JP6113981B2 - Screening method - Google Patents

Description

The present invention relates to a whitening agent having a novel mechanism of action suitable for cosmetics and the like, a screening method thereof, a whitening composition containing the whitening agent, and a method for producing the whitening composition. is there. In the description of the present invention, the term cosmetics includes quasi drugs.

  Skin aging symptoms such as skin pigmentation such as spots and dullness, changes in skin shape such as sagging and wrinkles, skin firmness, loss of elasticity, etc. are caused by accumulation of skin irritation over many years such as UV exposure and drying. Occurs and becomes apparent with aging. For the purpose of preventing or improving such skin aging symptoms, whitening agents, anti-aging agents, moisturizing agents and the like have been actively developed. In particular, the degree of attention to whitening agents for pigmentation symptoms such as spots and dullness is very high, and many whitening agents such as arbutin and ascorbic acid glucoside have been developed. However, although a certain effect is recognized in the conventional whitening agent, it cannot be said that the high whitening effect and durability which a user desires are achieved. This is due to the mechanism of pigmentation only by the action mechanism of melanin production, the inhibition of melanin transport to keratinocytes (keratinocytes), and the action of inhibiting melanin uptake into keratinocytes. This is because it is difficult to fully explain the introduction. Furthermore, some of the above-described whitening agents have problems in safety or stability. For this reason, a whitening agent having high effectiveness and safety is still required, and research and development on a whitening agent having a novel mechanism of action is actively conducted. In addition, if a whitening agent having a new mechanism of action can be found, an additive or synergistic effect can be expected when used in combination with an existing whitening agent, so the expectation for the appearance of a whitening agent having a new mechanism of action is very high. It can be said that it is very expensive.

In recent years, skin aging symptoms such as skin changes such as pigmentation such as spots and dullness, sagging and wrinkles, skin firmness and loss of elasticity, and epidermal basement membrane (hereinafter referred to as basement membrane) Attention has been focused on the relationship between function and structure. The keratinocyte stem cells and pigment cells (melanocytes) that make up the basal cell layer adhere to the basement membrane and adjacent cells, such as desmosomes, gap junctions, and hemidesmosomes. Have In addition, hemidesmosomes with integrin α6 / β4 as the main constituents anchor keratinocytes of the basal cell layer to the basement membrane, and control the movement of keratinocytes to the skin surface by the progress of keratinization. In addition to regulating turnover of cells, it is deeply involved in the expression of various cell functions such as maintenance of tissue structure and signal transduction (see, for example, Non-Patent Document 1 and Non-Patent Document 2). Furthermore, integrin, which is a cell adhesion molecule, is a heterodimer formed from an α chain and a β chain, and there are large differences in ligand specificity, expression function, and the like due to differences in combination and distribution of subtypes. As for the relationship between skin aging symptoms such as skin pigmentation, skin shape change or structural change and integrin molecules, integrin molecules of keratinocytes are involved in structural abnormalities and functional decline of basal cell layer and basement membrane, It has been reported to cause skin structure changes such as sagging and reduced elasticity (for example, see Patent Document 1). For such a skin structure change, an integrin α2β1 production promoter (see, for example, Patent Document 2) and a composition for promoting integrin α6β4 production (for example, Patent Document 3) containing plant extracts such as Japanese kizuta Integrin production promoters are effective. In addition, as the relationship between pigmentation symptoms such as stains and dullness and integrin molecules, it has been reported that the expression level of integrin β4 in skin sites such as stains and dullness is reduced. Integrin β4 production promoters such as integrin β4 production promoters (for example, see Patent Document 4) and chlorine-substituted alcohols (see, for example, Patent Document 5) composed of an aurene extract have been reported. Thus, in the relationship between skin aging symptoms and the function and structure of the basement membrane in the prior art, enhancing the cell adhesion function of cell adhesion molecules including integrins prevents or ameliorates skin aging symptoms. It was supposed to be effective.

JP 2009-535310 A JP 2009-227628 A JP 2003-171225 A JP 2006-045087 A JP 2006-045084 A

Dawling J et. Al., J. Cell Biol., 134, 559-572 (1996). Mainiero F et. Al., EMBO J., 16, 2365-2375 (1997).

  The present invention has been made in view of the situation as described above, and is a whitening agent having a novel action mechanism, which is difficult only by focusing on the whitening action based on the existing action mechanism. It is an object of the present invention to find a whitening agent having an inhibitory effect on cell adhesion of cell.

  As a result of intensive efforts to solve the above problems, the present inventor has enhanced cell adhesion ability of horny layer cells in the epidermis basal layer of the skin site where pigmentation such as blotches and dullness is observed. I found. That is, the present inventor has enhanced the cell adhesion ability of keratinocytes existing in the basal layer of the epidermis of the skin site where spots, dullness, etc. are observed compared to normal skin, specifically, keratinocyte integrins. Increases in the expression levels of α6 and β4 proteins were confirmed. Furthermore, the present inventor has found that the above-mentioned enhancement of cell adhesion in keratinocytes is caused by an increase in the accumulation amount of melanin and related substances produced by pigment cells in the epidermal basal layer and transferred to keratinocytes. I found it. Specifically, by adding melanin or its related substance to keratinocytes, increase in expression level of integrin α6 and / or β4 mRNA in keratinocytes, increase in expression level of integrin α6 and / or β4 protein in keratinocytes, An improvement in the adhesion of keratinocytes to the basement membrane (an improvement in adhesion of keratinocytes to Laminin-5) was found. Furthermore, it has been found that accumulation of melanin and related substances in keratinocytes suppresses upper layer movement of keratinocytes due to progress of keratinization. This fact suggests that in skin areas where pigmentation such as sunburn, spots and dullness due to increased melanin production is observed, the accumulation of melanin and related substances in keratinocytes increases, and the cell adhesion ability of keratinocytes increases. This indicates that the ability of keratinocytes to adhere to the basement membrane is enhanced, and the upper layer migration of keratinocytes is suppressed. Furthermore, since the keratinocytes are strongly anchored to the basement membrane due to the above-mentioned enhancement of cell adhesion of keratinocytes, the turnover of keratinocytes is impaired, and skin aging phenomena such as pigmentation symptoms appear. it is conceivable that. Based on such facts, the present inventor has clarified that a component having an inhibitory effect on cell adhesion of keratinocytes can be a whitening agent. The facts clarified by the present inventor are greatly different from conventional aging symptoms including pigmentation symptoms and the knowledge about cell adhesion ability of keratinocytes. Furthermore, since it has not been shown that the cell adhesion ability of keratinocytes in the basal cell layer of the skin site where sunburn, spots, dullness, etc. are observed, keratinocyte cell adhesion inhibitory action is used as an index. It can be said that studies on methods for screening whitening agents, whitening compositions containing such whitening agents, and methods for producing the same have not been actively conducted. Therefore, a component having a cell adhesion inhibitory action of keratinocytes can be a whitening agent having a new action mechanism.

The present inventor has sought a whitening agent having a novel mechanism of action, that is, a whitening agent having an inhibitory effect on cell adhesion of keratinocytes. A method for screening whitening agents with an order was established. Furthermore, it was confirmed that the whitening composition containing the whitening agent selected by the screening method exhibited an excellent whitening action, that is, the present invention is as follows.
<1> A screening method for a whitening agent, wherein a whitening agent is selected using the cell adhesion inhibitory action of keratinocytes as an index.
<2> A screening method for a whitening agent comprising a step of evaluating a cell adhesion inhibitory action of keratinocytes, the keratinocyte to which a test substance is added, and the integrin production inhibitory action of a keratinocyte to which no test substance is added, Comparing the action of inhibiting the adhesion of keratinocytes to the basement membrane, the action of one or more selected from the action of promoting the upper layer movement of keratinocytes, The screening method for a whitening agent according to <1>, wherein the whitening agent is selected using the action as an index.
<3> The method for screening a whitening agent according to <1> or <2>, comprising a step of applying a cell adhesion promoting factor.
<4> The whitening agent screening method according to <3>, wherein the cell adhesion promoting factor is melanin and / or a related substance thereof.
<5> The screening method for a whitening agent according to any one of <1> to <4>, wherein the integrin production inhibitory action of the keratinocytes is an integrin α6 and / or β4 gene expression inhibitory action.
<6> The screening method for a whitening agent according to <5>, wherein the integrin α6 and / or β4 gene expression inhibitory effect is an integrin α6 and / or β4 mRNA expression inhibitory effect.
<7> The method for screening a whitening agent according to any one of <1> to <4>, wherein the integrin production inhibitory action of the keratinocytes is an integrin α6 and / or β4 protein expression inhibitory action.
<8> The action of suppressing the adhesion of keratinocytes to the basement membrane is the action of suppressing the adhesion of keratinocytes to laminin-5. The screening method of the whitening agent in any one of <1>-<4>.
<9> A whitening composition comprising a whitening agent selected by the whitening agent screening method according to any one of <1> to <8>.
<10> The whitening composition according to <9>, which is an external preparation for skin.
<11> The whitening composition according to <9> or <10>, which is a cosmetic (including a quasi-drug).
<12> A whitening agent comprising a cell adhesion inhibitor for keratinocytes.
<13> The cell adhesion inhibitor of keratinocytes has an action selected from an action of inhibiting integrin production of keratinocytes, an action of inhibiting adhesion of keratinocytes to the basement membrane, and an action of promoting upper layer movement of keratinocytes. The whitening agent as described in <12> which has 1 type or 2 types or more.
<14> A skin external preparation containing the whitening agent according to <12> or <13>.

ADVANTAGE OF THE INVENTION According to this invention, the whitening agent which has a novel action mechanism based on the cell adhesion inhibitory action of the keratinocyte which was not clarified conventionally can be provided.

It is a figure regarding the integrin alpha6 protein expression level change in a human spot site. It is a figure regarding the integrin β4 protein expression level change in the human spot site. It is a figure regarding the integrin alpha6 and beta4 mRNA expression level change by the addition of melanin of a human normal keratinocyte. It is a figure regarding the integrin alpha6 and beta4 mRNA expression level change by the lipid melanin addition of a human normal keratinocyte. It is a figure regarding the integrin alpha6 and beta4 protein expression level change by the melanin addition of a human normal keratinocyte. It is a figure regarding the adhesiveness change of a keratinocyte with respect to a basement membrane component (laminin-5) by the melanin addition of a human normal keratinocyte. It is a figure which shows the upper layer mobility ability change of the keratinocyte by the melanin addition of a human normal keratinocyte. It is a figure regarding the integrin (alpha) 6 mRNA expression inhibitory effect in a human normal keratinocyte of a test substance (Yuzu extract (Yuzuceramide, the left figure)) or a Korean thistle extract (Artichoke extract, the right figure). It is a figure regarding the adhesion inhibitory effect | action of a keratinocyte with respect to the basement membrane component (laminin-5) in the human normal keratinocyte of a test substance (Yuzu extract (Yuzuceramide) or Datura extract (Artichoke extract)). It is a figure regarding the evaluation result of the upper layer movement promotion effect | action in a human normal keratinocyte of a test substance (Yuzu extract (Yuzuceramide) or Datura extract (Artichoke extract)).

  Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to these contents.

<Screening method of whitening agent including the step of evaluating the cell adhesion inhibitory action of keratinocytes of the present invention>
The whitening agent screening method of the present invention is a whitening agent screening method that evaluates the cell adhesion inhibitory action of keratinocytes and selects the whitening agent using the action as an index. When the present inventor compares normal skin with a skin site where spots, dullness, etc. are observed, the integrin α6 and β4 proteins of keratinocytes present in the basal cell layer of the skin site where spots, dullness, etc. are observed Increase in expression level, increase in integrin α6 and β4 mRNA expression level and integrin α6 and β4 protein expression level in keratinocytes by addition of melanin or related substances, and adhesion of keratinocytes to basement membrane by addition of melanin ( More specifically, it was found that the adhesion of keratinocytes to Laminin-5 is improved. Furthermore, it discovered that the upper layer movement of the keratinocyte by keratin advance was suppressed by melanin addition. This is because melanin and related substances are excessively transported and accumulated in keratinocytes at the skin site where pigmentation such as sunburn, blotches and dullness is observed and melanin production is increased. The cell adhesion ability of the keratinocytes is enhanced, the adhesion ability of the keratinocytes to the basement membrane is increased, and the upper layer movement of the keratinocytes is suppressed. This indicates that the cell adhesion inhibitory action of keratinocytes is deeply involved in the occurrence or worsening of pigmentation symptoms including sunburn, blotches and dullness. Based on the above facts, a whitening effect can be expected from a component having an inhibitory effect on cell adhesion of keratinocytes by suppressing cell adhesion of keratinocytes and normalizing turnover of keratinocytes. For this reason, the whitening agent which has a novel action mechanism can be screened by evaluating the cell adhesion inhibitory action of a keratinocyte.

  The whitening agent screening method of the present invention is a whitening agent screening method that evaluates the cell adhesion inhibitory action of keratinocytes and selects the whitening agent using the action as an index. Furthermore, the screening method of the whitening agent of the present invention is a screening method of a whitening agent including a step of evaluating the cell adhesion inhibitory action of keratinocytes, and a keratinocyte to which a test substance is added, and a corner to which no test substance is added. Inhibition of integrin production in keratinocytes, inhibition of adhesion of keratinocytes to the basement membrane, and action of promoting keratinocyte upper layer movement It is preferable to use a whitening agent screening method characterized by evaluating and selecting a whitening agent using the action as an index. As an index of the cell adhesion inhibitory action of keratinocytes in the screening method of the whitening agent of the present invention, any physical quantity related to the cell adhesion inhibitory action of keratinocytes can be evaluated. It can be applied without limitation. In the evaluation of the cell adhesion inhibitory action of keratinocytes of the present invention, the physical quantity can be measured, and the measured value or a value calculated from the measured value can be compared to evaluate the cell adhesion inhibitory action. Here, “compare” is a value calculated from a measured value or a measured value of a physical quantity related to a cell adhesion inhibitory action of a keratinocyte to which a test substance is not added in evaluating the cell adhesion inhibitory action of the test substance. Means that the test substance's cell adhesion inhibitory action is evaluated by comparing the measured value of the physical quantity related to the cell adhesion inhibitory action of the keratinocytes added with the test substance or a value calculated from the measured value. . Among the physical quantities related to the cell adhesion inhibitory action of the keratinocytes of the present invention, preferred are the physical quantities relating to the integrin α6 and / or β4 gene expression inhibitory action related to the keratinocyte integrin production inhibitory action, integrin α6 and In addition to the physical quantity related to the β4 protein expression inhibitory action, the keratinocytes and the basement membrane such as keratinocyte adhesion inhibitory action against the basement membrane (more preferably, laminin-5, the main constituent of the basement membrane). Preferred examples include physical quantities relating to the cell adhesion inhibitory action, physical quantities relating to the action of promoting the upper layer movement of keratinocytes due to the progress of keratinization, and the like. Furthermore, among the above integrin α6 and / or β4 gene expression levels, more preferable examples include integrin α6 and / β4 mRNA expression levels.

  In the present invention, “evaluating the cell adhesion inhibitory action of keratinocytes and selecting a whitening agent using the action as an index” means “integrin in keratinocytes to which a test substance is added and keratinocytes to which no test substance is added. Measure physical quantities related to cell adhesion inhibitory action of one or more keratinocytes selected from production inhibitory action, keratinocyte adhesion inhibitory action to basement membrane, keratinocyte upper layer movement promoting action, Comparing the measured value or a value calculated from the measured value, evaluating the cell adhesion inhibitory action of keratinocytes based on the comparison result, and selecting as a whitening agent using the cell adhesion inhibitory action as an index To do. Specifically, when the test substance shows a keratinocyte cell adhesion inhibitory action, it is determined that it can be selected as a whitening agent, and an evaluation standard is established in advance for the keratinocyte cell adhesion inhibitory action. When the standard is exceeded, the test substance is selected as a whitening agent. Furthermore, it is also possible to compare cell adhesion inhibitory action for a plurality of test substances, relatively evaluate these actions, and determine that a test substance having a higher cell adhesion inhibitory action is a better whitening agent. It is included in the “evaluating the cell adhesion inhibitory action of keratinocytes and selecting a whitening agent using the action as an index” of the invention.

  In the whitening agent screening method of the present invention, a cell adhesion promoting factor can be appropriately given to keratinocytes. The “cell adhesion promoting factor” in the present invention can be applied without particular limitation as long as it has an action of improving the cell adhesion ability of keratinocytes, and more preferably, the cell adhesion ability of keratinocytes. A factor having an action of improving keratin, a factor having an action of suppressing upper layer movement of keratinocytes due to progress of keratinization, and the like can be preferably exemplified. Among the keratinocyte cell adhesion promoting factors of the present invention, melanin and / or its related substances can be suitably exemplified as preferred examples. Among the keratinocyte cell adhesion promoting factors, melanin can be applied without particular limitation as long as it is a melanin pigment produced in animals, plants, protozoa, etc., and more preferably produced from pigment cells. Melanins such as eumelanin, pheomelanin, and the like, more preferably eumelanin. On the other hand, examples of the melanin-related substance include melanin-related substances that are derived in the melanin biosynthesis process of animals, plants, protozoa, and the like. A preferred example is lipidated melanin (lipidated melanin) produced by a reaction between the resulting lipid peroxide and melanin. Fatty melanin is a general term for substances generated by the reaction of lipid peroxide and melanin, and is not a single component. In addition, as a lipid as a biological component that produces glycated melanin, it can be applied without particular limitation as long as it is a lipid present in the living body, more preferably a phospholipid, and still more preferably a hydroxylated lecithin. I can do it. Lipidized melanin is generated not only by the reaction of lipid peroxide and melanin generated from active oxygen and lipid generated by exposure to ultraviolet rays or the like in vivo, but can also be generated chemically. As a method for producing oleated melanin, any method that produces oleated melanin can be applied without any particular limitation. If a preferred method is specifically mentioned, the methods described in Production Examples 1 and 2 described below are preferably exemplified. I can do it.

Regarding the application of the cell adhesion promoting factor of the keratinocytes of the present invention, the order and the number of times thereof are not particularly limited, and even if the cell adhesion promoting factor of keratinocytes is added after adding the test substance, The test substance may be added after the cell adhesion promoting factor is applied, or the test substance may be added and the cell adhesion promoting factor may be applied simultaneously. Furthermore, the addition of a cell adhesion promoting factor should be appropriately set according to the culture conditions of keratinocytes. For example, when melanin is added, the addition amount of the cell adhesion promoting factor of keratinocytes is usually 1 × 10 ⁻⁶ (w / v%) or more as the addition amount (total amount when added several times). 1 × 10 ⁻⁵ (w / v%) or more, more preferably 1 × 10 ⁻⁴ (w / v%) or more, and usually 3 × 10 ⁻¹ (w / v%) or less. Preferably, it is 3 × 10 ⁻² (w / v%) or less, more preferably 3 × 10 ⁻³ (w / v%) or less. In addition, in the case of addition of fat melanin, the addition amount (total amount in the case of adding multiple times) is usually 1 × 10 ⁻⁵ (w / v%) or more, preferably 1 × 10 ⁻⁴ (w / V%) or more, more preferably 1 × 10 ⁻³ (w / v%) or more, usually 3 (w / v%) or less, preferably 3 × 10 ⁻¹ (w / v%). Hereinafter, it is more preferable that it is 3 × 10 ⁻² (w / v%) or less. Within the above range, keratinocyte integrin production promoting action (integrin gene expression promoting action, integrin protein expression promoting action), keratinocyte adhesion promoting action to basement membrane, keratinocyte upper layer by keratinization progression A movement inhibitory action can be appropriately expressed. The whitening agent screening method of the present invention and the cell adhesion promoting factor used for the examination thereof were prepared according to the production methods described in Production Examples 1 and 2 described later in addition to commercially available melanin (Sigma Aldrich Japan Co., Ltd.). Oiled melanin was used. Moreover, although the lipidated melanin manufactured by the manufacture examples 1 and 2 mentioned later is used as a suspension, you may add the further refinement | purification process as needed.

<Production Example 1: Preparation Method of Lipid Peroxide Solution>
1 (w / v%) Resinol SH50 aqueous solution (Nippon Surfactant Kogyo Co., Ltd.) is heated at 90 ° C. for 10 minutes, and then returned to room temperature, so that lipoxygenase (Nacalai Tesque Co., Ltd.) has a final concentration of 1 (mg / mL) And shake at 37 ° C. overnight. The reaction solution was centrifuged (3000 rpm, 10 minutes), and the supernatant was collected and used as a lipid peroxide solution.

<Production Example 2: Preparation Method of Oiled Melanin Suspension>
A glycated melanin reaction solution containing the components listed in Table 1 was prepared, allowed to stand at room temperature for 24 hours, and then centrifuged (20,000 g, 10 minutes). After removing the supernatant, methanol (Wako Pure Chemical Industries, Ltd.) was added and pipetting was performed several times to remove lipids and lipid peroxide in the precipitate. The removal of lipid and lipid peroxide with methanol was repeated again, and then the precipitate was suspended in PBS (Takara Bio Inc.) 1 (mL) to prepare a lipidated melanin suspension.

  In the screening method for the whitening agent of the present invention, keratinocytes cultured under normal culture conditions can be used, and a cell adhesion promoting factor is added to the keratinocytes to improve the cell adhesion ability of the keratinocytes. Increased adhesion to keratinocytes, specifically keratinocytes with enhanced integrin α6 and / or β4 gene expression, keratinocytes with enhanced integrin α6 and / or β4 protein expression, and basement membrane It is also possible to use keratinocytes, keratinocytes in which the upper layer migration ability of keratinocytes is suppressed. In the whitening screening method of the present invention, keratinocytes that have been improved in cell adhesion ability by adding a cell adhesion promoting factor in terms of measurement sensitivity, accuracy, etc. of physical quantities related to the cell adhesion inhibitory action of keratinocytes. More preferably, it is used. Moreover, in the cell culture conditions that lead to the comparison of the cell adhesion inhibitory action of keratinocytes in the screening method of the whitening agent of the present invention, a normal medium for culturing the keratinocytes can be used, A medium obtained by adding a cell adhesion promoting factor for keratinocytes to a normal medium can also be used. As a medium used for the screening method of the whitening agent of the present invention, Humedia-KG2 (Keratinocyte growth medium, Kurashiki Boseki Co., Ltd.) can be preferably exemplified. In the screening method for the whitening agent of the present invention, it is preferable to use a single culture of keratinocytes, but a co-culture system of keratinocytes and pigment cells can also be used.

  The whitening agent selected by the whitening agent screening method of the present invention is characterized by having a cell adhesion inhibitory action of keratinocytes. In the basal cell layer of the skin site where pigmentation symptoms such as sunburn, blotches and dullness are observed, melanin production of pigment cells is increased or melanin production is increased, and the production of melanin and related substances is increased. ing. In addition, excessively produced melanin and related substances are transported to and accumulated in keratinocytes, thereby enhancing the cell adhesion ability of keratinocytes and firmly anchoring keratinocytes to the basement membrane. Causes cell turnover damage. In addition, keratinocytes that contain a large amount of melanin and related substances are firmly anchored in the basal layer due to the turnover failure of keratinocytes, causing the black color tone and pigmentation of the skin. As described above, the promotion of cell adhesion ability of keratinocytes is deeply involved in the occurrence of pigmentation symptoms, the establishment and deterioration of symptoms. The whitening agent having an inhibitory effect on cell adhesion of keratinocytes according to the present invention reduces the cell adhesion ability increased by excessive transfer and accumulation of melanin and related substances to keratinocytes, Whitening effect is achieved by normalizing over. For this reason, the whitening agent of the present invention has the effect of thinning or returning the pigmentation symptoms such as sunburn, blotches and dullness already formed by exposure to ultraviolet rays, sunburn, blotches and dullness formed in the future. It has an action to prevent pigmentation symptoms such as, and exhibits a whitening effect on such skin pigmentation symptoms. Moreover, since the whitening agent of this invention is a whitening agent through the mechanism of action different from the past, the outstanding whitening effect can be anticipated by containing in the composition for whitening. Furthermore, the whitening agent of the present invention can be expected to be additive or synergistically enhanced in the whitening effect when used in combination with conventional whitening agents.

  The test substance targeted by the screening method of the whitening agent of the present invention may be any of a chemical substance or a mixture of extracts from animals and plants. The extracts derived from animals and plants mean not only animal or plant-derived extracts themselves, but also generic terms such as extract fractions, purified fractions, solvent-removed products of purified products, and the like. As the plant-derived extract, an extract produced using a plant grown as a native or grown plant, a herbal medicine raw material, or the like, a commercially available extract, and the like are preferable. The plant part used for the extraction operation is not particularly limited. In addition to using the whole plant, the plant part, the above-ground part, the rhizome part, the tree trunk part, the leaf part, the stem part, the flower ear, the flower bud, etc. are used. I can do it. Moreover, it is preferable to improve the extraction efficiency by crushing or chopping the plant parts in advance. Extraction solvents include water, alcohols such as ethanol, isopropyl alcohol and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, ethers such as diethyl ether and tetrahydrofuran. One or two or more selected from polar solvents such as the above can be exemplified as preferable ones. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a plant or the like used for extraction of a plant or the like. If so, there may be mentioned a method of immersing for several hours, cooling to room temperature, removing insoluble matters and / or solvents if desired, and fractionating and purifying by column chromatography or the like.

The addition amount of the test substance in the whitening agent screening method of the present invention (the total amount when added multiple times) should be appropriately set according to the type of keratinocytes used for screening or the culture conditions. . The addition amount of the test substance in the whitening agent screening method of the present invention (total amount when added multiple times) is, for example, usually 0.0001 (v / v%) or more, preferably 0 as the final concentration in the medium. 0.005 (v / v%) or more, more preferably 0.001 (v / v%) or more, usually 50 (v / v%) or less, preferably 30 (v / v %) Or less, more preferably 20 (v / v%) or less.

1) Screening method for comparing integrin production inhibitory action of keratinocytes of the present invention Screening method of whitening agent for evaluating cell adhesion inhibitory action of keratinocytes of the present invention and selecting a whitening agent using the action as an index Among them, a screening method for a whitening agent including a step of comparing the integrin production inhibitory effect of keratinocytes will be described. The keratinocyte integrin production inhibitory action evaluated in the whitening agent screening method including the step of comparing the integrin production inhibitory action of the keratinocytes of the present invention includes a corner that can evaluate the cell adhesion inhibitory action of keratinocytes. If it is an integrin production inhibitory effect of a cell, it can be applied without particular limitation. Preferred examples of the integrin production inhibitory action of the keratinocytes include an integrin α6 and / or β4 gene expression inhibitory action, an integrin α6 and / or β4 protein expression inhibitory action, and the like. Among the integrin α6 and / or β4 gene expression inhibitory effects, a more preferable example is an integrin α6 and / or β4 mRNA expression inhibitory action. It is confirmed that integrin α6β4 protein is expressed in keratinocytes, and that the integrin α6 and β4 protein expression levels are increased in skin sites such as spots and dullness, and the relationship between the integrin α6β4 production inhibitory action and pigmentation Is shown. In evaluating the integrin production inhibitory action, an action highly related to the integrin α6 and / or β4 gene expression level or protein expression level may be substituted.

  The screening method for a whitening agent for comparing the integrin production inhibitory effect of keratinocytes and evaluating the cell adhesion inhibitory effect of keratinocytes of the present invention compares the integrin production inhibitory effect of keratinocytes, and It is characterized by evaluating and selecting the whitening agent using the action as an index. In the screening method of the whitening agent of the present invention, “comparing integrin production inhibitory effect of keratinocytes” refers to the integrin production inhibitory effect of keratinocytes to which a test substance is added and keratinocytes to which no test substance is added. Measures the physical quantity and suppresses the cell adhesion of keratinocytes added with the test substance based on the measured value of the physical quantity related to the cell adhesion inhibitory action of the keratinocytes without adding the test substance or the value calculated from the measured value This means that the physical adhesion measured value or the value calculated from the measured value is compared with the cell adhesion inhibitory action of the test substance.

  Integrin α6β4 is a cell adhesion molecule formed from two types of subunits, integrins α6 and β4. For this reason, the cell adhesion ability of keratinocytes decreases by decreasing the gene expression level or protein expression level of one or both of the two types of subunits of integrin α6 or β4. Therefore, when comparing the integrin production inhibitory effect of the present invention and evaluating the cell adhesion inhibitory effect of keratinocytes, the gene expression level or protein expression level of one or both of integrin α6 or β4 is measured, and the measured value Alternatively, the values calculated from the measured values can be compared and evaluated.

Specific steps in the screening method for the whitening agent for evaluating the integrin production inhibitory effect of keratinocytes are listed below.
(I) A test substance is added to the keratinocytes used in the evaluation (which may be activated or non-activated) (a keratinocyte to which no test substance is added is also prepared as a comparison control).
(Ii) Physical quantities related to the integrin production inhibitory action in keratinocytes to which a test substance is added and keratinocytes to which no test substance is added (for example, integrin α6 and / or β4 mRNA expression level, integrin α6 and / or β4 protein expression level) Etc.).
(Iii) The physical quantity related to the measured integrin production inhibitory action is compared, and the keratinocyte cell adhesion inhibitory action of the test substance is evaluated.
(Iv) The cell adhesion inhibitory action of the evaluated keratinocytes is determined based on preset criteria, and the test substance is determined.

  Regarding the method for screening a whitening agent for comparing the integrin production inhibitory action of the keratinocytes, the keratinocytes used in the step (i) include horns whose cell adhesion ability has been improved by adding a cell adhesion promoting factor or the like. Either keratinocytes or normal keratinocytes that do not have improved cell adhesion ability may be used, but keratinocytes with improved cell adhesion ability are preferably used in terms of measurement sensitivity and accuracy. As a cell adhesion promoting factor to be added to keratinocytes, melanin and related substances (for example, lipidated melanin produced from lipid peroxide and melanin) are preferable.

  The physical quantity relating to the integrin production inhibitory action measured by the step (ii) of the method for comparing the integrin production inhibitory action of the keratinocytes of the present invention is the above-mentioned keratinocyte integrin α6 and / or β4 gene expression level. Inhibition of integrin production of multiple types of keratinocytes is not limited to measuring and evaluating one type of physical quantity related to the integrin production suppression effect of keratinocytes, such as the expression level of integrin α6 and / or β4 protein Inhibition of cell adhesion of keratinocytes by measuring the physical quantity of keratinocytes, calculating the integrin production inhibitory effect of keratinocytes by any treatment method based on the measured value or the measured value, and comparing these values The effect may be evaluated. Furthermore, the criteria used for the determination in the step (iv) are not particularly limited, and can be set as appropriate according to the purpose. As a criterion (index) in the above step (iv), for example, when it is evaluated that the test substance has a cell adhesion inhibitory action of keratinocytes, it can be selected as a whitening agent. An evaluation standard is set in advance for the cell adhesion inhibitory action of cells, and when the standard is exceeded, the test substance is selected as a whitening agent. Furthermore, by evaluating the cell adhesion inhibitory action of keratinocytes on multiple test substances and comparing these actions relatively, it is determined that the test substance having a higher cell adhesion inhibitory action is a better whitening agent It is preferable to do. The whitening agent discriminated or selected through this process exhibits an excellent whitening effect.

Here, among the methods for screening whitening agents for comparing the integrin production inhibitory effect, an integrin α6 and / or β4 gene expression inhibitory effect, more specifically, a method for comparing integrin α6 and / or β4 mRNA expression levels will be described. Currently, methods for measuring integrin gene expression levels include Northern blotting, reverse transcription polymerase chain reaction (RT-PCR), and real-time PCR (real-time PCR). Time PCR) method, and further a measurement method combining real-time PCR and RT-PCR is known. In the measurement of the integrin α6 and / or β4 gene expression level in the present invention, in addition to the measurement method described above, any method that measures the integrin α6 and / or β4 gene expression level can be applied without particular limitation. From the viewpoint of measurement sensitivity and simplicity of measurement, a method of measuring the integrin α6 and / or β4 mRNA expression level by a real-time PCR method is preferable. By using the aforementioned method for measuring the expression level of integrin α6 and / or β4 gene, the expression level of integrin α6 and / or β4 gene in keratinocytes to which a test substance is added and keratinocytes to which no test substance is added, Specifically, the integrin α6 and / or β4 mRNA expression level can be measured. The integrin α6 and / or β4 mRNA expression level can be easily measured using a commercially available simple measurement kit. As such a commercially available simple measurement kit, for example, an RNA purification kit sold by QIAGEN can be suitably exemplified. After extracting total mRNA using such a commercially available simple measurement kit, the SuperScript® VILO ^™ cDNA synthesis kit ( Life Technologies Japan Co., Ltd., 11754-250) is used for reverse transcription reaction, PCR reaction is performed using real-time PCR analysis system (StepOnePlus ^™ Real-Time PCR Systems, Abride Biosystems), and the target integrin α6 and / Or β4 mRNA expression level can be measured. Among the methods for measuring the expression level of integrin α6 and / or β4 mRNA in stratum corneum of the present invention, a particularly preferred method is a method for measuring integrin α6 and / or β4 mRNA in stratum corneum described in Test Example 3 described later. Can be suitably exemplified.

  The component selected as the whitening agent in the screening method for the whitening agent for comparing the integrin α6 and / or β4 gene expression inhibitory action (integrin α6 and / or β4 mRNA expression inhibitory action) of the keratinocytes of the present invention is the above (iii ) When comparing the expression level of integrin α6 and / or β4 gene (inhibition effect on integrin α6 and / or β4 mRNA) in keratinocytes to which the test substance is added in the step) and keratinocytes to which no test substance is added, the test substance A component in which a decrease in the expression level of integrin α6 and / or β4 gene in keratinocytes is observed (decrease in expression level of integrin α6 and / or β4 mRNA), more preferably integrin α6 in keratinocytes to which a test substance is added and Tested against the β4 gene expression level (integrin α6 and / or β4 mRNA expression level) Component gene expression in keratinocytes without added quality has decreased by more than 20% preferably be exemplified. Among these components, the preferred is the integrin α6 and / or β4 gene expression level (integrin α6 and / or β4 mRNA in keratinocytes to which no test substance is added in the whitening agent screening method described in Example 1 described later. A component in which a decrease in integrin α6 and / or β4 gene expression level (integrin α6 and / or β4 mRNA expression level) of keratinocytes to which a test substance is added, more preferably, a test substance is added A component in which the gene expression level in keratinocytes to which no test substance is added is reduced by 20% or more relative to the integrin α6 and / or β4 gene expression level (integrin α6 and / or β4 mRNA expression level) in keratinocytes It can be illustrated.

  Here, among the methods for measuring the integrin production inhibitory action, a method for comparing the expression levels of integrin α6 and / or β4 protein will be described. Currently, methods for measuring the expression level of integrin α6 and / or β4 protein include direct and indirect fluorescent antibody methods, radioimmunoassay (RIA) using a labeled antibody, enzyme immunoassay (EIA), Western blotting, flow cytometry method. A method using an antibody such as is used. In the measurement of the integrin α6 and / or β4 protein expression level in the present invention, there is no particular limitation as long as it is a method capable of measuring the integrin α6 and / or β4 protein expression level in addition to the measurement method using the antibody. It can be applied. The method for measuring the expression level of integrin α6 and / or β4 protein of the present invention is preferably a method for measuring the expression level of integrin α6 and / or β4 protein by the indirect fluorescent antibody method from the viewpoint of measurement sensitivity and ease of measurement. . By the method for measuring the integrin α6 and / or β4 protein expression level described above, the integrin α6 and / or β4 protein expression level in the cornified cells with and without the test substance added can be accurately and simply measured. The amount of integrin α6 and / or β4 protein can be easily measured using a commercially available antibody or the like. Examples of such commercially available antibodies include anti-human Integrin alpha 6 antibody (Mouse monoclonal) (abcam, product number: ab20142) as the primary antibody of integrin α6 protein, and anti-human β4 protein as the primary antibody of anti-human Integrin alpha 6 antibody (Mouse monoclonal). Integrin beta 4 antibody (abcam, product number: ab110167), secondary antibodies of integrin α6 protein include Alexa Fluor 555 Goat Anti-mouse IgG (Life Technologies Japan, product number: A-21422), integrin β4 protein Preferred examples of the secondary antibody include Alexa Fluor 488 goat anti-rat IgG (H + L) (Life Technologies Japan, Inc., product number: A-11006). Furthermore, integrin α6 and / or β4 protein expression levels can be measured according to the method described in Test Example 4 described later. When measuring (comparing) the expression level of integrin α6 and / or β4 protein using such an antibody, it can be measured (compared) visually using a fluorescence microscope or the like, and the fluorescence intensity can be detected. Can be semi-quantified. Among the methods for measuring the expression level of integrin α6 and / or β4 protein in keratinocytes of the present invention, a particularly preferred method is keratinization in which a test substance is added based on the test method described in Test Example 4 described later. After reacting the primary antibody that recognizes integrin α6 or β4 in cells and keratinocytes to which no test substance is added, and a secondary antibody that reacts with the primary antibody and enables fluorescence detection, integrin α6 and A method of measuring (comparing) the β4 protein expression level and confirming the decrease in the protein expression level can be suitably exemplified.

  Among the screening methods for whitening agents for evaluating the integrin production inhibitory action of keratinocytes of the present invention, the component selected as the whitening agent in the whitening agent screening method for evaluating the integrin α6 and / or β4 protein expression inhibitory action When the expression levels of integrin α6 and / or β4 protein in the keratinocytes added with the test substance in the step (iii) and the keratinocytes not added with the test substance are compared, the integrin α6 of the keratinocytes is compared with the test substance. And / or a component in which a decrease in the expression level of β4 protein is preferred. Among the screening methods for whitening agents using the keratinocytes in comparison with the integrin α6 and / or β4 protein expression inhibitory action, evaluating the keratinocyte cell adhesion inhibitory action, and using the action as an index, Are the expression levels of integrin α6 and / or β4 protein in keratinocytes to which a test substance is added, and integrin α6 and / or β4 in keratinocytes to which no test substance is added, based on the method described in Test Example 4 described later. A method of comparing protein expression levels, evaluating the cell adhesion inhibitory action of keratinocytes, and selecting a whitening agent using the action as an index can be suitably exemplified. As a component selected as a whitening agent in the screening method for a whitening agent that compares the expression levels of integrin α6 and / or β4 protein in keratinocytes and evaluates the cell adhesion inhibitory action of keratinocytes, A component in which a decrease in the expression level of integrin α6 and / or β4 protein in keratinocytes can be suitably exemplified, and as such a component, when keratinocytes that have been reacted with an antibody and fluorescently stained are observed with a fluorescence microscope, Compared with the keratinocytes to which the test substance is added, a component in which a decrease in the expression level of the integrin α6 and / or β4 protein in the keratinocytes to which the test substance is not added, that is, a decrease in fluorescence coloration can be suitably exemplified. In addition to the visual observation using an apparatus such as a fluorescence microscope, the aforementioned fluorescent color development can be measured semi-quantitatively using an apparatus such as a fluorescence plate reader or image analysis software. A component in which a decrease in the expression level of α6 and / or β4 protein is observed can also be selected as a whitening agent. When using this method for (semi) quantitatively evaluating the expression level of integrin α6 and / or β4 protein, the component selected as the whitening agent of the present invention includes integrin α6 and / or in keratinocytes to which no test substance is added. Or a component in which a decrease in integrin α6 and / or β4 protein expression level is observed in keratinocytes to which a test substance is added relative to the β4 protein expression level, more preferably, the decrease in the protein expression level is 10% or more, more preferably Is preferably exemplified by a component having a decrease in the protein expression level of 20% or more.

2) Whitening agent screening method for evaluating the inhibitory effect of keratinocytes on the basement membrane Among the screening methods for whitening agents using the inhibitory effect of keratinocytes on cell adhesion as an index, A screening method for a whitening agent including a step of comparing the adhesion inhibitory action of keratinocytes will be described. In the screening method for a whitening agent including the step of comparing the inhibitory action of keratinocytes to the basement membrane of the present invention, the inhibitory action of keratinocytes to the basement membrane is a basal capable of evaluating the inhibitory action of keratinocytes. As long as the action of suppressing the adhesion of keratinocytes to the membrane, it can be applied without particular limitation. As an action of suppressing the adhesion of keratinocytes to the basement membrane, an action of inhibiting the adhesion of keratinocytes to laminin-5, which is a main component of the basement membrane, can be preferably exemplified.

Specific steps in the whitening agent screening method for comparing the inhibitory effect of keratinocytes on the basement membrane of the present invention are listed below.
(I) A test substance is added to cells used for evaluation (which may be activated or non-activated) (cells to which no test substance is added are also prepared as a comparative control).
(Ii) When the test substance is added and when the test substance is not added, the keratinocyte adhesion inhibitory action on the basement membrane in the keratinocytes (the keratinocyte adhesion inhibitory action on the basement membrane component laminin-5) taking measurement.
(Iii) The measured inhibitory effect of keratinocytes on the basement membrane (the inhibitory effect of keratinocytes on laminin-5) is compared to evaluate the inhibitory effect of keratinocytes on the test substance.
(Iv) The cell adhesion inhibitory action of the evaluated keratinocytes is determined based on preset criteria, and the test substance is determined.

  Regarding the method for screening a whitening agent for comparing the inhibitory effect of keratinocytes on the basement membrane, in the step (i), the keratinocytes whose cell adhesion ability is improved by adding a cell adhesion promoting factor, or the like, Any keratinocytes that do not have improved cell adhesion ability may be used, but keratinocytes with improved cell adhesion ability are preferably used in terms of measurement sensitivity and accuracy. As the cell adhesion promoting factor to be added, melanin and its related substances (for example, lipidated melanin produced from lipid peroxide and melanin) are preferable.

  The keratinocytes express biological activities such as cell and tissue structure maintenance and signal transduction by adhering to laminin-5, which is a major component of the basement membrane, via the cell adhesion molecule integrin α6β4. The whitening agent selected by the whitening agent screening method for comparing the inhibitory effect of keratinocytes on the basement membrane of the present invention and evaluating the inhibitory effect of keratinocytes on cell adhesion is the adhesion of keratinocytes to the basement membrane. A component with an inhibitory effect, more preferably a component with an inhibitory effect on the adhesion of keratinocytes to laminin-5, which is a constituent of the basement membrane, can be preferably exemplified. Such components normalize the turnover of keratinocytes and exert a whitening effect by suppressing the adhesion of keratinocytes to the basement membrane. Therefore, by comparing the inhibitory effect of keratinocytes on the basement membrane, the inhibitory effect of keratinocytes on cell adhesion can be evaluated, and a whitening agent can be selected.

  The keratinocyte adhesion inhibitory action on the basement membrane in the step (ii) is not particularly limited as long as the keratinocyte adhesion inhibitory action on the basement membrane can be evaluated. Preferably, it is preferably an action of inhibiting the adhesion of keratinocytes to laminin-5, a basement membrane component. Further, the physical quantity related to the inhibitory action of keratinocytes on the basement membrane is related to the inhibitory action of keratinocytes to the basement membrane of keratinocytes to which the test substance is added and keratinocytes to which no test substance is added. By measuring the physical quantity and comparing the measured value or a value calculated from the measured value, the cell adhesion inhibitory action of keratinocytes can be evaluated. Among the methods for comparing the inhibitory effect of keratinocytes on the basement membrane of the present invention, a preferable example is a method of comparing the inhibitory effect of keratinocytes on the basement membrane component laminin-5. For example, keratinocytes are cultured using a culture container coated with laminin-5 described in Example 2, and the cell nucleus is fluorescently stained with a fluorescent reagent such as Hoechst 33342, and then adhered cells using a measuring device such as a plate reader. A method of measuring the number and calculating and evaluating the ratio of the number of adherent cells can be suitably exemplified.

  Among the screening methods for whitening agents that evaluate the cell adhesion inhibitory action of keratinocytes of the present invention, the whitening agent that evaluates the adhesion inhibitory action of keratinocytes on the basement membrane (the inhibitory action of keratinocytes on laminin-5). In this screening method, the component selected as a whitening agent is a keratinocyte to which a test substance is added in the step (iii), and an inhibitory effect on the basement membrane in a keratinocyte to which no test substance is added (laminin-5) When the keratinocyte adhesion-suppressing action against keratinocytes is compared, a component in which the test substance has an inhibitory action on keratinocyte adhesion to the basement membrane (laminin-5) can be preferably exemplified. As a screening method for whitening agents for comparing the inhibitory effect of keratinocytes on the basement membrane (laminin-5) of the present invention, for example, the screening method for whitening agents described in Example 2 described later can be preferably exemplified. As a component selected as a whitening agent in the screening method of the whitening agent described in Example 2, a test substance is added to the adhesion of keratinocytes to laminin-5 of keratinocytes to which no test substance is added. A component in which a decrease in the adhesion of keratinocytes to laminin-5 is observed, more preferably a component in which the adhesion of keratinocytes to laminin-5 decreases with a statistically significant difference. Can be illustrated.

3) Screening method for whitening agent for comparing the keratinocyte upper layer migration promoting action of the present invention Among the screening methods for whitening agent comprising the step of evaluating the cell adhesion inhibitory action of the keratinocyte of the present invention, A method for screening a whitening agent including a step of comparing the upper layer movement promoting action will be described. The keratinocyte upper layer migration promoting action compared in the whitening agent screening method including the step of comparing the keratinocyte upper layer migration promoting action of the present invention is a keratinization capable of evaluating the cell adhesion inhibitory action of keratinocytes. It can be applied without particular limitation as long as it promotes cell upper layer movement. Specific steps in this screening method are listed below.
(I) A test substance is added to cells used for evaluation (which may be activated or non-activated) (cells to which no test substance is added are also prepared as a comparative control).
(Ii) The upper layer migration promoting action in keratinocytes when the test substance is added and when the test substance is not added is measured.
(Iii) Compare the measured keratinocyte upper layer migration promoting action and evaluate the test substance's keratinocyte cell adhesion inhibitory action.
(Iv) The cell adhesion inhibitory action of the evaluated keratinocytes is determined based on preset criteria, and the test substance is determined.

  Regarding the method for screening a whitening agent for comparing the above-mentioned keratinocyte upper layer movement promoting action, in the step (i), the keratinocytes whose cell adhesion ability is improved by adding a cell adhesion promoting factor or the like, or cell adhesion Any of the keratinocytes whose performance has not been improved may be used, but keratinocytes having improved cell adhesion ability are preferably used in terms of measurement sensitivity and accuracy. As the cell adhesion promoting factor to be added, melanin and its related substances (for example, lipidated melanin produced from lipid peroxide and melanin) are preferable.

  The keratinocytes in the basal cell layer receive and contain melanin and related substances produced by pigment cells, and then are pushed up to the skin surface by the progress of keratinization, and finally become plaque and discharged from the skin. In skin areas where sunburn, spots, dullness, etc. are observed, excessively produced melanin and related substances are transported to and accumulated in keratinocytes, thereby suppressing upper layer movement of keratinocytes. Disruption of cell turnover occurs. The whitening agent selected by the whitening agent screening method for comparing the keratinocyte upper layer migration promoting action of the present invention normalizes the turnover of keratinocytes by promoting the keratinocyte upper layer migration, and the whitening effect To demonstrate. Therefore, the cell adhesion inhibitory action of keratinocytes can be evaluated by comparing the upper layer movement promoting action of keratinocytes, and a whitening agent can be selected.

  Among the steps of the whitening agent screening method for comparing the keratinocyte upper layer migration promoting action of the present invention, the keratinocyte upper layer migration promoting action in the step (ii) described above is a keratinocyte cell adhesion inhibitory action. Any physical quantity relating to the action of promoting the upper layer movement of keratinocytes that can be evaluated can be applied without particular limitation. The keratinocyte upper layer migration promoting action is a measured value or a measured value by measuring a physical quantity related to the keratinocyte added with the test substance and the keratinocyte upper layer migration promoting action without adding the test substance. The cell adhesion inhibitory action of keratinocytes can be evaluated by comparing the calculated values. The method of evaluating the keratinocyte cell adhesion inhibitory effect by comparing the keratinocyte upper layer migration promoting action with and without the test substance added in the step (iii) is compared with the keratinocyte upper layer migration promoting action. Any method that can evaluate the cell adhesion inhibitory action of keratinocytes can be applied without particular limitation. Moreover, as a screening method of the whitening agent for comparing the keratinocyte upper layer migration promoting action of the present invention, for example, the whitening agent screening method for comparing the keratinocyte upper layer migration promoting action described in Example 3 to be described later Can be suitably exemplified. In the screening method for the whitening agent for comparing the keratinocyte upper layer migration promoting action described in Example 3, the test substance is used under the calcium-switched culture conditions using the culture vessel coated with the basement membrane component laminin-5. After culturing the added keratinocytes and keratinocytes to which no test substance is added, the nucleus is fluorescently stained with Hoechst 33342 or the like, and then the number of keratinocytes above the lowermost layer is evaluated by image analysis or the like. The action of promoting the upper layer movement of cells can be evaluated. Moreover, as a component selected as a whitening agent in the screening method for a whitening agent that compares the action of promoting the upper layer movement of keratinocytes described in Example 3 described later, the upper layer is lower than the lowermost layer of the keratinocytes to which the test substance is added. The number of keratinocytes in the keratinocytes to which the test substance is not added is increased compared to the number of keratinocytes in the uppermost layer of the keratinocytes, and more preferably, the test substance is added with a statistically significant difference. A component in which the number of keratinocytes in the upper layer is higher than the lowest layer of the added keratinocytes can be preferably exemplified.

<Test Example 1: Study 1 on enhancement of cell adhesion function in human stain tissue>
According to the following procedure, the cell adhesion function enhancement (integrin α6 protein expression level enhancement) in human skin tissue was confirmed. Formalin-fixed paraffin-embedded stain tissue section (BIOPREDIC International) was deparaffinized and rehydrated, immersed in 10 mM citrate buffer (pH 6.0) and heated in an autoclave at 120 ° C for 20 minutes to activate the antigen. It was. Non-specific reaction was blocked by incubating with 3% BSA-containing PBS at room temperature for 1 hour, and the primary antibody ((Anti-human Integrin alpha 6 antibody (Mouse monoclonal) (abcam, product) No. ab20142) was added dropwise to the sample, incubated overnight at 37 ° C., washed with PBS, and diluted 100-fold with PBS containing 1% BSA ((Alexa Fluor 555 Goat Anti-mouse IgG ( Life Technologies Japan Co., Ltd., product number: A-21422)) was added dropwise to the sample, incubated at 37 ° C. for 1 hour, washed with PBS, and an aqueous encapsulant (Fluoromount; Diagostic BioSystems, Cat. No. K024). And observed with a fluorescence microscope.

  A (fluorescence) micrograph of human stain tissue is shown in FIG. In FIG. 1, the photomicrograph on the left is a bright field image of human stain tissue, and the photo on the right is a fluorescence-stained image. In the bright-field image and the fluorescence-stained image in FIG. 1, white circles represent portions where differences in melanin amount are recognized. From the difference in color tone in each white circle portion of the bright field image of FIG. 1, it can be seen that the white circle portion in the upper left is a skin site with a small amount of melanin, and the white circle portion in the lower right is a skin site with a large amount of melanin. . In addition, the fluorescent staining image in FIG. 1 indicates that the upper left white circle portion has lower fluorescence intensity and lower integrin α6 protein expression level than the lower right white circle portion. Further, by comparing and examining the bright field image and the fluorescent staining image in FIG. 1, it is found that the integrin α6 protein expression level of keratinocytes is high in the skin region where the amount of melanin is large. For this reason, it is considered that the cell adhesion of keratinocytes is enhanced in the skin site where the amount of melanin is large.

<Test Example 2: Study 2 on enhancement of cell adhesion function in human stain tissue>
In accordance with the following procedure, cell adhesion function enhancement (integrin β4 protein expression level enhancement) in human blemis tissues was confirmed. A frozen stained tissue section (BIOPREDIC International) was fixed in formalin for 10 minutes at room temperature, and then washed with PBS (Takara Bio Inc.). Non-specific reaction was blocked by incubating with PBS containing 3% BSA at room temperature for 1 hour, and the primary antibody (Rat mAb to integrin b4 (abcam, product number: ab110167)) diluted 100 times with PBS containing 1% BSA was sampled. It was dripped in. Secondary antibody (Alexa Fluor 488 goat anti-rat IgG (H + L) (Life Technologies Japan, Inc., product) Number: A-11006)) was added dropwise to the sample. After incubation at 37 ° C. for 1 hour, the plate was washed with PBS, sealed with an aqueous mounting medium (Fluoromount; Diagostic BioSystems, Cat. No. K024), and observed with a fluorescence microscope.

  FIG. 2 shows a (fluorescence) micrograph of human stain tissue. In FIG. 2, the photomicrograph on the left is a bright field image of human stain tissue, and the photo on the right is a fluorescence-stained image. In the bright-field image and the fluorescence-stained image in FIG. 2, white circles represent portions where differences in melanin amount are recognized. From the difference in color tone in each white circle portion of the bright-field image in FIG. 2, it can be seen that the right white circle portion is a skin site with a small amount of melanin, and the left white circle portion is a skin site with a large amount of melanin. In addition, the fluorescence staining image in FIG. 2 shows that the right white circle portion has lower fluorescence intensity and the integrin β4 protein expression level is lower than the left white circle portion. In addition, by comparing and examining the bright field image and the fluorescent staining image in FIG. 2, it can be seen that the integrin β4 protein expression level of keratinocytes is high in the skin region where the amount of melanin is large. For this reason, it is considered that the cell adhesion of keratinocytes is enhanced in the skin site where the amount of melanin is large.

<Test Example 3: Examination of changes in integrin α6 and β4 mRNA expression levels by addition of cell adhesion promoting factor in human normal keratinocytes>
In accordance with the following procedure, examination was made regarding changes in integrin α6 and β4 mRNA expression levels due to the addition of a cell adhesion promoting factor in human normal keratinocytes. In addition, melanin or greasy melanin was used as a cell adhesion promoting factor. Using a keratinocyte growth medium (Humedia-KG2, hereinafter referred to as KG-2 medium, Kurashiki Boseki Co., Ltd.), newborn human normal keratinocytes (Kurashiki Boseki Co., Ltd.) were added at a density of 1.0 × 10 ⁵ (cells / well). Seeded in well plate. Next day, 10 (w / v%) melanin (melanin added group, Sigma Aldrich Japan Co., Ltd., final concentration 1.5 × 10 ⁻³ (w / v%))-containing dimethyl sulfoxide solution, or dimethyl sulfoxide (melanin non-added group) The medium was changed to KG-2 medium (1.0 mL / well) supplemented with Wako Pure Chemical Industries, Ltd., final concentration 1.5 × 10 ⁻² (w / v%)). 24 hours after the addition of melanin, the cells were collected with RLT buffer of QIAGEN RNeasy Mini Kit (QIAGEN) and analyzed for integrin α6 and β4 mRNA expression levels. The integrin α6 and β4 mRNA expression levels are calculated by comparative CT method using β-actin as an endogenous control, and the integrin α6 or β4 mRNA expression level in keratinocytes without melanin or lipidated melanin is set to “1” Indicated by value. In addition, the lipidated melanin used in this test was prepared by adjusting the lipidized melanin suspension of the cell adhesion promoting factor of keratinocytes according to the procedure described in the above Production Example 1 and Production Example 2, and then the above test. According to the procedure, the expression levels of integrin α6 and β4 mRNA by the addition of lipid melanin were measured. In addition, the addition density | concentration of fat-containing melanin is the density | concentration (final density | concentration 5 * 10 < ^-3 >) which added 0.5 (w / v%) fat-containing melanin suspension with respect to the culture medium 1 (mL). (W / v%)).

  The results are shown in FIG. 3 (changes in the expression levels of integrin α6 and β4 mRNA due to the addition of melanin) and FIG. 4 (changes in the expression levels of integrin α6 and β4 mRNA due to the addition of lipidated melanin). The vertical axis in FIGS. 3 and 4 is expressed as a ratio of the integrin α6 and β4 mRNA expression levels when the expression level of integrin α6 or β4 mRNA in keratinocytes without melanin or lipidated melanin is “1”. Yes. From the results of FIG. 3 and FIG. 4, the expression levels of integrin α6 and β4 mRNA in keratinocytes added with melanin or lipidated melanin are remarkable compared to the expression levels of integrin α6 and β4 mRNA in keratinocytes without melanin or lipidated melanin. Had increased. This suggests that keratinocyte integrin α6 and β4 mRNA expression levels are increased by the addition of melanin or lipidated melanin, and cell adhesion of keratinocytes is enhanced.

<Test Example 4: Study on change in expression level of integrin α6β4 protein by addition of cell adhesion promoting factor in human normal keratinocytes>
According to the following procedure, examination was made regarding changes in the expression level of integrin α6 and / or β4 protein by addition of cell adhesion factor in human normal keratinocytes. In addition, melanin or greasy melanin was used as a cell adhesion promoting factor. Using an 8-well culture slide (Falcon) with keratinocyte growth medium (Humedia-KG2, hereafter referred to as KG-2 medium, Kurashiki Boseki Co., Ltd.), normal human keratinocytes (Kurashiki Boseki Co., Ltd.) are 2.0 × 10 ⁴ (cells / Well). The next day, 10 (w / v%) melanin (melanin added group, Sigma Aldrich Japan Co., Ltd., final concentration: 1.5 × 10 ⁻³ (w / v%))-containing dimethyl sulfoxide solution, or dimethyl sulfoxide (without melanin added) Group, Wako Pure Chemical Industries, Ltd., and the medium was changed to KG-2 medium (0.35 mL / well) supplemented with a final concentration of 1.5 × 10 ⁻² (w / v%)). 24 hours after melanin addition, washed with PBS (Takara Bio Inc.), fixed with 4% paraformaldehyde / phosphate buffer (Wako Pure Chemical Industries, Ltd.), and then incubated with PBS containing 3% BSA for 1 hour at room temperature. Thus, non-specific reaction was blocked. Primary antibody adjusted to 30 (mg / mL) with PBS containing 1% BSA (integrin α6: anti-human Integrin alpha 6 antibody (Mouse monoclonal) (abcam, product number: ab20142), integrin β4: anti-human Integrin beta 4 antibody (Mouse monoclonal) (abcam, product number: ab78267)) was added dropwise to the sample, incubated at room temperature for 1 hour, washed with PBS, and diluted 200-fold with PBS containing 1% BSA (integrin α6 and β4 common: Alexa Fluor 555 Goat Anti-mouse IgG (Life Technologies Japan Co., Ltd., product number: A-21422)) was added dropwise to the sample. After incubation at room temperature for 45 minutes, the plate was washed with PBS, sealed with an aqueous mounting medium (Fluoromount; Diagostic BioSystems, Cat. No. K024), and observed with a fluorescence microscope.

  The results are shown in FIG. In addition, the left side of FIG. 5 (a total of 4 sheets for each of 2 sheets in the vertical and horizontal directions) shows the results of examination on the integrin α6 protein expression level, and the right side (2 sheets in the vertical and horizontal directions for a total of 4 sheets) shows the results of the study on the expression level of integrin β4 protein. Show. In the examination result of the expression level of integrin α6 protein, when comparing the left and upper bright field images, dark portions were observed by adding melanin, and it was found that melanin was accumulated in keratinocytes. Also, by comparing the bright-field image and the fluorescence-stained image, in the region where the color tone of the bright-field image is dark (dark) and the amount of melanin is high, the color tone due to the fluorescence development of the fluorescence-stained image is dark (red is dark). It was confirmed that the α6 protein expression level was high. That is, it can be seen that the expression level of the integrin α6 protein is increased at the site where the amount of melanin is large. On the other hand, the results similar to those of the integrin α6 protein expression level were obtained with respect to the result of examination on the integrin β4 protein expression level on the right side of FIG. That is, in a site where the color tone of the bright field image is dark (dark) and the amount of melanin is large, it is confirmed that the color tone due to the fluorescence development of the fluorescent stained image is also dark (red is dark), and the integrin β4 protein expression level is increased. It was. Thus, by comparing the bright-field image and the fluorescence-stained image, it was found that the expression level of integrin α6 and β4 protein increased and the cell adhesion ability of keratinocytes was improved in the region where the amount of melanin was large. .

<Test Example 5: Study on adhesion of keratinocytes to basement membrane by addition of cell adhesion promoting factor of human normal keratinocytes>
According to the following procedure, examination was made regarding the adhesion of keratinocytes to the basement membrane by addition of a cell adhesion promoting factor in human normal keratinocytes (specifically, the adhesion of keratinocytes to laminin-5). Note that melanin was used as a cell adhesion promoting factor. A human normal keratinocyte (Kurashiki Spinning Co., Ltd.) seeded with a keratinocyte growth medium (Humedia-KG2, hereafter referred to as KG-2 medium, Kurashiki Spinning Co., Ltd.) in a 75 (cm ² ) flask is brought into a subconfluent state and the next day. 1.0 (w / v%) melanin (melanin added group, Sigma Aldrich Japan Co., Ltd., final concentration: 6.0 × 10 ⁻⁴ (w / v%))-containing dimethyl sulfoxide solution, or dimethyl sulfoxide (melanin non- The medium was replaced with a KG-2 medium (10 mL / flask) to which an addition group, Wako Pure Chemical Industries, Ltd., final concentration 6.0 × 10 ⁻² (w / v%)) was added. 24 hours after the addition of melanin, the cells were collected by trypsin treatment and seeded in laminin-5 coated wells at a density of 3.0 × 10 ⁴ (cells / well). The next day, the medium was replaced with KG-2 medium containing 1.8 (mM) Ca ²⁺ . After 24 hours of medium exchange, the cells were washed with PBS (Takara Bio Inc.), fixed with 4% paraformaldehyde / phosphate buffer (Wako Pure Chemical Industries, Ltd.), and then added to the nuclear stain Hoechst 33342 (Dojindo Laboratories). And the ratio of the number of adherent cells was calculated using a fluorescent plate reader (Perkin Elmer, Wallac ARVO SX1420). In addition, the ratio of the number of adherent cells was measured by measuring the fluorescence intensity with a fluorescence plate reader (Perkin Elmer Japan Co., Ltd.) (excitation wavelength: 355 nm, fluorescence wavelength: 460 nm), and the fluorescence intensity of the melanin-free group was “1”. The ratio of the number of adherent cells (cell adhesion ratio) was calculated by calculating the fluorescence intensity when melanin was added as the ratio. The laminin-5 coated well is prepared by preparing a 96-well multiwell plate (Falcon). Each well contains 27 (μL) of laminin-5 (Reprocell Co., Ltd., catalog number: RCHEOT004) -containing PBS (10 μg / mL). / well), allowed to stand at 37 ° C. for 2 hours, washed with PBS (Takara Bio Inc.), and then added 100% (μL / PBS) containing 0.1% BSA (Takara Bio Inc.) well) was added and allowed to stand at room temperature for 30 minutes.

  The results are shown in FIG. The vertical axis in FIG. 6 represents the cell adhesion ratio. From the results of FIG. 6, it is confirmed that the addition of melanin (cell adhesion promoting factor) increases the ratio of the number of adherent cells and increases the adhesion of keratinocytes to laminin-5, which is a constituent of the basement membrane. It was done. This indicates that the adhesion of keratinocytes to laminin-5, which is a component of the basement membrane, is enhanced by increasing the amount of melanin, and the cell adhesion ability of keratinocytes is improved. .

<Test Example 6: Study on upper layer movement of keratinocytes by addition of cell adhesion promoting factor in normal human keratinocytes>
According to the following procedure, the investigation was made on the ability of human normal keratinocytes to migrate to the upper layer of keratinocytes by adding a cell adhesion promoting factor. Note that melanin was used as a cell adhesion promoting factor. It has been reported that cultured epidermis cells begin to move to the upper layer after 12 hours by performing a calcium switch while the cell density is confluent. Therefore, the upper layer migration ability can be quantitatively evaluated by counting the number of cells (Mol Biol Cell., 2009 Jan, 20 (1): 452-64). A human normal keratinocyte (Kurashiki Spinning Co., Ltd.) seeded in a 75 (cm ² ) flask using a keratinocyte growth medium (Humedia-KG2, hereafter referred to as KG-2 medium, Kurashiki Spinning Co., Ltd.) is brought into a subconfluent state, and the next day. 1.0 (w / v%) melanin (melanin added group, Sigma Aldrich Japan Co., Ltd., final concentration 6.0 × 10 ⁻⁴ (w / v%))-containing dimethyl sulfoxide solution, or dimethyl sulfoxide (without melanin added) Group, Wako Pure Chemical Industries, Ltd., medium exchange with KG-2 medium (10 mL / flask) added with final concentration 6.0 × 10 ⁻² (w / v%)). 24 hours after the addition of melanin, the cells were collected by trypsin treatment and seeded on laminin-5 coat well (8 well culture slide, Falcon) at a density of 1.5 × 10 ⁵ (cells / well). 24 hours after sowing, the cells were washed with PBS (Takara Bio Inc.), fixed with 4% paraformaldehyde / phosphate buffer (Wako Pure Chemical Industries, Ltd.), and then fluorescent with Hoechst 33342 (Dojin Chemical Laboratories), a nuclear stain. Stained and washed with PBS. After encapsulating with an aqueous mounting medium (Fluoromount; Diagostic BioSystems, Cat. No. K024), take a picture of the uppermost layer from the lowermost layer using a confocal laser microscope, and use the image analysis software ImageJ (National Institutes of Health) The number of keratinocytes in the upper layer from the lowest layer of keratinocytes was determined by measuring the number of nuclei.

When the keratinocytes were cultured under the culture conditions using the calcium switch, the upper layer migration of the keratinocytes was confirmed (data not shown). FIG. 7 shows the results relating to the ability of keratinocytes to move to the upper layer by adding melanin (cell adhesion promoting factor) in human normal keratinocytes. In addition, the vertical axis | shaft of FIG. 7 is a ratio of the number of keratinocytes above the lowest layer of a keratinocyte when the number of keratinocytes above the lowest layer of a keratinocyte in a melanin addition group is set to "1". (Upper cell number ratio). From the result of FIG. 7, it was confirmed that the upper layer movement of keratinocytes was suppressed by addition of a cell adhesion promoting factor (melanin). This indicates that, in the skin site where the amount of melanin is high, the cell adhesion ability of keratinocytes is improved, the upper layer movement of keratinocytes is suppressed, and the turnover of keratinocytes is impaired. .

<The composition for whitening containing the whitening agent of this invention, and its manufacturing method>
As described above, a whitening agent having a novel mechanism of action can be selected with high accuracy and efficiency by the screening method for a whitening agent using the keratinocyte cell adhesion inhibitory action of the present invention as an index. In addition, the component having an inhibitory effect on cell adhesion of keratinocytes of the present invention is useful as a whitening agent having a novel mechanism of action, and by being contained in the composition, pigmentation symptoms such as sunburn, blotches, dullness, etc. Demonstrate the prevention or improvement effect (whitening effect). For this reason, the method of screening the component which has the cell adhesion inhibitory action of a keratinocyte can be utilized in order to select the outstanding whitening agent. Whitening composition containing a whitening agent selected by the whitening agent screening method of the present invention (hereinafter sometimes abbreviated as “the whitening composition of the present invention”) and the whitening agent screening method of the present invention The method for producing a whitening composition comprising the step of blending the whitening agent selected by the whitening composition into the whitening composition (hereinafter sometimes abbreviated as “the production method of the whitening composition of the present invention”) is also included in the present invention. It is one aspect | mode. Hereinafter, the whitening composition of the present invention and the method for producing the whitening composition of the present invention will be described in detail.

  The whitening agent of the present invention may be any of simple chemical substances and mixed compositions such as extracts derived from animals and plants. The extracts derived from animals and plants mean not only the extracts derived from animals or plants per se, but also the generic name of the fraction of the extract, the extract or fraction, and the solvent-removed product of the purified product. As the plant-derived extract, an extract using a plant grown as a native or grown plant, a herbal medicine raw material or the like, or a commercially available extract (such as Maruzen Pharmaceutical Co., Ltd.) can be suitably exemplified. In addition to using whole plants as plant parts, plant parts, aerial parts, rhizome parts, tree trunk parts, leaf parts, stem parts, flower spikes, flower buds, etc. can be used. Thus, it is preferable to improve the extraction efficiency. Examples of the extraction solvent include alcohols such as water, ethanol, isopropyl alcohol and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, acetone, and methyl ethyl ketone. 1 type or 2 types or more selected from polar solvents, such as ketones, such as ketones, ethers, such as diethyl ether and tetrahydrofuran, can be illustrated as a suitable thing. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a plant or the like used for extraction of a plant or the like. If present, there may be mentioned a method of immersing for several hours, cooling to room temperature, removing insoluble matters and / or solvent if desired, and fractionating and purifying by column chromatography or the like.

  If the whitening composition of this invention and the manufacturing method of the whitening composition contain the whitening agent selected by the screening method of the whitening agent of this invention mentioned above (content), content of whitening agent The (mixing amount) and other configurations are not particularly limited, and the preparation method is not particularly limited. However, the content (blending amount) of the whitening agent in the whitening composition of the present invention is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, more preferably 0.001% by mass or more. It is 15 mass% or less, Preferably it is 10 mass% or less, More preferably, it is 5 mass%. This is because if the content (blending amount) of the whitening agent of the present invention is too small, the intended effect tends to decrease, and if it is too much, the effect tends to peak, and the degree of freedom of this system is reduced. This is because there is a case where it is damaged. Moreover, the kind of whitening agent contained in the composition for whitening of this invention is not limited to 1 type, You may select and contain 2 or more types.

  In formulating the whitening composition of the present invention, optional ingredients used in formulating normal foods, pharmaceuticals, cosmetics and the like can be contained. As such an optional component, if it is a composition for oral administration, for example, excipients such as lactose and sucrose, binders such as starch, cellulose, gum arabic, and hydroxypropyl cellulose, sodium carboxymethylcellulose, carboxymethylcellulose calcium, etc. Disintegrants, soy lecithin, surfactants such as sucrose fatty acid esters, sweeteners such as maltitol and sorbitol, sour agents such as citric acid, buffering agents such as phosphate, film forming agents such as shellac and zein, Lubricants such as talc and waxes, light anhydrous silicic acid, glidants such as dry aluminum hydroxide gel, diluents such as physiological saline and aqueous glucose solution, flavoring agents, coloring agents, bactericides, preservatives, A fragrance | flavor etc. can be illustrated suitably. If it is a composition for transdermal administration, hydrocarbons such as squalane, petrolatum and microcrystalline wax, esters such as jojoba oil, carnauba wax, octyldodecyl oleate, triglycerides such as olive oil, beef tallow and coconut oil , Fatty acids such as stearic acid, oleic acid, retinoic acid, higher alcohols such as oleyl alcohol, stearyl alcohol, octyldodecanol, anionic surfactants such as sulfosuccinic acid ester and sodium polyoxyethylene alkyl sulfate, Amphoteric surfactants such as alkylbetaine salts, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, their polyoxyethylene adducts, polyoxyethylene alkyl ethers, polyoxyethylenes Nonionic surfactants such as fatty acid esters, polyhydric alcohols such as polyethylene glycol, glycerin, and 1,3-butanediol, thickening / gelling agents, antioxidants, ultraviolet absorbers, colorants, antiseptics An agent, powder, etc. can be contained. Manufacture can be done without difficulty by treating these components according to conventional methods.

  As the whitening composition of the present invention, pharmaceuticals, cosmetics, foods, beverages and the like can be suitably exemplified, and since they can be taken on a daily basis, they are preferably applied to foods, cosmetics and the like. Either the oral route or the transdermal route can be used, but when a whitening effect is expected on the skin, the retention on the skin, the efficiency of reaching the target site, etc. should be taken into consideration. It is preferable to employ transdermal administration.

  The whitening agent selected by the screening method of the whitening agent of the present invention can be incorporated into cosmetics and utilized as a whitening cosmetic. Further, since the whitening agent of the present invention has a cell adhesion inhibitory action of keratinocytes, on the basis of such action, as a use other than whitening, a cancer metastasis inhibitor, a platelet aggregation inhibitor, a wound healing agent, an arteriosclerosis inhibitor It can also be used as an agent. Furthermore, by normalizing the turnover of keratinocytes due to the cell adhesion inhibitory action of keratinocytes, antiaging effects such as wrinkle improvement and prevention or improvement of rough skin can be expected.

  Preferred examples of the whitening agent having an inhibitory effect on cell adhesion of keratinocytes of the present invention include plant extracts obtained from Citrus citrus genus Yuzu and Asteraceae genus Chrysanthemum genus (Artichoke). Moreover, the plant extract used for the screening method as described in the Example mentioned later used the plant extract purchased from Ichimaru Falcos. Citrus citrus genus Yuzu is a citrus evergreen small tree that originates in China and is widely cultivated in the south of Tohoku. In addition, yuzu juice is used for food as a seasoning for Japanese cuisine, etc., and also has a blood circulation promoting action. Among the plant extracts obtained from Citrus citrus genus Yuz, a preferred example is a plant extract obtained from Citrus citrus genus Yuz (trade name: Yuzuceramide B), which can be purchased from Ichimaru Falcos Co., Ltd. The Asteraceae genus Chrysanthemum (Artichoke) is a perennial that originates in the Mediterranean coast. Asteraceae Chrysanthemum genus Chrysanthemum is cultivated mainly for ornamental use in Japan and partly for food use. Among the plant extracts obtained from Asteraceae, thistle plant is preferably a plant extract obtained from Ichimaru Falcos Co., Ltd. (product name: Biobenefiti). Preferably mentioned.

  Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to such examples.

<Test Example 7: Screening method 1 for the whitening agent of the present invention>
The whitening agent of the present invention was screened according to the test method described below. In addition, the cell adhesion inhibitory action of keratinocytes compared in this screening method is an integrin α6 mRNA expression inhibitory action. Moreover, melanin was used as a cell adhesion promoting factor. Using a keratinocyte growth medium (Humedia-KG2, hereinafter referred to as KG-2 medium, Kurashiki Boseki Co., Ltd.), newborn human normal keratinocytes (Kurashiki Boseki Co., Ltd.) were added at a density of 1.0 × 10 ⁵ (cells / well). Seeded in well plate. The next day, dimethyl sulfoxide (melanin-free group, Wako Pure Chemical Industries, Ltd., final concentration: 1.5 × 10 ⁻² (w / v%), 10 (w / v%) melanin (melanin-added group, Sigma Aldrich Japan) Corporation, final concentration: 1.5 × 10 ⁻³ (w / v%))-containing dimethyl sulfoxide solution, 10 (w / v%) melanin (Sigma Aldrich Japan Co., Ltd., final concentration: 1.5 × 10 ⁻³⁾ (W / v%))-containing dimethyl sulfoxide solution and test substance (final concentration: 1 × 10 ⁻² v / v%) (melanin addition and test substance addition group) added KG-2 medium (1.0 mL / well) 24 hours after addition of melanin, the cells were collected with RLT buffer of QIAGEN RNeasy Mini Kit (QIAGEN) and analyzed for integrin α6 mRNA expression level. Purchased A plant extract (trade name: Yuzuceramide B) obtained from the citrus citrus genus Yuzu and a plant extract (trade name: biobenefiti) obtained from the Asteraceae genus Chrysanthemum (artichoke) were used. The α6 mRNA expression level was calculated by the comparative CT method using β-actin as an endogenous control, and was expressed as a ratio when the integrin α6 mRNA expression level in the melanin added group was “1”.

  The results are shown in FIG. The vertical axis in FIG. 8 is expressed as a ratio when the integrin α6 mRNA expression level of the melanin added group is “1”. From the results of FIG. 8, it was confirmed that the expression level of integrin α6 mRNA was increased by the addition of a cell adhesion promoting factor (melanin), and a screening system was established. Furthermore, the plant extract obtained from the citrus family Citrus genus Yuzu or Asteraceae genus Chrysalis thistle significantly reduced the integrin α6 mRNA expression level increased by the addition of a cell adhesion promoting factor. According to the results shown in FIG. 8, the plant extract obtained from the citrus family Citrus genus Yuzu or Asteraceae genus Chrysanthemum genus Chrysanthemum has a keratinocyte integrin α6 mRNA expression inhibitory action (20% or more integrin compared to the control group). The expression level of α6 mRNA was reduced), and it was revealed that the cells have an inhibitory effect on cell adhesion of keratinocytes. For this reason, the plant extract obtained from the citrus family Citrus genus Yuzu or the Asteraceae genus Chrysanthemum genus Chosen thistle can be selected as the whitening agent of the present invention.

<Test Example 8: Screening method 2 for whitening agent of the present invention>
The whitening agent of the present invention was screened according to the test method described below. In addition, the cell adhesion inhibitory action of keratinocytes compared in this screening method is the adhesion inhibitory action of keratinocytes to laminin-5. Moreover, melanin was used as a cell adhesion promoting factor. A normal human keratinocyte (Kurashiki Spinning Co., Ltd.) seeded in a 75 (cm ² ) flask with a keratinocyte growth medium (Humedia-KG2, KG-2 medium, Kurashiki Spinning Co., Ltd.) is brought into a subconfluent state, and trypsin After recovering the cells by treatment, the cells were seeded in a 25 (cm ² ) flask at a density of 7.5 × 10 ⁵ (cells / flask). The next day, dimethyl sulfoxide (melanin non-added group, Wako Pure Chemical Industries, Ltd., final concentration: 6.0 × 10 ⁻² (w / v%)), 1.0 (w / v%) melanin (melanin added group, Sigma Aldrich Japan Co., Ltd., final concentration 6.0 × 10 ⁻⁴ (w / v%)), 1.0 (w / v%) melanin (Sigma Aldrich Japan Co., Ltd., final concentration 6.0 × 10 ⁻⁴ ( KG-2 medium (3.3 mL / flask) containing (w / v%) and test substance (final concentration: 1.0 × 10 ⁻² (v / v%)) (melanin addition and test substance addition group) 24 hours later, the cells were collected by trypsin treatment, and the collected cells were laminin-5 coated wells at a density of 3 × 10 ⁴ (cells / well) (refer to Production Examples 1 and 2 for the preparation method). ) 1 hour later, PBS (Takara Bio Inc.) After fixation with 4% paraformaldehyde / phosphate buffer (Wako Pure Chemical Industries, Ltd.), the cells were stained with Hoechst 33342 (Dojindo Laboratories), a nuclear stain, and a fluorescent plate reader (Perkin Elmer, Wallac ARVO SX1420) The ratio of the number of adherent cells was calculated by measuring the fluorescence intensity with a fluorescence plate reader (Perkin Elmer Japan Co., Ltd.) (excitation wavelength: 355 nm, fluorescence wavelength: 460 nm), When the fluorescence intensity of the melanin non-added group was “1”, the ratio of the number of adherent cells (cell adhesion ratio) was calculated by calculating the ratio of the fluorescence intensity when melanin was added and when the test substance was added. .

  The results are shown in FIG. The vertical axis in FIG. 9 indicates the ratio of the number of adherent cells (cell adhesion ratio) by calculating the ratio of the fluorescence intensity of melanin and test substance addition when the fluorescence intensity of the melanin-free group is “1”. As displayed. From the results of FIG. 9, it was confirmed that the addition of melanin increased the adhesion (cell adhesion ratio) of keratinocytes to laminin-5, and this test system was established. Moreover, the plant extract obtained from the citrus family Citrus genus Yuzu and Asteraceae genus Chrysanthemum thistle significantly decreased the adhesion of keratinocytes to laminin-5 enhanced by the addition of melanin. According to the results shown in FIG. 9, the plant extract obtained from the citrus family Citrus genus Yuzu or Asteraceae genus Chrysanthemum genus Chrysanthemum shows an adhesion inhibitory effect of keratinocytes on laminin-5, and cell adhesion of keratinocytes Has an inhibitory effect. For this reason, the said test substance can be selected as a whitening agent of this invention.

<Test Example 9: Screening method 3 for the whitening agent of the present invention>
The whitening agent of the present invention was screened according to the test method described below. In addition, the cell adhesion inhibitory action compared in this screening method is a keratinocyte upper layer movement promoting action. Moreover, melanin was used as a cell adhesion promoting factor. A normal human keratinocyte (Kurashiki Spinning Co., Ltd.) seeded in a 75 (cm ² ) flask with a keratinocyte growth medium (Humedia-KG2, KG-2 medium, Kurashiki Spinning Co., Ltd.) is brought into a subconfluent state, and trypsin After recovering the cells by treatment, the cells were seeded in a 25 (cm ² ) flask at a density of 7.5 × 10 ⁵ (cells / flask). The next day, dimethyl sulfoxide (melanin non-added group, Wako Pure Chemical Industries, Ltd., final concentration: 6.0 × 10 ⁻² (w / v%)), 1.0 (w / v%) melanin (melanin added group, Sigma Aldrich Japan Co., Ltd., final concentration: 6.0 × 10 ⁻⁴ (w / v%)), 1.0 (w / v%) melanin (Sigma Aldrich Japan Co., Ltd., final concentration: 6.0 × 10 ^{− 4} (w / v%)) and test substance (final concentration: 1.0 × 10 ⁻² (v / v%)) (melanin addition and test substance addition group) (KG-2 medium (3.3 mL / 24 hours later, cells were collected by trypsin treatment. The collected cells were seeded at a density of 1.5 × 10 ⁵ (cells / well) into laminin-5 coated wells (8-well chamber slide, see Production Examples 1 and 2 for the preparation method). On the next day, the medium was changed to KG-2 medium containing 1.8 (mM Ca ²⁺ ). After 24 hours of medium exchange, the medium was fixed with 4% paraformaldehyde / phosphate buffer (Wako Pure Chemical Industries, Ltd.), and the nucleus was fluorescently stained with the nuclear stain Hoechst 33342 (Dojindo Laboratories) and washed with PBS. After encapsulating with an aqueous mounting medium (Fluoromount; Diagostic BioSystems, Cat. No. K024), take a picture of the uppermost layer from the lowermost layer using a confocal laser microscope, and use the image analysis software ImageJ (National Institutes of Health) The number of keratinocytes in the upper layer from the lowest layer of keratinocytes was determined by measuring the number of nuclei.

The results are shown in FIG. In addition, the vertical axis | shaft of FIG. 10 represents as a ratio when the number of keratinocytes above the lowest layer of the keratinocytes of a melanin addition group is set to "1". According to the result of FIG. 10, the number of keratinocytes in the upper layer from the lowermost layer of keratinocytes decreased by the addition of melanin, and it was confirmed that this screening system was established. Furthermore, the plant extract obtained from the citrus citrus genus yuzu or the asteraceae daffodil genus chrysanthemum, the test substance, markedly increased the number of keratinocytes in the upper layer from the lowest layer of keratinocytes decreased by the addition of melanin. . According to the results of FIG. 10, the plant extract obtained from the citrus family Citrus genus Yuzu or Asteraceae genus Chrysanthemum genus Chrysanthemum exhibits an action of promoting keratinocyte upper layer movement and exhibits an inhibitory action on cell adhesion of keratinocytes. Have. For this reason, the said test substance can be selected as a whitening agent of this invention.

<Method 1 for manufacturing a whitening composition containing the whitening agent of the present invention (external preparation for skin)>
According to the following procedures, a whitening composition (skin external preparation) containing a whitening agent selected by the whitening agent screening method of the present invention was prepared. That is, each component described in the formulation component (A) in Table 2 was combined and dissolved at room temperature. On the other hand, each component described in the prescription component (B) is dissolved at room temperature, solubilized by adding (B) to the mixture of the prescription components described in (A) and containing the whitening agent of the present invention. Composition (skin external preparation) was prepared. In addition, in the prescription ingredients in Table 2, the “whitening agent of the present invention” is obtained from a plant extract (skin external preparation 1) obtained from Citrus citrus genus Yuzu, and Asteraceae genus Chrysanthemum (Artichoke). A plant extract (skin external preparation 2) was used. In addition, Comparative Example 1 in which “water whitening agent of the present invention” in the prescription components of Table 2 was replaced with “water” was also prepared.

<Whitening effect test of the whitening composition of the present invention>
With regard to the whitening composition of the present invention (external skin preparations 1 and 2) and Comparative Example 1 produced according to the method described in Example 4, the pigmentation inhibitory action was evaluated according to the following procedure. A total of 4 sites of 1.5 cm × 1.5 cm were provided on the inner side of the upper arm of the panelists who participated freely. The provided site was irradiated with UV radiation of a minimum erythema amount (1 MED) once a day for 3 consecutive days. From the end of the ultraviolet irradiation on the first day of the test (after the completion of the first irradiation), 50 μL of the topical skin preparations 1 and 2 and Comparative Example 1 were applied twice a day for 35 consecutive days. One of the four sites was an untreated site. 24 hours after the completion of 35 times of application, the skin lightness (L * value) of each test site was measured with a color difference meter (CR-300, Konica Minolta Co., Ltd.), and the L * of the treated site and the untreated site L * were measured. The difference (ΔL * = treatment site L * −untreated site L *) was calculated. The L * value decreases as the degree of pigmentation increases. Therefore, it can be determined that the greater the ΔL * value, the better the pigmentation. The results are shown in Table 3. Thereby, it turns out that the skin external preparations 1 and 2 of this invention show a high value compared with the comparative example 1, and have the outstanding pigmentation improvement effect (whitening effect). This is the whitening effect by the whitening agent having the cell adhesion inhibitory action of the keratinocytes of the present invention contained in the skin external preparations 1 and 2.

<Production Method 2 (Food) for Whitening Composition Containing Whitening Agent of the Present Invention>
A whitening composition (food product) containing a whitening agent selected by the whitening agent screening method of the present invention was prepared according to the following procedure. That is, the prescription ingredients listed in Table 4 were tumbled in phase with 10 parts by mass of water ("Numarumerizer" manufactured by Fuji Paudanil Co., Ltd.) and tableted to obtain tablet-like health foods. In addition, the unit of the numerical value in a table | surface represents a mass part.

  The present invention can be applied to foods, pharmaceuticals, cosmetics and the like.

Claims (10)

Comprising the step of evaluating keratinocytes (keratinocytes, keratinocyte) a reduction in the adhesion ability to the basal membrane,
In the keratinocytes to which the test substance is added and the keratinocytes to which the test substance is not added, the step comprises inhibiting the keratinocytes from producing integrin α6 and / or β4 and suppressing the adhesion of the keratinocytes to the basement membrane. Performed by comparing one or more actions selected from the group consisting of actions,
A screening method for a whitening agent, wherein the test substance is selected as a whitening agent when the action on the keratinocytes to which the test substance is added is greater than that of the keratinocytes to which the test substance is not added . The method for screening a whitening agent according to claim 1 , comprising a step of imparting a cell adhesion promoting factor to keratinocytes. The whitening agent screening method according to claim 2 , wherein the cell adhesion promoting factor is melanin and / or a related substance thereof. The method for screening a whitening agent according to any one of claims 1 to 3 , wherein the integrin α6 and / or β4 production inhibitory effect of the keratinocytes is an integrin α6 and / or β4 gene expression inhibitory effect. The whitening agent screening method according to claim 4 , wherein the integrin α6 and / or β4 gene expression inhibitory effect is an integrin α6 and / or β4 mRNA expression inhibitory effect. The method of screening for a whitening agent according to any one of claims 1 to 3 , wherein the integrin α6 and / or β4 production inhibitory effect of the keratinocytes is an integrin α6 and / or β4 protein expression inhibitory effect. The whitening agent screening according to any one of claims 1 to 3 , wherein the inhibitory action of keratinocytes on the basement membrane is an inhibitory action of keratinocytes on Laminin-5. Method. Step perform screening method of whitening agent according to any one of claim 1 to 7, and including also contain a whitening agent selected by the process, the design method of whitening composition. The design method according to claim 8 , wherein the whitening composition is a skin external preparation. The design method according to claim 8 or 9 , wherein the whitening composition is a cosmetic (including a quasi-drug).

Images