JP6142342B2 - Novel uses of Akinonogeshi extract - Google Patents

Description

  The present invention relates to a novel use of Akinonogushi extract, and in particular, a testosterone 5α-reductase inhibitor, a VEGF expression promoter, an ASIP expression inhibitor, a CSF2 expression promoter, and A composition for imparting gloss to hair, a composition for imparting hair elasticity, a composition for imparting firmness to hair, and a composition for suppleening hair, characterized by containing a hair growth promoter and an extract of Akinonogeshi The present invention relates to a composition for retaining moisture of hair and hair.

  Hair repeats a hair cycle called a hair cycle consisting of a growth phase, a regression phase, and a resting phase. Various cells act in the above cycle, and the hair papilla cell plays the most important role in controlling the cycle. The dermal papilla cell issues a command to the hair matrix cell and the cells existing around it to, for example, grow hair or stop hair growth.

  Among these, when the dermal papilla cell issues a command to stop hair growth or to drop hair, testosterone 5α-reductase (or simply referred to as 5α-reductase) may act. 5α-reductase has an action of converting an inactive male hormone “testosterone” into an active form “dihydrotestosterone (DHT)”, and DHT converted by 5α-reductase is a differentiation of hair matrix cells. In order to inhibit (growth of hair), the hair root is shrunken and hair loss is promoted.

  There are two types of 5α-reductase, type 1 and type 2. There are many types of 5α-reductase type 1 in the temporal region and occipital region. On the other hand, since 5α-reductase type 2 is present in large amounts in the frontal region and the parietal region, this 5α-reductase type 2 is also the main cause of male pattern baldness (AGA). So far, finasteride is known as a substance that inhibits the enzyme activity of 5α-reductase type 2, and is used as a therapeutic agent for AGA.

  Under such circumstances, an object of the present invention is to provide a novel testosterone 5α-reductase inhibitor capable of inhibiting the expression of 5α-reductase type 2 (SRD5A2). Another object of the present invention is to provide use of the extract of Akinonogeshi for new applications.

  As a result of intensive studies to achieve the above object, the present inventor has found that the expression of 5α-reductase type 2 can be inhibited by containing an extract of Akinonokeshi in a testosterone 5α-reductase inhibitor. The invention has been completed. In addition, as a result of further studies, the present inventor has also found that the Achinonogushi extract exhibits various actions such as a hair growth promoting action.

  That is, the testosterone 5α-reductase inhibitor of the present invention is characterized by comprising an Achinonogoshi extract as an active ingredient.

  In addition, the VEGF expression promoter, ASIP expression inhibitor, CSF2 expression promoter and hair growth promoter of the present invention are characterized in that the extract of Akinonageshi is used as an active ingredient, and further the composition for imparting gloss to the hair of the present invention A composition for imparting hair, a composition for imparting elasticity to hair, a composition for imparting firmness to hair, a composition for supple hair, and a composition for retaining moisture of hair are characterized by containing an extract of Akinonageshi.

  According to the testosterone 5α-reductase inhibitor of the present invention, it is possible to provide a testosterone 5α-reductase inhibitor that can inhibit the expression of 5α-reductase type 2 by using an extract of Akinonogoshi as an active ingredient.

  Further, by using an Achinonokeshi extract as an active ingredient, it is possible to promote the expression of VEGF, a VEGF expression promoter that can promote VEGF expression, an ASIP expression inhibitor that can inhibit ASIP expression, and CSF2. A CSF2 expression promoter and a hair growth promoter excellent in hair growth promoting action can be provided.

  Further, by adding an Akinonogoshi extract, the hair can be given luster, beam and stiffness, and the hair can be made supple and moisture of the hair can be retained.

It is a figure which shows the result of hair growth score evaluation. ** indicates p <0.01, * indicates p <0.05, and in these cases, there is a significant difference compared to the control group (Wilcoxon rank sum test). It is the photograph of the test 0th day of the mouse | mouth back which belongs to a control group. It is the photograph on the 29th day of the test of the back of the mouse belonging to the control group. It is the photograph on the 0th day of the test of the back of a mouse belonging to the 0.3% Achinonogeshi extract group. It is the photograph on the 29th day of the test of the back of a mouse belonging to the 0.3% Achinonogeshi extract group. It is the photograph on the 0th day of the test of the back of a mouse belonging to the 3% Achinonogeshi extract group. It is a photograph on the 29th day of the test on the back of a mouse belonging to the 3% Akinonogushi extract group. It is a figure which shows the relative expression level of CSF2 in a hair papilla cell. (n = 3, mean ± SD. *: p <0.05) It is a figure which shows the relative expression level of SRD5A2 in a hair papilla cell. (n = 3, mean ± SD. *: p <0.05) It is a figure which shows the relative expression level of ASIP in a hair papilla cell. (n = 3, mean ± SD. *: p <0.05) It is a figure which shows the relative expression level of VEGF in a hair papilla cell. (n = 3, mean ± SD. *: p <0.05)

<Testosterone 5α-reductase inhibitor of the present invention>
The present invention is described in detail below. The testosterone 5α-reductase inhibitor of the present invention is characterized by using an Achinonogeshi extract as an active ingredient, and can inhibit the expression of 5α-reductase type 2.

  In the testosterone 5α-reductase inhibitor of the present invention, the extract of Achinonogesi can inhibit the expression of 5α-reductase type 2 and promote the expression of colony stimulating factor 2 (CSF2). Colony stimulating factor 2 (CSF2), also known as granulocyte monocyte colony stimulating factor (GM-CSF), is a type of cytokine that promotes differentiation of hematopoietic stem cells into leukocytes such as neutrophils. CSF2 is secreted from epidermal keratinocytes in the skin, stimulates melanocytes, and promotes melanin synthesis. Therefore, when the testosterone 5α-reductase inhibitor of the present invention contains an Achinonokeshi extract as an active ingredient, the occurrence of gray hair can be suppressed while preventing or treating alopecia.

  Lactuca indica is a plant belonging to the genus Akinonokeshi, and in the present invention, the extract of Akinonogeshi can be prepared from the whole plant of Akinonogeshi. Roots, flowers, seeds, etc. are used.

  The above-mentioned Achinonogeshi extract is usually obtained by extracting the extraction site of Akinonogeshi with water and / or an organic solvent. Here, as the form of the extraction site of Akinonogeshi, for example, raw raw materials, those that have been cut finely, those that have been dried, those that have been dried and finely cut or pulverized, etc. are used. However, the present invention is not limited thereto, and other forms can be used as long as the effects of the present invention are not impaired. Moreover, when using water, warm water or hot water is preferable. On the other hand, examples of organic solvents used for extraction include alcohols such as ethanol, methanol, isopropanol, and 1,3-butylene glycol, hydrocarbons such as hexane, heptane, and cyclohexane, esters such as ethyl acetate, and ketones such as acetone. Can be mentioned. These extraction solvents may be used alone or in combination. In particular, ethanol, methanol, or a mixed solvent thereof is preferable. In addition, the mass ratio (A / B) of the extraction site (A) and the extraction solvent (B) is not particularly limited, but a range of 1/5 to 1/50 is preferable.

  In addition, for extraction from Akinonokeshi, means for immersing the extraction site of Akinonokeshi in an extraction solvent, means for heating and refluxing the extraction site of Akinonogeshi together with the extraction solvent, and the like are used. About extraction temperature, it does not specifically limit, Usually, it carries out in the range from normal temperature (about 15-25 degreeC) to the boiling point of the solvent under a normal pressure. The extraction time is not particularly limited and is performed for any time, but is preferably in the range of 1 to 72 hours, more preferably in the range of 12 to 48 hours, and in the range of 15 to 36 hours. It is more preferable that it is in the range of 18 to 30 hours. After extraction with a solvent, solids such as extraction residue are removed from the extract by a method such as filtration and centrifugation, and then the extraction solvent is removed to obtain an extract.

  In the testosterone 5α-reductase inhibitor of the present invention, the content of the extract of Akinonogushi is 0.01 to 500 ppm by mass from the viewpoint of exhibiting the action of inhibiting the expression of SRD5A2 and promoting the expression of CSF2. Is preferred. In the present invention, the content of the extract of Akinonogeshi is determined based on the mass of a dried product obtained by freeze-drying or vacuum-concentrating and drying the extract from Akinonokeshi.

  In addition, since the testosterone 5α-reductase inhibitor of the present invention can inhibit the expression of 5α-reductase type 2, it is useful for the prevention or treatment of male pattern baldness, but promotes the expression of colony stimulating factor 2 (CSF2). Therefore, it can be used as an anti-whitening agent. The testosterone 5α-reductase inhibitor of the present invention is also useful for the prevention or treatment of diseases involving male hormones such as acne and benign prostatic hyperplasia.

<CSF2 promoter of the present invention>
The CSF2 (colony stimulating factor 2) expression promoter of the present invention is characterized by using an Achinonogoshi extract as an active ingredient. As described above, the extract of Akinonogeshi can be used for a CSF2 (colony stimulating factor 2) expression promoter because it can promote the expression of colony stimulating factor 2 (CSF2). In addition, the Achinonokeshi extract used for the CSF2 promoter of the present invention is the same as that described in the testosterone 5α-reductase inhibitor of the present invention.
In the CSF2 expression promoter of the present invention, the content of the Achinonogoshi extract is preferably 10 to 50 μg / ml from the viewpoint of exerting an action of promoting the expression of CSF2.

<VEGF expression promoter of the present invention>
The VEGF expression promoter according to the present invention is characterized by using an Achinonogoshi extract as an active ingredient. The present inventor has found that the extract of Akinonogeshi exhibits a VEGF expression promoting action. Therefore, the Akinonogushi extract is suitable for use as a VEGF expression promoter. In addition, the Achinonokeshi extract used for the VEGF expression promoter of the present invention is the same as that described in the testosterone 5α-reductase inhibitor of the present invention.
In addition, VEGF is a vascular endothelial growth factor (Vascular endothelial growth factor).
In the VEGF expression promoter of the present invention, the content of the extract of Akinonogoshi is preferably 10 to 50 μg / ml from the viewpoint of exerting an action of promoting the expression of VEGF.

<ASIP expression inhibitor of the present invention>
The ASIP expression inhibitor of the present invention is characterized by using an Achinonogoshi extract as an active ingredient. The present inventor has found that the extract of Akinonogeshi exhibits an ASIP expression inhibitory action. Therefore, the extract of Akinonogeshi is suitable for use as an ASIP expression inhibitor. In addition, the Achinonokeshi extract used for the ASIP expression inhibitor of the present invention is the same as that described in the testosterone 5α-reductase inhibitor of the present invention.
Note that ASIP is an agoti signaling protein and is a paracrine signal molecule made of hair follicles. The melanocytes of hair follicles synthesize pheomelanin by antagonizing eumelanin.
In the ASIP expression inhibitor of the present invention, the content of the extract of Akinonogoshi is preferably 0.4 to 50 μg / ml from the viewpoint of exerting an action of inhibiting the expression of ASIP.

<Hair growth promoter of the present invention>
The hair growth-promoting agent of the present invention is characterized by comprising an Achinonogoshi extract as an active ingredient. The present inventor has found that the extract of Akinonogeshi exhibits a hair growth promoting action. Therefore, the Akinonage extract is suitable for use in a hair growth promoter. In addition, the Achinonogeshi extract used for the hair growth promoter of the present invention is the same as that described in the testosterone 5α-reductase inhibitor of the present invention.
In the hair growth-promoting agent of the present invention, the content of the above-mentioned Achinonogeshi extract may be 0.0001% by mass or more from the viewpoint of improving the hair-growth promoting effect, and may be 0.0001-10% by mass. Preferably, it is 0.0005-8 mass%, It is more preferable that it is 0.0005-5 mass%.

<Composition that imparts gloss to hair of the present invention, composition that imparts hair elasticity, composition that imparts firmness to hair, composition that softens hair, and composition that retains moisture in hair>
The composition for imparting gloss to the hair of the present invention, the composition for imparting hair elasticity, the composition for imparting firmness to the hair, the composition for suppleness of the hair, and the composition for retaining the moisture of the hair are extracted with Akinonageshi It is characterized by containing a product, and preferably an extract of Akinonogeshi is used as an active ingredient. The present inventor has found that, by containing an Akinonogoshi extract, the hair can be given luster, beam and stiffness, and the hair can be supple and retain moisture. Therefore, the Akinonogushi extract is suitable for use in compositions intended for these effects. In addition, the Achinonokeshi extract used for these compositions is the same as that described in the testosterone 5α-reductase inhibitor of the present invention.
The content of the extract of Akinonogeshi in these compositions should just contain 0.0001 mass% or more, it is preferable that it is 0.0001-10 mass%, and it is 0.0005-8 mass%. More preferably, it is 0.0005-5 mass% especially preferable.

  Testosterone 5α-reductase inhibitor, VEGF expression promoter, ASIP expression inhibitor, CSF2 expression promoter, hair growth promoter, composition for imparting gloss to hair, composition for imparting hair tension, hair As a composition for imparting hair, a composition for supple hair, and a composition for retaining moisture of hair, in addition to the extract of Akinonageshi, a combination agent commonly used in the food, cosmetic and pharmaceutical industries, for example, an excipient , Moisturizer, antiseptic, strengthening agent, thickener, emulsifier, antioxidant, sweetener, acidulant, seasoning, colorant, fragrance, whitening agent, moisturizer, oil component, UV absorber, surfactant Alcohols, powder components, colorants, aqueous components, water, various skin nutrients, and the like can be appropriately blended depending on the purpose. As these compounding agents, commercially available products can be suitably used.

  Testosterone 5α-reductase inhibitor, VEGF expression promoter, ASIP expression inhibitor, CSF2 expression promoter, hair growth promoter, composition for imparting gloss to hair, composition for imparting hair tension, hair A composition for imparting hair, a composition for supple hair, and a composition for retaining moisture of hair can be prepared by mixing an Akinonage extract and various compounding agents appropriately selected as necessary.

  Testosterone 5α-reductase inhibitor, VEGF expression promoter, ASIP expression inhibitor, CSF2 expression promoter, hair growth promoter, composition for imparting gloss to hair, composition for imparting hair tension, hair The composition for imparting hair, the composition for supple hair and the composition for retaining moisture of the hair are used in the form of a skin external preparation such as a solution, dispersion, emulsion, ointment, cream, gel, aerosol, pack, etc. It is particularly preferable to use it in the form of a scalp external preparation. These can also be suitably used as hair cosmetics such as shampoos and rinses.

  In addition, due to the action of the extract of Akinonogoshi as described above, various uses as quasi-drugs are expected. Efficacy effects such as hair promotion and hair restoration can be expected.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

<< Activation activity of dermal papilla cells and change in gene expression level in dermal papilla cells >>
1. 1. Material, sample preparation and test method 1-1. Test substance Akinonage extract manufactured by Sakamoto Bio Inc. was used. In addition, the Akinonogoshi extract is prepared by extracting 12.6 kg of dry raw material of whole Chinese Achinonomushi at room temperature using 99% ethanol of 10 times mass, and then performing filtration and concentration. (Yield: 0.22 kg).

1-2. Cell Human hair papilla cells (HFDPC, catalog No. 602-05a, lot No. 1710, Caucasian 40-year-old female origin) manufactured by CELL APPLICATION obtained from Toyobo Co., Ltd. were used.

1-3. Reagents and instruments, etc. (i) Papilla cell growth medium (hereinafter abbreviated as PCGM) (manufactured by Toyobo)
(Ii) Phosphate buffered saline (hereinafter abbreviated as PBS) (manufactured by Sigma)
(Iii) Trypsin EDTA (manufactured by Sigma)
(Iv) Collagen coating solution (Toyobo Co., Ltd.)
(V) RNeasy Mini Kit (Qiagen)
(Vi) 37 ° C. CO ₂ incubator (manufactured by ESPEC)
(Vii) Plate reader (VARIOSKAN, manufactured by Thermo Electron)
(Viii) 75 cm ² culture flask (hereinafter referred to as T75 flask) (manufactured by IWAKI)
(Ix) 6-well culture plate (IWAKI)
(X) 96-well culture plate (IWAKI)
(Xi) Microscope (OLYMPUS, IX-70)

1-4. Sample preparation Akinonage extract (test substance) was suspended in PCGM so as to be 10 mg / mL, then treated with a 0.2 μm filter, serially diluted with PCGM, and used for the test.

1-5. Test Method 1-5-1. Preparation of cells (i) Collagen coat solutions were placed in a T75 flask, 6-well culture plate, and 96-well culture plate, respectively, 1 ml, 0.5 ml, and 50 μl, and allowed to stand for 2 hours.
(Ii) The collagen coat solution was removed, washed twice with PBS, the flask and plate were sealed, and stored in a refrigerator until use.
(Iii) A collagen-coated T75 flask was seeded with HFDPC (P5) and cultured in a 5% CO ₂ incubator at 37 ° C. for 4 days.
(Iv) After washing the T75 flask with PBS, HFDPC was suspended by trypsin treatment to prepare a cell suspension of 1.2 × 10 ⁵ cells / mL, and then a collagen-coated 6-well culture plate, 96 1 ml and 0.1 ml of the cell suspension were placed in a well culture plate, respectively, and cultured overnight in a 5% CO ₂ incubator at 37 ° C.

1-5-2. Measurement of cell activation activity (i) According to the above “1-5-1. Preparation of cells”, the medium of HFDPC cultured in a 96-well culture plate was removed, and then washed twice with PBS.
(Ii) Samples having a concentration of Akinonage extract prepared according to the above “1-4. Sample preparation” of 300, 100, 33, 11, 3.7 μg / ml were added to HFDPC in a 96-well culture plate.
(Iii) After 24 hours of culture, the 96-well culture plate was washed twice with PBS.
(Iv) Cell Counting Kit-8 solution (manufactured by Dojindo Laboratories) diluted to 30 times volume with serum-free Dulbecco's modified Eagle's medium (DMEM) (manufactured by Sigma-Aldrich) at 100 μl / well in a 96-well culture plate Added.
(V) The sample was allowed to stand in a 5% CO ₂ incubator at 37 ° C. to cause appropriate color development, and then the absorbance at 450 nm was measured.
(Vi) Based on the obtained data, the ratio to the control (% control) was calculated according to the following formula.
% Control = [(data _sample −data _blank ) / (data _control −data _blank )] × 100

1-5-3. Cell recovery, RNA extraction (i) According to the above “1-5-1. Preparation of cells”, the hair papilla cells cultured in a 6-well culture plate were washed twice with PBS. A sample having a concentration of the extract of Akinonogoshi prepared according to “Preparation” of 3.7 μg / ml was added to a 6-well culture plate and cultured for 24 hours.
(Ii) After the culture, the 6-well culture plate was washed twice with ice-cooled PBS, and then the cell lysis buffer attached to the RNeasy Mini Kit was added. Subsequently, RNA was extracted according to the instruction of RNeasy Mini Kit.
(Iii) After extraction, the sample was immediately immersed in liquid nitrogen, frozen, and stored in a deep freezer.

1-5-4. Microarray Microarray analysis was performed at Miltenyi Biotech.

1-6. Analysis of results From the results of microarray analysis, genes expressed in hair papilla cells were extracted, and changes in the expression level of the genes were examined.

2. Results and discussion 2-1. Cell activation effect In order to determine the concentration of the extract of Akinonokeshi when performing microarray analysis, the activation activity of hair papilla cells by various concentrations of the extract of Akinonokeshi was examined. Table 1 shows the activation activity of dermal papilla cells when a sample containing the extract of Achinonogushi at each concentration was added. Since the highest concentration of cell activation activity was 3.7 μg / ml, the concentration in the microarray analysis was set to 3.7 μg / ml.

2-2. Microarray analysis Based on the results of microarray analysis of changes in gene expression in dermal papilla cells treated with Achinonomushi extract, the fold change (fold change) of SRD5A2 was observed in dermal papilla cells treated with Achinonogesi extract. ) Indicates −3.48, which indicates that the expression level of SRD5A2 is reduced. Moreover, the magnification change of CSF2 has shown 2.74, It turned out that the expression level of CSF2 increases in the hair papilla cell processed with the Achinonogeshi extract.

(References)
1. Stenn KS and Paus R, Control of hair follicle cycling, 2001, Physiol. Rev., 81, 449-494.
2. Satomi Itami, Onset mechanism and treatment strategy for male pattern baldness, 2004, Inflammation / Regeneration, 24, 118-120 (References for review of male pattern baldness)
3. Costin GE & Hearing VJ, Human skin pigmentation melanocytes modulate skin color in response to stress. 2007, FASEB J., 21, 976-994.

<< Evaluation of hair-growth promoting effect of Akinonogushi extract on mouse coat >>
1. Test substance Akinonage extract manufactured by Sakamoto Bio Inc. was used. In addition, the Akinonogoshi extract is prepared by extracting 12.6 kg of dry raw material of whole Chinese Achinonomushi at room temperature using 99% ethanol of 10 times mass, and then performing filtration and concentration. (Yield: 0.22 kg).

2. Test Contents 1) Experimental Animals (1) Donor Animals Male mice (C57BL / 6J strain) produced at Nippon SLC Co., Ltd. were used.
(2) Age of acquisition and number of animals 25 male C57BL / 6J mice aged 7 weeks were received.
(3) Age at start of study 8 weeks of age

2) Animal breeding management (1) Breeding method (i) Breeding environment
Acclimatization period Breeding period: 6 days Lighting time: 12 hours (8: 00-20: 00)
Cage: Polycarbonate flat bottom cage (W182 x D260 x H128mm)
Flooring: Wood chip accommodation: 2 to 3 animals were housed in a cage.
Test period Breeding period: 30 days Lighting time: 12 hours (8: 00-20: 00)
Cage: Polycarbonate flat bottom cage (W182 x D260 x H128mm)
Flooring: Wood chip accommodation: 2 to 3 animals were housed in a cage.
(Ii) Grouping of animals After the habituation period, healthy animals were divided into groups so that their body weights were almost uniform, and were used for the test.
(2) Feed From the start of the acclimatization period to the end of the test period, MF solid feed (Oriental Yeast Co., Ltd.) was freely ingested.
(3) Drinking water Tap water was used for drinking. It was ingested freely from the start of the acclimatization period to the end of the test period.

3) Test outline Male C57BL / 6J mice after the acclimation period were divided into groups based on body weight values. The hair on the entire back surface of the mouse was removed using a clipper and a shaver, and the test substance was applied to the back from which hair was removed the next day for 29 days.

4) Test substance administration method (1) Route of administration Transdermal (application)
(2) Group structure

5) Preparation method and administration method of test substance suspension (1) Preparation method of test substance suspension 70 volume% ethanol was used as a solvent. First, after dissolving in pure ethanol, distilled water was added and mixed well to prepare a test substance suspension shown in Table 2.
(2) Method of administering test substance suspension A hair removal treatment was applied to the back of the mouse, and 200 μL of the test substance suspension was dropped onto the exposed skin using a micropipette. It was applied using a micropartel.

6) Hair removal method on the back of the mouse The whole back was removed using a clipper and a shaver under diethyl ether anesthesia.

7) Inspection method (1) Body weight The body weight was measured on the animal arrival date and the test start date (at the time of grouping) with an animal electronic balance.
(2) Hair growth score evaluation (visual / photo judgment)
On the test start day (day 0), on the 8th day, 15th day, 22nd day and 29th day after the start of the test, the entire back of the mouse before application of the test substance suspension was photographed. The back of the mouse was observed using photographs, the hair growth state (growing area) was confirmed by visual observation, and was evaluated in 6 stages based on the table below. The evaluation was made with the average of the scores of the three people as the final evaluation in order to provide objectivity.

3. Statistical processing The obtained numerical values were averaged for each group. In addition, a Mann-Whitney U test (Wilcoxon rank sum test) was performed between the two groups of the control group and the other groups. Significance levels were 5% and 1%.

4). Results The results of hair growth score evaluation are shown in FIG. Moreover, regarding the mice belonging to each group, photographs of the back part of the test week 0 (day 0) and the test week 4 (day 29) are shown in FIGS.
In the 3% Akinonogeshi extract group, the hair growth scores in the second week of the test (day 15), the third week of the test (day 22) and the fourth week of the test (day 29) are significant compared to the control group. Showed a high value.
From the results, it was suggested that the hair growth-promoting effect of the Achinonogeshi extract.

<< Effects of Akinonogeshi extract on hair-growth / anti-white hair-related genes >>
1. Akinonokeshi extract The Akinonogeshi extract described in “1-1. Test substance” in “Activation activity of hair papilla cells and change in gene expression level in hair papilla cells” was used.

2. Cells Human hair papilla cells described in “1-2. Cells” of “Activation activity of hair papilla cells and change in gene expression level in hair papilla cells” described above were used.

3. Reagents and instruments, etc. The reagents and instruments described in “1-3. Reagents and instruments” of “Activation activity of hair papilla cells and changes in gene expression level in papilla cells” described above were used.

4). Preparation of test substance Akinonage extract was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mg / mL and diluted 200-fold with PCGM to obtain a DMSO concentration of 0.5% by volume and an Achinonogeni extract concentration of 50 μg / mL. A test substance was prepared. Similarly, test substances (DMSO concentration of 0.5 vol%) having a concentration of Akinonogushi extract of 10, 2, and 0.4 μg / mL were also prepared.

5). Test method 5-1. Cell culture (i) 7.5 mL of a collagen coat solution was placed in a 75 cm ² flask, and the flask was gently shaken so that the solution spread over the entire flask culture surface.
(Ii) After standing at room temperature for about 1 hour, the collagen coat solution was removed by suction and washed twice with 7.5 mL of PBS.
(Iii) HFDPC was cultured by PCGM using a collagen-coated 75 cm ² flask in a 37 ° C., 5% CO ₂ incubator.
(Iv) Cells suspended by trypsin treatment were added at 1.0 × 10 ⁵ cells / well to each well of a 6-well plate that was collagen-coated (collagen-coated solution 1 mL / well, PBS-washed 1 mL / well) as in the flask. Sowing.
(V) Pre-cultured for 24 hours in a CO ₂ incubator.
(Vi) After removing the medium from each well, 2 mL of a test substance having a concentration of Akinonogushi extract of 0.4, 2, 10, 50 μg / mL was added and cultured in a CO ₂ incubator for 24 hours (N = 3). In addition, 0.5 volume% DMSO containing PCGM which does not contain an Achinonokeshi extract was added to control.

5-2. Recovery of RNA, synthesis of cDNA and measurement of gene expression level After culture, the culture supernatant is removed, and RNA is recovered from hair papilla cells using RNeasy mini kit (manufactured by QIAGEN), and then Quantitect Reverse Transcribion kit (QIAGEN) is used to synthesize cDNA from the recovered RNA. Using the obtained cDNA, the mRNA expression level of each gene is measured.

6). Results The results are shown in FIGS. FIG. 8 shows the relative expression level of CSF2 in dermal papilla cells, FIG. 9 shows the relative expression level of SRD5A2 in dermal papilla cells, FIG. 10 shows the relative expression level of ASIP in dermal papilla cells, and FIG. Indicates the relative expression level of VEGF in dermal papilla cells. 8 to 11, the vertical axis represents the relative expression level of each gene, and the horizontal axis represents the concentration of Akinonage extract contained in the medium (unit: μg / mL).

  In FIG. 8, the relative expression level at 0.4 μg / ml is 0.84, the relative expression level at 2 μg / ml is 0.72, and the relative expression level at 10 μg / ml is 1. 63, and the relative expression level at 50 μg / ml is 6.39. In FIG. 9, the relative expression level at 0.4 μg / ml is 0.63, the relative expression level at 2 μg / ml is 0.67, and the relative expression level at 10 μg / ml is 0.8. 80, and the relative expression level at 50 μg / ml is 0.46. In FIG. 10, the relative expression level at 0.4 μg / ml is 0.87, the relative expression level at 2 μg / ml is 0.91, and the relative expression level at 10 μg / ml is 0.8. 60, and the relative expression level at 50 μg / ml is 0.40. In FIG. 11, the relative expression level at 0.4 μg / ml is 0.88, the relative expression level at 2 μg / ml is 0.88, and the relative expression level at 10 μg / ml is 1. The relative expression level at 50 μg / ml is 1.61.

<< Effects of Akinonogeshi extract on hair >>
1. Akinonokeshi extract The Akinonogeshi extract described in “1-1. Test substance” in “Activation activity of hair papilla cells and change in gene expression level in hair papilla cells” was used.

2. Preparation of Composition Containing Akinonage Extract According to the formulation shown in Table 4, a composition containing Akinonage extract was prepared. Specifically, to the aqueous phase in which the surfactant was dissolved, an oil phase in which Aquinonage extract was dissolved in ethanol and diethyl sebacate was added, stirred and emulsified, and then a thickener and a neutralizing agent were added.

  The composition obtained above is used twice a day for the scalp and hair, and a questionnaire for the change of the hair before and after use is conducted to 4 users, and the state of the hair before use is set to 3 in 5 stages. Evaluation (Table 5). The average value of 4 people was calculated and used as the evaluation results before and after use (Table 6).

  From Table 6, it turned out that it becomes a supple hair with a gloss, a stickiness, and a firmness by using the composition containing the extract of Akinonogeshi. Further, it was found that there was an effect of suppressing the hairiness of the hair, and the moisture of the hair could be retained. Moreover, in the results of continuous use for 2 months, the glossiness of the hair, the stiffness of the hair, and the lack of hairiness are maintained at a high average value. It was found that it was possible to maintain hair with no hairiness.

Claims (8)

  A testosterone 5α-reductase inhibitor comprising an extract of Akinonogoshi as an active ingredient.   A hair growth-promoting agent comprising an extract of Akinonogeshi as an active ingredient. A composition for imparting gloss to hair, characterized by comprising an Achinonogoshi extract as an active ingredient . A composition for applying a hair patch characterized by comprising an Achinonogoshi extract as an active ingredient . A composition for imparting stiffness to hair, characterized by comprising an Achinonogoshi extract as an active ingredient . A composition for suppleening hair, characterized by comprising an Achinonogoshi extract as an active ingredient . A composition for retaining moisture of hair, characterized by comprising an Achinonogoshi extract as an active ingredient . A composition for moisturizing hair, characterized by comprising an Achinonogeshi extract as an active ingredient.