JP6829636B2 - Anti-gray hair agent - Google Patents

Description

The present invention relates to an anti-white hair agent. It also relates to an activity promoter of MITF-M (Microphthalmia-Associated Transcription Factor type M) in melanocytes.

The occurrence of gray hair is a typical old symptom and has a great influence on the impression of appearance. Many consumers who want to be youthful respond to graying by dyeing with hair dyes and hair dyes. However, these hair dyeing methods have various problems such as time-consuming work, possibility of adversely affecting the hair and scalp, and difficulty in dyeing in a natural color. Therefore, there is a demand for the development of an anti-whitening method (for example, a method for preventing graying and a method for improving the hair) that does not rely on a hair dye or a hair dye.

The melanin pigment that determines the color of hair is a pigment component produced by melanocytes. Melanocytes are present in the hair bulb adjacent to the hair matrix cells, and when the hair matrix cells divide to produce hair, the melanin pigment is delivered from the melanocytes and taken into the hair. .. Therefore, if melanin synthesis in melanocytes is not performed normally, the hair cannot take up melanin, resulting in gray hair.

MITF-M plays an important role in melanin synthesis in melanocytes (Non-Patent Document 1). MITF-M is known to control transcription of tyrosinase, which is an enzyme involved in melanocyte synthesis, and its related proteins, TRP-1 (Tyrosinase protected protein-1) and TRP-2 (Tyrosinase protected protein-2). ing. In addition to the transcriptional regulation of the above-mentioned proteins, MITF-M is known to be involved in melanocyte differentiation induction, migration, and anti-apoptosis.

As mentioned above, research targeting the mechanism of graying hair is being actively conducted. However, these findings have not led to the discovery of a drastic solution for anti-white hair.

Emi K. Nishimura et al, Science, Vol. 307, Issue5710, February 4, 2005, p. 720-724

An object of the present invention is to provide an anti-whitening agent that targets the mechanism of graying hair. Another object of the present invention is to provide an activity promoter (MITF-M activity promoter) that activates MITF-M, which is known to play an important role in melanin synthesis.

In view of the above circumstances, the present inventor has focused on the mechanism of graying hair and conducted intensive studies. As a result, the Bergenia ligratha extract is a cell membrane receptor for melanocytes (pigment-forming cells), TGFBR2 (Transforming growth factor). We have found that it promotes the activity of beta receptor II), EDNRB (Endothelin receptor type B), and SCFR (Stem cell receptor receptor, also known as c-Kit, CD117), and promotes the activity of MITF-M. Completed the invention.

That is, the present invention provides an anti-white hair agent containing a Bergenia ligrata extract as an active ingredient.

The present invention also provides a MITF-M activity promoter containing a Bergenia ligrata extract as an active ingredient.

As a result of strongly promoting the activity of MITF-M, the anti-whitening agent of the present invention promotes melanin synthesis in melanocytes and exerts a high anti-whitening effect (prevention and improvement of graying).

Graph showing the expression levels of TGFBR2, EDNRB, and SCFR genes by Bergenia ligratha extract in in vitro test Graph showing expression levels of TGFBR2, EDNRB, and SCFR genes by Japanese pepper peel extract in in vitro test Graph showing the expression levels of TGFBR2, EDNRB, and SCFR genes by hop flower extract in in vitro test Graph showing the expression level of MITF-M gene by Bergenia ligratha extract, Japanese pepper peel extract, and hop flower extract in in vitro test Graph showing the expression level of MITF-M gene by Bergenia ligratha extract in in vivo test (subject 1) Graph showing the expression level of MITF-M gene by Bergenia ligratha extract in in vivo test (subject 2)

The present invention relates to an anti-white hair agent containing a Bergenia ligrata extract as an active ingredient. The present invention also relates to a MITF-M activity promoter containing a Bergenia ligratha extract as an active ingredient. In addition, these may be referred to as "the anti-white hair agent of this invention" or "the activity accelerator of this invention".

The anti-white hair agent and activity accelerator of the present invention are characterized in that the Bergenia ligratha extract is an active ingredient. Bergenia ligrata extract promotes the activity of melanocyte cell membrane receptors TGFBR2, EDNRB, and SCFR. It also promotes MITF-M activity in melanocytes by activating these cytoplasmic receptors. That is, the anti-white hair agent and activity promoter of the present invention promote the activity of MITF-M in melanocytes by activating TGFBR2, EDNRB, and SCFR, and as a result, various physiology including melanin synthesis. It works. One of the physiological actions is, for example, an anti-whitening action (prevention of graying or improvement thereof).

The reason why the anti-white hair agent and the activity promoter of the present invention strongly promote the activity of MITF-M is that the Bergenia ligratha extract is applied to all of the cell membrane receptors of melanocytes, TGFBR2, EDNRB, and SCFR. It is presumed that this is due to its activity. That is, it can be said that the anti-white hair agent and activity promoter of the present invention are anti-white hair agents and activity promoters that promote all the activities of TGFBR2, EDNRB, and SCFR, which are cell membrane receptors of melanocytes. In addition, the anti-white hair agent and activity promoter of the present invention are anti-white hair agents that promote the activity of MITF-M in melanocytes by promoting all the activities of TGFBR2, EDNRB, and SCFR, which are cell membrane receptors of melanocytes. And can be said to be an activity promoter.

The Bergenia ligratha extract is obtained by extracting from each site of the Bergenia ligratha using an extraction solvent. The extraction method is not particularly limited, and various known extraction methods can be used. As the extraction raw material, parts such as leaves, branches, timbers, bark, roots, etc. of Bergenia ligratha, and dried ones thereof are used. Of these, the roots of Bergenia ligratha are particularly preferable. Examples of the extraction method include extraction with an extraction solvent at room temperature or after heating.

The extraction solvent is not particularly limited as long as it is a solvent used for ordinary plant extraction, and for example, lower monohydric alcohols such as methanol and ethanol, glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol and the like. Examples thereof include liquid polyhydric alcohols, lower alkyl esters such as ethyl acetate, water, and the like, and one or more of these mixed solvents can be used. Among these, an extraction solvent composed of water and 1,3-butylene glycol is preferable.

The Bergenia ligrata extract may be used as it is, or may be filtered and concentrated if necessary. In addition, the Bergenia ligrata extract can also be used after being fractionated and purified by a column chromatography or the like. In addition, the Bergenia ligrata extract can be dried under reduced pressure or freeze-dried, then prepared in the form of powder or paste, and appropriately formulated and used. As the Bergenia ligratha extract, for example, a commercially available product such as "Pashanbe Extract SK" (manufactured by Koei Kogyo Co., Ltd.) can be used.

When applied to the scalp, the activity promoter of the present invention is percutaneously absorbed to reach the hair root site and promotes the activity of TGFBR2, EDNRB, SCFR, and MITF-M, which are cell membrane receptors of melanocytes. The activity accelerator of the present invention can be used as an anti-white hair agent by having these actions. Further, the anti-whitening agent and the activity promoter of the present invention can be blended with external skin products such as scalp care agents and hair products such as shampoos and treatment agents.

The skin external products and hair styling products of the present invention can be prepared and used in various dosage forms such as liquid, mist, mist, emulsion, cream, gel, wax and foam. Further, as long as the manifestation of the desired effect of the present invention is not inhibited, for example, lower alcohol, polyhydric alcohol, sugar alcohol, ultraviolet absorber, fragrance, preservative, chelating agent, antibacterial agent, antioxidant, moisturizing agent. Agents, refreshing agents, vitamins, cationic polymers, nonionic polymers, amphoteric polymers, anionic polymers, plant extracts, propellants, pH regulators, amino acids, anti-inflammatory agents, astringents, pigments, thickeners, etc. Other additives may be included.

The blending amount of the Bergenia ligratha extract in the external skin products or hair styling products is appropriately adjusted according to the mode of use, but it is preferably blended in the range of 0.00005 to 0.05% by mass of the entire preparation. It is more preferably blended in the range of 0.0001 to 0.025% by mass, and further preferably blended in the range of 0.0005 to 0.01% by mass. The blending amount of Bergenia ligratha extract is based on solid content conversion.

In addition to the anti-whitening agent, the activity-promoting agent of the present invention can also be suitably used as a skin cosmetic such as an anti-whitening agent for the skin, an anti-skin blackening agent, and a whitening agent.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples.

<In vitro test>
[Preparation of test substance 1 (medium containing 1.0% by volume of Bergenia ligrata extract)]
15 μL of Pashambe extract SK (Bergenia ligrata extract containing 0.5 mass% of Bergenia ligrata root extract; manufactured by Koei Kogyo Co., Ltd.) 1485 μL of Medium 254 (proliferation medium for epidermal melanocytes containing 1.0% by volume of HMGS) A medium containing 1.0% by volume of Bergenia ligrata extract was prepared by adding to (hereinafter referred to as "Melanocyte 254 (including HMGS)") and filtering after mixing.

[Preparation of test substance 2 (medium containing 1.0% by volume of Japanese pepper peel extract)]
Sansho extract-J (Sansho peel extract containing 0.18% by mass of Sansho peel extract; manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used instead of Pashanbe extract SK so that the final concentration was 1.0% by volume. A medium containing 1.0% by volume of Sansho peel extract was prepared by performing the same operation as the preparation of Test Substance 1 except that it was used in.

[Preparation of test substance 3 (medium containing 1.0% by volume of hop flower extract)]
Hop extract BG-JN (hop flower extract containing 1.8% by mass of hop flower extract; manufactured by Maruzen Pharmaceutical Co., Ltd.) was used instead of Pashanbe extract SK, and the final concentration of the hop flower extract was 1.0% by volume. A medium containing 1.0% by volume of hop flower extract was prepared by performing the same operation as the preparation of Test Substance 1 except that it was used so as to be.

[Preparation of negative control 1]
Butanediol was added to Medium 254 (including HMGS) to a final concentration of 1.0% by volume to prepare a negative control 1.

[Preparation of melanocytes]
Human melanocytes were cultured using Medium 254 (including HMGS) as a culture medium in a T75 flask in a CO ₂ incubator (5% by volume CO ₂ , 37 ° C.) until the required number of cells was reached. Then, remove the medium from the T75 flask, add 10 mL of HEPES, wash the cells, add 3 mL of trypsin / EDTA solution, immediately remove 2 mL of the added trypsin / EDTA solution, and allow to stand at room temperature for 1 to 2 minutes. And the cells were detached. Then, 10 mL of HEPES was added to collect the cells, and 10 mL of trypsin neutralized solution was further added and mixed, and the cell solution was centrifuged at 170 × g for 5 minutes. Medium 254 (including HMGS) was added to the precipitate (cells), and after suspension, the number of cells was measured with a hemocytometer to prepare human melanocytes.

[Activity test of test substances, etc.]
Human melanocytes obtained by culturing were seeded on a 24-well plate of 1.5 × 10 ⁵ cells / 0.5 mL / well, and after culturing in a CO ₂ incubator (5% by volume CO ₂ , 37 ° C.) for 24 hours. , Test substances 1 to 3 and negative control 1 were added to the wells in an amount of 0.5 mL each. Then, the cells were collected by culturing for 24 hours.

Total RNA was recovered from each cell obtained from the above test. RNA extraction was performed using ISOGEN (manufactured by Nippon Gene Co., Ltd.). Specifically, after lysing the sample cells with an ISOGEN solution, chloroform was added and centrifuged to obtain RNA dissolved in the aqueous phase. Then, isopropanol was added to the aqueous phase to precipitate RNA, and RNA was isolated.

cDNA synthesis was carried out using SuperScript III First-Strand Synthesis System (manufactured by Thermo Fisher Scientific Co., Ltd.). Specifically, after adding the reagents necessary for cDNA synthesis (random hexamers, dNTP mix, MgCl ₂ , DTT, RNaseOUT, SuperScript III RT) to the tube containing RNA, the temperature is adjusted to 50 ° C. for 10 minutes at 25 ° C. The cDNA was synthesized by sequentially reacting them for 50 minutes at 85 ° C. for 5 minutes. Samples (synthesized cDNA) were prepared in Real-time PCR tubes, and the expression levels of the TGFBR2, EDNRB, SCFR, and MITF-M genes were quantified using the Applied Biosystems real-time PCR system. After completion of the PCR reaction, the dissociation curve of the PCR product was determined. The expression level of each gene was calculated as a ΔΔCt value.

The Ct value (number of PCR cycles) was calculated from the intersection of the amplification curve and the threshold line. Ct (target gene) -Ct (GAPDH2) = ΔCt value obtained by subtracting the Ct value of the internal standard GAPDH2 gene from the Ct value of the target gene. Further, ΔCt (sample processing group) −ΔCt (blank group) = ΔΔCt value obtained by subtracting the average ΔCt value of the solvent control group from the ΔCt value. The 2-ΔΔCt value obtained by substituting the ΔΔCt value into the multiplier term is the relative expression level.

The above results are summarized in FIGS. 1 to 4. From the results obtained, the medium containing 1.0% by volume of Bergenia ligrata extract has activity against all of the cell membrane receptors of melanocytes, TGFBR2, EDNRB, and SCFR, and the synergistic effect thereof is a strong MITF. It was clarified that it exerts an action promoting -M activity. On the other hand, it was revealed that the MITF-M activity promoting action of the Japanese pepper peel extract and the hop flower extract was low.

<In vivo test>
[Preparation of test substance 4 (sample containing 2.0% by mass of Bergenia ligrata extract)]
Ethanol is 50.00% by mass, l-menthol is 0.40% by mass, sodium dihydrogen phosphate is 0.04% by mass, sodium monohydrogen phosphate is 0.03% by mass, and Pashanbe extract SK is 2.0% by mass. , And purified water so as to be 47.53% by mass, and the test substance 4 was prepared.

[Preparation of negative control 2]
Negative control 2 was prepared in the same manner as in Test substance 4 except that 49.53% by mass of purified water was used without using Pashanbe extract SK.

[Activity test of test substances, etc.]
After applying the test substance 4 to the scalp from two subjects (subjects 1 and 2), five hairs of each were removed with tweezers and placed in a tube. Total RNA was recovered from the obtained hair root cells. The same operation was performed for the negative control 2.

RNA extraction from the sample was performed using Power SYBRGreen Cell-to-Ct Kit (manufactured by Thermo Fisher Scientific Co., Ltd.). Specifically, a solution prepared by mixing Lysis solution and DNase I at a ratio of 99: 1 was added to the sample at a rate of 50 uL / tube, and mixed by pipetting. Then, after reacting at room temperature for 5 minutes, a solution prepared by mixing Xeno RNA control and Stop Solution at a ratio of 1: 5 was added to the sample by 5 uL / tube, mixed by pipetting, and reacted at room temperature for 2 minutes.

cDNA synthesis was carried out using SuperScript III First-Strand Synthesis System (manufactured by Thermo Fisher Scientific Co., Ltd.). Specifically, after adding the reagents necessary for cDNA synthesis (random hexamers, dNTP mix, MgCl ₂ , DTT, RNaseOUT, SuperScript III RT) to the tube containing RNA, the temperature is adjusted to 50 ° C. for 10 minutes at 25 ° C. The cDNA was synthesized by sequentially reacting them for 50 minutes at 85 ° C. for 5 minutes. A sample (synthesized cDNA) was prepared in a Real-time PCR tube, and the expression level of the MITF-M gene was quantified using the Applied Biosystems real-time PCR system. After completion of the PCR reaction, the dissociation curve of the PCR product was determined. The expression level of each gene was calculated as a ΔΔCt value.

The Ct value (number of PCR cycles) was calculated from the intersection of the amplification curve and the threshold line. Ct (target gene) -Ct (GAPDH2) = ΔCt value obtained by subtracting the Ct value of the internal standard GAPDH2 gene from the Ct value of the target gene. Further, ΔCt (sample processing group) −ΔCt (blank group) = ΔΔCt value obtained by subtracting the average ΔCt value of the solvent control group from the ΔCt value. The 2-ΔΔCt value obtained by substituting the ΔΔCt value into the multiplier term is the relative expression level.

The above results are summarized in FIG. 5 (subject 1) and FIG. 6 (subject 2). The relative expression levels of MITF-M in FIGS. 5 and 6 show the average values of the five samples. Further, "none" in the figure means that the negative control 2 is applied to the scalp, and "yes" means that the test substance 4 is applied to the scalp. From the obtained results, it was clarified that the sample containing 2.0% by mass of the Bergenia ligratha extract exerted a remarkable MITF-M activity promoting action.

From the above results, it was clarified that the Bergenia ligratha extract strongly promotes the activity of MITF-M even in the in vivo test. That is, it was shown that the Bergenia ligratha extract is a component that exerts an effective effect as a MITF-M activity promoter. Then, by activating MITF-M by the Bergenia ligratha extract, melanin synthesis in melanocytes is promoted, and as a result, a high anti-whitening effect (prevention and improvement of graying) is exhibited. .. That is, it was shown that the Bergenia ligratha extract is a component that exerts an effective effect as an anti-white hair agent.

Claims (1)

A MITF-M activity promoter containing a Bergenia ligrata extract as an active ingredient.

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