JP7464951B2 - Disuse muscle atrophy inhibitor and food composition for inhibiting disuse muscle atrophy - Google Patents

Description

The present invention relates to a disuse muscle atrophy inhibitor and a food composition for inhibiting disuse muscle atrophy.

By applying mechanical stress through muscle training, etc., muscle mass increases (muscle hypertrophy), but conversely, muscle mass decreases (muscle atrophy) when muscles are immobilized due to being bedridden or immobilized in a cast, or when mechanical stress continues to decrease in a weightless environment such as outer space. As muscle atrophy progresses, it can cause locomotive syndrome (abbreviated as locomo, Japanese name: musculoskeletal syndrome), which reduces motor functions such as walking, and increases the risk of becoming "needing nursing care." In Japan, which has become a super-aging society, a survey of people aged 40 and over estimated that there are approximately 47 million people with locomo, including those at risk. In addition, according to the Ministry of Health, Labor and Welfare's Basic Survey on National Living Conditions in 2013, the number one cause of needing nursing care or support was musculoskeletal disorders, i.e., locomotor syndrome. Therefore, it is believed that suppressing muscle atrophy due to aging or being bedridden will lead to the prevention of locomo and an improvement in quality of life (QOL).

For example, Patent Document 1 discloses an agent for inhibiting disuse muscle atrophy that is used to obtain a food or beverage composition for inhibiting disuse muscle atrophy by blending it with a food or beverage, and that contains at least one of Macaranga tanarius extract and 8-prenylnaringenin as active ingredients, and that the Macaranga tanarius extract contains nymphaeol-A, nymphaeol-B, isonymphaeol-B, nymphaeol-C, and 3'-geranylnaringenin.

JP 2016-136952 A

The objective of the present invention is to provide a novel agent for inhibiting disuse muscle atrophy.

The present inventors have newly discovered that eriocitrin has the effect of suppressing disuse muscle atrophy. Based on this novel finding, the present invention provides a new use of eriocitrin.

One aspect of the present invention is a disuse muscle atrophy inhibitor that contains eriocitrin as an active ingredient. Another aspect of the present invention is a disuse muscle atrophy inhibitor that contains a citrus extract containing eriocitrin as an active ingredient. The citrus extract may be a lemon peel extract.

The disuse muscle atrophy inhibitor of the present invention contains eriocitrin, and is therefore suitable for use in inhibiting disuse muscle atrophy. Furthermore, eriocitrin is an ingredient contained in citrus fruits such as lemons (especially lemon peel), and is therefore widely consumed as a dietary supplement. Therefore, the disuse muscle atrophy inhibitor of the present invention is highly safe to the living body and can be taken continuously over a long period of time.

One aspect of the present invention is a food composition for suppressing disuse muscle atrophy, which contains eriocitrin as an active ingredient. Another aspect of the present invention is a food composition for suppressing disuse muscle atrophy, which contains a citrus extract containing eriocitrin as an active ingredient. The citrus extract may be a lemon peel extract.

The food composition for inhibiting disuse muscle atrophy of the present invention contains eriocitrin, and is therefore suitable for use in inhibiting disuse muscle atrophy. Furthermore, eriocitrin is a component contained in citrus fruits such as lemons (especially lemon peel), and is therefore widely consumed as a food. Therefore, the food composition for inhibiting disuse muscle atrophy of the present invention is highly safe for the living body and can be taken continuously over a long period of time.

The present invention provides a novel agent for inhibiting disuse muscle atrophy.

1 shows the results of a disuse muscle atrophy inhibition test (Example 1) of lemon peel extract. (A) is a graph showing the evaluation results of gastrocnemius muscle mass (gastrocnemius muscle mass per body weight (mg/g body weight)). (B) is a graph showing the evaluation results of gastrocnemius muscle maintenance rate (the ratio (%) of denervated right hind leg gastrocnemius muscle mass to sham-operated left hind leg gastrocnemius muscle mass). 1 shows the results of a test of eriocitrin to inhibit disuse muscle atrophy (Example 2). (A) is a graph showing the evaluation results of gastrocnemius muscle mass (gastrocnemius muscle mass per body weight (mg/g body weight)). (B) is a graph showing the evaluation results of gastrocnemius muscle maintenance rate (the ratio (%) of denervated right hind limb gastrocnemius muscle mass to sham-operated left hind limb gastrocnemius muscle mass). 1 shows the results of a test (reference example) on the inhibition of disuse muscle atrophy by citric acid. (A) A graph showing the evaluation results of gastrocnemius muscle mass (gastrocnemius muscle mass per body weight (mg/g body weight)). (B) A graph showing the evaluation results of gastrocnemius muscle maintenance rate (the ratio (%) of denervated right hind leg gastrocnemius muscle mass to sham-operated left hind leg gastrocnemius muscle mass).

The following describes in detail the embodiments for implementing the present invention. However, the present invention is not limited to the following embodiments.

The disuse muscle atrophy inhibitor according to this embodiment contains eriocitrin as an active ingredient.

In this specification, "disuse muscle atrophy" is a concept that includes muscle atrophy due to disuse (e.g., muscle immobilization) and muscle atrophy due to a reduction in mechanical load in a weightless environment. The disuse muscle atrophy inhibitor according to this embodiment contains eriocitrin and is therefore capable of inhibiting disuse muscle atrophy.

Eriocitrin is a compound represented by the following formula (1): Eriocitrin is a flavonoid glycoside in which rutinose (L-rhamnosyl-D-glucose) is bound to eriodictyol.

The content of eriocitrin in the disuse muscle atrophy inhibitor of this embodiment may be 0.1 w/w% or more, 0.2 w/w% or more, 0.3 w/w% or more, or 0.5 w/w% or more. When the content of eriocitrin is within the above range, the disuse muscle atrophy inhibitory effect is more pronounced. In addition, the content of eriocitrin in the disuse muscle atrophy inhibitor of this embodiment may be 100 w/w% or less, or 95 w/w% or less.

Eriocitrin can be obtained, for example, by extraction or purification from citrus fruits, or by chemical synthesis. Commercially available eriocitrin may also be used.

The disuse muscle atrophy inhibitor according to this embodiment may contain a citrus extract containing eriocitrin as an active ingredient. By using a citrus extract containing eriocitrin as an active ingredient, the disuse muscle atrophy inhibitory effect becomes more excellent.

Examples of the citrus fruits include lemon, lime, shikuwasa, sudachi, yuzu, daidai, kabosu, and other fragrant citrus fruits, grapefruit, navel orange, Valencia orange, sour orange, hassaku, Satsuma mandarin, iyokan, ponkan, amagasaki, pomelo, and the like. The citrus extract may be derived from one or more of these citrus fruits. As the citrus fruit, at least one selected from the group consisting of lemon and lime is preferred, and lemon is more preferred, because it has a high content of eriocitrin.

Citrus fruits or their components are used as the raw material for extraction (extraction raw material). Fruit components include the peel, juice, pericarp, scutellaria, and seeds. Of the fruits or their components, it is preferable to use the peel as the extraction raw material because it is easy to obtain a large amount of eriocitrin. The above extraction raw material may be one that has been subjected to processing such as drying, freezing, processing, crushing, and sorting.

The citrus extract containing eriocitrin can be obtained by extracting the above-mentioned extraction raw material with an extraction solvent, performing solid-liquid separation to obtain an extract (extraction process), and, if necessary, purifying the extract with a synthetic adsorption resin or the like (purification process). The extraction solvent is preferably a polar solvent, and more preferably at least one extraction solvent selected from the group consisting of methanol, ethanol, and water.

The purification step is a step of purifying the citrus extract using, for example, a synthetic adsorption resin to obtain a citrus extract containing eriocitrin. The citrus extract may be a processed product that has been subjected to processing such as concentration and drying. Concentration and drying can be performed by known methods (for example, vacuum concentration and freeze drying).

The above citrus extract is preferably a lemon peel extract. This provides an even more excellent inhibitory effect on disuse muscle atrophy. The lemon peel extract is preferably an extract of lemon peel with at least one solvent selected from the group consisting of methanol, ethanol, and water, and is more preferably a water extract.

Eriocitrin can be analyzed by known methods, such as high performance liquid chromatography (HPLC) analysis.

The disuse muscle atrophy inhibitor according to this embodiment may consist of only the above-mentioned active ingredient, or may contain other ingredients acceptable for food, quasi-drugs, or pharmaceuticals in addition to the above-mentioned active ingredient. Examples of other ingredients include excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, suspending agents, etc.

Examples of excipients include lactose, sucrose, starch, dextrin, etc. Examples of binders include polyvinyl alcohol, gum arabic, tragacanth, gelatin, hydroxypropylmethylcellulose, hydroxypropylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, etc. Examples of lubricants include magnesium stearate, calcium stearate, talc, etc. Examples of disintegrants include crystalline cellulose, agar, gelatin, calcium carbonate, sodium bicarbonate, dextrin, etc. Examples of emulsifiers or surfactants include Tween 60, Tween 80, Span 80, glycerin monostearate, etc. Examples of bases include cetostearyl alcohol, lanolin, polyethylene glycol, rice bran oil, fish oil (DHA, EPA, etc.), olive oil, etc. Examples of solubilizing agents include polyethylene glycol, propylene glycol, sodium carbonate, sodium citrate, Tween 80, etc. Examples of suspending agents include Tween 60, Tween 80, Span 80, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxymethylcellulose, sodium alginate, etc.

The disuse muscle atrophy inhibitor according to this embodiment may be in any form, such as a solid (e.g., powder), liquid (a water-soluble or fat-soluble solution or suspension), or paste, and may take any dosage form, such as a powder, granules, tablets, capsules, liquid, suspension, emulsion, ointment, or plaster. It may also take the form of a controlled-release formulation.

The disuse muscle atrophy inhibitor according to this embodiment can be used as a product such as a medicine, a quasi-drug, a food composition, or a feed composition, or as an ingredient of these products. The food composition may be, for example, a health food, a functional food, a food for special dietary uses, a dietary supplement, a supplement, or a food for specified health uses. Specific examples of food compositions include bread, noodles, rice, tofu, dairy products, soy sauce, miso, confectionery, beverages, and the like. Examples of beverages include water, soft drinks, fruit juice drinks, milk drinks, alcoholic beverages, non-alcoholic beverages, sports drinks, and nutritional drinks. The above-mentioned products may be for inhibiting disuse muscle atrophy.

The content of eriocitrin in the product for inhibiting disuse muscle atrophy according to this embodiment (particularly, the food composition for inhibiting disuse muscle atrophy) may be 0.1 w/w% or more, 0.2 w/w% or more, 0.3 w/w% or more, or 0.5 w/w% or more. When the content of eriocitrin is within the above range, the disuse muscle atrophy inhibitory effect is more pronounced. In addition, the content of eriocitrin in the product for inhibiting disuse muscle atrophy according to this embodiment (particularly, the food composition for inhibiting disuse muscle atrophy) may be 100 w/w% or less, or 95 w/w% or less.

The disuse muscle atrophy inhibitor or product for inhibiting disuse muscle atrophy according to this embodiment can prevent or treat locomotive syndrome (musculoskeletal syndrome) by inhibiting disuse muscle atrophy, and therefore can also be considered as a locomotive syndrome preventive or therapeutic agent, or a locomotive syndrome preventive or therapeutic product.

The above-mentioned products may be labeled with, for example, claims that they prevent locomotive syndrome, that they have a function to support muscle building that helps maintain muscles that weaken with age and a function to help improve walking ability, that they have a function to support the maintenance of muscle mass and muscle strength that helps maintain the strength to support the body necessary for living an independent daily life, and that they help maintain leg muscle function (muscle strength necessary for movements such as standing and walking).

The disuse muscle atrophy inhibitor or product for inhibiting disuse muscle atrophy according to this embodiment may be ingested by humans or non-human mammals. The dosage (ingestion amount) of the disuse muscle atrophy inhibitor or product for inhibiting disuse muscle atrophy according to this embodiment may be, for example, 0.1 mg to 1 g, or 1 mg to 500 mg, per kg of body weight per day for an adult, converted into the amount of eriocitrin. The dosage amount may be appropriately determined depending on the condition, age, etc. of the individual.

The disuse muscle atrophy inhibitor or product for inhibiting disuse muscle atrophy according to this embodiment may be administered orally (ingested) or parenterally, but is preferably administered orally. The disuse muscle atrophy inhibitor or product for inhibiting disuse muscle atrophy may be administered once a day or in multiple doses per day, as long as the amount of eriocitrin per day is within the above range.

The disuse muscle atrophy inhibitor or product for inhibiting disuse muscle atrophy according to this embodiment can be taken continuously to achieve even better inhibition of disuse muscle atrophy. The disuse muscle atrophy inhibition according to this embodiment may be taken continuously for 4 weeks or more, 8 weeks or more, or 12 weeks or more.

The present invention will be described in more detail below with reference to the following examples. However, the present invention is not limited to the following examples. All reagents used in the examples are commercially available special grade reagents. Animal experiments were conducted in accordance with the Animal Experiment Regulations of the Sapporo Holdings Limited Value Creation Frontier Research Institute (approval number: 2017-008).

Example 1: Lemon peel extract inhibits disuse muscle atrophy
(Preparation of Lemon Peel Extract)
The lemon peel extract was prepared by the following method. After in-line juicing, twice the amount of water was added to the lemon peel, and extraction was performed at 25°C for 2 hours. The obtained extract was filtered through diatomaceous earth, and then the filtrate was filtered through MF. The obtained filtrate was concentrated under reduced pressure until the Brix value reached 40°. Dextrin (product name: NSD300, manufactured by Sanei Saccharomyces) was added to the obtained concentrate in an amount twice the amount of soluble solid content calculated with reference to the Brix value. The concentrate to which dextrin was added was then freeze-dried to obtain a powder. The obtained powder was used as a lemon peel extract (containing 0.5 w/w% eriocitrin) for testing.

(Laboratory animals and breeding conditions)
Male C57BL/6NCrSlc mice purchased from Japan SLC Co., Ltd. were used in the study. The mice were kept in an environment with a temperature of 24±1° C., humidity of 48±4%, and a 12-hour light/dark cycle (light period 8:00-20:00).

(Evaluation of the inhibitory effect of lemon peel extract on disuse muscle atrophy)
The sciatic nerve was resected in mice in which immobilization-induced muscle atrophy (disuse muscle atrophy) was induced, and the inhibitory effect of lemon peel extract on disuse muscle atrophy was evaluated.

Five-week-old male C57BL/6NCrSlc mice were pre-bred for one week on powdered feed (AIN-93M). After pre-bred, the mice were divided into groups based on body weight (test diet group and control diet group). The mice in the test diet group were fed a powdered diet in which 15.0 w/w% of AIN-93M was replaced with lemon peel extract (equivalent to a 5.0 w/w% lemon peel blend; equivalent to a 0.025 w/w% eriocitrin blend) as the test diet and bred for 14 days. The mice in the control diet group were fed a powdered diet in which 15.0 w/w% of AIN-93M was replaced with dextrin (product name: NSD300, Sanei Sugar Chemical Co., Ltd.) as the control diet and bred for 14 days.

The sciatic nerve of the right hind leg of mice in the test diet and control diet groups was resected under anesthesia to induce muscle atrophy (denervation treatment). The left hind leg of mice in the test diet and control diet groups was treated in the same way as the right hind leg, except that the sciatic nerve was not resected (sham operation). After denervation treatment and sham operation, the mice were fed the test diet and control diet, respectively, as described above, and kept for 6 days. After that, mice in both groups were dissected, and the gastrocnemius muscles of the left and right hind legs were removed. The weight of the removed gastrocnemius muscles was measured, and the gastrocnemius muscle mass (g/g body weight)) and gastrocnemius muscle retention rate (the ratio (%) of the gastrocnemius muscle mass of the denervated right hind leg to the gastrocnemius muscle mass of the sham-operated left hind leg) were calculated.

In order to eliminate differences in total food intake between the two groups, food amounts were adjusted by pair feeding.

(result)
The evaluation results of gastrocnemius muscle mass and gastrocnemius muscle maintenance rate are shown in Table 1 and Figure 1. The values of gastrocnemius muscle mass and gastrocnemius muscle maintenance rate shown in Table 1 and Figure 1 are mean ± standard error (n = 5 to 6). Statistical analysis was performed using JMP 13 (SAS) software. For gastrocnemius muscle mass, multiple comparisons were performed using Tukey's test, and for gastrocnemius muscle maintenance rate, two-group comparisons were performed using Student's t-test, with a risk rate of less than 5% considered significant.

As shown in Table 1 and Figure 1, it was confirmed that the intake of lemon peel extract inhibited the decrease in muscle weight (gastrocnemius muscle mass) caused by denervation treatment. Furthermore, the calf maintenance rate was significantly higher in the test diet group compared to the control diet group (p<0.001). These results demonstrated that lemon peel extract has an inhibitory effect on disuse muscle atrophy.

Example 2: Test for inhibiting disuse muscle atrophy with eriocitrin
(Preparation of Eriocitrin)
Eriocitrin was prepared by the following method. Ethyl acetate and distilled water were added to lemon peel extract (powder) and the mixture was partitioned to obtain an ethyl acetate fraction and a water fraction. After collecting the water fraction, n-butanol was added to the water fraction and the mixture was partitioned to obtain an n-butanol fraction and a water fraction. After collecting the n-butanol fraction, the n-butanol fraction was fractionated by medium pressure preparative liquid chromatography (LC) to collect a fraction rich in eriocitrin. The collected fraction was then fractionated by recycling preparative LC to purify eriocitrin. The purified eriocitrin was analyzed by high performance liquid chromatography (HPLC) and the purity was found to be 95% or more.

The HPLC analysis conditions for eriocitrin are shown in Table 2, the medium pressure preparative LC fractionation conditions are shown in Table 3, and the recycle preparative LC fractionation conditions are shown in Table 4.

(Evaluation of the inhibitory effect of eriocitrin on disuse muscle atrophy)
The gastrocnemius muscle mass and gastrocnemius muscle maintenance rate were evaluated in the same manner as in Example 1, except that the test diet used was a powdered feed in which 0.25 w/w% of AIN-93M was substituted with purified eriocitrin, and the control diet used was a powdered feed in which 0.25 w/w% of AIN-93M was substituted with dextrin (product name: NSD300, manufactured by Sanei Saccharomyces cerevisiae).

(result)
The evaluation results of gastrocnemius muscle mass and gastrocnemius muscle maintenance rate are shown in Table 5 and Figure 2. The values of gastrocnemius muscle mass and gastrocnemius muscle maintenance rate shown in Table 5 and Figure 2 are mean ± standard error (n = 7). Statistical analysis was performed using JMP 13 (SAS) software. For gastrocnemius muscle mass, multiple comparisons were performed using Tukey's test, and for gastrocnemius muscle maintenance rate, two-group comparisons were performed using Student's t-test, with a risk rate of less than 5% considered significant.

As shown in Table 5 and Figure 2, it was confirmed that the intake of eriocitrin inhibited the decrease in muscle weight (gastrocnemius muscle mass) caused by denervation treatment. Furthermore, the calf maintenance rate was significantly higher in the test diet group compared to the control diet group (p<0.05). These results demonstrated that eriocitrin has an inhibitory effect on disuse muscle atrophy.

[Reference example: Test to inhibit disuse muscle atrophy with citric acid]
(Evaluation of the inhibitory effect of citric acid on disuse muscle atrophy)
The gastrocnemius muscle mass and gastrocnemius muscle maintenance rate were evaluated in the same manner as in Example 1, except that the test diet was a powdered feed in which 1.0 w/w% of AIN-93M was substituted with citric acid, and the control diet was a powdered feed in which 1.0 w/w% of AIN-93M was substituted with dextrin (product name: NSD300, manufactured by Sanei Saccharomyces cerevisiae).

(result)
The evaluation results of gastrocnemius muscle mass and gastrocnemius muscle maintenance rate are shown in Table 6 and Figure 3. The values of gastrocnemius muscle mass and gastrocnemius muscle maintenance rate shown in Table 6 and Figure 3 are mean ± standard error (n = 7 to 8). Statistical analysis was performed using JMP 13 (SAS) software. For gastrocnemius muscle mass, multiple comparisons were performed using Tukey's test, and for gastrocnemius muscle maintenance rate, two-group comparisons were performed using Student's t-test, with a significance level of less than 5%.

As shown in Table 6 and Figure 3, ingestion of citric acid was not confirmed to inhibit the decrease in muscle weight (gastrocnemius muscle mass) caused by denervation treatment. In addition, no significant difference was observed in the calf maintenance rate between the test diet group and the control diet group. These results show that citric acid does not have an inhibitory effect on disuse muscle atrophy.

Lemon peel contains polyphenolic components such as eriocitrin, as well as sugars such as fructose and organic acids such as citric acid. Among these, citric acid is a component with a high content. Therefore, in Example 2 and Reference Example, in order to identify the component involved in the inhibitory effect of lemon peel extract on disuse muscle atrophy, the effects of ingestion of eriocitrin and citric acid on disuse muscle atrophy were evaluated. As a result, eriocitrin was found to have an inhibitory effect on disuse muscle atrophy (Table 5 and Figure 2), but citric acid did not have the same inhibitory effect (Table 6 and Figure 3). From these results, it was presumed that eriocitrin is the component involved in the inhibitory effect of lemon peel extract on disuse muscle atrophy.

Claims (4)

An agent that inhibits disuse muscle atrophy, containing eriocitrin as the active ingredient. An agent for inhibiting disuse muscle atrophy, comprising a citrus extract containing eriocitrin as an active ingredient, the citrus extract being a lemon peel extract, and the lemon peel extract being a water extract . A food composition for inhibiting disuse muscle atrophy, containing eriocitrin as an active ingredient. A food composition for inhibiting disuse muscle atrophy, comprising a citrus extract containing eriocitrin as an active ingredient, the citrus extract being a lemon peel extract, and the lemon peel extract being a water extract .

Images