WO2019131772A1 - 腸管バリア機能改善用組成物 - Google Patents
腸管バリア機能改善用組成物 Download PDFInfo
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- WO2019131772A1 WO2019131772A1 PCT/JP2018/047877 JP2018047877W WO2019131772A1 WO 2019131772 A1 WO2019131772 A1 WO 2019131772A1 JP 2018047877 W JP2018047877 W JP 2018047877W WO 2019131772 A1 WO2019131772 A1 WO 2019131772A1
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7024—Esters of saccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/74—Synthetic polymeric materials
- A61K31/765—Polymers containing oxygen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Definitions
- the present invention relates to a composition for improving intestinal barrier function.
- the invention also relates to methods of improving intestinal barrier function and the use of compounds having gallic acid residues for improving intestinal barrier function.
- the functions of the intestine are mainly the absorption function of nutrients and the barrier function (intestinal barrier function) for preventing the penetration (permeation) of harmful substances.
- the intestinal barrier function is closely related to the chronic inflammatory diseases which increase with age.
- intestinal epithelial cells Under the intestinal epithelial cells, there are many cells of the immune system such as macrophages, dendritic cells, T cells and B cells.
- intestinal epithelial cells are firmly attached to each other by a structure called tight junction, and high molecular weight substances are strictly controlled not to permeate cell gaps.
- transporters for draining hydrophobic foreign substances from intestinal epithelial cells These tight junction structures, transporters and the like are responsible for the intestinal barrier function to prevent the entry of foreign matter.
- EGF Epidermal growth factor
- Non-Patent Document 1 describes that flavonoids such as quercetin promote the formation of tight junctions and the like to prevent chronic inflammation.
- Patent Document 1 describes an absorption inhibitor comprising, as an active ingredient, one or more selected from lindane, octagon, marnies, black tea, black tea, or their processed products.
- Patent Document 2 describes that a hexapeptide having a specific sequence and tryptophan have an absorption suppressing activity of an allergen.
- Patent Document 3 describes a supplement for enteral administration to maintain or restore the intestinal barrier of the intestine, which comprises a combination of glutamine, a substance having antioxidant activity and a short chain fatty acid.
- An object of the present invention is to provide a composition for improving an intestinal barrier function which can improve an intestinal barrier function.
- the present inventors have intensively studied to solve the above-mentioned problems, and are capable of disrupting intestinal barrier function in humans by adding inflammatory cytokines to an intestinal permeation model using human intestinal cell culture Caco-2
- compounds having gallic acid residues such as flavan-3-ol polymers having galloyl groups are useful for improving intestinal barrier function.
- the present invention relates to the following composition for improving intestinal barrier function.
- An intestinal barrier comprising a compound having a gallic acid residue as an active ingredient, wherein the compound having a gallic acid residue is one or more compounds selected from the group consisting of (A1) to (A3) below: Composition for functional improvement.
- A1 Flavan-3-ol polymer having a galloyl group A2) Hydrolyzable tannin (A3) One or more compounds selected from the group consisting of catechin gallate, epicatechin gallate and gallocatechin gallate (2) Gallic acid
- composition for improving intestinal barrier function according to the above (5), wherein the gallotannin is a compound in which 3 to 5 molecules of gallic acid are bound to one glucose molecule.
- the above gallotannins are 1,4,6-tri-O-galoyl- ⁇ -D-glucose, 1,2,4,6-tetra-O-galoyl- ⁇ -D-glucose, 1,2,3 , 6-Tetra-O-galoyl- ⁇ -D-glucose, 2,3,4,6-tetra-O-galoyl- ⁇ -D-glucose and 1,2,3,4,6-penta-O-galoyl
- the composition for improving intestinal barrier function according to (5) or (6) above which is one or more compounds selected from the group consisting of - ⁇ -D-glucose.
- the above ellagitannins are terimaglandin I (Tellimagrandin I), pedounclazine (Pedunculagin), precoxin A (Praecoxin A), geraniin (Geraniin), stenophylanin A (Stenophyllin A), stenophyllanin B (Stenophyllin B)
- the composition for improving intestinal barrier function according to the above (5) which is one or more compounds selected from the group consisting of casuarinin and eugeniflorin D2.
- the composition for improving intestinal barrier function according to any one of the above (1) to (8) which is an oral composition.
- a method for improving intestinal barrier function which comprises administering to a subject a compound having one or more gallic acid residues selected from the group consisting of the following (A1) to (A3): (A1) Flavan-3-ol polymer having a galloyl group (A2) Hydrolyzable tannin (A3) One or more compounds selected from the group consisting of catechin gallate, epicatechin gallate and gallocatechin gallate (15) Intestinal barrier function Use of a compound having one or more gallic acid residues selected from the group consisting of the following (A1) to (A3) for improving (A1) Flavan-3-ol polymer having a galloyl group (A2) Hydrolyzable tannin (A3) One or more compounds selected from the group consisting of catechin gallate, epicatechin gallate and gallocatechin gallate
- the intestinal barrier function can be improved by using the composition for improving the intestinal barrier function of the present invention.
- the present invention can also contribute to the prevention or amelioration of a condition or disease associated with an abnormality in intestinal barrier function by improving the intestinal barrier function.
- FIG. 1 is a flow chart showing a procedure for purifying flavan-3-ol polymer (OPC) having a galloyl group from grape seed extract.
- FIG. 2 is a diagram showing a test schedule of Example 6.
- FIG. 3 is a graph showing the results of examining the influence of flavan-3-ol polymer having a galloyl group on the pain threshold of the large intestine, intestinal permeability of the large intestine and expression of Claudin-2 ((a) pain threshold, (B) Intestinal permeability of large intestine, (c) Claudin-2 relative expression level).
- the composition for improving intestinal barrier function of the present invention comprises a compound having a gallic acid residue as an active ingredient, and the compound having a gallic acid residue is selected from the group consisting of the following (A1) to (A3) One or more compounds.
- A1 Flavan-3-ol polymer having a galloyl group
- A2) Hydrolyzable tannin
- A3 At least one compound selected from the group consisting of catechin gallate, epicatechin gallate and gallocatenin gallate
- the intestinal barrier function of the present invention may contain only one compound selected from the group consisting of (A1) to (A3) above, or may contain two or more compounds.
- the gallic acid residue refers to a residue formed from a galloyl group or a gallic acid oligomer (gallic acid oligomer residue).
- the gallic acid oligomer is a gallic acid di- to tetramer in which gallic acid (3,4,5-trihydroxybenzoic acid) is bound by ether bond, ester bond, carbon-carbon bond, etc., preferably gallic acid 2 to It is a trimer, more preferably gallic acid dimer.
- HHDP group hexahydroxydiphenoyl group
- the compound having a gallic acid residue according to the present invention includes at least one gallic acid residue selected from the group consisting of a galloyl group, a residue of gallic acid dimer and a residue of gallic acid trimer
- the compound which has is preferable, and the compound which has the residue of a galloyl group and / or a gallic acid dimer is more preferable.
- the compounds of (A1) to (A3) above are compounds having gallic acid residues in the molecule.
- the compounds (A1) to (A3) are components contained in plants such as grapes, and they have few side effects and high safety even when taken over a long period of time. According to the present invention, it is possible to provide a composition for improving intestinal barrier function, which contains as the active ingredient a substance which is less in adverse side effects and high in safety even when taken over a long period of time.
- the compound having a gallic acid residue in the present invention is preferably a flavan-3-ol polymer having a galloyl group (A1), a hydrolyzable tannin (A2), and a flavan having a galloyl group (A1) 3-ol polymers are more preferred.
- the flavan-3-ol polymer in the present invention is a dimer or higher having flavan-3-ol as a constitutional unit, and flavan-3-ol bonded by condensation or polymerization at the 4-6 or 4-8 position. It is a polymer.
- Examples of flavan-3-ols include catechin and epicatechin.
- the flavan-3-ol polymer is a type of polyphenol and is a compound also referred to as condensed tannin.
- the flavan-3-ol polymer having a galloyl group (A1) is a polymer in which at least one structural unit is a flavan-3-ol having a galloyl group.
- the bonding position of the galloyl group in the flavan-3-ol of the constituent unit is not particularly limited, and may be, for example, bonded to a flavan skeleton.
- the flavan-3-ol polymer having a galloyl group in the present invention is a flavan-3-ol (a hydroxyl group at the 3-position of flavan skeleton is a carboxyl group of gallic acid) in which a galloyl group is bonded to the 3-position of flavan skeleton And an ester-linked flavan-3-ol) as a constitutional unit.
- the flavan-3-ol polymer having a galloyl group may be a mixture of two or more polymers having different degrees of polymerization and the like.
- the flavan-3-ol polymer having a galloyl group may have a gallic acid residue other than the galloyl group.
- the flavan-3-ol polymer having a galloyl group is not particularly limited depending on its origin or production method. For example, those derived from plants extracted from plants may be used, or those obtained by a synthetic method may be used.
- the flavan-3-ol polymer having a galloyl group can be obtained, for example, from plants such as grapes and pears.
- flavan-3-ol polymers having a galloyl group are preferably those derived from grapes, and are derived from one or more grapes selected from the group consisting of grape fruits, grape seed coats and grape seeds Those contained in the raw material (derived from the raw material) are more preferable, and those derived from grape seed (for example, derived from a grape seed extract) are more preferable.
- the resulting extract is filtered and concentrated to remove alcohol, and then column purification is performed.
- the resulting grape seed extract can be further purified to increase the purity of flavan-3-ol polymers having galloyl groups.
- the fact that the flavan-3-ol polymer has a galloyl group can be confirmed, for example, by formation of gallic acid when the flavan-3-ol polymer is treated with tannase or acid hydrolysis.
- the flavan-3-ol polymer which produces gallic acid by the above treatment has a galloyl group.
- the flavan-3-ol polymer having a galloyl group (A1) according to the present invention is a flavan-3-ol polymer that forms gallic acid by tannase treatment or acid hydrolysis treatment (in one embodiment, preferably by tannase treatment) It can also be said.
- the confirmation of the formation of gallic acid can be carried out by measuring the amount of gallic acid before and after the tannase treatment or acid hydrolysis treatment for the flavan-3-ol polymer.
- the amount of gallic acid in the flavan-3-ol polymer after the treatment is larger than the amount of gallic acid in the flavan-3-ol polymer before the treatment, it can be said that gallic acid is generated by the treatment.
- the acid hydrolysis treatment can be performed, for example, by the following method. Dissolve the sample powder in 20% acetonitrile to 2 mg / mL and add an equal volume of 6 N hydrochloric acid. Heat at 100 ° C. for 3 hours and allow to reach room temperature. The reaction solution that has reached room temperature can be used as a sample after acid hydrolysis treatment to measure the amount of gallic acid.
- the tannase treatment refers to reacting a compound having a gallic acid residue such as flavan-3-ol polymer with an enzyme having tannase (tannin acyl hydrolase) activity.
- an enzyme having tannase activity for example, tannase derived from Aspergillus oryzae is preferable.
- the tannase treatment can be performed under the conditions described in the examples below.
- gallic acid generated by tannase treatment is preferably 0.1 ⁇ g or more, more preferably 1 ⁇ g or more, and more preferably 10 ⁇ g, per 1 mg of flavan-3-ol polymer having a galloyl group.
- the above is more preferable.
- the flavan-3-ol polymer having an amount of gallic acid generated by the tannase treatment in the above range is used, higher intestinal barrier function improvement effect can be obtained.
- the upper limit of the amount of gallic acid generated by tannase treatment is not particularly limited for the flavan-3-ol polymer having a galloyl group.
- the flavan-3-ol polymer having a galloyl group may be, for example, 100 ⁇ g or less per mg of flavan-3-ol polymer having a galloyl group, 50 ⁇ g of gallic acid generated by tannase treatment. It may be the following.
- the flavan-3-ol polymer having a galloyl group is preferably 0.1 to 100 ⁇ g per mg of flavan-3-ol polymer having a galloyl group, wherein gallic acid produced by tannase treatment is preferably 1 to 100 ⁇ g. 100 ⁇ g is more preferable, 10 to 100 ⁇ g is more preferable, and 10 to 50 ⁇ g is particularly preferable.
- the tannase treatment can be performed under the conditions described in the examples.
- the measurement of the amount of gallic acid can be performed by external standard method by HPLC under the conditions described in the examples.
- the amount of gallic acid generated by the tannase treatment is as follows from the amount of gallic acid of the flavan-3-ol polymer (sample before treatment) before the tannase treatment and the flavan-3-ol polymer after the tannase treatment (sample after treatment): It is obtained by a formula.
- Amount of gallic acid generated by tannase treatment (amount of gallic acid of sample after treatment)-(amount of gallic acid of sample before treatment)
- the amount of gallic acid generated by the above-mentioned tannase treatment can be regarded as the amount of galloyl group converted to the weight of gallic acid generated by the tannase treatment.
- the content of the galloyl group is preferably in the above range in terms of the weight of gallic acid generated by the tannase treatment.
- the galloyl group content is preferably 0.1 ⁇ g / mg or more, more preferably 1 ⁇ g / mg or more, in terms of the weight of gallic acid generated by tannase treatment.
- the amount is preferably 10 ⁇ g / mg or more, and may be 100 ⁇ g / mg or less, and may be 50 ⁇ g / mg or less.
- the flavan-3-ol polymer preferably has a galloyl group content of 0.1 to 100 ⁇ g / mg, preferably 1 to 100 ⁇ g / mg, in terms of the weight of gallic acid produced by tannase treatment.
- 10 to 100 ⁇ g / mg is more preferable, and 10 to 50 ⁇ g / mg is particularly preferable.
- hydrolyzable tannin of the above (A2) one having a galloyl group and / or an HHDP group is preferable.
- the hydrolyzable tannin of (A2) may have gallic acid residues other than these in addition to the galloyl group and / or the HHDP group.
- examples of hydrolyzable tannins having a galloyl group and / or an HHDP group include gallotannins and ellagitannins.
- the hydrolyzable tannins may be used alone or in combination of two or more.
- the hydrolyzable tannin is preferably a compound that produces gallic acid and / or ellagic acid by tannase treatment or acid hydrolysis treatment.
- generates a gallic acid by the said process usually has a galloyl group.
- generates ellagic acid by the said process has a gallic acid oligomer residue, such as a HHDP group, normally.
- the confirmation of the formation of gallic acid and / or ellagic acid can be carried out by measuring the amount of gallic acid and / or ellagic acid before and after the treatment for the compound.
- the conditions and the like for the hydrolyzable tannin treatment or acid hydrolysis treatment are not particularly limited, and, for example, the same conditions and the like as the flavan-3-ol polymer described above can be adopted.
- Gallotannin refers to a compound in which one or more gallic acids are bound to glucose. More specifically, gallotannin is a compound in which a carboxyl group of gallic acid is ester-bonded to one or more hydroxyl groups of glucose. Gallotannin has one or more galloyl groups, and generates gallic acid by tannase treatment or acid hydrolysis treatment. In one aspect, a compound in which 3 to 5 molecules of gallic acid are linked to 1 glucose molecule as gallotannin (preferably, gallic acid is preferably linked to 3 to 5 hydroxyl groups of glucose), as gallotannin, because the effect of improving intestinal barrier function is high.
- Gallotannins having 3 to 5 galloyl groups in one molecule are preferred, and in which 4 molecules of gallic acid are linked to 1 glucose molecule (preferably, gallic acid wherein gallic acid is bound to 4 hydroxyl groups of glucose) Gallotannins having four groups are more preferred.
- Elagitannin is a hydrolyzable tannin which produces ellagic acid by tannase treatment or acid hydrolysis treatment.
- those having a galloyl group and / or an HHDP group are preferable as the ellagitannin, and more preferably they have an HHDP group or have a galloyl group and an HHDP group.
- the number of HHDP groups per molecule is preferably 1 or 2, and more preferably 2.
- ellagitannins terimaglandin I (Tellimagrandin I), pedounclazine (Pedunculagin), precoxin A (Praecoxin A), geraniin (Geraniin), stenophilanin A (Stenophyllanin A), stenophilanin
- One or more compounds selected from the group consisting of B (Stenophyllan B), Casalinin (Casuarinin) and Eugeniflorin D 2 (Eugeniflorin D2) are preferred, and Geraniin (Geraniin), Stenophylalanin A (Stenophyllanin A), Stenophylalanin B (Stenophyllin A) Stenophyllanin B), cassarinin (Casuarinin) is more preferable I'm sorry.
- the hydrolyzable tannin can be obtained, for example, by extracting the raw material containing hydrolyzable tannin with water or hydrous ethanol, filtering the extract to remove alcohol by concentration, and removing the alcohol, followed by column purification.
- Plants containing hydrolyzable tannins can be used as a source of hydrolyzable tannins.
- plants containing hydrolyzable tannins include plants of the family Fagaceae (Fagaceae), Lythaceae (Lythraceae), Myrtaceae (Myrtaceae), and Rosaceae (Rosaceae). These plants are rich in hydrolyzable tannins.
- Eucalyptus leaf extract contains a large amount of hydrolyzable tannins such as terimaglandin I and gallotannin.
- the catechin gallate, epicatechin gallate and gallocatechin gallate of the above (A3) are compounds having a galloyl group.
- the compound (A3) can be obtained, for example, by extraction from green tea leaves and purification.
- a commercial item can also be used as a compound of (A3).
- the intestinal barrier function refers to a function to prevent the entry (permeation) of foreign substances (for example, toxins such as endotoxin, proinflammatory substances, undigested substances, etc.) from the outside (inside the intestinal tract) of intestinal epithelial cells.
- the intestinal tract includes the large and small intestines.
- the state in which the invasion of foreign substances from outside the intestinal epithelial cells into the body is promoted compared to the normal state is referred to as the state in which the permeability of the foreign substances in the intestinal epithelial cells is increased.
- the improvement of the intestinal barrier function means both suppressing the increase (enhancement) of the permeability of foreign substances in intestinal epithelial cells and reducing the permeability of foreign substances in intestinal epithelial cells.
- intestinal barrier function improvement is used in the meaning including suppressing the fall of the intestinal barrier function and enhancing the lowered intestinal barrier function.
- intestinal barrier function is improved by normalizing or enhancing tight junctions that adhere intestinal epithelial cells to each other.
- the composition for improving intestinal barrier function of the present invention can be used to improve intestinal barrier function by normalizing or enhancing tight junctions in intestinal epithelial cells.
- the intestinal barrier function improvement effect is shown, for example, by the increase in the electrical resistance (transepithelial electric resistance (TEER)) of intestinal epithelial cells, or by suppressing the decrease in TEER.
- the substance that elevates the TEER or suppresses its reduction has an effect of normalizing or enhancing tight junctions in intestinal epithelial cells.
- the intestinal barrier function improving effect is also shown by a decrease in the amount of substance that permeates from the intestinal side of intestinal epithelial cells to the inside of the body.
- a method of measuring TEER can be used by using an intestinal permeation model using human intestinal epithelial cells (Caco-2 cells). Specifically, inflammatory cytokines (TNF ⁇ , IL-1 ⁇ , IFN ⁇ , etc.) are added to Caco-2 monolayer culture cells to create a state in which human intestinal barrier function can be disrupted in humans, and the test substance is added to the cells. If the decrease in TEER is suppressed as compared to the case where no substance is added, it can be evaluated that the test substance has an effect of improving the intestinal barrier function.
- inflammatory cytokines TNF ⁇ , IL-1 ⁇ , IFN ⁇ , etc.
- the compounds (A1) to (A3) having gallic acid residues are superior in suppressing the decrease in TEER by the addition of inflammatory cytokines in the intestinal permeation model using Caco-2 It was shown to have an intestinal barrier function improvement effect.
- the compound having a gallic acid residue showed an excellent intestinal barrier function improving effect as compared with the compound having no gallic acid residue.
- the compound (A3) showed an excellent intestinal barrier function improving effect as compared with a compound having no galloyl group (catechin, epicatechin, gallocatechin).
- Compounds of (A1) to (A3) having gallic acid residues can normalize or strengthen tight junctions in intestinal epithelial cells to improve intestinal barrier function.
- flavan-3-ol polymers having a galloyl group had an action of preventing or ameliorating the symptoms of colonic hyperalgesia by the intestinal barrier function improving action. Prevention or amelioration effect of abdominal discomfort is expected by prevention or amelioration of colonic hyperalgesia. Therefore, compounds having gallic acid residues such as flavan-3-ol polymers having galloyl groups are useful, for example, for preventing or improving abdominal discomfort by improving intestinal barrier function.
- composition for improving the intestinal barrier function of the present invention exhibits an excellent intestinal barrier function improving effect by containing the compound having a gallic acid residue as an active ingredient. Therefore, the composition for improving intestinal barrier function of the present invention is useful for the prevention or amelioration of a condition or disease in which the improvement of intestinal barrier function is effective, for example, a condition or disease associated with an abnormality in intestinal barrier function.
- Abnormalities in intestinal barrier function include reduction in intestinal barrier function.
- a condition or disease associated with an abnormality in intestinal barrier function includes a condition or disease caused by an abnormality in intestinal barrier function, or a condition or disease with an abnormality in intestinal barrier function.
- condition or disease associated with such an abnormality in intestinal barrier function for example, inflammatory bowel disease, irritable bowel syndrome, systemic autoimmune disease (rheumatoid arthritis, erythematosus etc.), allergy (food allergy, hay fever etc.) And lifestyle-related diseases (eg, obesity, type 1 or 2 diabetes, hypertension, hyperlipidemia, nonalcoholic fatty liver disease (NAFLD), arteriosclerosis, etc.) and the like (eg, Camilleri et al., Am J Physiol Gastrointest Liver Physiol 303: G775-G785, 2012; Mu et al., Front. Immunol., Vol. 8, Article 598, 2017; Bischoff et al., BMC Gastroenterolo y 2014 14: 189).
- lifestyle-related diseases eg, obesity, type 1 or 2 diabetes, hypertension, hyperlipidemia, nonalcoholic fatty liver disease (NAFLD), arteriosclerosis, etc.
- Camilleri et al. Am J Physio
- compositions for improving intestinal barrier function of the present invention have an effect of improving the condition of the intestine by improving the intestinal barrier function. Therefore, the composition for improving the intestinal barrier function of the present invention can adjust the condition of the intestine by the improvement of the intestinal barrier function, and is useful for preventing or ameliorating the above-mentioned intestinal symptoms.
- the composition for improving intestinal barrier function of the present invention is used for intestinal regulation (for example, for preventing or improving diarrhea, constipation, abdominal discomfort, etc.) by improving the intestinal barrier function. It can be done.
- abnormalities in intestinal barrier function are also associated with lifestyle-related diseases and the like (for example, Bischoff et al., BMC Gastroenterology 2014 14: 189 described above). Improving the intestinal barrier function is also effective in preventing or ameliorating lifestyle-related diseases.
- Symptoms of lifestyle-related diseases include glucose metabolism disorder, lipid metabolism disorder, body fat increase, visceral fat increase, abdominal circumference fat increase, high blood pressure and the like.
- the composition for improving intestinal barrier function improves glucose barrier function, improves lipid metabolism, and reduces or increases, suppresses, increases, and increases fat such as body fat, visceral fat and abdominal fat by improving intestinal barrier function. It can also contribute to the improvement of the blood pressure of
- prevention of a condition or disease refers to enhancing the subject's resistance to the condition or disease, delaying or preventing the onset of the condition or disease.
- aboration of a condition or disease recovering the subject from the condition or disease, alleviating the symptoms of the condition or disease, delaying or preventing the progression of the condition or disease Point to
- compositions of the invention can be applied for either therapeutic (medical) or non-therapeutic (non-medical) applications.
- the composition for improving intestinal barrier function of the present invention can be provided, for example, in the form of food and drink, medicine, quasi-drug, feed and the like, but is not limited thereto.
- the composition for improving intestinal barrier function of the present invention may itself be food and drink, medicine, quasi-drug, feed, etc., and may be a preparation such as an additive used for these, or a material.
- the composition for improving intestinal barrier function of the present invention can be provided, for example, in the form of an agent, but is not limited to this form.
- the agent can be provided as it is as a composition or as a composition containing the agent.
- the composition for improving intestinal barrier function of the present invention is preferably an oral composition. According to the present invention, it is possible to provide an oral composition having an excellent intestinal barrier function improving action.
- the composition for oral use includes food and drink, medicine, quasi-drug, and preferably food and drink.
- composition for improving intestinal barrier function of the present invention may contain one or more components (other components) other than the above-described compound having a gallic acid residue as long as the effect of the present invention is not impaired.
- lactic acid bacteria, bifidobacteria, dietary fiber, polysaccharides and the like may be contained as other components.
- the lactic acid bacteria and bifidobacteria are preferably bacteria which can be taken orally.
- the dietary fiber may be any of water-insoluble dietary fiber and water-soluble dietary fiber.
- water-insoluble dietary fibers include cellulose, lignin, hemicellulose, wheat bran, apple fiber, sweet potato fiber, chitin and the like.
- Water-soluble dietary fibers are roughly classified into high-viscosity substances and low-viscosity substances, and examples of high-viscosity substances include pectin, konjac mannan, alginic acid, sodium alginate, guar gum, agar and the like.
- the low-viscosity, water-soluble dietary fiber contains at least 50% by weight of dietary fiber and is a low-viscosity solution dissolved in normal temperature water, approximately 5% by weight It refers to a dietary fiber material that becomes a solution exhibiting a viscosity of 20 mPa ⁇ s or less in an aqueous solution.
- low-viscosity substances of water-soluble dietary fiber those of resistant digestive dextrin, polydextrose, guar gum degradation products, Lytes (polydextrose) and the like can be mentioned.
- any low-viscosity, water-soluble dietary fiber material is included.
- the dietary fiber may be used alone or in combination of two or more.
- polysaccharide examples include oligosaccharides such as galactooligosaccharides, xylooligosaccharides, mannooligosaccharides, agarooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, raffinose and the like. These may be used alone or in combination of two or more.
- the composition for improving intestinal barrier function of the present invention may contain any additive and any component other than the above.
- additives and components can be selected according to the form etc. of the composition for improving intestinal barrier function, and in general, those usable for food and drink, medicine, quasi-drug, feed and the like can be used.
- various additives which are orally or orally pharmaceutically acceptable, such as excipients, lubricants, stabilizers, dispersants, binders, diluents, flavors, sweeteners, flavors , Coloring agents and the like can be exemplified.
- compositions for improving intestinal barrier function of the present invention when used as a composition for oral use, vitamins, vitamin-like substances, proteins, amino acids, fats and oils, organic acids other than the above, as long as the effects of the present invention are not impaired.
- Ingredients that can be orally ingested such as carbohydrates, plant-derived materials, animal-derived materials, microorganisms, additives for foods and beverages, additives for pharmaceuticals, etc. can be appropriately contained.
- components such as materials used for food and drink, medicines, quasi-drugs, feeds and the like can be appropriately blended according to the use.
- composition for improving intestinal barrier function of the present invention is not particularly limited as long as it has the effect of the present invention, and for example, tablets, pills, granules, fine granules, lozenges, capsules (soft capsules Agents, including hard capsules), solutions, chewable agents, beverages and the like. Other food forms may be used. These dosage forms can be prepared using conventional methods commonly known in the art.
- the composition for improving intestinal barrier function of the present invention when used as a food or drink, the compound having the above gallic acid residue can be used as a component usable for food and drink (for example, food and drink materials, if necessary Additives and the like to be used can be blended to make various food and drink (food and drink composition).
- Food-drinks are not specifically limited, For example, general food-drinks, health food, functional indication food, food for specific health, food for sick people, food additive, these raw materials etc. are mentioned.
- the forms of food and drink are not particularly limited, and solid preparations for oral use such as tablets, coated tablets, fine granules, granules, powders, pills, capsules (including soft capsules and hard capsules), dry syrups, chewables, etc. It may be in the form of various preparations of liquid preparations for oral use such as internal liquid solutions and syrups.
- the food and drink may contain one or more of the above-mentioned lactic acid bacteria, bifidobacteria, dietary fiber, and polysaccharides.
- composition for improving intestinal barrier function of the present invention When used as a pharmaceutical or quasi-drug, an additive such as a pharmaceutically acceptable excipient is added to the compound having a gallic acid residue described above, It can be used as a pharmaceutical (pharmaceutical composition) or quasi-drug (quasi-drug composition) of various dosage forms.
- the dosage form of the pharmaceutical or quasi-drug is preferably orally administered.
- the pharmaceutical or quasi-drug dosage form may be a dosage form suitable for the administration form.
- a dosage form for oral pharmaceuticals or quasi-drugs for example, tablets, coated tablets, fine granules, granules, powders, pills, capsules (including soft capsules and hard capsules), dry syrups, chewables, etc.
- conventional coated forms may be used, for example, sugar-coated tablets, gelatin capsules, enteric-coated agents, film-coating agents, etc.
- the tablets can also be multi-layered tablets such as double tablets.
- the method for producing the same is not particularly limited, and the compound having the above gallic acid residue is used. It can be manufactured by a general method.
- the present invention also encompasses the use of one or more compounds selected from the group consisting of the above (A1) to (A3) for producing a composition for improving intestinal barrier function.
- the composition for improving intestinal barrier function of the present invention has one or more of applications such as packaging, containers or instructions, types of active ingredients, effects described above, methods of use (eg, intake method, administration method), etc. You may display it.
- the composition for improving intestinal barrier function of the present invention may be labeled as having an effect based on the intestinal barrier function improving action or the intestinal barrier function improving action. As such a display, for example, it may be displayed that it has an intestinal adjustment effect.
- the intestinal regulation action is not particularly limited as long as it is an intestinal regulation action based on the improvement of the intestinal barrier function.
- the content of the compound having a gallic acid residue of (A1) to (A3) in the composition for improving intestinal barrier function of the present invention can be appropriately set according to the form of the composition and the like.
- the composition for improving intestinal barrier function is a composition for oral use such as food and drink, medicine, quasi-drug, etc.
- the total content of the compounds (A1) to (A3) in the composition for improving intestinal barrier function is preferably 0.0001 to 80.0% by weight, preferably 0.01 to 20.0% by weight. More preferable.
- the total content is the total amount of two or more compounds having gallic acid residues of (A1) to (A3).
- the content of the compound having a gallic acid residue of (A1) to (A3) can be measured according to a known method, and for example, an HPLC method or the like can be used.
- composition for improving intestinal barrier function of the present invention can be taken or administered by an appropriate method according to the form.
- the composition for improving intestinal barrier function of the present invention is preferably orally administered or orally ingested.
- the intake amount (which can also be referred to as a dose) of the composition for improving intestinal barrier function of the present invention is not particularly limited, as long as it is an amount such that the effect of improving intestinal barrier function can be obtained. It may be set appropriately according to the situation.
- the intake amount of the composition for improving intestinal barrier function is the total intake of the compounds of (A1) to (A3)
- the amount is preferably 0.01 to 5000 mg, more preferably 0.1 to 4000 mg, and still more preferably 1 to 3000 mg per day. It is preferable that the above-mentioned amount is orally administered or taken, for example, once a day or divided into two or three times.
- the total intake of the compounds of (A1) to (A3) is the above
- the composition for improving intestinal barrier function can be orally ingested or administered to a subject within a range.
- the intake amount of the composition for improving intestinal barrier function is, in the case of a human (adult) (for example, a body weight of 60 kg),
- the intake amount of (all polymer) is preferably 1 to 2000 mg, more preferably 10 to 1500 mg, still more preferably 30 to 1000 mg, and particularly preferably 100 to 1000 mg.
- an excellent intestinal barrier function improving effect can be obtained.
- the above-mentioned amount is orally administered or taken, for example, once a day or divided into two or three times.
- the composition for improving intestinal barrier function of the present invention is an oral composition for allowing adults to take or administer the above-mentioned amount of the compound of the above (A1) per day per 60 kg body weight. Good.
- the composition for improving intestinal barrier function of the present invention is an amount such that the desired effect of the present invention can be obtained, that is, an effective amount of the above gallic acid residue, considering its administration form, administration method, etc. It is preferable to contain the compound which has these.
- the composition for improving intestinal barrier function is a composition for oral use such as food and drink, pharmaceutical for oral use, etc., per adult (for example, body weight 60 kg) intake per day of the composition.
- the total content of the compounds (A1) to (A3) is preferably 0.01 to 5000 mg, more preferably 0.1 to 4000 mg, and still more preferably 1 to 3000 mg.
- the content of the compound of (A1) in an adult (for example, 60 kg body weight) intake per day of the composition per one person Is preferably 1 to 2000 mg, more preferably 10 to 1500 mg, still more preferably 30 to 1000 mg, and particularly preferably 100 to 1000 mg.
- the compounds (A1) to (A3) which are active ingredients of the composition for improving intestinal barrier function of the present invention, are expected to enhance the effect of improving intestinal barrier function by being continuously taken (administered) .
- the composition for improving intestinal barrier function of the present invention is to be continuously ingested.
- the composition for improving intestinal barrier function is preferably taken continuously for one week or more.
- the subject (hereinafter, also simply referred to as a subject to be administered) to which the composition for improving intestinal barrier function of the present invention is administered or ingested is preferably a human or non-human animal, more preferably a mammal (human or non-human mammal) Humans are further preferred.
- a subject to be administered in the present invention a subject requiring or desired improvement in intestinal barrier function is preferable.
- a subject having a reduced intestinal barrier function, a subject that desires to prevent or ameliorate a condition or disease associated with the aforementioned intestinal barrier function abnormality, and the like can be mentioned as suitable subjects.
- the present invention also includes the following intestinal barrier function improvement methods and the like.
- a method for improving intestinal barrier function which comprises administering a compound having one or more gallic acid residues selected from the group consisting of the following (A1) to (A3) to a subject: (A1) Flavan-3-ol polymer having a galloyl group (A2) Hydrolyzable tannin (A3) One or more compounds selected from the group consisting of catechin gallate, epicatechin gallate and gallocatenin gallate Use of a compound having one or more gallic acid residues selected from the group consisting of A3) for improving intestinal barrier function.
- the above methods and uses may be therapeutic methods and uses, and may be non-therapeutic methods and uses.
- Non-therapeutic is a concept that does not include medical practice, i.e. surgery, treatment or diagnosis.
- the dose of the compound having a gallic acid residue is not particularly limited as long as it is an amount capable of obtaining an intestinal barrier function improving effect, that is, an effective amount, and for example, the above-mentioned amount is preferably administered.
- the administration route is preferably oral administration.
- the compound having gallic acid residues may be administered as it is, or a composition containing the compound having gallic acid residues described above may be administered.
- the composition for improving intestinal barrier function of the present invention described above can be administered.
- the compound having gallic acid residues, the subject (administration target), the administration method, the dose, and the preferable embodiments thereof are the same as those in the composition for improving intestinal barrier function described above.
- the daily dose of the compound (A1) is 1 per 60 kg body weight. -2000 mg is preferable, 10-1500 mg is more preferable, 30-1000 mg is more preferable, and 100-1000 mg is more preferable.
- flavan-3-ol polymer having a galloyl group (hereinafter, flavan-3-ol polymer is also referred to as OPC) 81% or more of grape having 80% or more specification of commercially available oligomeric procyanidin (flavan-3-ol polymer)
- Seed extract (grape seed extract) was dissolved in water and liquid-liquid separation (partition extraction) was performed three times using ethyl acetate. The obtained two fractions were concentrated under reduced pressure and lyophilized to obtain a dry powder.
- the water transfer part containing the OPC was fractionated by the method described in the literature (Biosci. Biotechnol. Biochem., 73, 1274-1279 (2009)) to obtain a further purified grape-derived OPC fraction with increased purity. .
- FIG. 1 is a flow chart showing a procedure for purifying flavan-3-ol polymer (OPC) having a galloyl group from grape seed extract.
- the transfer rate (%) means "100 x yield (g) / starting material (g)".
- the above-mentioned grape seed extract (20.00 g) was dissolved in water (200 mL) and partitioned and extracted three times with ethyl acetate (200 mL). The ethyl acetate transition was concentrated to dryness to give fraction 1 (Fr. 1) (transition rate: 20.8%). The water transfer was concentrated and dried to give fraction 2 (Fr. 2) (transition rate: 70.6%).
- Fraction 2 (10.06 g) is dissolved in methanol (200 mL), chloroform (200 mL) is added thereto, and the resulting solution is centrifuged (5000 rpm, 5 minutes) to precipitate (P1) and supernatant (S1) Divided into The precipitate (P1) was concentrated and dried to obtain fraction 3 (Fr. 3) (transition rate: 26.1%).
- Chloroform (100 mL) was added to the supernatant (S1), centrifuged (5000 rpm, 5 minutes), and the precipitate (P2) and the supernatant (S2) were separated.
- the precipitate (P2) was concentrated and dried to obtain fraction 4 (Fr. 4) (transition rate: 15.5%).
- Chloroform (168 mL) was added to the supernatant (S2), centrifuged (5000 rpm, 5 minutes), and the precipitate (P3) and the supernatant (S3) were separated.
- the precipitate (P3) was concentrated and dried to obtain fraction 5 (Fr. 5) (transition rate: 12.2%).
- Chloroform (132 mL) was added to the supernatant (S3), centrifuged (5000 rpm, 15 minutes), and the precipitate (P4) and the supernatant (S4) were separated.
- the precipitate (P4) was concentrated and dried to obtain fraction 6 (Fr. 6) (transition rate: 3.6%).
- the supernatant (S4) was concentrated and dried to obtain fraction 7 (Fr. 7) (transfer rate: 9.8%).
- the obtained fraction 5 (Fr. 5) was used as a grape-derived purified OPC fraction.
- Example 2 Analysis of OPC The purity of the grape-derived purified OPC fraction obtained in Example 1 was calculated according to the method described in Japanese Patent No. 4659407. The grape-derived purified OPC fraction was subjected to acid hydrolysis in accordance with Patent No. 4659407, and analyzed under the following analysis conditions.
- OPC purity (%) 100 ⁇ (Cyanidin concentration derived from acid-hydrolyzed purified grape fraction derived from grape) / (Cyanidin concentration derived from acid-hydrolyzed procyanidin B1) The purity of the OPC in the grape-derived purified OPC fraction prepared in Example 1 was 91% using the above formula.
- Example 3 Tannase treatment of grape-derived purified OPC 20 mg of the grape-derived purified OPC fraction prepared in Example 1 and 20 mg of tannase (Wako Pure Chemical Industries, Ltd., Aspergillus oryzae) each have a final concentration of 1 mg / mL. It was dissolved in an acid buffer (pH 5.5) and reacted overnight (about 16 hours) at 30 ° C. to perform tannase treatment. A part of the obtained reaction solution was used as a tannase-treated sample for measurement of gallic acid described later.
- this reaction solution is subjected to Sep-Pak Vac 20 cc (5 g) C18 Cartridges (manufactured by Waters), and after washing with 400 mL of distilled water to remove highly polar components, citrate, tannase and gallic acid, 100%
- the OPC was eluted with 60 mL of methanol. The obtained 100% methanol eluate was concentrated under reduced pressure and lyophilized to obtain a dry powder.
- This tannase-treated, grape-derived purified OPC was used as a grape-derived purified OPC (without a galloyl group).
- it carried out the same process as the above, prepared powder, and it was set as grape origin refinement
- the grape-derived purified OPC fraction prepared in Example 1 is dissolved in citrate buffer (pH 5.5) to a concentration of 1 mg / mL, and a sample before tannase treatment did.
- the sample after tannase treatment obtained above and the sample before tannase treatment were applied to a 0.45 ⁇ m filter, and then the amount of gallic acid was analyzed by HPLC.
- the quantification of gallic acid was performed by the external standard method.
- the amount of gallic acid generated by tannase treatment was determined from the amount of gallic acid from the tannase-treated sample and the sample before tannase treatment according to the following formula.
- Amount of gallic acid generated by tannase treatment (amount of gallic acid after treatment)-(amount of gallic acid before treatment)
- the amount of gallic acid after the treatment is the amount of gallic acid of the sample after the tannase treatment
- the amount of gallic acid before the treatment is the amount of gallic acid of the sample before the tannase treatment.
- gallic acid was not detected in the sample before tannase treatment, and 45 ⁇ g / mg of gallic acid was detected in the tannase treatment sample.
- This gallic acid (45 ⁇ g / mg) is a gallic acid generated from the above-mentioned grape-derived purified OPC (with a galloyl group) by tannase treatment (45 ⁇ g of gallic acid is formed per 1 mg of grape-derived purified OPC (with a galloyl group)). Therefore, the grape-derived purified OPC (with a galloyl group) had 45 ⁇ g / mg of galloyl group in terms of the weight of gallic acid generated by tannase treatment. In the grape-derived purified OPC (without galloyl group), the galloyl group has been removed by the above amount.
- Example 4 Comparative evaluation of intestinal barrier function improvement by the presence or absence of the galloyl group was performed.
- Evaluation method of intestinal barrier function improvement action Caco-2 cells were cultured at 37 ° C. for 3 weeks in Transwell (Millicell) using DMEM (Dulbecco's modified Eagle's medium). Media was removed from the plates of cultured Caco-2 cells, the wells were washed three times each with serum free DMEM and the wells were filled with the media.
- TEER transepithelial electrical resistance
- inflammatory cytokines TNF ⁇ , IL-1 ⁇ and IFN ⁇
- a sample was set as normal.
- inflammatory cytokines were added, and a well to which no sample was added was provided as a control.
- TEER was again measured to evaluate whether the sample suppressed the reduction (reduction) of TEER due to inflammatory cytokines.
- TEER reduction inhibition rate (%) 100 ⁇ ((TEER of the well to which the sample was added)-(TEER of control) / ((TEER of normal)-(TEER of control))
- TEER reduction inhibition rate (%) 100 ⁇ ((TEER of the well to which the sample was added)-(TEER of control) / ((TEER of normal)-(TEER of control))
- the samples evaluated are the compounds shown in Table 1 below.
- a gallate type compound of catechin, epicatechin and gallocatechin catechin gallate, epicatechin gallate and gallocatechin gallate were used respectively (all are Wako Pure Chemical Industries, Ltd.).
- the grape-derived purified OPC (with a galloyl group) and the grape-derived purified OPC (without a galloyl group) those prepared in Example 3 were used.
- the relative value in Table 2 indicates the relative value of the TEER decrease inhibition rate (%) of the gallate type compound when the TEER decrease inhibition rate (%) of the non-gallate type compound is 1 in each evaluation.
- the relative value of catechin gallate (gallate type compound) is a relative value of the TEER decrease suppression rate of catechin gallate when the TEER reduction suppression rate of catechin (non-gallate type compound) is 1.
- Evaluation No. 4 performed the said test by the sample (grape origin refinement
- Grape-derived purified OPC when the TEER decrease by inflammatory cytokines calculate 50% inhibition sample concentration (IC 50), was as shown in Table 3. For example, when the concentration of the grape-derived purified OPC is 1 ⁇ g / mL, the TEER reduction inhibition rate of the grape-derived purified OPC (with a galloyl group) is 65%, and the grape-derived purified OPC (without a galloyl group) was 51%.
- Example 5 Evaluation of Intestinal Barrier Function Improving Action of Hydrolyzable Tannin
- a hydrolytic tannin having a gallic acid residue was used as a sample to evaluate the intestinal barrier function improving action.
- the hydrolyzable tannin used what was refine
- terimaglandin I and gallotannin were purified from the leaves of Eucalyptus cypellocarpa.
- Geraniin was purified from the leaves of Geranium thunbergii.
- those purified from plants of the family Agronomidae such as leaves of Kunzea ambigua, tea trees (Melaleuca alternifolia) and the like were used.
- Example 5 The evaluation of the intestinal barrier function improving action was performed by the same method as in Example 4 (the evaluation method of the intestinal barrier function improving action described in Example 4).
- the sample was added to the test solution to have a sample concentration of 10 ⁇ mol / L.
- the TEER reduction inhibition rate (%) of the sample was determined from the above equation using the TEER values of the wells to which the sample was added, normal and control.
- the name of the compound (sample) evaluated, the evaluation concentration (sample concentration in the test solution), and the evaluation result (TEER reduction inhibitory rate) are shown in Table 4. Further, the number of galloyl groups and the number of hexahydroxydiphenoyl groups (HHDP groups) in one molecule of the compound are shown in Table 4 for the gallic acid residues contained in the compound.
- quercetin (Funakoshi Co., Ltd.) was added at 10 ⁇ mol / L to the test solution, and the intestinal barrier function improving action was evaluated by the above method.
- the TEER reduction inhibitory rate of quercetin (10 ⁇ mol / L) was 38%. With respect to the compounds evaluated, an intestinal barrier function improving effect higher than that of quercetin was found at the same concentration as quercetin.
- Example 6 Effects of grape seed-derived flavan-3-ol polymer (OPC) with galloyl group on intestinal permeability and stressed colonic hyperalgesia
- OPC grape seed-derived flavan-3-ol polymer
- Group 3 rats were administered the above-mentioned grape seed extract, and Group 1 and 2 rats were administered Vehicle (distilled water). Rats were orally administered Vehicle or grape seed extract for 1 week prior to stress application (both once daily). Grape seed extract was administered a 10 mg / mL solution dissolved in distilled water. The dose of the grape seed extract was 83 mg / kg (10 mL / kg) per body weight per day, converted to OPC having a galloyl group. Groups 1 and 2 rats received 10 mL / kg of distilled water per body weight. Feed (CRF-1, Oriental Yeast Co., Ltd.) and water were kept ad libitum during the test period.
- Colorectal hyperalgesia was evaluated by the following method. Insert a balloon catheter (silicone catheter, 2.0 mm, Terumo Corporation) 2 cm transanally into the rat under anesthesia and insert an electrode (Teflon (R) coated stainless steel, 0.05 mm, MT Giken Co., Ltd.) The left lateral oblique muscle was inserted 2 mm. Then I was awakened while placed in the ball man cage. Thirty minutes after the treatment, the catheter was dilated gradually by water injection, and electromyography was used to observe abdominal pain contraction induced by colon pain to measure pain threshold. Pain threshold measurements were taken twice before and after stress. The pre-stress measurement was performed on the first day of stress treatment, immediately before (pre-stress) stress treatment.
- the rat was inserted with a balloon catheter and an electrode as described above 24 hours after the last stress treatment (stress treatment on day 3), and the pain threshold was measured 30 minutes after that.
- Vehicle or grape seed extract was orally administered 1.5 hours before the pain threshold measurement after stress.
- the evaluation result of a pain threshold set the pain threshold before stress as 100%, and calculated the change of the pain threshold after stress (100 x pain threshold after stress / pain threshold before stress) (%).
- Example 6 The test schedule of Example 6 is shown in FIG.
- the arrow ( ⁇ ) is a surgery for inserting a balloon catheter into a rat
- the inverted triangle ( ⁇ ) is a measurement of pain threshold
- the circle ( ⁇ ) is WAS (Water avoidance stress) or no stress (Sham stress)
- Triangle ( ⁇ ) indicates the above-mentioned grape seed extract or vehicle administration.
- Treat. 1 and Meas. 1 shows balloon catheter insertion surgery before stress and pain threshold measurement, respectively
- Treat. 2 and Meas. 2 shows balloon catheter insertion surgery and pain threshold measurement after stress, respectively.
- intestinal permeability of the colon and expression level of tight junction protein were measured by the following method.
- the contents of the large intestine were washed under anesthesia, and the upper part of the large intestine was ligated at two places to make a 4 cm loop. 1 mL of 1.5% Evans blue solution was injected there, and it left still for 15 minutes. The ligation site was removed, washed with PBS and N-acetyl-cysteine, and the penetrated Evans blue was extracted with 2 mL N, N-dimethylformamide. Thereafter, the absorbance was measured to determine the Evans blue transmission amount.
- the intestinal permeability (mg / g tissue) of the large intestine was calculated by correcting the permeation amount (mg) of Evans blue with the weight (g) of the ligated site of the large intestine.
- the expression level of tight junction protein Claudin-2 was analyzed by the Wes system of proteinsimple. 1 cm tissue was collected from below the ligation site of the large intestine, and samples were prepared using tissue lysate (1% SDS, 1% Triton, 1% sodium deoxycholate in PBS).
- FIGS. 3 (a) to 3 (c) show the results of examining the effect of flavan-3-ol polymer having a galloyl group on the pain threshold of the large intestine, intestinal permeability of the large intestine and expression of Claudin-2.
- Fig. 3 (a) shows the evaluation results of pain threshold
- Fig. 3 (b) shows the evaluation results of intestinal permeability (large intestine permeability) of the large intestine
- Fig. 3 (c) shows the relative expression amount of Claudin-2.
- the relative expression level of Claudin-2 is the relative expression level with the expression level in group 1 being 100.
- Each graph is shown as mean ⁇ standard error, and Dunnett's test was performed on group 2 (stress + vehicle group) for statistically significant difference between groups (*: p ⁇ 0.05).
- the pain threshold decreased with stress treatment.
- the pain threshold was increased by the administration of the OPC having a galloyl group.
- stress treatment enhanced the permeability, and the OPC having a galloyl group suppressed or improved it.
- the expression level of tight junction protein Claudin-2 was increased by stress treatment, and the OPC having a galloyl group suppressed or improved it.
- Claudin-2 is known to increase intestinal permeability when expression is increased. The above-mentioned increase in intestinal permeability and the increase in the expression level of Claudin-2 suggested that stress reduced the function of tight junction in intestinal epithelial cells.
- the OPC having a galloyl group had an action to improve the intestinal barrier function.
- composition for improving intestinal barrier function of the present invention is useful in the field of food and drink, in the field of medicine and the like.
Abstract
Description
(1)没食子酸残基を有する化合物を有効成分として含み、上記没食子酸残基を有する化合物が、下記(A1)~(A3)からなる群より選択される1以上の化合物である、腸管バリア機能改善用組成物。
(A1)ガロイル基を有するフラバン-3-オール重合体
(A2)加水分解性タンニン
(A3)カテキンガレート、エピカテキンガレート及びガロカテキンガレートからなる群から選ばれる1以上の化合物
(2)上記没食子酸残基を有する化合物が、上記ガロイル基を有するフラバン-3-オール重合体である上記(1)に記載の腸管バリア機能改善用組成物。
(3)上記ガロイル基を有するフラバン-3-オール重合体が、ブドウ果実、ブドウ種皮及びブドウ種子からなる群より選択される1以上のブドウ由来の原料に含まれるものである上記(1)又は(2)に記載の腸管バリア機能改善用組成物。
(4)上記ガロイル基を有するフラバン-3-オール重合体は、タンナーゼ処理により生成する没食子酸が、上記ガロイル基を有するフラバン-3-オール重合体1mgあたり0.1μg以上である上記(1)~(3)のいずれかに記載の腸管バリア機能改善用組成物。
(5)上記加水分解性タンニンが、ガロタンニン又はエラジタンニンである上記(1)に記載の腸管バリア機能改善用組成物。
(6)上記ガロタンニンが、グルコース1分子に対し没食子酸が3~5分子結合した化合物である上記(5)に記載の腸管バリア機能改善用組成物。
(7)上記ガロタンニンが、1,4,6-トリ-O-ガロイル-β-D-グルコース、1,2,4,6-テトラ-O-ガロイル-β-D-グルコース、1,2,3,6-テトラ-O-ガロイル-β-D-グルコース、2,3,4,6-テトラ-O-ガロイル-β-D-グルコース及び1,2,3,4,6-ペンタ-O-ガロイル-β-D-グルコースからなる群より選択される1以上の化合物である上記(5)又は(6)に記載の腸管バリア機能改善用組成物。
(8)上記エラジタンニンが、テリマグランジンI(Tellimagrandin I)、ペドゥンクラジン(Pedunculagin)、プレコキシンA(Praecoxin A)、ゲラニイン(Geraniin)、ステノフィラニンA(Stenophyllanin A)、ステノフィラニンB(Stenophyllanin B)、カスアリニン(Casuarinin)及びユーゲニフロリンD2(Eugeniflorin D2)からなる群より選択される1以上の化合物である上記(5)に記載の腸管バリア機能改善用組成物。
(9)経口用組成物である上記(1)~(8)のいずれかに記載の腸管バリア機能改善用組成物。
(10)上記経口用組成物が、飲食品、医薬品又は医薬部外品である上記(9)に記載の腸管バリア機能改善用組成物。
(11)腸管バリア機能を改善することにより、整腸のために使用される上記(1)~(10)のいずれかに記載の腸管バリア機能改善用組成物。
(12)腸管バリア機能を改善することにより、腹部不快感を予防又は改善するために使用される上記(1)~(11)のいずれかに記載の腸管バリア機能改善用組成物。
(13)整腸作用を有する旨の表示を付した、上記(1)~(12)のいずれかに記載の腸管バリア機能改善用組成物。
(14)下記(A1)~(A3)からなる群より選択される1以上の没食子酸残基を有する化合物を対象に投与する、腸管バリア機能改善方法。
(A1)ガロイル基を有するフラバン-3-オール重合体
(A2)加水分解性タンニン
(A3)カテキンガレート、エピカテキンガレート及びガロカテキンガレートからなる群から選ばれる1以上の化合物
(15)腸管バリア機能を改善するための、下記(A1)~(A3)からなる群より選択される1以上の没食子酸残基を有する化合物の使用。
(A1)ガロイル基を有するフラバン-3-オール重合体
(A2)加水分解性タンニン
(A3)カテキンガレート、エピカテキンガレート及びガロカテキンガレートからなる群から選ばれる1以上の化合物
(A1)ガロイル基を有するフラバン-3-オール重合体
(A2)加水分解性タンニン
(A3)カテキンガレート、エピカテキンガレート及びガロカテキンガレートからなる群から選ばれる1以上の化合物
本発明の腸管バリア機能改善用組成物は、上記(A1)~(A3)からなる群より選択される化合物を1種のみ含んでいてもよく、2種以上含んでいてもよい。
(A1)ガロイル基を有するフラバン-3-オール重合体は、少なくとも1つの構成単位がガロイル基を有するフラバン-3-オールである重合体である。構成単位のフラバン-3-オールにおけるガロイル基の結合位置は特に限定されず、例えば、フラバン骨格に結合していてもよい。一態様において、本発明におけるガロイル基を有するフラバン-3-オール重合体は、フラバン骨格の3位にガロイル基が結合したフラバン-3-オール(フラバン骨格の3位の水酸基が没食子酸のカルボキシル基とエステル結合したフラバン-3-オール)を構成単位に有するフラバン-3-オール重合体であってよい。
ガロイル基を有するフラバン-3-オール重合体は、重合度等が異なる2種以上の重合体の混合物であってよい。ガロイル基を有するフラバン-3-オール重合体は、ガロイル基以外の没食子酸残基を有していてもよい。
没食子酸が生成したことの確認は、フラバン-3-オール重合体について、タンナーゼ処理又は酸加水分解処理の処理前と後の没食子酸量を測定することにより行うことができる。上記処理後のフラバン-3-オール重合体における没食子酸量が、処理前のフラバン-3-オール重合体における没食子酸量と比較して多い場合は、上記処理により没食子酸が生成したといえる。
一態様において、ガロイル基を有するフラバン-3-オール重合体は、タンナーゼ処理で生成する没食子酸が、該ガロイル基を有するフラバン-3-オール重合体1mg当たり0.1~100μgが好ましく、1~100μgがより好ましく、10~100μgがさらに好ましく、10~50μgが特に好ましい。
タンナーゼ処理により生成する没食子酸量の測定においては、タンナーゼ処理は、実施例に記載の条件で行うことができる。没食子酸量の測定は、実施例に記載の条件で、HPLCにより外部標準法にて行うことができる。
タンナーゼ処理により生成する没食子酸量は、タンナーゼ処理前のフラバン-3-オール重合体(処理前サンプル)及びタンナーゼ処理後のフラバン-3-オール重合体(処理後サンプル)の没食子酸量から、下記計算式により求められる。
タンナーゼ処理により生成する没食子酸量=(処理後サンプルの没食子酸量)-(処理前サンプルの没食子酸量)
没食子酸及び/又はエラグ酸が生成したことの確認は、化合物について、上記処理前後の没食子酸及び/又はエラグ酸量を測定することにより行うことができる。加水分解性タンニンをタンナーゼ処理又は酸加水分解処理する条件等は特に限定されず、例えば、上記のフラバン-3-オール重合体と同じ条件等を採用することができる。
一態様において、腸管バリア機能改善効果が高いことから、ガロタンニンとして、グルコース1分子に対し没食子酸が3~5分子結合した化合物(好ましくは、グルコースの3~5の水酸基に没食子酸が結合した、1分子中にガロイル基を3~5個有するガロタンニン)であることが好ましく、グルコース1分子に対し没食子酸が4分子結合した化合物(好ましくは、グルコースの4つの水酸基に没食子酸が結合した、ガロイル基を4個有するガロタンニン)がより好ましい。ガロタンニンとして、1,4,6-トリ-O-ガロイル-β-D-グルコース、1,2,4,6-テトラ-O-ガロイル-β-D-グルコース、1,2,3,6-テトラ-O-ガロイル-β-D-グルコース、2,3,4,6-テトラ-O-ガロイル-β-D-グルコース及び1,2,3,4,6-ペンタ-O-ガロイル-β-D-グルコースからなる群より選択される1以上の化合物が好ましく、中でも、1,2,4,6-テトラ-O-ガロイル-β-D-グルコース、1,2,3,6-テトラ-O-ガロイル-β-D-グルコース、2,3,4,6-テトラ-O-ガロイル-β-D-グルコースがより好ましい。
腸管バリア機能改善効果の点から、上記エラジタンニンとして、ガロイル基及び/又はHHDP基を有するものが好ましく、より好ましくは、HHDP基を有するか、又は、ガロイル基及びHHDP基を有する。エラジタンニンがHHDP基を有する場合、1分子あたりのHHDP基の数は、1又は2個が好ましく、2個がより好ましい。
腸管バリア機能改善効果の観点から、エラジタンニンとして、テリマグランジンI(Tellimagrandin I)、ペドゥンクラジン(Pedunculagin)、プレコキシンA(Praecoxin A)、ゲラニイン(Geraniin)、ステノフィラニンA(Stenophyllanin A)、ステノフィラニンB(Stenophyllanin B)、カスアリニン(Casuarinin)及びユーゲニフロリンD2(Eugeniflorin D2)からなる群より選択される1以上の化合物が好ましく、ゲラニイン(Geraniin)、ステノフィラニンA(Stenophyllanin A)、ステノフィラニンB(Stenophyllanin B)、カスアリニン(Casuarinin)がより好ましい。
加水分解性タンニンの原料として、加水分解性タンニンを含有する植物を使用することができる。加水分解性タンニンを含む植物として、例えば、ブナ科(Fagaceae)、ミソハギ科(Lythraceae)、フトモモ科(Myrtaceae)、バラ科(Rosaceae)の植物等が挙げられる。これらの植物には、加水分解性タンニンが多く含まれる。フトモモ科の植物として、フトモモ属(Syzygium)、ユーカリ属(Eucalyptus)、クンゼア属(Kunzea)等の植物等が好ましい。例えばユーカリ葉抽出物には、テリマグランジンI、ガロタンニン等の加水分解性タンニンが多く含まれている。
当業者は、目的に応じて腸管バリア機能改善効果の具体的な評価方法を選択することができる。例えば後述する実施例に示したように、ヒト腸管上皮細胞(Caco-2細胞)を用いた腸管透過モデルを用いて、TEERを測定する方法を用いることができる。具体的には、Caco-2単層培養細胞に炎症性サイトカイン(TNFα、IL-1β、IFNγ等)を添加してヒトで腸管バリア機能を破綻させ得る状態を作り出し、被験物質の添加により、該物質を添加しない場合と比べてTEERの低下が抑制されれば、その被験物質には腸管バリア機能改善効果があると評価することができる。
実施例に示されるように、没食子酸残基を有する(A1)~(A3)の化合物は、Caco-2を用いた腸管透過モデルにおいて、炎症性サイトカインの添加によるTEERの低下を抑制し、優れた腸管バリア機能改善効果を有することが示された。また、実施例に示されるように、没食子酸残基を有する化合物は、没食子酸残基を有さない化合物と比較して優れた腸管バリア機能改善効果を示した。例えば(A3)の化合物は、ガロイル基を有さない化合物(カテキン、エピカテキン、ガロカテキン)と比較して優れた腸管バリア機能改善効果を示した。没食子酸残基を有する(A1)~(A3)の化合物は、腸管上皮細胞におけるタイトジャンクションを正常化又は強化して、腸管バリア機能を改善することができる。
本発明の腸管バリア機能改善用組成物は、例えば、飲食品、医薬品、医薬部外品、飼料等の形態で提供することができるが、これらに限定されるものではない。本発明の腸管バリア機能改善用組成物は、それ自体が飲食品、医薬品、医薬部外品、飼料等であってもよく、これらに使用される添加剤等の製剤、素材であってもよい。本発明の腸管バリア機能改善用組成物は、一例として、剤の形態で提供することができるが、本形態に限定されるものではない。当該剤をそのまま組成物として、又は、当該剤を含む組成物として提供することもできる。
一態様において、本発明の腸管バリア機能改善用組成物は、好ましくは経口用組成物である。本発明によれば、優れた腸管バリア機能改善作用を有する経口用組成物を提供することができる。経口用組成物として、飲食品、医薬品、医薬部外品が挙げられ、好ましくは飲食品である。
一態様において、他の成分として、例えば、乳酸菌、ビフィズス菌、食物繊維、多糖類等を含有していてもよい。乳酸菌及びビフィズス菌は、経口的に摂取することができる菌であることが好ましい。
上記以外にも、その用途に応じて、飲食品、医薬品、医薬部外品、飼料等に使用される素材等の成分を適宜配合することができる。
整腸作用は、腸管バリア機能の改善に基づく整腸作用であればよく、特に限定されない。上記整腸作用を有する旨の表示の一例として、「便秘又は下痢気味な方に」、「おなかの調子が気になる方に」、「おなかの不快感を感じやすい方に」、「便通を改善」、「便の状態を改善」、「排便回数を改善」、「排便量を改善」、「おなかすっきり」、「おなかの調子を整える」、「腸の調子を整える」、「おなかの不快感を改善」、「ガスの発生を和らげる」、「おなかの張りを和らげる」、「おなかのごろごろ感を和らげる」等が挙げられる。本発明の腸管バリア機能改善用組成物には、このような表示の1又は2以上が付されていてもよい。
(A1)~(A3)の没食子酸残基を有する化合物の含量は、公知の方法に従って測定することができ、例えば、HPLC法等を用いることができる。
本発明の腸管バリア機能改善用組成物の摂取量(投与量ということもできる)は特に限定されず、腸管バリア機能改善効果が得られるような量であればよく、投与形態、投与方法等に応じて適宜設定すればよい。一態様として、ヒト(成人)(例えば、体重60kg)を対象に経口で投与する又は摂取させる場合、腸管バリア機能改善用組成物の摂取量は、(A1)~(A3)の化合物の総摂取量として、1日あたり、0.01~5000mgが好ましく、0.1~4000mgがより好ましく、1~3000mgがさらに好ましい。上記量を、例えば1日1回で又は2~3回に分けて経口投与又は摂取させることが好ましい。一態様において、ヒト(成人)を対象に腸管バリア機能改善効果を得ることを目的として腸管バリア機能改善用組成物を摂取させる場合は、(A1)~(A3)の化合物の総摂取量が上記範囲となるように、腸管バリア機能改善用組成物を対象に経口で摂取させる又は投与することができる。また本発明の一態様において、腸管バリア機能改善用組成物の摂取量は、ヒト(成人)(例えば、体重60kg)の場合、1日あたり、(A1)の化合物(ガロイル基を有するフラバン-3-オール重合体)の摂取量として1~2000mgが好ましく、10~1500mgがより好ましく、30~1000mgがさらに好ましく、100~1000mgが特に好ましい。(A1)の化合物の摂取量が上記範囲であると、優れた腸管バリア機能改善効果を得ることができる。上記量を、例えば1日1回で又は2~3回に分けて経口投与又は摂取させることが好ましい。一態様において、本発明の腸管バリア機能改善用組成物は、成人に、体重60kgあたり、1日あたり上記量の上記(A1)の化合物を摂取させる又は投与するための経口用組成物であってよい。
下記(A1)~(A3)からなる群より選択される1以上の没食子酸残基を有する化合物を対象に投与する、腸管バリア機能改善方法。
(A1)ガロイル基を有するフラバン-3-オール重合体
(A2)加水分解性タンニン
(A3)カテキンガレート、エピカテキンガレート及びガロカテキンガレートからなる群から選ばれる1以上の化合物
上記(A1)~(A3)からなる群より選択される1以上の没食子酸残基を有する化合物の、腸管バリア機能改善のための使用。
上記方法及び使用は、治療的な方法及び使用であってもよく、非治療的な方法及び使用であってもよい。「非治療的」とは、医療行為、すなわち手術、治療又は診断を含まない概念である。
没食子酸残基を有する化合物の投与量は、腸管バリア機能改善効果が得られる量、すなわち有効量であればよく、特に限定されず、例えば上述した量を投与することが好ましい。投与経路は、好ましくは経口投与である。没食子酸残基を有する化合物は、そのまま投与してもよいし、上述した没食子酸残基を有する化合物を含む組成物を投与してもよい。例えば、上述した本発明の腸管バリア機能改善用組成物を投与することができる。没食子酸残基を有する化合物、対象(投与対象)、投与方法、投与量及びそれらの好ましい態様等は、上述した腸管バリア機能改善用組成物におけるものと同じである。一態様において、例えば、ヒト(成人)を対象に経口投与する場合、(A1)の化合物(ガロイル基を有するフラバン-3-オール重合体)の1日あたりの投与量は、体重60kgあたり、1~2000mgが好ましく、10~1500mgがより好ましく、30~1000mgがさらに好ましく、100~1000mgがさらに好ましい。上記方法及び使用においては、(A1)~(A3)の1以上の没食子酸残基を有する化合物を1週間以上継続して投与することが好ましい。
ガロイル基を有するフラバン-3オール重合体(以下、フラバン-3-オール重合体を、OPCともいう)の精製
市販されているオリゴメリックプロシアニジン(フラバン-3-オール重合体)81%以上規格のブドウ種子エキス(ブドウ種子抽出物)を水で溶解し、酢酸エチルを用いて液々分離(分配抽出)を3回行った。得られた二つの画分を減圧濃縮、凍結乾燥し、乾燥粉末を得た。OPCを含有する水移行部を、文献(Biosci.Biotechnol. Biochem., 73, 1274-1279(2009))に記載の方法で分画し、さらに純度を高めたブドウ由来精製OPC画分を得た。
移行率(%)は、「100×収量(g)/出発原料(g)」を意味する。
上記ブドウ種子エキス(20.00g)を水(200mL)に溶解させた後、酢酸エチル(200mL)で3回分配抽出した。酢酸エチル移行部を、濃縮及び乾固し、フラクション1(Fr.1)とした(移行率:20.8%)。水移行部を、濃縮及び乾固し、フラクション2(Fr.2)を得た(移行率:70.6%)。フラクション2(10.06g)をメタノール(200mL)に溶解させ、これにクロロホルム(200mL)を添加し、得られた溶液を遠心分離し(5000rpm、5分)、沈殿(P1)と上清(S1)とに分けた。沈殿(P1)を濃縮及び乾固して、フラクション3(Fr.3)を得た(移行率:26.1%)。
上清(S1)に、クロロホルム(100mL)を添加し、遠心分離し(5000rpm、5分)、沈殿(P2)と上清(S2)とに分けた。沈殿(P2)を濃縮及び乾固して、フラクション4(Fr.4)を得た(移行率:15.5%)。
上清(S2)に、クロロホルム(168mL)を添加し、遠心分離し(5000rpm、5分)、沈殿(P3)と上清(S3)とに分けた。沈殿(P3)を濃縮及び乾固して、フラクション5(Fr.5)を得た(移行率:12.2%)。
上清(S3)に、クロロホルム(132mL)を添加し、遠心分離し(5000rpm、15分)、沈殿(P4)と上清(S4)と分けた。
沈殿(P4)を濃縮及び乾固して、フラクション6(Fr.6)を得た(移行率:3.6%)。上清(S4)を濃縮及び乾固して、フラクション7(Fr.7)を得た(移行率:9.8%)。
得られたフラクション5(Fr.5)をブドウ由来精製OPC画分とした。
OPCの分析
実施例1で得られたブドウ由来精製OPC画分について、特許第4659407号に記載の方法に従って純度を算出した。特許第4659407号に従ってブドウ由来精製OPC画分を酸加水分解処理し、下記の分析条件で分析した。
(高速液体クロマトグラフィー(HPLC)分析条件)
検出:520nm
カラム:YMC-Pack ODS-A(5μm、6.0mm i.d.×150mm、ワイエムシィ社製)
溶媒(移動相):酢酸:メタノール:蒸留水=15:17.5:67.5
カラム温度:40℃
流速:1mL/min
分析時間:12min
注入量:5μL
OPCの標準物質としてprocyanidinB1(AdooQ BioScience、純度99%以上)を用いた。
OPC純度(%)=100×(酸加水分解処理したブドウ由来精製OPC画分由来シアニジン濃度)/(酸加水分解処理したprocyanidinB1由来シアニジン濃度)
上記式を用いて、実施例1で調製したブドウ由来精製OPC画分中のOPCの純度を算出すると、91%であった。
ブドウ由来精製OPCのタンナーゼ処理
実施例1で作製したブドウ由来精製OPC画分20mgとタンナーゼ(和光純薬工業(株)製、Aspergillus oryzae由来)20mgの終濃度がそれぞれ1mg/mLとなるようにクエン酸緩衝液(pH5.5)に溶解し、30℃で一晩(約16時間)反応させてタンナーゼ処理を行った。得られた反応液の一部を、タンナーゼ処理後サンプルとして、後記する没食子酸の測定に用いた。また、この反応液をSep-Pak Vac 20cc(5g) C18 Cartridges(Waters社製)に供し、高極性成分、クエン酸塩、タンナーゼ及び没食子酸を除去するために蒸留水400mLで洗浄後、100%メタノール60mLでOPCを溶出させた。得られた100%メタノール溶出部を、減圧濃縮、凍結乾燥し、乾燥粉末を得た。このタンナーゼ処理をしたブドウ由来精製OPCを、ブドウ由来精製OPC(ガロイル基なし)とした。また、タンナーゼを添加しなかった以外は、上記と同様の工程を行い粉末を調製し、ブドウ由来精製OPC(ガロイル基あり)とした。
実施例1で作製したブドウ由来精製OPC画分を、濃度が1mg/mLとなるようにクエン酸緩衝液(pH5.5)に溶解し、タンナーゼ処理前サンプルとした。
上記で得たタンナーゼ処理後サンプル及びタンナーゼ処理前サンプルを、0.45μmフィルターに供したのち、HPLCで没食子酸量を分析した。没食子酸の定量は外部標準法にて行った。
検出波長:UV(280nm)
カラム:Capcell pak C18 (φ4.6×150.0mm,株式会社資生堂製)
溶媒A:0.05% トリフルオロ酢酸含有水溶液
溶媒B:0.05% トリフルオロ酢酸含有90%アセトニトリル水溶液
グラジエント:
溶媒B濃度(体積)%:10%、0-5 min;10-100%、5-20min;100%、20-25min;10%、25.01-30min
カラム温度:40℃
流速:1.0mL/min
インジェクション:10μL
タンナーゼ処理により生成した没食子酸量=(処理後の没食子酸量)-(処理前の没食子酸量)
上記式中、処理後の没食子酸量は、タンナーゼ処理後サンプルの没食子酸量であり、処理前の没食子酸量は、タンナーゼ処理前サンプルの没食子酸量である。
ガロイル基の有無による腸管バリア機能改善の比較評価を行った。
(腸管バリア機能改善作用の評価方法)
DMEM(ダルベッコ改変イーグル培地)を用いて、トランズウェル(Millicell社製)でCaco-2細胞を37℃で3週間培養した。培養したCaco-2細胞のプレートから培地を除去し、血清不含DMEMでウェルをそれぞれ3回洗浄し、その培地でウェルを満たした。その後、Millicell-ERS(ミリポア社製)によりCaco-2単層細胞の経上皮電気抵抗(TEER)を測定し、充分なタイトジャンクションが形成されていると判断される細胞(TEER≧1000Ω・cm2)を選抜して、次のスクリーニングに用いた。次いで、粘膜側、基底膜側両方の試験液(培地)にサンプルとTNFα(40ng/mL)、IL-1β(20ng/mL)及びIFNγ(10ng/mL)とを添加し、48時間培養した。なお、サンプルは、ジメチルスルホキシド(DMSO)に溶解後、試験液に添加した。この際に、炎症性サイトカイン(TNFα、IL-1β及びIFNγ)及びサンプルを添加しないウェルをノーマルとして設けた。また、炎症性サイトカインを添加し、サンプルを添加しないウェルをコントロールとして設けた。培養後、再びTEERを測定し、炎症性サイトカインによるTEERの低下(減少)をサンプルが抑制するかを評価した。
(TEER低下抑制率の計算式)
TEER低下抑制率(%)=100×((サンプルを添加したウェルのTEER)-(コントロールのTEER))/((ノーマルのTEER)-(コントロールのTEER))
この評価系では、TEER低下抑制率(%)が高いほど、腸管バリア改善作用が高い。
評価に用いたサンプルは、いずれも炎症性サイトカインによるTEERの低下を抑制した。また、上記評価の結果、カテキン、エピカテキン、ガロカテキンにおいて、非ガレート型化合物と比較し、ガレート型化合物は腸管バリア改善機能が高まることを見出した。結果を表2に示す。
ブドウ由来精製OPCは、例えば、試験液中の濃度を1μg/mLとした場合、ブドウ由来精製OPC(ガロイル基あり)のTEER低下抑制率は65%であり、ブドウ由来精製OPC(ガロイル基なし)は51%であった。
加水分解性タンニンの腸管バリア機能改善作用の評価
没食子酸残基を有する加水分解性タンニンをサンプルとして用いて、腸管バリア機能改善作用を評価した。加水分解性タンニンは、植物から精製したものを用いた(いずれも純度90%以上)。例えばテリマグランジンI及びガロタンニンは、ユーカリ・キエロカルパ(Eucalyptus cypellocarpa)の葉から精製した。ゲラニインはゲンノショウコ(Geranium thunbergii)の葉から精製した。その他の加水分解性タンニンについては、クンジア(Kunzea ambigua)の葉、ティーツリー(Melaleuca alternifolia)等のフトモモ科植物から精製したものを用いた。植物からの加水分解性タンニンの精製は、文献(笠島直樹、「Kunzea ambiguaおよびEucalyptus cypellocarpa の成分研究」(発刊2005年9月、博士論文、岡山大学)の76-95頁)に記載の方法で行った。
対比のため、サンプルに代えて、ケルセチン(フナコシ(株))を試験液中に10μmol/L添加して、上記の方法で腸管バリア機能改善作用を評価した。ケルセチン(10μmol/L)のTEER低下抑制率は、38%であった。評価した化合物について、ケルセチンと同濃度で、ケルセチンよりも高い腸管バリア機能改善作用が見出された。
腸管透過性及びストレス性大腸痛覚過敏に対するガロイル基を有するブドウ種子由来フラバン-3-オール重合体(OPC)の影響
一連の動物実験は、動物愛護管理法他関連法令を遵守し、社内動物実験委員会の審査を経て機関の長が承認した計画に基づき実施した。
ガロイル基を有するフラバン-3-オール重合体(OPC)の投与には、OPCを83%含有する市販のブドウ種子抽出物を使用した。このブドウ種子抽出物に含まれるOPCを実施例3に記載の方法でタンナーゼ処理すると、没食子酸が生成したことから、該OPCがガロイル基を有することが確認された。OPC1mgあたり没食子酸が25.5μg生成した。
群1:ストレス無+Vehicle(N=7)、群2:ストレス+Vehicle(N=6)、群3:ストレス+ブドウ種子抽出物(N=6)
群3のラットには上記ブドウ種子抽出物を投与し、群1及び2のラットには、Vehicle(蒸留水)を投与した。
ストレスを与える1週間前からラットにVehicle又はブドウ種子抽出物を経口投与した(いずれも1日1回)。ブドウ種子抽出物は、蒸留水に溶解させた10mg/mLの溶液を投与した。ブドウ種子抽出物の投与量は、ガロイル基を有するOPCに換算して、1日あたり体重あたり83mg/kg(10mL/kg)とした。群1及び2のラットには、蒸留水を体重あたり10mL/kg投与した。試験期間中、飼料(CRF-1、オリエンタル酵母株式会社)及び水は自由摂取できる状況で飼育を行った。
ストレスは、水槽の中央のプラットフォームにラットを1時間置くことで処置し、それを3日間(1日1回)行うことで大腸痛覚過敏を誘導した。ストレス処置期間(3日)は、Vehicle又はブドウ種子抽出物はストレスを与える1.5時間前に投与した。
痛覚閾値の評価結果は、ストレス前の痛覚閾値を100%とし、ストレス後の痛覚閾値の変化(100×ストレス後の痛覚閾値/ストレス前の痛覚閾値)(%)を算出した。
ストレス後の大腸痛覚過敏を評価した後に、麻酔下で大腸の内容物を洗浄し、大腸の上部を2ヶ所結紮し4cmのループを作製した。そこに1mLの1.5%エバンスブルー溶液を注入し、15分間静置した。結紮部位を取り出し、PBSとN-acetyl-cysteineで洗浄し、2mL N,N-dimethylformamideで透過したエバンスブルーを抽出した。その後、吸光度を測定してエバンスブルー透過量を求めた。大腸の腸管透過性(mg/g tissue)は、エバンスブルーの透過量(mg)を大腸の結紮部位の重量(g)で補正して算出した。
タイトジャンクションタンパク質Claudin-2の発現量は、proteinsimple社のWesシステムにより解析した。大腸の結紮部位の下から1cm組織を採取し、組織溶解液(1% SDS,1% Triton,1% sodium deoxycholate in PBS)を用いてサンプルを調製した。
図3(a)~(c)に、大腸の痛覚閾値、大腸の腸管透過性及びClaudin-2の発現に対するガロイル基を有するフラバン-3-オール重合体の影響を調べた結果を示す。図3(a)は痛覚閾値の評価結果、図3(b)は大腸の腸管透過性(大腸透過性)の評価結果、図3(c)はClaudin-2相対発現量である。Claudin-2の相対発現量は、群1における発現量を100とした相対発現量である。各グラフは平均±標準誤差で示し、群間の統計学的有意差については、群2(ストレス+Vehicle群)に対してDunnett testを実施した(*:p<0.05)。
Claims (15)
- 没食子酸残基を有する化合物を有効成分として含み、前記没食子酸残基を有する化合物が、下記(A1)~(A3)からなる群より選択される1以上の化合物である、腸管バリア機能改善用組成物。
(A1)ガロイル基を有するフラバン-3-オール重合体
(A2)加水分解性タンニン
(A3)カテキンガレート、エピカテキンガレート及びガロカテキンガレートからなる群から選ばれる1以上の化合物 - 前記没食子酸残基を有する化合物が、前記ガロイル基を有するフラバン-3-オール重合体である請求項1に記載の腸管バリア機能改善用組成物。
- 前記ガロイル基を有するフラバン-3-オール重合体が、ブドウ果実、ブドウ種皮及びブドウ種子からなる群より選択される1以上のブドウ由来の原料に含まれるものである請求項1又は2に記載の腸管バリア機能改善用組成物。
- 前記ガロイル基を有するフラバン-3-オール重合体は、タンナーゼ処理により生成する没食子酸が、前記ガロイル基を有するフラバン-3-オール重合体1mgあたり0.1μg以上である請求項1~3のいずれか一項に記載の腸管バリア機能改善用組成物。
- 前記加水分解性タンニンが、ガロタンニン又はエラジタンニンである請求項1に記載の腸管バリア機能改善用組成物。
- 前記ガロタンニンが、グルコース1分子に対し没食子酸が3~5分子結合した化合物である請求項5に記載の腸管バリア機能改善用組成物。
- 前記ガロタンニンが、1,4,6-トリ-O-ガロイル-β-D-グルコース、1,2,4,6-テトラ-O-ガロイル-β-D-グルコース、1,2,3,6-テトラ-O-ガロイル-β-D-グルコース、2,3,4,6-テトラ-O-ガロイル-β-D-グルコース及び1,2,3,4,6-ペンタ-O-ガロイル-β-D-グルコースからなる群より選択される1以上の化合物である請求項5又は6に記載の腸管バリア機能改善用組成物。
- 前記エラジタンニンが、テリマグランジンI(Tellimagrandin I)、ペドゥンクラジン(Pedunculagin)、プレコキシンA(Praecoxin A)、ゲラニイン(Geraniin)、ステノフィラニンA(Stenophyllanin A)、ステノフィラニンB(Stenophyllanin B)、カスアリニン(Casuarinin)及びユーゲニフロリンD2(Eugeniflorin D2)からなる群より選択される1以上の化合物である請求項5に記載の腸管バリア機能改善用組成物。
- 経口用組成物である請求項1~8のいずれか一項に記載の腸管バリア機能改善用組成物。
- 前記経口用組成物が、飲食品、医薬品又は医薬部外品である請求項9に記載の腸管バリア機能改善用組成物。
- 腸管バリア機能を改善することにより、整腸のために使用される請求項1~10のいずれか一項に記載の腸管バリア機能改善用組成物。
- 腸管バリア機能を改善することにより、腹部不快感を予防又は改善するために使用される請求項1~11のいずれか一項に記載の腸管バリア機能改善用組成物。
- 整腸作用を有する旨の表示を付した、請求項1~12のいずれか一項に記載の腸管バリア機能改善用組成物。
- 下記(A1)~(A3)からなる群より選択される1以上の没食子酸残基を有する化合物を対象に投与する、腸管バリア機能改善方法。
(A1)ガロイル基を有するフラバン-3-オール重合体
(A2)加水分解性タンニン
(A3)カテキンガレート、エピカテキンガレート及びガロカテキンガレートからなる群から選ばれる1以上の化合物 - 腸管バリア機能を改善するための、下記(A1)~(A3)からなる群より選択される1以上の没食子酸残基を有する化合物の使用。
(A1)ガロイル基を有するフラバン-3-オール重合体
(A2)加水分解性タンニン
(A3)カテキンガレート、エピカテキンガレート及びガロカテキンガレートからなる群から選ばれる1以上の化合物
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