AGENT FOR ENHANCING SKIN BARRIER FUNCTION, PROMOTER OF TRANSGLUTAMINASE-1 mRNA EXPRESSION, AND PROMOTER OF KAP5.1 mRNA EXPRESSION

Abstract

To identify a substance having an effect of enhancing skin barrier function, an effect of promoting transglutaminase-1 mRNA expression, or an effect of promoting KAP5.1 mRNA expression, from among highly safe natural materials, and to provide an agent for enhancing a skin barrier function, a promoter of transglutaminase-1 mRNA expression, and a promoter of KAP5.1 mRNA expression containing the identified substance as an active ingredient.SOLUTION: An agent for enhancing a skin barrier function, a promoter of transglutaminase-1 mRNA expression, and a promoter of KAP5.1 mRNA expression, contain a perilla extract as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a skin barrier function enhancer, a transglutaminase-1 mRNA expression promoter, and a KAP5.1 mRNA expression promoter.

Background Art

[0002] In the granular layer, the cell membrane thickens to form a thickened cell membrane, and by the action of transglutaminase-1, protein molecules are glutamyl-lysine cross-linked to form strong keratin protein fibers. Further, ceramide etc. are covalently bonded to a part thereof to take a hydrophobic structure, thereby supplying the base of the lamellar structure of the intercellular lipid and forming the basis of the stratum corneum barrier function and the skin moisturizing function.

[0003] However, as the production amount of transglutaminase-1 in the epidermis decreases with aging, the stratum corneum barrier function and the skin moisturizing function decline, so that skin aging symptoms such as rough skin and dry skin appear, or skin diseases such as ichthyosis, psoriasis, atopic dermatitis, xeroderma, and acne develop. Therefore, it is considered that by promoting the production of transglutaminase-1 in the epidermis, skin aging symptoms and the above skin diseases can be prevented, treated or improved. Conventionally, as those having a transglutaminase-1 production promoting action, an extract of the seeds of Annona squamosa (see Patent Document 1), an extract of Hunan sweet tea and its acid hydrolysate (see Patent Document 2), etc. are known.

[0004] Since hair has a great influence on people's impressions, its changes and abnormalities have been a major concern of people since ancient times. Problems related to hair include those related to the state of hair roots and hair follicles such as hair loss and thinning hair, as well as those related to the hair quality of hair such as hard, soft, thin, limp, split ends, and frizzy hair, and there are actually various ones. Furthermore, problems related to hair quality also include those caused by damage to hair due to daily hair care, hair makeup, ultraviolet exposure, etc., and those caused by problems in hair shaft formation, and extremely diverse factors are involved.

[0005] Conventionally, in order to improve the fineness of hair and the lack of stiffness, for example, hair tonics or hair conditioners containing hair growth active ingredients have been used. However, conventional hair tonics and hair conditioners have had the problem that the hair does not become thicker and the effect of imparting stiffness cannot be expected. In order to improve these, a method has been proposed in which a silanol compound produced by hydrolysis of an alkoxysilane is permeated into hair and polymerized inside the hair (see, for example, Patent Document 3). However, this method of modifying hair using chemical substances was not sufficient in terms of imparting natural stiffness to the hair.

[0006] On the other hand, attempts have also been made to improve hair quality biochemically and molecular biologically. Hair is composed of a cuticle (cuticula) that covers its surface, a cortex (cortex) inside it, and a medulla (medulla) that occupies the center of the hair. Among these, in hair damage, the cuticle that covers the surface is most easily affected, and it is known that the cuticle plays an important role in the hardness, stiffness, etc. of the hair (see, for example, Non-Patent Document 1). Therefore, if the cuticle can be effectively regenerated, it is considered possible to improve hair quality such as hair hardness and stiffness.

[0007] Among keratin-associated proteins (Keratin-associated protein; KAP), it is known that there is a correlation between the mRNA expression level of the KAP5 family gene and the strength of hair stiffness (see, for example, Patent Document 4). In addition, it has also been reported that KAP5.1, which is one of the KAP5 family, is localized in the growing cuticle (see, for example, Non-Patent Document 1). Therefore, if the expression of the KAP5 family, particularly KAP5.1 mRNA, can be promoted, it is expected to lead to the regeneration of the cuticle and, consequently, the improvement of hair quality such as hair hardness and stiffness. Serine, glycine, cysteine and their derivatives, etc. have been reported as promoters of KAP5.1 mRNA expression (see, for example, Patent Document 5).

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to find a substance having an action of enhancing the skin barrier function, an action of promoting the expression of transglutaminase-1 mRNA, or an action of promoting the expression of KAP5.1 mRNA from highly safe natural products, and to provide a skin barrier function enhancer, a transglutaminase-1 mRNA expression promoter, and a KAP5.1 mRNA expression promoter containing the same as an active ingredient.

Means for Solving the Problems

[0011] In order to solve the above problems, the skin barrier function enhancer, the transglutaminase-1 mRNA expression promoter, and the KAP5.1 mRNA expression promoter of the present invention contain an egoma extract as an active ingredient.

Effects of the Invention

[0012] According to the present invention, it is possible to provide a skin barrier function enhancing agent, a transglutaminase-1 mRNA expression promoting agent, and a KAP5.1 mRNA expression promoting agent, which have excellent skin barrier function enhancing effects, transglutaminase-1 mRNA expression promoting effects, and KAP5.1 mRNA expression promoting effects and are highly safe.

Embodiments for Carrying Out the Invention

[0013] Embodiments of the present invention will be described in detail. The skin barrier function enhancing agent, the transglutaminase-1 mRNA expression promoting agent, and the KAP5.1 mRNA expression promoting agent according to the present embodiment all contain perilla extract as an active ingredient.

[0014] The extraction raw material used to obtain the extract as an active ingredient of the skin barrier function enhancing agent, the transglutaminase-1 mRNA expression promoting agent, and the KAP5.1 mRNA expression promoting agent according to the present embodiment is perilla (sesame perilla, scientific name: Perilla frutescens).

[0015] Perilla frutescens is an annual herb belonging to the genus Perilla of the family Lamiaceae, which is distributed in Honshu, Shikoku, and Kyushu in Japan and can be easily obtained from these regions. There are no particular restrictions on the parts that can be used as the extraction raw material, and they can be appropriately selected according to the purpose. For example, above-ground parts such as leaf parts, branch parts, bark parts, trunk parts, stem parts, fruit parts, seed parts, flower parts, rhizome parts, or mixtures of these parts can be mentioned. In particular, leaf parts or a mixture of leaf parts and stem parts are preferred. In the present embodiment, the leaf part only needs to include at least a leaf blade and a leaf stalk. The leaf part is known as perilla leaf and is used for improving diarrhea and indigestion caused by cold. In addition, the seed part is used as a raw material for extracting perilla oil.

[0016] Although the details of the substances having the skin barrier function enhancing effect, the promoting effect on the expression of transglutaminase-1 mRNA, and the promoting effect on the expression of KAP5.1 mRNA contained in the extract from the above extraction raw material are unknown, an extract having the skin barrier function enhancing effect, the promoting effect on the expression of transglutaminase-1 mRNA, and the promoting effect on the expression of KAP5.1 mRNA can be obtained from the above extraction raw material by an extraction method generally used for plant extraction or the like. The extract includes any of an extract obtained from the extraction raw material by an extraction treatment, a diluted solution or a concentrated solution of the extract, a dried product obtained by drying the extract, or a crude purification product or a purified product thereof.

[0017] The above extract can be obtained by subjecting the extraction raw material as it is to extraction with an extraction solvent, or by drying the extraction raw material and then subjecting it to extraction with an extraction solvent as it is or after pulverizing it using a crusher. The drying of the extraction raw material may be carried out under the sun or using a commonly used dryer. Further, a pretreatment such as degreasing may be performed with a nonpolar solvent such as hexane and then used as an extraction raw material. By performing a pretreatment such as degreasing, the extraction treatment of the plant with a polar solvent can be efficiently performed.

[0018] Examples of the solvent used for extraction include water, a hydrophilic organic solvent, or a mixture thereof, and it is preferably used at room temperature or a temperature below the boiling point of the solvent. Components having the skin barrier function enhancing effect, the promoting effect on the expression of transglutaminase-1 mRNA, and the promoting effect on the expression of KAP5.1 mRNA contained in the extraction raw material can be easily extracted by an extraction treatment using a polar solvent as the extraction solvent.

[0019] Examples of the water that can be used as the extraction solvent include pure water, tap water, well water, mineral water, mineral water, hot spring water, spring water, fresh water, etc., and those obtained by subjecting these to various treatments are also included. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, etc. Therefore, the water that can be used as the extraction solvent in the present invention also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer solution, phosphate buffered saline, etc.

[0020] 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; polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol, and glycerin.

[0021] When a mixed solution of two or more polar solvents is used as the extraction solvent, the mixing ratio can be adjusted as appropriate. For example, when a mixed solution of water and a lower aliphatic alcohol is used, it is preferable to mix 1 to 90 parts by volume of the lower aliphatic alcohol with respect to 10 parts by volume of water. When a mixed solution of water and a lower aliphatic ketone is used, it is preferable to mix 1 to 40 parts by volume of the lower aliphatic ketone with respect to 10 parts by volume of water. When a mixed solution of water and a polyhydric alcohol is used, it is preferable to mix 1 to 90 parts by volume of the polyhydric alcohol with respect to 10 parts by volume of water.

[0022] For the extraction treatment, it is not necessary to adopt a special extraction method as long as the soluble components contained in the extraction raw material can be eluted into the extraction solvent, and the extraction can be carried out at room temperature or under reflux heating. For example, the extraction raw material is put into a treatment tank filled with the extraction solvent, and after standing for 30 minutes to 4 hours while stirring if necessary to elute the soluble components, the extract can be obtained by filtering to remove the solid matter. When the extraction solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and a dried product is obtained by further drying this concentrate. The extraction conditions may be about 1 to 4 hours at 50 to 95 °C when water is used as the extraction solvent. Also, when a mixed solvent of water and ethanol is used as the extraction solvent, it may be about 30 minutes to 4 hours at 40 to 80 °C.

[0023] The extract obtained as described above may be subjected to treatments such as dilution, concentration, drying, and purification according to conventional methods in order to obtain a diluted solution or concentrated solution of the extract, a dried product of the extract, or a crude or purified product thereof.

[0024] Incidentally, the obtained extract can be used as it is as an active ingredient of a skin barrier function enhancer, a transglutaminase-1 mRNA expression promoter, and a KAP5.1 mRNA expression promoter, 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.

[0025] In addition, since the above extract has a unique smell and taste, it is possible to perform purification for the purpose of decolorization, deodorization, etc. within a range that does not cause a decrease in its physiological activity. However, when added to cosmetics, etc., it is not used in large quantities, so there is no practical problem even if it remains unpurified. Purification can be carried out, for example, by activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, etc.

[0026] The perilla extract obtained as described above has a skin barrier function enhancing effect, a transglutaminase-1 mRNA expression promoting effect, and a KAP5.1 mRNA expression promoting effect, and thus can be used as an active ingredient of a skin barrier function enhancer, a transglutaminase-1 mRNA expression promoter, and a KAP5.1 mRNA expression promoter by utilizing these effects.

[0027] The skin barrier function enhancer, transglutaminase-1 mRNA expression promoter, and KAP5.1 mRNA expression promoter according to this embodiment may consist only of the perilla extract, or may be a formulation of the perilla extract.

[0028] The perilla extract can be formulated into any dosage form such as powder, granule, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin and any other auxiliary agent. At this time, as the auxiliary agent, for example, an excipient, a stabilizer, a flavoring agent, etc. can be used. The perilla extract can be used by being blended with other compositions (for example, skin external preparations, scalp external preparations, oral compositions, etc.).

[0029] In addition, the skin barrier function enhancer, the transglutaminase-1 mRNA expression promoter, and the KAP5.1 mRNA expression promoter of the present embodiment can, if necessary, be used as active ingredients by blending other natural extracts having a skin barrier function enhancing effect, a transglutaminase-1 mRNA expression promoting effect, and a KAP5.1 mRNA expression promoting effect together with the perilla extract.

[0030] The skin barrier function enhancer of the present embodiment can prevent, treat, or improve, through the skin barrier function enhancing effect of the perilla extract, for example, skin aging symptoms such as rough skin and dry skin, and skin diseases such as ichthyosis, psoriasis, atopic dermatitis, xeroderma, and acne. However, the skin barrier function enhancer of the present embodiment can be used for all applications that are meaningful in exerting the skin barrier function enhancing effect other than these applications.

[0031] The transglutaminase-1 mRNA expression promoter of the present embodiment can prevent, treat, or improve, through the transglutaminase-1 mRNA expression promoting effect of the perilla extract, for example, skin aging symptoms such as rough skin and dry skin, and skin diseases such as ichthyosis, psoriasis, atopic dermatitis, xeroderma, and acne. However, the transglutaminase-1 mRNA expression promoter of the present embodiment can be used for all applications that are meaningful in exerting the transglutaminase-1 mRNA expression promoting effect other than these applications.

[0032] The KAP5.1 mRNA expression promoter of the present embodiment can improve, through the KAP5.1 mRNA expression promoting effect of the perilla extract, for example, cuticle regeneration, and thus hair texture such as hair hardness and hair stiffness. However, the KAP5.1 mRNA expression promoter of the present embodiment can be used for all applications that are meaningful in exerting the KAP5.1 mRNA expression promoting effect other than these applications.

[0033] As the administration methods of the skin barrier function enhancer, the transglutaminase-1 mRNA expression promoter, or the KAP5.1 mRNA expression promoter of the present embodiment, generally transdermal administration, oral administration, etc. can be mentioned. However, depending on the type of disease, an administration method suitable for its prevention, treatment, etc. may be appropriately selected. Further, the dosage of the skin barrier function enhancer, the transglutaminase-1 mRNA expression promoter, or the KAP5.1 mRNA expression promoter of the present embodiment may also be appropriately increased or decreased depending on the type of disease, severity, individual differences of the patient, administration method, administration period, etc.

[0034] Since the skin barrier function enhancer, the transglutaminase-1 mRNA expression promoter, or the KAP5.1 mRNA expression promoter of the present embodiment has an excellent skin barrier function enhancing action, transglutaminase-1 mRNA expression promoting action, or KAP5.1 mRNA expression promoting action, it is suitable for blending, for example, in skin external preparations, scalp external preparations, or oral compositions. In this case, the perilla extract may be blended as it is, or a skin barrier function enhancer, a transglutaminase-1 mRNA expression promoter, or a KAP5.1 mRNA expression promoter formulated from the perilla extract may be blended.

[0035] Here, there is no limitation on the classification of skin external preparations and scalp external preparations, and they widely include skin cosmetics, hair cosmetics, quasi-drugs, pharmaceuticals, etc. that are used transdermally. Specifically, for example, ointments, creams, emulsions, lotions, beauty essences, lotions, packs, foundations, lip creams, bath agents, hair tonics, hair lotions, soaps, body shampoos, etc. can be mentioned.

[0036] The blending amount of the perilla extract in the skin external preparation or the scalp external preparation can be appropriately adjusted according to the type of the skin external preparation, but a suitable blending ratio is 0.0001 to 10% by mass, and a particularly suitable blending ratio is 0.001 to 1% by mass.

[0037] The external preparation for skin or scalp of this embodiment can be used in the production of ordinary cosmetics and the like, such as main agents, auxiliary agents or other components, for example, astringents, bactericidal and antibacterial agents, skin whiteners, ultraviolet absorbers, moisturizers, cell activators, anti-inflammatory and anti-allergic agents, antioxidants and reactive oxygen species scavengers, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, etc., as long as they do not interfere with the skin barrier function enhancing effect, the promoting effect on the expression of transglutaminase-1 mRNA, and the promoting effect on the expression of KAP5.1 mRNA possessed by the perilla extract. By using these components in combination, a more general product can be obtained, and a synergistic effect between the combined other active ingredients may bring about an excellent effect beyond the normal expectation.

[0038] An oral composition refers to something that has little risk of harming human health and is ingested by oral or gastrointestinal administration in normal social life, and is not limited to the classifications of foods, pharmaceuticals, quasi-drugs, etc. in administrative divisions. Therefore, the "oral composition" in this embodiment broadly includes general foods, feeds, health foods, health functional foods (foods for specified health uses, nutritional functional foods, foods with functional claims), quasi-drugs, pharmaceuticals, etc. that are orally ingested. The oral composition in this embodiment is preferably an oral composition capable of displaying the favorable effects possessed by the perilla extract on the oral composition or its packaging, and particularly preferably a health functional food (food for specified health use, nutritional functional food, food with functional claim), a quasi-drug, or a pharmaceutical.

[0039] The blending amount of the perilla extract in the oral composition can be appropriately changed in consideration of the purpose of use, symptoms, gender, etc., but in consideration of the general intake amount of the oral composition to which it is added, it is preferable that the intake amount of the perilla extract per adult per day is about 1 to 1000 mg. When the oral composition to which it is added is in the form of granules, tablets or capsules, the addition amount of the perilla extract is usually 0.0001 to 10% by mass, preferably 0.001 to 1% by mass, based on the oral composition to which it is added.

[0040] In addition, the skin barrier function enhancer, the transglutaminase-1 mRNA expression promoter, or the KAP5.1 mRNA expression promoter of the present embodiment has an excellent skin barrier function enhancing effect, a transglutaminase-1 mRNA expression promoting effect, or a KAP5.1 mRNA expression promoting effect, and thus can also be suitably used as a reagent for research on these action mechanisms.

[0041] Note that the skin barrier function enhancer, the transglutaminase-1 mRNA expression promoter, or the KAP5.1 mRNA expression promoter of the present embodiment is suitably applicable to humans, but can also be applied to animals other than humans as long as their respective effects are exhibited.

Example

[0042] Hereinafter, the present invention will be described in more detail with reference to production examples, test examples, etc., but the present invention is not limited to the following production examples, test examples, etc.

[0043] 〔Production Example 1〕Production of perilla extract 1500 mL of 30% by volume ethanol was added to 100 g of the leaf part of perilla, and extraction was performed at 80 to 90 °C for 2 hours using a reflux condenser, followed by filtration through filter paper to obtain an extract. The obtained extract was concentrated under reduced pressure and dried to obtain 20 g of perilla extract.

[0044] 〔Test Example 1〕Test for promoting transglutaminase-1 (TGM-1) mRNA expression Normal human neonatal epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte growth medium (KGM), and then recovered by trypsin treatment. The recovered cells were seeded in 6-well plates at 3.0×10 5 cells / 2 mL each using KGM and cultured overnight at 37 °C under 5% CO2. After completion of the culture, the medium was replaced with a medium (KBM) without added growth factors. After 24 hours, the culture solution was discarded, and 2 mL each of a test sample solution in which the perilla extract (Production Example 1) as the test sample was dissolved in KBM (see Table 1 below for the sample concentration) was added, and the cells were cultured at 37 °C under 5% CO2 for 24 hours.

[0045] After culturing, the culture medium was discarded, total RNA was extracted using ISOGEN II (manufactured by NIPPON GENE), the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared to a concentration of 200 ng / μL.

[0046] Using total RNA as a template, the expression levels of TGM-1 mRNA and GAPDH mRNA, which is an internal standard, were measured. Detection was performed using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) by a two-step real-time RT-PCR reaction using PrimeScriptTM RT Master Mix (Perfect Real Time, manufactured by Takara Bio Inc.) and TB Green (registered trademark) Fast qPCR Mix (manufactured by Takara Bio Inc.). The expression level of TGM-1 mRNA was corrected by the expression level of GAPDH mRNA, and a corrected value was calculated.

[0047] From the obtained corrected values, the promotion rate of TGM-1 mRNA expression (%) was calculated using the following formula. Promotion rate of TGM-1 mRNA expression (%) = A / B × 100 In the formula, A represents the "corrected value when the sample is added", and B represents the "corrected value when the sample is not added". The results are shown in Table 1.

[0048] [Test Example 2] Test for the effect of promoting KAP5.1 mRNA expression Normal human neonatal epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte growth medium (KGM), and then recovered by trypsin treatment. The recovered cells were seeded at 3.0×10 5 cells / 2 mL each in a 6-well plate and cultured overnight at 37°C under 5% CO2. After the culture was completed, the medium was replaced with a medium without growth factors (KBM). After 24 hours, the culture medium was discarded, and 2 mL each of a test sample solution in which the perilla extract (Production Example 1) as a test sample was dissolved in KBM (see Table 1 below for the sample concentration) was added, and the cells were cultured at 37°C under 5% CO2 for 24 hours.

[0049] After culturing, the culture medium was discarded, total RNA was extracted using ISOGEN II (manufactured by NIPPON GENE), the RNA amount was calculated from the absorbance at a wavelength of 260 nm, and total RNA was prepared to be 200 ng / μL.

[0050] Using total RNA as a template, the expression levels of KAP5.1 mRNA and GAPDH mRNA, which is an internal standard, were measured. Detection was performed by a two-step real-time RT-PCR reaction using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) and PrimeScriptTM RT Master Mix (Perfect Real Time, manufactured by Takara Bio Inc.) and TB Green (registered trademark) Fast qPCR Mix (manufactured by Takara Bio Inc.). The expression level of KAP5.1 mRNA was corrected by the expression level of GAPDH mRNA, and a corrected value was calculated.

[0051] From the obtained corrected value, the promotion rate (%) of KAP5.1 mRNA expression was calculated according to the following formula. Promotion rate of KAP5.1 mRNA expression (%) = A / B × 100 In the formula, A represents the "corrected value when the sample is added", and B represents the "corrected value when the sample is not added". The results are shown in Table 1.

[0052]

Table 1

[0053] As shown in Table 1, it was confirmed that the egoma extract has excellent effects of promoting the expression of transglutaminase-1 mRNA and KAP5.1 mRNA.

Industrial Applicability

[0054] The skin barrier function enhancer, the transglutaminase-1 mRNA expression promoter, and the KAP5.1 mRNA expression promoter according to this embodiment can greatly contribute to the prevention, treatment, or improvement of skin aging symptoms such as rough skin and dry skin, ichthyosis, psoriasis, atopic dermatitis, and skin diseases such as xeroderma and acne, and the improvement of hair quality such as hair hardness and stiffness.

Claims

1. A skin barrier function enhancer characterized by containing an egoma extract as an active ingredient.

2. A transglutaminase-1 mRNA expression promoter characterized by containing an egoma extract as an active ingredient.

3. A KAP5.1 mRNA expression promoter characterized by containing an egoma extract as an active ingredient.

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