JP2017075117A - Barrier function improver - Google Patents

Abstract

PROBLEM TO BE SOLVED: To find a compound having barrier function improvement action in natural product-derived compounds, and provide a barrier function improver comprising such a compound as an active ingredient.SOLUTION: A barrier function improver comprises astilbin as an active ingredient. Preferably, astilbin has one or more action 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.SELECTED DRAWING: Figure 1

Description

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

  One structure that separates the inside and the outside of a living body is epithelial tissue composed of epithelial cells. The epithelial tissue has a barrier function for controlling substance permeation, and thereby creates 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 adhesion structure that not only brings adjacent epithelial cells into close contact with each other, but also controls the permeation of substances by sealing 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 constitute the skeleton of TJ strands and have a TJ barrier function. It is thought that it controls (refer nonpatent literature 1).

  When the expression of claudin or occludin decreases for some reason, it causes a decrease in TJ function. For example, when the function of TJ decreases in the digestive tract, food allergens, pathogenic microorganisms and the like enter the body, which may contribute to inflammatory bowel diseases and various infectious diseases. Conventionally, it was thought that only the stratum corneum is responsible for the skin barrier function, but in recent years, when the TJ component protein present in the epidermal granule 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. decreases 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 infectious diseases.

  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 digestive tract, inflammatory bowel diseases, food allergies, various infectious diseases, etc. are prevented or improved. On the other hand, in the epidermis, it is considered that skin symptoms such as dry skin, rough skin, atopic dermatitis and various infectious diseases can be prevented or improved. Asparasine lineias extract (patent document 1) etc. are known as having a claudin production promoting action and an occludin production promoting action.

JP 2009-256244 A

Journal of the 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 a compound 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 astilbine as an active ingredient. The astilbine 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.

  ADVANTAGE OF THE INVENTION According to this invention, the barrier function improving agent excellent in the effect can be provided by containing astilbin which is a compound derived from a natural product as an active ingredient.

It is a graph showing the result of having evaluated the influence of astilbin with respect to mRNA expression of a TJ component factor by quantitative RT-PCR. It is a graph showing the result of having evaluated the influence of astilbin with respect to the protein expression of a TJ component factor by the immunoblot 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 the measurement of transepithelial electrical resistance.

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

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

  Astilbin can be produced by purification and isolation from a plant extract containing astilbin, or can be produced by synthesis.

  When producing astilbine by purification and isolation from a plant extract containing astilbine, such astilbine-containing plant extract can be obtained by a method generally used for plant extraction. Examples of plants containing astilbin include jaundice (scientific name: Engelhardtia chrysolepis Hance), hypericum (scientific names: Hypericum erectum, Hypericum perforatum) and the like.

  Twilight (Engelhardtia chrysolepis Hance) is an evergreen tree plant belonging to the genus Jatrobiaceae. Twilight has been drunk as a kind of sweet tea since ancient times and is a highly safe plant. Twilight is native to regions from southern China to Taiwan and is readily available from these regions. Examples of the constituent parts of jaundice that can be used as an extraction raw material include branches, leaves, branches and leaves, and the like, preferably leaves.

  Hypericum (Hypericum erectum, Hypericum perforatum) is a perennial herb belonging to the genus Hypericum, Hypericum, and is native to Honshu, Shikoku, Kyushu, etc., and can be easily obtained from these regions. Examples of the constituent part of hypericum that can be used as an extraction raw material include a leaf part, a stem part, a root part, a flower part, an above-ground part, or a mixture thereof, and the above-mentioned part is preferably an above-ground part or whole grass.

  The extract from the plant can be obtained by drying the extraction raw material, pulverizing it as it is or using a crusher, and subjecting it to extraction with an extraction solvent. Drying may be performed in the sun or using a commonly used dryer. Moreover, after performing pretreatment, such as degreasing, with a nonpolar solvent such as hexane, it may be used as an extraction raw material. By performing pretreatment such as degreasing, extraction with a polar solvent can be performed efficiently.

  As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These are used alone or in combination of two or more at room temperature or a temperature below 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 spring water, mineral water, hot spring water, spring water, fresh water, and the like, and those 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 this embodiment includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  Examples of hydrophilic organic solvents that can be used as extraction solvents 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 using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. 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), More preferably, it is 7: 3 to 2: 8 (capacity ratio). In addition, when a mixed liquid 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). When using the liquid mixture with a monohydric alcohol, it is preferable that the mixing ratio of water and a polyhydric alcohol is 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 5 to 15 times (mass ratio) of the extraction raw material, the soluble components are extracted at room temperature or under reflux, and then filtered to remove the extraction residue. A liquid can be obtained. When the solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and when this concentrate is further dried, a dried product is obtained.

  The method for purifying and isolating astilbine 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, or a porous resin such as styrene-divinylbenzene copolymer or polymethacrylate, and contains astilbine. Examples include a method for 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 etc. are mentioned. Further, the fraction containing astilbine obtained by column chromatography can be used for any organic phase such as reverse phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, column chromatography using ion exchange resin, etc. You may refine | purify using a compound refinement | purification means.

  On the other hand, in the case of producing astilbine by synthesis, examples of the method include the method by Suzuki et al. (Tetrahedron Lett., 41, 5537-5541 (2000)).

  Astilbine obtained as described above has an excellent barrier function improving action and can be used as an active ingredient of a barrier function improving agent. The barrier function improving agent of this embodiment can be used for a wide range of uses such as pharmaceuticals, quasi drugs, and cosmetics.

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

  When using a composition containing astilbine as an active ingredient of the barrier function improving agent according to the present embodiment, it is preferable to use a composition that has been purified to increase the purity of astilbine. A barrier function improver that is more excellent in action and effect can be obtained by using astilbine having an increased purity of astilbine as an active ingredient.

  The barrier function improving action of astilbin is, for example, 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 Based on. However, the barrier function improving action of astilbin is not limited to the barrier function improving action exhibited based on the above action.

  In addition, astilbine uses its tight junction function enhancing action, claudin-1 expression promoting action, occludin expression promoting action, or ZO-2 expression promoting action, respectively, and tight junction function enhancing agent and claudin-1 expression promoting action, respectively. You may use as an active ingredient of an agent, an occludin expression promoter, or a ZO-2 expression promoter.

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

  The barrier function-improving agent of this embodiment is an arbitrary pharmaceutically acceptable carrier such as dextrin and cyclodextrin, and any other auxiliary agent according to a conventional method, such as powder, granule, tablet, and liquid. It can be formulated into a dosage form. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring / flavoring agent, and the like can be used. The barrier function improving agent can be used by blending with other compositions (for example, skin cosmetics, etc.), and can also be used as an ointment, a liquid for external use, 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.

  In addition, the barrier function improving agent of this embodiment can mix | blend other natural extracts etc. which have a barrier function improvement effect | action with an astilbine as an active ingredient as needed.

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

  In addition, the dosage of the barrier function improving agent of the present embodiment may be appropriately increased or decreased depending on the type of disease, severity, individual differences among patients, 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, an claudin-1 expression promoting action, an occludin expression promoting action, and a ZO-2 expression promoting action possessed by the active ingredient astilbin. Through one or more actions, the barrier function in the gastrointestinal tract can be improved to prevent or improve inflammatory bowel disease, food allergies, various infectious diseases infecting from the gastrointestinal tract, and the like. However, the barrier function improving agent of the present embodiment is significant in that it exhibits a tight junction function enhancing action, a claudin-1 expression promoting action, an occludin expression promoting action, or a ZO-2 expression promoting action in addition to these uses. Can be used for all applications.

  Moreover, since the barrier function improving agent of this embodiment has an excellent barrier function improving action, it is suitable for blending into, for example, an oral pharmaceutical composition, food and drink, and the like. In this case, astilbine or an astilbine-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 a mucosal repair agent, an intestinal adjuster, a stomachic medicine, a gastrointestinal function promoter, a gastrointestinal function regulator and the like. In addition, in addition to these, the barrier function improving agent of the present embodiment is added to an oral pharmaceutical composition (for example, an anti-inflammatory agent, etc.) that has an effect other than barrier function improvement for the purpose of complementing the effect effect. You may mix | blend.

  The food and drink are not limited in its category, and include a wide range of foods that are taken orally, such as general foods, health foods (functional foods and drinks), health functional foods (food for specified health use, nutritional functional foods) and the like.

  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 as long as each effect is exhibited, animals other than humans (for example, mice, rats, hamsters, dogs). , Cats, cattle, pigs, monkeys, etc.).

  Hereinafter, although a manufacture example and a test example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all.

[Production Example] Production of Astilbin A 10-fold amount of 50% by volume ethanol was added to 100 g of dried yellow twig leaves, and the mixture was reflux extracted at 90 ° C. for 2 hours and filtered to obtain a filtrate. The obtained extract filtrate was concentrated under reduced pressure to obtain a yellow cocoon leaf part extract (20.8 g). Water was added to 20.0 g of the resulting yellow twig leaf extract, suspended, attached to a porous resin (Diaion HP-20, manufactured by Mitsubishi Chemical Corporation), in the order of 200 g of water, 200 g of 50% by volume methanol, and 200 g of methanol. Elute. Subsequently, the fraction eluted with 200 g of 50 volume% methanol was subjected to solvent distillation and lyophilization to obtain a 50% methanol elution fraction (11.3 g) of the yellow coconut leaf extract.

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

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

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

[Test Example 1] Measurement of transepithelial electrical resistance value The transepithelial electrical resistance (TER) value of the astilbine (sample 1) obtained in the production example was measured as follows.

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

About the obtained Caco-2 cell monolayer culture system, the value of transepithelial electrical resistance (TER) was measured using an electrical resistance measuring device (MILLIPORE, “ERS”). First, when TER was measured in a state where no test sample was added, 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 and the culture is continued. TER is measured 1, 3, 6, 12 and 24 hours after the addition, and the test sample is added. The ratio was calculated when the previous TER value was 100%. Quercetin was used as a positive control. The results are shown in Table 1.

  As shown in Table 1, astilbine (sample 1) increased the TER value in a monolayer culture system of Caco-2 cells, and thus was confirmed to have an excellent tight junction function enhancing action.

[Test Example 2] Evaluation of influence of tight junction constituent factor on mRNA expression Astyrubin (sample 1) obtained in the production example, the influence of tight junction (TJ) constituent factor on mRNA expression was evaluated as follows. .

  To the 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 incubated for 30 minutes. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Nippon Gene, Cat. No. 311-07361), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 ng / μL. Total RNA was prepared.

  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 performed using cDNA Synthesis Kit (manufactured by Takara Bio Inc.), and SYBR Green PCR Master Mix (manufactured by Takara Bio Inc.) using the obtained cDNA as a template and the primers shown in Table 2 below. Quantitative PCR was performed. Amplification was carried out under conditions of 40 cycles with a 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 component 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.

  As shown in FIG. 1, claudin-1 increased the mRNA expression level depending on the concentration of astilbine. Occludin, ZO-1 and ZO-2 showed increased mRNA expression levels when 50 μmol / L astilbine was treated. On the other hand, the expression level of claudin-4 did not change with or without astilbine.

[Test Example 3] Evaluation of influence of TJ constituent factor on protein expression Regarding astilbine (sample 1) obtained in the production example, the influence of TJ constituent factor on protein expression was evaluated as follows.

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

  The obtained cell lysate was developed on 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 using anti-clodin-1, anti-occludin and anti-ZO-2 antibodies derived from rabbits, and anti-ZO-1 antibodies derived from mice (all from Zymed Laboratories). A rabbit-derived anti-GAPDH antibody (purchased from Merck) was used as an internal standard. Next, by chemiluminescence method using anti-rabbit IgG-HRP (Jackson Immno Research) or anti-mouse IgG-HRP (Santa Cruz Biotechnology) and ECL Plus Western Blotting Detection Reagents (GE Healthcare), Bands were detected using an image capturing device (Bio-Rad, ChemiDoc XRS Plus), and quantitatively measured using Image J (NIH). The results are shown in FIG.

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

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

[Test Example 4] Effect of Astilbine on Damage of Gastrointestinal Barrier Function by Inflammatory Cytokines 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 the astilbine (sample 1) obtained in the production example on the damage to the gastrointestinal barrier function caused by inflammatory cytokines was examined.

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

  As shown in FIG. 3, the decrease in the TER value by TNF-α (FIG. 3A) and IL-6 (Same B) was recovered by simultaneous treatment with astilbin (sample 1). It became clear that astilbine restored functional damage.

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

Claims (2)

  A barrier function improving agent comprising astilbine as an active ingredient.   The astilbine 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. The barrier function improving agent according to claim 1.

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