JP6391959B2 - Non-alcoholic steatohepatitis ameliorating agent and ameliorating nutrition composition - Google Patents

Description

  The present invention relates to a preventive and / or ameliorating agent for nonalcoholic steatohepatitis and a nutritional composition for preventing and / or improving nonalcoholic steatohepatitis containing the same.

The liver is the largest glandular organ in the human body and an indispensable organ for living organisms. The liver is composed of a total of about 300 billion cells. Functional features of the liver include various biological reactions such as metabolic storage of nutrients, detoxification, phagocytosis of foreign substances and microorganisms, and regulation of blood circulation.
In recent years, due to an increase in obesity and diabetes accompanying changes in lifestyle, non-alcoholic fatty liver disease despite the absence of alcohol consumption, as well as pathological conditions that lead to liver inflammation, necrosis and liver fibrosis An increase in the number of “non-alcoholic steatohepatitis (hereinafter referred to as NASH)” patients who have been recognized has been reported (Non-patent Document 1). It is said that two steps are required to reach this NASH. The first step is to deposit fat in the liver to develop fatty liver, and then oxidative stress is applied to the fatty liver to damage hepatocytes. It is thought that it develops through the second stage of causing or inflammation. Currently, patients who have been pointed out to NASH are mainly treated with dietary improvement and exercise therapy, and other effective preventive and therapeutic methods have not been established yet. Prevention of NASH and the immediate treatment of NASH patients are at high risk of serious liver damage such as cirrhosis, and the development of prevention, improvement and treatment is an important medical issue. ing. However, the current situation is that drugs and treatment methods that are safe and have high preventive and therapeutic effects have not yet been established.

On the other hand, amino acids are highly safe compounds, and many nutritional reports have been made, but there are not so many reports of pharmacological effects on hepatocellular injury. In rats with liver damage caused by D-galactosamine or carbon tetrachloride, it has been reported that alanine suppresses the increase of blood aminotransferase and increases the survival rate. At this time, in the alanine administration group compared to the control group It has been reported that the proliferation rate of hepatocytes and the ATP content in the liver are significantly high (Non-patent Documents 2 and 3). It has also been reported that alanine inhibits hepatocyte damage caused by D-galactosamine in a concentration-dependent manner in vitro, and this action is suppressed by an inhibitor of the TCA cycle (Non-patent Document 4).
Alanine, an amino acid, is effective for alcoholic liver damage, viral hepatitis treatment, reports suggesting its effectiveness for liver regeneration (patent documents 1, 2 and 3), or for patients with primary biliary cirrhosis (PBC) There is a report that it is (patent document 4). As a report showing clinical improvement of NASH, a preventive, ameliorating and therapeutic agent for nonalcoholic steatohepatitis characterized by using L-alanine as an active ingredient has been proposed (Patent Document 5).
On the other hand, among the amino acids, branched-chain amino acids are used to improve encephalopathy during chronic liver injury and to improve hypoalbuminemia in decompensated cirrhosis patients with hypoalbuminemia despite adequate food intake. Often used. However, there has been a report that long-term administration of a nutrient containing a branched chain amino acid to a patient after liver resection for cirrhosis has no effect on either the cumulative cancer recurrence rate or survival rate (Non-patent Document) 7) On the other hand, a pharmaceutical for preventing and / or treating hepatocellular carcinoma comprising a combination of an acyclic retinoid and a branched chain amino acid has been disclosed (Patent Document 15), but the branched chain amino acid alone is disclosed. However, there is no suggestion that the effects of prevention, improvement, and treatment of nonalcoholic steatohepatitis are exhibited, and there is no suggestion that leucine and isoleucine among the branched chain amino acids are particularly effective.

  In addition, it attracts much attention because rare sugars have various functions. For example, Arnold et al. Suggest that D-allose and rare ketohexose are effective in suppressing the production of differentiated neutrophils and treating leukemia (Patent Document 6). However, D-allose is a rare sugar that rarely exists in nature, and since it was difficult to synthesize it by a chemical method, it was difficult to use for test studies. Recently, D-psicose has been produced in large quantities from D-fructose using D-tagatose-3-epimerase (Non-patent Document 5), and D-allose has been able to produce L-rhamnose isomerase from D-psicose. It has become possible to produce them by using them (Non-patent Document 6), and it has become possible to study the functionality and various uses of D-allose. Nitrous dopaminergic neuron oxidative injury inhibitor (Patent Document 7), angiogenesis inhibitor (Patent Document 8), blood pressure increase inhibitor (Patent Document 9), exercise caused by amyotrophic lateral sclerosis Pharmaceuticals for delaying onset or progression of disorder (Patent Document 10), liver cancer or skin cancer, transient ischemic stroke and stroke, flap necrosis, acute renal failure and uremia, retinal degeneration, premature infant Examples thereof include a medicine for treating retinopathy and ocular inflammation (Patent Document 11). However, the relationship between D-allose and the prevention or treatment of nonalcoholic steatohepatitis has not been reported.

  And, by continuously orally administering vitamin E to NASH patients with obesity, reports that the values of serum aminotransferase and alkaline phosphatase are reduced (Non-patent Document 8), containing pyruvic acid as an active ingredient, Furthermore, a non-alcoholic liver disease therapeutic agent characterized by containing vitamin E is disclosed (Patent Document 16), but non-alcoholic steatohepatitis is caused by the combination of vitamin E with D-allose and / or branched amino acids. There is no suggestion that it has a remarkable effect in the prevention or treatment of sulcer.

JP-A-5-213746 Japanese Patent Laid-Open No. 5-221858 JP-A-5-229940 Japanese Patent Laid-Open No. 11-853914 JP 2006-151937 A US Pat. No. 5,620,960 Japanese Patent No. 4724823 Japanese Patent No. 4943839 Japanese Patent No. 5158777 Japanese Patent No. 5317055 Japanese Patent No. 5330976 JP 2009-178143 A Japanese Patent No. 4499882 Japanese Patent No. 3975274 WO2012 / 020785 JP 2011-236160 A

Hepatology Praty, Volume 2 NASH / Alcoholic Liver Disorders (Bunkodo Co., Ltd.): 8-12 (2013) Hepatology, 24: 185-191 (1996). Hepatology, 24: 1211-1216 (1996). Biochem. Biophys. Res. Commun., 291: 738-743 (2002). J. of Biosci. and Bioengi. , 90, 453-455 (2000) J. of Ferment. and Bioengi. 85, 539-541. British Journal of Surgery vol. 84, p. 1525-1531 (1997) Journal of Psdiatrics vol. 136, p. 734-738 (2000)

  An object of the present invention is to provide a safe and effective preventive, ameliorating and therapeutic agent for NASH for which no effective drug therapy is currently known.

  Non-alcoholic steatohepatitis (hereinafter referred to as NASH) is a disease that subsequently occurs from fatty liver to NASH, and sufficient research has not been conducted on the prevention of onset and the therapeutic effect after onset. The present inventors have conducted various studies in order to find a medicine for preventing and / or treating nonalcoholic steatohepatitis. A pathological model very close to human NASH disease (see Patent Document 12) was verified, and as a result of using this model, administration of a feed supplemented with 2% by weight of D-allose is superior in NASH prevention and treatment effects, and It was newly found that administration of a branched chain amino acid (BCAA), particularly isoleucine (Ile) or leucine (leu) in a 2% weight ratio is excellent in NASH prevention and treatment effects. This model was also used for vitamin E, and it was newly found that a feed supplemented with only 50 or 100 mg of vitamin E per 100 g of feed has a higher NASH preventive effect than a normal feed supplemented with 20 mg per 100 g. . The rare sugar D-allose is a functional monosaccharide, and isoleucine and leucine are essential amino acids that contribute to the muscle part of the human body, among other amino acids. In addition, vitamin E resides in the cell membrane, stabilizes it by passing electrons to active oxygen as soon as possible to prevent the effects on unsaturated fatty acids, and changes itself into a mild reactive vitamin E radical. Either it loses its activity or transfers radicals to vitamin C, and itself has an excellent physiological action to become active vitamin E again. The present invention has been completed based on the above findings that the above components have the same effect on NASH.

The present invention provides a preventive and / or ameliorating agent for non-alcoholic steatohepatitis described in (1) to (6) below, and prevention of non-alcoholic steatohepatitis described in (7) and (8). The gist of the present invention is a nutritional composition for use and / or improvement.
(1) D-as an active ingredient allose, or, D- allose and to isoleucine and / or leucine and active ingredients, prophylactic and / or improving agent for nonalcoholic steatohepatitis, characterized in that.
(2) The preventive and / or improving agent for non-alcoholic steatohepatitis according to (1) above, wherein the dose per adult is 2.5 g to 20 g of D-allose.
(3) The preventive and / or improving agent for nonalcoholic steatohepatitis according to (1) or (2) above, further comprising vitamin E as an active ingredient.
(4) The preventive and / or ameliorating agent for nonalcoholic steatohepatitis according to (1), (2) or (3) above, which is a preventive and therapeutic agent.
(5) The preventive and / or ameliorating agent for nonalcoholic steatohepatitis according to (4) above, which is in the form of a pharmaceutical composition comprising a pharmaceutically acceptable pharmaceutical carrier.
(6) The preventive and / or ameliorating agent for nonalcoholic steatohepatitis according to any one of (1) to (5) above, which is a preparation for oral administration.
(7) A nutritional composition for preventing and / or improving nonalcoholic steatohepatitis comprising the preventive and / or improving agent for nonalcoholic steatohepatitis according to any one of (1) to (6) above .
(8) The nutritional composition for prevention and / or improvement of nonalcoholic steatohepatitis according to (7) above, which is a nutritional composition for prevention and treatment.

According to the present invention, a safe and effective improver for NASH can be provided. Since D-allose is used as an active ingredient, it is safe and has few side effects. Therefore, it is useful as a pharmaceutical for preventing or treating nonalcoholic steatohepatitis. Furthermore, since the monosaccharide of D-allose is a substance with established safety, the agent of the present invention is highly safe and can be used not only for pharmaceutical use but also for food. In addition, since a branched chain amino acid is used as an active ingredient, it is highly safe and has few side effects, and is therefore useful as a pharmaceutical for the prevention or treatment of nonalcoholic steatohepatitis. Furthermore, since two types of branched chain amino acids, isoleucine and leucine, are substances with established safety, the agent of the present invention is highly safe and can be used for food as well as for pharmaceutical use.
Furthermore, vitamin E is preferable because it is particularly safe as a food unless it is taken in a large amount. Vitamin E that can be used represents total vitamin E composed of one or more of α-tocopherol (α-vitamin E), β-tocopherol, γ-tocopherol, and δ-tocopherol.

NASH is a non-alcoholic fatty liver disease, and is a drawing explaining that it is a disease that exhibits liver dysfunction despite having no history of alcohol consumption. NASH is a disease that has a poor prognosis, and is a diagram that explains that an increase in NASH is a concern with an increase in lifestyle-related diseases. FIG. 6 is a diagram illustrating that STAM® mice are a model very similar to human NASH. The serum liver function test value of the STAM mouse | mouth of Example 1 (D-allose) is shown. The liver histological examination (NAS) result of the STAM mouse | mouth of Example 1 (D-allose) is shown. The liver histological examination (fat dyeing | staining) of the STAM mouse | mouth of Example 1 (D-allose) is shown. The primer list used for the gene expression fluctuation | variation analysis in the liver of the STAM mouse | mouth of Example 1 (D-allose) is shown. The result of having performed the gene expression fluctuation | variation analysis in the liver of the STAM mouse | mouth of Example 1 (D-allose) is shown. The serum liver function test value of the STAM mouse | mouth of Example 2 (Val, Leu, Ile) is shown. The liver histological examination (NAS) result of the STAM mouse | mouth of Example 2 (Val, Leu, Ile) is shown.

[NASH]
Non-alcoholic steatohepatitis (NASH) is a disease that causes necrosis / inflammation in liver tissue regardless of the history of alcohol consumption and exhibits liver dysfunction. At present, this disease is expected to increase further with the increase in the obese population and the westernization of lifestyle habits. FIG. 1 illustrates that NASH is a non-alcoholic fatty liver disease and is a disease that exhibits liver dysfunction despite having no history of alcohol consumption.
In addition, NASH is considered to be one of the important liver diseases that cannot be neglected in recent years because it progresses to cirrhosis or liver cancer and greatly affects the prognosis of life. FIG. 2 illustrates that NASH is a disease with a poor prognosis and is a disease in which an increase in NASH is a concern as lifestyle-related diseases increase. Moreover, the onset mechanism is not clear, and there is no established preventive and therapeutic methods at present. Since the onset mechanism, prevention method, and treatment method have not yet been clarified, research has been actively conducted in recent years, including basic research using animals.

  In Patent Document 6, an N-acetyl-β-D-glucosaminidase inhibitor is provided to mice for the purpose of providing a preparation technique of a model animal that becomes insulin resistant to fatty liver, steatohepatitis, liver fibrosis, cirrhosis, and liver cancer. Insulin resistance is induced by administration of sucrose, and further, the liver is induced using a high-fat diet to induce fatty liver, and it has been described that for the first time a mouse showing pathological findings similar to human NASH was successfully produced. .

  The present inventors have conducted intensive research to solve the problem of providing safe and effective improving agents and preventive / therapeutic agents for NASH. In the present invention, as shown in the examples, using STAM (registered trademark) mice and verifying this model, as a result of serum biochemical test values, it exhibits high blood sugar and high cholesterol, and is accompanied by liver dysfunction. Was recognized. From this, it was verified that STAM (registered trademark) mice (hereinafter referred to as “STAM mice”) are very similar to human NASH. By using a model animal, it is possible to evaluate whether it is a substance for prevention, improvement, or treatment of steatohepatitis. The evaluation of the preventive or ameliorating effect is made by determining whether steatohepatitis has been reduced by examining the pathological findings exhibited by the model animal.

[D-allose]
D-allose may be obtained by any production method, but according to the production method described in Patent Document 13, mass production is expected, so that it can be obtained more easily. According to the method for separating and recovering high-purity D-allose of Patent Document 14, D-allose crystals can be separated. In the above method, “L-rhamnose isomerase” is exemplified as an enzyme used for converting D-psicose into D-allose by an enzymatic reaction. L-rhamnose isomerase is a known enzyme published in the above-mentioned literature in 1998, and the same Pseudomonas stutzerii can be obtained from the Fermentation Research Institute of the strain Pseudomonas stutzerii LL172a. Pseudomonas stutzerii IFO 3773, Pseudomonas stutzerii IFO 13596 appear to have the same activity. A microorganism having the above-mentioned ability to produce L-rhamnose isomerase itself or a partially purified culture thereof can be used. By using it in the form of an immobilized enzyme or an immobilized microbial cell, it is possible to construct a reactor that has a low liquid feeding pressure and can be used continuously for a long period of time. The method for continuously producing high-purity D-allose allows desalting, deionization, concentration, and crystallization simultaneously with the separation of D-allose. Can be integrated into steps. Therefore, a large amount of processing is possible in a short time.

  D-allose may also be used as a mixed sugar mixed with a mixed sugar such as D-glucose, D-fructose, and other rare sugars (psicose, etc.).

  D-allose can be used as D-allose and / or a derivative thereof.

The derivative of D-allose used in the present invention will be described. A compound obtained by converting the structure of a molecule from a certain starting compound by a chemical reaction is called a derivative of the starting compound. There are various derivatives of hexose including D-allose. For example, amino sugars (such as those in which OH group of sugar molecule is substituted with NH ₂ group, glucosamine, chondrosamine, glycoside, etc.) However, it is not limited to them. In particular, in the case of the physiological action of monosaccharides, the specificity for the enzyme is the main cause of the action mechanism, and the three-dimensional structure of the sugar is particularly important. For example, an enzyme called fructokinase in the liver phosphorylates fructose as a main substrate. D-tagatose and D-psicose, which are isomers at the C-3 position of fructose, are also phosphorylated by this enzyme, but the phosphorylation rate varies depending on the three-dimensional structure. In this enzyme, the reaction rate is particularly affected depending on whether the carbon at the 2, 3, 4, 5 position is trans or axial. Therefore, even if it is a derivative | guide_body, if the three-dimensional structure is maintained to some extent, it is estimated that the effect | action of this invention is fully recognized. A derivative of D-allose is a sugar alcohol in which the ketone group of D-allose is an alcohol group, uronic acid in which the alcohol group of D-allose is oxidized, and an amino sugar in which the alcohol group of D-allose is substituted with an NH ₂ group D-allose derivatives selected from:

  As an action amount of D-allose, a single dose is in the range of 2.5 g to 20 g, and considering whether the body weight is within the range of the standard body weight depending on the severity of nonalcoholic steatohepatitis, etc. I can decide. Usually, it is desirable to be 5 g or more / once.

  In addition, the intake period of D-allose is about 1 week, preferably 2 weeks or longer.

  Furthermore, the form of D-allose referred to in the present invention may be any form such as powder, fine powder, granules, crystals, solids such as tablets, aqueous solutions, solutions and the like. Also, this production method is not particularly limited. Furthermore, it is possible to contain a bulking agent that is not another sweetening ingredient or taste ingredient, or a carrier that is used as a carrier, as long as the object of the present invention is not impaired.

[Isoleucine and / or leucine]
The branched chain amino acids used in the present invention, isoleucine and leucine, can be used in any of L-form, D-form and DL-form (mixture), preferably L-form and DL-form. Yes, and more preferably L-form. Isoleucine and / or leucine can be used not only in free form but also in salt form. Isoleucine and / or leucine in the present invention includes such salt forms.

  Examples of such salt forms include salts with acids (acid addition salts), salts with bases (base addition salts), and the like, and it is preferable to select a pharmaceutically acceptable salt. Such isoleucine and Examples of acids that are added to leucine and form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid, and phosphoric acid; acetic acid, lactic acid, citric acid, tartaric acid, Organic acids such as maleic acid, fumaric acid, monomethyl sulfuric acid and the like can be mentioned. Examples of the base which is added to isoleucine and / or leucine and forms a pharmaceutically acceptable base addition salt include, for example, metal hydroxides or carbonates such as sodium, potassium and calcium, or inorganic bases such as ammonia; Examples include organic bases such as ethylenediamine, propylenediamine, propylenediamine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine.

  When applied to humans, the content of two branched chain amino acids of isoleucine and / or leucine is 0.1 g or more, preferably 1 g or more as a single intake of the total amount. From the viewpoint of ease of ingestion, the single intake is preferably 100 g or less, more preferably 20 g or less, and most preferably 10 g or less. A single intake is the amount of active ingredient administered at a time, and in the case of food, it is the amount of active ingredient taken at a time. The amount of food intake varies from person to person, and it is difficult to define the amount of the active ingredient in the food as a whole. Therefore, it is recommended that the amount be taken as a single intake of the active ingredient. The single intake is an amount that varies appropriately depending on age, weight, sex, severity of non-alcoholic steatohepatitis, and the like.

Vitamin E refers to tocopherol (vitamin E) and its derivatives. Examples of those listed in the Japanese Pharmacopoeia include dL-α-tocopherol, tocopherol acetate (vitamin E acetate), and tocopherol succinate (vitamin E). Succinic acid ester). Non-Japanese Pharmacopoeia includes, for example, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, tocopherol nicotinate (vitamin E nicotinate ester), tocopherol phosphate (vitamin E phosphate ester), etc. Is mentioned.
As an action amount of vitamin E, a single dose is in the range of 0.5 g to 50 g, and is determined in consideration of whether the body weight is within the range of the standard body weight depending on the severity of nonalcoholic steatohepatitis. be able to. Usually, it is desirable to be 5 g or more / once. The content of vitamin E in the nutritional composition is preferably 0.5 to 50% by weight.

  The agent of the present invention is also useful as a medicine, food, and the like, and its application target includes mammals (eg, human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.). It is done. In addition, when adapted to mammals other than humans, the intake of the agent of the present invention may be appropriately adjusted according to the body weight or size of the animal.

The administration method in the case of using the agent of the present invention as a pharmaceutical is not particularly limited, but it can be via a general administration route such as oral administration, rectal administration, injection, administration by infusion.
Oral dosage forms include granules, fine granules, powders, coated tablets, tablets, suppositories, powders, (micro) capsules, chewables, syrups, juices, liquids, suspensions, emulsions, etc. Is mentioned. As dosage forms for administration by injection, general dosage forms of pharmaceutical preparations such as direct intravenous injection, infusion administration, and preparations that prolong the release of active substances can be employed.

  These medicaments are prepared by formulation according to a conventional method. Further, various pharmaceutically acceptable pharmaceutical substances can be blended as required in the preparation. The substance for the preparation can be appropriately selected depending on the dosage form of the preparation. For example, the excipient, the diluent, the additive, the disintegrant, the binder, the coating agent, the lubricant, the lubricant, the lubricant, and the flavoring agent. , Sweeteners, solubilizers and the like. Furthermore, specific examples of the pharmaceutical substance include magnesium carbonate, titanium dioxide, lactose, mannitol and other saccharides, talc, milk protein, gelatin, starch, cellulose and derivatives thereof, animal and vegetable oils, polyethylene glycol, and solvents, Examples include sterilized water and mono- or polyhydric alcohols such as glycerol.

  The dosage when using the medicament of the present invention varies depending on the age, weight or pathological condition of the subject patient, or the pharmaceutical dosage form or administration method, etc., but D-allose 0.015 g / kg body weight per adult day -1.2 g / kg body weight, isoleucine 0.015 g / kg body weight -1.2 g / kg body weight, leucine 0.03 g / kg body weight -2.4 g / kg body weight. The timing of administration is not particularly limited, and for example, it may be before meal, after meal, or between meals. Also, the administration period is not particularly limited.

Furthermore, the agent of the present invention can be easily used even in the form of food. When it is used as a food, it may be any general dietary form containing the active ingredient of the food of the present invention, that is, D-allose. For example, drinks such as soft drinks and powdered drinks can be prepared by adding an appropriate flavor. Specifically, for example, it can be eaten and eaten mixed with juice, milk, confectionery, jelly, yogurt, candy and the like.
It is also possible to provide such foods as health functional foods or dietary supplements. This health functional food includes food for specified health use and food with nutritional function. The food for specified health use is a food that can indicate that a specific health purpose can be expected, such as improvement of non-alcoholic steatohepatitis. In addition, functional nutritional food is a food that can indicate the function of the nutritional component when the amount of nutritional component included in the daily intake standard amount conforms to the national and national standards for upper and lower limits. is there. Dietary supplements include so-called nutritional supplements or health supplements. In the present invention, the food for specified health use is a food with a label indicating that it is used for applications such as the improvement of non-alcoholic steatohepatitis, and further, a so-called writing describing that it is used for such applications. Foods that include the above as a package are also included.

Furthermore, the agent of the present invention can be used as a concentrated liquid food or a food supplement. When used as a food supplement, it can be prepared in the form of tablets, capsules, powders, granules, suspensions, chewables, syrups and the like.
As used herein, “food supplement” refers to those taken for the purpose of supplementing nutrition in addition to those taken as food, and also includes nutritional supplements and supplements. Further, in the present invention, the “concentrated liquid food” is adjusted to a concentration of about 1 kcal / ml, and the qualitative composition of each nutrient is sufficiently taken into consideration so that a significant excess or deficiency of nutrients does not occur even after long-term single intake. It is a comprehensive nutritional food (liquid food) designed based on daily nutritional requirements.

  When ingested as food, the intake varies depending on the symptom, age or weight of the ingestion object, or the form or intake method of the food, but per adult day, D-allose 0.015 g / kg body weight to 1.2 g / kg The body weight, isoleucine 0.015 g / kg body weight to 1.2 g / kg body weight, and leucine 0.03 g / kg body weight to 2.4 g / kg body weight are used as a guide. The food can be ingested in a daily amount or divided into several times. The form of food, the method of intake, the intake period, etc. are not particularly limited.

D-allose has already been widely used in the pharmaceutical and food fields, and its safety has been established. Isoleucine and leucine have already been widely used in the pharmaceutical and food fields, and safety has been established.
Furthermore, the present invention includes foods and drinks obtained by using the above-described D-allose and / or isoleucine and / or leucine-containing materials of the present invention, particularly functional materials.

  The food and drink as used in the present invention refers to all foods that require sweetness such as beverages, candy, frozen desserts, yogurt, and chocolate. In addition, a therapeutic agent and a preventive agent for non-alcoholic steatohepatitis utilizing the effects of the nutritional composition of the present invention are included.

For example, soy sauce, powdered soy sauce, miso, powdered miso, moromi, horsetail, flicker, mayonnaise, dressing, vinegar, three cups of vinegar, powdered sushi vinegar, Chinese food, tentsuyu, noodle soup, sauce, ketchup, grilled meat sauce, curry roux, stew Useful as a sweetener for various seasonings such as Nomoto, soup, dashi, composite seasoning, mirin, new mirin, table sugar, coffee sugar, and as a taste improver, quality improver, etc. it can.
In addition, for example, various Japanese sweets such as rice crackers, hail, rice cakes, rice cakes, buns, eels, eels, sheepskin, water sheep rice cakes, brocade balls, jellies, castellas, rice cakes, bread, biscuits, crackers, cookies, pies, puddings , Butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy, etc. Paste such as paste, peanut paste, fruit paste, fruit such as jam, marmalade, syrup pickles, confectionery, processed foods of vegetables, breads, noodles, cooked foods such as cooked rice, artificial meat, Fukujin pickled, lettuce pickled , Senzuke, Lucky pickles, etc. Pickles, pickles, takuwanzuke, pickles such as Chinese cabbage, livestock products such as ham and sausage, fish ham, fish sausage, fish products such as seaweed, chikuwa, tempura, sea urchin, salted squid , Vinegared kombu, sakisume, dried delicacies such as blowfish mirin, paste, wild vegetables, sushi, small fish, shellfish made with shellfish, boiled beans, potato salad, side dish foods such as kombu rolls, dairy products, Fish, livestock, fruit, vegetable bottling, canned food, synthetic liquor, fruit liquor, western liquor, liqueur and other alcoholic beverages, coffee, cocoa, juice, carbonated beverages, lactic acid beverages, lactic acid bacteria beverages, pudding mix, hot It can be used as an additive to various foods such as premix powders such as cake mix, instant juice, instant coffee, instant juice powder and instant soup.
Examples of drinks that have particular functionality include carbonated drinks such as cola, sports drinks, fruit juice drinks, dairy drinks, and tea-based drinks. It can be said to be a good target.

  The above food and drink can also be used as functional foods, nutritional supplements or health foods. The form is not particularly limited, and examples of food production include milk proteins with high amino acid balance, soy protein, and proteins such as egg albumin, degradation products thereof, and egg white oligos. In addition to peptides, soybean hydrolysates, and the like, mixtures of amino acids alone can be used according to conventional methods. It can also be used in the form of a soft capsule, a tablet or the like.

  Examples of dietary supplements or functional foods include liquid foods, semi-digested nutritional foods, component nutritional foods, drinks, capsules, sugars, fats, trace elements, vitamins, agents, fragrances, etc. Examples of processing forms such as enteral nutrients. In the above-mentioned various foods, for example, foods and drinks such as sports drinks and nutritional drinks, nutritional additives and compositions such as amino acids, vitamins and minerals, spices and fragrances to improve nutritional balance and flavor , Pigments and the like can also be blended.

  The nutritional composition of the present invention is a food, health food, patient food, food material, health food material, patient food material, food additive, health food additive, patient food additive, beverage, health For liver function indicators and / or body fat reduction, such as beverages for patients, drinks for patients, drinking water for health, drinking water for patients, medicines, pharmaceutical ingredients, feeds, livestock and / or feeds for patients Can be used for everything you need.

When the nutritional composition of the present invention is used for food, it is prepared as it is, in a form diluted with oil, a milk form meal, or a form to which a carrier generally used in the food industry is added. May be. The form of the beverage is a non-alcoholic beverage or an alcoholic beverage. Non-alcoholic drinks include, for example, non-carbonated drinks such as carbonated drinks, fruit juice drinks, and nectar drinks, soft drinks, sports drinks, tea, coffee, cocoa, etc. The form of general foods, such as plum wine, beer, happoshu, and 3rd beer, can be mentioned.
The use form of the composition of the present invention as a food material or food additive for the purpose of improving the biological action includes solid preparations such as tablets or capsules, powders or granules dissolved in beverages, semi-forms such as jelly, etc. There are solids, liquids such as drinking water, and high-concentration solutions that are diluted.
Furthermore, the nutritional composition of the present invention can be appropriately added to foods to provide health foods or sick foods for the purpose of improving biological functions. As optional components, vitamins, carbohydrates, pigments, fragrances and the like that are usually added to foods can be appropriately blended. The food can be eaten in any form, liquid or solid. It can be eaten as a soft capsule encapsulated by gelatin or the like. The capsule is made of, for example, a gelatin film prepared by adding water to raw material gelatin and dissolving it, and adding a plasticizer (glycerin, D-sorbitol, etc.) thereto.

  The nutritional composition of the present invention can be applied to feed for livestock, poultry and pets. For example, it can mix | blend with livestock feeds, such as dry dog food, dry cat food, wet dog food, wet cat food, semi-moist dock food, poultry feed, cattle, and pigs. The feed itself can be prepared according to a conventional method. These therapeutic agents and prophylactic agents include non-human animals, for example, domestic mammals such as cattle, horses, pigs and sheep, poultry such as chickens, quails and ostriches, pets such as reptiles, birds and small mammals, It can also be used for farmed fish.

  The present invention will be described in more detail with reference to examples. However, the present invention is not limited to the examples.

[Reference example]
(Creation of a disease model very close to human NASH disease)
<1. STAM Mouse>
This is a pathological model for developing liver cancer from NASH without genetic modification (Patent Document 12). That is, the onset stage of fatty liver at 5 weeks of age, NASH at 7 weeks of age, liver fibrosis at 9 weeks of age, cirrhosis at 12 weeks of age, and liver cancer at 16 weeks of age is stably followed.
STAM mice induce insulin resistance by streptozotocin, which is the pathogenesis of human NASH, ie, streptozotocin, and cause systemic weak inflammation and hepatocyte apoptosis induction at high fat diet load.
The present inventors have studied the usefulness of STAM mice so far. As a result, from the results of liver function test values and histological examinations of STAM mice, it exhibits a pathological condition very similar to human NASH disease and develops stably, so it is an extremely useful pathological animal in NASH research. I have found that (Figure 3).
In this example, STAM mice were used. As a result of verifying this model, it was confirmed from the serum biochemical test values that hyperglycemia, high cholesterol, and liver dysfunction were accompanied. From this, it was verified that STAM mice are a model very similar to human NASH.

[Example 1]
[2. Experimental animal production and breeding schedule]
C57BL / 6J mice on the 14th day of pregnancy were purchased from Nippon Charles River Co., Ltd. The breeding was performed in an environment of a temperature of 24 ° C., a humidity of 50 ± 10%, and a light / dark cycle of 12 hours (light period 7:00 to 19:00). After confirming the birth, male mice were treated for pathogenesis according to Patent Document 7, and STAM mice were produced. Weaning was performed on the 4th week after birth, and the diet was switched to a high fat diet (HFD32: Nippon Claire Co., Ltd.). The STAM group (Control group) that performs grouping at 5 weeks of age, which is the stage of fatty liver onset, and continuously administers a high-fat diet (DA-STAM group that administers a high-fat diet supplemented with 2% D-allose) . In addition, after confirming birth, untreated male mice were administered a normal sample (AIN93G: Oriental Yeast Co.) after weaning to form a normal group. Fasting was performed at 8 weeks of age, the stage of NASH development, and the next day, dissection was performed.

[3. Serum transaminase activity measurement]
After blood collection, serum was collected by centrifugation. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using Fuji Dry Chem 7000 (Fuji Film Medical Co., Ltd.).
As a result, as shown in FIG. 4, although the liver function values (AST, ALT) of the STAM group (Control group) were significantly increased compared to normal (Normal group), D-allose administration (DA-STAM group) Tended to be low or significantly low. From the above, it was confirmed that D-allose is effective in suppressing the onset of NASH.

[4. Liver Histological Examination (NAS)]
A part of the liver was fixed with a 10% formalin solution after weighing, and a paraffin section was prepared. The prepared sections were stained with hematoxylin-eosin staining (HE staining), and hepatic steatosis (score: 0-3), intralobular inflammation (score: 0-3), liver using NAFLD activity score (NAS) Evaluation was made from the total score of cell balloon-like swelling (score: 0 to 2). The results are shown in FIG.
As shown in FIG. 5, as a result of examining the liver findings of STAM mice at NAS, which is a liver histological examination method for human NASH, D-allose administration group significantly reduces hepatic steatosis and lobular inflammation. A significant reduction was also observed in NAS, which is an index of severity of NASH.

[5. Liver Histological Examination (Fat Staining)]
In order to evaluate the degree of fat deposition in liver tissue, oil red O staining was performed as fat staining. That is, the frozen section was washed with water, immersed in 60% 2-propanol, oil red O solution (0.3 g of oil red O was dissolved in 100 ml of 99% 2-propanol, and a stock solution was prepared. Distilled water 4 was added to 6 and the mixture was stirred for 10 minutes and filtered, and then immersed in 60% 2-propanol. After lightly washing with water, it was immersed in Meyer's hematoxylin solution, washed with running water, and then sealed with a mounting agent (Mount Quick Aqueous, Cosmo Bio). An image of the stained tissue was obtained under a microscope, and the oil red positive area ratio was measured with dedicated image analysis software (imageJ). The results are shown in FIG.
As shown in FIG. 6, many fats observed in STAM mice are decreased in the D-allose administration group. Moreover, it was recognized that the D-allose administration group was significant for STAM mice even in the fat area after image analysis, and also in this result, suppression of hepatic steatosis was observed for D-allose. .

[6. Analysis of changes in the expression of inflammation-related genes and lipid metabolism-related genes in the liver]
The liver was partially frozen in dry ice acetone after weighing, and stored at −80 ° C. until total RNA was extracted. RNeasy Plus Universal Kit (QIAGEN) was used for the extraction of total RNA. CDNA was synthesized using River Tra Ace qPCR RT Kit (TOYOBO), and real-time PCR (LightCycler (registered trademark) 480, Roche) was performed using the primers shown in the primer list of FIG. Using GAPHD as an internal standard gene, gene expression fluctuation analysis was performed by the ΔCt method. The results are shown in FIG.
As shown in FIG. 8, SREBP-1 (sterol regulatory element-binding protein-1), which is a transcription factor related to fatty acid synthesis, is used as a lipid metabolism-related gene, and TNF-α, which is an inflammatory cytokine, is used as an inflammation-related gene. As a result of measuring the mRNA expression level in the liver for Tumor Necrosis Factor-α) and IL-1β (Interleukin-1β), the D-allose administration group showed a low value for the STAM mouse in both mRNA expression levels, Also from this result, D-allose was observed to inhibit fatty acid synthesis and inflammation induction in the liver.

[Statistical analysis]
JMP Pro 90.2 was used for statistical analysis of the data, and the numerical values were shown as mean ± standard error. 3. above. And 5. The results were analyzed using the Tukey-Kuramer method, and the results of 4. above were analyzed using the Steel L-Dwas method to obtain a significance level of p <0.05.

[Example 2]
[7. Experimental animal production and breeding schedule]
C57BL / 6J mice on the 14th day of pregnancy were purchased from Nippon Charles River Co., Ltd. The breeding was performed in an environment of a temperature of 24 ° C., a humidity of 50 ± 10%, and a light / dark cycle of 12 hours (light period 7:00 to 19:00). After confirming the birth, male mice were treated for pathogenesis according to Patent Document 12 to produce STAM mice. Weaning was performed on the 4th week after birth, and the diet was switched to a high fat diet (HFD32: Nippon Claire Co., Ltd.). STAM group (Control group), grouped at 5 weeks of age, which is the stage of fatty liver onset, and continuously administered a high-fat diet. High-fat diet supplemented with a total of 3 amino acids: 2% valine, isoleucine or leucine (Groups Val, Ile, and Leu, respectively). In addition, after confirming birth, untreated male mice were administered a normal sample (AIN93G: Oriental Yeast Co.) after weaning to form a normal group. Fasting was performed at 8 weeks of age, the stage of NASH development, and the next day, dissection was performed.

[8. Serum transaminase activity measurement]
After blood collection, serum was collected by centrifugation. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using Fuji Dry Chem 7000 (Fuji Film Medical Co., Ltd.).
As a result, as shown in FIG. 9, although the liver function values (AST, ALT) of the Control group and Val group were significantly increased as compared with the Normal group, the Ile group and Leu group had a low value or a significantly low value. It became. From the above, it was confirmed that Ile and Leu are effective in suppressing the onset of NASH.

[9. Liver histological examination (HE staining and fat staining)]
The liver collected by dissection at 8 weeks of age was partially weighed with a 10% formalin solution after weight measurement to prepare a paraffin section. The prepared sections were stained with hematoxylin-eosin staining (HE staining). In addition, for fat staining, frozen liver slices were washed with water, then immersed in 60% 2-propanol, and oil red O solution (0.3 g of oil red O was dissolved in 100 ml of 99% 2-propanol to prepare a stock solution. At the time of use, distilled water 4 was added to the preservation solution 6, and after stirring, left for 10 minutes and filtered, and then immersed in 60% 2-propanol. After lightly washing with water, it was immersed in Meyer's hematoxylin solution, washed with running water, and then sealed with a mounting agent (Mount Quick Aqueous, Cosmo Bio). Images of stained tissues were acquired under a microscope. All the photographs are shown in FIG.
From the results of HE staining, it was confirmed that the Control group and Val group exhibited histologically NASH images, and from the results of fat staining, the Control group and Val group exhibited fatty liver. On the other hand, it was recognized that the tissue images of the Ile group and Leu group alleviated NASH images and fatty liver.

[Statistical analysis]
JMP Pro 90.2 was used for statistical analysis of the data, and the numerical values were shown as mean ± standard error. 5. above. And 9. The results were analyzed using the Tukey-Kuramer method, and the results of 8 were analyzed using the Steel L-Dwas method to obtain a significance level of p <0.05.

Since monosaccharides and branched chain amino acids are used as active ingredients, they are safe and have few side effects, and are useful as pharmaceuticals for the prevention or treatment of nonalcoholic steatohepatitis. Furthermore, since the two types of branched chain amino acids D-allose and isoleucine and leucine contained in the agent of the present invention are substances having established safety, the agent of the present invention is highly safe and has pharmaceutical use. It can be used not only for food but also for industrial applicability.

Claims (8)

An active ingredient D- allose, or, D- allose and to isoleucine and / or leucine and active ingredients, prophylactic and / or improving agent for nonalcoholic steatohepatitis, characterized in that. The preventive and / or ameliorating agent for nonalcoholic steatohepatitis according to claim 1, wherein the dose per adult is contained in an amount of 2.5 to 20 g of D-allose. Furthermore, the preventive and / or ameliorating agent for nonalcoholic steatohepatitis according to claim 1 or 2, comprising vitamin E as an active ingredient. The preventive and / or ameliorating agent for nonalcoholic steatohepatitis according to claim 1, 2 or 3, which is a preventive and therapeutic agent. The preventive and / or ameliorating agent for nonalcoholic steatohepatitis according to claim 4, which is in the form of a pharmaceutical composition comprising a pharmaceutically acceptable pharmaceutical carrier. The preventive and / or ameliorating agent for non-alcoholic steatohepatitis according to any one of claims 1 to 5, which is a preparation for oral administration. A nutritional composition for preventing and / or improving nonalcoholic steatohepatitis comprising the preventive and / or improving agent for nonalcoholic steatohepatitis according to any one of claims 1 to 6. The nutritional composition for prevention and / or improvement of non-alcoholic steatohepatitis according to claim 7, which is a nutritional composition for prevention and treatment.