JP7034036B2 - Whitening agent screening method - Google Patents

Description

The present disclosure relates to screening methods, formulations, and fibrosis inhibitors for whitening agents.

Melanin is a pigment formed in animals including humans, plants, protozoa, some fungi, and eubacteria. In vertebrates, most of the melanin is produced by melanocytes (pigment cells) in the basal layer of the lowest layer of the epidermis of the skin and the hair matrix of the hair, and some are produced by the retinal pigment epithelial cells.
In melanocytes, melanin is synthesized in granules called melanosomes, and then
Melanosomes are transported to the surrounding keratinocytes (epidermal cells). Melanin is a medically important factor that absorbs UV light, which destroys DNA in the cell nucleus, and reduces the risk of skin cancer. However, excessive production of melanin in the skin may cause blemishes, age spots, freckles, and the like.

Formulations containing various whitening agents have been developed in order to suppress the above-mentioned blemishes, age spots, freckles and the like. Further, in the development of the pharmaceutical product containing the whitening agent, various methods are used as a screening method for the whitening agent for searching for the whitening agent.
For example, Patent Document 1 describes a method for screening a whitening agent using an action of suppressing the production of melanin fattening as an index.
Patent Document 2 describes a method for screening a whitening component, which comprises using fibroblasts.
Non-Patent Document 1 describes that the degradation of PMEL protein by BACE2, which is a kind of β-secretase, is associated with melanin production.

Japanese Unexamined Patent Publication No. 2014-408235 Japanese Unexamined Patent Publication No. 2015-204780

BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells., Rochin L et al., Proc. Natl. Acad. Sci. U.S.A. 2013 Jun 25; 110 (26): 10658-63.

Here, examples of currently known whitening agents include substances that inhibit melanin biosynthesis, substances that suppress signal transmitters that promote melanin biosynthesis, and the like.
Patent Documents 1 and 2 describe methods for screening whitening agents, respectively. However, there are still many unclear points in the mechanism by which blemishes, blemishes, freckles, etc. occur, such as melanin biosynthesis, and the screening method described in Patent Document 1 or 2 is not sufficient in searching for a whitening agent. , Development of further new screening methods is required.
Further, although Non-Patent Document 1 suggests a relationship between the enzyme BACE2 in pigment cells and melanin production, there is no description about a method for screening a whitening agent.

An object to be solved by the embodiment according to the present disclosure is to provide a novel whitening agent screening method and a preparation containing the whitening agent discovered by the above-mentioned whitening agent screening method.
Further, a problem to be solved by another embodiment according to the present disclosure is to provide a novel fibrosis inhibitor.

The means for solving the above problems include the following aspects.
<1> A method for screening a whitening agent, which comprises confirming the cleavage state of the β site of PMEL protein in cells having melanin-producing ability.
<2> The method for screening a whitening agent according to <1>, wherein the method for confirming the cleavage state is the quantification of PMEL-Mβ fragments.
<3> The method for screening a whitening agent according to <1> or <2>, wherein the cells are normal human cells.
<4> The method for screening a whitening agent according to any one of <1> to <3>, which further comprises imparting a test substance to the cells before confirming the cleavage state.
<5> A method for screening a whitening agent, which comprises measuring the inhibitory activity of β-secretase by a test substance.
<6> The method for screening a whitening agent according to <5>, wherein the β-secretase is BACE2.
<7> The method for screening a whitening agent according to any one of <4> to <6>, wherein the test substance does not have tyrosinase inhibitory activity.
<8> The method for screening a whitening agent according to any one of <1> to <7> above, which further comprises measuring the amount of melanin produced in cells.
<9> A preparation containing a whitening agent found by the method for screening a whitening agent according to any one of <1> to <8> above.
<10> The preparation according to <9> above, which is an external preparation for skin.
<11> A fibrosis inhibitor containing oryzanol.

According to the embodiment according to the present disclosure, it is possible to provide a novel whitening agent screening method and a preparation containing the whitening agent discovered by the above-mentioned whitening agent screening method.
Further, according to another embodiment according to the present disclosure, a novel fibrosis inhibitor can be provided.

It is a schematic explanatory diagram of the guessing mechanism that a fiber is formed by a part of PMEL protein. It is a graph which shows the result of the cutting state confirmation process when the BACE inhibitor was used. It is a graph which shows the melanin production inhibitory effect when a BACE inhibitor is used. It is a graph which shows the result of the inhibition activity measurement step when oryzanol was used. It is a graph which shows the melanin production inhibitory effect when oryzanol is used. It is a photograph which shows the result of the transmission electron microscope observation of the melanosomes when the BACE inhibitor was used. It is a graph which shows the melanin production inhibitory effect of BACE inhibition in a skin model.

The contents of the present disclosure will be described in detail below. The description of the constituents described below may be based on the representative embodiments of the present disclosure, but the present disclosure is not limited to such embodiments.
In the present disclosure, the numerical range represented by using "-" means a range including the numerical values before and after "-" as the lower limit value and the upper limit value.
In the numerical range described stepwise in the present disclosure, the upper limit value or the lower limit value described in one numerical range may be replaced with the upper limit value or the lower limit value of the numerical range described in another stepwise description. .. Further, in the numerical range described in the present disclosure, the upper limit value or the lower limit value of the numerical range may be replaced with the value shown in the examples.
In the present disclosure, the term "process" is included in this term not only as an independent process but also as long as the intended purpose of the process is achieved even if it cannot be clearly distinguished from other processes.
In the present disclosure, the amount of each component of the composition means the total amount of the plurality of substances present in the composition when a plurality of substances corresponding to each component are present in the layer, unless otherwise specified.
In the present disclosure, the combination of preferred embodiments is a more preferred embodiment.

(Screening method for whitening agents)
The first aspect of the method for screening a whitening agent according to the present disclosure includes confirming the cleavage state of the β site of PMEL protein in cells having a melanin-producing ability (cutting state confirmation step).
In addition, the second aspect of the method for screening a whitening agent according to the present disclosure includes measuring the inhibitory activity of β-secretase by a test substance (inhibitory activity measuring step).

The whitening agent screening method according to the present disclosure is a novel whitening agent screening method.
The first aspect of the screening method for a whitening agent according to the present disclosure focuses on the cleavage state of the β site of PMEL protein in cells having a melanin-producing ability.
In the present disclosure, the β site of PMEL protein refers to a site cleaved by β-secretase enzyme (β site cleavage enzyme) in PMEL protein, and is considered to be a site contained in gap domain 2 (GAP2) described later. ..
The second aspect of the method for screening a whitening agent according to the present disclosure focuses on the inhibitory activity of β-secretase in cells having a melanin-producing ability.
The present inventors presume that the formation of blemishes, blemishes, freckles and the like proceeds through, for example, the following four stages I to IV.
Stage I: Vesicles are formed from the Golgi apparatus in cells capable of producing melanin (for example, melanocytes), and some of the vesicles become melanosomes.
Stage II: PMEL protein is cleaved by an enzyme such as β-secretase, and fibers are formed by a part of the PMEL protein cleaved in the above melanosomes.
Stage III: Melanin synthesis by tyrosinase is initiated on the fibers.
Stage IV: The produced melanin is transported to keratinocytes and the like.

Here, the PMEL protein is a membrane protein of the above melanosomes, which is also called PMEL17, gp100, silver (SILV) or the like.
However, for example, when the activity of β-secretase is increased, the PMEL protein is cleaved at the β site in the melanin-producing cell, and a part of the cleaved PMEL protein undergoes further cleavage and the like. Furthermore, it is considered that the PMEL protein after cleavage acts as a scaffold for accumulating melanin by tyrosinase, and the fibers are formed in melanosomes.
The cause of the improvement in the activity of β-secretase may be irradiation with ultraviolet rays, aging, etc., but it is not clear.

In view of the above mechanism, as a result of diligent studies by the present inventors, a new criterion for a whitening agent screening method is provided by confirming the cleavage state of the β site of PMEL protein in cells having melanin-producing ability. We have found that we can do this, and have completed the method according to this disclosure.
Similarly, as a result of diligent studies by the present inventors, it was found that by measuring the inhibitory activity of β-secretase by the test substance, a new criterion can be provided in the screening method for whitening agents. The method for disclosure has been completed.
For example, when the cleavage of the β site of PMEL protein is suppressed or the activity of β-secretase is suppressed by applying a certain compound to the cells, for example, in the above speculation mechanism, the above-mentioned melanin is used. By the mechanism that the generation of PMEL protein fragments that can serve as an accumulated scaffold is suppressed and the subsequent melanin synthesis by tyrosinase is also suppressed, the formation of blemishes, age spots, freckles, etc. is also suppressed, and as a whitening agent. It is presumed to be useful.

That is, according to the first aspect or the second aspect of the whitening agent screening method according to the present disclosure, for example, a conventional whitening agent by inhibiting tyrosinase activity, the whitening agent described in the above-mentioned Patent Document 1 or Patent Document 2. It is considered possible to screen a whitening agent having a new mechanism of action, which is different from the above. That is, for example, by combining a conventional whitening agent (for example, a whitening agent having a tyrosinase inhibitory activity) and a whitening agent screened by the whitening agent screening method according to the present disclosure, a whitening agent exhibiting a further whitening effect can be achieved. The agent may be available.
At present, the cause of cleavage of the β site of the PMEL protein or activation of the β secretase is not clear, but if the cause of these causes is clarified in the future, the whitening according to the present disclosure will be made. Depending on the method of screening the agent, it may be possible to provide a whitening agent having a different application target or use from the conventional whitening agent, such as the timing of application to the living body, the method of application, and the amount of application.
Hereinafter, the details of the first aspect or the second aspect of the whitening agent screening method according to the present disclosure will be described.

<First aspect>
The first aspect of the method for screening a whitening agent according to the present disclosure comprises confirming the cleavage state of the β site of PMEL protein in cells having a melanin-producing ability.

[Cut status confirmation process]
In the cutting state confirmation step, any evaluation system that can confirm that the β site is cut can be used without particular limitation.
For example, when the test substance described later is applied to the cells and the cleavage of the β site is suppressed, the test substance is useful as a whitening agent, or when the cleavage of the β site is not suppressed. In addition, it can be determined that the test substance (or the dose of the test substance, etc.) may be unsuitable as a whitening agent.

FIG. 1 shows a schematic explanatory diagram of the speculation mechanism in which fibers are formed by a part of PMEL protein in the above-mentioned stage II.
FIG. 1 (A) shows an example of PMEL protein 10A present in the melanosome membrane 100.
In PMEL protein 10A, C indicates the C-terminal side and N indicates the N-terminal side.
Further, in the melanosome membrane 100, the C-terminal side of PMEL protein 10A is the cytoplasmic side, and the N-terminal side is the melanosome internal side.
The PMEL protein 10A contains a signal peptide domain 12, an N-terminal domain (NTD) 14, a polycystic kidney disease-like domain (PKD) 16, a gap domain 1 (GAP1) 18, and a proline. / Serine / threonine-rich repeat domain (RPT) 20, gap domain 2 (GAP2) 22, klingle-like domain (KLD) 24, and gap domain 3 (KLD) It has a GAP3) 26, a transmembrane domain (TM) 28, and a C-terminal domain (CTD) 30. Further, the disulfide bond 32 is a bond existing between NTD14 and KLD24 in PMEL protein 10.
GAP2 22 has a cleavage site 34 due to Kex2 protease, which is on the N-terminal side of the cleavage site 34 and contains a part of GAP2 22, RPT 20, GAP1 18, PKD 16, and NTD 14. The region is called the Mα region, the region on the C-terminal side of the cleavage site 34, and the region including a part of GAP2222, KLD 24, GAP3 26, TM 28, and CTD 30. Further, in the present disclosure, the fragment consisting of the Mα region (the fragment on the N-terminal side in FIG. 1C) is referred to as the PMEL-Mα fragment, and the fragment consisting of the Mβ region (the fragment on the C-terminal side in FIG. 1C) is referred to. Also called PMEL-Mβ fragment.
As shown in FIG. 1 (B), once the signal peptide domain 12 is cleaved in the melanosomes, it is believed that it is cleaved at the cleavage site 34 in GAP222 by a proprotein convertase.
After that, as shown in FIG. 1 (C), it is considered that the site (β site) shown by the broken line 36 in GAP3 26 is cleaved by an enzyme such as β-secretase (for example, BACE2).
After cleavage of the β site, as shown in FIG. 1 (D), the PMEL proteins 10B and 10C after cleavage of the β site were cleaved at the sites shown by the broken lines 38, 40, and 42, and the fragments 44, 46, 48, It is presumed to be decomposed into 50 and 52. Of these fragments, fragments 46 and / or 48 are presumed to form fibers that can serve as a scaffold for the above-mentioned melanin accumulation, but it is uncertain.

As an example of the method for confirming the cleavage state, there is a method of measuring (quantitating) the amount of PMEL protein fragment before cleavage at the β site.
It is considered that the larger the amount of the PMEL protein fragment before being cleaved at the β site, the more the formation of fibers that can serve as a scaffold for accumulating the above-mentioned melanin is suppressed, and the more useful as a whitening agent.
The fragment of the PMEL protein before being cleaved at the β site is not particularly limited, and examples thereof include the Mβ fragment (PMEL-Mβ fragment) before the β site is cleaved in FIG. 1 (C).
Specifically, the cleavage state can be evaluated based on, for example, the amount of PMEL-Mβ fragment (protein amount A) in the cells after the application of the test substance described later, and the application of the test substance is not performed. In this case, it is preferable to evaluate by comparing the amount of PMEL-Mβ fragment (protein amount B) in the cells with the protein amount A. In addition, the protein amount of the PMEL-Mβ fragment in cells to which a known β-secretase inhibitor has been applied may be measured and further compared with the protein amount A (and, if necessary, the protein amount B) for evaluation. ..
That is, the method for confirming the cleavage state in the first aspect of the whitening agent screening method according to the present disclosure is preferably quantification of PMEL-Mβ fragment.

As an example, the protein amount A and the protein amount B are measured at n = 3 or more, respectively, and the average of each sample value for the protein amount A in the sample group for the protein amount A and the sample group for the protein amount B. The null hypothesis was rejected at a significance level of 5% (preferably 1%) when the value was larger than the mean value of each sample value for protein amount B and the Student's t-test was performed for each group. If so, the cleavage is suppressed, and it can be determined that the test substance has the potential to be useful as a whitening agent. The statistical analysis processing method such as the Student's t-test is not particularly limited, and an appropriate method may be selected from known methods. The number of n can be changed as appropriate.

As another example of the method for confirming the cleavage state, there is a method of measuring (quantitating) the amount of PMEL protein fragment after cleavage at the β site.
The fragment of the PMEL protein after the β site is cleaved in the measurement of the protein amount B is not particularly limited, and any fragment in the sequence of the PMEL protein in which the β site of the PMEL protein is cleaved may be measured. Examples thereof include proteins 10B and 10C after β-site cleavage in FIG. 1D, and proteins represented by fragments 44, 46, 48, 50 or 52.
Among these proteins, for example, it is preferable to quantify the CTF fragment (protein 10C) and the HMB45 fragment (fragments 44, 46, 48 and / or 50).

Here, in the present disclosure, the amount of protein in the above cells may be measured by, for example, known SDS-PAGE (polyacrylamide gel electrophoresis) or the like. It is possible to confirm that the protein contained in the cell is each of the above-mentioned fragments, for example, by the antibody to which it binds, the molecular weight of the protein, or the like.
In addition, mass spectrometry such as Matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF MS), ELISA (Enzyme-Linked ImmunoSorbent Assay), biopsy The cleavage state of the β site in the PMEL protein may be confirmed by a known immunostaining method or the like for the sample obtained in.

-cell-
The cells used in the above-mentioned cleavage state confirmation step are not particularly limited as long as they have the ability to produce melanin, but are mammalian cells such as humans, mice, rats, guinea pigs, rabbits, sheep, pigs, goats, dogs, cats, and monkeys. It is preferable, and it is more preferable that it is a human cell.
Further, the cells may be normal cells or cancer cells.
For example, from the viewpoint of using the first aspect of the whitening agent screening method according to the present disclosure as a whitening agent screening for human use, the environment actually used as a whitening agent can be reproduced more accurately and closely, so that human normal cells can be reproduced. Is preferably used.

The cells from which the cells are derived and the state of differentiation are not particularly limited, but melanoma or human pigment cells (melanocytes) are preferable from the viewpoint of availability. In particular, normal human epidermal melanocytes are more preferable from the viewpoint of being able to more accurately and closely reproduce the environment actually used as a whitening agent.
In the first aspect of the whitening agent screening method according to the present disclosure, a system containing cells can be used without particular limitation, and for example, a living body, a tissue, a cell culture system, or the like may be used, but an experiment is performed. From the viewpoint of convenience, it is preferable to use a cell culture system.
As the cell culture system, a cell culture system containing one type of cell alone, a co-culture system containing two or more types of cells, or the like can be used without particular limitation. A known culture system such as a layer culture system or a model culture system in which a three-dimensional model of skin is constructed can be used without particular limitation, but from the viewpoint of simplicity of experiment, it is preferable to use a single layer culture system.
The cultured cells in the cell culture system may be either primary cultured cells or second-generation or later strain cells as long as they are normal cells, and the number of passages can be used without particular limitation. Generally, it is preferable that the cells have a passage number of 15 or less.
In addition, as for the detailed culture method and the like, a known method can be used without particular limitation.

[Test substance application process]
In the first aspect of the method for screening a whitening agent according to the present disclosure, it is preferable to include adding a test substance (test substance applying step) before the cutting state confirmation step.
The above-mentioned test substance is a substance considered as a candidate for a whitening agent. For example, when the cleavage of β site is suppressed in the above-mentioned cutting state confirmation step, it is determined that the above-mentioned test substance may be useful as a whitening agent. Will be done.
Further, in the test substance application step, the test substance may be applied alone or in combination of two or more.
The method for applying the test substance is not particularly limited, and a known method may be used.
For example, methods such as application to a living body, tissue, etc., addition to a medium in a cell culture system (a solvent such as DMSO (dimethyl sulfoxide) may be used if necessary) and the like can be considered.
The concentration of the test substance to be applied, the timing of application, the method of application, and the like may be appropriately set in consideration of the physical characteristics of the test substance to be used, the toxicity of the test substance to a living body, the type of object to be applied, and the like. For example, by performing the first aspect of the whitening agent screening method according to the present disclosure using a plurality of groups in which the application concentration, application timing, application method, etc. are changed, an appropriate application concentration, application timing, application method, etc. May be used to estimate.

As the test substance according to the present disclosure, a substance having no tyrosinase inhibitory activity, that is, a substance generally not known to have a tyrosinase inhibitory activity, or a substance having a tyrosinase inhibitory activity is determined by using a known screening method. You may use what you cannot do.
As the whitening agent, a compound having a tyrosinase inhibitory activity and the like are already known, but according to the first aspect of the whitening agent screening method according to the present disclosure, the test substance does not have the tyrosinase inhibitory activity. However, it will be possible to discover a test substance useful as a whitening agent by a new mechanism of action.

[Step of changing melanin production]
In the first aspect of the method for screening a whitening agent according to the present disclosure, the amount of melanin produced in the cells may be further changed (step of changing the amount of melanin produced).
For example, in the first aspect of the method for screening a whitening agent according to the present disclosure, melanin-producing cells are irradiated with ultraviolet rays after or while the above-mentioned test substance is applied to enhance melanin production. Etc. are conceivable.
Further, for example, the above-mentioned test substance may be imparted to cultured cells while previously imparting and / or imparting a compound that increases the amount of melanin produced. Examples of the compound that increases the amount of melanin produced include, but are not limited to, insulin, transferrin, cyclic adenosine monophosphate (cAMP), and the like.
In addition, the method for screening a whitening agent according to the present disclosure may be performed while suppressing the amount of melanin production, for example, by adding a compound that suppresses tyrosinase activity before or at the same time as adding the test substance.
The method for changing the amount of melanin produced is not particularly limited, but is known in the field of whitening agent screening such as application of a known compound, application of oxidative stress using an oxidizing agent such as hydrogen peroxide, and irradiation with ultraviolet rays. Method can be used without particular limitation.

[Melanin production measurement process]
The first aspect of the method for screening a whitening agent according to the present disclosure may further include measuring the amount of melanin produced in cells (step of measuring the amount of melanin produced).
The cell used in the melanin production amount measuring step may be a cell of the same type as the cell in the above-mentioned cleavage state confirmation step, or may be a cell of another type.
The preferred embodiment of the cell used in the melanin production amount measuring step is the same as the preferred embodiment of the cell in the above-mentioned cleavage state confirmation step.
By further including the step of measuring the amount of melanin produced, the first aspect of the screening method for a whitening agent according to the present disclosure makes it possible to add a criterion for determining whether or not it is useful as a whitening agent, and as a whitening agent. It may improve the accuracy of screening for useful compounds.
As a method for measuring the amount of melanin produced, a known method can be used without particular limitation.

[Other processes]
In addition, the first aspect of the method for screening a whitening agent according to the present disclosure may further include the step of measuring the inhibitory activity in the second aspect described later.
In addition, the first aspect of the whitening agent screening method according to the present disclosure may further include a whitening agent screening method showing a mechanism different from the above-mentioned mechanism of suppressing cleavage of the β site of PMEL protein. For example, an activity test of a gene involved in melanin production (for example, a MITF (Microphthalmia-associated transcription factor) gene, etc.), an activity measurement that contributes to melanin excretion (turnover) or degradation, and the like can be mentioned.
For example, a compound that suppresses cleavage of the β site of PMEL protein obtained by the first aspect of the screening method for a whitening agent according to the present disclosure and a compound that suppresses the expression of a gene involved in melanin production are used in combination. By doing so, it is considered that a preparation having a strong whitening effect may be obtained.
In addition, a compound that suppresses cleavage of the β site of PMEL protein and a compound that promotes melanin excretion or decomposition, which are obtained by the first aspect of the whitening agent screening method according to the present disclosure, are used in combination. Therefore, it is considered that a preparation having a strong whitening effect may be obtained.
Examples of such compounds include Japanese Patent No. 05530875, Japanese Patent No. 05635932, Japanese Patent No. 06121199, Japanese Patent No. 06626785, Japanese Patent Application Laid-Open No. 2015-051931, JP-A-2015-010071, and Japanese Patent Application Laid-Open No. 2015- Examples thereof include the compounds described in Japanese Patent Application Laid-Open No. 010070 and Japanese Patent Application Laid-Open No. 2014-210825.

<Second aspect>
A second aspect of the method for screening a whitening agent according to the present disclosure comprises measuring the inhibitory activity of β-secretase by a test substance.

[Inhibition activity measurement step]
In the inhibitory activity measuring step, an evaluation system capable of confirming that the activity of β-secretase is inhibited can be used without particular limitation.
For example, when the test substance, β-secretase, and the substrate of β-secretase are mixed and the activity of β-secretase is inhibited, the test substance is useful as a whitening agent, or the activity of β-secretase is inhibited. If not, or if the degree of inhibition is low, it can be determined that the test substance (or the dose of the test substance, etc.) may be unsuitable as a whitening agent.
As the test substance, it is preferable to use the test substance in the test substance application step in the above-mentioned first aspect.

When the activity of β-secretase is inhibited by the test substance, it is considered that when the test substance is added to the cells, the cleavage of the protein at the β site shown in FIG. 1 (C) above is suppressed. As a result, the generation of the fragments 46 and / or 48 shown in FIG. 1 (D) is suppressed, so that the formation of fibers that can serve as a scaffold for accumulating the above-mentioned melanin is suppressed, and spots and freckles are suppressed. , It is presumed that the occurrence of freckles, etc. is suppressed, but it is not certain.

Here, β-secretase in the present disclosure refers to an enzyme that cleaves at least one of the gap domains 2 of PMEL protein, and BACE1 known as an enzyme that cleaves amyloid β precursor protein (APP). Since PMEL protein is considered to be less involved in the cleavage of β-site, it is not included in β-secretase in the present disclosure.
BACE2 is an enzyme known at present that cleaves the β site of PMEL protein.
That is, the β-secretase whose activity is measured in the second aspect of the whitening agent screening method according to the present disclosure is preferably BACE2.
However, if an enzyme that cleaves the β-site of another PMEL protein is discovered in the future, the above-mentioned enzyme shall be included in the β-secretase in the present disclosure as long as it cleaves the β-site of the PMEL protein.
Further, as the BACE2, a recombinant protein may be used as long as it is an enzyme that cleaves the β site of the PMEL protein.
The recombinant protein may be used, for example, for the purpose of improving properties such as stability and solubility of β-secretase.
The origin of β-secretase is not particularly limited, and it may be a reagent synthesized via cells or a commercially available reagent. From the viewpoint of making the screening method simpler, reagents are preferable.

As a method for measuring the activity of β-secretase, a known method is used without particular limitation.
One example is a method of measuring a sample containing β-secretase by using a SensoLyte520 BACE2 Activity Assay Kit (manufactured by Funakoshi Co., Ltd.) or the like.
Specifically, a method for determining the presence or absence of cleavage by allowing a FRET substrate that emits fluorescence when cleaved by β-secretase acts on a sample containing a test substance and β-secretase (BACE2, etc.), β such as recombinant PMEL protein, etc. Examples thereof include a method of measuring the amount of fragment generated after the protein cleaved by secretase is allowed to act by a known method such as SDS-PAGE, but the present invention is not particularly limited.

In addition, inhibition of the above activity can be evaluated based on, for example, the activity of β-secretase in the presence of the above-mentioned test substance (activity A), and the activity (activity) of β-secretase in the absence of the above-mentioned test substance. It is preferable to evaluate by comparing B) with the activity A.
Further, it may be evaluated in comparison with the activity of β-secretase in the presence of a known β-secretase inhibitor such as BACE2 inhibitor.

As an example, the activity A and the activity B are measured at n = 3 or more, respectively, and in the sample group for the activity A and the sample group for the activity B, the average value of each sample value for the activity A is the activity B. If the null hypothesis is rejected at a significance level of 5% (preferably 1%) when the student's t-test is performed for each group, which is smaller than the mean value of each sample value for. Is suppressed, and it can be determined that the above-mentioned test substance has the potential to be useful as a whitening agent. The statistical analysis processing method such as the Student's t-test is not particularly limited, and an appropriate method may be selected from known methods. The number of n can be changed as appropriate.

[Test substance application step, melanin production amount change step, melanin production amount measurement step, and other steps]
The second aspect of the method for screening a whitening agent according to the present disclosure may further include a step using cells.
The step using cells may further include at least one step selected from the group consisting of a test substance application step, a melanin production amount change step, a melanin production amount measurement step, and other steps.
In the second aspect of the whitening agent screening method according to the present disclosure, the test substance application step, the melanin production amount change step, the melanin production amount measurement step, and the other steps are the above-mentioned whitening agent according to the present disclosure, respectively. It is synonymous with these steps in the first aspect of the screening method. Specifically, it can include a step in which the description of "the first aspect of the whitening agent screening method" in the description of each of the above steps is read as "the second aspect of the whitening agent screening method".

The second aspect of the method for screening a whitening agent according to the present disclosure preferably further comprises measuring the amount of melanin produced in cells (step of measuring the amount of melanin produced).
Examples of the cells include cells similar to the cells used in the above-mentioned cutting state confirmation step. Specifically, the description of "first aspect of the whitening agent screening method" in the description of the cells used in the above-mentioned cleavage state confirmation step is read as "second aspect of the whitening agent screening method". Can be used.
For example, in the second aspect of the whitening agent screening method according to the present disclosure, the test substance is added to the cells by the test substance application step after or while performing the melanin production amount change step as necessary. Then, the melanin production amount can be measured by the above-mentioned melanin production amount measurement step.
By further including the step of measuring the amount of melanin produced, the second aspect of the method for screening a whitening agent according to the present disclosure makes it possible to add a criterion for determining whether or not the whitening agent is useful as a whitening agent, and as a whitening agent. It may improve the accuracy of screening for useful compounds.

(Formula and method of designing the product)
The pharmaceutical product according to the present disclosure includes a whitening agent found by the screening method for the whitening agent according to the present disclosure.
The form of the pharmaceutical product according to the present disclosure is not particularly limited except that it contains the above-mentioned whitening agent, and examples thereof include an oil composition, an emulsified composition, and a powder composition. Further, the pharmaceutical product according to the present disclosure can be prepared according to a known method.
Further, the pharmaceutical product according to the present disclosure is preferably a cosmetic product. The form of the cosmetic is not particularly limited, and cosmetics such as lotion, beauty essence, cream, and milky lotion can be exemplified. Any of these can be prepared by a known method, and in the case of a milky lotion, for example, it can be produced by heating and dissolving the aqueous phase and the oil phase, respectively, emulsifying and dispersing, and cooling.

Examples of the pharmaceutical product according to the present disclosure include those administered orally or parenterally, and it is preferable to administer them parenterally. In addition, local administration that can be directly administered to the skin is preferable, and transdermal administration is more preferable.
That is, the preparation according to the present disclosure is preferably a skin external preparation.

The pharmaceutical product according to the present disclosure may contain other ingredients in addition to the above-mentioned whitening agent.
The other components may be appropriately selected depending on the form and purpose of the composition, and examples thereof include functional oily components, emulsifiers, and other additive components.

In addition, the pharmaceutical product according to the present disclosure may further contain a whitening agent other than the above-mentioned whitening agent.
Examples of other whitening agents include compounds in which suppression of β-site cleavage of PMEL protein is not observed, compounds in which β-secretase inhibitory activity is not observed, and the like, for example, compounds that suppress the expression of genes involved in melanin production. , Compounds that promote the excretion (turnover) or decomposition of melanin, and the like are preferable.

<Functional oil component>
The functional oil-based component is not particularly limited as long as it is an oil-soluble component that does not dissolve in an aqueous medium but dissolves in an oil-based medium, and a component having physical properties and functionality according to the purpose should be appropriately selected and used. Can be done. The functional oil component is a component other than the above-mentioned whitening agent, and is an ultraviolet absorber, an anti-inflammatory agent, a moisturizer, a hair protectant, a cell activator, an emollient agent, a keratolytic agent, an antistatic agent, and a fat-soluble component. Examples include those used as vitamins, metabolic syndrome improving agents, antihypertensive agents, sedatives, and the like.
Here, the functional oily component means an oily component that can be expected to exert a desired physiological action in a living body.

<Other additive ingredients>
In addition to the above-mentioned components, additive components usually used in the fields of pharmaceuticals, functional foods, cosmetics and the like may be appropriately contained in the composition according to the form thereof. The other additive components can be contained in the composition as oil-soluble or water-soluble additive components in consideration of their characteristics.

Other additives include polyhydric alcohols such as glycerin and 1,3-butylene glycol; glucose, sugar, lactose, maltose, sucrose, pectin, copper carrageenan, etc.
Monosaccharides or polysaccharides such as Locust Bean Gum, Gua Gum, Hydroxypropyl Gua Gum, Xanthan Gum, Karaya Gum, Tamarind Seed Polysaccharide, Arabian Gum, Tragacanth Gum, Hyaluronic Acid, Sodium Hyaluronate, Sodium Chondroitin Sulfate, Dextrin; Sugar alcohols such as lactose, maltotriitol, xylitol; inorganic salts such as sodium chloride and sodium sulfate; proteins with a molecular weight of over 5000 such as casein, albumin, methylated collagen, hydrolyzed collagen, water-soluble collagen, gelatin; glycine, valine , Leucine, isoleucine, serine, threonine, aspartic acid, glutamate, cystine, methionine, lysine, hydroxylysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, acetylhydroxyproline and other amino acids and their derivatives; Synthetic polymers such as vinyl polymers, sodium polyacrylates, polyvinyl alcohols, polyethylene glycols, ethylene oxide / propylene oxide block copolymers; water-soluble cellulose derivatives such as hydroxyethyl cellulose and methyl cellulose; Based on this, it may be contained, for example, as a functional ingredient, an excipient, a viscosity modifier, a radical trapping agent, or the like.
In addition, other additives usually used for that purpose, such as pH adjusters, pH buffers, preservatives, fragrances, and colorants, can be used in combination.

The method for designing a pharmaceutical product in the present disclosure includes incorporating a test substance selected as a whitening agent by the screening method for a whitening agent according to the present disclosure into the pharmaceutical product.
The method for designing a pharmaceutical product in the present disclosure may further include the inclusion of other components in the pharmaceutical product according to the present disclosure described above in the pharmaceutical product.
The method for designing the preparation in the present disclosure may include performing the screening method for the whitening agent according to the present disclosure described above, or the preparation may be based on the result of the screening method for the whitening agent according to the present disclosure performed by another person. It may be a design method to be designed.

(Fiber formation inhibitor)
The fibrosis inhibitor according to the present disclosure includes oryzanol.
Oryzanol is a ferulic acid ester in which ferulic acid and sterol are condensed, which is contained in lipids such as rice bran.
As constituents of oryzanol, cycloartenol ferulic acid ester, 24-methylenecycloartanol ferulic acid ester, campesterol ferulic acid ester, β-sitosterol ferulic acid ester, cyclobutanol ferulic acid ester and the like are known.
Here, oryzanol is a compound confirmed to suppress cleavage of the β site of PMEL protein in the first aspect of the screening method for a whitening agent according to the present disclosure.
Oryzanol is a compound confirmed to have β-secretase inhibitory activity in the second aspect of the whitening agent screening method according to the present disclosure.
The fibrosis inhibitor according to the present disclosure may be a whitening preparation, but it is also considered that it can be used as a therapeutic agent for Alzheimer's disease, for example.
Although the mechanism of action of Alzheimer's disease has not yet been completely elucidated, it has been reported that cleavage of amyloid β precursor protein by BACE1 or β-secretase and the accompanying formation of fibers are associated with the progression of Alzheimer's disease. There is.
Here, oryzanol, which has been confirmed by the whitening agent screening method according to the present disclosure to suppress the activity of β-secretase and suppress the cleavage of the β site of PMEL protein, suppresses the cleavage of the amyloid β precursor protein. Therefore, it is considered that there is a possibility of suppressing the formation of the above fibers. That is, the fibrosis inhibitor containing oryzanol is considered to be useful for, for example, treatment of Alzheimer's disease, suppression of progression, prevention, and the like.

The fibers in the present disclosure are not particularly limited, but are, for example, fibrous proteins having a specific structure and insoluble in water, tissues composed of long filamentous cells such as muscle fibers and nerve fibers, and fibrous. Contains proteins and the like. In the present disclosure, the fiber is preferably an amyloid fiber that forms the melanosome endoskeleton.
The content of oryzanol in the fibrosis inhibitor in the present disclosure is not particularly limited, but it is preferable to contain 0.0001 mg or more and 0.1 mg or less of oryzanol per 1 mL of the pharmaceutical product, and more preferably 0.003 mg or more and 0.1 mg or less. , 0.015 mg or more and 0.1 mg or less is most preferable.

Hereinafter, the present disclosure will be described in detail by way of examples. The materials, amounts, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be appropriately changed as long as they do not deviate from the gist of the embodiments of the present disclosure. Therefore, the scope of the embodiments of the present disclosure is not limited to the specific examples shown below. In this embodiment, "parts" and "%" mean "parts by mass" and "% by mass" unless otherwise specified.

<Cut status confirmation process>
After recovering human normal melanocytes (HEMn-DP, Thermo Fisher), they were suspended in a medium (containing Medium 254 manufactured by Gibco and Human Melanocyte Growth Supplement). Centrifuge (300 x g (1 g = 9.8 m / s ² ), 3 minutes, room temperature), remove the supernatant, resuspend in the above medium, and then place 3 x 10 ⁵ cells / cm ² per well on a 6-well plate. 1 mL was seeded so as to be, and 1 mL of a medium containing a BACE inhibitor (β-Secretase Inhibitor IV) manufactured by Merck at a concentration twice the target concentration was added, mixed well, and allowed to stand in a CO ₂ incubator (37 ° C.).
In the present disclosure, room temperature means 25 ° C.
The target concentration is 0.1 μM (mol / L), 1 μM, and 10 μM, and the medium containing twice the target concentration contains 1 μL of the BACE inhibitor dissolved in DMSO per 100 mL of the medium. Was given to. As a control, a medium containing DMSO containing no BACE inhibitor at the same ratio as described above was used instead of the medium containing twice the target concentration.
The test was performed using a total of 4 groups (n = 3 each) of 0.1 μM, 1 μM, 10 μM and control.

Three days after the seeding, the medium in each well was replaced with a medium containing a BACE inhibitor having the same composition, and one week after the seeding, each cell was recovered by trypsin treatment. Centrifuge (300 x g, 3 minutes, room temperature) to remove the supernatant, crush with a cytolytic solution containing 1% TrionX-100, place on ice for 5 minutes, and centrifuge (15000 x g, 10 minutes, 4 ° C.). The protein solution was extracted using the subsequent supernatant as a soluble fraction. The extracted protein solution was treated with SDS sample buffer (final concentration 1.67% SDS (sodium dodecyl sulfate), 1.55% DTT (dithiothreitol) included), and after heat denaturation at 95 ° C. for 5 minutes. It was applied to SDS-PAGE gel (e-PAGE E-T10L, ATTO) and electrophoresed. After migration, blotting is performed on a membrane, blocking is performed with a blocking agent (PVDF Blocking Reagent, Toyobo Co., Ltd.), a primary antibody reaction (anti-PMEL-Mβ fragment antibody, ab137062, Abcam) is followed by a secondary antibody reaction (HRP-labeled IgG antibody). The band was detected by chemiluminescence reaction with the HRP substrate. The detected band was quantified by the automatic concentration measurement of CSAnalizer (ATTO).

The results are shown in FIG. The vertical axis of FIG. 2 shows the amount of protein when the amount of PMEL-Mβ fragment in the control group is 100%. In addition, Cont. The description of 0.1 μM, 1 μM and 10 μM indicates the control group, and the description of 0.1 μM, 1 μM and 10 μM indicates the group to which the BACE inhibitor was applied at each concentration. In addition, the bar graph in each group represents the mean value, and the error bar represents the standard deviation.
From the results shown in FIG. 2, it can be seen that the amount of PMEL-Mβ fragment increased in a BACE inhibitor concentration-dependent manner. Assuming that the amount of PMEL-Mβ fragment in the control group was 100%, an increase of 300% or more was observed in the amount of PMEL-Mβ fragment when the BACE inhibitor 10 μM was applied.

<Confirmation of melanin production inhibitory effect by BACE inhibition>
Human normal melanocytes (HEMn-DP, Thermo Fisher) were recovered by trypsin treatment and then suspended in a medium (containing Medium 254 manufactured by Gibco and Human Melanocyte Growth Supplement). Centrifuge (300 x g, 3 minutes, room temperature), remove the supernatant, resuspend in the above medium, seed 1 mL on a 6-well plate at 3 x 10 ⁵ cells / cm ² per well, and further seed with Merck. 1 mL of a medium containing 2 times the target concentration of BACE inhibitor (β-Secretase Inhibitor IV) was added, mixed well, and allowed to stand in a CO ₂ incubator (37 ° C.).
The target concentration was set to 3 groups of 0.1 μM, 1 μM and 10 μM, and the BACE inhibitor dissolved in DMSO was added to the medium containing twice the target concentration so as to be 1 μL per 100 mL of the medium. As a control, a medium containing DMSO containing no BACE inhibitor at the same ratio as described above was used instead of the medium containing twice the target concentration.
The test was performed using a total of 4 groups (n = 3 each) of 0.1 μM, 1 μM, 10 μM and control.

Three days after the above seeding, the medium in each well was replaced with a medium containing a BACE inhibitor having the same composition, and one week after the seeding, the supernatant was removed and washed twice with PBS (phosphate buffered saline). Then, the number of living cells was detected with a WST reagent (Cell Counting Kit, manufactured by Dojin Chemical Laboratory Co., Ltd.). After measuring the number of living cells, each well was washed twice with PBS, melanin lysate (1 mol / l NaOH, 10% DMSO, aqueous solution) was added, and the wells were completely dissolved by ultrasonic treatment at 50 ° C. for 20 minutes. I let you.
After the above dissolution, 200 μL of the melanin solution after melanin dissolution in each well was transferred to a 96-well plate, the absorbance at 400 nm was measured at room temperature, and the amount of melanin was calculated from the absorbance obtained from the measurement. For the final amount of melanin, a value corrected by the above-mentioned number of living cells was adopted.

The results are shown in FIG. The vertical axis of FIG. 3 shows the amount of melanin produced when the amount of melanin produced in the control group is 100%. The description of Cont. Indicates the control group, and the description of 0.1 μM, 1 μM, and 10 μM indicates the group to which the BACE inhibitor was applied at each concentration. In addition, the bar graph in each group represents the mean value, and the error bar represents the standard deviation.
From the results shown in FIG. 3, it can be seen that the amount of melanin produced was suppressed in a BACE inhibitor concentration-dependent manner. Assuming that the melanin production amount in the control group was 100%, the melanin production amount was suppressed by 50% or more when the BACE inhibitor 10 μM was applied.

<Inhibition activity measurement step>
The inhibitory activity of β-secretase was measured using oryzanol.
The above-mentioned inhibitory activity was measured according to the protocol of SensoLyte520 BACE2 Activity Assay Kit (ANASPEC).
The DMSO stock was diluted 10-fold with assay buffer and 10 μL was placed on the plate.
Further, 40 μL of recombinant BACE2 protein prepared to 2.5 μg / mL was added, 50 μL of the substrate solution was added, and the mixture was allowed to stand at room temperature for 15 minutes.
The DMSO stock contains 100 times the target concentration of oryzanol (γ-oryzanol, Oryzanol, manufactured by Okayasu Shoten Co., Ltd.), ferulic acid or cholesterol in each of the following groups. used.
The target concentration was set to 2 groups of 3 μM and 30 μM for oryzanol, and 2 groups of 3 μM and 10 μM for ferulic acid and cholesterol, and DMSO was used as a control.
The test was performed using a total of 7 groups (n = 3 each) of oryzanol 3 μM, 30 μM, ferulic acid 3 μM, 10 μM, cholesterol 3 μM, 10 μM, and control.

After standing for 15 minutes, the fluorescence intensity at an excitation wavelength of 490 nm / fluorescence wavelength of 520 nm was detected by a plate reader Envision (PerkinElmer). The BACE2 activity was calculated with the BACE2 activity of the control group as 100% and the value of the BACE2 activity when the attached BACE inhibitor concentration was 0.5 μM (mol / L) as 0% (100% inhibition).

The results are shown in FIG. The vertical axis of FIG. 4 shows the BACE2 activity. In addition, Cont. The description of 3 μM, 30 μM, etc. indicates the control group, and the description of 3 μM, 30 μM, etc. indicates the group to which each of the above drugs was applied at each concentration. In addition, the bar graph in each group represents the mean value, and the error bar represents the standard deviation.
In addition, a homoscedasticity test was performed by F-test for each of the control group, the group to which 3 μM was given, or the group to which 30 μM was given, and then the student's t-test was performed. In FIG. 4, "***" indicates that the risk rate in the above t-test is 0.1% or less.
From the results shown in FIG. 4, BACE2 activity was inhibited depending on the oryzanol concentration, and 30 μM of oryzanol showed an inhibitory effect of almost 100%. Furthermore, as shown in the following structural formula, cholesterol and ferulic acid constituting oryzanol did not have an effect of suppressing BACE2 enzyme activity at high concentrations of any of the compounds, and this effect was caused by the specificity of the structure of oryzanol. It is presumed to do.

<Confirmation of melanin production inhibitory effect of oryzanol>
After recovering mouse melanoma cells (B-16, DS Pharma) by trypsin treatment, use medium (Gibco DMEM, 10% FBS (fetal bovine serum) contained, 1% PSN (penicillin-streptomycin-neomycin) contained). Suspended. Centrifuge (300 × g, 3 minutes, room temperature), remove the supernatant, resuspend in the above medium, and inoculate a 12-well plate with 0.5 mL so that the concentration is 5 × 10 ⁴ cells / cm ² per well. 0.5 mL of a medium containing twice the target concentration of γ-oryzanol manufactured by Okayasu Shoten Co., Ltd. was added, mixed well, and allowed to stand in a CO ₂ incubator (37 ° C.).
The target concentration was set to 2 groups of 30 μM and 100 μM, and the above-mentioned γ-oryzanol dissolved in DMSO was added to the medium containing twice the target concentration so as to be 1 μL per 100 mL of the medium. As a control, a medium containing DMSO containing no γ-oryzanol at the same ratio as described above was used instead of the medium containing twice the target concentration.
The test was performed using a total of 3 groups (n = 5 each) of 30 μM, 100 μM, and control.

Three days after seeding, the supernatant was removed from each well, washed twice with PBS, and the number of viable cells was detected with a WST reagent (Cell Counting Kit, manufactured by Dojin Chemical Research Institute Co., Ltd.).
After measuring the number of living cells, each well was washed twice with PBS, melanin lysate (1 mol / L NaOH, 10% DMSO, aqueous solution) was added, and the wells were completely dissolved by ultrasonic treatment at 50 ° C. for 20 minutes. I let you.
After the above dissolution, 200 μL of the melanin solution after dissolving melanin in each well was transferred to a 96-well plate, and the amount of melanin was measured at room temperature at an absorption wavelength of 400 nm. For the final amount of melanin, a value corrected by the above-mentioned number of living cells was adopted.

The results are shown in FIG. The vertical axis of FIG. 5 shows the amount of melanin produced when the amount of melanin produced in the control group is 100%. In addition, Cont. The description of 30 μM and 100 μM indicate the control group, and the description of 30 μM and 100 μM indicate the group to which the above-mentioned γ-oryzanol was given at the respective concentrations. In addition, the bar graph in each group represents the mean value, and the error bar represents the standard deviation.
In addition, a homoscedasticity test was performed by F-test for each of the control group, the group to which 30 μM was given, or the group to which 100 μM was given, and then the student's t-test was performed. In FIG. 5, "**" means that the risk rate in the above t-test exceeds 0.1% and is 0.5% or less, and "***" means that the risk rate in the above t-test is 0.1. It shows that it is less than%.
From the results shown in FIG. 5, the amount of melanin produced was suppressed in a manner dependent on the oryzanol concentration.
Assuming that the amount of melanin produced in the control group was 100%, suppression of melanin production by 20% or more was observed when oryzanol 100 μM was applied.

<Observation of melanosomes TEM (transmission electron microscope) after BACE inhibitor action>
Human normal melanocytes (HEMn-DP, Thermo Fisher) were recovered by trypsin treatment and then suspended in a medium (containing Medium 254 manufactured by Gibco and Human Melanocyte Growth Supplement). Centrifuge (300 x g, 3 minutes, room temperature), remove the supernatant, resuspend in the above medium, seed 1 mL on a 6-well plate at 3 x 10 ⁵ cells / cm ² per well, and further seed with Merck. 1 mL of a medium containing the BACE inhibitor manufactured by β-Secretase Inhibitor IV) at a concentration twice the target concentration was added, mixed well, and allowed to stand in a CO ₂ incubator (37 ° C.).
The target concentration was 10 μM, and the BACE inhibitor dissolved in DMSO was added to the medium containing twice the target concentration so as to be 1 μL per 100 mL of the medium. As a control, a medium containing DMSO containing no BACE inhibitor at the same ratio as described above was used instead of the medium containing twice the target concentration.
The test was performed using 10 μM and a total of 2 groups of controls (n = 3 each).

Three days after the seeding, the medium in each well was replaced with a medium containing a BACE inhibitor having the same composition, and one week after the seeding, each cell was recovered by trypsin treatment.
Centrifuge (300 x g, 3 minutes, room temperature) to remove the supernatant, treat with a prefix (containing 2% paraformaldehyde, 2% glutaraldehyde), wash and solidify with a 2% low temperature polymerized agar solution. rice field. The agar containing the cell mass was cut out, allowed to stand on ice for 60 minutes with a post-fixing solution (containing 2% osmium tetroxide), washed with ultrapure water and replaced with ethanol, and further n-butyl glycidyl ether and epoxy resin (Quetol-). After replacement with 651 (ethylene diglycidyl ether), thermal polymerization was performed at 60 ° C. for 24 hours to prepare a resin block. A thin section of 60 nm was collected on a TEM sample table, stained with gadolinium acetate for 60 minutes and with a lead stain for 4 minutes, and accelerated voltage using a transmission electron microscope (TEM: JEM-2100Plus, manufactured by JEOL Ltd.). CCD (Charge Coupled Device) photography was performed at 100 kV.

The results are shown in FIG. In FIG. 6, (A) is the appearance of immobilized and stained cells, (B) is the result of TEM observation in the control group, and (C) is TEM in the group to which the above BACE inhibitor was added at a concentration of 10 μM. The results of the observations are shown respectively.
The black arrowhead in (B) and the white arrowhead in (C) each indicate melanosomes.
From the results shown in FIG. 6, it was observed that the addition of 10 μM BACE inhibitor eliminated the fibrous structure of melanosomes and dramatically reduced the amount of melanin accumulated.

From the results shown in FIGS. 2 to 6, it was confirmed that according to the embodiment according to the present disclosure, a novel screening method for a whitening agent is provided.

<Confirmation of melanin production inhibitory effect of BACE inhibition in skin model>
A three-dimensional model of melanocyte-containing skin (MEL-300 kit, Kurabo Industries) was opened and then pre-incubated in a medium (EPI-100NMM113, Kurabo Industries). The next day, the BACE inhibitor (β-Secretase Inhibitor IV) manufactured by Merck was replaced with a medium containing the target concentration, 50 μL of the medium was further added to the stratum corneum side of the skin model, and the medium was allowed to stand in a CO ₂ incubator (37 ° C.). The medium was changed once every two days.
The target concentration was 3 groups of 1 μM, 10 μM, and 30 μM, and the BACE inhibitor dissolved in DMSO was added to the medium containing the target concentration so as to be 1 μL per 100 mL of the medium. As a control, instead of the medium containing the target concentration, a medium to which DMSO containing no BACE inhibitor was added at the same ratio as described above was used.
The test was performed using a total of 4 groups (n = 3 each) of 1 μM, 10 μM, 30 μM and control.

One week after the first medium exchange, the skin model was washed with PBS and the number of living cells was detected with an MTT reagent (MTT-100JP, Kurabo Industries Ltd.). After measuring the number of living cells, the product reacted with the MTT reagent with isopropanol for each model was completely removed, a melanin lysate (1 mol / l NaOH, 10% DMSO, aqueous solution) was added, and the mixture was heated at 80 ° C. for 1 hour. It was completely dissolved by the treatment.
After the above dissolution, 200 μL of the melanin solution after dissolution of melanin in each model was transferred to a 96-well plate, the absorbance at 400 nm was measured at room temperature, and the amount of melanin was calculated from the absorbance obtained from the measurement. For the final amount of melanin, a value corrected by the above-mentioned number of living cells was adopted.

The results are shown in FIG. The vertical axis of FIG. 7 shows the amount of melanin produced when the amount of melanin produced in the control group is 100%. In addition, Cont. The description of 1 μM, 10 μM, and 30 μM indicates the control group, and the description of 1 μM, 10 μM, and 30 μM indicates the group to which the above-mentioned BACE inhibitor was applied at each concentration. In addition, the bar graph in each group represents the mean value, and the error bar represents the standard deviation.
Further, after performing a homoscedasticity test by F-test for each of the control group, the group to which 1 μM was given, the group to which 10 μM was given, or the group to which 30 μM was given, the student's t-test was performed. Carried out. In FIG. 7, “*” indicates that the risk rate in the above t-test exceeds 1% and is 5% or less, and “**” indicates that the risk rate in the above t-test exceeds 0.1% and is 1% or less. It shows that they are.
From the results shown in FIG. 7, it can be seen that the amount of melanin produced was suppressed in a BACE inhibitor concentration-dependent manner. Assuming that the melanin production amount in the control group was 100%, the melanin production amount was suppressed by 20% or more when the BACE inhibitor 30 μM was applied.

<Preparation of astaxanthin-containing emulsified composition>
The following components were dissolved for 1 hour while heating at 70 ° C. to obtain an aqueous phase composition A.

[Aqueous phase composition A]
Sucrose stearic acid ester (HLB (Hydrophilic-Lipophilic Balance) value: 16): 33.0 g
Decaglyceryl monooleate (HLB value: 12): 67.0 g
Glycerin (alcohol): 450.0 g
Pure water: 300.0 g

The following components were dissolved for 1 hour while heating at 70 ° C. to obtain an oil phase composition A.

[Oil phase composition A]
Okiami extract (astaxanthin content: 20% by mass): 15.0 parts by mass Mixed tocopherol (RIKEN E Oil 800, manufactured by RIKEN Vitamin Co., Ltd.): 32.0 parts by mass Medium-chain fatty acid glyceride (Coconard MT, Kao Co., Ltd.) ): 93.0 parts by mass Lecithin (Resion P (derived from soybeans), manufactured by RIKEN Vitamin Co., Ltd.): 10.0 parts by mass

The aqueous phase composition A obtained above is stirred at 10,000 rpm (revolutions per minute) using a homogenizer (model name: HP93, manufactured by SMT Co., Ltd.) while keeping the aqueous phase composition A at 70 ° C. to form the aqueous phase composition. The oil phase composition A was added to the product A to obtain a preliminary emulsion A.
Subsequently, the obtained preliminary emulsion A was cooled to about 40 ° C., and high-pressure emulsification was performed at a pressure of 200 MPa using an ultra-high pressure homogenizer (Ultimizer HJP-25005, manufactured by Sugino Machine Limited). After high-pressure emulsification, the mixture was filtered through a microfilter having an average pore size of 1 μm to prepare an astaxanthin-containing emulsified composition (astaxanthin content: 0.3% by mass).
The obtained astaxanthin-containing emulsified composition was diluted to 1% by mass with Milli-Q water, and the particle size of the dispersed particles was measured using a particle size analyzer (model number: FPAR-1000, manufactured by Otsuka Electronics Co., Ltd.). However, it was 58 nm.

It is equivalent to replacing the above krill extract with "Astax-ST (product name)" (manufactured by Marine Daio Co., Ltd.) or haematococcus algae extract (product name: ASTOTS-S, manufactured by Fujifilm Co., Ltd.). The emulsified composition of can be obtained.

<Preparation of oryzanol-containing emulsified composition>
The following components were dissolved for 1 hour while heating at 70 ° C. to obtain an aqueous phase composition B (the same composition as the aqueous phase composition A).

[Aqueous phase composition B]
Sucrose stearic acid ester (HLB value: 16): 33.0 parts by mass Decaglyceryl monooleate (HLB value: 12): 67.0 parts by mass Glycerin (alcohol): 450.0 parts by mass Pure water: 300.0 Mass part

The following components were dissolved for 1 hour while heating at 70 ° C. to obtain an oil phase composition B.
[Oil phase composition B]
γ-Oryzanol (γ-oryzanol C: trade name, manufactured by Okayasu Shoten Co., Ltd.): 10.0 parts by mass Medium-chain fatty acid glyceride (Coconard MT, manufactured by Kao Co., Ltd.): 130.0 parts by mass Lecithin (Resion P ( (Derived from soybeans), manufactured by RIKEN Vitamin Co., Ltd.): 10.0 parts by mass

The aqueous phase composition B obtained above was stirred at 10,000 rpm (revolutions per minute) using a homogenizer (model name: HP93, manufactured by SMT Co., Ltd.) while keeping the aqueous phase composition B at 70 ° C. to form the aqueous phase composition. The oil phase composition B was added to the product B to obtain a preliminary emulsion B.
Subsequently, the obtained preliminary emulsion B was cooled to about 40 ° C., and high-pressure emulsification was performed at a pressure of 200 MPa using an ultra-high pressure homogenizer (Ultimizer HJP-25005, manufactured by Sugino Machine Limited). After high-pressure emulsification, the mixture was filtered through a microfilter having an average pore size of 1 μm to prepare an oryzanol-containing emulsified composition (oryzanol content: 1% by mass).
The obtained oryzanol-containing emulsified composition was diluted to 1% by mass with Milli-Q water, and the particle size of the dispersed particles was measured using a particle size analyzer (model number: FPAR-1000, manufactured by Otsuka Electronics Co., Ltd.). However, it was 61 nm.

<Preparation of Centella asiatica extract-containing aqueous dispersion composition>
The following components were mixed and dissolved while heating at 70 ° C. for 30 minutes with paddle stirring to obtain suspension A.

[Suspension A]
Glycerin: 67.0 parts by mass Pure water: 28.0 parts by mass Recitin (SLP-white (trade name); manufactured by Tsuji Oil Co., Ltd.): 450.0 parts by mass TECA ^{* 1} : 11.0 parts by mass * 1 : TECA (trade name) (manufactured by Bayer Healthcare, a mixture containing the pentacyclic triterpenes asiatinic acid, madecasinic acid and asiathicoside)

While keeping the suspension A obtained above at 60 ° C., it is passed through an ultra-high pressure disperser (model name: Starburst Mini, manufactured by Sugino Machine Co., Ltd.) 5 times at a pressure of 200 MPa for high-pressure dispersion treatment. To prepare an aqueous dispersion composition containing Centella asiatica extract.
The obtained aqueous dispersion composition containing Centella asiatica extract was diluted 10-fold with Milli-Q water and dispersed using a dynamic light scattering type particle size measuring machine (trade name: Nanotrack UPA, manufactured by Nikkiso Co., Ltd.). When the particle size of the particles was measured, it was 19 nm.

<Preparation of stearyl glycyrrhetinate dispersion composition>
The following components were dissolved for 1 hour while heating at 70 ° C. to obtain an aqueous phase composition C.
[Aqueous phase composition C]
Decaglyceryl monooleate (HLB value: 12): 18.9 parts by mass Glycerin: 35.0 parts by mass Pure water: Amount in which the total composition is 100 parts by mass

Further, the following components were dissolved for 1 hour while heating at 70 ° C. to obtain an oil phase composition C.
[Oil phase composition C]
Stearyl glycyrrhetinate: 3.33 parts by mass Tocopherol acetate: 3.33 parts by mass Tocopherol: 10.77 parts by mass Tri (caprylic acid / capric acid) Glyceryl: 6.0 parts by mass Purified lecithin (derived from soybeans): 4.2 parts by mass Department

While maintaining the aqueous phase composition C in a stirred state, the oil phase composition C was added and then dispersed for 4 minutes using a high-pressure homogenizer to obtain a preliminary dispersion C.
Subsequently, the obtained preliminary dispersion C was cooled to about 60 ° C., and high-pressure dispersion was performed at a pressure of 245 MPa using an ultra-high pressure homogenizer (Ultimizer HJP-25005, manufactured by Sugino Machine Limited) to obtain a dispersion composition. I got the thing C.
Then, the obtained dispersion composition C was filtered through a microfilter having an average pore size of 1 μm and sterilized by heating at 80 ° C. for 30 minutes to prepare a glycyrrhetinic acid derivative (stearyl glycyrrhetinate containing stearyl glycyrrhetinate). ..

A beauty essence having the following composition was prepared by a conventional method (total amount 100% by mass).
[Composition]: [Content (% by mass)]
Astaxanthin-containing emulsified composition (containing 0.3% by mass of astaxanthin): 0.5
Oryzanol dispersion composition (containing 1% by mass of oryzanol): 0.05
Dipropylene glycol: 4.0
Glycerin: 5.0
Diglycerin: 2.0
1,2-Pentanediol: 2.0
Polyoxyethylene hydrogenated castor oil (60EO (ethylene oxide)): 0.2
Phosphoric acid-L-ascorvir magnesium: 2.0
Dipotassium glycyrrhizinate: 1.0
Phenoxyethanol: 0.5
Methyl paraoxybenzoate: 0.1
Alcaligenes Ratorus B-16 Polymer: 0.05
Citric acid: Appropriate amount Sodium citrate: Appropriate amount N-Acetyl-L-Hydroxyproline: 1.0
Water-soluble collagen: 1.0
Hydrolyzed collagen: 1.0
Yeast extract: 1.0
Centella asiatica extract-containing aqueous dispersion composition (containing 1% by mass of Centella asiatica extract): 0.05
Stearyl glycyrrhetinate dispersion composition (containing 1.0% by mass of stearyl glycyrrhetinate): 0.5
Polyglyceryl monooleate: 0.2
Water: the rest

The obtained beauty essence was a beauty essence containing the whitening agent screened by the whitening agent screening method according to the present disclosure, and had a favorable usability as a whitening beauty essence.
The whitening agent screened by the whitening agent screening method according to the present disclosure can be widely applied to a lotion, a cream, a gel-like preparation containing the whitening agent.

10A PMEL protein 10B, 10C PMEL protein after cleavage of β site 12 Signal peptide domain 14 N-terminal domain (NTD)
16 polycystic kidney disease-like domain (PKD)
18 Gap domain 1 (GAP1)
20 proline / serine / threonine-rich repeat domain (RPT)
22 Gap Domain 2 (GAP2)
24 Kringle-like domain (KLD)
26 Gap Domain 3 (GAP3)
28 transmembrane domain (TM)
30 C-terminal domain (CTD)
32 Disulfide bond 34 Cleavage site 36, 38, 40, 42 Dashed line 44, 46, 48, 50, 52 Fragment 100 Melanosome membrane CC Terminal side NN Terminal side Mα Mα region Mβ Mβ region

Claims (5)

A method for screening a whitening agent, which comprises confirming the cleavage state of the β site of PMEL protein in cells capable of producing melanin.
The method for confirming the cleavage state is the quantification of PMEL-Mβ fragment.
Further comprising imparting the test substance to the cells prior to confirming the cleavage state.
Further including measuring the amount of melanin produced in the cells,
A method for screening a whitening agent, wherein the cells are epidermal melanoma or epidermal human pigment cells. A method for screening a whitening agent, which comprises measuring the inhibitory activity of β-secretase by a test substance.
Including the application of the test substance to the cells
Further including measuring the amount of melanin produced in the cells,
A method for screening a whitening agent, wherein the cells are epidermal melanoma or epidermal human pigment cells. The method for screening a whitening agent according to claim 2, wherein the β-secretase is BACE2. The method for screening a whitening agent according to claim 2 or 3, wherein the test substance does not have tyrosinase inhibitory activity. The method for screening a whitening agent according to any one of claims 1 to 4, wherein the cells are normal human cells.

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