JP5275537B2 - Oral preparation for preventing muscle oxidative stress - Google Patents

Description

  The present invention relates to an oral preparation for reducing oxidative stress in muscles containing a superoxide dismutase complex, and in particular, an oral agent capable of suppressing the degradation of muscle tissue due to oxidative stress and preventing muscle atrophy disorders such as disused muscle atrophy. It relates to the preparation.

Muscles are deeply involved in life-sustaining activities such as gastrointestinal motility, breathing, and heart pulsation, as well as spontaneous movement, and dysfunction due to muscle atrophy is known to cause serious symptoms. ing. Muscle atrophy refers to a decrease in muscle volume, and skeletal muscle atrophy is caused by atrophy of muscle fibers or a decrease in the number of muscle fibers. The cause of muscle atrophy is said to be associated with an increase in intracellular active oxygen.
A state in which active oxygen is increased more than the antioxidant capacity of a living body is called an oxidative stress state. In vivo, active oxygen (superoxide anion radical, hydrogen peroxide, hydroxy radical, nitrogen, for various purposes, such as degrading toxic or immunological foreign substances derived from physiological activities or entering the body. Oxides, etc.) are produced, which helps maintain physiological functions. Under normal conditions, excess active oxygen in the body is removed by a control system containing antioxidant enzymes and does not become excessive. However, muscular atrophy causes such an oxidative stress state.

Among muscle atrophys, disuse muscle atrophy is a phenomenon that is caused by resting state such as bedridden, or activity restriction such as long-term bed rest and cast fixation due to illness, and is a common phenomenon. Muscle weakness associated with muscle atrophy limits physical activity in daily life and reduces Quality of life (QOL).
Conventionally, in order to prevent or improve disuse muscular atrophy, it has been practiced to prevent moderate exercise etc. at normal times, or to carry out sufficient rehabilitation from the early stage after surgery. There was no effective solution other than these. Therefore, when disuse muscle atrophy occurs due to a bedridden state or a long-term bed after surgery, there is a problem that recovery of the body becomes more difficult.
In particular, since the ability to synthesize muscle protein decreases with age, it is considered that recovery of muscle function from retraction will be prolonged in old age. Therefore, in the present age of aging society, prevention of disuse muscle atrophy in the elderly is one of the important issues for ensuring healthy daily life activities.

Kondo et al. (Non-Patent Document 1) have reported that the tiobarbituric acid reactive substance (TBARS) of the soleus increases by restraining the lower limbs with casts. Lawler et al. (Non-Patent Document 2) also reported that oxidation of lipid peroxide and dichlorohydro-fluorescein diacetate (DCFH-DA) increases in the soleus due to tail suspension.
Although there are many unclear points about the mechanism of action of increased muscle atrophy due to an increase in active oxygen (oxidative stress state), one of them is thought to be an increase in intracellular calcium ions (Non-patent Document 3). When the cell membrane is damaged by lipid peroxidation and the calcium concentration in the cytoplasm increases (Non-patent Document 4), a calcium-dependent protease is activated and proteolysis is enhanced. As a result, it is considered that muscle tissue is decomposed to cause muscle atrophy.
Furthermore, proteins oxidized by free radicals are likely to be degraded in the living body (Non-patent Document 5), which is considered to further promote the degradation of muscle tissue and muscle atrophy.

As a means of suppressing such disuse muscle atrophy, administration of an antioxidant substance has been studied, and it has been reported that administration of vitamin E, an antioxidant vitamin, reduces the degree of disuse muscle atrophy ( Non-patent document 6, Non-patent document 7, Non-patent document 8).
Antioxidants such as vitamin E, vitamin C, and β-carotene, which are conventionally known, are absorbed into the body within a few hours after ingestion, like other nutrients, and quickly exert an effect to eliminate active oxygen. However, since they are oxidized by reacting with active oxygen, they are consumed stoichiometrically and do not have a sustained effect. Therefore, they need to be ingested frequently.

Superoxide dismutase (hereinafter sometimes abbreviated as “SOD”) is an antioxidant enzyme present in various living tissues, and is composed of proteins containing copper, zinc, manganese, etc., particularly in the liver, adrenal gland, Three types are known, Cu, Zn-SOD localized in the cytoplasm, Mn-SOD localized in mitochondria, and EC-SOD localized in body fluids, which are contained in the kidney and the like.
SOD decomposes a superoxide anion radical (hereinafter sometimes abbreviated as “O ₂ ⁻ ”), which is a highly reactive active oxygen generated in a living body. In the active oxygen control system in the living body, when O ₂ ^- is generated, it is disproportionated by the action of SOD to generate hydrogen peroxide, and the excess hydrogen peroxide is related to the removal of hydrogen peroxide such as catalase and glutathione peroxidase. It is decomposed by the enzyme. This series of schemes is shown below.

Action of SOD: 2O ₂ ⁻ + 2H ⁺ → H ₂ O ₂ + O ₂
Catalase action: 2H ₂ O ₂ → O ₂ + 2H ₂ O
Action of glutathione peroxidase: ROOH + 2GSH → ROH + 2H ₂ O + GSSG
It should be noted that hydrogen peroxide is more toxic according to the following scheme when the abundance of catalase and glutathione peroxidase is insufficient, and especially when the abundance of iron is very low due to cellular lesions. Converted to hydroxy radical (Fenton reaction).
Fenton reaction:
O ₂ ⁻ + Fe ³⁺ → Fe ²⁺ + O ₂
Fe ²⁺ + H ₂ O ₂ → Fe ³⁺ + OH ⁻ + OH ⁰
O ₂ ⁻ + H ₂ O ₂ → O ₂ + OH ⁻ + OH ⁰

Patent Document 1 describes a pharmaceutical composition for oral administration containing superoxide dismutase, which uses different inflammatory effects (especially rheumatism and fibrosis), viral effects (especially HIV infection), and It is said to help relieve toxic symptoms (central nervous system, ischemia, nonvascular gastrointestinal disorders, ocular disorders or suppression of undesirable effects of anticancer treatment) associated with the presence of large amounts of oxygen. However, there is no description that SOD effectively acts on muscle atrophy.
Patent Document 2 also discloses the use of SOD for the treatment of degenerative diseases selected from the group consisting of neurodegenerative diseases, cirrhosis, lentivirus infection, parasitic infections and iatrogenic diseases (drug addiction). Have been described. However, this document only describes the experimental results using cells, and does not describe that SOD is effective for muscle atrophy and that it can be administered orally.

Japanese National Patent Publication No. 10-511944 Special Table 2002-500236 Kondo et al., Acta Physiosol Scand 142, p527-528 (1991) Lawler et al., Free Radic Biol Med 35, 9-16 (2003) Masayasu Inoue, "Oxidative stress in disuse muscle atrophy. Reactive oxygen and exercise-in search of supple health and longevity-" Kyoritsu Shuppan (Tokyo), p115-119 (1999) Mourelle et al., J. Appl Toxicol 10, p23-27 (1990) Davies et al., J. Biol Chem 262, p8220-8226 (1987) Appell et al., Int J. Sports Med 18, p157-160 (1997) Kondo et al., Am J. Physiol 262, E583-590 (1992) Kondo et al., FEBS Lett 326, p189-919 (1993)

The present inventors considered that the oxidative stress of muscle contributes to the degradation of muscle tissue and muscle atrophy, and have conducted extensive research to develop an oral preparation useful for the prevention thereof.
An object of the present invention is to reduce muscle oxidative stress, which can induce in vivo SOD activity by ingestion, and in particular to prevent muscle tissue degradation and muscle atrophy, which are thought to be due to muscle oxidative stress. It is to provide an oral preparation to be obtained.

The oxidative stress- preventing oral agent for muscular oxidative stress provided by the present invention contains a complex of melon-derived superoxide dismutase and prolamin as an active ingredient, and is a oxidative stress- preventing oral muscular stress- derived disease prescribed for oral consumption. It is an agent.
The muscle oxidative stress- derived disease preventive oral preparation according to the present invention reduces the oxidative stress state of muscle by ingestion, and various diseases, dysfunctions of the living body caused by the oxidative stress state of muscle, or pre-onset stages thereof It is thought to prevent.
In particular, the oral muscular oxidative stress- preventing oral agent according to the present invention exerts a wide range of preventive and therapeutic effects on muscle tissue degradation or muscle atrophy, including disuse muscle atrophy frequently encountered clinically. To do.

  Since the SOD complex, which is an active ingredient, has digestion resistance, it is absorbed from the intestinal tract without being decomposed, and at the same time is recognized as a foreign substance to the living body and stimulates the immune system. For this reason, the SOD complex induces endogenous SOD and other antioxidant enzyme activities such as catalase and glutathione peroxidase as part of the defense reaction of the living body, and gives an overall improvement effect on the active oxygen suppression system. It is done.

The oral preparation for reducing oxidative stress of muscle according to the present invention has a simple administration route because an excellent reduction effect on the oxidative stress state of muscle is observed by ingestion.
In particular, in order to prevent, ameliorate, and treat disuse muscle atrophy by drug therapy, it is necessary to continue drug administration over a long-term activity-limiting state that causes atrophy and the subsequent recovery period. The oral preparation for reducing muscular oxidative stress according to the present invention is suitable for long-term administration because of its superior effect of preventing muscle atrophy by oral administration, and is very useful as a disuse muscle atrophy inhibitor.

The oral preparation for reducing oxidative stress in muscle according to the present invention contains an SOD complex having digestion resistance in the digestive tract as an active ingredient, and induces SOD activity in vivo by ingestion.
As the SOD that forms the complex, a heterogeneous SOD of any origin may be used as long as it is not a human-derived SOD, but it is preferable to use an SOD extracted from a melon fruit having a high SOD content.
SOD may be used as it is, or a pharmaceutically acceptable salt may be used. In the present invention, a natural SOD is subjected to a method such as site-directed mutagenesis to convert a part of the amino acid in the sequence to another amino acid, or a part of the amino acid is chemically modified. However, as long as it does not lose SOD activity, it can be used as SOD.

As a substance that forms a complex when added to SOD, a substance that has resistance to digestive enzymes when taken orally and is easily recognized as a foreign substance in vivo should be selected. For example, various lipids and proteins Can be used.
As a particularly preferred complex, a complex of SOD and one or more additional substances containing at least prolamin can be mentioned. Prolamin is a natural (ie non-denatured) insoluble protein derived from plant origin, especially various cereals (wheat, rye, barley, oats, rice, millet, corn, etc.), especially prolamin obtained from wheat ( Gliadin) is preferred.
The SOD complex can be produced by the method described in JP-T-10-511944, and a commercially available melon SOD-gliadin complex (trade name “Oxycaine (registered trademark)”, manufacturer ISOCELL SA). Can be used.

The oral preparation for reducing muscle oxidative stress according to the present invention reduces muscle oxidative stress state by ingestion and prevents various diseases, functional disorders or pre-onset stages due to muscle oxidative stress state I think that.
That is, since the SOD complex which is an active ingredient has digestion resistance when ingested orally, it is absorbed from the intestinal tract without being decomposed, and at the same time, it is recognized as a foreign substance to the living body and stimulates the immune system. . The SOD complex recognized as a foreign substance is decomposed by active oxygen or the like produced by macrophages or the like, but the SOD complex itself decomposes active oxygen, so that the SOD complex is essentially hardly decomposed. Therefore, the SOD complex is recognized as a strong foreign substance in the living body, and induces endogenous SOD and other antioxidant enzyme activities such as catalase and glutathione peroxidase as part of the defense reaction of the living body. It is thought that the improvement effect is given overall.

When the SOD derivative is administered directly into the body by injection, the active oxygen is only eliminated by its own enzyme action. However, the oral agent for reducing oxidative stress of muscle according to the present invention has an SOD complex as an immune system. In terms of improving the overall active oxygen suppression system by stimulating, the mechanism of effect expression is different.
Moreover, since the effect of the oral preparation for reducing oxidative stress of muscle according to the present invention is due to stimulation of the immune system, once it is expressed, it is persistent. Moreover, since the oral preparation for reducing oxidative stress of muscle according to the present invention is taken orally, it has less invasiveness to the living body at the time of administration than the injection.

The oral preparation for reducing oxidative stress of muscle according to the present invention is a pharmaceutical or health food for the purpose of prevention, improvement, treatment of muscle tissue degradation or muscle atrophy, including disuse muscle atrophy frequently encountered clinically. Can be used as
In actual administration, the oral preparation for reducing oxidative stress of muscle according to the present invention is appropriately determined in terms of the dosage, administration schedule and other conditions according to the progress of muscle tissue degradation or muscle atrophy.
In particular, in order to prevent, ameliorate, and treat disuse muscle atrophy with drug therapy, it is necessary to continue drug administration over a long-term activity-restricted state that causes atrophy and the subsequent recovery period. Such an oral preparation for reducing oxidative stress of muscle is effective for oral administration and has a long-lasting effect because of its long-lasting effect, and has a very high utility value.

The dose for oral administration varies depending on whether it is used prophylactically or healthily or therapeutically, but usually 1 mg to 25 mg or 100 to 2,500 units as SOD amount per day per adult. What is necessary is just to divide and administer to about 4 times.
In the present invention, one unit of SOD means 1 minute in a concentration range where the absorbance at 550 nm of the system that develops color by O ₂ ⁻ generated by xanthine / xanthine oxidase increases linearly according to the method of McCord and Fridovich. Is the amount of SOD contained in the test system (3.0 ml) when the absorbance change of 0.025 is inhibited to 50% by eliminating O ₂ ⁻ (McCord, JM & Fridovich, I .: J. Biol. Chem., 244, 6049-6055 (1969)).

  Preparation of oral dosage forms such as dosage forms and formulations is also appropriately designed in consideration of the administration route and administration conditions. As the dosage form for oral administration, known ones such as tablets, hard or soft capsules, granules such as granules or fine particles, powders and the like can be selected. As a prescription for oral administration, a known excipient, binder, lubricant, colorant, and other components may be appropriately blended in accordance with the above dosage form with the SOD complex as an active ingredient.

(1) Experimental method (a) Grouping and rearing conditions of experimental animals Randomized 5 week old Wister rats, (a) Water-administered group (hereinafter abbreviated as “W group”), (b) Compound SOD Administration group of certain oxycaine (trade name) (hereinafter abbreviated as “O group”), (c) Vitamin E administration group (hereinafter abbreviated as “E group”), (d) Control group (hereinafter abbreviated as “C group”) Were divided into 4 groups, and 8 to 10 animals were assigned to each group.
Rats in each group were fed with experimental animal chow MF (Oriental Yeast Industry) and water ad libitum for 5 weeks, and the following administration was performed during this breeding period.

In group O, oxycaine dissolved in water was orally administered at a frequency of 6 times a week using a small animal sonde. The dose was adjusted in consideration of changes in body weight. As a result, the dose was once a day. It was 5 to 6.25 mg per kg.
In group E, α-tocopherol acetate (Wako Pure Chemical Industries) dissolved in soybean oil was orally administered at a frequency of 6 times a week using a small animal sonde, but the dose was adjusted in consideration of changes in body weight. As a result, the dose was 30 to 35 mg per kg body weight once a day.
In group W, water was orally administered once a day at a frequency of 6 days a week with a small animal sonde at a dose of 0.2 ml per kg body weight. Group C was raised only with free intake of feed and water.

(B) Method of skeletal muscle atrophy Both lower limbs of rats of W group, O group, and E group in order to atrophy the muscles of the lower limbs from the day of the fifth week after the start of breeding (29 days from the start of breeding) The so-called “calf” muscles, soleus, plantaris, gastrocnemius, and so-called “shin” muscles, extensor digitorum longus It developed into a pulled state and was fixed with a plaster cast. On the other hand, group C was not cast. The above administration schedule was continued during the restraint period.

(C) Collection of skeletal muscle Five weeks after the start of breeding (35 days after the start of breeding), skeletal muscle was collected from rats under Nembutal anesthesia. The collected skeletal muscles are loosened by cast restraint, that is, soleus, plantar and gastrocnemius.
The collected skeletal muscles were immediately frozen in liquid nitrogen after measuring the muscle wet weight and stored frozen at -80 ° C. until analysis.

(2) Measuring method (a) Measurement of SOD activity value It measured using the SOD measuring kit (SOD Assay Kit-WST; Dojindo Molecular Technology). To a skeletal muscle sample of about 20 mg, the dilution buffer attached to the kit was added in a 10-fold amount and homogenized with a hand homogenizer. Thereafter, the supernatant was recovered by centrifugation (6,500 rpm × 10 min), and the supernatant diluted 10-fold with a dilution buffer was used for measurement. The reaction was performed using a 96-well plate, and the measurement procedure was in accordance with the protocol of the kit. The change in absorbance at 450 nm was measured with a plate reader (Multistan; Labsystems), and the inhibition rate of the increase in absorbance due to sample addition was determined. .

  At the same time, the SOD at a known concentration is measured in the same procedure as the sample, a calibration curve is created based on the inhibition rate at this time, and the SOD activity value of the sample is obtained by substituting the inhibition rate of the sample into this calibration curve. It was. Moreover, the protein concentration of the diluted sample used for SOD measurement was measured using Coomassie Plus Protein Assay Reagent Kit (Pierce). Finally, the SOD value of the sample was expressed as an SOD activity value (U / mg protein) per 1 mg of muscle protein.

(B) Measurement of α-tocopherol The amount of α-tocopherol in the tissue sample was determined by the method of Ueda et al. , Tokyo (1995)), and using high performance chromatography, the following procedure was used.
A tissue sample of about 20 mg is cut into pieces of 1 mm square or less with scissors, put into a tube and weighed. Then, 0.02 ml of 1% NaCl solution is added to disperse the cut sample, and then 0.2 ml. 6% pyrogallol / ethanol solution and 0.2 ml of PMC (2,2,5,7,8-pentamethyl-6-hydroxychroman) internal standard solution (1 μg / ml) were added and mixed. Further, 0.04 ml of 60% KOH solution was added, capped and mixed, and then saponified for about 30 minutes with occasional shaking in a 70 ° C. water bath. After the tube was cooled in ice water, 0.9 ml of 1% NaCl solution and 0.6 ml of 10% ethyl acetate / n-hexane solution were added and mixed vigorously for 1 minute to extract α-tocopherol. The tube was cooled again in ice water for about 5 minutes, and then centrifuged (3,000 rpm × 5 min), and 0.4 ml of the upper layer (ethyl acetate / n-hexane layer) was separated into another tube.

Thereafter, 0.2 ml of 5% n-decane solution was added, and N ₂ was vented while heating with a heat block (40 ° C. or less) to leave a small amount of n-decane, and the solution was distilled off. -0.04 ml of n-hexane was added to decane and dissolved. 0.05 ml of this solution was injected into high performance liquid chromatography to detect α-tocopherol. The measurement conditions of the high performance liquid chromatography at this time were as follows. The column was Inertosil-5NH ₂ (diameter 4.6 mm × length 250 mm), the column temperature was 30 ° C., the mobile phase was n-hexane / isopropanol (volume ratio 98: 2), The flow rate was 1.5 ml / min, and the detector was a fluorescence detector (excitation wavelength 297 nm, fluorescence wavelength 327 nm).

(C) Measurement of oxidized protein (Carbonyl protein) concentration
According to the method of Levin et al. (Levin RL et al., “Carbonyl assays for determination of oxidatively modified proteins”, Methods Enzymol 233, p346-357, 1994), the measurement was carried out by the following procedure.
15-20 mg of each muscle tissue sample is minced as finely as possible and 0.3 ml of homogenizing buffer (50 mM phosphate buffer, pH 7.4, 0.1% digitonin, 40 μg / ml phenylmethylsulfonyl fluoride, 5 μg / ml leupeptin, 7 μg / ml pepstatin) , 5 μg / ml aprotonin, 1 mM EDTA) and incubated at room temperature for 15 minutes, and then centrifuged (6,000 rpm × 10 min) to remove insolubles.

  In order to examine the presence or absence of nucleic acid, the absorbance ratio (280 nm / 260 nm) of the supernatant was measured. If this absorbance ratio was 1.0 or less, 0.03 ml of 10% streptomycin was added to remove the nucleic acid. After 15 minutes of incubation at room temperature, centrifugation (6,000 rpm × 10 min) was performed. The above operation was omitted when the extinction ratio was 1.0 or more. Aliquot 0.1 ml of the centrifugation supernatant into two 1.5 ml sample tubes, and add 0.4 ml of 10 mM dinitrophenylhydrazine (DNPH, in 2.5M HCl) to one of them. The same amount of 2.5M HCl was added to one. The tube was protected from light and incubated at room temperature for 1 hour. During this time, the tube was shaken every 15 minutes. 0.5 ml of 20% trichloroacetic acid (TCA) was added to both tubes, cooled in ice for 10 minutes, and centrifuged (1,000 rpm × 5 min) to remove the supernatant.

0.4 ml of 10% TCA was newly added to both tubes, the protein precipitate was crushed with a glass rod, and the supernatant was removed by centrifugation (1,000 rpm × 5 min). To remove free DNPH and lipid contamination, add 0.4 ml of ethanol / ethyl acetate solution (volume ratio 1: 1) to the precipitate and centrifuge (1,000 rpm x 5 min) to remove the supernatant. This washing operation was repeated three times. After adding 0.2 ml of 6M guanidine hydrochloride dissolved in 20 mM potassium phosphate (pH 2.3) and incubating at 37 ° C. for 10 minutes, the mixture was centrifuged (12,000 rpm × 5 min) to remove insoluble matters. Absorbance (370 nm) was measured for both the DNPH addition reaction solution and the 2.5 M HCl addition reaction solution. The oxidized protein concentration was calculated by the following formula.
<Calculation formula>
C (DNPH / ml) = Absorption (Abs) / ε = Abs (370nm) /2.2×10 4/10 6 = Abs (370nm) × 45.45 nmol / ml [ε = 22,000 / M = 22,000 / 10 ⁽⁶ nmol / ml)

  In order to measure the amount of protein finally recovered, the absorbance (280 nm) after adding 2.5 M HCl was measured. At the same time, BSA standard solutions (0.25-2.0 mg / ml) with various concentrations of bovine serum albumin (BSA) dissolved in 6M guanidine hydrochloride were prepared, and the absorbance was measured to create a calibration curve. Concentration was calculated. The amount of oxidized protein in the tissue sample was calculated as the amount of oxidized protein (DNPH concentration) per protein collected as a precipitate (display unit is nmol / mg protein).

(D) Measurement of lipid peroxide Measurement was performed using a lipid peroxide measurement kit (BIOXYTECH LPO-586; OXIS International). Each muscle was homogenized with phosphate buffered saline (PBS) and then centrifuged (6,500 rpm × 10 min) to recover the supernatant, which was used as a sample. The measurement was performed according to the manual attached to the kit, and the absorbance at 586 nm was measured with Ultrospec Plus (Pharmacia International).

(E) Statistical processing The obtained data were expressed as mean ± standard error. Two-way ANOVA was used for comparison of each measurement item, statistical processing was performed by Fisher's PLSD post hoc test, and the case where the risk rate was less than 5% was considered significant.

(3) Experimental results Table 1 shows the mean value ± standard error of each test group's body weight at the time of dissection, muscle wet weight, SOD activity, α-tocopherol amount, oxidized protein amount, and lipid peroxide amount for each group. In addition, these measured values are shown in a graph.

(A) Weight at the time of dissection FIG. 1 is a graph showing the weight of the rat at the time of dissection. Body weight showed a significant decrease in each group due to a one-week cast restriction. Stress due to cast restraint is thought to cause weight loss.

(B) Muscle wet weight FIG. 2 is a graph showing the muscle wet weight of the soleus muscle. The soleus muscle wet weight of group O was the closest to the muscle wet weight of group C, which was not restrained, and the effect of muscle atrophy due to restraint was the lightest. Further, the soleus muscle wet weight of the O group is effective in preventing muscle atrophy with a significant difference (P <0.05) in comparison with the other restraining groups E group and W group. It was.
FIG. 3 is a graph showing the muscle atrophy suppression rate of the O group and the E group when the change amount of the muscle atrophy of the W group compared with the unconstrained C group is used as a reference (suppression rate 0%). That is, the muscle atrophy suppression rate is calculated by the following equation.
<Calculation formula>
Muscle atrophy suppression rate (%) = 100-muscle atrophy change rate (%)
here,
Muscle atrophy change rate (%) = (Group muscle wet weight−Group O or group E muscle wet weight) ÷ (Group C muscle wet weight−Group W muscle wet weight) × 100
In the O group and the E group, inhibition of muscular atrophy was observed as compared with the muscular atrophy in the W group, which was a negative control. In particular, the O group was found to have a greater inhibitory effect than the E group.

(C) SOD activity value FIGS. 4a and 4b are SOD activity values of the soleus and plantar muscles, respectively. In the soleus and plantar muscles, the O group showed significantly higher values than the other three groups. On the other hand, there was no significant difference between the three groups of Group C, Group E, and Group W.

(D) α-Tocopherol Level FIGS. 5a and 5b are α-tocopherol levels of soleus and plantar muscles, respectively. In both the soleus and plantar muscles, the E group showed significantly higher values than the other 3 groups. On the other hand, there was no significant difference between the three groups of the C group, the O group, and the W group.

(E) Amount of oxidized protein FIGS. 6a and 6b are amounts of oxidized protein of soleus and plantar muscles, respectively. In the soleus shown in FIG. 6a, the E group and the W group showed significantly higher values than the C group. However, there was no significant difference between group C and group O. Moreover, in the comparison between the 3 groups restrained by the cast, the O group showed a significantly lower value than the W group and the E group.
On the other hand, in the plantar muscle shown in FIG. 6b, the 3 groups (O group, E group, W group) restrained by the cast showed a significantly higher value than the C group. Moreover, in the comparison between the three groups restrained by the cast, the O group showed a significantly lower value than the E group and the W group.

(F) Lipid peroxide amount FIGS. 7a and 7b are lipid peroxide amounts of soleus and plantar muscles, respectively. In the soleus shown in FIG. 7a, the 3 groups (O group, E group, and W group) restrained by the cast showed significantly higher values than the C group. Further, in the comparison between the three groups restricted by the cast, the O group showed a significantly lower value than the W group.
On the other hand, in the plantar muscle shown in FIG. 7b, as in the soleus muscle, the 3 groups (O group, E group, and W group) restricted by the cast showed significantly higher values than the C group. Moreover, in the comparison between the 3 groups restrained by the cast, the O group showed a significantly lower value than the E group.

(G) Summary of results From the measurement results of muscle wet weight, disuse muscle atrophy appeared in group W, which was a negative control to which cast was restricted and water was orally administered, as compared to group C, which was a control that was not cast. The O group which is an example in which oxycaine (SOD complex) was orally administered after being cast, and the E group which is a comparative example in which vitamin E was orally administered after being restricted to the cast were suppressed in the decrease in muscle wet weight as compared with the W group. The effect of preventing atrophy was confirmed.
Compared with the E group, the O group had a large muscle atrophy suppression rate, and a greater atrophy prevention effect was observed. This tendency was observed in all comparisons of the soleus, plantar and gastrocnemius muscles, but especially in the comparison of the soleus muscle (see FIGS. 2 and 3), the effect was significantly different (P <0.05). Differences were noted.

  From the measurement results of the amount of oxidized protein and the amount of lipid peroxide, it was recognized that the state of oxidative stress was further reduced in group O, which has a high effect of preventing muscle atrophy. That is, the amount of oxidized protein in group O which is an example in which oxycaine (SOD complex) was orally administered was significantly lower than that in group E as a comparative example and group W as a negative control (FIG. 6a and FIG. 6b). In particular, in the comparison of soleus muscle (see FIG. 6a), the amount of oxidized protein in group O was not significantly different from that in group C, which was a control that was not cast-restrained. The amount of lipid peroxide in the O group is significantly lower in the soleus muscle comparison (see FIG. 7a) than in the negative control group W, and the plantar muscle comparison (see FIG. 7b). The value was significantly lower than that of the E group as a comparative example.

From the results of the SOD activity value and the amount of α-tocopherol (FIGS. 4a, 4b, 5a, and 5b), the O group as an example has an effect of significantly increasing the SOD activity value by oral administration of the SOD complex. Admitted. On the other hand, in the E group as a comparative example, the amount of α-tocopherol was significantly increased by oral administration of vitamin E, but the SOD activity value was not so high.
In addition, the doses of the O group and the E group used in this experiment were 5 mg to 6.25 mg per kg of body weight once a day in terms of the SOD conversion in the O group. The dose of vitamin E in the group was 30 mg to 35 mg per kg of body weight once a day, and SOD was effective in preventing muscle atrophy with an amount smaller than vitamin E.
From these results, it was recognized that in the O group which is an example, SOD activity in the body was directly increased by oral administration of the SOD complex, and an excellent inhibitory effect against oxidative stress and disuse muscle atrophy was exhibited. . The inhibitory effect was significantly superior to that of the comparative group E (oral administration of vitamin E, which is an SOD-like substance).

It is a graph which shows the body weight at the time of dissection of a test rat. It is a graph which shows the soleus muscle wet weight of a test rat. It is a graph which shows the muscular-atrophy inhibitory rate with respect to the atrophy of W group about the soleus muscle wet weight of a test rat. It is a graph which shows the SOD activity value of the soleus muscle and plantar muscle of a test rat. It is a graph which shows the alpha-tocopherol amount of the soleus muscle and plantar muscle of a test rat. It is a graph which shows the amount of oxidized proteins of the soleus and plantar muscles of a test rat. It is a graph which shows the amount of lipid peroxide of the soleus muscle and plantar muscle of a test rat.

Claims (4)

An oral muscular oxidative stress- preventing disease-preventing agent containing a complex of melon-derived superoxide dismutase and prolamin as an active ingredient and prescribed for oral consumption. The oral preparation for preventing oxidative stress- derived diseases of muscle according to claim 1, which is a muscle degradation inhibitor. Oxidative stress from disease prevention oral muscles according to claim 1 or 2, an oral muscle atrophy inhibitor. The oral agent for preventing oxidative stress- derived diseases of muscle according to any one of claims 1 to 3, which is an oral disuse muscle atrophy inhibitor.

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