JP7328073B2 - CLSP and/or TINCR expression promoter - Google Patents

Description

TECHNICAL FIELD The present invention relates to CLSP and/or TINCR expression promoters, and skin anti-aging agents containing such expression promoters as active ingredients.

The epidermis of the skin is mainly composed of cells called keratinocytes. The keratinocytes proliferating in the basal layer, the innermost layer of the epidermis, migrate outward (toward the surface of the skin) as if extruded, and along with this migration, divide into the stratum spinosum, stratum granulosum, and stratum corneum in order. become. That is, the epidermis is composed of multiple layers of keratinocytes with different degrees of differentiation. Keratinocyte proliferation or cell division normally occurs only in the basal layer (including the deepest monolayer of the epidermis and the adjacent suprabasal layer), and cell differentiation occurs outside the stratum spinosum without cell division. only proceed.

Calmodulin-Like Skin Protein (CLSP) is a calcium-binding protein produced primarily in the skin. Non-Patent Document 1 states that although expression of CLSP is not detected in proliferating keratinocytes, in the later stage of keratinocyte differentiation, i.e., after differentiation day 6-7, which is after the time point when the expression of loricrin has already started. It is described that it is expressed. Loricrin is a protein whose expression starts when keratinocytes migrate to the granular layer. Similarly, Non-Patent Document 2 also describes that the CLSP protein is absent in the basal layer and abundantly present in the differentiated outer epidermal layer. Non-Patent Document 2 also describes that depletion of CLSP by shRNA had no effect on basal layer cell proliferation. Therefore, CLSP is believed to be involved in terminal differentiation (keratinization) of keratinocytes.

Patent Document 1 describes a full-length polypeptide of CLSP, "for example, in cosmetics, in the treatment of aging, in particular in the treatment of skin aging, in the treatment of skin damage associated with exposure to ultraviolet light, usually Alternatively, to modulate pathological epithelial proliferation dysfunction or epithelial hypodifferentiation (dry skin, hyperkeratosis, parakeratosis, psoriasis, ichthyosis, neoplasia, etc.). It is disclosed that the protein can be used.

In addition, TINCR, a type of long non-coding RNA, was known to stabilize CLSP mRNA (Non-Patent Document 2).

Here, the effectiveness of the marjoram extract has been studied in various ways, including promotion of type XVII collagen production (Patent Document 2), promotion of proton pump function (Patent Document 3), removal of active oxygen radicals (Patent Document 4), It is known to have many physiological activities such as heat shock protein expression induction (Patent Document 5), type VII collagen production promotion (Patent Document 6), hair growth (Patent Document 7), and platelet aggregation inhibitory effect (Patent Document 8). ing.

In addition, the effectiveness of the coltsfoot extract has also been investigated in various ways, including a sebum secretion promoting action (Patent Document 9), a kinesin suppressing action (Patent Document 10), a basement membrane stabilizing action (Patent Document 11), and an active oxygen scavenging action (Patent Document 11). Patent Document 12), lipase inhibitory action (Patent Document 13), melanocyte proliferation promoting action (Patent Document 14), and many other physiological activities are known.

Japanese Patent No. 3851818 JP 2018-108973 A Japanese Patent Application Laid-Open No. 2018-20970 JP 2017-197503 A JP 2015-042675 A JP 2014-221739 A JP 2014-185131 A JP 2012-153671 A JP 2017-066106 A JP 2013-166713 A Japanese Patent Application Laid-Open No. 2006-316050 JP 2006-117612 A JP 2005-008572 A JP 2004-161639 A

The Journal Of Biological Chemistry, Vol. 275, No. 17, April 28, pp. 12841-12847, 2000 Genes & Development, 2015, 29:2225-2230

An object of the present invention is to provide a plant extract that promotes the expression of TINCR and/or the expression of CLSP and is effective in preventing skin aging. Another object of the present invention is to provide a combination of plant extracts that synergistically enhances TINCR expression and/or CLSP expression.

Means for solving the problems of the present invention are as follows.
(1)
A CLSP and/or TINCR expression promoter containing a marjoram extract as an active ingredient.
(2)
2. The expression promoter according to claim 1, further comprising coltsfoot extract as an active ingredient.
(3)
A skin anti-aging agent comprising the expression promoter according to claim 1 or 2 as an active ingredient.

The marjoram extract has the effect of promoting CLSP expression in a concentration-dependent manner.
The marjoram extract has the effect of promoting the expression of TINCR in a concentration-dependent manner.
Combined use of the marjoram extract and the coltsfoot extract synergistically enhances the CLSP expression promoting effect.
Combined use of the marjoram extract and the coltsfoot extract synergistically enhances the effect of promoting the expression of TINCR.

FIG. 1 is a diagram showing the effect of marjoram extracts on enhancing the expression of CLSP in a concentration-dependent manner. FIG. 2 shows the effect of a marjoram extract on promoting the expression of TINCR in a concentration-dependent manner. FIG. 3 shows that the combined use of marjoram extract and coltsfoot extract synergistically enhances the effect of promoting CLSP expression. FIG. 4 shows that the combined use of marjoram extract and coltsfoot extract synergistically enhances the effect of promoting CLSP expression.

A mode for carrying out the present invention will be described below.

The marjoram extract used in the present invention is an extract obtained from marjoram (scientific name: Origanum majorana). The constituent parts of marjoram that can be used include, for example, leaves, stems, flowers, buds, rhizomes, aerial parts, and mixtures thereof, preferably leaves.

When preparing the extract, the raw plant is used as it is or dried.

Extraction solvents include lower alcohols such as methanol, ethanol, propanol and isopropanol; polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, dipropylene glycol and glycerin; ethers such as ethyl ether and propyl ether; , butyl acetate, and ketones such as acetone and ethyl methyl ketone. Physiological saline, phosphate buffer, phosphate buffered saline, etc. may also be used.

The extract from the above solvent can be used as it is, but after concentration and drying, it can be redissolved in water or a polar solvent, or decolorized, deodorized, or deodorized to the extent that it does not impair their skin physiological function-enhancing effects. It may be used after purification treatment of salt or the like or fractionation treatment by column chromatography. Alternatively, the extract may be hydrolyzed with an acid, an alkali, an enzyme, or the like. For storage, it can also be freeze-dried after purification and dissolved in a solvent before use. It can also be used by encapsulating it in vesicles such as liposomes or microcapsules.

The extraction treatment is not particularly limited as long as the soluble components contained in the raw material for extraction can be eluted into the extraction solvent, and can be carried out according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent of 5 to 30 times the amount (mass ratio) of the extraction raw material, the soluble component is extracted at room temperature or under reflux heating, and then filtered to remove the extraction residue. You can get the liquid. A paste-like concentrate is obtained by distilling off the solvent from the obtained extract, and a dried product is obtained by further drying the concentrate.

For example, the marjoram extract of the present invention can be obtained by immersing dried marjoram leaves in an 85% by volume ethanol aqueous solution, distilling off the solvent, and adding 50% by volume of a 1,3-butylene glycol aqueous solution to 1% by mass as a pure extract. can be used.

The coltsfoot extract used in the present invention is an extract obtained from coltsfoot (scientific name: Tussilago farfara). Constituent parts of coltsfoot that can be used include, for example, one or more parts selected from whole plants, leaves, stems, flowers, fruits, and roots, preferably from leaves, stems, and flowers. One or more selected species, more preferably flowers, are used.

The coltsfoot extract is extracted by the same method as for the marjoram extract described above.

For example, the coltsfoot extract of the present invention is obtained by immersing dried coltsfoot flowers in purified water, distilling off the solvent, and adding a 20% by volume aqueous solution of 1,3-butylene glycol so that the pure content of the coltsfoot is 1% by mass. can be used.

The skin anti-aging agent of the present invention contains a CLSP and/or TINCR expression promoter as an active ingredient.

In the present invention, the blending amount of each of the expression accelerators in the skin antiaging agent is preferably 0.00001 to 5% by mass.

The skin anti-aging agent of the present invention has a high CLSP and/or TINCR expression-promoting action and exhibits a high anti-aging effect.

The skin anti-aging agent of the present invention can contain, in addition to the above-described components, optional components used in ordinary cosmetics and quasi-drugs to the extent that the effects of the present invention are not impaired. Specific examples include oils, surfactants, thickeners, preservatives, fragrances, moisturizing agents, antioxidants, anti-inflammatory agents, antibacterial agents, and the like.

The dosage form of the skin anti-aging agent of the present invention is not particularly limited, and any dosage form such as water-based, oil-based, or emulsified type may be used.

The skin anti-aging agent of the present invention can be prepared by conventional methods.

The skin anti-aging agent of the present invention can be used, for example, in lotions, emulsions and ointments.

EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited by these examples. In addition, the compounding quantity is mass % unless otherwise specified.

First, the preparation method of the plant extracts used in Examples and the like will be described.

[Marjoram extract]
After immersing dried marjoram leaves in 85% by volume ethanol aqueous solution, the solvent was distilled off, and marjoram was extracted by adding to 50% by volume 1,3-butylene glycol aqueous solution so that the pure extract content was 1% by mass. I made it.

[Coltsfoot extract]
Dried coltsfoot flowers are immersed in purified water, the solvent is distilled off, and the coltsfoot extract is obtained by adding to 20% by volume of 1,3-butylene glycol aqueous solution so that the pure extract content is 1% by mass. and

[Expression promotion effect of CLSP and TINCR]
Human epidermal keratinocytes were seeded in 6-well plates at a cell density of 3×10 ⁵ cells/well and cultured overnight in HuMedia-KG2 medium. The medium was exchanged with a fresh medium to which the extract was added and dissolved so as to obtain the extract concentration shown in Table 1, or a fresh medium (blank) to which no extract was added, and the cells were further cultured for 24 hours. After that, total RNA was extracted from the harvested cells using a commercially available RNA extraction kit, and cDNA was synthesized. Expression levels were quantified by real-time PCR by the Cybergreen method using the following primers specific for CLSP or GAPDH. GAPDH was used as an internal standard. The expression level was shown as a relative value when the blank expression level was set to 1.

PCR primer sequence CLSP forward: GTTGACACGGATGGAAACG (SEQ ID NO: 1)
CLSP reverse: ACTCCTGGAAGCTGATTTCG (SEQ ID NO: 2)
TINCR forward: TGTGGCCCAAAACTCAGGGATACAT (SEQ ID NO: 3)
TINCR reverse: AGATGACAGTGGCTGGAGTTGTCA (SEQ ID NO: 4)
GAPDH Forward: CCACTCCTCCCACCTTTTGACG (SEQ ID NO: 5)
GAPDH reverse: CACCCTGTTGCTGTAGCCAA (SEQ ID NO: 6)

As shown in FIG. 1 and Table 1, the marjoram extract exhibited the effect of increasing the expression level of CLSP in a concentration-dependent manner.

As shown in FIG. 2 and Table 1, the marjoram extract exhibited the effect of increasing the expression level of TINCR in a concentration-dependent manner.

As shown in sample 6 in FIG. 3 and Table 1, by using marjoram extract and coltsfoot extract in combination, compared to sample 4 and sample 5, in which each extract alone was blended in double amounts, CLSP The expression level increased, and the combined use of the marjoram extract and the coltsfoot extract synergistically improved the effect of promoting the expression of CLSP.

As shown in sample 6 in FIG. 4 and Table 1, by using marjoram extract and coltsfoot extract in combination, compared to sample 4 and sample 5, in which each extract alone was blended in double amounts, TINCR The expression level increased, and the combined use of the marjoram extract and the coltsfoot extract synergistically enhanced the effect of promoting the expression of TINCR.

[Example 1] Emulsion (1) Squalane 10.0 (mass%)
(2) Methylphenylpolysiloxane 4.0
(3) Hydrogenated palm kernel oil 0.5
(4) Hydrogenated soybean phospholipid 0.1
(5) Polyoxyethylene monostearate
Sorbitan (20 E.O.) 1.3
(6) Sorbitan monostearate 1.0
(7) Glycerin 4.0
(8) Methyl paraoxybenzoate 0.1
(9) carboxyvinyl polymer 0.15
(10) Remaining purified water to 100 (11) Arginine (1% by mass aqueous solution) 20.0
(12) marjoram extract 0.5
(13) coltsfoot extract 0.5
Production method: The oil phase components (1) to (6) are heated and dissolved at 80°C. On the other hand, the water phase components (7) to (10) are heated and dissolved at 80°C. The oil phase components are added to this while stirring, and uniformly emulsified with a homogenizer. After cooling, at 40° C., (11) to (13) are sequentially added and uniformly mixed.

[Example 2] Lotion (1) Ethanol 15.0 (mass%)
(2) Polyoxyethylene (40E.O.) hydrogenated castor oil 0.3
(3) Perfume 0.1
(4) Remainder of purified water 100 (5) Citric acid 0.02
(6) sodium citrate 0.1
(7) Glycerin 1.0
(8) hydroxyethyl cellulose 0.1
(9) marjoram extract 0.3
(10) coltsfoot extract 0.5
Preparation method: (2) and (3) are dissolved in (1). Furthermore, after sequentially adding (4) to (10), the mixture is thoroughly stirred and uniformly mixed.

[Example 3] Cream (1) Squalane 10.0 (% by mass)
(2) stearic acid 2.0
(3) Hydrogenated palm kernel oil 0.5
(4) Hydrogenated soybean phospholipid 0.1
(5) Cetanol 3.6
(6) Lipophilic glyceryl monostearate 2.0
(7) Glycerin 10.0
(8) Methyl paraoxybenzoate 0.1
(9) Arginine (20% by mass aqueous solution) 15.0
(10) Remainder of purified water 100 (11) Carboxyvinyl polymer (1% by mass aqueous solution) 15.0
(12) marjoram extract 0.5
(13) coltsfoot extract 0.3
Production method: The oil phase components (1) to (6) are heated and dissolved at 80°C. On the other hand, the water phase components (7) to (10) are heated and dissolved at 80°C. The oil phase components are added to this while stirring, and uniformly emulsified with a homogenizer. After (11) is added and stirred, the mixture is cooled, and (12) and (13) are added at 40° C. and mixed uniformly.

[Example 4] Essence (1) Remainder of purified water 100 (% by mass)
(2) Glycerin 10.0
(3) Sucrose fatty acid ester 1.3
(4) Carboxyvinyl polymer (1% by mass aqueous solution) 17.5
(5) Sodium alginate (1% by mass aqueous solution) 15.0
(6) Polyglyceryl monolaurate 1.0
(7) macadamia nut oil fatty acid phytosteryl 3.0
(8) N-lauroyl-L-glutamic acid di(phytosteryl-2-octyldodecyl) 2.0
(9) Hydrogenated palm oil 2.0
(10) Squalane (derived from olive) 1.0
(11) behenyl alcohol 0.75
(12) Beeswax 1.0
(13) Jojoba oil 1.0
(14) 1,3-butylene glycol 10.0
(15) L-arginine (10 wt% aqueous solution) 2.0
(16) marjoram extract 1.0
(17) coltsfoot extract 1.0
Production method: The water phase components (1) to (6) are mixed and dissolved by heating at 75°C. On the other hand, the oil phase components (7) to (14) are mixed and dissolved by heating at 75°C. Next, the oil phase component is added to the water phase component and pre-emulsified, followed by uniform emulsification with a homomixer. After cooling, (15) is added at 50°C, and (16) and (17) are added at 40°C, and mixed uniformly.

[Example 5] Aqueous gel (1) Carboxyvinyl polymer 0.5 (mass%)
(2) Remaining purified water 100 (3) Sodium hydroxide (10% by mass aqueous solution) 0.5
(4) Glycerin 10.0
(5) 1,3-butylene glycol 10.0
(6) Ethanol 10.0
(7) methyl paraoxybenzoate 0.1
(8) Perfume 0.1
(9) marjoram extract 0.7
(10) coltsfoot extract 0.7
(11) Polyoxyethylene (60 E.O.) hydrogenated castor oil 1.0
Production method: Add (1) to (2), stir uniformly, and then add (3). After uniformly stirring, (5) dissolved in advance in (4) is added. After uniformly stirring, (6) to (11) mixed in advance are added and uniformly stirred and mixed.

Claims (1)

A CLSP and/or TINCR expression promoter comprising marjoram extract and coltsfoot extract as active ingredients.

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