Rage inhibitors

Abstract

The present invention discloses the use of an extract of a plant belonging to the tracheiaceae family or one or more compounds contained in the extract in the manufacture of a RAGE inhibitor for inhibiting the binding of AGE (advanced glycosylation end product) to an AGE-specific receptor (RAGE).

Description

Technical Field

[0001] The present invention relates to a novel RAGE (AGE-specific receptor) inhibitor. Background Art

[0002] Advanced Glycation Endproducts (AGE), also known as glycated proteins, Maillard reaction products, etc., are protein derivatives with various structures generated by non-enzymatic reactions between reducing sugars such as glucose and amino groups of proteins under hyperglycemia. AGE is associated with the onset and progression of various diseases as a result of glycation modification of extracellular matrix proteins, membrane proteins, and intracellular proteins, which leads to the destruction of the functions of these proteins and the cell functions that depend on them, or as a result of cellular responses caused by receptors with AGE as ligands.

[0003] For example, it is believed that if AGE is recognized by RAGE (Receptor for Advanced Glycation Endproducts, RAGE), which is one of the AGEs receptors, the generation of intracellular oxidative stress substances based on intracellular NADPH oxidase is enhanced, which changes the gene expression in epithelial cells, thereby causing various diabetic vascular disorders (see Non-Patent Document 1). In addition, in recent years, it has been revealed that AGE is not only related to diabetic vascular disorders, but also to cardiovascular disorders such as myocardial infarction and arteriosclerosis (see Non-Patent Document 2), neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (see Non-Patent Document 3), brain disorders and liver disorders caused by alcohol dependence (see Non-Patent Document 4), diabetic complications such as diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy (see Non-Patent Document 5), bone metabolism abnormalities such as osteoporosis (see Non-Patent Document 6), aging phenomena (see Non-Patent Document 7), insulin resistance (see Non-Patent Document 8), tumor proliferation and metastasis (see Non-Patent Document 9), etc. Furthermore, recent studies have shown that RAGE is present in various tissues such as blood vessels, kidneys, nerves, macrophages, and skin, and is involved not only in the above-mentioned diabetic complications but also in cancer and the like (see Non-Patent Document 10).

[0004] It is expected that the substance with the activity of inhibiting the binding of ligand to RAGE is effective as a therapeutic drug for the above-mentioned various diseases related to RAGE, and research related to its exploration and development is being actively carried out. As inhibitors of RAGE, compounds such as FPS-ZM1 and Azeliragon (Non-patent Document 11) are known. In addition, as ingredients derived from crude drugs, it is known that Mallotus japonicus extract (see Non-patent Document 12), Saururus chinensis Baill extract (see Patent Document 1), Flavangenol (see Patent Document 2) and the like have RAGE inhibitory activity.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2021-31424

[0008] Patent Document 2: Japanese Patent Application Publication No. 2020-33380

[0009] Non-patent literature

[0010] Non-patent document 1: Marie-Paule Wautier et al., Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE, Amarican Journal of Physiology-Endocrinology and Metabolism, May 2001, Vol. 280, E685-E694

[0011] Non-patent document 2: Yoshiteru Jinnouchi et al., Glycolaldehyde-modified low-density lipoprotein leads macrophages to foam cells via the macrophagescavenger receptor, The Journal of Biochemistry, 1998, Vol. 123, No. 6, pp. 1208-1217

[0012] Non-patent document 3: Nobuyuki Sasaki et al., Advanced Glycation End Products in Alzheimer's Disease and Other Neurodegenerative Diseases, The American Journal of Pathology, October 1998, Vol. 153, No. 4, pp. 1149-1155

[0013] Non-patent document 4: Keiko Iwamoto et al., Advanced glycation end products enhance the proliferation and activation of hepatic stellate cells, Journal of Gastroenterology, April 2008, Vol. 43, No. 4, pp. 298-304

[0014] Non-patent document 5: CW Yang et al., Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease, Proceedings of the National Academy of Sciences of the United States of America, September 27, 1994, Vol. 91, No. 20, p. 9436-9440

[0015] Non-patent document 6: James J. Tomasek et al., Diabetic and age-related enhancement of collagen-linked fluorescence in cortical bones of rats, Life Sciences, 1994, Vol. 55, p. 855-861

[0016] Non-patent document 7: Melpomeni Peppa et al., Aging and glycoxidant stress, Hormones, 2008, Vol. 7, No. 3, pp. 123-132

[0017] Non-patent literature 8: Masaya Mizuta, Insulin deficiency and oxidative stress, Japanese Journal of Pharmacology, 2005, Vol. 125, No. 3, pp. 125-128

[0018] Non-patent document 9: Riichiro Abe et al., Regulation of Human Melanoma Growth and Metastasis by AGE-AGE Receptor Interactions, Journal of Investigative Dermatology, February 2004, Vol. 122, No. 2, pp. 461-467

[0019] Non-patent document 10: Hiroshi Yamamoto, Basic research on the causes, pathology and overcoming of diabetic complications, Diabetes, Vol. 57, No. 10, 2014, pp. 765-771

[0020] Non-patent document 11: AHBurstein et al., Development of Azeliragon, an Oral SmallMolecule Antagonist of the Receptor for Advanced Glycation Endproducts, for the Potential Slowing of Loss of Cognition in Mild Alzheimer's Disease, The Journal of Prevention of Alzheimer's Disease, 2018, Volume 5, Issue 2, p.149-154

[0021] Non-patent literature 12: Teruki Matsumoto et al., Identification of pheophorbide as an inhibitor of receptor for advanced glycation end products in Mallotus japonicus, Journal of Natural Medicine, 2021, Vol. 75, No. 3, pp. 675-681 Summary of the invention

[0022] Problems to be solved by the invention

[0023] However, although azirige described in non-patent document 11 and the like has been confirmed to have binding inhibitory activity against various ligands and RAGE in in vitro tests, there are cases where clinical trials as Alzheimer's disease therapeutic drugs have failed due to confirmation of side effects. In addition, the RAGE inhibitors derived from crude drugs described in non-patent document 12 and patent documents 1 and 2 have the problem of low binding inhibitory activity between AGE and RAGE.

[0024] The object of the present invention is to provide a safe and highly active RAGE inhibitor.

[0025] Technical means to solve problems

[0026] The present inventors have repeatedly conducted in-depth studies to achieve the above-mentioned purpose, and as a result, have reached the following findings: the hot water extract of the peel of Trapa bispinosa, ellagic acid, 1,2,4,6-tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, and Tellimagrandin II contained in the extract have high inhibitory activity against the binding of AGE and RAGE. The inhibitory activity is higher than that of Azirig described in Non-Patent Document 11.

[0027] The present invention has been completed through further studies based on these findings, and provides the following RAGE inhibitors and the like.

[0028] Item 1. Use of an extract of a plant belonging to the family Trapaceae or one or more compounds contained in the extract for the manufacture of a RAGE inhibitor, wherein the RAGE inhibitor is used to inhibit the binding of AGE (advanced glycation end products) to an AGE-specific receptor (RAGE).

[0029] Item 2. The use according to Item 1, wherein the extract of the plant belonging to the family Trapezium is a pericarp extract of the plant belonging to the family Trapezium.

[0030] Item 3. The use according to Item 1 or 2, wherein the extract of the plant belonging to the family Nymphaceae or one or more compounds contained in the extract is at least one compound selected from the group consisting of one or more compounds belonging to the gallotannin or ellagitannin class and containing a β-D-glucose skeleton, and ellagic acid.

[0031] Item 4. The use according to Item 1 or 2, wherein the extract of the plant belonging to the family Trametes or one or more compounds contained in the extract is at least one compound selected from the group consisting of 1,2,4,6-tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, trematine II and ellagic acid.

[0032] Effects of the Invention

[0033] The active ingredient of the RAGE inhibitor of the present invention is a component contained in a plant belonging to the family Trapezium, which has been used as an edible food, and has been confirmed to be safe. As found in the present invention, it has a high inhibitory activity against the binding of AGE and RAGE. Thus, according to the present invention, a safe and highly active RAGE inhibitor can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a graph showing the results of measuring the AGE-RAGE binding inhibitory activity. The concentration unit on the horizontal axis is μg / ml, n=3, the values ​​are mean±SD, GG is the abbreviation for galloylglucose, and PentaGG is the abbreviation for 1,2,3,4,6-penta-O-galloyl-β-D-glucose. DETAILED DESCRIPTION

[0035] The RAGE inhibitor involved in one embodiment of the present invention (hereinafter sometimes simply referred to as "RAGE inhibitor") is a substance for inhibiting the binding of AGE to AGE-specific receptors, and is characterized in that an extract of a plant belonging to the family Trapezium or one or more compounds contained in the extract is used as an active ingredient.

[0036] The Trapaeaceae plant used as the extraction source of the RAGE inhibitor is not particularly limited, and specific examples thereof include Trapa japonica, Trapa natans L. ver. japonica, Trapaincisa, Trapa bispinosa Roxb., etc. The plant belonging to the Trapaeaceae is preferably a plant belonging to the genus Trapa, and among them, Trapa bispinosa Roxb. is preferred.

[0037] The extract can be obtained from the whole or various parts of the plant of the family Trapezium, such as flowers, flower spikes, peels, fruits, pulp, stems, leaves, roots, seeds, etc. As an extraction source, the peel of the plant of the family Trapezium is suitable. The extraction can be carried out by a commonly used method. Specifically, the peel of the plant is directly or cut into an appropriate size, extracted with a solvent, or extracted by homogenizing in a solvent. As an extraction solvent, for example, water, various organic solvents or mixed solvents thereof can be used. As an organic solvent for extraction, for example: lower alcohols (such as methanol, ethanol), chloroform, ethyl acetate, n-hexane, etc. can be cited. Among the extraction solvents, water, methanol, and ethanol are particularly preferred. In addition, these solvents can be used one or a mixture of two or more. The amount of the extraction solvent used varies depending on the part used, the extraction solvent, etc., and is preferably in the range of 1:2 to 1:30 (plant raw material: extraction solvent) in terms of mass ratio, preferably in the range of 1:3 to 1:20, and more preferably in the range of 1:5 to 1:10. The extraction time is preferably in the range of 1 hour to 15 days. The extraction temperature is preferably in the range of 5° C. to 100° C. A heated extraction solvent may be used as required to improve extraction efficiency (eg, hot water extraction). There are no particular restrictions on the extraction method, and any method such as intermittent extraction or continuous extraction using a chromatographic column may be used.

[0038] Before isolating one or more compounds from the solvent extract of the pericarp of a plant of the family Trametaceae, pretreatment may be performed by dialysis, ultrafiltration, filtration, column chromatography or the like in order to remove high molecular weight components, insoluble substances and the like.

[0039] When removing insoluble matter by filtration, adsorbents such as activated carbon, bentonite, diatomaceous earth, and filtration aids may be added as needed to remove impurities. In particular, when using the extract as a liquid, it is preferably sterilized by filtration using a membrane filter or the like.

[0040] Separation of the extract pretreated as required as described above can be carried out using any known method such as column chromatography, reverse phase chromatography, ion chromatography, etc., and can be carried out by fractionating a fraction having a high inhibitory activity against the binding of AGE and RAGE.

[0041] The active ingredient of the RAGE inhibitor can be one or more compounds contained in the extract of the plant belonging to the family Rhombaceae. As such a compound, for example, at least one compound selected from the group consisting of one or more compounds belonging to gallotannins or ellagitannins and containing a β-D-glucose skeleton and ellagic acid can be cited. Gallotannins and ellagitannins are a type of hydrolyzable tannin (the hydrolyzable tannin is hydrolyzed into polyphenolic acids and polyols under the action of acids, alkalis, enzymes, etc.), the former produces gallic acid as a polyphenolic acid, and the latter produces ellagic acid as a polyphenolic acid. The compounds belonging to gallotannins and ellagitannins used as the active ingredient of the RAGE inhibitor produce D-glucose as a polyol.

[0042] Examples of the compound belonging to the gallotannin class and containing a β-D-glucose skeleton include compounds represented by the following general formula (I) and (I′), and specific examples thereof include 2,6-di-O-galloyl-β-D-glucose, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,6-tri-O-galloyl-β-D-glucose, 2,3,6-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose, 1,2,4,6-tetra-O-galloyl-β-D-glucose, and 1,2,3,4,6-penta-O-galloyl-β-D-glucose.

[0043] [Chemical formula 1]

[0044]

[0045] [Chemical formula 2]

[0046]

[0047] In formula (I) and (I'), R, R' and R" each independently represent a hydrogen atom or a galloyl group represented by the following formula.

[0048] [Chemical formula 3]

[0049]

[0050] Examples of compounds belonging to the ellagitannins and containing a β-D-glucose skeleton include rugosin D represented by the following formula (II), cornusin G represented by the following formula (III), and telemax II represented by the following formula (IV).

[0051] [Chemical formula 4]

[0052]

[0053] [Chemical formula 5]

[0054]

[0055] [Chemical formula 6]

[0056]

[0057] In addition, ellagic acid is a compound represented by the following formula (V).

[0058] [Chemical formula 7]

[0059]

[0060] Particularly preferred compounds as active ingredients of RAGE inhibitors are 1,2,4,6-tetra-O-galloyl-β-D-glucose (a compound in which R and R" in the general formula (I) are galloyl groups and R' is a hydrogen atom), 1,2,3,4,6-penta-O-galloyl-β-D-glucose (a compound in which R, R' and R" in the general formula (I) are galloyl groups), tremaxine II (a compound represented by the formula (IV)), and ellagic acid (a compound represented by the formula (V)). These compounds may be used alone or in combination of any two or more thereof.

[0061] One or more compounds contained in the extract of plants belonging to the family Trapezaceae can be used in a free state or in a salt state. As salts, for example, salts with inorganic bases such as sodium salts, potassium salts, calcium salts, magnesium salts, aluminum salts, etc.; salts with organic bases such as methylamine, ethylamine, ethanolamine, etc.; salts with basic amino acids such as lysine, arginine, ornithine, and ammonium salts. The salt can be an acid addition salt, and as the above-mentioned salt, for example, acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid, glutamic acid, etc. can be mentioned. In addition, one or more compounds contained in the extract of plants belonging to the family Trapezaceae also include hydrates, solvates, polymorphs, etc. thereof.

[0062] The inhibitory activity against the binding of AGE to RAGE can be evaluated by studying the changes in the binding activity of AGE to RAGE in the presence or absence of a RAGE inhibitor.

[0063] RAGE inhibitors can be used as pharmaceutical compositions having one or both of therapeutic and preventive effects on diseases related to advanced glycation end products such as diabetes, diseases and symptoms related thereto, etc. As the form of administration of the pharmaceutical composition to humans or animals, oral administration, rectal administration, parenteral administration (e.g., intravenous administration, intramuscular administration, subcutaneous administration, etc.) and the like can be cited, and the dosage can be appropriately set according to the formulation form, administration method, purpose of use, and age, weight, symptoms, etc. of the subject to which it is applied, and it is difficult to clearly determine. In the case of humans, it is usually measured by the amount of the active ingredient contained in the preparation, preferably 0.1 mg / day to 2000 mg / day for adults per day. Of course, since the dosage varies according to various conditions, it is sometimes sufficient to administer with an amount less than the above dosage, or sometimes it is necessary to administer an amount exceeding the above range.

[0064] When prepared in the form of oral preparations, they can be prepared in the form of tablets, granules, powders, capsules, coatings, liquid preparations, suspensions, etc.; when prepared as non-oral preparations, they can be prepared in the form of injections, drops, suppositories, etc. Any known method can be used for formulation. For example, an extract of a plant belonging to the family Trapezium or one or more compounds contained in the extract can be combined with a pharmaceutically acceptable carrier or diluent, a stabilizer, and other required additives to prepare the above-mentioned required dosage form.

[0065] RAGE inhibitors can also be used as food and beverages (especially food and beverages for the purpose of health care, health maintenance, health promotion, etc. (such as health foods, functional foods, nutritional supplements, health foods, supplements, specific health foods, nutritional functional foods, or functional labeled foods)). Food and beverages can be in any form commonly used in food and beverages or health foods, such as capsules and tablets. The types of food and beverages are not particularly limited, and examples include liquid (flowing) foods such as coffee, juice, soft drinks, beer, milk, miso soup, soup, black tea, tea, nutrients, syrups, margarine, jam, rice, bread, potato products, mochi, candy, chocolate, furikake, ham, sausage, sugar (candy) and other solid foods such as staple foods, side dishes, cakes, seasonings, etc. It can also be formed into powders, granules, tablets, etc. according to the purpose. In addition, excipients, extenders, adhesives, thickeners, emulsifiers, colorants, spices, food additives, seasonings, etc. can be appropriately mixed as needed.

[0066] In addition, in addition to food and beverages for human consumption, RAGE inhibitors can also be used as functional feeds for livestock, pets and other animals. RAGE inhibitors can also be added to feeds for administration. That is, RAGE inhibitors can be used as functional feeds for livestock such as pigs, chickens, cattle, horses, sheep, fish, pets (dogs, cats, birds), etc., which are safe and have one or both of therapeutic effects and preventive effects on diseases related to advanced glycation end products such as diabetes, diseases and symptoms related thereto.

[0067] The ratio of the extract of the plant belonging to the family Trapezium contained in the RAGE inhibitor or one or more compounds contained in the extract is not particularly limited, and examples thereof include 0.01% by mass or more, 0.1% by mass or more, 1% by mass or more, 5% by mass or more, and 10% by mass or more. In addition, the upper limit thereof includes 99% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, and 10% by mass or less.

[0068] As shown in the examples described below, the present inventors have found that an extract of a plant belonging to the family Trapezium or one or more compounds contained in the extract exhibits an excellent RAGE inhibitory effect. Therefore, since an extract of a plant belonging to the family Trapezium or one or more compounds contained in the extract have an excellent RAGE inhibitory effect, they can be suitably used as an active ingredient of a RAGE inhibitor for inhibiting the binding of AGE to an AGE-specific receptor.

[0069] In addition, the extract of the plant belonging to the family Trapezium or one or more compounds contained in the extract are components contained in the plant belonging to the family Trapezium that has been eaten, and are highly safe.

[0070] It should be noted that, in the present specification, “comprising” also includes “consisting essentially of” and “consisting of.”

[0071] The various characteristics (properties, structures, functions, etc.) described for the various embodiments of the present invention described above can be combined in any manner to specify the subject matter included in the present invention. That is, the present invention includes all subject matter consisting of all combinations of the combinable characteristics described in this specification.

[0072] Example

[0073] Hereinafter, examples are given to explain the present invention in more detail, but the present invention is not limited to these examples and the like.

[0074] Example 1: Preparation of hot water extract of water chestnut peel (water chestnut extract)

[0075] The peel is separated from the fruit of Trapa bispinosa after harvest, cut and dried. Hot water extraction is performed by adding about 6 times the amount of 90°C hot water relative to the dried peel. The obtained extract is filtered and concentrated. Dextrin is added in a ratio of 33% by mass relative to 67% by mass of the concentrated solution, and spray drying is performed using a spray dryer to obtain a powdered Trapa bispinosa extract.

[0076] Test Example 1: Evaluation of the binding activity of AGE and RAGE

[0077] [method]

[0078] In the evaluation of the binding activity of AGE (glyceraldehyde-modified protein) and RAGE, the "CircuLex AGE-RAGE in vitro binding assay kit" (Medical Biology Research Institute Co., Ltd.) was used. The assay was carried out according to the operating procedure attached to the kit and the following steps. Add 85 μL of buffer and 5 μL of the assay sample solution (DMSO or distilled water is used as the solvent) and 10 μL of His-tag labeled sRAGE solution (final concentration 50 AU / mL) to the AGE-BSA solid phase plate and the BSA solid phase plate, and let stand at room temperature (24°C) for 60 minutes. Wash the wells 4 times with the washing buffer attached to the kit, add 100 μL of His-tag labeled monoclonal antibody HRP-bound solution, and let stand at room temperature (24°C) for 60 minutes. Wash the wells 4 times with the washing buffer, add 100 μL of the substrate solution, and react at room temperature for 10 minutes. After adding 100 μL of the reaction stop solution, measure the absorbance at a wavelength of 450 nm using an enzyme reader. After the measurement, the value of the BSA solid phase plate was subtracted from the value of the AGE-BSA solid phase plate, and the amount of sRAGE bound to AGE (A) was calculated using an sRAGE calibration curve prepared based on the attached His-tag labeled RAGE solution. Next, the AGE-RAGE binding inhibition rate was calculated by the following formula.

[0079] AGE-RAGE binding inhibition rate (%) = [sRAGE addition amount - (A)] / sRAGE addition amount × 100

[0080] [Measurement sample]

[0081] The measurement samples used in the evaluation are as follows. As a positive control, a compound reported to have AGE-RAGE binding inhibitory activity, i.e., azirig, was used. As the measurement object, eight compounds were used, including the water chestnut extract prepared in Example 1, dextrin used as a carrier when preparing the water chestnut extract, gallic acid, ellagic acid, trigalloyl glucose (1,2,3-tri-O-galloyl-β-D-glucose, 1,2,6-tri-O-galloyl-β-D-glucose), tetragalloyl glucose (1,2,3,6-tetra-O-galloyl-β-D-glucose, 1,2,4,6-tetra-O-galloyl-β-D-glucose), 1,2,3,4,6-penta-O-galloyl-β-D-glucose, and tremasin II as components of the water chestnut extract.

[0082] [result]

[0083] The results of the measurement of AGE-RAGE binding inhibitory activity are shown in Figure 1 Table 1. Trapa natans extract (IC 50 =0.11 μg / mL) showed a significantly stronger effect than the positive control azirige (IC 50 =4.7μg / mL). The inhibitory activities of the above 8 compounds as components of water chestnut extract were compared at a final concentration of 0.25μg / mL. As a result, 4 compounds (ellagic acid, 1,2,4,6-tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, and tremazone II) showed relatively strong inhibitory activities, so the IC values ​​of the compounds were calculated. 50 It should be noted that dextrin did not show inhibitory activity.

[0084] [Table 1]

[0085]

Claims

1. Use of an extract of a plant belonging to the family Trametaceae or one or more compounds contained in the extract in the manufacture of a RAGE inhibitor, wherein: The RAGE inhibitor is used to inhibit the binding of AGE (advanced glycation end products) to AGE-specific receptor (RAGE).

2. The use according to claim 1, wherein The extract of a plant belonging to the family Trapezium is an extract of a peel of a plant belonging to the family Trapezium.

3. The use according to claim 1 or 2, wherein The extract of the plant belonging to the family Trametaceae or the one or more compounds contained in the extract is at least one compound selected from the group consisting of one or more compounds belonging to gallotannins or ellagitannins and containing a β-D-glucose skeleton, and ellagic acid.

4. The use according to claim 1 or 2, wherein The extract of the plant belonging to the family Trametes or one or more compounds contained in the extract is at least one compound selected from the group consisting of 1,2,4,6-tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, tremelimine II and ellagic acid.

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