JP2013133313A - Therapeutic agent of non-alcoholic fatty liver disease (nafld) and/or non-alcoholic steatohepatitis (nash) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a therapeutic agent of NAFLD and/or NASH containing a fermented essence obtained by symbiotic fermentation with lactic acid bacteria and yeast, or melanoidin contained in the fermented essence as an active ingredient.SOLUTION: The therapeutic agent for the NAFLD and/or the NASH includes melanoidin as an active ingredient which is contained in the fermented essence of polysaccharides, vegetables and fruits, and seaweeds with the lactic acid bacteria and the yeast. The melanoidin contained in the fermented essence, obtained by fermenting brown rice as the polysaccharides, papaya as the vegetables and fruits, and sea tangle and Nemacystus decipiens as the seaweeds with the lactic acid bacteria and the yeast for six months, and removing the microbial cells, exhibits ameliorating effects on the NAFLD and/or the NASH.

Description

  The present invention relates to a therapeutic agent for nonalcoholic fatty liver disease and / or nonalcoholic steatohepatitis.

  Lifestyle-related diseases have increased due to changes in diet and lifestyle, and fatty liver has been observed in 20% of adults. Major causes of fatty liver are heavy drinking and overnutrition. Non-alcoholic fatty liver disease (hereinafter referred to as NAFLD), which presents symptoms of liver disease without drinking, has been considered benign and does not progress, but hepatitis to cirrhosis eventually The number of cases that shift to liver cancer has increased, making it a disease that cannot be overlooked. In addition, a disease that leads to cirrhosis through progression of NAFLD and fat deposition in the liver parenchyma, infiltration of inflammatory cells, liver parenchyma, and fibrosis is referred to as non-alcoholic steatohepatitis (NASH). ). It is a pathological condition in which hepatocyte degeneration and liver fibrosis findings such as inflammatory cell infiltration, Mallory bodies, hepatocyte balloon-like swelling are added to the liver with lipid droplets deposited. Although the number of people with severe obesity is increasing rapidly in the United States, it is expected that the number of NASH patients will increase in Japan as the lifestyle becomes westernized.

  NASH often has insulin resistance in the background, and periosome proliferator-activated receptor γ (hereinafter referred to as PPARγ) agonist, which is an insulin sensitizer, is an agent that improves insulin resistance. Has been reported to improve the pathological condition of the disease (Non-patent Document 1). There is also a report that the pathology of steatohepatitis has been improved by administration of an activator of AMP kinase involved in insulin resistance improvement (Non-patent Document 2). There is also a report that signal loss of adipocytokine involved in obesity / insulin resistance induces susceptibility to onset of steatohepatitis through suppression of induction of stress protein metallothionein (Non-patent Document 3). Furthermore, there is a report that TNFα, which is a kind of adipocytokine, promotes liver fibrosis in NASH pathology through enhanced expression of TIMP-1 (Non-patent Document 4). It means that the control of insulin resistance and the adipocytokine involved in it are deeply involved in the pathology of NASH that is based on the metabolic syndrome.

  Since NASH is a progressive disease, establishing a cure for it is urgent. As a liver disease preventive or therapeutic agent containing NASH, there is a liver disease preventive or therapeutic agent containing tranilast (N- (3,4-dimethoxycinnamoyl) anthranilic acid) or a pharmacologically acceptable salt thereof as an active ingredient. (Patent Document 1). Tranilast is an anti-allergic drug, but when tranilast was administered to obese diabetic rats fed a methionine / choline-deficient diet, its effect was examined and found to be effective in suppressing liver fibrosis, hepatic steatosis, and improving liver inflammation. It was said that there was.

  Oral intake effective for the prevention and / or treatment of non-alcoholic fatty liver disease, comprising as an active ingredient an extract extracted from at least one selected from the group consisting of Eucalyptus plants, Crape myrtle plants and Vangirou plants There is also a composition for use (Patent Document 2). As an active ingredient is an extract extracted from a natural product with a high food safety and high safety, it is effective for NAFLD, especially NASH resulting from a high-calorie diet, high in safety, and does not contain caffeine. There is an effect that it is easy to do.

  There is also a composition for the prevention and treatment of non-alcoholic steatohepatitis characterized by containing curcumin or tetrahydrocurcumin or a curcumin-containing product or tetrahydrocurcumin-containing product as an active ingredient (Patent Document 3). Curcumin is known to have antioxidant, bile secretion-promoting (biliary), visceral (liver, pancreas) function, carcinogenesis-inhibiting, and lipid metabolism-improving effects. The treated tetrahydrocurcumin is said to have a NASH healing effect, and in the Examples, it is administered to NASH patients and the decrease in GPT value is observed.

  In recent years, vigorous clinical research and basic research have been conducted on NAFLD and NASH, and the pathophysiology has been elucidated and therapies have been sought. However, effective treatments have not yet been developed, and further development is hoped for. It is rare.

JP 2009-120607 A JP 2011-231078 JP 2007-320864 A

Pioglitzone Prevents Alcohol-Induced Fatty Liver in Rats Through Up-regulation of c-Met, K Tomita et al. , Gastroenterology, vol. 126, p873-855, 2004 AICAR, an AMPK Activator, Has Protective Effects on Alcohol-Induced Fatty River in Rats, K Tomita et al. Alcohol Clin Exp Res. , Vol. 29, no. 12, pp240s-245s, 2005 Leptin efficacy enhances of rats to alcoholic steatohepatitis through suppression of metallothenine, K Tomita et al. , Am J Physiol Gastrointest Liver Physiol, vol. 287 ppG1078-G1085, 2004 Tumor necrosis factor alpha signaling through activation of Kupffer cells play an essential role in liver fibrosis of non-alcoholic diet. Gut, vol. 55, pp 415-424, 2006

  Liver diseases such as NAFLD and NASH are likely to become chronic, and therefore, it is desired to develop a therapeutic agent that is excellent in safety with few side effects as well as curative effects.

  On the other hand, microorganisms that are harmless and beneficial to humans, animals, crops and the natural environment are called useful microorganisms, and are photosynthetic bacteria, lactic acid bacteria, yeast, actinomycetes, filamentous fungi, basidiomycetes, Bacillus subtilis Etc. are known. Photosynthetic bacteria produce antioxidants, amino acids, saccharides, and various physiologically active substances using light as an energy source, and lactic acid and acetic acid produced by lactic acid bacteria can suppress the growth of harmful bacteria because of their strong antibacterial activity. In addition, yeast can enhance the activity of other useful bacteria in addition to changing sugar to alcohol. Furthermore, actinomycetes produce antibacterial substances, and filamentous fungi can degrade and saccharify persistent carbohydrates such as fiber. Fermentation using such useful microorganisms is an essential process for the production of miso, soy sauce, and other fermented foods.

  As fermented foods, in addition to miso and soy sauce, many fermented foods such as sake, beer, shochu and other alcoholic beverages, dairy products such as yogurt and cheese, fermented soybeans such as natto, sushi, and pickles have been used since ancient times. Has been. Fermented foods contain ingredients derived from raw materials and components derived from bacterial cells, and have advantages such as improved digestibility and flavor and improved storage as the raw materials are decomposed.

  Moreover, it is known that the fermented extract produced by decomposing the raw material by a microorganism contains a compound collectively called brown melanoidin, which is a Maillard reaction product of a reducing sugar and an amino compound. Although the production route has not been fully elucidated, the D-glucoside produced from the saccharide becomes an Amadori compound via enaminol, and is produced by condensation polymerization reaction with an amino compound using oson or furfural as an intermediate. It is considered. Melanoidin is an aggregate of various substances because different products can be produced depending on the type of reducing sugar or amino compound. The brown color of soy sauce and miso is thought to be due to this melanoidin.

  NASH is a disease in which NAFLD has become serious, and improvement in NAFLD means prevention of NASH. Most of NAFLD originates from lifestyle-related diseases, and it is preferable that NASH can be prevented and treated with components derived from fermented foods that are excellent in safety when long-term administration is considered.

  In view of the above-mentioned present situation, an object of the present invention is to provide a therapeutic agent for NAFLD or NASH containing melanoidin produced by fermentation using food as a raw material as an active ingredient.

  Another object of the present invention is to provide a therapeutic agent for NAFLD or NASH containing a natural product-derived fermented extract as an active ingredient.

  The inventors of the present invention describe in detail a fermented extract containing melanoidin produced by co-fermenting lactic acid bacteria and yeast with melanoidin and polysaccharides produced by symbiont fermentation of lactic acid bacteria and yeast, vegetables and fruits, and seaweed. As a result of the study, it was found that administration to obese diabetic rats fed a methionine / choline-deficient diet has an effect of improving liver tissue, and the present invention has been completed. It is not known that brown melanoidin is produced without heating by symbiont fermentation of lactic acid bacteria and yeast, and it is not known at all that such melanoidin has an excellent NASH healing effect.

  That is, the present invention provides a therapeutic agent for NAFLD and / or NASH containing melanoidin as an active ingredient.

  The present invention also provides the above therapeutic agent, wherein the melanoidin is an extract from a fermented extract obtained by co-fermenting lactic acid bacteria and yeast.

  The present invention also provides the above therapeutic agent, wherein the melanoidin is an extract from a fermented extract obtained by co-fermenting polysaccharides, vegetables / fruits, and seaweed. .

  Moreover, this invention provides the said therapeutic agent whose said vegetables and fruits are papayas.

  The present invention also provides the above therapeutic agent, wherein the polysaccharide is rice, rice bran and / or brown rice.

  The present invention also provides a therapeutic agent for NAFLD and / or NASH, which is obtained by co-fermenting polysaccharides, vegetables / fruits, seaweed, lactic acid bacteria and yeast, and containing as an active ingredient a fermented extract containing melanoidin. It is to provide.

  The present invention also provides the above therapeutic agent, wherein the polysaccharide is rice, rice bran and / or brown rice, and the vegetables and fruits are papayas.

  ADVANTAGE OF THE INVENTION According to this invention, the therapeutic agent of NAFLD and / or NASH which uses the melanoidin extracted from the fermented extract derived from a natural product or fermented extract as an active ingredient is provided.

It is a figure which shows the chromatogram of the gel filtration HPLC of the methanol elution fraction of the styrene-type synthetic adsorbent adsorbate of the fermentation extract manufactured in Manufacturing Example 2. 6 is a diagram showing a UV spectrum of a brown substance obtained in Production Example 2. FIG. 2 is a diagram showing a UV spectrum of melanoidin obtained in Production Example 1. FIG. (A) is a figure which shows the result of serum ALT of Example 1, (B) is a figure which shows the result of serum AST. It is a figure which shows the result of the liver PAI-1 of Example 1. It is a figure which shows the result of the liver TNF (alpha) of Example 1. It is a figure which shows the result of the liver TIMP-1 of Example 1. (A) is a figure which shows the result of serum ALT of Example 2, and (B) is a figure which shows the result of liver TG (Liver Triglyceride). It is a figure which shows the result of HE dyeing | staining of Example 2. FIG. (A) is a figure which shows the result of the liver TBARS of Example 2, (B) is a figure which shows the result of a liver SOD activity.

  The first of the present invention is a therapeutic agent for NAFLD and / or NASH containing melanoidin as an active ingredient. Since melanoidin is a general term for the reaction product of a saccharide and an amino compound, the melanoidin varies depending on the type of saccharide or amino compound. In addition, since fermentation is a process of processing organic compounds by the action of useful microorganisms, various melanoidins are produced if different microorganisms are used. According to the present invention, at least melanoidin produced by syrup fermentation of lactic acid bacteria and yeast has NAFLD and / or NASH healing effects.

  The melanoidin used in the present invention is an extract from a fermented extract obtained by co-fermenting lactic acid bacteria and yeast. Melanoidin can be produced by using at least lactic acid bacteria and yeast. Lactic acid bacteria produce lactic acid from carbohydrates, but the protein contained in the culture broth is broken down into peptides and amino acids by the lactic acid produced in the fermentation broth. In addition, lactic acid bacteria having amylase activity can decompose amylase contained in rice and brown rice into monosaccharides. Yeast produces alcohol and carbon dioxide from sugars under anaerobic conditions, but yeast containing proteolytic enzymes can break down proteins in the culture medium into peptides and amino acids, and the sugar that yeast ingests in the body. Can produce vitamins and amino acids. In the present invention, at least lactic acid and yeast may be used. Further, koji molds, Bacillus subtilis, filamentous fungi, photosynthetic bacteria, etc. are contained in the fermentation system as long as the effects of the present invention are not impaired. Also good. It is difficult to remove all bacteria attached to the raw material.

  In the present invention, “Kyoto fermentation” refers to fermentation performed by co-culturing lactic acid bacteria and yeast, and “fermentation extract” is obtained by removing microorganisms and raw material residues from the fermentation solution of Koji fermentation. .

  Nutrients for culturing lactic acid bacteria and yeast are polysaccharides, vegetables and fruits, and seaweed. As the polysaccharide, polysaccharides derived from natural raw materials such as rice, rice bran, brown rice, sake lees, distilled lees, wheat straw, beer lees, rice flour, wheat flour, starch and potatoes are preferred. More preferred are rice, rice bran, brown rice, sake lees, and rice flour, and particularly preferred are rice, rice bran and / or brown rice. Since brown rice and rice bran contain germs containing amylase, starch amylose and amylopectin can be converted into glucose, maltose, and oligosaccharide, and smooth fermentation using lactic acid bacteria and yeast using polysaccharides as raw materials Can do.

  The kind of vegetables and fruits is not particularly limited, but those containing a lot of minerals and enzymes can be preferably used. Those containing saccharides and proteins are preferred. For example, papaya, pineapple, apple, orange, banana, radish, carrot, cabbage, mushroom and the like can be suitably used. Papaya is preferable. Papaya contains a large amount of proteolytic enzymes such as papain, can break down proteins in the culture medium into peptides and amino acids, and participates in active melanoidin formation. The papaya is not limited to ripe as long as it is edible, and may be an immature green papaya. The amount of enzyme of green papaya is particularly preferable because it has a higher content than mature papaya.

  There are no particular limitations on the seaweed, and brown algae such as kelp, hijiki, mozuku, and young cloth, red algae such as cassava and primrose, green algae such as aosa, aonori, casanori, cactus, cypress, and mill are preferably used. More preferably, it is brown algae. In particular, kelp, hijiki and mozuku are preferred. These seaweeds contain amino acids in addition to minerals and polysaccharides.

  The lactic acid bacteria and yeast used in the present invention only need to be able to finally produce melanoidin in the fermented extract by mash fermentation. Examples of lactic acid bacteria include Lactobacillus genus, Bifidobacterium genus, Enterococcus genus, Lactococcus genus, Pediococcus genus, Leuconostoc genus, Streptococcus genus, and the like, which can be appropriately selected according to the raw materials used. In the present invention, since polysaccharides derived from natural raw materials such as rice, rice bran, brown rice, sake lees, distilled lees, wheat straw, beer lees, rice flour, wheat flour, starch are used, it is possible to use lactic acid bacteria that can assimilate sugars. preferable. Examples of lactic acid bacteria that can assimilate saccharides include lactic acid bacteria such as Lactobacillus, Streptococcus, and Leuconostoc.

Lactobacillus casei (Lactobacillus casei), Lactobacillus plantarum, Lactobacillus lactis, Lactobacillus amylophilus (Lactobacillus amylophilus), Lactobacillus cilophilus (Lactobacillus cilophilus) Lactobacillus sanfrancisco, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus paracasei, etc.
Examples of Streptococcus lactic acid bacteria include Streptococcus faecalis and Streptococcus faecium.
Examples of lactic acid bacteria belonging to the genus Leuconostoc include Leuconostoc mesenteroides and Leuconostoc lactis.

  Examples of yeast include Saccharomyces genus, Candida genus, Tolropsis genus, Digosaccharomyces genus, Schizosaccharomyces genus, Debariomyces genus, Geotrichum genus, Wickelhamia genus and Ferromyces genus. In the present invention, Saccharomyces genus that is a budding yeast, Schizosaccharomyces genus and Candida genus that are fission yeasts can be preferably used. Saccharomyces cerevisiae is preferable.

To Force fermentation using the lactic acid bacteria with yeast, charged with all the ingredients described above in Force culture of lactic acid bacteria and the yeasts at a time is also rather good, for uniformly adjusting the fermentation stage of each raw material, respectively The raw materials may be separately charged into a culture solution of lactic acid bacteria or yeast and pre-fermented, and then mixed and fermented together. It can be suitably selected according to the types of lactic acid bacteria and yeast, and the types of polysaccharides, vegetables / fruits, seaweeds and the like as nutrients for these.

The lactic acid bacteria and yeast used in the present invention are pre-cultured. The concentration of the lactic acid bacteria in the preculture solution is 10 ⁵ to 10 ¹⁰ cells / mL, and the concentration of the yeast in the preculture solution is 10 ⁵ to 10 ¹⁰ cells / mL. In addition, the preparation amount of a raw material (wet weight) is 0.01-100 w / v% of a preculture liquid, More preferably, it is 0.1-30 w / v%. The mixing ratio of vegetables and fruits to 100 parts by mass of polysaccharide is 1 to 100 parts by mass, more preferably 3 to 50 parts by mass. Moreover, the mixture ratio of the seaweed with respect to 100 mass parts of polysaccharides is 0.1-50 mass parts, More preferably, it is 1-30 mass parts.

A method for preparing a fermented extract using brown rice and rice bran as polysaccharides, papaya as vegetables and fruits, and kelp and mozuku as seaweed will be described. First, steamed brown rice is added to a syrup culture solution of lactic acid bacteria and yeast. As lactic acid bacteria to be used, lactic acid bacteria capable of assimilating polysaccharides are used. Thereby, the polysaccharide contained in brown rice can be decomposed into sucrose, glucose and the like. Lactic acid bacteria decompose polysaccharides and produce lactic acid. Since yeast has excellent acid resistance, it can grow in lactic acid acidic fermentation broth and produce amino acids and the like.
Separately from the above brown rice, papaya and seaweed are separately charged in a syrup culture medium of lactic acid bacteria and yeast, and then prefermented. Since papaya contains many proteolytic enzymes and the like, the components contained in the fermentation broth are further decomposed by these enzymes. When the degree of decomposition in each fermented liquid becomes substantially uniform, these are mixed and fermented together.

  Fermentation can be appropriately selected according to the type of microorganism used, the type of raw material, the amount of raw material charged, etc., and is generally 5 to 100 weeks, more preferably 10 to 50 weeks, and particularly preferably 20 to 40 weeks. is there. The above is the time from the start to the end of fermentation. Moreover, fermentation temperature is 20-40 degreeC. As the fermentation progresses, a culture solution of lactic acid bacteria or a culture solution of yeast may be added.

  Yeast and lactic acid bacteria can both grow under anaerobic conditions and aerobic conditions, and the products differ depending on the presence or absence of oxygen. For example, yeast produces alcohol using sugar as a raw material under anaerobic conditions, while lactic acid bacteria produce lactic acid under aerobic conditions. In the present invention, it is preferable to increase the activity of lactic acid bacteria in the first half of the fermentation, and to control the activity of other microorganisms in the second half of the fermentation. This is because by controlling the first half of the fermentation to an aerobic condition, it is possible to suppress the generation of alcohol by yeast, while it is possible to sterilize miscellaneous bacteria mixed by lactic acid produced by lactic acid bacteria. Such control can be performed by controlling pH, the amount of dissolved oxygen, and temperature. Thereby, long-lasting fermentation can be performed with lactic acid bacteria and yeast.

  The end of the fermentation can be known by the brown color of the fermentation broth. In addition, you may age | cure | ripen a fermented liquor on the conditions which do not supply oxygen after completion | finish of fermentation.

  In the above method, polysaccharides, vegetables / fruits, and seaweed are separately pre-fermented with a lactic acid bacteria / yeast syrup medium, but the total amount of polysaccharides, vegetables / fruits, and seaweeds is It may be prepared in a pre-fermented solution with yeast and co-cultured, and the polysaccharide is pre-fermented with a lactic acid bacterium and yeast co-cultured solution, and vegetables, fruits and seaweed are pre-fermented with a lactic acid bacterium culture solution. Fermentation liquid may be mixed and fermented together. Melanoidin is considered to be produced by microorganisms that coexist after the raw material is decomposed into reducing sugars and amino compounds by microorganisms. Therefore, it is not necessary that the entire fermentation process is symbiotic fermentation. In addition, in a fermentation process, preparation water may be added and a bacterial solution may be added as needed.

  After fermentation or subsequent aging, lactic acid bacteria, yeast and other microorganisms are removed by filtration, centrifugation, etc. to obtain a fermented extract. The pH is generally 4 to 8.5. The alcohol concentration is preferably 0 to 500 mg / L.

  The fermentation extract can be treated with an ion exchange resin, a synthetic adsorption resin, or the like to separate and recover the fermentation product melanoidin. An organic acid or the like contained in the fermented extract can be removed with an ion exchange resin, a synthetic adsorption resin, or the like to obtain brown melanoidin. As the ion exchange resin to be used, sulfonic acid groups, phosphoric acid groups, carboxymethyl groups, diethylamino groups, quaternary aminoethyl groups, and the like can be used as ion exchange groups, and both cation exchange resins and anion exchange resins can be used. Both can be used. Melanoidin can also be obtained by using a synthetic adsorption resin (Diaion, Sepabeads, etc., manufactured by Mitsubishi Chemical Corporation).

  The melanoidin used in the present invention has a molecular weight of 500 to 3000. This melanoidin was found to have NAFLD and / or NASH healing effects, as shown in the examples described below.

  The administration route of the therapeutic agent containing the melanoidin of the present invention as an active ingredient includes oral administration or parenteral administration such as intragastric administration, subcutaneous, intramuscular and intravenous. Desirable is oral administration. The dose varies depending on the administration route, and in the case of oral administration, it is 1 to 100 mg / day, and can be taken divided into a plurality of times per day.

  The therapeutic agent containing the melanoidin of the present invention as an active ingredient may be prepared in any form, and examples thereof include capsules, tablets, granules, syrups, fine granules, sprays, emulsions and injections.

  The therapeutic agent containing the melanoidin of the present invention as an active ingredient can be used not only for treatment of NAFLD and NASH but also as a prophylactic agent for these.

  A second aspect of the present invention is a treatment for NAFLD and / or NASH, which is obtained by co-fermenting polysaccharides, vegetables / fruits, seaweed, lactic acid bacteria and yeast, and containing as an active ingredient a fermented extract containing melanoidin. It is a medicine. This fermented extract can be prepared by the method described in the first invention. As described above, melanoidin is produced by co-fermenting polysaccharides, vegetables and fruits, seaweed, and lactic acid bacteria and yeast. Such a fermented extract contains various raw material degradation products and microbial production substances in addition to melanoidin, and it has been found that NAFLD and / or NASH can be cured without isolating melanoidin.

  The fermented extract used in the present invention uses rice, rice bran and / or brown rice as polysaccharides, but brown rice and rice bran contain amylase which decomposes starch, so that yeast can be cultured using starch as a raw material. . Further, by using papaya containing a large amount of proteolytic enzymes such as papain as vegetables and fruits, proteins in the culture solution can be decomposed into peptides and amino acids. The papaya is not limited to ripe as long as it is edible, and may be an immature green papaya. The amount of enzyme of green papaya is particularly preferable because it has a higher content than mature papaya.

  As the lactic acid bacteria and yeast to be used, those described in the first invention can be suitably used, and a fermentation extract can be produced by the fermentation method described in the first invention. The fermented extract used in the present invention preferably contains melanoidin used in the first invention and does not contain alcohol. This is to avoid the induction of alcoholic fatty liver disease by ingesting alcohol.

  The administration route of the therapeutic agent comprising the fermented extract of the present invention as an active ingredient includes oral administration or parenteral administration such as intragastric administration, subcutaneous, intramuscular and intravenous. Desirable is oral administration. The dose varies depending on the administration route, and in the case of oral administration, it is 5 to 500 ml / day, and can be taken divided into a plurality of times per day.

  The therapeutic agent containing the fermented extract of the present invention as an active ingredient may be prepared in any form. Furthermore, a taste-masking / flavoring agent, a stabilizer and a preservative may be added.

  The therapeutic agent comprising the fermented extract of the present invention as an active ingredient can be used not only for treatment of NAFLD and NASH but also as a prophylactic agent thereof.

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

(Production Example 1)
A solution containing 2 mol of glucose, 2 mol of glycine and 0.2 mol of sodium hydrogen carbonate was adjusted to pH 6.8. Melanoidin was synthesized by refluxing at 95 ° C. for 7 hours. After cooling the reaction solution, dialysis was performed for 1 week using a dialysis membrane (trade name “UC36-32-100”, manufactured by Viscose). The internal solution was lyophilized and pulverized in a mortar to form a powder. About the aqueous solution of this powdery substance, UV spectrum was measured using JASCO V-570. The results are shown in FIG. In the UV spectrum, no absorption peak was observed at a specific location, and the absorption gradually decreased from a wavelength of 300 nm to 700 nm.

(Production Example 2)
Lactobacillus casei, Lactobacillus plantarum, Lactobacillus lactis were used as lactic acid bacteria, and Saccharomyces cerevisia was used as yeast. A solution obtained by prefermenting this with a culture solution made of rice bran was used as a seed-fermented fermentation solution. Steamed brown rice was added to an inoculum fermentation solution containing lactic acid bacteria and yeast, and primary fermentation was performed under conditions that promoted production of lactic acid bacteria. Similarly, papaya and seaweed (kombu and mozuku) were separately added to the inoculum fermentation solution containing lactic acid bacteria and yeast, and primary fermentation was performed in the same manner as brown rice. The raw materials used were 25 parts by mass of papaya and 8 parts by mass of seaweed with respect to 100 parts by mass of steamed brown rice. Then, each symbiotic fermentation broth was mixed. Fermentation was continued under conditions where the amount of dissolved oxygen was adjusted to promote the activity of bacteria other than lactic acid bacteria. In addition, in the fermentation process, pure water or an inoculum fermentation liquid was added as needed. The end point of the fermentation was determined as the fermentation broth became clear and brown as the raw material progressed. It took 6 months from the start of primary fermentation to the end of fermentation. Subsequently, it was aged for 6 months from the end of fermentation. Bacteria were removed from the fermented liquid after aging using a filter having a diameter of 0.45 μm to obtain a fermented extract. The pH of this fermented extract was 6.0, and no alcohol was detected.

  The fermented extract was passed through a styrene-based synthetic adsorbent (manufactured by Mitsubishi Chemical Corporation, trade name “Diaion HP-20”) to adsorb the fermented product contained in the fermented extract.Then, the column was washed with water, Elution with methanol was carried out to fractionate a brown methanol elution.

  This brown methanol elution fraction was subjected to gel filtration HPLC analysis under the following conditions. A gel filtration chromatogram is shown in FIG.

HPLC analysis conditions Pump: manufactured by SHIMADZU, LC-10ADvp
Degasser: manufactured by SHIMADZU, DGU-14A
Auto injector: manufactured by SHIMADZU, SIL-10ADvp
System controller: SHIMADZU, SCL-10Avp
Diode array detector: manufactured by SHIMADZU, SPD-M10Avp
Evaporative light scattering detector: manufactured by SHIMADZU, ELSD-LT
Column: Shodex OH pak SB-803 HQ
Flow rate: 0.8mL / min
Mobile phase: 100 mM phosphate buffer

  When the molecular weight distribution of the brown fermentation product was measured under the same conditions as the above HPLC measurement conditions using pullulan as a molecular weight marker, a calibration curve was created thereby, and a size exclusion chromatography method for obtaining a relative molecular weight distribution was examined. ~ 3000.

  Subsequently, the UV spectrum of the brown substance was measured. The measurement was performed using JASCO V-570. The results are shown in FIG. In the UV spectrum, no absorption peak was observed at a specific location, and the absorption gradually decreased from a wavelength of 200 nm to 700 nm. This spectrum was similar in waveform to the melanoidin of Production Example 1.

  For brown fermentation products, protein, polysaccharides and monosaccharides are colorimetric using a spectrophotometer, organic acids are ion chromatographed, amino acids are amino acid analyzers, and lipids are gas chromatographed. Analysis. Proteins, polysaccharides, monosaccharides, organic acids, amino acids, and lipids were not detected in this fraction. Therefore, the main component of the methanol elution fraction was melanoidin.

Example 1
Eight-week-old C57BL6 / N male mice were divided into four groups, and methionine / choline-deficient diet, methionine / choline-deficient diet and melanoidin from Production Example 2, normal diet, normal diet, and melanoidin from Production Example 2 over 4 weeks. , Ingested.
Serum AST and serum ALT were measured after administration. The results are shown in FIG. In addition, livers were collected, RNA and proteins were extracted, and changes in various parameters such as adipocytokines and cytokines were quantitatively evaluated using real-time PCR, Western blotting, and immunoprecipitation. The results of liver PAI-1, liver TNFα, and liver TIMP-1 are shown in FIGS. 5, 6, and 7, respectively.

(result)
As shown in FIG. 4, in a group of mice that were fed a methionine / choline-deficient diet and induced a NASH-like condition, serum ALT increased to 220 IU / L and serum AST increased to 170 IU / L. However, in the group in which the melanoidin of Production Example 2 was administered to the mice in which NASH-like disease was induced, the serum ALT value was suppressed to 63% of 140 IU / L and 220 IU / L, the serum AST value was 105 IU / L, and 170 IU / L. It was suppressed to 62% of L. In the normal diet, melanoidin is considered to be excellent in safety because serum ALT and AST values were not changed by administration of melanoidin.

  As shown in FIG. 5, the above tendency was also observed in liver PAI-1. That is, the relative proportion of PAI-1 was high in the group of mice in which a NASH-like condition was induced, and these values were reduced by ingestion of melanoidin. In the group of mice fed with the normal diet, the relative ratio was low regardless of the presence or absence of melanoidin, and it was found that melanoidin can reduce PAI-1 only when a NASH-like pathological condition is induced.

  As shown in FIG. 6, hepatic TNFα increased in the group of mice in which the NASH-like pathological condition was induced, but decreased by administration of melanoidin. In addition, the amount of TNFα was lower in the mice administered with the normal diet and further administered with melanoidin than in the non-administered group. It was found that TNFα, which is an inflammatory cytokine, is decreased even in healthy mice.

  As shown in FIG. 7, the relative proportion of TIMP-1 was high in the group of mice in which the NASH-like pathology was induced, and these values were reduced by ingestion of melanoidin. In the group of mice fed with the normal diet, the relative ratio was low regardless of the presence or absence of melanoidin, and it was found that melanoidin can reduce TIMP-1 only when a NASH-like condition is induced.

  It is suggested that the administration of melanoidin suppresses the production of proinflammatory cytokines such as PAI-1 and TNFα in the liver, thereby exerting a liver protective action, and the administration of melanoidin also promotes the production of liver TIMP-1. Expression was suppressed, suggesting that this suppression suppresses liver fibrosis.

(Example 2)
A 4-week-old C57BL / 6 female mouse was administered with a methionine / choline-deficient diet for 4 weeks to create a mouse NASH model.
This mouse NASH model was divided into 4 groups, a normal diet and physiological saline (5 ml / kg body weight / day), a normal diet and the fermented extract of Production Example 2 (5 ml / kg body weight / day), a methionine / choline-deficient diet and physiological Saline (5 ml / kg body weight / day), a methionine / choline-deficient diet, and the fermented extract of Production Example 2 (5 ml / kg body weight / day) were each administered by gavage over 4 weeks.
After administration, mouse liver and serum were collected. A part of the liver tissue was fixed with 4% paraformaldehyde, and a paraffin section was prepared. Hematoxylin and eosin staining was performed, and liver tissue images were evaluated. Serum and tissue samples were stored at −80 ° C., and serum AST, liver TG, liver tissue immunohistochemical staining, measurement of lipid peroxide level in liver tissue, and measurement of superoxide dismutase activity in liver tissue were performed.
FIG. 8 shows the results of serum ALT and liver TG, FIG. 9 shows the results of HE staining, and FIG. 10 shows the results of liver TBARS and liver SOD. In FIG. 9, the results of the normal diet and physiological saline are Cont + C, the results of the normal diet and the fermented extract of Production Example 2 are Cont + M, and the results of methionine / choline-deficient diet and physiological saline are MCD + C and methionine / choline-deficient diet. The results of the fermented extract of Production Example 2 were expressed as MCD + M.

(result)
As shown in FIG. 8, the serum ALT was 20 IU / L and the liver TG was 10-13 mg / g regardless of whether or not the fermented extract was administered. In contrast, in the group of mice that were fed a methionine / choline-deficient diet and induced a NASH-like condition, serum ALT increased to 220 IU / L and liver TG increased to 122 mg / g. However, in the group in which the fermented extract of Production Example 2 was administered to mice in which a NASH-like disease state was induced, the serum ALT value was suppressed to 63% of 140 IU / L and 220 IU / L, and the liver TG was 70 mg / g, 122 mg / g. It was suppressed to 57% of g. Serum ALT levels and liver TG are increased by induction of NASH-like pathological conditions, and it has been found that these can be reduced by administration of a fermented extract. In the normal diet, serum ALT and AST values were not changed by administration of the fermented extract, and it was considered that there were few side effects on healthy mice.

  From the results of HE staining shown in FIG. 9, homogeneous liver tissue was observed in the group of mice ingested the normal diet, but adipocytes were scattered in the group of mice in which the NASH-like pathology was induced. However, when fermented extract was administered, the number of adipocytes and the size of adipocytes were reduced even in the group of mice in which a NASH-like condition was induced. In the group of mice that ingested the normal diet, there was little change in hepatocytes by administration of the fermented extract.

  As shown in FIG. 10, the liver TBARS in the mice fed the normal diet had a relative ratio of 0.5, but increased to 2 by feeding the methionine / choline-deficient diet. However, administration of the fermented extract reduced 1.3 to 65% with a relative ratio of 2. It should be noted that there is no change in liver TBARS even when the fermented extract is administered to mice fed with a normal diet. The fermented extract had no effect on healthy liver, and acted only in the group of mice in which the NASH-like pathological condition was induced, and was considered to be highly safe.

  In addition, liver SOD was lower in the group of mice in which the NASH-like pathological condition was induced than in the group fed with a normal diet. However, the administration of the fermented extract increased the relative ratio of SOD even in the NASH-like pathological group. In addition, even if fermented extract is administered to mice fed with a normal diet, there is no change in liver SOD values. The fermented extract had no effect on healthy liver, and acted only in the group of mice in which the NASH-like pathological condition was induced, and was considered to be highly safe.

  INDUSTRIAL APPLICABILITY The present invention uses melanoidins and fermented extracts extracted from natural fermentation products as therapeutic agents for NAFLD and NASH, and is useful.

Claims (7)

  A therapeutic agent for nonalcoholic fatty liver disease and / or nonalcoholic steatohepatitis, comprising melanoidin as an active ingredient.   The therapeutic agent according to claim 1, wherein the melanoidin is an extract from a fermented extract obtained by co-fermenting lactic acid bacteria and yeast.   The therapeutic agent according to claim 2, wherein the melanoidin is an extract from a fermented extract obtained by co-fermenting polysaccharides, vegetables / fruits, and seaweed.   The therapeutic agent of Claim 3 whose said vegetables and fruits are papayas.   The therapeutic agent according to claim 3 or 4, wherein the polysaccharide is rice, rice bran and / or brown rice.   Non-alcoholic fatty liver disease and / or non-alcoholic fat comprising a fermentation extract containing melanoidin as an active ingredient, which is produced by co-fermenting polysaccharides, vegetables / fruits, seaweed, lactic acid bacteria and yeast. Hepatitis treatment.   The therapeutic agent according to claim 6, wherein the polysaccharide is rice, rice bran and / or brown rice, and the vegetables and fruits are papaya.