JP2007022957A - TGF-beta RECEPTOR ANTAGONIST - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To discover a substance having TGF-β receptor binding inhibitory action from natural products, and to provide a new TGF-β receptor antagonist containing the substance as an active ingredient. <P>SOLUTION: This TGF-β receptor antagonist contains a mulberry root extract as the active ingredient. The TGF-β receptor antagonist may contain mulberry flavonoids, including kuwanon E, kuwanon G, kuwanon H, and molsin, as the active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a TGF-β receptor antagonist, and more particularly to a TGF-β receptor antagonist that can be used as a hair restorer, a nephritis therapeutic agent, a cirrhosis therapeutic agent, or the like.

  Mulberry has long been cultivated for silkworm feed in the sericulture industry, and its fruits have been used for food, and roots, leaves, fruits and juveniles have been used for medicinal purposes. Mulberry roots, excluding fine roots, are peeled off and dried, and are distributed as the herbal medicine "Sakuhakuhi". It is said that this Sakuhakuhi is good from white to light yellow except for the yellow-brown pericyte (cork skin). Sohakuhi is used as a cough and expectorant in Chinese herbal formulas or used alone to reduce urine volume, but it is also used as a raw material for hair restorer. (Patent Document 1). In addition, a hair growth assisting solution using the mesothelial portion of mulberry root has also been reported (Patent Document 2). However, in these reports, it has not been clarified which component in the mulberry root acts as a hair restorer by what mechanism.

  On the other hand, TGF-β is a cytokine having various actions such as growth control, generation and differentiation of various cells, and has been shown to be deeply involved in fibrosis of organs such as liver and kidney. Examples of diseases caused by such fibrosis include cirrhosis, arteriosclerosis, nephritis and the like, and studies on TGF-β receptor antagonists are being conducted as therapeutic agents for these diseases.

Among these TGF-βs, TGF-β2 has an effect of promoting the transition to the hair regression stage, and since Roman chamelette extract, Shimosukeso extract and the like suppress the action of TGF-β, these extracts are used as hair nourishing agents. It has been reported that it can be used as (Patent Document 3). However, in this report, mulberry is not mentioned, and it has not been known until now whether or not the extract of mulberry root has a TGF-β receptor binding inhibitory action.

JP-A-6-329517 JP 61-12607 A JP 2000-342296 A

  An object of the present invention is to find a substance having a TGF-β receptor binding inhibitory action from natural products, and to provide a novel TGF-β receptor antagonist containing the substance as an active ingredient.

  As a result of earnest search for natural products having TGF-β receptor binding inhibitory activity, the present inventors have found that the extract of mulberry root has an excellent inhibitory effect on TGF-β receptor binding. The TGF-β receptor binding inhibitory action of the extract was found to be exhibited by quaflavonoids such as quanon E, G, H and morsin contained therein, and the present invention was completed.

  That is, this invention is a TGF- (beta) receptor antagonist which uses the extract of a mulberry root as an active ingredient.

  Moreover, this invention is a TGF- (beta) receptor antagonist which uses quaflavonoids as an active ingredient.

  Furthermore, this invention is a TGF- (beta) receptor antagonist which is a hair restorer, a cirrhosis therapeutic agent, or a nephritis therapeutic agent.

  According to the present invention, a TGF-β receptor antagonist having high safety and excellent TGF-β receptor binding inhibitory activity can be obtained.

  The TGF-β receptor antagonist of the present invention has excellent effects on diseases caused by excessive production of TGF-β, hair loss prevention, hair restoration / hair growth / hair growth effect, nephritis and cirrhosis.

Furthermore, the hair restorer of the present invention also has a very high blood flow promotion, and not only improves hair loss, but also applies to hair, strains, restores damaged hair, prevents itching of the scalp, Furthermore, safety, solubility and storage stability are also good.

  The mulberry used in the present invention is a plant belonging to the genus Morus, such as Morus alba Linne, and examples of plants belonging to the genus include Morus bombycis Koidzumi, Morus multicaulis Perrottet, and Morus mongolica.

  Further, the mulberry flavonoids used in the present invention refer to substances having a flavonoid structure contained in mulberry.

  The TGF-β receptor antagonist of the present invention can be obtained by extracting the mulberry root. As the mulberry root used in the present invention, any of mulberry root bark including pericardium, persimmon peony from which mulberry root pericardum is removed, mulberry root pericarp and the like can be used, among which TGF-β receptor antagonist Mulberry root bark or mulberry root pericarp is preferably used because of its high content of components having an action.

  The mulberry root extract used in the present invention is produced by extracting the mulberry root with a suitable solvent. Preferable examples of the solvent used include water and alcohols such as ethanol, methanol and butanol; organic solvents such as ethyl acetate and hexane.

  As the mulberry root extract of the present invention, in addition to the extract obtained by directly extracting the mulberry root with a solvent, a solvent fraction obtained by further liquid-liquid partitioning of the extract is used. You can also. Examples of the solvent used for this liquid-liquid distribution include hexane, ethyl acetate, butanol and the like.

  Preferable examples of the mulberry root extract include an extract extracted with ethanol having a concentration of 30 to 100% by mass (hereinafter abbreviated as “%”), and this extract is further liquid-liquid distributed with ethyl acetate Fractions obtained in this way. Of these, the ethyl acetate fraction after liquid-liquid partitioning is particularly preferred because of its high TGF-β receptor binding inhibitory activity.

  The mulberry root extract described above may be used as it is in liquid form, or if necessary, purified by a known purification means, and further, if necessary, powdered by means such as freeze-drying. good.

  The TGF-β receptor antagonist of the present invention comprises, as an active ingredient, the mulberry root extract obtained as described above, and the TGF-β receptor in the mulberry root extract. A search was conducted for substances having body binding inhibitory activity, and it was found that the above-mentioned activity was possessed by quaflavonoids such as quanon E, quanon G, quanon H and morsin. Therefore, the TGF-β receptor antagonist of the present invention may be a mixture of mulberry flavonoids in addition to the mulberry root extract. These mulberry flavonoids may be isolated and purified from the above mulberry root extract, or extracted from plants other than mulberry, even if extracted from parts other than mulberry root. Or may be synthesized.

  Examples of the quaflavonoids include prenylflavonoids such as quanon E, quanon G, quanon H, morsin, sangenone C, sangenone D, morin, dihydrokaempferol, quercetin and the like. Of these, quanon E, quanon G, quanon H, and morsin represented by formulas 1 to 4 are preferable.

  The TGF-β receptor antagonist of the present invention obtained as described above can be used for prevention and treatment of diseases involving TGF-β. Specifically, it can be used as a hair restorer, a nephritis therapeutic agent, and a cirrhosis therapeutic agent.

  In order to use the TGF-β receptor antagonist of the present invention as a hair-growth agent, the mulberry root extract or its components, quaflavonoids such as quanon E, quanon G, quanon H, and morsin, are appropriately used as a base for the hair-growth agent. What is necessary is just to combine with an agent and formulate.

  In producing the hair restorer, for example, the mulberry root extract is preferably blended in an amount of 0.00001 to 1% in terms of dry matter, more preferably 0.001 to 0.5%, based on the whole hair restorer. 0.01 to 0.1% is particularly preferable. Moreover, when mix | blending the said mulberry flavonoids, you may mix | blend 1 type alone among these, but you may mix | blend 2 or more types in combination.

  As the base for the hair restorer, those commonly used for hair restorers, such as water, monohydric alcohols such as ethanol, polyhydric alcohols such as glycerin and ethylene glycol, etc. can be used, preferably water. It is a mixed solution of ethanol.

Further, the hair restorer of the present invention further includes components commonly used in hair restorers, such as hormones, vitamins, amino acids, moisturizers, antioxidants, cooling agents, gelling agents, chelating agents, preservatives, Additive components such as antibacterial agents, pH adjusters, oils, pigments, and fragrances can be blended. Specific examples include estradiol, nicotinamide, amino acid extract, soy peptide, vitamin E, disodium edetate, carboxyvinyl polymer, isopropylmethylphenol, diisopropanolamine, l-menthol, p-hydroxybenzoate ester, jojoba oil , Pigments, fragrances and the like.

  Furthermore, the hair-restoring agent of the present invention has an excellent hair-growth effect even if only the above TGF-β receptor antagonist is added as an active ingredient, but it can also be used in combination with other hair-restoring ingredients. Preferable examples of such hair-growth ingredients are button pi extract, carrot extract, assembly extract, ginger tincture, sensu extract, cucumber extract, safflower extract, capsicum tincture, mandarin extract, glycyrrhizic acid or its derivatives, cephalanthin , Tocopherols or derivatives thereof, hinokitiol, salicylic acid, resorcin, D-pantothenyl alcohol and the like.

  In addition, the dosage form of the hair restorer of this invention is not specifically limited, It can be set as a liquid agent, aerosol formulation, ointment, cream, gel, oil, pack, shampoo, rinse, treatment etc. Of these, liquid agents and aerosol preparations are particularly preferred.

  On the other hand, in order to use the TGF-β receptor antagonist of the present invention as a nephritis therapeutic agent or a cirrhosis therapeutic agent, the extract of mulberry root or mulberry flavonoids may be formulated by a conventional method. Examples of the dosage form include liquids, elixirs, injections, tablets, powders, capsules, granules, powders, troches and the like. Examples of the pharmaceutical carrier include ethanol, purified water, starch, lactose, glucose, sucrose, starch, and crystalline cellulose.

  The reason why the hair growth effect is obtained by the TGF-β receptor antagonist of the present invention is considered as follows. That is, when the secreted androgen binds to the androgen receptor of the hair papilla cell, TGF-β is produced. This TGF-β binds to the TGF-β receptor of the hair follicle cell and becomes a hair follicle cell. In order to induce apoptosis, hair regression proceeds. In contrast, when a hair-restoring agent containing a component having a TGF-β receptor binding inhibitory effect is administered to hair, the component moves to hair follicle cells, antagonizes TGF-β, and inhibits apoptosis of hair follicle cells. To do. Such a mechanism is considered to prevent the regression of hair.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.

Example 1
Method for producing mulberry root ethanol extract:
Mulberry root bark was cut, 1400 g of 70% ethanol aqueous solution was added to 200 g of mulberry root bark, and the mixture was stirred at 30 ° C. for about 1 day. Insoluble matter was filtered off, and the filtrate was allowed to stand at 4 ° C. for about 1 day. Again, insoluble matter was filtered off to obtain 1150 g of a 70% mulberry root extract.

Example 2
Mulberry root extract extraction method (1):
1100 g of mulberry root bark 70% ethanol extract obtained in Example 1 was concentrated under reduced pressure, and ethanol was distilled off. 1 L of water and 500 mL of hexane were added to the residue, and the mixture was allowed to stand after stirring in a separatory funnel, and the upper hexane soluble part was collected. Thereafter, the operation of adding 500 mL of hexane again to the remaining aqueous layer and distributing the mixture was repeated twice, and the upper layer liquids were combined and dried under reduced pressure to obtain 0.9 g of a hexane fraction.

Example 3
Mulberry root bark extract fraction method (2):
500 mL of ethyl acetate was added to the lower aqueous solution remaining after collecting the hexane-soluble part of Example 2, and the mixture was allowed to stand after stirring in a separatory funnel, and the upper ethyl acetate-soluble part was collected. This operation was performed three times, and the upper layer liquids were combined and dried under reduced pressure to obtain 8.8 g of an ethyl acetate fraction.

Example 4
Mulberry root extract extraction method (3):
1-Butanol 500mL was added to the lower layer aqueous solution which collected the ethyl acetate fraction of Example 3, and it left still after stirring with a separatory funnel, and the 1-butanol soluble part of the upper layer was extract | collected. This operation was performed three times, and the upper layer liquids were combined and dried under reduced pressure to obtain 0.8 g of a 1-butanol fraction.

Example 5
Mulberry root bark extract fraction method (4):
The lower layer aqueous solution remaining after collecting the 1-butanol soluble part of Example 4 was lyophilized to obtain 18.8 g of a water fraction.

Test example 1
TGF-β receptor binding inhibitory action test:
The extract or fraction obtained in Examples 1 to 4 was subjected to a TGF-β receptor binding inhibitory action test. The test was conducted as follows. That is, to the ELISA plate coated with TGF-β receptor, the extracts or fractions obtained in Examples 1 to 4 were added at respective predetermined concentrations, and after 1 hour, 4 ng / mL of TGF-β was added, Incubated for 3 hours at room temperature. After washing with a washing buffer, biotinylated anti-TGF-β antibody was added and incubated at room temperature for 2 hours. After washing, avidin-labeled peroxidase was added and incubated at room temperature for 30 minutes, each well was washed, ABTS as a chromogenic substrate was added, and incubated for 15 minutes. After adding the reaction stop solution, the absorbance at 405 nm was measured.

The results are shown by the binding inhibition rate calculated by the following formula for the binding inhibition rate to the TGF-β receptor of the extracts or fractions obtained in Examples 1 to 4. The result is shown in FIG.
Binding inhibition rate (%) = {1− (AC) / (BC)} × 100
A: Absorbance in the presence of the test substance B: Absorbance in the absence of the test substance C: Absorbance in the absence of TGF-β

Example 6
The ethyl acetate fraction obtained in Example 3 was added to a silica gel column (solvent system: hexane / ethyl acetate mixture (1: 0 to 1: 4)), Sephadex LH-20 column (solvent system: ethanol), ODS column. (TSK-gel80Ts and YMC-PackODSSH; solvent system: acetonitrile / water mixture (2: 3 to 1: 1) or methanol / water mixture (3: 2 to 7: 3)) are sequentially applied to the column chromatography. Gave 4 compounds. These compounds were identified as quanon E, quanon G, quanon H and morsin by various physical constants and various spectral data.

Test example 2
Each compound obtained by isolation and purification in Example 6 was subjected to a TGF-β receptor binding inhibitory action test in the same manner as in Test Example 1. The results are shown in FIG.

Test example 3
Hair growth test:
A hair growth test was conducted using the 1% solution of the mulberry root extract obtained in Example 1 as the test hair growth agent 1. First, the back of C57BL mice (8-week-old male) was depilated with hair clippers and a hair removal cream, and 100 μL of testosterone enanthate 5% solution was subcutaneously administered to the hair removal portion twice a week. Thereafter, 100 μL of test substance was applied to the hair removal part of the mouse once a day. The test was performed with 10 animals in each group, and the test substance was applied every day for 21 days. As a control, oxendron 2% solution was used. In addition, a group that did not receive testosterone enanthate and did not receive a test substance was defined as a normal control group, and a group that received testosterone enanthate but did not receive a test substance was defined as a control group. Oxendron is used as a prostatic hypertrophy drug, and there is a concern about side effects when it is used for hair growth.

  Photographs of the back skin of each group of mice photographed over time using a digital camera were analyzed using image analysis software (NIH image), and the ratio of the area of the hair regeneration area to the total area of the hair-cutting area was determined for each individual. It calculated as a hair regeneration rate (%). The results are shown in FIGS.

Example 7
A mulberry root pericarp extract was obtained in the same manner as in Example 1 except that the mulberry root pericarp of Example 1 was replaced with mulberry root pericarp.

Example 8
Except for replacing the mulberry root bark of Example 1 with Sakuhakuhi, a Sakuhakuhi extract was obtained in the same manner as Example 1.

Test example 4
Each extract obtained in Examples 1, 7, and 8 was subjected to a TGF-β receptor binding inhibitory test by the same method as in Test Example 1. The result is shown in FIG.

Test example 5
The contents (%) of quanon E, quanon G, quanon H and morsin contained in the extracts obtained in Examples 1, 7, and 8 were measured by HPLC analysis. The results are shown in FIG.

Example 9
Hair Nick:
Using the mulberry root extract obtained in Example 1, hair arts having the formulations shown in Table 1 below were produced by a conventional method.

Example 10
Hair growth aerosol products:
(Original formulation) (%)
Mulberry root extract of Example 1 1.00
Ethanol 50.00
D-pantothenyl alcohol 0.50
Edetate disodium 0.03
Fragrance (including l-menthol) 0.20
Preservative 0.10
Purified water balance
(100)
(Manufacturing method)
To the active ingredient 90.0% mixed with the above ingredients, 10.0% propellant consisting of dimethyl ether and nitrogen is added to form an aerosol formulation, which is filled into an aerosol can with a jet nozzle diameter of 0.5 mm, A product was manufactured (internal pressure: 5.8 kg / cm ² , at 25 ° C.).

Example 11
Hair growth aerosol products:
(Original formulation) (%)
Buttonpi extract 1.00
Carrot extract 2.00
Assembly extract 2.00
Showa tincture 1.00
Senkyu extract 1.00
Kujin extract 1.00
Mulberry root extract of Example 1 1.00
Safflower extract 0.10
Ethanol 21.00
D-pantothenyl alcohol 0.50
Edetate disodium 0.03
Carboxyvinyl polymer 2% aqueous solution 15.00
Neutralizer 0.30
Fragrance (including l-menthol) 0.20
Preservative 0.10
Purified water balance
(100)
(Manufacturing method)
Aerosol product for hair growth by adding 33.3% of dimethyl ether to the drug substance (viscosity 12,450 cp) 66.7% mixed with the above ingredients to form an aerosol formulation, and filling this into an aerosol can with a jet nozzle diameter of 0.5 mm (Internal pressure: 4.3 kg / cm ² , at 25 ° C.).

Example 12
Hair Liquid:
A hair liquid having the following formulation was produced by a conventional method.
(Prescription) (%)
Mulberry root extract of Example 1 0.50
Licorice extract 0.10
Pepper extract 0.50
Citrus extract 1.00
Tocopherol acetate 0.20
Minoxidil 1.00
Pentadecanoic acid glyceride 1.00
Polyoxypropylene (35) butyl ether phosphate 10.00
Polyoxypropylene (40) butyl ether 5.00
Polyvinylpyrrolidone / vinyl acetate copolymer 0.50
Polyoxyethylene (50) hydrogenated castor oil 1.00
Ethanol 60.00
Oxybenzone 0.05
Sodium hydroxide 0.16
Octyldodecanol 0.50
Fragrance 0.05
Dye 0.005
Purified water balance
(100)

Example 13
Hair shampoo:
A hair shampoo having the following formulation was produced by a conventional method.
(Prescription) (%)
Mulberry root extract of Example 1 0.05
Button pi extract 1.00
Dipotassium glycyrrhizinate 0.10
Polyoxyethylene lauryl ether sodium sulfate solution 20.00
N-coconut oil fatty acid acylglutamate triethanolamine solution 10.00
Palm oil fatty acid amidopropyl betaine solution 10.00
Palm fatty acid diethanolamide 4.00
Ethylene glycol distearate 2.00
Cationized cellulose 1.00
Cationized guar gum 0.20
Polyoxyethylene (10) stearyl ether 1.00
Methyl polysiloxane 0.50
Propylene glycol 5.00
Edetate disodium 0.30
P-Hydroxybenzoate ester 0.40
Citric acid 0.20
Fragrance 0.30
Purified water balance
(100)

Example 14
Hair Treatment:
A hair treatment having the following formulation was produced by a conventional method.
(Prescription) (%)
Mulberry root extract of Example 1 0.05
Button pi extract 1.00
Hinokitiol 0.10
Stearyltrimethylammonium chloride 1.00
Distearyldimethylammonium chloride 1.00
Alkyltrimethylammonium chloride 1.50
Polyoxyethylene (20) cetyl ether 0.50
Polyoxyethylene monostearate (20) sorbitan 1.00
Cetanol 1.50
Stearyl alcohol 1.50
Octyldodecanol 1.50
Lanolin derivative (lanolin cation) 0.50
Cationized cellulose 1.00
Cationized guar gum 0.20
Silicone emulsion 1.00
Glycerin 2.00
Diglycerin 4.00
Propylene glycol 1.00
P-Hydroxybenzoate 0.20
Fragrance 0.30
Purified water balance
(100)

  The TGF-β receptor antagonist of the present invention has an excellent effect in treating diseases caused by excessive production of TGF-β, hair loss prevention, hair restoration / hair growth / hair growth effect, nephritis and cirrhosis. In addition, the hair restorer of the present invention also has a very high blood flow promotion, not only improving hair loss, but also applying hair, straining, restoring damaged hair, preventing itching of the scalp, Furthermore, safety, solubility and storage stability are also good.

  According to the present invention, a TGF-β receptor antagonist having high safety and an excellent TGF-β inhibitory action can be obtained.

  The TGF-β receptor antagonist according to the present invention is useful as a hair restorer. Furthermore, it can be used as a therapeutic agent for diseases involving TGF-β such as a therapeutic agent for cirrhosis or nephritis.

It is a figure which shows the TGF- (beta) receptor binding inhibitory effect of the mulberry root extract obtained in Examples 1-4. FIG. 6 is a graph showing TGF-β receptor binding inhibitory action of quanon E, G, H and morsin obtained in Example 6. FIG. 2 is a graph showing the regeneration rate of mouse hair for each elapsed day by the mulberry root extract obtained in Example 1. FIG. 3 is a graph showing the regeneration rate of mouse hair after 21 days from the extract of mulberry root obtained in Example 1. FIG. 3 is a view showing a TGF-β receptor binding inhibitory action at each site of mulberry root obtained in Examples 1, 7 and 8. It is a figure which shows the content rate of quanon E, G, H, and morsin in each site | part of the mulberry root obtained in Example 1, 7 and 8. FIG.

Claims (8)

  A TGF-β receptor antagonist comprising an extract of mulberry root as an active ingredient.   The TGF-β receptor antagonist according to claim 1, wherein the extract contains one or more compounds selected from the group consisting of quanon E, quanon G, quanon H and morsin.   The TGF-β receptor antagonist according to claim 1 or 2, wherein the mulberry root is mulberry root bark or mulberry root pericarp.   A TGF-β receptor antagonist comprising quaflavonoids as active ingredients.   The TGF-β receptor antagonist according to claim 4, wherein the quaflavonoids are quanon E, quanon G, quanon H or morsin.   The TGF-β receptor antagonist according to any one of claims 1 to 5, which is a hair restorer.   The TGF-β receptor antagonist according to any one of claims 1 to 5, which is a nephritis therapeutic agent. The TGF-β receptor antagonist according to any one of claims 1 to 5, which is a therapeutic agent for cirrhosis.

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