JP6139937B2 - Dendritic negative regulator - Google Patents

Description

  The present invention relates to a dendrite negative control agent and a screening method for a dendrite control agent.

Dendrites are a plurality of protrusions that branch from a cell body like a tree branch in order to receive information sent from an external stimulus or an axon of another nerve cell.
In recent years, the role of dendrites of cells other than nerve cells has attracted attention. For example, melanosomes containing mature melanin granules migrate within cells and are passed from melanocyte dendrites to adjacent keratinocytes. At this time, formation of dendrites of melanocytes is considered to be important, but the mechanism of dendrite formation has not been fully elucidated.
For this reason, research on the control of dendrites has been further advanced.
For example, Patent Document 1 proposes to provide a technique for suppressing Rac activity and contracting dendrites to the same level in order to screen for Rac activity promoters useful for nerve cell regeneration and the like. ing.
Further, for example, Patent Document 2 proposes a melanocyte dendrite elongation inhibitor composed of the essence of Citrus agonia.
Moreover, for example, Patent Document 3 proposes a melanocyte dendrite formation inhibitor containing yeast extract as an active ingredient.
However, it is still unclear what process dendrite formation is formed through, and further research and development of substances capable of controlling dendrite formation is required.

JP 2006-067940 A JP 2001-199866 A JP 2003-252742 A

  Therefore, in view of such circumstances, the present invention intends to provide an excellent dendritic negative control agent and a screening method for a dendritic positive control agent using the same.

Therefore, as a result of intensive studies, the present inventors have found that an extract of the camellia family genus Chanoki, which is considered to be highly safe due to its natural origin, negatively controls dendrites, thereby completing the present invention. It was.
That is, this invention provides the dendrite negative control agent which uses the extract of the camellia tea genus chanoki as an active ingredient.
The extract of the camellia tea genus canoki may be a leaf extract.
It is preferable that the plant extract is selected from water, alcohols, and hydrous alcohols. The alcohols preferably have 1 to 4 carbon atoms. The alcohol may be selected from methanol, ethanol, and 1,3-butylene glycol. The water concentration may be 0 to 50 vol%.
The extract of the camellia tea genus may be oolong tea or green tea extract.
The oolong tea extract may be red oolong tea.
The green tea extract may be gyokuro.
The dendrite negative control may be suppression of dendrite formation, regression of dendrite, or suppression of dendrite activation.
The dendrites may be melanocyte dendrites.

In addition, the present invention is a dendritic control method characterized by applying an extract of Camellia genus Chamonaceae. The dendrite control method is preferably a method used for a dendrite control test or a method used for cells or tissues taken out of the body or artificially produced.
Moreover, this invention provides the screening method of the dendrite positive control agent which uses the extract of the camellia tea genus chanoki.

Here, dendrite control of the present disclosure refers to positive / negative control of dendrite. The negative control refers to control in a direction that suppresses the activity among the life phenomena, and the positive control refers to control in the direction that promotes the activity among the life phenomena.
Examples of dendrite negative control include suppression of dendrite formation, regression of dendrite, suppression of dendrite activation, and the like. The dendrite positive control includes, for example, that the formation of dendrite is promoted, the dendrite is elongated, and the dendrite is activated.

According to the present invention, it is possible to provide an excellent dendrite negative control agent and a screening method for a dendrite positive control agent using the same.

It is a photograph figure which shows the formation condition of the melanocyte dendrite after adding (alpha) -melanocyte stimulating hormone 30 minutes after adding oolong tea extract, and culture | cultivating for 3 days. Note that 0 (control), 0.1, 0.3, and 0.5 vol% of the activated state are final concentrations of the oolong tea extract. The length of the bar is 100 μm. It is a figure which shows the formation inhibitory effect of the dendrites of a melanocyte after 3 days culture | cultivation by adding (alpha) -melanocyte stimulating hormone 30 minutes after adding oolong tea extract. In addition, 0 vol% (αMSH not added), 0 (control) in the activated state, 0.1, 0.3, and 0.5 vol% are final concentrations of the oolong tea extract. In addition, the vertical axis represents the percentage (%) of cells having dendrites in all cells. It is a photograph figure which shows the formation condition of the melanocyte dendrites after adding alpha-melanocyte stimulating hormone 30 minutes after adding green tea extract, and culture | cultivating for 3 days. In addition, 0 (control), 0.1, 0.3, and 0.5 vol% of the activated state are final concentrations of the green tea extract. The length of the bar is 100 μm. It is a figure which shows the formation inhibitory effect of the dendrites of a melanocyte after adding alpha-melanocyte stimulating hormone 30 minutes after adding a green tea extract, and culturing for 3 days. The vertical axis shows the proportion of dendritic cells in all cells. Note that 0 vol% (without αMSH added), 0 (control) in the activated state, 0.1, 0.3, and 0.5 vol% are final concentrations of the green tea extract. The left figure is a photograph showing the formation of dendrites of melanocytes after 3 days of culture after adding α-melanocyte-stimulating hormone 30 minutes after addition of the cassis extract. The right figure is a figure which shows the formation condition of the dendrites of a melanocyte after adding (alpha) -melanocyte stimulating hormone after adding Sempukuka extract and culturing for 3 days. Note that 0 (no αMSH added), 0 (control of the activated state), and 1.0 vol% are the final concentrations of the cassis extract and Sempukuka extract, respectively. The length of the bar is 100 μm. The left figure is a figure which shows the formation inhibitory effect of the dendrites of a melanocyte after adding the alpha-melanocyte stimulating hormone 30 minutes after adding a cassis extract, and culture | cultivating for 3 days. The right figure is a figure which shows the formation inhibitory effect of the dendrites of a melanocyte after adding the alpha melanocyte stimulating hormone 30 minutes after adding a Sempukuka extract, and culture | cultivating for 3 days. Note that 0 (no αMSH added), 0 (control of the activated state), and 1.0 vol% are the final concentrations of the cassis extract and Sempukuka extract, respectively. In addition, the vertical axis represents the percentage (%) of cells having dendrites in all cells. It is a photograph figure which shows the retraction state of the dendrite of the melanocyte after adding oolong tea extract 3 days after adding (alpha) -melanocyte stimulating hormone, and culturing for 3 days. Note that 0 (control), 0.3, and 0.5 vol% of the activated state are final concentrations of the oolong tea extract. In addition, a photograph 3 days after the addition of α-melanocyte stimulating hormone showed one representative image. The length of the bar is 100 μm. It is a figure which shows the regression effect of the dendrite of a melanocyte after adding oolong tea extract 3 days after adding (alpha)-melanocyte stimulating hormone. Note that 0 (control), 0.3, and 0.5 vol% of the activated state are final concentrations of the oolong tea extract. The length of the bar is 100 μm. In addition, the vertical axis represents the percentage (%) of cells having dendrites in all cells. It is a photograph figure which shows the retraction state of the dendrite of a melanocyte after adding a green tea extract 3 days after adding (alpha)-melanocyte stimulating hormone, and culturing for 3 days. In addition, 0 (control), 0.1, 0.3, and 0.5 vol% of the activated state are final concentrations of the green tea extract. The length of the bar is 100 μm. It is a figure which shows the regression effect of the dendrite of a melanocyte after adding a green tea extract 3 days after adding (alpha) -melanocyte stimulating hormone, and culturing for 3 days. The vertical axis shows the proportion of dendritic cells in all cells. In addition, 0 (control), 0.1, 0.3, and 0.5 vol% of the activated state are final concentrations of the green tea extract.

There are no particular restrictions on the type or production area of the camellia family genus Chanoki used in the present disclosure. One or two or more selected from the following examples can be used.

Camellia sinensis (Theaaeenaceae) used in the present disclosure may be used for extraction of any part of leaves, stems, xylem, roots, fruits, flowers, etc. 2 The above parts may be mixed and used for extraction. From the viewpoint of the stability of the extract and the excellent dendrite negative control action, the stems are preferably stems or leaves, and leaves are particularly preferred.

The tea can be used for extraction regardless of whether it is fresh or dried, and it can also be used for fermented tea such as ready-made black tea, puerh tea, semi-fermented tea such as oolong tea, baked tea, green tea, potted green tea, roasted tea, You may use fermented tea etc. for extraction. From the viewpoint of the stability of the extract and excellent dendrite negative control action, it is preferable to use oolong tea or green tea for extraction.

Oolong tea used in the present disclosure is a semi-fermented product of the leaves of the Camellia family Thea sinensis L .. Oolong tea has a different degree of fermentation depending on the species and production area, and wushuiwa tea, iron kannon, single plexus, red oolong tea (also known as incense), frosted oolong tea, Wenshan Bao and Takayama tea are known. The degree of fermentation is the extent of the area where the leaves turn red as the fermentation progresses, and is not an accurate numerical value for visual observation, but is generally 15 to 80%. Among them, those having a high degree of fermentation with a degree of fermentation exceeding 50% are particularly called red oolong tea. Red oolong tea is preferable from the viewpoint of the stability of the extract and excellent dendrite negative control action.

  The green tea used in the present disclosure is a tea that has not been fermented with leaves of the Camellia family Thea sinensis L .. Green tea varies depending on the species, production area, production method, etc., and Gyokuro, Sencha, Shincha, Kabusecha, powdered tea, hojicha, matcha, Sayamacha, Ujicha, Tatsuicha, and Hiratsuharu are known. Among these, the one that covers the tea leaves before harvesting, blocks sunlight and increases amino acids is called Gyokuro. Gyokuro is preferred from the viewpoint of the stability of the extract and excellent dendrite negative control action.

Theaacenaceae tea genus (Thea sinensis L.) extract used in the present disclosure is subjected to an extraction operation after the above-mentioned chanoki as it is or pulverized, or is dried and pulverized as necessary. Can be prepared. The dried pulverized product can be prepared by drying and then pulverizing leaves, stems, xylem, roots, berries, flowers, etc. of cypress, or by cutting them into raw pieces and then drying them.

  The present invention uses an extract from chanoki (hereinafter abbreviated as chanoki extract). The method for preparing the chanoki extract is not particularly limited. For example, the leaves of chanoki are treated with a solvent at a low temperature (for example, less than 4 ° C.) or at a normal temperature (for example, 4 to 40 ° C.) to warming (for example, 40 to 100 ° C.). Obtained by extraction.

  Solvents used for extraction are generally water, lower monohydric alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (propylene glycol, 1,3-butylene glycol, etc.), lower esters (ethyl acetate, etc.), hydrocarbons (benzene, hexane, pentane, etc.), ketones (acetone, methyl ethyl ketone, etc.), ethers (diethyl ether, tetrahydrofuran, dipropyl ether) And acetonitrile. In addition, it is preferable that the number of carbons in "lower" is 1-4.

  As an example of a preferred extraction method, the leaves of Theaceae family genus Chaca (Thea sinensis L.) are exposed to sunlight, fried and then cooked in a kettle and dried, and a moisture content of 0 to 100 vol% (preferred) Is added with ethyl alcohol or 1,3-butylene glycol having a water content of 0 to 50 vol%, heated and extracted, filtered, and the filtrate obtained is concentrated under reduced pressure at 50 ° C. to give a soft extract. Get. A method may be mentioned in which water and 1,3-butylene glycol are added to this soft extract to form a 50 vol% 1,3-butylene glycol solution, left to mature for about one week, and filtered again. At the time of filtration, impurities such as impurities and colored substances may be removed using a filtering agent such as activated carbon. The water concentration 100 vol% is water.

  The chanoki extract may be used as it is as an active ingredient, or may be used after performing purification treatment such as removal of the extraction solvent, filtration, deodorization of an ion exchange resin, and decolorization as necessary. It is also possible to obtain a highly active fraction or the like by using a separation and purification means such as liquid chromatography.

  The chanoki extract can be used as it is by mixing the extract alone or the extract obtained by different extraction methods, or by diluting, concentrating or drying the extract to obtain a liquid, powder or paste. It can also be prepared and used.

By the way, since dendrites are thought to be involved in some kind of delivery between cells, the mechanism of dendrite formation has been studied also in the pharmaceutical field and the cosmetic field. For example, Purkinje cells and melanocytes are known as cells having dendrites, but by controlling these dendrites, prevention of symptoms or conditions caused by the formation of these dendrites, It can be used for treatment or improvement.
For example, Purkinje cells are cells involved in the nervous system, but if there is an agent that can control the dendrite of Purkinje cells, the agent can also be used for nerve cell regeneration and the like. In addition, when melanin production increases, melanin migration to keratinocytes can be suppressed by negatively controlling the dendrites of melanocytes, and finally pigmentation such as spots and buckwheat is suppressed. It is also possible to use it as an agent.

And, as shown in the examples below, in the dendrite formation inhibition test and the dendrite retraction test after dendrite formation, the tea extract of the present disclosure has a dendrite formation inhibitory action, a dendrite retraction action It was found that negative control of dendrites such as was possible.
Therefore, since the chanoki extract of the present disclosure has dendrite negative control (for example, dendrite formation inhibitory action, dendrite retraction action, etc.), the dendritic extract containing the chanoki extract as an active ingredient is used. It can be used as a protrusion negative control agent (for example, dendrite formation inhibitor, dendrite retraction agent, etc.).
The chanoki extract of the present disclosure may be used for dendritic negative control (for example, dendrite formation inhibitory action, dendritic retraction action, etc.), and dendritic negative control agents (for example, Can be used in various preparations intended for use as described above, such as dendrite formation inhibitors and dendritic retraction agents, and can also be used to produce these various preparations.
The tea tree extract of the present disclosure has dendrite negative control (dendrite formation inhibitory action, dendrite retraction action, etc.), and prevents, ameliorates and / or treats various symptoms and conditions caused by dendrite formation. Can be used in a method for Therefore, the tea tree extract of the present disclosure can be used in a method for preventing, ameliorating and / or treating various symptoms and conditions caused by dendrite formation by ingestion or administration to animals including humans. It is.

Therefore, the chanoki extract of the present disclosure can be used for external control of skin, cosmetics (preferably whitening cosmetics), pharmaceuticals for dendritic negative control (dendritic formation inhibitory action, dendritic regression action, etc.). It can be incorporated into quasi-drugs, foods, functional foods (eg, foods for specified health use) and the like, and the preparations of the present disclosure are useful as these external preparations for skin, cosmetics and the like.
The tea extract of the present disclosure is particularly suitable for use in external preparations for skin, cosmetics (preferably whitening cosmetics), quasi-drugs, pharmaceuticals, etc., and preparations that are brought into contact with the skin by application or the like are suitable. . Examples of the external preparation for skin and cosmetic include skin lotion, emulsion (oil-in-water type, etc.), cream (water-in-oil type, etc.), pack cosmetic, liquid foundation, ointment, hair nourishing agent and the like.

  The content of the tea extract of the present disclosure is preferably 0.00001 to 5% by mass, more preferably 0.0001 to 2% by mass as a dry solid content in the preparation. If it exists in this range, this plant extract can be mix | blended stably and the outstanding dendrite negative control effect | action can be exhibited. Moreover, when using an extract, if the content of the dry solid content which is a solute is in the said range, the extract concentration will not be limited at all.

The tea extract of the present disclosure is preferably used in combination with an agent that exhibits a whitening effect by a mechanism other than dendrite control, such as a tyrosinase inhibitor, a melanin production inhibitor, an anti-inflammatory agent, and a melanin excretion promoter. Thereby, it becomes possible to exhibit a synergistic whitening effect.
Examples of tyrosinase inhibitors and melanin production inhibitors include L-ascorbic acid and its derivatives, and salts thereof (ascorbic acid-2-glucoside, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, 3-O-ethylascorbic acid Etc.), hydroquinone and derivatives thereof (arbutin etc.), tranexamic acid and derivatives thereof (cetyl tranexamic acid, trans-4-aminomethylcyclohexanecarboxylic acid methylamide etc.), salicylic acid and derivatives thereof (4-methoxysalicylic acid etc.) and salts thereof Ellagic acid and its derivatives, kojic acid and its derivatives, adenosine-1-phosphate, linoleic acid, 5,5′-dipropyl-biphenyl 2,2′-diol, 4- (4-hydroxyphenyl) -2-butanol Or a derivative thereof, Zorushin and derivatives thereof (4-n-butyl resorcinol and the like), placental extract, chamomile extract, but nicotinamide, and the like, ascorbic acid 2-glucoside, 3-O-ethyl-ascorbic acid, or arbutin is preferred. Anti-inflammatory agents include tranexamic acid and derivatives thereof, glycyrrhizic acid derivatives such as dipotassium glycyrrhizinate, glycyrrhetinic acid derivatives such as stearyl glycyrrhetinate, salicylic acid and derivatives thereof, chamomile extract, etc., but tranexamic acid, dipotassium glycyrrhetinate or Stearyl glycyrrhetinate is preferred. Examples of the melanin excretion promoter include nicotinic acid derivatives such as nicotinic acid amide, adenosine-1-phosphate and derivatives thereof, and nicotinic acid amide or adenosine-1-phosphate is preferable.

In addition to the tea extract of this indication, you may use it for the said formulation combining arbitrary components as needed. Other components may be pharmaceutically acceptable components, such as cell activators, antioxidants, humectants, UV inhibitors, solvents (water, alcohols, etc.), oils, surfactants, Thickener, powder, chelating agent, pH adjuster, emulsifier, stabilizer, colorant, brightener, flavoring agent, flavoring agent, excipient, binder, disintegrant, lubricant, diluent, penetration A pressure regulator, a fragrance | flavor, etc. are mentioned, What is necessary is just to mix | blend according to the formulation for which these are aimed.
The form of the preparation is not particularly limited, and may be any form such as liquid, paste, gel, solid, and powder.

  Further, the tea tree extract of the present disclosure can be used for a dendrite control method, the dendrite control method can also be used for a dendrite control test, and a dendritic sample collected from an animal. Using a cell having a protrusion as a raw material, a medicine (cell medicine, etc.); a medical material (artificial substitute or substitute, etc.); a method for producing these intermediate products, or a method for analyzing them It is also possible to use it.

  Moreover, since the chanoki extract of this indication negatively controls dendrites, the said chanoki extract can be used for the screening method of the agent which can carry out positive control of dendrites. Thereby, it is possible to screen a dendrite controlling agent or a substance having a dendrite controlling action or to evaluate dendrite control. It is also possible to search for substances having different action mechanisms by adding the test substance and chanoki extract at the same time or separately.

Compared with the state in which the tea extract of the present disclosure and the test substance are added and the state in which the dendrite is not formed or the state in which the dendrite is negatively controlled, When dendrites are positively controlled, such as when formation of dendrites is promoted, the test substance can be determined as a dendritic positive control agent. In addition, it is also possible to make the dendrite negative control by adding the chanoki extract of the present disclosure and a known dendrite positive control agent.
Moreover, the dendrite formation state when adding the chanoki extract of the present disclosure and a known dendrite positive control agent, and the dendrite formation state when adding the dendrite positive control agent and the test substance In contrast, when dendrites are more negatively controlled than when adding the chanoki extract of the present disclosure, the test substance can be determined as a good dendritic negative inhibitor.

Although the said determination method is not specifically limited, The example is given to the following.
For example, a photo of cell morphology was taken with a phase contrast microscope (Olympus), and the number of dendritic cells (cells in which one or more dendrites were newly formed from bipolar cells) were determined. Count and calculate the number of dendritic cells / total number of cells × 100%. The positive / negative of the dendritic control of the test substance is judged by comparison with the control.

Below, a suitable example of the search method of the dendrite positive control agent using the tea tree extract of this indication is given.
By culturing melanocytes in the presence of the chanoki extract of the present disclosure, adding a test substance thereto, and observing morphological changes in dendrites of melanocytes, it is possible to screen for dendritic formation promoters.
At this time, a melanocyte activator may be added. Examples of the melanocyte activator include α-melanocyte stimulating hormone (α-MSH), stem cell factor (SCF), and endothelin-1 (Endothelin-1).
In the case of melanocytes, transfer to melanosomes to keratinocytes is promoted, and therefore can be applied to substance search such as improvement of gray hair.

The culture conditions in the above-mentioned dendrite control method, dendrite control agent screening method and dendrite control evaluation method may be the known culture conditions, medium conditions, etc. according to the cells having dendrites to be used. Good.
For example, when normal melanocytes are used, the culture may be performed under known culture conditions in which normal melanocytes can be cultured. As a culture growth medium, a commercially available melanocyte medium or the like may be used.
The culture temperature should just be the temperature which can be cultured corresponding to the origin of the melanocyte to be used, for example, 20 degreeC-40 degreeC, preferably about 37 degreeC in the case of human origin. The carbon dioxide concentration may be about 5 vol%.

  In addition, when using Purkinje cells, nerve regeneration is evaluated by culturing Purkinje cells by a known culture method in the presence of the chanoki extract of the present disclosure and evaluating that dendrites are positively controlled. It can be applied to substance search such as promotion.

In addition, this technique can also employ | adopt the following structures.
[1] A dendrite negative control agent containing an extract of Thea sinensis L. as an active ingredient.
[2] The dendritic negative control agent according to [1], wherein the extract of Theaceae family Thea sinensis L. is a leaf extract.
[3] In the above [1] or [2], the extract of the Theaeae family, Thea sinensis L., is extracted with water, alcohols, or hydrous alcohols. The dendritic negative control agent as described.
[4] The tree according to any one of [1] to [3], wherein the extract of theaceae family Thea sinensis L. is an extract of green tea or oolong tea Negative protrusion control agent.
[5] The dendrite negative control agent according to [4], wherein the green tea is gyokuro.
[6] The dendrite negative control agent according to [4], wherein the oolong tea is red oolong tea.
[7] The dendritic negative control agent according to any one of [1] to [6], wherein the dendritic negative control is melanocyte dendritic negative control.
[8] The dendrite negative control agent according to any one of [1] to [7], wherein the dendrite negative control is dendrite formation inhibition, dendrite retraction or dendrite activation inhibition .
[9] A screening method for a dendrite control agent using an extract of theaceae family Thea sinensis L.

  Examples, Reference Examples, Comparative Examples, Test Examples, Production Examples, etc. are given below to describe the present invention (present technique) more specifically. However, the present invention (present technique) is not limited to these examples. It is not something.

<Production Example 1: Production of red oolong tea extract>
100 g of red oolong tea (Toho Bijin Fermentation degree: about 70%: made in Taiwan) was pulverized, placed in 1 L of 50% aqueous ethanol solution, and extracted at 30 ° C. for 25 hours. After filtering insoluble matter, the filtrate was concentrated under reduced pressure to obtain 18 g of solid oolong tea extract.
<Production Example 2: Production of frosted oolong tea extract>
100 g of oolong tea (frozen oolong tea, fermentation degree: about 20%: made in Taiwan) was pulverized and extracted with 1 L of 50% dipropylene glycol solution at 40 ° C. for 20 hours with appropriate stirring. Then, it was left to stand for 24 hours, and the precipitate was removed by filtration to obtain an extract. The solid content concentration of this extract was 2.0%.
<Production Example 3: Production of Wuyi Oolong Tea Extract>
100 g of oolong tea (fermentation degree about 40% fermented tea: made in China) was pulverized and extracted and filtered in 1 L of purified water for 72 hours in a cool dark place with appropriate stirring. The filtrate was concentrated under reduced pressure until the volume was reduced to half, and 1,3-butylene glycol and purified water were added to adjust the concentration of 1,3-butylene glycol to 30%. The solid content concentration of this extract was 5.0% <Production Example 4: Production of gyokuro extract>
100 g of gyokuro (made in Japan) was pulverized, placed in 1 L of 80% ethanol aqueous solution, and gently heated and extracted. The insoluble material was filtered, and the filtrate was concentrated under reduced pressure to obtain 8.0 g of a solid gyokuro extract.
<Production Example 5: Production of kabuse tea extract>
100 g of kabusecha (made in Japan) was pulverized and extracted with 1 L of 50% dipropylene glycol solution at 40 ° C. for 20 hours with appropriate stirring. Thereafter, the precipitate was filtered and removed by leaving for 3 days to obtain an extract. The solid content concentration of this extract was 3.0%.
<Production Example 6: Production of roasted tea extract>
100 g of hojicha (made in China) was pulverized and extracted and filtered in 1 L of purified water for 72 hours in a cool dark place with appropriate stirring. The filtrate was concentrated under reduced pressure until the volume was reduced to half, and 1,3-butylene glycol and purified water were added to adjust the concentration of 1,3-butylene glycol to 30%. The solid content concentration of this extract was 4.0%.

<Test Example 1: Melanocyte dendrite formation inhibition test>
[Example 1: Oolong tea extract]
Using the solution prepared with 50% 1,3-butylene glycol aqueous solution so that the solid content concentration of the red oolong tea extract produced in Production Example 1 is 1.0%, Examined.
Specifically, an appropriate amount of epidermal melanocyte (melanocyte) medium containing HMGS growth additive is added to a 6-well plate, 1 × 10 ⁵ normal human epidermal melanocytes (melanocytes) are seeded, and the carbon dioxide concentration is 37 ° C. Static culture was performed in 5 vol%. After 2 days of culture, the medium was replaced with a medium in which BPE was removed from the HMGS growth additive, and the oolong tea extract produced in Production Example 1 had a concentration in the medium of 0 (control in activated state), 0.1, 0.3 30 minutes after adjusting to 0.5 vol%, α-MSH was added and mixed so that the final concentration was 10 ⁻⁸ mol / L.
Three days later, a photo of the cell morphology was taken with a phase contrast microscope (Olympus), and dendritic cells (cells in which one or more dendrites were newly formed from bipolar cells) and all The number of cells was counted and the number of dendritic cells / total number of cells × 100 was calculated, and the results are shown in FIG. 1, Table 1 and FIG.
[Reagents]
Epidermal melanocytes (melanocytes) basal medium: Medium 254 medium; sterile liquid medium M-254-500 (manufactured by Life Technologies) for human epidermal melanocyte culture
HMGS growth additive: HMGS growth additive dispensing kit KM-6350 (manufactured by Kurashiki Boseki Co., Ltd.)
Normal human epidermal melanocytes: Human Epidermal Melanocytes, neonatal (HEMn-LP); Neonatal lightly pigmented donor C-002-5C (Life Technologies)
α-MSH: α-melanocyte stimulating hormone M4135 (manufactured by Sigma)

[Example 2: Green tea extract]
Using a solution prepared with 50% 1,3-butylene glycol aqueous solution so that the solid content concentration of the gyokuro extract produced in Production Example 4 is 1.6%, the melanocyte dendrite formation inhibitory effect is examined. It was. Specifically, a melanocyte dendrite formation inhibition test was conducted in the same manner as in [Example 1] except that the sample “oolong tea extract” was replaced with “Gyokuro extract”. The results are shown in Table 2 and FIGS.

[Comparative Examples 1 and 2: Cassis extract, Sempukuka extract]
A melanocyte dendrite formation inhibition test was conducted in the same manner as in [Example 1] except that the sample “oolong tea extract” was replaced with “cassis extract” (Comparative Example 1).
The cassis extract was obtained by adding 50 g of purified water to 10 g of cassis (Ribes nigrum L.) fruit, followed by heating at 50 ° C. for 3 days, followed by filtration. The dry solid content of this extract was 3.1%.
A melanocyte dendrite formation inhibition test was carried out in the same manner as in [Example 1] except that the sample “Oolong tea extract” was replaced with “Sempukuka extract” (Comparative Example 2).
The Sempukuka extract was obtained by adding 500 g of an ethanol / water mixture containing 50 volumes of ethanol to 10 g of flowers of Inula britannica Linne 'var chinensis, followed by extraction at room temperature for 3 days, followed by filtration. The dry solid content of this extract was 0.3%.
The results of Comparative Examples 1 and 2 are shown in FIGS.

<Test Example 2: Melanocyte Dendritic Retraction Test>
[Example 3: Oolong tea extract]
The test was performed in the same manner as in Test Example 1 except that the addition / observation order / schedule / concentration of α-MSH / oolon tea extract was changed. Three days after the addition of α-MSH as an activator, it was confirmed that melanocyte dendrites were sufficiently formed, and then oolong tea extract was added, followed by further culturing for 3 days. The result is shown in FIG.
Even if the dendrites of the cells were already activated and elongated, it was found that the dendrites that had been elongated were regressed by adding oolong tea extract. The results of calculating the number of dendritic cells / total number of cells × 100 are shown in Table 3 and FIG.

[Example 4: Green tea extract]
Using a solution prepared with 50% 1,3-butylene glycol aqueous solution so that the solid concentration of the gyokuro extract produced in Production Example 4 was 1.6%, the melanocyte dendritic retraction action was examined. . Specifically, a melanocyte dendrite regression test was performed in the same manner as in [Example 3] except that the sample “oolong tea extract” was replaced with “Gyokuro extract”. The results are shown in Table 4 and FIGS.

Cassis extract and Sempukuka extract are known to have a whitening effect, but were found to have no dendrite formation-inhibiting action. In contrast, oolong tea extract was found to have a negative control action on dendrites. Specifically, when a dendritic activator was added at the same time as the oolong tea extract, formation of dendrites or suppression of elongation was observed. The same effect as that of the oolong tea extract was observed in the green tea extract.
Moreover, it was recognized that the extended dendrites were retracted even when the oolong tea extract was added after the dendritic activator was added first to activate and extend the dendrites. Moreover, the same effect as the oolong tea extract was recognized also in the green tea extract. In particular, retraction of the elongated dendrites makes it possible to develop a whitening effect even if the amount of melanin produced is large, and to improve already formed pigmentation. Such negative control of dendrites is considered to be an action different from a tyrosinase inhibitory action, a melanin production inhibitory action, an anti-inflammatory action, a melanin excretion promoting action, etc., which have been conventionally searched for as a whitening effect.
For this reason, the tea tree extract of the present disclosure and a drug that exhibits a whitening effect by an action mechanism other than dendrite control (for example, a tyrosinase inhibitor, a melanin production inhibitor, an anti-inflammatory agent, a melanin excretion promoter, etc.) In combination with, there is a high possibility of producing a synergistic whitening effect.
In addition, the action of suppressing the exchange of substances between cells via dendrites may lead to new cosmetic uses and pharmaceutical uses. It is considered promising as a method for searching for new whitening agents, white hair improving agents, nerve regeneration promoting agents, etc., that is not directed to suppressing the amount of melanin synthesis as in the past.

[Prescription Example 1: Solubilized lotion]
(Ingredient) (mass%)
1. POE (40 mol) hydrogenated castor oil 0.5
2. POE (12 mol) dioleate 0.3
3. Astaxanthin 0.05
4.1,3-Butylene glycol 2.0
5). Glycerin 2.0
6). Ethanol 15.0
7). Tranexamic acid 2.0
8). Red oolong tea extract of Production Example 1 0.1
9. Sodium lactate 0.2
10. Methyl paraoxybenzoate 0.1
11. Purified water balance (production method)
A. Components 1 to 6 are mixed and dissolved.
B. Components 7 to 11 are mixed and dissolved.
C. A was added to B to obtain a skin lotion.
The solubilized lotion of Formulation Example 1 moisturizes the skin, prevents drying of the skin over a long period of time, and has an excellent moisturizing effect, and when used continuously, the skin color becomes brighter and the skin is transparent. I felt a lot of feeling.

[Formulation Example 2: Emulsion lotion]
(Ingredient) (mass%)
1. Soybean-derived hydrogenated phospholipid 0.5
2. Cetostearyl alcohol 0.1
3. Polyoxyethylene (10 mol) cholesterol ether 0.2
4). Acetic acid-dl-α-tocopherol 0.1
5). Squalane 0.1
6). Hydroxyethyl cellulose 0.03
7). Purified water remaining amount 8. Stearyl glycyrrhetinate 0.25
9. Frozen oolong tea extract of Production Example 2 0.02
10. Disodium monohydrogen phosphate 0.1
11. Monosodium dihydrogen phosphate 0.1
12 Glycerin 3.0
13. Dipropylene glycol 2.0
14 Ethanol 7.0
15. Perfume appropriate amount (production method)
A. Ingredients 1-5 are heated to 75 ° C. and mixed and dissolved uniformly.
B. Ingredients 6 and 7 are heated to 75 ° C. and mixed and dissolved uniformly. Add B to A and emulsify.
D. C was cooled, components 8 to 15 were added, and an emulsified lotion was obtained.
The emulsified skin lotion of Formulation Example 2 moisturizes the skin, prevents the skin from drying for a long time, and has an excellent moisturizing effect. Also, when used continuously, the skin color becomes brighter and the skin is more transparent. It was a great thing.

[Prescription Example 3: Latex]
(Ingredient) (mass%)
1. Polyoxyethylene (20) sorbitan monooleate 1.0
2. Sorbitan sesquioleate 0.5
3. Glyceryl trioctanoate 0.5
4). Jojoba oil 0.5
5). Squalane 0.5
6). 6. Purified water remaining amount Edetate disodium 0.1
8). Methylparaben 0.2
9. Phenoxyethanol 0.5
10. Glycerin 5.0
11. Propanediol 1.0
12 Propylene glycol 2.0
13. Sodium lactate 0.5
14. 2-O-α-D-glucosyl-L-ascorbic acid (Note 1) 1.0
15. Wuyi oolong tea extract of Production Example 3 0.005
16. Xanthan gum 0.05
17. Purified water 10.0
18. Ethanol 3.0
19. Perfume appropriate amount (Note 1): Hayashibara Co., Ltd. (manufacturing method)
A: Swell component 16 with component 17 heated to 70 ° C.
B: Components 1 to 5 are heated and mixed at 70 ° C.
C: Components 6 to 13 are heated and dissolved at 70 ° C., then added to B and emulsified.
D: After cooling C to room temperature, ingredients 14, 15, 18, 19 and A were added to obtain a cosmetic liquid.
The emulsion of this Formulation 3 moisturizes the skin, prevents the skin from drying for a long time and has an excellent moisturizing effect, and when used continuously, the skin color becomes brighter and the skin feels transparent. Met.

[Prescription Example 4: Sunscreen]
(Ingredient) (mass%)
1. Stearic acid 1.0
2. Monooleic acid polyoxyethylene (20 mol) sorbitan 0.5
3. Sorbitan sesquioleate 0.5
4). Behenyl alcohol 0.5
5. Glyceryl 2-ethylhexanoate 2.0
6). 2-Ethylhexyl paramethoxycinnamate 10.0
7). Diethylaminohydroxybenzoyl hexyl benzoate 2.0
8). Dimethicodiethylbenzalmalonate 1.0
9. Tinosorb S (Note 2) 1.0
10. Octyl rearzone 0.5
11. Dipropylene glycol 10.0
12 Triethanolamine 1.0
13. Purified water remaining amount 14. Glycerin 5.0
15.1,3-butylene glycol 5.0
16. Tinosorb M (Note 2) 1.0
17. Acrylic acid / methacrylic acid alkyl ester copolymer (Note 3) 0.2
18. Purified water 5.0
19. Gyokuro extract of Production Example 4 0.1
20. Citric acid 0.1
21. Disodium succinate 0.1
22. Ethanol 10.0
23. Preservative appropriate amount 24. Perfume appropriate amount (Note 2) BASF (Note 3) CARBOPOL 1382 (LUBRIZOL ADVANCED MATERIALS)
(Production method)
A: Components 1 to 10 are uniformly dissolved at 80 ° C.
B: Components 11 to 19 are uniformly dissolved at 80 ° C.
C: A is added to B and emulsified.
D: C was stirred and cooled, and components 20 to 24 were added to obtain sunscreen.
The sunscreen of this Formulation 4 moisturizes the skin, prevents the skin from drying for an extended period of time, and has an excellent moisturizing effect. When used continuously, the skin color becomes brighter and the skin feels transparent. It was a thing.

[Prescription Example 5: Foundation]
(Ingredient) (mass%)
1. Polyoxyethylene methylsiloxane / polyoxypropylene oleyl methylsiloxane / dimethylsiloxane copolymer (Note 4) 2.0
2. PEG-3 Dimethicone (Note 5) 1.0
3. Dimethylpolysiloxane 5.0
4). Dimethylpolysiloxane-treated red iron oxide 1.0
5). Dimethylpolysiloxane-treated yellow iron oxide 1.5
6). Black iron oxide treated with dimethylpolysiloxane 0.5
7). Dimethylpolysiloxane-treated titanium oxide 10.0
8). Dimethylpolysiloxane treated talc 5.0
9. 2-Ethylhexyl paramethoxycinnamate 5.0
10. Stearyl glycyrrhetinate 0.5
11. Sorbitan sesquioleate 0.5
12 Purified water remaining amount 13.1,3-butylene glycol 15.0
14 Covered tea extract of Production Example 5 0.5
15. Sodium citrate 0.1
16. Monosodium dihydrogen phosphate 0.1
17. Phenylbenzimidazolesulfonic acid 0.3
18. Triethanolamine 1.7
19. Preservative appropriate amount20. Perfume appropriate amount (Note 4) KF-6026 (manufactured by Shin-Etsu Chemical Co., Ltd.)
(Note 5) KF-6015 (manufactured by Shin-Etsu Chemical Co., Ltd.)
(Production method)
A: Components 1 to 3 are mixed uniformly.
B: Components 4 to 11 are uniformly dispersed with a roller.
C: Add B to A and mix uniformly.
D: Components 12 to 20 are mixed and dissolved.
E: D was added to C and emulsified to obtain a foundation.
The foundation of this Formulation 5 moisturizes the skin, prevents the skin from drying for an extended period of time, and has an excellent moisturizing effect. Also, when used continuously, the skin color becomes brighter and the skin feels transparent. Met.

[Prescription Example 6: Nonwoven fabric impregnation type pack charge]
(Ingredient) (%)
1. Polyoxyethylene (25) Phytostanol (HLB14.5) 1.0
2. Stearyl alcohol 0.2
3. Cetanol 0.2
4). Hydrogenated soybean phospholipid 0.3
5). Propylene glycol 10.0
6). Diglycerin 8.0
7). Liquid paraffin 1.0
8). Meadow foam oil 0.5
9. Microcrystalline wax 5.0
10. Purified water 1.0
11. Ethanol 3.0
12 Purified water remaining
13. Ascorbic acid glucoside 2.0
14 Roasted tea extract from Production Example 6 0.3
15. Hydrolyzed hyaluronic acid (molecular weight 3000) 1.0
16. Sodium hydroxide
17. Fragrance 0.01
(Production method)
A: Components 1 to 6 are dissolved and mixed at 80 ° C.
B: Components 7 to 9 were heated to 80 ° C and added to A, and then component 10 heated to 75 ° C was added and emulsified.
C: This was cooled and degassed to prepare an oil-in-water liquid composition, and the non-woven fabric was impregnated with the stock solution mixed with components 11 to 17 to obtain a nonwoven fabric-impregnated type pack.
The non-woven fabric impregnated type packing material of this prescription example 6 moisturizes the skin, prevents the skin from drying for a long time, and has an excellent moisturizing effect. I felt a lot of feeling. The average emulsified droplet diameter of the stock solution impregnated in the nonwoven fabric impregnated type pack was 190 nm.

[Prescription Example 7: Hair Nourishing]
(Ingredient) (mass%)
1. Srutianin 1.5
2. Ginkgo biloba extract 0.5
3. Tranexamic acid 1.0
4). Red oolong tea extract of Production Example 1 1.0
5). Glycerin 2.0
6). 6. Purified water remaining amount D-pantothenyl alcohol 0.3
8). Hinokitiol 0.02
9. Cephalanthin 0.001
10. Tocopherol acetate 0.01
11. L-Menthol 0.2
12 Polyoxyethylene hydrogenated castor oil 0.2
13. Ethanol 60
(Production method)
A. Components 1 to 6 are mixed and dissolved.
B. Components 7 to 13 are mixed and dissolved.
C. B was added to A to obtain a hair nourishing agent.
The hair nourishing agent of Formulation Example 7 moisturized the scalp, prevented the scalp from drying for a long time, and was excellent in the moisturizing effect.

[Prescription Example 8: Ointment]
(Compounding ingredients) (mass%)
1. Stearyl alcohol 18.0
2. Owl 20.0
3. Polyoxyethylene (20) monooleate 0.25
4). Glycerin monostearate 0.3
5). Vaseline 40.0
6). 6. Purified water remaining amount Glycerin 10.0
8). Adenosine-1-phosphate 0.5
9. Gyokuro extract of Production Example 4 1.0
(Production method)
A. 1-5 are mixed uniformly at 70 degreeC.
B. Warm 6-8 to 70 ° C.
C. Add B to A and emulsify.
D. C was cooled and 9 was added to obtain an ointment.
The ointment of Formulation 8 moisturizes the skin, prevents the skin from drying for a long time, has an excellent moisturizing effect, and when used continuously, the skin color becomes brighter and the skin feels transparent. Met.

Claims (7)

Camellia (Theaceae) family tea genus tea tree (Thea sinensis L.) Gyokuro or dendritic retraction agent for the red oolong tea extract as an active ingredient (except, whitening by inhibiting melanin production caused by the alpha-MSH Except.) The extract of Camellia (Theaceae) family tea genus tea tree (Thea sinensis L.) is, dendritic retraction agent according to claim 1, wherein an extract of the leaves. The dendrite retraction according to claim 1 or 2, wherein the extract of the Camellia (Theaceae) family, Thea sinensis L., is extracted from water, alcohols or hydrous alcohols. Agent. The dendrites regression, dendritic retraction agent according to any one of claims 1 to 3 is a melanocyte dendrites retraction.   The dendritic retraction agent according to any one of claims 1 to 4, for blending into an external preparation for skin or a cosmetic.   The dendritic retraction agent according to any one of claims 1 to 5, which is used together with a drug that exhibits a whitening effect by a mechanism other than dendritic retraction. A method for screening a dendrite control agent, comprising a step of degenerating dendrite with an extract of gyokuro or red oolong tea from Theaceae family Thea sinensis L ..