method of the tyrosinase active material of Claim 10 containing this.
The method for producing a tyrosinase active substance according to claim 10 or 11, wherein the ethanol-soluble fraction obtained in the first fractionation step is a 30 to 100% ethanol-soluble fraction.
A tyrosinase active substance that activates tyrosinase, including a fat-soluble fraction obtained by further fractionating an ethanol-soluble fraction obtained by fractionating an extract of fermented stevia.
A pharmaceutical comprising the tyrosinase active substance produced by the production method according to any one of claims 10 to 12.