Skin moisturizing function improver, as well as aquaporin-3 gene expression promoter and hyaluronan synthase gene expression promoter

Abstract

To provide a skin moisturizing function improver that contains Sideritis scardica extract.SOLUTION: The present invention provides a skin moisturizing function improver that contains Sideritis scardica extract.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for improving or enhancing the skin's moisturizing function, and more specifically, to a mountain herb extract for enhancing the expression of aquaporin 3, a gene present in skin cells and involved in controlling water movement, and a gene involved in the synthesis of hyaluronic acid, which has the function of taking in water into intercellular spaces and retains moisture in the skin. [Background technology]

[0002] Human skin is composed of three layers, the epidermis, dermis, and subcutaneous tissue, from the outermost layer. Each of these layers plays a role in maintaining the barrier function of the skin. The epidermis is located on the surface of the skin and is composed of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. The dermis is the main body of the skin, and supports the elasticity of the skin by forming a network of fibers made of proteins such as collagen and elastin. In addition, fibroblasts present in the dermis produce these dermal components and are known to play an important role in the condition of the skin. The subcutaneous tissue contains adipose tissue and plays a role in maintaining body temperature and cushioning shocks.

[0003] The epidermis plays a major role in maintaining moisture in the skin, with epidermal keratinocytes making up more than 90% of the epidermis. Keratinocytes are formed in the basal layer of the epidermis, and differentiate into the spinous layer, granular layer, and stratum corneum while changing their morphology and function. In the process of differentiating from the basal layer to the granular layer, the cells actively express genes and perform biosynthesis of proteins necessary for the moisturizing function of the stratum corneum (see, for example, Non-Patent Document 1 below). Since the stratum corneum retains moisture, the skin becomes moisturized, and the skin can maintain its firmness, smoothness, and softness (see, for example, Non-Patent Document 2 and Non-Patent Document 3 below), it is important to improve and enhance the moisturizing function of the stratum corneum.

[0004] In addition, aquaporins (AQPs), proteins involved in the skin's moisturizing function, are water channels that promote the movement of water molecules through cell membranes, and 13 different isoforms (proteins with different structures but the same function) have been discovered in mammals. Of the 13 types, AQP3 is found mainly in large amounts in skin keratinocytes. Unlike other isoforms, AQP3 has the property of promoting the transport of not only water molecules but also glycerol, and is thought to be closely related to skin moisturizing (for example, see Non-Patent Document 4 below).

[0005] Hyaluronic acid synthase (HAS) is found in a wide range of organisms, from prokaryotes to eukaryotes. It is an enzyme that synthesizes hyaluronic acid on the cell membrane, and provides intercellular lubrication and buffering by retaining water. There are three isoforms of mammalian HAS: HAS1, HAS2, and HAS3 (see, for example, Non-Patent Document 5 and Non-Patent Document 6 below). Hyaluronic acid in the skin is produced by dermal fibroblasts and epidermal keratinocytes (see, for example, Non-Patent Document 7 below), and it has been revealed that HAS2 is mainly expressed in fibroblasts.

[0006] Meanwhile, dry skin occurs when the skin barrier function breaks down and the moisture inside evaporates. The normal moisture content of the stratum corneum is 10-20%. It is said that when the moisture content of the stratum corneum is lost and the moisture content falls below 10%, the stratum corneum cracks and rough skin occurs. The surface of the skin dries and cracks, and when it worsens, scales and excessive desquamation occur (see, for example, Non-Patent Document 2 below). Improving gene expression of AQP3 and HAS2, which are related to the skin's moisturizing function, is important for preventing dry skin and improving or maintaining the barrier function. As substances that promote the expression of AQP3 and HAS2, extracts of the genus Fragaria of the Rosaceae family (see, for example, Patent Document 1 below) and plant lipids such as acai oil (see, for example, Patent Document 2 below) are known. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2009-298765 A [Patent Document 2] Special Publication No. 2015-531380 [Patent Document 3] JP 2021-130652 A [Patent Document 4] JP 2023-013154 A [Non-patent literature]

[0008] [Non-Patent Document 1] Journal of the Japan Cosmetics Society Vol.37 No.2 pp.95-100(2013) [Non-Patent Document 2] Chemistry and Education Vol. 65 No. 2 pp.84-85(2017) [Non-Patent Document 3] Journal of the Adhesion Society of Japan Vol.52 No.5 pp.145-151(2016) [Non-Patent Document 4] Folia Pharmacol. Jpn. Vol.143 pp.115-119(2014) [Non-Patent Document 5] Chemistry and Biology Vol.26 No.5 pp.308-315(1988) [Non-Patent Document 6] Trends in Glycoscience and Glycotechnology. Vol.22 No.124 pp.68‐79(2010) [Non-Patent Document 7] Skin Pharmacology and Applied Skin Physiology. Vol.17 pp.77‐83(2004) [Non-Patent Document 8] Molecules. Vol.17 pp.2683‐2703(2012) Summary of the Invention [Problem to be solved by the invention]

[0009] As described above, aging, a decrease in the barrier function of the skin, and a decrease in the function of fibroblasts in the dermis are closely related to the formation of dry skin, loss of elasticity, wrinkles, and sagging. It is desired to provide a composition having an effect of enhancing the expression of the AQP3 gene, which is involved in the synthesis of a protein responsible for water transport in epidermal cells, and a composition having an effect of enhancing the expression of the HAS2 gene, which is synthesized in dermal fibroblasts and is involved in the synthesis of hyaluronic acid, which exerts the moisture retention function of the skin.

[0010] In view of the above problems, an object of the present invention is to provide a novel composition that has the effect of enhancing the expression of the AQP3 gene and the HAS2 gene. [Means for solving the problem]

[0011] The present inventors have conducted intensive research into the above-mentioned problems and have discovered that mountain herb extracts have the effect of enhancing the expression of the AQP3 gene and the HAS2 gene, thereby completing the present invention.

[0012] Mountain herbs are plants of the mint family, with the scientific name Sideritis scardica and the English names Mountain Herb and Mursalski. Mountain herbs are also called "mountain tea" because they grow in the harsh highlands of mountains over 1000 meters above sea level, and have been used since ancient times in Greece, the Mediterranean coast, Turkey, Bulgaria, and other places.

[0013] However, to date, mountain herbs have only been reported to have antibacterial effects against gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Pasteurella multocida, as well as blood pressure lowering effects, anti-glycation effects, antioxidant effects, anti-aging effects, anti-allergic effects, immunostimulating effects, periodontal disease bacteria suppression effects, and matrix metalloproteinase-1 production inhibitory effects (see, for example, the above-mentioned Non-Patent Document 8, Patent Document 3, and Patent Document 4), and no investigation has been conducted into their relationship with enhanced expression of the AQP3 gene or HAS2 gene.

[0014] That is, the skin moisturizing function enhancer according to one aspect of the present invention is characterized by containing an extract of mountain herb (Sideritis scardica), and preferably has the extract as an active ingredient.

[0015] Further, according to another aspect of the present invention, there is provided a food, pharmaceutical or cosmetic composition for improving the moisturizing function of the skin, which contains an extract of mountain herb (Sideritis scardica) as an active ingredient.

[0016] Furthermore, an aquaporin 3 gene expression promoter according to another aspect of the present invention contains a mountain herb (Sideritis scardica) extract as an active ingredient.

[0017] Another aspect of the present invention provides a hyaluronic acid synthase expression gene promoter that contains an extract of mountain herb (Sideritis scardica) as an active ingredient. Effect of the Invention

[0018] As described above, the present invention can provide a composition having an effect of enhancing the expression of the novel AQP3 gene and HAS2 gene. In particular, the skin moisturizing function improver of the present invention is safe because it can use a naturally derived product, and is suitable for use as a compound or additive in food compositions, pharmaceutical compositions, and cosmetic compositions. [Brief description of the drawings]

[0019] [Figure 1] FIG. 2 shows the change in the expression level of the AQP3 gene depending on whether or not a mountain herb extract was added in Example 1. [Diagram 2] FIG. 1 shows changes in the expression level of the HAS2 gene depending on whether or not a mountain herb extract was added in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present embodiment will be described in detail below. However, the present invention can be embodied in many different forms and is not limited to the examples described in the following embodiments and examples.

[0021] The skin moisturizing function enhancer according to this embodiment contains an extract of mountain herb as an active ingredient. As mentioned above, mountain herb is a plant of the Lamiaceae family, and its scientific name is Sideritis scardica, and its English name is Mountain Herb or Mursalski. Mountain herb is also called "mountain tea" because it grows in the highlands of mountains with harsh environments over 1000 meters, and has been cited since ancient times mainly in Greece, along the Mediterranean coast, Turkey, Bulgaria, etc.

[0022] The mountain herb extract used in this embodiment can be easily obtained by a method generally used for plant extraction. The mountain herb extract may include a mountain herb extract, a dried product obtained by drying a diluted solution of the extract, or a crude or purified product thereof.

[0023] In this case, the raw material for the mountain herb extract is not particularly limited and can be appropriately selected depending on the purpose, for example, the whole plant, above-ground part, stem, leaf, or flower of the mountain herb can be used. In addition, the mountain herb can be used as the raw material for the extraction in its raw form, or it can be dried or processed.

[0024] In addition, when dried mountain herbs are used as the raw material for extraction, they can be obtained by subjecting them to solvent extraction either directly or after crushing them using a crusher after drying. Drying can be performed in the sun or using a commonly used dryer.

[0025] In addition, the method of extracting mountain herb extract is not particularly limited, and can be performed according to a method well known to those skilled in the art. As the extraction solvent, water or hot water, alcoholic solvents, and other organic solvents such as acetone can be used. As the alcoholic solvent, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, and the like, and mixtures thereof can be exemplified. As the organic solvent other than acetone, esters such as ethyl acetate; polyhydric alcohols such as ethylene glycol, propylene glycol, and 1,3-butylene glycol; ethers such as diethyl ether, and the like can be exemplified. These solvents may be used alone or in combination. The extraction solvent may be used in the form of a water-containing hydrophilic solvent by combining water and a hydrophilic organic solvent. When the solvents are used in combination, the mixing ratio of the solvents can be set arbitrarily. Among these, it is preferable to use ethanol, and it is more preferable to use a water-containing ethanol having an ethanol content of 10% (w / w) or more and 95% (w / w) or less.

[0026] The amount of the extraction solvent is not limited as long as the composition having the above-mentioned effect can be obtained, but is preferably 2 to 100 parts by weight per 1 part by weight of the dried above-ground part of the mountain herb. The extraction temperature is preferably 4°C to 95°C. The extraction time is preferably 30 minutes to 1 week. The extraction method can be any method such as stirring extraction, immersion extraction, countercurrent extraction, ultrasonic extraction, and supercritical extraction.

[0027] The mountain herb extract may be any of the filtrate obtained by filtering the obtained extract, the concentrate obtained by concentrating the filtrate, the dried product obtained by drying the concentrate, the crude or purified product thereof, or a mixture of these. The concentration method may be any method such as vacuum concentration, evaporative concentration, or membrane concentration. The drying method may be any method such as vacuum drying, freeze drying, or spray drying. If necessary, an excipient such as dextrin may be added. When purification is performed, it may be performed according to the means known to those skilled in the art. For example, synthetic adsorption resins, activated carbon, ion exchange resins, gel filtration agents such as Sephadex and Biogel, column chromatography, recrystallization, etc. may be used alone or in combination.

[0028] In addition, the mountain herb extract according to this embodiment has the above or below-mentioned efficacy as an entire extract, but from the examples described below, it is presumed that it contains 8-hydroxyflavone glycoside as one of its main components, which can function as one of its active ingredients. As 8-hydroxyflavone glycoside, it is not limited, but preferred examples include 4'-O-methylisoscutellarein, 4'-O-methylhypolatein, and isoscutellarein glycosides, but is not limited thereto. In addition, the content of 8-hydroxyflavone glycoside is preferably 0.5 or more, more preferably 1 to 50, when the total weight of the mountain herb extract is 100. By containing it in this range, the effect of the present composition can be preferably exhibited.

[0029] As described above, mountain herb extracts can be included as an active ingredient in skin moisturizing function enhancers, and can also be used in various compositions, specifically food compositions, pharmaceutical compositions, and cosmetic compositions.

[0030] The form of the food composition is not particularly limited and can be appropriately selected depending on the purpose, and various forms are possible, such as beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, etc.; confectioneries such as candies, gums, chocolates, etc.; tablets, capsules, drinks, etc. More specific examples of food compositions include health foods, functional foods, and nutritional supplements.

[0031] In addition, the pharmaceutical composition can be used as a medicine for improving the skin's moisturizing function or as a skin moisturizing function enhancer. The form of the pharmaceutical composition is not particularly limited and can be appropriately selected according to the purpose, and examples thereof include tablets, capsules, drinks, troches, ointments, creams, external liquids, patches, etc.

[0032] The form of the cosmetic composition is not particularly limited and can be appropriately selected depending on the purpose, but examples include lotion, cosmetic liquid, cream, milky lotion, hair styling product, hair wash, finishing cosmetic product, etc.

[0033] This skin moisturizing function enhancer and food, pharmaceutical, or cosmetic compositions utilizing this effect are suitable for use in humans, but can also be applied to animals other than humans (non-human animals) as long as the respective functional effects are obtained.

[0034] Furthermore, in this embodiment, as will be apparent from the description of the examples below, the mountain herb extract can be used as an active ingredient in an aquaporin 3 synthase expression promoter or a hyaluronic acid synthase expression promoter, and as a result, the mountain herb extract serves as an active ingredient in an agent for improving the skin's moisturizing function.

[0035] As described above, this embodiment can provide a new skin moisturizing function enhancer. In particular, the mountain herb extract of the present invention is safe because it is derived from natural sources, and is suitable for use as a blend or addition to food compositions, pharmaceutical compositions, and cosmetic compositions. EXAMPLES

[0036] Here, examples in which the effects of the above-mentioned mountain herb and its extract were actually confirmed will be described. However, the present invention is not limited to these examples at all.

[0037] (Production Example) Production of 50% (w / w) ethanol extract of mountain herb 9 kg of a ground powder of the aerial part of mountain herb was put into 200 L of 50% (w / w) ethanol, heated under reflux with stirring for 1 hour, and then an extract was obtained. The extract was filtered, activated carbon was added to the filtrate, and after stirring for another 1 hour, it was filtered. An excipient was added to the filtrate, concentrated under reduced pressure, and then spray-dried to obtain an extract (powder form).

[0038] - Measurement of the content of 8-hydroxyflavone glycosides by HPLC analysis - In order to measure the contents of the three 8-hydroxyflavone glycosides shown in the production example, HPLC analysis was performed on the extract of the production example.

[0039] Approximately 0.05 g of the extract of this production example was precisely weighed, 20 mL of 11% hydrochloric acid and 10 mL of methanol were added. After dissolution, methanol was added to make exactly 50 mL. 10 mL of this solution was taken into a hydrolysis tube and hydrolyzed at 100 °C for 1 hour. After cooling to room temperature, it was filtered through a 0.45 μm membrane filter to obtain a sample solution. Separately, standards of 4'-O-methylisoscutellarein, 4'-O-methylhypolatein, and isoscutellarein (manufactured by Tokachi Phytochemical Research Institute) were dissolved in methanol to obtain a standard solution. Exactly 10 μL each of the sample solution and the standard solution were taken, and the test was conducted by liquid chromatography according to the method shown in Condition 1 below. The peak areas of 4'-O-methylisoscutellarein, 4'-O-methylhypolatein, and isoscutellarein in each solution were measured.

[0040] <HPLC analysis conditions> Detector: UV-Vis spectrophotometer Measurement wavelength: 280nm Column: CAPCELL PACK C18 UG120 4.6 x 250 nm Mobile phase: Solution A: TFA / water mixture (0.1:100) Solution B: TFA / acetonitrile mixture (0.1:100) [Table 1]

[0041] <Calculation of 8-hydroxyflavone glycoside content> The molecular weight ratio was used as a conversion factor to convert the contents of 4'-O-methylisoscutellarein, 4'-O-methylhypolatein, and isoscutellarein into the contents of each glycoside.

[0042] (Analysis results) As a result of the analysis, it was confirmed that the 50% (w / w) ethanol extract of mountain herb extracted in this manufacturing example contains the following amounts of 8-hydroxyflavone glycosides, including 4'-O-methylisoscutellarein, 4'-O-methylhypolatein, and isoscutellarein glycosides. [Table 2]

[0043] Example 1: Effect of Mountain Herb Extract on AQP3 Gene Expression Human neonatal dermal fibroblasts (NHDF) were cultured in serum-free medium for 24 hours, and the mountain herb extract described in the manufacturing example, which was prepared with DMSO to a final concentration of 50 μg / mL, or DMSO alone as a control, was added to the medium and cultured for another 2 hours. After culture, the medium was removed and the cultured cells were washed with PBS and collected. mRNA was extracted and purified from the cultured cells to prepare an mRNA solution. A cDNA solution was prepared from the prepared mRNA using reverse transcription PCR, and quantitative PCR was performed using the cDNA solution and various primers to quantify the gene expression level of AQP3. Actin was used as an internal standard.

[0044] The expression level of AQP3 gene relative to the expression level of actin gene was calculated as a percentage, and the change in the expression level of AQP3 gene was evaluated using cultured cells without mountain herb extract (only DMSO was added) as a comparison, as shown in Figure 1. The results shown in this figure indicate that the expression of AQP3 gene was significantly enhanced compared to the case without mountain herb extract, and that the expression of AQP3 gene was promoted.

[0045] Example 2: Effect of Mountain Herb Extract on HAS2 Gene Expression Human neonatal dermal fibroblasts (NHDF) were cultured in serum-free medium for 24 hours, and the mountain herb extract described in the manufacturing example, prepared with DMSO to a final concentration of 50 μg / mL, or DMSO alone as a control, was added to the medium and cultured for another 4 hours. After culturing, the medium was removed and the cultured cells were washed with PBS and collected. mRNA was extracted and purified from the cultured cells to prepare an mRNA solution. A cDNA solution was prepared from the prepared mRNA using reverse transcription PCR, and quantitative PCR was performed using the cDNA solution and various primers to quantify the gene expression level of HAS2. Actin was used as an internal standard.

[0046] The expression level of HAS2 gene relative to the expression level of actin gene was calculated as a percentage, and the change in the expression level of HAS2 gene was evaluated using cultured cells without mountain herb extract (only DMSO was added) as a comparison, as shown in Figure 2. The results shown in this figure indicate that the expression of HAS2 gene was significantly enhanced compared to the case without the addition of mountain herb extract, and promoted the expression of HAS2 gene.

[0047] As described above, this example demonstrates that the mountain herb extract of the present invention promotes the expression of the AQP3 gene and the HAS2 gene, which are involved in the skin's moisturizing function, and is useful for improving the skin's moisturizing function. [Industrial Applicability]

[0048] INDUSTRIAL APPLICABILITY The present invention has industrial applicability as an agent for improving the moisturizing function of skin, or an agent for enhancing the moisturizing function of skin, which contains an extract of mountain herb (Sideritis scardica) as an active ingredient.

Claims

1. A skin moisturizing function enhancer comprising an extract of mountain herb (Sideritis scardica) and promoting the expression of at least one of aquaporin 3 and hyaluronic acid synthase.

2. A food, pharmaceutical or cosmetic composition comprising the extract according to claim 1 as an active ingredient.

3. An aquaporin 3 gene expression promoter containing mountain herb (Sideritis scardica) extract as an active ingredient.

4. A hyaluronic acid synthase gene expression promoter containing mountain herb (Sideritis scardica) extract as an active ingredient.

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