JPH10265347A - Agent for extending hair growth period - Google Patents

Abstract

PROBLEM TO BE SOLVED: To find an ingredient having activities for extending a hair growth period and the obtain an agent for extending the hair growth period by using the ingredient as the active ingredient. SOLUTION: This agent for extending a hair growth period is obtained by using one or not less than two components selected from the group of extracts of Symphytum Officinale L., Curcuma domestica Valton, Zizyphus jujube Miller Var. inermis Rehder, Asarum sieboldii Miq., Coix lachryma-jobi L. var. ma-yuen Stapf, Stevia rebandiana Bertoni, Uncaria gambir Roxburgh, Gentiana lutea L., Humulus luplus L., Azukia angularis Ohwl, Isodon japonicus Hara, Arnica montana L., Mentha arvensis L., Glycyrrhiza glabra L., Poria cocos Wolf, Glycine max Merril, Perilla frutescens Britton var. acuta Kudo, Cinchona succirubra Pavon et Klitzch, Equisetum arvenes L., Prunus armeniaca L., Glycyrrhiza glabra L., Crataegus cuneata Siebold et Zuccarini, Cnidium Rhizome Makino, Cinnamoum cassia Blume, Ganoderma lucidum, Syzygum aromaticum Merrill et Perry and Citrus aurantium L. subsp. amara Engl. and royal jelly as an active ingredient.

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 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]

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 diligently studied the components having the above-described effect of extending the hair growth period in various substances using the hair growth drug assay method discovered by the present inventors, The present inventors have found that a desired hair growth period extending effect is observed in plant extracts and the like, and completed the present invention.

That is, the present invention relates to a comfrey extract, a turmeric extract, a syrup extract, a cysin extract, a yokinin extract, a stevia extract, an acacia extract, a royal jelly, a gentian extract, a hop extract and an adzuki bean extract. , Hikikoshi extract, Arnica extract, Mentha extract, Bakuryo extract, Soybean extract, Perilla extract,
1 selected from the group consisting of kina extract, horsetail extract, apricot kernel grain extract, liquorice extract, hawthorn extract, senkyu extract, calyx extract, litchi extract, clove extract and spruce extract It is an invention to provide a hair growth period prolonging agent containing one or more kinds of active ingredients as active ingredients.

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

This "hair growth period extending agent" shortens the anagen phase due to, for example, slow growth of hair follicle epithelial cells in the vicinity of the root of the hair, and thus the anagen of the telogen hair is relatively shorter than the anagen hair. It is a particularly effective drug for alopecia caused by an increased ratio. Also, by using in combination with other hair restorers having individual effects, comprehensive and synergistic effects can be achieved in a wide range of alopecia. That is, this "hair growth period extender"
A general purpose hair restoring application having the concept including the above-mentioned effects (1) to (4) is one that has a distinctive purpose.

[0010]

Embodiments of the present invention will be described below. The hair growth-prolonging agent of the present invention is a hair-related drug containing the above-listed plant extracts or the like as an active ingredient.

Comfrey is also known as Symphyllium
Also known as tum Officinale L.), it is a perennial plant native to Europe. Comfrey extract extracted from its leaves is known to have astringent and anti-inflammatory effects,
It is also commercially available as a compounding component of general cosmetics.

The comfrey used in the present invention is:
As long as the extract has the effect of extending the anagen phase in the hair cycle described below, it may belong to other species such as its variants and subspecies (in the present invention, these are collectively referred to as comfreys). Do).

Turmeric (Curcuma domestica Valton) is a perennial plant native to tropical Asia. The turmeric extract extracted from its roots has a yellow color, and the main component of the pigment is known as curcumin. This turmeric extract is known as a bile drug, a food additive or the like, and is also commercially available.

The turmeric used in the present invention may belong to other species such as its varieties and subspecies, as long as the extract has the effect of extending the anagen in the hair cycle described below ( In the present invention, these are collectively referred to as turmeric).

The narcissus is jujube (Zizyphus jujube Mil)
ler Var. inermis Rehder) or a fruit of a closely related plant thereof, and its extract is recognized as having a moisturizing effect and an anti-inflammatory effect, and is commercially available as a compounding component of general cosmetics.

Saishin refers to the root and rhizome of Keirinsaishin ( Asarum sieboldii Miq.), Which is a perennial plant that grows in the shade of mountain forests distributed in Honshu, Kyushu, the Korean Peninsula and China.

The cycin used in the present invention belongs to other species, such as its variants and subspecies, as long as its extract has the effect of extending the anagen in the hair cycle described later, as well as keirin cysin. Stuffs, for example, Asubarum heterotropoides Fr. Schmidt v
ar. mandshuricum (Maxim.) Kitagawa) and the like (in the present invention, these are collectively referred to as cycins).

It has been confirmed that the extract of cycin contains at least essential oil components such as methyl eugenol, asalyl ketone, α-binene, β-binene, eucarbon, safrole and cineol, and pungent components such as peritrine and higenamine. And is currently on the market.

Yokuinin is obtained from coix lachryma-j
obi L. var.ma -yuen Stapf) is the fruit obtained by removing the ginger, pericarp, and seed coat from the fruit.It has a grayish oval to wide egg shape with a deep groove on one side, and has anti-inflammatory and diuretic effects. It is known as a herbal medicine having many kinds of medicinal effects, and is currently commercially available. The extract of yoquinin is at least co
It is known to contain ixenolide and to further contain crude protein, crude fat, starch, ash and the like.

[0020] Stevia extract is obtained from Stevia reba
ndiana Bertoni) is extracted from water and dried, and is known to contain several kinds of sweet components such as stevioside, and is commercially available.

The extract of Acacia catechu is an Ascendatum (Uncaria)
gambir Roxburgh). It is collected by extracting leaves and shoots, and is also commercially available as a plant extract having moisturizing, astringent, and hemostatic effects.

The gentian extract is an extract obtained from roots and rhizomes of gentian (gentian, Gentiana lutea L.), and is commercially available as a plant extract component having a moisturizing action, an anti-inflammatory action and the like.

The hop extract is hop, a mulberry plant
(Humulus luplus L.) is a plant extract obtained by extracting from female ears (cones), and is also commercially available as a plant extract component having a moisturizing action, an astringent action, a bactericidal action and the like.

Hioki-koi refers to Hioki-koshi ( Isododon japonicus Hara) belonging to the genus Yamaha, and its foliage is known as a life-prolonging herb and a herbal medicine having an effect on the digestive system.

[0025] The chick sprouts used in the present invention are:
Of course, as long as the extract has the effect of extending the anagen in the hair cycle described below,
Those belonging to other species such as its varieties and subspecies, for example, turtle skull [( Isodon kameba Okuyama): whole plant is subject to extraction] and kurobana skull [( Isodon trichoc)
arpus Kudo): The foliage is the target of extraction] (in the present invention, these are collectively referred to as chickpeas). It is known that an extract of Hioki Koshi contains at least bitter tastant emmein, nodosine, etc., and is commercially available at present.

The adzuki bean extract used in the present invention,
Azukia angularis O
hw ₁ ) is an extract of seeds, and is sometimes used in Chinese medicine for such purposes as diuresis and anti-inflammation. Adzuki bean extract contains at least palmitic acid, stearic acid, arachidic acid and 3
It is known that it contains a kind of crystalline saponin and the like as one body, and is also commercially available as adzuki powder.

The arnica extract was prepared from arnica (Arnica mont
ana L.), which is known as a plant extract having anti-inflammatory and antipruritic effects, a blood flow promoting effect, a moisturizing effect, and the like, and is also commercially available.

The licorice extract is Glycyrrhiza
glabra L.) and other congeners of the same plant, which are extracted from roots and rhizomes, are known as plant components having an anti-inflammatory effect, and are commercially available.

The mint extract was obtained from Mentha arvensis
L. etc.) is obtained by steam distillation or the like, containing 50.0% or more of total menthol and commercially available.

[0030] The extract of Sengoku (Cendium)
Rhizome Makino) is a rhizome extract, is known as a plant extract having various medicinal effects, and is also commercially available.

The extract of Bakryo was obtained from pine hod (Poria coc
os Wolf), which is an extract obtained by extracting the outer layer from most of the sclerotia, is known as a component having an anti-inflammatory action and a skin activating action, and is also commercially available.

The soybean extract was obtained from soybean (Glycine max Mer
ril) is a plant extract extracted from boiled and pulverized seeds and is commercially available.

The perilla extract was obtained from perilla (Perilla frutescens)
Bcutton var. Acuta Kudo) or a plant extract that is extracted from the leaves and branches of related plants, and is known to have a whitening effect and an anti-inflammatory effect, and is also commercially available.

The kina extract was prepared from kina (Cinchona succirubra).
It is extracted from the bark of Pavon et Klotzch) or a closely related plant, and is known as a plant extract component having a skin stimulating action, an astringent action, and the like, and is also commercially available.

The horsetail extract is a horsetail (Equisetum arven)
es L.) is a plant extract known to have a moisturizing action, anti-inflammatory action, astringent action, etc., and is commercially available.

The apricot kernel extract was obtained from Prunus
armeniaca L.) or a plant extract extracted from a dry and crushed product of the core (endocarp) of a closely related plant, and is commercially available.

[0037] The hawthorn extract was obtained from hawthorn (Crataegus c.
uneata Siebold et Zuccarini) is a plant extract known to have a moisturizing effect, astringent effect, etc., and is commercially available.

The extract of cinnamon is Cinnamoum cassia Blume
Alternatively, it is a plant extract component that is extracted from the bark of a closely related plant and is known to have a tyrosinase activity inhibitory effect, and is also commercially available.

Ganoderma extract is an extract which is extracted from the fruit body of Ganoderma lucidum and is known to have a moisturizing action, a skin regeneration action and the like, and is also commercially available.

Clove extract was prepared from clove (Syzygium ar
omaticum Merrill et Perry) is extracted from dried buds, and is a plant extract known to have antibacterial action, blood flow promoting action, etc., and is also commercially available.

[0041] The spruce extract was obtained from the sea bream (Citrus aurant).
ium L.subsp. amara Engl.) a mature essential oil obtained by distillation from the pericarp, it is commercially available.

Extraction of these plant extracts can be carried out by a method generally used for extracting plant-derived extracts. 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; polyhydric alcohols such as propylene glycol and 1,3-butylene glycol; hydrates of these alcohols; and hydrocarbon solvents such as n-hexane and toluene. It is preferable to use the lower alcohol of the formula (1) as an extraction solvent. When these lower alcohols are used as the extraction solvent, the resulting extract can be directly blended as an active ingredient of the hair growth period prolonging agent of the present invention. However, the extraction solvent is once distilled off, and if necessary, blended after drying. Is also possible. Thus, an extract of the above plant can be obtained.

The royal jelly used in the present invention is a bait that a worker bee feeds to a queen bee.
Also called royal milk, it is secreted from the hypopharyngeal glands and the like that develop in the head of worker bees, and is also commercially available. It is known that this royal jelly integrally contains at least a protein as a main component, free fatty acids such as 10-hydroxydecenoic acid, vitamins and the like.

The above-mentioned various components can be produced according to the above-mentioned steps or can be obtained by collecting those existing in the natural world. Can be used as an active ingredient.

It is not clear which of the above-mentioned components, which are incorporated as the active ingredient of the agent for extending the hair growth period of the present invention, to proliferate the hair follicle epithelial cells described below is derived from any of these components. Has a strong hair follicle epithelial cell proliferation action as a whole.

The amount of the above components in the agent for extending hair growth period of the present invention can be appropriately selected according to the specific form of the agent for extending hair growth period of the present invention, and is not particularly limited. 0.00005% by weight or more and 20.0% by weight or less, preferably 0.01% by weight or more and 10.0% by weight or more as a dry matter of the extract with respect to the whole of the present agent.
It is blended as follows.

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

The above-mentioned components may be used alone or in combination of two or more, if necessary, as one active ingredient of the present invention.

Thus, the hair growth period prolonging agent of the present invention containing the above-mentioned components as an active ingredient has a hair growth period prolonging effect based on an excellent hair follicle cell proliferation action. Especially for alopecia caused by the slow growth of hair follicle epithelial cells in the vicinity of the follicles, resulting in a shorter anagen and a relatively higher proportion of telogen hairs than anagen hairs It is an effective drug. Also,
By using it in combination with other hair-growth agents with individual effects, it is also possible to achieve a synergistic effect in certain alopecia.

The means for specifying the effect of maintaining or extending the anagen phase in the hair cycle, which is an essential action in the expected effect of the hair anagen phase 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 . Below, this
A specific method for examining the growth effect of hair follicle epithelial cultured cells, which is one of the in vitro specific methods, will be briefly described (more specifically, described in Examples).

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 assay for assaying the effect of prolonging the anagen phase in the hair cycle ", that is, focusing on hair follicle epithelial cells that are directly involved in hair elongation, This is an in vitro hair growth drug assay method for determining the effect of extending the growth period.

In this method for assaying a hair-growth agent, the target substance is added to “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. Hair follicle epithelial cells particularly refer to cells such as outer root sheath cells and matrix cells near the hair root, and exclude the inner hair papilla cells.

The anagen phase in the hair cycle is exactly when the hair elongates, that is, when the hair follicle epithelial cells divide and proliferate, and the catagen and telogen phases are the times when they slow down and pause. . That is, the substance that prolongs the anagen phase in the hair cycle is a substance that prevents the hair from entering the catagen and telogen phases in the hair cycle by maintaining the division and proliferative activity of hair follicle epithelial cells by its administration, It is concluded that the substance is a substance that continues to promote or maintain the growth of hair follicle epithelial cells.

As an in vitro method for testing a hair growth drug in vitro, 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 also be used.

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-mentioned 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, 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. The specific formulation of the hair growth period extender of the present invention will be described later.

[0059]

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 thus grown were treated with 0.05 wt% trypsin-0.02% EDTA at 37 ° C. for 5 minutes,
The reaction was stopped with an equal volume of 0.1% trypsin inhibitor, and the cells were collected by centrifugation (800 × g, 5 minutes).

Next, 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 performing exchange and treating again with 0.05 wt% trypsin-0.02% EDTA at 37 ° C. for 5 minutes,
The reaction was stopped with an equal volume of 0.1% trypsin inhibitor, centrifuged (800 × g, 5 minutes), and the resulting human hair follicle epithelial cells were subjected to cell freezing (Cell Banker: Diatron) And add 1.0 × 10 ⁶ cell / m
l and add 1.0 × 10 ⁶ to each cryotube.
Each cell was placed and this was frozen and stored. In addition, these cell numbers were calculated with a blood cell counting plate.

2. Assay of target substance The fibroblast contamination rate (FB contamination rate) of the hair follicle epithelial cells obtained by the above steps was measured (3000 times, 5 visual fields). As a result, if the FB contamination rate was 3% or more, the assay was performed. Was excluded from the target. Then, after inoculating the hair follicle epithelial cells in a culture flask, the cells were mixed with 0.05% trypsin and 0.02%.
After treatment with% EDTA, the reaction was stopped with 0.1% trypsin inhibitor, the system was centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, 20 ml of KGM medium was added to the residue, and the cell suspension was removed. Was prepared.

At a rate of 0.2 ml / well, 96 well-plate
(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 obtained 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. 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. In addition, these cell numbers were calculated with a blood cell counting plate.

2. Preculture of hair follicle epithelial cells: To remove fibroblasts from the system as much as possible,
Preculture of hair follicle epithelial cells obtained by the above steps was performed. Hereinafter, the procedure will be described. The frozen cells obtained in the above steps were thawed in a thermostat at 37 ° C. Then F
AD medium [A mixture of Ham's F12 medium (described below) and MEN medium at a volume ratio of 3 to 1;
μg / ml), a medium containing hydrocortisone (0.45 μg / ml), epidermal growth factor (EGF) (10.0 ng / ml), cholera toxin (10 ⁻⁹ M) and fetal bovine serum (10%), The same applies to the following), the cell solution was diluted, and the system was centrifuged (10 ° C. or less, 1500
rpm, 5 minutes). After centrifugation, the supernatant was removed, 10 ml of FAD medium was added to the system, and pipetting was repeated until no cell mass was observed.

The number of cells obtained was calculated using a hemocytometer, and
It was adjusted to a concentration of 2.5 × 10 ⁵ cell / ml in AD medium. 75cm ³ coated with type I collagen
Cells were seeded in a flask of 37 ° C., 5% CO 2
₂ overnight. After the culture, the system was washed twice with 10 ml of PBS (-), 2 ml of PBS (-) containing 0.25% trypsin was added, and the mixture was incubated at 37 ° C and 5% CO ₂ for 4 minutes. Next, 2 fetal bovine serum (FBS) was added to the system.
After adding once and gently rocking, the supernatant was removed to remove fibroblasts contaminating the system.

Furthermore, KGM medium [Keratinocyto growth medium: Keratinocytob medium] was added to the system.
bovine pituitary extract (BPE) (0.4 vol%), insulin (0.5 μm / ml), hydrocortisone (0.5 μm / ml) in an asal medium (KBM medium (modified MCDB153 medium (Clonetics))) , h-EGF (0.1 ng / ml), the same applies to the following.
Cultured in O ₂ for 3 days.

3. Assay of target substance Measure the fibroblast contamination rate (FB contamination rate) of the culture flask in which the hair follicle epithelial cells obtained by the above steps were seeded (300).
(0 times, 5 fields of view), and as a result, those having a FB contamination rate of 3% or more were excluded from the subject of the assay. The system is PBS (-)
After washing twice with 10 ml, PBS containing 0.25% trypsin
2 ml of (-) was added, and this was incubated at 37 ° C. for 3 minutes. Taking advantage of the difference in reactivity between epithelial cells and fibroblasts with trypsin, trypsin was removed in order to remove fibroblasts from the system, and 0.2 times again.
2 ml of PBS (-) containing 5% trypsin was added, and 37
The mixture was shaken at 20 rpm for 5 minutes.

Next, after confirming the detachment of the cells under a microscope, 10 ml of 10% FBS-containing DMEM medium was added, pipetting was performed in a 50 ml centrifuge tube, and the system was centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, 20 ml of KGM medium was added, and pipetting was performed until there was no cell mass.

The suspension was added to a cell strainer (100 μm Nalg).
After filtration in a 50 ml centrifuge tube, the number of viable cells in the suspension was calculated using a hemocytometer, KGM medium was added to the system, and the cell concentration in the system was adjusted to 5.0. It was adjusted to be × 10 ⁴ cell / ml. Then, at a rate of 0.2 ml / well,
Seed into 96 well-plate (type I collagen coated plate: Falcon) (1.0 × 10 ⁴ cell / we
ll), the cells were left at room temperature for about 20 minutes until they settled to the bottom of the wells. Thereafter, the cells were cultured for 1 day at 37 ° C. and 5% CO ₂ to obtain desired human hair follicle epithelial cultured cells.

C. Preparation of test medium: (1) Preparation of extract 500 g of commercially available 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. As for other plant extracts, corresponding commercially available plants (in principle, dried products) were obtained and extracted by the extraction method shown in Table 1 below to prepare respective dry extracts. The royal jelly used was a commercially available royal jelly powder.

(2) Preparation of Medium Containing Target Substance The target substance was weighed in a screw tube of about 1.5 mg and prepared with an organic solvent (DMSO) so as to be 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, mint powder, soybean extract, kina extract, horsetail extract, clove extract, hop extract and perilla extract. Products, Acacia extract, Azuki powder, Saishin extract, Chimney extract, Cauliflower extract, Bucurio extract, Aloe extract, Reishi extract,
Gentian extract, Licorice extract, Shobu root. With turmeric extract, spruce extract, stevia extract, hawthorn extract (including liquorice flavonoids), senkyu extract, sisou extract, saffron extract, cypress extract, yokinin extract, apricot kernel extract and royal jelly there were.

Similarly, as a control, a 0.2% DMSO solution of tea extract (extraction method: extraction with water) and Bacmondou extract (extraction method: extraction with 10% ethanol) was 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. In addition, this medium was replaced with the KGM medium in the well.
This was performed by removing the cells attached to the bottom surface with an aspirator, taking care not to damage them, and then immediately adding a medium containing the target substance from both ends of the wells.

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.

[0082]

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

[0083]

(Equation 2)

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

[Table 1]

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

Hereinafter, the formulations of the hair growth period prolonging agent of the present invention are shown as examples, and their hair growth effects were further studied. [Example 1] Preparation of liquid hair growth period extender 0.1% of the above comfrey 30% ethanol dry matter
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 growth period extender. In the formulation of the liquid hair growth period extender, a liquid prepared by replacing 0.1% of the above-described dry extract of Bacmondou with 10% ethanol by the above-mentioned 30% ethanol-extract dry extract of Comfrey was used. Was prepared as a control (Comparative Example 1).

Example 2 Preparation of Emulsion Hair Growth Period Extender In the above-mentioned process for producing the comfrey extract, 30%
Obtaining an extract using methanol instead of ethanol,
This was used in the emulsion hair growth period extender of the following formulation. Ingredients Ingredients Amount (wt%) (A phase) Dried comfrey extract 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 (phase D) ion-exchanged water 4.5 (phase E) KOH 0.12 ion-exchanged water 5.0

<Production Method> A phase and B phase were each heated and dissolved at 60 ° C., mixed and homomixed 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 As in Example 2, Comfrey's dried methanol extract was used in a creamy hair growth extending agent having the following formulation. Ingredients Ingredients Amount (% by weight) (A phase) Liquid paraffin 5.0 cetostearyl alcohol 5.5 glyceryl monostearate 3.0 EO (20 mol) -2-octyldodecyl ether 8.0 propylparaben 0.3 Fragrance 0.1 (Phase B) Comfrey extract extract 5.0 Glycerin 8.0 Dipropylene glycol 20.0 Polyethylene glycol 4000 5.0 Sodium dodecyl sulfate 0.1 Sodium hexametaphosphate 0.005 Deionized water 39.995

<Production Method> Phases A and B were each dissolved by heating, mixed and emulsified with a homomixer to obtain a creamy hair growth period extender.

[Test Example 2] Examination of hair growth effect of hair growth period extender of the present invention In order to examine the hair growth effect such as hair loss preventing effect and hair growth effect of the 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 Before and after use of the above sample, the roots of the extracted hair were observed under a microscope, and the number of “resting roots”, which are the roots of the growth-stopped hair, were counted from the form of the roots. The hair growth effects of these samples were compared by increasing or decreasing the ratio. That is, the test sample and the control sample were each applied to the scalp of 10 male subjects twice a day, 2 ml at a time, continuously for 6 months, and immediately before the application and immediately after the end of the application for 6 months, 10 ml per subject was applied.
Hair was removed by 0 hairs, 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 2 below.

[Table 2]

From the results shown in Table 2, the hair growth-prolonging agent of the present invention containing the comfrey extract as an active ingredient showed a hair-growth effect based on the hair-growth prolongation effect of this comfrey extract. .

[0095] Further, in the case of plant extracts other than the above-mentioned comfrey extract, the effect of prolonging the hair growth period was observed as in the case of comfrey. It is clear that the hair growth-prolonging agent of the present invention also exhibits a hair-growth effect in the same manner as in the above Examples.

[0096]

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] 0065

[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.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 35/78 A61K 35/78 QHNK ADA ADAC 35/84 35/84 A (72) Inventor Masahiro Tajima Yokohama City, Kanagawa Prefecture 1050 Nippa-cho, Kohoku-ku, Shiseido Daiichi Research Center

Claims (1)

[Claims] (1) Comfrey extract, turmeric extract, syrup extract, saicin extract, yokunin extract, stevia extract, asenyaku extract, royal jelly, gentian extract, hop extract, adzuki extract, hioki koshi extract , Arnica extract, Mentha extract, Licorice extract, Bulberry extract, Soybean extract, Perilla extract, Kina extract, Horsetail extract, Apricot kernel extract, Licorice extract, Hawthorn extract, Senkyu extract A hair growth-prolonging agent comprising, as an active ingredient, one or more components selected from the group consisting of an extract, a cinnamon extract, a litchi extract, a clove extract and a spruce extract.