JP5858562B2 - Endothelin-1 production inhibitor, SCF production inhibitor, melanin production inhibitor and use thereof obtained from persimmon - Google Patents

Description

  The present invention relates to a composition that suppresses the production of endothelin-1, a composition that suppresses the production of SCF, a composition that suppresses the production of melanin, and a technical field related to the use thereof.

  With the increase in the amount of ultraviolet rays accompanying the destruction of the ozone layer, skin damage due to ultraviolet rays is becoming more and more conscious. When the skin is exposed to ultraviolet rays, particularly UV-B, melanocytes, which are cells that produce melanin present in the basal layer of the skin and the hair matrix, are stimulated and proliferated. In the melanocytes activated by this stimulation, melanin is produced from tyrosine through dopaquinone by the action of tyrosinase in melanosomes, which are intracellular organelles, which causes spots and dullness. Produced melanin is stored in melanosomes. The melanosome in which melanin is stored is transferred from the melanocyte dendrites to the keratinocytes and pushed up to the stratum corneum by the skin turnover.

  Conventionally, L-ascorbic acid and its derivatives, hydroquinone, arbutin, kojic acid, cysteine, glutathione, and the like are used as a measure against such spots and dullness, so-called whitening. Vitamin C also has the effect of reducing and whitening melanin, and hydroquine, arbutin, kojic acid, and cysteine are said to inhibit tyrosinase.

  In addition, aiming for a more fundamental solution, methods for intervening in the activation pathway so that melanocytes irradiated with UV-B are not activated are being investigated. For example, factors such as endothelin-1 and SCF (Stem cell factor, hereinafter referred to as “SCF”) are produced from epidermal keratinocytes after UV-B irradiation, and epidermal melanocytes proliferate in response to these stimuli, and melanin synthesis ability Has been reported (Non-patent document 1, Non-patent document 2). In particular, it has been reported that when endothelin-1 and SCF coexist, the proliferation of melanocytes and the expression of tyrosinase are synergistically promoted rather than acting independently (Non-patent Document 3). Endothelin is a strong vasoconstrictor peptide composed of 21 amino acids, and there are three types of peptide isomers, endothelin-1, endothelin-2 and endothelin-3. Although it is produced in various tissues of the body such as lung, kidney, brain, and placenta, endothelin-1 is produced in an extremely large amount particularly in the endothelium of blood vessels (Non-patent Document 4). On the other hand, SCF is a cytokine that acts at the early stage of hematopoiesis, and there are membrane-bound SCF composed of 273 amino acids and secreted SCF that is cleaved by the action of proteolytic enzymes and released from the membrane. SCF is mainly produced by fibroblasts, bone marrow endothelial cells and the like (Non-patent Document 5).

  Development of substances that suppress melanin production and exert a whitening effect by blocking the information transmission system related to endothelin-1 and SCF has been actively conducted. In particular, attempts have been made to suppress the production of melanocytes, suppress the expression of tyrosinase, and suppress the production of melanin by suppressing the production of endothelin-1 and SCF which are the most upstream side.

  Examples of endothelin-1 and SCF production inhibitors known to date include white orchid flower extract (Patent Document 1), black ginger extract (Patent Document 2), yucca extract (Patent Document 3), Extract of algae belonging to the genus Chlamydomonas (patent document 4), extract of western primrose, extract of lemon verbena, extract of commune, blueberry extract, extract of celery (patent document 5), flower of loquat An extract from the department (Patent Document 6), an extract of Koukatoken (Patent Document 7) and the like have been reported.

  The cocoon is a fossil that is mainly formed by the resin of a pine genus plant buried in the basement for a long time and condensed, and mainly contains resin, essential oil, succinic acid and the like. And it dissolves in a small amount in ethanol, diethyl ether or benzene (Non-patent Document 6). In addition to applications such as decorative crafts, jewelry and insulating materials, or incense scraps, it was used for wounds around the 19th century (Non-patent Document 7).

  In recent years, techniques for blending wrinkle powder into cosmetics to improve skin feel (Patent Document 8, Patent Document 9), techniques for blending an extract of wrinkle into an external preparation for skin (Patent Document 10, Patent Document 11), wrinkles Technology utilizing the melanin production inhibitory action and / or glutathione production promoting action of the hot water extract (Patent Document 12), technique utilizing a skin turnover promoting factor in the cocoon extract fraction (Patent Document 13), extraction of sputum A technology that uses the skin-farming effect of skin in a product (Patent Document 14), a technology that uses a hyaluronic acid production promoting factor in a sputum extract fraction (Patent Document 15), an angiogenesis promoting factor in a sputum extract fraction A technique to be used (Patent Document 16) has been reported.

JP2009-29708 JP2009-51790 JP2009-227619 JP2010-13413 JP2010-65009 JP2010-184967 JP2010-116350 Patent 3725848 JP2004-083478 Patent No. 4034839 Patent No. 4217718 JP2010-235551 JP2007-314522 JP2008-189669 JP2008-266260 Japanese Patent Application No. 2011-1106743

Yukihiro Yada, Kazuhiko Higuchi and Genji Imokawa, Effects of endothelins on signal transduction and proliferation in human melanocytes, The Journal of Biological Chemistry, 266, 18352-18357 (1991). Akira Hachiya, Akemi Kobayashi, Atsushi Ohuchi, Yoshinori Takema and Genji Imokawa, The paracrine role of stem cell factor / c-kit signaling in the activation of human melanocytes in ultraviolet-B-induced pigmentation, The Journal of Investigative Dermatology, 116, 578 -586 (2001). Penkanok Sriwiriyanont, Atsushi Ohuchi, Akira Hachiya, Marty O Visscher and Raymond E Boissy, Interaction between stem cell factor and endothelin-1: effects on melanogenesis in human skin xenografts, Laboratory Investigation, 86, 1115-1125 (2006). Oki Tamori, Takaoka Masanori, Matsumura Ikuo, Endothelin production regulation and involvement in pathology, Pharmaceutical Journal, 127 (9), 1319-1329 (2007). Kazuo Tsujimura, Kohei Miyazono, Keiji Miyazawa, Nobuyuki Tanaka, Cytokine / growth factor terminology library, Yodosha (2005). Chuyaku University Dictionary Vol.2 Shanghai Science and Technology Publishers (Jiangsu New Medical School “Chugaku Daikichi” Editorial Department) Shogakukan) Reference 1 K. Kaiserling Pathloge, 22 (4), 285-286 (2001).

  As described above, there has been an attempt to develop an endothelin-1 and SCF production inhibitor for whitening. Examples include white orchid flower extract (Patent Document 1) and black ginger extract (Patent Document 2). , Yucca extract (Patent Document 3), Western primrose extract, Lemon Verbena extract, Buttercup extract, Blueberry extract, Celery extract (Patent Document 5), Extract from loquat flower Product (Patent Document 6), extract of Koukatoken (Patent Document 7) and the like. However, although these confirmed the suppression of endothelin-1 and SCF mRNA expression increase at the gene level, the protein level did not confirm the suppression of endothelin-1 and SCF production, specifically endothelin-1 and Even though SCF production suppression has been confirmed at the protein level, the final objective, suppression of melanin production, has not been confirmed. Moreover, although there is a report which mentions a melanin production inhibitory action as one of various effects about a hot water extract, the inhibitory action was only about 30-40% suppression.

Then, this invention makes it the 1st subject to develop the endothelin-1 production inhibitor and / or SCF production inhibitor which can confirm concretely suppressing melanin production about the extract of persimmon.
This invention makes it the 2nd subject to develop the more effective melanin production inhibitor derived from a cocoon.

  The inventors of the present application have been searching for a novel endothelin-1 and SCF production inhibitor. From the specific fraction obtained by fractionating the koji extract by column chromatography or two-phase separation, the endothelin-1 and SCF production were inhibited. I found an agent. Furthermore, the inventors of the present invention have completed the present invention by confirming that these endothelin-1 and SCF production inhibitors have significant melanin production-inhibiting ability.

The present invention includes the following inventions.
1. A method for producing an endothelin-1 production inhibitor and / or an SCF production inhibitor comprising a step of extracting from straw with a lower alcohol.
2. A method for producing an endothelin-1 production inhibitor and / or an SCF production inhibitor comprising a step of pulverizing and washing with a hydrophobic organic solvent, a step of extracting with a lower alcohol, and a step of fractionating by column chromatography.
3. 3. The method according to 2 above, wherein the column chromatography uses silica gel chemically bonded with hydrocarbon as a filler.
4). A method for producing an endothelin-1 production inhibitor and / or an SCF production inhibitor comprising a step of pulverizing and washing with a hydrophobic organic solvent, a step of extracting with a lower alcohol, and a step of fractionating by two-phase separation.
5. The step of fractionation by the two-phase separation is a step of fractionation by two-phase separation with a lower ether and an aqueous phase, wherein an acidic aqueous solution and a basic aqueous solution are used for the aqueous phase, respectively. 5. The method according to 4 above, which comprises a step of removing a component and an acidic water-soluble component to obtain a neutral fraction.
6). The step of fractionation by the two-phase separation is a step of fractionation by two-phase separation with a lower ether and an aqueous phase, and an acidic water-soluble component is extracted using a basic aqueous solution in the aqueous phase, and then 5. The method according to 4 above, comprising a step of back-extracting with an organic solvent again to obtain an acidic fraction.
7). 7. The method according to any one of 1 to 6 above, wherein the extraction with a lower alcohol is carried out at a mild temperature to room temperature for 7 days or more.
8). Production of endothelin-1 production inhibitor and / or SCF production inhibitor according to any one of 2 to 7 above, wherein the hydrophobic organic solvent is one or more organic solvents selected from the group consisting of benzene, hexane and chloroform. Method.
9. 9. The method according to any one of 1 to 8 above, wherein the lower alcohol is ethanol, methanol, propanol or butanol, or a mixture of two or more alcohols selected from the group consisting of these alcohols.
10. An endothelin-1 production inhibitor and / or an SCF production inhibitor comprising an extract extracted from straw.
11. The endothelin-1 production inhibitor and / or SCF production inhibitor obtained by the method in any one of said 1-9.
12 The melanin production inhibitor containing the extract extracted by the method including the process of extracting with a lower alcohol from straw.
13. The melanin production inhibitor containing the endothelin-1 production inhibitor and / or SCF production inhibitor obtained by the method in any one of said 1-9.
14 14. A whitening cosmetic comprising the melanin production inhibitor according to 12 or 13 above.

  The present invention has an extremely excellent effect of providing a novel endothelin-1 and SCF production inhibitor and melanin production inhibitor from the koji component. Moreover, since the novel endothelin-1 and SCF production inhibitor and melanin production inhibitor according to the present invention are dissolved in ethanol, they can be incorporated into cosmetics without giving a feeling of roughness. Furthermore, the production of novel endothelin-1 and SCF production inhibitors and melanin production inhibitors of the present invention can be performed by just immersing them in ethanol at about 40 ° C. to room temperature for about 1 month, and equipment that emits high energy is prepared. Even if it does not, it can prepare and is convenient.

The results of endothelin-1 mRNA expression using HaCaT after UV-B irradiation are shown for the ethanol extract, F2 fraction, N fraction, and A fraction. The results of SCF mRNA expression using HaCaT after UV-B irradiation are shown for the ethanol extract, F2 fraction, N fraction, and A fraction. The influence of HaCaT on the endothelin-1 protein production after UV-B irradiation by the ethanol extract, F2 fraction, N fraction and A fraction is shown. The influence with respect to the melanin production using the co-culture of the HaCaT cell and normal mouse melanocytes by the ethanol extract, F2 fraction, and N fraction is shown. The influence with respect to melanin production in a normal human skin three-dimensional model by the ethanol extract, F2 fraction, N fraction, and A fraction is shown. 影響 Shows the effect of SCF protein expression suppression on ethanol extract, F2 fraction, N fraction and A fraction.

1. Introduction 1-1.琥珀 A fossil is a fossil formed mainly by condensing a resin of a pine genus plant underground for a long time, and mainly contains resin, essential oil, succinic acid, and the like. It is soluble in a small amount in ethanol, diethyl ether or benzene (Non-patent Document 1). In addition to the use of the casket itself for decorative crafts, jewelry, insulating materials, and the use of shaving shavings as an incense, it was used for scratches around the 19th century (Non-patent Document 2) . Furthermore, the effect of preventing various diseases associated with aging has been handed down (Yamano Beauty Mate HP http://www.yamanobeautymate.com).

1-2. Endothelin Endothelin is a strong vasoconstrictor peptide composed of 21 amino acids, and there are three types of peptide isomers, endothelin-1, endothelin-2 and endothelin-3. Although it is produced in various tissues of the body such as lung, kidney, brain, and placenta, endothelin-1 is produced in a large amount, particularly in the endothelium of blood vessels. The most important action of endothelin is vasoconstriction, but there are also actions of fibroblast activation and cell proliferation. Endothelin-1 activates various intracellular signal transduction systems such as activation of phospholipase C and opening of calcium channels via endothelin receptors (ETA receptor and ETB receptor) of target cells. As a result, various effects such as regulation of hormone / neurotransmitter secretion and cell proliferation / differentiation are exhibited. Furthermore, endothelin is regarded as an essential substance for the generation and proliferation of melanocytes.

1-3. SCF
SCF is a cytokine that acts at the early stage of hematopoiesis, and there are membrane-bound SCF composed of 273 amino acids and secreted SCF that is cleaved by the action of proteolytic enzymes and released from the membrane. SCF is mainly produced by fibroblasts, bone marrow endothelial cells, and the like.

1-4. Suppression of melanin synthesis stimulation by inhibiting production of endothelin-1 and SCF From many studies, epidermal keratinocytes produce factors such as endothelin-1 and SCF after UV-B irradiation, and epidermal melanocytes proliferate under these stimuli. It has been reported that the ability to synthesize melanin is increased. In particular, coexistence of endothelin-1 and SCF has been reported to synergistically promote the growth of melanocytes and the expression of tyrosinase rather than acting independently, and the signal transduction system that works downstream of these factors. Development of a substance that induces a whitening effect through suppression of melanin production due to blocking of the skin has been actively conducted.
The inventors of the present application have found a composition having the ability to suppress the production of endothelin-1 and SCF from the koji extract.

2. Method for preparing endothelin-1 production inhibitor and SCF production inhibitor from sputum The endothelin-1 production inhibitor and / or SCF production inhibitor of the present invention is an ability to suppress endothelin-1 and / or SCF production extracted from sputum Specifically, the following composition 2-1. And 2-2. And (1) an extract having the ability to suppress endothelin-1 and / or SCF production extracted from koji, and (2) an extract having the ability to suppress endothelin-1 and / or SCF production from koji, respectively. Of the composition to be purified (including crude product).

2-1. Preparation of an extract having the ability to inhibit endothelin-1 and / or SCF production from cocoons The method for extracting an extract having the ability to inhibit endothelin-1 and / or SCF production from the cocoons of the present invention includes: A step of extracting an active ingredient with alcohol. More specifically, the present invention includes at least a step of extracting an extract (composition) which is an active ingredient from the koji with a lower alcohol, and preferably a step of pulverizing the koji as a pretreatment step of extraction. And, if necessary, a step of washing the pulverized soot with a hydrophobic organic solvent. The invention of the present application may also include a step of fractionating the extract extracted by the step of extracting from the straw with a lower alcohol using a hydrocarbon-bonded silica gel column.

The process will be described below.
(i) Pretreatment step The soot is pulverized to an appropriate size so as to facilitate solvent extraction. As the pulverizing means, a file or a jet mill pulverizer can be used. Further, it is also possible to use a material obtained by pulverizing chips produced when processing a jar as a gemstone. The size to be pulverized is not particularly limited. From the viewpoint of extraction efficiency with a solvent, for example, the average particle size can be up to 100 μm, preferably about 10 to 30 μm. The ground soot is washed with a hydrophobic organic solvent as necessary. As the hydrophobic organic solvent, hexane, benzene, chloroform or a mixture thereof can be used. This washing can be omitted.

(ii) Extraction step The soot or the soot that has been crushed, ground and washed in the pretreatment step can be immersed in a lower alcohol to obtain an extract. For example, the soot or the soot that has been crushed or ground and washed in the pretreatment step is immersed in a lower alcohol for a long time at a low temperature or at room temperature to obtain an extract. Specifically, the soot or the soot that has been crushed or ground and washed in the pretreatment step is immersed in a lower alcohol at a temperature of, for example, 25 ° C. to 50 ° C. for 7 days or more, preferably 15 days or more. Obtain an extract. More specifically, for example, in the case of ethanol, the soot or the soot that has been crushed or ground and washed in the pretreatment step is at least 7 days at a temperature of 25 ° C. to 50 ° C., preferably about 40 ° C. Extracts are obtained by soaking for 1 month (15 to 30 days). The extract is concentrated and dried to a brownish candy-like dried product. As the lower alcohol, methanol, ethanol, butanol or a mixture thereof can be used.

  The thus obtained extract having endothelin-1 production inhibiting ability and / or SCF production inhibiting ability is a composition having endothelin-1 production inhibiting ability and / or SCF production inhibiting ability, or endothelin-1 production. It can be used as a composition that enhances the suppressive ability and / or SCF production inhibitory ability. The extract can also be used as a skin whitening agent.

2-2. Crude product of extract having endothelin-1 and / or SCF production inhibitory ability The extract having endothelin-1 and / or SCF production inhibitory ability obtained in the above 2-1 is obtained by column chromatography or two-layer separation. Fractionation can be performed to obtain a composition as a crude product. These compositions can be used as the endothelin-1 production inhibitor and / or the SCF production inhibitor of the present invention.

2-2-1. Fractionation by column chromatography The obtained extract (dried product) is dissolved in a lower alcohol, such as ethanol, and subjected to column chromatography using a hydrocarbon chemically bonded silica gel, such as octadecylsilylated silica gel, as a carrier. Thus, the eluate can be fractionated. In particular, a fraction which is a colorless and transparent liquid which is eluted after the colorless and transparent liquid which is eluted first (for example, 1 to 5% by volume of the total amount of elution) and becomes a dark yellow candy when dried. (For example, 10 to 15% by volume of the total elution amount) is excellent in storage stability and has endothelin-1 production inhibitory ability and / or SCF production inhibitory ability.

  This fraction can be used as a crude product of a composition containing a component having the ability to inhibit endothelin-1 production and / or SCF production from koji. Such a roughly purified product as this fraction can be used as an endothelin-1 production inhibitor and / or an SCF production inhibitor. The extract purified by the column chromatography includes the F2 fraction in the following examples. In addition, the present fraction can be used after being dried or dissolved in an appropriate solvent such as a lower alcohol, specifically ethanol.

Further, it can be further purified by adsorption column chromatography.
Specifically, column chromatography using silica gel as a carrier can be used as the adsorption column chromatography. As an elution solvent, for example, it is possible to elute with a mixed solution of benzene and ethyl acetate as a solvent, specifically, benzene: ethyl acetate = 50: 1.

2-2-2. Fractionation by two-phase separation (i) Removal of water-soluble components 2-1. The lower alcohol extract (dried product) of koji obtained by the above can be further fractionated into two phases, an aqueous phase and an organic solvent phase, using an appropriate organic solvent. Examples of the organic solvent include nonpolar organic solvents such as lower ether, dichloromethane and benzene, and specific examples include dimethyl ether and diethyl ether. The obtained lower alcohol extract of koji is dissolved in the organic solvent, and undissolved components are removed. Next, the organic solvent in which the soot extract is dissolved is washed with water. Specifically, pure water, for example, ultrapure water produced by an ultrapure water production apparatus is added to remove water-soluble components. If necessary, the removal of the water-soluble component using pure water is repeated 3 to 5 times.

(ii) Neutral fraction (N fraction)
Thereafter, an acidic aqueous solution and a basic aqueous solution are added several times as necessary, and mixed until the basic water-soluble component and the acidic water-soluble component are finally removed from the coloring component, respectively. ,Remove. First wash with acidic aqueous solution one or more times, for example, 3-5 times, then wash with basic aqueous solution several times, until the organic solvent in which the soot extract is dissolved is removed. Is desirable. Any acidic aqueous solution can be used as the acidic aqueous solution, and examples thereof include hydrochloric acid, aqueous citric acid solution, and acetic acid, and more specifically, dilute hydrochloric acid having a pH of 3.0. As the basic aqueous solution, any basic aqueous solution can be used. For example, sodium hydroxide aqueous solution, sodium hydrogen carbonate aqueous solution, calcium hydroxide aqueous solution, more specifically, sodium hydroxide having pH 12.0 An aqueous solution can be used. Finally, wash with pure water. The washing solution after washing with the basic solvent and the last pure water is collected for preparation (fraction A).

  The organic solvent is volatilized from the soot extract-dissolved organic solvent from which the basic water-soluble component and the acidic water-soluble component have been removed, and a composition having the ability to inhibit endothelin-1 and / or SCF production can be obtained.

  The neutral fraction (named as N fraction) can be used as a composition having the ability to inhibit endothelin-1 and / or SCF production, or as an endothelin-1 production inhibitor and / or an SCF production inhibitor. .

(Iii) Acidic fraction (A fraction)
The organic solvent in which the soot extract after removal of the water-soluble component in (i) is dissolved is first washed with an acidic aqueous solution as necessary. Although the cleaning with the acidic aqueous solution can be omitted, it is desirable to perform the cleaning first with the acidic cleaning solution. Next, it is washed with a basic aqueous solution. Collect the basic aqueous solution after washing. Further, when the organic solvent is finally washed with pure water, this washing solution can also be added to the basic aqueous solution after washing. Then, after washing, the basic aqueous solution is neutralized with an acid, and if necessary, a salt is added until it is saturated. The salt added here may be the same salt as the salt formed by the basic aqueous solution used in the washing and the acid added for neutralization. For example, when a sodium hydroxide aqueous solution is used as the basic aqueous solution and hydrochloric acid is used for neutralization, sodium chloride can be added as a salt to saturation. After saturation with a salt, back extraction with an organic solvent is performed, and the organic solvent is volatilized to obtain an acidic fraction. The acidic fraction can be a composition having the ability to suppress endothelin-1 and / or SCF production. Examples of the organic solvent used for the back extraction include lower ether, lower ether, dichloromethane, benzene and the like, and specifically, dimethyl ether and diethyl ether, as in the above (i).

  Any acidic aqueous solution can be used as the acidic aqueous solution, and examples thereof include hydrochloric acid, aqueous citric acid solution, and acetic acid, and more specifically, diluted hydrochloric acid having a pH of 3.0. As the basic aqueous solution, any basic aqueous solution can be used. For example, sodium hydroxide aqueous solution, sodium hydrogen carbonate aqueous solution, calcium hydroxide aqueous solution, more specifically, sodium hydroxide having pH 12.0 An aqueous solution can be used.

  The acidic fraction (named as A fraction) can be used as a composition having the ability to inhibit endothelin-1 and / or SCF production, or as an endothelin-1 production inhibitor and / or SCF production inhibitor. .

2-3. Test of endothelin-1 production and SCF production 2-1. The koji extract obtained in 1), the crude product obtained by column chromatography obtained in 2.2.1, and 2-2-2. Tests of endothelin-1 production and SCF production inhibition ability by examining endothelin-1 production and SCF production effects on cells for test samples such as N fraction and A fraction obtained in Can do.

(1) Endothelin-1
Regarding the expression level of endothelin-1, for example, a test sample is given to cells derived from human skin, for example, keratinocytes, and then mRNA is extracted from all cells. For example, mRNA of endothelin-1 is obtained using a commercially available PCR kit. The expression level can be examined.

  Direct quantification of endothelin-1 can also be quantified by, for example, the Elisa method using an anti-endothelin-1 antibody labeled with radiation, fluorescence, biotin, or enzyme.

(2) SCF
Regarding the expression level of SCF, for example, a test sample is given to cells derived from human skin, for example, keratinocytes, and then mRNA is extracted from all cells, and the expression level of SCF mRNA is examined using, for example, a commercially available PCR kit. (Journal of Investigative Dermatology, 116, 578-586 (2001)).

  Further, the direct quantification of SCF can be quantified by, for example, Elisa method or Western blot method using SCF antibody labeled with radiation, fluorescence, biotin, or enzyme.

3. Melanin production inhibitor 2. 2. The endothelin-1 production inhibitor and / or the SCF production inhibitor described in 1., specifically, 2-1. The koji extract obtained in 1), the crude product obtained by column chromatography obtained in 2.2.1, and 2-2-2. The N fraction and the A fraction obtained in the above can be used as a melanocyte stimulation inhibitor and / or a melanin production inhibitor.

3-1. Melanin production inhibition assay The amount of melanin produced can be measured by a colorimetric method after adding and culturing a sample in a co-culture system of keratinocytes and melanocytes or melanoma cells (Fragrance Journal, 33 (5), 60-66 (2005)).

  Alternatively, human skin cells can be added and cultured, and then stained by the Fontana-Masson method, and the degree of staining can be compared for examination.

4). Pharmaceutical preparation or cosmetic The endothelin-1 production inhibitor, SCF production inhibitor, and / or melanin production inhibitor of the present invention can be made into a pharmaceutical preparation or cosmetic, for example, using an appropriate excipient. . For example, it can be powdered into a preparation by absorption in an additive such as lactose, silica gel, crystalline cellulose, or starch.

  Additives usually used in these can be added to the pharmaceutical preparation or cosmetic of the present invention, for example, proteins such as bovine serum albumin, ovalbumin, milk casein, saccharides such as maltose, sucrose, trehalose, Polysaccharides such as heparin, chitosan, alginic acid, polyacrylic acid, polymethacrylic acid and carboxymethylcellulose can also be added as stabilizers.

Furthermore, as a thickener, water-soluble polysaccharides, such as sodium carboxymethylcellulose, sodium alginate, dextran, sodium hyaluronate, etc. are mentioned, for example. Examples of the oil phase thickener include various fatty acids and fats.
Examples of the preservative include alkyl parabens such as methyl paraben and propyl paraben.

4-1. Cosmetics The endothelin-1 production inhibitor, SCF production inhibitor, and / or melanin production inhibitor of the present invention can be added to cosmetics. For example, an extract from salmon having endothelin-1 production inhibitory ability and SCF production inhibitory ability, or a composition purified from the extract can be added to a whitening cosmetic. More specifically, (b) an extract having endothelin-1 production-inhibiting ability and SCF production-inhibiting ability extracted from the straw of the present invention using lower alcohol, (b) endothelin-1 production-inhibiting ability from straw Crude product of composition containing component having ability to suppress SCF production, or F2 fraction, (c) neutral fraction (N fraction) and / or (d) acidic fraction (A fraction) It can be set as the cosmetic for whitening containing this.

  The koji extract of the present invention can be added to cosmetics in a dried state or a state dissolved in an appropriate solvent, for example, a state dissolved in ethanol. The content of the endothelin-1 production inhibitor, the SCF production inhibitor, and / or the melanin production inhibitor of the present invention can be added in an amount of about 0.0001 to 50% by mass when the total cosmetic is 100. .

  For example, the above-described 2-1. Cocoon extract, 2-2-1. Fractionation by column chromatography and / or 2-2-2. For both fractions by two-phase separation, when the total cosmetic is 100, about 0.001 to 5.0 mass%, more preferably 0.001 to 1.0 mass% can be added. Specifically, the F2 fraction, the N fraction, the A fraction, or a mixture of two or more of the three types of fractions, when the total cosmetic is 100, about 0.001 to 5.0% by mass, Preferably, about 0.001 to 1.0 mass% can be added.

  Cosmetics to which endothelin-1 production inhibitor, SCF production inhibitor, and / or melanin production inhibitor obtained from the koji of the present invention are added, regardless of their dosage form, are emulsions, creams, ointments, solutions, Examples include dosage forms such as gels, packs, lotions, powders, and sticks. The cosmetics of the present invention can appropriately contain other additive components that are usually used as cosmetic raw materials. Further, the cosmetic of the present invention may contain other base components that are usually used as cosmetic raw materials. Base ingredients include liquid fats (olive oil, etc.), solid fats (shea fat, etc.), waxes (beeswax, etc.), hydrocarbon oils (liquid paraffin, paraffin, petrolatum, etc.), higher fatty acids (stearic acid, etc.), higher grades Oily components such as alcohol (cetanol, etc.), synthetic ester oil (octyldodecyl myristate, etc.), silicones (methylpolysiloxane, etc.), various surfactants, sequestering agents, water-soluble polymers (carboxyvinyl polymer, etc.) ), Thickeners, various powder components, fragrances, water and the like.

4-2. Skin external preparation The endothelin-1 production inhibitor, SCF production inhibitor, and / or melanin production inhibitor obtained from the sputum of the present invention can also be added to a skin external medicine. Such an external preparation for skin can be used for stains, dullness countermeasures, or removal and prevention of skin pigmentation.

  As an external preparation for skin, it can be produced in various forms such as liquid, paste, cream, ointment, powder and patch. In addition, other externally added ingredients such as mineral oils, higher alcohols, animal and vegetable oils, waxes, silicone oils, moisturizers, wetting agents, water-soluble polymers, lower alcohols, water Antioxidants, pH adjusters, dyes, pigments, antiseptics, antifungal agents and other medicinal agents, chelating agents, and the like can also be added.

  For example, the above-described 2-1. Cocoon extract, 2-2-1. Fractionation by column chromatography and / or 2-2-2. In any case of fractionation by two-phase separation, when the total amount of external preparation for skin is 100, about 0.001 to 5.0% by mass, more preferably 0.001 to 1.0% by mass can be added. Specifically, the F2 fraction, the N fraction, the A fraction, or a mixture of two or more of the three kinds of fractions, when the total amount of the external preparation for skin is 100, about 0.001 to 5.0% by mass, More preferably, about 0.001 to 1.0 mass% can be added.

[Example 1]
<How to extract cocoons>
Russian baltic sea bream was crushed to about 100 mesh, 10 g was immersed in 100 mL of hexane, allowed to stand at room temperature for a week, filtered through filter paper (No. 2), and the filtrate was discarded. The powder collected by filtration was immersed in 100 mL of ethanol and allowed to stand in a 40 ° C. water bath or in a thermostatic chamber for one week, followed by filtration with filter paper (No. 2), and the filtrate was stored in a dark place. The filtered powder was immersed in 50 mL of ethanol, covered with aluminum foil, etc., left on a 40 ° C. water bath or in a thermostat for one week, and then filtered again with filter paper (No. 2). Further, immersion in 50 mL of ethanol was repeated twice, and after filtration, all the filtrates were combined. While slightly warming (about 38 ° C.), the solution was concentrated and dried by an evaporator to obtain about 1.3 g of a brownish candy ethanol extract.

<Preparation of F2 fraction>
Column chromatography using 0.4 g of this ethanol extract in 2 mL of ethanol, 11 g of octadecylsilylated silica gel (Wakogel 100C18, manufactured by Wako Pure Chemical Industries, Ltd.) as a carrier, and methanol (reagent special grade manufactured by Wako Pure Chemical Industries, Ltd.) as a solvent ( Column size: 2.2 cm id × 4.7 cm), and fractionated according to the color of the eluate. Usually, it is judged and sorted by the absorbance at a specific wavelength using an ultraviolet visible spectrophotometer, but it is difficult to obtain information on the soot-containing component accurately, so that it is judged visually. Fractions in which the eluate was dark yellow and transparent (collected amount: 17 mL) were collected and dried by an evaporator to obtain 330 mg of a dark yellow and transparent candy-like fraction. This fraction was designated as F2.

<Preparation of neutral (N) and acidic (A) fractions>
Separately from F2, 0.5 g of ethanol extract was dissolved in 20 mL of diethyl ether (special grade reagent manufactured by Kokusan Kagaku Co., Ltd.) and the undissolved material was removed by filtration, and the filtrate was then removed with a 100 mL separatory funnel (manufactured by SIBATA) Add 5 mL of ultrapure water collected from the ultrapure water production system (made by Nihon Millipore), cap and shake, and shake for about 1 minute to reach equilibrium while opening the cock and returning the internal pressure occasionally. Extracted by mixing. Thereafter, the mixture was allowed to stand for about 5 minutes until it was separated into two layers, and the lower layer was discarded. The extraction was repeated by adding 5 mL of ultrapure water to the separating funnel again, and the work of removing the lower layer was repeated twice. Thereafter, the pH 3.0 hydrochloric acid prepared by adding 2 μL of hydrochloric acid (special grade reagent manufactured by Sigma) to 18 mL of ultrapure water in advance was extracted by adding to a 5 mL separating funnel, and the work of removing the lower layer was repeated three times. Again, 5 mL of ultrapure water was added to the separating funnel for extraction, and the lower layer was discarded. Then, the pH 12.0 sodium hydroxide aqueous solution prepared by adding 45 mg of sodium hydroxide (manufactured by Kanto Chemical Co., Inc.) to 62 mL of ultrapure water in advance was added to the 5 mL separatory funnel and extracted, and the work to remove the lower layer Repeated 6-12 times until the color disappeared. At this time, when it was shaken, sometimes it could not be separated into two layers, but in that case, it was left overnight to separate. When separation was not possible after standing overnight, the contents of the separatory funnel were taken out and separated by centrifugation (3,000 rpm, 30 minutes) with a centrifuge (manufactured by KUBOTA). The lower layer of this pH 12.0 sodium hydroxide aqueous solution was put together and stored in the dark. The remaining funnel was extracted by adding 5 mL of ultrapure water, and the lower layer was discarded. The organic layer remaining in the separatory funnel was collected and dried with an evaporator to obtain 105 mg of a pale yellow solid fraction. This fraction was designated N.

  In addition, add hydrochloric acid to the lower layer of the pH 12.0 aqueous sodium hydroxide solution stored in the dark to neutralize it, and then add sodium chloride (reagent grade manufactured by Junsei Kagaku Co.) until saturated, then add 100 mL Using a liquid funnel, 5 mL of diethyl ether was added three times to 20 mL of this lower layer solution, and back-extraction was performed. All the organic layers were combined and dried with an evaporator to obtain 233 mg of a pale yellow solid fraction. This fraction was designated as A.

[Example 2]
<Confirmation of expression of endothelin-1 gene and SCF gene in gene analysis by GeneChip (trademark) of F2 fraction, N fraction and A fraction>
Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) with 5% bovine serum (FETAL BOVINE SERUM, hereinafter referred to as FBS) (EQUITECH-BIO) and 1% antibacterial agent (Penicillin-Streptomycin Glutamine) (Invitrogen) Suspend human-derived immortalized epidermal keratinocytes (hereinafter referred to as HaCaT) at a concentration of 2.64 × 10 ⁶ cells / mL in a culture medium supplemented with DMEM (13 mL), and add 13 mL of DMEM medium in advance. 2 mL each was seeded in a dish (Nunc) having a diameter of 15 cm and cultured at 37 ° C. for 24 hours under 95% air-5% carbon dioxide gas. After removing the culture supernatant by suction, 15 mL each of a culture solution (hereinafter referred to as serum-free DMEM medium) containing 1% antibacterial agent (Penicillin-Streptomycin Glutamine) in phenol red-free Dulbecco's modified Eagle medium (manufactured by Invitrogen) The dish was further cultured for 48 hours. After removing the culture supernatant by suction, a culture solution containing 0.1% bovine serum albumin (manufactured by EQUITECH-BIO) and 1% antibacterial agent (Penicillin-Streptomycin Glutamine) in phenol red-free Dulbecco's modified Eagle's medium (hereinafter, for treatment) 10 mL of DMEM medium) was added. To this, 5 mL each of F2 fraction, N fraction, and A fraction dissolved in ethanol so that the final concentration was 30 μg / mL was mixed with DMEM medium for treatment (ethanol final concentration: 0.33%) was added. . In addition, a treatment obtained by treating a DMEM medium for treatment with ethanol added to a final ethanol concentration of 0.33% was used as control. These dishes were cultured at 37 ° C. for 24 hours under 95% air-5% carbon dioxide gas.

  Total RNA was extracted from cells treated with the RNA purification kit RNeasy Mini Kit (manufactured by QIAGEN) (the product is hereinafter referred to as total RNA). The total RNA amount was determined by absorbance at 260 nm using a spectrophotometer (Nano Drop). This total RNA was converted into GeneChip (trademark) Eukaryotic Poly-A RNA Control Kit (AFFYMETRIX), GeneChip (trademark) Expression 3'-Amplification One-Cycle cDNA Synthesis Kit (manufactured by AFFYMETRIX), GeneChip (trademark) Sample Cleanup Module ( AFFYMETRIX) and GeneChip (trademark) Expression 3'-Amplification Reagents (AFFYMETRIX) were used to prepare Biotin-Labeled complementary RNA (hereinafter referred to as cRNA) according to the protocol recommended by Affymetrix. The cRNA was hybridized with GeneChip (trademark) using GeneChip (trademark) Hybridazation Oven 640 (manufactured by Affymetrix), washed with GeneChip (trademark) Fluidics Station 450 (manufactured by Affymetrix), stained and then GeneChip (trademark) Scanner 3000 The fluorescence intensity was read with (Affymetrix). Using microarray data analysis software GeneSpring GX Ver. 9. 0.5 (manufactured by Agilent Technologies), the ratio of luminescence was measured based on the obtained image data, and the expression level of endothelin-1 mRNA in the control sample was 100 The ratio of the expression level of endothelin-1 mRNA and the ratio of the expression level of SCF mRNA when the SCF mRNA expression level of the control sample was 100 were calculated.

  The results are shown in Table 1. In HaCaT, endothelin-1 mRNA expression when the F2 fraction is added is 8.4% of the control sample, 9.7% when the N fraction is added similarly, and when the A fraction is added similarly It was suggested that the expression was significantly suppressed at 48.4%. The SCF mRNA expression when the F2 fraction was added was 27.1% with respect to the control sample, 12.0% when the N fraction was added similarly, and 28.7% when the A fraction was added similarly. It was suggested that the expression was significantly suppressed.

[Example 3]
<Confirmation of suppression of endothelin-1 mRNA expression and SCF mRNA expression in the ethanol extract of HaCaT after UV-B irradiation and the F2 fraction, N fraction, and A fraction>
Suspend HaCaT at a concentration of 9.25 × 10 ⁵ cells / mL in DMEM medium, and inoculate 865 μL each in a 6.0 cm diameter dish (manufactured by Nunc) containing 4.1 mL of DMEM medium in advance, 95% air-5 The cells were cultured at 37 ° C. for 24 hours under% carbon dioxide gas. After removing the culture supernatant by aspiration, 5.0 mL each of serum-free DMEM medium was added to the dish and further cultured for 48 hours. After removing the culture supernatant by suction, 2.0 mL of Phosphate-Buffered Saline (hereinafter referred to as PBS) was added. Thereto, the discharge tube of 6W having a peak at 302 nm (UVP Inc.) UVSL-26P ultraviolet irradiation apparatus fitted with a (UVP Inc.) of 125μW / cm ² so that the irradiation dose of 90 mJ / cm ² using UV-B was irradiated for 12 minutes and immediately replaced with 3.75 mL of DMEM medium for treatment. Next, 琥珀 ethanol extract dissolved in ethanol to a final concentration of 25 μg / mL, F2 fraction, N fraction, A fraction each mixed with DMEM medium for treatment (ethanol final concentration: 0.25%), 1.25 mL each was added. In addition, a UV-B non-irradiated sample was obtained by treating a culture solution in which ethanol was added to a treatment DMEM medium to a final ethanol concentration of 0.25% without being irradiated with UV-B. Further, a UV-B irradiated sample was prepared by treating a culture solution in which ethanol was added to a treatment DMEM medium to a final ethanol concentration of 0.25% after UV-B irradiation. These dishes were cultured at 37 ° C. for 24 hours under 95% air-5% carbon dioxide gas.

From these, total RNA was extracted by the method shown in Example 2 above, and the amount of total RNA was determined. Subsequently, based on this total RNA, RT reaction was performed according to the attached document using PrimeScript ™ RT reagent Kit (manufactured by TaKaRa). Furthermore, using SYBR (trademark) Premix Ex Taq ^™ II (manufactured by TaKaRa), a reaction solution was prepared according to the package insert. This reaction solution was reacted with each primer (manufactured by Invitrogen) of endothelin-1, SCF and GAPDH as an internal standard by a quantitative RT-PCR method. And the relative change in the sample addition sample when the endothelin-1 mRNA expression level of the UV-B non-irradiated sample was 100 was calculated. At that time, it was corrected with the value of GAPDH which is an internal standard. The SCF mRNA expression level was calculated in the same manner.

  The results of endothelin-1 mRNA expression are shown in FIG. 1-1. In HaCaT, endothelin-1 mRNA expression after UV-B irradiation increased to 147% of the UV-B non-irradiated sample, whereas when ethanol extract was added after UV-B irradiation 29.1% of UV-B unirradiated sample, 31.1% when F2 fraction was added in the same manner, 32.3% when N fraction was added in the same manner, and A fraction was added in the same way In this case, 60.8%, it was confirmed that endothelin-1 mRNA expression was significantly suppressed despite UV-B irradiation.

  Next, the result of SCF mRNA expression is shown in FIG. In HaCaT, the expression of SCF mRNA after UV-B irradiation increased to 120% of the UV-B unirradiated sample, whereas when the ethanol extract was added after UV-B irradiation, -B. 62.3% of unirradiated sample, 63.0% when F2 fraction is added in the same way, 62.0% when N fraction is added in the same way, and A fraction when added in the same way 88.8%, it was confirmed that SCF mRNA expression was significantly suppressed despite UV-B irradiation.

[Example 4]
<Confirmation of inhibition of endothelin-1 protein production in ethanol extract using HaCaT after UV-B irradiation and F2 fraction, N fraction, and A fraction>
Suspend HaCaT at a concentration of 5.40 × 10 ⁶ cells / mL in DMEM medium, and inoculate 83 μL each in a 6-well plate (manufactured by TPP) containing 2 mL of DMEM medium. 95% air-5% carbon dioxide And cultured at 37 ° C. for 24 hours. After removing the culture supernatant by aspiration, 2 mL each of serum-free DMEM medium was added and further cultured for 48 hours. After removing the culture supernatant by suction, 2.0 mL of PBS was added. Irradiate it with UV-B of 125μW / cm ² for 4 minutes so that the irradiation dose is 30mJ / cm ² using a UVSL-26P UV irradiation device equipped with a 6W discharge tube with a peak at 302nm, and then Immediately PBS was removed. The final concentration of 25 and 50 μg / mL of ethanol extract and F2 fraction, N fraction, and A fraction, respectively, dissolved in ethanol were mixed with DMEM medium for treatment (ethanol final concentration: 0.25% ) Was added in 1.5 mL portions. In addition, a control solution obtained by treating a DMEM medium for treatment with ethanol added to a final treatment concentration of 0.25% after UV-B irradiation was used as a control. These plates were cultured for 24 hours at 37 ° C. under 95% air-5% carbon dioxide gas, and then the amount of endothelin-1 produced in the culture supernatant was transferred to Endothelin-1 Human ELISA Kit (R & D Systems) It measured by ELISA method using. Then, the ratio of the endothelin-1 production amount in the sample-added sample when the endothelin-1 production amount in the control sample was 100 was calculated.

  The results are shown in FIG. In HaCaT after UV-B irradiation, the amount of endothelin-1 produced when the ethanol extract was added to a final concentration of 25 μg / mL was 62.2% of the control sample, and the F2 fraction was similarly added. In the case of 51.4%, when N fraction was added similarly, it was suppressed to 50.9%, and when A fraction was added similarly, it was suppressed to 74.4%, respectively, so that the ethanol extract and F2 fraction, N fraction and A fraction were confirmed to have significant endothelin-1 production inhibitory ability. It was also found that the ethanol extract, F2 fraction, N fraction and A fraction suppress endothelin-1 production in a concentration-dependent manner.

[Example 5]
<Confirmation of suppression of melanin production in ethanol extract and F2 and N fractions using co-culture of HaCaT cells and normal mouse melanocytes>
10% FBS, 1% antibacterial agent (Penicillin-Streptomycin Glutamine), 0.1 mM β-mercaptoethanol (manufactured by Sigma-Aldrich Japan), and 200 nM 12-O-tetradecanoylphorbol 13- Suspend normal mouse melanocytes (hereinafter referred to as Melan-A) at a concentration of 1.27 × 10 ⁶ cells / mL in a culture solution (hereinafter referred to as RPMI1640 medium) supplemented with acetate (TPA) (Roche). 79 μL each was seeded in a 6-well plate (manufactured by TPP) containing 821 μL of RPMI1640 medium in advance. Separately, prepare HaCaT suspended in DMEM medium at a concentration of 3.01 × 10 ⁶ cells / mL and seeded 17 μL each in a cell culture insert (Falcon) containing 333 μL DMEM medium. I put it on the 6-hole plate. This was cultured at 37 ° C. for 24 hours under 95% air-5% carbon dioxide gas. After removing the culture supernatant by aspiration, a culture solution (hereinafter referred to as serum-free RPMI1640 medium) in which 1% antibacterial agent (Penicillin-Streptomycin Glutamine) is added to phenol red-free RPMI1640 medium (manufactured by Invitrogen Corporation) Were placed in 900 μL each and 350 μL each in a cell culture insert, and further cultured for 48 hours. After removing the culture supernatant by suction, a culture solution containing 0.1% bovine serum albumin (manufactured by EQUITECH-BIO) and 1% antibacterial agent (Penicillin-Streptomycin Glutamine) in RPMI1640 medium without phenol red (hereinafter referred to as RPMI1640 medium for treatment) ) Was added to each 6-well plate at 900 μL. Cell culture inserts were prepared by mixing the ethanol extract, F2 fraction, and N fraction dissolved in ethanol to a final concentration of 30 μg / mL in RPMI1640 medium for treatment (ethanol final concentration: 0.25%). 350 μL was added to each. Further, a control solution obtained by treating a RPMI1640 medium for treatment with ethanol added to a final ethanol concentration of 0.25% was used as control.

  After culturing the above plate under 95% air-5% carbon dioxide gas at 37 ° C for 24 hours, only Melan-A was recovered using trypsin-EDTA (manufactured by Immunobiological Laboratories) and the number of cells was counted. 1% IGEPAL CA-630 (manufactured by Sigma Aldrich Japan), 0.1 mM ethylenediaminetetraacetic acid (EDTA) (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) (manufactured by Roche) 20 μL of a 10 mM phosphate buffer solution (pH 6.8) to which was added was added and centrifuged (18,000 rpm, 5 minutes) with a centrifuge (manufactured by KUBOTA). After the supernatant was aspirated, 100 μL of diethyl ether (manufactured by Kokusan Chemical Co., Ltd.) was added and further centrifuged (12,000 rpm, 5 minutes), and the supernatant was aspirated. Thereto, 8 μL of 0.85 N KOH per cell was added to dissolve the precipitate, and the absorbance at 405 nm was measured with a multi-label counter (manufactured by PerkinElmer Japan). The ratio of the melanin production amount in the sample added sample when the melanin production amount in the control sample was 100 was calculated.

The results are shown in FIG. The amount of melanin produced in HaCaT is 22.2% when the ethanol extract is added to the control sample to a final concentration of 30 μg / mL, 35.2% when the F2 fraction is added in the same manner, and the N fraction When the fractions were added in the same manner, they were suppressed to 49.3%, respectively. Thus, it was confirmed that the ethanol extract, F2 fraction, and N fraction had a significant ability to suppress melanin production.

[Example 6]
<Confirmation of suppression of melanin production in ethanol extract and F2, N and A fractions using normal human skin 3D model>
Normal human skin three-dimensional tissue model MEL-300A (manufactured by Kurabo Industries Co., Ltd.) was cultured at 37 ° C. for 1 hour in 95% air-5% carbon dioxide gas. Thereafter, UV-B of 125 μW / cm ² was irradiated for 4 minutes so as to obtain an irradiation dose of 30 mJ / cm ² using a UVSL-26P ultraviolet irradiation device equipped with a 6 W discharge tube having a peak at 302 nm. Subsequently, the ethanol extract and F2 fraction, N fraction, and A fraction dissolved in ethanol to a final concentration of 25 and 50 μg / mL, respectively, were added to Hanks' Balanced Salt Solutions (hereinafter referred to as HBSS) (Invitrogen). 100 μL of ethanol (final ethanol concentration: 0.25%) was added onto the tissue. Further, after UV-B irradiation, a control solution in which ethanol was added to HBSS to a final ethanol concentration of 0.25% was added to the tissue. Furthermore, what was cultured without administering anything on the tissue after UV-B irradiation was defined as a negative control. These were cultured for 15 days at 37 ° C. under 95% air-5% carbon dioxide. At that time, the EPI-100-LLMM maintenance medium (manufactured by Kurabo Industries Co., Ltd.) was replaced every other day, and the ethanol extract, F2 fraction, N fraction, and A fraction were added.

The results are shown in FIG. When melanin was stained black by Fontana-Masson staining, ethanol-treated tissue (b), which is the control, showed a lot of black stained images, compared with 琥珀 ethanol extract, F2 fraction, N fraction And tissue treated with a solution in which the fractions A and A were dissolved in ethanol to a final concentration of 25 μg / mL and 50 μg / mL (c, e, g, and i [25 μg / mL]; d, f, h, and j, respectively) In [50 μg / mL]), a dose-dependent decrease in the black stained image was observed. From the above results, it was found that the ethanol extract and the F2 fraction, the N fraction, and the A fraction have melanin production inhibitory activity.

[Example 7]
<Confirmation of SCF expression suppression in salmon ethanol extract, F2 fraction, N fraction and A fraction using HaCaT after UV-B irradiation>
Suspend HaCaT at a concentration of 8.95 × 10 ⁵ cells / mL in DMEM medium, and inoculate 391 μL each in a 3.5 cm diameter dish (CORNING) containing 1.1 mL of DMEM medium in advance, 95% air-5 The cells were cultured at 37 ° C. for 24 hours under% carbon dioxide gas. After removing the culture supernatant by aspiration, 1.5 mL of serum-free medium was added to the dish and further cultured for 48 hours. After removing the culture supernatant by aspiration, 1.0 mL of PBS was added. Then, 90 mJ / cm ² of UV-B was irradiated once using a UVSL-26P ultraviolet irradiation device equipped with a 6 W discharge tube having a peak at 302 nm, and immediately replaced with 1.125 mL of the treatment medium. Next, the ethanol extract, F2 fraction, N fraction and A fraction dissolved in ethanol so that the final concentration was 25 and 50 μg / mL were mixed with the processing medium (ethanol final concentration: 0.25%). 375 μL each was added. Further, a control solution obtained by treating a culture medium in which ethanol was added to a treatment medium to a final ethanol concentration of 0.25% after UV-B irradiation was used as a control.

  After culturing the above dishes under 95% air-5% carbon dioxide gas at 37 ° C for 48 hours, 0.1M Tris-HCl (pH7.5), 0.01% sodium lauryl sulfate (Sigma Aldrich Japan) in ultrapure water ), 1% NP-40 (manufactured by Merck), and complete tablets (manufactured by Roche) were used to extract intracellular proteins. Subsequently, Western blotting was performed according to a conventional method, and SCF protein expressed in the cells was detected. At that time, electrophoresis was performed using 10% SDS polyacrylamide gel, and the protein was transferred onto a polyvinylidene difluoride membrane (Millipore). When detecting SCF protein, anti-human SCF (K089) rabbit antibody (IBL) is used as the primary antibody, and horseradish peroxidase-labeled anti-rabbit goat antibody (Jackson ImmunoResearch) is used as the secondary antibody. When detecting the standard GAPDH protein, an anti-human GAPDH mouse antibody (Millipore) was used as the primary antibody, and a horseradish peroxidase-labeled anti-mouse goat antibody (Jackson ImmunoResearch) was used as the secondary antibody. Band detection is performed by reacting ECL Plus Western Blotting Detection Reagent (GE Healthcare Japan) with a secondary antibody, chemiluminescence, exposing Hyperfilm ECL (GE Healthcare Japan), and developing. went. For the analysis, ImageJ software was used to calculate the relative change in the sample-added sample when the SCF protein expression level of the control sample was 1.00. At that time, it was corrected with the value of GAPDH which is an internal standard.

The results are shown in FIG. The expression of SCF protein is 0.59 times that of the control sample when the ethanol extract is added to a final concentration of 25 μg / mL after irradiating 90 mJ / cm ² UV-B in HaCaT. When it was added in the same manner, it decreased 0.40 times when the N fraction was added in the same manner, and 0.46 times when the A fraction was added in the same manner, and 0.56 times when the A fraction was added in the same manner. F2 fraction, N fraction, and A fraction were confirmed to have significant SCF expression-inhibiting ability despite being irradiated with UV-B. Moreover, it was found that the ethanol extract, F2 fraction and A fraction suppress the expression of SCF protein in a concentration-dependent manner.

  The present invention relates to the manufacture of cosmetics such as whitening cosmetics containing endothelin-1 production inhibitors, SCF production inhibitors, and / or melanin production inhibitors, and the manufacture of pharmaceuticals such as skin color improvers containing the same ingredients. Can be used.

Claims (7)

Endothelin-, comprising a step of pulverizing the cocoon and washing with a hydrophobic organic solvent, a step of immersing the cocoon washed in the washing step in a lower alcohol to obtain an extract , and a step of fractionating the extract by two-phase separation A production method of 1 production inhibitor and / or SCF production inhibitor ,
The step of fractionation by two-phase separation is a step of fractionation by two-phase separation with a lower ether and an aqueous phase, wherein water, an acidic aqueous solution and a basic aqueous solution are used for the water phase, The method for producing the endothelin-1 production inhibitor and / or the SCF production inhibitor, which is a step of removing a basic water-soluble component and an acidic water-soluble component to obtain a neutral fraction . Endothelin-, comprising a step of pulverizing the cocoon and washing with a hydrophobic organic solvent, a step of immersing the cocoon washed in the washing step in a lower alcohol to obtain an extract , and a step of fractionating the extract by two-phase separation A production method of 1 production inhibitor and / or SCF production inhibitor ,
The step of fractionation by the two-phase separation is a step of fractionation by two-phase separation with a lower ether and an aqueous phase, wherein water and an acidic aqueous solution are used for the aqueous phase, respectively, and water-soluble components and basic After removing the organic components, the basic aqueous solution is used to extract the components that become water-soluble under basic conditions, and then the solution is neutralized and then back-extracted again with a hydrophobic organic solvent to remove the acidic fraction. The manufacturing method of the said endothelin-1 production inhibitor and / or SCF production inhibitor which is a process to obtain . The method according to claim 1 or 2 , wherein the extraction with a lower alcohol is carried out at 25-50 ° C or at room temperature for 7 days or more. Hydrophobic organic solvent in the step of washing with pulverized hydrophobic organic solvent amber, benzene, hexane, and any one of claims 1 to 3 is one or more organic solvents selected from the group consisting of chloroform The manufacturing method of the endothelin-1 production inhibitor and / or SCF production inhibitor of description. The method according to claim 2, wherein the hydrophobic organic solvent in the step of back-extracting to obtain the acidic fraction is one or more organic solvents selected from the group consisting of dichloromethane, benzene, dimethyl ether, and diethyl ether. The method according to any one of claims 1 to 5 , wherein the lower alcohol is ethanol, methanol, propanol or butanol, or a mixture of two or more alcohols selected from the group consisting of these alcohols. The endothelin-1 production inhibitor and / or SCF production inhibitor obtained by the method of any one of Claims 1-6 .

Images