Transglutaminase 3 expression-promoting composition

Abstract

To provide a transglutaminase 3 expression-promoting composition excellent in transglutaminase 3 expression-promoting effects.SOLUTION: The transglutaminase 3 expression-promoting composition of the present invention contains, as an active ingredient, at least one selected from among Panax ginseng extract, peony extract, citrus peel extract, isodon extract, lotus extract, Rehmannia glutinosa extract, sage extract, watercress extract, comfrey extract, Ganoderma lucidum extract, gardenia extract, Zanthoxylum fruit extract, Morus alba extract, swallow nest extract, and pea extract.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for promoting transglutaminase 3 expression, which contains a plant extract such as ginseng extract as an active ingredient. [Background technology]

[0002] Generally, transglutaminase is known as an enzyme that crosslinks glutamic acid and lysine by covalent bonds. Transglutaminase exists in a wide variety of organisms, including animals, plants, insects, and microorganisms. In addition, various isozymes are known, and for example, eight isozymes are known in humans. As disclosed in Patent Document 1, transglutaminase 1 is known to be abundant in the skin and to play a role in increasing the physical strength of the skin surface and improving the moisturizing function by the crosslinking activity of proteins.

[0003] By the way, "hair keratinization" is a boundary area between biochemistry and physicochemistry, and little research has been done on it. In addition, aging care focusing on hair "keratinization" is also almost unknown. Recently, the relationship between aging and transglutaminase 3, a keratinization enzyme, is becoming clear. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2022-161273 A Summary of the Invention [Problem to be solved by the invention]

[0005] Provided is a composition for promoting transglutaminase 3 expression, which has an excellent transglutaminase 3 expression promoting effect. [Means for solving the problem]

[0006] As a result of research conducted by the present inventors to solve the above problems, it was confirmed that transglutaminase 3 is characteristically decreased in women with weak hair. It was suggested that promoting the production of transglutaminase 3 contributes to increasing the strength and volume of hair. It was also discovered that active ingredients such as ginseng extract, which is a natural ingredient, have an excellent transglutaminase 3 expression promoting effect.

[0007] Various aspects for solving the above problems will be described below. The composition for promoting transglutaminase 3 expression of embodiment 1 contains at least one active ingredient selected from ginseng extract, peony extract, citrus peel extract, mountain mint extract, lotus extract, rehmannia extract, sage extract, watercress extract, comfrey extract, Ganoderma lucidum extract, gardenia extract, Japanese pepper extract, mulberry extract, bird's nest extract, and pea extract.

[0008] Aspect 2 relates to the composition for promoting transglutaminase 3 expression according to Aspect 1, wherein the active ingredient comprises at least one selected from the group consisting of ginseng extract, peony extract, citrus peel extract, mountain mint extract, lotus extract, and pea extract.

[0009] Aspect 3 relates to the composition for promoting transglutaminase 3 expression according to Aspect 1, wherein the active ingredients include ginseng extract and citrus peel extract, ginseng extract and lotus extract, or peony extract and lotus extract.

[0010] Aspect 4 is the composition for promoting transglutaminase 3 expression according to Aspect 1, wherein the active ingredients include ginseng extract, citrus peel extract, and peony extract. Effect of the Invention

[0011] According to the present invention, an excellent transglutaminase 3 expression promoting effect can be exhibited. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] An embodiment of the composition for promoting expression of transglutaminase 3 (hereinafter referred to as "TGase3") according to the present invention will be described. The composition for promoting expression of TGase3 according to this embodiment contains at least one active ingredient selected from ginseng extract, peony extract, citrus peel extract, mountain mint extract, lotus extract, Rehmannia pedunculosa extract, sage extract, watercress extract, comfrey extract, Ganoderma lucidum extract, gardenia extract, Japanese pepper extract, mulberry extract, bird's nest extract, and pea extract.

[0013] (Active ingredient) Specific examples of raw materials for ginseng extract include Korean ginseng and Korean ginseng. Specific examples of raw materials for peony extract include peony root bark (myophyte) and peony root. Specific examples of raw materials for citrus peel extract include mandarin orange peel, satsuma mandarin peel, and tachibana peel. Specific examples of raw materials for mountain mint extract include Japanese laurel (Emmeisou) and black hawthorn Japanese laurel. Specific examples of raw materials for lotus extract include lotus germ. Specific examples of raw materials for Rehmannia japonica extract include Rehmannia japonica, which is the root of Rehmannia japonica. Specific examples of raw materials for comfrey extract include comfrey leaves. Specific examples of raw materials for Ganoderma lucidum extract include Reishi mushroom. Specific examples of raw materials for Gardenia extract include Sanshishi, which is the fruit of Gardenia. Specific examples of raw materials for mulberry extract include, for example, mulberry root bark, etc. Specific examples of raw materials for bird's nest extract include, for example, swiftlet nests, etc. Specific examples of raw materials for pea extract include, for example, germinated peas, etc. These may be used alone or in combination of two or more.

[0014] Among these, it is preferable to contain at least one selected from ginseng extract, peony extract, citrus peel extract, mountain mint extract, lotus extract, and pea extract as an active ingredient, which can exert a more excellent TGase3 expression promoting effect.

[0015] It is more preferable that the active ingredients include a combination of ginseng extract and citrus peel extract, ginseng extract and lotus extract, or peony extract and lotus extract, which can exert a more excellent TGase3 expression promoting effect.

[0016] It is also preferable that the active ingredients contain at least three components, namely, ginseng extract, citrus peel extract, and peony extract, which can exert a particularly excellent TGase3 expression promoting effect.

[0017] These extract components may be commercially available, or may be extracted from each raw material. The extract may be extracted by a known method. For example, known extraction methods such as stirring extraction, kneader extraction, drip extraction, and column extraction may be used. Examples of the extraction solvent include water such as hard water, soft water, ion-exchanged water, and distilled water, as well as organic solvents such as ethanol, and mixtures of the above water and organic solvents such as ethanol. The extraction solvent may contain an extraction aid or the like as necessary. Examples of the extraction aid include pH adjusters such as ascorbic acid, potassium carbonate, and sodium bicarbonate.

[0018] The extraction temperature and extraction time are appropriately set depending on each raw material. The obtained extract is concentrated by evaporating water, or dried and powdered. The concentration and drying of the treated liquid can be performed by known methods such as reduced pressure concentration, membrane concentration, freeze concentration, vacuum drying, spray drying, or freeze drying.

[0019] The content of the active ingredient in the composition for promoting TGase3 expression is appropriately set depending on the dosage form and the type of active ingredient, but is preferably 0.1% by mass to 50% by mass, more preferably 0.5% by mass to 20% by mass. By being specified within such a range, the composition can be efficiently administered to a living body while maintaining its stability, etc.

[0020] (Usage form) Specific application forms of the composition for promoting TGase3 expression of the present invention include cosmetics, external skin preparations, quasi-drugs, foods and beverages, pharmaceuticals, etc., intended to achieve the above-mentioned functional effects.

[0021] When applied to cosmetics, topical skin preparations, or quasi-drugs, they can be produced by blending them into a base material for cosmetics, topical skin preparations, or quasi-drugs. The form of the cosmetics, topical skin preparations, or quasi-drugs may be any of an aqueous solution, emulsion, cream, powder, etc. The base material may be appropriately blended with at least one selected from known materials such as oil, purified water, and alcohol as main components, surfactants, moisturizers, antioxidants, thickeners, antiseborrheic agents, blood circulation promoters, pH adjusters, color pigments, preservatives, and fragrances.

[0022] The food and drink can be used as various food materials or beverage materials, for example, health foods, supplements, etc. The form of the food and drink is not particularly limited and may be any of liquid, powder, gel, solid, etc., and the dosage form may be any of tablets, capsules (hard capsules, soft capsules, etc.), granules, drinks, etc. Among them, in the case of capsules, hygroscopicity is suppressed and storage stability can be improved.

[0023] When applied to food and beverages, additives such as excipients, sugars, flavorings, sweeteners, oils and fats, base materials, food additives, sub-ingredients, bulking agents, etc. may be appropriately blended as the contents. The uses of food and beverages are not particularly limited, and include so-called (1) general foods, (2) health foods such as nutritional supplements, dietary supplements, nutritionally adjusted foods, and supplements, (3) health functional foods such as foods for specified health uses, foods with nutritional functions, foods with functional claims, and functional foods, and (4) foods for special uses such as foods for sick people. When a use indication is attached to a food or beverage, examples of the indication include indications stipulated by various laws, enforcement regulations, guidelines, etc. In the case of food and beverages, indication of use includes indication on packages such as wrapping and containers, as well as indication on advertising media such as pamphlets.

[0024] The contents of the indication of the use of the composition for promoting TGase3 expression include indication of promotion of TGase3 expression, etc. Also included are indications suggesting promotion of TGase3 expression. In addition, examples of indications include indications of increase or inhibition of decrease in hair firmness associated with promotion of TGase3 expression, inhibition or delay of decrease in hair firmness due to aging, etc.

[0025] When used as a pharmaceutical composition, a known administration method can be appropriately adopted depending on the purpose, etc. For example, in addition to application to the skin surface and oral ingestion (ingestion), any administration method such as intravascular administration and transdermal administration can be adopted. When used as a pharmaceutical composition, pharma- ceutically acceptable additives such as bases, excipients, emulsifiers, solvents, stabilizers, carriers, dispersants, buffers, binders, disintegrants, lubricants, etc. may be further blended as the contents.

[0026] When applied as a cosmetic, skin external preparation or quasi-drug, the amount of application per day is not particularly limited. From the viewpoint of the expression of an excellent TGase3 expression promoting effect, the dosage is preferably 10 mg or more and 5000 mg or less per day, more preferably 100 mg or more and 1000 mg or less per day, as the solid content of the active ingredient. In addition, the administration time or period is not particularly limited, but it is preferable to apply several times a day and continuously apply for several months.

[0027] (action) The composition for promoting TGase 3 expression exerts the effect of promoting the expression of the TGase 3 gene by the above-mentioned active ingredient. This increases the amount of TGase 3 protein in the living body to which it is applied. The present inventors have confirmed that TGase 3 is specifically expressed in the keratinized zone, an area where keratin fibers are formed. TGase 3 is important for the formation of keratin fibers, and its reduction is believed to be one of the causes of the loss of hair strength and stiffness due to aging. Therefore, it is expected that promoting the production of TGase 3 will contribute to increasing hair strength and stiffness.

[0028] The effect of the composition for promoting TGase3 expression of this embodiment will be described. (1) The composition for promoting TGase3 expression of this embodiment contains the above-mentioned ginseng extract and the like as active ingredients. Therefore, it exerts an excellent TGase3 expression promoting effect. Therefore, it is expected to contribute to the increase in hair elasticity and strength associated with the promotion of TGase3 expression.

[0029] The above embodiment may be modified as follows: The above embodiment and the following modifications may be combined with each other to the extent that no technical contradiction occurs. The composition for promoting TGase3 expression in the above embodiment can be applied not only to humans but also to domestic animals such as pets and livestock. EXAMPLES

[0030] The configuration and effects of the present invention will be specifically described below using examples, but the present invention is not limited to these examples. <Test Example 1: TGase3 activation test by plant extracts using normal human epidermal keratinocytes 1> (cell) NHEK (Normal human epidermal keratinocytes: Kurabo Industries, Ltd.) were used in this experiment. After cell thawing, the entire contents of the tube were added to Humedia-KB2 medium (Kurabo Industries, Ltd.) (hereinafter referred to as "KB medium") and then centrifuged (1,000 rpm, 4°C, 5 minutes). After that, the supernatant was removed and fresh medium was added, after which the cells were seeded on a 10 cm petri dish (Corning Industries, Ltd.) and cultured (37°C, 5% CO2). During the culture, the medium was replaced in its entirety approximately once every two days.

[0031] The medium was aspirated from the culture dish, 2 mL of HEPES buffer was added, and the mixture was allowed to mix thoroughly. The HEPES buffer was removed, and 2 mL of trypsin / EDTA solution was added and mixed thoroughly to detach the cells. 5 mL of HEPES buffer was then added to the dish, and the cells were then collected in a 15 mL tube containing 5 mL of trypsin neutralizing solution. After centrifugation (1,000 rpm, 4°C, 5 minutes), the supernatant was removed and an appropriate amount of medium was added. The number of cells was counted using a hemocytometer. The cell suspension was diluted with medium and then diluted to 2 × 10 4 The cells were dropped onto a 12-well plate (Corning) at 12 cells / well, and then cultured for 4 days (37°C, 5% CO2) before being used in the tests using the plant extracts shown below.

[0032] (Plant extract) The plant extract powders shown in Tables 1 and 2 were used. Test KB medium was prepared as a composition for promoting TGase 3 expression by adding the following raw materials to the KB medium at 1% by mass. N=2 was cultured. The control was KB medium without the addition of the plant extract.

[0033] After removing the medium from the cultured cells, test KB medium containing plant extract powder was added. Six hours after medium replacement, the medium was removed and the cells were washed with PBS. Then, 500 μL of TRIzol (Thermo Fisher) was added and left for about 15 minutes. The TRIzol solution in which the cells had dissolved was collected and transferred to a 1.5 mL tube. It was stored at -80°C.

[0034] (Method for measuring the expression level of TGase3) RNA extraction and cDNA synthesis 0.2 mL of chloroform was added per 1 mL of TRIzol, mixed by inversion for 15 seconds, and then allowed to stand at room temperature for 3 minutes. After centrifugation at 12,000 × g and 4 °C for 15 minutes, the aqueous layer was collected.

[0035] The collected aqueous layer was mixed with 0.25 mL of isopropanol per mL of TRIzol, vortexed, and then left at room temperature for 3 minutes. The mixture was centrifuged at 12,000 × g and 4 ° C for 15 minutes, and the supernatant was removed. The precipitate obtained at this stage was mixed with 1 mL or more of 75% ethanol per mL of TRIzol, vortexed, and then centrifuged at 7,500 × g and 4 ° C for 5 minutes.

[0036] After removing as much of the supernatant as possible, the precipitate was air-dried. RNAse-free water was added to the precipitate and mixed by pipetting, and the amount of RNA obtained was measured using a nano drop (Thermo Fisher). The obtained RNA was subjected to reverse transcription PCR using a SuperScript VILO cDNA Synthesis Kit (Invitrogen) and stored as stable cDNA.

[0037] · Measurement of mRNA quantity (real-time PCR) Real-time PCR was carried out using TaqMan Gene Expression Assay, which was a premix of primers and probes for each gene synthesized by Applied Biosystems. The genes analyzed were transglutaminase 3 (TGase 3) and 18S, an endogenous control. Quantitative real-time PCR was carried out using Taq-man Fast Advanced Master Mix (Applied Biosystems). The relative expression level (Relative Quantification: RQ) to the control was calculated as ΔΔC tThe expression level was calculated relative to the untreated control only in Example 4. The calculation method is as follows.

[0038]

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[0039]

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[0040]

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[0041] [Table 1]

[0042] [Table 2] As shown in Tables 1 and 2, the plant extracts of each Example were confirmed to have a higher TGase3 expression promoting effect than the control. In particular, it was confirmed that ginseng extract, Peony extract, Mandarin orange extract, citrus peel extract, Japanese peppermint extract, lotus germ extract, and pea extract were more effective in promoting TGase3 expression.

[0043] <Test Example 2: TGase3 activation test 2 by plant extracts using normal human epidermal keratinocytes> In Test Example 1, five types of plant extracts, namely, ginseng extract, Peony extract, Mandarin orange extract, Japanese peppermint extract, and lotus germ extract, were tested for their preferred combinations, as they had a relatively high TGase3 expression promoting effect in Test Example 1. The test method was the same as in Test Example 1, except that the amount of each plant extract used was changed to 0.5% by mass. The same types of plant extracts as those in Test Example 1 were used. In Examples 18 to 22, one type of plant extract was used, and the amount of each plant extract was 0.5% by mass. In Examples 23 to 31, two types of plant extracts were used in combination, and the amount of each plant extract was 0.5% by mass (total 1% by mass). The relative expression amount (RQ) to the control was calculated based on the above formulas 1 to 3, as ΔΔC t The mean and standard deviation were calculated for N=2 tests. The results are shown in the "TGase3 expression level" column in Table 3.

[0044] [Table 3] As shown in Table 3, the combination of Panax ginseng extract and mandarin orange extract, which is a citrus peel extract, in Example 24 was confirmed to have a superior TGase3 expression promoting effect to those of Example 3, in which 1% by mass of Panax ginseng extract was used, and Example 8, in which 1% by mass of mandarin orange extract was used.

[0045] It was confirmed that the combination of ginseng extract and lotus germ extract, which is a lotus extract, in Example 26 had a superior TGase3 expression promoting effect to those of Example 3, in which 1% by mass of ginseng extract was used, and Example 15, in which 1% by mass of lotus germ extract was used.

[0046] The combination of the peony extract, Peony pisum sativum extract, in Example 28, and the lotus extract, Lotus germ extract, was confirmed to have a superior TGase3 expression promoting effect to that of Example 7, in which 1% by mass of Peony pisum extract was used, and Example 15, in which 1% by mass of lotus germ extract was used.

[0047] <Test Example 3: TGase3 activation test by plant extracts using normal human epidermal keratinocytes 3> In Test Example 1, three types of plant extracts, namely, ginseng extract, peony extract, and mandarin orange extract, which are citrus peel extracts, were relatively highly effective in promoting the expression of TGase3, and their combinations were tested. The test method was the same as in Test Example 1, except that the amount of each plant extract used was changed to 0.3 mass%. The same types of plant extracts as in Test Example 1 were used. In Examples 32 to 34, one type of plant extract was used, and the amount of each plant extract was 0.3 mass%. In Example 35, three types of plant extracts were used in combination, and the amount of each plant extract was 0.3 mass% (total 0.9 mass%). The relative expression amount (RQ) to the control was calculated based on the above formulas 1 to 3 as ΔΔC t The mean and standard deviation were calculated for N=4 tests. The results are shown in the "TGase3 expression level" column in Table 4.

[0048] [Table 4] As shown in Table 4, the combination of ginseng extract, mandarin orange (a citrus peel extract), and peony extract (Moutan Pea Extract) was confirmed to have a superior TGase3 expression promoting effect to Example 3, which used 1% by mass of ginseng extract, Example 8, which used 1% by mass of mandarin orange extract, and Example 7, which used 1% by mass of peony extract.

[0049] <Test Example 4: TGase3 activation test by plant extracts in humans> A test was carried out on TGase3 activation when plant extracts were administered to humans. ·subject 12 women aged 20-49 Test sample A scalp care agent with TGase3 activation was prepared by dissolving 3.3% by mass of ginseng extract powder (Korean ginseng: Shinhongginisin LV (Ichimaru Falcos)), 3.3% by mass of peony extract powder (Peony root: Falcorex Peony root E (Ichimaru Falcos)), and 3.3% by mass of citrus peel extract powder (Mandarin orange: Mandarin Cria (Ichimaru Falcos)) in 95% aqueous ethanol.

[0050] ·Usage Apply 10 to 15 pumps (each pump is approximately 0.15 mL) to the entire scalp twice a day (morning and night).

[0051] ·TGase3 mRNA detection method Before and one month after the test, 12 hair roots were removed from the temporal region of each subject and the hair roots were collected. RNA was extracted from the collected samples using TRIzol. mRNA was extracted from the hair roots and cDNA was synthesized using reverse transcription PCR. After that, the amount of TGase3 and the amount of 18S as a control gene were measured using real-time PCR.

[0052] RNA extraction, cDNA synthesis, and mRNA amount measurement (real-time PCR) were performed according to the method described in Test Example 1. The relative expression amount (RQ) to the untreated (before test) was calculated based on the above formulas 1 to 3 as ΔΔC t The results are shown in the "TGase3 expression level" column of Table 5.

[0053] [Table 5] As shown in Table 5, the expression of TGase3 in the hair roots was confirmed in 9 out of 12 subjects. Therefore, it is expected that the application of ginseng extract etc. will improve the firmness and strength of human hair by promoting the expression of TGase3.

[0054] Next, the technical ideas that can be understood from the above-described embodiment and other examples will be described below. (i) An agent for improving hair firmness and strength by promoting the expression of transglutaminase 3, comprising as an active ingredient at least one selected from ginseng extract, peony extract, citrus peel extract, mountain mint extract, lotus extract, rehmannia extract, sage extract, watercress extract, comfrey extract, Ganoderma lucidum extract, gardenia extract, Japanese pepper extract, mulberry extract, bird's nest extract, and pea extract.

Claims

1. A composition for promoting expression of transglutaminase 3, comprising as an active ingredient at least one selected from ginseng extract, peony extract, citrus peel extract, mountain mint extract, lotus extract, rehmannia extract, sage extract, watercress extract, comfrey extract, Ganoderma lucidum extract, gardenia extract, Japanese pepper extract, mulberry extract, bird's nest extract, and pea extract.

2. The composition for promoting transglutaminase 3 expression according to claim 1 , wherein the active ingredient comprises at least one selected from the group consisting of ginseng extract, peony extract, citrus peel extract, mountain mint extract, lotus extract, and pea extract.

3. The composition for promoting transglutaminase 3 expression according to claim 1 , wherein the active ingredients include ginseng extract and citrus peel extract, ginseng extract and lotus extract, or peony extract and lotus extract.

4. The composition for promoting transglutaminase 3 expression according to claim 1 , wherein the active ingredients include ginseng extract, citrus peel extract, and peony extract.

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