JPH10265350A - Hair tonic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a hair tonic capable of synergistically manifesting further hair growing activities by using the hair tonic with a component recognized as having activities for extending a hair growth period in combination. SOLUTION: This hair tonic contains an ingredient having activities for inhibiting testosterone-5α-reductase and an ingredient having activities capable of maintaining and extending the growth period in hair cycle, especially the ingredient having activities capable of maintaining and extending the growth period in the hair cycle as active ingredients. The hair tonic manifests very superior hair growing activities because both ingredients synergistically manifests the activities.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention is an invention belonging to the technical field related to a hair restorer. More specifically, the present invention relates to a hair restorer having a synergistic hair-growth effect by using a component having an activity of inhibiting testosterone-5α-reductase and a component having an activity of maintaining or prolonging the anagen phase in the hair cycle as active ingredients. .

[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 restorer”. The main effects of the hair restorer on the hair include a hair growth-inducing effect (a hair growth-promoting effect, a growth period-inducing effect), a thickening effect, a hair growth period extending effect, a testosterone-5α-reductase inhibitory effect,
The effects include a blood circulation promoting effect, a bactericidal effect, a dandruff preventing effect, a moisturizing effect, an antioxidant effect, and the like.

[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. 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.

[0004]

The inventor of the present invention has shown that a hair-growth component that has been conventionally tended to be intuitively blended can be further improved if it can be formulated in consideration of the above-mentioned hair loss mechanism. It was thought that a hair restorer could be provided. One of the ideas is to establish a hair growth stimulating method for assaying the above-described hair growth period extending effect, find a component having this hair growth period extending effect, and find this component and a component having other effects. By providing a hair restorer as an active ingredient in combination with the above, the idea of providing a hair restorer exhibiting a synergistic effect in which the respective effects simultaneously act on the hair is provided.

[0005] In such a technical flow, the present inventors have established a hair-growth drug assay method capable of simply and accurately assaying the above-described effect of extending the hair growth period. Therefore, the problem to be solved by the present invention is that a hair growth-increasing effect can be synergistically exhibited by combining the above-mentioned hair growth-prolonging effect in the hair-growth-testing method and the like with an active ingredient in combination with the active ingredient. An object of the present invention is to provide a hair restorer.

[0006]

Means for Solving the Problems Further, the present inventors have conducted intensive studies to solve this problem, and as a result, the above-mentioned hair growth stimulating method has the above-mentioned components which have the above-mentioned effect of extending the hair growth period and the above-mentioned components. It has been found that this problem can be solved by providing a hair restorer as an active ingredient by combining a component having a testosterone-5α-reductase inhibitory effect.

That is, the present inventor provides the following invention in the present application. The testosterone according to claim 1,
Provided is a hair restorer comprising, as an active ingredient, a component having an action of inhibiting 5α-reductase and a component having an action of maintaining or prolonging the anagen phase in the hair cycle.

[0008] In the second aspect, the hair restorer according to the first aspect, wherein the component having an action of maintaining or prolonging the anagen phase in the hair cycle is a component having an activity of expanding hair follicle epithelial cells. .

[0009] In the third aspect, the component having an activity of growing hair follicle epithelial cells is a comfrey extract, a turmeric extract, a syrup extract, a coriander extract, a ginseng extract, a cycin extract, or a yoquinin extract. Extract, Stevia extract, Acacia extract, Gentian extract, Hop extract, Hioki sorghum extract, Azuki extract, Arnica extract, Mentha extract, Licorice extract,
Bakuryo extract, soybean extract, perilla extract, kina extract, horsetail extract, apricot kernel extract, hawthorn extract, senkyu extract, cinnamon extract, litchi extract,
The hair restorer according to claim 2, wherein the hair restorer is one or more components selected from the group consisting of a clove extract, a spruce extract and royal jelly.

[0010]

Embodiments of the present invention will be described below. A. The hair restorer according to the present invention (hereinafter referred to as the hair restorer of the present invention) is a "hair restorer comprising a component having an action of maintaining or prolonging the anagen phase in the hair cycle as an active ingredient".
A component having an action of maintaining or prolonging the anagen phase in the hair cycle (hereinafter, also referred to as a anagen component) can promote hair elongation by this action.

[0011] As the growth period extending component, for example, Comfrey [also known as Symphytum Officinale L.]
It is a perennial plant native to Europe. Extract; turmeric (Curcuma domestica Valton) extract (usually extracted from roots); diatom (jujube (Zizyphus ju
jube Miller Var. inermis Rehder) or a related plant thereof; an extract of a plant belonging to the genus Cilantro [eg,
Coriandrum sativum L.
Extract (usually extracted from whole plants or immature fruits) [hereinafter referred to as coriander extract]; plants belonging to the genus Datura (eg, Datura stramonium L.), Datura stramonium L. var.chalybea Ko
ch) etc.) (usually extracted from leaves or seeds)
[Hereinafter referred to as Datura morning glory extract]; an extract of cycin ( Asarum sieboldii Miq. Or a variant or subspecies thereof) (usually extracted from the root or rhizome); Yokuinin [ Coix lachryma- job
. i L. var ma-yuen Stapf ) reward and pericarp from the fruit of the, extract minus the seed coat]; Stevia (Stevia re
bandiana Bertoni) (usually extracted from above-ground parts); Acacia (Uncaria gambir Roxburgh) extract (usually extracted from leaves or shoots); Gentian (gentian, Gentiana lutea L.) extract (usually Extract from hops (Humulus luplus L.) [usually extracted from female cotyledons (cones)]; Hioki-koi [ Hioki-koi ( Isodon japonicus Hara) belonging to the genus Yamanaka or a variant or sub-species thereof Seed] extract (usually extracted from leaves called life-prolonging grass);

An extract of Azukia (Azukia angularis Ohw ₁ ) (usually extracted from seeds); Arnica (Arnica montana )
L.) extract (usually extracted from flowers); Licorice (Glycy
rrhiza glabra L.) extract (usually extracted from roots or rhizomes); Mint extract (Mentha arvensis L. etc.);
Bulky extract (extract of poria cocos wolf, which is customary and is obtained by extracting the outer layer from most sclerotia ); soybean (Glycine max Merril) extract (usually extracted from seed) ; Perilla frutescens
Extract of Britton var. Acuta Kudo etc. (usually extracted from leaves or branches); kina (Cinchona succirubra Pavon et.
Extracts such as Klotzch (usually extracted from bark);
(Equisetum arvenes L.) extract (usually extracted from whole plant); plant extract (hereinafter apricot kernel grain) extracted from dried and ground material of nucleus (endocarp) of Hong apricot (Prunus armeniaca L. etc.) Extract); hawthorn (Crataegus cuneata )
Extract of Siebold et Zuccarini (usually extracted from fruits); extract of Cnidium (Cnidium Rhizome Makino) (usually extracted from rhizomes); cinnamon extract extracted from bark of Cinnamoum cassia Blume or its closely related plants; Ganoderma oleracea extract extracted from Ganoderma lucidum fruit body;
(Syzygium aromaticum Merrill et Perry) extract (usually extracted from buds); Daidai (Citrus aurantium L.
subsp.amara Engl.), a spruce extract which is an essential oil obtained by distillation from mature pericarp; royal jelly, cyclosporin A, retinol, and the like, but are not limited to these components. These components can be used alone as the hair growth period extending component in the hair restorer of the present invention, but they can also be used in combination of two or more.

[0013] whether a anagen extension component of the determination is not particularly limited as long as the determination method itself is reasonable to identify the growth-life extending effect, for example, in
Identification Method in vi tro also can be used, but a specific method in the in vivo, considering its simplicity and effectiveness, it is preferable to use a specific method of in vitro.

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

[0015] That is, this identification method is as follows: "A culture of hair follicle epithelial cells 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-testing method for testing the effect of maintaining or prolonging the anagen phase in the hair cycle, that is, focusing on hair follicle epithelial cells directly related to hair elongation, and using the cultured cells to obtain desired hair This is an in vitro hair growth drug assay method for determining the effect of maintaining or prolonging the growth phase in a cycle.

In this method for assaying a hair-growth agent, the target substance is added to a "hair follicle epithelial cell culture" 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 cell refers to a cell in which the outer root sheath cell and the matrix cell in the vicinity of the hair root are combined, and the inner hair papilla cell and the basement membrane covering the cell 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 in the hair cycle is the period when the hair is elongating, that is, the period when the hair follicle epithelial cells are dividing and proliferating. It is time to do it. In other words, the substance that maintains or prolongs the anagen phase in the hair cycle is a substance that prevents the hair from entering the catagen and telogen phases of the hair cycle by maintaining the division and proliferation activity of hair follicle epithelial cells by its administration. That is, it is concluded that the substance is a substance that continues to promote or maintain the proliferation 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 the dermal papilla fat of an animal to determine its growth promoting effect, etc.

As a method of in vivo determination, for example, “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 growth period of the target substance in the hair cycle is determined. A hair growth drug assay method for assessing the effect of maintaining or prolonging hair growth, i.e., hair growth in nude mice that are hairless in principle, but whose hair growth site moves to the body surface over time with characteristic hair growth. A method of testing the length of the anagen phase in the hair cycle by specifying the width of the hair site and the moving speed of the hair growth site can be mentioned.

B. Further, the hair restorer of the present invention is a hair restorer containing, as an active ingredient, "a component having an action of inhibiting testosterone-5α-reductase" together with the above-mentioned components. A component having an effect of inhibiting testosterone-5α-reductase (hereinafter also referred to as a 5α-reductase inhibitory component) can inhibit hair loss by the action of 5α-reductase in the head by this effect.

As the 5α-reductase inhibitory component, for example, a peony [ Paeonia albiflora
Trichocarpa Bunge) or a variant or subspecies thereof (extractable from whole plant); mint extract;
Hawthorn extract; Jancan [ Sterculiaceae (Sterculiaceae)
ae ) belonging to the star clear foeti (Sterculia foeti
da L.), etc., and its pericarp is the main object of extraction]; Woro (Bolassus belonging to the family Palmae)
Flaverifare (Borassus flabellifera L.) and its flowers are the main target of extraction]
Torauzu [Lauraceae (Lauraceae) belonging to Rittsuae Odorifera (Litsea odorifera Val.) Derived from, etc.,
Extracts of which are the main extraction targets]; coriander extract and the like.

In addition, peach extract, goldfish extract,
Bokeh extract, Laceae extract, Ombo extract, Hikawa extract, Yamazakura extract, Snake strawberry extract, Dutch strawberry extract, Bonuntilla extract, Tormentilla extract, Almond extract, Apricot extract, Tonin extract, Spruce Extract, plum extract, capsicum extract, Japanese radish extract, black bean extract, terry high rose extract, rowan extract, Atlantic rose extract, raspberry extract, tokli strawberry extract, simabarai strawberry extract, shrimp strawberry extract , Waremokou extract, Spiraea extract, Chishima cherry extract, strawberry extract,
Fukubon extract, Karin extract, Oak extract, Tenmondou extract, Bakumondou extract, Engosaku extract,
Kobushi extract, Colombo extract, Konzulango extract, Sanshuyu extract, Sanzukon extract, Paniculata extract, Achillea millefolium extract, Tokonomi extract, Honeysuckle extract, Selenoa extract, Acacia extract, Fennel extract, Onji Extract, licorice extract, cinnamon extract, goby extract, peonies extract, shazens extract, senso extract, rhubarb extract, clove extract, areca extract, rosin extract, cuckoo thistle extract, gennoshoko extract , Sprouts extract, psycho extract, intin extract, oedum extract, yokuinin extract, soybean extract, nigaki extract,

Extracts of oyster scallop, catalpa extract, japonicus extract, valerian extract, velvet extract, primrose extract, burdock extract, honey locust extract, sanshishi extract, primrose extract, sedron extract, yerbarisa extract , Achicoria extract, Machiko extract, Cardosant extract, Ortiganigra extract, Chancabiedra extract, Chinese tea leaf or stem extract, Cotton seed extract, Taxus leaf or bark extract , Guarana seed extract, epicaquitene, epigallocaquiten, epigallocotechin galade, glycyrrhetinic acid, sinol, sinol analogues, unsaturated fatty acids such as specific unsaturated fatty acids (linoleic acid, petroselinic acid), etc. However, it is not limited to these components. These components can be used alone as the α-reductase inhibiting component in the hair restorer of the present invention, but they can also be used in combination of two or more.

The determination as to whether or not the component is an α-reductase inhibitor is not particularly limited as long as the determination method itself is appropriate for specifying the inhibitory effect of 5α-reductase. For example, there can be exemplified a method for identifying in vitro an inhibitory activity on testosterone-5α-reductase, which is involved in the conversion of testosterone into 5α-dihydrotestosterone, which is called a so-called action androgen.

Extraction of a plant-derived extract from the above components can be carried out by a method generally used for extracting a plant-derived extract. That is, it can be obtained by subjecting the above-mentioned plant as it is or after drying it as necessary, and then subjecting it to solvent extraction as it is or pulverized. 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; propylene glycol, 1,
Polyhydric alcohols such as 3-butylene glycol; hydrates of these alcohols; hydrocarbon solvents such as n-hexane and toluene; and lower alcohols such as methanol and ethanol as extraction solvents. Is preferred. When these lower alcohols are used as the extraction solvent, the resulting extract can be directly blended as an active ingredient of the hair growth period extender of the present invention,
It is also possible to once remove the extraction solvent and mix it after drying if necessary. In the hair restorer of the present invention, it is a matter of course that commercially available products of the above-mentioned various components can be used.

The amounts of the above components (the sum of the amounts of the growth-prolonging component and the 5α-reductase inhibitor) in the hair restorer of the present invention can be appropriately selected according to the specific form of the hair restorer of the present invention. Although it is not particularly limited, it is generally 0.000 as a dry matter with respect to the whole of the present drug.
It is blended so as to be at least 05% by weight and at most 20.0% by weight, preferably at least 0.01% by weight and at most 10.0% by weight.

[0027] 0.000 as a dry matter with respect to the whole agent
If the amount is less than 05% by weight, the desired effect of the present invention, the hair growth effect based on the hair follicle cell proliferation effect, is not sufficiently exhibited, which is not preferable. In addition, not only is it not possible to expect an increase in the effect corresponding to the increase in the amount of the compound, but also the tendency to cause troubles in the preparation becomes remarkable, which is not preferable.

The mixing ratio of the growth-prolonging component and the 5α-reductase-inhibiting component is to be appropriately selected according to the specific form of the hair restorer of the present invention and specifically selected components, and is not particularly limited. Although not necessary, it is preferable to mix them in approximately equal amounts. When the above components are blended in the hair growth agent of the present invention within a range deviating from this blending ratio, the action of one of them becomes too strong, and the synergistic hair growth is achieved by combining and combining components having desired different actions. The effect tends to stop being exhibited, which is not preferable.

As described above, the hair restorer of the present invention is a hair restorer containing the above-mentioned growth period extending component and 5α-reductase inhibitory component as active ingredients, and has an excellent hair growth epithelial cell proliferation activating action and the like. It has a prolongation promoting action and a hair loss preventing action by inhibiting 5α-reductase, and these two components act synergistically on hair, that is, the proliferation activity of hair follicle epithelial cells is maintained, As a result, the hair growth effect is extremely improved by improving the hair fixation rate on the scalp.

The hair restorer of the present invention can be used, for example, for alopecia caused by relatively higher proportion of telogen hair than anagen hair or alopecia caused by excessively increased 5α-reductase. It is a particularly effective drug. It is also possible to achieve a synergistic effect in other alopecia by using it in combination with other hair restorers having individual effects.

The dosage form that the hair growth agent of the present invention can take 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. Further, the form of the hair restorer of the present invention is arbitrary, and for example, tonic, hair cream, mousse, shampoo, rinse, cream, emulsion, lotion,
It can take the form of a pack or the like.

The hair restorer of the present invention is generally used in cosmetics, quasi-drugs, pharmaceuticals, etc., in addition to the above essential components, if necessary and as long as the intended effect of the present invention is not impaired. Various oily or aqueous components,
A humectant, a thickener, a preservative, an antioxidant, a fragrance, a coloring agent, various chemicals and the like can be added.

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.

[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, "%" and the content are indicated by weight unless otherwise specified. First, in v to evaluate the effect of the target substance on promoting hair growth
The cell proliferation test of itro 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. In this case, the medium was a serum-free medium [Keratinocyte G
rowth Medium (KGM)] (Keratinocyte Ser
um 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 growth of the cells were confirmed, the medium was changed, and thereafter the medium was changed every two days. The cells grown in this manner were transformed with 0.05 wt% trypsin-0.02% E
After treatment with DTA at 37 ° C. for 5 minutes, the reaction was stopped with an equal volume of 0.1% trypsin inhibitor, and centrifuged (800 ×
g, for 5 minutes) to collect the cells. Next, the cells were suspended in the above serum-free medium, and seeded on a collagen-coated (Type I) culture dish at a density of 5000 cells / cm ² ,
The medium was changed every two days until the cells became subconfluent, and again with 0.05 wt% trypsin-0.02% EDTA at 37 ° C.
After 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 freeze-up ( Cell Banker: manufactured by Diatron).
The concentration was adjusted to 0 × 10 ⁶ cells / ml, and 1.0 × 10 ⁶ cells were added to each cryotube, and the cells were 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, 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.

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.

Further, a 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 filtered through a cell strainer (100 μm, manufactured by Nalgene), put into 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. Cell concentration in 5.0 × 1
0 ⁴ was adjusted to cell / ml.

Then, at a rate of 0.2 ml / well, 96we
The cells were seeded on an ll-plate (type I collagen coated plate: manufactured by Falcon) (1.0 × 10 ⁴ cells / well), and left at room temperature for about 20 minutes until the cells sank to the bottom of the well. Thereafter, the cells were cultured at 37 ° C. and 5% CO ₂ for 1 day,
The desired cultured human hair follicle epithelial cells were obtained.

C. Preparation of test medium: (1) Preparation of extract 500 g of commercially available comfrey (dry matter) was added to 7.5 l of 3
It was immersed in 0% ethanol at room temperature (23 ° C.) for 5 days. The solvent was distilled off from the extract to obtain 50 g of a comfree 30% ethanol extract dry product. Further, 500 g of commercially available chamomile (dried product) was immersed in 7.5 l of water at room temperature (23 ° C.) for 5 days. The solvent was distilled off from the extract and then dried to obtain 100 g of an aqueous extract of chamomile.

Regarding other plant extracts, commercially available corresponding plants (in principle, dried products) were obtained and
Each dried extract was prepared by extraction according to the extraction method shown in the table. The royal jelly used was a commercially available royal jelly powder.

(2) Preparation of Target Substance-Added Medium About 1.5 mg of the target substance was weighed into a screw tube, and prepared with an organic solvent (DMSO) to a 0.2% solution. Next, a DMSO solution of the crude drug extract was added to the KBM medium in a 1000-fold amount [extract concentration: 2.0 × 1
0 ^-4 % (DMSO 0.1%)].

The crude drug extract used as the target substance includes comfrey extract, arnica extract, peppermint powder, soybean extract, kina extract, horsetail extract, clove extract, hop extract, perilla extract. Products, Acacia extract, Azuki powder, Saishin extract, Chimney extract, Cauliflower extract, Bucurio extract, Aloe extract, Reishi extract,
Gentian extract, licorice extract (including licorice flavonoids), shobu root. Turmeric extract, Spruce extract, Stevia extract, Hawthorn extract, Senkyu extract, Tiso extract, Saffron extract, Hioki sorghum extract, Yokunin extract, Apricot kernel particle extract, Coriander extract, Datura extract And royal jelly.

Similarly, as a control, a 0.2% DMSO solution of a tea extract (extraction method: water extraction) and a Bacmondou extract (extraction method: 10% ethanol extraction) were prepared. 0.2 ml of these target substance-supplemented media was added to 0.1% DMSO
It was added to 1.8 ml of the KBM medium containing the mixture, and the concentration of the target substance was adjusted to 2.0 × 10 ⁻⁵ % (10-fold dilution).

(2) 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 culture medium exchange: 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 a target substance-added medium and a control medium (200 μl / well). After the exchange, the cells were cultured at 37 ° C. and 5% CO ₂ for 2 days.

The exchange of the medium was performed using 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.

[0059]

(Equation 1)

The cell growth promotion index was calculated according to the following equation based on the cell growth degree calculated here.

(Equation 2)

F. Results: Table 1 below shows the cell proliferation degree of each of the measured target substances.

[Table 1]

From these results, it was confirmed that the above-mentioned components showed the growth activity of hair follicle epithelial cultured cells. 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.

Test Example 2 Measurement of testosterone-5α-reductase inhibitory activity: The inhibitory effect of the target substance on 5α-reductase is involved in the conversion of testosterone to 5α-dihydrotestosterone, which is a so-called active androgen. A test to be specified by evaluating the inhibitory activity on testosterone-5α-reductase in vitro will be described.

The effect of the target substance on testosterone-5α-reductase inhibitory activity was determined by the method of Takayasu et al. [S.
yasu, K. Adachi, J. Clin. Endrocrinol. Metab., 34, 1
098-1101 (1972)]. That is, the amount of testosterone reduced to 5α-dihydrotestosterone (also referred to as 5α-DHT) was measured using human hair roots, and 0.1% and 70% ethanol solutions of each target substance (dry matter) were tested. It can be specified by measuring the 5α-reductase inhibitory activity and determining the inhibition rate according to the following formula.

[0065]

(Equation 3)

As the target substances, extracts of Jankan, Woro, Legro, Down troars, Coriander, Peony, Yokuinin, Bokeh and Hikawa were used. In addition,
Each extract was extracted by the aforementioned extraction method. For example, as an extract of a peony, 500 g of a commercially available peony (dried product) is immersed in 7.5 l of water for 5 days, water is distilled off from the extract, and then dried to obtain 100 g of a dried aqueous extract of a peony. I got In addition, a psycho extract was used as a control. The results are shown in Table 2.

[0067]

[Table 2]

According to this result, Jankan, Woro, Legro, Down Trawas, Coriander, Peony,
The extract of yokuinin, bokeh and mulberry clearly has 5
An α-reductase inhibitory action was observed.

Hereinafter, the formulations of the hair restorer of the present invention are shown as examples, and their hair restorer effects were further examined. [Comparative Example] Preparation of Liquid Hair Restorative as Comparative Example 0.1% of 30% ethanol dried product of Comfrey described above
Are prepared by adding 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 restorer ( Comparative Example 2 ).

In the formulation of this liquid hair restorer, the dried product of the above-mentioned 10% ethanol extract of Bacmondou was prepared.
A liquid agent prepared by replacing 1% with 0.1% of the above comfrey 30% ethanol dried product was prepared as a control ( Comparative Example 1 ). Also, 0.1% of the above-mentioned dried water-dried peony is 70% ethanol 90%, sodium oleate 0.1%, dodecylbenzenesulfonic acid 0.4%.
9%, 0.5% of hydrogenated castor oil ethylene oxide (40 mol) adduct and ion-exchanged water (residual) were mixed and dissolved by stirring. Further, ion-exchanged water (10%) was added and mixed to obtain a liquid hair restorative ( Comparative Example 3 ).

Example 1 Preparation of Liquid Hair Restoration Agent The above comfrey 30% ethanol dried product 0.05
% And dried peony with water extract 0.05%, 70%
Mix with 90% ethanol, 0.1% sodium oleate, 0.49% dodecylbenzenesulfonic acid, 0.5% hydrogenated castor oil ethylene oxide (40 mol) adduct and 0.5% ion-exchanged water (residual) to dissolve. Further, ion-exchanged water (10%) was added and mixed to obtain a liquid hair restorer.

Test Example 3 Examination 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 carried out on humans by the following method and the test was carried out. A usage test was conducted. The test sample and the control sample were the hair growth agent of the present invention of Example 1, the agents of Comparative Examples 1 to 3, and 7
0% 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 growth-stopped hairs, were counted from the morphology 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 are shown in Table 3 below.

[Table 3]

From the results shown in Table 3, not only Comparative Example 1 but also Comfrey extract or 5α-
Compared with Comparative Examples 2 and 3 in which one of the extracts of the peony which is a reductase inhibitory component was used, the hair growth effect of Example 1 in which the two types of extracts were combined in the same amount in total was synergistically superior. There was found. From this, the hair restorer of the present invention, which has a synergistically improved hair restorer effect by using two types of hair restorer components having different action mechanisms in combination, is provided.

Hereinafter, hair growth agents of the present invention other than the above-mentioned dosage forms will be exemplified as examples. [Example 2] Preparation of emulsion type hair restorer (Blending components) Blending amount (% by weight) (Phase A) Comfrey extract dried product 0.5 Peony extract dried product 0.5 Polyoxyethylene (60 mol) addition-cured castor Oil 2.0 Glycerin 10.0 Dipropylene glycol 10.0 1,3-butylene glycol 5.0 Polyethylene glycol 1500 5.0 (Phase B) Cetyl isooctanoate 10.0 Squalane 5.0 Vaseline 2.0 Propyl parapen 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) Kaseikari 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, and phase D was gradually added thereto and dispersed with a homomixer. Next, dissolved C phase was added thereto, and finally dissolved E
The phase was added and emulsified with a homomixer to obtain an O / W emulsion type hair restorer.

[Example 3] Preparation of creamy hair restorer 0.5% of the above comfrey 30% ethanol dried product
And 0.5% of a dried water-extracted product of a peony was used to prepare a creamy Example 7 as follows. (Blending components) Blending 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 3 Fragrance 0.1 (Phase B) Comfrey extract dried product 0.5 Peony extract dried product 0.5 Glycerin 8.0 Dipropylene glycol 20.0 Polyethylene glycol 4000 5.0 Sodium dodecyl sulfate 0.1 Sodium hexametaphosphate 0 0.005 ion-exchanged water 43.995

<Production Method> The phases A and B were each dissolved by heating, mixed and emulsified by a homomixer to prepare a creamy hair restorer.

[0080]

Industrial Applicability According to the present invention, there is provided a hair restorer exhibiting a synergistic hair restorer action by combining hair nourishing components having different action mechanisms.

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[Procedure amendment]

[Submission date] January 30, 1998

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0039

[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.

Continuation of the front page (51) Int.Cl. ⁶ Identification code FI A61K 35/78 A61K 35/78 JQHNKADA ADAR 35/84 35/84 A (72) Inventor Yosuke Nakazawa Kohoku-ku, Yokohama-shi, Kanagawa 1050 Nippacho, Shiseido First Research Center Co., Ltd. (72) Inventor Masahiro Tajima 1050 Nippacho, Kohoku-ku, Yokohama, Kanagawa Prefecture, Shiseido No.1 Research Center

Claims (3)

[Claims] 1. A hair restorer comprising as an active ingredient a component having an action of inhibiting testosterone-5α-reductase and a component having an action of maintaining or prolonging the anagen phase in the hair cycle. 2. The hair restorer according to claim 1, wherein the component having an action of maintaining or prolonging the anagen phase in the hair cycle is a component having an activity of expanding hair follicle epithelial cells. 3. A component having an activity of proliferating hair follicle epithelial cells is a comfrey extract, a turmeric extract, a scallop extract, a coriander extract, a ginseng extract, a cycin extract, a yoquinin extract, or stevia. Extracts, Acacia catechu extract, Gentian extract, Hop extract, Hioki sorghum extract, Adzuki bean extract, Arnica extract, Mentha extract, Licorice extract, Bucurio extract, Soybean extract, Perilla extract, Kina extract One or two or more selected from the group consisting of radish extract, horse chestnut extract, apricot kernel extract, hawthorn extract, rose extract, cinnamon extract, litchi extract, clove extract, spruce extract and royal jelly 3. The component of claim 2,
The hair restorer according to the above.