JP2022119581A - Intercellular adhesion protective agent - Google Patents

Abstract

To provide an intercellular adhesion protective agent.SOLUTION: Provided is an intercellular adhesion protective agent that contains at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β-1,3-glucan.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to intercellular adhesion protective agents and the like.

Cell-to-cell adhesion plays an important role in vivo. For example, intestinal epithelial cells are tethered by intestinal tight junctions, which play an important role in intestinal barrier function. Specifically, intestinal tight junctions physically prevent the intrusion of harmful foreign substances, pathogenic bacteria, allergens, etc. into the body under normal conditions, but when they are disrupted, they invade. In recent years, the condition in which the intestinal tight junction is damaged is called leaky gut, which causes various diseases. Patent Document 1 reports that a eucalyptus leaf extract has the ability to promote tight junction formation. However, since the raw material is a cultivated tree, complicated processes such as cultivation, harvesting, transportation and extraction of the raw material are required.

Euglena is a microalga belonging to the genus Euglena (= Euglena genus) and is used as a food material. Euglena extracts have also been applied to the skin. Paramylon is a β-1,3-glucan produced by Euglena, and is reported to be useful for wound healing and allergy suppression. However, the relationship between euglena or β-1,3-glucan and cell-cell adhesion is still unknown.

JP 2018-197217 A

An object of the present invention is to provide an intercellular adhesion protective agent.

As a result of intensive research in view of the above problems, the present inventors have found an intercellular adhesion protective agent containing at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β-1,3-glucan. If so, it was found that the above problems can be solved. As a result of further research based on this knowledge, the present invention was completed. That is, the present invention includes the following aspects.

Section 1. An agent for protecting intercellular adhesion containing at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β-1,3-glucan.

Section 2. Item 2. The intercellular adhesion protective agent according to Item 1, containing at least one selected from the group consisting of euglena, paramylon, and processed products of paramylon.

Item 3. Item 3. The intercellular adhesion protective agent according to Item 1 or 2, which contains the Euglena, and the Euglena is Euglena gracilis.

Section 4. Item 3. The intercellular adhesion protective agent according to Item 1 or 2, which contains the Euglena, and the Euglena is Euglena gracilis EOD-1 strain (accession number FERM BP-11530).

Item 5. 5. The intercellular adhesion protective agent according to Item 1, 2 or 4, which contains the paramylon, and the paramylon is derived from Euglena gracilis.

Item 6. Item 6. The intercellular adhesion protective agent according to any one of Items 1 to 5, for use in protecting intestinal tight junctions.

Item 7. Item 7. The intercellular adhesion protective agent according to any one of Items 1 to 6, which is used for suppressing leaky gut.

Item 8. Item 8. The intercellular adhesion protective agent according to any one of Items 1 to 7, which is used for suppressing deterioration of intestinal barrier function caused by alcohol and/or inflammatory cytokines.

Item 9. Item 9. The agent for protecting intercellular adhesion according to any one of Items 1 to 8, which is used for suppressing intrusion into the body of at least one species selected from the group consisting of allergens, viruses and pathogenic bacteria.

Item 10. Item 10. The intercellular adhesion protective agent according to any one of Items 1 to 9, which is used for suppressing diarrhea or loose stools.

Item 11. Item 11. The intercellular adhesion protective agent according to any one of Items 1 to 10, which is a food composition, food additive, cosmetic, cosmetic additive, or pharmaceutical.

Item 12. Item 12. The intercellular adhesion protective agent according to any one of Items 1 to 11, which is an oral composition.

According to the present invention, an intercellular adhesion protective agent can be provided.

1 shows the results of an evaluation test for the intercellular adhesion protective action of Test Example 1. FIG. The vertical axis indicates the TER relative value when the TER (Transepithelial Electrical Resistance) at the time of TNF-α addition (0 hours) is set to 100. The horizontal axis indicates the elapsed time from the addition of TNF-α. PM indicates paramylon. For the paramylon-added groups (groups C and D), ** indicates that the p-value for the control group (group B) (TNF-α added (5 ng/ml), PM not added (0 μg/ml)) is less than 0.01. indicates 2 shows the results of an evaluation test for the intercellular adhesion protective action of Test Example 2. FIG. The vertical axis indicates the TER relative value when the TER at the time of ethanol addition (0 hour) is set to 100. The horizontal axis indicates the elapsed time from the addition of ethanol. EtOH indicates ethanol and PM indicates paramylon. For the paramylon-added group (group C), * indicates that the p-value for the control group (group B) (ethanol added (5%), PM not added (0 μg/ml)) is less than 0.05; Indicates a p-value less than 0.01.

As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

In this specification, the expression "and/or" includes the meaning when either "and" or "or" is selected. That is, the expression "A and/or B" includes both the meanings of "A or B" and "A and B."

In one aspect of the present invention, an intercellular adhesion protective agent (herein, It may be indicated as "the agent of the present invention"). This will be explained below.

1. Euglena Euglena is a microalgae belonging to the genus Euglena (= Euglena genus), and is not particularly limited as long as it is. Specific examples of Euglena include Euglena gracilis , Euglena longa , Euglena caudata , Euglena oxyuris , Euglena tripteris , Euglena proxima , Euglena viridis , Euglena sociabilis , Euglena ehrenbergii , Euglena deses , Euglena pisciformis , Euglena spirogyra , Euglena acus , Euglena geniculata , Euglena intermedia , Euglena mutabilis , Euglena sanguinea , Euglena stellata , Euglena terricola , Euglena klebsi , Euglena rubra , Euglena cyclopicola and the like. Among these, from the viewpoint that the effects of the present invention can be more reliably exhibited, Euglena gracilis is preferable, and Euglena gracilis EOD-1 strain [as of June 28, 2013 by Independent Administrative Agency Product Evaluation Under the provisions of the Budapest Treaty, the Patent Organism Depositary Center, National Institute of Technology, NITE-IPOD (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Room 120) under the provisions of the Budapest Treaty, with the accession number FERM BP-11530. Internationally deposited].

The form of Euglena is not particularly limited as long as it contains the cell body of Euglena or most of its components. Examples of the form of Euglena include dry powder form of Euglena, suspension of Euglena, Euglena extract, etc. Among them, dry powder form of Euglena is preferred.

The paramylon content of Euglena in a dry state is, for example, 50% or more, preferably 60% or more, and more preferably 70% or more.

Euglena may be used singly or in combination of two or more.

2. β-1,3-Glucan and paramylon β-1,3-glucan have a single sugar chain (or sugar chain structure) consisting of glucose linked only by β1,3 bonds as the main chain. There are no particular restrictions. β-1,3-Glucans include not only straight-chain ones but also branched-chain ones.

The weight average molecular weight of the β-1,3-glucan derivative is not particularly limited, but is, for example, 1×10 ⁴ to 2×10 ⁶ , preferably 5×10 ⁴ to 1×10 ⁶ , more preferably 1×10 ⁵ to There are 1×10 ⁶ . In addition, the weight average molecular weight can be measured by the GPC method.

The β-1,3-glucan may be one obtained by chemical synthesis, but natural β-1,3-glucan produced by various organisms is preferable from the viewpoint of availability. Examples of natural β-1,3-glucans include paramylon, curdlan, laminaran, callose, lentinan, schizophyllan and the like. Among these, paramylon is preferred. Paramylon will be described below.

Paramylon is a β-1,3-glucan derived from Euglena, and is not particularly limited as far as it is concerned.

Euglena from which paramylon is derived is the same as described in the above "1. Euglena".

The mass average molecular weight of paramylon is not particularly limited, but is, for example, 1×10 ⁴ to 5×10 ⁶ , preferably 2×10 ⁴ to 1×10 ⁶ , more preferably 5×10 ⁴ to 1×10 ⁶ , still more preferably. is between 1×10 ⁵ and 5×10 ⁵ .

The mass average molecular weight can be measured by SEC-MALS analysis under the following conditions:
Detector: Multi-angle scattering detector (DAWN HELEOS II manufactured by Wyatt Technology)
Differential refractometer detector (Optilab T-rEX from Wyatt Technology)
Columns used: 2 TSKgel α-M (manufactured by Tosoh)
Mobile phase: DMSO with 0.05M potassium bromide
Flow rate: 0.5 mL/min.

In Euglena cells, paramylon normally exists as paramylon particles, which are highly-accumulated triple-helix structures formed by β-1,3-glucan chains on certain regular bases.

The shape of the paramylon particles is not particularly limited, but is usually a flattened spheroid.

The particle size distribution of paramylon particles is not particularly limited, but is, for example, 0.5 to 15 μm, preferably 1 to 6 μm. Also, the average particle size of the paramylon particles is not particularly limited, but is, for example, 1 to 10 μm, preferably 2 to 4 μm.

The form of paramylon is not particularly limited as long as it contains paramylon. Examples of the form of Euglena include a dry powder form of paramylon, a suspension of paramylon, etc. Among them, a dry powder form of paramylon is preferred.

Paramylon may be used singly or in combination of two or more.

3. Method for Producing Euglena and Paramylon Euglena can be prepared in large quantities by a method including a step of culturing Euglena contained in a liquid (culturing step). The culture step can be performed, for example, according to a known method (eg, the method described in Japanese Patent No. 5883532). In the culturing step, typically, Euglenoid microalgae are cultured under aerobic conditions while stirring a liquid (culture solution) containing water, Euglena, and nutrients that Euglena can use.

Nutrients include sugars (monosaccharides such as glucose (glucose) and fructose (fructose)), minerals (such as sodium, potassium, magnesium, calcium, iron, zinc, molybdenum, copper, phosphorus, nitrogen, sulfur, or boron). etc.), B vitamins (e.g. vitamin B1 (thiamine), vitamin B2 (riboflavin), niacin, pantothenic acid, vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine), vitamin B12 (cyanocobalamin), folic acid, biotin, etc.) be done. The concentration of nutrients in the culture medium is not particularly limited as long as it is a concentration that allows Euglena to survive, proliferate, and the like.

The light conditions for the culturing step are not particularly limited, and the culturing step may be performed under either light or dark conditions. When cultured in heterotrophic culture, it is cultured under dark conditions. As a light condition, a normal light intensity for growing algae can be adopted. Dark conditions include, for example, less than 10 μmol/m ² /s, preferably completely dark conditions with no light.

The culture temperature in the culture step is not particularly limited as long as it is a temperature at which Euglena can grow. As the culture temperature (temperature of the culture solution), for example, 20°C to 35°C is adopted.

The pH of the liquid in the culturing step is not particularly limited as long as it allows Euglena to proliferate. A pH of 3.0 to 5.5, for example, is adopted as the pH at which Euglena can grow.

After the culturing step, it is preferable to concentrate Euglena by liquid centrifugation, gravity separation, or the like. The obtained Euglena can be subjected to additional processing (for example, suspension in liquid, dispersion in water or oil, extract extraction, dry powderization, etc.) depending on the desired form.

Paramylon particles can be produced by separating, isolating, or purifying from Euglena according to or according to known methods (eg, the method described in Japanese Patent No. 5883532). Paramylon particles can be easily obtained, for example, by recovering intracellular components obtained by disrupting Euglena cell membranes. Moreover, if necessary, the paramylon particles may be purified. Various methods for purifying paramylon particles are known (eg, Japanese Patent No. 5883532), and can be performed according to those methods. Examples of the purification step include a surfactant treatment step and a washing step. The resulting Euglena can be subjected to additional processing (eg, suspension in liquid, dispersion in water or oil, dry powderization, etc.) depending on the desired form.

Four. Paramylon-processed product The paramylon-processed product is obtained by subjecting paramylon to a processing treatment, such as a physical treatment or a chemical treatment, and is not particularly limited as long as it is processed. Examples of paramylon-processed products include fiberized paramylon and amorphous paramylon. Amorphal paramylon can be obtained by chemical treatment according to or in accordance with a known method, for example, using the method described in JP-A-2011-184592.

Fiberized paramylon is preferable as the processed paramylon. Fiberized paramylon will be described below.

Fibrosis paramylon is Euglena-derived β-1,3-glucan, and is not particularly limited as long as it is in a fibrous form. So far, amorphous paramylon obtained by chemically treating paramylon particles (alkali treatment, etc.) has been reported. Since it is an amorphous mass, it is not included in fibrillated paramylon.

The weight average molecular weight of fiberized paramylon is not particularly limited, but is, for example, 1×10 ⁴ to 2×10 ⁷ , preferably 1×10 ⁵ to 5×10 ⁵ .

The weight average molecular weight can be measured by SEC-MALS analysis using the following method. Detector: multi-angle scattering detector (DAWN HELEOS II manufactured by Wyatt Technology)
Differential refractometer detector (Optilab T-rEX from Wyatt Technology)
Columns used: 2 TSKgel α-M (manufactured by Tosoh)
Mobile phase: DMSO with 0.05M potassium bromide
Flow rate: 0.5 mL/min.

The fiber diameter of the fiberized paramylon is not particularly limited, but is, for example, 10 to 500 nm, preferably 20 to 300 nm, more preferably 50 to 200 nm. The diameter of the fibers of fibrillated paramylon can usually be measured based on an electron microscope image of fibrillated paramylon.

The settling volume of fibrillated paramylon in water is not particularly limited, but is, for example, 30 to 300 mL/g, preferably 50 to 250 mL/g, more preferably 70 to 200 mL/g.

Settling volume in water can be measured according to or in accordance with the following method:
Measure according to the method described in "Supervised by the Japan Dietary Fiber Society, Edited by the Japan Dietary Fiber Society Editorial Committee (2008) Dietary Fiber -Basics and Applications- 3rd Edition, p.111 Daiichi Publishing, Tokyo" . Specifically, it is as follows. Weigh 125 mg of the sample slurry test sample into a 25 mL volume plastic tube, calculated as dry mass, and shake the plastic tube vigorously by hand to agitate the contents. Then transfer the contents to a 25 mL graduated cylinder and add pure water to make 25 mL. After stirring the liquid in the graduated cylinder, leave it at 37°C for 24 hours. This causes the sample to settle, resulting in two layers, one containing mainly the precipitated sample (lower layer) and the other containing mainly water (upper layer), separated by an interface. Calculate the volume of the lower layer from the scale of the graduated cylinder, divide the obtained volume by the sample mass (dry mass), and calculate the volume settled in water (mL/g). Perform the test 3 or 4 times and calculate the average value and standard deviation.

Fibrillating paramylon has a relatively high resistance to enzymatic degradation. For example, the amount of monomer (glucose) produced by the decomposition of β-glucanase is, for example, 0.1-50 mg, preferably 1-10 mg, per 1 g of fibrillated paramylon.

This amount can be measured according to or according to the following methods:
Reaction solution [test substance 30 mg (dry weight), buffer solution (B0156 manufactured by Tokyo Chemical Industry Co., Ltd., potassium hydrogen phthalate-sodium hydroxide buffer (pH 4.0)) 5 mL, enzyme solution (endo- 1,3-β-Glucanase (enzyme content: 50 units/mL)) 0.1 mL, pure water, reaction volume 10 mL], shake horizontally at 40°C for 24 hours at 45 rpm. After shaking, immediately store frozen and lyophilize for concentration. After freeze-drying, add 0.5 mL of pure water to each sample and stir (20-fold concentration). The operation of centrifuging (10000G, 5 minutes, 4°C) and collecting the supernatant is repeated twice. The glucose concentration in the collected supernatant is measured using a measurement kit (Glucose CII-Test Wako manufactured by Wako Pure Chemical Industries, Ltd.). Based on the measured values, calculate the amount of glucose produced (mg) per 1 g of the test substance.

Fiberized paramylon has relatively low solubility in alkaline solutions. For example, fiberized paramylon does not dissolve in 0.1-0.3M sodium hydroxide aqueous solution. Here, "does not dissolve" means, for example, that the absorbance (660 nm) of the solution after suspending the fibrillated paramylon in the aqueous solution (for example, immediately after to 1 hour later) is, for example, 0.1 or more, preferably 1.0. means greater than or equal to

Solubility can be measured according to or in accordance with the following methods:
Suspend 250 mg (dry weight) of the test substance in 10 mL of test solution (pure water, 0.1 M NaOH aqueous solution, 0.3 M NaOH aqueous solution) in a vial. The absorbance at 660 nm of the liquid in the vial is measured after shaking the vial vigorously by hand for 20 seconds and after shaking on a shaker at 80 rpm for 1 hour. The absorbance is measured using a spectrophotometer V-730 manufactured by JASCO Corporation.

The relative value of crystallinity of fiberized paramylon to granular paramylon (crystallinity of fiberized paramylon/crystallinity of granular paramylon) is, for example, 0.60 to 0.90, preferably 0.65 to 0.80.

Crystallinity can be measured according to or according to the following methods:
XRD measurement is performed on the test substance. The conditions are as follows. Equipment: PANalytical X'Pert3 Powder, tube voltage: 45kV, tube current: 40mA, measurement range: 5.005 to 50.018°, measurement interval: 0.013°, analysis software: HighScore. The degree of crystallinity is analyzed by the ratio of the intensity of the amorphous part to the intensity of the crystalline part at 2θ=5 to 80°. The analysis was performed after removing the background from each measurement sample (background setting Auto, venting factor 0, granularity 100), and the amorphous part was determined by the tangent line passing through 2θ = 14, 29°. do. The pending factor and granularity condition for determining each amorphous portion is 0/20.

Fiberized paramylon may be in a form dispersed in a solvent such as water, or may be in a dry form. Fiberized paramylon, even in dry form, can be redispersed in water.

As used herein, the term "dry form" indicates that the water content is 15% by mass or less, preferably 10% by mass or less, and more preferably 5% by mass or less.

As the fibrillated paramylon, a fibrillated product of paramylon particles obtained by physically fibrillating the paramylon particles can be preferably used. In addition, a fibrillated product of Euglena obtained by applying this fibrillation treatment to Euglena can also be used as fiberized paramylon.

The fibrillation treatment hardly breaks the hydrogen bonds of β-1,3-glucans present in the paramylon particles (for example, 10% or less, 5% or less, 2% or less of the hydrogen bonds of β-1,3-glucans , 1% or less), or unraveling part or all of the β-1,3-glucan chains present in the paramylon particles or the triple helix structure formed by them. is not particularly limited as long as the processing can be performed. Preferably, the β-1,3-glucan present in the paramylon particles is fibrillated without breaking the hydrogen bonds to form fibrils. Any known treatment capable of grinding (shearing) or pulverizing (preferably grinding (shearing)) fine particles such as paramylon particles can be employed as defibration treatment.

The fibrillation treatment can be performed using a known device such as a grinder (shearer) or a grinder. Examples of apparatuses used for fibrillation treatment include a stone grinder, a jet mill, a twin-screw kneader, a high-pressure homogenizer, a high-pressure emulsifier, a twin-screw extruder, and a bead mill. Among these, stone mills and bead mills are preferred.

The defibration treatment can be performed wet or dry. A wet fibrillation treatment is preferable because the fiberized paramylon can be more efficiently dispersed in the solution. The solvent in the wet method is not particularly limited as long as it can disperse the fiberized paramylon, and water can be preferably used.

The defibration treatment may be performed singly or in combination of two or more. In addition, it may be a partially fibrillated paramylon, and is intended in the present invention as long as it contains fibrillated paramylon.

Five. Since euglena , paramylon, processed paramylon, and β-1,3-glucan have an action to protect intercellular adhesion, specifically, these components are effective for intercellular adhesion, preferably tight junctions, especially It has a protective effect on intestinal tight junctions, tight junctions between intestinal epithelial cells, etc., that is, an effect of promoting intercellular adhesion, an effect of strengthening intercellular adhesion, an effect of suppressing dissociation of intercellular adhesion, and the like. Therefore, at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β-1,3-glucan can be used as an active ingredient such as an intercellular adhesion protective agent.

In addition, at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β-1,3-glucan is used in compositions and compositions of the present invention for various uses based on the action of protecting intercellular adhesion. It can also be used as an active ingredient such as a drug. Such uses include, for example, suppression of leaky gut, suppression of intestinal barrier dysfunction (especially, suppression of intestinal barrier dysfunction due to alcohol and/or inflammatory cytokines), suppression of diarrhea and loose stools, allergens, viruses, and pathogenic bacteria. Inhibition of intrusion into the body of at least one selected from the group consisting of

If the intestinal tight junction is normal, it can prevent viruses and pathogenic bacteria from entering from the intestinal tract like allergens, but if it is damaged, it allows viruses and pathogenic bacteria to enter. Restoring damaged intercellular adhesion or maintaining (protecting) intercellular adhesion with at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β-1,3-glucan (repair, maintenance, and protection of intestinal tight junctions), the invasion of viruses and pathogenic bacteria into the body can be suppressed by decreasing or suppressing the decrease of the intestinal barrier function. It is also expected to maintain the health of healthy people by maintaining intestinal function.

Viruses include, for example, influenza virus (e.g., type A, type B, etc.), rubella virus, Ebola virus, coronavirus, measles virus, varicella-zoster virus, mumps virus, arbovirus, respiratory syncytial virus, SARS virus, hepatitis virus ( enveloped viruses such as hepatitis B virus, hepatitis C virus, etc.), yellow fever virus, AIDS virus, rabies virus, hantavirus, dengue virus, nipah virus, lyssa virus; adenovirus, norovirus, rotavirus , human papillomavirus, poliovirus, enterovirus, coxsackievirus, human parvovirus, encephalomyocarditis virus, poliovirus, rhinovirus, and other non-enveloped viruses (viruses without envelopes).

Pathogenic bacteria include, for example, Staphylococci (e.g. Staphylococcus aureus, Staphylococcus epidermidis), Enterococci (e.g. Enterococcus), Streptococci (e.g. cocci, Streptococcus hemolyticus), Bacillus (e.g. Anthrax, Bacillus subtilis), Clostridia (e.g. Tetanus, Clostridium botulinum, Clostridium perfringens), Corynebacterium (e.g. Diphtheria), Listeria, Lactobacillus Bifidobacterium, Propionibacterium (e.g. acne-causing bacteria), Actinomycetes, Escherichia (e.g. enterohemorrhagic Escherichia coli (e.g. serotype O157, serotype O111, serum type O26, etc.), enteropathogenic E. coli, enteroinvasive E. coli, enterotoxigenic E. coli, enterodiffusive E. coli, enteroaggregative E. coli, etc.), Salmonella spp., Pseudomonas spp. (e.g. Pseudomonas aeruginosa), Helicobacter genus Haemophilus influenzae, Neisseria genus (eg, Neisseria gonorrhoeae, Meningococcus), and the like.

Examples of allergens include the viruses and pathogenic bacteria themselves, substances derived from the viruses and pathogenic bacteria (e.g., nucleic acids, sugar chains, peptides, proteins, etc.), various foods (e.g., eggs, milk, wheat, buckwheat, peanuts, shrimp, etc.). , crab, soybeans, squid, salmon roe, salmon, mackerel, beef, chicken, pork, walnuts, yams, oranges, kiwifruit, peaches, apples, bananas, gelatin, abalone, matsutake mushrooms, sesame seeds, cashew nuts, etc.) (eg, nucleic acids, sugar chains, peptides, proteins, etc.) and the like.

In addition to these, there are also previous reports (for example, Japanese Journal of Food Microbiology Jpn. J. Food Microbiol., 36(1), 32-35, 2019, Curr Opin Gastroenterol. 2016 Mar;32(2):74-9 .) reported various uses.

Preferably, multiple (two or more, more preferably three or more, still more preferably four or more, even more preferably five or more, even more preferably six or more) of the above uses are used. It can be used for a comprehensive range of applications including:

The agent of the present invention is used in various fields, for example, food compositions (including health foods, health promoting agents, dietary supplements (such as supplements)), food additives, cosmetics, cosmetic additives, pharmaceuticals, reagents, feeds, etc. can be used as The agent of the present invention is preferably an oral composition.

The form of the agent of the present invention is not particularly limited, and can take a form commonly used in each application depending on the application.

As for the form of the agent of the present invention, when the application is a food composition, liquid, gel or solid foods such as juices, soft drinks, teas, soups, beverages such as soy milk, salad oils, dressings, yoghurts and jellies. , puddings, sprinkles, powdered milk for infants, cake mixes, dairy products (eg, powder, liquid, gel, solid, etc.), bread, confectionery (eg, cookies, etc.), and the like.

Examples of the form of the agent of the present invention include emulsions, cosmetic liquids, face creams, hand creams, lotions, body soaps, shampoos, rinses, cosmetic gels, packs, foundations, lip balms, and facial cleansers when the application is cosmetics. agents and the like.

Forms of the agent of the present invention include ointments, liquids for external use (liniments, lotions, etc.), sprays (aerosols for external use, pump sprays, etc.), creams, gels, etc., when the application is a medicine. , patches (plasters, tapes such as plasters (reservoir type, matrix type, etc.), cataplasms, patches, microneedles, etc.), eye drops, ophthalmic ointments, nasal drops, suppositories, semi-rectal Formulations suitable for parenteral intake such as solid formulations and enemas (especially formulations for external use); Formulations suitable for oral intake such as pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), and jellies ( oral dosage forms).

As the form of the agent of the present invention, when the application is an additive, a health promoting agent, a nutritional supplement (such as a supplement), for example, a tablet (orally disintegrating tablet, chewable tablet, effervescent tablet, troche, jelly) drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including suspensions and syrups), jellies, and the like.

The agent of the present invention may further contain other ingredients as necessary. Other components include food compositions (including health foods, health-promoting agents, dietary supplements (supplements, etc.)), food additives, cosmetics, cosmetic additives, pharmaceuticals, reagents, ingredients that can be added to feeds, etc. Although not particularly limited as long as it ingredients, fragrances, chelating agents, and the like.

The content of the active ingredient in the agent of the present invention depends on the application, the mode of use, the state of the application target, etc., and is not limited, but for example, 0.0001 to 100% by mass, preferably 0.001 to 50% by mass. can do.

The amount of application (e.g., administration, ingestion, inoculation, etc.) of the agent of the present invention is not particularly limited as long as it is an effective amount that exhibits an intercellular adhesion protective action. 0.1 to 10000 mg/kg body weight. The above dosage is preferably applied in divided doses once or more (eg, 1 to 3 times) per day, and can be adjusted as appropriate depending on age, disease state, and symptoms.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

Reference example 1
Euglena gracilis EOD-1 strain (NITE-IPOD, National Institute of Technology and Evaluation) dry powder (manufactured by Kobelco Eco-Solutions, paramylon content of 70% or more) was prepared as Euglena.

Reference example 2
Paramylon particles were prepared as follows.

Five flasks of liquid from the prepared Euglena gracilis EOD-1 strain (after culture but before drying) were collected, and the collected liquid was centrifuged in a centrifuge tube (500 xg, 4 minutes, room temperature). . The supernatant in the centrifugal tube was once removed and collected. The recovered supernatant was placed in a centrifuge tube to disperse the sediment in the centrifuge tube, and the whole was transferred to a graduated cylinder with a volume of 100 mL. Furthermore, the collected supernatant was added to a graduated cylinder to make up to 90 mL.

[Enzyme treatment step]
The liquid made up to 90 mL was transferred to a 200 mL beaker, and the pH of the liquid was adjusted to 3 by adding aqueous hydrochloric acid with stirring. A proteolytic enzyme (acidic protease, product name “Protease YP-SS” manufactured by Yakult Pharmaceutical Industry Co., Ltd., optimum pH 2.5 to 3.0) was added to the liquid at a concentration of 5 g/L. Enzymatic treatment was performed at 50° C. for 2 hours while stirring the liquid.

[Surfactant treatment step]
An aqueous solution of sodium dodecylsulfate was added to the liquid that had undergone the enzyme treatment step so that the concentration of sodium dodecylsulfate was 3.0% by mass/volume (w/v). While stirring the liquid containing sodium dodecyl sulfate, the pH of the liquid was adjusted to 3 by adding aqueous hydrochloric acid. Further, the liquid was stirred with a propeller stirrer (rotation speed 200 rpm) at 60° C. for 30 minutes.

[Separation process]
Paramylon was precipitated by centrifugation (1000×g, 2 minutes, room temperature) and separated from the liquid that had undergone the surfactant treatment step. A surfactant treatment step was further performed in the same manner, except that the concentration of sodium dodecyl sulfate was changed to 1.0 mass/volume % and the pH was not adjusted. After that, the separation step was performed in the same manner as described above. In this manner, the surfactant treatment step and separation step were each performed three times.

[Washing process]
Paramylon precipitated by centrifugation in the separation step was suspended in pure water and allowed to stand at 40° C. for 10 minutes. Paramylon was then precipitated by centrifugation (1000×g, 2 minutes, room temperature). Such operations were performed a total of three times.

[Drying process]
The paramylon precipitated by centrifugation in the washing step was dried at 50° C. to obtain paramylon particles. The obtained paramylon particles were used as paramylon in the following test examples.

Test example 1. Evaluation Test 1 for Intercellular Adhesion Protection Effect
<Test Example 1-1. Preparation of intestinal epithelial cell layer model>
Caco-2 cells derived from human colon cancer were used as model cells of human intestinal epithelium. Caco-2 cells were complement inactivated by heating in Dalbecco's Modified Eagle Medium (DMEM) medium (Sigma, D6046) with 10% inactivated Fetal Bovine Serum (FBS) (56°C incubator for 40 minutes). ), subcultured in medium supplemented with 1% antibiotic-antimycotic (Sigma, A5955) at 37°C in the presence of 5% CO ₂ .

Put 750 μL of medium in a 24-well plate for cell culture inserts (Corning, 353504), and set the insert (Apical (AP) side) (Cell Culture Insert, Transparent PET Membrane 24 Well 0.4 μm pore size, 353095) there. , made a transwell. 250 μL of 2×10 ⁵ cells/ml Caco-2 cell suspension was seeded in the insert (5×10 ⁴ cells/insert). The cells were cultured for 14 days, and the medium was changed every 3 days. As a result, Caco-2 cells were differentiated to obtain an intestinal epithelial cell layer model in which tight junctions were formed.

<Test example 1-2. Evaluation test>
The medium on the insert side was replaced with 250 µL of medium with a paramylon concentration of 0, 100, or 500 µg/mL, and after 30 minutes, the medium on the plate side was replaced with 750 µL of medium with a TNF-α concentration of 0 or 5 ng/mL, Incubated for 48 hours. Combinations of the paramylon (PM) concentration in the insert-side medium and the TNF-α concentration in the plate-side medium are shown in the table below.

After 24 hours and 48 hours from the addition of TNF-α, the electrical resistance value was measured using an electrical resistance measurement system Merck Millicell ERS-2, and TER (Transepithelial Electrical Resistance) was calculated. The measurement method and TER calculation method followed the attached instructions. The relative value of TER was calculated by setting the TER at the time of TNF-α addition (0 hours) as 100. For the paramylon-added group, Williams' multiple comparison test was performed with respect to the control group, and the p-value was calculated.

The results are shown in FIG. Addition of paramylon inhibited the decrease in TER (decrease in intercellular adhesion (mainly intercellular adhesion due to tight junctions)) caused by inflammatory cytokine (TNF-α). From this, it was found that paramylon has an intercellular adhesion protective effect.

Test example 2. Evaluation test 2 of intercellular adhesion protection effect
An intestinal epithelial cell layer model prepared in the same manner as in Test Example 1-1 was used. The medium on the insert side was replaced with 225 μL of medium having a paramylon concentration of 0 or 550 μg/mL, and after 30 minutes, 25 μL of 50% ethanol (diluted with PBS) was added to the insert side and cultured for 2 hours. Combinations of paramylon (PM) concentration and ethanol concentration in the insert-side medium are shown in the table below.

After 20 minutes, 40 minutes, 60 minutes, and 2 hours from the addition of ethanol, the electrical resistance value was measured using an electrical resistance measurement system Merck Millicell ERS-2, and TER was calculated. The measurement method and TER calculation method followed the attached instructions. The TER at the time of ethanol addition (0 hour) was set to 100, and the relative value of TER was calculated. A t-test was performed to determine if there was a difference between the paramylon-added group and the control group, and the p-value was calculated.

The results are shown in FIG. The ethanol-induced decrease in TER (decrease in intercellular adhesion (mainly intercellular adhesion due to tight junctions)) was significantly suppressed by the addition of paramylon. From this, it was found that paramylon has an intercellular adhesion protective effect.

Claims (12)

An agent for protecting intercellular adhesion containing at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β-1,3-glucan. The intercellular adhesion protective agent according to claim 1, containing at least one selected from the group consisting of euglena, paramylon, and paramylon processed products. The intercellular adhesion protective agent according to claim 1 or 2, which contains the Euglena, and the Euglena is Euglena gracilis. The intercellular adhesion protective agent according to claim 1 or 2, which contains the Euglena, and the Euglena is Euglena gracilis EOD-1 strain (accession number FERM BP-11530). 5. The intercellular adhesion protective agent according to claim 1, 2 or 4, which contains the paramylon and the paramylon is derived from Euglena gracilis. The intercellular adhesion protective agent according to any one of claims 1 to 5, for use in protecting intestinal tight junctions. The intercellular adhesion protective agent according to any one of claims 1 to 6, for use in suppressing leaky gut. The agent for protecting intercellular adhesion according to any one of claims 1 to 7, for use in suppressing deterioration of intestinal barrier function caused by alcohol and/or inflammatory cytokines. 9. The agent for protecting intercellular adhesion according to any one of claims 1 to 8, which is used for suppressing intrusion into the body of at least one species selected from the group consisting of allergens, viruses and pathogenic bacteria. The intercellular adhesion protective agent according to any one of claims 1 to 9, for use in suppressing diarrhea or loose stools. The intercellular adhesion protective agent according to any one of claims 1 to 10, which is a food composition, food additive, cosmetic, cosmetic additive, or pharmaceutical. The intercellular adhesion protective agent according to any one of claims 1 to 11, which is an oral composition.

Images