JP2022006397A - Barrier function enhancer - Google Patents

Abstract

To provide a barrier function enhancer having an excellent action.SOLUTION: A barrier function enhancer contains corosolic acid as an active ingredient.SELECTED DRAWING: Figure 1

Description

The present invention relates to a barrier function enhancer.

One of the structures that separates the inside and the outside in a living body is epithelial tissue composed of epithelial cells. The epithelial tissue has a barrier function that controls the permeation of substances, thereby creating an internal environment different from the outside world in the living body. Such a barrier function is mainly formed by adhesion between cells, and one of such adhesions is a tight junction (hereinafter sometimes referred to as "TJ"). TJ is an intercellular adhesion structure that not only brings adjacent epithelial cells into close contact with each other, but also controls the permeation of substances by sealing the gaps between cells. TJ is composed of cell membrane proteins such as claudin and occludin, and lining proteins such as ZO-1 and ZO-2. These proteins form the skeleton of TJ strands and function as a barrier for TJ. It is considered to be controlled (see Non-Patent Document 1).

To date, more than 20 types of claudin molecules have been reported, forming the claudin family. These are known to exhibit tissue-specific expression patterns, with claudin-1 and claudin-4 being expressed in the epidermis. Claudin-4 is also highly expressed in the mucosal epithelium and functions as a barrier to prevent the invasion of foreign substances inside and outside the body.

If the expression of claudin or occludin is reduced for some reason, it causes a decrease in TJ function. For example, when the function of TJ in the gastrointestinal tract is reduced, food allergens, pathogenic microorganisms and the like invade the body, which is considered to contribute to inflammatory bowel disease and various infectious diseases. In addition, conventionally, it was thought that the barrier function of the skin is borne only by the stratum corneum, but in recent years, when the constituent protein of TJ existing in the stratum granulosum of the epidermis is deleted at the gene level, the barrier function of the skin is disrupted. It has been found that TJ also plays an important role in the barrier function of the skin (see Non-Patent Document 2). Here, when the expression of claudin, occludin, etc. is reduced for some reason, structural destruction of TJ occurs and it does not function as a permeation barrier for substances, resulting in dry skin, rough skin, atopic dermatitis and various infections. It is thought to contribute to skin symptoms such as illness.

Therefore, by strengthening the function of TJ by promoting the production of claudin and occludin, the barrier function in epithelial tissue is strengthened, and in the gastrointestinal tract, inflammatory intestinal diseases, food allergies, various infectious diseases, etc. are prevented or improved. On the other hand, in the epithelial skin, it is considered that skin symptoms such as dry skin, rough skin, atopic dermatitis and various infectious diseases can be prevented or ameliorated. Asparagus linearis extract (Patent Document 1) and the like are known to have a claudin production promoting action and an occludin production promoting action.

Japanese Unexamined Patent Publication No. 2009-256244

Journal of Japanese Society of Cosmetic Science, 2007, vol.31, pp.296-301 J. Cell Biol., 2002, vol.156, pp.1099-1111

An object of the present invention is to provide a barrier function enhancer having an excellent action and effect.

In order to achieve the above object, the present invention provides a barrier function enhancer characterized by containing corosolic acid as an active ingredient (Invention 1).

In the above invention (Invention 1), corosolic acid is used for improving barrier function, suppressing barrier function decline, promoting claudin-1 expression, promoting claudin-4 expression, promoting ocludin expression, and promoting ZO-1 expression. And, it is preferable to use it for one or more uses selected from the group consisting of ZO-2 expression promoting uses (Invention 2).

According to the present invention, by using corosolic acid as an active ingredient, it is possible to provide a barrier function enhancer having an excellent action and effect.

It is a graph which shows the result of having evaluated the barrier function improving effect of corosolic acid by the transepithelial electric resistance (TER) value measurement with time.

Hereinafter, embodiments of the present invention will be described.
The barrier function enhancer according to the present embodiment contains corosolic acid as an active ingredient.

Corosolic acid is a kind of triterpenoid compound having a chemical structure represented by the following formula.

Since the corosolic acid used in this embodiment is commercially available, it may be used. Further, corosolic acid can be produced by isolation and purification from a plant extract containing corosolic acid, or can be produced by synthesis. In the case of production by synthesis, the synthesis method is not particularly limited, and synthesis can be performed by a known method (see JP-A-2005-29570).

A plant extract containing corosolic acid can be obtained by an extraction method commonly used for extracting plants. The plant extract includes an extract obtained by using a plant containing corosolic acid as an extraction raw material, a diluted solution or a concentrated solution of the extract, a dried product obtained by drying the extract, or a crude product thereof. Both purified and refined products are included.

Examples of plants containing corosolic acid include loquat (scientific name: Eriobotrya japonica) and banaba (scientific name: Lagerstroemia speciosa).

Loquat (Eriobotrya japonica) is an evergreen tree of the Rosaceae family. The fruit part is edible, and the leaf part is used for the treatment of gastrointestinal weakness, neuralgia, diarrhea and the like. Loquat grows naturally in Japan, Taiwan, China, etc. and can be easily obtained from these regions. The constituent parts of the biwa that can be used as the extraction raw material are not particularly limited, and for example, the above-ground parts such as the leaves, branches, bark, stems, stems, fruits, seeds, and flowers, and roots. Alternatively, a mixture of these parts may be mentioned, but it is preferable to use the leaf part among them.

Banaba (Lagerstroemia speciosa) is a Lythraceae plant that is widely distributed in tropical and subtropical regions such as the Philippines and Australia, and can be easily obtained from these regions. The constituent parts of vanaba that can be used as an extraction raw material are not particularly limited, and are, for example, above-ground parts such as leaves, branches, bark, stems, stems, fruits, seeds, and flowers, and roots. Alternatively, a mixture of these parts may be mentioned, but it is preferable to use the leaf part among them.

The plant extract containing corosolic acid can be obtained by drying the extraction raw material, pulverizing it as it is or using a coarse crusher, and subjecting it to extraction with an extraction solvent. Drying may be carried out in the sun or by using a commonly used dryer. Further, it may be used as an extraction raw material after being subjected to pretreatment such as degreasing with a non-polar solvent such as hexane. By performing pretreatment such as degreasing, extraction treatment with a polar solvent of plants can be efficiently performed.

As the extraction solvent, it is preferable to use a polar solvent, for example, water, a hydrophilic organic solvent and the like, and these are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. Is preferable.

Water that can be used as an extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmoregulation, buffering and the like. Therefore, the water that can be used as the extraction solvent in the present embodiment includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.

Hydrophilic organic solvents that can be used as the extraction solvent 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; 1,3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.

When a mixed solution of two or more polar solvents is used as an extraction solvent, the mixing ratio can be appropriately adjusted. For example, when a mixed solution of water and a lower aliphatic alcohol is used as an extraction solvent, the mixing ratio of water and the lower aliphatic alcohol is preferably 9: 1 to 1: 9 (volume ratio). It is more preferably 7: 3 to 2: 8 (volume ratio). When a mixed solution of water and a lower aliphatic ketone is used, the mixing ratio of water and the lower aliphatic ketone is preferably 9: 1 to 2: 8 (volume ratio), and water and a large amount are used. When a mixed solution with a valent alcohol is used, the mixing ratio of water and the polyhydric alcohol is preferably 8: 2 to 1: 9 (volume ratio).

The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be carried out according to a conventional method. For example, the extraction material is immersed in an extraction solvent having an amount (mass ratio) of 5 to 50 times that of the extraction material, the soluble component is extracted at room temperature or under reflux heating, and then the extract is filtered to remove the extraction residue. You can get the liquid. Distilling off the solvent from the obtained extract gives a paste-like concentrate, and further drying the concentrate gives a dried product.

The method for purifying and isolating corosolic acid from the extract obtained as described above, the concentrate of the extract, or the dried product of the extract is not particularly limited, and may be carried out by a conventional method. can. For example, the extract is dissolved in a developing solvent and subjected to column chromatography using a porous substance such as silica gel or alumina, or a porous resin such as a styrene-divinylbenzene copolymer or polymethacrylate to obtain corosolic acid. Examples thereof include a method of collecting the contained fraction. In this case, the developing solvent may be appropriately selected according to the stationary phase to be used. For example, when the extract is separated by normal phase chromatography using silica gel as the stationary phase, the developing solvent is chloroform: methanol = 20 :. 1st grade can be mentioned. Further, the fraction containing corosolic acid obtained by column chromatography can be subjected to any arbitrary method such as reverse phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, and column chromatography using an ion exchange resin. It may be purified by using an organic compound purification means.

Since the corosolic acid obtained as described above has an excellent barrier function enhancing action, it can be used as an active ingredient of the barrier function enhancing agent. The barrier function enhancer of the present embodiment can be used in a wide range of applications such as pharmaceuticals, quasi-drugs, cosmetics, and foods and drinks.

As the active ingredient of the barrier function enhancer according to the present embodiment, a composition containing corosolic acid may be used instead of the purified / isolated corosolic acid. Here, the "composition containing corosolic acid" in the present embodiment includes a plant extract obtained by using a plant containing corosolic acid as an extraction raw material.

When a composition containing corosolic acid is used as the active ingredient of the barrier function enhancer according to the present embodiment, it is preferable to use one that has been purified to increase the purity of corosolic acid. Specifically, the content of corosolic acid in the composition is preferably 1% by mass or more, more preferably 5% by mass or more, and particularly preferably 10% by mass or more. By using a substance having an increased purity of corosolic acid as an active ingredient, a barrier function enhancer having a further excellent action and effect can be obtained.

Here, the barrier function enhancing action of corosolic acid is a barrier function improving action, a barrier function lowering inhibitory action, a claudin-1 expression promoting action, a claudin-4 expression promoting action, an occludin expression promoting action, and a ZO-1 expression promoting action. It is preferably exerted based on the action and the ZO-2 expression promoting action. However, the barrier function enhancing action possessed by corosolic acid is not limited to the barrier function enhancing action exerted based on the above action.

In addition, corosolic acid has a barrier function improving action, a barrier function lowering suppressing action, a claudin-1 expression promoting action, a claudin-4 expression promoting action, an occludin expression promoting action, a ZO-1 expression promoting action and a ZO-2 expression. Utilizing the promoting action, barrier function improving use, barrier function deterioration suppressing use, claudin-1 expression promoting use, claudin-4 expression promoting use, occludin expression promoting use, ZO-1 expression promoting use and ZO-2 use, respectively. It can be used for expression promotion. That is, the barrier function enhancer of the present embodiment is a barrier function improver containing corosolic acid as an active ingredient, a barrier function decrease inhibitor, a claudin-1 expression promoter, a claudin-4 expression promoter, and an occludin expression promoter. , ZO-1 expression promoter and ZO-2 expression promoter.

The barrier function enhancer of the present embodiment may consist only of corosolic acid or a composition containing corosolic acid, or may be a formulation containing corosolic acid.

The barrier function enhancer of the present embodiment is an arbitrary powder, granule, tablet, liquid or the like according to a conventional method using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent. It can be formulated into a dosage form. At this time, as the auxiliary agent, for example, excipients, binders, disintegrants, lubricants, stabilizers, flavoring / odorizing agents and the like can be used. The barrier function enhancer can be used by blending with other compositions (for example, an external preparation for skin, etc.), and can also be used as an ointment, an external liquid, a patch, or the like.

When the barrier function enhancer of the present embodiment is formulated, the content of corosolic acid is not particularly limited and can be appropriately set according to the purpose.

The barrier function enhancer of the present embodiment can be used as an active ingredient by blending other natural extracts having a barrier function enhancing action together with corosolic acid, if necessary.

Examples of the method for administering the barrier function enhancer of the present embodiment to a patient include oral administration, transdermal administration, and the like, and a method suitable for prevention / treatment thereof may be appropriately selected according to the type of disease.

In addition, the dose of the barrier function enhancer of the present embodiment may be appropriately increased or decreased depending on the type of disease, severity, individual difference of patients, administration method, administration period and the like.

The barrier function enhancer of the present embodiment can enhance the barrier function in epithelial tissue through the barrier function enhancing action of corosolic acid, which is an active ingredient. For example, it can enhance the barrier function in the gastrointestinal tract and prevent, treat or improve inflammatory bowel disease, food allergies, various infectious diseases transmitted from the gastrointestinal tract, and the like. In addition, it is possible to enhance the barrier function and the water retention function in the epidermis, and prevent, treat or improve skin symptoms such as dry skin, rough skin, atopic dermatitis and various infectious diseases. However, the barrier function enhancer of the present embodiment can be used for all uses other than these uses, which are significant in exerting the barrier function enhancer action.

When the barrier function enhancer of the present embodiment is used for a barrier function improving application or a barrier function deterioration suppressing application (when used as a barrier function improving agent or a barrier function deterioration suppressing agent), through a barrier function improving effect and a barrier function deterioration suppressing effect. , Enhances barrier function in epithelial tissue, prevents, treats or improves inflammatory bowel diseases, food allergies, various infectious diseases transmitted from the digestive tract; skin symptoms such as dry skin, rough skin, atopic dermatitis and various infections It can prevent, treat or improve diseases. However, in addition to these uses, it can be used for all uses that are significant in exerting a barrier function improving action or a barrier function deterioration suppressing action.

The barrier function enhancer of the present embodiment is used for claudin-1 expression promoting use, claudin-4 expression promoting use, occludin expression promoting use, ZO-1 expression promoting use or ZO-2 expression promoting use (claudin). -1 When used as an expression promoter, claudin-4 expression promoter, occludin expression promoter, ZO-1 expression promoter or ZO-2 expression promoter), claudin-1 expression promoter action, claudin-4 expression Through promoting action, occludin expression promoting action, ZO-1 expression promoting action and ZO-2 expression promoting action, the barrier function is enhanced by promoting the formation of tight junctions in epithelial tissue, and inflammatory bowel disease, food allergy, and gastrointestinal tract. Prevention, treatment or improvement of various infectious diseases transmitted from the skin; skin symptoms such as dry skin, rough skin, atopic dermatitis and various infectious diseases can be prevented, treated or ameliorated. However, in addition to these uses, it is significant to exert a claudin-1 expression promoting action, a claudin-4 expression promoting action, an occludin expression promoting action, a ZO-1 expression promoting action or a ZO-2 expression promoting action. It can be used for all purposes.

In addition, the barrier function enhancer of the present embodiment has an excellent barrier function enhancer effect, and is therefore suitable for blending in, for example, an external skin preparation or an oral composition. In this case, corosolic acid or a composition containing corosolic acid may be blended as it is, or a barrier function enhancer formulated from corosolic acid or a composition containing corosolic acid may be blended.

Here, the external skin preparation is not limited in its classification, and includes a wide range of skin lotions, non-medicinal products, pharmaceuticals, etc. that are used transdermally, and specifically, for example, an ointment. Examples include creams, milky lotions, serums, lotions, packs, foundations, lip creams, bathing agents, hair tonics, hair lotions, soaps, body shampoos and the like.

The blending amount of corosolic acid in the external skin preparation can be appropriately adjusted according to the type of the external skin preparation, but the suitable blending ratio is 0.0001 to 10% by mass, and the particularly suitable blending ratio is It is 0.001 to 1% by mass.

Oral compositions are those that are less likely to harm human health and are ingested by oral or gastrointestinal administration in normal social life, such as foods, pharmaceuticals, quasi-drugs, etc. under administrative divisions. It is not limited to classification. Therefore, the "oral composition" in the present embodiment includes general foods, feeds, health foods, foods with health claims (foods for specified health use, foods with nutritional claims, foods with functional claims), and non-pharmaceutical products that are orally ingested. , Pharmaceuticals, etc. are widely included. The oral composition according to the present embodiment is preferably an oral composition capable of displaying the preferable action of corosolic acid or a composition containing corosolic acid on the oral composition or its packaging, and has a health function. Foods (foods for specified health use, foods with nutritional function, foods with functional claims), non-pharmaceutical products or pharmaceutical products are particularly preferable.

The blending amount of corosolic acid in the oral composition can be appropriately changed in consideration of the purpose of use, symptoms, gender, etc., but in consideration of the general intake of the oral composition to be added, adult 1 It is preferable that the daily intake of the extract is about 1 to 1000 mg. When the oral composition to be added is in the form of granules, tablets or capsules, the amount of corosolic acid added is usually 0.0001 to 10% by mass, preferably 0.001 with respect to the oral composition to be added. ~ 1% by mass.

Further, since the barrier function enhancer of the present embodiment has an excellent barrier function enhancer action, it can be suitably used as a reagent for research on these action mechanisms.

Although the barrier function enhancer of the present embodiment is suitably applied to humans, animals other than humans (for example, mice, rats, hamsters, dogs) as long as the respective actions and effects are exhibited. , Cats, cows, pigs, monkeys, etc.).

Hereinafter, the present invention will be specifically described with reference to test examples, but the present invention is not limited to the following examples. The corosolic acid (Sample 1) used in the following test example is a commercially available product (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[Test Example 1] Skin barrier function improving action test For corosolic acid (Sample 1), the value of transepithelial electrical resistance (TER) was measured as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM), and then the cells were recovered by tripsin treatment. After diluting the collected cells with KGM to a concentration of 2.2 × 10 ⁵ cells / mL, 0.5 mL per well is placed on the upper layer of a 12-well transwell (Corning, 12 mm in diameter, 0.4 μm pore). The seeds were sown, and 1.5 mL each of KGM was further added to the lower layer and cultured for 2 days.

After completion of culture (0 hours), 0.5 mL of the test sample dissolved in KGM (Sample 1, see Table 1 for sample concentration) was added to the upper layer of each well, treated for 15 minutes, and then 0.5 mL of the upper layer was added. Was replaced with KGM and the culture was continued. Twenty-four hours after the treatment, the transepithelial electrical resistance (TER) was measured using the Millicell-ERS resistance measurement system (manufactured by Millipore), and then the sample for 15 minutes (Sample 1, sample concentration is shown in Table 1). The treatment was repeated 24 hours later (48 hours after the first treatment), and the TER was measured to evaluate the effect of improving the skin barrier function over time. The measured TER values are shown in Table 1 and FIG.

As shown in Table 1 and FIG. 1, it was found that the treatment with corosolic acid (Sample 1) increased the TER value and improved the barrier function of the skin.

[Test Example 2] Skin barrier function deterioration inhibitory effect test Normal human neonatal epidermal keratinocytes (NHEK) were cultured in a normal human epidermal keratinocyte proliferation medium (KGM), and then the cells were recovered by tripsin treatment. After diluting the collected cells with KGM to a concentration of 2.2 × 10 ⁵ cells / mL, 0.5 mL per well is placed on the upper layer of a 12-well transwell (Corning, 12 mm in diameter, 0.4 μm pore). The seeds were sown, and 1.5 mL of KGM was further added to the lower layer for culturing. Two days later, 0.5 mL of CaCl ₂ (final concentration 1.8 mM) dissolved in KGM was added to the upper layer of each well, and 1.5 mL of KGM was added to the lower layer, and the cells were cultured for 4 days to induce tight junction formation.

After the culture is completed (0 hours), the high CaCl ₂ medium is removed, and 0.5 mL of the test sample (Sample 1, sample concentration is shown in Table 2) dissolved in KGM is added to the upper layer of each well and treated for 15 minutes. After that, the upper layer was replaced with 0.5 mL of KGM and the culture was continued. After 24 hours and 48 hours, the same 15-minute sample treatment (see Table 2 for sample 1 and sample concentration) was repeated, and 72 hours after the treatment, a Millicell-ERS resistance value measurement system (manufactured by Millipore) was used. , Transepithelial electrical resistance value (TER) was measured and the effect of suppressing the decrease in skin barrier function was evaluated. The measured TER values are shown in Table 2.

As shown in Table 2, it was found that the treatment with corosolic acid (Sample 1) suppressed the decrease in TER value and suppressed the decrease in the barrier function of the skin.

[Test Example 3] Claudin-1 expression promoting action test With respect to corosolic acid (Sample 1), the claudin-1 expression promoting action was tested as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM), and then the cells were recovered by tripsin treatment. Using KGM, 30 × 10 ⁴ cells / 2 mL were seeded on 6-well plates and cultured overnight at 37 ° C. under 5% CO ₂ . After culturing, the medium was replaced with a medium (KBM +) from which BPE and EGF were removed from the growth additive in KGM.

After 24 hours, the culture broth was discarded, 2 mL of a test sample dissolved in KBM + (Sample 1, see Table 3 for sample concentration) was added to each well, and the mixture was treated at 37 ° C. under 5% CO ₂ for 15 minutes. After the treatment, the cells were replaced with 2 mL of KBM + again and cultured for 4 hours and 8 hours. After culturing, total RNA was extracted with ISOGEN II (manufactured by NIPPON GENE), the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared so as to be 200 ng / μL.

Using this total RNA as a template, the expression level of mRNA was measured for claudin-1 and GAPDH, which is an internal standard. Detection is performed by PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc.) and TB Green Fast qPCR Mix (Takara Bio Inc.) using a real-time PCR device Thermal Cycler Dice Real Time System III (manufactured by Takara Bio Inc.). It was performed by a 2-step real-time RT-PCR reaction. The primer used was manufactured by Takara Bio. The expression level of each gene mRNA was calculated by adjusting the expression level of GAPDH mRNA based on the total RNA standard prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the expression promotion rate of claudin-1 mRNA by adding the test sample was calculated by the following formula.

Claudin-1 mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with sample addition B: Correction value without sample addition Claudin-1 The results of mRNA expression promotion rate are shown in Table 3.

As shown in Table 3, it was confirmed that corosolic acid (Sample 1) has an excellent claudin-1 expression promoting action.

[Test Example 4] Claudin-4 expression promoting action test With respect to corosolic acid (Sample 1), the claudin-4 expression promoting action was tested as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM), and then the cells were recovered by tripsin treatment. Using KGM, 30 × 10 ⁴ cells / 2 mL were seeded on 6-well plates and cultured overnight at 37 ° C. under 5% CO ₂ . After culturing, the medium was replaced with a medium (KBM +) from which BPE and EGF were removed from the growth additive in KGM.

After 24 hours, the culture broth was discarded, 2 mL of a test sample dissolved in KBM + (Sample 1, see Table 4 for sample concentration) was added to each well, and the mixture was treated at 37 ° C. under 5% CO ₂ for 15 minutes. After the treatment, the cells were replaced with 2 mL of KBM + again and cultured for 4 hours and 8 hours. After culturing, total RNA was extracted with ISOGEN II (manufactured by NIPPON GENE), the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared so as to be 200 ng / μL.

Using this total RNA as a template, the expression level of mRNA was measured for claudin-4 and GAPDH, which is an internal standard. Detection is performed by PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc.) and TB Green Fast qPCR Mix (Takara Bio Inc.) using a real-time PCR device Thermal Cycler Dice Real Time System III (manufactured by Takara Bio Inc.). It was performed by a 2-step real-time RT-PCR reaction. The primer used was manufactured by Takara Bio. The expression level of each gene mRNA was calculated by adjusting the expression level of GAPDH mRNA based on the total RNA standard prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the expression promotion rate of claudin-4 mRNA by adding the test sample was calculated by the following formula.

Claudin-4 mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with sample addition B: Correction value without sample addition Claudin-4 The results of mRNA expression promotion rate are shown in Table 4.

As shown in Table 4, it was confirmed that corosolic acid (Sample 1) has an excellent claudin-4 expression promoting action.

[Test Example 5] Occludin expression promoting action test The occludin expression promoting action was tested for corosolic acid (Sample 1) as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM), and then the cells were recovered by tripsin treatment. Using KGM, 30 × 10 ⁴ cells / 2 mL were seeded on 6-well plates and cultured overnight at 37 ° C. under 5% CO ₂ . After culturing, the medium was replaced with a medium (KBM +) from which BPE and EGF were removed from the growth additive in KGM.

After 24 hours, the culture broth was discarded, 2 mL of a test sample dissolved in KBM + (Sample 1, see Table 5 for sample concentration) was added to each well, and the mixture was treated at 37 ° C. under 5% CO ₂ for 15 minutes. After the treatment, the cells were replaced with 2 mL of KBM + again and cultured for 4 hours and 8 hours. After culturing, total RNA was extracted with ISOGEN II (manufactured by NIPPON GENE), the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared so as to be 200 ng / μL.

Using this total RNA as a template, the expression level of mRNA was measured for occludin and GAPDH, which is an internal standard. Detection is performed by PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc.) and TB Green Fast qPCR Mix (Takara Bio Inc.) using a real-time PCR device Thermal Cycler Dice Real Time System III (manufactured by Takara Bio Inc.). It was performed by a 2-step real-time RT-PCR reaction. The primer used was manufactured by Takara Bio. The expression level of each gene mRNA was calculated by adjusting the expression level of GAPDH mRNA based on the total RNA standard prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the expression promotion rate of ocludin mRNA by adding the test sample was calculated by the following formula.

Occludin mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with sample addition B: Correction value without sample addition Table 5 shows the results of the occludin mRNA expression promotion rate.

As shown in Table 5, it was confirmed that corosolic acid (Sample 1) has an excellent occludin expression promoting action.

[Test Example 6] ZO-1 expression promoting action test Corosolic acid (Sample 1) was tested for ZO-1 expression promoting action as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM), and then the cells were recovered by tripsin treatment. Using KGM, 30 × 10 ⁴ cells / 2 mL were seeded on 6-well plates and cultured overnight at 37 ° C. under 5% CO ₂ . After culturing, the medium was replaced with a medium (KBM +) from which BPE and EGF were removed from the growth additive in KGM.

After 24 hours, the culture broth was discarded, 2 mL of a test sample dissolved in KBM + (Sample 1, see Table 6 for sample concentration) was added to each well, and the mixture was treated at 37 ° C. under 5% CO ₂ for 15 minutes. After the treatment, the cells were replaced with 2 mL of KBM + again and cultured for 4 hours and 8 hours. After culturing, total RNA was extracted with ISOGEN II (manufactured by NIPPON GENE), the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared so as to be 200 ng / μL.

Using this total RNA as a template, the expression level of mRNA was measured for ZO-1 and GAPDH, which is an internal standard. Detection is performed by PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc.) and TB Green Fast qPCR Mix (Takara Bio Inc.) using a real-time PCR device Thermal Cycler Dice Real Time System III (manufactured by Takara Bio Inc.). It was performed by a 2-step real-time RT-PCR reaction. The primer used was manufactured by Takara Bio. The expression level of each gene mRNA was calculated by adjusting the expression level of GAPDH mRNA based on the total RNA standard prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the expression promotion rate of ZO-1 mRNA by adding the test sample was calculated by the following formula.

ZO-1 mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with sample addition B: Correction value without sample addition Table 6 shows the results of the ZO-1 mRNA expression promotion rate.

As shown in Table 6, it was confirmed that corosolic acid (Sample 1) has an excellent ZO-1 expression promoting action.

[Test Example 7] ZO-2 expression promoting action test Corosolic acid (Sample 1) was tested for ZO-2 expression promoting action as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM), and then the cells were recovered by tripsin treatment. Using KGM, 30 × 10 ⁴ cells / 2 mL were seeded on 6-well plates and cultured overnight at 37 ° C. under 5% CO ₂ . After culturing, the medium was replaced with a medium (KBM +) from which BPE and EGF were removed from the growth additive in KGM.

After 24 hours, the culture broth was discarded, 2 mL of a test sample dissolved in KBM + (Sample 1, see Table 7 for sample concentration) was added to each well, and the mixture was treated at 37 ° C. under 5% CO ₂ for 15 minutes. After the treatment, the cells were replaced with 2 mL of KBM + again and cultured for 4 hours. After culturing, total RNA was extracted with ISOGEN II (manufactured by NIPPON GENE), the amount of RNA was measured with a spectrophotometer, and total RNA was prepared so as to be 200 ng / μL.

Using this total RNA as a template, the expression level of mRNA was measured for ZO-2 and GAPDH, which is an internal standard. Detection is performed by PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc.) and TB Green Fast qPCR Mix (Takara Bio Inc.) using a real-time PCR device Thermal Cycler Dice Real Time System III (manufactured by Takara Bio Inc.). It was performed by a 2-step real-time RT-PCR reaction. The primer used was manufactured by Takara Bio. The expression level of each gene mRNA was calculated by adjusting the expression level of GAPDH mRNA based on the total RNA standard prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the expression promotion rate of ZO-2 mRNA by adding the test sample was calculated by the following formula.

ZO-2 mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with sample addition B: Correction value without sample addition Table 7 shows the results of the ZO-2 mRNA expression promotion rate.

As shown in Table 7, it was confirmed that corosolic acid (Sample 1) has an excellent ZO-2 expression promoting action.

[Formulation Example 1]
A cream having the following composition was produced by a conventional method.
Corosolic acid 0.05g
Kujin extract 0.1g
Scutellaria extract 0.1g
Liquid paraffin 5.0g
Sarashi beeswax 4.0g
Squalene 10.0g
Cetanol 3.0g
Lanolin 2.0g
Stearic acid 1.0g
Polyoxyethylene sorbitan oleate (20E.O.) 1.5g
Glyceryl monostearate 3.0 g
Oil-soluble licorice extract 0.1g
1,3-butylene glycol 6.0 g
Methyl paraoxybenzoate 1.5g
Fragrance 0.1g
Purified water balance (total amount is 100 g)

[Formulation Example 2]
A milky lotion was produced by a conventional method according to the following composition.
Corosolic acid 0.01g
Jojoba oil 4.00g
1,3-butylene glycol 3.00g
Arbutin 3.00g
Polyoxyethylene cetyl ether (20E.O.) 2.50 g
Olive oil 2.00g
Squalan 2.00g
Cetanol 2.00g
Glyceryl monostearate 2.00 g
Polyoxyethylene sorbitan oleate (20E.O.) 2.00 g
Methyl paraoxybenzoate 0.15 g
Stearyl glycyrrhetinate 0.10 g
Yellow 杞 extract 0.10g
Dipotassium glycyrrhizinate 0.10 g
Ginkgo leaf extract 0.10g
Conchiolin 0.10g
Phellodendron extract 0.10g
Chamomile extract 0.10g
Fragrance 0.05g
Purified water balance (total amount is 100 g)

[Formulation Example 3]
A beauty essence having the following composition was produced by a conventional method.
Corosolic acid 0.1g
Chamomile extract 0.1g
Carrot extract 0.1g
Xanthan gum 0.3g
Hydroxyethyl cellulose 0.1g
Carboxyvinyl polymer 0.1g
1,3-butylene glycol 4.0 g
Dipotassium glycyrrhizinate 0.1g
Glycerin 2.0g
Potassium hydroxide 0.25g
Fragrance 0.01g
Preservative (methyl paraoxybenzoate) 0.15 g
Ethanol 2.0g
Purified water balance (total amount is 100 g)

[Formulation Example 4]
Tablets having the following composition were produced by a conventional method.
Corosolic acid 5.0 mg
Dolomite (containing 20% calcium and 10% magnesium) 83.4 mg
Casein phosphopeptide 16.7 mg
Vitamin C 33.4 mg
Maltitol 136.8 mg
Collagen 12.7 mg
Sucrose fatty acid ester 12.0 mg

[Formulation Example 5]
An oral liquid preparation having the following composition was produced by a conventional method.
<Composition in 1 ampoule (100 mL per ampoule)>
Corosolic acid 0.1% by mass
Sorbitol 12.0% by mass
Sodium benzoate 0.1% by mass
Fragrance 1.0% by mass
Calcium sulfate 0.5% by mass
Purified water balance (100% by mass)

The barrier function enhancer of the present invention enhances the barrier function in epithelial tissues (prevention, treatment or improvement of inflammatory intestinal diseases, food allergies, various infectious diseases transmitted from the digestive tract, etc .; dry skin, rough skin, atopic dermatitis). It can greatly contribute to the prevention, treatment or improvement of skin symptoms such as and various infectious diseases.

Claims (2)

A barrier function enhancer characterized by containing corosolic acid as an active ingredient. The corosolic acid is used for improving barrier function, suppressing barrier function decline, promoting claudin-1 expression, promoting claudin-4 expression, promoting occludin expression, promoting ZO-1 expression, and promoting ZO-2 expression. The barrier function enhancer according to claim 1, wherein the agent is used for one or more uses selected from the group consisting of.

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