JP2014162730A - Superoxide dismutase activity enhancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel superoxide dismutase (SOD) activity enhancer.SOLUTION: A superoxide dismutase (SOD) activity enhancer comprises Miso extracts as an active ingredient.

Description

  The present invention relates to a superoxide dismutase activity enhancer.

  Miso, the representative of Japanese food culture, was originally a luxury and valuable food that was valued by temples and aristocratic classes, and was used as a side dish and medicine rather than being cooked as miso soup. Since the samurai's dietary habit of “one soup one dish” was established during the Kamakura period and it became popular to eat in the form of miso soup, it is now an indispensable item for the Japanese diet.

  The main ingredient of miso is soybean, which is rich in nutrients by mixing koji and salt and fermenting it, soy protein is easy to digest, and amino acids that are the source of umami are released in large quantities. In addition, many nutrients such as saponins, isoflavones, enzymes, vitamins, potassium, and fiber are included, and effects on health maintenance and prevention of lifestyle-related diseases are expected. On the other hand, there is a disagreement that the health effects of miso have been underestimated due to concerns about the salt contained. Miso contains many nutrients in addition to salt, and is expected to suppress salt absorption in the gastrointestinal tract, dilate blood vessels, protect endothelial cells, and protect blood vessels and organs beyond the effects of salt. It is considered high. However, the health benefits of miso have often been inferred from the constituent nutrients of miso so far, and long-term ingestion of miso exerts a protective effect on vascular disorders that are actually seen in hypertension, and contributes to brain, heart and kidney function. It is not always clear whether it is useful or not.

  Miso is known to contain antioxidants such as saponins and isoflavones. Moreover, the free radical elimination effect | action is known as an effect | action of a miso extract (patent documents 1, 2).

  Of the active oxygens, hydroxyl radicals, which are free radicals, are most likely to react and have strong oxidative power, and therefore tend to give oxidative stress to living tissue and cause tissue damage. For example, hydroxyl radicals react with biomolecules such as proteins, DNA, lipids, enzymes and the like to change their structures and impair cell functions. In addition, hydroxyl radicals react with lipids such as cholesterol and neutral fat to produce lipid peroxides, which damage and destroy cells themselves constituting tissues and organs.

  A superoxide anion exists upstream of the hydroxy radical, and the superoxide anion is detoxified to oxygen and hydrogen peroxide by superoxide dismutase (SOD).

  Therefore, if the biological tissue SOD activity is enhanced, it becomes possible to suppress the peroxidation reaction caused by the human oxyl radical and the accompanying biological tissue oxidative stress.

  However, the antioxidant effects such as saponins and isoflavones contained in miso and the free radical scavenging action (free radical scavenger action) of the miso extract are independent of the SOD activity.

JP 2006-69949 A JP 2006-70015 A

  An object of the present invention is to provide a novel superoxide dismutase (SOD) activity enhancer.

In order to solve the above-mentioned problems, the present invention provides a superoxide dismutase (SOD) activity enhancer containing a miso extract as an active ingredient.
In a preferred embodiment of the SOD activity enhancer of the present invention, the miso extract is a fraction having a molecular weight of less than 3 kDa obtained by filtering the miso extract with a filtration membrane having a fractional molecular weight of less than 3 kDa.
A preferred embodiment of the SOD activity enhancer of the present invention is a SOD activity enhancer for enhancing brain tissue SOD activity or heart tissue SOD activity.
A preferred embodiment of the SOD activity enhancer of the present invention is a SOD activity enhancer for suppressing brain tissue oxidative stress or heart tissue oxidative stress.

  According to the present invention, as a novel SOD activity enhancer, an SOD activity enhancer containing a miso extract as an active ingredient is provided.

It is a figure which shows the measurement result of the rat brain lipid peroxide (LPO) amount in each experimental group. It is a figure which shows the measurement result of the rat brain SOD activity in each experimental group. It is a figure which shows the measurement result of the amount of lipid peroxide (LPO) produced | generated from the rat brain homogenate which added the centrifugal supernatant of the miso aqueous solution, or its MW <3kDa fraction. It is a figure which shows the measurement result of DPPH radical scavenging action. It is a figure which shows the measurement result of the rat heart tissue lipid peroxide (LPO) amount in each experimental group, and the area of intramyocardial collagen fibers. It is a figure which shows the measurement result of the rat heart tissue SOD activity in each experimental group. It is a figure which shows the relationship between myocardial collagenous fibrosis and the amount of heart tissue lipid peroxide (LPO).

  The SOD activity enhancer of the present invention contains a miso extract as an active ingredient. Here, the “miso extract” includes any form of a miso extract, a diluted solution of the miso extract, a concentrated solution or a dried product, or a purified product thereof.

  The miso used as an extraction raw material when obtaining the miso extract is not particularly limited as long as it is an edible miso. As miso, for example, rice miso (soybeans (excluding defatted soybeans; the same applies hereinafter) cooked in rice and cultured with koji mold (hereinafter referred to as “rice koji”) is added. Salt mixed), barley miso (soybean soup, barley or bare wheat steamed and koji mold cultured (hereinafter referred to as “wheat cake”) mixed with salt), Bean miso (soybean boiled and cultured with koji mold (hereinafter referred to as “bean koji”) mixed with salt), mixed miso (rice miso, wheat miso or bean miso mixed, rice koji and wheat Rice miso, wheat miso, and beans miso, etc., etc. (classified based on raw materials), sweet miso, dry miso (classified based on taste), red Miso, light-colored miso, white miso (classified based on color) Of these, US miso are preferred, more preferably dry miso of rice miso. Miso can be used as an extraction raw material alone or in combination of two or more.

The miso used as an extraction raw material when obtaining the miso extract may be either a homemade miso brewed according to a conventional method or a commercially available miso.
A general method for brewing miso is a koji-making process in which various koji such as rice koji, wheat koji, and soybean cake are obtained by adding koji fungus to rice or wheat and other cereals or beans cooked and cultivated, and soybean Steamed rice (If necessary, you may add rice or wheat steamed grains or beans), various rice cakes obtained in the koji making process, salt, water, other materials in the desired ratio And a fermentation process for obtaining a brewed product by fermenting and aging the koji mixed material.

  Examples of cereals used for brewing miso include rice, wheat (for example, barley, bare wheat, hard wheat, rye, wheat, etc.), corn, buckwheat, millet, millet, and the like. Examples of beans include soybeans, mung beans, peas, black beans, red beans, broad beans, and the like. There are no particular limitations on the varieties and production areas of cereals and beans.

  The koji mold added and cultured to obtain various koji is not particularly limited as long as it is a koji mold generally used for brewing miso. Examples of Aspergillus include Aspergillus oryzae, Aspergillus sojae, Aspergillus tamari and the like. The koji mold may be added to steamed cereals and / or beans in the form of seed pods. The temperature at which the koji mold is added and cultured depends on the optimum growth temperature of the koji mold used, but is usually 20 to 45 ° C., preferably 25 to 40 ° C.

  The salt used for brewing miso is not particularly limited as long as it is an edible salt, and examples thereof include salt, special grade salt, rock salt, common salt, and white salt.

  Examples of other materials used for brewing miso include salt-tolerant yeast and salt-tolerant lactic acid bacteria, and these may be further added and mixed in the preparation step to prepare a koji-mixed material. . Examples of the salt-tolerant yeast include Zygosaccharomyces rouxii, Candida versatilis, Candida etchelsi, and the like, Examples thereof include Tetragenococcus halophilus and Pediococcus acidilactici.

  Various materials (cereals, beans, salt, other materials, etc.) used for brewing miso can be blended in proportions appropriately adjusted according to the type of miso to be brewed. For example, the blending ratio of koji is usually 50% to 20%, preferably 50%. The compounding ratio of the salt is usually 5% by weight or more, preferably 8 to 13% by weight. The amount of water contained in the soot mixed material is usually adjusted to a range of 35 to 55% by weight in consideration of the amount of water contained in all materials.

  A brewed product, that is, miso is brewed by fermenting and aging the koji mixed material obtained in the preparation process in the fermentation and aging process. The fermentation / ripening step is usually performed at 10 to 80 ° C, preferably 25 to 35 ° C or 45 to 60 ° C. You may stir suitably as needed in the middle of a fermentation / ripening process. The period of the fermentation / ripening step depends on the ripening temperature, but is usually about 1 month to 1 year at 25 to 35 ° C. and usually about 1 to 7 days at 45 to 55 ° C.

  The solvent used as the extraction solvent when obtaining the miso extract is preferably a polar solvent. Examples of the polar solvent include water, a polar organic solvent, and a mixed solution thereof, and water is preferable. Examples of water include tap water, deionized water, and distilled water. Examples of polar organic solvents include monohydric alcohols, polyhydric alcohols, and ketones. Examples of the monohydric alcohol include lower fats having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butyl alcohol, isobutyl alcohol, sec-butyl alcohol, tert-butyl alcohol, pentyl alcohol, and isopentyl alcohol. Examples of the polyhydric alcohol include C2-C5 polyhydric alcohols such as ethylene glycol, propylene glycol, 1,3 butylene glycol, and glycerin. Examples of the ketone include acetone. , Lower aliphatic ketones such as methyl ethyl ketone and diethyl ketone. These solvents are usually used as extraction solvents at room temperature or at temperatures below the boiling point of the solvent. When using the liquid mixture of 2 or more types of solvents as an extraction solvent, the mixing ratio can be adjusted suitably.

  The extraction method for obtaining the miso extract is not particularly limited as long as the miso component soluble in the polar solvent can be eluted, and a normal extraction method used when separating the component from the food may be used. it can. For example, the extraction raw material is put into a treatment tank filled with an extraction solvent, and after elution of soluble components with occasional stirring as necessary, solids are removed by standing, filtration or centrifugation to obtain an extract. be able to. The amount of extraction solvent is usually 5 to 30 times (weight ratio), preferably 10 to 25 times (weight ratio) of the extraction raw material. When water is used as the extraction solvent, the extraction temperature is usually 4 to 40 ° C, preferably 15 to 35 ° C. Centrifugation conditions are usually 5000-15000 g, preferably 8000-12000 g, and usually 5-15 minutes, preferably 8-12 minutes.

  When the solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and when this concentrate is further dried, a solid extract is obtained. The miso extract may be purified by a method such as activated carbon treatment, adsorption resin treatment, ion exchange resin, liquid-liquid counter-current distribution, etc., as long as the physiological activity is not lowered.

  The miso extract is preferably a fraction having a molecular weight of less than 3 kDa obtained by filtering the miso extract with a filter membrane having a fractional molecular weight of less than 3 kDa. Examples of the filtration membrane include a molecular sieve membrane (ultrafiltration membrane), a reverse osmosis membrane and the like, and a molecular sieve membrane (ultrafiltration membrane) is preferable. Filtration is preferably carried out at 4 ° C.

  Miso extract has an SOD activity enhancing action. Although the details of the active ingredient have not been clarified at present, it is clear from the examples described later that the extract of miso has SOD activity enhancing action.

  The miso extract can be used as it is as an SOD activity enhancer, but it can be used as a tablet, capsule, powder, granule, syrup, troche using a pharmaceutically acceptable carrier or other auxiliary agent. It can also be used by formulating it into a desired dosage form such as an agent, a drink, an injection, a drip, a suppository. Examples of the auxiliary agent used for the formulation include excipients, binders, disintegrants, lubricants, stabilizers, extenders, diluents, flavoring and flavoring agents, and the like.

  The administration route of the SOD activity enhancer of the present invention is not particularly limited, but is preferably oral administration. The dose and frequency of administration of the SOD activity enhancer of the present invention can be appropriately adjusted according to the administration subject, administration route and the like. The miso extract, which is the active ingredient of the SOD activity enhancer of the present invention, is non-toxic to the human body, so there is no particular limitation on the amount of intake thereof. In terms of the amount of the product, it can be administered about 1 to 3 times a day so that the daily dose is 8 to 24 g / day.

  The SOD activity enhancer of the present invention can enhance in vivo SOD activity (particularly brain tissue SOD activity and heart tissue SOD activity). Here, “enhancement of SOD activity” means that SOD activity is enhanced with a statistically significant difference compared to the control. The SOD activity enhancer of the present invention preferably enhances SOD activity to 115% or more, more preferably 120% or more, and further to 130% or more when the control is 100%. Even more preferred. Examples of methods for measuring SOD activity used for comparing SOD activity include absorptiometry (eg, cytochrome c reduction, nitroblue tetrazolium (NBT) reduction, etc.), chemiluminescence (eg, chemiluminescence). Examples of the probe include a sea firefly luciferin analog (MCLA) or lucigenin), an electron spin resonance spectroscopy (ESR) method, and the like.

  The SOD activity enhancer of the present invention enhances the SOD activity that renders the superoxide anion harmless to oxygen and hydrogen peroxide, thereby reducing the human oxyl radical existing downstream of the superoxide anion and the peroxidation by the hydroxy radical. Reaction and accompanying biological tissue oxidative stress (particularly brain tissue oxidative stress and heart tissue oxidative stress) can be suppressed.

[Example 1]
In this example, the amount of lipid peroxide in the brain (hereinafter referred to as “LPO amount”) and superoxytodismutase activity (hereinafter referred to as “SOD activity”) were measured ex vivo. In addition, the DPPH radical scavenging action was measured in vitro.

1. Experimental Method (1) Reagents The following reagents were purchased from Wako Pure Chemical Industries, Ltd.
2-amino-2-hydroxymethyl-1,3-propanediol 999
Trichloroacetic acid Sucrose 4,6-dihydroxy-2-mercaptopyrimidine 1,1,3,3-tetraethoxypropane rabbit albumin (4X Crystallized)
· Sodium hydroxide · Ethylenediaminetetraacetic acid · L (+)-sodium ascorbate · 1,1-diphenyl-2-picrylhydrazyl (DPPH)
・ Ethanol (99.5%)
The following kits were purchased from Dojindo Laboratories.
・ SOD assay kit-WST
Bio-Rad protein assay (BIO-RAD) was used for protein amount measurement.

(2) Apparatus The following apparatus was used for the measurement of lipid peroxide amount.
・ Homogenizer Kinematica CH-6010 (manufactured by POLYTRON)
・ Small high-speed cooling centrifuge MX-301 (manufactured by Tommy)
・ Genius Dry Bath Incubator MD-01N (manufactured by Major Science)
・ V-520-SR type spectrophotometer (manufactured by JASCO Corporation)
・ Lacom tester-tabletop PH meter PH510 (manufactured by ASONE Co., Ltd.)
The following equipment was used for the measurement of SOD activity.
・ Homogenizer Kinematica CH-6010 (manufactured by POLYTRON)
・ Model 680 Microplater Reader (BIO-RAD)
The following equipment was used for measuring the DPPH radical scavenging action.
・ V-520-SR type spectrophotometer (manufactured by JASCO Corporation)

(3) Experimental group 20 6-week-old female salt-sensitive Dahl rats (purchased from Sankyo Lab Service Co., Ltd.)
(A) A tap water administration group (hereinafter referred to as “control group”),
(B) 0.5% saline administration group (hereinafter referred to as “0.5% DS group”), and (C) 4% miso aqueous solution (corresponding to 0.5% saline) administration group (hereinafter “MISO group”). Called.)
The animals were reared for 12 weeks under constant temperature and humidity (23 ± 2 ° C.) and automatic lighting (8:00 am to 8:00 pm).
Rats of any experimental group were allowed to freely feed the solid feed CE-7 (Japan Clare Co., Ltd.) for long-term breeding.
The 0.5% saline was prepared by dissolving sodium chloride (Wako Pure Chemical Industries, Ltd.) in water and freely ingested by the 0.5% DS group.
A 4% miso aqueous solution was prepared by dissolving 40 g of miso with 1000 mL of tap water at about 25 ° C., pulverizing with a homogenizer, and applying to an autoclave, and the MISO group freely ingested. As the miso, commercially available dry rice miso (boiled rice paste: 5 steps, salt content: 12.5%, raw materials: rice (domestic), salt (domestic), soybean (produced in North America)) was used.
The water in the control group and the saline in the 0.5% DS group were changed once every two days, and the aqueous miso solution in the MISO group was changed daily.
After rearing for 12 weeks, brains were removed from Dahl rats under pentobarbital anesthesia, immediately immersed in liquid nitrogen, and stored at −80 ° C. until measurement of lipid peroxide (LPO) level and SOD activity.

(4) Method for measuring the amount of LPO in the brain (4-1) Method for measuring the amount of LPO in the brain 2 mL of D-PBS (pH 7.4) was added to the brain tissue extracted from the rats of each experimental group and homogenized. After centrifugation at 1,000 g for 10 minutes, the supernatant was recovered, and 1 mL of the supernatant was used for measurement. The collected brain homogenate supernatant was incubated at 37 ° C., and lipid peroxide produced in 30 minutes was converted into malondialdehyde under acidic conditions, and then developed with thiobarbituric acid, and the absorbance at 532 nm was measured.

(4-2) Influence of Miso Aqueous Solution Centrifugal Supernatant and its MW <3 kDa Fraction on Lipid Peroxide Formation Rat Membrane Aqueous Membrane Supernatant, Miso Aqueous Solution Centrifugal Supernatant (0.1 g / mL) or its MW <3 kDa Fraction (0.1 g / mL) was added to an equal volume, incubated at 37 ° C., and lipid peroxide produced in 30 minutes was converted to malondialdehyde, then developed with thiobarbituric acid, and absorbance at 532 nm Was measured. The centrifugal supernatant of the miso aqueous solution is a supernatant obtained by adding 100 mL of water at about 4 ° C. to 10 g of miso and stirring sufficiently, followed by centrifugation at 10,000 g for 10 minutes. The MW <3 kDa fraction is a fraction having a molecular weight of less than 3 kDa (MW <3 kDa) obtained by filtering the centrifugal supernatant of a miso aqueous solution with a molecular sieve membrane having a fractional molecular weight of less than 3 kDa (ultrafiltration membranes NMWL3,000 manufactured by MILLIPORE). Minutes.

(4-3) Method for Measuring Protein Amount A protein amount in a measurement sample was measured using a Bio-Rad protein assay (manufactured by BIO-RAD). 100 μL each of the standard solution, the measurement sample diluted 50 times, and distilled water (blank) were added, and 5 mL reaction reagent was added and immediately mixed well. After incubating for 15 minutes, the absorbance at 595 nm was measured using a spectrophotometer. The concentration and amount of protein in the measurement sample were calculated using a calibration curve.

(5) Measurement method of SOD activity in brain 5 mL sucrose buffer (0.25 mol / L sucrose, 10 mmol / L tris-HCl buffer (pH 7.4), 1 mmol / L EDTA) was added, homogenized well, centrifuged at 16,000 g for 30 minutes at 4 ° C., and the resulting supernatant was used as a measurement sample. After incubating at 37 ° C. for 20 minutes using SOD assay kit-WST, the absorbance was measured with a Microplate Reader (416 nm).

(6) Measuring method of DPPH radical scavenging action 20% ethanol was added to a 0.4 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution and dissolved. Add 1 mL of the prepared DPPH solution to six test tubes, add 80% ethanol 300, 270, 240, 210, 180 and 150 μL to each, and add 10% miso aqueous solution to 0, 30, 60, 90, 120. 150 μL each was added and the absorbance at 520 nm was followed for 20 minutes. As a standard solution, 0.2 mM L (+)-sodium ascorbate was used.

(7) Statistical analysis Data were expressed as mean ± standard deviation (SD). Tested by analysis of variance, Post-hoc (LSD) using STATISTICA. Two-sided test determined p <0.05 as significant.

2. Result (1) Measurement result of LPO amount in the brain (Fig. 1)
As shown in FIG. 1, the amount of LPO produced in brain tissue homogenate did not change in the 0.5% DS group compared to the control group, but significantly increased in the MISO group compared to the 0.5% DS group. Decreased (0.323 ± 0.085 vs 0.177 ± 0.111 ng / mg protein / 30 min, p <0.05).

(2) Measurement results of SOD activity in the brain (Fig. 2)
As shown in FIG. 2, the SOD activity in brain tissue homogenate was not changed in the 0.5% DS group compared to the control group, but was significantly higher in the MISO group than in the 0.5% DS group. (4.336 ± 0.187 vs 11.173 ± 6.396 U / mg protein, p <0.05).

(3) Effect of centrifugal supernatant of miso aqueous solution and its MW <3 kDa fraction on lipid peroxide production (FIG. 3)
When the amount of LPO produced from the centrifugal supernatant (0.1 g / mL) of the miso aqueous solution or the brain tissue homogenate added with the MW <3 kDa fraction (0.1 g / mL) was examined, as shown in FIG. , All showed significantly lower values compared to the control with buffer added (0.110 ± 0.015 vs 0.032 ± 0.008 ng / mg protein / 30 min, p <0.05). 0.102 ± 0.007 vs 0.034 ± 0.003 ng / mg protein / 30 min, p <0.05)).
Moreover, as shown in FIG. 3, the MW <3 kDa fraction showed the lipid peroxide production inhibitory effect comparable to the centrifugal supernatant of the miso aqueous solution.

(4) DPPH radical scavenging action measurement results (Fig. 4)
In vitro, when the presence or absence of the active oxygen scavenging action of the miso component was examined, as shown in FIG. The DPPH radical scavenging effect was shown, but it was shown that MISO's DPPH radical scavenging action (“MISO with DPPH” in the figure) was 14% stronger than vitamin C.

3. Discussion Membrane lipids in brain cell membranes in the brain are rich in unsaturated acids that tend to produce lipid peroxides. In addition, active oxygen produced in mitochondria and not eliminated by antioxidant enzymes such as SOD generates hydroxyl radicals, and the generated hydroxyl radicals react with membrane lipids to produce lipid peroxides (Eguchi H, Fujiwara N, Ookawara T, Suzuki K, Taniguchi N. Oxidative stress and health.Biological Sample Analysis 2009; 32: 247-256., Ji LL, Dillon D, Wu E. Alteration of antioxidant enzymes weith aging in rat skeletal muscle and liver Am J Physiol 1990; 2: 918-923.). Accumulation of a large amount of lipid peroxide is thought to damage brain blood vessels and cerebral neurons and cause brain dysfunction such as memory impairment and movement disorder.
The results of this example showed that oral administration of miso significantly reduced lipid peroxide produced from the brain tissue of salt-sensitive Dahl rats as compared to the salt intake group at the same concentration. Moreover, the brain tissue SOD activity was significantly higher in the miso ingestion group than in the saline group.
From these results, it was shown that the miso component enhances brain tissue SOD activity and exerts a brain tissue oxidative stress lowering action through suppression of active oxygen in the brain. In addition, since the fraction with a molecular weight of less than 3 kDa showed the same level of lipid peroxide inhibitory effect as the centrifugal supernatant of the miso aqueous solution, the oxidative stress inhibitory factor (particularly the brain tissue SOD activity-enhancing component) is a fraction less than 3 kDa. Presumed to exist in minutes.

The results and discussion of Example 1 are summarized below.
・ Ingestion of miso suppresses oxidative stress in the brain.
・ It is thought that this involves the SOD activity maintenance and hydroxyl radical scavenging action by the miso component.
-Miso's oxidative stress inhibitor is thought to exist in fractions with a molecular weight of less than 3 kDa.

[Example 2]
The heart is one of the important target organs of hypertension, and pressure overload causes myocardial necrosis and collagen fibrosis, leading to heart failure. Increased systolic and diastolic blood pressure burden results in myocardial remodeling. Myocardial fibrosis has been reported to progress through oxidative stress (Barnes PJ, Karin M. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997; 336: 1066- 1071.). In Example 1, it was found that the miso component suppresses oxidative stress by direct scavenger action (capture action) of hydroxyl radical and SOD activity enhancement action. If this is correct, it is expected that the effects of suppressing myocardial damage and collagen fiber proliferation will be observed when ingesting miso. Salt-sensitive Dahl rats have the property that myocardial damage is likely to occur with salt-dependent hypertension. Thus, the purpose of this example is to clarify the relationship between the influence of miso intake on oxidative stress in heart tissue and myocardial collagen fibrosis using salt-sensitive Dahl rats.

1. Experimental Method (1) Reagents The following reagents were purchased from Wako Pure Chemical Industries, Ltd.
2-amino-2-hydroxymethyl-1,3-propanediol 999
Trichloroacetic acid Sucrose 4,6-dihydroxy-2-mercaptopyrimidine 1,1,3,3-tetraethoxypropane rabbit albumin (4X Crystallized)
Sodium hydroxide / ethylenediaminetetraacetic acid The following kits were purchased from Dojindo Laboratories.
・ SOD assay kit-WST
Bio-Rad protein assay (BIO-RAD) was used for protein amount measurement.

(2) Apparatus The following apparatus was used for the measurement of lipid peroxide amount.
・ Homogenizer Kinematica CH-6010 (manufactured by POLYTRON)
・ Small high-speed cooling centrifuge MX-301 (manufactured by Tommy)
・ Genius Dry Bath Incubator MD-01N (manufactured by Major Science)
・ V-520-SR type spectrophotometer (manufactured by JASCO Corporation)
・ Lacom tester-desktop PH meter PH510 type (manufactured by ASONE Co., Ltd.)
-The following apparatus was used for the measurement of SOD activity.
・ Homogenizer Kinematica CH-6010 (manufactured by POLYTRON)
・ Model 680 Microplater Reader (BIO-RAD)
The following instruments were used to measure the area of intramyocardial collagen fibers.
・ Model BX51TE microscope (OLYMPUS)
・ Win ROOF V3.0, Viewfinder Lite Version 1.0

(3) Experimental group Thirty 4-week-old male salt-sensitive Dahl rats
(A) Tap water administration group (hereinafter referred to as “control group”),
(B) 0.9% saline administration group (hereinafter referred to as “DS-Low group”),
(C) 1.3% saline administration group (hereinafter referred to as “DS-High group”) and (D) 10% miso aqueous solution (corresponding to 1.3% NaCl) administration group (hereinafter referred to as “MISO group”)
And were reared for 8 weeks under constant temperature and humidity (23 ± 2 ° C.) and automatic lighting (8:00 am to 8:00 pm).
Rats of any experimental group were allowed to freely feed the solid feed CE-7 (Japan Clare Co., Ltd.) for long-term breeding.
0.9% and 1.3% saline were prepared by dissolving sodium chloride (Wako Pure Chemical Industries, Ltd.) in water and allowed to freely ingest into the DS-Low group and DS-High group, respectively.
A 10% miso aqueous solution prepared by dissolving 10 g of miso in 100 mL of water at about 25 ° C. was pulverized with a homogenizer, autoclaved, and freely taken into the MISO group. As the miso, commercially available dry rice miso (boiled rice paste: 5 steps, salt content: 12.5%, raw materials: rice (domestic), salt (domestic), soybean (produced in North America)) was used.
Water in the control group and saline in the DS-Low group and DS-High group were changed once every two days, and the miso aqueous solution in the MISO group was changed daily.
After rearing for 8 weeks, hearts were removed from Dahl rats under pentobarbital anesthesia, immediately immersed in liquid nitrogen, and stored at −80 ° C. until measurement of lipid peroxide amount and SOD activity.

(4) Method for measuring amount of heart tissue LPO Lipid peroxide amount was measured in the same manner as in Example 1 using heart tissue extracted from rats of each experimental group.

(5) Method of measuring heart tissue SOD activity SOD activity was measured in the same manner as in Example 1 using heart tissue extracted from rats of each experimental group.

(6) Method for measuring the area of intramyocardial collagen fibers (Yoshinaga M et al., Japanese Traditional Miso Soup Attenuates Salt-induced Hypertension and its Organ Damage in Dahl Salt-sensitive Rats. Nutrition 2012; 28: 924-931.)
For the intramyocardial collagen fibers stained with Azan Mallory staining, the dark blue wavelength was analyzed, and the area of the same wavelength region was measured to determine the amount of intramyocardial collagen fibers (Model BX51TE microscope, Win ROOF V3.0, Viewfinder Lite Version 1.0). The area of 10 measurement areas was measured from the left ventricle to the ventricular septum, and the total area of collagen fibers at the site was measured and expressed as a ratio per examination area.

(6) Statistical analysis The significance test was performed using STATISTICA to perform statistical significance tests with ANOVA and Post-hoc (LSD).

2. Result (1) Measurement result of heart tissue LPO amount (FIG. 5)
As shown in FIG. 5, the amount of LPO produced in the heart tissue homogenate was significantly higher in the DS-High group than in the control group (0.158 ± 0.100 vs 0.542 ± 0.086 ng / mg protein / 30 min, p <0.05), significantly lower in the MISO group compared to the DS-High group (0.542 ± 0.086 vs 0.281 ± 0.119 ng / mg) Protein / 30 min, p <0.05).

(2) Measurement result of heart tissue SOD activity (FIG. 6)
As shown in FIG. 6, the SOD activity in heart tissue homogenate was significantly lower in the DS-High group compared to the control group (10.572 ± 7.422 vs. 6.045 ± 1.074 U / mg protein, p <0.05), significantly higher in the MISO group than in the DS-High group (6.045 ± 1.074 vs 18.321 ± 12.651 U / mg protein, p < 0.05).

(3) Measurement results of area of intramyocardial collagen fibers (Fig. 5)
Optically measured intramyocardial collagen fiber amount was significantly higher in the DS-High group than in the control group (0.033 ± 0.021 vs 0.153 ± 0.043 mm ^{2 /} 1.234 mm). ² , p <0.05), the increase in collagen fiber amount in the DS-High group disappeared completely by ingestion of miso (MISO group) (0.074 ± 0.061 mm ^{2 /} 1.234 mm ² , p <0 .05).

3. Discussion In the miso intake group, the activity of the antioxidant enzyme SOD in the heart tissue of rats was maintained, and the amount of lipid peroxide produced was also low. From this result, it was shown that, like brain oxidative stress, miso suppresses myocardial oxidative stress. The decrease in oxidative stress is thought to suppress the myocardial injury. Actually, the amount of intrafibrillar collagen fibers measured optically was significantly lower than that in the saline ingestion group due to ingestion of miso.
When the relationship between lipid peroxide produced from cardiac tissue homogenate and intramyocardial collagen fiber amount was examined, a significant positive correlation was observed between the two (r = 0.676, p <0.05). (FIG. 7). It shows that the smaller the active oxygen, the smaller the amount of collagen fibers. It was strongly suggested that miso suppresses the active oxygen produced from the heart tissue and reduces the amount of collagen fibers in the myocardium.
Miso intake increases the antioxidant enzyme SOD activity in heart tissue, lowers active oxygen, and suppresses oxidative stress. Reduction of oxidative stress may reduce the amount of collagen fibers in the heart and protect myocardial function by suppressing myocardial fibrosis.

Claims (4)

  A superoxide dismutase (SOD) activity enhancer containing a miso extract as an active ingredient.   The SOD activity enhancer according to claim 1, wherein the miso extract is a fraction having a molecular weight of less than 3 kDa obtained by filtering the miso extract with a filter membrane having a fractional molecular weight of less than 3 kDa.   The SOD activity enhancer according to claim 1 or 2, which enhances brain tissue SOD activity or heart tissue SOD activity.   The SOD activity enhancer according to claim 3 for suppressing brain tissue oxidative stress or heart tissue oxidative stress.

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