JP5464730B2 - Whitening agent, anti-aging agent, profilagrin mRNA expression increase promoter, hair restorer and hair papillary cell proliferation promoter - Google Patents

Description

  The present invention relates to a whitening agent, an anti-aging agent, a profilagrin mRNA expression increase promoter, a hair restorer and a hair papillary cell growth promoter.

  Melanin also plays a role in protecting the body from ultraviolet rays in the skin, but overproduction and uneven accumulation cause skin darkening and spots. Therefore, in order to prevent, treat, or improve skin darkness (skin pigmentation), spots, buckwheat, etc., it is conceivable to suppress the production of melanin.

  From such a point of view, conventionally, as one having an inhibitory action on melanin production, for example, an extract from roots of castor bean (see Patent Document 1), an extract from a plant belonging to the genus Southus (Saussurea) (see Patent Document 2) Etc. are known.

  Glutathione is a tripeptide consisting of three amino acids, glutamic acid, cysteine and glycine, and is a compound having a major cysteine residue in the cell. Intracellular glutathione functions as a radical donor, regulation of cell function by redox, and an SH donor for various enzymes, and is also known as an antioxidant component. Its action expression is thought to be derived from cysteine residues. However, it has been reported that the amount of glutathione in the skin decreases with aging, and this is one factor that reduces the oxidative defense ability in the skin and damages cellular components such as DNA and proteins. It is believed that.

  That is, promoting the production of glutathione in the skin increases the defense against oxidative stress that declines with aging and suppresses damage to oxidative stress caused by ultraviolet rays. It is considered that improvement for pigmentation can be expected. Based on such an idea, bilberry extract, walnut extract (see Patent Document 3), gardenia plant extract (see Patent Document 4), and the like are known as having glutathione production promoting action.

  An amino acid which is a main component of natural moisturizing factors (NMF) is produced by degrading filaggrin derived from keratohyalin granules in the stratum corneum. This filaggrin is expressed as profilagrin in epidermal keratinocytes present in the granule layer immediately below the stratum corneum. It is then phosphorylated immediately, accumulated in keratohyalin granules, decomposed into filaggrin through dephosphorylation and hydrolysis, and then transferred to the stratum corneum, increasing the aggregation efficiency of keratin filaments and participating in the internal construction of keratinocytes It is known (see Non-Patent Document 1).

  In recent years, it has been known that this filaggrin is very important and indispensable for moisture retention of the skin, and that the synthetic power of filaggrin is reduced by conditions such as drying, and the amount of amino acids in the stratum corneum is reduced ( Non-patent document 2). It is known that when the amount of amino acid in the stratum corneum decreases and the water content of the stratum corneum decreases, the stratum corneum becomes hard, causing cracks in the stratum corneum and causing skin wrinkles and the like.

  Therefore, in the epidermal keratinocytes, by promoting the production of profilagrin, the production of filaggrin and thereby increasing the amount of amino acids in the stratum corneum can essentially improve the water environment of the stratum corneum. It is expected that wrinkles can be prevented.

  As what has such a profilagulin production promotion effect and a filaggrin production promotion effect, for example, a liquorice extract (refer patent document 5), the liquiritin known as flavanone glycoside contained in a natural plant (refer patent document 6) For example, a plant extract or yeast extract belonging to the genus Citrus (see Patent Document 7) or the like is known as one having profilaggrin and filaggrin protein production promoting action.

  Hair growth repeats growth and loss according to a periodic hair cycle (hair cycle) consisting of a growth period, a regression period, and a rest period. Of these hair cycles, the stage at which new hair follicles are formed from the resting stage to the growing stage is considered to be the most important for hair growth. It is thought that hair papilla cells play an important role in differentiation. The dermal papilla cell is a cell located inside the hair follicle epithelial cell composed of outer root sheath cells and matrix cells in the vicinity of the hair root, and located in the root portion of the hair root wrapped in the basement membrane. It plays an important role in differentiation into hair, such as by acting on epithelial cells to promote their proliferation (see Non-Patent Document 3).

  Thus, dermal papilla cells play the most important role in the proliferation and differentiation of hair follicle epithelial cells and the formation of hair. Conventionally, the target substance is brought into contact with dermal papilla cells, and the proliferation activity of the cells is controlled. A method for testing the hair-growth effect of the target substance by specifying the presence and / or strength is proposed (see Patent Document 8). Conventional herbal medicines having a dermal papilla cell growth promoting action include, for example, pearl barley extract, wild thyme extract, cedar extract, shobu extract, rosemary extract, turmeric extract, birch extract and kocha extract. A thing etc. are proposed (refer patent document 9).

JP 2001-213757 A JP 2002-201222 A JP 2006-241062 A JP 2006-347934 A JP 2002-363054 A JP 2003-146886 A JP 2001-261568 A JP-A-10-229978 JP 2006-219407 A

"Fragrance Journal Extra Edition", 2000, Vol.17, p.14-19 “Arch. Dermatol. Res.”, 1996, Vol.288, p.442-446 "Trends Genet", 1992, Vol. 8, pp.56-61

  An object of the present invention is to provide a whitening agent, an anti-aging agent, a profilagrin mRNA expression increase promoter, a hair restorer, and a hair papillary cell growth promoter comprising a highly safe natural extract as an active ingredient.

  In order to achieve the above object, the whitening agent, anti-aging agent, profilagulin mRNA expression increase promoter, hair restorer or hair papilla cell growth promoter of the present invention contains a hot water extract as an active ingredient. Features.

  In the whitening agent of this invention, it is preferable that the said hot water extract has a melanin production inhibitory effect and / or a glutathione production promotion effect.

  According to the present invention, it contains a natural hot water extract as an active ingredient, and is a highly safe whitening agent, anti-aging agent, profilagrin mRNA expression increase promoter, hair restorer, and hair papillary cell proliferation promoter. Can be provided.

It is a chromatogram which shows the analysis result analyzed about the hot water extract using the liquid chromatography. It is a chromatogram which shows the analysis result analyzed using the liquid chromatography about succinic acid.

Hereinafter, an embodiment of the present invention will be described.
The whitening agent, anti-aging agent, profilagrin mRNA expression increase promoter, hair restorer or hair papilla cell growth promoter of this embodiment contains a hot water extract as an active ingredient.

  Here, in the present embodiment, the “extract” refers to an extract obtained by using koji as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a rough product thereof. Both purified products and purified products are included.

  The extraction raw material used in the present embodiment is straw. A cocoon is a resin made of wood (mainly a resin belonging to the pine family, such as pine, cedar, cypress, etc.) buried in the ground and solidified over the years. Although it is known that the resin contains a large amount of diterpenes, most of the diterpenes are hardly soluble polymers. The cocoons are usually irregular, lump, stalactite, or dusty, and some of them contain plant or insect fossils inside. The color is yellow, yellow-brown or red-yellow, transparent or translucent, and the streak is white or pale yellow and has a pine-like luster. The cross section has a shell shape. Amber is conventionally used as a decorative item (jewel) because of its beauty. The hardness is 2 to 2.5, and the specific gravity is 1.05 to 1.09. Coral is produced from clay layers, sand layers, coal beds or sedimentary rocks, and is produced from the Baltic Sea coast, Dominican Republic, Kuji region in Iwate Prefecture, etc., and can be easily obtained from these regions.

  The hot water extract can be obtained by pulverizing the raw material as an extraction raw material as it is, or subjecting it to extraction with hot water as an extraction solvent. The soot may be used as an extraction raw material after pretreatment such as washing with a nonpolar solvent such as hexane.

  Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as water that has been subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, the water that can be used as the extraction solvent in this embodiment includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  The hot water extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted into the hot water, and can be performed according to a conventional method. For example, the extraction raw material is immersed in water, which is an extraction solvent in an amount 5 to 25 times (mass ratio) of the extraction raw material, and heated at a temperature of usually 40 to 100 ° C., preferably 60 to 100 ° C. After extraction, an extract can be obtained by filtering to remove the extraction residue. The obtained extract is diluted, concentrated, dried, purified, etc. according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude purified product or a purified product thereof. Processing may be performed.

  Purification can be performed by, for example, activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, or the like. The obtained extract can be used as an active ingredient of a whitening agent, an anti-aging agent, a profilagrin mRNA expression increase promoter, a hair restorer, or a hair papillary cell growth promoter, but it can be a concentrated solution or a dried product. Things are easier to use.

  琥珀 Hot water extract has a peculiar smell and can be purified for the purpose of decolorization, deodorization, etc. within the range that does not cause a decrease in its physiological activity. When blended in cosmetics, etc., it is not used in large quantities, so there is no practical problem even if it is not purified.

  The scorched water extract obtained as described above has a whitening effect, an anti-aging effect, a profilagrin mRNA expression increase promoting effect, a hair growth effect or a hair papillary cell growth promoting effect, and therefore uses each action. Then, it can be used as an active ingredient of a whitening agent, an anti-aging agent, a profilagrin mRNA expression increase promoter, a hair restorer or a hair papillary cell proliferation promoter. In addition, succinic acid is known as a main component contained in koji, and it is known that the succinic acid has a whitening effect. However, the hot water extract is as shown in the following examples. Because it contains substantially no succinic acid, the whitening action (melanin production suppression action, glutathione production promotion action), anti-aging action (profilaggrin mRNA expression increase promotion action), hair growth action ( It is speculated that the dermal papilla cell proliferation promoting action) is not exhibited by succinic acid.

  The whitening action of the hot water extract is exhibited based on, for example, a melanin production inhibitory action and / or a glutathione production promoting action. However, the whitening action of the hot water extract is not limited to the whitening action exhibited based on these actions.

  The anti-aging effect of the hot water extract is exhibited based on, for example, a profilagrin mRNA expression promoting effect. However, the anti-aging effect of the hot water extract is not limited to the anti-aging action exhibited based on this action.

  The hair-growth action of the hot water extract is exhibited based on, for example, the action of promoting hair papillary cell proliferation. However, the hair-growth effect of the hot water extract is not limited to the hair-growth effect exhibited based on these effects.

  In addition, since a scorching water extract has a melanin production inhibitory effect or a glutathione production promotion effect, you may use those effects as an active ingredient of a melanin production inhibitor or a glutathione production promoter.

  The whitening agent, anti-aging agent, profilagrin mRNA expression increase promoter, hair restorer or hair papilla cell growth promoter of this embodiment may consist of only a hot water extract or hot water extraction. A product may be formulated.

  琥珀 Hot water extract is used in any dosage form such as powder, granule, tablet, liquid, etc., using a pharmaceutically acceptable carrier such as dextrin, cyclodextrin, etc. It can be formulated. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent and the like can be used. The hot water extract can be used by blending with other compositions (for example, skin cosmetics, hair cosmetics, etc.).

  In addition, the whitening agent, anti-aging agent, profilagrin mRNA expression increase promoter, hair restorer or hair papillary cell proliferation promoter of the present embodiment, if necessary, whitening action, anti-aging action, profilagrin mRNA expression increase promotion Other natural extracts having an action, a hair-growth action or a hair papillary cell growth-promoting action can be blended and used as an active ingredient.

  Examples of the method for administering the whitening agent, anti-aging agent, profilagrin mRNA expression increase promoter, hair restorer or hair papillary cell growth promoter of this embodiment generally include transdermal administration, depending on the type of disease. A suitable method for the prevention / treatment or the like may be appropriately selected.

  Further, the dosage of the whitening agent, anti-aging agent, profilagulin mRNA expression increase promoter, hair restorer or hair papilla cell growth promoter of this embodiment is also the type of disease, severity, individual differences of patients, administration method, What is necessary is just to increase / decrease suitably according to an administration period etc.

  The whitening agent of this embodiment can prevent or improve pigmentation such as skin darkening, spots, and freckles through the melanin production inhibitory action and / or glutathione production promoting action of the hot water extract. However, the whitening agent of the present embodiment can be used for all purposes that are meaningful in exhibiting a melanin production inhibitory action and / or a glutathione production promoting action in addition to these uses.

  The anti-aging agent or profilagrin mRNA expression increase promoter of the present embodiment can prevent or improve skin aging symptoms and the like through the action of promoting profilaggrin mRNA expression increase of the scorched water extract. However, the anti-aging agent of this embodiment can be used for all the uses meaningful in exhibiting the profilaggrin mRNA expression increase promotion effect besides these uses.

  The hair-restoring agent or dermal papilla cell proliferation promoting action of the present embodiment can prevent, treat or ameliorate alopecia through the dermal papilla cell proliferation promoting action of the scorched water extract. However, the hair-restoring agent of the present embodiment can be used for all uses other than these uses, which are meaningful for exerting the effect of promoting hair papillary cell proliferation.

  The whitening agent, anti-aging agent, profilagrin mRNA expression increase promoter, hair restorer or hair papilla cell growth promoter of the present embodiment are suitably applied to humans, but each effect is Can be applied to animals other than humans as long as

  Hereinafter, although a manufacture example and a test example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all.

[Production Example 1] Production of hot water extract 50 g of ground mash was added to 1000 mL of hot water at 90 ° C, extracted for 2 hours, and filtered while hot. The residue was further extracted in the same manner. The obtained extracts were combined, concentrated under reduced pressure, and further dried to obtain 0.0005 g of a scintillating water extract (Sample 1).

  The hot water extract (sample 1) and succinic acid (manufactured by Wako Pure Chemical Industries, Ltd.) obtained as described above were analyzed using liquid chromatography under the following conditions. The analysis result about a hot water extract is shown in FIG. 1, and the analysis result about a succinic acid is shown in FIG.

<Liquid chromatography conditions>
Product name: Agilent 1100 (Agilent Technologies)
Stationary phase: Wakosil-II 5C18 HG (manufactured by Wako Pure Chemical Industries, Ltd.)
Column diameter: 4.6 mm
Column length: 150mm
Mobile phase: 85% phosphate buffer Mobile phase flow rate: 1.0 mL / min Detector: UV
Detection wavelength: 210 nm

  As shown in FIG. 2, when analyzed using liquid chromatography under the above conditions, the retention time of succinic acid was 3.044 min, but peaked at the retention time in the hot water extract (sample 1). Was not seen. From this result, it was confirmed that the hot water extract (sample 1) does not substantially contain succinic acid.

[Test Example 1] Test for inhibiting melanin production on B16 melanoma cells The scorched water extract (Sample 1) obtained in Production Example 1 was tested for melanin production inhibitory action on B16 melanoma cells as follows.

B16 melanoma cells were cultured using Dulbecco MEM containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with Dulbecco MEM containing 10% FBS and 1 mmol / L theophylline to a cell density of 25.0 × 10 ⁴ cells / mL, then seeded on a 48-well plate at 300 μL per well and cultured for 6 hours.

  After completion of the culture, 300 μL of a sample solution (Sample 1, see Table 1 below for sample concentration) in which the sample was dissolved in 10% FBS and 1 mmol / L theophylline-containing Dulbecco MEM was added to each well and cultured for 4 days. After completion of the culture, the medium was removed from each well, 200 μL of 1 mol / L NaOH solution was added, the cells were disrupted with an ultrasonic disrupter, and the absorbance at a wavelength of 475 nm was measured to obtain the amount of melanin produced.

  In addition, in order to evaluate the melanin production inhibitory action per unit cell, after culturing in the same manner as above, the culture solution was removed by washing with 400 μL of PBS (−), and the final concentration was 0.05 mg / mL. 200 μL of neutral red dissolved in 10% FBS-containing Dulbecco MEM was added to each well and cultured for 2.5 hours. After incubation, the neutral red solution was discarded, and 200 μL of ethanol / acetic acid solution (ethanol: acetic acid: water = 50: 1: 49) was added to each well to extract the dye. After extraction, the absorbance at a wavelength of 540 nm was measured.

  Furthermore, as a blank test, the absorbance at a wavelength of 475 nm and the absorbance at 540 nm were measured for cells cultured in the same manner as described above without adding a sample. From the obtained results, the melanin production inhibition rate (%) per unit cell was calculated by the following formula.

Melanin production inhibition rate (%) = {1− (B / D) / (A / C)} × 100
In the formula, A represents “absorbance at 475 nm when no sample is added”, B represents “absorbance at 475 nm when no sample is added”, C represents “absorbance at 540 nm when no sample is added”, and D represents “ The absorbance at 540 nm at the time of sample addition is expressed.
The results are shown in Table 1.

  As shown in Table 1, it was confirmed that the hot water extract (sample 1) has a melanin production inhibitory effect.

[Test Example 2] Glutathione production promoting action test in B16 melanoma cells The hot water extract (sample 1) obtained in Production Example 1 was tested for glutathione production promoting action as follows.

B16 melanoma cells were cultured using D-MEM medium containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with 10% FBS-containing D-MEM medium to a cell density of 1.0 × 10 ⁵ cells / mL, then seeded at 200 μL per well in a 48-well plate and cultured overnight.

  After culturing, 200 μL of a sample solution dissolved in 1% FBS-containing D-MEM medium (Sample 1, see Table 2 below for sample concentration) was added to each well and cultured for 24 hours. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS (−), and cells were lysed using 150 μL of M-PER (PIERCE).

  Of these, 100 μL was used to quantify total glutathione. That is, after adding 100 μL of cell extract dissolved in 96-well plate, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH and 25 μL of glutathione reductase (final concentration 17.5 units / mL), the mixture was heated at 37 ° C. for 10 minutes. 25 μL of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm until 5 minutes was measured to determine ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione (manufactured by Wako Pure Chemical Industries, Ltd.). After correcting the obtained value to the amount of glutathione per total protein amount, the glutathione production promotion rate (%) was calculated by the following formula.

Glutathione production promotion rate (%) = B / A × 100
In the formula, A represents “the amount of glutathione per total protein in the cell when no sample is added (control)”, and B represents “the amount of glutathione per total amount of protein in the cell when added to the sample”.
The results are shown in Table 2.

  As shown in Table 2, it was confirmed that the hot water extract (sample 1) has an excellent glutathione production promoting action.

[Test Example 3] Profilaggrin mRNA expression increase promoting action test The hot water extract (sample 1) obtained in Production Example 1 was tested for profilagrin mRNA expression increase promoting action as follows.

Normal human epidermal keratinocyte (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes in an 80 cm ² flask at 37 ° C., 5% CO ₂ -95% air The cells were precultured under the above conditions and cells were collected by trypsin treatment.

EpiLife-KG2 was used to seed 40 × 10 ⁴ cells / 2 mL / dish in 35 mm dishes (FALCON) and cultured overnight at 37 ° C. and 5% CO ₂ -95% air. After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, UV-B irradiation (50 mJ / cm ² ) was performed, and then the test sample dissolved in EpiLife-KG2 to the required concentration (sample 1, sample concentration is shown in Table 3 below) 2 mL) was added to each petri dish and cultured at 37 ° C. under 5% CO ₂ -95% air for 24 hours. After culturing, the culture solution is discarded, and total RNA is extracted with ISOGEN (NIPPON GENE, Cat. No. 311-02501), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 ng / μL. Total RNA was prepared.

  Using this total RNA as a template, the expression level of profilaggrin and the internal standard GAPDH mRNA was measured. Detection was performed by real-time 2-step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR Kit (Perfect Real Time, code No. RR063A) using a real-time PCR device Smart Cycler (Cepheid). The expression level of mRNA for profilagrin is calculated based on the total RNA preparation prepared from the cells cultured with and without sample addition. The correction value of sample addition when 100 was calculated. From the obtained results, the profilaggrin mRNA expression increase promotion rate (%) was calculated by the following formula.

Profilaggrin mRNA expression increase suppression rate (%) = A / B × 100
In the formula, A represents “correction value when sample is added”, and B represents “correction value when sample is not added”.
The results are shown in Table 3.

  As shown in Table 3, it was confirmed that the hot water extract (sample 1) has an excellent effect of promoting the increase in profilagrin mRNA expression.

[Test Example 4] Hair papilla cell proliferation promoting action test The scorched water extract (sample 1) obtained in Production Example 1 was tested for hair papilla cell proliferation promoting action as follows.

Normal human hair hair papilla cells were cultured using a hair papilla cell growth medium containing 1% fetal bovine serum (FBS) and growth additives, and then cells were collected by trypsinization. The collected cells were diluted to a cell density of 2.0 × 10 ⁴ cells / mL using Dulbecco MEM containing 10% FBS, then seeded at 100 μL per well on a collagen-coated 96-well plate, and cultured for 3 days. After the culture, the medium was removed, and a sample solution (sample 1, refer to Table 4 below for sample concentration) dissolved in serum-free DMEM was added to each well in an amount of 200 μL, and further cultured for 4 days.

  The dermal papilla cell proliferation promoting effect was measured using MTT assay. After completion of the culture, the medium was removed, and 100 μL of MTT (final concentration 0.4 mg / mL) dissolved in serum-free DMEM was added to each well. After culturing for 2 hours, blue formazan produced in the cells was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used as the amount of blue formazan produced. As a control, the same measurement was performed when serum-free DMEM was added instead of the sample solution. From the obtained results, the hair papilla cell proliferation promotion rate (%) was calculated by the following formula.

Hair papilla cell growth promotion rate (%) = A / B × 100
In the formula, A represents “absorbance when a sample is added”, and B represents “absorbance when no sample is added”.
The results are shown in Table 4.

  As shown in Table 4, it was confirmed that the hot water extract (sample 1) has an excellent dermal papilla cell proliferation promoting action.

  The whitening agent of the present invention is used for the prevention and improvement of skin pigmentation and the like, and the anti-aging agent and the profilagrin mRNA expression increase promoter of the present invention are used for the prevention and improvement of skin aging. The papillary cell proliferation promoter can greatly contribute to the prevention and improvement of alopecia.

Claims (6)

A whitening agent characterized by containing, as an active ingredient, a hot water extract which is extracted at 40 to 100 ° C. at normal pressure and contains substantially no succinic acid .   The whitening agent according to claim 1, wherein the hot water extract has a melanin production inhibitory action and / or a glutathione production promotion action. An anti-aging agent characterized by containing, as an active ingredient, a hot water extract that is extracted under normal pressure using water at 40 to 100 ° C. and contains substantially no succinic acid . A profilaggrin mRNA expression increase promoter characterized by containing, as an active ingredient, a hot water extract that is extracted under normal pressure using water at 40 to 100 ° C. and contains substantially no succinic acid . A hair restorer characterized by containing, as an active ingredient, a hot water extract that is extracted at 40 to 100 ° C. at normal pressure and contains substantially no succinic acid . A hair papilla cell growth promoter characterized by containing, as an active ingredient, a hot water extract that is extracted at 40 ° C to 100 ° C at normal pressure and substantially does not contain succinic acid .

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