JP2005080655A - New composition containing isoflavone and activated oxygen inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an activated oxygen inhibitor from a composition containing isoflavone. <P>SOLUTION: The composition containing the isoflavone is obtained by fermenting a soybean protein, rice bran, and rice germs by using Bacillus natto to form a fermented liquid, subjecting the fermented liquid to protease degradation to form a degradation liquid, adding a fermented material of soybean curd lees, Malta jute, a Siberian ginseng extract, and tea catechin to the degradation liquid to form a reaction product, and extracting the reaction product by isopropyl alcohol, so as to obtain the composition. The activated oxygen inhibitor contains the composition. The inhibitor has antioxidant action and activated oxygen free radical extinguishing action, and toxicity of the inhibitor is extremely low. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a novel isoflavone-containing composition and an activated oxygen inhibitor.

In the prior art, SOD as an activated oxygen inhibitor is difficult to produce and the availability of raw materials is limited. Vitamin E, vitamin C, catechins, flavonoids and peptides are active in experiments using living organisms. Many of the active oxygen inhibitors exhibiting the active oxygen free radical scavenging action and the antioxidant action are mostly produced by chemical synthesis. Even if it is derived from natural products using materials from plants and animals, it is made by using chemical substances that cause harm to the human body during the production process and reacting some of the products with chemical substances There are many. Under such circumstances, there is a demand for an activated oxygen inhibitor having a more potent action.
Japanese Patent Laid-Open No. 06-128168 Japanese Patent Laid-Open No. 08-009892 JP 09-059297 A JP 09-157291 A JP 09-157292 A Japanese Patent Laid-Open No. 11-035598 Japanese Patent Laid-Open No. 11-035599 Japanese Patent Laid-Open No. 11-292897 McCord, J. et al. M.M. & Fridovic, I.D. : J. Biol. Chem. , 244, 6049 (1969) Yamaguchi et al., New Food Industry, Vol. 31, 18-22 (1989) Takushoku, Journal of Japanese Society for Agricultural Chemistry, 65, 1635 (1991) Chen et al. Agric. Food Chem. 46 卷, 49 pages (1998) Kudo et al., Journal of Japan Society for Food Science and Technology, 48, 44 (2001) Seida et al., Journal of Japanese Society for Agricultural Chemistry, 72, 1181 (1998)

Diseases involving activated oxygen include a wide range of diseases such as inflammation such as burns and arthritis, reperfusion injury, side effects of anticancer drugs, radiation injury, peptic ulcer, bacterial shock, cachexia, and autoimmune diseases. All diseases caused by activation of neutrophils and macrophages caused by a large amount of activated oxygen generated are targeted. In general, oxygen includes oxygen essential for animals (triplet oxygen molecule: ³ O ₂ ) and radicals (activated oxygen) generated at specific conditions or when the body is upset. Radicals directly or indirectly (in the form of peroxidation) alter or damage various cell components including cell membranes, intracellular granule membranes, and DNA. The radicals are produced in the body, the type superoxide anion ^(- _O 2 ·), singlet oxygen ⁽¹ _O 2 ·), there is such a radical hydroxide (· OH). Among superoxide anion ^(- O 2 _·) causes peroxidation acts on unsaturated fatty acids in cell membranes, the oxidizing power to lipid is said to be higher several thousand times the essential oxygen to animals. Superoxide dismutase (SOD, enzyme number EC1.15.1.1) as an activated oxygen inhibitor is an enzyme whose action was discovered in 1969 by McCold et al. one-electron reduction has been caused superoxide anion ^(- _{O 2} ·) the disproportionation _{^{2 - O 2 · + 2H +}} → H 2 O 2 + O 2
To catalyze. When the human body is normal, SOD works to suppress the generation of superoxide anions. This SOD activity decreases with aging, that is, the activity decreases from the middle age to the old age, and the increase or decrease in SOD activity is said to be a barometer of aging and canceration of the living body. When such SOD activity decreases, generation of radicals is difficult to suppress, and it becomes necessary to reinforce intake of SOD or to ingest an activated oxygen inhibitor that captures and removes radicals.
On the other hand, the activated oxygen inhibitory action of polypeptides from amino acids to proteins as water-soluble antioxidants inhibits the contact between oxygen molecules and unsaturated fatty acids by encapsulating fats and oils, resulting in lipid peroxy radicals (LOO).・) Is considered to suppress the generation of, and acts on radicals (LOO.) Generated during the oxidation of fats and oils (L), such as the antioxidant action of BHA (butylhydroxyanisole) and BHT (dibutylhydroxytoluene). Thus, it is distinguished from the radical scavenging action that stops the chain reaction of oxidation.
LOO ・ + AH ₂ → LOOH + AH ・
2AH · → 2AH ₂ + A or LOO · AH · → LOOH + A
(AH ₂ ; antioxidant)
Today, in the absence of anti-cancer and anti-aging drugs, DNA damage factors, mutation factors, carcinogenic factors, aging factors, etc. are removed from the environment to inactivate, showing activated oxygen free radical scavenging action and antioxidant action Studies and studies on activated oxygen inhibitors are ongoing. On the other hand, as a water-soluble antioxidant, there is very little knowledge about the amino acid sequence and antioxidant power of polypeptides ranging from amino acids to proteins. Yamaguchi et al. [Non-Patent Document 2] show that dipeptides are more antioxidant than amino acids and proteins. Recently, Takushoku et al. [Non-patent Document 3] recently published Ala-His-Lys, Val-His-His, Val-His-His-Ala-Asn-Glu-Asn, Chen et al. [Non-Patent Document 4] describes Leu-Leu-Pro-His-His and other five types of antioxidant peptides, and Kudo et al. [Non-Patent Document 5] describes Ala-Arg-His-Pro-His-Pro-His-Leu- Ser-Phe-Met is reported. In contrast, the inventors have reported many catechins [Patent Document 1] and activated oxygen-scavenging peptides (antioxidant peptides) [Patent Documents 2 to 8]. In addition, there is no report that a fertility oxygen inhibitor having an antioxidant action or the like has been developed as a pharmaceutical.

The present inventor obtained an isoflavone obtained by subjecting soy protein, rice bran, and rice germ to fermented with natto bacteria with protease, followed by further adding okara fermented product, moroheiya, sorghum extract and tea catechin, followed by isopropyl alcohol extraction It has been found that the containing composition has a strong activated oxygen free radical scavenging action as well as an antioxidant action. And they have intensively studied to put these novel isoflavone-containing compositions into practical use as pharmaceuticals. As a result, the novel isoflavone-containing composition has an activated oxygen free radical scavenging action and an antioxidant action, and has found usefulness as a natural product-derived activated oxygen inhibitor. The present invention is based on such knowledge. The present invention is described in detail below. The novel isoflavone-containing composition according to the present invention is a white powder at room temperature.

When preparing the novel isoflavone-containing composition according to the present invention, soy protein, rice bran and rice germ are used as materials. Natto bacteria (for example, Bacillus NATTO) is added to a mixture obtained by adding fresh water to the mixture and then culturing with stirring. Furthermore, after adding a proteolytic enzyme (for example, Protease) to the culture solution, the enzyme is decomposed with stirring in the same manner. After the enzymatic decomposition is completed, boiling is performed to deactivate the proteolytic enzyme. After cooling the enzymatic decomposition solution, fermented okara, rice bran, sorghum extract and tea catechin are added and heated with stirring. Then, after adjusting the pH of the reaction solution to the acidic side (for example, pH 3 to 4), using a continuous centrifuge (for example, DeLaval, decanter, etc.), and further using an ultra-high speed centrifuge (for example, Sharpless). Centrifugation (for example, 4,000 rpm to 15,000 rpm) to obtain a centrifugal supernatant. This centrifugal supernatant is freeze-dried and spray-dried to obtain an isoflavone-containing crude powder. The isoflavone-containing crude powder is fractionated with an alcohol (for example, isopropyl alcohol). After removing alcohols (for example, isopropyl alcohol) from these fractions containing a lot of isoflavones, the low-molecular-weight fractions are removed with a reverse osmosis membrane (RO membrane) and a microfiltration membrane (MF membrane), and then lyophilized. And spray-dried to obtain an isoflavone-containing composition powder.

The novel isoflavone-containing composition according to the present invention does not cause antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. In addition, since these isoflavone-containing compositions are gradually decomposed by a degrading enzyme in vivo after administration, the toxicity is very low and the safety is very high (LD ₅₀ > 5000 mg / kg: oral administration in rats).
These isoflavone-containing compositions can be prepared into injections, tablets, capsules, granules, powders and the like using commonly used additives such as excipients. Examples of the administration method include injection into a mammal (for example, human, dog, rat, etc.) usually having oxidative stress caused by activated oxygen, or oral administration. The dose is, for example, 0.1 to 100 mg of these isoflavone-containing compositions per kg of animal weight. The number of administration is usually about 1 to 4 times a day, but can be appropriately adjusted depending on the administration route. The types of excipients, binders, and lubricants used in the above various preparations are not particularly limited, and those used in ordinary injections, powders, granules, tablets, or capsules can be used. .

Examples of additives used for tablets, capsules, granules, and powders include the following. As excipients, sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate, etc .; starches, hydroxylpropylmethylcellulose, etc. as binders; carboxymethylcellulose and potassium salts thereof as disintegrants; Examples of lubricants include stearic acid and its salts, talc, and waxes. In preparation of the preparation, flavoring agents such as menthol, citric acid and salts thereof, and fragrance can be used as necessary. The sterile composition for injection is prepared by dissolving or suspending the isoflavone-containing composition in water for injection, physiological saline, sugar alcohol injection such as xylitol or mannitol, or glycol such as propylene glycol or polyethylene glycol by a conventional method. Can be used as an injection. At this time, a buffer solution, a preservative, an antioxidant and the like can be added as necessary. A preparation containing these isoflavone-containing compositions is in the form of a lyophilized product or a dry powder, and can be used after being dissolved in an ordinary solubilizer such as water or physiological saline at the time of use.

Activated oxygen is produced in phagocytic cells such as macrophages and has the role of decomposing foreign substances that the phagocytic cells prey on. Cause trouble. The novel isoflavone-containing composition according to the present invention has an excellent activated oxygen free radical scavenging action and an antioxidant action and exhibits an activated oxygen inhibitory action. Furthermore, because it has the action of decomposing excess active oxygen that causes tissue damage and protecting the tissue, it is effective for arthritis, rheumatism, etc. as an anti-inflammatory agent, and also useful for Behcet's disease, myocardial infarction, etc. It is.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a novel isoflavone-containing composition according to the present invention described below, which has utility as a pharmaceutical product, and an activated oxygen inhibitor having an activated oxygen free radical scavenging action and an antioxidant action, comprising the isoflavone-containing composition as an active ingredient About.
As examples, production examples and test examples will be described below to further explain the present invention. However, the present invention is not limited to these examples.

Production Example 1
To 100 kg of soy protein, 25 kg of rice bran and 25 kg of rice germ, 3,000 liters of fresh water was added and stirred. While stirring, sterilization was performed at a liquid temperature of 95 ° C. for 1 hour. After cooling, 4.5 kg of Bacillus Natto (3% relative to soy protein, rice bran and rice germ) was added, and the mixture was cultured at 40 to 45 ° C. for 28 hours with stirring. Next, 4.5 kg of protease (Protease) (3% of soybean protein, rice bran, and rice germ) is added to the culture solution of Bacillus natto, and enzymatic degradation is performed with stirring at a liquid temperature of 45-50 ° C. for 15 hours. It was. For enzyme deactivation, after boiling at 95 ° C. for 10 minutes, after cooling, add 100 kg of Okara fermented product, 25 kg of rice germ, 10 kg of Sorghum extract and 130 g of tea catechin, and stir at a liquid temperature of 55-60 ° C. for 2 hours. The reaction was carried out. Next, after adjusting the pH of the reaction solution to pH 3.8 using citric acid, continuous centrifuge DeLaval (4,000 rpm to 6,000 rpm), and further, ultra-high speed centrifuge shear press (10,000 rpm to (15,000 rpm) to obtain a centrifugal supernatant. Next, the centrifugal supernatant was lyophilized to obtain 50.3 kg of coarse powder containing isoflavone. The isoflavone-containing crude powder was subjected to 5% to 95% isopropyl alcohol fractionation in a cold place (5 ° C.). After removing isopropyl alcohol from the fraction containing high isoflavones using the vacuum evaporator Evapor, filtered through a reverse osmosis membrane (RO membrane) and precision filtration membrane (MF membrane) to remove the low molecular fraction At the same time, a higher fraction fraction containing isoflavones was obtained. This was freeze-dried to obtain 42.7 kg of an isoflavone-containing powder.
The novel isoflavone-containing composition according to the present invention obtained by the above production examples was confirmed to have an activated oxygen inhibitory action such as an activated oxygen free radical scavenging action and an antioxidant action by the following tests.

Test example 1
(Measurement of activated oxygen free radical scavenging action)
The measurement was carried out according to the method of Takeda et al. That is, 0.1 mL each of 3 mM xanthine, 3 mM EDTA, 1 mM XTT, and the sample solution was added to a test tube containing 2.5 mL of a buffer solution (50 mM), and 0.1 mL of 57 mU / mL XOD was immediately added as a trigger. After reacting accurately at 25 ° C. for 20 minutes, the absorbance at 470 nm was measured. The concentration at which the reduction of XTT by activated oxygen free radicals (superoxide anion) was inhibited by 50% was defined as the IC ₅₀ value. The results are shown in FIG. The activated oxygen free radical scavenging activity, that is, the activated oxygen inhibitory activity (IC ₅₀ value) of the novel isoflavone-containing composition according to the present invention is 1.26 × 10 ⁻³ .
Test example 1
(Measurement of antioxidant effect)
For the measurement of the antioxidant effect, the reaction solution was added to a mixture of linoleic acid 51.1 mg, ethanol 4.052 mL, 0.05 M phosphate buffer (pH 7.0) 4.0 mL, and deionized water 1.948 mL. 1-3 mg of the peptide having an action was added, and the total amount was adjusted to 10 mL. The solution was sealed with a threaded test tube and allowed to stand in a thermostat at 50 ° C., and the peroxide value of linoleic acid was measured by the rodan iron method every 24 hours.
That is, 0.1 mL of reaction solution, 9.7 mL of 75% ethanol solution, 0.1 mL of 30% rhodanonium ammonium solution, and 0.1 mL of 3.5% hydrochloric acid solution containing 0.02M ferric chloride were added and reacted for 3 minutes. Thereafter, an absorbance of 500 nm was measured. At that time, the number of days until the absorbance value at 500 nm reached 0.300 was defined as the induction period (days). The results are shown in FIG. The antioxidant activity (induction days) of 0.1 mg of the novel isoflavone-containing composition according to the present invention is 63.7 days, 0.2 mg; 72.4 days, 0.3 mg; 81.9 days.
As a result of the above tests, it was confirmed that the novel isoflavone-containing composition according to the present invention has an activated oxygen inhibitory action such as an activated oxygen free radical scavenging action and an antioxidant action. Therefore, the novel isoflavone-containing composition according to the present invention is useful as a therapeutic or prophylactic agent for patients with ischemic heart disease, rheumatoid arthritis, and severe burn patients who are targets of activated oxygen inhibitors. The novel isoflavone-containing composition according to the present invention can also be employed in the structure of the flavonoids structurally.

It is a figure which shows the activated oxygen free radical scavenging action of the isoflavone containing powder in manufacture example 1 of the novel isoflavone containing composition which concerns on this invention. It is a figure which shows the antioxidant effect | action of the isoflavone containing powder in manufacture example 1 of the novel isoflavone containing composition which concerns on this invention.

Claims (2)

  An isoflavone-containing composition obtained by subjecting a fermented solution fermented with natto to soybean protein, rice bran, and rice germ to protease decomposition, and further extracted with isopropyl alcohol by adding an okara fermented product, moroheiya, ezokogi extract and tea catechin. As an active ingredient, an isoflavone-containing composition obtained by subjecting soy protein, rice bran, and rice germ to fermented with natto bacteria with protease, followed by protease decomposition, addition of okara fermented product, moroheiya, sorghum extract and tea catechin, and isopropyl alcohol extraction An activated oxygen inhibitor characterized by containing.

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