JP2000157205A - Fractionated substance of extract from mycelium of lentinus edodes sing. and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject fraction having protecting effects on hepatic dysfunction without adverse effects, usable with the administration of a drug causing the hepatic dysfunction and excellent in safety by treating an ethanol- insoluble fraction of an extract from a mycelium of Lentinus edodes Sing. under specific conditions. SOLUTION: This fractionated substance is obtained by treating an ethanol- insoluble fraction prepared by treating an extract from a mycelium of Lentinus edodes Sing. with an ethanol-containing solution with an ion exchanger such as a diethylaminoethyl synthetic resin ion exchanger and is discharged without being adsorbed when eluted with water. The resultant fractionated substance is preferably formulated with a pharmaceutically acceptable carrier as an optional ingredient to prepare a therapeutic and/or a prophylactic composition for hepatic dysfunction. The daily dose of the fraction is preferably within the range of 30-200 mg.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel fraction obtained from a Shiitake mushroom mycelium extract and a liver damage-protecting agent containing the same.

[0002]

2. Description of the Related Art Shiitake (Lentinus edodes) is
It is a typical edible mushroom in China, and has been cultivated artificially in Japan for about 300 years. Mushrooms that are consumed daily are called fruiting bodies, and they are germ bodies that produce spores because fungi leave their offspring, and vegetative mycelium cells form mycelia over a long period of time in the ground and in raw wood. I do.

[0003] Shiitake has long been said to be effective for various diseases and symptoms, but it has been relatively recently that its pharmacological action has been elucidated. Shiitake mushroom mycelium extract inhibited tumor formation and growth of transplanted tumor cells in the large intestine and liver of animals and increased the survival rate of animals in carcinogenicity experiments in rats and mice (N. Sugano eta).
l., Cancer Letter, 17: 109, 1982; Yasumasa Suzuki et al., Journal of the Japanese College of Colitis, 43: 178, 1990, etc., and showed mitogenic activity (T. Tabata et al., Immunopharmacology, 2
4:57, 1992; Y. Hibino et al., Immunopharmacology,
28:77, 1994, etc.), which enhance antibody production and allow antibody-mediated ADCC (antibody-dependent cell-mediated cytotox
Various reports have been reported, such as those showing an inhibitory effect on immunological hepatocellular injury due to icity) (Yasuhiro Mizoguchi et al., Hepatobiliary pancreas, 15: 127, 1987). In addition, the extract of shiitake mushroom mycelium also has phytohormonal effects such as promotion of rooting and growth of crops (M. Mitsuhashi-Kato et al., Plant Cell Physiol.
6: 221, 1985) and anti-plant virus activity (Yasuo Komuro: Report of the Institute of Plant Virus Research, Ministry of Agriculture, Forestry and Fisheries, 1977).

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to obtain a novel fraction from Shiitake mushroom mycelium extract, elucidate its pharmacological action in more detail, and obtain a new pharmaceutical use of the shiitake mushroom mycelium extract fraction. And / or exploring health applications.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the insoluble fraction obtained by treating a shiitake mushroom mycelium extract with an ethanol-containing solution (hereinafter referred to as ethanol-insoluble fraction) Fraction, which is treated with an ion exchanger and eluted with water. The fraction of Shiitake mushroom mycelium extract, which flows out without adsorption, has a remarkable protective effect against liver damage. Discovered and completed the present invention.

[0006] That is, the present invention provides a fraction of Shiitake mushroom mycelium extract which is not adsorbed when the ethanol-insoluble fraction of the mushroom mycelium extract is treated with an ion exchanger and eluted with water. I do.

[0007] The present invention further provides a liver containing a fraction of Shiitake mushroom mycelium extract, which is not adsorbed when the ethanol-insoluble fraction of the shiitake mushroom mycelium extract is treated with an ion exchanger and eluted with water. Provide a protective agent.

[0008] The agent for protecting liver damage according to the present invention is a fraction of a shiitake mushroom mycelium extract which flows out without being adsorbed when an ethanol-insoluble fraction of a shiitake mushroom mycelium extract is treated with an ion exchanger and eluted with water. It may be in the form of a composition for the treatment and / or prophylaxis of liver disorders, comprising a product and optionally a pharmaceutically acceptable carrier.

[0009] The liver injury protective agent of the present invention may be in the form of a food or a beverage.

Furthermore, the liver injury protective agent of the present invention is not limited to these forms.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a shiitake mushroom mycelium extract refers to a mycelium obtained by culturing shiitake mushroom on a solid medium, preferably a solid medium containing the mycelium in the presence of water and an enzyme. An extract obtained by pulverization and decomposition.

The shiitake mushroom mycelium extract preferably used is one obtained by the following method, but is not limited thereto. That is, Shiitake mushrooms are inoculated on a solid medium based on bagasse (sugar cane squeezer) and defatted rice bran, and then a solid medium containing mycelium obtained by growing mycelium is 30 weight per 30 mesh passing. % Of water and one or more enzymes selected from cellulase, protease or glucosidase, and the solid medium is mixed with 30 to 55% of water.
The solid medium is crushed and crushed in the presence of the enzyme so that at least 70% by weight of the Bacass fiber passes through 12 mesh, and then the solid medium is heated to 95 ° C. The enzyme is inactivated by heating and sterilized, and the resulting suspension is filtered to obtain a shiitake mushroom mycelium extract. Shiitake mushroom mycelium extract may be used as it is in the protective agent of the present invention, but it is concentrated, freeze-dried and stored as a powder,
It is convenient to use it in various forms at the time of use. The powder obtained by freeze-drying is a brown powder, is hygroscopic and has a unique taste and odor.

The thus-obtained shiitake mushroom mycelium extract contains 15-50%, preferably 20-40% (w / w) of saccharide by Lo sugar analysis by a phenol-sulfuric acid method.
10- protein by wry protein analysis
40%, preferably 13-30% (w / w) of polyphenol by the Folin-Denis method using gallic acid as a standard.
-5%, preferably 2.5-3.5% (w / w).
Shiitake mushroom mycelium extract also contains about 0.1% lipid,
Contains about 0.4% fiber and about 20% ash.

The constituent sugar composition (%) of the shiitake mushroom mycelium extract was as follows: xylose: 15.2;
Arabinol: 8.2; Mannose: 8.4; Glucose: 39.4; Galactose: 5.4; Amino sugar: 1
2.0; uronic acid: 11.3.

[0015] The fraction of Shiitake mushroom mycelium extract which is not adsorbed when the ethanol-insoluble fraction of the Shiitake mushroom mycelium extract according to the present invention is treated with an ion exchanger and eluted with water (hereinafter referred to as ion exchange) The non-body-adsorbed fraction is sometimes used as a solid particle filter agent (preferably, a shiitake mushroom mycelium extract, preferably a shiitake mushroom mycelium extract powder as an aqueous solution of an appropriate concentration to remove impurities. After filtration using Celite 545), 1) ethanol is added to the Lentinus edodes mycelium extract to separate it into an insoluble fraction and a soluble fraction; 2) the ethanol-insoluble fraction obtained in step 1 is treated with an ion exchanger A non-adsorbed non-adsorbed fraction which is not adsorbed when eluted with water and adsorbed to the column with 0.05-3M N
aCl, preferably a fraction eluted with 2M NaCl (hereinafter sometimes referred to as an ion-exchanger-adsorbed fraction).

In the above step 1), ethanol (preferably 4 volumes) is added to the Lentinus edodes mycelium extract to separate into an ethanol-insoluble fraction and a soluble fraction. The ethanol-insoluble fraction is a fraction containing a crude polysaccharide, and contains saccharide as a main component.
Contains 4.3%, 14.6% protein.

The ethanol-insoluble fraction was obtained from the shiitake mushroom mycelium extract in a yield of 20%. The constituent sugar composition (%) of the ethanol-insoluble fraction was as follows: xylose:
15.0; Arabinose: 9.3; Mannose: 12.
9; glucose: 28.7; galactose: 10.7;
Amino sugar: 12.5; uronic acid: 10.9.

Next, in the step 2), the ethanol-insoluble fraction obtained in the step 1) is redissolved in distilled water, treated with an ion exchanger, and an ion-exchanger non-adsorbed fraction flowing out without being adsorbed; The adsorbent fraction is adsorbed on the column and eluted with 0.05-3M NaCl, preferably 2M NaCl.
As an ion exchanger, for example, diethylaminoethyl
Anions such as synthetic resin ion exchanger (Cl- type), diethylaminoethyl cellulose, diethylaminoethyl agarose, diethyl- (2-hydroxypropyl) aminoethyl cellulose, and diethyl- (2-hydroxypropyl) aminoethyl agarose An exchanger can be used, and a column type of a diethylaminoethyl-toyopearl ion exchanger (manufactured by Tosoh Corporation) is preferable. FIG. 1 shows an example of the chromatogram obtained in the second step.

The fraction of the present invention (the fraction not adsorbed by the ion exchanger) was obtained in a yield of 9.5% in terms of Shiitake mushroom mycelium extract. The fraction not adsorbed by the ion exchanger had a sugar content of 21.6.
%, 2.6% protein, and 0.1% polyphenol (all w / w).

The constituent sugar composition (%) of the non-adsorbed fraction of the ion exchanger was as follows: mannose: 14.3;
Glucose: 63.9; galactose: 21.8; amino sugar: 3.6; uronic acid: 1.1.

On the other hand, the ion-exchanger-adsorbed fraction was obtained in a yield of 10.3% in terms of Shiitake mushroom mycelium extract.
The ion-exchanger-adsorbed fraction contains 47.4% of carbohydrate and 1 protein
8.2%, Polyphenol 2.7% (all w / w)
Was included.

Saccharide composition (%) of the ion-exchanger adsorbed fraction
Was as follows: xylose: 23.2; arabinose: 13.5; mannose: 18.7; glucose: 31.9; galactose: 12.7;
9.1; uronic acid: 3.0.

The ethanol-insoluble fraction of the shiitake mushroom mycelium extract according to the present invention is treated with an ion exchanger and eluted with water. When the protective effect of the adsorbed fraction) on liver damage was tested by the following method, a remarkable protective effect was observed in an in vitro test using rat hepatocytes.

The protective agent of the present invention is a therapeutic agent and / or a prophylactic agent and / or a health agent. The protective agent of the present invention includes cefemazines such as cefloxazine, cefuroxime sodium and latamoxef sodium, aminoglycosides such as amikacin sulfate, antitumor systems such as aclarubicin, mycobacterial antibiotics such as rifampicin, and tetracyclines. ,
Antibiotics such as macrolides and penicillins, drugs for central nervous system, antipyretic analgesic and anti-inflammatory systems such as aspirin, mental and nervous systems such as cloxazolam, antiepileptic agents such as sodium valproate, and general anesthetics such as halothane , Hepatic disorders caused by administration of drugs such as sedative hypnotics such as phenobarbital, general cold remedy, and arrhythmia such as pindolol, vasodilators such as travidil, and circulation of nicardipine hydrochloride Organ system, antihypertensive system such as labetalol hydrochloride, cardiovascular drugs known in the arteriosclerotic system such as clofibrate, antimetabolites such as tegafur, hormonal agents such as estrasite, and sulfamethoxazole Anti-tuberculosis drugs such as sulfa drugs, isoniazid, and synthetic antibacterial drugs such as norfloxacin It is useful not only for liver damage caused by the administration of drugs, but also for liver damage caused by environmental harmful substances such as bisphenol, phthalate ester, chlorine compound, alkylphenol, and alcohol present in the living environment. Liver disease, viral liver injury, fatty liver, liver cancer, alcoholic liver injury, cirrhosis, and the like. Therefore, the ethanol-insoluble fraction of the Lentinula edodes mycelium extract is treated with an ion exchanger and eluted with water. It can be used as a composition for treating and / or preventing liver damage, comprising a carrier.

The administration route is most preferably oral administration, but may be intravenous administration, subcutaneous administration and the like. Formulations suitable for oral administration include, but are not limited to, tablets, capsules, powders, granules, solutions, syrups and the like.

Pharmaceutically acceptable carriers include suitable excipients, binders, disintegrants, lubricants, flavoring agents, and the like known in the art.
Examples include, but are not limited to, colorants, solubilizing agents, suspending agents, coating agents, and the like.

The dose of the protective agent of the present invention is determined by a physician, pharmacist, dietitian or the like in consideration of the patient's age, body weight, symptoms, administration route and the like. The shiitake mushroom mycelium extract contained in the protective agent of the present invention has been used as a food, and since it is extremely safe, it is not necessary to strictly limit the dose, but usually the ion-exchange non-adsorbed fraction 3 mg-600 mg per day, preferably 30
mg-200 mg. Furthermore, there is no problem even when administered in combination with a drug that causes liver damage.

The protective agent of the present invention can be provided in the form of a food. Preferred foods include granules, noodles, candies, jellies, cookies and the like. further,
The protective agent of the present invention can also be provided in the form of a beverage.
Such foods and beverages contain, in addition to shiitake mushroom mycelium extract, vitamins, inorganic components such as calcium, dietary fiber such as chitosan, proteins such as soybean extract, lipids such as lecithin, and sugars such as lactose. May be added.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto. It is possible for those skilled in the art to use the method of the present invention with various changes and modifications, and these are also included in the scope of the present invention.

[0030]

EXAMPLES Example 1 Method for Preparing Shiitake Mushroom Mycelium Extract A solid medium consisting of 90 parts by weight of bagasse and 10 parts by weight of rice sugar was made to contain moderately pure water. Was allowed to stand in a conditioned culture room to grow mycelium. After the mycelium spread on the solid medium, the fibrous material of the bagasse base material was unbundled so that the amount passed through the 12 mesh was 24% by weight or less. To 1.0 kg of the unbundled medium, 3.5 L of pure water was added.
g was added to obtain a mixture containing the medium.

Next, while circulating the medium-containing mixture with a gear pump having a variable speed, the solid medium was subjected to a pulverizing and crushing action in the gear portion for about 200 minutes so that about 80% by weight of the Bacass fiber passed 12 mesh. The pulverization and crushing of the medium-containing mixture were performed while gradually increasing the temperature of the mixture. Thereafter, the medium-containing mixture was further heated to 90 ° C. and left for 30 minutes.
By heating to 90 ° C., the enzyme was inactivated and sterilized. The obtained culture medium-containing mixture was filtered using a 60-mesh filter cloth to obtain a shiitake mushroom mycelium extract, which was concentrated to obtain a lyophilized powder.

Example 2 Preparation of Ethanol Insoluble Fraction Shiitake Mushroom Mycelium Extract Dry Powder 12 Obtained in Example 1
g was dissolved in 2 L of water and treated with Celite 545 to remove impurities. 8 L of ethanol was added thereto, and the mixture was stirred and allowed to stand. The resulting precipitate was centrifuged to obtain a precipitate. The precipitate fraction (ethanol-insoluble fraction) was a crude polysaccharide fraction, which was 20% (lyophilized weight) with respect to the shiitake mushroom mycelium extract.

Example 3 Ion Exchange Chromatography 1 g of the ethanol-insoluble fraction obtained in Example 2 was distilled water 2
After dissolving in 00 mL, diethylaminoethyl-Toyopearl ion exchange column (2.5 x 50 cm: manufactured by Tosoh Corporation)
And eluted with distilled water. After the non-ion exchanger non-adsorbed fraction that was not adsorbed to the column flowed out, the ion exchanger-adsorbed fraction adsorbed to the column was eluted with 2M NaCl. Each fraction obtained by elution was subjected to phenol-sulfuric acid method and Lowry method at wavelengths of 490 nm and 6 nm.
The chromatogram measured spectrophotometrically at 60 nm is shown in FIG. The obtained ion exchanger non-adsorbed fraction was 47.5 mg, and the ion exchanger adsorbed fraction was 51.5 mg.

Example 4: Composition analysis Shiitake mushroom mycelium extract obtained in Example 1, ethanol-insoluble fraction obtained in Example 2, ion-exchanger non-adsorbed fraction obtained in Example 3, and ion exchanger The adsorbed fraction was subjected to carbohydrate analysis by the phenol-sulfuric acid method, protein analysis by the Lowry method, and polyphenol analysis by the Folin-Denis method using gallic acid as a standard. Table 1 shows the obtained results.
Shown in

[0035]

[Table 1]

Example 5: Constituent sugar composition Hydrolyzate tube (for 3 mL, manufactured by Pierce) was put into the shiitake mushroom mycelium extract obtained in Example 1, the shiitake mushroom mycelium extract obtained in Example 2 ethanol-insoluble fraction, 1 mg of each of the ion-exchanger-non-adsorbed fraction and the ion-exchanger-adsorbed fraction obtained in Example 3 was taken, 1 mL of 4M trifluoroacetic acid was added, and the mixture was vacuum-sealed and subjected to acid hydrolysis at 100 ° C. for 3 hours. Was. Transfer the hydrolyzed liquid to an eggplant flask and concentrate under reduced pressure (rotary evaporator) until it becomes a viscous oil.
After dissolving in 2 mL of pure water, the solution was filtered with a 0.45 μm filter, and the constituent monosaccharide was quantified by a post-column labeling method using high performance liquid chromatography. Amino sugar was measured by the nitrite-indole method, and uronic acid was measured by the carbazole method. Table 2 shows the obtained results.

[0037]

[Table 2]

Example 6 In Vitro Using Primary Cultured Hepatocytes
ro test 1) Isolation and primary culture of rat hepatocytes Hepatocytes of rat (Wistar, male, 6-8 weeks old) were isolated,
The cells were cultured overnight at 37 ° C. and 5% CO ₂ . 2) Treatment of hepatocytes with CCl ₄ and test substance To the hepatocytes prepared in 1), CCl ₄ (DMSO solution: liver injury inducer) and test substance (ion exchanger non-adsorbed fraction) were prepared at various concentrations. 500 μl of the serum-free medium thus obtained was added, and the cells were cultured at 37 ° C. under 5% CO ₂ conditions. 3) GOT activity measurement Transfer the culture to an Eppendorf tube and
pm for 5 minutes at room temperature. The GOT activity in the culture supernatant was measured using a GOT measurement reagent (Liquitec GOT IF
CC: Boehringer Mannheim). The ratio of the GOT activity of each culture supernatant was determined when the average value of the GOT activity of the culture supernatant to which the test substance was not added was taken as 100, and the ratio was defined as liver injury intensity. The result is shown in FIG.

As is clear from the figure, the ion-exchanger-non-adsorbed fraction of the shiitake mushroom mycelium extract was observed to have a protective effect against liver damage.

[0040]

EFFECTS OF THE INVENTION The fraction of the shiitake mushroom mycelium extract which is not adsorbed when the ethanol-insoluble fraction of the shiitake mushroom mycelium extract of the present invention is treated with an ion exchanger and eluted with water is remarkable. It has a protective effect on liver damage and can be used for prevention and treatment of liver damage. The protective agent of the present invention can be used in combination with the administration of a drug that causes liver damage, and has no side effects. Therefore, it can be used safely, and great industrial applicability can be expected.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of ion exchange column chromatography, which is the second step of separation.

FIG. 2 is a graph showing the results of a liver damage protection test of the fraction of the present invention in an in vitro test using primary cultured rat hepatocytes.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) A61K 35/84 A23L 2/00 F (72) Inventor Reiko Sugino 3-13-35 Minitsuya Minami, Yodogawa-ku, Osaka-shi, Osaka Kobayashi Inside Pharmaceutical Co., Ltd. (72) Inventor Hideki Shirogane 3-13-35 Minitsuya Minami, Yodogawa-ku, Osaka, Osaka Prefecture Inside Kobayashi Pharmaceutical Co., Ltd. (72) Toyomi Takeuchi 3-13-35 Minitsuya Minami, Yodogawa-ku, Osaka, Osaka Kobayashi Pharmaceutical Co., Ltd. F-term (reference) 4B017 LC03 LG19 LP01 LP03 LP08 4B018 LB08 LE03 MD83 ME14 MF01 MF06 MF07 4C088 AA08 AC17 BA05 BA12 BA16 CA14 CA19 MA16 MA52 NA14 ZA75

Claims (6)

[Claims] An ethanol-insoluble fraction obtained by treating a shiitake mushroom mycelium extract with an ethanol-containing solution is treated with an ion exchanger and eluted with water. Fractions. 2. A method according to claim 1, wherein the ethanol-insoluble fraction obtained by treating the Lentinus edodes mycelium extract with an ethanol-containing solution is treated with an ion exchanger and eluted with water. Liver injury protective agent containing fractions. 3. An extract of a shiitake mushroom mycelium extract, which is obtained by treating a shiitake mushroom mycelium extract with an ethanol-containing solution, treating the ethanol-insoluble fraction with an ion exchanger, and eluted with water when eluted with water. For the treatment of liver damage and / or comprising a fraction and optionally a pharmaceutically acceptable carrier.
3. The protective agent according to claim 2, which is a prophylactic composition. 4. The protective agent according to claim 2, which is administered orally. 5. The protective agent according to claim 2, which is a food. 6. The protective agent according to claim 2, which is a beverage.