JP2000063242A - Hair tonic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a hair tonic having hair growth period extension effect, namely, capable of maintaining and extending the hair growth period in the hair cycle through promoting hair growth by including a sugar glyceride as active ingredient. SOLUTION: This hair tonic is obtained by including pref. 0.01-10.0 wt.% of a sugar glyceride pref. of the formula (R1 is a sugar residue; R2 and R3 are each H or a saturated fatty acid residue or the like) as active ingredient; wherein the sugar is pref. galactose, glucose or fructose. The formulation of this hair tonic is e.g. a liquid, milky lotion, ointment, being in the form of tonic, hair cream moose, shampoo, etc. Furthermore, this hair tonic may be formulated with a surfactant (e.g. higher saturated fatty acid), moisturizer (e.g. glycerol), thickening agent (e.g. quince mucous matter), antiseptic (e.g. salicylic acid), vasodilator (e.g. nicotinamide), etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention belongs to the technical field relating to hair-growth agents. More specifically, it relates to a hair-growth agent having a hair-growth-prolonging effect of maintaining or prolonging the growth phase in the hair cycle by promoting hair growth.

[0002]

2. Description of the Related Art In modern society, which is said to be an aging society and a stress society, head hair is increasingly exposed to hair loss due to various causes. In response to this, various attempts have been made to provide a superior “hair growth agent”. The main effects of hair restorers on hair are: hair growth inducing effect (hair growth promoting effect, growth phase inducing effect), hair thickening effect, hair growth phase extending effect, 5α-reductase inhibitory effect, blood circulation promoting effect. ，
It has effects such as bactericidal effect, anti-dandruff effect, moisturizing effect, and antioxidant effect.

However, in spite of various attempts as described above, conventional hair-growth agents have not always had sufficient hair-growth effects such as hair loss prevention and hair growth effects. This is probably because there are various causes of hair loss and the mechanism of hair growth is very complicated. The "hair-growth agents" that have been provided so far have been developed with a relatively rough concept of hair loss, in other words, the phenomenon of "hair loss", which was deliberately grasped. Is never many. The major reason for this is that it cannot be denied that the hair-growth drug assay method capable of simply assaying 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 hair growth-prolonging effect, and as a result, the hair-growth agents that have been provided so far are the above-mentioned hair growth that induces hair to the growth phase of the hair cycle. Many focused on the inducing effect and on the 5α-reductase inhibitory effect.

[0004]

The object of the invention is to solve is therefore an object of the present invention have in
Establishing a simple hair-growing period assay method for assaying the above hair growth period-prolonging effect performed in vitro , and using the hair-growing period assay method to find a hair-growing agent containing the above-mentioned component having a hair growth period-prolonging effect as an active ingredient. I aimed at that.

[0005] Of the series of problems, the present application aims to find, in particular, the above-mentioned component having an effect of extending the hair growth period, and to provide a hair restorer containing this as an active ingredient.

[0006]

Means for Solving the Problems The present inventor diligently studied various substances having the above hair growth period-prolonging effect by using the hair-growing drug assay method found by the inventor. In addition, they found that the desired effect of extending the hair growth period was recognized, and completed the present invention.

That is, the present invention provides a hair restorer containing a sugar glyceride as an active ingredient.

This sugar glyceride has the formula (I)

[Chemical 2] [In the formula, R ¹ is a sugar residue, and R ² and R ³ may be the same or different from each other and are a hydrogen atom, a saturated fatty acid residue or an unsaturated fatty acid residue. ] It is preferable that it is a sugar glyceride represented by these. In the formula (I), the saturated fatty acid or the unsaturated fatty acid is represented by the formula (II) C _n H _m O ₂ (II) [wherein 6 ≦ n ≦ 28 and n + 1 ≦ m ≦ 2n−2. ] It is preferable that it is a saturated fatty acid or unsaturated fatty acid represented by these. Further, in the formula (I), R ² and R ³ are preferably hydrogen atoms or unsaturated fatty acid residues. Further, the saturated fatty acid is preferably a cis unsaturated fatty acid.

Among these unsaturated fatty acids, particularly preferred unsaturated fatty acids are petroselinic acid, oleic acid and linoleic acid. The sugar in the sugar glyceride is preferably galactose, glucose or fructose.

[0010] As described above, in the present invention, the "hair-growth agent" means that the hair growth-promoting activity of at least the hair follicle epithelial cells is maintained or promoted by the hair-growing agent assay method described below. It is a hair-related drug containing a component having an effect of prolonging the growth period as an active ingredient, particularly focusing on the effect of prolonging the hair growth period,
So-called "individually effective hair-growth agent".

This "hair-growth agent" has a short growth period due to, for example, slow growth of hair follicle epithelial cells in the vicinity of hair roots, and thus has a relatively large proportion of quiescent hair rather than anagen hair. It is a particularly effective drug for alopecia caused by the fact that In addition, by using it in combination with other hair-growth agents having individual effects, it is possible to achieve a comprehensive and synergistic effect in a wide range of alopecia. That is, this "hair-growth agent" has an application that is distinct from the general use of a hair-growth agent that has the concept including the above-mentioned effects (1) to (3).

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. The hair restorer of the present invention is a hair restorer containing a sugar glyceride as an active ingredient thereof as described above.

The sugar glyceride which can be an active ingredient of the hair-growing agent of the present invention is not particularly limited as long as the safety to the human body is ensured, but the formula (I)

[Chemical 3] A sugar glyceride represented by In formula (I), R ¹ is a sugar residue. The sugar may be either a monosaccharide or a polysaccharide. Examples of the monosaccharide include galactose, glucose, fructose, mannose, and the like, and the polysaccharide includes sucrose,
Maltose, lactose, trehalose, hyaluronic acid, etc. can be mentioned. Of these sugars, galactose, glucose or fructose are preferable, and galactose or glucose is particularly preferable.

In the formula (I), R ² and R ³ may be the same or different and each is a hydrogen atom, a saturated fatty acid residue or an unsaturated fatty acid residue. This saturated fatty acid or unsaturated fatty acid has the formula (II) C _n H _m O ₂ (II) [wherein 6 ≦ n ≦ 28 and n + 1 ≦ m ≦ 2n−2. ] Saturated fatty acid or unsaturated fatty acid represented by Further, in the formula (I), R ² and R ³ are preferably hydrogen atoms or unsaturated fatty acid residues, and in particular, at least one of R ² and R ³ is an unsaturated fatty acid residue. preferable. Further, the unsaturated fatty acid is preferably a cis unsaturated fatty acid.

Specific examples of the saturated fatty acid include caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, serotic acid, heptacosanoic acid. Among these, palmitic acid is preferable.

Specific examples of unsaturated fatty acids include hydrosorbic acid, undecylenic acid, dodecenoic acid, tridecenoic acid, heptadecenoic acid, petroselinic acid (cis-6-octadecenoic acid), and 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, Examples thereof include docosatetraenoic acid, docosapentaenoic acid, arachidonic acid, nervonic acid (cis-15-tetracosenoic acid) and docosahexaenoic acid.

Among these unsaturated fatty acids, petroselinic acid, palmitoleic acid, oleic acid, linoleic acid, α
-Linolenic acid, γ-linolenic acid, arachidonic acid and / or docosahexaenoic acid are preferred, and petroselinic acid, oleic acid and / or linoleic acid are particularly preferred. In addition,
The source of these sugar glycerides is a plant such as comfrey, and it is also preferable to select a sugar glyceride (plant extract) obtained from such a plant as the active ingredient of the hair-growing agent of the present invention.

Each of these sugar glycerides can be produced by a commonly known method. Also, it goes without saying that a commercially available product can be used. These sugar glycerides have a strong hair follicle epithelial cell proliferation action and hair shaft elongation action, and these may be used alone or in combination of two or more as an active ingredient of the hair restorer of the present invention. it can.

The blending amount of the above-mentioned sugar glyceride 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 and is not particularly limited. On the other hand, it is preferably 1.0 × 10 ^{−12 to} 20.0% by weight, and particularly preferably 0.01 to 10.0% by weight.

When the content of the sugar glyceride is less than 1.0 × 10 ⁻¹² % by weight based on the whole hair-growing agent, the desired effect of the present invention is to prolong the hair growth period based on the hair follicle epithelial cell proliferation action. The effect is not sufficiently exerted, which is not preferable.
When the content exceeds the weight%, not only the effect corresponding to the increase of the compounding amount cannot be expected, but also the tendency of causing a trouble in the formulation becomes remarkable, which is not preferable.

In this way, the hair-growing agent of the present invention containing the above-mentioned sugar glyceride as an active ingredient has an excellent hair-growth-prolonging effect based on the excellent hair follicle epithelial cell proliferation action, and as described above, for example, hair roots. Particularly effective for alopecia due to the fact that the growth period is shortened due to slow growth of hair follicle epithelial cells in the vicinity, and the proportion of resting hair is relatively higher than that of growing hair. It is a drug. It is also possible to increase the synergistic effect in specific alopecia by using it in combination with other hair-growth agents having individual effects.

The means for identifying the effect of maintaining or extending the growth phase in the hair cycle, which is the essential effect of the desired effect of the hair-growth agent of the present invention, on the effect of extending the hair growth period, is determined by the identification method itself. is not particularly limited as far as it is reasonable to identify, also specific methods in example vitro, i
Although the in vivo identification method can be used, the in vitro identification method is preferably used in consideration of its simplicity and effectiveness.

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

That is, this identification method is performed by "contacting a hair follicle epithelial cultured cell with a target substance in a serum-free medium and specifying the presence or absence and / or the strength of the proliferation activity of the cell, Perform a primary screening to test the effect of prolonging the growth period in the hair cycle, and for the target substance for which an effect was recognized by this primary screening,
Human hair follicles are cultured in a serum-free medium to which the target substance is added, and a secondary screening is performed to further select the target substance in which hair shaft elongation is observed. This is an in vitro method for assaying hair-growing agents, which focuses on hair follicle epithelial cells that are directly related to the elongation of hair and identifies the effect of extending the growth phase in the desired hair cycle by using these cultured cells.

In the primary screening of this hair-growth drug assay method, in the hair-follicle epithelial cell culture obtained by isolating hair follicle epithelial cells of animals (including humans, the same applies hereinafter) Contact the target substance with the "cell",
The presence and / or strength of the proliferation is specified.

The hair follicle epithelial cells refer to epithelial cells derived from hair follicles, such as outer root sheath cells in the vicinity of hair roots and matrix cells, and exclude inner dermal papilla cells.

The growth phase in the hair cycle is just the elongation of the hair shaft, that is, the period when the hair follicle epithelial cells divide and proliferate, and the catagen period and the resting period are the periods when they become blunted and rest. is there.

That is, the substance that prolongs or maintains the growth phase in the hair cycle maintains the division and proliferative activity of hair follicle epithelial cells by its administration, whereby the hair shifts to the catagen phase and the telogen phase in the hair cycle. It is concluded that it is a substance that prevents the above, that is, a substance that continues to promote or maintain the proliferation of hair follicle epithelial cells.

Further, in the secondary screening, human hair follicles are cultured, and a target substance in which hair shaft elongation is recognized is further selected. In this secondary screening, it is necessary to first prepare hair follicle fragments. Usually, hair in a growing stage is selected from the hairs of human healthy hair, and a hair follicle fragment can be prepared by cutting about 2 mm from the hair bulb of this hair.

This hair follicle fragment is maintained in a growth phase for a relatively long period of time when cultured in a basal medium containing insulin, but when cultivated in a basal medium containing no insulin, it takes about a week or so. The condition resembles a hair follicle in the catagen period.

Therefore, when culturing in a basal medium that does not contain insulin, it is preferable to evaluate within 1 week, and when using a medium that contains insulin, the culturing period can be extended to about 2 weeks. It will be possible.
In both media, the extent of hair shaft elongation can be observed by culturing the hair follicles in the presence of the test substance of interest.

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

The hair follicles can be cultured under the usual conditions for animal culture such as 5% CO ₂ · 37 ° C. Culturing is usually performed for 5 to 14 days. The extension of the hair shaft in the human hair follicle can be visually confirmed by, for example, a stereoscopic microscope equipped with a micrometer.

By carrying out the above-mentioned primary screening and secondary screening in this manner, it is possible to identify the action of maintaining or extending the growth phase in the hair cycle and the action of extending the hair shaft.

As an in vitro method for assaying hair-growth agents, a method in which a target substance is allowed to act on animal's hair papilla cells to determine its growth-promoting effect can also be used.

Further, 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 this nude mouse is specified, and the effect of the target substance to extend the growth phase in the hair cycle is assayed", In principle, it is hairless, but the hair growth site moves over time to the body surface. To identify the width of the hair growth site and the migration speed of the hair growth site in nude mice that have characteristic hair growth. The method of assaying the growth period length in the hair cycle can be mentioned.

The dosage form of the hair restorer of the present invention is not particularly limited as long as it can be applied to the outer skin, and for example, liquid, emulsion, ointment and the like can be selected. The hair restorer of the present invention may have any form, for example, tonic, hair cream, mousse, shampoo, rinse, cream, milky lotion, lotion,
It can be in the form of a pack or the like.

In addition to the above essential ingredients, the hair restorer of the present invention is generally used in cosmetics, quasi-drugs, pharmaceuticals, etc. as needed and as long as it does not impair the intended effects of the present invention. Various oily or aqueous components, surfactants, moisturizers, thickeners, preservatives, antioxidants, fragrances, coloring materials, various chemicals and the like can be added.

For example, higher saturated fatty acids, solid paraffin, liquid paraffin, oily components such as silicone oil, squalane, glyceryl monooleate, olive oil, higher alcohols; surface active agents such as polyoxyethylene alkyl ether, polyoxyethylene hydrogenated castor oil, etc. Moisturizers such as glycerin, hyaluronic acid, propylene glycol, maltitol, atelocollagen, sodium lactate, etc .;
Quince mucilage, carboxyvinyl polymer, thickener such as xanthan gum; preservative such as salicylic acid; nicotinamide, benzyl nicotinate, vitamin E acetate,
Assemblage extract, vasodilators such as carpronium chloride and acetylcholine derivatives; amino acids such as serine, methionine and arginine; vitamins such as vitamin B ₆ , vitamin E or its derivatives, biotin, pantothenic acid or its derivatives; nicotinic acid, Nicotinic acid esters such as methyl nicotinate and tocopherol nicotinate; Skin function enhancers such as cepharanthin; Female hormone agents such as estradiol; Anti-inflammatory agents such as glycyrrhetinic acid or its derivatives; Hinokitiol, hexachlorophen,
Antibacterial agents such as benzalkonium chloride and bithionol;
A cooling agent such as menthol; zinc or a derivative thereof; lactic acid or an alkyl ester thereof; organic acids such as citric acid can be blended. The specific formulation of the hair restorer of the present invention will be described later.

[0040]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention should not be limitedly interpreted by these examples. In addition, in the following Examples and the like, "%" and the content indicates the weight% unless otherwise specified. First, an in vitro cell proliferation test for evaluating the hair growth period-prolonging effect of the target substance used in the present Examples and the like will be described.

Test Example 1 Cell Proliferation Test Using Hair Follicle Epithelial Cultured Cells A. Human hair follicle epithelial cells 1. Collection of Human Hair Follicle Epithelial Cells From the human male scalp obtained as a by-product of surgery, hair follicles in the growing phase of the hair cycle were mechanically collected under a stereomicroscope. 1000 U / mL dispase ・ 0.2% of hair follicles in this growth period
Dulbecco's modified MEM containing collagenase (DME
M) for 30 minutes at 37 ° C and using the tip of a needle
Phosphate buffer solution containing 0.05% trypsin and 0.02% EDTA by removing dermal sheath, dermal papilla, and epithelial tissue of hair bulb part [PBS (−): (−) means not containing calcium ion or magnesium ion) Yes] for 5 minutes at 37 ° C.

Next, the hair follicles were allowed to stand on a culture dish coated with collagen (Type I), and explant culture was carried out. The medium used at this time was serum-free medium [Keratinocyte Growth Medi
um (KGM)] (Keratinocyte Serum Free Me
You can also use dium). After 4 to 5 days of this culture, the medium was exchanged when the adhesion of the hair follicles to the culture dish and the growth of cells could be confirmed, and the medium was exchanged every two days thereafter.

The cells grown in this manner were mixed with 0.05 wt.
After treatment with PBS (-) containing 10% trypsin-0.02% for 5 minutes at 37 ° C, the reaction was stopped with an equal amount of 0.1% trypsin inhibitor, and the cells were recovered 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 Replace with PBS (-) containing 0.05wt% trypsin-0.02% EDTA again at 37 ℃.
After treatment for 1 minute, the reaction was stopped with an equal volume of 0.1% trypsin inhibitor, and the cells were centrifuged (800 × g, 5 minutes), and the resulting human hair follicle epithelial cells were frozen (cell banker). : Made by Diatron) and added 1.0
The concentration was adjusted to × 10 ⁶ cells / mL, 1.0 × 10 ⁶ cells were put in each freezing tube, and the frozen tubes were stored. The number of these cells was calculated using a hemocytometer.

2. Assay of target substance The fibroblast contamination rate (FB contamination rate) of hair follicle epithelial cells obtained by the above steps was measured (3000 times, 5 fields of view), and as a result, the FB contamination rate of 3% or more was assayed. Excluded from the subject. Then, the hair follicle epithelial cells were seeded in a culture flask, and then 0.25% trypsin-0.02.
After treatment with PBS (-) containing 10% EDTA, the reaction was stopped with 0.1% trypsin inhibitor, and then the system was run at 1500 rp.
Centrifuge at m for 5 minutes, remove the supernatant, and leave KG in the residue.
A cell suspension was prepared by adding 20 mL of M medium.

96 well-plate at a rate of 0.2 mL / well
(Type I collagen coating plate: manufactured by Falcon) (3.0 × 10 ³ cells / well), and left at room temperature for about 20 minutes until the cells sink to the bottom of the well. Then, the cells were cultured at 37 ° C. and 5% CO ₂ for 1 day to obtain desired human hair follicle epithelial cells.

B. Rat hair follicle epithelial cells 1. Collection of rat hair follicle epithelial cells: (1) Collection of hair follicles After immersing the dorsal skin of newborn (3-4 day old) rats in PBS (-) containing 5% PSF, The film was removed with scissors for dissection. Then, the back skin was again soaked in PBS (-) containing 1% PSF, which was further soaked in PBS (-) containing 0.25% trypsin-0.02% EDTA at 4 ° C overnight.

After immersion in this trypsin solution, the dermis layer and epidermal layer of the back skin were peeled off with tweezers, and then the dermis layer was treated with Ham's F12 medium containing 0.35% collagenase [composition (mg / L): 1- 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), Vitamin B12 (1.36), folic acid (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
atl.Acad.Sci.USA, 53,288 (1965) "and so on], and was cut with scissors. The medium containing this cut was 37 ° C.
For 35 minutes at 60 rpm. After permeation, pipetting was performed until no lumps could be seen in the collagenase reaction product, and Ha containing DNase (10000 unit).
m's F12 medium was added and left for 5 minutes.

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

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

(2) Collection of hair follicle epithelial cells To the hair follicles obtained by the above operation, 5 mL of PBS (-) containing 0.25% trypsin-0.02% EDTA was added to prepare a cell suspension. Incubated at 37 ° C for 5 minutes.

After completion of the incubation, an equal amount of 0.1% trypsin inhibitor and Ham's F12 medium were added, and the cell suspension was added to a cell strainer (100 μm Nalgene).
(Manufactured by K.K.), and the cell 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 It was put in and stored frozen.

2. Pre-culture of hair follicle epithelial cells: In order to remove as much fibroblasts contaminating the system as possible from the system,
The hair follicle epithelial cells obtained in the above step were pre-cultured. The procedure will be described below.

The frozen cells obtained in the above step were thawed in a 37 ° C. thermostat. Then FAD medium [Ham's F1
Insulin (5.0 μg / mL), hydrocortisone (0.45 μg / mL), epidermal growth factor were added to a mixture of 2 medium (described later) and MEN medium in a volume ratio of 3: 1.
(EGF) (10.0 ng / mL), cholera toxin (10 ^-9 M) and fetal calf serum (10%)-containing medium, the same shall apply hereinafter]
Was added, the cell solution was diluted, 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.

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

Furthermore, in the system, KGM medium [Keratinocyto growth medium: Keratinocyto b
Asal medium (KBM medium (modified MCDB153 medium (manufactured by Clonetics))), bovine pituitary extract (BPE) (0.4 vol%), insulin (0.5 μm / mL), hydrocortisone (0.5 μm / mL) , h-EGF (0.1 ng / mL) -added medium, the same applies hereinafter] was added, and the cells were cultured at 37 ° C., 5% CO ₂ for 3 days.

3. Assay of Target Substance The fibroblast contamination rate (FB contamination rate) of the culture flask seeded with the hair follicle epithelial cells obtained in the above step was measured (300
Those with a FB contamination rate of 2% or more were excluded from the assay targets.

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. Then, by utilizing the difference in the reactivity of epithelial cells and fibroblasts to trypsin, trypsin was removed to remove fibroblasts from the system, and 0.25% trypsin- was added again.
PBS (-) containing 0.02% EDTA was added to give 37
The mixture was shaken at 20 rpm at 5 ° C for 5 minutes.

Then, after confirming the detachment of cells under a microscope, DMEM medium containing 10% FBS was added for pipetting, and the system was centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, KGM medium was added, and pipetting was performed until there were no cell clumps.

The suspension was added to a cell strainer (100 μm Nalg
After filtering with ene), the number of viable cells in the suspension was calculated with a hemocytometer, and KGM medium was added to the system to make the cell concentration in the system 2.0 × 10 ⁴ cells / mL. I adjusted it so that. Then, it was seeded at a rate of 0.2 mL / well on a 96-well plate (type I collagen coating plate: manufactured by Falcon) (4.0 × 10 ³ cells / well), and about 20 cells were allowed to sink to the bottom of the well. It was left at room temperature for a minute. Then 37
Culturing was carried out 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 target substance-added medium The target substance shown in Table 1 below was 1.0 × with respect to the entire medium.
10 ^{−17 to} 1.0 × 10 ⁻⁴ mol / L, solvent (DMSO)
It was added to the KBM medium so that the concentration was 0.1%.

[0062]

[Table 1]

(2) Preparation of control medium-Negative control: KBM medium with DMSO
A medium was prepared by adding (solvent) to a final concentration of 0.1%. -Positive control: Negative control medium with a final concentration of 5 μg / mL insulin, 0.5 μg / mL
Prepared by adding mL of hydrocortisone.

D. Exchange of target substance medium: KGM medium in 96 well-plate prepared with human hair follicle epithelial cultured cells and rat hair follicle epithelial cultured cells in the above A and B was replaced with target substance-added medium and control medium (200 μl / well) After exchange, the cells were cultured at 37 ° C. and 5% CO ₂ for 2 days after the exchange.

The replacement of this medium was carried out by using KGM in the well.
The medium was removed with a multi-pipette, taking care not to damage the cells attached to the bottom surface, and then the target substance-containing medium and the like were immediately added from both ends of the well.

E. Measurement of cell proliferation: alamar blue (ala
mar blue: Alamar Bioscience Co., Ltd.) was added at 1/10 volume to the volume (volume) of the medium, and the mixture was incubated at 37 ° C. (5
% CO ₂ ) and incubated for 6 hours. After the incubation, the absorbance of the system at 595 nm and 570 nm was measured using a micro plate reader (Micro plate reader: manufactured by Bio RAD).
Was measured, the cell proliferation rate was calculated according to the following calculation formula, and the significant difference test was performed.

[0067]

[Equation 1]

F. Results: Table 2 below shows the concentration [LOG] of each of the above-mentioned target substances in which a significant hair follicle epithelial cell growth promoting effect was observed with respect to the negative control.

[0069]

[Table 2]

From these results, it was confirmed that the above-mentioned sugar glycerides, which are the target substances, had a proliferative activity on hair follicle epithelial cultured cells at a low concentration. That is, it was revealed that the above-mentioned sugar glyceride is capable of maintaining and prolonging the growth phase of hair by maintaining the mitotic proliferation activity of hair epithelial cells.

G. Examination of Hair Shaft Extension (Secondary Screening) Furthermore, the hair extension effect was examined for the target substances shown in Table 3 below.

[0072]

[Table 3]

Human Hair Follicle Organ Cultures Growing hair follicles were isolated from human scalp using a microblade under a stereomicroscope. The isolated hair follicle is "Williams
E-medium (Gibco) plus penicillin, streptomycin, and fungizone (Fungizone) "(hereinafter, also referred to as (-) medium) was washed, and the length was measured, and 10 ng / mL was added to this (-) medium. In a medium containing hydrocortisone, 10 μg / mL insulin, 10 ng / mL sodium selenite and 10 μg / mL transferrin (hereinafter also referred to as (+) medium) (use of 24-well microplate: 1 mL per well) Let it sink 5%
It was cultured under CO ₂ at 37 ° C. overnight (preculture). After this pre-culture, the length of the hair follicles cultured again was measured, and the elongation was 0.
Hair follicles of 25 mm or more were selected and divided into 3 groups of 9 hairs so that the degree of extension was uniform. The extension of the hair shaft in the hair follicle was visually observed using an inverted microscope equipped with the above microplate equipped with a micrometer.

Evaluation of Target Substances Each of the above sugar glycerides, which are target substances, has a final concentration of 1.0.
× 10 ^-5 mol / L (DMSO 0.1%),
It 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 at a final concentration of 0.1%.

The medium of the three groups of hair follicles described above was replaced with the above medium, and the medium was further incubated for 5 days under 5% CO ₂ .
Cultured at ° C. The length of each hair follicle was measured 5 days after the start of culture, and the elongation of the hair shaft in the hair follicle of the control group and that of the target substance-added group were compared. The results are shown in Table 4.

[0076]

[Table 4]

From these results, the above-mentioned sugar glyceride not only promotes the proliferation of hair follicle epithelial cells, but also promotes the elongation of the hair shaft in the hair follicle, and has a harmonious hair growth period extending effect in the hair follicle organ. It became clear to have. Hereinafter, the formulations of the hair-growing agent of the present invention are shown as examples, and the hair-growing effects thereof were examined.

[Example 1] Preparation of liquid hair restorer 0.01% galactosylglyceride linoleate and 0.01% galactosylglyceride oleate

[Chemical 4] 70% ethanol 90%, dodecylbenzene sulfonic acid 0.49%, hydrogenated castor oil ethylene oxide (40%
(Mol) adduct 0.5% and ion-exchanged water (residual) with stirring to dissolve. Further, ion-exchanged water (10%) was added and mixed to obtain a liquid hair restorer. As a comparative example 1, a liquid agent prepared by removing the galactosyl glyceride of linoleic acid and the galactosyl oleic acid in the formulation of the liquid hair-growing agent was prepared.

[Example 2] Preparation of liquid hair restorer 0.02% of galactosyl glyceryl palmitate was added to 7
90% of 0% ethanol, 0.49% of dodecylbenzenesulfonic acid, 0.5% of hydrogenated castor oil ethylene oxide (40 mol) adduct and ion-exchanged water (residual) were mixed with stirring to dissolve. Further, ion-exchanged water (10%) was added and mixed to obtain a liquid hair restorer.

Example 3 Preparation of Emulsion Hair Growth Agent An emulsion-type hair restorer was prepared according to the following formulation. Blending ingredients Blending amount (wt%) (Phase A) Linoleic acid galactosyl glyceride 1.0 Polyoxyethylene (60 mol) addition hydrogenated 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 Propylparaben 2.0 (Phase C) Carboxyvinyl polymer 1% aqueous solution 30.0 Sodium hexametaphosphate 0.03 Ion-exchanged water 8.35 (Phase D) Ion-exchanged water 4.5 (E phase) KOH 0.12 Ion-exchanged water 5.0

<Manufacturing Method> Phases A and B are each 60 ° C.
The mixture was heated and dissolved by heating, mixed and treated with a homomixer to form a gel, to which phase D was gradually added and dispersed with a homomixer. Next, the dissolved phase C was added to this, and finally the dissolved E
The phases were added and emulsified with a homomixer to obtain an O / W emulsion type hair restorer.

Example 4 Preparation of Cream Hair Growth Agent A creamy hair restorer was prepared according to the following formulation.   Blending ingredients Blending amount (wt%) (Phase A) Liquid paraffin 5.0 Cetostearyl alcohol 5.5 Glyceryl monostearate 3.0 EO (20 mol) -2-octyldodecyl ether 8.0 Propylparaben 0.3 Perfume 0.1 (Phase B) Linoleic acid galactosyl glyceride 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> Phases A and B were melted by heating, mixed, and emulsified with a homomixer to obtain a creamy hair-growing agent.

[Test Example 2] Examination of hair-growth action of the hair-growth agent of the present invention To examine hair-growth effects such as hair loss prevention and hair-growth effect of the hair-growth agent of the present invention, a trichogram test was conducted on humans by the following method. Carried out. The test sample and the control sample are Examples 1 to 4.
Of the present invention, the agent of Comparative Example 1 and 70% ethanol.

Test method The roots of the removed hair before and after the use of the above sample were observed under a microscope, and from the morphology of the roots, the number of "resting hair roots", which were the roots of hairs that had stopped growing, Is found to have a higher proportion of this hairy root than in normal people.)
Were counted and the hair growth effect of these samples was compared by increasing or decreasing the ratio.

That is, the test sample and the control sample were respectively applied to the scalp of 10 male subjects twice a day, and 2 mL each time 6 mL.
The hair was continuously applied for a month, and 100 hairs were removed from each subject immediately before the application and immediately after the application for 6 months, and each hair root was observed under a microscope. In addition, a practical use test was conducted on whether the hair-growth effect in the above sample was effective or ineffective. The results of these tests are shown in Table 5 below.

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[Table 5]

From the results shown in Table 5, it is found that the hair-growing agent of the present invention containing linoleic acid galactosyl glyceride which is a sugar glyceride, palmitic acid galactosyl glyceride, or linoleic acid galactosyl glyceride and oleic acid galactosyl glyceride as active ingredients. , It was revealed that the hair-growth effect based on the hair-growth-prolonging effect of these sugar glycerides was recognized. Further, similar sugar glycerides other than the above-mentioned sugar glycerides were also found to have similar hair-growth-prolonging effects. Therefore, the hair-growing agent of the present invention containing these sugar glycerides as an active ingredient is similar to the above-mentioned examples. It is clear that the hair-growth effect is observed in.

[0089]

Industrial Applicability According to the present invention, there is provided a hair-growing agent that maintains or extends the growth phase of the hair cycle by promoting hair growth.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Chika Hamada             1050 Shinba-cho, Kohoku Ward, Yokohama City, Kanagawa Prefecture             Shiseido 1st Research Center (72) Inventor Jun Suzuki             1050 Shinba-cho, Kohoku Ward, Yokohama City, Kanagawa Prefecture             Shiseido 1st Research Center (72) Inventor Tsutomu Soma             2-12-1, Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa               Shiseido 2nd Research Center Co., Ltd. (72) Inventor Masahiro Tajima             1050 Shinba-cho, Kohoku Ward, Yokohama City, Kanagawa Prefecture             Shiseido 1st Research Center (72) Inventor Toshihiro Nohara             2861-27 Nagaminecho, Kumamoto City, Kumamoto Prefecture F-term (reference) 4C083 AB282 AC022 AC102 AC122 AC122                       AC131 AC182 AC241 AC421                       AC432 AC482 AC792 AD042                       AD092 AD112 AD191 AD202                       CC37 DD23 DD31 EE22

Claims (7)

[Claims] 1. A hair restorer containing sugar glyceride as an active ingredient. 2. The sugar glyceride has the formula (I): [In the formula, R ¹ is a sugar residue, and R ² and R ³ may be the same or different from each other and are a hydrogen atom, a saturated fatty acid residue or an unsaturated fatty acid residue. ],
The hair restorer according to claim 1. 3. The saturated or unsaturated fatty acid has the formula (I
I) C _n H _m O ₂ (II) [wherein 6 ≦ n ≦ 28 and n + 1 ≦ m ≦ 2n−2. ] The hair restorer of Claim 2 represented by these. 4. The hair restorer according to claim 2 or 3, wherein in the formula (I), R ² and R ³ are hydrogen atoms or unsaturated fatty acid residues. 5. The hair restorer according to any one of claims 2 to 4, wherein the unsaturated fatty acid is a cis type unsaturated fatty acid. 6. The hair restorer according to any one of claims 2 to 5, wherein the unsaturated fatty acid is petroselinic acid, oleic acid and / or linoleic acid. 7. The hair restorer according to any one of claims 1 to 6, wherein the sugar is galactose, glucose or fructose.