JP5847975B1 - FGF-7 production promoter and hair papilla cell proliferation promoter - Google Patents

Abstract

[PROBLEMS] To provide a new FGF-7 production promoter that is excellent in FGF-7 production promoting effect and highly safe against scalp. An FGF-7 production promoter contains a proteoglycan. [Selection figure] None

Description

  The present invention relates to an FGF-7 production promoter and a hair papilla cell proliferation promoter that increase the amount of FGF-7 production and promote the growth of hair papilla cells.

  Recently, an increasing number of men and women suffer from hair loss symptoms such as hair loss and thinning of the head. In order to improve such symptoms, research on the mechanism of hair growth and hair cycle has been carried out, and the development of hair-growth ingredients that promote hair growth and suppress hair loss has been promoted. At present, typical hair growth ingredients include minoxidil (see Patent Document 1) and adenosine (see Patent Document 2). In developing such hair-growth components, FGF-7 (fibroblast growth factor-7), which is a factor involved in the hair growth mechanism, has attracted attention in recent years. This FGF-7 is one member of the FGF (fibroblast growth factor) family, and is known to be produced in hair papilla cells and act on hair matrix cells to promote hair growth. Therefore, hair growth promotion by increasing the production amount of FGF-7 in hair papilla cells is expected. According to Patent Document 2, it is disclosed that the above-mentioned adenosine enhances the expression of FGF-7 in hair papilla cells and maintains and promotes hair thickening.

  On the other hand, proteoglycan is a kind of glycoprotein in which a sugar chain (glycosaminoglycan) is bound to a protein. Proteoglycans are abundantly contained in cartilage tissue as one of the main components of the extracellular matrix of animals, and are said to have functions such as water retention, bone formation, joint lubrication, and intercellular communication. For this reason, in recent years, research and development on the functions of proteoglycans have been promoted. For example, Patent Document 3 describes that proteoglycan has a TNF-α production inhibitory action, an IFN-γ production inhibitory action, an IL-10 production promoting action, and the like.

Japanese Patent No. 2733980 International Publication No. 2005/44205 JP 2007-131548 A

  However, it has been reported that the hair-growth ingredient described in Patent Document 1 causes itching and rash on the scalp as a side effect. Moreover, although the component which has the effect | action which raises the FGF-7 production amount is proposed variously including the component described in patent document 2, the component which can further raise the FGF-7 production amount in a hair papilla cell. Was desired.

  On the other hand, although proteoglycan has a specific function as described in Patent Document 3, there is no report on a function relating to hair papilla cells, and its effectiveness is unknown.

  The present invention has been devised in view of the above points, and an object thereof is to provide a new FGF-7 production promoter that is excellent in FGF-7 production promoting effect and highly safe against the scalp.

  As a result of intensive studies in view of such circumstances, the present inventors have found that proteoglycan has a very excellent FGF-7 production-promoting action in dermal papilla cells, and further has an action to proliferate dermal papilla cells themselves. The present invention was completed.

  In order to solve the above problems, the FGF-7 production promoter of the present invention is characterized by containing a proteoglycan. Thereby, the amount of FGF-7 production in the hair papilla cell can be remarkably increased. Proteoglycans are naturally derived and are widely used in foods and pharmaceuticals, and thus have high safety.

  In the FGF-7 production promoter of the present invention, the proteoglycan preferably has a molecular weight of 1 million daltons or more. Thereby, the molecular weight of proteoglycan that shows stable activity and significantly increases the amount of FGF-7 production in the hair papilla cells is selected.

  In the FGF-7 production promoter of the present invention, the proteoglycan is also preferably chondroitin sulfate proteoglycan. Thereby, a suitable type of proteoglycan is selected.

  In the FGF-7 production promoter of the present invention, it is also preferable that the proteoglycan is obtained from fish cartilage tissue. Thereby, a suitable raw material of proteoglycan having FGF-7 production promoting activity is selected.

  In the FGF-7 production promoter of the present invention, it is also preferred that the fish cartilage tissue is salmon nasal cartilage. Thereby, the raw material which contains many proteoglycans which have FGF-7 production promotion activity and can be obtained efficiently is selected.

  Furthermore, the dermal papilla cell proliferation promoter of the present invention contains a proteoglycan. Thereby, hair papilla cells can be remarkably increased. Proteoglycans are naturally derived and are widely used in foods and pharmaceuticals, and thus have high safety.

According to the present invention, an FGF-7 production promoter and hair papilla cell growth promoter having the following excellent effects can be obtained.
(1) A highly safe FGF-7 production promoter derived from a natural product having an excellent FGF-7 production promoting action can be obtained.
(2) A stable effect can be obtained by selecting a proteoglycan of 1 million daltons or more.
(3) A hair dermal papilla cell proliferation promoter having an excellent dermal papilla cell proliferation promoting action and a high safety derived from a natural product can be obtained.
(4) An FGF-7 production promoter and a hair papilla cell proliferation promoter that promotes the proliferation of dermal papilla cells and promotes the amount of FGF-7 produced by each of the grown dermal papilla cells.

2 is a graph showing the relative number of viable dermal papilla cells on day 1 of culture in Example 1. FIG. 2 is a graph showing the relative viable cell number of dermal papilla cells on the third day of culture in Example 1. 2 is a graph showing the amount of FGF-7 produced on the first day of culture in Example 2. 2 is a graph showing the amount of FGF-7 produced on the third day of culture in Example 2. It is a graph which shows the relative viable cell number and relative FGF-7 production amount of the hair papilla cell of the culture | cultivation 1st day. It is a graph which shows the relative viable cell number and the relative amount of FGF-7 production of the dermal papilla cells on the third day of culture.

  Hereinafter, the present invention will be described in detail. The FGF-7 production promoter and hair papilla cell growth promoter of the present invention contain proteoglycan as an active ingredient. This proteoglycan is obtained from a biological tissue containing proteoglycan. Any biological tissue may be used as long as it contains proteoglycan, but from the viewpoint of containing proteoglycan in a large amount and being inexpensively available as a site usually discarded in the field of food processing, fish, mollusks, birds or mammals. Preferred are cartilage tissues or fish, mollusks, birds or mammal skins. For example, salmon nasal cartilage (ice head), ray cartilage or shark cartilage, avian cartilage tissue as chicken cartilage or knee cartilage, mammalian cartilage tissue Is preferably bovine throat cartilage or bronchial cartilage or whale cartilage, and more preferably salmon nasal cartilage or ray cartilage, which is a fish cartilage tissue. The above-mentioned cartilage tissue refers to a tissue having hyaline cartilage, elastic cartilage or fibrocartilage, cartilage matrix, chondrocyte or perichondrium, and also includes a tissue around a cartilage.

  As a method for producing proteoglycan from a biological tissue, it is obtained by immersing the biological tissue in an aqueous solvent such as water, an acidic aqueous solution or a basic aqueous solution and extracting it into the solvent. As an example, JP 2012-201614 A Can be suitably produced by the method described in 1. above. This publication describes that chondroitin sulfate proteoglycan can be efficiently obtained from salmon, chicken, shark, ray cartilage and the like. Furthermore, it is also suitable by immersing biological tissues such as salmon, chicken, shark, and ray cartilage in a sodium hydroxide aqueous solution with a low concentration of 0.2% by weight or less and extracting it at room temperature of about 15 to 20 ° C. Can be manufactured. According to this method, chondroitin sulfate proteoglycans having a relatively large molecular weight of 1 to 1.4 million daltons are efficiently recovered. In addition, in order to make the molecular weight of the recovered proteoglycan within a suitable range, dialysis treatment, ultrafiltration treatment, or the like can be further performed. Since the molecular weight of proteoglycan is affected by the number of glycosaminoglycan (in this case, chondroitin sulfate) bound to the core protein, the glycosaminoglycan bound to proteoglycan is almost completely intact without being degraded. Is extracted and recovered, it is considered to exhibit a molecular weight of 1 to 1.4 million daltons. As a chondroitin sulfate proteoglycan produced based on this method, there is “Proteoglycan LS” (derived from salmon nasal cartilage, molecular weight 1 million to 1.4 million daltons), which is preferably used as the proteoglycan of the present invention.

  Moreover, the proteoglycan used in the present invention is not limited to those produced by the above production method, and proteoglycans obtained by a known production method can also be used. In addition, as a proteoglycan, a biological tissue itself containing proteoglycan can be used.

  The molecular weight of proteoglycan is preferably 1 million daltons or more, and particularly preferably 1.2 million daltons or more, from the viewpoint of stably obtaining excellent FGF-7 production promoting action and hair papillary cell proliferation promoting action. preferable. Since proteoglycans are obtained from biological materials such as biological cartilage tissue, the quality of the produced proteoglycans may vary depending on the production method and the state of the biological tissue. The present inventors have found that a relatively small proteoglycan having a molecular weight of 500,000 daltons or less, that is, a proteoglycan in which the amount of glycosaminoglycan bound is small or the core protein itself has been degraded has an FGF-7 production promoting action and It has been confirmed that the dermal papilla cell proliferation promoting action is reduced. Therefore, the molecular weight of proteoglycan can be used as a criterion for ensuring excellent FGF-7 production promoting action and hair papillary cell proliferation promoting action. In addition, it is preferable that the molecular weight of proteoglycan is determined by chromatography, and particularly determined by gel filtration chromatography.

  The FGF-7 production promoter of the present invention increases the secretion amount of FGF-7 in hair papilla cells, activates hair matrix cells, and promotes hair growth. Further, the hair papilla cell proliferation promoter of the present invention activates hair papilla cells, promotes the proliferation of hair papilla cells, and promotes hair growth. These FGF-7 production promoters and hair papilla cell proliferation promoters can be used on the scalp and hair as hair growth agents, hair nourishing agents, and hair growth promoters, and can be used to improve or prevent hair loss symptoms such as hair loss and thinning hair. Moreover, in the range which does not lose the effect of this invention, it is also possible to use it as a compounding agent or a combination agent in combination with another active ingredient. Furthermore, it is also possible to apply these FGF-7 production promoter and hair papilla cell proliferation promoter to scalp preparations, hair cosmetics, and the like. Scalp preparations and hair cosmetics include creams, emulsions, ointments, gels, powders, scalp care products, shampoos, conditioners, tonics, hair dyes, lotions, styling agents and the like.

  The proteoglycan contained in the composition of the present invention is a component obtained from natural biomaterials as described above, and is widely used in foods such as supplements and cosmetics. Therefore, since it is highly safe and has little irritation to the skin, it can be used with confidence. In addition, since proteoglycan is water-soluble, it is not necessary to use an organic solvent as a solvent. Even when the FGF-7 production promoter and hair papilla cell growth promoter of the present invention are used as liquids, water is used as a solvent. A hypoallergenic prescription can be realized.

  The preferable blending amount of proteoglycan in the FGF-7 production promoter of the present invention is not particularly limited because it varies depending on the target action effect, administration method, administration subject and dosage form, but 0.01 to 10 in the dosage form. % By weight is preferred, 0.1 to 5% by weight is more preferred, and 0.1 to 3% by weight is particularly preferred. Moreover, since the preferable compounding quantity of the proteoglycan in a dermal papilla cell growth promoter changes with target effect, administration method, administration object, and dosage form, it is not specifically limited, 0.001-3 weight in a dosage form % Is preferable, 0.01 to 1% by weight is more preferable, and 0.01 to 0.5% by weight is particularly preferable.

  Furthermore, the FGF-7 production promoter and dermal papilla cell growth promoter of the present invention are used as pharmaceuticals for parenteral administration (transdermal absorption agent) such as bop, tape, ointment, cream, lotion, suppository, etc. Is also possible. Moreover, it can also be set as oral preparations, such as a tablet, a granule, a capsule, or a liquid for internal use. Moreover, it is also possible to use the FGF-7 production promoter and hair papillary cell proliferation promoter of the present invention as a reagent for studying the FGF-7 production promoting effect and the hair papillary cell proliferation promoting effect.

  When formulating the FGF-7 production promoter and hair papilla cell proliferation promoter of the present invention as a hair restorer, cosmetics, or pharmaceuticals, they can be prepared in various forms by conventionally used methods. In this case, it can be formulated using additives that are accepted as additives for cosmetics and pharmaceuticals, such as carriers and excipients for normal formulations. In order to improve the bioavailability and stability of proteoglycan, a drug delivery system including a preparation technique such as microcapsule, micronized powder, inclusion using cyclodextrin and the like can also be used.

  EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited at all by these Examples.

[Example 1]
1. Hair papilla cell proliferation test Human hair papilla cells (white race, 22-year-old male, derived from the top of the head, product number: CA60205a, product of Toyobo Co., Ltd.) are 1.2 × 10 ⁴ cells / 0.3 mL per well. Inoculated into a 48-well culture plate (Product No. 677180, manufactured by Greiner Japan Co., Ltd.). In addition, the medium only for a hair papilla cell (Product number: TMTPGM-250, Toyobo Co., Ltd. product) was used for the culture medium. After culturing for one day in a CO ₂ incubator (5% CO ₂ , 37 ° C.) to make it 50% confluent, it was replaced with a medium containing the test substance shown in Table 1 below.

  Of the test substances in Table 1 above, proteoglycan was dissolved in a medium to a concentration of 10 mg / mL and sterilized by filter, and then diluted to each test concentration with a medium and adjusted. Minoxidil was dissolved in ethanol to make a 60 mM solution, and then added to the medium so that the final concentration was 60 μM. Adenosine was dissolved in DMSO to make a 25 mg / mL solution, sterilized by filter, and added to the medium so that the final concentration was 25 μg / mL. In addition, a test group (0.1% DMSO) consisting only of DMSO was provided as a solvent control for the adenosine solution. On the other hand, ethanol, which is the solvent of the minoxidil solution, has a concentration of 0.1% as a solvent, and generally has no effect on cells if the ethanol concentration is 0.1% or less. Was not provided.

  After replacing with the medium containing the test substance shown in Table 1 above, the culture was cultured for 1 day, and the culture supernatant was collected. Using this culture supernatant, the proliferation of each cell was measured with a living cell count reagent SF (product of Nacalai Tesque, Inc.). The measurement was performed at n = 5. Further, the culture supernatant cultured for 3 days after substituting the medium containing the test substance was also collected and the number of viable cells was measured. The collected culture supernatant was stored at −80 ° C. and used for measurement of FGF-7 production later. Specifically, the number of viable cells was measured as follows. After the cells were washed once with 300 μL of DPBS, 300 μL of a medium containing 10% of live cell measurement reagent SF was added per well. The absorbance of the formazan dye of WST-8 produced 30 minutes and 90 minutes after the addition was measured with a microplate reader at a measurement wavelength of 450 nm. The amount of change in absorbance per hour was calculated from the value of 90 minutes to 30 minutes, and the relative viable cell number was calculated as a relative value when the value of no test substance addition (control) was 100. In addition, a significant difference test was performed in the comparative test section. The test was performed as a student's T-test, and those with P <0.05 (the null hypothesis was less than 5%) were judged to be significant, and marked with * in the figure. The results on the first day of culture are shown in Table 2 and FIG. 1 below, and the results on the third day of culture are shown in Table 3 and FIG. 2 below. The “sample concentration” in the figure and table indicates the concentration of proteoglycan.

  From the results shown in Table 2 and FIG. 1, when the results of the number of viable cells on the first day of culture were observed, the number of viable dermal papilla cells was significantly increased in the test group having a proteoglycan concentration of 0.1 mg / mL or more. In addition, the rate of increase in the number of viable cells increased in a concentration-dependent manner as the proteoglycan concentration increased. For example, when the proteoglycan concentration in the medium was 10 mg / mL, the number of viable cells reached 206% of the test group without addition. Thus, it was found that proteoglycan has a very high cell growth promoting effect. Further, from the results of Table 3 and FIG. 2, the number of living cells on the third day of culture shows that the number of living hair papillary cells is significantly increased in the test group having a proteoglycan concentration of 1 mg / mL or more. In the test group of / mL concentration, the number of viable cells increased to about 173% of the test group without addition. From these results, it was found that proteoglycan has a very high dermal papilla cell growth promoting effect. Moreover, inhibition of cell proliferation was not observed in the whole concentration range where proteoglycan was added, and cytotoxicity due to proteoglycan was not observed. In addition, the result of the first day of culture is wider than that of the third day of culture, and the proteoglycan concentration range in which the number of viable cells is significantly increased is larger and the cell increase rate is higher. It was considered that the difference was reduced because the dermal papilla cells became confluent on the day and could not grow any more as a whole.

[Example 2]
2. FGF-7 Production Test Using the culture supernatant of each test group collected on Example 1 and Culture 3 collected in Example 1, the amount of FGF-7 contained in the culture supernatant was measured. The amount of FGF-7 was measured using FGF7 Human ELISA Kit (product number: ab100519, manufactured by Abcam Corporation). With respect to the obtained measured values, a significant difference test was performed in the comparative test section. The test was performed as a student's T-test, and those with P <0.05 (the null hypothesis was less than 5%) were judged to be significant, and marked with * in the figure. The results on the first day of culture are shown in Table 4 and FIG. 3 below, and the results on the third day of culture are shown in Table 5 and FIG. 4 below. The “sample concentration” in the figure and table indicates the concentration of proteoglycan.

  From Table 4 and FIG. 3, when the measurement result of the FGF-7 production amount on the first day of the culture is seen, in the test group in which the proteoglycan concentration is 0.01 to 1 mg / mL, the significant FGF-7 production amount is There was an increase. Moreover, from Table 5 and FIG. 4, on the third day of culture, the FGF-7 production amount significantly increased in the test group in which the proteoglycan concentration ranged from 0.1 mg / mL to 1 mg / mL. On both the first and third days of culture, the test group with a proteoglycan concentration of 1 mg / mL showed a very high increase in FGF-7 production, which was about 6 times that of the test group without addition (culture The production amount increased about 14.6 times (on the first day) and on the third day of culture. Thus, it was found that proteoglycan has a very high FGF-7 production promoting effect.

  Furthermore, the relative FGF-7 production amount was calculated as a relative value with respect to the measured value of the FGF-7 production amount on the first day of culture shown in Table 4 when the value of no addition (control) was 100. FIG. 5 shows a comparison between the relative FGF-7 production amount and the relative viable cell number shown in Table 2. Similarly, relative FGF-7 production was calculated as a relative value when the value of no addition (control) was set to 100 for the measured value of FGF-7 production on the third day of culture shown in Table 5. FIG. 6 shows a comparison between this relative FGF-7 production amount and the relative viable cell number shown in Table 3. According to these results, in the test group to which adenosine was added, the increase rate of FGF-7 production was almost the same as the cell growth rate, and the increase in FGF-7 production by adenosine was due to the increase in the number of cells. It is considered a thing. On the other hand, in the test group to which proteoglycan was added, the increase rate of FGF-7 production was larger than the cell growth rate in the concentration range of 0.01 to 1 mg / mL, and particularly 0.1 to 1 mg / mL. In the concentration range, it was found that the increase rate of FGF-7 production was significantly greater than the cell growth rate.

  The embodiments described above are all illustrative and do not limit the present invention, and the present invention can be implemented in various other variations and modifications. Therefore, the scope of the present invention is defined only by the claims and their equivalents.

  Since the FGF-7 production promoter and hair papilla cell proliferation promoter according to the present invention have excellent FGF-7 production promotion effect and hair papilla cell proliferation effect, the pharmaceutical, research reagent, hair restorer, scalp preparation, It is widely useful in industries such as hair cosmetics and foods (including supplements, health foods, functional foods, foods for specified health use, etc.).

Claims (1)

An agent for promoting FGF-7 production of dermal papilla cells, comprising proteoglycan derived from vomeronasal cartilage having a molecular weight of 1 million to 1.4 million daltons .

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