WO2022146029A1 - Composition, for relieving muscular disease and increasing muscle mass, comprising sesame dregs - Google Patents

Abstract

The present invention relates to a composition, for relieving muscular disease and increasing muscle mass, comprising sesame dregs. An ethanol fraction of a hot-water sesame dreg extract exhibits effects including sarcopenia relief, increased muscle mass and enhanced motor ability due to same, and thus can be effectively used in relieving, preventing or treating muscle disease, increasing muscle mass and enhancing motor ability.

Description

Composition for improving muscle disease and increasing muscle mass containing sesame seed extract

The present invention was made by the project specific number 1711123462 and detailed project number E0160500-05 under the support of the Ministry of Science and ICT. ) (Main Project Expenses)”, the research project title is “Research on improvement of muscle loss in the elderly using useful food ingredients”, the lead institution is the Korea Food Research Institute, and the research period is from January 1, 2020 to December 31, 2020.

This patent application is based on Korean Patent Application No. 10-2020-0186797, filed with the Korean Intellectual Property Office on December 29, 2020, and Korean Patent Application No. 10-2021-0190006, filed with the Korean Intellectual Property Office on December 28, 2021 Priority is claimed to, and the disclosure of the above patent application is incorporated herein by reference.

The present invention relates to a composition for improving muscle disease and increasing muscle mass, including a sesame seed extract extract, and more particularly, to an improvement in muscle loss, an increase in muscle mass, and the effect of improving exercise performance due to the sesame seed extract extract.

As the quality of life of the elderly in an aging society is emerging as an important issue due to the rapid increase of the elderly population aged 65 and over, the demand for healthy lifespan extension is increasing. Skeletal muscle is the largest organ in the human body and accounts for 40 to 50% of the total body weight and is responsible for various functions of the human body. In other words, it is involved in physical activity such as maintaining the physique and walking, as well as energy homeostasis, thermogenesis, and sugar and amino acid metabolism.

Sarcopenia is one of the representative symptoms of aging, which is accompanied by a decrease in skeletal muscle mass and a decrease in muscle function. Such muscle loss is integrally manifested by an imbalance in the production and decomposition of muscle protein, an increase in muscle cell death, a decrease in muscle regeneration, and the like. Although there are some differences between individuals, it is known that the human muscle gradually decreases after the age of 40, and approximately 50% of the maximum muscle mass decreases at the age of 80. It has been reported that such muscle loss in the elderly is accompanied by a decrease in overall physical function, which lowers the quality of life of individuals, increases the death rate of the elderly without muscle, and has a much higher mortality rate in the elderly with sarcopenia even with the same disease.

The causes of sarcopenia include lack of hormones such as growth hormone and sex hormone, imbalance in the synthesis and degradation of muscle protein, inactivity, obesity, increase in inflammatory cytokines, decreased mitochondrial function, and insulin resistance. have. In particular, it is reported that obesity and visceral fat increase with increasing age, and sarcopenia is induced as inflammatory cytokines increase with this.

Exercise and diet are recommended to prevent sarcopenia. Various studies are being conducted at home and abroad for treatment and improvement materials using natural products to overcome sarcopenia, but there is no clear material yet.

On the other hand, sesame seeds are an annual herb belonging to the genus Pedaliaceae . If you look at the nutritional components of sesame, it contains 51% lipid, 20% protein, and 15% carbohydrate. Compared to rice and wheat, it is rich in calcium, phosphorus, zinc, iron, vitamins B1, B2, and unsaturated fatty acids. In addition, sesame contains 0.1- and sesaminol 0.3- and sesamin, which exhibit antioxidant effects.

In addition, after the sesame is roasted at an appropriate temperature and milked by pressing, the milking meal (hereinafter referred to as sesame meal), which is an essential by-product, contains a large amount of ingredients effective for the human body. The main active ingredients present in sesame seeds include lignan components such as sesamin, sesaminol, sesaminol, and pinoreisnol. They exist mainly in the form of water-soluble glycosides bound to glucose, and the content is about 1%.

It has been reported that the lignan glycosides have an inhibitory effect on lipid peroxidation, and that the lipid peroxidation inhibitory effect increases depending on the concentration of the lignan glycoside. In addition, lignan components of sesame seeds are known to prevent oxidation of low density lipoprotein (LDL). In particular, among the lignan components, sesaminol glucosides have been reported to have strong antioxidant and anti-atherosclerotic properties.

However, so far, the fact that the extract from the sesame seeds has an effect of treating or preventing muscle disease or increasing exercise performance is not known.

Accordingly, the present inventors confirmed that the sesame seed extract exhibits an inhibitory effect on muscle atrophy under the conditions of inducing muscle loss, and has an excellent degree of promoting differentiation from myoblasts to myotubes.

Accordingly, an object of the present invention is sarcopenia, muscular atrophy, muscle dystrophy, cardiac atrophy, atony, muscular dystrophy, muscle containing sesame seed extract It is to provide a food composition for improving at least one muscle disease selected from the group consisting of dystrophy, and myasthenia gravis.

Another object of the present invention is to provide a food composition for increasing muscle mass comprising a sesame seed extract.

Another object of the present invention is to provide a food composition for improving exercise performance comprising a sesame seed extract.

Another object of the present invention is to prevent at least one muscle disease selected from the group consisting of sarcopenia, muscular atrophy, muscular dystrophy, cardiac atrophy, dystonia, muscular dystrophy, muscular dystrophy, and myasthenia gravis, including sesame seed extract, or To provide a pharmaceutical composition for treatment.

Another object of the present invention is to improve, prevent or treat muscle diseases of the sesame seed extract; gain muscle mass; And it relates to the use of improving exercise performance.

The present invention relates to a composition for improving muscle disease and increasing muscle mass, comprising the sesame seed extract, and the ethanol fraction of the hot water extract from sesame seed according to the present invention has the effect of improving muscle loss, increasing muscle mass and improving exercise performance. indicates.

The present inventors confirmed that the ethanol fraction of hot water extract from sesame seeds exhibited an inhibitory effect on muscle atrophy under the condition of inducing muscle loss, and also increased the expression level of differentiation-related important proteins in the differentiation of myoblasts into myotubes.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention includes sesame seed extract for sarcopenia, muscular atrophy, muscle dystrophy, cardiac atrophy, atony, muscular dystrophy, muscle degeneration It is a food composition for improving at least one type of muscle disease selected from the group consisting of myasthenia gravis and myasthenia gravis.

As used herein, the term "extract" includes a solvent fraction of a crude solvent extract, a specific solvent soluble extract (solvent fraction), and a solvent fraction of a crude solvent extract, and the sesame seed extract may be in a solution, concentrate or powder state.

In the present invention, the sesame seed extract may be extracted with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid, preferably water, an aqueous methanol solution and It may be extracted with one or more solvents selected from the group consisting of an aqueous ethanol solution, for example, it may be a hot water extract obtained by extracting water as a solvent, but is not limited thereto.

The sesame seed extract hot water extract may be obtained as a water-soluble fraction after hot water extraction of defatted sesame seeds or centrifugation by dissolving dry powder of sesame seeds in water.

In one embodiment of the present invention, hot water was added to defatted sesame to prepare an extract. Specifically, a sesame extract was prepared by pulverizing the sesame meal obtained after milking sesame, and then extracting it with hot water at 100° C. for 1 hour, followed by centrifugation to obtain a supernatant. The sesame extract obtained by the above method contains sesaminol glycosides, and their content is 1 to 5% by weight, preferably 2 to 4% by weight, based on the total weight of the extract.

One aspect of the present invention relates to a method for preparing a sesame seed extract.

The sesame meal extract includes a solvent crude extract and a solvent fraction of sesame meal, as described above.

The manufacturing process of the sesame meal extract according to the present invention will be described in more detail as follows: It is extracted with an extraction solvent in an amount of about 10 to 50 times the weight of the sesame meal. After extraction, the filtrate is collected by filtration. The extraction temperature is not particularly limited, but is preferably 15 to 110°C, preferably 20 to 100°C.

The extraction process can be repeated once or several times, and a method of re-extraction after the first extraction can be adopted. This is to prevent this because the extraction efficiency may be lowered only by the first extraction.

The filtered extract thus obtained may be prepared as a powdered sesame seed extract by freeze-drying to be suitable for use.

The extraction method used in the present invention may be any method commonly used, for example, cold extraction, hot water extraction, ultrasonic extraction, or reflux cooling extraction method, but is not limited thereto.

In the present invention, the sesame meal extract may be an alcohol fraction obtained by fractionating the sesame meal hot water extract with alcohol.

Prior to fractionation of the sesame seed extract hot water extract with alcohol, a process of eluting and purifying using an ion exchange resin may be performed, and the ion exchange resin may be one selected from the group consisting of a cation exchange resin and an anion exchange resin. It may be more than one, but is not limited thereto.

In the case of using a mixture of water and alcohol as a solvent for use in preparing the alcohol fraction, the solvent is 10% to 100% (v/v), 20% to 100% (v/v), 30% to 100 % (v/v), 40% to 100% (v/v), 50% to 100% (v/v), 10% to 80% (v/v), 20% to 80% (v/v) , 30% to 80% (v / v), 40% to 80% (v / v) or 50% to 80% (v / v) of a linear or branched alcohol solution having 1 to 4 carbon atoms, for example, It may be an aqueous solution of 60% (v/v) straight-chain or branched alcohol having 1 to 4 carbon atoms, but is not limited thereto.

In addition, the alcohol aqueous solution may be at least one selected from the group consisting of methanol aqueous solution, ethanol aqueous solution, propanol aqueous solution, and butanol aqueous solution, for example, may be an ethanol aqueous solution, but is not limited thereto.

The alcohol fraction of the sesame meal hot water extract may be an ethanol fraction of the sesame meal hot water extract, and the ethanol fraction may contain sesaminol glycosides.

As used herein, the term "sesaminol glycoside" refers to a form in which sesaminol is combined with glucose. Sesaminol glycosides are classified into sesaminol-1 glycosides (sesaminol monoglucoside), sesaminol-2 glycosides (sesaminol diglucoside) and sesaminol-3 glycosides (sesaminol triglucoside) according to the number of glucose bound to sesaminol.

Preferably, the sesaminol glycoside according to the present invention can be isolated and purified from nature. Most preferably, the sesaminol glycoside can be isolated and purified from sesame. Sesame seeds contain lignan components such as sesaminol, sesaminol glycosides, sesamin and sesamolin. Since most of the above lignan components are water-soluble, in order to separate them, it is preferable to separate the oil-soluble component from the sesame and then separate the water-soluble material.

In one embodiment of the present invention, sesaminol glycosides were separated from the extract using a cation exchange resin after hot water extraction of the sesame meal obtained after milking sesame. However, the method for isolating the sesaminol glycoside according to the present invention is not limited thereto, and any method capable of separating the sesaminol glycoside from sesame may be used.

In the present invention, sarcopenia may be obese sarcopenia or senescent sarcopenia.

When the food composition of the present invention is used as a food additive, the food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, and the like, and includes all foods in a conventional sense.

The beverage may contain various flavoring agents or natural carbohydrates as additional ingredients. As the above-mentioned natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used. . The ratio of the natural carbohydrate may be appropriately determined by the selection of those skilled in the art.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.

Another aspect of the present invention is a food composition for increasing muscle mass comprising a sesame seed extract.

In the present invention, the sesame seed extract may be extracted with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid, for example, water as a solvent It may be an extracted hot water extract, but is not limited thereto.

In the present invention, the sesame meal extract may be an alcohol fraction obtained by fractionating the sesame meal hot water extract with alcohol.

The alcohol may be at least one selected from the group consisting of methanol, ethanol, propanol and butanol, for example, may be ethanol, but is not limited thereto.

Prior to fractionation of the sesame seed extract hot water extract with alcohol, a process of eluting and purifying using an ion exchange resin may be performed, and the ion exchange resin may be one selected from the group consisting of a cation exchange resin and an anion exchange resin. It may be more than one, but is not limited thereto.

The composition for increasing muscle mass is from the group consisting of transversus abdominis (TA), extensor Digitorum Longus (EDL), calf (Gastroc), soleus, quadriceps, and triceps (Triceps). It may be to increase one or more selected muscles, preferably to increase the quadriceps or triceps, but is not limited thereto.

Another aspect of the present invention is a food composition for improving exercise performance comprising a sesame seed extract.

In the present invention, the sesame seed extract may be extracted with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid, for example, water as a solvent It may be an extracted hot water extract, but is not limited thereto.

In the present invention, the sesame meal extract may be an alcohol fraction obtained by fractionating the sesame meal hot water extract with alcohol.

The alcohol may be at least one selected from the group consisting of methanol, ethanol, propanol and butanol, for example, may be ethanol, but is not limited thereto.

Prior to fractionation of the sesame seed extract hot water extract with alcohol, a process of eluting and purifying using an ion exchange resin may be performed, and the ion exchange resin may be one selected from the group consisting of a cation exchange resin and an anion exchange resin. It may be more than one, but is not limited thereto.

Another aspect of the present invention is sarcopenia, muscular atrophy, muscular dystrophy, cardiac atrophy, dystonia, muscular dystrophy, muscle dystrophy, and one or more muscle diseases selected from the group consisting of myasthenia gravis, including sesame seed extract, prevention or treatment It is a pharmaceutical composition for

In the present invention, the sesame seed extract may be extracted with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid, for example, water as a solvent It may be an extracted hot water extract, but is not limited thereto.

In the present invention, the sesame meal extract may be an alcohol fraction obtained by fractionating the sesame meal hot water extract with alcohol.

The alcohol may be at least one selected from the group consisting of methanol, ethanol, propanol and butanol, for example, may be ethanol, but is not limited thereto.

Prior to fractionation of the sesame seed extract hot water extract with alcohol, a process of eluting and purifying using an ion exchange resin may be performed, and the ion exchange resin may be one selected from the group consisting of a cation exchange resin and an anion exchange resin. It may be more than one, but is not limited thereto.

In the present invention, sarcopenia may be obese sarcopenia or senescent sarcopenia.

The pharmaceutical composition of the present invention may be used as a pharmaceutical composition comprising a pharmaceutically effective amount of a sesame seed extract and/or a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the above-mentioned sesame seed extract.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

The pharmaceutical composition according to the present invention may be administered to mammals including humans by various routes. The administration method may be any method commonly used, for example, it may be administered by routes such as oral, skin, intravenous, intramuscular, subcutaneous, and the like.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity of the patient, An ordinarily skilled physician can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. Or it can be prepared by introducing into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule, or gel (eg, hydrogel), and may additionally include a dispersant or stabilizer. .

The present invention relates to a composition for improving muscle disease and increasing muscle mass comprising a sesame seed extract, wherein the sesame seed extract exhibits an effect on improving muscle loss, increasing muscle mass and improving exercise performance thereby, effectively treating muscle disease improvement, prevention or treatment; gain muscle mass; And it can be used to improve exercise performance.

1 is a sesaminol-2 glycoside (sesaminol diglucoside; SDG) and sesaminol-3 glycoside (sesaminol It is a graph showing the HPLC chromatogram result of triglucoside; STG).

Figure 2 is a western blotting showing the effect of the sesame seed ethanol fraction on protein expression in C2C12 myotubes treated with palmitic acid (PA) according to an embodiment of the present invention; This is the result picture.

Figure 3a is a graph showing the mRNA expression level of MURF according to the treatment amount of the sesame seed ethanol fraction in C2C12 myotube cells treated with PA according to an embodiment of the present invention.

Figure 3b is a graph showing the mRNA expression level of Atrogin-1 according to the treatment amount of the sesame seed ethanol fraction in C2C12 myotube cells treated with PA according to an embodiment of the present invention.

Figure 4a is a fluorescence micrograph of myotube cells according to the treatment amount of each concentration of sesame seed ethanol fraction in C2C12 myotube cells treated with dexamethasone (DEX) according to an embodiment of the present invention.

Figure 4b is a graph showing the fusion index (fusion index) according to the treatment amount of each concentration of sesame seed ethanol fraction in C2C12 myotube cells treated with DEX according to an embodiment of the present invention.

5 is a graph showing the mRNA expression level of Atrogin-1 according to the treatment amount by concentration of the sesame seed ethanol fraction in C2C12 myotube cells treated with DEX according to an embodiment of the present invention.

6 is a western blotting result photograph showing the protein expression level according to the amount of treatment of the ethanol fraction from sesame seed during differentiation of C2C12 myotube cells according to an embodiment of the present invention.

7A is a graph showing the mRNA expression levels of Total MHC and MHC2A according to the amount of ethanol fraction treated in C2C12 myotube cells according to an embodiment of the present invention.

7B is a graph showing the mRNA expression levels of MyoD, MyoG, and Myf5 according to the amount of treatment of the sesame seed ethanol fraction upon differentiation of C2C12 myotube cells according to an embodiment of the present invention.

Figure 8a is a graph showing the change in body weight according to the sesame seed ethanol fraction addition diet for the aging muscle loss model prepared according to an embodiment of the present invention.

Figure 8b is a graph showing the change in muscle weight according to the sesame seed ethanol fraction addition diet for the aging muscle loss model prepared according to an embodiment of the present invention.

Figure 9a is a graph showing the change in grip strength (Grip strength) according to the sesame seed ethanol fraction addition diet for the aging muscle reduction model prepared according to an embodiment of the present invention.

Figure 9b is a graph showing the change in total running time (Total running time) according to the sesame seed ethanol fraction addition diet for the aging muscle loss model prepared according to an embodiment of the present invention.

Figure 9c is a graph showing the change in the distance to exhaustion (Distance to exhaustion) according to the sesame seed ethanol fraction addition diet for the aging muscle loss model prepared according to an embodiment of the present invention.

9d is a graph showing the change in stride length according to the sesame seed ethanol fraction addition diet for the aging muscle loss model prepared according to an embodiment of the present invention.

Figure 10a is a graph showing the change in the mRNA expression level of the muscle atrophy marker gene MuRF1 according to the sesame seed ethanol fraction addition diet for the aging muscle loss model prepared according to an embodiment of the present invention.

Figure 10b is a graph showing the change in the mRNA expression level of the muscle atrophy marker gene Atrogin-1 according to the sesame seed ethanol fraction addition diet for the aging muscle loss model prepared according to an embodiment of the present invention.

The present invention includes sesame seed extract for sarcopenia, muscular atrophy, muscle dystrophy, cardiac atrophy, atony, muscular dystrophy (muscular dystrophy), muscle dystrophy, and It relates to a food composition for improving at least one muscle disease selected from the group consisting of myasthenia gravis.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight)% solid/solid, (weight/volume)%, and (weight/volume)% for solid/solid, and Liquid/liquid is (vol/vol) %.

Experimental results are expressed as mean ± standard error per experimental group. Statistical analysis was performed using GraphPad Prism, version 8.3.1 software, and comparison between two groups was evaluated by unpaired t-test. After way ANOVA analysis, duncan's multiple range test was used.

Example 1: Preparation of Sesame Seed Extract

1-1. Preparation of hot water extract

Domestic sesame seeds were purchased from the market, selected, washed, dried, roasted, and then milked. After preparing sesame oil, the obtained sesame meal was powdered to pass through 40 mesh, stored in a -20°C freezer, and used as a sample.

Hot water was added to the sesame meal, followed by hot water extraction at 100° C. for 1 hour, and then the extract was filtered with three layers of gauze and centrifuged at 6,000 rpm for 30 minutes (Dupont Sorvall RC-5C, SORVALL, USA) to obtain a supernatant, The supernatant was filtered again with Whatman filter paper (Whatman filter paper NO. 2).

1-2. Preparation of ethanol fractions from hot water extracts

The obtained sesame meal hot water extract was concentrated by heating at 100° C., then it was filled in a cation exchange resin (Diaion HP 20 column, Mitsubishi Co., Ltd.) and allowed to stand overnight to elute. After that, 60% ethanol was added to the eluate, extracted twice, and the extracted 60% ethanol extract was collected and concentrated under reduced pressure (Rotary vacuum evaporator R-114, Swiss), then freeze-dried using a freeze dryer and powdered as an ethanol fraction. did

The hot water extract of sesame meal and the separated and purified ethanol fraction were analyzed using HPLC and mass spectrometry. At this time, μ-bondapak C18 column was used as HPLC analysis conditions, and a solvent in which MeOH:H ₂ O was mixed in a ratio of 8:2 as a mobile phase was used at a flow rate of 0.8 mL/min, and the detector was UV detector. A 290 nm signal was measured.

As can be seen in FIG. 1 , the measurement result shows that the hot water extract and the separated It was confirmed that the ethanol fraction contained sesaminol-2 glycosides (sesaminol diglucoside) and sesaminol-3 glycosides (sesaminol triglucoside).

Example 2: Inhibitory effect of palmitic acid-induced muscle atrophy by sesame seed extract

2-1. Cell culture and differentiation of myotube cells

Mouse C2C12 myoblasts (Mouse C2C12 myoblast, CRL1772, American type cell collection (ATCC), USA) were maintained with DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% Fetal bovine serum (FBS). After reaching 95%-confluence in a 6-well plate, the medium was replaced with DMEM medium containing 2% horse serum every 2 days. It was cultured for 4 days and differentiated into myotube cells.

2-2. Confirmation of the effect of improving muscle atrophy induced by palmitic acid

C2C12 myotube cells on the 4th day of differentiation were treated with 1.0, 2.5 or 5.0 ug/ml (solid content) of sesame seed meal ethanol fraction (SG) for 24 hours, and then 0.5 mM bound with bovine serum albumin (BSA). of palmitic acid (hereinafter PA) was treated to induce muscle loss in muscle cells. After 24 hours, cells were harvested and RT-qPCR and western blotting were performed.

In performing Western blotting, proteins were extracted from the cells cultured using a buffer (RIPA buffer, Thermo Scientific, Waltham, MA, USA), and then electrophoresis was performed using an SDS-PAGE gel. After blocking the protein-transferred membrane with 5% skim milk for 1 hour, primary antibodies MHC (Cell Signaling, Danvers, MA, USA), MHC1, MHC2A, MHC2B (Developmental) Studies Hybridoma Bank, Iowa City, Iowa, USA), MuRF1, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and secondary antibody (Goat anti-Rabbit IgG (H+L) Secondary Antibody [HRP] After attachment, the expression of the protein was measured using an ECL solution.

As can be seen in FIG. 2, protein expression of total MHC (myosin heavy chain) and MHC2A (oxidative, fast type) was inhibited by treatment with palmitic acid, but was effectively increased by treatment with ethanol fraction from sesame seeds.

In addition, in performing RT-qPCR, palmitic acid-treated C2C12 myotube cells were washed twice with PBS, then the cells were obtained, and the user protocol of the RNA extraction kit (Nucleo RNA Spin Ⅱ, Macherey-Nagel, GmBH&Co, GA) ( protocol), and cDNA was synthesized from the extracted RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) and qPCR was performed. Real-time PCR analysis was performed according to the manufacturer's recommended cycling conditions of the equipment (Applied Biosystems 7900 HT thermal cycler (Applied Biosystems)), and the expression level of each mRNA was 18s as a standard (endogeneous control). The fold change was calculated by calculation of 2 ^-ΔΔCt .

SEQ ID NO:	designation	order
One	MuRF1 Forward Primer	TGT CTC ACG TGT GAG GTG CCT A
2	MuRF1 reverse primer	CAC CAG CAT GGA GAT GCA GTT AC
3	Atrogin-1 Forward Primer	AAG GCT GTT GGA GCT GAT AGC A
4	Atrogin-1 reverse primer	CAC CCA CAT GTT AAT GTT GCC C
5	18s forward primer	CTC AAC ACG GGA AAC CTC AC
6	18s reverse primer	CGC TCC ACC AAC TAA GAA CG

Relative expression level of mRNA	BSA	PA	PA + SG 1.0 ug/ml	PA + SG 2.5 ug/ml	PA + SG 5.0 ug/ml
MURF1	1.00 ± 0.03	2.16 ± 0.16	1.78 ± 0.01	1.33 ± 0.05	1.14 ± 0.01
Atrogin-1	1.00 ± 0.03	1.48 ± 0.00	1.35 ± 0.10	1.24 ± 0.11	1.22 ± 0.03

As can be seen in Table 2, Figures 3a and 3b, the expression of MURF1 and Atrogin-1 (muscle-specific ubiquitin ligase) mRNA involved in muscle loss was significantly increased by treatment with palmitic acid, but sesame seed ethanol It was shown that the increased expression of MuRF1 mRNA was significantly inhibited in a concentration-dependent manner by the treatment of the fraction.

2-3. Confirmation of the effect of improving muscle atrophy induced by dexamethasone

In the C2C12 cell line, it was confirmed whether the ethanol fraction (SG) from sesame seeds at each concentration relieved dexamethasone (DEX)-induced muscle atrophy.

Specifically, on the 4th day of differentiation, C2C12 cells were treated with 1.0, 2.5, or 5.0 ug/ml of sesame seed ethanol fraction (SG) and 50 μM of dexamethasone for 24 hours to obtain cells, followed by immunofluorescence and quantitative RT- qPCR was performed.

In performing the immunofluorescence method, the obtained cells were fixed with 4% formaldehyde at room temperature for 15 minutes, then permeablized with a 0.05% saponin solution for 30 minutes at room temperature, and then with 1% BSA solution. Blocked for 30 minutes. After reacting with the primary antibody (Total MHC) at 4°C overnight (overnight) conditions, washing with PBS, reacting the secondary fluorescent antibody at room temperature for 30 minutes, staining the nucleus with DAPI for 1 minute, and then using a fluorescence microscope The morphology of myotube cells was observed, and the fusion index was derived as an index indicating the degree of cell differentiation by expressing the number of nuclei in the root canal from the total nucleus as a percentage.

As can be seen in FIG. 4a , as a result of microscopic imaging after fluorescence staining of total MHC, it was observed that the atrophy of myotube cells by DEX treatment was improved by treatment with sesame seed ethanol fraction (SG).

As can be seen in FIG. 4b , it was confirmed that the DEX-treated group significantly inhibited the degree of cell differentiation, whereas the SG-treated group improved the inhibition of cell differentiation compared to the DEX control group.

RT-qPCR was performed in the same manner as in Example 2-2 to synthesize cDNA, then primers and SYBR Green master mix were added, and amyotrophic markers were used using Step One Plus Real Time PCR system (Applied Biosystems) equipment. The mRNA expression of the Atrogin-1 gene, one of the genes, was analyzed.

As can be seen in FIG. 5 , the expression level of Atrogin-1 gene in cells was significantly increased by DEX treatment, but DEX-induced muscle atrophy in cells treated with 5 μg/ml of sesame seed ethanol fraction (SG) was observed. was confirmed to be inhibited.

Example 3: Confirmation of myotube cell differentiation activity

On the fourth day of differentiation of C2C12 myotube cells prepared according to Example 2-1, 1.0, 2.5 or 5.0 ug/ml of sesame seed ethanol fraction (SG) was treated for 24 hours and cultured. Western blotting was performed in the same manner as in Example 2-2, but primary antibodies were used as MHC, MHC2A, MyoD, MyoG, and Myf5.

As can be seen in FIG. 6 , in the cells treated with the sesame seed ethanol fraction, the protein expression of total MHC and MHC2A was increased in a concentration-dependent manner, compared to the no-addition control group, and MyoD, MyoG and Myf5, important proteins related to the early differentiation of myotube cells, also It was found to increase with the addition of the ethanol fraction.

Also, mRNA expression level was measured in the same manner as in Example 2-2.

SEQ ID NO:	designation	order
7	MHC2A Forward Primer	AAG CGA AGA GTA AGG CTG TC
8	MHC2A reverse primer	GTG ATT GCT TGC AAA GGA AC
9	MyoD Forward Primer	CCA CTC CGG GAC ATA GAC TTG
10	MyoD reverse primer	AAA AGC GCA GGT CTG GTG AG
11	MyoG Forward Primer	GAG ACA TCC CCC TAT TTC TAC CA
12	MyoG reverse primer	GCT CAG TCC GCT CAT AGC C
13	Myf5 Forward Primer	ATC CAG GTA TTC CCA CCT GCT
14	Myf5 reverse primer	ACT GGT CCC CAA ACT CAT CCT

Relative expression level of mRNA	untreated group	SG 1.0 ug/ml	SG 2.5 ug/ml	SG 5.0 ug/ml
Total MHC	1.00 ± 0.13	1.28 ± 0.08	1.12 ± 0.04	1.81 ± 0.09
MHC2A	1.00 ± 0.11	1.41 ± 0.13	1.58 ± 0.03	2.42 ± 0.19
MyoD	1.00 ± 0.11	1.43 ± 0.17	1.36 ± 0.04	1.70 ± 0.05
MyoD	1.00 ± 0.12	1.25 ± 0.10	1.32 ± 0.07	1.96 ± 0.05
Myf5	1.00 ± 0.19	1.55 ± 0.11	1.54 ± 0.05	2.58 ± 0.38

Table 4, As can be seen in FIGS. 7A and 7B , the relative mRNA expression levels of total MHC, MHC2A, MyoD, MyoG and Myf5 relative to the untreated group were also increased by treatment with the ethanol fraction from sesame seeds.

Example 4: Confirmation of the effect of sesame seed extract on the aging muscle loss model

4-1. Preparation of senescent muscle loss model

Animals were subjected to natural aging up to 16 months of age using C57BL/6J mice and used in the experiment. The experiment was carried out for a total of 9 weeks, and as described above, the sesame seed ethanol fraction (SG) was administered and reared for 9 weeks for the aging muscle reduction model due to natural aging.

The experimental diet was prepared according to the composition of the AIN-93 diet, and the experimental group was a normal mouse group (14 weeks old, Young), an aging control group (16 months old, Old), and a SG supplemented diet group (16 months old, 0.25SG: 0.25% diet). group; 0.5SG: 0.5% SG added diet group). Water and experimental diet were allowed to be freely consumed. During the experimental period, food intake was measured twice a week and body weight was measured once a week for a certain period of time.

4-2. Effect of improving muscle loss on aging muscle loss model

Four experimental groups prepared according to Example 4-1 were bred for 9 weeks. Thereafter, blood was collected from the abdominal aorta of the experimental animal, and the collected blood was centrifuged to separate serum. Each muscle (TA: Transversus Abdominis, EDL: Extensor Digitorum Longus, Gastroc: calf, Soleus: soleus, Quadriceps: quadriceps, Triceps: triceps) was weighed and separated from serum - Stored at 80° C. until analysis.

As can be seen in Figures 8a and 8b, in the aging control group (Old), compared to the normal mouse group (Young), the weight was significantly increased by aging, but it was confirmed that the muscle weight was decreased. In the SG-added diet group (0.25G, 0.5G), there was no change in body weight compared to the aging control group (Old), but it was confirmed that the muscle weight increased.

4-3. Effect of improving muscle function on aging muscle loss model

Changes in muscle function were confirmed through grip strength test, exercise load test, and gait test for the four experimental groups prepared according to Example 4-1.

The grip strength test was performed by continuously performing the grip test 5 times on the experimental animal, and the average value excluding the highest and lowest values among the 5 trials was used as data, and the numerical value on the data indicates the value of the measured force. Values were normalized to body weight (g) to indicate grip strength.

In the exercise load test using a treadmill, after exercising at 10 m/min for 4 minutes, the speed is increased to 2 m/min every 4 minutes, and the maximum speed is 20 m/min for experimental animals that have been adapted to the test with prior training. It was done in a way that didn't pass. The maximum exercise performance was converted to the running distance until staying on the grid for more than 10 seconds after stopping the exercise, and total running time and distance to exhaustion were measured.

The gait test was performed with footstep analysis. The forelimbs and hind legs of each mouse were coated in distinct colors using non-toxic paint, and the mice were allowed to walk on a white sheet attached to a high platform, leaving footprints. As a result of the experiment, stride length was measured at 4 to 6 consecutive strides (the distance between the forelimbs and hind legs).

As can be seen in Figures 9a to 9d, in the aging control group (Old), compared to the normal mouse group (Young), the grip strength, the total running time, the distance to exhaustion, and the stride length all significantly decreased, but the SG-added diet group (0.25G) , 0.5G) significantly increased compared to the aging control group (Old), confirming that SG had an improvement effect on the decrease in muscle function due to aging.

4-4. Improvement effect of muscle atrophy markers on aging muscle loss model

The mRNA expression of the muscle atrophy marker genes MuRF1 and Atrogin-1 was analyzed in the muscles from the four experimental groups prepared according to Example 4-2.

Specifically, RNA was extracted from muscle tissue with the Qiagen RNeasy Fibrous Tissue Mini Kit (Qiagen Inc., Hilden, Germany), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan), followed by SYBR Green master mix was added, and mRNA expression of the gene was measured using the Step One Plus Real Time PCR system (Applied Biosystems) equipment.

As can be seen in FIGS. 10a and 10b, the expression of MuRF1 and Atrogin-1 was significantly increased by aging in the aging control group (Old) compared to the normal mouse group (Young), but in the SG-added diet group (0.25G, 0.5) In G), compared to the aging control group (Old), the expression of MuRF1 and Atrogin-1 decreased in a concentration-dependent manner of SG, thereby significantly improving muscle atrophy.

The present invention relates to a composition for improving muscle disease and increasing muscle mass, comprising a sesame seed extract extract, and more particularly, to an improvement in muscle loss, an increase in muscle mass, and the effect of improving exercise performance due to the sesame seed extract extract.

Claims (14)

1. Sacopenia, muscular atrophy, muscle dystrophy, cardiac atrophy, atony, muscular dystrophy, muscle dystrophy, and myasthenia gravis containing sesame seed extract A food composition for improving at least one muscle disease selected from the group.
2. The food composition for improving muscle disease according to claim 1, wherein the sesame meal extract is extracted with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid.
3. The food composition for improving muscle disease according to claim 1, wherein the sesame meal extract is an alcohol fraction obtained by fractionating the sesame meal hot water extract with alcohol.
4. According to claim 1, wherein the sarcopenia is obese sarcopenia or senile sarcopenia, the food composition for improving muscle disease.
5. A food composition for increasing muscle mass comprising a sesame seed extract.
6. The food composition for increasing muscle mass according to claim 5, wherein the sesame meal extract is extracted with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid.
7. The food composition for increasing muscle mass according to claim 5, wherein the sesame seed extract is an alcohol fraction obtained by fractionating the sesame seed extract hot water extract with alcohol.
8. A food composition for improving exercise performance comprising a sesame seed extract.
9. The food composition for improving exercise performance according to claim 8, wherein the sesame meal extract is extracted with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid. .
10. The food composition for improving exercise performance according to claim 8, wherein the sesame meal extract is an alcohol fraction obtained by fractionating the sesame meal hot water extract with alcohol.
11. Sacopenia, muscular atrophy, muscle dystrophy, cardiac atrophy, atony, muscular dystrophy, muscle dystrophy, and myasthenia gravis containing sesame seed extract A pharmaceutical composition for preventing or treating at least one muscle disease selected from the group.
12. The agent for preventing or treating muscle disease according to claim 11, wherein the sesame seed extract is extracted with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, subcritical fluids and supercritical fluids. academic composition.
13. The pharmaceutical composition for preventing or treating muscle disease according to claim 11, wherein the sesame seed extract is an alcohol fraction obtained by fractionating the sesame seed extract hot water extract with alcohol.
14. The pharmaceutical composition for preventing or treating muscle disease according to claim 11, wherein the sarcopenia is obese sarcopenia or senescent sarcopenia.

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