JPH1192343A - Agent for prolonging hair growth period - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an agent for prolonging hair growth period which can maintain or prolong the growth period in the hair cycle by means of promoting the hair growth and is useful for depilation by formulating an unsaturated fatty acid and/or its derivative as active ingredients. SOLUTION: This agent is obtained by formulating an unsaturated fatty acid, e.g. preferably petroselinic acid, docosahexaenoic acid and oleic acid, which is shown by the formula Cn Hm O2 [the number (n) of carbon atoms is 12-28; the number (m) of hydrogen atoms is n+1-2n-2] and possesses cis configuration of double bond, and/or its derivative preferably in an amount of 0.01-10.0 wt.% based on the whole weight. It is preferable that the raw material of the above-stated unsaturated fatty acid is a plant, e.g. coriander and comfrey. This agent for prolonging hair growth period is used in various forms, e.g. tonic, hair cream and mousse.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention belongs to the technical field related to a hair growth period extending agent. More specifically,
The present invention relates to a hair growth period extending agent that maintains or prolongs the anagen phase in the hair cycle by promoting hair elongation.

[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. 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 extension effect, and as a result, the hair growth materials provided so far induce the hair to the growth period of the hair cycle, and the hair growth described above. Many focused on the induction effect and many focused on the 5α-reductase inhibitory effect.

[0004]

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 period extending agent containing the same as an active ingredient.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies on the components having the above-mentioned effect of extending the hair growth period in various substances using the hair growth drug assay method found by the present inventors. It has been found that a desired hair growth period extending effect is recognized in fatty acids and derivatives thereof,
The present invention has been completed.

That is, the present invention provides a hair growth period prolonging agent comprising an unsaturated fatty acid and / or a derivative thereof as an active ingredient. The unsaturated fatty acids are generally of the formula C _n H _m O ₂ [number n of carbon atoms 12 to 28, hydrogen atoms m = n +. 1 to
2n-2].

Further, it is preferable to select, from these unsaturated fatty acids, unsaturated fatty acids whose double bond part is a cis form. As unsaturated fatty acids satisfying these conditions, for example, dodecenoic acid, tridecenoic acid, heptadecenoic acid, petroselinic acid, palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, α-linolenic acid, γ-
Linolenic acid, eicosenoic acid, eicosatrienoic acid, erucic acid (cis-13-docosenoic acid), docosadienoic acid, docosatrienoic acid, docosatetraenoic acid, docosapentaenoic acid,
One or more unsaturated fatty acids selected from the group consisting of arachidonic acid, nervonic acid and docosahexaenoic acid can be mentioned.

[0009] Among these unsaturated fatty acids, it is particularly preferable to select petroselinic acid, docosahexaenoic acid or oleic acid.

As described above, in the present invention, the "hair growth period prolonging agent" is used to maintain or promote at least the mitotic proliferation activity or hair shaft elongation activity of hair follicle epithelial cells by the hair growth drug assay method described below. It is a hair-related drug that has been combined with a component having an effect of extending the growth period of hair as an active ingredient, and particularly focuses on the above-described effect of extending the growth period of hair, and has the characteristics of a so-called “individually effective hair restorer”.

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, so that 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.

[0012]

Embodiments of the present invention will be described below. The hair growth period extending agent of the present invention is a hair-related agent containing an unsaturated fatty acid and / or a derivative thereof as an active ingredient as described above.

The unsaturated fatty acid which can be used as an active ingredient of the hair growth period prolonging agent of the present invention is not particularly limited as long as its safety against the human body is ensured. Unsaturated fatty acids having a double bond in the cis form are preferred. Specifically, for example, dodecenoic acid, tridecenoic acid, heptadecenoic acid, petroselinic acid (cis-6-octadecenoic acid), palmitoleic acid (cis-9-
Hexadecenoic acid), oleic acid, vaccenic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, eicosenoic acid,
Eicosatrienoic acid, erucic acid (cis-13-docosenoic acid),
Docosadienoic acid, docosatrienoic acid, docosatetraenoic acid, docosapentaenoic acid, arachidonic acid, nervonic acid (cis-15-tetracosenoic acid) or docosahexaenoic acid.

[0014] Of these unsaturated fatty acids, it is particularly preferred to select petroselinic acid, docosahexaenoic acid and / or oleic acid.

[0015] It is also preferable that the unsaturated fatty acid used as a raw material of the plant is, for example, an unsaturated fatty acid such as coriander or comfrey, as an active ingredient of the present invention.

It is also possible to select derivatives of these unsaturated fatty acids as active ingredients of the hair growth period extender of the present invention.

Specific examples of the derivative include unsaturated fatty acid monoglyceride, unsaturated fatty acid diglyceride, unsaturated fatty acid triglyceride, pharmaceutically acceptable unsaturated fatty acid salt, unsaturated fatty acid ester, unsaturated fatty acid amide, Examples include unsaturated fatty acid dibasic acids or pharmaceutically acceptable salts thereof. Each of these unsaturated fatty acids or derivatives thereof can be produced by a generally known method. Of course, a commercially available product can also be used.

These unsaturated fatty acids or derivatives thereof are
It has strong hair follicle epithelial cell proliferation activity and hair shaft elongation activity, and these can be used alone or in combination of two or more as active ingredients of the hair growth period extending agent of the present invention.

The amount of the unsaturated fatty acid and / or its derivative in the hair growth period extender of the present invention can be appropriately selected according to the specific form of the hair growth period extender of the present invention, and is particularly limited. Although not to be done, it is generally not less than 1.0 × 10 ⁻⁹ wt% and not more than 20.0 wt%, preferably not less than 0.01 wt% and not more than 10.0 wt% based on the whole agent.
% By weight or less.

When the amount is less than 1.0 × 10 ^-9 % by weight based on the whole preparation, the effect of extending the hair growth period based on the hair follicle epithelial cell proliferation effect, which is the desired effect of the present invention, is sufficiently achieved. It is not preferred because it is not exhibited, and if it is added in excess of 20.0% by weight, not only is it not possible to expect an increase in the effect corresponding to the increase in the amount added, but also there is a marked tendency to cause problems in the formulation, which is not preferable.

Thus, the hair growth-prolonging agent of the present invention containing the unsaturated fatty acid or its derivative as an active ingredient has a hair growth period-prolonging effect based on an excellent hair follicle epithelial cell proliferation action. As described above, for example, the growth of hair follicle epithelial cells in the vicinity of the hair root is slow, so that the anagen is shortened and the ratio of telogen hair is relatively larger than that of anagen hair. It is a particularly effective drug for alopecia caused by it. It is also possible to increase the synergistic effect in specific alopecia by using it in combination with other hair restorers having individual effects.

The means for specifying the maintenance or prolongation of the growth period in the hair cycle, which is the essential effect of the desired effect of the hair growth period prolonging agent of the present invention, is the method itself. is not particularly limited as far as it is reasonable to identify its action, also specific method in example in vitro, can be used, but a specific method in the in vivo, considering its simplicity and effectiveness, in Vitr
It is preferable to use the identification method in o .

The following is a brief description of one of the in vitro identification methods, which is characterized by examining the effect of proliferating cultured follicular epithelial cells (more specifically, in Example 1). Described in.).

[0024] 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. Perform a primary screening to test the effect of prolonging the anagen phase in the hair cycle, and for the target substance that has been confirmed to be effective by this primary screening,
A hair-growth drug assay method comprising culturing a human hair follicle in a serum-free medium to which the target substance has been added, and performing a secondary screening to further select a target substance in which hair shaft elongation has been observed. This is an in vitro hair growth drug assay method that focuses on hair follicle epithelial cells that are directly involved in hair growth and specifies the effect of extending the anagen phase in a desired hair cycle by using the cultured cells.

In the primary screening of this method for assaying a hair-growth agent, in the first step, "hair follicle epithelial culture," which is a cultured cell obtained by isolating hair follicle epithelial cells of animals (including humans, the same applies hereinafter) Contact the target substance with the cells,
The presence or absence and / or strength of the proliferation is specified.

Hair follicle epithelial cells particularly refer to epidermal cells derived from hair follicles such as outer root sheath cells and matrix cells near the hair root, and the inner dermal papilla cells are excluded.

The anagen in the hair cycle is exactly the elongation of the hair shaft, that is, the time when the hair follicle epithelial cells are dividing and proliferating, and the catagen and the telogen are the periods when they slow down and pause. is there.

In other words, a substance that prolongs or maintains the anagen phase in the hair cycle maintains the division and proliferative activity of the hair follicle epithelial cells by its administration, whereby the hair shifts to the catagen and telogen phases in the hair cycle. It is concluded that this is a substance that prevents the growth of hair follicle epithelial cells, ie, a substance that continues to promote or maintain the proliferation of hair follicle epithelial cells.

In the secondary screening, human hair follicles are cultured, and a target substance in which hair shaft elongation is observed is further selected. In this secondary screening, it is necessary to prepare a hair follicle fragment first. Normally, hair in the anagen phase is selected from the hair of human healthy hair, and the upper part of the hair bulb of this hair is cut about 2 mm to prepare a hair follicle fragment.

This hair follicle fragment can maintain its growth phase for a relatively long time when cultured in a basal medium containing insulin, but can be cultured within a short period of about one week when cultured in a basal medium containing no insulin. It changes to a state resembling a catagen follicle.

Therefore, when culturing in a basal medium not containing insulin, it is preferable to evaluate within about one week, and when using a medium containing insulin, it is preferable to extend the culturing period to about two weeks. Will be possible.
In either medium, the degree of hair shaft elongation can be observed by culturing this hair follicle in the presence of the target test substance.

As the basal medium used in this secondary screening, a commonly used medium for culturing animal cells can be used, and antibiotics or antibacterial agents can be added to prevent contamination. it can.

The hair follicles can be cultured under normal conditions for animal culture such as 5% CO ₂ and 37 ° C. The culture is usually performed for 5 to 14 days. Elongation of the hair shaft in the human hair follicle can be visually confirmed, for example, with a stereomicroscope equipped with a micrometer.

As described above, by performing the primary screening and the secondary screening, it is possible to specify the action of maintaining or prolonging the anagen phase and the action of elongating the hair shaft in the hair cycle.

In addition, as an in vitro method for assaying a hair growth drug, a method in which a target substance is allowed to act on hair papilla cells of an animal to determine its growth promoting effect can be used.

In addition, 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.

The dosage form that the hair growth period extender 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 growth period prolonging agent of the present invention is arbitrary, and for example, can be in the form of tonic, hair cream, mousse, shampoo, rinse, cream, milky lotion, lotion, pack and the like.

Hair growth period extender of the present invention Generally used in cosmetics, quasi-drugs, pharmaceuticals, etc., in addition to the above essential components, if necessary 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 added.

For example, oils such as higher saturated fatty acids, solid paraffin, liquid paraffin, silicone oil, squalane, glyceryl monooleate, olive oil, higher alcohols; glycerin, hyaluronic acid, propylene glycol, maltitol, atelocollagen, sodium lactate Humectant; thickener such as quince, carboxyvinyl polymer, xanthan gum; nicotinamide;
Vasodilators such as benzyl nicotinate, vitamin E acetate, assembly extract, carpronium chloride, and acetylcholine derivatives; amino acids such as serine, methionine, and arginine; vitamin B ₆ , vitamin E (or derivatives thereof), biotin, pantothenic acid ( Or derivatives thereof); nicotinic acid, methyl nicotinate,
Nicotinic acid esters such as tocopherol nicotinate;
Skin function enhancers such as cepharanthin; female hormones such as estradiol; anti-inflammatory agents such as glycyrrhetinic acid (or a derivative thereof); antibacterial agents such as hinokitiol, hexachlorophen, benzalkonium chloride, bithionol; , Zinc (or a derivative thereof), lactic acid (or an alkyl ester thereof), and the like; and organic acids such as citric acid, and the like. The specific formulation of the hair growth period extender of the present invention will be described later.

[0040]

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, an in vitro cell proliferation test for evaluating the effect of the plant extract used in the Examples and the like on extending the hair growth period 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 grown in this manner were added to 0.05 wt.
After treatment with PBS (-) containing 5% trypsin-0.02% at 37 ° C for 5 minutes, the reaction was stopped with an equal amount of 0.1% trypsin inhibitor, and the cells were collected by centrifugation (800 xg, 5 minutes). .

Next, cells were resuspended in serum-free medium described above were seeded in culture dishes collagen-coated (Type I) at a density of 5000 cells / cm ^2, culture medium every two days until the cells became subconfluent After the exchange, the cells were re-exchanged with PBS (−) containing 0.05 wt% trypsin-0.02% EDTA at 37 ° C. for 5 hours.
After treatment for 1 min, the reaction was stopped with an equal volume of 0.1% trypsin inhibitor, centrifuged (800 xg, 5 min), and the resulting human hair follicle epithelial cells were subjected to cell freezing (Cell Banker). : Diatron) and add 1.0
The concentration was adjusted to × 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. After inoculating the hair follicle epithelial cells into a culture flask, the cells were cultured at 0.25% trypsin-0.02%.
After treatment with PBS (-) containing 0.1% EDTA, the reaction was stopped with 0.1% trypsin inhibitor, and the system was incubated at 1500 rp.
centrifugation at 5 m for 5 minutes, remove the supernatant, and add KG to the residue.
A cell suspension was prepared by adding 20 ml of M medium.

A 96-well plate at a rate of 0.2 ml / well
(Type I collagen coated plate: manufactured by Falcon) (3.0 × 10 ³ 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 Newborn (3-4 day old) rats were sacrificed with carbon dioxide, disinfected with ethanol, and the back skin of these rats was collected with scissors. . Then, the collected back skin was
After immersion in PBS (-) containing% PSF, the subcutaneous fat, film, and the like below the skin fat layer were removed with scissors for dissection. Then, the back skin was immersed again in PBS (-) containing 1% PSF, and further immersed in 0.25% trypsin-0.02%.
It was immersed in PBS (-) containing EDTA at 4 ° C overnight.

After immersion in this trypsin solution, the dermis layer and the epidermis layer of the back skin were peeled off with forceps, and the dermis layer was replaced with a Ham's F12 medium containing 0.35% collagenase [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-Glutamin
e (146), Glycine (7.5), l-Histidine (HCl: 19), l-Isoleu
cine (3.9), l-leucine (13.1), l-Lysine (HCl: 36.5), l-Met
hionine (4.5), l-Phenylalanin (5.0), Proline (34.5), lS
erine (10.5), l-Threonine (11.9), l-Tryptophane (2.0), l
-Tyrosine (5.4), l-Valine (11.7), Biotine (0.0073), Chol
ine (Cl: 14.0), VitaminB12 (1.36), folate (1.32), Inositol
(18.0), Nicotinamide (0.037), Pantothenic acid (Ca: 0.477),
VitaminB6 (HCl: 0.062), VitaminB2 (0.038), VitaminB1 (HC
l: 0.337), CaCl ₂ (2H ₂ O: 44.0), CuSO ₄・ 5H ₂ O (0.0025), FeSO
_{4 · 7H 2 O (0.834)} , KCl (224.0), MgCl 2 (6H 2 O: 122), "Proc.N
USA, 53, 288 (1965) ", the same applies hereinafter). The medium containing the cut product was cultured at 37 ° C.
For 35 minutes (60 rpm). After infiltration, pipetting was performed until no clumps were seen in the collagenase reaction product, and the DNase (10000 unit) -containing Ha
m's F12 medium was added and left for 5 minutes.

After standing, the obtained suspension was further pipetted and filtered through a nylon mesh (Nytex 157 mesh). The suspension was diluted with PBS (−), and the diluted suspension was centrifuged (4 ° C., 400 rpm).
, 5 minutes). After centrifugation, the fat was removed from the system except the supernatant.

Next, PBS (-) was added to the residue to suspend it, which 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-0.02% EDTA was added, and the cell suspension was added. Incubate at 37 ° C for 5 minutes.

After completion of the incubation, equal amounts of 0.1% trypsin inhibitor and Ham's F12 medium were added, and the cell suspension was washed with a cell strainer (100 μm Nalgene).
The suspension was centrifuged (4 ° C., 1500 rpm, 5 minutes). The supernatant was removed from this system to obtain the desired hair follicle epithelial cells as a residue. The hair follicle epithelial cells in cell freezing solution (Cell Banker: Dia Tron Ltd.) was added, was adjusted to a concentration of ^{1.5 × 10 7 cell / ml,} 1.5 × each 10 ⁷ cell to each cryotubes And stored frozen.

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, FAD medium [Ham's F1
2 medium (to be described later) and MEN medium at a volume ratio of 3: 1 mixed with insulin (5.0 μg / ml), hydrocortisone (0.45 μg / ml), epidermal growth factor
A medium containing (EGF) (10.0 ng / ml), cholera toxin (10 ^-9 M) and fetal calf serum (10%), and so on.]
Was added to dilute the cell solution, and the system was centrifuged (10 ° C. or lower, 1500 rpm, 5 minutes). After centrifugation, the supernatant was removed, FAD medium was added to the system, and pipetting was repeated until no cell mass was observed.

Next, the cell concentration was 2.5 × in the FAD medium.
It was adjusted to be 10 ⁵ cells / ml. The cells were seeded in a 75 cm ³ culture flask coated with type I collagen, and cultured overnight at 37 ° C. and 5% CO ₂ . After culturing, the system was washed with PBS (-), and PBS (-) containing 0.25% trypsin-0.02% EDTA was added.
This was incubated at 37 ° C., 5% CO ₂ for 4 minutes. Next, 0.1% trypsin inhibitor of the same volume as the trypsin solution was added to the system, and the mixture was slightly shaken once, and then the supernatant was removed to remove fibroblasts contaminating the system.

Furthermore, KGM medium [Keratinocyto growth medium: Keratinocytob) 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 hereinafter), and cultured at 37 ° C. and 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 with a FB contamination rate of 2% or more were excluded from the subject of the assay.

The system was washed with PBS (-), PBS (-) containing 0.25% trypsin-0.02% EDTA was added, and this was incubated at 37 ° C for 3 minutes. Taking advantage of the difference in reactivity of epithelial cells and fibroblasts to trypsin, trypsin was removed and 0.25% trypsin-
PBS (-) containing 0.02% EDTA was added, and 37
The mixture was shaken at 20 rpm for 5 minutes.

Next, after confirming the detachment of the cells under a microscope, 10% FBS-containing DMEM medium was added, pipetting was performed, and the system was centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, KGM medium was added, and pipetting was performed until the cell mass disappeared.

The suspension was added to a cell strainer (100 μm Nalg).
After filtration with a hemocytometer, the KGM medium was added to the system to bring the cell concentration in the system to 2.0 × 10 ⁴ cells / ml. It was adjusted to become. Subsequently, the cells were seeded at a rate of 0.2 ml / well on a 96-well-plate (type I collagen coated plate: manufactured by Falcon) (4.0 × 10 ³ cells / well), and about 20 × 10 ³ cells / well until the cells sank to the bottom of the well. It was left at room temperature for minutes. Then 37
Culturing was performed at 5 ° C. and 5% CO ₂ for 1 day to obtain desired human hair follicle epithelial cells.

C. Preparation of test medium: (1) Preparation of culture medium containing target substance The target substance was adjusted to 1.0 × 10 ⁻⁶ mol / l based on the whole medium, and the KB was adjusted so that the concentration of the solvent (DMSO) became 0.1%.
M medium.

(2) Preparation of control medium • Negative control: DMSO was added to KBM medium.
(Solvent) was added to the medium to prepare a final concentration of 0.1%. -Positive control: In a negative control medium, a final concentration of 5 μg / ml insulin, 0.5 μg / ml
It was prepared by adding ml of hydrocortisone.

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 medium was replaced with the KGM in the well.
The medium was removed with a multipipette while taking care not to damage the cells adhering to the bottom surface, 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 ° C).
% 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.

[0066]

(Equation 1)

Further, the cell growth promotion index was calculated according to the following formula.

(Equation 2)

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

[Table 1]

From these results, it was confirmed that the unsaturated fatty acids had a growth activity of hair follicle epithelial cells in culture. In other words, it was found that the unsaturated fatty acids exhibited the effect of maintaining and prolonging the anagen phase in hair by maintaining the mitotic proliferation activity of hair epithelial cells.

G. Examination of hair shaft elongation (secondary screening) Further, the hair elongation effect of the above unsaturated fatty acids was examined. Human Hair Follicle Organ Cultures The growing hair follicles were isolated from human scalp using a micro-blade under a stereomicroscope. The isolated hair follicle is "Williams
After washing with an E medium (Gibco) plus a medium containing penicillin, streptomycin and fungizone (hereinafter also referred to as (-) medium), the length was measured, and 10 ng / ml was further added to the (-) medium. In a medium supplemented with hydrocortisone, 10 μg / ml insulin, 10 ng / ml sodium selenite and 10 μg / ml transferrin (hereinafter also referred to as (+) medium) (using a 24-well microplate: 1 ml per well) Sink 5%
The cells were cultured overnight at 37 ° C. under CO ₂ (pre-culture). The length of the hair follicles cultured again after this pre-culture was measured to be 0.
Hair follicles of 25 mm or more were selected and divided into three groups of nine hair follicles so that the degree of elongation was uniform. The elongation of the hair shaft in the hair follicle was visually observed on the microplate using an inverted microscope equipped with a micrometer.

The above unsaturated fatty acid, which is a target substance for evaluation , is added at a final concentration of 1.0 ×
10 ^-5 mol / l (DMSO 0.1%)
(-) The medium was prepared by adding a DMSO solution of the target substance to the medium. At the same time, a medium (control medium) was prepared by adding an organic solvent (DMSO) to the (-) medium to a final concentration of 0.1%.

The medium of the three hair follicle groups described above was replaced with the above medium, respectively, and further cultured for 5 days under 5% CO ₂ under 37%.
Incubated at ℃. The length of each hair follicle on the 5th day from the start of culture was measured, and the elongation of the hair follicles in the hair follicles of the control group and the control substance-added group was compared. The results are shown in Table 2.

[0073]

[Table 2]

From these results, it is found that unsaturated fatty acids not only promote the growth of hair follicle epithelial cells, but also promote the growth of hair shafts in hair follicles, and have a prolonged hair growth period as a hair follicle organ. Was found to be recognized.
Hereinafter, the formulations of the hair growth period prolonging agent of the present invention are shown as examples, and the hair growth effects thereof were further studied.

[Example 1] Preparation of liquid hair growth period prolonging agent Petroseric acid 0.1% and oleic acid 0.1%
The mixture was dissolved by stirring and mixing with 0% ethanol 90%, dodecylbenzenesulfonic acid 0.49%, hydrogenated castor oil ethylene oxide (40 mol) adduct 0.5%, and ion-exchanged water (residual). Further, ion-exchanged water (10%) was added and mixed to obtain a liquid hair growth period extender. In the formulation of the liquid hair growth period prolonging agent, a liquid agent prepared by removing petroseric acid and oleic acid was prepared as a control (Comparative Example 1).

Example 2 Preparation of Emulsion Hair Growth Prolonging Agent An emulsion liquid hair growth prolonging agent was prepared according to the following formulation. Ingredients Ingredients Amount (% by weight) (A phase) Petroseric acid 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 Exchanged water 8.35 (D phase) Ion exchanged water 4.5 (E phase) KOH 0.12 Ion exchanged water 5.0

<Production Method> A phase and a B phase are each heated and dissolved at 60 ° C., mixed, and homogenized to form a gel.
Phase D was gradually added to this 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 Prolonging Agent A creamy hair growth extending agent was prepared according to 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 (B phase) Petroselinic acid 5.0 Glycerin 8.0 Dipropylene glycol 20.0 Polyethylene glycol 4000 5.0 Sodium dodecyl sulfate 0.1 Sodium hexametaphosphate 0.005 Ion-exchanged 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 growth period extender.

[Test Example 2] Examination of hair growth effect of hair growth period extender of the present invention To perform a trigram test. The test sample and the control sample are the hair growth period extending agent of the present invention of Examples 1 to 3, the agent of Comparative Example 1, and 70% ethanol.

Test Method The hair roots of the extracted hair before and after use of the above sample were observed under a microscope, and the number of “resting hair roots”, which are the hair roots of hair that had stopped growing, was determined based on the shape of the hair root (for those who report hair loss). Has been found to have a higher proportion of this telogen root than a normal person.)
Was counted, and the hair growth effect of these samples was 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, 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.

[0083]

[Table 3]

From the results shown in Table 3, the hair growth-prolonging agent of the present invention containing petrothenic acid, which is an unsaturated fatty acid, as an active ingredient has a hair-growth effect based on the hair growth-prolonging effect of petroselinic acid. Was. In addition, the unsaturated fatty acids other than petroselinic acid described above also exhibited a hair growth period extending effect similar to that of petroselinic acid. It is clear that the hair growth effect can be observed in the same manner as in the above Examples.

[0085]

Industrial Applicability According to the present invention, there is provided a hair growth-prolonging agent that maintains or prolongs the anagen in the hair cycle by promoting hair elongation.

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[Procedure amendment]

[Submission date] January 30, 1998

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0047

[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 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
F-containing PBS (-), and further immersed in PBS (-) containing 0.25% trypsin-0.02% EDTA.
Soaked overnight at ° C.

Continued on the front page (72) Inventor Jun Suzuki 2-12-1 Fukuura, Kanazawa-ku, Yokohama, Kanagawa Prefecture Inside Shiseido Second Research Center Co., Ltd. (72) Tsutomu Soma 2-12-, Fukuura, Kanazawa-ku, Yokohama, Kanagawa 1 Shiseido 2nd Research Center Co., Ltd. (72) Inventor Masashi Ogo 2-12-1 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa Prefecture 2nd Shiseido 2nd Research Center Co., Ltd. (72) Inventor Masahiro Tajima Kohoku, Yokohama-shi, Kanagawa 1050 Nippa-cho, Tokyo-ku Shiseido First Research Center

Claims (5)

[Claims] 1. A hair growth period extending agent comprising an unsaturated fatty acid and / or a derivative thereof as an active ingredient. Wherein the unsaturated fatty acid has the formula C _n H _m O ₂ [number of carbon atoms and n is 12 to 28, the number of hydrogen atoms m = n +. 1 to
The hair growth prolonging agent according to claim 1, which is an unsaturated fatty acid represented by 2n-2]. 3. The hair growth period extending agent according to claim 1, wherein the double bond of the unsaturated fatty acid is a cis form. 4. The unsaturated fatty acid is dodecenoic acid, tridecenoic acid, heptadecenoic acid, petroselinic acid, palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, eicosenoic acid, eicosatrienoic acid. One or two unsaturated fatty acids selected from the group consisting of, erucic acid (cis-13-docosenoic acid), docosadienoic acid, docosatrienoic acid, docosatetraenoic acid, docosapentaenoic acid, arachidonic acid, nervonic acid and docosahexaenoic acid The hair growth period prolonging agent according to any one of claims 1 to 3, which is at least one unsaturated fatty acid. 5. The hair growth extender according to claim 4, wherein the unsaturated fatty acid is petroselinic acid, docosahexaenoic acid and / or oleic acid.