JP2006199651A - Fibroblast growth factor 5 inhibitor, method for producing the same and hair growth agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a non-peptide-based inexpensive FGF-5 inhibitor. <P>SOLUTION: This fibroblast growth factor 5 inhibitor is provided by containing ≥1 kind of an extract of a plant selected from a group consisting of the genus Euphorbia of Euphorbiaceae, the genus Crassocephalum of Comppositae, the genus Ipomoea of Convolvulus, the genus Toddaria of Rutaceae and the genus Galaxaura of Red algae. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an inhibitor of fibroblast growth factor 5 (hereinafter referred to as FGF-5) containing an extract extracted from a specific plant.

  Among fibroblast growth factors, FGF-5 has been reported to be involved in hair growth, for example (Non-patent Document 1). In addition, FGF-5 antagonists are known to inhibit the hair growth inhibitory effect of FGF-5 (Non-patent Document 2). Furthermore, FGF-5 antagonist is a polypeptide comprising a part of FGF-5, and is an FGF for hair growth. A polypeptide consisting of 10 amino acids that inhibits the inhibitory action of -5 has been reported as an FGF-5 antagonist (Patent Document 1, Non-Patent Document 3). However, in order to express the inhibitory action of FGF-5 on the hair growth of the peptides used above, intradermal administration at a high concentration of 10 mg / 100 ml is required. Moreover, since a peptide is expensive, when putting a FGF-5 inhibitor into a hair-restoration agent, the non-peptide thing is desired.

J. et al. Cell 78: 1017-1025. J. et al. Invest. Dearmatol. 114: 456-463. J. et al. Cell. Physiol. 197: 272-283. Japanese Patent Laid-Open No. 2002-293720

  The object of the present invention is to provide a non-peptide inexpensive FGF-5 inhibitor.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a part of plants existing in nature has FGF-5 inhibitory activity, and completed the present invention.

Specifically, it was achieved by the following means.
(1) Fibroblast growth, comprising an extract of at least one plant selected from the group consisting of Euphorbia spp. Euphorbia spp. Factor 5 inhibitor.
(2) An extract of at least one kind of plant selected from the group consisting of Euphorbia spp. Euphorbia spp., Asteraceae safflower spp. The fibroblast growth factor 5 inhibitor according to (1), which is an extract of at least one kind of plant selected from the group consisting of Yaeyamakazura, Sarkekekan and Velvet Rattle.
(3) Extracting at least one plant selected from the group consisting of Euphorbia spp. Euphorbia spp. A method for producing a fibroblast growth factor 5 inhibitor according to 1) or (2).
(4) The method for producing a fibroblast growth factor 5 inhibitor according to (3), wherein the plant is extracted at a temperature of 10 ° C. before and after the boiling point of the solvent.
(5) A hair nourishing agent comprising an extract of at least one kind of plant selected from the group consisting of Euphorbia spp. Euphorbia spp.
(6) A hair nourishing agent comprising an extract of at least one kind of plant selected from the group consisting of Hanakirin, safflower ragwort, Yaeyama katsura, Sarca kemican and velvet rattle.
(7) renal cell carcinoma, adenocarcinoma, breast cancer, prostate cancer, fibroblastoma, hemangioma, osteoblastoma, neuroblastoma, neuronal cell death, Alzheimer's disease, Parkinson's disease, amnesia, dementia, myocardial infarction, The fibroblast growth factor 5 inhibitor according to (1) or (2), which is used for prevention and / or treatment of at least one disease of thrombocytopenia and thrombocytopenic purpura.

  The FGF-5 inhibitor of the present invention has made it possible to inhibit the function of FGF-5. As a result, it became significant for prevention and treatment of various diseases caused by FGF-5. In particular, it is significant as a hair restorer.

  Hereinafter, the contents of the present invention will be described in detail. In the present specification, “to” is used to mean that the numerical values described before and after it are included as a lower limit value and an upper limit value. The extract referred to in the present invention is intended to include dried powder of plants and the like in addition to those eluted with a liquid or the like.

Plant used for FGF-5 inhibitor The FGF-5 inhibitor of the present invention is at least selected from the group consisting of Euphorbia spp. Euphorbia spp. Contains one or more plant extracts.
The Euphorbia genus Euphorbia is preferably hanakirin ( Euphorbia milliMoul. Var. Splendens (Boj.) Ursen et Leandri ). As the Asteraceae safflower genus, preferably safflower ragweed ( Crassocephalum crepidioides S. Moora ). As the convolvulaceae sweet potato, Ipomoea batatas L. is preferable. As the genus Salmiaceae , the citrus family is preferably Toddaria asiatica (L.) Lamk . The red alga Rattlera is preferably galaxaura fasciculata Kjellman .

These plants may be used as a whole, or roots, stems, leaves, flowers and other parts may be used alone or in appropriate mixtures. In addition, the plant body used as a raw material may be in a dry state or in a non-dry state, more preferably in a dry state, and even more preferably freeze-dried.
In particular, for honey giraffe, stems and whole trees are preferable, for safflower ragwort, whole grasses are preferable, for salchemica, leaves and whole trees are preferable, for prunus japonica, for whole plants, and for velvet rattles, all algae are preferable.

Extraction Method The extraction method of the FGF-5 inhibitor from the plant is not particularly defined, and examples thereof include extraction with a solvent and drying.
(1) Extraction with a solvent Examples of preferable solvents for extraction with a solvent include water, organic solvents, and mixtures thereof. Monohydric alcohols, polyhydric alcohols or derivatives thereof, ketones, esters, ethers, petroleum Ethers, aliphatic hydrocarbons and their halides, aromatic hydrocarbons, and mixtures of two or more thereof are more preferred, and water, methanol and ethanol, and combinations thereof are more preferred. Although the usage-amount of a solvent is not specifically limited, Preferably it is 0.1 to 1000 weight times with respect to a plant, More preferably, it is 1 to 100 weight times, More preferably, it is 10 to 70 weight times.
The plant is preferably extracted after the raw material is chopped or ground. The pulverization is preferably performed, for example, after lyophilization. The pulverized plant is preferably capable of passing through a 1.0 mm mesh.

The extraction temperature is not particularly defined, but is preferably room temperature to a temperature around the boiling point of the solvent, more preferably a temperature around 10 ° C. around the boiling point of the solvent used, and a temperature around 5 ° C. around the boiling point of the solvent used. preferable. Thereby, it becomes possible to extract the active ingredient for FGF-5 inhibition from a smaller amount of plants. In particular, when a plant extract is extracted at a high temperature, its effect is often inactivated. However, the FGF-5 inhibitor of the present invention does not have such a problem, and is extremely significant from this viewpoint. In addition, when it is a mixed solvent etc. and a boiling point is not clear, the temperature which starts vaporization or the temperature which completes vaporization is contained in the said temperature range. Of course, it goes without saying that, for example, extraction at room temperature (for example, 20 to 30 ° C.) is not excluded.
Further, a reflux extraction method can also be preferably used.
The extraction time is not particularly defined, but is preferably 1 hour to 30 days, and preferably 10 to 60 minutes. In particular, extraction with a high temperature solvent is advantageous in that the extraction time can be shortened.
The extraction may be stationary extraction or shaking extraction. Moreover, you may extract using a high-speed solvent extraction apparatus etc.
Moreover, you may extract the plant after extraction again. In addition, the precipitate may be removed by centrifugation or the like after extraction.

(2) Drying An extract can be obtained by drying a plant that can be used as an FGF-5 inhibitor. In this case, it is preferable to cut and dry the plant. In this case, it is preferable to further grind the dried product.

Physiological functions of FGF-5 include hair growth or hair growth regulation, brain nervous system nutrition or function regulation, platelet regulation, vascular endothelial cells, fibroblasts, myoblasts, chondrocytes, osteoblasts, It is an effect of promoting or suppressing the proliferation and differentiation of glial cells.
Therefore, the FGF-5 inhibitor of the present invention includes various hair growth promoters such as hair, kidney cell cancer, adenocarcinoma, breast cancer, prostate cancer, fibroblastoma, hemangioma, osteoblastoma, neuroblastoma and the like. Useful for prevention and treatment of diseases. Also, prevention and treatment of various diseases such as neuronal cell death, Alzheimer's disease, Parkinson's disease, amnesia, dementia, myocardial infarction, thrombocytopenic purpura, and thrombocytopenia caused by cancer chemotherapy and severe infections It is also useful for prevention and treatment.
In particular, it can be preferably used as an active ingredient of a hair nourishing agent. Here, the hair nourishing agent is intended to include a hair growth promoting agent, a hair growth promoting agent, a hair loss preventing agent and the like.

  When the FGF-5 inhibitor of the present invention is used as a pharmaceutical product or a quasi-drug, the extract of the present invention may be administered as it is, but preferably a pharmaceutical composition that can be produced by methods well known to those skilled in the art. Can be administered as a product. Examples of the pharmaceutical composition include tablets, capsules, powders, fine granules, granules, liquids, and syrups. Said pharmaceutical composition can be manufactured by adding a pharmacologically and pharmaceutically acceptable additive. Examples of pharmacologically and pharmaceutically acceptable additives include, for example, excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, and dissolution. Agents, disintegration aids, tonicity agents, pH adjusters, stabilizers, propellants, adhesives and the like can be mentioned. You may mix | blend 1 type (s) or 2 or more types with another FGF-5 inhibitor within the range which does not deviate from the meaning of this invention in said pharmaceutical composition. The administration method of the FGF-5 inhibitor of the present invention is not particularly limited, and any of an internal preparation and an external preparation may be used, and an internal preparation is preferred. The internal preparation may be administered by intravenous injection as an injection, an infusion, or the like, or may be administered orally. Further, the dosage of the pharmaceutical agent of the present invention is not particularly limited and can be appropriately selected according to the type of active ingredient, and should generally be taken into consideration such as patient weight and age, disease type and symptoms, administration route, etc. Depending on various factors, it can be appropriately increased or decreased. In general, when used as an internal preparation, it can be used preferably in the range of 0.1 to 10 g, more preferably 0.5 to 5 g per day for an adult.

  In particular, when used as a hair nourishing agent, in addition to the above, it is preferably used as a cream, spray, or coating agent. These may be used as a hair nourishing agent or may be blended in hair styling agents, shampoos, rinses, treatments, hair dyes and the like.

  The present invention will be described more specifically with reference to the following examples. The materials, amounts used, ratios, processing details, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the specific examples shown below.

Example 1
Hanakirin stems, safflower ragwort, yamakazura (whole plant) and sorghum citrus leaves were cut to a suitable size and then air-dried at 60 ° C. for 12 hours, followed by ultracentrifugation mill (manufactured by MRK-Retsch, ZM100 ) And passed through a 0.5 mm mesh. Each obtained plant was extracted using the high-speed solvent extraction apparatus (Nippon Dionex company make, ASE-200). That is, 2 g of the plant was added to each extraction cell together with 5 g of diatomaceous earth, and an extraction operation was performed (solvent: 50% ethanol, solvent amount: 25 ml, extraction temperature: 82 ° C., extraction time: 10 minutes, number of extractions: Twice).
The velvet rattle algae are freeze-dried and then crushed by an ultracentrifugation mill, 2 g of the dried powder is immersed in 40 ml of 50% ethanol, and extracted by shaking at 290 rpm for 48 hours at room temperature. Then, the precipitate was removed to obtain an extract.

Example 2
About 100 liters of 50% ethanol aqueous solution was added to 100 g of dried plant bodies of honey beetle, safflower, yamakazura, sarca kemican and velvet rattle, respectively, and immersed and extracted at room temperature for 3 days with light agitation 3 times a day. The extract was filtered and the filtrate was taken to obtain an extract.

Example 3 Inhibition rate of R1c / Ba / F3 cell proliferation R1c / Ba / F3 cells were obtained from Nat. Biotec. 18 (6): 641-644. It was produced according to the method described in 1. That is, J. et al. Biol. Chem. 271 (25): 15292-15297, the human FGFR-1 (III) c plasmid was inserted into Ba / F3 cells by electroporation. Here, FBS represents fetal bovine serum (manufactured by Hycron). Next, 10% FBS, 500 μg / ml anti-antibiotic G-418 Sulfate (Promega), 10 ng / ml FGF-1 (Research Diaact Stick) and 10 μg / ml heparin (Sigma) Stable R1c / Ba / F3 cells subcultured in RPMI medium were obtained.
The obtained R1c / Ba / F3 cells were suspended in RPMI medium containing 10% FBS and 500 μg / ml anti-antibiotic G-418 Sulfate, and then added to a 96-well cell culture plate at 5 × 10 ⁴ cells / well. It was.
Next, the sample obtained in Example 1 was diluted with 50% ethanol to the concentrations shown in Table 1 below, and 1.54 × 10 ⁻⁹ M FGF-5 and 5 μg / ml heparin / 10% FBS / 500 μg / 100 μl / well was added together with ml of Antibiotic G-418 Sulfate / RPMI medium and cultured for 72 hours. The proliferation activity of R1c / Ba / F3 cells was determined by adding 5 μl of WST-8 solution (manufactured by Wako Pure Chemical Industries, Ltd.) to each well, further culturing for 3 hours, and coloring at 450 nm accompanying the production amount of WST-8 formazan. Was measured. The results are shown in FIG.

  As shown in FIG. 1, these plant extracts showed cell proliferation activity by FGF-5. Furthermore, as is evident from Samples 1-1 and 1-2 or Samples 2-1 and 2-2, they were shown to inhibit depending on the concentration.

Example 4 Receptor Binding Inhibition Rate The inhibition of receptor binding activity of 50% ethanol extract was measured by measuring the binding activity of FGF-5 ligand to FGFR-1 (III) c-Fc-chimera. Here, PBS represents a phosphate buffer solution of pH (7.4), BSA represents bovine albumin serum (manufactured by Sigma), and DDW represents (Milli-Q water). The FGFR-1 (III) c-Fc-chimera was a product manufactured by Research Diaacts. FGF-5 was prepared according to the description on page 273 of Non-Patent Document 3 above.
DDW containing 5 μg / ml Protein A (Sigma) was added to a 96-well microtiter plate at 50 μl / well and left overnight at 4 ° C. After washing with PBS / 0.05% Tween 20, 100 μl of 1% BSA / PBS was added and left at room temperature for 2 hours, and then 50 μl of 0.5 μg / ml FGFR-1 (III) c-Fc-chimera / 1% BSA / PBS was added and left at room temperature for 1 hour. After washing with PBS / 0.05% Tween 20, 1.54 × 10 ⁻⁹ M FGF-5 and 5 μg / ml heparin / 10% FBS / RPMI medium were added at 50 μl / well. Further, each sample obtained in Example 2 as a specimen was diluted to the concentration shown in Table 2, added at 10 μl / well, and allowed to stand at room temperature for 1 hour. After washing with PBS / 0.05% Tween 20, 50 μl / well of 2 μg / ml sheep-derived anti-human FGF-5 polyclonal antibody (R & D System) was added and left at room temperature for 1 hour. After washing with PBS / 0.05% Tween 20, horse radish peroxidase-conjugated horse-derived anti-sheep immunoglobulin antibody (manufactured by Funakoshi) diluted 500-fold was added at 50 μl / well and allowed to stand at room temperature for 1 hour. After washing with PBS / 0.05% Tween 20, 50 μl / well of 0.1 M citrate buffer (pH 5.2) containing 3 mg / ml peroxidase substrate (Sigma) and 0.01% H ₂ O ₂ was added. After standing at room temperature for 30 minutes, 50 μl / well of 1 M H ₂ SO ₄ was added, and the color development amount at 450 nm was measured.
The color development amount when only 50% ethanol was added was defined as 100% control, and the color development amount of each sample with respect to the control was determined as an inhibition rate (%). The results are shown in FIG.

  As shown in FIG. 2, all of the samples obtained in Example 2 inhibited the binding of FGF-5 ligand to the FGFR-1 (III) c-Fc-chimera, and both samples were at the receptor level. It was shown to be an FGF-5 antagonist that inhibits binding. Furthermore, these receptor binding inhibitions were also found to be concentration dependent.

Example 5
A hair shampoo was produced according to the following formulation and production method.
Component Weight%
1. Extract obtained in Example 1 0.1
2. Sodium lauryl ether sulfate ethanol 20
3. Sodium lauryl sulfate 10
4). 1,3 Butylene glycol 1
5. Perfume appropriate amount 6. Purified water remaining amount (100 in total)
The prescription ingredients were heated to 80 ° C., stirred and mixed, and then cooled by stirring to produce the hair shampoo of the present invention. These shampoos were found to be more effective for nourishing hair than shampoos that do not contain Component 1 above.

Example 6
A hair liquid was produced according to the following formulation and production method.
Component Weight%
1. Extract obtained in Example 1 0.1
2. Ethanol 40
3. Glycerin 1
4). Perfume appropriate amount 5. Purified water remaining amount (100 in total)
After adding the formulation components and dissolving with stirring, purified water was added to produce a hair liquid. These hair liquids were found to be more effective for nourishing hair than the hair liquids that do not contain Component 1 above.

Example 7
A hair cream was produced according to the following formulation and manufacturing method.
Component Weight%
1. Extract obtained in Example 1 0.1
2. Liquid paraffin 40
3. Vaseline 1
4). Cetostearyl alcohol 1
5. Methyl polysiloxane 1
6). Methyl paraoxybenzoate 0.2
7). Propylene glycol 5
4). Perfume appropriate amount 5. Purified water remaining amount (100 in total)
The formulation ingredients were mixed with stirring to produce the hair cream of the present invention. These hair creams were found to be more effective for nourishing hair than hair creams that did not contain Component 1 above.

FIG. 1 is a graph showing the results of measuring inhibitory activity on R1c / Ba / F3 cell proliferation activity. FIG. 2 is a graph showing the results of measuring the inhibitory activity of FGF-5 on receptor binding.

Claims (7)

Inhibition of fibroblast growth factor 5 comprising an extract of at least one plant selected from the group consisting of Euphorbia spp. Euphorbia spp. Agent. The extract of at least one kind of plant selected from the group consisting of Euphorbia spp. Euphorbia spp. The fibroblast growth factor 5 inhibitor according to claim 1, which is an extract of at least one kind of plant selected from the group consisting of salka kemican and velvet rattle. Or at least one kind of plant selected from the group consisting of Euphorbia spp. Euphorbia spp., Asteraceae Succulenta spp. 2. A method for producing a fibroblast growth factor 5 inhibitor according to 2. The method for producing a fibroblast growth factor 5 inhibitor according to claim 3, wherein the plant is extracted at a temperature of 10 ° C before and after the boiling point of the solvent. A hair nourishing agent comprising an extract of at least one plant selected from the group consisting of Euphorbia spp. Euphorbia spp. A hair nourishing agent comprising an extract of at least one kind of plant selected from the group consisting of Hanakirin, safflower ragwort, Yaeyama quail, Salke chemican and velvet rattle. Renal cell carcinoma, adenocarcinoma, breast cancer, prostate cancer, fibroblastoma, hemangioma, osteoblastoma, neuroblastoma, neuronal cell death, Alzheimer's disease, Parkinson's disease, amnesia, dementia, myocardial infarction, thrombocytopenia and The fibroblast growth factor 5 inhibitor according to claim 1 or 2, which is used for prevention and / or treatment of at least one disease of thrombocytopenic purpura.

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