JP5634087B2 - Endothelin-1 mRNA expression increase inhibitor, stem cell growth factor mRNA expression increase inhibitor, basic fibroblast growth factor mRNA expression increase inhibitor, and proopiomelanocortin mRNA expression increase inhibitor - Google Patents

Description

  The present invention relates to an endothelin-1 mRNA expression increase inhibitor, a stem cell growth factor mRNA expression increase inhibitor, a basic fibroblast growth factor mRNA expression increase inhibitor, and a proopiomelanocortin mRNA expression increase inhibitor.

  Spots, buckwheat, skin pigmentation after sunburn, etc. occur as a result of markedly increased production of melanin due to activation of pigment cells (melanocytes) present in the skin. It has become one of the troubles.

  The development of conventional whitening agents has been focused on tyrosinase, which is the rate-limiting enzyme for melanin production. Recently, α-melanocyte is produced as a cytokine that is produced from epidermal keratinocytes after UV-B irradiation and activates melanocytes. Stimulating hormone (α-MSH), endothelin-1, nitric oxide (NO), etc. are known, and various actions leading to the whitening effect by inhibiting the production of melanin by blocking the information transmission system involved. The development of agents has been actively conducted.

  It is also known that blood endothelin-1 levels are elevated in patients with cardiovascular diseases (eg, myocardial infarction, hypertension, ischemic heart disease, etc.) and renal diseases (eg, acute renal failure). Therefore, it is considered that the prevention / treatment effect of these diseases can be expected by suppressing the excessive secretion of endothelin-1, that is, suppressing the increase in the expression of endothelin-1 mRNA.

  Based on this idea, chamomile extract, artea extract (see Non-Patent Document 1) and the like are known as herbal medicines that inhibit the action of endothelin-1 on melanocytes, and endothelin-1 from epidermal keratinocytes is known. Known herbal medicines that suppress the production include citrus extract (see Non-patent Document 1), β-glycyrrhetinic acid stearyl (see Patent Document 1), and the like.

  Stem cell factor (SCF), also called Mast Cell Growth Factor, C-Kit Ligand, Steel Factor, etc., is a protein produced from keratinocytes, fibroblasts, vascular endothelial cells, bone marrow stromal cells, etc. It is. SCF is known to have an action of promoting proliferation and differentiation of pluripotent hematopoietic stem cells, germ cells, mast cells, megakaryocyte precursor cells, granulocyte / macrophage precursor cells, pigment cells and the like. Moreover, it is known that the expression of SCF is enhanced by a spot site, ultraviolet irradiation or the like (see Non-Patent Document 2).

  As SCF, a membrane-bound SCF composed of 273 amino acid residues and a secreted SCF that is cleaved by the action of a proteolytic enzyme and released from the membrane are known. Membrane-bound SCF binds to the SCF receptor of the pigment cell while bound to keratinocytes and promotes the proliferation of the pigment cell. Secreted SCF is cleaved at the binding site, released from the cell membrane, and bound to the SCF receptor of the pigment cell, thereby promoting proliferation of the pigment cell. Furthermore, SCF is a bone marrow in patients with acute myeloid leukemia in the presence of interleukin-3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF). It is known to promote the proliferation of blasts (see Non-Patent Document 3).

  Basic fibroblast growth factor (bFGF), also called FGF-2, is released from keratinocytes by UV irradiation, and the released bFGF acts on pigment cells to synthesize melanin. It is thought that this also promotes cell division of pigment cells (see Non-Patent Document 4). Further, bFGF is known as an angiogenesis promoting factor, and is known to promote angiogenesis in tumor cells (particularly malignant tumor cells).

  Therefore, abnormal production of SCF and bFGF may lead to abnormal proliferation of pigment cells, increase melanin production, and cause stains, freckles, dullness, and the like. In addition, abnormal production of SCF is thought to lead to abnormal proliferation of myeloblasts, thereby causing diseases such as myelodysplastic syndrome, acute myeloid leukemia (AML), and abnormal production of bFGF occurs in tumor cells. It is thought to promote angiogenesis, thereby leading to the growth of tumor cells.

  Therefore, suppressing the increase in expression of SCF mRNA and bFGF mRNA suppresses pigment cell proliferation, suppresses excessive production of melanin in the skin, and is useful for prevention or suppression of pigmentation, sun spots, buckwheat etc. after sunburn. it is conceivable that. Moreover, suppressing the increase in the expression of SCF suppresses the abnormal proliferation of myeloblasts and is considered useful for the prevention or treatment of myelodysplastic syndrome, acute myeloid leukemia, etc., and suppresses the increase in the expression of bFGF. This is considered to be useful for cancer treatment and the like by suppressing angiogenesis in tumor cells and suppressing the growth of tumor cells.

  Based on such an idea, for example, rose extract water, tea extract, hop extract, hawthorn extract, azuki bean powder, birch extract, caihi extract, clove extract, arnica extract, Button extract, Bodaiju, Chlorella extract, Roman chamomile extract, Black tea extract, Eucalyptus extract, Sojutsu extract powder, Peony extract powder, Oolong tea extract powder, Onionis extract, Asenya extract, Grape leaf extract, Bowfish extract, Mulberry extract, Parietaria extract, Anne Known are persica extract, stevia extract, cypress, ginger root extract, soybean extract, scallop extract, bonito extract, altea extract, hypericum extract, and mugwort extract (see Patent Document 2). Moreover, as a thing which can suppress the effect | action of bFGF, onony extract etc. are known, for example (refer patent document 3).

  It is known that α-MSH as a cytokine that activates ACTH and melanocytes, which are secreted from skin epidermis cells and are involved in the production of melanin, is produced using proopiomelanocortin (POMC) as a precursor. . Therefore, by suppressing the production of POMC, that is, by suppressing the increase in the expression of POMC mRNA, it is possible to suppress the production of melanin as a result, and to prevent or improve pigmentation, blemishes, freckles, etc. after sunburn It is thought that you can.

  Conventionally, what has the effect | action which suppresses the expression of POMC is known, for example, panthenol, pyridoxine hydrochloride, nicotinamide, and the like (see Patent Document 4).

JP 2004-300048 A JP 2003-194809 A JP 2005-104904 A JP 2007-176810 A

“Fragrance Journal”, 2000, Vol. 28, No. 9, p. 65-71 Hachiya A et al., J. Invest. Dermatol., No. 116, 2001, p. 578-586 Virginia C. Broudy et al., Blood, Vol.80, No.1, 1992, p.60-67 Halaban R. et al., J. Cell. Biol., No. 107, 1988, p. 1611-1619

  The present invention finds a substance having an endothelin-1 mRNA expression increase inhibitory action, an SCF mRNA expression increase suppressive action, a bFGF mRNA expression increase suppressive action, and a POMC mRNA expression increase suppressive action, and an endothelin-1 mRNA expression increase suppressor comprising the same as an active ingredient, SCF mRNA Expression increase inhibitor, bFGF mRNA expression increase inhibitor, POMC mRNA expression increase inhibitor, disease prevention / treatment agent caused by endothelin-1 mRNA expression increase, disease prevention / treatment agent caused by SCF mRNA expression increase, bFGF mRNA expression increase It is an object of the present invention to provide a preventive / therapeutic agent for a disease to be prevented and a preventive / therapeutic agent for a disease caused by increased expression of POMC mRNA.

  The present inventors succeeded in isolating and identifying a flavone compound having an endothelin-1 mRNA expression increase inhibitory action, an SCF mRNA expression increase suppressive action, a bFGF mRNA expression increase suppressive action and a POMC mRNA expression increase suppressive action from a star fruit extract, The present invention has been completed.

  That is, the present invention relates to an endothelin-1 mRNA expression increase inhibitor, an SCF mRNA expression increase inhibitor, a bFGF mRNA expression increase inhibitor, a POMC mRNA expression increase inhibitor, endothelin, which contains a flavone compound represented by the following formula (I) as an active ingredient. -1 Preventive / Therapeutic Agent for Disease Caused by Increased Expression of mRNA, Preventive / Therapeutic Agent for Disease Caused by Increased Expression of SCF mRNA, Preventive / Therapeutic Agent for Disease Caused by Increased Expression of bFGF mRNA, and Preventing / Provide a therapeutic agent.

  The flavone compound represented by the above formula (I) obtained from star fruit, which is a highly safe natural product, suppresses endothelin-1 mRNA expression increase, suppresses SCF mRNA expression, suppresses bFGF mRNA expression, and POMC mRNA expression. In order to show the action, this is used as an endothelin-1 mRNA expression increase inhibitor, an SCF mRNA expression increase inhibitor, a bFGF mRNA expression increase inhibitor, and a POMC mRNA expression increase inhibitor, thereby causing spots, freckles, and skin darkness (skin pigmentation). Can be prevented, treated or ameliorated.

The present invention will be described below.
The flavone compound of the present invention is represented by the following formula (I). Hereinafter, the flavone compound represented by the following formula (I) is referred to as “carambola flavone”.

  Carambola flavones can be isolated from star fruit, but may also be present in plants other than star fruit. Therefore, the source of isolation of carambola flavones is not limited to star fruit, and any plant containing carambola flavones may be used.

  Carambola flavones are, for example, fixed to polyvinyl alcohol polymers after being subjected to purification operations effective for concentrating flavone compounds such as liquid-liquid partition extraction, various types of chromatography, and membrane separation. It can be obtained by processing by liquid chromatography used as a phase.

  Specifically, first, the star fruit is subjected to an extraction treatment with water, a hydrophilic organic solvent or a mixed solvent thereof to obtain a star fruit extract.

  The constituent part of the star fruit used as the extraction raw material is not particularly limited, and examples thereof include a leaf part, a stem part, a fruit part, and a root part. Among these, it is preferable to use a leaf part as an extraction raw material especially, and these may be used independently and may be used together. Here, the “leaf part” includes not only a complete leaf but also a part of the leaf (eg, leaf blade, petiole, cocoon leaf, leaf sheath, etc.). Star fruit (Averrhoa carambola L.) belongs to the family Oxalis, fresh fruit is edible, and the fruit is called star fruit because its cross section is star-shaped. Star fruit is cultivated in Okinawa, southeastern China, Yunnan and other tropical regions, and can be easily obtained from these regions.

  The star fruit extract can be obtained by drying an extraction raw material, pulverizing the raw material as it is or using a crusher, and subjecting it to extraction with an extraction solvent. Drying may be performed in the sun or using a commonly used dryer. Moreover, after performing pretreatment, such as degreasing, with a nonpolar solvent such as hexane, it may be used as an extraction raw material. By performing pretreatment such as degreasing, the extraction treatment of the star fruit with a polar solvent can be performed efficiently.

  As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. Is preferred.

  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, and those 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 the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.

  When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when using a mixed solution of water and a lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by volume of a lower aliphatic alcohol with respect to 10 parts by volume of water. When using a mixed solution, it is preferable to mix 1 to 40 parts by volume of a lower aliphatic ketone with 10 parts by volume of water, and when using a mixed solution of water and a polyhydric alcohol, water 10 It is preferable to mix 10 to 90 parts by volume of a polyhydric alcohol with respect to the volume part.

  The extraction treatment is not particularly limited as long as at least carambola flavone contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent 5 to 15 times (mass ratio) of the extraction raw material, the soluble components are extracted at room temperature or under reflux, and then filtered to remove the extraction residue. A liquid can be obtained. When the solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and when this concentrate is further dried, a dried product is obtained.

  Next, after the obtained star fruit extract is adsorbed on an adsorbent, it is eluted with water, a water-soluble solvent or a mixed solvent thereof to fractionate a fraction containing a flavone compound.

  The hydrophilic organic solvent that can be contained in the star fruit extract is distilled off as necessary before adsorbing to the adsorbent. The extract obtained by distilling off the hydrophilic organic solvent is dissolved or suspended in, for example, water, a water-soluble solvent or a mixed solvent thereof, and then adsorbed on an adsorbent.

  As the water-soluble solvent used for dissolution, suspension or elution, the above-mentioned hydrophilic organic solvent can be used, and it is preferable to use the lower alcohol having 1 to 5 carbon atoms, particularly methanol and ethanol among these. When a mixed solvent of water and a water-soluble solvent is used, the mixing ratio can be adjusted as appropriate. For example, when using a mixed solvent of water and methanol, the mixing ratio of water and methanol should be 0: 100 to 100: 0 (volume ratio), preferably 40:60 to 60:40 (volume ratio). Can do.

  The adsorbent is not particularly limited as long as it can adsorb the flavone compound, but known adsorbents such as ion exchange resin, synthetic adsorption resin, activated carbon, chelate resin, silica gel, alumina gel-based adsorbent, porous glass, etc. Can be used alone or in combination. Preferably, column chromatography using a porous synthetic adsorbent such as Diaion HP-20 (all manufactured by Mitsubishi Chemical Corporation), which is a porous synthetic adsorption resin, and Chromatrex, which is octadecylsilylated silica gel (ODS). Combined with reverse phase column chromatography using ODS DM1020T (manufactured by Fuji Silysia Chemical Ltd.) as a filler.

  Finally, the obtained eluate is fractionated by liquid column chromatography using water, a water-soluble solvent or a mixed solvent thereof as a mobile phase to obtain a purified flavone compound.

The fractionation method by liquid chromatography is not particularly limited, and can be performed by a usual method. For example, as the stationary phase, a chemically bonded porous spherical silica gel whose surface is modified with a silica gel (for example, silica gel 60 (product name), manufactured by Merck & Co., Inc.) or an octadecyl silyl (C ₁₈ H ₃₇ Si) group. (For example, Chromatorex ODS DM1020T (product name), manufactured by Fuji Silysia Chemical Ltd.), gels such as hydroxypropylated dextran (for example, SEPHADEX LH-20 (product name), manufactured by GE Healthcare Japan), etc. Or in combination.

  As the water-soluble solvent, the above hydrophilic organic solvent can be used, and methanol, ethanol, acetonitrile and the like are particularly preferable. When water and a water-soluble solvent are used, the mixing ratio can be adjusted as appropriate. For example, when a mixed solvent of water and acetonitrile is used, the mixing ratio of water and acetonitrile can be 20:80 to 80:20 (volume ratio), preferably 60:40 (volume ratio).

  In this way, a purified carambola flavone can be obtained by fractionating a fraction containing carambola flavone using liquid chromatography.

  The carambola flavones obtained as described above have an endothelin-1 mRNA expression increase suppressive action, an SCF mRNA expression increase suppressive action, a bFGF mRNA expression increase suppressive action, or a POMC mRNA expression increase suppressive action. Thus, it can be used as an active ingredient of an endothelin-1 mRNA expression increase inhibitor, an SCF mRNA expression increase inhibitor, a bFGF mRNA expression increase inhibitor, or a POMC mRNA expression increase inhibitor.

  Also, carambola flavone uses its endothelin-1 mRNA expression increase inhibitory action to prevent or treat diseases caused by increased endothelin-1 mRNA expression (for example, myocardial infarction therapeutic agent, hypertensive therapeutic agent, ischemic heart It can also be used as an active ingredient of disease therapeutic agents, acute renal failure therapeutic agents, etc.).

  Furthermore, carambola flavone uses its SCF mRNA expression increase inhibitory action to prevent or treat diseases caused by increased expression of SCF mRNA (for example, prevention and treatment of myelodysplastic syndrome, prevention and treatment of acute myeloid leukemia) Agent, antitumor agent, etc.).

  Furthermore, carambola flavone uses its bFGF mRNA expression increase inhibitory action to prevent or treat diseases caused by increased bFGF mRNA expression (for example, angiogenesis inhibitors, anticancer agents, antitumor agents, It can also be used as an active ingredient of a pharmaceutical composition or the like that suppresses metastasis of cancer cells.

  Carambola flavone can also be used as an active ingredient of a prophylactic / therapeutic agent for diseases caused by increased expression of POMC mRNA (for example, stress pruritus etc.) by utilizing its POMC mRNA expression increase suppressing action. .

  The endothelin-1 mRNA expression increase inhibitor, SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor or POMC mRNA expression increase inhibitor of the present invention may be composed solely of carambola flavone, or may be formulated with carambola flavone. Good.

  Carambola flavone can be formulated into any dosage form such as powder, granule, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin, cyclodextrin and any other auxiliary agent. it can. At this time, as an auxiliary agent, for example, an excipient, a stabilizer, a flavoring agent and the like can be used. Examples of the endothelin-1 mRNA expression increase inhibitor, SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor or POMC mRNA expression increase inhibitor formulated with carambola flavone include ointments, liquids for external use, patches and the like. It is done.

  In addition, the endothelin-1 mRNA expression increase suppressor, the SCF mRNA expression increase suppressor, the bFGF mRNA expression increase suppressor, or the POMC mRNA expression increase suppressor of the present invention are, as necessary, an endothelin-1 mRNA expression increase suppressive action or an SCF mRNA expression increase suppressive action. A natural extract or the like having an inhibitory effect on bFGF mRNA expression or POMC mRNA expression can be blended with carambola flavone and used as an active ingredient.

  As an administration method to the patient of the endothelin-1 mRNA expression increase inhibitor, SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor or POMC mRNA expression increase inhibitor of the present invention, subcutaneous tissue administration, intramuscular administration, intravenous administration, oral administration Administration, transdermal administration, and the like can be mentioned, and a suitable method for the prevention / treatment or the like may be appropriately selected according to the type of disease. In addition, the dosage of the endothelin-1 mRNA expression increase inhibitor, SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor or POMC mRNA expression increase inhibitor of the present invention is also the type of disease, severity, individual differences among patients, administration methods, What is necessary is just to increase / decrease suitably according to an administration period etc.

  The endothelin-1 mRNA expression increase inhibitor of the present invention suppresses endothelin-1 production through an inhibitory effect on the increase in endothelin-1 mRNA expression possessed by carambola flavones, stains, buckwheat, skin darkness (skin pigmentation), etc. Can be prevented and improved. Moreover, the endothelin-1 mRNA expression increase inhibitor of the present invention prevents, treats, or improves diseases such as myocardial infarction, hypertension, ischemic heart disease, and acute renal failure through an inhibitory effect on the increase in endothelin-1 mRNA expression of carambolaflavone. can do. However, the endothelin-1 mRNA expression increase inhibitor of the present invention can be used for all purposes that are meaningful in exhibiting the endothelin-1 mRNA expression increase suppressive action in addition to these uses.

  The SCF mRNA expression increase inhibitor of the present invention can suppress the increase in SCF expression through the SCF mRNA expression increase suppressive action of carambola flavone, thereby suppressing pigment cell proliferation and melanin production, Buckwheat, skin pigmentation, etc. can be prevented or improved, and a whitening effect can be obtained. In addition, the SCF mRNA expression increase inhibitor of the present invention can suppress abnormal proliferation of myeloblasts through the suppressive action of carambola flavone on SCF mRNA expression, and thereby, such as myelodysplastic syndrome, acute myeloid leukemia, etc. The disease can be prevented, treated or ameliorated. However, the SCF mRNA expression increase inhibitor of the present invention can be used for all purposes that are meaningful for exhibiting the SCF mRNA expression increase suppression action in addition to these applications.

  The bFGF mRNA expression increase inhibitor of the present invention can suppress the increase in bFGF expression through the bFGF mRNA expression increase suppressive action possessed by carambola flavone, thereby suppressing the proliferation of pigment cells and the production of melanin, Buckwheat, skin pigmentation, etc. can be prevented or improved, and a whitening effect can be obtained. In addition, the bFGF mRNA expression increase inhibitor of the present invention may suppress abnormal angiogenesis in tumor cells through the bFGF mRNA expression increase suppression action of carambola flavone, and prevent, treat or improve diseases such as cancer. it can. However, the bFGF mRNA expression increase inhibitor of the present invention can be used for all purposes that are meaningful for exhibiting the bFGF mRNA expression increase suppression action in addition to these applications.

  The POMC mRNA expression increase inhibitor of the present invention can suppress the increase in POMC expression through the inhibitory action of POMC mRNA expression increase possessed by carambola flavone, thereby biosynthesis of α-MSH as a cytokine that activates melanocytes. Can be suppressed, and spots, buckwheat, skin pigmentation, etc. can be prevented or improved, and a whitening effect can be obtained. Moreover, the POMC mRNA expression increase inhibitor of the present invention can prevent, treat or improve stress-related pruritus and the like through the POMC mRNA expression increase suppression action of carambola flavone. However, the POMC mRNA expression increase inhibitor of the present invention can be used for all purposes other than these applications that are meaningful for exhibiting the POMC mRNA expression increase suppression action.

  In addition, although the endothelin-1 mRNA expression increase inhibitor, the SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor or the POMC mRNA expression increase inhibitor of the present invention are suitably applied to humans, their effects are as follows. 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 carambola flavone 5 kg of pulverized star fruit leaf as an extraction raw material was placed in 20 L of 50% ethanol by volume and heated and extracted at 80 ° C for 3 hours with gentle stirring. Filtered hot. The same extraction operation was repeated for the residue, and the obtained filtrates were combined, concentrated under reduced pressure at 40 ° C., and further dried with a vacuum dryer to obtain 1815 g of a powdery star fruit extract (yield 36.3). %).

  1815 g of the obtained star fruit extract was suspended by adding 1000 mL of water, attached to a porous resin (Diaion HP-20, 6 kg), 30 L of water, 30 L of 40 volume% methanol, 30 L of 80 volume% methanol, Elution was carried out in the order of 30 L of methanol. Subsequently, 80 volume% methanol contained in the fraction eluted with 30 L of 80 volume% methanol was distilled off to obtain 169.8 g of an 80 volume% methanol eluted fraction. 169.8 g of the 80 volume% methanol elution fraction obtained was dissolved in a mixed solvent of chloroform: methanol: water = 10: 5: 1 (volume ratio), and silica gel (trade name: silica gel 60, manufactured by Merck & Co., Inc.) was used. It was injected from the top of a packed glass column and adsorbed onto silica gel. Subsequently, chloroform: methanol: water = 10: 5: 1 (volume ratio) was passed as the mobile phase, and the eluate was collected and desolvated to obtain 42.8 g of a flavone compound concentrate.

  42.8 g of the obtained flavone compound concentrate was dissolved in a mixed solvent of acetonitrile: water = 2: 5 (volume ratio) and filled with ODS (trade name: Chromatorex ODS DM1020T, manufactured by Fuji Silysia Chemical Ltd.). It was injected from the top of the manufactured column and adsorbed on ODS. Next, acetonitrile: water = 2: 5 (volume ratio) was passed as a mobile phase, and the eluate was collected and desolvated to obtain 22.0 g of a flavone compound concentrate.

  22.0 g of the obtained flavone compound concentrate was dissolved in methanol and injected from the top of a glass column filled with a gel having a hydrophobic group (Sephadex LH-20, manufactured by GE Healthcare Japan), Adsorbed. Subsequently, methanol was passed as a mobile phase, and the eluate was collected and desolvated to obtain 16.2 g of a flavone compound concentrate.

  The obtained flavone compound concentrate (16.2 g) was fractionated using high performance liquid chromatography under the following conditions to isolate carambola flavone (1.2 g).

<High performance liquid chromatography condition 1>
Stationary phase: 2 JAIGEL GS-310 (manufactured by Nippon Analytical Industrial Co., Ltd.) Connection column diameter: 20 mm
Column length: 500mm
Mobile phase: Methanol Mobile phase flow rate: 5 mL / min
Detection: RI

<High performance liquid chromatography condition 2>
Stationary phase: Develosil RPAQUEOUS-AR-5 (manufactured by Nomura Chemical Co., Ltd.)
Column diameter: 20mm
Column length: 250mm
Mobile phase: acetonitrile: water = 3: 7 (volume ratio)
Mobile phase flow rate: 9 mL / min
Detection: RI

The isolated carambola flavone was subjected to ¹ H-NMR and ¹³ C-NMR analysis. The results are shown below.

< ¹ H-NMR chemical shift δ (assigned hydrogen):>
0.54 (3H, d, J = 5.6Hz, Rha-6-H), 1.14 (3H, d, J = 6.0H1, Fuc-6-H), 2.38 (1H, m, Rha-5-H), 2.92 (1H, br.s, Rha-4-H), 3.17 (1H, overlapped, Rha-3-H), 3.54 (1H, br.s, Fuc-4-H), 3.62 (1H, overlapped, Rha- 2-H), 3.62 (1H, overlapped, Fuc-3-H), 3.72 (1H, br.q, J = 6.0Hz, Fuc-5-H), 4.16 (1H, br.t, J = 8.8Hz , Fuc-2-H), 4.39 (1H, d, J = 9.6Hz, Fuc-1-H), 5.01 (1H, s, Rha-1-H), 6.51 (1H, s, 8-H), 6.74 (1H, s, 3-H), 6.92 (2H, d, J = 8.8Hz, 3 ', 5'-H), 7.90 (2H, d, J = 8.8Hz, 2', 6'-H)

< ¹³ C-NMR chemical shift δ (assigned hydrogen):>
16.8 (Fuc-6-C), 17.5 (Rha-6-C), 68.0 (Rha-5-C), 70.3 (Rha-3-C), 70.4 (Rha-2-C), 71.2 (Fuc-1 -C), 71.3 (Rha-4-C), 72.0 (Fuc-4-C), 73.6 (Fuc-2-C), 73.6 (Fuc-5-C), 75.7 (Fuc-3-C), 94.4 (8-C), 100.3 (Rha-1-C), 102.7 (3-C), 103.4 (10-C), 108.8 (6-C), 115.8 (3 ', 5'-C), 120.9 (1 '-C), 128.1 (2', 6'-C), 156.3 (9-C), 158.9 (5-C), 160.9 (4'-C), 162.5 (7-C), 163.3 (2-C ), 181.5 (4-C)

  From the above results, it was confirmed that the carambola flavone isolated from the star fruit extract was a flavone compound represented by the following formula (I).

[Test Example 1] Endothelin-1 mRNA expression increase inhibitory action test The above-mentioned carambola flavone (sample 1) was tested for the endothelin-1 mRNA expression increase suppressive 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 1 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 levels of mRNA for endothelin-1 and GAPDH as an internal standard were measured. The 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 endothelin-1 mRNA is the value of GAPDH based on the total RNA preparation prepared from cells cultured without UV irradiation / sample addition, UV irradiation / sample addition and UV irradiation / sample addition, respectively. Then, the correction values were calculated, and the correction values for UV irradiation / no sample addition and UV irradiation / sample addition when the correction value for UV non-irradiation / no sample addition was 100 were calculated. From the obtained results, the endothelin-1 mRNA expression increase suppression rate (%) was calculated by the following formula.

mRNA expression increase suppression rate (%) = {(AB) − (AC)} / (AB) × 100
In the formula, A represents “correction value when no UV irradiation is performed and no sample is added”, B represents “correction value when UV irradiation is performed and no sample is added”, and C is “correction value when UV irradiation is performed and sample is not added”. Represents.
The results are shown in Table 1.

  As shown in Table 1, it was confirmed that carambola flavone has an excellent inhibitory effect on endothelin-1 mRNA expression increase.

[Test Example 2] SCF mRNA expression increase inhibitory action test The carambola flavone (sample 1) was tested for SCF mRNA expression increase inhibitory action as follows.

Normal human neonatal foreskin epidermal keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes in an 80 cm ² flask under conditions of 37 ° C., 5% CO ₂ -95% air. The cells were cultured and collected by trypsinization.

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 2 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 mRNA of SCF and GAPDH as an internal standard was measured. The 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 SCF mRNA was corrected with the value of GAPDH based on the total RNA preparation prepared from cells cultured without UV irradiation / sample addition, UV irradiation / sample addition and UV irradiation / sample addition, respectively. Values were calculated, and correction values for UV irradiation / no sample addition and UV irradiation / sample addition were calculated when the correction value for non-UV irradiation / no sample addition was 100. From the obtained results, the SCF mRNA expression increase suppression rate (%) was calculated by the following formula.

mRNA expression increase suppression rate (%) = {(AB) − (AC)} / (AB) × 100
In the formula, A represents “correction value when no UV irradiation is performed and no sample is added”, B represents “correction value when UV irradiation is performed and no sample is added”, and C is “correction value when UV irradiation is performed and sample is not added”. Represents.
The results are shown in Table 2.

  As shown in Table 2, it was confirmed that carambola flavone has an excellent SCF mRNA expression increase inhibitory action.

[Test Example 3] bFGF mRNA expression increase inhibitory action test The carambola flavone (Sample 1) was tested for bFGF mRNA expression increase suppressive action as follows.

Normal human neonatal foreskin epidermal keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes in an 80 cm ² flask under conditions of 37 ° C., 5% CO ₂ -95% air. The cells were cultured and collected by trypsinization.

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 bFGF and mRNA of GAPDH as an internal standard was measured. The 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 bFGF mRNA is corrected with the value of GAPDH based on the total RNA preparation prepared from cells cultured without UV irradiation / sample addition, UV irradiation / sample addition and UV irradiation / sample addition, respectively. Values were calculated, and correction values for UV irradiation / no sample addition and UV irradiation / sample addition were calculated when the correction value for non-UV irradiation / no sample addition was 100. From the obtained results, the bFGF mRNA expression increase suppression rate (%) was calculated by the following formula.

mRNA expression increase suppression rate (%) = {(AB) − (AC)} / (AB) × 100
In the formula, A represents “correction value when no UV irradiation is performed and no sample is added”, B represents “correction value when UV irradiation is performed and no sample is added”, and C is “correction value when UV irradiation is performed and sample is not added”. Represents.
The results are shown in Table 3.

  As shown in Table 3, carambola flavone was confirmed to have an excellent inhibitory effect on the increase in bFGF mRNA expression.

[Test Example 4] POMC mRNA expression increase inhibitory action test The above-mentioned carambola flavone (sample 1) was tested for POMC mRNA expression increase suppressive action as follows.

Normal human neonatal foreskin epidermal keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes in an 80 cm ² flask under conditions of 37 ° C., 5% CO ₂ -95% air. The cells were cultured and collected by trypsinization.

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 4 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 POMC and the internal standard GAPDH mRNA was measured. The 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). POMC mRNA expression level was corrected with GAPDH values based on total RNA preparations prepared from cells that were cultured without UV irradiation / sample addition, UV irradiation / sample addition and UV irradiation / sample addition, respectively. Values were calculated, and correction values for UV irradiation / no sample addition and UV irradiation / sample addition were calculated when the correction value for non-UV irradiation / no sample addition was 100. From the obtained results, the POMC mRNA expression increase suppression rate (%) was calculated by the following formula.

mRNA expression increase suppression rate (%) = {(AB) − (AC)} / (AB) × 100
In the formula, A represents “correction value when no UV irradiation is performed and no sample is added”, B represents “correction value when UV irradiation is performed and no sample is added”, and C is “correction value when UV irradiation is performed and sample is not added”. Represents.
The results are shown in Table 4.

  As shown in Table 4, it was confirmed that carambola flavone has an excellent POMC mRNA expression increase inhibitory action.

  The endothelin-1 mRNA expression increase inhibitor, the SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor, and the POMC mRNA expression increase inhibitor of the present invention are useful for preventing and improving spots, freckles, skin darkness (skin pigmentation) and the like. It can contribute greatly.

Claims (5)

An endothelin-1 mRNA expression increase inhibitor (excluding use of food and drink ), comprising a flavone compound represented by the following formula (I) as an active ingredient.

A stem cell growth factor (SCF) mRNA expression increase inhibitor (except for use in food and drink ), which comprises the flavone compound represented by formula (I) according to claim 1 as an active ingredient. A basic fibroblast growth factor (bFGF) mRNA expression increase inhibitor comprising the flavone compound represented by formula (I) according to claim 1 as an active ingredient (excluding use of food and drink) . A proopiomelanocortin (POMC) mRNA expression increase inhibitor (except for use in food and drink ), which comprises the flavone compound represented by formula (I) according to claim 1 as an active ingredient. A whitening agent (except for the use of food and drink), which contains the flavone compound represented by the formula (I) according to claim 1 as an active ingredient.