MITF gene expression inhibitor, fibroblast proliferation promoter, SOD-like activator

Abstract

To provide components having the actions of MITF gene expression inhibition, fibroblast growth stimulation and SOD-like activity.SOLUTION: An MITF gene expression inhibitor has a degradation product of a yellow dye derived from Pinctada martensii as an active ingredient. There are also provided methods for producing an MITF gene expression inhibitor, a fibroblast growth stimulator, and an SOD-like activator, including decalcifying a pearl layer and degrading a yellow dye extracted from the insoluble portion.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an MITF gene expression inhibitor, a fibroblast proliferation promoter, and an SOD-like activator that contain, as an active ingredient, a decomposition product of a yellow pigment derived from pearl oysters, and also to methods for producing the MITF gene expression inhibitor, the fibroblast proliferation promoter, and the SOD-like activator. [Background technology]

[0002] The skin is broadly divided into three layers: the epidermis, the dermis, and the subcutaneous tissue. Each layer plays a different role, such as maintaining the body's homeostasis and protecting it from the outside world.

[0003] [1. MITF gene expression] Melanocytes are present in the basal layer of the skin epidermis and contain melanosomes responsible for melanin production. The MITF gene (microphtalmia-associated transcription factor) is a regulatory gene that controls the transcription of tyrosinase, which is involved in pigment synthesis in melanocytes, as well as TRP1 and TRP2. However, it is known that it is not only involved in pigment synthesis, but also functions as a master gene involved in the overall activity of melanocytes, such as melanocyte differentiation, proliferation, and location. Therefore, the expression level of the MITF gene is a comprehensive indicator of melanocyte activity, and it is believed that by identifying substances that can suppress MITF gene expression, it is possible to identify substances that can comprehensively suppress not only melanin synthesis activity but also melanocyte activity.

[0004] 2. Fibroblast proliferation The dermis also functions as a sort of cushion that absorbs shocks from the outside world. When this cushioning effect, that is, the thickness and flexibility of the dermis, is lost, wrinkles and sagging occur, which are considered to be one of the skin problems. The dermis is mainly composed of collagen, elastin, and fibroblasts, and the deterioration, reduction, or denaturation of collagen and elastin leads to aging symptoms such as loss of elasticity, wrinkles, and sagging. In healthy dermis, old collagen and elastin are absorbed and excreted, while new collagen and elastin are produced by fibroblasts, maintaining a constant quality and quantity. However, it is known that as we age, this homeostasis is disrupted by external and internal factors, and it is thought that these problems can be alleviated by proliferation of fibroblasts.

[0005] [3. SOD-like action] Furthermore, in recent years, there has been concern about the effects of active oxygen, which has strong oxidizing power, on living organisms. However, the skin is far more affected by active oxygen than other organs, not only because of oxygen stress caused within the body, but also because it is in contact with air and directly exposed to ultraviolet rays. This oxidative stress causes various skin problems, such as rough skin, hypersensitivity, and even skin inflammation. Therefore, suppressing the production of active oxygen is an essential element in alleviating skin problems. Several mechanisms for eliminating reactive oxygen species in the body have been elucidated. The first to be produced is O2 - It is known that superoxide (superoxide) is enzymatically dismutated into H2O2 and O2 by SOD (superoxide dismutase). However, the amount of SOD decreases with age, and the decrease in SOD amount increases the concentration of superoxide, which causes damage. Therefore, substances that have the same effect as SOD are thought to be effective as antioxidants in the skin.

[0006] [4. Pearl Cultivation and Akoya Oyster Nacre] Pearl farming, which is carried out throughout Japan, mainly uses Akoya oysters (Pinctada fucata) as mother shells. The purpose of this farming is to produce gemstones, so after the gemstone pearls are extracted from the oysters, the adductor muscle is used for food, while the pearls, shells, and flesh that are not commercially available are discarded as having no usable value. These unused resources may contain valuable substances that we do not yet know about. Pearls made from Akoya oysters come in a variety of colors, including white, yellow, and blue, each with its own distinctive hue. Of these, it is known that the color of yellow pearls comes from pigments, and pigments generally have antioxidant and other functional properties. Therefore, we focused on the yellow pigment contained in the nacre of pearl oysters. It is known that the yellow pigment contains iron-containing calcite as its structure (Patent Document 1), but the other components are unknown, and the biochemical properties are also unknown. We aimed to elucidate these and utilize them effectively. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2017-137225 A Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present invention is to effectively utilize waste materials from pearl farming and to provide a component that suppresses MITF gene expression, a component that promotes fibroblast proliferation, and a component that has SOD-like activity. [Means for solving the problem]

[0009] The inventors hypothesized that by obtaining a component that suppresses MITF gene expression, a component that promotes fibroblast proliferation, and a component that has SOD-like activity, these components could be applied to a variety of diseases and could enable effective use of waste materials from pearl farming.

[0010] In addition, they focused on the yellow nacre of Akoya oysters, a waste product from pearl farming, and searched for components that suppress MITF gene expression, promote fibroblast proliferation, and have SOD-like activity. They found that the decomposition product of the yellow pigment contained in the nacre of Akoya oysters has these effects.

[0011] That is, the present invention relates to a MITF gene expression inhibitor, a fibroblast proliferation promoter, and an SOD-like activator that contain as an active ingredient a decomposition product of a yellow pigment derived from Akoya oysters, and further relates to a method for producing the MITF gene expression inhibitor, the fibroblast proliferation promoter, and the SOD-like activator, which is characterized by decalcifying the nacreous layer and decomposing the yellow pigment extracted from the insoluble portion. Effect of the Invention

[0012] The present invention can provide an agent for suppressing MITF gene expression, an agent for promoting fibroblast proliferation, and an SOD-like activator. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the MITF gene expression inhibitory effect (confirmation test-2) of a decomposition product of a yellow pigment derived from a pearl oyster (Example-1).

[0014] [Diagram 2] FIG. 1 shows the fibroblast proliferation promoting effect (confirmation test-3) of the decomposition product of a yellow pigment derived from pearl oysters (Example-1).

[0015] [Diagram 3] FIG. 1 is a graph showing the SOD-like activity (confirmation test-4) of the decomposition product of a yellow pigment derived from a pearl oyster (Example-1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention relates to the inhibitory effect of MITF gene expression, the promotion of fibroblast proliferation, and the SOD-like activity of a degradation product of a yellow pigment derived from pearl oysters.

[0017] The color of pearls or shell nacre is determined by the actual color and interference color (structural color). The actual color is determined by the amount of yellow pigment contained in the pearl, and pearls are broadly divided into yellow and white. Yellow Akoya oysters are oysters that have the potential to produce yellow pearls, and can be selected by visually inspecting the nacreous layer on the inside of the shell. The selected pearls and / or yellow pearl oyster shells are preferably treated by removing the nucleus in the case of pearls and removing the outer layer from the prismatic layer in the case of pearl oyster shells. Methods for removing the outer layer from the prismatic layer include a method using a grinder, a method using hydrochloric acid (see JP-A-62-120319), and a method using sodium hypochlorite (see JP-A-2006-052183).

[0018] The mixture is pulverized to a size required for extracting the pigment by using a cutter mill, hammer mill, roller mill, crusher, rotary cutter, ball mill, screen mill, jet mill, cyclone mill, or other suitable combination.

[0019] In addition, pigments are extracted from powdered pearls and nacre of shells and used. There are methods to extract only the pigment, but there are also methods to only demineralize and extract the pigment while still containing the effective substances such as conchiolin. Either way, demineralization is necessary. The demineralization method involves dissolving calcium carbonate using one or more of inorganic acids such as hydrochloric acid, acetic acid, citric acid, and ethylenediaminetetraacetic acid, organic acids, and chelating agents, and collecting the insoluble portion, which is then used.

[0020] This is further purified. Extraction is performed with an aqueous solution of one or more acids selected from inorganic acids and organic acids, or with a hydrophilic organic solvent, or with a mixed solvent of water and one or more hydrophilic organic solvents. Various acids can be used, but since the acid is often removed in a subsequent process, hydrochloric acid, which is easy to remove, is often used. Examples of hydrophilic organic solvents include methanol, ethanol, and acetone. The extraction operation is not limited to a single operation. Fresh solvent can be added to the residue after extraction and the extraction operation can be performed, or the extraction solvent can be brought into contact with the extraction raw material multiple times. A yellow pigment can be obtained by this method.

[0021] The yellow pigment thus obtained is decomposed using one or more selected from inorganic acids, organic acids, and enzymes. These may also be used in combination. When an inorganic acid is used for extraction, the decomposition may be performed using the same type of inorganic acid, or a different type of inorganic acid. When the same type of inorganic acid is used, the decomposition is performed at a higher concentration than that used for extraction.

[0022] If necessary, further purification treatment such as deodorization may be carried out within a range that does not affect the effect of the extract, and the extract may be concentrated by using a vacuum concentration device such as an evaporator or by removing the solvent by heating. In addition, this extract can be further purified using synthetic adsorbents (Diaion HP20, Sepabise SP825, Amberlite XAD4, MCIgel CHP20P, etc.), dextran resins (Sephadex LH-20, etc.), ultrafiltration, etc.

[0023] In addition, yellow pigments from pearl oysters of other colors, such as white, can also be used. EXAMPLES

[0024] The present invention will be specifically described below by way of examples, but the present invention is not limited to these examples in any way.

[0025] Example 1 From the shells from which the pearls and shell flesh were removed, shells with a deep yellow nacre layer and shells with a less yellow nacre layer were selected, and the yellow shells were roughly polished using a barrel polishing machine. The prismatic layer was then removed using a grinder, and the shells were pulverized to obtain a shell nacre powder. This powder was decalcified using a 0.5M EDTA solution (pH 8.0), and the residue was further extracted with 90% acetone to obtain a residue. The resulting residue was dissolved in 1N hydrochloric acid methanol, and the extract was neutralized with sodium hydroxide, causing a yellow pigment to precipitate. This precipitate was washed with water etc., and then freeze-dried to obtain a yellow pigment.

[0026] The yellow dye was dissolved in 6N hydrochloric acid and kept at 120° C. for 72 hours to obtain a yellow dye decomposition product.

[0027] Confirmation Test-1 1 g of a powder of a yellow pigment or a decomposition product of a yellow pigment was added to 1 mL of a solvent, and the solubility was confirmed (temperature: 25° C.).

[0028] According to the confirmation test 1, the yellowish pigment before decomposition was insoluble in ethanol, methanol, acetone and water, but the decomposition products were soluble in the above-mentioned solvents. The results are shown in Table 1.

[0029] [Table 1] 1 is a table showing the solubility (confirmation test-1) of a yellow pigment derived from pearl oysters and its decomposition products (Example-1) in various solvents.

[0030] Confirmation Test-2 Human melanocytes were cultured in a 6-well plate at 4.8 × 10 4 After 24 hours in culture, the cells were cultured in a medium without additives for 24 hours. After that, the cells were exposed to the test medium containing 500μM IBMX and various concentrations of yellow pigment decomposition products, and cultured for 3 days. After 3 days, the cells were disrupted, RNA was extracted, reverse transcribed, and expression levels were measured by real-time PCR. Equipment used: Applied Biosystems 7500 real-time PCR system Cells used: Human melanocytes (Kurabo)

[0031] From the confirmation test 2, it was confirmed that the yellow pigment decomposition product derived from pearl oyster (Example 1) has an inhibitory effect on MITF gene expression (Confirmation Test 2). The results are shown in FIG.

[0032] Confirmation Test-3 Fibroblast cells were cultured in a 24-well plate at 1.6 × 10 3 The cells were seeded at 1000 cells / well. The medium (DMEM) was replaced with 1 ml of test product solution and cultured for 5 days. After that, the culture medium was removed, 0.3 ml of MTT solution was added, and cultured for 4 hours. Then, 0.3 ml of 2-propanol containing 0.04 N HCl was added, and the plate was shaken for 5 minutes on a plate shaker, and the absorbance at 540 nm (control 630 nm) was measured using a plate reader. Cells used: Human fibroblast cells (Kurabo)

[0033] From the confirmation test 3, it was confirmed that the yellow pigment decomposition product derived from pearl oyster (Example 1) has a fibroblast proliferation promoting effect (Confirmation Test 3). The results are shown in FIG.

[0034] Confirmation Test-4 Test method: SOD-like activity was determined according to the protocol of SOD Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).

[0035] From the confirmation test 4, it was confirmed that the yellow pigment decomposition product derived from the pearl oyster (Example 1) has SOD-like activity (Confirmation Test 4). The results are shown in FIG.

[0036] The decomposition product of the yellow pigment derived from pearl oysters can be used as an external preparation as it is, but it is also possible to create an external preparation by mixing it with other ingredients.

[0037] The yellow pigment decomposition product was found to be soluble in ethanol and water, and was suggested to be compatible with ingredients commonly used in topical preparations, making it an easy-to-handle substance. It was also found to function as a naturally derived yellow pigment and to be stable against heat.

Claims

1. An MITF gene expression inhibitor for use in an external preparation, the active ingredient of which is an acid decomposition product of a yellow pigment, which is an acid-containing solvent extract of the residue of a hydrophilic organic solvent extraction of a demineralization residue derived from a pearl oyster (wherein the hydrophilic organic solvent is acetone and the acid-containing solvent is hydrochloric acid / methanol).

2. A method for producing an MITF gene expression inhibitor for use in topical preparations, comprising the steps of: demineralizing the nacre; obtaining a residue by extracting the insoluble portion of the demineralized residue with a hydrophilic organic solvent; and extracting a yellow pigment from the obtained residue with an acid-containing hydrophilic organic solvent, and decomposing the yellow pigment with the acid (wherein the hydrophilic organic solvent used for extraction from the demineralized residue is acetone, and the hydrophilic organic solvent containing an acid used for extraction of the yellow pigment is hydrochloric acid-methanol).

3. The manufacturing method described in claim 2, characterized in that the acid that decomposes the yellow pigment is hydrochloric acid and has a high concentration of 6N.

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