JP2017057155A - Method for preparing composition for preventing or treating nonalcoholic fatty liver disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective drug or food for the prevention or treatment of nonalcoholic steatohepatitis (NASH).SOLUTION: The present invention provides a method for producing a composition, comprising the steps of: (1) fermenting a placenta using a fermenting microorganism to obtain a placenta fermentation product; (2) separating the placenta fermentation decomposition product into a fermented extracted placenta extract, which is a soluble fraction, and an insoluble fraction; (3) subjecting the insoluble fraction to a protease treatment to obtain a protease-decomposed extracted placenta extract; and (4) mixing the fermented extracted placenta extract and the protease-decomposed extracted placenta extract to obtain a composition. The present invention also provides a pharmaceutical composition obtained by such a production method. The fermenting microorganism is one or more microorganisms selected from lactic acid bacteria, yeast, Bacillus subtilis, or koji mold. The protease is one or more selected from acid protease, neutral protease, alkaline protease, pepsin, trypsin, chymotrypsin, elastase, collagenase, etc.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a composition for preventing or treating nonalcoholic fatty liver disease, particularly nonalcoholic steatohepatitis. According to the production method of the present invention, it is possible to provide a composition effective for the prevention or treatment of nonalcoholic fatty liver disease, particularly nonalcoholic steatohepatitis. In addition, the composition obtained by the production method of the present invention can be administered not only as a pharmaceutical product, but also in various forms, for example, health foods and functional foods (including beverages), or food and drink as feed. It is also possible to give.

  With the westernization of meal content, high fat diets have become a problem in Japan in recent years. Conventionally, the main cause of fatty liver has been considered to be excessive intake of alcohol, but with such changes in diet content, non-alcoholic fatty liver disease (NAFLD) is often seen It has become.

  Among NAFLD, non-alcoholic steatohepatitis (NASH) is a non-alcoholic steatohepatitis (NASH: Non-Alcoholic), which is caused by fat deposition in the liver parenchyma, infiltration of inflammatory cells, hepatic parenchyma, fibrosis, and cirrhosis and liver cancer. Steatohepatitis). NAFLD / NASH is a metabolic syndrome because obesity, insulin resistance, diabetes, dyslipidemia are frequently combined, and adipocytokines and free fatty acids secreted from visceral fat are deeply involved in the onset and progression of NASH Is considered a phenotype in the liver.

  The detailed development mechanism of NASH is not clear, and the therapeutic means has not been established. From previous studies, obesity and increased visceral fat caused fat to accumulate ectopically in the liver, causing lipotoxicity (lipotoxicity), and increased lipid peroxidation (oxidative stress). It is thought that it will progress to NASH. The importance of oxidative stress has been pointed out in the process of inflammation from fatty liver, which is the key to NASH development. From such a point of view, anti-NASH action has been studied for various antioxidants, such as vitamin E (Non-patent Document 1), astaxanthin (Patent Document 1), β-cryptoxanthin (Patent Document 2, Non-patent Document 2). Has been reported to have a certain effect. There is also a report that pioglitazone, which is a PPARγ agonist that improves insulin resistance in the background of NASH, improves the symptoms of steatohepatitis (Non-patent Document 1).

International Publication 2012 / 157289A1 JP 2014-118390 A JP 2005-185242 A

“The New England Journal of Medicine” (USA) 2010, vol. 156, p. 1675-85 “Endocrinology” (USA) 2015, Vol. 362, p. 987-99 “Biological & Pharmaceutical Bulletin” (Japan) 2014, Vol. 37, p. 1853-9 “Hepatology Research” (Japan) 2014, DOI: 10.11111 / hepr. 12432

In Non-Patent Documents 3 and 4, it has been attempted to treat NASH using a medicine containing a placental extract obtained by enzymatic degradation of the placenta as an active ingredient. However, the therapeutic effect was not sufficient. That is, a method for treating NASH has not been established, and no therapeutic drug for NASH has been approved. The most important treatment or prevention for NAFLD / NASH is diet and weight loss with limited fat intake, but it is often difficult to continue, so there is strong development of effective medicines or foods to supplement it. It is desired.
Accordingly, an object of the present invention is to provide a drug or food for effective prevention or treatment of NASH.

As a result of diligent research on a method for producing a composition for preventing or treating NAFLD / NASH, the present inventors have surprisingly found that a fermented extract obtained by fermenting a placenta using a fermenting microorganism. A composition comprising a placenta extract and a protease-degraded and extracted placenta extract obtained by subjecting the residue of the fermented and extracted placenta extract to protease treatment is effective for treating nonalcoholic fatty liver disease, particularly nonalcoholic steatohepatitis. Have been found to be preventable or treatable.
The present invention is based on these findings.
Therefore, the present invention
[1] A method for producing a composition comprising the following four steps: (1) a step of fermenting a placenta using a fermenting microorganism to obtain a placental fermented degradation product, (2) a soluble fraction of the placental fermented degradation product (3) a step of treating the insoluble fraction with a protease to obtain a protease-degraded extracted placenta extract, and (4) the fermented-extracted placenta extract and the insoluble fraction. Proteolytic degradation placenta extract is mixed to obtain a composition,
[2] The method according to [1], wherein the composition is a pharmaceutical composition,
[3] The method according to [1] or [2], wherein the composition is for prevention or treatment of non-alcoholic fatty liver disease.
[4] The method according to [3], wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.
[5] The method according to any one of [1] to [4], wherein the fermentation microorganism is one or more microorganisms selected from the group consisting of lactic acid bacteria, yeast, Bacillus subtilis, and koji molds.
[6] The protease is one or more proteases selected from the group consisting of acidic protease, neutral protease, alkaline protease, pepsin, trypsin, chymotrypsin, elastase, collagenase, carboxypeptidase, and aminopeptidase [1] ] To the method according to any one of [5],
[7] The composition produced by the production method according to any one of [1] to [6], and [8] The composition is a health food or a functional food according to [7]. Composition,
About.
For example, Patent Document 3 discloses a health food containing placenta fermented with fermenting bacteria. However, the effect of this health food was improvement of low back pain and joint pain.

  According to the production method of the present invention, a composition effective for the prevention or treatment of non-alcoholic fatty liver disease, particularly non-alcoholic steatohepatitis can be provided. In addition, the composition obtained by the production method of the present invention can prevent or treat nonalcoholic fatty liver disease, particularly nonalcoholic steatohepatitis.

It is the graph which showed the weight change of the mouse | mouth when the composition of this invention is orally administered to the NASH model mouse which ingested the NASH induction diet. It is the graph which showed the average food intake of the normal food intake group (NC), the NASH induction food intake group (CL), or the NASH induction food and the composition intake group of the present invention (CL + LPL or CL + HPL). It is the graph which showed the improvement of AST and ALT in plasma when the composition of this invention is orally administered to the NASH model mouse | mouth which ingested the NASH induction diet. It is the graph which showed the improvement of TG, TC, and NEFA in a liver tissue when the composition of this invention is orally administered to the NASH model mouse | mouth which ingested the NASH induction diet. Photograph showing liver appearance and HE-stained image of liver tissue section of normal diet intake group (NC), NASH induced diet intake group (CL), or NASH induced diet and composition intake group of the present invention (CL + LPL or CL + HPL) It is. It is the graph which showed the fall of the expression of SREBP-1c gene, FAS gene, and SCD1 gene in a liver tissue when the composition of this invention is orally administered to the NASH model mouse which ingested the NASH induction diet. It is the graph which showed the increase in the expression of the PPAR alpha gene and Cptla gene in a liver tissue when the composition of this invention is orally administered to the NASH model mouse which ingested the NASH induction diet. Graph showing a decrease in the expression of F4 / 80 gene, TNF alpha gene, IL1 beta gene, and CD11c gene in liver tissue when the composition of the present invention was orally administered to NASH model mice ingesting a NASH-induced diet It is.

[1] Method for Producing Composition The method for producing the composition of the present invention comprises: (1) a step of fermenting the placenta using a fermentation microorganism to obtain a placental fermentation decomposition product (hereinafter referred to as a fermentation treatment step). (2) a step of separating the placental fermentation degradation product into a fermentation extract placenta extract which is a soluble fraction and an insoluble fraction (hereinafter sometimes referred to as a separation step), (3) the insoluble fraction A step of treating with protease to obtain a protease-degraded and extracted placenta extract (hereinafter also referred to as a protease-treating step), and (4) mixing the fermentation-extracted placenta extract and the protease-degrading and extracting placenta extract, A step of obtaining (hereinafter sometimes referred to as a mixing step).

(1) Fermentation treatment step The fermentation treatment step in the production method of the present invention is (1) a step of fermenting the placenta using a fermentation microorganism, and a placental fermentation decomposition product can be obtained.

"placenta"
The origin of the placenta used in the fermentation treatment process is not particularly limited, but is derived from human, monkey, cow, horse, pig, sheep, goat, rabbit, cat, dog, mouse, rat, guinea pig, whale, or hamster From the viewpoint of commercial use, it is preferable to use a placenta obtained by postpartum delivery of a normal pig or horse.

《Fermenting microorganisms》
The fermenting microorganism is not limited as long as a fermented and extracted placenta extract that can be used in the present invention is obtained, and examples thereof include lactic acid bacteria, yeasts, Bacillus subtilis, and koji molds.

  Examples of lactic acid bacteria include, but are not limited to, Bulgarian bacteria (Lactobacillus delbruecki), Thermophilus bacteria (Streptococcus thermophilus), Lactococcus lactis, or various Lactobacillus bacteria of the genus Lactobacillus, There may be mentioned lactic acid bacteria belonging to the genus Leuconostoc, Pediococcus, Tetragenococcus or Enterococcus. Lactic acid bacteria can produce amino acids obtained by fermentation such as GABA and / or ornithine or branched chain amino acids such as leucine, isoleucine, and / or valine by lactic acid fermentation.

  Examples of yeast include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces pombe, Saccharomyces pastorianu, Saccharomyces bayanus, or yeast of the genus Digosaccharomyces (Digosaccharomyces roxy, etc.). The yeast can produce amino acids obtained by fermentation such as GABA and / or ornithine or branched chain amino acids such as leucine, isoleucine, and / or valine by alcohol fermentation.

  Examples of the koji mold include, but are not limited to, koji molds used in the production of miso soy sauce, such as Aspergillus oryzae, Aspergillus soja, Aspergillus luchuensis, and the like. Aspergillus has strong protein and carbohydrate resolution and can produce amino acids obtained by fermentation such as GABA and / or ornithine, or branched chain amino acids such as leucine, isoleucine, and / or valine.

  Examples of Bacillus subtilis include, but are not limited to, Bacillus subtilis, Bacillus amuroliquefaciens, and the like. Bacillus subtilis has strong protein and carbohydrate resolution and can produce amino acids obtained by fermentation such as GABA and / or ornithine, or branched chain amino acids such as leucine, isoleucine, and / or valine. .

(Fermentation treatment)
The fermentation treatment is not particularly limited as long as γ-aminobutyric acid (GABA) and ornithine (Orn) can be extracted from the placenta.
Although it does not specifically limit as an energy source of the fermentation microorganisms in a fermentation process, Sugar (for example, glucose, white sugar, brown sugar, molasses) can be mentioned. Although the addition amount of saccharide | sugar is not specifically limited, For example, 1-50 g of saccharide | sugar can be added with respect to 1 kg of placenta, and a fermentation process can be performed.
Accordingly, the fermentation temperature is not particularly limited as long as a placental fermentation decomposition product that can be used in the present invention is obtained. For example, the fermentation temperature may be 15 ° C to 45 ° C, and preferably 20 ° C to 35 ° C. ° C, more preferably 25 ° C to 30 ° C.
In addition, the fermentation time is not particularly limited as long as a placental fermentation decomposition product that can be used in the present invention is obtained. For example, the fermentation time can be 6 to 5 days, preferably 12 to 4 hours. Day, more preferably 1 to 3 days. By combining a relatively short fermentation with the protease degradation described below, effective amounts of γ-aminobutyric acid (GABA) and / or ornithine (Orn) and branched chain amino acids such as leucine, isoleucine and / or valine Can be obtained.
In order to obtain a sufficient amount of γ-aminobutyric acid (GABA) and ornithine (Orn), generally, the reaction time may be short when the fermentation temperature is high, and conversely when the fermentation temperature is low, the reaction time is reduced. It can be adjusted by making it longer. For example, it is preferable that the obtained fermented extract placenta extract is fermented so that it contains 1% by weight or more of γ-aminobutyric acid by free amino acid analysis or 0.4% by weight or more of ornithine.

(Placement fermentation products)
The placental fermentation degradation product contains an extract obtained by fermentation, and includes a fermentation extract placenta extract which is a soluble fraction described later and a residue of an insoluble fraction.

(2) Separation step The separation step (2) is a step of separating the placental fermentation degradation product into a fermentation extract placenta extract which is a soluble fraction and an insoluble fraction. Examples of the method for separating the placental fermentation broth into a soluble fraction and an insoluble fraction include, but are not limited to, centrifugation, filtration, sieving separation, and a combination thereof. The insoluble fraction is subjected to the next step (3) of obtaining a protease-degraded and extracted placenta extract.

《Fermentation extracted placenta extract》
Fermentation-extracted placenta extract contains many kinds of amino acids, but includes γ-aminobutyric acid (GABA) and ornithine (Orn) as characteristic amino acids. The content of γ-aminobutyric acid (GABA) in the fermentation-extracted placenta extract is not particularly limited, but is preferably 1% by weight or more, more preferably 2% by weight or more based on the total amount of amino acids. More preferably 3% by weight or more, still more preferably 4% by weight or more. In addition, the content of ornithine (Orn) in the fermentation-extracted placenta extract is preferably 0.4% by weight or more, more preferably 0.8% by weight or more, and still more preferably 1%, based on the total amount of amino acids. .2% by weight or more, more preferably 1.6% by weight or more. In addition, the upper limit of the content of γ-aminobutyric acid (GABA) and ornithine (Orn) depends on the upper limit of the content of γ-aminobutyric acid (GABA), ornithine (Orn), and their precursors contained in the placenta, Since it is determined, it is not particularly limited. Although it is not limited, non-alcoholic fatty liver disease, especially non-alcoholic steatohepatitis can be effectively prevented or treated by including γ-aminobutyric acid (GABA) and ornithine (Orn). it can. The fermentation-extracted placenta extract also contains branched chain amino acids such as leucine, isoleucine, and / or valine. These branched chain amino acids can further enhance the effect of preventing or treating nonalcoholic fatty liver disease, particularly nonalcoholic steatohepatitis.
In addition, the fermentation extract placenta extract preferably contains a high molecular weight component having a molecular weight of 10,000 or more. The content of the high molecular weight component having a molecular weight of 10,000 or more is not limited, but is preferably 3% by weight or more, more preferably 5% by weight or more, still more preferably 8% by weight or more, Most preferably, it is 10 weight% or more. A high molecular weight fraction having a molecular weight of 10,000 or more can suppress reabsorption of bile acids by binding to bile acids in the digestive tract. Thereby, the onset and progress of non-alcoholic fatty liver disease, particularly non-alcoholic steatohepatitis can be suppressed.
The upper limit of the content of the high molecular weight component having a molecular weight of 10,000 or more is not particularly limited, but is preferably 50% by weight or less, more preferably 40% by weight or less, and still more preferably 35% by weight or less. It is. When the content of the high molecular weight component is high, the fermentation degradation of the protein into amino acids is reduced, and the content of leucine, isoleucine, and / or valine may be reduced.

<Insoluble fraction>
The insoluble fraction of the placental fermentation degradation product is a solid content obtained by centrifugation, filtration, and / or sieving separation. That is, it contains solid components that are difficult to be decomposed by fermenting microorganisms.

(3) Protease treatment step The protease treatment step (3) is a step of subjecting the insoluble fraction to a protease treatment to obtain a protease-degraded and extracted placenta extract. Specifically, water or a buffer solution is added to the insoluble fraction, protease is added, and incubation is performed. Water or buffer solution is not particularly limited because it can be appropriately selected depending on the protease to be used, but water, phosphate buffer, bicarbonate buffer solution, citrate buffer solution, lactate buffer solution, acetate buffer solution And adipic acid buffer, tartaric acid buffer, fumarate buffer, malic acid buffer, Tris buffer, and HEPES buffer. In either case, the pH may be adjusted to an optimum pH with an appropriate amount of acid or alkali. The amount of water or buffer can be appropriately determined depending on the amount of insoluble fraction and the type of protease.

《Protease》
The protease is not particularly limited as long as a protease-degraded and extracted placenta extract that can be used in the present invention can be obtained. For example, various enzymes used for food processing can be used. Specifically, acidic proteases produced by microorganisms such as Rhizopus sp. And Aspergilus sp. (A. saitoi, A. niger, A. etc.), Aspergillus sp. (A. oryzae etc.), Bacillus sp. subtilis, B. amyloliquefaciens, etc.), neutral proteases produced by microorganisms such as Geobacillus caldoproteolyticus, Bacillus sp. (B. subutilis, B. licheniformis, B. amyloliquefaciens, etc.), Aspergillus sp. (A. niger, A. oryzae, etc., and other microorganisms such as Streptomyces sp. (Streptomyces griseus, etc.), Rhizomucor sp. (Rhizomucor miehei, Rhizomucor miehei, etc.), Kluyveromyces sp. (Kluyveromyces lactis, etc.), and papaya , Pineapple, and other plant-produced proteases or various proteases produced by animals as digestive enzymes (pepsin, trypsin, chymotrypsin, elastase, collagenase, carboxypeptider , Can be used aminopeptidase, etc.) alone or in combination.

The protease used in the present invention is not limited as long as a low molecular weight component having a molecular weight of 500 or less contained in the obtained protease-degraded and extracted placenta extract is sufficiently obtained.
Moreover, the addition amount of protease can also be suitably adjusted according to the titer and decomposition performance of each protease. Specifically, for example, the amount of protease added can be adjusted so that the content of low molecular weight components having a molecular weight of 500 or less contained in the protease-degraded and extracted placenta extract is 30% by weight or more.
Furthermore, the temperature and time of the protease treatment can be appropriately adjusted depending on the type of protease used or the titer of the protease. Specifically, for example, the temperature and time of protease treatment of the protease can be adjusted so that the content of low molecular weight components having a molecular weight of 500 or less contained in the protease-degraded and extracted placenta extract is 30% by weight or more. For example, the protease treatment can be performed at a temperature of 25 ° C to 65 ° C, preferably 30 ° C to 60 ° C, more preferably 40 to 55 ° C. Also, the time for protease treatment is not particularly limited, but for example, it can be carried out for 0.5 hour to 72 hours, preferably 1 hour to 48 hours, more preferably 1 hour to 24 hours.

《Protease Degradation Extraction Placenta Extract》
The protease-decomposing and extracting placenta extract obtained by subjecting the insoluble fraction (residue) of the placental fermentation degradation product to protease treatment is not particularly limited as long as the effects of the present invention can be obtained. It is preferable that the molecular weight component is sufficiently contained. The content of the low molecular weight component having a molecular weight of 500 or less is not particularly limited, but is preferably 30% by weight or more, more preferably 40% by weight or more, still more preferably 45% by weight or more, Most preferably, it is 50 weight% or more. When the low molecular weight component is 30% by weight or less, protease decomposition is not sufficient, and the effects of the present invention may not be obtained.

  Further, the protease-degraded extraction placenta extract is not limited, but preferably contains γ-aminobutyric acid (GABA) and ornithine (Orn). When the placenta is treated with protease as it is, the extraction efficiency of γ-aminobutyric acid (GABA) and ornithine (Orn) is not high, but the residue (insoluble fraction) after fermentation of the placenta with fermenting microorganisms is treated with protease. Thus, γ-aminobutyric acid (GABA) and ornithine (Orn) can be efficiently extracted. By including γ-aminobutyric acid (GABA) and ornithine (Orn), the effects of the present invention can be efficiently obtained. Further, branched chain amino acids such as leucine, isoleucine, and / or valine that could not be sufficiently extracted only by the fermentation process with the fermenting microorganisms can be extracted effectively by carrying out this process, and the effects of the present invention can be more efficiently achieved. Leads to gain.

(4) Mixing step The mixing step (4) is a step of mixing the fermentation extraction placenta extract and the protease-decomposing extraction placenta extract to obtain a composition. By mixing the fermented extract placenta extract and the protease-degraded extract placenta extract, the composition of the present invention has a sufficient amount of γ-aminobutyric acid (GABA) and ornithine (Orn) and leucine, isoleucine, and / or It can contain branched chain amino acids such as valine. Further, a high molecular weight component having a molecular weight of 10,000 or more can be sufficiently contained.
As the mixing method, a mixing method used in this field can be used without limitation. Moreover, the process of removing an insoluble matter by centrifugation or filtration etc. can be included before mixing as needed.

"mixing ratio"
The mixing ratio of the fermented extracted placenta extract and the protease-decomposed extracted placenta extract is not particularly limited as long as the effects of the present invention can be obtained. Is preferably 1: 9 to 9: 1, more preferably 2: 8 to 8: 2, still more preferably 3: 7 to 7: 3, and most preferably 4: 6 to 6: 4. It is. When the mixing ratio is as described above, γ-aminobutyric acid (GABA) and ornithine (Orn) and a high molecular weight component having a molecular weight of 10,000 or more can be contained in a balanced manner.

<Action>
The mechanism by which the composition containing the fermented and extracted placenta extract obtained by the production method of the present invention can effectively prevent or treat nonalcoholic fatty liver disease has been clearly analyzed. However, it can be estimated as follows. However, the present invention is not limited by the following estimation.
In the production method of the present invention, the placenta is first fermented with a fermenting microorganism. The fermentation-extracted placenta extract (placental fermentation decomposition product) obtained by this fermentation treatment contains a large amount of γ-aminobutyric acid (GABA) and ornithine (Orn). These γ-aminobutyric acid (GABA) and ornithine (Orn) are considered to act effectively on nonalcoholic fatty liver disease. On the other hand, the extract obtained by the conventional protease treatment does not contain γ-aminobutyric acid (GABA) and ornithine (Orn), and the composition of the present invention is effective for nonalcoholic fatty liver disease. Conceivable.
Further, in the present invention, the protease-degraded and extracted placenta extract obtained by subjecting the residue of the placental fermentation degradation product to protease treatment also contains a large amount of γ-aminobutyric acid (GABA) and ornithine (Orn). As described above, the extract obtained by subjecting the placenta to direct protease treatment does not contain γ-aminobutyric acid (GABA) and ornithine (Orn), whereas the protease-degrading extraction placenta extract of the present invention has γ-aminobutyric acid (GABA). ) And ornithine (Orn) are not clear, but GABA and Orn are contained in the residue fermented by the fermenting microorganism, and by using it for protease treatment, γ-aminobutyric acid (GABA) is efficiently contained. ) And ornithine (Orn) are considered to have been extracted. Therefore, in the present invention, it is important to perform the protease treatment step (3) after the fermentation treatment step (1).
Furthermore, the fermentation extract placenta extract contains a high molecular weight component having a molecular weight of 10,000 or more, but the extract obtained by directly treating the placenta with protease does not contain a high molecular weight component having a molecular weight of 10,000 or more. It is considered that the remarkable effects of the present invention can be obtained by including this polymer component.
In addition, the fermentation / protease-degrading placenta extract obtained by the production method of the present invention contains branched chain amino acids (leucine, isoleucine, valine) equivalent to the conventional protease-degrading protease extract, and is expected in conventional products. It is expected that the anti-NASH action is also retained. These branched chain amino acids such as leucine, isoleucine, and / or valine can be efficiently extracted by performing fermentation treatment with a fermentation microorganism and treatment with a protease.
In the production method of the present invention, the placenta extract extracted by protease decomposition cannot be further subjected to fermentation using microorganisms. This is because the composition of the placenta extract component extracted by protease decomposition is altered by the proteolytic enzyme due to fermentation using microorganisms.

[2] Composition Although the composition of this invention can be manufactured with the said manufacturing method, it is not limited to what was manufactured with the said manufacturing method. Specifically, the composition of the present invention can contain the fermentation extract placenta extract and the protease-degraded extract placenta extract, but may contain other components. Moreover, the composition of this invention can be used as a pharmaceutical composition or a health food. The pharmaceutical composition is effective for non-alcoholic fatty liver disease (NAFLD), and particularly effective for non-alcoholic steatohepatitis (NASH).

  The dosage form of the composition obtained by the production method of the present invention is not particularly limited. For example, powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts Oral preparations such as pills, or parenteral preparations such as injections, solutions for external use, ointments, suppositories, topically applied creams, or eye drops, etc. can be mentioned, and oral preparations are particularly preferred.

  Oral preparations include, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid ester, talc, magnesium stearate, Excipients such as polyethylene glycol, magnesium silicate, anhydrous silicic acid, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, preservatives, colorants, It can be produced according to a conventional method using a fragrance, a flavoring agent, a stabilizer, a moisturizing agent, an antiseptic, or an antioxidant.

Examples of parenteral administration methods include injection (subcutaneous, intravenous, etc.) or rectal administration. Of these, the injection is most preferably used.
For example, in the preparation of injections, in addition to the fractions as active ingredients, for example, water-soluble solvents such as physiological saline or Ringer's solution, water-insoluble solvents such as vegetable oil or fatty acid esters, glucose or sodium chloride, etc. An isotonic agent, a solubilizer, a stabilizer, an antiseptic, a suspending agent, an emulsifier, or the like can be arbitrarily used.
In addition, the composition according to the present invention may be administered using a method of sustained release preparation using a sustained release polymer or the like. For example, a composition according to the present invention can be incorporated into a pellet of ethylene vinyl acetate polymer and the pellet can be surgically implanted into the tissue to be treated or prevented.

"healthy food"
The composition obtained by the production method of the present invention can be provided in various forms, for example, as a health food (preferably a functional food) or a feed. The food includes a beverage.

In the present specification, the “health food” means a food that has some effect on health or can be expected to have an effect, and the “functional food” includes various “health foods”. Means food that is designed and processed so that it can fully express the biological regulation functions of the body (for example, the regulation function of physiological systems such as digestive system, circulatory system, endocrine system, immune system, or nervous system) To do.
As the health food of the present invention, those having a function of preventing or treating nonalcoholic steatohepatitis and / or nonalcoholic fatty liver disease are preferable.

  The health food of the present invention can be prepared in the same manner as a general health food except that it contains a bacterial fermentation extract placenta extract and a protease-degradation extract placenta extract as active ingredients. For example, the health food of the present invention can be prepared by containing an effective amount of the active ingredient in a substance that can be ingested as a food. The effective amount can be appropriately determined according to various factors such as the shape or type of food, or the symptoms, age, weight, etc. of the individual taking the food.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1: Production of composition for preventing or treating non-alcoholic fatty liver disease
Yeast (Zygosaccharomyces sp.) And lactic acid bacteria (Pediococcus sp.) Were added to about 20 kg of placenta obtained as a postpartum during normal delivery of pigs, and after further addition of about 300 g of glucose, it was allowed to stand for 1 day. A placental fermentation broth was obtained. The culture supernatant and the residue were separated by sieving and centrifuging. The supernatant was concentrated by an evaporator and then sterilized by filtration with a 0.2 μm filter to obtain a fermentation-extracted placenta extract (about 2.6 kg). To the residue (about 4.5 kg), equal amounts of water and protease (manufactured by Amano Enzyme) were added, and protease was degraded for 2 hours. When sieving after heat sterilization, the residue was 27 g, which was about 0.5% of the 4.5 kg residue of the previous step used for the preparation. The protease-degraded extracted placenta extract obtained by removing the residue is mixed with the aforementioned fermentation-extracted placenta extract and freeze-dried to prevent powdery nonalcoholic steatohepatitis and / or nonalcoholic fatty liver disease or About 1.1 kg of therapeutic composition was obtained.
In addition, when the fermentation-extracted placenta extract was freeze-dried as it was, about 500 g of a powdery composition was obtained, so that about 50% of the placenta extract was extracted in each of the fermentation process and the protease decomposition process. In other words, it was found that an extract having both the characteristics of fermentation extraction and the characteristics of protease decomposition can be fully obtained by subjecting the residue to protease decomposition after the fermentation step with bacteria.

<< Comparative Example 1 >>
To about 20 kg of pig placenta, 6.7 kg of water and protease (manufactured by Amano Enzyme) were added and reacted for 5 hours. After heating and sterilization, sieving, concentrating with an evaporator, and freeze-drying, a direct protease-degraded placenta extract, about 1.2 kg, was obtained.

<< Reference Examples 1-12 and Comparative Example 2 >>
Extract extraction with various fermenting microorganisms was performed on a 100 g or 500 g scale of porcine placenta. As fermentation microorganisms, baker's yeast (Saccharomyces cerevisiae) (Reference Examples 1 to 4), lactic acid bacteria mixture for yogurt (5 lactic acid bacteria + 1 yeast) (Reference Example 5), koji mold for soy sauce (Aspergillus soya) Reference Examples 6-9), lactic acid bacteria for soy sauce (Pediococcus genus) (Reference Examples 6-9), yeast for soy sauce (Gigosaccharomyces genus) (Reference Examples 6-9), inoculum for kefir (Reference Examples 10-12) . Fermentation was carried out for 1 or 2 days depending on the bacterial species, and the extract after fermentation was sterilized by heating and then subjected to centrifugal filtration, and subjected to total nitrogen content measurement and free amino acid analysis. Examples 7 and 8 were cultured for 1 day in lactic acid bacteria for soy sauce and yeast for soy sauce after culturing for 1 day in koji mold for soy sauce. Examples 9 and 10 were cultured for one day in a mixture of koji mold for soy sauce, lactic acid bacteria for soy sauce, and yeast for soy sauce. Moreover, the comparative example 2 extracted by boiling water from a pig placenta, without performing culture | cultivation by microorganisms.

<< Analysis Example 1: Analysis of free amino acids >>
Obtained in the fermented extract placenta extract (A) of Example 1 and the freeze-dried powder (B), (A) and (B) of the protease-decomposed extract placenta extract of the present invention (C) and Comparative Example 1 Free amino acid composition analysis was performed for each of the freeze-dried powder (D) of the direct protease-degraded placenta extract obtained by directly protease-degrading the obtained placenta. The results are shown in Table 1. 0.1 g of each sample was taken, sulfosalicylic acid test solution was added to make 20 mL, shaken vigorously for 10 minutes, and then allowed to stand at room temperature for 4 hours. After taking 2 mL of this solution and adding lithium citrate buffer (pH 2.98) to 20 mL, it was filtered through a 0.22 μm membrane filter to obtain a sample solution. It measured using the amino acid automatic analyzer using 50 microliters of this liquid. It was found that the distribution of the amount of free amino acids (per powder weight; nmole / g) was not significantly different in each of A, B, C, and D samples. Further, the composition (C) of the present invention contained all amino acids contained in the direct protease-degraded placenta extract (D). On the contrary, in (A), (B), and (C) including the fermentation step, γ-aminobutyric acid (GABA) and ornithine (Orn) not included in (D) were detected. Although the amount of arginine contained in a large amount in (D) is small in each of (A), (B), and (C), this may have been consumed for the synthesis of ornithine by microorganisms.

  Free amino acid analysis was performed on the placenta extract prepared using various fermented microorganisms obtained in Reference Examples 1 to 12 and Comparative Example 2. At the same time, the analysis of total nitrogen amount by Kjeldahl method was carried out. The results are shown in Table 2. Table 2 also shows the amount of GABA and ornithine among the free amino acids. It was found that the total amount of nitrogen and the total amount of free amino acids were remarkably increased as compared with the case of no fermentation even if any fermentative bacteria were used. From this, it can be seen that a soluble placenta extract typified by a free amino acid is extracted using any bacterial species. In addition, the addition of glucose as a carbon source during fermentation tends to increase the total amount of nitrogen and the total amount of free amino acids. In particular, in the study with baker's yeast and kefir seeds, the increase in GABA and / or ornithine is significant Met.

<< Analysis Example 2: Molecular Weight Distribution Analysis >>
The molecular weight of the composition of the present invention (C), fermentation extract placenta extract (A), fermentation residue protease degradation product (B), and direct protease degradation placenta extract (D) using the gel filtration HPLC method The distribution was examined. Each sample was dissolved in purified water at a concentration of about 5%, and insoluble matters were removed by filter filtration, and then subjected to analysis. Molecular weight standard substances include cytochrome C (molecular weight 12327), insulin B chain (molecular weight 3496), Gly-Gly-Gly-Gly-Gly-Gly (molecular weight 360.32), Gly-Gly-Gly (molecular weight 189.17). , Gly (molecular weight 75.07) (concentrations were 0.192 mg / mL, 0.2 mg / mL, 0168 mg / mL, 0.28 mg / mL, and 0.86 mg / mL, respectively). The gel filtration HPLC analysis was performed by implanting 20 μL of the sample solution and 10 μL of the standard solution. The measurement conditions are as follows.

Equipment used: Agilent 1100 (detection wavelength = 214 nm)
Column: SuperdexTM Peptide 10 / 300GL (GE Healthcare Japan)
Column temperature: room temperature Mobile phase: 20 mM phosphate buffer pH 7.2 (including 250 mM sodium chloride)
Flow rate: 9.5 mL / min
Analysis time: 45 minutes

The molecular weight was determined from a calibration curve created using retention time data in a standard solution.
Table 3 shows the chromatographic area in each molecular weight range. Regarding the region of 500 or less corresponding to the molecular weight region of free amino acids, all samples accounted for nearly 50% or more of the total. This result is consistent with the free amino acid analysis showing that the same free amino acid is observed. In (A), the ratio of 500 or less is even more likely, but it is probably because it contains low molecules accompanying fermentation in addition to amino acids. On the other hand, about the area | region (area | region corresponded to the molecular weight of an oligopeptide, a peptide, and a low molecular protein) of molecular weight 500-10,000, the fraction obtained by protease decomposition | disassembly is included rather than a fermentation extract (A) (B ), (C), and (D) tended to be more common. However, as for the component (protein region) having a molecular weight of greater than 10,000, the composition (C) of the present invention and the component (A) and (B) containing the component that has undergone the fermentation process are more directly proteolytic degradation placenta. It was found that the extract contained more than the lyophilized powder (D). This result indicates that a placenta extract containing a component of a high molecular region that cannot be obtained by directly protease-degrading the placenta by directly extracting the extract from the placenta using a microorganism can be produced. Such a placenta extract cannot be obtained by a method in which the order of the fermentation process and the protease decomposition process is reversed.

<< Analysis Example 3: Metabolomic Analysis of Fermentation Decomposition Solution >>
In order to clarify the low-molecular components produced characteristically in the fermentation process, metabolomic analysis was performed on the fermentation-extracted placenta extract (A) and the direct protease-degraded placenta extract (D). 100 mg of each sample was dissolved in 10 mL of ultrapure water to prepare a metabolome analysis solution. 100 μL of a metabolome analysis solution was added to 450 μL of a methanol solution prepared so that the concentration of the internal standard substance was 10 μM and stirred. To this, 500 μL of chloroform and 100 μL of ultrapure water were added and stirred, followed by centrifugation. After centrifugation, the aqueous layer was transferred to an ultrafiltration tube (Milipore, Ultra Free MC PLHCC HMT centrifugal filter unit 5 kDa) by 400 μL × 1. This was centrifuged and subjected to ultrafiltration treatment. The filtrate was dried and dissolved again in 50 μL of ultrapure water and used for measurement.
The measurement was performed in both the cation mode and the anion mode by the method of capillary electrophoresis / time-of-flight mass spectrometry (CE-TOFMS). The analysis conditions are as shown below.

Cationic metabolite (cation mode)
apparatus
Agilent CE-TOFMS system (Agilent Technologies)
Capillary: Fused silica capillary id 50μm × 80 cm
Measurement condition
Run buffer: Cation Buffer Solution (p / n: H3301-1001)
Rinse buffer: Cation Buffer Solution (p / n: H3301-1001)
Sample injection ： Pressure injection 50 mbar, 10sec
CE voltage: Positive, 27 kV
MS ionization: ESI positive
MS capillary voltage: 4,000 V
MS scan range: m / z 50-1,000
Sheath liquid: HMT Sheath Liquid (p / n: H3301-1020)

Anionic metabolites (anion mode)
apparatus
Agilent CE-TOFMS system (Agilent Technologies)
Capillary: Fused silica capillary id 50μm × 80 cm
Measurement condition
Run buffer: Anion Buffer Solution (p / n: H3302-1021)
Rinse buffer: Anion Buffer Solution (p / n: H3302-1022)
Sample injection: Pressure injection 50 mbar, 25sec
CE voltage: Positive, 30 kV
MS ionization: ESI negative
MS capillary voltage: 3,500 V
MS scan range: m / z 50-1,000
Sheath liquid: HMT Sheath Liquid (p / n: H3301-1020)

Peaks detected by CE-TOFMS were automatically extracted by automatic integration software, and mass-to-charge ratio (m / z), migration time (MT), and peak area were calculated as peak information. The obtained peak area value was converted into a relative area value (= [target peak area value] / [internal standard substance area value × sample amount]) using the area of the internal standard substance. After examining artifacts such as adduct ions such as Na + and K +, dehydration, and deammonium, the values of m / z and MT were used to collate and align peaks between samples. The detected peaks were compared with the HMT metabolite database (Human Metabolome Technologies) to identify the peaks. As a result of this analysis, the relative peak area with respect to the standard substance is 1 or more in the fermented extracted placenta extract (A), and 10 times more than the direct protease-decomposed placenta extract (D) is detected in the fermented extracted placenta extract (A). The metabolites are summarized in Table 4. By including a fermentation step, for example, a placenta extract containing components that cannot be obtained only by protease degradation, such as γ-aminobutyric acid, ornithine, 2-aminobutyric acid, N-acetylglucosamine, succinic acid, and lactic acid, exemplified here. It was found that it can be prepared. The detection of γ-aminobutyric acid and ornithine in the fermentation extract placenta extract (A) is consistent with the results of free amino acid analysis.

<< Analysis Example 4: Bile acid binding activity >>
The bile acid binding activity of each of the dry powder (A) and the dry powder (D) was examined.
70 mg of each lyophilized powder was mixed with 1.4 mL of a 1.25 mM sodium taurocholate aqueous solution (10 mM phosphate buffer, pH 6.8) in a 1.5 mL microtube and incubated at 37 ° C. for 2.5 hours. After incubation, the mixture was centrifuged (12,000 rpm, 10 minutes, 4 ° C.), filtered through a 0.22 μm filter, and then a polymer fraction was obtained with a centrifugal ultrafiltration membrane having a molecular weight cut-off of 10,000. . The polymer fraction was washed 3 times with phosphate buffer, redissolved in 500 μL phosphate buffer, and the remaining sodium taurocholate concentration was determined using an enzyme reaction method (Wako Pure Chemicals, Bile Acid Test Wako). ).
The results are shown in Table 5. The concentration of bile acid remaining in the polymer fraction when treated with the placenta extract produced only with the prosthesis was not different from the control, but in the fermentation and protease-degraded placenta extract (D), the bile remaining in the polymer fraction The acid concentration was significantly higher.

<< Analytical Example 5: Evaluation of NASH Onset Suppressing Activity by Mouse Model >>
Materials and methods (materials)
A high cholesterol / high fat diet (hereinafter abbreviated as CL), which is a special feed for inducing NASH, was used. CL is made of oriental yeast (cocoa butter 40%, cholesterol 1.25%, sodium cholate 0.5%, CRF-1 58.25%), and based on this, the composition of the present invention is 0.1%. Alternatively, 0.3% was mixed and solidified.

(Animal test design)
After acclimating male 7-week-old C57BL / 6L mice (Japan SLC) for 1 week, normal diet group (NC, n = 8), CL diet group (CL, n = 10), CL diet + 0.1% placenta extract (CL + L-PL, n = 8) and CL diet + 0.3% placenta extract (CL + H-PL, n = 8) were divided into 4 groups, and each feed was freely ingested for 12 weeks.

(Evaluation item)
Body weight (up to 12 weeks after food intake) and food intake (first 8 days) were measured. After euthanasia 12 weeks after feeding, blood (plasma) and liver tissue were collected. The following items were measured using each sample.
Plasma: Liver function (alanine aminotransferase (ALT), aspartate aminotransferase (AST).
Liver tissue: lipids (triglycerides (TG), total cholesterol (TC), non-esterified fatty acids (NEFAs)), various gene expressions (measured by real-time PCR method; used primers are shown in Table 6).

(Statistical analysis)
Comparison between experimental groups was performed by t-test.

Results (weight change and food consumption)
FIG. 1 and FIG. 2 show the change in body weight and the average amount of food consumed per day, respectively. Regarding the change in body weight, the CL intake group tended to have a slower weight gain than the NC intake group regardless of the FP placenta extract combination, but there was no significant difference between the CL intake groups. .

  The average food intake was significantly less in the CL group than in the NC group. By blending FP placenta extract there, there was a tendency for decreased food intake to increase. The above results indicate that changes in food intake (so-called overeating) cannot explain the onset of the fatty liver state and steatohepatitis state described below.

(Measurement of AST and ALT using plasma)
The results of measuring AST and ALT in blood after ingesting various foods for 12 weeks are shown in FIG. Both AST and ALT increased significantly in the CL group compared to the NC group, indicating that hepatitis symptoms were caused. However, such changes were improved in a dose-dependent manner by adding FP placenta extract to CL.

(Liver fat status)
After ingesting various diets for 12 weeks, the liver was taken out, and the amounts of triglyceride (TG), total cholesterol (TC), and non-esterified fatty acid (NEFA) in the tissue were measured. The results are shown in FIG. In addition, representative liver surface images and hematoxylin / eosin-stained tissue section images of each group are shown in FIG.

  As shown in FIG. 4, it was found that in the CL group, all of TG, TC, and NEFA in the liver tissue were significantly increased as compared with the NC group. However, compounding FP placenta extract in CL improved these increases in a dose-dependent manner.

As shown in the upper part of FIG. 5, the above results can be seen from the appearance image of the liver. The NC group has a clean red liver surface color, while the CL group has a distinctly whitish color reflecting the fatty liver state. However, when FP placenta extract was blended with CL, this whiteness faded, and the 0.3% blended group exhibited a redness similar to that of the NC group.
These changes were also confirmed by microscopic observation of liver tissue sections (FIG. 5, bottom). After liver tissue was cut out and sliced, hematoxylin and eosin staining and microscopic examination revealed that white voids reflecting adipose tissue were hardly observed in the NC group, whereas many white voids were observed in the CL group. Was observed. However, this gap was drastically reduced by blending FP placenta extract with CL.

(Changes in the expression of various genes in the liver)
After ingesting various diets for 12 weeks, the liver was taken out and the expression of various genes in the tissues was examined by real-time PCR. FIG. 6 shows the results of gene expression related to lipid synthesis and triglyceride synthesis, FIG. 7 shows the results of gene expression related to fatty acid metabolism, and FIG. 8 shows the results of gene expression of inflammatory cytokines.

  In the CL group, among the genes involved in lipid and triglyceride synthesis, the expression of SREBP-1c and SCD1 was significantly increased, and FAS was also increasing (FIG. 6). However, when FP placenta extract was added to CL, their expression tended to decrease in a dose-dependent manner. On the other hand, the expression of PPARα and Cpt1, which are genes involved in fat metabolism, was significantly decreased in the CL group as compared to the NC group (FIG. 7). However, this decrease was recovered in a dose-dependent manner by adding FP placenta extract to CL. Furthermore, regarding the expression of inflammatory cytokine genes (F4 / 80, TNFα, IL-1β, and CD11c) measured as hepatitis indicators, it was found that all of them were elevated in the CL group (FIG. 8). However, these increases in expression were also reduced in a dose-dependent manner by adding FP placenta extract to CL.

  The above results indicate that the NASH state is induced by a high cholesterol diet (CL), and that the NASH state is markedly improved in a dose-dependent manner by adding FP placenta extract to CL. That is, it was suggested that FP placenta exhibits the effect of preventing the onset of NASH by oral ingestion.

  The composition produced by the production method of the present invention is effective for preventing or treating nonalcoholic steatohepatitis and / or nonalcoholic fatty liver disease.

Claims (8)

A method for producing a composition comprising the following four steps:
(1) A step of fermenting the placenta using a fermenting microorganism to obtain a placental fermentation decomposition product;
(2) a step of separating the placental fermentation degradation product into a fermentation extract placenta extract which is a soluble fraction and an insoluble fraction;
(3) A step of subjecting the insoluble fraction to protease treatment to obtain a protease-degraded extracted placenta extract, and (4) a step of mixing the fermentation-extracted placenta extract and the protease-degraded extracted placenta extract to obtain a composition.   The method of claim 1, wherein the composition is a pharmaceutical composition.   The method according to claim 1 or 2, wherein the composition is for prevention or treatment of non-alcoholic fatty liver disease.   4. The method of claim 3, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.   The method according to any one of claims 1 to 4, wherein the fermenting microorganism is one or more microorganisms selected from the group consisting of lactic acid bacteria, yeast, Bacillus subtilis, and koji molds.   6. The protease is one or more proteases selected from the group consisting of acidic protease, neutral protease, alkaline protease, pepsin, trypsin, chymotrypsin, elastase, collagenase, carboxypeptidase, and aminopeptidase. The method as described in any one of.   The composition manufactured by the manufacturing method as described in any one of Claims 1-6.   The composition according to claim 7, wherein the composition is a health food or a functional food.