WO2005072763A1

WO2005072763A1 - Liver fibrosis inhibitor

Info

Publication number: WO2005072763A1
Application number: PCT/JP2004/018606
Authority: WO
Inventors: Kiyohito Yagi; Masaya Kawase; Aiko Watanabe; Makoto Tamasada
Original assignee: Kobayashi Pharmaceutical Co., Ltd.; Nagaoka, Hitoshi
Priority date: 2004-01-30
Filing date: 2004-12-14
Publication date: 2005-08-11
Also published as: JP2005239699A

Abstract

[PROBLEMS] To provide a preventive means or a therapeutic means efficacious against liver fibrosis and to provide a liver fibrosis inhibitor, a medicinal composition, a food and a drink usable as the therapeutic means. [MEANS FOR SOLVING PROBLEMS] It is intended to provide a liver fibrosis inhibitor which contains a shiitake mushroom mycelium extract or a fraction thereof. It is also intended to provide a liver protecting agent having an effect of inhibiting liver fibrosis which contains a shiitake mushroom mycelium extract or a fraction thereof. It is also intended to provide a medicinal composition, a food, a drink and so on containing the above-described fraction.

Description

Specification

Liver fibrosis inhibitor

Technical field

The present application relates to a hepatoprotective agent, particularly a hepatic fibrosis inhibitor, comprising a shiitake mycelium extract or a fraction thereof. Furthermore, the present invention relates to a composition for oral ingestion, a pharmaceutical composition, a food composition and the like containing the hepatic fibrosis inhibitor.

Background art

[0002] Progression of liver fibrosis is observed in liver diseases such as acute hepatitis, chronic hepatitis, and cirrhosis. Liver fibrosis is caused by the deposition of extracellular matrix and is one of the causes of the transition from chronic hepatitis to cirrhosis.

[0003] The production of extracellular matrix in liver fibrosis involves the activation of hepatic stellate cells (Ito cells, also referred to as fat intake cells). That is, the hepatic stellate cells are activated by various site forces produced by the necrosis of hepatocytes caused by the inflammation associated with the above-mentioned liver disease, liver damage by drugs and the like, and change into myofibroblasts, Liver fibrosis progresses as the myofibroblasts produce extracellular matrix.

[0004] There have been several reports on pathological models of liver fibrosis using experimental animals to search for substances that inhibit hepatic stellate cell activation. For example, a series of pathologies of liver fibrosis caused by hepatocellular necrosis are caused by carbon tetrachloride (C

C1) can be reproduced by long-term administration of C1) to experimental animals to maintain inflammatory necrosis of hepatocytes for a long period of time (see Non-Patent Document 1). In such a pathological model, administration of an antioxidant having a scavenging activity for the CC1 radical, which is a causative agent of liver cell necrosis, suppresses the production of various cytokins and subsequent activation of hepatic stellate cells. It is believed that the progress of liver fibrosis can be suppressed as a result.

[0005] On the other hand, it is known that administration of dimethylnitrosamine (DMN) to experimental animals for a certain period of time causes liver fibrosis without hepatocellular necrosis (see Non-Patent Document 1). The mechanism by which liver fibrosis progresses in this disease model using DMN is clearly different from that using CC1, and in the case of CC1, liver fibrosis progresses due to necrosis of hepatocytes. On the other hand, DMN is thought to act on hepatic stellate cells in the quiescent phase and promote liver fibrosis by promoting activation. Therefore, substances that inhibit the activation of hepatic stellate cells are considered to be useful in the prevention and treatment of hepatic fibrosis, and also in the prevention and treatment of liver diseases associated with hepatic fibrosis. A substance that suppresses hepatic fibrosis in a liver injury model following administration may be a hepatic fibrosis inhibitor that acts directly on hepatic stellate cells.

[0006] Naturally derived foods and herbal medicines generally have advantages such as fewer side effects, and their usefulness against various intractable diseases, which are increasing in number in recent years, is attracting attention. Has been done. Amatani et al. Demonstrated that the herbal medicine `` Sho-saiko-to '', which has a crude drug as an active ingredient, has an effect on suppressing liver fibrosis in both liver injury models in which carbon tetrachloride was administered to experimental animals and in liver injury models in which DMN was administered. We report that (see Non-Patent Document 1). However, Sho-saiko-to is a traditional Chinese medicine, but many side effects have been reported, and it has been reported to be fatal. Also, as a contraindication to the use of rusho-saiko-to, which is used for medical purposes, administration to patients with liver cirrhosis and liver cancer is mentioned. Therefore, administration to patients with liver disease requires higher safety and higher demand for drugs.

[0007] On the other hand, shiitake mushrooms (shiitake mushrooms), which are representative edible mushrooms from Japan and China and have been cultivated artificially in Japan for 300 years, have been ingested as food for many years. The active substance is considered to have excellent safety when administered to humans, especially when administered orally for a long period of time. Several reports have been reported on the pharmacological effects of shiitake mushrooms, for example, in carcinogenicity experiments in rats and mice, inhibition of tumor formation in the large intestine and liver of experimental animals and growth of transplanted tumor cells And enhances the survival rate (see Non-patent Documents 2 and 3), enhances antibody production and activates antibody-mediated ADC (antiDody-dependent cell-mediated cytotoxicity). An effect exhibiting an effect (see Non-patent Document 4), an immunomodulating effect (see Non-Patent Document 5), a biomembrane protecting effect and an antioxidant effect of Shiitake mycelium extract (see Non-Patent Document 6), Macrophage activation (see Patent Documents 1 and 2), in vivo antioxidant function-enhancing activity (see Patent Document 3), and infection-inhibiting and growth-inhibiting effects of AIDS virus (see Patent Document 4) The report has been done. However, the effect of Shiitake mycelium extract on hepatic fibrosis and the effect on hepatic stellate cells have not been reported.

Patent Document 1: JP-A-62-270532

Patent Document 2: JP-A-2-237934

Patent Document 3: JP-A-11-228441

Patent Document 4: JP-A-2-134325

Non-Patent Document 1: Ametani et al., Kampo Medicine, Vol. 13, No. 8, 1989

Non-Patent Document 2: Cancer Letters, Vol. 17, pp. 109-114, 1982

Non-Patent Document 3: Cancer Letters, Vol. 27, pp. 1-6, 1985

Non-Patent Document 4: Hepatobiliary, Vol. 15, pp. 127-135, 1987

Non-patent document 5: Journal of the Japanese Society of Pharmaceutical Sciences, Vol. 8, pp. 162-166, 1991

Non-Patent Document 6: Japanese and Chinese Pharmaceutical Magazine, Vol. 19 (Extra Issue), p. 161, 2002

Disclosure of the invention

Means for solving the problem

[0008] The present inventors have conducted intensive studies in order to solve such problems, and have found that an extract of Shiitake mycelium has a hepatic fibrosis-suppressing action, thereby completing the present invention.

An object of the present invention is to provide an extract derived from Shiitake mycelium having a hepatic fibrosis inhibitory action, and a hepatic fibrosis inhibitor, a hepatoprotective agent, a composition for oral ingestion, a pharmaceutical composition, and a food containing the extract It is to provide compositions, foods and beverages.

[0009] That is, according to one aspect of the present invention, there is provided a liver fibrosis inhibitor comprising a shiitake mycelium extract or a fraction thereof.

According to still another aspect of the present invention, there is provided a hepatic fibrosis inhibitor comprising a fraction of Shiitake mycelium extract, wherein the fraction is used.

1) adding a solution containing ethanol to the shiitake mycelium extract to obtain a soluble fraction with respect to the solution; and

2) subjecting the soluble fraction to gel filtration chromatography, eluting with water, and then eluting with a solution containing methanol to obtain a fraction eluted with the solution. Is a fraction containing polyphenols, The above-mentioned hepatic fibrosis inhibitor is provided.

[0010] According to another aspect of the present invention, there is provided a hepatoprotective agent having a hepatic fibrosis-suppressing effect and comprising a shiitake mycelium extract or a fraction thereof.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing a disease associated with hepatic fibrosis, comprising the hepatic fibrosis inhibitor.

[0011] According to still another aspect of the present invention, there is provided the pharmaceutical composition, wherein the disease associated with liver fibrosis is fibrosis, hepatitis or cirrhosis.

Further, according to another aspect of the present invention, there is provided a food composition comprising the hepatic fibrosis inhibitor.

Hereinafter, the present invention will be described more specifically.

The shiitake mycelium in the present invention is in the state of mycelia in the stage before the edible fruiting body, and may be produced by culture or collected from nature. For example, a mycelium obtained by cultivating Shiitake fungi on a solid medium can be used. The shiitake mycelium extract used in the present invention can be prepared by a method that can be prepared by a method known in the art.For example, the extract is obtained by pulverizing and decomposing a solid medium containing mycelium in the presence of water and an enzyme. The extract can be used. The Shiitake mushroom mycelium extract is not limited, but for example, those obtained by the following method can be used. That is, Shiitake mushrooms are inoculated on a solid medium based on bagasse (sugar cane pulp) and defatted rice bran, and then the solid medium containing mycelium obtained by growing the mycelium is passed through 12 mesh. 30% by weight or less, and water and one or more enzymes selected from cellulase, protease or dalcosidase are added to the unbound solid medium at a temperature of 30 to 55 ° C. While adding, the solid medium is crushed and ground in the presence of the enzyme so that at least 70% by weight or more of the bagasse fiber passes through the 12 mesh, and then heated to a temperature of up to 90 ° C. Then, the enzyme is inactivated and sterilized, and the resulting suspension is filtered to obtain a shiitake mycelium extract. The extract may be used as a shiitake mycelium extract, but it is convenient to concentrate, freeze-dry and store it as a powder and use it in various forms at the time of use. The powder obtained by freeze-drying is a brown powder, With a unique taste and smell. The shiitake mushroom mycelium extract obtained in this way contains 15 to 50%, preferably 20 to 40% / ^^ of carbohydrate in the sugar analysis by the phenolic sulfuric acid method, and protein analysis by the Lowry method. 10 to 40%, preferably 13 to 30% (w / w), 1 to 5%, preferably 2.5 to 3.5% (w / w) of polyphenols by the Folin-Denis method using gallic acid as a standard. w) included. Shiitake mycelium extract also includes, but is not limited to, a force S containing about 0.1% lipid, about 0.4% fiber, and about 20% ash.

[0013] An example of the constituent sugar composition (%) of the shiitake mushroom mycelium extract was as follows, but this composition may vary depending on the culture conditions and the like: xylose: 15.2; arabinose: 8. 2; mannose: 8.4; glucose: 39.4; galactose: 5.4; amino sugars: 12.0; Uron acid: 11.3 ₀ '

[0014] The "fraction" in the present invention is not particularly limited as long as it can obtain the extract of Shiitake mycelium extract by a fractionation method usually used in the technical field to which the present invention belongs. Examples of the fractionation method include fractionation by extraction using an arbitrary solvent, gel filtration column chromatography, fractionation using an ion exchange column, and silica gel column chromatography. The fractionation method may be a single method or a combination of a plurality of means. Among the above-mentioned fractionation methods, in extraction with an arbitrary solvent, for example, a solution containing ethanol, a solution containing methanol, or the like can be used. Here, the `` solution containing methanol '' is a solution containing 20 to 80%, preferably 30 to 70%, more preferably 40 to 60%, and most preferably 45 to 55% of methanol in a solvent by volume. means. The solvent is not particularly limited, but for example, water, ethanol, n-propanol, isopropanol, acetic acid, acetone, and a mixture thereof can be used, and preferably water can be used. By "a solution containing ethanol" is meant a solution containing 20-80%, preferably 30-70%, more preferably 40-60%, and most preferably 45-55% ethanol by volume in the solvent. I do. Although the solvent is not particularly limited, for example, water, methanol, n-propanol, isopropanol, acetic acid, acetone, and a mixture thereof can be used, and preferably, water can be used. . The mixture of the solution and the shiitake mycelium extract can be left, for example, for a certain period of time to obtain a fraction containing a soluble component with respect to the solution. Let it rest here Although the time is not limited, it can be, for example, 6 hours to 100 hours, preferably 24 hours to 84 hours, and more preferably 60 hours to 84 hours. In addition, the mixture may be heated and / or stirred as necessary in order to reduce the time required for the preparation. The soluble fraction obtained by removing insolubles in the mixture by filtration or decantation can be used as it is as a solution.However, if necessary, concentration and / or lyophilization may be performed. The resulting concentrate, viscous substance or solid can be used as the soluble fraction.

[0015] Among the above fractionation methods, gel filtration column chromatography can be performed by a usual method known in the technical field to which the present invention belongs, and using a carrier commonly used in the technical field. It can be carried out. Specific examples of the carrier include Sephadex LH-20, Sephadex G-20, Sephacryl S-100HR, and the like. As an eluate for gel filtration column chromatography, water such as distilled water, pure water, ultrapure water, a solution containing methanol, and a solution containing ethanol are used. Here, the solution containing methanol means a solution containing 50 to 100%, preferably 70 to 100%, and more preferably 90 to 100% by volume of methanol in a solvent, and is preferably commercially available. 99.8% methanol solution can be used. Although the solvent is not particularly limited, for example, water, ethanol, _n -propanol, isopropanol, acetic acid, acetone, and a mixture thereof can be used, and preferably water can be used. The fraction of Shiitake mycelium extract is obtained, for example, as an elution fraction of the above-mentioned solution containing methanol in gel filtration column chromatography. The eluted fraction can be used in the present invention as a concentrate, a viscous substance, and a fraction obtained by performing a treatment such as concentration under reduced pressure, freeze-drying, and further purification such as dialysis. The fraction contained in the present invention is obtained as an eluted fraction of the solution containing methanol. Concentrated liquids, viscous substances, and solids obtained by subjecting the eluted fraction to a treatment such as concentration under reduced pressure, lyophilization, and further purification such as dialysis are also included in the present invention.

[0016] Generally, a fraction containing a polar substance having a relatively low molecular weight can be obtained by the purification by gel filtration column chromatography in the present invention. It is not limited to such fractions. Extraction contained in the liver fibrosis inhibitor of the present invention The output or fraction may include, for example, polyphenols (including phenols and phenols), proteins, and saccharides. As used herein, the term "polyphenols" refers to the total phenolic component determined by the Folin-Denis method, which is a known method for detecting phenols, and includes phenol. The term "phenol" is a generic term for compounds in which a hydrogen atom of an aromatic hydrocarbon nucleus is substituted with a hydroxyl group.

[0017] The fraction contained in the present invention contains, for example, 11 to 35%, preferably 12 to 30% (wZw), more preferably 12 to 30% of polyphenols determined by the Folin-Denis method using catechin as a standard substance. May include lS SOO / wZw), for example, 12% or more. Although not particularly limited, for example, polyphenols include 5-hydroxymethylfurfural, phenols such as protocatechuic acid (3,4-dihydroxybenzoic acid) and methyl gallate (polyphenol or polyphenol), and syringic acid And phenols such as vanillic acid. In addition, the fraction may contain, for example, 60 to 80%, preferably 65 to 85% (%) of protein quantified by protein analysis by the Lowry method, for example, saccharide quantified by carbohydrate analysis by the phenol-monosulfuric acid method. From 0 to 20%, preferably 5 to 15% (w / w).

[0018] As used herein, the term "hepatic fibrosis inhibitor" generally refers to a substance that suppresses hepatic stellate cell activation, suppresses collagen production, or inhibits matrix production. It has the function of suppressing liver fibrosis. Diseases associated with liver fibrosis include, but are not limited to, various liver diseases such as acute hepatitis, chronic hepatitis, cirrhosis and liver cancer. For example, the liver fibrosis-suppressing agent of the present invention can be expected to have an effect in preventing and / or treating the above-mentioned various liver diseases and a liver-protecting effect.

[0019] The term "hepatoprotective agent" as used herein generally refers to liver damage caused by drugs or harmful substances, immunological liver damage, viral liver damage, fatty liver, liver cancer. It refers to those that suppress liver function decline due to alcoholic liver disease and cirrhosis, and those that protect the liver.

[0020] Since the liver fibrosis inhibitor of the present invention contains a plurality of shiitake-derived physiologically active substances, it is expected to exert a liver fibrosis inhibitory effect by a plurality of mechanisms of action and a hepatoprotective effect resulting therefrom. Is done. Examples of the mechanism of action include: i) inhibiting inflammatory hepatocyte necrosis Inhibits the release of cytodynamics in liver tissue and the resulting activation of hepatic stellate cells, and suppresses the production of extracellular matrix; ii) direct action on hepatic stellate cells Iii) activate the metabolism of the produced extracellular matrix, and the like. However, the effect of the hepatic fibrosis inhibitor of the present invention has other effects. It may be based on a mechanism. On the other hand, the hepatic fibrosis inhibitor of the present invention may exert its effect mainly by a single mechanism of action.

[0021] The liver fibrosis inhibitor of the present invention can be used as an active ingredient of a pharmaceutical composition. The pharmaceutical composition can be in various dosage forms, for example, for oral administration, tablets, capsenoles, powders, granules, pills, solutions, emulsions, suspensions, solutions, spirits, syrups , An extract and an elixir, but are not limited thereto. The pharmaceutical composition may contain various components commonly used, for example, one or more pharmaceutically acceptable excipients, disintegrants, diluents, lubricants, flavoring agents, Coloring agents, sweetening agents, flavoring agents, suspending agents, wetting agents, emulsifying agents, dispersing agents, adjuvants, preservatives, buffers, binders, stabilizers, coating agents and the like can be included. Further, the pharmaceutical composition of the present invention may be in a sustained or sustained release form.

[0022] The dose of the hepatic fibrosis inhibitor of the present invention can be appropriately selected depending on the patient's body type, age, physical condition, degree of disease, elapsed time after onset, etc., and the pharmaceutical composition of the present invention , A therapeutically effective amount and / or a prophylactically effective amount of a hepatic fibrosis inhibitor. For example, as a fraction of the shiitake mycelium extract of the present invention, it is generally used at a dose of 10 to 50,000 mgZ adult, preferably 100 to 5000 mgZ adult. The pharmaceutical composition may be administered in a single dose or in multiple doses.For example, it may be used in combination with another drug such as another therapeutic agent for liver disease, a liver protectant, or a liver fibrosis inhibitor. You can also.

[0023] The food composition of the present invention includes a liquid beverage such as a functional beverage. The food composition can be used not only as a functional food, but also as a quasi-drug, a component of food and drink, a food additive, and the like. In addition, the composition for oral ingestion in the present specification can be used as it is as a functional food, and can also be used as a drug, a quasi-drug, a component of food and drink, a food additive, and the like. By the use, the food composition or the composition for oral ingestion of the present invention having a liver fibrosis-suppressing effect and a liver-protecting effect of the present invention Regular and continuous ingestion is possible, and it is possible to improve the constitution by effective liver fibrosis inhibitory effect and liver protection effect, and to treat and prevent the onset of various liver diseases. Examples of foods or beverages containing the hepatic fibrosis inhibitor or hepatoprotective agent of the present invention include functional foods, health foods, and general foods (jiuse, confectionery, Processed foods, etc.), dietary supplements (energy drinks, etc.) and the like. Food or beverages in this specification include, but are not limited to, mushroom mycelium extracts or fractions, inorganic components such as iron and calcium, various vitamins, dietary fiber such as oligosaccharides and chitosan, and the like. Proteins such as soy extracts, lipids such as lecithin, and sugars such as sucrose and lactose can be included.

The invention's effect

As shown in the following examples, the hepatic fibrosis inhibitor of the present invention has a hepatic fibrosis inhibitory action. Therefore, the present invention provides an effective means for treating and / or preventing various liver diseases associated with liver fibrosis.

Brief Description of Drawings

[FIG. 1] An example of the measurement results of AST in the serum of a mouse model of liver fibrosis caused by long-term administration of dimethylnitrosamine (DMN), eluted with Shiitake mycelium extract (LEM :) and methanol FIG. 9 shows that the fraction (ES-Me) expresses a liver fibrosis inhibitory effect.

[Fig. 2] An example of the measurement results of ALT in the serum of a mouse model of liver fibrosis caused by long-term administration of dimethylnitrosamine (DMN). The fraction eluted with shiitake mycelium extract (LEM :) and methanol was eluted. (ES-Me) shows that hepatic fibrosis is suppressed.

FIG. 3 is an example of RT-PCR results showing the expression levels of HSP47 and SMA in the liver of a model fibrosis model mouse with long-term administration of dimethylnitrosamine (DMN), and is an example of a result of shiitake mushroom mycelium extract (LEM) This shows that the fraction eluted with methanol (ES-Me) exhibits the effect of suppressing hepatic fibrosis.

[Figure 4] An example of the results of LEM activation inhibitory effect (transformation inhibitory effect) on hepatic stellate cells, showing that LEM suppresses hepatic stellate cell activation and exerts an inhibitory effect on liver fibrosis. is there.

[Figure 5] An example of the results of ES-Me activation inhibitory effect (transformation inhibitory effect) on hepatic stellate cells Yes, it shows that ES-Me suppresses the activation of hepatic stellate cells and exerts an inhibitory effect on hepatic fibrosis.

[Figure 6] This is an example of the results of LEM and ES-Me inhibiting the DNA synthesis of hepatic stellate cells. LEM and ES-Me suppress the DNA synthesis of hepatic stellate cells, suppress their activation, and suppress liver fibrosis This shows that the effect is exhibited.

BEST MODE FOR CARRYING OUT THE INVENTION

[0026] Hereinafter, preferred embodiments of the present invention will be described in further detail. The present invention is not limited to these embodiments.

[Example 1] Method for preparing shiitake mycelium extract

After appropriately adding pure water to a solid medium consisting of 90 parts by weight of bagasse and 10 parts by weight of rice bran, Shiitake mycelium was inoculated, and allowed to stand in a temperature- and humidity-controlled culture room to grow mycelium. After the mycelium spread on the solid medium, the bagasse-based fibrous material was unbundled so that the amount of .12 mesh passed was less than 24% by weight. To 1.0 kg of the unbound medium, 3.5 L of pure water was added, and while maintaining the temperature at 40 ° C., 2.Og of purified cellulase was added to obtain a medium-containing mixture. Then, while circulating the mixture containing the medium with a gear-pump with a variable speed, the solid medium was subjected to a grinding and grinding action in a portion of the gear for about 200 minutes so that about 80% by weight of the bagasse fiber passed through 12 mesh. Grinding and grinding of the medium-containing mixture was performed while gradually increasing the temperature of the mixture. Thereafter, the mixture containing the medium was further heated and left at 90 ° C. for 30 minutes to inactivate the enzyme and sterilize. The resulting medium-containing mixture was filtered using a 60-mesh filter cloth and concentrated, and then a freeze-dried powder was obtained as a shiitake mycelium extract (LEM).

Example 2 Preparation of Fractions of Shiitake Mycelium Extract

2- 1 Preparation of a fraction of Shiitake mycelium extract

Shiitake mycelium extract (LEM) was added to a 50% aqueous ethanol solution and allowed to stand for 3 days to perform extraction. The insoluble fraction was separated by filtration, and the extract, which was a soluble fraction, was filtered under reduced pressure and freeze-dried to extract a brownish 50% aqueous ethanol solution at a yield of about 90% by weight with respect to the Shiitake mycelium extract. A fraction (ES) was obtained. The insoluble fraction was dispersed in an appropriate amount of distilled water, and then freeze-dried to obtain a brown 50% ethanol aqueous solution insoluble fraction (EI). ES fraction obtained Was dissolved in a small amount of ethanol and subjected to gel filtration chromatography. Sephadex LH-20 was used as a carrier, and distilled water was used first as a mobile phase, and then elution was performed using methanol. The fraction eluted with methanol was concentrated under reduced pressure and freeze-dried to obtain a methanol-eluted fraction (ES-Me) at a yield of about 10% by weight based on the shiitake mycelium extract.

2-2 Measurement of the component ratio of the fraction of Shiitake mycelium extract

The component composition ratio of each obtained fraction was calculated by the following quantitative analysis method. That is, sugars are obtained by the phenolic sulfate method (see Methods in Carbohydrate Chemistry. RL Whistler and MLML Wolfrom, edited by Academic Press, New York, Vol. 1, p. 388, 1962), and proteins are obtained by the Lowry method. An improved method, the BCA method (see Anal. Biochem. 150, 76, 1985), polyphenols were prepared using the Folin-Denis method (Dietary Tannins: onsequwnces and remedies, CRC Press, Volume 13, 1989.). The measurement conditions of the Folin-Denis method are as follows.

[0028] Standard substance: catechin

Creation of calibration curve: Created from one point of catechin of arbitrary concentration and origin

Measurement wavelength: 760 / m

Test solution concentration: 0.2% (W / W)

Table 1 shows an example of component measurement results for the shiitake mycelium extract (L.E.M.), a 50% aqueous ethanol extract (ES), and a methanol eluted fraction (ES-Me). From the measurement results shown in Table 1, the Shiitake mycelium extract and the 50% ethanol aqueous extract fraction have almost the same component ratio.The methanol-eluted fraction has a significantly higher ratio of protein and polyphenols. Became evident. However, this composition may vary depending on the culture conditions of the shiitake mycelium and the like.

[0029] Table 1. Component ratios (% by weight) of Shiitake mycelium extract (L. E. M.), 50% aqueous ethanol extraction fraction (ES) and methanol elution fraction (ES-Me)

[Table 1] Sugar protein Polyphenol Others EM 33.96 34.00 3.34 28.69

ES 31.20 33.54 3.38 31.88

ES-Me 9.48 76.79 13.73 0.00

[Example 3] Hepatoprotective effect test by L.E.M., ES-I Me using long-term administration of dimethylnitrosamine (DMN) mice

Six-week-old, male, BALBZc mice were given dimethylnitrosamine (DMN) lOmgZkg by i.p. administration for the first 3 days of the week and continued for 4 weeks to produce a liver fibroid model. In addition, a model was prepared in which DMN dOmgZkg) and L.E.M. or ES-Me (10 mg / kg) as a hepatoprotective substance were simultaneously administered i.p. At the end of the fourth week, blood was collected from the fundus, and AST and ALT were measured in the serum. As shown in FIGS. 1 and 2, AST and ALT were increased by DMN administration, and the increase was suppressed by LEM, ES-Me administration. This suggested that L.E.M. and ES-Me have a protective effect on DMN-damaged mice.

[Example 4] RT-PCR method using mRNA extracted from liver

Livers were collected 4 weeks after DMN administration, and half of them were homogenized and treated with cepazole to extract riiRNA. After mRNA extraction, an RT reaction was performed to obtain cDNA. Thereafter, expression of HSP47, an a-smooth muscle actin (c-SMA), which is an indicator of liver fibrosis, was confirmed using a PCR kit (TAKA A).

FIG. 3 shows the results. DMN administration increased the expression of both HSP47 and α-SMA. In addition, L-E.M. Administration suppressed the expression of both a-SMA and ES-Me administration of both HSP47 and α-SMA. It was suggested that both L. E. M. and ES-Me suppressed liver fibrosis caused by DMN.

[Example 5] Test of hepatic stellate cell activation inhibitory effect by L. E. M., ES-Me

Using Sprague Dawley (SD) male rats, Collagenase 'Pronase digestion method

Replacement paper «26) Hepatic stellate cells were isolated. The isolated hepatic stellate cells were seeded at a concentration of ⁵ × 10 ⁵ cell _S / ml in a plastic Petri dish, cultured in a DMEM medium containing 10% serum for 4 hours, and non-implanted cells were removed with a pipette. Each sample culture solution was supplemented with LEM (100, 500, 1000 ig Zml) and ES-Me (100, 500, 1000 μg Zml) as a protective substance for moonlight. Approximately 7 days after the start of culturing in the sample culture solution, morphological changes of hepatic stellate cells were observed with a phase contrast microscope.

[0033] One test was performed using stellate cells isolated from one rat, and the test was repeated several times to confirm reproducibility. Representative examples of the results are shown in FIGS. Both L. E. M. and ES-Me inhibited the transformation of hepatic stellate cells and showed an effect of suppressing hepatic fibrosis.

[Example 6] Test of hepatic stellate cell DNA synthesis inhibitory effect by L. E. M. and ES-Me

Using Sprague Dawley (SD) male rats, hepatic stellate cells were isolated by Collagenase'Pronase digestion. The isolated hepatic stellate cells are seeded on a plastic petri dish at a concentration of 2 × 10 ⁵ cellsZml, cultured in a DMEM medium containing 10% serum for 4 hours, and the non-implanted cells are removed with a pipette. LEM as _{(100, 500, 1000 μ §} / ml), ES -Me (100, 500, 1000 g / ml), / JヽSaiko hot (100, 500, 1000 ^ g / ml) of each sample was added The medium was exchanged. After culturing in a sample culture solution for 4 days, BrdU was added, followed by culturing for 24 hours, and the DNA uptake ability of BrdU was measured to confirm the DNA synthesis inhibitory effect of hepatic stellate cells.

[0034] One test was carried out using stellate cells isolated from one rat, and the test was repeated twice to confirm reproducibility. Figure 6 shows the results based on the average of two test data. L.E.M., ES-Me, and Sho-saiko-to all inhibited DNA synthesis in hepatic stellate cells and showed an effect of suppressing hepatic stellate cell activation. In particular, ES-Me exhibited an effect at a lower concentration than Sho-saiko-to, which was used as a prescription drug, and a high effect of ES-Me on liver fibrosis was confirmed.

[0035] When the cytotoxicity was confirmed in the specimens used in this test, no toxicity was confirmed at any concentration of the specimen, and this result was not due to cytotoxicity. It was confirmed.

Claims

The scope of the claims

[1] A hepatic fibrosis inhibitor comprising a shiitake mycelium extract or a fraction thereof.

[2] A liver fibrosis inhibitor comprising a fraction of Shiitake mycelium extract, wherein the fraction is

1) a step of mashing a solution containing ethanol with the shiitake mycelium extract to obtain a soluble fraction with respect to the solution; and

2) subjecting the soluble fraction to gel filtration chromatography, eluting with water, eluting with a solution containing methanol, and obtaining an eluted fraction with the solution. The hepatic fibrosis inhibitor according to claim 1, which is a fraction containing polyphenols, which can be used.

[3] A pharmaceutical composition comprising the hepatic fibrosis inhibitor according to claim 1 or 2 for treating or preventing a disease associated with liver fibrosis. '

[4] The pharmaceutical composition according to claim 3, wherein the disease associated with liver fibrosis is fibrosis, hepatitis or cirrhosis.

[5] A food composition comprising the liver fibrosis inhibitor according to claim 1 or 2.

[6] A hepatoprotective agent having an effect of suppressing hepatic fibrosis, comprising a shiitake mycelium extract or a fraction thereof.