JP2022075294A - Agent that lowers blood ammonia concentration - Google Patents

Abstract

To provide techniques that safely and effectively lower blood ammonia concentration.SOLUTION: Provided is an agent containing as an active ingredient clostridium butyricum, 1-kestose, or clostridium butyricum and 1-kestose, which lowers blood ammonia concentration. According to the present invention, blood ammonia concentration can be safely and effectively lowered. Therefore, hyperammonemia and hepatic encephalopathy can be prevented or ameliorated.SELECTED DRAWING: Figure 1

Description

The present invention relates to an agent containing Clostridium butyricum and / or 1-kestose as an active ingredient, which lowers the blood ammonia concentration.

In the living body, ammonia is constantly produced in the process of metabolism of dietary proteins, proteins constituting the body and nucleic acids. Since ammonia is a toxic substance, there are a plurality of systems for removing ammonia in the living body as shown in the following (i) to (iv).
(I) Urea cycle (ornithine cycle) in the liver.
(Ii) Acid amide formation in all tissues.
(Iii) Reaction with keto acid, mainly in the liver.
(Iv) Direct excretion into urine in the kidney.

The urea cycle of (i) above is the center of the ammonia removal system. Ammonia taken into the urea cycle is converted to non-toxic urea and excreted as urine. In the acid amide formation of (ii), glutamine is produced from ammonia and glutamic acid by the action of glutaminase. In addition, asparagine is produced from ammonia and aspartic acid by the action of asparaginase. (Iii) In the reaction with keto acid, glutamic acid is produced from ammonia and α-ketoglutaric acid by the action of glutamic acid dehydrogenase. In the direct excretion of (iv), ammonia is produced from glutamic acid in the kidney and directly excreted in urine (Non-Patent Document 1).

By working these multiple systems, even if a part of the ammonia removal system is slightly damaged, the ammonia in the blood is kept at a low concentration (about 10 to 70 μg / dL). .. On the other hand, if severe liver disease such as liver cirrhosis, liver cancer, fulminant hepatitis, or congenital urea cycle enzyme deficiency occurs in this system, ammonia removal will be insufficient and blood will be removed. Ammonia in the liver becomes high concentration (hyperammonemia). If hyperammonemia progresses, it inhibits the function of the central nervous system and presents neuropsychiatric symptoms mainly due to impaired consciousness, and in severe cases, it leads to coma and death.

Therefore, in order to avoid a situation where the blood ammonia concentration becomes high, a technique for lowering the blood ammonia concentration has been researched and developed. For example, in Patent Document 1, the blood ammonia concentration containing a collagen peptide is described. Disclosed are foods and drinks or pharmaceutical compositions for oral administration that reduce.

Japanese Unexamined Patent Publication No. 2009-149541

S. Terrada, Faculty of Science, Faculty of Science, Functional Biochemistry Laboratory, Lecture Materials, Metabolism Map, Amino Acid Metabolism, Urea Cycle, Last Updated 2006/6/3, [online], [Search on June 12, 2nd year of Reiwa], Internet <URL: http://133.100.212.50/~bc1/Biochem/ureacycl.htm>

However, it cannot be said that the technology for reducing the blood ammonia concentration safely and effectively is still sufficiently supplied. The present invention has been made to solve the above problems, and an object of the present invention is to provide a technique for safely and effectively reducing the blood ammonia concentration.

As a result of diligent research, the present inventors have found that Clostridium butyricum and 1-kestose reduce blood ammonia levels. Therefore, based on the above findings, the following inventions were completed.

(1) The first aspect of the agent for lowering the blood ammonia concentration according to the present invention (hereinafter, may be referred to as "the agent for lowering the blood ammonia concentration" or "the present agent") is effective with Clostridium butyricum. It is an ingredient.

(2) In the second aspect of the agent for lowering the blood ammonia concentration according to the present invention, 1-kestose is used as an active ingredient.

(3) The third aspect of the agent for lowering the blood ammonia concentration according to the present invention comprises Clostridium butyricum and 1-kestose as active ingredients.

(4) The agent for lowering the blood ammonia concentration according to the present invention can be used for prevention or amelioration of hyperammonemia.

(5) The agent for lowering the blood ammonia concentration according to the present invention can be used for the prevention or amelioration of hepatic encephalopathy.

According to the present invention, the blood ammonia concentration can be lowered.

Clostridium butyricum is an anaerobic gram-positive bacillus that forms spores. It is widely marketed as an intestinal regulator and feed additive, and has high safety because it has been ingested by mammals such as humans and livestock for a long period of time. Therefore, according to the present invention, the blood ammonia concentration can be safely lowered.

In addition, Clostridium butyricum has high resistance to various external environments such as heat and acid in the state of spores. In addition, it can be dried and processed into a powder form, and does not exhibit a strong odor or taste. From these facts, Clostridium butyricum can be easily ingested on a daily basis as it is or by easily blending it with various foods, pharmaceuticals, feeds and the like. Therefore, according to the present invention, it is possible to obtain an agent for lowering blood ammonia concentration, which is highly safe and can be easily ingested on a daily basis by simply blending it as it is or in various foods, pharmaceuticals, feeds and the like. be able to.

1-Kestose is a trisaccharide oligosaccharide consisting of one molecule of glucose and two molecules of fructose. 1-Kestose is also contained in vegetables and grains such as onions, garlic, barley, and rye, and has a long history of eating, mutagenicity test, acute toxicity test, subchronic toxicity test, and chronic toxicity test. Since no toxicity has been observed in any of the above, the safety is high. Therefore, according to the present invention, the blood ammonia concentration can be safely lowered.

In addition, 1-kestose has high solubility and heat resistance equivalent to that of sugar, and has a sweetness of about one-third that of sugar, and exhibits a good sweetness similar to that of sugar. Therefore, it can be easily ingested as it is or as a seasoning such as a sweetener on a daily basis, and can be easily blended in various foods, pharmaceuticals, feeds and the like. Therefore, according to the present invention, it is possible to obtain an agent for lowering blood ammonia concentration, which is highly safe and can be easily ingested on a daily basis by simply blending it as it is or in various foods, pharmaceuticals, feeds and the like. be able to.

Hyperammonemia is a condition in which the blood concentration of ammonia deviates from the reference value (15 to 70 μg / dL) and becomes high. Hyperammonemia occurs when the ammonia removal system is severely impaired due to liver damage, drugs, infectious diseases, etc., and if it progresses, it causes various disturbances of consciousness and may lead to death in the worst case. According to the present invention, it is possible to contribute to the prevention or improvement of hyperammonemia by lowering the blood ammonia concentration.

Hepatic encephalopathy is associated with severe hepatic dysfunction and is associated with neuropsychiatric symptoms (impairment of sleep-wake rhythm, impaired memory and sensibleness, abnormal behavior, depression, somnolence, excitement / delirium, coma, epileptic seizures). , Flapping tremor), myoclonus, muscle atrophy, etc.). The main cause of hepatic encephalopathy is that the above-mentioned ammonia removal system such as (i) and (iii) becomes dysfunctional due to the functional deterioration of the liver, and high concentration of ammonia reaches the brain and deteriorates the brain function. Be done. According to the present invention, it is possible to contribute to the prevention or improvement of hepatic encephalopathy by lowering the blood ammonia concentration.

It is a bar graph which shows the serum ammonia concentration in the mouse which ingested Clostridium butyricum and / or 1-kestose.

Hereinafter, the present invention will be described in detail. The agent for lowering the blood ammonia concentration according to the present invention has the following aspects 1 to 3;
First aspect: Clostridium butyricum is used as an active ingredient.
Second aspect: 1-kestose is used as an active ingredient.
Third aspect: Clostridium butyricum and 1-kestose are used as active ingredients.

"Blood ammonia concentration" means the concentration of ammonia in blood. The blood ammonia concentration can be measured according to a known measuring method (direct colorimetric method, dry chemistry method, enzyme method (GIDH-UV method), enzyme method (NADS-UV method), enzyme cycling method, etc.). For measurement, blood is collected from the subject, and depending on the measurement method, whole blood or plasma or serum is prepared and used as a sample. Conveniently, measurement can be performed using a commercially available measurement kit or measurement reagent based on the principle of the above measurement method.

As Clostridium butyricum, commercially available ones may be used, or those deposited in the culture collection may be used. Commercially available products include, for example, live bacterial agents commercially available from Miyarisan Pharmaceutical Co., Ltd. (trade name: Miyarisan BM (registered trademark) fine granules, Miyarisan BM (registered trademark) tablets, and new Miyarisan Aiji (registered trademark) preparation. Examples thereof include enteric drugs, Miyarisan (registered trademark) tablets, and strong Miyarisan (registered trademark) tablets.

The items deposited in the Culture Collection include, for example, Clostridium butyricum Miyairi 588 (FERM BP-2789), Clostridium Butyricum Miyairi 585 (FERM BP-6815), and Clostridium Butyricum Miyairi 595 (FERM BP-). 6816), Clostridium butyricum Miyairi 630 (FERM BP-6817), Clostridium butyricum NIP1020 (FERM BP-5794), Clostridium butyricum NIP1021 (FERM BP-5795), Clostridium butyricum (FERM P-1868), Examples include Clostridium butyricum (FERM P-11869), Clostridium butyricum (FERM P-11870), Clostridium butyricum (ATCC859), Clostridium butyricum (NBRC3315), Clostridium butyricum (ATCC860), Clostridium butyricum (ATCC19398). be able to.

In the present invention, only one type of Clostridium butyricum may be used alone, or two or more types may be used in combination. Further, Clostridium butyricum may be used in the form of a culture solution obtained by culturing it, a residue containing Clostridium butyricum obtained by centrifuging the culture solution, or a dried product thereof.

The culture of Clostridium butyricum can be carried out by a known culture, for example, the method disclosed in Japanese Patent Application No. 08-252808. The culture conditions can be appropriately set according to the microbial strain to be used. For example, Clostridium butyricum is used as a medium (1.0 (w / v)% peptone, 1.0 (w / v)% yeast extract, 1). .0 (w / v)% corn starch and 0.2 (w / v)% precipitated calcium carbonate, pH ^6.5-7.5 ) were inoculated to ^105-106 cells / mL and at 37 ° C. It may be statically cultured for 48 hours. Since Clostridium butyricum is obligately anaerobic, it is cultured under anaerobic conditions by not aerating it, or by lowering the redox potential by aerating nitrogen or carbon dioxide gas or by adding a reducing agent to the medium. Is preferable. By centrifuging the culture broth thus obtained and collecting the precipitate, a culture broth residue containing Clostridium butyricum can be obtained. Further, if this residue is treated by air drying or vacuum drying, a dried product can be obtained. Examples of the air-drying condition include 0 to 80 ° C., preferably 10 to 20 ° C. for 1 to 24 hours, preferably 5 to 18 hours. Examples of the conditions for drying under reduced pressure include 0 to 80 ° C., preferably 10 to 20 ° C., 0.05 to 500 Torr, preferably 1 to 100 Torr, and 1 to 24 hours, preferably 2 to 15 hours.

1-Kestose can be produced by using sucrose as a substrate and performing an enzymatic reaction with an enzyme as disclosed in JP-A-58-201980. Specifically, first, β-fructofuranosidase is added to a sucrose solution, and the mixture is allowed to stand at 37 ° C. to 50 ° C. for about 20 hours to carry out an enzymatic reaction to produce 1-kestose. By subjecting the generated enzyme reaction solution containing 1-kestose to a chromatographic separation method as disclosed in JP-A-2000-232878, 1-kestose and other sugars (glucose, fructose, sucrose, etc.) (Oligosaccharides of 4 or more sugars) are separated and purified. The resulting solution containing 1-kestose with high purity is concentrated and then crystallized by a crystallization method as disclosed in Japanese Patent Publication No. 6-70075 to crystallize 1-kestose. Alternatively, a composition containing 1-kestose with a purity of 98% by mass or more can be obtained.

Further, since 1-kestose is contained in a commercially available fructooligosaccharide, 1-kestose may be used as it is, or 1-kestose may be separated and purified from fructooligosaccharide by the above-mentioned method. That is, as the 1-kestose of the present invention, a 1-kestose-containing composition such as an oligosaccharide containing 1-kestose may be used. When a 1-kestose-containing composition is used, the purity of 1-kestose is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more. In the present invention, the "purity" of 1-kestose means the mass% of 1-kestose when the total mass of sugar is 100.

The agent of the present invention can be used by ingesting it orally by humans or animals, or by adding an active ingredient to an enteral nutritional supplement and inserting it into a gastrointestinal tract such as the stomach or small intestine via a tube. Then, it may be used by a method of administration by enteral nutrition.

The agent of the present invention comprises only the active ingredients crotridium butyricum and / or 1-kestose, as well as pharmaceuticals, non-pharmaceutical products, food additives, supplements, food compositions (beverages, seasonings, breads, confectionery). , Side dishes, health foods, infant foods, other processed foods, etc.), feeds, feed additives, etc.

When the agent of the present invention is in the form of a pharmaceutical product, a food additive, or a supplement, the dosage form includes, for example, powder, tablet, sugar coating, capsule, granule, dry syrup, liquid, syrup, drop, etc. Examples thereof include solid or liquid dosage forms such as drinks. Each dosage form can be produced by a method known to those skilled in the art, for example, in the case of a powder (second embodiment), 800 g of 1-kestose and 200 g of lactose are well mixed, and then 300 mL of 90% ethanol is added. And moisten. Subsequently, after granulating the wet powder, it is air-dried at 60 ° C. for 16 hours, and then granulated to obtain 1000 g (1-kestose content 800 mg / 1 g) of a powder having an appropriate fineness. In the case of tablets, 300 g of 1-kestose, 380 g of powdered reduced starch syrup, 180 g of rice starch and 100 g of dextrin are well mixed, and then 300 mL of 90 (v / v)% ethanol is added and moistened to obtain a wet powder. The wet powder is extruded and granulated, and then air-dried at 60 ° C. for 16 hours to obtain granules. The granules are sized using a 850 μm sieve, and then 50 g of sucrose fatty acid ester is added to 470 g of the granules and mixed. By subjecting this to a rotary tableting machine (6B-2, Kikusui Seisakusho) for tableting, 5000 tablets (1-kestose content 60 mg / tablet) having a diameter of 8 mm and a weight of 200 mg can be obtained.

When the agent of the present invention is in the form of a food composition, it may be produced by adding Clostridium butyricum and / or 1-kestose in the process of producing a normal food or drink. Clostridium butyricum is spore-forming and has high heat resistance and acid resistance. In addition, it can be dried and processed into a powder form, and does not exhibit a strong odor or taste. 1-Kestose also has high heat resistance and has taste, physical characteristics and processability similar to those of sugar. From this, Clostridium butyricum and / or 1-kestose can be treated in the same manner as seasonings such as sugar in the process of producing various foods and drinks to produce various foods and drinks.

When this drug is used for humans and animals, the intake (dose) of the active ingredient may be, for example, 0.04 g / kg body weight or more per day in the case of 1-kestose. In the case of Clostridium butyricum, the culture solution, the residue containing bacteria obtained by centrifuging the culture solution, or the dried product of the residue is 20 to 360 mg / day in terms of solid content, 1 adult. 40-200 mg / one adult, or 60-120 mg / one adult can be exemplified. The amount of such intake is not limited to once a day, and may be divided into a plurality of times.

Hereinafter, the present invention will be described based on each embodiment. The technical scope of the present invention is not limited to the features shown by these examples.

In this example, as "1-kestose", a composition containing 1-kestose with a purity of 98% by mass or more (Busan Food Science Co., Ltd.) was used. Further, as "Clostridium butyricum", a powder containing Clostridium butyricum Miyairi 588 having an accession number of FERM BP-2789 (number of bacteria: 10 ⁹ to 10 ¹⁰ / g) was used.

Melanocortin type 4 receptor-deficient mice (MC4R-KO mice) were transferred by Dr. Joel K. Elmquist of the Southwestern Medical Center, University of Texas, Texas, USA. MC4R-KO mice are genetically background C57BL / 6J mice that lack the melanocortin type 4 receptor (MC4R), which plays an important role in central feeding regulation, and are non-alcoholic when fed with a high-fat diet. Develops steatohepatitis (M. Itoh et al., The American Journal of Pathology, Vol. 179, No. 5, 2454-2463, November 2011).

Eight-week-old male MC4R-KO mice were divided into four groups, I-IV, eight by eight. Mice from each group were placed in individual cages and bred for 12 weeks in an environment of temperature 22-26 ° C, humidity 40-60%, light period 12 hours and dark period 12 hours, free drinking, and free feeding. bottom. The feed should be a high-fat diet (D12492; 524kcal / 100g, 60% of total energy with fat; Research Diets Inc., New Zealand), or a high-fat diet with Clostridium butyricum and / or 1-. The one to which Kestose was added was given.
Group I: High-fat diet Group II: High-fat diet with Clostridium butyricum added (Clostridium butyricum concentration 2.0 g / feed 100 g)
Group III: High-fat diet with 1-kestose added (1-kestose concentration 2.5 g / feed 100 g)
Group IV: Clostridium butyricum and 1-kestose added to a high-fat diet (Clostridium butyricum concentration 2.0 g / feed 100 g, 1-kestose concentration 2.5 g / feed 100 g)

Based on the feed intake, the average value of each group was calculated for the daily intake of Clostridium butyricum and 1-Kestose. In addition, after the end of the breeding period, blood was collected to prepare serum, and the ammonia concentration was measured by the blood ammonia concentration measurement kit "Shikarikid NH ₃ " (Kanto Chemical Co., Inc.) based on the enzyme cycling method. For the measured ammonia concentration, the mean and standard deviation were calculated for each group. Table 1 shows the average values of feed intake, Clostridium butyricum intake, 1-kestose intake, and ammonia concentration. Table 2 shows the measurement results of the ammonia concentration. Further, a bar graph of the average value of the ammonia concentration is shown in FIG.

As shown in Table 1, Table 2 and FIG. 1, the serum ammonia concentration was lower in the II group, the III group and the IV group as compared with the I group. In particular, the serum ammonia concentration was significantly lower in the IV group than in the I group. From this result, it was clarified that Clostridium butyricum and 1-kestose reduce the blood ammonia concentration.

Claims (5)

An agent that lowers the blood ammonia concentration, containing Clostridium butyricum as an active ingredient. 1-An agent containing kestose as an active ingredient that lowers the blood ammonia concentration. An agent containing Clostridium butyricum and 1-Kestose as active ingredients to reduce blood ammonia concentration. The agent according to any one of claims 1 to 3, which is used for prevention or amelioration of hyperammonemia. The agent according to any one of claims 1 to 3, which is used for prevention or amelioration of hepatic encephalopathy.

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