JP2010030955A - Antimutagenic agent, production process and usage therefore - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition in the form capable of developing an antimutagenic agent containing an ingredient which can strongly inhibit mutagenic properties due to mutagenic substances, and capable of using the antimutagenic agent industrially effectively. <P>SOLUTION: The antimutagenic agent contains, as an effective ingredient, an ampholyte-containing fraction obtained through ion exchange chromatofocusing of a soluble material separated from a hot water-extract of Agaricus blazei Murrill with hydrous ethanol, the ampholyte-containing fraction having more preferably a molecular weight in a range of 120-280, or a fraction containing saccharides and an acidic substance. Foods and beverages, feedstuffs and medicines each containing the antimutagenic agent are also provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an antimutagenic agent comprising a specific antimutagenic component, a method for producing the same, and use thereof.

  Factors that cause mutations in the genes and chromosomes of living organisms and cause abnormalities in cells and tissues in the body, diseases such as cancer are called mutagens, mutagens, mutagens, or genotoxic substances. The nature and action of is called mutagenicity. There are many mutagens in the daily living environment, for example, there are radiation, ultraviolet rays, active oxygen, etc. in nature, and there are dioxins and phthalates as chemical substances. Nitroso compounds, benzpyrene, tryptophan degradation products, and the like generated by cooking, food intake, smoking and the like are known.

  On the other hand, the development of substances and components that have the action of suppressing or preventing the mutagenicity of such mutagens has been studied in the past, and such useful substances are used in foods and foods that are consumed daily. It is also known that ingredients are included. Vitamin C, β-carotene, quercetin, and the like of vegetables and fruits are examples, and basidiomycetes represented by edible mushrooms are said to contain components having a mutagenicity-inhibiting effect. That is, free unsaturated fatty acid extracted from the fruiting body of Agaricus brazi has an antimutagenic action on benzo [a] pyrene (Patent Document 1), edible basidiomycetes such as Ganoderma lucidum or its extract and supplement The combination with a substance has antimutagenicity against Trp-P2 (3-amino-1-methyl-5H-pyrido [4,3-b] indole) (Patent Document 2), and The fraction obtained by chromatographic fractionation of the external liquid obtained by dialyzing the hot water extract of the substance is a carcinogen such as urethane (ethyl carbamate), NNK [(4-N-methyl-N-nitrosamino) -1 It has been proposed to suppress the mutagenicity of-(3-pyridyl) -1-butanone] and suppress tumors caused by AOM (azoxymethane) (Patent Document 3).

Agaricus mushroom is a kind of agaric mushroom, belongs to the genus Agaricus, and typical examples include Agaricus blazei Murrill, mushroom (Agaricus bisporus) and the like. Agaricus blazei murril is a potato native to Brazil, also known as Kawariharatake, Himematsutake, etc., and has recently been artificially cultivated in Japan, and is used as a food supplement for food. It is known that polysaccharides (β-glucan) and polysaccharide protein complexes, which are contained components, have actions such as antitumor growth inhibition, immune enhancement, and blood glucose level reduction. Mushrooms have other names such as tsutsutake mushrooms and champignons and are used as general foods, and the extract is said to have an active oxygen scavenging ability.
Japanese Unexamined Patent Publication No. 6-90773 JP-A-8-266246 Republished WO2004 / 004748

  Those having the above-mentioned mutagenicity-inhibiting action generally have the effect of suppressing the mutagenicity of a specific mutagen, but exhibit the desired effect for other types of mutagens. In many cases, the mechanism of action that inhibits the mutagenicity of mutagens has not been universally elucidated, and it is difficult to predict the versatility of antimutagenic mutagenicity-inhibiting effects. It was supposed to be. In addition, even though it is known that useful ingredients showing antimutagenicity are present in food materials such as vegetables, fruits, mushrooms, etc., the mutagenicity suppressing component contained in the raw materials is usually extremely small, There is little knowledge that the component is specified, and because it is a natural product, there is a large variation in the useful component depending on the production area and harvest time, and the manufacturing cost increases even when processed into an extract or purified product. The fact is that useful components have not necessarily been effectively utilized industrially.

  In view of the current situation, the present inventors have developed a mutagenic inhibitor that can remarkably suppress or prevent mutagenicity caused by mutagens particularly related to food and drink, and effectively utilize this industrially. It was made into the subject to provide the composition of the aspect which can be performed.

  In order to achieve the above-mentioned problems, the present inventors have made extensive studies on the anti-mutagenic component contained in Agaricus moth, and as a result, treated with Agaricus moth in a specific manner, has a remarkable mutagenicity suppressing effect. It has been found that a component exhibiting the above can be obtained, and it has been found that it can be effectively used in industrial fields such as foods, drinks, feeds, pharmaceuticals, etc., and the present invention has been completed.

  That is, the object of the present invention is to extract (a) the first step of extracting agaricus koji with hot water, (b) extracting the hot water extract obtained in the first step with water-containing ethanol having a water content of 10 to 60% by mass. The second step, (c) the third step of fractionating the water-soluble ethanol-soluble material obtained in the second step by ion exchange chromatography, and (d) the amphoteric electrolyte-containing fraction fractionated in the third step, Alternatively, it is achieved by providing an amphoteric electrolyte obtained through the fourth step of collecting a saccharide and acidic substance-containing fraction, or an antimutagenic agent containing saccharide and acidic substance as active ingredients.

  Here, the water content of the water-containing ethanol in the second step is preferably 20 to 40% by mass. The main component of the amphoteric electrolyte-containing fraction in the fourth step preferably has a molecular weight in the range of 120 to 280. Further, the mutagen targeted by the anti-mutagenic agent is N-nitrosodimethylamine (hereinafter abbreviated as NDMA) or N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter abbreviated as MNNG). It is desirable that

  Moreover, according to this invention, in order to achieve the said subject, the process shown to following (a)-(d), ie, (a) 1st process of extracting Agaricus soup with hot water, (b) 1st The second step of extracting the hot water extract obtained in one step with water-containing ethanol having a water content of 10 to 60% by mass, (c) fractionation of the water-soluble ethanol soluble product obtained in the second step by ion exchange chromatography Step (3), and then (d) the amphoteric electrolyte-containing fraction collected in the third step, or the amphoteric electrolyte-containing fraction or saccharide obtained through the fourth step of collecting the saccharide and acidic substance-containing fraction. And a method for producing an antimutagenic agent, comprising producing an acidic substance-containing fraction as an active ingredient.

  In this production method, the water content of the water-containing ethanol in the second step is preferably 20 to 40% by mass, and the main component of the amphoteric electrolyte fraction in the fourth step belongs to a molecular weight range of 120 to 280. It is desirable that the mutagen targeted by the antimutagenic agent is NDMA or MNNG.

  Furthermore, according to this invention, the food-drinks, feed, or pharmaceutical characterized by including the said antimutagenic agent are provided.

  According to the present invention, a fraction containing an amphoteric electrolyte contained in agaricus koji that has been conventionally used for food and drink, more preferably a fraction having a molecular weight in the range of 120 to 280, more preferably a molecular weight. 123 and / or 137, 242, or 260, and most preferably, an antimutagenic agent comprising a component having a molecular weight of 260 or a fraction containing a saccharide and an acidic substance as an active ingredient is provided. When the active ingredient is the amphoteric electrolyte-containing fraction, this antimutagenic agent is contained especially in the burned portions of livestock meat and seafood, and carcinogenic initiation of the liver and kidneys if continuously ingested in large quantities. It has the effect of remarkably suppressing the mutagenicity of NDMA acting as an agent. In addition, when the active ingredient is the above-mentioned fraction containing saccharides and acidic substances, MNNG mutagen, which is a kind of nitroso compound as well as NDMA, and is known to be a carcinogen that induces gastric cancer and colon cancer. Has the effect of inhibiting sex. Therefore, the antimutagenic agent of the present invention is induced by NDMA or MNNG, particularly in the fields of foods and drinks, feeds, pharmaceuticals, etc., in the form as it is or contained in various conventional products. It can be effectively used to prevent mutagenicity, chromosomal abnormalities, body organ abnormalities, carcinogenesis, and the like.

The present invention is described in detail below. First, the antimutagenic agent of the present invention contains an amphoteric electrolyte-containing fraction collected through the following steps (a) to (d), or a saccharide and acidic substance-containing fraction as an active ingredient. There are features.
(A) First step of extracting Agaricus koji with hot water (b) Second step of extracting the hot water extract obtained in the first step with hydrous ethanol having a water content of 10 to 60% by mass (c) Second Fraction containing the amphoteric electrolyte fractionated in the third step (d) third step, or fraction containing saccharides and acidic substances, fractionating the aqueous ethanol-soluble material obtained in the step by ion exchange chromatography The fourth step to collect

  In the first step, the fruit body of a basidiomycete belonging to the genus Agaricus such as Agaricus brazeimuril or mushroom or mycelium can be used as the Agaricus spp., But the fruit body of Agaricus brazeimril is more preferred.

  In the first step, agaricus koji is extracted with hot water to produce a hot water extract. In more detail, the raw or dried product of the fruit body of Agaricus persimmon is appropriately subjected to freezing crushing, cutting, impact breaking, enzymatic cell wall degradation, etc., and crushing, chopping, crushing or cell wall partial degradation, and this mass 1 to 50 times the volume of water, more preferably 2 to 20 times the volume of water and about 40 ° C. or higher, preferably 80 to 95 ° C., or hot water of the same temperature is added to Alternatively, in a state where 0.1 to 5 atmospheres is pressurized to normal pressure, more preferably in a state where 0.5 to 3 atmospheres is pressurized to normal pressure, with appropriate stirring, about 10 minutes to 24 hours, Preferably, extraction is performed for 30 minutes to 5 hours. At this time, if the treatment is performed under pressure under the above conditions, the yield of the extract is increased, and it is possible to obtain an extract having a high desired effect of the present invention by using water from neutral to pH 9 or so. Become. After such extraction treatment, the residue is filtered to obtain an extract. In addition, a secondary extract can be obtained by adding water to the extraction residue and treating in the same manner, and the operation may be repeated several times. The obtained extract is combined, neutralized as necessary, and subjected to treatment such as concentration, spray drying, or freeze drying to obtain the hot water extract according to the present invention.

  In the second step, water-containing ethanol having a water content of 10 to 60% by mass is added to the hot water extract for extraction treatment, and components soluble in the water-containing ethanol are collected. That is, water-containing ethanol having the water content with respect to the mass of the hot water extract, more preferably the water content is 20 to 40% by weight, more preferably 25 to 35% by weight, and most preferably the water content is 30% by weight. 2-10 times the volume of water-containing ethanol is added, and the mixture is stirred appropriately at normal temperature to a temperature below the boiling point for 10 minutes to 12 hours, and the resulting precipitate is subjected to processing such as filter filtration and centrifugation. The soluble component is collected, and the solvent is removed to obtain the water-soluble ethanol-soluble material according to the present invention.

In the third step, the water-containing ethanol-soluble material obtained in the previous step is subjected to ion exchange chromatography and fractionated. In this ion exchange chromatography, the components are fractionated using a cation exchange resin and an anion exchange resin. The raw material is supplied to a column packed with the ion exchange resin, and a solvent is passed through to separate the fraction components. A mode in which elution is carried out sequentially is preferred. At this time, known cation exchange resins and anion exchange resins can be used, and each of them is activated in advance by a conventional method, and is used after being packed in an appropriate column made of glass or stainless steel.
In the fourth step, the fraction collected in the above step is subjected to solvent removal, concentration, lyophilization and the like, thereby collecting an amphoteric electrolyte-containing fraction or a saccharide and acidic substance-containing fraction.

  As the third step and the fourth step, for example, the following means and procedures can be employed. That is, the aqueous ethanol-soluble material obtained in the second step is an aqueous solution in which the concentration is 0.1 to 10% by mass, which is applied to an activated cation exchange resin column, and has a predetermined concentration (pH 1 to 3). Ingredients that elute with an acidic solvent are collected, removed by solvent, and lyophilized, and collected as a saccharide and acidic substance fraction. Next, a non-eluting component that was not eluted with the above acidic solvent was eluted and collected using a basic solvent (pH 10 to 13) at a predetermined concentration, and the solvent was removed, concentrated, and lyophilized. A sample for painting.

  Next, an aqueous solution in which the sample is dissolved at a concentration of 10 to 60% by mass is applied to a column filled with activated anion exchange resin, and components eluted with a basic solvent at a predetermined concentration (pH 10 to 13) are collected. Remove the solvent and freeze-dry to collect as a basic substance fraction. Thereafter, the non-eluting components that were not eluted with the basic solvent were eluted using ion-exchanged water and an acidic solvent having a predetermined concentration (pH 1 to 3), and the solvent was removed and freeze-dried in the same manner to obtain an amphoteric electrolyte fraction. Collect as

  The amphoteric electrolyte-containing fraction obtained by such a technique does not contain saccharides or lipids at all, and the molecular weight of the main component belongs to the range of 120 to 280 as described later in Test Example 2, and is more desirable. Has a molecular weight of 123 and / or 137, 242, or 260, and most preferably has a molecular weight of 260, and particularly exerts an action of strongly suppressing mutagenicity induced by NDMA. In addition, the saccharide and acidic substance-containing fraction expresses an antimutagenic action particularly on MNNG.

  The amphoteric electrolyte-containing fraction or saccharide and acidic substance-containing fraction thus obtained is mixed, adsorbed, emulsified, dispersed or dissolved as it is or in a known additive such as a stabilizer, excipient or binder. And can be used as the antimutagenic agent of the present invention. As long as this additive does not violate the gist of the present invention, it can be used without any problem. Starch, modified starch, dextrin, indigestible dextrin, powdered cellulose, Crystalline cellulose, cellulose derivative, polyhydric alcohol fatty acid ester, sucrose fatty acid ester, lactose, gum arabic, mannitol, trehalose, glucose, gelatin, calcium stearate, fine silicon dioxide, purified water, physiological saline, etc. alone or in combination Can be used. The content of the amphoteric electrolyte-containing fraction or the saccharide and acidic substance-containing fraction in the antimutagenic agent of the present invention is about 0.01% by mass or more in consideration of the usage of the agent. preferable.

  The antimutagenic agent of the present invention can be used as a product in various forms in the field of food and drink, pharmaceuticals, feed and other industrial fields, or can be used in an aspect in which it is a part of the blended raw material of such a product. be able to. For practical applications, food and drink are good.

  The aspect of the food and drink to which the antimutagenic agent of the present invention can be applied is not particularly limited, and for example, powder, granule, pill, tablet, soft capsule, hard capsule, paste or liquid Shaped dietary supplements, foods for specified health use, functional nutritional foods, health foods, beverages such as tea, fruit juice drinks, vegetable juices, soft drinks, fermented milk drinks, and powdered products thereof, bread, cakes, cookies, candies , Confectionery such as gum, chocolate, gummy, noodle pasta such as udon, soba, spaghetti, macaroni, jam, spread, sprinkle, boiled, miso, soy sauce, sauce, tomato ketchup, sauce, soy sauce, dashi sauce, General processed foods and beverages such as mayonnaise, dressings and other seasonings or seasoning products, meat and fish products such as hams and sausages, therapeutic foods for liquid foods and foods for dysphagia It can be mentioned. In particular, it is suitable to process into shapes such as granules, tablets, capsules, drinks, etc. and use them as functional foods, nutritional supplements, health foods, and the like.

In order to produce these foods and drinks, the antimutagenic agent of the present invention and known raw materials are used, or a part of the known raw materials are replaced with the antimutagenic agent of the present invention and manufactured by a conventional method. Good.
In the food and drink, the mixing ratio of the antimutagenic agent of the present invention is the active ingredient in the agent, that is, the concentration of the amphoteric electrolyte-containing fraction, or the sugar and acidic substance-containing fraction, the type and form of the food and drink. Although it is difficult to define it generally due to factors such as the above, the content of the amphoteric electrolyte-containing fraction or the fraction containing the saccharide and acidic substance is about 0.01% by mass to about 90% by mass, more preferably about 1% by mass. To about 50% by weight. If the amount is less than 0.01% by mass, a large amount of food or drink must be ingested in order to achieve the desired effect of the present invention. It may be denatured and it may be difficult to create a normal processed food or drink form. The standard of intake of food and drink according to the present invention is, in the case of adults, the daily intake of the amphoteric electrolyte-containing fraction or the saccharide and acidic substance-containing fraction is about 10 mg to about 1,000 mg, preferably Ingestion or tube administration may be performed so that the dose is about 30 mg to about 500 mg, more desirably about 50 mg to about 200 mg.

  When the antimutagenic agent of the present invention is used in the form of a pharmaceutical product, it is preferable to combine the antimutagenic agent with a pharmaceutically acceptable carrier, and use a known excipient or additive in combination. Therefore, tablets, pills, capsules, granules, powders, injections and the like can be prepared by conventional methods, and oral administration, tube administration or intradermal administration is preferred. The content of the antimutagenic agent of the present invention in this pharmaceutical composition is generally 0.001 to 50% by mass based on the amphoteric electrolyte-containing fraction which is the active ingredient, or the saccharide and acidic substance-containing fraction. It is. When the amphoteric electrolyte-containing fraction is the main component, NDMA is targeted as a mutagen, and when the saccharide and acidic substance-containing fraction is the main component, MNNG is targeted as a mutagen. It is desirable to apply for the suppression of mutagenicity induced by substances, the prevention of gene and chromosome damage and abnormalities, the prevention of cancer and other diseases associated with the mutagenicity.

  Next, the present invention will be described in more detail with reference to examples. In each example,%, part and ratio are based on mass unless otherwise specified. The technical scope of the present invention is not limited by the following examples.

[Production example]
Into a stainless steel pressure vessel with a stirrer (50L capacity), add 1kg of Agaricus brazeimuril dried fruit body roughly crushed to a size of about 1cm square and add 10L of water (pH 8). After extraction under pressure at 95 ° C. for 3 hours, the residue was separated by filtration and an extract was collected. Further, 5 L of water (pH 8) was added to the residue and treated in the same manner to collect an extract. The two extracts were combined, concentrated under reduced pressure, and lyophilized to obtain a hot water extract of Agaricus koji. Next, 3 times (volume / mass) of 70% ethanol (= water content 30 mass%) is added to the hot water extract, heated to 35 ° C. and stirred for 10 minutes, and then the precipitate is filtered off. The supernatant was collected, concentrated and freeze-dried to obtain 215 g of powdered water-soluble ethanol soluble material (referred to as a supernatant fraction).

Next, in order to perform fractionation treatment by ion exchange chromatography at the analytical level, the cation exchange resin and the anion exchange resin were activated in advance by the method described below. That is, a strongly acidic cation exchange resin (Muromachi Technos Co., Ltd., Dowex 50WX4 (H ⁺ type)) is packed in a glass tube (φ10 mm × 12 cm), 2N aqueous sodium hydroxide solution, pure water, 2N hydrochloric acid, An activated cation exchange column was prepared by sequentially flowing pure water and 0.01N hydrochloric acid, and a strongly basic anion exchange resin (Muromachi Technos Co., Ltd., Dowex) was placed in a glass tube (φ10 mm × 12 cm). 1 × 8 (Cl ⁻ type)) was packed, and 5% ammonia water, pure water and 5% ammonia water were sequentially flowed to prepare an activated anion exchange column.

  25 mL of 2N hydrochloric acid was added to 5 mL of the aqueous solution (100 mg / mL) of the above water-soluble ethanol soluble substance (supernatant fraction) to make a final concentration 0.01N hydrochloric acid aqueous solution, and this was applied to the activated cation exchange column. The fraction eluted with 50 mL of 0.01N hydrochloric acid was designated as fraction 1 (a fraction containing saccharides and acidic substances). The residue in the column was eluted with 50 mL of pure water and then with 70 mL of 5% aqueous ammonia, concentrated under reduced pressure, and lyophilized. To this freeze-dried product, 10 mL of pure water and 2.5 mL of 25% aqueous ammonia were added to make a final aqueous solution of 5% ammonia, which was applied to the activated anion exchange column thus prepared and eluted with 5% aqueous ammonia. This was designated as fraction 2 (basic substance-containing fraction). Further, 50 mL of pure water and then 70 mL of 0.05N hydrochloric acid were passed through the same column to elute the fraction 3 (amphoteric electrolyte-containing fraction), and the fraction eluted with 50 mL of 0.2N hydrochloric acid was fraction 4 (other components). Containing fraction). Each fraction was concentrated under reduced pressure and freeze-dried by a conventional method to obtain a powdery solid. The yield was fraction 1: 62.4%, fraction 2: 12.8%, fraction 3: 22.3%, fraction 4: 2.5%.

[Test Example 1]
About the solids of each fraction (the samples corresponding to the solids of fractions 1 to 4 are sampled 1 to 4, respectively) obtained by performing the above-described ion exchange column fractionation in the same manner on a 10-fold scale. The mutagenicity-inhibiting action of was tested and evaluated using the reverse mutation test method (Ames test method). That is, each of the samples 1 to 4 is dissolved in sterilized water, and the final concentration is 1, 2, 5 in terms of the equivalent amount of Agaricus sputum supernatant (amount converted to the supernatant fraction in consideration of the yield of each sample). Or each solution was prepared so that it might become 10 mg / mL, and 0.1 mL of each was dispensed to the test tube. Next, 0.1 mL of a solution in which NDMA is dissolved in dimethylsulfonic acid amide as a mutagen is added, and S9mix (rat liver S9, which contains a drug metabolizing enzyme because NDMA is a mutagen that requires metabolic activation. 0.4 mol / L magnesium chloride aqueous solution, 1.65 mol / L potassium chloride aqueous solution, 1.0 mol / L glucose-6-phosphate aqueous solution, 0.1 mol / L reduced nicotinamide adenine dinucleotide phosphate aqueous solution and 0.2 mol / L mixed solution of sodium phosphate buffer, 0.5 mL of pH 7.4) and 0.1 mL of culture solution of Salmonella typhimurium TA-100 strain were added to prepare a mixed solution.

  After shaking the mixed solution at 37 ° C. for 20 minutes, 2 mL of soft agar solution was added, and Ames plate (20 g of D-glucose, 15 g of agar, 100 mL of Vogel-Bonner medium, 0.62 mg of histidine and 0. 98 mg), and incubated at 37 ° C. for 48 hours, and the number of back-mutated colonies that appeared on the plate was counted. Mutagenicity suppression rate of mutagen by each sample is: Mutation rate (%) = (Number of mutations when mutagen and sample are added-Number of spontaneous mutations) / (Number of mutations when mutagen is added-Natural The number of mutations generated) was evaluated by comparing the numerical values obtained from the formula of 100. Here, the number of spontaneous mutations is the number of mutant colonies generated when only the sample is added. Some of these test results are shown in Table 1. In Table 1, the amount of sample added is equivalent to the added amount of the supernatant fraction (2.0 mg / plate) in terms of the yield of each sample, and the numerical value of mutation occurrence is only 0.2 mmol for NDMA. In this case, the relative value is 100 (control test). Table 2 shows the test results when the addition amount of sample 3 was changed under the same conditions.

  From the data in Table 1, it was confirmed that the mutation rate due to NDMA was remarkably suppressed when Sample 3, that is, the amphoteric electrolyte-containing fraction, was added, and the effect was not observed in other fraction samples. From Table 2, it was also confirmed that Sample 3 inhibited the NDMA mutation activity in a concentration-dependent manner.

[Test Example 2]
In order to elucidate the substantial active ingredient of the sample 3, it was subjected to fractionation treatment by high performance liquid chromatography under the following conditions. Apparatus: L-7100 series manufactured by Hitachi High-Technologies Corporation, column: Hydrosphere C18 manufactured by YMC Corporation, eluent: 0.5% formic acid and 10% methanol, flow rate: 2 mL / min, UV at 250 nm detection. 20 μL of a solution prepared by dissolving the sample 3 in sterilized water (containing 5 mg as an equivalent amount of Agaricus sputum supernatant) was injected, fractionated into nine fractions (Fr1 to Fr9), freeze-dried, and then tested for each fraction. The test was conducted in the same manner as in Example 1 to evaluate the NDMA mutagenicity inhibitory action. The results are shown in Table 3. In Table 3, the amount of sample added is the amount corresponding to the added amount of the supernatant fraction (5.0 mg / plate) in terms of the yield of each sample. In this case, the relative value is 100 (control test).

  From Table 3, it was revealed that addition of fraction 3 (HPLC fraction Fr3 of sample 3) strongly suppressed the mutagenicity of NDMA, and it was assumed that the active ingredient was contained in the fraction. This fraction 3 consisted of two types of peak components on the HPLC chart.

  Therefore, the fraction 3 of the sample 3 was subjected to the HPLC fractionation under the same conditions except that the eluent was replaced with 0.5% formic acid and 5% methanol, and six types of fractions (fr1 to fr6) were used. Was sorted. After freeze-drying each, the test was conducted in the same manner as in Test Example 1 and the NDMA mutagenicity inhibitory action was evaluated. As shown in Table 4, the third eluted fraction 3 (fraction Fr3 of sample 3) When the HPLC fraction fr3) was added, the strongest effect was exhibited. This fraction 3 was composed of a single peak component on the HPLC chart.

The molecular weight of the fraction consisting of the single peak (HPLC fraction fr3 of the fraction Fr3 of the sample 3) is analyzed using a high performance liquid chromatography / tandem mass spectrometer (LC / MS / MS). The ion peaks at m / z 124 and 138 were detected.
Apparatus: AP13000LC / MS / MS system manufactured by Applied Biosystems, eluent: 1 mM ammonium formate / methanol (20/80), flow rate: 0.2 mL / min, ionization method: electrospray ionization method, positive ion mode.
From this finding, the active ingredient that suppresses the mutagenicity of NDMA belongs to the range of molecular weight 120 to 280, and the molecular weight of the higher certainty is 123 and / or 137, 242, or 260. It was revealed that the molecular weight of the most reliable one was 260.

[Test Example 3]
Inhibition of mutagenicity of Samples 1 to 4 collected in Test Example 1 based on the method described in the above Production Example when the mutagen is N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) The action was evaluated according to the test method described in Test Example 1. However, in this test method, since MNNG is a direct mutagen that does not require metabolic activation, the addition of S9mix was omitted. A part of the results is shown in Table 5. In Table 5, the amount of sample added is equivalent to the amount of supernatant fraction added (1.0 mg / plate) in terms of the yield of each sample. It is a relative value when (control test) is 100. Table 6 shows the test results when the addition amount of Sample 1 was changed under the same conditions.

  From the data in Tables 5 and 6, it was revealed that Sample 1, that is, the fraction containing saccharides and acidic substances, expresses an inhibitory effect on the mutagenicity of MNNG in a concentration-dependent manner. Sample 3 showed an inhibitory effect on the mutagenicity of NDMA, but almost no effect was observed on the mutagenicity of MNNG.

[Prototype Example 1]
Fraction 3 (sample 3) prepared by the method described in Production Example and Test Example 1, that is, agaricus koji was extracted with hot water, and the extract was extracted with 70% ethanol. Exchange chromatography fractionated amphoteric electrolyte-containing fraction (sample 3 in Table 1): 150 parts, beeswax: 40 parts, and edible vegetable oil: 60 parts were mixed and homogenized at about 60 ° C., followed by a capsule filling machine Then, a gelatin-coated soft capsule type composition having an inner volume of 250 mg per grain was prepared by a conventional method. This composition can be used as a dietary supplement, pet health food or medicine that can be taken orally, and is especially applied to prevent the development of mutagenicity by ingestion of NDMA or to prevent the occurrence of cancer induced by NDMA. It is desirable to do.

[Prototype Example 2]
The HPLC fraction (Fr3) of Fraction 3 (Sample 3) prepared by the method described in Production Example and Test Example 2, that is, the 70% ethanol soluble matter of the hot water extract of Agaricus sp. Fractions obtained by further HPLC fractionating the fraction containing amphoteric electrolytes (Fr3 in Table 3): 100 parts, ascorbic acid: 30 parts, starch: 50 parts, calcium stearate: 30 parts, and crystals Cellulose: 50 parts mixed and homogenized, then applied to a direct compression tablet machine to produce uncoated tablets with a diameter of 7 mm, a height of 4 mm, and a mass of 200 mg / piece, and then applied to a coating machine to form a shellac film Thus, a tablet-type oral composition was produced as a prototype. This composition can be used as a dietary supplement, a health food for animals, or a pharmaceutical, and is applied especially to prevent the development of mutagenicity due to the intake of NDMA or to prevent the occurrence of cancer induced by NDMA. Is desirable.

[Prototype Example 3]
Fraction 1 (sample 1) prepared by the method described in Production Example and Test Example 1, that is, agaricus koji was extracted with hot water, and the extract was extracted with 70% ethanol. Fractionated sugar and acidic substance-containing fraction (sample 1 in Table 5): 150 parts, beeswax: 40 parts, and edible vegetable oil: 60 parts were mixed at about 60 ° C. and homogenized. A gelatin-coated soft capsule composition having an inner volume of 250 mg per grain was prepared by a conventional method using a filling machine. This composition can be used as an orally ingestible dietary supplement, pet health food, or pharmaceutical, and is especially applied to prevent the occurrence of mutagenicity by ingestion of MNNG or to prevent the occurrence of cancer induced by MNNG. It is desirable to do.

[Prototype Example 4]
In Prototype Example 2, the HPLC fraction (Fr3) of Fraction 3 (Sample 3) prepared by the method described in Production Example and Test Example 2 was used as fraction 1 (Fr3) prepared by the method described in Production Example and Test Example 1. Sample 1), that is, a soluble substance obtained by extracting agaricus koji with hot water and extracting the extract with 70% ethanol, and a fraction containing a saccharide and an acidic substance obtained by further ion-exchange chromatography fractionation ( A tablet-type oral composition was prepared in the same manner except that it was replaced with sample 1) in Table 5. This composition can be used as a dietary supplement, animal health food, or pharmaceutical, and is especially applied to prevent the occurrence of mutagenicity by ingesting MNNG or to prevent the occurrence of cancer induced by MNNG. Is desirable.

[Prototype Example 5]
Pork loin: 100 parts, salt: 5%, sodium phosphate: 1.3%, vitamin C: 0.7%, spices and chemical seasonings: 0.7%, sodium nitrite as color former: 0.3% and fraction 3 (sample 3) prepared by the method described in Production Example and Test Example 1, that is, Agaricus koji was extracted with hot water, and the extract was extracted with 70% ethanol. Amphoteric electrolyte-containing fraction obtained by further ion-exchange chromatography fractionation of the soluble matter (sample 3 in Table 1): 30% injection solution containing 30% was injected, and 7 ° C. using a rotary massage machine. I massaged for 24 hours. After that, it was filled into an 11 cm casing, subjected to smoking treatment at 60 ° C. for 30 minutes, and further subjected to boiling treatment at 75 ° C. for 120 minutes to produce a ham-like food. The taste and texture of this ham-like food were comparable to commercially available loin ham.

  Since the antimutagenic agent of the present invention has an action of suppressing the mutagenic expression of a specific mutagen such as NDMA or MNNG, gene damage induced by ingestion of the mutagen, chromosomal It can be effectively used for foods and drinks, medicines, feeds, etc. for preventing abnormalities and the occurrence of abnormalities of the body or cancer caused by these abnormalities.

Claims (6)

The antimutagenic agent which uses as an active ingredient the amphoteric electrolyte containing fraction obtained through the process shown to the following (a)-(d), or a sugar and an acidic substance containing fraction.
(A) a first step of extracting Agaricus koji with hot water;
(B) a second step of extracting the hot water extract obtained in the first step with water-containing ethanol having a water content of 10 to 60% by mass;
(C) a third step of fractionating the water-soluble ethanol soluble material obtained in the second step by ion exchange chromatography;
(D) The 4th process which extract | collects the amphoteric electrolyte containing fraction fractionated at the 3rd process, or a saccharide | sugar and an acidic substance containing fraction.   The antimutagenic agent according to claim 1, wherein the water content of the water-containing ethanol in the second step is 20 to 40% by mass.   The antimutagenic agent according to claim 1 or 2, wherein the molecular weight of the main component of the amphoteric electrolyte-containing fraction in the fourth step belongs to the range of 120 to 280.   The ampholyte targets N-nitrosodimethylamine as a mutagen, and the saccharide and acidic substance target N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen. 4. The antimutagenic agent according to any one of 3. An antimutagenic agent characterized by containing an amphoteric electrolyte-containing fraction obtained through the steps shown in the following (a) to (d), or containing a saccharide and an acidic substance-containing fraction as active ingredients Manufacturing method.
(A) a first step of extracting Agaricus koji with hot water;
(B) a second step of extracting the hot water extract obtained in the first step with water-containing ethanol having a water content of 10 to 60% by mass;
(C) a third step of fractionating the water-soluble ethanol soluble material obtained in the second step by ion exchange chromatography;
(D) The 4th process which extract | collects the amphoteric electrolyte containing fraction fractionated at the 3rd process, or a saccharide | sugar and an acidic substance containing fraction.   A food, beverage, feed or pharmaceutical comprising the antimutagenic agent according to any one of claims 1 to 4 or the antimutagenic agent obtained by the production method according to claim 5.