JP2016204356A - Hepatic fibrogenesis inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide hepatic fibrogenesis inhibitors, therapeutic and/or prophylactic agents for diseases attributed to hepatic fibrogenesis, and the like, comprising components that have demonstrated to be excellent in safety or taste due to long history of use, the inhibitors, agents, and the like being ingestible for a long period of time.SOLUTION: We confirmed that hepatic fibrogenesis is inhibited in a rat administered with feed containing the protein of which 50% by weight is α-lactalbumin and 50% by weight is casein, as compared with a rat administered with feed containing the protein of which 100% by weight is casein, and that the increase of ALT (alanine amino transferase), AST (aspartic acid amino transferase), bilirubin total, and MMP (matrix metalloproteinase)-2 in serum that are indicators of hepatic cirrhosis is inhibited. From such findings, we found that hepatic fibrogenesis can be inhibited by administering α-lactalbumin, and also found the potential of the treatment and/or prevention of diseases attributed to hepatic fibrogenesis.SELECTED DRAWING: None

Description

  The present invention relates to an inhibitor of liver fibrosis containing α-lactalbumin and the like. The present invention also relates to a method for imparting an effect of suppressing liver fibrosis to food and drink, including a step of blending α-lactalbumin into the food and drink.

  Non-steroidal anti-inflammatory agents (NSAIDs) inhibit cyclooxygenase 2 (COX-2) and are useful as anti-inflammatory agents. However, since NSAIDs are metabolized in the liver, the burden on the liver is large. Therefore, patients with liver diseases such as cirrhosis cannot take NSAIDs. On the other hand, Patent Document 1 describes that α-lactalbumin (α-La) selectively inhibits COX-2 and is useful as an anti-inflammatory agent.

  Several compositions containing a hydrolyzate of α-La are known. For example, Patent Document 2 describes a method for producing a peptide degradation product obtained by hydrolyzing α-La, and a nutritional supplement composition for patients with liver diseases containing the peptide degradation product. Patent Document 3 describes a nutritional composition for patients with liver diseases, which contains an α-La hydrolyzate. However, Patent Document 2 and Patent Document 3 do not describe the use of α-La itself.

  Patent Document 4 describes that a complex having α-La biological activity is used for treatment of liver cancer and the like. However, Patent Document 4 describes that the α-La complex is a mutant, and does not describe the use of α-La itself.

WO 2008/140041 JP 05-276896 Special table 2006-515287 Special table 2007-524708 JP 09-110686

  In recent years, the number of patients with cirrhosis has been increasing. Cirrhosis occurs when liver fibrosis progresses in liver disorders such as hepatitis. When liver damage such as hepatitis occurs, hepatic stellate cells are activated and collagen fibers are synthesized. When liver damage becomes chronic, hepatic stellate cells are always activated, collagen fibers accumulate in the liver, and liver fibrosis further progresses. Liver fibrosis causes cirrhosis, which can become hepatocellular carcinoma if cirrhosis worsens further. Accordingly, there is a need to establish a method for suppressing liver fibrosis and an effective method for treating and preventing liver diseases such as cirrhosis.

  The present invention has been made in view of such a current situation, has proven to be excellent in safety and taste from a long eating experience, and contains α-La that can be ingested for a long period of time. It is an object to provide a fibrosis inhibitor and the like.

The inventors of the present invention have intensively studied to solve the above problems. Specifically, α-La was administered to cirrhosis model rats that were orally administered DMN (dimethylnitrosamine) for a short period of time to suppress liver fibrosis and liver cirrhosis caused by hepatocyte damage. The effect was examined. Here, since α-La has a small burden on the liver, it may be possible for patients with liver disease to take it.
As a result, the present inventors administered a diet in which 100% by weight of protein was casein in rats administered with a diet in which 50% by weight of protein was α-La and 50% by weight of protein was casein. Compared with rats, liver fibrosis is suppressed, and serum ALT (alanine aminotransferase), AST (aspartate aminotransferase), total bilirubin, MMP (matrix metalloproteins) are indicators of cirrhosis.・ It was clarified that the rise of proteinase-2 was suppressed. From these findings, it was found that administration of α-La can suppress liver fibrosis and can treat and / or prevent diseases caused by liver fibrosis. In addition, the present inventors may be concerned with nitric oxide (NO) in the process of inhibiting liver fibrosis by α-La and treating and / or preventing diseases caused by liver fibrosis. I found out.

That is, the present invention provides the following.
[1] An inhibitor of liver fibrosis, comprising α-lactalbumin as an active ingredient.
[2] The agent according to [1], wherein the fibrosis of the liver is due to hepatocyte damage.
[3] The agent according to [2], wherein the hepatocyte damage is caused by methylation of hepatocyte nucleic acid.
[4] The agent according to any one of [1] to [3], comprising a unit packaging form containing α-lactalbumin in an effective amount.
[5] The agent according to [4], wherein the effective amount of α-lactalbumin is at least 4.67 g / kg / day.
[6] A therapeutic and / or prophylactic agent for diseases caused by fibrosis of the liver, comprising α-lactalbumin as an active ingredient.
[7] Diseases caused by liver fibrosis are cirrhosis, hepatocellular carcinoma, chronic hepatitis B, chronic hepatitis C, alcoholic liver injury, nonalcoholic fatty liver disease, drug-induced liver injury, autoimmunity The agent according to [6], which is selected from the group consisting of hepatitis, cholestasis, biliary atresia, porphyria, and Wilson disease.
[8] The agent according to [6] or [7], comprising a unit packaging form containing an effective amount of α-lactalbumin.
[9] The agent according to [8], wherein the effective amount of α-lactalbumin is at least 4.67 g / kg / day.
[10] A method for imparting a liver fibrosis-suppressing effect to a food or drink, comprising a step of blending the food or drink with α-lactalbumin as an active ingredient.
[11] The method according to [10], wherein the fibrosis of the liver is due to hepatocyte damage.
[12] The method according to [11], wherein the hepatocyte damage is caused by nucleic acid methylation in the hepatocyte.
[13] A method for imparting a therapeutic and / or preventive effect on a disease caused by fibrosis of the liver to a food or drink, comprising a step of incorporating α-lactalbumin as an active ingredient into the food or drink.
[14] Diseases caused by liver fibrosis are cirrhosis, hepatocellular carcinoma, chronic hepatitis B, chronic hepatitis C, alcoholic liver disorder, non-alcoholic fatty liver disease, drug-induced liver disorder, autoimmunity The method according to [13], which is selected from the group consisting of hepatitis, cholestasis, biliary atresia, porphyria, and Wilson disease.

  The present invention provides an inhibitor of liver fibrosis and the like, which contains α-La as an active ingredient and can be taken by patients with liver diseases. In particular, the agent of the present invention can effectively suppress liver fibrosis due to hepatocyte damage.

It is a graph which shows transition of ALT and AST 6 days, 13 days after administration of DMN. It is a photograph which shows the tissue image of a liver. It is a graph which shows the density | concentration of serum ALT at the time of necropsy. It is a graph which shows the density | concentration of AST of the serum at the time of necropsy. It is a graph which shows the density | concentration of the serum ALP at the time of necropsy. It is a graph which shows the density | concentration of the serum total bilirubin at the time of necropsy. It is a graph which shows the density | concentration of the hyaluronic acid of the serum at the time of necropsy. It is a graph which shows the density | concentration of the serum MMP-2 at the time of necropsy. Results of measurement of ALT concentrations 3 and 10 days after DMN administration in 5 groups of normal, DMN-control, DMN-αLA, DMN-LA-LN, DMN-LN groups It is a graph which shows. In the statistical analysis, Dunnett's test or Steel's test was performed using the DMN-control group as a control. Results of measurement of AST concentration 3 and 10 days after DMN administration in 5 groups of normal, DMN-control, DMN-αLA, DMN-LA-LN, DMN-LN groups It is a graph which shows. In the statistical analysis, Dunnett's test or Steel's test was performed using the DMN-control group as a control.

  Hereinafter, the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention.

  The present invention relates to a liver fibrosis inhibitor comprising α-La as an active ingredient. Since α-La is contained in the milk of mammals, it is recognized as safe from a long eating experience. In fact, the safety of α-La has been scientifically verified. In addition, since α-La is a substance present in foods, it can be expected to be processed and distributed not only as pharmaceuticals but also as foods and drinks. Furthermore, since α-La can be obtained from relatively inexpensive raw materials such as milk, it can be mass-produced.

  In the present invention, α-La is not particularly limited in origin, but is preferably derived from humans or mammals other than humans (such as cows, sheep, goats, horses, etc.), more preferably derived from humans or cows. It is. The amino acid sequence and base sequence of α-La are already known and are registered in databases such as EMBL, DDBJ, NCBI. For example, α-La derived from bovine is registered as ACCESSION No. J05147 in DDBJ. The base sequence of α-La derived from bovine is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. In the amino acid sequence shown in SEQ ID NO: 2, positions 1 to 19 are signal peptides. In addition, in the amino acid sequence shown in SEQ ID NO: 2, it is known that there is a mutant in which arginine (R) at position 29 is replaced with glutamine (Q) (“Milk General Dictionary” p.35, Asakura Shoten) ). Α-La secreted into milk is a mature peptide obtained by cleaving the signal peptide. Α-La used in the examples of the present invention is the above-described bovine mature peptide.

  In the present invention, α-La is derived from the milk of mammals such as humans, cows, sheep, goats, etc., by ammonium sulfate precipitation ("Latest revised dairy technology manual", Dairy Technology Promotion Society, pages 122-125, 1975), It can be prepared by a known technique such as an ultrafiltration method (Japanese Patent Laid-Open No. 5-28879) or an ion exchange method (Japanese Patent No. 2916047). The content of α-La in milk varies depending on the production area and feed, but is usually about 1.2 g / kg. If milk is used as a raw material, α-La can be easily prepared in large quantities. can do. Further, α-La can be synthesized by a chemical method based on an amino acid sequence and a base sequence, and α-La can be produced by a genetic engineering method known to those skilled in the art. Commercially available α-La (for example, manufactured by Davisco) can be used.

  In the present invention, α-La is not limited to the above-mentioned known amino acid sequence, and is a mutant polypeptide having a mutation in the amino acid sequence as long as it has an action of suppressing fibrosis of the liver. Also good. Examples of such polypeptides include natural mutants, homologs, and artificial mutants. Specifically, a polypeptide containing a sequence in which one or more amino acids are added, deleted, substituted, or inserted into a known amino acid sequence of α-La, or a complementary strand of a polynucleotide encoding α-La, A polypeptide encoding a polynucleotide that hybridizes under stringent conditions can be mentioned. In addition, one or more amino acids (for example, 2, 3, 4, 5, 10, 20, 30, 40, 50, or 100) are added to or deleted from the amino acid sequence set forth in SEQ ID NO: 2. Polypeptides having an action of suppressing liver fibrosis, including substituted and inserted sequences, can also be mentioned, and stringent conditions can be applied to the complementary strand of the polynucleotide containing the coding region of the base sequence described in SEQ ID NO: 1. And a polypeptide encoding a hybridizing polynucleotide and having an action of suppressing liver fibrosis. At this time, these mutant polypeptides can be prepared by techniques known to those skilled in the art.

  The present invention provides an inhibitor of liver fibrosis containing α-La as an active ingredient. On the other hand, the present invention provides an inhibitor of liver fibrosis caused by hepatocyte damage, comprising α-La as an active ingredient. In the present invention, damage to hepatocytes means that the structure of the liver is changed by some external action such as a physical action on the liver, a chemical action such as exposure to a chemical substance, or a biological action such as an infection. Or that the function of the liver is reduced. In general, liver fibrosis is the result of a response to hepatocyte damage. In the present invention, liver fibrosis refers to the fibrosis state of liver tissue. Examples include, but are not limited to, accumulation of extracellular matrix such as collagen in liver tissue and activation of fibroblasts. In the present invention, fibrosis of the liver can also be expressed as fibrosis of hepatocytes, fibrosis of liver tissue, liver fibrosis and the like. The liver fibrosis inhibitor of the present invention may be one that suppresses the above-described hepatocyte damage itself in addition to liver fibrosis. That is, the present invention also provides an inhibitor of hepatocyte damage comprising α-La as an active ingredient.

  The agent of the present invention can effectively suppress liver fibrosis. In the present invention, “suppressing fibrosis” means reducing or eliminating fibrotic lesions in a tissue in which fibrosis has occurred, or delaying or preventing the progression of fibrosis (suppressing the increase or increase of fibrotic lesions). To do).

  In the present invention, the degree of liver fibrosis can be evaluated by methods known to those skilled in the art. For example, a method of evaluating the findings of fibrosis by liver biopsy can be mentioned. Specifically, there can be mentioned a method of specially staining a liver biopsy sample (Masson-trichrome staining, silver staining, etc.) and histologically evaluating an image of the emphasized fibrotic tissue. Histological assessment of fibrosis is performed according to, for example, immunohistology according to Dai K, et al. World J Gactroenterol. 31: 4822-4826, 2005, Hillebrandt S, et al. Nature Genetics 37: 835-843, 2005. Based on the dynamic staining, the fibrosis level of the hepatocytes of each sample can be expressed as the fibrosis frequency. It is also possible to evaluate the degree of liver fibrosis using markers such as hyaluronic acid, type I, type III, type IV collagen, fibroblasts, macrophages, etc., which are indicators of liver fibrosis. The degree of liver fibrosis can be evaluated using liver imaging such as platelet count measurement and abdominal ultrasonography. In addition, non-invasive measuring instruments (such as Fibro Scan502) based on the transition end elastography technology developed by EcoSense (EcoSence, France) in recent years can also be used to assess the extent of liver fibrosis. . In the present invention, the degree of suppression of liver fibrosis can be determined based on the evaluation of the degree of liver fibrosis obtained by these methods.

  In the present invention, hepatocyte damage is preferably caused by methylation of hepatocyte nucleic acid. Methylation of hepatocyte nucleic acids includes methylation-specific PCR (MSP) method, HELP assay, ChIP (chromatin immunoprecipitation) -on-chip assay, restriction enzyme landmark genome scanning, methylated DNA immunoprecipitation, etc. Can be detected by techniques known to those skilled in the art.

  The present invention is an agent that can be orally administered to a subject where liver fibrosis is observed. That is, the present invention provides an inhibitor of liver fibrosis, which is used for administration to a subject where liver fibrosis is observed. The present invention is an inhibitor of liver fibrosis containing α-La as an active ingredient, and includes a step of determining whether or not liver fibrosis has occurred in a subject to be administered, It is also an agent for oral administration to a subject determined to have fibrosis. In the present invention, “administration” can also be referred to as “intake”.

  The present invention provides a therapeutic and / or prophylactic agent for diseases caused by fibrosis of the liver and damage of hepatocytes, comprising α-La as an active ingredient. The present invention is also an agent for oral administration to a subject in which a disease caused by liver fibrosis is observed. That is, the present invention provides a therapeutic and / or preventive agent for a disease caused by liver fibrosis, which is used for oral administration to a subject in which a disease caused by liver fibrosis is observed. The present invention is a therapeutic and / or prophylactic agent for diseases caused by fibrosis of the liver, containing α-La as an active ingredient, wherein a disease caused by fibrosis of the liver occurs in the subject of administration. And an agent for oral administration to a subject determined to have developed a disease caused by liver fibrosis. In the present invention, “administration” can also be referred to as “intake”.

  In the present invention, the disease caused by liver fibrosis is not particularly limited as long as it is a disease accompanied by liver fibrosis, but chronic hepatitis (such as chronic hepatitis B and chronic hepatitis C), cirrhosis, liver failure, Chronic liver diseases such as hepatocellular carcinoma, alcoholic liver disease, nonalcoholic fatty liver disease, drug-induced liver injury, autoimmune hepatitis, cholestasis, biliary atresia, porphyria, Wilson disease, and these diseases And preferably include chronic liver diseases such as chronic hepatitis (such as chronic hepatitis B and chronic hepatitis C), cirrhosis, liver failure, and hepatocellular carcinoma.

  The present invention is an agent particularly useful for the treatment and / or prevention of cirrhosis. In the present invention, cirrhosis is particularly a condition of the liver in which extensive fibrosis occurs in the liver and normal liver lobule structure is lost and reconstructed into an abnormal structure (pseudolobule / regeneration nodule). Without limitation, viral cirrhosis, parasitic cirrhosis, toxic cirrhosis, dystrophic cirrhosis, alcoholic cirrhosis, congestive cirrhosis, hepatic sclerosis, primary biliary cirrhosis, secondary biliary cirrhosis, monolobal cirrhosis, Liver cirrhosis, obstructive cirrhosis, biliary cirrhosis, biliary cirrhosis, atrophic cirrhosis, post-necrotic cirrhosis, post-hepatic cirrhosis, nodular cirrhosis, mixed cirrhosis, nodular Cirrhosis, compensatory cirrhosis, decompensated cirrhosis, major nodular cirrhosis, septal cirrhosis, idiopathic cirrhosis, periportal cirrhosis, portal cirrhosis and the like. Cirrhosis is classified into many types according to its etiology and the characteristics of pseudolobule.

  In the present invention, the administration (intake) amount and administration (intake) method can be selected according to the age, symptoms, and body weight of the subject requiring the administration (intake). In the present invention, although not limited thereto, the active ingredient α-La is administered (ingested) per kg body weight per day, for example, 0.002 mg to 10 g, preferably It can be set to 0.01 mg to 8 g, more preferably 0.2 mg to 7 g, still more preferably 10 mg to 6 g, and particularly preferably 40 mg to 5 g. However, since the safety of α-La has been scientifically verified, it is not always necessary to set the upper limit of the dose (intake). Therefore, in the present invention, although not limited thereto, α-La, which is an active ingredient, is administered (ingested) per 1 kg body weight per day, for example, 1 mg or more, more preferably 20 To provide an agent to be administered in mg or more, more preferably 1 g or more, particularly preferably 4 g or more (for example, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 g or more). Can do.

  The present invention is packaged in a unit amount (one intake amount) from the viewpoint of hygiene at the time of administration (intake) and ease of handling during transport and storage (consisting of a unit package form). It is preferable that it is an agent. Therefore, this invention can provide the agent for suppression of the fibrosis of the liver which consists of a unit package form. In the present invention, the unit packaging form can be set to a form packaged for each unit dose per administration or meal. In the present invention, although not limited to these, α-La, which is the active ingredient, is administered per unit or in a unit amount per serving, for example, 5 g to 500 g (or 5 mL to 500 mL), preferably Can be set to 10 g to 300 g (or 10 mL to 300 mL), more preferably 50 g to 200 g (or 50 mL to 200 mL). In the present invention, α-La, which is the active ingredient, can be set to a daily dose as a unit amount per serving, and the daily dose can be subdivided to ½ to 1/10 of the daily dose. Can be set to

  The present invention relates to pharmaceuticals (pharmaceutical compositions, nutritional pharmaceutical compositions, drugs, enteral nutrients, pharmaceutical additives (pharmaceutical additives), etc.), foods and beverages (food and beverage compositions, nutritional compositions, special-purpose foods, nutrition It is an agent that can be used in any form such as functional foods, health foods, food additives (food additives) and the like, and cosmetics. In addition, the present invention is an agent that can be added (blended) to pharmaceuticals, foods and drinks, cosmetics, etc. and / or used in combination with pharmaceuticals, foods and drinks, cosmetics, and the like. This makes it possible to suppress the effect of liver fibrosis (suppression function), treatment effect (treatment function) and / or prevention effect (prevention function) of diseases caused by liver fibrosis, etc. on pharmaceuticals, foods and drinks, cosmetics, etc. Can be granted.

  When the agent of the present invention is used as a medicine (form of a medicine), it can be administered in various forms. Examples of the administration form include oral administration or enteral administration (nasal tube, gastric fistula, intestinal fistula, etc.) of pharmaceuticals such as enteral nutrients and liquids. At this time, the agent of the present invention can be processed into a tablet, capsule, granule, powder, syrup or the like for administration. And as these various preparations, according to conventional methods, the main ingredients are excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, solvents, isotonic agents. Examples thereof include, but are not limited to, those produced using known adjuvants in the technical field of pharmaceutical preparations such as In addition, these various preparations can contain an appropriate amount of calcium. Further, it can contain appropriate amounts of vitamins, minerals, organic acids, sugars, amino acids, peptides and the like.

  When the agent of the present invention is used as a pharmaceutical, the dosage and administration method can be selected according to the age, weight, administration purpose (symptoms), etc. of the subject (patient etc.) requiring its administration. At this time, in the present invention, although not limited to these, the dose of the agent is, for example, 0.05 g to 250 g, preferably 0.5 g to 200 g, more preferably 1 g per patient at a time. ˜150 g, more preferably 1.5 g to 125 g, particularly preferably 2.5 g to 100 g. The dosage and administration method of the agent of the present invention are not limited to these, but per kg body weight at a time, for example, 0.001 mg to 5,000 mg, preferably 0.01 mg to 4,000 mg, more preferably Is 0.02 mg to 3,000 mg, more preferably 0.03 mg to 2,500 mg, particularly preferably 0.05 mg to 2,000 mg, for example, 1 to 6 times a day, preferably 1 to 5 times a day, more preferably 1 to 4 times a day, more preferably 1 to 3 times a day, for example 1 week or more, preferably 1 week to 12 months, more preferably 2 weeks to 6 months, more preferably 3 weeks to It can be administered for 4 months, particularly preferably for 4 weeks to 3 months. Furthermore, the agent of the present invention can be administered to a subject (patient or the like) in need of administration in a lump sum, before meals, after meals, between meals and / or before going to bed, etc., or divided and administered. You can also In addition, the agent of the present invention can be adjusted individually according to the state of each subject while observing the state after actual administration to the subject and the degree of fibrosis of the liver. Can do.

  When using the agent of this invention as food / beverage products (it is set as the form of food / beverage products), it can be ingested with a various form. Examples of the ingestion form include oral intake or enteral intake (eg, nasal tube, gastric fistula, intestinal fistula) of foods and drinks such as dairy products and liquid foods. At this time, the agent of the present invention can be processed and ingested into foods and drinks such as liquids, pastes, solids, and powders, mixed (added) into various foods and drinks, and / or Or it can also take in combination with various food-drinks. And as these food-drinks, Preferably, it is a drink, confectionery (including dessert etc.), dairy products, liquid food (including foods and drinks with high nutritional value), infant formula, and instant food, and more preferably Are water, beverages, confectionery, dairy products, liquid foods, infant formula, more preferably confectionery, dairy products, liquid foods, and particularly preferably dairy products. It is not limited. That is, more specifically, milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pudding, jelly, ice cream, pizza crust, prepared milk powder, liquid food, food for the sick, nutritional food, Examples include frozen foods, food compositions, processed foods, and other commercially available foods. These various foods and drinks can also be provided as meals at various schools, nursing homes, and welfare facilities.

  When the agent of the present invention is used as a food or drink, the intake amount or intake method can be selected according to the age, weight, intake purpose, etc. of the subject requiring the intake. Examples of the form of the agent of the present invention include dairy products, liquid foods, etc. For example, it may be a fruit juice type beverage, a milk shake type beverage, etc. -It may be a powdered food that can be dispersed. The effective dose of the agent of the present invention is, for example, 0.05 g to 1000 g, preferably 0.05 g to 500 g, more preferably 0.1 g to 500 g, still more preferably 1 g to 300 g, and particularly preferably 2 g per day. ~ 200g. The agent of the present invention can be taken in a lump before or after a meal, between meals and / or before going to bed, etc. in a subject that needs to be taken, or can be taken in divided portions. In addition, the agent of the present invention can be used as a substitute for a meal, or can be used as an auxiliary to a meal.

  In order to adjust the nutritional composition, foods and drinks containing the agent of the present invention include water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like. Nutrients can be added (added). Specifically, as protein, for example, whole milk powder, skim milk powder, partially skimmed milk powder, whole fat concentrated milk, skim concentrated milk, partially skimmed concentrated milk, casein, whey powder, whey protein, whey protein concentrate, whey protein Isolates, α-casein, β-casein, κ-casein, lactoferrin, soy protein, chicken egg protein, meat protein, etc., their degradation products, various milk-derived components, and the like can be used. Peptides such as casein phosphopeptide, arginine, and lysine, amino acids, and the like can also be used. As the saccharide, for example, saccharides, modified starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber and the like can be used. Examples of lipids include animal oils such as lard and fish oil, fractionated oils thereof, hydrogenated oils and transesterified oils; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, hydrogen Vegetable oils such as additive oils and transesterified oils can be used. As vitamins, for example, vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, Folic acid or the like can be used. As minerals, for example, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium and the like can be used, and trace elements derived from natural products, such as copper, zinc, selenium, manganese of mineral yeast, Chromium, copper gluconate, zinc glucone and the like can also be used. Examples of organic acids that can be used include malic acid, citric acid, lactic acid, and tartaric acid. These components can be used even if they are synthetic products or derived from natural products, foods containing many of them can be used as raw materials, and two or more types can be used in combination.

  When the agent of the present invention is used as a food or drink, a champignon extract having a fecal odor reducing effect, for example, 0.005% to 0.5% by weight, a carotenoid preparation having a nutrition enhancing effect (for example, α-carotene, β-carotene, lycopene) , Including lutein) can be blended (added) at, for example, 0.00001% to 0.0002% by weight. Moreover, catechin, polyphenol, etc. can also be mix | blended as an antioxidant. Furthermore, carnitine having an effect of enhancing lipid metabolism can be added. Carnitine is a biological trace component produced in the liver and kidney from lysine and methionine, and it is known that the production amount thereof decreases with age. Carnitine has been shown to play an important role in the metabolism of nutrients, such as the delivery of long-chain fatty acids to muscle cells.

  Dietary fiber is classified into water-soluble dietary fiber and insoluble dietary fiber, both of which can be used in the agent of the present invention. As water-soluble dietary fiber, the indigestible oligosaccharides lactulose, lactitol, and raffinose can be used. Physiological functions of indigestible oligosaccharides include the effect of improving the intestinal environment by reaching the large intestine as it is undigested, contributing to the activation and proliferation of intestinal bifidobacteria. Lactulose is a synthetic disaccharide composed of galactose and fructose and is used as a basic drug for hyperammonemia (Bircher, J. et al., Lancet i: 890, 1965). Chronic recurrent hepatic encephalopathy due to chronic liver failure responds well to lactulose administration, special amino acid infusion for liver failure (Fischer solution), etc. Lactitol (β-galactosyl-sorbitol) is the second generation of lactulose and is comparable to lactulose in clinical effects of chronic hepatic encephalopathy (Lanthier, PL. And Morgan, M., Gut, 26 : 415, 1985; Uribe, M., et al., Dig. Dis. Sci., 32: 1345, 1987; Heredia, D. et al., J. Hepatol, 7: 106, 1988; Riggio, O., et al., Dig. Dis. Sci., 34: 823, 1989), used as a drug for the treatment of hyperammonemia.

  Use water-soluble dietary fiber such as pectin (protopectin, pectinic acid, pectinic acid), guar gum enzyme degradation product, tamarind seed gum, etc. that have an effect of improving lipid metabolism (lowering action of cholesterol and lowering action of neutral fat) Can do. Guar gum enzyme degradation products also have an inhibitory effect on blood sugar level increase and an insulin saving effect (Kazuhiko Yamatoya et al., Journal of Japanese Society of Nutrition and Food, 46: 199, 1993). Furthermore, as water-soluble dietary fiber, konjac glucomannan, alginic acid, low-molecular alginic acid, psyllium, gum arabic, seaweed polysaccharide (cellulose, lignin-like substance, agar, carrageenan, alginic acid, fucodyne, laminarin) ), Microbial gums (welan gum, curdlan, xanthan gum, gellan gum, dextran, pullulan, lambzan gum), other gums (locust bean gum derived from seeds, tamarind gum, tara gum, caraya gum derived from sap, tragacanth gum), etc. For dietary fiber, polydextrose, indigestible dextrin, maltitol and the like can be used.

  As the insoluble dietary fiber, cellulose, hemicellulose, lignin, chitin, chitosan, soybean dietary fiber, wheat bran, pine fiber, corn fiber, beet fiber and the like can be used. As a physiological function of insoluble dietary fiber, there is an effect of increasing the number of defecations and increasing the amount of stool by increasing the amount of indigestible material in the large intestine and shortening the passage time.

  The agent of the present invention can be produced by methods known to those skilled in the art in the fields of beverages, dairy products, liquid foods, enteral nutrients, medicines, and the like. If it is an expert, the compounding process of this invention or those processed materials will be mixed with other components, a molding process, a sterilization process, a fermentation process, a firing process, a drying process, a cooling process, a granulation process, a packaging process. Etc. can be selected and combined to produce a desired food or beverage or medicine. Specifically, for example, when the usage form of the agent of the present invention is liquid, the agent (raw material solution) is preliminarily heat sterilized and then cooled as necessary, and then aseptically filled into a container. (E.g., ultra high temperature (UHT) sterilization (e.g., 130-145C, 1-10 seconds) or high temperature (HTST) sterilization (e.g., 100-130C, 1-60 seconds) and aseptic filling (aseptic) ( A method in which a packaging method is used in combination), a method in which the agent (raw material solution) is filled into a container and then heat-sterilized with the container (for example, a retort method, an autoclave method), or the like can be used. In addition, when the use form of the agent of the present invention is liquid, a homogenized product based on the agent (a material obtained by homogenizing a raw material solution or a sterilizing solution) is again heat-sterilized (for example, ultra-high temperature (UHT)) as necessary. A method of filling aseptically after cooling after sterilization or high temperature (HTST) sterilization), a method of heating and sterilizing with a container after filling into a can container or a soft bag can be used. At this time, when the usage form of the agent of the present invention is an acidic liquid, the pH can be set to 2.0 to 6.0, preferably 3.0 to 5.0. Further, when the usage form of the agent of the present invention is a powder, a homogenized product based on the agent is, as necessary, for example, vacuum evaporation concentration or freeze concentration, followed by spray drying or freeze drying to obtain a powder, etc. Can be used.

  When the agent of the present invention is used as various dairy products, it can be produced by methods known to those skilled in the art. For example, in the case of yogurt, a preparation process for a starter for culturing lactic acid bacteria, blending (adding) α-La and then a raw milk preparation process for mixing milk components, etc. It can be manufactured by a method that goes through a fermentation process that holds at a certain temperature, a cooling process that lowers the fermented milk to a predetermined temperature, a flavoring process that blends flavors and flavoring substances as needed, a filling process, etc. it can. In this case, for example, a mixture of lactic acid bacteria belonging to the genus Lactobacillus and lactic acid bacteria belonging to the genus Streptococcus can be used as the starter, and preferably a mixture of Lactobacillus delbrueckii subsp. Bulgaricus and Streptococcus thermophilus can be used. Moreover, in the case of cheese, for example, the preparation process of the starter as described above, the preparation process of the raw material milk as described above, the fermentation process as described above, the production process of the cheese curd containing the rennet, the cutting process of the cheese curd The cheese whey can be produced by a method such as a cheese whey discharging process, a salting process, and an aging process. At this time, for example, lactic acid bacteria belonging to the genus Lactobacillus can be used as the starter, and preferably, Lactobacillus delbrueckii subsp. Bulgaricus or Lactobacillus helveticus can be used.

  The present invention provides a method for imparting a liver fibrosis inhibitory effect (suppression function) to a food or drink, including a step of blending the food and drink with α-La as an active ingredient. In addition, the present invention includes a step of incorporating α-La as an active ingredient into a food or drink, and has a therapeutic effect (therapeutic function) and / or a preventive effect (preventive function) for a disease caused by fibrosis of the liver in the food or drink. Provide a way to grant. In the present invention, the method of blending α-La into a food or drink is not particularly limited, and can be blended by methods known to those skilled in the art.

The present invention can also be expressed as (1) to (24) below.
(1) A method for suppressing fibrosis of the liver, comprising a step of administering α-La to a subject.
(2) Use of α-La in the production of an inhibitor of liver fibrosis.
(3) α-La for use in suppressing liver fibrosis.
(4) A method for producing an inhibitor of liver fibrosis, comprising a step of blending α-La and a pharmaceutically acceptable carrier.
(5) An agent containing α-La for addition to a pharmaceutical composition or food and drink and for suppression of liver fibrosis.
(6) A method for treating and / or preventing a disease caused by fibrosis of hepatocytes, comprising a step of administering α-La to a subject.
(7) Use of α-La in the manufacture of a therapeutic and / or prophylactic agent for diseases caused by hepatocyte fibrosis.
(8) α-La for use in treatment and / or prevention of diseases caused by fibrosis of hepatocytes.
(9) A method for producing an agent for treating and / or preventing a disease caused by fibrosis of hepatocytes, comprising a step of blending α-La and a pharmaceutically acceptable carrier.
(10) An agent for addition to a pharmaceutical composition or food or drink, containing α-La, and for treating and / or preventing a disease caused by fibrosis of hepatocytes.
(11) A method for suppressing damage to hepatocytes, comprising a step of administering α-La to a subject.
(12) Use of α-La in the production of an inhibitor of hepatocyte damage.
(13) α-La for use in suppressing damage to hepatocytes.
(14) A method for producing an inhibitor of hepatocyte damage, comprising a step of blending α-La and a pharmaceutically acceptable carrier.
(15) An agent containing α-La for addition to a pharmaceutical composition or food and drink and for inhibiting damage to hepatocytes.

(16) Use of α-La in the manufacture of a composition for suppressing liver fibrosis.
(17) A method for producing a composition for inhibiting fibrosis of the liver, comprising a step of blending α-La and a pharmaceutically acceptable carrier.
(18) A composition containing α-La for addition to a pharmaceutical composition or food and drink, and for suppressing liver fibrosis.
(19) Use of α-La in the manufacture of a composition for treating and / or preventing a disease caused by fibrosis of hepatocytes.
(20) A method for producing a composition for treating and / or preventing a disease caused by fibrosis of hepatocytes, comprising a step of combining α-La and a pharmaceutically acceptable carrier.
(21) A composition for use in addition to a pharmaceutical composition or food and drink, and for treating and / or preventing a disease caused by fibrosis of hepatocytes, comprising α-La.
(22) Use of α-La in the manufacture of a composition for inhibiting damage to hepatocytes.
(23) A method for producing a composition for inhibiting damage to hepatocytes, comprising a step of blending α-La and a pharmaceutically acceptable carrier.
(24) A composition containing α-La for addition to a pharmaceutical composition or food and drink, and for inhibiting damage to hepatocytes.

In addition, the present inventors have clarified that production of nitric oxide is necessary for the suppression of liver fibrosis by α-LA and the treatment and / or prevention of diseases caused by liver fibrosis. That is, α-LA may have an effect of improving or recovering synthesis of nitric oxide whose synthesis is inhibited, or an effect of promoting synthesis of nitric oxide. Therefore, the present invention also provides the following inventions.
(101) An agent for improving or restoring the synthesis of nitric oxide whose synthesis is inhibited, or for promoting the synthesis of nitric oxide, containing α-La as an active ingredient.
(102) The agent according to 101, comprising a unit package form containing an effective amount of α-La.
(103) The agent according to 102, wherein the effective amount of α-La is at least 4.67 g / kg / day.
(104) An effect of improving or recovering the synthesis of nitric oxide inhibited by the food or drink, or an effect of promoting the synthesis of nitric oxide, including a step of blending α-La into the food or drink as an active ingredient Method.
(105) A method for improving or restoring the synthesis of nitric oxide whose synthesis has been inhibited, or for promoting the synthesis of nitric oxide, comprising a step of administering α-La to a subject.
(106) Use of α-La in the manufacture of an agent for improving or restoring the synthesis of nitric oxide whose synthesis has been inhibited, or for promoting the synthesis of nitric oxide.
(107) α-La for use in improving or restoring the synthesis of nitric oxide whose synthesis has been inhibited, or for promoting the synthesis of nitric oxide.
(108) For improving or restoring the synthesis of nitric oxide whose synthesis has been inhibited, or for promoting the synthesis of nitric oxide, comprising the step of combining α-La and a pharmaceutically acceptable carrier Manufacturing method.
(109) Addition of α-La to a pharmaceutical composition or food and drink, and to improve or restore the synthesis of nitric oxide whose synthesis has been inhibited, or to promote the synthesis of nitric oxide Agent for.
(110) Use of α-La in the manufacture of a composition for improving or restoring the synthesis of nitric oxide whose synthesis has been inhibited, or for promoting the synthesis of nitric oxide.
For improving or restoring the synthesis of nitric oxide whose synthesis has been inhibited, or for promoting the synthesis of nitric oxide, comprising the step of combining (111) α-La with a pharmaceutically acceptable carrier A method for producing the composition.
(112) Addition of α-La to a pharmaceutical composition or food and drink, and to improve or restore the synthesis of nitric oxide whose synthesis has been inhibited, or to promote the synthesis of nitric oxide Composition.

  In addition, all the prior literatures cited in this specification are incorporated in this specification as a reference.

1. Example 1
Animal experiment [Experimental method]
Seven-week-old SD female rats (Japan SLC) were used in the experiment. At this time, the rats were divided into three groups (each group: n = 9), that is, a normal group, a DMN-control group, and a DMN-αLA group so that the average body weights of rats were equal. After the arrival of the rats, AIN-93M diet was fed to the normal group and DMN-control group in a day / night 12 hour cycle setting, and DMN-αLA group was fed 50 weights of protein of AIN-93M diet. The feed was replaced with α-lactalbumin (α-La) (α-La of the total amount of feed: 7% by weight). In this animal experiment, the average daily intake of food by rats was 20 g. Therefore, the daily intake of α-La by rats was about 1.4 g. Since the average body weight of the rat used was about 300 g, the daily intake of α-La of the rat was about 4.67 g / kg body weight.

  One week after the arrival of rats, normal groups were orally administered with 1 mL / kg of normal saline, and DMN-control and DMN-αLA groups were treated with dimethylnitrosamine (DMN) in saline. (1% by weight) was orally administered at 1 mL / kg. At this time, in the DMN-control group and DMN-αLA group, a cirrhosis model rat was prepared by orally administering DMN physiological saline continuously at a frequency of 3 days / week for 3 weeks. DMN has the effect of causing methylation of biological macromolecules such as nucleic acids in hepatocytes to damage the cells. It is known that a cirrhosis model animal can be produced by orally administering DMN to a laboratory animal in a short period of time.

  Then, 75 μL of blood was collected from the tail vein of the rat 3 days after the final administration day of each week, and necropsied 12 days after the final administration day of DMN. Furthermore, at the end of the experimental period, whole blood was collected from the abdominal aorta under isoflurane anesthesia, and after blood death, the blood was dissected and the weight of each organ (liver, kidney, spleen) was measured. In addition, the liver tissue was fixed in formalin (10%) and stored for staining of the liver tissue. At this time, blood was centrifuged (3000 rpm, 10 minutes), and serum was collected. For drinking water, UV sterilized water was freely ingested throughout the test period.

Measurement of serum and organ parameters Serum ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase) and bilirubin were measured with Fuji Dry Chem NX500 (Fuji Film). Hyaluronic acid was measured with an ELISA kit (Biotech Trading Partners), and MMP (matrix metalloproteinase) -2 was measured with a fluorescence method kit (Biotech Trading Partners).

Staining of liver tissue Hematoxylin and eosin staining and Masson trichrome staining were performed.

Statistical analysis For the measured values of organ weight, ALT, AST, ALP, bilirubin, hyaluronic acid, and MMP-2, equivariance was confirmed by the Bartlett test using the statistical analysis software “Stat Light” (Yukmus) did. Here, when equidispersity was observed, a Dunnet test was performed. When equidispersibility was not observed, Steel test was performed. For data obtained by grading liver tissue images, a cumulative chi-square test (likelihood ratio test) was performed.

[Experimental result]
Changes in ALT and AST after DMN administration
The transition of AST and ALT 6 days, 13 days and 20 days after administration of DMN is shown in FIG. ALT and AST increased with time in any of the normal group, DMN-control group, and DMN-αLA group. In the DMN-αLA group, ALT and AST were suppressed as compared with the DMN-control group, and this phenomenon was particularly remarkable at 13 days after administration of DMN. Therefore, it became clear that ALT and AST can be suppressed by α-La salary.

  It is known that when cirrhosis progresses, ALP and AST rise due to damage to hepatocytes. Therefore, it was clarified that the progression of cirrhosis caused by liver cell damage can be suppressed by feeding α-La.

Organ Weight at Necropsy Table 1 shows the organ weight at necropsy. In the DMN-control group and DMN-αLA group, body weight, liver weight, and relative liver weight decreased, and spleen weight, relative spleen weight, and relative kidney weight increased compared to the normal group. In the DMN-αLA group, a decrease in relative liver weight was suppressed and an increase in spleen weight and relative spleen weight was suppressed as compared with the DMN-control group.

  In cirrhosis, hepatocytes are damaged and die, and the entire liver tissue shrinks, reducing the body weight and liver weight. In addition, it is known that blood flow in the liver deteriorates and portal pressure increases, resulting in an increase in blood flow to the spleen and enlargement of the spleen. Therefore, it was clarified that cirrhosis model by administration of DMN is valid and that cirrhosis can be suppressed by α-La feeding.

The results of grading the findings of liver cirrhosis tissue using fibrosis of liver tissue as an index are shown in Table 2. Moreover, the organization image of each representative group was shown in FIG. Fibrosis was exacerbated by the action of DMN, but it became clear that exacerbation of fibrosis can be significantly suppressed by feeding α-La.

Serum ALT, AST, ALP, total bilirubin, hyaluronic acid, MMP-2 at necropsy
Serum ALT, AST, ALP, total bilirubin, hyaluronic acid, and MMP-2 concentrations at the time of necropsy were measured, and the results are shown in FIGS. The effects of DMN increased ALT, AST, total bilirubin, hyaluronic acid, and MMP-2, but it became clear that the increase in total bilirubin and MMP-2 could be suppressed by feeding α-La.

  It is known that when cirrhosis progresses, ALP, AST, ALP, and total bilirubin are increased by damaging hepatocytes. In addition, hyaluronic acid and MMP-2 are known to increase as liver fibrosis progresses. Therefore, it was clarified that α-La feeding can suppress the progression of cirrhosis caused by hepatocyte damage and the progression of liver fibrosis.

2. Example 2
In Objective Example 1, it was revealed that α-LA has an inhibitory effect on cirrhosis in dimethylnitrosamine (DMN) cirrhosis model rats. It is known that α-LA has an anti-inflammatory action through cyclooxygenase-2 inhibition, an anti-inflammatory action through production (synthesis) of nitric oxide (NO), and an antioxidant action. In Example 2, focusing on NO production, the action mechanism of the effect of suppressing cirrhosis was examined. Specifically, the NO synthesis inhibitor N-nitro-L-arginine methyl ester (L-NAME) was administered together with α-LA, and the inhibitory effect of α-LA on the suppression of cirrhosis was examined.

Method
Six-week-old SD male rats (Japan SLC) were used in the experiment. RA-2 feed was ad libitum and acclimated in one week. At this time, normal group, DMN-control group, DMN-αLA group, DMN-LA-LN group, DMN-LN group (5 groups, normal group: n = 6) 4 groups other than the normal group: n = 9). After the arrival of the rats, the normal group, DMN-control group, DMN-LN group were fed with AIN-93M diet, and DMN-αLA group, DMN-LA-LN group under the setting of 12 hours day and night cycle Was fed a diet in which 50% by weight of the protein of the AIN-93M feed was replaced with α-La (α-La of the total amount of the feed: 7% by weight).
For 3 days after acclimation (1 week) of rats, normal group was orally administered with physiological saline at 1 mL / kg, and 4 groups other than normal group were treated with saline DMN solution ( 1% by weight) was orally administered at 1 mL / kg (first administration). Then, the rats were orally administered in the same manner for 3 days after 1 week (2 weeks) after acclimatization (1 week) (second administration).
During the test period, the normal group, DMN-control group, and DMN-αLA group were allowed to freely ingest UV sterilized water, and the DMN-LA-LN group and DMN-LN group contained L-NAME aqueous solution ( 0.2 mg / mL) was ad libitum.
75 μL of blood was collected from the tail vein of the rat 3 days after the first administration day and 3 days after the second administration day (10 days after the first administration day). And AST were measured.

Results FIG. 9 shows the ALT concentration, and FIG. 10 shows the AST concentration.
In the white DMN-control group, ALT and AST increased (increased) compared to the gray normal group, and it was confirmed that liver function deteriorated when DMN was administered.
In contrast, in the black DMN-αLA group compared to the white DMN-control group, ALT and AST decreased (decreased), so even if DMN was administered, α-La was administered, It was confirmed that liver function was improved.
On the other hand, in the DMN-LN group indicated by thick shading compared to the gray normal group, ALT and AST increased (increased), so when L-NAME was administered together with DMN, liver function deteriorated I was able to confirm that.
On the other hand, in the DMN-LA-LN group shown by thin shading, the ALT and AST did not decrease (decrease) compared to the DMN-LN group shown by thick shading. It was confirmed that liver function was not improved even when α-La was administered.

  From the above, administration of α-LA improved the deterioration of liver function caused by DMN, but administration of L-NAME, known as a NO synthesis inhibitor, caused DMN even after administration of α-La. The resulting deterioration of liver function was not improved. In other words, it was suggested that NO synthesis may be involved in the improvement of liver function of α-LA.

  By taking the agent containing α-La provided based on the present invention, liver fibrosis and hepatocyte damage can be suppressed. In addition, by ingesting an agent containing α-La provided based on the present invention, a disease caused by liver fibrosis can be treated and / or prevented. The agent containing α-La provided in accordance with the present invention is composed of components that have been proven to be excellent in safety and taste from a long dietary experience, with a low physical burden, and a long-term You can ingest it over time.

Claims (14)

  An inhibitor of liver fibrosis, containing α-lactalbumin as an active ingredient.   The agent according to claim 1, wherein the fibrosis of the liver is due to damage of hepatocytes.   The agent according to claim 2, wherein the hepatocyte damage is caused by methylation of nucleic acid in the hepatocyte.   The agent according to any one of claims 1 to 3, comprising a unit packaging form containing an effective amount of α-lactalbumin.   The agent according to claim 4, wherein the effective amount of α-lactalbumin is at least 4.67 g / kg / day.   A therapeutic and / or prophylactic agent for diseases caused by liver fibrosis, comprising α-lactalbumin as an active ingredient.   Diseases caused by liver fibrosis include cirrhosis, hepatocellular carcinoma, chronic hepatitis B, chronic hepatitis C, alcoholic liver injury, nonalcoholic fatty liver disease, drug-induced liver injury, autoimmune hepatitis, bile The agent according to claim 6, which is selected from the group consisting of stasis, biliary atresia, porphyria, and Wilson disease.   The agent according to claim 6 or 7, comprising a unit packaging form containing an effective amount of α-lactalbumin.   The agent according to claim 8, wherein the effective amount of α-lactalbumin is at least 4.67 g / kg / day.   A method for imparting an effect of suppressing liver fibrosis to a food or drink, comprising a step of blending α-lactalbumin into the food or drink as an active ingredient.   11. The method of claim 10, wherein the liver fibrosis is due to hepatocyte damage.   The method according to claim 11, wherein the hepatocyte damage is due to nucleic acid methylation in the hepatocyte.   A method for imparting a therapeutic and / or prophylactic effect for a disease caused by fibrosis of a liver to a food or drink, comprising a step of blending α-lactalbumin as an active ingredient in the food or drink.   Diseases caused by liver fibrosis include cirrhosis, hepatocellular carcinoma, chronic hepatitis B, chronic hepatitis C, alcoholic liver injury, nonalcoholic fatty liver disease, drug-induced liver injury, autoimmune hepatitis, bile 14. The method of claim 13, wherein the method is selected from the group consisting of stasis, biliary atresia, porphyria, and Wilson disease.