HK1178433B - Epithelial cell-cell adhesion enhancer, and ameliorating, therapeutic or prophylactic agent for allergic diseases using same - Google Patents

Description

Epithelial cell-cell adhesion enhancer and ameliorating, therapeutic or prophylactic agent for allergy using same

Technical Field

The present invention relates to an epithelial cell-cell adhesion enhancer and an agent for ameliorating, treating or preventing allergy using the enhancer. In particular, it relates to an epithelial cell-cell adhesion enhancer which can enhance the intercellular adhesion of epithelial cells (epithelial cell-cell adhesion) which is one of the barrier functions against allergens, and can improve, treat and prevent allergy, and an agent for improving, treating or preventing allergy using the enhancer.

Background

Allergies are caused by an abnormal reaction of the immune system to foreign bodies. The organs of the human body that come into contact with foreign bodies include the epidermis, respiratory organs, and digestive organs. Among them, especially in the digestive organs, since they have a unique immune system and are immune organs affecting the systemic immune system, attention has been paid in recent years.

At present, as a first option of allergy treatment, a synthetic steroid oral drug having an effect of inhibiting the expression of inflammatory cytokines produced by the binding of an allergen and a nuclear glucocorticoid receptor is used, but there are reported side effects of inducing an infectious disease, causing an arteriosclerotic lesion, pararenal insufficiency, digestive tract disorders, female menstrual disorder, and the like. In the case where severe side effects are observed, it is considered to use other treatment methods or to use a combination therapy.

In recent years, there have been many reports that a certain therapeutic effect can be obtained not only by synthetic steroid oral drugs but also by combining a method of improving the balance of cells responsible for immunity in the intestinal tract and activating immune cells.

Conventionally, a method for improving the balance of intestinal bacterial flora has been mainly used as an allergy prevention method, based on the fact that allergy-causing bacteria in the intestine of children are fewer than those in healthy children.

A method based on the idea of improving or preventing allergy by adjusting the balance of the intestinal bacterial flora is described in japanese patent application laid-open No. 8-157379 (patent document 1) as a method of using an allergy preventive agent containing fructo-oligosaccharide. Japanese patent application laid-open No. 2003-201239 (patent document 2) describes an immunoactivated food containing fructooligosaccharide. Further, methods for suppressing allergy are described in japanese patent No. 4162147 (patent document 3) and japanese patent application laid-open No. 2008-280354 (patent document 4).

Patent document 1 describes a method of taking a composition containing a magnesium source, fructooligosaccharide, and an indigestible sugar alcohol against allergy induced by magnesium deficiency. The method can prevent allergy induction by producing organic acids from fructooligosaccharides by intestinal bacteria and promoting magnesium absorption by the action of the organic acids and indigestible sugar alcohols, and can treat allergy induced by magnesium deficiency, but the method cannot treat allergy caused by other reasons and limits the treatment target. Patent document 2 describes a method of activating intestinal immunity by inducing production of IgA in Peyer's patch (Peyer's patch) through intake of fructooligosaccharide. However, the effective amount of this method is large and it is difficult to take it every day.

Patent document 3 describes an allergy inhibitor for the composition 1 in which 1-kestose in fructooligosaccharide is the maximum composition ratio; patent document 4 describes an allergy-inhibiting oligosaccharide containing a composition 1 having 1-kestose as the maximum composition ratio among fructooligosaccharides. These allergy inhibitors and allergy-inhibiting oligosaccharides have a higher allergy-inhibiting effect than the effect of the allergy symptoms described in patent documents 1 to 2. However, it is also described in Clinical effects of ketose, allergic oligomeric oligosaccharides, on the treatment of atopicimpairment in infants (non-patent document 1) that the improvement of allergic symptoms does not strongly correlate with the proliferation of bifidobacteria as a result of the test with 1-kestose intake by young infants.

In recent years, it has been found that cysteine/serine protease activity commonly possessed by substances that become allergens can decompose tight junction proteins between epithelial cells and further reduce adhesion between epithelial cells (non-patent documents 2 and 3). Therefore, a method based on the following concept was attempted: the method for improving, treating or preventing allergy comprises repairing and promoting the formation of tight junction protein between epithelial cells to enhance adhesion between epithelial cells and inhibit the invasion of allergen into body.

To date, many studies have been conducted on models using intestinal epithelial cells to search for substances that enhance adhesion between epithelial cells. Examples of the nucleic acid include nucleic acids derived from fish sperm and yeast (patent document 5), peptides derived from cellulase preparations (patent document 6), monosialoganglioside 3 (patent document 7), lipid membrane acids derived from lactic acid bacteria (patent document 8), and whey proteins (patent document 9). These substances have been confirmed to have an effect of suppressing allergy by promoting the formation of tight junction protein between epithelial cells.

However, the production steps of the substances described in patent documents 5 to 8 are complicated, and it is difficult to mass-produce them industrially. Further, although the substance described in patent document 9 is easy to produce, it is necessary to ingest a large amount to achieve a therapeutic effect, and it is difficult to ingest daily. For the above reasons, a preparation for ameliorating, treating or preventing allergy by the above substances has not been realized.

Documents of the prior art

Patent document

Patent document 1: japanese laid-open patent publication No. 8-157379

Patent document 2: japanese patent laid-open publication No. 2003-201239

Patent document 3: japanese patent No. 4162147

Patent document 4: japanese patent laid-open No. 2008-280354

Patent document 5: japanese patent No. 4050799

Patent document 6: japanese laid-open patent publication No. 2002-275196

Patent document 7: japanese patent No. 4034364

Patent document 8: japanese patent laid-open No. 2008-212006

Patent document 9: japanese patent No. 4330088

Non-patent document

Non-patent document 1: shibata R, et al, clin. exp. allergy,2009 Sep,39(9), P1397-1403

Non-patent document 2: wan H.et.al., J.Clin.invest.1999,104, P123-133

Non-patent document 3: runwick S, et.al., j.allergy clin.immnol.,2003, 111, P704-713

Summary of the invention

Problems to be solved by the invention

The present invention is based on the idea of improving, treating or preventing allergy by enhancing the adhesion between epithelial cells and inhibiting the invasion of allergens into the body. The present inventors have found that a composition of a metal cation having a valence of 2 and a specific oligosaccharide can further enhance adhesion between epithelial cells as compared with the metal cation having a valence of 2, and have completed the following invention. That is, the object of the present invention is to provide a composition containing a specific oligosaccharide and a metal cation having a valence of 2, which is generally known as food and the metal cation having a valence of 2 is present in a living body and is not toxic to living bodies in a physiological concentration range, but is very safe and effective in ameliorating, treating or preventing allergy or in treating a local inflammatory reaction as compared with a synthetic steroid oral drug.

Means for solving the problems

(1) An intercellular adhesion enhancer for epithelial cells, which comprises 1-kestose and/or nystose and a metal cation having a valence of 2 as active ingredients.

(2) The enhancer according to (1), which comprises 1-kestose and a metal cation having a valence of 2 as active ingredients.

(3) The enhancer according to (2), wherein the 1-kestose is an oligosaccharide containing 1-kestose having a purity of 95% by weight or more, and the metal cation having a valence of 2 is 1 part by weight or more based on 10 parts by weight of the oligosaccharide containing 1-kestose.

(4) The enhancer according to any one of (1) to (3), wherein the 2-valent metal cation is a calcium ion.

(5) The enhancer of any one of (1) to (4), wherein the epithelial cells are intestinal epithelial cells.

(6) An ameliorating, therapeutic or prophylactic agent for allergy, which uses the enhancing agent according to any one of (1) to (5).

ADVANTAGEOUS EFFECTS OF INVENTION

By using the enhancer for enhancing adhesion between epithelial cells and the agent for improving, treating or preventing allergy using the enhancer of the present invention, it is possible to inhibit the destruction of the tight junction protein between epithelial cells, which is a cause of the invasion of an allergen into the body, and it is also possible to enhance adhesion between epithelial cells by repairing the tight junction protein between epithelial cells or promoting the formation thereof, thereby effectively improving, treating or preventing allergic symptoms.

Drawings

FIG. 1 shows the results of measuring the change in TEER with time by adding 1-kestose or nystose in example 1.

FIG. 2 shows the results of measuring the change in TEER with time by adding 1-kestose in example 2.

FIG. 3 shows the results of measuring the change in TEER with time by adding 1-kestose in example 3.

FIG. 4 shows the results of measuring the change in TEER with time by adding 1-kestose in example 4.

FIG. 5 shows the results of measuring the change in TEER with time by adding 1-kestose in example 4.

FIG. 6 shows the results of measurement of the change in the amount of substance permeation by the addition of 1-kestose, sucrose or nystose in example 5.

FIG. 7 shows the results of measuring the change in TEER with time by adding 1-kestose in example 6.

Detailed Description

The following is a detailed description of the epithelial intercellular adhesion enhancer of the present invention and an agent for ameliorating, treating or preventing allergy using the enhancer. The epithelial intercellular adhesion enhancer of the present invention is an intercellular adhesion enhancer located in epithelial cells, and the effective components thereof are 1-kestose and/or nystose and 2-valent metal cations, or 1-kestose and 2-valent metal cations.

From the above, it is known that cysteine/serine protease activity commonly possessed by substances that become allergens degrades tight junction proteins between epithelial cells, thereby reducing adhesion between epithelial cells (non-patent documents 2 and 3). The adhesion enhancer between epithelial cells of the present invention can repair or promote the formation of tight junction protein between epithelial cells, and as a result, enhance the adhesion energy between epithelial cells, and can suppress the invasion of allergens into the body.

In the present invention, examples of the epithelial cells include absorptive epithelial cells, keratinized epithelial cells, wet-barrier epithelial cells (wet-barrier epithelial cells), coated epithelial cells (lining epithelial cells), exocrine epithelial cells, endocrine epithelial cells, extracellular matrix-secreting epithelial cells, and contractile epithelial cells. Specifically, examples of the cells include intestinal epithelial cells such as small intestinal epithelial cells, large intestinal epithelial cells, and duodenal epithelial cells, gastric mucosal epithelial cells, esophageal epithelial cells, corneal epithelial cells, conjunctival epithelial cells, amniotic epithelial cells, skin epithelial cells, and palatal epithelial cells.

The 2-valent metal cation may be any 2-valent metal cation that exists in a living body and is not toxic to the living body in a physiological concentration range, and examples thereof include 2-valent ions such as calcium ion, magnesium ion, barium ion, iron ion, copper ion, and zinc ion. Calcium ions are used in this example as suitable metal cations of valence 2.

Previous studies have shown that intercellular adhesion is plastic by the so-called calcium switch action that carries out the extracellular detachment and addition of calcium ions (Sarah l.d.et.al, b.b.a.,2008,1778, P2318-2324; geogena c.et.al, j. membrane Biol. (J. Membrane Biol., 2010,237, P115-123). Recent studies have shown that 2-valent metal cations other than calcium ions, such as magnesium ions and zinc ions, have a positive effect on the formation of intercellular adhesion in a physiological concentration range (Sarah L.D.et. al., B.B.A.,2008,1778, P2318-2324; Geoga C.et. al., J.Membrane Biol. ("J.Membrane Biol., 2010,237, P115-123), suggesting that there are cellular mechanisms of 2-valent metal ions that exert the same action as calcium ions as their common cellular mechanisms. Therefore, 1-kestose and/or nystose and calcium ion, or 1-kestose and calcium ion described in the present invention can promote the formation of intercellular adhesion, and 1-kestose and/or nystose and 2-valent metal cation, or 1-kestose and 2-valent metal cation can promote the formation of intercellular adhesion.

The oligosaccharide which can be used in the present invention may be, for example, fructooligosaccharides other than sucrose, such as 1-kestose, nystose, fructosyl nystose (fructosyl-nystose), and the like. In this example, 1-kestose and nystose were used as suitable oligosaccharides.

1-kestose is a fructotriose composed of 1 molecule of glucose residues and 2 molecules of fructose residues. A preferred form of 1-kestose that can be used in the present invention is, for example, an oligosaccharide containing 1-kestose, which is prepared by subjecting sucrose as a raw material to an enzymatic reaction, removing monosaccharides and sucrose by chromatography to improve the purity, and then crystallizing the resulting 1-kestose to obtain 1-kestose having a purity of 95 wt% or more. Such oligosaccharides containing 1-kestose are low energy substances with readily soluble, indigestible properties. Further, they have an IgA production-enhancing action and an IgE production-inhibiting action (patent documents 3 and 4).

The preparation method of the above-mentioned enzyme reaction using sucrose as a raw material is described in Japanese patent application laid-open No. 58-201980, the method of removing monosaccharides by chromatography to improve the purity of sucrose is described in Japanese patent application laid-open No. 11-34787 (Japanese patent application laid-open No. 2000-232878), and the crystallization method is described in Japanese patent application laid-open No. 2-224312 (Japanese patent application laid-open No. 4-235192), and these described methods can be used respectively. The contents of Japanese patent application No. Hei 11-34787, Japanese patent application No. Hei 2-224312, Japanese application No. 2005-371005 (cited reference 3) and Japanese application No. 2008-166463 (cited reference 4) are included in the present specification.

The daily intake of 1-kestose in the present invention is preferably 0.015g/kg body weight or more, and in the case of infants, the daily intake of 1-kestose is preferably about 2.5g in terms of 1 part by weight of 2-valent metal ions per 10 parts by weight of 1-kestose.

In addition, the adhesion between epithelial cells in the present invention can be achieved by repairing the epithelial cell-to-cell tight junction protein or promoting the formation of the epithelial cell-to-cell tight junction protein.

The term "enhancing" as used herein may be replaced with "activating", "promoting", "strengthening", "enhancing", or the like.

Examples of the allergy include atopic reaction, allergic rhinitis, allergic conjunctivitis, allergic enterogastritis, bronchial asthma, infantile asthma, urticaria, animal allergy, food allergy, metal allergy, drug allergy, and allergy. In the present invention, it is preferable to target atopic reaction, allergic rhinitis, allergic conjunctivitis, and food allergy.

The method for preparing the epithelial cell-to-cell adhesion enhancer and the allergy ameliorating, treating or preventing agent using the enhancer of the present invention may be any method known to those skilled in the art. The administration form may be appropriately selected and used according to the requirements of those skilled in the art, and examples of such administration forms include forms such as tablets, granules, powders, capsules, coating agents (coating agents), liquids, and suspensions for preparing oral preparations, and forms such as inhalants, injections, drops, suppositories, coatings, sprays, and patches for preparing non-oral preparations; preferably, the application is to the itchy or eruptive areas of the skin, or the administration is to the mucous membranes of the nose or eyes. The dose can be appropriately set according to the form of the pharmaceutical composition, the method of administration, the purpose of use, and the age, body weight, and symptoms of the subject to which the pharmaceutical composition is to be administered.

Hereinafter, the adhesion enhancer between epithelial cells and the agent for improving, treating or preventing allergy using the enhancer according to the present invention will be described based on examples. The technical scope of the present invention is not limited to the features shown in these embodiments.

Examples

"example 1: test for confirming that 1-kestose has an effect of promoting recovery from decreased adhesion between epithelial cells due to extracellular calcium deficiency "

In example 1, it was confirmed that 1-kestose has an effect of promoting recovery from decrease in adhesion between epithelial cells due to extracellular calcium deficiency by an experiment using cultured cells.

The oligosaccharide containing 1-kestose used in example 1 had a composition of oligosaccharide containing 98 wt% of 1-kestose, 1 wt% of nystose and 1 wt% of sucrose, that is, oligosaccharide containing 98 wt% of 1-kestose. Furthermore, epithelial intercellular resistance (TEER) was used as an index of intercellular adhesion strength of cultured cells. This measurement method utilizes the principle that the stronger the adhesion between epithelial cells, the greater the resistance value of the upper medium and the lower medium in which the cells are cultured, and is also used in experiments using epithelial cells as a model, and the like.

Colonic epithelial cancer cells, Caco-2 cells, were serially subcultured in DMEM medium (GIBCO) containing 10% FBS, and the 48 th generation cultured cells were cryopreserved for use in the next experiment. Caco-2 cells were seeded on the upper layer of a Transwell double-layer cell culture plate (Corning Life Science) and placed in 5% CO in the presence of DMEM medium containing 10% FBS₂And culturing at 37 deg.C. Performing cell culture until the resistance (TEER) between the upper layer and the lower layer of the Transwell double-layer cell culture plate reaches 400-500 omega cm²。

The DMEM medium containing 10% FBS is replaced by the DMEM medium lacking calcium, and the TEER cultured to the Transwell double-layer cell culture plate reaches 400-500 omega cm²Latter Caco-2 cellsThe culture was carried out for 2 hours. Thereafter, 1-kestose-containing oligosaccharide or nystose was added to the upper layer of the DMEM medium containing calcium at a concentration of 1 wt%, TEER was measured every 30 minutes, and TEER was measured up to 22 hours after the culture (1% kestose group, 1% nystose group, n = 3). As control groups, a group cultured only in a calcium-deficient DMEM medium (negative control group, n = 3) and a group cultured only in a calcium-deficient DMEM medium after 2 hours of culture (positive control group, n = 3) in a calcium-containing DMEM medium were prepared, and the temporal change of TEER was compared.

Hereinafter, fig. 1 shows the measurement results of the TEER ratio in example 1. The recovery of TEER was hardly seen in the negative control group, but significant TEER recovery was seen in the 1% kestose group, the 1% nystose group and the positive control group, compared with the negative control group. Meanwhile, compared with the positive control group, the 1% kestose group and the 1% nystose group also have obvious TEER recovery promoting effects. Specifically, in the 1% kestose group after 30 minutes from the replacement of the calcium-deficient DMEM medium with the calcium-containing DMEM medium, the recovery promoting effect was found to be 2.4 times on average as compared with the positive control group. In addition, the 1% kestose group showed a stronger tendency of recovery promotion than the 1% kestose group. From this result, it was confirmed that 1-kestose has a strong recovery promoting effect on decreased adhesion between epithelial cells.

"example 2: confirmation that 1-kestose has an effect of promoting recovery from decreased adhesion between epithelial cells due to extracellular calcium deficiency "

In example 2, it was confirmed that 1-kestose is calcium-dependent in its effect of promoting recovery from decreased adhesion between epithelial cells due to extracellular calcium deficiency, as measured by experiments using cultured cells.

The purity of the oligosaccharide containing 1-kestose used was the same as in example 1, and the culture was carried out under the same cell culture conditions as in example 1.

The DMEM medium containing 10% FBS is replaced by the DMEM medium lacking calcium, and the TEER cultured to the Transwell double-layer cell culture plate reaches 400-500 omega cm²The latter Caco-2 cells were cultured for 2 hours. Then, 1-kestose-containing oligosaccharide was added to the upper layer of the DMEM medium containing calcium at a concentration of 1 wt%, TEER measurement was performed every 30 minutes, and culture was performed until 3 hours later (calcium (+) 1% kestose group, n = 3) to measure TEER. Further, the following group was made: directly on the upper layer of the calcium-deficient medium, 1 wt.% oligosaccharide containing 1-kestose was added, cultured and TEER was measured until 3 hours later (calcium (-) 1% kestose group, n = 3). As the control group, a group cultured only in a calcium-deficient DMEM medium (negative control group, n = 3) and a group cultured only in a calcium-deficient DMEM medium for 2 hours and then replaced with a calcium-containing DMEM medium (positive control group, n = 3) were prepared, and the temporal change of TEER was compared.

Hereinafter, fig. 2 shows the measurement results of the TEER ratio in example 2. Recovery of TEER was hardly seen in the negative control group and the calcium (-) 1% kestose group. However, as in example 1, significant TEER recovery was seen for both the calcium (+) 1% kestose group and the positive control group; in the calcium (+) 1% kestose group, a significant TEER recovery-promoting effect was also seen in the same manner as in example 1, as compared with the positive control group. From this, it was confirmed that 1-kestose does not exhibit the TEER recovery effect if no extracellular calcium is present, and that the recovery promoting effect of 1-kestose on reduced adhesion between epithelial cells depends on extracellular calcium.

"example 3: concentration-dependent assay to confirm that 1-kestose has a restorative effect on decreased adhesion between epithelial cells due to extracellular calcium deficiency "

In example 3, it was confirmed that 1-kestose has a concentration-dependent effect on recovery promotion of decreased adhesion between epithelial cells due to extracellular calcium deficiency, as measured by experiments using cultured cells.

The purity of the oligosaccharide containing 1-kestose used was the same as in example 1, and the culture was carried out under the same cell culture conditions as in example 1.

The DMEM medium containing 10% FBS is replaced by the DMEM medium lacking calcium, and the TEER cultured to the Transwell double-layer cell culture plate reaches 400-500 omega cm²The latter Caco-2 cells were cultured for 2 hours. Thereafter, 1-kestose-containing oligosaccharide was added to the upper layer of the DMEM medium containing calcium at a concentration of 1 wt% or 0.1 wt%, TEER measurement was performed every 30 minutes, and culture was performed until 3.5 hours later (1% kestose group, 0.1% kestose group, n = 3) to perform TEER measurement. As control groups, a group cultured only in a calcium-deficient DMEM medium (negative control group, n = 3) and a group cultured only in a calcium-deficient DMEM medium for 2 hours (positive control group, n = 3) in which the culture was changed to a calcium-containing DMEM medium were prepared, and the temporal change of TEER was compared.

Hereinafter, fig. 3 shows the measurement results of the TEER ratio in example 3. As in example 1, little recovery of TEER was seen in the negative control group. However, significant TEER recovery was seen in the 1% kestose group, the 0.1% kestose group and the positive control group, compared to the negative control group. In addition, the 1% kestose group showed a stronger tendency of TEER recovery promoting effect than the 0.1% kestose group. From the above, it was confirmed that the recovery promoting effect of 1-kestose on reduced adhesion between epithelial cells is concentration-dependent.

"example 4: test for investigating the inhibitory Effect of 1-kestose administration in advance on the decrease in adhesion between epithelial cells due to extracellular calcium deficiency "

In example 4, it was confirmed whether the decrease in adhesion between epithelial cells due to extracellular calcium deficiency can be suppressed by the test using cultured cells by administering 1-kestose in advance.

The purity of the oligosaccharide containing 1-kestose used was the same as in example 1, and the culture was carried out under the same cell culture conditions as in example 1.

TEER in a double-layer cell culture plate cultured in a Transwell reaches 400-500 omega cm²Then, the Caco-2 cells were subjected to TEER measurement every 30 minutes by changing the DMEM medium containing 10% FBS to a DMEM medium containing calcium, adding 1-kestose-containing oligosaccharide to the upper layer at a concentration of 1 wt%, changing the medium to a medium lacking calcium after 1 hour or 6 hours, and measuring 3 cycles (1 hour calcium (+) 1% kestose group, 6 hour calcium (+) 1% kestose group, n = 3). As a control group, a group cultured on a DMEM medium lacking calcium (1 hour calcium (+) negative control group, 6 hour calcium (+) negative control group, n = 3) after 1 hour or 6 hours of culture on a DMEM medium containing calcium was prepared, and a group cultured only on a DMEM medium containing calcium (positive control group, n = 3) was prepared, and the change with time of TEER was compared.

Hereinafter, the measurement results of the TEER ratio in example 4 are shown in fig. 4 and 5. FIG. 4 shows the results of comparison of the 1-hour calcium (+) 1% kestose group, the 1-hour calcium (+) negative control group and the positive control group. It was confirmed that in the TEER assay immediately before the 1-hour calcium (+) 1% kestose group was changed to the calcium-deficient DEME medium, TEER was significantly increased as compared with the 1-hour calcium (+) negative control group and the positive control group. In addition, it was confirmed that the decrease in TEER in the 1-hour calcium (+) 1% kestose group was suppressed in the state where 1-kestose was removed by replacement with the calcium-deficient DMEM medium, as compared with the 1-hour calcium (+) negative control group.

Next, fig. 5 shows the results of comparison between the 6-hour calcium (+) 1% kestose group, the 6-hour calcium (+) negative control group, and the positive control group. As in fig. 4, it was confirmed that the TEER was significantly increased in the TEER assay immediately before the 6-hour calcium (+) 1% kestose group was changed to the calcium deficient DEME medium, as compared with the 6-hour calcium (+) negative control group and the positive control group. Similarly, it was confirmed that the decrease in TEER in the 6-hour calcium (+) 1% kestose group was suppressed in the state where the oligosaccharide containing 1-kestose was removed by changing to the calcium-deficient DMEM medium, as compared with the 6-hour calcium (+) negative control group.

From the above results, it was confirmed that 1-kestose administered in advance can prevent the decrease in adhesion between epithelial cells, and that 1-kestose can play a role in promoting adhesion between epithelial cells in the presence of the same.

"example 5: test for confirming the Effect of inhibiting the permeation of a substance that counteracts the enhancement of cell Membrane permeability due to extracellular calcium deficiency "

In example 5, the effect of 1-kestose cells in inhibiting the permeation of substances that resist the increase in cell membrane permeability due to extracellular calcium deficiency was confirmed by an experiment using cultured cells including comparison with other fructooligosaccharides.

The purity of the oligosaccharide containing 1-kestose used was the same as in example 1, and the culture was carried out under the same cell culture conditions as in example 1.

The DMEM medium containing 10% FBS was replaced with calcium-deficient colorless HBSS medium (GIBCO), and TEER cultured on a Transwell double-layer cell culture plate was 400 to 500. omega. cm²After 2 hours of the subsequent Caco-2 cell culture, 1-kestose-containing oligosaccharide, sucrose, or nystose was added to the upper layer of the calcium-deficient colorless HBSS medium (GIBCO) at a concentration of 1 wt%, while fluorescein (Lucifer Yellow) as an intercellular permeation substance was added to the upper layer at a concentration of 100 μ M (MP biomedicalal), and after 3 hours of the culture, the HBSS medium in the lower layer was collected and the residual concentration of the fluorescein (Lucifer Yellow) permeated from the upper layer to the lower layer was measured by a fluorometric microplate reader (1% kestose group, 1% sucrose group, 1% nystose group, n = 3). As control groups, a group cultured only in the calcium-deficient HBSS medium (negative control group, n = 3) and a group cultured only in the calcium-deficient HBSS medium for 2 hours and then replaced with the calcium-deficient HBSS medium (positive control group, n = 3) were prepared, and the group was further combinedComparison of intercellular permeate quality.

FIG. 6 shows the results of measuring the residual concentration of fluorescing Yellow (Lucifer Yellow) in the lower layer in example 5. The residual concentrations in the 1% kestose group, the 1% nystose group and the positive control group were significantly lower than those in the negative control group. In addition, the 1% kestose group and the 1% nystose group showed less residual concentration compared to the positive control group, with the residual concentration of the 1% kestose group being the lowest.

From the above results, it was found that 1-kestose can inhibit the permeability of proteins and the like which become allergens by promoting adhesion between epithelial cells.

"example 6: test for confirming inhibitory Effect on decrease in adhesion between epithelial cells caused by IL-1. beta. inflammatory cytokine "

In example 6, it was confirmed that 1-kestose has an effect of suppressing decrease in adhesion between epithelial cells caused by inflammatory cytokines by an experiment using cultured cells.

The purity of the oligosaccharide containing 1-kestose used was the same as in example 1, and the culture was carried out under the same cell culture conditions as in example 1.

Adding 1 wt% oligosaccharide containing 1-kestose to the upper layer of DMEM medium to obtain TEER of 400-500 Ω -cm cultured on Transwell double-layer cell culture plate²After 24 hours of culture of the latter Caco-2 cells, the inflammatory cytokine IL-1. beta. was added to the lower layer at a concentration of 10ng/mL, and TEER measurement was performed 48 hours later, and the ratio to the value measured at the beginning of TEER (1% kestose (+), IL-1. beta. +), n = 3) was calculated. As control groups, groups to which only IL-1. beta. was added (1% kestose (-), IL-1. beta. (+), and n = 3) and groups to which no oligosaccharide containing 1-kestose and IL-1. beta. were added (1% kestose (-), IL-1. beta. (-), and n = 3) were prepared, and TEER ratios were compared.

Hereinafter, the measurement results of the TEER ratio in example 6 are shown in fig. 7. Compared with the mean value of the TEER ratios of the 1% kestose (-), IL-1. beta. (-), 0.914 standard deviation 0.031, the mean value of the TEER ratios of the 1% kestose (-), IL-1. beta. (-), 0.585 standard deviation 0.002, the mean value of the TEER ratios of the 1% kestose (+), IL-1. beta. (+), 0.849 standard deviation 0.0003. From this result, it can be concluded that TEER reduction by IL-1. beta. can be suppressed by about 26% by adding oligosaccharide containing 1-kestose. From the above results, it was found that 1-kestose has an effect of suppressing the decrease in adhesion between epithelial cells caused by local inflammatory reactions.

Possibility for use in industry

The present invention can contribute to the improvement, treatment and prevention of allergy by repairing or promoting the formation of tight junction protein between epithelial cells, which is a cause of allergy.

Claims (2)

1. Use of a composition comprising 1-kestose and calcium ions as active ingredients for the preparation of an agent for ameliorating, treating or preventing allergy, wherein the 1-kestose is an oligosaccharide containing 1-kestose having a purity of 95% by weight or more, and the calcium ions are 1 part by weight or more relative to 10 parts by weight of the oligosaccharide containing 1-kestose.

2. Use of an intercellular adhesion enhancer for epithelial cells comprising 1-kestose and calcium ions as active ingredients for the preparation of an agent for ameliorating, treating or preventing allergy, wherein the 1-kestose is an oligosaccharide containing 1-kestose having a purity of 95% by weight or more, and the calcium ions are 1 part by weight or more relative to 10 parts by weight of the oligosaccharide containing 1-kestose.