SMAD6 expression inhibitor

Abstract

To provide an SMAD6 expression inhibitor capable of reducing expression of SMAD6.SOLUTION: An SMAD6 expression inhibitor comprising Geranium thunbergii or an extract thereof as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an SMAD6 expression inhibitor, a TGF-β signaling enhancer, an NEP expression inhibitor, a microfibril formation promoter, and an elastic fiber formation promoter. [Background technology]

[0002] Transforming growth factor-β (TGF-β) is a cytokine expressed in various cells and is known to regulate cell proliferation, cell differentiation, immune regulation, cell motility, angiogenesis, and other functions. Stimulation by TGF-β is transmitted via the TGF-β / SMAD signaling pathway, which is composed of the TGF-β receptor and SMAD proteins. When dimerized TGF-β binds to receptor 2, which consists of two molecules present on the cell membrane, receptor 1, which also consists of two molecules, is recruited, and the two receptors form a heterotetramer. Receptor 2 then phosphorylates serine / threonine residues in receptor 1, and activated receptor 1 phosphorylates SMAD2 and SMAD3 (SMAD2 / 3) via the Smad anchor for receptor activation (SARA), thereby transmitting signals intracellularly. Activated SMAD2 / 3 form a heterotrimer with SMAD4, translocate to the nucleus, and cooperate with transcription factors to regulate the transcription of various target genes (Non-Patent Document 1). For example, it has been reported that in dermal fibroblasts, TGF-β stimulation promotes elastin gene expression via SMAD, promoting the formation of elastic fibers (Non-Patent Documents 2 and 3).

[0003] On the other hand, the TGF-β / SMAD signaling pathway is suppressed intracellularly by inhibitory factors called I-SMADs (inhibitory Smads). I-SMADs include SMAD6 and SMAD7, and it has been reported that SMAD6 inhibits the phosphorylation of SMAD2 and the subsequent complex formation with SMAD4, thereby suppressing the TGF-β signal transduction pathway (Non-Patent Document 4). Therefore, suppressing SMAD6 is thought to be useful for enhancing TGF-β / SMAD signaling.

[0004] Geranium herb is a perennial plant of the Geraniaceae family, and for example, Patent Document 1 reports that an extract of Geranium herb has a blood vessel strengthening effect and an effect of inhibiting vascular endothelial cell contraction. Furthermore, Patent Document 2 reports that an extract of Geranium herb has an effect of increasing the expression level of microfibrillar-associated protein 4 (MFAP4). However, it is unknown what effect Geranium Herb has on SMAD6 expression and the formation of microfibrils and elastic fibers. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-36673 [Patent Document 2] Japanese Patent Publication No. 2021-100977 [Non-patent literature]

[0006] [Non-Patent Document 1] Kohei Miyazono Journal of the Japanese Society of Internal Medicine 105, 9, 1558-156 [Non-patent document 2] Kahari VM, et al., Lab Invest. 1992 May;66(5):580-8 [Non-patent document 3] Neumann C, et al., Invest Ophthalmol Vis Sci. 2008 Apr;49(4):1464-72 [Non-patent document 4] Imamura T, et al., Nature. 1997 Oct 9;389(6651):622-6 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing an SMAD6 expression inhibitor that inhibits the expression of SMAD6. [Means for solving the problem]

[0008] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that an extract of Geranium Herb suppresses the expression of SMAD6 and also suppresses the expression of elastic fiber degradation-related factors, while promoting the expression of elastic fiber formation-related factors and promoting the formation of microfibrils and elastic fibers.

[0009] That is, the present invention relates to the following 1) to 5). 1) An SMAD6 expression inhibitor containing Geranium herb or its extract as an active ingredient. 2) A TGF-β signal transduction enhancer containing Geranium herb or its extract as an active ingredient. 3) An NEP expression inhibitor containing Geranium herb or its extract as an active ingredient. 4) A microfibril formation promoter containing Geranium Herb or an extract thereof as an active ingredient. 5) An elastic fiber formation promoter containing Geranium herb or its extract as an active ingredient. [Effects of the Invention]

[0010] According to the present invention, it is possible to suppress the expression of SMAD6. Furthermore, according to the present invention, it is possible to control the expression of elastic fiber-related factors and promote the formation of microfibrils and elastic fibers. [Brief explanation of the drawings]

[0011] [Figure 1] Microfibril formation promoting effect of Geranium Herb extract (immunofluorescence staining image). [Figure 2] The binarized image in Fig. 1. [Figure 3] The effect of Geranium Herb extract in promoting elastic fiber formation (immunofluorescence staining image). [Figure 4] The binarized image in Fig. 3. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, "Geranium thunbergii" refers to Geranium thunbergii, a plant of the Geraniaceae family, Geranium genus. Examples of the parts of the plants to be used include the whole plant, leaves, stems, buds, flowers, buds, roots, rhizomes, pseudocorms, tuberous roots, lichens, thallus, seeds, fruits, kernels, and mixtures thereof. The parts of the plants to be used are preferably leaves, stems, flowers, and roots, and more preferably leaves, stems, and flowers.

[0013] Such plants can be used as they are, or as juice obtained by squeezing them, or as a dried or pulverized product of the plant itself, or as an extract extracted from these, but it is preferable to use them as an extract.

[0014] Examples of extracts include various solvent extracts obtained by extracting the above-mentioned plants at room temperature or under heating, or by extracting using extraction equipment such as a Soxhlet extractor, extracts obtained by supercritical extraction using supercritical carbon dioxide or the like, diluted solutions thereof, concentrated solutions thereof, and dried powders thereof. The extraction method is not particularly limited, but may be any of the usual methods such as immersion, decoction, percolation, reflux extraction, ultrasonic extraction, microwave extraction, stirring, etc.

[0015] The solvent for extraction can be either polar or nonpolar. Specific examples of solvents include water; monohydric, dihydric, or polyhydric alcohols; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear or cyclic ethers such as diethyl ether and tetrahydrofuran; polyethers such as polyethylene glycol; saturated or unsaturated hydrocarbons such as hexane; aromatic hydrocarbons such as benzene and toluene; halogenated hydrocarbons such as dichloromethane, chloroform, dichloroethane, and carbon tetrachloride; pyridines; dimethyl sulfoxide; acetonitrile; carbon dioxide; supercritical carbon dioxide; fats and oils, waxes, and other oils; and mixtures thereof. Suitable solvents include water, alcohols, and aqueous solutions thereof. Examples of alcohols include methanol, ethanol, 1,3-butylene glycol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, and t-butanol. Ethanol and 1,3-butylene glycol are preferred, and ethanol is more preferred.

[0016] The concentration of the alcohols in the aqueous solution of the alcohols (volume percentage at 25°C, hereinafter referred to as % (v / v)) is preferably 12% (v / v) or more, more preferably 15% (v / v) or more, even more preferably 18% (v / v) or more, and is preferably 99.8% (v / v) or less, more preferably 90% (v / v) or less, even more preferably 85% (v / v) or less. The concentration of the alcohol in the aqueous solution of the alcohol is preferably 12 to 99.8% (v / v), more preferably 15 to 90% (v / v), and even more preferably 18 to 85% (v / v). The extraction solvent in the present invention is preferably an 18 to 85% (v / v) aqueous ethanol solution, more preferably a 30 to 70% (v / v) aqueous ethanol solution, and even more preferably a 40 to 60% (v / v) aqueous ethanol solution.

[0017] The amount of solvent used in extraction is preferably 1 to 100 mL, more preferably 3 to 50 mL, per 1 g of the plant (converted to dry mass). The extraction conditions are not particularly limited as long as they allow sufficient extraction. For example, the extraction time is preferably 1 hour or more, more preferably 3 hours or more, and is preferably 2 months or less, more preferably 5 weeks or less, and more preferably 3 weeks or less. The extraction temperature is preferably 0° C. or higher, more preferably 5° C. or higher, and is preferably below the boiling point of the solvent, more preferably below 90° C. Generally, extraction is performed for a long time at low temperatures and for a short time at high temperatures.

[0018] The extract may be a crude product as long as it meets the standards acceptable for cosmetics and pharmaceuticals and exhibits the effects of the present invention. If necessary, the extract may be subjected to treatments such as removal of inactive contaminants, deodorization, decolorization, etc., using known techniques such as liquid-liquid partitioning, solid-liquid partitioning, filtration membranes, activated carbon, adsorption resins, ion exchange resins, and precipitation. Furthermore, the purity of these compounds may be increased by appropriately combining known separation and purification methods, such as organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatography, and column chromatography.

[0019] The extract may be used as is, diluted with an appropriate solvent, or prepared as a concentrated extract, dried powder, or paste. It may also be lyophilized and diluted with solvents commonly used in extractions, such as water, ethanol, 1,3-butylene glycol, a water-ethanol mixture, or a water-1,3-butylene glycol mixture, before use. It may also be encapsulated in vesicles such as liposomes or microcapsules.

[0020] As shown in the Examples below, Geranium Herb extract suppresses the gene and protein expression of SMAD6 in dermal fibroblasts. As mentioned above, SMAD6 has been reported to have an inhibitory effect on the TGF-β signaling pathway. Therefore, it is thought that suppression of SMAD6 expression will enhance TGF-β / SMAD signaling.

[0021] Furthermore, as shown in the Examples below, Geranthes herb extract suppresses the gene expression of neprilysin (NEP), a factor related to elastic fiber degradation, while promoting the gene expression of fibrillin-1 (FBN1) and tropoelastin (ELN), factors related to elastic fiber formation, thereby promoting the formation of microfibrils and elastic fibers. Neprilysin (NEP) is a type II membrane glycoprotein that has been reported to degrade elastic fibers (Morisaki N, et al., J Biol Chem. 2010 Dec 17;285(51):39819-27). "Fibrillin-1 (FBN1)" is the main glycoprotein constituting microfibrils. "Tropoelastin (ELN)" is a precursor to elastin. "Microfibrils" are glycoprotein complexes that, together with tropoelastin, make up elastic fibers. "Elastic fibers" are extracellular matrices formed by cross-linking elastin, an aggregate of tropoelastin, onto microfibrils (Kielty CM, et al., J Cell Sci. 2002 Jul 15;115(Pt 14):2817-28). Elastic fibers are contained in the heart, and a decrease in the amount of elastic fibers in the heart leads to a decrease in elasticity, leading to systolic and diastolic dysfunction, which has been suggested to be one of the causes of heart failure (Mujumdar VS, et al., Int J Cardiol. 2001 Jul;79(2-3):277-86). In addition, fascia is a connective tissue containing elastic fibers (Thomas F, et al., J Bodyw Mov Ther. 2012 Jan;16(1):67-75). Elastic fibers are an important component that contributes to the elasticity of fascia, but the amount of elastic fibers in fascia decreases with age, leading to a decrease in elasticity (JP 2022-80107 A). It has been suggested that a decrease in fascial elasticity can cause decreased muscle or joint mobility and pain (JP 2018-135272 A). Therefore, it is believed that the elasticity of the heart and the elasticity of the fascia can be improved by controlling the expression of elastic fiber-related factors and promoting the formation of microfibrils and elastic fibers as described above.

[0022] Therefore, Gennosho or an extract thereof can serve as an SMAD6 expression inhibitor, a TGF-β signaling enhancer, an NEP expression inhibitor, a microfibril formation promoter, and an elastic fiber formation promoter (hereinafter also referred to as "SMAD6 expression inhibitor, etc."), and can be used to suppress SMAD6 expression, enhance TGF-β signaling, suppress NEP expression, promote microfibril formation, and promote elastic fiber formation, and can also be used to produce SMAD6 expression inhibitors, etc. Therefore, the SMAD6 expression inhibitors of the present invention can be used for purposes such as inhibiting the decline in elasticity, promoting elasticity, strengthening or restoring elasticity of the heart, and inhibiting the decline in elasticity, promoting elasticity, strengthening or restoring elasticity of fascia. Here, "use" can refer to use on humans or non-human animals, and can be therapeutic or non-therapeutic. "Non-therapeutic" does not include medical procedures, i.e., methods of surgery, treatment, or diagnosis on humans, and more specifically, does not include methods of surgery, treatment, or diagnosis performed on humans by physicians, medical professionals, or persons under the direction of a physician. In the present invention, non-therapeutic use includes the use of the above-mentioned plants or extracts thereof for cosmetic or aesthetic purposes.

[0023] As used herein, "suppression of SMAD6 expression" encompasses suppression of SMAD6 expression at the gene level and suppression of SMAD6 expression at the protein level. Suppression of expression at the gene level includes inhibition of transcription of mRNA encoding SMAD6, while suppression of expression at the protein level includes inhibition of mRNA translation. "SMAD6" is a protein with a molecular weight of 60 kDa that belongs to the SMAD family and is classified as an I-SMAD.

[0024] "Enhanced TGF-β signaling" means that stimulation by TGF-β via the TGF-β / SMAD signal pathway is enhanced.

[0025] "Suppression of NEP expression" encompasses suppression of NEP expression at the gene level and suppression of NEP expression at the protein level. Suppression of expression at the gene level includes inhibition of transcription of mRNA encoding NEP, while suppression of expression at the protein level includes inhibition of mRNA translation. Of these, suppression of expression at the gene level is preferred in the present invention.

[0026] "Promotion of microfibril formation" means promotion of polymerization of fibrillin-1 (FBN1), a glycoprotein that is the main constituent of microfibrils. In the present invention, promotion of microfibril formation in dermal fibroblasts is preferred.

[0027] "Promotion of elastic fiber formation" means that tropoelastin (ELN) binds to microfibrils, promoting the formation of mature elastic fibers. In the present invention, promotion of elastic fiber formation in dermal fibroblasts is preferred.

[0028] The SMAD6 expression inhibitors of the present invention may themselves be pharmaceuticals, quasi-drugs, or cosmetics for suppressing SMAD6 expression, enhancing TGF-β signaling, suppressing NEP expression, promoting microfibril formation, or promoting elastic fiber formation, or may be materials or preparations to be incorporated into such pharmaceuticals, quasi-drugs, or cosmetics.

[0029] The pharmaceutical (including quasi-drugs, the same applies hereinafter) contains Geranium Herb or an extract thereof as an active ingredient for suppressing SMAD6 expression, enhancing TGF-β signaling, suppressing NEP expression, promoting microfibril formation, or promoting elastic fiber formation. Furthermore, the pharmaceutical may contain a pharmaceutically acceptable carrier or other active ingredient, medicinal ingredient, etc. as needed, as long as the function of the active ingredient is not lost. The administration form of the pharmaceutical product is not particularly limited, but is preferably parenteral. Dosage forms for parenteral administration include various preparations for topical application to the skin, transdermal, transmucosal, nasal, rectal, injection, suppository, injection, inhalation, patch, etc. Among these, the preferred preparation form is a topical application to the skin, specifically, ointment, emulsion, cream, milky lotion, lotion, gel, aerosol, etc.

[0030] The cosmetic contains Geranium Herb or an extract thereof as an active ingredient for suppressing SMAD6 expression, enhancing TGF-β signaling, suppressing NEP expression, promoting microfibril formation, or promoting elastic fiber formation. Furthermore, the cosmetic may contain a cosmetically acceptable carrier, other active ingredients, cosmetic ingredients, etc., as needed, as long as the function of the active ingredient is not lost. Preferred examples of cosmetics include face and body cosmetics (for example, lotions, gels, creams, packs, etc.), make-up cosmetics, and face or body cleansers.

[0031] Such pharmaceutical and cosmetic preparations can be produced according to conventional methods by combining Geranium Herb or an extract thereof with a pharmaceutically or cosmetically acceptable carrier, the other active ingredients, medicinal ingredients, cosmetic ingredients, etc., as needed. Examples of the pharmaceutically or cosmetically acceptable carrier include various oils, surfactants, gelling agents, buffers, preservatives, antioxidants, solvents, dispersants, chelating agents, thickeners, UV absorbers, emulsion stabilizers, pH adjusters, pigments, fragrances, etc. Examples of such other active ingredients, medicinal ingredients, and cosmetic ingredients include plant extracts, disinfectants, moisturizers, anti-inflammatory agents, antibacterial agents, keratolytic agents, cooling agents, antiseborrheic agents, cleansers, and makeup ingredients.

[0032] The content of Geranium Herb or an extract thereof in the above-mentioned pharmaceutical or cosmetic formulations cannot be generalized because it varies depending on the form of the formulation. However, for example, based on the total amount of the formulation, the content is preferably 0.00006% by mass or more, more preferably 0.0003% by mass or more, even more preferably 0.0006% by mass or more, and is preferably 10% by mass or less, more preferably 5% by mass or less, and even more preferably 1% by mass or less, calculated as solid content. Furthermore, the amount is preferably 0.00006 to 10% by mass, more preferably 0.0003 to 5% by mass, and even more preferably 0.0006 to 1% by mass, calculated as solid content, based on the total amount of the preparation.

[0033] The dosage or use amount of Geranium Herb or an extract thereof may be an amount that can achieve the effects of the present invention. The dosage or use amount may vary depending on the species, body weight, sex, age, condition, and other factors of the subject, but in the case of parenteral administration such as an external skin preparation, the mass of Geranium Herb or an extract thereof used in solid content terms per adult (60 kg) is preferably 0.000006 g or more, more preferably 0.00003 g or more, even more preferably 0.00006 g or more, and preferably 0.1 g or less, more preferably 0.05 g or less, and even more preferably 0.01 g or less. The mass of Geranium Herb or an extract thereof used per adult (60 kg) in solid content is preferably 0.000006 to 0.1 g, more preferably 0.00003 to 0.05 g, and even more preferably 0.00006 to 0.01 g. In the present invention, such an amount can be administered or used repeatedly or continuously, once or in divided doses per day, for one day or more, preferably seven days or more, more preferably 14 days or more, and even more preferably 42 days or more.

[0034] The subjects to which the SMAD6 expression inhibitors of the present invention are administered or used are not particularly limited, as long as they are humans or non-human animals that require or desire them. Preferred examples of subjects include humans who desire the suppression of SMAD6 expression, the enhancement of TGF-β signaling, the suppression of NEP expression, and the promotion of microfibril formation or the promotion of elastic fiber formation. Non-human animals include non-human mammals such as apes and other primates. Furthermore, the site to which the SMAD6 expression inhibitor of the present invention is administered or used is not particularly limited. [Example]

[0035] Example 1 Suppression of SMAD6 expression by Geranium Herb extract (1)Cell culture Normal human dermal fibroblasts (fibroblasts) were purchased from KAC Co., Ltd. Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) containing 10% (v / v) fetal bovine serum (FBS) (Sigma-Aldrich) at 37°C and 5% CO2.

[0036] (2) Plant extract Geranium Herb Extract-J (Geranium Herb extract; extraction solvent is 50% (v / v) aqueous ethanol, dry residue 1.4% (w / v)) was obtained from Maruzen Pharmaceutical Co., Ltd.

[0037] (3) Effect of Geranium Herb extract on SMAD6 gene expression 5 x 10 fibroblasts 4Cells were seeded at a density of 1000 cells / 500 μl / well in 24-well plates using DMEM medium supplemented with 10% (v / v) FBS. The following day, the medium was replaced with 500 μl / well of FBS-free DMEM medium supplemented with a final concentration of 0.1% (v / v) Geranthes herb extract (with the same volume of 50% (v / v) aqueous ethanol as a control). After two days of culture, the cells were washed with PBS and total RNA was extracted using an RNeasy Mini Kit (QIAGEN) according to standard procedures. cDNA was synthesized by reverse transcription using the extracted total RNA as a template with a High Capacity RNA to cDNA Kit (Thermo Fisher Scientific). The reaction was carried out using a ProFlex PCR System (Thermo Fisher Scientific). Gene expression analysis was then performed by quantitative PCR using the synthesized cDNA and TaqMan® probes. The probe and primer specific to the SMAD6 gene were TaqMan® Gene Expression Assays (P / N 4331182) manufactured by Thermo Fisher Scientific. The expression level of the SMAD6 gene was normalized based on the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The reaction was performed using the StepOnePlus System (Thermo Fisher Scientific) under standard conditions.

[0038] The results are shown in Table 1. The results are expressed as relative values, with the expression level in the solvent control, to which only the extract solvent was added, set at 1. The Geranium Herb extract significantly suppressed the expression of the SMAD6 gene. Statistical analysis was performed using SPSS software. Significance with respect to the solvent control was analyzed using t-test or Dunnett's multiple comparison test. A P value of less than 0.05 was considered statistically significant.

[0039] [Table 1]

[0040] (4) Effect of Geranium Herb extract on SMAD6 protein expression 2 x 10 fibroblasts 5Cells were seeded at a density of 1 ml / well in 6-well plates using DMEM medium supplemented with 2% (v / v) FBS. The next day, the medium was replaced with 1 ml / well of FBS-free DMEM medium supplemented with 0.05% (v / v) or 0.1% (v / v) final concentrations of Geranius herb extract (as a control, an equal volume of 50% (v / v) aqueous ethanol). After 2 days of culture, the supernatant was removed, the cells were washed with PBS, and PMSF (Cell Signaling Technology) was added to a concentration of 1 mM. Cells were harvested using cell lysis buffer (Cell Signaling Technology) and disrupted by sonication. The cells were then centrifuged at 15,000 rpm for 15 minutes. Protein content of the supernatant was quantified by the bicinchoninic acid (BCA) method using bovine serum albumin (BSA) as a standard. Protein amounts for each group were then equalized and subjected to SDS-PAGE and Western blotting according to standard methods. The primary antibody was an anti-SMAD6 antibody (Sigma-Aldrich; SAB4200383) diluted 200-fold in blocking solution containing 5% skim milk powder (Fujifilm Wako Pure Chemical Industries, Ltd.; 198-10605) in DPBS (Life Technologies; 14190144). The internal standard was an anti-GAPDH antibody (Santa Cruz Biotechnology; sc-25778) diluted 2000-fold in blocking solution. The secondary antibody was an HRP-Conjugated Polyclonal Goat Anti-Rabbit (Dako; P0448) diluted 2000-fold in blocking solution. The cells were then illuminated using SuperSignal® West Dura Extended Duration Substrate (Thermo Fisher Scientific), and expression levels were visualized using an Amersham Imager 600 (GE Healthcare Life Science). The SMAD6 (60 kDa) band was then detected using the same imager system to quantify the expression level of SMAD6 protein, and the value corrected with the expression level of GAPDH, an internal standard, was used as the quantitative expression level. The results are shown in Table 2. The results are expressed as relative values, with the protein expression level in the solvent control, to which only the extract solvent was added, set at 1. The Geranium Herb extract significantly suppressed the expression of SMAD6 protein (molecular weight 60 kDa) in a concentration-dependent manner. Statistical analysis was performed as in (3).

[0041] [Table 2]

[0042] Example 2 Regulation of gene expression of elastic fiber-related factors by Geranium Herb extract (1)Cell culture The same procedure as in Example 1 was carried out.

[0043] (2) Plant extract The same Geranium Herb extract as in Example 1 was used.

[0044] (3) Effect of Geranius herb extract on the expression of elastase degradation-related genes (neprilysin (NEP)) and elastase formation-related genes (fibrillin-1 (FBN1) and tropoelastin (ELN)) 5 x 10 fibroblasts 4Cells were seeded at a density of 1000 cells / 500 μl / well in 24-well plates using DMEM medium supplemented with 10% (v / v) FBS. The following day, the medium was replaced with 500 μl / well of FBS-free DMEM medium supplemented with a final concentration of 0.1% (v / v) Geranthes herb extract (with the same volume of 50% (v / v) aqueous ethanol as a control). After two days of culture, the cells were washed with PBS and total RNA was extracted using an RNeasy Mini Kit (QIAGEN) according to standard procedures. cDNA was synthesized by reverse transcription using the extracted total RNA as a template with a High Capacity RNA to cDNA Kit (Thermo Fisher Scientific). The reaction was carried out using a ProFlex PCR System (Thermo Fisher Scientific). Gene expression analysis was then performed by quantitative PCR using the synthesized cDNA and TaqMan® probes. The probes and primers specific to each gene were TaqMan® Gene Expression Assays (P / N 4331182) manufactured by Thermo Fisher Scientific. The expression level of each target gene was normalized by the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The reaction was performed using the StepOnePlus System (Thermo Fisher Scientific) according to standard procedures.

[0045] The results are shown in Table 3. The results are expressed as relative values, with the expression level in the solvent control, to which only the extraction solvent was added, set at 1. The Geranium Herb extract significantly suppressed the gene expression of neprilysin (NEP) and significantly promoted the gene expression of fibrillin-1 (FBN1) and tropoelastin (ELN). Statistical analysis was performed in the same manner as in Example 1.

[0046] [Table 3]

[0047] Example 3 Promotion of microfibril formation by Geranium herb extract (1)Cell culture Foreskin-derived normal human dermal fibroblasts (fibroblasts) were purchased from Kurabo Industries, Ltd. Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) containing 10% (v / v) fetal bovine serum (FBS) (Sigma-Aldrich) at 37°C and 5% CO .

[0048] (2) Plant extract The same Geranium Herb extract as in Example 1 was used.

[0049] (3) Effect of Geranium Herb Extract on Microfibril Formation A cover glass was placed on the bottom of each well of a 24-well plate, and 3 × 10 fibroblast cells were added. 5The cells were seeded at a density of 1000 cells / 500 μl / well in DMEM medium supplemented with 5% (v / v) FBS. The next day, the plates were replaced with 500 μl / well of DMEM / F12 medium (Sigma-Aldrich) containing 0.05% (v / v) FBS supplemented with a final concentration of 0.05 or 0.1% (v / v) Gelatinum herb extract (or an equivalent volume of 50% (v / v) aqueous ethanol solution as a control). After 3 days of culture, the fibroblasts cultured on the coverslips were fixed by incubating in ice-cold methanol for 20 minutes. After blocking with 5% (v / v) BSA in PBS at room temperature for 1 hour, rabbit anti-human Fibrillin-1 antibody (Elastin Products Company; EPC-PR217) was added at a 1:200 dilution in 2.5% (v / v) BSA in PBS and incubated at 4°C for 16 hours. After washing with PBS, Alexa Fluor 594-conjugated anti-rabbit IgG antibody (Thermo Fisher Scientific; A11012) was added at a 1:200 dilution in PBS containing 2.5% (v / v) BSA and incubated for 2 hours at room temperature. After washing with PBS, the cells attached to the coverslips were mounted on slides using ProLong Gold antifade reagent with DAPI (Thermo Fisher Scientific). Immunofluorescence was observed using a confocal laser scanning microscope (LSM710, Carl Zeiss).

[0050] The results are shown in Figure 1. It was observed that the Geranius herb extract increased the density of fibrillin-1 and promoted microfibril formation.

[0051] Next, the image shown in Figure 1 was binarized using ImageJ software to obtain an image in which only the fibrillin-1 signal was visualized, and the signal area was calculated. The results are shown in Figure 2 and Table 4. The binarized image in Figure 2 also showed that the Geranius herb extract increased the density of fibrillin-1 and promoted microfibril formation. Furthermore, as shown in Table 4, it was confirmed that the fibrillin-1 signal area increased significantly in a concentration-dependent manner compared to the solvent control. Statistical analysis was performed as in Example 1.

[0052] [Table 4]

[0053] Example 4 Promotion of elastic fiber formation by Geranium herb extract (1)Cell culture The same procedure as in Example 3 was carried out.

[0054] (2) Plant extract The same Geranium Herb extract as in Example 1 was used.

[0055] (3) Effect of Geranium Herb Extract on Elastic Fiber Formation A cover glass was placed on the bottom of each well of a 24-well plate, and 3 × 10 fibroblast cells were added. 5The cells were seeded at a density of 1000 cells / 500 μl / well in DMEM medium supplemented with 5% (v / v) FBS. The next day, the cells were replaced with 500 μl / well of DMEM / F12 medium (Sigma-Aldrich) containing 2% (v / v) FBS supplemented with a final concentration of 0.05 or 0.1% (v / v) Gelatin extract (or an equivalent volume of 50% (v / v) aqueous ethanol solution as a control). After further culture for 14 days, the fibroblasts cultured on the coverslips were fixed by incubation in ice-cold methanol for 20 minutes. After blocking with 5% (v / v) BSA in PBS at room temperature for 1 hour, a mouse anti-human elastin antibody (Merck Millipore; MAB2503) diluted 200-fold in 2.5% (v / v) BSA in PBS was added and incubated at 4°C for 16 hours. After washing with PBS, Alexa Fluor 594-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific; A11030) was added at a 1:200 dilution in PBS containing 2.5% (v / v) BSA and incubated for 2 hours at room temperature. After washing with PBS, the cells attached to the coverslips were mounted on slides using ProLong Gold antifade reagent with DAPI (Thermo Fisher Scientific). Immunofluorescence was observed using a confocal laser scanning microscope (LSM710, Carl Zeiss).

[0056] The results are shown in Figure 3. It was observed that the Geranius herb extract increased the density of tropoelastin and promoted elastic fiber formation.

[0057] Next, the image shown in Figure 3 was binarized using ImageJ software to obtain an image in which only the tropoelastin signal was visualized, and the signal area was calculated. The results are shown in Figure 4 and Table 5. The binarized image in Figure 4 also confirmed that the Geranius herb extract increased tropoelastin density and promoted elastic fiber formation. Furthermore, as shown in Table 5, it was confirmed that the tropoelastin signal area significantly increased in a concentration-dependent manner compared to the solvent control. Statistical analysis was performed as in Example 1.

[0058] [Table 5]

Claims

1. An SMAD6 expression inhibitor containing Geranium herb or an extract thereof as an active ingredient.

2. A TGF-β signal transduction enhancer containing Geranium herb or an extract thereof as an active ingredient.

3. An NEP expression inhibitor containing Geranium herb or an extract thereof as an active ingredient.

4. A microfibril formation promoter containing Geranium herb or an extract thereof as an active ingredient.

5. An elastic fiber formation promoter containing Geranium herb or an extract thereof as an active ingredient.

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