TWI788377B - Hair papilla cell proliferation enhancer, fibroblast growth factor-7(fgf-7) production promoter, vascular endothelial growth factor(vegf) production promoter, insulin-like growth factor-1(igf-1) production promoter, hepatocyte growth factor(hgf) production promoter, and hair growth tonic - Google Patents

Abstract

Provide a novel hair follicle dermal papilla cell (follicle dermal papilla cell) proliferation promoter, fibroblast growth factor-7 (FGF-7) production promoter, vascular endothelial growth factor ( vascular endothelial growth factor (VEGF) production enhancer, insulin-like growth factor-1 (insulin-like growth factor-1, IGF-1) production enhancer, hepatocyte growth factor (HGF) production enhancer and containing these enhancers at least one hair restorer. The hair tonic contains lanosterol, 3β-hydroxylanosta-8,24-diene-21-al (3β-hydroxylanosta-8,24-diene-21-al), inotodiol ), 3β,21-dihydroxylanost-8,24-diene (3β,21-dihydroxylanost-8,24-diene), and at least one of trametenolic acid (trametenolic acid) as an active ingredient.

Description

Hair follicle dermal papilla cell proliferation accelerator, fibroblast growth factor-7 (FGF-7) production accelerator, vascular endothelial growth factor (VEGF) production accelerator, insulin-like growth factor-1 (IGF-1) production accelerator, Hepatocyte growth factor (HGF) production promoter and hair growth agent

The present invention relates to a follicle dermal papilla cell (follicle dermal papilla cell) proliferation accelerator, a fibroblast growth factor-7 (fibroblast growth factor-7; FGF-7) production accelerator, and a vascular endothelial growth factor (vascular endothelial growth factor; VEGF) production promoters, insulin-like growth factor-1 (insulin-like growth factor-1; IGF-1) production promoters, hepatocyte growth factor (HGF) production promoters, and hair growth agents.

As factors related to hair growth, fibroblast growth factor-7 (FGF-7), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (insulin-like growth factor; IGF-1) and liver Cell growth factor (hepatocyte growth factor; HGF), etc.

It is said that minoxidil and adenosine promote the production of factors such as the above-mentioned FGF-7 and VEGF, and hair growth agents containing these as active ingredients are known (Patent Documents 1 and 2). Also, it is known that a certain lanostane triterpene (triterpene) extracted from Ganoderma lucidum has an effective 5α-reductase (5α-reductase) inhibitory effect on the prevention and treatment of androgenic alopecia (patent Document 3).

[Prior Art Literature] [Patent Document]

[Patent Document 1] Japanese Patent Application Laid-Open No. 5-4908

[Patent Document 2] Japanese Unexamined Patent Publication No. 2000-297015

[Patent Document 3] Japanese International Publication No. 2005/079164

In recent years, in order to obtain a hair growth agent that exhibits a superior hair growth effect, a growth promoter for dermal papilla cells, a fibroblast growth factor-7 (FGF-7) production promoter, a vascular endothelial growth factor (VEGF) production promoter, and similar Insulin growth factor-1 (IGF-1) production promoters and hepatocyte growth factor (HGF) production promoters require higher effects.

Therefore, the object of the present invention is to provide a novel hair follicle dermal papilla cell proliferation promoter, a fibroblast growth factor-7 (FGF-7) production promoter, a vascular endothelial growth factor (VEGF) production promoter, etc. A hair growth agent, an insulin-like growth factor-1 (IGF-1) production promoter, a hepatocyte growth factor (HGF) production promoter, and a hair growth agent containing at least one of these promoters.

The hair follicle dermal papilla cell proliferation accelerator system related to the present invention is characterized by containing lanosterol (lanosterol), 3β-hydroxylanosterol-8,24-diene-21-aldehyde (3β-hydroxylanosta-8,24-diene-21 -al), inotodiol, 3β,21-dihydroxylanoster-8,24-diene (3β,21-dihydroxylanost-8,24-diene) and at least one of trametenolic acid (trametenolic acid) as an active ingredient.

The fibroblast growth factor-7 (FGF-7) production accelerator system related to the present invention is characterized by containing lanosterol, 3β-hydroxylanoster-8,24-diene-21-aldehyde, inonotofatiol and At least one kind of 3β,21-dihydroxylanostan-8,24-diene is used as an active ingredient.

The vascular endothelial growth factor (VEGF) production-promoting agent related to the present invention is characterized by containing tranexamic acid as an active ingredient.

The insulin-like growth factor-1 (IGF-1) production accelerator system related to the present invention is characterized by containing inotolin as an active ingredient.

The hepatocyte growth factor (HGF) production-promoting agent related to the present invention is characterized by containing at least one of 3β-hydroxylanostan-8,24-dien-21-al and inotolinol as an active ingredient.

The hair restorer system related to the present invention is characterized by containing lanosterol, 3β-hydroxylanoster-8,24-dien-21-aldehyde, inotolinol, 3β,21-dihydroxylanoster-8,24- At least one of diene and Tranolic acid is used as an active ingredient.

The hair follicle dermal papilla cell proliferation accelerator system related to the present invention is characterized by containing ergosterol (ergosterol), star fish sterol (stellasterol), inotolactone B (inotolactone B), ergosterol peroxide (ergosterol peroxide) and at least one of inonotusin C (inonotusin C) as the active ingredient.

The fibroblast growth factor-7 (FGF-7) production accelerator system related to the present invention is characterized in that it contains ergosterol, astral sterol, inonotrolactone B, betulin (betulin), β-phyton Sterol (β-sitosterol), ergosterol peroxide, inonotsutriol C, 3β,22R,25-trihydroxylanoster-8,23E-diene and inonotsutriol A (inonotsutriol At least one of A) is used as an active ingredient.

The vascular endothelial growth factor (VEGF) production promoter related to the present invention is characterized by containing at least one of ergosterol, inonotiolide B and betula as active ingredients.

The insulin-like growth factor-1 (IGF-1) production accelerator system related to the present invention is characterized by containing ergosterol, sterol, inonotiolide B, β-phytosterol, and obliquulin At least one of C and 3β, 22R, 25-trihydroxylanostan-8, 23E-diene as an active ingredient.

The hepatocyte growth factor (HGF) production-promoting agent related to the present invention is characterized by containing at least one of ergosterol, stestrosterol, and Inonotus obliquilide B as an active ingredient.

The hair restorer system related to the present invention is characterized by containing ergosterol, sterol, inonotolactone B, betulin, β-phytosterol, ergosterol peroxide, and obliquulin C , 3β, 22R, 25-trihydroxylanosta-8, 23E-diene and at least one of obulintriol A as the active ingredient.

The hair restorer related to the present invention is characterized by containing a lanostane-type triterpene as an active ingredient.

The hair follicle dermal papilla cell proliferation accelerator of the present invention contains lanosterol, 3β-hydroxylanoster-8,24-dien-21-aldehyde, inotolinol, 3β,21-dihydroxylanoster-8,24 - at least one of diene and Trametesic acid, or at least one of ergosterol, staurosterol, inonotiolide B, ergosterol peroxide, and oblin C as active ingredients Therefore, it can play a more excellent role in promoting the proliferation of hair follicle dermal papilla cells than the conventional hair follicle dermal papilla cell proliferation promoter.

The fibroblast growth factor-7 (FGF-7) production accelerator of the present invention contains lanosterol, 3β-hydroxylanoster-8,24-diene-21-aldehyde, inotolinol and 3β,21- Dihydroxylanosterol- At least one of 8,24-diene, or containing ergosterol, staurosterol, inonotolactone B, betulin, β-phytosterol, ergosterol peroxide, obliquulin Since at least one of C, 3β, 22R, 25-trihydroxylanoster-8, 23E-diene and statuulin triol A is used as an active ingredient, it can perform better than the conventional FGF-7 production accelerator FGF-7 promotes production.

The vascular endothelial growth factor (VEGF) production accelerator of the present invention contains tranexamic acid, or contains at least one of ergosterol, inonotiolide B and betulin as active ingredients, and can play a more conventional role. The VEGF production accelerator has a more excellent VEGF production promotion effect.

The insulin-like growth factor-1 (IGF-1) production accelerator of the present invention contains inotorol, or contains ergosterol, stellosterol, inonotrolactone B, β-phytosterol, betula Due to at least one of fumacin C and 3β, 22R, 25-trihydroxylanosta-8, 23E-diene as the active ingredient, it can exert better IGF-1 production than conventional IGF-1 production accelerators enhancement.

The hepatocyte growth factor (HGF) production accelerator of the present invention contains at least one of 3β-hydroxylanoster-8,24-dien-21-aldehyde and inotorol, or contains ergosterol, starfish Since at least one of sterol and Inonotolidin B is used as an active ingredient, it can exert a more excellent HGF production-promoting effect than conventional HGF production-promoting agents.

The hair restorer of the present invention contains hair follicle dermal papilla cell proliferation promotion, fibroblast growth factor-7 (FGF-7) production promotion, vascular endothelial growth factor (VEGF) production promotion, insulin-like growth factor-1 ( Lanosterol, 3β-hydroxylanoster-8,24-diene-21-aldehyde, chaga, which have any one or more effects of IGF-1) production promotion and hepatocyte growth factor (HGF) production promotion Conofol, at least one of 3β,21-dihydroxylanosta-8,24-diene and Trametesic acid, ergosterol, astral sterol, inonotrolactone B, betulin, β-phytene Sterols, ergosterol peroxide, statuulin C, 3β,22R,25-trihydroxylanoster-8,23E-diene, and statuulin triol A At least one of them, or because it contains lanostane-type triterpenes as an active ingredient, it can exert a more excellent hair growth effect than conventional hair growth agents.

[ Fig. 1] Fig. 1 is a graph showing the promotion rate of dermal papilla cell proliferation of five kinds of lanostane-type triterpenoids and minoxidil.

[ Fig. 2 ] A graph showing the promotion rate of dermal papilla cell growth of ergosterol, sterol, inonotiolide B, betulin, β-phytosterol, lanosterol, and minoxidil.

[ Fig. 3] Fig. 3 is a graph showing the promotion rate of dermal papilla cell proliferation of ergosterol peroxide, statuulin C, lanosterol, and minoxidil.

[Form used to practice the invention]

Embodiments related to the present invention will be described in detail below.

The inventors of the present invention have discovered that lanosterol (CAS registration number: 79-63-0), 3β-hydroxylanosterol-8,24-diene-21-aldehyde (CAS registration number: 96574-03-7), Inotolinol (CAS registration number: 35963-37-2), 3β,21-dihydroxylanosta-8,24-diene (CAS registration number: 267649-99-0) and Trametes acid (CAS registration No.: 24160-36-9), the five kinds of lanostane-type triterpenoids have the effect of promoting the proliferation of hair follicle dermal papilla cells, promoting the production of fibroblast growth factor-7 (FGF-7), and promoting the growth of vascular endothelial cells. Any one or two or more effects of growth factor (VEGF) production promotion, insulin-like growth factor-1 (IGF-1) production promotion and hepatocyte growth factor (HGF) production promotion. These five kinds of lanostane-type triterpenes can be represented by the following general formula (LTT).

In the above general formula (LTT), the combination of R ¹ and R ² is selected from the following a), b), c), d) and e).

a) R ¹ =CH ₃ and R ² =H; b) R ¹ =CHO and R ² =H; c) R ¹ =CH ₃ and R ² =OH; d) R ¹ =CH ₂ OH and R ² = H; e) R ¹ =COOH and R ² =H.

The five lanostane-type triterpenoids represented by the above general formula (LTT) can be obtained, for example, by extraction and isolation from plants. Extraction from plants can be performed by a general method. For plants, Chaga (Inonotus obliquus) can be cited. The extract obtained by solvent extraction from the fruiting body of Chaga can be separated and purified by using appropriate separation and purification means such as column chromatography, ion exchange chromatography, high-speed liquid chromatography, etc., to obtain the above-mentioned The five represented by the general formula (LTT) Types of lanostane-type triterpenoids. In addition, in this example, the term "plant" is used in a broad sense including fungi, and the plant includes fruiting bodies of fungi and the like.

Since Chaga extract contains all the above five types of lanostane terpenoids (lanostane type terpenoids), by extracting and isolating from Chaga, these five types of lanostane terpenoids can be efficiently obtained alkene.

The aforementioned five compounds do not necessarily need to be extracted and isolated from a single plant, and can be obtained by any method. For example, each of the five kinds of lanosterane-type triterpenoids can be extracted and isolated from individual plants. The five kinds of lanostane-type terpenoids can also be chemically synthesized.

In addition, the present inventors have found that in ergosterol (CAS registration number: 57-87-4), astral sterol (CAS registration number: 2465-11-4), inonotrolactone B (CAS registration number No.: 1587735-79-2), betulin (CAS registration number: 473-98-3), β-phytosterol (CAS registration number: 83-46-5), ergosterol peroxide (CAS registration number : 2061-64-5), statuulin C (CAS registration number: 1889275-09-5), 3β,22R,25-trihydroxylanoster-8,23E-diene (CAS registration number: 106483- 69-6), Chatulintriol A (CAS registration number: 374797-72-5) (hereinafter, these nine types of compounds are summarized and sometimes referred to as nine compounds), also have hair follicles related to hair growth Dermal papilla cell proliferation promotion, fibroblast growth factor-7 (FGF-7) production promotion, vascular endothelial growth factor (VEGF) production promotion, insulin-like growth factor-1 (IGF-1) production promotion and liver One or two or more of the promoting effects of cell growth factor (HGF). Among the nine kinds of compounds, inonotolactone B, obliquulin C, 3β,22R,25-trihydroxylanoster-8,23E-diene, obliquulin triol A are lanosteranes type terpenoids.

Each of the nine compounds is also associated with the five types of lanostane-type triterpenoids represented by the above general formula (LTT) (hereinafter, these five types of compounds are sometimes collectively referred to as compound five species), for example, it can be obtained by extracting from plants, and can be extracted from plants by a general method. Regarding the nine compounds, Chaga can also be cited as far as plants are concerned. The extract obtained from the solvent extraction of the fruiting body of Chaga was separated and purified by using appropriate separation and purification methods such as column chromatography, ion exchange chromatography, and high-speed liquid chromatography to obtain nine compounds . The Chaga extract contains all of the nine compounds, and each of the nine compounds can be efficiently obtained by extracting from Chaga. Since Chaga can obtain all five compounds and all nine compounds at the same time, it is very useful to use Chaga as a material.

Each of the nine compounds does not necessarily need to be obtained from a single plant, and can be obtained by any method. For example, each of the nine compounds can be obtained by extracting each of the nine compounds from individual plants. In addition, the nine kinds of compounds may also be chemically synthesized.

It is also possible to use a combination of the five types of compounds and the nine types of compounds described above. In this case, the combined compound of five compounds and compound of nine compounds may be extracted from different plants, or one or both of them may be chemically synthesized.

Hereinafter, the fibroblast growth factor-7 production-promoting agent may be referred to as an FGF-7 production-promoting agent or fibroblast growth factor-7 (FGF-7). Also, a vascular endothelial growth factor production accelerator is sometimes called a VEGF production accelerator or a vascular endothelial growth factor (VEGF) production accelerator, and an insulin-like growth factor-1 production accelerator is called an IGF-1 production accelerator or a similar Insulin growth factor-1 (IGF-1) production promoters and hepatocyte growth factor production promoters are referred to as HGF production promoters or hepatocyte growth factor (HGF) production promoters.

By using lanosterol, 3β-hydroxylanoster-8,24-diene-21-al, obliqurol, 3β,21-dihydroxylanoster-8,24-diene and Trametes acid, Angosterol, astrasterol, inonotrolactone B, betulin, β-phytosterol, ergosterol peroxide, betulin C, 3β,22R,25-trihydroxy Based on at least one of lanosta-8,23E-diene, chatuulin triol A, the hair follicle dermal papilla cell proliferation promoter, fibroblast growth factor-7 ( FGF-7) production promoter, vascular endothelial growth factor (VEGF) production promoter, insulin-like growth factor-1 (IGF-1) production promoter, hepatocyte growth factor (HGF) production promoter and hair growth agent.

Five kinds of lanostane-type triterpenoids represented by the general formula (LTT) among the above fourteen kinds of compounds, and Inonotrolactone B, Inonotolide C, 3β, 22R, 25-3 Hydroxylanostane-8,23E-diene and chamatulin triol A are lanosterane-type terpenes (terpenes), and the present inventors have discovered that among lanosterane-type terpenoids, especially lanosterane-type terpene Oxene is useful as a hair restorer. That is, it was found that lanostane-type terpenes have a growth-promoting effect on hair follicle dermal papilla cells, a role in promoting the production of fibroblast growth factor-7 (FGF-7), a role in promoting the production of vascular endothelial growth factor (VEGF), insulin-like Either one or two or more effects of growth factor-1 (IGF-1) production promotion and hepatocyte growth factor (HGF) production promotion.

Also, in the following description, lanosterol may be referred to as compound Z1, 3β-hydroxylanoster-8,24-dien-21-al may be referred to as compound Z2, and inotolinol may be referred to as compound Z3, 3β, 21-dihydroxylanosta-8,24-diene is called compound Z4, Trametesic acid is called compound Z5, ergosterol is called compound Z6, star sterol is called compound Z7, and Inonotrolactone B is called compound Z8, betulin is called compound Z9, β-phytosterol is called compound Z10, ergosterol peroxide is called compound Z11, and betulin C It is referred to as compound Z12, 3β,22R,25 hydroxylanosta-8,23E-diene is referred to as compound Z13, and statuulin triol A is referred to as compound Z14 or its name is indicated.

<Follicular dermal papilla cell proliferation promoter>

The hair follicle dermal papilla cell proliferation promoter related to the present invention contains lanosterol (compound Z1), 3β-hydroxylanosterol-8,24-diene-21-aldehyde (compound Z2), inotolinol (compound Z3 ), 3β,21-dihydroxylanoster-8,24-diene (Compound Z4), Trametesic acid (Compound Z5), Ergosterol (Compound Z6), Stestrosterol (Compound Z7), Inonotus obliquus At least one of lactone B (compound Z8), ergosterol peroxide (compound Z11), and statuulin C (compound Z12) is used as an active ingredient, and other ingredients are contained as necessary. As an active ingredient, one of the aforementioned ten compounds may be used alone, or two or more of them may be used in combination. When two or more compounds are used in combination, five compounds, nine compounds may be used in combination, or five compounds and nine compounds may be used in combination. Also, the same applies to other accelerators and hair restorers regarding the combination of such compounds.

The content of the active ingredient in the dermal papilla cell proliferation accelerator can be appropriately selected according to the purpose and is not particularly limited. The content of the active ingredient, for example, can be made into a dosage form of about 1 μg to 100 mg per unit dosage. The hair follicle dermal papilla cell proliferation promoter related to the present invention can be the aforementioned active ingredient itself.

The other components in the dermal papilla cell proliferation promoter are not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected according to the purpose. Other components include, for example, pharmacologically acceptable carriers. The carrier, for example, when using the above-mentioned active ingredient in various dosage forms, can be appropriately selected according to the dosage form.

The dosage form is not particularly limited, and can be appropriately selected according to the administration method. The dosage form includes, for example, oral solids (tablets, coated tablets, granules, powders, capsules, buccal tablets, etc.), oral liquids (internal liquids, syrups, elixirs, etc.) etc.), injections (solutions, suspensions, solid preparations for dissolving when used, etc.), inhalation powders, ointments, liquids for external use, patches, coatings, eye drops, nose drops, and ear drops.

Oral solid preparations include, for example, adding excipients to the aforementioned active ingredients, adding additives such as binders, disintegrating agents, lubricants, coloring agents, and flavoring and odoring agents as necessary, and can be prepared by general methods. manufacture. Excipients include, for example, lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid.

The additives are not particularly limited, and may be appropriately selected according to the purpose. Binders include, for example, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose , ethyl cellulose, shellac (shellac), calcium phosphate and polyvinylpyrrolidone (polyvinylpyrrolidone), etc.

Examples of disintegrants include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceryl stearate, and lactose. Examples of lubricants include purified talc, stearates, borax, and polyethylene glycol. As a coloring agent, titanium oxide, iron oxide, etc. are mentioned, for example. Examples of flavoring and odor correcting agents include sugar, orange peel, citric acid, and tartaric acid.

Oral liquid preparations can be produced, for example, by conventional methods by adding additives such as flavoring and odor correcting agents, buffering agents, and stabilizing agents to the above-mentioned active ingredients. The additives are not particularly limited, and may be appropriately selected according to the purpose. Examples of flavoring and odor correcting agents include sugar, orange peel, citric acid, and tartaric acid. As the buffer, for example, sodium citrate and the like can be cited. Examples of stabilizers include tragacanth, gum arabic, and gelatin.

Injections can be produced, for example, by a common method by adding a pH adjuster, buffer, stabilizer, isotonic agent, local anesthetic, etc. to the above-mentioned active ingredient. Injection is Subcutaneous injection, intramuscular injection, or intravenous injection. Examples of pH adjusters and buffers include sodium citrate, sodium acetate, and sodium phosphate.

Examples of stabilizers include sodium metabisulfite, EDTA, thioglycolic acid, and thiolactic acid. As an isotonic agent, sodium chloride, glucose, etc. are mentioned, for example. Examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride.

The hair follicle dermal papilla cell growth-promoting agent related to the present invention contains an active ingredient having a hair follicle dermal papilla cell growth-promoting effect, and thus activates the hair papilla by the hair follicle dermal papilla cell growth-promoting effect. The hair follicle dermal papilla cell growth-promoting agent related to the present invention can be used, for example, in the study of the mechanism of growth promotion of hair follicle dermal papilla cells, the improvement of symptoms by the growth promotion of hair follicle dermal papilla cells, Prophylaxis or treatment of symptoms improved by promoting proliferation.

Specific examples of the dermal papilla cell proliferation accelerator include, for example, an accelerator for pharmaceutical use, and an accelerator for prevention and treatment of symptoms (including "unfavorable symptoms" such as diseases and hair loss). Symptoms include, for example, thinning hair, hair loss, androgenic baldness, and the like.

The method of using the dermal papilla cell proliferation promoter related to the present invention is not particularly limited, and can be appropriately selected according to the purpose. For example, it can be used in direct contact with dermal papilla cells of hair follicles by any method. The hair follicle dermal papilla cell proliferation promoter related to the present invention can be used by contacting one or both of the scalp hair and the scalp by any method.

<Fibroblast growth factor-7 (FGF-7) production promoter>

The fibroblast growth factor-7 (FGF-7) production accelerator related to the present invention contains lanosterol (compound Z1), 3β-hydroxylanoster-8,24-diene-21-al (compound Z2), Inonotol (compound Z3), 3β, 21-dihydroxylanosta-8,24-diene (compound Z4), ergosterol (compound Z6), star sterol (compound Z7), obliquus Mycolide B (compound Z8), betulin (compound Z9), β-phytosterol (compound Z10), ergosterol peroxide (compound Z11), betulin C (compound Z12), 3β, At least one of 22R,25-trihydroxylanostan-8,23E-diene (Compound Z13) and Chatulintriol A (Compound Z14) is used as an active ingredient, and other ingredients are contained as described above as necessary. As an active ingredient, one of the aforementioned thirteen compounds may be used alone, or two or more of them may be used in combination.

The content of the active ingredient in the FGF-7 production accelerator can be appropriately selected according to the purpose, and is not particularly limited. The content of the active ingredient, for example, can be made into a dosage form of about 1 μg to 100 mg per unit dosage. The FGF-7 production accelerator related to the present invention can be the aforementioned active ingredients themselves.

The FGF-7 production-promoting agent related to the present invention contains an active ingredient having a FGF-7 production-promoting effect. Accordingly, the FGF-7 production-promoting agent related to the present invention can be used, for example, in the study of the promotion mechanism of FGF-7 production, the improvement of symptoms by the promotion of FGF-7 production, the study of symptoms produced by FGF-7 The prevention or treatment of symptoms that are improved by promoting them.

<Vascular endothelial growth factor (VEGF) production promoter>

The vascular endothelial growth factor (VEGF) production accelerator related to the present invention contains tranexamic acid (compound Z5), ergosterol (compound Z6), inonotrolactone B (compound Z8) and betula (compound At least one of Z9) as an active ingredient, if necessary, contains other ingredients as above. As an active ingredient, one of the aforementioned four compounds may be used alone, or two or more of them may be used in combination. VEGF The content of the active ingredient in the production accelerator can be appropriately selected according to the purpose. The content of the active ingredient can be, for example, about 1 μg to 100 mg per unit dose. The VEGF production accelerator related to the present invention can be the aforementioned active ingredients themselves.

The VEGF production-promoting agent related to the present invention contains an active ingredient having a VEGF production-promoting effect. Accordingly, the VEGF production-promoting agent related to the present invention can be used, for example, in the study of the promotion mechanism of VEGF production, the study of symptoms improved by the promotion of VEGF production, and the study of symptoms improved by the promotion of VEGF production. prevention or treatment.

<Insulin-like growth factor-1 (IGF-1) production promoter>

The insulin-like growth factor-1 (IGF-1) production accelerator related to the present invention contains inotolin (compound Z3), ergosterol (compound Z6), star sterol (compound Z7), chaga Poriolide B (compound Z8), β-phytosterol (compound Z10), obulin C (compound Z12) and 3β, 22R, 25-trihydroxylanoster-8,23E-diene (compound Z13 ) as an active ingredient, and if necessary, contain other ingredients as mentioned above. As an active ingredient, one of the aforementioned seven compounds may be used alone, or two or more of them may be used in combination. The content of the active ingredient in the IGF-1 production accelerator can be appropriately selected according to the purpose. The content of the active ingredient can be made, for example, about 1 μg to 100 mg per unit dosage dosage form. The IGF-1 production accelerator related to the present invention can be the aforementioned active ingredients themselves.

The IGF-1 production promoter related to the present invention contains an active ingredient having an IGF-1 production promoting effect. Accordingly, the IGF-1 production promoter related to the present invention can be used, for example, in the study of the mechanism of promoting IGF-1 production, the study of symptoms improved by the promotion of IGF-1 production, the study of Prophylaxis or treatment of symptoms to be improved.

<Hepatocyte growth factor (HGF) production promoter>

The hepatocyte growth factor (HGF) production-promoting agent related to the present invention contains 3β-hydroxylanosta-8,24-diene-21-aldehyde (compound Z2), obetol (compound Z3), ergot At least one of sterol (compound Z6), sterol (compound Z7) and inonotiolide B (compound Z8) is used as an active ingredient, and other ingredients as mentioned above are contained as necessary. As an active ingredient, one of the aforementioned five compounds may be used alone, or two or more of them may be used in combination. The content of the active ingredient in the HGF production accelerator can be appropriately selected according to the purpose. The content of the active ingredient can be, for example, about 1 μg to 100 mg per unit dose. The HGF production accelerator related to the present invention can be the aforementioned active ingredients themselves.

The HGF production-promoting agent related to the present invention contains an active ingredient having an HGF production-promoting effect. Accordingly, the HGF production-promoting agent related to the present invention can be used, for example, in the study of the mechanism of promoting HGF production, the study of symptoms improved by the promotion of HGF production, and the study of symptoms improved by the promotion of HGF production. prevention or treatment.

<hair restorer>

The hair restorer related to the present invention contains lanosterol (compound Z1), 3β-hydroxylanoster-8,24-diene-21-al (compound Z2), obofamol (compound Z3), 3β,21 -dihydroxylanoster-8,24-diene (Compound Z4), Trametesic Acid (Compound Z5), Ergosterol (Compound Z6), Stestrosterol (Compound Z7), Inonotus obliquactone B ( Compound Z8), betulin (compound Z9), β-phytosterol (compound Z10), ergosterol peroxide (compound Z11), betulin C (compound Z12), 3β,22R,25-three At least one of hydroxylanostan-8,23E-diene (compound Z13) and statuulin triol A (compound Z14) is used as an active ingredient. In terms of active ingredients, one of the aforementioned fourteen compounds can be used alone, Alternatively, two or more types may be used in combination. For example, in the case of using Chaga extract containing five compounds Z1 to Z5, the content of the extract in the hair growth agent may be about 0.1 to 5.0% by mass. Also, when using Chaga extract containing fourteen compounds Z1 to Z14, the content of the extract in the hair growth agent can be made about 0.1 to 10% by mass.

Also, the hair restorer related to the present invention contains a lanostane-type triterpene as an active ingredient. The compound which is a lanostane-type triterpene as an active ingredient may contain 1 type alone, or may contain 2 or more types.

In the hair restorer related to the present invention, water, lower alcohols (methanol, ethanol, denatured ethanol, isopropanol, etc.), dissolution aids, surfactants, emulsion stabilizers, gelling agents, In addition to the adhesive, in order to obtain a desired dosage form, commonly used base components can be blended.

Depending on the purpose of use of the hair restorer related to the present invention, other ingredients commonly used in hair restorers may be blended within the range not impairing the effects of the present invention. For other ingredients, for example, minoxidil, carpronium chloride (carpronium chloride) and other vasodilators; spironolactone (spironolactone), flutamide (flutamide), cyproterone acetate ( Androgen receptor inhibitors such as cyproterone acetate and oxendolone; steroid-5α-reductase inhibitors such as finasteride and epristeride; glycyrrhetinic acid (glycyrrhetinic acid) anti-inflammatory agents such as acid) and azulene; adrenal corticosteroids such as hydrocortisone acetate and dexamethasone acetate; keratolytic agents such as urea and salicylic acid; Alcohols such as propylene glycol, 1,3-butanediol, macrogol, glycerin, dipropylene glycol, ethanol, and isopropanol; fatty acid glycerides such as glyceryl monopentadecanoate; hydrochloric acid Antihistamines such as diphenhydramine hydrochloride, etc.

Furthermore, examples include chlorhexidine gluconate, isopropylmethylphenol, fourth grade ammonium salt, hinokitiol, piroctone olamine, etc. Fungicides; moisturizing agents such as sodium hyaluronate, glycerin, chondroitin sulfate, etc.; Loquat (Eriobotrya japonica), Artemisia capillaris, Comfrey, Angelica keiskei, saffron, Gardenia fruit, Rosemary, Medicinal mouse Animal and plant extracts such as Sage, Saussureae Radix, Aristolochia debilis, Hop, and placenta; retinol acetate, Vitamins such as pyridoxine hydrochloride, ascorbic acid, thiamine nitrate, cyanocobalamin, biotin, and tocopherol acetate ; Oils of isopropyl myristate, isopropyl palmitate, squalane, liquid paraffin, lecithin, etc.; polyoxyethylene sorbitan Polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene hardened castor oil, etc. surfactants; antioxidants such as dibutyl hydroxy toluene, isopropyl gallate, etc., ethylenediaminetetraacetic acid (ethylenediaminetetraacetic acid) Chelating agents such as nediamine tetraacetate, cooling agents such as menthol and camphor. Depending on the case, colorants, fragrances, transdermal absorption enhancers, etc. may be blended.

The hair restorer related to the present invention is preferably an external preparation, and any dosage form generally used as an external preparation can be used. In terms of dosage forms, examples include cleansers, emulsions, creams, tonics (tonic agent), ointment, gel, aerosol, etc., but not limited thereto. The hair restorer related to the present invention can be used by applying an appropriate amount to the scalp once to several times a day, for example.

The hair growth agent related to the present invention can be used, for example, in the study of hair growth mechanism, symptoms improved by hair growth, and prevention or treatment of symptoms improved by hair growth promotion. Specific aspects of the hair restorer related to the present invention include, for example, a hair restorer for pharmaceutical use, and a hair restorer for prevention and treatment of symptoms. Symptoms include, for example, male pattern baldness and the like.

The hair restorer related to the present invention contains lanosterol (compound Z1), 3β-hydroxylanosterol-8,24-dien-21-aldehyde (compound Z2), inonotofaciol (compound Z3), 3β, 21-Dihydroxylanosta-8,24-diene (Compound Z4), Trametesic Acid (Compound Z5), Ergosterol (Compound Z6), Strigosterol (Compound Z7), Inonotrolactone B (compound Z8), betulin (compound Z9), β-phytosterol (compound Z10), ergosterol peroxide (compound Z11), betulin C (compound Z12), 3β,22R,25- At least one of trihydroxylanoster-8,23E-diene (compound Z13) and chatolintriol A (compound Z14) as active ingredients has the effect of promoting the proliferation of hair follicle dermal papilla cells and promoting the production of FGF-7 Any one or two or more of the effects of VEGF production promotion, IGF-1 production promotion and HGF production promotion.

Inonotofatiol (compound Z3) and 3β,21-dihydroxylanosta-8,24-diene (compound Z4) have the activity of testosterone 5α-reductase in addition to the above-mentioned effects inhibition. According to this, for example, by including all five kinds of compounds Z1 to Z5 which are lanostane-type triterpenoids, it is possible to obtain excellent dermal papilla cell proliferation-promoting action, FGF-7 production-promoting action, and VEGF production-promoting action. , A hair growth agent that promotes the production of IGF-1 and HGF as well as inhibits the activity of testosterone 5α-reductase. Of course, by combining inotolinol (compound Z3) and one of 3β, 21-dihydroxylanosta-8,24-diene (compound Z4) or Both, and one or more compounds other than the five and nine compounds, can also obtain a hair growth agent with an inhibitory effect on testosterone 5α-reductase activity.

[Example]

Examples are described below, but the present invention is not limited to these Examples at all.

(Example 1)

Example 1 is an example of extract from chaga extract.

1) Add 80% ethanol to 5.0kg of cut fruiting bodies of Chaga (produced in Russia; batch No. 131008, Chihaya Co., Ltd.), and extract at room temperature.

2) The extract obtained in the above 1) was dissolved in 50% ethanol‧for suction filtration to obtain the insoluble fraction (56.1g) and the soluble fraction (183.0g). That is, the treated liquid obtained by dissolving and treating the extract in 50% ethanol was subjected to suction filtration to separate into a soluble part and an insoluble part. Also, the treatment liquid obtained through the dissolution treatment is a suspension in a suspended state.

Also, both 80% ethanol and 50% ethanol are aqueous ethanol solutions, and the volume of ethanol is 80% and 50% relative to the total volume of the aqueous ethanol solution. Hereinafter, it shows similarly.

A compound with low polarity contained in the insoluble part, that is, a low-polarity compound. On the other hand, compounds with high polarity, that is, highly polar compounds, are contained in the soluble portion because the solubility of inositol in water at 25° C. is 14 g/100 mL, for example.

(Example 2)

Example 2 is the extract of chaga obtained in Example 1, lanosterol, 3β-hydroxylanoster-8,24-dien-21-al, chafol, 3β,21-dihydroxy Examples of isolating lanosta-8,24-diene and Tranolic acid (compounds Z1~Z5).

From 1 g of the obtained 50% ethanol insoluble fraction, Compound A (84.8 mg), Compound B (55.0 mg), Compound C (219.9 mg), Compound D (28.1 mg) and Compound E (117.6 mg) were isolated. Specifically, using silica gel column chromatography, using a mixed solvent of hexane:ethyl acetate (12:1→10:1→9:1→8:1→4:1→2:1), a gradient Gradient elution (gradient operation 1).

The molecular structures of the isolated compounds A, B, C, D and E were analyzed by ¹³ C-NMR and ¹ H-NMR, and the measured values were compared with the literature values. The results are as described below, and it was confirmed that all of the compounds A, B, C, D, and E are lanostane-type triterpenoids represented by the above-mentioned general formula (LTT).

Also, in this example, the soluble part means the soluble part containing substances (compounds) that are actually dissolved in ethanol aqueous solution, and the insoluble part or 50% ethanol insoluble part means the part that contains practically undissolved substances (compounds). insoluble part. In this example, although the compounds Z1-Z5 were extracted from the insoluble part, the extraction from the soluble part was not negated. The same applies to the extraction of compounds Z6 to Z14 described later.

The measured values of ¹³ C-NMR and ¹ H-NMR of compounds A, B, C, D and E are shown in Tables 1, 2, 3, 4 and 5 below.

[Table 2]

[table 3]

[Table 4]

[table 5]

Compounds A~D were identified by comparing the above measured values with the values in the following documents 1)~4).

1) Toshihiro Akihisa, Taro Matsumoto, ¹³ C-NMR spectra of sterols and triterpene alcohols, Oil Chemistry, Vol.36(5): 301-319, 1987

2)Satoru Sawai, Tomoyoshi Akashi, Nozomu Sakurai, Hideyuki Suzuki, Daisuke Shibata, Shin-ichi Ayabe, and Toshio Aoki Satoru, Plant Lanosterol Synthase: Divergence of the Sterol and Triterpene Biosynthetic Pathways in Eukaryotes, Plant Cell Physiol (May 2006) 47 (5): 673-677, supplementary data

3) Zheng-Fei Yan, Yang Yang, Feng-Hua Tian, Xin-Xin Mao, Yu Li, and Chang-Tian Li, Inhibitory and Accelerator Effects of Inonotus obliquus on Tyrosinase Activity and Melanin Formation in B16 Melanoma Cells, Evidence-Based Complementary and Alternative Medicine, Vol.2014, 1-11.

4) Kahlos, Kirsti; Hiltunen, R.; Von Schantz, M., 3β-Hydroxylanosta-8,24-dien-21-al, a new triterpene from Inonotus obliquus, Planta Medica (1984), 50(2), 197 -8.

The measured value of compound A was compared with the value recorded in literature 1)~3), and the measured value of compound B and D was compared with the value recorded in literature 4). The measured values of compounds C and E were compared with the values recorded in documents 3) and 4).

Compound A is a compound of the above-mentioned general formula (LTT), R ¹ =CH ₃ and R ² =H, that is, it is identified as lanosterol represented by the following chemical formula (S1) (compound Z1).

Compound B is a compound of the above-mentioned general formula (LTT), R ¹ =CHO and R ² =H, that is, identified as 3β-hydroxylanoster-8,24-diene-21 represented by the following chemical formula (S2) - Aldehydes (compound Z2).

[Chem 3]

Compound C is a compound of the above-mentioned general formula (LTT), R ¹ =CH ₃ and R ² =OH, that is, it is identified as chafol (compound Z3) represented by the following chemical formula (S3).

Compound D is a compound of the above general formula (LTT), R ¹ =CH ₂ OH and R ² =H, that is, identified as 3β,21-dihydroxylanoster-8,24 represented by the following chemical formula (S4) - Diene (compound Z4).

[chemical 5]

Compound E is a compound of the above-mentioned general formula (LTT), R ¹ =COOH and R ² =H, that is, identified as Tranolic acid represented by the following chemical formula (S5) (compound Z5).

(Example 3)

Example 3 is an example of carrying out a test for promoting the proliferation of dermal papilla cells of hair follicles for the five kinds of lanosterane-type triterpenoids (compounds Z1 to Z5) isolated in Example 2.

First, normal human hair follicle dermal papilla cells (manufactured by Toyobo Co., Ltd., trade name "Human Follicle Dermal Papilla Cells (HFDPC) (code name CA60205a)") were cultured. The medium used was 250 mL of a basal medium of a low-serum medium, a hair follicle dermal papilla cell proliferation medium (manufactured by Toyobo Co., Ltd., trade name "Follicular Dermal Papilla Cell Proliferation Medium" (PCGM) (code: TMTPGM-250)), Fetal calf serum (FCS) 2.5mL, insulin‧transferrin‧triiodothyronine (insulin‧transferrin‧triiodothyronine) mixture (ITT) 1.25mL, bovine pituitary gland extract (BPE) added as medium supplements ) 2.5mL and 1.25mL cyproterone acetate (Cyp) dermal papilla cell proliferation medium.

The cells cultured at 37°C, 5% CO ₂ were treated with trypsin by trypsin/EDTA solution (0.05% concentration). The treated cells were diluted with Dulbecco's Modified Eagle DMEM (manufactured by Nacalai Tesque Co., Ltd., hereinafter abbreviated as "DMEM") containing 10% fetal bovine serum (FBS). The concentration is 1.0×10 ⁴ cells/mL. The diluted cells were inoculated into a 96-well microplate Falcon (registered trademark) (manufactured by Becton Dickinson Labware, trade name "MICROTEST ^™ 96") at 200 µL/well. After culturing for 3 days, the medium was aspirated off.

Compounds Z1 to Z5 serving as test samples were dissolved in serum-free DMEM at a predetermined concentration. The concentration was set to 0.01953 μmol/L, 0.078125 μmol/L, 0.3125 μmol/L, 1.25 μmol/L, 5 μmol/L, and 20 μmol/L. Each dissolved test material was added in an amount of 200 μL/well to each well of the 96-well microplate in which the above-mentioned cells were inoculated. After further culturing for 4 days, the above medium was aspirated. Follicular dermal papilla cell proliferation was measured using the MTT assay. That is, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-Dimethylthiazol-2-yl)-2 ,5- diphenyltetrazolium bromide)), dissolved in serum-free DMEM at a final concentration of 0.4 mg/mL, and added at 100 μL/well.

(formazan)生成。將此甲

，以50μL之2-丙醇萃取。萃取後，測定波長570nm中的吸光度，獲得第一吸光度。同時，測定作為濁度之波長650nm中的吸光度，獲得第二吸光度。以第一吸光度及第二吸光度的差作為甲

生成量。毛囊真皮乳頭細胞增殖促進率(%)係利用下述式(1)算出。 After incubating for 2 hours, confirm that there is formazan in the cells

(formazan) generated. put this armor

, extracted with 50 μL of 2-propanol. After the extraction, the absorbance at a wavelength of 570 nm was measured to obtain the first absorbance. Simultaneously, absorbance at a wavelength of 650 nm as turbidity was measured to obtain a second absorbance. The difference between the first absorbance and the second absorbance was used as a

Generation volume. The dermal papilla cell proliferation promotion rate (%) was calculated by the following formula (1).

Hair follicle dermal papilla cell proliferation promotion rate (%)=A1/B1×100‧‧‧Formula (1)

生成量 A1: A when the test material is added

Generation

生成量 B1: A when the test material is not added

Generation

The dermal papilla cell proliferation promotion rates calculated for each of the compounds Z1 to Z5 are summarized in Table 6 below. The promotion rate of dermal papilla cell proliferation is determined by each concentration of compounds Z1 to Z5, and 6 points are obtained for each. The average value and standard error of the 6 points are shown in Table 6 as (mean ± standard error). For reference, minoxidil known to have a proliferation-promoting effect on dermal papilla cells of hair follicles was used.

*：ρ<0.05，***：ρ<0.001，※：細胞生存率<80%

*: ρ<0.05, ***: ρ<0.001, ※: cell viability <80%

Reference) Minoxidil 80μmol/L 116.1±3.5*

As shown in Table 6 above, a low concentration of 3β,21-dihydroxylanosta-8,24-diene (compound Z4) was observed at 0.3125 μmol/L, showing a comparable degree of activity to 80 μmol/L minoxidil Follicular dermal papilla cell proliferation promotion. Also, at a concentration of 1.25 μmol/L of 3β,21-dihydroxylanosta-8,24-diene (compound Z4), the proliferation-promoting effect of minoxidil exceeding 80 μmol/L was obtained.

In addition, at a low concentration of 0.3125 μmol/L, Trametesic acid (Compound Z5) obtained the same degree of promoting effect on the proliferation of dermal papilla cells as 80 μmol/L minoxidil. At 0.3125 μmol/L of inoforol/L (compound Z3) and 20 μmol/L of lanosterol (compound Z1), it was also confirmed that minoxidil, which is close to 80 μmol/L, can promote the proliferation of hair follicle dermal papilla cells.

The results of the growth-promoting effect test on dermal papilla cells of five kinds of lanostane-type triterpenoids (compounds Z1 to Z5) at low concentrations are shown in Table 7 below and FIG. 1 . In Table 7 and Figure 1 of these, the results of minoxidil are also combined for comparison.

平均值±S.E.，n=4，*：ρ<0.05，**：ρ<0.01，***：ρ<0.001

Mean ± SE, n=4, *: ρ<0.05, **: ρ<0.01, ***: ρ<0.001

Also, 3β-hydroxylanosta-8,24-diene-21-aldehyde (compound Z2) also obtained the hair follicle dermis of minoxidil exceeding 80 μmol/L at low concentrations of 0.01953 μmol/L and 0.078125 μmol/L. Papillary cell proliferation-promoting effect.

From the results of Example 3, it was confirmed that lanosterol, 3β-hydroxylanoster-8,24-diene-21-aldehyde, obetrol, 3β,21-dihydroxylanoster-8,24-diene and Trametesic acid (compounds Z1~Z5) can be an active ingredient of a proliferator for dermal papilla cells of hair follicles. These compounds Z1~Z5 showed a promoting effect on the proliferation of dermal papilla cells exceeding minoxidil at a lower concentration than minoxidil. Accordingly, lanosterol, 3β-hydroxylanoster-8,24-diene-21-aldehyde, obliqurol, 3β,21-dihydroxylanoster-8,24-diene and Trametes acid ( Compounds Z1~Z5) are better than minoxidil in promoting the proliferation of hair follicle dermal papilla cells.

(Example 4)

Example 4 is an example of carrying out a test for promoting the proliferation of hair follicle dermal papilla cells for the mixture of five kinds of lanosterane-type triterpenoids (compounds Z1-Z5) isolated in Example 2.

The concentration (selection concentration) of each compound in the mixture is set as lanosterol (20μmol/L), 3β-hydroxylanoster-8,24-diene-21-al (0.3125μmol/L), obetulinol (0.3125μmol/L), 3β,21-dihydroxylanoster-8,24-diene (5μmol/L) and Trametes acid (1.25μmol/L).

Using this mixture, a test for promoting the growth of follicle dermal papilla cells was carried out by the same method as above. The calculated dermal papilla cell proliferation promotion rate is summarized in Table 8 below together with the results of various concentrations of minoxidil.

As shown in the above Table 8, the mixtures of compounds Z1 to Z5 containing five kinds of lanostanoid terpenoids at specified concentrations were observed to exhibit a growth-promoting effect of minoxidil exceeding 20 μmol/L on hair follicle dermal papilla cells.

(Example 5)

Example 5 is an example of performing a test for promoting the production of various growth factor genes related to the hair cycle for the five kinds of lanostanoid triterpenoids (compounds Z1 to Z5) isolated in Example 2. The production promotion test was performed by an mRNA expression promotion test for evaluating the expression of mRNA.

Human normal hair follicle dermal papilla cells (HFDPC: derived from the top of the head) were inoculated on 60mm Petri dishes and cultured using human normal hair follicle dermal papilla cells medium (PCGM). After the cells were fused, the medium was exchanged with DMEM medium containing 10% FBS and cultured for 2 hours. Compounds Z1~Z5, which were the test materials, were dissolved in serum-free DMEM medium at a specified concentration. The concentration system was set to 1.25 μmol/L, 5 μmol/L, and 20 μmol/L.

Add 3 mL of each dissolved test sample to each Petri dish after incubation. After further culturing for 6 hours, total RNA was prepared by a general method. Cells cultured without supplementation in the test material Cells also modulated total RNA in the same way. The amount of each RNA was measured with a spectrophotometer, and the total RNA was adjusted to 200 ng/μL.

Using this total RNA as a template, the expression levels of mRNAs of various growth factors related to the hair cycle and GAPDH as an internal standard were measured. The detection system uses real-time PCR (Real time PCR) device Smart Cycler (registered trademark) (Cepheid company), by using the real-time 2 Step RT-PCR reaction was carried out.

The mRNA expression levels of various growth factors were calculated by correcting the expression level of GAPDH mRNA. The expression rate of each growth factor mRNA was calculated by the following formula (2).

Various growth factor mRNA discovery rate (%)=A2/B2×100‧‧‧Formula (2)

A2: The correction value when the test material is added

B2: The corrected value when the test material is not added (control)

As the above growth factors, fibroblast growth factor-7 (FGF-7), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) were investigated.

The mRNA expression rates of various growth factors of compounds Z1 to Z5 are summarized in Tables 9 to 13 below. The mRNA expression rates shown in Tables 9 to 13 are relative values when the control is set as 100% in each point. Furthermore, the mRNA expression ratios of adenosine mononucleotides known to have a vascular endothelial growth factor (VEGF) production-promoting effect are summarized in Table 14 below.

As shown in the table above, in lanosterol (compound Z1), 3β-hydroxylanoster-8,24-diene-21-al (compound Z2), obetrol (compound Z3) and 3β,21-dihydroxy Lanosta-8,24-diene (compound Z4) was observed to promote the production of FGF-7 mRNA by about 1.2~1.6 times that of the test sample without addition.

From the results of Example 5, it was confirmed that lanosterol, 3β-hydroxylanoster-8,24-diene-21-aldehyde, inotorol and 3β,21-dihydroxylanoster-8,24-diene ( Compounds Z1~Z4) can each be an active ingredient of an FGF-7 production accelerator. It can be seen that any of these compounds Z1~Z4, at a low concentration of 5 μmol/L, exhibits a production-promoting effect equal to or more than that of 25 μmol/L adenylic acid. The promotion effect is more excellent.

As shown in the above Table 13, 20 μmol/L Trametesic acid (compound Z5) can promote the expression of VEGF mRNA about 1.5 times that of the test sample without addition. Tranexamic acid (compound Z5) can be used as an active ingredient of a VEGF production accelerator as shown in Example 5. In addition, even at 20 μmol/L, Trametesic acid (compound Z5) exhibits a VEGF mRNA expression promoting effect of more than 25 μmol/L adenosine, which is more excellent than the conventional adenosine in promoting VEGF production.

Furthermore, inotolin (compound Z3), it was confirmed that about 1.2 times the IGF-1 mRNA expression promotion effect of the test sample without addition was confirmed. In 3β-hydroxylanosta-8,24-dien-21-al (compound Z2) and inotolinol (compound Z3), it was confirmed that HGF mRNA expression was promoted. Inotolinol (compound Z3) is an active ingredient that can be used as an IGF-1 production accelerator. 3β-Hydroxylanosta-8,24-dien-21-al (compound Z2) and inotolinol (compound Z3) can each be used as an active ingredient of the HGF production accelerator.

(Example 6)

Example 6 is an example of carrying out an inhibitory effect test on testosterone 5α-reductase activity for the five kinds of lanostane-type triterpenoids (compounds Z1-Z5) isolated in Example 2.

Dissolve testosterone in propylene glycol to prepare a solution with a concentration of 4.2 mg/mL. Mix 20 μL of this solution and 825 μL of 5 mmol/L Tns-HCl buffer (pH 7.13) containing 1 mg/mL NADPH in a V-bottom test tube with a cap. Compounds Z1 to Z5 as test samples were dissolved in ethanol, 50% ethanol or pure water to prepare solutions.

Add 80 μL of the test sample solution and 75 μL of S-9 rat liver homogenate to the mixture in the aforementioned V-bottom test tube with a cap, and mix again. After reacting the contents in the test tube at 37° C. for 60 minutes, 1 mL of dichloromethane was added to stop the reaction. The reaction mixture was centrifuged (1600×g, 10 minutes), and the dichloromethane phase was analyzed by gas chromatography under the following conditions. Further blank test was carried out in the same way.

In order to obtain the conversion rate from testosterone to 3α-androstanediol (androstanediol) and dihydrotestosterone, the ethanol solution of the standard products of 3α-androstanediol, dihydrotestosterone and testosterone was used as a gas phase layer in advance. The analysis method was used to obtain the peak areas of these three compounds.

The relative ratio of the peak areas of 3α-androstanediol, dihydrotestosterone, and testosterone after the reaction using S-9 to the peak areas of the standard was obtained according to the following formula (3). After that, the conversion rate of the test sample is calculated according to formula (4).

Relative ratio = peak area of tested material/peak area of standard product‧‧‧Formula (3)

Conversion rate (%)=(A3+B3)/(A3+B3+C3)×100‧‧‧Formula (4)

A3: Relative comparison of 3α-androstanediol

B3: Relative comparison of dihydrotestosterone

C3: Relative comparison of testosterone

Based on the calculated conversion rate, the testosterone 5α-reductase activity inhibition rate was obtained according to the following formula (5).

Inhibition rate of testosterone 5α-reductase activity (%)=(1-E/D)×100‧‧‧Formula (5)

D: conversion rate in blank test

E: Conversion rate added to the tested material

Also, the conditions of gas chromatography are as follows.

Machine used: Shimadzu GC-2010

String: DB-1701

(φ0.53mm×30m. Thickness: 1.0μm)

Column/injection temperature: 240°C/300°C

Detector: FID

Carrier gas: nitrogen gas

The testosterone 5α-reductase activity inhibition rate and 50% inhibitory concentration (IC ₅₀ ) of compounds Z1-Z5 are summarized in Table 15 below together with the results of β-estradiol as a positive control.

From the results of Example 6, it was confirmed that inotolinol (compound Z3) and 3β,21-dihydroxylanosta-8,24-diene (compound Z4) had an inhibitory effect on testosterone 5α-reductase activity.

(Example 7)

Example 7 is the extract of chaga obtained in free example 1, each extracting ergosterol (compound Z6), astral sterol (compound Z7), inonotolidin B (compound Z8), betula (compound Z9), β-phytosterol (compound Z10), ergosterol peroxide (compound Z11), obulin C (compound Z12), 3β,22R,25-trihydroxylanoster-8, Example of extraction of 23E-diene (compound Z13), statuulin triol A (compound Z14).

From the 50% ethanol insoluble fraction obtained in Example 1, compound F, compound G, compound H1, compound H2, compound I, compound J, compound K, compound L, compound M, compound N.

Among the 18 fractions FA1 to FA18 obtained in the fractionation operation 1 of Example 2, the 5th to 7th fractions FA5 to FA7 were respectively removed from the eluting solvent to obtain fractions. The mixture of fractions obtained from fractions FA5~FA7 was fractionated into 9 fractions FB1~ FB9 (grading operation 2).

Fractions FB6 and FB7 at the 6th and 7th positions among the fractions FB1 to FB9 obtained in the fractionation operation 2 were respectively removed from the eluting solvent to obtain fractions. The mixture of fractions obtained from fractions FB6 and FB7 was subjected to silica gel column chromatography (dissolution solvent: toluene → "mixed solvent of toluene:acetone (30:1 → 15:1)" → ethyl acetate) , classified into 10 fractions FC1~FC10 (fractionation operation 3).

In addition, the arrows in the above description of the eluting solvent indicate that the composition of the eluting solvent and the ratio of the mixed solvent used therein are changed, and it means that gradient elution is performed. The same applies to eluents used in high-speed liquid chromatography described later.

Among the fractions FC1 to FC10 obtained in the fractionation operation 3, the third fraction FC3 was obtained from which the eluting solvent was removed as compound F (12.0 mg). Also, the compound G (4.3 mg) obtained from the 4th fraction FC4 after removing the eluting solvent was obtained.

In addition, 39.4 g of the 50% ethanol insoluble portion obtained in Example 1 was subjected to silica gel column chromatography (dissolution solvent: "mixed solvent of hexane: ethyl acetate (12:1 → 11:1 → 10:1 → 9:1→8:1→6:1→4:1→2:1)"), classified into 8 fractions FD1~FD8 (grading operation 4).

Remove the eluting solvent from the 4th fraction FD4 in the fractions FD1~FD8, and perform silica gel column chromatography (dissolving solvent: "hexane: ethyl acetate mixed solvent (15:1→13:1→10: 1→8:1→6:1→4:1)”) into 11 fractions FE1~FE11 (fractionation operation 5).

Fractions were obtained by removing eluting solvents from the 8th and 9th fractions FE8 and FE9 of the fractions FE1-FE11. The mixture of fractions obtained from fractions FE8 and FE9 was fractionated into 6 fractions by silica gel column chromatography (dissolution solvent: "mixed solvent of toluene:acetone (20:1→19:1→18:1)") Fractions FF1~FF6 (fractionation operation 6).

The eluting solvent is removed from the 6th and 7th fractions FE6 and FE7 among the fractions FE1 to FE11 through the fractionation operation 5, and the first fraction FF1 among the fractions FF1 to FF6 through the fractionation operation 6. , to obtain splits respectively. The mixture of the fractions obtained from fractions FE6, FE7 and fraction FF1 was subjected to silica gel column chromatography (dissolution solvent: "mixed solvent of hexane:ethyl acetate (12:1→10:1→9 :1→8:1→7:1)”) into 14 fractions FG1~FG14 (grading operation 7).

Among the fractions FG1 to FG14 obtained in the above-mentioned fractionation operation 7, the 8th fraction FG8 was removed from the eluting solvent as compound J (113.9 mg).

From fractions FD1~FD8 of fractions FD1~FD8 after fractionation operation 4, fractions from the 6th fraction FD6 were removed from the eluting solvent, and silica gel column chromatography (dissolving solvent: mixed solvent of "toluene: acetone (30: 1→ 25:1→20:1→15:1)”), classified into 10 fractions FH1~FH10 (fractionation operation 8).

Among the fractions FH1 to FH10 obtained in the fractionation operation 8, the fifth fraction FH5 was obtained from which the eluting solvent was removed as compound H1 (209 mg). Also, the compound H2 (120.8 mg) was obtained from the 6th FH6 by removing the eluting solvent.

Obtained from fractions FH1~FH10 in the 7th and 8th fractions FH7, FH8 respectively remove the fractionation solvent. The mixture of fractions obtained from fractions FH7 and FH8 was analyzed by high-speed liquid chromatography (column: N-2 type, eluent: "hexane: ethyl acetate mixed solvent (8:2)", flow rate 7.0mL/min), graded into 8 fractions FI1~FI8 (grading operation 9).

Among the fractions FI1 to FI8 obtained in the fractionation operation 9, the fourth fraction FI4 was obtained after removing the eluting solvent as compound I (10.2 mg).

Also, the fraction obtained from fractions FH1 to FH10 obtained through the fractionation operation 8 obtained from fractions FH9 at the 9th position in which the eluting solvent was removed, was subjected to silica gel column chromatography (elution solvent: "hexane: The mixed solvent of ethyl acetate (6:1 → 5:1 → 4:1 → 3:1)") was classified into 12 fractions FJ1~FJ12 (fractionation operation 10).

Compound K (9.4 mg) was obtained by removing the eluent from the ninth fraction FJ9 among the fractions FJ1 to FJ12 obtained in the fractionation operation 10.

From the 8th fraction FD8 in the fractions FD1~FD8 of the fractionation operation 4, remove the eluting solvent, and use silica gel column chromatography (dissolving solvent: "hexane: ethyl acetate mixed solvent (6 :1→4:1→2:1→1:1)”), classified into 13 fractions FK1~FK13 (grading operation 11).

The fraction obtained from fraction FK10 at the 10th position among the fractions FK1 to FK13 obtained through the fractionation operation 11 from which the eluting solvent was removed. The fraction obtained from this fraction FK10 was subjected to high-speed liquid chromatography (column: N-6 type, eluent: "hexane: ethyl acetate mixed solvent (6:1)", flow rate 3.0mL/ points), classified into 18 fractions FL1~FL18 (grading operation 12).

The 17th fraction FL17 among fractions FL1~FL18 was subjected to high-speed liquid chromatography (column: R-41 type, eluent: mixed solvent of "acetonitrile (CH ₃ CN): water (H ₂ O) (7:3)", flow rate 5.0mL/min), classified into 12 fractions FL1~FL12 (fractionation operation 13).

Of the fractions FL1 to FL12 obtained from fractionation operation 13, the second fraction FL2 from which the eluent was removed was taken as compound M (7.7 mg), and the twelfth fraction FL12 from which the eluent was removed was taken as the compound N (47.3mg) and obtained.

The molecular structures of compounds F, G, H1, H2, I~N obtained as above were analyzed by ¹³ C-NMR and ¹ H-NMR. As a result, it was confirmed that compound H1 and compound H2 were extracted from the same compound. Also, it was confirmed that compound L (47.3 mg) was the same compound extracted from fraction FA10 at the 10th position among fractions FA1 to FA18 obtained from fractionation operation 1. Furthermore, it was confirmed that compound I is the same compound as the compound extracted from the 12th fraction FA12, and compound N is the same compound as the compound extracted from the 16th fraction FA16.

The measured values of ¹³ C-NMR and ¹ H-NMR of compounds F, G, H1, H2 and I~N were compared with literature values. As a result, as described below, it was confirmed that compounds F, G, H1, H2, and I~N were extracting ergosterol (compound Z6), astral sterol (compound Z7), and inonotiolide B (compound Z8) , betulin (compound Z9), β-phytosterol (compound Z10), ergosterol peroxide (compound Z11), betulin C (compound Z12), lanosterol-8,23E-diene- 3β-, 22R, 25-triol (compound Z13), statuulin triol A (compound Z14).

The measured values of ¹³ C-NMR and ¹ H-NMR of compounds F, G, H1, H2 and I~N are shown in Table 16~Table 24. Also, Table 21 shows the measured value of Compound K0, which was confirmed to be the same as Compound K obtained by other fractionation operations, and Table 22 shows the measured value of Compound L0 obtained from fraction FA10.

[Table 16]

[Table 17]

[Table 18]

[Table 19]

[Table 20]

[Table 21]

[Table 22]

[Table 23]

[Table 24]

The identification of the compound was carried out by comparing the measured values with the literature values described in the above literature 1) and the following literature 5) to 14).

5) Seo, Hyo Won; Arch. Pharm. Res., 2009, 32(11), 1573-1579

6) Jeffrey L.C. Wright, Can. J. Chem., 1979, 57, 2569-2571

7) Alejandro F. Barrero, et. al., A.C.S. and A.S.P, 1998, 1491-1496

8) You-Min Ying, et.al., Phytochemistry, 2014, 108, 171-176

9) Mochammad Sholichin, Kazuo Yamasaki, Ryoji Kasai and Osamu, Chem. Pharma. Bull., 1980, 28(3), 1006-1008

10) Kuo-Ching Kao, Yu-Ling Ho, I-Hsin Lin, Li-Kang Ho and Yuan-Shiun Chang, J. Chin. Chem. Soc., 2004, 51(1), 199-204

11) Garg. V.K., and Nes, W.R., Phytochemistry, 1984, 23, 2925-2929

12) T. Akihisa, S. Thakur, F.U. Rosenstein, T. Matsumoto, Lipids, 1986, 21, 39-47

13) Zhao Fenqin, et. al. Fitoterapia, 2015, 101, 34-40

14) S Taji, et al., European Journal of Medicinal Chemistry 43 (2008), 2373-2379

15) Sayaka Taji, Takeshi Yamada and Reiko Tanaka, Helvetica Chimica Acta, 2008, 91, 1513-1524

The measured value of compound F, G is compared with the value recorded in documents 1), 5) ~ 7), the measured value of compound H is compared with the value recorded in document 8), and the measured value of compound I is compared with the value recorded in document 9), 10 ) to compare with the value recorded. Also, the measured value of compound J is compared with the value recorded in documents 1), 11), and 12), the measured value of compound K is compared with the value recorded in document 5), and the measured value of compound L is compared with the value recorded in document 13). value comparison. In addition, the measured value of compound M is compared with the value described in document 14), and the measured value of compound N is compared with the value described in document 15).

Compound F can be judged to be ergosterol (compound Z6) represented by the following chemical formula (S6).

Compound G can be judged to be stestrosterol (compound Z7) represented by the following chemical formula (S7).

Compounds H1 and H2 can be determined to be Inonotus obliquilide B (compound Z8) represented by the following chemical formula (S8).

Compound I can be determined to be betulin (compound Z9) represented by the following chemical formula (S9).

[chemical 10]

Compound J can be determined to be β-phytosterol (compound Z10) represented by the following chemical formula (S10).

Compound K can be determined to be an ergosterol peroxide (compound Z11) represented by the following chemical formula (S11).

[chemical 12]

Compound L can be judged to be chacilin C (compound Z12) represented by the following chemical formula (S12).

Compound M can be judged to be 3β,22R,25-trihydroxylanostan-8,23E-diene (compound Z13) represented by the following chemical formula (S13).

[chemical 14]

Compound N can be judged to be statuulin triol A (compound Z14) represented by the following chemical formula (S14).

(Embodiment 8)

Example 8 is an example of carrying out a test for promoting the proliferation of hair follicle dermal papilla cells for the compounds Z6-Z12 obtained in Example 7.

Utilize the method identical with above-mentioned embodiment 3, carry out dermal papilla cell proliferation promotion test, for the ergosterol (compound Z6) that embodiment 7 obtains, astral sterol (compound Z6) obligate Z7), inonotrolactone B (compound Z8), betulin (compound Z9), β-phytosterol (compound Z10), ergosterol peroxide (compound Z11) and betulin C (compound Z12), and obtain the promotion rate of dermal papilla cell proliferation of hair follicles. The concentrations of the compounds Z6~Z12 in the test samples were 0.3125 μmol/L, 1.25 μmol/L, 5 μmol/L, and 20 μmol/L. The tests of compounds Z6~Z10 were carried out separately from the tests of compounds Z1 and Z12. In each test, compound Z1 and minoxidil were used as references.

The dermal papilla cell proliferation promotion rate (%) was calculated from the aforementioned formula (1). Table 25 and the graph of FIG. 2 show the promotion rate of dermal papilla cell proliferation for compounds Z6 to Z10, and Table 26 and the graph of FIG. 3 for the promotion rate of dermal papilla cell proliferation for compounds Z11 and Z12. In Table 25 and Table 26, the results for each concentration of lanosterol (compound Z1) and minoxidil are shown together. The promotion rate of dermal papilla cell proliferation of each concentration of compounds Z6~Z12, Z1 and minoxidil shown in Table 25 and Table 26 is the mean ± standard error of 3 points respectively.

As shown in Table 25, it was confirmed that ergosterol (compound Z6), sterol (compound Z7) and Inonotus obliquus lactone B (compound Z8) at a concentration of 1.25 μmol/L showed higher levels than those at the same concentration and above The concentration of lanosterol (compound Z1) and minoxidil have a stronger effect on promoting the proliferation of dermal papilla cells. Also, as shown in Table 26, it was confirmed that ergosterol peroxide (Compound Z11) and Chatulin C (Compound Z12) with a concentration of 0.315 μmol/L were more effective than lanosterol (Compound Z1) and the same concentration. Minoxidil with a higher concentration has a stronger effect on promoting the proliferation of hair follicle dermal papilla cells. These imply that the lanostane backbone is important for activity.

From the results of Example 8, it was confirmed that ergosterol (compound Z6), sterol (compound Z7), Inonotus obliquus lactone B (compound Z8), ergosterol peroxide (compound Z11) and betula Fustatin C (compound Z12) can be an active ingredient of a proliferative agent for dermal papilla cells of hair follicles. These compounds Z6~Z8, Z11, and Z12, at a lower concentration than minoxidil, showed a promoting effect on the proliferation of dermal papilla cells exceeding minoxidil. Therefore, it was found that compounds Z6 to Z8, Z11, and Z12 were superior to minoxidil in promoting the growth of dermal papilla cells.

(Example 9)

Example 9 is an example in which the production promotion test of various growth factor genes related to the hair cycle was carried out with respect to the compounds Z6 to Z14 obtained in Example 2. The production promotion test was carried out using an mRNA expression promotion test for evaluating the expression of mRNA.

Ergosterol (compound Z6), sterol (compound Z7), inonotrolactone B (compound Z8), betula (compound Z9), β-phytosterol (compound Z10), ergosterol Peroxide (compound Z11), statuulin C (compound Z12), 3β, 22R, 25-trihydroxylanoster-8,23E-diene (compound Z13), statuulin triol A ( Compound Z14) was used as a test sample, and the mRNA expression promoting effect test was carried out by the same method as the above and Example 5. The concentration of each tested sample was set at 1.25 μmol/L, 5 μmol/L, and 20 μmol/L.

Fibroblast growth factor-7 (FGF-7), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) The expression level of each mRNA. The expression level of growth factor mRNA was corrected by the expression level of GAPDH mRNA, and the expression rate (%) of growth factor mRNA was calculated using the aforementioned formula (2). Tables 27-35 summarize the expression rates of various growth factor mRNAs for each of the compounds Z6-Z14. The mRNA expression rates (unit: %) shown in Tables 27 to 35 are each at 1 point, and the control group is set as a relative value of 100%. Table 36 also shows the mRNA expression rates (%) of fibroblast growth factor-7 (FGF-7) and vascular endothelial growth factor (VEGF) with respect to adenosine at a concentration of 25 μmol/L.

As shown in Table 27 to Table 35, in compounds Z6 to Z14, it was confirmed that the FGF-7 mRNA production promotion effect was about 1.15 to 1.6 times that of the case where the test sample was not added. Based on the results of Example 9, it was confirmed that compounds Z6-Z14 could be effective components of FGF-7 production accelerators. In particular, it was confirmed that 20 μmol/L of compound Z6, compound Z8, and compound Z10 were equivalent to 25 μmol/L of adenylic acid 1.01 to 1.08 times the FGF-7 mRNA production-promoting effect, it is known that the FGF-7 production-promoting effect is superior to that of the conventional adenosine monophosphate.

As shown in Table 27, Table 29, and Table 30, it is confirmed that Compound Z6, Compound Z8, and Compound Z9 exhibit a promoting effect on VEGF mRNA at 1.25 μmol/L, 1.1 to 1.2 times that of the test material, and at 1.25 μmol/L. 20μmol/L, 1.2~2.1 times of the VEGF mRNA expression without the addition of the tested sample showed a promoting effect. From the results of Example 9, it was confirmed that compound Z6, compound Z8 and compound Z9 can be effective ingredients of VEGF production promoters. In addition, Compound Z6, Compound Z8, and Compound Z9 showed a VEGF mRNA expression-stimulating effect equivalent to 25 μmol/L of adenylic acid to more than 20 μmol/L. produce a promotional effect.

As shown in Table 27 to Table 29, Table 31, Table 33, and Table 34, it was confirmed that compounds Z6 to Z8, Z10, Z12, and Z13 had an IGF-1 mRNA expression promoting effect of about 1.1 to 1.55 times that of the test sample without addition. Accordingly, it is known that compounds Z6~Z8, Z10, Z12, and Z13 can be used as active ingredients of IGF-1 production accelerators.

As shown in Table 27 to Table 29, it was confirmed that compounds Z6 to Z8 had an HGF mRNA expression promoting effect of about 1.3 to 1.4 times that of the test samples without addition. From this, it was found that the active ingredient can be used as an HGF production accelerator.

From the results of the above examples, it is known that compounds Z1 to Z14 are used as dermal papilla cell proliferation promoters, fibroblast growth factor-7 (FGF-7) production promoters, and vascular endothelial growth factor (VEGF) production promoters. It is a novel active ingredient of insulin-like growth factor-1 (IGF-1) production enhancer and hepatocyte growth factor (HGF) production enhancer. In addition, it is speculated that compounds Z1~Z14 can stimulate the expression of FGF-7, VEGF, IGF-1 and HGF mRNA through different mechanisms. growing up. This effect seems to have nothing to do with the presence or absence of the 8-position double bond and the 4-position dimethyl group of the lanosterane backbone, but has something to do with the side chain of the lanosterane-type triterpenes.

(Example 10)

Example 10 is the result of an activity evaluation test related to hair growth for the 80% ethanol extract (50% ethanol insoluble part and soluble part) obtained in Example 1.

Table 37 below shows the results of the growth-promoting effect test on dermal papilla cells of hair follicles. The test for promoting the proliferation of dermal papilla cells was carried out by the method described in Example 3.

As shown in Table 37 above, in the 50% ethanol insoluble fraction, the proliferative effect of minoxidil at a low concentration of 3.125 μg/mL exceeding 4.185 μg/mL was observed on dermal papilla cells of the follicle. The dermal papilla cell proliferation effect of the insoluble portion at this time was equivalent to the dermal papilla cell proliferation promoting effect of the soluble portion at 12.5 μg/mL.

Tables 38 and 39 below show the results of the test for promoting the production of various proliferation genes related to the hair cycle. The test for promoting the production of various proliferation genes was carried out by the method described in Example 5. Table 38 shows the results when the concentration was 3.125 μg/mL, and Table 39 shows the results when the concentration was 12.5 μg/mL. In addition, the results with respect to adenosine acid are collated in Table 40 below.

As shown in the above table, the 50% ethanol insoluble fraction was observed at a low concentration of 3.125 μg/mL, showing the promotion of FGF-7 production and the promotion of VEGF equivalent to 13.4 μg/mL of adenosine monophosphate.

Table 41 below shows the results of the test for the testosterone 5α-reductase activity inhibitory effect. The test of testosterone 5α-reductase activity inhibition was carried out by the method described in Example 6.

In Table 41 above, it is shown that the 50% ethanol-insoluble fraction has an inhibitory effect on testosterone 5α-reductase activity.

Furthermore, an androgen receptor (androgen receptor) antagonistic inhibition test was performed on the 50% ethanol insoluble fraction and the soluble fraction. The test method will be described below.

First, SC-3 cells, which were isolated from naturally occurring breast cancer in mice (Shionogi carcinoma, SC-115), were cultured in MEM medium containing 2% DCC-FBS and 10 ^-8 mol/L testosterone ( After culturing in MEM/2 medium), the cells were recovered by treatment with trypsin. The recovered cells were diluted with MEM/2 medium to a concentration of 1.0×10 ⁵ cells/mL. The diluted cells were seeded in a 96-well dish at 100 μL/well.

After culturing overnight, the culture medium was removed. Add 100 μL of 10 ⁻⁹ mol/L dihydrotestosterone dissolved in Ham F12+MEM (HMB) containing 0.5% BSA and the test sample. After culturing for 48 hours, MTT dissolved in MEM/2 at a final concentration of 0.4 mg/mL was added at 100 μL/well.

之生成。以2-丙醇200μL萃取此甲

。萃取後，測定波長570nm中的吸光度，獲得第一吸光度。同時，測定波長650nm中的吸光度作為濁度，獲得第二吸光度。以第一吸光度與第二吸光度的差作為甲

生成量。 After incubating for 2 hours, confirm that A

of generation. Extract the formazan with 200 μL of 2-propanol

. After the extraction, the absorbance at a wavelength of 570 nm was measured to obtain the first absorbance. At the same time, absorbance at a wavelength of 650 nm was measured as turbidity to obtain a second absorbance. Take the difference between the first absorbance and the second absorbance as the formazan

Generation volume.

Cells cultured with only HMB were used as a blank test, and cells cultured with HMB containing only 10 ^-9 mol/L DHT were used as a positive control, and the same method was used for correction. The obtained results are shown in Table 42 below.

In the above Table 42, it is shown that the 50% ethanol insoluble fraction has a moderate androgen receptor antagonistic inhibitory effect.

(Embodiment 8)

Hereinafter, the aspect example of the hair restorer related to this invention is shown.

Claims (6)

A use for preparing hair restorer with active ingredients, characterized in that the above-mentioned active ingredients include at least one of the following ingredients: lanosterol, 3β-hydroxylanosterol-8,24-diene-21-aldehyde (3β- hydroxylanosta-8,24-diene-21-al), inotodiol, 3β,21-dihydroxylanost-8,24-diene (3β,21-dihydroxylanost-8,24-diene) , Trametenolic acid (trametenolic acid), ergosterol, star sterol, inonotrolactone B, ergosterol peroxide, betulin C, 3β, 22R, 25-trihydroxy wool Stero-8,23E-diene and statuulin triol A. The use of preparing hair restorer as described in Claim 1, wherein the above-mentioned active ingredients lanosterol, 3β-hydroxylanoster-8,24-diene-21-al, obetol, 3β,21-diene At least one of hydroxylanoster-8,24-diene, trametesic acid, ergosterol, sterol, inonotiolide B, ergosterol peroxide, and betulin C It is included as an active ingredient of the hair follicle dermal papilla cell proliferation promoter in the aforementioned hair restorer. The use of preparing hair restorer as described in Claim 1, wherein the above-mentioned active ingredients lanosterol, 3β-hydroxylanoster-8,24-diene-21-al, obetol, 3β,21-diene Hydroxylanosterol-8,24-diene, ergosterol, star sterol, inonotrolactone B, ergosterol peroxide, betulin C, 3β,22R,25-tri At least one of Hydroxylanoster-8,23E-diene and Chatulintriol A is used as an active ingredient of the fibroblast growth factor-7 (FGF-7) production accelerator in the aforementioned hair restorer Include. The use of hair tonic as described in Claim 1, wherein at least one of Tranexamic acid, ergosterol and Inonotus obliquactone B in the above-mentioned active ingredients is used as the vascular endothelial growth factor in the aforementioned hair tonic (VEGF) is contained as an active ingredient of a production accelerator. The use of preparing hair restorer as described in claim 1, wherein the above-mentioned active ingredients include inotolin, ergosterol, sterol, inonotolactone B, inonotin C and 3β , At least one of 22R, 25-trihydroxylanosta-8, 23E-diene is included as an active ingredient of the insulin-like growth factor-1 (IGF-1) production accelerator in the aforementioned hair growth agent. The use of preparing hair restorer as described in Claim 1, wherein the 3β-hydroxylanosta-8,24-diene-21-aldehyde, inotolinol, ergosterol, and astral sterol in the above-mentioned active ingredients and at least one of Inonotiolide B is included as an active ingredient of the hepatocyte growth factor (HGF) production promoter in the aforementioned hair restorer.

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