Aquaporin 3 mRNA expression promoter

Abstract

To provide an aquaporin 3 mRNA expression promoter that has superior aquaporin 3 mRNA expression promoting action and is highly safe; a hyaluronan synthase 3 mRNA expression promoter that has superior hyaluronan synthase 3 mRNA expression promoting action and is highly safe; and a β defensin 3 mRNA expression promoter that has superior β defensin 3 mRNA expression promoting action and is highly safe.SOLUTION: The present invention provides an aquaporin 3 mRNA expression promoter, a hyaluronan synthase 3 mRNA expression promoter, and a β defensin 3 mRNA expression promoter, each of which contains Saxifraga stolonifera extract as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an aquaporin 3 mRNA expression promoter, a hyaluronan synthase 3 mRNA expression promoter, and a beta-defensin 3 mRNA expression promoter.

Background Art

[0002] In skin cells, aquaporin, known as a water channel, is expressed on the cell membrane and is known to play a role in taking up low-molecular-weight substances such as water in the intercellular space into the cell. In humans, the existence of 13 types of aquaporin (AQP0 to AQP12) is known. In epidermal cells, mainly AQP3 exists, and it is considered to play a role in taking up, in addition to water, low-molecular compounds such as glycerol and urea involved in the water retention function.

[0003] However, since AQP3 decreases with aging, and this is suggested to be one of the causes of the decline in the water retention function, it is considered possible to control the water retention ability and barrier function due to aging by promoting the expression of AQP3 (see, for example, Non-Patent Document 1).

[0004] So far, as those having an aquaporin 3 (AQP3) expression promoting action, for example, an extract from the leaf part of star fruit (see, for example, Patent Document 1) and the like are known.

[0005] Hyaluronic acid is a kind of mucopolysaccharide, and has a function of holding cells by filling the intercellular space. Furthermore, it has many functions such as retention of water in the intercellular space, imparting lubricity and flexibility to tissues, and resistance to external forces such as mechanical damage. If the production of hyaluronic acid can be promoted by promoting the expression of hyaluronan synthase 3 (HAS3) involved in the promotion of epidermal hyaluronic acid synthesis, it is considered possible to prevent, treat or improve skin aging symptoms such as skin roughness, wrinkles, dullness, change in texture, decrease in elasticity and decrease in moisturizing function.

[0006] To date, substances known to promote hyaluronic acid synthase 3 (HAS3) mRNA expression include, for example, licorice leaf extract (see Patent Document 2).

[0007] The skin is the outermost layer of the body and is an important tissue that protects the organism from external stimuli, possessing a defense mechanism that prevents harmful microorganisms from entering the body. One of these is antimicrobial peptides (AMPs). More than 20 types of AMPs have been identified in the skin, with β-defensin 3 being a representative example. AMPs are produced in epidermal cells, stored in lamellar granules, and released into stratum corneum cells (see, for example, Non-Patent Document 2). AMPs are thought to protect the organism from external stimuli by inhibiting the growth of harmful microorganisms as antimicrobial agents.

[0008] To date, butcher's broom extract (see, for example, Patent Document 3) and others have been known to promote β-defensin 3 mRNA expression.

[0009] As described above, various studies have been conducted to date. However, there remains a strong demand for new materials that are highly safe, derived from natural products, and possess at least one of the following effects: aquaporin 3 mRNA expression promoting effect, hyaluronic acid synthase 3 mRNA expression promoting effect, and β-defensin 3 mRNA expression promoting effect, and that can therefore be widely used as components in topical compositions such as cosmetics and research reagents. The rapid development of such materials is therefore urgently needed. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Japanese Patent Publication No. 2009-191039 [Patent Document 2] Japanese Patent Publication No. 2010-090035 [Patent Document 3] Japanese Patent Publication No. 2018-104364 [Non-patent literature]

[0011] [Non-Patent Document 1] "Fragrance Journal," 2006, Vol. 34, No. 10, pp. 19-23 [Non-Patent Document 2] Dermatology to Read When Considering the Efficacy of Cosmetics, 2020, pp. 37-39 [Overview of the project] [Problems that the invention aims to solve]

[0012] The present invention aims to solve the aforementioned conventional problems and achieve the following objectives. Specifically, the present invention aims to provide an aquaporin 3 mRNA expression promoter that has excellent aquaporin 3 mRNA expression promoting activity and is highly safe. Furthermore, the present invention aims to provide a hyaluronic acid synthase 3 mRNA expression promoter that has excellent hyaluronic acid synthase 3 mRNA expression promoting activity and is highly safe. Furthermore, the present invention aims to provide a β-defensin 3 mRNA expression promoter that has excellent β-defensin 3 mRNA expression promoting activity and is highly safe. [Means for solving the problem]

[0013] In order to solve the aforementioned problems, the present inventors conducted extensive research and found that Saxifraga stolonifera extract, a highly safe natural material, has excellent aquaporin 3 mRNA expression promoting activity, hyaluronic acid synthase 3 mRNA expression promoting activity, and β-defensin 3 mRNA expression promoting activity, thus completing the present invention.

[0014] The present invention is based on the inventors' aforementioned findings, and the means for solving the aforementioned problems are as follows: <1> An aquaporin 3 (AQP3) mRNA expression promoter characterized by containing a saxifraga extract as an active ingredient. <2> A hyaluronan synthase 3 (HAS3) mRNA expression promoter characterized by containing a saxifraga extract as an active ingredient. <3> A β - defensin 3 mRNA expression promoter characterized by containing a saxifraga extract as an active ingredient.

Effects of the Invention

[0015] According to the aquaporin 3 mRNA expression promoter of the present invention, the above - mentioned various problems in the prior art can be solved, the above - mentioned object can be achieved, an aquaporin 3 mRNA expression promoter having an excellent aquaporin 3 mRNA expression promoting effect and high safety can be provided. According to the hyaluronan synthase 3 mRNA expression promoter of the present invention, the above - mentioned various problems in the prior art can be solved, the above - mentioned object can be achieved, a hyaluronan synthase 3 mRNA expression promoter having an excellent hyaluronan synthase 3 mRNA expression promoting effect and high safety can be provided. According to the β - defensin 3 mRNA expression promoter of the present invention, the above - mentioned various problems in the prior art can be solved, the above - mentioned object can be achieved, a β - defensin 3 mRNA expression promoter having an excellent β - defensin 3 mRNA expression promoting effect and high safety can be provided.

Modes for Carrying Out the Invention

[0016] (Aquaporin 3 mRNA expression promoter, hyaluronan synthase 3 mRNA expression promoter, and β - defensin 3 mRNA expression promoter) The aquaporin 3 (AQP3) mRNA expression promoter, hyaluronan synthase 3 (HAS3) mRNA expression promoter, and β - defensin 3 mRNA expression promoter according to the present embodiment contain a saxifraga extract as an active ingredient and further contain other components as required.

[0017] <Saxifraga stolonifera extract> The extraction raw material as the active ingredient in this embodiment is Saxifraga stolonifera (scientific name: Saxifraga stolonifera).

[0018] Saxifraga stolonifera , Yu is an evergreen perennial herb belonging to the genus Saxifraga in the family Saxifragaceae be .

[0019] The above-mentioned Saxifraga stolonifera extract may be prepared from the extraction raw material or a commercially available product may be used.

[0020] There is no particular limitation on the part that can be used as the extraction raw material, and it can be appropriately selected according to the purpose. For example, leaves, stems, flowers, buds, seeds, roots, above-ground parts or mixtures thereof can be mentioned. Among these, the whole herb is preferred. There is no particular limitation on the shape, structure and size of the above-mentioned extraction raw material, and it can be appropriately selected according to the purpose.

[0021] Although the details of the substances having the promoting effect on AQP3 mRNA expression, the promoting effect on HAS3 mRNA expression, and the promoting effect on β - defensin 3 mRNA expression contained in the Saxifraga stolonifera extract are unknown, an extract having the promoting effect on AQP3 mRNA expression, the promoting effect on HAS3 mRNA expression, and the promoting effect on β - defensin 3 mRNA expression can be obtained from Saxifraga stolonifera by an extraction method generally used for plant extraction, etc.

[0022] In the present invention, the "extract" includes any of an extract obtained from an extraction raw material by an extraction treatment, a diluted solution or a concentrated solution of the extract, a dried product obtained by drying the extract, or a crude purified product or a purified product thereof.

[0023] The above extract can be obtained by drying the raw material, then using it as is, or by grinding it using a crusher, and then subjecting it to extraction with an extraction solvent. Drying may be done in the sun or using a commonly used drying oven. Alternatively, the raw material may be pretreated by degreasing with a non-polar solvent such as hexane before use. Pretreatment such as degreasing allows for more efficient extraction of the raw material with a polar solvent.

[0024] There are no particular restrictions on the solvent used for extraction, and it can be appropriately selected depending on the purpose. Examples include water, hydrophilic organic solvents, or mixtures thereof. The solvent is preferably used at room temperature or at a temperature below the boiling point of the solvent. Components in the extracted raw material that promote AQP3 mRNA expression, HAS3 mRNA expression, and β-defensin 3 mRNA expression can be easily extracted by extraction using a polar solvent.

[0025] There are no particular restrictions on the type of water that can be used as an extraction solvent, and it can be appropriately selected depending on the purpose. Examples include pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, etc., as well as water that has undergone various treatments. There are no particular restrictions on the treatments that can be applied to the water, and it can be appropriately selected depending on the purpose. Examples include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc. Therefore, in this embodiment, the water that can be used as an extraction solvent also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate-buffered physiological saline, etc.

[0026] There are no particular restrictions on the hydrophilic organic solvents that can be used as extraction solvents, and they can be appropriately selected depending on the purpose. Examples include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; and polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol, and glycerin. These may be used individually or in combination of two or more.

[0027] When using a mixture of two or more polar solvents as an extraction solvent, the mixing ratio can be adjusted as appropriate. For example, when using a mixture of water and a lower aliphatic alcohol as an extraction solvent, it is preferable to mix 1 to 90 parts by volume of the lower aliphatic alcohol with 10 parts by volume of water. When using a mixture of water and a lower aliphatic ketone as an extraction solvent, it is preferable to mix 1 to 40 parts by volume of the lower aliphatic ketone with 10 parts by volume of water. When using a mixture of water and a polyhydric alcohol as an extraction solvent, it is preferable to mix 1 to 90 parts by volume of the polyhydric alcohol with 10 parts by volume of water. Among these, it is preferable to use a mixture of 50% water by volume and 50% 1,3-butylene glycol by volume as the extraction solvent.

[0028] The extraction process is not particularly limited as long as it allows the soluble components contained in the raw material to be eluted into the extraction solvent, and can be carried out according to conventional methods. For example, extraction can be performed at room temperature or under reflux heating. For example, the raw material can be placed in a processing tank filled with the extraction solvent, and after standing for 30 minutes to 4 hours with stirring as needed to elute the soluble components, the extract can be obtained by filtering to remove solid matter. By distilling off the extraction solvent from the obtained extract, a paste-like concentrate can be obtained, and by further drying this concentrate, a dried product can be obtained. When water is used as the extraction solvent, the extraction conditions are approximately 1 to 4 hours at 50 to 95°C. When a mixed solvent of water and ethanol is used as the extraction solvent, the conditions are approximately 30 minutes to 4 hours at 40 to 80°C. When a mixed solvent of water and 1,3-butylene glycol is used as the extraction solvent, the conditions are approximately 2 hours at 80 to 90°C. The extraction time and temperature mentioned above are examples and may be changed as appropriate. Furthermore, there are no particular restrictions on the amount of extraction solvent used; it can be selected as appropriate depending on the purpose.

[0029] The extract obtained as described above may be subjected to dilution, concentration, drying, purification, and other treatments according to conventional methods in order to obtain a diluted or concentrated extract, a dried extract, or a crude or purified product thereof.

[0030] The obtained extract can be used as is as an AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter, but a concentrated solution or dried product is preferred. When obtaining a dried product, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity.

[0031] Furthermore, because Saxifraga stolonifera has a distinctive odor, it is possible to purify it for purposes such as decolorization and deodorization, provided that it does not reduce its physiological activity. However, since it is not used in large quantities when added to cosmetics, there is no practical problem even if it is left unpurified. Purification can be carried out by methods such as activated carbon treatment, adsorption resin treatment, and ion exchange resin treatment.

[0032] The Saxifraga extract obtained as described above has excellent AQP3 mRNA expression promoting activity, HAS3 mRNA expression promoting activity, and β-defensin 3 mRNA expression promoting activity. Therefore, it can be used as an active ingredient in AQP3 mRNA expression promoters, HAS3 mRNA expression promoters, and β-defensin 3 mRNA expression promoters by utilizing these activities.

[0033] The AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter according to this embodiment may consist solely of Saxifraga stolonifera extract, or they may be formulations of Saxifraga stolonifera extract.

[0034] The above-mentioned Saxifraga stolonifera extract can be formulated into any dosage form, such as powder, granules, or liquid, according to conventional methods, using pharmaceutically acceptable carriers such as dextrin or cyclodextrin, or other optional auxiliary agents. In this case, auxiliary agents such as excipients, stabilizers, and deodorizers can be used. Examples of forms of the AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter formulated from the above-mentioned Saxifraga stolonifera extract include ointments and topical solutions.

[0035] There are no particular restrictions on the content of Saxifraga stolonifera extract in the formulated AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter, and it can be appropriately selected depending on the purpose.

[0036] <Other ingredients> The aforementioned other components are not particularly limited and can be appropriately selected depending on the form of use of the AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter. Examples include components that can be used when formulating the above-mentioned formulations. The aforementioned other components may be used individually or in combination of two or more.

[0037] There are no particular restrictions on the content of the other components in the AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter, and they can be appropriately selected depending on the purpose.

[0038] There are no particular restrictions on the method of administering the AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter of this embodiment to the target, and can be appropriately selected according to the purpose, for example, transdermal administration.

[0039] There are no particular restrictions on the dosage, duration of administration, or interval of administration of the AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter of this embodiment, and they can be appropriately selected according to the purpose.

[0040] The AQP3 mRNA expression promoter according to this embodiment can promote AQP3 mRNA expression through the AQP3 mRNA expression-promoting effect of Saxifraga stolonifera extract. This can improve age-related moisture retention and barrier function. However, the AQP3 mRNA expression promoter of this embodiment can be used in any application where exhibiting an AQP3 mRNA expression-promoting effect is significant, in addition to these applications.

[0041] The HAS3 mRNA expression promoter in this embodiment can promote HAS3 mRNA expression through the HAS3 mRNA expression-promoting effect of Saxifraga stolonifera extract. This promotes hyaluronic acid production, which can prevent, treat, or improve skin aging symptoms such as rough skin, wrinkles, dullness, changes in skin texture, decreased elasticity, and decreased moisturizing function. However, the HAS3 mRNA expression promoter in this embodiment can be used in any application where it is meaningful to exhibit HAS3 mRNA expression-promoting effects, in addition to these applications.

[0042] The β-defensin 3 mRNA expression promoter in this embodiment can promote the expression of β-defensin 3 mRNA through the β-defensin 3 mRNA expression-promoting effect of Saxifraga stolonifera extract. This can prevent infections and strengthen the skin barrier by suppressing the growth of harmful microorganisms. However, the β-defensin 3 mRNA expression promoter in this embodiment can be used in any other application where the β-defensin 3 mRNA expression-promoting effect is significant.

[0043] Furthermore, the AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter according to this embodiment have excellent AQP3 mRNA expression promoting effects, HAS3 mRNA expression promoting effects, and β-defensin 3 mRNA expression promoting effects, and are therefore suitable for incorporation into cosmetics such as skin cosmetics, scalp cosmetics, and hair cosmetics. In this case, the Saxifraga stolonifera extract may be incorporated as is, or a formulation of the Saxifraga stolonifera extract may be incorporated.

[0044] There are no particular restrictions on the cosmetics that can contain AQP3 mRNA expression promoters, HAS3 mRNA expression promoters, and β-defensin 3 mRNA expression promoters, and they can be appropriately selected according to the purpose. Examples include ointments, creams, emulsions, lotions, masks, foundations, hair tonics, hair lotions, shampoos, conditioners, and soaps.

[0045] When AQP3 mRNA expression promoters, HAS3 mRNA expression promoters, and β-defensin 3 mRNA expression promoters are incorporated into cosmetics, there are no particular restrictions on the amount they are incorporated, and they can be adjusted as appropriate depending on the type of cosmetic. However, a suitable formulation ratio is approximately 0.0001 to 10% by mass when converted to a standard extract, and a particularly suitable formulation ratio is approximately 0.001 to 1% by mass when converted to a standard extract.

[0046] Cosmetics may be used in combination with main ingredients, auxiliary ingredients, or other components commonly used in the manufacture of cosmetics, such as astringents, bactericides / antibacterial agents, whitening agents, UV absorbers, moisturizers, cell activators, anti-inflammatory / anti-allergic agents, antioxidants / reactive oxygen species scavengers, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, etc., as long as they do not interfere with the AQP3 mRNA expression promoting effect, HAS3 mRNA expression promoting effect, and β-defensin 3 mRNA expression promoting effect of the above extract. By using such combinations, a more general product may be produced, and the synergistic effect between the combined active ingredients may result in superior effects beyond what is normally expected.

[0047] There are no particular restrictions on the method for manufacturing the cosmetic composition, and known methods can be appropriately selected.

[0048] While the AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter according to this embodiment are suitably applied to humans, they can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cattle, pigs, monkeys, etc.) as long as the AQP3 mRNA expression promoting effect, HAS3 mRNA expression promoting effect, and β-defensin 3 mRNA expression promoting effect are achieved.

[0049] Furthermore, the AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter according to this embodiment can also be suitably used as reagents for research on the mechanisms of action of AQP3 mRNA expression, HAS3 mRNA expression, and β-defensin 3 mRNA expression, respectively.

[0050] As described above, the AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter of this embodiment have excellent AQP3 mRNA expression promoting effects, HAS3 mRNA expression promoting effects, and β-defensin 3 mRNA expression promoting effects, respectively. Accordingly, the present invention also relates to a method for promoting AQP3 mRNA expression, characterized by administering an AQP3 mRNA expression promoter to a subject; a method for promoting HAS3 mRNA expression, characterized by administering a HAS3 mRNA expression promoter; or a method for promoting β-defensin 3 mRNA expression, characterized by administering a β-defensin 3 mRNA expression promoter. [Examples]

[0051] The present invention will be described in more detail below with reference to manufacturing examples, test examples, and formulation examples, but the present invention is not limited in any way to the manufacturing examples, test examples, and formulation examples below. In the test examples and formulation examples below, the Saxifraga stolonifera extract used was the extract obtained in Manufacturing Example 1 below.

[0052] [Manufacturing Example 1] Production of Saxifraga stolonifera extract 100 g of dried whole Saxifraga stolonifera was mixed with 1,500 mL of 50% butylene glycol, and extracted using a reflux extractor at 80-90°C for 2 hours. The mixture was then filtered while hot. The resulting extract was dried to obtain Saxifraga stolonifera extract (10 g).

[0053] [Test Example 1] Aquaporin 3 (AQP3) mRNA expression promotion test The Saxifraga stolonifera extract obtained in Production Example 1 was tested for its AQP3 mRNA expression-promoting effect using the following method.

[0054] Normal human neonatal epidermal keratinocytes (NHEKs) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsin treatment. The harvested cells were then plated in a 6-well plate using KGM in a 3.0 × 10⁶ format. 5 The cells were seeded to a cell density of 2 mL and cultured overnight at 37°C under 5% CO2. After overnight incubation, the culture medium was replaced with KBM without growth factors. After 24 hours, the culture medium was discarded, and 2 mL of the test sample dissolved in KBM to the required concentration (see Table 1 below for the concentration) was added to each well. The cultures were then incubated at 37°C under 5% CO2 for 24 hours. As a control, the same incubation procedure was performed using KBM without the test sample. After culturing, the culture medium was removed, and total RNA was extracted using ISOGEN II (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 a concentration of 200 ng / μL. Using this total RNA as a template, mRNA expression levels for AQP3 and the internal standard GAPDH were measured. Detection was performed using a real-time PCR instrument, Thermal Cycler Dice® Real Time System III (manufactured by Takara Bio Inc.), with PrimeScript. TM The reaction was performed using a real-time two-step RT-PCR reaction with RT Master Mix (Perfect Real Time) (Takara Bio Inc.) and TB Green® Fast qPCR Mix (Takara Bio Inc.). The expression level of AQP3 mRNA was corrected for the expression level of GAPDH mRNA and then calculated. Based on the obtained results, the AQP3 mRNA expression enhancement rate was calculated using the following formula. The results are shown in Table 1. Note that in the formula below, the AQP3 mRNA expression enhancement rate for the sample without the test sample is 100%. AQP3 mRNA expression enhancement rate (%) = A / B × 100 In the above formula, A to B represent the following, respectively. A: Correction value when adding the test sample B: Corrected value when no test sample is added.

[0055] [Table 1]

[0056] As shown in Table 1, it was confirmed that Saxifraga stolonifera extract has excellent AQP3 mRNA expression-promoting activity.

[0057] [Test Example 2] Hyaluronic acid synthase 3 (HAS3) mRNA expression promotion test The Saxifraga stolonifera extract obtained in Production Example 1 was tested for its HAS3 mRNA expression-promoting effect using the following method.

[0058] Normal human neonatal epidermal keratinocytes (NHEKs) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsin treatment. The harvested cells were then plated in a 6-well plate using KGM in a 3.0 × 10⁶ format. 5 The cells were seeded to a cell density of 2 mL and cultured overnight at 37°C under 5% CO2. After overnight incubation, the culture medium was replaced with KBM without growth factors. After 24 hours, the culture medium was discarded, and 2 mL of the test sample dissolved in KBM to the required concentration (see Table 2 below for the concentration) was added to each well. The culture was then incubated at 37°C under 5% CO2 for 24 hours. As a control, the same incubation was performed using KBM without the test sample. After culturing, the culture medium was removed, and total RNA was extracted using ISOGEN II (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 a concentration of 200 ng / μL. Using this total RNA as a template, mRNA expression levels for HAS3 and the internal standard GAPDH were measured. Detection was performed using a real-time PCR instrument, Thermal Cycler Dice® Real Time System III (manufactured by Takara Bio Inc.), with PrimeScript. TMThe reaction was performed using a real-time two-step RT-PCR reaction with RT Master Mix (Perfect Real Time) (Takara Bio Inc.) and TB Green® Fast qPCR Mix (Takara Bio Inc.). The expression level of HAS3 mRNA was corrected for the expression level of GAPDH mRNA and then calculated. The HAS3 mRNA expression enhancement rate was calculated from the obtained results using the following formula. The results are shown in Table 2. Note that in the formula below, the HAS3 mRNA expression enhancement rate without the test sample is 100%. HAS3 mRNA expression enhancement rate (%) = A / B × 100 In the above formula, A to B represent the following, respectively. A: Correction value when adding the test sample B: Corrected value when no test sample is added.

[0059] [Table 2]

[0060] As shown in Table 2, it was confirmed that Saxifraga stolonifera extract has excellent HAS3 mRNA expression-promoting activity.

[0061] [Test Example 3] β-Defensin 3 mRNA Expression Promotion Test The Saxifraga stolonifera extract obtained in Production Example 1 was tested for its β-defensin 3 mRNA expression-promoting effect using the following method.

[0062] Normal human neonatal epidermal keratinocytes (NHEKs) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsin treatment. The harvested cells were then plated in a 6-well plate using KGM in a 3.0 × 10⁶ format. 5 The cells were seeded to a cell density of 2 mL and cultured overnight at 37°C under 5% CO2. After overnight incubation, the culture medium was replaced with KBM without growth factors. After 24 hours, the culture medium was discarded, and 2 mL of the test sample dissolved in KBM to the required concentration (see Table 3 below for the concentration) was added to each well. The cultures were then incubated at 37°C under 5% CO2 for 24 hours. As a control, the same incubation was performed using KBM without the test sample. After culturing, the culture medium was removed, and total RNA was extracted using ISOGEN II (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 a concentration of 200 ng / μL. Using this total RNA as a template, mRNA expression levels for β-defensin 3 and the internal standard GAPDH were measured. Detection was performed using a real-time PCR instrument, Thermal Cycler Dice® Real Time System III (Takara Bio Inc.), with PrimeScript. TM The reaction was performed using a real-time two-step RT-PCR reaction with RT Master Mix (Perfect Real Time) (Takara Bio Inc.) and TB Green® Fast qPCR Mix (Takara Bio Inc.). The expression level of β-defensin 3 mRNA was corrected for the expression level of GAPDH mRNA and then calculated. Based on the obtained results, the β-defensin 3 mRNA expression enhancement rate was calculated using the following formula. The results are shown in Table 3. Note that in the formula below, the β-defensin 3 mRNA expression enhancement rate without the test sample is 100%. β-defensin 3 mRNA expression enhancement rate (%) = A / B × 100 In the above formula, A to B represent the following, respectively. A: Correction value when adding the test sample B: Corrected value when no test sample is added.

[0063] [Table 3]

[0064] As shown in Table 3, it was confirmed that Saxifraga stolonifera extract has excellent β-defensin 3 mRNA expression promoting activity.

[0065] [Formulation example 1] An emulsion with the following composition was prepared by a conventional method. • Saxifraga stolonifera extract (Manufacturing Example 1) 0.01g • Jojoba oil 4.00g • 1,3-Butylene glycol 3.00g Arbutin 3.00g • Polyoxyethylene cetyl ether (20 E.O.) 2.50g • Olive oil 2.00g • Squalane 2.00g Cetanol 2.00g Glyceryl monostearate 2.00g • Polyoxyethylene sorbitan oleate (20E.O.) 2.00g • Methyl parahydroxybenzoate 0.15g • Stearyl glycyrrhetinate 0.10g • Scutellaria baicalensis extract 0.10g • Dipotassium glycyrrhizinate 0.10g Ginkgo biloba extract 0.10g Conchiolin 0.10g • Phellodendron bark extract 0.10g • Chamomile extract 0.10g · Fragrance 0.05g • Purified water (assuming a total volume of 100g)

[0066] [Formulation example 2] A cream with the following composition was manufactured by conventional methods. • Saxifraga stolonifera extract (Manufacturing Example 1) 0.05g • Sophora flavescens extract 0.1g • Scutellaria baicalensis extract 0.1g • Liquid paraffin 5.0g • Bleached beeswax 4.0g • Squalane 10.0g • Cetanol 3.0g • Lanolin 2.0g • Stearic acid 1.0g • Polyoxyethylene sorbitan oleate (20E.O.) 1.5g Glyceryl monostearate 3.0g • Oil-soluble licorice extract 0.1g • 1,3-Butylene glycol 6.0g • Methyl parahydroxybenzoate 1.5g · Fragrance 0.1g • Purified water (assuming a total volume of 100g)

[0067] [Formulation example 3] A beauty serum with the following composition was manufactured by conventional methods. • Saxifraga stolonifera extract (Manufacturing Example 1) 0.01g • Chamomile extract 0.1g • Carrot extract 0.1g • Xanthan gum 0.3g • Hydroxyethylcellulose 0.1g • Carboxyvinyl polymer 0.1g • 1,3-Butylene glycol 4.0g • Dipotassium glycyrrhizinate 0.1g Glycerin 2.0g • Potassium hydroxide 0.25g · Fragrance 0.01g • Preservative (methyl parahydroxybenzoate) 0.15g • Ethanol 2.0g • Purified water (assuming a total volume of 100g) [Industrial applicability]

[0068] The AQP3 mRNA expression promoter, HAS3 mRNA expression promoter, and β-defensin 3 mRNA expression promoter according to the present invention can be suitably used, for example, as a component of cosmetics, and further as a reagent for research purposes.

Claims

[Claim 1] It contains Saxifraga stolonifera extract as an active ingredient, An aquaporin 3 (AQP3) mRNA expression promoter characterized in that the aforementioned Saxifraga stolonifera extract is an extract obtained using a mixed solvent of water and butylene glycol.

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