JP2009007313A - Amyotrophy inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an amyotrophy inhibitor having a further effective and also safe amyotrophy inhibitory effect which can be used for a long period of time as a beverage and food. <P>SOLUTION: This amyotrophy inhibitor is characterized by being safe even on taking for a long period of time, having both of the multiplication effect and differentiation effect of myoblasts and also amyotrophy-preventing effect, and containing α-glycosylated hesperidin exhibiting an excellent amyotrophy inhibitory effect based on these effects as an active ingredient. The amyotrophy inhibitor is preferably used as a peroral agent or a skin agent for external use. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a muscle atrophy inhibitor that effectively suppresses muscle atrophy.

  When a long-term bed rest or rest is forced due to an injury or morbidity such as a fracture, muscle atrophy occurs, and it takes time to return to daily life, and in some cases, there is a risk of disturbing daily life. Even when healthy, due to lack of daily exercise and aging, muscle atrophy may occur, as well as trouble with daily life. Furthermore, in a special case, it is known that people who are placed in a weightless environment such as space flight also cause muscle atrophy and take time to adapt to daily life.

  When muscle atrophy occurs, exercise becomes difficult and so-called quality of life decreases. When exercise becomes difficult, the sick can make the condition worse, and the elderly can shorten their lives. When muscle atrophy occurs, it is important to recover a certain amount of exercise continuously while taking proper nutrition. In order to recover muscles by such exercise therapy, the intake of nutrients and the amount of exercise are appropriately set, and the person needs the perseverance to continue. Therefore, healthy people who have been temporarily inadequate due to injury, etc. can recover muscles by exercise therapy as long as they have patience. I have to say that it is difficult.

  Therefore, the development of drugs and supplements to prevent the sick and elderly, as well as healthy people who have suffered from lack of exercise for some reason, from causing muscle atrophy that causes a decline in the quality of life has been studied. Conventionally, several proposals have been made.

  For example, Patent Literature 1 proposes a muscle atrophy-suppressing composition containing fruit polyphenol as an active ingredient.

  Further, Patent Document 2 proposes a creatine oral replacement therapy agent containing creatine as an active ingredient for the purpose of treating or preventing a muscle nonuse syndrome.

JP 2001-089387 A Japanese translation of PCT publication No. 2002-530330

  The composition for inhibiting muscle atrophy disclosed in Patent Document 1 was developed based on information suggesting that oxidative stress is involved in disuse muscle atrophy induced by lack of exercise and the like, and has an antioxidant effect. It is a muscular atrophy inhibitor that attempts to suppress muscular atrophy by reducing the oxidative stress presumed to be the cause by using a fruit polyphenol having an active ingredient. However, although the cause of muscle atrophy is considered to be almost apparently due to lack of exercise clinically, it has not yet been clarified physiologically or pathologically. As described in Patent Document 1, oxidative stress may induce muscle atrophy, or may be based on other causes. Ingestion of such specific polyphenols can suppress all muscle atrophy. It is not possible, and there are limits to effectiveness.

  The creatine oral replacement therapy disclosed in Patent Document 2 is a medical agent for treating or preventing muscle disuse syndrome including muscle atrophy by supplementing creatine as an important material for muscle energy production. . This medical drug was developed based on the suggestion that the decline in exercise capacity associated with aging is due to a decrease in the body's creatine level. To promote muscle regeneration and maintenance. However, the cause of muscle atrophy has not yet been fully clarified, so it cannot be said that creatine supplementation can cope with all muscle atrophy, and its effect seems to be limited, except for creatine supplementation. There may be other approaches that are more effective.

  This invention is made | formed in view of the said conventional problem, Comprising: The subject is that a muscle atrophy inhibitory action is more effective and safe, and provides the muscle atrophy inhibitor which can be used for a long term. is there.

  In order to solve the above-mentioned problems, the inventors of the present application have made extensive studies, experiments, and studies. As a result, they have obtained the following knowledge.

That is, ingredients that are safe for long-term ingestion and have a higher muscle atrophy inhibitory effect than before were screened widely for food groups and plant extract groups.
As a result, it was confirmed that α-glycosylated hesperidin has a high inhibitory effect on muscle atrophy.

  In the present invention, muscular atrophy includes strict muscle atrophy which means a decrease in muscle mass, and further decreases the maximum strength of the muscle, decreases the maximum ability, increases the muscle relaxation time, premature muscle fatigue, It is used in a broad sense, including a decrease in muscle energy retention, a decrease in sugar intake, an increase in lipid content, a decrease in muscle blood flow, and a decrease in muscle ability such as impaired motor control. And in this invention, suppression of a muscular atrophy means reducing or preventing the reduction | restoration symptom which arises in these muscles.

  As described above, the present inventors have found that α-glycosylated hesperidin has a muscle atrophy inhibitory effect as a result of various experimental studies. As is well known, α-glycosylated hesperidin Is known to have various effects, and there is a concern that α-glycosylated hesperidin may be disclosed or suggested to have an effect of suppressing muscle atrophy prior to the inventors' knowledge. Therefore, we conducted an intensive investigation on this point. As a result, it was confirmed that such knowledge was not disclosed nor suggested.

  For example, Japanese Patent Application Laid-Open No. 10-218777 discloses a sag improving agent and a slimming agent containing α-glycosylated hesperidin as an active ingredient. The effect of α-glycosylated hesperidin disclosed herein is as follows. There is no description or suggestion about the effect of suppressing muscle atrophy, which is an effect of preventing swelling of the lower body that occurs naturally due to standing or sitting for a long time.

  Japanese Patent Application Laid-Open No. 2002-234844 discloses a bone density improving agent containing α-glycosylated hesperidin as an active ingredient and use thereof. The effect of α-glycosylated hesperidin disclosed herein is An object of the present invention is to provide a bone density improver that is effective in preventing osteoporosis, and thereby, a food and drink, and a medicine that contains the bone density improver, and that suppresses muscle atrophy. Not only is there no mention of the effect, it is not even suggested.

  As a result of repeated screening as to whether or not to have a muscle atrophy inhibitory effect on many food groups and plant extract groups, and how much effect can be expected, the present inventors have mentioned above. As described above, the present inventors have found that α-glucosylated hesperidin has a particularly remarkable muscular atrophy inhibitory effect. As a result of further investigation of the action mechanism of the muscle atrophy by this α-glucosylated hesperidin, as shown in the examples described later, the proliferation action and differentiation of myoblasts resulting in the enhancement of muscle cells. It has been found that there is a possibility that a muscle atrophy-suppressing action may be brought about based on both actions.

  The muscle atrophy inhibitor according to the present invention has been made based on the above-mentioned findings, and is characterized by containing α-glycosylated hesperidin as an active ingredient.

  The muscle atrophy inhibitor according to the present invention has α-glucosylated hesperidin as an active ingredient, which has both an action of proliferation and differentiation of myoblasts and exhibits an excellent effect of inhibiting muscle atrophy based on such action. is doing. Therefore, by appropriately taking the muscle atrophy inhibitor according to the present invention, it effectively suppresses muscle atrophy that occurs in the case of lack of exercise for a long time due to morbidity or injury, and the quality of life is improved by muscle atrophy. It becomes possible to reduce or prevent damage.

  As described above, the muscle atrophy inhibitor according to the present invention is characterized by containing α-glucosylated hesperidin as an active ingredient.

  In the present invention, the effect of inhibiting muscle atrophy includes a decrease in muscle mass, a decrease in maximum muscle strength, a decrease in maximum ability, an increase in muscle relaxation time, premature muscle fatigue, a decrease in glucose uptake, an increase in lipid content, and muscle energy. It is an action that reduces or prevents the symptoms of decline that occur in muscles such as a decrease in holding power, a decrease in muscle blood flow, and a movement control disorder. This muscle atrophy action is effectively brought about by α-glucosylated hesperidin which is an active ingredient. This α-glucosylated hesperidin inhibitory effect on muscle atrophy is obtained when α-glucosylated hesperidin has both an effect of promoting myoblast proliferation and an effect of promoting myoblast differentiation. I think that the.

  The action of promoting the proliferation of myoblasts means that the cell proliferation in the case where a sample aqueous solution of a muscle atrophy inhibitor is added to cultured myoblasts is superior to that without addition. The degree of proliferation of this myoblast can be evaluated by measuring how much proliferation has been promoted compared to the case of no addition.

  On the other hand, the action of promoting the differentiation of myoblasts means that the cell proliferation in the case of adding a sample aqueous solution of a muscle atrophy inhibitor to cultured myoblasts is differentiating compared to those without addition. means. The degree of differentiation of this myoblast can be evaluated by measuring the creatine kinase activity of the cell.

  Next, α-glucosylated hesperidin, which is an active ingredient of the muscle atrophy inhibitor of the present invention, will be described.

  Hesperidin is a rutinose glycoside of hesperetin and is also called vitamin P. Hesperidin is contained in fruit skins such as citrus fruits, fruit juices, and seeds, and has been known as a substance with various physiological effects. However, its solubility in water is extremely low, and it is also used in solvents such as alcohol. Since it hardly dissolves, its use has been very limited.

  The α-glycosylated hesperidin used as an essential active ingredient of the muscle atrophy inhibitor of the present invention is obtained by binding a sugar to the rutinose part of hesperidin with an α-glycosyl bond, and its adjustment method is not particularly limited, and any known method May be used. A typical method is a method obtained by allowing a glycosyltransferase such as glucosidase to act in the presence of an α-glycosylation compound as described in JP-A-3-7593. By this method, α-glycosylated hesperidin in which the sugar moiety of hesperidin is α-glycosylated is obtained.

  The α-glycosylated hesperidin is represented by the following general formula (1).

（式（１）中、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４、Ｒ^５及びＲ^６のうち少なくとも１個は単糖又はオリゴ糖残基を示し、残余は水素原子を示す）

(In formula (1), at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ represents a monosaccharide or oligosaccharide residue, and the remainder represents a hydrogen atom)

In the general formula (1), the monosaccharide or oligosaccharide residue represented by R ^{1 to} R ⁶ is preferably a monosaccharide residue, and particularly preferably a glucose residue.

  As the α-glycosylated hesperidin used in the muscle atrophy inhibitor of the present invention, the above-mentioned sugar conversion reaction mixture can be used as it is, but if necessary, one purified by a known method may be used. Furthermore, you may use the existing commercial item. Among the existing commercial products, α-glucosylated hesperidins manufactured by Toyo Seika Co., Ltd. (trade name “αG Hesperidin PS”) and Esaki Glico Co., Ltd. (trade name “αG Hesperidin”) are particularly preferable.

  The recommended intake for obtaining a muscle atrophy effect using the muscle atrophy inhibitor of the present invention is preferably 0.1 mg / day to 100 g / day, more preferably 0 in terms of 100% α-glucosylated hesperidin. .1 g / day to 50 g / day. In such a range, the optimum intake amount, intake method, number of intakes, and intake period may be appropriately determined depending on the age, sex, and health status of the user. Even if the intake amount is increased from 100 g / day, the enhancement of the effect becomes a plateau state, which is uneconomical. If it is less than 0.1 mg / day, the effect of suppressing muscle atrophy cannot be expected.

  The preparation of the muscle atrophy inhibitor of the present invention can be performed by a known preparation technique, and appropriate additives can be added to the preparation. The muscle atrophy inhibitor of the present invention is desirably administered orally.

  Examples of the muscle atrophy inhibitor according to the present invention will be described below. In addition, the Example shown below is only a suitable illustration for demonstrating this invention, and does not limit this invention at all.

Example 1
In this Example 1, myoblast proliferation as described below with respect to α-glycosylated hesperidin, which is an essential active ingredient constituting the muscle atrophy inhibitor according to the present invention, and IGF-1 (comparative control). The promotion effect was evaluated. In addition, IGF-1 used for comparison is insulin-like growth factor-1 and is a pharmaceutical product exhibiting a myoblast proliferation action.

  As a target myoblast, a mouse-derived myoblast (trade name “C2C12”) manufactured by Dainippon Sumitomo Pharma Co., Ltd. was used. In addition, as a sample of α-glycosylated hesperidin, which is an essential active ingredient of the muscle atrophy inhibitor of the present invention, “αG Hesperidin PS (trade name)” (α-glycosylated hesperidin content: about 83%) manufactured by Toyo Seiki Co., Ltd. Prepared. The medium used was DMEM (Dulbecco's Modified Eagle's Medium) manufactured by Invitrogen.

5 × 10 ⁴ myoblasts were suspended in 400 μL medium (DMEM containing 10% FBS, 100 units / mL penicillin, 100 μg / mL streptomycin), seeded in a 25-well plate, and then incubated at 37 ° C., 5 ° C. It was fixed by culturing for 3 hours under% CO ₂ .

  Thereafter, a medium for evaluation [DMEM containing 0.5% FBS (fetal bovine serum), 100 units / mL penicillin, 100 μg / mL streptomycin, each component sample (the α-glycosylated hesperidin and IGF-1)] 400 μL Was replaced. The evaluation concentration of each component sample was 0.1, 1, 10, and 100 ppm as final concentrations. For each component sample, a 1% aqueous solution (hydrophobic sample was an ethanol solution) was prepared and added as appropriate to a final concentration.

After culturing the aforementioned medium for 2 days, the medium was removed, 200 μL of DMEM supplemented with 10% Alamar Blue, 0.5% FBS, 100 units / mL penicillin, 100 μg / mL streptomycin was added, and after 3 hours of incubation, fluorescence was added. The fluorescence value (Ex544, Em590) was measured with a plate reader (fluorescence intensity was proportional to the number of cells).
The relative degree of proliferation was calculated by the following formula (1).
(Relative growth rate) = (Fluorescence intensity when sample is added) ÷ (Fluorescence intensity when sample is not added)
(1)

  The fluorescence intensity when each sample was added was measured with respect to the fluorescence intensity 1.00 with no sample added. The fluorescence intensity of the comparison control added with IGF-1 was 1.48, whereas the fluorescence intensity of the addition of α-glycosylated hesperidin as an active ingredient of the muscle atrophy inhibitor of the present invention was 1.41. Met. Thereby, it was confirmed that the myoblast proliferation action of α-glycosylated hesperidin, which is an active ingredient of the muscle atrophy inhibitor of the present invention, is almost the same as that of IGF-1, which is a highly effective drug. .

(Example 2)
In Example 2, the myoblast differentiation promoting effect was evaluated as follows with respect to α-glycosylated hesperidin, which is an essential active ingredient constituting the muscle atrophy inhibitor according to the present invention.

  As a target myoblast, a mouse-derived myoblast (trade name “C2C12”) manufactured by Dainippon Sumitomo Pharma Co., Ltd. was used. In addition, as a sample of α-glycosylated hesperidin, which is an active ingredient of the muscle atrophy inhibitor of the present invention, αG hesperidin PS (trade name, manufactured by Toyo Seiki Co., Ltd .: α-glycosylated hesperidin content: about 83%) was prepared. As a comparative sample, creatine (manufactured by Degussa Bioactives, trade name “creatine citrate”) was prepared. The medium used was DMEM (Dulbecco's Modified Eagle's Medium) manufactured by Invitrogen.

  The myoblasts (C2C12) were seeded on a 12-well cell culture plate, and cultured to 90% confluence in 1 mL of culture medium DMEM + 10% FBS + 1% penicillin-streptomycin. The myoblasts were washed twice with 1 mL of DMEM + 1% penicillin-streptomycin. Thereafter, 10 μL of an evaluation component sample having concentrations of 100 ppm, 1000 ppm, and 10000 ppm of the above two types of samples were added to 1 mL of DMEM + 1% penicillin-streptomycin containing the myoblasts to obtain final concentrations of 1 ppm, 10 ppm, and 100 ppm. A culture solution for evaluation was used.

  The culture medium for evaluation was replaced with the same culture medium after 3 days, and cultured for 5 days to induce differentiation. Each evaluation component sample was dissolved in distilled water or ethanol at a concentration of 1%, sterilized by filter, diluted with DMEM + 1% penicillin-streptomycin 1-fold, 10-fold, 100-fold, and 10000 ppm, 1000 ppm, and 100 ppm solutions, respectively. did.

  In order to quantify the degree of differentiation of myoblasts, creatine kinase activity used as a differentiation marker in the literature (Z-Q cheng, Jornal of Endcrinology, 167, 175-182 (2000)) was measured. The creatine kinase activity is measured in 100 μL of a cell lysate (trade name “Cellytic M”: manufactured by Sigma) supplemented with 1% of a protease inhibitor (trade name “protease inhibitor cocktail for bacterial cells”: manufactured by Sigma). The sample was diluted 100 times with PBS (phosphate buffered saline) and the creatine kinase measurement kit (trade name “CPKII Test Wako” manufactured by Wako Pure Chemical Industries, Ltd.) was used as a sample. Measured according to As an index indicating the effect of the evaluation component sample, the differentiation promotion rate (= creatine kinase activity when the component sample was added / creatine kinase activity when no component sample was added) was used.

  The differentiation promotion rate measured as described above is 1.21 times in the sample of α-glycosylated hesperidin, which is the active ingredient of the muscle atrophy inhibitor of the present invention, and 1.1 in the creatine sample of the control comparison. It was 25 times. Thus, it was confirmed that α-glycosylated hesperidin has the same myoblast differentiation promoting effect as creatine used as a medical drug for muscle nonuse syndrome.

(Example 3)
In Example 3, the muscle atrophy prevention effect of the muscle atrophy inhibitor according to the present invention was evaluated.
Rats (Wistar strain, rabbit, 8 weeks old, 10 animals in each group) are fed standard food “AIN-93G” (trade name, manufactured by Japan SLC Co., Ltd.) with the sample rate shown below (Table 1) at a rate of 1%. The added food was ingested at 15 g / day for 3 weeks.

  As an evaluation sample, “αG Hesperidin PS (trade name)” (sample 1) manufactured by Toyo Seiki Co., Ltd. was used as α-glycosylated hesperidin, which is an active ingredient of the muscle atrophy inhibitor of the present invention, and hesperidin (sample 2). ) Using hesperidin manufactured by Wako Pure Chemical Industries, Ltd., glucose manufactured by Wako Pure Chemical Industries, Ltd. as glucose (sample 3), and “creatine cytos” manufactured by Degussa Bioactives as creatine (sample 4). "Rate (trade name)" was used, and "Gravinol Super (trade name)" manufactured by Kikko-Man Co., Ltd. was used as the fruit polyphenol (sample 5). On the other hand, the control group was fed with a standard bait mixed with 1% corn starch (manufactured by Japan SLC).

  From the first week of the test to the final day of the test (the 14th day from the start of the test), the right hind leg of the rat was casted to induce muscle atrophy. On the last day of the test, the right hind limb was dissected and the wet weight of the soleus was measured. The percentage of muscle atrophy was determined by the mean relative value of the right hind leg soleus wet weight when the left hind leg soleus wet weight of the control group was taken as 100. The measurement results are listed below (Table 1).

  As is clear from the above (Table 2), it was confirmed that α-glycosylated hesperidin has a high effect of preventing muscle atrophy in each evaluation sample.

  The use form of the muscle atrophy inhibitor of the present invention is preferably used as an oral preparation or an external preparation for skin. Below, the combination example (2 examples) which used the muscular atrophy inhibitor of this invention as an oral preparation, and the combination example (2 examples) used as a skin external preparation are illustrated.

(Formulation example 1: oral solution (oral))
“ΑG Hesperidin PS” manufactured by Toyo Seika Co., Ltd .: 1000 mg, “αG Hesperidin” manufactured by Ezaki Glico Co., Ltd .: 1000 mg, vitamin B ₁ nitrate: 10 mg, ascorbic acid: 500 mg, maltitol: 15000 mg, vitamin B ₆ : 10 mg, Inositol: 50 mg, anhydrous caffeine: 20 mg, malic acid: 150 mg, citric acid: 700 mg, sodium citrate: appropriate amount, glycerin: 60 mg, sodium benzoate: 70 mg, and fragrance: trace amounts of internal liquid components, which are purified water And dissolved to obtain 100 ml of an internal solution (pH = 4.5).

(Formulation Example 2: Tablet (oral))
“ΑG Hesperidin PS” manufactured by Toyo Seika Co., Ltd .: 20 g, gelatin: 130 g, glycerin: 70 g, water: 100 g, and ethyl paraoxybenzoate: 0.5 g were heated and stirred to obtain a uniform gelatin dispersion (L1 )
On the other hand, 100 g of enzyme-decomposed lecithin and 1.0 g of dipotassium glycyrrhizinate were dispersed in wheat germ oil: 200 g using a homomixer to obtain a uniform solution-like wheat germ oil liquid (L2).
The gelatin dispersion (L1) was extruded into a 12 mm diameter aluminum capsule mold to obtain gelatin capsules.
Next, the wheat germ oil liquid (L2) was extruded through a nozzle and poured into the gelatin capsule, filled, cooled and dried. Thereby, a solid preparation (tablet oral preparation: gel composition) having a gelatinous outer layer of gelatin filled with liquid wheat germ oil liquid (L2) was obtained.

The composition and composition amount per one above-mentioned solid preparation are shown below.
Gelatin: 130 mg (outer layer)
Glycerin: 70mg (outer layer)
Ethyl paraoxybenzoate: 0.5 mg (outer layer)
αG Hesperidin PS: 20 mg (muscle atrophy inhibitor, outer layer)
Enzymatic degradation lecithin: 50 mg: (flavoring, flavoring ingredients, inner layer)
Dipotassium glycyrrhizinate: 0.5 mg (flavoring, flavoring ingredients, inner layer)
Wheat germ oil 100mg: (inner layer)
Or more, total: 371 mg

(Formulation example 3: patch (external preparation for skin))
The oil phase component and the water phase component shown below were prepared.
<Oil phase component>
Squalane: 10.0 g, liquid paraffin: 3.0 g, beeswax: 2.0 g, cetostearyl alcohol: 4.0 g, glyceryl monostearate: 2.0 g.
<Water phase component>
“ΑG Hesperidin PS” manufactured by Toyo Seika Co., Ltd .: 5.0 g, POE (20) sorbitan monolaurate: 2.0 g, glycolic acid: 0.2 g, salicylic acid: 0.1 g, pharmacopoeia sulfur: 5. 0 g, diglycerin: 5.0 g, resorcinol-labonite complex: 1.0 g, sodium hydroxide: appropriate amount, purified water: (so that the sum of the oil phase and the aqueous phase is 100 g).

  The oil phase component and the aqueous phase component were separately heated at 70 ° C., then mixed and emulsified. While cooling this, fragrance | flavor (a trace amount) was added on the way, and it further cooled to room temperature, and obtained 100-g patch liquid (external skin solvent).

(Formulation 4: Patch sheet (external preparation for skin))
Polyethylene glycol 400 (mass average molecular weight 400): 10.00 g, “αG Hesperidin PS” manufactured by Toyo Seika Co., Ltd .: 3.75 g, L-menthol: 2.00 g are mixed and dissolved to obtain a mixed solution (L3): 15. 75 g was obtained.
Next, rubber-based pressure-sensitive adhesive SIS (trade name “Clayton D-1107”, manufactured by Kraton Polymer Japan Co., Ltd.) heated to 150 ° C .: 73.7 g and alicyclic hydrocarbon (trade name “Arcon P-100”, Arakawa Chemical Co., Ltd.): 110.55 g was added and mixed to obtain an adhesive component (M): 184.25 g.
Next, the mixed solution (L3): 15.75 g and the adhesive component (M): 184.25 g were added and mixed to obtain a plaster composition: 200 g.
Next, the plaster composition was coated on polyethylene terephthalate (PET, liner) by a hot melt method so that the coating amount (paste mass) was 100 g / m ² after drying. A plaster was obtained.
Finally, the plaster surface of the plaster is coated with a polyurethane film / polyester knit laminate support (knit weight: 30 g / m ² , thickness: 180 μm, moisture permeability: 2200 g, transparency: 49), and a patch A sheet was obtained.

  As described above, the muscular atrophy inhibitor according to the present invention has both effects of proliferating and differentiating myoblasts, and exhibits an excellent muscular atrophy-suppressing effect based on such effects. Is contained as an active ingredient. Therefore, by appropriately taking the muscle atrophy inhibitor according to the present invention, it effectively suppresses muscle atrophy that occurs in the case of lack of exercise for a long time due to morbidity or injury, and the quality of life is improved by muscle atrophy. It becomes possible to reduce or prevent damage.

Claims (2)

  A muscle atrophy inhibitor comprising α-glycosylated hesperidin as an active ingredient.   The muscular atrophy inhibitor according to claim 1, which is for oral or external use on the skin.