JP2005132812A - Fraction of extract from mycelium of lentinus edodes and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide fractions that contains active ingredients in Lentinus edodes, provide prophylactic and therapeutic means effective for diseases associated with oxidation substances and provide medicinal compositions, foods and beverages that can become the therapeutic means. <P>SOLUTION: The fractions of extracts from the mycelia of Lentinus edodes (Shiitake mushroom) is prepared by a method including the following steps: (1) an ethanol containing solution is added to a Shiitake mushroom mycelium extract to obtain a soluble fraction corresponding to the extract and (2) the soluble fraction is subjected to gel filtration chromatography, eluted with water to obtain an elution fraction by the water. This invention further provides medical compositions, foods, beverages, and the like, containing the fraction. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present application relates to a fraction derived from shiitake mycelium having a pharmacological effect, and a method for producing the fraction. Furthermore, the present invention relates to an oral intake composition, a pharmaceutical composition, an antioxidant, a biological membrane protective agent, a liver protective agent and the like containing the fraction.

  The usefulness of dietary fiber and edible mushrooms for increasing the incidence of various intractable diseases, adult diseases, cancer and malignant tumors in recent years has attracted attention, and various studies have been reported in this field. Among them, shiitake mushroom is a typical edible mushroom in Japan and China, and has been artificially cultivated in Japan for 300 years.

  Specific ingredients having a pharmacological action contained in shiitake mushroom include cholesterol-lowering action of eritadenine separated from the edible fruit body (see Non-Patent Document 1), anticancer action of lentinan (Non-Patent Document 2 and 3) is well known. In 1985, formulated lentinan was launched as an injection for gastric cancer.

The mycelium of shiitake mushroom is in the state of mycelium in the previous stage of the edible fruiting body, but it has attracted attention because of its good digestion and absorption and high nutritional value, and many reports on its pharmacological action have been made. Yes. Among them, in carcinogenesis experiments in rats and mice, the action of suppressing the formation of tumors in the large intestine and liver of experimental animals and the growth of transplanted tumor cells and increasing the survival rate (see Non-Patent Documents 4 and 5), antibodies An action that exhibits an inhibitory effect on immunological hepatocellular injury caused by antibody-mediated ADCC (antibody-dependent cell-mediated cytotoxicity) (see Non-Patent Document 6), an immunomodulatory action (see Non-Patent Document 7) ) Etc., there are notable reports. In addition, it has been reported that there are plant hormone-like actions (see Non-Patent Document 8) and anti-plant virus actions (see Non-Patent Documents 9 and 10) such as rooting promotion and growth promotion of crops. Yes. However, regarding the mycelium of shiitake mushroom, there have been few reports on the mechanism of its pharmacological action and specific active ingredients. In fact, the extract of cultured Lentinus edodes mycelia contains only a very small amount of 1,3-β-D-glucan, which is the main chain of lentinan, of about 0.0018%, and eritadenine. Therefore, it is highly possible that the pharmacological action of shiitake mycelium is expressed by involvement of other than these components.

  Furthermore, some reports have been made regarding the protective action and antioxidant action of the shiitake mycelium extract (see Non-Patent Document 11). In particular, regarding antioxidant activity, oxidizing substances such as active oxygen (eg, hydroxyl radical, superoxide anion, hydrogen peroxide) in vivo are known to be associated with many diseases. These include cancer, inflammation, allergic diseases, cardiovascular diseases such as arteriosclerosis and ischemic heart disease, respiratory diseases such as emphysema and asthma, gastrointestinal diseases such as gastric and duodenal ulcers, and the like. Shiitake mycelium is relatively inexpensive and economical, and has been ingested as a food for many years. Therefore, the component derived from shiitake mycelium having a useful pharmacological action is considered to be excellent in safety when administered to humans, and particularly excellent in safety when administered orally for a long period of time.

Therefore, in order to efficiently ingest active ingredients for useful pharmacological action such as antioxidant activity or biological membrane protective activity, for example, fractions of shiitake mycelium components containing active ingredients at high concentrations are used in various diseases. Therefore, there is a need for a method for efficiently preparing the fraction.
JP-A-11-228440 Journal of Atherosclerosis and Thrombosis, Vol. 9, pp. 149-156, 2002 Int. J. et al. Immunopharmamac, 10, 855-861, 1988 Immune Modulation Agents and Their Mechanisms, 409-435, 1984. Cancer Letters, 17, 109-114, 1982 Cancer Letters, 27, 1-6, 1985 Hepatobiliary pancreas, Vol. 15, pp. 127-135, 1987 Journal of the Japan Society for Pharmaceutical Sciences, Vol. 8, pp. 162-166, 1991 Plant Cell Physiol. 26, 221-228, 1985 Med. Microbiol. Immunol. 177, 235-244, 1988 Agric. Biol. Chem. 54, 1337-1339, 1990. Japanese and Chinese Journal of Pharmaceutical Sciences, Volume 19 (Special Issue), page 161, 2002

  The present inventor has intensively studied to solve such problems, and as a result, has found a method for preparing a fraction of shiitake mycelium having specific physiological activity, and further, the fraction has an antioxidant action and a biological body. The present invention was completed by discovering that it has a membrane protecting action as well as a liver protecting action.

  An object of the present invention is to provide a fraction derived from shiitake mycelium having an antioxidant action and / or biological membrane protective action, and a therapeutic and / or preventive action for liver damage, and an antioxidant containing the fraction, It is to provide a biological membrane protective agent, a liver protective agent and a composition for oral consumption, and a pharmaceutical composition containing the antioxidant, a biological membrane protective agent or a liver protective agent, a composition for oral consumption, a food and a beverage. .

  That is, according to one aspect of the present invention, it is a fraction of a shiitake mycelium extract containing polyphenols, and 1) a solution containing ethanol is added to the shiitake mycelium extract and is soluble in the solution. A step of obtaining a fraction; and 2) a step of subjecting the soluble fraction to gel filtration chromatography and elution with water, followed by elution with a solution containing methanol to obtain an elution fraction from the solution. Said fractions prepared by methods comprising are provided.

  According to the other aspect of this invention, the antioxidant containing the said precipitation fraction is provided. Furthermore, according to the other aspect of this invention, the pharmaceutical composition containing the said antioxidant is provided. Although this pharmaceutical composition is not specifically limited, For example, it can be used for the treatment and / or prevention of a liver disorder. Furthermore, according to the other aspect of this invention, the composition for food intake, a foodstuff, and a drink containing the said antioxidant are provided.

  According to another aspect of the present invention, there is provided a biological membrane protective agent comprising the precipitate fraction. Furthermore, according to the other aspect of this invention, the pharmaceutical composition containing the said biological film protective agent is provided. Furthermore, according to the other side surface of this invention, the composition for food intake, foodstuff, and a drink containing the said biological film protective agent are provided.

  According to another aspect of the present invention, a hepatoprotectant comprising the precipitate fraction is provided. Furthermore, according to the other aspect of this invention, the pharmaceutical composition containing the said liver protective agent is provided. Furthermore, according to the other aspect of this invention, the composition for food intake, a foodstuff, and a drink containing the said liver protective agent are provided.

According to still another aspect of the present invention, a method for preparing a fraction of a shiitake mycelium extract containing polyphenols, comprising:
1) a step of adding a solution containing ethanol to a shiitake mycelium extract to obtain a soluble fraction in the solution; and 2) subjecting the soluble fraction to gel filtration chromatography and elution with distilled water. Elution with a solution containing methanol to obtain an elution fraction from the solution;
Is provided.

Hereinafter, the present invention will be described more specifically.
The shiitake mycelium in the present invention may be produced by culture or collected from nature. For example, a mycelium obtained by cultivating shiitake fungus in a solid medium can be used. The shiitake mycelium extract used in the present invention can be prepared by a method known in the art. For example, it is obtained by pulverizing and decomposing a solid medium containing mycelium in the presence of water and an enzyme. An extract can be used. The shiitake mycelium extract is not limited, but for example, an extract obtained by the following method can be used. In other words, a solid medium containing mycelia obtained by inoculating shiitake fungus on a solid medium based on bagasse (sugar cane scum) and defatted rice bran, and then growing mycelium has a weight of 30 mesh. %, And one or more of water and one or more enzymes selected from cellulase, protease, or glucosidase are added to the unbound solid medium while maintaining the solid medium at a temperature of 30 to 55 ° C. In addition, the solid medium is pulverized and ground in the presence of the enzyme so that at least 70% by weight or more of the bagasse fiber is passed through 12 mesh, and then the enzyme is deactivated by heating to a temperature up to 90 ° C. And sterilizing, 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 as a powder, and use it in various forms when used. The powder obtained by freeze-drying is a brown powder, is hygroscopic and has a unique taste and smell. The thus obtained shiitake mycelium extract has a saccharide content of 15 to 50%, preferably 20 to 40% (w / w) by saccharide analysis by the phenol-sulfuric acid method, and protein by 10% by protein analysis by the Lowry method. -40%, preferably 13-30% (w / w), 1-5% polyphenols by the Folin-Denis method with gallic acid as standard, preferably 2.5-3.5% (w / w) Including. Shiitake mycelium extract includes, but is not limited to, about 0.1% lipid, about 0.4% fiber, and about 20% ash.

  An example of the constituent sugar composition (%) of the shiitake 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 sugar: 12.0; uronic acid: 11.3.

  The “solution containing ethanol” in the present invention is a solution containing ethanol in a volume ratio of 20 to 80%, preferably 30 to 70%, more preferably 40 to 60%, and most preferably 45 to 55%. Means. Although it does not specifically limit as said solvent, For example, water, methanol, n-propanol, isopropanol, acetic acid, acetone, and mixtures thereof can be used, 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 in the solution. The time for standing still is not limited, but may be, for example, 6 hours to 100 hours, preferably 24 hours to 84 hours, and more preferably 60 hours to 84 hours. Moreover, in order to shorten the time required for preparation, the mixture may be heated and / or stirred as necessary. The soluble fraction obtained by removing the insoluble matter in the mixture by a method such as filtration or decantation can be used as a solution as it is, but can be obtained by concentrating and / or lyophilizing as necessary. The resulting concentrate, viscous material or solid can be used as the soluble fraction in the next step.

  The gel filtration column chromatography in the present invention can be performed by a usual method known in the technical field of the present invention. The gel filtration column chromatography in the present invention can be performed using a carrier usually used in the technical field. Specific examples of the carrier include Sephadex LH-20, Sephadex G-20, Sephacryl S-100HR, and the like. The water used as the eluent of the gel filtration column chromatography is distilled water, pure water, ultrapure water or the like, and preferably distilled water is used. The “solution containing methanol” in the present invention means a solution containing 50 to 100% by volume of methanol in a solvent, preferably 70 to 100%, more preferably 90 to 100%, preferably commercially available. A 99.8% methanol solution can be used. Although it does not specifically limit as said solvent, For example, water, ethanol, n-propanol, isopropanol, acetic acid, acetone, and mixtures thereof can be used, Preferably water can be used. The fraction of the present invention is obtained as an elution fraction of the solution containing methanol. Concentrated liquids, viscous substances and solids obtained by subjecting the eluted fractions to treatment such as vacuum concentration, lyophilization, and further purification such as dialysis are also included in the present invention.

  Although a fraction containing a polar substance having a relatively low molecular weight can be generally obtained by purification by gel filtration column chromatography in the present invention, the present invention is not limited to such a fraction. The fraction of the present invention may contain, for example, polyphenols (including phenols and phenol), proteins, and saccharides. As used herein, the term “polyphenols” means all phenol components quantified by the Folin-Denis method, which is a known method for detecting phenols, and includes phenol. The term “phenol” means a general term for compounds in which a hydrogen atom of an aromatic hydrocarbon nucleus is substituted with a hydroxyl group.

  The fraction of the present invention comprises, for example, 11 to 35%, preferably 12 to 30% (w / w), more preferably 13 to 20% of polyphenols quantified by the Folin-Denis method using catechin as a standard substance. (W / w), for example, 12% or more. Although not particularly limited, for example, polyphenols include 5-hydroxymethylfurfural, protocatechuic acid (3,4-dihydroxybenzoic acid) and phenols such as methyl gallate (polyphenol or polyhydric phenol), syringic acid and vanillin Phenols such as acids are included.

  The fraction is, for example, 60-80%, preferably 65-85% (w / w) of protein quantified by protein analysis by Lowry method, for example, saccharide quantified by carbohydrate analysis by phenol-sulfuric acid method. It may contain 0-20% quality, preferably 5-15% (w / w).

  The term “antioxidant” as used herein generally eliminates or attenuates the action of oxidizing substances such as active oxygen (eg, hydroxyl radicals, superoxide anions, hydrogen peroxide) in vivo. Means a drug. Diseases resulting from tissue damage due to active oxygen are not limited, but include cancer, inflammation, allergic diseases, cardiovascular diseases such as arteriosclerosis and ischemic heart disease, respiratory diseases such as emphysema and asthma, gastric ulcers And gastrointestinal diseases such as duodenal ulcer, diabetic complications and skin diseases. For example, the antioxidant of the present invention can be expected to be effective in preventing and / or treating liver damage, skin diseases and aging.

  As used herein, the term “biological membrane” means a membrane surrounding various cells and organelles. Cells include various organs, organs, tissue cells, blood cells, germ cells and the like. An organelle is a cytoplasmic component having a certain function, and includes the Golgi apparatus, mitochondria, central body, ribosome, endoplasmic reticulum, lysosome, nuclear membrane, and the like.

  As used herein, “biological membrane protection” means stabilization of biological membranes, protection of biological membranes from injuries and damages originating from various causes, prevention and treatment of injuries and damages of biological membranes, etc. Including doing. Diseases that are thought to result from damage or damage to biological membranes include, but are not limited to, hypercholesterolemia, coronary artery disease, and the like. For example, the increase in serum AST and ALT, which is an index at the time of liver injury, is considered to be the result of intracellular AST and ALT flowing out of the cell due to liver cell membrane injury. Improvement can be expected.

  As used herein, “hepatoprotective agent” generally refers to liver damage caused by drugs or harmful substances, immunological liver damage, viral liver damage, fatty liver, liver cancer, alcoholic liver damage, cirrhosis, etc. It means anything that suppresses the decrease in liver function due to, and everything that protects the liver.

  The antioxidant, biological membrane protective agent and liver protective agent of the present invention can be used as an active ingredient of a pharmaceutical composition. The pharmaceutical composition can be used in various dosage forms such as tablets, capsules, powders, granules, pills, solutions, emulsions, suspensions, solutions, spirits, syrups, for oral administration. Although it can be set as an extract and an elixir, it is not limited to these. The pharmaceutical composition may contain various commonly used components, such as 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, buffering agents, binders, stabilizers, coating agents and the like can be included. The pharmaceutical composition of the present invention may be in a sustained or sustained release dosage form.

  The dosage of the antioxidant, biological membrane protective agent and liver protective agent of the present invention can be appropriately selected depending on the patient's body shape, age, physical condition, degree of disease, elapsed time after onset, etc. The pharmaceutical composition can comprise a therapeutically effective amount and / or a prophylactically effective amount of an antioxidant, a biomembrane protecting agent and / or a liver protecting agent. For example, the fraction of the shiitake mycelium extract of the present invention is generally used at a dose of 10 to 50000 mg / day / adult, preferably 100 to 5000 mg / day / adult. The pharmaceutical composition may be administered in a single dose or multiple doses, and may be used in combination with other drugs such as other antioxidants, biological membrane protective agents, and liver protective agents.

  In addition, the composition for oral consumption of the present invention can be used as a functional food as it is, as well as as a component of a pharmaceutical product, quasi-drug, food and drink, a food additive, and the like. The use enables daily and continuous ingestion of the composition for oral intake having the antioxidant effect, biological membrane protecting effect and liver protecting effect of the present invention, and effective antioxidant action and biological membrane protecting action. In addition, it is possible to improve the constitution by protecting the liver, treat diseases caused by oxidants, and prevent their onset. Examples of foods or beverages containing the antioxidant, biological membrane protective agent or liver protective agent of the present invention include functional foods, antioxidants, biological membrane protective effects or liver protective effects, health foods, general foods (juices , Sweets, processed foods, etc.), nutritional supplements (nutrient drinks, etc.). The food or beverage of the present invention is not limited, but in addition to fractions of shiitake mycelium extract, inorganic components such as iron and calcium, various vitamins, dietary fibers such as oligosaccharides and chitosan, soybean extract Proteins such as food, lipids such as lecithin, and sugars such as sucrose and lactose.

  As shown in the following examples, it was revealed that the fraction of the present invention has an antioxidant effect, a biological membrane protecting effect, and a liver protecting effect. In particular, the present invention also has a hepatocyte protective effect against various hepatotoxins (necrosis-inducing hepatotoxin, apoptosis-inducing hepatotoxin, etc.) and a liver protecting effect resulting from the effect. Accordingly, the present invention provides an effective means for the treatment and / or prevention of oxidant-related diseases, diseases caused by biological membrane damage, and liver diseases.

EXAMPLES Hereinafter, although the preferable Example of this invention is described in detail, this invention is not limited to these Examples.
[Example 1] Preparation method of 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, inoculation with shiitake mycelia and controlling the temperature and humidity The mycelium was allowed to grow in the room. After the mycelium spread on the solid medium, the bagasse-based fibrin was bunched so that the 12-mesh passage was 24% by weight or less. To 1.0 kg of the unbundled medium, 3.5 L of pure water was added, and 2.0 g of purified cellulase was added while maintaining the temperature at 40 ° C. to obtain a medium-containing mixture. Next, the medium-containing mixture was circulated by a gear pump with a speed change, and the grinding and grinding action in the gear portion was added to the solid medium for about 200 minutes, so that about 80% by weight of bagasse fibers became 12 meshes. The medium-containing mixture was pulverized and ground while gradually raising the temperature of the mixture. Thereafter, the medium-containing mixture was further heated to 90 ° C. for 30 minutes to inactivate the enzyme and sterilized. The obtained medium-containing mixture was filtered using a 60 mesh filter cloth and concentrated, and then a lyophilized powder was obtained as a shiitake mycelium extract (LEM).

[Example 2] Preparation of fractions of shiitake mycelium extract and identification of components 2-1 Preparation of fractions of shiitake mycelium extract 50% of shiitake mycelium extract (LEM) Extraction was performed by adding to an aqueous ethanol solution and allowing to stand for 3 days. The insoluble fraction was filtered off, and the extract, which was a soluble fraction, was filtered under reduced pressure, and then freeze-dried, and then the extract of Shiitake mycelium was extracted at a yield of about 90% by weight with a brown 50% aqueous ethanol solution. (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). The obtained ES fraction 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 first used as a mobile phase, followed by elution with 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 component composition ratio of fraction of shiitake mycelium extract The component composition ratio of each obtained fraction was calculated by the following quantitative analysis method. That is, the sugar is the phenol sulfuric acid method (Methods in Carbohydrate Chemistry RL Whistler and ML Wolfrom, Academic Press, New York, Vol. 1, page 388, 1962), protein is Low. The BCA method (see Anal. Biochem. Vol. 150, p. 76, 1985), which is an improved method of the method, and the polyphenols are the Folin-Denis method (Dietary Tannins: Consequences and remedias, CRC Press, Vol. 13, 1989). Quantified by year). The measurement conditions of the Folin-Denis method are as follows.

Standard substance: Catechin Preparation of calibration curve: Created from one point of catechin of arbitrary concentration and origin Measurement wavelength: 760 μm
Test solution concentration: 0.2% (W / W)
Table 1 shows an example of component measurement results for shiitake mycelium extract (LEM), 50% aqueous ethanol solution extract (ES), and methanol-eluted fraction (ES-Me). From the measurement results shown in Table 1, the shiitake mycelium extract and the 50% aqueous ethanol extract fraction have almost the same component composition ratio, but the composition ratio of protein and polyphenols is remarkably high in the methanol elution fraction. Became clear. However, this composition may vary depending on shiitake mycelium culture conditions.

  Table 1. Component ratio (wt%) for shiitake mycelium extract (LEM), 50% aqueous ethanol extract fraction (ES) and methanol elution fraction (ES-Me)

2-3 Identification of components of fraction of shiitake mycelium extract The obtained ES-Me fraction was dissolved in a solution of ethanol: methanol = 1: 1 and used as a sample, and open column chromatography (LH-20gel, flow rate) : 1.5 ml / min), and dispensed into 122 test tubes. About these test tubes, the light absorbency and DPPH radical scavenging activity in wavelength 280nm were measured, and it divided into four fractions based on the result. The fraction was subjected to HPLC analysis under the following conditions, and each phenol was identified from the retention time.
Column: Develosil ODS HG-5 (4.6 mm ID x 250 mm)
Flow rate: 10 ml / min Temperature: 35 ° C
Eluent: Methanol / 0.01% TFA = 5/95 to 90/10 (45 minutes)
As a result, in the fraction having the strongest DPPH radical scavenging activity, syringic acid (RT: 20.7 min), vanillic acid (RT: 19.8 min), protocatechuic acid (3,4-dihydroxybenzoic acid), gallic acid It was confirmed that methyl was contained. In addition, it was confirmed that 5-hydroxymethylfurfural (RT: 10.9 minutes) was contained in the second most active fraction.

[Example 3] Effect of fraction of shiitake mycelium extract on lipid peroxidation of liver microsomes 3-1 Separation and preparation of liver microsomes 10-week-old Wistar male rats fasted for 24 hours were removed under light ether anesthesia. After the blood was sacrificed and the liver was refluxed with chilled physiological saline, a liver homogenate solution was prepared with physiological saline. The liver homogenate was centrifuged at 10,000 × g for 10 minutes, the supernatant was centrifuged at 100,000 × g for 60 minutes, the precipitate was washed with deionized distilled water, and then 0.1 M Tris-HCl buffer. After suspending in a liquid (pH 7.4), it was uniformly dispersed again with a homogenizer to obtain a microsomal fraction suspension. The protein concentration was measured by the BCA method (see Anal. Biochem. Vol. 150, p. 76, 1985) to prepare a suspension of 5 mg protein / ml.
3-2 Lipid Peroxidation by Liver Microsomes The specific experimental procedure was carried out with reference to the method of Fukuzawa et al. (See Vitamins, 63, 215, 1989). 0.1 ml of a test substance solution, 0.1 ml of 0.1 M Tris-HCl buffer (pH 7.4), 0.6 ml, and 0.1 ml of a microsome solution (5 mg protein / ml) prepared in 3-1. 5 mg) 0.1 ml of ADP · Fe ³⁺ solution consisting of a mixed solution of 1.7 mM ADP · Na and 0.1 mM FeCl ₃ (final concentration ADP 0.17 mM FeCl ₃ 0.01 mM), and 1 mM NADPH Was mixed with 0.1 ml (final concentration: 0.1 mM), incubated at 37 ° C., and the lipid peroxide value was measured at the start of incubation (0 minutes) and 5 minutes, 15 minutes and 30 minutes after the start.
3-3 Lipid Peroxide Amount Measurement Buege and Aust's method (see Meth. Enzymol. 52, 302-310, 1978), which is an improvement on the TBA method, is used with the absorbance at 535 nm as an index. The amount of oxidized lipid was measured. That is, 1 ml of the total reaction solution, 2 ml of TBA reagent, and 30 μl of 50 mM BHT ethanol solution were added to the centrifuge tube and incubated at 100 ° C. for 15 minutes. After incubation, the mixture was quickly cooled and centrifuged at 3000 rpm for 10 minutes, and then malondialdehyde (MDA) produced in the supernatant was measured using a spectrophotometer (OD 535 nm). The measurement results are shown in FIG. The value of the control MDA markedly increased to about 3 nmol / ml 5 minutes after the start of the reaction, and was almost the same value until the end of the reaction 30 minutes later. L. E. M.M. And ES-Me suppressed the lipid peroxidation reaction of this microsome, and suppression by ES-Me was especially remarkable. That is, L. E. M.M. However, the final concentration of 1 mg / ml showed almost 100% inhibition, but decreased to 85% at 0.5 mg / ml, 60% at 0.25 mg / ml, and 45% at 0.125 mg / ml. On the other hand, ES-Me showed almost 100% inhibition even in the solution with the final concentration of 0.125 mg / ml, which is the lowest concentration.
Example 4 Measurement of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity S. It was measured by a modification of the method of Blois et al. (Nature, 181; 1199-1200, 1958). A reaction solution in which 50 mM 2-morpholinoethanesulfonic acid (MES) buffer, ethanol, and 0.5 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) were mixed at a ratio of 2: 2: 1, respectively. After incubating at 25 ° C., the samples were mixed so that the final concentrations were 0.05, 0.1, 0.2, and 0.4 mg / ml, respectively, and the absorbance at 517 nm was measured. The absorbance when the sample was added to the reaction solution was Abs (sample), and the absorbance when water was added instead of the sample solution was Abs (control).

The measurement results are shown in FIG. L. E. M.M. The DPPH radical scavenging activity is less than 10% at a concentration lower than the final concentration of 0.1 mg / ml, whereas ES-Me shows about 80% radical scavenging activity up to a concentration of 0.1 mg / ml, 0.05 mg An activity of about 40% was observed even in a / ml solution.
[Example 5] Measurement of O ₂ radical (O ₂ ·-) scavenging activity R. This was carried out with reference to the method of Buettner (see Free. Rad. Biol. Med. Vol. 3, pp. 259-303, 1987). Using an ESR apparatus (JES-RE1X ESR spectrometer: manufactured by JEOL Ltd.), the O ₂ radical (O ₂ .−) scavenging activity generated by hypoxanthine and xanthine oxidase in vitro was determined by L. E. M.M. , ES and ES-Me.

Take 50 μl of 2.0 mM hypoxanthine (phosphate buffer solution, pH 7.4) and 35 μl of 5.5 mM diethylenetriaminepentaacetic acid (DETAPAC) (phosphate buffer solution, pH 7.4) in a test tube to various concentrations. L. E. M.M. , ESR ES, an aqueous suspension 50 [mu] l of ES-Me addition, further the 4.0 M DMPO20myueru, and added 0.4unit / ml of xanthine oxidase (XOD) 50 [mu] l, the value of O ₂ radicals generated after stirring It measured using. Measurement conditions are magnetic field 335.1 ± 5 mT, Mn: 566, field modulation: 0.79 × 0.1, time constant: 0.03 seconds, sweep time: 1 minute, Power: 8, Gain: × 500 did. L. E. M.M. , ES and ES-Me were added to final concentrations of 0.4, 0.2, 0.1 and 0.05 mg / ml, respectively. The value of O ₂ radical scavenging activity was converted to the activity value of superoxide dismutase (SIGMA chemical co. USA). The measurement results are shown in FIG. O ₂ radicals generated from hypoxanthine and xanthine oxidase in vitro are described in L. E. M.M. , ES and ES-Me were added, and it became clear that ES-Me showed remarkable activity. The titer is converted into the value of SOD, E. M.M. , ES and ES-Me were added at 0.05 mg / ml (final concentration), respectively, and were 1.70, 1.59, 8.00 unit / ml. Thereafter, when the addition concentration of the sample was increased, the O ₂ radical scavenging activity increased in a concentration-dependent manner, and the activity in the ES-Me 0.4 mg / ml solution was 42.10 unit / ml.
[Example 6] Measurement of erythrocyte hemolytic protection 6-1 Preparation of erythrocyte suspension Blood was collected from 10-week-old Wistar male rats (heparin added as an anticoagulant), washed twice with physiological saline, and isotonic. Dilute with buffer (NaH ₂ PO ₄ .2H ₂ O: 0.26 g, NaHPO ₄ : 1.15 g, NaCl: 8.00 g of H ₂ O: 1000 ml, pH 7.4) to 40% (V / V) The erythrocyte suspension was prepared.
6-2 Hypotonic hemolytic protective action According to the method of Abe et al. (See Naunyn-Schmiedebergs Arch. Pharmacol. 316, 262-265, 1981), various concentrations of L. E. M.M. Hypotonic buffer containing ES and ES-Me (sodium phosphate (10 mM) buffer containing 56 mM NaCl, pH 7.4) and L. at the same concentration. E. M.M. 3.0 ml each of isotonic buffer containing ES and ES-Me, and 3.0 ml each of hypotonic buffer containing no test drug and isotonic buffer containing no test drug as controls Four sets of 4 were simultaneously added with 20 μl of erythrocyte suspension and incubated at room temperature for 10 minutes. Immediately after the incubation, the mixture was centrifuged at 3000 rpm for 3 minutes, and the absorbance (540 nm) of the supernatant was measured. Absorbance of the hypotonic buffer with the sample added is Abs (sample hypotonic buffer), and the absorbance of the isotonic buffer with the sample added is Abs (sample isotonic buffer). Absorbance when water is added is Abs (control hypotonic buffer), and absorbance when water is added to isotonic buffer instead of sample solution is Abs (control isotonic buffer). The hemolysis rate in each solution when the hemolysis rate in the hypotonic buffer not containing was defined as 100 was calculated from the following equation.

The measurement results are shown in FIG. In each graph of FIG. 4, a hemolysis rate of 100 or less indicates a hemolysis protection effect, and a value of 100 or more indicates a hemolysis promoting effect. L. E. M.M. No effect on hypotonic hemolysis was observed at any of the 6 concentrations from the final concentration of ES fraction to 0.4 to 0.013 mg / ml. On the other hand, in ES-Me, about 6% in a 0.025 mg / ml solution, about 11% in a 0.05 mg / ml solution, about 12% in a 0.10 mg / ml solution, and in a 0.20 mg / ml solution. About 43% of hemolytic protection was observed. When the osmotic pressure of the hypotonic solution at this time was measured, the control was 143 mOsM, ES-Me 0.4 mg / ml added hypotonic solution was 143 mOsM, and the addition of ES-Me did not affect the osmotic pressure. I understood it. From the above results, ES-Me E. M.M. It was confirmed to have a biological membrane protective effect in a hypotonic solution not found in ES and ES.
6-3 Protective effect on heat hemolysis According to the method of Abe et al. (See Naunyn-Schmiedebergs Arch. Pharmacol. 316, 262-265, 1981), various concentrations of LEM, ES and ES-Me were added to test tubes. 2 test tubes containing 3.0 ml of isotonic buffer solution (10 mM phosphate buffer containing 154 mM NaCl, pH 7.4) (a) and, as a control, isotonic solution containing no test drug Prepare a total of 4 tubes (b) with 2 tubes (3.0 ml) containing 3.0 ml of buffer solution, and simultaneously add 20 μl of erythrocyte suspension, each of (a) and (b) is 54. Incubate for 20 minutes in a 5 ° C. water bath and the remaining one at room temperature. Immediately after the incubation, the mixture was ice-cooled, centrifuged at 3000 rpm for 3 minutes, and the absorbance (540 nm) of the supernatant was measured. Abs (sample heating) is the absorbance when the sample solution is heated, Abs is the absorbance of the sample solution treated at room temperature, Abs is the absorbance of the solution heated by adding water instead of the sample solution (control) Heat), and the absorbance of the solution treated at room temperature by adding water instead of the sample solution is Abs (control non-heated), and the hemolysis rate in the isotonic buffer solution not containing the test drug is 100 The hemolysis rate in the solution was calculated from the following formula.

L. E. M.M. In ES and Me, a hemolytic protective effect was observed at a concentration of 0.05 mg / ml or higher, whereas in ES-Me, hemolytic protective activity was observed at a concentration of 0.013 mg / ml or higher, and 0.025 mg / ml. And about 39% of hemolytic protection was observed. Moreover, the result of FIG. 5 suggests that there is an optimum concentration for the hemolytic protective action of ES-Me.
[Example 7] Measurement of hepatocellular protective effect against toxicity of bromobenzene Hepatocytes isolated from rats were seeded on polystyrene culture plates at a density of 1 x 10 ⁵ cells / cm ² , and 24% in WE medium containing 10% serum. Primary culture for hours. When the medium was exchanged for 24 hours after seeding, 1 mM of bromobenzene (BB), a necrosis-inducing liver toxin, and shiitake mycelium extract (LEM) (0.2, 0.00) as a hepatoprotective substance. 1 and 0.05 mg / ml) and methanol elution fractions (ES-Me) (0.1, 0.05 and 0.01 mg / ml) were added. 24 hours after the addition of the added solvent, the number of viable cells was counted using a cell count method using trypan blue.

  The measurement results are shown in FIG. L. E. M.M. As for ES-Me, no hepatocyte protective effect was confirmed at 0.05 mg / ml, whereas for ES-Me, a protective effect was confirmed up to a minimum concentration of 0.01 mg / ml.

It is a graph which shows the measured value of the generation amount of lipid peroxide by the lipid peroxidation reaction of a liver microsome, Compared with a shiitake mycelium extract (LEM), a methanol elution fraction (ES-Me) Indicates that the lipid peroxidation reaction is more remarkably suppressed. It is a graph which shows the measurement result of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, and compared with a shiitake mycelium extract (LEM), a methanol elution fraction ( ES-Me) has higher radical scavenging activity. O ₂ radical (O ₂ ^{· -)} is a graph showing the conversion value measurements of scavenging activity to activity of superoxide dismutase scavenging activity, shiitake mycelium extract (L.E.M.) and 50% ethanol This shows that the methanol-eluted fraction (ES-Me) has higher O ₂ radical scavenging activity than the aqueous solution extracted fraction (ES). FIG. 4 is a graph showing the results of measuring the erythrocyte hemolysis rate in a hypotonic solution, and the methanol-eluted fraction (ES-Me) is a shiitake mycelium extract (LEM) or 50% ethanol. It shows that it has a biological membrane protective effect in a hypotonic solution not in the aqueous solution extraction fraction (ES). It is a graph which shows the result of having measured the erythrocyte hemolysis rate on heating conditions, and a methanol elution fraction (ES-Me) is a shiitake mycelium extract (LEM) or 50% ethanol aqueous solution extraction fraction ( It shows that the biological membrane protecting effect is exhibited under heating conditions at a lower concentration than ES). FIG. 6 is a graph showing the results of measuring the effect of protecting hepatocytes against the toxicity of bromobenzene, in which the methanol elution fraction (ES-Me) is compared with the shiitake mycelium extract (LEM). This shows that the effect is exhibited at a lower concentration.

Claims (17)

A fraction of a shiitake mycelium extract containing polyphenols,
1) a step of adding a solution containing ethanol to a shiitake mycelium extract to obtain a soluble fraction in the solution; and 2) subjecting the soluble fraction to gel filtration chromatography and elution with water. Elution with a solution containing methanol to obtain an elution fraction from the solution;
Said fraction which can be prepared by a method comprising   The antioxidant containing the fraction of Claim 1.   A pharmaceutical composition for treating or preventing a liver disorder, comprising the antioxidant according to claim 2.   A composition for oral consumption comprising the antioxidant according to claim 2.   A food comprising the antioxidant according to claim 2.   A beverage comprising the antioxidant according to claim 2.   A biofilm protective agent comprising the fraction according to claim 1.   A pharmaceutical composition for biological membrane protection comprising the biological membrane protective agent according to claim 7.   A composition for oral consumption comprising the biofilm protective agent according to claim 7.   A food comprising the biological membrane protective agent according to claim 7.   A beverage comprising the biofilm protective agent according to claim 7.   A hepatoprotectant comprising the fraction according to claim 1.   A pharmaceutical composition comprising the hepatoprotective agent according to claim 12.   A composition for oral consumption comprising the liver protective agent according to claim 12.   A food comprising the liver protective agent according to claim 12.   A beverage comprising the liver protective agent according to claim 12. A method for preparing a fraction of a shiitake mycelium extract containing polyphenols, comprising:
1) a step of adding a solution containing ethanol to a shiitake mycelium extract to obtain a soluble fraction in the solution; and 2) subjecting the soluble fraction to gel filtration chromatography and elution with water. Elution with a solution containing methanol to obtain an elution fraction from the solution;
Including said method.

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