JP2023149544A - TESTOSTERONE 5α-REDUCTASE ACTIVITY INHIBITORS, ANTI-ANDROGENIC AGENTS, HGF MRNA EXPRESSION PROMOTING AGENTS, HGF PROTEIN PRODUCTION PROMOTING AGENTS AND KAP5.1 MRNA EXPRESSION PROMOTING AGENTS - Google Patents

Abstract

To provide testosterone 5α-reductase activity inhibitors, anti-androgenic agents, HGF mRNA expression promoting agents, HGF protein production promoting agents and KAP5.1 mRNA expression promoting agents by finding those having testosterone 5α-reductase inhibiting activity, anti-androgenic activity, HGF mRNA expression promoting activity, HGF protein production promoting activity and KAP5.1 mRNA expression promoting activity from compositions derived from highly safe natural products and using them as effective ingredients.SOLUTION: The testosterone 5α-reductase activity inhibitors, anti-androgenic agents, HGF mRNA expression promoting agents, HGF protein production promoting agents and KAP5.1 mRNA expression promoting agents contain a ginkgo leaf extract as an effective ingredient.SELECTED DRAWING: None

Description

The present invention relates to a testosterone 5α-reductase activity inhibitor, an anti-androgen, an HGF mRNA expression promoter, an HGF protein production promoter, and a KAP5.1 mRNA expression promoter.

Most steroid hormones are molecular forms secreted from the producing organ and bind to receptors to express their effects. However, in the case of male hormones collectively known as androgens, for example, testosterone enters the cells of the target organ. After being reduced to 5α-dihydrotestosterone (5α-DHT) by testosterone 5α-reductase, it binds to a receptor and exerts its action as an androgen.

Androgens are important hormones, but if they act excessively, they can cause various problems such as male pattern baldness, hirsutism, seborrhea, acne, benign prostatic hyperplasia, prostate tumors, and premature male puberty. It induces no symptoms. Therefore, in order to improve these various symptoms, there is a method to suppress the action of excessive androgen, specifically, by inhibiting the action of testosterone 5α-reductase, which reduces testosterone to the active form 5α-DHT. There are known methods for suppressing the production of 5α-DHT and methods for preventing the expression of androgenic activity by inhibiting the binding of 5α-DHT produced from testosterone to receptors.

Conventionally, as substances having an inhibitory effect on testosterone 5α-reductase activity, extracts of Aedes albopictus (see Patent Document 1), plants belonging to the Quercus genus or extracts thereof (see Patent Document 2), and the iris plant Hiruginosa or its rhizomes have been used. A dried product of (see Patent Document 3) and the like are known.

Hair repeatedly grows and falls out according to a cyclical hair cycle consisting of a growth phase, a regression phase, and a resting phase. Of this hair cycle, the stage from the telogen phase to the anagen phase, when new hair follicles are formed, is considered to be the most important for hair growth. Dermal papilla cells are thought to play an important role in the proliferation and differentiation of hair follicle epithelial cells at this stage. Dermal papilla cells are cells located inside the hair follicle epithelial cells, which are composed of outer root sheath cells and matrix cells, near the hair root, and are located in the root part of the hair root, which is surrounded by a basement membrane. It acts on epithelial cells and promotes their proliferation, playing an important role in the differentiation into hair (see Non-Patent Document 1).

Hepatocyte growth factor (HGF) was discovered in the plasma of patients with fulminant hepatitis as a factor that promotes the proliferation of hepatocytes. HGF is known to promote the proliferation of not only the liver but also epithelial cells, endothelial cells, hematopoietic cells, etc. derived from various organs. It has been reported that the blood concentration of HGF increases in various diseases such as liver disease, pneumonia, leukemia, cancer, and myocardial infarction. Furthermore, HGF has been recognized to have the effect of suppressing organ damage and fibrosis and promoting regeneration. Patent Document 4 discloses a technique of using HGF as an active ingredient of a pulmonary fibrosis preventive agent.

Furthermore, it has been shown that HGF is expressed in dermal papilla cells of hair follicles (see Non-Patent Document 2), and it has been revealed that HGF has a hair growth effect through the activation of hair roots. Until now, rice and the like have been known as plant extracts that have an effect of promoting HGF production (see Patent Document 5).

In addition to problems related to hair, such as those related to the condition of the hair roots and follicles such as hair loss and thinning, hair quality (hair quality) such as hard, soft, thin, lack of elasticity, split ends, frizzy hair, etc. There are things related to this. Furthermore, problems related to hair quality include those caused by damage to the hair due to daily hair care, hair makeup, UV exposure, etc., and those caused by problems in hair shaft formation. factors are involved.

BACKGROUND ART Conventionally, in order to improve the thinness and lack of elasticity and stiffness of hair, it has been considered to use, for example, hair restorers and hair tonics containing active ingredients for hair growth. However, when using hair restorers, hair tonics, etc., there is a problem in that the hair does not become thicker and the effect of providing firmness and stiffness cannot be expected. In order to improve these problems, a method has been proposed in which a silanol compound produced by hydrolysis of alkoxysilane is permeated into the hair and polymerized inside the hair (see Patent Document 6). However, this method of modifying hair using chemical substances was not sufficient in terms of imparting natural strength and stiffness to hair.

On the other hand, attempts have been made to improve hair quality biochemically and molecularly biologically. Hair is composed of a cuticle that covers its surface, a fur substance (cortex) inside the cuticle, and a medulla that occupies the center of the hair. Among these, it is known that the cuticle that covers the surface of hair plays an important role in hair damage (see Non-Patent Document 3). Therefore, it is believed that if the cuticle can be effectively regenerated, it will be possible to improve hair quality such as hair hardness, elasticity, and stiffness.

Recently, it has been revealed that there is a correlation between the mRNA expression level of the KAP5 family gene among keratin-associated proteins (KAPs) and the strength of hair stiffness (see Patent Document 7). ). It has also been reported that KAP5.1, a member of the KAP5 family, is localized in the cuticle during the growth process (see Non-Patent Document 4). Therefore, if the expression of the KAP5 family, particularly KAP5.1 mRNA, can be promoted, it is expected to lead to regeneration of the cuticle and improvement of hair quality such as hair hardness, elasticity, and stiffness.

JP2008-088076A Japanese Patent Application Publication No. 2003-238435 Japanese Patent Application Publication No. 09-301884 Japanese Patent Application Publication No. 2006-131649 Japanese Patent Application Publication No. 2004-99503 Japanese Patent Application Publication No. 2005-320314 Japanese Patent Application Publication No. 2006-014721

"Trends Genet", Volume 8, 56-61 (1992) Anti-Aging Series (1), Facts about gray hair, hair loss, and hair growth, NTS, 1-18 (2005) Journal of the Japan Cosmetic Engineers Association, Volume 36, No. 3, 207-216 (2002) Int. J. Trichology, 2(2), 89-95 (2010)

The present invention has discovered, among compositions derived from highly safe natural products, that have an effect of inhibiting testosterone 5α-reductase activity, an effect of promoting HGF mRNA expression, an effect of promoting HGF protein production, and an effect of promoting KAP5.1 mRNA expression. The object of the present invention is to provide a testosterone 5α-reductase activity inhibitor, an anti-androgen agent, an HGF mRNA expression promoter, an HGF protein production promoter, and a KAP5.1 mRNA expression promoter as active ingredients.

In order to solve these problems, the present invention provides a testosterone 5α-reductase activity inhibitor and an anti-androgen agent containing ginkgo biloba extract as an active ingredient.

The present invention also provides an HGF mRNA expression promoter containing ginkgo biloba extract as an active ingredient.

The present invention also provides an HGF protein production promoter containing ginkgo biloba extract as an active ingredient.

The present invention also provides a KAP5.1 mRNA expression promoter containing ginkgo biloba extract as an active ingredient.

According to the present invention, a highly safe testosterone 5α-reductase activity inhibitor, an anti-androgen agent, an HGF mRNA expression promoter, an HGF protein production promoter, and a KAP5.1 mRNA expression promoter, which contain ginkgo biloba extract as an active ingredient. can be provided.

Embodiments of the present invention will be described.
The testosterone 5α-reductase activity inhibitor, anti-androgen agent, HGF mRNA expression promoter, HGF protein production promoter, and KAP5.1 mRNA expression promoter according to the present embodiment contain ginkgo biloba extract as an active ingredient.

The extraction raw material as an active ingredient in this embodiment is Ginkgo biloba (scientific name: Ginkgo biloba L.) leaves.

Ginkgo biloba (scientific name: Ginkgo biloba L.) is a deciduous tree belonging to the genus Ginkgo in the Ginkgo family that grows naturally in Hokkaido, Honshu, Shikoku, Kyushu, etc., and can be easily obtained from these areas. The constituent part of Ginkgo biloba used as an extraction raw material is the leaf part.

Although the details of the substance contained in ginkgo biloba extract that inhibits testosterone 5α-reductase activity, promotes HGF mRNA expression, promotes HGF protein production, and promotes KAP5.1 mRNA expression are unknown, it is generally used for plant extraction, etc. Using the disclosed extraction method, an extract having the activity of inhibiting testosterone 5α-reductase activity, promoting HGF mRNA expression, promoting HGF protein production, and promoting KAP5.1 mRNA expression can be obtained from ginkgo leaves. Note that extracts include extracts obtained from extraction raw materials through extraction processing, diluted or concentrated extracts, dried products obtained by drying extracts, or any of these crude or purified products. is included.

The above-mentioned extract can be obtained by drying the extraction raw material and then pulverizing it as it is or using a coarse pulverizer and subjecting it to extraction with an extraction solvent. Drying may be performed in the sun or using a commonly used dryer. Alternatively, it may be used as an extraction raw material after being subjected to pretreatment such as degreasing with a nonpolar solvent such as hexane. By performing pre-treatment such as degreasing, extraction treatment of plants with a polar solvent can be efficiently performed.

Examples of the solvent used for extraction include water, a hydrophilic organic solvent, or a mixture thereof, and it is preferable to use it at room temperature or a temperature below the boiling point of the solvent. Components contained in the extraction raw material that inhibit testosterone 5α-reductase activity, promote HGF mRNA expression, promote HGF protein production, and promote KAP5.1 mRNA expression can be easily extracted by an extraction process using a polar solvent as the extraction solvent. I can do it.

Examples of water that can be used as an extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and those obtained by subjecting these to various treatments. Examples of treatments applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. Therefore, water that can be used as an extraction solvent in the present invention includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; - Polyhydric alcohols having 2 to 5 carbon atoms, such as butylene glycol, propylene glycol, and glycerin.

When a mixture of two or more polar solvents is used as an extraction solvent, the mixing ratio can be adjusted as appropriate. For example, when using a mixed solution of water and lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by volume of the lower aliphatic alcohol to 10 parts by volume of water. When using a mixed solution of water and a lower aliphatic ketone, it is preferable to mix 1 to 40 parts by volume of the lower aliphatic ketone to 10 parts by volume of water. When using a mixed solution of water and polyhydric alcohol, it is preferable to mix 1 to 90 parts by volume of polyhydric alcohol to 10 parts by volume of water.

In the extraction process, there is no need to employ a special extraction method as long as the soluble components contained in the extraction raw material can be eluted into the extraction solvent, and extraction can be performed at room temperature or under reflux heating. For example, by putting the extraction raw material into a processing tank filled with an extraction solvent, stirring if necessary, leaving it to stand for 30 minutes to 4 hours to elute the soluble components, and then filtering to remove solids. Extracts can be obtained. A paste-like concentrate is obtained by distilling off the extraction solvent from the obtained extract, and a dry product is obtained by further drying this concentrate. Extraction conditions are 50 to 95°C for about 1 to 4 hours when water is used as the extraction solvent. Further, when a mixed solvent of water and ethanol is used as the extraction solvent, the time is about 30 minutes to 4 hours at 40 to 80°C.

The extract obtained as described above is diluted, concentrated, It may be subjected to treatments such as drying and purification.

The obtained extract can be used as it is as a testosterone 5α-reductase activity inhibitor, an anti-androgen, an HGF mRNA expression promoter, an HGF protein production promoter, and a KAP5.1 mRNA expression promoter, but the concentrated solution Alternatively, it is preferable to use a dried product. When obtaining a dried product, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity.

In addition, since ginkgo biloba leaves have a unique odor, it is possible to purify them for purposes such as decolorization and deodorization as long as this does not reduce their biological activity, but when adding them to cosmetics, Since it is not used in large quantities, there is no practical problem even if it remains unrefined. Purification can be performed, for example, by activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, etc.

The ginkgo biloba extract obtained as described above has an effect of inhibiting testosterone 5α-reductase activity, an effect of promoting HGF mRNA expression, an effect of promoting HGF protein production, and an effect of promoting KAP5.1 mRNA expression. It can be used as an active ingredient of a testosterone 5α-reductase activity inhibitor, an anti-androgen agent, an HGF mRNA expression promoter, an HGF protein production promoter, and a KAP5.1 mRNA expression promoter.

The testosterone 5α-reductase activity inhibitor, anti-androgen agent, HGF mRNA expression promoter, HGF protein production promoter, and KAP5.1 mRNA expression promoter according to the present embodiment may be formulated from ginkgo biloba extract. good.

The above extract can be formulated into any desired dosage form such as powder, granules, or liquid according to a conventional method using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent. can. In this case, as the auxiliary agent, for example, an excipient, a stabilizer, a flavoring agent, etc. can be used. Forms of the testosterone 5α-reductase activity inhibitor, anti-androgen agent, HGF mRNA expression promoter, HGF protein production promoter, and KAP5.1 mRNA expression promoter prepared from the above extract include, for example, ointment, external liquid, etc. can be mentioned.

The testosterone 5α-reductase activity inhibitor and anti-androgen agent according to the present embodiment can inhibit the activity of testosterone 5α-reductase through the inhibitory effect of the testosterone 5α-reductase activity possessed by the ginkgo biloba extract. This makes it possible to suppress the effects of androgens, which can be used to treat various diseases related to male hormones, such as androgenetic alopecia, hirsutism, seborrhea, acne, benign prostatic hyperplasia, and prostate tumors. , precocious male puberty, etc. can be prevented, treated, or improved. Therefore, ginkgo biloba extract can also be used as an active ingredient in hair growth cosmetics and the like. However, the testosterone 5α-reductase activity inhibitor and anti-androgen agent according to the present embodiment can be used for all other purposes in addition to these uses that have the effect of inhibiting testosterone 5α-reductase activity.

The HGF mRNA expression promoter and HGF protein production promoter in this embodiment can promote HGF mRNA expression and HGF protein production through the HGF mRNA expression promoting effect and HGF protein production promoting effect of the ginkgo biloba extract. This can, for example, promote the proliferation of epithelial cells, endothelial cells, hematopoietic cells, etc. derived from each organ, suppress organ damage and fibrosis, promote regeneration, and prevent alopecia, fibrosis, and liver diseases. , arteriosclerosis obliterans, etc. can be improved or treated. Therefore, ginkgo biloba extract can also be used as an active ingredient in hair growth cosmetics and the like. However, the HGF mRNA expression promoter and the HGF protein production promoter in this embodiment can be used for all purposes other than these applications that have significance in exerting the HGF mRNA expression promoting effect and the HGF protein production promoting effect.

The KAP5.1 mRNA expression promoter in this embodiment can promote the expression of KAP5.1 mRNA through the KAP5.1 mRNA expression promoting effect of the ginkgo biloba extract. Thereby, it is possible to regenerate the cuticle and improve hair quality such as hair hardness, elasticity, and stiffness. Therefore, ginkgo biloba extract can also be used as an active ingredient in hair quality improving agents and the like. However, the KAP5.1 mRNA expression promoter according to the present embodiment can be used for all other meaningful uses in which it exhibits a KAP5.1 mRNA expression promoting effect in addition to these uses.

In addition, the testosterone 5α-reductase activity inhibitor, anti-androgen agent, HGF mRNA expression promoter, HGF protein production promoter, and KAP5.1 mRNA expression promoter according to the present embodiment have excellent testosterone 5α-reductase activity inhibitory action, HGF mRNA expression promoter, and HGF mRNA expression promoter. Since it has an expression-promoting effect, an HGF protein production-promoting effect, and a KAP5.1 mRNA expression-promoting effect, it is suitable for inclusion in cosmetics such as skin cosmetics, scalp cosmetics, and hair cosmetics.

Examples of cosmetics that can contain testosterone 5α-reductase activity inhibitors, anti-androgen agents, HGF mRNA expression promoters, HGF protein production promoters, and KAP5.1 mRNA expression promoters include ointments, creams, milky lotions, lotions, and packs. , foundation, hair tonic, hair lotion, shampoo, conditioner, soap, etc. When compounding a testosterone 5α-reductase activity inhibitor, an anti-androgen agent, an HGF mRNA expression promoter, an HGF protein production promoter, and a KAP5.1 mRNA expression promoter into a cosmetic, the amount to be added depends on the type of cosmetic. Although it can be adjusted as appropriate, a suitable blending ratio is about 0.0001 to 10% by mass in terms of standard extract, and a particularly suitable blending ratio is about 0.0001 to 10% by mass in terms of standard extract. It is about 0.001 to 1% by mass. Cosmetics may contain base ingredients commonly used in the production of cosmetics, as long as they do not interfere with the testosterone 5α-reductase activity inhibition, HGF mRNA expression promotion, HGF protein production promotion, and KAP5.1 mRNA expression promotion effects of the above-mentioned extract. Auxiliary agents or other ingredients, such as astringents, bactericidal/antibacterial agents, whitening agents, ultraviolet absorbers, humectants, cell activators, anti-inflammatory/antiallergic agents, antioxidants/active oxygen scavengers, oils and fats, waxes , hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, etc. can be used in combination. By using them in combination in this way, the product becomes more general in use, and the synergistic effect with other active ingredients used in combination may bring about better effects than would normally be expected.

Note that the testosterone 5α-reductase activity inhibitor, anti-androgen agent, HGF mRNA expression promoter, HGF protein production promoter, and KAP5.1 mRNA expression promoter according to this embodiment are suitably applied to humans. However, it can also be applied to animals other than humans as long as the effects of inhibiting testosterone 5α-reductase activity, promoting HGF mRNA expression, promoting HGF protein production, and promoting KAP5.1 mRNA expression are achieved.

Hereinafter, the present invention will be explained in more detail by showing production examples, test examples, etc., but the present invention is not limited to the following production examples, test examples, etc. In addition, in the following test example, the ginkgo biloba leaf extract obtained in the production example was used as a test sample.

[Production Example] Production of Ginkgo Biloba Leaf Extract 150 mL of a 70% by volume ethanol solution was added to 10 g of dried Ginkgo biloba leaves, and the mixture was heated and extracted at 80 to 90° C. for 2 hours using a reflux extractor, followed by filtration. The residue was further subjected to the same extraction process. The obtained extracts were combined and concentrated under reduced pressure, and further dried in a vacuum drier to obtain a ginkgo biloba extract (2.5 g).

[Test Example 1] Test for inhibitory effect on testosterone 5α-reductase activity A test for inhibitory effect on testosterone 5α-reductase activity was conducted on ginkgo biloba extract by the following method.

In a V-bottomed test tube with a lid, 20 μL of 4.2 mg/mL testosterone prepared with propylene glycol and 5 mmol/mL Tris-HCl buffer (pH 7. 13) Mixed 825 μL. To this, 80 μL of a test sample solution prepared with 50% ethanol and 75 μL of S-9 (manufactured by Oriental Yeast Co., Ltd., rat liver homogenate) were added and mixed, and the mixture was incubated at 37° C. for 60 minutes. Thereafter, 1 mL of methylene chloride was added to stop the reaction. This was centrifuged (1600 x g, 10 minutes), the methylene chloride layer was separated, and the separated methylene chloride layer was subjected to gas chromatography analysis under the following conditions. - Dihydrotestosterone (5α-DHT) and testosterone concentrations were determined. As a control, the same amount (80 μL) of the sample solvent (50% ethanol) was used instead of the test sample solution, and the sample was treated in the same manner and subjected to gas chromatography analysis.

<Gas chromatography conditions>
Equipment used: Shimadzu GC-2014 (manufactured by Shimadzu Corporation)
Column: DB-1701 (inner diameter: 0.53 mm, length: 30 m, film thickness: 1.0 μm) (manufactured by J&W Scientific)
Column temperature: 240℃
Inlet temperature: 300℃
Detector: FID
Sample injection volume: 1μL
Split ratio: 1:2
Carrier gas: Nitrogen gas Carrier gas flow rate: 3mL/min

The concentrations of 3α-androstanediol, 5α-DHT, and testosterone were determined by the following method. Specifically, standard products of 3α-androstanediol, 5α-DHT, and testosterone were dissolved in methylene chloride, and the solution was subjected to gas chromatography analysis, and the concentration (μg/mL) and peak area of these compounds were determined. , the correspondence between the peak area and the concentration of the compound was determined in advance. Then, the concentrations per peak area of 3α-androstanediol, 5α-DHT, and testosterone after the reaction between testosterone and S-9 are determined based on the following formula (1) using the correspondence relationship determined in advance. I asked.

A=B×C/D...(1)
In the formula, "A" represents the concentration of 3α-androstanediol, 5α-DHT or testosterone, "B" represents the peak area of 3α-androstanediol, 5α-DHT or testosterone, and "C" , represents the concentration of the standard, and "D" represents the peak area of the standard.

Using the concentrations of 3α-androstanediol, 5α-DHT and testosterone calculated based on the above formula (1), the conversion rate (formed by reduction of testosterone by testosterone 5α-reductase) is determined based on the following formula (2). The concentration ratios between the concentrations of 3α-androstanediol and 5α-DHT and the initial concentration of testosterone were calculated.

Conversion rate = (E+F)/(E+F+G)...(2)
In the formula, "E" represents the concentration of 3α-androstanediol, "F" represents the concentration of 5α-DHT, and "G" represents the concentration of testosterone.

Using the conversion rate calculated based on the above formula (2), the inhibition rate (%) of testosterone 5α-reductase activity was calculated based on the following formula (3).

Testosterone 5α-reductase activity inhibition rate (%) = (1-H/I) x 100... (3)
"H" in the formula represents the conversion rate when a sample is added, and "I" represents the conversion rate when no sample is added.

The results of the above test are shown in Table 1.

As shown in Table 1, the ginkgo biloba extract was confirmed to have an excellent inhibitory effect on testosterone 5α-reductase activity.

[Test Example 2] HGF mRNA expression promoting effect test Ginkgo biloba leaf extract was tested for HGF mRNA expression promoting effect by the following method.

Normal human dermal papilla cells (HFDPC: derived from the top of the head) were seeded in a 60 mm petri dish, and cultured at 37° C. under 5% CO ₂ using a medium for human normal dermal papilla cells (DPCGM).

After culturing, 3 mL of the test sample dissolved in dermal papilla cell basic medium (DPCBM) containing no growth additive was added to each Petri dish, and cultured at 37° C. and 5% CO ₂ for 2 hours. As a control, DPCBM to which no sample was added was used and cultured in the same manner. After culturing, the medium was removed, total RNA was extracted using ISOGEN II (manufactured by Nippon Gene), the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was adjusted to 200 ng/μL.

Using this total RNA as a template, the expression levels of mRNA for HGF and GAPDH, which was an internal standard, were measured. Detection was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by Takara Bio Inc.), and PrimeScript ^TM RT Master Mix (Perfect Real Time) (manufactured by Takara Bio Inc.) and TB Green (registered trademark) Fast. The real-time 2-Step RT-PCR reaction was performed using qPCR Mix (manufactured by Takara Bio Inc.). The expression level of each mRNA was calculated by correcting the expression level of GAPDH mRNA. The HGF mRNA expression promotion rate (%) was calculated using the following formula.

HGF mRNA expression promotion rate (%) = A / B × 100
"A" in the formula represents the correction value when the test sample is added, and "B" represents the correction value when the test sample is not added.

The results of the above test are shown in Table 2. In addition, in the above formula, the HGF mRNA expression promotion rate without the addition of the test sample is 100%.

As shown in Table 2, the ginkgo biloba extract showed a high HGF mRNA expression promotion rate. From this result, it was confirmed that the ginkgo biloba extract has an excellent effect of promoting HGF mRNA expression.

[Test Example 3] HGF protein production promoting effect test Ginkgo biloba leaf extract was tested for HGF protein production promoting effect by the following method.

Normal human dermal papilla cells (HFDPC: derived from the top of the head) were seeded in a 60 mm petri dish, and cultured at 37° C. under 5% CO ₂ using a medium for human normal dermal papilla cells (DPCGM).

After culturing, 3 mL of the test sample dissolved in dermal papilla cell basic medium (DPCBM) containing no growth additive was added to each Petri dish and cultured at 37° C. and 5% CO ₂ for 72 hours. After culturing, the amount of HGF in the medium was quantified using Human HGF Quantikine ELISA Kit (manufactured by R&D Systems). From the obtained results, the HGF protein production promotion rate (%) was calculated using the following formula.

HGF protein production promotion rate (%) = A / B × 100
"A" in the formula represents the correction value when the test sample is added, and "B" represents the correction value when the test sample is not added.

The results of the above test are shown in Table 3. In addition, in the above formula, the HGF protein production promotion rate without the addition of the test sample is 100%.

As shown in Table 3, the ginkgo biloba extract showed a high HGF protein production promotion rate. From this result, it was confirmed that the ginkgo biloba extract has an excellent effect of promoting HGF protein production.

[Test Example 4] KAP5.1 mRNA expression promoting effect test Ginkgo biloba leaf extract was tested for KAP5.1 mRNA expression promoting effect by the following method.

Normal human neonatal epidermal keratinocytes (NHEK) were precultured using normal human epidermal keratinocyte growth medium (KGM) and collected by trypsinization. The collected cells were seeded in a 6-well plate using KGM at a cell density of 3.0×10 ⁵ cells/2 mL, and cultured overnight at 37° C. and 5% CO ₂ .

After culturing, the medium was replaced with a growth factor-free medium (KBM). After 24 hours, the culture medium was discarded, and 2 mL of the test sample dissolved in KBM to the required concentration was added to each well, and cultured at 37° C. and 5% CO ₂ for 24 hours. As a control, KBM to which no sample was added was used and cultured in the same manner. After culturing, the medium was removed, total RNA was extracted using ISOGEN II (manufactured by Nippon Gene), the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was adjusted to 200 ng/μL.

Using this total RNA as a template, the expression levels of mRNA for KAP5.1 and GAPDH as an internal standard were measured. Detection was performed using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by Takara Bio Inc.), and PrimeScript ^TM RT Master Mix (Perfect Real Time) (manufactured by Takara Bio Inc.) and TB Green (registered trademark) Fast. The real-time 2-Step RT-PCR reaction was performed using qPCR Mix (manufactured by Takara Bio Inc.). The expression level of KAP5.1 mRNA was calculated by correcting the expression level of GAPDH mRNA. The KAP5.1 mRNA expression promotion rate (%) was calculated using the following formula.

KAP5.1 mRNA expression promotion rate (%) = A / B × 100
"A" in the formula represents the correction value when the test sample is added, and "B" represents the correction value when the test sample is not added.

The results of the above test are shown in Table 4. In addition, in the above formula, the KAP5.1 mRNA expression promotion rate without the addition of the test sample is 100%.

As shown in Table 4, the ginkgo biloba extract showed a high rate of promotion of KAP5.1 mRNA expression. From this result, it was confirmed that the ginkgo biloba extract has an excellent effect of promoting KAP5.1 mRNA expression.

The testosterone 5α-reductase activity inhibitor, antiandrogen, HGF mRNA expression promoter, HGF protein production promoter, and KAP5.1 mRNA expression promoter of the present invention can be used as a component of cosmetics, etc., and further as a research reagent. It can be suitably used.

Claims (5)

A testosterone 5α-reductase activity inhibitor containing ginkgo biloba extract as an active ingredient. An anti-androgen agent containing ginkgo biloba extract as an active ingredient. An HGF mRNA expression promoter containing ginkgo biloba extract as an active ingredient. An HGF protein production promoter containing ginkgo biloba extract as an active ingredient. A KAP5.1 mRNA expression promoter containing ginkgo biloba extract as an active ingredient.