Fibroblast proliferation promoter, SOD-like activator

Abstract

To provide a component having MITF gene expression inhibition, fibroblast proliferation promotion and SOD-like activating effect.SOLUTION: An MITF gene expression inhibitor, a fibroblast proliferation promoter and an SOD-like activator have a decomposed product of a yellow-based pigment derived from pearl oyster as an active ingredient. Further, as a method for producing the MITF gene expression inhibitor, the fibroblast proliferation promoter and the SOD-like activator, a pearl layer is decalcified, and a yellow-based pigment extracted from an insoluble part is decomposed.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a MITF gene expression inhibitor, a fibroblast growth promoter, and a SOD-like activator containing a decomposition product of a yellow pigment derived from Pinctada fucata as an active ingredient, and also to a method for producing a MITF gene expression inhibitor, a fibroblast growth promoter, and a SOD-like activator.

Background Art

[0002] The composition of the skin is roughly classified into three categories, and from the outer layer, it is formed by the epidermis, dermis, and subcutaneous tissue, each of which plays various roles such as maintaining the body's homeostasis and protecting from the external environment.

[0003] [1. MITF Gene Expression] Melanocytes are present in the basal layer of the skin epidermis and have melanosomes responsible for melanin production within the cells. The MITF gene (microphtalmia-associated transcription factor) is a regulatory gene that controls the transcription of tyrosinase and TRP1 and TRP2 involved in melanocyte pigment synthesis. However, it is known to function not only in pigment synthesis but also as a master gene involved in the overall activity of melanocytes, such as differentiation, proliferation, and arrangement. Therefore, the expression level of the MITF gene is a comprehensive indicator of melanocyte activity, and it is considered that by identifying substances that can suppress MITF gene expression, substances that can comprehensively suppress not only melanin synthesis activity but also melanocyte activity can be identified.

[0004] [2. Fibroblast Growth] In addition, the function of the skin dermis plays a role of a cushion that absorbs impacts from the external environment. When the effect of this cushion, that is, the thickness and flexibility of the dermis are lost, wrinkles and sagging occur, which is one of the concerns of the skin. The dermis is primarily composed of collagen, elastin, and fibroblasts, which make up the majority of its structure. Deterioration, reduction, or degeneration of collagen and elastin leads to aging symptoms such as loss of firmness, 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, as we age, this homeostasis is known to be disrupted by external and internal factors, and it is thought that these problems can be mitigated by increasing the proliferation of fibroblasts.

[0005] [3. SOD-like effect] Furthermore, in recent years, there has been concern about the effects of reactive oxygen species, which possess strong oxidizing power, on living organisms. However, the skin is far more susceptible to the effects of reactive oxygen species than other organs, not only because of oxygen stress originating 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, including rough skin, hypersensitivity, and even inflammatory skin diseases. Therefore, suppressing the production of these reactive oxygen species is an essential element in alleviating skin problems. Several mechanisms have been elucidated for the scavenging of reactive oxygen species in living organisms, and the first to be produced is ·O2 - It is known that superoxide is enzymatically disproportionated into H2O2 and O2 by SOD (superoxide dismutase). However, the amount of SOD decreases with age, and this decrease in SOD leads to an increase in superoxide concentration, causing damage. Therefore, substances that have a similar effect to SOD are considered to be effective as antioxidants in the skin.

[0006] [4. Pearl Cultivation and the Nacreous Layer of the Akoya Oyster] Pearl farming, which takes place throughout Japan, primarily uses the Akoya oyster (Pinctada fucata) as the mother-of-pearl. Since the purpose of this farming is to produce gemstones, after extracting the pearls from the oysters, the adductor muscle is used for food, while the pearls, shells, and meat that do not meet commercial sales standards are discarded as having no further value. These underutilized resources may contain valuable substances that we are yet unaware of. Pearls derived from Akoya oysters come in a variety of colors, including white, yellow, and blue, each exhibiting its own distinctive hue. Of these, the yellow color of pearls is known to be derived from pigments, and these pigments generally possess functional properties such as antioxidant properties. Therefore, we focused on the yellow pigment contained in the nacre of the Akoya oyster. It is clear that the yellow pigment contains iron-containing calcite (Patent Document 1), but the other constituent components and biochemical properties are unknown. We aimed to utilize it effectively by elucidating these properties. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2017-137225 [Overview of the project] [Problems that the invention aims to solve]

[0008] The object of this invention is to effectively utilize waste products from pearl cultivation and to provide components that suppress MITF gene expression, components that promote fibroblast proliferation, and components that have SOD-like activity. [Means for solving the problem]

[0009] The inventors believed that by obtaining components that suppress MITF gene expression, components that promote fibroblast proliferation, and components with SOD-like activity, they could be applied to various diseases and enable the effective utilization of waste products from pearl farming.

[0010] Furthermore, focusing on the yellow nacreous layer of Akoya oysters, a waste product from pearl cultivation, we searched for components that suppress MITF gene expression, promote fibroblast proliferation, and have SOD-like activity. We found that the degradation products of the yellow pigment contained in the nacreous layer of Akoya oysters have these effects.

[0011] In other words, the present invention relates to a MITF gene expression inhibitor, a fibroblast proliferation promoter, and an SOD-like activator, all of which contain a degradation product of a yellow pigment derived from pearl oysters as an active ingredient. Furthermore, the present invention relates to a method for producing the MITF gene expression inhibitor, fibroblast proliferation promoter, and SOD-like activator, characterized by decalcifying the nacreous layer and decomposing the yellow pigment extracted from the insoluble portion. [Effects of the Invention]

[0012] The present invention provides a MITF gene expression inhibitor, a fibroblast proliferation promoter, and an SOD-like activator. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows the inhibitory effect of a degradation product of a yellow pigment derived from pearl oyster (Example-1) on MITF gene expression (Confirmation Test-2).

[0014] [Figure 2] This figure shows the fibroblast proliferation-promoting effect (confirmation test-3) of a degradation product of a yellow pigment derived from pearl oyster (Example-1).

[0015] [Figure 3] This figure shows the SOD-like activity (confirmation test-4) of a degradation product of a yellow pigment derived from pearl oyster (Example-1). [Modes for carrying out the invention]

[0016] The present invention relates to an inhibitory effect on MITF gene expression, a promoting effect on fibroblast proliferation, and a SOD-like activity effect by a decomposition product of a yellow pigment derived from Pinctada fucata.

[0017] The color of pearls or nacre of shells is determined by the body color and interference color (structural color). The body color is determined by the amount of yellow pigment contained in the pearl, and pearls are roughly classified into yellow and white types. The yellow Akoya oyster refers to an Akoya oyster that has the potential to produce yellow pearls. To select it, the nacre on the inner surface of the shell can be visually observed. As a treatment for the selected pearls and / or yellow Akoya oyster shells, it is desirable to remove the nucleus from the pearls and remove the outer layer from the Akoya oyster shells from the prismatic layer. Examples of methods for removing the outer layer from the prismatic layer include methods using a grinder, methods using hydrochloric acid (see Japanese Patent Laid-Open No. 62-120319), methods using sodium hypochlorite (see Japanese Patent Laid-Open No. 2006-052183), and the like.

[0018] This is pulverized. The pulverization method appropriately combines a cutter mill, a hammer mill, a roller mill, a crusher, a rotary cutter, a ball mill, a screen mill, a jet mill, a cyclone mill, etc. and pulverizes it to the size required for pigment extraction.

[0019] Furthermore, pigments are extracted from the powders of pearls and nacre of shells and used. There is a method of extracting only the pigment, but there is also a method of performing only demineralization and extracting and using the pigment while containing effective substances such as conchiolin. In any case, demineralization is necessary. The demineralization method uses one or more of inorganic acids, organic acids, and chelating agents such as hydrochloric acid, acetic acid, citric acid, and ethylenediaminetetraacetic acid to dissolve calcium carbonate, collects the insoluble part, and uses this insoluble part.

[0020] This is further purified. Extraction is performed with an aqueous solution of one or more acids selected from inorganic and organic acids, a hydrophilic organic solvent, or a mixed solvent of water and one or more hydrophilic organic solvents. Various acids can be used, but hydrochloric acid is often used because it is easy to remove in subsequent steps. Examples of hydrophilic organic solvents include methanol, ethanol, and acetone. Note that the extraction operation is not limited to a single operation. The residue after extraction can be treated again with fresh solvent, or the extraction solvent can be brought into contact with the raw material multiple times. Yellow pigments can be obtained by this method.

[0021] The resulting yellow pigment is decomposed. Decomposition is carried out using one or more selected from inorganic acids, organic acids, or enzymes. These can also be used in mixtures. If an inorganic acid is used for extraction, decomposition can be carried out with the same type of inorganic acid or with a different type of inorganic acid. If the same type of inorganic acid is used, decomposition should be carried out at a higher concentration than that used for extraction.

[0022] If necessary, further purification processes such as deodorization may be added to the extent that it does not affect the effect, and concentration can also be achieved by vacuum concentration devices such as evaporators or by removing the solvent by heating. Furthermore, this extract can be further purified using synthetic adsorbents (such as Diaion HP20, Sepabies SP825, Amberlite XAD4, MCIgel CHP20P), dextran resins (such as Sephadex LH-20), ultrafiltration, etc.

[0023] In addition, yellow pigments from other colored Akoya oysters, such as white ones, can also be used. [Examples]

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

[0025] Example 1 From the shells of Akoya oysters, after removing the pearls and flesh, shells with a deep yellow nacreous layer and shells with a less yellow nacreous layer were selected. The yellowish shells were then roughly polished using a barrel polishing machine. Subsequently, the prismatic layer was removed with a grinder and the shells were crushed to obtain shell nacreous powder. This powder was decalcified with a 0.5M EDTA solution (pH 8.0), and the residue was further extracted with 90% acetone to obtain a residue. The obtained residue was dissolved in 1N hydrochloric acid methanol, and the extract was neutralized with sodium hydroxide, causing a yellow pigment to precipitate. After washing this precipitate with water, it was freeze-dried to obtain a yellow pigment.

[0026] A yellow pigment was dissolved in 6N hydrochloric acid and maintained at 120°C for 72 hours to obtain a decomposition product of the yellow pigment.

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

[0028] Confirmation Test-1 showed that the yellow pigment before decomposition was insoluble in ethanol, methanol, acetone, and water, but the decomposition products were soluble in the aforementioned solvents. The results are shown in Table 1.

[0029] [Table 1] This table shows the solubility (confirmation test-1) of yellow pigments derived from pearl oysters and their decomposition products (Example-1) in various solvents.

[0030] Confirmation Test - 2 Human melanocytes were placed in a 6-well plate in a 4.8 × 10⁶ size. 4 Cells were placed in a cell / well and cultured for 24 hours in a culture medium without additives. Subsequently, cells were exposed to a test medium prepared by adding yellow pigment decomposition products to a medium containing 500 μM IBMX and culturing for 3 days. After 3 days, the cells were lysed, 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 (manufactured by Kurabo)

[0031] Confirmation Test-2 confirmed that the yellow pigment decomposition product derived from pearl oysters (Example-1) suppresses MITF gene expression (Confirmation Test-2). The results are shown in Figure 1.

[0032] Confirmation Test - 3 Fibroblasts are placed in a 24-well plate in a 1.6 × 10⁶ arrangement. 3 Cells were seeded to a density of cells / well. The culture medium was replaced with 1 ml of the test solution prepared in DMEM and incubated for 5 days. After that, the culture medium was removed, 0.3 ml of MTT solution was added, and the culture was incubated for 4 hours. Then, 0.3 ml of 2-propanol containing 0.04 N HCl was added, the mixture was shaken in a plate shaker for 5 minutes, and the absorbance at 540 nm (control 630 nm) was measured using a plate reader. Cells used: Human fibroblasts (Kurabo)

[0033] Confirmation Test-3 confirmed that the yellow pigment decomposition product derived from pearl oysters (Example-1) promotes fibroblast proliferation (Confirmation Test-3). The results are shown in Figure 2.

[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] Confirmation Test-4 confirmed that the yellow pigment decomposition product derived from pearl oysters (Example-1) exhibited SOD-like activity (Confirmation Test-4). The results are shown in Figure 3.

[0036] The decomposition products of the yellow pigment derived from pearl oysters can be used directly as topical preparations, but it is also possible to create topical preparations by combining them with other raw materials.

[0037] The yellow pigment decomposition product was found to be soluble in ethanol and water, and to have good compatibility with ingredients commonly used in topical preparations, suggesting it is an easy-to-handle substance. Furthermore, it was found to function as a naturally derived yellow pigment and to be stable against heat.

Claims

1. A fibroblast proliferation promoter comprising, as an active ingredient, an acid hydrolysis product of a yellow pigment, which is an acid-containing solvent extract of the hydrophilic organic solvent extraction residue of the demineralized residue of Akoya oyster, wherein the hydrophilic organic solvent is acetone, the acid-containing solvent is methanol hydrochloride, and the acid in the acid hydrolysis product of the yellow pigment is hydrochloric acid at a higher concentration than methanol hydrochloride.

2. A method for producing a fibroblast proliferation promoter, comprising: decalcifying nacreous layer; obtaining a residue extracted from the decalcified residue, which is the insoluble portion, with a hydrophilic organic solvent; and decomposing a yellow pigment extracted from this residue with an acid-containing hydrophilic organic solvent, wherein the hydrophilic organic solvent used for extraction from the decalcified residue is acetone; the acid-containing hydrophilic organic solvent used for extraction of the yellow pigment is methanol hydrochloride; and the acid used to decompose the yellow pigment is hydrochloric acid at a higher concentration than methanol hydrochloride.

Images