JP2009227619A - ENDOTHELIN-1 mRNA EXPRESSION ELEVATION INHIBITOR, STEM CELL GROWTH FACTOR mRNA EXPRESSION ELEVATION INHIBITOR, BASIC FIBROBLAST GROWTH FACTOR mRNA EXPRESSION ELEVATION INHIBITOR, AND PROPIOMELANOCORTIN mRNA EXPRESSION ELEVATION INHIBITOR - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find out a substance having an endothelin-1 mRNA expression elevation inhibitory activity, an SCFmRNA expression elevation inhibitory activity, a bFGFmRNA expression elevation inhibitory activity or a POMCmRNA expression elevation inhibitory activity, and to provide an endothelin-1 mRNA expression elevation inhibitor, an SCFmRNA expression elevation inhibitor, a bFGFmRNA expression elevation inhibitor or a POMCmRNA expression elevation inhibitor by using the above substance as an active ingredient. <P>SOLUTION: This extract of yucca is incorporated to the endothelin-1 mRNA expression elevation inhibitor, SCFmRNA expression elevation inhibitor, bFGFmRNA expression elevation inhibitor or POMCmRNA expression elevation inhibitor as the active ingredient. <P>COPYRIGHT: (C)2010,JPO&INPIT

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.

  Conventional whitening agent development has been focused on tyrosinase, the rate-limiting enzyme for melanin production. Recently, alpha-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 (ET-1), nitric oxide (NO), and the like are known, and the production of melanin is suppressed by blocking the information transmission system involved in these and whitening. Various agents that lead to the effect have been actively developed.

  In patients with cardiovascular diseases (for example, myocardial infarction, hypertension, ischemic heart disease, etc.) or renal diseases (for example, acute renal failure, etc.), blood endothelin-1 (ET-1) concentration increases. Since it is known that, by inhibiting excessive secretion of endothelin-1 (ET-1), that is, by suppressing the increase in the expression of endothelin-1 mRNA, the prevention / treatment effect of these diseases can be expected. Conceivable.

  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 (ET-1) on melanocytes, and epidermal keratinocytes are known. As herbal medicines that suppress endothelin-1 (ET-1) production from cucumber, diu extract (see Non-patent Document 1), β-glycyrrhetinic acid stearyl (see Patent Document 1) and the like are known.

  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 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 There are known persimmon 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, etc. (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 or a POMC mRNA expression increase suppressive action, and an endothelin-1 mRNA expression increase suppressor comprising the substance as an active ingredient, SCF mRNA expression elevation inhibitor, bFGF mRNA expression elevation inhibitor, POMC mRNA expression elevation inhibitor, endothelin-1 mRNA expression elevation preventive / therapeutic agent, SCF mRNA expression elevation disease prevention / treatment agent, bFGF mRNA expression elevation An object of the present invention is to provide a preventive / therapeutic agent for a disease caused by the disease and a prophylactic / therapeutic agent for a disease caused by an increased expression of POMC mRNA.

  In order to solve the above-mentioned problems, the endothelin-1 mRNA expression increase inhibitor, the SCF mRNA expression increase inhibitor, the bFGF mRNA expression increase inhibitor, the POMC mRNA expression increase inhibitor, and the prevention / treatment of diseases caused by the increase in endothelin-1 mRNA expression of the present invention. Agent, preventive / therapeutic agent for diseases caused by increased expression of SCF mRNA, preventive / therapeutic agent for diseases caused by increased expression of bFGF mRNA, or preventive / therapeutic agent for diseases caused by increased expression of POMC mRNA, containing Yucca extract as an active ingredient It is characterized by doing.

  According to the present invention, an endothelin-1 mRNA expression increase inhibitor, an SCF mRNA expression increase inhibitor, a bFGF mRNA expression increase inhibitor, and a POMC mRNA that can prevent, treat, or improve spots, buckwheat, skin darkness (skin pigmentation), and the like. A prophylactic / therapeutic agent for diseases caused by increased endothelin-1 mRNA expression, which can provide an expression increase inhibitor, and can prevent or treat various diseases caused by increased expression of endothelin-1 mRNA, SCF mRNA, bFGF mRNA, POMC mRNA, It is possible to provide a prophylactic / therapeutic agent for diseases caused by increased SCF mRNA expression, a prophylactic / therapeutic agent for diseases caused by increased bFGF mRNA expression, and a prophylactic / therapeutic agent for diseases caused by increased POMC mRNA expression.

The present invention will be described below.
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 contain a yucca extract as an active ingredient.

  In the present invention, the “extract” refers to an extract obtained from 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 or a purified product thereof. Any of the objects are included.

  The extraction raw material used in the present invention is yucca. Yucca is an evergreen tree belonging to the genus Yucca belonging to the agave family distributed in the southeastern part of Mexico, and can be easily obtained from these areas. Examples of the constituent parts of yucca that can be used as an extraction raw material include leaf parts, branch parts, stem parts, fruit parts, seed parts, flower parts, root parts, or a mixture of these parts, preferably stem parts. .

  There are various kinds of yucca such as Yucca arizonica, Yucca brevifolia, Yucca elata, Yucca intermdia, Yuccomahavensis, Yucca schidigera, Yucca peninsularis, Yucca schottii, Yucca whipplei. Of these, extract any kind of yucca Although it may be used as a raw material, it is particularly preferable to use Yucca schidigera as an extraction raw material.

  Although details of substances having an inhibitory effect on endothelin-1 mRNA expression, SCF mRNA expression increase, bFGF mRNA expression increase or POMC mRNA expression increase in Yucca extract are unknown, they are generally used for plant extraction. Extracts having these actions can be obtained from yucca by the extraction method.

  The yucca 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 yucca extract obtained as described above has an endothelin-1 mRNA expression increase inhibitory action, an SCF mRNA expression increase suppressive action, a bFGF mRNA expression increase suppressive action or a POMC mRNA expression increase suppressive action, and therefore uses these actions. 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.

  Further, the yucca extract uses its endothelin-1 mRNA expression increase inhibitory action to prevent or treat a disease caused by increased endothelin-1 mRNA expression (for example, prevention of cardiovascular diseases such as myocardial infarction and hypertension). -It can also be used as an active ingredient in therapeutic agents.

  Furthermore, the yucca extract uses its SCF mRNA expression increase inhibitory action to prevent or treat a disease caused by increased SCF mRNA expression (for example, prevention or treatment of myelodysplastic syndrome, prevention or treatment of acute myeloid leukemia) Agent, antitumor agent, etc.).

  Furthermore, the yucca extract 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.

  Moreover, the yucca extract 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-related pruritus etc.) using its POMC mRNA expression increase suppressing action. .

  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 may be composed only of a yucca extract, or a preparation of a yucca extract. Good.

  The yucca extract 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. 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 yucca extract include, for example, ointments, external liquids, 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 may be, if necessary, an endothelin-1 mRNA expression increase suppressor, an SCF mRNA expression increase suppressor. A natural extract having a bFGF mRNA expression increase inhibitory action or POMC mRNA expression increase inhibitory action can be blended with a yucca extract 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 the production of endothelin-1 through the action of suppressing the increase in endothelin-1 mRNA expression of the yucca extract, and stains, buckwheat, skin darkness (skin pigmentation), etc. Can be prevented and improved. Moreover, the endothelin-1 mRNA expression increase inhibitor of the present invention can prevent, treat or improve cardiovascular diseases such as myocardial infarction and hypertension through the endothelin-1 mRNA expression increase suppressive action of the yucca extract. However, the endothelin-1 mRNA expression increase inhibitor of the present invention can be used for all uses other than these uses, which are meaningful for exhibiting the endothelin-1 mRNA expression increase suppressive action.

  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 the yucca extract, 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 growth of myeloblasts through the SCF mRNA expression increase inhibitory action possessed by the Yucca extract, 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 of the yucca extract, 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 can suppress abnormal angiogenesis in tumor cells through the bFGF mRNA expression increase suppression action of the yucca extract, and can 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 POMC mRNA expression increase suppressive action of the yucca extract, and 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 the yucca extract. However, the POMC mRNA expression increase inhibitor of the present invention can be used for all purposes that are meaningful for exhibiting the POMC mRNA expression increase suppression action in addition to these uses.

  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 test example is shown and this invention is demonstrated concretely, this invention is not restrict | limited to the following test example at all. In this test example, a yucca extract (product name: Yucca Sarasaponin 80-M, sample 1) manufactured by Mitsuba Trading Co., Ltd. was used.

[Test Example 1] Endothelin-1 mRNA expression increase inhibitory action test The above-mentioned yucca extract (sample 1) was tested for its endothelin-1 mRNA expression increase suppressive action as follows.

Normal human epidermis keratinocyte (NHEK) in an 80 cm ² flask in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermis keratinocytes (EpiLife-KG2) 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 the table 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). The amount of each RNA is measured with a spectrophotometer, and the total amount is adjusted to 200 ng / μL. 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. 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 the yucca extract has an excellent endothelin-1 mRNA expression increase inhibitory action.

[Test Example 2] SCF mRNA expression increase inhibitory action test The Yucca extract (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 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 the table 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 UV non-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 the yucca extract has an excellent inhibitory effect on the increase in SCF mRNA expression.

[Test Example 3] bFGF mRNA expression increase inhibitory action test The Yucca extract (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 the table 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, it was confirmed that the yucca extract has an excellent inhibitory effect on the increase in bFGF mRNA expression.

[Test Example 4] POMC mRNA expression increase inhibitory action test The Yucca extract (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 the table 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 the yucca extract 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 (8)

  An endothelin-1 mRNA expression increase inhibitor comprising a yucca extract as an active ingredient.   A stem cell growth factor (SCF) mRNA expression increase inhibitor comprising a yucca extract as an active ingredient.   A basic fibroblast growth factor (bFGF) mRNA expression increase inhibitor comprising a yucca extract as an active ingredient.   A proopiomelanocortin (POMC) mRNA expression increase inhibitor comprising a yucca extract as an active ingredient.   A prophylactic / therapeutic agent for diseases caused by increased expression of endothelin-1 mRNA, comprising a yucca extract as an active ingredient.   A prophylactic / therapeutic agent for a disease caused by elevated expression of stem cell growth factor (SCF) mRNA, comprising a yucca extract as an active ingredient.   A prophylactic / therapeutic agent for diseases caused by increased expression of basic fibroblast growth factor (bFGF) mRNA, comprising a yucca extract as an active ingredient.   A prophylactic / therapeutic agent for diseases caused by elevated expression of proopiomelanocortin (POMC) mRNA, comprising a yucca extract as an active ingredient.