JP6582322B2 - Hair 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, liver Cell growth factor (HGF) production promoter and hair restorer - Google Patents

Description

  The present invention relates to a dermal papilla cell growth promoter, fibroblast growth factor-7 (FGF-7) production promoter, vascular endothelial growth factor (VEGF) production promoter, and insulin-like growth factor-1 (IGF-1) production. The present invention relates to a promoter, a hepatocyte growth factor (HGF) production promoter and a hair restorer.

  Known factors relating to hair growth include fibroblast growth factor-7 (FGF-7), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF). It has been.

  Minoxidil and adenosine are said to promote the production of the above-mentioned factors such as FGF-7 and VEGF, and hair restorers containing these as active ingredients are known (Patent Documents 1 and 2). In addition, it is known that certain lanostane-type triterpenes extracted from garlic mushroom have a 5α-reductase inhibitory action effective for prevention and treatment of male pattern baldness (Patent Document 3).

Japanese Patent Laid-Open No. 5-4908 JP 2000-297015 A International Publication No. 2005/079164

  In recent years, in order to obtain a hair growth agent exhibiting a more excellent hair growth effect, a hair papilla cell growth promoter, a fibroblast growth factor-7 (FGF-7) production promoter, a vascular endothelial growth factor (VEGF) production promoter, Higher effects are required for insulin-like growth factor-1 (IGF-1) production promoters and hepatocyte growth factor (HGF) production promoters.

  Accordingly, the present invention provides a novel dermal papilla cell growth promoter, fibroblast growth factor-7 (FGF-7) production promoter, vascular endothelial growth factor (VEGF) production promoter, insulin, which exhibits an effect superior to conventional ones. An object of the present invention is to provide a growth-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 dermal papilla cell proliferation promoter according to the present invention comprises lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol, 3β, 21-dihydroxylanosto-8,24-diene and trametenolic acid. It contains at least one kind as an active ingredient.

  The fibroblast growth factor-7 (FGF-7) production promoter according to the present invention includes lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol and 3β, 21-dihydroxylanosto-8. , 24-diene is contained as an active ingredient.

  The vascular endothelial growth factor (VEGF) production promoter according to the present invention is characterized by containing trametenolic acid as an active ingredient.

  The insulin-like growth factor-1 (IGF-1) production promoter according to the present invention is characterized by containing inotodiol as an active ingredient.

  The hepatocyte growth factor (HGF) production promoter according to the present invention is characterized by containing at least one of 3β-hydroxylanosto-8,24-dien-21-al and inotodiol as an active ingredient.

  The hair restorer according to the present invention comprises at least one of lanosterol, 3β-hydroxylanosto-8,24-diene-21-al, inotodiol, 3β, 21-dihydroxylanosto-8,24-diene and trametenolic acid. It is contained as an active ingredient.

  The dermal papilla cell proliferation promoter according to the present invention is characterized by containing at least one of ergosterol, stellaterol, inotolactone B, ergosterol peroxide, and inonotsan C as an active ingredient.

  The fibroblast growth factor-7 (FGF-7) production promoter according to the present invention includes ergosterol, stellaterol, inotolactone B, betulin, β-sitosterol, ergosterol peroxide, inonotsan C, 3β, 22R, 25-tri It contains at least one of hydroxylanosto-8,23E-diene and inonotutriol A as an active ingredient.

  The vascular endothelial growth factor (VEGF) production promoter according to the present invention is characterized by containing at least one of ergosterol, inotolactone B and betulin as an active ingredient.

  Insulin-like growth factor-1 (IGF-1) production promoters according to the present invention include ergosterol, stellaterol, inotolactone B, β-sitosterol, inonotsan C, and 3β, 22R, 25-trihydroxylanosto-8,23E. -It contains at least one kind of diene as an active ingredient.

  The hepatocyte growth factor (HGF) production promoter according to the present invention is characterized in that it contains at least one of ergosterol, stellaterol and inotolactone B as an active ingredient.

  The hair restorer according to the present invention includes ergosterol, stellaterol, inotolactone B, betulin, β-sitosterol, ergosterol peroxide, inonotsan C, 3β, 22R, 25-trihydroxylanosto-8,23E-diene and inonotriol. It contains at least one kind of A as an active ingredient.

  The hair restorer according to the present invention contains lanostane type triterpene as an active ingredient.

  The dermal papilla cell proliferation promoter of the present invention comprises at least lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol, 3β, 21-dihydroxylanosto-8,24-diene and trametenolic acid. Since it contains at least one of ergosterol, stellaterol, inotolactone B, ergosterol peroxide, and inonotsan C as an active ingredient, it is more effective in promoting dermal papilla cell proliferation than conventional dermal papilla cell proliferation promoters. Can be demonstrated.

  The fibroblast growth factor-7 (FGF-7) production promoter of the present invention includes lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol and 3β, 21-dihydroxylanosto-8, At least one of 24-dienes, or ergosterol, stellaterol, inotolactone B, betulin, β-sitosterol, ergosterol peroxide, inonotsan C, 3β, 22R, 25-trihydroxylanosto-8,23E-diene and inono Since at least one type of Tutriol A is contained as an active ingredient, it can exert an FGF-7 production promoting action superior to conventional FGF-7 production promoting agents.

  Since the vascular endothelial growth factor (VEGF) production promoter of the present invention contains trametenolic acid or at least one of ergosterol, inotolactone B and betulin as an active ingredient, it is superior to conventional VEGF production promoters. A VEGF production promoting action can be exhibited.

  Insulin-like growth factor-1 (IGF-1) production promoter of the present invention is inotodiol, or ergosterol, stellaterol, inotolactone B, β-sitosterol, inonotsan C, and 3β, 22R, 25-trihydroxylanosto- Since at least one of 8,23E-diene is contained as an active ingredient, it can exhibit an IGF-1 production promoting action superior to conventional IGF-1 production promoters.

  The hepatocyte growth factor (HGF) production promoter of the present invention is at least one of 3β-hydroxylanosto-8,24-dien-21-al and inotodiol, or at least one of ergosterol, stellaterol and inotolactone B. Since the seed is contained as an active ingredient, it can exhibit an HGF production promoting action superior to conventional HGF production promoters.

  The hair-restoring agent of the present invention has a hair papillary cell growth promoting action, a fibroblast growth factor-7 (FGF-7) production promoting action, a vascular endothelial growth factor (VEGF) production promoting action, insulin-like growth factor-1 (IGF- 1) Lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol, 3β, which has one or more of production promoting activity and hepatocyte growth factor (HGF) production promoting activity , 21-dihydroxylanosto-8,24-diene and trametenolic acid, ergosterol, stellaterol, inotolactone B, betulin, β-sitosterol, ergosterol peroxide, inotosan C, 3β, 22R, 25-tri Low levels of hydroxylanosto-8,23E-diene and innotutriol A Because it contains Kutomo one or a lanostane-type triterpene as an active ingredient, it can exhibit an excellent hair growth effect than conventional hair tonic.

It is a graph which shows the hair dermal papilla cell proliferation promotion rate of five types of lanostane type | mold triterpenoids and minoxidil. It is a graph which shows the hair papillary cell proliferation promotion rate of ergosterol, stellaterol, inotolactone B, betulin, β-sitosterol, lanosterol and minoxidil. It is a graph which shows the dermal papilla cell proliferation promotion rate of ergosterol peroxide, inonotsan C, lanosterol and minoxidil.

  Hereinafter, embodiments according to the present invention will be described in detail.

  The inventors have identified lanosterol (CAS registration number: 79-63-0), 3β-hydroxylanosto-8,24-dien-21-al (CAS registration number: 96574-03-7), inotodiol (CAS registration). Number: 35963-37-2), 3β, 21-dihydroxylanosto-8,24-diene (CAS registration number: 267649-99-0) and trametenolic acid (CAS registration number: 24160-36-9) Various types of lanostane-type triterpenoids promote the growth of dermal papilla cells involved in hair growth, promote the production of fibroblast growth factor-7 (FGF-7), promote the production of vascular endothelial growth factor (VEGF), insulin-like growth factor- 1 (IGF-1) production promoting action and hepatocyte growth factor (HGF) production promoting action were found to have any one or more actions. These five types of lanostane-type triterpenoids can be represented by the following general formula (LTT).

In the 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 types of lanostane type triterpenoids represented by the above general formula (LTT) can be obtained by extraction and isolation from a plant body, for example. Extraction from a plant can be performed by a general method. An example of the plant body is birch. By separating and purifying the extract obtained by solvent extraction from the fruit body of the birch tree using an appropriate separation and purification means such as column chromatography, ion exchange chromatography, high performance liquid chromatography, etc., the above general formula (LTT) is obtained. 5 types of lanostane-type triterpenoids represented by In this example, the plant is used in a broad sense including fungi, and the plant includes a fruit body of the fungus.

  The birch anatake extract contains all of the above-mentioned five types of lanostane terpenoids. Therefore, these five types of lanostane terpenoids can be efficiently obtained by extraction and isolation from birch moss.

  The above five compounds do not necessarily have to be extracted and isolated from a single plant, and can be obtained by any method. For example, each of the five types of lanostane-type triterpenoids, You may acquire by extracting and isolating from a separate plant body. The five types of lanostane type terpenoids may be chemically synthesized.

  In addition, the present inventors include ergosterol (CAS registration number: 57-87-4), stellaterol (CAS registration number: 2465-11-4), inotolactone B (CAS registration number: 1587735-79-2), Betulin (CAS registration number: 473-98-3), β-sitosterol (CAS registration number: 83-46-5), ergosterol peroxide (CAS registration number: 2061-64-5), Inonotsan C (CAS registration number: 1889275-09-5), 3β, 22R, 25-trihydroxylanosto-8,23E-diene (CAS registration number: 106483-69-6), Inonottriol A (CAS registration number: 374797-72-5) (Hereinafter, these nine types of compounds may be collectively referred to as nine types of compounds), the hair papillary cell growth promoting action involved in hair growth, and the fibroblast growth factor-7 (FGF-7) production promoting action. , Vascular endothelial growth factor (VEGF) production promoting action, insulin-like growth factor -1 it was found (IGF-1) have either 1 or 2 or more working of the production promoting effect and hepatocyte growth factor (HGF) production promoting effect. Of the nine compounds, inolactone B, inonotsan C, 3β, 22R, 25-trihydroxylanosto-8,23E-diene and inonottriol A are lanostane terpenoids.

  For each of the nine compounds, similarly to the five types of lanostane-type triterpenoids represented by the above general formula (LTT) (hereinafter, these five types of compounds may be collectively referred to as five types of compounds), for example, It can be obtained by extracting from the plant body, and can be extracted from the plant body by a general method. As for the nine types of compounds, the plant body includes birch. Nine compounds are obtained by separating and purifying the extract obtained by solvent extraction from the fruit body of the birch tree using suitable separation and purification means such as column chromatography, ion exchange chromatography, high performance liquid chromatography and the like. It is done. Since the extract of birch moth contains all nine compounds of compound, each compound of nine compounds can be efficiently obtained by extracting from birch moth. Since all compounds of 9 compounds can be obtained together with all compounds of 5 compounds from birch, it is very useful to use birch.

  Each of the nine compounds is not necessarily obtained from a single plant, and can be obtained by any method. For example, each of the nine compounds can be obtained from a separate plant. You may extract and acquire. Further, the nine compounds may be chemically synthesized.

  You may use combining the said compound of 5 types of compounds, and the compound of 9 types of compounds. In this case, the five compounds to be combined and the nine compounds of the compounds may be extracted from different plants, or one or both of them may be chemically synthesized.

  Hereinafter, the fibroblast growth factor-7 production promoter may be referred to as FGF-7 production promoter or fibroblast growth factor-7 (FGF-7). Also, the vascular endothelial growth factor production promoter is referred to as VEGF production promoter or vascular endothelial growth factor (VEGF) production promoter, and the insulin-like growth factor-1 production promoter is referred to as IGF-1 production promoter or insulin-like growth factor— 1 (IGF-1) production promoter, and hepatocyte growth factor production promoter may be referred to as HGF production promoter or hepatocyte growth factor (HGF) production promoter.

  Lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol, 3β, 21-dihydroxylanosto-8,24-diene and trametenolic acid, ergosterol, stellaterol, inotolactone B, betulin, β -By using at least one of sitosterol, ergosterol peroxide, inonotsan C, 3β, 22R, 25-trihydroxylanosto-8,23E-diene, inonottriol A, the present invention as described below Hair 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, liver Cell growth factor (HGF) production promoter and hair restorer can be obtained .

  Of the above 14 compounds, 5 types of lanostane type triterpenoids represented by the general formula (LTT), inotolactone B, inonotsan C, 3β, 22R, 25-trihydroxylanosto-8,23E-diene, inonotriol A is a lanostane type terpene, and the present inventors have found that a lanostane type terpenoid is particularly useful as a hair-restoring agent among lanostane type terpenoids. That is, lanostane-type terpene promotes the growth of dermal papilla cells involved in hair growth, promotes the production of fibroblast growth factor-7 (FGF-7), promotes the production of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 It has been found that it has one or more of (IGF-1) production promoting action and hepatocyte growth factor (HGF) production promoting action.

  In the following description, lanosterol is compound Z1, 3β-hydroxylanosto-8,24-diene-21-al is compound Z2, inotodiol is compound Z3, 3β, 21-dihydroxylanosto-8,24-diene. Compound Z4, trametenolic acid compound Z5, ergosterol compound Z6, stellaterol compound Z7, inotlactone B compound Z8, betulin compound Z9, β-sitosterol compound Z10, ergosterol peroxide compound Z11, innothansan C Compound Z12, 3β, 22R, 25-trihydroxylanosto-8,23E-diene may be referred to as compound Z13, and inonotriol A may be referred to as compound Z14, and the names may be appended.

<Propagation promoter of hair papilla cells>
The dermal papilla cell proliferation promoter according to the present invention includes lanosterol (compound Z1), 3β-hydroxylanosto-8,24-dien-21-ar (compound Z2), inotodiol (compound Z3), 3β, 21-dihydroxylano. Str-8,24-diene (Compound Z4) Trametenolic acid (Compound Z5), Ergosterol (Compound Z6), Stellaterol (Compound Z7), Inotolactone B (Compound Z8), Ergosterol peroxide (Compound Z11), Inotssan C It contains at least one of (Compound Z12) as an active ingredient, and further contains other components as necessary. As an active ingredient, one of the ten compounds described above can be used alone, or two or more can be used in combination. When using 2 or more types together, you may use the compound of 5 types compounds, the compound of 9 types compounds, and may use it combining 5 types of compounds and 9 types of compounds. In addition, about the combination of such a compound, it is the same also about another promoter and hair restorer.

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

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

  The dosage form is not particularly limited and can be appropriately selected depending on the administration method. Examples of dosage forms include oral solids (tablets, coated tablets, granules, powders, capsules, troches, etc.), oral solutions (internal solutions, syrups, elixirs, etc.), injections (solutions, suspensions) Liquids, solid preparations for dissolution, etc.), inhaled powders, ointments, liquids for external use, patches, coating agents, eye drops, nasal drops and ear drops.

  Oral solid preparations, for example, by adding additives such as excipients, disintegrants, lubricants, colorants, flavoring and flavoring agents to the above-mentioned active ingredients, if necessary, by a general method Can be manufactured. Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid.

  An additive is not specifically limited, According to the objective, it can select suitably. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone. .

  Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include purified talc, stearate, borax, and polyethylene glycol. Examples of the colorant include titanium oxide and iron oxide. Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid and tartaric acid.

  The oral liquid preparation can be produced by a general method, for example, by adding additives such as a corrigent / flavoring agent, a buffering agent and a stabilizer to the above-mentioned active ingredient. An additive is not specifically limited, According to the objective, it can select suitably. Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid and tartaric acid. Examples of the buffer include sodium citrate. Examples of the stabilizer include tragacanth, gum arabic, and gelatin.

  An injection can be produced by a general method, for example, by adding a pH adjuster, a buffer, a stabilizer, a tonicity agent, a local anesthetic, or the like to the above-mentioned active ingredient. The injection is a subcutaneous injection, an intramuscular injection, or an intravenous injection. Examples of the pH adjuster and buffer include sodium citrate, sodium acetate, and sodium phosphate.

  Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid and thiolactic acid. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

  Since the dermal papilla cell proliferation promoter according to the present invention contains an active ingredient having a dermal papilla cell proliferation promoting action, it can activate the dermal papilla through the proliferation promoting action of the dermal papilla cells. The dermal papilla cell proliferation promoter according to the present invention is, for example, a mechanism for promoting the proliferation of dermal papilla cells, the study of symptoms improved by promoting the proliferation of dermal papilla cells, the prevention of symptoms improved by promoting the proliferation of dermal papilla cells, Can be used for treatment.

  Specific examples of the dermal papilla cell proliferation promoter include, for example, a promoter for use as a reagent and a promoter for prevention / treatment of symptoms (including diseases and “unfavorable symptoms” such as hair loss). . Symptoms include, for example, thinning hair, hair loss, androgenetic alopecia.

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

<Fibroblast growth factor-7 (FGF-7) production promoter>
The fibroblast growth factor-7 (FGF-7) production promoter according to the present invention includes lanosterol (compound Z1), 3β-hydroxylanost-8,24-dien-21-al (compound Z2), inotodiol (compound Z3), 3β, 21-dihydroxylanosto-8,24-diene (compound Z4), ergosterol (compound Z6), stellaterol (compound Z7), inotolactone B (compound Z8), betulin (compound Z9), β- Sitosterol (compound Z10), ergosterol peroxide (compound Z11), inonotsan C (compound Z12), 3β, 22R, 25-trihydroxylanosto-8,23E-diene (compound Z13) and inonotriol A (compound Z14) Containing at least one of the above as an active ingredient, if necessary It contains other components, such as. As an active ingredient, one of the aforementioned 13 compounds can be used alone, or two or more can be used in combination.

  The content of the active ingredient in the FGF-7 production promoter can be appropriately selected according to the purpose, and is not particularly limited. The content of the active ingredient can be, for example, about 1 μg to 100 mg per unit dosage form. The FGF-7 production promoter according to the present invention may be the aforementioned active ingredient itself.

  The FGF-7 production promoter according to the present invention contains an active ingredient having an FGF-7 production promotion action. Therefore, the FGF-7 production promoter according to the present invention is, for example, a mechanism for promoting FGF-7 production, a study of symptoms improved by promoting FGF-7 production, or a symptom improved by promoting FGF-7 production. Can be used for prevention and treatment.

<Vascular endothelial growth factor (VEGF) production promoter>
The vascular endothelial growth factor (VEGF) production promoter according to the present invention comprises at least one of trametenolic acid (compound Z5), ergosterol (compound Z6), inotolactone B (compound Z8) and betulin (compound Z9) as an active ingredient. And other components as described above if necessary. As an active ingredient, one of the aforementioned four compounds can be used alone, or two or more can be used in combination. The content of the active ingredient in the VEGF production promoter 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 dosage form. The VEGF production promoter according to the present invention may be the aforementioned active ingredient itself.

  The VEGF production promoter according to the present invention contains an active ingredient having a VEGF production promoting action. Therefore, the VEGF production promoter according to the present invention can be used, for example, for the promotion mechanism of VEGF production, the study of symptoms improved by the promotion of VEGF production, and the prevention or treatment of symptoms improved by the promotion of VEGF production. it can.

<Insulin-like growth factor-1 (IGF-1) production promoter>
Insulin-like growth factor-1 (IGF-1) production promoters according to the present invention include inotodiol (compound Z3), ergosterol (compound Z6), stellaterol (compound Z7), inotolactone B (compound Z8), β-sitosterol. (Compound Z10), Inonotsan C (Compound Z12) and 3β, 22R, 25-trihydroxylanosto-8,23E-diene (Compound Z13) as an active ingredient, and as described above if necessary Contains other ingredients. As an active ingredient, one of the aforementioned seven compounds can be used alone, or two or more can be used in combination. The content of the active ingredient in the IGF-1 production promoter 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 dosage form. The above-mentioned active ingredient itself may be sufficient as the IGF-1 production promoter which concerns on this invention.

  The IGF-1 production promoter according to the present invention contains an active ingredient having an IGF-1 production promotion action. Therefore, the IGF-1 production promoter according to the present invention is, for example, a mechanism for promoting IGF-1 production, a study of symptoms improved by promoting IGF-1 production, or a condition improved by promoting IGF-1 production. Can be used for prevention and treatment.

<Hepatocyte growth factor (HGF) production promoter>
The hepatocyte growth factor (HGF) production promoter according to the present invention includes 3β-hydroxylanosto-8,24-dien-21-al (compound Z2), inotodiol (compound Z3), ergosterol (compound Z6), stellar. At least one of sterol (compound Z7) and inotolactone B (compound Z8) is contained as an active ingredient, and other ingredients as described above are contained as necessary. As an active ingredient, one of the aforementioned five compounds can be used alone, or two or more can be used in combination. The content of the active ingredient in the HGF production promoter 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 dosage form. The HGF production promoter according to the present invention may be the aforementioned active ingredient itself.

  The HGF production promoter according to the present invention contains an active ingredient having an HGF production promoting action. Therefore, the HGF production promoter according to the present invention can be used, for example, for the promotion mechanism of HGF production, the study of symptoms improved by promoting HGF production, and the prevention or treatment of symptoms improved by promoting HGF production. it can.

<Hair restorer>
The hair restorer according to the present invention includes lanosterol (compound Z1), 3β-hydroxylanosto-8,24-dien-21-al (compound Z2), inotodiol (compound Z3), 3β, 21-dihydroxylanosto-8, 24-Diene (Compound Z4), Trametenolic acid (Compound Z5), Ergosterol (Compound Z6), Stellaterol (Compound Z7), Inotolactone B (Compound Z8), Betulin (Compound Z9), β-Sitosterol (Compound Z10) , Ergosterol peroxide (compound Z11), inonotsan C (compound Z12), 3β, 22R, 25-trihydroxylanosto-8,23E-diene (compound Z13), and inonot triol A (compound Z14). Contains as an active ingredient. As an active ingredient, one of the aforementioned 14 compounds can be used alone, or two or more can be used in combination. For example, when using an extract of birch salmon containing 5 compounds Z1 to Z5, the content of the extract in the hair restorer can be about 0.1 to 5.0% by mass. Moreover, when using the extract of birch salmon containing 14 compounds Z1-Z14, content of the extract in a hair restorer can be about 0.1-10 mass%.

  The hair restorer according to the present invention contains lanostane type triterpene as an active ingredient. The compound which is a lanostane type triterpene as an active ingredient can contain 1 type individually or 2 types or more.

  For the hair restorer according to the present invention, water, lower alcohol (methanol, ethanol, denatured ethanol, isopropyl alcohol, etc.), solubilizer, surfactant, emulsion stabilizer, gelling agent, adhesive, etc. In order to obtain a desired dosage form, base components usually used can be blended.

  Depending on the intended use of the hair restorer according to the present invention, other components commonly used in hair restorers may be blended within a range that does not impair the effects of the present invention. Examples of other components include vasodilators such as minoxidil and carpronium chloride; androgen receptor inhibitors such as spironolactone, flutamide, cyproterone acetate, and oxendron; steroid-5α-reductase inhibitors such as finasteride and epilisteride Anti-inflammatory agents such as glycyrrhetinic acid and azulene; corticosteroids such as hydrocortisone acetate and dexamethasone acetate; keratolytic agents such as urea and salicylic acid; propylene glycol, 1,3-butylene glycol, macrogol, glycerin, dipropylene glycol, Examples include alcohols such as ethanol and isopropanol; fatty acid glycerin esters such as glyceryl monopentadecanoate, and antihistamines such as diphenhydramine hydrochloride.

  Furthermore, bactericides such as chlorhexidine gluconate, isopropyl petylphenol, quaternary ammonium salts, hinokitiol, piroctone olamine; moisturizers such as sodium hyaluronate, glycerin, chondroitin sulfate; yew, button birch, licorice, hypericum, appendix , Loquat, sagebrush, comfrey, ashitaba, saffron, sanshishi, rosemary, sage, mokko, seimokko, hops, placenta and other animal and plant extracts; retinol acetate, pyridoxine hydrochloride, ascorbic acid, thiamine nitrate, cyanocobalamin, biotin, acetic acid Vitamins such as tocophenol; oils such as isopropyl myristate, isopropyl palmitate, squalane, liquid paraffin, lecithin; polyoxyethylene sorbidan fatty acid ester Surfactants such as bidane fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene hydrogenated castor oil; antioxidants such as dibutylhydroxytoluene and isopropyl gallate; chelating agents such as ethylenediaminetetraacetate; refreshing agents such as menthol and camphor Is mentioned. Depending on the case, you may mix | blend a pigment | dye, a fragrance | flavor, a transdermal absorption promoter, etc.

  The hair restorer according to the present invention is preferably an external preparation and can be used as any dosage form that is usually used as an external preparation. Examples of the dosage form include, but are not limited to, lotions, emulsions, creams, tonics, ointments, gels and aerosols. The hair restorer according to the present invention can be used, for example, by applying an appropriate amount to the scalp 1 to several times a day.

  The hair restorer according to the present invention can be used for, for example, a hair growth mechanism, research on symptoms improved by hair growth, and prevention or treatment of symptoms improved by promotion of hair growth. Specific embodiments of the hair restorer according to the present invention include, for example, a hair restorer for reagents and a hair restorer for prevention / treatment of symptoms. Examples of symptoms include male pattern baldness.

  The hair restorer according to the present invention includes lanosterol (compound Z1), 3β-hydroxylanosto-8,24-dien-21-al (compound Z2), inotodiol (compound Z3), 3β, 21-dihydroxylanosto-8, 24-Diene (Compound Z4), Trametenolic acid (Compound Z5), Ergosterol (Compound Z6), Stellaterol (Compound Z7), Inotolactone B (Compound Z8), Betulin (Compound Z9), β-Sitosterol (Compound Z10) , Ergosterol peroxide (compound Z11), inonotsan C (compound Z12), 3β, 22R, 25-trihydroxylanosto-8,23E-diene (compound Z13), and inonot triol A (compound Z14). Since it is contained as an active ingredient, it promotes hair papillary cell proliferation , FGF-7 production promoting action, VEGF production promoting action, and includes any one or more of the effects of IGF-1 production-promoting effect and HGF production promoting action.

  Inotodiol (compound Z3) and 3β, 21-dihydroxylanosto-8,24-diene (compound Z4) have a testosterone 5α-reductase activity inhibitory action in addition to the actions described above. Therefore, for example, by including all five types of lanostane-type triterpenoid compounds Z1 to Z5, hair papillary cell proliferation promoting action, FGF-7 production promoting action, VEGF production promoting action, IGF-1 production promoting action and HGF production It is possible to obtain a hair restorer that is excellent in promoting action and has testosterone 5α-reductase activity inhibiting action. Of course, depending on the combination of one or both of inotodiol (compound Z3) and 3β, 21-dihydroxylanosto-8,24-diene (compound Z4) with one or more compounds of the five and nine compounds In addition, it is possible to obtain a hair restorer having an inhibitory action on testosterone 5α-reductase activity.

  Examples will be described below, but the present invention is not limited to these examples.

Example 1
Example 1 is an example in which an extract was extracted from birch salmon.
1) 80% ethanol was added to 5.0 kg (product of Russia; lot number No. 131008, Chihaya Co., Ltd.) of the fruit body of the birch fruit extract and extracted at room temperature.
2) The extract obtained in 1) was dissolved and suction filtered with 50% ethanol to obtain an insoluble part (56.1 g) and a soluble part (183.0 g). That is, the processing solution obtained by dissolving the extract with 50% ethanol was subjected to suction filtration to separate the soluble portion and the insoluble portion. The treatment liquid obtained by the dissolution treatment was a suspension in a suspended state.
80% ethanol and 50% ethanol are both ethanol aqueous solutions, and the volume of ethanol with respect to the total volume of the ethanol aqueous solution is 80% and 50%. Hereinafter, the same is used.

  The insoluble part contains a low polarity compound, that is, a low polarity compound. On the other hand, a highly polar compound, that is, a highly polar compound, for example, inositol, is contained in the soluble part because its solubility in water at 25 ° C. is 14 g / 100 mL.

(Example 2)
Example 2 was prepared by extracting lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol, 3β, 21-dihydroxylanosto-8,24 from the extract from birch salmon obtained in Example 1. -Examples of diene and trametenolic acid (compounds Z1 to Z5) isolated.

  From 1 g of the 50% ethanol-insoluble part obtained, 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) Was isolated. Specifically, gradient elution using a mixed solvent of hexane: ethyl acetate (12: 1 → 10: 1 → 9: 1 → 8: 1 → 4: 1 → 2: 1) in silica gel column chromatography. (Fractionation 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 actually measured values were compared with literature values. As a result, as described below, it was confirmed that all of the compounds A, B, C, D and E are lanostane type triterpenoids represented by the above general formula (LTT).

  In this example, the soluble part is a dissolved part composed of a substance (compound) actually dissolved in an ethanol aqueous solution, and the insoluble part or 50% ethanol insoluble part is not actually dissolved (compound). Means a non-dissolved part consisting of In this example, the compounds Z1 to Z5 were extracted from the insoluble part, but extraction from the soluble part is not denied. The same applies to the extraction of compounds Z6 to Z14 described below.

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

The above measured values were compared with the values of the following documents 1) to 4) to identify compounds A to D.
1) Toshihiro Akiku, Taro Matsumoto, ¹³ C-NMR spectrum of sterol and triterpene alcohol, 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 Acceleratory 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 actual measurement value of Compound A was compared with the values described in References 1) to 3), and the actual measurement values of Compounds B and D were compared with the values described in Reference 4). The measured values of compounds C and E were compared with the values described in documents 3) and 4).

Compound A was identified as a compound having R ¹ = CH ₃ and R ² = H in the general formula (LTT), that is, lanosterol (compound Z1) represented by the following chemical formula (S1).

Compound B is a compound in which R ¹ = CHO and R ² = H in the above general formula (LTT), that is, 3β-hydroxylanosto-8,24-dien-21-al ( Compound Z2) was identified.

Compound C was identified as a compound having R ¹ = CH ₃ and R ² = OH in the above general formula (LTT), that is, inotodiol (compound Z3) represented by the following chemical formula (S3).

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

Compound E was identified as a compound having R ¹ = COOH and R ² = H in the general formula (LTT), that is, trametenolic acid (compound Z5) represented by the following chemical formula (S5).

Example 3
Example 3 is an example in which the dermal papilla cell proliferation promoting action test was conducted on the five types of lanostane type triterpenoids (compounds Z1 to Z5) isolated in Example 2.

  First, normal human hair papilla cells (trade name “Human Hair Dermal Papilla Cells (HFDPC) (Code No. CA60205a)” manufactured by Toyobo Co., Ltd.) were cultured. The medium used was added to 250 mL of a basal medium of a dermal papilla cell growth medium (trade name “Papilla Cell Growth Medium” (PCGM) (Code.TMTPGM-250) manufactured by Toyobo Co., Ltd.), which is a low serum medium. Fetal bovine serum (FCS) 2.5 mL, insulin transferrin triiodothyronine mixed solution (ITT) 1.25 mL, bovine pituitary extract (BPE) 2.5 mL and cyproterone acetate (Cyp) It is a dermal papilla cell growth medium supplemented with 25 mL.

Cells cultured at 37 ° C. and 5% CO ₂ were trypsinized with a trypsin / EDTA solution (0.05% concentration). The treated cells were treated with 1.0 × 10 ⁴ cells / mL using Dulbecco's modified Eagle medium DMEM (manufactured by Nacalai Tesque, Inc., hereinafter abbreviated as “DMEM”) containing 10% fetal bovine serum (FBS). Diluted to a concentration of The diluted cells were seeded at 200 μL / well in a 96-well microplate Falcon (registered trademark) (trade name “MICROTEST ^™ 96” manufactured by Becton Dickinson Laboratories). After culturing for 3 days, the medium was removed by suction.

  Compounds Z1 to Z5 as test samples were dissolved in serum-free DMEM at a predetermined concentration. The concentrations were 0.01953 μmol / L, 0.078125 μmol / L, 0.3125 μmol / L, 1.25 μmol / L, 5 μmol / L, and 20 μmol / L. Each lysed test sample was added in an amount of 200 μL / well to each well of a 96-well microplate seeded with the above cells. After further culturing for 4 days, the medium was removed by suction. Hair papillary cell proliferation was measured using MTT assay. That is, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) was dissolved in serum-free DMEM at a final concentration of 0.4 mg / mL and added at 100 μL / well. did.

  After 2 hours of culture, the formation of blue formazan was confirmed in the cells. This blue formazan was extracted with 50 μL of 2-propanol. After the extraction, the absorbance at a wavelength of 570 nm was measured to obtain a first absorbance. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity to obtain a second absorbance. The difference between the first absorbance and the second absorbance was used as the amount of blue formazan produced. The dermal papilla cell proliferation promotion rate (%) was calculated by the following formula (1).

Hair papilla cell proliferation promotion rate (%) = A1 / B1 × 100
... Formula (1)
A1: Amount of blue formazan produced when test sample is added
B1: Blue formazan production when no test sample is added

  The dermal papilla cell proliferation promotion rate calculated for each of the compounds Z1 to Z5 is summarized in Table 6 below. The dermal papilla cell growth promotion rate was determined for each concentration of the compounds Z1 to Z5 at 6 points. The average value and standard error of 6 points are shown in Table 6 as (average value ± standard error). As a reference, minoxidil known to have a hair papillary cell proliferation promoting action was used.

  As shown in Table 6 above, 3β, 21-dihydroxylanosto-8,24-diene (compound Z4) has a low concentration of 0.3125 μmol / L, and dermal papilla cell growth comparable to 80 μmol / L minoxidil. It was observed to show a promoting effect. In addition, 3β, 21-dihydroxylanosto-8,24-diene (compound Z4) has a hair papillary cell growth promoting action exceeding 80 μmol / L minoxidil at a concentration of 1.25 μmol / L.

  Trametenolic acid (compound Z5) also has a hair papillary cell growth promoting effect similar to 80 μmol / L minoxidil at a low concentration of 0.3125 μmol / L. 0.3125 μmol / L inotodiol (compound Z3) and 20 μmol / L lanosterol (compound Z1) were also confirmed to have a hair papillary cell growth promoting effect close to 80 μmol / L minoxidil.

  Table 7 and FIG. 1 show the results of the test for promoting the growth of dermal papilla cells at low concentrations of five types of lanostane-type triterpenoids (compounds Z1 to Z5). In Table 7 and FIG. 1, the results for minoxidil are also shown for comparison.

  3β-Hydroxylanosto-8,24-dien-21-al (compound Z2) also promotes dermal papilla cell proliferation over 80 μmol / L minoxidil at low concentrations of 0.01953 μmol / L and 0.078125 μmol / L The effect is obtained.

  From the results of Example 3, lanosterol, 3β-hydroxylanosto-8,24-diene-21-al, inotodiol, 3β, 21-dihydroxylanosto-8,24-diene and trametenolic acid, (compounds Z1 to Z5) ) Was confirmed to be an active ingredient of a hair papilla cell proliferation promoter. These compounds Z1 to Z5 have an action to promote hair papillary cell proliferation that exceeds minoxidil at a lower concentration than minoxidil. Therefore, lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol, 3β, 21-dihydroxylanosto-8,24-diene and trametenolic acid (compounds Z1 to Z5) are more hairy than minoxidil. It can be seen that the papillary cell proliferation promoting action is excellent.

Example 4
Example 4 is an example in which a hair papillary cell proliferation promoting action test was performed on a mixture of five types of lanostane type triterpenoids (compounds Z1 to Z5) isolated in Example 2.

  The concentration (selective concentration) of each compound in the mixture is lanosterol (20 μmol / L), 3β-hydroxylanosto-8,24-dien-21-al (0.3125 μmol / L), inotodiol (0.3125 μmol / L). , 3β, 21-dihydroxylanosto-8,24-diene (5 μmol / L) and trametenolic acid (1.25 μmol / L).

  About this mixture, the hair papillary cell proliferation promotion effect test was done by the method similar to the above-mentioned. The calculated dermal papilla cell proliferation promotion rate is summarized in Table 8 below together with the results for various concentrations of minoxidil.

  As shown in Table 8 above, it was recognized that a mixture containing the compounds Z1 to Z5, which are five kinds of lanostane type terpenoids, at a predetermined concentration exhibits an action of promoting the proliferation of dermal papilla cells exceeding 20 μmol / L minoxidil. .

(Example 5)
Example 5 is an example in which the production promotion test for various growth factor genes involved in the hair cycle was performed on the five types of lanostane type triterpenoids (compounds Z1 to Z5) isolated in Example 2. The production promotion test was performed by an mRNA expression promoting action test for evaluating the expression of mRNA.

  Human normal hair papilla cells (HFDPC: derived from the top of the head) were seeded in a 60 mm petri dish and cultured using a medium for human normal hair papilla cells (PCGM). After the cells became confluent, the medium was replaced with DMEM medium containing 10% FBS and cultured for 2 hours. Compounds Z1 to Z5 as test samples were dissolved in serum-free DMEM medium at a predetermined concentration. The concentration was 1.25 μmol / L, 5 μmol / L, and 20 μmol / L.

  3 mL of each dissolved test sample was added to each petri dish after culture. After further incubation for 6 hours, total RNA was prepared by a general method. Total RNA was similarly prepared for cells cultured without addition of the test sample. The amount of each RNA was measured with a spectrophotometer, and total RNA was prepared so that it might become 200 ng / microliter.

  Using this total RNA as a template, the expression levels of various growth factors involved in the hair cycle and GAPDH mRNA as an internal standard were measured. For detection, real-time PCR device Smart Cycler (registered trademark) (Cepheid) was used, and real-time 2Step RT-PCR using TaKaRa SYBR (registered trademark) Prime Script ™ RT-PCR Kit (Perfect Real Time) (code No. RR063A). Performed by reaction.

The mRNA expression levels of various growth factors were calculated by correcting the expression levels of GAPDH mRNA. The expression rate of various growth factor mRNAs was calculated by the following formula (2).
Various growth factor mRNA expression rates (%) = A2 / B2 × 100
... Formula (2)
A2: Correction value when test sample is added B2: Correction value when test sample is not added (control)

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

  The various growth factor mRNA expression rates for each of the 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 with respect to 100% for one point. Furthermore, Table 14 below summarizes the mRNA expression rates of adenosine that is conventionally known to have a vascular endothelial growth factor (VEGF) production promoting action.

  As shown in the above table, lanosterol (compound Z1), 3β-hydroxylanosto-8,24-dien-21-al (compound Z2), inotodiol (compound Z3) and 3β, 21-dihydroxylanosto-8, 24-Diene (compound Z4) has been observed to promote the production of FGF-7 mRNA by about 1.2 to 1.6 times that of the test sample without addition.

  From the results of Example 5, each of lanosterol, 3β-hydroxylanosto-8,24-diene-21-al, inotodiol and 3β, 21-dihydroxylanosto-8,24-diene (compounds Z1 to Z4) It was confirmed that it can be an active ingredient of an FGF-7 production promoter. All of these compounds Z1 to Z4 exhibit a production promoting effect equivalent to or better than that of 25 μmol / L adenosine at a low concentration of 5 μmol / L. Therefore, they have an excellent FGF-7 production promoting effect than conventionally known adenosine. You can see that

  As shown in Table 13 above, 20 μmol / L of trametenolic acid (Compound Z5) has been observed to promote VEGF mRNA expression by about 1.5 times that of the test sample without addition. The results of Example 5 show that trametenolic acid (Compound Z5) can be an active ingredient of a VEGF production promoter. In addition, trametenolic acid (compound Z5) exhibits a VEGF mRNA expression promoting effect that exceeds 20 μmol / L adenosine even at 20 μmol / L, and is superior to the conventionally known adenosine in promoting VEGF production.

  Furthermore, inotodiol (compound Z3) was confirmed to have an IGF-1 mRNA expression promoting effect about 1.2 times that of the test sample without addition. 3β-hydroxylanosto-8,24-dien-21-al (compound Z2) and inotodiol (compound Z3) were confirmed to promote HGF mRNA expression. Inotodiol (Compound Z3) can be used as an active ingredient of an IGF-1 production promoter. Each of 3β-hydroxylanosto-8,24-dien-21-al (compound Z2) and inotodiol (compound Z3) can be used as an active ingredient of an HGF production promoter.

(Example 6)
Example 6 is an example in which the testosterone 5α-reductase activity inhibitory action test was performed on the five types of lanostane type triterpenoids (compounds Z1 to Z5) isolated in Example 2.

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

  80 μL of the test sample solution and 75 μL of S-9 rat liver homogenate were added to the mixed solution in the aforementioned V-bottom tube with lid, and mixed again. After the contents in the test tube were reacted at 37 ° C. for 60 minutes, 1 mL of methylene chloride was added to stop the reaction. The mixture after the reaction was centrifuged (1600 × g, 10 minutes), and the methylene chloride phase was subjected to gas chromatography analysis under the following conditions. Furthermore, a blank test was performed in the same manner.

  In order to determine the conversion rate of testosterone to 3α-androstanediol and dihydrotestosterone, a standard ethanol solution of 3α-androstanediol, dihydrotestosterone and testosterone was analyzed by gas chromatography in advance, and the peak areas of these three compounds were analyzed. Asked.

  With respect to the peak areas of 3α-androstanediol, dihydrotestosterone and testosterone after the reaction with S-9, the relative ratio to the peak area of the standard product was determined according to the following formula (3). Thereafter, the conversion rate of the test sample was calculated according to the equation (4).

Relative ratio = peak area of test sample / peak area of standard product (3)
Conversion rate (%) = (A3 + B3) / (A3 + B3 + C3) × 100
... Formula (4)
A3: Relative ratio of 3α-androstanediol B3: Relative ratio of dihydrotestosterone C3: Relative ratio of testosterone

Based on the calculated conversion rate, the testosterone 5α-reductase activity inhibition rate was determined according to the following formula (5).
Testosterone 5α-reductase activity inhibition rate (%)
= (1-E / D) × 100 (5)
D: Conversion rate in blank test E: Conversion rate in addition of test sample

The conditions for gas chromatography are as follows.
Equipment used: Shimadzu GC-2010
Column: DB-1701
(Φ0.53 mm × 30 m. Film thickness: 1.0 μm)
Column / injection temperature: 240 ° C./300° C.
Detector: FID
Carrier gas: Nitrogen gas

Testosterone 5α-reductase activity inhibition rate and 50% inhibitory concentration (IC ₅₀ ) for compounds Z1 to 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 inotodiol (compound Z3) and 3β, 21-dihydroxylanosto-8,24-diene (compound Z4) have testosterone 5α-reductase activity inhibitory action.

(Example 7)
Example 7 was prepared by extracting ergosterol (compound Z6), stellaterol (compound Z7), inotolactone B (compound Z8), betulin (compound Z9), β-sitosterol (extracted from the birch salmon obtained in Example 1). Compound Z10), ergosterol peroxide (compound Z11), inonotsan C (compound Z12), 3β, 22R, 25-trihydroxylanosto-8,23E-diene (compound Z13), inonotriol A (compound Z14), respectively It is the extracted Example.

  From the 50% ethanol-insoluble part obtained in Example 1, Compound F, Compound G, Compound H1, Compound H2, Compound I, Compound J, Compound K, Compound L, Compound M, Compound N were prepared according to the procedure described below. Got.

  The eluting solvent was removed from each of the fifth to seventh fractions FA5 to FA7 of the 18 fractions FA1 to FA18 obtained in the fractionation operation 1 of Example 2 to obtain fractions. The mixture of fractions obtained from fractions FA5 to FA7 was fractionated into nine fractions FB1 to FB9 by silica gel column chromatography (eluent: “hexane: ethyl acetate mixed solvent (12: 1)”). (Fractionation operation 2).

  The eluted solvent was removed from each of the sixth and seventh fractions FB6 and FB7 among the fractions FB1 to FB9 obtained in the fractionation operation 2 to obtain fractions. The mixture of the fractions obtained from the fractions FB6 and FB7 was subjected to 10 chromatography on silica gel column chromatography (elution solvent: toluene → “toluene: acetone mixed solvent (30: 1 → 15: 1)” → ethyl acetate). Fractions were fractionated into FC1 to FC10 (fractionation operation 3).

  The arrow in the description of the elution solvent indicates that the composition of the elution solvent or the ratio of the mixed solvent used for it is changed, and means that gradient elution is performed. The same applies to an eluent used for high performance liquid chromatography, which will be described later.

  From the fractions FC1 to FC10 obtained in the fractionation operation 3, the third fraction FC3 from which the elution solvent had been removed was obtained as compound F (12.0 mg). Further, the product obtained by removing the elution solvent from the fourth fraction FC4 was obtained as Compound G (4.3 mg).

  Further, 39.4 g of the 50% ethanol-insoluble part obtained in Example 1 was subjected to silica gel column chromatography (elution solvent: “hexane: ethyl acetate mixed solvent (12: 1 → 11: 1 → 10: 1 → 9: 1 → 8: 1 → 6: 1 → 4: 1 → 2: 1) ”) and fractionated into 8 fractions FD1 to FD8 (fractionation operation 4).

  The fraction obtained by removing the eluting solvent from the fraction FD4 of the fraction FD1 to FD8 was subjected to silica gel column chromatography (elution solvent: “hexane: ethyl acetate mixed solvent (15: 1 → 13: 1 → 10: 1 → 8: 1 → 6: 1 → 4: 1) ”) and fractionated into 11 fractions FE1 to FE11 (fractionation operation 5).

  The eluted solvent was removed from each of the eighth and ninth fractions FE8 and FE9 among the fractions FE1 to FE11 to obtain fractions. A mixture of fractions obtained from fractions FE8 and FE9 was subjected to silica gel column chromatography (eluent: “toluene: acetone mixed solvent (20: 1 → 19: 1 → 18: 1)”) to obtain 6 fractions FF1. -Fractionated to FF6 (fractionation operation 6).

  The elution solvent is respectively removed from the sixth and seventh fractions FE6 and FE7 of the fractions FE1 to FE11 by the fractionation operation 5 and the first fraction FF1 of the fractions FF1 to FF6 by the fractionation operation 6. Each fraction was obtained. A mixture of each fraction obtained from fractions FE6 and FE7 and fraction FF1 was subjected to silica gel column chromatography (eluent: “hexane: ethyl acetate mixed solvent (12: 1 → 10: 1 → 9: 1 → 8: 1 → 7: 1) ”) and fractionated into 14 fractions FG1 to FG14 (fractionation operation 7).

  Of the fractions FG1 to FG14 obtained in the fractionation operation 7, a product obtained by removing the elution solvent from the eighth fraction FG8 was obtained as Compound J (113.9 mg).

  A fraction obtained by removing the elution solvent from the sixth fraction FD6 of fractions FD1 to FD8 by fractionation operation 4 was subjected to silica gel column chromatography (elution solvent: “mixed solvent of toluene: acetone (30: 1 → 25: 1 → 20: 1 → 15: 1) ”) and fractionated into 10 fractions FH1 to FH10 (fractionation operation 8).

  A fraction obtained by removing the elution solvent from the fifth fraction FH5 of the fractions FH1 to FH10 obtained in the fractionation operation 8 was obtained as compound H1 (209 mg). Moreover, what removed the elution solvent from 6th FH6 was acquired as compound H2 (120.8 mg).

  A fraction obtained by removing the eluting solvent from each of the seventh and eighth fractions FH7 and FH8 among the fractions FH1 to FH10 was obtained. A mixture of the fractions obtained from fractions FH7 and FH8 was subjected to high performance liquid chromatography (column: N-2 type, eluent: “hexane: ethyl acetate mixed solvent (8: 2)”, flow rate 7.0 mL / The fraction was fractionated into 8 fractions FI1 to FI8 (fractionation operation 9).

  Among the fractions FI1 to FI8 obtained in the fractionation operation 9, a solution obtained by removing the elution solvent from the fourth fraction FI4 was obtained as Compound I (10.2 mg).

  Further, a fraction obtained by removing the elution solvent from the ninth fraction FH9 among the fractions FH1 to FH10 obtained by the fractionation operation 8 was obtained, and this fraction was obtained by silica gel column chromatography (elution solvent: “hexane: Fractionation into 12 fractions FJ1 to FJ12 with a mixed solvent of ethyl acetate (6: 1 → 5: 1 → 4: 1 → 3: 1) ”(fractionation operation 10).

  Of the fractions FJ1 to FJ12 obtained in the fractionation operation 10, a product obtained by removing the elution solvent from the ninth fraction FJ9 was obtained as Compound K (9.4 mg).

  The fraction obtained by removing the elution solvent from the eighth fraction FD8 of fractions FD1 to FD8 by fractionation operation 4 was subjected to silica gel column chromatography (elution solvent: “hexane: ethyl acetate mixed solvent (6: 1 → 4 : 1 → 2: 1 → 1: 1) ”) and fractionated into 13 fractions FK1 to FK13 (fractionation operation 11).

  A fraction obtained by removing the eluting solvent from the 10th fraction FK10 of the fractions FK1 to FK13 obtained by the fractionation operation 11 was obtained. The fraction obtained from this fraction FK10 was subjected to high performance liquid chromatography (column: N-6 type, eluent: “hexane: ethyl acetate mixed solvent (6: 1)”, flow rate: 3.0 mL / min). Fractions were fractionated into 18 fractions FL1 to FL18 (fractionation operation 12).

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

  Of the fractions FL1 to FL12 obtained in fractionation operation 13, the product obtained by removing the eluting solvent from the second fraction FL2 is designated as compound M (7.7 mg), and the product obtained by removing the eluting solvent from the twelfth fraction FL12 is designated as compound. Obtained as N (47.3 mg), respectively.

The molecular structures of the compounds F, G, H1, H2, and I to N obtained as described 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. Compound L (47.3 mg) was confirmed to have been extracted from the same compound as the compound obtained from the 10th fraction FA10 of fractions FA1 to FA18 obtained in fractionation operation 1. Further, it was confirmed that Compound I was extracted from the compound obtained from the 12th fraction FA12, and Compound N was extracted from the same compound as the compound obtained from the 16th fraction FA16.

The measured values of ¹³ C-NMR and ¹ H-NMR of compounds F, G, H1, H2 and I to N were compared with literature values. As a result, as described below, compounds F, G, H1, H2, and I to N are ergosterol (compound Z6), stellaterol (compound Z7), inotolactone B (compound Z8), betulin (compound Z9). , Β-sitosterol (compound Z10), ergosterol peroxide (compound Z11), inonotsan C (compound Z12), lanosta-8,23 E-diene-3β-, 22R, 25-triol (compound Z13), innotutriol A It was confirmed that (Compound Z14) was extracted.

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

The compound was identified by comparing the measured values with the literature values described in the above literature 1) and the following literatures 5) to 14).
5) Seo, Hyo Won; Arch. Pharm. Res., 2009, 32 (11), 1573-1579
6) Jeffrey LC Wright, Can. J. Chem., 1979, 57, 2569-2571
7) Alejandro F. Barrero, et.al., ACSand ASP, 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. VK, and Nes, WR, Phytochemistry, 1984, 23, 2925-2929
12) T. Akihisa, S. Thakur, FU 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 values of compounds F and G are compared with the values described in documents 1), 5) to 7), and the measured values of compound H are compared with the values described in document 8). The values were compared with the values described in documents 9) and 10). In addition, the actual measurement value of Compound J is compared with the values described in References 1), 11), and 12), the actual measurement value of Compound K is compared with the value described in Reference 5), and the actual measurement value of Compound L The value was compared with the value described in Reference 13). Furthermore, the actual measurement value of Compound M was compared with the value described in Reference 14), and the actual measurement value of Compound N was compared with the value described in Reference 15).

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

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

  Compounds H1 and H2 could be judged as inotolactone B (compound Z8) represented by the following chemical formula (S8).

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

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

  Compound K could be judged to be ergosterol peroxide (compound Z11) represented by the following chemical formula (S11).

  Compound L could be determined to be Inonotsan C (Compound Z12) represented by the following chemical formula (S12).

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

  Compound N could be determined as Inonot Triol A (Compound Z14) represented by the following chemical formula (S14).

(Example 8)
Example 8 is an example in which hair dermal papilla cell proliferation promoting action test was performed on the compounds Z6 to Z12 obtained in Example 7.

  In the same manner as in Example 3 described above, a test for promoting dermal papilla cell proliferation was performed, and the dermal papilla cell proliferation promotion rate was determined according to ergosterol (compound Z6), stellaterol (compound Z7), inotolactone B obtained in Example 7. (Compound Z8), betulin (Compound Z9), β-sitosterol (Compound Z10), ergosterol peroxide (Compound Z11) and Inonotsan C (Compound Z12) were determined. The concentrations of the compounds Z6 to Z12 as test samples were 0.3125 μmol / L, 1.25 μmol / L, 5 μmol / L, and 20 μmol / L. The test of compounds Z6 to Z10 and the test of compounds Z1 and Z12 were performed separately. In each test, compound Z1 and minoxidil were used as a reference.

  The dermal papilla cell proliferation promotion rate (%) was calculated by the above-mentioned formula (1). The growth rate of dermal papilla cells for compounds Z6 to Z10 is shown in Table 25 and the graph of FIG. 2, and the growth rate of dermal papilla cells for compounds Z11 and Z12 is shown in the graph of Table 26 and FIG. Tables 25 and 26 also show the results for each concentration of lanosterol (compound Z1) and minoxidil. The hair follicle cell growth promotion rate for each concentration of compounds Z6 to Z12, Z1 and minoxidil shown in Tables 25 and 26 is an average value of three points ± standard error.

  As shown in Table 25, ergosterol (compound Z6), stellaterol (compound Z7), and inotolactone B (compound Z8) at a concentration of 1.25 μmol / L are lanosterol (compound Z1) at the same or higher concentration. ) And a stronger dermal papilla cell growth promoting effect than minoxidil. In addition, as shown in Table 26, ergosterol peroxide (compound Z11) and inonotsan C (compound Z12) having a concentration of 0.315 μmol / L are more than lanosterol (compound Z1) having the same concentration and minoxidil having the same concentration or more. It was recognized that the hair papillae cell proliferation promoting action was strong. These suggest that the lanostane skeleton is important for activity.

  From the results of Example 8, ergosterol (compound Z6), stellaterol (compound Z7), inotolactone B (compound Z8), ergosterol peroxide (compound Z11), and inonotsusan C (compound Z12) are hair papillary cell proliferation promoters. It was confirmed that it can be an active ingredient. These compounds Z6 to Z8, Z11, and Z12 show hair papillary cell proliferation promoting action exceeding minoxidil at a lower concentration than minoxidil. Therefore, it can be seen that the compounds Z6 to Z8, Z11, and Z12 are more effective in promoting dermal papilla cell proliferation than minoxidil.

Example 9
Example 9 is an example in which the production promotion test for various growth factor genes involved in the hair cycle was performed on the compounds Z6 to Z14 obtained in Example 2. The production promotion test was performed by an mRNA expression promoting action test for evaluating the expression of mRNA.

  Ergosterol (Compound Z6), Stellaterol (Compound Z7), Inotolactone B (Compound Z8), Betulin (Compound Z9), β-Sitosterol (Compound Z10), Ergosterol Peroxide (Compound Z11), Inonotsan C (Compound Z12), 3β, 22R, 25-trihydroxylanosto-8,23E-diene (compound Z13), innotutriol A (compound Z14) was used as a test sample, and the mRNA expression promoting action test was performed in the same manner as described above and in Example 5. Went. The concentration of each test sample was 1.25 μmol / L, 5 μmol / L, and 20 μmol / L.

  According to the above test, the growth factors include fibroblast growth factor-7 (FGF-7), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF). Each mRNA expression level was determined. 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 by the above-described equation (2). Tables 27 to 35 summarize the expression rates of various growth factors mRNA for each of the compounds Z6 to Z14. The mRNA expression rate (unit:%) shown in Tables 27 to 35 is a relative value with respect to 100% for one point. Table 36 shows the mRNA expression rate (%) of fibroblast growth factor-7 (FGF-7) and vascular endothelial growth factor (VEGF) for adenosine at a concentration of 25 mol / L.

  As shown in Tables 27 to 35, the compounds Z6 to Z14 have a FGF-7 mRNA production promoting effect of about 1.15 to 1.6 times that in the case where the test sample is not added. From the results of Example 9, it was confirmed that the compounds Z6 to Z14 can be active ingredients of the FGF-7 production promoter. In particular, 20 μmol / L of compound Z6, compound Z8, and compound Z10 were found to have an FGF-7 mRNA production-promoting effect of about 1.01-1.08 times that of 25 μmol / L of adenosine. It can be seen that the FGF-7 production promoting action is excellent.

  As shown in Table 27, Table 29, and Table 30, Compound Z6, Compound Z8, and Compound Z9 are about 1.1 to 1.2 times the addition of the test sample at 1.25 μmol / L, and 20 μmol. In / L, a VEGF mRNA expression promoting effect of about 1.2 to 2.1 times that of no test sample added is observed. From the results of Example 9, it was confirmed that Compound Z6, Compound Z8, and Compound Z9 can be active ingredients of VEGF production promoters. In addition, compound Z6, compound Z8 and compound Z9 show VEGF mRNA expression promoting action comparable to or exceeding 25 μmol / L adenosine even at 20 μmol / L or less, and VEGF production promoting action from conventionally known adenosine. It is understood that is superior.

  As shown in Table 27 to Table 29, Table 31, Table 33, and Table 34, the compounds Z6 to Z8, Z10, Z12, and Z13 are about 1.1 to 1.55 times as much IGF-1 as the test sample is not added. The effect of promoting mRNA expression was confirmed. Thereby, it turns out that compound Z6-Z8, Z10, Z12, Z13 can be used as an active ingredient of an IGF-1 production promoter.

  As shown in Tables 27 to 29, the compounds Z6 to Z8 were confirmed to have an HGF mRNA expression promoting effect of about 1.3 to 1.4 times that of the test sample not added. Thereby, it turns out that it can be used as an active ingredient of an HGF production promoter.

  From the results of the above Examples, the compounds Z1 to Z14 are the dermal papilla cell growth promoter, fibroblast growth factor-7 (FGF-7) production promoter, vascular endothelial growth factor (VEGF) production promoter, insulin, It can be seen that it is a novel active ingredient as a growth-like growth factor-1 (IGF-1) production promoter and a hepatocyte growth factor (HGF) production promoter. In addition, it is speculated that the compounds Z1 to Z14 can stimulate hair growth through different mechanisms for mRNA expression of FGF-7, VEGF, IGF-1 and HGF. This effect seems to be related to the side chain of the lanostane-type triterpenoid, although it seems to be independent of the presence or absence of the double bond at the 8-position of the lanostane skeleton and dimethyl at the 4-position.

(Example 10)
Example 10 is the result of performing an activity evaluation test on 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 dermal papilla cell proliferation promoting action test. The dermal papilla cell proliferation-promoting action test was carried out by the method described in Example 3.

  As shown in Table 37 above, it was confirmed that the 50% ethanol-insoluble part showed a hair papillary cell proliferation action exceeding 4.185 μg / mL minoxidil at a low concentration of 3.125 μg / mL. The hair papilla cell proliferation action of the insoluble part at this time is comparable to the hair papilla cell proliferation promoting action of the soluble part of 12.5 μg / mL.

  Tables 38 and 39 below show the results of production promotion tests for various growth genes involved in the hair cycle. Production promotion tests for various growth genes were performed 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 for adenosine are summarized in Table 40 below.

  As shown in the above table, the 50% ethanol-insoluble part shows FGF-7 production promoting action and VEGF production promoting action comparable to 13.4 μg / mL adenosine at a low concentration of 3.125 μg / mL. Admitted.

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

  Table 41 shows that the 50% ethanol-insoluble part has a testosterone 5α-reductase activity inhibitory action.

  Furthermore, an androgen receptor antagonistic inhibitory effect test was performed on the 50% ethanol insoluble part and the soluble part. The test method is described below.

First, SC-3 cells cloned from mouse spontaneous breast cancer (Shionogi cancer, SC-115) were used in a MEM medium (MEM / 2 medium) containing 2% DCC-FBS and 10 ⁻⁸ mol / L testosterone. Then, the cells were collected by trypsin treatment. The collected cells were diluted with MEM / 2 medium to a concentration of 1.0 × 10 ⁵ cells / mL. Diluted cells were seeded in 96 well plates at 100 μL / well.

After culturing overnight, the medium was extracted. 100 μL of 10 ⁻⁹ mol / L dihydrotestosterone and a test sample dissolved in Ham F12 + MEM (HMB) containing 0.5% BSA were added. 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.

  After culturing for 2 hours, formation of blue formazan was confirmed. This blue formazan was extracted with 200 μL of 2-propanol. After the extraction, the absorbance at a wavelength of 570 nm was measured to obtain a first absorbance. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity to obtain a second absorbance. The difference between the first absorbance and the second absorbance was used as the amount of blue formazan produced.

A cell cultured with only HMB was used as a blank test, and a cell cultured with HMB containing only 10 ⁻⁹ mol / L DHT was used as a positive control. The obtained results are shown in Table 42 below.

  Table 42 shows that the 50% ethanol-insoluble part has a moderate androgen receptor antagonistic inhibitory action.

(Example 8)
Below, the prescription example of the hair restorer which concerns on this invention is shown.
Birch extract bamboo shoot 0.5g
0.1g dipotassium glycyrrhizinate
Carrot extract 0.2g
D-pantothenyl alcohol 0.1g
Sodium hyaluronate 0.3g
Assembly extract 0.2g
Biwa leaf extract 0.1g
1,3-butylene glycol 5.0 g
Ethanol 25.0g
Perfume appropriate amount Purified water balance Total 100.0g

Claims (12)

Lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol, 3β, 21-dihydroxylanosto-8,24-diene , trametenolic acid , ergosterol, stellaterol, inotolactone B, ergosterol peroxide A hair restorer containing at least one of Inonotsan C, 3β, 22R, 25-trihydroxylanosto-8,23E-diene and Inonottriol A as an active ingredient. Lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol, 3β, 21-dihydroxylanosto-8,24-diene , trametenolic acid , ergosterol, stellaterol, inotolactone B, ergosterol peroxide And a hair papilla cell proliferation promoter comprising at least one of Inotsusan C as an active ingredient. Lanosterol, 3β-hydroxylanosto-8,24-dien-21-al, inotodiol , 3β, 21-dihydroxylanosto-8,24-diene , ergosterol, stellaterol, inotolactone B, ergosterol peroxide, inotosan C, Fibroblast growth factor-7 (FGF-7) production promotion characterized by containing at least one of 3β, 22R, 25-trihydroxylanosto-8,23E-diene and inotutriol A as an active ingredient Agent. A vascular endothelial growth factor (VEGF) production promoter comprising at least one of trametenolic acid , ergosterol, and inotolactone B as an active ingredient. Insulin-like growth factor characterized in that it contains at least one of inotdiol , ergosterol, stellaterol, inotolactone B, inonotsan C and 3β, 22R, 25-trihydroxylanosto-8,23E-diene as an active ingredient 1 (IGF-1) production promoter. Hepatocyte growth factor (HGF) production promotion characterized by containing at least one of 3β-hydroxylanosto-8,24-dien-21-al , inotodiol , ergosterol , stellaterol and inotolactone B as an active ingredient Agent. The hair restorer according to claim 1, wherein the active ingredient is obtained by extraction / isolation from birch moss. The dermal papilla cell growth promoter according to claim 2, wherein the active ingredient is obtained by extraction / isolation from birch. The fibroblast growth factor-7 (FGF-7) production promoter according to claim 3, wherein the active ingredient is obtained by extraction and isolation from birch salmon. 5. The vascular endothelial growth factor (VEGF) production promoter according to claim 4, wherein the active ingredient is obtained by extraction / isolation from birch moss. The insulin-like growth factor-1 (IGF-1) production promoter according to claim 5, wherein the active ingredient is obtained by extraction and isolation from birch moss. The hepatocyte growth factor (HGF) production promoter according to claim 6, wherein the active ingredient is obtained by extraction / isolation from birch moss.

Images