Foods that prevent non-alcoholic steatohepatitis

Abstract

Currently, there is no established treatment strategy to prevent progression to non-alcoholic steatohepatitis (NASH / MASH) in individuals diagnosed with non-alcoholic fatty liver disease (NAFL). Therefore, this report provides a preventative measure. [Solution] This invention provides brown rice and rice bran koji, and by having individuals diagnosed with non-alcoholic fatty liver disease (NAFL) consume food containing brown rice and rice bran koji (FBRA), it is possible to prevent progression to non-alcoholic steatohepatitis (NASH / MASH).

Description

Technical Field

[0001] The present invention relates to a food for preventing non-alcoholic steatohepatitis, and particularly to brown rice and rice bran koji (FBRA) that enables a person diagnosed with non-alcoholic fatty liver (NAFL) to prevent progression to non-alcoholic steatohepatitis (NASH).

Background Art

[0002] Non-alcoholic fatty liver (NAFL) refers to a disease in which fat accumulates in hepatocytes due to overeating or unbalanced diet without alcohol, and if it becomes chronic, it may progress to non-alcoholic steatohepatitis (NASH), and further progress in a chain to liver cirrhosis and hepatocellular carcinoma. NAFL and NASH together are called non-alcoholic fatty liver disease (NAFLD).

[0003] In Japan, the morbidity rate of NAFLD has been increasing against the background of the increase in the obese population, from 18% in 2001 to 29.7% in 2010. Although the change in the morbidity rate of NASH has not been published, it is considered to be increasing in parallel with the morbidity rate of NAFLD. The incidence of liver cancer from NAFLD is reported to be 0.43 - 0.44 per 1000 person-years, and from NASH is 5.46 per 1000 person-years. In 2023, the Japanese Liver Society etc. changed non-alcoholic steatohepatitis from NASH: Nonalcoholic steatohepatitis / NAFLD: Nonalcoholic fatty liver disease to MASH: Metabolic dysfunction associated steatohepatitis / MAFLD: Metabolic dysfunction associated liver disease. Therefore, in the specification of this application, non-alcoholic steatohepatitis is denoted as NASH / MASH.

[0004] Fermented brown rice and rice bran with Aspergillus oryzae (FBRA) is a food product made by fermenting brown rice and rice bran with Aspergillus oryzae. Rice bran contains many beneficial components such as vitamins, ferulic acid, minerals, phytic acid, and dietary fiber. A method for producing FBRA is disclosed, for example, in Patent Document 1. Specifically, the process involves milling brown rice to a milling ratio of 96% or more using a rice milling machine, adding water to the milled brown rice, separately creating a hydrated mixture of rice bran and a calcium compound using a mixer, mixing the hydrated milled brown rice with the hydrated mixture and steaming it, inoculating it with koji starter and allowing it to germinate in a culture bed, and stirring the substrate to release heat during fermentation and maintain an appropriate temperature. The matured koji is then dried at the substrate's temperature to a moisture content of 4% or less, and powdered while maintaining enzyme activity to produce brown rice and rice bran koji (FBRA).

[0005] Numerous studies have been conducted on the efficacy of brown rice and rice bran koji (FBRA), and it has been shown that feeding FBRA to disease model mice has cancer-preventive, anti-inflammatory, and anti-inflammatory carcinogenic effects. For example, Non-Patent Document 1 studied the modifying effect of dietary intake of FBRA on N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN)-induced bladder carcinogenesis in male ICR mice. The group fed a diet containing FBRA had a significantly lower incidence of cancer than the group fed a diet without FBRA. This indicates that FBRA is a potent anti-carcinogenic compound and exerts a chemopreventive effect against chemically induced bladder carcinogenesis through an antiproliferative mechanism.

[0006] For example, Non-Patent Document 2 studied the inhibitory effect of brown rice and rice bran ferment (FBRA) on the induction of acute colitis by dextran sulfate sodium (DSS) in rats. When rats were fed a diet containing FBRA, the ulcer and erosion areas in the colon of rats stained with Alcian blue were significantly reduced. In addition, the ulcer index (the ratio of the total length of ulcers to the total length of the colon) and the colitis score, as determined by macroscopic observation, decreased. Furthermore, myeloperoxidase activity in the colonic mucosa also decreased. These results indicate that FBRA has an effect of suppressing the induction of colitis by DSS.

[0007] For example, Non-Patent Document 3 established an inflammation-related carcinogenesis model in mice. Specifically, when degenerative QR-32 cells were transplanted subcutaneously along with a foreign material, gelatin sponge, massive infiltration of inflammatory cells into the sponge led to the formation of a lethal tumor. When mice were fed a diet containing brown rice and rice bran ferment (FBRA) (5% or 10%), the tumor incidence in the diet group was lower than in the untreated group (70%) (35% and 20%, respectively), indicating that FBRA reduced the number of inflammatory cells infiltrating the sponge. This suggests that FBRA acts by inhibiting the infiltration of inflammatory cells into inflammatory lesions, making it an effective chemopreventive agent against inflammation-related carcinogenesis. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Patent No. 6420083

[0009] [Non-Patent Document 1] TOSHIYA KUNO et al. "Chemological prevention of bladder carcinogenesis in mice with fermented brown rice and rice bran." ONCOLOGY REPORTS 15, 2006, pp. 533-538. [Non-Patent Document 2] Keiko Kataoka et al. "Inhibitory effect of fermented brown rice on the induction of acute colitis induced by dextran sulfate sodium in rats." Dig Dis Sci 53, 2006, pp. 1601-1608. [Non-Patent Document 3] Kunishige Onuma et al. "Fermented brown rice and rice bran (FBRA) suppress inflammation-related carcinogenesis in mice by inhibiting inflammatory cell infiltration." Nutrients 2015, Vol. 7, pp. 10237-10250. [Overview of the project] [Problems that the invention aims to solve]

[0010] However, to date, there has been no research whatsoever on whether brown rice / rice bran koji (FBRA) suppresses the development of non-alcoholic steatohepatitis (NASH / MASH). While non-alcoholic fatty liver (NAFL) can potentially return to a normal liver state through lifestyle changes and exercise, it becomes extremely difficult to return to a normal liver state once it progresses to non-alcoholic steatohepatitis (NASH / MASH). Therefore, there has been a need for measures to prevent progression to NASH / MASH or to treat NASH / MASH. However, even now, no effective measures to prevent progression to NASH / MASH or to treat NASH / MASH have been established.

[0011] Currently, preventative measures against progression to NASH / MASH include lifestyle modifications, drug therapy, and iron-reducing therapy. Lifestyle modifications involve correcting underlying conditions such as obesity, diabetes, dyslipidemia, and hypertension through dietary and exercise therapy. Drug therapy involves treatment with antioxidants such as vitamin C and vitamin E, thiazolidinediones, biguanides, diabetes medications such as sitagliptin, fibrates, and lipid metabolism disorder medications such as ezetimibe and epadel to prevent progression to cirrhosis and liver cancer. Iron-reducing therapy involves reducing iron in the body, as excess iron burdens the liver. This can be achieved through methods such as phlebotomy (regularly removing a certain amount of blood) or a low-iron diet. However, at present, there is no established effective method to prevent progression to NASH / MASH. [Means for solving the problem]

[0012] Therefore, since NASH / MASH develops as a result of chronic nasolabial flaccid (NAFL) / masolabial flaccid (MAFL) causing inflammation, we hypothesized that consuming FBRA, which has been shown to have anti-inflammatory and anti-inflammatory carcinogenic effects, could reduce inflammation and prevent progression to NASH / MASH.

[0013] The specific details of this invention are as follows: (1) This brown rice and rice bran malt product is characterized by preventing the progression to non-alcoholic steatohepatitis (NASH / MASH) when consumed by individuals diagnosed with non-alcoholic fatty liver disease (NAFL / MAFL).

[0014] (2) A brown rice and rice bran malt product characterized in that, when consumed by a person diagnosed with non-alcoholic fatty liver disease (NAFLD / MAFLD), the progression of the said non-alcoholic fatty liver disease (NAFLD / MAFLD) is suppressed.

[0015] (3) The brown rice and rice bran koji described in item (1) or (2) above, characterized in that the content of the brown rice and rice bran koji (FBRA) in the food containing the brown rice and rice bran koji (FBRA) is 5% by weight to 100% by weight.

[0016] (4) In item (1) or (2) above, the brown rice / rice bran koji (FBRA) is provided in the form of a powder, granules, tablets, or solid.

[0017] (5) A method for creating diseased mice is provided in order to develop therapeutic responses to prevent progression to non-alcoholic steatohepatitis (NASH / MASH) in individuals diagnosed with non-alcoholic fatty liver disease (NAFL), characterized by feeding leptin receptor-deficient mice (db / db mice) a high-fat and high-cholesterol diet (HFHCD) to create NAFL / MAFL or NASH / MASH diseased mice. [Effects of the Invention]

[0018] According to the present invention, the following effects can be achieved. According to the invention described in (1) above, there is currently no established method for preventing progression to non-alcoholic steatohepatitis (NASH / MASH) or for treating NASH / MASH in individuals diagnosed with non-alcoholic fatty liver disease (NAFL / MAFL). Therefore, consuming foods containing brown rice and rice bran ferment (FBRA) can have the advantageous effect of preventing progression to non-alcoholic steatohepatitis (NASH / MASH).

[0019] According to the invention described in (2) above, there is currently no established method for suppressing the progression of non-alcoholic fatty liver disease (NAFLD / MAFLD) in individuals diagnosed with NAFLD / MAFLD. Therefore, by consuming foods containing brown rice and rice bran koji (FBRA), it is possible to achieve the advantageous effect of suppressing the progression of non-alcoholic fatty liver disease (NAFLD).

[0020] According to the invention described in (3) above, for a person corresponding to (1) or (2) above, as a measure to prevent progression to non-alcoholic steatohepatitis (NASH / MASH) or suppress the progression of non-alcoholic fatty liver disease (NAFLD), by indicating an effective content rate of brown rice and rice bran koji (FBRA) in a food containing brown rice and rice bran koji (FBRA), the brown rice and rice bran koji (FBRA) can be used without waste.

[0021] According to the invention described in (4) above, when a person corresponding to (1) or (2) above ingests a food containing brown rice and rice bran koji (FBRA) as a measure, by providing the brown rice and rice bran koji in powder, or granules, or tablets, or in solid form, it is possible to achieve the effect that it becomes easy to prepare a food containing brown rice and rice bran koji (FBRA).

[0022] According to the invention described in (5) above, by establishing a method for creating NAFL or NASH / MASH pathological state mice, it is possible to achieve an advantageous effect that the effect of brown rice and rice bran koji (FBRA) can be verified more easily and effectively.

Brief Description of Drawings

[0023] [Figure 1] It is a diagram showing an experimental protocol including the composition of various mice, the content of food intake, and the inspection process. [Figure 2] It is a diagram showing the change in the liver weight ratio along the experimental protocol of FIG. 1. [Figure 3] It is a diagram showing the pathological tissue image of the liver by hematoxylin and eosin (HE) staining along the experimental protocol of FIG. 1. [Figure 4] It is a diagram showing the pathological tissue image of the liver by azan staining observing the fibrosis of the liver along the experimental protocol of FIG. 1. [Figure 5] It is a table showing a clinical evaluation measure for scoring the degree of progression of NAFLD / MAFLD by observing pathological specimens of the liver. [Figure 6]This table shows the evaluation of pathological specimens of various mice based on Figure 5. [Figure 7] This diagram shows the results of a blood biochemistry test using serum ALT levels. [Figure 8] This diagram shows the results of blood biochemistry tests using serum AST. [Modes for carrying out the invention]

[0024] The present invention will be described below based on examples using disease model mice. Non-alcoholic steatohepatitis (NASH / MASH) develops when non-alcoholic fatty liver disease (NAFL) becomes chronic and causes inflammation. On the other hand, many studies have been conducted on the efficacy of brown rice and rice bran ferment (FBRA), and it has been shown that feeding FBRA to disease model mice has cancer-preventive, anti-inflammatory, and anti-inflammatory carcinogenic effects. Therefore, we hypothesized that feeding FBRA, which has been shown to have anti-inflammatory and anti-inflammatory carcinogenic effects, could reduce inflammation and prevent progression to NASH / MASH.

[0025] Figure 1 shows the composition of various mice, their diet, and the experimental protocol including the testing process. As a NASH / MASH disease model, BKS.Cg-+ Lepr is a 6-week-old leptin receptor-deficient mouse that develops obesity and type 2 diabetes due to overeating. db / + Lepr db / Jcl (i.e., db / db mice; all experiments used were female mice) were fed a high-fat and high-cholesterol diet (HFHCD). The high-fat and high-cholesterol diet used in this experiment contained 45% fat and 1% cholesterol by weight. This high-fat and high-cholesterol diet is called the "Control diet (HFHCD)". HFHCD was fed to db / db mice to induce NAFL and subsequent NASH / MASH disease. Leptin is a hormone that controls appetite. Leptin, secreted from adipocytes, plays a role in maintaining a healthy body weight. Therefore, leptin is also called the "appetite-suppressing hormone" and the "anti-obesity hormone".

[0026] The experimental groups are classified into three groups according to the type of diet. The first is the "control group," in which 6-week-old db / db mice are fed only HFHCD; the second is the "5%FBRA group," in which 6-week-old db / db mice are fed a diet supplemented with 5% by weight of FBRA added to HFHCD; and the third is the "10%FBRA group," in which 6-week-old db / db mice are fed a diet supplemented with 10% by weight of FBRA added to HFHCD.

[0027] On the other hand, normal mice that did not overeat (C57BL / 6) were used as a control group. All female mice were used in the experiment. The control groups were classified into two groups according to the type of diet. The first was the "HFHCD control group," in which 6-week-old normal mice were fed only HFHCD, and the second was the "10% FBRA control group," in which 6-week-old normal mice were fed a diet supplemented with HFHCD and 10% by weight of FBRA.

[0028] Furthermore, for individuals diagnosed with non-alcoholic fatty liver disease (NAFLD / MAFLD), the amount of brown rice and rice bran fermented into foods containing FBRA can be between 5% and 100% by weight. In other words, FBRA can be consumed as is. FBRA is a food product made by fermenting brown rice and rice bran with Aspergillus oryzae, and it is commonly consumed as is.

[0029] Mice were raised with free feeding and free watering. As indicated by the circles in Figure 1, blood biochemistry tests and liver pathology specimens were prepared for the "control group" and "10% FBRA group" at weeks 4 and 12 after the start of the experiment, and blood biochemistry tests and liver pathology specimens were prepared for all "experimental groups" and "comparative control groups" at week 20 after the start of the experiment.

[0030] Pathological specimens are prepared as follows: First, a living mouse is anesthetized, the abdomen is opened, and then all blood is collected from the inferior vena cava (at this stage, the mouse will bleed to death), and the cervical vertebrae are dislocated. After that, the entire liver is removed, weighed, and a portion is fixed with formalin to prepare the pathological specimen. Blood biochemistry tests analyze various components of the collected blood to determine which part of the body is diseased, whether there is inflammation, etc. Details of blood biochemistry tests are explained in Figures 7 and 8.

[0031] Figure 2 shows the changes in liver weight ratio in mice according to the experimental protocol in Figure 1. Liver pathology specimens were prepared for the "control group" and the "10% FBRA group" at weeks 4 and 12 after the start of the experiment, and for all "experimental groups" and the "comparison group" at week 20 after the start of the experiment. At weeks 4 and 12 after the start of the experiment, the "10% FBRA group" was compared with the "control group". At week 4, the liver weight ratio (g / 100g body weight) of the "control group" was 5.69 ± 0.96, while the liver weight ratio of the "10% FBRA group" was 4.68 ± 0.67. The liver weight ratio of the "10% FBRA group" was lower than that of the "control group," indicating that the increase in liver weight due to fatty liver was suppressed by FBRA intake. When the liver weight ratio of the "10% FBRA group" was compared to the liver weight ratio of the "control group" using Student's t-test, the result was p<0.05, indicating a statistically significant difference. This suggests that if the same experiment were performed 20 times, there might be one instance where the data shows no difference, indicating that the difference is not merely a matter of chance but has a meaningful significance.

[0032] Similarly, at week 12, the liver weight ratio in the "10% FBRA group" was 4.75 ± 1.34 compared to 6.76 ± 1.04 in the "control group," indicating that the liver weight ratio in the "10% FBRA group" was lower than that of the "control group," with Student's t-test showing p < 0.01. Furthermore, at week 20, the liver weight ratio in the "5% FBRA group" was 7.42 ± 1.02 and in the "10% FBRA group" was 5.47 ± 1.86, compared to 8.03 ± 1.45 in the "control group." From these results, it can be seen that the liver weight ratio (g / 100g body weight) in the "5% FBRA group" and the "10% FBRA group" was lower than that of the "control group," indicating that the increase in liver weight due to fatty liver was suppressed by FBRA intake. No difference in liver weight ratio was observed in the "HFHCD comparison control group" and the "10% FBRA comparison control group," which are normal mice.

[0033] Figure 3 shows the histopathological images of the liver stained with HE according to the experimental protocol in Figure 1. In the "control group," fat accumulation was observed within hepatocytes in the liver pathology specimens (HE stained) (corresponding to the white areas in Figure 3 for the "control group"). On the other hand, in the "10% FBRA group," the fat droplets remained small at all stages throughout the experimental period, whereas in the "control group," as time progressed, the fat droplets became larger, and many cells became deformed, exhibiting a balloon-like appearance. Hematoxylin and eosin staining (HE staining) is a basic staining method necessary for histopathological diagnosis.

[0034] Figure 4 shows the histopathological image of the liver using Azan staining, observing liver fibrosis according to the experimental protocol in Figure 1. In the pathological specimens of liver fibrosis, the "10% FBRA group" showed some collagen (corresponding to the blue-stained areas; in Figure 4, although it is actually the "blue-stained area," it is shown as "dark black" due to the color constraints of the drawings in the patent application) from week 12, but it had not progressed significantly even at week 20. In contrast, the "control group" also showed collagen from week 12, and at week 20, it was observed that it had strongly cross-linked and fibrotic. Liver fibrosis refers to scarring, or wounds, that occur due to chronic damage or continued inflammation of the liver. As this fibrosis progresses, the liver gradually hardens and liver function declines, and in cases of cirrhosis or liver failure, fibrosis becomes severe. Azan staining is a staining method that distinguishes collagen fibers from muscle fibers, and it is a staining method that stains collagen fibers in fibrous connective tissue with aniline blue.

[0035] Figure 5 shows a table illustrating a clinical assessment method for scoring the progression of NAFLD based on observation of liver pathology specimens. Reported by Kleiner et al. in the journal Hepatology in 2005, the assessment items include "degree of steatosis," "degree of intralobular inflammation," "frequency of balloon-like hepatocytes," and "degree of fibrosis," with scoring based on the degree of each item. In other words, it is a clinical assessment method for scoring the progression of NAFLD and the progression to NASH / MASH based on observation of liver pathology specimens.

[0036] Figure 6 is a table evaluating pathological specimens of various mice using Figure 5. Although the scores increased over time, at week 4, the NAFLD score for the "10% FBRA" group was 1.00 ± 0.50, compared to 2.75 ± 0.96 for the "control group." This indicates that the NAFLD score for the "10% FBRA" group was lower than that of the "control group," suggesting that the progression to NASH / MASH was suppressed by FBRA intake. Student's t-test yielded a value of p<0.005, indicating a very significant difference.

[0037] In Figure 6, at week 12, the NAFLD score for the "control group" was 5.86±1.27, while the "10% FBRA group" showed an NAFLD score of 3.75±1.64, with Student's t-test yielding a p<0.05 result. Furthermore, at week 20, the NAFLD score for the "control group" was 8.38±0.86, while the "5% FBRA group" showed an NAFLD score of 6.50±0.86 and the "10% FBRA group" showed an NAFLD score of 4.50±2.06, with Student's t-test yielding a p<0.01 result. The NAFLD score results at week 12 and week 20 also indicate that the progression to NASH / MASH is suppressed by FBRA intake.

[0038] Figure 7 shows the results of blood biochemistry tests, specifically serum ALT levels. Serum ALT (alanine aminotransferase), a liver injury marker, is an enzyme found in the liver and is most abundant there. Therefore, if there is any liver damage, ALT leaks into the bloodstream. In other words, it is one indicator of the degree of inflammation in the liver. At every stage during the experiment, the "10% FBRA group" or the "5% FBRA group" showed significantly lower levels or a tendency toward lower levels compared to the "control group," indicating that FBRA intake suppresses liver cell damage. In normal mice, there was no difference in levels between the "HFHCD control group" and the "10% FBRA control group," both of which showed low levels.

[0039] Similarly, Figure 8 shows the results of blood biochemistry tests using serum AST. Serum AST (aspartate aminotransferase), a liver injury marker, is found in large quantities in the liver. Therefore, when liver cells are damaged, AST is released into the bloodstream. At every stage throughout the experiment, the "10% FBRA group" or the "5% FBRA group" showed significantly lower or lower levels compared to the "control group," indicating that FBRA intake suppresses liver cell damage. In normal mice, there was no difference between the "HFHCD control group" and the "10% FBRA control group," as both showed normal values.

[0040] From the above, in mouse experiments using a NASH / MASH disease model, feeding brown rice and rice bran koji (FBRA) resulted in significantly lower liver weight ratios, significantly lower NAFLD scores, and milder progression to steatohepatitis in the 10% FBRA and 5% FBRA groups compared to the HFHCD control group. Blood biochemistry tests showed significantly lower serum ALT and serum AST levels. These results clearly demonstrate that suppressing fat accumulation in the liver and reducing the resulting inflammation protects hepatocytes and prevents progression to NASH / MASH. The present invention provides a solution to prevent the progression to NASH / MASH, which has been a challenge until now. It is expected that daily intake of brown rice and rice bran koji (FBRA) can prevent progression to NASH / MASH.

Claims

1. This brown rice and rice bran fermented rice product is characterized by its ability to prevent progression to non-alcoholic steatohepatitis (NASH / MASH) in individuals diagnosed with non-alcoholic fatty liver disease (NAFL) by consuming foods containing brown rice and rice bran fermented rice (FBRA).

2. Brown rice and rice bran koji (FBRA) is characterized in that, when consumed by a person diagnosed with non-alcoholic fatty liver disease (NAFLD / MAFLD), it suppresses the progression of said non-alcoholic fatty liver disease (NAFLD / MAFLD).

3. The brown rice / rice bran koji according to claim 1 or 2, characterized in that the content of the brown rice / rice bran koji (FBRA) in the food containing the brown rice / rice bran koji (FBRA) is 5% by weight to 100% by weight.

4. The brown rice and rice bran koji (FBRA) described above is provided in the form of a powder, granules, tablets, or solid, as described in claim 1 or 2.

5. A method for creating diseased mice is described, characterized by feeding leptin receptor-deficient mice (db / db mice) a high-fat and high-cholesterol diet (HFHCD) to create mice with NAFL or NASH / MASH disease, in order to develop therapeutic strategies to prevent progression to non-alcoholic steatohepatitis (NASH / MASH) in individuals diagnosed with non-alcoholic fatty liver disease (NAFL).

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