JP5525511B2 - Drugs for non-alcoholic fatty liver disease and / or non-alcoholic steatohepatitis - Google Patents

Description

  The present invention relates to a therapeutic agent for non-alcoholic fatty liver disease and / or non-alcoholic steatohepatitis, comprising as an active ingredient a complex bacterium comprising photosynthetic bacteria, lactic acid bacteria and yeast.

  So-called metabolic syndrome means that even if individual risk factors are not severe, epidemiological findings that the risk of developing cardiovascular diseases such as arteriosclerosis is significantly increased by accumulation, hyperlipidemia, diabetes In addition, it is considered that effective management is a comprehensive management of these as risk factors. Currently, the number of individuals who develop metabolic syndrome is increasing due to changes in diet and lifestyle, and in many of them, fat accumulation in the liver is observed. Adipose tissue is closely related not only to fat accumulation but also to the expression of functions such as hormones, inflammation, and immune system. Therefore, in metabolic syndrome, adipose tissue dysfunction occurs with an increase in adipocytes, and the secretion of adiponectin, which is an adipokine, decreases, and conversely, the inflammatory cytokines TNF-α, IL-6, IL-8, MCP- The production of 1 increases, and as a result, the adipose tissue is in an inflammatory state.

  Currently, 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 alcohol, has been neglected as a benign disease but does not progress to hepatitis. The number of cases that have shifted 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. NASH is considered a phenotype in the liver of the metabolic syndrome described above. 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 pioglitazone, which is an agonist for improving insulin resistance, is a peroxisome proliferator-activated receptor gamma (hereinafter referred to as PPARγ). 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).

  Since NASH is a progressive disease, establishing a cure for it is urgent. Here, the animal protein is at least one lactic acid bacterium selected from the group of lactic acid bacteria belonging to any of the genus Streptococcus, Enterococcus, Leuconostoc, Lactococcus, Lactobacillus, and Pediococcus. An animal protein lactic acid fermented product obtained by lactic acid fermentation and having an activity of reducing the amount of lipid in the liver is disclosed (Patent Document 1). It has been found that a lactic acid fermented product obtained by fermenting fish meat and livestock meat with lactic acid bacteria has a bile acid binding ability.

  There is also a therapeutic agent for chronic hepatitis B containing lactoferrin as an active ingredient as a therapeutic agent for hepatitis (Patent Document 2). A mixture containing probiotics consisting of lactoferrin, microorganisms belonging to the genus Bifidobacterium, microorganisms belonging to the genus Lactobacillus and microorganisms belonging to the genus Streptococcus and oligosaccharides as prebiotics is temporarily relaxed. After an increase in alanine aminotransferase levels and confirmation of immune activation of the host, a therapeutic effect with a high efficacy rate for hepatitis B is derived, and there are no side effects on the treatment.

  On the other hand, there is an arteriosclerotic index lowering agent containing a photosynthetic bacterium as an active ingredient (Patent Document 3). It is a dry powder of photosynthetic bacteria with disrupted cell walls as an active ingredient and is said to have an excellent effect in preventing and improving arteriosclerosis. It is said that it is preferable to use a material obtained by crushing the cell wall because the components in the fungus body act more effectively. In the examples, Wistar male rats were bred on a normal diet for 3 days, then a modified diet supplemented with 1% cholesterol, and a lyophilized powder of photosynthetic bacteria added at 0.2% by weight or 2.0% by weight. When total cholesterol, TG, and the like were measured, it was found that the addition of 0.2 wt% and 2.0 wt% lowered the total cholesterol value and TG.

JP 2008-178398 A Japanese Patent No. 4087249 Special table 2008-56450 gazette

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

  In recent years, due to the criticism of the side effects of antibiotics (antibiotics) and the development of resistant bacteria born of antibiotics, the balance of the digestive system has been improved by ingesting useful bacteria such as lactic acid bacteria from food to prepare the body environment. Probiotics that can improve and reduce the incidence of illness are in the spotlight. For example, 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. For example, photosynthetic bacteria are widely distributed in nature, have the advantage of using sunlight as an energy source and photosynthesis using hydrogen and carbon dioxide as nutrients, and have an excellent ability to assimilate organic matter and are rich in amino acids and vitamins. .

  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 a microbial preparation having excellent safety when long-term administration is considered. However, there are still no probiotics that use microorganisms and are effective for NASH treatment.

  For example, the lactic acid fermented product used in Patent Document 1 has a bile acid-binding ability in a state that does not contain lactic acid bacteria, and has an effect of reducing the amount of neutral fat and cholesterol in the liver in rats and the like. In addition, it has the effect of reducing the content of neutral fat and cholesterol in the adipocytes in the liver. However, there is no description regarding the therapeutic effect of hepatitis.

  Patent Document 2 is for taking lactoferrin and Bifidobacterium longum powder together with lactoferrin. However, immunostimulation is observed by using Bifidobacterium longum fungus powder in combination with lactoferrin alone, not the hepatitis healing effect of Bifidobacterium longum powder itself. .

  Furthermore, in Patent Document 3, a freeze-dried product of photosynthetic bacteria is orally ingested and a decrease in total cholesterol level and TG is observed in the photosynthetic bacteria-administered group. In the photosynthetic bacteria-administered group, the amount of food consumed and the efficiency of feeding were reduced, and the direct effect of photosynthetic bacteria on liver function is unknown.

  In view of the above-described present situation, an object of the present invention is to provide a therapeutic agent for NAFLD and / or NASH, which comprises a complex bacterium composed of photosynthetic bacteria, lactic acid bacteria, and yeast as an active ingredient.

  An object of the present invention is to provide a therapeutic agent for NAFLD and / or NASH in the form of a solution, a gel, or a capsule.

  The present inventors have examined in detail a complex bacterium composed of photosynthetic bacteria, lactic acid bacteria, and yeast. As a result, the inventors have found that hepatitis can be improved and thus completed the present invention. Moreover, when the said therapeutic agent was administered to the db / db mouse which is a diabetes model animal with obesity, the gluconeogenesis inhibitory effect was confirmed.

That is, the present invention is a therapeutic agent for NAFLD and / or NASH containing a composite bacterium composed of a photosynthetic bacterium, a lactic acid bacterium, and a yeast as an active ingredient , wherein the photosynthetic bacterium is Rhodopseudomonas pultris, Lactobacillus casei and / or Lactobacillus plantarum, wherein the yeast is a Saccharomyces cerevisiae NAFLD and / or NASH therapeutic agent .

  ADVANTAGE OF THE INVENTION According to this invention, the therapeutic agent of NAFLD and / or NASH which use the composite microbe which consists of photosynthetic bacteria, lactic acid bacteria, and yeast as an active ingredient is provided.

(A) is the figure of the serum AST of Example 1, (B) is a figure which shows the result of serum ALT. (A) is the figure of liver TG of Example 1, (B) is the figure of the HE dyeing | staining image of a control group, (C) is a figure which shows the result of the HE dyeing | staining image of the therapeutic agent of manufacture example 1. FIG. (A) is the figure of liver PAI-1 of Example 1, (B) is a figure which shows the result of liver TNF (alpha). (A) is the figure of the fasting blood glucose of Example 2, (B) is a figure which shows the result of an insulin concentration. (A) is a figure which shows the total cholesterol value of Example 2, (B) is a figure which shows the result of serum TG. (A) is the figure of the serum AST of Example 2, (B) is a figure which shows the result of serum ALT. (A) is the figure of the liver pyruvate carboxylase of Example 2, (B) is a figure which shows the result of glucose 6-phosphatase.

A first aspect of the present invention is a therapeutic agent for NAFLD and / or NASH containing a composite bacterium comprising a photosynthetic bacterium, a lactic acid bacterium, and a yeast as an active ingredient , wherein the photosynthetic bacterium is Rhodopseudomonas pultris, Is Lactobacillus casei and / or Lactobacillus plantarum, and the yeast is a therapeutic agent for NAFLD and / or NASH, which are Saccharomyces cerevisiae . Most of the metabolic syndrome is fatty liver, and NAFLD and NASH are common to fatty liver. Also, many of the metabolic syndromes are common to NAFLD and NASH in that they have insulin resistance due to obesity, and therefore NASH is regarded as a phenotype in the liver of metabolic syndrome. Therefore, a drug capable of preventing and treating NAFLD and NASH means that it is a preventive and therapeutic agent for metabolic syndrome. In the present invention, when a complex bacterium containing a photosynthetic bacterium is administered to a mouse in which a NASH-like condition is induced by feeding a methionine / choline-deficient diet, a healing effect on hepatitis is observed. It is useful as a therapeutic agent for alcoholic steatohepatitis.

  The present invention is characterized in that a photosynthetic bacterium is used. This is because photosynthetic bacteria can be obtained at low cost, and as shown in Patent Document 3, serum TG can be lowered and is excellent in safety. However, Patent Document 3 uses a dry powder obtained by crushing the cell walls of photosynthetic bacteria, and there is no example in which photosynthetic bacteria are used as probiotics in a complete form or as a therapeutic agent for NASH. .

  The photosynthetic bacteria used in the therapeutic agent of the present invention is a general term for eubacteria that perform photosynthesis broadly, and includes red bacteria and red non-sulfur bacteria. Photosynthesis is carried out by cyanobacteria, red bacteria, green sulfur bacteria, green non-sulfur bacteria, and heliobacteria, and red bacteria are classified into red sulfur bacteria and red non-sulfur bacteria by nutritional classification. Crimson bacteria do not generate oxygen among photosynthetic bacteria, and appear red or brown due to the accumulation of carotenoids. In the present invention, red non-sulfur bacteria can be preferably used. Preferred is Rhodopseudomonas palustris. More preferably, as the photosynthetic bacterium used in the present invention, it is described in the trust certificate (Notification Date December 5, 2011, Notification No. 11-309) of the National Institute of Technology and Evaluation Microorganisms. As mentioned above, it is Rhodopseudomonas pallistris TPR001 strain (hereinafter simply referred to as NITE P-1166) deposited as deposit number NITE P-1166 based on receipt number NITE AP-1166 deposited on November 24, 2011. This is because it is facultative anaerobic and can grow under anaerobic and aerobic conditions. Note that synthesis of photosynthetic pigments and proteins involved in photosynthesis is not performed under aerobic conditions, and synthesis is not started until the growth environment is anaerobic.

  Examples of lactic acid bacteria that can be incorporated into the therapeutic agent of the present invention include Lactobacillus, Bifidobacterium, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, Streptococcus, and the like. . In the present invention, lactic acid bacteria such as Lactobacillus, Streptococcus, and Leuconostoc are particularly preferable because they can assimilate saccharides.

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.

  In the therapeutic agent of the present invention, a plurality of lactic acid bacteria can be used in combination. The number of lactic acid bacteria to be used is 0.5 to 1000 times lactic acid bacteria, more preferably 1 to 100 times lactic acid bacteria with respect to one cell of photosynthetic bacteria. This is because the healing effect of NAFLD and NASH is excellent within this range.

Examples of yeast that can be incorporated into the therapeutic agent of the present invention include the genus Saccharomyces, Candida, Tolropsis, Digosaccharomyces, Schizosaccharomyces, Debaryomyces, Geotrichum, Wickelhamia, and Ferromyces. 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. Yeast contains proteolytic enzymes, can break down proteins into peptides and amino acids, and can produce vitamins and amino acids from the sugars that yeast ingests.
In the therapeutic agent of the present invention, 0.01 to 100 times of yeast, more preferably 0.1 to 10 times of yeast is blended with respect to one cell of photosynthetic bacteria.

  The therapeutic agent of the present invention may further include koji molds, Bacillus subtilis, filamentous fungi and the like as long as the effects of the present invention are not impaired.

The therapeutic agent of this invention uses the said microbe in a living microbe state. Since all the above-mentioned bacteria are marketed, this can be mixed and the complex microbe used by this invention can be prepared. For example, the above-mentioned photosynthetic bacteria, lactic acid bacteria, and yeast are separately precultured in a culture solution containing dairy products, black honey, yeast extract, and the like, or in a YM medium and MRS medium, and the bacterial concentration in the culture liquid is measured. For a photosynthetic bacterium, a lactic acid bacterium and yeast are mixed so as to be in the above range to prepare a complex bacterial solution. This complex bacterial solution is concentrated by centrifugation or filtration as necessary, and adjusted to a concentrated complex bacterial solution having 1 × 10 ⁷ to 1 × 10 ¹² bacteria per ml.
This complex bacterial solution or concentrated complex bacterial solution can be used as it is as a therapeutic agent for NAFLD or NASH, but may be formulated by adding other dispersion medium or the like. In addition, the above-mentioned preculture liquid and concentrated complex bacteria can be preserve | saved by freezing or freeze-drying.

  The therapeutic agent of the present invention may be a dried product as long as the complex bacterium used is a living bacterium. Therefore, a powder obtained by freeze-drying the above complex bacterial solution or concentrated complex bacterial solution can be used as the therapeutic agent of the present invention. Such powders can be added with taste-masking agents, flavoring agents and the like as long as the effects of the present invention are not impaired. Further, excipients such as starch and lactose can be added to such powders to form powdered powders, tableted into tablets, or granulated into granules to form granules. Furthermore, the above powder can be filled into a capsule to form a capsule.

  A syrup or jelly agent can be prepared by adding a taste-masking or flavoring agent to the complex or concentrated complex. It can be suitably used for infants and elderly people who have difficulty swallowing.

The therapeutic agent of the present invention is only required to be administered into the digestive organs, and may be introduced into the digestive organs such as the stomach and intestine by oral administration in addition to oral administration. The dose can be appropriately selected depending on the composition of the above complex bacteria, but the number of photosynthetic bacteria contained in the therapeutic agent of the present invention is 1 × 10 ⁵ to 1 × 10 ¹⁵ cells / day, more preferably 1 × 10 ⁶ to 1 × 10. ^The dose is ¹² / day.

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

  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 photosynthetic bacterium (Rhodopseudomonas palustris: NITE P-1166) was cultured in a medium containing 10 g of polypeptone, 2 g of yeast extract, 1 g of MgSO ₄ .7H ₂ O, and 1 liter of distilled water at 27 ° C. under irradiation of 5000 lux fluorescent light. Pre-cultured for a week under static anaerobic conditions.
As lactic acid bacteria, lactic acid bacteria (Lactobacillus casei: IFO (NBRC) 15883) and lactic acid bacteria (Lactobacillus plantarum: ATCC 8014) were separately pre-cultured in MRS medium at 37 ° C. for 3 days under static anaerobic conditions. .
In addition, yeast (Saccharomyces cerevisiae: ATCC No. 11909) was precultured in YM medium at 330 ° C. for 3 days under static anaerobic conditions.
Each of the bacteria pre-cultured as described above was mixed in a microtube so that the total amount was 1.2 ml and the number of bacteria in Table 1 per 200 μL.
Thereafter, centrifugation was performed at 7000 rpm for 15 minutes, 0.9 ml of the supernatant was removed, and then the same amount of dispersion medium (Morinaga skim milk 20% w / v) as the removed liquid medium was added to make 1.2 ml. This was used as a therapeutic agent.

Example 1
8-week-old C57BL6 / N male mice are divided into 2 groups, methionine / choline-deficient diet and dispersion medium (Morinaga Milk Industry, Morinaga skim milk 20% (w / v)), methionine / choline-deficient diet and production example 1 Each of the therapeutic agents was ingested for 12 weeks.
After administration, liver was collected and liver TG was measured. 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 subjected to serum AST, serum ALT, serum TG, and liver tissue immunohistochemical staining. The results of serum AST and serum ALT are shown in FIG. Further, the results of liver TG and HE stained images are shown in FIG. Furthermore, RNA and protein were extracted from liver cells, and changes in various parameters such as adipocytokine and cytokine were quantitatively evaluated using a real-time PCR method. The results of liver PAI-1 and liver TNFα are shown in FIG.

(result)
As shown in FIG. 1 (A) and FIG. 1 (B), when the therapeutic agent of Production Example 1 is administered to a group of mice fed with a methionine / choline-deficient diet and induced a NASH-like disease state, serum AST and serum ALT However, liver function was improved.

  As shown in FIG. 2 (A), the amount of liver TG was 115 IU / L in the group of mice induced with a methionine / choline-deficient diet to induce NASH-like pathology, but in mice that induced NASH-like pathology. The group to which the therapeutic agent of Production Example 1 was administered was about 90 IU / L. By administration of the therapeutic agent of the present invention, liver TG could be reduced to 78%, and a tendency to decrease the triglyceride content was observed.

  Furthermore, as shown in FIGS. 2 (B) and 2 (C), an improvement tendency of the liver pathology was also observed in the examination of the liver histology. That is, from the results of HE staining shown in FIG. 2 (B), in mice in which NASH-like pathology was induced by feeding a methionine / choline-deficient diet, adipocytes were scattered, but when the therapeutic agent of Production Example 1 was administered As shown in FIG. 2 (C), the number of fat cells and the size of fat cells were also reduced in the group of mice in which the NASH-like pathological condition was induced.

  As shown in FIGS. 3 (A) and 3 (B), hepatic PAI-1 and liver TNFα, which are inflammatory cytokines, were administered with the therapeutic agent of Production Example 1 from a group of mice in which a NASH-like disease state was induced. There was a significant difference in the group. It is suggested that administration of the therapeutic agent of Production Example 1 suppresses the production of proinflammatory cytokines such as PAI-1 and TNFα in the liver, thereby exerting a liver protective action.

(Example 2)
Seven-week-old female, leptin receptor-deficient mice (db / db) were divided into two groups, one with a dispersion medium (Morinaga Milk Products, Morinaga skim milk 20% (w / v)) and the other with Production Example 1 Was administered for 4 weeks.
After administration, blood was collected and liver was collected after an overnight fast. Serum and tissue samples were stored at −80 ° C., and fasting blood glucose, insulin concentration, total cholesterol level, serum TG, serum AST, and serum ALT were performed. The results of fasting blood glucose and insulin concentration are shown in FIG. Moreover, the result of a total cholesterol level and serum TG is shown in FIG. The results of serum AST and serum ALT are shown in FIG. Furthermore, RNA and protein were extracted from liver cells, and changes in various parameters such as adipocytokines and cytokines were quantitatively evaluated using real-time PCR, Western blotting, and immunoprecipitation. The results for liver pyruvate carboxylase and glucose 6-phosphatase are shown in FIG.

(result)
As shown in FIG. 4 (A), even when the therapeutic agent of Production Example 1 was administered, there was no change in the fasting blood glucose level. However, as shown in FIG. 4B, the serum insulin level decreased, suggesting an improvement in insulin resistance. As shown in FIGS. 5 (A) and 5 (B), the total cholesterol level and TG level do not change, but as shown in FIGS. 6 (A) and 6 (B), the serum AST level and ALT level The value is somewhat reduced. Furthermore, as shown in FIGS. 7A and 7B, the expression levels of hepatic pyruvate carboxylase and glucose 6-phosphatase, which are enzymes relating to gluconeogenesis, are reduced. Although the mechanism of action of the therapeutic agent of the present invention is not clear, it is presumed that reduction of the expression level of an enzyme involved in gluconeogenesis is also involved.

  The present invention uses photosynthetic bacteria, lactic acid bacteria, and yeast as probiotics, and is useful as a therapeutic agent for NAFLD and NASH.

Claims (1)

A therapeutic agent for non-alcoholic fatty liver disease and / or non-alcoholic steatohepatitis, comprising a composite bacterium composed of photosynthetic bacteria, lactic acid bacteria and yeast as active ingredients,
The photosynthetic bacterium is Rhodopseudomonas pultris,
The lactic acid bacterium is Lactobacillus casei and / or Lactobacillus plantarum,
The said yeast is Saccharomyces cerevisiae, The therapeutic agent of nonalcoholic fatty liver disease and / or nonalcoholic steatohepatitis.