WO2019225707A1 - Differentiation inducer of myeloid stem cells, agent for improving epidermis, skin external agent, and method for improving epidermis - Google Patents

Abstract

Provided is a differentiation inducer of myeloid stem cells, said agent comprising a neem leaf extract and a black tea extract. Also provided are an agent for improving epidermis and a skin external agent both further comprising an inducer of myeloid stem cells. The aforesaid differentiation inducer, agent for improving epidermis and skin external agent may further comprise an eggplant extract.

Description

Bone marrow stem cell differentiation inducer, epidermis improving agent, skin external preparation, and epidermis improving method Cross-reference of related applications

This application claims the priority of Japanese Patent Application No. 2018-099443, which is incorporated herein by reference.

The present invention relates to a differentiation inducer for bone marrow stem cells, an epidermis improving agent, an external preparation for skin, and an epidermis improving method.

Stem cells that have not yet differentiated and can differentiate into various tissue cells include embryonic stem cells (ES cells), which are cells in a state where fertilized eggs are not differentiated, and are not differentiated yet contained in differentiated tissues. Somatic stem cells, which are cells in a state, are known.
Somatic stem cells are present in various tissues in the body. As somatic stem cells, for example, bone marrow stem cells existing in the bone marrow are known.

Bone marrow stem cells are attracting attention as being able to restore tissue function by differentiating into tissue cells in a tissue that has lost function. Specifically, bone marrow stem cells are attracted and accumulated in the bloodstream in inflamed or damaged tissues, and differentiate into the tissue cells under the influence of differentiation-inducing agents that induce differentiation. It is attracting attention as something to gain.

By the way, conventionally, as a differentiation-inducing agent capable of differentiating bone marrow stem cells into specific tissue cells, for example, PDGF-BB as a platelet-derived growth factor (Platelet-DerivedrivGrowth Factor) is included, and bone marrow stem cells are differentiated into muscle tissue cells. A differentiation-inducing agent to be obtained is known (Patent Document 1).

International Publication No. 2005/063967

However, although the differentiation inducer described in Patent Document 1 can induce differentiation of bone marrow stem cells into muscle tissue cells, there is a problem that it does not have the ability to induce differentiation of bone marrow stem cells into epidermal cells, for example.

In view of the above-mentioned problems and the like, an object of the present invention is to provide a bone marrow stem cell differentiation inducer capable of inducing differentiation of bone marrow stem cells into epidermal cells.
Another object of the present invention is to provide an epidermis improving agent, an external preparation for skin, and a method for improving epidermis, which can improve epidermal function by attracting bone marrow stem cells to the epidermis to differentiate into epidermal cells.

The differentiation-inducing agent for bone marrow stem cells according to the present invention is characterized by containing a neem leaf extract and a black tea extract. It is preferable that said differentiation inducer further contains eggplant fruit extract.

The epidermis improving agent and external preparation for skin according to the present invention are characterized by containing an attractant for bone marrow stem cells, a neem leaf extract, and a black tea extract. The above-mentioned epidermis improving agent and external preparation for skin preferably further contain eggplant fruit extract.

The method for improving the epidermis according to the present invention comprises attracting bone marrow stem cells to the epidermis by applying the above-mentioned epidermis improving agent or the above-mentioned external preparation for skin to the epidermis, and differentiating the attracted bone marrow stem cells into epidermal cells. Prepare.

The graph showing the evaluation test result in connection with differentiation induction. The graph showing the evaluation test result in connection with differentiation induction. The graph showing the evaluation test result in connection with differentiation induction. The graph showing the evaluation test result in connection with differentiation induction. The graph showing the evaluation test result in connection with differentiation induction. The graph showing the evaluation test result in connection with differentiation induction. The graph showing the evaluation test result in connection with differentiation induction. The graph showing the evaluation test result in connection with differentiation induction. The graph showing the evaluation test result of the epidermis improvement effect in a human. The figure which showed the mode of the cell attraction test method typically.

Hereinafter, embodiments of the differentiation inducer for bone marrow stem cells of the present invention will be described.

The differentiation-inducing agent for bone marrow stem cells of the present embodiment includes a neem leaf extract and a black tea extract. According to the bone marrow stem cell differentiation inducer of this embodiment, bone marrow stem cells can be induced to differentiate into epidermal cells.

Bone marrow stem cells are one type of mesenchymal stem cells. Bone marrow stem cells are stem cells produced in the bone marrow. Bone marrow stem cells are also contained in blood and can be carried to each tissue in the body along the bloodstream.

The neem leaf extract is obtained by subjecting the leaves of neem belonging to the genus Azadiracta to the extraction process with an extraction solvent. In other words, the neem leaf extract contains components extracted from neem leaf by the extraction solvent. The scientific name of neem is Melia azadirachta L. (Meliceae) . Neem is called Neem in English.

The neem leaf extract can be obtained, for example, by subjecting heat-treated neem leaves to extraction with an extraction solvent.

The black tea extract is obtained by subjecting the leaves of standard varieties belonging to the camellia family Camellia (including varieties Assamcha) to extraction with an extraction solvent. In other words, the black tea extract contains components extracted from the leaves of tea tree by the extraction solvent. The scientific name of the standard variant, Chanoki , is Thea sinensis Linne . Chanoki includes toucha and safflower. The scientific name of Assamcha is Thea sinensis Linne var. Assamica Pierre (Theaceae) . In this embodiment, the black tea extract is an extract of Assamcha leaves.

The black tea extract is obtained by subjecting tea leaves (black tea) that has been completely fermented (oxidative fermentation process) to extraction with an extraction solvent. Examples of completely fermented black tea include Assam black tea, Darjeeling black tea, and Ceylon black tea.

It is preferable that the bone marrow stem cell differentiation inducer of this embodiment further includes an eggplant fruit extract. When the differentiation inducer further contains eggplant fruit extract, there is an advantage that differentiation of bone marrow stem cells into epidermal cells can be more fully induced.

The eggplant fruit extract is obtained by subjecting an eggplant ( Solanum melongena ) fruit belonging to the genus Solanum to an extraction solvent with an extraction solvent. As eggplant, Mizunas is preferable. The scientific name of Miznas is Solanum melongena (Mizunasu) .

The above eggplant fruit extract can be obtained by subjecting eggplant fruit to extraction with an extraction solvent.

Each extract described above can be in the state of an extract containing an extraction solvent or a diluting solvent, or in the form of a dried product from which the extraction solvent has been removed. Specifically, each extract can be in a state of, for example, a solution, a paste, a gel, or a powder.

Examples of the extraction solvent include water; aliphatic monohydric alcohols such as methanol, ethanol, propanol, and butanol (organic compounds having 1 to 4 carbon atoms and one OH group (including structural isomers)); glycerin, propylene Aliphatic polyhydric alcohols such as glycol and 1,3-butylene glycol (organic compounds having 1 to 5 carbon atoms and having a plurality of OH groups); ketones such as acetone; diethyl ether, dioxane, acetonitrile, ethyl acetate, etc. Esters; aromatics such as xylene, benzene and toluene; and organic solvents such as halogenated alkyls such as chloroform.
These extraction solvents may be used alone or in combination of two or more. The mixing ratio of the mixed extraction solvent is not particularly limited, and is appropriately adjusted.

As the extraction solvent for the neem leaf extract, a solvent containing an aliphatic polyhydric alcohol is preferable, an extraction solvent containing 50% by mass or more of an aliphatic polyhydric alcohol (1,3-butylene glycol) is more preferable, and 1,3 -A solvent containing 90% by mass or more of butylene glycol is more preferable.
As an extraction solvent for the black tea extract, a solvent containing an aliphatic monohydric alcohol is preferable, an extraction solvent containing an aliphatic monohydric alcohol (ethanol) at 50% by mass or more is more preferable, and a solvent containing ethanol at 90% by mass or more is preferable. Further preferred. The extract extracted with the aliphatic monohydric alcohol may be dissolved with an aliphatic polyhydric alcohol such as 1,3-butylene glycol.
As an extraction solvent for the eggplant fruit extract, a mixed solvent containing water and an aliphatic polyhydric alcohol (for example, 1,3-butylene glycol) is preferable. In the mixed solvent, the mixing ratio of water and the aliphatic polyhydric alcohol (1,3-butylene glycol) is preferably 7: 3 (water: aliphatic polyhydric alcohol).

The extraction method is not particularly limited, and a conventionally known general extraction method can be employed. In the extraction process, the extraction site (leaves and fruits) of each plant can be used as it is or after drying as an extraction raw material. Usually, the amount of extraction solvent is 5 to 15 times the mass of the extraction raw material (mass ratio), the extraction temperature is 20 ° C. to 80 ° C., and the extraction time is 2 hours to 3 days. After the extraction treatment, purification treatment such as filtration, deodorization, and decolorization can be performed as necessary.

In the bone marrow stem cell differentiation inducer, the mass ratio of neem leaf extract to black tea extract (neem leaf extract / black tea extract) is preferably 1/1 or more and 23/1 or less in terms of dry matter. It is more preferably 2/1 or more and 5/1 or less, and further preferably 3/1 or more and 5/1 or less.
The mass ratio of the total amount of neem leaf extract and black tea extract (neem leaf extract and black tea extract / eggplant extract) to the mass of eggplant fruit extract is 1/1 or more and 7/1 or less. It is preferable that the ratio is 1/1 or more and 5/1 or less.
In addition, dry matter conversion is converting into the mass of the dry matter which removed the solvent contained in an extract from the extract.

The concentration of each of the extracts contained in the bone marrow stem cell differentiation inducer is not particularly limited. For example, the concentration of neem leaf extract is 0.0005% by mass or more and 0.075% by mass or less in terms of dry matter. The concentration of the black tea extract is 0.00015% by mass or more and 0.03% by mass or less in terms of dry matter. Moreover, the density | concentration of eggplant fruit extract is 0.0001 mass% or more and 0.015 mass% or less in conversion of a dry matter.

As each of the above-mentioned extracts, for example, those commercially available as cosmetic raw materials and food additives can be used.

The differentiation-inducing agent for bone marrow stem cells of the present embodiment may contain water, ethanol, polyhydric alcohol, oil, surfactant, thickener, preservative, pH adjuster, etc. in addition to the above-mentioned extracts. Good. The differentiation-inducing agent for bone marrow stem cells of the present embodiment may be in a liquid, gel-like, cream-like or solid state, for example.

The differentiation inducing agent for bone marrow stem cells of the present embodiment may be, for example, an external preparation for skin application or skin application. The bone marrow stem cell differentiation inducer of the present embodiment may be used in applications such as cosmetics, pharmaceuticals, and quasi drugs.

Next, embodiments of the epidermis improving agent and the external preparation for skin of the present invention will be described. Each of the epidermis improving agent and the external preparation for skin of the present embodiment contains an attractant for bone marrow stem cells, the above neem leaf extract, and the above black tea extract. It is preferable that the epidermis improving agent and skin external preparation of this embodiment further contain said eggplant fruit extract.

The above-mentioned attractant for bone marrow stem cells is not particularly limited as long as it is applied to the epidermis and attracts bone marrow stem cells in the bloodstream to the epidermis. Although the density | concentration of the said attractant (total amount of compound amount and dry matter conversion amount) contained in an epidermis improving agent or a skin external preparation is not specifically limited, For example, it is 0.00001 mass% or more and 10 mass% or less. Moreover, the concentration of the neem leaf extract contained in the epidermis improving agent or the external preparation for skin is, for example, 0.0005 mass% or more and 0.075 mass% or less in terms of dry matter, and the concentration of the tea extract is, for example, It is 0.00015% by mass or more and 0.03% by mass or less in terms of dry matter.

The above attractants include bodaiju extract, button pipi extract, coxnohagashi extract, asenyaku extract, pashanbe extract, cinnamtannin B1, pentagalloyl glucose, borkensiflavone, moreroflavone, kaempferitrin, quercetin-3 , 7-ziramnoside, procyanidin A2, procyanidin B1, deacetylcytochalasin C, a culture of Eupenicillium ludwigii , and at least one selected from the group consisting of a culture of Talaromyces trachyspermus Preferably there is.

The above body extract is obtained by subjecting a plant of the family Tiliaceae to extraction with an extraction solvent. Plants of the family Tiliaceae include tree swordfish ( Tilia platyphyllos Scop. ), Tree swordfish ( Tilia.cordata Mill. ), Linden tree ( Tilia.europaea L. ), or other related plants. Among these, the plant of the family Tiliaceae is preferably a coral mill ( Tilia.cordata Mill. ) In that it can attract bone marrow stem cells more reliably. That is, as the body extract, the body extract is preferable. The extraction site of the lindenaceae plant is not particularly limited, and examples thereof include flowers, fruits, and bark. Among them, the extracted site is preferably a flower (flower part) in that bone marrow stem cells can be attracted more reliably.
As an extraction solvent for the above-mentioned Bodaige extract, an extraction solvent containing ethanol and water is preferable, and an extraction solvent containing water and 50% by volume or more of ethanol is more preferable.

The button pi extract is obtained by subjecting the root bark of a button family button ( Paeonia suffruticosa Andrews ) to extraction with an extraction solvent.
As the extraction solvent for the button pi extract, an extraction solvent containing ethanol and water is preferable, and an extraction solvent containing water and 50% by volume or more of ethanol is more preferable.

The above-mentioned Kusunohagashi extract is obtained by subjecting Kusnohagashi wrinkles ( Mallotus philippinensis ) belonging to Euphorbiaceae to extraction treatment with an extraction solvent. There are no particular limitations on the extraction site of Kusunohagashi, and examples include leaves, branches, timber parts, bark, and roots. Of these, bark is preferable because bone marrow stem cells can be attracted more reliably.
As an extraction solvent for the above-mentioned Kusunohagashi extract, an extraction solvent containing ethanol and water is preferable, and an extraction solvent containing water and 50% by volume or more of ethanol is more preferable.

The asenyaku extract is obtained by subjecting asenyaku ( Uncaria gambir ) to an extraction treatment with an extraction solvent. There are no particular limitations on the extraction site of Acacia yak, and examples include leaves and branches. As the Acacia yak extract, an extract obtained by subjecting both leaves and branches to extraction with an extraction solvent is preferable in that bone marrow stem cells can be attracted more reliably.
As the extraction solvent for the asenyaku extract, an extraction solvent containing ethanol and water is preferable, and an extraction solvent containing water and 50% by volume or more of ethanol is more preferable.

The above-mentioned Pashanbe extract is obtained by subjecting one or more Pashanbheda belonging to the genus Himalaya Yukinoshita to an extraction treatment with an extraction solvent. As pashambe, Bergenia ligulata (Wall.) Engl., Bergenia stracheyi (Hook. F. & Thoms.) Engl. Or Bergenia ciliata (Haw.) Sternb. Etc. A Pashambe extract obtained by subjecting Bergenia ligulata (Wall.) Engl. To extraction treatment is preferable in that it can attract bone marrow stem cells more reliably. The extraction site of the pashambe is not particularly limited, but a rhizome is preferable in that bone marrow stem cells can be attracted more reliably.
As an extraction solvent for the Pasambe extract, an extraction solvent containing ethanol and water is preferable, and an extraction solvent containing water and 50% by volume or more of ethanol is more preferable.

The cinnamtannin B1 is a compound having a molecular structure represented by the following formula (1). In the above-mentioned epidermis improving agent or external preparation for skin, the concentration of cinnamtannin B1 is not particularly limited, and is, for example, 0.001 to 10% by mass.

The pentagalloyl glucose is a compound having a molecular structure represented by the following formula (2). In the above-mentioned epidermis improving agent or skin external preparation, the concentration of pentagalloyl glucose is not particularly limited, and is, for example, 0.001 to 10% by mass.

The above-mentioned Volkensiflavone (Talbotaflavone) is a compound having a molecular structure represented by the following formula (3). In the above-mentioned epidermis improving agent or skin external preparation, the concentration of Borkensiflavone is not particularly limited, and is, for example, 0.001 to 10% by mass.

The above morelloflavone “Morelloflavone” is a compound having a molecular structure represented by the following formula (4). In the epidermis improving agent or the external preparation for skin, the concentration of moreroflavone is not particularly limited, and is, for example, 0.001 to 10% by mass.

The kaempferitrin (or kaempferol-3,7-dirhamnoside) is a compound having a molecular structure represented by the following formula (5). In the above-mentioned epidermis improving agent or skin external preparation, the concentration of kaempferitrin is not particularly limited, and is, for example, 0.001 to 10% by mass.

The quercetin-3,7-dirhamnoside [Quercetin-3,7, -di-O-α-L-rhamnoside] is a compound having a molecular structure represented by the following formula (6). In the above-mentioned epidermis improving agent or skin external preparation, the concentration of quercetin-3,7-dirhamnoside is not particularly limited, and is, for example, 0.001 to 10% by mass.

The procyanidin A2 is a compound having a molecular structure represented by the following formula (7). In the above-mentioned epidermis improving agent or skin external preparation, the concentration of procyanidin A2 is not particularly limited, and is, for example, 0.001 to 10% by mass.

The procyanidin B1 is a compound having a molecular structure represented by the following formula (8). In the above-mentioned epidermis improving agent or skin external preparation, the concentration of procyanidin B1 is not particularly limited, and is, for example, 0.001 to 10% by mass.

The deacetylcytochalasin C is a compound having a molecular structure represented by the following formula (9). In the above-mentioned epidermis improving agent or skin external preparation, the concentration of deacetylcytochalasin C is not particularly limited, and is, for example, 0.001 to 10% by mass.

The deacetylcytochalasin C is included in, for example, a product generated by culturing a black rot fungus ( Metarhizium anisopliae ) classified as a baby fungus microorganism.

The culture of the above-mentioned Eupenicillium ludwigii is included in the product produced by culturing Eupenicillium ludwigii classified as, for example, an aspergillus fungus microorganism ( Eupenicillium ludwigii ).

The culture of the above-mentioned Talaromyces trachyspermus ( Talaromyces trachyspermus ) is included in, for example, a product generated by culturing Talaromyces trachyspermus classified as an irregular fungus microorganism.

By applying the epidermis improving agent or external preparation for skin of the present embodiment to the epidermis, for example, bone marrow stem cells can be attracted to the epidermis and differentiated into epidermal cells, and the epidermal function can be improved.
The epidermis has a stratum corneum layer, a granule layer, a spiny layer, and a basal layer in order from the outermost side, and epidermal cells existing in the basal layer are pushed up by metabolism to form cells of each layer. The induced bone marrow stem cells can differentiate into basal layer epidermal cells.

The epidermis improving agent or external preparation for skin of this embodiment further includes water, ethanol, polyhydric alcohol, oil, surfactant, thickener, preservative, pH adjuster, and the like, as with the differentiation inducer described above. May be included. The skin-improving agent of the present embodiment may be in a liquid, gel-like, cream-like, or solid state, for example.

The epidermis improving agent and external preparation for skin of this embodiment may be used, for example, for skin application or skin application. The epidermis improving agent and external preparation for skin of this embodiment may be used in applications such as cosmetics, pharmaceuticals, and quasi drugs. The epidermis improving agent and skin external preparation of this embodiment are used like the epidermis improving method mentioned later, for example.

Subsequently, an embodiment of the skin improvement method according to the present invention will be described.

The epidermal improvement method of this embodiment comprises attracting bone marrow stem cells to the epidermis by applying the above-mentioned epidermis improving agent or the above-mentioned external preparation for skin to the epidermis and differentiating the bone marrow stem cells into epidermal cells.
The epidermal function is worsened by a decrease in the number of cells constituting the epidermis and the disruption of the cell arrangement, as the epidermal stem cells that form the epidermal cells decrease with age. By increasing the number of epidermal stem cells in the epidermis, the once deteriorated epidermal function can be recovered. Therefore, the epidermis function can be recovered by attracting bone marrow stem cells to the epidermis by the epidermis improving agent or the above-mentioned external preparation for skin, and differentiating the bone marrow stem cells into epidermal cells in the epidermis.

The part of the epidermis to which the above-mentioned epidermis improving agent or external preparation for skin is applied is not particularly limited, and is, for example, the stratum corneum, granule layer, spiny layer, or basal layer. Such a portion of the epidermis may be a healthy part or a wound part.

In the above-mentioned epidermis improvement method, bone marrow stem cells can be attracted to the epidermis by the above-mentioned epidermis improving agent or the skin external preparation attractant.

For example, by applying the above-described epidermis improving agent (external preparation for skin) to the epidermal tissue, bone marrow stem cells in the blood can be attracted to the epidermal tissue.

The above-described method for improving the epidermis can be performed in situ on animals other than humans. In humans, it can be carried out non-therapeutically for the cosmetic purpose of improving the epidermal function. Beauty purposes include, for example, the purpose of improving skin firmness, the purpose of suppressing skin wrinkles, the purpose of suppressing skin drying, the purpose of suppressing skin dullness, the purpose of removing skin spots, or The purpose is to lighten the skin.

The composition of differentiation-inducing agent, epidermis-improving agent, external preparation for skin and method for improving epidermis according to this embodiment are as exemplified above, but the present invention is not limited to the above-exemplified embodiment. . Moreover, in this invention, the various form employ | adopted in a general differentiation-inducing agent, an epidermis improving agent, an external preparation for skin, and an epidermis improving method can be employ | adopted in the range which does not impair the effect of this invention.

[Neem leaf extract]
The product name “Neem Leaf Liquid B” (manufactured by Ichimaru Falcos) was used as the neem leaf extract. The method for producing such neem leaf extract is as follows.
Extraction of neem leaf ( Melia azadirachta L. (Meliceae) ) after heat treatment, followed by extraction using 1,3-butylene glycol as an extraction solvent, followed by filtration It was a thing. The neem leaf extract was found to contain 1.1% by mass of dry matter when calculated from the remaining amount of evaporation after 5 mL of neem leaf extract was evaporated to dryness at 105 ° C. for 6 hours on a water bath ( Solid content concentration conversion).

[Black tea extract]
The product name “Tea Liquid” (manufactured by Ichimaru Falcos) was used as the tea extract. The manufacturing method of such a black tea extract is as follows.
Assamcha ( Thea sinensis L. var. Assamica Pierre (Theaceae) ) leaves were extracted using ethanol as an extraction solvent, frozen, and filtered to obtain a filtrate. 1,3-butylene glycol was added to this filtrate and the resultant was filtered to obtain a black tea extract (Assam black tea extract). As calculated from the remaining amount of evaporation after 10 mL of black tea extract was evaporated to dryness at 105 ° C. for 6 hours on a water bath, the black tea extract contained 2.4% by mass of the dried product (solid content) Concentration conversion).

[Eggplant fruit extract]
A commercially available eggplant fruit extract was used. The production method of such eggplant fruit extract is as follows.
To 100 g of dried fruit of Solanum melongena (Mizunasu ), 1200 g of a mixture of purified water and 1,3-butylene glycol (mass ratio water: 1,3-BG = 7: 3) was mixed. After extracting at 2 degreeC for 2 hours, the filtration process was performed and the mizunas fruit extract was obtained. The obtained Mizuna fruit extract contained 1.01% by mass of a dried product (in terms of solid content concentration).

(Test Example 1)
The above neem leaf extract and the above black tea extract were mixed and diluted such that the total amount of the extract in terms of dry matter was 0.01% by mass (the value after rounding off), and Test Example 1 Samples were prepared. As a dilution solvent, a differentiation induction medium (culture supernatant obtained by culturing epidermal cells for a long period of time) that induces differentiation of bone marrow stem cells into epidermal cells was used. In terms of dry matter, the mass ratio of the neem leaf extract to the black tea extract (neem leaf extract / tea extract) was 1/1 after rounding off.

(Test Example 2)
The above neem leaf extract and the above black tea extract were mixed and diluted such that the total amount of the extract in terms of dry matter was 0.01% by mass (number after rounding off), and Test Example 2 Samples were prepared. As a dilution solvent, a differentiation induction medium (culture supernatant obtained by culturing epidermal cells for a long period of time) that induces differentiation of bone marrow stem cells into epidermal cells was used. In terms of dry matter, the mass ratio of neem leaf extract to black tea extract (neem leaf extract / black tea extract) was 2/1 after rounding off.

(Test Example 3)
The above neem leaf extract and the above black tea extract were mixed and diluted so that the total amount of the extract in terms of dry matter was 0.01% by mass (number after rounding off), and Test Example 3 Samples were prepared. As a dilution solvent, a differentiation induction medium (culture supernatant obtained by culturing epidermal cells for a long period of time) that induces differentiation of bone marrow stem cells into epidermal cells was used. In terms of dry matter, the mass ratio of the neem leaf extract to the black tea extract (neem leaf extract / tea extract) was 3/1 after rounding off.

(Test Example 4)
The above neem leaf extract and the above black tea extract were mixed and diluted so that the total amount of the extract in terms of dry matter was 0.01% by mass (number after rounding off), and Test Example 4 Samples were prepared. As a dilution solvent, a differentiation induction medium (culture supernatant obtained by culturing epidermal cells for a long period of time) that induces differentiation of bone marrow stem cells into epidermal cells was used. In terms of dry matter, the mass ratio of neem leaf extract to black tea extract (neem leaf extract / tea extract) was 5/1 after rounding off.

(Test Example 5)
The above neem leaf extract and the above black tea extract were mixed and diluted so that the total amount of the extract in terms of dry matter was 0.01% by mass (number after rounding off), and Test Example 5 Samples were prepared. As a dilution solvent, a differentiation induction medium (culture supernatant obtained by culturing epidermal cells for a long period of time) that induces differentiation of bone marrow stem cells into epidermal cells was used. In terms of dry matter, the mass ratio of neem leaf extract to black tea extract (neem leaf extract / black tea extract) was 23/1 as a value after rounding off.

(Test Example 6)
The above-described eggplant fruit extract was added to epidermal cells, and the supernatant was collected after 24 hours of culture to prepare a differentiation induction medium (with eggplant fruit extract) for differentiating bone marrow stem cells into epidermal cells. Using this differentiation-inducing medium (with eggplant fruit extract), the above neem leaf extract and black tea extract were diluted so that the total amount of the extract in terms of dry matter was 0.02% by mass. Six samples were prepared. The ratio of the total mass of neem leaf extract and black tea extract ((neem leaf extract and Assam black tea extract) / Miznas fruit extract) to the mass of eggplant fruit extract in terms of dry matter is the value after rounding off. It was 7/1.

(Test Example 7)
The above-described eggplant fruit extract was added to epidermal cells, and the supernatant was collected after 24 hours of culture to prepare a differentiation induction medium (with eggplant fruit extract) for differentiating bone marrow stem cells into epidermal cells. Using this differentiation-inducing medium (with eggplant fruit extract), the above neem leaf extract and black tea extract were diluted so that the total amount of the extract in terms of dry matter was 0.02% by mass. Seven samples were prepared. The ratio of the total mass of neem leaf extract and black tea extract ((neem leaf extract and Assam black tea extract) / Miznas fruit extract) to the mass of eggplant fruit extract in terms of dry matter is the value after rounding off. It was 4/1.

(Test Example 8)
The above-described eggplant fruit extract was added to epidermal cells, and the supernatant was collected after 24 hours of culture to prepare a differentiation induction medium (with eggplant fruit extract) for differentiating bone marrow stem cells into epidermal cells. Using this differentiation-inducing medium (with eggplant fruit extract), the above neem leaf extract and black tea extract were diluted so that the total amount of the extract in terms of dry matter was 0.02% by mass. Eight samples were prepared. The ratio of the total mass of neem leaf extract and black tea extract ((neem leaf extract and Assam black tea extract) / Miznas fruit extract) to the mass of eggplant fruit extract in terms of dry matter is the value after rounding off. It was 1/1.

(Reference Example 1, Reference Example 2)
The neem leaf extract was diluted so that the amount of the neem leaf extract in terms of dry matter was 0.006% by mass and 0.011% by mass, respectively. Samples were prepared. As a dilution solvent, a differentiation induction medium (culture supernatant obtained by culturing epidermal cells for a long period of time) that induces differentiation of bone marrow stem cells into epidermal cells was used.

(Reference Examples 3 to 5)
The above black tea extract was diluted so that the dry tea equivalents of the black tea extract were 0.0002 mass%, 0.0024 mass%, and 0.0036 mass%, respectively. Five samples were prepared. As a dilution solvent, a differentiation induction medium (culture supernatant obtained by culturing epidermal cells for a long period of time) that induces differentiation of bone marrow stem cells into epidermal cells was used.

<Evaluation test for differentiation induction>
Bone marrow stem cells were seeded at 6,000 cells / well in a 96-well plate and cultured for 3 days. Separately, epidermal cells were seeded in a petri dish having a diameter of 10 cm, and when 90% confluent was reached, the medium was replaced with a new medium, and the supernatant culture solution was collected after 24 hours of culture. The solution obtained by centrifuging and filtering the collected supernatant culture solution was used as a differentiation induction medium (differentiation induction medium for inducing differentiation of bone marrow stem cells into epidermal cells). In the test using eggplant fruit extract, eggplant fruit extract was added to epidermal cells, the supernatant was collected after 24 hours of culture, and the solution obtained by centrifugation and filtration was used as a differentiation-inducing medium. According to each test, neem leaf extract and black tea extract were diluted using the prepared differentiation induction medium and added to cultured bone marrow stem cells, and then differentiation induction culture was performed for 9 to 10 days. After culturing, the degree of differentiation was evaluated by immune cell staining. Differentiation-induced bone marrow stem cells (MSC) were fixed with 4% paraformaldehyde (manufactured by WAKO), and then washed three times with PBS (−). Thereafter, it was treated with a 0.25% Triton solution and washed with TBS-T. Furthermore, a reaction with a 2% BSA / PBS (−) (manufactured by SIGMA) solution was carried out at room temperature for 1 hour, followed by a reaction with a primary antibody at 4 ° C. overnight. After the reaction, the primary antibody solution was removed, and after washing 5 times with TBS-T, the secondary antibody reaction and the reaction with Hoechst solution were performed at room temperature for 30 minutes. After removing the secondary antibody solution, the plate was washed 5 times with TBS-T and replaced with PBS (−), and the degree of differentiation was observed. As the primary antibody for immune cell staining, keratin 14 (K14) antibody and keratin 5 (K5) antibody expressed only in epidermal cells were used. The degree of staining was evaluated by using a numerical value of the stained image (Dens level sum × Area) / Hoches) using an In cell analyzer device (GE Healthcare).

The evaluation test results relating to differentiation induction using Test Examples 1 to 5 are shown in FIG. The evaluation test results in Reference Examples 1 and 2 are shown in FIGS. 2A and 2B. The evaluation test results in Reference Examples 3 to 5 are shown in FIGS. 3A and 3B. The evaluation test results using the eggplant fruit extract alone are shown in FIGS. 4A and 4B. The evaluation test results of Test Examples 3 and 6 to 8 are shown in FIG. As can be seen from the results in each figure, bone marrow stem cells could be differentiated into epidermal cells by using a bone marrow stem cell differentiation inducer containing neem leaf extract and black tea extract. Neem leaf extract and black tea extract have a specific mass ratio (for example, in terms of dry matter, neem leaf extract / black tea extract = 2: 1 to 5: 1, more preferably 3: 1 to 5: 1), the differentiation-inducing performance was remarkably exhibited. This effect is a synergistic effect of the neem leaf extract and the black tea extract.
Moreover, as shown in FIG. 5, differentiation induction performance could be further improved by further combining eggplant fruit extract with neem leaf extract and black tea extract. Eggplant fruit extract is considered to have an action of increasing secretion of differentiation-inducing factors for epidermal cells.

A bone marrow stem cell attractant was prepared as follows. Using the prepared attractant, an in vitro cell attraction test was performed.

"Attractant 1"
A Bodaiju extract of Attractant 1 (Fuyubodaiju extract) was prepared. Specifically, 1L of a 50% ethanol aqueous solution is added to 100g of dried and finely crushed flowers of Tilia.cordata Mill , followed by extraction at room temperature (20 ° C) for 3 days, followed by filtration. went. Then, a dried product was obtained from the filtered solution by drying under reduced pressure, and this dried product was diluted with 1,3-butylene glycol to prepare a jujube extract. When calculated from the amount of the dry substance after the extraction solvent was removed by drying under reduced pressure, this body extract contained 0.45% by mass of the dried product.

"Attractant 2"
A button pea extract of attractant 2 was prepared. In detail, 1L of 50 vol% ethanol aqueous solution was added to 100g of dried root bark of a button ( Paeonia suffruticosa Andrews ), followed by extraction at room temperature (20 ° C) for 3 days, followed by filtration. It was. A dried product was obtained from the filtered solution by drying under reduced pressure, and the dried product was diluted with 1,3-butylene glycol to prepare a button pi extract. This button pi extract, when calculated from the amount of dry substance after removal of the extraction solvent by drying under reduced pressure, contained 0.90% by mass of the dried product.

"Attractant 3"
A Kusunohagashi extract of attractant 3 was prepared. For details, add 2L of 50% ethanol aqueous solution to 200g of dried and finely crushed bark of Kusonohagashi ( Mallotus philippinensis Mueller-Argoviensis ), and perform extraction for 2 days while maintaining 60-80 ° C. went. Then, a dried product was obtained from the filtered liquid by drying under reduced pressure, and this dried product was diluted with 1,3-butylene glycol to prepare a Kusunohagashi extract. This Kusunohagashi extract contained 0.20% by mass of the dried product, as calculated from the amount of dry substance after the extraction solvent was removed by drying under reduced pressure.

"Attractant 4"
An asenyaku extract of attractant 4 was prepared. Specifically, 2 L of a 50% ethanol aqueous solution was added to 100 g of dried and finely crushed leaves and young branches of Acacia yam ( Uncaria gambir Roxburgh ), and the extraction operation was performed for 3 hours while maintaining 50 to 70 ° C. with stirring. Filtration was performed. A dried product was obtained from the filtered solution by drying under reduced pressure, and the dried product was diluted with 1,3-butylene glycol to prepare an asenyaku extract. This asenyaku extract was found to contain 4.10% by mass of the dried product when calculated from the amount of dry substance after the extraction solvent was removed by drying under reduced pressure.

"Attractant 5"
A pashambe extract of attractant 5 was prepared. Specifically, 3 kg of a 50 vol% ethanol aqueous solution was added to 200 g of dried and finely crushed rhizomes of Pashambe ( Bergenia ligulata (Wall.) Engl. ), And extraction was performed at 50 ° C. for 8 hours while stirring. The crude extract was cooled, filtered, concentrated, and passed through a column packed with a synthetic adsorbent (trade name “Diaion HP-20” manufactured by Mitsubishi Chemical Corporation). After washing with water, the eluate eluted with a 30% by volume ethanol aqueous solution was dried under reduced pressure and redissolved in 1,3-butylene glycol to prepare a Pashambe extract. When calculated from the amount of dry substance after the extraction solvent was removed by drying under reduced pressure, this Pashambe extract contained 0.50% by mass of the dried product.

"Attractant 6"
As attractant 6, commercially available cinnamtannin B1 was used.
"Attractant 7"
As the attractant 7, commercially available pentagalloyl glucose was used.
"Attractant 8"
As attractant 8, commercially available Borkensiflavone was used.
"Attractant 9"
As the attractant 9, a commercially available moreroflavone was used.
"Attractant 10"
As the attractant 10, commercially available kaempferitrin was used.
"Attractant 11"
As the attractant 11, commercially available quercetin-3,7-dirhamnoside was used.
"Attractant 12"
As the attractant 12, commercially available procyanidin A2 was used.
"Attractant 13"
As the attractant 13, commercially available procyanidin B1 was used.

"Attractant 14"
As shown below, an attractant containing deacetylcytochalasin C was prepared.
That is, black rot fungus ( Metarhizium anisopliae ) was cultured, and a crude culture containing the cultured medium and cells was obtained. A culture extract was prepared by subjecting this crude culture to an extraction treatment. Details will be described below.
First, in order to perform pre-culture of Metarhizium anisopliae , inoculum extracted from the glycerol stock was cultured according to the following culture conditions.
Pre-culture conditions: Temperature 28 ° C., 40 days Pre-culture medium composition: LCA agar medium shown below 1 g glucose
KH ₂ PO ₄ 1g
MgSO ₄ · 7H ₂ O 0.2g
KCl 0.2g
NaNO ₃ 2g
Bacto-yeast extract 0.2g
(Becton, Dickinson and Company)
Agar (Wako Pure Chemical Industries) 20g
Tap water 1L
NaOH 64.8mg
After pre-culture, the agar medium containing mycelium was punched with a straw. Meanwhile, 3.5 mL of sterilized tap water was dispensed into a 15 mL centrifuge tube. Five pieces of the punched agar medium were placed in a centrifuge tube, and the agar medium was crushed with a cotton swab to prepare a suspension.
Subsequently, as a medium for main culture, a main culture medium having the following composition is prepared in a 50 mL capacity centrifuge tube, and 1 mL of the above suspension is added to the main culture centrifuge tube. Main culture was performed.
・ Main culture conditions: Temperature 28 ° C., 2 weeks ・ Main culture medium composition: Solid medium shown below Vegetable juice 10 mL
(Product name “V8 Juice” manufactured by Campbell Soup Company)
Oatmeal 3g
(Snow Brand Milk Products (Quaker))
An 80 vol% acetone aqueous solution as an extraction solvent was added to the centrifuge tube after the main culture, and the crude culture was extracted with the extraction solvent.
Specifically, after adding the extraction solvent to the centrifuge tube, the centrifuge tube was vigorously shaken up and down to stir the contents, and allowed to stand at room temperature for 30 minutes. Thereafter, the centrifuge tube was centrifuged at 3500 rpm and 23 ° C. for 5 minutes, and about 15 mL of the supernatant after centrifugation was collected by decantation.
Subsequently, the supernatant was allowed to stand for about 15 hours in a draft to evaporate the extraction solvent and concentrated, and then stored at −20 ° C. until the concentrated supernatant reached about 10 mL.
The stored supernatant was placed in a microtube, and a precipitate and a sample solution other than the precipitate were obtained by centrifugation at 13,000 rpm.
225.6 μL of the sample solution (corresponding to 200 μL of main culture broth) was dried to dryness with a centrifugal evaporator and stored at −20 ° C. The dried sample is dissolved in 100 μL of 80% by volume methanol aqueous solution and suspended for 30 minutes using a vortex mixer, and then the supernatant is obtained by centrifugation at 13,500 rpm, 4 ° C. for 5 minutes, followed by culture extraction A product was prepared and an attractant was produced.
The attractant contained 15% by mass of a dried product as calculated from the amount of dry substance after removing the solvent.
It was confirmed by nuclear magnetic resonance (NMR) and mass spectrometry (MS) that the attractant 14 produced as described above contained deacetylcytochalasin C.

" Attractant 15" (culture of Eupenicillium ludwigii )
By culturing Eupenicillium ludwigii , a crude culture containing the cultured medium and cells was obtained. The crude culture was subjected to an extraction treatment to obtain a culture extract, and the attractant 15 was produced. Details thereof will be described below.
First, the microbial cells taken out from the glycerol stock were planted in an inclined medium (malt extract agar medium (MA medium)) and pre-cultured for 1 week. Next, the cells after pre-culture are removed with a platinum loop and suspended in 5 mL of sterile physiological saline, and then the entire suspension is put into a 150 mL polypropylene flask containing a medium having the following composition. Inoculated and statically cultured (25 ° C., 12 days).
・ Medium composition Buckwheat flour 10g
SB solution 10mL (total 20g / flask)
-Composition of SB solution Sodium tartrate 0.005%
KH ₂ PO ₄ 0.005%
MgSO ₄ · _7H 2 O 0.005%
FeSO ₄ · _7H 2 O 0.0005%
ZnSO ₄ · _7H 2 O 0.0005%
Yeast extract (Oriental Yeast Co., Ltd.) 0.01%
Water remainder Subsequently, the supernatant after separation by centrifugation was performed on 240 μL of the culture medium and bacterial cell mixture (crude culture) after culture using 100% butanol (100 μL) as an extraction solvent. Was lyophilized to remove the solvent.
Furthermore, 50 μL of methanol was added to the lyophilized product after extraction with butanol, and the mixture was suspended by stirring for 30 minutes with a vortex mixer, and then a supernatant was obtained by centrifugation at 13,500 rpm, 4 ° C. for 5 minutes. .
In this way, an attractant containing a culture extract of Eupenicillium ludwigii was produced.
The attractant contained 2% by mass of the dried product when calculated from the amount of dry substance after removing the solvent.

“ Attractant 16” (culture of Talaromyces trachyspermus )
The attractant 16 was produced in the same manner as the attractant 15 except that Talaromyces trachyspermus was used instead of Eupenicillium ludwigii . The attractant contained 2% by mass of a dried product.

<In vitro cell attraction test>
FIG. 7 schematically shows the state of the test method. Hereinafter, the details of the test method will be described with reference to FIG.
Each of the above attractants was prepared as a test sample. Diluted so that the concentration of the attractant is a predetermined concentration (1%, 0.1%, etc.) was prepared as a test sample.
On the other hand, as a negative control sample, DMEM (Dulbecco's modified Eagle medium) “FBS (−), P / S (−)” was prepared.
As a positive control sample, a DMEM solution having a concentration of 20 ng / mL PDGF-BB (platelet-derived growth factor PEPRO TECH) was prepared.
First, mouse bone marrow stem cells (hereinafter also referred to as mMSC) are cultured and collected to confluence, suspended in 10% FBS / DMEM “P / S (−)” to 1 × 10 7 cells / ml, and cell suspension A liquid was prepared. FBS represents 10% fetal bovine serum, and P / S represents 100 unit penicillin and 0.1 mg / mL streptomycin. The symbol (-) indicates that no blending is performed.
Next, a Boyden chamber B (manufactured by Neuro Probe) having a plurality of independent wells, in which the well is partitioned into an upper well P and a lower well Q by a membrane M as shown in FIG. Prepared. One kind of each test sample was set so that a negative control sample and a positive control sample could be tested in the same Boyden chamber B, and 28 μL of each sample was applied to each lower well of the chamber B. As the membrane M of Boyden chamber B, the trade name “Polycarbonate Membranes” (pore size 8 μm, manufactured by Neuro Probe) was used.
Subsequently, 50 μL of the cell suspension was seeded in each upper well P, and cultured for 4 hours under conditions of 37 ° C. and 5% CO ₂ (see (b) in FIG. 7).
After culturing for 4 hours, as shown in FIG. 7 (c), the unmigrated mMSC was removed with the attached filter wiper R, and only the mMSC that had moved to the lower side of the membrane M was subjected to Diff-Quick staining (Sysmex kit). Used).
Then, the stained image is digitized and loaded into a computer, and the image is converted to a white portion of the blue-stained portion so that the image is binarized into black and white. The measurement was performed using the function of editing software (trade name “Photoshop”). The mMSC attracting activity of each attractant was evaluated by comparing the brightness in the negative and positive control samples with the brightness in each test sample.

The results (brightness) of the cell attraction test are shown in Table 1. Of the results obtained using the sample in which the attractant was diluted to a predetermined concentration such as 1% concentration or 0.1% concentration, the result of the highest luminance is shown. Moreover, the result of a positive control and a negative control when the test is performed simultaneously is also shown. As can be seen from Table 1, each of the above attractants can attract bone marrow stem cells.

<Evaluation test of human skin improvement>
An evaluation test was conducted with seven subjects. In a preliminary test, the minimum erythema amount (MED) was determined for each subject by irradiating the inner part of the forearm with ultraviolet rays in the UV-B region. Six spots (1 × 1 cm) on the inner side of the upper arm were irradiated with ultraviolet rays having a minimum erythema dose (MED) once a day for 3 days to artificially generate pigment spots.
One of the following was continuously applied to this pigment spot twice a day for two months, and the degree of pigment spot disappearance was evaluated.
・ Only 3% by mass of Kusunohagashi extract, which is an attractant for bone marrow stem cells (MSC) ・ 3% by mass of Kusunohagashi extract (0.006% by mass in terms of dry matter)
+ Differentiation-inducing agent (neem leaf extract, dry matter equivalent 0.011% by mass,
Assam black tea extract 0.012% by mass in terms of dry matter,
Eggplant fruit extract as dry matter 0.00505% by mass)
Neem leaf extract as dry matter: Assam black tea extract: Eggplant fruit extract =
2.2: 2.4: 1.0
The evaluation was performed by measuring with a color difference meter, and calculating ΔL ^* (change with time) of the sample application part from the obtained Munsell value. The larger the value, the greater the amount of color disappearance. The results are shown in FIG.

The differentiation-inducing agent of the present invention is suitable for the purpose of, for example, attracting bone marrow stem cells circulating in the body in the bloodstream to the epidermis tissue and differentiating the induced bone marrow stem cells into epidermal cells in the epidermis tissue. used. The epidermis improving agent, external preparation for skin, and epidermal improvement method of the present invention, as described above, induces bone marrow stem cells to epidermal tissue, and further differentiates the induced bone marrow stem cells into epidermal cells to improve epidermal function. It is preferably used for the purpose. For example, the epidermis improving agent and the external preparation for skin of the present invention are applied to the epidermis to attract bone marrow stem cells to the epidermis, and the induced bone marrow stem cells are differentiated into epidermal cells to improve the appearance of the skin. And is preferably used.

B: Boyden chamber, P: upper well, Q: lower well, M: membrane, R: filter wiper.

Claims (7)

1. A bone marrow stem cell differentiation inducer comprising neem leaf extract and black tea extract.
2. The differentiation inducer according to claim 1, further comprising eggplant fruit extract.
3. An epidermis improving agent containing an attractant for bone marrow stem cells, neem leaf extract, and black tea extract.
4. The epidermis improving agent according to claim 3, further comprising eggplant fruit extract.
5. An external preparation for skin containing an attractant for bone marrow stem cells, a neem leaf extract, and a tea extract.
6. The skin external preparation according to claim 5, further comprising eggplant fruit extract.
7. By applying the epidermis improving agent according to claim 3 or 4 or the external preparation for skin according to claim 5 or 6 to the epidermis, the bone marrow stem cells are attracted to the epidermis, and the attracted bone marrow stem cells are converted into epidermal cells. A method for improving the epidermis, comprising differentiating.

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