JP2009209050A - Stem cell factor mrna expression increase inhibitor and basic fibroblast growth factor mrna expression increase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a SCFmRNA expression increase inhibitor, a bFGFmRNA expression increase inhibitor, an agent for preventing-treating diseases caused by SCFmRNA expression increase, and an agent for preventing-treating diseases caused by bFGFmRNA expression increase, which each contains, as an active ingredient, a found substance having a SCFmRNA expression increase-inhibiting action or a bFGFmRNA expression increase-inhibiting action. <P>SOLUTION: Provided are the SCFmRNA expression increase inhibitor, the bFGFmRNA expression increase inhibitor, the agent for preventing-treating diseases caused by SCFmRNA expression increase, and the agent for preventing-treating diseases caused by bFGFmRNA expression increase, each of which contains ampelopsin as an active ingredient. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a stem cell growth factor mRNA expression increase inhibitor and a basic fibroblast growth factor mRNA expression increase inhibitor.

  Stem Cell Factor (SCF), also called Must 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 progenitor cells, granulocyte / macrophage progenitor 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 1).

  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 2).

  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 3). 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, Japanese cypress, camphor root extract, soybean extract, scallop extract, bonito extract, altea extract, hypericum extract and mugwort extract (see Patent Document 1). Moreover, as a thing which can suppress the effect | action of bFGF, onony extract etc. are known, for example (refer patent document 2).
JP 2003-194809 A JP 2005-104904 A 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 SCF mRNA expression increase inhibitory action or bFGF mRNA expression increase inhibitory action, and an SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor, and prevention of diseases caused by SCF mRNA expression increase using the substance as an active ingredient. An object is to provide a therapeutic agent and a prophylactic / therapeutic agent for a disease caused by increased expression of bFGF mRNA.

  In order to solve the above problems, the SCF mRNA expression increase suppressor, bFGF mRNA expression increase suppressor, the disease preventive / therapeutic agent caused by the increased SCF mRNA expression or the disease preventive / therapeutic agent caused by the increased bFGF mRNA expression of the present invention, It contains amperopsin as an active ingredient.

  According to the present invention, an SCF mRNA expression increase inhibitor, a bFGF mRNA expression increase inhibitor, a disease caused by an increase in SCF mRNA expression, which can prevent, treat or ameliorate spots, freckles, skin darkness (skin pigmentation), and the like. A therapeutic agent and a prophylactic / therapeutic agent for a disease caused by increased bFGF mRNA expression can be provided.

The present invention will be described below.
The SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor, the disease preventive / therapeutic agent caused by the increased SCF mRNA expression or the disease preventive / therapeutic agent caused by the increased bFGF mRNA expression of the present invention contains amperopsin as an active ingredient.

  Amperopsin is a kind of flavonoid and can be produced by isolation and purification from a plant extract containing ampelopsin. In the present invention, the “extract” refers to an extract obtained by using a plant as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a roughly purified product thereof. Alternatively, any purified product is included.

  A plant extract containing amperopsin can be obtained from a plant containing amperopsin by a method generally used for extraction of plants. Examples of plants containing amperopsin include wisteria tea (scientific name: Ampelopsis grossedentata).

  Fuji tea (Ampelopsis grossedentata) is a perennial plant belonging to the grape family. In China, there are areas where the leaves of this plant are used as beverages, and the roots or whole plants are used as folk medicines for the treatment of jaundice hepatitis, colds, sore throats, acute conjunctival inflammation and the like. Examples of the parts that can be used as the extraction raw material include flower parts, flower parts, fruit skin parts, fruit parts, cocoon parts, stem parts, leaf parts, branch parts, branch leaves parts, trunk parts, bark parts, root parts, and above-ground parts. However, among these, it is preferable to use branches and leaves.

  The Fuji tea extract can be obtained by drying the raw material for extraction and then 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, extraction treatment with a polar solvent of a plant 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. It is preferable.

  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 the soluble component 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.

  The method for isolating and purifying amperopsin from the Fuji tea extract obtained as described above is not particularly limited, and can be performed by a conventional method. For example, the Fuji tea extract is subjected to column chromatography using a porous material such as silica gel or alumina, or a porous resin such as styrene-divinylbenzene copolymer or polymethacrylate, followed by water and alcohol in this order. Elute and obtain as a fraction eluted with alcohol.

  The alcohol used as the eluent in the column chromatography is not particularly limited, and examples thereof include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, isopropyl alcohol, and their aqueous solutions. Can be mentioned.

  Further, the alcohol fraction obtained by column chromatography can be purified by any organic compound such as reverse phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, column chromatography using ion exchange resin, etc. It may be purified using means.

  Since the amperopsin obtained as described above has an SCF mRNA expression increase inhibitory action or a bFGF mRNA expression increase inhibitory action, the active ingredient of the SCF mRNA expression increase inhibitor or bFGF mRNA expression increase inhibitor is utilized using these actions. Can be used as

  In addition, amperopsin uses its SCF mRNA expression increase inhibitory action to prevent or treat diseases caused by increased SCF expression (for example, myelodysplastic syndrome preventive or therapeutic agent, acute myeloid leukemia preventive or therapeutic agent, It can also be used as an active ingredient of antitumor agents and the like.

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

  The SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor, the disease preventive / therapeutic agent caused by the increased SCF mRNA expression or the disease preventive / therapeutic agent caused by the increased bFGF mRNA expression of the present invention may be composed of amperopsin alone. Alternatively, a preparation of ampelopsin may be used.

  Amperpsin 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 and cyclodextrin and any other auxiliary agent. 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 form of an SCF mRNA expression increase inhibitor, a bFGF mRNA expression increase inhibitor, an agent for preventing or treating a disease caused by an increase in SCF mRNA expression, or a drug for preventing or treating a disease caused by an increase in bFGF mRNA expression, for example, include ointment Agents, external solutions, patches and the like.

  The SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor, the disease preventive / therapeutic agent caused by the increased SCF mRNA expression or the disease preventive / therapeutic agent caused by the increased bFGF mRNA expression according to the present invention is used as necessary. A natural extract or the like having an inhibitory effect on the increase in expression or an inhibitory effect on the increase in the expression of bFGF mRNA can be used as an active ingredient by blending with amperopsin.

  As an administration method to a patient of the SCF mRNA expression increase inhibitor, bFGF mRNA expression increase suppressor, the disease preventive / therapeutic agent caused by the increased SCF mRNA expression or the disease preventive / therapeutic agent caused by the increased bFGF mRNA expression of the present invention, the subcutaneous tissue Intramuscular administration, intramuscular administration, intravenous administration, oral administration, transdermal administration, and the like can be mentioned. A suitable method for the prevention / treatment or the like may be appropriately selected depending on the type of disease. In addition, the dosage of the SCF mRNA expression increase inhibitor, bFGF mRNA expression increase inhibitor, the disease preventive / therapeutic agent caused by the increased SCF mRNA expression or the disease preventive / therapeutic agent caused by the increased bFGF mRNA expression of the present invention is also determined depending on the type of the disease. The dose may be increased or decreased appropriately depending on the severity, individual differences among patients, administration methods, administration periods, and the like.

  The SCF mRNA expression increase inhibitor of the present invention can suppress the increase in SCF expression through the inhibitory action of SCF mRNA expression that amperopsin has, thereby suppressing the proliferation of pigment cells and the production of melanin. Skin pigmentation or the like can be prevented or improved, and a whitening effect can be obtained. In addition, the abnormal growth of myeloblasts can be suppressed through the inhibitory effect of amperopsin on the increase in SCF mRNA expression, whereby diseases such as myelodysplastic syndrome and acute myeloid leukemia can be prevented, treated or improved. 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 an increase in bFGF expression through an inhibitory effect on the increase in bFGF mRNA expression possessed by amperopsin, thereby suppressing the proliferation of pigment cells and the production of melanin. Skin pigmentation or the like can be prevented or improved, and a whitening effect can be obtained. Moreover, the bFGF mRNA expression increase inhibitor of the present invention can suppress abnormal angiogenesis in tumor cells through the bFGF mRNA expression increase suppressive action of amperopsin, and can prevent, treat or improve diseases such as cancer. 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 prophylactic / therapeutic agent for diseases caused by increased SCF mRNA expression of the present invention uses the inhibitory action of SCF mRNA expression increased by ampelopsin to cause diseases caused by increased SCF mRNA expression (for example, myelodysplastic syndrome, acute myelogenous leukemia, etc. ) Can be prevented and treated.

  The prophylactic / therapeutic agent for diseases caused by increased bFGF mRNA expression of the present invention can prevent or treat diseases (for example, cancer, etc.) caused by increased bFGF mRNA expression by using the inhibitory effect on the increase in bFGF mRNA expression possessed by ampelpsin. it can.

  The SCF mRNA expression increase inhibitor or bFGF mRNA expression increase inhibitor of the present invention is preferably applied to humans, but may be applied to animals other than humans as long as each effect is exhibited. You can also

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

  1000g of the ground portion of the leaves of Fuji tea was put into 5000mL of 50% ethanol (volume ratio of water to ethanol = 1: 1), kept at 80 ° C for 3 hours with gentle stirring, and then filtered while hot did. The filtrate was concentrated under reduced pressure at 40 ° C., and further dried with a vacuum dryer to obtain a powdery Fuji tea leaf part extract. The extract yield (% by mass) was 17.6% by mass.

The obtained Fuji tea 50 volume% ethanol extract 5.5g was melt | dissolved in the methanol: water = 3: 7 (volume ratio) mixed solvent, and ODS (trade name: Chromatorex ODS DM1020T, Fuji Silysia Chemical Co., Ltd. product) was obtained. It was injected from the top of the packed glass column and adsorbed on ODS. Subsequently, methanol: water = 3: 7 (volume ratio) was passed as a mobile phase, and the eluate was collected, desolvated, recrystallized with water / methanol, and the crystals were separated by filtration. The obtained crystals were dried with a vacuum dryer to obtain 2.2 g of a purified product. The result of analyzing the obtained purified product (sample 1) by ¹³ C-NMR is shown below.

< ¹³ C-NMR chemical shift δ (assigned carbon): DMSO-d ₆ >
71.9 (d, C-3), 83.6 (d, C-2), 95.4 (d, C-8), 96.4 (d, C-6), 100.7 (s, C-10), 107.4 (d, C -2 ', C-6'), 127.6 (s, C-4 '), 133.6 (s, C-1'), 145.9 (s, C-3 ', C-5'), 162.9 (s, C -9), 163.4 (s, C-5), 167.0 (s, C-7), 197.4 (s, C-4)

  From the above results, it was confirmed that the obtained purified product was amperopsin (sample 1).

[Test Example 1] SCF mRNA expression increase inhibitory action test The above-described amperopsin (sample 1) was tested for SCF 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 petri dishes (manufactured by 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: 10 μg / mL) 2 mL was added to each petri dish, and cultured for 24 hours under conditions of 37 ° C., 5% CO ₂ -95% air. 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, manufactured by Takara Bio Inc.) 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 UV non-irradiation / no sample addition was 100. From the obtained results, the SCF mRNA expression promotion 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.

  As a result of the above test, the inhibition rate (%) of amperopsin SCF mRNA expression was 54.4%. Thus, it was confirmed that amperopsin has an excellent inhibitory effect on the increase in SCF mRNA expression.

[Test Example 2] bFGF mRNA expression increase inhibitory action test The above-described amperopsin (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 petri dishes (manufactured by 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 level of bFGF and mRNA of GAPDH as an internal standard was measured. Detection was performed by real-time 2-step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR Kit (Perfect Real Time, code No. RR063A, manufactured by Takara Bio Inc.) 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 promotion 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 amperopsin has an excellent inhibitory effect on the increase in bFGF mRNA expression.

  The SCF mRNA expression increase inhibitor and bFGF mRNA expression increase inhibitor of the present invention can greatly contribute to the prevention and improvement of spots, buckwheat, skin darkness (skin pigmentation) and the like.

Claims (4)

  An inhibitor of stem cell growth factor (SCF) mRNA expression increase, characterized by containing amperopsin as an active ingredient.   A basic fibroblast growth factor (bFGF) mRNA expression increase inhibitor comprising amperopsin as an active ingredient.   A prophylactic / therapeutic agent for a disease caused by elevated expression of stem cell growth factor (SCF) mRNA, comprising amperopsin as an active ingredient.   A prophylactic / therapeutic agent for a disease caused by elevated expression of basic fibroblast growth factor (bFGF) mRNA, characterized by containing amperopsin as an active ingredient.