JPH10265348A - Hair tonic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a hair tonic and an agent for extending a hair growth period capable of maintaining and extending the growth period in the hair cycle by improving the hair growth by using a specific plant extract as an active ingredient. SOLUTION: This hair tonic and agent for extending a hair growth period comprises an extract of a plant belonging to the genus Datura of the family Solanaceae as an active ingredient. The plant belonging to the genus Datura is exempified by Datura stramonium L. and Datura stramonium L. var. chalybea Koch. The formulating amount of the active ingredient is 0.00005-20.0 wt.% especially 0.1-10.0 wt.% in terms of dry matter of the extract based on the nearly whole preparation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention belongs to the technical field related to a hair restorer and a hair growth period extender. More specifically, the present invention relates to a hair restorer and a hair growth period extender that maintain or extend the growth period in the hair cycle by promoting hair growth.

[0002]

2. Description of the Related Art In a modern society referred to as an aging society or a stressed society, there are more and more opportunities for head hair to be exposed to the risk of hair loss due to various causes. In response to this, various attempts have been made to provide a more excellent “hair restoration”. The main effects of hair restoration on hair are hair growth inducing effect (hair growth promotion effect, growth period induction effect), hair thickening effect, hair growth period extension effect, 5α-reductase inhibitory effect, and blood circulation promotion effect. ,
The effects include a bactericidal effect, an anti-dandruff effect, a moisturizing effect, and an antioxidant effect.

[0003] However, despite various attempts as described above, conventional hair restorers have not always had sufficient hair restoring effects such as hair loss prevention and hair growth effects. This is probably due to various causes of hair loss and the very complex mechanism of hair growth. The `` hair restoration '' provided so far has been developed by capturing only the relatively rough concept of hair loss, in other words, simply and simply the phenomenon of `` hair loss '', and was developed by focusing on the mechanism Is not much.

[0004] The major reason is that it cannot be denied that a hair-growth drug assay method 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 drug assay method for assaying the above-described hair growth period prolonging effect. As a result, the hair growth products provided so far induce the hair growth to the growth period of the hair cycle and the hair growth induction. Many focused on the effects. Today, as the causes of hair loss are gradually elucidated, it is considered preferable to individually use a hair restorer having a formulation corresponding to these various causes, and in this respect, a hair restorer having the above-mentioned effects is provided. It is required to be.

[0005]

Therefore, the present inventor has proposed in
Establish a simple hair growth drug assay method for assaying the above-described hair growth period extending effect carried out in vitro , and use the hair growth agent assay method to extend the hair growth period using a component having the above-described hair growth period extending effect as an active ingredient. The aim was to find an agent.

The object of the present invention is to find a component having the above-mentioned effect of extending the hair growth period, and to provide a hair growth agent and a hair growth period extending agent containing the same as an active ingredient. .

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies on the components having the above-described effect of extending the hair growth period in various substances 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 Datura such as morning glory has a desired effect of extending the hair growth period, and completed the present invention.

[0008] That is, the present invention is an invention which provides a hair restorer and a hair growth period extender comprising an extract of a plant belonging to the genus Datura as an active ingredient.

As described above, in the present invention, the "hair growth period prolonging agent" is mainly used to maintain or promote at least the mitotic proliferation activity of hair follicle epithelial cells by the hair growth drug assay method described below to thereby enhance the hair growth period. It is a hair-related drug which is obtained by blending a component having an effect of maintaining or prolonging the hair as an active ingredient, and particularly focuses on the above-described effect of extending the hair growth period, and has a characteristic as a so-called "individually effective hair growth agent".

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 hair root, so that the anagen of the telogen hair is relatively shorter than 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, comprehensive and synergistic effects can be achieved in a wide range of alopecia. That is, this "hair growth period extender"
A general purpose hair restoring application having the concept including the above-mentioned effects (1) to (4) is one that has a distinctive purpose.

[0011]

Embodiments of the present invention will be described below. The hair restorer according to the present invention and the hair extender according to the present invention (hereinafter referred to as the hair restorer according to the present invention) include Solanaceae (Solanacea).
It is a hair-related drug containing, as an active ingredient, an extract of a plant belonging to the genus Datura (Datura ) of e) . Plants belonging to the genus Datura include, for example, Datura stramonium L.,
Datura stramonium L.
var. chalybea Koch) and the like, as long as the extract has the action of extending the anagen phase in the hair cycle described below, and belongs to other species such as their variants and subspecies. Is also good.

Further, the extract of the plant belonging to the genus Datura can be advantageously obtained from the leaves or seeds of the plant (all of the raw materials of these extracts are collectively referred to as " It is sometimes referred to as a plant belonging to the genus Datura.).

[0013] Extraction of a plant belonging to the genus Datura can be performed by a method generally used for extracting a plant-derived extract. That is, it can be obtained by subjecting the plant belonging to the genus Datura as raw or dried as necessary, and then directly or pulverizing it for solvent extraction. As a solvent that can be used at this time, a common solvent used when extracting a component of the plant from the plant can be used and is not particularly limited. Examples thereof include hot water; methanol, ethanol, isopropanol, and n-butanol. Lower alcohols; polyhydric alcohols such as propylene glycol and 1,3-butylene glycol; hydrates of these alcohols; and hydrocarbon solvents such as n-hexane and toluene. It is preferable to use the lower alcohol of the formula (1) as an extraction solvent. When these lower alcohols are used as an extraction solvent, the resulting extract can be directly blended as an active ingredient such as the hair restorer of the present invention. However, the extraction solvent can be distilled off once and, if necessary, blended after drying. It is.

It has been confirmed that the extract of the plant belonging to the genus Datura thus obtained contains at least tropine alkaloids such as scopolamine and atropine and fatty oils such as oil and linoleic acid. . It is not known which of the above-mentioned components, which are incorporated as active ingredients such as the hair restorer of the present invention, the proliferative action of hair follicle epithelial cells described below is derived from any of these components. It has the action of proliferating the epithelial cells.

The amount of the above components in the hair restorer of the present invention and the like can be appropriately selected according to the specific form of the hair restorer of the present invention and the like, and is not particularly limited.
In general, 0.00005% by weight or more and 20.0% by weight or less, preferably 0.01% by weight or more and 10.0% by weight of a dry extract (including adzuki bean powder) based on the whole product
It is blended as follows.

When the amount of the dried extract is less than 0.00005% by weight based on the whole preparation, the desired effect of the present invention, ie, the effect of extending the hair growth period based on the hair follicle cell proliferation effect, is sufficient. If the amount exceeds 20.0% by weight, not only the effect corresponding to the increase in the amount is not expected to increase, but also the tendency to cause troubles in the preparation is remarkable, which is not preferable.

The above-mentioned components may be used alone as one type of active ingredient such as the hair restorer of the present invention, but may be used in combination of two or more types as appropriate. As described above, the hair restorer of the present invention, which contains an extract of a plant belonging to the genus Datura as an active ingredient, has an excellent hair growth period extending effect based on an excellent hair follicle cell proliferation effect, as described above. Alopecia due to, for example, slow growth of hair follicle epithelial cells near the hair root, shortening the anagen phase and relatively increasing the proportion of telogen hairs compared to anagen hairs It is a particularly effective drug.

The hair restorer of the present invention can be formulated, for example, by combining it with other components having individual effects.
It is possible to prevent hair loss from various viewpoints. Means for specifying the maintenance or prolonging action of the anagen phase in the hair cycle, which is an essential action in the hair growth term prolonging effect, which is the main effect of the hair restorer of the present invention, is because the specifying method itself specifies the action. The method is not particularly limited as long as it is appropriate. For example, an in vitro method and an in vivo method can be used, but considering its simplicity and effectiveness, the in vitro method should be used. Is preferred.

Hereinafter, a specific method for examining the growth effect of hair follicle epithelial cultured cells, which is one of the in vitro specific methods, will be briefly described (more specifically, Examples Described in.).

[0020] That is, this identification method is as follows: "A cultured substance of hair follicle epithelial system is brought into contact with a target substance in a serum-free medium, and the presence or absence and / or strength of the proliferation activity of the cell is specified. A hair growth assay for assaying the effect of prolonging the anagen phase in the hair cycle ", that is, focusing on hair follicle epithelial cells that are directly involved in hair elongation, This is an in vitro hair growth drug assay method for determining the effect of extending the growth period.

In this method for assaying a hair-growth agent, the target substance is added to “cultured cells of hair follicle epithelial system” which is a cultured cell obtained by isolating hair follicle epithelial cells of animals (including humans, the same applies hereinafter). By contact, the presence or absence and / or strength of the proliferation is specified.

The hair follicle epithelial cells particularly refer to cells in which the outer root sheath cells and matrix cells in the vicinity of the hair root are combined, and the inner hair papilla cells and the basement membrane covering the cells are excluded. Hair papilla cells located at the root of the hair root act on these hair follicle epithelial cells to promote their proliferation, thereby promoting hair elongation. The anagen phase in the hair cycle is exactly when this hair is growing, that is, when the hair follicle epithelial cells are dividing and proliferating.
This is the time to slow down and pause.

In other words, the substance that prolongs the anagen in the hair cycle prevents the hair from shifting to the regression phase and the telogen phase in the hair cycle by maintaining the dividing and proliferating activity of the hair follicle epithelial cells by its administration. It is concluded that the substance is a substance that continues to promote or maintain the growth of hair follicle epithelial cells.

As another in vitro method for assaying a hair growth drug in vitro , for example, a method in which a target substance is allowed to act on hair papilla cells of an animal and the effect of promoting the proliferation thereof can be determined.

[0025] As a specific method in vivo ,
For example, a "hair growth drug assay method in which a target substance is administered to a nude mouse, the state of the hair growth site on the body surface of the nude mouse is specified, and the effect of the target substance on the growth cycle in the hair cycle is assayed" To specify the width of hair growth sites and the speed of hair growth sites in nude mice that are hairless in principle, but whose hair growth sites move over time on the body surface over time. For example, a method of testing the length of the anagen phase in the hair cycle can be mentioned.

By using the hair restorer of the present invention in combination with another hair restorer having an individual effect, a synergistic effect can be achieved in a specific alopecia, and together with other hair growth period extending components. It is also possible to mix.
Hereinafter, one of the in vitro specific methods, which is characterized by examining the proliferation effect of hair follicle epithelial cultured cells, will be briefly described (more specifically, described in Examples. .).

The dosage form that can be used by the hair restorer of the present invention is not particularly limited as long as it is a dosage form applicable to the outer skin.
Emulsion, ointment, etc. can be selected. Further, the form of the hair restorer of the present invention and the like is arbitrary, and for example, it can take the form of tonic, hair cream, mousse, shampoo, rinse, cream, milky lotion, lotion, pack and the like.

The hair restorer of the present invention is generally used in cosmetics, quasi-drugs, pharmaceuticals, etc., in addition to the above-mentioned essential components, if necessary and as long as the intended effect of the present invention is not impaired. Various oily or aqueous components, humectants, thickeners, preservatives, antioxidants, fragrances, coloring agents, various chemicals, and the like can be used.

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 ; serine, methionine, amino acids such as arginine; vitamin B _6, vitamin E (or derivatives thereof), biotin, vitamins such as pantothenic acid (or derivatives thereof); nicotinic acid, Nico 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. Specific formulations of the hair restorer of the present invention will be described later.

[0030]

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, "%" and the content are indicated by weight unless otherwise specified. First, in for assessing hair growth life extending effect of the extract of a plant belonging to Datura used in the Examples, etc.
An in vitro cell proliferation test will be described.

Test Example 1 Cell Proliferation Test Using Hair Follicle Epithelial Cultured Cells Human hair follicle epithelial cells 1. 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. 1000 U / ml dispase 0.2%
Dulbecco's modified MEM containing collagenase (DME
M) for 30 minutes at 37 ° C., using the tip of a syringe needle.
Phosphate buffer solution containing 0.05% trypsin and 0.02% EDTA after removing dermal sheath, dermal papilla and epithelium of hair bulb [PBS (-): (-) means that it does not contain calcium or magnesium ions Yes] for 5 minutes at 37 ° C.

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 Medication).
um (KGM)] (Keratinocyte Serum Free Me
dium can 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 thus grown were treated with 0.05 wt% 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).

Next, the cells were suspended in the above-mentioned serum-free medium, seeded on a culture dish coated with collagen (Type I) at a density of 5000 cells / cm ² , and cultured every two days until the cells became subconfluent. After performing exchange and treating again with 0.05 wt% trypsin-0.02% EDTA at 37 ° C. for 5 minutes,
The reaction was stopped with an equal volume of 0.1% trypsin inhibitor, centrifuged (800 × g, 5 minutes), and the resulting human hair follicle epithelial cells were subjected to cell freezing (Cell Banker: Diatron) And add 1.0 × 10 ⁶ cell / m
l and add 1.0 × 10 ⁶ to each cryotube.
Each cell was placed and this was frozen and stored. 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, after inoculating the hair follicle epithelial cells in a culture flask, the cells were mixed with 0.05% trypsin and 0.02%.
After treatment with% 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 removed. Was prepared.

A 96-well plate at a rate of 0.2 ml / well
(Type I collagen coated plate: manufactured by 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 1. Collection of rat hair follicle epithelial cells: (1) Collection of hair follicles After disinfecting neonatal (3-4 day old) rats with ethanol,
The rats were sacrificed with carbon dioxide and the back skin of these rats was collected with scissors. Then, the collected back skin was added with 1% P
Two sheets were immersed in SF-containing PBS (-) at a time. Thereafter, the subcutaneous fat, skin, and the like below the skin fat layer were removed with scissors for dissection. Then, the back skin was again cleaned with P containing 1% PSF.
This was immersed in BS (-), and further immersed in PBS (-) containing 0.25% trypsin (containing 0.02% EDTA; the same applies hereinafter) 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 'containing 0.35% collagenase.
sF12 medium [composition (mg / L): l-Alanin (8.9), l-Arginine
(HCl: 211), l-Asparagine (13.2), l-Asparatic acid (13.
3), l-Cysteine (HCl: 31.5), l-Glutamic acid (14.7), l-
Glutamine (146), Glycine (7.5), l-Histidine (HCl: 19),
l-Isoleucine (3.9), l-leucine (13.1), l-Lysine (HCl: 36.
5), l-Methionine (4.5), l-Phenylalanin (5.0), Proline (3
4.5), l-Serine (10.5), l-Threonine (11.9), l-Tryptophan
e (2.0), l-Tyrosine (5.4), l-Valine (11.7), Biotine (0.00
73), Choline (Cl: 14.0), Vitamin B12 (1.36), folic acid (1.32), I
nositol (18.0), Nicotinamide (0.037), pantothenic acid (Ca:
0.477), VitaminB6 (HCl: 0.062), VitaminB2 (0.038), Vitam
inB1 (HCl: 0.337), CaCl ₂ (2H ₂ O: 44.0), CuSO ₄・ 5H ₂ O (0.002
_{5), FeSO 4 · 7H 2} O (0.834), KCl (224.0), MgCl 2 (6H 2 O: 122),
Acad. Sci. USA, 53,288 (1965) "). A 100 mm dish containing the same was transferred and cut with scissors. The medium containing the cut product was permeated at 37 ° C. for 35 minutes (60 rpm). After infiltration, pipetting was carried out until no clumps could be seen in the collagenase reaction product, and this was transferred to a 50 ml centrifuge tube, Ham's F12 medium containing DNase (10000 units) was added, and the mixture was allowed to stand for 5 minutes. .

After standing, the resulting suspension was further pipetted, filtered through a nylon mesh (Nytex 157 mesh), and transferred to a 50 ml centrifuge tube. The suspension was divided into half volumes, and PBS (-) was added to each with a volume of 30%.
The suspension was diluted to a volume of 1 ml and then 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) × 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 F12
Add medium and add cell suspension to cell strainer (100
μm Nalgene), and put into a 50 ml centrifuge tube.
This cell suspension was centrifuged (4 ° C., 1500 rp).
m, 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 ce
Adjust to a concentration of ll / ml and add 1.5x1 to each cryotube.
Put one by 0 ⁷ cell, was frozen save it. In addition, these cell numbers were calculated with a blood cell counting plate.

2. Preculture of hair follicle epithelial cells: To remove fibroblasts from the system as much as possible,
Preculture of hair follicle epithelial cells obtained by the above steps was performed. Hereinafter, the procedure will be described. The frozen cells obtained in the above steps were thawed in a thermostat at 37 ° C. Then F
AD medium [A mixture of Ham's F12 medium (described below) and MEN medium at a volume ratio of 3 to 1;
μg / ml), a medium containing hydrocortisone (0.45 μg / ml), epidermal growth factor (EGF) (10.0 ng / ml), cholera toxin (10 ⁻⁹ M) and fetal bovine serum (10%), The same applies to the following), the cell solution was diluted, and the system was 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 cell / ml in 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 fetal bovine serum (FBS) was added to the system.
After adding once and gently rocking, the supernatant was removed to remove fibroblasts contaminating the system.

Furthermore, KGM medium [Keratinocyto growth medium: Keratinocytob medium] was added to the system.
bovine pituitary extract (BPE) (0.4 vol%), insulin (0.5 μm / ml), hydrocortisone (0.5 μm / ml) in an asal medium (KBM medium (modified MCDB153 medium (Clonetics))) , h-EGF (0.1 ng / ml), the same applies to the following.
Cultured in O 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 is PBS (-)
After washing twice with 10 ml, PBS containing 0.25% trypsin
2 ml of (-) was added, and this was incubated at 37 ° C. for 3 minutes. Taking advantage of the difference in reactivity between epithelial cells and fibroblasts with trypsin, trypsin was removed in order to remove fibroblasts from the system, and 0.2 times again.
2 ml of PBS (-) containing 5% trypsin was added, and 37
The mixture was shaken at 20 rpm for 5 minutes.

Then, after confirming the detachment of the cells under a microscope, 10 ml of 10% FBS-containing DMEM medium 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 there was no cell mass.

The suspension was added to a cell strainer (100 μm Nalg).
After filtration in a 50 ml centrifuge tube, the number of viable cells in the suspension was calculated using a hemocytometer, KGM medium was added to the system, and the cell concentration in the system was adjusted to 5.0. It was adjusted to be × 10 ⁴ cell / ml. Then, at a rate of 0.2 ml / well,
Seed into 96 well-plate (type I collagen coated plate: Falcon) (1.0 × 10 ⁴ cell / we
ll), 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 cultured cells.

C. Preparation of test medium: (1) Preparation of extract 500 g of commercially available datura (dry matter) was used for 7.
Two immersions in 5 l of 70% methanol at room temperature (23 ° C.) for 24 hours were performed. The solvent was distilled off from the extract to obtain 50.0 g of a 70% methanol-extracted dried product of Korean morning glory.

(2) Preparation of Medium Containing Target Substance The above-mentioned Datura extract was weighed in a screw tube of about 1.5 mg, and prepared with an organic solvent (DMSO) to a 0.2% solution. Similarly, as a control, a 0.2% DMSO solution of a tea extract (extraction method: water extraction) and a bagwort extract (extraction method: 10% ethanol extraction) were prepared.

Next, a DMSO solution of the crude drug extract was added to a 1000-fold amount of the KBM medium (manufactured by Kronetics) [extract concentration: 2.0 × 10 ^-4 % (D
MSO 0.1%)]. These target substance-supplemented media 0.
2 ml was added to 1.8 ml of a 0.1% DMSO-containing KBM medium to adjust the concentration of the target substance to 2.0 × 10 ⁻⁵ % (10-fold dilution).

(3) Preparation of control medium Negative control: DMS was added to 2 ml of KBM medium.
O (2 μl) was added (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. Object substance medium exchange: KGM medium in a 96-well-plate in which human hair follicle epithelial cell culture cells and rat hair follicle epithelial cell culture cells were prepared in A and B above was prepared in the above C, and the target substance addition medium and control medium ( 200μl / well), 37 ° C, 5%
Cultured for 2 days in CO ₂ .

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. Cell proliferation measurement: alamar blue (ala
mar blue (manufactured by Alamar Biosciences, Inc.) was added at 1/10 the volume (volume) of the medium, and the mixture was added at 37 ° C (5
% CO ₂ ) for 6 hours. After the incubation, the absorbance of the system at 595 nm and 570 nm was measured using a microplate reader (Micro RAD).
The cell proliferation was calculated according to the following formula.

[0054]

(Equation 1) Further, the cell growth promotion index was calculated according to the following formula.

[0055]

(Equation 2)

F. Results: The determined cell proliferation degree was 0.7 in rat hair follicle epithelial cultured cells and 0.5 in human hair follicle epithelial cultured cells. From these results, it was found that the extract of the Aspergillus niger, which is an extract of a plant belonging to the genus Datura, clearly showed the growth activity of hair follicle epithelial cultured cells.
That is, it was revealed that the extract of a plant belonging to the genus Datura has a hair growth period extending effect.

Hereinafter, the formulations of the hair restorer of the present invention and the like are shown as examples, and their hair restorer effects were further examined. [Example 1] Preparation of liquid hair restorer, etc. 0.1% of the above-mentioned 70% methanol-extracted dried ginseng was converted to 70% ethanol 90%, sodium oleate 0.1%, dodecylbenzenesulfonic acid 0.1%.
49%, 0.5% of hydrogenated castor oil ethylene oxide (40 mol) adduct and ion-exchanged water (residual) were mixed and stirred to dissolve. Further, ion-exchanged water (10%) was added and mixed to obtain a liquid hair restorer and the like. In the formulation of this liquid hair restorer, etc., a liquid agent prepared by replacing 0.1% of the above dried 10% ethanol extract of Bacmondou with the 70% methanol extracted dried product of Datura as a control. Prepared.

Example 2 Preparation of Emulsion Hair Restoration Agent, etc. In the above-mentioned step of producing a Datura morning glory extract, an extract was prepared by using ethanol instead of 70% methanol, and this was used as the emulsion of the following formula. Used in preparations and the like. Ingredients Ingredients (Weight%) (A phase) Datura Asagao 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 .03 Ion exchange water 8.35 (D phase) Ion exchange water 4.5 (E phase) KOH 0.12 Ion exchange water 5.0

<Production Method> Phases A and B were each heated and dissolved at 60 ° C., mixed and treated with a homomixer to prepare a gel. Phase D was gradually added thereto and dispersed with a homomixer. Next, dissolved C phase was added thereto, and finally dissolved E
The phases were added and emulsified with a homomixer to obtain an O / W emulsion type hair restorer and the like.

[Example 3] Preparation of cream-like hair restorer, etc. As in Example 2, an ethanol-extracted dried product of Korean Asagao was used in a cream-like hair restorer having the following formulation. Ingredients Ingredients Amount (% by weight) (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 Fragrance 0.1 (Phase B) Ginseng Asagao extract dried product 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 by a homomixer to obtain a creamy hair restorer and the like.

Test Example 2 Investigation of the Hair Restoring Effect of the Hair Restoration Agent of the Present Invention In order to examine the hair restoring effect such as the hair loss preventing effect and the hair growth effect of the hair restoring agent of the present invention, a tricogram test was performed on humans by the following method. And an actual use test. The test sample and the control sample are the agent of Comparative Example 1, such as the hair restorer of the present invention of Examples 1 to 3, and 70% ethanol.

Test Method The roots of the extracted hair before and after use of the above sample were observed under a microscope, and the number of “resting roots”, which are the roots of the hairs that had stopped growing, 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, each of the test sample and the control sample was applied to the scalp of 10 male subjects twice a day, 2 ml at a time, continuously for 6 months.
The hair was removed by 00 hairs, and each hair root was observed under a microscope. In addition, an actual use test was performed on whether the hair growth effect of the sample was effective or ineffective. The results of these tests
The results are shown in Table 1 below.

[0064]

[Table 1]

[0065]

Industrial Applicability According to the present invention, there are provided a hair growth agent and a hair growth period extending agent for extending the growth period in the hair cycle.

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[Procedure amendment]

[Submission date] January 30, 1998

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0036

[Correction method] Change

[Correction contents]

B. Rat hair follicle epithelial cells 1. Collection of rat hair follicle epithelial cells: (1) Collection of hair follicle The back skin of a newborn (3 to 4 day old) rat was collected, and the collected back skin was added to PBS (-) containing 1% PSF for 2 hours.
Soaked one by one. Thereafter, the subcutaneous fat, skin, and the like below the skin fat layer were removed with scissors for dissection. Next, the back skin was immersed again in PBS (-) containing 1% PSF, and further immersed in PBS (-) containing 0.25% trypsin (0.02).
% EDTA included. Hereinafter, the same applies. ) At 4 ° C. overnight.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Masahiro Tajima 1050 Nippa-cho, Kohoku-ku, Yokohama-shi, Kanagawa Inside Shiseido First Research Center Co., Ltd.

Claims (2)

[Claims] 1. A hair restorer comprising an extract of a plant belonging to the genus Datura as an active ingredient. 2. A hair growth-prolonging agent comprising an extract of a plant belonging to the genus Datura as an active ingredient.