JP4524296B2 - Hair growth promoter - Google Patents

Description

  The present invention relates to a hair growth promoter containing a glycosaminoglycan. More specifically, the present invention relates to a hair growth promoter containing a specific glycosaminoglycan having a specific molecular weight or molecular size.

  So far, hair growth agents, hair growth promoters and the like mainly composed of various glycosaminoglycans and plant extracts have been proposed.

  Patent Document 1 describes a hair treatment agent containing hyaluronic acid having a molecular weight of 500,000 to 2,000,000 and / or salts thereof.

  Patent Document 2 describes a hair nourishing cosmetic characterized by containing hyaluronic acid and an assembly extract or a ginseng extract. The molecular weight of hyaluronic acid that can be used may be a normal range of 500,000 to 2,000,000, and may be a low molecular weight of 500,000 or less, but a statement that 100,000 or less is particularly preferable. is there.

  Patent Document 3 describes a hair growth promoter composed of a sulfated polysaccharide and / or a physiologically acceptable salt. The sulfated polysaccharide is a polysulfated mucopolysaccharide (chondroitin sulfate, keratan sulfate, keratan). (Sulfuric acid etc.) is preferably used.

  Patent Document 4 describes a hair nourishing agent containing bamboo extract as an active ingredient.

  Patent Document 5 describes cosmetics containing, as an active ingredient, a specific polysaccharide contained in medicinal carrots such as a carrot (Tanachi carrot), which also describes that it gives vitality to hair. is there.

  Patent Document 6 describes a cosmetic or dermatological composition aimed at promoting hair growth, characterized by containing a plant extract containing a specific saponin in a specific ratio. In addition, there is a description of a Panax notoginseng (Panax notoginseng) extract as a plant extract, and it is also described that 0.01 to 1% by weight of hyaluronic acid may be contained.

Patent Document 7 describes a hair nourishing cosmetic containing at least one of low molecular weight chondroitin sulfate having an average molecular weight of 2000 to 9000 and a salt thereof.
Patent Document 8 describes a composition having hair stimulating activity, which contains heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate A + B, keratan sulfate, hyaluronic acid, nucleic acid and protein.

  However, in these conventional techniques, a hair growth promoter containing hyaluronic acid having a weight average molecular weight of 200,000 to 400,000, dermatan sulfate having a weight average molecular weight of 20,000 to 50,000, and low molecular weight keratan sulfate is described. Not.

JP 59-110612 A JP-A-6-9349 JP-A-9-188607 JP-A-4-18012 JP 61-1115013 Special table hei 8-503931 Japanese Patent Publication 5-38724 JP-A-1-93513

  An object of the present invention is to provide a novel hair growth promoter having a remarkable hair growth effect, high safety and high economic efficiency.

As a result of intensive studies to achieve the above object, the present inventors have found that a specific glycosaminoglycan having a specific molecular weight or molecular size has a remarkable hair growth effect. Further, these glycosaminoglycans can be used in combination with plant extracts, other glycosaminoglycans, or combined with hyaluronidase treatment of the skin where hair growth is desired. It has been found that a hair growth promoter or a method for promoting hair growth can be provided which is highly safe and has a high economic efficiency with a wide range of hair growth effects with a small amount of use. The present invention has been completed based on these findings.
That is, this invention provides the hair growth promoter (henceforth the hair growth agent of this invention) containing 1 or more types of substances chosen from the following substance group (A).
Substance group (A): Hyaluronic acid having a weight average molecular weight of 200,000 to 400,000, dermatan sulfate having a weight average molecular weight of 20,000 to 50,000, and low molecular weight keratan sulfate.

In the hair growth agent of the present invention, those containing one or more substances selected from the following substance group (B) can be mentioned as preferred embodiments.
Substance group (B): Sasa extract, Tabuchi carrot extract, keratan sulfate, and chondroitin sulfate.

As a preferred embodiment of the hair growth agent of the present invention, the hair growth agent of the present invention described above containing a combination or component of any of the following components (a) to (f) can be mentioned.
(a) hyaluronic acid having a weight average molecular weight of 200,000 to 400,000 and dermatan sulfate having a weight average molecular weight of 20,000 to 50,000, (b) hyaluronic acid having a weight average molecular weight of 200,000 to 400,000, and carrot extract (C) hyaluronic acid having a weight average molecular weight of 200,000 to 400,000 and sasa extract, (d) hyaluronic acid having a weight average molecular weight of 200,000 to 400,000, dermatan sulfate having a weight average molecular weight of 20,000 to 50,000, Carrot extract and Sasa extract, (e) Hyaluronic acid having a weight average molecular weight of 200,000 to 400,000, dermatan sulfate, keratan sulfate, paddy carrot extract, Sasa extract having a weight average molecular weight of 20,000 to 50,000 And chondroitin sulfate, (f) low molecular weight keratan sulfate.

  In the hair growth agent of the present invention, the substance selected from the substance group (A) contains hyaluronic acid having a weight average molecular weight of 200,000 to 400,000, and the hyaluronic acid has a weight average molecular weight of 250,000 to 350,000 Dermatan sulfate having a weight average molecular weight of 30,000 to 40,000 and low molecular weight keratan sulfate are products obtained by degrading keratan sulfate with endo-β-N-acetylglucosaminidase type keratan sulfate decomposing enzyme Some are listed as preferred embodiments.

  Furthermore, the present invention provides a method for promoting hair growth comprising treating the skin desired for hair growth with hyaluronidase and then applying the hair growth agent of the present invention to the skin treated with hyaluronidase, comprising hyaluronidase as an active ingredient. The hair growth-enhancing agent of the hair growth agent of the present invention, and the hair growth promoting kit comprising the hair growth agent of the present invention and hyaluronidase are provided.

According to the hair growth agent of the present invention, (1) a remarkable hair growth effect is obtained, (2) the hair growth effect over a wide area with a small application area, and (3) the hair growth effect and moisturizing effect (prevention of scalp dryness Effect), (4) hair loss is also prevented, and (5) since natural ingredients are blended, it is possible to provide a hair growth promoter that does not adversely affect the scalp and hair (high safety).
Further, the hair growth agent of the present invention has a hair growth effect over a wide area with a narrow application area, so that the amount of use can be reduced, safety is further enhanced, and economic efficiency is also high.

Hereinafter, the hair growth agent of the present invention will be described in more detail.
<1> Component of hair growth agent of the present invention The hair growth agent of the present invention is a hair growth promoter containing one or more substances selected from the following substance group (A).
Substance group (A): Hyaluronic acid having a weight average molecular weight of 200,000 to 400,000, dermatan sulfate having a weight average molecular weight of 20,000 to 50,000, and low molecular weight keratan sulfate.

The hair growth agent of the present invention preferably further contains one or more substances selected from the following substance group (B).
Substance group (B): Sasa extract, Tabuchi carrot extract, keratan sulfate, and chondroitin sulfate.

  Hereinafter, each substance included in the substance groups (A) and (B) and other components that can be added as necessary will be described in detail.

(1) Substance group (A)
(1-1) Hyaluronic acid having a weight average molecular weight of 200,000 to 400,000 The origin of hyaluronic acid having a weight average molecular weight of 200,000 to 400,000 used in the hair growth agent of the present invention is not particularly limited. Hyaluronic acid separated and purified from the microorganisms to be produced can be used, but those derived from chicken crowns are preferred.

  The separation and purification of hyaluronic acid can be carried out by crushing, extraction, enzymatic degradation (for example, degradation by protease), precipitation with organic solvent (for example, alcohol), salting out, salt solution, various chromatographies or combinations thereof, glycosamino It can be performed by a method usually used for separation and purification of glycans. In addition, when the hyaluronic acid thus separated and purified has a molecular weight larger than the above molecular weight, the hyaluronic acid is removed by high-temperature / high-pressure treatment with an autoclave, shearing treatment with high rotational speed, treatment with alkali, acid or enzyme, etc. The molecular weight can be reduced and adjusted to the above molecular weight range.

  The hyaluronic acid having a weight average molecular weight of 200,000 to 400,000 used in the hair growth agent of the present invention may be a pharmaceutically acceptable salt thereof. Examples of pharmaceutically acceptable salts of hyaluronic acid include salts with inorganic bases such as alkali metal salts (sodium salt, lithium salt, potassium salt, etc.), alkaline earth metal salts, ammonium salts, or diethanolamine salts, A salt with an organic base such as a cyclohexylamine salt or an amino acid salt can be used. Of these, sodium salts are preferred.

  The weight average molecular weight of the hyaluronic acid used in the hair growth agent of the present invention is not particularly limited as long as it is included in the range of 200,000 to 400,000, but is preferably about 250,000 to 350,000, and more preferably about 300,000.

(1-2) Dermatan sulfate having a weight average molecular weight of 20,000 to 50,000 The origin of the dermatan sulfate having a weight average molecular weight of 20,000 to 50,000 used in the hair growth agent of the present invention is not particularly limited. Although dermatan sulfate separated and purified from biological materials such as a meat crown (eg, a chicken crown), skin, umbilical cord, and viscera can be used, those derived from a chicken crown are preferred.

  Separation / purification of dermatan sulfate includes glycosamino, such as crushing, extraction, enzymatic degradation (for example, degradation with protease), precipitation with organic solvent (for example, alcohol), salting out, salt solution, various chromatographies, or combinations thereof. It can be performed by a method usually used for separation and purification of glycans.

  The dermatan sulfate having a weight average molecular weight of 20,000 to 50,000 used in the hair growth agent of the present invention may be a pharmaceutically acceptable salt thereof. Examples of the pharmaceutically acceptable salt of dermatan sulfate include salts with inorganic bases such as alkali metal salts (sodium salt, lithium salt, potassium salt, etc.), alkaline earth metal salts or ammonium salts, or organic amines. A salt with an organic base such as a salt (diethanolamine salt, cyclohexylamine salt, triethylamine salt, triethanolamine salt, etc.) or an amino acid salt (arginine salt, lysine salt, etc.) can be used. Of these, sodium salts are preferred.

  Although the weight average molecular weight of the dermatan sulfate used for the hair growth agent of this invention is not specifically limited as long as it is included in the range of 20,000-50,000, About 30,000-40,000 are preferable.

  Of the dermatan sulfate having a weight average molecular weight of 20,000 to 50,000 that can be used in the hair growth agent of the present invention, dermatan sulfate having the following properties is particularly preferred (hereinafter, this dermatan sulfate is abbreviated as DS1).

(A) 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hexy- in unsaturated disaccharides analyzed by high performance liquid chromatography when digested with chondroitinase ABC The proportion of 4-enopyranosyluronic acid) -6-sulfo-D-galactose (ΔDi-6S) is about 4-25%, preferably about 7-16%.

(B) When chondroitinase B is allowed to act, about 3 wt% to about 17 wt%, preferably about 5 wt% to 15 wt% is not digested.

(C) The molecular weight range measured by the multi-angle light scattering method is 20,000 Dalton to 200,000 Dalton.

  The dermatan sulfate (DS1) preferably further has the following characteristics.

(D) The molecular weight distribution measured by the multi-angle light scattering method is a fraction having a molecular weight of 40,000 daltons or less, 79 to 99%, a fraction having a molecular weight of 40,000 to 100,000 daltons, 1 to 20%, a fraction having 100,000 daltons or more. The fraction composition is detected as a fraction composition of 1% or less.

(E) The weight average molecular weight measured by the multi-angle light scattering method is about 28,000 to about 43,000 daltons, preferably about 32,000 to about 38,000 daltons.

(F) The weight average molecular weight measured by gel filtration high performance liquid chromatography (GPC-HPLC) method is about 30,000 to 46,000 daltons, preferably about 35,000 to about 41,000 daltons. .

(G) The molecular weight distribution measured by GPC-HPLC method is a fraction with a molecular weight of 20,000 daltons or less, 5-30%, a fraction with 20,000-80,000 daltons is 70-95%, a fraction with 80,000 daltons or more Is detected as a fraction composition with 0%.

(H) Contents of various unsaturated disaccharides The composition of unsaturated disaccharides when DS1 is digested with chondroitinase ABC is as follows.

-Content of 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-sulfo-D-galactose (ΔDi-6S) Is about 4% to about 25%, preferably about 7% to about 16%.

The content of 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -D-galactose (ΔDi-0S) is about 4 % To about 10%, preferably about 4.5% to about 10%.
2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D-galactose (ΔDi-4S) Is about 63% to about 85%, preferably about 68% to about 84%.

2-acetamido-2-deoxy-3-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D- the content of galactose (ΔDi-diS _D) is from about 0.5% to about 2.2%, preferably about 0.8% to about 1.7%.
2-acetamido-2-deoxy-3-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D- The content of galactose (ΔDi-diS _B ) is about 2% to about 8%, preferably about 3% to about 7%.
2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4,6-bis-O-sulfo-D-galactose ( The content of ΔDi-diS _E is about 0% to about 2%.

  The names and abbreviations of the various unsaturated disaccharides were as described in Yoshida, K. et al., Analytical Biochemistry, 177, 327-332 (1989).

Among such DS1, the most preferable characteristics of DS1 are as follows.
Average molecular weight (kDa) by GPC-HPLC method 38
Average molecular weight (kDa) by multi-angle light scattering method 35.1
Intrinsic viscosity (dl / g) 1.06
Chondroitinase B digestion resistant part (%) 5.3
Disaccharide part (%) 25.4
Disaccharide composition ratio (%)
ΔDi-0S 6.8
ΔDi-6S 12.0
ΔDi-4S 74.1
ΔDi-diS _D 1.3
ΔDi-diS _B 4.4
ΔDi-diS _E 1.4
ΔDi-triS 0.0

This DS1 can be manufactured, for example, by the following method.
About 2-3 times the amount of water is added to 1 weight of chicken crown, minced and then boiled. After cooling, protease (Pronase, Kaken Pharmaceutical Co., Ltd.) is added and hydrolyzed overnight. After adding the benzalkonium chloride solution to the hydrolyzate, the solution is filtered through diatomaceous earth, the filtered supernatant is discarded, and the remaining diatomaceous earth is obtained. This diatomaceous earth is suspended in a sodium chloride solution, ethanol is added, the resulting precipitate is allowed to stand, and the resulting precipitate is dried to obtain a powder.

  This powder is dissolved in water to prepare a 10% solution, and heparin / heparan sulfate is removed by performing nitrous acid treatment by Shively and Conrad methods. That is, a solution in which the powder is dissolved is mixed with a 0.1% nitrous acid aqueous solution and left at room temperature, and then the precipitate is removed by filtration. The pH of the filtrate is adjusted to 10.5, sodium chloride is added to a final concentration of 1%, and ethanol is added with stirring to a final concentration of 48%. Activated charcoal is added to the resulting precipitate and suction filtered. The filtrate is desalted through an ion exchange resin Diaion SA-12A (Mitsubishi Chemical Corporation), and ethanol is added to the filtrate to obtain DS1.

  The obtained DS1 is preferably subjected to molecular ultrafiltration using a membrane having a molecular exclusion limit of 100,000 Dalton (manufactured by Amicon) and washed with a sodium chloride solution of about 0.2M.

In addition, the said characteristic of this DS1 can be confirmed with the following method.
(a) Measurement of average molecular weight by GPC-HPLC method The average molecular weight of DS1 can be measured according to the method of Arai et al. (Biochem. Biophys. Acta, 1117, 60-70, 1992) and the molecular weight is known. (Molecular weight 39100, 18000, 8050) and sodium hyaluronate (molecular weight 104000) as standard products can be determined by elution time in gel filtration (GPC-HPLC) using high performance liquid chromatography. As the column, a column in which TSK gel G4000PW _XL , G3000PW _XL and G2500PW _XL (each φ7.8 × 300 mm, Tosoh Corporation) are connected can be used. A 0.2 mol / L sodium chloride solution is used as a solvent, a flow rate is 0.6 ml / min, and a differential refractive index detector (RI-8000, Tosoh Corporation) can be used as a detector.

(b) Measurement of molecular weight by the multi-angle light scattering method Using a differential refractive index detector (Shodex RI-71, Showa Denko KK) and a multi-angle light scattering detector (DAWN, Wyatt Technology Corporation) as the detector, Analysis is performed using the Jim Plot module of ASTRA software (Wyatt Technology Corporation).

  The column is SB-806HQ (φ8 × 300 mm, Showa Denko KK), the solvent is 0.1 mol / L sodium nitrate solution, and the flow rate is 1.0 mL / min.

(c) Analysis of chondroitinase B digestion pattern 100 μL of DS1 solution, 0.03 U of chondroitinase B (raw) in 10 μL of buffer A (0.001 mol / L calcium acetate, 0.02 mol / L Tris-HCl, pH 7.5) Chemical dissolved) is added and digested at 37 ° C. for 2 hours. The reaction is stopped by heating in a boiling water bath for 1 minute, and 10 μL corresponding to 100 μg of digest is analyzed using GPC-HPLC at 40 ° C. As the column, TSK gel G4000PW _XL , G3000PW _XL and G2500PW _XL (each φ7.8 × 300 mm, Tosoh Corporation) are connected. The solvent is a 0.2 mol / L sodium chloride solution, the flow rate is 0.6 mL / min, and the detector is a differential refractive index detector (RI-8020, Tosoh Corporation) and an ultraviolet-visible detector (UV-8020, A230 nm, Tosoh Corporation) can be used.

(d) Digestion with chondroitinase ABC Dissolve 0.5 U of chondroitinase ABC in 10 μL of buffer B (0.01 mol / L sodium acetate, 0.05 mol / L Tris-HCl, pH 7.5) in 50 μL of 1% DS1 solution. And digest for 2 hours at 37 ° C. The reaction is stopped by heating in a boiling water bath for 1 minute, and the insoluble matter is removed by centrifugation.

  To this degradation product, 0.5 U of chondroitinase ABC dissolved in 10 μL of buffer B is added and digested at 37 ° C. for 6 hours. The reaction is stopped by heating in a boiling water bath for 1 minute, and the insoluble matter is removed by centrifugation.

(e) Digestion with chondro-6-sulfatase Dissolve 0.25 U of chondro-6-sulfatase in 50 μL of buffer solution C (0.02 mol / L Tris-AcOH, pH 7.0) with respect to 100 μg of the digest of (c) above. The digested product is added and digested at 37 ° C. for 24 hours to remove insolubles.

(f) Disaccharide composition analysis Each of the digests (d) or (e) is analyzed using HPLC. As the column, a YMC-Pack PA-120-S5 ion exchange column (φ2.6 × 250 mm, YMC Corporation) can be used. The flow rate is 1.5 mL / min, and 0.8 mol / L sodium hydrogen phosphate is flowed in a linear concentration gradient from 2% to 100% in 60 minutes. Based on the elution position of the unsaturated chondro-disaccharide kit (Seikagaku Corporation), various unsaturated disaccharides eluted during this period are identified at 232 nm, and the sum of the peak areas identified as unsaturated disaccharides is calculated. The disaccharide composition ratio can be determined by calculating as 100%.

(g) Measurement of intrinsic viscosity The intrinsic viscosity of DS1 can be measured according to the 13th revised Japanese Pharmacopoeia. As the measuring device, an automatic viscosity measuring device (VMC-052, Kogaisha Co., Ltd.) can be used. A 0.2 mol / L sodium chloride solution is used as the solvent, and the same solution can also be used for measuring the flow time of the Ubbelohde viscosity tube. Viscosity is measured at 30 ± 0.1 ° C., 1/100 second of the flow-down time is rounded off, and the measured value within 3 seconds is used for the calculation of limiting viscosity.

The intrinsic viscosity is the reduced viscosity (= (t _S / t ₀ −1) / C, t _S : solution flow time, t ₀ : solvent flow time, C: sample concentration (% by weight)). The straight line obtained by plotting on the horizontal axis can be obtained from the intercept when the concentration is extrapolated to zero.

(1-3) Low-molecular-weight keratan sulfate The low-molecular-weight keratan sulfate used in the hair growth agent of the present invention is a substance obtained by degrading keratan sulfate to reduce its molecular weight, and the galactose residue (Gal) constituting keratan sulfate And N-acetylglucosamine residues (GlcNAc) are β-glycosidically linked structural units (Gal-GlcNAc) of 1 to 10, preferably 1 to 4, ie 2 to 20, preferably 2 to 8 It is a substance consisting of a number of sugar residues (part or all of the 6-positions of galactose residues and N-acetylglucosamine residues are sulfated). An N-acetylneuraminic acid residue may be added to the non-reducing end of this low molecular weight keratan sulfate via a glycosidic bond.

  The low molecular weight keratan sulfate used in the hair growth agent of the present invention is not particularly limited as long as keratan sulfate is reduced in molecular weight as described above, but keratan sulfate is endo-β-N-acetyl. It is preferably a product obtained by degrading with a glucosaminidase-type keratan sulfate degrading enzyme, and more preferably a product obtained by degrading keratan polysulfate with an endo-β-N-acetylglucosaminidase-type keratan sulfate degrading enzyme. . The definition of keratan polysulfate will be described later.

  The origin of keratan sulfate that can be used as a raw material for the low molecular weight keratan sulfate is not particularly limited, and it is separated and purified from cartilaginous fish such as sharks, mammalian cartilage such as whales and cows, and raw materials such as bone and cornea. The keratan sulfate produced can be used, but those derived from cartilaginous fish such as sharks are preferred, and those derived from sharks are more preferred. Keratan sulfate obtained from cartilaginous fish such as sharks is highly sulfated keratan sulfate containing 1.5 to 2 molecules of sulfate group per constituent disaccharide, and such keratan sulfate is referred to herein as “ It is called "keratane polysulfate".

  Further, an endo-β-N-acetylglucosaminidase-type keratan sulfate degrading enzyme that can be used for lowering the molecular weight of keratan sulfate is not particularly limited. For example, Keratanase II derived from Bacillus bacteria (JP-A-2-57182), Examples thereof include keratan sulfate degrading enzyme (WO96 / 16973) produced by Bacillus circulans KsT202 strain.

  The low molecular weight keratan sulfate used in the hair growth agent of the present invention may be a pharmaceutically acceptable salt thereof. Examples of pharmaceutically acceptable salts of low molecular weight keratan sulfate include salts with inorganic bases such as alkali metal salts (sodium salt, lithium salt, potassium salt, etc.), alkaline earth metal salts, ammonium salts, or the like. Among salts with organic bases such as diethanolamine salt, cyclohexylamine salt and amino acid salt, pharmaceutically acceptable salts can be used. Of these, a sodium salt is preferable.

The low molecular weight keratan sulfate is not particularly limited as long as it is a low molecular weight keratan sulfate as described above, but it may be one or more sugar chains selected from the following formulas (1) to (3). preferable.
Gal (6S) β1-4GlcNAc (6S) ・ ・ ・ (1)
Gal (6S) β1-4GlcNAc (6S) β1-3Gal (6S) β1-4GlcNAc (6S) ・ ・ ・ (2)
NeuAc〜Gal (6S) β1-4GlcNAc (6S) β1-3Gal (6S) β1-4GlcNAc (6S) ・ ・ ・ (3)
In the formula, Gal is a galactose residue, GlcNAc is an N-acetylglucosamine residue, NeuAc is an N-acetylneuraminic acid residue, β1-4 is a β1,4 glycosidic bond, β1-3 is β1,3 A glycoside bond, (6S) represents a 6-O-sulfate ester, and ˜ represents an α2,3 glycoside bond or an α2,6 glycoside bond.

  The sugar chains of the above formulas (1) to (3) can be obtained by degrading keratan polysulfate from cartilaginous fish such as sharks with endo-β-N-acetylglucosaminidase-type keratan sulfate degrading enzyme. Specifically, it can be produced by the method described in WO96 / 16973 or WO96 / 16166.

  The hair growth agent of the present invention contains one or more substances selected from these substances. The most preferable of these substances is hyaluronic acid having a weight average molecular weight of 200,000 to 400,000. The concentration of these substances in the hair growth agent of the present invention is not particularly limited, but is usually preferably about 0.01 to 5% (W / V), preferably 0.1 to 3% (W / V) per substance. Is more preferable.

(2) Substance group (B)
(2-1) Sasa Extract The Sasa extract used in the hair growth agent of the present invention is not particularly limited as long as it is a Sasa extract, but it is preferable to use a Kumazasa extract. The solvent used for the extraction is not particularly limited. For example, one selected from water, alcohols such as ethanol and methanol, ketones such as acetone and methyl ethyl ketone, ethers such as diethyl ether and tetrahydrofuran, halogenated hydrocarbons such as dichloromethane and chloroform, and nitriles such as acetonitrile, or Two or more solvents are exemplified. According to these solvents, components desired for the hair growth agent of the present invention are extracted from Sasa. Among them, alcohols are preferable and ethanol is particularly preferable from the viewpoint of the toxicity of the solvent.

  The extraction conditions are not particularly limited, and can be easily selected by those skilled in the art in consideration of the desired extract concentration, operability, and the like.

  Sasa extract contains various substances, but the main components are copper chlorofin sodium, crude fibers (lignin, etc.) and the like.

(2-2) Tabuchi carrot extract The Tabuchi carrot extract used for the hair growth agent of this invention will not be specifically limited if it is an extract of Tabuchi carrot. The solvent used for the extraction is not particularly limited, for example, water, alcohols such as ethanol and methanol, ketones such as acetone and methyl ethyl ketone, ethers such as diethyl ether and tetrahydrofuran, halogenated hydrocarbons such as dichloromethane and chloroform, Examples thereof include one or more solvents selected from nitriles such as acetonitrile. According to these solvents, the components desired for the hair growth agent of the present invention are extracted from the carrot, but water or alcohol is preferable among them from the viewpoint of toxicity of the solvent, and water or ethanol is particularly preferable. .

  The extraction conditions are not particularly limited, and can be easily selected by those skilled in the art in consideration of the desired extract concentration, operability, and the like.

  Tabuchi carrot extract contains various substances, but the main component is saponin, and it contains other proteins, neutral sugars and the like.

(2-3) Keratan sulfate In the components used in the hair growth agent of the present invention, the keratan sulfate included in the substance group (B) is the low molecular weight keratan sulfate included in the substance group (A). Unlike, it is a polymeric keratan sulfate having a higher molecular weight. The molecular weight is not particularly limited, but the weight average molecular weight is preferably about 5000 to 15000, more preferably about 5000 to 10,000.

  The origin of the keratan sulfate included in this substance group (B) is not particularly limited, and keratan sulfate separated and purified from tissues such as sharks, whales, cows and pigs can be used. preferable. The keratan sulfate is preferably keratan polysulfate.

  Separation / purification of keratan sulfate includes glycosamino, such as crushing, extraction, enzymatic degradation (for example, degradation by protease), precipitation with organic solvent (for example, alcohol), salting out, salt solution, various chromatographies, or combinations thereof. It can be performed by a method usually used for separation and purification of glycans.

  The keratan sulfate may be a pharmaceutically acceptable salt thereof. Examples of pharmaceutically acceptable salts of keratan sulfate include salts with inorganic bases such as alkali metal salts (sodium salt, lithium salt, potassium salt, etc.), alkaline earth metal salts, ammonium salts, or diethanolamine salts, A salt with an organic base such as a cyclohexylamine salt or an amino acid salt can be used. Of these, sodium salts are preferred.

(2-4) Chondroitin sulfate The type of chondroitin sulfate used in the hair growth agent of the present invention is not particularly limited, and any of chondroitin sulfate A and chondroitin sulfate C may be used except for chondroitin sulfate B (dermatan sulfate).

  The origin of chondroitin sulfate used in the hair growth agent of the present invention is not particularly limited, and chondroitin sulfate separated and purified from tissues such as shark, cow, pig, and whale can be used. Preferably, those derived from sharks are more preferable.

  Chondroitin sulfate separation / purification includes glycosamino, such as crushing, extraction, enzymatic degradation (for example, degradation by protease), precipitation with organic solvent (for example, alcohol), salting out, salt solution, various chromatographies, or combinations thereof. It can be performed by a method usually used for separation and purification of glycans.

  The chondroitin sulfate may be a pharmaceutically acceptable salt thereof. Examples of pharmaceutically acceptable salts of chondroitin sulfate include salts with inorganic bases such as alkali metal salts (sodium salts, lithium salts, potassium salts, etc.), alkaline earth metal salts, ammonium salts, or diethanolamine salts, Among salts with organic bases such as cyclohexylamine salts and amino acid salts, pharmaceutically acceptable salts can be used. Of these, a sodium salt is preferable.

  Although the molecular weight of this chondroitin sulfate is not particularly limited, it is preferably about 5000 to 50000, more preferably about 5000 to 25000.

  In the hair growth agent of this invention, it is preferable to mix | blend 1 type, or 2 or more types of substances chosen from said (2-1)-(2-4). The concentration in the hair growth agent of the present invention is not particularly limited, but is preferably about 0.01 to 5% (W / V), more preferably 0.1 to 3% (W / V) per one kind of substance. . However, the amount of dry carrot extract is about dry weight. As for the extract of Sasa, it is preferably about 0.01 to 5% (V / V), more preferably 0.1 to 3% (V / V) as an extract immediately before the components are precipitated when it is concentrated. Degree.

(3) Components that can be added as needed The hair growth agent of the present invention does not adversely affect the skin, hair, etc., and does not adversely affect the above-described components in the hair growth agent of the present invention. Insofar as possible, substances other than those described above may be added. Examples of such substances include hydrocarbons, waxes, fats and oils, esters, higher fatty acids, higher alcohols, surfactants, fragrances, pigments, preservatives, and the like used in normal hair cosmetics. Examples include antioxidants, ultraviolet absorbers, alcohols, pH adjusters, various medicinal ingredients, and one or more substances selected from these can be appropriately selected and blended. Of course, hair growth and hair growth components other than the hair growth agent of the present invention may be further blended.

  Examples of such substances include ethanol, glycerin, olive oil, l-menthol, panthenol, vitamin E derivatives, minoxidil and the like.

<2> Providing Form and Manufacturing Method of Hair Growth Agent of the Present Invention The hair growth agent of the present invention can provide one or more substances selected from the substance group (A) as they are. It is also possible to provide in the form of a mixture of one or two substances selected from the above-mentioned substance group (B), and also in the form of a mixture obtained by further mixing these and other components that can be added as necessary. it can. The form of provision is not particularly limited, such as a solution state, a frozen state, a lyophilized state, or the like. The hair growth agent of the present invention can be produced by a method known per se according to these forms of provision.

  For example, when the hair growth agent of the present invention is provided in a solution state, one or more substances selected from the substance group (A) are not adversely affected on the skin, hair, etc. The above-mentioned components in the hair growth agent of the present invention are not adversely affected, and the components in the above-described hair growth agent of the present invention are dissolved or suspended in a solvent that can be dissolved or suspended. it can. At this time, it is preferable to further dissolve or suspend one or more substances selected from the above-mentioned substance group (B) and other components that can be added as necessary. Examples of such a solvent include water (distilled water), but are not limited thereto.

  In the hair growth agent of this invention, the preferable component or combination of components which shows the remarkable hair growth effect is illustrated below.

  Here, hyaluronic acid having a weight average molecular weight of 200,000 to 400,000 is “HA (200,000 to 400,000)”, and dermatan sulfate having a weight average molecular weight of 20,000 to 50,000 is “DS (20,000 to 50,000)”. The low molecular weight keratan sulfate is abbreviated as “LMKS”, the Sasa extract as “Sasa”, the Tanachi carrot extract as “Tanachi”, the keratan sulfate as “KS”, and the chondroitin sulfate as “CS”.

(a) HA (200,000 to 400,000) + DS (20,000 to 50,000)
(b) HA (200,000 to 400,000) + Tabanachi
(c) HA (200,000 to 400,000) + Sasa
(d) HA (200,000 to 400,000) + DS (20,000 to 50,000) + Tabuchi + Sasa
(e) HA (200,000 to 400,000) + DS (20,000 to 50,000) + KS + Tabuchi + Sasa + CS
(f) LMKS

  The components of the combinations (a) to (f) preferably further contain ethanol and glycerin, and in addition, one or more selected from olive oil, panthenol and vitamin E derivatives. It is preferable to contain a substance.

  Preferred concentrations of these substances in the hair growth agent of the present invention are about 50-60% for ethanol, about 5% for glycerin, about 5% for olive oil, about 1% for panthenol, and 0.05% for vitamin E derivatives. However, it is not limited to these.

  Moreover, the more preferable component or combination of components in the hair growth agent of this invention is shown below.

(a) HA (200,000 to 400,000) + DS (20,000 to 50,000)
(c) HA (200,000 to 400,000) + Sasa
(d) HA (200,000 to 400,000) + DS (20,000 to 50,000) + Tabuchi + Sasa
(e) HA (200,000 to 400,000) + DS (20,000 to 50,000) + KS + Tabuchi + Sasa + CS
(f) LMKS

Among these components or combinations of components, the following combinations of components are particularly preferable because they exhibit a hair growth effect over a wide area with a small application area.
(d) HA (200,000 to 400,000) + DS (20,000 to 50,000) + Tabuchi + Sasa
(e) HA (200,000 to 400,000) + DS (20,000 to 50,000) + KS + Tabuchi + Sasa + CS

  The dosage form of the hair growth agent of the present invention is not particularly limited as long as it can be expected to act on the hair. Hair tonic, shampoo, rinse, pomade, hair lotion, hair cream, hair liquid, hair treatment, hair Conditioners, hair gels, hair mists, hair foams and the like are exemplified, and any of them can be produced by a known method.

<3> Use of the hair growth agent of the present invention The application target of the hair growth agent of the present invention is not particularly limited as long as it is a person who desires hair growth or a person who desires prevention of hair loss. Examples are those who suffer from hair loss, thin hair, and hairlessness on the whole or the head.

  Moreover, the usage amount, usage frequency, and the like of the hair growth agent of the present invention are not particularly limited, and are appropriately determined depending on the degree of hair removal or the like of the person to whom the hair growth agent of the present invention is applied and the dosage form of the hair growth agent of the present invention. The amount used can be about 50 to 500 mg / person / time as the total weight of the substance groups (A) and (B) in the hair growth agent of the present invention. Moreover, as a use frequency, the frequency of the grade which is used once a day for every 3 days from 1 to 3 times a day is illustrated.

  In addition, as shown in the examples described later, before applying the hair growth agent of the present invention, the hair growth effect of the hair growth agent of the present invention is obtained by treating the skin desired for hair growth with hyaluronidase. It has been found that a higher hair growth effect can be obtained.

  Therefore, as described above, the present invention provides a method for promoting hair growth comprising treating the skin desired for hair growth with hyaluronidase and then applying the hair growth agent of the present invention to the skin treated with hyaluronidase. .

  The hyaluronidase used in the above method is not particularly limited as long as it is an enzyme that depolymerizes hyaluronic acid. It includes any of hyaluronoglucosaminidase, hyaluronic acid lyase and the like. The origin of these hyaluronidases is not particularly limited, and for example, those produced by microorganisms such as Streptococcus dysgalactiae, those derived from sheep testicles, those derived from medicinal leeches can be used.

  The treatment with hyaluronidase can be performed by applying a hyaluronidase solution to the skin to be treated. The amount of hyaluronidase used for the treatment with hyaluronidase is not particularly limited, but is usually about 0.001 to 1 U, preferably about 0.01 to 0.2 U. Such a level of hyaluronidase may be applied to the skin at one time or divided into several times.

  Thereafter, the hair growth agent of the present invention is applied. In this case, the hair growth agent of the present invention can be applied in accordance with the application of the hair growth agent of the present invention described above. The time until the hair growth agent of the present invention is applied after the above hyaluronidase treatment is not particularly limited, but is usually applied after about 6 hours to 3 days, preferably about 12 hours to 1 day.

  The present invention also provides a hair growth effect enhancer for the hair growth agent of the present invention, comprising hyaluronidase as an active ingredient for use in the above method, and a hair growth comprising the hair growth agent of the present invention and hyaluronidase. Provide promotional kits.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this Example shows an example of this invention and is not limited to this.

(1) Materials The materials commonly used in the examples will be described below.
(1-1) Hyaluronic acid Hyaluronic acid (weight-average molecular weight 800,000) extracted and purified from chicken caps was autoclaved at 121 ° C. for 60 minutes to produce hyaluronic acid with a weight-average molecular weight of 300,000 (hereinafter referred to as this hyaluronic acid). The acid is called "HA (300,000)").

  Further, hyaluronic acid having a weight average molecular weight of 800,000 produced as described above was subjected to alkali hydrolysis using sodium hydroxide to produce hyaluronic acid having a weight average molecular weight of 50,000 (hereinafter, this hyaluronic acid was referred to as “HA (50,000)”. ")

(1-2) Dermatan sulfate Dermatan sulfate was produced by the method described in the above “Best Mode for Carrying Out the Invention”. The weight average molecular weight of this dermatan sulfate was 35,000 (hereinafter, this dermatan sulfate is referred to as “DS (35,000)”)

(1-3) Low molecular weight keratan sulfate Low molecular weight keratan sulfate is obtained by digesting keratan polysulfate extracted and purified from shark cartilage at 37 ° C. for 12 hours using keratanase II (Seikagaku Corporation). Manufactured. This low molecular weight keratan sulfate contained sugar chains represented by the following formulas (1) to (3).
Gal (6S) β1-4GlcNAc (6S) (1)
Gal (6S) β1-4GlcNAc (6S) β1-3Gal (6S) β1-4GlcNAc (6S) (2)
NeuAc〜Gal (6S) β1-4GlcNAc (6S) β1-3Gal (6S) β1-4GlcNAc (6S) ・ ・ ・ (3)

  In the formula, Gal is a galactose residue, GlcNAc is an N-acetylglucosamine residue, NeuAc is an N-acetylneuraminic acid residue, β1-4 is a β1,4 glycosidic bond, β1-3 is β1,3 A glycoside bond, (6S) represents a 6-O-sulfate ester, and ˜ represents an α2,3 glycoside bond or an α2,6 glycoside bond (hereinafter, this low molecular weight keratan sulfate is referred to as “LMKPS”).

(1-4) Keratan sulfate Keratan sulfate was produced by extraction and purification from shark cartilage. This keratan sulfate was keratan polysulfate and had a weight average molecular weight of 6,000 (hereinafter, this keratan sulfate is referred to as “KPS”).

(1-5) Tabuchi carrot water extract Tabuchi carrot water extract was prepared by crushing tabuchi carrot, adding water to this, stirring at room temperature for 5 hours, filtering and drying ( Hereinafter, this extract is referred to as “Nanachi Water Extract”).

(1-6) Tabuchi carrot ethanol extract Tabuchi carrot ethanol extract was produced in the same manner as the tabuchi water extract, except that ethanol was used instead of water (hereinafter this extract was Seven Et extracts ”).

(1-7) Kumazasa Ethanol Extract Kumazasa Ethanol Extract was extracted using a evaporator after pulverizing Kumazasa, adding a large excess of ethanol and stirring at room temperature for 1 hour, filtering this extract, and It was manufactured by concentrating until the components were precipitated in the extract (hereinafter, this extract is referred to as “Kumazasa Et extract”).

(1-8) Chondroitin sulfate Chondroitin sulfate was produced by extraction and purification from shark cartilage. The weight average molecular weight of this chondroitin sulfate was 30,000 (hereinafter, this chondroitin sulfate is referred to as “CS”).

(2) Hair growth experiment in mice
(2-1) Preparation of test solution A test solution was prepared according to the following formulation.
・ Sample (Substances used will be described in detail in Experiments 1 to 4 below. The final concentration in the test solution is 1% (W / V) unless otherwise specified)
・ Olive oil 5% (2.5mL)
・ Ethanol 60% (30mL)
・ Glycerin 5% (2.5mL)
Add purified water to 50 mL.

(2-2) Method of hair growth experiment The experiment was conducted according to the hair growth effect determination test using mice by Ogawa et al. (Fragrance Journal, 17 (5), 20 (1989)). That is, 6 C3H / HeN male mice (7 weeks old) were prepared for each group and preliminarily raised for 7 days. Thereafter, the back was shaved using an electric eyebrow shave and an electric razor. From the day after shaving, the test solution was applied to the shaved site (about 2 cm × about 5 cm) at a daily rate of 0.2 mL / animal. On the determination day, the shaved part of the back of each individual was photographed with a digital camera, and the hair growth area was visually evaluated based on this image, and the hair growth score for each individual was determined according to the determination method shown in Table 1 below. Was attached. The hair growth area was visually evaluated by two judges in consideration of objectivity.

(2-2-1) Hair growth experiment 1
In accordance with the above, hair growth experiment 1 was conducted from spring to early summer. The test solutions used are as follows.

・ Test solution 1: HA (300,000) (final concentration: 0.75% (W / V))
・ Test solution 2: HA (50,000) (final concentration: 0.75% (W / V))
・ Control: Sample-free solution (base only)
The results of the average hair growth score on the 17th day (determination day) after application are shown in Table 2. The ratio when the average hair growth score of the control was 100% was also calculated and is shown in Table 2.

  In this experiment, no skin or hair abnormalities were observed after administration of the test solution.

  As shown in Table 2, HA (50,000) was less effective than the control, whereas HA (300,000) was significantly higher than the control. Therefore, in the following experiment, HA (300,000) was used as HA.

(2-2-2) Hair growth experiment 2
Similarly, hair growth experiment 2 was conducted in the summer. The test solutions used are as follows.

・ Test solution 3: DS (35,000)
・ Test solution 4: Tanachi water extract ・ Test solution 5: Kumazasa Et extract (final concentration 1% (V / V))
・ Test liquid 6: LMKPS
・ Control: Sample-free solution (base only)

  Table 3 shows the results of the average hair growth score on the 18th day (determination date) after application. The ratio when the average hair growth score of the control was 100% was also calculated and is shown in Table 3.

  In this experiment, no skin or hair abnormalities were observed after administration of the test solution.

  As shown in Table 3, LMKPS showed a significantly higher hair growth effect than the control.

(2-2-3) Hair growth experiment 3
Hair growth experiment 3 was conducted from spring to early summer. The test solutions used are as follows.

・ Test solution 7: HA (300,000) (final concentration 0.75%) + Tabana Et extract ・ Test solution 8: HA (300,000) (final concentration 0.75%) + Tabana water extract ・ Test solution 9: HA (300,000) (final concentration 0.75%) + Kumazasa Et extract (final concentration 1% (V / V))
・ Test solution 10: HA (300,000) (final concentration 0.75%) + DS (35,000)
・ Test solution 11: HA (300,000) (final concentration: 0.75%) + KPS
・ Control: Sample-free solution (base only)

  Moreover, the test which apply | coats the test liquid 7 was done one day after apply | coating the hyaluronidase (hyaluronidase SD, Seikagaku Corporation, 0.5 U) which is a hyaluronic acid lyase.

  The results of the average hair growth score on the 17th day (determination day) after application are shown in Table 4. The ratio when the average hair growth score of the control was 100% was also calculated and is shown in Table 4 together.

  In this experiment, no skin or hair abnormalities were observed after administration of the test solution.

  As shown in Table 4, when Kumazasa Et extract or DS (35,000) was added in addition to HA (300,000), a markedly higher hair growth effect was seen compared to the control. Further, when the hyaluronidase treatment was performed before the test solution 7 was applied, the hair growth effect was further increased as compared with the case where the test solution 7 alone was used. As a cause of the increase in hair growth effect by this hyaluronidase treatment, hyaluronic acid present in the skin is decomposed by the action of hyaluronidase, which is caused by the action of hyaluronidase, which exerts the hair growth effect. There is a possibility that absorption of an active ingredient applied later is improved.

(2-2-4) Hair growth experiment 4
Hair growth experiment 4 was conducted in the summer. In hair growth experiment 4, the left and right sides (two places) of the back of the mouse were shaved to approximately the same area (about 2 cm × about 5 cm per place). From the day after shaving, the test solution is applied to the shaved site on the right side of the back 0.2 mL / animal daily, and the left and right shaved parts of each individual are photographed with a digital camera on the 21st day (judgment day) after application. Then, a hair growth score was given by the above-described method based on this image.

  The test solutions used are as follows.

・ Test solution 12: HA (300,000) + DS (35,000) + KPS + CS
・ Test solution 13: HA (300,000) + DS (35,000) + Tanashi water extract + Kumazasa Et extract (final concentration 1% (V / V))
・ Test solution 14: HA (300,000) + DS (35,000) + KPS + Tabanamizu extract + Kumazasa Et extract (final concentration 1% (V / V)) + CS
・ Control: Sample-free solution (base only)

  Table 5 shows the results of the average hair growth score for the right back (application surface) and the left back (non-application surface). The ratio when the average hair growth score of the control was 100% was also calculated and is shown in Table 5 together.

  In this experiment, no skin or hair abnormalities were observed after administration of the test solution.

  From Table 5, it was shown that the hair growth effect was further increased by adding the above components in addition to HA (300,000) and DS (35,000).

  Surprisingly, the test solution 13 (HA (300,000) + DS (35,000) + Tanamizu extract + Kumazasa Et extract) and the test solution 14 (HA (300,000) + DS (35,000) + KPS + In Tanashi Water Extract + Kumazasa Et Extract + CS), a remarkable hair growth promoting effect was seen not only on the coated surface but also on the non-coated surface. From this, it was suggested that the hair growth agent of the present invention has a hair growth effect over a wide area with a small application area. In addition, since this effect is presumed to be an effect obtained by absorbing the test solution into the body, it suggests that the active ingredient of the hair growth agent of the present invention may be effective even by internal use. Is.

(3) Hair growth experiments in humans
(3-1) Preparation of test solution A test solution was prepared according to the following formulation.

・ HA (300,000), DS (35,000), KPS, CS, Tanashi Water Extract and Kumazasa Et Extract 1% each (W / V) (However, Kumazasa Et Extract 1% (V / V) )
・ Panthenol 1%
・ Vitamin E derivative 0.05%
・ Ethanol 50%
Add purified water to 100%.

(3-2) Hair growth experiment Fourteen people who wanted hair growth or hair growth were massaged once a day with 2 to 3 mL of the test solution applied to their scalp after washing their hair. After continuing this for 6 to 7 months, the answers to the following questions were obtained.

  The contents and results of the questions (number of respondents, answer contents) are shown below.

  (Contents of questions) Regarding the results of actual use of the test solution, answer the corresponding number for each item below.

Item 1. About hair restoration
1) Hair increased (6 people)
2) No change (8 people)
3) Hair decreased (0 people)
Item 2. About hair loss
1) Fewer hair loss (10 people)
2) No change (4 people)
3) Hair loss increased (0 people)
Item 3. About Dandruff
1) Decreased (7 people)
2) No change (7 people)
3) Increased (0)

Item 4. Can be used instead of opinions and hair styling about the feeling of use (can be set, not disturbed).

・ You may feel medicinal.

・ No particular discomfort. Stickiness promotes the image of the effect and gives you a sense of expectation for hair growth.

Item 5. Other noticed points / hair became thicker.

・ It was said that the hair growth diagnosis by the barber improved (the area around the hair root was very clean).

  As a result of using the test solution, no one complained of an abnormality on the scalp or hair.

  From this result, it was confirmed that the hair growth agent of the present invention not only has a hair growth effect but also has a hair growth effect and a scalp drying prevention effect. In addition, since no abnormality was observed on the scalp and hair, it was shown that the hair growth agent of the present invention is very safe.

Claims (2)

  A hair growth promoter comprising dermatan sulfate having a weight average molecular weight of 35,000 and hyaluronic acid having a weight average molecular weight of 300,000 as active ingredients. The hair growth-promoting agent according to claim 1, comprising the component according to any of the following (a) or (b):
(a) Tabuchi carrot extract and Sasa extract, (b) Keratan sulfate having a weight average molecular weight of 5000 to 15,000, Tabuchi carrot extract, Sasa extract, and chondroitin sulfate.