JP2002205922A - Prolongation agent for anagen of hair - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a prolongation agent for anagen of hair comprising a component having a prolonging effect on anagen of hair as an active ingredient. SOLUTION: This prolongation agent for anagen of hair comprises an extract of a plant of the genus Sophora of the family Leguminosae as an active ingredient. The prolongation agent for anagen of hair comprises maackiain and/or medicarpin as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scalp and hair preparation having an effect of extending the hair growth period.

[0002]

2. Description of the Related Art In a modern society, which is also referred to as an aging society or a stressed society, head hair is increasingly exposed to danger of hair loss from various causes. In response to this, various attempts have been made to provide a more excellent “hair restorer”. The main effects of the hair restorer on the hair are: hair growth inducing effect (hair growth promoting effect, growth period inducing effect), hair thickening effect, hair growth period prolonging effect, 5α-reductase inhibitory effect, blood circulation promoting effect,
Examples include a bactericidal effect, an anti-dandruff effect, a moisturizing effect, and an antioxidant effect.

[0003] As described above, despite various attempts, the hair loss preventing effect and the hair growth effect of the conventional hair tonics are not always sufficient. This is probably due to various causes of hair loss and the very complex mechanism of hair growth. The `` hair nourishing agent '' provided so far has been developed by capturing only a relatively rough concept of hair loss, in other words, simply and simply the phenomenon of `` hair loss '', and the one developed by focusing on the mechanism is focused on Not much.

[0004] As a major reason, it cannot be denied that a method for testing a hair restorer capable of simply testing the hair growth effect focusing on these mechanisms has not been sufficiently provided. In particular, it is difficult to establish a hair-growth test method for testing the effect of extending the hair growth period, and as a result, the hair restoration agents provided so far induce the hair into the growth period of the hair cycle and grow the hair. Many focused on the induction effect and 5α-reductase.

[0005]

Therefore, the present inventor has proposed:
A hair growth prolonging agent comprising a component having the above-described hair growth period prolonging effect as an active ingredient is found by using the established hair growth prolongation effect test method for testing the above-mentioned hair growth period prolonging effect which is established in vitro. I aimed for that.

[0006]

Means for Solving the Problems The present inventors diligently studied the components having the above-described effect of extending the hair growth period in various substances by using the hair growth drug assay method found by the present inventors. The present inventors have found that an extract of a plant belonging to the genus Clara belonging to the legume family has an effect of extending a desired hair growth period, thereby completing the present invention.

Further, the present inventors have found that substances in the extracts belonging to the genus Clara that can exert the desired hair growth period extending action include merchiain and medicarpi, which are a kind of pterocarpan derivatives.
n).

That is, the present inventor has proposed a hair growth period prolonging agent containing an extract of a plant belonging to the genus Clara ( Sophora L.) as an active ingredient, and a hair containing merchiain and / or medical pin as an active ingredient. The present invention provides a growth prolonging agent (hereinafter, the hair growth prolonging agent according to the present invention is also referred to as the present prolonging agent).

In the present invention, the "hair growth period extender"
Mainly, as an active ingredient, a component having an effect of prolonging the growth period of hair by maintaining or promoting at least the mitotic proliferation activity of hair follicle epithelial cells by the hair growth drug assay method described below, especially the above hair A hair-related drug that focuses on the effect of prolonging growth, so to speak
It has the feature as.

[0010] The "hair growth period extending agent" shortens the anagen phase due to, for example, slow growth of hair follicle epithelial cells in the vicinity of the root of the hair, and relatively reduces the telogen hair compared to the anagen hair. It is a particularly effective drug for alopecia caused by an increased ratio. Also, by using in combination with other hair restorers having individual effects, it is possible to achieve a comprehensive and synergistic effect in a wide range of alopecia. That is, the "hair growth period extending agent" has a use that is different from a general hair restorer application having the concept including the above-mentioned effects (1) to (4).

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described. A. Active ingredient of this extender The active ingredient of this extender is legume Clara ( Sophora L.)
It is an extract of a plant belonging to.

[0012] Plants belonging to the genus Clara of the legume family (hereinafter also referred to as plants of the genus Clara) are deciduous or evergreen trees, a few are perennial herbs, and are plants distributed from the tropical to warm temperate zones.

[0013] Specifically, as plants of the genus Clara, Clara (alias: Sophora.flavescens Ait.),
Enju (S. japonica L.) and the like. In particular, Clara extract is preferred as the active ingredient of the present extender. In addition, as long as the extract has the effect of extending the anagen phase in the hair cycle described below, even extracts of plant varieties of Clara genus, subspecies, etc., can be used as an active ingredient of the present extender. .

[0014] The extraction site of the plant of Clara genus is not limited, but it is particularly preferable to use the root portion as it is or without the perimeter. Extraction of an extract of a plant of the genus Clara can be performed by a method generally used for extracting an extract derived from a plant.

That is, it can be obtained by extracting an extract of a plant belonging to the genus Clara such as Clara, as it is or after drying it as necessary, and then subjecting it to solvent extraction as it is or after pulverization. As a solvent that can be used at this time, a common solvent used when extracting a component of a plant from a plant can be used, and is not particularly limited. For example, water, hot water; methanol, ethanol, and isopropanol , N-
Lower alcohols such as butanol; polyhydric alcohols such as propylene glycol and 1,3-butylene glycol;
Examples of hydrates of these alcohols include hydrocarbon solvents such as ethyl acetate, n-hexane, and toluene. Water is preferred, and hot water is more preferred.
When these extraction solvents are used, the resulting extract can be directly blended as an active ingredient of the present extender. It is also possible to once remove the extraction solvent and mix it after drying if necessary.

In addition to using the extract of the plant of the genus Clara extracted according to such an extraction step as an active ingredient of the present extender, it is also possible to use a commercial product. Furthermore, when examining the content of the extract of Clara sp., Which is the active ingredient of the present extender, the substances with the effect of extending the hair growth period in this extract are merchiain (maackiain) and medical pin (medicarpin). There was found.

Both components are pterocarpan
It is a derivative (hereinafter also referred to as a specific pterocarpan derivative) and is a kind of flavonoid (isoflavonoid) that exists only in leguminous plants, and is known to be contained in soybeans and alfalfa in addition to Clara plants. Have been.

[0018]

Embedded image

The steps for separating and purifying the specific pterocarpan derivative from the extract of the plant of the genus Clara and the test examples are described in the Examples section. The means for detecting the essential action of hair extracts or specific pterocarpan derivatives of Clara spp. On the hair, which is the action of maintaining or prolonging the anagen phase in the hair cycle, is appropriate for the detection method itself to be able to detect the action. is not particularly limited as far as it is such, even detection in vitro, it can be used detection in in vivo. However, in general, it is preferable to use an in vitro detection method in consideration of simplicity and effectiveness.

A detection method characterized by examining the proliferation effect of hair follicle epithelial cultured cells, which is one of the in vitro detection methods, will be briefly described below (more specifically, in Example 1). Described in.).

This detection method comprises the steps of: "contacting a hair follicle epithelial cell culture with a target substance in a serum-free medium and identifying the presence or absence and / or strength of the proliferation activity of the cell; Hair growth agent detection method for detecting the effect of prolonging the anagen phase in the cycle, that is, focusing on hair follicle epithelial cells that are directly related to hair elongation, This is an in vitro method for detecting a hair restorer, which detects the effect of prolongation.

This method for detecting a hair-growth agent comprises the steps of: isolating a hair follicle epithelial cell of an animal (including a human being; the same applies hereinafter) to a "hair follicle epithelial cell culture" obtained by isolating a target substance; It is performed by contacting and specifying the presence or absence and / or strength of the proliferation.

The hair follicle epithelial cells particularly refer to cells such as outer root sheath cells and matrix cells in the vicinity of the hair root, and the dermal papilla cells, which are inner mesenchymal cells, are excluded. The anagen phase in the hair cycle is exactly when the hair is growing, i.e., when the hair follicle epithelial cells are dividing and proliferating. is there.

[0024] The substance that prolongs the anagen phase in the hair cycle is a substance that, by its administration, maintains the division and proliferative activity of hair follicle epithelial cells, thereby preventing the hair from entering the catagen and telogen phases in the hair cycle. That is, it is a substance that continues to promote or maintain the growth of hair follicle epithelial cells.

[0025] As an in vitro detection method,
A method in which a target substance is allowed to act on hair papilla cells of an animal to detect its growth promoting effect can also be used. Also, in
In vivo detection methods include, for example, `` administering a target substance to a nude mouse, specifying the state of the hair growth site on the body surface of the nude mouse, and detecting the effect of extending the anagen phase in the hair cycle of the target substance. Hair growth agent detection method ", that is, the hair growth area and hair growth in a nude mouse with a characteristic hair growth that is hairless in principle, but whose hair growth site moves over time on its body surface A method for detecting the length of the anagen phase in the hair cycle by specifying the moving speed of the site can be cited.

B. Specific forms of the present prolonging agent As described above, the prolonging agent mainly uses the extract of a plant of the genus Clara or a specific pterocarban derivative as an active ingredient to mainly increase the effect of extending the anagen phase of the hair cycle. It is a hair-related drug that exerts a hair-growing effect when exerted.

The amount of the extract of the plant of the genus Clara in the present extender can be appropriately selected according to the specific form of the present extender, and is not particularly limited. 0.0% of the total extract
0005 to 20% by mass, preferably 0.01 to 10% by mass. When the amount is less than 0.00005% by mass of the extract as a dry matter of the whole preparation, the effect of extending the hair growth period and the effect of restoring hair based on the action of hair follicle cell proliferation are sufficiently exhibited. In addition, if the amount is more than 20% by mass, not only the effect corresponding to the increase in the amount is not expected to increase, but also the tendency to cause trouble in the preparation becomes remarkable.

The amount of the specific pterocarpan derivative in the present extender is not particularly limited as in the case of the extract of the plant of the genus Clara, but it is generally 1 × 10 ⁻¹⁰ to the present agent. -20% by mass, preferably 0.0% by mass
0001 to 10% by mass. If this compounding amount is less than 1 × 10 ^-10 % by mass with respect to the whole of the present agent, the effect of extending the hair growth period and the effect of nourishing hair based on the hair follicle cell proliferation effect will not be sufficiently exerted, and 20% by mass of the same. Even if the amount is more than that, the effect cannot be expected in proportion to the increase in the amount.

As described above, the present prolonging agent containing the specific pterocarpan derivative or the plant extract of the genus Clara as an active ingredient has a hair growth period extending effect based on an excellent hair follicle cell proliferation effect. Thus, for example, the growth of hair follicle epithelial cells in the vicinity of the hair root is slow, so that the anagen of the hair cycle is shortened, and the proportion of telogen hair is relatively higher than that of the anagen hair It is a particularly effective drug for alopecia caused by the above. In addition, by using in combination with other hair growth agents that have individual effects,
It is also possible to have a synergistic effect on certain alopecia.

The present extender may take a form as a hair restorer having an individual effect, and may be combined with other hair restorer components to form a “total hair restorer having a hair restore effect in various aspects. In some cases.

The dosage form that can be taken by the present extender is not particularly limited as long as it is a dosage form applicable to the outer skin, and for example, liquid, emulsion, ointment and the like can be selected. The form of the present extender is arbitrary, and for example, it can be in the form of a tonic, a hair cream, a mousse, a shampoo, a rinse, a cream, a milky lotion, a lotion, a pack and the like.

The present extender is generally used in cosmetics, quasi-drugs, pharmaceuticals, etc. in addition to plant extracts of the genus Clara as needed and as long as the desired effects of the present invention are not impaired. Various oily or aqueous components used. A humectant, a thickener, a preservative, an antioxidant, a fragrance, a coloring agent, various chemicals and the like can be compounded.

For example, higher fatty acids, solid paraffin, liquid paraffin, silicone oil, squalane, glyceryl monooleate, olive oil, isopropyl myristate, higher fatty acids, higher alcohols and other oils; glycerin, hyaluronic acid, propylene glycol, maltitol, Moisturizing agents such as atelocollagen and sodium lactate; thickeners such as quince, carboxyvinyl polymer and xanthan gum; vasodilators such as nicotinamide, benzyl nicotinate, vitamin E acetate, assembly extract, carpronium chloride and acetylcholine derivatives Amino acids such as serine, methionine and arginine; vitamins such as vitamin B6, vitamin E (or a derivative thereof), biotin, pantothenic acid (or a derivative thereof); nicotinic acid, nicotine Nicotinic acid esters such as methyl phosphate and tocopherol nicotinate; skin function enhancers such as cepharanthin; female hormones such as estradiol; anti-inflammatory agents such as glycyrrhetinic acid (or a derivative thereof); hinokitiol, hexachlorophen, benzalkonium chloride Antibacterial agents such as thiol, bitionol, etc .; cooling agents such as menthol; salicylic acid, zinc (or a derivative thereof), lactic acid (or an alkyl ester thereof); and organic acids such as citric acid. The specific formulation of the present extender will be described later.

[0034]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples and the like, but the technical scope of the present invention should not be construed as being limited by these Examples and the like. In the following Examples and the like, “%” is indicated and the content is indicated by mass% based on the compounding object and the like unless otherwise specified.
Means

First, an in vitro cell proliferation test for evaluating the effect of the plant extract used in this example and the like on the prolongation of the hair growth period will be described. [Test Example 1] Cell proliferation test using hair follicle epithelial cell culture cells Human hair follicle epithelial cells Collection of Human Hair Follicle Epithelial Cells Hair follicles during the anagen phase of the hair cycle were mechanically collected from a human male scalp obtained as a by-product of surgery under a stereoscopic microscope. The hair follicles during this growth period are dispensed at 1000 U / mL dispase
Dulbecco's modified MEM containing 0.2% collagenase
(DMEM) for 30 minutes at 37 ° C, and remove the dermal sheath, dermal papilla, and epithelium of the hair bulb using the tip of a syringe needle.
The cells were treated at 37 ° C. for 5 minutes with a phosphate buffer solution containing EDTA (PBS (−): (−) means no calcium or magnesium ions).

Next, the hair follicle was allowed to stand on a culture dish coated with collagen (Type I), and explant culture was performed. The medium used in this case was a serum-free medium (Keratinocyte Growth Me
dium (KGM)] (Keratinocyte Serum Free
Medium can also be used). After 4 to 5 days of this culture, when the attachment of the hair follicle to the culture dish and the proliferation of the cells were confirmed, the medium was replaced, and thereafter the medium was replaced every two days.

The cells grown in this way were
After treatment with 5% trypsin-0.02% EDTA at 37 ° C. for 5 minutes, the reaction was stopped with an equal volume of 0.1% trypsin inhibitor, and the cells were collected by centrifugation (800 × g, 5 minutes). did.

Next, the cells were suspended in the above-mentioned serum-free medium and seeded on a collagen-coated (Type I) culture dish at a density of 5000 cells / cm ² , and the cells were subconflued.
The medium was changed every two days until it reached
% Trypsin-0.02% EDTA at 37 ° C. for 5 minutes, the reaction was stopped with an equal amount of 0.1% trypsin inhibitor, and centrifugation (800 × g, 5 minutes) was performed. A cell frozen solution (Cell Banker: manufactured by Diatron) was added to the obtained human hair follicle epithelial cells,
After adjusting to a concentration of × 10 ⁶ cells / mL, 1.0 × 10 ⁶ cells were added to each cryotube, and this was cryopreserved. In addition, these cell numbers were calculated with a blood cell counting plate.

2. Assay of target substance The fibroblast contamination rate (FB contamination rate) of the hair follicle epithelial cells obtained by the above steps was measured (3000 times, 5 visual fields). As a result, if the FB contamination rate was 3% or more, the assay was performed. Was excluded from the target.

Then, the hair follicle epithelial cells were seeded in a culture flask, and then inoculated with 0.05% trypsin and 0.02%.
After treatment with 0.1% EDTA, the reaction was stopped with 0.1% trypsin inhibitor, the system was centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, 20 mL of KGM medium was added to the residue, and the cell suspension was added. Was prepared.

96 well-platform at a rate of 0.2 mL / well
e (Type I collagen coated plate: Falcon) (1.0 × 10 ⁴ cells / well), and allowed to stand at room temperature for about 20 minutes until the cells sank to the bottom of the well. Thereafter, the cells were cultured for 1 day at 37 ° C. and 5% CO ₂ to obtain desired human hair follicle epithelial cultured cells.

B. Rat hair follicle epithelial cells Collection of rat hair follicle epithelial cells: (1) Collection of hair follicle The back skin of a newborn (3-4 day old) rat was collected, and the collected back skin was immersed in PBS (-) containing 5% PSF. Then, the subcutaneous fat, skin, and the like below the skin fat layer were removed with scissors for dissection. Then, again this back skin was 1% PS
It is immersed in PBS (-) containing F, and further contains PBS (-) containing 0.25% trypsin (containing 0.02% EDTA).
Hereinafter, the same applies. ) At 4 ° C. overnight.

After immersion in this trypsin solution, the dermis layer and epidermis layer of the back skin were peeled off with tweezers, and the dermis layer was replaced with Ham's F1 containing 0.35% collagenase.
2 Medium (Composition (mg / L): l-Alanin (8.9), l-Arginine (HCl: 21
1), l-Asparagine (13.2), l-Asparatic acid (13.3), l-Cys
teine (HCl: 31.5), l-Glutamic acid (14.7), l-Glutamine
(146), Glycine (7.5), l-Histidine (HCl: 19), l-Isoleuci
ne (3.9), l-leucine (13.1), l-Lysine (HCl: 36.5), l-Methi
onine (4.5), l-Phenylalanin (5.0), Proline (34.5), l-Ser
ine (10.5), l-Threonine (11.9), l-Tryptophane (2.0), lTy
rosine (5.4), l-Valine (11.7), Biotine (0.0073), Choline
(Cl: 14.0), VitaminB12 (1.36), folic acid (1.32), Inositol (1
8.0), Nicotinamide (0.037), pentenoic acid (Ca: 0.477), Vitam
inB ₆ (HCl: 0.062), VitaminB ₂ (0.038), VitaminB ₁ (HCl: 0.3
37), CaCl ₂ (2H ₂ O: 44.0), CuSO ₄・ 5H ₂ O (0.0025), FeSO ₄ 7H2O
(0.834), KCl (224.0), MgCl ₂ (6H ₂ O: 122), Proc.Natl.Aca
d.Sci.USA, 53,288 (1965). The same applies hereinafter.), and cut with scissors. The medium containing the cut product was permeated at 37 ° C. for 35 minutes (60 rpm).
). After infiltration, pipetting was performed until no clumps were visible in the collagenase reaction.
The mixture was transferred to a mL centrifuge tube, Ham's F12 medium containing DNase (10000 units) was added, and the mixture was left for 5 minutes.

After standing, the resulting suspension was further pipetted, filtered through a nylon mesh (Nytex 157mesh), and transferred to a 50 mL centrifuge tube. The suspension was divided into half volumes, and the volume of PBS (-) was 30 mL for each.
The suspension was diluted until the suspension became centrifuged (4 ° C., 400 rpm, 5 minutes). After centrifugation, the fat was removed from the system except the supernatant.

Next, 25 mL of PBS (-) was added to the residue to suspend it, and this was further centrifuged [(4 ° C.,
400 rpm, 5 minutes) x 3 times]. The residue obtained by this centrifugation is a hair follicle in the back skin of the rat.

(2) Collection of hair follicle epithelial cells To the hair follicle obtained by the above operation, 5 mL of PBS (-) containing 0.25% trypsin was added, and the cell suspension was heated at 37 ° C.
For 5 minutes. After the incubation,
5 mL equal volumes of fetal bovine serum (FBS) and Ham's F1
2 medium was added and the cell suspension was added to the cell strainer (1
After filtration through Nalgene (00 μm), the cells were placed in a 50 mL centrifuge tube, and the cell suspension was centrifuged (4 ° C., 150
0 rpm for 5 minutes). The supernatant was removed from this system to obtain the desired hair follicle epithelial cells as a residue.

A cell frozen solution (Cell Banker: manufactured by Diatron) was added to the hair follicle epithelial cells, and 1.5 × 10 ⁷ cels were added.
Adjust to a concentration of ls / mL and add 1.5x1 to each cryotube.
Put one by 0 ⁷ cells, were stored frozen this.

The numbers of these cells were calculated using a hemocytometer. 2. Preculture of hair follicle epithelial cells: In order to remove fibroblasts contaminating the system as much as possible from the system, the hair follicle epithelial cells obtained by the above steps were precultured. Hereinafter, the procedure will be described.

The frozen cells obtained in the above steps were thawed in a thermostat at 37 ° C. Then, FAD medium [Ham's F
12 medium (described later) and MEN medium at a volume ratio of 3: 1 were mixed with insulin (5.0 μg / mL), hydrocortisone (0.45 μg / mL), and epidermal growth factor (EGF) (10. 0 ng / mL), a medium containing cholera toxin (10-9 M) and fetal calf serum (10%), the same applies hereinafter), and the cell solution was diluted and centrifuged. 10 ° C or less,
1500 rpm, 5 minutes). After centrifugation, the supernatant was removed, 10 mL of FAD medium was added to the system, and pipetting was repeated until no cell mass was observed.

The number of cells obtained was calculated using a hemocytometer, and
It was adjusted to a concentration of 2.5 × 10 ⁵ cells / mL in the AD medium. 75cm ³ coated with type I collagen
Cells were seeded in a flask of 37 ° C., 5% CO 2
₂ overnight.

After the culture, the system was washed twice with 10 mL of PBS (-), 2 mL of PBS (-) containing 0.25% trypsin was added, and the mixture was incubated at 37 ° C and 5% CO ₂ for 4 minutes. Next, 2 mL of fetal bovine serum (FBS) was added to the system, and the mixture was slightly shaken once, and the supernatant was removed to remove fibroblasts contaminating the system.

Further, a KGM medium [Keratinocyto growth medium: Keratinocyto ba ba]
bovine pituitary extract (BPE) (0.4 vol%), insulin (0.5%) in sal medium (KBM medium (modified MCDB153 medium (manufactured by Kronetics))).
μm / mL), hydrocortisone (0.5 μm / mL),
h-EGF (0.1 ng / mL), the same applies hereinafter), and cultured at 37 ° C., 5% CO ₂ for 3 days.

3. Assay of target substance Measure the fibroblast contamination rate (FB contamination rate) of the culture flask in which the hair follicle epithelial cells obtained by the above steps were seeded (300).
(0 times, 5 fields of view), and as a result, those having a FB contamination rate of 3% or more were excluded from the subject of the assay.

The system was washed twice with 10 mL of PBS (-)
2 mL of PBS (-) containing 0.25% trypsin was added, and this was incubated at 37 ° C for 3 minutes. Next, in order to remove fibroblasts from the system by utilizing the difference in reactivity between epithelial cells and fibroblasts with trypsin, trypsin was removed, and 2 mL of 0.25% trypsin-containing PBS (-) was again added. Add, 5 rpm at 37 ° C and 20 rpm.
Shake for minutes.

Then, after confirming the detachment of the cells under a microscope, 10 mL of DMEM medium containing 10% FBS was added, pipetting was performed in a 50 mL centrifuge tube, and the system was centrifuged at 1500 rpm for 5 minutes.

The supernatant was removed, 20 mL of KGM medium was added, and pipetting was performed until no cell mass was found. The suspension was filtered through a cell strainer (100 μm, manufactured by Nalgene), placed in a 50 mL centrifuge tube, the number of viable cells in the suspension was calculated using a hemocytometer, and KGM medium was added to the system. The cell concentration was adjusted so as to be 5.0 × 10 ⁴ cells / mL.

Next, at 96 mL of 0.2 mL / well.
Seed on ll-plate (type I collagen coated plate: Falcon) (1.0 × 10 ⁴ cells / weld)
l) The cells were left at room temperature for about 20 minutes until they settled to the bottom of the wells.

Thereafter, the cells were cultured for 1 day at 37 ° C. and 5% CO ₂ to obtain desired human hair follicle epithelial cells. C. Preparation of test medium : (1) Preparation of extract 500 g of commercially available roots (dry matter) of Clara were immersed in 7.5 L of 30% ethanol at room temperature (23 ° C) for 5 days.
The solvent was distilled off from the extract to obtain 50 g of dried Clara 30% ethanol extract. As for other plant extracts, corresponding commercially available plants (in principle, dried products) were obtained and extracted by the following extraction method to prepare respective dry extracts.

(2) Preparation of medium containing target substance The target substance was weighed in a screw tube at about 1.5 mg, and prepared with an organic solvent (DMSO) to a 0.2% solution.

Next, a DMSO solution of the above crude drug extract was added to a 1,000-fold amount of the KBM medium [extract concentration: 2.0 × 10 ⁻⁴ % (DMSO 0.1%)]. Similarly, as a control, a 0.2% DMSO solution of a tea extract (extraction method: extraction with water) and a Bacmondou extract (extraction method: extraction with 10% ethanol) were prepared.

[0061] 0.2 mL of the medium containing these target substances was
It was added to 1.8 mL of 0.1% DMSO-containing KBM medium, and the concentration of the target substance was adjusted to 2.0 × 10 ⁻⁵ % (10-fold dilution).

(3) Preparation of control medium Negative control: DMS was added to 2 mL of KBM medium.
It was prepared by adding 2 μL of O (DMSO 0.1%). Positive control: A negative control medium was prepared by adding 2 μL of insulin (5 mg / mL) and 2 μL of hydrocortisone (0.5 mg / mL).

D. Replacement of target substance medium : The KGM medium in the 96-well-plate prepared with human hair follicle epithelial cell culture cells and rat hair follicle epithelial cell culture cells in A and B above was replaced with the target substance-supplemented medium and control medium (200 μL / wel
After the replacement, the cells were cultured at 37 ° C. and 5% CO ₂ for 2 days.

The medium was replaced with the KGM in the well.
The medium was removed with an aspirator, taking care not to damage the cells adhering to the bottom, and then the medium containing the target substance was immediately added from both ends of the well.

E. Measurement of cell proliferation : alamar blue (al
amar blue: manufactured by Alamar BioSciences Co., Ltd.), 1/10 of the volume (volume) of the medium, and then added at 37 ° C. (5
% CO ₂ ) for 6 hours.

After incubation, the system at 595 nm and 57
The absorbance at 0 nm is measured using a microplate reader (Micro pl
ate reader: Bio RAD) and the cell proliferation was calculated according to the following formula.

[0067]

## EQU1 ## Calculation method of cell proliferation degree Further, the cell growth promotion index was calculated according to the following formula.

[0068]

(Equation 2) The cell proliferation index of the negative control is 0, and the cell proliferation index of the positive control is 1.

F. Results : Table 1 below shows the cell growth promotion index of each of the measured target substances.

[0070]

[Table 1] Table 1 ──────────────────────────────────── Cell growth promotion index Target substances細胞 Rat-derived cells Human-derived cells ───────────────────────── ─────────── Dried extract of Clara 112% 115% (extracted with 30% ethanol) Dried extract of tea 99% 97% (extracted with water) Dried extract of Bakmondou 101% 96% (10 % Ethanol extract dried product) よ り Based on this result, Proliferation activity of the enveloped epithelial cells was certainly observed. In other words, it was revealed that the above-mentioned components have the effect of maintaining and prolonging the anagen phase in hair by maintaining the mitotic proliferation activity of hair epithelial cells.

G. Fineness in specific pterocarpan derivatives
Examination of vesicle growth promoting action Hot water extract 10 of the above-mentioned commercially available Clara root (dry matter) 10
0 g (dry residue amount) was subjected to solvent extraction with methylene chloride, ethyl acetate and n-butanol, and the above-mentioned cell growth promotion test was performed on each fraction. As a result, proliferation activity of hair follicle epithelial cells was observed in the methylene chloride fraction.

Then, this methylene chloride fraction was separated by a silica gel column (Wako silica gel 60) to isolate two kinds of active components, and they were analyzed by ¹ H-NMR and ¹³ C-NMR (analysis chart of both these NMR). Is shown in FIG. 1), HMB
The structure was determined by C (analysis chart is shown in FIG. 2), HMQC (analysis chart is shown in FIG. 3) and FAB-MAS (analysis chart is shown in FIG. 4). As a result, it was presumed that one of the active components in the isolated methylene chloride fraction was merchiain and the other was medical pin.

From this, it is presumed that the active ingredients in the above methylene chloride fraction are two kinds of pterocarpan derivatives of medical pin and merchiain. The cell proliferation test on cultured human hair follicle epithelial cells was performed according to the method (described above) performed on plant extracts. Table 2 shows the results.

[0074]

[Table 2] Table 2 指標 Cell proliferation promotion index Target substances ─ ─────────────────── Human-derived cells ────────────────────────────マ ー Merchiain 10 ng / mL 119% 0.1 ng / mL 113% Medical pin 10 ng / mL 126% 0.1 ng / mL 115% ──────── ───────────────────────── Therefore, it was confirmed that these specific pterocarpan derivatives exert the proliferation activity of cultured hair follicle epithelial cells. Was.
In other words, it was revealed that the specific pterocarpan derivative exhibited the effect of maintaining and prolonging the anagen phase of hair by maintaining the mitotic proliferation activity of hair epithelial cells.

Hereinafter, the formulation of the present extender is shown as an example, and the hair growth effect thereof was further examined. [Example 1] Preparation of liquid hair growth period prolonging agent 30% ethanol dry product of Clara extract described above 0.1
%, 70% ethanol 90%, sodium oleate 0.1%, dodecylbenzenesulfonic acid 0.49%, hydrogenated castor oil ethylene oxide (40 mol) adduct 0.5
% And ion-exchanged water (residual) and dissolved by stirring. Further, ion-exchanged water (10%) was added and mixed to obtain a liquid hair growth period extender.

In the formulation of this liquid hair growth-prolonging agent, 0.1% of the above-mentioned extract of Bacmondou using 10% ethanol was replaced with 30% of the above Clara extract.
A liquid agent prepared in place of the dried ethanol extract was used as a control (Comparative Example 1).

Example 2 Preparation of Emulsion Hair Growth Prolongation Agent In the above-mentioned Clara extract production process, an extract was prepared using methanol instead of 30% ethanol, and this was used as an emulsion having the following formulation. Used in hairy hair growth extenders. Ingredients Compounding amount (% by mass) (A phase) Clara extract dried product 1.0 Polyoxyethylene (60 mol) addition-hardened castor oil 2.0 Glycerin 10.0 Dipropylene glycol 10.0 1,3-butylene glycol 5.0 Polyethylene glycol 1500 5.0 (B phase) Cetyl isooctanoate 10.0 Squalane 5.0 Vaseline 2.0 Propyl paraben 2.0 (C phase) Carboxyvinyl polymer 1% aqueous solution 30.0 Sodium hexametaphosphate 0 0.03 ion-exchanged water 8.35 (phase D) ion-exchanged water 4.5 (phase E) KOH 0.12 ion-exchanged water 5.0 <Production method> A phase and B phase were each heated and dissolved at 60 ° C.
The mixture was mixed and treated with a homomixer to form a gel, to which phase D was gradually added and dispersed with a homomixer. Next, the dissolved C phase was added thereto, and finally, the dissolved E phase was added and emulsified with a homomixer to obtain an O / W emulsion type hair growth period extender.

[Example 3] Preparation of creamy hair growth period prolonging agent In the same manner as in Example 2, Clara methanol extract dried
The following formulation was used in the creamy hair growth extender. Ingredients Amount (% by mass) (A phase) Liquid paraffin 5.0 cetostearyl alcohol 5.5 glyceryl monostearate 3.0 EO (20 mol) -2-octyldodecyl ether 8.0 propylparaben 0.3 Perfume 0.1 (Phase B) Clara extract 5.0 Glycerin 8.0 Dipropylene glycol 20.0 Polyethylene glycol 4000 5.0 Sodium dodecyl sulfate 0.1 Sodium hexametaphosphate 0.005 Deionized water 39.995 <Production method > The phases A and B were each dissolved by heating, mixed and emulsified with a homomixer to obtain a creamy hair growth period extender.

[Test Example 2] Examination of the hair growth effect of the present prolonging agent In order to examine the hair growth effect such as the hair loss preventing effect and the hair growth effect of the present prolonging agent, a tricogram test was performed on humans by the following method. The test sample and the control sample are the present extender of Examples 1 to 3, the agent of Comparative Example 1, and 70% ethanol.

Test Method Before and after use of the above sample, the roots of the extracted hair were observed under a microscope, and the number of “resting roots”, which are the roots of the growth-stopped hair, were counted from the form of the roots. The hair growth effects of these samples were compared by increasing or decreasing the ratio.

That is, a test sample and a control sample were applied to the scalp of 10 male subjects twice a day, 2 mL at a time, for 6 times.
The hair was applied continuously for months, and immediately after the application and immediately after the completion of the application for six months, 100 hairs were removed from each subject and each hair root was observed under a microscope. In addition, a practical use test was performed to determine whether the hair growth effect of the sample was effective or ineffective. The results of these tests are shown in Table 3 below.

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[Table 3] Table 3 ──────────────────────────────────── Sample Ratio of resting roots ( %) Evaluation of hair growth effect ───────────── 20% or more ± 20% 20% or more Decrease Increase ──────────────────── ──────────────── 70% ethanol (target) 0 60 40 Ineffective Example 1 60 20 20 Effective Example 2 40 30 30 Effective Example 3 50 30 20 Effective Comparative Example 1 20 50 30 Invalid ク ラ Extract Clara from the results in Table 3. The hair growth effect based on the effect of Clara extract on prolonging the hair growth period was observed in the present prolonging agent containing the product as an active ingredient.

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Industrial Applicability According to the present invention, there is provided a hair growth period extending agent which can maintain or prolong the growth period in the hair cycle and suppress hair loss and the like.

[Brief description of the drawings]

FIG. 1 is an analysis chart of ¹ H-NMR and ¹³ C-NMR of an active component of a methylene chloride fraction of Clara extract.

FIG. 2 is an HMBC analysis chart of the active component of the methylene chloride fraction of Clara extract.

FIG. 3 is an HMQC analysis chart of an active component of a methylene chloride fraction of Clara extract.

FIG. 4 is an FAB-MAS analysis chart of the active component of the methylene chloride fraction of Clara extract.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07D 493/04 106 C07D 493/04 106A 493/14 493/14 (72) Inventor Jun Suzuki Yokohama, Kanagawa Prefecture 2-2-1 Hayabuchi, Tsuzuki-ku Shiseido Research Center (Shin-Yokohama) Co., Ltd. (72) Inventor Masahiro Tajima 2-2-1 Hayabuchi, Tsuzuki-ku, Yokohama-shi, Kanagawa Shiseido Research Center (Shin-Yokohama) F term (for reference) 4C071 AA01 BB01 BB02 BB06 CC12 CC14 DD14 EE04 EE05 FF15 FF17 HH05 KK17 LL01 4C083 AA111 AA112 AB032 AB282 AC012 AC022 AC072 AC102 AC122 AC182 AC252 AC352 AC422 AC432 AC482 AC37 AC2423 DD27 AC042 AC042 ACDD DD838 AA01 AA02 CA01 MA01 MA04 NA14 ZA92 ZB22 4C088 AB59 AC02 BA09 BA10 BA23 BA32 CA05 CA06 CA07 CA08 MA04 M A17 MA22 MA28 NA14 ZA92 ZB22

Claims (4)

[Claims] 1. A hair growth-prolonging agent comprising an extract of a plant belonging to the genus Clara ( Sophora L.) as an active ingredient. 2. The hair growth prolonging agent according to claim 1, wherein the plant belonging to the leguminous genus Clara ( Sophora L.) is Clara (Sophora.flavescens Ait.). Wherein extract of plants, leguminous Clara genus (Sophor
The hair growth period prolonging agent according to claim 1 or 2, which is an aqueous extract of the root of a plant belonging to a L.). 4. A hair growth-prolonging agent which comprises maackiain and / or medical pin as an active ingredient.