JP3284097B2 - Biological antioxidant function enhancer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antioxidant function enhancer for a living body extracted from a mycelium culture of basidiomycetes and ascomycetes.

[0002]

2. Description of the Related Art Oxygen is indispensable for survival for human beings, which are aerobic organisms. Humans have efficiently generated ATP by oxidative phosphorylation in intracellular mitochondria during the evolution process, and efficiently gained energy. Even in normal cases, several percent of consumed oxygen is produced in the form of highly reactive active oxygen It is a harmful substance for living organisms.

[0003] However, the living body originally has a protective ability to protect itself from the toxicity of active oxygen, but exceeds the erasing ability by vigorous exercise, drugs such as certain anticancer drugs, smoking, radioactivity or ultraviolet irradiation. When free radicals, particularly active oxygen, are produced, nucleic acids, proteins, lipids, and the like that make up the body are oxidatively damaged. As a result, recent studies have revealed that biological membranes and genes are damaged and are involved in cancer, adult diseases, and sometimes serious diseases.

[0004] Here, active oxygen generally means oxygen species that are more active than ordinary oxygen in the atmosphere. Superoxide radicals (O ₂ ⁻ ), excess oxygen Hydrogen oxide water (H ₂ O ₂ ), hydroxyl radical (.OH), and singlet oxygen ( ¹ O ₂ ) can be given.

[0005] Free radicals are atoms or molecules having unpaired electrons, which are unstable and highly active, and most active oxygens are also free radicals.
As free radicals in living bodies, hypochloride (HOCl), lipid hydroperoxide (LOOH), and peroxy radical (LOO)
・), Alkoxy radical (LO ・), nitric oxide (NO)
And the like.

[0006] Such active oxygen is produced in various metabolic processes in a living body, and generally has a short life span, often acts locally and develops biotoxicity. Its production systems include mitochondrial and microsomal electron transport systems, intracellular oxygen systems, activated leukocytes, and vascular endothelial cells.

[0007] The active oxygen most easily produced in these systems is the superoxide radical. Hydroxyl radicals have the highest reactivity and are generated by reduction with transition metal ions, but are also generated by irradiation with radiation or ultraviolet rays, and damage genes.

On the other hand, there are enzymes such as superoxide dismutase (SOD) and peroxidase (glutathione peroxidase, catalase) as protective substances on the living body side, but these enzymes act in hydrophilic compartments. Very unlikely to block lipid peroxidation reactions that occur in a hydrophobic environment. Although there are enzymes that scavenge superoxide radicals and aqueous hydrogen peroxide, there is no enzyme that scavenges the most reactive hydroxyl radical.

Therefore, an erasing agent for capturing these radicals is required. Α as an eraser present in cells
-Tocopherol (vitamin E), ascorbic acid (vitamin C), carotene, glutathione, ubiquinone, phenols, sugars, flavonoids, metallothionein,
Chelators and the like.

For example, ascorbic acid is a water-soluble fraction,
When present in mitochondria, cytoplasm, blood, etc., when superoxide radicals are generated, they are oxidized enzymatically or non-enzymatically to produce monodehydroascorbic acid radicals, which become dishydroascorbic acid by a disproportionation reaction, resulting in glutathione It is regenerated to ascorbic acid by the action of (GSH) and reductase. In the adult body, about
Although it contains 1.5 g of ascorbic acid, 3% of it is lost every day and cannot be biosynthesized in the body, so it must be taken as a vitamin.

In order to stop the chain reaction of free radical and lipid peroxide formation in the fat-soluble space, α-
Tocopherol plays a central role. In cells, α-tocopherol mainly exists in the membrane, reacts with radicals generated in a hydrophobic environment to form α-tocopheroxy radicals, and α-tocopheroxy radicals are reduced by ascorbic acid, It becomes α-tocopherol. Thus, α-tocopherol acts on free radicals in cooperation with ascorbic acid.

By the way, plants grow without being damaged by ultraviolet rays even when exposed to strong sunlight. this is,
This is because the leaves and the like of plants have a mechanism to suppress the generation of active oxygen and a function of eliminating active oxygen are much higher than other organisms, and have abundant substances that can eliminate active oxygen. For example,
Green vegetables include vitamin A, vitamin B, vitamin C,
It is rich in substances that scavenge active oxygen such as vitamin E, flavonoids, and catechins, and the importance of abundant consumption of colored vegetables is important here.

[0013]

As described above, active oxygen is produced in a small amount even in respiration for life support, but it is involved in various disease states, so that it is generated in a large amount. Requires the ingestion of antioxidants, and it is important to enhance the antioxidant function of the living body by ingesting antioxidants from foods such as colored vegetables even during normal times.

However, in recent years, the industrialization of agriculture has progressed, and vitamins and minerals content of vegetables grown under low light, such as artificially controlled plastic greenhouses, have been reduced to less than half that of those grown in the open field. It is becoming difficult to take enough antioxidants.

[0015] Accordingly, an object of the present invention is to provide free radicals generated in a living body by daily ingestion,
In particular, it is an object of the present invention to provide a biological antioxidant function enhancer capable of easily and safely eliminating active oxygen.

[0016]

The present inventors have conducted various studies on the mycelium of edible mushrooms belonging to basidiomycetes such as shiitake mushrooms for many years. As a result, the substance in the mycelium culture of basidiomycete was The inventors have found that they have a strong antioxidant action for eliminating free radicals, and have completed the present invention.

That is, the antioxidant function enhancer for a living body of the present invention is obtained by culturing mycelia of basidiomycetes and / or ascomycetes using a medium containing a plant fiber component and extracting from the culture. It is characterized by containing carbohydrates, proteins and water-soluble lignin as main components.

In the present invention, the medium containing the plant fiber component is preferably prepared from a grass plant.

The antioxidant function-enhancing agent of the present invention is a water-soluble agent in which hemicellulose and lignin contained in plant tissue are denatured by oxidative degradation and condensation by enzymes such as peroxidase and cellulase produced by basidiomycetes or ascomycetes. It is composed of a substance in which a polysaccharide mainly composed of pentose, which is a main component of hemicellulose, is intricately bound to a component rich in soluble lignin, and as shown in the following examples, strong free radical scavenging is performed in an electron spin resonance (ESR) test. It shows activity and also scavenges the most active hydroxy radical among free radicals. Furthermore, macrophages of immunocompetent cells produce nitric oxide, a kind of free radical, when stimulated by a mitogen such as lipopolysaccharide, and the antioxidant function enhancer of the present invention is derived from macrophages. Nitric oxide also has the activity of erasing.

Therefore, taking the antioxidant function enhancer of the present invention is expected to have the effect of eliminating free radicals in the living body and suppressing the development of various diseases involving active oxygen. In addition, the antioxidant function enhancer of the present invention is obtained from natural products, and can be easily taken in daily life without worrying about side effects and the like seen in synthetic chemicals and the like.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Basidiomycetes used in the present invention include edible mushrooms such as shiitake mushrooms, spatula mushrooms, maitake mushrooms, enoki mushrooms, shimeji mushrooms, yamabushi mushrooms, etc .; And various kinds of medicinal mushrooms. Examples of the ascomycetes used in the present invention include Amigasa mushrooms.

In the present invention, the mycelium of these basidiomycetes and / or ascomycetes is cultured, and the active ingredient is extracted from the culture. In this case, as the medium, any of a solid medium and a liquid medium can be used, but a medium containing a plant fiber component,
Preferably, a medium prepared from a plant containing lignin is used. As the plant containing lignin, a botanical plant, for example, bagasse (sugar cane squeezed cake), corn stover, wheat bran, rice straw, kaya, and the like are preferably used. In addition, bamboo grass and bamboo can also be used. Further, a medium to which rice bran, dried yeast, yeast extract, and the like are added and mixed as other nutrients as necessary is preferably used.

The culture of basidiomycete and ascomycete mycelium is carried out by inoculating a spore or a germinated hypha into a medium containing the above-mentioned plant as a main raw material. For solid media, 60-80% moisture
Adjusted to become, after high-pressure steam sterilization according to the usual method,
The mycelium or spores are inoculated, and cultured for 3 to 6 months in a culture room air-conditioned at, for example, 18 to 25 ° C. It is preferable that the medium in which the mycelium spreads is transferred to a temperature treatment chamber and subjected to a temperature change treatment. The temperature change treatment is performed, for example, first at 30 to 34 ° C for 24 hours.
Heat for ~ 48 hours, then transfer to cold room and treat for 3-5 days. Thereafter, when the cells are transferred to a culture room, fruiting bodies start to be generated. At this point, the culture is terminated and the culture is crushed by a crusher.

On the other hand, in the case of a liquid medium, the plant material is finely crushed and cultured by aeration culture or shaking culture, preferably at a temperature of 20 to 25 ° C. for about 2 weeks to 2 months. The culture ends when the mycelium spreads in the medium.

After completion of the cultivation, the mycelium is autolyzed by utilizing enzymes present in the mycelium, and the culture is extracted.
As a preferred method, in the case of a solid medium, the culture after cultivation is crushed, and a small amount of water is added as necessary to obtain a solid medium.
The mixture is treated at ~ 90 ° C for 3 to 6 hours to advance the enzymatic reaction of the mycelium and autolyze. Next, this crushed material is infiltrated with warm water or hot water of 50 ° C. or more to extract an active ingredient. Extraction, for example
It can be carried out under a high pressure and high temperature such as 120 ° C. under a vapor pressure of 1.2 kg / cm ² . In a preferred embodiment of the extraction method, about 4 L (liter) of water is added to 800 g of the crushed material, and the mixture is heated to 70 ° C. and extracted for 4 to 16 hours. As the extraction method, a method of circulating the extract for 12 to 16 hours, or a method of stirring for 4 to 6 hours is preferably employed. The extract suspension thus obtained is preferably filtered or centrifuged to collect a filtrate or a supernatant to obtain a metabolite of mycelium and an active ingredient contained in mycelial cells.

On the other hand, in the case of liquid culture, the whole culture
The mixture is treated at 9090 ° C. for 3 to 6 hours to autolyze the mycelium to obtain a liquid suspension culture. Then, if necessary, a small amount of water is added, and at 50 ° C. or higher, and optionally under high pressure (for example,
Heat under a vapor pressure of 1.2 kg / cm ² ) to obtain an extract. The extract is filtered or centrifuged as necessary to collect a filtrate or a supernatant to obtain an active ingredient.

The antioxidant function-enhancing agent of the present invention can be used as it is in the form of a liquid product by directly or concentrating the extract obtained by the above method. It can be powdered. The powder obtained from this extract, as shown in Examples described later, carbohydrate,
It was mainly composed of protein and water-soluble lignin, and was confirmed to have an antioxidant effect.

The antioxidant function enhancer of the present invention may be obtained by further purifying the extract obtained by the above-mentioned method or a dry powder thereof into a component rich in water-soluble lignin. Products with higher oxidizing action can be obtained. As the purification method, for example, the following method is employed.

That is, the brown powder obtained by freeze-drying the above extract is converted into an aqueous solution having a concentration of 10%, and ethyl alcohol is added so that the alcohol concentration becomes 80% to obtain a precipitate. The obtained precipitate is redissolved in water and purified by gel filtration using column chromatography. In this case, agarose gel or dextran gel is used for gel filtration.

The purified active ingredient was powdered, and the powder was analyzed. As a result, it was confirmed that the powder had the following molecular weight and composition. Molecular weight: 200,000-2,000,000 Chemical composition Carbohydrate: 20-30% Protein: 8-10% Water-soluble lignin: 50-70%

The results of safety tests on the above-mentioned cultured extract (powder) obtained using Shiitake fungi are as follows. (A) Acute toxicity (LD ₅₀ mg / kg) Oral administration for 7 days Mouse Male: 19600 Female: 17700 rat Male: 16400 Female: 15600 Intraperitoneal administration Mouse Male: 5500 Female: 4920 Rat Male: 2490 Female: 2270 (B) Subacute toxicity (mg / kg), maximum no effect dose Mouse Male: 6740 Female 9100 Rat Male: 3840 Female 7910

The effective dosage of the antioxidant function enhancer of the present invention is 1 to 10 g per day for an adult when taken orally. If the dose is smaller than this, sufficient antioxidant activity in vivo is not obtained, and if the dose is larger than this,
Loose stools or abdominal distension may occur. However, there is no problem in safety even if the dose is higher than the above.

[0033]

EXAMPLES Example 1 (Solid culture of Shiitake mushroom using bagasse) 80% of bagasse and 20% of defatted rice bran were blended to adjust the water content to 70%, and a solid medium was prepared. This was inoculated with a Shiitake mushroom inoculum. After culturing the medium for 4 months in a room where the temperature of the medium has been adjusted to 22 to 24 ° C., after the mycelium has spread in the medium, the medium is transferred to a temperature treatment room and heated at 32 to 34 ° C. for 24 hours. Treated in room for 5 days. Thereafter, the cells were cultured in a culture room for 2 days, and the medium was crushed twice with a crusher. The crushed medium was treated at 50 ° C. for 4 hours to promote autolysis, and then packed in a tank and extracted for 16 hours while circulating hot water at 60 ° C.
The obtained suspension was preliminarily filtered through a diatomaceous earth filter cake, further filtered and sterilized with a membrane filter, and then concentrated.
Lyophilization gave a brown powder.

The analysis of the components of this powder showed that the carbohydrate:
35%, protein: 12%, water-soluble lignin: 35%, minerals:
13%. The components were analyzed by the phenol sulfate method for carbohydrates, the semi-micro Kjeldahl method for proteins, the acetyl bromide method for water-soluble lignin, and the direct incineration method for inorganics.

Test Example 1 (Free Radical Scavenging Activity) The free radical scavenging activity of the powder obtained in Example 1 was measured. 2,2-diphenyl-1-picrylhydrazyl (DPPH)
Radical scavenging activity. Stable and most commonly used as a standard sample for paramagnetic resonance.
The scavenging activity for DPPH radical was measured. The reaction solution is
30 μM (micromol) in 100 μl ethyl alcohol
Is dissolved, and 100 μl of an aqueous solution of 2.5 mg of the present powder per 1 ml is mixed. Start the magnetic field sweep after one minute,
Electron spin resonance (ESR) was measured using "Radical Monitor FR-30" (trade name, manufactured by JEOL Ltd.). As a control, the signal intensity when 100 μl of water was added instead of the sample solution was measured. The result is shown in FIG. FIG. 1 (a)
Indicates the ESR (control) when water was added, and FIG. 1 (b)
Indicates the ESR of the sample solution. As a result, it was confirmed that the Shiitake mushroom mycelium culture extract had a DPPH radical scavenging activity.

In the same manner as the hydroxyl radical scavenging activity, the scavenging activity of this sample for hydroxy radical was measured by ESR. However, the sample solution was prepared as follows. Diethlene triaminopentaacetic acid (DETAPAC) 1
50 μm of a mixed solution of mM (mmol) and 1 mM ferrous sulfate
l, 50 μl of a 100-fold diluted solution of 5,5-dimethyl-1-pyrroline-N-oxide (5,5-Dimethyl-1-pyrroline-N-oxide, DMPO), and a sample solution (2.5 mg of this powder sample) / Ml H
₂ O) are mixed with 50 [mu] l. Then, 50 μl of a 10000-fold diluted aqueous solution of hydrogen peroxide was added to the mixed solution, and after 60 seconds, the magnetic field sweep was started. The result is shown in FIG. Fig. 2 (a) shows the ESR of the hydroxyl radical when water was added, and Fig. 2 (b) shows the ESR of this sample solution.
Is shown. As a result, it was confirmed that the Shiitake mushroom mycelium culture extract had an effect of eliminating even highly reactive hydroxyl radicals.

Test Example 2 (Superoxide scavenging activity) In vivo, superoxide was used as nicotinamide adenine dinucleotide phosphate.
leotidephosphate, NADPH) ・ cytochrome C reductase, xanthine oxidase, aldehyde oxidase and NAD
Produced by enzymatic reactions such as PH oxidase. The resulting superoxide varies toxic to low triplet enzyme ⁽³ O ₂₎ and hydrogen peroxide by SOD. Therefore, a calibration curve was prepared with the SOD standard solution for the erasing activity of superoxide, and the erasing activity in the sample was measured.

That is, 50 μl of 2 mM hypoxanthine-phosphate buffer, 35 μm of 5.5 mM DATAPAC-phosphate buffer
l, DMPO 15 μl, 0.4 unit / ml xanthine oxidase-phosphate buffer 50 μl and test solution 50 μl or
After preparing two types prepared by mixing 50 μl of the SOD standard solution, the sample was set in the ESR device, and after 40 seconds, the sweep was started, and the Mn ²⁺
The ratio of the peak height of (internal standard) to the peak height of the measured signal was determined. The value obtained from the test solution was interpolated from the calibration curve of the SOD standard solution to determine the erasing activity in the sample and the reduction of cytochrome C by 50%.
As a result, the amount of SOD required for controlling the% SOD was determined as 1 unit, and the amount of the cultured extract of Shiitake mycelium was 6.9 × 10 ³ units / g.

Test Example 3 (Nitrogen oxide scavenging activity) The macrophages had an inducible nitric oxide synthase.
Using arginine, one of the amino acids, as a substrate, it is stimulated and induced by cytokines to produce nitric oxide.
The produced nitric oxide has an effect of killing bacteria taken up by macrophages and killing cancer cells. On the other hand, if the immune system continues to increase due to severe infection, advanced cancer, etc., excessive nitric oxide is produced, causing inflammation and exerting a damaging action as a radical. By the way, lipopolysaccharide, which is an outer membrane component of Gram-negative bacteria, is known to exhibit various biotoxicities and physiological activities. One of them is to activate macrophages. Therefore, the effects on the production of nitric oxide by macrophages were investigated by the following procedure.

The cells were established as macrophages.
J774.1 cells (available from RIKEN, Cell Bank) were used. This J774.1 cell suspension was seeded in a 96-well microplate at 100 μl / well, and 100 μl / well of RPMI1640 medium containing 10% fetal bovine serum (FBS).
(200 μl / well in total).

In the RPMI1640 medium, 1 μg / ml of lipopolysaccharide (Bacto ^R lipopolysaccharide), 100 units / ml of interferon-γ (trade name “Recombinant Mouse Interferon-γ”, manufactured by Wako Pure Chemical Industries) and a sample ( Shiitake mushroom mycelium culture extract obtained in Example 1) was added at a concentration of 0 to 500 μg / ml.

Since interferon-γ is known to be a cytokine that enhances the production of nitric oxide from macrophages, lipopolysaccharide was added at 1.0 μg / ml here.
As an increase in the production of nitric oxide from J774.1 cells under the condition of 100 units / ml of interferon-γ,
The effect of this sample (Shitake mushroom mycelium culture extract) was examined.

Next, the microplate prepared in the above was cultured in a 5.0% CO ₂ incubator (37 ° C.) for 3 days.

Next, 100 μl of the culture supernatant was collected from each well and transferred to another 96-well microplate. The Grace (Greiss) reagent (0.1% naphthylethylenediamide.2HCl, 2.5% phosphoric acid) After adding 100 μl / well of the mixture and leaving the mixture at room temperature for 10 minutes, _A540 was measured using a microplate reader. Separately, the nitrogen oxides in the samples were calculated from a calibration curve developed with the Greiss reagent using NaNO ₂ . Macrophages produce nitric oxide when activated, but nitric oxide is unstable and easily oxidized to nitrite ions (N
O ₂ ^- since in), actually Greiss this nitrite
Measure with reagents.

As a result, the yield of nitrite ions from J774.1 cells when this sample (shiitake mycelium culture extract) was not added (only lipopolysaccharide 1.0 μg / ml, interferon-γ 100 units / ml) was determined. , 20 nM / ml. On the other hand, 7.8 μg /
As shown in FIG. 3,
Nitric oxide production was suppressed in a dose-dependent manner. As a result,
It was confirmed that the Shiitake mushroom mycelium culture had an action of eliminating excessively induced nitric oxide radicals from macrophages.

Example 2 (Purification of active substance) The powder obtained in Example 1 was converted into a 10% aqueous solution, and the insoluble substance was reduced to 30%.
After spinning down at 00 rpm for 30 minutes, dissolve in water again,
At 5 ° C., 4 times the volume of ethyl alcohol was added to a concentration of 80%, the resulting precipitate was collected, dissolved again in water, and freeze-dried.

The obtained powder was dissolved in a 0.05 M (molar) phosphate buffer (pH 6.5), and the mixture was dissolved in “Sephadex G-200”.
Gel filtration was performed using a column (trade name, manufactured by Pharmacia). Elution was carried out with the same buffer, and void fractions were collected using the absorbance at a wavelength of 280 nm as an index, concentrated under reduced pressure, dialyzed with water, and freeze-dried.

The obtained purified substance was composed of 28% saccharide and 10% protein.
%, And water-soluble lignin was 58%. When the superoxide scavenging activity of the purified substance was measured, 1.6 × 10 ⁵
Unit / g, and an activity 20 times higher than before purification was obtained. In addition, FIG. 4 shows the result of ESR measurement of the hydroxy radical scavenging activity of this purified substance.

Example 3 (Solid culture of Shiitake fungi using corn stalks) Corn stalks were washed with water, dried, crushed, and rice bran was added at a weight ratio of 20% to adjust the water content to 70%. Then, a solid medium was prepared, and after sterilization, the mycelium of Shiitake was cultured in the same manner as in Example 1 to obtain a culture extract. The resulting extract was 40% saccharide, 12% protein, 35% water-soluble lignin, and 12% mineral. This extract exhibited the same scavenging activity against DPPH radicals as the culture extract of Example 1.

Example 4 (Solid culture of Shiitake mushroom using Kumasa bamboo leaves and stems) The leaves and stems of Kumasa bamboo were thoroughly washed, dried and crushed, and rice bran was mixed at a weight ratio of 20% to prepare a solid medium. In the same manner as in Example 1, the hypha of Shiitake mushroom was cultured to obtain a culture extract. This extract exhibited the same scavenging activity against DPPH radicals as the culture extract of Example 1.

Example 5 (Solid Culture of Shiitake Mushroom Using Kaya, Rice Straw, and Bamboo) Kaya, rice straw, and bamboo were washed, dried, crushed, and mixed with rice bran in a weight ratio of 20% for each. Was prepared, and the mycelia of Shiitake mushrooms were cultured on these solid media in the same manner as in Example 1 to obtain a culture extract. Each of the obtained extracts exhibited the same scavenging activity against DPPH radicals as the culture extract of Example 1.

Example 6 (Solid Culture of Mannensis Mushrooms Using Bagasse) In a solid medium prepared in the same manner as in Example 1 and mixed with bagasse and defatted rice bran at a ratio of 9: 1, the hypha of Mannensis mushrooms was added. After culturing, a culture extract was obtained in the same manner as in Example 1.
The components of the powder obtained by freeze-drying this extract were 28% saccharide, 15% protein, 38% water-soluble lignin, and 14% inorganic.
This powder sample eliminated DPPH radicals, superoxide radicals, and nitric oxide radicals as shown below.

DPPH Radical Scavenging Activity The effect of this sample on the DPPH radical was measured by the method described in Example 1, and as a result, as shown in FIG. 5, the same scavenging activity as that of the Shiitake mushroom mycelium culture extract was obtained.

Superoxide radical scavenging activity A similar test was carried out for the SOD-like activity shown in Test Example 2. As a result, the superoxide radical scavenging activity of this sample was
4.3 was × 10 ⁴ Units / g.

Nitrogen monoxide radical scavenging activity A similar experiment was carried out in the production of nitric oxide induced by lipopolysaccharide stimulation from J774.1 cells shown in Test Example 3. As shown in FIG. As in the case of the Shiitake mushroom mycelium culture extract, an effect of eliminating nitric oxide radicals was obtained.

Example 7 (Solid culture of Mannensis fungi using Kumasa bamboo) A solid prepared by mixing rice bran in a weight ratio of 10% by crushing the fiber residue obtained by extracting the pigment and aroma of Kumasa bamboo with ethyl alcohol. The hypha of Mannen mushroom was cultured in the medium and extracted in the same manner as in Example 1. The obtained extract was freeze-dried to give 30% carbohydrate, 15% protein, and 35% water-soluble lignin.
(31-42%), inorganic ions 10%, and others 10%.
This sample eliminated DPPH radical, superoxide radical and nitric oxide radical as shown below.

DPPH radical scavenging activity The effect of this sample on DPPH radicals was tested by the method described in Example 1. As a result, the same radical scavenging activity as in Example 1 was obtained at 2.5 mg / ml.

Superoxide radical scavenging activity The SOD-like activity was tested in the same manner as in Test Example 2, and as a result, the superoxide radical scavenging activity of this sample was 5.2 × 10 ⁴ units / g.

Nitric oxide radical scavenging activity In an experiment of inducing nitric oxide production by lipopolysaccharide-interferon-γ from J774.1 cells, this sample was prepared in Example 6.
Nitrogen monoxide radical scavenging activity similar to that described above was obtained.

Example 8 (Liquid cultivation of Shiitake fungi using bagasse) To 1 L (liter) of water, 60 g of bagasse finely crushed by a mixer, 20 g of dried okara, 20 g of rice bran, and 2 g of yeast extract were added. The mycelia of Shiitake mushrooms were inoculated into the steam-sterilized liquid medium. After aeration culture at 23 ° C. for 3 weeks, the culture was heated at 60 ° C. for 1 hour and at 80 ° C. for 2 hours to allow the mycelia to undergo autolysis by enzymes and to extract metabolites. The culture is centrifuged at 3000 rpm for 30 minutes,
The precipitate was removed to obtain a brown liquid.

The extract was freeze-dried to give carbohydrate 38
%, Protein 16%, water-soluble lignin 35%, and other 11%. As a result of investigating the effect of this sample on the DPPH radical, the same radical scavenging activity as in Example 1 was obtained. Further, the SOD-like activity was tested by the method described in Example 1, and as a result, the superoxide radical scavenging activity of this sample was 5.1 × 10 ³ units / g.

Example 9 (Liquid culture of Mannensis fungi using Kumasa bamboo) The fiber residue obtained by extracting the pigment and aroma of Kumasa bamboo with ethyl alcohol was finely crushed with a mixer, and the resulting mixture was mixed with 90 L / water.
g, 10 g of rice bran, and 5 g of yeast extract were added, and a liquid medium that had been subjected to high-pressure steam sterilization according to a conventional method was inoculated with hyphae of Mannen mushrooms, and aerated culture was performed at 23 ° C. for 2 weeks. The culture was heated and centrifuged in the same manner as in Example 7 to obtain a dark brown liquid. The extract obtained by freeze-drying contained 33% of carbohydrate, 18% of protein, 40% of water-soluble lignin and 9% of others.
In this sample, DPPH radical and hydroxy radical scavenging activities (the results are shown in FIG. 7) were obtained. The SOD-like activity was 8.5 × 10 ⁴ units / g.

Example 10 (Solid culture of yamabushi mushroom using bagasse) Bagasse (65%), wheat bran (20%), rice bran (10%), and dry yeast (5%) were blended and adjusted to a water content of 70%. And sterilized according to a conventional method. An extract obtained by inoculating mycelia of yamabushi mushrooms on the solid medium and culturing the mycelium by the method described in Example 1 below was 43% saccharide and 12% protein.
%, Water-soluble lignin 28%, inorganic substances 10%, and other 7%. This sample showed the activity of scavenging DPPH radicals and hydroxy radicals as in Example 1. SOD of this sample
Activity was 4.5 × 10 ³ units / g.

Example 11 (Solid Culture of Amigasa Mushroom Using Rice Straw and Bagasse) Rice straw was washed with water, dried and crushed to 40%, and bagasse 30.
%, Wheat bran 15%, rice bran 10%, dry yeast 5%, make a solid medium by adjusting the water content to 70%,
It was sterilized according to a conventional method. The mycelia of Amigasa mushrooms were cultured on this solid medium, and the extract obtained by the method described in Example 1 below was used to obtain 46% of carbohydrate, 16% of protein, 26% of water-soluble lignin,
Others were 12%. In this sample, DPPH radicals were eliminated at a concentration of 10.0 mg / ml in the same manner as in Example 1.

Example 12 (Solid culture of Hiratake mushroom using bagasse) An extract obtained by cultivating the mycelia of Hiratake mushroom in the same bagasse / rice bran medium as in Example 1 was subjected to DPPH as in Example 1. Radicals eliminated.

Example 13 (Solid culture of Maitake mushrooms using bagasse) An extract obtained by culturing mycelia of Maitake mushrooms in the same bagasse / rice bran medium as in Example 1 was subjected to DPPH as in Example 1. Radicals eliminated.

[0067]

As described above, the antioxidant function enhancer of the present invention is obtained by culturing the mycelium of basidiomycetes and / or ascomycetes using a medium containing a plant fiber component and extracting from the culture. It has carbohydrates, proteins, and water-soluble lignin as main components, and shows strong free radical scavenging activity in electron spin resonance (ESR) test, and also scavenges the most active hydroxy radical in free radicals. . Furthermore, it also has an activity of eliminating nitric oxide induced and produced from macrophages. Therefore, ingestion of the antioxidant function enhancer of the present invention is expected to have the effect of eliminating free radicals produced in the living body and suppressing the occurrence of various diseases involving active oxygen. In addition, the antioxidant function enhancer of the present invention is obtained from natural products, and can be easily taken in daily life without worrying about side effects and the like seen in synthetic chemicals and the like.

[Brief description of the drawings]

FIG. 1 shows the DP of the sample of Example 1 (Shitake mushroom mycelium culture extract)
5 is a chart showing the results of ESR measurement of PH elimination activity, in which (a) shows the case where water was added instead of the sample as a control, and (b) shows the case where 2.5 mg / ml of the sample was added.

FIG. 2 is a chart showing the results of ESR measurement of hydroxy radical scavenging activity of a sample (a Shiitake mushroom mycelium culture extract) of Example 1; (a) shows a case where water was added instead of the sample as a control; (B) shows the case where 2.5 mg / ml of the sample was added.

FIG. 3 is a table showing the rate of inhibition of nitric oxide by the sample of Example 1 (Shitake mushroom mycelium culture extract).

FIG. 4 is a table showing the results of ESR measurement of the hydroxy radical scavenging activity of the sample of Example 2 (purified product of a Shiitake mushroom mycelium culture extract).

FIG. 5 is a table showing the results of ESR measurement of DPPH scavenging activity of the sample of Example 6 (Mannensis mushroom mycelium culture extract).

FIG. 6 is a table showing the rate of inhibition of nitric oxide by the sample of Example 6 (Mannensis mushroom mycelium culture extract).

FIG. 7 is a table showing the results of ESR measurement of hydroxy radical scavenging activity of a sample of Example 9 (a culture extract obtained by cultivating Mannen mushroom hyphae on Kumasa bamboo grass).

Continuation of the front page (56) References Hepatobiliary pancreas, 1987, Vol. 16, No. 1 pp. 127-135 Quarterly "Science of Health", Mushroom Health Reader, 1,1995, No. 1, pp. 42-47 Monthly Food Chemical, 1996, Vol. 6, pp. 35-43 Journal of Clinical Electron Microscopy, 1995, Vol. 28, No. 1, pp. 35-43 Annual Review Gastroenterology, 1997, pp. 37-45 (58) Field surveyed (Int. Cl. ⁷ , DB name) A61K 35/84 C09K 15/34 BIOSIS (DIALOG) CA (STN) MEDLINE (STN)

Claims (2)

(57) [Claims] 1. A basidiomycete and / or ascomycete mycelium is cultured using a medium containing a plant fiber component, and carbohydrates, proteins, and water-soluble lignin obtained by extracting from the culture are used as main components. A biological antioxidant function enhancer, characterized in that: 2. The antioxidant function enhancer for a living body according to claim 1, wherein the medium containing the plant fiber component is prepared from a grass plant.