Epidermal keratinocyte growth stimulator and transglutaminase-1 mRNA expression promoter

Abstract

To discover, from among safe compositions of natural origin, ones having epidermal keratinocyte growth promoting action and transglutaminase-1 mRNA expression promoting action, and provide an epidermal keratinocyte growth stimulator and a transglutaminase-1 mRNA expression promoter containing the discovered compositions as an active ingredient.SOLUTION: An epidermal keratinocyte growth stimulator or a transglutaminase-1 mRNA expression promoter contains Citrus junos extract as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for promoting epidermal keratinocyte proliferation and an agent for promoting transglutaminase-1 mRNA expression. [Background technology]

[0002] The epidermis, which is composed of the basal layer, spinous layer, granular layer, and stratum corneum, functions to absorb external stimuli and control the loss of body components such as water. Keratinocytes divide and proliferate in the basal layer, differentiate as they pass through the spinous layer and granular layer, and become the stratum corneum, which is composed of keratin protein fibers with strong cross-links, and are eventually shed from the stratum corneum as sebum.

[0003] The stratum corneum, the outermost layer of the skin, constantly turns over to maintain a certain thickness and moisture content, acting as a barrier against external stimuli. To maintain this barrier function, keratinocytes are born in the basal layer and then turn into keratinocytes, which are then shed, typically every four weeks, carrying out epidermal metabolism. However, the metabolic function of the stratum corneum declines with age, resulting in skin aging symptoms such as fine wrinkles, dullness, pigmentation, and rough skin. Therefore, promoting keratinocyte proliferation and restoring skin metabolism may prevent and improve these skin aging symptoms.

[0004] Conventionally, substances known to have the effect of promoting epidermal keratinocyte proliferation include vitamin B3, astaxanthin, and shell ginger extract (see Patent Document 1), Japanese laurel extract (see Patent Document 2), and extract from thinned kiwi fruit (see Patent Document 3).

[0005] In the granular layer, the cell membrane thickens to form a thickened cell membrane, and the action of transglutaminase-1 causes glutamyl-lysine crosslinking between protein molecules to form strong keratin protein fibers. Furthermore, ceramides and other molecules are covalently bonded to some of these fibers, forming a hydrophobic structure that provides a foundation for the lamellar structure of intercellular lipids, forming the basis for the stratum corneum barrier function and the skin's moisturizing function.

[0006] However, as the production of transglutaminase-1 in the epidermis decreases with age, the stratum corneum barrier function and the skin's moisturizing function decline, leading to the development of skin aging symptoms such as rough skin and dry skin, and skin diseases such as ichthyosis, psoriasis, atopic dermatitis, xerosis, acne, etc. Therefore, it is believed that promoting the production of transglutaminase-1 in the epidermis can prevent, treat, or improve skin aging symptoms and the above-mentioned skin diseases.

[0007] Conventionally, known substances that have the effect of promoting the production of transglutaminase-1 include a fermentation liquid of thyme by Aspergillus oryzae and a fermentation liquid of cornflower by Aspergillus oryzae (see Patent Document 4), and an extract of gardenia treated by a process including a steam heating process and a drying process (see Patent Document 5). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 2019-214533 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-088075 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-144113 [Patent Document 4] Japanese Patent Application Publication No. 2020-164486 [Patent Document 5] Japanese Patent Publication No. 2020-105126 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to discover highly safe compositions derived from natural products that have the effect of promoting epidermal keratinocyte proliferation and transglutaminase-1 mRNA expression, and to provide an epidermal keratinocyte proliferation promoter and a transglutaminase-1 mRNA expression promoter that contain the same as an active ingredient. [Means for solving the problem]

[0010] In order to solve these problems, the present invention provides an epidermal keratinocyte proliferation promoter and a transglutaminase-1 mRNA expression promoter, each containing a yuzu extract as an active ingredient. [Effects of the Invention]

[0011] According to the present invention, highly safe compositions derived from natural products have been found to have the effect of promoting epidermal keratinocyte proliferation and transglutaminase-1 mRNA expression, and an epidermal keratinocyte proliferation promoter and a transglutaminase-1 mRNA expression promoter containing the same as an active ingredient can be provided. DETAILED DESCRIPTION OF THE INVENTION

[0012] An embodiment of the present invention will be described. The epidermal keratinocyte proliferation promoter and transglutaminase-1 mRNA expression promoter according to this embodiment contain a yuzu extract as an active ingredient.

[0013] The raw material used for extraction to obtain the active ingredient in this embodiment is yuzu (scientific name: Citrus junos).

[0014] Yuzu (scientific name: Citrus junos) is an evergreen tree of the genus Rutaceae that has been cultivated in China and Japan for a long time and is readily available in these regions. The parts of yuzu that can be used as the raw material for extraction can be appropriately selected depending on the purpose, and examples include leaves, trunks, branches, flowers, buds, fruit, peel, pit, seeds, roots, and mixtures thereof, with the fruit being preferred.

[0015] Although the details of the substance contained in the extract from the above-mentioned extraction raw material that has the effect of promoting epidermal keratinocyte proliferation or transglutaminase-1 mRNA expression are unknown, an extract having the effect of promoting epidermal keratinocyte proliferation and transglutaminase-1 mRNA expression can be obtained from the above-mentioned extraction raw material by an extraction method generally used for plant extraction, etc. The extract includes the extract obtained from the extraction raw material by extraction treatment, a diluted or concentrated extract, a dried product obtained by drying the extract, or any of these roughly purified or purified products.

[0016] The extract can be obtained by drying the raw material, either directly or after crushing using a crusher, and then subjecting it to extraction with an extraction solvent. Drying can be performed in the sun or using a commonly used dryer. The raw material can also be used after pretreatment, such as degreasing, using a nonpolar solvent such as hexane. Pretreatment, such as degreasing, allows for efficient extraction of plants with a polar solvent.

[0017] The solvent used for extraction may be water, a hydrophilic organic solvent, or a mixture thereof, and is preferably used at room temperature or a temperature below the boiling point of the solvent. Components contained in the extraction material that promote epidermal keratinocyte proliferation and transglutaminase-1 mRNA expression can be easily extracted by extraction using a polar solvent as the extraction solvent.

[0018] Examples of water that can be used as an extraction solvent include pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, and water that has undergone various treatments. Treatments that can be applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, etc. Therefore, water that can be used as an extraction solvent in the present invention also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate-buffered physiological saline, etc.

[0019] Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones, such as acetone and methyl ethyl ketone; and polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin.

[0020] When a mixture of two or more polar solvents is used as the extraction solvent, the mixing ratio can be adjusted appropriately. For example, when using a mixture of water and a lower aliphatic alcohol, it is preferable to mix 10 parts by volume of water with 1 to 90 parts by volume of the lower aliphatic alcohol. When using a mixture of water and a lower aliphatic ketone, it is preferable to mix 10 parts by volume of water with 1 to 40 parts by volume of the lower aliphatic ketone. When using a mixture of water and a polyhydric alcohol, it is preferable to mix 10 parts by volume of water with 1 to 90 parts by volume of the polyhydric alcohol.

[0021] The extraction process does not require a special extraction method as long as the soluble components contained in the extraction material can be dissolved into the extraction solvent. Extraction can be performed at room temperature or under reflux heating. For example, the extraction material is placed in a treatment tank filled with the extraction solvent, stirred as needed, and left to stand for 30 minutes to 4 hours to dissolve the soluble components, followed by filtration to remove solids, to obtain an extract. Distilling the extraction solvent from the resulting extract yields a paste-like concentrate, which can then be further dried to obtain a dried product. When water is used as the extraction solvent, extraction conditions are 50 to 95°C for approximately 1 to 4 hours. When a mixture of water and ethanol is used as the extraction solvent, extraction conditions are 40 to 80°C for approximately 30 minutes to 4 hours.

[0022] The extract obtained as described above may be subjected to treatments such as dilution, concentration, drying, purification, etc. according to conventional methods to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude or purified product thereof.

[0023] The obtained extract can be used as an epidermal keratinocyte proliferation promoter or transglutaminase-1 mRNA expression promoter as is, but a concentrated solution or a dried product is preferred. When obtaining a dried product, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity.

[0024] Furthermore, because the extract has a unique odor and taste, it is possible to purify it for the purpose of bleaching, deodorizing, etc., to the extent that this does not result in a decrease in its physiological activity, but since it is not used in large quantities when added to cosmetics, there is no practical problem in using it unpurified. Purification can be carried out, for example, by treatment with activated carbon, treatment with adsorption resins, treatment with ion exchange resins, etc.

[0025] The yuzu extract obtained in the manner described above has the effect of promoting epidermal keratinocyte proliferation and transglutaminase-1 mRNA expression, and can therefore be used as an active ingredient in an epidermal keratinocyte proliferation promoter and a transglutaminase-1 mRNA expression promoter by utilizing these effects.

[0026] The epidermal keratinocyte proliferation promoter or transglutaminase-1 mRNA expression promoter according to this embodiment may be a formulation of a yuzu extract.

[0027] The extract can be formulated into any dosage form, such as powder, granules, or liquid, using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin, or any other auxiliary agent, according to a conventional method. In this case, examples of auxiliary agents that can be used include excipients, stabilizers, and odor masking agents. Examples of the form of the epidermal keratinocyte proliferation promoter and transglutaminase-1 mRNA expression promoter formulated from the extract include ointments and external liquids.

[0028] The epidermal keratinocyte proliferation promoter according to this embodiment can promote the proliferation of epidermal keratinocytes through the epidermal keratinocyte proliferation-promoting activity of the yuzu extract. This restores the metabolic function of the skin and can prevent and improve skin aging symptoms such as fine wrinkles, dullness, pigmentation, and rough skin. However, in addition to these uses, the epidermal keratinocyte proliferation promoter according to this embodiment can also be used for any other use where it is meaningful to exert the epidermal keratinocyte proliferation-promoting activity.

[0029] The transglutaminase-1 mRNA expression promoter according to this embodiment can promote the expression of transglutaminase-1 through the transglutaminase-1 mRNA expression promoting effect of the yuzu extract. This can prevent, treat, or improve skin aging symptoms such as rough skin and dry skin, as well as skin diseases such as ichthyosis, psoriasis, atopic dermatitis, xerosis, and acne. However, in addition to these uses, the transglutaminase-1 mRNA expression promoter according to this embodiment can also be used for any other uses where the transglutaminase-1 mRNA expression promoting effect is significant.

[0030] The method of administering the epidermal keratinocyte proliferation promoter or transglutaminase-1 mRNA expression promoter according to this embodiment to a patient may include subcutaneous administration, intramuscular administration, intravenous administration, oral administration, transdermal administration, etc., and a method suitable for the prevention, treatment, etc. of the disease may be appropriately selected depending on the type of disease. Furthermore, the dosage of the epidermal keratinocyte proliferation promoter or transglutaminase-1 mRNA expression promoter according to this embodiment may be appropriately increased or decreased depending on the type and severity of the disease, individual patient differences, administration method, administration period, etc.

[0031] Furthermore, the epidermal keratinocyte proliferation promoter and transglutaminase-1 mRNA expression promoter of this embodiment have excellent epidermal keratinocyte proliferation promoting effects and transglutaminase-1 mRNA expression promoting effects, and are therefore suitable for incorporation into cosmetics such as skin cosmetics, scalp cosmetics, and hair cosmetics, as well as into foods, beverages, and the like.

[0032] Cosmetics that can contain the epidermal keratinocyte proliferation promoter or the transglutaminase-1 mRNA expression promoter include, for example, ointments, creams, emulsions, lotions, packs, foundations, hair tonics, hair lotions, shampoos, rinses, soaps, etc. When the epidermal keratinocyte proliferation promoter or the transglutaminase-1 mRNA expression promoter is contained in a cosmetic, the amount of the promoter can be adjusted appropriately depending on the type of cosmetic, but a preferred blending ratio is about 0.0001 to 10% by mass, calculated as a standard extract, and a particularly preferred blending ratio is about 0.001 to 1% by mass, calculated as a standard extract. As long as the cosmetic preparation does not interfere with the epidermal keratinocyte proliferation-promoting activity or transglutaminase-1 mRNA expression-promoting activity of the extract, it can be used in combination with the main ingredients, auxiliary ingredients, and other ingredients typically used in the manufacture of cosmetics, such as astringents, bactericides / antibacterial agents, whitening agents, UV absorbers, moisturizers, cell activators, anti-inflammatory / antiallergic agents, antioxidants / active oxygen scavengers, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, etc. Such combinations can result in more versatile products, and the synergistic effect with the other active ingredients used in combination can sometimes produce superior effects beyond those normally expected.

[0033] Foods and beverages refer to those that are unlikely to pose a risk to human health and are taken orally or by gastrointestinal administration in normal social life, and are not limited to administrative classifications such as food, medicine, or quasi-drug. Therefore, the "foods and beverages" of this embodiment broadly include compositions that constitute orally ingested general foods, health foods (functional foods and beverages), health functional foods (foods for specified health uses, foods with nutrient claims, foods with functional claims), quasi-drugs, medicines, etc. The foods and beverages of this embodiment may be those that display on the food or beverage or its packaging the effect of the extract in promoting epidermal keratinocyte proliferation or transglutaminase-1 mRNA expression, or may be health functional foods (foods for specified health uses, foods with functional claims, foods with nutrient claims), quasi-drugs, or medicines.

[0034] When the extract or the epidermal keratinocyte proliferation promoter or transglutaminase-1 mRNA expression promoter formulated from the extract is incorporated into a food or beverage, the amount of active ingredient can be varied as appropriate, taking into account the intended use, symptoms, gender, etc. However, taking into account the general intake of the food or beverage to be added, it is preferable to adjust the amount of extract to be approximately 1 to 1000 mg per adult per day. When the food or beverage to be added is in the form of granules, tablets, or capsules, the amount of the extract or the epidermal keratinocyte proliferation promoter or transglutaminase-1 mRNA expression promoter added is typically 0.1 to 100% by mass, and preferably 5 to 100% by mass, of the food or beverage to be added.

[0035] Although the epidermal keratinocyte proliferation promoter or transglutaminase-1 mRNA expression promoter according to this embodiment is preferably applied to humans, it can also be applied to animals other than humans as long as the respective effects are achieved. [Example]

[0036] The present invention will be explained in more detail below by showing production examples, test examples, etc., but the present invention is not limited to the following production examples, test examples, etc.

[0037] [Production Example] Production of Yuzu extract 1500 mL of 50% by volume ethanol solution was added to 100 g of yuzu fruit, and reflux extraction was carried out at 80 to 90°C for 2 hours using a reflux extractor, followed by hot filtration. The resulting extract was dried to obtain yuzu extract (24 g).

[0038] [Test Example 1] Epidermal keratinocyte proliferation promoting activity test The yuzu extract obtained in the above Production Example was added to a 50% by volume 1,3-butylene glycol solution to prepare a yuzu extract solution with a solids concentration of 1.2% by mass. This yuzu extract solution was used as a test sample and tested for its effect of promoting epidermal keratinocyte proliferation using the method described below.

[0039] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM) and harvested by trypsinization. The harvested cells were collected at a concentration of 3.0 × 10 4 The cells were diluted with KGM to a cell density of 100 cells / mL, and then seeded at 100 μL per well on a collagen-coated 96-well plate and cultured overnight.

[0040] 100 μL of KGM containing the test sample dissolved at a predetermined concentration (see Table 1 for the added concentration) was added to each well and cultured for 3 days. As a control, culture was performed in the same manner using KGM without the sample added.

[0041] The epidermal keratinocyte proliferation-promoting activity was measured using the MTT assay. Specifically, after incubation, the medium was removed, and 100 μL of MTT dissolved in PBS(-) at a final concentration of 0.4 mg / mL was added to each well and incubated for 2 hours. After incubation, blue formazan produced within the cells was extracted with 100 μL of 2-propanol. After extraction, absorbance at a wavelength of 570 nm was measured. At the same time, absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used as the amount of blue formazan produced. From the obtained values, the epidermal keratinocyte proliferation promotion rate (%) was calculated using the following formula.

[0042] Epidermal keratinocyte proliferation promotion rate (%) = A / B x 100 In the formula, "A" represents the amount of blue formazan produced when a test sample is added, and "B" represents the amount of blue formazan produced when no test sample is added.

[0043] The results of the above test are shown in Table 1. In the above formula, the rate of promotion of epidermal keratinocyte proliferation without the addition of the test sample is 100%.

[0044] [Table 1]

[0045] As shown in Table 1, the yuzu extract showed a high rate of promoting epidermal keratinocyte proliferation. These results confirmed that the yuzu extract has an excellent effect of promoting epidermal keratinocyte proliferation.

[0046] [Test Example 2] Transglutaminase-1 mRNA expression promoting effect test The yuzu extract obtained in the above Production Example was added to a 50% by volume 1,3-butylene glycol solution to prepare a yuzu extract solution with a solids concentration of 1.2% by mass. This yuzu extract solution was used as a test sample and tested for transglutaminase-1 mRNA expression activity by the following method.

[0047] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM) and then harvested by trypsinization. The harvested cells were placed in a 6-well plate at a density of 3.0 × 10 5 The cells were diluted with KGM to a cell density of 100 cells / 2 mL and then cultured overnight at 37°C in 5% CO2-95% air.

[0048] After incubation, the medium was removed and replaced with growth factor-free medium (normal human epidermal keratinocyte basal medium; KBM) and cultured for 24 hours. After incubation, the medium was removed, and 2 mL of KBM containing the test sample dissolved at the specified concentration (see Table 2 for the concentration) was added to each well and cultured for 24 hours under conditions of 37°C, 5% CO2, and 95% air. As a control, KBM without the sample was cultured in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene Co., Ltd.). The amount of RNA was calculated from the absorbance at 260 nm and adjusted to 200 ng / μL.

[0049] Using this total RNA as a template, the mRNA expression levels of transglutaminase-1 and GAPDH as an internal standard were measured. Detection was performed using a real-time PCR device, Thermal Cycler Dice® Real Time System III (manufactured by Takara Bio Inc.), with PrimeScript PCR. TM Real-time 2-step RT-PCR was performed using RT Master Mix (Perfect Real Time) (Takara Bio) and TB Green® Fast qPCR Mix (Takara Bio). The expression level of transglutaminase-1 mRNA was calculated by correcting it with the GAPDH mRNA level. From the obtained values, the transglutaminase-1 mRNA expression promotion rate (%) was calculated using the following formula.

[0050] Transglutaminase-1 mRNA expression promotion rate (%) = C / D x 100 In the formula, "C" represents the corrected value when the test sample was added, and "D" represents the corrected value when the test sample was not added.

[0051] The results of the above test are shown in Table 2. In the above formula, the transglutaminase-1 mRNA expression promotion rate without addition of the test sample is 100%.

[0052] [Table 2]

[0053] As shown in Table 2, yuzu extract showed a high transglutaminase-1 mRNA expression promotion rate. In particular, when the test sample concentration was 1% by mass, it was confirmed that the transglutaminase-1 mRNA expression promotion effect was approximately twice as high as the transglutaminase-1 mRNA expression promotion rate when no test sample was added. These results confirmed that yuzu extract has an excellent transglutaminase-1 mRNA expression promotion effect. [Industrial Applicability]

[0054] The epidermal keratinocyte proliferation promoter or transglutaminase-1 mRNA expression promoter of the present invention can be suitably used as an ingredient in cosmetics, foods and beverages, etc., and further as a research reagent.

Claims

[Claim 1] A transglutaminase-1 mRNA expression promoter containing yuzu extract as an active ingredient.

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