JP2019077621A - DNA methylation regulator - Google Patents

Abstract

To find a modulator of DNA methylation level that is easy to introduce into a living body and can be routinely used and to provide DNA methylation regulators comprising it as an active ingredient.SOLUTION: The present invention provides a DNA methylation regulator and a Wnt expression inhibitor in cells which contain Rosa rugosa extract as an active ingredient. The present invention enables Wnt signal expression inhibition that works to maintain stem cell functions by regulating the level of DNA methylation, and is effective in the treatment, improvement, and prevention of diseases and pathologies associated with Wnt signal enhancement.SELECTED DRAWING: None

Description

   The present invention relates to intracellular DNA methylation regulators.

  Cells present in various tissues have a tissue-specific DNA methylation profile, and promoter regions of genes that are not required to construct a tissue are methylated, thereby suppressing gene transcription. ing. Such genomic DNA methylation modifications pass through cell division and are inherited to the next generation of cells, and thus function as a long-term stable cellular phenotype memory mechanism. Therefore, an abnormality in the DNA methylation profile originally means a change in the characteristic of the cell, and is considered to be involved in the onset of various diseases such as cancer (Non-patent Document 1). Ultraviolet light is known as a factor that changes the methylation state of such DNA. It has also been reported that in skins repeatedly irradiated with ultraviolet light, the methylation profile of DNA is abnormal and the gene expression pattern changes (Non-patent documents 2 and 3).

  Methylation modification (5-methylated cytosine, 5mC) at carbon 5 position of cytosine base of DNA is a physiological DNA modification mechanism, and most mammalian DNA methylation modifications are phosphodiester linked cytosine And guanine occur in a continuous sequence (CpG sequence) (Non-patent Document 4). Regions called CpG islands in which CpG sequences are accumulated are scattered on the genome, and are found in many gene promoter regions and gene regulatory regions (Non-patent Document 5). When this CpG island is methylated, the structure of DNA changes and gene expression is suppressed.

On the other hand, Wnt, which is a secreted protein, is an important factor involved in the development of the living body, cell differentiation, proliferation and the like, and 19 types have been identified in mammals (Non-patent Document 6). In the absence of Wnt, β-catenin phosphorylated intracellularly by GSK-3β (glycogen synthase kinase-3β) is further ubiquitinated and degraded by the proteasome. In the β-catenin pathway (Wnt / β-catenin signal), which is one of the Wnt signaling pathways, binding of Wnt to a receptor (Frizzled) on cell membrane suppresses phosphorylation by GSK-3β and escapes degradation -It is known that catenin translocates into the nucleus and promotes cell proliferation and differentiation via gene expression (Non-patent Document 7). Wnt 1, 2, 3, 7A, etc. are known as Wnt involved in Wnt / β-catenin signal (Non-patent Document 8). On the other hand, β-catenin independent pathways that are not mediated by β-catenin are classified into planar cell polarity pathways and Ca ²⁺ pathways, and act to suppress cell motility, cell polarity, or β-catenin pathway (non-patent literature) 9, 10). As Wnt involved in the β-catenin independent pathway, Wnt4, 5a, 11 and the like are known (Non-patent Document 8).

  Thus, while the Wnt signaling pathway is essential for the maintenance of the function of the cells that make up the tissues of the living body, its abnormality is associated with various pathological conditions. Melanins in the skin and hair are synthesized by melanocytes. Wnt / β-catenin signaling regulates melanocyte differentiation from melanocytes, which are the origin of melanocytes present near the bulge region of hair follicles, and melanin synthesis of differentiated melanocytes, senile pigmented spots, melasma spots, It is involved in pigment abnormalities such as sputum egg spots, lentigo, spots and sunburn. A polynucleotide for suppressing the expression of Wnt is known as a method of appropriately controlling Wnt / β-catenin signal and improving such pigmentation (Patent Document 1). In addition, Wnt / β-catenin signaling is also enhanced in cancers such as breast cancer, colon cancer, stomach cancer, esophagus cancer, glioblastoma, and fibroma, and these also suppress Wnt expression. Polynucleotides and monoclonal antibodies that inhibit the function of Wnt have been developed (Patent Document 2). Furthermore, Wnt signaling acts on differentiation, proliferation and undifferentiation maintenance of embryonic stem cells, small intestine stem cells, pigmented stem cells, hematopoietic stem cells, epidermal stem cells, hair follicle stem cells, hematopoietic stem cells, bone marrow and adipose tissue-derived mesenchymal stem cells. In addition, polynucleotides that suppress the expression of Wnt genes have also been developed to control the function of these stem cells (Patent Document 3).

  One of the important mechanisms among various Wnt expression mechanisms is the aforementioned DNA methylation. Also in the promoter region of Wnt, CpG sequences are present, and it has been found that DNA methylation suppresses expression (Non-patent Document 11), and Wnt expression can also be suppressed through control of DNA methylation. It can be expected. So far, development of a method for demethylating DNA which has been excessively methylated with a DNA methylation inhibitor or the like as a means for controlling DNA methylation has been advanced (Patent Documents 4 to 7). However, since the polynucleotide is easily degraded by the enzyme present in the living body and the means for introducing it into the living body is complicated, there is a problem that daily use is difficult. In addition, in the methods for controlling the methylation of DNA developed so far, DNA methylation levels can be brought closer to normal by demethylating DNA that has been excessively methylated, but conversely demethylation It has not been possible to develop a method for suppressing or remethylating DNA that is overly demethylated.

  Hamanas (Scientific name: Rosa rugosa) belongs to the genus Rosaceae, and is a deciduous shrub native to Japan, the Korean peninsula, northern China, and so on. There are many varieties of Hamanasu, and it is used as a raw material for dyes and perfumes, as well as for ornamental use, and the fruits are edible as rosehips and jams and the like. Hermanus, which is rich in vitamin C, has been known to have a beautiful skin action and an antioxidant action, as well as a testosterone 5α-reductase inhibitory action (Patent Document 8), and hair-raising effects such as hair loss prevention and hair growth effects (Patent Document 9) ), Α-amylase activity inhibitory action and α-glucosidase activity inhibiting action (patent document 10), sebum synthesis inhibiting action (patent document 11) and the like are known. However, nothing has been known so far regarding the Wnt expression suppression effect.

JP, 2013-67582, A Japanese Patent Publication No. 2007-536938 Japanese Patent Publication No. 2008-526229 JP, 2010-248223, A JP-A-2016-529257 JP 2013-126412 A Japanese Patent Application Publication No. 2013-514779 Japanese Patent Laid-Open No. 5-17365 JP-A-7-277930 JP 2005-306801 A JP 2015-124189 A

Esteller M, N. Engl. J. Med., 2008, 358, 1148-1159 Mittal A, et al., Neoplasia, 2003, 5, 555-565 Yang J, et al., 2014, 113, 45-54 Bird A, Genes Dev., 2002, 16, 6-21 Tamura Taura, Experimental Medicine, 2010, 28, 2346-2353 Nusse R. et al., Cell Res., 2008, 18, 523-527 Kikuchi A. et al., Cell. Signal., 1999, 11, 777-788 Sastre-Perona A., Santisteban P., Front Endocrinol., 2012, doi: 10.3389 / fendo.2012.00031. ECollection. Kikuchi A., et al., Cancer Sci., 2008, 99, 202-208 Veemam M. T., et al., Dev. Cell, 2003, 5, 367-377 Wang Z, et al., Anat Rec (Hoboken), 2010, 293, 1947-1953

  Therefore, an object of the present invention is to find a regulator of DNA methylation level that can be easily introduced into a living body and can be used routinely, and to provide a DNA methylation regulator that uses this as an active ingredient.

  The inventors of the present invention conducted intensive studies to solve the above problems, and as a result, found that the extract of Hamanasu has the function of regulating the level of DNA methylation in cells and suppressing the expression of Wnt. We came to complete the invention.

That is, the present invention includes the following inventions.
(1) An intracellular DNA methylation regulator containing an extract of Hamanasu as an active ingredient.
(2) The DNA methylation regulator according to (1), wherein the cell is an epithelial cell.
(3) The DNA methylation regulator according to (1), wherein the DNA is a DNA of a region involved in the regulation of expression of Wnt gene.
(4) The method according to any one of (1) to (3), wherein the regulation of DNA methylation is any of suppression of DNA demethylation, promotion of DNA methylation, or promotion of remethylation of DNA demethylation. DNA methylation regulator.
(5) A Wnt expression inhibitor comprising an extract of Hamanasu as an active ingredient.

  The DNA methylation regulator of the present invention can be controlled to increase DNA methylation levels to normal levels by suppressing the decrease in DNA methylation levels in cells caused by ultraviolet light etc. Gene expression can be suppressed. Therefore, according to the DNA methylation regulator of the present invention, a disease associated with hypomethylation of DNA, for example, a dye associated with enhancement of Wnt signal by hypomethylation of a region on DNA involved in the regulation of expression of Wnt gene It can treat, ameliorate, and prevent diseases such as deposition and cancer. Since the DNA methylation regulator of the present invention uses plant extracts as an active ingredient, it can be easily and routinely used in vivo.

The DNA methylation regulator in cells of the present invention contains an extract of Hamanasu as an active ingredient.
Hermanus used in the present invention includes Hamanas (scientific name: Rosa rugosa Thunb.), Yaehmanasu (scientific name: Rosa rugosa Thunb. Var. Plena), Mykai (scientific name: Rosa rugosa Thunb. Var. Plena Regel), and the like. In the present invention, an extract of an eggplant is an extract of whole plant (whole grass) or a part of plant such as leaves, stems, flowers, shoots, fruits, seeds, roots or mixtures thereof , Leaf extract is preferred. In addition, these plants may be used as they are for extraction, and may be subjected to treatments such as drying, crushing, shredding, and the like.

  The extraction method is not particularly limited, but can be carried out by stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. As the organic solvent, alcohols, ethers, esters and the like can be used, but water-soluble organic solvents such as ethanol, methanol, acetone, n-propanol, t-butanol, propylene glycol, 1,3-butylene glycol and the like One or two or more of these may be used, and for example, a 30 to 70 v / v% aqueous solution of ethanol can also be used.

  Particularly preferred extraction solvents include water or mixed polar solvents of water-ethanol type. The amount of the solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more to the leaf part (dry weight) of the above-mentioned Hamanasu, but concentration after extraction or isolation may be carried out It is preferable that it is 100 times or less for convenience of operation in the case of performing. Moreover, although extraction temperature and time are based on the kind of solvent to be used, they can be illustrated at, for example, 10 to 100 ° C., preferably 30 to 90 ° C., for 30 minutes to 24 hours, preferably 1 to 10 hours. In addition, the extract may be used as it is in the extracted solution, but if necessary, concentration (organic solvent, concentration by vacuum concentration, concentration by membrane concentration, etc.), dilution, filtration, to the extent that the effect is not affected. It may be used after processing such as decolorization with activated carbon, deodorization, ethanol precipitation and the like. Furthermore, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, lyophilization and the like, and used as a dried product.

  In the present invention, "modulation of DNA methylation in cells" includes suppression of DNA demethylation, promotion of DNA methylation, and promotion of remethylation of DNA demethylation.

  The cells to be subjected to DNA methylation are not particularly limited, but epithelial cells are preferable. The epithelial cell is not particularly limited as long as it is a cell present in a living tissue such as skin, hair root, oral mucosa, cornea, digestive organ, trachea, liver, mammary gland, prostate etc. For example, epidermal cells, hair mother cells, Examples thereof include epithelial cells in the digestive tract (esophagus, stomach, small intestine, large intestine), epithelial cells in respiratory tract (nasal cavity, pharynx, airway, lung), corneal epithelial cells, and the like. The cells are preferably derived from human, mouse, rat, hamster, rabbit, dog, cat, pig, cow, horse, monkey, and other mammalian cells. The cells may be cells in vivo or isolated cells. The isolated cells can be maintained and cultured by known methods.

The extract of Hamanasu can suppress the expression of Wnt gene by the regulation of DNA methylation in cells, and therefore can be used also as a Wnt expression inhibitor. In addition to suppression of Wnt expression by promoting methylation of the region on DNA involved in the expression regulation of Wnt gene, typically the region containing CpG island, the Wnt expression suppression by the extract of Hamanasu is a positive control gene for Wnt. Also included is the suppression of Wnt expression by promoting methylation. Here, Wnt refers to a protein involved in the Wnt signaling pathway, and up to now, 19 types of human Wnt proteins have been identified (Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16). The Wnt signaling pathway includes the β-catenin pathway that induces gene expression through stabilization of β-catenin by binding to Wnt receptors on the cell surface, the PCP pathway that activates JNK and Rho kinase, PKC, etc. Although there is an activating Ca ²⁺ pathway, Wnt in the present invention refers to a protein involved in β-catenin pathway (hereinafter sometimes referred to as “Wnt / β-catenin signal”). Thus, Wnt against which the Wnt expression inhibitor of the present invention acts refers to Wnt involved in Wnt / β-catenin signal, and examples include Wnt1, Wnt2, Wnt3, Wnt3a, Wnt7a, Wnt7a, Wnt10a, Wnt10b and the like. In the present invention, “suppressing Wnt expression” refers to suppressing Wnt mRNA expression and protein expression.

  The Wnt expression inhibitor of the present invention can suppress Wnt expression, as the extract of the Hamanasu, which is an active ingredient, can suppress Wnt expression. It is effective for Here, as a disease or pathological condition related to the enhancement of Wnt / β-catenin signal, skin diseases in which pigmentation by promotion of melanin synthesis is recognized (senile pigment spots, melasma spots, sparrow spots, lentigo, seborrhoeic Keratosis, inflammatory pigmentation, lentigo (Nevi cell nevus, pigmented nevus), Acquired dermis melanocytosis (Late Ota nevus), Squatorial nevus, Riehl's melasma, rubbing black skin Disease, hereditary contralateral pigmentary disorder, Addison's disease, actinic petaloid pigment spot, pigmentary pigmentation fixed erythema, sunburn, etc., cancer (colorectal cancer, melanoma, gastric cancer, hepatocellular carcinoma, prostate cancer, esophageal cancer) , Pancreatic cancer, lung cancer, breast cancer, kidney cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, head and neck cancer, lymphoma, glioma, glioblastoma and the like, but not limited thereto.

  The DNA methylation regulator or Wnt expression inhibitor of the present invention can be used as it is, but it can be used together with appropriate additives as long as the effects of the present invention are not impaired. And the like. The pharmaceutical preparation of the present invention is intended to include pharmaceuticals for animals, ie, veterinary medicine.

  When a DNA methylation regulator or a Wnt expression inhibitor is added to cosmetics or quasi-drugs, its dosage form is an aqueous solution system, a solubilization system, an emulsion system, a powder system, a powder dispersion system, an oil liquid system, a gel It may be any of a system, an ointment system, an aerosol system, a water-oil two-layer system, or a water-oil-powder three-layer system. In addition, the cosmetics and quasi-drugs are selected from the various components, additives, bases, etc. that are usually used in skin care compositions together with DNA methylation regulators or Wnt expression inhibitors according to their types. The composition can be appropriately formulated and manufactured according to a method known in the art. The form thereof may be any of liquid, emulsion, cream, gel, paste, spray and the like. Ingredients include, for example, oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil etc.), waxes (lanolin, beeswax, carnauba wax etc.), hydrocarbons (liquid paraffin, squalene, Squalane, vaseline etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol etc.), esters (myristin Isopropyl acetate, isopropyl palmitate, cetyl octanoate, glycerin trioctanoate, octyldodecyl myristate, octyl stearate, octyl stearate, etc., organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone cal Acids, sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various interfaces Activators, moisturizers, UV absorbers, antioxidants, stabilizers, preservatives, bactericides, perfumes and the like can be mentioned.

  Types of cosmetics and quasi-drugs include, for example, lotions, emulsions, gels, cosmetic solutions, general creams, sunscreens, packs, masks, face cleansers, cosmetic soaps, foundations, lotions, body lotions, etc. .

  When the DNA methylation regulator or Wnt expression inhibitor of the present invention is incorporated into a pharmaceutical, various pharmaceutical forms suitable for application to the affected area by mixing with pharmacologically and pharmaceutically acceptable additives. It can be formulated into a formulation. As pharmacologically and pharmaceutically acceptable additives, depending on the dosage form and application, a substrate for preparation, carrier, excipient, diluent, binder, lubricant, coating appropriately selected Agents, disintegrants or disintegrants, stabilizers, preservatives, preservatives, preservatives, extenders, dispersants, wetting agents, buffers, solubilizers or solubilizers, tonicity agents, pH adjusters, propellants A coloring agent, a sweetening agent, a flavoring agent, a flavoring agent, etc. may be added as appropriate to prepare various formulations which can be orally or parenterally administered systemically or topically by various known methods. When the medicament of the present invention is provided in each of the above-mentioned forms, it can be produced by a production method usually used by those skilled in the art, such as production methods shown in the respective sections of the general formula of pharmaceutical preparation [2] preparation of Japanese Pharmacopoeia.

  Preparations for oral administration include, for example, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate or dextrin; carboxymethylcellulose, carboxymethylcellulose calcium, Disintegrants or disintegrants such as starch or hydroxypropyl cellulose; binders such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate or talc Coating agents such as hydroxypropyl methylcellulose, sucrose, polyethylene glycol or titanium oxide; vaseline, liquid paraffin, polyethylene glycol Call, gelatin, kaolin, glycerin, purified water, or the like can be used base hard fat, but are not limited to.

  Preparations for parenteral administration include solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum water, vegetable oil, etc .; isotonicity agents such as glucose, sodium chloride, D-mannitol, etc .; Although pH adjusters, such as an organic acid, an inorganic base, or an organic base, etc. can be used, limitation is not carried out to these.

  The form of the pharmaceutical preparation of the present invention is not particularly limited. For example, tablets, coated tablets, capsules, troches, granules, powders, solutions, pills, emulsions, syrups, suspensions, elixirs Agents such as oral agents, injections (eg subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, suppositories, ointments, lotions, eye drops, sprays, Examples include parenteral agents such as skin absorbents, transmucosal absorbents and patches. It may also be a dry product which is redissolved upon use, and in the case of an injectable preparation, provided in the form of unit dose ampoules or multiple dose containers.

  A suitable form for using the DNA methylation regulator or Wnt expression inhibitor of the present invention as a medicament for treating, ameliorating or preventing pigmentation is an external preparation, for example, ointment, cream, gel Agents, solutions, patches and the like. An ointment refers to a homogeneous semi-solid external preparation, and includes an oleaginous ointment, an emulsion ointment and a water-soluble ointment. The gel agent refers to an external preparation in which a water-insoluble compound of a water-insoluble component is suspended in an aqueous liquid. The liquid preparation refers to a liquid external preparation, and includes lotions, suspensions, emulsions, liniments and the like.

  The pharmaceutical agent of the present invention functions as a preventive agent for suppressing the onset of the above-mentioned disease or condition and / or as a therapeutic agent for improving to a normal state. The active ingredient of the medicament of the present invention is derived from a natural product, so it is very safe and has no side effects. Therefore, when used as a medicine for treating, ameliorating and preventing the aforementioned diseases, human, mouse, rat, rabbit Can be administered orally or parenterally at a wide range of dosages to mammals such as dogs, cats and the like.

  When the extract of Hamanasu is incorporated into the above-mentioned cosmetics and pharmaceuticals, its content is not particularly limited, but it is 0.001 to 30 wt in terms of the dry solid content of the extract of Hamanasu based on the total weight of the preparation % Is preferable, and 0.01 to 10% by weight is more preferable. If the amount is less than 0.001% by weight, the effect is low, and if it exceeds 30% by weight, a large increase in the effect is hardly observed. In addition, the method of adding the active ingredient in formulation may be added in advance or may be added during the production, and may be appropriately selected in consideration of workability.

  The present invention will be described in more detail based on examples given below, but the present invention is not limited to these examples.

Example 1
An extract of Hamanasu was prepared as follows.
Production Example 1 Preparation of Hot Water Extract of Hamanas Leaf One liter of purified water was added to 50 g of Hamanas (dried product of leaf), and extracted at 90 to 100 ° C. for 2 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain 5.2 g of a hot water extract of Hamanas leaf.

Preparation Example 2 Preparation of 50% Ethanol Extract of Hamanas Leaf One liter of 50% ethanol aqueous solution was added to 50 g of Hamanasu (dried product of leaves), and extracted for 1 week at room temperature. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain 4.8 g of a 50% ethanol extract of Hamanasu leaf.

Preparation Example 3 Preparation of Ethanolic Extract of Hamanas Leaf One liter of ethanol was added to 50 g of Hamanas (dried product of leaf) and extracted at room temperature for 1 week. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain 4.1 g of an ethanol extract of Hamanas leaf.

Preparation Example 4 Preparation of Hot Water Extract of Hamanas Fruit 1 L of purified water was added to 50 g of Hamanas (dried product of fruit), and extracted at 90 to 100 ° C. for 2 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure, and freeze-dried to obtain 4.7 g of a hot water extract of Hemera canis.

Preparation Example 5 Preparation of 50% Ethanol Extract of Hamanas Fruit 1 L of 50% ethanol aqueous solution was added to 50 g of Hamanas (dried product of fruit), and extracted for 1 week at room temperature. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain 4.1 g of a 50% ethanol extract of an eggplant seed.

Preparation Example 6 Preparation of Ethanolic Extract of Hamanas Fruit 1 L of ethanol was added to 50 g of Hamanas (dried product of fruit) and extracted at room temperature for 1 week. The obtained extract was filtered, and then the filtrate was concentrated under reduced pressure and freeze-dried to obtain 3.1 g of an ethanol extract of the fruit of Hamanasu.

Preparation Example 7 Preparation of Hot Water Extract of Hamanas Flower 1 L of purified water was added to 50 g of Hamanas (dried product of flower), and extracted at 90 to 100 ° C. for 2 hours. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain 4.0 g of a hot water extract of an eggplant flower.

Preparation Example 8 Preparation of 50% Ethanolic Extract of Hamanas Flower One liter of 50% aqueous ethanol solution was added to 50 g of Hamanasu (dried product of flower), and extracted for 1 week at room temperature. The obtained extract was filtered, and then the filtrate was concentrated under reduced pressure and freeze-dried to obtain 3.5 g of a 50% ethanol extract of an eggplant flower.

Preparation Example 9 Preparation of Ethanolic Extract of Hamanas Flower 1 L of ethanol was added to 50 g of Hamanasu (dried product of flower), and extracted for 1 week at room temperature. The obtained extract was filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain 2.0 g of an ethanol extract of an eggplant flower.

Example 2
(Test Example 1) Evaluation of Wnt1 expression suppression effect
Hamanasu using human squamous cell carcinoma cell HSC-1 (obtained from JCRB cell bank) known to express Wnt1 (Yamada et al., Biosci Biotechnol Biochem. 2016 Jul; 80: 1321-6.) It was examined whether the extract of E. coli suppresses the expression of Wnt1.

HSC-1 cultured with Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich) containing 20% fetal bovine serum (FBS, Nichirei Bio) is suspended in DMEM containing 2% FBS, and then 8-well The culture slide was seeded with 1 × 10 ³ cells. After culturing for 24 hours, the extract of Hamanasu (Manufacturing Example 1-9) was replaced with DMEM containing 2% FBS added so that the final concentration was 0.01%, and culture was further continued for 72 hours. The cells were washed twice with PBS (−), 4% paraformaldehyde was added, and the cells were fixed by incubation for 10 minutes at room temperature.

  The cells were washed twice with PBS (-), and 2% bovine serum albumin (BSA, manufactured by Wako Pure Chemical Industries, Ltd.) in PBS (-) containing anti-Wnt 1 antibody (manufactured by Spring Bioscience) and anti-β-actin antibody (manufactured by Spring Bioscience) The primary antibody solution to which Abcam's product was added was added and incubated at 37 ° C. for 1 hour. The cells are washed twice with PBS (-), and a secondary antibody solution in which Alexa 488 labeled anti-rabbit IgG antibody and Alexa 594 labeled anti-mouse IgG antibody are added to PBS (-) containing 2% BSA is added, and 1 hour at 37 ° C. Incubated. The cells were washed twice with PBS (−), observed using a fluorescence microscope (Olympus), and images were taken with Wnt1 expression as green fluorescence and β-actin expression as red fluorescence. About the acquired image, the expression amount of each protein was measured using fluorescence intensity as an index using Image J (NIH), and the relative expression amount of Wnt 1 (green fluorescence intensity / red fluorescence intensity) was calculated. The relative expression level of Wnt1 in cells cultured without addition of sample was 1.0, and the value of the relative expression level of Wnt1 in cells cultured with addition of sample was calculated and evaluated. The results are shown in Table 1.

  As shown in Table 1, a remarkable Wnt1 expression suppression effect was observed in all of the extracts of Hamanasu (Production Example 1-9). From the above, the extremely excellent Wnt1 expression suppression effect of the extract of Hamanasu became clear.

(Experimental Example 2) Evaluation of Demethylation of DNA by UV Irradiation and Evaluation of Upregulation of Wnt1 From the previous researches, repeated irradiation of UVB to epidermal cells causes demethylation of DNA and expression of Wnt1 Is known to be induced (Yamada et al., 2015, The 38th Annual Meeting of the Japan Molecular Biology Society 88th Annual Meeting of the Biochemical Society of Japan, 1P0981). Therefore, the following test was performed to examine whether the extract of Hamanasu can suppress UVB-induced excessive DNA demethylation of epidermal cells and Wnt1 expression.

Normal human epidermal keratinocytes (manufactured by Kurabo) were suspended in HuMedia-KG2 (manufactured by Kurabo) and seeded at 1 × 10 ^{5 in} 6-well plates. After culturing for 24 hours, the medium was changed to PBS (-) and irradiated with ultraviolet light (UVB) to 10 mJ / cm ² using a Toshiba FL20S E lamp. PBS (−) was replaced with HuMedia-KG2 in which the extract of Hamanasu (Production Example 1-9) was added to a final concentration of 0.01%, and the culture was further continued for 24 hours. This operation was performed 4 times a week for 2 weeks, for a total of 8 UVB irradiations. Twenty four hours after the last UVB irradiation, cells were seeded at 1 × 10 ⁴ in 8-well chamber slides. After 24 hours, cells were washed twice with PBS (−), 4% paraformaldehyde was added, and the cells were fixed by incubation for 10 minutes at room temperature.

  The cells are washed twice with PBS (-), and in order to analyze the relative amount of DNA methylation, a primary antibody solution to which anti 5-methylated cytosine (5 mC, EPIGENTEK) antibody has been added is added, 37 Incubate at 1 ° C for 1 hour, further wash the cells twice with PBS (-), add a secondary antibody solution in which Alexa 594 labeled anti-mouse IgG antibody is added to PBS (-) containing 2% BSA, and 1 at 37 ° C. Incubated for time. Furthermore, a DNA staining reagent 4 ', 6-Diamidino-2-phenylindole dihydrochloride (DAPI, manufactured by Sigma) was added to 1 μg / mL, and incubation was performed for 5 minutes at room temperature.

  On the other hand, for the purpose of relative expression level analysis of Wnt1, a primary antibody solution obtained by adding anti-Wnt1 antibody and anti-β-actin antibody to PBS (-) containing 2% BSA is added and incubated at 37 ° C. for 1 hour. After washing twice with (-), a secondary antibody solution to which Alexa 488 labeled anti-rabbit IgG antibody and Alexa 594 labeled anti-mouse IgG antibody were added was added to PBS (-) containing 2% BSA, and incubated at 37 ° C for 1 hour .

  The cells were washed twice with PBS (−) and observed using a fluorescence microscope, and for methylated cytosine, an image was taken with 5 mC as red fluorescence and DAPI as blue fluorescence. In addition, for Wnt1 and β-actin expression, images were taken as green fluorescence and red fluorescence, respectively. About the acquired image, 5 mC amount and total DNA amount were measured using fluorescence intensity as an index using Image J (NIH), relative DNA methylation amount (red fluorescence intensity / blue fluorescence intensity) was calculated. The relative DNA methylation amount in cells cultured without irradiation of UVB and without addition of sample was 1.0, whereas cells irradiated with UVB and UVB were irradiated, and cells cultured with addition of sample The value of relative DNA methylation was calculated and evaluated. With regard to the expression of Wnt1 and β-actin, the amount of expression of each protein was measured using the image J (NIH) with the fluorescence intensity as an index for the acquired image, and the relative expression amount of Wnt1 (green fluorescence intensity / red The fluorescence intensity was calculated. The relative expression level of Wnt1 in cells cultured without UVB irradiation and without addition of sample was 1.0, whereas UVB-irradiated cells and UVB were irradiated, and samples were added and cultured The value of the relative expression level of Wnt1 in cells was calculated and evaluated. The results are shown in Table 2 (relative DNA methylation (relative methylated cytosine)) and Table 3 (relative Wnt1 expression).

  As shown in Table 2, all of the extract of Hamanasu (Production Example 1-9) had an inhibitory effect on the demethylation of DNA induced by UVB irradiation. In addition, as shown in Table 3, all extracts of Hamanasu (Production Example 1-9) had a remarkable inhibitory effect on the enhancement of Wnt1 expression induced by UVB irradiation.

(Test Example 3) Evaluation of remethylation promoting effect on DNA demethylation by ultraviolet irradiation and expression suppressive effect of Wnt 1 Next, excessive UV DNA demethylation of epidermal cells and expression of Wnt 1 induced by UVB once The following tests were conducted to examine whether the extract of Hamanasu could be returned to normal levels of enhancement.

Normal human epidermal keratinocytes were suspended in HuMedia-KG2 and seeded at 1 × 10 ^{5 in} 6-well plates. After culturing for 24 hours, the medium was changed to PBS (-) and irradiated with ultraviolet light (UVB) to 10 mJ / cm ² using a Toshiba FL20S E lamp. The PBS (-) was replaced with HuMedia-KG2 and cultured for another 24 hours. This operation was performed 4 times a week for 2 weeks, for a total of 8 UVB irradiations. After 24 hours from the last UVB irradiation, cells are suspended in HuMedia-KG2 to which a final extract of Hamanasu extract (Production Example 1-9) has been added to a final concentration of 0.01%, and 1 x 10 ⁴ cells each in an 8-well chamber slide Sowed. After 72 hours, cells were washed twice with PBS (-), 4% paraformaldehyde was added, and the cells were fixed by incubation for 10 minutes at room temperature. The relative amounts of DNA methylation and the amount of expression of Wnt1 were analyzed using the same method as in Test Example 2. The results are shown in Table 4 (relative DNA methylation (relative methylated cytosine)) and Table 5 (relative Wnt1 expression).

  As shown in Table 4, all of the extract of Hamanasu (Production Example 1-9) had a remethylation promoting effect on the demethylation of DNA induced by UVB irradiation. Further, as shown in Table 5, all of the extract of Hamanasu (Production Example 1-9) showed a remarkable inhibitory effect on the enhanced expression of Wnt1 induced by UVB irradiation.

  By using an extract of Hamanasu, it is possible to regulate the level of DNA methylation in cells and suppress the expression of Wnt. Therefore, the present invention relates to pharmaceuticals, quasi-drugs, cosmetics manufacturing fields and DNA methylation mechanisms for the purpose of preventing, ameliorating or treating diseases and pathological conditions such as pigmentation and malignancy associated with enhanced expression of Wnt. Can be used in basic research on

Claims (5)

  An intracellular DNA methylation regulator comprising an extract of Hamanasu as an active ingredient.   The DNA methylation regulator according to claim 1, wherein the cell is an epithelial cell.   The DNA methylation regulator according to claim 1, wherein the DNA is a DNA of a region involved in expression regulation of a Wnt gene.   The DNA methylation according to any one of claims 1 to 3, wherein the regulation of DNA methylation is any of suppression of DNA demethylation, promotion of DNA methylation, or promotion of remethylation of DNA demethylation. Modifier.   A Wnt expression inhibitor comprising an extract of Hamanasu as an active ingredient.