JP6729913B2 - Anti-inflammatory agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to an anti-inflammatory agent, and more particularly to an anti-inflammatory agent that exerts an anti-inflammatory action by suppressing the production of tumor necrosis factor α (TNF-α) and interleukin 8 (IL-8). Is.

Inflammation is a local transient reaction that a living body shows when a substance that causes some harmful irritation (a stimulating substance) acts on a living tissue, and is a kind of biological defense reaction. However, since it also damages biological tissues, when inflammation becomes chronic, an excessive inflammatory reaction is rather harmful. In recent years, the number of patients related to various inflammatory diseases has been increasing as a whole due to changes in lifestyle, air pollution, and increase in oxidative stress due to exposure to exhaust gas and excessive ultraviolet rays.

BACKGROUND ART Dipotassium glycyrrhizinate (hereinafter sometimes abbreviated as "GK2") is widely used as an anti-inflammatory component of cosmetics such as skin care products, quasi-drugs such as toothpaste, and drugs for treating stomatitis. However, GK2 is a derivative of glycyrrhizic acid contained in the roots of licorice, which is a plant of the legume family, and has an excellent anti-inflammatory effect, but has a steroid-like action. Is strictly regulated. Under such circumstances, there is a demand for an anti-inflammatory agent that can be used with peace of mind for the prevention and treatment of inflammatory diseases, in addition to alleviation and improvement of inflammatory symptoms.

Generally, carbohydrates are recognized as highly safe compounds, and in recent years, utilization of various carbohydrates as anti-inflammatory agents has been proposed. In Patent Document 1, nigerose (3-O-α-glucosylglucose), which is a glucodisaccharide, and nigerooligosaccharide, which contains a large amount thereof, produce TNF-α or interleukin 6 (IL-6), which are inflammatory cytokines. It is disclosed that it can be suppressed and used as an anti-inflammatory agent. Further, in Patent Document 2, a saccharide consisting of neo-agarobiose, neo-agarotetraose and neo-agarohexaose obtained by decomposing agarose (agar) with β-agarase suppresses the production of IL-6. , It can be used as an anti-inflammatory agent. Further, Non-Patent Document 1 discloses that D-psicose, which is a kind of ketohexose, has an anti-inflammatory effect. In addition, Patent Document 3 discloses an anti-inflammatory food containing trehalose and chondroitin sulfate.

However, while the above-mentioned anti-inflammatory agent containing a sugar as an active ingredient is highly safe, it has a relatively weak anti-inflammatory effect, and if it is not used in a large amount, the desired effect may not be obtained. was there. Under these circumstances, development of safer and more effective anti-inflammatory agents with less side effects is desired for the prevention and treatment of inflammatory diseases in addition to alleviation and improvement of inflammatory symptoms.

Japanese Patent No. 5444425 Japanese Patent No. 5154762 Japanese Patent No. 3247873

K. Murao et al., "Life Sciences", Vol. 81, 592-599 (2007).

It is an object of the present invention to provide a safe anti-inflammatory agent that is effective in alleviating and improving inflammatory symptoms, and further preventing and treating inflammatory diseases, and that contains a safe anti-inflammatory agent with few side effects. It is an object of the present invention to provide cosmetics, quasi-drugs, foods, and pharmaceuticals that meet the requirements. Another object is to provide a method for improving the anti-inflammatory action of a carbohydrate having an aldohexose structure in its molecule.

The present inventors have searched for sugars which are generally expected to have high safety, and have searched for substances having an anti-inflammatory action stronger than those of the above-mentioned saccharides which are conventionally known to have an anti-inflammatory action. .. Specifically, many of the excessive inflammatory reactions in the living body are caused by excessive inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and interleukin 8 (IL-8). Focusing on the cause of production, we searched for carbohydrates having a strong anti-inflammatory action by using the suppression of production of these inflammatory cytokines as an index. As a result, a carbohydrate having a ketoaldohexose structure in the molecule strongly shows an action of suppressing the production of inflammatory cytokines, but at the same time, it is not a cytotoxic agent at an effective concentration and is a safe anti-inflammatory agent with few side effects. The present invention has been completed by finding that it can be used as an active ingredient of.

That is, the present invention solves the above problem by providing an anti-inflammatory agent containing a sugar having a ketoaldohexose structure in the molecule as an active ingredient.

The present invention also solves the above problems by providing cosmetics, quasi drugs, foods and pharmaceuticals containing an anti-inflammatory agent containing a sugar having a ketoaldohexose structure in the molecule as an active ingredient. Is.

Furthermore, the present invention, in a sugar having an aldohexose structure in the molecule, a molecule characterized by converting a free hydroxyl group which is not bonded to other sugars or substituents in the aldohexose structure into a keto group. It is intended to solve the above problems by providing a method for improving the anti-inflammatory action of a carbohydrate having an aldohexose structure therein.

According to the present invention, a safe anti-inflammatory agent that suppresses the production of inflammatory cytokines and has few side effects can be provided. Further, according to the anti-inflammatory agent of the present invention, since the production of inflammatory cytokines such as TNF-α and IL-8 can be remarkably suppressed, the inflammatory symptoms caused by the overproduction of these inflammatory cytokines are alleviated. It can be used for improvement, and also for prevention and treatment of inflammatory diseases. Therefore, the anti-inflammatory agent of the present invention can be advantageously used as an oral agent or external preparation for cosmetics, quasi drugs, foods, pharmaceuticals and the like, and particularly, as an anti-inflammatory agent for skin inflammation, it has a sufficient anti-inflammatory effect. And has high safety, it can be advantageously used.

3 is an HPLC chromatogram of a 3-ketotrehalose preparation. It is a ¹³ C-NMR spectrum of a 3-ketotrehalose preparation.

The present invention relates to an anti-inflammatory agent containing a sugar having a ketoaldohexose structure in its molecule as an active ingredient.

The term "sugar having a ketoaldohexose structure in the molecule" as used in the present invention refers to an aldohexose (hexose having an aldehyde group at the 1-position) which is a free sugar that is not bound to another sugar or a substituent. It means all sugars and derivatives thereof having a structure in which one of the hydroxyl groups is oxidized and converted into a keto group in the molecule. A "carbohydrate having a ketoaldohexose structure in the molecule" is a method in which a microbial enzyme is allowed to act on a raw sugar or a derivative thereof, regardless of its origin or production method, even if it is organically synthesized. Method) or a method obtained by culturing and converting a microorganism using the starting sugar or its derivative as a carbon source (fermentation method) in the culture solution. Incidentally, hexose (hexose) is classified into an aldohexose having an aldehyde group at the 1-position and a ketohexose having a ketone group at the 2-position, and as the aldohexose, allose, altrose, glucose, mannose, gulose, Eight kinds of idose, galactose and talose are known. Further, as ketohexose, four kinds of psicose, fructose, sorbose and tagatose are known. The “sugar having a ketoaldohexose structure in the molecule” in the present invention is usually composed of a D-form having a high abundance ratio in nature.

The “sugar having a ketoaldohexose structure in the molecule” used as an active ingredient in the anti-inflammatory agent of the present invention is not particularly limited, but the 3-position hydroxyl group or 2-position hydroxyl group of aldohexose is oxidized and converted to a keto group. A sugar having a ketoaldohexose structure or a 2-ketoaldohexose structure in its molecule and a derivative thereof are preferably used. A carbohydrate having a 3-ketoaldohexose structure in the molecule and a derivative thereof (hereinafter abbreviated as "3-ketosugar" in the present specification), or a carbohydrate having a 2-ketoaldohexose structure and a derivative thereof (Hereinafter, it is abbreviated as “2-keto sugar” in the present specification.) It is preferable to use a monosaccharide, a homo- or hetero-disaccharide oligosaccharide or more, and a polysaccharide. it can.

<3-keto sugar>
Examples of the monosaccharide included in the 3-keto sugar in the present invention include 3-ketoglucose in which the 3-hydroxy group of glucose is oxidized and converted into a keto group, and 3-hydroxy group of galactose is oxidized and converted into a keto group. 3-ketogalactose and the like. In addition, as a monosaccharide derivative, for example, methyl α-3-ketoglucoside or methyl β- in which the 3-position hydroxyl group of each glucose residue of methyl α-glucoside or methyl β-glucoside is oxidized and converted into a keto group. 3-ketoglucoside, methyl α-galactoside or methyl β-galactoside, such as methyl α-3-ketogalactoside or methyl β-3-ketogalactoside in which the 3-position hydroxyl group of each galactose residue is oxidized and converted into a keto group, Can be mentioned. Examples of other monosaccharide derivatives include 1,5-anhydro-3-ketoglucitol in which the 3-hydroxyl group of 1,5-anhydroglucitol is oxidized and converted into a keto group.

Examples of the disaccharide included in the 3-keto sugar in the present invention include, for example, kojibiose (2-O-α-glucosyl glucose), which is a reducing gluco disaccharide having two glucose molecules bound thereto, and nigerose (3-O-α). -Glucosyl glucose), maltose (4-O-α-glucosyl glucose), isomaltose (6-O-α-glucosyl glucose) and cellobiose (4-O-β-glucosyl glucose) on the non-reducing end side. 3-ketocorgibiose (2-O-α-3-ketoglucosylglucose), 3-ketonigerose (3-O-α-3-ketoglucosylglucose) in which the 3-position hydroxyl group of each group was oxidized to be converted into a keto group ), 3-ketomaltose (4-O-α-3-ketoglucosylglucose), 3-ketoisomaltose (6-O-α-3-ketoglucosylglucose) and 3-ketocellobiose (4-O-β-). 3-ketoglucosyl glucose));
Non-reducing glucodisaccharide trehalose (α-glucosyl α-glucoside, α,α-trehalose), neotrehalose (α-glucosyl β-glucoside, α,β-trehalose), or isotrehalose (β-glucosyl β- Glucoside, β,β-trehalose), 3-ketotrehalose, 3-ketoneotrehalose, or 3-ketoisotrehalose in which the 3-position hydroxyl group of one of the glucose residues constituting the same is converted into a keto group;
3,3′-diketotrehalose, 3,3′-diketoneotrehalose in which two hydroxyl groups at three positions of two glucose residues constituting trehalose, neotrehalose, or isotrehalose are converted into keto groups Or 3,3'-diketoisotrehalose;
A heterodisaccharide in which different monosaccharides are bound to each other, for example, the 3-position hydroxyl group of the glucose residue of sucrose or isomaltulose, which is a non-reducing heterodisaccharide composed of glucose and fructose, is converted into a keto group. 3-ketosucrose (O-α-3-ketoglucosyl (1→2)β-fructoside) or 3-ketoisomaltulose (6-O-α-3-ketoglucosylfructose);
3-Ketolactose (4-O- in which the 3-position hydroxyl group of the galactose residue of lactose (4-O-β-galactosyl glucose), which is a reducing heterodisaccharide composed of galactose and glucose, is converted to a keto group. β-3-ketogalactosyl glucose));
Examples of the disaccharide derivative included in the 3-keto sugar include maltitol, which is a sugar alcohol obtained by reducing the aldehyde group of glucose at the reducing terminal of maltose or isomaltose, or the 3-position hydroxyl group of the glucose residue of isomaltitol. Of 3-ketomaltitol (4-O-α-3-ketoglucosyl sorbitol) or 3-ketoisomaltitol (6-O-α-3-ketoglucosyl sorbitol) further converted into a keto group; Lactitol, which is a sugar alcohol obtained by reducing the aldehyde group of reducing terminal glucose, has 3-ketraactitol (4-O-β-3-ketogalactosylsorbitol) in which the 3-position hydroxyl group of the galactose residue is further converted to a keto group; And so on.

The trisaccharide subsumed 3-keto sugars in the present invention, for example, melezitose ⁽³ F -O-alpha-glucosyl sucrose), 3 of terminal glucose residues Erurosu ⁽⁴ G ^-O-α- glucosyl sucrose) position hydroxyl group converted 3- Ketomerechitosu the keto group ⁽³ F ^-O-α-3- keto glucosyl sucrose) and 3- Ketoerurosu ⁽⁴ G ^-O-α-3- keto glucosyl sucrose) or maltotriose Examples thereof include 3-ketomaltotriose (4-O-α-3-ketoglucosylmaltose) in which the 3-position hydroxyl group of the non-reducing terminal glucose residue is converted to a keto group.

When the carbohydrate having an aldohexose structure in the molecule has a plurality of aldohexose structures in the molecule, usually, only the 3-position hydroxyl group of the aldohexose at the non-reducing end is keto-oxidized to be a 3-keto sugar. In 3-keto sugars, the 3-keto aldohexose structure is considered to be involved in the exertion of the anti-inflammatory effect, and the higher the proportion of the 3-keto aldohexose structure in the molecule, the stronger the anti-inflammatory effect. It is thought that. From this point of view, as the 3-keto sugar, a disaccharide or a monosaccharide having a large proportion of the 3-keto aldohexose structure in the molecule is particularly useful as an active ingredient of the anti-inflammatory agent.

However, when the 3-keto sugar is a monosaccharide, the hydroxyl group at the 1-position shows a reducing power because it may have the structure of an aldehyde group, and the keto group at the 3-position further shows a reducing power, and thus is highly reactive. , Becomes unstable as a compound. As the active ingredient of the anti-inflammatory agent of the present invention, a more stable 3-keto sugar is desirable, and in the case of a monosaccharide, the hydroxyl group at the 1-position is bonded to a substituent other than sugar to form a structure of an aldehyde group. It is more preferable that only the keto group at the 3-position shows a reducing power without being taken. Examples of such 3-keto sugar monosaccharides include methyl α-3-ketoglucoside and methyl β-3-ketoglucoside in which the hydroxyl group at the 1-position is methylated.

When the 3-keto sugar is a reducing disaccharide, usually, only the 3-position hydroxyl group of the aldohexose on the non-reducing terminal side is converted to a keto group, and the 1-position of the aldohexose is bonded to another sugar. Since there is no aldehyde group showing a reducing power in the same aldohexose molecule and it is more stable, it can be preferably used. Furthermore, 3-ketotrehalose and 3-ketosucrose obtained from trehalose and sucrose, which are non-reducing disaccharides, and 3,3′-diketotrehalose obtained from trehalose are also preferably used.

For the same reason, a sugar alcohol obtained by reducing the aldehyde group of glucose at the reducing terminal of disaccharide does not exhibit reducibility and is more stable as a compound, and thus can be preferably used. Examples of such sugar alcohols of disaccharides of 3-keto sugars include 3-keto maltitol, 3-ketoisomaltitol, and 3-ketolactitol.

The 3-keto sugar in the present invention is not particularly limited as long as it is a sugar having a 3-keto aldohexose structure, but sugars having a glucose structure or a galactose structure in the molecule are naturally present in large numbers, and Since the production rate of 3-keto sugars from these sugars by an enzymatic method or a fermentation method is high, for the purpose of industrially carrying out the present invention, as a 3-keto sugar, a 3-keto glucose structure or Carbohydrates having a 3-ketogalactose structure are particularly preferably used.

Usually, the preparation of 3-keto sugar is carried out by an enzymatic method or fermentation using a 3-keto sugar-forming enzyme capable of producing the desired 3-keto sugar by selectively oxidizing the 3-hydroxyl group of aldohexose. Done by law.

As an enzyme that catalyzes the reaction of oxidizing the 3-hydroxyl group of aldohexose to convert it to a keto group and producing a 3-keto sugar, glucoside 3-dehydrogenase (EC.1.1.99) is used. .13) and pyranose dehydrogenase (EC.1.1.9.99.29).

In addition, as a microorganism having the ability to produce glucoside 3-dehydrogenase, Rhizobium radiobacter, in the former classification, Agrobacterium tumefaciens, Flavobacterium saccharophilum (Flavovacti), Examples thereof include microorganisms belonging to the genus Sphingobacterium faecium, the genus Halomonas, the genus Cytophaga, and the genus Agaricus.

Examples of microorganisms having the ability to produce pyranose dehydrogenase include Agaricus bisporus, Agaricus meleagris, Agaricus xanthotherma, and Macrolepiota rocota lacodola lacodos lacodo Lacodolactos lacodolactos. .. Pyranose dehydrogenase may not only oxidize the 3-position hydroxyl group of the sugar aldohexose but also oxidize the 2-position and 4-position hydroxyl groups depending on the reaction conditions and the substrate to be reacted. When it is desired to selectively prepare a keto sugar, a glucoside 3-dehydrogenase that selectively oxidizes only the 3-position hydroxyl group of aldohexose is more preferably used.

The 3-keto sugar is usually converted (oxidized) by an enzymatic reaction by reacting a solution containing a starting sugar or a derivative thereof with a 3-keto sugar-forming enzyme such as the above-mentioned glucoside 3-dehydrogenase or pyranose dehydrogenase. The glucoside 3-dehydrogenase described above is a liquid medium produced by a method of recovering and purifying the resulting 3-keto sugar from a reaction solution or containing a sugar or its derivative as a raw material as a carbon source. Alternatively, a 3-ketosugar produced by culturing a microorganism capable of producing a 3-ketosugar-forming enzyme such as pyranose dehydrogenase and microbially converting (oxidizing) a raw material sugar or a derivative thereof in the culture solution It is manufactured by a method of further recovery and purification. Recovery and purification of 3-keto sugar from a reaction solution or a culture solution is carried out by removing bacterial cells and enzymes by centrifugation, removing proteins and the like which are insolubilized by heat treatment of the culture solution and the reaction solution supernatant, alcohol precipitation treatment, etc. Various operations such as removal of high-molecular polysaccharides by deionization, decolorization by activated carbon, desalting using ion exchange resin or electrodialyzer, chromatography using ion exchange resin, crystallization of 3-keto sugar using alcohol Can be combined as appropriate.

Among the various 3-keto sugars described above, 3-keto disaccharides such as 3-ketomaltose, 3-ketosucrose, 3-ketolactose, and 3-ketotrehalose are commonly used for maltose, sucrose, lactose, and trehalose. It is known that it is obtained from a disaccharide as a raw material and has a 3-ketoglucose structure or a 3-ketogalactose structure in the molecule, and that both are obtained in a crystalline form. Since it can be efficiently produced, it can be more suitably used as an active ingredient of the anti-inflammatory agent of the present invention.

<2-keto sugar>
The 2-keto sugar in the present invention is not particularly limited as long as it is a sugar having a 2-keto aldohexose structure, but as the 2-keto monosaccharide or 2-keto disaccharide preferably used in the present invention, for example, , 2-ketoglucose (2-dehydro-D-glucose), 2-ketotrehalose, 2-ketoneotrehalose, 2-ketoisotrehalose and the like.

The 2-keto sugar was usually prepared by using pyranose 2-oxidase (EC 1.1.3.10) capable of producing 2-ketoglucose by selectively oxidizing the 2-hydroxyl group of glucose. It is performed by an enzymatic method. In addition, examples of the microorganisms having the ability to produce pyranose 2-oxidase include mushrooms, such as Trametes versicolor, Trametes multicolor, and Basidiomycete fungus. 2-ketoglucose, and 2-ketosugar such as 2-ketotrehalose obtained by the transglycosylation reaction using 2-ketoglucose as an acceptor are also purified by the same method as described in the above-mentioned section of 3-ketosugar, It can be isolated.

As shown in Experiment 3 and the like described later, 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose, 3-ketoisomaltose, 3-ketomaltose, 3-ketosucrose, 3 which are 3-ketodisaccharides. -3-ketomaltitol, which is a sugar alcohol of keto disaccharide, and 2-keto glucose and 2-keto trehalose, which are 2-keto sugars, significantly exert a production inhibitory effect on TNF-α or IL-8. Since the concentration does not adversely affect cell growth and cell viability, saccharides having a ketoaldohexose structure in the molecule such as 3-keto sugar and 2-keto sugar are safe to be applied to the living body. It is considered to be a simple sugar.

As described above, many of the excessive inflammatory reactions in the living body are caused by excessive inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and interleukin 8 (IL-8). It is said that the cause is the production.

TNF-α is an inflammatory cytokine mainly produced by activated macrophages. Excessive production of TNF-α enhances the inflammatory response and strongly activates cytotoxic cells such as neutrophils. It has also been reported that vascularization induces damage to vascular endothelial cells and tissues, leading to organ dysfunction. It is also known that TNF-α is also produced from adipocytes enlarged by obesity and is involved in the onset and pathogenesis of type II diabetes by inducing insulin resistance. In general, overproduction of TNF-α is said to be involved in inflammation such as dermatitis, rheumatoid arthritis, psoriasis, and inflammatory bowel disease. On the other hand, IL-8 is known as an inflammatory cytokine that infiltrates neutrophils into inflamed areas. Neutrophils infiltrated in the inflamed area undergo degranulation, whereby enzymes such as myeloperoxidase and elastase having a tissue-damaging effect are released. Therefore, if the inflammatory reaction is prolonged, tissue damage will also be seen. Generally, overproduction of IL-8 is said to be involved in inflammation such as rheumatoid arthritis, psoriasis, bronchial asthma, sepsis, vascular inflammation and hepatitis. That is, suppressing the excessive production of inflammatory cytokines that cause the onset or exacerbation of inflammation is expected to be effective not only for alleviating and improving inflammatory symptoms but also for preventing and treating inflammatory diseases.

The carbohydrate having a ketoaldohexose structure in the molecule of the present invention is, as shown in the experimental section described later, TNF-α in human skin fibroblasts, epidermal keratinocytes, gingival fibroblasts, intestinal epithelial cells and the like. , Suppresses the production of IL-8 and the like, and therefore exerts its action effects even when ingested or applied to the skin, and is involved in overproduction of inflammatory cytokines such as TNF-α and IL-8. It can be advantageously used as an anti-inflammatory agent against inflammation.

In addition, it has been reported that oxidative stress caused by reactive oxygen species causes cell damage and is directly involved in various diseases including inflammatory diseases and aging phenomenon. It is also considered to be effective in the prevention and treatment of sexual disorders. The 3-keto sugar, which is an active ingredient of the anti-inflammatory agent of the present invention, has an antioxidant effect, and is also useful as an anti-inflammatory agent in this respect as well.

In general, inflammation is roughly divided into acute inflammation and chronic inflammation. Acute inflammation appears as the main signs of redness, swelling, fever, and pain in response to an external invasive stimulus, and if the causative agent of inflammation and infection are eliminated and the damaged tissues and cells are restored to their original state. Symptoms tend to converge, so they end in a short period of time. On the other hand, in chronic inflammation, structural changes and functional defects of tissues are caused by an incomplete or excessive biological immune response, so that the symptoms are maintained for a long period of time as compared with acute inflammation.

Examples of diseases corresponding to chronic inflammation include allergy such as hay fever, chronic respiratory disease, asthma, sepsis, rheumatoid arthritis, ARDS (acute respiratory distress syndrome), hepatitis, gastritis, inflammatory bowel disease, pancreatitis, Arthritis, arteriosclerosis, ischemia-reperfusion injury, uveitis, endotoxin shock, burn, viral myocarditis, idiopathic dilated cardiomyopathy, SIRS (systemic inflammatory response syndrome), multiple organ failure, hemolytic uremic syndrome And diseases caused by vascular endothelial cell damage such as hemorrhagic colitis, hyperγ-globulinemia, systemic lupus erythematosus (SLE), atherosclerosis, multiple sclerosis, monoclonal B cell abnormality, polyclonal B-cell abnormalities, atrial myxoma, Castorman's syndrome, primary glomerulonephritis, mesangial proliferative nephritis, nephritis such as diabetic nephritis, postmenopausal osteoporosis, gingivitis, periodontal disease, allergic dermatitis, atopic skin Inflammation of the skin, such as a flame. Incidentally, examples of the hepatitis include alcoholic hepatitis, viral hepatitis, drug-induced hepatitis, non-alcoholic fatty liver, autoimmune hepatitis, liver fibrosis, cirrhosis and fulminant hepatitis. Examples of inflammatory bowel disease include ulcerative colitis and Crohn's disease. Furthermore, examples of neurodegenerative diseases caused by inflammation of nerve cells include Parkinson's disease and Alzheimer's disease. Chronic inflammation is not only the above-mentioned inflammatory diseases but also a common basis of lifestyle-related diseases such as obesity, metabolic syndrome, diabetes and arteriosclerotic diseases.

The carbohydrate having a ketoaldohexose structure in the molecule, which is an active ingredient of the anti-inflammatory agent of the present invention, is a highly safe substance and can be advantageously used as an oral preparation or an external preparation for a long period of time. Can be effectively used as an anti-inflammatory agent against When a sugar having a ketoaldohexose structure is compounded in the molecule as an active ingredient, 0.1 to 90% by mass (hereinafter, unless otherwise specified, the mass% is referred to as “% It is desirable that the content is 10 to 90%.

The carbohydrate having a ketoaldohexose structure in the molecule, which is an active ingredient of the anti-inflammatory agent of the present invention, affects safety, anti-inflammatory effect, and suppressive action on the production of inflammatory cytokines such as TNF-α and IL-8. Unless otherwise specified, it may be a standard product containing components derived from manufacturing raw materials and by-products generated in the manufacturing process. However, it is desirable to use a material having a purity as high as possible, and normally, a material having a purity of 80% or more, preferably 90% or more, more preferably 95% or more, in terms of solid matter, is preferably used.

Further, the anti-inflammatory agent of the present invention can be incorporated into various compositions such as cosmetics, quasi drugs, foods, and pharmaceuticals. The amount of the sugar having a ketoaldohexose structure in the molecule, which is the active ingredient, in various compositions may be an amount capable of exerting an anti-inflammatory effect, and is not particularly limited, but is usually cosmetics or quasi-drugs. It is suitable to contain 0.01% or more, preferably 0.1% or more, and more preferably 0.2% or more, in terms of anhydride, based on the total mass of the product, food or medicine. Usually, if it is less than 0.01%, it may be insufficient to exert an anti-inflammatory effect.

Although the anti-inflammatory agent of the present invention can exert an anti-inflammatory action alone, it can be advantageously used in combination with a known component having an anti-inflammatory action.

Known components having an anti-inflammatory action include, for example, allantoin or a derivative thereof, glycyrrhetin or a derivative thereof, pantothenic acid or a derivative thereof, vitamin E or a derivative thereof, L-ascorbic acid or a derivative thereof, pyridoxine hydrochloride, menthol, biotin, Camphor, turpentine oil, zinc oxide, azulene, guaiazulene and its derivatives, mefenamic acid and its derivatives, phenylbutazone and its derivatives, indomethacin and its derivatives, ibuprofen and its derivatives, ketoprofen and its derivatives, ε-aminocaproic acid, diclofenac sodium. , Diphenhydramine, tranexamic acid and its derivatives, dexamethasone, cortisone and its ester, hydrocortisone and its ester, prednisone, adrenocorticosteroids such as prednisolone, indigo, Athene yak, amacha, artea, arnica, aloe, ibutranoo, nettle, turmeric, agets, Echinashi, Trillium vulgaris, Ogon, Otaku, Barley, Hypericum perforatum, Odorichosou, orange, valerian, chamomile, chamomile extract, carrot, kawara wormwood, licorice, cucumber, goldfish, gardenia, sardine, sardine, genus, gentian, genus, gentian, genus Comfrey, salvia, salamander, perilla, perilla, syringa, peony, birch, horsetail, St. John's wort, oak ivy, oat mouse, oat mint, sage senkyu, senburi, sohakuhi, taiso, thyme, cha, chimpi, tencha, chili, Touki, pearl millet, tonnin, Dokudami, tormentilla, elderberry, carrot, parsley, peppermint, rose, sandalwood, loquat, butterbur, butcherbroom, grape, broccoli, safflower, salamander, bodice, button, horse chestnut, sprout, peach, yam Examples thereof include plants or plant-derived components such as cornflower, eucalyptus extract, eucalyptus, mugwort, lavender, rosehip, rosemary, and chamomile.

In the production of the composition containing the anti-inflammatory agent of the present invention, at least one kind of sugar having a ketoaldohexose structure in the molecule, which is an active ingredient, and cosmetics, quasi drugs, foods, pharmaceuticals, etc. The optional components that are usually used in the case of producing are preferably added. The form of the agent is not particularly limited, and may be an external preparation or an oral preparation.

When the composition containing the anti-inflammatory agent of the present invention is a cosmetic product, purified water, a pH adjusting agent, a lower alcohol, a polyhydric alcohol, oils and fats, waxes, hydrocarbons, fatty acids, esters are optional components. , Organic solvents, silicones, surfactants, thickeners, softeners, emulsifiers, defoamers, humidifiers, fragrances, sequestering agents, dyes, colorants, water-soluble polymers, preservatives, buffers A liquid, a dye, an ultraviolet absorber, an astringent, a bactericidal/antibacterial agent, a moisturizer, a cell activating agent, an anti-inflammatory/anti-allergic agent, an antioxidant/active oxygen removing agent, a fragrance, and various medicinal ingredients can be added.

Cosmetics containing the anti-inflammatory agent of the present invention include toilet soap, facial cleansing cream, facial cleansing foam, cleansing cream, cleansing milk, cleansing lotion, cleansing oil, massage cream, cold cream, moisture cream, vanishing cream, hand cream, moisture. Lotion, cosmetic oil, liquid foundation, powder foundation, cake foundation, stick foundation, oily compact foundation, cream foundation, cheek blusher, emulsification foundation, foundation cosmetic, body powder, creamy white powder, white powder, water white powder, solid Mold white powder, talcum powder, paste white powder, loose shadow, baby powder, blusher, eyebrow, mascara, lipstick, lip balm, pack, shaving cream, after shaving cream, lotion, hand lotion, shaving lotion, after shaving lotion, sunscreen Cream, suntan oil, sunscreen lotion, suntan lotion, softening lotion, astringent lotion, wash lotion, multi-layer lotion, facial shampoo, body shampoo, hair shampoo, hair wash powder, hand soap, facial rinse, Hodgy Rinse, Hair Rinse, Hair Treatment, Chick, Pomade, Hair Cream, Hair Liquid, Hair Tonic, Set Lotion, Ski Oil, Bale Oil, Hair Spray, Hair Mousse, Hair Tonic, Hair Dye, Hair Bleach, Color Rinse, Color Spray, Permanent Wave liquid, press powder, loose powder, eye cream, eye shadow, cream eye shadow, powder eye shadow, eyeliner, eyebrow pencil, mascara, hair removal cream, general perfume, kneading perfume, powder perfume, cologne, deodorant, bath agent , Bath oil, bath salt, cosmetic oil, baby oil, nail color, enamel, enamel remover, nail treatment and the like.

When the composition containing the anti-inflammatory agent of the present invention is a quasi drug, as an optional component, an excipient, a base, a surfactant, a dispersant, a solubilizer, a solvent, an alkaline agent, a viscosity modifier. , Thickener, film forming agent, foaming agent, defoaming agent, flavoring agent, coloring agent, stabilizer, preservative, bactericide, anti-fading agent, antioxidant, hair treatment agent, wetting agent, hair protecting agent , Hair follicle activator, antistatic agent, auxiliary agent, solvent, dissolving agent, solubilizing agent, fluidizing agent, suspension agent, buffer agent, binder, adsorbent, propellant, coating agent, chewable agent, filler , Conventional additives such as softeners, modifiers, sequestering agents, anti-fading agents, fats and oils, oil-soluble polymers, analgesics, disintegrants, lubricants, emulsifiers, isotonic agents, etc. can be added. ..

As a quasi drug containing the anti-inflammatory agent of the present invention, a quasi drug used for the oral cavity, for example, toothpaste, mouthwash, mouth rinse, drink, quasi drug used for the scalp, for example, hair nourishing agent , Hair-growth agents, hair loss prevention agents, depilatory agents, massage agents, and other insect repellents, ointment-treating ointments, antibacterial creams, steroid ointments, sheet-like or film-like patches to be applied to affected areas of the oral cavity or skin. Examples include agents, beauty essences, medicated soaps, clean cotton, bath agents, baby powders, and disinfectants for soft contact lenses.

When the composition containing the anti-inflammatory agent of the present invention is a food, as an optional component, water, alcohol, starch, protein, fiber, sugar, lipid, vitamin, mineral, flavoring agent, colorant, sweetener Raw materials or raw materials usually added to foods such as seasonings, stabilizers and preservatives can be added.

Examples of foods containing the anti-inflammatory agent of the present invention include beverages, soups, alcoholic beverages, jellies, hard candy, soft candy, gummy candy, chewing gum, chocolates, tablets and frozen desserts. Foods also include functional foods, health foods, health-oriented foods, and the like.

The form of the drug containing the anti-inflammatory agent of the present invention includes solid preparations such as powders, tablets, pills, capsules, fine granules and granules, liquid preparations such as water preparations, suspension preparations and emulsions, gel preparations. Etc. If necessary, tablets, pills, granules, and granules of capsules containing granules may be sugar-coated with sugars such as sucrose, sugar alcohols such as maltitol, or with gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc. It may be coated or may be covered with a film of gastric or enteric substance. Further, in order to improve the solubility of the preparation, the above-mentioned preparation may be subjected to a known solubilization treatment. Based on a conventional method, the above liquid preparation may be used by mixing it with an injection or a drip.

When using a drug containing the anti-inflammatory agent of the present invention, the dose or intake is not particularly limited as long as the desired improvement, therapeutic or prophylactic effect is obtained, usually its aspect, patient It is appropriately selected according to the age, sex, constitution and other conditions, type of disease and its degree. For example, a sugar having a ketoaldohexose structure in the molecule which is an active ingredient in the drug is blended so as to have a content of 0.01 to 30% in terms of an anhydride, and a ketoaldohexose structure is incorporated in the molecule. The sugars to be contained may be ingested in the range of about 0.1 mg to 1,000 mg per day.

Further, the anti-inflammatory agent of the present invention is excellent in safety, not only for humans, for example, non-human animals, for example, rat, mouse, guinea pig, rabbit, sheep, pig, cow, You may mix|blend with therapeutic agents, mammals, such as horses, cats, dogs, monkeys, and chimpanzees, birds, amphibians, reptiles, etc., feed, or feed. As the feed or feed, for example, sheep, pig, cow, horse, feed for livestock used for chicken, etc., feed for small animals used for rabbits, rats, mice, etc., eel, Thailand, yellowtail, feed for seafood used for shrimp, etc., Examples include pet foods used for dogs, cats, small birds, squirrels, and the like.

Further, according to the method for improving the anti-inflammatory effect of the present invention, by converting a free hydroxyl group which is not bonded to another sugar or a substituent in a sugar having an aldohexose structure in the molecule into a keto group. Since it is possible to improve the anti-inflammatory action of a carbohydrate having a weak anti-inflammatory action, it is also useful as a method for improving the anti-inflammatory action of a carbohydrate having an aldohexose structure.

Examples of a suitable method for improving the anti-inflammatory action include a method of converting the 3-position hydroxyl group or 2-position hydroxyl group in the aldohexose structure into a keto group by an enzymatic method or a fermentation method. In the case of the method, the aldohexose structure is used. The 3-position hydroxyl group or the 2-position hydroxyl group of the aldohexose at the non-reducing end of the sugar having in the molecule is oxidized, and the anti-inflammatory action of the sugar is improved as compared with that before conversion.

Hereinafter, the present invention will be specifically described by experiments.

<Experiment 1: Preparation of various 3-keto sugars>
Trekerose, kojibiose, isomaltose, lactose, maltose, sucrose and maltitol are used as raw material sugars, respectively, and 3-ketotrehalose, 3-ketocodybiose, 3-ketoisomaltose, 3-ketolactose, 3-by fermentation. Ketomaltose, 3-ketosucrose and 3-ketomaltitol were prepared. In addition, 3,3′-diketotrehalose and methyl α-3-ketoglucoside were prepared by an enzymatic method using trehalose and methyl α-glucoside as starting sugars, respectively.

<Experiment 1-1: 3-Preparation of 3-ketotrehalose>
Trehalose (registered trademark "Treha", sold by Hayashibara Co., Ltd., trehalose purity 98.0% or more) 20 (w/v)%, polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.) 1 (w/v)%, yeast extract S (Japan Pharmaceutical Co., Ltd.) 0.2 (w/v)%, magnesium sulfate heptahydrate 0.01 (w/v)%, and a liquid medium (pH 6.8) consisting of water, put 3 mL per test tube. After sterilizing in an autoclave at 121°C for 20 minutes and cooling, one platinum loop of Flavobacterium saccharophilum NBRC15944 strain cultivated at 27°C in an agar slant medium was inoculated and shaken at 27°C for 72 hours at 270 rpm. This was used as the seed culture solution.

A liquid medium having the same composition as the above, 50 mL of which was placed in a 500 mL Erlenmeyer flask was prepared, adjusted to pH 6.8, sterilized at 121° C. for 20 minutes, cooled, and then the above seed culture solution was added. 0.5 mL each was inoculated to each Erlenmeyer flask, and then cultured at 27° C. for 72 hours with rotary shaking tow to obtain a main culture solution. When the sugar composition of the culture broth was analyzed by HPLC, a new sugar peak other than the raw material trehalose was confirmed.

After completion of the culture, the resulting culture solution of about 2,300 mL was centrifuged at 10,000 rpm to remove the cells, and the obtained culture supernatant solution was heat-treated in a water bath at 100°C. Insoluble matter was removed by centrifugation at 10,000 rpm, and the supernatant was collected. Ethanol (9,200 mL) was added to the obtained supernatant and centrifuged at 10,000 rpm to collect the supernatant. The recovered solution was concentrated to 1,100 mL under reduced pressure with an evaporator, filtered with a 0.2 μm membrane filter, and then applied to an electrodialyzer (trade name “Micro Acylyzer G0”, manufactured by Asahi Kasei Kogyo Co., Ltd.) to remove the solution. Salted The obtained electrodialysate was filtered through a 0.2 μm membrane filter and subjected to preparative HPLC under the following conditions to collect a peak fraction of a sugar newly formed from the raw material trehalose, which was concentrated and freeze-dried. It was pulverized to obtain 76.5 g of a standard product.

(Preparative HPLC conditions)
Column: YMC-Pack ODS-AQ
(Inner diameter 50 mm, length 500 mm, made by YMC Co., Ltd.)
Eluent: Ultrapure water Flow rate: 5.0 mL/min Column temperature: 25°C
Detection: Differential refractometer

The obtained preparation was subjected to HPLC analysis under the following conditions, and the purity was determined to be 98.1% based on the HPLC chromatogram (Fig. 1). Further, when the sample was subjected to LC/MS analysis, it was found to have a molecular weight 340 that was 2 (two hydrogen atoms) smaller than the molecular weight 342 of trehalose. Furthermore, when the NMR ( ¹ H-NMR and ¹³ C-NMR) spectrum was measured using the sample as a sample, the glucose of one of the two glucose residues constituting trehalose in the ¹³ C-NMR spectrum (FIG. 2) was measured. It was confirmed that the signal of the 3rd carbon of the residue was largely shifted to the low magnetic field side (Spectral database (SDBS) of organic compounds provided by the National Institute of Advanced Industrial Science and Technology (SDBS) "http://sdbs .db.aist.go.jp/sdbs/cgi-bin/cre_index.cgi” was compared with the ¹³ C-NMR data of the raw material trehalose), and it was found that this product was 3-ketotrehalose. ..

(HPLC conditions for analysis)
Device: Prominence (Shimadzu Corporation)
Column: Shodex SUGAR KS-801
(Inner diameter 8 mm, length 300 mm, Showa Denko KK)
Eluent: Ultrapure water Flow rate: 0.4 mL/min Column temperature: 75°C
Detection: Differential refractometer

<Experiment 1-2: Preparation of 3-ketocorgi biose>
Maltose 5.0% (w/v), yeast extract S (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.1 (w/v), fish meat extract (manufactured by Maruha Co., Ltd.) 0.3 (w/v)%, phosphoric acid Dipotassium hydrogen 0.01 (w/v)%, sodium dihydrogen phosphate monohydrate 0.06 (w/v)%, magnesium sulfate heptahydrate 0.05 (w/v)%, iron sulfate Liquid medium consisting of 0.001 (w/v) heptahydrate, 0.001 (w/v)% manganese sulfate n hydrate, 0.3 (w/v)% calcium carbonate and water (pH 6. 8 mL) was placed in a test tube at 3 mL, sterilized in an autoclave at 121° C. for 20 minutes, cooled, and inoculated with one platinum loop of Rhizobium radiobacter G37 strain cultured at 27° C. in an agar slant medium, and 27° C. What was cultivated with shaking tow for 72 hours was used as a seed culture solution.

A liquid medium having the same composition as described above except that maltose was replaced with kojibiose (Hayashibara Co., Ltd., purity: 95.9%) was prepared by mixing 50 mL in a 500 mL Erlenmeyer flask to pH 6.8. After the adjustment, it was sterilized at 121° C. for 20 minutes and cooled. Then, 0.5 mL of each of the above-mentioned seed cultures was inoculated into each Erlenmeyer flask, followed by rotary shaking tow culture at 27° C. for 54 hours to obtain a main culture. When the sugar composition of the culture solution was analyzed by HPLC, a new sugar peak other than the raw material kojibiose was confirmed.

After completion of the culturing, 1,200 mL of deionized water was added to the obtained culture solution of about 500 mL to dilute it, and the cells were removed by centrifugation at 8,000 rpm, and ethanol 5 was added to the obtained supernatant. , 100 mL was added, and the precipitate was removed by filtration. Next, the filtrate was concentrated to 250 mL under reduced pressure by an evaporator, 500 mL of ethanol was added to the obtained concentrated solution, and the supernatant was recovered by centrifugation at 8,000 rpm. The collected solution was concentrated to 300 mL under reduced pressure with an evaporator, centrifuged at 11,000 rpm to collect the supernatant, filtered with a 0.2 μm membrane filter, and then electrodialyzed (trade name “Micro Acylizer G1", manufactured by Asahi Kasei Kogyo Co., Ltd.) for desalting. The resulting electrodialysate was concentrated to about 20 mL under reduced pressure with an evaporator, filtered through a 0.2 μm membrane filter, and preparative HPLC was repeated under the following conditions to obtain a peak fraction of sugar newly generated from raw material Kojibiose. Fractions were collected, concentrated, and lyophilized to give a powder (8.7 g). When the purity of the obtained standard product was analyzed by HPLC, it was 91.2%. When analyzed by the same LC/MS analysis and ¹³ C-NMR spectrum analysis as those performed for 3-ketotrehalose in Experiment 1-1, the obtained product was found to be 3-ketocorgibiose (2-O-α-3). -Ketoglucosyl glucose).

(Preparative HPLC conditions)
Column: YMC-Pack R&D ODS-A
(Inner diameter 20 mm, length 250 mm, made by YMC Co., Ltd.)
Eluent: Ultrapure water Flow rate: 3.5 mL/min Column temperature: 35°C
Detection: Differential refractometer

<Experiment 1-3: Preparation of 3-ketoisomaltose>
Rhizobium radiobacter G37 strain was cultured in the same manner as in Experiment 1-2, except that the sugar in the liquid medium used for the main culture was replaced with isomaltose (Hayashibara Co., Ltd., purity 97.7%), and the same operation was performed. When purified by, crystals were precipitated from the concentrated solution after desalting. Crystals were recovered from the crystal suspension by centrifugation, washed with special grade ethanol, and dried in an oven (40° C.) to obtain 6.0 g of a standard product. When the purity of the obtained standard product was analyzed by HPLC, it was 98.5%. Analysis by a method similar to that performed for 3-ketotrehalose in Experiment 1-1 revealed that the obtained sample was 3-ketoisomaltose (6-O-α-3-ketoglucosylglucose). ..

<Experiment 1-4: Preparation of 3-ketolactose>
A liquid medium (pH 6.8) consisting of lactose 2.0% (w/v), yeast extract (manufactured by Difco Co., Ltd.) 0.1 (w/v)% and water was used for seed culture and main culture, and a volume of 500 mL was used. Rhizobium radiobacter G37 strain was cultured in the same manner as in Experiment 1-2, except that three Erlenmeyer flasks each containing 100 mL of the medium were used. When the sugar composition of the culture solution was analyzed by HPLC, a new sugar peak other than the raw material lactose was confirmed.

After completion of the culture, about 300 mL of the obtained culture solution was filtered through diatomaceous earth to remove bacterial cells, the filtrate was concentrated under reduced pressure with an evaporator, and the obtained concentrate was subjected to preparative HPLC according to Experiment 1-2. Then, the peak fraction of newly produced sugar was collected. The separated solution was subjected to decolorization filtration with activated carbon, the filtrate was concentrated under reduced pressure with an evaporator, 5 times the amount of methanol of the concentrated solution was added, and the mixture was allowed to stand at room temperature, and crystals were precipitated in about 1 week. The crystal suspension was filtered to collect crystals, and the crystals were vacuum dried to obtain 3.8 g of a standard product. When the purity of the obtained standard product was analyzed by HPLC, it was 98.6%. Analysis by a method similar to that performed for 3-ketotrehalose in Experiment 1-1 revealed that the obtained standard product was 3-ketolactose (4-O-β-3-ketogalactosylglucose).

<Experiment 1-5: Preparation of 3-ketomaltose>
Maltose 5.0% (w/v), beer yeast extract (Oriental Yeast Co., Ltd.) 0.02 (w/v), Sollis 095E (Oriental Yeast Co., Ltd.) 0.04 (w/v)% , Diammonium citrate 0.45 (w/v)%, diammonium hydrogen phosphate 0.45 (w/v)%, ammonium succinate 0.60 (w/v)%, dipotassium hydrogenphosphate 0. 10(w/v)%, calcium chloride dihydrate 0.05(w/v)%, magnesium sulfate heptahydrate 0.05(w/v)%, iron sulfate heptahydrate 0.005( w/v)%, mineral yeast Zn 0.001 (w/v)% and water in a liquid medium (pH 6.8) containing 3 mL per test tube, sterilized in an autoclave at 121° C. for 20 minutes, and cooled One platinum loop of Rhizobium radiobacter G37 strain cultivated at 27° C. in an agar slant medium was inoculated, and cultured with shaking tow at 27° C. for 48 hours as a seed culture solution.

Twenty liquid medium having the same composition as the above was put in a 500 mL Erlenmeyer flask in an amount of 50 mL, adjusted to pH 6.8, sterilized at 121° C. for 20 minutes, and cooled. Then, 0.5 mL of each of the above-mentioned seed culture solutions was inoculated into each Erlenmeyer flask, and then rotary shaking tow culture was carried out at 27° C. for 72 hours to obtain a main culture solution. When the sugar composition of the culture broth was analyzed by HPLC, new sugar peaks other than the raw material maltose were confirmed.

After culturing, about 1 L of the obtained culture solution was centrifuged at 10,000 rpm to remove the cells, and the obtained centrifugation supernatant was heat-treated at 100°C for 10 minutes, and then again at 10,000 rpm. It was centrifuged. Ethanol (2,700 mL) was added to the obtained centrifugation supernatant, and the mixture was further centrifuged at 10,000 rpm. Then, the centrifugal product was concentrated to 500 mL under reduced pressure with an evaporator, the obtained concentrated solution was filtered with a 0.22 μm membrane filter, and then preparative HPLC under the same conditions as in Experiment 1-1 was repeated to prepare the raw material maltose. The newly-produced peak fraction of sugar was collected, concentrated, and lyophilized to give powder, giving 5.1 g of a standard product. When the purity of the obtained standard product was analyzed by HPLC, it was 96.2%. Analysis by a method similar to that performed for 3-ketotrehalose in Experiment 1-1 revealed that the obtained preparation was 3-ketomaltose (4-O-α-3-ketoglucosylglucose).

<Experiment 1-6: Preparation of 3-ketosucrose>
Rhizobium radiobacter G37 strain was cultured in the same manner as in Experiment 1-5, except that sucrose (Wako Pure Chemical Industries, Ltd., reagent special grade) was used as a sugar in the liquid medium used for seed culture and main culture. When the sugar composition of the culture broth was analyzed by HPLC, a new sugar peak other than the raw material sucrose was confirmed. After completion of the culture, the product was purified by almost the same operation as for 3-ketomaltose to obtain 7.5 g of a standard product. When the purity of the obtained standard product was analyzed by HPLC, it was 90.3%. When analyzed by a method similar to that performed for 3-ketotrehalose in Experiment 1-1, the obtained standard product is 3-ketosucrose (O-α-3-ketoglucosyl(1→2)β-fructoside). It has been found.

<Experiment 1-7: Preparation of 3-keto maltitol>
Rhizobium as in Experiment 1-5, except that the sugar in the liquid medium used for seed culture and main culture was changed to maltitol (Tokyo Kasei Kogyo Co., Ltd., purity 93.0% or more) and the concentration was changed to 3%. Radiobacterium G37 strain was cultured. When the sugar composition of the culture broth was analyzed by HPLC, a new sugar peak other than the raw material maltitol was confirmed. After completion of the culture, the product was purified by the same operation as for 3-ketomaltose to obtain 9.4 g of a standard product. When the purity of the obtained standard product was analyzed by HPLC, it was 90.2%. Analysis by a method similar to that performed for 3-ketotrehalose in Experiment 1-1 revealed that the obtained sample was 3-ketomaltitol (4-O-α-3-ketoglucosyl sorbitol). ..

<Experiment 1-8: Preparation of 3,3′-diketotrehalose>
Sucrose 1.0 (w/v)%, trehalose 4.0 (w/v)%, urea 0.09 (w/v)%, magnesium sulfate heptahydrate 0.015 (w/v)%, sulfuric acid Iron heptahydrate 0.001 (w/v)%, citric acid monohydrate 0.017 (w/v)%, potassium dihydrogen phosphate 0.107 (w/v)%, dihydrogen phosphate After adjusting the liquid medium containing sodium dihydrate 0.217 (w/v)% to pH 6.8, put 3 mL per test tube, sterilize in an autoclave at 121° C. for 20 minutes, cool, and then flavobacterium. Um Saccharophilum strain NBRC15944 was inoculated in an amount of 30 μL from a glycerol stock. What was subjected to shake tow culture at 27° C. for 96 hours was used as a seed culture.

40 liquid medium having the same composition as described above was placed in a 500-mL Erlenmeyer flask in an amount of 100 mL, adjusted to pH 6.8, sterilized at 121° C. for 20 minutes, and cooled. Next, 1 mL of each of the above-mentioned seed culture solutions was inoculated into each Erlenmeyer flask, followed by rotary shaking tow culture at 27° C. for 24 hours to obtain a main culture solution.

The main culture solution was centrifuged at 10,000 rpm. The centrifugation supernatant was removed, Triton X-100 and lysozyme were added to the obtained cells, and the mixture was stirred at room temperature for 1 hour. Next, it was frozen by placing it at −80° C. for 1 hour and thawed in a 38° C. water bath for 1 hour. Then, ultrasonication was performed and centrifugation was performed at 10,000 rpm. The obtained centrifugation supernatant was used as a crude enzyme solution of glucoside 3-dehydrogenase.

The activity of glucoside 3-dehydrogenase was roughly measured based on the following principle. That is, flavin that is a coenzyme of glucoside 3-dehydrogenase when glucoside 3-dehydrogenase oxidizes methyl-α-glucoside and converts it into methyl-α-3-ketoglucoside using methyl α-glucoside as a substrate. Since adenine dinucleotide (FAD) is reduced to produce reduced FAD (FADH ₂ ), phenazine methosulfate (PMS) and dichloroindophenol (DCIP) are coupled to each other, and finally, reduction of oxidized DCIP ( The oxidation activity of methyl-α-glucoside was evaluated by measuring the absorbance of the reduced DCIP) at a wavelength of 660 nm. Specifically, 195 μL of an appropriately diluted enzyme solution is placed in a well of a microplate, and 50 μL of a substrate solution containing methyl-α-glucoside, PMS, DCIP, and a phosphate buffer solution (pH 7.0) is added to the well. As a result, the reaction liquid volume was adjusted to 200 μL, and the final concentrations of methyl-α-glucoside, PMS, DCIP and phosphate buffer were adjusted to 10 mM, 1 mM, 60 μM and 50 mM, respectively, to prepare an enzyme reaction liquid. .. The enzyme reaction was performed at pH 7.0, 30° C. in the dark for 10 minutes, and the absorbance at a wavelength of 660 nm was measured with a plate reader over time. In addition, 1 unit of activity of glucoside 3-dehydrogenase was defined as the activity of oxidizing 1 μmol of substrate per minute under the above-mentioned conditions.

The final concentrations of trehalose, potassium ferricyanide, and phosphate buffer (pH 7.0) were 40 mM, 100 mM, and 250 mM, respectively, and the crude enzyme solution obtained above was prepared to have 18 units per 1 g of trehalose, 390 mL of reaction solution. Was reacted at 27° C. for 8 hours. 910 mL of ethanol was added to the obtained reaction solution, and the mixture was centrifuged at 10,000 rpm. The centrifugal supernatant was concentrated to 60 mL under reduced pressure by an evaporator, the obtained concentrated solution was filtered through a 0.22 μm membrane filter, and then preparative HPLC was repeated under the same conditions as in Experiment 1-1 to recover the trehalose starting material. The peak fraction of newly produced sugar was collected and concentrated. Then, the fractionation was repeated under the following conditions, and the peak fraction of the sugar newly formed from the raw material trehalose was fractionated, concentrated and lyophilized to give 4.3 g of a standard product. When the purity of the obtained standard product was analyzed by HPLC, it was 98.1%. When a part of the preparation was analyzed by the same method as that for the 3-ketotrehalose in Experiment 1-1, it was found that the obtained preparation was 3,3'-diketotrehalose.

(Preparative HPLC conditions)
Column: Shodex SUGAR KS-801
(Inner diameter 8.0 mm, length 300 mm, Showa Denko KK)
Eluent: Ultrapure water Flow rate: 0.4 mL/min Column temperature: 75°C
Detection: Differential refractometer

<Experiment 1-9: Preparation of methyl α-3-ketoglucoside>
The final concentrations of methyl α-glucoside, potassium ferricyanide, and phosphate buffer (pH 7.0) were 40 mM, 100 mM, and 250 mM, respectively, and the crude enzyme solution prepared by the same method as in Experiment 1-8 was methyl α-glucoside. 240 mL of the reaction liquid prepared so as to have 8.1 units per 1 g was reacted at room temperature for 2 hours. 560 mL of ethanol was added to the obtained reaction solution, and the mixture was centrifuged at 10,000 rpm. The centrifugal supernatant was concentrated to 40 mL under reduced pressure with an evaporator, the obtained concentrated solution was filtered through a 0.22 μm membrane filter, and then preparative HPLC was repeated under the same conditions as in Experiment 1-1 to obtain methyl α as a raw material. -The peak fraction of sugar newly generated from glucoside was collected, concentrated, and lyophilized to give powder (0.9 g). When the purity of the obtained standard product was analyzed by HPLC, it was 98.1%. A part of the preparation was analyzed by a method similar to that performed for 3-ketotrehalose in Experiment 1-1, and it was found that the obtained preparation was methyl α-3-ketoglucoside.

<Experiment 2: Preparation of 2-ketoglucose and 2-ketotrehalose>
While 2-ketoglucose was prepared from D-glucose by an enzymatic method using pyranose 2-oxidase, the obtained 2-ketoglucose was used as an acceptor, and β-glucose-1-phosphate (hereinafter, “β-G1P 2) was prepared by the transglycosylation reaction of trehalose phosphorylase using glucosyl donor.

<Experiment 2-1: Preparation of 2-ketoglucose>
In 100 mL of a substrate solution in which D-glucose was dissolved in 50 mM acetate buffer (pH 5.5) to a final concentration of 3% (w/v), 31 units of pyranose 2-oxidase (Tarametes sp. ) Origin, sold by Sigma-Aldrich Japan), and 2,700 units of catalase (derived from beef liver, sold by Wako Pure Chemical Industries, Ltd.) per 1 g of the substrate, and shaken at 240 rpm for 24 hours at 27° C. It was made to react. In addition, the reason for using catalase together is that hydrogen peroxide is generated in the reaction process in which pyranose 2-oxidase acts on D-glucose to generate 2-ketoglucose, and therefore hydrogen peroxide which may inactivate the enzyme is used. This is to disassemble. After the enzymatic reaction, the obtained reaction solution was heated at 100° C. for 10 minutes to deactivate the enzyme. When the purity of 2-ketoglucose in the obtained reaction liquid was analyzed by HPLC, it was 96.4%. The fact that the obtained standard product was 2-ketoglucose was confirmed by comparing a commercially available 2-ketoglucose standard product (reagent, sold by Sigma-Aldrich Japan) with a chromatogram in HPLC analysis. Finally, 2.86 g of 2-ketoglucose was obtained from 3 g of D-glucose.

<Experiment 2-2: Preparation of 2-ketotrehalose>
Β-G1P (prepared by Hayashibara) and 50 mM acetate buffer (pH 5.5) were added to the 2-ketoglucose-containing reaction solution obtained by the method of Experiment 2-1, and the final concentration of 2-ketoglucose was 1% ( w/v), in 200 mL of a substrate solution adjusted to a final concentration of β-G1P of 10%, 10 units (100 units per 2-ketoglucose) of trehalose phosphorylase (derived from Thermoanaerobacter, Hayashibara preparation) was added and reacted at 40° C. for 24 hours. After the enzymatic reaction, the obtained reaction solution was heated at 100° C. for 10 minutes to deactivate the enzyme. The reaction solution was subjected to LC/MS analysis to confirm the production of 2-ketotrehalose having a molecular weight of 2 smaller than trehalose. The 2-ketotrehalose content in the reaction solution was 0.37 g. After desalting the obtained reaction solution, preparative HPLC was performed under the same conditions as in Experiment 1-1 to collect the 2-ketotrehalose fraction, and the 2-under the same analytical conditions as in Experiment 1-1. When the purity of ketotrehalose was measured, it was 95.0%. Finally, 0.36 g of 2-ketotrehalose was obtained using 2 g of 2-ketoglucose.

<Experiment 3: Effect of 3-keto sugar or 2-keto sugar on TNF-α production of human monocytic cells>
The human monocytic cell line THP-1 cells produce large amounts of TNF-α when stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Therefore, the effect of 3-keto sugar or 2-keto sugar on TNF-α production was examined using this experimental system.

It has been reported that the THP-1 cells used in this experiment have increased responsiveness to LPS when treated with sodium butyrate, which is also known as a cell differentiation inducer. What was cultivated for a day was used. Sodium butyrate-treated THP-1 cells were prepared at a cell concentration of 1×10 ⁶ cells/mL using RPMI1640 medium containing 10% fetal bovine serum, and 0.1 mL was added to a 96-well microplate. Next, in the same medium, 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose, 3-ketoisomaltose, 3-ketomaltose, 3-ketosucrose, 3-ketomaltitol, 3 obtained in Experiment 1 were added. , 3'-diketotrehalose, methyl α-3-ketoglucoside, 2-ketoglucose obtained in Experiment 2 and 2-ketotrehalose were added at 0.05 mL per well so that each final concentration shown in Table 1 was obtained. Conditioned and incubated at 37° C. for 2-3 hours. Then, 0.1 mL per well of a medium in which LPS prepared at a concentration of 12.5 μg/mL and human IFN-γ prepared at a concentration of 1,250 IU/mL were added to the same medium to add THP-1 cells. Stimulated. After culturing THP-1 cells at 37° C. for 18 hours, the amount of TNF-α produced in the culture supernatant was measured by a specific ELISA (enzyme-linked immunoassay) method. The amount of TNF-α produced was relatively evaluated by the following formula, with the amount of TNF-α produced in the culture supernatant of the control without addition of the test substance taken as 100%. Further, the action was compared with that of dipotassium glycyrrhizinate (GK2) which has been known as an anti-inflammatory agent.

Table 1 shows the effect of 3-keto sugar or 2-keto sugar on TNF-α production of THP-1 cells. The experiment was performed three times, and Dunnett's multiple comparison test was performed on the control. Table 1 shows the average value of three experiments, and when the risk rate p<0.01, it was determined that there was a significant difference, and indicated by *.

As is clear from Table 1, GK2, which is known as an anti-inflammatory agent, suppresses TNF-α production of THP-1 cells in a concentration-dependent manner, and at 1.0 mM, 53.3% of the control, 2.0 mM. Was suppressed to 17.4%. On the other hand, 3-keto disaccharides such as 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose, 3-ketoisomaltose, 3-ketomaltose, 3-ketosucrose, 3-ketomaltitol and 3, In the concentration range of 0.5 mM to 2.0 mM, 3'-diketotrehalose also suppressed the TNF-α production of THP-1 cells to 1.5 to 87.8% of the control, and the suppression was any. Was also concentration-dependent and significant.

Methyl α-3-ketoglucoside, which is a derivative of 3-keto monosaccharide, has a weak inhibitory effect on TNF-α production and showed no significant inhibition even at 4.0 mM, but it was 79.7% at 8.0 mM, 16 It was suppressed to 49.5% at 0.0 mM. On the other hand, 2-ketoglucose suppressed the production of TNF-α up to 63.8% at 2.0 mM and 53.8% at 4.0 mM, and 2-ketotrehalose at a concentration range of 0.5 to 2.0 mM. It was suppressed to 69.6 to 41.3%.

In addition, in this experiment, when the number of cells was simultaneously measured at each concentration of each test sample, it was found that 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose, and 3-ketoiso were 3-ketosugars. When maltose, 3-ketomaltose, 3-ketosucrose, 3-ketomaltitol and methyl α-3-ketoglucoside, 2-ketosugars 2-ketoglucose and 2-ketotrehalose are used at any concentration However, the cell number was almost the same as that of the control, and it was confirmed that the addition of these 3-keto sugars or 2-keto sugars did not reduce the viable cell number of THP-1 cells. In the case of 3,3′-diketotrehalose, the number of viable cells was slightly reduced at a concentration of 1.0 to 2.0 mM, but TNF was also reduced at a low concentration of 0.25 mM, which did not reduce the number of viable cells. An inhibitory effect on the production of α was confirmed.

The results of this experiment are the 3-ketosugars 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose, 3-ketoisomaltose, 3-ketomaltose, 3-ketosucrose, 3-ketomaltitol. , 3,3'-diketotrehalose and methyl α-3-ketoglucoside, and 2-ketosugars 2-ketoglucose and 2-ketotrehalose all suppress the production of inflammatory cytokine TNF-α. However, it is shown to be useful as an active ingredient of an anti-inflammatory agent. The inflammatory cytokine TNF-α secreted by THP-1 cells is diabetic, chronic respiratory disease, rheumatoid arthritis, gastritis, inflammatory bowel disease, atherosclerosis, cardiovascular disorder, acne, atopic dermatitis, It is known that it is deeply involved in the pathogenesis of neurodegenerative diseases and the like, and the results of this experiment show that 3-keto sugar or 2-keto sugar is used as an active ingredient of an anti-inflammatory agent against a wide range of inflammation including these diseases. It shows that sugar is available.

<Comparative Experiment 1: Effect of raw sugar on TNF-α production of human monocytic cells>
In addition to the raw material sugars of 3-keto sugar used above, that is, trehalose, lactose, kojibiose, isomaltose, maltose, sucrose, maltitol, and methyl-α-glucoside, they have an anti-inflammatory action in Non-Patent Document 1. Using D-psicose, which was reported to be a test sample, the TNF-α production inhibitory effect was similarly examined using the experimental system used in Experiment 3. The concentration of each test sample in the medium was 4.8 mM, 19.2 mM and 57.2 mM for trehalose, lactose, kojibiose, isomaltose, maltose, sucrose, maltitol, methyl-α-glucoside, D-. For psicose, three levels of 4.8 mM, 14.4 mM and 43.2 mM were used.

Table 2 shows the effects of the starting sugar of 3-keto sugar and D-psicose on the production of TNF-α by THP-1 cells. The experiment was performed three times, and Dunnett's multiple comparison test was performed on the control. Table 2 shows the average value of three experiments, and when the risk rate p<0.01, it was determined that there was a significant difference, and indicated by *.

As is clear from Table 2, trehalose, lactose, kojibiose, maltose, sucrose and maltitol showed a TNF-α production inhibitory action only when the concentration was increased to 57.6 mM or 43.2 mM, but isomaltose was There was no effect even at a concentration of 57.6 mM. Compared with the results in Table 1 above, in which all 3-keto disaccharides exhibited a TNF-α production inhibitory action at a concentration of 2 mM or less, the action of these raw material disaccharides was very weak, and a concentration of at least 50 times or more must be used. It was found that, for example, the same TNF-α production inhibitory action was not exhibited. On the other hand, D-psicose, which has been reported to have an anti-inflammatory action, and methyl-α-glucoside exhibited a TNF-α production inhibitory action at a concentration of 43.2 mM. However, the actions of D-psicose and methyl-α-glucoside are the effects of 3-keto disaccharides such as 3-ketotrehalose, 3-ketolactose, 3-ketocodibiose, 3-ketoisomaltose, 3-ketomaltose, and 3-ketomaltose. It was weaker than the actions of -ketosucrose, 3-ketomaltitol, 3,3'-diketotrehalose, and 2-ketosugars 2-ketoglucose and 2-ketotrehalose.

From these results, in the carbohydrate having an aldohexose structure in the molecule, the anti-inflammatory action of the carbohydrate having an aldohexose structure is remarkable by converting the 3-position hydroxyl group or 2-position hydroxyl group in the aldohexose structure into a keto group. It became clear that it can be improved.

<Experiment 4: Effect of 3-keto sugar on IL-8 production of human skin fibroblasts>
The effect of 3-ketosugar on the production of the inflammatory cytokine IL-8 was investigated in human normal skin fibroblasts (NHDF cells).

To a 96-well microplate, 0.2 mL of NHDF cells prepared at a concentration of 1×10 ⁵ cells/mL was added using Dulbecco's modified Eagle's medium containing 10% fetal bovine serum until the whole bottom surface of the well was occupied. Cultured. After removing the culture supernatant by suction, 0.1 mL of fresh Dulbecco's modified Eagle medium containing 10% fetal bovine serum was added to each well. Next, 0.05 mL of 3-ketotrehalose, 3-ketolactose, 3-ketocordibiose or 3-ketoisomaltose appropriately diluted in the same medium was added to adjust the final concentration to the concentration shown in Table 3. The cells were cultured at 37° C. for 24 hours, and human IL-1β that stimulates the production of IL-8 was added thereto so that the final concentration was 1 ng/mL, and the cells were further cultured for 24 hours. For comparison, the same procedure was performed for GK2, which is known as an anti-inflammatory agent.

The amount of IL-8 in the culture supernatant was detected using a specific ELISA kit (manufactured by Affymetrix), developed with tetramethylbenzidine, and then the absorbance at 450 nm was measured. The same treatment as that described above except that the test sample was not added was used as a control, and the amount of IL-8 produced in the culture supernatant in the control was set to 100%, and the amount of IL-8 produced was relatively evaluated by the following formula.

Table 3 shows the effect of 3-keto sugar on the IL-8 production of NHDF cells. The experiment was performed three times, and Dunnett's multiple comparison test was performed on the control. Table 3 shows the average value of three experiments, and when the risk rate p<0.01, it was determined that there was a significant difference, and indicated by *.

As is clear from Table 3, in comparison with the control, 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose and 3-ketoisomaltose had an inhibitory effect on IL-8 production in the tested concentration range. Admitted. It should be noted that at the concentration at which the IL-8 production inhibitory action was observed, there was no effect on the cell number except for a slight decrease in the cell number at 3.0 mM of 3-ketotrehalose. -It was confirmed that the suppression of IL-8 production by keto sugar was not due to the damaging effect of the cells themselves. Further, GK2 at 3.0 mM showed a significant suppression of IL-8 production as compared with the control, but at 0.5 mM and 1.0 mM, on the contrary, an increase in IL-8 production was observed.

The results of this experiment indicate that 3-ketosugars 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose, and 3-ketoisomaltose all suppress the production of inflammatory cytokine IL-8. , That is useful as an active ingredient of anti-inflammatory agents. More specifically, since 3-keto sugar suppressed IL-8 production from human normal skin fibroblasts, alleviation of skin inflammatory symptoms such as allergic dermatitis, atopic dermatitis, acne and psoriasis. In addition to improvement, it is also useful for prevention and treatment of various inflammatory diseases.

<Experiment 5: Effect of 3-keto sugar on IL-8 production of human epidermal keratinocyte cells (NHEK cells)>
The effect of 3-keto sugar on the production of IL-8, which is an inflammatory cytokine, was examined in human epidermal keratinocyte cells (NHEK cells).

Normal NHEK cells prepared in a concentration of 5.0×10 ⁴ cells/mL using a growth factor-containing keratinocyte medium (trade name “EpiLife”, manufactured by Life Technologies, Inc.) were added to a collagen-coated 96-well microplate. .2 mL was added, and the cells were cultured at 37° C. for 3 days while changing the medium every 1 to 2 days. Then, the medium was replaced with a keratinocyte medium containing no growth factor, and the cells were further cultured for 24 hours to prepare cells. After suction-removing the culture supernatant, 0.2 mL per well of 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose or 3-ketoisomaltose appropriately diluted in a growth factor-free medium for keratinocytes. Then, the final concentration was adjusted to the concentration shown in Table 4 and cultured at 37° C. for 24 hours, and human TNF-α that stimulates the production of IL-8 was added to the final concentration of 10 ng/mL and human IFN-γ. 0.05 mL was added to each well so as to be 100 IU/mL, and the cells were further cultured for 18 to 20 hours. For comparison, the same procedure was performed for GK2, which is known as an anti-inflammatory agent.

The amount of IL-8 in the culture supernatant was detected using a specific ELISA kit (manufactured by Affymetrix), developed with tetramethylbenzidine, and then the absorbance at 450 nm was measured. IL-8 production was relatively evaluated in the same manner as in Experiment 4, using the same treatment as the control except that the test sample was not added.

Table 4 shows the effect of 3-keto sugar on the IL-8 production of NHEK cells. The experiment was performed three times, and Dunnett's multiple comparison test was performed on the control. Table 4 shows the average value of three experiments, and when the risk rate p<0.01, it was determined that there was a significant difference, and indicated by *.

As is clear from Table 4, in comparison with the control, the 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose and 3-ketoisomaltose had an inhibitory effect on IL-8 production in the tested concentration range. Admitted. In addition, at the concentration at which the IL-8 production inhibitory action was observed, there was no effect on the cell number except for a slight decrease in the number of 3-ketotrehalose at 3.0 mM. -It was confirmed that the inhibition of IL-8 production by keto sugar is not due to the damaging effect on cells. Further, GK2 at 0.5 mM and 1.0 mM showed a significant suppression of IL-8 production as compared with the control, but conversely at 3.0 mM, a significant increase in IL-8 production was observed.

The results of this experiment indicate that 3-ketosugars 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose, and 3-ketoisomaltose all suppress the production of inflammatory cytokine IL-8. , That is useful as an active ingredient of anti-inflammatory agents. More specifically, since 3-keto sugar suppressed IL-8 production from human epidermal keratinocyte cells, alleviation and improvement of skin inflammatory symptoms such as allergic dermatitis, atopic dermatitis, acne and psoriasis. In addition, it shows that it is useful for the prevention and treatment of various inflammatory diseases.

<Experiment 6: Effect of 3-keto sugar and 2-keto sugar on prostaglandin E2 and IL-6 production of human gingival fibroblasts>
In periodontal disease, infection of periodontopathic bacteria activates macrophages and fibroblasts in periodontal tissues, and IL-1, IL-6, TNF-α and prostaglandin E2 (PGE2), which are inflammation-related cytokines. ) Is produced in excess and is thought to induce tissue destruction. Especially, in the gingival tissue and gingival crevicular fluid of patients with periodontal disease, the PGE2 level was higher than that in healthy subjects, and when the nonsteroidal anti-inflammatory drug indomethacin or ibuprofen was actually administered to the periodontitis model. It has also been reported that the progression of periodontal disease is suppressed (Kazuyuki Noguchi, Periodontal disease and prostaglandins, Bulletin of Shikato, 28, 39-48 (2008)). Therefore, if production of inflammatory mediators such as PGE2 and IL-6 from activated gingival fibroblasts can be suppressed, it is considered to be effective in the prevention and treatment of periodontal disease.

Using the PGE2 and IL-6 production system from human gingival fibroblasts (HGF cells) activated by IL-1β stimulation, the effect of 3-ketotrehalose and 2-ketoglucose on PGE2 and IL-6 production was examined. , GK2.

0.2 mL of HGF cells prepared to a concentration of 5×10 ⁴ cells/mL was added to a collagen-coated 96-well microplate (manufactured by ASAHI Glass Co., Ltd.) using Dulbecco's modified Eagle medium containing 10% fetal bovine serum. The culture was performed until the whole bottom surface of the well was occupied. After the culture supernatant was removed by suction, 0.15 mL of fresh Dulbecco's modified Eagle medium containing 10% fetal bovine serum was added to each well. Next, 0.05 mL of 3-ketotrehalose, 2-ketoglucose or GK2 appropriately diluted in the same medium was added, and the mixture was cultured at 37°C for 24 hours. Then, human IL-1β was added so that the final concentration was 1 ng/mL, and the cells were further cultured for 24 hours.

PGE2 and IL-6 in the culture supernatant were each detected using a specific ELISA kit (PGE2; manufactured by Enzo Lifesciences, IL-6; manufactured by R&D), colored with tetramethylbenzidine, and then developed at 450 nm. It was quantified by measuring the absorbance. As a control, the same treatment was carried out except that the test substance was not added, and PGE2 and IL-6 in the culture supernatant in the control were set to 100%, according to the same formula as that shown for the IL-8 production amount in Experiment 4, Relative evaluation of PGE2 and IL-6 production was performed. The experiment was performed three times, and Dunnett's multiple comparison test was performed on the control. The risk rate p<0.01 was regarded as a significant difference and indicated by *. The results are shown in Table 5.

As is clear from Table 5, 3-ketotrehalose was found to have a significant PGE2 production inhibitory effect in a concentration-dependent manner, as compared with the control in which the test substance was not added. Moreover, at a concentration of 1 mM or less, a stronger PGE2 production suppressing effect than GK2 was observed. On the other hand, 2-ketoglucose showed a significant PGE2 production inhibitory effect at a concentration of 2 mM. Further, both 3-ketotrehalose and 2-ketoglucose exhibited an IL-6 production inhibitory effect at a concentration of 2 mM, which was similar to that of GK2. No cytotoxic effect was observed in the tested concentration range.

From these results, it was found that 3-ketotrehalose and 2-ketoglucose have an action of suppressing the production of inflammatory mediators from activated gingival fibroblasts and are effective in the prevention or treatment of periodontal disease. ..

<Experiment 7: Effect of 3-keto sugar on IL-8 production of human intestinal epithelial cell line Caco-2>
At the time of inflammation due to invasion of bacteria into the intestinal tract, IL-1 and TNF-α produced from macrophages in the lamina propria act on epithelial cells as a defense reaction of the body to produce chemokines such as IL-8. IL-8 infiltrates and accumulates neutrophils in the inflamed area to eliminate microorganisms. However, as in the case of inflammatory bowel disease, when inflammation is prolonged or chronic for some reason, tissue destruction by infiltrated neutrophils as a result of excessive inflammatory response (eg, persistent increase in IL-8 production). Is also reported to occur. Therefore, suppressing the production of IL-8 as an inflammatory mediator from intestinal epithelial cells by IL-1 and TNF-α is considered to lead to the reduction or treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. Therefore, the effect of 3-keto sugar on the production of IL-8 from human intestinal epithelial cells was investigated.

0.2 mL of Caco-2 cells prepared in a concentration of 3×10 ⁴ cells/mL using a Dulbecco's modified Eagle medium containing 10% fetal bovine serum on a 96-well microplate (IWAKI Glass Co., Ltd.) coated with collagen. The mixture was added and cultured at 37° C. for 3 days. After removing the culture supernatant by suction, 0.1 mL of fresh Dulbecco's modified Eagle medium containing 1% fetal bovine serum was added to each well. Next, 0.05 mL of 3-ketotrehalose, 3-ketomaltitol, or GK2 appropriately diluted in the same medium was added to each well, and the mixture was incubated at 37° C. for 7 hours. After the incubation, 0.1 mL per well of a medium containing IL-1β adjusted to a concentration of 2.5 ng/mL and TNF-α adjusted to a concentration of 62.5 ng/mL in the same medium was added for stimulation. After 16 to 18 hours from stimulation, the amount of IL-8 produced in the culture supernatant was detected using a specific ELISA kit (manufactured by R&D), developed with tetramethylbenzidine, and then the absorbance at 450 nm was measured. .. The amount of IL-8 produced was treated in the same manner except that the test substance was not added, was used as a control, and the amount of IL-8 produced in the culture supernatant in the control was set to 100%. evaluated.

As is clear from Table 6, in the activated intestinal epithelial cell line Caco-2 cells, GK2 did not show any inhibition of IL-8 production by stimulation with IL-1β and TNF-α. In contrast, 3-ketotrehalose and 3-ketomaltitol were found to have a concentration-dependent and significant IL-8 production inhibitory effect.

This result indicates that 3-ketotrehalose and 3-ketomaltitol have a preventive or therapeutic effect on inflammatory bowel disease.

<Experiment 8: Effect of pretreatment with 3-keto sugar on resistance of human skin fibroblasts to oxidative damage>
Using normal human skin fibroblasts (NHDF cells), the effect of 3-keto sugar on the resistance of human skin fibroblasts to oxidative damage, in particular, pretreatment of cells in the presence of 3-keto sugar is active The effect on cell damage caused by treatment with hydrogen peroxide, which is one of the oxygen species, was investigated.

To a 96-well microplate, 0.2 mL of NHDF cells prepared at a concentration of 1×10 ⁵ cells/mL was added using Dulbecco's modified Eagle's medium containing 10% fetal bovine serum until the whole bottom surface of the well was occupied. Cultured. After removing the culture supernatant by suction, 0.1 mL of fresh Dulbecco's modified Eagle medium containing 10% fetal bovine serum was added to each well. Next, 0.1 mL of 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose or 3-ketoisomaltose appropriately diluted in the same medium was added, and the mixture was cultured at 37°C for 24 hours. For comparison, the same procedure was performed for GK2, which is known as an anti-inflammatory agent.

After the culture, the culture supernatant was removed by suction, 0.2 mL of hydrogen peroxide adjusted to a concentration of 150 μM with HANKS buffer was added to each well, and the mixture was incubated at 37° C. for 2 hours. Then, the hydrogen peroxide solution was removed by suction, 0.2 mL of Dulbecco's modified Eagle medium containing 10% fetal bovine serum was added to each well, and the mixture was further cultured at 37° C. for 48 hours.

The number of cells after culturing was measured using a cell counting kit (manufactured by Dojindo Laboratories Co., Ltd.). That is, after the culture supernatant of cells cultured for 48 hours was removed by suction, 0.1 mL of fresh Dulbecco's modified Eagle medium containing 10% fetal bovine serum was added to each well, and the cell counting was further diluted 10-fold with the same medium. The kit was added at 0.1 mL per well and incubated at 37° C. for 2 hours. Finally, the absorbance at 450 nm corresponding to the number of cells was measured with a spectrophotometer. Similarly, cells treated with hydrogen peroxide were used as a control except that the pretreatment with the test substance (3-keto sugar) was not performed, and the absorbance in the control was set to 100%, and the cell number was evaluated as a relative cell number by the following calculation formula. ..

Table 5 shows the effect of the pretreatment in the presence of 3-keto sugar on the resistance of human dermal fibroblasts to the cytotoxicity caused by the treatment with hydrogen peroxide. The experiment was performed three times, and Dunnett's multiple comparison test was performed on the control. Table 7 shows the average value of three experiments, and when the risk rate p<0.01, it was determined that there was a significant difference, and indicated by *.

As is apparent from Table 7, human dermal fibroblasts pretreated with 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose and 3-ketoisomaltose were tested at the concentrations tested compared to controls. Since the number of cells was significantly concentration-dependent and significantly increased in the range, it was revealed that 3-keto sugar has an effect of imparting resistance to cell damage of human skin fibroblasts due to hydrogen peroxide. It should be noted that such an action was not observed in GK2.

The results of this experiment show that 3-ketotrehalose, 3-ketolactose, 3-ketocorgibiose, and 3-ketoisomaltose, which are 3-ketosugars, are resistant to cell damage caused by reactive oxygen species in human skin fibers. It has the effect of imparting to blast cells and is useful as an active ingredient of an anti-inflammatory agent against inflammation caused by reactive oxygen species. More specifically, the involvement of reactive oxygen species has been reported for surgical invasion, ischemia-reperfusion injury, ulcerative colitis, and even skin inflammatory diseases such as atopic dermatitis and skin aging due to UV exposure. It shows that it is useful for prevention and treatment.

From the results of Experiments 3 to 8, saccharides having a ketoaldohexose structure in the molecule, such as 3-keto sugar or 2-keto sugar, did not adversely affect cells and exhibited inflammatory cytokines such as TNF-α and IL. -8, PGE2, IL-6 and the like are suppressed, and since 3-keto sugar also has an action of preventing cell damage caused by reactive oxygen species, these 3-keto sugar and 2-keto sugar are It has been found that can be used as an active ingredient of anti-inflammatory agents. In addition, the anti-inflammatory agent of the present invention containing a carbohydrate having a ketoaldohexose structure in the molecule as an active ingredient suppresses the production of inflammatory cytokines at a lower concentration than GK2, and thus has an antioxidant effect. It can be said that it is an effective and safe anti-inflammatory agent with few side effects.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

<Anti-inflammatory agent>
The following compositions were mixed, and 0.5 g each was tabletted by a conventional method to prepare an oral anti-inflammatory agent.
(Composition) (% by mass)
3-ketotrehalose 10.0
Trehalose 90.0
Magnesium stearate 0.2

Since this product contains 3-ketotrehalose as an active ingredient for anti-inflammatory action, it is effective for oral administration which can improve sepsis, rheumatoid arthritis, ARDS, hepatitis, inflammatory bowel disease, active oxygen related diseases and the like. It is an inflammatory agent.

<Cleansing cream>
A cleansing cream having the following composition was prepared by a conventional method.
(Composition) (% by mass)
Stearic acid 2.0
Stearyl alcohol 3.0
Lipophilic type glyceryl monostearate 2.0
Beeswax 1.5
Vaseline 6.0
Liquid paraffin 40.0
Dimethyl polysiloxane (100CS) 0.5
Sorbitan sesquioleate 1.0
Preservative Appropriate amount Triethanolamine 1.0
Propylene glycol 10.0
Polyethylene glycol 20000 0.5
Carboxy vinyl polymer 0.05
Purified water Remaining amount 3-Ketotrehalose 5% (w/v) aqueous solution 1.0
Fragrance suitable amount

Since this product contains 3-ketotrehalose, it can suppress the production of inflammatory cytokines in dermal fibroblasts and epidermal keratinocytes in human skin to suppress and improve skin inflammation. In addition, this product is a cleansing cream that smoothes the skin, and it spreads lightly and cleans makeup well.

<Cleanser>
A face wash having the following composition was prepared by a conventional method.
(Composition) (% by mass)
Lauric acid 5.0
Myristic acid 18.5
Stearic acid 6.0
Glycerin 12.0
Polyethylene glycol 1500 5.0
Potassium hydroxide 6.5
Purified water Remaining amount Coconut oil fatty acid diethanolamide 5.0
Coconut oil fatty acid methyl taurine sodium 1.8
Polyoxyethylene lauryl ether (7.5 EO) 2.0
Ethylene glycol lauryl ether distearate 1.0
Hydroxypropyl methylcellulose 1% (w/v) aqueous solution 5.0
3-Ketolactose 5% (w/v) aqueous solution 2.0
Fragrance suitable amount

Since this product contains 3-ketolactose, it can suppress the production of inflammatory cytokines in dermal fibroblasts and epidermal keratinocytes in human skin to suppress and improve skin inflammation. In addition, this product is a facial cleanser that keeps the skin fine and gives a rich lather and a refreshing feeling of use.

<Pack>
A pack having the following composition was prepared by a conventional method.
(Composition) (% by mass)
Polyvinyl alcohol 15.0
Polyethylene glycol 3.0
Propylene glycol 7.0
Ethanol 10.0
Royal Jelly Extract 1.0
3-ketokojibiose 0.5
Preservative Suitable amount Perfume Suitable amount Purified water Remaining amount

Since this product contains 3-ketocorgibiose, it can suppress the production of inflammatory cytokines in dermal fibroblasts and epidermal keratinocytes in human skin, and can suppress and improve skin inflammation. .. This product is a pack that can improve skin inflammation and maintain a good skin condition according to its typical use, and also has an excellent whitening effect.

<Sunscreen gel cream>
A sunscreen gel cream having the following composition was prepared by a conventional method.
Ingredients (mass%)
Octyl paramethoxycinnamate 4.0
Oxybenzone 3.0
Liquid paraffin 16.0
Olive oil 9.0
Dibutyl hydroxytoluene 0.01
3-ketoisomaltose 1.0
Acrylic acid/alkyl methacrylate copolymer 0.6
Carboxy vinyl polymer 0.4
Ascorbic acid 2-glucoside 2.0
Allantoin 1.0
Triethanolamine 1.0
Preservative Suitable amount Purified water Remaining amount

Since this product contains 3-ketoisomaltose, it can suppress the production of inflammatory cytokines in dermal fibroblasts and epidermal keratinocytes in human skin to suppress and improve skin inflammation. In addition, this product is a gel that has an excellent anti-inflammatory effect that suppresses sunburn and suppresses hot flashes due to the anti-inflammatory action of 3-ketoisomaltose and allantoin.

<Emulsion>
0.5 parts by mass of polyoxyethylene behenyl ether, 1 part by mass of polyoxyethylene sorbitol tetraoleate, 1 part by mass of lipophilic glyceryl monostearate, 0.5 parts by mass of pyruvic acid, 0.5 parts by mass of behenyl alcohol, avocado oil 1 part by mass, 1 part by mass of 3-ketomaltose, suitable amounts of vitamin E and a preservative are dissolved by heating according to a conventional method, and 1 part by mass of L-sodium lactate, 5 parts by mass of 1,3-butylene glycol and carboxyvinyl are added thereto. 0.1 parts by mass of polymer and 85.3 parts by mass of purified water were added, and the mixture was homogenized and emulsified. Further, an appropriate amount of perfume was added and mixed with stirring to produce an emulsion.

Since this product contains 3-ketomaltose, it suppresses the production of inflammatory cytokines in dermal fibroblasts and epidermal keratinocytes in human skin, suppresses and improves inflammation of the skin, and at the same time, the skin condition. Can be maintained satisfactorily. This product can be advantageously used as an emulsion for sunscreen, prevention of stains/freckles, beautiful skin and fair skin.

<Juice>
A juice having the following composition was produced by a conventional method.
(Composition) (% by mass)
Frozen concentrated Unshu mandarin orange juice 5.0
Fructose glucose liquid sugar 11.0
Citric acid 0.2
L-ascorbic acid 0.02
Fragrance 0.2
Pigment 0.1
3-keto lactose 0.2
Water 89.28

Since this product contains 3-ketolactose, it is a juice useful for preventing periodontal disease and gingivitis. In addition, this product is a juice in which the flavor of mandarin orange is unlikely to deteriorate for a long time because the vitamin C action is enhanced.

<Grapefruit jelly>
Grapefruit jelly having the following composition was produced by a conventional method.
(Composition) (% by mass)
3-ketotrehalose 6.0
Stabilizer 1.2
Grapefruit sand cake 1.0
pH adjuster 0.9
Grapefruit juice (6 times concentrated juice) 0.5
Ascorbic acid 2-glucoside 2.0
Enzyme-treated stevia 0.04
Safflower yellow 0.01
Purified water Remaining amount

Since this product contains 3-ketotrehalose, it is a grapefruit jelly useful for preventing periodontal disease and gingivitis. In addition, this product is a jelly whose flavor of grapefruit is unlikely to deteriorate for a long period of time because its vitamin C action is enhanced.

<chewing gum>
3 parts by mass of gum base is heated and melted until it becomes soft, 7 parts by mass of powdered green-yellow vegetable having a trehalose content of about 50% (w/w) is added to the gum base, and 3-ketosucrose is added together with an appropriate amount of a coloring agent and a flavoring agent. After adding so as to have a solid content weight content of 0.1%, the chewing gum containing 3-ketosucrose was obtained by kneading, molding and packaging by a conventional method.

The product containing 3-ketosucrose is a chewing gum that suppresses skin inflammation and has an excellent effect of preventing periodontal disease and gingivitis, and has good texture and taste.

<Liquid formulation>
According to the standard method, the following components were mixed and subjected to membrane filtration, and then 30 mL each was filled in a sterilized plastic container to obtain a liquid agent.
(Composition) (% by mass)
Sodium chloride 0.6
Potassium chloride 0.03
Calcium chloride 0.02
Sodium lactate 0.31
Trehalose 4.4
Palatine 0.2
3-ketotrehalose 0.05
Purified water Remaining amount

This product containing 3-ketotrehalose is useful as an eye drop or an injection for treating conjunctivitis and inflammatory diseases, and also has a vitamin, calorie and mineral supplementing action.

<Mouthwash>
A mouthwash having the following composition was produced by a conventional method.
(Composition) (% by mass)
Glycerin 10.0
1,3-propanediol 3.0
Low molecular weight agar 5.0
Polyoxyethylene hydrogenated castor oil 0.2
3-ketoisomaltose 0.5
Ascorbic acid 2-glucoside 1.0
Cetylpyridinium chloride 0.05
l-menthol 0.05
Lanexamic acid 0.05
Saccharin sodium 0.02
Methyl paraoxybenzoate 0.01
Fragrance 0.05
Citric acid qs water 89.28

Since this product contains 3-ketoisomaltose, it can be advantageously used as a mouthwash excellent in the prevention of periodontal disease and gingivitis.

<Toothpaste>
A toothpaste having the following composition was produced by a conventional method.
(Composition) (% by mass)
Dicalcium phosphate 30.0
Hydroxyapatite 10.0
Calcium carbonate 5.0
3-ketotrehalose 30.0
Sodium lauryl sulfate 1.5
Sodium monofluorophosphate 0.7
Polyoxyethylene sorbitan laurate 0.5
Diphenhydramine hydrochloride 0.5
Preservative 0.05
Purified water 22.0

Since this product contains 3-ketotrehalose, it can be advantageously used as a dentifrice excellent in the prevention and treatment of inflammation in the oral cavity, swelling of the gum due to alveolar pyorrhea, inflammation and bleeding.

<Foundation>
A foundation having the following composition was prepared by a conventional method.
(Composition) (% by mass)
Talc 20.0
Mica 33.0
Kaolin 7.0
Nylon powder 10.0
Titanium dioxide 10.0
Mica titanium 3.0
Zinc stearate 1.0
Red iron oxide 1.0
Yellow iron oxide 3.0
2-ketoglucose 1.5
Gardenia yellow/safflower red treated cellulose 2.0
Glycosyl transfer hesperidin 3.0
Squalane 6.0
Lanolin acetate 1.0
Octyldodecyl myristate 2.0
Perfume Suitable amount Preservative Suitable amount

Since this product contains 2-ketoglucose, it suppresses skin inflammation, maintains good skin condition, and can be used as a foundation for sunscreen, stain/freckle prevention, beautiful skin and fair skin. it can. In addition, since this product contains cellulose powder supporting gardenia yellow/safflower red, which has antioxidant action, radical scavenging action, elastase inhibitory action, lipase inhibitory action, etc., it suppresses the generation of acne and wrinkles, Can hold firm skin.

The anti-inflammatory agent of the present invention, which contains a carbohydrate having a ketoaldohexose structure in the molecule as an active ingredient, is a palliative or ameliorative agent for various inflammatory symptoms, and as a preventive or therapeutic agent for various inflammations, It can be used in the fields of cosmetics, quasi drugs, foods and pharmaceuticals.

In FIG.
←: Signal of C-3 carbon shifted to the lower magnetic field side compared to ¹³ C-NMR spectrum of trehalose

Claims (19)

An anti-inflammatory agent containing a sugar having a ketoaldohexose structure in its molecule as an active ingredient. The anti-inflammatory agent according to claim 1, wherein the ketoaldohexose structure is a 3-ketoaldohexose structure or a 2-ketoaldohexose structure. The anti-inflammatory agent according to claim 1 or 2, wherein the sugar is a monosaccharide, a disaccharide or a sugar alcohol thereof. 4. The sugar according to claim 1, wherein the sugar is a sugar in which the 1-position hydroxyl group of the ketoaldohexose structure is bonded to another sugar or a substituent other than sugar. Inflammatory agent. The anti-inflammatory agent according to any one of claims 2 to 4, wherein the 3-ketoaldohexose structure is a 3-ketoglucose structure or a 3-ketogalactose structure. The sugar having a 3-ketoaldohexose structure is 3-ketotrehalose, 3-ketocorgibiose, 3-ketoisomaltose, 3-ketolactose, 3-ketomaltose, 3-ketosucrose and 3-ketomultis. The anti-inflammatory agent according to any one of claims 2 to 5, which is one kind or two or more kinds selected from Toll. The anti-inflammatory agent according to any one of claims 2 to 4, wherein the 2-keto aldohexose structure is a 2-keto glucose structure. The anti-inflammatory agent according to any one of claims 2 to 4 and 7, wherein the sugar having a 2-ketoaldohexose structure is 2-ketoglucose or 2-ketotrehalose. The anti-inflammatory agent according to claim 1, which suppresses the production of tumor necrosis factor α (TNF-α) or interleukin-8 (IL-8) which are inflammatory cytokines. The anti-inflammatory agent according to any one of claims 1 to 9, wherein the inflammation targeted by the anti-inflammatory agent is chronic inflammation. 11. The chronic inflammation is diabetes, chronic respiratory disease, rheumatoid arthritis, gastritis, hepatitis, inflammatory bowel disease, neurodegenerative disease, cardiovascular disorder, psoriasis, atopic dermatitis, or periodontal disease. Anti-inflammatory agent. The anti-inflammatory agent according to any one of claims 1 to 11, which contains 0.1% to 90% of a sugar having a ketoaldohexose structure in the molecule. Cosmetics, foods, quasi drugs or pharmaceuticals containing the anti-inflammatory agent according to any one of claims 1 to 12. In a sugar having an aldohexose structure in the molecule, it is characterized by converting a free hydroxyl group which is not bonded to other sugars or substituents in the aldohexose structure into a keto group, which has an aldohexose structure in the molecule. And a method for improving the anti-inflammatory action of carbohydrates having 15. The method according to claim 14, wherein the free hydroxyl group that is not bound to the other sugar or the substituent is the 3-position hydroxyl group or the 2-position hydroxyl group. The method according to claim 14 or 15, wherein the conversion into a keto group is performed by an enzymatic method or a fermentation method. The method according to claim 14, wherein the sugar having an aldohexose structure in the molecule is a monosaccharide or a disaccharide. 18. The method of claim 17, wherein the disaccharide is trehalose, kojibiose, isomaltose, lactose, maltose, sucrose and maltitol. 18. The method of claim 17, wherein the monosaccharide is glucose.

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