JP5189754B2 - S100A8 expression promoter - Google Patents

Description

  The present invention relates to an S100A8 expression promoter that promotes the expression of S100A8.

  S100A8 (also known as calgranulin A or MRP-8) is a calcium binding protein having calcium responsiveness (Non-patent Document 1). S100A8 has a three-dimensional structure called EF-hand that is commonly found in the calcium-binding site of calcium-binding proteins.

  In recent years, it has been reported that the expression of S100A8 increases with the acute phase response, wound healing, and increased keratinization such as psoriasis (Non-Patent Documents 2 to 4). In addition, S100A8 has been reported to be involved in extracellular and intracellular intermolecular interactions such as binding to keratin fibers (Non-Patent Documents 5 to 6). Furthermore, it has been reported that S100A8 can be used as a marker for UV damage and atopic dermatitis because its expression is increased by UV irradiation and increased expression is observed in atopic dermatitis (Patent Documents 1 and 2).

  On the other hand, it has been reported that substances that promote the expression of the S100A8 gene and S100A8 are effective in promoting skin diseases, wounds, wound healing delay, and hair growth associated with the decrease in S100A8 (Patent Documents 3 to 4).

  Furthermore, as functions of S100A8, it has been reported that the migration activity of neutrophils and monocytes is increased (Non-patent Document 7) and antibacterial activity is exhibited (Non-Patent Document 8).

  It has been found that granule release reaction can be controlled by controlling active S100A8 in a cell line having granule release ability (Patent Document 5). By subjecting the cell line to a treatment to increase active S100A8, the cell line is released into granules, whereas by subjecting it to a treatment to reduce active S100A8, granule release from the cell line is reduced. For example, it has been reported that neutrophils, which are cell lines with the ability to release granules, are subjected to a treatment that reduces active S100A8, thereby suppressing granule release reactions of neutrophils and suppressing damage to the intima. ing.

  Recently, it has been reported that S100A8 is a marker for epidermal regeneration in human skin epidermis (Non-patent Document 9).

Special Table 2005-520483 Japanese Patent Laid-Open No. 2005-110602 US Patent Application Publication No. 2003/0003482 Special Table 2000-501115 International Publication No. 00/18970 Pamphlet Donato, R., Biochim. Biophys. Acta., 1450, 191-231, 1999 Thorey, I.S., et al., J. Biol. Chem., 276, 35818-25, 2001 Siegenthaler, G. et al., J. Biol. Chem., 272, 9371-77, 1997 Broome, AM. Et al., J. Histochem. Cytochem., 51, 675-685, 2003 Donato, R., International J. Biochem. & Cell Biology, 33, 637-668, 2001 Goebeler, M. et al., Biochem. J., 309, 419-424, 1995 Geczy, C.L., Biochim. Biophys. Acta., 1313, 246-253, 1996 Murthy, A.R.K., et al., J. Immunol., 151, 6291-6301, 1993 Marionnet C et al., J. Invest. Dermatol. 121, 1447-58, 2003

  An object of the present invention is to provide an S100A8 expression promoter that promotes the expression of S100A8 in vivo, which is useful for the prevention or treatment of various symptoms and diseases that occur in connection with the decreased expression of S100A8, for example.

  As a result of diligent research to solve the above problems, the present inventors have examined natural substances that regulate the expression of S100A8, and found that a specific plant has an action of promoting the expression of S100A8. The invention has been completed.

  That is, the S100A8 expression promoter according to the present invention contains, as an active ingredient, one or more selected from plants belonging to the family Mulaceae, Leguminosae, Chrysomelidae, Thripsaceae, or Gurlidaceae. Here, as the plant belonging to the mulberry family, Artocarpus lakoocha Roxb., As the plant belonging to the legume family, Burmanem (Albizia lebbeck (Linn.) Benth), Alocasia odora (Roxb.) C Koch), the plant belonging to the family Trichomyceae includes the spinach (Impatiens balsamina L); it can.

  In particular, the above-mentioned active ingredients include a solvent extract of lacchachapanki using water as a solvent, a solvent extract of burmanem using water as a solvent, a solvent extract of a candle using 50% ethanol as a solvent, and 50% ethanol as a solvent. It is preferable that the solvent extract is at least one selected from the group consisting of a sesame solvent extract and a cattle solvent extract containing 50% ethanol as a solvent.

  ADVANTAGE OF THE INVENTION According to this invention, the S100A8 expression promoter useful for the prevention or treatment of the various symptoms and diseases which arise in relation to the fall of S100A8 expression can be provided. Since the S100A8 expression promoter according to the present invention contains a natural substance as an active ingredient, it can be safely used as a medicine, quasi drug or cosmetic.

Hereinafter, the present invention will be described in detail.
The S100A8 expression promoter according to the present invention comprises one or more selected from plants belonging to the family Mulaceae, Leguminosae, Chrysomelidae, Trichogumaceae, or Gurlidaceae, as active ingredients. By administering the S100A8 expression promoter according to the present invention to humans, the expression of S100A8 can be promoted in vivo.

  Here, “S100A8 expression promotion” means expression promotion at the gene level encoding S100A8 and / or S100A8 protein level.

The mulberry family, legume family, gentian family, echidaceae family, or genus family plant used in the S100A8 expression promoter according to the present invention is not particularly limited, but the following plants are preferred.
Mulberry family: Rakuchapannoki (Scientific name: Artocarpus lakoocha Roxb.,: Herbal medicine name: Rakdi)
Legumes: Birmanem (scientific name: Albizia lebbeck (Linn.) Benth, crude drug name: Sirisha)
Chrysomelidae: Roodok (scientific name: Alocasia odora (Roxb.) C Koch, crude drug name: wolf poison)
Thrips medusae: Hosenka (scientific name: Impatiens balsamina L, crude drug name: 鳳 senka)
Catfish family: Catfish (scientific name: Typha latifolia L., T. angustifolia L, crude drug name: Hou, jade yellow)

  In the S100A8 expression promoter according to the present invention, the whole plant, leaves, bark, branches, fruits or roots of the plant can be used as they are. Alternatively, the whole plant, leaves, bark, branches, fruits or roots of the plant may be pulverized.

Sites that are preferably used for the plants specifically listed above are shown below.
Lacchu Chapanki: Bark Birmanem: Bark Wadoku: Root Butter: Flower Catama: Pollen

  On the other hand, in the S100A8 expression promoter according to the present invention, a plant extract is obtained by extracting a plant at room temperature or under heating, or by using an extraction instrument such as a Soxhlet extractor. In the present invention, the plant extract means various solvent extracts obtained by the above extraction method, diluted solutions thereof, concentrated solutions thereof or dried powders thereof.

  As an extraction solvent used for obtaining a plant extract, either a polar solvent or a nonpolar solvent can be used. Examples of the extraction solvent include water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate. Chain and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as toluene; dichloromethane, chloroform and dichloroethane And halogenated hydrocarbons such as carbon dioxide and the like. Alternatively, a mixture obtained by combining two or more of the above solvents can be used as the extraction solvent.

  In particular, when a solvent extract of lacucha bread is used as an active ingredient, the solvent is preferably a polar solvent, particularly preferably an aqueous solvent, and most preferably water. When the solvent extract of burmanem is used as an active ingredient, the solvent is preferably a polar solvent, particularly preferably an aqueous solvent, and most preferably water. When a wax extract is used as the active ingredient, the solvent is preferably a polar solvent, particularly preferably an alcohol solvent, and most preferably a 50% ethanol solvent. When the solvent extract of bosenka is used as an active ingredient, the solvent is preferably a polar solvent, particularly preferably an alcohol solvent, and most preferably a 50% ethanol solvent. In the case of using an extract of cattail as an active ingredient, the solvent is preferably a polar solvent, particularly preferably an alcohol solvent, and most preferably a 50% ethanol solvent.

  As the S100A8 expression promoter according to the present invention, the above plant extract can be used as it is, but after the extract is diluted, concentrated or lyophilized, it can also be used in the form of powder or paste. . In addition, it is also possible to use an extract obtained by removing the inactive contaminants from the extract by subjecting the extract of the plant or the like to a separation technique such as chromatographic liquid-liquid distribution. In the S100A8 expression promoter according to the present invention, two or more extracts of the above-mentioned plants and the like may be mixed and used.

  When the S100A8 expression promoter according to the present invention is used as a medicine or quasi-drug, the dosage form is not particularly limited, but for example, oral preparations such as tablets and capsules, ointments, water Preparations, extracts, lotions and emulsions, and injections. In addition to the plant extract, the pharmaceutical or quasi-drug is optionally combined with pharmaceutically acceptable carriers such as auxiliaries, stabilizers, wetting agents, emulsifiers, absorption enhancers and surfactants. Can be blended.

  On the other hand, when the S100A8 expression promoter according to the present invention is used as a cosmetic, the dosage form is not particularly limited. For example, a water-in-oil type or oil-in-water type emulsified cosmetic, cream , Lotions, gels, foams, essences, foundations, packs, sticks and powders. In addition to plant extracts, the cosmetics include oils, surfactants, ultraviolet absorbers, alcohols, chelating agents, pH adjusters, preservatives, thickeners, which are commonly used as cosmetic ingredients. Pigments, fragrances, various skin nutrients, and the like can be combined in any combination.

  When the S100A8 expression promoter according to the present invention is used as a medicine, quasi-drug or cosmetic, the amount of plant extract is usually calculated as a dry product and is usually a drug, quasi-drug or cosmetic. The total composition is preferably 0.0001 to 5% by weight, more preferably 0.0001 to 0.1% by weight. In addition, when the S100A8 expression promoter according to the present invention is used as a medicine, it is desirable that the dose of the plant extract is 0.01 mg to 1 g / day as a solid residual amount in a normal adult.

  The S100A8 expression inhibitor and S100A8 expression promoter according to the present invention can be pharmacologically evaluated in vitro as follows, for example.

  Examples of the in vitro pharmacological evaluation include a method using a cell line expressing S100A8. The S100A8 expression inhibitor or S100A8 expression promoter according to the present invention is allowed to act on or contact a cell line such as normal human neonatal foreskin-derived epidermal keratinocytes. Next, the cells that have been acted or contacted are cultured, and after the culture, protein or mRNA is extracted from the cells. Further, the obtained protein is subjected to, for example, ELISA or Western blotting. Alternatively, the obtained mRNA is subjected to, for example, PCR or Northern hybridization.

  Compared to cells not acting or contacting the S100A8 expression inhibitor according to the present invention, the amount of S100A8 protein or the amount of mRNA encoding S100A8 is 1.5 or less in the cells acted or contacted by the S100A8 expression promoter according to the present invention. It can be judged that the expression could be promoted at the in vitro level when increased by -10 times, preferably 2-5 times.

  The plant extract contained as an active ingredient in the S100A8 expression regulator according to the present invention promotes the expression of S100A8, as shown in the examples below. Can promote the expression of S100A8.

  As already mentioned, it has been reported that substances that promote the expression of S100A8 gene or S100A8 are effective for promoting skin growth, wound healing delay and hair growth associated with a decrease in S100A8 (Patent Document 1). ~ 2). Furthermore, as functions of S100A8, it has been reported that the migration activity of neutrophils and monocytes is increased (Non-patent Document 7) and antibacterial activity is exhibited (Non-Patent Document 8). In addition, it has been reported that the granule release reaction can be controlled in cell lines (neutrophils, etc.) by increasing or decreasing the expression level of S100A8 (Patent Document 3).

  In view of the above facts, the S100A8 expression promoter according to the present invention is a symptom or disease associated with decreased expression of S100A8, for example, protection or treatment of skin properties such as prevention of wounds or promotion of wound healing, infection It can be used for prevention or treatment of diseases, promotion of hair growth, release of granules (elastase, lactoferrin, etc.) and the like. In addition, plants belonging to the family Mulaceae, Legumes, Chrysomelidae, Thripsaceae, or Legumaceae are used to produce agents for the prevention, amelioration, and treatment of symptoms or diseases associated with decreased expression of S100A8. can do.

  Furthermore, according to the present invention, the expression of S100A8, comprising the step of administering an S100A8 expression promoter comprising one or more selected from plants belonging to the family Mulaceae, Legumes, Glyphaceae, Trichodaceae, or Gurlidaceae. It is possible to provide a method for preventing or treating various symptoms and diseases that occur in relation to the decrease.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Production of plant extract 1
(1) Manufacture of rachuchapannoki extract:
1,000 g of water was added to 100 g of raccoon bread bark, extracted at 70 ° C. for 5 hours, and filtered to obtain an extract. The yield of the extract was 903 mL and the evaporation residue was 1.18 w / v%.
(2) Production of burmanem extract:
To 100 g of Burmanem bark, 1,000 mL of water was added, extracted at 70 ° C. for 5 hours, and then filtered to obtain an extract. The yield of the extract was 887 mL, and the evaporation residue was 0.87 w / v%.
(3) Manufacture of a wax extract A 1,000 g of 50% ethanol was added to 100 g of a candle root, extracted at room temperature for 7 days, and then filtered to obtain an extract. The yield of the extract was 796 mL, and the evaporation residue was 1.24 w / v%.
(4) Manufacture of extract of spinach A 100 mL of spinach flower was added with 1,000 mL of 50% ethanol, extracted at room temperature for 7 days, and then filtered to obtain an extract. The yield of the extract was 772 mL, and the evaporation residue was 2.18 w / v%.
(5) Manufacture of catfish extract To 100 g of catfish pollen, 1,000 mL of 50% ethanol was added, extracted at room temperature for 7 days, and filtered to obtain an extract. The yield of the extract was 642 mL and the evaporation residue was 1.45 w / v%.

[Example 2] S100A8 expression regulation effect 1
In this example, the extract produced in Example 1 was used to verify the expression regulation effect of S100A8.
(1) Materials and Methods Normal human neonatal foreskin-derived epidermal keratinocytes (KK-4009, Strain No 3C0660, Kurabo Industries) were used for the test.
First, cells were plated in a 6-well plate containing 2 ml of Defined-keratinocyte-SFM medium per well and cultured until 50-60% confluent. The culture was performed under conditions of 5% CO 2 and 37 ° C. After reaching 50 to 60% confluence, the medium is added to the Defined-Keratinocyte-SFM medium (hereinafter, “Defined-Keratinocyte-SFM (- The medium was replaced with 1 ml) and acclimated.

  The medium is then replaced with Defined-keratinocyte-SFM (-) medium containing 1.5 millimolar calcium, and the test substance is added directly to the medium to a 0.1% vol concentration (ie, 2 μl added). The test was started. After culturing for 24 hours from the start of the test, the medium was removed by suction, and the cells were washed twice with PBS (−). After washing, 300 μl of CelLytic-M (C-2978, Sigma) was added per well, and the protein was extracted from the cells.

  10 μg of protein obtained from cells subjected to each test substance was subjected to Western blotting. The Western blot was transferred to a PVDF membrane by wet method after SDS-PAGE. The membrane after transfer was subjected to western blotting using S100A8 specific antibody (anti-MRP8 antibody T-1030, BMA Biomedicals), peroxidase-labeled anti-mouse IgG antibody and ECL Advance Reagent (GE Healthcare), and the S100A8 protein band was obtained. Detection was performed with an autoradiography film (GE Healthcare). The signal integrated value of the detected band was measured by Lane & Spot Analyzer (Ato) and the amount of S100A8 protein in the sample was calculated. As a control, cells cultured in the same manner as described above were used except that the test substance was not added.

(2) Results The test results are shown in FIG. In FIG. 1, the amount of S100A8 protein is expressed as a relative value with respect to the case where the amount of S100A8 protein obtained from the control cells is 1.
As is clear from FIG. 1, lachachapanki, burmanem, candle, spinach and cattail promoted the expression of S100A8 protein in normal human neonatal foreskin-derived epidermal keratinocytes. From this result, it is demonstrated that the solvent extract of Lacchachapanki belonging to the mulberry family, Burmeseum belonging to the leguminous family, Rhododendron belonging to the Glyphaceae family, spinach belonging to the Trichodactaceae family, and the cattail belonging to the catfish family can be used as S100A8 expression promoters. It was done.

[Example 3] Production of plant extract 2
In Example 1, an extract was produced using water for rachuchapanki and burmanem, but in this example, an extract was produced using various solvents different from water.
(1) Manufacture of lacucha bread extract (50% ethanol extract):
1,000 mL of 50% ethanol was added to 100 g of bark of Laccha bread and extracted at room temperature for 7 days, followed by filtration to obtain an extract. The yield of the extract was 833 mL and the evaporation residue was 3.21 w / v%.
(2) Manufacture of raccoon bread extract (ethanol extract):
1,000 mL of ethanol was added to 100 g of the bark of Lacchacha bread, extracted at room temperature for 7 days, and then filtered to obtain an extract. The extract yield was 937 mL and the evaporation residue was 1.98 w / v%.
(3) Production of lacucha bread extract (hexane extract):
1,000 mL of hexane was added to 100 g of bark of Laccha bread and extracted for 7 days at room temperature, followed by filtration to obtain an extract. The yield of the extract was 944 mL, and the evaporation residue was 0.22 w / v%.
(4) Production of burmanem extract (50% ethanol extract):
To 100 g of Burmanem bark, 1,000 mL of 50% ethanol was added, extracted at room temperature for 7 days, and filtered to obtain an extract. The yield of the extract was 861 mL, and the evaporation residue was 0.99 w / v%.
(5) Production of burmanem extract (ethanol extract):
To 100 g of Burmanem bark, 1,000 mL of ethanol was added, extracted at room temperature for 7 days, and filtered to obtain an extract. The yield of the extract was 681 mL, and the evaporation residue was 0.05 w / v%.
(6) Production of burmanem extract (hexane extract):
To 100 g of Burmanem bark, 1,000 mL of hexane was added, extracted at room temperature for 7 days, and filtered to obtain an extract. The yield of the extract was 931 mL and the evaporation residue was less than 0.01 w / v%.

[Example 4] S100A8 expression regulation effect 2
In this example, the expression-regulating effect of S100A8 was verified using the extract produced in Example 1 and the extract produced in Example 3 for lachacha bread and burmanem.

(1) Materials and Methods Normal human neonatal foreskin-derived epidermal keratinocytes (KK-4009, Strain No 3C0660, Kurabo Industries) were used for the test.
First, cells were plated in a 6-well plate containing 2 ml of Defined-keratinocyte-SFM medium per well and cultured until 50-60% confluent. The culture was performed under conditions of 5% CO 2 and 37 ° C. After reaching 50 to 60% confluence, the medium is added to the Defined-Keratinocyte-SFM medium (hereinafter, “Defined-Keratinocyte-SFM (- The medium was replaced with 1 ml) and acclimated.

  The medium is then replaced with Defined-keratinocyte-SFM (-) medium containing 1.5 millimolar calcium, and the test substance is added directly to the medium to a 0.1% vol concentration (ie, 2 μl added). The test was started. After culturing for 24 hours from the start of the test, the medium was removed by suction, and the cells were washed twice with PBS (−). After washing, 300 μl of CelLytic-M (C-2978, Sigma) was added per well, and the protein was extracted from the cells.

  10 μg of protein obtained from cells subjected to each test substance was subjected to Western blotting. The Western blot was transferred to a PVDF membrane by wet method after SDS-PAGE. The membrane after transfer was subjected to western blotting using S100A8 specific antibody (anti-MRP8 antibody T-1030, BMA Biomedicals), peroxidase-labeled anti-mouse IgG antibody and ECL Advance Reagent (GE Healthcare), and the S100A8 protein band was obtained. Detection was performed with an autoradiography film (GE Healthcare). The signal integrated value of the detected band was measured by Lane & Spot Analyzer (Ato) and the amount of S100A8 protein in the sample was calculated. As a control, cells cultured in the same manner as described above were used except that the test substance was not added.

(2) Results Table 1 shows the test results. As is clear from Table 1, lachachapanki and burmanem promoted the expression of S100A8 protein more than twice in normal human neonatal foreskin-derived epidermal keratinocytes only in the water extract. On the other hand, the hexane extract of lacucha bread and the 50% ethanol extract of burmanem suppressed the expression of S100A8 protein more than 1.5 times. From this result, it was clarified that, in the case of rachuchapannoki and burmanem, when an extract was produced using water as a solvent, a better expression promoting effect of S100A8 could be achieved.

It is a characteristic figure which shows the expression promotion effect | action of S100A8 protein.

Claims (2)

An S100A8 expression promoter containing a water extract or 50% ethanol extract of Artocarpus lakoocha Roxb. As an active ingredient and excluding use as an antibacterial agent and wound healing agent.   An S100A8 expression promoter containing an aqueous extract of bilmanem (Albizia lebbeck (Linn.) Benth) as an active ingredient and excluding use as an antibacterial agent and wound healing agent.

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