JP3854657B2 - Intestinal protective agent - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an intestinal protective agent containing casein and / or a casein hydrolyzate obtained by hydrolyzing casein with a protein hydrolase as an active ingredient. The intestinal protective agent of the present invention has an action of counteracting relaxation of tight junctions present in the intestinal tract and suppresses permeation of allergen substances and the like, and thus is useful for the prevention and treatment of allergic diseases and the like.
[0002]
[Prior art]
Between the epithelial cells of the intestinal tract, there is a tight junction that seals the mucosal surface cells without leakage and surrounds the surface cells in a headband shape. It is known that it is absorbed by the body through the space. In addition, macromolecules such as bacteria, viruses, and proteins that remain undigested may pass through the epithelial cells of the intestinal tract and be absorbed into the body, which causes infections and food allergies and is a cause of exacerbations. It is considered one. Especially in infants, because the intestinal tract and its associated organs are not sufficiently developed, macromolecules often pass between the epithelial cells of the intestinal tract and are absorbed into the body, which causes infections and food allergies. ing. Therefore, the present inventors previously proposed an intestinal permeation inhibitor containing a substance that can be taken orally and promotes the formation of tight junctions (Japanese Patent Application No. 6-234282). (Publication).
[0003]
On the other hand, some food ingredients have an action of relaxing tight junctions such as glucose and cytochalasin D. In allergic inflammation, it is considered that tight junctions are also relaxed by inflammatory actions on the living body side such as inflammatory cytokines. Thus, tight junctions are constantly exposed to the danger of being relaxed by food components and inflammatory effects.
[0004]
[Problems to be solved by the invention]
The present inventors prevent the relaxation of tight junctions from the viewpoint of preventing tight junctions between intestinal epithelial cells due to food components and inflammatory effects, and preventing and treating infections and food allergies. As a result of diligent research in search of a substance capable of achieving this, it was found that casein and / or a casein hydrolyzate obtained by hydrolyzing casein with a protein hydrolase has an action of preventing relaxation of tight junctions. As a result of animal experiments using these substances, it was found that tight junctions can be prevented from loosening and permeation of allergen substances can be suppressed, and the present invention has been completed. Therefore, the present invention uses casein and / or a casein hydrolyzate obtained by hydrolyzing casein with a protein hydrolase as an active ingredient, prevents relaxation of tight junctions due to food components and inflammatory action, and suppresses permeation of allergen substances. It is an object of the present invention to provide an intestinal protective agent having an effect of preventing and treating infectious diseases and food allergies.
[0005]
[Means for Solving the Problems]
In the present invention, as an active ingredient of an intestinal protective agent that prevents the relaxation of tight junctions due to food components and inflammatory action, suppresses the permeation of allergen substances, and prevents and treats infections and food allergies, β- Casein such as casein and / or a casein degradation product obtained by hydrolyzing casein with a protein hydrolase is used. Further, casein and / or casein casein hydrolyzate hydrolysis with proteolytic enzymes, as is normal practice, you hydrolyzing protein with proteolytic enzyme.
[0006]
The intestinal protective effect of these substances was carried out using an experimental system for quantitatively determining the substance permeability of tight junctions previously established by the present inventors. The experimental system will be described below.
[0007]
Permeable membrane coated with Caco-2 (ATCC HTB37), a cultured cell line derived from human colon cancer, with a type I collagen (made by Nitta Gelatin) solution at a concentration of 100 μg / ml (Millicell CM, pore size; 0.4 μm, 0.6 cm) ² ) After culturing on DMEM medium (manufactured by Nissui Pharmaceutical) containing 10% fetal bovine serum (manufactured by Bioserum) for a certain period of time, the medium was washed and replaced with Hanks' solution, and Millicell ERS (Millipore) Manufactured), the transepithelial electrical resistance (TEER) was measured to be 550 Ω · cm ² 48 hours after the start of the culture. This indicates that a tight junction was formed and stabilized.
[0008]
Next, when the tight junction formed by adding 10 mM hydrogen peroxide to Hanks' liquid was treated for 30 minutes, the TEER decreased to 70% or less, and the tight junction was in a relaxed state. The reason for using hydrogen peroxide as a substance that relaxes the tight junction is that a situation similar to that in which the tight junction is relaxed by a chemical mediator caused by inflammation occurring in the intestinal tract is assumed. That is, in inflammation, cells around the inflammatory site release inflammatory cytokines and chemical mediators. In particular, the hydrogen peroxide solution released from the cells affects the intestinal tract wall and relaxes tight junctions. To do. Then, the allergen is absorbed into the body through the relaxed tight junction, and food allergy is developed or amplified.
[0009]
Therefore, before adding the hydrogen peroxide solution to the Hanks solution, after processing the tight junction formed by adding to the Hanks solution a substance that serves to search for substances that have the action of preventing the relaxation of the tight junction, A 10 mM hydrogen peroxide solution was added to Hanks' solution to treat the tight junction for 30 minutes, and the TEER was measured to determine whether the relaxation of the tight junction was suppressed.
An experimental system model for measuring TEER is shown in FIG.
[0010]
[Test Example 1]
Using β-casein, casein of the present invention, and β-lactoglobulin, bovine serum albumin (BSA) and whey protein concentrate (WPC) as comparative examples, the effect of preventing tight junction relaxation was examined. That is, after the above-described experimental system, a tight junction formed by adding 10 μM β-casein, 2% casein, 10 μM β-lactoglobulin, 10 μM BSA or 2% WPC to Hanks solution for 30 minutes, A 10 mM hydrogen peroxide solution was added to the solution to treat the tight junction for 30 minutes, and the TEER was measured. Moreover, what did not process before adding hydrogen peroxide solution to Hanks liquid was used as a control. The result is shown in FIG. The relative TEER in the figure is the TEER value after the tight junction is treated with the hydrogen peroxide solution relative to the TEER value before the treatment with the hydrogen peroxide solution.
[0011]
Thus, the treatment of the formed tight junction with β-casein, casein, β-lactoglobulin, BSA, or WPC suppressed the decrease in TEER.・ It was found that the relaxation of the junction was suppressed.
[0012]
[Test Example 2]
Using β-lactoglobulin, the concentration dependency of the effect of preventing relaxation of tight junctions was examined. That is, by treating the tight junction formed by adding β-lactoglobulin adjusted to each concentration of 5 to 500 μM to Hank's solution by the same method as in Test Example 1, it was treated with 10 mM hydrogen peroxide in Hank's solution for 30 minutes. Water was added to treat the tight junction for 30 minutes and the TEER was measured. Moreover, what did not process before adding hydrogen peroxide solution to Hanks liquid was used as a control. The result is shown in FIG. In addition, the width | variety of TEER value in a figure is a standard error of the experiment performed 6 times repeatedly.
[0013]
Thus, the treatment of the formed tight junction with 5 μM β-lactoglobulin can suppress the decrease in TEER, and the treatment with 500 μM β-lactoglobulin can completely suppress the decrease in TEER. understood. In addition, the same tendency was seen also in the experiment conducted using BSA and β-casein of the present invention .
[0014]
[Test Example 3]
Trypsin with a substrate concentration of 1/100 was added to ammonium formate buffer solution (pH 8.0) in which BSA was dissolved to a concentration of 10%, reacted at 37 ° C for 12 hours, and then an equivalent amount of soybean with trypsin. The reaction was stopped by adding trypsin inhibitor and a BSA degradation product was prepared. Then, the concentration dependency of the effect of preventing relaxation of tight junctions using this BSA decomposition product was examined. That is, in the same manner as in Test Example 1, a tight junction formed by adding a BSA degradation product adjusted to each concentration of 5 to 500 μM in terms of BSA to Hanks solution was treated for 30 minutes, and then 10 mM excess was added to Hanks solution. Hydrogen oxide water was added to treat the tight junction for 30 minutes, and the TEER was measured. Moreover, what did not process before adding hydrogen peroxide solution to Hanks liquid was used as a control. The result is shown in FIG.
[0015]
Thus, it was found that treatment of the formed tight junction with 5 μM BSA degradation product also suppressed the decrease in TEER, and treatment with 500 μM BSA degradation product completely suppressed the decrease in TEER. . In addition, the same tendency was seen also in the experiment conducted using the β-lactoglobulin degradation product prepared in the same manner and the β-casein degradation product of the present invention . In addition, the soybean trypsin inhibitor used to stop the enzyme reaction was not effective in preventing relaxation of tight junctions.
[0016]
[Test Example 4]
The intestinal protective effect was examined by animal experiments using WPC, BSA, BSA degradation product, β-lactoglobulin degradation product and β-casein degradation product of the present invention . The BSA degradation product, β-lactoglobulin degradation product, and β-casein degradation product were prepared according to the method for preparing the BSA degradation product shown in Test Example 3.
[0017]
7 weeks old DBA / 2 mice (CLEA Japan) were preliminarily raised for 1 week, and each group consisted of 3 mice, 1% WPC, 1% BSA, 0.1% BSA, 1% BSA degradation product, 0.1% BSA degradation product, Feeds prepared by adding 0.1% β-lactoglobulin degradation product or 0.1% β-casein degradation product to commercially available CE-2 powder feed (manufactured by CLEA Japan, Inc.) were fed and bred for 7 days. Then, after fasting and drinking for 24 hours, 200 μl of 10 mM hydrogen peroxide was administered five times every 2 hours with an oral sonde. In addition, CE-2 powdered diet containing 0.5% ovalbumin (OVA, Sigma) was freely ingested and OVA was orally administered, and then 100 μl was collected from the fundus venous plexus every 0.5 weeks (3 days or 4 days). Changes in serum OVA-specific antibody titers were measured. In addition, a group fed only with CE-2 powder feed was used as a control. The result is shown in FIG.
[0018]
The serum OVA-specific antibody titer was measured as follows.
(1) After adding 100 μl of OVA dissolved in physiological phosphate buffer (PBS) 10 μg / ml to each well of an ELISA plate, the OVA was fixed by leaving it at room temperature for 2 hours.
(2) Each well to which OVA was fixed with a physiological phosphate buffer solution (PBST) containing 0.05% Tween 20 was washed three times, and then 0.1 M citrate buffer solution (pH 6.4) containing BSA at a concentration of 10 mg / ml. 200 μl was added to each well and allowed to block for 2 hours at room temperature.
(3) After washing each well blocked with PBST three times, 100 μl of a sample diluted with a physiological phosphate buffer solution (PBSTB) containing 1% BSA and 0.05% Tween 20 was added to each well. did.
(4) The plate was allowed to stand at 4 ° C. for 17 hours, and each well was washed with PBST three times. Then, 100 μl of a biotin-labeled anti-mouse IgG (Sigma) diluted 10,000 times with PBSTB was added to each well.
(5) The plate was allowed to stand at room temperature for 1 hour, and each well was washed three times with PBST. Then, 100 μl of a solution in which an avidin-labeled alkaline phosphatase solution was diluted 5,000 times with PBSTB was added to each well.
(6) Leave at room temperature for 0.5 hour, wash each well 3 times with PBST, and dissolve substrate solution in 0.02% magnesium chloride and p-nitrophenyl phosphate 1mg / ml in 1M diethanolamine-hydrochloric acid buffer (pH 9.8) 100μl Was added to each well, the color was developed at room temperature, and the absorbance (415 nm) was measured in duplicate.
[0019]
In order to observe the influence of nonspecific adsorption of the sample, a well in which BSA was fixed by adding 100 μl of a solution in which BSA was dissolved in PBS to a concentration of 10 mg / ml instead of the OVA solution was also prepared. . The antibody titer was the geometric mean of the absorbance values of 3 animals per group.
[0020]
Thus, in the control group, the OVA-specific antibody titer started to increase one week after the start of OVA administration, whereas 1% WPC, 1% BSA, 0.1% BSA, 1% BSA degradation product, 0.1% In each experimental group to which a feed supplemented with a BSA degradation product, 0.1% β-lactoglobulin degradation product or 0.1% β-casein degradation product was administered, the time when the OVA-specific antibody titer began to rise was delayed by 0.5 to 1 week, The antibody titer was also low. Therefore, it was found that these substances reduced the permeability of the mouse intestine and prevented the invasion of OVA from the intestine into the living body, thereby preventing food allergies.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, casein and / or a casein hydrolyzate obtained by hydrolyzing casein with a protein hydrolase is used as an active ingredient of the intestinal protective agent. And it can be formulated in the form of powders, granules, tablets, capsules, drinks, etc., and used as a medicine having an intestinal protective effect, or it can be blended in various nutritional compositions and foods and drinks, etc. You may give and use. Furthermore, although it varies depending on age, therapeutic effect, pathological condition, etc., in order to exert an intestinal protective effect on casein and / or a casein hydrolyzate obtained by hydrolyzing casein with a protein hydrolase , it is usually 130 mg / kg body weight at a time. It may be administered orally once to several times a day within a range of ˜13 g.
[0022]
[Example 1]
Casein and / or casein hydrolyzed with protein hydrolase 100g casein hydrolyzate 100g and lactose 65g were mixed, passed through a 60 mesh sieve, then moistened with 20 g of alcoholic polyvinylpyrrolidone and dried through a 12 mesh sieve . Then, 25 g of talc and 10 g of starch were added, and tableting was carried out by a conventional method to produce a tablet having an intestinal tract protecting action with a weight of 300 mg.
[0023]
[Example 2]
Casein and / or casein hydrolyzate 1 200 g, methylcellulose 75 g, after mixed with a little cornstarch 40g and perfume, through a 60 mesh sieve, moistened with an alcoholic polyvinyl pyrrolidone 15 g, a sieve of 0.7 mm diameter stainless steel Through this, a granule having an intestinal protective action was produced.
[0024]
【The invention's effect】
Casein and / or a casein hydrolyzate obtained by hydrolyzing casein with a protein hydrolase has an action of preventing relaxation of tight junctions, and is an allergen or other substance that passes through tight junctions and is absorbed into the body. It has the effect of preventing transmission. Therefore, by using an intestinal protective agent containing these substances as active ingredients, it is possible to exert the effects of preventing and treating food allergies and the like. In addition, these substances are components usually used as food materials, and there is no problem in terms of safety.
[Brief description of the drawings]
FIG. 1 shows a model of an experimental system for measuring TEER.
2 shows the effect of preventing tight junction relaxation of β-casein, casein, β-lactoglobulin, BSA and WPC in Test Example 1. FIG.
FIG. 3 shows the effect of preventing tight junction relaxation due to the difference in β-lactoglobulin concentration in Test Example 2.
4 shows the effect of preventing tight junction relaxation due to the difference in the concentration of the BSA degradation product in Test Example 3. FIG.
FIG. 5 shows intestinal protective effects of WPC, BSA, BSA degradation product, β-lactoglobulin degradation product and β-casein degradation product in Test Example 4.

Claims (3)

An intestinal protective agent comprising casein and / or a casein hydrolyzate obtained by hydrolyzing casein with a protein hydrolase .   The intestinal protective agent according to claim 1, wherein the casein is β-casein. The intestinal protective agent according to claim 1 or 2, wherein the protein hydrolase is trypsin.

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