Involucrin mRNA expression promoter, transglutaminase-1 mRNA expression promoter, and filaggrin mRNA expression promoter

Abstract

To discover, from among compositions of natural origin having high safety, ones having filaggrin mRNA expression promoting action, involucrin mRNA expression promoting action, and transglutaminase-1 mRNA expression promoting action, and provide a filaggrin mRNA expression promoter, an involucrin mRNA expression promoter, and a transglutaminase-1 mRNA expression promoter containing the same as an active ingredient.SOLUTION: A filaggrin mRNA expression promoter, an involucrin mRNA expression promoter, and a transglutaminase-1 mRNA expression promoter, which are used to promote the expression of filaggrin mRNA, involucrin mRNA, and transglutaminase-1 mRNA in epidermal keratinocytes, are made to contain, as an active ingredient, a water extract from the leaf pulp portion of Aloe vera and / or a hydrophilic organic solvent extract from the leaf pulp portion of Aloe vera.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an ATP production promoter, a glutathione production promoter, a filaggrin mRNA expression promoter, an involucrin mRNA expression promoter, and a transglutaminase-1 mRNA expression promoter.

Background Art

[0002] ATP (Adenosine triphosphate) plays an important role as an energy substance in living organisms. It is considered that increasing the production amount of ATP in vivo leads to the activation of functions such as cell proliferation, metabolism, and repair.

[0003] When the ability to produce ATP, which plays an important role as an energy substance in vivo, decreases, cell functions decline, leading to the promotion of aging and cell death. In order to promote the reduced cell functions, it is important to supply the cells with the energy necessary for cell division. Therefore, if the production of ATP in cells can be promoted, it is considered that the cells can be activated to promote cell division and restore the functions of the cells. In addition, promoting the production of ATP is also expected to have effects such as activating functions such as cell proliferation, metabolism, and repair, and anti-aging effects. Furthermore, ATP is known to have a vasodilatory effect, and it is considered that promoting the production of ATP can prevent, treat, or improve eye fatigue, dizziness, gastritis, etc. Conventionally, as those having an ATP production promoting effect, young barley leaves (see Patent Document 1), a Cistus monspeliensis extract extracted from Cistus monspeliensis of the family Cistaceae (see Patent Document 2), an extract of one or more plants selected from plants of the genus Cryptomeria (see Patent Document 3), etc. are known.

[0004] Glutathione is a tripeptide composed of three amino acids: glutamic acid, cysteine, and glycine. Glutathione in cells is known to play roles such as scavenging radicals and regulating cell functions through redox reactions, as well as serving as an SH donor for various enzymes and an antioxidant component.

[0005] However, when the amount of glutathione in the skin decreases with ultraviolet irradiation or the progression of aging, the antioxidant defense ability of the skin decreases, which can damage cell components such as DNA and proteins, leading to a decline in the barrier function of the epidermis, the elasticity of the dermis, etc., and presenting skin aging symptoms such as fine wrinkles, dullness, pigmentation, and rough skin. In particular, when the production amount of glutathione in epidermal keratinocytes in the epidermis decreases, aging symptoms such as a decline in the barrier function, rough skin, and the formation of fine wrinkles are likely to appear. On the other hand, when the production amount of glutathione in fibroblasts in the dermis decreases, aging symptoms such as a decline in skin elasticity and water retention function, and pigmentation are likely to appear. Therefore, it is considered that by promoting the production of glutathione in the skin, the above-mentioned skin aging symptoms can be prevented, treated, or improved. In addition, glutathione is present in large amounts in the liver and is known to play a role in excreting harmful substances in the liver out of the body. Therefore, it is considered that by promoting the production of glutathione, it is possible to improve liver function in chronic liver diseases and improve fatty liver, etc. Conventionally, as those having a glutathione production-promoting effect, the solvent-soluble components of Polygonum multiflorum Thunb., the solvent-soluble components of Glycyrrhiza glabra L. (see Patent Document 4), the celery seed extract extracted from unsprouted celery seeds (see Patent Document 5), the extract of Alpinia speciosa (Wendl.) K. Schum. of the genus Alpinia in the family Zingiberaceae (see Patent Document 6), etc. are known.

[0006] Amino acids, which are the main components of the Natural Moisturizing Factor (NMF), are produced by the degradation of filaggrin derived from keratohyalin granules in the stratum corneum. Filaggrin is expressed as profilaggrin in epidermal keratinocytes present in the granular layer located immediately below the stratum corneum. Subsequently, it is immediately phosphorylated, accumulated in keratohyalin granules, and decomposed into filaggrin through dephosphorylation and hydrolysis, migrating to the stratum corneum to enhance the aggregation efficiency of keratin filaments and being reported to be involved in the internal construction of stratum corneum cells (see Non-Patent Document 1).

[0007] In recent years, it has been reported that filaggrin is very important and indispensable for skin moisture retention, and that the synthetic ability of filaggrin decreases under conditions such as dryness, resulting in a decrease in the amount of amino acids in the stratum corneum (see Non-Patent Document 2). Therefore, it is considered that by promoting the production of filaggrin in epidermal keratinocytes, the amount of amino acids in the stratum corneum can be increased, and the decrease in the skin's moisture retention function can be prevented or improved. Conventionally, as those having a filaggrin production-promoting effect, extracts of gardenia (see Patent Document 7), extracts of purple root (see Patent Document 8), extracts of safflower, extracts of cimicifuga, extracts of black tea, and extracts of amur corktree (see Patent Document 9), etc. are known.

[0008] Stratum corneum cells are composed of keratin fibers as the main component and cornified envelope (CE) cells that surround them. Cornified envelope cells are formed by cross-linking and insolubilization of a plurality of cornified envelope cell precursor proteins produced by the differentiation of epidermal keratinocytes by the enzyme transglutaminase. Furthermore, ceramides, etc. covalently bond to form a hydrophobic structure, supplying the base of the lamellar structure of intercellular lipids to a part of the cornified envelope cells, thereby forming the basis of the stratum corneum barrier function.

[0009] Involucrin is known as one of the precursor proteins of the cornified envelope. By promoting the production of involucrin, it is considered possible to 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. Conventionally, as substances having an involucrin production-promoting effect, an extract of the seeds of Annona squamosa (see Patent Document 10), an extract obtained from Gnaphalium uliginosum L. of the genus Gnaphalium of the Asteraceae family (see Patent Document 11), Betula platyphylla Sukatchev var. japonica Hara sap (see Patent Document 12), etc. are known.

[0010] In the granular layer, the cell membrane thickens to form a thickened cell membrane, and due to the action of transglutaminase-1, protein molecules are cross-linked by glutamyl-lysine bridges to form strong keratin protein fibers. Furthermore, ceramides, etc. are covalently bonded to a part of them, taking a hydrophobic structure, thereby supplying the basis for the lamellar structure of the intercellular lipids and forming the basis for the stratum corneum barrier function and the skin moisturizing function.

[0011] 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, xerosis, and acne develop. Therefore, it is considered possible to prevent, treat, or improve skin aging symptoms and the above skin diseases by promoting the production of transglutaminase-1 in the epidermis. Conventionally, as substances having a transglutaminase-1 production-promoting effect, an extract of the seeds of Annona squamosa (see Patent Document 10), an extract of Hunan sweet tea and its acid hydrolysate (see Patent Document 13), etc. are known.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

Non-Patent Document

[0013]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0014] The present invention aims to find a composition derived from a highly safe natural product that has an effect of promoting involucrin mRNA expression, an effect of promoting transglutaminase-1 mRNA expression, and an effect of promoting filaggrin mRNA expression, and to provide an involucrin mRNA expression promoter, a transglutaminase-1 mRNA expression promoter, and a filaggrin mRNA expression promoter containing the same as an active ingredient.

Means for Solving the Problems

[0015] In order to solve such problems, the present invention provides an involucrin mRNA expression promoter, a transglutaminase-1 mRNA expression promoter, and a filaggrin mRNA expression promoter containing an extract of Aloe vera as an active ingredient.

Effects of the Invention

[0016] According to the present invention, it is possible to find a composition derived from a highly safe natural product that has an effect of promoting involucrin mRNA expression, an effect of promoting transglutaminase-1 mRNA expression, and an effect of promoting filaggrin mRNA expression, and to provide an involucrin mRNA expression promoter, a transglutaminase-1 mRNA expression promoter, and a filaggrin mRNA expression promoter containing the same as an active ingredient.

Modes for Carrying Out the Invention

[0017] Embodiments of the present invention will be described. The ATP production promoter, glutathione production promoter, filaggrin mRNA expression promoter, involucrin mRNA expression promoter, and transglutaminase-1 mRNA expression promoter according to the present embodiment contain an extract of Aloe vera as an active ingredient.

[0018] The extraction raw material used to obtain the active ingredient in the present embodiment is Aloe vera (scientific name: Aloe vera).

[0019] Aloe vera (scientific name: Aloe vera) is a succulent plant belonging to the genus Aloe in the family Asphodelaceae. It is cultivated in Africa, India, etc. and can be easily obtained from these regions. As the constituent parts of aloe vera that can be used as extraction raw materials, they can be appropriately selected according to the purpose. For example, leaf parts, mesophyll parts, stem parts, flower parts, fruit parts, pericarp parts, fruit core parts, seed parts, root parts, mixtures thereof, or whole plants, or juices (pressing liquids) obtained by pressing these, concentrates, dried products, etc. can be mentioned. However, it is preferable to use concentrates, dried products, etc. of the pressing liquid of the mesophyll part.

[0020] Although the details of the substances having the ATP production promoting effect, glutathione production promoting effect, filaggrin mRNA expression promoting effect, involucrin mRNA expression promoting effect, and transglutaminase-1 mRNA expression promoting effect contained in the extract from the above extraction raw materials are unknown, by the extraction methods generally used in plant extraction and the like, extracts having the ATP production promoting effect, glutathione production promoting effect, filaggrin mRNA expression promoting effect, involucrin mRNA expression promoting effect, and transglutaminase-1 mRNA expression promoting effect can be obtained from the above extraction raw materials. In addition, the extract includes any of the extraction liquid obtained from the extraction raw material by the extraction treatment, the diluted or concentrated liquid of the extraction liquid, the dried product obtained by drying the extraction liquid, or the crude purification product or purification product thereof.

[0021] The above extract can be obtained by drying the extraction raw material and then using it as it is or pulverizing it using a crusher and subjecting it to extraction with an extraction solvent. The drying may be carried out in the sun or using a commonly used dryer. Also, it may be used as an extraction raw material after performing pretreatment such as degreasing with a non-polar solvent such as hexane. By performing pretreatment such as degreasing, the extraction treatment of the plant with a polar solvent can be carried out efficiently.

[0022] Examples of the solvent used for extraction include water, hydrophilic organic solvents, or mixtures thereof, etc., and it is preferably used at room temperature or a temperature below the boiling point of the solvent. Components having an ATP production promoting effect, a glutathione production promoting effect, a filaggrin mRNA expression promoting effect, an involucrin mRNA expression promoting effect, and a transglutaminase-1 mRNA expression promoting effect contained in the extraction raw material can be easily extracted by an extraction treatment using a polar solvent as the extraction solvent.

[0023] Examples of water that can be used as the extraction solvent include, for example, pure water, tap water, well water, mineral water, mineral water, hot spring water, spring water, fresh water, etc., and also those obtained by subjecting these to various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, etc. Therefore, 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, phosphate buffered saline, etc.

[0024] Examples of hydrophilic organic solvents that can be used as the extraction solvent include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, isopropyl alcohol; lower aliphatic ketones such as acetone, methyl ethyl ketone; polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol, glycerin, etc.

[0025] When using a mixed solution of two or more polar solvents as the extraction solvent, the mixing ratio can be adjusted as appropriate. For example, when using a mixed solution of water and a lower aliphatic alcohol, 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 using a mixed solution of water and a lower aliphatic ketone, 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 using a mixed solution of water and a polyhydric alcohol, it is preferable to mix 1 to 90 parts by volume of the polyhydric alcohol with respect to 10 parts by volume of water.

[0026] The extraction process does not need 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 it can be extracted 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 as necessary to elute the soluble components, the extract can be obtained by filtration to remove the solid matter. When the extraction solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and the dry matter is obtained by further drying this concentrate. The extraction conditions are 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 is about 30 minutes to 4 hours at 40 to 80 °C.

[0027] 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 dry matter of the extract, or a crude purification product or purified product thereof.

[0028] In addition, the obtained extract can be used as it is as an ATP production promoter, a glutathione production promoter, a filaggrin mRNA expression promoter, an involucrin mRNA expression promoter, and a transglutaminase-1 mRNA expression promoter, but it is preferably in the form of a concentrated solution or a dry matter. When obtaining the dry matter, carriers such as dextrin and cyclodextrin may be added to improve hygroscopicity.

[0029] Also, 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. The purification can be carried out, for example, by activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, etc.

[0030] The aloe vera extract obtained as described above has the effects of promoting ATP production, promoting glutathione production, promoting filaggrin mRNA expression, promoting involucrin mRNA expression, and promoting transglutaminase-1 mRNA expression. Therefore, it can be used as an active ingredient of an ATP production promoter, a glutathione production promoter, a filaggrin mRNA expression promoter, an involucrin mRNA expression promoter, and a transglutaminase-1 mRNA expression promoter by utilizing these effects.

[0031] The ATP production promoter, glutathione production promoter, filaggrin mRNA expression promoter, involucrin mRNA expression promoter, and transglutaminase-1 mRNA expression promoter according to this embodiment may be those in which the aloe vera extract is formulated.

[0032] The above extract can be formulated into any dosage form such as powder, granule, liquid, etc. according to a conventional method by using a pharmaceutically acceptable carrier such as dextrin, 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. Examples of the forms of the ATP production promoting effect, glutathione production promoting effect, filaggrin mRNA expression promoting effect, involucrin mRNA expression promoting effect, and transglutaminase-1 mRNA expression promoting effect obtained by formulating the above extract include ointments, external liquids, etc.

[0033] The ATP production promoter according to this embodiment can promote the production of ATP through the ATP production promoting effect of the aloe vera extract. As a result, it leads to the activation of functions such as cell growth, metabolism, and repair, and an anti-aging effect is achieved. In addition, the ATP production promoter in this embodiment can also be used as an active ingredient of a preventive, therapeutic, or improving agent for diseases and symptoms caused by a decrease in the amount of ATP production (for example, a preventive, therapeutic, or improving agent for eye fatigue, dizziness, gastritis, swelling, etc.). However, the ATP production promoter according to this embodiment can be used for all applications that are meaningful in exerting the ATP production promoting effect other than these applications.

[0034] The glutathione production promoter according to this embodiment can promote the production of glutathione through the glutathione production promoting action of the extract of Aloe vera. Thereby, it is possible to prevent, treat, or improve skin aging symptoms such as fine wrinkles, dullness, pigmentation, and rough skin. In particular, the glutathione production promoter in this embodiment utilizes the glutathione production promoting action of the above extract. In the epidermis, it can be used as an active ingredient of an agent for preventing, treating, or improving aging symptoms such as a decrease in the barrier function, rough skin, and the formation of fine wrinkles due to a decrease in the production amount of glutathione in epidermal keratinocytes. In the dermis, it can be used as an active ingredient of an agent for preventing, treating, or improving aging symptoms such as a decrease in skin elasticity and moisture retention function and pigmentation due to a decrease in the production amount of glutathione in fibroblasts. Therefore, it is considered that the above skin aging symptoms can be prevented, treated, or improved by promoting the production of glutathione in the skin. Further, the glutathione production promoter in this embodiment can also be used as an active ingredient of an agent for preventing, treating, or improving diseases, symptoms, etc. (for example, an agent for improving liver function in chronic liver disease, an agent for improving fatty liver, etc.) caused by a decrease in the production amount of glutathione by utilizing the glutathione production promoting action of the above extract. However, the glutathione production promoter according to this embodiment can be used for all applications that are meaningful in exerting the glutathione production promoting action other than these applications.

[0035] The filaggrin mRNA expression promoter according to this embodiment can promote the production of filaggrin through the filaggrin mRNA expression promoting action of the extract of Aloe vera. Thereby, it is possible to prevent or improve the decrease in the moisture retention function of the stratum corneum. However, the filaggrin mRNA expression promoter in this embodiment can be used for all applications that are meaningful in exerting the filaggrin mRNA expression promoting action other than these applications.

[0036] The involucrin mRNA expression promoter in this embodiment can promote the production of involucrin through the involucrin mRNA expression promoting action of the extract of Aloe vera. Thereby, it is possible to prevent, treat, or improve skin aging symptoms such as rough skin and dry skin, and skin diseases such as ichthyosis, psoriasis, atopic dermatitis, xeroderma, and acne. However, the involucrin mRNA expression promoter in this embodiment can be used for all applications that are meaningful for exerting the involucrin mRNA expression promoting action, in addition to these applications.

[0037] The transglutaminase-1 mRNA expression promoter according to this embodiment can promote the production of transglutaminase-1 through the transglutaminase-1 mRNA expression promoting action of the extract of Aloe vera. Thereby, it is possible to prevent, treat, or improve 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 according to this embodiment can be used for all applications that are meaningful for exerting the transglutaminase-1 mRNA expression promoting action, in addition to these applications.

[0038] Examples of the administration methods for patients of the ATP production promoter, glutathione production promoter, filaggrin mRNA expression promoter, involucrin mRNA expression promoter, and transglutaminase-1 mRNA expression promoter according to this embodiment include subcutaneous administration, intramuscular administration, intravenous administration, oral administration, transdermal administration, etc. However, according to the type of disease, a method suitable for its prevention, treatment, etc. may be appropriately selected. In addition, the dosage of the ATP production promoter, glutathione production promoter, filaggrin mRNA expression promoter, involucrin mRNA expression promoter, and transglutaminase-1 mRNA expression promoter according to this embodiment may be appropriately increased or decreased according to the type of disease, severity, individual differences of the patient, administration method, administration period, etc.

[0039] In addition, the ATP production promoter, glutathione production promoter, filaggrin mRNA expression promoter, involucrin mRNA expression promoter, and transglutaminase-1 mRNA expression promoter according to the present embodiment have excellent ATP production promoting action, glutathione production promoting action, filaggrin mRNA expression promoting action, involucrin mRNA expression promoting action, and transglutaminase-1 mRNA expression promoting action. Therefore, they are suitable for being formulated into cosmetics such as skin cosmetics, scalp cosmetics, and hair cosmetics, as well as food and beverages.

[0040] Examples of cosmetics that can incorporate an ATP production promoter, a glutathione production promoter, a filaggrin mRNA expression promoter, an involucrin mRNA expression promoter, and a transglutaminase-1 mRNA expression promoter include ointments, creams, emulsions, lotions, packs, foundations, hair tonics, hair lotions, shampoos, rinses, soaps, and the like. When formulating an ATP production promoter, a glutathione production promoter, a filaggrin mRNA expression promoter, an involucrin mRNA expression promoter, and a transglutaminase-1 mRNA expression promoter into cosmetics, the blending amount can be appropriately adjusted according to the type of cosmetics. However, a preferred blending ratio is about 0.0001 to 10% by mass in terms of a standard extract, and a particularly preferred blending ratio is about 0.001 to 1% by mass in terms of a standard extract. As long as the cosmetics do not interfere with the ATP production promoting action, glutathione production promoting action, filaggrin mRNA expression promoting action, involucrin mRNA expression promoting action, and transglutaminase-1 mRNA expression promoting action of the above extract, they can be used in combination with the main agents, auxiliaries, or other components used in the production of ordinary cosmetics, such as astringents, bactericidal and antibacterial agents, whitening agents, ultraviolet absorbers, moisturizing agents, cell activators, anti-inflammatory and anti-allergic agents, antioxidants and active oxygen scavengers, oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, and the like. By combining them in this way, a more general product can be obtained, and there may be an excellent effect beyond the synergistic effect usually expected between the combined other active ingredients.

[0041] Food and drink refers to substances that pose little risk of harm to human health and are usually ingested orally or through gastrointestinal administration in normal social life, and are not restricted by the classifications of foods, pharmaceuticals, quasi-drugs, etc. in administrative divisions. Therefore, the "food and drink" according to this embodiment broadly includes compositions constituting general foods, health foods (functional foods and drinks), health functional foods (foods for specified health use, nutritional functional foods, foods with function claims), quasi-drugs, pharmaceuticals, etc. that are ingested orally. The food and drink in this embodiment may display the ATP production promoting effect, glutathione production promoting effect, filaggrin mRNA expression promoting effect, involucrin mRNA expression promoting effect, and transglutaminase-1 mRNA expression promoting effect possessed by the extract on the food and drink or its packaging, or the food and drink may be a health functional food (foods for specified health use, foods with function claims, nutritional functional foods), a quasi-drug, or a pharmaceutical.

[0042] When the above extract, or an ATP production promoter, glutathione production promoter, filaggrin mRNA expression promoter, involucrin mRNA expression promoter, and transglutaminase-1 mRNA expression promoter formulated from the above extract are incorporated into food and drink, the blending amount of the active ingredients in them can be appropriately changed considering the purpose of use, symptoms, gender, etc., but considering the general intake amount of the food and drink to which they are added, it is preferable that the extract intake amount per adult per day is about 1 to 1000 mg. When the food and drink to which they are added is in the form of granules, tablets, or capsules, the addition amount of the above extract, or an ATP production promoter, glutathione production promoter, filaggrin mRNA expression promoter, involucrin mRNA expression promoter, and transglutaminase-1 mRNA expression promoter formulated from the above extract is usually 0.1 to 100% by mass, preferably 5 to 100% by mass, based on the food and drink to which they are added.

[0043] The ATP production promoter, glutathione production promoter, filaggrin mRNA expression promoter, involucrin mRNA expression promoter, and transglutaminase-1 mRNA expression promoter according to this embodiment are preferably applicable to humans, but can also be applied to animals other than humans as long as their respective effects are achieved.

Example

[0044] Hereinafter, the present invention will be described more specifically by showing production examples, test examples, etc., but the present invention is not limited to the following production examples, test examples, etc.

[0045] 〔Production Example〕Production of Aloe vera extract To 1 g of the solid obtained by concentrating the squeezed juice of the mesophyll of Aloe vera, 100 mL of a 30% by volume 1,3-butylene glycol solution was added, and heating extraction was performed at 50°C for 1 hour followed by filtration. The obtained extract was dried to obtain an Aloe vera extract (0.8 g).

[0046] 〔Test Example 1〕Test for ATP production promoting effect Regarding the Aloe vera extract obtained in the above production example, a test for the ATP production promoting effect was carried out by the following method.

[0047] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured using normal human epidermal keratinocyte growth medium (KGM), and the cells were recovered by trypsin treatment. The recovered cells were diluted with KGM to a cell density of 2.0×10 5 cells / mL, and then 100 μL per well was seeded into a collagen-coated 96-well plate and cultured overnight.

[0048] After culturing, the medium was removed, and 100 μL of KGM in which the test sample (Aloe vera extract of the production example) was dissolved was added to each well and cultured for 2 hours. As a control, culturing was performed in the same manner using KGM without addition of the sample.

[0049] The ATP production promoting effect was evaluated by measuring the amount of ATP in cells using the firefly luciferase luminescence method. Specifically, after the completion of the culture, 100 μL of the 'ATP measurement reagent for [cells]' (manufactured by Toyo B-net Co., Ltd.) was added to each well, and the chemiluminescence amount after the reaction was measured. From the measurement results, the ATP production promotion rate (%) was calculated according to the following formula.

[0050] ATP production promotion rate (%) = A / B × 100 In the formula, 'A' represents the chemiluminescence amount in the cells to which the test sample was added, and 'B' represents the chemiluminescence amount in the cells without the test sample.

[0051] The results of the above test are shown in Table 1. In the above formula, the ATP production promotion rate without the test sample is 100%.

[0052]

Table 1

[0053] As shown in Table 1, the aloe vera extract showed a high ATP production promotion rate. From this result, it was confirmed that the aloe vera extract has an excellent ATP production promoting effect.

[0054] 〔Test Example 2〕Test for promoting glutathione production in epidermal keratinocytes Regarding the aloe vera extract obtained in the above production example, a test for promoting glutathione production in epidermal keratinocytes was carried out by the following method.

[0055] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured using normal human epidermal keratinocyte growth medium (KGM), and the cells were collected by trypsin treatment. The collected cells were diluted with KGM to a cell density of 1.0×10 5 cells / mL, and then seeded at 200 μL per well in a collagen-coated 48-well plate and cultured for 48 hours.

[0056] After culturing, the medium was removed, and 200 μL of KGM in which the test sample (the aloe vera extract of the production example) was dissolved was added to each well, followed by culturing for 24 hours. As a control, culturing was performed in the same manner using KGM without addition of the sample. After culturing, the medium was removed, the cells were washed with 300 μL of PBS(-), and then the cells were lysed using 150 μL of M-PER(R) (manufactured by PIERCE).

[0057] Quantification of total glutathione was performed using 100 μL of this. Specifically, 100 μL of the cell extract dissolved in a 96-well plate, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH, and 25 μL of 3.2 units / mL glutathione reductase were added, and after heating at 37 °C for 10 minutes, 25 μL of 10 mM 5,5′-dithiobis(2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm up to 5 minutes later was measured to obtain ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione. After correcting the obtained value to the amount of glutathione per total protein amount, the glutathione production promotion rate (%) was calculated by the following formula.

[0058] Glutathione production promotion rate (%) = C / D × 100 In the formula, "C" represents the amount of glutathione per total protein amount in the cells to which the test sample was added, and "D" represents the amount of glutathione per total protein amount in the cells without addition of the test sample.

[0059] The results of the above test are shown in Table 2. In the above formula, the glutathione production promotion rate without addition of the test sample is 100%.

[0060]

Table 2

[0061] As shown in Table 2, the aloe vera extract showed a high glutathione production promotion rate in epidermal keratinocytes. From this result, it was confirmed that the aloe vera extract has an excellent glutathione production promotion effect in epidermal keratinocytes.

[0062] 〔Test Example 3〕Test for promoting glutathione production in dermal fibroblasts Regarding the aloe vera extract obtained in the above Production Example, a test for promoting glutathione production in dermal fibroblasts was carried out by the following method.

[0063] Normal human dermal fibroblasts (NB1RGB) were pre-cultured using α-MEM containing 10% FBS, and the cells were collected by trypsin treatment. The collected cells were diluted with α-MEM containing 10% FBS to a cell density of 2.0×10 5 cells / mL, and then seeded at 200 μL per well in a 48-well plate and cultured for 48 hours.

[0064] After culturing, the medium was removed, 200 μL of D-MEM containing 1% FBS in which the test sample (aloe vera extract of the Production Example) was dissolved was added to each well, and cultured for 24 hours. As a control, the same culture was carried out using D-MEM containing 1% FBS without addition of the sample. After culturing, the medium was removed, washed with 300 μL of PBS(-), and the cells were lysed using 150 μL of M-PER(R) (manufactured by PIERCE).

[0065] Using 100 μL of this, the total glutathione was quantified. Specifically, 100 μL of the cell extract dissolved in a 96-well plate, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH, and 25 μL of 3.2 units / mL glutathione reductase were added, and after heating at 37 °C for 10 minutes, 25 μL of 10 mM 5,5′-dithiobis(2-nitrobenzoic acid) was added. The absorbance at a wavelength of 412 nm up to 5 minutes later was measured, and ΔOD / min was determined. The total glutathione concentration was calculated based on the calibration curve prepared using oxidized glutathione. After correcting the obtained value to the amount of glutathione per total protein amount, the glutathione production promotion rate (%) was calculated by the following formula.

[0066] Glutathione production promotion rate (%) = C / D × 100 In the formula, "C" represents the amount of glutathione per total protein amount in the cells to which the test sample was added, and "D" represents the amount of glutathione per total protein amount in the cells without the test sample added.

[0067] The results of the above test are shown in Table 3. In the above formula, the glutathione production promotion rate without the test sample added is 100%.

[0068]

Table 3

[0069] As shown in Table 3, the aloe vera extract showed a high glutathione production promotion rate in dermal fibroblasts. From this result, it was confirmed that the aloe vera extract has an excellent glutathione production promotion effect in dermal fibroblasts.

[0070] 〔Test Example 4〕 Filaggrin mRNA Expression Promotion Effect Test Regarding the aloe vera extract obtained in the above Production Example, a test for the filaggrin mRNA expression promotion effect was carried out by the following method.

[0071] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured using normal human epidermal keratinocyte growth medium (KGM), and the cells were recovered by trypsin treatment. The recovered cells were diluted with KGM to a cell density of 3.0×10 5 cells / 2 mL in a 6-well plate and then cultured overnight under the conditions of 37 °C and 5% CO2 - 95% air.

[0072] After culturing, the medium was removed and replaced with a medium without added growth factors (normal human epidermal keratinocyte basal medium; KBM), and the cells were cultured for 24 hours. After culturing, the medium was removed, and 2 mL of KBM in which the test sample (aloe vera extract of the production example) was dissolved was added to each well, and the cells were cultured for 24 hours under the conditions of 37 °C and 5% CO2 - 95% air. As a control, the same culture was performed using KBM without addition of the sample. After culturing, the medium was removed, and total RNA was extracted with ISOGEN II (manufactured by Nippon Gene Co., Ltd.), and the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was prepared to be 200 ng / μL.

[0073] Using this total RNA as a template, the expression levels of mRNA for filaggrin and GAPDH as an internal standard were measured. Detection was performed by real-time 2Step RT-PCR reaction using a real-time PCR device Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by Takara Bio Inc.) with PrimeScript TM 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 filaggrin mRNA was calculated after correction with the value of GAPDH mRNA. From the obtained values, the filaggrin mRNA expression promotion rate (%) was calculated by the following formula.

[0074] Filaggrin mRNA expression promotion rate (%) = E / F × 100 In the formula, "E" represents the corrected value of the sample added with the test sample, and "F" represents the corrected value of the sample without addition of the test sample.

[0075] The results of the above test are shown in Table 4. In the above formula, the promoting rate of filaggrin mRNA expression without the addition of the test sample is 100%.

[0076]

Table 4

[0077] As shown in Table 4, the aloe vera extract showed a high promoting rate of filaggrin mRNA expression. From this result, it was confirmed that the aloe vera extract has an excellent effect of promoting filaggrin mRNA expression.

[0078] 〔Test Example 5〕Test for the effect of promoting involucrin mRNA expression For the aloe vera extract obtained in the above Production Example, a test for the effect of promoting involucrin mRNA expression was carried out by the following method.

[0079] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured using normal human epidermal keratinocyte growth medium (KGM), and the cells were recovered by trypsin treatment. The recovered cells were diluted with KGM to a cell density of 3.0×10 5 cells / 2mL, and then cultured overnight under the conditions of 37°C and 5% CO2 - 95% air.

[0080] After culturing, the medium was removed, and the cells were replaced with a medium without added growth factors (normal human epidermal keratinocyte basal medium; KBM) and cultured for 24 hours. After culturing, the medium was removed, and 2 mL of KBM in which the test sample (the aloe vera extract of the Production Example) was dissolved was added to each well, and the cells were cultured for 24 hours under the conditions of 37°C and 5% CO2 - 95% air. As a control, the same culture was carried out using KBM without the addition of the sample. After culturing, the medium was removed, total RNA was extracted with ISOGEN II (manufactured by Nippon Gene Co., Ltd.), the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was prepared to be 200 ng / μL.

[0081] Using this total RNA as a template, the expression levels of mRNA for involucrin and GAPDH, 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.), with PrimeScript TM RT Master Mix (Perfect Real Time) (manufactured by Takara Bio Inc.) and TB Green (registered trademark) Fast qPCR Mix (manufactured by Takara Bio Inc.) by a real-time 2Step RT-PCR reaction. The expression level of involucrin mRNA was calculated after correction with the value of GAPDH mRNA. From the obtained values, the promotion rate (%) of involucrin mRNA expression was calculated by the following formula.

[0082] Promotion rate of involucrin mRNA expression (%) = E / F × 100 In the formula, "E" represents the corrected value of the sample with the test sample added, and "F" represents the corrected value of the sample without the test sample added.

[0083] The results of the above test are shown in Table 4. In the above formula, the promotion rate of involucrin mRNA expression without the test sample added is 100%.

[0084]

Table 5

[0085] As shown in Table 5, the aloe vera extract showed a high promotion rate of involucrin mRNA expression. From this result, it was confirmed that the aloe vera extract has an excellent effect of promoting involucrin mRNA expression.

[0086] 〔Test Example 6〕Test for the effect of promoting transglutaminase-1 mRNA expression For the aloe vera extract obtained in the above Production Example, a test for the effect of promoting transglutaminase-1 mRNA expression was carried out by the following method.

[0087] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured using normal human epidermal keratinocyte growth medium (KGM), and the cells were recovered by trypsin treatment. The recovered cells were diluted with KGM to a cell density of 3.0×10 5 cells / 2 mL in a 6-well plate and then cultured overnight under the conditions of 37 °C and 5% CO2 - 95% air.

[0088] After culturing, the medium was removed and replaced with a medium without added growth factors (normal human epidermal keratinocyte basal medium; KBM), and the cells were cultured for 24 hours. After culturing, the medium was removed, and 2 mL of KBM in which the test sample (aloe vera extract of the production example) was dissolved was added to each well, and the cells were cultured for 24 hours under the conditions of 37 °C and 5% CO2 - 95% air. As a control, the same culture was performed using KBM without addition of the sample. After culturing, the medium was removed, and total RNA was extracted with ISOGEN II (manufactured by Nippon Gene Co., Ltd.), and the amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was prepared to be 200 ng / μL.

[0089] Using this total RNA as a template, the expression levels of mRNA for transglutaminase-1 and GAPDH as an internal standard were measured. Detection was performed by real-time 2Step RT-PCR reaction using the PrimeScript TM RT Master Mix (Perfect Real Time) (manufactured by Takara Bio Inc.) and TB Green (registered trademark) Fast qPCR Mix (manufactured by Takara Bio Inc.) using a real-time PCR apparatus Thermal Cycler Dice (registered trademark) Real Time SystemIII (manufactured by Takara Bio Inc.). The expression level of transglutaminase-1 mRNA was calculated after correction with the value of GAPDH mRNA. From the obtained values, the promotion rate (%) of transglutaminase-1 mRNA expression was calculated by the following formula.

[0090] Promotion rate (%) of transglutaminase-1 mRNA expression = E / F × 100 In the formula, "E" represents the corrected value of the sample added with the test sample, and "F" represents the corrected value of the sample without addition of the test sample.

[0091] The results of the above test are shown in Table 6. In the above formula, the promotion rate of transglutaminase-1 mRNA expression without the test sample added is 100%.

[0092]

Table 6

[0093] As shown in Table 6, the aloe vera extract showed a high promotion rate of transglutaminase-1 mRNA expression. From this result, it was confirmed that the aloe vera extract has an excellent effect of promoting transglutaminase-1 mRNA expression.

Industrial Applicability

[0094] The ATP production promoter, glutathione production promoter, filaggrin mRNA expression promoter, involucrin mRNA expression promoter, and transglutaminase-1 mRNA expression promoter of the present invention can be suitably used as a component of cosmetics, foods and drinks, etc., and further as a reagent for research.

Claims

1. An involucrin mRNA expression promoter used for promoting the expression of involucrin mRNA in epidermal keratinocytes, comprising: An involucrin mRNA expression promoter comprising as an active ingredient a water extract from the mesophyll of Aloe vera and / or a hydrophilic organic solvent extract from the mesophyll of Aloe vera.

2. A transglutaminase-1 mRNA expression promoter for promoting expression of transglutaminase-1 mRNA in epidermal keratinocytes, comprising: A transglutaminase-1 mRNA expression promoter comprising, as an active ingredient, a water extract from the mesophyll of Aloe vera and / or a hydrophilic organic solvent extract from the mesophyll of Aloe vera.

3. A filaggrin mRNA expression promoter used for promoting the expression of filaggrin mRNA in epidermal keratinocytes, comprising: A filaggrin mRNA expression promoter comprising as an active ingredient a water extract from the mesophyll of Aloe vera and / or a hydrophilic organic solvent extract from the mesophyll of Aloe vera.

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