JP2025018445A - Skin barrier function improver - Google Patents

Abstract

[Problem] To provide a tight junction formation promoter, a TRPV4 activator, a skin barrier function improver that maintains skin elasticity and improves the skin to a smooth and moisturized state, and a cosmetic containing the same.
[Solution] Aromatic water obtained from sudachi and shekwasha is used.
[Selection diagram] None

Description

The present invention relates to a skin barrier function improver that contains sudachi and/or shekwasha aromatic water and acts as a tight junction formation promoter, a TRPV4 activator, and a skin barrier function improver that maintains skin elasticity and improves the skin to a smooth, moisturized state, and to a cosmetic that contains the same.

The skin is often visible to the public, so keeping the skin healthy is a major concern for many people.

The skin, which constitutes the skin, is a sensory organ that transmits external stimuli such as heat and pain to the body, and also has an important barrier function that prevents evaporation of water from the body, prevents the invasion of bacterial infections and harmful substances from the external environment, and maintains the body's biological functions.
It is known that the barrier function of the skin is reduced by temperature, humidity, excessive friction of the skin, changes in daily rhythm, stress, dryness in winter, exposure to excessive ultraviolet rays, etc. Furthermore, it is said that a reduction in the barrier function can lead to the onset of skin disorders such as atopic dermatitis and seborrheic eczema due to direct damage caused by the invasion of bacteria or harmful substances from outside the body, or due to triggers such as allergic reactions.
To keep skin beautiful, it is important to maintain and improve the skin's barrier function as well as to improve skin condition through moisturizing.

It is known that the barrier function of the skin is strengthened by the formation of an adhesive structure due to the adhesion between cells. The adhesive structure due to the adhesion between cells means an intercellular adhesive structure in which adjacent cells are brought into close contact with each other to seal the intercellular gaps. When the formation of an intercellular adhesive structure is promoted in the epidermal tissue (epithelial tissue) of the skin, epidermal cells such as keratinocytes are adhered to each other, preventing external foreign substances from entering the tissue through the intercellular gaps, and at the same time, preventing body fluids from leaking out through the intercellular gaps. In other words, the barrier function of the skin is strengthened by promoting the formation of an intercellular adhesive structure in the epidermal tissue of the skin.
Tight junctions are widely known as one of the representative intercellular adhesion structures present in vertebrates.
Tight junctions exist in the form of a belt around cells and serve to tightly attach adjacent cells to each other and seal the intercellular spaces, so promoting the formation of tight junctions strengthens the barrier function of the skin.

Furthermore, with regard to improving the skin barrier function, it has been reported that the TRPV4 receptor present in epidermal cells is activated by 4α-phorbol ester (hereinafter referred to as "4α-PDD"), which increases the Ca concentration in skin keratinocytes and enhances the skin barrier function (e.g., Non-Patent Document 1).
TRPV4 is one of nine types of temperature-sensitive receptors belonging to the TRP (Transient receptor potential) channel, a non-selective cation channel, and functions as a transmembrane calcium influx channel. TRPV4 is expressed in various tissues, such as the sensory nerves, hypothalamus, kidney, lung, and inner ear, as well as in the epidermal cells of the skin, and plays an important role in transmitting pain caused by not only temperature stimuli but also osmotic and mechanical stimuli and inflammatory pain (Patent Document 4). Activated TRPV4 interacts with a substance called β-catenin to promote the development and formation of intercellular adhesion structures such as tight junctions between keratinocytes (Non-Patent Document 2). In addition, Non-Patent Document 1 describes that activation of TRPV4 promotes the formation of tight junctions, and Non-Patent Document 7 describes that TRPV4 plays an important role in epidermal barrier function.

Other known attempts to improve the barrier function of the skin include the use of extracts from the dried rhizomes and roots or whole plants with roots of Asiasarum heterotropoides F. or Asiasarum sieboldi F., which belong to the Aristolochiaceae family, and/or the dried leaves and stems of Apocynum venetum L., which belongs to the Apocynaceae family (Patent Document 1), the use of an extract obtained by solvent extraction of mandarin orange peel (Patent Document 2), and the use of β-cryptoxanthin extracted from citrus fruits (Patent Document 3).
However, Patent Document 2 does not mention the effectiveness of extracts of citrus fruits other than mandarin orange, and does not show any method other than solvent extraction as a method for obtaining active ingredients. Furthermore, Patent Document 3 does not specifically describe a method for extracting β-cryptoxanthin from citrus fruits, but β-cryptoxanthin is a carotenoid pigment that has low solubility in water and is easily soluble in chloroform, benzene, pyridine, and carbon disulfide (Non-Patent Document 3).

Furthermore, Patent Document 5 describes a cosmetic composition containing steam distilled water of a plant of the genus Citrus of the Rutaceae family, and also describes that application of the cosmetic composition improves dry skin and gives the skin a glossy, firm skin. However, it does not describe or suggest that the cosmetic composition has an effect of improving the barrier function of the skin. In addition, it does not describe or suggest that the cosmetic composition has an effect of promoting the formation of tight junctions or activating TRPV4.
Patent Document 6 describes a moisturizer containing cell water from citrus peels, and also describes that such cell water is obtained by distilling citrus peels under reduced pressure at a temperature of 25 to 40° C. and a pressure of −98 to −93 kPa. According to the method described in Patent Document 6, such cell water also contains water-insoluble components. Patent Document 6 does not describe or suggest that it has an effect of improving the barrier function of the skin, nor does it describe or suggest that it has an effect of promoting the formation of tight junctions or activating TRPV4.
Patent Document 7 describes a tight junction formation promoter containing mandarin orange extract as an active ingredient. The mandarin orange extract is obtained by crushing and squeezing the raw or dried mandarin orange peel or fruit, or by crushing the raw or dried mandarin orange peel or fruit and extracting it with a solvent. Patent Document 7 does not state or suggest that a tight junction formation promoter is obtained by using an extract from a citrus plant other than mandarin orange, nor does it state or suggest that a tight junction formation promoter can be obtained by a method other than extraction.

Because the skin is very sensitive, cosmetic ingredients to be applied to the skin must not only be non-irritating and safe, but also naturally derived and contain as few pesticides and other chemicals as possible. For this reason, it is important to use only agricultural products of a certain quality that have been properly managed from the time of cultivation to ensure that no pesticides or other chemicals are used for the cosmetic ingredients to be applied to the skin, and it is desirable to manufacture products using agricultural products that have been consistently managed from cultivation, harvesting, and processing.

In addition, it is known that sudachitin, contained in sudachi, has anti-inflammatory effects, suppresses weight gain, and improves sugar and lipid metabolism, and nobiletin, contained in shekwasha, has anti-inflammatory effects, suppresses hepatitis, and has anti-obesity and anti-diabetic effects (Non-Patent Documents 4-6).

JP 2007-001914 A WO2019/078370 Brochure JP 2014-12718 A WO2012/144080 Brochure JP 2001-213719 A JP 2009-196898 A JP 2016-222558 A

Skin Pharmacol Physiol.: 26, 15-21 (2013) J. Biol. Chem.: 285(24), 18749-18758 (2010) Wikipedia: "β-cryptoxanthin" (https://ja.wikipedia.org/wiki/%CE%92-%E3%82%AF%E3%83%AA%E3%83%97%E3%83%88%E3%82%AD%E3%82%B5%E3%83%B3%E3%83%81%E3%83%B3) Tokushima Prefectural Industrial Technology Center Report: Vol. 23, pp. 15-19 (2014) Journal of the Japanese Society for Food Science and Technology: Vol. 66, No. 7, pp. 223-237 (2019) Wikipedia: "Nobiletin" entry (https://ja.wikipedia.org/wiki/Nobiletin) J. Soc. Cosmet. Chem. Jpn.: 47 (2), 108-118 (2013) Japan Journal of Aromatherapy: 17(1), 39-47(2016)

The object of the present invention is to provide a tight junction formation promoter, a TRPV4 activator, a skin barrier function improver, and a fragrance composition and cosmetic that utilize these, all of which are highly safe when taken orally and applied transdermally. In particular, the object is to normalize the skin barrier function by activating the function of the skin itself.

The present invention relates to a tight junction formation promoter and a TRPV4 activator that contain, as an active ingredient, a water-soluble fraction of a steam distillate obtained from sudachi (Citrus sudachi) and shikuwasa (Citrus sudachi) (hereinafter abbreviated as "aroma water").
Furthermore, the present invention also relates to a skin barrier function improving agent containing aromatic water as an active ingredient.
Specifically, it includes at least the following aspects.

[1] A tight junction formation promoter containing, as an active ingredient, aromatic water obtained from sudachi and/or shekwasha.
[2] A TRPV4 activator having as an active ingredient aromatic water obtained from sudachi and/or shekwasha.
[3] A skin barrier function improving agent containing aromatic water obtained from sudachi and/or shekwasha as an active ingredient.
[4] A fragrance composition comprising aromatic water obtained from sudachi and/or shekwasha.
[5] Any of the agents [1] to [3], wherein the aroma water obtained from sudachi and/or tangerine is a filtrate obtained by adding 1 to 10 parts by mass of water to 1 part by mass of sudachi peel and/or tangerine peel, performing steam distillation at a steam flow rate of 150 to 3,000 g/h per 1 kg of peel, cooling the obtained distillate to 0 to 10°C, and filtering it through diatomaceous earth.
[6] The fragrance composition according to [4], wherein the aroma water obtained from sudachi and/or tangerine is a filtrate obtained by adding 1 to 10 parts by mass of water to 1 part by mass of sudachi peel and/or tangerine peel, steam distilling the mixture at a steam flow rate of 150 to 3,000 g/h per kg of peel, cooling the resulting distillate to 0 to 10°C, and filtering it through diatomaceous earth.
[7] A cosmetic preparation for improving skin barrier function, comprising: 1 to 10 parts by mass of water added to 1 part by mass of sudachi peel and/or tangerine peel; steam distillation performed at a steam flow rate of 150 to 3,000 g/h per 1 kg of peel; cooling the resulting distillate to 0 to 10°C; and filtering the resulting filtrate through diatomaceous earth, as an aromatic water.
[8] A method for producing a cosmetic preparation for improving skin barrier function, comprising adding 1 to 10 parts by mass of water to 1 part by mass of sudachi peel and/or tangerine peel, performing steam distillation at a steam flow rate of 150 to 3,000 g/h per 1 kg of peel, cooling the resulting distillate to 0 to 10°C, and filtering it through diatomaceous earth to obtain a filtrate, and blending the filtrate as aroma water.
[9] An aroma water obtained from sudachi and/or tangerine, which is obtained by adding 1 to 10 parts by mass of water to 1 part by mass of sudachi peel and/or tangerine peel, performing steam distillation at a steam flow rate of 150 to 3,000 g/h per kg of peel, cooling the obtained distillate to 0 to 10°C, and filtering through diatomaceous earth.

The present invention provides raw materials that are highly safe when taken orally or applied transdermally, and that promote tight junction formation, activate TRPV4, and improve skin barrier function, as well as various products that contain them.

The present invention will now be described based on its preferred embodiments.
The present invention relates to an agent for promoting tight junction formation, a TRPV4 activator, and an agent for improving skin barrier function (hereinafter, these agents are collectively referred to as the agent of the present invention).
The following description applies to any of the agents of the present invention unless otherwise specified.

The sudachi (Citrus sudachi) used in the agent of the present invention is a citrus fruit closely related to "Yuzu". In addition to being eaten raw, sudachi is also consumed as a raw material for processed foods, but the peel has not yet been found to be used effectively. As mentioned above, sudachitin contained in sudachi is known to have anti-inflammatory effects, suppress weight gain, and improve sugar and lipid metabolism, but it was not known that sudachi promotes the formation of tight junctions, activates TRPV4, and improves skin barrier function.
Citrus depressa is used as a seasoning by pouring directly squeezed juice over grilled fish or garnishing sashimi, due to its sour taste and unique mellow aroma. As mentioned above, it is also known that nobiletin contained in Citrus depressa has anti-inflammatory, hepatitis-suppressing, anti-obesity and anti-diabetic effects. In recent years, it has been widely used as a beverage, such as Citrus depressa juice and drinks. However, Citrus depressa is small among citrus fruits, and when it is used as juice, the ratio of residues such as peel and seeds as squeezed pomace to the juice is high, and these peels have not been used effectively. Furthermore, it was not known that Citrus depressa also promotes the formation of tight junctions, activates TRPV4, and improves skin barrier function.

The agent of the present invention contains sudachi and/or shekwasha aroma water as an active ingredient, and more specifically, provides a cosmetic raw material containing, as an active ingredient, aroma water obtained by steam distilling sudachi and/or shekwasha peels that have been cultivated and harvested under consistent quality control from cultivation onwards, and clarifying the water-soluble fraction of the distillate.
In the present invention, aroma water means a product obtained by further clarifying the water-soluble fraction from the fraction obtained by steam distillation of the peels of sudachi and/or shikwasha. Because of this production method, the components contained in aroma water are diverse, and it is difficult to identify the properties of a specific component.
In order to characterize the aromatic water of the present invention, there exist impossible and impractical circumstances that require specification of the manufacturing method for obtaining it.
The fact that sudachi and shekwasha aromatic waters have the effect of promoting tight junction formation and have TRPV4 activity, and that these in turn improve skin barrier function, was discovered for the first time through detailed studies by the inventors of the present application, which will be described later.

Taking advantage of the above-mentioned properties, the aromatic water of the present invention can be used as a raw material for various products, either alone or in combination with other ingredients. Such products include products in which the aromatic water is taken percutaneously, such as skin cosmetics, transdermal medicines or quasi-drugs, bath additives, and cleaning agents such as soap and shampoo, as well as products taken orally, such as oral or buccal compositions. The skin cosmetics referred to here may be intended for humans, or may be intended for animals other than humans, particularly mammals. By using the aromatic water in these products, the desired effect of improving the skin barrier function can be imparted to these products.

When the aromatic water is made into a product for transdermal ingestion, it may be in any form, whether liquid or solid, including lotion, emulsion, gel, cream, ointment, powder, granules, capsule, sheet, foam, spray, aerosol, paste, pack, etc.

Specific fields of application of the aromatic water in the form of a product to be taken percutaneously include, for example, various topical preparations (including preparations for use in animals other than humans). Specifically, the aromatic water can be used in the following forms: powder, granule, solid, liquid, gel, foam, emulsion, sheet, mist, spray, etc., and can be used in cosmetics for local or whole body use, particularly cosmetics for the skin (for example, basic cosmetics such as skin lotion, milky lotion, cream, ointment, lotion, oil, and pack, face wash and skin cleanser, massage agent, cleansing agent, hair remover, depilatory agent, shaving treatment, aftershave lotion, preshave lotion, shaving cream, foundation, lipstick, blusher, eye shadow, eyeliner, mascara, and other makeup cosmetics, perfumes, nail beautifiers, nail polish ... enamel, nail enamel remover, poultices, plasters, tapes, sheets, patches, aerosols, etc.), medicinal and/or cosmetic preparations to be applied to the scalp (for example, shampoos, rinses, hair treatments, pre-hair treatments, permanent solutions, hair dyes, hair styling products, hair tonics, hair growth and care products, poultices, plasters, tapes, sheets, patches, aerosols, etc.), bath additives to be added to bath water, underarm odor prevention agents, deodorants, deodorants, antiperspirants, sanitary products, sanitary cotton, wet tissues, etc.

When the agent of the present invention is used in a form for transdermal ingestion, the content of the aromatic water described above in various products will vary depending on the processing method of the aromatic water used, the purpose and formulation of the agent, the ingestion method, the level of effect desired, etc., but generally, the aromatic water of the present invention is preferably 0.00001 parts by mass or more, more preferably 0.00005 parts by mass or more, even more preferably 0.0001 parts by mass or more, and particularly preferably 0.001 parts by mass or more, per 100 parts by mass of product. Furthermore, the content of aromatic water in the agent of the present invention is preferably 30 parts by mass or less, more preferably 20 parts by mass or less, even more preferably 15 parts by mass or less, and particularly preferably 10 parts by mass or less.

The cosmetic composition of the present invention may contain an appropriate amount of physiologically active ingredients, provided that the purpose of the present invention is not impaired. A physiologically active substance is a substance that imparts some kind of physiological activity to the skin when applied to the skin, and examples of such substances include anti-aging agents, UV protection agents, tightening agents, antioxidants, moisturizing agents, blood circulation promoters, antibacterial agents, germicides, drying agents, cooling agents, warming agents, vitamins, amino acids, wound healing promoters, irritation mitigators, analgesics, cell activators, enzyme components, etc.

The cosmetic of the present invention can be produced according to conventional methods. Examples of the cosmetic of the present invention include basic cosmetics, makeup cosmetics, and body cosmetics. The dosage form can also be selected arbitrarily depending on the purpose. That is, examples include liquid, cream, gel, emulsion, sheet, stick, and aerosol forms. The skin cosmetic of the present invention is not limited to general cosmetics, but includes quasi-drugs, designated quasi-drugs, and topical medicines.

The agent of the present invention can be used as a tight junction formation promoter, a TRPV4 activator, and an agent for improving skin barrier function.
<Tight junction formation promoter>
The agent of the present invention can effectively promote the formation of intercellular adhesion structures such as tight junctions in the epidermis and suppress the permeation of substances through the gaps between keratinocytes, thereby enhancing the skin barrier function.
<TRPV4 activator>
As will be apparent from the description of the Examples below, the agent of the present invention can enhance an increase in intracellular calcium ion concentration in epidermal cells by activating TRPV4.
<Skin barrier function improver>
The improvement of skin barrier function in the present invention means improving the deterioration of the moisture retention function and barrier function of the skin caused by dryness, ultraviolet rays, and other physical and chemical stimuli, and making the skin smooth and moisturized. The agent of the present invention can improve the skin barrier function.
In the present application, the TER value (transepithelial electrical resistance) described below is used as an index of improvement in the skin barrier function (see https://www.yodosha.co.jp/jikkenigaku/keyword/3319.html). An improvement in the TER value indicates an improvement in the skin barrier function.

The aroma water obtained from the sudachi and/or shekwasha of the present invention can also be used as a fragrance. The concentration of the aroma water in the fragrance composition is preferably 0.0001 parts by mass or more, more preferably 0.0005 parts by mass or more, even more preferably 0.001 parts by mass or more, and particularly preferably 0.01 parts by mass or more. The content of the aroma water in the fragrance composition is preferably 70 parts by mass or less, more preferably 50 parts by mass or less, even more preferably 30 parts by mass or less, and particularly preferably 10 parts by mass or less.

The present invention will now be described in detail with reference to examples, but is not limited thereto.

<Production of aromatic water>
The aromatic water of the present invention is obtained by steam distilling sudachi and/or shekwasha peel and clarifying the water-soluble fraction of the distillate.
The steam distillation method may be any known method using a steam distillation apparatus and is not particularly limited, but in order to obtain a tight junction formation promoter, a TRPV4 activator, and a skin barrier function improver, the ratio of water to sudachi and/or tangerine peel is preferably 1 to 10 parts by mass, more preferably 1.5 to 8 parts by mass, and most preferably 2 to 6 parts by mass per part by mass of peel. Also, the ratio of the amount of distillate to the raw material (peels) is preferably 0.2 to 2.0 parts by mass, more preferably 0.3 to 1.5 parts by mass, and most preferably 0.4 to 1.0 parts by mass per part by mass of peel.
In order to suppress thermal changes of the active ingredients, the steam flow rate is preferably 150 to 3000 g/h, more preferably 200 to 2000 g/h, and most preferably 250 to 1000 g/h per kg of peel. Furthermore, it is important to cool and filter the distillate at 0 to 10°C in order to remove fat-soluble ingredients.

[Production Example 1] Manufacturing method of Sudachi aroma water 2 kg of Sudachi peel was charged into a steam distiller, and 8 kg of water was added. Steam distillation was performed at a steam flow rate of 720 g/h, and 2 kg of distillate was distilled. The distillate was filtered through diatomaceous earth at 5°C to obtain 1.8 kg of Sudachi aroma water.
The obtained Sudachi aroma water was a colorless and transparent liquid that was indistinguishable from water in appearance. It had a slight Sudachi fragrance and a pH of 8.1. When the inventor applied the aroma water to his skin, he felt no irritation at all.

[Production Example 2] Production method of Citrus depressa aroma water 2 kg of Citrus depressa peel was charged into a steam distiller, and 8 kg of water was added. Steam distillation was performed at a steam flow rate of 720 g/h, and 2 kg of distillate was distilled. The distillate was filtered through diatomaceous earth at 5°C to obtain 1.8 kg of Citrus depressa aroma water.
The obtained Citrus depressa aroma water was a colorless and transparent liquid that was indistinguishable from water in appearance, but had a slight Citrus depressa fragrance and a pH of 8.2. When the inventor applied the aroma water to his skin, he felt no irritation whatsoever.

As is clear from the above description of the production examples, the aromatic water of the present invention is characterized by having many water-soluble components, unlike essential oils generally used as fragrances. It has been reported that essential oils of citrus plants contain furocoumarins with phototoxicity (Non-Patent Document 8). Among these furocoumarins, bergapten and angelicin, which are particularly phototoxic, can be removed or reduced to very small amounts by a steam distillation process or a process of separating the water-soluble fraction. Therefore, the aromatic water of the present application has a function of preventing the adverse effects on the skin caused by light (ultraviolet rays) after application to the skin, in addition to direct irritation to the skin. In terms of production method and ingredients, the aromatic water is also different from the extract obtained by extracting fruit with water, alcohol, organic solvent, or supercritical fluid. In this respect, the aromatic water of the present invention is different from those described in the above Patent Documents 2, 3, and 5 to 7.
Thus, aromatic water does not contain non-volatile dietary fiber, polyphenols, flavanone glycosides, fat-soluble nobiletin, naringin, polymethoxyflavonoids, hesperidin, neohesperidin, hesperetin, narirutin, flavonoids, sudachitin, cryptoxanthin, carotenoids, sphingolipids, flavanones, etc., or even if they are contained, the amounts are so small that the respective functions cannot be expected. The agent of the present invention differs from those described in the prior patent documents in that it contains aromatic water containing only a water-soluble fraction as an active ingredient.

<TER value>
In the present invention, the improvement of the skin barrier function is evaluated using the TER value (transepithelial electrical resistance) as an index, as described above.

Test Example 1: TER value measurement test HEC human epidermal keratinocytes (Total Kit; neonatal, TOYOBO, CA102K05n) were cultured using growth medium (GROWTH MEDIUM, TOYOBO, CA141K500). 1.5 mL of medium was added to each well of a 12-well plate, and a cell suspension was adjusted to 5 x ¹⁰⁵ cells/insert (1.1 ^cm2 ). 0.5 mL of the medium was seeded on each insert, and the cells were incubated at 37 °C _in 5% CO2.
C. and cultured for 24 hours. The inserts seeded with cells were transferred to a plate containing 1.5 mL of 1.5 mM calcium chloride-containing medium, the medium (supernatant) in the inserts was removed with an aspirator, 0.5 mL of 1.5 mM calcium chloride-containing medium was added to each insert, and cultured at 28.degree. C. for 24 hours. The inserts seeded with cells were transferred to a medium containing 10 .mu.M 4.alpha.-PDD and 0.1% of Production Example 1 or 2, the medium (supernatant) in the inserts was removed with an aspirator, 0.5 mL of each medium was added, the inserts were sealed, and culture was started at 28.degree. C., and the TER value after 24 hours was measured. TER measurement was performed three times per well, and the average value was taken as the TER value ( ^{.OMEGA.cm.sup.2} ) of that well.
The change in TER value was calculated as (TER value after 24 hours)−(TER value immediately after the addition of the sample), and the results are shown in FIG.

4α-PDD (Non-Patent Document 1) (Comparative Example 2), which has been reported to have the effect of promoting the formation of intercellular adhesion structures (tight junctions and/or adherens junctions), significantly increased the TER value compared to the additive-free product (Comparative Example 1).
By adding 0.1% of the Sudachi aroma water of Production Example 1 (Example 1) and by adding 0.1% of the Shiikuwasa aroma water of Production Example 2 (Example 2), the TER value was significantly increased compared to the product without any additives (Comparative Example 1), and the effect was higher than that of 4α-PDD (Comparative Example 2), which is known as a TRPV4 activator.

Cultivation was carried out in the same manner, except that the amount of the aromatic water of Production Example 1 or 2 added to the medium was changed to 10%, and the TER value after 30 hours was measured. TER measurement was performed three times per well, and the average value was regarded as the TER value of that well.
The change in TER value was calculated as (TER value after 30 hours)−(TER value immediately after the addition of the sample), and the results are shown in FIG.

By adding 10% of the Sudachi aroma water from Production Example 1 (Example 3) and 10% of the Shikuwasa aroma water from Production Example 2 (Example 4), the TER value increased significantly compared to the additive-free product (Comparative Example 3).

The above tests demonstrated that the agent of the present invention promotes the formation of intercellular adhesion structures and is effective as an agent for improving skin barrier function.

<TPRV4 activity>
[Test Example 2] Measurement of TRPV4 activity (1) Preparation of a human TRPV4 stable expression strain A human TRPV4 expression vector was purchased from GeneCopoeia, Inc. The expression vector was introduced into HEK293 cells using Lipofectamine LTX Reagent (Thermo Fisher Scientific, Inc.), and cells into which the gene had been introduced were selected using 1 mg/L puromycin.

(2) Measurement of TRPV4 Activity The activity of TRPV4 transduced into HEK293 cells was measured using a fluorescent calcium imaging method.
First, the cultured TRPV4-expressing cells were seeded on a poly-D-lysine-coated 96-well plate and incubated overnight at 37°C. The culture medium was removed, and 100 μL of Hanks' buffer solution containing Fluo-8NW (1.3 × 10-3 μL) and surfactant (0.11 μL) was added, followed by incubation at 37°C for 60 minutes. Then, the plate was set in a fluorescent plate reader Flexstation 3 (Molecular Devices). The fluorescence image obtained when excited at an excitation wavelength of 480 nm with the temperature inside the device at 37°C was detected at a detection wavelength of 520 nm. The measurement was performed for 60 seconds, and 20 seconds after the start of the measurement, 25 μL of a test solution prepared by diluting the aroma water of Production Example 1 or 2 with Hanks' buffer solution so that the final concentration was 1% was added, and the TRPV4 activity was evaluated based on the change in fluorescence intensity thereafter. As a positive control, a similar evaluation was carried out using the TRPV4 agonist GSK1016790A adjusted to 50 nM.
TRPV4 activity was expressed as the fluorescence intensity ratio (Ratio; Fpeak/F0) obtained by dividing the peak fluorescence intensity (Fpeak) after addition of the test solution by the fluorescence intensity (F0) in the absence of any stimulation inhibitor.
Next, the TRPV4 activity of GSK1016790A and the aroma waters of Examples 1 and 2 in the presence of a TRPV4 inhibitor was evaluated using the same method as above, except that 25 μL of Hank's buffer solution containing 2.36 ppm (5 μM) of a TRPV4 inhibitor, HC-067047 (Sigma-Aldrich), was added.

The results measured using the above method are shown in Table 1.

As shown in Table 1, in Comparative Example 4 in which GSK1016790A, a positive control, was added, the fluorescence intensity ratio was high, and in the presence of an inhibitor, the fluorescence intensity ratio was significantly reduced. In other words, it was confirmed that TRPV4 activity could be accurately measured in this test, and therefore, measurements were made using this test system hereinafter.
By this measurement, the TRPV4 activity of the Sudachi aroma water of Production Example 1 (Example 5) and the Shikwasha aroma water of Production Example 2 (Example 6) was measured. An increase in the fluorescence intensity ratio equal to or greater than that of GSK1016790A, and a decrease in the fluorescence intensity ratio in the presence of inhibitors were confirmed, confirming that the Sudachi aroma water of Production Example 1 and the Shikwasha aroma water of Production Example 2 have a TRPV4 activation effect.

In order to verify the TRPV4 activity effects of other citrus fruits, the TRPV4 activity of aroma waters produced from lemon and yuzu peels (Comparative Example 5 and Comparative Example 6, respectively) prepared in the same manner as in Production Examples 1 and 2 was measured in the same manner. In both tests with and without inhibitors, the fluorescence intensity ratio was low, confirming the absence of TRPV4 activity.
From the above, it was understood that the species with TRPV4 activity are limited among citrus fruits, and that this activity is unique to Sudachi and Shikwasha.

Formulation Example 1 Milky Lotion According to Table 2, the aqueous phase was prepared by blending purified water with moisturizers such as 1,3-butylene glycol and glycerin, and the aromatic water of Production Example 1 or 2. The oily phase was prepared by blending solid oils such as beeswax, cetyl alcohol, and petrolatum, liquid oils such as squalane, preservatives, surfactants, and other oily components, and heating the mixture to approximately 80° C. A thickener such as quince seed was added to the aqueous phase heated to approximately 80° C., and the oily phase was gradually added while stirring with a homomixer to emulsify the mixture, thereby preparing an emulsion.

(Improves skin barrier function)
The emulsion using the aromatic water of Preparation Example 1 or 2 of Formulation Example 1 was used continuously for one week, and the smoothness of the skin after the continuous use was evaluated, and it was confirmed that the skin condition had improved.

Formulation Example 2: Face pack According to Table 3, purified water was mixed with a moisturizer such as 1,3-butylene glycol or glycerin, and the aromatic water of Production Example 1 or 2. This was heated to 70 to 80°C, a thickener such as carboxymethyl cellulose, and a film-forming agent such as polyvinyl alcohol were added and dissolved by stirring, and a solution of a preservative, a surfactant, etc. dissolved in ethyl alcohol was further added to solubilize the mixture, to prepare a face pack.

Formulation Example 3: Lotion According to Table 4, the lotion of the present invention was prepared by mixing at room temperature a cosmetic base prepared by dissolving a moisturizing agent such as 1,3-butylene glycol or glycerin, a skin nutrient such as oleyl alcohol, and a preservative in ethyl alcohol in purified water, and adding 0.1% of the aromatic water of Production Example 1 or 2 to the cosmetic base.

Formulation Example 4: Body soap As the aqueous phase, purified water was mixed with moisturizing agents such as propylene glycol and glycerin. As the oil phase, oily components such as sodium polyoxyethylene lauryl ether sulfate, triethanolamine lauryl sulfate, coconut oil fatty acid amidopropyl betaine, and ethylene glycol distearate were mixed. The aqueous phase was gradually heated to about 80°C, and the oil phase heated to about the same temperature was gradually added thereto, and 0.1% of the aroma water of Production Example 1 or 2 was mixed and homogenized to prepare a body soap.

Formulation Example 5: Fragrance composition A fragrance composition was prepared according to the formula in the table below. The addition of the aromatic water of Preparation Example 1 or 2 did not affect the balance of the fragrance, and the fragrance did not feel strange after application to the skin after mixing with a milky lotion.

The sudachi and/or shekwasha aromatic water used in the present invention has an effect of improving skin barrier function by promoting the formation of tight junctions, an effect of increasing TRPV4 expression, and an excellent effect of improving skin condition. Therefore, it can be preferably used in products such as lotions, milky lotions, beauty essences, creams, packs, shampoos, conditioners, tablets, soft capsules, hard capsules, granules, and liquid beverages.

Claims (9)

A tight junction formation promoter whose active ingredient is aromatic water obtained from sudachi and/or shekwasha. A TRPV4 activator whose active ingredient is aromatic water obtained from sudachi and/or shekwasha. A skin barrier function improver that contains aromatic water extracted from sudachi and/or shekwasha as an active ingredient. A fragrance composition containing aromatic water obtained from sudachi and/or shekwasha. The agent according to any one of claims 1 to 3, wherein the aroma water obtained from sudachi and/or tangerine is a filtrate obtained by adding 1 to 10 parts by mass of water to 1 part by mass of sudachi peel and/or tangerine peel, performing steam distillation at a steam flow rate of 150 to 3000 g/h per kg of peel, cooling the obtained distillate to 0 to 10°C, and filtering through diatomaceous earth. The fragrance composition according to claim 4, wherein the aroma water obtained from sudachi and/or tangerine is a filtrate obtained by adding 1 to 10 parts by mass of water to 1 part by mass of sudachi peel and/or tangerine peel, performing steam distillation at a steam flow rate of 150 to 3000 g/h per kg of peel, cooling the obtained distillate to 0 to 10°C, and filtering through diatomaceous earth. A cosmetic preparation for improving skin barrier function, characterized by adding 1 to 10 parts by mass of water to 1 part by mass of sudachi peel and/or shekwasha peel, performing steam distillation at a steam flow rate of 150 to 3,000 g/h per 1 kg of peel, and incorporating the filtrate obtained by cooling the distillate to 0 to 10°C and filtering through diatomaceous earth as an aroma water. A method for producing a cosmetic for improving skin barrier function, comprising adding 1 to 10 parts by mass of water to 1 part by mass of sudachi peel and/or shekwasha peel, performing steam distillation at a steam flow rate of 150 to 3,000 g/h per 1 kg of peel, cooling the distillate obtained to 0 to 10°C, and filtering through diatomaceous earth to obtain a filtrate, and blending the filtrate as aroma water. Aromatic water obtained from sudachi and/or shekwasha, which is obtained by adding 1 to 10 parts by mass of water to 1 part by mass of sudachi peel and/or shekwasha peel, performing steam distillation at a steam flow rate of 150 to 3000 g/h per kg of peel, and cooling the resulting distillate to 0 to 10°C and filtering it through diatomaceous earth.