JP6688585B2 - Barrier function improver - Google Patents

Description

  The present invention relates to a barrier function improving agent containing a compound derived from a natural product as an active ingredient.

  One of the structures that separate the inside and the outside of a living body is an epithelial tissue composed of epithelial cells. The epithelial tissue has a barrier function of controlling substance permeation, thereby creating an internal environment different from the external environment in the living body. Such a barrier function is mainly formed by adhesion between cells, and one of such adhesion is a tight junction (hereinafter sometimes referred to as “TJ”). TJ is an intercellular adhesive structure that controls the permeation of substances by not only making adjacent epithelial cells adhere to each other but also sealing the gap 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 the TJ strand and have a TJ barrier function. It is considered to control (see Non-Patent Document 1).

  When the expression of claudin or occludin is decreased for some reason, the function of TJ is lowered. For example, if the function of TJ is lowered in the digestive tract, food allergens, pathogenic microorganisms, etc. may invade the body, which may contribute to inflammatory bowel disease and various infectious diseases. Further, conventionally, it was considered that only the stratum corneum is responsible for the skin barrier function, but in recent years, when the TJ constituent protein present in the epidermal granular layer is deleted at the gene level, the skin barrier function 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 substance permeation barrier, 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 tissues is strengthened, and in the digestive tract, inflammatory bowel disease, food allergy, various infectious diseases, etc. are prevented or improved. On the other hand, it is considered that the epidermis can prevent or improve skin symptoms such as dry skin, rough skin, atopic dermatitis and various infectious diseases. Aspalas linealis extract (Patent Document 1) and the like are known as those having a claudin production promoting action and an occludin production promoting action.

JP, 2009-256244, A

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

  An object of the present invention is to find compounds having a barrier function improving action from compounds derived from natural products, and to provide a barrier function improving agent containing the compound as an active ingredient.

  The barrier function improving agent of the present invention is characterized by containing astilbin as an active ingredient. The astilbin preferably has one or more actions selected from the group consisting of a tight junction function enhancing action, a claudin-1 expression promoting action, an occludin expression promoting action, and a ZO-2 expression promoting action.

  According to the present invention, by containing astilbin, which is a compound derived from a natural product, as an active ingredient, it is possible to provide a barrier function improving agent having excellent action and effect.

It is a graph showing the result of having evaluated the influence of astilbin on the mRNA expression of a TJ constituent factor by quantitative RT-PCR. It is a graph showing the result of having evaluated the influence of astilbin on the protein expression of a TJ constituent factor by the immunoblotting method. It is a graph showing the result of having evaluated the effect of astilbin with respect to the damage of the digestive tract barrier function by an inflammatory cytokine by measuring transepithelial electrical resistance.

Hereinafter, embodiments of the present invention will be described.
The barrier function improving agent according to the present embodiment contains astilbin as an active ingredient.

  Astilbin, also called taxifolin 3-rhamnoside, is a dihydroflavonol glycoside having a chemical structure represented by the following formula.

  Astilbin can be produced by purifying and isolating from a plant extract containing astilbin, or can be produced synthetically.

  When astilbin is produced by purifying and isolating from an astilbin-containing plant extract, such an astilbin-containing plant extract can be obtained by a method generally used for plant extraction. Examples of plants containing astilbin include yellow tan (scientific name: Engelhardtia chrysolepis Hance), Hypericum (scientific name: Hypericum erectum, Hypericum perforatum) and the like.

  Yellow 杞 (Engelhardtia chrysolepis Hance) is an evergreen tree plant belonging to the genus Yellow genus of the walnut family. Yellow mud is a highly safe plant that has been used as a kind of sweet tea since ancient times. Huangshu grows naturally in regions from southern China to Taiwan and is readily available from these regions. Examples of the constituent parts of the yellow frost that can be used as the extraction raw material include a branch portion, a leaf portion, and a branch leaf portion, and the leaf portion is preferable.

  Hypericum erectum (Hypericum erectum, Hypericum perforatum) is a perennial herb belonging to the genus St. John's wort, which is native to Honshu, Shikoku, Kyushu, and can be easily obtained from these areas. Examples of the constituent parts of Hypericum vulgaris that can be used as an extraction raw material include leaves, stems, roots, flowers, above-ground parts, and mixtures thereof, but above-ground parts or whole grasses are preferable.

  The extract from the above plant 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 performed in the sun or using a commonly used dryer. Further, it may be used as an extraction raw material after being subjected to a pretreatment such as degreasing with a non-polar solvent such as hexane. By performing the pretreatment such as degreasing, the extraction treatment with the polar solvent can be efficiently performed.

  As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water, a hydrophilic organic solvent, and the like. These are used alone or in combination of two or more, and are used at room temperature or a temperature not higher than the boiling point of the solvent. It is preferable.

  Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those that have been subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering and the like. Therefore, the water that can be used as the extraction solvent in the present embodiment also 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 the extraction solvent, the mixing ratio can be adjusted appropriately. For example, when a mixed liquid of water and lower aliphatic alcohol is used as an extraction solvent, the mixing ratio of water and 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 liquid of water and lower aliphatic ketone is used, it is preferable that the mixing ratio of water and lower aliphatic ketone is 9: 1 to 2: 8 (volume ratio). When a mixed liquid with a polyhydric alcohol is used, the mixing ratio of water and polyhydric alcohol is preferably 5: 5 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 performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent in an amount of 5 to 15 times (mass ratio) of the extraction raw material, the soluble component is extracted at room temperature or under reflux heating, and then extraction is performed by removing the extraction residue. A liquid can be obtained. The solvent is distilled off from the obtained extract to obtain a paste-like concentrate, and this concentrate is further dried to obtain a dry product.

  The method for purifying and isolating astilbin from the extract obtained as described above, the concentrate of the extract or the dried product of the extract is not particularly limited and can be performed by a conventional method. . For example, the extract is dissolved in a developing solvent and subjected to column chromatography using a porous material such as silica gel or alumina, a porous resin such as styrene-divinylbenzene copolymer or polymethacrylate, and containing astilbin. Examples include a method of collecting fractions. 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 = 95: 5 and the like. Furthermore, the fraction containing astilbin obtained by column chromatography is subjected to any organic phase such as reverse-phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, column chromatography using an ion exchange resin, and the like. It may be purified using a compound purification means.

  On the other hand, when astilbin is produced by synthesis, for example, the method by Suzuki et al. (Tetrahedron Lett., 41, 5537-5541 (2000)) can be mentioned.

  The astilbin obtained as described above has an excellent barrier function improving action, and thus can be used as an active ingredient of a barrier function improving agent. The barrier function improving agent of the present embodiment can be used in a wide range of applications such as pharmaceuticals, quasi drugs, and cosmetics.

  In addition, as the active ingredient of the barrier function improving agent according to the present embodiment, a composition containing astilbin may be used instead of the purified and isolated astilbin. Here, the “composition containing astilbin” in the present embodiment includes an extract obtained using a plant containing astilbin as an extraction raw material. In addition, the “extract” includes an extract obtained by the extraction treatment, a diluted solution or a concentrated solution of the extract, or a dried product obtained by drying the extract.

  When a composition containing astilbin is used as the active ingredient of the barrier function improving agent according to the present embodiment, it is preferable to use a composition that is purified to increase the purity of astilbin. By using astilbin with high purity as an active ingredient, a barrier function improving agent having a further excellent action and effect can be obtained.

  The barrier function improving action of astilbin is, for example, one or more actions selected from the group consisting of tight junction function enhancing action, claudin-1 expression promoting action, occludin expression promoting action, and ZO-2 expression promoting action. Is demonstrated based on. However, the barrier function improving action of astilbin is not limited to the barrier function improving action exerted based on the above action.

  Astilbin utilizes its tight junction function-enhancing action, claudin-1 expression promoting action, occludin expression promoting action, or ZO-2 expression promoting action, astilbin enhances tight junction function enhancing agent and claudin-1 expression promoting action, respectively. Agent, occludin expression promoter, or ZO-2 expression promoter may be used as an active ingredient.

  The barrier function improving agent of the present embodiment may be composed of only astilbin or an astilbin-containing composition, or may be a formulation of astilbin.

  The barrier function improving agent of the present embodiment, using a pharmaceutically acceptable carrier such as dextrin, cyclodextrin or any other auxiliary, according to a conventional method, in the form of powder, granules, tablets, liquid, etc. It can be formulated into a dosage form. At this time, as the auxiliary agent, for example, an excipient, a binder, a disintegrating agent, a lubricant, a stabilizer, a flavoring agent, a flavoring agent and the like can be used. The barrier function improving agent can be used by being mixed with other compositions (for example, skin cosmetics and the like), and can also be used as an ointment, an external preparation, a patch and the like.

  When the barrier function-improving agent of the present embodiment is formulated, the content of astilbin is not particularly limited and can be appropriately set according to the purpose.

  The barrier function-improving agent of the present embodiment can be used as an active ingredient, if necessary, by blending another natural extract having a barrier function-improving effect together with astilbin.

  Examples of the method of administering the barrier function-improving agent of the present embodiment to a patient include oral administration and transdermal administration. Depending on the type of disease, a method suitable for prevention / treatment thereof may be appropriately selected. Since the barrier function improving agent of the present embodiment is particularly effective in improving the barrier function of the digestive tract, oral administration is preferable.

  Further, the dose of the barrier function improving agent of the present embodiment may be appropriately increased or decreased depending on the type of disease, severity, individual difference of patient, administration method, administration period and the like.

  The barrier function-improving agent of the present embodiment is selected from the group consisting of a tight junction function-enhancing action, a claudin-1 expression promoting action, an occludin expression promoting action, and a ZO-2 expression promoting action possessed by astilbin which is an active ingredient. Through one or more actions, it is possible to improve the barrier function in the digestive tract and prevent or improve inflammatory bowel disease, food allergy, various infectious diseases transmitted from the digestive tract, and the like. However, the barrier function improving agent of the present embodiment is significant in that it exerts a tight junction function enhancing action, claudin-1 expression promoting action, occludin expression promoting action, or ZO-2 expression promoting action in addition to these applications. It can be used for all applications with

  Further, the barrier function improving agent of the present embodiment has an excellent barrier function improving action, and therefore is suitable for incorporation into, for example, oral pharmaceutical compositions, foods and drinks and the like. In this case, astilbin or the astilbin-containing composition may be blended as it is, or a barrier function improving agent formulated from astilbin may be blended.

  Here, examples of the oral pharmaceutical composition include mucosal repair agents, intestinal agents, stomachic agents, gastrointestinal function promoters, gastrointestinal function regulators and the like. In addition to these, in addition to the barrier function improvement, the oral pharmaceutical composition that advocates an effect other than the barrier function improvement (for example, an anti-inflammatory agent), for the purpose of complementing the effect, the barrier function improving agent of the present embodiment. You may mix.

  There is no limitation on the category of food and drink, and a wide range of foods such as ordinary foods, health foods (functional foods and drinks), foods with health claims (foods for specified health use, foods with nutritional function), etc., are widely included.

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

  The barrier function-improving agent of the present embodiment is preferably applied to humans, but animals other than humans (for example, mice, rats, hamsters, dogs) can be used as long as the respective effects are exhibited. , Cats, cattle, pigs, monkeys, etc.).

  Hereinafter, the present invention will be specifically described with reference to production examples and test examples, but the present invention is not limited to the following examples.

[Production Example] Production of astilbin To 100 g of dried yellow and yellow leaflets, 10 volumes of 50% by volume of ethanol was added, and the mixture was subjected to reflux extraction at 90 ° C for 2 hours and filtered to obtain a filtrate. The obtained extract filtrate was concentrated under reduced pressure to obtain an extract of yellow-yellow eyelid (20.8 g). Water was added to 20.0 g of the obtained yellow-yellow-leaf extract and suspended, and the suspension was applied to a porous resin (Diaion HP-20, manufactured by Mitsubishi Chemical Corporation), and 200 g of water, 200 g of 50% by volume methanol, and 200 g of methanol in this order. It was eluted. Then, the solvent was distilled off from the fraction eluted with 200 g of 50% by volume methanol and freeze-drying was carried out to obtain a 50% methanol elution fraction (11.3 g) of the yellow-leaf extract.

  70% by volume of methanol was added to and suspended in 10.0 g of the obtained 50% methanol elution fraction, and the suspension was subjected to reverse phase chromatography using an ODS (Chromatorex ODS DM1020T, manufactured by Fuji Silysia Chemical Ltd.) column (mobile phase. 70% by volume methanol, 300 g), and the eluate was fractionated and desolvated to give a crudely purified fraction (dihydroflavonol fraction, 3.5 g). The obtained crudely purified fraction was further subjected to recycle HPLC under the following conditions to separate and purify to isolate a purified product (2.2 g, sample 1).

<High performance liquid chromatography conditions>
Equipment used: JAI LC-9201 (Nippon Analytical Industry Co., Ltd.)
Stationary phase: JAIGEL GS310 (Nippon Analytical Industry Co., Ltd.)
Column diameter: 20 mm
Column length: 250 mm
Mobile phase: Methanol Mobile phase flow rate: 0.8 mL / min

The specific rotation ([α] _D ) of the obtained purified product, infrared absorption spectrum (IR), and ¹ H- and ¹³ C-nuclear magnetic resonance spectrum (NMR) were measured, and the results were compared with known samples. It was confirmed that the purified product (Sample 1) was astilbin.

[Test Example 1] Measurement of transepithelial electrical resistance value The transepithelial electrical resistance (TER) value of the astilbin (Sample 1) obtained in the Production Example was measured as follows.

Human gastrointestinal epithelial cells (Caco-2) were treated with trypsin using a high glucose (4.5 g / L glucose) DMEM medium containing 10% FBS to recover the cells. The collected cells were diluted with 10% FBS-containing high glucose DMEM medium so as to have a cell density of 13.5 × 10 ⁴ cells / mL, and then plated in a 12-well Transwell insert (manufactured by Coster) at 250 μL per well. And cultured for 13 to 14 days to form a monolayer of Caco-2 cells. In the following test examples, Caco-2 cells thus monolayer-cultured were used.

The value of transepithelial electrical resistance (TER) of the obtained Caco-2 cell monolayer culture system was measured using an electrical resistance meter (MILLIPORE, "ERS"). First, when the TER was measured without adding the test sample, it was 800 to 1000 V_cm ² . Then, a test sample (Sample 1, see Table 1 below for sample concentration) is added to each well to continue the culture, and TER is measured 1, 3, 6, 12 and 24 hours after the addition, and the test sample is added. The ratio when the previous TER value was set to 100% was calculated. Quercetin was used as a positive control. The results are shown in Table 1.

  As shown in Table 1, since astilbin (Sample 1) increased the TER value in the Caco-2 cell monolayer culture system, it was confirmed that it has an excellent effect of enhancing tight junction function.

[Test Example 2] Evaluation of influence of tight junction constituent factor on mRNA expression The influence of tight junction (TJ) constituent factor on mRNA expression of the astilbin (Sample 1) obtained in Production Example was evaluated as follows. .

  To Caco-2 cells cultured in the same manner as in Test Example 1, a test sample (Sample 1, see Fig. 1 for sample concentration) was added to each well and cultured for 30 minutes. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Nippon Gene Co., Cat. No. 311-07361), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 ng / μL. The total RNA was prepared in.

  Using this total RNA as a template, the expression level of mRNA was measured for SPT and GAPDH as an internal standard. Specifically, reverse transcription was carried out using a cDNA Synthesis Kit (Takara Bio), and the obtained cDNA was used as a template and the primers shown in Table 2 below, and SYBR Green PCR Master Mix (Takara Bio). Quantitative PCR was performed by. Amplification was carried out under conditions of 40 cycles with denaturation treatment at 95 ° C. for 10 minutes, followed by 95 ° C. for 15 seconds and 60 ° C. for 1 minute, and a real-time PCR device Smart Cycler II (Cepheid) was used for detection.

  The mRNA expression level of the TJ constituent factor was quantified by the ΔΔCt method based on the total RNA preparation prepared from the cells cultured under each culture condition, and corrected with the β-actin value. The results are shown in Fig. 1.

  As shown in FIG. 1, claudin-1 increased the mRNA expression level depending on the concentration of astilbin. In addition, occludin, ZO-1 and ZO-2 had increased mRNA expression levels when treated with 50 μmol / L astilbin. On the other hand, the expression level of claudin-4 was not changed with or without astilbin.

[Test Example 3] Evaluation of influence of TJ constituent factor on protein expression The influence of TJ constituent factor on protein expression was evaluated for the astilbin (Sample 1) obtained in Production Example as follows.

  To Caco-2 cells cultured in the same manner as in Test Example 1, a test sample (Sample 1, see Fig. 2 for sample concentration) was added to each well and cultured for 3, 6 and 24 hours, respectively. After culturing, the medium was removed, the cells were washed with 0.5 mL of PBS (-) buffer, and then cells were lysed using 150 μL of M-PER (manufactured by PIERCE) to extract proteins.

  The resulting cell lysate was developed by SDS-PAGE (Bio-Rad, Mini-Protean 4-20%) and transferred to a PVDF membrane (Bio-Rad, Trans Blot Pack). The transferred PVDF membrane was blotted with rabbit anti-claudin-1, anti-occludin and anti-ZO-2 antibodies, and mouse anti-ZO-1 antibody (all manufactured by Zymed Laboratories). In addition, a rabbit-derived anti-GAPDH antibody (purchased from Merck) was used as an internal standard. Then, by chemiluminescence method using anti-rabbit IgG-HRP (manufactured by Jackson Immno Research) or anti-mouse IgG-HRP (manufactured by Santa Cruz Biotechnology) and ECL Plus Western Blotting Detection Reagents (manufactured by GE Healthcare), Bands were detected using an image capturing device (Bio-Rad, ChemiDoc XRS Plus), and quantitatively measured with Image J (NIH). The results are shown in Figure 2.

  As shown in FIG. 2, claudin-1 (FIG. 2A), occludin (same B), and ZO-2 (same C) showed an increase in the amount of protein by the treatment with astilbin.

  From the results of Test Examples 2 and 3 (FIGS. 1 and 2), it was confirmed that astilbin has an excellent claudin-1 expression promoting action, an occludin expression promoting action, and a ZO-2 expression promoting action.

[Test Example 4] Effect of astilbin on damage of digestive tract barrier function by inflammatory cytokine Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) It is known to damage the barrier function. Therefore, the influence of astilbin (Sample 1) obtained in Production Example on damage to the gastrointestinal barrier function due to inflammatory cytokines was examined.

A test sample (Sample 1, 50 μmol / L) and / or TNF-α (100 ng / mL) was added to each well of the Caco-2 cell monolayer culture system obtained in the same manner as in Test Example 1. The TER was measured 12 hours after the addition, and the TER was measured in the same manner as in Test Example 1.
IL-6 (100 ng / mL) was also tested in the same manner as TNF-α, and the TER was measured.
The results are shown in Fig. 3.

  As shown in FIG. 3, the decrease in the TER value due to TNF-α (FIG. 3A) and IL-6 (the same B) was recovered by the simultaneous treatment with astilbin (Sample 1). Astilbin was found to recover functional damage.

  The barrier function improving agent of the present invention can greatly contribute to the improvement of the barrier function in the digestive tract (prevention or improvement of inflammatory bowel disease, food allergy, various infectious diseases transmitted from the digestive tract, etc.).

Claims (2)

Barrier function improving agent characterized by the astilbin an active ingredient (except barrier function improving agent containing as an active ingredient an extract of Hypericum perforatum or yellow杞葉containing astilbin). It is used for one or more applications selected from the group consisting of a tight junction function enhancing application, a claudin-1 expression promoting application, an occludin expression promoting application, and a ZO-2 expression promoting application. The barrier function improving agent according to (excluding the barrier function improving agent containing, as an active ingredient, an extract of St. John's wort or Aspergillus ores containing astilbin) .

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