JPH0885789A - Preparation of oxidation-resistant substance and its use - Google Patents

Abstract

PURPOSE: To improve the oxidation resistance, safety, mass productivity, and hepatopathy inhibitory activity by culturing a microorganism belonging to the genus Monascus and then harvesting the product by extraction. CONSTITUTION: A microorganism belonging to the genus Monascus is inoculated into a culture medium comprising a starch raw material, a carbon source, a nitrogen source, a mineral, a vitamin and the like and cultured at 20 to 40 deg.C for about 10 days or longer. The cultured product is comminuted, a solvent having a high polarity is added thereto, soaking and stirring are conducted, followed by solid-liquid separation. The extract is brought to a concentrate or a dry powder.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an antioxidant from a microorganism belonging to the genus Monascus, an antioxidant containing the antioxidant as an active ingredient, and a preventive / therapeutic agent for liver damage.

[0002]

2. Description of the Related Art Oils and fats present in foods contain oxygen,
The phenomenon of deterioration and deterioration under the influence of light, heat, etc. (oxidation phenomenon) is generally well known, but this phenomenon occurs not only in fats and oils in food but also in fats and oils in the living body, causing diseases and It has been known in recent years that it causes aging.

In order to prevent such an oxidation phenomenon, conventionally, antioxidant substances extracted from natural products such as plants, microorganisms and marine products, and antioxidant substances obtained by chemical synthesis have been used as antioxidants. ing. For example, butylhydroxyl anisole (BHA), vitamin E (tocopherol), vitamin C (L-ascorbic acid) and the like are used as antioxidants. Among them, vitamin C is a processed meat product, fruit, vegetable processed product. It is used as an additive for foods such as beverages, processed marine products, wine and pickles, and as a compounding ingredient for medicines and cosmetics.

However, each of these antioxidants has its own problems, and it has been required to provide new antioxidants. For example, BHA, which is a typical existence of antioxidants obtained by chemical synthesis, has been pointed out to have a carcinogenic problem, and has a problem that it is currently used only in the dried sardines field. Further, there is a problem that many antioxidants derived from natural products such as vitamins are limited in mass production from the viewpoint of resources.

[0005]

Therefore, there has been a demand for the development of a method capable of industrially mass-producing an antioxidant substance which is highly safe and can be advantageously used as an antioxidant.

[0006]

[Means for Solving the Problems] The inventors of the present invention have searched for antioxidants existing in natural products, and found that among the microorganisms known as Red mold Aspergillus, substances with extremely high antioxidant properties were found. We found that there is something to produce and succeeded in separating and obtaining the substance.

That is, a first object of the present invention is to provide a method for producing an antioxidant, which comprises extracting the antioxidant from a culture of a microorganism belonging to the genus Monascus. Another object of the present invention is to provide an antioxidant and a preventive / therapeutic agent for liver damage, which has an antioxidant as an active ingredient, which is present in a culture of a microorganism belonging to the genus Monascus or an extract thereof.

The microorganisms belonging to the genus Monascus used in the present invention include Monascus anchor (Monascus).
anka), Monascus louver (M. ruber), Monascus vitreus (M. vitreus), Monascus rubiginosus (M.rubiginosus), Monascus albidus (M. albidus), Monascus purpueus (M. purpu)
reus), Monascus pirosus (M. pilosus) and the like.

Cultivation of these microorganisms may be carried out according to a conventional method for producing koji. That is, as a starch-based raw material for koji production, for example, various grains such as polished rice, brown rice, wheat, millet, kouryan, buckwheat, corn, soybeans, adzuki beans, and one kind of bran, bran, germ, chaff, etc. Alternatively, two or more or more of them, in which various carbon sources, nitrogen sources, inorganic substances, vitamins, etc. are added, and cultured by a known method such as solid koji method (rose koji method, co-koji method), liquid koji method, etc. Just do it. Generally, it may be cultured at a temperature of about 20 to 40 ° C.

In the case of a Monascus genus microorganism, the culture period may be about 1 week, but preferably about 10 days or more, for example, about 2 to 3 weeks.

The culture obtained as described above can be used as an antioxidant in the case of solid culture, as dried as it is, or in the case of liquid culture as a culture solution obtained by removing cells from the culture. Although it can be utilized, it is preferable to take out an antioxidant from the culture by means such as extraction and obtain an antioxidant using this as an active ingredient.

In order to extract the antioxidant from the culture, the culture may be crushed, then an appropriate solvent may be added thereto, and the mixture may be sufficiently immersed and stirred, and then the solid-liquid may be separated by centrifugation or the like.

As the solvent used for extracting the antioxidant, a highly polar solvent such as water, a buffer solution, methanol, ethanol, acetone, or a mixed solvent of these and water can be used.

The presence of antioxidants in the obtained extract may be confirmed by the thiobarbital acid reaction (TBA method), but it is more preferable that the presence of an antioxidant is used.

The extract containing the antioxidant thus obtained can be used as it is as an antioxidant, but generally, a concentrated solution or dry powder thereof is combined with a known carrier according to a conventional method. It is preferably an oxidizing agent. In the case of concentrating, an evaporation concentration method or the like may be used.
Further, when a dry powder is obtained, a freeze-drying method or a method using a spray dryer may be used. Since the antioxidant substance of the present invention is stable to heat, various concentration and drying methods in the range of 0 to 100 ° C can be used.

The antioxidants obtained as described above have excellent antioxidant properties as shown in the examples below, and from koji mold which has been widely used for the production of foods, alcoholic beverages, etc. since ancient times. Since it was obtained, it is highly safe.

Therefore, for the purpose of preventing the oxidation of fats and oils, it is added to, for example, processed meat products, fats and oils products such as margarine, fruits, processed vegetables products, beverages, processed marine products, foods such as wine, pickles, miso and soy sauce. Besides, it can be added to cosmetics and the like.

The antioxidant obtained as described above is a medicine for preventing and treating diseases which are considered to be caused by oxidative disorders in the living body, such as liver disorders, cranial nerve disorders, lung disorders, cardiovascular disorders and the like. It can also be used as a compounding ingredient in cosmetics that prevents skin aging. When the above antioxidant is used as a medicine, the purity of the antioxidant is listed by using appropriate purification means alone or in combination, and then combined with a known pharmaceutically acceptable carrier for oral or parenteral use. The preparation of

[0019]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Production of Antioxidant from Monascus fungus (microorganism of the genus Monascus): (1) Strains used Monascus fungus shown in Table 1 below was used. (2) Manufacture of koji-making and koji-extract A test red yeast mold is inoculated into a slant of PGA medium ^* at 30 ° C.
A preculture is obtained by culturing for 7 days.

* PGA medium: (composition) Potato 200 g Glucose 20 g Yeast extract 2 g Distilled water 1000 ml pH 5.6 (Preparation) Peel potatoes, cut into dicers, and boil until fully boiled with prescribed half amount of water. And filter. Distilled water is added to the filtrate to a specified amount.

After soaking the rice in water for 24 hours, it is drained for about 1 hour. 50 g of this soaked rice is put into a 200 ml Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes.
After autoclaving, add about 25 steamed rice in the Erlenmeyer flask.
Cool to ℃. Next, add 5 ml of sterile water to the preculture,
Spores were scraped off with an ase to prepare a spore suspension. Aseptically inoculate the steamed rice with all of this spore suspension.
Incubate at 14 ° C for 14 days. During the culture period, the inoculated rice is occasionally stirred.

The rice koji obtained was freeze-dried and then pulverized with a sample mill to obtain rice koji powder. For 1 g of this powder,
4 ml of 0.05 M Tris-hydrochloric acid (pH 7.4)
A buffer solution was added, and the mixture was shaken at 37 ° C for 60 minutes. Then
Cool and centrifuge at 4 ° C for 30 minutes (12,000 rp
m), and the resulting supernatant was used as a koji extract.

(3) Antioxidant action The antioxidative action of the koji extract obtained in (2) above was measured by DPP.
It was investigated by the scavenging action of H (1,1-diphenyl-2-picrylhydrazyl) radical. As the reaction liquid, 10
Ethanol solution is added to 1 ml of 0 μM DPPH ethanol solution.
ml, 0.05M Tris-hydrochloric acid (pH 7.4) buffer
Using 0.95 ml of the extract and 50 μl of the extract to make 3 ml, the absorbance at 517 nm 30 seconds after the addition of the extract was measured.

The radical scavenging action is 100 μM DPP
The absorbance was calculated as the absorbance in the presence of each extract, assuming that the absorbance of H was 100%, and the antioxidant activity was 50% of this absorbance.
% Or less was ++, 70% was ++, and 80% was +. The absorbance of the extract alone was subtracted as a blank. The results are shown in Table 1 along with the color of the extract.

[0026]

Example 2 Hepatopathy-improving effect of antioxidant substances from Aspergillus oryzae: The hepatic-disorder improving action of antioxidant substances produced by a bacterium belonging to the genus Monascus was examined by reducing the toxicity of carbon tetrachloride.

(1) Production of Koji and Koji Extract Monascus Anka I as a bacterium belonging to the genus Monascus
Using FO 6085, the rice koji powder obtained in the same manner as in Example 1 was used as a raw material. 4g for 1g of this powder
0.05 M Tris-hydrochloric acid (pH 7.4) buffer solution was added at the ratio of, and shaken at 37 ° C. for 60 minutes. Then cool to 4 ° C and centrifuge at the same temperature for 30 minutes (12,000 rp
m), and the resulting supernatant was used as a koji extract.

(2) Drug toxicity reducing action: Three groups of male SD rats, each group consisting of four rats, were used as experimental animals. Of the three groups of experimental animals, one group was the group of the present invention, and 7.5 mg / 100 sodium phenobarbital was administered 48 hours and 24 hours before the intraperitoneal administration of carbon tetrachloride.
The rice koji extract was intraperitoneally administered 0.4 ml / 100 g 12 hours and 1 hour before. On the other hand, the other group was a comparative group in which only 7.5 mg / 100 g of phenobarbital sodium was intraperitoneally administered 48 hours before and 24 hours before the intraperitoneal administration of carbon tetrachloride, and the other group was carbon tetrachloride. Was used as a control group. Carbon tetrachloride was intraperitoneally administered in 0.08 ml / 100 g as a 30% corn oil solution.

One hour after the administration of carbon tetrachloride, the experimental animals were sacrificed, and the livers of the experimental animals were perfused with a 1.15% potassium chloride solution and then excised, and 2 ml of 1.15% potassium chloride solution was added per 1 g of the liver. And homogenized. The resulting homogenate was cooled, centrifuged at 12,000 rpm for 30 minutes, the supernatant was filtered, and the homogenate was kept at 4 ° C. for 60 minutes at 35,000.
Centrifuge at 0 rpm.

The obtained supernatant was used as a cytosol fraction,
The precipitate was 0.15M Tris-HCl buffer (pH
After resuspension and washing in 8.0), it is further centrifuged under the above conditions,
The precipitate was used as the microsome fraction.

Using these fractions, glutathione (G
SH) amount, microsomal lipid peroxide (TBARS)
The amount, cytosolic glutathione S-transferase (GSTc) activity and aniline hydrooxidase (AN) activity were measured. The results are shown in Tables 3 and 4.

[0033]

[0034]

As is clear from these results, the antioxidant of the present invention has the action of reducing the toxicity of carbon tetrachloride,
In particular, the effect of reducing the decrease in GSH amount was great. Therefore, the antioxidant of the present invention is effective in treating, preventing or reducing liver damage.

Example 3 Inhibitory Action of Lipid Peroxidation by Antioxidants from Monascus: The inhibitory action of lipid peroxidation of antioxidative substances produced by bacteria belonging to the genus Monascus was examined by the following method. (1) Production of strains used and koji extract The same strains as in Example 2 were used, and the koji extract obtained by the same method was used.

(2) Assay method Microsomes were prepared from rat liver by a standard method, and koji extract of various concentrations was allowed to act on the microsomes to inhibit the lipid peroxidation reaction by hydrogen peroxide (1 mM). It was measured by the TBA (thiobarbituric acid) method. As a result, as shown in FIG. 1, the amount of microsomal lipid peroxide (TBARS) decreased depending on the concentration of koji extract, and it was found that hydrogen peroxide-induced lipid peroxidation reaction was suppressed. It was

Example 4 Hepatotoxicity-reducing action of antioxidative substances from Aspergillus oryzae: The hepatotoxicity-reducing action of antioxidant substances produced by a bacterium belonging to the genus Monascus was reduced by reducing the hepatotoxicity of acetaminophen and galactosamine. Examined.

(1) Production of koji and koji extract Using the same strain as in Example 2, a red koji extract was obtained as follows. First, 25 ml of distilled water was added to 5 g of dried red yeast rice, and the mixture was incubated at 37 ° C. for 60 minutes. This culture was centrifuged at 12,000 rpm for 30 minutes at 4 ° C. to separate the supernatant and the precipitate. The precipitate was further added with 25 ml of distilled water, cultured again at 37 ° C. for 60 minutes, and centrifuged under the same conditions as above to obtain a supernatant. This supernatant and the supernatant initially obtained were combined and concentrated to obtain 1.7 g of red yeast rice extract.

Then, the red yeast rice extract was subjected to Sephadex G-75 column chromatography (column: φ2.6).
× 79 cm (420 ml), solvent; distilled water, fractionation; 4
00 drop (13 ml) / tube)
Fraction Nos. 28-33 having an H radical scavenging action were collected and concentrated to obtain 1.48 g of a red yeast rice extract fraction.
The red malt extract fraction was dissolved in 20 ml of distilled water and further diluted 30 times to obtain a red malt fraction solution.

(2) Acetaminophen Liver Injury-Reducing Action One group of three male SD rats was used as an experimental animal. Of the 3 groups of experimental animals, 1 group was the group of the present invention, and 25 mg / kg of 3-methylcholanthrene was intraperitoneally administered 24 hours before the intraperitoneal administration of acetaminophen,
Further, 12 hours and 1 hour before, 0.4 ml / 100 g of the red yeast rice fraction solution was intraperitoneally administered. On the other hand, the other group was a comparative group in which only 25 mg / kg of 3-methylcholanthrene was intraperitoneally administered 24 hours before the intraperitoneal administration of acetaminophen, and the other group was a control group in which acetaminophen was not administered. In groups. The dose of acetaminophen was 180 mg / kg.

Three hours after the administration of acetaminophen, the rat was killed by decapitation, and blood was collected from the cut end. Then, the rat liver was perfused with a 1.15% potassium chloride solution, excised, homogenized by adding a double amount of the same potassium chloride solution, and then centrifuged at 12,000 rpm for 30 minutes at 4 ° C. The resulting supernatant is 60 at 35,000 rpm.
After centrifuging for minutes, the supernatant was used as cytosol and the precipitate was used as microsome. The microsomes used were further centrifuged twice with 0.1 M Tris-HCl buffer (pH 8.0).

(3) Galactosamine liver injury-reducing action Galactosamine was administered in the same manner as in (2) above except that 400 mg / kg of galactosamine was administered instead of 180 mg / kg of acetaminophen, and blood and liver were collected 24 hours later. Hepatotoxicity was investigated.

(4) Measurement of hepatotoxicity In accordance with a conventional method, transaminase (GOT) and glutathione S-transferase (GST) activities in serum, GST activity in liver cytosol and microsomes,
The amount of glutathione (GSH) in the liver homogenate was measured. GS in serum when acetaminophen is administered
Table 5 for T and GOT, Table 6 for GST in cytosol and microsomes and GSH in hepatic homodonate.
Table 7 shows GST and GOT in serum when galactosamine was administered, and Table 8 shows GST in cytosol and microsomes and GSH in liver fomodnate.

[0045]

[0046]

[0047]

[0048]

As shown in Tables 5 and 7, the serum GOT and GST activities showing hepatotoxicity were increased by the administration of acetaminophen and galactosamine (comparative group), and this increase was observed in the red yeast rice fraction solution. It was suppressed by the pre-administration (the present invention group). Further, as shown in Table 8, the amount of glutathione was decreased by the administration of galactosamine, but it was restored to the control level by the administration of the red yeast rice fraction solution.

Hepatotoxicity is GOT, GS, which is a blood divergent enzyme.
With an increase in T as an index, the higher the value of T, the greater the degree of liver damage, and the decrease of these values indicates that liver damage is suppressed. Then, as shown in Tables 5 and 7 above, the increase in these enzymes due to the administration of acetaminophen or galactosamine was suppressed by the red yeast rice fraction solution in each case. When tested with galactosamine, serum G was obtained by pre-administration of the red yeast rice fraction solution.
The ST value has returned to almost the normal level, and glutathione has also recovered to the normal level.

Liver glutathione acts as a reducing agent in the body, and its low level indicates that it was subjected to oxidative stress. Therefore, acetaminophen and galactosamine cause strong oxidative stress. It is understood that pre-administration of the fraction solution increases the amount of glutathione and suppresses oxidative stress-induced liver damage caused by these drugs. In addition, liver damage caused by galactosamine is used as a model of hepatitis, and it is shown that the present oxidant has an anti-hepatitis action. As described above, it was confirmed that the red koji extract component has a strong hepatoprotective effect and is useful as a preventive and / or therapeutic agent for liver damage.

[0052]

According to the method of the present invention, an antioxidant having excellent antioxidant properties and safety can be obtained. Further, since the raw material thereof also utilizes microorganisms, it is easy to mass-produce it. Then, the obtained antioxidant has an action of suppressing liver damage due to causes such as lipid peroxides and drugs in addition to the usual antioxidant action, so that it can be used as an antioxidant for food additives or cosmetic ingredients. In addition, it can be advantageously used as a prophylactic / therapeutic agent for liver disorders such as so-called hepatoprotective agents and hepatitis therapeutic agents.

[Brief description of drawings]

FIG. 1 is a drawing showing the relationship between the concentration of koji extract and the amount of microsomal lipid peroxide. that's all

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location // (C12P 1/02 Z C12R 1: 645) (72) Inventor Teruichi Teruya Gushikawa City, Okinawa Prefecture 5 Kyushuzaki 1 Tropical Techno Center Co., Ltd.

Claims (5)

[Claims] 1. A culture of a microorganism belonging to the genus Monascus,
A method for producing an antioxidant, which comprises collecting an antioxidant from a culture. 2. The method for producing an antioxidant according to claim 1, wherein the antioxidant is collected by extraction. 3. The method for producing an antioxidant according to claim 2, wherein the extraction is performed with a highly polar solvent. 4. An antioxidant comprising an antioxidant contained in a culture of a microorganism belonging to the genus Monascus or an extract thereof as an active ingredient. 5. A prophylactic / therapeutic agent for liver damage, which comprises an antioxidant contained in a culture of a microorganism belonging to the genus Monascus or an extract thereof as an active ingredient.