JP2018162235A - Anti-poliosis agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anti-poliosis agent targeting the mechanism of poliosis, and also provide an activity promoter (MITF-M activity promoter) that activates MITF-M, known to play an important role in melanin synthesis.SOLUTION: An anti-poliosis agent contains Bergenia ligulata extract as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to an anti-white hair agent. Moreover, it is related with the activity promoter of MITF-M (Microphthalmia-Associated Transcription Factor type M) in a melanocyte.

  The occurrence of gray hair is a typical sensation and has a great impact on the appearance impression. Many consumers who want to be youthful respond to whitening by dyeing with a hair dye or hair dye. However, these hair dyeing methods have various problems such as being troublesome, having the possibility of adversely affecting the hair and scalp, and being difficult to dye natural colors. For this reason, development of an anti-white hair method (for example, a method for preventing or improving gray hair) that does not depend on a hair dye or a hair dye is required.

  Melanin pigments that determine the color of hair are pigment components produced in melanocytes. Melanocytes are present in the hair bulb adjacent to the hair matrix cells, and when the hair matrix cells divide and produce hair, the melanocytes are transferred from the melanocytes and taken into the hair. . Therefore, when melanin synthesis in melanocytes is not normally performed, hair cannot take up melanin, and gray hair is generated.

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

  As described above, research targeting the mechanism of gray hair has been actively conducted. However, these findings have not led to the discovery of a fundamental solution for anti-white hair.

Emi K. Nishimura et al, Science, Vol. 307, Issue 5710, 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. Another object 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 circumstances as described above, the present inventor has conducted extensive research focusing on the mechanism of graying. As a result, the extract of Bergenia ligrata is a cell membrane receptor of melanocytes (pigmented cells), TGFBR2 (Transforming growth factor). to promote the activity of beta receptor II), EDNRB (Endothelin receptor type B), and SCFR (Stem cell factor receptor, also known as c-Kit, CD117), and to promote the activity of MITF-M Completed the invention.

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

  Moreover, this invention also provides the MITF-M activity promoter which uses 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 exhibits a high anti-whitening action (prevention and improvement of graying).

Graph showing expression levels of TGFBR2, EDNRB, and SCFR genes by Bergenia ligrata extract in in vitro test Graph showing expression levels of TGFBR2, EDNRB, and SCFR genes by salamander skin extract in in vitro test Graph showing expression levels of TGFBR2, EDNRB, and SCFR genes by hop flower extract in in vitro test The graph which shows the expression level of the MITF-M gene by the Bergenia ligrata extract, the salmon peel extract, and the hop flower extract in an in vitro test The graph which shows the expression level of the MITF-M gene by the Bergenia ligrata extract in an in vivo test (subject 1) The graph which shows the expression level of MITF-M gene by the Bergenia ligrata extract in an in vivo test (subject 2)

  The present invention relates to an anti-whitening agent containing a Bergenia ligrata extract as an active ingredient. Moreover, it is related with the MITF-M activity promoter which uses a Bergenia ligrata extract as an active ingredient. These may be referred to as “the anti-whitening agent of the present invention” or “the activity promoter of the present invention”.

  The anti-whitening agent and activity promoter of the present invention is characterized in that a Bergenia ligrata extract is an active ingredient. Bergenia ligrata extract promotes the activity of TGFBR2, EDNRB, and SCFR, which are cell membrane receptors for melanocytes. In addition, activation of these cytoplasmic receptors promotes the activity of MITF-M in melanocytes. That is, the anti-gray hair agent and activity promoter of the present invention activates TGFBR2, EDNRB, and SCFR, thereby promoting the activity of MITF-M in melanocytes, and as a result, various physiology including melanin synthesis. Demonstrate the effect. As one of the physiological actions, for example, an anti-white hair action (prevention of gray hair and improvement thereof) can be mentioned.

  The reason why the anti-whitening agent and activity promoter of the present invention strongly promotes the activity of MITF-M is that the extract of Bergenia ligrata is used for all of TGFBR2, EDNRB, and SCFR, which are cell membrane receptors of melanocytes. It is assumed that this is due to the activity. That is, the anti-whitening agent and activity promoter of the present invention can be said to be an anti-whitening agent and activity promoter that promotes all the activities of TGFBR2, EDNRB, and SCFR, which are cell membrane receptors of melanocytes. In addition, the anti-whitening agent and activity promoter of the present invention is an anti-whitening agent that promotes 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 an activity promoter.

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

  The extraction solvent is not particularly limited as long as it is a solvent used for normal plant extraction, 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 a mixture of two or more of these 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, but may be filtered and concentrated as necessary. Further, the Bergenia ligrata extract can be used after being fractionated and purified by column chromatography or the like. Further, the Bergenia ligrata extract can be used after being dried under reduced pressure or lyophilized, then prepared into a powder or paste, and appropriately formulated. In addition, as a Bergenia ligrata extract, commercial items, such as "Pashanbe extract SK" (made by Koei Kogyo Co., Ltd.), can be used, for example.

  When applied to the scalp, the activity promoter of the present invention is absorbed transcutaneously to reach the hair root site, and promotes the activity of melanocyte cell membrane receptors TGFBR2, EDNRB, SCFR, and MITF-M. The activity promoter of the present invention can be used as an anti-whitening agent by having these actions. Furthermore, the anti-whitening agent and the activity promoter of the present invention can be used by blending with external skin products such as a scalp care agent and hair preparations such as a shampoo and a treatment agent.

  The external skin product and hair preparation of the present invention can be prepared and used in various dosage forms such as liquid, mist, mist, emulsion, cream, gel, wax and foam. Moreover, as long as expression 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, fresheners, vitamins, cationic polymers, nonionic polymers, amphoteric polymers, anionic polymers, plant extracts, propellants, pH adjusters, amino acids, anti-inflammatory agents, astringents, pigments, thickeners, etc. Other additives may be included.

  The blending amount of the Bergenia ligrata extract in the skin external product or hair preparation is appropriately adjusted according to the use mode, but it is preferably blended in the range of 0.00005 to 0.05% by mass of the whole preparation, It is more preferable to mix | blend in 0.0001-0.025 mass%, and it is still more preferable to mix | blend in 0.0005-0.01 mass%. In addition, the compounding quantity of Bergenia ligrata extract is based on solid content conversion.

  In addition to the anti-whitening agent, the activity promoter of the present invention can also be suitably used as a skin cosmetic such as a skin anti-white spot therapeutic agent, anti-skin blackening agent, and 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)]
Epidermal melanocyte growth medium containing 1485 μl of Medium 254 (HMGS 1.0 vol%) Hereinafter referred to as “Medium 254 (including HMGS)”, and a filter filtration treatment was performed after mixing to prepare a medium containing 1.0% by volume of Bergenia ligrata extract.

[Preparation of test substance 2 (medium containing 1.0% by volume salamander extract)]
In place of Pashanbe extract SK, salamander extract-J (a salamander extract containing 0.18% by mass of salamander extract; manufactured by Maruzen Pharmaceutical Co., Ltd.) is used so that the final concentration is 1.0% by volume. A medium containing 1.0% by volume of salamander peel extract was prepared by performing the same operation as in the preparation of test substance 1 except that it was used in the above.

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

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

[Preparation of melanocytes]
Human melanocytes were cultured using Medium 254 (including HMGS) as a culture solution and using a T75 flask in a CO ₂ incubator (5 vol% CO ₂ , 37 ° C.) until the required number of cells was reached. Thereafter, the medium is removed from the T75 flask, 10 mL of HEPES is added, the cells are washed, 3 mL of trypsin / EDTA solution is added, and 2 mL of the added trypsin / EDTA solution is immediately removed, and left at room temperature for 1-2 minutes. And the cells were detached. Thereafter, 10 mL of HEPES was added to recover the cells, and 10 mL of a trypsin neutralizing 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, human melanocytes were prepared by measuring the number of cells with a hemocytometer.

[Activity test for test substances]
Human melanocytes obtained by culturing were seeded in a 24-well plate of 1.5 × 10 ⁵ cells / 0.5 mL / well and cultured for 24 hours in a CO ₂ incubator (5 vol% CO ₂ , 37 ° C.). The test substances 1 to 3 and the negative control 1 were added to the wells 0.5 mL each. Then, it culture | cultivated for 24 hours and collect | recovered cells.

  Total RNA was collected from each cell obtained from the above test. RNA extraction was performed using ISOGEN (Nippon Gene Co., Ltd.). Specifically, sample cells were lysed with an ISOGEN solution, and chloroform was added and centrifuged to obtain RNA dissolved in the aqueous phase. Thereafter, isopropanol was added to the aqueous phase to precipitate RNA and to isolate RNA.

cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (manufactured by Thermo Fisher Scientific Co., Ltd.). Specifically, reagents necessary for cDNA synthesis (random hexamers, dNTP mix, MgCl ₂ , DTT, RNaseOUT, SuperScript III RT) are added to a tube containing RNA, and then at 25 ° C. for 10 minutes at 50 ° C. For 50 minutes at 85 ° C. for 5 minutes to synthesize cDNA. Samples (synthesized cDNA) were prepared in Real-time PCR tubes, and the expression levels of TGFBR2, EDNRB, SCFR, and MITF-M genes were quantified using an Applied Biosystems real-time PCR system. After completion of the PCR reaction, a PCR product dissociation curve was obtained. 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 treatment 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. From the obtained results, 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 its synergistic effect makes it a powerful MITF. It became clear that -M activity promotion effect was exhibited. On the other hand, it was revealed that the action of promoting the MITF-M activity of the salamander 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 Ligurata Extract)]
50.00% by mass of ethanol, 0.40% by mass of l-menthol, 0.04% by mass of sodium dihydrogen phosphate, 0.03% by mass of sodium monohydrogen phosphate, 2.0% by mass of Pashanbe extract SK , And purified water were mixed so as to be 47.53 mass% to prepare Test Substance 4.

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

[Activity test for test substances]
After the test substance 4 was applied to the scalp from two subjects (subjects 1 and 2), 5 hairs were each removed with tweezers and placed in a tube. Total RNA was recovered from the cells of the obtained hair root. The same operation was performed for negative control 2.

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

cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (manufactured by Thermo Fisher Scientific Co., Ltd.). Specifically, reagents necessary for cDNA synthesis (random hexamers, dNTP mix, MgCl ₂ , DTT, RNaseOUT, SuperScript III RT) are added to a tube containing RNA, and then at 25 ° C. for 10 minutes at 50 ° C. For 50 minutes at 85 ° C. for 5 minutes to synthesize cDNA. A sample (synthesized cDNA) was prepared in a Real-time PCR tube, and the expression level of the MITF-M gene was quantified using an Applied Biosystems real-time PCR system. After completion of the PCR reaction, a PCR product dissociation curve was obtained. 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 treatment 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). In addition, the relative expression level of MITF-M in FIGS. 5 and 6 shows an average value of five specimens. 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 Bergenia ligrata extract exerts a remarkable MITF-M activity promoting action.

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

Claims (2)

  An anti-whitening agent containing Bergenia ligrata extract as an active ingredient.   A MITF-M activity promoter comprising a Bergenia ligrata extract as an active ingredient.

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